Aproveitamento do soro do queijo coalho para produção e ...

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RENORBIO

Universidade Federal do Rio Grande do Norte

Programa de Pós-Graduação em Biotecnologia

Aproveitamento do soro do queijo “coalho” para produção

e aplicação da -galactosidase

Catherine Teixeira de Carvalho

Natal – RN

2019

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Catherine Teixeira de Carvalho

Aproveitamento do soro do queijo “coalho” para produção

e aplicação da -galactosidase

Orientadora: Profa. Drª. Gorete Ribeiro de Macedo

Co-orientador: Prof. Dr. Francisco Canindé de Sousa Junior

Tese de Doutorado submetida ao Programa de

Pós-Graduação em Biotecnologia–RENORBIO,

como um dos pré-requisitos necessários à

obtenção do título de doutora.

Área de concentração: Biotecnologia Industrial

Linha de Pesquisa: Bioprocessos

Natal – RN

2019

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Catherine Teixeira de Carvalho – Dezembro / 2019

Universidade Federal do Rio Grande do Norte - UFRN

Sistema de Bibliotecas - SISBI

Catalogação de Publicação na Fonte. UFRN - Biblioteca Central Zila Mamede

Carvalho, Catherine Teixeira de.

Aproveitamento do soro do queijo "coalho" para produção e aplicação

da -galactosidase / Catherine Teixeira de Carvalho. - 2020.

136 f.: il.

Tese (doutorado) - Universidade Federal do Rio Grande do Norte,

Centro de Ciências da Saúde, Programa de Pós-Graduação em

Biotecnologia, RENORBIO, Natal, RN, 2020.

Orientadora: Profa. Dra. Gorete Ribeiro de Macedo.

Coorientador: Prof. Dr. Francisco Canindé de Sousa Júnior.

1. Kluyveromyces sp. - Tese. 2. -galactosidase - Tese. 3. Hidrólise

lactose - Tese. I. Macedo, Gorete Ribeiro de. II. Sousa Júnior,

Francisco Canindé de. III. Título.

RN/UF/BCZM CDU 608.7

Elaborado por Ana Cristina Cavalcanti Tinoco - CRB-15/262

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CATHERINE TEIXEIRA DE CARVALHO

Aproveitamento do soro do queijo “coalho” para produção e aplicação da -galactosidase

Tese apresentada à Rede Nordeste de Biotecnologia (RENORBIO) como requisito para

obtenção do título de Doutora em Biotecnologia.

Área de Concentração: Biotecnologia Industrial

Aprovada em 18 de dezembro de 2019 por:

________________________________________

Profª. Dra. Gorete Ribeiro Macedo

Presidente - Orientadora

Universidade Federal do Rio Grande do Norte – UFRN

________________________________________

Prof. Dr. Francisco Canindé de Sousa Júnior

Titular – Coorientador


_____________________________________

Prof Dr. Everaldo Silvino dos Santos

Titular


____________________________________

Prof.ª Dra. Cristiane Fernandes de Assis

Titular


_____________________________________

Profª. Dra. Ana Lúcia Figueiredo Porto

Titular

Universidade Federal Rural de Pernambuco– UFRPE

____________________________________

Dr. Sérgio Dantas de Oliveira Júnior

Titular

Instituto Nacional de Pesquisa da Amazônia - INPA

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AGRADECIMENTOS

Aos meus pais, Carlos César (in memorian) e em especial Arlete Teixeira, por todo carinho,

apoio, dedicação e torcida em todas as etapas da minha vida, minha imensa gratidão.

À minha família meus agradecimentos.

Ao meu Coorientador Prof. Francisco Canindé de Sousa Júnior, pelo direcionamento e apoio

oferecido durante este trabalho, meu muito obrigado.

À Profa. Gorete Ribeiro, minha orientadora, pelo incentivo, amizade e energia positiva que eu

sempre pude contar, minha gratidão.

Ao Prof. Everaldo Silvino, coordenador do Laboratório de Engenharia Bioquímica, pelo

incentivo, amizade e energia positiva que eu sempre pude contar, muito obrigado

Em especial a Fábio Macedo pela constante disposição e paciência em contribuir no

desenvolvimento deste trabalho, minha eterna gratidão.

À Sérgio Dantas pelo carinho e disponibilidade em contribuir com o trabalho, todo o meu

carinho e gratidão

À profa. Márcia Pedrini por disponibilizar o laboratório de bioprocessos, sempre que

necessário, meu muito obrigado

.

À Waleska, Ana Laura, Júlia, Carlos Padilha e Wildson pela valiosa ajuda e apoio durante o

desenvolvimento do trabalho.

Aos Colegas do Laboratório Sinara, Cleitiane, Vanessa, Petrucia, Victor, Willyan, Pedro,

Marcelo pelo convívio harmonioso, companheirismo e incentivo.

À servidora do Programa de Pós-graduação em Biotecnologia/RENORBIO (Ponto focal RN)

Paula Peroba, pela disponibilidade e atenção.

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SUMÁRIO

LISTA DE FIGURAS .............................................................................................................. v

LISTA DE TABELAS ........................................................................................................... vii

LISTA DE ABREVIATURAS E SIGLAS ........................................................................... viii

RESUMO ................................................................................................................................. x

ABSTRACT ............................................................................................................................ xi

CAPÍTULO 1 – INTRODUÇÃO ........................................................................................... 13

CAPÍTULO 2– OBJETIVOS ................................................................................................. 16

2.1 Objetivo geral ........................................................................................................... 16

2.2 Objetivos específicos ................................................................................................ 16

CAPÍTULO 3 – REVISÃO A LITERATURA ...................................................................... 18

3.1. Setor de laticínios ...................................................................................................... 18

3.1.1 Produção de leite ................................................................................................ 18

3.1.2 Soro do queijo .......................................................................................................... 20

3.1.3 Soro do queijo um resíduo valioso .................................................................... 21

3.2. Lactose importância e aplicação ................................................................................ 23

3.2.1 Hidrólise da lactose ........................................................................................... 24

3.3. Enzima -galactosidase ............................................................................................ 25

3.4. Biotecnologia aplicada a produção de -galactosidase.................................................. 29

3.4.1. Gênero Kluyveromyces e Saccharomyces: metabolismo e produção de -gal .. 29

3.5. Recuperação e purificação da β-galactosidase .......................................................... 32

3.5.1 Sistemas de Cromatografia ................................................................................. 33

3.6. Imobilização de Enzimas ........................................................................................... 35

3.7. Aplicações da β-galactosidase na indústria alimentícia ............................................ 38

REFERÊNCIAS BIBLIOGRÁFICAS ................................................................................... 43

CAPÍTULO 4 – ARTIGOS DERIVADOS DA TESE .......................................................... 54

ARTIGO 1 - Potential of “coalho” cheese whey as lactose source for β-galactosidase and

ethanol co-production by Kluyveromyces spp. yeasts ........................................................... 54

ARTIGO 2 - Recovery of β-galactosidase produced by Kluyveromyces lactis by ion

exchange chromatography: Optimization of pH and ionic strength conditions using

experimental design .............................................................................................................. 85

ARTIGO 3- Lactose hydrolysis using β-Galactosidase from Kluyveromyces lactis

immobilized with sodium alginate for potential commercial applications .......................... 105

References ........................................................................................................................... 126

CAPÍTULO 5 – CONSIDERAÇÕES FINAIS .................................................................... 134

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LISTA DE FIGURAS

Figura 1: Evolução da produção mundial do leite de vaca................................................ 17

Figura 2: Evolução da produção do leite no Brasil 2006- 2018......................................... 18

Figura 3: Estrutura química da lactose............................................................................... 22

Figura 4: Estrutura molecular da -galactosidase.............................................................. 25

Figura 5: Métodos de imobilização de enzima...................................................................

35

ARTIGO 1

Figure 1: Biomass growth profile for K. marxianus ATCC 36907 (●) and K. lactis

NRRLY-8279 (○), and lactose consumption profile for K. marxianus ATCC 36907 (▲)

and K. lactis NRRLY-8279 (△) in the orbital shaker cultivations. (A) C:N 1,5:1; (B) C:N

2,5:1. Mean values ± Standard deviation…………………………………………

64

Figure 2: Co-production profile of β-galactosidase for K. marxianus ATCC 36907 (■)

and K. lactis NRRL Y-8279 (□) and of ethanol for K. marxianus ATCC 36907 (▼) and

K. lactis NRRLY-8279 (▽) in the orbital shaker cultivations. (A) C/N 1,5:1; (B) C/N

2,5:1. Mean values ± Standard deviation………………………………………………...

65

Figure 3: (A) Biomass (cells) growth (●) and substrate (lactose) consumption (▲)

profiles; and (B) Co-production of β-galactosidase (○) and ethanol (□) for the bioreactor

cultivations using K. lactis NRRLY-8279 and the C:N ratio of 1.5:1. Mean values ±

Standard deviation……………………………………………………………

70

Figure 4: Enzymatic activity stability of β-galactosidase produced by K. lactis NRRL Y-

8279, using the C:N ratio of 1.5:1 in bioreactor cultivations. (A) Stability in relation to

the pH; (B) Stability in relation to metallic ions; (C) Stability in relation to temperature.

Mean values ± standard deviation. Different lowercase letters (a, b, c, d) indicate

statistical differences (p < 0.05) for different pH/metallic ion/temperature, at the same

incubation time. Different capital letters (A, B, C) indicate statistical differences (p <

0.05) for different incubation periods within the same pH/metallic

ion/temperature…………………………………………………………………………...

73

ARTIGO 2

Figure 1: Adsorption of β-gal on 4 resins in the pH range of 6.0-8.0. Different lowercase

letters (a, b, c, d) indicate statistical differences (p < 0.05) for different pH with the same

adsorbent. Different uppercase letters (A, B, C) indicate statistical differences (p < 0.05)

for different adsorbents considering the same pH…………………

91

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Figure 2: Pareto Charts of the standardized effects for the responses yield (Y) and fold

purification of β-gal (FP), respectively………………………………………………….

94

Figure3: Response surface (RS) of Y and FP. (A) RS of the yield and (B) of the fold

purification of β-gal as function of pH x IS………………………………………………

97

Figure 4: Purification chromatogram of β-gal produced by K. lactis in a fixed bed,

submitted to a NaCl gradient through elution, where (□) protein concentration and (■)

enzymatic activity……………………………………………………………………….

98

Figure 5: PAGE gel of polyacrylamide 9%. Lane M – Maker protein (MM), Lane 1–

crude extract (EB), Lane 2 – elution sample of 0,1 to 0,4 M of NaCl…………………

100

ARTIGO 3

Figure 1: Stability of the enzymatic activity of immobilized β-galactosidase with sodium

alginate at different pH values. Mean values ± Standard deviation. Different low case

letters (a, b, c) indicate statistical difference (p < 0.05) for different pH values at the same

incubation time according to Tukey’s test. Different capital letters (A, B, C) indicate

statistical difference (p < 0.05) for different incubation times at the same pH according

to Tukey’s test……………………………………………………………….

115

Figure 2: Stability of the enzymatic activity of immobilized β-galactosidase with sodium

alginate at different temperatures. Mean values ± Standard deviation. Different low case

letters (a, b, c) indicate statistical difference (p < 0.05) for different temperatures at the

same incubation time according to Tukey’s test. Different capital letters (A, B, C)

indicate statistical difference (p < 0.05) for different incubation times at the same

temperature according to Tukey’s test………………………………………

117

Figure 3: Stability of the enzymatic activity of immobilized β-galactosidase with

sodium alginate at different metallic ions. Mean values ± Standard deviation. Different

low case letters indicate statistical difference (p < 0.05) for different metallic ions at the

same incubation time according to Tukey’s test. Different capital letters indicate

statistical difference (p < 0.05) for different incubation times at the same metallic ion

according to Tukey’s test…………………………………………………………………

118

Figure 4: FT-IR spectra of sodium alginate, immobilization support, free −gal and

Immobilized −gal, respectively…………………………………………………………

120

Figure 5: SEM of immobilization support (A, B) and immobilized -gal (C, D), pH 6,6

and fixed alginate concentration of 0,7%...........................................................................

121

Figure 6: Comparative lactose hydrolysis conversion (%) between the immobilized

system (○) and the crude enzymatic extract (●). Mean values for three repetitions (n =

3)………………………………………………………………………………………..

122

Figure 7: Gastroinstestinal stability of immobilized β-gal on simulated gastrical fluid

(SFG, pH 2.0; ●) and simulated intestinal fluid (SFI, pH 7.5; ○)………………………

124

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LISTA DE TABELAS

Tabela 1: Alguns microrganismos produtores de −galactosidase .................................

ARTIGO 1

Table 1: Complete composition of the culture media used for the two carbon/nitrogen

(C: N) ratios ratio) in this study. Lactose from partially deproteinized cheese whey

(CWD) was used as a sole source of carbon…………………………………………….

28

58

Table 2: Physicochemical parameters for cheese whey (CW) and cheese whey partially

deproteinized (CWD)…………………………………………………………………….

63

Table 3: Kinetic parameters for the orbital shaker and bioreactor cultivations for the co-

production of ethanol and β-galactosidase using cheese whey as substrate…………….

66

ARTIGO 2

Table 1: Factors and levels used in the experimental design 22……………………… 89

Table 2: Experimental design 22 with coding and levels of variables used to purify the β-

gal enzyme as a function of pH and ionic strength……………………………………

93

Table 3: Analysis of variance (ANOVA) of the adjusted models to the experimental

responses FP and ……………………………………………………………………….

96

ARTIGO 3

Table 1. Immobilization conditions of the β-gal enzyme for the different concentrations

of sodium alginate………………………………………………………………………

113

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LISTA DE ABREVIATURAS E SIGLAS

A Valor da atividade enzimática em equilíbrio

A0 Valor inicial da atividade enzimática

ad Atividade da enzima imobilizada

ANOVA Análise de variância

ar Atividade enzimática recuperada

at Atividade enzimática teórica

BSA Albumina sérica bovina

C:N Razão carbono/nitrogênio

CO2 Gás carbônico

CW Cheese whey

CWD Deproteinized cheese whey

EI Eficiência de imobilização

FDA Food and drug Administration

FP Fator de purificação

g/L gramas por litro

gl graus de liberdade

GOS Galactooligossacarídeos

GRAS Generally Recognized as safe

IS Força iônica

kDa Kilodalton

mg Miligrama

mL Mililitro

mM Milimolar

MQ Média quadrática

ºC Graus celsius

ONP O- nitrofenil- piranose

ONPG O- nitrofenil- -D galactopiranosídeo

PAGE Eletroforese em gel de poliacrilamida

Px Máxima produtividade de células

q Capacidade de adsorção

R2 Coeficiente de determinação

rpm Rotações por minuto

SFI Fluido intestinal simulado

SGF Fluido gástrico simulado

SQ Soro do queijo

SQD Soro do queijo desproteinizado

SS Soma quadrática

T Temperatura

U/g Unidade de enzima por grama

U/mL Unidade de enzima por mililitro

V Volume do extrato enzimático

Vads Volume do adsorvente

Y Rendimento

-gal - galactosidase

max Velocidade máxima específica de crescimento

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𝑋𝑜 Concentração inicial de células (g/L)

% Percentual

𝑋𝑚𝑎𝑥 Concentração máxima de biomassa

𝑌𝑋 𝑆⁄ Rendimento de células

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RESUMO

O presente estudo teve como objetivo avaliar o potencial do soro do queijo “coalho” como

substrato para produção de biomoléculas por leveduras Kluyveromyces sp. e investigar

estratégias de purificação e imobilização da enzima β-galactosidade (β-gal) visando aplicação

industrial. Na primeira etapa, observou-se o comportamento das leveduras para síntese de β-gal

e a produção de etanol por fermentação submersa em frascos agitados e biorreator utilizando

diferentes razões carbono/nitrogênio C:N (1,5:1 e 2,5:1). A melhor eficiência foi obtida com

Kluyveromyces lactis NRRL Y- 8279, que produziu 21,09 ± 0,69 U/mL de β-gal e 7,10 ± 0,09

g/L de etanol em 16 horas de cultivo. Diante dos resultados iniciais, avaliou-se as condições de

purificação da β-gal em cromatografia em leito fixo utilizando um planejamento experimental

22. Os parâmetros pH e força iônica foram avaliados considerando o fator de purificação, sem

prejuízo ao rendimento. Os níveis mais altos de ambos os parâmetros no estudo aumentaram o

fator de purificação (FP) de β-gal para 2,00, com maior influência da força iônica na resposta

para FP. A enzima purificada parcialmente foi submetida a eletroforese, que apresentou uma

banda com massa molecular na faixa entre 66 e 140 kDa, configurando a enzima de interesse.

Na última etapa do estudo, observou-se as condições de hidrólise da lactose no soro de queijo

“coalho” com a forma imobilizada de β-gal em alginato de sódio a 1% (p/v). A eficiência da

imobilização atingiu 66%. Além disso, a forma imobilizada da enzima apresentou maior

estabilidade às mudanças de pH e temperatura e uma conversão de lactose (46%) sem maiores

diferenças estetíticas quando comparada ao extrato bruto de β-gal (53%). Para as simulações

gástricas e intestinais, cerca de 40% da atividade enzimática foi preservada após 2 horas de

exposição a condições gastrointestinais simulados. No geral, os resultados aqui descritos são

promissores para as aplicações industriais da β-galactosidase de K. Lactis NRRL Y- 8279.

Palavras-chave: Kluyveromyces sp., Etanol, β-Galactosidase, Imobilização, Hidrólise da

lactose.

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ABSTRACT

The present study aimed to produce the enzyme β-galactosidase (β-gal) using “coalho” cheese

whey as biotechnological substrate by yeasts of the genus Kluyveromyces and to evaluate

processing strategies that enable its application in the food industry. In the first stage of this

study, the co-production of β-gal and ethanol by submerged fermentation in shake flasks and

bioreactors using different carbon/nitrogen C:N rations (1.5:1 and 2.5: 1). The best efficiency

was obtained with Kluyveromyces lactis NRRL Y-8279, which produced 21.09 ± 0.69 U / mL

β-gal and 7.10 ± 0.09 g / L ethanol in 16 hours of cultivation. Based on the initial results, the β-

gal purification conditions in fixed bed chromatography were evaluated using experimental

design 22. The pH and ionic strength parameters were evaluated considering the purification

factor, without prejudice to yield. Higher levels of both parameters in the study increased the

β-gal purification factor (PF) to 2.00, with greater influence of ionic strength on the PF

response. The partially purified enzyme was submitted to electrophoresis, which presented a

band with molecular mass in the range between 66 and 140 kDa, configuring the enzyme of

interest. In the last stage of the study, lactose hydrolysis conditions were observed in the curd

cheese whey with the immobilized form of β-gal in 1% (w/v) sodium alginate. The

immobilization efficiency reached 66% and high recovered activity was achieved. In addition,

the immobilized form of the enzyme presented higher stability to pH and temperature changes

and a lactose conversion (46%) without major aesthetic differences when compared to the crude

enzyme extract (53%). For the gastrointestinal simulations, around 40% of the enzymatic

activity was preserved after 2 hours of exposure to simulated gastrointestinal environments.

Overall, the results described here are promising for the industrial applications of β-

galactosidase from K. lactis.

Key-words: Cheese whey, Kluyveromyces sp; β-galactosidase; Ethanol; Lactose hydrolysis

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Capítulo 1

Introdução

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CAPÍTULO 1 – INTRODUÇÃO

A indústria de laticínios desempenha um papel economicamente importante no setor

agroalimentar, e sua produção anual crescente está diretamente relacionada à maior geração de

resíduos e subprodutos, como o soro de queijo (SQ) (Akhlaghi et al., 2017; Escalante et al.,

2018). A geração global do soro do queijo é estimada em cerca de 200 milhões de toneladas

por ano, com tendência de crescimento linear (Domingos et al., 2017; Treu et al., 2019).

No nordeste do Brasil, a produção de soro de queijo está intrinsecamente relacionada à

indústria do queijo de coalho. O queijo de coalho é um produto brasileiro altamente tradicional,

que assume componentes socioeconômicos e nutricionais relevantes nessa região (Fontenele et

al., 2017; Soares et al., 2017). Entretanto, o soro do queijo é um subproduto com significativo

potencial poluidor devido ao seu elevado teor de matéria orgânica e alta demanda bioquímica

de oxigênio (DBO) (Andrade et al., 2017). No entanto, em base seca, a composição de SQ pode

atingir até 80% de lactose (Albuquerque et al., 2018; Zhou et al., 2019). Considerado um

subproduto valioso, o soro do queijo é o principal componente das águas residuais de produtos

lácteos, sendo avaliado como um material recuperável em relação à disponibilidade de

nutrientes (Aydiner et al., 2013).

