Enzymatic preparation of casein hydrolysates with high digestibility and low bitterness studied by peptidomics and random forests analysis

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Cite this: DOI: 10.1039/d3fo01222k

Received 29th March 2023,

Accepted 25th June 2023

DOI: 10.1039/d3fo01222k

rsc.li/food-function

Enzymatic preparation of casein hydrolysates with

high digestibility and low bitterness studied by

peptidomics and random forests analysis†

Yixin Hu,

a,c

Chenyang Wang,

a,c

Mingtao Huang,

Lin Zheng*

a,c

and

Mouming Zhao *

a,b,c

Enzymatic hydrolysis can not only increase the digestibility of casein, but also cause bitterness. This study

aimed to investigate the eﬀect of hydrolysis on the digestibility and bitterness of casein hydrolysates and

provided a novel strategy for the preparation of high-digestibility and low-bitterness casein hydrolysates

based on the release pattern of bitter peptides. Results showed that with the increase of the degree of

hydrolysis (DH), the digestibility and bitterness of hydrolysates increased. However, the bitterness of

casein trypsin hydrolysates rapidly increased in the low DH range (3%–8%), while the bitterness of casein

alcalase hydrolysates rapidly increased in a higher DH range (10.5%–13%), indicating the discrepancy in

the release pattern of bitter peptides. Peptidomics and random forests revealed that peptides containing

>6 residues with hydrophobic amino acids (HAAs) at the N-terminal and basic amino acids (BAAs) at the

C-terminal (HAA–BAA type) obtained from trypsin contributed more to the bitterness of casein hydroly-

sates than those containing 2–6 residues. On the other hand, peptides containing 2–6 residues with

HAAs at both N- and C-terminals (HAA–HAA type) released by alcalase contributed more to the bitterness

of casein hydrolysates than those containing >6 residues. Furthermore, a casein hydrolysate with a signiﬁ-

cantly lower bitter value containing short-chain HAA–BAA type peptides and long-chain HAA–HAA type

peptides from the combination of trypsin and alcalase was obtained. The digestibility of the resultant

hydrolysate was 79.19% (52.09% higher than casein). This work is of great signiﬁcance for the preparation

of high-digestibility and low-bitterness casein hydrolysates.

1. Introduction

Protein is an important macronutrient for humans as a struc-

tural and functional material, and the best protein source for

infants is breast milk. When breast milk is inadequate, bovine

milk infant formulas (IFs) are often oﬀered as a substitute.

Casein plays a significant role in milk as a supplier of essential

amino acids and a carrier of calcium, phosphate and mag-

nesium,

which makes it an essential component in IFs.

However, bovine casein is hard to digest by infants because of

the coagulation formed in their stomach and the weak digest-

ing capacity of their digestive tract.

2–6

The undigested protein

in the feces

would further cause gastrointestinal symptoms

such as bloating, diarrhea and colic in infants.

Furthermore,

indigestible protein curds in the distal ileum and colon may

damage gut barrier function, causing an inflammatory

response.

Therefore, the development of highly digestible

modified casein ingredients is of great interest.

A previous study proved that enzymatic hydrolysis increased

the digestibility of casein.

Pre-hydrolysis using a commercial

protease could break down the native structure of casein, making

it easier to be digested in the infant gastrointestinal tract.

However, the hydrolysis process of casein leads to the develop-

ment of bitterness,

11–15

resulting in food refusal, which limits the

application of casein hydrolysates in infant formulas. Therefore,

hydrolysis techniques for preparing casein hydrolysates with low

bitterness and high digestibility need to be explored. However,

recent studies have mainly focused on the bioactivities of casein

hydrolysates.

16–18

There is limited understanding on the eﬀect of

enzymatic hydrolysis on the digestibility and bitterness of casein

hydrolysates. Although some studies have compared the bitter-

ness values of diﬀerent casein hydrolysates,

12–15

the release

pattern of bitter peptides during casein hydrolysis by diﬀerent

enzymes was not studied.

†Electronic supplementary information (ESI) available. See DOI: https://doi.org/

10.1039/d3fo01222k

School of Food Science and Engineering, South China University of Technology,

Guangzhou 510640, China. E-mail: femmzhao@scut.edu.cn, linzheng18@163.com;

Fax: +86 20 87113914; Tel: +86 20 87113914

Chaozhou Branch of Chemistry and Chemical Engineering, Guangdong Laboratory,

Chaozhou 521000, China

Guangdong Food Green Processing and Nutrition Regulation Technologies Research

Center, Guangzhou 510640, China

This journal is © The Royal Society of Chemistry 2023 Food Funct.

Published on 12 July 2023. Downloaded by HeFei University of Technology on 7/12/2023 12:51:07 PM.

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Research proved that the bitter taste of hydrolysates was

mainly caused by bitter peptides rather than free amino

acids.

The sequence, length and spatial structure influence

the bitterness of peptides.

20–22

Diﬀerent enzymes with varied

specificities produced diﬀerent types of bitter peptides.

Therefore, it was expected that low-bitterness hydrolysates can

be produced by controlling the release of bitter peptides.

However, the release pattern of bitter peptides by diﬀerent

enzymes remains unknown. Daher et al.

established a

method to screen bitter peptides using a heat map and

random forests. The random forests model is a tree-based

approach, which was used to model the bitterness descriptor

as a function of a number of predictors (presence, absence or

normalised abundance of peptides) and quantify the contri-

bution of each predictor in the study above. Therefore, we

expected to reveal the dominant bitter peptides and their

release patterns in casein hydrolysates using peptidomics and

random forests, in order to develop a novel strategy to prepare

casein hydrolysates with low bitterness and high digestibility.

