RUANN JANSER SOARES DE CASTROrepositorio.unicamp.br/.../Castro_RuannJanserSoaresde_D.pdfCastro,...

i

RUANN JANSER SOARES DE CASTRO

EXPERIMENTAL MIXTURE DESIGN AS A TOOL FOR PROTEASES

PRODUCTION BY Aspergillus niger AND OBTAINING OF PROTEIN

HYDROLYSATES WITH MULTIPLE FUNCTIONAL AND BIOLOGICAL

PROPERTIES

APLICAÇÃO DA FERRAMENTA DE PLANEJAMENTO EXPERIMENTAL DE

MISTURAS COMO ESTRATÉGIA PARA PRODUÇÃO DE PROTEASES POR Aspergillus

niger E OBTENÇÃO DE HIDROLISADOS PROTEICOS COM MÚLTIPLAS

PROPRIEDADES FUNCIONAIS E BIOLÓGICAS

CAMPINAS

2015

iii

UNIVERSIDADE ESTADUAL DE CAMPINAS

FACULDADE DE ENGENHARIA DE ALIMENTOS

RUANN JANSER SOARES DE CASTRO

EXPERIMENTAL MIXTURE DESIGN AS A TOOL FOR PROTEASES PRODUCTION

BY Aspergillus niger AND OBTAINING OF PROTEIN HYDROLYSATES WITH

MULTIPLE FUNCTIONAL AND BIOLOGICAL PROPERTIES

APLICAÇÃO DA FERRAMENTA DE PLANEJAMENTO EXPERIMENTAL DE

MISTURAS COMO ESTRATÉGIA PARA PRODUÇÃO DE PROTEASES POR Aspergillus

niger E OBTENÇÃO DE HIDROLISADOS PROTEICOS COM MÚLTIPLAS

PROPRIEDADES FUNCIONAIS E BIOLÓGICAS

Orientadora: Prof.ª Dr.ª Helia Harumi Sato

CAMPINAS

2015

Tese apresentada à Faculdade de Engenharia de Alimentos da

Universidade Estadual de Campinas como parte dos requisitos

exigidos para a obtenção do título de Doutor em Ciência de Alimentos

Thesis presented to the School of Food Engineering of the

University of Campinas in partial fulfillment of the requirements

for the degree of Doctor in the area of Food Science

ESTE EXEMPLAR CORRESPONDE À VERSÃO

FINAL DA TESE DEFENDIDA PELO ALUNO RUANN

JANSER SOARES DE CASTRO E ORIENTADA PELA

PROF.ª DR.ª HELIA HARUMI SATO

Assinatura da orientadora

Ficha catalográfica

Universidade Estadual de Campinas

Biblioteca da Faculdade de Engenharia de Alimentos

Helena Joana Flipsen - CRB 8/5283

Castro, Ruann Janser Soares de, 1987-

C279e CasExperimental mixture design as a tool for proteases production by Aspergillus

niger and obtaining of protein hydrolysates with multiple functional and biological

properties / Ruann Janser Soares de Castro. – Campinas, SP : [s.n.], 2015.

CasOrientador: Hélia Harumi Sato.

CasTese (doutorado) – Universidade Estadual de Campinas, Faculdade de

Engenharia de Alimentos.

Cas1. Protease. 2. Hidrolisados proteicos. 3. Atividade biológica. 4. Propriedades

funcionais. 5. Planejamento experimental de misturas. I. Sato, Hélia Harumi,1952-.

II. Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos.

III. Título.

Informações para Biblioteca Digital

Título em outro idioma: Aplicação da ferramenta de planejamento experimental de misturas

como estratégia para a produção de proteases por Aspergillus niger e obtenção de hidrolisados

proteicos com múltiplas propriedades funcionais e biológicas

Palavras-chave em inglês:Proteases

Protein hydrolysates

Biological activity

Functional properties

Experimental mixture design

Área de concentração: Ciência de Alimentos

Titulação: Doutor em Ciência de Alimentos

Banca examinadora:Hélia Harumi Sato [Orientador]

Juliano Lemos Bicas

Júnio Cota Silva

Luciana Francisco Fleuri

Marcela Pavan Bagagli

Data de defesa: 02-03-2015

Programa de Pós-Graduação: Ciência de Alimentos

iv

v

Banca examinadora

Profa. Dra. Hélia Harumi Sato

Orientadora – DCA/FEA/UNICAMP

Prof. Dr. Juliano Lemos Bicas

Membro Titular – DCA/FEA/UNICAMP

Dr. Júnio Cota Silva

Membro Titular – VTT Brasil Pesquisa e Desenvolvimento

Profa. Dra. Luciana Francisco Fleuri

Membro Titular – UNESP

Dra. Marcela Pavan Bagagli

Membro Titular – Lanagro/MAPA

Prof. Dr. Alexandre Leite Rodrigues de Oliveira

Membro Suplente – Instituto de Biologia/UNICAMP

Dr. Francisco Fábio Cavalcante Barros

Membro Suplente - INPI

Profa. Dra. Luciana Ferracini dos Santos

Membro Suplente – UNIARARAS

vii

Dedico este trabalho à minha família e aos

meus amigos por todo apoio, carinho e companheirismo.

ix

Agradecimentos

Ao meu querido e bom Deus por estar comigo em todos os momentos me dando força e a certeza

de que Ele está no comando de tudo.

À mainha e ao “pain”, Ivoneide e Antonio, por serem as pessoas que mais se alegram pelas

minhas conquistas, por todo amor, compreensão e apoio!

À minha querida irmã, Ruanna, pelo companheirismo, apoio e pelos ótimos momentos

compartilhados.

Aos meus tios, tias, primos e avós por serem pessoas tão maravilhosas e que me fazem tão bem.

À professora Hélia, pelo cuidado, carinho, atenção, preocupação, paciência, dedicação e por todo

conhecimento compartilhado ao longo desses anos de muito aprendizado e crescimento.

Aos meus amigos da Engenharia de Alimentos da UFC: Renata, Moara, Talita, Thiago, Monique,

Millena, Delane, Tatiane, Karina, Niédila e Cinthia por estarem sempre presentes apesar da

distância, pelo incentivo, carinho e amizade sincera.

Às professoras Suzana Cláudia e Claudia Martins da UFC, por terem me ensinado a base da

pesquisa e pelo conhecimento imprescindível que tem me acompanhado ao longo desses anos.

À professora Maria do Carmo Passos Rodrigues da UFC, pelos ensinamentos, apoio, carinho,

torcida e pelas longas horas de conversa. Por ser uma pessoa tão especial, que me incentiva a ir

cada vez mais longe e se alegra por cada conquista alcançada.

À professora Elizabeth Mary Cunha da Silva da UFC pelo enorme carinho e consideração.

Ao Dr. Gustavo Saavedra da Embrapa Agroindústria Tropical de Fortaleza pela oportunidade de

estágio, incentivo e conhecimento compartilhado.

Aos meus amigos da Embrapa Agroindústria Tropical: Adriana, Ana Paula, Andréa, Carina,

Carol, Cyntia, Genilton, Helder, Janaína, Kally, Leise, Luciana, Manuella, Mariza, Millena,

Myrella, Natália Lima, Natália Moura, Rakel e Virna, por sempre me receberem de portas

abertas, pela amizade sincera e por todos os momentos de apoio, incentivo e força.

À delegação cearense da FEA: Aliciane, Ana Laura, Bruna, Carine, Carol, Jessika, Mirela e

Wellington por trazerem um pouco do Ceará para Campinas.

x

Aos meus amigos do Laboratório de Bioquímica de Alimentos da FEA: André, Bia, Bruna,

Camilo, Dani, Débora, Elaine, Erica, Eulália, Fabíola, Fernanda, Gilberto, Giulia, Isabela,

Jessika, Joelise, Lívia, Marcela, Paula Menezes, Paula Speranza, Ricardo, Tati, Val e Viviane,

pelos momentos de descontração, ajuda e acolhimento.

Agradecimento especial à Fabíola pela demonstração diária de carinho, cuidado e

companheirismo. À Paulinha pelas horas de conversa e descontração, pelos conselhos e apoio. À

Val e à Bia por todo o carinho, atenção e força. À Vivi pela companhia diária nos almoços, pelo

cuidado e amizade.

Às minhas alunas de iniciação científica Juliana Albernaz, Marília Soares e Tânia Nishide por me

darem a oportunidade de orientá-las, pelo trabalho desenvolvido sempre com muita

responsabilidade e competência e pela ótima convivência.

À Bianca Pelici, à Paula Okuro e ao Tainan pela amizade, parceria nas corridas e treinos e por

estarem sempre a postos para ajudar.

Aos amigos adquiridos ao longo desses anos na república, disciplinas e outros laboratórios:

Alaíde, Angélica, Cyntia Cabral, David, Janclei, Luiz Vieira, Manu, Renata, Rodrigo, Tiago e

Verônica.

Ao Dr. Marcio Schmiele e ao Laboratório de Cereais, Raízes e Tubérculos pelo auxílio nas

análises de granulometria e composição centesimal dos resíduos agroindustriais utilizados neste

estudo.

Ao professor Alexandre Oliveira e ao Rodrigo Fabrizzio do Laboratório de Regeneração Nervosa

(IB-Unicamp) pelo suporte e auxílio dados para execução dos experimentos de atividade anti-

adipogênica.

Ao Gepea pela grande oportunidade de participar como orientador em alguns projetos, pela

competência e seriedade sempre presentes durante os serviços prestados.

Aos alunos de graduação da FEA que tive o privilégio de ser PED nas disciplinas de Bioquímica

e que foram tão importantes para o meu amadurecimento profissional.

A todos os professores que fazem parte da FEA pela competência e contribuição com valiosos

conhecimentos repassados durante as disciplinas.

xi

Aos funcionários da FEA: Cosme, Marcos Sampaio, Marcos A. de Castro, Guiomar e Jardette

pela competência e auxílio prestados.

Aos funcionários da Biblioteca da FEA: Sueli de Fátima Faria, Cláudia Romano, Monica

Wohnrath e José Carlos Marcondes pelo auxílio prestado. Agradecimento especial à Bianca

Fernandes, Geraldo Silva e Márcia Sevillano pela atenção e disposição em ajudar sempre.

Às funcionárias da limpeza, D. Vilani e Elizângela, por manterem a organização e limpeza do

nosso ambiente de trabalho e pela simpatia dos “bons dias” de todas as manhãs.

À Bunge Alimentos S.A., Cooper Ovos e Alibra pela doação de material para execução deste

trabalho.

Aos membros da banca examinadora: Dr. Alexandre Leite Rodrigues de Oliveira, Dr. Francisco

Fábio Cavalcante Barros, Dr. Juliano Lemos Bicas, Dr. Júnio Cota Silva, Dra. Luciana Francisco

Fleuri, Dra. Luciana Ferracini dos Santos e Dra. Marcela Pavan Bagagli pela valorosa

contribuição neste trabalho.

Ao CNPq pela concessão da bolsa de estudos.

À FAPESP pelo apoio financeiro necessário ao desenvolvimento deste trabalho.

Ao Departamento de Ciência de Alimentos, à Faculdade de Engenharia de Alimentos e à

Unicamp pela grande oportunidade de desenvolvimento, aprimoramento dos conhecimentos

científicos e formação profissional.

xiii

Sempre que possível, não deixes de cooperar com quem precisa de ajuda.

Não respondas simplesmente ao teu próximo:

“Vai e volta amanhã, e eu te darei algo”, se o tens disponível agora e podes ajudar.

(Provérbios 3:27-28)

xv

Resumo

O presente trabalho teve como objetivo utilizar a técnica de delineamento experimental de

misturas como estratégia para a produção de proteases por Aspergillus niger LBA02 em

fermentação semissólida utilizando formulações contendo diferentes resíduos agroindustriais e

produção de hidrolisados proteicos com atividades biológicas e funcionais utilizando a hidrólise

enzimática simultânea de proteínas de diferentes fontes. Efeitos sinérgicos e significativos entre

as misturas quaternárias de farelo de trigo, farelo de soja, farelo de algodão e casca de laranja

foram observados durante a fermentação de A. niger LBA02, atingindo aumentos de 33,7, 7,6,

30,8 e 581,7%, respectivamente, na produção de proteases em comparação com os substratos

utilizados de forma isolada. O estudo das características bioquímicas das preparações enzimáticas

mostrou que a linhagem de A. niger LBA02 foi capaz de secretar diferentes tipos de proteases em

resposta a cada substrato. De um modo geral, as proteases apresentaram atividade ótima a 50 °C e

na faixa de pH de 3 a 4. As maiores diferenças entre as preparações de proteases foram

observadas para os parâmetros cinéticos e termodinâmicos de ativação e inativação térmica. Na

hidrólise enzimática de misturas contendo proteína isolada de soja, proteínas do soro de leite e da

clara de ovo utilizando a preparação comercial Flavourzyme® 500L foram observados efeitos

sinérgicos entre as formulações contendo misturas binárias ou ternárias, para vários parâmetros.

Para atividade antioxidante determinada pelo método DPPH, a mistura contendo proteínas do

soro de leite e proteínas da clara de ovo apresentaram aumentos de 45,1 e 37,3% na atividade,

quando comparada aos hidrolisados obtidos com as duas fontes de forma isolada,

respectivamente. Entre as propriedades funcionais, a capacidade emulsificante foi a que

apresentou maior efeito sinérgico, onde os hidrolisados contendo a mistura ternária de proteína

isolada de soja, proteínas do soro de leite e da clara de ovo, alcançaram valores 2 a 12 vezes

superiores, em relação aos hidrolisados obtidos de forma isolada. A determinação da atividade

anti-adipogênica dos hidrolisados revelou que o tratamento de células pré-adipócitas 3T3-L1 com

a mistura binária de proteínas do soro de leite e da clara de ovo na concentração de 1.200 ppm

reduziu o acúmulo relativo de lipídeos nas células em até 47,9%. Em relação à atividade

antimicrobiana, a linhagem de Staphylococcus aureus ATCC 6538 foi a única que apresentou

inibição do crescimento quando cultivada em meio suplementado com uma mistura binária de

proteína isolada de soja e proteínas da clara de ovo não hidrolisadas, resultando em inibição de

16,82%. Os hidrolisados obtidos com misturas binárias de proteínas do soro de leite e da clara de

xvi

ovo ou proteína isolada de soja e soro de leite estimularam o crescimento de bactérias lácticas e

probióticas, resultando em aumentos de 29,4 a 100% comparados aos meios não suplementados.

A utilização de composições contendo diferentes preparações comerciais de proteases para

hidrólise de proteína isolada de soja e estudo da atividade antioxidante mostrou diferentes

resultados para cada método utilizado. Para inibição dos radicais DPPH, os hidrolisados obtidos

com Flavourzyme® 500L combinada com Alcalase

® 2.4L mostraram o maior efeito sinérgico,

com aumentos de 10,9 e 13,2% da atividade antioxidante, em comparação aos hidrolisados

produzidos com as enzimas isoladas. Os hidrolisados obtidos utilizando a mistura ternária de

Flavourzyme® 500L, Alcalase

® 2.4L e YeastMax

® A apresentaram o maior poder de inibição da

auto-oxidação do ácido linoleico.

Palavras-chave: proteases, hidrolisados proteicos, atividade biológica, propriedades funcionais,

planejamento experimental de misturas.

xvii

Abstract

This study aimed to use the mixture experimental design technique as a strategy to produce

proteases by Aspergillus niger LBA02 under solid state fermentation, using formulations

containing different agroindustrial wastes. It also aimed to produce protein hydrolysates with

biological and functional activities using the simultaneous enzymatic hydrolysis of proteins from

the different sources. Synergistic and significant effects between the quaternary mixtures of

wheat bran, soybean meal, cottonseed meal and orange peel were observed during fermentation

by A. niger LBA02, reaching increases of 33.7, 7.6, 30.8 and 581.7%, respectively, for the

production of proteases as compared to the isolated substrates. The study of the biochemical

properties of the enzyme preparations showed that the strain of A. niger LBA02 was able to

secrete different types of proteases in response to each substrate. In general, the proteases showed

optimal activity at 50 °C in the pH range from 3 to 4. The major differences between the protease

preparations were observed for the kinetic and thermodynamic parameters of thermal activation

and inactivation. In the enzymatic hydrolysis of mixtures containing soy protein isolate, bovine

whey protein and egg white protein using the commercial preparation FlavourzymeTM

500L,

synergistic effects were observed for various parameters between formulations containing binary

or ternary mixtures,. For antioxidant activity as determined by the DPPH assay, the mixture

containing bovine whey protein and egg white protein showed increases of 45.1 and 37.3% in

their activities as compared to hydrolysates obtained with the isolated proteins, respectively. Of

the functional properties, the emulsifying capacity showed the greatest synergistic effect, the

hydrolysates containing the ternary mixture of soy protein isolate, bovine whey protein and egg

white protein, showing increases ranging from 2 to 12-fold as compared to the hydrolysates

obtained using isolated substrates. The determination of the anti-adipogenic activity of the

hydrolysates indicated that the treatment of 3T3-L1 preadipocyte cells with 1200 ppm of the

mixture containing bovine whey protein and egg white protein reduced the relative lipid

accumulation to 47.9%. With respect to antimicrobial activity, the strain of Staphylococcus

aureus ATCC 6538 was the only one which showed growth inhibition when cultivated in a

medium supplemented with a non-hydrolyzed binary mixture of soy protein isolate and egg white

protein, resulting in inhibition of 16.82%. The hydrolysates obtained with binary mixtures of

bovine whey protein and egg white protein or soy protein isolate and bovine whey protein

stimulated the growth of probiotic and lactic acid bacteria, reaching increases from 29.4 to 100%

xviii

when compared to non-supplemented media. The use of formulations containing various

commercial preparations of proteases for the hydrolysis of soy protein isolate and the study of

antioxidant activities showed different results for each method. For DPPH radical scavenging, the

hydrolysates obtained with FlavourzymeTM

500L combined with AlcalaseTM

2.4L showed greater

synergistic effects, with increases of 10.9 and 13.2% in antioxidant activity as compared to the

hydrolysates produced with individual enzymes. The hydrolysates obtained from ternary mixtures

of FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A showed the greatest power of

inhibition of linoleic acid autoxidation.

Keywords: proteases, protein hydrolysates, biological activities, functional properties,

experimental mixture design.

xix

Sumário

Introdução ........................................................................................................................................ 1

Referências ...................................................................................................................................... 4

Capítulo I: Produção de enzimas por fermentação semissólida: aspectos gerais e uma

avaliação direcionada às características físico-químicas dos substratos para otimização de

processos. ........................................................................................................................................ 7

Resumo ............................................................................................................................................ 8

1. Introdução .................................................................................................................................... 9

2. Avaliação de parâmetros de cultivo para FSS ...........................................................................14

2.1. Tamanho de partículas... .................................................................................................14

2.2. Capacidade de absorção de água.. ..................................................................................17

2.3. Composição química.......................................................................................................19

3. Considerações finais.............................................................................................................. ....20

Referências .................................................................................................................................... 21

Capítulo II: Peptídeos com atividade biológica: processos de obtenção, purificação,

identificação e potenciais aplicações. ......................................................................................... 27

Resumo .......................................................................................................................................... 28

1. Introdução .................................................................................................................................. 29

2. Principais processos de obtenção de peptídeos bioativos..........................................................32

2.1. Fermentação....................................................................................................................32

2.2. Hidrólise enzimática........................................................................................................35

3. Concentração, purificação e identificação de peptídeos bioativos............................................37

4. Propriedades biológicas de peptídeos bioativos........................................................................39

4.1. Peptídeos com atividade antimicrobiana.........................................................................39

4.2. Peptídeos com atividade antioxidante.............................................................................42

4.3. Peptídeos com atividade antiadipogênica.......................................................................48

4.4. Peptídeos com atividade anti-hipertensiva......................................................................49

4.5. Indução do crescimento de bactérias ácido lácticas e probióticas..................................52

5. Conclusão .................................................................................................................................. 53

Referências .................................................................................................................................... 54

xx

Capítulo III: Improving the functional properties of milk proteins: focus on the specificities

and mechanisms of action of proteolytic enzymes .................................................................... 67

Abstract ......................................................................................................................................... 68

1. Introduction ............................................................................................................................... 69

2. Functional properties of milk proteins ...................................................................................... 71

2.1. Solubility ........................................................................................................................ 71

2.2. Gelation properties ......................................................................................................... 72

2.3. Emulsifying properties ................................................................................................... 73

3. Conclusion ................................................................................................................................. 75

References ..................................................................................................................................... 76

Capítulo IV: Improving the protease production by Aspergillus niger under solid state

fermentation by substrate formulation using statistical mixture design ............................... 79

Abstract ......................................................................................................................................... 80

1. Introduction ............................................................................................................................... 81

2. Materials and Methods .............................................................................................................. 82

2.1. Agroindustrial wastes and centesimal composition ........................................................ 82

2.2. Microorganism culture ................................................................................................... 82

2.3. Protease production and sampling .................................................................................. 83

2.4. Statistical mixture design................................................................................................ 83

2.5. Determination of protease activity ................................................................................. 84

2.6. Calculations and statistics ............................................................................................... 85

3. Results and Discussion .............................................................................................................. 85

3.1. Chemical composition of the agroindustrial wastes ....................................................... 85

3.2. Synergistic and antagonistic effects of the agroindustrial wastes on protease

production……………………………………………………………………………………..88

3.3. Interpretation of contour plots ........................................................................................ 90

3.4. Model fitting, regression analysis and validation tests ................................................... 93

4.Conclusion .................................................................................................................................. 96

References ..................................................................................................................................... 96

Capítulo V: A new approach for proteases production by Aspergillus niger based on the

kinetic and thermodynamic parameters of the enzymes obtained ......................................... 99

xxi

Abstract ....................................................................................................................................... 100

1. Introduction ............................................................................................................................. 101

2. Materials and Methods ............................................................................................................ 102

2.1. Chemical composition of the agroindustrial wastes ..................................................... 102

2.2. Microorganism culture ................................................................................................. 102

2.3. Protease production and sampling ................................................................................ 103

2.4. Determination of protease activity ............................................................................... 103

2.5. Activation energy and temperature quotient (Q10) ....................................................... 103

2.6. Determination of the kinetic parameters Km and Vmax ............................................... 104

2.7. Determination of kinetic and thermodynamic parameters for thermal inactivation ..... 104

2.7.1. Kinetic parameters for thermal inactivation ......................................................... 104

2.7.2. Thermodynamic parameters for thermal inactivation .......................................... 105

2.8. Substrate specificity of the proteases ............................................................................ 105

2.9. Calculations and statistics ............................................................................................. 106

3. Results and Discussion ............................................................................................................ 106


3.2. Biochemical properties of the proteases from A. niger LBA02 ................................... 108

3.2.1. Activation energy and temperature quotient (Q10) ................................................ 108

3.2.2. Kinetic parameters Km and Vmax ........................................................................ 111

3.2.3. Thermal inactivation ............................................................................................. 112

3.2.4. Substrate specificity of the enzyme ........................................................................ 119

4. Conclusions ............................................................................................................................. 120

Acknowledgements ..................................................................................................................... 121

References ................................................................................................................................... 121

Capítulo VI: Production, biochemical properties of proteases secreted by Aspergillus niger

under solid state fermentation in response to different agroindustrial substrates and their

application for production of whey protein hydrolysates with antioxidant activities ........ 125

Abstract ....................................................................................................................................... 126

1. Introduction ............................................................................................................................. 127


xxii

2.1. Physical–chemical characterization of the agroindustrial wastes ................................. 128

2.1.1. Chemical composition of the agroindustrial wastes ............................................. 128

2.1.2. Determination of the water absorption index (WAI) of the agroindustrial wastes128

2.1.3. Particle size ........................................................................................................... 129

2.1.4. Packing density ..................................................................................................... 129

2.2. Microorganism culture ................................................................................................. 129

2.3. Determination of the microorganism growth: radial growth rate and biomass estimation

by glucosamine level ............................................................................................................... 129

2.4. Protease production and sampling ................................................................................ 130

2.5. Effects of pH and temperature on the activity and stability of the protease determined

using an experimental design .................................................................................................. 131


2.7. Determination of milk-clotting activity ........................................................................ 132

2.8. Application of the proteases to protein hydrolysis ....................................................... 132

2.9. Determination of antioxidant activities ........................................................................ 133

2.9.1. DPPH radical-scavenging activity ........................................................................ 133

2.9.2. Total antioxidant capacity ..................................................................................... 133




3.2. The influence of the water absorption index (WAI) on protease production ............... 135

3.3. The influence of the granulometric distribution and the apparent density of the

agroindustrial wastes on protease production .......................................................................... 136

3.4. Determination of the microorganism growth ............................................................... 139

3.5. Biochemical characteristics of protease from A. niger LB02 ....................................... 140

3.5.1. Effects of pH and temperature on the activity and stability of the protease

determined using an experimental design ............................................................................ 140

3.5.2. Determination of milk-clotting activity ................................................................. 146

3.6. Application of the proteases from A. niger to bovine whey protein hydrolysis and

antioxidant activities of the hydrolysates ................................................................................ 147

4. Conclusion ............................................................................................................................... 150

xxiii

References ................................................................................................................................... 150

Capítulo VII: Comparison and synergistic effects of intact proteins and their hydrolysates

on the functional properties and antioxidant activities in a simultaneous process of

enzymatic hydrolysis ................................................................................................................. 155

Abstract ....................................................................................................................................... 156

1. Introduction ............................................................................................................................. 157


2.1. Reagents........................................................................................................................ 158

2.2. Preparation of protein hydrolysates .............................................................................. 158

2.3. Statistical mixture design.............................................................................................. 158

2.4. TCA soluble protein content ......................................................................................... 159

2.5. Antioxidant activities .................................................................................................... 159

2.5.1. ORAC assay ........................................................................................................... 159


2.5.3. Inhibition of linoleic acid autoxidation ................................................................. 160

2.6. Functional properties .................................................................................................... 161

2.6.1. Solubility ................................................................................................................ 161

2.6.2. Heat stability ......................................................................................................... 161

2.6.3. Emulsifying property ............................................................................................. 161

2.6.4. Foaming capacity .................................................................................................. 162



3.1. Comparison of the functional properties between the intact proteins and their

hydrolysates ............................................................................................................................. 163

3.2. Comparison of the antioxidant activities between the intact proteins and their

hydrolysates ............................................................................................................................. 165

3.3. Comparison of the TCA soluble protein content between the intact proteins and their

hydrolysates ............................................................................................................................. 165

3.4. Synergistic effects and antagonistic effects of the intact proteins and their hydrolysates

on functional properties, antioxidant activities and TCA soluble protein content .................. 166

3.5. Mixture contour plots for functional properties, antioxidant activities and TCA soluble

protein contents........................................................................................................................ 168

xxiv

3.6. Analysis of variance (ANOVA) and models for the functional properties, antioxidant

activities and TCA soluble protein contents of the intact proteins and their hydrolysates ..... 170

4. Conclusions ............................................................................................................................. 173

Acknowledgments ....................................................................................................................... 173

References ................................................................................................................................... 173

Capítulo VIII: Synergistic effects of protein hydrolysates on the suppression of lipid

accumulation in 3T3-L1 adipocytes ......................................................................................... 177

Abstract ....................................................................................................................................... 178

1. Introduction ............................................................................................................................. 179


2.1. Reagents........................................................................................................................ 180



2.4. Determination of the TCA-soluble protein ................................................................... 182

2.5. Inhibition of the relative lipid accumulation in the 3T3-L1 adipocytes ....................... 182

2.5.1. Cell culture ............................................................................................................ 182

2.5.2. Assay for the relative lipid accumulation (RLA) ................................................... 182

2.5.3. Effect of the concentration of the protein hydrolysates and various treatments on

the RLA. ............................................................................................................................... 183

2.5.4. Fractionation of the hydrolysates by ultrafiltration .............................................. 183



3.1. Comparative analysis of the TCA-soluble protein and the RLA (%) between the intact

proteins and their hydrolysates. ............................................................................................... 184

3.2. Synergistic and antagonistic effects of the intact proteins and their hydrolysates on the

TCA-soluble protein and the RLA (%) ................................................................................... 185

3.3. Mixture-contour plots for TCA-soluble protein and RLA (%) .................................... 187

3.4. Analysis of variance (ANOVA) and models for the TCA-soluble protein and the RLA

(%) of the intact proteins and their hydrolysates ..................................................................... 189

3.5. Effect of the concentration of protein hydrolysates and various treatments on the

RLA…………………………………………………………………………………………..191

3.6. Fractionation of the hydrolysates by ultrafiltration. ..................................................... 193

xxv

4. Conclusions ............................................................................................................................. 195

Acknowledgments ....................................................................................................................... 195

References ................................................................................................................................... 195

Capítulo IX: Atividade antimicrobiana de hidrolisados de proteína isolada de soja, soro de

leite e clara de ovo. .................................................................................................................... 199

Resumo ........................................................................................................................................ 200

1. Introdução ................................................................................................................................ 201

2. Material e métodos .................................................................................................................. 202

2.1. Protease ......................................................................................................................... 202

2.2. Determinação da atividade de protease ........................................................................ 203

2.3. Obtenção dos hidrolisados proteicos ............................................................................ 203

2.4. Determinação da atividade antimicrobiana .................................................................. 205

2.4.1. Micro-organismos e condições de cultivo ............................................................. 205

2.4.2. Determinação da atividade antimicrobiana ......................................................... 205

2.5. Análises estatísticas ...................................................................................................... 206

3. Resultados e Discussão ........................................................................................................... 206

4. Conclusões .............................................................................................................................. 216

Referências bibliográficas ........................................................................................................... 217

Capítulo X: Growth promotion of bifidobacteria and lactic acid bacteria strains by protein

hydrolysates using a statistical mixture design ....................................................................... 221

Abstract ....................................................................................................................................... 222

1. Introduction ............................................................................................................................. 223


2.1. Reagents........................................................................................................................ 224


2.3. Determination of the TCA-soluble proteins ................................................................. 225

2.4. Growth performance of bifidobacteria and lactic acid bacteria strains in the media

supplemented with intact and hydrolyzed proteins ................................................................. 225

2.4.1. Microorganisms and culture conditions ............................................................... 225

2.4.2. Bacterial growth in the media supplemented with intact and hydrolyzed proteins226

xxvi

2.4.3. Effect of concentration of protein hydrolysates on cell growth ............................ 226



3.1. Comparative analysis of the TCA-soluble proteins and bacteria growth between the

intact proteins and their hydrolysates. ..................................................................................... 227

3.2. Synergistic and antagonistic effects of the intact proteins and their hydrolysates on the

TCA-soluble proteins and bacteria growth (%) ....................................................................... 229

3.3. Mixture contour plots for TCA-soluble proteins and bacteria growth (%) .................. 230

3.4. Analysis of variance (ANOVA) and models for the TCA-soluble proteins and bacteria

growth (%) ............................................................................................................................... 231

3.5. Effect of the concentration of the protein hydrolysates on cell growth ........................ 233

4. Conclusion ............................................................................................................................... 235

Acknowledgements ..................................................................................................................... 235

References ................................................................................................................................... 235

Capítulo XI: Synergistic actions of proteolytic enzymes for production of soy protein

isolate hydrolysates with antioxidant activities: an approach based on enzymes

specificities.. ............................................................................................................................... 239

Abstract ....................................................................................................................................... 240

1. Introduction ............................................................................................................................. 241

2. Material and Methods .............................................................................................................. 243

2.1. Reagents........................................................................................................................ 243

2.2. Enzymes........................................................................................................................ 243


2.4. Kinetic parameters for thermal inactivation ................................................................. 243



2.7. Determination of TCA soluble protein content ............................................................ 246

2.8. Determination of antioxidant activities ........................................................................ 246


2.8.2. Inhibition of linoleic acid autoxidation ................................................................. 246

2.8.3. Reducing power assay ........................................................................................... 247

xxvii

2.8.4. Total antioxidant capacity ..................................................................................... 247



3.1. Investigation of thermal inactivation modulation of the enzyme by the substrate or

products from soy protein isolate hydrolysis using kinetic parameters................................... 248

3.2. Synergistic and antagonistic effects of the proteases on production of soy protein isolate

hydrolysates with antioxidant activities .................................................................................. 252

4. Conclusion ............................................................................................................................... 259

References ................................................................................................................................... 260

Conclusões gerais ....................................................................................................................... 265

Sugestões para trabalhos futuros ............................................................................................. 269

xxviii

1

Introdução

As proteases constituem um dos grupos de enzimas mais importantes comercialmente,

respondendo por aproximadamente 60% do mercado mundial de enzimas, sendo amplamente

utilizadas nas indústrias de detergentes, couro, produtos farmacêuticos, alimentos e biotecnologia

(Vijayaraghavan et al., 2014). Estas enzimas estão amplamente distribuídas na natureza e podem

ser obtidas a partir de uma grande diversidade de fontes, tais como plantas, animais e micro-

organismos. Destas fontes, os micro-organismos apresentam um grande potencial para a

produção de proteases, devido à sua diversidade bioquímica e susceptibilidade à manipulação

genética. Além disso, as proteases microbianas são predominantemente extracelulares,

diminuindo a necessidade de etapas complexas para a recuperação da enzima a partir do meio

fermentativo (Muthulakshmi et al., 2011).

Diversas espécies de fungos filamentosos têm sido exploradas em processos fermentativos

para a produção de metabólitos e enzimas industriais. Aspergillus niger possui uma longa

tradição de utilização industrial na produção de enzimas e ácidos orgânicos. Muitos destes

produtos foram listados como ''Geralmente Reconhecidos como Seguros (GRAS)'' pelo FDA

(Food and Drug Administration) (Schuster et al., 2002). De acordo com Pel et al. (2007), o

sequenciamento do genoma de A. niger identificou cerca de 198 genes envolvidos na codificação

de proteases, tornando-se assim uma das mais importantes fontes de proteases fúngicas.

Nos últimos anos, processos biotecnológicos inovadores têm explorado a fermentação

semissólida (FSS) como uma tecnologia promissora. No caso específico do cultivo de fungos

filamentosos, a FSS mostra-se um processo atraente visto que os substratos sólidos apresentam

características semelhantes ao habitat natural dos fungos, resultando em melhor crescimento e

secreção de uma ampla variedade de enzimas. Características da FSS, como menor risco de

contaminação, maior produtividade, utilização de substratos de baixo custo, simplicidade de

processamento, maior facilidade de separação e purificação de produtos, requisitos mais baixos

de energia e menor produção de águas residuais tornam esse processo mais atrativo quando

comparado à fermentação submersa (Chutmanop et al. , 2008;. Chen et al, 2014).

Na literatura, diferentes resíduos agroindustriais têm sido utilizados para a produção de

protease por FSS, como farelos de trigo, soja, arroz e lentilha e cascas de laranja, maçã e banana

(Chutmanop et al, 2008; Monton et al, 2013; Karatas et al, 2013).

2

A expressão e secreção de diferentes conjuntos de proteases e outras enzimas pelo micro-

organismo podem ser reguladas pelo tipo de substrato utilizado como fonte de carbono e

nitrogênio. Este é um aspecto particularmente importante, sendo uma nova abordagem para a

obtenção de preparações enzimáticas com propriedades bioquímicas desejáveis.

A caracterização bioquímica de enzimas é importante para avaliar o seu potencial

biotecnológico. O estudo de propriedades, tais como especificidade de substrato, pH ótimo de

atuação, perfis de temperatura para atividade e estabilidade, características cinéticas e

termodinâmicas pode ser utilizado para direcionar a aplicação destas enzimas em processos

industriais específicos (Castro e Sato, 2013).

Dentre as aplicações de proteases, processos envolvendo a hidrólise de proteínas têm sido

estudados para a produção de peptídeos com atividade biológica. Peptídeos bioativos são

definidos como frações específicas de proteínas com sequência de aminoácidos que promovem

um impacto positivo em várias funções biológicas, incluindo efeitos como atividades:

antioxidante, anti-hipertensiva, antitrombótica, antiadipogênica e antimicrobiana (Biziulevicius et

al., 2006; Zhang et al., 2010; Tsou et al., 2010; Tavares et al., 2011). Estes peptídeos apresentam

sequências de 2-20 aminoácidos e massas moleculares inferiores a 6000 Da.

A bioatividade é definida principalmente pela composição e sequência de aminoácidos

(Sarmadi e Ismail, 2010). Essa enorme diversidade funcional coloca os peptídeos e as proteínas

em posição de destaque no campo das aplicações biotecnológicas (Miranda e Liria, 2008), sendo

apontados por alguns autores como possíveis substitutos de substâncias químicas utilizadas como

fármacos ou conservadores de alimentos (Hong et al., 2008).

Os processos de produção de proteases assim como os de hidrólise enzimática de

proteínas para obtenção de peptídeos bioativos têm sido extensivamente relatados na literatura

utilizando substratos de forma individual. A formulação de meios fermentativos utilizando a

mistura de diferentes resíduos agroindustriais, bem como a hidrólise enzimática simultânea de

formulações contendo mais de um tipo de proteína pode ser utilizada como estratégia para

balancear componentes específicos, resultando em produtos com características mais atrativas.

O planejamento de misturas é uma classe especial de delineamento experimental, onde as

proporções entre os componentes ou fatores, assim como as interações entre os mesmos e os seus

efeitos sobre a variável resposta podem ser utilizados para maximizar resultados e aperfeiçoar

3

processos. A utilização desta técnica permite um melhor entendimento dos dados experimentais,

pois inclui avaliação estatística e geração de gráficos e modelos que facilitam a interpretação dos

resultados assim como a verificação de efeitos sinérgicos ou antagônicos entre os componentes

das misturas.

Nesse contexto, o presente trabalho visou utilizar a técnica de delineamento experimental

de misturas como estratégia para a produção de proteases por Aspergillus niger LBA02 em

fermentação semissólida utilizando formulações contendo diferentes resíduos agroindustriais e

produção de hidrolisados com propriedades multifuncionais utilizando a hidrólise enzimática

simultânea de formulações contendo proteínas de diferentes fontes. O estudo da obtenção de

hidrolisados proteicos com atividade antioxidante utilizando diferentes preparações comerciais de

proteases de forma isolada ou combinada também foi relatado. O trabalho encontra-se dividido

em forma de capítulos como descrito a seguir.

O Capítulo I consiste em uma Revisão Bibliográfica sobre aspectos gerais relacionados

aos processos de fermentação semissólida (FSS), incluindo a produção de diversos metabólitos

com foco principalmente na produção de enzimas e uma discussão de parâmetros ligados às

características físico-químicas dos resíduos agroindustriais utilizados para FSS e a influência

destes sobre a produção de enzimas.

O Capítulo II visa discutir os principais processos utilizados para obtenção de peptídeos

bioativos, com destaque para a fermentação e hidrólise enzimática. Métodos de isolamento,

purificação e caracterização destes peptídeos, aspectos relacionados às diferentes atividades

biológicas destes peptídeos, incluindo atividade antioxidante, antimicrobiana, anti-hipertensiva,

antiadipogênica e indução do crescimento de bactérias probióticas também foram apresentados.

O Capítulo III apresenta uma abordagem especial com foco na discussão dos mecanismos

de ação e especificidades de diferentes proteases e a influência destas características sobre as

propriedades funcionais de hidrolisados obtidos a partir de proteínas do leite, incluindo

solubilidade, capacidade de gelificação e propriedades emulsificantes.

O Capítulo IV foi composto pelo estudo da produção de proteases por A. niger LBA02

por fermentação semissólida utilizando como substratos farelo de trigo, farelo de soja, farelo de

algodão e casca de laranja individualmente ou combinados em misturas binárias, ternárias ou

quaternárias.

4

O Capítulo V inclui a determinação das características bioquímicas das preparações

enzimáticas de proteases produzidas em farelo de trigo, farelo de soja, farelo de algodão, casca de

laranja e a mistura quaternária destes substratos, com ênfase em parâmetros cinéticos e

termodinâmicos e especificidade quanto ao substrato.

O Capítulo VI relata a produção de proteases por A. niger LBA02 em farelo de trigo,

farelo de soja e farelo de algodão com uma discussão focada principalmente nos aspectos físico-

químicos dos substratos e o impacto dos mesmos sobre a produção da enzima. As características

bioquímicas das preparações de proteases, incluindo pH e temperatura ótimos para atividade

catalítica e estabilidade e atividade coagulante do leite foram determinadas. O perfil de atividade

antioxidante de hidrolisados de proteínas do soro de leite utilizando as diferentes preparações de

proteases também foi investigado.

Os Capítulos VII, VIII, IX e X relatam o estudo da hidrólise enzimática simultânea de

diferentes fontes de proteínas, incluindo proteína isolada de soja, proteínas do soro de leite e da

clara de ovo de forma individual e combinadas em formulações binárias e ternárias. A avaliação

de efeitos sinérgicos e antagônicos entre as diferentes fontes de proteínas sobre as propriedades

funcionais (solubilidade, estabilidade térmica, capacidade emulsificante e de formação de

espuma) e atividades biológicas (atividade antioxidante, anti-adipogênica, estímulo do

crescimento de bactérias lácticas e probióticas e atividade antimicrobiana) foi apresentada.

O Capítulo XI aborda a hidrólise enzimática de proteína isolada de soja utilizando

diferentes proteases comerciais de forma individual ou combinadas em formulações binárias e

ternárias. A determinação da atividade antioxidante dos hidrolisados obtidos foi discutida dando

especial atenção aos mecanismos de ação de cada preparação enzimática.

Referências

BIZIULEVICIUS, G. A., KISLUKHINA, O. V., KAZLAUSKAITE, J., ZUKAITE, V. Food-

protein enzymatic hydrolysates possess both antimicrobial and immunostimulatory activities: a

“cause and effect” theory of bifunctionality. FEMS Immunology and Medical Microbiology, v.

46, p. 131-138, 2006.

CASTRO, R. J. S., SATO, H. H. Synergistic effects of agroindustrial wastes on simultaneous

production of protease and α-amylase under solid state fermentation using a simplex centroid

mixture design. Industrial Crops and Products, v. 49, p. 813-821, 2013.

5

CHEN, H.-Z., LIU, Z.-H., DAI, S.-H. A novel solid state fermentation coupled with gas stripping

enhancing the sweet sorghum stalk conversion performance for bioethanol. Biotechnology for

Biofuels, v. 7, p. 1-13, 2014.

CHUTMANOP, J., CHUICHULCHERM, S., CHISTI, Y., SIRINOPHAKUN, P. Protease

production by Aspergillus oryzae in solid-state fermentation using agroindustrial substrates.

Journal of Chemical Technology and Biotechnology, v. 83, p. 1012-1018, 2008.

HONG, F. MING, L., YI, S., ZHANXIA, L., YONGQUAN, W., CHI, L. The antihypertensive

effect of peptides: a novel alternative to drugs? Peptides, v. 29, p. 1062-1071, 2008.

KARATAS, H., UYAR, F., TOLAN, V., BAYSAL, Z. Optimization and enhanced production of

α-amylase and protease by a newly isolated Bacillus licheniformis ZB-05 under solid-state

fermentation. Annals of Microbiology, v. 63, p. 45–52. 2013.

MIRANDA, M. T. M., LIRIA, C. W. Técnicas de análise e caracterização de peptídeos e

proteínas. In: PESSOA JR., A. e KILIKIAN, B. V. Purificação de Produtos Biotecnológicos.

Barueri: Manole, 2008, cap. 21, p. 411 – 427.

MONTON, S., UNREAN, P., PIMSAMARN, J., KITSUBUN, P., TONGTA, A. Fuzzy logic

control of rotating drum bioreactor for improved production of amylase and protease enzymes by

Aspergillus oryzae in solid-state fermentation. Journal of Microbiology and Biotechnology, v.

23, n. 3, p. 335–342, 2013.

MUTHULAKSHMI, C., GOMATHI, D., KUMAR, D. G., RAVIKUMAR, G., KALAISELVI,

M., UMA, C. Production, purification and characterization of protease by Aspergillus flavus

under solid state fermentation. Jordan Journal of Biological Sciences, v. 4, p. 137-148, 2011.

PEL, H. J. et al. Genome sequencing and analysis of the versatile cell factory Aspergillus niger

CBS 513.88. Nature Biotechnology, v. 25, p. 221-231, 2007.

SARMADI, B. H., ISMAIL, A. Antioxidative peptides from food proteins: a review. Peptides, v.

31, p. 1949-1956, 2010.

SCHUSTER, E., DUNN-COLEMAN, N., FRISVAD, J. C., VAN DIJCK, P. W. On the safety of

Aspergillus niger – a review. Applied Microbiology and Biotechnology, v. 59, p. 426–435,

2002.

6

TAVARES, T. G., CONTRERAS, M. M., AMORIM, M., MARTÍN-ÁLVAREZ, P. J.,

PINTADO, M. E., RECIO, I., MALCATA, F. X. Optimisation, by response surface

methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey

protein hydrolysates obtained with cardoon extract. International Dairy Journal, v. 21, p. 926-

933, 2011.

TSOU, M. J., KAO, F. J., TSENG, C. K., CHIANG, W. D. Enhancing the anti-adipogenic

activity of soy protein by limited hydrolysis with Flavourzyme and ultrafiltration. Food

Chemistry, v. 122, p. 243–248, 2010.

VIJAYARAGHAVAN, P., LAZARUS, S., VINCENT, S. G. P. De-hairing protease production

by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung:

biosynthesis and properties. Saudi Journal of Biological Sciences, v. 21, p. 27–34, 2014.

ZHANG, L., LI, J., ZHOU, K. Chelating and radical scavenging activities of soy protein

hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation.

Bioresource Technology, v. 101, p. 2084–2089, 2010.

7

Capítulo I: Produção de enzimas por fermentação semissólida: aspectos gerais

e uma avaliação direcionada às características físico-químicas dos

substratos para otimização de processos.

8

Resumo

A fermentação semissólida (FSS) vem sendo utilizada como uma tecnologia promissora para a

produção de diversos metabólitos microbianos, como as enzimas. Características deste processo,

como menor risco de contaminação, maior produtividade, utilização de substratos de baixo custo,

simplicidade de processamento, maior facilidade de separação e purificação de produtos, menor

requerimento de energia e menor produção de água residual tornam esse processo mais atrativo

quando comparado à fermentação submersa (FSm). Nesse contexto, o presente trabalho teve

como objetivo apresentar aspectos gerais relacionados aos processos de FSS traçando um

paralelo com processos de FSm. O potencial de aplicação da FSS foi fundamentalmente

embasado em dados sobre a produção de diversos metabólitos em uma análise comparativa com a

FSm com foco principalmente na produção de enzimas. A discussão de importantes parâmetros

relacionados essencialmente às características físico-químicas dos resíduos agroindustriais

utilizados para FSS e a influência destes sobre a produção de diversas enzimas também foi

apresentada.

Palavras-chave: fermentação semissólida; resíduos agroindustriais; enzimas; parâmetros físico-

químicos.

9

1. Introdução

A fermentação semissólida (FSS) vem ganhando muita credibilidade na indústria

biotecnológica nos últimos anos pelo potencial de aplicação na produção de metabólitos

biologicamente ativos, além de possuir uma grande gama de aplicações nas indústrias de

alimentos, combustível, química e farmacêutica. Além disso, a busca por processos sustentáveis e

ecologicamente corretos em substituição aos tradicionais processos químicos para a fabricação de

produtos transformou fortemente o setor industrial. Nesse contexto, a FSS alcançou muita

relevância, por apresentar diversas características que a torna ecologicamente correta (Thomas et

al., 2013).

A FSS envolve o crescimento de micro-organismos em materiais sólidos úmidos em que

os espaços entre as partículas destes materiais encontram-se preenchidos com uma fase gasosa

contínua. É importante ressaltar que a palavra “fermentação” dentro do conceito de “fermentação

semissólida” é geralmente utilizada no sentido mais amplo de “processos microbianos

controlados” e não implica que o micro-organismo está necessariamente utilizando vias

metabólicas fermentativas durante o seu cultivo (Mitchell et al., 2006).

Nos últimos anos, processos biotecnológicos inovadores têm explorado a FSS como uma

tecnologia promissora para a produção de metabólitos secundários de alto valor agregado, como

antibióticos, enzimas, ácidos orgânicos, biopraguicidas, biossurfactantes, biocombustíveis e

bioaromas (Pandey et al., 2000; Abraham et al., 2013).

Dentre estes metabólitos e devido à alta demanda industrial, as enzimas apresentam

grande potencial de aplicação. As enzimas de origem microbiana possuem grande notoriedade e

consequentemente interesse nos campos de pesquisa, pelo fato de os micro-organismos serem

excelentes fontes e apresentarem ampla diversidade bioquímica e susceptibilidade a manipulação

genética (Rao et al., 1998). Fungos filamentosos, leveduras e bactérias são amplamente utilizados

para a produção de enzimas por FSS. No caso específico do cultivo de fungos filamentosos, a

FSS mostra-se um processo atraente visto que os substratos sólidos apresentam características

semelhantes ao habitat natural dos fungos, resultando em melhor crescimento e secreção de uma

ampla variedade de enzimas. Características da FSS, como menor risco de contaminação

bacteriana (baixa atividade de água), maior produtividade, utilização de substratos de baixo custo,

simplicidade de processamento, maior facilidade de separação e purificação de produtos,

10

requisitos mais baixos de energia e menor produção de água residual tornam esse processo mais

atrativo quando comparado à fermentação submersa (FSm) (Chutmanop et al., 2008; Chen et al.,

2014). É importante ressaltar, que embora a FSS possua algumas vantagens em relação à FSm, a

avaliação do processo mais adequado será função de parâmetros como o tipo de micro-organismo

utilizado (bactérias, leveduras ou fungos filamentosos), suas exigências nutricionais e morfologia

de crescimento e consequentemente dos produtos de interesse, não existindo assim uma indicação

de qual o melhor processo. Um resumo comparativo sobre as principais características da FSm e

FSS, no qual é possível observar vantagens e desvantagens para cada tipo de processo, é

apresentado na Tabela 1. Dados relativos a alguns processos para a produção de diversos

metabólitos por FSm e FSS são apresentados na Tabela 2.

Tabela 1 – Comparação entre as principais características dos processos de fermentação

semissólida (FSS) e submersa (FSm).

A FSS desperta maior interesse econômico em regiões, como o Brasil, com abundância

em biomassa e resíduos agroindustriais de baixo custo (Castilho et al., 2000), como algodão,

arroz, laranja, soja e trigo que atingiram juntos no anos de 2012-2013, uma produção agrícola

nacional de aproximadamente 117 milhões de toneladas e uma produção mundial de 1,84 bilhão

de toneladas (FAO, 2014) (Figura 1). O processamento destes insumos dá origem a subprodutos

de baixo valor agregado, como farelos e tortas, mas de alto valor nutritivo, sendo grande parte

Características FSm FSS

Meio de cultivo Complexo Simples

Geração de efluentes Alta Baixa

Espaço requerido Grande Pequeno

Contaminação bacteriana Risco alto Baixo risco

Solubilidade e difusão de O2 Menor Maior

Energia necessária Demanda alta Demanda baixa

Controle de temperatura Simples Complexo

Controle de pH Simples Complexo

Controle da agitação Simples Complexo

Controle de nutrientes e produtos Simples Complexo

Custo Maior Menor

Recuperação e purificação de produtos Mais complexo Maior facilidade

Produtividade Menor Maior

11

destinada à alimentação animal. A utilização destes resíduos como substrato para o

desenvolvimento de processos biotecnológicos, como a produção de enzimas por FSS é um

exemplo promissor da obtenção de biomoléculas de alto valor agregado, como as enzimas, a

partir de substratos de baixo custo (Tabela 3).

Figura 1 – Dados sobre a produção brasileira e mundial (milhões de toneladas) de alguns

produtos agroindustriais que dão origem a resíduos utilizados em processos fermentativos

semissólidos (FAO, 2014).

0

10

20

30

40

50

60

70

80

90

Algodão Arroz Laranja Soja Trigo

Pro

duçã

o b

rasi

leir

a (m

ilhões

de

tonel

adas

)

0

100

200

300

400

500

600

700

800

Algodão Arroz Laranja Soja Trigo

Pro

du

ção

mu

nd

ial

(mil

hõ

es d

e to

nel

adas

)

12

Tabela 2 – Comparação entre os processos de fermentação semissólida (FSS) e submersa (FSm) para a produção de diversos

metabólitos por micro-organismos.

Metabólito

de interesse

Micro-

organismo

Meios de Cultivo Produção Referência

FSm FSS FSm FSS

Feruloil

esterase

Aspergillus

niger

Concentração em g L-1

: tartarato de

amônio (1,842), extrato de levedura (0,5),

KH2PO4 (0,2), CaCl2 (0,0132), MgSO4

(0,5), polpa de beterraba (15,0), maltose

(2,5)

Concentração em g por 100 g de polpa de

beterraba seca: tartarato de amônio (12,3),

extrato de levedura (3,4), KH2PO4 (1,3),

CaCl2 (0,09), MgSO4 (3,3) e maltose (2,5)

2,2

nkat g−1

9,6

nkat g−1

Asther et al.,

2002

Proteases Aspergillus

oryzae


: KH2PO4 (1,0),

MgSO4 (5,0), NaCl (5,0) e FeSO4 (0,04);

farelo de trigo (2,0%)

Farelo de trigo suplementado com solução

de sais com composição semelhante à

utilizada para o FSm

8,7

U g-1

31,2

U g-1

Sandhya et

al., 2005

Lipases Aspergillus

spp.


: farelo de trigo

(10,0), extrato de levedura (45,0), óleo de

soja (20,0), KH2PO4 (2,0) e MgSO4 (1,0).

Solução de elementos traço (mg L−1

):

FeSO4 (0,63), MnSO4 (0,01) e ZnSO4

(0,62)

Mistura de farelo de soja (85,7%) e casca

de arroz (14,3%) suplementada com

solução salina contendo (g L-1

): KH2PO4

(2,0), MgSO4 (1,0). Solução de elementos

traço (mg L−1

): FeSO4 (0,63), MnSO4

(0,01) e ZnSO4 (0,62). Azeite de oliva

(2,0% p/p) e nitrato de sódio (2,0% p/p)

4,52

U

25,22

U

Colla et al.,

2010

Tanino acil

hidrolase

Lactobacillus

plantarum


: ácido tânico

(13,16), glicose (1,5), NH4Cl (1,0), CaCl2

(1,0), K2HPO4 (0,5), KH2PO4 (0,5),

MgSO4 (0,5) e MnSO4 (0,03)

Casca de café suplementada com solução

mineral contendo (g L-1

): ácido tânico

(10,0); NH4NO3, (5,0); KH2PO4 (1,0);

NaCl (1,0), MgSO4 (1,0) e CaCl2 (0,5)

9,13

U mL-1

5,32

U mL-1

Natarajan e

Rajendran,

2012

Proteases Aspergillus

oryzae

Polpa de tomate (40 g L-1

) suplementada

com farelo de trigo (7,92 g L-1

) e NaCl

(1,18 g L-1

)

10g de polpa de tomate suplementada com

caseína (19,79 g L-1

) e NaCl (0,92 g L-1

)

2.343,5

U g-1

21.309

U g-1

Belmessikh

et al., 2013

Queratinases Aspergillus

niger


: (NH4)2SO4 (3,5),

KH2PO4 (1,0), MgSO4 (0,5), KCl (0,1),

ZnSO4 (5×10-3

) e pena de galinha (10

penas por L)

Mistura de penas de galinha (0,4 g) e farelo

de trigo (40 g) umedecida com solução de

(NH4)2SO4 (0,9%)

21,3

U mL-1

172,7

U mL-1

Mazotto et

al., 2013

Monacolina

K

Monascus

purpureus


: glicerol (180,0),

farelo de soja (20,0), NaNO3 (2,0),

MgSO4 (1,0), K2HPO4 (1,0), ZnSO4 (2,0)

e milhocina (10 mL L-1

)

Composição semelhante à utilizada para a

FSm com adição de agar (4,0%)

2.047,03

mg L-1

458,37

mg L-1

Zhang et al.,

2013

13

Tabela 3 – Produção de enzimas com aplicações industriais utilizando diferentes resíduos agroindustriais e fermentação semissólida

(FSS).

Enzima Micro-organismo Substratos Condições de cultivo Referência

Protease Populações microbianas nativas Resíduos de fibra de soja

Relação sólido:líquido: 1:1

Temperatura de incubação: 37 °C

Tempo de fermentação: 96 h

Abraham et al.,

2013

Elagitanase Aspergillus niger Bagaço de cana, sabugo de

milho e casca de coco


Temperatura de incubação: 35-40 °C

Inóculo: 2×107 esporos g

-1

Buenrostro-

Figueroa et al.,

2014

Protease Aspergillus niger Farelos de trigo, soja e

algodão

Umidade inicial do meio: 50%

Tempo de fermentação: 24-96 h


Inóculo: 107 esporos g

-1

Castro et al.,

2014

Poligalacturonase Aspergillus sojae Farelo de trigo

Umidade inicial do meio: 62%



Inóculo: 107 esporos g

-1

Demir e Tari,

2014

Xilanase Trichoderma viride

Farelos de trigo, soja, e

girassol, casca de arroz,

bagaço de cana e sabugo de

milho

Relação líquido:sólido: 11:10

Tempo de fermentação: 7 dias


Inóculo: 10%

Irfan et al., 2014

Peroxidase Phanerochaete chrysosporium Resíduos de mandioca

Relação líquido:sólido: 2:1

Tempo de fermentação: 10 dias


Li et al., 2014

Quitinase Penicillium ochrochloron MTCC 517 Farelos de trigo e arroz

Umidade inicial do meio: 70-74%



Patil e Jadhav,

2014

Xilanase Sporotrichum thermophile Torta de pinhão manso

Relação sólido:líquido: 1:1,5



Inóculo: 6%

Sadaf e Khare,

2014

Protease e lipase Aspergillus versicolor Torta de pinhão manso

Umidade inicial do meio: 20-70%


Temperatura de incubação: 20-35 °C

Inóculo: 103-10

8 esporos mL

-1

Veerabhadrappa

et al., 2014

14

O presente trabalho teve como objetivo apresentar aspectos importantes para a otimização

de processos e produção de enzimas por FSS, incluindo parâmetros físico-químicos dos

substratos e algumas estratégias que vêm sendo utilizadas no meio científico.

2. Avaliação de parâmetros de cultivo para FSS

Vários aspectos importantes devem ser considerados para o desenvolvimento e otimização

de bioprocessos em FSS. Estes incluem principalmente a seleção adequada das variáveis a serem

utilizadas no processo, como a linhagem microbiana, substratos, umidade inicial do meio,

temperatura de incubação e inóculo. Nesse trabalho, será dada especial atenção aos parâmetros

físico-químicos dos substratos, como tamanho de partícula, capacidade de absorção de água e

composição química.

A seleção adequada do substrato é um aspecto de suma importância para FSS, pois o

material sólido vai atuar como suporte físico e fonte de nutrientes. Diversos materiais podem ser

utilizados como suportes para FSS, dentre os quais podemos citar suportes inertes como

vermiculita, perlita e polietileno, que podem ser embebidos com uma solução nutriente adequada

para o crescimento microbiano e suportes naturais, como os resíduos agroindustriais, que por si

só apresentam características suficientes para o desenvolvimento satisfatório dos micro-

organismos e para os quais será dada especial atenção neste trabalho. Características físico-

químicas destes substratos vêm sendo utilizadas como importantes parâmetros para o estudo da

produção de enzimas por FSS. A influência do tamanho de partículas, capacidade de absorção de

água e composição química de substratos sobre a produção de enzimas por FSS será discutida a

seguir.

2.1. Tamanho de partículas

O tamanho das partículas do substrato é um importante fator para a produção de enzimas

por FSS uma vez que está diretamente relacionado com a porosidade do meio de cultivo. A

avaliação deste parâmetro pode ser realizada por técnicas de distribuição granulométrica

utilizando peneiras com diâmetros de abertura conhecidos ou pela determinação da densidade

aparente.

Uma avaliação mais criteriosa deste parâmetro inclui a classificação das propriedades das

partículas com base nas estruturas porosas dos substratos que afetarão diretamente o crescimento

microbiano e a bioconversão dos substratos. Estas propriedades podem ser classificadas em

15

intrapartículas (propriedades térmicas, teor de umidade, granulometria, porosidade e cinética de

processos biológicos) e extrapartículas (propriedades de transferência de calor, permeabilidade e

condições de transferência de massa). É importante notar que as características das partículas dos

substratos possuem impacto direto em vários outros parâmetros, já que a FSS é um sistema

combinado de três fases principais: o substrato propriamente dito que é a fase sólida, a retenção

de água na matriz e nos espaços interpartículas que é a fase líquida e o gás presente nos espaços

ou poros que é a fase gasosa (Figura 2) (Mitchel et al., 2006; Chen e He, 2012).

Figura 2 – Esquema ilustrativo do arranjo de partículas sólidas e dos componentes principais do

sistema de FSS durante o cultivo de fungos filamentosos (Figura adaptada de Mitchel et al.,

2006).

Hifas de fungos

filamentosos

Partícula

sólida úmida

Gotículas de água nos

espaços interpartículas

Fase

gasosa

Água e nutrientes absorvidos

pela partícula

16

Em geral, partículas de substrato com tamanho reduzido proporcionam uma maior área de

superfície para o ataque microbiano, o que é considerado um fator desejável. É importante

ressaltar que partículas muito pequenas podem ter um efeito contrário, resultando em

aglomeração, redução da difusão de oxigênio e consequentemente do crescimento dos micro-

organismos, enquanto partículas maiores fornecem maiores espaços entre si, melhores condições

de transferência de calor e massa, mas podem resultar em superfície limitada para o ataque

microbiano (Pandey et al., 2001; Wong et al., 2011; Chen e He, 2012; Ruiz et al., 2012). Nesse

contexto, meios de cultivos com distribuição de tamanho de partículas heterogêneo ou com

tamanhos de partículas intermediários, seriam mais adequados para a produção de enzimas por

FSS, pois apresentariam os requisitos necessários para uma difusão satisfatória de oxigênio

aliados a uma maior área superficial para o crescimento microbiano.

Melikoglu et al., (2013) estudaram a influência de diversos parâmetros de cultivo sobre a

produção de proteases e glicoamilases pelo micro-organismo Aspergillus awamori por FSS

utilizando resíduos de pão. As partículas dos resíduos de pão fracionadas em tamanhos variando

de 5 a 50 mm foram utilizadas como meios de cultivo e apresentaram um efeito direto sobre o

crescimento microbiano e produção das enzimas durante as fermentações. Os maiores valores

para produção simultânea de protease e glicoamilase foram 56,4 e 73,6 U g-1

, respectivamente,

detectados para as fermentações conduzidas com partículas com diâmetro de 20 mm. O micro-

organismo respondeu de maneira diferente em relação à produção das enzimas em partículas de

tamanhos extremos. Para a produção de glicoamilase, os menores valores de atividade foram

detectados para as fermentações conduzidas com partículas de 5 e 10 mm, enquanto para

protease, os menores valores foram detectados para tamanho de partículas de 50 mm.

Buenrostro-Figueroa et al., (2014) avaliaram a utilização de resíduos lignocelulósicos

incluindo bagaço de cana, sabugo de milho, casca de coco e caule de candelila (Euphorbia

antisyphilitica) para produção de elagitanase por A. niger em FSS. Um dos parâmetros avaliados

incluiu a determinação da densidade aparente dos resíduos e a influência sobre a produção da

enzima. Segundo Chávez-González et al., (2010), a densidade aparente fornece informações

sobre o grau de compactação dos materiais, estando diretamente relacionado ao espaço disponível

para a transferência de massa e energia. Valores mais altos de densidade aparente implicam em

menor relação entre área e volume, o que pode resultar em problemas de difusão de oxigênio pela

redução dos espaços vazios entre as partículas. Os autores detectaram uma influência direta da

17

densidade aparente sobre a produção de elagitanase, onde os menores valores de atividade

enzimática foram detectados nos meios compostos por caule de candelila, com densidade

aparente de 0,86 g cm-3

, ao passo que, os maiores valores foram obtidos para os meios

fermentados com casca de coco, que apresentou densidade de 0,82 g cm-3

, um valor intermediário

entre os resíduos avaliados.

2.2. Capacidade de absorção de água

Outro parâmetro importante a ser considerado é a capacidade de retenção de água pelo

substrato. Essa medida indica a capacidade das partículas do substrato em absorver água e está

ligada diretamente à disponibilidade de grupos hidrofílicos para ligação com as moléculas de

água (Mussatto et al., 2009). Essa capacidade é crucial para o crescimento microbiano e

fermentação, pois apresenta impacto direto sobre características físicas do meio de cultivo, como

por exemplo, sobre as dimensões dos poros que podem ser alteradas pelo inchaço das partículas

sólidas após a absorção da água, tornando-se favoráveis ou não para a biodegradação e

bioconversão da biomassa (Chen e He, 2012). O conteúdo adequado de água no meio de cultivo

apresenta também uma importante função ligada à disponibilidade e difusão de nutrientes e aos

mecanismos de troca gasosa entre dióxido de carbono e oxigênio durante a fermentação (Torrado

et al., 2011).

A determinação da umidade crítica dos substratos também pode ser utilizada

paralelamente à estimativa da capacidade de absorção de água dos substratos. Esse parâmetro

representa a quantidade de água fortemente ligada ao suporte, e que, portanto não está disponível

para utilização pelos micro-organismos; sendo assim, recomenda-se a utilização de substratos

com baixos valores de umidade crítica (Melikoglu et al., 2013).

A determinação da capacidade de absorção de água e da umidade crítica de bagaço de

malte (resíduo da indústria cervejeira), palha de trigo, sabugos de milho, cascas de café, cortiça

de carvalho e esponja vegetal foi utilizada como parâmetro de seleção do substrato mais

adequado para produção de β-frutofuranosidase por Aspergillus japonicus ATCC 20236

(Mussatto et al., 2009). Os valores mais altos para capacidade de absorção de água foram

observados para palha de trigo, bagaço de malte e casca de café, com valores estimados em 9,95,

9,03 e 8,30 g de água por g de material, respectivamente. Para umidade crítica, bagaço de malte,

cortiça de carvalho e palha de trigo apresentaram valores de 60, 58 e 57%, respectivamente;

18

sendo estes, os mais elevados. De acordo com os autores, uma avaliação isolada destes

parâmetros não pode ser utilizada para uma indicação precisa de qual substrato induziria uma

maior produção da enzima β-frutofuranosidase por A. japonicus ATCC 20236. No entanto, estes

parâmetros podem ser usados para estimar em qual substrato, a adesão celular e o crescimento

microbiano serão favorecidos. A maior produção da enzima foi detectada quando o micro-

organismo foi cultivado em sabugo de milho, que apresentou o menor valor de umidade crítica

(50%) e o segundo menor valor para capacidade de absorção de água (3,77 g de água/g de

material) (Mussatto et al., 2009).