Várias estratégias têm sido investigadas para lidar com o descarte de resíduos de soro

do queijo, dentre as quais se destaca o uso de processos biotecnológicos, como alternativa viável

para converter tal subproduto em compostos com maior valor agregado (Carota et al., 2017).

Embora difícil de degradar no meio ambiente, a lactose do soro do queijo pode ser utilizada

como substrato para a fermentação visando a obtenção de etanol (Ariyanti e Hadiyanto, 2013;

Beniwal et al., 2018), ácido galactônico (Zhou et al., 2019), β-galactosidase (Perini, et al., 2013;

Rao e Dutta, 1977) entre outros bioprodutos.

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A enzima β-galactosidase (β-gal; EC 3.2.1.23), também conhecida como lactase, é responsável

pela hidrólise de ligações glicosídicas da lactose (Panesar et al., 2018) sendo um produto de

grande interesse, e com diversas aplicações, na indústria alimentícia. Além de contribuir para a

crescente participação, no mercado, de produtos sem lactose para consumidores com restrição

de dieta (Suri et al., 2019), a β-gal também é usada para a produção enzimática de prebióticos

alimentares, como lactulose e diferentes galactooligossacarídeos (GOS) (Nooshkam et al, 2018;

Panesar et al., 2018).

Neste sentido, o presente estudo teve como objetivo geral avaliar o potencial do resíduo

soro do queijo “coalho” como substrato para produção de biomoléculas por leveduras

Kluyveromyces sp., bem como avaliar estratégias de purificação e imobilização da enzima β-

gal visando a sua aplicação na indústria alimentícia.

A tese encontra-se dividida em cinco capítulos, apresentados da seguinte forma: O

Capítulo 1 consiste em uma introdução, na qual a importância da tese é contextualizada. O

Capítulo 2 mostra os objetivos propostos e o Capítulo 3 traz uma revisão da literatura, na qual

são apresentados os fundamentos teóricos. No Capítulo 4, a metodologia, os resultados e as

discussões são apresentados na forma de artigos. Destaca-se que os artigos são apresentados

seguindo as regras de formatação exigidas pelos periódicos. Apresenta-se, em inglês, os artigos

“Potential of “coalho” cheese whey as lactose source for β-galactosidase and ethanol co-

production by Kluyveromyces spp. yeasts”, submetido no periódico Journal of Dairy Science,

o artigo “Recovery of β-galactosidase produced by Kluyveromyces lactis in ion exchange

chromatography: Optimization of pH and ionic strength conditions using experimental design”

a ser submetido ao periódico Biotechnology Progress e o artigo “Lactose hydrolysis using

sodium alginate immobilized Kluyveromyces lactisβ-galactosidase for use in food industry”,

submetido ao periódico Process Biochemistry. No Capítulo 5, apresenta-se as considerações

finais.

15


Capítulo 2

Objetivos

16


CAPÍTULO 2– OBJETIVOS

2.1 Objetivo geral

• Avaliar o potencial do soro do queijo “coalho” como substrato para produção de

biomoléculas por leveduras Kluyveromyces sp. e investigar estratégias de purificação e

imobilização da enzima -gal visando aplicação na indústria alimentícia.

2.2 Objetivos específicos

• Realizar a caracterização físico-química do soro do queijo coalho determinando os

componentes do sobrenadante rico em lactose;

• Avaliar as condições de co-produção de β-galactosidase (β-Gal) e etanol com diferentes

razões de C:N em frascos agitados;

• Otimizar a co-produção de β-gal e etanol em biorreator de bancada utilizando a melhor

condição obtida nos ensaios em frascos agitados;

• Determinar os parâmetros cinéticos das leveduras do gênero Kluyveromyces sp. em

cultivo submerso nas diferentes razões de C:N nos ensaios em frascos agitados

• Selecionar diferentes adsorventes para obter melhor condição de adsorção baseado nos

valores de recuperação e fator de purificação da β-gal obtida por Kluyveromyces lactis;

• Otimizar o processo de purificação da enzima empregando sistema de adsorção por

cromatografia em leito fixo;

• Imobilizar a enzima β-gal produzida para hidrólise da lactose em soro do queijo;

• Avaliar a estabilidade da enzima livre e imobilizada em diferentes condições de pH,

temperatura e íons metálicos;

• Determinar a estabilidade da enzima imobilizada em condições de simulações gástricas

e intestinais.

17


Capítulo 3

Revisão a Literatura

18


CAPÍTULO 3 – REVISÃO A LITERATURA

Neste capítulo será apresentado uma breve revisão com abordagem inicial sobre o leite,

o soro do queijo e suas potenciais aplicações. Posteriormente, se fará uma explanação sobre a

lactose e as reações de interesse com ênfase na produção e utilização da -galactosidase (-gal)

e por último, uma pequena síntese sobre as técnicas de recuperação e purificação da enzima de

interesse e os métodos de imobilização utilizados atualmente com a finalidade de aumentar a

eficiência para aplicação industrial.

3.1. Setor de laticínios

3.1.1 Produção de leite

A produção mundial de leite vem crescente há décadas e essa tendência se mantém nos

anos recentes (Figura 1). O crescimento não é linear principalmente devido a situações

climáticas que atrapalham pontualmente a produção. Ainda assim, a produção aumentou 11%

entre 2010 e 2017, passando de 603 bilhões de toneladas para 668,4 em 7 anos como

evidenciado na Figura 1.

Fonte: IBGE (2018)

Figura 1: Evolução da produção mundial do leite de vaca

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No setor agropecuário o leite ocupa terceiro lugar em produção total e o primeiro em

valor monetário, fornecendo 5% de energia, 9% de gordura e 10% da proteína total consumida

globalmente (Global Dairy Platform, 2016).O Brasil ocupa o quinto lugar na produção mundial

de leite e apresenta grande relevância socioeconômica, sendo quase 1,2 milhão de produtores,

distribuídos em 99% dos municípios (Sorio, 2018).

Os dados do diagnóstico da cadeia Industrial do leite no Brasil de 2018 (Figura 2)

revelam que o leite proporciona a produção de uma infinidade de derivados lácteos, mas os

produtos mais importantes, além do próprio leite fluido pasteurizado ou UHT, são o leite em

pó, a manteiga e os queijos. A produção mundial de queijos, de 19,5 milhões de toneladas em

2017 foi amplamente dominada pela União Europeia e pelos EUA, sendo o Brasil o 4º maior

fabricante de queijos.

Figura 2: Evolução da produção de leite no Brasil 2006-2017

De acordo com o Regulamento Técnico de Identidade e Qualidade de Queijos (Brasil,

1996), o queijo é definido como um produto fresco ou maturado que se obtém por meio da

separação parcial do soro do leite ou leite reconstituído (integral, parcial ou totalmente

desnatado), ou de soros lácteos, coagulados pela ação física do coalho, de enzimas específicas,

de bactéria específica, de ácidos orgânicos isolados ou combinados, todos de qualidade aptos

20


ao uso alimentar. O soro do queijo e seu aproveitamento é uma das alternativas viáveis para

indústria de laticínios, podendo ser utilizado como substrato para elaboração de subprodutos de

interesse industrial com a obtenção de etanol (Ariyanti e Hadiyanto, 2013; Beniwal et al., 2018),

ácido galactônico (Zhou et al., 2019), β-galactosidase (Perini, et al., 2013; Rao e Dutta, 1977),

entre outros.

3.1.2 Soro do queijo

Com a industrialização do leite para a produção de seus derivados, ocorre a geração de

coprodutos, destacando-se o soro de queijo. Na fabricação do queijo, em torno de 85 a 95% do

volume do leite utilizado resulta em soro de queijo, originado após a separação da coagulação

das micelas de caseínas, de cor amarelo-esverdeada, com sabor ligeiramente ácido ou doce

(Bald et al., 2014).

Segundo Suzart e Dias (2006),o soro do queijo é um líquido opaco, de coloração amarelo

esverdeado, que apresenta na sua composição aproximadamente 55% de sólidos existentes no

leite integral, sendo a lactose o maior constituinte presente. Além disso, é composto por sais

minerais, proteínas hidrossolúveis dentre elas a α-Lactoalbumina e a β-Lactoglobulina,

respectivamente com massas molares (MM) em torno de 14.000 e 18.000 Daltons; e menor

quantidade de gordura, com ácidos graxos de baixo ponto de fusão (em torno de 29ºC) (Nath

et al., 2014).

O soro do queijo pode apresentar sabor ligeiramente doce, porém vai depender do tipo

de queijo que está sendo fabricado; contém nutrientes importantes como: aminoácidos

facilmente digeríveis (Barbosa et al., 2010) no qual esses contribuem aproximadamente com

60% do teor proteico total do soro. O soro possui ainda concentrações elevadas de leucina e

lisina, além de constituir uma boa fonte de aminoácidos contendo enxofre, tais como: cisteína

e metionina, e de ser uma fonte concentrada de cálcio, sendo rico em vitaminas, tais como:

21


tiamina, riboflavina, vitamina B6 e B12 e ácido pantotênico (Barbosa et al., 2010; Saudades et

al., 2017).

A composição do soro do queijo varia de acordo com o procedimento de separação da

caseína e pode ser classificado em dois tipos: Soro ácido que apresenta uma acidez titulável de

0,5 a 0,6% em ácido láctico (Barbosa et al, 2010). Esse tipo de soro é obtido da precipitação

ácida no pH isoelétrico (pH = 4,6) durante a manufatura de caseína e queijos com leites

coagulados inicialmente por ácidos, como o tipo coalho, cottage, minas, prato, requeijão e ricota

(Sgarbieri, 2004). Soro doce que apresenta pH entre 6,0-7,0 e uma acidez titulável de 0,15 a

0,18% em ácido lático. Esse tipo de soro é obtido do processo de coagulação enzimática do

leite com o uso de uma enzima a quimosina e do processamento para a fabricação de queijos

tipo cheddar, mussarela e suíço. O soro doce normalmente contém maior teor de minerais e

menor concentração de proteínas do que o soro ácido e o uso desse último é mais limitado na

alimentação e na indústria devido ao seu sabor ácido e ao elevado teor salino (Siso, 1996). As

bebidas lácteas à base de soro são de grande valor dietético e de fácil digestão. São produzidas

com o uso de soro desmineralizado ou soro concentrado (Barbosa et al., 2010; Schmidell et al.,

2001).

Por meio de inúmeros processos químicos, o soro pode ser desidratado e utilizado como

alimento ou aditivo alimentar, ou ainda, ser convertido em combustíveis e outros produtos

(Albuquerque et al., 2016; Fernández-Gutiérrez et al., 2017). O concentrado proteico

representa a maior parte da produção oriunda do soro, ultrapassado pelo soro em pó comum e

desmineralizado (Barbosa et al, 2010).

3.1.3 Soro do queijo um resíduo valioso

O processo deficiente de gerenciamento de resíduos e subprodutos da indústria

alimentícia tem gerado sérios impactos na sustentabilidade ambiental. As matérias-primas e

22


coadjuvantes entram na cadeia de produção e completam o ciclo com o produto desejado, porém

também geram produtos não específicos ou um resíduo destes produtos. Esses últimos são

acumulados como resultado do processamento de matérias-primas e, muitas vezes, o descarte

ocorre de forma inapropriada no meio ambiente (Jayathilakan et al., 2012).

Diante do exposto, observa-se que na questão relacionada ao aproveitamento de

resíduos, a engenharia de bioprocessos surge como uma área estratégica e útil na pesquisa

acadêmica industrial, agregando valor com o desafio de produção de biomoléculas de grande

interesse mercadológico (Doran, 2012). A grande vantagem e desafio nesta área é converter os

resíduos e subprodutos utilizando os processos biotecnológicos, que muitas vezes englobam:

pré-tratamentos com agentes físicos e biológicos, com posteriores etapas de produção e

purificação controladas, obtendo-se biomoléculas de alto valor agregado como antioxidantes

naturais, agentes antimicrobianos, vitaminas, além de enzimas, celulose, amido, lipídeos,

proteínas e pigmentos, sendo estes de grande interesse para as indústrias química, petroquímica,

farmacêutica, cosmética e alimentícia (Murthy e Madhava, 2012).

A indústria de laticínios está entre as maiores geradoras de resíduos, sendo uma das mais

importantes do setor agroindustrial (Fernández-Gutiérrez et al., 2017). Com o intuito de atender

as leis ambientais, que estão se tornando cada vez mais rigorosas, as indústrias têm buscado

alternativas biotecnológicas, inovadoras e viáveis para o tratamento dos resíduos

agroindustriais e o desenvolvimento de novos produtos. Com a indústria de laticínios não é

diferente, setor no qual o principal efluente (soro do queijo) apresenta elevado volume de

produção, uma rica composição nutricional e alto poder poluente, tornando-se alvo de estudos

para transformá-lo de poluidor ambiental em fonte de bioprodutos com alto valor agregado

(Prazeres et al., 2012).

23


O soro do queijo, considerado um subproduto valioso, é o principal componente das

águas residuais de produtos lácteos que é avaliado como um material recuperável em relação à

disponibilidade de alimentos (Aydiner et al., 2013).

A composição do soro de queijo pode atingir até 80% de lactose (Albuquerque et al.,

2018; Zhou et al., 2019). Porém sua produção está diretamente relacionada a geração de

resíduos de alto teor poluente (Andrade et al., 2017). A produção do soro do queijo é mais

relevante no nordeste do Brasil, no qual o queijo de coalho é um produto tradicional que compõe

os aspectos socioeconômicos e nutricionais da região (Fontenele et al., 2017; Soares et al.,

2017).

3.2. Lactose importância e aplicação

A utilização do soro do queijo pela a indústria é dificultada pela grande quantidade de

lactose ( figura 3 ) que esse componente apresenta, a qual contribui para sua baixa solubilidade,

baixo poder de doçura, baixa digestibilidade quando utilizado como alimento, sendo ainda,

pouco fermentável quando comparado a outros açúcares (Carminatti, 2001; Guimarães et al,

2010).

O teor de lactose é inversamente proporcional à concentração de lipídios e da caseína

presentes no leite variando, consideravelmente, entre as espécies e de acordo com a alimentação

do animal. Além disso, é responsável por 50% da pressão osmótica do leite, que é isotônico

com o sangue (Oliveira et al., 2008).

Fonte:https://www.infoescola.com/bioquimica/lactose/

Figura 3: Estrutura química da lactose

https://www.infoescola.com/bioquimica/lactose/

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A lactose é um carboidrato encontrado no leite e seus derivados. Trata-se de um

dissacarídeo redutor formado por um radical D-glicose e outro D- galactose unidos por uma

ligação glicosídica β-1,4, considerado o componente mais importante dos sólidos não

gordurosos do leite.(Pereira et al., 2012).

Com baixo poder edulcorante e baixa solubilidade em água quando comparada a outros

açúcares em virtude das suas formas anoméricas α e β (Guimarães et al., 2010), a lactose em

sua forma comercial, α-lactose, tem um poder edulcorante quatro vezes menor que o da

sacarose. A β-lactose apresenta um poder edulcorante maior, além de ser mais solúvel que a α-

lactose. Também é possível aumentar o poder edulcorante da lactose pela hidrólise que resulta

em glicose e galactose (Oliveira et al., 2012; Panesar et al., 2018). Essa conversão pela ação da

enzima β- galactosidase é interessante tanto no aspecto tecnológico, pois aumenta o teor de

doçura, facilita o processo fermentativo, diminuindo a cristalização da lactose e aumenta o

período de estocagem, quanto nutricional, pois possibilita a obtenção de produtos lácteos com

baixo teor de lactose (Carota et al., 2017; Suri et al., 2019).

Embora difícil de degradar no meio ambiente, a lactose do soro do queijo pode ser

utilizada como substrato para a fermentação visando a obtenção de etanol (Ariyanti e

Hadiyanto., 2013; Beniwal et al., 2018), ácido galactônico (Zhou et al., 2019), β-galactosidase

(Perini, 2013; Rao e Dutta, 1977), entre outros.

3.2.1 Hidrólise da lactose

A hidrólise da lactose é um pré-requisito para a conversão de soro do queijo em diversos

produtos de valor agregado, no entanto, a maioria das espécies de leveduras, como S. cerevisiae,

não possuem a capacidade de hidrolisar a lactose (Gänzle, et al., 2008).

25


A hidrólise da lactose realizada por via ácida ou enzimática, utilizando a enzima β-

galactosidase. A hidrólise ácida exige pH ácido e elevada temperatura. Por outro lado, a

hidrólise enzimática ocorre em condições mais brandas, tanto de temperatura quanto de pH,

porém, exige uma etapa posterior para separação dos produtos formados (Carminatti, 2001).

Na hidrólise enzimática a β-galactosidase que está localizada na superfície chamada

borda em escovado intestino delgado promove a quebra da lactose em dois monossacarídeos a

glicose e a galactose, hexoses que são facilmente absorvidas pelo organismo (Zhao et al., 2018)

Em humanos, a intolerância à lactose ocorre em 75% da população, sendo causada pela

deficiência da β-galactosidade no organismo, que resulta em uma diminuição daatividade dessa

enzima na membrana da borda da mucosa no intestino delgado em adultos.Preparações

enzimáticas de lactases em produtos lácteos pode reduzir a lactose e promover um efeito

benéfico para os intolerantes à lactose (Braga et al., 2014; Zhao et al., 2018).

Almeida et al (2015) estudaram a hidrólise enzimática da lactose de formulações de

permeado de soro, em concentração de 0,2%, 0,7% e 1% nos tempos 30, 60 e 90 minutos com

o pH do meio de 6,3 e temperatura de 37 ºC e constataram que na concentração enzimática de

0,7% no tempo de 30 minutos, as formulações se tornaram seguras para o consumo de

intolerantes à lactose, de acordo com níveis mínimos estabelecidos pela legislação.

Torres et al (2016) analisaram o efeito de diferentes níveis de hidrólise enzimática da

lactose (0%, 25%, 50%, 75% e 99%), na produção e estocagem de leite em pó integral. Todos

os resultados indicam que a hidrólise da lactose afeta a produção do leite em pó por aumentar

absorção de umidade durante a estocagem.

3.3. Enzima -galactosidase

A enzima β-galactosidase (E.C.3.1.23) (β-gal) também chamada de lactase, é classificada como

hidrolase, capaz de hidrolisar a lactose em glicose e galactose. Essa biomolécula é encontrada

26


na forma de tetrâmeros com quatro subunidades de cadeias de polipeptídios de

aproximadamente 90 a 120 KDa, cada unidade dimérica contribui com um sítio ativo de união

Mg+2 e dois resíduos catalíticos (um de cada cadeia polipeptídica), conforme pode ser

evidenciado na figura 4 (Klen, 2010)

Fonte: (https://www.rcsb.org/structure/4V40, [s.d.]) Figura 4: Estrutura molecular β-galactosidase

Essa enzima pode estar presente entre alguns vegetais como amêndoas, pêssego,

damasco, em órgãos de animais e são produzidas por grande quantidade de microrganismos,

tais como fungos filamentosos, bactérias e leveduras (Suzart e Dias, 2006). No entanto, as suas

propriedades variam de acordo com a fonte respectivamente (Lima, 2012).

As β-galactosidases em sua maioria são sintetizadas intracelular por microrganismos

que utilizam a lactose como substrato para produção de energia. Rajoka et al., (2004)

produziram β-gal utilizando leveduras Kluyveromyces marxianus na presença de lactose,

sacarose e outras fontes de carbono e observaram que o tipo de substrato e a temperatura

influenciam no crescimento especifico e na taxa de produção da enzima. Para Lima et al.

(2011), o soro do leite desproteinizado e suplementado serviu como fonte de carbono para

Kluyveromyces lactis NRRLY1564 na síntese da β-galactosidase obtendo-se 3,5 U/mL de

enzima em 12h de fermentação.

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A reação da hidrólise enzimática da β-galactosidase ocorre com a utilização do substrato

cromogênico O-nitrofenil-β-D-galactopiranosídeo (ONPG) sendo este processo uma das

formas de confirmar a atividade desta enzima, trata-se de um teste colorimétrico, no qual após

hidrólise, o ONPG que é um líquido incolor produz a galactose (incolor) e o O-nitrofenil-

piranose (ONP) que tem coloração amarela (Braga et al., 2014; Machado et al., 2015).

