Ultrasound-assisted enzymatic hydrolysis of goat milk casein: Effects on hydrolysis kinetics and on the solubility and antioxidant activity of hydrolysates

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Food Research International 157 (2022) 111310

Available online 29 April 2022

Ultrasound-assisted enzymatic hydrolysis of goat milk casein: Effects on

hydrolysis kinetics and on the solubility and antioxidant activity

of hydrolysates

Isabela Soares Magalh˜

aes

, Al´

ecia Daila Barros Guimar˜

aes

, Alline Artigiani Lima Tribst

Eduardo Basílio de Oliveira

, Bruno Ricardo de Castro Leite Júnior

Department of Food Technology, Federal University of Viçosa, Av. Peter Henry Rolfs, S/n, University Campus, 36570-900 Viçosa, MG, Brazil

Center for Food Studies and Research (NEPA). University of Campinas (UNICAMP), Albert Einstein, 291, 13083-852 Campinas, SP, Brazil

ARTICLE INFO

Keywords:

Biological properties

Goat milk protein

Hydrolysis degree

Peptides

Proteases

Protein solubility

Techno-functional properties

Ultrasounds

ABSTRACT

This study evaluated the ability of ultrasound (US) bath to improve the hydrolysis of goat milk casein (GMC) by

three commercial proteases (Alcalase, Brauzyn and Flavourzyme) using assisted reactions at 60 ◦C for up to 300

min. Process performance was evaluated based on the rate reaction, nal hydrolysis degree, solubility, and

antioxidant activity of the hydrolysates. For all enzymes, the US-assisted reaction increased the rate of GMC

hydrolysis (up to 120%), the hydrolysis degree (23–48%), and the small peptides formed, i.e., those soluble in

trichloroacetic acid (TCA) (up to 40%). Consequently, US-assisted GMC hydrolysis improved the solubility of the

hydrolyzed product (up to a 35.7% increase after 300 min of reaction) and, compared to conventional hydrolysis,

reduced the time to achieve the maximum solubility by up to 10 times. Regarding the in vitro antioxidant activity,

especially for Alcalase, the technology promoted a higher scavenging capacity of 2,2′-azino-bis(3-ethyl-

benzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (p <0.05), thus 10-fold

accelerating the production of antioxidant peptides, according to ABTS assays (p <0.05). In conclusion, the

US-assisted enzymatic reaction is a promising technology to improve the hydrolysis rate and yield of the process

of obtaining hydrolysates from GMC. Moreover, these hydrolysates showed high solubility and good in vitro

antioxidant activity, which demonstrates the potential to be used as food ingredient with nutritional and techno-

functional appeal.

1. Introduction

Goat milk has a high nutritional value due to its composition.

Worldwide, its production has grown in recent years, and there is an

expectation to increase by approximately 53% until 2030 (Pulina et al.,

2018; Silva & Favarin, 2020).

The main proteins in goat milk are divided between caseins and

whey proteins, with caseins representing 60 to 80% of the total protein

concentration (Park, 2017). In addition to the nutritional and techno-

functional properties of caseins in dairy products, these proteins have

the potential to be used in different applications, including the pro-

duction of peptides with improved biological and techno-functional

properties, such as antioxidant, antihypercholesterolemic, and antihy-

pertensive activity (Li et al., 2013; Ibrahim, Ahmed, & Miyata, 2017;

Kalyan et al., 2018), and solubility and emulsifying capacity (Luo, Pan,

& Zhong, 2014). However, to exert these properties, native protein must

be hydrolyzed to release the peptides (Ulug, Jahandideh, & Wu, 2021).

Currently, peptides are mainly obtained by enzymatic hydrolysis,

which is a method that has high specicity and does not use organic

solvents. However, this process has disadvantages, such as the high cost

of enzymes, long hydrolysis time, and low yield. Thus, there is a research

eld focused on nding alternatives to reduce these limitations (Ulug

et al., 2021).

Recently, new technologies, such as high-pressure processing (HPP)

(Leite Júnior et al., 2018; Leite Júnior et al., 2019; Carullo, Barbosa-

C´

anovas, & Ferrari, 2021), high-pressure homogenization (HPH) (Leite

Júnior, Tribst, & Cristianini, 2015; Carullo, Donsì, & Ferrari, 2020),

pulsed electric elds (PEF) (Giteru, Oey, & Ali, 2018), high-intensity

pulsed light (HILP) (Siddique et al., 2016), microwaves (MW) (Dong,

Wang, & Raghavan, 2021) and ultrasound (US) (Soares et al., 2019;

* Corresponding author at: Avenue Peter Henry Rolfs, s/n, University Campus, Viçosa, MG 36570-900, Brazil.