Orzua et al., (2009) avaliaram a viabilidade de utilização de 10 resíduos agroindustriais

como suportes para o crescimento de uma linhagem de A. niger em FSS. Os autores incluíram os

parâmetros de capacidade de absorção de água e umidade crítica dos resíduos como

determinantes para uma seleção mais adequada. O estudo apontou que, casca de coco, polpa de

maçã, cascas de limão e laranja foram os materiais de maior potencial para utilização em FSS,

uma vez que apresentaram alta capacidade de absorção de água, níveis de umidade crítica

considerados adequados, além de terem permitido uma boa taxa de crescimento da linhagem de

A. niger.

Embora haja uma indicação de que substratos com maior capacidade de absorção de água

sejam mais adequados para o cultivo de fungos filamentos em FSS, é possível inferir que quando

estes valores superam um determinado limite, os mecanismos envolvidos no desenvolvimento

dos micro-organismos e consequentemente na secreção de enzimas ficam comprometidos. Duas

considerações importantes devem ser feitas acerca disso; a primeira avaliando os substratos com

altos índices de absorção de água e a segunda, os substratos com baixa capacidade. Materiais com

alta capacidade de absorção de água apresentam um efeito desejável que é a manutenção dos

níveis de umidade ao longo do processo fermentativo. No entanto, se a quantidade de água

adicionada ao meio de cultivo for alta o suficiente para ser totalmente absorvida pelo material,

mas resulte em valores de umidade inicial muito elevados, pode haver diminuição da porosidade,

perda da estrutura da partícula, redução das trocas gasosas e maior susceptibilidade à

contaminação bacteriana; fenômenos estes que impactam negativamente o desenvolvimento e a

produção de enzimas pelos micro-organismos. Por outro lado, os substratos com baixa

capacidade de absorção de água possuem uma limitação com relação à quantidade de água a ser

adicionada aos meios de cultivo, o que pode resultar em baixa umidade inicial e

19

consequentemente redução na solubilidade de nutrientes, menor grau de inchaço do substrato e

maior tensão superficial das partículas de água, dificultando o crescimento microbiano.

2.3. Composição química

A produção de enzimas por FSS pode ser afetada pela presença de componentes químicos

específicos que podem ser adicionados ou estarem presentes naturalmente nos substratos e que

atuam como indutores. É válido ainda ressaltar que os substratos devem apresentar uma

proporção adequada entre os nutrientes que serão utilizados como fontes de carbono e nitrogênio

(relação C:N) para um crescimento satisfatório dos micro-organismos durante a fermentação

(Castro et al., 2014).

Ghanem et al., (2000) avaliaram a produção de xilanase por Aspergillus terreus utilizando

palha e farelo de trigo, sabugo de milho, casca de arroz e bagaço de cevada como substratos para

FSS. A investigação incluiu a avaliação entre a produção e o teor de celulose nos substratos, o

qual pode ser indutor da secreção da enzima. Dentre os substratos avaliados, a palha de trigo

apresentou maior quantidade de celulose (50,7%) resultando também em maior produção de

xilanase por A. terreus (16,16 U mL-1

).

Resíduos agroindustriais obtidos a partir de extração de óleo de sementes têm sido

relatados como potenciais substratos para a produção de lipases por micro-organismos devido ao

conteúdo residual de lipídeos que podem ser indutores da produção destas enzimas (Azeredo et

al., 2007; Rigo et al., 2010). Ferraz et al., (2012) estudaram a produção de lipases por

Sporobolomyces ruberrimus utilizando farelo de soja, farelo de arroz e bagaço de cana como

substratos em FSS. Dentre os resíduos avaliados, o farelo de arroz apresentou o maior conteúdo

de lipídeos (16,43%) e permitiu maior produção de lipases pelo micro-organismo.

Thanapimmetha et al., (2012) realizaram uma análise comparativa com o trabalho de

Chutmanop et al., (2008) sobre a produção de proteases pela mesma linhagem de A. oryzae

utilizando como substratos torta de pinhão-manso, farelo de trigo e farelo de arroz. A avaliação

mostrou que a produção de proteases em farelo de arroz e farelo de trigo foi 22 e 30% menor,

respectivamente, que a observada em torta de pinhão-manso. O aumento da produção de

proteases em torta de pinhão-manso foi relacionado ao alto teor de proteínas presente nesse

substrato. Quando os teores de proteína foram comparados entre os substratos, a torta de pinhão-

manso apresentou 60%, enquanto os valores estimados para os farelos de arroz e trigo foram 13-

20

14% e 12-17%, respectivamente. Segundo estes autores, as proteases secretadas pelos micro-

organismos podem ser estimuladas pelos aminoácidos presentes nas proteínas, o que resultou na

maior produção de proteases no substrato com maior quantidade de proteína. Resultados

semelhantes foram observados por Castro et al. (2014), onde o conteúdo de proteína apresentou

uma forte e significativa correlação com a produção de proteases por A. niger utilizando farelos

de trigo, soja e algodão durante as primeiras 48h de cultivo semissólido.

3. Considerações finais

A fermentação semissólida (FSS) é um processo promissor para a produção de diversas

biomoléculas, dentre elas, as enzimas. Uma análise comparativa entre este processo e a

fermentação submersa mostra as diversas vantagens da FSS. A otimização de processos para

produção de enzimas por FSS inclui o estudo de diversos parâmetros de cultivo, os quais são

extremamente variáveis dependendo da enzima que se deseja obter, do substrato utilizado e do

micro-organismo. Na presente revisão foi dada especial atenção aos parâmetros físico-químicos

dos resíduos agroindustriais, incluindo tamanho de partícula, capacidade de absorção de água e

composição química. Embora estes parâmetros tenham sido discutidos de forma isolada para uma

melhor compreensão do impacto de cada um sobre os processos fermentativos, torna-se

necessária uma avaliação conjunta de diversos fatores assim como da contribuição particular de

cada um para a definição das condições mais adequadas de cultivo microbiano para atingir altos

níveis de produtividade das moléculas de interesse.

21

Referências

ABRAHAM, J., GEA, T., SÁNCHEZ, A. Potential of the solid-state fermentation of soy fibre

residues by native microbial populations for bench-scale alkaline protease production.

Biochemical Engineering Journal, v. 74, p. 15-19, 2013.

ASTHER, M., HAON, M., ROUSSOS, S., RECORD, E., DELATTRE, M., LESAGE-

MEESSEN, L., LABAT, M., ASTHER, M. Feruloyl esterase from Aspergillus niger a

comparison of the production in solid state and submerged fermentation. Process Biochemistry,

v. 38, p. 685-691, 2002.

AZEREDO, L.A.I., GOMES, P.M., SANT’ANNA JR. G.L., CASTILHO, L.R., FREIRE,

D.M.G. Production and regulation of lipase activity from Penicillium restrictum in submerged

and solid-state fermentations. Current Microbiology, v. 54, p. 361–365, 2007.

BELMESSIKH, A., BOUKHALFA, H., MECHAKRA-MAZA, A., GHERIBI-AOULMI, Z.,

AMRANE, A. Statistical optimization of culture medium for neutral protease production by

Aspergillus oryzae. Comparative study between solid and submerged fermentations on tomato

pomace. Journal of the Taiwan Institute of Chemical Engineers, v. 44, p. 377–385, 2013.

BUENROSTRO-FIGUEROA, J., ASCACIO-VALDÉS, A., SEPÚLVEDA, L., DE LA CRUZ,

R., PRADO-BARRAGÁN, A., AGUILAR-GONZÁLEZ, M.A., RODRÍGUEZ, R., AGUILAR,

C.N. Potential use of different agroindustrial by-products as supports for fungal ellagitannase

production under solid-state fermentation. Food and Bioproducts Processing, v. 92, 376–382,

2014.

CASTILHO, L. R., MEDRONHO, R. A., ALVES, T. L. M. Production and extraction of

pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger.

Bioresource Technology, v. 71, p.45-50, 2000.

CASTRO, R.J.S., NISHIDE, T.G., SATO, H.H. Production and biochemical properties of

proteases secreted by Aspergillus niger under solid state fermentation in response to different

agroindustrial substrates. Biocatalysis and Agricultural Biotechnology, 2014. doi:

10.1016/j.bcab.2014.06.001i.

CHÁVEZ-GONZÁLEZ, M.L., RODRÍGUEZ-DURÁN, L.V., CRUZ-HERNÁNDEZ, M.,

PRADO-BARRAGÁN, A., AGUILAR, C.N. 2010. Packing density and aeration rate effect

http://dx.doi.org/10.1016/j.bcab.2014.06.001i

http://dx.doi.org/10.1016/j.bcab.2014.06.001i

22

on tannase production by Aspergillus niger GH1 in solid state fermentation. In: SABU, A.,

AGUILAR, C.N., ROUSSOS, S. (Org.). Chemistry and Biotechnology of Polyphenols:

Fundamentals and Application. Kerala: Cibet Publishers, p. 199.

CHEN, H.-Z., LIU, Z.-H., DAI, S.-H. A novel solid state fermentation coupled with gas stripping

enhancing the sweet sorghum stalk conversion performance for bioethanol. Biotechnology for

Biofuels, v. 7, p. 1-13, 2014.

CHEN, H-Z., HE, Q. Value-added bioconversion of biomass by solid-state fermentation. Journal

of Chemical Technology and Biotechnology, v. 87, p. 1619–1625, 2012.

CHUTMANOP, J., CHUICHULCHERM, S., CHISTI, Y., SIRINOPHAKUN, P. Protease

production by Aspergillus oryzae in solid-state fermentation using agroindustrial substrates.

Journal of Chemical Technology and Biotechnology, v. 83, p. 1012-1018, 2008.

COLLA, L.M., RIZZARDI, J., PINTO, M.H., REINEHR, C.O., BERTOLIN, T.E., COSTA,

J.A.V. Simultaneous production of lipases and biosurfactants by submerged and solid-state

bioprocesses. Bioresource Technology, v. 101, p. 8308–8314, 2010.

DEMIR, H., TARI, C. Valorization of wheat bran for the production of polygalacturonase in SSF

of Aspergillus sojae. Industrial Crops and Products, v. 54, p. 302–309, 2014.

FAO. Disponível em: http://faostat.fao.org/. Acesso em 04 de dezembro de 2014.

FERRAZ, L.R., OLIVEIRA, D.S., SILVA, M. F., RIGO, E., DI LUCCIO, M., OLIVEIRA, J.V.,

OLIVEIRA, D., TREICHEL, H. Production and partial characterization of multifunctional

lipases by Sporobolomyces ruberrimus using soybean meal, rice meal and sugarcane bagasse as

substrates. Biocatalysis and Agricultural Biotechnology, v. 1, p. 243–252, 2012.

GHANEM, N.B., YUSEF, H. H., MAHROUSE, H.K. Production of Aspergillus terreus xylanase

in solid-state cultures: application of the Plackett-Burman experimental design to evaluate

nutritional requirements. Bioresource Technology, v. 73, p. 113-121, 2000.

IRFAN, M., NADEEM, M., SYED, Q. One-factor-at-a-time (OFAT) optimization of xylanase

production from Trichoderma viride-IR05 in solid-state fermentation. Journal of Radiation

Research and Applied Sciences, v. 7, p. 317-326, 2014.

http://faostat.fao.org/

23

LI, H., ZHANG, R., TANG, L., ZHANG, J., MAO, Z. Manganese peroxidase production from

cassava residue by Phanerochaete chrysosporium in solid state fermentation and its

decolorization of indigo carmine. Chinese Journal of Chemical Engineering, 2014. doi:

10.1016/j.cjche.2014.11.001.

MAZOTTO, A.M., COURI, S., DAMASO, M.C.T., VERMELHO, A.B. Degradation of feather

waste by Aspergillus niger keratinases: comparison of submerged and solid-state fermentation.

International Biodeterioration & Biodegradation, v. 85, p. 189-195, 2013.

MELIKOGLU, M., LINA, C.S.K., WEBB, C. Kinetic studies on the multi-enzyme solution

produced via solid state fermentation of waste bread by Aspergillus awamori. Biochemical

Engineering Journal, v. 80, p. 76–82, 2013.

MITCHELL, D. A., LUZ, L. F. L., KRIEGER, BEROVIC, N. M. Bioreactors for solid-State

fermentation. Heidelberg: Springer, 2006. 447p.

MUSSATTO, S.I., AGUILAR, C.N., RODRIGUES, L.R., TEIXEIRA, J.A.

Fructooligosaccharides and β-fructofuranosidase production by Aspergillus japonicus

immobilized on lignocellulosic materials. Journal of Molecular Catalysis B: Enzymatic, v. 59,

p. 76–81, 2009.

NATARAJAN, K., RAJENDRAN, A. Evaluation and optimization of food-grade tannin acyl

hydrolase production by a probiotic Lactobacillus plantarum strain in submerged and solid state

fermentation. Food and Bioproducts Processing, v. 90, p. 780–792, 2012.

ORZÚA, M.C., MUSSATTO, S.I., CONTRERAS-ESQUIVEL, J.C., RODRÍGUEZ, R., DE LA

GARZA, H., TEIXEIRA, J.A., AGUILAR, C.N. Exploitation of agroindustrial wastes as

immobilization carrier for solid-state fermentation. Industrial Crops and Products, v. 30, p. 24–

27, 2009.

PANDEY, A., SOCCOL, C. R., MITCHELL, D. New developments in solid state fermentation:

I-bioprocesses and products. Process Biochemistry, v. 35, p. 1153–1169, 2000.

PANDEY, A., SOCCOL, C.R., RODRIGUEZ-LEON, J.A., NIGAM, P. Solid-state

fermentation in biotechnology—fundamentals and applications. New Delhi: Asiatech

Publishers, 2001. 221 p.

24

PATIL, N.S., JADHAV, J.P. Enzymatic production of N-acetyl-D-glucosamine by solid state

fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues.

International Biodeterioration & Biodegradation, v. 91, p. 9-17, 2014.

RAO M.B., TANKSALE A.M., GHATGE M.S., DESHPANDE V.V. Molecular and

biotechnological aspect of microbial proteases. Microbiology and Molecular Biology Reviews,

v. 62, p. 597-635, 1998.

RIGO, E., NINOW, J.L., DI LUCCIO, M., OLIVEIRA, J.V., POLLONI, A.E., REMONATTO,

D., ARBTER, F., VARDANEGA, R., OLIVEIRA, D., TREICHEL, H. Lipase production by

solid fermentation of soybean meal with different supplements. LWT - Food Science and

Technology, v. 43, p. 1132-1137, 2010.

RUIZ, H.A., RODRIGUEZ-JASSO, R.M., RODRIGUEZ, R., CONTRERAS-ESQUIVEL, J.C.,

AGUILAR, C.N. Pectinase production from lemon peel pomace as support and carbon source in

solid-state fermentation column-tray bioreactor. Biochemical Engineering Journal, v. 65, p. 90–

95, 2012.

SADAF, A., KHARE, S.K. Production of Sporotrichum thermophile xylanase by solid state

fermentation utilizing deoiled Jatropha curcas seed cake and its application in

xylooligosachharide synthesis. Bioresource Technology, v. 153, 126–130, 2014.

SANDHYA, C., SUMANTHA, A., SZAKACS, G., PANDEY, A. Comparative evaluation of

neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation.

Process Biochemistry, v. 40, p. 2689–2694, 2005.

THANAPIMMETHA, A., LUADSONGKRAM, A., TITAPIWATANAKUN, B.,

SRINOPHAKUN, P. Value added waste of Jatropha curcas residue: optimization of protease

production in solid state fermentation by Taguchi DOE methodology. Industrial Crops and

Products, v. 37, p. 1–5, 2012.

THOMAS, L., LARROCHE, C., PANDEY, A. Current developments in solid-state fermentation.

Biochemical Engineering Journal, v. 81, p. 146–161, 2013.

TORRADO, A.M., CORTES, S., SALGADO, J.M., MAX, B., RODRIGUEZ, N., BIBBINS,

B.P., CONVERTI, A., DOMINGUEZ, J.M. Citric acid production from orange peel wastes by

solid-state fermentation. Brazilian Journal of Microbiology, v. 42, p. 394–409, 2011.

25

VEERABHADRAPPA, M.B., SHIVAKUMAR, S.B., DEVAPPA, S. Solid-state fermentation of

Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in

Jatropha seed cake using Aspergillus versicolor CJS-98. Journal of Bioscience and

Bioengineering, v. 117, n. 2, p. 208-214, 2014.

WONG, Y., SAW, H., JANAUN, J., KRISHNAIAH, K., PRABHAKAR, A. Solid-state

fermentation of palm kernel cake with Aspergillus flavus in laterally aerated moving bed

bioreactor. Applied Biochemistry and Biotechnology, v. 164, p. 170–182, 2011.

ZHANG, B-B., LU, L-P., XIA, Y-J., WANG, Y-L., XU, G-R. Use of agar as carrier in solid-state

fermentation for Monacolin K production by Monascus: a novel method for direct determination

of biomass and accurate comparison with submerged fermentation. Biochemical Engineering

Journal, v. 80, p. 10-13, 2013.

27

Capítulo II: Peptídeos com atividade biológica: processos de obtenção,

purificação, identificação e potenciais aplicações.

28

Resumo

Avanços tecnológicos recentes têm despertado grande interesse para o uso de peptídeos com

atividade biológica. Peptídeos bioativos podem ser definidos como frações específicas de

proteínas com sequência contendo de 2 a 20 aminoácidos que promovem um impacto positivo em

várias funções biológicas, incluindo efeitos como atividades: antioxidante, anti-hipertensiva,

antitrombótica, antiadipogênica, antimicrobiana e anti-inflamatória. Características especiais

como a baixa toxicidade e alta especificidade colocam estas moléculas em posição de destaque

para aplicação na indústria alimentícia e farmacêutica. Nesse contexto, o presente trabalho visa

discutir os principais processos utilizados para obtenção de peptídeos bioativos, com destaque

para a fermentação e hidrólise enzimática, sendo abordados também a associação de diferentes

tecnologias e o uso de processos auxiliares. Um levantamento sobre métodos de isolamento,

purificação e caracterização destes peptídeos, como cromatografia e espectrometria de massas,

foi realizado no sentido de mostrar as principais técnicas de identificação das estruturas dos

peptídeos bioativos. Por fim, foram discutidos aspectos relacionados à atividade antioxidante,

antimicrobiana, anti-hipertensiva, antiadipogênica e indução do crescimento de bactérias

probióticas por peptídeos obtidos através de diferentes processos e variadas fontes proteicas.

Palavras-chave: proteínas, hidrólise enzimática, fermentação, peptídeos bioativos.

29

1. Introdução

As proteínas têm relevância fundamental como componentes dos alimentos.

Nutricionalmente, são fontes de aminoácidos essenciais, indispensáveis para o crescimento,

manutenção do organismo e também fonte de energia. Em alimentos proteicos, possuem a

capacidade de afetar propriedades físico-químicas e sensoriais, como a solubilidade, viscosidade,

gelificação e estabilidade da emulsão.

Algumas proteínas da dieta possuem propriedades biológicas específicas, fazendo destas,

ingredientes potenciais de alimentos funcionais (Korhonen et al., 1998). Um alimento funcional

pode ser definido como qualquer alimento, que além das funções nutritivas básicas, fornece

benefícios adicionais à saúde, regulando uma ou mais funções no organismo (Diplock et al.,

1999; Hernández-Ledesma et al., 2011).

Estudos recentes têm relacionado a prevalência de doenças cardiovasculares, obesidade,

hipertensão, diabetes e câncer à fatores alimentares. Em resposta ao aumento na percepção sobre

a relação entre alimentos e saúde, o mercado de alimentos funcionais sofreu um grande impulso.

As propriedades funcionais das proteínas alimentares decorrem do fato de que, durante a

digestão gastrointestinal, elas são hidrolisadas gerando uma grande variedade de peptídeos.

Alguns destes peptídeos apresentam características estruturais que permitem a interação com

peptídeos endógenos, os quais são responsáveis por funções vitais no organismo, podendo atuar

como neurotransmissores, hormônios ou agentes reguladores (Hernández-Ledesma et al., 2014).

Processos envolvendo a hidrólise de proteínas têm sido estudados para a produção de

peptídeos com atividade biológica. Mellander (1950) foi responsável pelo primeiro estudo

relacionando a ingestão de peptídeos bioativos derivados de proteínas hidrolisadas de caseína ao

aumento da calcificação óssea em recém-nascidos raquíticos. Desde então, peptídeos com

inúmeras bioatividades foram identificados. De acordo com os Bancos de Dados Biopep e BioPD

(Bioactive peptide database), mais de 1200 diferentes peptídeos bioativos encontram-se

registrados em suas bases (Singh et al., 2014).

Peptídeos bioativos são definidos como frações específicas de proteínas, com sequência

de aminoácidos que promovem um impacto positivo em várias funções biológicas, incluindo

atividades: antioxidante, anti-hipertensiva, antitrombótica, antiadipogênica, antimicrobiana, anti-

inflamatória e imunomoduladoras (Biziulevicius et al., 2006; Tsou et al., 2010; Zhang et al.,

30

2010; Tavares et al., 2011; Ahn et al., 2015). Estes peptídeos apresentam sequências de 2-20

aminoácidos e massas moleculares inferiores a 6000 Da. A bioatividade é definida

principalmente pela composição e sequência de aminoácidos (Sarmadi e Ismail, 2010). Essa

enorme diversidade funcional coloca os peptídeos e as proteínas em posição de destaque no

campo das aplicações biotecnológicas (Miranda e Liria, 2008), sendo apontados por alguns

autores como possíveis substitutos de substâncias químicas utilizadas como fármacos ou

conservadores de alimentos (Hong et al., 2008; Uhlig et al., 2014).

De acordo com Uhlig et al., (2014) há uma perspectiva muito importante para a utilização

de peptídeos bioativos na área farmacêutica. Alguns peptídeos em fase de ensaios clínicos têm

apresentado resultados muito promissores no tratamento de doenças cardiovasculares, infecciosas

e de origem metabólica. Os peptídeos apresentam uma importante vantagem competitiva com os

medicamentos tradicionais devido à algumas características, como: 1) são moléculas que

apresentam alta especificidade pelas células ou tecidos alvos, resultando em baixo ou nenhum

efeito tóxico e exigindo baixas concentrações para uma atuação efetiva (característica

extremamente importante, principalmente para os tratamentos de doenças durante um tempo

prolongado); 2) as moléculas químicas presentes nos medicamentos tradicionais muitas vezes

apresentam efeito cumulativo no organismo. Em casos de metabolismo deficiente, incluindo os

mecanismos de biotransformação, transporte, absorção e excreção, no qual estas moléculas

seriam excretadas ainda na forma ativa, a possibilidade de danos ao ambiente tornaria-se alta. Por

outro lado, peptídeos sofrem pouco ou nenhum acúmulo no organismo e são facilmente

degradados no ambiente (Uhlig et al., 2014).

Diferentes vias são utilizadas na obtenção de peptídeos bioativos, dentre as quais podemos

citar: fermentação, hidrólise enzimática ou a associação dos dois processos (Figura 1). No

processo de fermentação, a aplicação de culturas de bactérias lácticas com atividade proteolítica,

leva à formação de peptídeos bioativos, principalmente durante a fabricação de produtos lácteos.

A hidrólise enzimática envolve a aplicação de enzimas proteolíticas digestivas, vegetais ou de

origem microbiana em um processo de hidrólise limitada, levando a redução de fatores

alergênicos, assim como melhoria da digestibilidade e formação de peptídeos com atividade

biológica (Korhonen, 2009). Uma estratégia utilizada em algumas pesquisas científicas

demonstrou que a fermentação utilizando bactérias ácido lácticas em associação com a aplicação

de enzimas de grau alimentício resultou em produtos finais com características mais interessantes

31

quando comparados aos processos isolados. A combinação das técnicas, além de aumentar o teor

de peptídeos dos produtos fermentados, resultou em efeitos biológicos e funcionais diversificados

(Hafeez et al., 2014).

Figura 1 – Principais vias de obtenção de peptídeos e ensaios de bioatividade.

Em adição aos processos convencionais citados anteriormente, a associação de diferentes

tecnologias vem mostrando resultados eficazes na geração de peptídeos funcionais (Korhonen,

2009). O uso de ultrafiltração e nanofiltração são exemplos de tecnologias que têm sido estudadas

para refinar e fracionar peptídeos bioativos, permitindo uma separação em tamanhos selecionados

e direcionando para aplicações específicas (Quirós et al., 2007; Picot et al., 2010).

Bactérias ou fungos

produtores de proteases

Homogeneização

à alta pressão

Peptídeos

bioativos

Hidrolisados proteicos

Ultrafiltração ou nanofiltração

Proteases:

vegetais, animais ou

microbianas.

Fermentação microbiana

Proteínas animais ou vegetais

Isolamento, purificação

e identificação

Atividades biológicas:

métodos in vitro e in vivo

Anti-hipertensiva Antimicrobiana Antiadipogênica

Anti-inflamatória Imunomoduladoras Antioxidante

32

Peptídeos bioativos podem ser obtidos a partir de proteínas de origem animal ou vegetal.

As fontes vegetais geralmente incluem cereais, como o trigo, arroz, aveia, centeio e milho e

algumas leguminosas, como soja, ervilha e grão de bico. Entre as fontes vegetais, a soja é uma

das mais estudadas para obtenção de peptídeos por ser uma importante fonte de proteína

alimentar (Ortiz-Martinez et al., 2014). As fontes de proteínas animais por sua vez, também

apresentam grande potencial de aplicação. Uma das linhas de pesquisa mais estudadas e

promissoras é a produção de hidrolisados proteicos a partir de proteínas derivadas de carne, que

além de possuírem importantes atividades biológicas e serem excelentes fontes de nutrientes

como aminoácidos essenciais, minerais e vitaminas, podem ser utilizados como intensificadores

de sabor e agentes emulsificantes (Mora et al., 2014; Lafarga e Hayes, 2014). Outras fontes de

proteínas animais como ovo e peixe também foram estudadas quanto às suas propriedades

biológicas (Sakanaka et al., 2004; Theodore et al., 2008).

O conhecimento dos parâmetros críticos de processo é de fundamental importância para

obtenção de hidrolisados proteicos com características biológicas e funcionais desejáveis. Estes

parâmetros incluem: a fonte de proteína utilizada e suas características como composição química

e variações sazonais; a preparação enzimática e os aspectos relacionados à pureza, especificidade

quanto ao substrato, atividade específica, condições de pH e temperatura para a atividade e

estabilidade e as condições de processo, incluindo as concentrações de enzima e substrato, pH,

temperatura, tempo de reação. Um conhecimento prévio e a identificação destes parâmetros

podem ser utilizados como ferramentas para a obtenção de produtos com funções distintas, no

âmbito de produzir peptídeos multifuncionais, ou ainda diferentes peptídeos com funções

específicas e consequentemente com uma contribuição particular (Li-Chan, 2015).

Nesse contexto, o presente trabalho visou abordar avanços da pesquisa científica

envolvendo os processos de obtenção, purificação e identificação, atividades biológicas e

potencial de aplicação de peptídeos bioativos.

2. Principais processos de obtenção de peptídeos bioativos

2.1. Fermentação

A aplicação de processos fermentativos para obtenção de peptídeos bioativos está

relacionada principalmente com a fabricação de produtos derivados de leite, o qual possui

naturalmente proteínas precursoras de moléculas bioativas (Schanbacher et al., 1997; Akalın et

33

al., 2014). A fermentação do leite envolve uma série de vias metabólicas, que são responsáveis

pela geração de metabólitos que contribuem de forma significativa na obtenção de atributos

químicos, bioquímicos e nutricionais de produtos fermentados. O sistema proteolítico de

bactérias ácido lácticas (BAL) é complexo e constituído por três componentes principais:

proteases ligadas à parede celular que promovem a hidrólise inicial da caseína do leite a

oligopeptídeos; transportadores específicos que conduzem os oligopeptídeos para o citoplasma e

peptidases intracelulares que finalizam o processo de hidrólise dos oligopeptídeos a aminoácidos

livres e/ou peptídeos de menor massa molecular (Chaves-López et al., 2014). A capacidade

destes micro-organismos em produzir enzimas proteolíticas faz delas potenciais produtoras de

peptídeos bioativos, os quais podem ser liberados durante o processo de fabricação de produtos

fermentados. Alguns micro-organismos são extensivamente relatados na literatura por possuírem

um sistema proteolítico eficaz na hidrólise de proteínas e liberação de peptídeos com atividade

biológica, entre eles merecem destaque: Lactobacillus helveticus, Lactobacillus delbrueckii ssp.

bulgaricus, Lactococcus lactis ssp. diacetylactis, Lactococcus lactis ssp. cremoris e

Streptococcus salivarius ssp. thermophylus (Hernández-Ledesma et al., 2011). Além da

utilização de micro-organismos vivos, as enzimas proteolíticas isoladas de BAL também têm sido

utilizadas com sucesso em processos de hidrólise enzimática e produção de peptídeos bioativos

(Choi et al., 2012).

Embora os produtos lácteos tenham destaque nas pesquisas científicas que envolvem a

produção destes peptídeos por fermentação, foi demonstrado que produtos fermentados derivados

de soja, feijão, arroz e trigo também apresentaram atividade biológica (Inoue et al., 2009;

Nakahara et al., 2010; Hati et al., 2014; Limón et al., 2015) (Tabela 1).

34

Tabela 1 - Obtenção de peptídeos com diferentes atividades biológicas por meio de processo fermentativo utilizando diversas fontes

de proteína.

Micro-organismo Fonte proteica Condições do processo fermentativo Peptídeos Bioatividade Referência

Streptococcus thermophilus e

Lactobacillus bulgaricus

+

Protease Flavourzyme®

Leite de soja Processo de fermentação submersa

conduzido durante 5 h a 43 °C Tyr-Pro-Tyr-Tyr Anti-hipertensiva

Tsai et al.,

2008

Aspergillus oryzae Arroz, soja e

caseína

Processo de fermentação em estado

sólido conduzido durante 40 h a 30 °C Val-Pro-Pro; Ile-Pro-Pro Anti-hipertensiva

Inoue et al.,

2009

Aspergillus sojae Soja e trigo


sólido conduzido durante 192 h a

20-45 °C e umidade de 95%

Gly-Tyr; Ala-Phe; Val-

Pro; Ala-Ile; Val-Gly Anti-hipertensiva

Nakahara et

al., 2010

Enterococcus faecalis

TH563

Lactobacillus delbrueckii

subsp. bulgaricus LA2

Leite bovino

Processo de fermentação submersa

conduzido durante 24 h a 37 °C

(Enterococcus faecalis) ou 44 °C

(Lactobacillus delbrueckii)

Peptídeos com massa

molecular inferior a 5000

Da

Anti-hipertensiva e

imunomodulatória

Regazzo et

al., 2010

L. acidophilus ATCC 4356 e

Lc. lactis subsp. lactis GR5

Caseinato de

sódio


conduzido durante 5h a 30 °C

(Lactococcus lactis) ou 37 °C (L.

acidophilus) com agitação de 140 rpm


molecular inferior a 3000

Da

Imunomodulatória Stuknyte et

al., 2011

B. subtilis 10160 Colza


sólido conduzido durante 6 dias a 32 °C

e umidade relativa de 85 ± 5%


molecular entre 180 e

5500 Da

Antioxidante He et al.,

2012

Bifidobacterium longum

KACC91563 Caseína


conduzido durante 24 h Val-Leu-Pro-Val-Gln Antioxidante

Chang et

al., 2013

Lactobacillus casei spp.

pseudoplantarum

Proteína

concentrada de

soja


conduzido durante 36 h a 37 °C Leu-Ile-Val-Thr-Gln Anti-hipertensiva

Vallabha e

Tiku, 2014

B. subtilis ATCC 6051 Feijão


sólido conduzido durante 96 h a 30 °C e

umidade relativa de 90%


molecular entre 6,2 e

201,2 kDa

Antioxidante

Anti-hipertensiva

Limón et

al., 2015

35

2.2. Hidrólise enzimática

A hidrólise enzimática é uma das técnicas mais rápidas, seguras e de fácil controle para a

produção de peptídeos bioativos, podendo ser utilizada para melhorar propriedades funcionais e

biológicas de proteínas, assim como agregar valor a subprodutos de baixo valor comercial (Zarei

et al., 2014).

As proteases catalisam a reação de hidrólise das ligações peptídicas das proteínas e ainda

podem apresentar atividade sobre ligações éster e amida. Todas as proteases apresentam certo

grau de especificidade quanto ao substrato, em geral relacionado aos aminoácidos envolvidos na

ligação peptídica a ser hidrolisada e àqueles adjacentes a eles (Santos e Koblitz, 2008). Essa

especificidade em adição às condições de hidrólise (pH, temperatura, tempo) afetam o tamanho e

a sequência de aminoácidos na cadeia peptídica, além da quantidade de aminoácidos livres, que

por sua vez influenciam a atividade biológica dos hidrolisados (Tsou et al., 2010a; Sarmadi e

Ismail, 2010). A utilização de uma protease com atividade específica ou a combinação de

diversas proteases não específicas tem sido utilizada como estratégia para a produção de

peptídeos bioativos mais efetivos e estáveis por implicarem em reduzidos tempos de reação para

obtenção do grau de hidrólise requerido assim como a obtenção de diferentes perfis,

principalmente relacionados à composição e distribuição de massa molecular dos peptídeos. Estes

processos são especialmente utilizados em indústrias alimentícias e farmacêuticas utilizando

proteases de origem animal, vegetal e microbiana (Singh et al., 2014).

Um grande número de estudos demonstrou a liberação de peptídeos com atividade

biológica após a hidrólise de proteínas (Tabela 2).

36

Tabela 2 - Aplicação de proteases para obtenção de peptídeos com atividade biológica a partir de diferentes fontes de proteína.

Protease Condições de

hidrólise Fonte proteica

Bioatividade dos

peptídeos Peptídeos Método de identificação Referência

Alcalase®

pH 8,0; 50 °C; 3 h

E:S = 1:20

[S] = 5,0%

Soja Antiadipogênese

Peptídeos com

massa molecular

entre 754 e 3897

Da

Cromatografia líquida acoplada

à espectrometria de massas

Mejia et al.,

2010

Flavourzyme®

pH 7,0; 50 °C; 2 h

E:S = 1:100

[S] = 2,5%

Proteína isolada

de soja Antiadipogênese

Peptídeos com

massa molecular

inferior a 1300 Da

Cromatografia de exclusão

molecular de alta performance

Tsou et al.,

2010a

Neutrase®

pH 6,0; 45 °C; 4 h

E:S = 1:100

[S] = 2,5%

Proteína isolada

de soja Antiadipogênese

Peptídeos com

massa molecular

entre 1300 e 2200

Da


molecular de alta performance

Tsou et al.,

2010b

Pepsina pH 5,5; 23 °C

[S] = 1,0%

Hemoglobina

bovina

Antimicrobiana

Anti-hipertensiva

Peptídeos com

massa molecular

entre 668 e 4430

Da

Espectrometria de massas com

ionização por “electrospray”

(ESI/MS)

Adje et al.,

2011

Alcalase®

pH 8,0; 50 °C; 3 h

[E] = 0,2 mg mL-1

[S] = 8,0%

Feijão Antioxidante

Anti-inflamatória

Peptídeos com

massa molecular

entre 445 e 2148

Da

Espectrometria de massas com

ionização/dessorção a laser

assistida por matriz

(MALDI-TOF)

Oseguera-

Toledo et al.,

2011

Alcalase®

Flavourzyme®

Protamex®

Neutrase®

Pepsina

Tripsina

pH 7,0; 50 °C; 8 h

pH 7,0; 50 °C; 8 h

pH 7,0; 50 °C; 8 h

pH 7,0; 50 °C; 8 h

pH 2,0; 37 °C; 8 h

pH 8,0; 37 °C; 8 h

Salmão Antioxidante

Anti-inflamatória

Peptídeos com

massa molecular

entre 1000 e 2000

Da


molecular de alta performance Ahn et al., 2012

37

3. Concentração, purificação e identificação de peptídeos bioativos

Diversas técnicas podem ser utilizadas para o isolamento, identificação e caracterização

de proteínas e peptídeos. As técnicas cromatográficas estão entre as mais aplicadas, destacando-

se a cromatografia líquida de alta eficiência (CLAE) como a mais utilizada (Singh et al., 2014). A

técnica de CLAE de fase reversa, por exemplo, pode ser utilizada para fracionar peptídeos com

base em suas propriedades hidrofóbicas (Pownall et al., 2010). Técnicas de eletroforese em gel e

ultrafiltração também foram utilizadas como métodos auxiliares na caracterização estrutural e

composição química de peptídeos (Roblet et al., 2012; Singh et al., 2014).

A espectrometria de massas, por sua vez, representou um grande avanço na identificação

de sequências peptídicas e estudos sobre os perfis de proteínas e seus produtos de hidrólise. O

desenvolvimento de interfaces, nas quais a ionização do analito por métodos que permitem a

obtenção de íons a partir de moléculas sensíveis à temperatura e/ou pouco voláteis, como a

ionização por eletronebulização ("electrospray ionization") e a ionização/dessorção a laser

assistida por matriz (MALDI-TOF) surgiram recentemente como importantes ferramentas para a

identificação e caracterização de proteínas e peptídeos bioativos utilizando espectrometria de

massas. A técnica de cromatografia líquida acoplada à espectrometria de massas é comumente

utilizada para identificar sequências peptídicas (Chiaradia et al., 2008; Contreras et al., 2008;

Singh et al., 2014).

Peptídeos com atividade anticoagulante obtidos a partir do peixe “goby” (Awaous

guamensis) e uma protease de Bacillus licheniformis foram fracionados por cromatografia de

exclusão molecular e cromatografia líquida de alta eficiência de fase reversa e identificados por

espectrometria de massas. A solução de hidrolisados contendo os peptídeos foi aplicada à coluna

de filtração em gel Sephadex G-25 (5,2 x 56 cm) pré-equilibrada e eluída com água destilada.

Frações de 4,5 mL foram coletadas utilizando-se fluxo de 0,5 mL min-1

e a absorbância

mensurada a 220 nm para determinar o perfil de eluição dos peptídeos. As frações com maior

atividade anticoagulante foram recolhidas e purificadas em coluna de fase reversa Vydac C18 (10

x 250 mm, Grace-Vydac) e eluídas por gradiente linear de acetonitrila (0 a 40% v/v) e fluxo de

0,6 mL min-1

. A massa molecular e a sequência de aminoácidos dos peptídeos foram

determinadas utilizando um espectrômetro de massas triploquadrupolo com fonte de ionização

por electrospray (Applied Biosystems API 3000, PE Sciex, Toronto, Canadá). Quatro sequências

38

peptídicas apresentaram alta atividade anticoagulante e foram identificadas como Leu-Cys-Arg,

His-Cys-Phe, Cys-Leu-Cys-Leu-Arg e Cys-Arg-Arg (Nasri et al., 2012).

Tsou et al., (2013) utilizaram como estratégia para purificação e identificação de

peptídeos bioativos obtidos a partir de proteína isolada de soja e protease Flavourzyme®, o

fracionamento sequencial com membranas de ultrafiltração de diferentes tamanhos,

cromatografia em gel, cromatografia líquida de alta eficiência de fase reversa e espectrometria de

massas. Os hidrolisados foram inicialmente fracionados em membranas de ultrafiltração de 30,

10 e 1 kDa. A fração retentada da membrana de 1 kDa foi selecionada para purificação com base

em sua capacidade em estimular a lipólise em células pré-adipócitas 3T3-L1. O retentado de 1

kDa foi então aplicado em coluna Superdex® peptide 10/300 GL (10 x 300 mm; GE Healthcare)

equilibrada e eluída com acetonitrila 30% e fluxo de 0,5 mL min-1

. Frações de 1,0 mL foram

coletadas e curvas de eluição foram construídas a partir das medidas de absorbância a 214 nm. As

frações com maior atividade antiadipogênica foram recolhidas e purificadas em coluna de fase

reversa Develosil® ODS-HG-5 (4,6 x 250 mm, Nomura Chemical) e eluídas por um gradiente

linear de acetonitrila (5 a 75%) e fluxo de 1,0 mL min-1

. A fração com maior atividade anti-

adipogênica foi submetida a uma segunda etapa de purificação em coluna de fase reversa

utilizando gradientes lineares de acetonitrila de 10 a 40%. Os peptídeos foram finalmente

identificados por cromatografia líquida acoplada à espectrometria de massas. Três peptídeos com

as seguintes sequências de aminoácidos Ile-Leu-Leu, Leu-Leu-Leu e Val-His-Val-Val foram

identificados como os responsáveis pela atividade anti-adipogênica dos hidrolisados de proteína

isolada de soja.

Peptídeos com atividade anti-hipertensiva foram isolados e identificados a partir de

hidrolisados de gelatina extraída de pele de arraia (Okamejei kenojei). Inicialmente, os

hidrolisados foram submetidos à ultrafiltração em membranas de 1 kDa e os peptídeos com

massa molecular inferior à este corte foram recolhidos. A etapa de purificação consistiu na

aplicação de etapas sequenciais de isolamento por cromatografia líquida rápida de proteínas

(FPLC) (AKTA, Amersham Bioscience Co., Uppsala, Suécia) utilizando a coluna de troca iônica

de fluxo rápido HiPrep 16/10 (16 x 100 mm, Amersham Biosciences, Piscataway, NJ, EUA) e a

coluna de filtração em gel GE Healthcare Superdex® Peptide 10/300 GL (10 x 300 mm). Os

peptídeos purificados foram então identificados utilizando a técnica de espectrometria de massas

39

MALDI-TOF. Dois peptídeos purificados mostraram alta atividade anti-hipertensiva e foram

identificados como Leu-Gly-Pro-Leu-Gly-His-Gln com massa molecular estimada em 720 Da e

Met-Val-Gly-Ser-Ala-Pro-Gly-Val-Leu com massa molecular de 829 Da (Ngo et al., 2015).

4. Propriedades biológicas de peptídeos bioativos

Peptídeos bioativos de proteínas alimentares têm sido estudados extensivamente ao longo

da última década para elucidar seu potencial biológico e influência sobre os principais sistemas

do corpo humano, como: digestivo, cardiovascular, nervoso e imunológico. Alguns peptídeos

bioativos apresentaram atividades biológicas com impacto positivo sobre a saúde, dentre as quais

podemos citar: atividade antimicrobiana (Adje et al., 2011), anti-hipertensiva (Alemán et al.,

2011), antioxidante (Zhang et al., 2009) anticancerígena (Alemán et al., 2011), antiadipogênica

(Tsou et al., 2010), imunomoduladoras (Huang et al., 2010) e anti-inflamatória (Ahn et al.,

2015); portanto têm perspectivas de serem incorporados como ingredientes em alimentos

funcionais, nutracêuticos e medicamentos, onde essas bioatividades podem ser aliadas no

controle e prevenção de doenças (Agyei e Danquah, 2012).

A obtenção e características de peptídeos com atividade antimicrobiana, antioxidante,

antiadipogênica, anti-hipertensiva e aplicação na indução do crescimento de bactérias lácticas e

probióticas estão descritas neste trabalho.

4.1. Peptídeos com atividade antimicrobiana

Ao longo das últimas décadas, um número crescente de micro-organismos patogênicos

desenvolveu resistência aos antibióticos convencionais, gerando sérios problemas no tratamento

de infecções, principalmente de indivíduos imunocomprometidos. Aliado a este fato, o

desenvolvimento de novos antibióticos apresentou uma diminuição neste mesmo período. Duas

causas principais justificam o aumento da resistência de micro-organismos aos antibióticos: o uso

indiscriminado destes medicamentos em condições não recomendadas de uso, como tempo ou

dosagem inferiores para um tratamento efetivo, e a capacidade de mutação genética dos micro-

organismos, que aumenta a dificuldade de desenvolvimento de drogas baseadas em mecanismos

específicos de ação (Harrison et al., 2014). Nesse contexto, a utilização de fontes naturais de

compostos antimicrobianos possui um enorme potencial de aplicação, visto que possuem

características interessantes como baixa toxicidade e alta especificidade. Esses mecanismos

podem ser melhor compreendidos ao compararmos o modo de ação de peptídeos antimicrobianos

40

sobre células bacterianas (unicelulares) e animais (pluricelulares). As membranas bacterianas

possuem uma camada rica em fosfolipídeos carregados negativamente e com essas porções

voltadas para meio externo, o que facilita a interação com os peptídeos que em sua maioria estão

carregados positivamente. Por outro lado, as células animais são compostas, principalmente, por

lipídeos não carregados na camada mais externa, enquanto as porções carregadas negativamente

estão voltadas para o interior celular (citoplasma) (Matsuzaki, 1999) (Figura 2).

Figura 2 – Especificidade de peptídeos antimicrobianos mediante membranas de organismos

pluricelulares (células animais) e unicelulares (bactérias) (Figura adaptada de Zasloff, 2002).

Peptídeos antimicrobianos estão amplamente distribuídos na natureza e representam um

componente essencial do sistema imunológico. Eles são reconhecidamente, a primeira linha de

defesa do organismo contra a colonização de micro-organismos exógenos, com papel

fundamental na regulação de populações bacterianas em mucosas e outras superfícies epiteliais

(Boman e Hultmark, 1987; Bevins e Zasloff, 1990; Zasloff, 2002). Mais de 800 peptídeos

antimicrobianos já foram descritos em plantas e animais (Boman, 2003). Apesar da diversidade

na estrutura primária, a grande maioria dos peptídeos antimicrobianos possui cadeias curtas de

41

aminoácidos, que são caracterizadas pela predominância de aminoácidos catiônicos e

hidrofóbicos (Zasloff, 2002; Dashper et al., 2007) (Figura 2). A reduzida massa molecular das

frações peptídicas, com consequente maior exposição dos resíduos de aminoácidos e suas cargas,

e a formação de pequenos canais na bicamada lipídica foram relacionados com o poder

antimicrobiano, pois causam modificações que aumentam a interação peptídeo-membrana

(Gobbetti et al., 2004; Patrzykat e Douglas, 2005; Gómez-Guillén et al., 2010).

O mecanismo exato de ação de muitos peptídeos antimicrobianos ainda não está bem

estabelecido, e devido ao grande número de peptídeos já conhecidos, acredita-se na probabilidade

de existirem mecanismos distintos de ação (Dashper et al., 2007).

Além dos naturalmente presentes nos sistemas de defesa de plantas e animais, peptídeos

com atividade antimicrobiana já foram identificados em diversos hidrolisados proteicos.

Hidrolisados de caseína de leite bovino obtidos a partir da hidrólise enzimática com

quimosina foram avaliados quanto ao poder antimicrobiano. Cinco diferentes peptídeos

antibacterianos foram isolados a partir da extremidade carboxílica da αs2-caseína. As frações de

peptídeos f (181-207), f (175-207) e f (164-207) apresentaram amplo espectro de ação e foram

capazes de inibir diversas bactérias Gram+ e Gram-; os valores de concentração inibitória mínima

(CIM) de cada fração variaram de 21,0 a 168,0 mg mL-1

, 10,7 a 171,2 mg mL-1

e 4,8 a 76,2 mg

mL-1

, respectivamente. É válido ainda ressaltar que o potencial de inibição destes peptídeos

contra bactérias Gram+ foi tão alto quanto o dos conhecidos peptídeos antimicrobianos nisina e

lactoferricina B (McCann et al., 2005).

Peptídeos com atividade antimicrobiana foram preparados a partir de gelatina hidrolisada

com Alcalase® 2.4L (Sigma-Aldrich, Estados Unidos). As frações obtidas por ultrafiltração em

membranas de 1 e 10 kDa foram utilizadas para testes antimicrobianos contra 18 bactérias. As

bactérias mais sensíveis na presença das frações testadas foram: Lactobacillus acidophilus,

Bifidobacterium lactis, Shewanella putrafaciens e Photobacterium phosphoreum (Gómez-Guillén

et al., 2010). Hidrolisados de hemoglobina bovina tratada com pepsina foram purificados por

CLAE e testados quanto ao poder antimicrobiano contra duas linhagens Gram- (Escherichia coli,

Salmonella enteritidis) e três Gram+ (Kocuria luteus A270, Staphylococcus aureus e Listeria

innocua). Os resultados obtidos mostraram que as frações peptídicas purificadas apresentaram

amplo espectro de ação, agindo contra 4 das 5 bactérias testadas (Kocuria luteus A270, Listeria

42

innocua, Escherichia coli e Staphylococcus aureus) com CIM variando entre 35,2 e 187,1 µM

(Adje et al., 2011).

Tellez et al. (2011) mostraram a eficiência de uma fração peptídica, isolada a partir de

leite fermentado com Lactobacillus helveticus, contra uma infecção proposital com Salmonella

enteritidis em ratos. A taxa de sobrevivência no grupo alimentado com a fração peptídica (0,02

µg por dia) foi superior ao grupo alimentado com metade da dose (0,01 µg por dia) e ao grupo

controle.

O potencial antimicrobiano de proteína isolada de soro de leite hidrolisada por diferentes

enzimas gastrointestinais foi verificado por Théolier et al., (2013). Os resultados obtidos por

estes autores mostraram que hidrolisados proteicos obtidos com tripsina e quimotripsina não

apresentaram atividade antibacteriana contra Listeria ivanovii HPB28 e Escherichia coli

MC4100, enquanto os hidrolisados produzidos por ação da enzima pepsina apresentaram

atividade significativa. Os hidrolisados foram fracionados por cromatografia líquida de alta

eficiência de fase reversa, resultando em cinco frações com alta atividade antibacteriana e CIM

variando de 20,0 a 35,0 µg mL-1

. Uma fração peptídica obtida a partir de água residuária

proveniente do cozimento de anchovas (Engraulis japonicus) e ação da enzima Protamex®,

apresentou alta atividade antimicrobiana contra Staphylococcus aureus. A fração identificada

apresentou a sequência peptídica Gly-Leu-Ser-Arg-Leu-Phe-Thr-Ala-Leu-Lys e massa molecular

estimada em 1,1 kDa (Tang et al., 2015).

4.2. Peptídeos com atividade antioxidante

A formação de radicais livres, tais como superóxido (O˙2-) e hidroxila (˙OH), é uma

consequência inevitável em organismos aeróbios durante a respiração. Estes radicais são muito

instáveis e reagem rapidamente com outros grupos ou substâncias no organismo, ocasionando

lesões celulares ou nos tecidos (Zhang et al., 2009). Uma quantidade excessiva desses radicais no

organismo foi associada ao desenvolvimento de várias doenças, como aterosclerose, artrite,

diabetes e câncer (Gu et al., 2015). Por serem espécies altamente reativas, os radicais livres

podem causar danos às proteínas e mutações no DNA, oxidação de fosfolipídeos de membrana e

modificação em lipoproteínas de baixa densidade (LDL) (Pihlanto, 2006). Em alimentos, a

oxidação também afeta diretamente a qualidade, comprometendo caraterísticas como sabor,

43

aroma e coloração. Nesse contexto, a presença de substâncias que inibem reações oxidativas que

comprometem a qualidade dos alimentos é de suma importância.

É importante ressaltar que a capacidade de eliminação de radicais livres por compostos

antioxidantes é determinada por vários fatores, como: reatividade química e consequentemente a

taxa de eliminação destes radicais, o destino do produto derivado após a reação antioxidante-

radical, interação com outras substâncias antioxidantes, concentração e mobilidade no meio

ambiente e mecanismos de absorção, distribuição, retenção e metabolismo (Niki, 2010).

Antioxidantes são considerados importantes nutracêuticos apresentando diversos

benefícios à saúde, e são definidos como quaisquer substâncias que retardam ou inibem

significativamente a oxidação de um substrato. Atualmente, alguns antioxidantes sintetizados

artificialmente como hidroxitolueno butilado (BHT), hidroxianisol butilado (BHA) e

tertbutilhidroquinona (TBHQ), têm sido empregados para prevenir os danos oxidativos em

alimentos e biosistemas. No entanto, estes produtos químicos sofrem uma tendência de uso cada

vez mais limitada por apresentar riscos potenciais à saúde humana, como danos ao DNA,

toxicidade e efeitos colaterais (Wang et al., 2014; Chi et al., 2015). Consequentemente, há um

interesse crescente entre os pesquisadores para obtenção de moléculas antioxidantes mais seguras

a partir de fontes naturais, como os peptídeos provenientes de proteínas hidrolisadas (Senphan e

Benjakul, 2014; Chi et al., 2015).

Alguns peptídeos com atividade antioxidante têm ocorrência natural em alimentos. A

glutationa (γ-Glu-Cys-Gly) e a carnosina (β-alanil-L-histidina) são antioxidantes naturalmente

presentes em tecidos musculares e apresentam capacidade de doar elétrons, quelar metais e íons e

inibir a peroxidação lipídica (Samaranayaka e Li-Chan, 2011). Além dos naturalmente presentes,

peptídeos obtidos a partir de alimentos proteicos hidrolisados têm sido relatados por terem

capacidade antioxidante similar ou superior a antioxidantes sintéticos como o BHT, sendo assim

uma fonte segura para aplicação em alimentos (Yasufumi et al., 2001).

Os mecanismos de ação que explicam a atividade antioxidante de peptídeos não são

totalmente compreendidos, mas vários estudos mostraram a capacidade de peptídeos em inibir a

peroxidação lipídica (Sakanaka et al., 2004), eliminar radicais livres (Gómez-Guillén, 2010),

quelar íons metálicos (Alemán et al., 2011) e eliminar espécies reativas de oxigênio (Zhuang e

Sun, 2011). Assim como para outras atividades biológicas, as propriedades antioxidantes dos

44

peptídeos estão relacionadas com sua composição, estrutura e hidrofobicidade (Chen et al.,

1998). A presença dos aminoácidos tirosina, triptofano, metionina, lisina e cisteína, foi relatada

como importante fator para a ação antioxidante dos peptídeos, especialmente pela capacidade de

redução do Fe3+

a Fe2+

e atividade quelante de íons Fe2+

e Cu2+

(Wang e De Mejia, 2005; Huang

et al., 2010; Carrasco-Castilla et al., 2012). Deve-se ressaltar, que não só a presença, mas também

a sequência destes aminoácidos na cadeia peptídica desempenha papel importante no poder

antioxidante (Rajapakse et al., 2005). A capacidade antioxidante de peptídeos pode ser avaliada

por diversos métodos in vitro, nos quais estão envolvidos diferentes mecanismos de ação e

consequentemente medem atividades distintas (Tabela 3).

Além da avaliação in vitro, a atividade antioxidante pode ser avaliada por métodos in

vivo, os quais utilizam modelos animais. A capacidade antioxidante in vivo é determinada por

vários fatores, já que estas substâncias devem ser absorvidas, transportadas, distribuídas e retidas

adequadamente nos fluidos biológicos, células e tecidos. A biodisponibilidade destes compostos,

o efeito da dosagem e a duração dos tratamentos têm sido estudados por análise de fluidos

biológicos e tecidos humanos e animais após a ingestão (Niki, 2010; Alam et al., 2013). A Tabela

4 mostra alguns destes métodos e o princípio de cada avaliação.

45

Tabela 3 – Principais métodos de determinação de atividade antioxidante in vitro de peptídeos e respectivos mecanismos de cada

método.

Método Mecanismo Reação Medida realizada Referência

DPPH Captura do

radical DPPH

O radical DPPH (2,2-difenil-1-picril-hidrazil) reage com antioxidantes

doadores de hidrogênio, com mudança de coloração violeta para amarela.

Redução da absorbância

a 517 nm

Sharma e

Bhat, 2009

ORAC

Captura de

radical

peroxila

O radical peroxila, gerado pela decomposição do AAPH [dicloreto de 2,2’-

azobis (2-amidinopropano)] na presença de oxigênio atmosférico, reage

com um indicador fluorescente formando um produto não fluorescente. Na

presença de antioxidantes, a fluorescência é preservada.

Redução de

fluorescência (excitação

a 485 nm e emissão a

520 nm)

Dávalos et

al., 2004

FRAP

Poder de

redução de

ferro

Na presença de antioxidantes doadores de elétrons, o complexo Fe3+

-TPTZ

[2,4,6-tri(2-piridil)-1,3,5-triazina] é reduzido a Fe2+

-TPTZ, com mudança

de coloração azul clara para azul escura

Aumento da absorbância

a 593 nm

Ou et al.,

2002

ABTS Captura do

radical ABTS

O radical ABTS (ácido 2,2'-azinobis-(3-etilbenzotiazolino-6-sulfônico) é

estabilizado na presença de antioxidantes doadores de hidrogênio, com

mudança de coloração verde escura para verde clara.


a 734 nm

Gómez-

Guillén et

al., 2010

Habilidade em

quelar metais de

transição (Cu²+)

Quelação de

Cu²+

Reação de complexação de Cu2+

com violeta de pirocatecol gerando um

produto colorido. A presença de antioxidantes diminui a formação do

complexo Cu2+

-pirocatecol com consequente redução da intensidade de

cor.


a 620 nm

Theodore et

al., 2008

Habilidade em

quelar metais de

transição (Fe²+)

Quelação de

Fe²+

Reação de complexação de Fe2+

com ferrozina gerando um produto

colorido. A presença de antioxidantes diminui a formação do complexo

Fe2+

-ferrozina com consequente redução da intensidade de cor.


a 562 nm

Nazeer e

Kulandai,

2012

TBARS

Quantificação

de produtos de

peroxidação

de lipídica

Reação do ácido tiobarbitúrico com produtos da decomposição dos

hidroperóxidos, sendo o malonaldeído, o principal elemento quantificado.

Absorbância e atividade antioxidante são inversamente proporcionais.

Aumento da absorbância

a 532 nm

Raghavan e

Kristinsson,

2008

46

Tabela 4 – Principais métodos de determinação de atividade antioxidante de peptídeos e proteínas hidrolisadas in vivo e respectivos

mecanismos de ação e princípios de cada determinação.

Método Amostra avaliada Animais Tecido/órgão

avaliado Mecanismo de ação e princípios de cada determinação Referência

Superóxido

dismutase (SOD)

Peptídeo isolado a

partir de hidrolisado

de plasma suíno

Ratos da

linhagem Wistar,

machos e adultos

Fígado

A SOD é uma enzima que catalisa a dismutação do

radical superóxido em peróxido de hidrogênio e

oxigênio, tendo assim um importante papel de proteção

celular contra espécies reativas de oxigênio

Liu et al.,

2011

Catalase (CAT)

Peptídeo isolado de

proteína hidrolisada

de peixe

Ratos da

linhagem Wistar,

albinos, machos e

adultos

Lisado de

eritrócitos

(sangue)

A CAT é uma enzima responsável pela conversão de

peróxido de hidrogênio em água e oxigênio,

apresentando assim um dos principais mecanismos de

eliminação e radicais livres no organismo

Nazeer et al.,

2012

Teor de glutationa

reduzida (GSH)

Isolado proteico de

sementes de arruda

da Síria (Peganum

harmala)

Ratos albinos e

machos

Fígado e

plasma

sanguíneo

GSH é um redutor intracelular que apresenta um

importante papel de proteção celular contra radicais

livres, peróxidos e outros compostos tóxicos

Soliman et

al., 2013

Glutationa-S-

transferase (GST)

Peptídeo isolado de

proteína hidrolisada

de mexilhão

Ratos machos e

adultos Fígado

GST é um sistema enzimático localizado no citosol que

catalisa a conjugação de moléculas eletrofílicas reativas

com a glutationa, facilitando o metabolismo e excreção

de toxinas e consequentemente reduzindo a ocorrência de

danos celulares como mutações no DNA

Kim et al.,

2013

Glutationa

peroxidase (GPx)

Hidrolisados de

glúten de milho

Camundongos

Kunming machos

e fêmeas

Fígado e

plasma

sanguíneo

A GPx é uma enzima que catalisa a reação entre

hidroperóxidos com glutationa reduzida levando à

formação de glutationa dissulfeto e o produto de redução

de hidroperóxidos

Liu et al.,

2014

Determinação do

teor de

malonaldeído

Hidrolisados de

proteína de peixe

(Salaria basilisca)

Ratos da

linhagem Wistar,

machos e adultos

Fígado e

plasma

sanguíneo

O malonaldeído é um produto intermediário da

peroxidação lipídica e, portanto sua quantificação pode

ser utilizada como indicativo da presença de radicais

livres

Ktari et al.,

2014

47

Os métodos in vitro são os mais utilizados para a avaliação da atividade antioxidante de

proteínas hidrolisadas. Nazeer e Kulandai (2012) avaliaram as propriedades antioxidantes de

hidrolisados proteicos de peixe obtidos por tratamento enzimático utilizando diferentes proteases

(papaína, pepsina, tripsina e quimotripsina). A atividade antioxidante foi avaliada pela redução do

radical DPPH, poder de redução do ferro e habilidade em quelar metais. Todos os hidrolisados

apresentaram atividade antioxidante, sendo que os obtidos com pepsina e tripsina mostraram

maior atividade. Li et al. (2012) verificaram que hidrolisados proteicos de carpa preparados com

Alcalase® 2.4L e papaína apresentaram atividade antioxidante utilizando-se as metodologias

ABTS, DPPH, poder de redução do Fe3+

e habilidade em quelar Fe2+

. Najafian e Babji (2015)

estudaram a atividade antioxidante de proteína miofibrilar de peixe (Pangasius sutchi)

hidrolisada utilizando as preparações enzimáticas de proteases papaína, Alcalase® e

Flavourzyme®. Os graus de hidrólise variaram de acordo com a enzima utilizada e atingiram

valores de 36,53 a 89,17%. Os hidrolisados obtidos a partir de 60 min de hidrólise utilizando

papaína apresentaram atividade antioxidante superior aos demais no método TBARS e nos

métodos que mediram a habilidade em quelar Fe2+

e o poder de redução do Fe3+

. Os hidrolisados

obtidos com a enzima Alcalase® mostraram maior atividade contra o radical DPPH enquanto os

obtidos com Flavourzyme® apresentaram atividade superior contra o radical ABTS.

A determinação da atividade antioxidante por métodos in vivo é baseada principalmente

em medidas de atividade enzimática. A diminuição da atividade de enzimas antioxidantes como a

catalase (CAT), glutationa peroxidase (GPx), superóxido dismutase (SOD) e glutationa-S-

transferase (GST) influenciam criticamente a susceptibilidade de vários tecidos ao estresse

oxidativo e está associada ao desenvolvimento de diversas doenças (Ktari et al., 2014). Liu et al

(2014) investigaram as propriedades antioxidantes de hidrolisados proteicos de glúten de milho

preparados utilizando as proteases Alcalase®

, Flavourzyme®

e Protamex®. A atividade

antioxidante dos hidrolisados foi avaliada por métodos in vivo. Os experimentos foram realizados

com 40 camundongos da linhagem Kunming (25,0 ± 2,0 g) com 4-5 semanas de idade. Os

camundongos foram aleatoriamente divididos em cinco grupos experimentais contendo oito

animais cada, sendo igualmente divididos em machos e fêmeas. Os grupos foram tratados da

seguinte maneira: o grupo I foi utilizado como controle e recebeu a dieta de base comum a todos

os grupos; o grupo II foi tratado diariamente com padrão de vitamina E (83 mg/kg/dia) durante

10 dias; os grupos III, IV e V foram tratados com 300, 700 e 1000 mg/kg/dia dos hidrolisados,

48

respectivamente, durante 10 dias. Os resultados obtidos mostraram que os hidrolisados obtidos

utilizando a combinação das enzimas Alcalase e Protamex produziram peptídeos com alta

atividade antioxidante. O perfil de distribuição da massa molecular dos hidrolisados mostrou

peptídeos com tamanho entre 250 e 1200 Da. A ingestão de 300 mg kg-1

de peptídeos resultou em

aumento das atividades enzimáticas de superóxido dismutase, glutationa-peroxidase e reduziu os

teores de malonaldeído no fígado e sangue dos camundongos em comparação ao grupo controle e

ao tratado com vitamina E, indicando um grande potencial antioxidante.

4.3. Peptídeos com atividade antiadipogênica

A obesidade é resultado de um desequilíbrio entre a ingestão e a real necessidade de

energia, levando a um crescimento patológico de células adipócitas (Aoyama et al., 2000). A

quantidade de tecido adiposo pode ser controlada por inibição da adipogênese em células

precursoras ou pré-adipócitas, como os pré-adipócitos 3T3-L1, que são os modelos mais bem

caracterizados para o estudo de adipogênese. Muitos fatores de transcrição estão envolvidos na

diferenciação de células pré-adipócitas em adipócitos, e a inibição ou regulação destes fatores

pode levar a uma diminuição do acúmulo de gordura no organismo (Tsou et al., 2010). A

glicerol-3-fosfato desidrogenase (GPDH) é uma enzima que ocupa uma posição chave no

metabolismo da glicose, e está ligada à biossíntese de fosfolipídeos e triglicerídeos (Harding et

al., 1975; Tsou et al., 2010b). A supressão da atividade GPDH pode resultar na inibição da

diferenciação bem como na redução do acúmulo de lipídeos em células 3T3-L1, assim a

determinação da atividade desta enzima pode ser empregada para avaliar o efeito antiadipogênico

(Hirai et al., 2005). Outra enzima envolvida no processo de adipogênese é a ácido graxo sintetase

(FAS), a qual participa da síntese endógena de ácidos graxos saturados de cadeia longa a partir

dos precursores acetil-CoA e malonil-CoA (Rahman et al., 2008; Maier et al., 2008). Tem sido

relatado que certas frações de proteínas hidrolisadas possuem a capacidade de inibir a ação destas

enzimas, regulando assim o processo de diferenciação celular e o acúmulo relativo de lipídeos.

De acordo com Kim et al. (2007), estes hidrolisados apresentam grande potencial em tratamentos

antiobesidade por diminuírem o acúmulo de gordura no organismo.

Martinez-Villaluenga et al., (2008) estudaram a produção de hidrolisados obtidos a partir

de proteínas de soja utilizando a preparação comercial de proteases Alcalase®. Os hidrolisados

foram avaliados quanto ao efeito sobre o acúmulo relativo de lipídeos em células pré-adipócitas

49

3T3-L1 e mostraram supressão variando de 29 a 46%. Os autores também avaliaram a atividade

anti-adipogênica de diferentes frações da proteína de soja e detectaram que as unidades da fração

de β-conglicinina apresentaram um maior número de peptídeos responsáveis pela inibição do

acúmulo de lipídeos em células 3T3-L1 quando comparadas às subunidades de glicinina.