As β-galactosidases produzidas por bactérias e leveduras apresentam um melhor

desempenho em pH neutro, enquanto que as expressas por fungos são mais ativas em pH ácido.

Desta forma, a escolha da enzima dependerá da reação que será catalisada e do substrato. Na

hidrólise de produtos lácteos, e preferencialmente, o leite geralmente são utilizadas as enzimas

produzidas por leveduras que apresentam atividade ótima de pH na faixa de 6,0 – 7,0. As

enzimas expressas por leveduras parecem se tornar mais ativa na presença de íons Mg+2 e Mn+2

como cofatores (Nath et al., 2014).

Um fator limitante na atividade hidrolítica da β-galactosidase está relacionado a sua

inibição por um dos produtos formados na reação, a galactose e glicose, e a formação dos

isômeros. Inibidores competitivos e não competitivos respectivamente, a galactose e glicose

impedem a hidrólise por completo da lactose, sendo a inibição por glicose bem menor quando

comparada a galactose (Klein, 2010).

A lactose não consegue ser digerida na ausência da β-galactosidase. Diante deste

aspecto, quando os alimentos que contém lactose como leite e derivados são consumidos pelo

ser humano, e não existe a presença da β-galactosidase em concentrações suficientes no

organismo, a lactose, conforme passa pelo cólon, é fermentada por bactérias produzindo ácidos

orgânicos de cadeia curta, principalmente lático e acético e libera gases, como o dióxido de

carbono, nitrogênio e metano (Chanalia et al., 2018; Suri et al., 2019). Os produtos obtidos por

ação das bactérias acidificarem o meio, aumenta a osmolaridade e as contrações peristálticas

do músculo circular do cólon e leva a uma diminuição da reabsorção de água pelo intestino

28


grosso o que resulta em diversos sintomas. Esses sintomas são cólicas, flatulência, desconforto

abdominal e diarreia osmótica (Fani, 2010).

A intolerância a lactose é uma síndrome clínica de desconforto intestinal, também

conhecida como deficiência de lactase do adulto. Ela compromete o indivíduo devido aos

baixos níveis, ou até a ausência da atividade da enzima β-galactosidase no aparelho digestivo,

por consequência de uma deficiência congênita desta enzima ou de uma diminuição gradativa

de sua atividade com o avanço da idade (Bosso et al., 2019;Suri et al., 2019).

A β-galactosidase vem sendo utilizada amplamente como suplemento de ingestão oral

por pessoas intolerantes à lactose. Outra forma diferente de utilização é a utilização desta

enzima em produtos na indústria de laticínios para promover a hidrólise da lactose obtendo-se

assim, alimentos com baixos teores de lactose, melhorando a solubilidade e digestibilidade do

leite e derivados lácteos, ideais para consumidores intolerantes à lactose (Martarello et al.,

2019; Suri et al., 2019).

A preocupação com uma alimentação com efeitos benéficos para a saúde tem aumentado

a demanda por produtos alimentícios com essas características, dando suporte para novas

pesquisas nesta área (Gosling et al., 2010; Chanalia et al., 2018).

A β-galactosidase vem sendo ressaltada por sua propriedade de gerar derivados de

lactose através de transgalactosilação para formar galactooligossacarídeos (GOS), com um

novo alcance de utilização como alimentos funcionais. Por não serem digeridos, os GOS podem

alcançar a microbiota no cólon e promover a proliferação de Bifidobacterium, o que faz deles

importantes aditivos em fórmulas infantis e em outros produtos lácteos (González-Delgado et

al., 2016; Chanalia et al., 2018; Panesar et al., 2018).

A legislação brasileira específica, por meio da resolução RDC nº 205/2006, ressalta que

29


a enzima lactase utilizada na indústria de alimentos deve ser de origem microbiana, proveniente

dos seguintes microrganismos: Aspergillus niger, Aspergillus oryzae, Candida

pseudotropicalis, Kluyveromyces lactis, Kluyveromyces flagilis, Kluyveromyces marxianus

uma vez que tais espécies são classificadas como GRAS (Generally Recognized as Safe) pela

Food and Drug Administration (FDA), sendo esse um importante critério para aplicações

alimentícias (Alvarenga et al., 2017; Brasil, 2006), conforme é evidenciado na tabela 1:

Tabela 1: Alguns microrganismos produtores de β-galactosidase

Microrganismo Referências

Aspergillus niger (Martarello et al.,2019)

Aspergillus oryzae (Araujo et al., 2015)

Candida pseudotropicalis (Adalberto et al., 2010)

Guehomyces pullulans (Song et al., 2010)

Kluyveromyces fragilis (Vieira, 2009)

Kluyveromyces lactis (Bosso et al., 2016; Mörschbächeet al.,

2016; Sun et al., 2016; Alvarenga et al.,

2017; Darif, 2018)

Kluyveromyces marxianus (Duarte et al., 2012; Medeiroset al.,

2012; Ariyanti e Hadiyanto, 2013;

Beniwal et al., 2018b;) Fonte: Adaptado (Lima, 2012)

3.4. Biotecnologia aplicada a produção de -galactosidase

3.4.1. Gênero Kluyveromyces e Saccharomyces: metabolismo e produção de -gal

Kluyveromyces spp. são leveduras que apresentam uma via metabólica respiratório-

fermentativa que pode gerar energia através do ciclo de Krebs (ciclo do ácido cítrico) ou por

fermentação exclusiva, na qual o etanol é o principal produto com efeito Crabtree negativo. Em

contraste com a Saccharomyces spp., leveduras com metabolismo respiratório-fermentativo que

não exploram plenamente sua capacidade de absorver glicose durante o crescimento oxidativo,

ou seja, apresentando efeito Crabtree positivo (Petrova et al, 2010). De fato, elas são capazes

de realizar simultaneamente fermentação e processos respiratórios, e o equilíbrio entre essas

30


duas vias metabólicas depende da especificidade da linhagem (Lane e Morrissey., 2010). Além

disso, elas também são termotolerantes e podem ser encontradas em uma variedade de habitat,

representando uma ampla diversidade metabólica, atingindo uma variedade de aplicações

biotecnológicas (Petrova et al., 2010; Fonseca et al., 2008).

Duarte et al.,(2012) realizaram a caracterização cinética e termodinâmica da β-gal

produzida por K. marxianus com o objetivo de analisar o comportamento metabólico da

levedura frente a temperatura e o pH. Os resultados mostraram a influência dos parâmetros no

crescimento celular e na síntese enzimática. Em estudos realizados por Bansal et al (2008) com

a finalidade de avaliar a melhor condição de produção e extração de β-gal por K. marxianus,

foi observado uma produção máxima de β-gal em 30 horas de fermentação com um crescimento

celular de 2,54 mg/mL, ainda foi possível identificar que o uso do clorofórmio foi o método

mais eficiente e barato de extração, mantendo a atividade da enzima.

Estudos realizados por Lima (2011) utilizando o soro de leite desproteinizado e

suplementado como fonte de carbono alternativa para produção da enzima β-gal por

Kluyveromyces lactis NRRL Y1564 mostrou uma capacidade interessante de expressão dessa

biomolécula com resultados de atividade em torno de 3,5 U/mL com 12 h de fermentação.

Perini et al. (2013) realizaram estudos similares utilizando o soro do queijo como fonte de

carbono e a milhocina como fonte de nitrogênio e por meio de um planejamento experimental

reportaram atividade de 6,59 U/mL.

K. lactis possui os genes LAC 12 e LAC 4, que são responsáveis pela codificação das

enzimas lactose-permease e β-galactosidade, respectivamente, as quais desempenham papéis

diferenciados. A lactose-permease participa do transporte da lactose através da membrana

citoplasmática para o interior da célula, e a β-galactosidase é responsável pela hidrólise da

31


lactose (dissacarídeo) em dois monossacarídeos, a glicose e a galactose (Rubio-Texeira, 2005;

Guimarães et al., 2010).

A β-galactosidase de K. lactis apresenta massa molar em torno de 120 a 150 kDa (Zhou

e Dill, 2001), sendo assim considerada uma proteína de alta massa molecular. Por ter uma

elevada atividade hidrolítica, tem sido utilizada para produzir alimentos livres de lactose

(Fischer et al., 2013). Esse microrganismo não é muito utilizado para produção de etanol, mas

tem sido muito usada na produção de proteínas e, principalmente, da lactase usando o soro do

leite como meio de cultura (Spohner et al., 2016).

Várias estratégias têm sido investigadas para lidar com o descarte de resíduos de soro

do queijo dentre as quais destaca-se o uso de processos biotecnológicos como uma maneira

interessante de converter tal subproduto em compostos com maior valor agregado (Carota et

al., 2017).

A técnica de fermentação submersa tem se tornada atrativa para indústria, em virtude

dos avanços na instrumentação e controle de processos, sendo bem adaptada para o cultivo de

microrganismos recombinantes, empregados de forma cada vez mais crescente na produção de

enzimas e outras biomoléculas de interesse (Lima, 2012; Schmidell et al., 2001).

A fermentação submersa utiliza substratos líquidos como meio de cultivo nutritivo,

facilitando o controle de parâmetros como: temperatura, pH e aeração, além de outros fatores

que podem interferir no processo. Os nutrientes presentes, o pH, temperatura e agitação podem

exercer influência na taxa de crescimento dos microrganismos, no rendimento da biomassa e

na produção de metabólitos. Os componentes nutritivos do meio com as fontes de carbono e

nitrogênio, estão diretamente ligados ao crescimento e a produção enzimática microbiana

(Alvarenga et al., 2017; Sundarram et al, 2014).

A fermentação deve ser rápida a fim de maximizar a produtividade do processo. No

32


entanto, na concepção de um processo para produção de -galactosidase e etanol a partir do

soro do leite, esta tem que ser acompanhada para maximização do título/produtividade das

biomoléculas de interesse e minimização da concentração de açúcar residual do efluente, uma

vez que a finalidade do processo é normalmente o tratamento de resíduos (Guimarães et al.,

2010; Martarello et al, 2019).

Em um estudo recente You et al. (2017), propôs a utilização da lactose comercial como

substrato para uma produção por fermentação submersa de baixo custo de β-Gal usando uma

estratégia de co-produção na qual o etanol, um subproduto da fermentação, também foi

recuperado, usando a lactose comercial com um meio enriquecido os autores conseguiram

maximizar a produção de duas biomoléculas de grande interesse industrial.

3.5. Recuperação e purificação da β-galactosidase

Inúmeras são as técnicas utilizadas para a recuperação e purificação de enzimas de

origem animal, vegetal ou microbiana, porém existem muitas dificuldades, do ponto de vista

técnico que exigem um elevado número de etapas (Braga et al., 2014).

Ao iniciar um processo de recuperação e purificação de uma enzima é importante saber

o grau de pureza exigido. As enzimas que são usadas para fins terapêuticos ou de uso direto em

seres humanos necessitam de um alto grau de pureza, o que não é necessário para as enzimas

que serão aplicadas em outros processos industriais. Na purificação em larga escala,

dependendo do grau de pureza durante a etapa de downstream diversas operações unitárias são

aplicadas para promover a purificação da biomolécula de interesse (Pessoa Jr e Kilikian, 2008).

A escolha das técnicas a serem empregadas no processo de recuperação e purificação

está vinculada às propriedades moleculares inerentes a cada enzima. Dessa forma, a

combinação correta de várias etapas que exploram estas propriedades permitirá a recuperação

33


e purificação a partir de uma mistura (Pessoa Jr e Kilikian, 2008).

Nas primeiras etapas de purificação quase sempre é desejável reduzir o volume, e para

isto é frequentemente utilizada à precipitação fracionada com sais ou solventes orgânicos.

Posteriormente são utilizadas técnicas que exploram interações eletrostáticas (cromatografia de

troca iônica) pela sua relativa alta capacidade. Para as etapas finais, o objetivo quase sempre é

um aumento de resolução, e para isto utilizam-se técnicas como cromatografia em gel filtração

ou cromatografia de afinidade. Análise via eletroforese em gel de poliacrilamida (PAGE) pode

indicar a pureza e o número de contaminantes presentes (Harris, 2001).

3.5.1 Sistemas de Cromatografia

O processo de purificação de proteínas tem como objetivo principal alcançar um

rendimento máximo com alta seletividade, levando sempre em consideração os custos da

operação. Em se tratando de enzima o desempenho de qualquer técnica de purificação é

evidenciado pelas variáveis rendimento e fator de purificação. A primeira estabelece o

percentual da molécula que foi recuperado durante a etapa, e a outra mensura o aumento da

atividade específica da enzima ao usar etapas que promovam a purificação (Padilha, 2013).

A cromatografia é uma das muitas técnicas de purificação que tem se destacado por seu

alto desempenho e eficiência na separação de misturas complexas. A adsorção de biomoléculas

da fase fluida para uma superfície sólida é um fenômeno comum utilizado em várias áreas como

biologia, biotecnologia e processamento de alimentos (Lima, 2014).

A cromatografia em leito fixo se fundamenta na propriedade de alguns materiais

(adsorventes) que possuem a capacidade de reter moléculas (adsorbato) sobre sua superfície. A

busca por adsorventes com elevada capacidade de adsorção e seletividade tem sido um desafio

no desenvolvimento de recuperação e purificação de subprodutos (Villeneuve et al., 2000).

34


Medeiros et al., (2012) no estudo de purificação da β-galactosidase comercial por meio

da técnica de cromatografia de troca iônica utilizando a resina sepharose Q obtiveram valores

de recuperação de 88%.

Boeris et al., (2012) obtiveram valores de recuperação próximos a 65% quando

submeteram a enzima à cromatografia de troca iônica em leite expandido usando a resina

Streamline DEAE.

Lima et al., (2016) analisando as condições de recuperação e purificação da β-gal

comercial usando a técnica de cromatografia multimodal e alcançaram 48% de rendimento e

um fator de purificação de 1,17.

Ji et al., (2019) sobre a cinética de purificação, caracterização e inativação térmica da

-gal Lactobacillus leichmannii 313 as proeteínas foram separadas por meio de um sistema de

cormatografia de alta eficiência (FPLC) utilizando uma coluna de troca iônica alcançando

condições favoráveis de purificação.

Portanto, independentemente do método utilizado no processo de purificação, deve ser

considerado dois fatores importantes o rendimento e o fator de purificação, conforme equações

1 e 2, respectivamente . Para enzimas, a concentração total de proteínas e atividade enzimática

são analisadas normalmente. A partir da atividade enzimática do extrato bruto e purificado, se

obtém o rendimento do processo (Padilha, 2013).

Rendimento (%) = 𝐴𝑡𝑖𝑣𝑖𝑑𝑎𝑑𝑒 𝑒𝑛𝑧𝑖𝑚á𝑡𝑖𝑐𝑎 𝑑𝑜 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑝𝑢𝑟𝑖𝑓𝑖𝑐𝑎𝑑𝑜

𝐴𝑡𝑖𝑣𝑖𝑑𝑎𝑑𝑒 𝑒𝑛𝑧𝑖𝑚á𝑡𝑖𝑐𝑎 𝑑𝑜 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑏𝑟𝑢𝑡𝑜 x 100 (1)

Fator de Purificação =𝐴𝑡𝑖𝑣𝑖𝑑𝑎𝑑𝑒 𝑒𝑠𝑝𝑒𝑐í𝑓𝑖𝑐𝑎 𝑑𝑜 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑝𝑢𝑟𝑖𝑓𝑖𝑐𝑎𝑑𝑜

𝐴𝑡𝑖𝑣𝑖𝑑𝑎𝑑𝑒 𝑒𝑠𝑝𝑒𝑐í𝑓𝑖𝑐𝑎 𝑑𝑜 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑏𝑟𝑢𝑡𝑜 x 100 (2)

35


3.6. Imobilização de Enzimas

Uma estratégia biotecnológica que está sendo frequentemente utilizada com a finalidade

de melhorar a aplicação industrial da -galactosidase permitindo redução de custos é a

imobilização de enzimas (Klein, 2010). Cada enzima possui características especificas e requer

um critério minucioso na escolha dos métodos com a proposta de fornecer procedimentos

simples, econômicos e que resultem em uma enzima imobilizada com boa retenção de atividade

e alta estabilidade operacional (Fai et al., 2015; Mendes et al., 2011).

O desenvolvimento dessas técnicas de imobilização é de grande avanço para a indústria

alimentícia, uma vez que proporciona a reutilização das enzimas, pode aumentar a estabilidade

térmica e melhorar a separação dos produtos (Lemos, 2018; Vieira, 2009).

Existem alguns tipos de imobilização de enzimas, por aprisionamento físico ou por

adsorção na superfície. O primeiro método faz o encapsulamento das enzimas em esferas ou

matrizes de polissacarídeos, proteínas ou polímeros sintéticos. As enzimas ainda podem ser

fixadas ao suporte de imobilização diretamente por ligações químicas (iônicas ou covalentes)

(Meersman, 1992). O método de adsorção física para imobilização de enzimas é baseado na

interação física da enzima na superfície de suportes insolúveis. A enzima pode ser retida na

superfície do suporte insolúvel e essa interação pode ser ocasionada por ligações hidrofóbicas,

interações de Van der Waal, pontes de hidrogênio e interações específicas (Cunha et al., 2007;

Muguruma et al., 2006; Souza et al., 2019). A Figura 5 mostra esquematicamente os métodos

de imobilização enzimática.

36


Figura 5: Métodos de imobilização de enzimas Fonte:(Fischer, 2010)

O encapsulamento em matrizes de biopolímeros é um tipo de imobilização de enzima

que pode aumentar a estabilidade da biomolécula, sendo esse um dos fatores primordiais em

aplicações industriais. Neste método, as enzimas são mantidas dentro de um espaço confinado,

com restrita mobilidade que contribui para sua maior estabilidade (Escobar et al., 2014; Zhou

e Dill, 2001). Esse processo confere uma maior proteção da enzima contra condições extremas

de pH e a matriz pode evitar a evasão da enzima durante a catálise. Essas vantagens são

importantes tanto economicamente quanto do ponto de vista ambiental (Albuquerque et al.,

2018).

Entre os vários suportes utilizados para o método de aprisionamento enzimático, o

alginato de sódio tem mostrado características adequadas para encapsulação. O alginato é um

polímero aniônico solúvel em água, obtido de algas marrons e composto por ácidos β-1-4-D-

manurônico e α-1,4-gulurônico (Blandino et al., 2001; Bustamante-Vargas et al., 2015). O

processo de geilificação do alginato ocorre quando há troca de íons sódio por cátions divalentes

tais como Ca2+, Cu2+, Zn2+ ou Mn2+ (Darif, 2018).

A imobilização com alginato de sódio consiste em dissolver a enzima em

uma solução aquosa contendo alginato, que é gotejada em uma solução

37


aquosa contendo íons bivalentes (Ca2+, por exemplo). Em contato com a solução

CaCl2, a gota de alginato de sódio forma esfera de alginato de cálcio na qual a

enzima fica aprisionada (Freitas, 2007; Schmidell et al., 2001). A malha de gel formada,

induzida pela ligação do íon Ca2+, por exemplo, formam junções estáveis (uma rede

tridimensional) (Roy e Gupta, 2003).

Estudos realizados por Shen et al., (2011) que testaram uma matriz híbrida de alginato-

gelatina-fosfato de cálcio para a imobilizar de β-galactosidase de K. lactis constataram uma

menor atividade relativa (58,6 %) de β-galactosidase imobilizada em esferas de alginato-

gelatina-fosfato de cálcio, quando comparada à β-galactosidade imobilizada em esferas do

matriz controle (62,3 %) utilizando apenas alginato. Os autores atribuíram este resultado a

problemas de transferência de massa ocasionados pela camada de fosfato de cálcio e gelatina

formada no suporte.

Ansari e Husain,(2012) analisaram a imobilização de β-galactosidase de amêndoa em

alginato de cálcio-celulose revestido com concanavalina A, empregando glutaraldeído como

agente reticulante. O derivado obtido reteve 72% de sua atividade inicial e as condições ótimas

de pH (5,5) e temperatura (50 °C) não sofreram alterações em relação à enzima livre, sendo que

a enzima imobilizada mostrou um alargamento notável nestes perfis. Por outro lado, o derivado

obtido sofreu maior inibição aos produtos formados e a taxa de conversão de lactose foi menor

em relação à enzima livre.

