Mitigation of malondialdehyde-induced protein lipoxidation by epicatechin in whey protein isolate

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Food Chemistry 456 (2024) 139954

Available online 3 June 2024

Mitigation of malondialdehyde-induced protein lipoxidation by epicatechin

in whey protein isolate

Wenhua Yao

, Xingya Hao

, Zhangjie Hu

, Zhenghao Lian

, Yue Cao

, Rong Liu

Xiaoying Niu

, Jun Xu

, Qin Zhu

Key Laboratory for Quality and Safety of Agricultural Products of Hangzhou City, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou

311121, China

Jiaxing Key Laboratory for Research and Application of Green and Low-carbon Advanced Materials, School of Advanced Materials & Engineering, Jiaxing Nanhu

University, 572 South Yuexiu Road, Jiaxing 314001, China

ARTICLE INFO

Keywords:

Malondialdehyde

Lipoxidation

Epicatechin

Whey protein isolate

ABSTRACT

Malondialdehyde (MDA) can induce lipoxidation in whey protein isolate (WPI). The physicochemical changes in

this reaction with or without the presence of a phenolic compound epicatechin (EC) were characterized in this

study. Results suggested the content of MDA was signicantly reduced during co-incubation of MDA and EC. The

addition of EC dose-dependently alleviated MDA-induced protein carbonylation, Schiff base formation and loss

of tryptophan uorescence. The interruption of MDA-binding to WPI was directly visualized by immunoblotting

analysis. Observation of the surface microstructure of WPI showed that MDA-induced protein aggregation was

partially restored by EC. Meanwhile, EC was found to promote loss of both protein sulfhydryls and surface

hydrophobicity due to possible phenol-protein interactions. These observations suggested the potential of EC in

the relief of MDA-mediated protein lipoxidation.

1. Introduction

Malondialdehyde (MDA, OCH-CH

-CHO, MW 72; pKa 4.45), is the

most intensively studied secondary products produced in both foodstuff

and living organisms as a consequence of lipid peroxidation. Being one

of the most abundant aldehydes produced in oxidative degradation of

unsaturated fatty acids (both

-3 and

-6) with three and more double

bonds, MDA is widely accepted as a typical marker of oxidative stress

(Esterbauer, 1993; Zhou et al., 2020). MDA can be found in almost all

lipid-containing foods, especially in high-fat foods such as vegetable

oils, animal oils, meat products, and fried foods, with a content ranging

from 0.1 to 10 mg/kg (Zhou et al., 2020). However, only free MDA is

estimated in this well-accepted lipid oxidation marker, without

considering the existence of bound forms of MDA (Vandemoortele,

Babat, Yakubu, & De Meulenaer, 2020). The formation of bound MDA is

ascribed to its high electrophilic reactivity towards biological nucleo-

philes (proteins, nucleic acids, and phospholipids, especially amino

groups), with adverse impacts on the structure and function of bio-

molecules (Esterbauer, 1993). Nucleophilic protein degradation

mediated by MDA was implicated in decreasing the bioavailability and

nutritional value of dietary proteins (Zamora, Navarro, Aguilar, & Hi-

dalgo, 2015). Furthermore, MDA-modied proteins were also suggested

to be involved in the pathogenesis of some human diseases such as

atherosclerosis, diabetes, Alzheimer's disease, and cancer, as well as

aging (Kanner, 2007; Lankin, Tikhaze, & Melkumyants, 2022). Although

there are no ofcially published detailed MDA limits, the European Food

Safety Authority (EFSA) Scientic Committee recommended a 30

g/kg

bw/day threshold of toxicological concern (TTC) for MDA (Zhou et al.,

2020).

Upon formation, MDA can diffuse and react with adjacent proteins.

Such post-translational non-enzymatic modication of proteins by

reactive or electrophilic lipid species is termed as protein lipoxidation.

The process involves the formation of Schiff's bases or Micheal adducts

by reactions between lipid oxidation-derived carbonyls (aldehydes or

ketones) and primary amines (Zhou et al., 2020). MDA covalently binds

to protein

-amino and thiol groups with modication on protein side

chains, leading to protein cross-linking, misfolding, aggregation, and

loss of enzymatic activity (Huang, Sarkhel, Roy, & Mohan, 2023).