E-mail addresses: isabela.magalhaes@ufv.br (I.S. Magalh˜

aes), bruno.leitejr@ufv.br (B.R.C. Leite Júnior).

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

https://doi.org/10.1016/j.foodres.2022.111310

Received 11 February 2022; Received in revised form 8 April 2022; Accepted 26 April 2022

Food Research International 157 (2022) 111310

Soares et al., 2020), have been studied to enhance enzymatic perfor-

mance. Strategically, these technologies can be applied alone in en-

zymes or substrates and, for some processes, through assisted reactions,

aiming to improve biocatalysis (Ulug et al., 2021). However, some of

these technologies have limitations for industrial application, such as

high cost and scalability limitations.

Among these technologies, the US is considered an economically

viable technology, and advances in its efciency and versatility have

expanded its applications in the food industry (Kadam et al., 2015). The

effect of US on proteins, including casein, has already been studied

(mainly using probe ultrasound), and the results showed that high en-

ergy conditions (high power and high temperature) can disrupt hydro-

phobic interactions and hydrogen bonds of protein molecules (Zhang

et al., 2018; Wu et al., 2018; Koirala, Prathumpai, & Anal, 2021),

inducing molecular unfolding or aggregation, as well as protein de-

fragmentation (Wu et al., 2018; Zhang et al., 2018) due to changes in

protein secondary and tertiary structures (Wu et al., 2018; Koirala,

Prathumpai, & Anal, 2021). These changes may lead to an increase in

surface hydrophobicity through exposure of sulfhydryl and hydrophobic

groups (Wu et al., 2018), contributing to improvements in the techno-

functional properties (Zhang et al., 2018).

However, the effect of bath US-assisted reactions on the production

of bioactive and techno-functional peptides from the hydrolysis of casein

from goat milk has not yet been explored. Compared with the use of

probe US, bath US has advantages such as lower equipment cost, greater

resistance for operating in milder conditions, and greater scalability

potential (Soares et al., 2019; Soares et al., 2020). Therefore, this work

aimed to evaluate the effect of bath ultrasound-assisted enzymatic hy-

drolysis of goat milk casein (GMC) and the impact on the solubility and

antioxidant activity of the hydrolysates obtained.

2. Material and methods

2.1. Enzymes and goat milk casein (GMC)

Three commercial proteases were used in the study: Brauzyn® 100 L

(papain) donated from Prozyn Biosolutions (S˜

ao Paulo, Brazil) and

Alcalase® (Bacillus lincheniformis) and Flavourzyme® (Bacillus oryzae)

donated from Novozymes Latino Americana Ltda (Paran´

a, Brazil).

Fresh goat milk was obtained from the goat sector of the Federal

University of Viçosa (Viçosa, MG, Brazil). Goat milk was obtained from

80 lactating animals of the Saanen breed. These animals (lot of 12 goats)

are kept in pens with wood shaving bedding. The goats have free access

to solarium, water, mineral salt, and corn silage (corn, straw, cob). Their

diet was supplemented with a mixture of corn grain, soybean meal, salt,

lime, urea, and vitamin complex supplied 3 times daily. The average

milk composition was 3.5% lactose, 4.8% protein, 4.5% fat, and 13.7%

total dry extract.

Casein extraction was carried out by isoelectric precipitation ac-

cording to the method described by Kalyan et al. (2018) with some

modications. After obtaining, the goat milk was skimmed by centri-

fugation at 2000 rpm for 20 min at 4 ◦C. The skim milk was heated to

37 ◦C, and the pH was adjusted to 4.1 (isoelectric point) under constant

stirring using HCl (1 M). After the isoelectric precipitation of the caseins,

centrifugation was carried out at 3500 rpm for 30 min to separate the

casein and whey. The obtained pellet (casein) was lyophilized to obtain

goat milk casein (GMC) with 1.6% moisture and 88.6% protein and

9.8% nonprotein dry extract from milk, mainly minerals and residues of

fat and lactose.