Freitas et al., (2012) utilizando a β-galactosidase de K. lactis imobilizada em alginato

de sódio, gelatina, e ligação cruzada com glutaraldeído. Constataram que as condições ótimas

para a imobilização da enzima foram 6,60 % de alginato (m/v), 4,05 % de gelatina (m/v) e 3,64

% de glutaraldeído (v/v). A enzima imobilizada obtida nas condições otimizadas de adsorção

38


permaneceu com 80 % da sua atividade inicial após 25 utilizações. Nas concentrações de lactose

estudadas (10 a 100 g/L), não houve inibição pelo produto para enzima imobilizada.

Estudo mais recente realizado por Darif, (2018) sobre a imobilização de β-galactosidase

de Aspergillus oryzae, Kluyveromyces lactis e Saccharomyces marxianus var lactis em matriz

de alginato de cálcio, gelatina e transglutaminase (T-Gase). Mostrou que as β-galactosidases A

e B neutras, obtidas de Kluyveromyces lactis, nas formas livre e imobilizadas em alginato de

cálcio-gelatina-TGase, apresentaram temperatura ótima de atividade a 35 °C em pH 7,0

enquanto que a β-galactosidase C, obtida de Saccharomyces marxianus var. lactis, na forma

livre e imobilizada em alginato de cálcio-gelatina-TGase apresentou temperatura ótima de

atividade a 40 °C em pH 7,0. A β-galactosidase ácida D obtida de Aspergillus oryzae na forma

livre apresentou temperatura ótima de atividade a 50 °C em pH 4,5 e a sua forma imobilizada,

em pH 4,5, apresentou atividade máxima a 45 °C. Temperaturas acima de 50 °C mostrou

alteração da textura dos grânulos de alginato de cálcio-gelatina-TGase e diminuição da

atividade enzimática. Essa técnica permite uma infinidade de aplicações da β-galactosidase na

indústria alimentícia em virtude da maior estabilidade proporcionada a biomolécula.

3.7. Aplicações da β-galactosidase na indústria alimentícia

A β-galactosidase vem sendo amplamente utilizada como suplemento de ingestão oral

por pessoas intolerantes à lactose (Khan e Husain, 2019). Outra forma diferente de utilização

além da ingestão direta é a utilização desta enzima na indústria de laticínios promovendo a

hidrólise da lactose em produtos com alto teor deste açúcar e obtendo-se assim, alimentos com

baixos teores de lactose, melhorando a solubilidade e digestibilidade do leite e derivados

lácteos, ideais para consumidores com esse tipo de restrição alimentar (Cavalcante et al, 2015;

Suri et al., 2019).

39


A preocupação com uma alimentação e seus efeitos benéficos para a saúde tem

aumentado a demanda por produtos alimentícios com essas características, dando suporte para

novas pesquisas nesta área (Bosso et al., 2019; Gosling et al., 2010).

A β-galactosidase vem sendo ressaltada por sua propriedade de gerar derivados de

lactose através de transgalactosilação para formar galactooligossacarídeos (GOS), com um

novo alcance de utilização como alimentos funcionais (Silvério et al., 2018). Por não serem

digeridos, os GOS podem alcançar a microbiota no cólon e promover a proliferação de

Bifidobacterium, o que faz deles importantes aditivos em fórmulas infantis e em outros produtos

lácteos (Panesar et al., 2018).

Atualmente os insumos produzidos por Kluyveromyces spp e Aspergillus são

comumente utilizados nos processos industriais (Carota et al., 2017; Eskandarloo e

Abbaspourrad, 2018; Husain, 2010; Oliveira et al., 2012). O fungo Aspergillus spp produz

lactase extracelular, que apresenta valores ácidos de pH ótimo entre 2,5 - 5,4 e uma temperatura

ótima alta de 50 °C (Panesar et al., 2018). Sua principal aplicação é na hidrólise ácida de soro

de queijo resultando em queijos frescos (Chiara Mollea, 2018). A lactase de Kluyveromyces

spp. é produzida intracelularmente, a lactose é transportada para o interior da levedura por uma

permease, onde é hidrolisada em glicose e galactose, que seguem então a via glicolítica ou o

caminho de Leloir, respectivamente (Guimarães et al., 2010). Essa lactase possui um pH neutro

(6,0 – 7,0), o que permite uma gama de aplicações mais ampla. Uma de suas aplicações é a

hidrólise de leite (Panesar et al., 2018; Suri et al., 2019).

Inúmeras são as pesquisas desenvolvidas sobre a produção de β-galactosidase com a

utilização de inúmeros microrganismos, porém sobre otimização das condições de produção

desta enzima poucos trabalhos são relatados. O efeito da temperatura nos parâmetros cinéticos

que quantificam a reprodução e morte celular, o consumo de substrato e a produção da enzima

40


são informações importantes para a elaboração de estratégias de controles mais eficientes nas

indústrias (Schmidell et al., 2001).

Mariotti et al., (2008) realizaram o estudo referente a hidrólise da lactose do soro de

leite em reator com a β-galactosidase proveniente de A. oryzae imobilizada em sílica.

Verificaram que os melhores resultados de imobilização foram alcançados usando o

glutaraldeído como ativante do suporte e estabilizador da enzima. A proporção otimizada entre

enzima e suporte foi 15-20 mg/g. A atividade de β-galactosidase imobilizada em torno de 650

U/g.

Klein, (2010) analisou o efeito da utilização da enzima β-galactosidase frente o

processo de cristalização da lactose no doce de leite. Foram avaliadas as seguintes

concentrações de enzimas: 0 a 0,4g/L. A verificação do grau de cristalização do produto foi

realizada por análise sensorial após 30, 60, 90 e 180 dias de armazenamento à temperatura

ambiente, por provadores previamente treinados. Constatou-se que a concentração de 0,2 g/L

de β-galactosidase utilizada (23,16 % de hidrólise da lactose) foi suficiente para que a

arenosidade no doce de leite não fosse percebida sensorialmente, durante todo o período

considerado.

De acordo com Chanalia et al., (2018) que analisaram a utilização da β-galactosidase

para hidrólise da lactose e síntese de GOS, mostram que a enzima apresentou uma ótima

estabilidade de pH e temperatura com uma leve ativação na presença do íon Ca+2 o que revela

ser adequada para o processamento do leite na produção de alimentos com baixo teor de lactose

e consequente síntese de prebióticos.

Outra aplicação de extrema necessidade e com grande avanço na indústria farmacêutica

se refere à utilização ampla de enzimas como agentes terapêuticos. Por administração oral em

uso clinico ou como coadjuvantes no tratamento de patologias especificas como doença celíaca

e a fenilcetonúria (Fuhrmann e leroux, 2014). Como medicamento essas biomoléculas são de

41


grande interesse para indústria em virtude da sua elevada atividade, seletividade e pela a

possibilidade de manipulação das suas propriedades. No entanto, o uso de enzimas em grau

farmacêutico administrada via oral é um aspecto desafiador devido ao potencial de inativação

desta molécula em ambiente hostil gastrointestinal (Wang et al., 2009).

Diante do exposto, a busca de alternativas biotecnológicas viáveis para o tratamento dos

resíduos agroindustriais e o desenvolvimento de novos produtos, marca a relevância desta

pesquisa por estudar técnicas acessíveis e eficientes que permitam o aproveitamento do soro do

queijo “coalho” para produção, recuperação e aplicação da β-galactosidase na indústria de

alimentícia.

O capítulo a seguir por meio de artigos evidencia as técnicas de bioprocessos

utilizadas para obtenção, recuperação e aplicação da enzima. O artigo 1 contextualiza o perfil

de co-produção de β-galactosidase e etanol por leveduras Kluyveromyces marxianus ATCC

36907 e Kluyveromyces lactis NRRL Y-8279 usando lactose do soro de queijo "coalho" como

fonte de carbono. O artigo 2 utiliza o extrato enzimático de β-galactosidase produzido por

Kluyveromyces lactis NRRL Y-8279 com a finalidade de recuperar e purificar a enzima por

cromatografia de troca iônica, em coluna de leito fixo, avaliando a influência do pH e da força

iônica. E por fim o artigo 3 utiliza a β-galactosidase produzida por Kluyveromyces lactis NRRL

Y-8279, parcialmente purificada por cromatografia de troca iônica e imobilizada com alginato

de sódio para avaliar as condições de hidrólise da lactose do soro de queijo "coalho" e sua

possivel aplicação na indústria alimentícia.

42


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.

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Capítulo 4

Artigos derivados da tese

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CAPÍTULO 4 – ARTIGOS DERIVADOS DA TESE

ARTIGO 1 - Potential of “coalho” cheese whey as lactose source for β-galactosidase and

ethanol co-production by Kluyveromyces spp. yeasts

Catherine Teixeira de CARVALHOa, Sérgio Dantas de OLIVEIRA JÚNIORa, Wildson

Bernardino de Brito LIMAa, Fábio Gonçalves Macêdo de MEDEIROSb, Ana Laura Oliveira

de Sá LEITÃO a, Everaldo Silvino dos SANTOSa, Gorete Ribeiro de MACÊDOa, Francisco

Canindé de SOUSA JÚNIORc *

a Laboratory of Biochemical Engineering, Chemical Engineering Department, Federal

University of Rio Grande do Norte (UFRN), 59078-970, Natal-RN, Brazil

b Bioprocess Laboratory, Chemical Engineering Department, Federal University of Rio Grande

do Norte (UFRN), 59078-970, Natal-RN, Brazil

c Laboratory of Bromatology, Department of Pharmacy, Health Sciences Center, Federal


*Corresponding author:

Francisco Canindé de Sousa Júnior

Laboratory of Bromatology, Department of Pharmacy, Health Sciences Center, Federal

University of Rio Grande do Norte (UFRN), 59012-970, Natal, Brazil. E-mail:

[email protected]

55


Abstract

The present study evaluated the co-production of β-galactosidase and ethanol by yeasts

Kluyveromyces marxianus ATCC 36907 and Kluyveromyces lactis NRRL Y-8279 using lactose

from "coalho" cheese whey as a carbon source. Cheese whey was subjected to partial

deproteinization and physicochemical parameters were assessed. Submerged cultivations were

carried out in an orbital shaker to evaluate two carbon/nitrogen (C:N) ratios, 1.5:1 and 2.5:1.

The best C:N ratio (1.5:1) was carried to 1.5-L bioreactor cultivation in order to increase co-

production yields. Stability of β-galactosidase was assessed against different temperatures and

pH, and in the presence of metal ions. Concerning the co-production of β-galactosidase and

ethanol, Kluyveromyces lactis NRRL Y-8279 proved to be more efficient in both the C:N ratios,

reaching 21.09 U/mL of total enzyme and 7.10 g/L of ethanol in 16 h, for bioreactor

cultivations. This study describes the development of a viable, value-adding biotechnological

process using regional cheese by-product from northeast Brazil for co-production of

biomolecules of industrial interest.

Key-words: Cheese whey, β-galactosidase, Kluyveromyces lactis, Kluyveromyces marxianus.

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1. Introduction

The dairy industry plays an economically important role on the agri-food sector, and its

annually growing production is directly related to the increasing generation of wastes and by-

products, such as cheese whey (CW) (Escalante et al., 2018). The global CW generation is

estimated at around 200 million tons per year, with a linear increasing tendency (Domingos et

al., 2018; Treu et al., 2019). In Northeast Brazil, the CW production is intrinsically related to

the “coalho” cheese industry. The “coalho” cheese is a highly traditional Brazilian cheese,

which assumes relevant socio-economical and nutritional components in the Northeast region

(Fontenele et al., 2017; Soares et al., 2017). The CW is a by-product with significant pollutant

potential due to its organic matter content and high biological oxygen demand (BOD; Andrade

et al., 2017). On a dry basis, the composition of CW can reach up to 80% of lactose, one of the

most environmentally harmful sugars (Zhou et al., 2019).

Several strategies have been investigated for dealing with the CW waste disposal and

the use of biotechnological processes figures as an interesting way of converting such by-

product into a valuable feedstock (Carota et al., 2017). Although difficult to degrade on the

environment, the lactose content of CW can be used as platform for the fermentation of value-

added products such as ethanol (Beniwal et al., 2018), galactonic acid (Zhou et al., 2019), and

β-galactosidase (Perini, et al., 2013; Rao e Dutta, 1977). The enzyme β-galactosidase (β-gal;

EC 3.2.1.23), also known as lactase, is a product of great interest and several applications in the

food industry, as it is responsible for the hydrolysis of lactose glycosidic bonds (Panesar et al.,

2018a). In addition, to the increasing market share of lactose-free products for diet-restricted

consumers (Suri et al., 2019), β-gal is also used for the enzymatic production of food prebiotics

such as lactulose and different galactooligossacarídeos (GOS) ( Nooshkam et al., 2018; Panesar

et al., 2018).

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Yeasts from the genus Kluyveromyces spp. have been used for the production of β-gal

enzyme from lactose-based substrates for generally recognized as safe (GRAS) applications for

human consumption (González-Delgado et al., 2016; Perini, et al., 2013). Kluyveromyces spp.

yeasts present a respiratory-fermentative metabolic pathway that can generate energy through

the Krebs cycle (citric acid cycle) or by exclusive fermentation, in which ethanol is the main

product. In contrast to Saccharomyces spp. genus, yeasts with respiratory-fermentative

metabolism do not fully exploit their ability to uptake glucose during oxidative growth and,

therefore, present a Crabtree effect. In fact, Kluyveromyces spp. yeasts are able to

simultaneously perform fermentation and respiratory processes, and the balance between these

two metabolic pathways depends on the specificity of the lineage (Lane e Morrissey, 2010).

In a recent study, You et al. (You, Chang, et al., 2017) proposed the utilization of whey

powder as a substrate for low-cost production of β-gal enzyme. In addition, the authors used a

co-production strategy in which the ethanol, a fermentation by-product, could also be

recovered. Thus, the present study investigates the use of “coalho” cheese whey as a substrate

for the low-cost co-production of β-gal enzyme and ethanol by Kluyveromyces marxianus and

Kluyveromyces lactis. To the best of our knowledge, this is the first report in the literature that

describes the use of this regional industrial by-product for co-production of these biomolecules

thus showing an interesting biotechnological process.

2. Material and methods

2.1. Reagents

Yeast extract was acquired from Exôdo Científica (Brazil), dextrose, potassium

phosphate and magnesium sulfate were acquired from Synth (Brazil), peptone was acquired

from Kasvi (Brazil), agar was acquired from Vetec (Brazil) and ammonium sulphate was

acquired from Cinética (Brazil).

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2.2 Cheese whey (CW) obtaining and treatment

The cheese whey (CW) used in this study was obtained from a small "coalho" cheese

producer in the city of Ceará Mirim (Rio Grande do Norte, Brazil) and kept at -20° C until use.

For use in the process, the cheese whey partially deproteinized (CWD) was obtained by acid

precipitation followed by thermal coagulation. For this process, CW (250 mL) was heated to

85 °C for 15 min and 3 mL of citric acid 10% was added. After that, the acidified mixture was

heated to 90 °C for 15 minutes. The CWD was filtered with cheese cloth and kept at -20 °C

until further use (Koushki et al., 2012; Florêncio et al., 2013).

2.3 Yeast strains and inoculum preparation

Kluyveromyces marxianus ATCC 36907, donated by the Federal University of Ceará

(UFC, Brazil), and Kluyveromyces lactis NRRL Y-8279, provided by the ARS Collection

culture (Peoria, Illinois, USA) were used in this study.

Both yeasts were stored in 30% (v/v) glycerol at -20 °C. For the preparation of the

inoculum, the microorganisms were cultured in YEPD medium (10 g/L yeast extract, 20 g/L

dextrose, 20 g/L peptone e 20 g/L agar) for 24 h, at 30 °C. Three isolated colonies were

transferred to 50 mL of the culture medium containing 10 g/L lactose, 5 g/L of potassium

phosphate, 1.2 g/L of ammonium sulfate, 1.0 g/L yeast extract 0.4 g/L of magnesium sulfate

prepared in potassium phosphate buffer (0.2 M, pH 5.5) according to Lima et al.(2013), and

incubated for 16 h, at 180 rpm and 30 °C. Lactose used in the growth medium was exclusively

from QWD.

2.4. Carbon/nitrogen ratio assessment

The co-production of β-galactosidase (β-gal) and ethanol was evaluated in submerged

cultivation using orbital shaker and bioreactor. Two carbon/nitrogen ratios (C:N) were

evaluated for the co-production process (1.5:1 and 2.5:1) according to the conditions described

59


by (You et al., 2017b). For all cultivations, lactose from CWD was used as the sole source of

carbon available in the growth medium. Table 1 describes the complete compositions of culture

media for the two C:N ratios in this study.

For each of the co-production experiment, the media pH, lactose concentration (g/L),

ethanol concentration (g/L), total enzymatic activity of β-gal (U/mL), β-gal specific enzymatic

activity (U/mg) and total protein (mg/mL) were evaluated.

Table 1 - Complete composition of the culture media used for the two carbon/nitrogen (C: N)

ratios ratio) in this study. Lactose from partially deproteinized cheese whey (CWD) was used

as a sole source of carbon.

C: N ratioa

Lactose

(g/L)

(NH4)2SO4

(g/L)

Yeast

extract

(g/L)

KH2PO4

(g/L)

MgSO4.7H2O

(g/L)

1.5:1 20.0 1.3 12.0 5.0 0.4

2.5:1 40.0 1.6 14.4 5.0 0.4

2.4.1 Culture conditions for the orbital shaker

Orbital shaker (model TE-241, Tecnal, Brazil) cultivations were conducted in 250 mL

flasks with 50 mL growth medium, at 30 °C and 180 rpm. The inoculum was prepared as

described in Section 2.2 and corresponded to 10% (v/v) of the final media volume. The culture

medium was previously sterilized. CWD (lactose source) was filtered through a 0.22 μm

membrane and added to the media under sterile conditions. Samples were taken after 4, 8, 12,

16, 20, 24, 36 and 48 h of fermentation

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2.4.2. Culture conditions in the bioreactor

Bioreactor cultivations were conducted in a 2-L fermenter (model Biostat B, B-Braun,

USA). The bioreactor was sterilized with 1.5-L of culture media and lactose from CWD was

filtered through a 0.22 μm membrane and added moments before the start of the fermentation.

The inoculum was prepared as described in Section 2.2 and corresponded to 10% (v/v) of the

final media volume (Perini et al., 2013). The experiments were conducted in the following

conditions: 30° C, pH 5.5 (controlled with solutions of 1.0 M HCl and 1.0 M NaOH), 200 rpm

agitation and aeration of 1.33 vvm. Samples were taken after 4, 8, 12, 16, 20, 24, 36 and 48 h

of fermentation.

2.4.3 Enzyme extraction

Due to the intracellular production of β-gal, the enzymatic extract was obtained from

mechanical disruption of the yeasts cellular structure, as described by Braga et al.(2014), with

minor modifications. The procedure was performed in 50 mL falcon tubes with 25 mL of cell

suspension and 27.5 g of glass beads (diameter ranging from 0.95 to 1.05 mm), agitated in a

vortex for 5 min, followed by 1 min of an ice bath. This procedure was repeated four times. The

suspension was then centrifuged at 5200 ×g for 10 min, 4 °C (model CT-5000R, Solab, Brazil).

The enzymatic extract was collected and stored at -20° C until use.

2.5 β-galactosidase stability assays

The β-gal stability was assayed at different pHs, in the presence of metal ions and at

different temperatures based on the remaining activity after exposure, according to Oliveira et

al.(2018). After each treatment, the β-gal activity was determined as described in Section 2.4,

and the results were expressed as the enzymatic activity retention.

For pH stability, the enzymatic extract was exposed to pH 4.0 to 10.0, at 25 °C, for 30,

60 and 90 min. The following buffer solutions were used: sodium acetate (0.2 M, pH 4.0 – 5.0),

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sodium phosphate (0.2 M, pH 6.0-8.0), glycine-NaOH (0.2 M, pH 9.0-10.0). For thermal

stability, the enzymatic extract was exposed to temperatures of 40, 45 and 50 °C, at pH 6.6, for

15, 30 and 60 min. The effect of metal ions on the β-gal activity was assessed as described by

Dias et al.(Dias et al., 2002), with minor modification. The enzymatic extract was exposed to

solutions containing ions Mg2+ (MgSO4·7H2O, 5 mM), Cu2+ (CuSO4 ·5H2O, 5 mM),

Zn2+(ZnSO4 ·7H2O, 5 mM), Mn2+ (MnCl2, 5 mM), Fe2+ (FeSO4, 5 mM) e Co2+ (CoCl2, 5 mM),

at 25 °C, for 30, 60 and 120 min.

