Disulfide bond cleavage combined with critical pH induced unfolding and assembly of soy protein and its encapsulation effect on curcumin

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Food Hydrocolloids 157 (2024) 110358

Available online 26 June 2024

Disulde bond cleavage combined with critical pH induced unfolding and

assembly of soy protein and its encapsulation effect on curcumin

Yuying Wang

, Siyu Sun

, Jing Shen

, Bowen Zou

, Ling Zhang

Xianbing Xu

, Chao Wu

College of Food Science, Dalian Polytechnic University, Dalian, 116034, China

State Key Laboratory of Marine Food Processing and Safety Control, China

National Engineering Research Center of Seafood, China

Ningjin Market Supervision Administration, Dezhou, 253400, China

ARTICLE INFO

Keywords:

Soy protein

Unfolding

Curcumin

ABSTRACT

The limited water solubility and bioactivity of hydrophobic phytochemicals can be improved and preserved by

encapsulation technologies. In this research, a low-cost and low-energy encapsulation method was explored

based on the self-assembly characteristics of soy protein (SP). The disulde bond of SP was broken by Na

and then the pH of SP was adjusted to the critical pH 10.5 with NaOH, which was the critical point of protein

unfolding. The treated protein was added to curcumin (Cur), which was encapsulated in self-assembled protein

nanoparticles during the subsequent neutralization process to obtain the soy protein-curcumin complex (SP-Cur).

This process was known as the disulde bond breaking combined with critical pH-driven technology. The

cleavage of SP disulde bonds was studied by the methods of 4, 4

′

-dithiopyridine (DPS) and sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When the SP:Cur mass ratio was 1:1, the encapsulation

efciency (EE%) of Cur in SP-Cur obtained by disulde bond breaking combined with the critical pH-driven

method increased to 85.36 ±1.03%. It was notably greater than the EE% of Cur in SP-Cur prepared by the

pH-driven method (61.82 ±1.54%) and the EE% of Cur in SP-Cur prepared by disulde bond breaking treatment

(66.38 ±1.2%). The encapsulation of Cur in SP particles exhibited signicant resistance to light and tempera-

ture, leading to enhanced stability. Soy protein treated with disulde bond cleavage combined with critical pH-

driven method can serve as a potential functional carrier to further incorporate hydrophobic compounds into

food systems.

1. Introduction

Hydrophobic polyphenols and avonoid compounds present in

edible plants, fruits, and vegetables have been shown to display a wide

range of biological activities, including anti-inammatory, antioxidant,

antiproliferative, anti-hypertensive, anti-brotic, anti-bacterial, anti-

coagulative and anti-tumor properties (Mukhopadhyay & Prajapati,

2015; Hazra, Mandal, Mandal, Bhuniya, & Ghosh, 2015). Nevertheless,

despite their extensive pharmacological properties, hydrophobic poly-

phenols and avonoids face challenges in clinical applications due to

their low water solubility, poor absorption and instability in the physi-

ological media (Altunbas, Lee, Rajasekaran, Schneider, & Pochan, 2011;

Caballero, Li, McClements, & Davidov-Pardo, 2022). A variety of

nanotechnology-mediated strategies are currently being tried to get

around these restrictions using micelles, polymeric material, liposomes,

hydrogels, biodegradable microspheres and solid-lipid nanoparticles,

whereby the hydrophobic polyphenols and avonoids can be stabilized,

made more bioavailable, and adsorbed by being encapsulated, wrapped,

or adsorbed in the polymer matrix (Kumari, 2017; Maan, Bajpai, &

Bajpai, 2016; Putra, Siswanta, & Suratman, 2016). Therefore, the

designing of non-toxic, stable and biocompatible state-of-the-art

encapsulating media for the hydrophobic polyphenols and avonoids

based on naturally occurring materials is a highly valued study objective

for the scholars working in this eld (Das et al., 2017).

The pH-driven encapsulation is a reported effective encapsulation

method, where protein is unfolded at either pH 12 or 2 and reassembled

to form new nanostructures when pH is readjusted to 7 (Pan, Luo, Gan,

Baek, & Zhong, 2014). If the protein is introduced with the hydrophobic

* Corresponding author. National Engineering Research Center of Seafood, China.

E-mail address: wuchao@dlpu.edu.cn (C. Wu).