Tsou et al. (2010a) estudaram a aplicação da preparação comercial de proteases

Flavourzyme®

na hidrólise de proteína isolada de soja e avaliaram a capacidade antiadipogênica

das frações dos hidrolisados obtidas por ultrafiltração. Os resultados revelaram que a hidrólise

limitada de proteína isolada de soja permitiu a obtenção de hidrolisados com grande capacidade

antiadipogênica, e que as frações obtidas por ultrafiltração inibiram mais eficientemente a

atividade GPDH, sendo a fração obtida com membranas de 1 kDa, a mais efetiva (59% de

inibição). A atividade antiadipogênica de hidrolisados de proteína isolada de soja após tratamento

enzimático com Neutrase e o efeito do fracionamento por ultrafiltração sobre a bioatividade

foram estudados por Tsou et al. (2010b). Assim como no estudo anterior, os resultados

mostraram que peptídeos com baixa massa molecular (entre 1.300 e 2.200 Da) foram mais

efetivos na inibição da atividade GPDH.

Mejia et al. (2010) avaliaram o efeito de hidrolisados proteicos de soja enriquecidos com

β-conglicinina (proteína naturalmente presente na soja) sobre a atividade da FAS e adipogênese

em adipócitos humanos in vitro. Os resultados mostraram que alterações genotípicas nas

subunidades da proteína de soja (enriquecimento com β-conglicinina) produziram perfis

peptídicos que levaram à inibição da FAS e diminuição do acúmulo de lipídeos in vitro. A

quantidade de hidrolisados de proteína de soja necessária para inibir 50% da atividade da FAS

(IC50) variou de 50-175 µM. Um peptídeo com capacidade antiadipogênica foi isolado por

ultrafiltração, filtração em gel e CLAE a partir de hidrolisados proteicos de soja. A capacidade

antiadipogênica foi confirmada por meio da inibição da diferenciação de células pré-adipócitas

3T3-L1. O inibidor de adipogênese foi identificado com um tripeptídeo (Ile-Gln-Asn), tendo um

valor de IC50 de 0,014 mg de proteína mL-1

(Kim et al., 2007).

4.4. Peptídeos com atividade anti-hipertensiva

A hipertensão arterial afeta cerca de 25% da população adulta em todo o mundo, e há uma

previsão de que este número atinja 29% da população até 2025, o que representa um total de 1,56

bilhão de pessoas (Ngo et al., 2015). Embora seja um distúrbio controlável, a hipertensão está

50

associada ao desenvolvimento de doenças cardiovasculares, como arteriosclerose, infarto de

miocárdio e acidente vascular cerebral (Sheih et al., 2009). A enzima conversora de angiotensina

(ECA) desempenha um papel importante na regulação da pressão arterial porque catalisa a

conversão da angiotensina I (forma inativa) em angiotensina-II (vasoconstritor), além de inativar

a bradicinina (vasodilatador). Consequentemente inibidores sintéticos da ECA, tais como

captopril e enalapril são muitas vezes utilizados para tratar a hipertensão e outras doenças

relacionadas com o coração. No entanto, os inibidores sintéticos podem causar diversos efeitos

colaterais, como tosse, alteração do paladar, erupções cutâneas e angioedema (Alemán et al.,

2011).

É bem reconhecido, que proteínas alimentares contêm sequências primárias de peptídeos

capazes de modular funções fisiológicas específicas (Hong et al., 2008). Muitos tipos de

peptídeos bioativos com atividade inibidora da ECA foram isolados de hidrolisados proteicos e

produtos fermentados. O dipeptídeo Ala-Pro e o tripeptídeo Phe-Ala-Pro, por exemplo,

apresentam estruturas análogas às drogas captopril e enalapril, respectivamente (Figura 3).

Figura 3 - Estruturas de medicamentos inibidores da ECA e os seus peptídeos análogos (Figura

adaptada de Matsui e Matsumoto, 2006).

H

H C

2

N C

H

CH 3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

C NC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro

51

Frações peptídicas de proteína de soja hidrolisada com pepsina foram separadas por

cromatografia de troca iônica, filtração em gel e CLAE e apresentaram atividade inibitória sobre

a ECA. Quatro sequências de aminoácidos foram identificadas como potenciais inibidoras da

ECA: Ile-Ala (IC50 153 µM), Tyr-Leu-Ala-Gly-Asn-Gln (IC50 14 µM), Phe-Phe-Leu (IC50 37

µM) e Ile-Tir-Leu-Leu (IC50 42 µM). Quando administrados em uma dose de 2,0 g de peso

corporal kg-1

em ratos hipertensos durante 15 semanas, as frações de peptídeos reduziram

consideravelmente a pressão arterial (Chen et al., 2003).

Peptídeos com atividade anti-hipertensiva foram isolados de hidrolisados proteicos de

leite após fermentação com bactérias lácticas e hidrólise enzimática com a protease comercial

Prozyme® 6. Os peptídeos foram identificados como Gly-Thr-Trp e Gly-Val-Trp, e apresentaram

atividade inibitória da ECA com valores de IC50 de 464,4 e 240,0 µM, respectivamente (Chen et

al., 2007). Hernández-Ledesma et al. (2007) hidrolisaram proteínas do leite humano com pepsina

e pancreatina para estudo das propriedades anti-hipertensivas de peptídeos e verificaram que os

hidrolisados derivados da β-caseína mostraram potente ação inibidora da ECA, com IC50 de 21

µM.

Chaves-López et al., (2014) estudaram o efeito da combinação de culturas microbianas

previamente selecionadas como proteolíticas e a capacidade de liberação de peptídeos com

atividade inibidora da ECA durante produção de leite fermentado. Foram utilizadas as linhagens

de leveduras Torulaspora delbruekii KL66A, Galactomyces geotrichum KL20B, Pichia

kudriavzevii KL84A e Kluyveromyces marxianus KL26A e linhagens de bactérias ácido lácticas

Lactobacillus plantarum LAT03, Lb. plantarum KLAT01 e Enterococcus faecalis KE06 (não

virulenta). Os resultados obtidos indicaram que a combinação de diferentes culturas pode

aumentar significativamente os teores de peptídeos com atividade anti-hipertensiva. A

combinação mais eficaz para a produção destes peptídeos foi P. kudriavzevii KL84A, Lb.

plantarum LAT3 e E. faecalis KL06, com IC50 para atividade de inibição da ECA de 30,63 µg

mL-1

.

O efeito anti-hipertensivo de um peptídeo de caseína bovina previamente identificado

como Met-Lys-Pro foi investigado in vitro e in vivo. Os ensaios in vitro basearam-se na

capacidade de inibição da ECA e os estudos in vivo foram conduzidos utilizando grupos de ratos

naturalmente hipertensos. Os animais foram tratados com soluções de peptídeos (10 mg kg-1

)

52

duas vezes ao dia e enalapril (10 mg kg-1

) em única dose diária durante 28 dias consecutivos. Os

ensaios in vitro mostraram que o peptídeo apresentou atividade inibidora da ECA com IC50 de

0,43 µM. Já para os ensaios in vivo, a pressão arterial dos animais apresentou valores de 171,7,

163,3 e 139,7, respectivamente, para os grupos controle, tratado com solução de peptídeos e

tratado com enalapril, indicando diferenças e reduções significativas na pressão arterial entre os

grupos controle e os tratados com peptídeos (p < 0,05) e enalapril (p < 0,01) (Yamada et al.,

2015).

4.5. Indução do crescimento de bactérias ácido lácticas e probióticas

Bactérias ácido lácticas não possuem capacidade para sintetizar todos os aminoácidos

necessários para o seu crescimento. Assim, estes micro-organismos devem hidrolisar proteínas

durante o processo de fermentação de produtos lácteos para obtenção de aminoácidos livres e

pequenos peptídeos como fontes nutricionais essenciais para seu crescimento. O sistema

proteolítico de bactérias ácido lácticas compreende três mecanismos básicos e é composto por: 1)

uma ou mais enzimas proteolíticas presentes na parede celular, também conhecida como

proteases do envelope celular, as quais têm a capacidade de hidrolisar proteínas do leite a

peptídeos contendo de 4 a 30 resíduos de aminoácidos; 2) sistema de transporte de peptídeos

composto por proteínas de ligação, duas permeases responsáveis pela formação dos canais de

transporte e duas ATPases que fornecem energia ao sistema e 3) um conjunto de peptidases

intracelulares que promoverão a hidrólise dos peptídeos inicialmente transportados para o interior

das células a aminoácidos (Hafeez et al., 2014).

Apesar de todo o aparato proteolítico, vários estudos têm demonstrado que a

suplementação do leite com fontes de proteínas previamente hidrolisadas apresentaram impacto

positivo sobre o crescimento de bactérias lácticas e probióticas. Estes estudos são impulsionados

principalmente em virtude de duas características principais deste grupo de micro-organismos: 1)

bactérias lácticas e probióticas são bastante exigentes nutricionalmente, especialmente com

relação ao aporte de aminoácidos e 2) o conjunto de aminoácidos e peptídeos livres no leite não é

suficiente para garantir o crescimento bacteriano ideal neste substrato, podendo assim dificultar

processos de fermentação nos quais estes micro-organismos estejam envolvidos (Zhang et al,

2011). Assim, diferentes fontes de proteína têm sido avaliadas na suplementação de meios de

53

cultura para estudo da indução do crescimento de espécies de bactérias ácido lácticas e

probióticas (McComas Jr. e Gilliland, 2003; Zhang et al , 2011; Prasanna et al., 2012).

McComas Jr. e Gilliland (2003) investigaram o crescimento de bactérias lácticas e

probióticas em amostras de leite bovino suplementadas com hidrolisados de proteína de soro de

leite. Os resultados obtidos mostraram que os hidrolisados não apresentaram efeitos sobre o

crescimento de L. delbrueckii ssp. bulgaricus e S. thermophilus; no entanto, aumentos

significativos sobre o crescimento de Bifidobacterium longum e L. acidophilus foram observados.

Prasanna et al., (2012) estudaram a suplementação de leite bovino desnatado com

diferentes fontes de proteínas hidrolisadas e os efeitos sobre o crescimento de bactérias

probióticas e observaram que o tipo ou fração da proteína utilizada influenciava diretamente no

crescimento dos micro-organismos. A concentração final de células de B. longum subsp. infantis

CCUG 52486 e B. infantis NCIMB 702.205 foi superior quando estas linhagens foram cultivadas

em amostras de leite suplementadas com hidrolisados de caseína, em comparação com outras

frações proteicas hidrolisadas a partir de lactoalbumina, concentrado proteico de soro de leite ou

isolado proteico de soro de leite.

5. Conclusão

Peptídeos com atividade biológica podem ser definidos como sequências específicas de

aminoácidos que promovem efeitos fisiológicos benéficos. As tecnologias para obtenção de

peptídeos bioativos envolvem a hidrólise de proteínas por enzimas exógenas de origem

microbiana, vegetal ou animal e processos fermentativos utilizando-se fungos ou bactérias. A

ampla diversidade bioquímica das proteases, assim como a existência de fontes proteicas com

composições variadas de aminoácidos, torna possível a obtenção de peptídeos com funções

biológicas distintas e/ou até mesmo com multifuncionalidade, assim como as condições de

processo. Entre as técnicas de purificação e identificação, os métodos cromatográficos e a

espectrometria de massas surgem como importantes ferramentas. O estudo sobre os processos de

obtenção assim como o entendimento da sua multifuncionalidade e avaliação utilizando métodos

in vitro e in vivo tornam-se aliados na aplicação de peptídeos bioativos como potentes agentes

biológicos naturais que podem ser utilizados em conjunto ou até mesmo em substituição a

substâncias sintéticas em processos de conservação de produtos alimentícios, na administração de

alimentos funcionais e na produção de fármacos.

54

Referências

ADJE, E. Y., BALTI, R., KOUACH, M., GUILLOCHON, D., NEDJAR-ARROUME, N. α 67-

106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme

inhibitory peptides. European Food Research and Technology, v. 232, p. 637–646, 2011.

AGYEI, D., DANQUAH, M. K. Rethinking food-derived bioactive peptides for antimicrobial

and immunomodulatory activities. Trends in Food Science & Technology, v. 23, p. 62-69,

2012.

AHN, C-B., CHO, Y-S, JE, J-Y. Purification and anti-inflammatory action of tripeptide from

salmon pectoral fin byproduct protein hydrolysate. Food Chemistry, v. 168. p. 151–156, 2015.

AHN, C-B., JE, J-Y. , CHO, Y-S. Antioxidant and anti-inflammatory peptide fraction from

salmon byproduct protein hydrolysates by peptic hydrolysis. Food Research International, v.

49, p. 92-98, 2012.

AKALIN, A. S. Dairy-derived antimicrobial peptides: Action mechanisms, pharmaceutical uses

and production proposals. Trends in Food Science & Technology, v. 36, p. 79-95, 2014.

ALAM, M. N., BRISTI, N. J., RAFIQUZZAMAN, M. Review on in vivo and in vitro methods

evaluation of antioxidant activity. Saudi Pharmaceutical Journal, v. 21, p. 143–152, 2013.

ALEMÁN, A., PÉREZ-SANTÍN, E., BORDENAVE-JUCHEREAU, S., ARNAUDIN, I.,

GÓMEZ-GUILLÉN, M.C., MONTERO, P. Squid gelatin hydrolysates with antihypertensive,

anticancer and antioxidant activity. Food Research International, v. 44, p. 1044–1051, 2011.

AOYAMA, T., FUKUI, K., TAKAMATSU, K., HASHIMOTO, Y., YAMAMOTO, T. Soy

protein isolate and its hydrolysate reduce body fat of dietary obese rats and genetically obese

mice (yellow KK). Nutrition, v. 16, p. 349-354, 2000.

BEVINS, C. L., M. ZASLOFF. Peptides from frog skin. Annual Review of Biochemistry, v. 59,

p. 395-414, 1990.



‘cause and effect’ theory of bifunctionality. FEMS Immunology & Medical Microbiology, v.

46, p. 131-138, 2006.

55

BOMAN, H. G. Antibacterial peptides: basic facts and emerging concepts. Journal of Internal

Medicine, v. 254, p. 197-215, 2003.

BOMAN, H. G., HULTMARK, D. Cell-free immunity in insect. Annual Review of

Microbiology, v. 41, p. 103-126, 1987.

CARRASCO-CASTILLA, J., HERNÁNDEZ-ÁLVAREZ, A. J., JIMÉNEZ-MARTÍNEZ, C.,

JACINTO-HERNÁNDEZ, C., ALAIZ, M., GIRÓN-CALLE, J., VIOQUE, J., DÁVILA-ORTIZ,

GLORIA. Antioxidant and metal chelating activities of Phaseolus vulgaris L. var. Jamapa protein

isolates, phaseolin and lectin hydrolysates. Food Chemistry, v. 131, 1157–1164, 2012.

CHANG, O. K., SEOL, K. H., JEONG, S. G., OH, M. H., PARK, B. Y., PERRIN, C., HAM, J.

S. Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of

peptides derived therefrom. Journal of Dairy Science, v. 96, n. 9, p. 5544–5555, 2013.

CHAVES-LÓPEZ, C., SERIO, A., PAPARELLA, A., MARTUSCELLI, M., CORSETTI, A.,

TOFALO, R., SUZZI, G. Impact of microbial cultures on proteolysis and release of bioactive

peptides in fermented milk. Food Microbiology, v. 42, p. 117-121, 2014.

CHEN, G., TSAI, J., PAN, B. Purification of angiotensin I-converting enzyme inhibitory

peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation.

International Dairy Journal, v. 17, p. 641–647, 2007.

CHEN, H. M., MURAMOTO, K., YAMAUCHI, F., FUJIMOTO, K., NOKIHARA, K.

Antioxidative properties of histidine-containing peptides designed from peptide fragments found

in the digests of a soybean protein. Journal of Agricultural and Food Chemistry, v. 46, p. 49-

53, 1998.

CHEN, J., OKADA, T., MURAMOTO, K., SUETSUNA, K., YANG, S.. Identification of

angiotensin I-converting enzyme inhibitory peptides derived from the peptic digest of soybean

protein. Journal of Food Biochemistry, v. 26, p. 543–554, 2003.

CHI, C-F., WANG, B., WANG, Y-M., ZHANG, B., DENG, S-G. Isolation and characterization

of three antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon

septentrionalis) heads. Journal of Functional Foods, v. 12, p. 1-10, 2015.

56

CHIARADIA, M. C., COLLINS, C. H., JARDIM, I. C. S. F. O estado da arte da cromatografia

associada à espectrometria de massas acoplada à espectrometria de massas na análise de

compostos tóxicos em alimentos. Química Nova, v. 31, n. 3, p. 623-636, 2008.

CHOI, J., SABIKHI, L., HASSAN, A., ANAND, S. Bioactive peptides in dairy products.

International Journal of Dairy Technology, v. 65, n. 1, 2012.

CONTRERAS, M. M; LÓPEZ-EXPÓSITO, I.; HERNÁNDEZ-LEDESMA, B.; RAMOS, M.;

RECIO, I. Application of mass spectrometry to the characterization and quantification of food-

derived bioactive peptides. Journal of AOAC International, v. 91, n. 4, p. 981-994, 2008.

DASHPER, S. G., LIU, S. W., REYNOLDS, E. C. Antimicrobial peptides and their potential as

oral therapeutic agents. International Journal of Peptide Research and Therapeutics, v. 13, n.

4, p. 505–516, 2007.

DÁVALOS, A., GÓMEZ-CORDOVÉS, C., BARTOLOMÉ, B. Extending applicability of the

oxygen radical absorbance capacity (ORAC-fluorescein) assay. Journal of Agricultural and

Food Chemistry, v. 52, p. 48-54, 2004.

DIPLOCK, A. T., AGGETT, P. J., ASHWELL, M., BORNET, F., FERN, E.B., ROBERFROID,

M. B. Scientific concepts of functional foods in Europe: consensus document. British Journal of

Nutrition, v. 81, p. 51, 1999.

GOBBETTI, M., MINERVINI, F., RIZZELLO, C.G. Angiotensin I-converting enzyme-

inhibitory and antimicrobial bioactive peptides. International Journal Dairy Technology, v. 57,

p. 173-188, 2004.

GÓMEZ-GUILLÉN, M. C., LÓPEZ-CABALLERO, M. E., LÓPEZ DE LACEY, A., ALEMÁN,

A., GIMÉNEZ, B., MONTERO, P. Antioxidant and antimicrobial peptide fractions from squid

and tuna skin gelatin. In Bihan, E. L., Koueta, N. Sea by-products as a real material: new ways

of application. Kerala: Transworld Research Network Signpost, 2010. p. 89-115.

GU, M., CHEN, H-P., ZHAO, M-M., WANG, X., YANG, B., REN, J-Y., SU, G-W.

Identification of antioxidant peptides released from defatted walnut (Juglans Sigillata Dode)

meal proteins with pancreatin. LWT - Food Science and Technology, v. 60 p. 213-220, 2015.

57

HAFEEZ, Z., CAKIR-KIEFER, C., ROUX, E., PERRIN, C., MICLO, L., DARY-MOUROT, A.

Strategies of producing bioactive peptides from milk proteins to functionalize fermented milk

products. Food Research International, v. 63, p. 71–80, 2014.

HARDING, J. W., PYERITZ, E. A., COPELAND, E. S., WHITE, H. B. Role of glycerol 3-

phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken

liver. Biochemistry Journal, v. 146, p. 223–229, 1975.

HARRISON, P. L., ABDEL-RAHMAN, M. A., MILLER, K., STRONG, P. N. Antimicrobial

peptides from scorpion venoms. Toxicon, v. 88, p. 115-137, 2014.

HATI, S., VIJ, S., MANDAL, S., MALIK, R.K., KUMARI, V., KHETRA, Y. α-galactosidase

activity and oligosaccharides utilization by lactobacilli during fermentation of soy milk. Journal

of Food Processing and Preservation, v. 38, p. 1065–1071, 2014.

HE, R., JU, X., YUAN, J., WANG, L., GIRGIH, A. T., ALUKO, R. E. Antioxidant activities of

rapeseed peptides produced by solid state fermentation. Food Research International, v. 49, p.

432–438, 2012.

HERNÁNDEZ-LEDESMA, B., CONTRERAS, M. M., RECIO, I. Antihypertensive peptides:

Production, bioavailability and incorporation into foods. Advances in Colloid and Interface

Science, v. 165, p. 23–35, 2011.

HERNÁNDEZ-LEDESMA, B., GARCÍA-NEBOT, M. J., FERNÁNDEZ-TOMÉ, S., AMIGO,

L., RECIO, I. Dairy protein hydrolysates: peptides for health benefits. International Dairy

Journal, v. 38, p. 82-100, 2014.

HERNÁNDEZ-LEDESMA, B., QUIRÓS, A., AMIGO, L., RECIO, I. Identification of bioactive

peptides after digestion of human milk and infant formula with pepsin and pancreatin.

International Dairy Journal, v. 17, p. 42–49, 2007.

HIRAI, S., YAMANAKA, M., KAWACHI, H., MATSUI, T., YANO, H. Activin A inhibits

differentiation of 3T3-L1 preadipocyte. Molecular and Cellular Endocrinology, v. 232, p. 21–

26, 2005.

HONG, F., MING, L., YI, S., ZHANXIA, L., YONGQUAN, W., CHI, L. The antihypertensive

effect of peptides: A novel alternative to drugs? Peptides, v. 29, p. 1062-1071, 2008.

58

HUANG, S., CHEN, K-N., CHEN, Y-P., HONG, W-S., CHEN, M-J. Immunomodulatory

properties of the milk whey products obtained by enzymatic and microbial hydrolysis.

International Journal of Food Science and Technology, v. 45, p. 1061–1067, 2010.

INOUE, K., GOTOU, T., KITAJIMA, HIROKUNI, MIZUNO, S., NAKAZAWA, T.,

YAMAMOTO, N. Release of antihypertensive peptides in miso paste during its fermentation, by

the addition of casein. Journal of Bioscience and Bioengineering, v. 108, n. 2, p. 111–115,

2009.

KIM, E-K., OH, H-J., KIM, Y-S., HWANG, J-W., AHN, C.-B., LEE, J. S., JEON, Y-J., MOON,

S-H., SUNG, S. H., JEON, B-T., PARK, P-J. Purification of a novel peptide derived from

Mytilus coruscus and in vitro/in vivo evaluation of its bioactive properties. Fish & Shellfish

Immunology, v. 34, p. 1078-1084, 2013.

KIM, H. J., BAE, I. Y., AHN, C., LEE, S., LEE, H. G. Purification and identification of

adipogenesis inhibitory peptide from black soybean protein hydrolysate. Peptides, v. 28, p. 2098-

2103, 2007.

KORHONEN, H. Milk-derived bioactive peptides: From science to applications. Journal of

Functional Foods I, p. 177-187, 2009.

KORHONEN, H., PIHLANTO-LEPPALA, A., RANTAMAKI, P., TUPASELA, T. Impact of

processing on bioactive proteins and peptides. Trends in Food Science & Technology, v. 9, p.

307-319, 1998.

KTARI, N., NASRI, R., MNAFGUI, K., HAMDEN, K., BELGUITH, O., BOUDAOUARA, T.,

FEKI, A. E., NASRI, M. Antioxidative and ACE inhibitory activities of protein hydrolysates

from zebra blenny (Salaria basilisca) in alloxan-induced diabetic rats. Process Biochemistry, v.

49, p. 890-897, 2014.

LAFARGA, T., HAYES, M. Bioactive peptides from meat muscle and by-products: generation,

functionality and application as functional ingredients. Meat Science, v. 98 p. 227–239, 2014.

LI, X., LUO, Y., SHEN, H., YOU, J. Antioxidant activities and functional properties of grass

carp (Ctenopharyngodon idellus) protein hydrolysates. Journal of the Science of Food and

Agriculture, v. 92, p. 292–298, 2012.

59

LI-CHAN, E. C. Bioactive peptides and protein hydrolysates: research trends and challenges for

application as nutraceuticals and functional food ingredients. Current Opinion in Food Science,

v. 1, p. 28-37, 2015.

LIMÓN, R. I., PEÑAS, ELENA, TORINO, M. I., MARTÍNEZ-VILLALUENGA, C., DUEÑAS,

M., FRIAS, J. Fermentation enhances the content of bioactive compounds in kidney bean

extracts. Food Chemistry, v. 172, p. 343-352, 2015.

LIU, Q., KONG, B., LI, G., LIU, N., XIA, X. Hepatoprotective and antioxidant effects of

porcine plasma protein hydrolysates on carbon tetrachloride-induced liver damage in rats. Food

and Chemical Toxicology, v. 49, p. 1316–1321, 2011.

LIU, X., ZHENG, X., SONG, Z., LIU, X., KOPPARAPU, N. K., WANG, X., ZHENG, Y.

Preparation of enzymatic pretreated corn gluten meal hydrolysate and in vivo evaluation of its

antioxidant activity. Journal of Functional Foods, 2014, doi: 10.1016/j.jff.2014.10.013.

MAIER, T., LEIBUNDGUT, M., BAN, N. The crystal structure of mammalian fatty acid

synthase. Science, v. 321, p. 1315-1322, 2008.

MARTINEZ-VILLALUENGA, C., BRINGE, N. A., BERHOW, M. A., MEJIA, E. G. β-

conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro.

Journal of Agricultural and Food Chemistry, v. 56, p. 10533–10543, 2008.

MATSUI, T., MATSUMOTO, K. Antihypertensive peptides from natural resources. In: Khan,

M. T. H., Ather, A. Lead Molecules from Natural Products, Elsevier, p. 255-271, 2006.

MATSUZAKI, K. Why and how are peptide-lipid interactions utilized for self-defense?

Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta, v. 1462, p. 1-10,

1999.

McCANN, K. B., SHIELL, B. J., MICHALSKI, W. P., LEE, A., WAN, J., ROGINSKI, H.,

COVENTRY, M. J. Isolation and characterisation of antibacterial peptides derived from the f

(164–207) region of bovine αS2-casein. International Dairy Journal, v. 15, p. 133–143, 2005.

McCOMAS JR., K. A., GILLILAND, S. E. Growth of probiotic and traditional yogurt cultures in

milk supplemented with whey protein hydrolysate. Journal of Food Science, v. 68, p. 2090-

2095, 2003.

60

MEJIA, E. G., MARTINEZ-VILLALUENGA, C., ROMAN, M., BRINGE, N. A. Fatty acid

synthase and in vitro adipogenic response of human adipocytes inhibited by α and α’ subunits of

soybean β-conglycinin hydrolysates. Food Chemistry, v. 119, p. 1571–1577, 2010.

MELLANDER, O. The physiological importance of the casein phosphopeptides calcium salts. II.

Per oral calcium dosage of infants. Acta Society Medicine Uppsala, v. 55, p. 247-255, 1950.




MORA, L., REIG, M., TOLDRÁ, F. Bioactive peptides generated from meat industry by

products. Food Research International, 2014, doi.org/10.1016/j.foodres.2014.09.014.

NAJAFIAN, L., BABJI, A. S. Isolation, purification and identification of three novel

antioxidative peptides from patin (Pangasius sutchi) myofibrillar protein hydrolysates. LWT -

Food Science and Technology, v. 60, p. 452-461, 2015.

NAKAHARA, T., SANO, A., YAMAGUCHI, H., SUGIMOTO, K., CHIKATA, H.,

KINOSHITA, E., UCHIDA, R. Antihypertensive effect of peptide-enriched soy sauce-like

seasoning and identification of its angiotensin I-converting enzyme inhibitory substances.

Journal of Agricultural and Food Chemistry, v. 58, p. 821–827, 2010.

NASRI, R., AMOR, I. B., BOUGATEF, A., NEDJAR-ARROUME, N., DHULSTER, P.,

GARGOURI, J., CHÂABOUNI, M. K., NASRI, M. Anticoagulant activities of goby muscle

protein hydrolysates. Food Chemistry, v. 133, p. 835–841, 2012.

NAZEER, R. A., KULANDAI, K. A. Evaluation of antioxidant activity of muscle and skin

protein hydrolysates from giant kingfish, Caranx ignobilis (Forsskal, 1775). International

Journal of Food Science and Technology, v. 47, p. 274–281, 2012.

NAZEER, R. A., KUMAR, N.S. S., GANESH R. J. In vitro and in vivo studies on the antioxidant

activity of fish peptide isolated from the croaker (Otolithes ruber) muscle protein hydrolysate.

Peptides, v. 35, p. 261–268, 2012.

NGO, D-H., KANG, K-H., RYU, B., VO, T-S., JUNG, W-K., BYUN, H-G., KIM, S-K.

Angiotensin-I converting enzyme inhibitory peptides from antihypertensive skate (Okamejei

61

kenojei) skin gelatin hydrolysate in spontaneously hypertensive rats. Food Chemistry, v. 174, p.

37–43, 2015.

NIKI, E. Assessment of antioxidant capacity in vitro and in vivo. Free Radical Biology &

Medicine, v. 49, 503–515, 2010.

ORTIZ-MARTINEZ, M., WINKLER, R., GARCÍA-LARA, S. Preventive and therapeutic

potential of peptides from cereals against cancer. Journal of Proteomics, v. 111, p. 165–183,

2014.

OSEGUERA-TOLEDO, M. E., MEJIA, E. G., DIA, V. P., AMAYA-LLANO, S. L. Common

bean (Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macrophages

through suppression of NF-κB pathways. Food Chemistry, v. 127, p. 1175–1185, 2011.

OU, B., HUANG, D., HAMPSCH-WOODILL, M., FLANAGAN, J. A., DEEMER, E. K.

Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance

capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study.

Journal of Agricultural and Food Chemistry, v. 50, p. 3122-3128, 2002.

PATRZYKAT, A., DOUGLAS, S.E. Antimicrobial peptides: cooperative approaches to

protection. Protein and Peptide Letters, v. 12, p. 19-25, 2005.

PICOT, L. RAVALLEC, R., FOUCHEREAU-PÉRON, M., VANDANJON, L., JAOUEN, P.,

CHAPLAIN-DEROUINIOT, M., GUÉRARD, F., CHABEAUD, A., LEGAL, Y., ALVAREZ,

O. M., BERGÉ, J. P., PIOT, J. M., BATISTA, I., PIRES, C., THORKELSSON, G.,

DELANNOY, C., JAKOBSEN, G., JOHANSSON, I., BOURSEAU, P. Impact of ultrafiltration

and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties. Journal of

the Science of Food and Agriculture, v. 90, 1819–1826, 2010.

PIHLANTO, A. Antioxidative peptides derived from milk proteins. International Dairy

Journal, v. 16, p. 1306–1314, 2006.

POWNALL, T. L., UDENIGWE, C. C., ALUKO, R. E. Amino acid composition and antioxidant

properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. Journal of

Agricultural and Food Chemistry, v. 58, p. 4712–4718, 2010.

62

PRASANNA, P. H. P., GRANDISON, A. S., CHARALAMPOPOULOS, D. Effect of dairy-

based protein sources and temperature on growth, acidification and exopolysaccharide production

of Bifidobacterium strains in skim milk. Food Research International, v. 47, p. 6–12, 2012.

QUIRÓS, A., CHICHÓN, R., RECIO, I., LÓPEZ-FANDIÑO, R. The use of high hydrostatic

pressure to promote the proteolysis and release of bioactive peptides from ovalbumin. Food

Chemistry, v. 104, p. 1734-1739, 2007.

RAGHAVAN, S., KRISTINSSON, H. G. Antioxidative efficacy of alkali-treated tilapia protein

hydrolysates: a comparative study of five enzymes. Journal of Agricultural and Food

Chemistry, v. 56, p. 1434–1441, 2008.

RAHMAN, M. A., KUMAR, S. G., KIM, S. W., HWANG, H. J., BAEK, Y. M., LEE, S. H., et

al. Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte

differentiation. Proteomics, v. 8, p. 569-581, 2008.

RAJAPAKSE, N., MENDIS, E., JUNG, W., JE, J., KIM, S. Purification of a radical scavenging

peptide from fermented mussel sauce and its antioxidant properties. Food Research

International, v. 38, p. 175–182, 2005.

REGAZZO, D., DA DALT, L., LOMBARDI, A., ANDRIGHETTO, C., NEGRO, A., GABAI,

G. Fermented milks from Enterococcus faecalis TH563 and Lactobacillus delbrueckii subsp.

bulgaricus LA2 manifest different degrees of ACE-inhibitory and immunomodulatory activities.

Dairy Science & Technology, v. 90, p. 469-476, 2010.

ROBLET, C., AMIOT, J., LAVIGNE, C., MARETTE, A., LESSARD, M., JEAN, J.,

RAMASSAMY, C., MORESOLI, C., BAZINET, L. Screening of in vitro bioactivities of a soy

protein hydrolysate separated by hollow fiber and spiral-wound ultrafiltration membranes. Food

Research International, v. 46, p. 237–249, 2012.

SAKANAKA, S.; TACHIBANA, Y.; ISHIHARA, N.; JUNEJA, L.R. Antioxidant activity of

egg-yolk protein hydrolysates in linoleic acid oxidation system. Food Chemistry, v. 86, n. 1, p.

99-103, 2004.

SAMARANAYAKA, A. G. P., LI-CHAN, E. C. Y. Food-derived peptidic antioxidants: a review

of their production, assessment, and potential applications. Journal of Functional Foods, v. 3, p.

229-254, 2011.

63

SANTOS, L. F., KOBLITZ, M. G. B. Proteases. In: KOBLITZ, M. G. B. Bioquímica de

Alimentos. Rio de Janeiro: Guanabara Koogan, 2008. p. 78-103.

SARMADI, B. H., ISMAIL, A. Antioxidative peptides from food proteins: A review. Peptides,

v. 31, p. 1949–1956, 2010.

SCHANBACHER, F.L., TALHOUK, R.S., MURRAY, F.A. Biology and origin of bioactive

peptides in milk. Livestock Production Science, v. 50, p. 105-123, 1997.

SENPHAN, T., BENJAKUL, S. Antioxidative activities of hydrolysates from seabass skin

prepared using protease from hepatopancreas of Pacific white shrimp. Journal of Functional

Foods, v. 6, p. 147-156, 2014.

SHARMA, O. P., BHAT, T. K. DPPH antioxidant assay revisited. Food Chemistry, v. 113, p.

1202–1205, 2009.

SHEIH, I. C., FANG, T. J., WU, T. K. Isolation and characterisation of a novel angiotensin I-

converting enzyme (ACE) inhibitory peptide from the algae protein waste. Food Chemistry, v.

115, p. 279-284, 2009.

SINGH, B. P., VIJ, S., HATI, S. Functional significance of bioactive peptides derived from

soybean. Peptides, v. 54, p. 171–179, 2014.

SOLIMAN, A. M., ABU-EL-ZAHAB, H. S., ALSWIAI, G. A. Efficacy evaluation of the protein

isolated from Peganum harmala seeds as an antioxidant in liver of rats. Asian Pacific Journal of

Tropical Medicine, p. 285-295, 2012.

STUKNYTE, M., NONI, I. D., GUGLIELMETTI, S., MINUZZO, M., MORA, D. Potential

immunomodulatory activity of bovine casein hydrolysates produced after digestion with

proteinases of lactic acid bacteria. International Dairy Journal, v. 21, p. 763-769, 2011.

TANG, W., ZHANG, H., WANG, L., QIAN, H., QI, X. Targeted separation of antibacterial

peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis. Food

Chemistry, v. 168, p. 115–123, 2015.

TAVARES, T. G., Contreras, M. M., Amorim, M., Martín-Álvarez, P. J., Pintado, M. E., Recio,

I., Malcata, F. X. Optimization, by response surface methodology, of degree of hydrolysis and

64

antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon

extract. International Dairy Journal, v. 21, p. 926-933, 2011.

TELLEZ, A., CORREDIG, M., TURNER, P. V., MORALES, R., GRIFFITHS, M. A peptidic

fraction from milk fermented with Lactobacillus helveticus protects mice against Salmonella

infection. International Dairy Journal, v. 21, 607-614, 2011.

THEODORE, A.E., RAGHAVAN, S., KRISTINSSON H. G. Antioxidative activity of protein

hydrolysates prepared from alkaline-aided channel catfish protein isolates. Journal of

Agricultural and Food Chemistry, v. 56, p. 7459–7466, 2008.

THÉOLIER, J., HAMMAMI, R., LABELLE, P., FLISS, I., JEAN, J. Isolation and identification

of antimicrobial peptides derived by peptic cleavage of whey protein isolate. Journal of

Functional Foods, v. 5, p. 706-714, 2013.

TSAI, J-S., CHEN, T-J., PAN, B. S., GONG, S-D., CHUNG, M-Y. Antihypertensive effect of

bioactive peptides produced by protease-facilitated lactic acid fermentation of milk. Food

Chemistry, v. 106, p. 552–558, 2008.



Chemistry, v. 122, p. 243–248, 2010a.

TSOU, M., LIN, W., LU, H., TSUI, Y., CHIANG, W. The effect of limited hydrolysis with

Neutrase and ultrafiltration on the anti-adipogenic activity of soy protein. Process Biochemistry,

v. 45, p. 217–222, 2010b.

TSOU, M-J., KAO, F-J., LU, H-C., KAO, H-C., CHIANG, W-D. Purification and identification

of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein. Food

Chemistry, v. 138, p. 1454–1460, 2013.

UHLIG, T., KYPRIANOU, T., MARTINELLI, F. G., OPPICI, C. A., HEILIGERS, D., HILLS,

D., CALVO, X. R., VERHAERT, P. The emergence of peptides in the pharmaceutical business:

from exploration to exploitation. EuPA Open Proteomics, v. 4, p. 58-69, 2014.

VALLABHA, V. S., TIKU, P. K. Antihypertensive peptides derived from soy protein by

fermentation. International Journal of Peptide Research and Therapeutics, v. 20, p. 161–168,

2014.

65

WANG, W. Y., DE MEJIA, E. G. A new frontier in soy bioactive peptides that may prevent age-

related chronic diseases. Comprehensive Reviews in Food Science and Food Safety, v. 4, p.

63-78, 2005.

WANG, B., GONG, Y. D., LI, Z. R., YU, D., CHI, C. F., MA, J. Y. Isolation and characterisation

of five novel antioxidant peptides from ethanol-soluble proteins hydrolysate of spotless

smoothhound (Mustelus griseus) muscle. Journal of Functional Foods, v. 6, p. 176–185, 2014.

YAMADA, A., SAKURAI, T., OCHI, D., MITSUYAMA, E., YAMAUCHI, K., ABE, F.

Antihypertensive effect of the bovine casein-derived peptide Met-Lys-Pro. Food Chemistry, v.

172, p. 441–446, 2015.

YASUFUMI, K., SHIGEKI, M., KEIJI, I., TOMOYUKI, O. Antioxidative peptides from milk

fermented with Lactobacillus delbrueckii subsp. bulgaricus IFO 13953. Journal of the Japanese

Society for Food Science and Technology, v. 48, p. 44-50, 2001.

ZAREI, MOHAMMAD, EBRAHIMPOUR, A., ABDUL-HAMID, A., ANWAR, F., BAKAR, F.

A., PHILIP, R., SAARI, N. Identification and characterization of papain-generated antioxidant

peptides from palm kernel cake proteins. Food Research International, v. 62, p. 726–734, 2014.

ZASLOFF, M. Antimicrobial peptides of multicellular organisms. Nature, v. 145, p. 389-395,

2002.

ZHANG, J., ZHANG, H., WANG, L., GUO, X., WANG, X., YAO, H. Antioxidant activities of

the rice endosperm protein hydrolysate: identification of the active peptide. European Food

Research and Technology, v. 229, p. 709-719, 2009.




ZHANG, Q., REN, J., ZHAO, H., ZHAO, M., XU, J. & ZHAO, Q. Influence of casein

hydrolysates on the growth and lactic acid production of Lactobacillus delbrueckii subsp.

bulgaricus and Streptococcus thermophilus. International Journal of Food Science and

Technology, v. 46, p. 1014–1020, 2011.

66

ZHUANG, Y., SUN, L. Preparation of reactive oxygen scavenging peptides from tilapia

(Oreochromis niloticus) skin gelatin: optimization using response surface methodology. Journal

of Food Science, v. 76, n. 3, p. 483-489, 2011.

67

Capítulo III: Improving the functional properties of milk proteins: focus on

the specificities and mechanisms of action of proteolytic enzymes

Revista: Current Opinion in Food Science

68

Abstract

The modification of milk proteins by enzymatic hydrolysis has great potential for optimizing

functional properties. The most common method of improving the functional properties of milk

proteins is to evaluate the hydrolysis parameters. However, a limited number of studies correlated

their results with the biochemical properties of the proteolytic enzymes to understand the

mechanism of action by which the functional properties of the milk proteins are affected by the

specificity of the proteases. In this review, the primary focus is the use of enzymatic hydrolysis of

milk proteins as a tool for enhancing their functional properties, emphasizing protein solubility,

gelation, and emulsion capacities. In particular, there is a discussion of the specificities and

mechanisms of action of the proteolytic enzymes.

Keywords: milk proteins, proteases, specificities, functional properties.

69

1. Introduction

Milk proteins are key functional components and provide desirable characteristics to food

products and food systems to which they are added [1]. The functionality of food proteins refers

to their physicochemical properties and is generally classified into two main groups,

hydrodynamic or hydration-related, including water absorption, solubility, viscosity, and

gelation, and surface-active properties, such as emulsification, foaming, and film formation [2,

3].

During the last decade, industrial-scale technologies suitable for the industrial production

of milk-derived proteins have been developed, resulting in many different grades and types of

protein enriched products [3, 4]. Milk proteins have enhanced functional properties by altering

the protein and non-protein composition, and/or modifying the proteins by enzymatic treatments

[5, 6].

Enzymatic hydrolysis disrupts the protein tertiary structure and reduces the molecular

weight of the protein, enhancing the interaction of peptides with themselves and with the

environment, and consequently altering their functional properties [7]. Notably, the nature of the

protein modification is influenced by several hydrolysis parameters, including the reaction

conditions, such as pH, temperature, degree of hydrolysis, and enzyme specificities, and intrinsic

characteristics of each food, such as ionic strength, concentration of calcium and other polyvalent

ions, sugars, and hydrocolloids [3, 8, 9].

The modification of milk proteins based on enzymatic hydrolysis has broad potential and

are likely an innovative tool in food protein processing for optimizing the techno-functional and

tropho-functional properties of proteins in food products [10, 11, 12]. Several studies showed that

the enzymatic hydrolysis of milk proteins resulted in increased protein solubility, heat stability,

emulsion capacity, foaming properties and surface hydrophobicity, which make hydrolysates

suitable for ingredients in other foods, including dairy products (Table 1).

70

Table 1 – Enzymatic hydrolysis of milk proteins using proteases with different mechanisms of action and parameters of hydrolysis and

the effects on the functional properties of the hydrolysates.

Protein

source Enzymes

Mechanism

of action Parameters of hydrolysis

Degree of

hydrolysis Changes in functional properties Reference

Milk protein

concentrates

Chymotrypsin

Trypsin

Pepsin

Papain

Endoproteases

pH 8.0 / 50 °C / 5-10 min

pH 8.0 / 37 °C / 10-60 min

pH 2.0 / 37 °C / 240-720 min

pH 6.8 / 60 °C / 30-180 min

24.3-24.4%

14.8-15.1%

5.0-5.7%

7.2-9.8%

Solubility was improved in the pH range

of 4.6 to 7.0

Surface hydrophobicity and gel strength

were reduced

Emulsification capacity was increased

[13]

Whey

protein

isolate

AlcalaseTM

2.4L Endoprotease pH 8.0 / 45 °C / 5-120 min 10.0-20.3%

The gelling properties showed an increase

with increase in the degree of hydrolysis [14]

Whey

protein

concentrate

AlcalaseTM

2.4L

Prolyve TM

1000

Endoproteases pH 7.0 / 50 °C / 30-300 min 12.0-19.2%

10.0-15.9%

AlcalaseTM

2.4L hydrolysates formed a soft

gel at DH of 16.9%

Prolyve TM 1000 hydrolysates showed no

evidence of gel formation

[15]

Whey

protein

concentrate

FlavourzymeTM

500L

Endoprotease /

Exoprotease pH 5.0 / 50 °C / 120 min 19.4%

Increase in solubility and decrease in heat

stability and emulsion activity index [16]

Sodium

caseinate

Papain

Pancreatin

Trypsin

Endoproteases

pH 7.0 / 37 °C / 10 min – 24 h

pH 8.0 / 37 °C / 10 min – 24 h

pH 8.0 / 37 °C / 10 min – 24 h

13.32-22.06%

9.42-20.91%

14.86-20.68%

The solubility of hydrolysates varied with

the enzyme type. Papain-treated

hydrolysates exhibited higher solubility,

followed by trypsin and pancreatin

hydrolysates

For most hydrolysate samples, the

emulsifying properties were improved

[17]

Whey

protein

isolate

Chymotrypsin

Pepsin Endoproteases

pH 7.8 / 37 °C / 180 min

pH 2.6 / 37 °C / 180 min

10.6%

11.0%

Chymotrypsin hydrolysates formed

nanoemulsions

Pepsin hydrolysates did not form

nanoemulsions

[18]

71

There are a few detailed studies about the specificity and mechanisms of action of the

proteolytic enzymes and the manner in which these characteristics affect the functional properties

of the milk protein hydrolysates. Therefore, information of these mechanisms is valuable for

optimizing the enzymatic hydrolysis of milk proteins with different properties directed to specific

applications.

In this review, the primary focus is on the enzymatic hydrolysis of milk proteins as a tool

to enhance their functional properties, with emphasis on protein solubility, gelation, and emulsion

capacities. A specific discussion of the specificities and mechanisms of action of the proteolytic

enzymes and how these characteristics affect the functional properties of milk protein

hydrolysates will be reviewed.

2. Functional properties of milk proteins

2.1. Solubility

Solubility is one of the most important functional properties of protein hydrolysates and

largely determines their use in foods. This parameter was highly correlated with the reduction in

molecular weight and an increase in the number of smaller, more hydrophilic, and more solvated

polypeptide units via enzymatic hydrolysis, resulting in an increase in the exposed net charge

density [19]. A decrease in solubility after enzymatic hydrolysis may occur when the protein

molecule exposes more hydrophobic groups, therefore, to improve the solubility of milk proteins,

limited and selective hydrolysis is a critical point [7, 16]. A comparative study using three

different proteases showed that the solubility of sodium caseinate hydrolysates varied with the

enzyme type, presenting an interesting correlation with the enzymes specificities. Of the three

enzymes, the hydrolysates obtained with papain exhibited a greater capability of improving the

solubility of sodium caseinate, followed by trypsin and pancreatin [17]. Papain is a cysteine

endopeptidase that exhibits broad substrate specificity and specifically hydrolyzes arginine,

lysine and phenylalanine bonds [20, 21] (Figure 1). The open conformation of papain allows it

rapidly and extensively degrade larger bovine β-casein molecules, an abundant milk protein, to

smaller peptides originating predominantly from the C-terminus, which results in the formation

of a hydrophilic surface and in increase of protein solubility [22]. Trypsin is a serine protease

with a narrow specificity that only hydrolyzes peptide bonds involving the carboxylic group of

lysine and arginine residues [23] (Figure 1). Pancreatin is an enzymatic complex comprising

72

lipase, amylase and proteolytic enzymes, including trypsin, chymotrypsin and elastase, which has

more target cleavage sites in the proteins and may hydrolyze the protein more randomly.

Pancreatin preferentially cleaves N-terminal phosphorylated regions and the C-terminal

hydrophobic regions of casein molecules, showing similar profiles with casein hydrolysates

obtained via trypsin [24] (Figure 1). Because of their specificities, the interactions between the

peptides generated from enzymatic hydrolysis with pancreatin and/or trypsin may increase,

resulting in hydrolysates with lesser protein solubility compared to hydrolysates obtained using

papain [17, 24, 25].

Figure 1 – Proteolytic enzymes and their specificities for cleavage of peptide bonds.

2.2. Gelation properties

Enzymatic hydrolysis can result in the release of peptides with less hydrophobic

properties and more charge than the intact protein, decreasing the interactions between peptide

fragments and consequently, gel formation. In this case, the selection of the most suitable enzyme

plays a central role in protein hydrolysis and it is essential to thoroughly investigate the substrate

73

specificity using specific milk protein substrates and proteases with different mechanisms of

action [26]. The gelation properties of milk proteins hydrolysates are due to the hydrophobic

interactions between hydrophobic peptide aggregates, with a minor contribution by hydrogen

bonds and electrostatic interactions. Spellman et al. [15] showed that whey protein concentrate

hydrolyzed with two commercial preparations of proteases under the same hydrolysis conditions

resulted in protein hydrolysates with different capacities for gel formation, attributed to the

enzymes specificities. In their studies, the main proteolytic component of both enzymes Alcalase

2.4LTM

and Prolyve 100TM

that were used for whey protein concentrate hydrolysis is subtilisin

Carlsberg (EC 3.4.21.62). Subtilisin proteases are serine proteases with relatively low substrate

specificity, but they preferentially cleave peptide bonds after large non-β-branched hydrophobic

residues [15]. Although both enzymes had subtilisin activities, Alcalase 2.4L

TM presents a

specific activity of glutamyl endopeptidase that is not present in Prolyve 100TM

and is responsible

for cleaving peptide bonds after a glutamic acid and to a lesser extent, aspartic acid residues. This

explains its ability to digest substrates with glutamic acid residues at the C-terminus, such as

casein phosphopeptides and whey proteins [15, 26]. The whey protein hydrolysates obtained with

Alcalase 2.4L

TM resulted in the formation of a soft gel, whereas the Prolyve 100

TM hydrolysates

showed no evidence of gel formation, suggesting that the glutamyl endopeptidase activity in

Alcalase 2.4L

TM improved the gelation properties of the whey protein concentrate [15].

2.3. Emulsifying properties

Emulsions, specifically nanoemulsions, are widely used in food and pharmaceutical

industries to deliver poorly water-soluble bioactive compounds and drugs [27, 28]. Conventional

emulsions are a dispersion of two completely or partially immiscible liquids formed by

mechanical shear. Proteins have been used as emulsifiers because of their amphiphilic

characteristics and because they can unfold and re-orientate at the interface [29]. These

macromolecules can be substituted for common emulsifiers that confer weak stabilization and

have toxicological concerns for long-term use [28, 29]. Milk proteins have good emulsifying

capacity [28, 30], and the hydrolysis of these proteins can expose groups buried inside the tertiary

structure allowing new interactions at the interface of the emulsions droplets, such as disulfide

and non-polar bonds to improve the emulsification capacity [28, 31]. Luo et al. [17] observed that

papain and trypsin treatment of casein reduced the surface hydrophobicity of the hydrolyzed

protein; however, pancreatin treatment increased this parameter during the first 4 h of the

74

treatment and significantly reduced it after 24 h. The emulsion activity (EAI) and emulsion

stability index (ESI) were increased for the three proteases, except for the papain treatment after

1 h and pancreatin treatment after 24 h. Trypsin treatment of casein resulted in a higher EAI and

ESI after 24 h compared to papain and pancreatin, although the degree of hydrolysis was nearly

the same (approximately 20%), reinforcing the high substrate specificity of trypsin compared to

papain and pancreatin. Castro & Sato [16] observed that bovine whey protein hydrolyzed with

FlavourzymeTM

500L protease for 2 h had an emulsion activity index reduced by 12%.

FlavourzymeTM

500L is a neutral protease from Aspergillus oryzae that hydrolysis a variety of

peptide bonds, promoting a high degree of hydrolysis [32] (Figure 1). Banach et al. [13] observed

that chymotrypsin and trypsin hydrolysis of milk protein concentrate increased the EAI and ESI

of the hydrolyzed protein, but papain did not change these indices. These observations indicate

that the protease substrate specificity leads to different hydrolysate products with specific

interactive sites on the surface and specific molecular masses that improve the ability of the

peptide to adsorb to the interface of an immiscible liquid emulsion. Extensive hydrolysis can

produce large amounts of free amino acids and short-chain peptides that decrease the emulsifying

properties of proteins (Figure 2). By contrast, limited proteolysis exposes hydrophobic and

hydrophilic residues, enhances the amphiphilic characteristics of proteins, and improves

emulsification [20, 30, 33, 34].

75

Figure 2 - Proteolytic enzymes with high and low specificities and their effects on production of

peptides with emulsifying properties. Red dots are hydrophobic moieties and green “x” are free

amino acids.

3. Conclusion

The enzymatic hydrolysis of milk proteins has great potential for enhancing their

functional properties and is becoming one of the most frequently used tools in food protein

processing. Several studies showed the effect of the parameters of hydrolysis, such as pH,

temperature, time, and enzyme:substrate ratio, which affected the functional properties of the

milk proteins. A particular focus on the specificities and mechanisms of action of proteolytic

enzymes is critical for obtaining milk protein hydrolysates with highly desirable characteristics.

The evaluation of hydrolysis sites, including the composition, distribution and interactions of the

amino acid residues of milk proteins, is fundamental for regulating their functional properties,

such as solubility, gelation and emulsification. Based on the mechanisms of action and substrate

specificities of different proteolytic enzymes, it is possible to modulate milk protein hydrolysates

to balance their functional properties for specific applications in the food and pharmaceutical

industries.

Inta

ct P

rote

in

Water

Em

uls

ion Water

Oil Oil

High specificity

protease (eg. trypsin)

Low specificity

protease (eg. subtilisin)

Migration and reorientation

of protein fragments to the

oil-water interface

Small peptides and free

amino acids can not bind to

the oil-water interface and

reorientate

Hy

dro

lyze

d

Pro

tein

76

References

1. Ye A: Functional properties of milk protein concentrates: Emulsifying properties,

adsorption and stability of emulsions. Int Dairy J 2011, 21:14-20.

2. Kilara A, Panyam D: Peptides from milk proteins and their properties. Crit Rev Food Sci

Nutr 2003, 43(6):607–633.

3. Singh H: Milk protein products: functional properties of milk proteins. In Encyclopedia of

Dairy Sciences. Edited by John WF, Patrick FF, Paul LHM. Academic Press; 2011:887-893.

4. Korhonen H. 2009: Milk-derived bioactive peptides: from science to applications. J Funct

Foods 1:177–187.

5. Sinha R, Radha C, Prakash J, Kaul P: Whey protein hydrolysate: functional properties,

nutritional quality and utilization in beverage formulation. Food Chem 2007, 101:1484–

1491.

6. Guan X, Yao H, Chen Z, Shan L, Zhang M: Some functional properties of oat bran protein

concentrate modified by trypsin. Food Chem 2007, 101:163–170.

7. Liu Q, Kong B, Xiong YL, Xi X: Antioxidant activity and functional properties of porcine

plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chem 2010,

118:403–410.

8. Fernández A, Riera F: β-Lactoglobulin tryptic digestion: A model approach for peptide

release. Biochem Eng J 2013, 70:88–96.

9. Segura-Campos MR, Espinosa-García L, Chel-Guerrero LA, Betancur-Ancona DA: Effect of

enzymatic hydrolysis on solubility, hydrophobicity, and in vivo digestibility in cowpea

(Vigna unguiculata). Int J Food Prop 2012, 15:770–780.

10. Foegeding EA, Davis JP, Doucet D, McGuffey MK: Advances in modifying and

understanding whey protein functionality. Trends Food Sci Tech 2002, 13:151–159.

11. Gerrard JA: Protein–protein crosslinking in food: methods, consequences, applications.

Trends Food Sci Tech 2002, 13:391–399.

12. Hiller B, Lorenzen PC: Functional properties of milk proteins as affected by enzymatic

oligomerisation. Food Res Int 2009, 42:899–908.

77

13. Banach JC, Lin Z, Lamsal BP: Enzymatic modification of milk protein concentrate and

characterization of resulting functional properties. LWT - Food Sci Technol 2013, 54:397-

403.

14. Doucet D, Gauthier SF, Foegeding EA: Rheological characterization of a gel formed

during extensive enzymatic hydrolysis. J Food Sci 2001, 66(5):711-715.

15. Spellman D, Kenny P, O’Cuinn G, Fitzgerald RJ: Aggregation properties of whey protein

hydrolysates generated with Bacillus licheniformis proteinase activities. J Agric Food Chem

2005, 53:1258-1265.

16. Castro RJS, Sato HH: Comparison and synergistic effects of intact proteins and their

hydrolysates on the functional properties and antioxidant activities in a simultaneous

process of enzymatic hydrolysis. Food Bioprod Process 2014, 92:80-88.

17. Luo Y, Pan K, Zhong Q: Physical, chemical and biochemical properties of casein

hydrolyzed by three proteases: partial characterizations. Food Chem 2014, 155:146-154.

18. Adjonu R, Doran G, Torley P, Agboola S: Formation of whey protein isolate hydrolysate

stabilised nanoemulsions. Food Hydrocoll 2014, 41:169-177.

19. Zhao Q, Xiong H, Selomulya C, Chen XD, Zhong H, Wang S, Sun W, Zhou Q: Enzymatic

hydrolysis of rice dreg protein: effects of enzyme type on the functional properties and

antioxidant activities of recovered proteins. Food Chem 2012, 134:1360–1367.

20. Nongonierma AB, FitzGerald RJ: Enzymes Exogenous to Milk in Dairy Technology -

Proteinases. In Encyclopedia of Dairy Sciences. Edited by John WF, Patrick FF, Paul LHM.

Academic Press; 2011:289-296.

21. Storer AC, Ménard R: Papain. In Handbook of Proteolytic Enzymes. Edited by Neil D R,

Guy S. Academic Press; 2013:1858-1861.

22. Yao J, Lin C, Tao T, Lina F: The effect of various concentrations of papain on the

properties and hydrolytic rates of β-casein layers. Colloids Surf B 2013, 101:272– 279.

23. Bougatef A: Trypsins from fish processing waste: characteristics and biotechnological

applications - Comprehensive review. J Clean Prod 2013, 57:257-265.

78

24. Su R, Liang M, Qi W, Liu R, Yuan S, He Z: Pancreatic hydrolysis of bovine casein:

peptide release and time-dependent reaction behavior. Food Chem 2012, 133:851–858.

25. Su R, He Z, Qi W: Pancreatic hydrolysis of bovine casein: changes in the aggregate size

and molecular weight distribution. Food Chem 2008, 107:151–157.

26. Kalyankar P, Zhu Y, O’Keeffe M, O’Cuinn G, FitzGerald RJ: Substrate specificity of

glutamyl endopeptidase (GE): hydrolysis studies with a bovine α-casein preparation. Food

Chem 2013, 136:501–512.

27. Donsì F, Annuziata M, Vincensi M, Gerrari G: Design of nanoemulsion-based delivery

system of natural antimicrobials: Effect of the emulsifier. J Biotechnol 2012, 159:342-350.

28. He W, Lu Y, Qi J, Chen L, Hu F, Wu W: Nanoemulsion-templated shell-crosslinked

nanocapsules as drug delivery systems. Int J Pharm 2013, 445:69-78.

29. Lam RSH, Nickerson MT: Food proteins: A review on their emulsifying properties using

a structure-function approach. Food Chem 2013, 141:975-984.

30. Singh H: Aspects of milk-protein-stabilised emulsions. Food Hydrocolloid 2011b, 8:1938-

1944.

31. He TanY, Tian Z, Chen L, Hu F, Wu W: Food protein-stabilized nanoemulsions as

potential delivery systems for poorly water-soluble drugs: preparation, in vitro

characterization, and pharmacokinetics in rats. Int J Nanomedicine 2011, 6:521-533.

32. Rao M B, Tanksale A M, Ghatge M S, Deshpand V V: Molecular and biotechnological

aspects of microbial proteases. Microbiol Mol Biol Rev 1998, 62:5970635.

33. Scherze I, Muschiolik G: Effects of variuous whey protein hydrolysates on the

emulsifying and surface properties of hydrolysed lecithin. Colloids Surf B 2001, 21:107-117

34. Tavano OL: Protein hydrolysis using proteases: An important tool for food

biotechnology. J Mol Catal B Enzym 2013, 90:1-11.

79

Capítulo IV: Improving the protease production by Aspergillus niger under

solid state fermentation by substrate formulation using statistical mixture

design

Revista: Industrial Crops and Products

80

Abstract

Wheat bran, soybean meal, cottonseed meal and orange peel, alone or in combinations, were used

as the substrates for protease production by Aspergillus niger LBA02 under solid state

fermentation using a simplex-lattice mixture design. Correlation analysis suggested that protein

and ash contents exerted relevant and positive impact on proteases secretion in the first 48 h

fermentation, while higher carbohydrates content negatively influenced the production at the

initial hours of fermentation. Synergistic and antagonistic effects of agroindustrial wastes were

observed during the fermentation time. The highest protease activity was found using the medium

containing the binary mixture of wheat bran (1/2) and soybean meal (1/2), reaching 262.78 U g-1

after 48 h fermentation. The most prominent synergetic effects were observed on fermentations

performed using the medium composed by the four agroindustrial wastes in equal proportions at

48 and 72 h fermentation, reaching increases of 33.7, 7.6, 30.8 and 581.7% and 11.6, 131.4, 69.5

and 547.0%, respectively, in protease production as compared to the individual substrates. The

results suggest that the application of the statistical mixture designs is an attractive method for

improving protease production and identifying optimum formulations using different substrates.

Keywords: protease; solid state fermentation; agroindustrial wastes; statistical mixture design.

81

1. Introduction

Proteases are multifunctional enzymes that catalyze the hydrolysis of proteins to

polypeptides and oligopeptides to amino acids. These enzymes accounting for nearly 60% of the

whole enzyme market and have been used in a wide variety of applications including the

production of pharmaceuticals, detergents, fertilizers or textiles and in processes in leather, food

and biotechnology industries (Ramakrishna et al., 2010; Yin et al., 2013; Abraham et al., 2014).

They can be isolated from plants, animals and microorganisms. Among these sources, the

microorganisms show great potential for protease production due to their broad biochemical

diversity and their susceptibility to genetic manipulation. It has been estimated that microbial

proteases represent approximately 40% of the total worldwide enzyme sales (Rao et al., 1998).

Several species of filamentous fungi have been exploited in industrial processes for the

production of metabolites and industrial enzymes. A. niger has a long tradition of safe use in the

production of enzymes and organic acids. Many of these products have listed as a ‘‘Generally

Recognized as Safe (GRAS)’’ by the US Food and Drug Administration (Schuster et al., 2002).

A. niger is one of the most important sources of fungal proteases. According Pel et al., (2007),

genome sequencing shows that A. niger has 198 proteins involved in proteolytic degradation

process.

Proteolytic enzymes can be produced by submerged and solid state fermentation. For the

growth of fungi, solid state fermentation is most appropriate method because the solid substrates

resemble the natural habitat of the fungi and improving their growth and the secretion of a wide

range of extracellular enzymes. Some characteristics make solid state fermentation more

attractive than submerged fermentation: simplicity, low cost, high yields and concentrations of

the enzymes and the use of inexpensive and widely available agricultural residues as substrates

(Chutmanop et al., 2008). This process arouses most economical interest in regions such as

Brazil, which has abundant biomass and agroindustrial wastes with low cost, such as the residues

from the processing of soybeans, wheat, cottonseed and oranges, that reached a world production

of approximately 1.1 billion tons in 2013 (FAO, 2014). The use of these wastes as substrates for

the development of biotechnological processes such as the enzymes production by solid state

fermentation is a promising example of obtaining biomolecules with high added value from low

cost substrates.

82

The biochemical characterization of enzymes is important to evaluate their

biotechnological potential. The study of the protease properties, such as the substrate specificity,

the optimum catalytic pH conditions and the temperature and stability profiles, can be used to

predict the successful application of the enzyme to particular industries or processes (Castro and

Sato, 2013).

Statistical methods were applied to different engineering problems for improving the

performance and to find the optimum process variables. Statistical mixture designs are special

class of response surface designs where the proportions of the components or factors are

considered important. It involves the use of different combinations between the components for

changing mixture composition and exploring how such changes will affect a specific response.

The interactions between the components of a mixture for maximizing the response are studied

using mixture design approach (Rao and Baral, 2011).

In this work, a simplex-lattice mixture design has been applied to investigate the presence

of synergistic or antagonistic effects of different agroindustrial wastes for protease production by

Aspergillus niger LBA02 under solid state fermentation. The correlation between the chemical

components of the agroindustrial wastes with the protease production was further studied.

2. Materials and Methods

2.1. Agroindustrial wastes and centesimal composition

Wheat bran, soybean meal, and cottonseed meal were kindly provided by Bunge Foods

S/A. Orange peel was purchased from local market of Campinas (Sao Paulo, Brazil). To be used

as matrix support, the orange peel was grinded, washed three times with distilled water and dried

at 50 °C for 24-48 h.

Moisture, protein content, lipids and ash of the agroindustrial wastes were determined by

AOAC methods (AOAC, 2010). The carbohydrate content was measured by difference between

the total value of 100% and the sum of the other components. The tests were performed in

triplicate and the results were expressed as the mean ± standard deviation.

2.2. Microorganism culture

The strain used in this study was A. niger LBA02, previously selected as a proteolytic

strain from the culture collection of the Laboratory of Food Biochemistry, School of Food

83

Engineering, University of Campinas. The strain was periodically subcultured and maintained on

potato dextrose agar slants. To produce fungal spores, the microorganism was inoculated into a

medium composed of 10 g wheat bran and 5 mL of solution containing 1.7% (w/v) NaHPO4 and

2.0% (w/v) (NH4)2SO4 and incubated for 3 days at 30 °C. The fungal spores were dispensed into

sterile Tween 80 solution (0.3%) to prepare the inoculum for fermentation. The number of spores

per milliliter in the spore suspension was determined with a Neubauer cell counting chamber.

2.3. Protease production and sampling

The protease production was performed under solid state fermentation using the individual

substrates, binary, ternary or quaternary mixtures of their in various proportions in 250 mL

Erlenmeyer flasks containing 20 g medium. The initial cultivation parameters, defined in

previous studies in our laboratory (data no shown), as the most appropriate conditions for

protease production by A. niger LBA02, were 50 % moisture, temperature set at 30 °C, and an

inoculum level of 107 spores g

-1.

The protease activity was tested at 24 h intervals during 96 h fermentation. The crude

extract was obtained by the addition of 100 mL distilled water. After 1h at rest, the solution was

filtered through a filter membrane to obtain an enzyme solution free of any solid material.

2.4. Statistical mixture design

The mixture design of experiment was used to obtain the optimum composition between

the agroindustrial wastes for maximum protease production and to evaluate the interaction effects

in a blend of components. A four component augmented simplex-lattice design has been

employed in which each components is studied in six levels, namely 0 (0%), 1/8 (12.5%), 1/4

(25%), 1/2 (50%), 5/8 (62.5%) and 1 (100%) (Table 1).

Special cubic regression models were fitted for variations of all studied responses as

function of significant (p < 0.05) interaction effects between the proportions, with acceptable

determination coefficients (R2 > 0.90). Eq. (1) represents these models:

where Yi is the predicted response; ‘q’ represents the number of components in the system; ‘Xi,

Xj, Xk’ are the coded independent variables; ‘βi’ is the regression coefficient for each linear effect

𝑌𝑖 = 𝛽𝑖𝑋𝑖 + 𝛽𝑖𝑗

𝑞

𝑖<𝑗

𝑞

𝑖=1

𝑋𝑖𝑋𝑗 + 𝛽𝑖𝑗𝑘𝑋𝑖𝑋𝑗𝑋𝑘

𝑞

𝑖<𝑗<𝑘

84

term; and ‘βij’ and ‘βijk’ are the binary and ternary interaction effect terms, respectively.