2.6 Analytical methods

The physicochemical parameters of CW and CWD samples were analyzed according to

AOAC methods (AOAC, 2006). Briefly, total protein content was assessed by Kjeldahl method

(method 991.20), ash content was analyzed by incineration at 550 °C (method 900.02), and pH

was determined using a digital potentiometer (model pH 2600, Instrutherm, Brazil; method

981.12). Lipid content was determined by the Bligh & Dyer method (Gusso et al., 2012). The

moisture content was determined by the gravimetric method at 105 °C (Bueno et al., 2017).

The lactose content was evaluated by High-performance Liquid Chromatography (HPLC;

model Ultimate 3000, Thermo Fisher Scientific, USA) using a Shim-Pack column (model SCR-

101H, Shimadzu, Japan) with refraction index detector (RID), at 40 °C. Sulfuric acid (0.005 M)

was used as a mobile phase, under a flow of 1.0 mL/min. All samples were filtered through

0.22 µm membrane before analysis.

For all the cultivations, the biomass monitoring was carried through optical density

(Spectrophotometer - model Genesys 10, Thermo Spectronic, USA), with reading at 620 nm

(OD620; Lima et al., 2013) using an external calibration, and the results were expressed in g/L.

The concentrations of ethanol, lactose, glucose, and galactose were evaluated by HPLC, as

described previously.

62


The enzymatic activity of β-gal was determined using the synthetic substrate O-

nitrophenyl-β-D-galactopyranoside (ONPG), as described by Braga et al.(2014). The

enzymatic activity of β-gal was expressed in U/mL. The enzymatic activity unit (U) was defined

as the enzyme necessary to produce 1.0 μmol of O-nitrophenol per min at the assay’s conditions

(37 °C, pH 6.6). The total protein was assessed by the Bradford method (Bradford, 1976).

Bovine Serum Albumin (BSA) was used as standard and reads were performed at 595 nm. The

protein concentration was expressed in mg/mL and used to calculate the specific activity of the

enzyme β-gal (U/mg).

2.7 Kinetic parameters

The maximum specific growth velocity (𝜇𝑚𝑎𝑥), the maximum productivity of cells (𝑃𝑋)

and products (𝑃𝑝𝑚𝑎𝑥; either 𝑃𝛽−𝐺𝑎𝑙 or 𝑃𝑒𝑡ℎ𝑎𝑛𝑜𝑙), and yield of cells (𝑌𝑋 𝑆⁄ )and products (𝑌𝑃 𝑆⁄ ;

either 𝑌𝛽−𝑔𝑎𝑙 𝑆⁄ or 𝑌𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝑆⁄ ) based on substrate (lactose) consumption were calculated using

Equations 1 – 5 (Schmidell et al., 2001; Vasconcelos et al., 2018).

ln𝑋𝑚𝑎𝑥

𝑋0= 𝜇𝑚𝑎𝑥 ∙ (𝑡𝑓 − 𝑡0) (1)

𝑃𝑋 =𝑋𝑚𝑎𝑥−𝑋0

𝑡𝑓 (2)

𝑃𝑝𝑚𝑎𝑥 =𝑃𝑚𝑎𝑥−𝑃0

𝑡𝑓 (3)

𝑌𝑋 𝑆⁄ =𝑋𝑚𝑎𝑥−𝑋0

𝑆0−𝑆𝑚𝑎𝑥 (4)

𝑌𝑃 𝑆⁄ =𝑃𝑚𝑎𝑥−𝑃0

𝑆0−𝑆𝑚𝑎𝑥 (5)

Where 𝑋𝑚𝑎𝑥 is the maximum cells (biomass) concentration (g/L), 𝑋0 is the initial cell

concentration (g/L), 𝑡𝑓 is the fermentation time to reach 𝑋𝑚𝑎𝑥 (h), 𝑆0 is the initial lactose

63


concentration (g/L), 𝑆𝑚𝑎𝑥 is the lactose concentration found when 𝑋𝑚𝑎𝑥 was achieved (g/L),

and 𝑃𝑝𝑚𝑎𝑥 is the maximum concentration of the products, either β-gal (𝑃𝛽−𝑔𝑎𝑙) or ethanol

(𝑃𝑒𝑡ℎ𝑎𝑛𝑜𝑙).

From the maximum specific growth (𝜇𝑚𝑎𝑥), the generation time (GT) was calculated

with the following equation:

𝑡𝑔 =𝑙𝑛𝑥

µ𝑚𝑎𝑥 (6)

2.8 Statistical analysis

All experiments were performed as three independent replicates (n = 3). Statistical

analysis was performed using the software Statistica v. 8.0 (TIBCO Statistica, Palo Alto, CA,

USA). One-way ANOVA combined with Tukey HSD post hoc test was applied to establish

statistical significance (p < 0.05).

3. Results and Discussion

3.1 Physicochemical characterization of cheese whey

The results for the physical-chemical parameters of the cheese whey (CW) and cheese

whey partially deproteinized (CWD) are presented in Table 2.

The differences in the composition of CW and CWD can be related to the partial

deproteinization process, which altered the moisture and lactose contents, in addition to the

protein composition of CW (p < 0.05). According to the pH, the CW used here is featured as

"sweet whey" in agreement to Brazilian regulations (Brasil, 2013), but the acid treatment of the

deproteinization lowed the pH of CWD to around 4.6. The partial deproteinization aims to

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reduce the influence of the protein content of CW during the cultivations, besides, to increase

the lactose concentration (Koushki. et al, 2012).

Table 2: Physicochemical parameters for cheese whey (CW) and cheese whey partially

deproteinized (CWD).

Parameter CW CWD

pH 6.81 ± 0.50a 4.60 ± 0.50b

Ash (%) 6.91± 0.04a 6.46 ± 0.06a

Protein (%) 10.39 ± 0.02a 5.40 ± 0.02b

Fat (%) 2.31 ± 0.41a 2.16 ±0.33a

Moisture (%) 94.71 ± 0.06b 97.17 ± 0.06a

Lactose content (%) 2.70 ± 0.01b 4.40± 0.01a

Mean values ± Standard deviation. Different letters in the same line indicate statistical

difference (p < 0.05) according to Tukey’s test.

The cheese whey is a cloudy liquid, yellow-green, which carries approximately 55% of

the composition of the solids found in the whole milk. Lactose is the principal sugar found in

the CW and is related to its high chemical oxygen demand (COD; Alves et al., 2014). Jacinto

et al. (2012) evaluated the physicochemical composition of the CW used in the production of a

fermented dairy drink and found similar results to the presented here, for moisture (92.13%),

carbohydrates (lactose, 5.54%) and pH (6.46)

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3.2 Co-production of β-galactosidase and ethanol in the orbital shaker

The co-production of β-gal and ethanol in the orbital shaker was investigated using two

C:N ratios. Lactose from the CWD was the single carbon source used for all cultivations. K.

lactis NRRL Y-8279 and K. marxianus ATCC 36907 were used for the co-production using

C:N ratios of 1.5:1 and 2.5:1. The biomass and substrate (lactose) concentration profiles during

the cultivations are presented in Fig. 1. Fig. 2 shows the co-production profiles of the two

products, β-gal and ethanol. The kinetic parameters for all cultivations are presented in Table

3. The pH from the culture media during all the orbital shaker cultivations ranged from 4.95 to

5.47.

Fig. 1. Biomass growth profile for K. marxianus ATCC 36907 (●) and K. lactis NRRLY-8279

(○), and lactose consumption profile for K. marxianus ATCC 36907 (▲) and K. lactis NRRLY-

8279 (△) in the orbital shaker cultivations. (A) C/N 1,5:1; (B) C/N 2,5:1. Mean values ±

Standard deviation.

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Fig. 2. Co-production profile of β-galactosidase for K. marxianus ATCC 36907 (■) and K.

lactis NRRL Y-8279 (□) and of ethanol for K. marxianus ATCC 36907 (▼) and K. lactis

NRRLY-8279 (▽) in the orbital shaker cultivations. (A) C:N 1,5:1; (B) C/N 2,5:1. Mean values

± Standard deviation

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Table 3: Kinetic parameters for the orbital shaker and bioreactor cultivations for the co-production of ethanol and β-galactosidase using cheese

whey as substrate.

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aC: N: Carbon/nitrogen ratio.

bt: Cultivation time for the best co-production condition.

cXa: Maximum cell concentration at the best co-production condition.

dSf: Final concentration of substrate (lactose).

eS0: Initial concentration of substrate (lactose).

fμmax: Maximum specific growth velocity

gPx, Pβ-gal, and Pethanol: Maximum productivity of cells, β-gal and ethanol

The biomass growth for both strains and lactose consumption profiles (Fig. 1A-B)

presented a similar pattern, suggesting good metabolic adaptation to the culture medium based

on CWD. The growth profile also suggested a high rate of cell growth, with generation time

(GT) ranging from 4.62 h to 4.95 h, for the C:N ratios of 1.5:1 and 2.5:1, respectively, for K.

lactis NRRL Y-8279. The generation time presented by the K. marxianus ATCC 36907 for both

C:N ratios was similar (4.95 h), as shown in Table 3. In addition, the increase in the carbon

concentration (C:N ratio from 1.5:1 to 2.5:1) resulted in a decrease of ethanol and β-gal in all

cultivations. You et al. (2017a) conducted a similar study for the co-production of β-gal and

ethanol from commercial whey powder and observed a similar relationship between the C:N

ratio and the co-production yields, i.e., the increase of C:N ratio reduced the co-production of

β-gal and ethanol. Also, Sampaio et al. (2019) recently conducted a similar study for the

bioethanol production from cheese whey permeate and observed that ethanol production was

increased after the yeast exponential growth.

The maximum specific growth velocity (μmax) ranged from 0.12 to 0.15 h-1 for both

strains in the orbital shaker cultivations. Sampaio et al. (2019) also investigated the co-

production of ethanol and β-gal from cheese whey, using initial concentrations of lactose of 55

and 75 g/L in the culture medium and observed μmax between 0.46 and 0.48 h-1. On the other

69


hand, those authors observed low substrate conversion factors between 0.083g.g-1 and 0.027g.g-

1, which can be related to the high initial concentration of lactose in the culture media, due to

substrate inhibition.

For the K. marxianus ATCC 36907 cultivations, no significant difference (p > 0.05) was

observed for the biomass concentrations between the two C:N ratios studied (14.38 ± 0.16 g/L

and 13.89 ± 0.05 g/L, for C: N 1.5:1 and 2.5:1, respectively). However, for the C:N ratio of

1.5:1, the maximum co-production of β-gal and ethanol was observed after 16 h of cultivation,

which accounted for a higher productivity factor (Px; 0.76 ± 0.01 g/L.h). Nonetheless, the

overall production yields of β-gal for the K. marxianus ATCC 36907 cultivations were

relatively low (Fig. 2A-B), reaching 92.49 and 66.23 U/g cells to the C:N ratios of 1.5:1 and

2.5:1, respectively. In addition, the yield of cells based on substrate (lactose) consumption (Yx/s)

for the K. marxianus ATCC 36907 cultivations ranged from 0.42 ± 0.01 to 0.99 ± 0.30 g.g-1,

which are superior to the results found by Manera et al. (2008) in a study for the production of

β-gal with K. marxianus using synthetic medium.

For the K. lactis NRRL Y-8279 cultivations, the maximum co-production of β-gal and

ethanol were achieved after 20 h and 16 h of cultivation, for the C:N ratios of 1.5:1 and 2.5:1,

respectively. After these peak productions, a decline in ethanol and β-gal production is

observed. This behavior may be related to the batch strategy adopted, which did not involve

further lactose retro-feeding strategy. The co-production yields for both products were

significant different (p < 0.05) in the two C:N ratios. The lower C:N ratio (1.5:1) favored the

production of β-gal, yielding 18.5 ± 0.01 U/mL of total enzyme concentration and 2340.08 U/g

cells, for 18.58 g/L of lactose consumption and 9.01 ± 0.23 g/L of ethanol production, while

the higher C:N ratio (2.5:1) favored the ethanol production (9.70 ± 0.29 g/L), for 38.98 g/L of

lactose consumption and 770.10 U/g cells and 8.91 ± 0.38 U/mL.

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You et al. (2017a) reported the best C:N ratio for the co-production of β-gal and ethanol

of 2.5:1, using an initial concentration of 55 g/L of lactose from commercial whey powder.

Additionally, they obtained 37.43 ± 2.6 U/mL of total β-gal activity and 21.42 ± 0.15 g/L of

ethanol concentration. Although such results are higher than those obtained in the present study,

it is important to note that the use of CWD takes into consideration the composition of an

industrial waste as a source of nutrients, which may have been responsible for the differences

in the results. In addition, the results of co-production presented here are quite promising since

it proposes the use of cheese whey from small and mid-sized companies, which usually drain

this kind of waste into the environment, for an innovative, viable biotechnological application,

as a treatment for agro-industrial wastes (Silva et al., 2018).

From the results presented in Table 3, the best co-production of β-gal and ethanol

condition was achieved for the cultivation in which the lowest biomass concentration was

reached. Therefore, the bioreactor cultivations were carried using the yeast K. lactis NRRL Y-

8270 in the C:N ratio of 1.5:1.

3.3 Co-production of β-galactosidase and ethanol in bioreactor using K. lactis NRRL

Y-8270

In order to increase the co-production yields of β-galactosidase and ethanol, the culture

medium with C:N ratio of 1.5:1, with 20 g/L of lactose from the CWD, was used for the

cultivation in a bioreactor with useful volume of 1.5 L. Biomass (cells) concentration and

substrate consumption profiles are shown in Fig. 3. The kinetic parameters for the cultivations

in bioreactor were calculated and are presented in Table 3. The pH of the culture medium in the

bioreactor cultivations was controlled at 5.5, with no significant difference from the cultivations

carried in the orbital shaker (from 4.95 to 5.5). According to Parazzi et al. (2017), this pH range

favors the reproduction of yeasts and inhibits the growth of contaminating bacteria.

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Fig. 3. (A) Biomass (cells) growth (●) and substrate (lactose) consumption (▲) profiles; and

(B) Co-production of β-galactosidase (○) and ethanol (□) for the bioreactor cultivations using

K. lactis NRRLY-8279 and the C:N ratio of 1.5:1. Mean values ± Standard deviation. .

The profile of cellular growth of the K. lactis NRRLY-8279 strain in the bioreactor

cultivations was similar to the observed for the orbital shaker cultivations for the same

processing time (48 h). The maximum co-production time in the bioreactor was 16 h (Fig. 3A),

reducing the optimum co-production time obtained in the orbital shaker cultivations (20 h,

Table 3), and reaching 12.47 ± 0.10 g/L of biomass concentration and 1691.26 U/g cells. Since

in the cultivations carried in the bioreactor the parameters such as pH and medium aeration

were strictly controlled, the β-gal productivity was increased from 0.85 ± 0.08 to 1.27 ± 0.03

U/mL.h, when compared to the orbital shaker cultivations. In addition, the GT observed for the

bioreactor cultivation was also reduced, from 4.62 to 3.15 h, when compared to the orbital

shaker.

The lactose content found in the culture medium from the CWD, was totally consumed

around 12 h of fermentation, but cellular growth was still observed after that. This behavior is

a result of the high content of glucose found in the culture medium after lactose hydrolysis,

72


which was used as carbon source by the yeast to keep cell multiplication, which eventually

declined.

The genes LAC12 and LAC4 are found in the strain K. lactis NRRLY-8279, and they

are responsible for the codification of lactose-permease and ß-galactosidade enzymes,

respectively, which play different roles in this process. Lactose-permease enzymes promote the

lactose transport through the plasma membrane into the yeast cells, while the β-gal is

responsible for the hydrolysis of lactose (disaccharide) into two monosaccharides, glucose and

galactose. These two sugars are metabolized via glycolysis, however, before attending this

metabolic route, galactose is converted into a glycolytic intermediate, the glucose-6-phosphate,

via the Leloir pathway, by the action of three enzymes galactoquinase, galactose-1-P-uridil

transferase and UDP-galactose 4-epimerase (Guimarães et al., 2010; Rubio-Texeira, 2005).

The decline in biomass concentration can also be related to the accumulation of the

ethanol produced. This kind of cell growth inhibition by the product occurs due to structural

and metabolic changes that lead to the reduction of cell viability during the fermentation

(Bleoanca et al., 2013; Ricci et al., 2012; Wang et al., 2007). Some yeasts suffer from damages

in the plasma membrane caused by ethanol, resulting in changes in the phospholipidic structure

(Henderson et al., 2011), increasing the content of fatty acids and ergosterol to stabilize the

fluidity (Vanegas et al., 2012; Zhao e Bai, 2009).

The activity of β-gal and the concentration of ethanol in the cultivations conducted in

bioreactor reached maximum values simultaneously after 16 h of fermentation, reaching 21.09

± 0.69 U/mL of total enzyme activity, 16.69 ± 0.02 U/mg protein for the maximum specific

activity and 7.10 ± 0.09 g/L of ethanol produced. Fig. 3B shows the co-production profile.

You et al. (2017) conducted studies in 7-L bioreactor using Kluyveromyces lactis

CICC1773 to propose an integrated process of co-production of β-gal and ethanol using lactose

73


from commercial whey powder as substrate. In that study, the authors proposed that the ethanol

produced could be used to permeabilize the yeast cells and obtain the enzymatic extract, in the

batch feed. Using this strategy, they obtained 105.91 U/mL of total β-gal concentration and

32.16 g/L of ethanol. Thus, whereas in the present study the substrate used was a dairy residue

with a low degree of purity, with a concentration of initial lactose 20 g/L, in batch cultivation,

and that the cell disruption method for obtaining of the enzyme was mechanical, the results

obtained in this study are promising.

3.4 Stability of β-gal enzymatic activity against pH, temperature and metallic ions

The enzymatic activity of β-gal may be influenced by several factors, such as

temperature, pH, presence and concentration of metallic ions (Fischer et al., 2013). In addition,

β-gal may also present different properties, depending on the source (plant, animal, or

microbial), which can lead to different technological applications (Braga et al, 2014). In this

study, the stability of the enzymatic activity of β-gal, produced by K. lactis NRRL Y-8279 in

bioreactor cultivations, was evaluated against different temperatures, pH ranges and in the

presence of several metallic ions.

For the pH stability (Fig. 4A), the enzymatic activity of β-gal presented the highest

stability in the pH range between 6.0 and 9.0, being the best results achieved for the pH 7.0 in

30 min of exposure (67.76%). In a study carried out by Bosso et al. (2016), the highest

enzymatic stability of β-gal produced by K. lactis and Aspergillus oryzae was observed for pH

6.0 and 7.0. At pH 7.0 and 40 °C the K. lactis-produced β-gal presented 97.9% of lactose

hydrolysis. These results are in agreement with the showed here, in which higher stability was

achieved in neutral to alkaline pH.

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Fig. 4. Enzyme stability of β-galactosidase produced by K. lactis NRRL Y-8279, using the C:N

ratio of 1.5:1 in bioreactor cultivations. (A) pH Stability in relation to the pH; (B) Stability in

relation to metallic ions; (C) Stability in relation to temperature. Mean values ± standard

deviation. Different lowercase letters (a, b, c, d) indicate statistical differences (p < 0.05) for

different pH/metallic ion/temperature, at the same incubation time. Different capital letters (A,

B, C) indicate statistical differences (p < 0.05) for different incubation periods within the same

pH/metallic ion/temperature.

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As to the effect of the presence of metal ions on the stability of β-gal, highest stability

(p < 0.05) was achieved for a 60 min incubation period in the presence of Mg2+ ions (74.19%

of retained activity). On the other hand, the β-gal was inactivated during the 60 min incubation

period in the presence of the ions Zn2+, Cu2+ and Ag+, reaching values of relative activity of

2%, 0.68% and 0%, respectively (Fig. 4B).

