Characterization of ultrasound-assisted covalent binding interaction between β-lactoglobulin and dicaffeoylquinic acid: Great potential for the curcumin delivery

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Food Chemistry 441 (2024) 138400

Available online 9 January 2024

Characterization of ultrasound-assisted covalent binding interaction

between β-lactoglobulin and dicaffeoylquinic acid: Great potential for the

curcumin delivery

Gongshuai Song

, Fang Li

, Xiaotong Shi

, Jiayuan Liu

, Yong Cheng

, Yuhan Wu

Zexu Fang

, Yuxiao Zhu

, Danli Wang

, Tinglan Yuan

, Ruikang Cai

, Ling Li

Jinyan Gong

Zhejiang Provincial Key Lab for Biological and Chemical Processing Technologies of Farm Product, School of Biological and Chemical Engineering, Zhejiang University of

Science and Technology, Hangzhou 310023, Zhejiang, China

Zhejiang Skyherb Biotechnology Inc., Huzhou 313300, Zhejiang, China

ARTICLE INFO

Keywords:

β-lactoglobulin

Dicaffeoylquinic acids

Ultrasound

Covalent binding

Curcumin

ABSTRACT

The low bioavailability and poor gastrointestinal instability of curcumin hampers its application in pharma-

ceutical and food industries. Thus, it is essential to explore efcient carrier (e.g. a combination of polyphenols

and proteins) for food systems. In this study, covalent β-lactoglobulin (LG)-dicaffeoylquinic acids (DCQAs)

complexes were prepared by combining ultrasound and free radical induction methods. Covalent interactions

between LG and DCQAs were conrmed by analyzing reactive groups. Variations in secondary or tertiary

structure and potential binding sites of covalent complexes were explored using Fourier transform infrared

spectroscopy and circular dichroism. Results showed that the β-sheet content decreased and the unordered

content increased signicantly (P <0.05). The embedding rate of curcumin in prepared LG-DCQAs complexes

using ultrasound could reach 49 % −62 %, proving that complexes could embed curcumin effectively. This study

highlights the benet of ultrasound application in fabrication of protein–polyphenol complexes for delivering

curcumin.

1. Introduction

In recent years, bioactive compounds have drawn widespread

attention as promising functional ingredients for food development and

formulation. These bioactive compounds are benecial to humans, as

they reduce the risk of cardiovascular disease, diabetes, cancer, and

obesity (Zolqadri et al., 2023; Malekjani & Jafari, 2021; Li, Wei, & Xue,

2021). Curcumin is the main pigment in turmeric, and it possesses

numerous medicinal functions and in vivo pharmacological activities

(Yang et al., 2023). Curcumin has been approved by the US Food and

Drug Administration as a preservative and colorant. However, the low

bioavailability (low blood plasma content), poor gastrointestinal insta-

bility (rapidly metabolized into sulfate or glucuronide conjugates), and

poor solubility (11 ng/mL, pH 5) of curcumin hamper its application in

pharmaceutical and food industries (Meng et al., 2020). The combina-

tion of polyphenols with proteins as a carrier can effectively improve the

solubility and stability of polyphenols, and the presence of polyphenols

effectively improves the antioxidant property of proteins (Poojary et al.,

2023). Non-covalent and covalent interactions are the main pathways

for protein–polyphenol interactions. Liu et al. (2021) demonstrated that

covalent complexes exhibit superior polyphenol protection, oxidation

resistance, and thermal stability compared with non-covalent complexes

(Liu et al., 2021). Notably, the cross-linking site and structure of pro-

tein–polyphenol complexes can be affected by the type, structure, and

weight of proteins and polyphenols.

Chlorogenic acids (CQAs) are a class of natural non-avonoids

formed by the condensation of quinic acid with a variable number of

caffeic acids by esterication reactions. They are widely distributed in

nature (e.g., in medicinal and edible plants) (Wang et al., 2023).

Dicaffeoylquinic acids (DCQAs), mainly including 3,4-DCQA, 3,5-

DCQA, and 4,5-DCQA, are a hot topic in functional food research as they

have various biological activities, such as antioxidant, free radical

* Corresponding authors.

E-mail addresses: liling1113@zust.edu.cn (L. Li), jygong@zust.edu.cn (J. Gong).

Contents lists available at ScienceDirect

Food Chemistry

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

https://doi.org/10.1016/j.foodchem.2024.138400

Received 5 September 2023; Received in revised form 30 December 2023; Accepted 6 January 2024

Food Chemistry 441 (2024) 138400

scavenging, and antiviral activities (Meinhart et al., 2019; Moglia et al.,

2014). β-lactoglobulin (LG) is the key component of whey protein,

which is a by-product of the cheese production process. It is widely

available and relatively inexpensive (Pham et al., 2019; Liu et al.,

2023a). Xu et al. (2019) found that the IgE-binding capacity of LG was

reduced signicantly after the interaction of LG with three common

polyphenols (mono-CQA, delphinidin-3-O-glucoside, and theaavin)

because IgE linear epitopes were obscured by polyphenol-binding sites,

which played an important role in the structure and potential function of

LG. Covalent binding of polyphenols and proteins can be conrmed by

determining contents of sulfhydryl groups and free amino groups and by

sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

(Xu et al., 2019).