StatisticaTM

10 software from Statsoft Inc. (Tulsa, Oklahoma, USA) was employed for the

experimental design, data analysis and model building.

Table 1 – Matrix of the simplex-lattice mixture design for protease production by A. niger

LBA02 under solid state fermentation using different agroindustrial wastes and their mixtures as

substrates.

*All the formulated medium had the moisture level adjusted to 50% according to the initial moisture.

2.5. Determination of protease activity

The protease activity was measured using azocasein as the substrate according to Charney

and Tomarelli (1947) and described by Castro and Sato (2013). The reaction mixture containing

0.5 mL 0.5% (w/v) azocasein (Sigma), pH 5.0, and 0.5 mL of the enzyme solution was incubated

for 40 min. The reaction was stopped by adding 0.5 mL 10% TCA and the test tubes were

centrifuged at 17,000 x g for 15 min at 25 °C. A 1.0 mL aliquot of the supernatant was

neutralized with 1.0 mL 5 M KOH. One unit of enzyme activity (U) was defined as the amount of

enzyme required to increase the absorbance at 428 nm by 0.01 under the assay conditions

described.

Run

Independent variables

Wheat bran Soybean meal Cottonseed meal Orange peel

x1 x2 x3 x4

1 1 0 0 0

2 0 1 0 0

3 0 0 1 0

4 0 0 0 1

5 1/2 1/2 0 0

6 1/2 0 1/2 0

7 1/2 0 0 1/2

8 0 1/2 1/2 0

9 0 1/2 0 1/2

10 0 0 1/2 1/2

11 5/8 1/8 1/8 1/8

12 1/8 5/8 1/8 1/8

13 1/8 1/8 5/8 1/8

14 1/8 1/8 1/8 5/8

15 1/4 1/4 1/4 1/4

85

2.6. Calculations and statistics

The Tukey test was used to check the significant differences between the groups analyzed

that were considered significant when p-value ≤ 0.05. Pearson correlation coefficient was used to

measure the strength of linear dependence between two responses. The correlation coefficient

ranges from – 1 to 1. A value of 1 implies that a linear equation describes the relationship

between the responses was perfectly and positive, while a value of -1 indicate a perfectly and

negative correlation. A value of 0 implies that there is no linear correlation between the

responses. The correlations between analyzed parameters were considered significant when the p-

value ≤ 0.10.

3. Results and Discussion

3.1. Chemical composition of the agroindustrial wastes

The centesimal compositions of the agroindustrial wastes used as fermentation substrates

for production of protease by A. niger LBA02 under solid state fermentation are showed in Table

2.

Table 2 – Average values of the centesimal composition (%) of the agroindustrial wastes used for

protease production by A. niger LBA02 under solid state fermentation.

Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. Tukey

tests were applied between the chemical components of each substrate (not between different substrates). Carbohydrate content

(%) was measured by difference between the total value of 100% and the sum of the other components.

The enzymes production under solid state fermentation can be affected by the composition

of the substrates and various cultivation factors. On protease production, for example, the

presence of protein sources can induce the enzyme secretion by the microorganism. On the other

hand, the substrate must have a carbon to nitrogen ratio (C:N) suitable for the fermentation

(Castro and Sato, 2013). Soybean meal and cottonseed meal were the materials with higher

protein content and orange peel showed the major C:N ratio (Table 3).

Chemical components Wheat bran Soybean meal Cottonseed meal Orange peel

Moisture (%) 12.77 ± 0.08a 11.93 ± 0.02

a 6.42 ± 0.01

a 7.92 ± 0.08

a

Protein (%) 14.74 ± 0.51b 49.24 ± 0.07

b 25.91 ± 0.60

b 7.01 ± 0.08

b

Carbohydrates (%) 63.04c 31.53

c 55.80

c 79.68

c

Lipids (%) 4.47 ± 0.22d 1.40 ± 0.02

d 7.83 ± 0.01

d 1.95 ± 0.13

d

Ash (%) 4.98 ± 0.06e 5.90 ± 0.04

e 4.04 ± 0.01

e 3.44 ± 0.11

e

86

Table 3 - Correlation analysis between the chemical components of the agroindustrial wastes with the protease production by A. niger

LBA02 under solid state fermentation at 24, 48, 72 and 96 h.

Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. Tukey tests were applied between the runs for each

fermentation time (not between different fermentation time). *The correlations between analyzed parameters were considered significant when the p-value ≤ 0.10.

Runs C:N Protein

(%)

Carbohydrate

(%)

Lipids

(%)

Ash

(%)

Protease production (U g-1

)

24 h 48 h 72 h 96 h

1 4.28 16.89 72.27 5.12 5.72 101.10 ± 5.73b 183.98 ± 5.65

f, g 178.00 ± 9.17

b, c 184.08 ± 6.84

a

2 0.64 55.91 35.80 1.59 6.70 130.10 ± 3.45a 228.60 ± 7.30

b, c 85.85 ± 3.10

f, g 63.77 ± 1.70

i

3 2.15 27.69 59.63 8.36 4.32 66.18 ± 1.05e 188.03 ± 7.65

f, g 117.17 ± 7.18

e 125.20 ± 2.28

c, d

4 11.37 7.61 86.53 2.12 3.74 40.62 ± 4.54f 36.08 ± 3.33

j 30.70 ± 3.40

h 14.00 ± 1.64

j

5 1.48 36.4 54.04 3.36 6.21 99.58 ± 1.47b 262.78 ± 10.39

a 90.20 ± 1.21

f, g 58.40 ± 1.14

i

6 2.96 22.29 65.95 6.74 5.02 69.22 ± 1.99d, e

176.27 ± 9.88g 137.92 ± 2.66

d 135.95 ± 5.76

c

7 6.48 12.25 79.40 3.62 4.73 74.18 ± 3.10c, d, e

99.75 ± 4.26i 97.55 ± 4.68

f 122.28 ± 8.83

c, d

8 1.14 41.8 47.72 4.98 5.51 105.63 ± 2.58b 221.73 ± 4.07

c, d 90.95 ± 4.69

f, g 105.42 ± 6.98

e, f

9 1.93 31.76 61.17 1.86 5.22 79.50 ± 6.81c, d

133.35 ± 5.60h 75.32 ± 1.69

g 71.37 ± 2.62

h, i

10 4.14 17.65 73.08 5.24 4.03 65.12 ± 3.95e 201.42 ± 9.94

d, e, f 179.07 ± 7.08

b 165.92 ± 7.55

b

11 3.09 21.96 67.91 4.71 5.42 84.25 ± 4.63c 218.92 ± 6.03

c, d, e 119.85 ± 4.97

e 83.75 ± 3.18

g, h

12 1.20 41.47 49.68 2.94 5.91 131.95 ± 4.57a 202.95 ± 5.55

d, e, f 116.68 ± 4.88

e 62.82 ± 2.45

i

13 2.25 27.36 61.59 6.33 4.72 103.93 ± 3.36b 198.22 ± 4.81

e, f, g 161.85 ± 8.71

c 111.45 ± 6.37

d, e

14 1.20 41.47 49.68 2.94 5.91 71.18 ± 1.50d, e

125.62 ± 8.80h 141.18 ± 5.63

d 163.27 ± 8.54

b

15 2.35 27.03 63.56 4.30 5.12 99.70 ± 1.49b 245.97 ± 13.35

a, b 198.63 ± 5.32

a 91.13 ± 2.48

f, g

Correlation analysis between the protease production and C:N

Pearson coefficient -0.67 -0.75 -0.33 -0.21

p-value 0.007* 0.001

* 0.234 0.462

Correlation analysis between the protease production and the protein content (%)

Pearson coefficient 0.71 0.52 -0.10 -0.17

p-value 0.003* 0.049

* 0.723 0.558

Correlation analysis between the protease production and the carbohydrates content (%)

Pearson coefficient -0.71 -0.58 0.03 0.08

p-value 0.003* 0.023

* 0.927 0.758

Correlation analysis between the protease production and the lipids content (%)

Pearson coefficient -0.19 0.27 0.50 0.54

p-value 0.491 0.322 0.056* 0.036

*

Correlation analysis between the protease production and the ash content (%)


p-value 0.001* 0.047

* 0.978 0.828

87

The Pearson coefficient was used to verify the correlation between the chemical

components and the C:N ratio in the substrates with the protease production. The analysis

indicated positive and significant correlations between the protein and the ash contents (%) in the

substrates with the protease production at 24 h and 48 h fermentation. In contrast, negative and

significant correlations between the C:N ratio and the carbohydrate content with the protease

production were observed at 24h and 48 h fermentation. Positive and significant correlations

were detected at 72 and 96 h fermentation between the protease production and the lipids content

(Table 3).

These results suggested that an adequate supply of proteins as nitrogen source can induce

the protease production at the first hours of fermentation. On the other hand, high content of

carbon source can cause catabolic repression, a mechanism particularly important in the

regulation of the extracellular enzymes that degrade complex substrates in organisms exposed to

changing environments, which probably happened in the present study. An interesting

observation included the positive and significant impact of the ash content on the protease

production. The ash content can be defined as the measurement of the mineral content and other

inorganic matter in biomass. The presence of considerable amounts of minerals such as

potassium, phosphorus and calcium is a relevant characteristic for use of substrates in solid state

fermentation (Chutmanop et al., 2008). It is important to note that the lipids only presented

significant effects on protease production at the last hours of fermentation, as it is considered a

nutrient source not readily metabolizable. In addition to the content of each chemical component,

their specific structure, molecular weight, residues and chain length, especially for proteins and

carbohydrates, can influence the enzymes production. Chutmanop et al., (2008) reported the

protease production by A. oryzae using wheat bran and rice bran with similar content of proteins.

These authors observed higher protease production in rice bran and associated it to the easy

digestion of rice bran proteins compared to wheat bran proteins. The presence of diferent types of

carbohydrates, such as non-starch polysaccharides (pectin, cellulose, hemicellulose) or starch,

can be hardly or easily assimilated, respectively, by microorganisms for use as a carbon source.

88

Several studies describe the use of agroindustrial wastes as potent substrates for the

production of proteases by filamentous fungi of the genus Aspergillus. Thanapimmetha et al.,

(2012) observed maximum yields of protease production by a strain of A. oryzae using Jatropha

curcas seed cake as a substrate under solid state fermentation. The highest secretion of protease

was measured to be 3,094 U g DM−1

(units of protease per gram of dry material). Shivakumar

(2012) screened 11 different substrates, including 8 cereals and 3 agroindustrial residues, for

protease production by Aspergillus sp. under solid state fermentation and found that wheat flour,

wheat bran and soya flour proved superior protease production, reaching activities of 320, 280

and 160 U g-1

, respectively. Veerabhadrappa et al., (2014) evaluated the protease production by

A. versicolor CJS-98 under solid state fermentation using Jatropha seed cake and reached a

maximum protease activity of 3,366 U g-1

at 96 h. Castro et al., (2015) investigated the protease

production under solid state fermentation by A. awamori IOC-3914 using babassu cake as

substrate and observed maximum production of 31.8 U g-1

at 168 h fermentation.

3.2. Synergistic and antagonistic effects of the agroindustrial wastes on protease

production

The interactions amongst the four substrates in the protease production were studied in the

15 assays using a simplex-lattice mixture design (Table 1 and Table 3). The highest protease

activities were found using the medium containing wheat bran (1/2) and soybean meal (1/2) (run

5) and the formulation composed by the quaternary mixture of the substrates in equal proportions

(run15), reaching 262.78 and 245.97 U g-1

, respectively, after 48 h fermentation.

Synergistic and antagonistic effects between the different agroindustrial wastes in the

protease production were detected during all the fermentation time. The highest and similar

protease activities at 24 h fermentation were observed in runs 2 and 12, enabling the application

of isolated or mixture of substrates. A synergistic effect was detected in the medium composed of

wheat bran (1/8), soybean meal (5/8), cottonseed meal (1/8) and orange peel (1/8) (run 12),

resulting in increases of 30.5, 1.42, 99.4 and 224.8% of protease production as compared to the

individual substrates, respectively. At 48 h fermentation, the medium formulated with the binary

mixture of wheat bran (1/2) and cottonseed meal (1/2) (run 5) presented increases of 42.8 and

14.9% in protease production as compared to the individual substrates, respectively. The medium

composed by the four agroindustrial wastes in equal proportions (run 15) showed strong and

89

synergetic effects at 48 h fermentation, reaching increases of 33.7, 7.6, 30.8 and 581.7% and at

72 h reaching increases of 11.6, 131.4, 69.5 and 547.0%, respectively, in protease production as

compared to the individual substrates (Table 3).

The lowest values for protease activities were detected when orange peel was used as

isolated substrate, however, important synergistic effects were observed when combining it with

cottonseed meal. The protease production in cottonseed meal (run 3) and orange peel (run 4)

reached 117.17 and 30.70 U g-1

, respectively, while in the mixture of these two substrates in

equal proportions (run 10), the protease production reached 179.08 U g-1

at 72 h fermentation,

which represented increases of 52.8 and 483.3% in protease production as compared to the

individual substrates. This same formulation (run10) showed similar results at 96 h fermentation,

resulting in increases of 32.5 and 1,085% of the protease production as compared to the isolated

substrates (runs 3 and 4, respectively) (Table 3).

Antagonistic effects between the agroindustrial wastes were observed too, characterized by

the decrease in protease production when mixtures were used for fermentation. In general, the

combination of orange peel with wheat bran or soybean meal decreased the protease production.

For example, at 48 h fermentation, the protease production in the medium composed by wheat

bran (1/2) and orange peel (1/2) (run 7) was 99.75 U g-1

, while the production in wheat bran (run

1) and orange peel (run 4) as isolated substrates were 183.98 and 36.08 U g-1

, respectively.

Although, this result represents an increase in protease production compared to the assay

performed using orange peel as isolated substrate (run 4), a decrease of 45.8% in protease

production was observed compared to the fermentation using wheat bran in isolated form (run 1).

The same occurred in run 9 at 48 h, that presented a decrease of 71.4% in protease production for

the fermentation medium composed by soybean meal (1/2) and orange peel (1/2) as compared to

the assay performed using soybean meal (run 2) as an isolated substrate (Table 3).

The application of statistical mixture designs for enzymes production under solid state

fermentation is a scarce process described in the scientific literature. Some researchers used

predefined combinations of substrates to optimize enzyme production by microorganisms, so

there was no use of a statistical tool that allowed the construction of models and prediction of

responses at different substrate formulations. Chutmanop et al., (2008) studied the protease

production under solid state fermentation using a strain of A.oryzae (Ozykat-1) and reported a

90

protease production of 1,200 U g-1

within 96 h fermentation using a substrate mixture of 75% rice

bran and 25% wheat bran. Lazim et al., (2009) studied the production of thermophilic alkaline

protease by Streptomyces sp. CN902 using different agroindustrial residues individually or in

combination as the substrate under solid state fermentation. Different solid substrates such as

wheat bran, barley bran, rice bran, olive spinet, oats bran, chopped date stones and chopped dried

fish were tested. The results showed the binary mixture of wheat bran with chopped date stones

(5:5) proved to be the best as it gave the highest enzyme activity (90.5 U g−1

), which represented

increases of 21.5 and 30.2% when compared to individual substrates wheat bran (74.5 U g−1

) and

chopped date stones (69.5 U g−1

), respectively. Delabona et al., (2013) used the experimental

mixture design as a tool to enhance glycosyl hydrolases (xylanase, β-glucosidase and filter paper

activity) production by Trichoderma harzianum P49P11 under submerged fermentation. The

components studied were delignified steam-exploded bagasse, sucrose and soybean flour and

their combinations in the culture media. It was found that a mixed culture medium could

significantly maximize the enzymes production. These results corroborated with the present study

and showed that the statistical mixture design is an attractive process for find optimum

formulations and production of high amounts of enzymes.

3.3. Interpretation of contour plots

The variation of the amounts protease produced by A. niger LBA02 using different

agroindustrial wastes proportions is also shown using mixture contour plots, in which, each factor

(pure mixture component) is represented in a corner of an equilateral triangle; each point within

this triangle refers to a different proportion of components in the mixture (Figures 1 and 2). The

maximum percentage of each ingredient considered by the regression is placed at the

corresponding corner while the minimum is positioned at the middle of the opposite side of the

triangle. The center of the triangle represents the mixture in equal parts (Martinello et al., 2006).

A contour plot provides a two-dimensional view where all points that have the same response are

connected to produce contour lines of constant responses (Rao and Baral, 2011).

Figures 1 and 2 show the contour plots for protease production at 48 h fermentation

(maximum production) and for the runs selected to validation tests in each fermentation time,

respectively. It was observed profile changes in protease production in response to different

substrate formulations. The contour plots for 48 h fermentation indicated maximum responses in

91

three different formulations with predicted values above 260 U g-1

. In the medium contained the

ternary mixtures of wheat bran, soybean meal and cottonseed meal or wheat bran, soybean meal

and orange peel, the zones of maximum response variables were located towards the side of

triangle having mixtures of wheat bran and soybean meal as the substrates. This indicates that the

addition of wheat bran and soybean meal helps to improve the response variables whereas the

addition of orange peel has negative effects on the response variable. The addition of equal

proportions of wheat bran, cottonseed meal and orange peel helped to improve the response

variables, reaching values up to 260 U g-1

, as indicated by the zone of maximum protease activity

that was located in the center of the contour plot (Figure 1). From the contour plots for 48 h

fermentation, the protease production was decreased when the medium contained the ternary

mixture of soybean meal, cottonseed meal and orange peel, with a maximum predicted value of

180 U g-1

(Figure 1).

Figure 1 - Mixture contour plots for protease production by A. niger LBA02 at 48 h fermentation

as function of significant (p < 0.05) interaction effects of agroindustrial wastes proportions.

> 260 < 260 < 196 < 156 < 116 < 76 < 36

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Wheat

bran

0.00 0.25 0.50 0.75 1.00

Cottonseed

meal

> 260 < 260 < 192 < 152 < 112 < 72 < 32

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Wheat

bran

0.00 0.25 0.50 0.75 1.00

Soybean

meal

> 260 < 260 < 240 < 220 < 200 < 180

0.00

0.25

0.50

0.75

1.00

Cottonseed

meal0.00

0.25

0.50

0.75

1.00

Wheat

bran

0.00 0.25 0.50 0.75 1.00

Soybean

meal

> 220 < 220 < 152 < 112 < 72 < 32

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Soybean

meal

0.00 0.25 0.50 0.75 1.00

Cottonseed

meal

92

Figure 2 shows the variation of the protease activity in the fermentations performed using

the formulations selected to validation tests. These assays were randomly selected and not

necessarily indicate the maximum protease production at the fermentation time. Two

formulations showed maximum protease production when equal proportions of soybean meal,

cottonseed meal and orange peel or wheat bran, cottonseed meal and orange peel were used as the

substrates, reaching values above 170 U g-1

at 24 h fermentation and above 180 U g-1

at 96 h

fermentation. At 48 h fermentation, the binary mixture of soybean meal and cottonseed meal

favoured the enzyme production, while at 72 h fermentation, the medium composed by

cottonseed meal and orange peel was more appropriate with predicted values above 150 U g-1

.

This can be verified by observing the mixture surface plots (Figure 2).

Figure 2 - Mixture contour plots for protease production by A. niger LBA02 during 24, 48, 72

and 96 h fermentation as function of significant (p < 0.05) interaction effects of agroindustrial

wastes proportions. These assays were randomly selected for validation tests.

> 260 < 260 < 192 < 152 < 112 < 72 < 32

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Wheat

bran

0.00 0.25 0.50 0.75 1.00

Soybean

meal

> 170 < 170 < 144 < 124 < 104 < 84 < 64 < 44

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Soybean

meal

0.00 0.25 0.50 0.75 1.00

Cottonseed

meal

> 170 < 170 < 152 < 132 < 112 < 92 < 72 < 52 < 32

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Wheat

bran

0.00 0.25 0.50 0.75 1.00

Cottonseed

meal

> 180 < 180 < 136 < 96 < 56 < 16

0.00

0.25

0.50

0.75

1.00

Orange

peel0.00

0.25

0.50

0.75

1.00

Wheat

bran

0.00 0.25 0.50 0.75 1.00

Cottonseed

meal

24 h 48 h

72 h 96 h

93

3.4. Model fitting, regression analysis and validation tests

The response data based on the independent variables was obtained from the experiments

and recorded in Table 4. The experiments were conducted with triplicates and found that there

was good agreement between the replicates. All the independent and response variables were

fitted to special cubic models.

The coefficient of determination R2 and the F test (analysis of variance-ANOVA) were

used to verify the quality of fit of the models. Table 4 contains the models, corresponding R2

of

the regression equations for the responses as well as the corresponding F-ratio and p-values. The

high R2, which were above 0.90, indicate that all response functions adequately fit the

experimental data, and the models can be used for predictive purposes in the protease production

by A. niger LBA02 under solid state fermentation using different substrates and their mixtures.

As showed in Table 4, the predicted regression equations represent the models with the

significant factors for production protease at 24, 48, 72 and 96 h fermentation. The negative

quadratic (binary) and cubic (ternary) terms of fitted regression equation showed the antagonistic

effects as well the positive quadratic and cubic terms indicated synergistic effects of the

agroindustrial wastes on the protease production. The highest significant effects of the

independent variables showed changes in the profile during the fermentation time. In the first 48

h fermentation, the soybean meal (x2) exerted the highest significant effect on the protease

production followed by wheat bran (x1) at 24 h fermentation or cottonseed meal (x3) at 48 h

fermentation. On the other hand, after 72 and 96 h fermentation, the highest significant effect was

caused by wheat bran (x1), followed by cottonseed meal (x3), soybean meal (x2) and orange peel

(x4) (Table 4). At 24 h fermentation, the ternary interaction between soybean meal, cottonseed

meal and orange peel (x2x3x4) showed the highest positive significant effect on protease

production, while the interaction between wheat bran, cottonseed meal and orange peel (x1x3x4)

presented the highest negative significant effect. At 48 h fermentation, the ternary interaction of

wheat bran (x1), cottonseed meal (x3) and orange peel (x4) showed strong positive and significant

effect on protease production, while at 72 and 96 h fermentation, this effect was detected for the

mixture composed by soybean meal, cottonseed meal and orange peel (x2x3x4) (Table 4).

94

Table 4 - Analysis of variance (ANOVA) including models, R2 and probability values for the

final reduced models for protease production at 24, 48, 72 and 96 h fermentation.

*F-ratio = Fcalculated/Ftabulated

Response: protease production at 24 h fermentation

Source of variation Sum of

squares

Degrees of

freedom

Mean of

squares F-ratio

* R² p-value

Regression 26,255.46 11 2,386.86 57.12 0.97 <0.001

Residual 768.81 33 23.29

Total 27,024.27

Special cubic model: Y = 103.1x1 + 128.4x2 + 66.6x3 + 40.5x4 - 61.5x1x2 – 59.3x1x3 + 35.7x2x3 + 49.4x3x4 +

1,500.5x1x2x3 – 1,212.1x1x2x4 – 1,254.1x1x3x4 + 2,470.5x2x3x4



squares

Degrees of

freedom

Mean of

squares F-ratio R² p-value

Regression 146,303.9 7 20,900.55 59.94 0.95 <0.001

Residual 6,887.1 37 186.14

Total 153,191.0

Special cubic model: Y = 177.4x1 + 233.5x2 + 185.1x3 + 30.4x4 + 235.7x1x2 + 353.0x3x4 + 3,672.6x1x3x4 –

1,961.5x2x3x4



squares

Degrees of

freedom

Mean of


Regression 84,089.51 8 10,511.19 58.04 0.96 <0.001

Residual 3,564.69 36 99.02

Total 87,654.20

Special cubic model: Y = 172.6x1 + 80.8x2 + 109.2x3 + 27.9x4 – 153.8x1x2 + 76.1x2x4 + 433.1x3x4 – 1,438.1x1x2x3

+ 5,249.0x2x3x4



squares

Degrees of

freedom

Mean of


Regression 92,031.84 13 7,079.37 117.72 0.99 <0.001

Residual 1,073.36 31 34.62

Total 93,105.20

Special cubic model: Y = 184.5x1 + 64.1x2 + 125.6x3 + 14.4x4 – 260.6x1x2 – 73.2x1x3 + 94.5x1x4 + 45.3x2x3 +

131.5x2x4 + 386.8x3x4 – 9,431.2x1x2x3 + 2,286.7x1x2x4 + 1,038.7x1x3x4 + 4,815.7x2x3x4

95

Validation tests were performed to determine the accuracy of the polynomial models

obtained for protease production by A. niger LBA02 using different agroindustrial wastes and

their mixtures (Table 5).

Table 5 - Validation tests performed to determine the adequacy of the polynomial models

obtained for the protease production using agroindustrial wastes in different formulations.

Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. RSD (%):

relative standard deviation. x1 – wheat bran; x2 – soybean meal; x3 – cottonseed meal; x4 – orange peel.

The validation tests were conducted in two ways: 1) assays randomly selected for each time

fermentation and 2) assays selected from the matrix of the statistical mixture design which the

protease production was maximum for each fermentation time. The tests were performed in

triplicate and the results were presented in Table 5. According to the regression models (Table 4),

the most of experimental values agreed with the values predicted by the models within a 95%

confidence interval, thereby confirming the validity of the models for the evaluated responses

(Table 5). Although the predicted value for maximum protease production at 72 h fermentation

did not agree with the experimental response (p < 0.05), the relative standard deviation (RSD)

was low and this model can be considered as predictive (Table 5).

Randomized tests used for models validation

Protease production Independent variables Predicted

response

Experimental

response

RSM

(%) x1 x2 x3 x4

24 h fermentation 0.000 0.333 0.333 0.333 179.08a 179.80 ± 5.55

a 0.40

48 h fermentation 0.000 0.333 0.333 0.333 188.66b 182.63 ± 5.38

b -3.30

72 h fermentation 0.333 0.000 0.333 0.333 151.16c 148.53 ± 8.92

c -1.77

96 h fermentation 0.333 0.000 0.333 0.333 191.67d 184.80 ± 5.02

d -3.93

Tests performed for maximum protease production

24 h fermentation 0.125 0.625 0.125 0.125 128.82e 130.25 ± 4.95

e 1.09

48 h fermentation 0.500 0.500 0.000 0.000 259.83f 258.98 ± 11.35

f -0.33

72 h fermentation 0.250 0.250 0.250 0.250 180.40g 195.65 ± 1.77

h 7.79

96 h fermentation 1.000 0.000 0.000 0.000 184.46i 181.68 ± 7.67

i -1.53

96

4. Conclusion

The results obtained in the present study suggested that the application of the statistical

mixture designs for protease production by A. niger LBA02 using different agroindustrial wastes

under solid state fermentation is an attractive method for improving the performance and to find

the optimum formulations. Binary mixtures containing wheat bran (1/2) and soybean meal (1/2)

resulted in maximal protease production during all fermentation time, reaching 262.7 U g-1

at 48

h fermentation. Increases ranging from 7.6 to 581.7% in protease production, compared to

individual substrates, were observed when quaternary mixtures of wheat bran, soybean meal,

cottonseed meal and orange peel in equal proportions were used as substrate at 48 h fermentation,

reaching a maximum protease production of 245.9 U g-1

. The process proposed in this study can

be extended to other enzymes groups produced by microorganisms, such as lipases, pectinases,

cellulases and invertases allowing the obtaining of multi-enzyme complexes in a simplified

combination of agroindustrial wastes with different characteristics for maximizing the enzymes

production using mixture designs.

References

Abraham, J., Gea, T., Sánchez, A., 2014. Substitution of chemical dehairing by proteases from

solid-state fermentation of hair wastes. J. Clean. Prod., 74, 191-198.

Association of Official Analytical Chemists (AOAC), 2010. In: Horwitz, W. (Ed.), Official

Methods of Analysis of the Association of Official Agriculture Chemistry. AOAC, Washington,

DC.

Castro, A. M., Castilho, L. R., Freire, D. M.G., 2015. Performance of a fixed-bed solid-state

fermentation bioreactor with forced aeration for the production of hydrolases by Aspergillus

awamori. Biochem. Eng. J., 93, 303–308.

Castro, R.J.S., Sato, H.H., 2013. Synergistic effects of agroindustrial wastes on simultaneous


mixture design. Ind. Crops Prod., 49, 813-821.

Charney, J., Tomarelli, R. M., 1947. A colorimetric method for the determination of the

proteolytic activity of duodenal juice. J. Biol. Chem., 170, 501-505.

97

Chutmanop, J., Chuichulcherm, S., Chisti, Y., Sirinophakun, P., 2008. Protease production by

Aspergillus oryzae in solid-state fermentation using agroindustrial substrates. J. Chem. Technol.

Biotechnol., 83, 1012-1018.

Delabona, P. S., Farinas, C. S., Lima, D. J. S., Pradella, J. G. C., 2013. Experimental mixture

design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum

P49P11 strain cultivated under controlled bioreactor submerged fermentation. Bioresource

Technol., 132, 401–405.

FAO. (2014, November 20). Retrieved from http://faostat.fao.org/.

Lazim, H., Mankai, H., Slama, N., Barkallah, I., Limam, F., 2009. Production and optimization of

thermophilic alkaline protease in solid-state fermentation by Streptomyces sp. CN902. J. Ind.

Microbiol. Biotechnol., 36, 531–537.

Martinello, T., Kaneko, T.M., Velasco, M.V.R., Taqueda, M.E.S., Consiglieri, V.O., 2006.

Optimization of poorly compactable drug tablets manufactured by direct compression using the

mixture experimental design. Int. J. Pharm. 322, 87–95.

Pel, H. J. et al., 2007. Genome sequencing and analysis of the versatile cell factory Aspergillus

niger CBS 513.88. Nature Biotechnol., 25 (2), 221-231.

Ramakrishna, V., Rajasekhar, S., Reddy, L. S., 2010. Identification and purification of

metalloprotease from dry grass pea (Lathyrus sativus L.) seeds. Appl. Biochem. Biotechnol., 160,

63-71.

Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V., 1998. Molecular and

biotechnological aspect of microbial proteases. Microbiol. Mol. Biol. Res., 62, 597–635.

Rao, P. V., Baral, S. S., 2011. Experimental design of mixture for the anaerobic co-digestion of

sewage sludge. Chem. Eng. J., 172, 977-986.

Schuster, E., Dunn-Coleman, N., Frisvad, J.C., Van Dijck, P.W.M., 2002. On the safety of

Aspergillus niger – a review. Appl. Microbiol. Biotechnol., 59, 426–435.

Shivakumar, S., 2012. Production and characterization of an acid protease from a local

Aspergillus sp. by solid substrate fermentation. Archives Appl. Sci. Res., 4 (1), 188-199.

98

Thanapimmetha, A., Luadsongkram, A., Titapiwatanakun, B., Srinophakun, P., 2012. Value

added waste of Jatropha curcas residue: optimization of protease production in solid state

fermentation by Taguchi DOE methodology. Ind. Crop. Prod., 37, 1-5.

Veerabhadrappa, M. B., Shivakumar, S. B., Devappa, S., 2014. Solid-state fermentation of

Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in

Jatropha seed cake using Aspergillus versicolor CJS-98. J. Biosc. Bioeng., 117 (2), 208-214.

Yin, L. J., Hsu, T. H., Jiang, S. T., 2013. Characterization of acidic protease from Aspergillus

niger BCRC 32720. J. Agric. Food Chem., 61, 662−666.

99

Capítulo V: A new approach for proteases production by Aspergillus niger

based on the kinetic and thermodynamic parameters of the enzymes

obtained

Revista: Biocatalysis and Agricultural Biotechnology

100

Abstract

This study reports the proteases production by Aspergillus niger LBA02 under solid state

fermentation using different agroindustrial wastes and the variation of the biochemical properties

of these proteases in response to each substrate. The biochemical properties of the proteases

varied widely when produced in wheat bran (PWB), soybean meal (PSM), cottonseed meal

(PCM), orange peel (POP) and the quaternary mixture of them (PQM). The lower value for

activation energy (Ea) was detected for protease POP (16.32 kJ mol-1

) and the higher for protease

PQM (19.48 kJ mol-1

). The temperature quotient (Q10) values ranging from 1.20 to 1.28 at

temperatures between 35-55 °C. The higher Vmax/Km ratio was 562.79 U mL g-1

mg-1

for the

protease PSM. The order of thermal stability of the proteases at temperatures ranging from 40 to

60 °C as revealed from t1/2 and D values and thermodynamic parameters Ead (activation energy

for irreversible deactivation), ΔH (enthalpy), ΔG (Gibbs free energy) and ΔS (entropy) was:

PWB > PQM > POP > PSM > PCM. In the study of the substrate specificity, the best substrate

was hemoglobin from bovine blood. Our study provides a new point of view for proteases

production under solid state fermentation, which was possible to evaluate the most suitable

substrate for secretion of enzymes with more attractive characteristics based on their biochemical

properties, such as high thermal stability.

Keywords: protease; substrate specificities; thermodynamic and kinetic parameters.

101

1. Introduction

Proteases constitute one of the commercially important groups of extracellular microbial

enzymes, accounting for nearly 60% of the whole enzyme market and are frequently used in

detergent, leather, pharmaceuticals, food and biotechnology industries (Vijayaraghavan et al.,

2014). These enzymes are found in a wide diversity of sources such as plants, animals and

microorganisms. Among these sources, the microorganisms show great potential for protease

production due to their broad biochemical diversity and their susceptibility to genetic

manipulation. In addition, microbial proteases are predominantly extracellular and can be

secreted in the fermentation medium, decreasing the requirement of complex steps for enzyme

recovery (Muthulakshmi et al., 2011). It has been estimated that microbial proteases represent

approximately 40% of the total worldwide enzyme sales (Rao et al., 1998).





A. niger is one of the most important sources of fungal proteases. According Pel et al. (2007),


process.

In the past years, new and innovative biotechnological processes have explored solid state

fermentation (SSF) as a promising technology. For the growth of fungi, SSF is an attractive

process because the solid substrates resemble the natural habitat of the fungi and improving their

growth and the secretion of a wide range of extracellular enzymes. Some characteristics make

solid state fermentation more attractive than submerged fermentation: lower risk of

contamination, higher productivity, use of inexpensive substrates, simplicity on downstream

processing, easier separation and purification of products, lower energy requirements and lesser

production of wastewater (Chutmanop et al., 2008; Chen et al., 2014).

The biochemical characterization of enzymes is important to evaluate their

biotechnological potential. The study of the protease properties, such as the substrate specificity,

the optimum catalytic pH conditions, the temperature and stability profiles, and kinetic and

102

thermodynamic characteristics can be used to predict the successful application of the enzyme to

particular industries or processes (Castro and Sato, 2013).

Several studies use the high levels of production or productivity as a criterion for selecting

the most suitable substrate for obtaining of enzymes under solid state fermentation. However, the

expression and secretion of different sets of proteases and other enzymes, as well as their

biochemical properties, can be regulated by the type of substrate used as carbon and nitrogen

source (Speranza et al., 2011; Farnell et al., 2012). In this context, the main objectives of the

present study were to evaluate the proteases production by A. niger LBA02 under solid state

fermentation using different agroindustrial wastes and to determine the biochemical properties of

the proteases produced in each agroindustrial waste, with emphasis on the kinetic and

thermodynamic parameters and substrate specificities. This evaluation provides a new point of

view to select the most suitable substrate for proteases production under solid state fermentation.



Wheat bran, soybean meal and cottonseed meal were kindly provided by Bunge Foods

S/A. Orange peel was purchased from local market of Campinas (Sao Paulo, Brazil). To be used

as matrix support, the orange peel was grinded, washed three times with distilled water and dried

at 50 °C for 24-48 h.

Moisture, protein content, lipids and ash of the agroindustrial wastes were determined by

AOAC methods (AOAC, 2010). The carbohydrate content was determined by difference between

the total value of 100% and the sum of the other components. The tests were performed in

triplicate and the results were expressed as the mean ± standard deviation.


The microorganism used in this study was A. niger LBA02, previously selected as a

proteolytic strain from the culture collection of the Laboratory of Food Biochemistry, School of

Food Engineering, University of Campinas. The strain was periodically subcultured and

maintained on potato dextrose agar slants. To produce fungal spores, the microorganism was

inoculated into a medium composed of 10 g wheat bran and 5 mL of solution containing 1.7%

(w/v) NaHPO4 and 2.0% (w/v) (NH4)2SO4 and incubated for 3 days at 30 °C. The fungal spores

103

were dispensed into sterile Tween 80 solution (0.3%) to prepare the inoculum for fermentation.

The number of spores per milliliter in the spore suspension was determined with a Neubauer cell

counting chamber.


The protease production was performed under solid state fermentation using the individual

substrates and the quaternary mixture of their in equal proportions in 250 mL Erlenmeyer flasks

containing 20 g medium. The cultivation parameters were 50% moisture, temperature set at

30 °C, and an inoculum level of 107 spores g

-1. The protease activity was tested at 24 h intervals

during a 96 h fermentation. The crude extract was obtained by the addition of 100 mL distilled

water. After 1h at rest, the solution was filtered through a filter membrane to obtain an enzyme

solution free of any solid material.









described.

2.5. Activation energy and temperature quotient (Q10)

The activation energy (Ea) was determined by incubating the protease with 0.5%

azocasein at various temperatures ranging from 40 to 60 °C in 50 mM acetate buffer (pH 5.0).

The dependence of the rate constants with temperature was assumed to follow the Arrhenius Law

and Ea was calculated from the slope of the plot of 1000/T vs. ln (protease activity), where

Ea = -slope×R, R (gas constant) = 8.314 J K-1

mol-1

and T is the absolute temperature in Kelvin

(K) (Jakób et al., 2010).

The effect of temperature on the rate of reaction was expressed in terms of temperature

quotient (Q10), which is the factor by which the rate increases due to a rise in the temperature by

104

10 °C. Q10 was calculated by the equation given by Dixon and Webb (1979), as shown in

Equation 1:

Q10 = antilogɛ (Ea × 10/RT2) (1)

2.6. Determination of the kinetic parameters Km and Vmax

Azocasein (pH 5.0) was used over the concentration ranges 1.0-10.0 mg mL-1

to

determine the kinetic parameters Km and Vmax of the proteases from A. niger LBA02. The

Michaelis-Menten constant (Km) and maximum velocity (Vmax) values were determined as the

reciprocal absolute values of the intercepts on the x and y axes, respectively, of the linear

regression curve.

2.7. Determination of kinetic and thermodynamic parameters for thermal inactivation

2.7.1. Kinetic parameters for thermal inactivation

The protease stability as a function of incubation time was evaluated. For this, the enzyme

was incubated for 300 min at temperatures ranging from 40 to 60 °C and the samples were

collected at various times for determination of the residual protease activity. The value of the

deactivation rate constant (kd) for the protease produced in different agroindustrial substrates,

expressed as an exponential decay, was found by plotting ln (A/A0) vs. time using the

experimental data as shown in Equation 2:

A = A0 × e-k

dt (2)

Where t is time, A0 is the initial enzyme activity and A is the enzyme activity at a determined

time t.

The activation energies for denaturation (Ead) of protease were calculated by plotting

ln (kd) vs. −1/RT as shown in Equation 3:

kd = Ae−Ead/RT

(3)

Where R is the universal gas constant = 8.314 J K-1

mol-1

and T is the absolute temperature in

Kelvin (K).

The apparent half-life of the enzyme, defined as the time where the residual activity

reaches 50%, was estimated as shown in Equation 4:

105

t1/2 = ln (0.5) / kd (4)

Decimal reduction time (D value) was defined as the time required for a one-log10

reduction or 90% reduction in the initial enzyme activity at a specific temperature. The D value is

related to the first-order deactivation rate constant (kd) and it was calculated as shown in Equation

5:

D = 2.303 / kd (5)

2.7.2. Thermodynamic parameters for thermal inactivation

Thermodynamic parameters for proteases produced in different agroindustrial substrates

were estimated using the Eyring absolute rate, as shown in Equation 6:

kd = (kB×T/h)×e(-ΔH/RT)

×e(ΔS/R)

(6)

Where, kB is the Boltzmann constant (1.38×10−23

J K-1

); T is the absolute temperature in Kelvin;

h is the Planck constant (6.63 × 10−34

J s); ΔH is the enthalpy of activation, kJ mol-1; and ΔS is

the entropy of activation, J/mol K. The enthalpy of activation, ΔH, given in Equation 7, can be

calculated using the activation energies for denaturation as shown in Equation 3.

Similarly, the free energy of activation, ΔG, can be calculated using Equation 8. Finally,

entropy of activation, ΔS, represented in Equation 9, can be calculated using the enthalpy (ΔH)

and free energies of activation (ΔG).

ΔH = Ead – RT (7)

ΔG = -RT ln [kd×h/kb×T] (8)

ΔS = (ΔH – ΔG)/T (9)

2.8. Substrate specificity of the proteases

The proteases produced by A. niger LBA02 in wheat bran, soybean meal, cottonseed

meal, orange peel and the quaternary mixture were assayed for substrate specificity by using

different substrates: casein, whey protein, soybean protein concentrate, soy protein isolate,

hemoglobin from bovine blood, gelatin and albumin from egg white. The enzyme concentrations

were adjusted to 20.0 U per mL of reaction, according to the activity of each protease, as

previously determined. The substrates were dissolved in 50 mM acetate buffer (pH 5.0) at the

concentration of 10 mg mL-1

. The reaction mixture containing 1.0 mL of each substrate and

106

1.0 mL of the enzyme solution was incubated at 50 °C for 60 min. The reaction was stopped by

adding 1.0 mL 10% TCA. The test tubes were centrifuged at 17,000 x g for 15 min at 5 °C and

the absorbance of the supernatant was measured at 280 nm. The results were expressed as relative

activity (%) using the substrate casein as the standard (100% relative activity).


Values were expressed as the arithmetic mean. The Tukey test was used to check the

significant differences between the groups analyzed. The differences were considered significant

when p-value ≤ 0.05.

Pearson correlation coefficient was used to measure the strength of linear dependence

between two responses. The correlation coefficient ranges from – 1 to 1. A value of 1 implies that

a linear equation describes the relationship between the responses was perfectly and positive,

while a value of -1 indicate a perfectly and negative correlation. A value of 0 implies that there is

no linear correlation between the responses. The correlations between analyzed parameters were

considered significant when the p-value ≤ 0.10.





1. The enzymes production under solid state fermentation can be affected by the composition of

the substrates and various cultivation factors. On protease production, for example, the presence

of protein sources can induce the enzyme secretion by the microorganism. On the other hand, the

substrate must have a carbon to nitrogen ratio (C:N) suitable for the fermentation (Castro and

Sato, 2013). Soybean meal and cottonseed meal were the materials with higher protein content

and wheat bran showed the major C:N ratio (Table 2).

107




tests were applied between the chemical components of each substrate (not between different substrates). Carbohydrate content


Table 2 - Correlation analysis between the ratio (C:N) and the protein content of the

agroindustrial wastes in dry basis with the protease production by A. niger LBA02 under solid

state fermentation at 24, 48, 72 and 96 h.


tests were applied between the runs for each fermentation time (not between different fermentation time). *The correlations

between analyzed parameters were considered significant when the p-value ≤ 0.10.

The Pearson coefficient was used to verify the correlation between the C:N ratio and the

protein content in the substrates with the protease production. The correlation analysis indicated a

positive and significant correlation between the protein content (%) in the substrates and the

protease production at 24 h (Pearson coefficient = 0.80; p = 0.10). In contrast, it was observed a

negative and significant correlation between the C:N ratio and the protease production at 24 h

(Pearson coefficient = -0.80; p = 0.10) and 48 h fermentation (Pearson coefficient = -0.96;

Chemical components Wheat bran Soybean meal Cottonseed meal Orange peel

Moisture (%) 12.77 ± 0.08a 11.93 ± 0.02

a 6.42 ± 0.01

a 7.92 ± 0.08

a

Protein (%) 14.74 ± 0.51b 49.24 ± 0.07

b 25.91 ± 0.60

b 7.01 ± 0.08

b


c 55.80

c 79.68

c

Lipids (%) 4.47 ± 0.22d 1.40 ± 0.02

d 7.83 ± 0.01

d 1.95 ± 0.13

d

Ash (%) 4.98 ± 0.06e 5.90 ± 0.04

e 4.04 ± 0.01

e 3.44 ± 0.11

e

Substrates C:N Protein

content (%)


)

24 h 48 h 72 h 96 h

Wheat bran 4.28 16.89 101.10 ± 5.73a 183.98 ± 5.65

b 178.00 ± 9.17

b 184.08 ± 6.84

a

Soybean meal 0.64 55.91 130.10 ± 3.45b 228.60 ± 7.30

a 85.85 ± 3.10

c 63.77 ± 1.70

b

Cottonseed meal 2.15 27.69 66.18 ± 1.05c 188.03 ± 7.65

b 117.17 ± 7.18

d 125.20 ± 2.28

c

Orange peel 11.37 7.61 40.62 ± 4.54d 36.08 ± 3.33

c 30.70 ± 3.40

e 14.00 ± 1.64

d

Quaternary mixture 2.35 27.03 99.70 ± 1.49a 245.97 ± 13.35

a 198.63 ± 5.32

a 91.13 ± 2.48

e


Pearson coefficient -0.80* -0.96

* -0.56 -0.48

p-value 0.10 0.01 0.32 0.42


Pearson coefficient 0.80* 0.70 0.03 -0.05

p-value 0.10 0.19 0.96 0.94

108

p = 0.01) (Table 2). These results confirm that an adequate supply of proteins as nitrogen source

can induce the protease production at the first hours of fermentation.


production of proteases by filamentous fungi of the genus Aspergillus (Esparza et al., 2011; Leng

and Xu, 2011). It is important to note that a standard assay is essential for comparing the results

of different studies. In several studies, research groups used different methodologies to determine

the protease activities, which may result in differences in the substrates, incubation times, pH and

temperatures of the reaction mixtures. The experimental data variability complicates the

comparisons among different studies. As such, a report of higher protease activities values does

not necessarily suggest a higher production. Negi and Banerjee (2006) observed maximum yields

of protease production by a strain of A. awamori MTCC 6652 using wheat bran as a substrate

under solid state fermentation. The highest secretion of protease was measured to be 1,930 U g-1

.

Chutmanop et al. (2008) studied the protease production under solid state fermentation using

strain of A. oryzae (Ozykat-1) and reported a protease production of ∼1,200 U g−1

within 96 h

fermentation using a substrate mixture of 75% rice bran and 25% wheat bran. Shivakumar (2012)

screened 11 different substrates, including eight cereals and three agroindustrial residues, for

protease production by Aspergillus sp. under solid state fermentation and found that wheat flour,

wheat bran and soy flour proved superior protease production, reaching activities of 320, 280 and

160 U g-1

, respectively.

3.2. Biochemical properties of the proteases from A. niger LBA02

3.2.1. Activation energy and temperature quotient (Q10)

The influence of temperature on activity of the proteases produced in different

agroindustrial wastes were shown in Fig. 1. Accordingly, the proteases exhibited optimal activity

at 50 °C, except the protease POP, which presented maximum activity in a range of temperatures

between 50 and 55 °C. However, a rapid loss of activity was observed over 55 °C.

The activation energies of the proteases were calculated at temperatures between 35 and

65 °C. The Arrhenius plots in temperature range from 35 °C to up to the optimum reaction

temperatures, 55 °C for protease POP and 50 °C for other proteases, showed a linear variation

with temperature increase, suggesting that the proteases from A. niger LBA02 have single

conformations up to the transition temperatures (Fig. 2). Based on Arrhenius plots, inflection

109

points were observed in temperature range from 35 °C to up to the optimum reaction

temperatures and deflections above these points, indicating that catalysis reactions suppress

enzymatic deactivation below the inflection points. Thus, Ea presented positive values at

temperatures ranging from 35 °C to up to the optimum temperature for activity of each enzyme.

The lower Ea value was detected for protease POP (16.32 kJ mol-1

) and the higher for protease

PQM (19.48 kJ mol-1

). Negative Ea values were observed above the optimum temperatures for

activity (Table 3). Melikoglu et al. (2013) reported similar results for a protease from Aspergillus

awamori, which presented activation energies (Ea) for bread protein hydrolysis of 36.8 kJ mol-1

in the temperature range from 30 to 55 °C and − 62.0 kJ mol-1

in the temperature range from 55

to 65 °C.

Fig. 1. Effect of reaction temperature, between 35 and 65 °C with +5 °C increments, on enzyme

activities of the proteases from A. niger LBA02 produced under solid state fermentation using

wheat bran (PWB), soybean meal (PSM), cottonseed meal (PCM), orange peel (POP) and the

quaternary mixture of these agroindustrial wastes (PQM).

The effect of temperature on rate of reaction was measured in terms of temperature

quotient (Q10). The Q10 values ranging from 1.20 to 1.23 between temperatures 35-55 °C for

protease POP, and other proteases showed Q10 values ranging from 1.22 to 1.28 at temperatures

between 35-50 °C (Table 3).

0

50

100

150

200

250

35 40 45 50 55 60 65

Pro

teas

e (U

g-1

)

Temperature ( C)

PWB PSM PCM POP PQM

110

Fig. 2. Arrhenius plots for the determination of activation energies (Ea) of the proteases from A.

niger LBA02 produced under solid state fermentation using wheat bran (PWB), soybean meal

(PSM), cottonseed meal (PCM), orange peel (POP) and the quaternary mixture of these

agroindustrial wastes (PQM).

3.08 3.12 3.16 3.20 3.24 3.28

1000/T (K)

4.7

4.8

4.9

5.0

5.1

ln (

Vm

ax)

3.08 3.12 3.16 3.20 3.24 3.28

1000/T (K)

4.9

5.0

5.1

5.2

5.3

ln (

Vm

ax)

3.08 3.12 3.16 3.20 3.24 3.28

1000/T (K)

4.7

4.8

4.9

5.0

5.1

ln (

Vm

ax)

3.00 3.04 3.08 3.12 3.16 3.20 3.24 3.28

1000/T (K)

3.0

3.1

3.2

3.3

3.4

3.5

ln (

Vm

ax)

3.08 3.12 3.16 3.20 3.24 3.28

1000/T (K)

5.0

5.1

5.2

5.3

5.4

ln (

Vm

ax)

PWB PSM

PCM POP

PQM

111

Table 3 - Activation energies for azocasein hydrolysis and Q10 of the proteases from A. niger

LBA02 produced under solid state fermentation of wheat bran (PWB), soybean meal (PSM),

cottonseed meal (PCM), orange peel (POP) and the quaternary mixture of these substrates in

equal proportions (PQM).

3.2.2. Kinetic parameters Km and Vmax

Enzyme kinetics parameters Km and Vmax were calculated from the Lineweaver-Burk

graphs for the proteases form A. niger LBA02 produced in different agroindustrial wastes and are

presented in Table 4. The Km value for a given enzyme provides an indication of the binding

strength of that enzyme to its substrate, thus, a low Km indicates a higher affinity for the

substrate. The Vmax can be defined as the maximum velocity as the total amount of enzyme

participates in the reaction. This measurement is theoretical and has an approximate value

because at given time, it would require all enzyme molecules to be tightly bound to their

substrates (Bisswanger, 2002; Goyeneche et al., 2013).

The proteases from A. niger LBA02 showed different values for Km and Vmax in

response to the agroindustrial wastes used as the substrates for enzyme production under the solid

Temperature range (°C) PWB

Ea (kJ moL-1

) R² Q10

35-50 17.33 0.99 1.25-1.22

50-65 -63.23 0.96

Temperature range (°C) PSM

Ea (kJ moL-1

) R² Q10

35-50 19.44 0.93 1.28-1.25

50-65 -67.53 0.98

Temperature range (°C) PCM

Ea (kJ moL-1

) R² Q10

35-50 18.38 0.96 1.26-1.24

50-65 -68.19 0.99

Temperature range (°C) POP

Ea (kJ moL-1

) R² Q10

35-55 16.32 0.92 1.23-1.20

55-65 -111.60 0.96

Temperature range (°C) PQM

Ea (kJ moL-1

) R² Q10

35-50 19.48 0.97 1.28-1.25

50-65 -70.28 0.99

112

state fermentation. The greatest affinity for the substrate azocasein was observed for PSM, with

Km value estimated at 0.44 mg mL-1

, followed by PCM, PWB, PQM and POP. The maximum

reaction rate (Vmax) value was 344.83 U g-1

for protease PQM (Table 4). Vmax/Km ratio was

taken as the criterion to evaluate substrates specificity (Altunkaya and Gokmen, 2008). The

higher Vmax/Km ratio was 562.79 U mL g-1

mg-1

for the protease PSM, followed by the

proteases PCM and PQM, which showed values of 225.45 and 212.85 U mL g-1

mg-1

(Table 4).

Table 4 - Kinetic parameters Km and Vmax for the proteases from A. niger LBA02 produced

under solid state fermentation of wheat bran (PWB), soybean meal (PSM), cottonseed meal

(PCM), orange peel (POP) and the quaternary mixture of these substrates in equal proportions

(PQM).

*Protease activity determined using azocasein as the substrate at 50 °C and pH 5.0.

3.2.3. Thermal inactivation

The residual protease activities after treatment at different temperatures are presented in

Fig. 3. It can be observed that the deactivation rates of the proteases depend on the substrate in

which they were produced (Table 5). The proteases from A. niger LBA02 produced in wheat bran

(PWB), orange peel (POP) and the quaternary mixture of substrates (PQM) showed higher

stability in temperatures ranging from 40 to 50 °C, retaining above 70% of the initial activity

after 30 min incubation, but they lose 50-65% of their original activities after 15 min of

incubation at 55 °C. The proteases produced using soybean meal (PSM) and cottonseed meal

(PCM) as substrates presented low stability, reducing about 70% of initial activity in the first

minutes of the heat treatment at 40 °C and up to 90% at temperatures ranging from 55 to 60 °C

(Fig. 3). These data were used to estimate the kinetic and thermodynamic parameters for thermal

inactivation of the proteases using Arrhenius plots (Fig. 4).

Protease Km (mg mL-1

) Vmax (U g-1

) R² Vmax / Km (U mL g-1

mg-1

)

PWB 1.32 248.65 0.98 188.37

PSM 0.44 247.63 0.99 562.79

PCM 1.05 236.70 0.99 225.43

POP 1.92 42.74 0.97 22.26

PQM 1.62 344.83 0.98 212.85

113

Fig. 3. Thermal deactivation of proteases from A. niger LBA02 produced under solid state

fermentation using wheat bran (PWB), soybean meal (PSM), cottonseed meal (PCM), orange

peel (POP) and the quaternary mixture of these agroindustrial wastes (PQM).

0

20

40

60

80

100

0 30 60 90 120

Res

idu

al a

ctiv

ity (

%)

Time (min)

40°C 45°C 50°C 55°C 60°C

0

20

40

60

80

100

0 30 60 90 120

Res

idu

al a

ctiv

ity (

%)

Time (min)

40°C 45°C 50°C 55°C 60°C

0

20

40

60

80

100

0 30 60 90 120

Res

idu

al a

ctiv

ity (

%)

Time (min)

40°C 45°C 50°C 55°C 60°C

0

20

40

60

80

100

0 30 60 90 120

Res

idu

al a

ctiv

ity (

%)

Time (min)

40°C 45°C 50°C 55°C 60°C

0

20

40

60

80

100

0 30 60 90 120

Res

idu

al a

ctiv

ity (

%)

Time (min)

40°C 45°C 50°C 55°C 60°C

PWB PSM

PCM POP

PQM

114

Table 5 - Thermodynamic and kinetic parameters for thermal deactivation of proteases from A.

niger LBA02 produced under solid state fermentation of wheat bran (PWB), soybean meal

(PSM), cottonseed meal (PCM), orange peel (POP) and the quaternary mixture of these substrates

in equal proportions (PQM).

Temperature (°C) PWB

kd (min-1

) t1/2 (min) D (min) R²

Ead (kJ moL-1

) 40 0.0005 1386.29 4605.20 0.95

45 0.0016 433.22 1439.13 0.95

50 0.0051 135.91 451.49 0.94

197.08 55 0.0160 43.32 143.91 0.84

60 0.0463 14.97 49.73 0.97

Temperature (°C) PSM

kd (min-1

) t1/2 (min) D (min) R²

Ead (kJ moL-1

) 40 0.0126 55.01 182.75 0.80

45 0.0162 42.79 142.14 0.89

50 0.0234 29.62 98.40 0.85

74.20 55 0.0401 17.29 57.42 0.90

60 0.0684 10.13 33.66 0.97

Temperature (°C) PCM

kd (min-1

) t1/2 (min) D (min) R²

Ead (kJ moL-1

) 40 0.0124 55.90 185.69 0.77

45 0.0181 38.30 127.22 0.85

50 0.0248 27.95 92.85 0.88

69.21 55 0.0476 14.56 48.37 0.95

60 0.0562 12.33 40.97 0.88

Temperature (°C) POP

kd (min-1

) t1/2 (min) D (min) R²

Ead (kJ moL-1

) 40 0.0019 364.81 1211.89 0.94

45 0.0053 130.78 434.45 0.98

50 0.0067 103.45 343.67 0.97

107.92 55 0.0122 56.82 188.74 0.97

60 0.0281 24.67 81.94 0.99

Temperature (°C) PQM

kd (min-1

) t1/2 (min) D (min) R²

Ead (kJ moL-1

) 40 0.0011 630.13 2093.27 0.92

45 0.0023 301.37 1001.13 0.96

50 0.0086 80.60 267.74 0.98

178.48 55 0.0335 20.69 68.73 0.97

60 0.0491 14.12 46.90 0.97

115

Fig. 4. Pseudo-first-order plots for irreversible thermal denaturation of proteases from A. niger

LBA02 produced under solid state fermentation using wheat bran (PWB), soybean meal (PSM),

cottonseed meal (PCM), orange peel (POP) and the quaternary mixture of these agroindustrial

wastes (PQM).

-2

-1

0

1

2

3

4

5

0 15 30 45 60 75 90 105 120

ln (

Ut/

U0)

Time (min)

40°C 45°C 50°C 55°C 60°C

-5

-4

-3

-2

-1

0

1

2

3

4

5

0 15 30 45 60 75 90 105 120

ln (

Ut/

U0)

Time (min)

40°C 45°C 50°C 55°C 60°C

-4

-3

-2

-1

0

1

2

3

4

5

0 15 30 45 60 75 90 105 120

ln (

Ut/

U0)

Time (min)

40°C 45°C 50°C 55°C 60°C

1

2

3

4

5

0 15 30 45 60 75 90 105 120

ln (

Ut/

U0)

Time (min)

40°C 45°C 50°C 55°C 60°C

-2

-1

0

1

2

3

4

5

0 15 30 45 60 75 90 105 120

ln (

Ut/

U0)

Time (min)

40°C 45°C 50°C 55°C 60°C

PWB PSM

PCM POP

PQM

116

The half-life (t1/2) of an enzyme, at a given temperature, is the time it takes for the activity

to reduce to a half of its original/initial activity. The decimal reduction time (D value) is defined

as the time required for a 90% reduction in the initial enzyme activity. Higher values of these

parameters at the specific operating temperature are important and desirable for industrial

applications since indicate the resistance of the enzyme to thermal inactivation. The protease

PWB showed the highest thermal resistance when compared to the other proteases preparations,

reaching D values ranging from 4,605.20 to 49.73 min, t1/2 ranging from 1,386.29 to 14.97 min at

temperatures between 40 and 60 C. It’s important to note that the protease PWB showed the

highest thermal resistance at the optimum temperature for activity (50 °C), presenting D value

and t1/2 estimated in 451.49 and 135.91 min, respectively. It can be seen that the inactivation rate

constants (kd) increased with increased temperature for all proteases. The kd values ranged from

slowest for protease PWB of 5×10-3

min-1

at 40 °C to fastest for protease PSM of 0.0684 min-1

at

60 °C (Table 4). The Ead for proteases deactivation were calculated using Arrhenius plots (Fig. 5)

and showed values in the order of PCM < PSM < POP < PQM < PWB, indicating thermal

stability in reverse order (Table 5). A neutral protease produced by Aspergillus oryzae CICIM

F0899 was kinetically characterized and the results showed half-lives (t1/2) of 20.4 and 14.2 min

at 55 and 60 °C, respectively (Wang et al., 2013). Sant’Anna et al. (2013) studied the kinetic

modeling of thermal inactivation of a protease from Bacillus sp. and showed D values of 432.54,

131.41, 17.98 and 7.82 min at 45, 50, 55 and 65 °C, respectively.

The stability of a protein is the result of a balance between stabilizing and destabilizing

forces, which are influenced by hydrophobic and electrostatic interactions, hydrogen and

disulfide bonds and folding degree of the molecule (Ortega et al., 2004). Thus, the investigation

of thermodynamic parameters such as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) of the

proteases from A. niger LBA02 was performed to understand the behavior of these molecules in

different conditions and the results are given in Table 6. These thermodynamic parameters varied

widely when the proteases produced in different agroindustrial wastes were compared.

117

Fig. 5. Arrhenius plots to calculate activation energy ‘Ea(d)’ for irreversible thermal

inactivation/denaturation of proteases from A. niger LBA02 produced under solid state



3.00 3.05 3.10 3.15 3.20

1000/T (K)

-4.4

-4.0

-3.6

-3.2

-2.8

ln (

kd)

3.00 3.05 3.10 3.15 3.20

1000/T (K)

-4.4

-4.0

-3.6

-3.2

-2.8

ln (

kd)

3.00 3.05 3.10 3.15 3.20

1000/T (K)

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

-3.0

ln (

kd)

3.00 3.05 3.10 3.15 3.20

1000/T (K)

-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

-3.0

-2.5

ln (

kd)

3.00 3.05 3.10 3.15 3.20

1000/T (K)

-8.0

-7.5

-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

-3.0ln

(k

d)

PWB PSM

PCM POP

PQM

118

Table 6 - Thermodynamic parameters for thermal deactivation of proteases from A. niger LBA02

produced under solid state fermentation of wheat bran (PWB), soybean meal (PSM), cottonseed

meal (PCM), orange peel (POP) and the quaternary mixture of these substrates in equal

proportions (PQM).

Temperature (°C) PWB

ΔH (kJ moL-1

) ΔG (kJ moL-1

) ΔS (J moL-1

K-1

)

40 194.48 107.28 278.45

45 194.43 105.95 278.12

50 194.39 104.55 278.02

55 194.35 103.09 278.11

60 194.31 101.76 277.80

Temperature (°C) PSM

ΔH (kJ moL-1

) ΔG (kJ moL-1

) ΔS (J moL-1

K-1

)

40 71.60 98.87 -87.09

45 71.55 99.83 -88.87

50 71.51 100.45 -89.55

55 71.47 100.58 -88.70

60 71.43 100.68 -87.80

Temperature (°C) PCM

ΔH (kJ moL-1

) ΔG (kJ moL-1

) ΔS (J moL-1

K-1

)

40 66.61 98.91 -103.16

45 66.56 99.53 -103.61

50 66.52 100.30 -104.52

55 66.48 100.11 -102.48

60 66.44 101.22 -104.40

Temperature (°C) POP

ΔH (kJ moL-1

) ΔG (kJ moL-1

) ΔS (J moL-1

K-1

)

40 105.32 103.80 4.84

45 105.27 102.79 7.82

50 105.23 103.81 4.39

55 105.19 103.83 4.16

60 105.15 103.14 6.04

Temperature (°C) PQM

ΔH (kJ moL-1

) ΔG (kJ moL-1

) ΔS (J moL-1

K-1

)

40 175.88 105.22 225.62

45 175.83 104.99 222.66

50 175.79 103.14 224.82

55 175.75 101.07 227.58

60 175.71 101.59 222.47

119

ΔH is seen as a measure of the number of non-covalent bounds broken in forming a

transition state for enzyme inactivation. In general, large values of ΔH are associated with

increased enzyme stability (Olusesan et al., 2011; Batista et al., 2014). ΔH showed the lowest

value (66.44 kJ mol-1

) at 60 °C for protease PCM and the highest (194.48 kJ mol

-1) for protease

PWB at 40 C. Gibbs free energy (ΔG) measures the spontaneity of a reaction. Therefore, the

protein stability is directly related to ΔG values, where high ΔG values indicate higher thermal

stability of the enzyme (Batista et al., 2014). The higher ΔG values were observed for protease

PWB, which presented ΔG of 107.28 kJ mol-1

at 40 °C and 101.76 kJ mol-1

at 60 °C, followed by

proteases PQM and POP (Table 5). ΔS represent the variation in the extent of local disordering

between transition state and the ground state (Subhedar and Gogate, 2014). Thus, an increase in

ΔS implies an increase in the number of protein molecules in the transition active state and

increase in disorder (of the active site or of the structure), which is the main driving force of heat

denaturation (Singh and Chhatpar, 2011; Melikoglu et al., 2013). According to Olusesan et al.

(2011), positive ΔS values are found if the rate-limiting reaction is the protein unfolding, as result

of moderately high values of ΔH and low values of ΔG. On the other hand, negative ΔS values

are result of moderately low values of ΔH and high values of ΔG, which is in well agreement

with the present study. The proteases PSM and PCM presented negative values for ΔS, which

indicate that the rate-limiting reaction probably involves the aggregation of partially unfolded

enzyme molecules which predominate during the exposure of protein to high temperatures

(Olusesan et al., 2011).

3.2.4. Substrate specificity of the enzyme

The activities of the proteases from A. niger LBA02 produced in wheat bran (PWB),

soybean meal (PSM), cottonseed meal (PCM), orange peel (POP) and the quaternary mixture of

these substrates (PQM) against various proteinaceous substrates were examined (Table 7). PWB,

PSM and PCM showed a high level of catalytic activity against all substrates evaluated, with

relative activities superior to casein, used as standard (100% relative activity). However, the

protease POP presented the lower proteolytic activities against the substrates, reaching a

maximum relative activity of 83.32% for hemoglobin from bovine blood and a minimum value of

22.39% for soy protein concentrate. It can be noted that the substrate specificity demonstrated

different values in response to the different agroindustrial wastes used for protease production.

The use of quaternary mixture of agroindustrial wastes for enzymes production resulted in

120

proteases with higher catalytic activities for the substrates hemoglobin from bovine blood, gelatin

and egg albumin compared to the proteases obtained by fermentation of the isolated

agroindustrial wastes. The best substrate for the proteases was hemoglobin from bovine blood

with the highest relative activities of 183.84, 147.06, 186.81, 83.32 and 496.47% for PWB, PSM,

PCM, POP and PQM, respectively (Table 7).