Fan et al. (2015) reported that ions Fe3+ and Zn2+ presented inhibitory effects on the

activity of β-gal, in which the Zn2+ had a more significant effect. However, the authors reported

that the ions Mg2+ and Mn2+ promoted an increase in enzyme activity. These results are similar

to the presented here. An inhibitory effect was observed for the presence of the ion Cu2+, which

agrees with the results presented by Hoyuox et al. (2001). However, they considered that the

inhibition/activation effects of the metal ions are concentration-dependent. Divalent metal ions

are of great importance to the catalytic efficiency of most β-gal enzymes, that present important

binding sites to Mg2+ (Sutendra et al., 2007), since this ion forms a complex with protein

structure. Although this process is not yet fully understood, it has been reported that the ion

participates in the enzyme catalysis, acting as a Lewis acid (Adalberto et al., 2010).

Concerning to the effect of temperature on the stability of the enzymatic extract, it was

observed that the higher stability was obtained at the temperature of 40 °C for a 15 min

incubation period, with a relative activity of 93.14%. The increase in temperature to values

above 45 °C significantly reduced enzymatic activity (p < 0.05), reaching 1.97% when

subjected to 50 °C for 15 min (Fig. 4B). In a study by Chanalia et al. (2018), enzymatic stability

of β-gal was reported to maintain 80% of initial activity in the range between 45 °C and 55 °C.

However, above 55 °C, the enzyme activity decreased abruptly, with no activity evidenced at

60 °C.

76


4. Conclusions

In this study, the yeast Kluyveromyces lactis NRRL Y-8279 was shown to be more

efficient for co-production of β-gal and ethanol using "coalho" cheese whey as substrate. The

maximum production of β-gal was 21.09 ± 0.69 U/mL with ethanol concentration reaching 7.10

± 0.09 g/L in 16 h of submerged cultivation. The enzymatic activity of β-gal was more stable

in the presence of Mg2+, after 60 min of exposure, at 40 °C, after 15 min of exposure, and at pH

7.0, after 30 min of exposure. The CW presented itself as a viable alternative source for the co-

production process, evidencing that the Kluyveromyces lactis NRRL Y-8279 metabolize more

efficiently the lactose and can generate simultaneously two products of biotechnological

interest. Thus, this process adds value to the dairy sector allowing the use of the cheese whey

to leverage the development of such industries.

Acknowledgments

The authors would like to thank the Federal University of Rio Grande do Norte

(UFRN), especially the Laboratory of Biochemical Engineering and the Laboratory of Food

Engineering. Fábio Medeiros was supported by Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq/Brazil), grant number 144415/2017-8. The present work was

carried out with support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES/Brazil) - Funding Code 001.

Conflict of interests

The authors declare no conflict of interest regarding the funding sources or the materials used

in the presented study.

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ARTIGO 2 - Recovery of β-galactosidase produced by Kluyveromyces lactis by ion

exchange chromatography: Optimization of pH and ionic strength conditions using

experimental design

Catherine Teixeira de CARVALHOa, Sérgio Dantas de OLIVEIRA JÚNIORa, Wildson

Bernardino de Brito LIMAa, Fábio Gonçalves Macêdo de MEDEIROSb, Ana Laura Oliveira

de Sá LEITÃO a, Everaldo Silvino dos SANTOSa, Gorete Ribeiro de MACÊDOa, Francisco

Canindé de SOUSA JÚNIORc *




do Norte (UFRN), 59078-970, Natal-RN, Brazil

c Laboratory of Bromatology, Department of Pharmacy, Health Sciences Center, Federal


*Corresponding author:

Francisco Canindé de Sousa Júnior



[email protected]

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Abstract

The use of β-galactosidase in food products has been a major focus of the industry. Therefore,

the development of efficient and inexpensive methodologies to purificate this product is

extremely important. Thus, the aim of this study was to partially purified the enzyme β-

galactosidase (β-gal) by ion exchange chromatography, in a fixed bed column, evaluating the

influence of the parameter’s pH and ionic strength, in order to obtain a high purification factor.

To determine the initial adsorption conditions of the β-gal, four types of adsorbents resins were

tested in rotary incubators: Capto MMC multimodal, Streamline DEAE, Streamline SP and

Amberlite XAD polymeric-XDA 7. The pH influence was also studied, so the systems were

exposed to different pHs 6-8. Then, after determined the best adsorption conditions, an

experimental design 22 with three central points was applied to optimize the purification using

a FPLC, evaluating the ionic strength (50 and 200 mM) and the pH (6.0 and 7.0). Subsequently,

the β-gal enzyme fractions of the best purification assay were submitted to a qualitative analysis

by electrophoresis. The enzyme adsorption capacity in the rotary incubator was favored by the

high pH and the Streamline DEAE resin had the best selectivity to the target molecule, with a

β-gal retention capacity of 5.79 ± 0.79 U/g at pH 7.0. The design results showed the passage

from the lower level (-1) to the superior (+ 1) of the ionic strength and pH increases the

purification fold (PF) of the β-gal enzyme to 2.00 ± 0.42, however the ionic strength exerted

greater influence on the PF response The fraction of the β-gal enzyme in the best elution range

using between 0.1 and 0.4 M of NaCl in the and purification condition with a buffer with

200mM and pH 7.0 was submitted to electrophoresis, where an evident band with molecular

weight in the range between 60 and 120 kDa was observed, configuring the enzyme of interest.

These results point to the recovery of a stable β-galactosidase of K. lactis with potential

industrial application from low-cost residue.

Key-words: Purification, Kluyveromyces lactis, Adsorption, β –galactosidase

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1. Introduction

β-galactosidase (β-gal) is an enzyme widely used in the food industry, it acts as a catalyst

in the hydrolysis of lactose in milk and its derivatives, and in the synthesis of Galacto-

Oligosaccharides (GOS) with prebiotic properties (Zhao et al., 2018).

Lactose, the carbohydrate found in milk and derivatives, is a disaccharide formed by

glucose and galactose. In humans, lactose intolerance occurs in 75% of the population, being

caused by the insufficiency of β-gal in the body, which results in a decrease of the enzyme

activity in the membrane of the mucosa edge located in the small intestine of adults. Enzyme

formulations of β-gal in dairy products can reduce lactose and promote a beneficial effect for

lactose intolerant (Braga et al., 2014; Zhao et al., 2018)

The lactases or β-gal of biotechnological interest that are produced by yeasts of the

genus Kluyveromyces, are generally intracellular and obtained by submerged fermentation. This

fermentation, as in most biotechnological process, involves the purification of proteins and

peptides from different strategies (Machado et al., 2015). In this context, one of the promising

techniques used for this purpose is chromatography, where the protein binds to an adsorbent by

ionic or hydrophobic interactions being subsequently eluted for recovery.

The chromatography using an ion exchange matrix is widely used for protein adsorption,

in which the concept is based on the attraction between the proteins molecules and the resin

that presents opposite charges. One of the advantages of this process is that it provides smooth

separation conditions, allowing proteins to maintain their conformation. (Braga et al., 2014;

Medeiros et al, 2012).

Thus, the aim of this study was to evaluate the best condition of purification using a

single chromatographic step varying the adsorbent, pH and ionic strength, using purification

fold and yield as reponses.

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2. Materials and methods

2.1 Microorganism and inoculum production

Kluyveromyces lactis NRRL Y-8279 used in this study was provided by the ARS

Collection culture (Peoria, Illinois, USA). The strain was maintained in a 30% (v/v) solution of

glycerol at -20 °C. For pre-inoculum preparation, K. lactis was cultivated in YEPD medium

containing 10 g/L of yeast extract, 20 g/L of dextrose, 20 g/L of peptone and 20 g/L of agar for

24 h, at 30 °C. Then, three isolated colonies (inoculum) were transferred into 50 mL of the

culture medium containing 10 g/L of lactose, 5.0 g/L of potassium phosphate, 1.2 g/L of

ammonium sulfate, 1.0 g/L of yeast extract and 0.4 g/L of magnesium sulfate, prepared in

potassium phosphate buffer (0.2 M, pH 5.5) according to (Lima et al., 2013). The Lactose used

in the growth medium was exclusively from the cheese whey partially deproteinized (CWD).

The inoculums were cultivated in Erlenmeyers under agitation of 180 rpm, at 30 °C, for 16 h in

a rotating incubator.

2.2 Production of enzymatic extract

The enzyme β-galactosidase (β-gal) was produced through submerged cultivation por

Kluyveromyces lactis NRRL Y-8279 using rotary incubators (Tecnal, model TE-241), with 250

mL Erlenmeyers flasks containing 50 mL of culture medium (20 g/L of QWD, 1.3 g/L of

(NH4)2SO4, 12 g/L of yeast extract, 5.0 g/L of KH2PO4 and 0.4 g/L MgSO4.7H2O, in 0.2 M

potassium phosphate buffer at pH 5.5, according to the adapted methodology of (Braga et al.,

2014). The fermentation was performed with 10% of inoculum (v/v) prepared as described in

section 2.1. The culture medium was previously sterilized and the lactose from the QWD was

filtered in a 0.22 μm membrane. The cultivations in rotary incubators were performed at 30 °C,

180 rpm. Samples were removed after 20 h of fermentation.

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The enzymatic extract was obtained from the yeast cell disruption after the fermentation,

according to (Braga et al., 2014). The procedure was performed in centrifuge tubes of 50 mL,

containing 25 mL of the cell suspension and 27.5 g of glass pearls (diameter ranging from 0.95

to 1.05 mm) under vortex agitation for 5 min, followed by 1 minute in an ice bath. This

procedure was repeated 4 times. Then, the suspension was centrifuged (Cientec, CT-5000R

model, Brazil) at 5200 × g during 10 min at 4 ºC. The enzyme extract was collected and stored

at -20 °C.

2.3. Determination of adsorption conditions for -gal purification

In order to determine the initial adsorption conditions of the β-Gal enzyme, four types

of adsorbents (Multimodal capto MMC, Streamline DEAE, Streamline SP and Amberlite XAD

polymeric-XDA 7) and three pHs (6.0, 7.0 and 8.0) were tested. The assays were performed in

batch, at 30 ºC and 150 rpm, in a rotary incubator for 60 minutes. In 25 mL Erlenmeyer vials,

0.2 g of resin, 2 mL of sodium phosphate buffer 200 mM in the respective pH, and 2 mL of

enzymatic extract were added (Padilha et al., 2017). The enzymatic activity retained in the solid

phase calculated according to equation 1:

𝑞 (𝑈/𝑔) =𝑉 (𝑐0−𝑐)

𝑉𝑎𝑑𝑠 (1)

Being equation 1: q the amount of enzyme adsorbed on the resin, V the volume of the enzyme

extract, C0 the value of the initial activity, C the value of the activity in the equilibrium, Vads

the volume of the adsorbent.

2.4. -gal purification

The resin that presented the best adsorption capacity to the target molecule was selected

for the purification assays using a fast protein liquid chromatography (FPLC) system (AKTA

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start, GE healthcare Bio-Science, New Jersey, USA) using a fixed bed column HR 16/5 with a

bed volume of 6 mL (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

The process was performed with a superficial velocity of the mobile phase of 100 cm.h-

1. The column containing the DEAE streamline resin was initially equilibrated for 30 minutes

with buffer A (sodium phosphate). Then, 4.5 mL of the crude extract was injected into the

system. After the load, the non-bonded or weakly bonded molecules were removed from the

system in the washing step, in which a volume of 7.5 mL of buffer A was used. And later,

during the elution stage, a mobile phase ionic strength was linearly increased by adding the

buffer B (sodium phosphate 50 mM, 125 mM and 200 mM at pH 6, 6.5 and 7 and NaCl 1 M)

from 0 to 100%, aiming to separate the proteins according to the strength with which they were

adsorbed in the resin. Samples were collected at each 1.5 mL. Two runs were performed with

the same parameters to atone to the reproducibility of the system.

The influence of the factors (independent variables) ‘ionic strength’ and ‘pH’ on the

dependent variables ‘fold purification’ (FP) and the ‘yield’ (Y) of β-Gal was analyzed through

an experimental design 22 with 3 central points, resulting in 7 assays. The factors and levels

chosen for the design are presented in Table 1.

Table 1: Factors and levels used in the experimental design 22

Factor Levels

-1 0 +1

Ionic

strength

50 mM 125 mM 200 mM

pH 6.0 6.5 7.0

The analysis of the experimental design data was performed by of Analysis of Variance

(ANOVA), at a significance level of p ≤ 0.05, and the extent of Variance explained by each

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model was given by the coefficient of determination (R2). The Software Statistica® 7.0, was

used for the graphical analysis of the data.

2.5 Analytical methods

To determine the hydrolytic activity of the β-gal enzyme, ortho-nitrophenyl-β-D-

galactoside (ONPG) was used according to (Braga et al., 2014). The β-Gal hydrolytic activity

was determined at 37 ºC and pH 6.6 for 10 minutes and expressed in U/mL. A unit (U) of

enzymatic activity corresponds to the amount of enzyme that catalyzes a reaction with the

formation velocity of 1.0 µmol of ortho-nitrophenol (o-nitrophenol) for 1 minute, in the

conditions aforementioned. The total protein concentration was determined using the method

proposed by (Bradford, 1976). Bovine serum albumin (BSA) was used as a standard protein

from a calibration curve that ranged from 0 to 1.5 mg/ml of protein, in which the measurements

were performed in triplicate. The protein concentration was expressed in mg/mL and used to

calculate the specific activity of the enzyme β-Gal (U/mg).

From the enzymatic activity of the crude and partially purified extract, the purification

yield was obtained according to equation 2 and the fold purification calculated from the specific

activities of the crude and purified extract according to equation 3 (Leitão et al., 2018).

Yield (%) = 𝐸𝑛𝑧𝑦𝑚𝑎𝑡𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑝𝑢𝑟𝑖𝑓𝑖𝑒𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡

𝐸𝑛𝑧𝑦𝑚𝑎𝑡𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑐𝑟𝑢𝑑𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 x 100 (2)

Fold purification =𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑝𝑢𝑟𝑖𝑓𝑖𝑒𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡

𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑐𝑟𝑢𝑑𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (3)

The β-gal enzyme fractions of the best purification range were subjected to qualitative

analysis using polyacrylamide gel electrophoresis (PAGE) at 9%, according to the methodology

adapted from (General Eletric Conpany, 2000)

3. Results and discussion

3.1. Determination of adsorption conditions for -gal partially purification

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The results obtained in the adsorption experiments of the β-gal enzyme in different

adsorbents and pHs using the sodium phosphate buffer are shown in Figure 1.

In Figure 1, it was observed that the retention capacity of the enzyme rises in all the

basic regions tested, and the Streamline DEAE resin presents the best selectivity to the target

molecule, which promotes a better performance of the chromatographic system with a β-gal

enzyme retention capacity of 18.77 ± 0.14 U/g for pH 6.0, 5.79 ± 0.79 U/g at pH 7.0 and 13.50

± 1.14 U/g at pH 8.0.

Figure 1 – Adsorption of β-gal on 4 resins in the pH range of 6.0-8.0, 30° C, 150rpm and 0,2g

support. Different lowercase letters (a, b, c, d) indicate statistical differences (p < 0.05) for

different pH with the same adsorbent. Different uppercase letters (A, B, C) indicate statistical

differences (p < 0.05) for different adsorbents considering the same pH.

For proteins, purification in its active form was dependent on the solubility, load and

size of the molecule (Thadathil and Velappan, 2014). In most cases, the number of steps in the

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entire purification process will increase according to the required degree of purity (Wilken and

Nikolov, 2012). Each process requires resins with specific characteristics, they are usually

polymers of high chemical, mechanical and biological stability such as: cellulose, agarose,

dextran, polyacrylamide and polystyrene. In addition to the type of polymer, for the ion-

exchange chromatography, another important factor is the ionized groups on the surface of the

enzymes. These groups are generated from amino acid residues and their load balance (between

negative and positive groups) resulting in the enzyme load. The presence of these groups varies

with the pH of the medium and when they are present in equal number it is called isoelectric

point (IP). Therefore, there are matrices loaded with positive groups, such as the DEAE type

(diethylaminoethyl) called anionics, and those with negative groups, such as those of type CM

(Carboxy-methyl), called cationic (Lovato et al., 2017).

3.2 Central composite design using FLPC

After identification of the best adsorbent and pH for the β-gal adsorption, a experimental

design 22, with three central points, was carried out to optimize the enzyme purification. The

DEAE streamline resin was tested in the automated chromatography system FPLC (AKTA

Start in the pH Ranges 6.0, 6.5 and 7.0.). It was investigated the influence of the factors

(independent variables) pH and ionic strength (IS) on the Fold Purification (FP) of the enzyme

and the Yield (Y) of the process (dependent variables). The experimental conditions and the

responses for linear experimental design are presented in Table 2. One can observe that the

results related to FP ranged from 0.56 (assay 2) to 2.00 (assay 3), while those obtained for the

Y ranged from 49.08% (assay 1) to 91.07% (assay 2).

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Table 2: Experimental design 22 with coding and levels of variables used to purify the β-gal

enzyme as a function of pH and ionic strength.

Run pH IS (mM) FP Y (%)

1 -1 (6.0) +1 (200) 1.29 ± 0.29 49.08 ± 0.75

2 -1 (6.0) -1 (50) 0.56 ± 0.52 91.07 ± 0.12

3 +1 (7.0) +1 (200) 2.00 ± 0.42 51.64 ± 0.35

4 +1 (7.0) -1 (50) 0.64 ± 0.13 79.95± 0.45

5* 0 (6.5) 0 (125) 1.21± 0.01 65.73± 0.45

6* 0 (6.5) 0 (125) 1.05 ± 0.85 66.47± 0.77

7* 0 (6.5) 0 (125) 1.08 ± 0.01 68.11± 0.41

*Central points

Caption: IS - Ionic strength; FP- Fold Purification e Y- Yield of activity purified enzyme

The use of statistical methods and mathematical models contributed to the development

and optimization of processes over time. The response surface methodology (RSM) is a tool to

evaluate and model experimental data in order to identify independent and combined influences

of independent variables in the output variables of the process (Câmara junior et al., 2016).

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Figure 2: Pareto Charts of the standardized effects for the responses yield (Y) of activity

purified enzyme and fold purification of β-gal (FP), respectively

Based on the adjusted regression coefficients obtained by the software Statistica 7.0, a

statistical model that correlates the responses yield (Y%) and fold purification (FP) with the

variables ionic strength and pH were constructed, as observed in equations 2 and 3, in which

X1 is the pH and X2 the ionic strength.

A)

B)

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Y= 67.427 – 2.142.X1 – 17.576.X2 + 3.422.X1.X2 (2)

FP = 1.114 + 0.186.X1 + 0.512.X2 + 0.148.X1.X2 (3)

One can observe on the Pareto is charts obtained that the Y and FP responses were

influenced by both linear effects of the selected variables. The concept of interaction terms

indicates that changes in a combination of process variables had a synergistic effect on the

experimental response (Zhao et al., 2018). The passage from the lower level (-1) to the higher

(+ 1) of the ionic strength and pH increases the purification degree of the β-gal enzyme to 2.00,

but the ionic strength exerts greater influence on the FP. The contributions of ionic strength and

pH for enzyme recovery were -2,142 and -17,576, respectively.

The analysis of variance (ANOVA) performed in the obtained models confirmed the

significance (p< 0.05) evidenced in Table 3 and the Lack-of-Fit test (LoF) was significant (p>

0.05), suggesting the capacity to predict the models. High correlation coefficients (R²; 0.98858

and 0.99594, for FP and Y respectively, indicate a strong concordance of the adjusted data with

the empirical results, responsible for robust empirical models (Câmara Junior et al., 2016).

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Table 3: Analysis of variance (ANOVA) of the adjusted models to the experimental reponses

FP and Y.

Y

VARIANCE

FACTOR

gl SS MQ F p-valor

Model 3 1300.497 433.499 245.192 0.000

Residual 3 5.305 1.768

Lack of fit 1 2.327 2.327 1.563 0.033

Pure error 2 2.978 1.489

Total 6 1305.802

R2 0.98858

Caption: Y- β-gal yield; FP- fold purification; gl – degrees of freedom; SS – Square sum; MQ

– Mean square; R² – correlation coefficient.

Through the F test, it can be observed that the models proposed in equations 2 and 3 are

statistically significant and useful for predictive purposes. For the FP, the Fcal value was 15.26

times higher than the Ftab (9.28), and for the Y the calculated value was 26.42 times higher than

the tabulated.

FP

VARIANCE

FACTOR

gl SS MQ F p-valor

Model 3 1.274 0.425 141.666 0.042

Residual 3 0.015 0.003

Lack of fit 1 0 0 0 0.000

Pure error 2 0.015 0.007

Total 6 1.289

R2 0.99594

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The 3D representations of the adjusted models (equations 2 and 3) are shown in the

Figures 3 A e 3B. The highest region found on the response surfaces indicates the ideal

conditions for maximizing the responses of the purification process of β-galactosidase.