Ultrasound, a non-isothermal technology, has been used in many

food processing elds. The high mechanical energy and shear stress

generated by high-intensity ultrasound (ca. 20–100 kHz) can induce

microstreaming currents and cavitation bubbles. Modications to

physicochemical properties of food components have been applied to

improve the efciency of different food processing methods (e.g.,

ltration, homogenization/emulsication, freezing/thawing, and

extraction) (Wang et al., 2023; Baba, Abdelrahman, & Maqsood, 2021;

Zhang et al., 2021). In our previous study, we found that the physico-

chemical property of LG-CQA complexes prepared under ultrasound and

non-covalent bonding conditions were superior to those of complexes

prepared without ultrasound treatment (Liu et al., 2023b). Character-

ization of the conjugation of LG and polyphenols is complicated. How-

ever, few studies have reported the ultrasound treatment of cross-linking

sites and the structure of the conjugate prepared using the free radical

method.

The aim of this study was to explore the potential effect of covalent

interactions between LG and three DCQAs (3,4-DCQA, 3,5-DCQA, and

4,5-DCQA) using the free radical method. The fabricated LG-DCQA

complexes were selected as carriers for delivering curcumin. The

bioavailability of LG-DCQA-curcumin complexes was further investi-

gated. The obtained results contribute to a better understanding of the

application of various protein–polyphenol covalent complexes as

transport carriers in the functional food eld.

2. Materials and methods

2.1. Materials and reagents

LG (purity ≥95 %, from milk) was purchased from Macklin

(Shanghai, China); 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA (purity ≥99 %,

HPLC) were provided by Munster (Chengdu, China); dialysis bags with

cut-off MW of 3500 Da were bought from Vake (Beijing, China); cur-

cumin with analytical grade was purchased from Beijing Wokai

Biotechnology Co, Ltd. (Beijing, China); Other reagents with analytical

grade were purchased from Lingfeng (Shanghai, China). Ultra-pure

deionized water was ltered by Millipore Milli-Q system (Millipore,

Bedford, MA, USA).

2.2. Preparation of LG-DCQAs complex

According to the method of Liu et al. (2023c) with some slight

modications, protein–polyphenol covalent complexes were prepared

by combining ultrasound (270 W, 2 h) and free radical induction (Liu

et al., 2023c). All reactions were performed at room temperature. The

samples were obtained by frozen in a refrigerator (-80

◦

C) for 24 h and

dried under vacuum for 48 h. For control, LG samples were prepared by

the same procedure.

2.3. Embedding curcumin

Referring to the method of Liu et al. (2023c) with some slight

modications, the sample was re-dissolved with deionized water to 10

mg/mL, mixed with curcumin ethanol solution according to the mass

ratio of 1:7.5, stirred for 1 h in a magnetic stirrer, centrifuged at 7104 ×

g for 10 min, and the supernatant was freeze-dried under vacuum at

−40

◦

C for 48 h to obtain LG-DCQAs-curcumin complexes (Liu et al.,

2023c).

2.4. Determination of encapsulation efciency

The absorbance value of the prepared supernatant was measured at

426 nm by UV spectrophotometer (Nicolet 5700, Thermo Electron Co.,

Waltham, MA, USA) according to the reported method with some slight

modications (Liu et al., 2023c). In this study, the encapsulation ef-

ciency of the sample was calculated according to the Equation (1):

Encapsulationefficiency =Concentrationofcurcumininsupernatant

Totalcurcurcuminconcentration (1)

2.5. Determination of complex turbidity

According to the previous research, LG-DCQAs/LG-DCQAs-curcumin

complexes systems were diluted 500 times with deionized water, and the

absorbance value was measured at 500 nm by UV spectrophotometer

(Nicolet 5700, Thermo Electron Co., Waltham, MA, USA) (Sahu et al.,

2008). The turbidity of systems was calculated according to the Equa-

tion (2):

T=2.303 ×A500 ×V

l(2)

where T is the turbidity, A

500

is the absorbance value at 500 nm, V is the

sample dilution, and l is the cuvette diameter (cm).

2.6. Binding capacity of the complex

2.6.1. Detection the binding equivalents of DCQAs

The binding equivalent of DCQAs was determined by the Folin

phenol method based on the previous method with some slight modi-

cations (Fan et al., 2018). The LG sample was prepared as the control

solution for absorbance measurement. Results are expressed as nmol/

mg.

2.6.2. Detection the content of free amino group

For analyzing the integration degree of LG-DCQAs complexes, the

content measurement of free amino acids in samples was performed

using the o-phthalaldehyde (OPA) method (Liu et al., 2015). The

absorbance value at 340 nm was determined immediately. The standard

curve was made with glycine. The OPA aqueous solution was prepared

as the control solution for absorbance measurement. Results are

expressed as nmol/mg.

2.6.3. Detection the content of free tryptophan

According to our previous method, the content of free tryptophan

was measured (Liu et al., 2023c). The content of free tryptophan was

calculated by the Equation (3):

C=0.61905A360 −0.2619A430 (3)

where C is the content of free tryptophan; A: Absorbance value. Results

are expressed as nmol/mg.

2.6.4. Detection the content of free sulfhydryl group

The content of free sulfhydryl groups in samples was assayed based

on the method of Wu et al. (2018) with some slight modications (Wu

et al., 2018). The content of free sulfhydryl groups was calculated by the

Equation (4):

Cfreesulfhydryl =73.53A412/Csample (4)

G. Song et al.

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Characterization of ultrasound-assisted covalent binding interaction between β-lactoglobulin and dicaffeoylquinic acid: Great potential for the curcumin delivery

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