Effect of ultrasound and alkali-heat treatment on the thermal gel properties and catechin encapsulation capacity of ovalbumin

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Food Hydrocolloids 145 (2023) 109069

Available online 14 July 2023

Effect of ultrasound and alkali-heat treatment on the thermal gel properties

and catechin encapsulation capacity of ovalbumin

Xiaohan Zhang, Zhao Huang, Dong An, Huajiang Zhang

, Jing Wang

, Ning Xia, Yanqiu Ma,

Siyao Han, Afeng Wei

College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang, 150030, PR China

ARTICLE INFO

Keywords:

Ovalbumin

Polyphenol

Antioxidant

Molten globule

Molecular docking

ABSTRACT

Ovalbumin (OVA) is widely used to prepare nanocomposites for the delivery of bioactive compounds. During

long-term storage of shell eggs, natural OVA is slowly transformed into a more heat-stable form called S-OVA,

which can also be obtained articially through alkali-heat treatment. This study aimed to reveal the effects of

alkali-heat and ultrasonic treatment on the structure, stability and polyphenol encapsulation ability of OVA by

analyzing the interaction mechanism through molecular docking and multispectral analysis. The results showed

that the proteins treated with alkali-heat and ultrasound had higher stability and surface hydrophobicity,

reecting the change in their functional properties. The S-OVA generated by alkali-heat treatment had higher

thermal stability and formed softer gels than those generated by ultrasound treatment. The results of infrared

spectroscopy and molecular docking indicated that hydrophobic interactions and hydrogen bonds were the main

forces stabilizing the formation of catechin-OVA complexes. X-ray diffraction showed that OVA was a good

carrier for catechins, which existed in an amorphous state in the hydrophobic core of proteins. Both ultrasound

and alkali-heat treatments exposed hydrophobic groups in the protein and signicantly enhanced the ability of

the protein to bind polyphenols (increased from 15.72 nmol/mg to 44.10 nmol/mg and 43.18 nmol/mg,

respectively), resulting in greater antioxidant capacity. This study mainly revealed the binding mode of OVA

with polyphenols and explained the reasons for the changes in the functional properties of OVA by revealing the

structural changes in proteins under different treatments, providing a basis for the advantages of alkali-heat and

ultrasonic treatment of OVA in the food industry.

1. Introduction

Eggs are the most common source of protein in people’s daily diet,

and their nutritional value is higher than that of other edible proteins.

Ovalbumin (OVA) is the most abundant protein in egg whites, ac-

counting for more than half of the egg white composition by weight

(approximately 54–58%) (Liu et al., 2018; Rostamabadi et al., 2023). As

a typical high-quality globulin in egg white protein, OVA has charac-

teristics such as water solubility, easy digestion, self-assembly, and

amphiphilicity. Because its hydrophobic amino acid content is more

than 50%, OVA is usually used in the food industry to produce strong

gels and as an emulsier or foaming agent (Chen et al., 2018; Rosta-

mabadi et al., 2023). Similarly, OVA can interact with food ingredients,

such as polyphenols, through electrostatic attraction, hydrophobic in-

teractions, and hydrogen bonding (Niu et al., 2018), thus providing a

biocompatible environment for engineering biopolymer carriers. Due to

the low price and easy availability of OVA, it has been widely used to

prepare nanoparticles for delivering bioactive compounds such as cur-

cumin (Liu et al., 2018), resveratrol (Y. Y. Xu et al., 2021), and tannic

acid (Tao, Shi, Cao, & Cai, 2022) to solve problems such as poor water

solubility of embedded substances, low bioavailability, and chemical

instability (Jin, Zeng, Geng, & Ma, 2020).

Catechin (CT), one of the main components of tea polyphenols, is a

hydrophilic polyphenolic substance (Han et al., 2023). Due to its

excellent antioxidant properties and free radical scavenging activity, it

can eliminate free radicals, reduce inammation, and prevent cardio-

vascular diseases. Therefore, CT is often used as a food additive and

dietary supplement (Al-Shabib et al., 2020). However, CT has poor

stability, with susceptibility to air humidity, temperature, oxygen, pH,

and high chemical reactivity, which greatly limits its bioavailability (Dai

* Corresponding author.