Figure 3: Response surface (RS) of Y and FP. (A) RS of the yield and (B) of the fold

purification of β-gal as function of pH x IS.

It is observed in Figure 3(A) that lower values of ionic strength (IS) and pH favor the

degree of recovery of the enzyme, reaching 91.07% ± 0.12 of yield. In contrast, the purity

A)

B)

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degree of the biomolecule of interest is not significant, achieving a 0.56 ± 0.52 fold

purification. It is also evident that higher values of IS and pH increase the degree of purity for

the enzyme reaching a FP of 2.00 ± 0.43 with a significant Y of 51.64% ± 0.35.

The results of Y and FP obtained in this work are promising and superior to those

reported by (Lima et al., 2016) who used the technique of multimodal chromatography for

recovery and purification of β-gal and reached around 48% of recovery with a FP of 1.17. In

the case of (Medeiros et al, 2012), they used sepharose Q resin and obtained recovery values

close to 88%, however it is important to highlight that in this study only commercial enzyme

solutions were used, which favors the process, given the higher degree of purity of samples. In

this way, Figure 4 shows the variation of protein concentration and enzymatic activity through

stages of chromatography (load, elution and washing) of the run with the best performance.

Figure 4: Purification chromatogram of β-gal produced by K. Lactis in a fixed bed, submitted

to a NaCl gradient through elution, where (□) protein concentration and (■) enzymatic activity.

100


The variation of the two responses analyzed during the entire run was observed. At the

first moment, during the load, there was a slight concentration of protein, with little significant

enzymatic activity. In the course of washing, it can be identified a slight increase in the amount

of enzyme present in the mobile phase, considering that the function of this stage is to carry out

the molecules not adsorber on to the matrix of the column and due to the high hydrophilic

character of biomolecule of interest.

During the elution phase, it was evidenced that the total protein and enzyme

concentrations did not increase proportionally to the ionic strength of the mobile phase, showing

a purification range using between 0.1 and 0.4 M NaCl presenting similar results to (Lima et

al., 2016). The enzyme recovery from the crude broth is considered significant, even with the

presence of contaminants after cell lysis.

The chromatography using as matrix an ion exchange resin is well used in protein

adsorption, in which the concept is based on the attraction between the molecules of proteins

that are charged electrostatically and the resin that presents opposite loads. One of the

advantages of this process is that it provides smooth separation conditions allowing proteins to

maintain their conformation(Braga et al., 2014; Medeiros et al, 2012).

3.3 PAGE gel

The fraction of the β-gal enzyme in the best elution range using 0.1 to 0.4 M NaCl in

the optimum purification condition with IS of 200mM and pH 7.0 were subjected to qualitative

analysis by means of a 9% polyacrylamide PAGE gel under non-denatured conditions as shown

in Figure 5.

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Figure 5: Native PAGE gel of polyacrylamide 9%. Lane M – Maker protein (MM), Lane 1–

crude extract (EB), Lane 2 – elution sample of 0,1 to 0,4 M of NaCl.

The wide-band marker protein used contained molecular mass ranging from 66 to 669

kDa, serving as a standard to identify the molar mass of the β-gal enzyme used in this research.

The β-gal enzyme may vary its properties according to its source. Molecular mass may range

from 850 kDa of enzyme produced by Escheriria coli to 201 kDa for those produced by

Kluyveromyces Marxianus (Gekas, V, and Lopez-Leiva, 1985). The literature reports that β-gal

molecules originating from Kluyveromyces lactis have a molecular mass varying between 120

and 140 kDa, values calculated by particle size exclusion chromatography (Boeris et al., 2012;

Carminatti, 2001). It is observed in Figure 4 an evident band with molecular weight in the range

between 60 and 120 kDa was observed, configuring the enzyme of interest.

4. Conclusion

In the present study, the enzyme adsorption capacity of the resind in the rotary incubator

rises in all the basic regions tested, and the Streamline DEAE resin presented the best selectivity

to the target molecule, with a β-gal retention capacity of 18.77 U /g ± 0.14 for pH 6.0, 5.79 U/g

± 0.79 at pH 7.0 and 13.50 U/g ± 1.14 for pH 8.0. The results of the experimental design showed

102


the passage from the lower (-1) to the higher (+1) level of Ionic strength and pH increases the

purification degree of the β-gal enzyme to 2.00 ± 0.42, but the ionic strength exerts greater

influence on the FP response. The contributions of ionic strength and pH for enzyme recovery

were -2,142 and -17,576, respectively. The fraction of the β-gal enzyme in the best elution range

using 1 M of NaCl was between 0.1 and 0.4 M in the optimum purification condition with IS

200mM and pH 7.0 was submitted to electrophoresis, where a well-evident band with molecular

weight in the range between 60 and 120 kDa was observed, configuring the enzyme of interest.

These results suggest the partially purification of a stable β-galactosidase of K. lactis with

potential industrial application from low-cost residue.

Acknowledgement

The authors thank the Federal University of Rio Grande do Norte (UFRN), especially

the laboratory of Biochemical Engineering and the Laboratory of Food Engineering. The

present work was carried out with the financial support of the Coordination for the Improvement

of Higher Education Personnel-Brazil (CAPES)-Financing Code 001.

Interest conflicts

The authors declare that there is no conflict of interest in relation to the sources of funding or

the materials used in the present study.

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ARTIGO 3- Lactose hydrolysis using β-Galactosidase from Kluyveromyces lactis

immobilized with sodium alginate for potential commercial applications

Catherine Teixeira de CARVALHO a, Wildson Bernardino de Brito LIMA a, Fábio Gonçalves

Macêdo de MEDEIROS b, Julia Maria de Medeiros DANTAS a, Carlos Eduardo de Araújo

Padilhaa , Everaldo Silvino dos SANTOS a, Gorete Ribeiro de MACÊDOa, Francisco Canindé

de SOUSA JÚNIOR* a,c


University of Rio Grande do Norte (UFRN), Natal, Brazil


do Norte (UFRN), Natal, Brazil

c Laboratory of Bromatology, Pharmaceutical Department, Health Sciences Centre, Federal

University of Rio Grande do Norte (UFRN), Natal, Brazil

Corresponding author

*Francisco Canindé de Sousa Júnior



[email protected]

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Abstract

The present study aimed to evaluate the hydrolysis conditions of lactose from "coalho" cheese

whey using β-galactosidase (β-gal) produced by Kluyveromyces lactis immobilized with

sodium alginate. Three sodium alginate-based immobilization systems were evaluated (0.5, 0.7

and 1% w/v) for maximizing the immobilization yield (Y), efficiency (EM) and recovered

activity (ar). The stability of the immobilized β-gal was investigated against different pH values,

temperatures and in the presence of metallic ions. The lactose hydrolysis capacity of the

immobilized form of β-gal was determined and simulated environments were used in order to

assess the preservation of the immobilized enzyme in the gastrointestinal tract. The results

showed that β-gal immobilization with 1% (w/v) sodium alginate presented the best results (EM

of 66%, Y of 41% and ar of 65%). The immobilization system sustained the highest pH stability

for the range between 5.0 – 7.0, and temperature stability was also favored by immobilization.

In 6 h of hydrolysis, the immobilized β-gal was able to hidrolyze 46% of the initial lactose

content. For the gastrointestinal simulations, around 40% of the activity was preserved after 2

h of exposure. Overall, the results described here are promising for the industrial applications

of β-galactosidase from K. lactis.

Key-words: cheese whey, lactose hydrolysis, Kluyveromyces lactis, lactase.

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1. Introduction

The use of cheese whey by the dairy industry is limited by its high content of lactose,

which reduces its solubility and impacts on its low sweetness capacity and digestibility, when

incorporated into food products. In addition, lactose is less fermentable when compared to other

sugars [1,2].

Lactose is a disaccharide formed by glucose and galactose, a type of carbohydrate found

in milk and dairy products. In humans, lactose intolerance occurs in 75% of the population,

being caused by the insufficiency of β-galactosidase (lactase) in the body, which compromises

the activity of this enzyme in the membrane of the mucosa edge in the small intestine of adults.

Lactase enzyme preparations is used in dairy products for reducing the lactose content and

promote a beneficial effect for lactose intolerants [3,4]

The β-galactosidase enzymes are widely found in nature, being present in animals,

plants and microorganisms. Kluyveromyces spp. yeast are capable of fermenting lactose by

producing an intracellular form of β-galactosidase. The lactase obtained from Kluyveromyces

spp. yeasts presents optimum activity in pH range around 6.0 – 7.0 [5,6]. As a drug and for the

food industry, these biomolecules are of great interest to industry due to their high activity,

selectivity and the possibility of manipulating their properties. However, the use of enzymes in

pharmaceutical grade administered orally is a challenging aspect due to the potential of

inactivation of this molecule in a hostile gastrointestinal environment [7].

Enzyme immobilization is an efficient biotechnological strategy for increasing enzyme

stability and enhancing industrial applications of such labile biomolecules, which allows cost

reduction [8]. However, a strict criteria selection is required for efficient enzyme

immobilization, in order to achieve a simple and economical immobilization strategy that

results in high enzyme activity retention and high operation stability [9,10].

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Therefore, the present study aimed to evaluate the hydrolysis conditions of the cheese

whey lactose using β-galactosidase (β-Gal) of Kluyveromyces lactis immobilized with sodium

alginate with potential for industrial application. The viability of this process would help

avoiding the inappropriate disposal of the "coalho" cheese whey, a regional industrial by-

product, through a value-adding strategy.

2. Materials and methods

2.1. Reagents and enzymes

The synthetic substrate o-nitrophenyl-β-D-galactopyranosid (ONPG) was acquired from

Sigma-Aldrich (St. Louis, MO, USA). Pepsin, pancreatin, sodium chloride, lactose, sodium

alginate, calcium chloride and all other reagents were of analytical grade.

2.2. Microorganism and inoculum production

Kluyveromyces lactis NRRL Y-8279 used in this study was provided by the collection

of cultures. The strain was maintained in a 30% (v/v) solution of glycerol at -20 °C. For pré-

inoculum preparation, K. lactis was cultivated in YEPD medium containing 10 g/L of yeast

extract, 20 g/L of dextrose, 20 g/L of peptone and 20 g/L of agar for 24 h, at 30 °C. Then, three

isolated colonies (inoculum) were transferred into 50 mL of the culture medium containing 10

g/L of lactose, 5.0 g/L of potassium phosphate, 1.2 g/L of ammonium sulfate, 1.0 g/L of yeast

extract and 0.4 g/L of magnesium sulfate, prepared in potassium phosphate buffer (0.2 M, pH

5.5) according to (Lima et al., 2013). The Lactose used in the growth medium was exclusively

from the cheese whey partially deproteinized (CWD). The inoculums were cultivated in

Erlenmeyers under agitation of 180 rpm, at 30 °C, for 16 h in a rotating incubator.

2.3. Production of enzymatic extract

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The enzyme β-galactosidase (β-Gal) was produced through submerged cultivation por

Kluyveromyces lactis NRRL Y-8279 using rotary incubators (Tecnal, model TE-241), with 250

mL Erlenmeyers flasks containing 50 mL of culture medium (20 g/L of QWD, 1.3 g/L of

(NH4)2SO4, 12 g/L of yeast extract, 5.0 g/L of KH2PO4 and 0.4 g/L MgSO4.7H2O, in 0.2 M

potassium phosphate buffer at pH 5.5, according to the adapted methodology of (Braga et al.,

2014). The fermentation was performed with 10% of inoculum (v/v) prepared as described in

section 2.1. The culture medium was previously sterilized and the lactose from the QWD was

filtered in a 0.22 μm membrane. The cultivations in rotary incubators were performed at 30 °C,

180 rpm. Samples were removed after 20 h of fermentation.

The enzymatic extract was obtained from the yeast cell disruption after the fermentation,

according to (Braga et al., 2014). The procedure was performed in centrifuge tubes of 50 mL,

containing 25 mL of the cell suspension and 27.5 g of glass pearls (diameter ranging from 0.95

to 1.05 mm) under vortex agitation for 5 min, followed by 1 minute in an ice bath. This

procedure was repeated 4 times. Then, the suspension was centrifuged (Cientec, CT-5000R

model, Brazil) at 5200 × g during 10 min at 4 ºC. The enzyme extract was collected and stored

at -20 °C.

2.4. β-galactosidase immobilization with sodium alginate

For the immobilization of β-gal, three concentrations of sodium alginate (0.5%, 0.7% and

1% w/v) were tested. The appropriate amount of sodium alginate was mixed into 10 mL of

potassium phosphate buffer 50 mM (pH 6.6) and heated under magnetic agitation until complete

solubilization. The sodium alginate solution were cooled to room temperature and 0.2 g of

freeze-dried β-gal (3 U/mL) were added and homogenized for 10 minutes under magnetic

stirring. The enzyme-sodium alginate dispersion was added drop-wise to a solution of calcium

chloride 0.25 M, with the aid of a peristaltic pump (TE-BP01 dosing system, TECNAL, Brazil),

for the formation of the hydrogel granules. The enzyme-sodium alginate granules were kept

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immersed in the calcium chloride solution for 10 minutes under constant agitation. After that,

the granules were stored for 10 minutes at 4 °C and subsequently washed with distilled water

for removal of excess calcium chloride. The granules were stored at 4 °C in distilled water until

further use, following the method adapted from [11,12].

2.4.1. Evaluation of immobilization parameters

In order to evaluate the enzyme immobilization process, the immobilization yield, the recovered

enzymatic activity and the immobilization efficiency were assessed. The immobilization yield

(Yi) was calculated from the enzymatic activity prior (a0) and after to immobilization (af),

according to Equation 1 [13,14].

Yi (%) = 𝑎0−𝑎𝑓

𝑎0 x 100 (1)

The recovered enzymatic activity (ar) is defined as the ratio between the theoretical enzymatic

activity (at) that the immobilized enzyme should present after the process, and the real

enzymatic activity (ad) of the immobilized system. This parameter evaluates if the active sites

of the enzyme immobilized form are available equations 2 and 2.1:[16,18]

𝑎𝑡 = 𝑎0 − 𝑎𝑓 (2)

𝑎𝑟(%) = 𝑎𝑑

𝑎𝑡 x 100 (2.1)

The immobilization efficiency (EI) is determined as the ratio between the enzymatic activity of

the immobilized system and the enzymatic activity of the free β-gal enzyme (al), according to

Equation 3 [13].

𝐸𝐼 (%) = 𝑎𝑑

𝑎𝑙 x 100 (3)

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The best immobilization condition was selected for the stability, lactose hydrolysis and

gastrointestinal simulation tests. All experiments were performed in triplicates.

2.5. Enzymatic stability of immobilized β-galactosidase and operational stability

The β-Gal stability was assessed at different pHs, in the presence of metal ions and at

different temperatures based on the remaining activity after exposure according to [19]. For pH

stability, the immobilized β-gal was exposed to pH 5.0 to 10.0, at 30 °C, for 30, 60 and 120

minutes. The following buffer solutions were used: sodium acetate (0.2 M, pH 5.0), sodium

phosphate (0.2 M, pH 6.0-8.0), glycine-NaOH (0.2 M, pH 9.0-10.0). For thermal stability, the

enzymatic extract was exposed to temperatures of 40 °C, 45 °C and 50 °C, at pH 6.6, for 15, 30

and 60 minutes [20].

The effect of metal ions on the β-gal activity was assessed as described by [21], with

minor modification. The immobilized β-gal was exposed to solutions containing ions Mg2+

(MgSO4·7H2O, 5 mM), Cu2+ (CuSO4 ·5H2O, 5 mM), Zn2+ (ZnSO4 ·7H2O, 5 mM), Mn2+

(MnCl2, 5 mM), Fe2+ (FeSO4, 5 mM) e Co2+ (CoCl2, 5 mM), at 25 °C, for 30, 60 and 120

minutes. After each treatment, the β-gal activity was determined and the results were expressed

as the enzymatic activity retention [22].

The enzyme β-galactosidase immobilized in alginate according to item 1.2 was further

submitted to the operational storage stability study, being stored in potassium phosphate buffer

pH 6.6 and temperature of 4 °C for a period of 62 days. Residual activity was determined by

the initial rates method according to [4]. Activity was calculated relative to initial activity [23].

2.6. Scanning Electron microscopy (SEM)

For evaluating the microstructure of the immobilization system, SEM images of immobilized

enzyme and the enzyme-free biopolymer (sodium alginate 1%) were acquired with a scanning

electron microscope (Hitachi Tabletop Microscope TM-3000, USA). Samples were freeze dried

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prior to the analysis. Images were acquired accelerating voltage of 5kV and 15kV, and the

electron micrographs were taken at 150x and 1000x magnification [16,19].

2.7. Fourier transformer infrared spectroscopy analysis (FT-IR)

For the study of polymers, the FT-IR technique allows analyzing the compounds

present in the structure and the interactions between them. Biopolymer (sodium alginate 1 %)

and free recovered enzyme were initially freeze dried for 24 h. Then, the immobilized enzyme

complex, and the biopolymer without enzyme were analyzed by Fourier transform infrared

spectroscopy (model IRTracer-100, Shimadzu, USA). Measurements were performed by ATR

(Total Attenuated Reflectance) with zinc selenide crystal. FT-IR spectra were obtained in the

range of 400 to 4000 cm-1 [24]

2.8. Analytical methods

The enzymatic activity of β-gal was determined using the synthetic substrate o-

nitrophenyl-β-D-galactopyranosid (ONPG), as described by [4]. The enzymatic activity of β-

gal was expressed in U/mL. The enzymatic activity unit (U) was defined as the enzyme

necessary to produce 1.0 μmol of o-nitrophenol per minute at the assay’s conditions (37 °C, pH

6.6). The total protein was assessed by Bradford method [25]. Bovine Serum Albumin (BSA)

was used as standard and reads were performed at 595 nm. The protein concentration was

expressed in mg/mL and used to calculate the specific activity of the enzyme β-Gal (U/mg).

2.9. Application of β-galactosidase enzyme

2.9.1. Hydrolysis of cheese whey lactose

Lactose from the "coalho" cheese whey (10 g/L) was used for the hydrolysis

experiments. The hydrolysis of lactose was performed in order to compare the immobilized

enzyme and crude enzyme extract. Cheese whey aliquots (1 mL) were added to test tubes with

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either 0.1 g of alginate-immobilized β-Gal or 1 mL of crude enzyme extract, at pH 6.0, 40 °C

and 100 rpm. Samples were collected in triplicate after 0, 120, 240 and 360 minutes of

hydrolysis. For inactivation of the β-Gal enzyme, the samples were subjected to heating in a

water bath at 100 °C for 5 minutes [11]. The lactose and glucose contents after hydrolysis were

evaluated by High-performance Liquid Chromatography (HPLC; model Ultimate 3000,

Thermo Fisher Scientific, USA) using a Shim-Pack column (model SCR-101H, Shimadzu,

Japan) with refraction index detector (RID), at 40 °C. Sulfuric acid 0.005 M was used as a

mobile phase, under flow of 1.0 mL/min. All samples were filtered through 0.22 µm membrane

before analysis [11,18].

2.9.2. Stability of β-galactosidase in simulated gastrointestinal conditions

The stability of the immobilized enzyme under simulated gastric and intestinal

conditions was performed using the methodology described by [26]. For simulation of gastric

conditions, 0.2 g of immobilized enzyme was incubated with 2 mL of simulated gastric fluid

(SGF, pH 2.0; 2.0 g sodium chloride, 3.2 g pepsin, 7.0 mL HCl 37%, for 1000 mL) at 37 °C,

100 rpm. For intestinal conditions, 0.2 g of immobilized enzyme was incubated with 2 mL of 2

mL of simulated intestinal fluid (SIF, pH 7.5; 6.8 g monobasic potassium phosphate, 10 g

pancreatin, 190 mL NaOH 0.2 N, for 1000 mL), at the same previous conditions. Samples were

taken after 5, 10, 30, 45, 60 and 120 minutes and enzymatic activity was evaluated [27].

2.10. Statistical analysis

All experiments were performed as three independent replicates (n = 3). Statistical

analysis was performed using the software Statistica v. 8.0 (TIBCO Statistica, Palo Alto, CA,

USA). One-way ANOVA combined with Tukey HSD post hoc test was applied to establish

statistical significance (p < 0.05).