* Corresponding authors.

E-mail addresses: xujun@jxnhu.edu.cn (J. Xu), zhuqin@hznu.edu.cn (Q. Zhu).

The authors contribute equally to this paper.

Contents lists available at ScienceDirect

Food Chemistry

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

https://doi.org/10.1016/j.foodchem.2024.139954

Received 19 December 2023; Received in revised form 12 May 2024; Accepted 1 June 2024

Food Chemistry 456 (2024) 139954

Various intervention strategies were developed to delay and interrupt

the lipoxidation process. Antioxidant phenolic additives have been

demonstrated to behave as a triple barrier (Zamora & Hidalgo, 2016),

whereas the roles of chelator, free radical scavenger and lipid-derived

carbonyl scavenger, are all responsible for minimizing the conse-

quence of lipoxidation. The reaction mechanisms of phenolics towards

lipid peroxidation-derived carbonyls including

, β-unsaturated alde-

hyde (4-hydroxy-trans-2-nonenal and acrolein), di-aldehyde (glyoxal)

have been elucidated with identication of aldehyde-phenol adducts in

chemical and food models. However, the interaction mechanism be-

tween phenolic compounds and MDA has not yet been described clearly

due to the complexity of reaction pathways. Until recently, the main

adducts formed in the reaction between the phenolic compounds (2,5-

dimethylresorcinol, orcinol, olivetol, and alkylresorcinol) and MDA

were produced via the reaction of one molecule of the phenol, one

molecule of MDA, and additional one molecule of acetaldehyde (which

is absent in other carbonyl-phenol reaction) (Zamora, Alcon, & Hidalgo,

2023).

The recognition of phenols as MDA-trapping agents justies their

application in retarding MDA-induced lipoxidation. Theoretically, fast

scavenging of MDA by phenol contributes to protection on proteins by

inactivation or removal of MDA. Nevertheless, in a complicated food

system with co-existence of protein, MDA, and phenol, multiple re-

actions happen among these compounds. The A ring of phenol, with OH

groups at meta-positions, is involved in trapping of MDA through

nucleophilic addition. The B ring of phenols, with OH groups at the

ortho-positions, is often engaged in antioxidant activities donating H-

atoms to free radicals, leading to the formation of corresponding qui-

nones with further reactivity towards nucleophilic groups on proteins

(Farooq, Abdullah, Zhang, & Weiss, 2021). Hence, the variations in

protein physicochemical properties introduced by phenolic MDA-

trapping agents need to be evaluated to avoid potential adverse effects

on protein quality.

Epicatechin (EC), as a major ingredient in popular tea beverages, was

selected as the potential MDA-trapping agent in this study. The position

OH groups in A ring of EC which meets the structural requirement (meta-

diphenols) in trapping of MDA. However, the ortho-positions OH groups

in B ring may implicate possible phenol-protein interaction which could

cause protein cross-linking and aggregation. Therefore, the effects of EC

in a typical dietary protein (whey protein isolate, WPI) under exposure

to MDA were investigated. The efciency of EC in eliminating MDA was

measured by HPLC and the resultant mitigation on MDA-induced lip-

oxidation was visualized by Western blot analysis. The physiochemical

changes were revealed by monitoring protein carbonylation, sulfhydryl

content, surface hydrophobicity as well as uorescence proles of

intrinsic tryptophan and Schiff base adducts. The inuence of EC on

protein surface microstructure and protein hydrolysis were also evalu-

ated. The ndings of the present study may provide a theoretical basis

for the development of phenolic MDA-trapping agents in controlling

lipid oxidation product-mediated protein damage in food system.