** Corresponding author.

E-mail addresses: hjthzhang@163.com (H. Zhang), wangjing_cau@163.com (J. Wang).

Contents lists available at ScienceDirect

Food Hydrocolloids

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

https://doi.org/10.1016/j.foodhyd.2023.109069

Received 28 April 2023; Received in revised form 14 June 2023; Accepted 10 July 2023

Food Hydrocolloids 145 (2023) 109069

et al., 2023). Therefore, many researchers have intensively investigated

methods to increase the bioavailability of CT and curcumin, including

solid nanoparticles, microemulsions, liposomes, and encapsulation

technology (H. Wang, Gao, et al., 2022). To date, studies have shown

that using proteins to embed polyphenols can not only improve the

functional properties of the proteins but also increase the stability and

bioavailability of the polyphenols (Han et al., 2023). Dai et al. (2023)

conducted in vitro simulated digestion experiments on soy protein-CT

covalent complexes and found that the complexes signicantly

improved the bioaccessibility of CT, enabling better development of

bioactivity and improvement of thermal and storage stability. Feng, Cai,

Wang, Li, and Liu (2018) synthesized and studied OVA-CT conjugates by

free radical copolymerization and found that they had better storage

stability and oxidation stability in emulsion delivery systems. Although

complexes of catechins with proteins have been prepared, there are still

many physicochemical factors that may promote or inhibit their

formation.

During the long-term storage of shelled eggs, whether at room tem-

perature or low temperature, natural OVA will slowly and spontane-

ously transform into S-OVA with higher thermal stability (Deleu et al.,

2015; Rostamabadi et al., 2023). S-OVA can also be prepared in vitro by

using separated OVA under alkaline conditions and at higher tempera-

tures. Studies have demonstrated that S-OVA exhibits minor alterations

in conformational conguration, while retaining the principal compo-

sitional and structural characteristics (Shitamori & Nakamura, 1983;

Takahashi et al., 2005). Researchers have also used the term “molten

globule” to describe the structure of S-OVA (Y. T. Xu, Yang, Liu, & Tang,

2020). Hu et al. (2023) promoted the formation of dense network

structures in thermally induced protein hydrogels through succinylation

combined with changes in the pH to cause substantial OVA unfolding

into a molten globule conformation. This conrmation could trap water

more tightly in network structures and effectively improve the water

retention capacity of OVA. Moreover, studies have shown that soy

protein isolates formed the same molten globule conformation, exposing

more hydrophobic groups, while some polyphenols such as curcumin

bound to proteins through hydrophobic interactions, making it easier for

these hydrophobic polyphenols to be embedded and achieve better ef-

fects (H. Li, Zhang, Zhao, et al., 2022). Therefore, we hypothesize that

S-OVA may also have higher thermal stability and a more stable struc-

ture to encapsulate and protect polyphenols and can thus be used to

embed CT to better improve the stability and bioavailability of CT and

expand its practical application range. Interestingly, we believe OVA is

more suitable as a carrier for polyphenols than other proteins since it

spontaneously forms a fused globule conformation. The preparation of

protein-polyphenol complexes using S-OVA is cost effective and reduces

the environmental footprint problems associated with manual

processing.

In recent years, high-intensity ultrasound technology has also been

widely used to change the molecular characteristics of food proteins to

obtain better functional properties (Xiong et al., 2016). As described by

Sun, Zhang, et al. (2022), ultrasonic treatment can induce protein

unfolding by cavitation effects to expose more buried hydrophobic re-

gions to the water phase, thereby increasing protein surface hydrophi-

licity. Similarly, ultrasonic waves destroy protein aggregates and

enhance protein-water interactions, which can also increase protein

solubility. The study by J. Sun, Zhang, et al. (2022) also showed that

ultrasonic waves promoted the interaction between hydroxyl radicals

and polyphenols, signicantly improving their antioxidant activity and

emulsication properties and further improving the in vitro digestion

efciency of ovalbumin. It is expected that sonication of proteins may

disrupt the aggregation of S-OVA, which may be more conducive to the

formation of more homogeneous and stable polyphenol complexes of

proteins. However, to the best of our knowledge, there are few reports

on the effects of alkali-heat in combination with sonication on protein

properties.