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3. Results and discussion

The results regarding the immobilization conditions of the β-gal enzyme in relation to

the different concentrations of sodium alginate are shown in Table 1.

Table 1: Immobilization parameters of β-gal enzyme for the different concentrations of sodium

alginate

Sodium alginate

(% w/v)

Y

(%)

rar

(%)

EI

(%)

0,5 66.95 ± 1.48 a 17.40 ± 0.51 c 39.54 ± 0.02 c

0,7 17.98 ± 0.26 c 73.28 ± 0.11 a 45.71 ± 0.09 b

1,0 41.77 ± 0.23 b 65.02 ± 0.10 b 66.03 ± 0.10 a

Where Y – Yield, ar – recovered activity and EI -immobilization efficiency, respectively. Mean

values ± Standard deviation. Different letters (a, b, c) in the same column indicate statistical

differences (p < 0.05) according to Tukey’s test.

The enzyme concentration for alginate ratio was established at 2.6 U/g of granules. For

the three concentrations of sodium alginate tested, the granules obtained at 0.7% sodium

alginate (w/v) presented the highest recovered activity (73.28 ± 0.11%). On the other hand,

when the three key parameters (Y, EI, ar) are taken all together, the granules obtained at 1.0%

sodium alginate (w/v) presented better higher EM (66.03 ± 0.10%) with low reduction of Y

(41.77 ± 0.23%) and ar (65.02 ± 0.10%).

Shen et al. [28] developed a hybrid matrix of sodium alginate-gelatin-calcium phosphate

for immobilization of the β-gal enzyme of K. lactis. The authors observed a lower relative

activity of the enzyme immobilized with the hybrid matrix (58.6%) when compared to the

control matrix with only sodium alginate (62.3%). These results were attributed by the authors

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to problems of mass transfer caused by the calcium phosphate layer and gelatin in the

immobilization matrix, which indicates the correct choice of support for this research.

Freitas et al. [29] investigated the development of a biocatalysts system using β-gal of

K. lactis immobilized in sodium alginate-gelatin-cross-linking with glutaraldehyde, and

observed that the immobilized enzyme retained 80% of its initial activity after 25 cycles of use.

In addition, the hydrolysis of lactose using 10 and 100 g/L of substrate did not present inhibition

by the product for the immobilized enzyme.

In a study conducted by Souza et al. [12] using a polysaccharide complexed with sodium

alginate for immobilization of β-gal, the authors obtained 69.4% of recovered activity, very

similar to the results presented here. Souza et al. [15] observed in their study that the formation

of the alginate complex and β-gal promoted changes in the enzyme structure, however the

conformational modification was reversible after the dissociation of the complex which allowed

the enzyme to recover its activity. This fact may contribute to expanding the functional

applications of enzymatic immobilization.

3.1. Effect of pH value, temperature and metallic ions on stability of

immobilized β-galactosidase

The immobilization of β-gal in sodium alginate was performed with the objective of

increasing enzymatic activity, enzyme stability and reduction of operation costs by allowing

the recycle use [13]. Some factors such as temperature, pH, presence and concentration of metal

ions may influence the activity of the enzyme β-Gal [30]. In the present study, the stability of

the β-Gal enzyme immobilized with alginate was evaluated in different pH values, temperature

and in the presence of several metal ions. 1 shows that the complex enzyme/support showed

higher stability in the pH range between 5.0-7.0, with the highest relative activity obtained at

pH 5.0 remaining stable for 30 and 60 minutes, conditions in which the enzyme maintained an

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average of 105.50 % ± 0.25 of the relative activity. In addition, the immobilized form of the

enzyme presented higher values for relative activity when compared to the same pH range in

the free enzyme.

Albuquerque et al. [31] analyzed the stability of the enzymatic activity of β-

galactosidase immobilized with agarose at pH 4.5, 7.0 and 9.0. In the immobilized enzyme the

pH 9.0 was less stable when compared to the free enzyme. On the other hand, when the

immobilized form of the enzyme in solutions with pH 5.0 or 7.0 was clearly more stable than

the free enzyme.

Figure 1: Stability of the enzymatic residual activity of immobilized β-galactosidase with

sodium alginate at different pH values. Mean values ± Standard deviation. Different low case

letters (a,b,c) indicate statistical difference (p < 0.05) for different pH values at the same

incubation time according to Tukey’s test. Different capital letters (A, B, C) indicate statistical

difference (p < 0.05) for different incubation times at the same pH according to Tukey’s test.

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It was observed that of higher pH values (8-10), there was a significant reduction of the

enzymatic activity. Depending on the nature and charges on the surface of the immobilization

matrix, and the source of the enzyme, the optimum pH is changed between free and

immobilized forms [32]. According to Vasileva et al. [33], the optimum immobilization pH for

one enzyme depends on the chosen immobilization method and the type of matrix used. The

optimum pH can be maintained or displaced to a more basic or more acidic region in relation

to the free enzyme, this change in the profile of the pH curve occurs by the unequal distribution

of charges in the microenvironment of the immobilized enzyme form. This modification may

occur by the introduction of negative charges, such as amine groups, promoting displacement

to the acidic pH range, or carboxyl groups that promote this displacement to the basic range.

When it comes to the effect of temperature on the stability of the immobilized enzyme,

it was observed that the highest stability was obtained at 50 ºC for 30 minutes, with a relative

activity of 180.0 ± 1.37% (Figure 2). According to Sousa Junior et al. [34] there is a strong

dependence on the stability of the immobilized enzyme with the temperature. For temperatures

of 55, 57 and 60 ºC, the authors reported the results similar to this work. Therefore, it is clear

that the immobilization strategy also favors the increase of enzyme stability against

temperature.

According to Souza et al. [18] the denaturation of the immobilized enzyme is less

observed because of the protective effect credited to the increased structural stiffness of the

enzyme, thus promoting the protection of amino acids in the active site, as well as on the

surface.

In a study produced by Freitas et al. [35] on the immobilization of the β-gal enzyme of

Aspergillus oryzae in sodium alginate-reticulated gelatin with glutaraldehyde, it was observed

that the optimal enzymatic activity of the immobilized enzyme increased from 55 ºC to 60 ºC,

and the immobilized form also presented higher tolerance to high temperatures. In addition, in

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studies conducted by El-Masry et al. [36], the authors observed that the change of stability to

higher temperatures indicates that the immobilization process provides a higher resistance

against thermal inactivation, preserving the structure of the enzyme when compared with the

free form, which was also observed in this study.


sodium alginate at different temperatures. Mean values ± Standard deviation. Different low case

letters (a, b, c) indicate statistical difference (p < 0.05) for different temperatures at the same

incubation time according to Tukey’s test. Different capital letters (A,B,C) indicate statistical

difference (p < 0.05) for different incubation times at the same temperature according to

Tukey’s test.

As for the effect of metallic ions on the stability of the immobilized β-Gal, Figure 3

shows that during the incubation time of 60 minutes the enzyme was stable for most of the ions

tested. A pronounced reduction was observed only when the enzyme/support complex was in

contact with the Co2+ and Cu2+ ions with a relative activity of 59.82 ± 0.93% and 52.29 ±

0.13%.In contrast, in the presence of Ag+, Mg2+ and Fe2+ there was a significant increase in the

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relative activity in the immobilized enzyme exceeding the percentages between 144.75 ±

1.53%, 154.59 ± 0.60% and 158.90 ± 1.25% in 2 hours of incubation.


sodium alginate at different metallic ions. Mean values ± Standard deviation. Different low case

letters indicate statistical difference (p < 0.05) for different metallic ions at the same incubation

time according to Tukey’s test. Different capital letters indicate statistical difference (p < 0.05)

for different incubation times at the same metallic ion according to Tukey’s test.

The activation of the enzyme by metallic ions can be explained by measuring samples using

identical parameters, favorable conformational changes are observed in the enzymatic structure

that leads to stabilization, with a marked increase in activity [37]. The metallic ion forms a

complex with the structure of the protein, which must have a second binding site to Mg2+, which

is important of the catalytic point. Although this process is not yet fully understood, it is

assumed that the ion participates in the enzymatic catalysis, acting as a Lewis acid [38].

Khan et al. [37] reported the action of the Zn2+, Ca2+, Mn2+ and Mg2+ metallic ions

commonly found in milk on the enzymatic activity of the β-Gal of Aspergillus oryzae

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immobilized with nanocomposites of chitosan, polyaniline and silver. A multiple increase in

the catalytic activity was observed in the presence of the ion cocktail, although fluorescence

and FT-IR studies showed significant conformational alterations in the secondary structure of

the enzyme linked to the silver nanocomposite, chitosan and polyaniline, when compared with

the free enzyme. This improved catalytic efficiency of the immobilized enzyme in the presence

of metallic ions can promote its economical use in milk lactose hydrolysis. These results

converge with those achieved by this research that presented a high catalysis performance in its

immobilized form when in the presence of the same metallic ions with a highlight for the metal

ion Ag+.

Albuquerque et al. [20] analyzed the production of lactulose by lactose hydrolysis using

the β-Gal immobilized in a chitosan and glyceraldehyde complex, and reported that the

production of this prebiotic increased with the presence of some metallic ions such as the Mn2+,

Zn2+ and Ca2+, which enhanced lactose hydrolysis to 88.4%, 85% and 84%, respectively.

Regarding the influence of storage time on the activity of immobilized -gal enzyme,

the results show that the sample analyzed after 62 days its activity dropped to 34 ± 0.33%,

indicating conditions oscillations over longer storage periods

3.2. Scanning Electron microscopy (SEM)

The morphological structure of the immobilization support and immobilized enzyme

was observed by scanning electron microscopy (SEM). Figure 5 shows th micrographs at 150x

and 1000x magnifications of the support and immobilized enzyme respectively. The

immobilized enzyme is rougher compared to the micrograph of the immobilization support at

150x magnification. According to Souza et al. [18], a three-dimensional network may have been

formed after the complexation between −gal and alginate, suggesting that the enzyme plays a

cross-linking role along the polymer chain, as can be evidenced in the 1000x magnified

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micrographs. However further studies are needed to clarify to clarify the internal structure of

this complex.

Figure 4: SEM of immobilization support (A, B) and immobilized -gal with (C, D) at pH 6,6

and fixed alginate concentration of 1% with 150 x and 1000 x magnification respectively

3.3. Fourier transformer infrared spectroscopy analysis (FT-IR)

The identification of functional groups of sodium alginate, immobilization support, free

−gal, and immobilized −gal were performed by FT-IR analysis between 400 and 4000 cm-1

(Figure 4). The peaks between 3200 and 3300 cm-1 in the spectra represent the elongation

vibration of the O─H bonds on the immobilization support (calcium alginate), free lactase, and

the immobilized −gal/alginate complex. Absorption ranges around 1385 and 1560 cm-1 were

identified as representing the vibrations of COO- and C─O groups, respectively [39]. The

characteristics observed in the −gal spectra were identified in the peaks recorded at 1627 cm -

1 for group amide II, while the peaks between 1057 and 983 cm-1 represent the vibration of the

C─N elongation bands present in the enzyme. The absorption range around 2893 cm-1 is

attributed to the asymmetrical and symmetrical elongation of CH2 [40,41]

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Figure 5: FT-IR spectra of sodium alginate, immobilization support, free −gal and

Immobilized −gal, respectively

The vibrational changes can be observed when comparing the spectra of immobilization

support, free enzyme and −gal/alginate complex. In the immobilization support, there is no

region of evident vibrations in the range between 800 to 1110 cm-1. In the free enzyme, a

displacement of the C─N peak in 1057 cm-1 to 1103 cm-1 in the −gal/alginate complex is

evidenced. In the −gal/alginate complex, there was a decrease in the transmittance intensity of

the bands relative to the C─N groups. This fact can be explained by the composition of the

complex formed, indicating changes in the structure of the immobilized enzyme [37].

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3.4. Application of β-galactosidase

3.4.1. Cheese whey lactose hydrolysis

Figure 6 shows the results related to the conversion of lactose from the "coalho" cheese

whey. The experiments were conducted comparing the hydrolysis efficiency of immobilized β-

gal enzyme and crude enzyme extract during 6 hours of incubation, with analysis of lactose

degradation every 2 hours. It was observed that the immobilized enzyme presented a lower

percentage of lactose hydrolysis (46.92 ± 0.65%) when compared to the crude enzyme extract

(53.08 ± 0.65%). However, when the higher enzymatic stability and enzymatic activation of the

immobilized form are taken into account, the lower hydrolysis rate does not jeopardize the

immobilization strategy.

Figure 6: Comparative lactose hydrolysis conversion (%) between the immobilized system (○)

and the crude enzymatic extract (●). Mean values for three repetitions (n = 3).

Fischer et al. [30] investigated the hydrolysis of lactose using β-gal of Aspergillus

oryzae immobilized with ion exchange resin Dualite A568 with glutaraldehyde cross-linking.

It was found that the average conversion rate reached 65%, using 50g/L of lactose.

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Mörschbächer et al. [13] investigated the effect of pre-treating β-galactosidase

Lactozym® 3000l with concanavalin A in the immobilization of the enzyme in a calcium

alginate-gelatin matrix, and the effect of the addition of glutaraldehyde in the preparation of the

immobilized enzyme granules. The enzyme without pre-treatment with concanalin A and

immobilized in calcium alginate with glutaraldehyde presented 12.36% conversion of lactose.

Immobilized β-galactosidase without pre-treatment and without addition of glutaraldehyde

presented 54% conversion of lactose from whey after 360 minutes of reaction at 37 ºC.

Immobilization in a hybrid support favors the conversion of lactose, improving the efficiency

of the enzyme, similar results were found in the present study.

3.4.2. Enzyme stability in simulated gastrointestinal conditions

Enzymes are being widely used as therapeutic agents. By oral administration in clinical

use or as part in the treatment of specific pathologies such as celiac disease and phenylketonuria

[43]. As a medicine, these biomolecules are of great interest to industry due to their high

activity, selectivity and the possibility of manipulating their properties. However, the use of

enzymes for oral administration is a challenging aspect due to the potential of inactivation of

this molecule in the hostile gastrointestinal environment [7]. For a possible application of

immobilized β-gal as a digestive supplement for lactose intolerant individuals, a simulation test

was performed in gastric and intestinal conditions.

For the treatment to be bioavailable after oral administration, the enzyme needs to keep

its activity after the acidic and enzymatic conditions of the stomach, and reaches the intestine

with its preserved catalytic activity. Therefore, the stability of the immobilized β-gal in

125


simulated gastric fluid (SFG) and simulated intestinal fluid (SFI) was tested for 2 hours as

recommended by [44].

Figure 7: Gastroinstestinal stability of immobilized β-gal on simulated gastrical fluid (SFG,

pH 2.0; ●) and simulated intestinal fluid (SFI, pH 7.5; ○).

The enzymatic activity is represented with a percentage relative activity expressed by

the control that was 0,35 U/mL. It is observed that the enzyme reached a residual activity level

of 48.42 ± 1.40% within 2 hours when submitted to SFG. When submitted to SFI the residual

activity decreased to 36.18 ± 1.45% at the same time. This study managed to partially preserve

the catalytic activity of the enzyme up to the intestine during the 2 hours of incubation, which

is a promising result for possible industrial application.

Enzymes are particularly sensitive because proteolysis can lead to their inactivation. For

this reason, the current efforts to overcome this deficiency involve the application of

gastroresistent delivery systems and the modification of enzymatic structures by conjugation of

polymers, bringing recent progress in the administration of enzymes treatment, whose substrate

is located in the gastrointestinal tract [43].

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4. Conclusion

The present study investigated the performance of the immobilization process of β-

galactosidase using a sodium alginate matriz and described that the use of 1% (w/v) sodium

alginate presented the best combined results for immobilization efficiency and recovered

activity.

This study showed that the use of the whey residue in the cheese process fermentation

is promising for the synthesis of β-gal with potential application to industrial scale. It is also

emphasized the importance of a step reduction purification technique, contributing to a

significant degree of purity of the biomolecule of interest. In addition, the immobilization

support provided greater stability to the enzyme, enabling its reuse and improving product

separation with application in various industrial processes. Finally, the use of this regional

industrial by-product allows to add value and leverage the development of dairy industries,

bringing low cost alternative to the lactose intolerant public.

Acknowledgements

The authors would like to thank the Federal University of Rio Grande do Norte

(UFRN), specially the Laboratory of Biochemical Engineering and the Laboratory of Food

Engineering. The present work was carried out with support of the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Brazil) - Funding Code 001.

Conflict of interests

The authors declare no conflict of interest regarding the funding sources or the materials

used in the presented study.

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Capítulo 5

Considerações Finais

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CAPÍTULO 5 – CONSIDERAÇÕES FINAIS

O estudo sobre o aproveitamento do queijo de coalho para produção e aplicação da β-

galactosidase com potencial uso industrial foi realizado em três etapas. Na primeira etapa da

pesquisa observou-se o comportamento de duas leveduras para síntese de β-gal e etanol por

fermentação submersa, utilizando diferentes razões C:N, obtendo-se melhor eficiência com

Kluyveromyces lactis NRRL Y- 8279 em ambas as razões C:N testadas, alcançando 21,09

U/mL de β-gal e 7,10 g/L de etanol em 16 horas de cultivo.

Diante dos resultados iniciais no qual o processo fermentativo aplicado favoreceu a rota

metabólica enzimática, na segunda etapa avaliou-se as condições de purificação da β-gal em

cromatografia em leito fixo utilizando um planejamento experimental 22. Os parâmetros pH e

força iônica foram avaliados para obter um alto fator de purificação, sem prejuízo no

rendimento. Os níveis mais altos de ambos os parâmetros no estudo aumentaram o fator de

purificação de β-gal para 2,00, com maior influência da força iônica na resposta do fator de

purificação. A recuperação da enzima β-gal foi maior faixa de eluição entre 0,1 a 0,4 M de

NaCl com FI 20 0mM e pH 7,0, e em seguida, confirmada por eletroforese.

Na terceira e última etapa foram avaliados três sistemas de imobilização à base de

alginato de sódio (0,5, 0,7 e 1% p/v) para maximizar o rendimento, a eficiência e a atividade

recuperada da imobilização. A imobilização de β-gal com alginato de sódio a 1% (p/v)

apresentou os melhores resultados nas condições testadas (66% de eficiência de imobilização,

41% de rendimento de imobilização e 65% de atividade recuperada). O sistema de imobilização

promoveu a maior estabilidade da enzima na faixa de pH entre 5,0 - 7,0, com a maior atividade

relativa para pH 5,0. A estabilidade da enzima a temperatura também foi favorecida pela

imobilização e a atividade enzimática relativa da β-galactosidase atingiu 180% a 50°C após

30 minutos de exposição. Após 2 h de contato com os íons Ag+, Mg2+ e Fe2+, a atividade

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enzimática residual atingiu 144%, 154% e 158%, respectivamente. Após 6 horas de hidrólise,

a β-galactosidase imobilizada foi capaz de converter 46% do conteúdo inicial de lactose em

glicose e galactose. Para as simulações gastrointestinais, cerca de 40% da atividade enzimática

foi preservada após 2 horas de exposição a ambientes gastrointestinais simulados. Assim,

conseguiu-se manter a atividade catalítica da enzima parcialmente preservada até o intestino.

O referido estudo mostrou que a utilização do resíduo do soro do queijo no processo

fermentativo é promissor para síntese da β-gal com potencial aplicação para escala industrial.

Ressalta-se, ainda, a importância de uma técnica de purificação com redução de etapas,

contribuindo para um grau de pureza significativo da biomolécula de interesse. Ademais, o

suporte de imobilização proporcionou maior estabilidade a enzima, possibilitando sua

reutilização e melhorando a separação de produtos com aplicação em vários processos

industriais. Por fim, o uso desse subproduto industrial regional permite agregar valor e

alavancar o desenvolvimento das indústrias de laticínios, trazendo alternativa de baixo custo

para o público com intolerância à lactose.

Para pesquisas futuras sugere-se otimizar a produção em maior escala utilizando a

batelada alimentada a fim de minimizar os efeitos de inibição pelo produto e substrato no

processo fermentativo, avaliar outras temperaturas para o cultivo de Kluyveromyces lactis,

investigar outros métodos de imobilização utilizando nanopartículas, aplicar a enzima

imobilizada na síntese de galactooligossacarídeos (GOS) prebióticos e avaliar a eficiência dos

GOS sintetizados in vitro, utilizando cultura de células.

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