In this study, trypsin, alcalase, papain, neutrase, and fla-

vourzyme were used to prepare casein hydrolysates at the DH

of 3%, 5.5%, 8%, 10.5%, and 13%. The digestibility character-

istics in an in vitro infant digest model and the bitterness of

each hydrolysate were evaluated. PCA was used to reveal the

eﬀect of enzymatic hydrolysis on the digestibility and bitter-

ness of casein hydrolysates. Furthermore, peptides in each

hydrolysate were characterized by UPLC-MS/MS and random

forests were used to screen peptides which contribute to the

bitterness. The peptidomics analysis was carried out to reveal

the mechanism of diﬀerent bitterness increase behaviors

during hydrolysis in hydrolysates obtained using diﬀerent

enzymes. Finally, casein hydrolysates with low bitterness and

high digestibility were prepared.

2. Materials and methods

2.1 Materials

Casein from bovine milk was purchased from Fonterra Co-

operative Group (Richmond, Victoria, Australia). Pepsin from

porcine gastric mucosa (P7012) was purchased from Sigma-

Aldrich (St Louis, MO, USA). Trypsin was provided by Pangbo

Biological Engineering Co. Ltd (Guangxi, China). Alcalase, neu-

trase and flavourzyme were provided by Novozymes

Biotechnology Co., Ltd (Tianjin, China). Papain was provided

by Solarbio Science & Technology Co. Ltd (Beijing, China).

Methanol (MS grade), acetonitrile (MS grade) and formic acid

(HPLC grade) for UPLC-MS were purchased from Sigma-

Aldrich (St Louis, MO, USA). Other chemicals used were of

analytical grade.

2.2 Enzymatic hydrolysis of casein

Trypsin, alcalase, papain, neutrase, and flavourzyme were used

to hydrolyze casein. The hydrolysis was carried out according

to Yu et al.

with some modifications. Briefly, casein was dis-

persed in distilled water by the ratio of 1 : 10 (w/w). The mix-

tures were adjusted to the optimum pH of enzymes before

enzyme addition and placed in a THZ-82A shaking water bath

(Jintan Ronghua Instrument Manufacture Co., Ltd, Jiangsu,

China) at the optimum temperature of enzymes for 4 h. The

optimal conditions for each enzyme used were as follows:

trypsin, 37 °C, pH 7.5; alcalase, 55 °C, pH 8.0; papain, 55 °C,

pH 7.0; neutrase, 50 °C, pH 7.0; and flavourzyme, 50 °C, pH

7.0. The enzyme/substrate ratio was adjusted to obtain hydroly-

sates with DH of 3%, 5.5%, 8%, 10.5%, and 13%, respectively.

To terminate the hydrolysis, the mixture was heated in boiling

water for 15 min. Subsequently, the mixtures were centrifuged

for 15 min at 8000gand 4 °C. Then, the supernatants were col-

lected, freeze-dried, and stored at −20 °C until use.

2.3 Degree of hydrolysis (DH)

The DH of casein hydrolysates was measured by an o-phthalal-

dehyde (OPA) assay according to Wang et al.

with modifi-

cations. Briefly, 180 μL of the OPA reagent was mixed with

24 μL of samples. After 2 min of incubation, the absorbance

was measured using a ReadMax 1900 microplate reader

(Shanpu Biotechnology Co., Ltd, Shanghai, China) at 340 nm.

Distilled water or serine solution (0.97 mM) was used as the

control or standard, respectively.

2.4 SDS-PAGE analysis of hydrolysates

Hydrolysate-lyophilized powders were dissolved in reducing

loading buﬀer at 2 mg mL

−1

and heated in boiling water for

5 min. After centrifugation at 10 000gfor 5 min, 10 μL of the

supernatant was loaded for electrophoretic analysis. The separ-

ation gel and concentration gel were 12% (w/w) and 5% (w/w),

respectively. Electrophoretic separations were carried out at 80

V for about 2 h. The gels were stained with 0.25% Coomassie

blue R-250 solution for about 1 h and then decolored with a

decoloring solution (10% glacial acetic acid, 10% methanol

and 80% distilled water) until the band was clear.

2.5 Precipitation ratio and soluble protein content of

hydrolysates at pH 4.5

Hydrolysate-lyophilized powders were diluted in sodium

acetate buﬀer (0.2 M, pH 4.5) at a concentration of 2% (w/v).

The mixture was centrifuged for 15 min at 10 000gand the

soluble protein content in the supernatant was determined

using a BCA Protein Assay kit (Dingguo Biotechnology Co. Ltd,

Beijing, China). Meanwhile, the precipitate was collected,

washed with buﬀer and then centrifuged for 15 min at

10 000g, repeated twice and then weighed after being freeze-

dried. The precipitation ratio was calculated as follows:

Precipitation ratio ð%Þ¼Wprecipitate=Wtotal 100;ð1Þ

where W

precipitate

is the weight of the precipitate and W

total

the weight of the hydrolysate-lyophilized powder.

2.6 In vitro simulated gastric digestion

The in vitro simulated gastric digestion was carried out accord-

ing to the INFOGEST model

with some modifications. The

Paper Food & Function

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