2. Materials and methods

2.1. Material

WPI (>92% protein in dry basis) was supplied by Hilmar Ingredients

(Hilmar, CA, USA). 1,1,3,3-tetramethoxypropane, the enzymes used for

simulated gastrointestinal digestion, including pepsin (≥250 units/mg,

solid), trypsin (8 ×USP specications), were the products of Sigma-

Aldrich Co., LLC (United States). The rabbit polyclonal specically

binds to MDA-modied proteins (Ab27642) and the goat anti-rabbit IgG

H&L (HRP) secondary antibody were purchased from Abcam (Cam-

bridge, UK). 1,1,3,3-tetramethoxypropane,1-anilino-8-naphthalenesul-

fonate, trichloroacetic acid, acetonitrile, 5,5

′

-dithiobis (2-

nitrobenzoicacid), 2,4-dinitrophenylhydrazine (DNPH), guanidine hy-

drochloride, Brilliant blue R250, and other reagents were all purchased

from Sinopharm Chemical Reagent Co., Ltd. (China) or Tokyo Chemical

Industry Co., Ltd. (Japan). All chemicals used in the study were of

analytical grade and acetonitrile was of HPLC grade.

2.2. Preparation of MDA

For each experiment, MDA was freshly prepared by acidic hydrolysis

of 1,1,3,3-tetramethoxypropane (TMP) according to the procedure

described in a previous study (Kikugawa, Tsukuda, & Kurechi, 1980).

Briey, acid hydrolysis was carried out by the incubation of 845

L TMP

and 1 mL HCl (1 M) in 3.16 mL ultrapure water for 2.5 min at 40 ◦C. 6 M

NaOH was used to adjust the pH to 7.4. The obtained MDA solution was

diluted 200 times with 10 mM phosphate buffer (pH 7.4) and the MDA

concentration was measured at the UV absorbance of 267 nm and

calculated with a molar extinction coefcient value of 31,500 mol

−1

2.3. Retained MDA content after the incubation with EC

To understand the interactions between MDA and EC, MDA (1 mM)

was incubated with EC (0.1, 1, and 2 mM) in 10 mM PBS (pH 7.4) at

25 ◦C for 24 h. The residual MDA was monitored by the Agilent 1260

Innity II HPLC system with a separation module (G7111B), an auto-

sampler (G7129E), and a multiwavelength UV detector (G7115A). Re-

action mixtures were chromatographically separated by a YMC Pro C18

column (250 ×4.6 mm, 5

m). The ow rate was 1 mL/min, and the

column temperature was kept at 25 ◦C. The mobile phase was composed

of deionized (DI) water with 0.1% formic acid (solvent A) and aceto-

nitrile (solvent B). Gradient elution (ow rate: 1 mL/min) was carried

out for 98% solvent A in 0 to 8 min, 80% solvent A in 8 to 20 min, and

98% solvent A in 20 to 30 min. MDA was monitored the at the wave-

length of 245 nm according to a previous study of Cai et al. (2011).

2.4. MDA-induced protein lipoxidation

MDA-induced protein lipoxidation reaction system was established

according to a previous report by Zheng et al. (2022). In 10 mM phos-

phate buffer (pH 7.4), MDA (1 mM) was co-incubated with WPI (6 mg/

mL) and EC (0, 0.1, 1, and 2 mM). The reaction was carried out at 25 ◦C

for 24 h in the darkness with the addition of 0.02% sodium azide as

germicide.

2.5. Protein carbonylation

Protein carbonyl content in WPI was measured according to the

procedure of Levine (Levine, Wehr, Williams, Stadtman, & Shacter,

2000). Briey, 0.2 mL DNPH derivatization reagent (0.1% w/v dissolved

in 2 M HCl) was added to 0.1 mL of the reaction mixture. The deriva-

tization process was last for 1 h at room temperature. Carbonylated

proteins were then precipitated by 20% TCA solution (w/v) and washed

with ethanol/ethyl acetate (1:1, v/v) at least 3 times. Finally, the ob-

tained protein precipitate was dissolved in guanidine hydrochloride (8

M). The UV absorption was monitored at 370 nm and the protein

carbonyl content (nmol carbonyl/protein) was calculated as follows:

Protein carbonyl content (nmol carbonyl/protein) = A370

×106

where A

370

is the UV absorbance at 370 nm, C is the MP concentration,

and

is the absorption coefcient of 22,000 mol

−1

2.6. Protein sulfhydryls

The total sulfhydryl content of WPI was measured using Ellman's

method (Ellman, 1959). Briey, urea-SDS solution was prepared by

dissolving 8.0 M urea and 0.03 g/mL SDS in 0.1 M phosphate buffer (pH

W. Yao et al.

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