Table 7 - Substrate specificity of the proteases from A. niger LBA02 produced under solid state




tests were applied between the relative activities of substrates for each protease preparation (not between different proteases).

Yossan et al. (2006) reported that cytochrome C, soybean protein isolate and casein were

good substrates for protease from Bacillus megaterium with the relative protease activity of 114,

109 and 100%, respectively. A protease from Aspergillus sp. showed relative protease activity of

130, 121 and 114% for gelatin, casein and egg albumin, respectively, when bovine serum

albumin was used as control (100%) (Shivakumar et al., 2012).

4. Conclusions

The results obtained in our study showed that the protease production by A. niger

LBA02 and the biochemical properties of these proteases can be regulated by the agroindustrial

waste used as the substrate in solid state fermentation. Higher levels of protein in the

agroindustrial wastes showed as important factor, inducing the protease production in the first 24

h of fermentation. Kinetic and thermodynamic parameters indicated that the enzymes showed

different profiles for thermal inactivation, where the proteases produced in wheat bran (PWB)

Substrate Relative activity (%)

PWB PSM PCM POP PQM

Casein 100.00 ± 4.37a 100.00 ± 6.39a 100.00 ± 1.33a 100.00 ± 3.01a 100.00 ± 8.88a

Whey protein 106.26 ± 0.36b 102.21 ± 2.60a 108.79 ± 4.63b, e 57.99 ± 0.27b 96.83 ± 2.66a

Soy protein concentrate 126.26 ± 9.08c, d, f 105.88 ± 6.11a, b 108.79 ± 2.05b 22.39 ± 2.24c 30.69 ± 1.84b

Soy protein isolate 126.26 ± 2.79c 101.47 ± 3.39a 106.59 ± 3.66b 42.08 ± 2.75d 37.04 ± 5.52b

Hemoglobin from

bovine blood 183.84 ± 2.22e 147.06 ± 2.97c 186.81 ± 1.50d 83.32 ± 7.04e 496.47 ± 3.58c

Gelatin 115.15 ± 5.47d 114.71 ± 2.22b 114.29 ± 0.81e 54.98 ± 3.37b 179.89 ± 4.76d

Egg albumin 137.37 ± 3.42f 112.50 ± 1.77b 127.47 ± 2.12f 50.42 ± 8.04b, d 215.17 ± 2.41e

121

and using the quaternary mixture of the substrates (PQM) presented the most thermal resistance,

while the protease produced in cottonseed meal (PCM) had the lowest thermal resistance. The

protease produced using orange peel as the substrate (POP) showed the lowest value for

activation energy, indicating that there was a lower energy requirement for azocasein hydrolysis.

The proteases produced in different substrates also exhibit large differences for the kinetic

parameters Km and Vmax and substrate specificities. Our study showed interesting results about

the secretion of proteases by A. niger LBA02 in response to different agroindustrial wastes,

providing a new point of view for enzymes production under solid state fermentation, such as

selecting of the most suitable substrate for obtaining enzymes with more attractive properties.

Acknowledgements

The work described in this paper was substantially supported by the Department of Food

Science, School of Food Engineering, University of Campinas, which are gratefully

acknowledged. Acknowledgements to the National Counsel of Technological and Scientific

Development – CNPq by the granting of scholarship.

References

Altunkaya, A., Gokmen, V., 2008. Effect of various inhibitors on enzymatic browning,

antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa). Food Chem., 107,

1173-1179.



DC.

Batista, K.A., Batista, G.L.A., Alves, G.L., Fernandes, K.F., 2014. Extraction, partial purification

and characterization of polyphenol oxidase from Solanum lycocarpum fruits. J. Mol. Catal. B:

Enzym., 102, 211–217.

Bisswanger, H., 2002. Enzyme kinetics: principles and methods, third ed., Wiley-VCH,

Weinheim.




122

Charney, J., Tomarelli, R.M., 1947. A colorimetric method for the determination of the


Chen, H.-Z., Liu, Z.-H., Dai, S.-H., 2014. A novel solid state fermentation coupled with gas

stripping enhancing the sweet sorghum stalk conversion performance for bioethanol. Biotechnol.

Biofuels, 7, 1-13.


Aspergillus oryzae in solid-state fermentation using agroindustrial substrates, J. Chem. Technol.

Biotechnol., 83, 1012-1018.

Dixon, M., Webb, E.C., 1979. Enzyme kinetics, third ed., Academic Press, New York.

Esparza, Y., Huaiquil, A., Neira, L., Leyton, A., Rubilar, M., Salazar, L., Shene, C., 2011.

Optimization of process conditions for the production of a prolylendopeptidase by Aspergillus

niger ATCC 11414 in solid state fermentation. Food Sci. Biotechnol., 20, 1323-1330.

Farnell, E., Rousseau, K., Thornton, D.J., Bowyer, P., Herrick, S.E., 2012. Expression and

secretion of Aspergillus fumigatus proteases are regulated in response to different protein

substrates. Fungal Biol. 116, 1003-1012.

Goyeneche, R., Di Scala, K., Rour, S., 2013. Biochemical characterization and thermal

inactivation of polyphenol oxidase from radish (Raphanus sativus var. sativus). LWT - Food Sci.

Technol., 54, 57-62.

Jakób, A., Bryjak, J., Wójtowicz, H., Illeová, V., Annus, J., Polakovic, M., 2010. Inactivation

kinetics of food enzymes during ohmic heating. Food Chem., 123, 369-376.

Leng, Y., Xu, Y., 2011. Improvement of acid protease production by a mixed culture of

Aspergillus niger and Aspergillus oryzae using solid-state fermentation technique. African J.

Biotechnol., 10, 6824-6829.

Melikoglu, M., Lina, C.S.K., Webb, C., 2013. Kinetic studies on the multi-enzyme solution

produced via solid state fermentation of waste bread by Aspergillus awamori. Biochem. Eng. J.,

80, 76–82.

123

Muthulakshmi, C., Gomathi, D., Kumar, D.G., Ravikumar, G., Kalaiselvi, M., Uma, C., 2011.

Production, purification and characterization of protease by Aspergillus flavus under solid state

fermentation. Jordan J. Biol. Sci., 4, 137-148.

Negi, S., Benerjee, R., 2006. Optimization of amylase and protease production from Aspergillus

awamori in single bioreactor through EVOP factorial design technique. Food Technol.

Biotechnol. 44, 257–261.

Olusesan, A.T., Azura, L.K., Forghani, B., Bakar, F.A., Mohamed, A.K.S., Radu, S., Manap,

M.Y.A., Saari, N., 2011. Purification, characterization and thermal inactivation kinetics of a non-

regioselective thermostable lipase from a genotypically identified extremophilic Bacillus subtilis

NS 8, New Biotechnol., 28, 738-745.

Ortega, N., Diego, S., Perez-Mateos, M., Busto, M.D., 2004. Kinetic properties and thermal

behaviour of polygalacturonase used in fruit juice clarification. Food Chem., 88, 209–217.

Pel, H.J. et al., 2007. Genome sequencing and analysis of the versatile cell factory Aspergillus

niger CBS 513.88, Nature Biotechnol., 25, 221-231.


biotechnological aspect of microbial proteases. Microbiol. Mol. Biol. Rev., 62, 597–635.

Sant’Anna, V., Corrêa, A.P.F., Daroit, D.J., Brandelli, A., 2013. Kinetic modeling of thermal

inactivation of the Bacillus sp. protease P7. Bioproc. Biosyst. Eng., 36, 993–998.

Schuster, E., Dunn-Coleman, N., Frisvad, J.C., van Dijck, P.W., 2002. On the safety of



Aspergillus sp. by solid substrate fermentation. Archives Appl. Sci. Res., 4, 188-199.

Singh, A.K., Chhatpar, H.S., 2011. Purification, characterization and thermodynamics of

antifungal protease from Streptomyces sp. A6. J Basic Microbiol. 51, 424–432.

Speranza, P., Carvalho, P.O., Macedo, G.A., 2011. Effects of different solid state fermentation

substrate on biochemical properties of cutinase from Fusarium sp., J. Mol. Catal. B: Enzym., 72,

181-186.

124

Subhedar, P.B., Gogate, P.R., 2014. Enhancing the activity of cellulase enzyme using ultrasonic

irradiations. J. Mol. Catal. B: Enzym., 101, 108– 114.

Vijayaraghavan, P., Lazarus, S., Vincent, S.G.P., 2014. De-hairing protease production by an

isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: biosynthesis

and properties. Saudi J. Biol. Sci., 21, 27–34.

Wang, D., Zheng, Z.Y., Feng, J., Zhan, X.B., Zhang, L.M., Wu, J.R., Lin, C.C., Zhu, L., 2013.

Influence of sodium chloride on thermal denaturation of a high-salt-tolerant neutral protease from

Aspergillus oryzae. Food Sci. Biotechnol., 22, 1359-1365.

Yossan, S., Reungsang, A., Yasuda, M., 2006. Purification and characterization of alkaline

protease from Bacillus megaterium isolated from Thai fish sauce fermentation process. Sci. Asia,

32, 377-383.

125

Capítulo VI: Production, biochemical properties of proteases secreted by

Aspergillus niger under solid state fermentation in response to different

agroindustrial substrates and their application for production of whey

protein hydrolysates with antioxidant activities

Revista: Biocatalysis and Agricultural Biotechnology

126

Abstract

This study reports the proteases production by Aspergillus niger LBA02 under solid state

fermentation (SSF) using different agroindustrial wastes as substrates and the correlation between

the protease production and some physical-chemical parameters. The biochemical properties of

the proteases produced in each substrate and their application in enzymatic hydrolysis of bovine

whey protein for obtaining antioxidant hydrolysates were further investigated. The highest

protease production was obtained using wheat bran as the substrate at 96 h fermentation. The

results for chemical composition showed that the substrates with higher protein content induced

the protease production at the first 48 h of fermentation. The crude extracts of proteases from A.

niger LBA02 produced in wheat bran (PWB), soybean meal (PSM) and cottonseed meal (PCM)

showed different biochemical properties. The biochemical characterization showed that the

enzymes were most active over the pH range 3.0-4.0 and was stable from pH 2.5-4.5. The

optimum temperature for activity was approximately 50 °C, and the enzymes were stable at 40-

50 °C. The PWB showed higher ratio milk-clotting/protease activities (15.24) compared to PSM

(0.38) and PCM (6.28). Bovine whey protein hydrolysates showed different antioxidant activities

in response to each protease. The highest DPPH radical scavenging was observed for PCM

hydrolysates while PWB hydrolysates showed maximum activity in total antioxidant capacity

assay.

Keywords: protease; solid state fermentation; agroindustrial wastes; biochemical properties;

enzymatic hydrolysis; antioxidant activities.

127

1. Introduction

Proteases are multifunctional enzymes accounting for nearly 60% of the whole enzyme

market and are frequently used in detergent, leather, pharmaceuticals, food and biotechnology

industries (Ramakrishna et al., 2010; Yin et al., 2013). They can be isolated from plants, animals

and microorganisms. Of these sources, the microorganisms show great potential for protease

production due to their broad biochemical diversity and their susceptibility to genetic

manipulation. It has been estimated that microbial proteases represent approximately 40% of the

total worldwide enzyme sales (Rao et al., 1998).





A. niger is one of the most important sources of fungal proteases. According to Pel et al., (2007),


process.

Proteolytic enzymes can be produced by submerged and solid state fermentation. For the

growth of fungi, solid state fermentation is most appropriate method because the solid substrates

resemble the natural habitat of the fungi and improving their growth and the secretion of a wide

range of extracellular enzymes. Some characteristics make solid state fermentation more

attractive than submerged fermentation: simplicity, low cost, high yields and concentrations of

the enzymes and the use of inexpensive and widely available agricultural residues as substrates

(Chutmanop et al., 2008).

The biochemical characterization of enzymes is important to evaluate their biotechnological

potential. The study of the protease properties, such as the substrate specificity, the optimum

catalytic pH conditions and the temperature and stability profiles, can be used to predict the

successful application of the enzyme to particular industries or processes (Castro and Sato,

2014a). Previous work has shown that the expression and secretion of different sets of proteases

and other enzymes, as well as their biochemical properties, can be regulated by the type of

substrate used as carbon and nitrogen source (Speranza et al., 2011; Farnell et al., 2012).

128

The application of proteases to the hydrolysis of animal and plant proteins to increase their

biological and functional properties has attracted much attention. The antioxidant activities of

protein hydrolysates are extensively reported in several studies. It is postulated that the

antioxidant characteristics of peptides comes from their abilities to inactivate reactive oxygen

species (ROS), scavenge free radicals, chelate prooxidative transition metals, and reduce

hydroperoxides (Zhou et al., 2012). Thus, studies of the application of new proteolytic enzyme

sources are critical to advancing the knowledge concerning bioactive peptides.

In this context, the main objectives of the present study were to evaluate the production of

the protease by A. niger LBA02 under solid state fermentation using different agroindustrial

wastes and to verify the correlation between some physical–chemical parameters, including

chemical composition, water absorption index, particle size and packing density with the protease

production. In addition, the biochemical properties of the proteases produced in each

agroindustrial waste, including the optimum pH and temperature for activity and stability and

milk-clotting activities were investigated. After the biochemical characterization, the application

of the different preparations of proteases to protein hydrolysis for the study of the antioxidant

properties of the hydrolysates was evaluated.


2.1. Physical–chemical characterization of the agroindustrial wastes

2.1.1. Chemical composition of the agroindustrial wastes

Moisture, protein content, lipids and ash of the agroindustrial wastes wheat bran, soybean

meal and cottonseed meal were determined by AOAC methods (AOAC, 2010). The carbohydrate

content was determined by difference between the total value of 100% and the sum of the other

components. The tests were performed in triplicate and the results were expressed as the mean ±

standard deviation.

2.1.2. Determination of the water absorption index (WAI) of the agroindustrial wastes

Their water absorption index (WAI) of the agroindustrial wastes was determined using the

method of Anderson et al. (1969) with slight modifications. Briefly, the sample (1.25 g) was

suspended in 15 mL of distilled water in a 50 mL centrifuge tube. The slurry was manually

stirred for 1 min at room temperature (25 °C) and centrifuged at 8000 x g and 25 °C for 15 min.

129

The supernatants were discarded, and the WAI was calculated from the weight of the remaining

gel and expressed as g gel g-1

dry weight.

2.1.3. Particle size

The particle sizes of the agroindustrial wastes were determined using AOAC method

965.22 (AOAC, 2010). The sieves used had the following opening values: 1.680, 0.841, 0.595,

0.250, 0.177 and 0.149 mm. One hundred g of the material were transferred to top of set of sieves

(opening value: 1.680 mm) assembled and fixed in a sieve shaker (Telastem, Produtest Model T,

Sao Paulo, Brazil). The sieves were kept under constant shaking at 3,600 vpm for 5 minutes to

separate the fractions and the retained material on each sieve was weighed. The experiments were

performed in triplicate and the results expressed as percentage.

2.1.4. Packing density

The packing densities of the agroindustrial wastes were quantified by apparent densities

using the dry substrates and the substrates with initial moisture adjusted to 50%. One hundred g

of each sample was transferred to standard graduated plastic cylinders and vertically agitated

until no change in volume. Apparent densities were calculated as the ratio between the sample

mass and its total volume and expressed in g cm-³.


The strain used in this study was A. niger LBA02, previously selected as a proteolytic

strain from the culture collection of the Laboratory of Food Biochemistry, School of Food

Engineering, University of Campinas. The strain was periodically subcultured and maintained on

potato dextrose agar slants. To produce fungal spores, the microorganism was inoculated into a

medium composed of 10 g wheat bran and 5 mL of solution containing 1.7% (w/v) NaHPO4 and

2.0% (w/v) (NH4)2SO4 and incubated for 3 days at 30 °C. The fungal spores were dispensed into

sterile Tween 80 solution (0.3%) to prepare the inoculum for fermentation. The number of spores

per milliliter in the spore suspension was determined with a Neubauer cell counting chamber.

2.3. Determination of the microorganism growth: radial growth rate and biomass

estimation by glucosamine level

The assays for determination of the microorganism growth rate were performed in Petri

dishes. The agroindustrial wastes have the initial humidity adjusted to 50% and an aliquot of

130

100 µL spore suspension containing 107 spores mL

-1 was inoculated in the central region of the

Petri dishes containing the substrates. The dishes were incubated at 30 °C for the time required to

completely cover all materials. The fungal radial growth was monitored and the results were

expressed in mm.

Fungal biomass estimation was carried out according to described by Ramachandran et al.

(2005) with slight modifications. This determination was based in the N-acetyl glucosamine

released by the acid hydrolysis of the chitin, present in the cell wall of the fungi. Dried fermented

sample (0.5 g) was mixed with concentrated sulphuric acid (2.0 mL) and the reaction mixture was

kept for 24 h at 30 °C. The mixture was diluted with 10 mL of distilled water, autoclaved at 15

psi pressure for l h and filtered through a filter membrane to obtain a solution free of any solid

material. The filtered solution was neutralized with 5 M NaOH and made to 50 mL with distilled

water. The reaction mixture containing 1.0 mL of the sample (resulting from the extractions

described above) and 1.0 mL of acetyl acetone reagent (1.0 mL of acetyl acetone and 50 mL of

0.5 M sodium carbonate solution) was incubated for 20 min in a boiling water bath. After

cooling, a 6.0 mL aliquot of ethanol was added followed by the addition of 1.0 mL Ehrlich

reagent (2.67 g of p-dimethylamino benzaldehyde in 1:1 mixture of analytical reagent grade

ethanol and concentrated hydrochloric acid and made up to 100 mL) and incubated at 65 °C for

10 min. After cooling, the absorbance of the reaction mixture was read at 530 nm against the

blank reagent. The reference standard was a glucosamine (Sigma) solution prepared daily in

distilled water and diluted (14.0–2.0 mg L-1

) for the preparation of the standard curve. The

biomass estimation was tested at 12 h intervals during 96 h fermentation and the results were

expressed as mg glucosamine per gram dry substrate.


Wheat bran, soybean meal, and cottonseed meal were kindly provided by Bunge Foods

S/A. These agroindustrial wastes were used for the protease production by A. niger LBA02. The

protease production was performed under solid state fermentation in 250 mL Erlenmeyer flasks

containing 20 g medium. The cultivation parameters were 50% moisture, temperature set at

30 °C, and an inoculum level of 107 spores g

-1. The protease activity was tested at 24 h intervals

during a 96 h fermentation. The crude extract was obtained by the addition of 150 mL distilled

131

water. After 1h at rest, the solution was filtered through a filter membrane to obtain an enzyme

solution free of any solid material.

2.5. Effects of pH and temperature on the activity and stability of the protease

determined using an experimental design

The optimum pH and temperature for activity and stability were determined using a

central composite rotatable design (CCRD) with three replicates at the central point and four

axial points (a total of 11 runs). To study the protease stability, the enzyme was incubated for 1 h

at various pH values and temperatures.

The experiments were randomized to maximize the variability in the observed responses

caused by extraneous factors. A second-order model equation was used for this model,

represented by Equation 1:

𝑌 = 𝛽0 + 𝛽𝑖𝑥𝑖 + 𝛽𝑖𝑗𝑥𝑖𝑥𝑗

𝑛

𝑗=𝑖+1

𝑛−1

𝑖=1

𝑛

𝑖=1

(1)

where Y is the estimated response, i and j equal values from 1 to the number of variables (n), β0 is

the intercept term, βi values are the linear coefficients, βij values are the quadratic coefficients,

and xi and xj are the coded independent variables. The coefficient of determination R2 and the F

test [analysis of variance (ANOVA)] were used to verify the quality of the fit of the second-order

model equation. The relationships between the responses and the variables were determined using

the StatisticaTM

10.0 software package from Statsoft Inc.









described.

132

2.7. Determination of milk-clotting activity

Milk-clotting activity was determined according to the methods described by Ahmed et

al., (2009). The substrate skim milk was dissolved in 200 mM phosphate buffer pH 6.5

containing 0.01 M CaCl2 at a final concentration of 100 mg mL-1

. A 2.0 mL aliquot of the

substrate was pre-incubated for 10 min at 37 °C, to which 0.2 mL of enzyme solutions were

added, and the curd formation was observed while manually rotating the test tube from time to

time. A comparative evaluation was performed using different preparations of commercial

proteases. The identification of discrete particles indicated the end point of the reaction. One

milk-clotting unit is defined as the amount of enzyme that clots 10 mL of the substrate within 40

min.

MCA (U mL-1

) = (2400 / clotting time (s)) × dilution factor.

2.8. Application of the proteases to protein hydrolysis

Bovine whey protein was used as the substrate for enzymatic hydrolysis and was kindly

provided by Alibra Ingredients Ltd. (Campinas, Brazil). The crude extracts of proteases produced

in wheat bran, soybean meal and cottonseed meal by A. niger LBA02 were concentrated by

ammonium sulfate (80%) precipitation, dialysis and freeze-drying. The partial purified

preparations were used for protein hydrolysis experiments. The enzyme concentrations were

adjusted to 50 U per mL of reaction mixture. The proteins were suspended in acetate buffer to a

final concentration of 100 mg mL-1

, and 50 mL aliquots of the mixtures were distributed in 125

mL Erlenmeyer flasks. Hydrolysis was performed at the optimum temperature and pH value of

the enzyme activity for 240 min. After hydrolysis, the samples were incubated in a water bath at

100 °C for 20 min for protease inactivation. The mixtures were centrifuged at 17,000 x g at 5 °C

for 20 min, and the supernatants containing the peptides were collected and freeze-dried for the

determination of their antioxidant activities and functional properties.

133

2.9. Determination of antioxidant activities

2.9.1. DPPH radical-scavenging activity

The DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, Steinheim, Germany)

radical-scavenging activity of the protein hydrolysates was determined as described by Bougatef

et al. (2009). A 500 µL aliquot of the protein hydrolysates at different concentrations was mixed

with 500 µL 99.5% ethanol and 125 µL 0.02% DPPH in 99.5% ethanol. The mixture was then

kept at room temperature in the dark for 60 min, and the reduction of the DPPH radical was

measured at 517 nm using a UV-visible spectrophotometer (Beckman DU 70 spectrophotometer,

Beckman-Coulter, Inc., Fullerton, CA, USA). The DPPH radical-scavenging activity was

calculated as follows:

The control reaction was performed in the same manner, except that distilled water was

used instead of sample. The tests were performed in triplicate.

2.9.2. Total antioxidant capacity

Total antioxidant capacity of the hydrolysates was performed according to the method

described by Prieto et al., (1999). An aliquot of 0.1 mL of the protein hydrolysates solutions at 10

mg mL-1

was mixed with 1.0 mL of the reagent solution containing 0.6 M sulphuric acid, 28 mM

sodium phosphate and 4 mM ammonium molybdate. The reaction mixtures were then incubated

at 90 °C and kept in the dark for 90 min. The samples were cooled to room temperature and the

absorbance was measured at 695 nm. An appropriate control was prepared with 1.0 mL of the

reagent solution and 0.1 mL distilled water. The results were expressed in function of the

absorbance considering that the absorbance was directly proportional to the total antioxidant

capacity.


Values are expressed as the arithmetic mean. The Tukey test was used to check the

significant differences between the groups analyzed. The differences were considered significant

when p-value ≤ 0.05.

𝑅𝑎𝑑𝑖𝑐𝑎𝑙 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 % = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ∗ 100

134






considered significant when the p-value ≤ 0.10.





1. The enzymes production under solid state fermentation can be affected by the composition of

the substrates and various cultivation factors. The presence of protein sources can induce the

enzyme secretion by the microorganism. On the other hand, the substrate must have a carbon to

nitrogen ratio (C:N) suitable for the fermentation (Castro and Sato, 2014a). Soybean meal and

cottonseed meal were the materials with higher protein content and wheat bran showed the major

C:N ratio (Table 1).




tests were applied between the chemical components for each substrate (not between different substrates). Carbohydrate content


The Pearson coefficient was used to verify the correlation between the C:N ratio and the

protein content in the substrates with the protease production. The correlation analysis indicated a

strong, positive and significant correlation between the protein content (%) in the substrates and

the protease production at 24 h (Pearson coefficient = 0.99; p = 0.08) and 48 h fermentation

(Pearson coefficient = 0.99; p = 0.06). In contrast, it was observed a negative and significant

Chemical components Wheat bran Soybean meal Cottonseed meal

Moisture (%) 12.77 ± 0.08a 11.93 ± 0.02

a 6.42 ± 0.01

a

Protein (%) 14.74 ± 0.51b 49.24 ± 0.07

b 25.91 ± 0.60

b


c 55.80

c

Lipids (%) 4.47 ± 0.22d 1.40 ± 0.02

d 7.83 ± 0.01

d

Ash (%) 4.98 ± 0.06e 5.90 ± 0.04

e 4.04 ± 0.01

e

135

correlation between the C:N ratio and the protease production at 24 h fermentation (Pearson

coefficient = -0.98; p = 0.10) (Table 2). These results confirm that an adequate supply of proteins

as nitrogen source can induce the protease production at the first hours of fermentation.

Table 2 – Correlation analysis between the ratio (C:N) and the protein content of the

agroindustrial wastes with the protease production by A. niger LBA02 under solid state

fermentation at 24, 48, 72 and 96 h.

*The correlations between analyzed parameters were considered significant when the p-value ≤ 0.10.

3.2. The influence of the water absorption index (WAI) on protease production

The WAI indicates the quantity of water that can be absorbed by the support. Materials

with high WAI are preferred for solid state fermentation since their moisture content can be

modified during the solid state culturing (Robledo et al., 2008). Wheat bran showed higher WAI

values; whereas the soybean meal showed the lowest WAI values (Fig. 1a). The highest values

for production of protease were observed in the substrates with higher WAI values, indicating a

positive impact of this physical–chemical parameter on enzymes production, probably caused by

the maintenance of the moisture throughout the fermentation process (Fig. 1a). Orzua et al.,

(2009) studied ten agroindustrial wastes for their suitability as fungus immobilization carrier for

solid-state fermentation and pointed out the materials with high water absorption capacity as the

most appropriate for use in SSF.

Substrate C:N Protein content

(%)


)

24 h 48 h 72 h 96 h

Wheat bran 4.27 14.74 13.40 81.46 176.21 186.42

Soybean meal 0.64 49.24 20.74 152.38 138.04 94.04

Cottonseed meal 2.15 25.91 16.61 110.25 111.71 118.83


Pearson coefficient -0.98 -0.98 0.67 0.99

p-value 0.10* 0.13 0.54 0.10

*



p-value 0.08* 0.06

* 0.73 0.29

136

3.3. The influence of the granulometric distribution and the apparent density of the

agroindustrial wastes on protease production

An important parameter in SSF is the particle size of the substrate since it is directly

related to porosity, the material compaction degree, therefore, the available space for mass and

energy transfer (Figueroa et al., 2013). In this work, the determination of particle size distribution

and the apparent density were used to evaluate the characteristics of the agroindustrial wastes and

their impact on protease production by A. niger LBA02.

Generally, small substrate particles provide larger surface area for microbial attack and

this is considered as a desirable factor. However, too small particles may result in agglomeration

and poor growth, whereas larger particles provide better inter-particle space but limited surface

for microbial attack (Pandey et al., 2001). Therefore, it is necessary to arrive at a compromised

particle size for a particular process. Wheat bran showed more heterogeneous particle size

distribution, with a predominance of particles ranging from 0.258 to 1.68 mm (Fig. 1b) and the

highest protease production was achieved at 96 h fermentation when this agroindustrial waste

was used as the substrate, reaching 186.42 U g-1

(Fig. 2a). Soybean and cottonseed meals showed

particle sizes greater, with granulometric distribution of 72.9 and 83.5% of particles larger than

1.68 mm, respectively (Fig. 1b). Although the protease acitivities (U g-1

) were lower in the latter

two substrates, the productivity indicated higher values when soybean meal was used as the

substrate, reaching 3.47 U g-1

h-1

at 48 h fermentation, while the maximum productivity detected

when wheat bran was used as the substrate was 2.45 U g-1

at 72 h fermentation, followed by the

cottonseed meal (2.30 U g-1

h-1

at 48 h fermentation) (Fig. 2). Important factors can be considered

from these results: when the particle size exceeded a particular value, the enzymes production can

be affected owing to reduction of contact surface between substrate’s particles and fungus.

However, small particle sizes affect the air supplied in terms of the aeration which can hinder the

microbial growth (Vaseghi et al., 2013).

The lowest apparent density values were obtained with wheat bran (0.32 g cm-3

) and

cottonseed meal (0.33 g cm-3

) as dry substrates (Fig. 1c). When the substrates have the initial

moisture adjusted to 50% for simulation of the real fermentation conditions, increases in the

apparent densities were observed. A high value of apparent density was found in soybean meal as

dry or moist substrate, showing values of 0.72 and 0.80 g cm-3

, respectively, which might to

137

result in problems as compactation, impairing the mass and energy transference (Fig. 1c). No

significant correlations (p-value ≤ 0.10) were observed between the apparent densities and the

protease production.

Fig. 1 - Water absorption index (WAI) (a), the granulometric fractions (% retained) (b) and

apparent density (g cm-3

) (c) of the agroindustrial wastes used for protease production by A. niger

LBA02 under solid state fermentation; fungal growth (d) and biomass estimation (glucosamine)

(e).

0

1

2

3

4

5

6

Wheat bran Soybean meal Cottonseed meal

WA

I (g

of

wat

er/g

dri

ed s

ubst

rate

)

0

15

30

45

60

75

90

1.68 0.841 0.595 0.250 0.177 0.149 <0.149

Gra

nulo

met

ric

frac

tio

ns

(% r

etai

ned

)

Opening (mm)


0.32

0.72

0.33

0.63

0.80

0.55

Wheat bran Soybean meal Cottoseed meal

Ap

par

ent

den

sity

(g

/cm

³)

Dry substrate Moist substrate

0

15

30

45

60

75

90

0 24 48 72 96 120 144

Fu

ngal

rad

ial

gro

wth

(m

m)

Time (h)


a

c

b

d

0

20

40

60

80

100

0 24 48 72 96 120

Glu

cosa

min

e (m

g g

-1)

Time (h)

Wheat bran Soybean meal Cottonssed meal

e

138

Fig. 2 - The protease production (U g-1

) and productivity (U g-1

h-1

) by A. niger LBA02 under

solid state fermentation using agroindustrial wastes: wheat bran (a), soybean meal (b) and

cottonseed meal (c).

a

b

c

0

1

2

3

4

0

40

80

120

160

200

0 24 48 72 96

Pro

ductiv

ity (U

g-1

h-1)

Pro

duct

ion (

U g

-1)

Fermentation time (h)

Production Productivity

0

1

2

3

4

0

40

80

120

160

200

0 24 48 72 96

Pro

ductiv

ity (U

g-1

h-1)

Pro

duct

ion (

U g

-1)



0

1

2

3

4

0

40

80

120

160

200

0 24 48 72 96

Pro

ductiv

ity (U

g-1

h-1)

Pro

duct

ion (

U g

-1)



139


production of proteases by filamentous fungi of the genus Aspergillus (Mukhtar and Haq, 2009;

Esparza et al., 2011; Leng and Xu, 2011). It is important to note that a standard assay is essential

for comparing the results of different studies. In several studies, research groups used different

methodologies to determine the protease activities, which may result in differences in the

substrates, incubation times, pH and temperatures of the reaction mixtures. The experimental data

variability complicates the comparisons among different studies. As such, a report of higher

protease activities does not necessarily suggest a higher production. Negi and Banerjee (2006)

observed maximum yields of protease production by a strain of A. awamori MTCC 6652 using

wheat bran as a substrate under solid state fermentation. The highest secretion of protease was

measured to be 1,930 U g-1

. Chutmanop et al., (2008) studied the protease production under solid

state fermentation of wheat bran, rice bran and their mixtures using strain of A. oryzae (Ozykat-1)

and reported a protease production of ∼1,200 U g−1

within 96 h fermentation using a substrate

mixture of 75% rice bran and 25% wheat bran. Shivakumar (2012) screened 11 different

substrates, including 8 cereals and 3 agroindustrial residues, for protease production by

Aspergillus sp. under solid state fermentation and found that wheat flour, wheat bran and soya

flour proved superior protease production, reaching activities of 320, 280 and 160 U g-1

,

respectively.

3.4. Determination of the microorganism growth

Filamentous fungi are the most widely microorganisms used in SSF because of their

ability to grow in solid substrates even in the absence of free water (Prakash et al., 2008; Orzua et

al., 2009). In the present work, wheat bran, soybean meal and cottonseed meal were used as

substrates for solid-state cultivation and evaluation of A. niger LBA02 growth into the

agroindustrial wastes. Fig. 1d shows the fungal radial growth (mm) in each substrate for 144 h

cultivation. In general, the microorganism had also good growth when cultivated in all substrates.

It can be noted that A. niger LBA02 exhibited a slight tendency to decrease their growth rate

when cultivated in soybean meal, reaching the maximum growth (80 mm) after 120h cultivation,

while the time required to completely cover wheat bran and cottonseed meal was 96 h. No

significant correlations (p-value ≤ 0.10) were observed between the protease production and the

water absorption index with the radial growth. However, the apparent densities showed a strong,

negative and significant correlation (Pearson coefficient ≤ − 0.99; p-value ≤ 0.10) with the fungal

140

radial growth, indicating a decrease in A. niger LBA02 growth with increasing of the apparent

densities values of the agroindustrial wastes.

Fig. 1e shows the evolution of fungal cellular growth as estimated by glucosamine level in

each substrate for 120 h cultivation. A. niger LBA02 exhibited a maximum growth when

cultivated in wheat bran, reaching glucosamine level of 90.33 mg g-1

, followed by soybean meal

(83.35 mg g-1

) and cottonseed meal (73.61 mg g-1

) after 96 h cultivation. Incubation beyond this

period did not result any further increase in glucosamine content. The glucosamine level showed

a strong, positive and significant correlation (Pearson coefficient ≥ 0.92; p-value ≤ 0.01) with the

fungal radial growth, confirming the agreement between these parameters as an important tool to

estimate the fungal growth in solid state fermentation. The protease production in the three

substrates showed a good and significant correlation with the glucosamine content, resulting in

Pearson coefficients of 0.91 (p-value = 0.03), 0.83 (p-value = 0.08) and 0.90 (p-value = 0.04) for

wheat bran, soybean meal and cottonssed meal, respectively, indicating an increase of the

protease production with increase of the glucosamine level.

3.5. Biochemical characteristics of protease from A. niger LB02

3.5.1. Effects of pH and temperature on the activity and stability of the protease determined

using an experimental design

The crude extracts of proteases produced in wheat bran (PWB), soybean meal (PSM) and

cottonseed meal (PCM) were biochemically characterized. Table 3 shows the CCRD matrix with

its independent variables (pH and temperature) and the results for protease activity and stability.

In the study of the determination of the optimum pH and temperature for activity, the highest

values obtained for PSB and PCM, were observed in the central points (runs 9-11) (50 °C, pH

4.5), averaging 148.04 and 106.36 U g-1

, respectively, while the PWB showed higher protease

activity (163.35 U g-1

) when incubated at 50 °C pH 2.0 (run 5). For protease stability, the highest

values obtained for PWB, PSM and PCM were observed when the enzymes were incubated at

50 °C pH 4.5 (runs 9-11).

141

Table 3 - The CCRD matrix used to determine the pH and temperature for optimum activity and

stability of the proteases from A. niger LBA02 produced in wheat bran, soybean meal and

cottonseed meal, with the coded and real values for the variables and responses.

1Proteases from A. niger LBA02 produced in wheat bran (PWB), soybean meal (PSM) and cottonseed meal (PCM). 2Residual

protease activity (%) of the proteases from A. niger LB02 produced in wheat bran (PWB), soybean meal (PSM) and cottonseed

meal (PCM) after incubation for 1h at different pH and temperatures.

Optimum pH and temperature for the protease activity

Run x1 / pH x2 /Temperature (°C) PWB1 (U g

-1) PSM

1 (U g

-1) PCM

1 (U g

-1)

1 -1.0 (2.73) -1.0 (39.4) 143.83 ± 1.70 44.12 ± 0.51 64.95 ± 0.56

2 +1.0 (6.27) -1.0 (39.4) 23.92 ± 0.67 23.23 ± 0.69 12.20 ± 0.73

3 -1.0 (2.73) +1.0 (60.6) 148.03 ± 1.36 41.63 ± 0.33 49.37 ± 1.56

4 +1.0 (6.27) +1.0 (60.6) 26.95 ± 0.13 25.38 ± 1.33 17.92 ± 0.53

5 -1.41 (2.0) 0.0 (50.0) 163.35 ± 10.10 77.12 ± 0.78 83.80 ± 4.09

6 +1.41 (7.0) 0.0 (50.0) 12.95 ± 0.13 13.72 ± 1.32 10.33 ± 1.50

7 0.0 (4.5) -1.41 (35.0) 120.58 ± 2.58 56.92 ± 1.48 66.92 ± 1.27

8 0.0 (4.5) +1.41 (65.0) 81.02 ± 2.90 68.40 ± 0.93 60.28 ± 2.19

9 0.0 (4.5) 0.0 (50.0) 149.93 ± 2.33 102.55 ± 2.65 104.60 ± 2.38

10 0.0 (4.5) 0.0 (50.0) 148.45 ± 1.98 102.28 ± 0.90 107.65 ± 1.23

11 0.0 (4.5) 0.0 (50.0) 145.75 ± 5.24 101.08 ± 2.42 106.83 ± 0.85

pH and temperature for the protease stability

Run x1 / pH x2 /Temperature (°C) PWB (%)2 PSM (%)

2 PCM (%)

2

1 -1.0 (2.73) -1.0 (39.4) 63.20 ± 0.33 73.17 ± 3.42 79.02 ± 2.89

2 +1.0 (6.27) -1.0 (39.4) 11.99 ± 4.33 35.92 ± 6.21 12.01 ± 0.96

3 -1.0 (2.73) +1.0 (60.6) 25.58 ± 0.48 31.58 ± 2.07 1.17 ± 0.57

4 +1.0 (6.27) +1.0 (60.6) 1.43 ± 0.80 8.27 ± 1.00 2.78 ± 1.32

5 -1.41 (2.0) 0.0 (50.0) 56.31 ± 1.26 46.51 ± 6.42 74.75 ± 3.89

6 +1.41 (7.0) 0.0 (50.0) 2.42 ± 3.62 4.54 ± 0.15 4.61 ± 1.17

7 0.0 (4.5) -1.41 (35.0) 62.86 ± 1.59 71.13 ± 5.96 80.07 ± 0.93

8 0.0 (4.5) +1.41 (65.0) 4.81 ± 1.65 10.42 ± 0.36 2.76 ± 1.87

9 0.0 (4.5) 0.0 (50.0) 97.48 ± 2.09 100.00 ± 3.36 96.32 ± 1.30

10 0.0 (4.5) 0.0 (50.0) 98.73 ± 2.28 96.78 ± 5.01 95.57 ± 1.02

11 0.0 (4.5) 0.0 (50.0) 100.00 ± 0.86 91.92 ± 3.28 100.00 ± 2.27

142

The proteases PWB and PSM demonstrated lower stability at temperatures above 60 °C

and pH range 6.0-7.0, reaching residual activities of 1.43 (run 4) and 4.54% (run 6); while for

PCM, the minimum value was detected when the enzyme was incubated at 60.6 °C and pH 2.73

(run 3), reaching residual activity of 1.17% (Table 3). The limited variability of the central points

(runs 9-11) indicated good reproducibility of the experimental data (Table 3).

Table 4 shows the models, R2, F-values, probability values for the final reduced models

and the validation tests performed under the conditions predicted by the models of the pH and

temperature for optimum activity and stability of the proteases from A. niger produced in wheat

bran (PWB), soybean meal (PSM) and cottonseed meal (PCM). For protease activity, the linear

and quadratic terms for the pH (x1) and temperature (x2) demonstrated a significant effect (p <

0.05) and the interaction pH x temperature (x1x2) showed no significant effects (p < 0.05) (Table

4). The estimated regression coefficients for the protease stability showed high statistical

significance (p < 0.05). The linear terms of pH (x1) and temperature (x2) showed positive effects

on the protease stability, while the quadratic terms indicated negative effects. These results

showed that the increasing pH and temperature positively influenced the stability of proteases,

but from a certain value, this effect started to be negative and quadratic. The interaction term

(pH×T) demonstrated positive effect for PWB and PCM stability but was not statistically

significant for PSM stability (Table 4).

An analysis of variance (ANOVA) showed that 89-98% of the total variation was

explained by the models. All F-values calculated for the regressions were greater than the

tabulated F-values (p-value < 0.01), reflecting the statistical significance of the model equations

(Table 4).

143

Table 4 - Models, R2, F-test, probability values and validation tests for the final reduced models of the pH and temperature for

optimum activity and stability of the proteases from A. niger LBA02 produced in wheat bran, soybean meal and cottonseed meal.

1Proteases from A. niger LBA02 produced in wheat bran (PWB), soybean meal (PSM) and cottonseed meal (PCM). 2Residual protease activity (%) of the proteases from A. niger

produced in wheat bran (PWB), soybean meal (PSM) and cottonseed meal (PCM) after incubation for 1h at different pH and temperatures. x1 – pH; x2 – temperature. The proteases

used to determine the pH and temperature for optimum activity and stability were obtained under the following fermentation conditions: initial moisture content of 50%, inoculum

level of 107 spores g-1, incubation temperature at 30 °C for 72 h fermentation.

Optimum pH and temperature for the protease activity

Responses Equations F-test F tabulated R² p-value

PWB (U g-1

) Y = – 460.68 + 60.50pH – 10.28pH2 + 22.36T – 0.23T

2 67.30 4.53 0.98 <0.001

PSM (U g-1

) Y = – 633.49 + 87.21pH – 10.68pH2 + 22.19T – 0.22T

2 11.97 4.53 0.89 0.005

PCM (U g-1

) Y = – 627.29 + 85.94pH – 11.02pH2 + 23.03T – 0.23T

2 21.22 4.53 0.93 0.005

pH and temperature for the protease stability

PWB (%) Y = – 684.14 + 75.49pH – 11.58pH2 + 27.02T – 0.30T

2 + 0.36pH×T 72.84 3.45 0.98 <0.001

PSM (%) Y = – 589.94 + 90.36pH – 10.98pH2 + 21.90T – 0.24T

2 131.32 4.01 0.98 <0.001

PCM (%) Y = – 437.07 + 79.55pH – 10.35pH2 + 16.16T – 0.18T

2 + 0.13pH×T 23.97 3.45 0.96 0.002

Validation tests

Maximum protease activity Predicted response (U g-1

) Experimental response (U g-1

)

PWB pH 3.0 Temperature 48.7 °C 173.46 181.20 ± 13.72

PSM pH 4.0 Temperature 50.4 °C 103.97 102.55 ± 1.78

PCM pH 4.0 Temperature 49.5 °C 110.49 106.36 ± 4.58

Maximum protease stability Predicted response Experimental response

PWB pH 3.5 – 4.5 Temperature: 45-50 °C ≥ 95% / ≥ 170.49 U g-1

101.27 ± 1.55% / 172.67 ± 2.69 U g-1

PSM pH 3.5 – 4.5 Temperature: 45-50 °C ≥ 95% / ≥ 89.67 U g-1

101.63 ± 3.37% / 91.13 ± 3.07 U g-1

PCM pH 3.0 – 4.5 Temperature: 40-50 °C ≥ 95% / ≥ 109.10 U g-1

102.78 ± 2.37% / 112.13 ± 2.66 U g-1

144

The surface responses were generated from the models. In general, all proteases were

more active in the acidic pH range (2.0-5.0) and at temperatures above 45 °C. However, the

proteases from A. niger LBA02 obtained by cultivation in wheat bran (PWB), soybean meal

(PSM) and cottonseed meal (PCM) showed some different characteristics in response to each

substrate. PSM and PCM were more active in the pH range 3.5-4.5 and temperature range 45-55

°C, while PSM showed higher activity in the pH range 2.0-4.5 and temperature range 45-60 °C

(Fig. 3). In the study of the protease stability, an evaluation of the response surfaces demonstrated

that the PWB, PSM and PCM showed different ranges for pH and temperature and stability.

PWB and PSM were more stable in the pH range 3.0-5.0 and in the temperature below 50 °C

after 1 h, retaining above 64% of the protease activity. PCM showed a higher range of stability,

with retention of the protease activity above 64% at pH range 2.0-5.0 and temperatures below

50 °C (Fig. 4).


obtained for the protease activity and stability with three assays (Table 4). The optimum

conditions for PWB, PSM and PCM activities determined according to the CCRD analysis were:

pH 3.0 and 48.7 °C, pH 4.0 and 50.4 °C, pH 4.0 and 49.5 °C, respectively. The optimum

conditions for protease stability of PWB, PSM and PCM showed similar profiles; for retention of

residual protease activity superior to 95%, the incubation conditions were: pH range 3.5 to 4.5

and 40-50 °C. The Tukey test showed that the experimental values agreed with the values

predicted by the models within a 95% confidence interval, thereby confirming the validity of the

models for the evaluated responses (Table 4).

The protease produced by A. niger LBA02 demonstrated pH and temperature activity and

stability profiles similar to those of the acid proteases from A. niger. An acid protease synthesized

by A. niger NRRL 1785 showed retention of 60% relative activity and above over a wide range

of pH 2.5-5.5 and was also stable up when incubated at 50 °C pH 4.0 for 1 h (Olajuyigbe et al.,

2003). A protease from A. niger ATCC 11414 exhibited maximum activity at pH 4.0 and 50 °C

and it was stable in a wide pH range (2.2-10.0) and at temperatures lower than 70 °C (Esparza et

al., 2011). An acidic protease from A. niger BCRC 32720 showed the optimum pH and

temperature activity at 2.5 and 50 C, respectively and was stable at pH 2.0−4.0 and temperatures

below 40 °C (Yin et al., 2013).

145

Fig. 4 – Response surfaces for maximum enzymes activities (U g-1

) and stability (% residual

activity) of the proteases from A. niger LBA02 produced in wheat bran (a, A), soybean meal (b,

B) and cottonseed meal (c, C) as a function of the pH and the temperature (°C).

> 160

< 160

< 124

< 104

< 84

< 64

< 44

2

3

4

5

6

7

pH

35

40

45

50

55

60

65

Temperature (°C)

20

40

60

80100120140160180

Protease (U

g-1

)

> 95 < 95 < 64 < 44 < 24 < 4

2

3

4

5

6

7

pH

3540

4550

5560

65

Temperatura (°C)

20

40

60

80

100

Pro

tease (%)

> 90

< 90

< 63

< 48

< 33

< 18

< 3

2

3

4

5

6

7

pH

3540

4550

5560

65

Temperature (°C)

15

30

45

60

75

90

105

Pro

tease (U g

-1)

> 95 < 95 < 64 < 44 < 24 < 4

2

3

4

5

6

7

pH

3540

4550

5560

65

Temperature (°C)

20

40

60

80

100

Pro

tease (%)

> 105

< 105

< 78

< 63

< 48

< 33

< 18

2

3

4

5

6

7

pH

3540

4550

5560

65

Temperature (°C)

15

30

45

60

75

90

105

120

Pro

tease (U g

-1)

> 95 < 95 < 64 < 44 < 24 < 4

2

3

4

5

6

7

pH

3540

4550

5560

65

Temperature (°C)

20

40

60

80

100

Pro

tease (%)

a

b

c

A

B

C

146

3.5.2. Determination of milk-clotting activity

A summary with the results for milk-clotting and protease activities of the enzymes

produced by A. niger LBA02 compared to other proteases preparations is presented in Table 5.

As shown, the commercial rennet from A. niger had the higher ratio clotting/protease activities

compared to other proteases. It was found that the clotting and protease activities of the

commercial rennet from A. niger were 3,908.79 U mL-1

and 26.46 U mL-1

, respectively. The

milk-clotting activity demonstrated different values in response to the different agroindustrial

wastes used for protease production. The PWB showed higher ratio clotting/protease activities

(15.24) compared to other commercial preparations of proteases, such as FlavourzymeTM

500L

(0.13) and Alcalase 2.4LTM

(8.01).

Table 5 - Ratio of milk-clotting activity/protease activity of the proteases from A. niger LBA02

produced under solid state fermentation using wheat bran (PWB), soybean meal (PSM) and

cottonseed meal (PCM) as substrates and other coagulants.

¹Protease activity determined using azocasein as the substrate at 37 °C and pH 6.5. The proteases used to determine the ratio of

milk-clotting activity/protease activity were obtained under the following fermentation conditions: initial moisture content of

50%, inoculum level of 107 spores g-1, incubation temperature at 30 °C for 72 h fermentation.

Fazouane-Naimi et al., (2010) showed that an acid protease produced by solid state

fermentation of A. niger FFB1 exhibited specific proteolytic and milk-clotting activities of

3,020.0 and 1,858.0 U mg-1

, respectively.

Enzymes Clotting activity

(U mL-1

)

Protease activity¹

(U mL-1

)

Ratio

(Clotting/Protease)

PWB 22.22 1.46 15.24

PSM 0.56 1.44 0.38

PCM 5.80 0.92 6.28

FlavourzymeTM

500L 1,136.36 8,513.00 0.13

AlcalaseTM

2.4L 111,627.91 13,944.67 8.01

Rennet from A. niger 3,908.79 26.46 147.72

147

3.6. Application of the proteases from A. niger to bovine whey protein hydrolysis and

antioxidant activities of the hydrolysates

Bovine whey proteins were separately hydrolyzed with the proteases from A. niger

LBA02 produced in the different agroindustrial wastes. The antioxidant activity of the

hydrolysates was evaluated using DPPH radical scavenging and total antioxidant activity

methods.

As shown in Figure 5, the bovine whey protein exhibited increase in antioxidant activities

after enzymatic hydrolysis. The antioxidant activities showed a strong positive and significant

correlation with the protein concentration (Pearson coefficient > 0.95, p-value < 0.01), with

maximum activities detected at 10 mg mL-1

. It can be noted that the antioxidant activities

demonstrated different values in response to the proteases produced in each agroindustrial waste.

DPPH is a stable free radical that shows maximal absorbance at 517 nm in ethanol. When

DPPH encounters a hydrogen-donating substance, such as an antioxidant, the radical is

scavenged and the absorbance is reduced by changing color from purple to yellow (Chen et al.,

2012). The highest DPPH radical scavenging was observed for PCM hydrolysates, followed by

PWB and PSM hydrolysates, which presented maximum activities of 84.5, 82.8 and 64.1% at 10

mg mL-1

. These values represented increases between 7.0 and 9.2-fold higher than non-

hydrolyzed protein.

Total antioxidant capacity is a method based on the reduction of Mo (VI) to Mo (V) by

the antioxidant agent (electron donation capacity) and the subsequent formation of a green

phosphate/Mo (V) complex at acidic pH (Prieto et al., 1999; Bougatef et al., 2009). For total

antioxidant activity, the hydrolysates showed maximum activity in following order: PWB > PSM

> PCM hydrolysates at 10 mg mL-1

, as was determined by their absorbance at 695 nm (Fig. 5).

148

Fig. 5 – Antioxidant activities (DPPH radical scavenging (a) and total antioxidant activity (b)) for

whey protein non-hydrolyzed and hydrolysates obtained with the proteases from A. niger LBA02

produced in wheat bran (PWB), soybean meal (PSM) and cottonseed meal (PCM).

These results demonstrate that the bovine whey protein hydrolysates possess hydrogen-

and electron-donating abilities, exhibiting adequate potency to react with free radicals.

Table 6 contains the correlation analysis between the different methods used to assess the

antioxidant activities and showed that non-hydrolyzed protein and all hydrolysates were

positively correlated (Pearson coefficient = 0.96–0.99; p-values < 0.01).

.

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0 2 4 6 8 10

Abso

rban

ce 6

95 n

m

Protein concentration (mg mL-1)

Non-hydrolyzed PWB PSM PCM

0

15

30

45

60

75

90

0 2 4 6 8 10

DP

PH

rad

ical

sca

ven

gin

g (

%)

Protein concentration (mg mL-1)

Non-hydrolyzed PWB PSM PCM

a

b .

.

.

.

.

. .

.

149

According Adjonu et al., (2013), the relatively high correlation between methods with

different reaction pathways suggests that the results complemented each other when it came to

assessing the antioxidant activities of proteins and their hydrolysates, and may also provide some

insight into their mechanisms of action. These authors observed correlation coefficients ranging

from 0.56 to 0.99 between ORAC and ABTS radical scavenging methods for samples of whey

protein isolate hydrolysates, which is consistent with the results reported in our study.

Table 6 – Correlations analysis between DPPH radical scavenging and total antioxidant activity

methods for non-hydrolyzed and hydrolyzed bovine whey protein.

Bovine whey protein Pearson correlation coefficients (R

2)

DPPH radical scavenging vs. total antioxidant activity

Non-hydrolyzed 0.96

PWB-hydrolysates 0.99

PSM-hydrolysates 0.98

PCM-hydrolysates 0.97

Luo et al., (2014) studied the enzymatic hydrolysis of sodium caseinate using papain,

pancreatin and trypsin and investigated the antioxidant properties of the hydrolysates using

different methods. The antioxidant activities showed different results in response to the proteases

used for enzymatic hydrolysis and in function of the methods. DPPH scavenging activity of

papain hydrolysates was higher than the other two groups, reaching a maximum value about 50%

after 24 h reaction. The hydrolysates obtained using pancreatin after 24 h hydrolysis, exhibited

the greatest reducing power, which was 2-fold that of non-hydrolyzed protein.

Castro and Sato (2014b) reported a comparative study with a pre-purified protease from

A. oryzae produced under solid state fermentation using wheat bran as substrate and two

commercial proteases (FlavourzymeTM

500L and AlcalaseTM

2.4L) for bovine whey protein

hydrolysis and study of the antioxidant properties of the hydrolysates. The results indicated that

the antioxidant capacity of whey protein increased after enzymatic hydrolysis. For the proteases

that were evaluated, the antioxidant activity increased approximately 2-fold, and the highest

DPPH radical scavenging (73.62% at 5 mg mL-1

) was observed in whey protein hydrolysates that

were prepared with the pre-purified enzyme from A. oryzae. Hydrolysates obtained with

150

FlavourzymeTM

500L and AlcalaseTM

2.4L reached maximum DPPH radical scavenging of 61.24

and 57.22% at 5 mg mL-1

.

4. Conclusion

The results obtained in our study showed that the protease production by A. niger

LBA02 can be affected by some physical chemical parameters, as the particle size and the water

absorption index of the agroindustrial wastes used in solid state fermentation. Higher levels of

protein in the agroindustrial wastes showed as important factor, inducing the protease production

in the first 48 h of fermentation. The biochemical characterization showed that the proteases

produced in different agroindustrial wastes exhibited different properties. The enzymes were

most active in the pH range 3.0-4.0 at 50 °C and stable from pH 2.5 to 4.5 at 40-50 °C. The

application of the proteases produced using different agroindustrial wastes as substrates to bovine

whey protein hydrolysis increased the antioxidant properties of the protein and resulted in

hydrolysates with different profiles of antioxidant activity.

References

Adjonu, R., Doran, G., Torley, P., Agboola, S., 2013. Screening of whey protein isolate

hydrolysates for their dual functionality: influence of heat pre-treatment and enzyme specificity.

Food Chem., 136, 1435–1443.

Ahmed, I.A.M., Morishima, I., Babiker, E.E., Mori, N., 2009. Characterisation of partially

purified milk-clotting enzyme from Solanum dubium Fresen seeds. Food Chem., 116, 395–400.

Anderson, R.A., Conway, H.F., Pfeifer, V.F., Griffin, E., 1969. Gelatinization of corn grits by

roll and extrusion cooking. Cereal Sci. Today, 14, 11–12.



DC.

Bougatef, A., Hajji, M., Balti, R., Lassoued, I., Triki-Ellouz, Y., Nasri, M., 2009. Antioxidant and

free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein

hydrolysates obtained by gastrointestinal proteases. Food Chem., 114, 1198–1205.

151




Castro, R.J.S., Sato, H.H., 2014a. Production and biochemical characterization of protease from

Aspergillus oryzae: an evaluation of the physical-chemical parameters using agroindustrial

wastes as supports. Biocat. Agric. Biotechnol., 3, 20-25.

Castro, R.J.S., Sato, H.H., 2014b. Advantages of an acid protease from Aspergillus oryzae over

commercial preparations for production of whey protein hydrolysates with antioxidant activities.

Biocat. Agric. Biotechnol., 3, 58–65.

Charney, J., Tomarelli, R. M., 1947. A colorimetric method for the determination of the


Chen, C. Chi, Y-J., Xu, W., 2012. Comparisons on the functional properties and antioxidant

activity of spray-dried and freeze-dried egg white protein hydrolysate. Food Bioprocess.

Technol., 5, 2342–2352.


Aspergillus oryzae in solid-state fermentation using agroindustrial substrates. J. Chem. Technol.

Biotechnol., 83, 1012-1018.

Esparza, Y., Huaiquil, A., Neira, L., Leyton, A., Rubilar, M., Salazar, L., Shene, C., 2011.

Optimization of process conditions for the production of a prolylendopeptidase by Aspergillus

niger ATCC 11414 in solid state fermentation. Food Sci. Biotechnol., 20 (5), 1323-1330.

Farnell, E., Rousseau, K., Thornton, D.J., Bowyer, P., Herrick, S.E., 2012. Expression and

secretion of Aspergillus fumigatus proteases are regulated in response to different protein

substrates. Fungal Biol., 116, 1003-1012.

Fazouane-Naimi, F., Mechakra, A., Abdellaoui, R., Nouani, A., Daga, S. M., Alzouma, A. M.,

Gais, S., Penninckx, M. J., 2010. Characterization and cheese-making properties of rennet-like

enzyme produced by a local algerian isolate of Aspergillus niger. Food Biotechnol., 24, 258–269.

Figueroa, J.B., Valdés, A.A., Sepúlveda, L., De la Cruz, R., Barragán, A.P., González, M.A.A.,

Rodríguez, R., Aguilar, C.N., 2013. Potential use of different agroindustrial by-products as

152

supports for fungal ellagitannase production under solid-state fermentation. Food Bioprod.

Process., http://dx.doi.org/10.1016/j.fbp.2013.08.010.

Leng, Y., Xu, Y., 2011. Improvement of acid protease production by a mixed culture of

Aspergillus niger and Aspergillus oryzae using solid-state fermentation technique. African J.

Biotechnol., 10 (35), 6824-6829.

Luo, Y., Pan, K., Zhong, Q., 2014. Physical, chemical and biochemical properties of casein

hydrolyzed by three proteases: partial characterizations. Food Chem., 155, 146-154.

Mukhtar, H., Haq, I.U. 2009. Production of acid protease by Aspergillus niger using solid state

fermentation. Pakistan J. Zool., 4, 253-260.

Negi, S., Benerjee, R., 2006. Optimization of amylase and protease production from Aspergillus

awamori in single bioreactor through EVOP factorial design technique. Food Technol.

Biotechnol., 44, 257–261.

Olajuyigbe, F. M., Ajele, J. O., Olawoye, T. L., 2003. Some physicochemical properties of acid

protease produced during growth of Aspergillus niger (NRRL 1785). Global J. Pure Appl. Sci.,

9(4), 523-528.

Orzua, M.C., Mussatto, S.I., Contreras-Esquivel, J.C., Rodriguez, R., Garza, H., Teixeira, J.A.,

Aguilar, C.N., 2009. Exploitation of agroindustrial wastes as immobilization carrier for solid-

state fermentation. Ind. Crops Prod., 30, 24–27.

Pandey, A., Soccol, C.R., Rodriguez-Leon, J.A., Nigam, P., 2001. Solid-state fermentation in

biotechnology—fundamentals and applications. Asiatech Publishers, New Delhi, 21–31.

Pel, H.J. et al., 2007. Genome sequencing and analysis of the versatile cell factory Aspergillus

niger CBS 513.88. Nature Biotechnol., 25 (2), 221-231.

Prakash, B.G.V.S., Padmaja, V., Kiran, S.R.R., 2008. Statistical optimization of process variables

for the large-scale production of Metarhizium anisopliae conidiospores in solid-state

fermentation. Bioresour. Technol., 99, 1530–1537.

Prieto, P., Pineda, M., Aguilar, M., 1999. Spectrophotometric quantitation of antioxidant capacity

through the formation of a phosphomolybdenum complex: specific application to the

determination of vitamin E1. Anal. Biochem., 269, 337–341.

http://dx.doi.org/10.1016/j.fbp.2013.08.010

153

Ramachandran, S., Roopesh, K., Nampoothiri, K. M., Szakacs, G., Pandey, A. 2005. Mixed

substrate fermentation for the production of phytase by Rhizopus spp. using oilcakes as

substrates. Process Biochem., 40, 1749–1754.

Ramakrishna, V., Rajasekhar, S., Reddy, L. S., 2010. Identification and purification of

metalloprotease from dry grass pea (Lathyrus sativus L.) seeds. Appl. Biochem. Biotechnol., 160,

63-71.


biotechnological aspect of microbial proteases. Microbiol. Mol. Biol. Res., 62, 597–635.

Robledo, A., Aguilera-Carbó, A., Rodriguez, R., Martinez, J.L., Garza, Y., Aguilar, C.N., 2008.

Ellagic acid production by Aspergillus niger in solid state fermentation of pomegranate residues.

J. Ind. Microbiol. Biotechnol., 35, 507–513.

Schuster, E., Dunn-Coleman, N., Frisvad, van Dijck, J.C.P.W.M., 2002. On the safety of



Aspergillus sp. by solid substrate fermentation. Archives Appl. Sci. Res., 4 (1), 188-199.

Speranza, P., Carvalho, P.O., Macedo, G.A., 2011. Effects of different solid state fermentation

substrate on biochemical properties of cutinase from Fusarium sp. J. Mol. Catal. B: Enzym., 72,

181-186.

Vaseghi, Z., Najafpour, G.D., Mohseni, S., Mahjoub, S., 2013. Production of active lipase by

Rhizopus oryzae from sugarcane bagasse: solid state fermentation in a tray bioreactor. Int. J. Food

Sci. Technol., 48, 283–289.

Yin, L. J., Hsu, T. H., Jiang, S. T., 2013. Characterization of acidic protease from Aspergillus

niger BCRC 32720. J. Agric. Food Chem., 61, 662−666.

Zhou, D., Qin, L., Zhu, B., Li, D., Yang, J., Dong, X., Murata, Y., 2012. Optimisation of

hydrolysis of purple sea urchin (Strongylocentrotus nudus) gonad by response surface

methodology and evaluation of in vitro antioxidant activity of the hydrolysate. J. Sci. Food

Agric., 92, 1694–1701.

155

Capítulo VII: Comparison and synergistic effects of intact proteins and

their hydrolysates on the functional properties and antioxidant activities in

a simultaneous process of enzymatic hydrolysis

Revista: Food and Bioproducts Processing

156

Abstract

Soy protein isolate (SPI), bovine whey protein (BWP) and egg white protein (EWP) were

hydrolyzed with the FlavourzymeTM

500L protease, and the interactions of these substrates and

their mixtures on their functional properties and antioxidant activities were studied using a

simplex centroid mixture design. Synergistic effects between the formulations containing binary

or ternary mixtures were observed for several parameters, especially the DPPH radical-

scavenging activity and emulsion activity index, which exhibited increases of up to 45.0 and

1,200%, respectively, after enzymatic hydrolysis compared to the isolated substrates. The results

suggest that the application of the statistical mixture designs in a simultaneous process of

enzymatic hydrolysis using different protein sources is an attractive method for improving

enzyme performance and identifying optimum formulations.

Keywords: enzymatic hydrolysis; protein hydrolysates; functional properties; antioxidant

activities; mixture design.

157

1. Introduction

Processes involving protein hydrolysis have been studied for bioactive peptide

production. Bioactive peptides can be defined as specific amino acid sequences that promote

beneficial biological activities. Bioactive peptides can be produced by enzymatic hydrolysis

using digestive, microbial and plant enzymes. Limited and controlled proteolysis unfolds protein

chains, reduces the incidence of allergenic factors and increases the formation of small peptides

with biological activities (Korhonen, 2009).

In the last decade, enzymatic hydrolysis of proteins from animal and plant sources for

the production of bioactive peptides have attracted much attention, and the antioxidant activities

of peptides have been extensively reported in several studies. The action mechanism of peptides

with antioxidant properties is related to the inactivation of reactive oxygen species (ROS),

scavenging of free radicals, chelation of prooxidative transition metals and reduction of

hydroperoxides (Zhou et al., 2012a).

In addition to their antioxidant activities, protein hydrolysates have shown interesting

functional properties, such as high solubility, resulting in increases in the concentration of free

amino and carboxyl groups. Hydrolysis disrupts the protein tertiary structure and reduces the

molecular weight of the protein consequently altering its functional properties (Liu et al., 2010).

Different protein sources have been used for enzymatic hydrolysis, such as rice, egg

white protein and whey protein (Zhao et al, 2012; Naik et al., 2013; Hoppe et al., 2013).

However, these reports investigated enzymatic hydrolysis using separate substrates, and no

investigation using statistical mixture designs has been reported.

Mixture designs are a special class of response surface designs where the proportions of

the components or factors are considered important rather than their magnitude and are useful in

the design of mixtures. The interactions between the components of a mixture can be studied

using the mixture design approach aiming to maximize the response. Statistical methods have

been applied to different engineering problems to improve performance and to find the optimum

process variables (Rao and Baral, 2011).

In the present study, a simplex centroid mixture design was used to produce

hydrolysates from different protein sources by enzymatic hydrolysis to study the effects of these

mixtures on functional properties and antioxidant activities.

158


2.1. Reagents

Ammonium thiocyanate, ferrous chloride, linoleic acid, trichloroacetic acid (TCA), 2,2′-

azobis(2-methylpropionamidine) dihydrochloride (97%) (AAPH), fluorescein, (±)-6-hydroxy-

2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2-diphenyl-1-picrylhydrazyl

(DPPH) were purchased from Sigma-Aldrich (Steinheim, Germany). All other purchased

chemicals were of commercially available grade.

2.2. Preparation of protein hydrolysates

The soy protein isolate (SPI), bovine whey protein (BWP) and egg white protein (EWP)

used as the substrates for enzymatic hydrolysis were kindly provided by Bunge Foods S/A

(Gaspar, Brazil), Alibra Ingredients Ltd. (Campinas, Brazil) and Cooperovos (Mogi das Cruzes,

Brazil), respectively. The commercial protease, FlavourzymeTM

500L from Aspergillus oryzae

(Novozymes Latin America Ltd., Araucária, Brazil) was used for enzymatic hydrolysis. The

enzyme concentrations were adjusted to 0 (control) or 50 U per mL of reaction mixture according

to the previously determined protease activity. The proteins were suspended in a buffer to a final


, and 50-milliliter aliquots of the mixtures were distributed in 125

mL Erlenmeyer flasks. Hydrolysis was performed for 120 min at the optimum temperature and

pH value of the enzyme according to the supplier's information: 50 °C and pH 5.0. After

hydrolysis, the samples were incubated in a water bath at 100 °C for 20 min for protease

inactivation. The mixtures were centrifuged at 17,000 x g at 5 °C for 20 min, and the supernatants

containing the peptides were collected and freeze-dried for the determination of their antioxidant

activities, functional properties and TCA soluble protein contents.