Therefore, in addition to using the alkali-heat method to prepare S-

OVA in this experiment, ultrasonic treatment was introduced to study

the combined effects of these two treatments on the structural and

functional properties of OVA, especially its performance in the presence

of polyphenols. This information provides a new strategy to provide

stable and highly encapsulated protein-polyphenol complexes, facili-

tating the widespread use of OVA in nanoscale bio-delivery systems.

2. Material and methods

2.1. Materials

Catechins (≥98%) were purchased from Yuanye Biotechnology Co.,

Ltd. (Shanghai, China). Ovalbumin (≥90%), 2,2-diphenyl-1-picrylhy-

drazyl (DPPH), and 2,2

′

-azinobis (3-ethylbenzothiazoline-6-sulfonic

acid) (ABTS) were provided by Sigma‒Aldrich (St. Louis, MO, USA). All

other chemicals and solvents used were of analytical grade. Deionized

water was used throughout the investigation.

2.2. Preparation of samples

OVA was rst treated by alkali-heat and/or ultrasound, followed by

CT encapsulation to produce the complexes. S-OVA was prepared ac-

cording to the method reported by Y. T. Xu et al. (2020) with slight

modications as follows. OVA was magnetically stirred in deionized

water at a concentration of 30 mg/mL for 2 h and hydrated overnight at

4 ◦C. The solution was subsequently stirred at pH 10 for 24 h at a stirring

temperature of 55 ◦C and then immediately brought to room tempera-

ture in an ice bath. Then, the solution was dialyzed for 48 h after using 4

M HCl to adjust the pH back to neutral and lyophilized, and the S-OVA

powder was used for the assay.

OVA/S-OVA-catechin complexes were prepared according to the

method of Dai et al. (2022) with slight modications. The OVA/S-OVA

powder was magnetically stirred in deionized water at a concentration

of 10 mg/mL for 2 h. Equal amounts of catechin were added to the

protein solution to reach a catechin concentration of 0.5 mg/mL, and

then the complexes were stirred for 2 h at 20 ◦C in an oxygen-free

environment to allow noncovalent binding of catechin. Subsequently,

the complexes were processed at 20 kHz under an ultrasound processor

(SCIENTZ-IID, Lichen Technology Co., Ltd, Shanghai, China) at a power

ratio of 40% for 20 min (pulse duration of 5 s for on-time and 2 s for

off-time) (Xiong et al., 2016). The samples were then dialyzed for 24 h to

remove the free catechins that were not bound to proteins and lyophi-

lized. Protein solutions without catechin addition were used as the

control group (SC and OC). The nomenclature, treatment methods, and

detailed composition of the samples are shown in Table 1.

2.3. Particle properties of proteins

The particle size distribution and ζ-potential were measured ac-

cording to D. Li et al. (2020). A Zetasizer Nano ZS90 (Malvern In-

struments Ltd, Worcestershire, UK) was used to determine the

hydrodynamic diameter distribution and ζ-potential of protein solutions

and nanocomplexes. Each sample was diluted 20 times with deionized

water adjusted to different pH values (12.0, 8.0, 7.0, 6.0, and 3.0). The

Table 1

Abbreviation, treatment methods, and composition of the samples.

Sample abbreviation Alkali-heat treatment Ultrasonic treatment Catechin

OVA – – –

S-OVA ✓ – –

O/U – ✓ –

S/U ✓ ✓ –

O/CT – – ✓

S/CT ✓ – ✓

O/U/CT – ✓ ✓

S/U/CT ✓ ✓ ✓

X. Zhang et al.

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