2.2. Evaluation of enzyme activity in GMC at different temperatures

The optimal temperature (ranging from 25 to 65 ◦C) of each enzyme

was determined according to the methodology described by Leite Jú-

nior, Tribst, & Cristianini (2014). An aliquot of 50 mL of GMC protein

solution (1% w/v) was dispersed under agitation at 100 rpm in sodium

phosphate buffer solution (pH 6.8; 0.1 M) and kept in a thermostatic

bath for 10 min to equilibrate the reaction temperature. After this, 0.5

mL of the enzyme solution (Alcalase 1% v/v, Brauzyn 2% w/v, and

Flavourzyme 3% v/v – concentrations dened based on Km values and

within the dilution range recommended by the manufacturers) diluted

in the same buffer was added, and hydrolysis was carried out for 30 min.

Then, 1 mL of the hydrolysate was removed and added to 1 mL of tri-

chloroacetic acid (TCA) (20% w/v) to stop the reaction. The solution

was centrifuged at 7500g for 15 min at 4 ◦C, and the supernatant was

measured in a UV light spectrophotometer at an absorbance of 280 nm.

The UV absorbance at 280 nm is suitable for determining peptides that

have phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp) in their

composition. The use of this technique in this study is justied by the

relatively high amount of these amino acids present in goat casein (Park,

2017). A control sample (blank) was prepared by adding TCA at time 0,

and the nal value was determined from the difference in absorbance

between the sample and the blank. An activity unit (U) was arbitrarily

dened as the amount of enzyme required to promote a 0.1 increase in

absorbance at 280 nm under the assay conditions. Enzyme activity was

calculated according to Eq. (1).

ml =ΔAbs280nm •10 •dilutionfactor

0,5•30 (1)

The temperature condition with the highest activity was established

as optimal, with 100% of the enzymatic activity. The relative enzyme

activity (REA) was calculated according to equation (2) (2).

REA(%) = activity non optimal condition

activity optimal condition •100 (2)

2.3. US-assisted enzymatic hydrolysis of GMC

The US-assisted hydrolysis of the GMC was performed in an ultra-

sonic bath (Unique, USC 2800 A model, Indaiatuba, Brazil) with tem-

perature control, nominal capacity 9.5 L, internal dimensions of 30 ×24

×15 cm, equipped with ve transducers of 25 kHz arranged below the

vat and rated power of 450 W.

Enzyme solutions (Alcalase 1% v/v, Brauzyn 2% w/v, and Fla-

vourzyme 3% v/v) and GMC protein solution (1% w/v) were used at an

enzyme-substrate ratio of 1%. The US bath was previously lled with a

volume of 6.5 L of distilled water, and a glass beaker containing the

solution was positioned at the point of maximum exposure to ultrasonic

intensity. The process was carried out at pH 6.8 with a volumetric power

of 38 W/L (previously determined by the aluminum foil method

(Vinatoru, 2015)) at the optimal enzyme temperature (60 ◦C, for all

enzymes) for 300 min. A stainless steel heat exchanger was coupled to

the ultrasonic bath and to an external water bath to control the tem-

perature of the samples. For comparison of the processes, conventional

hydrolysis was carried out in a thermostatic bath under the same con-

ditions of pH, time, and temperature of the samples hydrolyzed under

US. In addition, GMC without enzyme addition was subjected to US

processing under the same conditions of pH, time, and temperature to

determine whether US or residual activity of any endogenous/microbial

enzyme of GMC could hydrolyze the substrate under the processing

conditions. The hydrolysis was measured by the degree of hydrolysis

(DH) and the TCA soluble protein concentration.

2.3.1. Degree of hydrolysis

The degree of hydrolysis (DH) was determined by the pH-stat

method according to Adler-Nissen (1986). For this, the enzyme solu-

tions and the GMC protein solution were prepared in distilled water, and

the initial pH was adjusted to 6.8 and maintained at this value for 300

min of hydrolysis at 60 ◦C by adding NaOH (0.1 M). The DH is dened as

the percent ratio between the number of peptide bonds broken (h) and

the total number of peptide bonds in the substrate studied (h

tot

calculated according to equation (3):

I.S. Magalh˜

aes et al.

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Ultrasound-assisted enzymatic hydrolysis of goat milk casein: Effects on hydrolysis kinetics and on the solubility and antioxidant activity of hydrolysates

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