The protein content in the freeze-dried supernatants was determined using the Biuret

method, and the results were expressed in milligrams of protein per grams of freeze-dried sample.


The experimental mixture design was used to obtain the optimum mixture compositions

of the different protein sources for maximum antioxidant activity and to investigate the presence

of either synergistic or antagonistic effects in a blend of the components. A three component

augmented simplex centroid design was employed in which each component was studied at four

159

levels, namely 0 (0%), 1/3 (33%), 1/2 (50%) and 1 (100%) (Table 1). Quadratic or special cubic

regression models were fitted for the variations of all the responses studied as a function of

significant (p < 0.05) interaction effects between the proportions, thereby obtaining acceptable

determination coefficients (R² > 0.70). Equation 1 represents these models as follows:

where ‘Yi’ is the predicted response; ‘q’ represents the number of components in the system; ‘Xi,



StatisticaTM



To confirm the validity of the models, three assays were performed under randomly

selected test conditions, and the experimental values were compared with the predicted values by

the models within a 95% confidence interval.

2.4. TCA soluble protein content

The TCA soluble protein content of the hydrolysates was determined using a modified

version of the method described by Pericˇin et al. (2009). A 1.0 mL aliquot of the hydrolysate

was added to an equal volume of 0.44 mol L-1

trichloroacetic acid (TCA). The mixture was

incubated for 30 min at room temperature and then centrifuged at 17,000 x g for 15 min. A 0.22

mol L-1

TCA soluble protein fraction was obtained, and the supernatant of the hydrolysate

mixture (without the addition of TCA) was analyzed using the Lowry method (1951), which uses

bovine serum albumin as the standard protein to determine the protein content. The results were

expressed as a percentage and were calculated as the ratio of the 0.22 mol L-1

TCA soluble

protein content to the total protein content in the supernatant of the hydrolysate mixture.

2.5. Antioxidant activities

2.5.1. ORAC assay

The antioxidant activity of the hydrolysates was estimated by the ORAC method as

developed by Dávalos et al. (2004) and described by Macedo et al. (2011), which uses


𝑞

𝑖<𝑗

𝑞

𝑖=1


𝑞

𝑖<𝑗<𝑘

??

160

fluorescein (FL) as the ‘‘fluorescent probe’’. The automated ORAC assay was performed using a

Novo Star MicroplateTM

reader (BMG LABTECH, Germany) with fluorescence filters for an

excitation wavelength of 485 nm and an emission wavelength of 520 nm. The measurements

were made in a COSTARTM

96 plate. The reaction was performed at 37 °C and was started by the

thermal decomposition of AAPH in a 75 mM phosphate buffer (pH 7.4) due to the sensitivity of

FL to the pH value. A solution of FL (0.4 µg mL-1

) in phosphate buffered saline (PBS) (75 mM;

pH 7.4) was prepared daily and stored in complete darkness. The reference standard was a 75 µM

Trolox solution prepared daily in distilled water, and the standard was diluted (1,500 - 1.5 µmol

L-1

) for the preparation of the Trolox standard curve. In each well, 120 µL of the FL solution was

mixed with either 20 µL of sample, distilled water (blank) or standard (Trolox solutions) before

adding 60 µL of AAPH (108 mg mL-1

). The fluorescence was measured immediately after the

addition of AAPH, and measurements were taken every minute for 75 min. The ORAC values

were calculated from the difference between the area under the FL decay curve and that of the

blank (net AUC). Regression equations for the net AUC and antioxidant concentration were

calculated for all samples. The ORAC values were expressed as µmol of Trolox equivalent g-1

of

protein hydrolysate (Trolox EQ µmol g-1

).


The DPPH radical-scavenging activity of the hydrolysates was determined as described

by Bougatef et al. (2009). An aliquot (500 µL) of each sample (5 mg mL-1

) was mixed with 500

µL of 99.5% ethanol and 125 µL of DPPH (0.2 mg mL-1

) in 99.5% ethanol. The mixture was

then kept at room temperature in the dark for 60 min, and the reduction of the DPPH radical was

measured at 517 nm using a UV-Visible spectrophotometer. The DPPH radical-scavenging

activity was calculated as follows (Equation 2):

Radical scavenging activity (%) = [(Absorbance of control - Absorbance of sample) /

(Absorbance of control)] * 100.

2.5.3. Inhibition of linoleic acid autoxidation

The lipid peroxidation inhibition activity was measured in a linoleic acid emulsion system

according to the method described by Nazeer and Kulandai (2012) with slight modifications. A

20 mg aliquot of each hydrolysate was dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0)

and was later added to 130 µL of a linoleic acid solution and 10 mL of 99.5% ethanol. The total

161

volume was then adjusted to 25 mL with distilled water. The mixture was incubated in a 50-mL

assay tube with a screw cap at 42 ± 1 °C for 5 days in a dark room. The degree of oxidation of

linoleic acid was measured using the ferric thiocyanate method of Sakanaka et al. (2004) with

slight modifications. To 10 µL of the reaction mixture, 4.7 mL of 75% ethanol, 0.1 mL of 30%

ammonium thiocyanate and 0.1 mL of 20 mM ferrous chloride solution in 3.5% HCl were added.

After a 3-min incubation, the color development, which represents the linoleic acid oxidation,

was measured at 500 nm. The antioxidative capacity of inhibiting peroxide formation in the

linoleic acid system was expressed as follows: Inhibition (%) = [(Absorbance of control -

Absorbance of sample) / (Absorbance of control)] * 100.

2.6. Functional properties

2.6.1. Solubility

Protein solubility was determined according to a method described by Li et al. (2012)

with slight modifications. Solutions containing 100 mg of the protein hydrolysates were dispersed

in 10 mL of distilled water (pH 6.5 ± 0.3) at room temperature and centrifuged at 17000 x g for

10 min. The total protein content of the samples was determined by dissolving the sample in 0.5

mol L−1

NaOH and determining the protein content using the Biuret method. The protein

solubility was calculated as follows: solubility (%) = (protein content in supernatant / total

protein content in sample) × 100.

2.6.2. Heat stability

To determine the heat stability of the non-hydrolyzed proteins and their hydrolysates, 100

mg protein samples were dispersed in 10 mL of distilled water (pH 6.5 ± 0.3). After heating at

93 °C for 1 min, the solution was cooled in an ice water bath and centrifuged at 17,000 x g for 10

min. The protein content was determined using the Biuret method. The heat stability was

calculated as follows: stability (%) = (protein content in supernatant after heat treatment / total

protein content before the heat treatment) × 100.

2.6.3. Emulsifying property

The emulsifying property was measured using a modified version of the method described

by Pearce and Kinsella (1979). Vegetable oil (0.5 mL) and 1.5 mL aliquots of 10 mg mL−1

protein solutions were mixed and homogenized for 1 min. A 50 μL aliquot of the emulsion was

162

removed from the bottom of the container at 0 min after homogenization and mixed with 4.95 mL

of a 1 mg mL−1

sodium dodecyl sulfate (SDS) solution, and the absorbance was measured at 500

nm. The emulsifying activity index (EAI) was calculated as follows: EAI (m2 g

−1) = (2 × 2.303 ×

A500) / (0.25 × protein weight (g)).

2.6.4. Foaming capacity

Foaming capacity was measured using a modified version of the method described by

Klompong et al. (2007). Protein solutions (10 mL; 10 mg mL−1

) were homogenized for 1 min.

The foaming capacity, which was expressed as a percentage, was calculated as the ratio of the

volume after whipping (mL) to the volume before whipping (mL).


The values were expressed as the arithmetic means. Tukey’s test was used to test for

significant differences between the groups analyzed, and the differences were considered to be

significant at p < 0.05.

163


3.1. Comparison of the functional properties between the intact proteins and their

hydrolysates

The results for all the parameters were evaluated from two points of view as follows: 1) a

comparative analysis of the functional properties, antioxidant activities and TCA soluble protein

contents of the hydrolyzed and non-hydrolyzed proteins in their respective runs of the statistical

mixture design to verify any changes caused by enzymatic hydrolysis; and 2) evaluation of the

synergistic or antagonistic interactions of the hydrolyzed proteins and their mixtures on the

functional properties, antioxidant activities and TCA soluble protein contents.

The effects of the interactions among the three substrates on the functional properties,

antioxidant activities and TCA soluble protein contents were studied for the 7 described assays

using a simplex centroid mixture design (Table 1). Selective enzymatic hydrolysis under

controlled conditions has been used to improve the solubility, heat stability, emulsifying

properties and foaming properties of proteins. However, the present study showed that enzymatic

hydrolysis increased protein solubility, except for EWP and its mixtures. Several other studies

have also shown that enzymatic hydrolysis increases solubility. This result may be due to the

decrease in molecular size of the protein creating small peptides and unfolding the protein

molecule leading to the exposition of more polar and ionizable groups on the protein surface,

which could improve the ability of the protein molecule to form hydrogen bonds with water,

thereby augmenting solubility. However, other studies have reported a decrease in solubility after

hydrolysis, which may occur when the protein molecule exposes more hydrophobic groups (Liu

et al., 2010). For most of the formulations evaluated, the hydrolysates exhibited a tendency to

decrease their foaming capacities and heat stability. In contrast, the emulsion activity index

showed increases of 61.4, 107.0 and 155.2% for the SPI (1/2) plus EWP (1/2) binary formulation,

BWP (1/2) plus EWP (1/2) binary formulation and the ternary mixture (SPI, BWP and EWP in

equal proportions), respectively. The presence of short peptides, as shown by the increase in TCA

soluble protein content, may be associated with the change in these functional properties. In

addition, although the small peptides have the ability to rapidly diffuse towards the interface, they

are less efficient in stabilizing emulsions because they do not readily agglomerate to produce a fat

globule membrane due to charge repulsion (Turgeon et al., 1991).

164

Table 1 - Matrix of the simple centroid mixture design used to study the functional properties and antioxidant activities of the different

protein sources and their hydrolysates obtained by enzymatic hydrolysis.

a, b, c...The results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. Tukey tests were applied between the runs for each

parameter (not between different parameters). x1 – soy protein isolate (SPI); x2 – bovine whey protein (BWP); x3 – egg white protein (EWP).

Mixture design Functional properties

Run

Independent

variables Solubility (%) Heat stability (%) Emulsion activity index (m² g

-1) Foaming capacity (%)

x1 x2 x3 Non-

hydrolyzed Hydrolysates Non-hydrolyzed Hydrolysates

Non-

hydrolyzed Hydrolysates

Non-


1 1 0 0 11.19 ± 0.62a 99.17 ± 1.67

a 100.44 ± 2.30

a 100.49 ± 4.24

a 30.67 ± 0.09

a 4.26 ± 0.22

a 20.00 ± 5.00

b, c 22.50 ± 2.50

a

2 0 1 0 21.09 ± 1.02b 86.52 ± 4.00

b 101.90 ± 1.39

a 88.02 ± 1.64

b, c 15.31 ± 0.09

b 13.53 ± 0.07

b 0.00 ± 0.00

d 0.00 ± 0.00

b

3 0 0 1 82.14 ± 2.55c 26.92 ± 0.48

c 91.68 ± 1.15

d 86.56 ± 0.92

c 26.55 ± 0.09

c 27.41 ± 0.07

c 52.33 ± 2.52

a 37.50 ± 2.50

c

4 1/2 1/2 0 41.44 ± 1.58d 88.16 ± 3,50

b 95.01 ± 1.53

d 90.76 ± 4.13

b, c 14.62 ± 0.12

d 13.86 ± 0.09

b 0.00 ± 0.00

d 0.00 ± 0.00

b

5 1/2 0 1/2 89.53 ± 2.29e 39.27 ± 0.61

d 99.72 ± 1.66

a, b 94.78 ± 1.37

a, b 22.95 ± 0.07

e 37.05 ± 0.15

d 28.33 ± 2.89

b 27.50 ± 2.50

a

6 0 1/2 1/2 94.78 ± 1.84f 36.09 ± 2.01

d 81.41 ± 0.69

e 99.23 ± 1.90

a 23.95 ± 0.06

f 49.58 ± 0.07

e 45.00 ± 5.00

a 5.83 ± 1.44

d

7 1/3 1/3 1/3 86.99 ± 1.67c, e

49.46 ± 5.25e 96.15 ± 0.70

b, c 99.70 ± 2.59

a 22.19 ± 0.20

g 56.62 ± 0.06

f 12.33 ± 2.52

c 8.33 ± 1.44

d

Antioxidant activities

TCA soluble protein (%) Protein content in freeze-dried

supernatants (mg g-1

)

Run

ORAC (µmol Trolox EQ g-1

) DPPH radical scavenging (%) Inhibition of linoleic acid

autoxidation (%)

Non-


Non-


Non-


Non-


Non-


1 229.54 ± 8.04a 1157.18 ± 134.66

a 27.18 ± 0.15

a 51.55 ± 0.56

a 24.95 ± 0.02

a 22.01 ± 0.15

a 30.70 ± 0.71

a 62.47 ± 1.91

a, c 525.0 ± 11.78

a 579.0 ± 11.97

b

2 52.64 ± 0.69e 160.72 ± 26.26

d 17.13 ± 2.33

b 29.81 ± 0.48

b 49.63 ± 0.06

b 3.99 ± 0.02

b 37.24 ± 1.63

b 56.70 ± 1.67

b 301.5 ± 17.74

b 679.1 ± 91.98

a

3 125.56 ± 4.39b 546.45 ± 55.75

b, c 33.39 ± 0.26

c 31.50 ± 0.24

b 65.72 ± 0.01

c 29.85 ± 0.03

c 16.42 ± 0.13

c 63.79 ± 2.78

a, c 896.1 ± 28.51

c 982.8 ± 15.29

c

4 90.71 ± 3.45c 530.02 ± 48.12

b, c 21.06 ± 0.10

d 38.53 ± 0.63

c 17.77 ± 0.01

d 2.55 ± 0.01

d 36.03 ± 0.99

b 65.07 ± 1.67

a 424.5 ± 15.44

d 548.6 ± 28.6

b

5 79.17 ± 1.63c, d

595.88 ± 80.74b 45.19 ± 1.86

e 47.84 ± 0.59

d 72.78 ± 0.01

e 32.68 ± 0.01

e 17.42 ± 0.24

c 61.72 ± 0.71

a, c 713.2 ± 21.95

e 742.6 ± 25.6

a

6 71.41 ± 0.41d 347.16 ± 64.81

c, d 29.41 ± 1.40

a 43.26 ± 0.94

e 47.95 ± 0.02

f 43.16 ± 0.04

f 16.99 ± 0.11

c 59.58 ± 2.06

b, c 605.9 ± 7.46

f 687.8 ± 12.38

a

7 82.96 ± 14.47c, d

403.33 ± 86.51b, c

36.60 ± 1.12c 35.42 ± 0.61

f 42.55 ± 0.06

g 27.59 ± 0.01

g 17.65 ± 0.12

c 62.28 ± 1.41

a, c 582.0 ± 12.69

g 639.6 ± 4.51

a

165

3.2. Comparison of the antioxidant activities between the intact proteins and their

hydrolysates

The results obtained in the ORAC and DPPH assays showed no significant correlation

(data not shown) because the ORAC and DPPH-scavenging assays have different reaction

mechanisms. The DPPH compound is a stable free radical that has an unpaired valence electron

on one atom of the nitrogen bridge, and it shows maximum absorbance at 517 nm in ethanol.

When DPPH encounters a hydrogen-donating substance, such as an antioxidant, the radical is

scavenged, and the absorbance is reduced. Scavenging of the DPPH radical is the basis of the

popular DPPH antioxidant assay (Guerard et al., 2007; Sharma and Bath, 2009). The ORAC

assay measures the antioxidant-scavenging activity against the peroxyl radical induced by AAPH

at 37 °C (Ou et al., 2001). This free radical causes damage to a fluorescent probe, thereby

decreasing the fluorescence intensity. The capacity of antioxidants to inhibit free radical damage

is measured as the degree of protection against the change in probe fluorescence in the ORAC

assay (Huang et al., 2002; Macedo et al., 2011). Thus, a report of higher ORAC activity does not

necessarily suggest greater DPPH-scavenging ability. In the ORAC assay, the SPI hydrolysates

showed an increase in antioxidant activity of up to 400% followed by the EWP (335.2%) and

BWP (205.3%) hydrolysates compared to the intact proteins. The increases were even greater in

the mixtures reaching up to 600% for the formulation containing SPI (1/2) and EWP (1/2)

compared to the intact protein mixture. In the DPPH assay, the SPI hydrolysates showed an

increase in antioxidant activity of up to 89.7% compared to the intact proteins followed by the

BWP hydrolysates (74.1%). However, the DPPH-radical scavenging of the EWP hydrolysates

and the intact proteins showed no statistically significant difference (p < 0.05). For the inhibition

of linoleic acid autoxidation, the enzymatic hydrolysis showed a negative impact because the

non-hydrolyzed proteins showed greater inhibition (%) than the hydrolysates. The formulation

containing SPI (1/2) and EWP (1/2) (non-hydrolyzed) (2 mg mL-1

) showed the maximum

inhibition of linoleic acid autoxidation reaching 72.78% after 5 days of storage at 42 °C.

3.3. Comparison of the TCA soluble protein content between the intact proteins and

their hydrolysates

The size of the peptides is known to be a significant factor in the overall antioxidant

activity and functional properties of protein hydrolysates. Proteolysis levels are often assessed by

166

global quantification of the soluble peptides in certain concentrations of trichloroacetic acid

(TCA). This parameter has been used as an indication of the amount of small peptides in protein

hydrolysates and has a positive correlation with the degree of hydrolysis (DH) (Zhou et al.,

2012b). As expected, the TCA soluble protein content of the hydrolysates showed significant

changes after enzymatic hydrolysis with increases in the values ranging from 52.2 (BWP) to

288.5% (EWP) compared to the intact proteins. In some studies, an increase in the TCA soluble

protein content of the protein hydrolysates increases the antioxidant activity. However, other

studies have reported a decrease in antioxidant activity with an increase in TCA soluble protein

content. During hydrolysis, peptides with antioxidant properties may be continuously formed and

degraded depending on their molecular structure, which is primarily affected by the hydrolysis

conditions (Vastag et al., 2010).

3.4. Synergistic effects and antagonistic effects of the intact proteins and their

hydrolysates on functional properties, antioxidant activities and TCA soluble protein

content

The analysis of the interaction between the different protein sources and their mixtures

after enzymatic hydrolysis showed synergistic and antagonistic effects. For functional properties,

most formulations containing the protein mixtures showed antagonistic effects, except for the

emulsion activity index, which showed a synergistic effect between the binary and ternary

mixtures. Increases of up to 12-, 4- and 2-fold were detected in the ternary mixture (run 7) (SPI,

BWP and EWP in equal proportions) compared to the respective isolated hydrolysates (runs 1, 2

and 3, respectively). Concerning to the antioxidant activities, the ORAC assay of the seven

formulations containing the SPI, BWP and EWP hydrolysates (alone or in combination) showed

an antagonistic effect for all the mixtures. The SPI hydrolysates showed the strongest antioxidant

activity reaching 1,157.18 µmol Trolox EQ g-1

. However, for the DPPH assays, the mixture

composed of BWP (1/2) plus EWP (1/2) (run 6) showed a synergistic effect with increases of

45.1 and 37.3% in DPPH-radical scavenging compared to the respective isolated hydrolysates

(runs 2 and 3, respectively). The TCA soluble protein content showed a similar profile in the 7

runs of the statistical mixture design ranging from 56.70 to 65.07%. The interactions among the

following formulations were not statistically significant (p < 0.05): SPI (1/2) plus EWP (1/2) (run

5), BWP (1/2) plus EWP (1/2) (run 6) and SPI, BWP and EWP (in equal proportions) (run 7)

(Table 1).

167

This study demonstrated that proteins, protein mixtures and their hydrolysates prepared

with FlavourzymeTM

500L showed different functional properties and antioxidant activities.

These differences could be attributed to the physicochemical characteristics, including size,

shape, amino acid composition, sequence, net charge, charge distribution,

hydrophobicity/hydrophilicity ratio, secondary structure, tertiary structure, quaternary structure,

molecular rigidity and molecular flexibility. Therefore, the enzymatic protein hydrolysates

obtained using binary or ternary mixtures of proteins may contain different concentrations and

compositions of peptides and free amino acids compared to the isolated substrates, which can be

associated to the synergistic or antagonistic effects. Marcuse (1962) and Liu et al. (2010)

reported that peptides containing certain amino acids, such as Tyr, Met, His, Lys, Gly and Trp,

can present antioxidant or prooxidative effects. Therefore, a variation in the levels of these amino

acids in the peptides in protein mixtures may result in higher (synergistic effect) or lower

(antagonistic effect) values of antioxidant activities. The emulsifying properties of soluble

proteins depend upon the hydrophilic/lipophilic balance, which is affected by the presence of

hydrophilic and hydrophobic groups in peptides. In the present study, synergistic effects were

observed when binary and ternary protein mixtures were used resulting in increased emulsion

activity indexes, which may be due to the appropriate balance and distribution of peptides with

hydrophobic and hydrophilic residues.

In addition, it is important to note that the simultaneous enzymatic hydrolysis of soy

protein isolate, bovine whey protein and egg white protein was investigated in the present study.

However, the process proposed in this study can be extended to other protein sources allowing

multifunctional hydrolysates to be obtained in a simplified combination process of proteins with

different physicochemical characteristics and chemical compositions for maximizing the

bioactivities using the statistical mixture designs.

Intarasirisawat et al. (2012) studied the antioxidative and functional properties of

protein hydrolysates from defatted skipjack tuna (Katsuwonous pelamis) roe hydrolyzed by

AlcalaseTM

2.4 L with different degrees of hydrolysis (DH 5-50%) using different assays (DPPH

radical-scavenging, ABTS radical-scavenging and superoxide anion radical-scavenging activities

as well as reducing power and chelating capacity). The results showed that the DPPH radical-

scavenging and ABTS radical-scavenging activities as well as the reducing power decrease with

increasing DH. In contrast, the metal-chelating activity and superoxide-scavenging activity

168

increase with increasing DH. With regard to the functional properties, enzymatic hydrolysis

increases the protein solubility to above 80%. However, the highest emulsion activity indexes and

foam stabilities of the hydrolysates are observed at low DH (5%). Liu et al. (2010) investigated

the functional properties of porcine blood plasma protein hydrolysates prepared with AlcalaseTM

at 6.2, 12.7 and 17.6% degrees of hydrolysis (DH), and they reported that hydrolysis increases

the protein solubility and decreases the emulsifying and foaming capacities of the plasma protein.

Zhou et al. (2012b) studied the antioxidant activities of hydrolysates prepared from sea urchin

(Strongylocentrotus nudus) gonads using DPPH radical-scavenging activity, reducing power,

hydroxyl radical-scavenging activity, hydrogen peroxide-scavenging activity, lipid peroxidation-

inhibiting activity and Fe2+

-chelating activity, and they observed the highest values for protein

hydrolysates with TCA soluble protein contents of up to 50%.

3.5. Mixture contour plots for functional properties, antioxidant activities and TCA

soluble protein contents

The variations in the functional properties and antioxidant activities of the hydrolysates

obtained from SPI, BWP and EWP were also depicted using mixture contour plots (Fig. 1 and

Fig. 2). On the response surfaces, each factor (pure mixture component) is represented in the

corner of an equilateral triangle, and each point within this triangle refers to a different proportion

of the components in the mixture. The maximum percentage of each ingredient considered by the

regression is placed at the corresponding corner, and the minimum percentage is positioned at the

middle of the opposite side of the triangle (Martinello et al., 2006). A contour plot provides a

two-dimensional view where all points that have the same response are connected to produce

contour lines of constant responses (Rao and Baral, 2011).

For the functional properties, high solubility was detected in the SPI hydrolysates with

values up to 90%. However, the zones of maximum response variables for heat stability were

located towards the side of the triangle having mixtures of SPI/EWP or BWP/EWP on the

vertices (Fig. 1). These results indicated that to certain extent, these protein proportions may be

added to improve the heat stability of the hydrolysates. The EWP hydrolysates showed the

maximum foaming capacity. The emulsion activity index showed more possibilities of mixture

applications to maximize the response variable. The addition of equal proportions of SPI, BWP

and EWP helped to improve the response variables reaching values up to 50 m² g-1

. The

169

antioxidant activities evaluated in the ORAC and DPPH-radical scavenging assays showed

similar profiles with maximum responses detected in the SPI hydrolysates. However, the

inhibition of linoleic acid autoxidation was greater in the hydrolysates prepared with mixtures of

BWP and EWP reaching values up to 40%. High TCA soluble protein contents were observed in

the mixtures containing SPI and BWP, which was verified by the mixture surface plots (Fig. 2).

Fig. 1 - Mixture contour plots for the functional properties (a: solubility; b: heat stability; c:

foaming capacity; d: emulsion activity index) of the protein hydrolysates.

> 90 < 90 < 80 < 70 < 60 < 50 < 40 < 30

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 99 < 99 < 97 < 95 < 93 < 91 < 89 < 87

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 35 < 35 < 30 < 25 < 20 < 15 < 10 < 5

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 50 < 50 < 40 < 30 < 20 < 10

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

c

a b

d

170

Fig. 2 - Mixture contour plots for the antioxidant activities (a: ORAC; b: DPPH; c: inhibition of

linoleic acid autoxidation (%)) and TCA soluble protein (d) of the protein hydrolysates.

3.6. Analysis of variance (ANOVA) and models for the functional properties, antioxidant

activities and TCA soluble protein contents of the intact proteins and their

hydrolysates


and recorded in Table 1. The experiments were conducted with triplicates. In almost all cases, a

good agreement existed between the original and triplicates. All the independent and response

variables were fitted to quadratic or special cubic models. The coefficient of determination (R2)

and the F-test (analysis of variance; ANOVA) were used to verify the quality of fit of the models.

Table 2 shows the models and corresponding R2, F-test and p-values of the regression equations

for the responses.

a b

d c

> 1000 < 1000 < 800 < 600 < 400 < 200

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 50 < 50 < 46 < 42 < 38 < 34 < 30

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 40 < 40 < 35 < 30 < 25 < 20 < 15 < 10 < 5

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 65 < 65 < 63 < 61 < 59 < 57

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

171

Table 2 - Models, R2, and probability values for the final reduced models of the functional properties and antioxidant activities.

Responses Models Equations (non-hydrolyzed proteins) F-test R² p-value

Solubility (%) Quadratic Y = 11.23x1 + 21.13x2 + 82.18x3 + 100.35x1x2 + 170.58x1x3 + 171.80x2x3 526.6 0.99 <0.001

Heat stability (%) Special cubic Y = 101.66x1 + 98.88x2 + 92.90x3 – 21.05x1x2 – 57.94x2x3 + 191.96x1x2x3 5.7 0.85 <0.001

Emulsion activity index (m² g-1) Special cubic Y = 30.67x1 + 15.31x2 + 26.55x3 – 33.49x1x2 – 22.64x1x3 + 12.08x2x3 + 78.55x1x2x3 2,686.6 0.99 <0.001

Foaming capacity (%) Special cubic Y = 20.00x1 + 52.33x3 – 40.00x1x2 – 31.33x1x3 + 75.33x2x3 – 330.00x1x2x3 44.1 0.98 <0.001

ORAC (µmol Trolox EQ g-1) Special cubic Y = 229.54x1 + 52.64x2 + 125.56x3 – 201.52x1x2 – 393.52x1x3 – 70.75x2x3 + 567.71 x1x2x3 84.2 0.99 <0.001

DPPH radical scavenging (%) Special cubic Y = 26.81x1 + 16.76x2 + 33.40x3 + 60.35x1x3 + 17.33x2x3 + 62.34x1x2x3 62.3 0.98 <0.001

Inhibition of linoleic acid

autoxidation (%) Special cubic Y = 24.95x1 + 49.63x2 + 65.72x3 – 78.07x1x2 + 109.80x1x3 – 38.91x2x3 – 92.41x1x2x3 416,715.7 0.99 <0.001

TCA soluble protein (%) Special cubic Y = 30.70x1 + 37.24x2 + 16.42x3 + 8.25x1x2 – 24.55x1x3 – 39.35x2x3 – 115.61x1x2x3 215.3 0.99 <0.001

Equations (hydrolysates)

Solubility (%) Quadratic Y = 99.14x1 + 86.49x2 + 26.89x3 – 18.16x1x2 – 94.53x1x3 – 81.98x2x3 101.6 0.99 <0.001

Heat stability (%) Quadratic Y = 100.33x1 + 86.86x2 + 87.30x3 + 52.75x2x3 6.4 0.80 <0.001

Emulsion activity index (m² g-1) Special cubic Y = 4.26x1 + 13.53x2 + 27.41x3 + 19.86x1x2 + 84.87x1x3 + 116.44x2x3 + 458.51x1x2x3 29,255.8 0.99 <0.001

Foaming capacity (%) Quadratic Y = 21.63x1 + 36.63x3 – 44.29x1x2 – 50.96x2x3 86.5 0.98 <0.001

ORAC (µmol Trolox EQ g-1) Quadratic Y = 1,160.81x1 + 156.15x2 + 541.89x3 – 572.01x1x2 – 1,080.05x1x3 20.4 0.95 <0.001

DPPH radical scavenging (%) Special cubic Y = 51.55x1 + 29.81x2 + 31.49x3 – 8.62x1x2 + 25.27x1x3 + 50.42x2x3 – 260.64x1x2x3 191.6 0.99 <0.001

Inhibition of linoleic acid

autoxidation (%) Special cubic Y = 22.01x1 + 3.99x2 + 29.85x3 – 41.78x1x2 + 26.99x1x3 + 104.94x2x3 – 28.18x1x2x3 59,133.3 0.99 <0.001

TCA soluble protein (%) Quadratic Y = 62.00x1 + 56.51x2 + 63.05x3 + 22.04x1x2 5.9 0.70 <0.001

172

The high coefficients of determination (R2), which were greater than 0.70 (Table 2),

indicated that all the response functions adequately fitted the experimental data and that the

models could be used for predictive purposes in the determination of the functional properties,

antioxidant activities and TCA soluble protein contents using the different protein sources and

their mixtures.


obtained for the functional properties and antioxidant activities of the different protein sources

and their hydrolysates using different formulations with three assays (Table 3). According to the

regression models (Table 2), the experimental values agreed with the values predicted by the

models within a 95% confidence interval, thereby confirming the validity of the models for the

evaluated responses (Table 3).


obtained for the functional properties and antioxidant activities of the different protein sources

and their hydrolysates in different formulations.

Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05.

Comparisons were made between the observed and predict values for each correspondent response.

Responses

Non-hydrolyzed proteins

Independent variables Predicted

response

Experimental

response x1 (SPI) x2 (BWP) x3 (EWP)

Solubility (%) 0.66 0.17 0.17 60.34a 58.28 ± 2.18

a

Heat stability (%) 0.17 0.66 0.17 93.13b 94.62 ± 1.95

b

Emulsion activity index (m² g-1

) 0.17 0.17 0.66 24.68c 23.85 ± 1.32

c

Foaming capacity (%) 0.17 0.17 0.66 35.42d 32.50 ± 3.53

d


) 0.66 0.17 0.17 123.81e 130.90 ± 7.93

e

DPPH radical scavenging (%) 0.17 0.66 0.17 26.17f 24.45 ± 2.87

f

Inhibition of linoleic acid autoxidation (%) 0.17 0.17 0.66 59.99g 58.41 ± 5.21

g

TCA soluble protein (%) 0.66 0.17 0.17 24.21h 24.95 ± 3.23

h

Hydrolysates

Solubility (%) 0.66 0.17 0.17 69.69i 68.92 ± 2.48

i

Heat stability (%) 0.33 0.33 0.34 97.37j 99.48 ± 3.62

j

Emulsion activity index (m² g-1

) 0.34 0.33 0.33 56.37l 56.92 ± 0.97

l

Foaming capacity (%) 0.17 0.17 0.66 20.86m 19.75 ± 2.77

m


) 0.66 0.17 0.17 699.44n 641.56 ± 86.55

n

DPPH radical scavenging (%) 0.66 0.17 0.17 42.79o 42.12 ± 3.35

o

Inhibition of linoleic acid autoxidation (%) 0.17 0.17 0.66 37.18p 40.99 ± 4.23

p

TCA soluble protein (%) 0.33 0.34 0.33 62.95q 61.07 ± 2.85

q

173

4. Conclusions

The results suggested that the application of statistical mixture designs for the enzymatic

hydrolysis of different protein sources is an attractive process for improving their performance

and for finding the optimum mixture formulations of proteins with specific characteristics. The

maximum increases in antioxidant activities were observed in the formulations containing SPI

(1/2) and EWP (1/2) reaching up to 600%. Synergistic effects between the formulations

containing binary or ternary mixtures were observed for several parameters, especially for the

DPPH-radical scavenging and emulsion activity indexes, which showed increases of up to 45 and

1,200%, respectively, in their activities after enzymatic hydrolysis compared to the isolated

substrates. The functional properties of the hydrolysates, such as high solubility, suggested that

the hydrolysates could have wider applications in formulated food systems.

Acknowledgments

The authors gratefully acknowledge the São Paulo Research Foundation (FAPESP;

Project No. 2011/10429-9) and the Department of Food Science, School of Food Engineering,

University of Campinas for the substantial grants received.

References

Bougatef, A., Hajji, M., Balti, R., Lassoued, I., Triki-Ellouz, Y., Nasri, M., 2009. Antioxidant and

free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein

hydrolysates obtained by gastrointestinal proteases. Food Chem., 114, 1198–1205.

Dávalos, A., Gómez-Cordovés, C., Bartolomé, B., 2004. Extending applicability of the oxygen

radical absorbance capacity (ORAC-fluorescein) assay. J. Agr. Food Chem., 52, 48-54.

Guerard, F., Sumaya-Martinez, M. T., Laroque D. et al., 2007. Optimization of free radical

scavenging activity by response surface methodology in the hydrolysis of shrimp processing

discards. Process Biochem., 42, 1486–1491.

Hoppe, A., Jung, S., Patnaik, A., Zeece M. G., 2013. Effect of high pressure treatment on egg

white protein digestibility and peptide products. Innovat. Food Sci. Emerg. Tech., 17, 54–62.

Huang, D., Ou, D., Hampsch-Woodi, M., 2002. Development and validation of oxygen radical

absorbance capacity assay for lipophilic antioxidants using randomly methylated β-cyclodextrin

as the solubility enhancer. J. Agr. Food Chem., 50, 1815–1821.

174

Intarasirisawat, R., Benjakul, S., Visessanguan, W., Wu, J., 2012. Antioxidative and functional

properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe. Food

Chem., 135, 3039–3048.

Klompong, V., Benjukal, S., Kantachote, D., Shahidi, F., 2007. Antioxidative activity and

functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as

influenced by the degree of hydrolysis and enzyme type. Food Chem., 102, 1317–1327.

Korhonen, H., 2009. Milk-derived bioactive peptides: from science to applications. J. Funct.

Foods, 1, 177-187.

Li, X., Luo, Y., Shen, H., You, J., 2012. Antioxidant activities and functional properties of grass

carp (Ctenopharyngodon idellus) protein hydrolysates. J. Sci. Food Agr., 92, 292–298.

Liu, Q., Kong, B., Xiong, Y. L., Xi, X., 2010. Antioxidant activity and functional properties of

porcine plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chem., 118,

403–410.

Lowry, O. H., Rosenbrough, N. J., Fair, A. L., Randall, R. J., 1951. Protein measurement with the

Folin-phenol reagents. The J. Biol. Chem., 193, 265–275.

Macedo, J. A., Battestin, V., Ribeiro, M. L., Macedo, G. A., 2011. Increasing the antioxidant

power of tea extracts by biotransformation of polyphenols. Food Chem., 126, 491–497.

Marcuse, R. (1962). The effect of some amino acids on the oxidation of linoleic acid and its

methyl ester. JAOCS, 39, 97–103.

Martinello, T., Kaneko, T. M., Velasco, M. V. R., Taqueda, M. E. S. & Consiglieri, V. O. (2006).


mixture experimental design. Int. J. Pharm., 322, 87-95.

Naik, L., Mann, B., Bajaj, R., Sangwan, R. B., Sharma, R., 2013. Process optimization for the

production of bio-functional whey protein hydrolysates: adopting response surface methodology.

Int. J. Pept. Res. Therapeut., DOI 10.1007/s10989-012-9340-x.

Nazeer, R. A., Kulandai, K. A., 2012. Evaluation of antioxidant activity of muscle and skin

protein hydrolysates from giant kingfish, Caranx ignobilis (Forsska° l, 1775). Int. J. Food Sci.

Tech., 47, 274–281.

175

Ou, B., Huang, D., Hampsch-Woodill, M. et al., 2002. Analysis of antioxidant activities of

common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing

antioxidant power (FRAP) assays: a comparative study. J. Agr. Food Chem., 50, 3122-3128.

Pearce, K. N., Kinsella, J. E., 1978. Emulsifying properties of proteins: evaluation of a

turbidimetric technique. J. Agr. Food Chem., 26, 716–723.

Pericˇin, D., Radulovic´-Popovic´, L., Vaštag, Zˇ., Madarev-Popovic´, S., Trivic, S., 2009.

Enzymatic hydrolysis of protein isolate from hull-less pumpkin oil cake: application of response

surface methodology. Food Chem., 115, 753–757.

Rao, P. V., Baral, S. S., 2011. Experimental design of mixture for the anaerobic co-digestion of

sewage sludge. Chem. Eng. J., 172, 977-986.

Sakanaka, S., Tachibana, Y., Ishihara, N., Juneja, L. R., 2004. Antioxidant activity of egg-yolk

protein hydrolysates in a linoleic acid oxidation system. Food Chem., 86 (1), 99-103.

Sharma, O. P., Bhat, T. K., 2009. DPPH antioxidant assay revisited. Food Chem, 113, 1202–

1205.

Turgeon, S. L., Gauthier, S. F., Paquin, P., 1991. Interfacial and emulsifying properties of whey

peptides fraction obtained with a two-step ultrafiltration process. J. Agr. Food Chem., 39(4), 637–

676.

Vastag, Z., Popovic, L., Popovic, S. et al., 2010. Hydrolysis of pumpkin oil cake protein isolate

and free radical scavenging activity of hydrolysates: Influence of temperature, enzyme/substrate

ratio and time. Food Bioprod. Process., 88, 277-282.

Zhao, Q., Selomulya, C., Wang, S., Xiong, H., Chen, X. D., Li, W., Peng, H., Xie, J., Sun, W.,

Zhou, Q., 2012. Enhancing the oxidative stability of food emulsions with rice dreg protein

hydrolysate. Food Res. Int., 48, 876–884.

Zhou, D., Zhu, B., Qiao, L., Wu, H., Li, D., Yang, J., Murata, Y., 2012a. In vitro antioxidant

activity of enzymatic hydrolysates prepared from abalone (Haliotis discushannai Ino) viscera. .

Food Bioprod. Process., 90, 148-154.

Zhou, D., Qin, L., Zhu, B., Li, D., Yang, J., Dong, X., Murata, Y., 2012b. Optimisation of


176

methodology and evaluation of in vitro antioxidant activity of the hydrolysate. J. Sci. Food Agr.,

92, 1694–1701.

177

Capítulo VIII: Synergistic effects of protein hydrolysates on the suppression of

lipid accumulation in 3T3-L1 adipocytes

Revista: LWT Food Science and Technology

178

Abstract


hydrolyzed with the protease FlavourzymeTM

500L, and the interactions of these substrates for

the inhibition of the relative lipid accumulation (RLA) in 3T3-L1 preadipocytes during

differentiation was studied using a simplex-centroid mixture design. The results indicated that

there were synergistic effects for mixtures of intact and hydrolyzed proteins. The hydrolyzed

mixture containing BWP (1/2) plus EWP (1/2) at 800 ppm showed increases of up to 220 and

27% in their activities, respectively, compared to the isolated substrates, reaching a maximum

RLA suppression of 15.5%. The treatment in which the two-day postconfluent 3T3-L1

preadipocytes received 1200 ppm of the mixture containing BWP (1/2) plus EWP (1/2) at day

zero and every two days afterwards until the end of the experiment at day eight was demonstrated

to be more effective, reaching an RLA suppression of 47.9%. The results from the fractionation

by ultrafiltration indicated that the non-fractionated sample of BWP (1/2) and EWP (1/2) was the

most active for the anti-adipogenic activity and indicated that there is an important contribution

of peptide fractions with various molecular sizes in the inhibition of lipid accumulation in 3T3-

L1 cells.

Keywords: protein hydrolysates; synergistic effects; anti-adipogenic activity

179

1. Introduction

Bioactive peptides can be defined as specific amino-acid sequences that promote

beneficial biological activities. The size of active sequences may vary from 2 to 20 amino acid

residues, and many peptides are known to reveal multi-functional properties, such as antioxidant,

anti-adipogenic, antihypertensive and antimicrobial activities. In general, these bioactive peptides

are inactive within the sequence of parent protein and can be released by limited and controlled

hydrolysis using digestive, microbial and plant enzymes (Korhonen, 2009; Chen, Chi, Zhao &

Xu, 2012).

Accumulation of body fat arises from a chronic imbalance between energy acquisition and

expenditure that may lead to a pathologic growth of adipocytes, characterized by increased fat-

cell size and number (Shimomura, Hammer, Richardson, Ikemoto & Bashmakov, 1998; Mejia,

Martinez-Villaluenga, Roman & Bringe, 2010). It is known that the amount of adipose tissue can

be regulated by the inhibition of adipogenesis from precursor cells (Rahman et al., 2008).

Preadipocytes that do not yet contain a significant amount of lipid and resemble fibroblasts can

be cultured, and after differentiation is induced, the cell cultures may be used for metabolic

studies (Poulos, Dodson & Hausman, 2010). The preadipocyte differentiation can be stimulated

by treatment with adipogenic agents, including 3-isobutyl-1-methylxanthine, dexamethasone and

insulin, which induces postconfluent mitotic clonal expansion and begins to express adipocyte-

specific genes (Rubin, Hirsch, Fung & Rosen, 1978; Tsou, Kao, Tseng & Chiang, 2010). The

mature adipocytes contain single, large lipid droplets that appear to comprise the majority of the

cell volume and can be quantified as intracellular lipid or triglyceride content by staining with Oil

Red O (Green & Kehinde, 1975; Poulos, Dodson & Hausman, 2010). Thus, determining the

relative lipid accumulation in preadipocyte cells during differentiation could be used as a

convenient tool to evaluate the anti-adipogenesis effects of protein hydrolysates. The 3T3-L1

preadipocytes are one of the most well characterized and reliable models for studying

adipogenesis (Tsou, Kao, Tseng & Chiang, 2010). Several studies reported the reduction in body

fat and food intake when proteins were hydrolyzed into bioactive peptides (Martinez-Villaluenga,

Bringe, Berhow & Mejia, 2008; Tsou, Lin, Lu, Tsui & Chiang, 2010).

In the past decade, the enzymatic hydrolysis of proteins from animal and plant sources for

the production of bioactive peptides has attracted much attention. Among the biological

180

activities, the anti-adipogenic effects have been reported. In the literature, various protein sources

have been used for the enzymatic hydrolysis, such as rice, egg white protein and whey protein

(Zhao et al., 2012; Naik, Mann, Bajaj, Sangwan & Sharma, 2013; Hoppe, Jung, Patnaik & Zeece,

2013). However, these reports show many studies on enzymatic hydrolysis using distinct

substrates; no investigations were found using formulations containing mixtures of different

protein sources.

Mixture design is a special class of response surface design in which the proportions of

the components or factors are considered important, rather than their magnitude, and are useful in

the mixture design. The interactions between the components of a mixture for maximizing the

response are studied using a mixture-design approach. Statistical methods were applied to various

engineering problems to improve the performance and to find the optimum process variables

(Rao & Baral, 2011).

In this study, a simplex-centroid mixture design was used for the production of

hydrolysates of various protein sources by enzymatic hydrolysis, and the effects on the inhibition

of the relative lipid accumulation (RLA) in 3T3-L1 preadipocytes during differentiation were

studied. The effect of different fractions of the protein hydrolysates obtained by ultrafiltration on

RLA suppression in 3T3-L1 preadipocytes was also investigated.


2.1. Reagents

FlavourzymeTM

500L, 3-isobutyl-1-methylxanthine, water-soluble dexamethasone,

insulin, Oil Red O, sodium dodecyl sulfate (SDS) and trichloroacetic acid (TCA) were purchased

from Sigma-Aldrich (Steinheim, Germany). All other chemicals were purchased in the grade

commercially available.



used as substrates for enzymatic hydrolysis. The commercial protease FlavourzymeTM

500L from

Aspergillus oryzae was used for enzymatic hydrolysis. The protease activity was determined

using azocasein as the substrate, as described by Castro & Sato (2013). The enzyme

concentrations were adjusted to zero (control) and 50 U per mL of reaction, according to the

181

protease activity, as previously determined. The proteins were suspended in a 200-mM acetate

buffer at pH 5.0 to a final concentration of 100 mg mL-1

. Fifty-milliliter aliquots of the mixtures

were distributed in 125-mL Erlenmeyer flasks, and the hydrolysis was performed under the

optimum temperature and pH of the enzyme (50 °C, pH 5.0) for 120 min. After hydrolysis, the

samples were incubated in a water bath at 100 °C for 20 min for protease inactivation. The

mixtures were centrifuged at 17,000 x g and 5 °C for 20 min, and the supernatants containing

peptides were collected and freeze-dried for the determination of the TCA-soluble protein and the

suppression of the relative lipid accumulation (RLA) in the 3T3-L1 preadipocytes.


The mixture design of the experiment was used to obtain the optimal mixture composition

of the various protein sources for the maximal inhibition of the relative lipid accumulation (RLA)

in 3T3-L1 preadipocytes during differentiation and to investigate the presence of either

synergistic or antagonistic effects in a blend of components. A three-component augmented

simplex-centroid design has been employed, in which each component is studied in four levels,

namely 0 (0%), 1/3 (33%), 1/2 (50%) and 1 (100%) (Table 1). Quadratic- or special-cubic-

regression models were fitted for variations of all of the studied responses as a function of the

significant (p < 0.05) interaction effects between the proportions, with acceptable determination

coefficients (R² > 0.70). Equation 1 represents these models:




StatisticaTM



To confirm the validity of the models, three assays were performed under randomly

selected test conditions, and the experimental values were compared with the predicted values by

the models within a 95% confidence interval.


𝑞

𝑖<𝑗

𝑞

𝑖=1


𝑞

𝑖<𝑗<𝑘

??

182

2.4. Determination of the TCA-soluble protein

The TCA-soluble protein of the hydrolysates was determined with a modified version of

the method described by Pericˇin, Radulovic´-Popovic´, Vaštag, Madarev-Popovic´ & Trivic,

2009. A 1.0 mL aliquot of the protein hydrolysates was added to an equal volume of 0.44 mol L-1

trichloroacetic acid (TCA). The mixture was incubated for 30 min at room temperature. Then, the

mixture was centrifuged at 17,000 x g for 15 min. The obtained 0.22 mol L-1

TCA-soluble protein

fraction and the supernatant of the hydrolysate mixture (without the addition of TCA) were each

analyzed to determine the protein content using the Lowry method (Lowry, Rosenbrough & Fair,

1951), which uses bovine serum albumin as the standard protein. The TCA-soluble protein value,

expressed as a percentage, was calculated as the ratio of 0.22 mol L-1

TCA-soluble protein to the

total protein in the supernatant of the hydrolysate mixture. The assays were performed with four

replicates.

2.5. Inhibition of the relative lipid accumulation in the 3T3-L1 adipocytes

2.5.1. Cell culture

The 3T3-L1 preadipocytes (Rio de Janeiro Cell Bank, Rio de Janeiro, Brazil) were seeded

in a 24-well plate at a density of 103 cells/well. The cells were grown in Dulbecco's Modified

Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5%

CO2 humidified atmosphere. To induce preadipocytes differentiation, two days after reaching

100% confluence, the cells were stimulated for 48 h with 0.5 mM 3-isobutyl-1-methylxanthine,

0.1 µM dexamethasone, and 10 µg mL-1

insulin in DMEM supplemented with 10% FBS

(differentiation medium). Then, the preadipocytes were maintained and were fed every two days

with DMEM supplemented with 10% FBS and 10 µg mL-1

insulin (maturation medium) for an

additional eight days. For selection of the samples with the highest inhibition of the relative lipid

accumulation (RLA), the cells received 800 ppm of the intact or hydrolyzed proteins two days

postconfluence for 48 h and were fed two days with maturation medium for an additional eight

days. The control assays were performed using the cultivation medium without the protein

samples. The assays were performed in three replicates.

2.5.2. Assay for the relative lipid accumulation (RLA)

On day eight after differentiation, 3T3-L1 cells were stained with Oil Red O to determine

the intracellular lipid or triglyceride content (Green & Kehinde, 1975). The cells were fixed with

183

10% formaldehyde for 1 h at room temperature. After fixation, the cells were washed twice with

distilled water and twice with 60% isopropanol and allowed to air-dry. Then, the cells were

stained using 0.3% Oil Red O in isopropanol for 10 min at room temperature. After being stained

with Oil Red O lipid, the cells were washed with distilled water four times and were air-dried.

The Oil Red O dye-lipids complex was eluted by adding 100% isopropanol after 10 min of

incubation at room temperature. The absorbance of the complex was measured at 520 nm and

expressed as RLA (%): [(Ac-As)/(Ac)]×100, where Ac denotes the absorbance of the control cell

culture and As denotes the absorbance of the treated cell culture. The RLA suppression (%) was

calculated as follows: RLA suppression (%) = RLA (%) in the control assay - RLA (%) in the test

assay. The RLA (%) in the control assay was considered to be 100%.

2.5.3. Effect of the concentration of the protein hydrolysates and various treatments on the

RLA.

The protein hydrolysates with major RLA suppression at 800 ppm were evaluated at

various concentrations (from 400 to 1400 ppm). The assays were performed as described in

Section 2.5.1.

Two treatments were employed to examine the effect of the hydrolysates on the RLA

during preadipocyte differentiation. For treatment 1, the two-day postconfluent 3T3-L1

preadipocytes received 1200 ppm of the hydrolysates only at day zero during the early phase of

differentiation. For treatment 2, the two-day postconfluent 3T3-L1 preadipocytes received 1200

ppm of the protein hydrolysates at day zero and every two days afterwards until the end of the

experiment at day eight (from day zero to day eight). The assays were performed in three

replicates.

2.5.4. Fractionation of the hydrolysates by ultrafiltration

The hydrolysates with the highest stimulator effects on the inhibition of the relative lipid

accumulation (RLA) were subjected to ultrafiltration to assess the partitioning of active

compounds. The fractionation was performed in a series of centrifugal ultrafiltration filters with

molecular weight cut-off (MWCO) membranes of 30, 10 and 3 kDa (Millipore CorporationTM

Ultrafiltration Membranes, Billerica, USA). The effect of the fractions on the RLA (%) was

determined only using a cell culture with treatment 2. The assays were performed in three

replicates.

184


The statistical analyses were performed using the MinitabTM

16.1.1 software package

from Minitab Inc. (USA). The values were expressed as the arithmetic mean. The Tukey test was

used to test for significant differences between the groups analyzed. The differences were

considered significant at p < 0.05.

The Pearson correlation coefficient was used to measure the strength of the linear

dependence between the two responses. The correlation coefficient ranges from - 1 to 1. A value

of 1 implies that a linear equation describing the relationship between the responses was perfect

and positive, and a value of -1 indicate a perfect and negative correlation. A value of zero implies

that there is no linear correlation between the responses. The correlations between the analyzed

parameters were considered significant when p < 0.10.


3.1. Comparative analysis of the TCA-soluble protein and the RLA (%) between the

intact proteins and their hydrolysates.

The results for all of the parameters were evaluated according to two points of view: 1)

comparative analysis of the TCA-soluble protein and the RLA (%) in 3T3-L1 preadipocytes

treated with hydrolyzed and non-hydrolyzed proteins in respective runs of the statistical-mixture

design to verify the changes caused by the enzymatic hydrolysis and 2) evaluation of the

synergistic or antagonistic interactions of the hydrolyzed and non-hydrolyzed proteins and their

mixtures on the TCA-soluble protein and the RLA (%).

The interactions among the three substrates in the TCA-soluble protein and the RLA (%)

were studied in the seven assays using a simplex-centroid mixture design (Table 1). The size of

the peptides is known to be a significant factor in the overall bioactivities and functional

properties of protein hydrolysates. The assessment of proteolysis levels is often achieved by

global quantification of the peptides soluble at certain concentrations of trichloroacetic acid

(TCA). This parameter has been used as an indication for the amount of small-sized peptides in

the protein hydrolysates and has a positive correlation with the degree of hydrolysis (DH) (Zhou

et al., 2012). As expected, the TCA-soluble protein of the hydrolysates showed significant

185

changes after the enzymatic hydrolysis with increased values ranging from 52.2% (BWP) to

288.5% (EWP) compared to intact proteins (Table 1).

After enzymatic hydrolysis, the mixture containing BWP (1/2) and EWP (1/2) showed the

highest increase in the RLA suppression (258.6%), followed by EWP (84.9%) and SPI (20.6%)

mixture compared to the intact proteins (Table 1). However, the mixtures containing SPI (1/2)

plus BWP (1/2) and SPI (1/2) plus EWP (1/2) showed a decrease in the RLA suppression after

enzymatic hydrolysis. The Pearson coefficient indicates a positive correlation between the TCA-

soluble protein and the RLA (%) (Pearson coefficient = 0.64; p = 0.12).

3.2. Synergistic and antagonistic effects of the intact proteins and their hydrolysates on

the TCA-soluble protein and the RLA (%)

The analysis of the interaction between the various protein sources and their mixtures

after enzymatic hydrolysis showed synergistic and antagonistic effects. The TCA-soluble protein

showed a similar profile in the seven runs of the statistical-mixture design, ranging from 56.70 to

65.07%. Interactions between the formulations: SPI (1/2) plus EWP (1/2) (run 5), BWP (1/2) plus

EWP (1/2) (run 6) and SPI, BWP and EWP (in equal proportions) (run 7) were not statistically

significant (p < 0.05).

For RLA suppression (%), synergistic effects were observed for mixtures of intact and

hydrolyzed proteins. The mixture containing BWP (1/2) plus EWP (1/2) hydrolysates (run 6)

showed increases of up to 220 and 27% in their activities compared to those of the isolated

substrates (runs 2 and 3) after enzymatic hydrolysis, reaching a maximum RLA suppression of

15.5%. The mixture of intact proteins in equal proportions of SPI and BWP (run 4) showed RLA

suppressions of 14.1%, indicating a synergistic effect between these proteins sources, with

increases of 81.1 and 195.6%, respectively, compared to the values for the isolated substrates

(runs 1 and 2).

186

Table 1 - Matrix of the simplex-centroid mixture design for study of the suppression of the relative lipid accumulation (RLA%) and

the RLA suppression (%) of various protein sources and their hydrolysates obtained by enzymatic hydrolysis and results for the TCA-

soluble protein (%).

a, b, c...Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. Tukey tests were applied between the runs for each

response.

Mixture design TCA-soluble protein (%) RLA (%) RLA suppression (%)

Runs


x1

(SPI)

x2

(BWP)

x3

(EWP) Non-hydrolyzed Hydrolysates Non-hydrolyzed Hydrolysates Non-hydrolyzed Hydrolysates

1 1 0 0 30.70 ± 0.71a 62.47 ± 1.91

a, c 92.20 ± 2.67

a, b, c 90.59 ± 1.96

b, c 7.80 ± 2.67

a, b, c 9.41 ± 1.96

b, c

2 0 1 0 37.24 ± 1.63b 56.70 ± 1.67

b 95.22 ± 0.31

a 95.20 ± 2.34

b 4.78 ± 0.31

c 4.80 ± 2.34

c

3 0 0 1 16.42 ± 0.13c 63.79 ± 2.78

a, c 93.42 ± 2.45

a, b 87.83 ± 2.24

c, d 6.58 ± 2.45

b, c 12.17 ± 2.24

a, b

4 1/2 1/2 0 36.03 ± 0.99b 65.07 ± 1.67

a 85.87 ± 4.17

c 103.81 ± 0.67

a 14.13 ± 4.17

a -3.81 ± 0.67

d

5 1/2 0 1/2 17.42 ± 0.24c 61.72 ± 0.71

a, c 86.61 ± 3.01

b, c 93.71 ± 2.03

b, c 13.39 ± 3.01

a, b 6.29 ± 2.03

b, c

6 0 1/2 1/2 16.99 ± 0.11c 59.58 ± 2.06

b, c 95.66 ± 2.92

a 84.46 ± 1.17

d 4.34 ± 2.92

c 15.54 ± 1.17

a

7 1/3 1/3 1/3 17.65 ± 0.12c 62.28 ± 1.41

a, c 88.82 ± 0.48

a, b, c 89.70 ± 3.39

b, c, d 11.18 ± 0.48

a, b, c 10.30 ± 3.39

a, b, c

187

The limited hydrolysis of dietary proteins may give rise to particularly interesting

functional, organoleptic food properties, and it was required to maintain the structure or sequence

of the active peptides. Several studies have indicated that hydrolysis led to a reduction or increase

in the specific functionality, as was observed in our study. Tsou, Kao, Tseng & Chiang, (2010)

studied the effect of soy protein hydrolyzed with FlavourzymeTM

with DH 5.46–17.86% on the

suppression of the RLA in 3T3-L1 preadipocytes and showed that extensive hydrolysis (DH >

8.06%) resulted in a decrease of the RLA inhibition. Tsou, Lin, Lu, Tsui & Chiang (2010)

evaluated the effect of the limited hydrolysis of isolated soy protein with Neutrase on RLA

suppression in 3T3-L1 cells during differentiation and showed that the RLA suppression

increased with the DH and TCA-soluble protein increased, reaching approximately 13% of the

maximum RLA inhibition with DH 9.94-15.19%. On the other hand, with DH 18.08%, the RLA

inhibition decreases to 2.5%. Martinez-Villaluenga, Bringe, Berhow & Mejia (2008) tested the

effects of soy hydrolysates on RLA in 3T3-L1 adipocytes and showed the inhibition of lipid

accumulation of soy hydrolysates ranged from 29 to 46%.

3.3. Mixture-contour plots for TCA-soluble protein and RLA (%)

The variations in the TCA-soluble protein and the RLA (%) of the hydrolysates obtained

from SPI, BWP and EWP are also shown using mixture-contour plots (Fig. 1). On the response

surfaces, each factor (pure mixture component) is represented in the corner of an equilateral

triangle, and each point within this triangle refers to a different proportion of the components in

the mixture. The maximal percentage of each ingredient considered by the regression was placed

at the corresponding corner, and the minimal percentage was positioned at the middle of the

opposite side of the triangle (Martinello, Kaneko, Velasco, Taqueda & Consiglieri, 2006). A

contour plot provides a two-dimensional view in which all points that have the same response are

connected to produce contour lines of constant responses (Rao & Baral, 2011). The high levels of

TCA-soluble protein were observed in the mixtures contained SPI and BWP. This can be verified

by observing the mixture-surface plots (Fig. 1a).

The maximum responses for RLA suppression (%) were detected in the EWP

hydrolysates and mixtures containing BWP and EWP, reaching values of 12.2 and 15.5%,

respectively (Fig. 1c). The plots of the experimental versus the predicted responses suggested that

the experimental points were reasonably aligned, an indicator of the normal distribution (Fig. 1).

188

Fig. 1. Mixture-contour plots and fitted line plots between the experimental and the predicted

values for the TCA-soluble protein (a), the RLA (%) (b) and the RLA suppression (%) (c) of the

protein hydrolysates.

a

b

c

> 65 < 65 < 63 < 61 < 59 < 57

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

52 54 56 58 60 62 64 66 68

Experimental values

55

56

57

58

59

60

61

62

63

64

65

66

Pre

dic

ted v

alues

> 99 < 99 < 97 < 93 < 89 < 85

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

80 85 90 95 100 105 110

Experimental values

82

84

86

88

90

92

94

96

98

100

102

104

106

Pre

dic

ted v

alu

es

> 14 < 14 < 12 < 10 < 8 < 6 < 4 < 2

0.00

0.50

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

0 2 4 6 8 10 12 14 16 18 20

Experimental values

0

2

4

6

8

10

12

14

16

18

Pre

dic

ted v

alues

189

3.4. Analysis of variance (ANOVA) and models for the TCA-soluble protein and the

RLA (%) of the intact proteins and their hydrolysates


and recorded in Table 1. The experiments were conducted in triplicate, and it was found that in

nearly all cases, there exists good agreement between the replicates. All of the independent and

response variables were fitted to linear, quadratic or special-cubic models. The coefficient of

determination R2 and the F test (analysis of variance-ANOVA) were used to verify the quality of

the fit of the models. Table 2 shows the models and the corresponding R2

of the regression

equations for the responses, as well as the corresponding F-ratio and p-values for each term in the

predicted regression equations. The high coefficients of determination (R2), which were above

0.70 (Table 2), indicate that all of the response functions adequately fitted the experimental data

and that the models could be used for predictive purposes in the determination of the TCA-

soluble protein and RLA (%), using the different protein sources and their mixtures. The negative

quadratic (binary) and cubic (ternary) terms of the fitted regression equation showed the

antagonistic effects, and the positive quadratic and cubic terms indicated synergistic effects of the

protein sources on the TCA-soluble protein and the RLA (%).

The highest values for the RLA (%) were detected in the 3T3-L1 pre-adipocytes treated

with non-hydrolyzed bovine whey protein (x2) followed by egg white protein (x3) and soy protein

isolate (x1). Regarding to the hydrolysates, the regression coefficients indicated higher values for

RLA (%) in the 3T3-L1 pre-adipocytes treated with bovine whey protein (x2), followed by soy

protein isolate (x1) and egg white protein (x3) (Table 2). It is important to note that, in both cases,

the higher values of the RLA (%) indicate a lower anti-adipogenic activity and so a lower

coefficient is more desirable. The binary and the ternary interactions of the protein hydrolysates

had significant (p < 0.05) effects, whereas for the non-hydrolyzed proteins, only the interactions

of soy protein isolate (x1) with bovine whey protein (x2) and bovine whey protein (x2) with egg

white protein (x3) were found to be significant (Table 2).

190

Table 2 - Analysis of variance (ANOVA), including models, R2 and probability values for the

final reduced models for the TCA-soluble proteins and the RLA (%).

Response: TCA-soluble proteins (non-hydrolyzed proteins)


squares

Degrees of

freedom

Mean of

squares Fcalculated/Ftabulated R² p-value

Regression 2,208.97 6 368.16 613.60/2.07 0.99 <0.001

Residual 12.69 21 0.60

Total 2,221.66

Special cubic model: Y = 30.70x1 + 37.24x2 + 16.42x3 + 8.25x1x2 – 24.55x1x3 – 39.35x2x3 – 115.61x1x2x3

Response: TCA-soluble proteins (protein hydrolysates)


squares

Degrees of

freedom

Mean of


Regression 184.62 5 36.92 11.43/2.13 0.70 <0.001

Residual 71.12 22 3.23

Total 255.74

Quadratic model: Y = 62.00x1 + 56.51x2 + 63.05x3 + 22.04x1x2

Response: RLA % (non-hydrolyzed proteins)


squares

Degrees of

freedom

Mean of


Regression 283.62 4 70.90 10.87/2.33 0.73 <0.001

Residual 104.41 16 6.52

Total 388.03

Quadratic model: Y = 92.09x1 + 95.73x2 + 93.94x3 – 30.48x1x2 – 23.93x1x3

Response: RLA % (protein hydrolysates)


squares

Degrees of

freedom

Mean of


Regression 701.83 6 116.97 25.76/2.24 0.92 <0.001

Residual 63.50 14 4.54

Total 765.33

Special cubic model: Y = 90.59x1 + 95.20x2 + 87.83x3 + 43.66 x1x2 + 17.98x1x3 – 28.22x2x3 – 140.89x1x2x3

191


obtained for the TCA-soluble protein and the RLA (%) of the protein hydrolysates using various

formulations with three assays (Table 3). According to the regression models (Table 2), the

experimental values agreed with the values predicted by the models within a 95% confidence

interval, thereby confirming the validity of the models for the evaluated responses (Table 3).


obtained for the TCA-soluble protein of the protein hydrolysates and the RLA (%) for the 3T3-L1

preadipocytes treated with various formulations of the protein hydrolysates at 800 ppm.


Comparisons were made between the observed and predict values for each correspondent response.

3.5. Effect of the concentration of protein hydrolysates and various treatments on the

RLA.

The binary mixture of intact proteins containing SPI (1/2) and BWP (1/2) and the

hydrolyzed mixture of BWP (1/2) and EWP (1/2) showed the greatest RLA suppression in 3T3-

L1 cells at 800 ppm, so the effect of the sample concentration was evaluated. The 3T3-L1

preadipocytes were treated with 400-1400 ppm of the SPI and BWP (non-hydrolyzed) and the

hydrolyzed mixture of BWP and EWP during differentiation and a dose-dependent reduction of

the RLA was observed. The Pearson coefficient indicated a positive and significant correlation

between the concentration and the RLA inhibition (%) for both samples (Pearson coefficient >

0.84; p < 0.04). For the mixture containing SPI plus BWP (non-hydrolyzed), the higher RLA

inhibitions were 20.08 and 20.94% at 1000 and 1400 ppm, respectively. As for the hydrolyzed

mixture of BWP and EWP, the maximum RLA inhibition (32.78%) was detected at a

concentration of 1200 ppm (Fig. 2).

Responses


response

Experimental

response x1

(SPI)

x2

(BWP)

x3

(EWP)

TCA-soluble protein (%) 0.33 0.34 0.33 62.95a 61.07 ± 2.85

a

RLA (%) 1 1.00 0.00 0.00 90.59b 83.57 ± 8.07

b

RLA (%) 2 0.00 0.50 0.50 84.46c 87.24 ± 9.25

c

RLA (%) 3 0.33 0.33 0.34 89.56d 85.47 ± 5.82

d

192

Fig. 2. Effect of the concentration of intact proteins containing SPI (1/2) and BWP (1/2) and the

protein hydrolysates of BWP (1/2) and EWP (1/2) on the RLA suppression (%) in 3T3-L1

preadipocytes.

To study the effect of various treatments on the RLA (%) in the 3T3-L1 cells, two

treatments were performed using the hydrolyzed mixture of BWP (1/2) and EWP (1/2). The

results showed that the RLA (%) decreased significantly for both treatments. For treatment 1, in

which the two-day postconfluent 3T3-L1 preadipocytes received 1200 ppm of the hydrolysates

only at day zero during the early phase of differentiation, an RLA of 69.43% was observed,

resulting in the suppression of 30.57%. Treatment 2, in which the two-day postconfluent 3T3-L1

preadipocytes received 1200 ppm of the protein hydrolysates at day zero and every two days

afterwards until the end of the experiment at day eight, proved to be more effective, reaching an

RLA of 52.07% and, consequently, a suppression of 47.93% (Fig. 3).

0

5

10

15

20

25

30

35

400 600 800 1000 1200 1400

RL

A s

upp

ress

ion (

%)

Concentration (ppm)

Non-hydrolyzed Hydrolysates

193

Fig. 3. Effect of the various treatments on the RLA (%) in 3T3-L1 preadipocytes using the

hydrolyzed mixture of BWP (1/2) and EWP (1/2) at 1200 ppm.

3.6. Fractionation of the hydrolysates by ultrafiltration.

The hydrolyzed mixture of BWP (1/2) and EWP (1/2) was sequentially ultrafiltered to

obtain three fractions: (i) permeate 30 kDa (<30 kDa), (ii) permeate 10 kDa (<10 kDa) and (iii)

permeate 3 kDa (<3 kDa). Fig. 4 shows the effect of 1200 ppm of various fractions on the RLA

(%) at day eight during the 3T3-L1 cell differentiation with treatment 2. All of the fractions and

the non-fractionated sample showed a much lower RLA (%) than the control. The RLA varied

significantly ranging between 52.07 and 83.75%, resulting in suppressions of 47.93 and 16.25%

for the non-fractionated sample and the 3 kDa permeate, respectively. These results showed that

there is an important contribution of fractions of different sizes in the inhibition of lipid

accumulation in the 3T3-L1 cells during differentiation; thus, the use of ultrafiltration as a

concentration step subsequently became unnecessary, which was a positive aspect for the

process.

The sequential fractionation of protein hydrolysates by ultrafiltration has been reported to

separate and concentrate active compounds, enhancing their biological and functional properties.

Tsou, Kao, Tseng & Chiang (2010) investigated the anti-adipogenic activity of soy-protein-

isolate hydrolysates obtained by enzymatic hydrolysis using FlavourzymeTM

and further

separated by sequential ultrafiltration to obtain fractions ranging from 1 to 30 kDa. These authors

0

20

40

60

80

100

Control Treatment 1 Treatment 2

RL

A (

%)

194

reported that, among these fractions, the 1-kDa permeate had the highest anti-adipogenic effect,

resulting in a reduction of 59% in the activity of glycerol-3-phosphate dehydrogenase (GPDH),

an enzyme that has an important function in the metabolism linking glycolysis to phospholipid

and triglyceride biosynthesis (Harding, Pyeritz, Copeland & White, 1975). The sequential

ultrafiltration using membranes of 10 and 3 kDa was reported as an auxiliary step in the

purification of an adipogenesis-inhibitory peptide from black-soybean-protein hydrolysate by

Kim, Bae, Ahn, Lee & Lee (2007). Tsou, Lin, Lu, Tsui & Chiang (2010) reported that the

sequential fractionation of isolated soy protein hydrolyzed with Neutrase using ultrafiltration

proved to be a useful way to increase its anti-adipogenic activity, resulting in a reduction in the

GPDH activity and lipid accumulation in the 3T3-L1 preadipocyte cells treated with a 1-kDa

concentrate at 400 ppm.

Fig. 4. Effect of various ultrafiltered fractions of the hydrolyzed mixture of BWP (1/2) and EWP

(1/2) at 1200 ppm on the RLA (%) at day eight during 3T3-L1 preadipocyte differentiation with

treatment 2.

0

20

40

60

80

100

Control Not fractionated < 30 kDa < 10 kDa < 3 kDa

RL

A (

%)

Fractions

195

4. Conclusions

The results suggest that the application of the statistical-mixture design for the enzymatic

hydrolysis of various protein sources is an attractive process for improving the performance by

identifying the optimum mixture formulations of proteins with specific characteristics, for

example, increased RLA (%) suppression. The mixture containing BWP (1/2) plus EWP (1/2)

showed increases of up to 220 and 27% in their activities compared to those of the isolated

substrates after enzymatic hydrolysis, reaching a maximum RLA suppression of 15.5% at 800

ppm. The correlation analysis indicated a positive and significant correlation between the samples

concentration and the RLA suppression (%), reaching a maximum value of 47.93% for the

hydrolyzed mixture of BWP (1/2) and EWP (1/2) at 1200 ppm. Sequential fractionation of the

hydrolyzed mixture of BWP (1/2) and EWP (1/2) using ultrafiltration became unnecessary for

this process because the non-fractionated sample had the highest anti-adipogenic activity.

Acknowledgments

The work described in this paper was substantially supported by grants from São Paulo

Research Foundation – FAPESP (Project No. 2011/10429-9), the Department of Food Science,

the School of Food Engineering and the Department of Anatomy, Institute of Biology, University

of Campinas, which are gratefully acknowledged.

References

Castro, R. J. S. & Sato, H. H. (2013). Synergistic effects of agroindustrial wastes on simultaneous


mixture design. Industrial Crops and Products, 49, 813-821.

Chen, C., Chi, Y-J., Zhao, M.-Y. & Xu, W. (2012). Antioxidant and ACE inhibitory activities of

egg white protein hydrolysate. Food Science and Biotechnology, 21(1), 27-34.

Green, H. & Kehinde, O. (1975). An established preadipose cell line and its differentiation in

culture. II. Factors affecting the adipose conversion. Cell, 5, 19–27.

Harding, J. W., Pyeritz, E. A., Copeland, E. S. & White, H. B. (1975). Role of glycerol 3-

phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken

liver, Biochemical Journal, 146, 223–229.

196

Hoppe, A., Jung, S., Patnaik, A. & Zeece, M. G. (2013). Effect of high pressure treatment on egg

white protein digestibility and peptide products. Innovative Food Science and Emerging

Technologies, 17, 54–62.

Kim, H. J., Bae, I.Y., Ahn, C-W., Lee, S. & Lee, H. G. (2007). Purification and identification of

adipogenesis inhibitory peptide from black soybean protein hydrolysate, Peptides, 28, 2098-2103.

Korhonen, H. (2009). Milk-derived bioactive peptides: from science to applications. Journal of

Functional Foods, 1, 177-187.

Lowry, O. H., Rosenbrough, N. J., Fair, A. L. & Randall, R. J. (1951). Protein measurement with

the Folin-phenol reagents. The Journal of Biological Chemistry, 193, 265–275.

Martinello, T., Kaneko, T. M., Velasco, M. V. R., Taqueda, M. E. S. & Consiglieri, V. O.

(2006). Optimization of poorly compactable drug tablets manufactured by direct compression

using the mixture experimental design. International Journal of Pharmaceutics, 322, 87-95.

Martinez-Villaluenga, C., Bringe, N. A., Berhow, M. A. & Mejia, E. G. (2008). β-conglycinin

embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro. Journal of

Agricultural and Food Chemistry, 56, 10533–10543.

Mejia, E. G., Martinez-Villaluenga, C., Roman, M. & Bringe, N. A. (2010). Fatty acid synthase

and in vitro adipogenic response of human adipocytes inhibited by α and α´ subunits of soybean

β-conglycinin hydrolysates. Food Chemistry, 119, 1571–1577.

Naik, L., Mann, B., Bajaj, R., Sangwan, R. B. & Sharma, R. (2013). Process optimization for the


International Journal of Peptide Research and Therapeutics, 19, 231-237.

Pericˇin, D., Radulovic´-Popovic´, L., Vaštag, Zˇ., Madarev-Popovic´, S. & Trivic, S. (2009).


surface methodology. Food Chemistry, 115, 753–757.

Poulos, S. P., Dodson, M. V. & Hausman, G. J. (2010). Cell line models for differentiation:

preadipocytes and adipocytes. Experimental Biology and Medicine, 235, 1185–1193.

197

Rahman, M. A., Kumar, S. G., Kim, S. W., Hwang, H. J., Baek, Y. M., Lee, S. H. et al. (2008).

Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte

differentiation. Proteomics, 8, 569–581.

Rao, P. V. & Baral, S. S. (2011). Experimental design of mixture for the anaerobic co-digestion

of sewage sludge. Chemical Engineering Journal, 172, 977-986.

Rubin, C. S., Hirsch, A., Fung, C. & Rosen, O. M. (1978). Development of hormone receptors

and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte

and adipocyte forms of 3T3-L1 cells, The Journal of Biological Chemistry, 253, 7570–7578.

Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S. & Bashmakov, Y. (1998). Insulin

resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose

tissue: Model for congenital generalized lipodystrophy. Genes and Development, 12, 3182–3194.

Tsou, M. J., Kao, F. J., Tseng, C. K. & Chiang, W. D. (2010). Enhancing the anti-adipogenic


Chemistry, 122, 243–248.

Tsou, M. J., Lin, W. T., Lu, H. C., Tsui, Y. L. & Chiang, W. D. (2010b). The effect of limited

hydrolysis with Neutrase and ultrafiltration on the anti-adipogenic activity of soy protein. Process

Biochemistry, 45, 217–222.

Zhao, Q., Selomulya, C., Wang, S., Xiong, H., Chen, X. D., Li, W., Peng, H., Xie, J., Sun, W. &

Zhou, Q. (2012). Enhancing the oxidative stability of food emulsions with rice dreg protein

hydrolysate. Food Research International, 48, 876–884.

Zhou, D., Qin, L., Zhu, B., Li, D., Yang, J., Dong, X. & Murata, Y. (2012). Optimisation of


methodology and evaluation of in vitro antioxidant activity of the hydrolysate. Journal of the

Science of Food and Agriculture, 92, 1694–1701.

199

Capítulo IX: Atividade antimicrobiana de hidrolisados de proteína isolada

de soja, soro de leite e clara de ovo.

200

Resumo

Processos envolvendo a hidrólise de proteínas têm sido estudados para a produção de peptídeos

com atividade biológica. No presente trabalho, hidrolisados de proteína isolada de soja, soro de

leite e clara de ovo foram preparados utilizando uma preparação comercial de protease de

Aspergillus oryzae (Flavourzyme® 500L) para a obtenção de peptídeos com atividade

antimicrobiana utilizando planejamento experimental de misturas. As hidrólises enzimáticas

foram conduzidas em frascos Erlenmeyers contendo 50 mL de solução de proteína (100 mg mL-1

)

durante 2 h a 50 °C e pH 5,0. Para a avaliação da atividade antimicrobiana das amostras de

proteínas hidrolisadas e não hidrolisadas foram utilizadas três culturas de leveduras: Candida

albicans ATCC 10231, Saccharomyces cerevisiae KL 88 e Kluyveromyces marxianus NRRL

7571 e três culturas bacterianas: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC

11229 e Salmonella choleraesuis ATCC 14028. Os resultados mostraram que na maior parte dos

ensaios, a suplementação dos meios de cultivo com fontes de proteínas estimulou o crescimento

das bactérias patogênicas. A linhagem de S. aureus ATCC 6538 foi a única que apresentou

inibição significativa do crescimento quando cultivada em meio suplementado com uma mistura

binária de proteína isolada de soja (1/2) e proteínas da clara de ovo (1/2) não hidrolisadas,

resultando em inibição de 16,82%. Para as linhagens de leveduras, não foram observadas

mudanças nos perfis de inibição do crescimento quando comparadas as amostras hidrolisadas e

não hidrolisadas. A maior inibição observada foi detectada para a linhagem de S. cerevisiae KL

88 cultivada em meio suplementado com a mistura ternária de proteínas hidrolisadas em

proporções iguais, resultando em inibição de 15,42%.

Palavras-chave: proteases; hidrólise enzimática; hidrolisados proteicos; atividade

antimicrobiana.

201

1. Introdução

Processos envolvendo hidrólise de proteínas têm sido estudados para a produção de

peptídeos com atividade biológica. Peptídeos bioativos são definidos como frações específicas de

proteínas com sequência de aminoácidos que promovem um impacto positivo em várias funções

biológicas, incluindo efeitos como atividades: antioxidante, anti-hipertensiva, antitrombótica,

antiadipogênica e antimicrobiana (Zhang et al., 2010; Biziulevicius et al., 2006; Tsou et al.,

2010; Tavares et al., 2011). Estes peptídeos apresentam sequências de 2-20 aminoácidos e

massas moleculares inferiores a 6000 Da. A bioatividade é definida principalmente pela

composição e sequência de aminoácidos (Sarmadi e Ismail, 2010). Essa enorme diversidade

funcional coloca os peptídeos e as proteínas em posição de destaque no campo das aplicações

biotecnológicas (Miranda e Liria, 2008), sendo apontados por alguns autores como possíveis

substitutos de substâncias químicas utilizadas como fármacos ou conservadores de alimentos

(Hong et al., 2008).

Uma das formas mais comuns e rentáveis de produzir peptídeos bioativos é através da

hidrólise enzimática de proteínas (Hernández-Ledesma et al., 2011). Esse processo oferece

algumas vantagens, como: emprego de enzimas em concentrações muito baixas, reações rápidas

em condições suaves e alta especificidade, gerando um produto livre de resíduos químicos e com

melhores propriedades funcionais e nutricionais (Adler-Nissen, 1981).

Dentre as várias atividades biológicas de peptídeos, encontram-se os que apresentam a

capacidade de inibir o crescimento de micro-organismos. Peptídeos antimicrobianos estão

amplamente distribuídos na natureza e representam um componente essencial do sistema

imunológico. Eles são reconhecidamente, a primeira linha de defesa do organismo contra a

colonização de micro-organismos exógenos, com papel fundamental na regulação de populações

bacterianas em mucosas e outras superfícies epiteliais (Boman e Hultmark, 1987; Bevins e

Zasloff, 1990). Mais de 800 peptídeos antimicrobianos já foram caracterizados em plantas e

animais (Boman, 2003). Apesar da diversidade na estrutura primária, a grande maioria dos

peptídeos antimicrobianos possui cadeias curtas de aminoácidos, que são caracterizadas pela

predominância de aminoácidos catiônicos e hidrofóbicos. Embora haja diferenças significativas

nas estruturas secundária e terciária, peptídeos antimicrobianos são geralmente compostos por

uma superfície hidrofóbica e uma hidrofílica. O caráter anfipático destas moléculas é definitivo

202

no mecanismo de ação antimicrobiana permitindo uma maior interação com a membrana

bacteriana (Dashper et al., 2007). Em adição a característica anfipática, a reduzida massa

molecular das frações peptídicas, com consequente maior exposição dos resíduos de aminoácidos

e suas cargas, e a formação de pequenos canais na bicamada lipídica, foram relacionados com o

poder antimicrobiano, pois causam modificações que aumentam a interação peptídeo-membrana

(Gobetti et al., 2004, Patrzykat e Douglas, 2005, Gómez-Guillén et al., 2010).

Na literatura, diferentes fontes de proteínas têm sido utilizadas para a hidrólise

enzimática, tal como arroz, proteínas de clara de ovo e do soro de leite (Zhao et al., 2012; Naik et

al., 2013; Hoppe et al., 2013). No entanto, estas pesquisas relatam processos de hidrólise

enzimática utilizando substratos separadamente; nenhum trabalho foi encontrado utilizando

formulações contendo misturas de diferentes fontes de proteína.

O planejamento de misturas é uma classe especial de delineamento experimental, no qual

as proporções entre os componentes ou fatores, assim como as interações entre os mesmos e os

seus efeitos sobre a variável resposta podem ser utilizados para maximizar resultados e

aperfeiçoar processos. A utilização desta técnica permite um melhor entendimento dos dados

experimentais, pois inclui avaliação estatística e geração de gráficos e modelos que facilitam a

interpretação dos resultados assim como a verificação de efeitos sinérgicos ou antagônicos entre

os componentes das misturas.

Neste contexto, o objetivo do presente trabalho foi avaliar a atividade antimicrobiana de

proteínas de diferentes fontes utilizando a técnica de planejamento experimental de misturas.

Amostras de proteína isolada de soja, proteínas do soro de leite e da clara de ovo isoladas ou

combinadas em formulações binárias e/ou ternárias não hidrolisadas e hidrolisadas

enzimaticamente foram utilizadas na suplementação de meios de cultivo para avaliação do efeito

no crescimento de linhagens de bactérias e leveduras.

2. Material e métodos

2.1. Protease

A protease comercial Flavourzyme® 500L de Aspergillus oryzae foi utilizada para

obtenção dos hidrolisados proteicos (Sigma Aldrich).

203

2.2. Determinação da atividade de protease

A atividade proteolítica utilizando azocaseína como substrato foi determinada segundo a

metodologia de Charney e Tomarelli (1947), com modificações. A mistura reacional contendo

0,5mL de azocaseína (0,5% p/v), pH 5,0 e 0,5mL de solução enzimática foi incubada por 40 min

na temperatura ótima de atividade da preparação enzimática (pH 5,0 e 50 °C). A reação foi

paralisada pela adição de 1,0 mL de ácido tricloroacético (TCA 10%). A mistura reacional foi

centrifugada a 17.000 x g e o sobrenadante coletado. A formação do composto cromóforo

ocorreu pela adição de 1,0 mL de solução de hidróxido de potássio 5M a 1,0mL do sobrenadante

da mistura reacional centrifugada e a leitura de absorbância foi realizada a 428 nm. Uma unidade

de atividade proteolítica foi definida como a quantidade de enzima que produz uma diferença de

0,01 na absorbância a 428 nm por minuto de reação entre o branco reacional e a amostra nas

condições do ensaio.

2.3. Obtenção dos hidrolisados proteicos

Proteína isolada de soja, proteínas do soro de leite e proteínas da clara de ovo foram

utilizadas para a preparação de hidrolisados utilizando planejamento experimental de misturas.

Amostras de 50 mL das soluções de proteínas 10% (p/v) e protease Flavourzyme® 500L (50 U

por mL de mistura reacional) foram incubadas em pH 5,0, 50 °C sob agitação de 100 rpm durante

2h. Após a hidrólise, as soluções foram submetidas a tratamento térmico (100 °C por 20 min)

para inativação das proteases. As amostras foram centrifugadas a 17.000 x g a 5 °C por 20 min e

os sobrenadantes contendo os peptídeos bioativos foram coletados, congelados e liofilizados para

determinação da atividade antimicrobiana. O processo de hidrólise enzimática utilizando o

planejamento experimental de misturas englobou sete ensaios com os componentes avaliados em

4 níveis: 1) 0 (0%), 2) 1/3 (33%), 3) 1/2 (50%) e 4) 1 (100%) como mostrado na Tabela 1. Todos

os experimentos realizados foram analisados comparativamente com as suas respectivas amostras

não hidrolisadas.

204

Tabela 1 – Matriz do planejamento experimental de misturas para obtenção de peptídeos com

atividade antimicrobiana através da hidrólise enzimática de proteína isolada de soja (x1),

proteínas do soro de leite (x2) e proteínas da clara de ovo (x3).

Equações quadráticas ou cúbicas foram utilizadas para definição de modelos para cada

variável estudada, como mostrado abaixo:


𝑞

𝑖<𝑗

𝑞

𝑖=1


𝑞

𝑖<𝑗<𝑘

onde 𝑌𝑖 representa a resposta estimada pelo modelo, q corresponde ao número de componentes

no sistema, ‘Xi, Xj, Xk’ correspondem às variáveis independentes codificadas, βi representa os

coeficientes de regressão linear para o efeito de cada termo, βij and βijk correspondem ao efeito de

interação entre as misturas binárias e ternárias. O coeficiente de correlação múltipla (R2) e o teste

de Fisher (análise de variância-ANOVA) foram utilizados para verificar a adequação estatística

dos modelos propostos codificados aos pontos reais. O software Statistica®

10 da Statsoft Inc.

(Tulsa, Oklahoma, EUA) foi utilizado para o planejamento experimental, análise de dados e

construção de modelos.

A variação dos resultados utilizando diferentes proporções de proteínas também foi

avaliada através de curvas de contorno, nas quais, cada fator é representado em um canto de um

triângulo equilátero, sendo cada ponto dentro do triângulo referente a uma proporção diferente de

componentes na mistura. A porcentagem máxima de cada componente considerado pela

regressão é colocada no canto correspondente, enquanto o mínimo é posicionado no meio do lado

oposto do triângulo e o centro representa a mistura dos três componentes em partes iguais.

Ensaios

Variáveis independentes

Proteína isolada de

soja

Proteínas do soro de

leite

Proteínas da clara de

ovo

x1 x2 x3

1 1 0 0

2 0 1 0

3 0 0 1

4 1/2 1/2 0

5 1/2 0 1/2

6 0 1/2 1/2

7 1/3 1/3 1/3

205

2.4. Determinação da atividade antimicrobiana

2.4.1. Micro-organismos e condições de cultivo

Para a avaliação da atividade antimicrobiana dos hidrolisados de proteína isolada de soja,

soro de leite e clara de ovo, foram utilizadas três culturas de leveduras: Candida albicans ATCC

10231, Saccharomyces cerevisiae KL 88 e Kluyveromyces marxianus NRRL 7571 e três culturas

bacterianas: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229 e Salmonella

choleraesuis ATCC 14028, previamente cultivadas em Ágar YM (para as linhagens de leveduras)

ou Ágar Nutriente (para as linhagens bacterianas) e mantidas em estoque a 4-8 °C. As culturas

foram inicialmente reativadas em 50 mL de caldo YM ou caldo nutriente durante 24 h a 30 °C

(para as linhagens de leveduras) ou 37 °C (para as linhagens bacterianas) sob agitação de 100

rpm. Em seguida, alíquotas de 1 mL das suspensões microbianas reativadas foram transferidas

para frascos Erlenmeyer de 125 mL com 50 mL de caldo YM ou caldo nutriente e incubados a 30

ou 37 °C e 100 rpm durante 8h. As suspensões microbianas foram previamente diluídas no meio

de cultura correspondente para obtenção da concentração final de inóculo desejada para os testes

de atividade antimicrobiana.

2.4.2. Determinação da atividade antimicrobiana

O crescimento microbiano foi monitorado utilizando-se o leitor de microplacas Novo

Star® (BMG LABTECH, Alemanha) a 600 nm. As medições foram realizadas em microplacas de

96 poços (TPP). Cada poço continha 100 µL da suspensão microbiana diluída em caldo YM ou

caldo nutriente com densidade óptica (DO) inicial ajustada para 0,2 a 600 nm e 100 µL das

soluções de proteína (0,5 mg mL-1

), previamente dissolvidas no meio de cultivo correspondente e

esterilizadas por filtração em membranas de 0,2 μm. As microplacas foram incubadas durante

24h a 30 ou 37 °C com posterior medida da absorbância a 600 nm. O experimento controle

consistiu no cultivo de 100 µL das suspensões microbianas e 100 µL de caldo YM ou caldo

nutriente (sem a adição dos hidrolisados proteicos) nas mesmas condições descritas

anteriormente. Os ensaios foram realizados com seis replicatas. O crescimento microbiano,

expresso em termos percentuais, foi calculado como a razão da DO600 dos meios contendo as

amostras de hidrolisados proteicos e a DO600 do ensaio controle.

206

2.5. Análises estatísticas

Os resultados foram analisados estatisticamente pelo teste de Tukey, realizado com

auxílio do software Minitab® 16.1.1 de Minitab Inc. (EUA). Os valores foram expressos como

média aritmética e considerados estatisticamente diferentes quando os valores de p foram

inferiores a 0,05.

3. Resultados e Discussão

Os resultados obtidos para atividade antimicrobiana de proteína isolada de soja, proteínas

do soro de leite e da clara de ovo hidrolisadas e não hidrolisadas estão apresentados nas Tabelas 2

e 3. Em termos gerais, as proteínas avaliadas neste estudo, nas condições de ensaio descritas,

apresentaram baixa ou nenhuma capacidade de inibição dos micro-organismos. Na maior parte

dos ensaios, a suplementação dos meios de cultivo com fontes de proteínas estimulou o

crescimento das bactérias patogênicas. É válido ressaltar que embora este estímulo tenha sido

observado, foi possível detectar mudanças nos perfis de crescimento das linhagens bacterianas

em uma análise comparativa entre os meios suplementados com proteínas não hidrolisadas e

hidrolisadas. A hidrólise enzimática das proteínas diminuiu o estímulo ao crescimento, embora os

valores ainda tenham sido superiores aos respectivos ensaios controle (100%) (Tabela 2) (Figura

1).

A linhagem de S. aureus ATCC 6538 mostrou o maior crescimento percentual dentre os

micro-organismos avaliados, atingindo 186,78%, na presença de proteína isolada de soja não

hidrolisada. Por outro lado, quando cultivada em meio suplementado com uma mistura binária de

proteína isolada de soja e proteínas da clara de ovo (ensaio 5) não hidrolisadas, esta mesma

linhagem apresentou crescimento de 83,18%, resultando em inibição de 16,82% do crescimento,

quando comparada ao ensaio controle (100%). Para as linhagens de E. coli ATCC 11229 e S.

choleraesuis ATCC 14028 não foram observadas inibições significativas no crescimento em

nenhum dos ensaios realizados (Tabela 2).

207

Para as linhagens de leveduras avaliadas, não foram observadas mudanças nos perfis de

inibição do crescimento quando comparadas as amostras hidrolisadas e não hidrolisadas. O

crescimento médio para as linhagens avaliadas variou de 84,5 a 95,5%, indicando assim níveis de

inibição entre 4,5 e 15,5%. A maior inibição observada foi detectada para a linhagem de S.

cerevisiae KL 88 cultivada em meio suplementado com a mistura ternária de proteínas

hidrolisadas em proporções iguais (ensaio 7), resultando em crescimento relativo de 84,58% e

consequente inibição de 15,42% (Tabela 3).

208

Tabela 2 - Estudo da atividade antimicrobiana de hidrolisados de proteína isolada de soja (x1), proteínas do soro de leite (x2) e

proteínas da clara de ovo (x3): composições das misturas e crescimento percentual de linhagens de bactérias patogênicas.

a, b, c Os resultados estão apresentados como média aritmética (n = 6) ± desvio padrão e as letras diferentes indicam diferença estatística entre os diferentes ensaios (p < 0,05).

Ensaios

Escherichia coli ATCC 11229 Salmonella choleraesuis ATCC 14028 Staphylococcus aureus ATCC 6538

Não hidrolisados Hidrolisados Não hidrolisados Hidrolisados Não hidrolisados Hidrolisados

1 127,98 ± 7,53a, b, c

116,08 ± 8,65b 119,25 ± 4,92

a 109,38 ± 2,25

a, b 186,78 ± 2,42

e 132,31 ± 6,35

d

2 133,43 ± 6,65a 130,43 ± 4,26

a 121,69 ± 0,90

a 114,51 ± 4,15

a 112,04 ± 4,03

c 159,62 ± 1,66

e

3 113,04 ± 6,66e 127,48 ± 7,56

a 105,95 ± 1,63

b 110,59 ± 2,96

a, b 110,56 ± 6,35

b, c 117,97 ± 5,66

b

4 131,44 ± 2,48a, b

122,78 ± 3,82a, b

118,43 ± 2,41a 107,06 ± 2,12

b, c 155,25 ± 8,06

d 93,77 ± 4,16

a

5 117,90 ± 3,06d, e

122,50 ± 2,08a, b

105,38 ± 2,80b 99,95 ± 4,05

d 83,18 ± 1,32

a 128,77 ± 6,23

c, d

6 120,13 ± 4,51c, d, e

123,42 ± 8,74a, b

105,78 ± 5,25b 101,52 ± 3,51

c, d 106,25 ± 6,31

b, c 92,86 ± 1,88

a

7 123,36 ± 7,37b, c, d

113,30 ± 4,67b 103,55 ± 4,35

b 94,13 ± 3,60

e 104,32 ± 2,64

b, c 121,03 ± 5,06

b, c

209

Tabela 3 - Estudo da atividade antimicrobiana de hidrolisados de proteína isolada de soja (x1), proteínas do soro de leite (x2) e

proteínas da clara de ovo (x3): composições das misturas e crescimento percentual de linhagens de leveduras.

a, b, c Os resultados estão apresentados como média aritmética (n = 6) ± desvio padrão e as letras diferentes indicam diferença estatística entre os diferentes ensaios (p < 0,05).

Mistura Candida albicans ATCC 10231 Kluyveromyces marxianus NRRL 7571 Saccharomyces cerevisiae KL 88

Não hidrolisados Hidrolisados Não hidrolisados Hidrolisados Não hidrolisados Hidrolisados

1 92,86 ± 1,40a 92,93 ± 0,32

a 91,24 ± 1,01

a 91,85 ± 0,34

a 93,51 ± 0,96

a 90,28 ± 1,09

a

2 92,48 ± 0,90a 95,26 ± 0,33

b 90,40 ± 0,93

a 92,05 ± 0,47

a 92,24 ± 0,17

a, c 93,21 ± 1,02

b

3 92,76 ± 1,10a 95,40 ± 0,61

b 90,08 ± 1,02

a 92,57 ± 1,31

a 93,24 ± 0,34

a 93,68 ± 1,82

b

4 94,12 ± 0,97a 95,05 ± 0,5

b 91,64 ± 0,81

a 91,75 ± 1,05

a 93,89 ± 0,56

a 92,74 ± 1,11

b

5 94,30 ± 0,57a 94,65 ± 0,46

b 93,07 ± 0,85

b 92,34 ± 0,94

a 95,43 ± 0,89

b 92,83 ± 1,51

a, b

6 94,19 ± 0,84a 95,25 ± 0,55

b 92,15 ± 0,83

a, b 94,99 ± 0,67

b 95,47 ± 2,00

a, b 92,98 ± 2,79ª

, b

7 94,74 ± 1,52a 92,51 ± 0,38

a 93,38 ± 1,05

b 91,97 ± 1,02

a 96,25 ± 1,54

b 84,58 ± 3,97

c

210

As equações geradas a partir das variáveis independentes e respostas foram ajustadas a

modelos lineares, quadráticos ou cúbicos de acordo com a significância estatística dos

coeficientes de regressão (Tabelas 4 e 5). O coeficiente de correlação múltipla (R2) e o teste F

(Análise de variância-ANOVA) foram utilizados para verificar a qualidade do ajuste dos modelos

aos valores reais. As Tabelas 4 e 5 mostram os modelos, os valores de R2 e de F, assim como os

valores de p para cada equação. As equações mostraram valores de R2, variando entre 0,42 e 0,98,

indicando que as equações foram capazes de explicar de 42 a 98% da variação dos resultados.

Valores de R² inferiores a 0,70 indicam baixa adequação estatística dos modelos propostos, o que

compromete a utilização dos mesmos para prever respostas em diferentes condições de ensaios.

Assim, alguns modelos apresentados não são indicados para prever as respostas para crescimento

microbiano. Os baixos valores dos coeficientes de correlação (R²) podem ser devido à pequena

variação nos valores das respostas mesmo em condições experimentais totalmente distintas.

Em adição aos resultados já apresentados, a análise das curvas de contorno facilita a

interpretação dos dados, assim como a verificação dos efeitos das variáveis independentes e de

suas interações. No planejamento experimental de misturas, a variação dos resultados utilizando

diferentes proporções de proteínas também foi avaliada através das curvas de contorno, nas quais,

cada fator é representado em um canto de um triângulo equilátero, sendo cada ponto dentro do

triângulo referente a uma proporção diferente de componentes na mistura. A porcentagem

máxima de cada componente considerado pela regressão é colocada no canto correspondente,

enquanto o mínimo é posicionado no meio do lado oposto do triângulo e o centro representa a

mistura dos três componentes em partes iguais. As curvas de contorno para o crescimento das

linhagens de bactérias e leveduras em meios suplementados com diferentes fontes de proteínas

hidrolisadas e não hidrolisadas são apresentadas nas Figuras 1 e 2.

211

Tabela 4 – Análise de variância (ANOVA) para verificação da adequação estatística dos modelos codificados aos pontos reais para as

diferentes respostas de crescimento de bactérias patogênicas em meios suplementados com proteínas não hidrolisadas e hidrolisadas.

Proteínas não hidrolisadas

Resposta Unidade Modelo Equação Fcalculado/

Ftabelado R² p-valor

Escherichia coli

ATCC 11229

Crescimento

(%) Linear Y = 127,64x1 + 132,89x2 + 111,16x3 30,71/3,23 0,61 <0,001

Salmonella

choleraesuis ATCC

14028

Crescimento

(%) Cúbico

especial

Y = 118,57x1 + 121,01x2 + 105,95x3 – 27,52x1x3 – 30,80x2x3 –

139,15x1x2x3 35,44/2,44 0,83 <0,001

Staphylococcus

aureus ATCC 6538

Crescimento

(%) Quadrático Y = 188,66x1 + 113,92x2 + 110,66x3 – 267,45x1x3 – 25,69x2x3 279,46/2,79 0,98 <0,001

Proteínas hidrolisadas

Escherichia coli

ATCC 11229

Crescimento

(%) Cúbico

especial Y = 116,15x1 + 130,23x2 + 127,75x3 – 22,28x2x3 – 241,09x1x2x3 8,84/2,60 0,49 <0,001

Salmonella

choleraesuis ATCC

14028

Crescimento

(%) Cúbico

especial

Y = 109,38x1 + 114,51x2 + 110,59x3 – 19,54x1x2 – 40,16x1x3 –

44,15x2x3 – 157,33x1x2x3 27,23/2,33 0,82 <0,001

Staphylococcus

aureus ATCC 6538

Crescimento

(%) Cúbico

especial

Y = 133,52x1 + 159,61x2 + 119,18x3 – 211,20x1x2 – 186,14x2x3 +

748,90x1x2x3 109,58/2,66 0,96 <0,001

212

Tabela 5 – Análise de variância (ANOVA) para verificação da adequação estatística dos modelos codificados aos pontos reais para as

diferentes respostas de crescimento de linhagens de leveduras em meios suplementados com proteínas não hidrolisadas e hidrolisadas.

Proteínas não hidrolisadas

Resposta Unidade Modelo Equação Fcalculado/

Ftabelado R² p-valor

Candida albicans

ATCC 10231

Crescimento

(%) Quadrático

Y = 92,86x1 + 92,48x2 + 92,76x3 + 5,84x1x2

+ 6,00x1x3 + 6,33x2x3 5,13/2,45 0,42 0,001

Kluyveromyces

marxianus

NRRL 7571

Crescimento

(%) Quadrático

Y = 91,20x1 + 90,36x2 + 90,04x3 + 3,99x1x2

+ 10,37x1x3 + 8,36x2x3 12,76/2,45 0,64 <0,001

Saccharomyces

cerevisiae

KL 88

Crescimento

(%) Quadrático

Y = 93,46x1 + 92,20x2 + 93,19x3 + 4,97x1x2

+ 9,15x1x3 + 11,84x2x3 12,04/2,45 0,63 <0,001

Proteínas hidrolisadas

Candida albicans

ATCC 10231

Crescimento

(%) Cúbico especial

Y = 92,93x1 + 95,23x2 + 95,37x3 + 3,85x1x2

+ 2,01x1x3 – 71,59x1x2x3 49,33/2,45 0,87 <0,001

Kluyveromyces

marxianus

NRRL 7571

Crescimento


Y = 91,83x1 + 91,97x2 + 92,62x3 +

10,76x2x3 – 36,93x1x2x3 15,16/2,60 0,62 <0,001

Saccharomyces

cerevisiae

KL 88

Crescimento


Y = 90,88x1 + 93,28x2 + 93,70x3 –

216,99x1x2x3 27,18/2,84 0,68 <0,001

213

Figura 1 - Curvas de contorno para crescimento percentual de E. coli ATCC 11229 (a, A), S.

choleraesuis ATCC 14028 (b, B) e S. aureus proteína isolada de soja, proteínas do soro de leite e

proteínas da clara de ovo não hidrolisadas (letras minúsculas) e hidrolisadas (letras maiúsculas)

com protease comercial Flavourzyme® 500L de A. oryzae.

> 132

< 132

< 128

< 124

< 120

< 116

< 112

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 130

< 130

< 128

< 126

< 124

< 122

< 120

< 118

< 116

< 114

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 120

< 120

< 114

< 110

< 106

< 102

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 114

< 114

< 110

< 106

< 102

< 98

< 94

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 180

< 180

< 160

< 140

< 120

< 100

< 80

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 150

< 150

< 132

< 122

< 112

< 102

< 92

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

a

b

c

A

B

C

214

Figura 2 - Curvas de contorno para crescimento percentual de C. albicans ATCC 10231 (a, A),

K. marxianus NRRL 7571 (b, B) e S. cerevisiae KL 88 (c, C) na presença de proteína isolada de

soja, proteínas do soro de leite e proteínas da clara de ovo não hidrolisadas (letras minúsculas) e

hidrolisadas (letras maiúsculas) com protease comercial Flavourzyme®

500L de A. oryzae.

Não hidrolisadas Não hidrolisadas Não hidrolisadas

> 94,6 < 94,5 < 94,1 < 93,7 < 93,3 < 92,9 < 92,5

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína

isolada de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 95

< 95

< 94,5

< 94

< 93,5

< 93

< 92,5

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína isolada

de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 93

< 92,6

< 92,1

< 91,6

< 91,1

< 90,6

< 90,1

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína

isolada de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 94,5

< 94,1

< 93,6

< 93,1

< 92,6

< 92,1

< 91,6

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína

isolada de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 95,5

< 95,2

< 94,7

< 94,2

< 93,7

< 93,2

< 92,7

< 92,2

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína

isolada de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

> 92

< 91

< 89

< 87

< 85

0,00

0,25

0,50

0,75

1,00

Proteínas da

clara de ovo0,00

0,25

0,50

0,75

1,00

Proteína

isolada de soja

0,00 0,25 0,50 0,75 1,00

Proteínas do

soro de leite

a

b

c

A

B

C

215

Peptídeos com atividade antimicrobiana já foram identificados em diversos hidrolisados

proteicos. Biziulevicius et al., (2006) avaliaram o potencial antimicrobiano, frente a cepas de

bactérias e leveduras (Escherichia coli, Proteus vulgaris, Bacillus subtillis, Candida lambica e

Saccharomyces cerevisiae) de hidrolisados protéicos obtidos a partir do tratamento enzimático de

caseína, α-lactoalbumina, β-lactoglobulina, ovalbumina e albumina com proteases (tripsina, α-

quimiotripsina, pepsina e pancreatina) e verificaram que todos os hidrolisados obtidos

apresentaram atividade antimicrobiana contra as linhagens testadas. Hayes et al., (2006)

verificaram a presença de três frações peptídicas produzidas durante fermentação de caseinato de

sódio utilizando Lactobacillus acidophilus DPC6026, com atividade antibacteriana contra

linhagens patogênicas de Enterobacter sakazakii ATCC 12868 e Escherichia coli DPC5063. Liu

et al., (2008) em seus estudos com hidrolisados proteicos obtidos por digestão combinada com

alcalase e bromelina, observaram amplo espectro de ação de uma fração denominada CgPep33,

obtida após purificação por ultrafiltração, cromatografia de troca iônica, filtração em gel e

Cromatografia Líquida de Alta Eficiência (CLAE), contra diversos tipos de micro-organismos,

incluindo bactérias Gram positivas e negativas e fungos. CgPep33 foi capaz de inibir o

crescimento de todas as bactérias estudadas (Escherichia coli, Pseudomonas aeruginosa, Bacillus

subtilis e Staphylococcus aureus) e fungos (Botrytis cinerea e Penicillium expansum). Os valores

de IC50 (concentração necessária para inibir 50% do crescimento) variaram de 18,6-48,2 µg mL-1

.

As bactérias Gram positivas foram as que apresentaram maior sensibilidade, com valores de CIM

(concentração inibitória mínima) entre 40 e 60 µg mL-1

. Gómez-Guillén et al., (2010)

hidrolisaram gelatina com uma preparação comercial de proteases (Alcalase®

2.4L) e

submeteram os hidrolisados a fracionamento por ultrafiltração em membranas de 1 e 10 kDa. As

frações obtidas foram utilizadas para testes antimicrobianos contra 18 cepas bacterianas. As

bactérias mais sensíveis na presença das frações testadas foram: Lactobacillus acidophilus,

Bifidobacterium lactis, Shewanella putrafaciens e Photobacterium phosphoreum. Adje et al.,

(2011) estudaram a aplicação de pepsina na hidrólise de hemoglobina bovina para obtenção de

peptídeos com atividade antimicrobiana. Os hidrolisados foram purificados por CLAE e testados

quanto ao poder antimicrobiano contra duas linhagens Gram negativas (Escherichia coli,

Salmonella enteritidis) e três Gram positivas (Kocuria luteus A270, Staphylococcus aureus e

Listeria innocua). Os resultados obtidos mostraram que as frações peptídicas purificadas

apresentaram amplo espectro de ação, agindo contra quatro das cinco bactérias testadas (Kocuria

216

luteus A270, Listeria innocua, Escherichia coli e Staphylococcus aureus) com CIM variando

entre 35,2 e 187,1 µM. Tellez et al., (2011) mostraram a eficiência de uma fração peptídica,

isolada a partir de leite fermentado com Lactobacillus helveticus, contra uma infecção proposital

com Salmonella enteritidis em ratos. A taxa de sobrevivência no grupo alimentado com a fração

peptídica (0,02 µg por dia) foi superior ao grupo alimentado com uma dose inferior (0,01 µg por

dia) e ao grupo controle.

4. Conclusões

Os resultados obtidos neste estudo mostraram que a suplementação dos meios de cultivo

com fontes de proteínas estimulou o crescimento das bactérias E. coli ATCC 11229, S.

choleraesuis ATCC 14028 e S. aureus ATCC 6538. A linhagem de S. aureus ATCC 6538 foi a

única que apresentou inibição significativa do crescimento quando cultivada em meio

suplementado com uma mistura binária de proteína isolada de soja (1/2) e proteínas da clara de

ovo (1/2) não hidrolisadas, resultando em inibição de 16,82%. Para as linhagens de leveduras C.

albicans ATCC 10231, K. marxianus NRRL 7571 e S. cerevisiae KL 88, não foram observadas

mudanças nos perfis de inibição do crescimento quando comparadas as amostras hidrolisadas e

não hidrolisadas. A maior inibição observada foi detectada para a linhagem de S. cerevisiae KL

88 cultivada em meio suplementado com a mistura ternária de proteínas hidrolisadas em

proporções iguais, resultando em inibição de 15,42%. Embora não tenham sido detectados níveis

significativos de inibição do crescimento microbiano nas condições de ensaio utilizadas no

presente estudo, foi possível observar que a utilização de misturas de proteínas de diferentes

fontes apresentaram efeitos positivos sobre os resultados, atingindo maiores níveis de inibição do

crescimento quando comparadas às proteínas utilizadas isoladamente. Estudos posteriores são

necessários para determinar as melhores condições de obtenção dos hidrolisados e condições

específicas de aplicação dos mesmos em testes de atividade antimicrobiana.

217

Referências bibliográficas

ADJE, E. Y., BALTI, R., KOUACH, M., GUILLOCHON, D., NEDJAR-ARROUME, N. α 67-

106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme

inhibitory peptides. European Food Research and Technology, v. 232, p. 637–646, 2011.

ADLER-NISSEN, J. Procesamiento enzimático de las proteínas alimenticias. Alimentos, v. 6, p.

29-33, 1981.

BEVINS, C. L., M. ZASLOFF. Peptides from frog skin. Annual Review of Biochemistry, v. 59,

p. 395-414, 1990.



‘cause and effect’ theory of bifunctionality. FEMS Immunology & Medical Microbiology, v.

46, p. 131-138, 2006.

BOMAN, H. G. Antibacterial peptides: basic facts and emerging concepts. Journal of Internal

Medicine, v. 254, p. 197-215, 2003.

BOMAN, H. G., HULTMARK, D. Cell-free immunity in insect. Annual Review of

Microbiology, v. 41, p. 103-126, 1987.

CHARNEY, J., TOMARELLI, R.M., A colorimetric method for the determination of the

proteolytic activity of duodenal juice. Journal of Biological Chemistry, v.170, n. 23, p.501-505,

1947.

DASHPER, S. G., LIU, S. W., REYNOLDS, E. C. Antimicrobial peptides and their potential as

oral therapeutic agents. International Journal of Peptide Research and Therapeutics, v. 13, n.

4, p. 505–516, 2007.

GOBBETTI, M., MINERVINI, F. & RIZZELLO, C.G. Angiotensin I-converting enzyme-

inhibitory and antimicrobial bioactive peptides. International Journal Dairy Technology, v. 57,

p. 173-188, 2004.

GÓMEZ-GUILLÉN, M. C., LÓPEZ-CABALLERO, M. E., LÓPEZ DE LACEY, A., ALEMÁN,

A., GIMÉNEZ, B., MONTERO, P. Antioxidant and antimicrobial peptide fractions from squid

218

and tuna skin gelatin. In Bihan, E. L., Koueta, N. Sea by-products as a real material: new ways

of application. Kerala: Transworld Research Network Signpost, 2010. p. 89-115.

HAYES, A., ROSS, R. P., FITZGERALD, G. F., HILL, C., STANTON, C. Casein-derived

antimicrobial peptides generated by Lactobacillus acidophilus DPC6026. Applied and

Environmental Microbiology, v. 72, p. 2260-2264, 2006.

HERNÁNDEZ-LEDESMA, B., CONTRERAS, M. M., RECIO, I. Antihypertensive peptides:

Production, bioavailability and incorporation into foods. Advances in Colloid and Interface

Science, v. 165, p. 23–35, 2011.

HONG, F., MING, L., YI, S., ZHANXIA, L., YONGQUAN, W., CHI, L. The antihypertensive

effect of peptides: A novel alternative to drugs? Peptides, v. 29, p. 1062-1071, 2008.

HOPPE, A., JUNG, S., PATNAIK, A., ZEECE, M. G. Effect of high pressure treatment on egg


Technologies, 17, 54–62, 2013.

LIU, Z., DONG, S., XU, J., ZENG, M., SONG, H., ZHAO, Y. Production of cysteine-rich

antimicrobial peptide by digestion of oyster (Crassostrea gigas) with alcalase and bromelin.

Food Control, v. 19, p. 231–235, 2008.




NAIK, L., MANN, B., BAJAJ, R., SANGWAN, R. B., SHARMA, R. Process optimization for

the production of bio-functional whey protein hydrolysates: adopting response surface

methodology. International Journal of Peptide Research and Therapeutics, 2013. DOI

10.1007/s10989-012-9340-x.

PATRZYKAT, A., DOUGLAS, S.E. Antimicrobial peptides: cooperative approaches to

protection. Protein and Peptide Letters, v. 12, p. 19-25, 2005.

SARMADI, B. H., ISMAIL, A. Antioxidative peptides from food proteins: A review. Peptides,

v. 31, p. 1949–1956, 2010.

219

TAVARES, T. G., CONTRERAS, M. M., AMORIM, M., MARTÍN-ÁLVAREZ, P. J.,

PINTADO, M. E., RECIO, I., MALCATA, F. X. Optimization, by response surface

methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey

protein hydrolysates obtained with cardoon extract. International Dairy Journal, v. 21, p. 926-

933, 2011.

TELLEZ, A., CORREDIG, M., TURNER, P. V., MORALES, R., GRIFFITHS, M. A peptidic

fraction from milk fermented with Lactobacillus helveticus protects mice against Salmonella

infection. International Dairy Journal, v. 21, 607-614, 2011.



Chemistry, v. 122, p. 243–248, 2010a.




ZHAO, Q., SELOMULYA, C., WANG, S., XIONG, H., CHEN, X. D., LI, W., PENG, H., XIE,

J., SUN, W., ZHOU, Q. Enhancing the oxidative stability of food emulsions with rice dreg

protein hydrolysate. Food Research International, 48, 876–884, 2012.

221

Capítulo X: Growth promotion of bifidobacteria and lactic acid bacteria

strains by protein hydrolysates using a statistical mixture design

Revista: Food Bioscience

222

Abstract

The use of different protein sources as supplement for cultivation of probiotic and lactic acid

bacteria species has been reported as an important way to guarantee optimum bacterial growth. In

this work, soy protein isolate (SPI), bovine whey protein (BWP) and egg white protein (EWP)

were hydrolyzed with the protease FlavourzymeTM

500L, and the effects of the media

supplemented with these different proteins and their mixtures on the growth performance of

bifidobacteria and lactic acid bacteria strains using a simplex centroid mixture design were

investigated. The results showed that the protein hydrolysis positively stimulated the bacteria

growth and that the mixtures of hydrolyzed proteins from different sources showed synergistic

effects on cell growth promotion. Compared with control, the cell growth of the mix culture of

Streptococcus thermophilus and Lactobacillus delbrueckii, Lactobacillus acidophilus and

Bifidobacterium lactis were increased with the supplementation of the media with mixtures of

BWP (1/2) plus EWP (1/2) and SPI (1/2) plus BWP (1/2) at 25 mg mL-1

in 100.0, 29.4 and

86.2%, respectively.

Keywords: protein hydrolysates, growth-stimulating, lactic acid bacteria, probiotic, mixture

design.

223

1. Introduction

Processes involving protein hydrolysis have been studied for bioactive peptide

production. Bioactive peptides can be defined as specific amino acid sequences that promote

beneficial biological activities. Bioactive peptides can be produced by enzymatic hydrolysis

using digestive, microbial and plants proteases. The limited and controlled proteolysis unfolds the

protein chains, can reduce the incidence of allergenic factors and also increase the formation of

small peptides with biological activities (Korhonen, 2009).

In the last decade, the enzymatic hydrolysis of proteins from animal and plant sources

for the production of bioactive peptides has attracted much attention. Among the biological

activities, the growth stimulation of probiotic bacteria has been reported. A characteristic of

bifidobacteria and lactic acid bacteria strains is their fastidious requirements for growth and

biological activities, particularly amino acids (Rajagopal & Sandine, 1990). The pool of free

amino acids and peptides in milk is not enough to guarantee optimum bacterial growth in this

substrate (Mills and Thomas, 1981; Zhang et al., 2011). Many ingredients have been evaluated to

stimulate the growth and activity of probiotic and lactic acid bacteria species, for example the

protein hydrolysates (Prasanna, Grandison & Charalampopoulos, 2012). As a result, much

interest has been focused in utilizing different protein sources as additives, such as whey protein

concentrate, whey protein isolate and casein hydrolysate, studying mainly the effect of these

compounds on the growth promotion of probiotic and lactic acid bacteria species (McComas Jr.

& Gilliland, 2003; Zhang et al., 2011; Prasanna, Grandison & Charalampopoulos, 2012).

In the literature, different protein sources have been used for enzymatic hydrolysis, such

as rice, egg white protein and whey protein (Zhao et al, 2012; Naik et al., 2013; Hoppe et al.,

2013). However, these reports show studies on enzymatic hydrolysis using distinct substrates; no

investigations were found using formulations containing mixtures of different protein sources as

well as their interaction effects.

Statistical methods have been applied for improving the performance, to find the

optimum process variables and formulations in different engineering problems (Rao & Baral,

2011). Statistical mixture designs are an interesting class of experimental designs where the

components or factors distributed in different proportions are used to verify the interactions

224

between the components of a mixture and maximizing the responses studied using mixture design

approach.

The general purpose of mixture design is to make possible estimates, through a contour

plots analysis of evaluated responses of a multicomponent system from a limited number of

experiments (Anarjan & Tan, 2013). In this experimental design, the total amount of material is

held constant because the response depends only on the proportions of the components present,

but not on the total amount of the mixture (Rao & Baral, 2011; Anarjan & Tan, 2013). In the

simplex centroid design, 2k

- 1 observations are taken, where k is the amount of pure components,

(k/2) is the binary mixtures with equal proportions and (k/3) is the ternary mixtures with equal

proportions (Scheffe, 1963).

In this work, a simplex centroid mixture design was used for production of hydrolysates

of different protein sources by enzymatic hydrolysis. The effects on the performance of

bifidobacteria and lactic acid bacteria strains grown in the media supplemented with different

protein sources and their mixtures were studied.


2.1. Reagents

FlavourzymeTM

500L, trichloroacetic acid (TCA) and MRS culture broth were purchased

from Sigma-Aldrich (Steinheim, Germany). All other chemicals were purchased in the grade

commercially available.


The soy protein isolate (SPI), bovine whey protein (BWP) and egg white protein (EWP)

used as the substrates for enzymatic hydrolysis were kindly provided by Bunge Foods S/A

(Gaspar, Brazil), Alibra Ingredients Ltd. (Campinas, Brazil) and Cooperovos (Mogi das Cruzes,

Brazil), respectively. The commercial protease obtained from Aspergillus oryzae (FlavourzymeTM

500L) was used for enzymatic hydrolysis. The enzyme concentrations were adjusted to 0

(control) and 50 U per mL of reaction, according to the protease activity, as previously

determined (Charney & Tomarelli, 1947). The proteins were suspended in a buffer to a final


. Fifty-milliliter aliquots of the mixtures were distributed in 125 mL

Erlenmeyer flasks and the hydrolysis was carried out under the optimum temperature and pH of

225

the enzyme (50 °C, pH 5.0) for 120 min. After hydrolysis, the samples were incubated in a water

bath at 100 °C for 20 min for protease inactivation. The mixtures were centrifuged at 17,000 x g

at 5 °C for 20 min and the supernatants containing peptides were collected and freeze-dried for

the determination of the TCA-soluble proteins and in the growth promotion of the bacteria

cultures.

2.3. Determination of the TCA-soluble proteins

The TCA-soluble proteins of the protein hydrolysates were determined with a modified

version of the method described by Pericˇin et al. (2009). A 1.0 mL aliquot of the hydrolysates



incubated for 30 min at room temperature. Then, the mixture was centrifuged at 17,000 x g for 15

min. The obtained 0.22 mol L-1

TCA-soluble proteins fraction and the supernatant of the

hydrolysate mixture (without the addition of TCA) were each analyzed to determine the protein

content using the Lowry method (1951), which uses bovine serum albumin as the standard

protein. The results were expressed as mg of TCA-soluble proteins per 100 mg of total protein in

sample.

2.4. Growth performance of bifidobacteria and lactic acid bacteria strains in the media

supplemented with intact and hydrolyzed proteins

2.4.1. Microorganisms and culture conditions

YFL811 (Streptococcus thermophilus and Lactobacillus delbrueckii), LA5 (Lactobacillus

acidofilus) and BB12 (Bifidobacterium lactis) freeze dried commercial cultures (from Chr.

Hansen China Company, Guangzhou, China) were used throughout this work. The stock cultures

were initially reactivated in 20 mL MRS broth under anaerobic conditions for 24 hr at 40 °C. The

reactivated cultures were sub-cultured twice at 24 hr intervals with 0.5% volume transfer to the

same medium. Then, 1 mL aliquot of the sub-cultured suspension was transferred to 125 mL

Erlenmeyer flasks with 50 mL of MRS broth and incubated at 40 °C and 50 rpm for 8 hr. The

cultures suspensions were diluted in the same medium to obtain the final concentration required

for inoculation of micro-assay plate wells.

226

2.4.2. Bacterial growth in the media supplemented with intact and hydrolyzed proteins

The study of the effects of the media supplemented with different protein sources and

their mixtures on the growth performance of the cultures was monitored using a Novo Star

MicroplateTM

reader (BMG LABTECH, Germany) at 600 nm. The measurements were made in a

96-well micro titer cell culture plates (TPP). To each well were added 100 μL of the culture

suspension diluted in MRS broth to optical density (OD) 0.2 at 600 nm and an equal volume of

protein solutions (0.5 mg mL-1

dissolved in the same medium); the mixtures were incubated for

24 hr at 40 °C and then OD at 600 nm were measured. As a control experiment, 100 μL of culture

suspension and 100 μL of MRS broth were applied into the wells. All assays were performed in

six replicate. The growth, expressed as a percentage, was calculated as the ratio of OD600 of the

samples to OD600 of the control.

2.4.3. Effect of concentration of protein hydrolysates on cell growth

The protein hydrolysates with major stimulator effects at 0.5 mg mL-1

on the bacteria

growth were evaluated at different concentrations: 0.5, 2.5, 5, 10, 15, 20 and 25 mg mL-1

. The

culture growths were carried out as described in Section 2.4.2, at 40 °C for 24 hr.


The statistical analyzes were performed using the MinitabTM


from Minitab Inc. (USA). Values are expressed as the arithmetic mean. The Tukey test was used

to test for significant differences between the groups analyzed. The differences were considered






no linear correlation between the responses. The correlations between the analyzed parameters

were considered significant when p < 0.10.

227


3.1. Comparative analysis of the TCA-soluble proteins and bacteria growth between the

intact proteins and their hydrolysates.

The results for all parameters were evaluated on two points of view: 1) comparative

analysis of the TCA-soluble proteins and stimulation of the bacteria growth in the media

supplemented with hydrolyzed and non-hydrolyzed proteins in respective runs of the statistical

mixture design to verify the changes caused by the enzymatic hydrolysis and 2) evaluation of the

synergistic or antagonistic interactions of hydrolyzed, non-hydrolyzed proteins and their mixtures

on the TCA-soluble proteins and stimulation of the bacteria growth.

The interactions amongst the three substrates in the TCA-soluble proteins and bacteria

growth (%) were studied in the 7 assays using a simplex centroid mixture design (Table 1). The

size of peptides is known to be a significant factor in the overall bioactivities of protein

hydrolysates. Assessment of proteolysis levels is often achieved by global quantification of the

peptides soluble at certain concentrations of trichloroacetic acid (TCA). This parameter has been

used as an indication for the amount of small-sized peptides in the protein hydrolysates and has a

positive correlation with the degree of hydrolysis (DH) (Zhou et al., 2012). The TCA-soluble

proteins of the different protein sources showed significant changes after the enzymatic

hydrolysis. For SPI (run 2), the TCA-soluble proteins ranged from 30.70 mg (intact protein) to

62.47 mg (protein hydrolysates), resulting in an increase of 103.5%. For BWP (run 2) and EWP

(run 3), the observed increases were 52.2 and 288.5%, respectively, as compared to intact

proteins (Table 1).

The results showed that the enzymatic hydrolysis positively stimulated the bacteria

growth. The strain LA5 (L. acidophilus) showed increased growth ranging 12.9 to 19.4% higher

than control in the presence of the hydrolyzed proteins at 0.5 mg mL-1

. On the other hand, in the

medium supplemented with EWP (run 3), BWP (1/2) plus EWP (1/2) (run 6) and the ternary

mixture (run 7) as the non-hydrolyzed proteins, the LA5 growth was inhibited in 22.9, 28.3 and

6.7%, respectively. For the strain BB12 (B. lactis), the non-hydrolyzed proteins had a higher

stimulating effect on bacterial growth compared to the protein hydrolysates. The greatest

increases were observed in the medium supplemented with EWP (non-hydrolyzed) (run 3) and

BWP hydrolysates (run 2), which grew 28.8 and 25.2% higher, respectively, compared to control.

228

The mixed culture YFL811 (S. thermophilus and L. delbrueckii) showed increased growth

ranging 6.8 to 19.4% higher than control when the medium was supplemented with protein

hydrolysates. However, the EWP (run 3), the binary mixtures SPI (1/2) plus EWP (1/2) (run 5)

and BWP (1/2) plus EWP (1/2) (run 6) and the ternary mixture contained the three proteins in

equal proportions (run 7) as the non-hydrolyzed form, inhibited the growth from 6.8 to 10.8%

(Table 1). The Pearson coefficient indicates a positive and significant correlation between the

TCA-soluble proteins and the BB12 growth (%) in the presence of protein hydrolysates (Pearson

coefficient = 0.79; p = 0.04); for LA5 and YFL811, the correlations analysis between the TCA-

soluble proteins and the growth (%) were not statistically significant (p < 0.10).

Table 1 - Matrix of the simple centroid mixture design used to study the growth promotion of

bifidobacteria and lactic acid bacteria species in media supplemented with different protein

sources and their hydrolysates obtained by enzymatic hydrolysis and results for TCA-soluble

proteins.

a, b, c...The results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05.

Tukey tests were applied between the runs for each response (not between different responses). YFL811 (Streptococcus

thermophilus and Lactobacillus delbrueckii), LA5 (Lactobacillus acidophilus) and BB12 (Bifidobacterium lactis). The controls

assays were considered 100%.

Run

Independent variables YFL811 growth (%) LA5 growth (%)

x1

(SPI) x2

(BWP) x3

(EWP)

Non-


Non-


1 1 0 0 108.35 ± 1.45a 106.85 ± 2.45

c, d 102.19 ± 1.54

a, b 119.44 ± 0.98

a

2 0 1 0 107.85 ± 3.82a 112.23 ± 1.83

a, b 104.52 ± 2.06

a 116.32 ± 1.83

b, c

3 0 0 1 89.17 ± 1.66b 107.87 ± 2.38

c, d 77.14 ± 2.34

d 116.89 ± 1.85

a, b

4 1/2 1/2 0 106.54 ± 2.19a 110.77 ± 1.68

b, c 103.62 ± 2.94

a, b 116.75 ± 1.16

a, b

5 1/2 0 1/2 93.13 ± 3.77b 106.73 ± 2.52

d 98.23 ± 2.83

b, c 113.51 ± 1.46

c, d

6 0 1/2 1/2 89.41 ± 2.53b 114.95 ± 2.51

a 71.66 ± 6.33

d 116.60 ± 1.94

a, b

7 1/3 1/3 1/3 92.87 ± 2.20b 107.62 ± 2.42

c, d 93.27 ± 1.99

c 112.92 ± 2.31

d

BB12 growth (%) TCA-soluble proteins (mg/100mg)

Non-


Non-


1 1 0 0 107.09 ± 7.37c, d

109.23 ± 3.38c 30.70 ± 0.71

a 62.47 ± 1.91

a, c

2 0 1 0 113.32 ± 9.12b, c

125.23 ± 5.45a 37.24 ± 1.63

b 56.70 ± 1.67

b

3 0 0 1 128.88 ± 7.15a 114.85 ± 1.89

b, c 16.42 ± 0.13

c 63.79 ± 2.78

a, c

4 1/2 1/2 0 104.14 ± 4.84c, d

117.06 ± 4.00b 36.03 ± 0.99

b 65.07 ± 1.67

a

5 1/2 0 1/2 122.85 ± 3.42a, b

100.69 ± 2.68d 17.42 ± 0.24

c 61.72 ± 0.71

a, c

6 0 1/2 1/2 119.65 ± 9.37a, b

100.12 ± 4.62d 16.99 ± 0.11

c 59.58 ± 2.06

b, c

7 1/3 1/3 1/3 124.30 ± 3.21a, b

96.89 ± 3.70d 17.65 ± 0.12

c 62.68 ± 1.41

a, c

229

It is well documented that various substances can stimulate the growth of certain bacteria

strains, among these, the protein hydrolysates have been investigated by provide a more readily

available source of peptides or amino acids needed for growth of the probiotic cultures

(McComas Jr. & Gilliland, 2003; Zhang et al., 2011; Prasanna, Grandison & Charalampopoulos,

2012). McComas Jr. & Gilliland (2003) investigated the growth of probiotic bacteria in milk

supplemented with whey protein hydrolysates. The results obtained in their research showed that

the hydrolysates had no effect on the growth of L. delbrueckii ssp. bulgaricus and S.

thermophilus; however, caused significant increases in growth of Bifidobacterium longum and L.

acidophilus. Prasanna, Grandison & Charalampopoulos (2012) studied the supplementation of

skim milk and the effects on the growth of probiotic bacteria. Their results showed that the type

of protein source had a clear effect on the cell growth of the tested strains, as the observed in our

work. The final cell concentration of B. longum subsp. infantis CCUG 52486 and B. infantis

NCIMB 702205 were higher when grown in milk supplemented with casein hydrolysates

compared with the other protein sources (lactalbumin hydrolysate, whey protein concentrate and

whey protein isolate).

3.2. Synergistic and antagonistic effects of the intact proteins and their hydrolysates on

the TCA-soluble proteins and bacteria growth (%)

The analysis of the interaction between the different protein sources and their mixtures

after enzymatic hydrolysis showed synergistic and antagonistic effects. The TCA-soluble proteins

showed a similar profile in the 7 runs of the statistical mixture design, ranging from 56.70 to

65.07 mg per 100 mg of total protein in sample. The interactions between the formulations: SPI

(1/2) plus EWP (1/2) (run 5), BWP (1/2) plus EWP (1/2) (run 6) and SPI, BWP and EWP (in

equal proportions) (run 7) were not statistically significant (p < 0.05).

In the study of the growth promotion of bifidobacteria and lactic acid bacteria strains,

the protein hydrolysates obtained as mixtures, showed antagonistic effects, resulting in a decrease

of the microorganisms growth (Table 1). The major growth reductions were observed in the

medium supplemented with the hydrolysates obtained from the ternary mixture (run 7), which

showed decreases of 11.3, 22.6 and 15.6% in BB12 growth as compared to the individual

substrates, respectively (Table 1).

230

3.3. Mixture contour plots for TCA-soluble proteins and bacteria growth (%)

The variations in the growth promotion of bifidobacteria and lactic acid bacteria strains

and TCA-soluble proteins of the hydrolysates obtained from SPI, BWP and EWP are also shown

using mixture contour plots (Fig. 1). Each factor or isolated component of the mixture is

represented on the response surface, in the corner of an equilateral triangle and the different

proportions of the components in the mixture are distributed at each point within this triangle.

The minimum concentration of each component of the mixture was located at the middle of the

opposite side of the triangle while the maximum was positioned at the corresponding corner

(Martinello et al., 2006). A contour plot provides a two-dimensional view where all points that

have the same response are connected to produce contour lines of constant responses (Rao &

Baral, 2011).

Fig. 1 - Mixture contour plots for growth of YFL 811 (a), LA5 (b) and BB12 (c) in the media

supplemented with protein hydrolysates and TCA-soluble proteins (d) of the protein

hydrolysates.

a b

> 119

< 119

< 118

< 117

< 116

< 115

< 114

< 113

0.00

0.25

0.50

0.75

1.00

EWP

0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 114 < 114 < 112 < 110 < 108 < 106

0.00

0.25

0.50

0.75

1.00

EWP0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

> 121

< 121

< 116

< 111

< 106

< 101

< 96

0.00

0.25

0.50

0.75

1.00

EWP

0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

c

> 65

< 65

< 63

< 61

< 59

< 57

0.00

0.25

0.50

0.75

1.00

EWP

0.00

0.25

0.50

0.75

1.00

SPI

0.00 0.25 0.50 0.75 1.00

BWP

d

231

Profile changes in growth promotion were observed for each microorganism. For

YFL811, the maximum growth stimulation was observed in the medium supplemented with EWP

and BWP. However, from the contour plots for LA5 and BB12, the zones of maximum response

variables were located in the corner of triangle having BWP and SPI, respectively, as the vertices

(Fig. 1). The high levels of TCA-soluble proteins were observed in the mixtures containing SPI

and BWP. This can be verified by observing the mixture surface plots (Fig. 1).

3.4. Analysis of variance (ANOVA) and models for the TCA-soluble proteins and

bacteria growth (%)

The responses data based on the independent variables were obtained from the

experiments and recorded in Table 1. In the study of the growth promotion of bifidobacteria and

lactic acid bacteria species, the experiments were conducted with six replicates and for TCA-

soluble proteins, the experiments were performed in triplicates. In most all cases there was good

agreement between the original and replicates. All the independent and response variables were

fitted to linear, quadratic or special cubic models. The coefficient of determination R2 and the F

test (analysis of variance-ANOVA) were used to verify the quality of fit of the models. Table 2

shows the models and corresponding R2

of the regression equations for the responses, as well as

the corresponding F-ratio and p-values for each term in the predicted regression equations. Most

equations showed high coefficients of determination (R2), which were above 0.70 (Table 2),

indicate that all the response functions adequately fitted the experimental data, and the models

could be used for predictive purposes in the determination of the TCA-soluble proteins using the

different protein sources and their mixtures. Specially, in the study of the growth promotion of

probiotic and lactic acid bacteria species, some R2

obtained were below 0.70; however the models

were validated experimentally and were predictive (Table 3).

The negative quadratic (binary) and cubic (ternary) terms of fitted regression equation

showed the antagonistic effects as well the positive quadratic and cubic terms indicated

synergistic effects of the protein sources on the TCA-soluble proteins and bacteria growth (%).

The growth of probiotic and lactic acid bacteria species showed different responses

between the tested microorganisms. When the medium was supplemented with soy protein isolate

(x1), bovine whey protein (x2) and egg white protein (x3) (non-hydrolyzed), the highest positive

and significant effects on growth (%) were detected for YFL811, LA5 and BB12, respectively. In

232

the medium supplemented with hydrolysates, most all the interactions showed significant and

negative effects, except for YFL811 cultivated in medium supplemented with bovine whey

protein (x2) and egg white protein (x3), which stimulated positively the microorganism growth

(Table 2).

Table 2 – Models, R2, F-test and probability values for the final reduced models for TCA-soluble

proteins and bacterial growth.

YFL811 (Streptococcus thermophilus and Lactobacillus delbrueckii), LA5 (Lactobacillus acidophilus) and BB12

(Bifidobacterium lactis).


obtained for TCA-soluble proteins and bacterial growth of the protein hydrolysates using

different formulations with three assays (Table 3). According to the regression models (Table 2),

the experimental values agreed with the values predicted by the models within a 95% confidence

interval, thereby confirming the validity of the models for the evaluated responses (Table 3).

Responses Models Equations (non-hydrolyzed proteins) F-test R² p-value

TCA-soluble

proteins (%) Special cubic

Y = 30.70x1 + 37.24x2 + 16.42x3 + 8.25x1x2 – 24.55x1x3 – 39.35x2x3 – 115.61x1x2x3

215.30 0.99 <0.001

YFL811 growth (%) Quadratic Y = 107.71x1 + 107.21x2 + 89.34x3 –

24.41x1x3 – 38.29x2x3 35.99 0.91 <0.001

LA5 growth (%) Special cubic Y = 102.28x1 + 104.61x2 + 77.14x3 +

34.08x1x3 – 76.86x2x3 + 90.35x1x2x3 48.41 0.95 <0.001

BB12 growth (%) Special cubic Y = 108.84x1 + 112.54x2 + 129.86x3 –

26.22x1x2 – 273.72x1x2x3 6.08 0.63 <0.001

Equations (hydrolysates)

TCA-soluble

proteins (%) Quadratic Y = 62.00x1 + 56.51x2 + 63.05x3 + 22.04x1x2 5.92 0.70 <0.001

YFL811 growth (%) Special cubic Y = 107.02x1 + 112.69x2 + 107.58x3 +

19.24x2x3 – 97.76x1x2x3 6.48 0.65 <0.001

LA5 growth (%) Special cubic Y = 119.05x1 + 115.99x2 + 116.95x3 –

17.97x1x3 – 65.09x1x2x3 5.65 0.62 <0.001

BB12 growth (%) Special cubic Y = 109.17x1 + 125.17x2 + 114.85x3 –

45.27x1x3 – 79.57x2x3 – 152.33x1x2x3 21.13 0.88 <0.001

233


obtained for bacterial growth and TCA-soluble proteins of the protein hydrolysates in different

formulations.


Comparisons were made between the observed and predict values for each correspondent response. YFL811 (Streptococcus

thermophilus and Lactobacillus delbrueckii), LA5 (Lactobacillus acidophilus) and BB12 (Bifidobacterium lactis).

3.5. Effect of the concentration of the protein hydrolysates on cell growth

In order to study the possible effects of different concentrations of the protein

hydrolysates on the growth stimulation of the bifidobacteria and lactic acid bacteria strains, levels

ranging from 0.5 to 25 mg mL-1

were used for supplementation of the culture media. The specific

hydrolysates with the highest stimulation growth, considering the difference between the

hydrolyzed and the non-hydrolyzed samples on growth promotion, were selected for each culture;

for YFL811 and LA5 were used the medium supplemented with the mixture of EWP (1/2) and

BWP (1/2) hydrolyzed and for BB12 was used the medium supplemented with the mixture of SPI

(1/2) and BWP (1/2) hydrolyzed. Significant (p < 0.05) differences on BB12 growth were

observed when the medium was supplemented with SPI and BWP from 0.5 up to 25 mg mL-1

.

The Pearson coefficient showed a positive and significant correlation between the concentration

and the BB12 growth (%) in the presence of protein hydrolysates (Pearson coefficient = 0.95; p <

0.001), indicating that the growth increased as the concentration increased (Fig. 2). However, the

effects on LA5 growth of the medium supplemented with EWP and BWP hydrolysates at

different concentrations were less noticeable. The LA5 growth ranged from 123.3 to 129.4% at

0.5 and 25 mg mL-1

, respectively; no significant differences in the growth were observed when

the concentration ranged from 10 to 25 mg mL-1

(Fig. 2).

Responses


response

Experimental

response x1

(SPI)

x2

(BWP)

x3

(EWP)

TCA-soluble proteins (%) 0.33 0.34 0.33 62.95a 61.07 ± 2.85

a

YFL811 growth (%) 0.00 0.50 0.50 114.95b 117.52 ± 17.63

b

LA5 growth (%) 0.00 0.50 0.50 116.47c 123.26 ± 9.04

c

BB12 growth (%) 0.50 0.50 0.00 117.17d 118.47 ± 6.21

d

234

Fig. 3 - Influence of different concentrations of protein hydrolysates in supplemented media on

the growth of YFL811 (S. thermophilus and L. delbrueckii), LA5 (L. acidophilus) and BB12 (B.

lactis).

0

50

100

150

200

250

0 0.5 2.5 5 10 15 20 25

YF

L811 g

row

th (

%)

Concentration (mg mL-1)

0

20

40

60

80

100

120

140

0 0.5 2.5 5 10 15 20 25

LA

5 g

row

th (

%)


0

20

40

60

80

100

120

140

160

180

200

0 0.5 2.5 5 10 15 20 25

BB

12

gro

wth

(%

)


235

The Pearson coefficient was 0.59 (p = 0.12), indicating that there is not a linear

correlation between the LA5 growth and the concentration of the protein hydrolysates. Overall,

there was a good correlation between the YFL811 growth (%) and the EWP and BWP

concentrations (Pearson coefficient = 0.85; p = 0.008). More specifically, there was 2-fold

increase in the YFL811 growth as the EWP and BWP hydrolysates supplementation was

increased from 0 (control) to 20 mg mL-1

; no significant differences in the YFL811 growth were

observed when the medium was supplemented with EWP and BWP hydrolysates ranged from 5

to 25 mg mL-1

(Fig. 2).

4. Conclusion

The results suggest that the application of the statistical mixture designs for enzymatic

hydrolysis of different protein sources is an attractive process for improving the performance and

to find the optimum mixture formulations of proteins for growth promotion of bifidobacteria and

lactic acid bacteria strains. It was possible to observe the maximization of responses when the

mixtures were used compared to the isolated substrates. Compared with control, the cell growth

of L. bulgaricus plus S. thermophilus, L. acidophilus and B. lactis were increased with the

supplementation of the media with mixtures of BWP (1/2) plus EWP (1/2) and SPI (1/2) plus

BWP (1/2) at 25 mg mL-1

in 100.0, 29.4 and 86.2%, respectively.

Acknowledgements

The work described in this paper was substantially supported by grants from São Paulo

Research Foundation – FAPESP (Project No. 2011/10429-9), the Department of Food Science,

School of Food Engineering, University of Campinas, which are gratefully acknowledged.

References

Anarjan, N. & Tan, C. P. (2013). Developing a three component stabilizer system for producing

astaxanthin nanodispersions. Food Hydrocolloids, 30, 437-447.

Charney, J. & Tomarelli, R. M. (1947). A colorimetric method for the determination of the

proteolytic activity of duodenal juice. The Journal of Biological Chemistry, 170, 501-505.

Hoppe, A., Jung, S., Patnaik, A. & Zeece, M. G. (2013). Effect of high pressure treatment on egg


Technologies, 17, 54–62.

236

Korhonen, H. (2009). Milk-derived bioactive peptides: from science to applications. Journal of

Functional Foods, 1, 177-187.



Martinello, T., Kaneko, T. M., Velasco, M. V. R., Taqueda, M. E. S. & Consiglieri, V. O. (2006).


mixture experimental design. International Journal of Pharmaceutics, 322, 87-95.

McComas Jr., K. A. & GILLILAND, S. E. (2003). Growth of probiotic and traditional yogurt

cultures in milk supplemented with whey protein hydrolysate. Journal of Food Science, 68, 2090-

2095.

Naik, L., Mann, B., Bajaj, R., Sangwan, R. B. & Sharma, R. (2013). Process optimization for the


International Journal of Peptide Research and Therapeutics, DOI 10.1007/s10989-012-9340-x.




Prasanna, P. H. P., Grandison, A. S. & Charalampopoulos, D. (2012). Effect of dairy-based

protein sources and temperature on growth, acidification and exopolysaccharide production of

Bifidobacterium strains in skim milk. Food Research International, 47, 6–12.

Rajagopal, S. N., & Sandine, W. E. (1990). Associative growth and proteolysis of Streptococcus

thermophilus and Lactobacillus bulgaricus in skim milk. Journal of Dairy Science, 73 (4), 894–

899.

Rao, P. V. & Baral, S. S. (2011). Experimental design of mixture for the anaerobic co-digestion

of sewage sludge. Chemical Engineering Journal, 172, 977-986.

Scheffe, H. (1963). The Simplex-Centroid Design for experiments with mixtures. Journal of the

Royal Statistical Society. Series B (Methodological), 25(2), 235-263.

Zhang, Q., Ren, J., Zhao, H., Zhao, M., Xu, J. & Zhao, Q. (2011). Influence of casein

hydrolysates on the growth and lactic acid production of Lactobacillus delbrueckii subsp.

237

bulgaricus and Streptococcus thermophiles. International Journal of Food Science and

Technology, 46, 1014–1020.

Zhao, Q., Selomulya, C., Wang, S., Xiong, H., Chen, X. D., Li, W., Peng, H., Xie, J., Sun, W. &

Zhou, Q. (2012). Enhancing the oxidative stability of food emulsions with rice dreg protein

hydrolysate. Food Research International, 48, 876–884.

Zhou, D., Qin, L., Zhu, B., Li, D., Yang, J., Dong, X. & Murata, Y. (2012). Optimisation of




239

Capítulo XI: Synergistic actions of proteolytic enzymes for production of

soy protein isolate hydrolysates with antioxidant activities: an approach

based on enzymes specificities

Revista: Food Chemistry

240

Abstract

The objective of this study was investigate the enzymatic hydrolysis of soy protein isolate by

screening individual and blended commercial protease preparations using a statistical mixture

design. Information about the modulation of thermal inactivation of the enzyme by substrate or

products of hydrolysis and the determination of synergistic effects between the enzymes on

production of soy protein hydrolysates with antioxidant activities were reported. The kinetic

parameters for thermal inactivation measured under reactive and non-reactive conditions

indicated that product inhibition was not significant on soy protein hydrolysis using the

commercial proteases FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A. The

antioxidant activities showed different results in each method used. For DPPH radical

scavenging, the hydrolysates obtained with FlavourzymeTM

500L combined with AlcalaseTM

2.4L

showed the higher synergistic effect with increases of 10.9 and 13.2% in antioxidant activity as

compared to the hydrolysates produced with individual enzymes. The hydrolysates obtained

using the ternary mixtures of FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A showed

the highest power of inhibition of linoleic acid autoxidation. On the other hand, for reducing

power assay and total antioxidant activity, the most of all interactions was antagonistic with high

antioxidant activity detected for the hydrolysates produced using FlavourzymeTM

500L,

individually.

Keywords: proteases, enzyme specificity, antioxidant activities, mixture design

241

1. Introduction

Proteases constitute the most important category of industrial enzymes that catalyze the

hydrolysis of proteins to polypeptides and oligopeptides to amino acids (Abraham et al., 2014).

These enzymes can be classified according with their biochemical characteristics such as

mechanisms of action, catalytic sites or based on the pH for maximum activity. Proteases that

cleave peptide bonds within the polypeptide chain are called endopeptidases and those that cleave

peptides bonds at the N or C termini of polypeptide chains are classified as exopeptidases

(López-Otín & Bond, 2008; Hsiao et al., 2014). According to the catalytic sites, these enzymes

are classified in six groups: aspartic, cysteine, glutamic, serine and threonine proteases,

depending upon the amino acids present in the active site, or as metalloproteases if a metal ion is

required for catalytic activity (Li et al., 2013). They may further classified in acidic, neutral and

alkaline proteases depending on the pH at which they show the maximum activity (Ktari et al.,

2014). Through structural and functional diversity, proteases carry out a vast array of

applications, including food production (eg. baking and brewing), leather processing,

pharmaceutical manufacture, detergent formulations and protein modification (eg. protein

hydrolysis and peptide synthesis) (Anbu, 2013).

Enzymatic hydrolysis disrupts the protein tertiary structure and reduces the molecular

weight of the protein, enhancing the interaction of peptides with themselves and with the

environment, and consequently altering their functional and biological properties (Liu et al.,

2010). Notably, the nature of the protein modification is influenced by several hydrolysis

parameters, including the reaction conditions, such as pH, temperature, degree of hydrolysis, and

enzyme specificities, and intrinsic characteristics of each protein source, such as amino acids

profile (Singh, 2011; Segura-Campos et al., 2012; Fernández & Riera, 2013). The modification

of proteins based on enzymatic hydrolysis have broad potential and are likely an innovative tool

in food protein processing for optimizing the functional and biological properties of proteins

(Hiller & Lorenzen, 2009; Adjonu et al., 2013).

The combined use of proteases with different specificities and mechanisms of action can

be applied as a valuable tool to improving the functional and biological properties of protein

hydrolysates. Prior knowledge about enzyme characteristics such as purity, substrate specificity,

specific activity, single or multiple enzymatic activity have been used to obtain products

242

containing multifunctional peptides) or a mixture of different peptides with each contributing to a

specific function (Rui et al., 2012; Betancur-Ancona et al., 2014; Li-Chan, 2015).

Statistical methods have been applied for improving the performance, to find the optimum

process variables and formulations in different engineering problems (Rao & Baral, 2011).

Statistical mixture designs are an interesting class of experimental designs where the components

or factors distributed in different proportions are used to verify the interactions between the

components of a mixture and maximizing the responses studied using mixture design approach.

The general purpose of mixture design is to make possible estimates, through a contour

plots analysis of evaluated responses of a multicomponent system from a limited number of

experiments (Anarjan & Tan, 2013). In this experimental design, the total amount of material is

held constant because the response depends only on the proportions of the components present,

but not on the total amount of the mixture (Rao & Baral, 2011; Anarjan & Tan, 2013). In the

simplex centroid design, 2k

- 1 observations are taken, where k is the pure components, (k/2) is

the binary mixtures with equal proportions and (k/3) is the ternary mixtures with equal

proportions (Scheffe, 1963).

In this context, the soy protein isolate hydrolysis was investigated by screening

individual and blended commercial protease preparations using a statistical mixture design. The

study of thermal inactivation modulation of the enzyme by the substrate or products from soy

protein isolate hydrolysis using kinetic parameters was reported. The synergistic or antagonistic

effects between the different proteolytic enzymes on generation of protein hydrolysates with

antioxidant activities were further assessed.

243

2. Material and Methods

2.1. Reagents

Ammonium thiocyanate, ferrous chloride, linoleic acid, azocasein, trichloroacetic acid

(TCA), 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Steinheim,

Germany). All other chemicals were purchased in the grade commercially available.

2.2. Enzymes

Three commercial preparations of proteolytic enzymes were used in this study. The

proteases FlavourzymeTM

500L from Aspergillus oryzae and AlcalaseTM

2.4L from Bacillus

licheniformis were purchased from Sigma Aldrich (Steinheim, Germany). The protease

YeastMaxTM

A was kindly provided by Prozyn (Sao Paulo, Brazil).


The protease activity was measured using azocasein as a substrate, according to Charney

and Tomarelli (1947), with modifications proposed by Castro and Sato (2013). The reaction

mixtures containing 0.5 mL 0.5% (w/v) azocasein, pH 7.0, and 0.5 mL of the enzyme solutions

were incubated for 40 min at 50 °C. The reaction was stopped by adding 0.5 mL 10% TCA and

the test tubes were centrifuged at 17,000 x g for 15 min at 25 °C. A 1.0 mL aliquot of the

supernatant was neutralized with 1.0 mL 5 M KOH. One unit of enzyme activity (U) was defined

as the amount of enzyme required to increase the absorbance at 428 nm by 0.01 under the assay

conditions described.

2.4. Kinetic parameters for thermal inactivation

The thermal stability of the commercial proteases as a function of the time was evaluated

at reactive (50 U mL-1

of protease and 100 mg mL-1

soy protein isolate solution pH 7.0) e non-

reactive (50 U mL-1

of protease and 0.2M phosphate buffer pH 7.0) conditions. For this, the

samples were incubated at 50 °C and aliquots were collected at various times for determination of

the residual protease activity. The value of the deactivation rate constant (kd) for the proteases,

expressed as an exponential decay, was found by plotting ln (A/A0) vs. time using the

experimental data as follows:

A = A0 × e-kdt

244

Where t is time, A0 is the initial enzyme activity and A is the enzyme activity at a determined

time t.

The apparent half-life of the proteases, defined as the time where the residual activity

reaches 50%, was estimated as follows:

t1/2 = ln (0.5) / kd

Decimal reduction time (D value) was defined as the time required for a one-log10

reduction or 90% reduction in the initial enzyme activity at a specific temperature. The D value is

related to the first-order deactivation rate constant (kd) and it was calculated as follows:

D = 2.303 / kd


The soy protein isolate was kindly provided by Bunge Foods S/A (Gaspar, Brazil). The

commercial proteases, FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A were used for

enzymatic hydrolysis. The enzyme concentrations were adjusted to 0 (control) or 50 U per mL of

reaction mixture according to the previously determined protease activity. The soy protein isolate

was suspended in phosphate buffer pH 7.0 to a final concentration of 100 mg mL-1

, and 50 mL

aliquots of the mixtures were distributed in 125 mL Erlenmeyer flasks. Hydrolysis was

performed at 50 °C and pH 7.0 for 120 min. After hydrolysis, the samples were incubated in a

water bath at 100 °C for 20 min for proteases inactivation. The mixtures were centrifuged at

17,000 x g at 5 °C for 20 min, and the supernatants containing the peptides were collected and

freeze-dried for the determination of their antioxidant activities and TCA soluble protein

contents.


The experimental mixture design was used to obtain the optimum mixture compositions

of the different proteolytic enzymes for maximum antioxidant activities and to investigate the

presence of either synergistic or antagonistic effects in a blend of the components. A three

component augmented simplex centroid design was employed in which each component was

studied at six levels, namely 0 (0%), 1/6 (16.67%), 1/3 (33%), 1/2 (50%), 2/3 (66.67%) and 1

(100%) (Table 1).

245

Table 1 – Matrix of the simplex centroid mixture design for soy protein isolate hydrolysis using

different sources of commercial proteases and their mixtures and study of the antioxidant

properties of the hydrolysates.

Quadratic or special cubic regression models were fitted for the variations of all the

responses studied as a function of significant (p < 0.05) interaction effects between the

proportions, thereby obtaining acceptable determination coefficients (R² > 0.75). Equation 1

represents these models as follows:




StatisticaTM




𝑞

𝑖<𝑗

𝑞

𝑖=1


𝑞

𝑖<𝑗<𝑘

Run


FlavourzymeTM

500L AlcalaseTM

2.4L YeastMaxTM

A

x1 x2 x3

1 1 0 0

2 0 1 0

3 0 0 1

4 1/2 1/2 0

5 1/2 0 1/2

6 0 1/2 1/2

7 2/3 1/6 1/6

8 1/6 2/3 1/6

9 1/6 1/6 2/3

10 1/3 1/3 1/3

246

2.7. Determination of TCA soluble protein content

The TCA soluble protein content of the hydrolysates was determined using a modified

version of the method described by Pericˇin et al. (2009). A 1.0 mL aliquot of the hydrolysate



incubated for 30 min at room temperature and then centrifuged at 17,000 x g for 15 min. The

protein content of the supernatant (0.22 mol L-1

TCA soluble protein fraction) and the supernatant

of the hydrolysate mixture (without the addition of TCA) was analyzed by the Lowry method

(1951), using bovine serum albumin as the standard protein. The results were expressed as a

percentage and were calculated as the ratio of the 0.22 mol L-1

TCA soluble protein content to the

total protein content in the supernatant of the hydrolysate mixture.

2.8. Determination of antioxidant activities


The DPPH radical-scavenging activity of the protein hydrolysates was determined as

described by Bougatef et al. (2009). An aliquot (500 µL) of each sample (5 mg mL-1

) was mixed

with 500 µL of 99.5% ethanol and 125 µL of DPPH (0.2 mg mL-1

) in 99.5% ethanol. The

mixture was then kept at room temperature in the dark for 60 min, and the reduction of the DPPH

radical was measured at 517 nm using a UV-Visible spectrophotometer. The DPPH radical-

scavenging activity was calculated as follows:

Radical scavenging activity (%) = [(Absorbance of control - Absorbance of sample) / (Absorbance of

control)] * 100.

2.8.2. Inhibition of linoleic acid autoxidation

The lipid peroxidation inhibition activity was measured in a linoleic acid emulsion system

according to the method described by Nazeer & Kulandai (2012) with slight modifications. A 20

mg aliquot of each hydrolysate was dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0) and

was later added to 130 µL of a linoleic acid solution and 10 mL of 99.5% ethanol. The total

volume was then adjusted to 25 mL with distilled water. The mixture was incubated in a 50-mL

assay tube with a screw cap at 42 ± 1 °C for 24 h in a dark room. The degree of oxidation of

linoleic acid was measured using the ferric thiocyanate method of Sakanaka et al. (2004) with

slight modifications. An aliquot of 0.1 mL of the reaction mixture was mixed with 4.7 mL of

247

75% ethanol, 0.1 mL of 30% ammonium thiocyanate and 0.1 mL of 20 mM ferrous chloride

solution in 3.5% HCl and after a 3-min incubation at room temperature, the color development,

which represents the linoleic acid oxidation, was measured at 500 nm. The antioxidant capacity

of inhibiting peroxide formation in the linoleic acid system was expressed as follows:

Inhibition (%) = [(Absorbance of control - Absorbance of sample) / (Absorbance of control)] * 100.

2.8.3. Reducing power assay

The capacity of the protein hydrolysates to reduce iron (III) was determined according to

the method described by Yildirim et al., (2001) with slight modifications. A 0.2 mL aliquot of

each protein hydrolysate at 10 mg mL-1

was mixed with 0.5 mL of 0.2 M phosphate buffer (pH

6.6) and 0.5 mL of 1% potassium ferricyanide. The reaction mixtures were incubated at 50 °C in

the dark and after 30 min, 0.5 mL of 10% (w/v) trichloroacetic acid was added. The reaction

mixtures were centrifuged at 8000 x g for 10 min at 5 °C and 0.75 mL of the supernatant

solutions were collected and mixed with 0.75 mL of distilled water and 0.15 mL of 0.1% (w/v)

ferric chloride. The absorbance of the final solution was measured after 10 min reaction at 700

nm. The results were expressed in function of the absorbance considering that the absorbance was

directly proportional to the reducing power.

2.8.4. Total antioxidant capacity

Total antioxidant capacity of the hydrolysates was performed according to the method

described by Prieto et al., (1999). An aliquot of 0.1 mL of the protein hydrolysates solutions at 10

mg mL-1

was mixed with 1.0 mL of the reagent solution containing 0.6 M sulphuric acid, 28 mM

sodium phosphate and 4 mM ammonium molybdate. The reaction mixtures were then incubated

at 90 °C and kept in the dark for 90 min. The samples were cooled to room temperature and the

absorbance was measured at 695 nm. An appropriate control was prepared with 1.0 mL of the

reagent solution and 0.1 mL distilled water. The results were expressed in function of the

absorbance considering that the absorbance was directly proportional to the total antioxidant

capacity.

248


The statistical analyzes were performed using the MinitabTM


from Minitab Inc. (USA). Values are expressed as the arithmetic mean. The Tukey test was used

to test for significant differences between the groups analyzed. The differences were considered







considered significant at p-value < 0.10.


3.1. Investigation of thermal inactivation modulation of the enzyme by the substrate or

products from soy protein isolate hydrolysis using kinetic parameters

The commercial enzymes FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A

showed 5,489.33 U mL-1

, 327,760.00 U mL-1

and 10,900.67 U g-1

of protease activity,

respectively.

In order to assess the modulation of the thermal inactivation of the enzyme by the

substrate or products during enzymatic hydrolysis, experiments were carried out at reactive

conditions, in which the residual protease activities were measured in the presence of soy protein

isolate at 100 mg mL-1

pH 7.0, and non-reactive conditions, performed in 0.2 M phosphate buffer

solution pH 7.0. The protease activities were determined as a function of time at 50 °C and the

results were presented in Figure 1. The protease activities decreased with increasing reaction

times, retaining approximately 21.43, 26.37 and 18.59% of the initial activity after 180 min under

reactive conditions and residual activities of 9.10, 20.83 and 10.69% after the same time under

non-reactive conditions for FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A,

respectively (Figure 1). These data were used to estimate the kinetic parameters for thermal

inactivation of the proteases using Arrhenius plots (Figure 2).

249

Figure 1 - Residual protease activities for commercial enzymes FlavourzymeTM

500L (a), AlcalaseTM

2.4L (b) and YeastMaxTM

A (c) under non-reactive and reactive conditions.

a

b

c

0

20

40

60

80

100

0 30 60 90 120 150 180

Res

idu

al a

ctiv

ity (

%)

Time (min)

Non-reactive Reactive

0

20

40

60

80

100

0 30 60 90 120 150 180

Res

idu

al a

ctiv

ity (

%)

Time (min)


0

20

40

60

80

100

0 30 60 90 120 150 180

Res

idu

al a

ctiv

ity (

%)

Time (min)


250

Figure 2 – Arrhenius plots for FlavourzymeTM

500L (a, A), AlcalaseTM

2.4L (b, B) and

YeastMaxTM

A (c, C) inactivation under non-reactive and reactive conditions, respectively.

0 30 60 90 120 150 180

Time (min)

-2.0

-1.6

-1.2

-0.8

-0.4

0.0

ln (

A/A

0)

0 30 60 90 120 150 180

Time (min)

-2.5

-2.0

-1.5

-1.0

-0.5

0.0ln

(A

/A0)

0 30 60 90 120 150 180

Time (min)

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

ln (

A/A

0)

0 30 60 90 120 150 180

Time (min)

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

ln (

A/A

0)

0 30 60 90 120 150 180

Time (min)

-2.4

-2.0

-1.6

-1.2

-0.8

-0.4

0.0

ln (

A/A

0)

0 30 60 90 120 150 180

Time (min)

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

ln (

A/A

0)

y = -0.0124x – 0.2314

R² = 0.98

y = -0.0083x – 0.0825

R² = 0.98

y = -0.0084x – 0.1806

R² = 0.94 y = -0.0066x – 0.2076

R² = 0.92

y = -0.0115x – 0.4521

R² = 0.91

y = -0.0086x – 0.2162

R² = 0.95

a A

b B

c C

251

The half-life (t1/2) of an enzyme, at a given temperature, is the time it takes for the activity

to reduce to a half of its original/initial activity. The decimal reduction time (D value) is defined

as the time required for a 90% reduction in the initial enzyme activity. Higher values of these

parameters at the specific operating temperature are important and desirable for industrial

applications since indicate the resistance of the enzyme to thermal inactivation. AlcalaseTM

2.4L

showed the highest thermal resistance when compared to the other proteases, reaching D value of

348.88 min and t1/2 of 105.02 min at 50 °C under reactive conditions. There is a clear indicative

that in the presence of soy protein isolate, the commercial proteases showed higher thermal

stability. FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A presented an increase in the

half-life from 55.90 min to 83.51 min, 82.52 to 105.02 min and 60.27 to 80.60 min, respectively,

in the presence of substrate protein (Table 2).

Table 2 - Kinetic parameters for thermal deactivation of commercial preparations of proteases

under non-reactive and reactive conditions.

It was observed that residual protease activities of AlcalaseTM

2.4L under non-reactive

conditions were higher than reactive conditions during the first 45 min reaction at 50 °C. After

this time, a positive modulation increased progressively with the soy protein isolate hydrolysis

(reactive conditions), suggesting that smaller protein/peptides had a greater stabilizing effect for

this enzyme. The loss of protease activity for FlavourzymeTM

500L and YeastMaxTM

A presented

lower rates when considering the same reaction times under reactive and non-reactive conditions

until 120 min, indicating that the presence of smaller protein molecules was also important to

stabilize these enzymes (Table 2). These results evidenced that the product inhibition phenomena

was not significant on soy protein hydrolysis using these commercial preparations.

Protease Non-reactive conditions

kd (min-1

) t1/2 (min) D (min) R²

FlavourzymeTM

500L 0.0124 55.90 185.69 0.98

AlcalaseTM

2.4L 0.0084 82.52 274.12 0.94

YeastMaxTM

A 0.0115 60.27 200.23 0.91

Reactive conditions

FlavourzymeTM

500L 0.0083 83.51 277.42 0.98

AlcalaseTM

2.4L 0.0066 105.02 348.88 0.92

YeastMaxTM

A 0.0086 80.60 267.74 0.95

252

3.2. Synergistic and antagonistic effects of the proteases on production of soy protein

isolate hydrolysates with antioxidant activities

The interactions amongst the three commercial proteases on production of soy protein

isolate hydrolysates and study of their antioxidant properties were studied in the 10 assays using a

simplex centroid mixture design (Table 3). The antioxidant activities of the hydrolysates were

evaluated using a DPPH radical-scavenging, inhibition of linoleic acid autoxidation, reducing

power assay and total antioxidant capacity.

The variations in the antioxidant activities of the hydrolysates obtained from different

proteases were depicted using mixture contour plots (Figure 3 and Figure 4). On the response

surfaces, each factor (pure mixture component) is represented in the corner of an equilateral

triangle, and each point within this triangle refers to a different proportion of the components in

the mixture. The maximum percentage of each ingredient considered by the regression is placed

at the corresponding corner, and the minimum percentage is positioned at the middle of the

opposite side of the triangle (Martinello et al., 2006). A contour plot provides a two-dimensional

view where all points that have the same response are connected to produce contour lines of

constant responses (Rao & Baral, 2011).

The correlation analysis between the antioxidant activities measured by different methods

showed the following results: DPPH vs. inhibition of linoleic acid autoxidation (Pearson

coefficient = 0.41; p-value = 0.02), DPPH vs. total antioxidant capacity (Pearson coefficient =

0.07; p-value = 0.71), DPPH vs. reducing power (Pearson coefficient = 0.31; p-value = 0.09),

inhibition of linoleic acid autoxidation vs. total antioxidant capacity (Pearson coefficient = -0.09;

p-value = 0.61), inhibition of linoleic acid autoxidation vs. reducing power (Pearson coefficient =

0.03; p-value = 0.88) and total antioxidant capacity vs. reducing power (Pearson coefficient =

0.91; p-value < 0.01). It can be observed that no significant correlations were detected for most of

the analyzed responses, because the antioxidant methods have different reaction mechanisms and

consequently measure particular antioxidant capacities. Methods based on the same reaction

mechanism, as total antioxidant capacity and reducing power that measure the electron donation

capacity of antioxidant molecules, showed a positive and significant correlation.

DPPH is a relatively stable organic radical that is characterized by a deep purple color and

a maximum absorbance at 515–520 nm. When DPPH encounters a hydrogen-donating substance,

253

the radical is scavenged, and the absorbance is reduced. Therefore, DPPH is widely used as a

substrate to evaluate the efficacy of antioxidants (Gao et al., 2010). DPPH-radical scavenging

showed synergistic effects for all assays performed using binary mixtures of proteases (runs 4-6).

The hydrolysates obtained with FlavourzymeTM

500L (0.5) combined with AlcalaseTM

2.4L (0.5)

(run 4) showed the higher synergistic effect with increases of 10.9 and 13.2% in antioxidant

activity as compared to the hydrolysates produced with individual enzymes (runs 1-2),

respectively, and reaching a maximum DPPH radical scavenging of 70.84% at 5 mg mL-1

(Table

3). From the contour plots for DPPH assay, the zones of maximum response variables were

located towards the side of triangle having mixtures of FlavourzymeTM

500L and AlcalaseTM

2.4L as the vertices (Figure 3a). This indicates that to certain extent, these enzyme proportions

may be added to improve the antioxidant activities of the soy protein hydrolysates.

A complex process that involves formation and propagation of free radicals in the

presence of oxygen is the lipid peroxidation. Antioxidant peptides can inhibit this process afford

their protective actions in lipid peroxidation by scavenging the lipid-derived radicals (R•, RO• or

ROO•) (Nazeer et al., 2012). For the inhibition of linoleic acid autoxidation, the ternary mixtures

of proteases presented important synergistic effects, while the binary mixtures showed no

significant effects. The contour plot analysis indicated that the soy protein isolate hydrolysis

using the ternary mixtures of the proteases in equal proportions resulted in the highest inhibition

of linoleic acid autoxidation (Figure 3b). This can also be confirmed in run 10 that showed

increase of approximately 3-fold in the ability to inhibit the linoleic acid autoxidation as

compared to the hydrolysates obtained using individual enzymes, and reached maximum values

about 60% inhibition at 0.8 mg mL-1

(Table 3).

Soy protein hydrolysates demonstrated electron-donating capacity and thus they may act

as radical chain terminators, transforming reactive free radical species into more stable non-

reactive products (Dorman et al., 2003; Arabshahi-Delouee & Urooj, 2007). This mechanism is

the basis for total antioxidant capacity and reducing power methods. For the reducing power

assay, the presence of antioxidant in tested samples results in the reduction of Fe3+

/ferricyanide

complex to ferrous form (Yildirim et al., 2000; Bougatef et al., 2009). Whereas, total antioxidant

capacity is based on the reduction of Mo (VI) to Mo (V) by the antioxidant agent and the

subsequent formation of a green phosphate/Mo (V) complex at acidic pH (Prieto et al., 1999;

254

Bougatef et al., 2009). The results for both assays were similar and showed maximum responses

for the hydrolysates obtained using FlavourzymeTM

500L (run 1) (Figure 3c and 3d). Since the

values obtained for run 1 in these assays were too high compared to the other runs, most of the

observed interaction effects were antagonistic, resulting in decreased of the antioxidant capacity

of the hydrolysates (Table 3).

Table 3 - Matrix of the simplex centroid mixture design used to study the antioxidant activities of

soy protein isolate hydrolysates obtained different proteases and their mixtures.

a, b, c...The results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05.

Tukey tests were applied between the runs for each response (not between different responses). 1The non-hydrolyzed sample was

used as the control. 2Results presented as the absorbance at 700 nm. 3Results presented as the absorbance at 695 nm.

Runs DPPH radical

scavenging (%)

Inhibition of linoleic

acid autoxidation (%)

Reducing power

assay2

Total antioxidant

capacity3

Control1 51.14 ± 1.30

f 17.48 ± 6.23

a 0.1287 ± 0.01

h 0.2186 ± 0.01

g

1 63.83 ± 1.03c, d, e

15.39 ± 4.95a 0.3993 ± 0.03

a 0.7032 ± 0.01

a

2 62.57 ± 2.42d, e

15.59 ± 4.77a 0.1761 ± 0.01

f, g 0.3060 ± 0.01

f

3 59.91 ± 1.56e 15.00 ± 4.16

a 0.1653 ± 0.01

g 0.3215 ± 0.02

e, f

4 70.84 ± 1.55a 16.07 ± 4.91

a 0.2804 ± 0.01

b, c 0.3924 ± 0.03

b, c, d

5 64.53 ± 1.36c, d, e

16.28 ± 5.95a 0.2398 ± 0.02

c, d 0.3780 ± 0.01

b, c, d

6 65.23 ± 0.95c, d

15.91 ± 3.98a 0.1914 ± 0.01

e, f, g 0.3506 ± 0.01

d, e, f

7 67.76 ± 1.14a, b, c

59.80 ± 1.76b 0.3044 ± 0.02

b 0.4177 ± 0.01

b

8 65.21 ± 2.11c, d

60.50 ± 2.58b 0.2247 ± 0.01

d, e 0.3699 ± 0.02

c, d

9 65.54 ± 1.28b, c, d

60.19 ± 2.32b 0.2179 ± 0.01

d, e, f 0.3540 ± 0.02

d, e

10 69.92 ± 2.07a, b

60.21 ± 2.72b 0.2504 ± 0.01

c, d 0.4117 ± 0.03

b, c

255

Figure 3 - Mixture contour plots for antioxidant activities of soy protein isolate hydrolysates as

function of significant (p < 0.05) interaction effects of different commercial proteases

proportions: DPPH radical scavenging (a), inhibition of linoleic acid autoxidation (b), reducing

power assay (c) and total antioxidant capacity (d).

The size of the peptides is known to be a significant factor in the overall antioxidant

activity and functional properties of protein hydrolysates. Proteolysis levels are often assessed by

global quantification of the soluble peptides in certain concentrations of trichloroacetic acid

(TCA). This parameter has been used as an indication of the amount of small peptides in protein

hydrolysates and has a positive correlation with the degree of hydrolysis (DH) (Zhou et al.,

2012).

> 70 < 70 < 60 < 50 < 40 < 30 < 20

0.00

0.25

0.50

0.75

1.00

YeastMax A0.00

0.25

0.50

0.75

1.00

Flavourzyme®

500L

0.00 0.25 0.50 0.75 1.00

Alcalase®

2.4L

> 70 < 70 < 67 < 65 < 63 < 61

0.00

0.25

0.50

0.75

1.00

YeastMax A0.00

0.25

0.50

0.75

1.00

Flavourzyme®

500L

0.00 0.25 0.50 0.75 1.00

Alcalase®

2.4L

> 0.38 < 0.38 < 0.33 < 0.29 < 0.25 < 0.21 < 0.17

0.00

0.25

0.50

0.75

1.00

YeastMax A0.00

0.25

0.50

0.75

1.00

Flavourzyme®

500L

0.00 0.25 0.50 0.75 1.00

Alcalase®

2.4L

> 0.65 < 0.65 < 0.56 < 0.51 < 0.46 < 0.41 < 0.36 < 0.31

0.00

0.25

0.50

0.75

1.00

YeastMax A0.00

0.25

0.50

0.75

1.00

Flavourzyme®

500L

0.00 0.25 0.50 0.75 1.00

Alcalase®

2.4L

a b

c d

256

Figure 4 shows the distribution profile of TCA soluble protein for the assays performed

using the statistical mixture design as well as the contour plot for this response as function of

significant interaction effects of different proteases proportions. The results showed that the

hydrolysis performed using the protease YeastMaxTM

A (run 3) resulted in highest TCA soluble

protein value, reaching 90.5%, while the hydrolysates obtained with the binary mixture of

FlavourzymeTM

500L (0.50) and YeastMaxTM

A (0.50) (run 5) presented the lowest value

(69.9%). In some studies, an increase in the TCA soluble protein content of the protein

hydrolysates increases the antioxidant activity. However, other studies have reported a decrease

in antioxidant activity with an increase in TCA soluble protein content. For our study, the

correlation analysis indicated no significant relationship between the antioxidant activities and

TCA soluble protein. This can be justified by the complexity involved when the different

protease sources were combined in binary or ternary mixtures.

Figure 4 – Variation of TCA soluble protein content in the runs 1-10 performed using statistical

mixture design (a) and contour plot for TCA soluble protein of the hydrolysates as function of

significant (p < 0.05) interaction effects of different proteases proportions (b).


and recorded in Table 3. The experiments were conducted with triplicates. In almost all cases, a

good agreement existed between the original and triplicates. All the independent and response

variables were fitted to quadratic or special cubic models. The coefficient of determination (R2)

and the F-test (analysis of variance; ANOVA) were used to verify the quality of fit of the models.

> 90 < 90 < 86 < 82 < 78 < 74 < 70

0.00

0.25

0.50

0.75

1.00

YeastMax A0.00

0.25

0.50

0.75

1.00

Flavourzyme®

500L

0.00 0.25 0.50 0.75 1.00

Alcalase®

2.4L

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10

TC

A s

olu

ble

pro

tein

(%

)

Runs

a b

257

Table 4 shows the models, corresponding R2, F-ratio and p-values of the regression

equations for antioxidant activities. The high coefficients of determination (R2), which were

greater than 0.75 (Table 4), indicated that all the response functions adequately fitted the

experimental data and that the models could be used for predictive purposes in the determination

of the antioxidant activities of soy protein isolate hydrolysates using the different proteases

sources and their mixtures.

Table 4 - Analysis of variance (ANOVA) including models, R2 and probability values for the

final reduced models for antioxidant activities of soy protein isolate hydrolysates.

*F-ratio = Fcalculated/Ftabulated

DPPH radical scavenging


squares

Degrees of

freedom

Mean of

squares F-ratio

* R² p-value

Regression 265.75 5 53.15 7.85 0.77 <0.01

Residual 77.26 24 3.22

Total 343.01

Quadratic model: Y = 63.79x1 + 61.89x2 + 60.14x3 + 30.32x1x2 + 12.22x1x3 + 16.25x2x3

Inhibition of linoleic acid autoxidation


squares

Degrees of

freedom

Mean of

squares F-ratio

* R² p-value

Regression 1,1870.77 3 3,956.92 16.54 0.81 <0.01

Residual 2,695.65 26 103.68

Total 14,566.42

Special cubic model: Y = 18.64x1 + 18.89x2 + 18.47x3 + 1,601.25x1x2x3

Reducing power assay


squares

Degrees of

freedom

Mean of

squares F-ratio

* R² p-value

Regression 0.1300 4 0.0325 60.20 0.95 <0.01

Residual 0.0062 25 0.0002

Total 0.1362

Quadratic model: Y = 0.39x1 + 0.17x2 + 0.17x3 – 0.14x1x3 + 0.12x2x3

Total antioxidant capacity


squares

Degrees of

freedom

Mean of

squares F-ratio

* R² p-value

Regression 0.320 5 0.064 29.14 0.93 <0.01

Residual 0.025 24 0.001

Total 0.345

258

The specificity of each enzyme on protein hydrolysis could be attributable to the

differences observed in antioxidant activities and TCA soluble protein, releasing peptides with

different sizes, amino acid sequences and amounts. The enzyme specificities can be used to

predict the type of peptides produced and therefore their application.

The protease FlavourzymeTM

500L is a fungal protease/peptidase complex obtained from

A. oryzae that contains endoprotease and exopeptidase activities and cleaves amino acids on the

C-terminal side (Luna-Vital et al., 2014). On the other hand, AlcalaseTM

2.4L is an endoprotease

of the serine type obtained from B. licheniformis that has a high specificity for aromatic (Phe,

Trp, Tyr), acidic (Glu), sulfur containing (Met), aliphatic (Leu, Ala), hydroxyl (Ser) and basic

(Lys) residues (Doucet et al., 2003). Information about the source, mechanisms of action and

specificities for YeastMaxTM

A were not found in the literature and not provided by the enzyme

supplier. This commercial preparation is described as a mixture of proteases indicated for yeast

hydrolysis with maximum activity at pH 6-10 and temperature ranging from 50 to 60 °C. Based

on the results obtained in our study, YeastMaxTM

A probably has a broad specificity, hydrolyzing

most peptide bonds, which was characterized by the high TCA soluble protein content of the

hydrolysates (Figure 4a).

Studies about specificities of proteolytic enzymes as well as the application of systems

based on combination of proteases from different sources have been used for production of

bioactive peptides.

Adjonu et al., (2013) studied the enzymatic hydrolysis of whey protein isolate using α-

chymotrypsin, pepsin and trypsin with a focus on enzyme specificities in order to obtain peptides

with antioxidant activities. The results showed maximum DH for all samples ranging from 11.8

to 14.1%, with high values for the no heat treated protein hydrolyzed by chymotrypsin, followed

by pepsin and trypsin. Regarding to antioxidant activities, protein hydrolysates obtained using

pepsin were less effective in scavenging peroxyl radicals (ORAC assay) compared to the ABTS˙+

free radicals (FRSA assay). The ORAC and the FRSA values suggested that high DH values may

not be suitable for generating antioxidant peptides, since may result in release of high proportions

of free amino acids, which can negatively affect the antioxidant activity by acting as prooxidants

(Pihlanto, 2006; Adjonu et al., 2013). The profile changes in antioxidant activities and DH were

associated to the enzyme-peptide bond specificity. Both chymotrypsin and trypsin are alkaline

259

endoproteases with specificity by peptide bonds at the C-terminal side containing aromatic amino

acids (Trp, Tyr and Phe) and at the C-terminal side with Arg and Lys residues, respectively. On

the other hand, pepsin is an acidic endoprotease that act on peptide bonds at the N-terminal side

containing aromatic and hydrophobic amino acids (Adjonu et al., 2013).

Betancur-Ancona et al., (2014) reported that the enzymatic treatment of protein

concentrate of beans using mixture of proteases increased DH and had a positive impact on

antioxidant properties of the hydrolysates. The protein concentrate of beans reached DH values

ranging from 10 to 20% using AlcalaseTM

and FlavourzymeTM

after 120 min hydrolysis,

respectively, while the combination of them resulted in DH value of 33.29%. The combined use

of pepsin and pancreatin also increased DH of the protein, reaching a maximum value of 28.47%.

The hydrolysates obtained with AlcalaseTM

-FlavourzymeTM

system showed higher antioxidant

activity compared to Pepsin-Pancreatin hydrolysates. According these authors, intestinal enzymes

such as pepsin and pancreatin generate a large amount of proteins and oligopeptides, while

enzymes from bacterial and fungal such as AlcalaseTM

and FlavourzymeTM

generate a large

amount of small peptides and free amino acids, which exhibit greater activity.

4. Conclusion

The enzymatic hydrolysis of proteins based on their specificities and mechanisms of

action can be used as a valuable tool to improve the antioxidant properties of soy protein isolate

hydrolysates. Kinetic parameters obtained for thermal inactivation showed that the commercial

preparations of proteases were not inhibited by the products from soy protein isolate hydrolysis.

The use a binary mixture of FlavourzymeTM

500L and AlcalaseTM

2.4L increased the capacity of

DPPH radical scavenging reaching a maximum value of 70.84% at 5 mg mL-1

. Strong and

significant synergistic effects were observed in the hydrolysates obtained using the ternary

mixtures of FlavourzymeTM

500L, AlcalaseTM

2.4L and YeastMaxTM

A, reaching about 60%

inhibition of linoleic acid autoxidation against values of approximately 15% inhibition for the soy

protein hydrolysates obtained using individual enzymes. The results obtained in our study suggest

that the combined use of proteases with broad enzymatic specificities will release different

peptides and thus increase the number of cleavage sites in the protein and hydrolysates, resulting

in high antioxidant activities.

260

References

Abraham, J., Gea, T. & Sánchez, A. (2014). Substitution of chemical dehairing by proteases from

solid-state fermentation of hair wastes. Journal of Cleaner Production, 74, 191-198.

Adjonu, R., Doran, G., Torley, P. & Agboola, S. (2013). Screening of whey protein isolate

hydrolysates for their dual functionality: influence of heat pre-treatment and enzyme specificity.

Food Chemistry, 136, 1435–1443.

Anarjan, N. & Tan, C. P. (2013). Developing a three component stabilizer system for producing

astaxanthin nanodispersions. Food Hydrocolloids, 30, 437-447.

Anbu, P. (2013). Characterization of solvent stable extracellular protease from Bacillus koreensis

(BK-P21A). International Journal of Biological Macromolecules, 56, 162– 168.

Arabshahi-Delouee, S., & Urooj, A. (2007). Antioxidant properties of various solvent extracts of

mulberry (Morus indica L.) leaves. Food Chemistry, 102, 1233–1240.

Betancur-Ancona, D., Sosa-Espinoza, T., Ruiz-Ruiz, J., Segura-Campos, M. & Chel- Guerrero,

L. (2014). Enzymatic hydrolysis of hard-to-cook bean (Phaseolus vulgaris L.) protein

concentrates and its effects on biological and functional properties. International Journal of Food

Science and Technology, 49, 2-8.

Bougatef, A., Hajji, M., Balti, R., Lassoued, I., Triki-Ellouz, Y. & Nasri, M. (2009). Antioxidant

and free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein

hydrolysates obtained by gastrointestinal proteases. Food Chemistry, 114, 1198–1205.

Charney, J. & Tomarelli, R. M. (1947). A colorimetric method for the determination of the

proteolytic activity of duodenal juice. The Journal of Biological Chemistry, 170, 501-505.

Dorman, H. J. D., Kosar, M., Kahlos, K., Holm, Y., & Hiltunen, R. (2003). Antioxidant

properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and

cultivars. Journal of Agricultural and Food Chemistry, 51, 4563–4569.

Doucet, D., Otter, D. E., Gauthier, S. F., & Foegeding, E. A. (2003) Enzyme-induced gelation of

extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of

enzyme specificity. Journal of Agricultural and Food Chemistry, 51, 6300-6308.

261

Fernández, A. & Riera, F. (2013). β-Lactoglobulin tryptic digestion: a model approach for

peptide release. Biochemical Engineering Journal, 70, 88–96.

Gao, D., Cao, Y., & Li, H. (2010). Antioxidant activity of peptide fractions derived from

cottonseed protein hydrolysate. Journal of the Science of Food and Agriculture, 90, 1855–1860.

Hiller, B. & Lorenzen, P. C. (2009). Functional properties of milk proteins as affected by

enzymatic oligomerisation. Food Research International, 42, 899–908.

Hsiao, N-W., Chen, Y., Kuan, Y-C., Lee, Y-C., Lee, S-K., Chan, H-H. & Kao, C-H. (2014).

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease

extract, Peptidase R. Electronic Journal of Biotechnology, 17, 89–94.

Ktari, N., Bkhairia, I., Jridi, M., Hamza, I., Riadh, B. S. & Nasri, M. (2014). Digestive acid

protease from zebra blenny (Salaria basilisca): characteristics and application in gelatin

extraction. Food Research International, 57, 218–224.

Li, Q., Yi, L., Marek, P. & Iverson, B. L. (2013). Commercial proteases: present and future.

FEBS Letters, 587, 1155–1163.

Li-Chan, E. C. (2015). Bioactive peptides and protein hydrolysates: research trends and

challenges for application as nutraceuticals and functional food ingredients. Current Opinion in

Food Science, 1, 28-37.

Liu, Q., Kong, B., Xiong, Y. L. & Xi, X. (2010). Antioxidant activity and functional properties of

porcine plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chemistry,

118, 403–410.

López-Otín, C. & Bond, J. S. (2008). Proteases: multifunctional enzymes in life and disease. The

Journal of Biological Chemistry, 283, 30433-30437.



Luna-Vital, D.A., Mojica, L., González de Mejía, E., Mendoza, S. & Loarca-Piña, G., (2014).

Biological potential of protein hydrolysates and peptides from common bean (Phaseolus vulgaris

L.): a review. Food Research International, doi: 10.1016/j.foodres.2014.11.024

262

Martinello, T., Kaneko, T.M., Velasco, M.V.R., Taqueda, M.E.S., & Consiglieri, V.O., (2006).


mixture experimental design. International Journal of Pharmaceutics, 322, 87-95.

Nazeer, R. A. & Kulandai, K. A. (2012). Evaluation of antioxidant activity of muscle and skin

protein hydrolysates from giant kingfish, Caranx ignobilis (Forsska° l, 1775). International

Journal of Food Science and Technology, 47, 274–281.

Nazeer, R. A., Kumar, N.S. S., & Ganesh, R. J. (2012). In vitro and in vivo studies on the

antioxidant activity of fish peptide isolated from the croaker (Otolithes ruber) muscle protein

hydrolysate. Peptides, 35, 261–268.




Pihlanto, A. (2006). Antioxidative peptides derived from milk proteins. International Dairy

Journal, 16 (11), 1306–1314.

Prieto, P., Pineda, M. & Aguilar, M. (1999). Spectrophotometric quantitation of antioxidant

capacity through the formation of a phosphomolybdenum complex: specific application to the

determination of vitamin E1. Analytical Biochemistry, 269, 337–341.

Rao P.V., & Baral S.S. (2011). Experimental design of mixture for the anaerobic co-digestion of

sewage sludge. Chemical Engineering Journal, 172, 977-986.

Rui, X., Boye, J. I., Simpson, B. K. & Prasher, S. O. (2012). Angiotensin I-converting enzyme

inhibitory properties of Phaseolus vulgaris bean hydrolysates: effects of different thermal and

enzymatic digestion treatments. Food Research International, 49, 739-746.

Scheffe, H. (1963). The Simplex-Centroid Design for experiments with mixtures. Journal of the

Royal Statistical Society. Series B (Methodological), 25(2), 235-263.

Segura-Campos, M. R., Espinosa-García, L., Chel-Guerrero, L. A. & Betancur-Ancona, D. A.

(2012). Effect of enzymatic hydrolysis on solubility, hydrophobicity, and in vivo digestibility in

cowpea (Vigna unguiculata). International Journal of Food Properties, 15, 770–780.

263

Singh, H. (2011). Milk protein products: functional properties of milk proteins. In W. F. John, F.

F. Patrick, & L. H. M. Paul (Eds.), Encyclopedia of Dairy Sciences (pp. 887-893). Waltham:

Elsevier Inc.

Yildirim, A., Mavi, A., & Kara, A. A. (2001). Determination of antioxidant and antimicrobial

activities of Rumex crispus L. extracts. Journal of Agriculture and Food Chemistry, 49, 4083–

4089.

Zhou, D., Qin, L., Zhu, B., Li, D., Yang, J., Dong, X., & Murata, Y. (2012). Optimisation of




265

Conclusões gerais

Os resultados obtidos mostraram que a aplicação da técnica de planejamento experimental

de misturas é um método atrativo para o aumento da produção de proteases e obtenção de

preparações com diferentes propriedades bioquímicas. Esse tipo de planejamento também

mostrou-se como uma importante ferramenta para o estudo da hidrólise simultânea de misturas de

proteínas em um processo simplificado assim como a utilização de composições contendo

proteases de diferentes fontes, permitindo a maximização de diversas atividades biológicas e

propriedades funcionais das proteínas.

O estudo da produção de proteases por A. niger LBA02 em fermentação semissólida

utilizando diferentes resíduos agroindustriais como substratos e a utilização de formulações

contendo misturas binárias, ternárias ou quaternárias dos substratos mostrou-se um processo

atrativo para maximização da produção. Os resultados obtidos mostraram que a utilização de uma

mistura contendo farelo de trigo, farelo de soja, farelo de algodão e casca de laranja em iguais

proporções resultou em aumentos de 33,7, 7,6, 30,8 e 581,7% na produção de proteases, quando

os valores são comparados aos obtidos utilizando cada substrato de forma isolada. As maiores

atividades de protease foram detectadas quando utilizada a mistura quaternária dos substratos e a

mistura binária de farelo de trigo (1/2) e farelo de soja (1/2), atingindo 245,97 e 262,78 U g-1

após

48h de fermentação.

O estudo de aspectos físico-químicos dos resíduos agroindustriais revelou que parâmetros

como a capacidade de retenção de água, distribuição granulométrica, densidade aparente e

composição química apresentaram forte influência sobre a produção de proteases por A. niger

LBA02 em fermentação semissólida. A composição química dos resíduos agroindustriais exerceu

um dos efeitos mais notórios, onde a produção de proteases foi induzida nas primeiras 48h de

fermentação nos substratos que continham maior quantidade de proteína e inibida pelos

substratos com alto teor de carboidratos.

A caracterização bioquímica das preparações enzimáticas obtidas a partir de A. niger

LBA02 por fermentação semissólida utilizando farelo de trigo, farelo de soja, farelo de algodão,

casca de laranja e a mistura quaternária destes substratos permitiu avaliar os aspectos de

produção sob um novo ponto de vista, permitindo a seleção de uma formulação ou substrato que

266

permita a produção de enzimas com características mais atrativas, como maior estabilidade

térmica. O micro-organismo apresentou a capacidade de secretar diferentes tipos de protease

quando cultivado nos diferentes substratos. Parâmetros cinéticos e termodinâmicos para

inativação térmica incluindo valores de t1/2, D, Ead, ΔH, ΔG e ΔS mostraram que as proteases

produzidas em farelo de trigo foram as mais estáveis termicamente ao passo que as proteases

produzidas em farelo de algodão foram as mais sensíveis. Para ativação térmica, as proteases

produzidas em casca de laranja apresentaram o menor valor para energia de ativação, atingindo

16,32 kJ mol-1

, enquanto o maior valor foi detectado para as proteases produzidas utilizando a

mistura quaternária dos substratos (19,48 kJ mol-1

). As preparações enzimáticas também

apresentaram diferentes perfis quando a especificidade de substrato foi determinada frente a

diferentes substratos proteicos. A maior atividade relativa (496,4%), considerando o substrato

caseína como padrão (100%) foi observada para hemoglobina de sangue bovino hidrolisada com

a preparação enzimática de proteases de A. niger LBA02 obtida a partir da fermentação

utilizando a mistura quaternária dos resíduos agroindustriais.

A determinação do pH e temperatura ótima para atividade e estabilidade utilizando

delineamento composto central rotacional (DCCR), mostrou que as proteases de A. niger LBA02

foram mais ativas na faixa de pH 3 a 4 e temperaturas de 45 a 50 °C. As proteases mostraram-se

estáveis na faixa de pH de 2,5 a 4,5 e na faixa de 40 a 50 °C após 1h de incubação.

As preparações de proteases produzidas em farelo de trigo, farelo de soja e farelo de

algodão apresentaram diferentes valores para atividade coagulante do leite atingindo 22,22, 0,56

e 5,80 U mL-1

, respectivamente. Hidrolisados de proteínas do soro de leite obtidos a partir destas

preparações, também apresentaram diferentes perfis de atividade antioxidante, sendo as proteases

produzidas em farelo de trigo e farelo de algodão as mais adequadas para obtenção de

hidrolisados com forte capacidade de inibição de radicais DPPH, atingindo 82,8 e 84,5% de

inbição, respectivamente, na concentração de 10 mg mL-1

.

O estudo da hidrólise enzimática simultânea utilizando diferentes fontes de proteínas de

forma isolada ou em combinadas em misturas e a protease comercial Flavourzyme® 500L

mostrou diversos efeitos sinérgicos para propriedades funcionais, atividade antioxidante, anti-

adipogênica e na indução do crescimento de bactérias lácticas e probióticas.

267

Dentre as propriedades funcionais avaliadas, a atividade emulsificante foi a que

apresentou o maior efeito sinérgico entre as diferentes fontes de proteínas. A hidrólise enzimática

da mistura ternária contendo proteína isolada de soja, proteínas do soro de leite e da clara de ovo

em iguais proporções aumentou o índice de atividade emulsionante de 2 a 12 vezes comparado

aos índices obtidos para os hidrolisados produzidos de forma isolada.

Para atividade antioxidante, o maior destaque foi para a capacidade de inibição de radicais

DPPH, onde a mistura hidrolisada de proteínas do soro de leite e da clara de ovo, resultaram em

aumentos de 45,1 e 37,3%, respectivamente, em comparação aos hidrolisados obtidos utilizando

as fontes isoladas.

Para atividade anti-adipogênica, os resultados obtidos mostraram que após a hidrólise

enzimática, a mistura contendo proteínas do soro de leite (1/2) e proteínas da clara de ovo (1/2)

mostrou o maior aumento na supressão do acúmulo relativo de lipídeos (ARL) nas células pré-

adipócitas 3T3-L1. A mistura hidrolisada contendo proteínas do soro de leite (1/2) e proteínas da

clara de ovo (1/2) na concentração de 800 ppm mostraram aumentos de 220 e 27% na supressão

do ARL, respectivamente, quando comparadas aos substratos isolados, atingindo uma supressão

máxima de ARL de 15,5%. Na avaliação de diferentes concentrações dos hidrolisados, o maior

nível de supressão do ARL (%) foi de 47,9% quando as células 3T3-L1 foram tratadas com os

hidrolisdos contendo a mistura de proteínas do soro de leite (1/2) e da clara de ovo (1/2) na

concentração de 1200 ppm. A ultrafiltração mostrou que frações de massas moleculares inferiores

a 30 kDa exercem forte influência sobre a supressão do ARL.

Para atividade antimicrobiana, os resultados obtidos mostraram que na maior parte dos

ensaios, a suplementação dos meios de cultivo com fontes de proteínas estimulou o crescimento

das bactérias patogênicas. A linhagem de S. aureus ATCC 6538 foi a única que apresentou

inibição significativa do crescimento quando cultivada em meio suplementado com uma mistura

binária de proteína isolada de soja (1/2) e proteínas da clara de ovo (1/2) não hidrolisadas,

resultando em inibição de 16,82%. As linhagens de leveduras não apresentaram mudanças nos

perfis de inibição do crescimento quando comparadas as amostras hidrolisadas e não hidrolisadas.

A maior inibição observada foi detectada para a linhagem de S. cerevisiae KL 88 cultivada em

meio suplementado com a mistura ternária de proteínas hidrolisadas em proporções iguais,

resultando em inibição de 15,42%.

268

Os hidrolisados proteicos também apresentaram um efeito positivo sobre o estímulo do

crescimento de linhagens de bactérias lácticas e probióticas. A suplementação do meio de cultivo

com a mistura binária de hidrolisados de proteínas do soro de leite (1/2) e da clara de ovo (1/2) na

concentração de 25 mg mL-1

resultou em aumentos de 100,0 e 29,4% no crescimento celular de

uma cultura mista de Streptococcus thermophilus e Lactobacillus delbrueckii e de Lactobacillus

acidophilus, respectivamente. Para a linhagem de Bifidobacterium lactis, um crescimento 86,2%

superior ao controle foi observado quando os meios foram suplementados com 25 mg mL-1

dos

hidrolisados contendo a mistura binária de proteína isolada (1/2) e proteínas do soro de leite

(1/2).

O estudo da obtenção de hidrolisados de proteína isolada de soja utilizando proteases de

diferentes fontes de forma isolada ou em uso combinado permitiu uma interessante discussão

sobre os mecanismos de ação e especificidades de enzimas proteolíticas e os seus efeitos sobre a

atividade antioxidante dos hidrolisados. Os resultados obtidos mostraram que a atividade

antioxidante apresentou respostas variáveis quando avaliada por diferentes métodos. Para

inibição de radicais DPPH, os hidrolisados obtidos com a protease Flavourzyme® 500L

combinada com Alcalase® 2.4L mostrou o maior efeito sinérgico, atingindo aumentos de 10,9 e

13,2% na atividade antioxidante, em comparação com os hidrolisados produzidos com enzimas

isoladas. Os hidrolisados obtidos utilizando as misturas ternárias de Flavourzyme® 500L,

Alcalase® 2.4L e YeastMax

® A apresentaram o maior poder de inibição da auto-oxidação do

ácido linoleico. Já para os ensaios baseados no poder redutor das moléculas antioxidantes, que

incluíram o poder de redução de Fe3+

e a capacidade antioxidante total (redução de Mo (VI) a Mo

(V)), os hidrolisados produzidos usando Flavourzyme® 500L apresentaram maior atividade.

269

Sugestões para trabalhos futuros

- Caracterizar os resíduos agroindustriais de forma mais aprofundada e propor mecanismos de

indução da produção de proteases baseados na presença de fontes de proteínas com diferentes

massas moleculares, digestibilidade e composição de aminoácidos, assim como verificar a

correlação entre a presença de diferentes fontes de carbono (glicose, sacarose, amido, celulose,

hemicelulose, pectina, etc.) e/ou o balanço entre elas e a produção de diversas enzimas;

- Caracterizar os extratos enzimáticos obtidos por fermentação semissólida utilizando meios de

cultivo baseados na mistura de diferentes resíduos, quanto à presença de outros grupos de

enzimas e propor formulações para a produção simultânea de enzimas ou produção preferencial

de uma determinada enzima;

- Verificar a produção simultânea de enzimas com perfis bioquímicos complementares utilizando

culturas mistas de micro-organismos, como por exemplo, para atuação em uma ampla faixa de

pH e temperatura;

- Realizar um estudo mais aprofundado de caracterização das matrizes proteicas utilizadas para a

hidrólise enzimática, e propor formulações para a produção de hidrolisados com propriedades

multifuncionais, englobando aspectos tecnológicos e biológicos;

- Purificar os peptídeos com atividade biológica por cromatografia de troca iônica e filtração em

gel;

- Identificar as sequências peptídicas responsáveis pelas bioatividades utilizando cromatografia

líquida de alta eficiência e espectrometria de massas;

- Verificar as alterações no perfil de peptídeos após a hidrólise enzimática correlacionando com

as atividades biológicas;

- Avaliar outras atividades biológicas, incluindo atividade antitumoral, antitrombótica e anti-

hipertensiva, dos hidrolisados proteicos.

RUANN JANSER SOARES DE CASTROrepositorio.unicamp.br/.../Castro_RuannJanserSoaresde_D.pdfCastro,...

Documents

Transcript of RUANN JANSER SOARES DE CASTROrepositorio.unicamp.br/.../Castro_RuannJanserSoaresde_D.pdfCastro,...