Contents lists available at ScienceDirect

Food Hydrocolloids

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

https://doi.org/10.1016/j.foodhyd.2024.110358

Received 7 March 2024; Received in revised form 6 June 2024; Accepted 25 June 2024

Food Hydrocolloids 157 (2024) 110358

bioactive substance at either pH 12 or 2 (when the protein is unfolded),

the hydrophobic bioactive substance will co-assemble with the protein

to produce colloidal particles. Li et al. (Li et al., 2022) prepared a

nanocomposite by adding egg white-derived peptides (EWDP) into

protein-polysaccharide complexes, triggering the self-assembly of the

EWDP for packaging curcumin by a pH-driven method. According to this

study, EWDP could interact with protein-polysaccharide composites,

exhibiting excellent colloidal qualities, good aqueous solubility, dis-

persibility, and physical stability. Disulde bonds play a key role in

protein stability, and their cleavage may promote or inhibit intermo-

lecular interactions (Chang & Li, 2002; Maeda, Ado, Takeda, & Tani-

guchi, 2007; Silvers et al., 2012). Studies have shown that the cleavage

of disulde bonds can reveal additional hydrophobic sites, thereby

further promoting protein self-assembly via hydrophobic interactions

(Ewbank & Creighton, 1993; Li, Xie, Liu, Xu, & Zhang, 2021). Hassanin

et al. (Hassanin & Elzoghby, 2020) also proposed novel strategies to

induce the non-covalent self-assembly of proteins to produce

drug-carrying proteins by utilizing hydrophobic interactions or manip-

ulating disulde bonds. Although protein nanocarriers have biocom-

patibility and the ability to encapsulate hydrophobic active ingredients,

traditional preparation techniques for protein nanoparticles often

incorporate a large number of toxic solvents, crosslinking agents, and

polar acid/base reagents, which may lead to signicant toxicity or

damage the stability of active ingredients (Hassanin et al., 2020).

Ghasemi et al. (Ghasemi & Abbasi, 2014) claried the effect of the

combined use of alkaline pH and ultrasound on the release of protein

hydrophobic regions, so as to embed polyunsaturated fatty acids in

natural casein micelles. The outcomes demonstrated that the encapsu-

lation efciency of casein micelles obtained by the combined treatment

was higher than that by the single treatment, and the obtained nano-

capsules showed good oxidation stability to UV light. Fang et al. (Fang

et al., 2021) also studied the structural changes of soy protein isolate and

its impact on the creation of sodium alginate and resveratrol complexes

by combining ultrasound and pH-shifting. Additionally, following the

combined ultrasonication/pH-shifting treatment, the encapsulation ef-

ciency of resveratrol in soy protein isolate increased to 91.4 ±4.3%,

demonstrating that the simultaneous treatment was clearly superior to

ultrasound alone, which was thought to be an effective protein modi-

cation method. The ndings above suggested that a combination of

treatments could more effectively modify the structure and functionality

of protein, and combined technology achieved higher encapsulation

efciency and application stability than the single technology.

This study used edible reducing agents (such as potassium/sodium

metabisulte) combined with a pH-driven (critical pH) method to

encapsulate hydrophobic bioactive substances. The critical pH referred

to the critical point of protein unfolding. In a system where the pH was

above the critical pH, the protein unfolded, and when the pH was below

the critical pH, the protein tended to refold. The selection of critical pH

was based on previous research (Wang et al., 2024). Sodium meta-

bisulte (Na

) is extensively used in the food business as a food

additive (preservative, bleach, loosening agent). Curcumin (Cur) was

investigated as a model for encapsulation in soy protein (SP) nano-

particles by rst breaking the disulde bond to dissociate the protein,

then by increasing the pH to further unfold the protein structure, at

which time Cur was added, and nally the protein solution was

neutralized to complete the encapsulation. Cur is a representative hy-

drophobic polyphenol with various physiological effects, but it has

certain drawbacks such as poor stability and water solubility (Cuomo

et al., 2022). SP was selected as the encapsulation wall material due to

its exceptional features such as biodegradability and amphiphilicity

(Mariotti, 2019). The two primary protein components of SP are 11S

(globulin) and 7S (β-conglycinin). The hexameric structure of 11S is

formed by disulde connections between basic and acidic subunits in

each monomer (Wang et al., 2022; Wu et al., 2021). Furthermore, due to

its excellent stability and safety prole, SP is given preference over other

animal proteins and polymers when used as nanocarriers (Wang et al.,

2022). The aim was to achieve effective protein unfolding and improve

the encapsulation efciency, and further elucidate the mechanism of

protein encapsulating to Cur based on combined treatment. The use of

disulde bond breaking combined with critical pH-driven treatment has

not been previously reported.

2. Materials and methods

2.1. Materials

Low temperature defatted soy meal was purchased from Shandong

Yuwang Industrial Co., Ltd. The 98% pure curcumin was bought from

Sangon Biotech Co., Ltd. All other chemicals are analytical grade.

2.2. SP preparation

The defatted soy meal was ground into ne powder to prepare pro-

tein. The protein was prepared with the alkaline solution acid precipi-

tation method according to the previous research with slight

modications (Wang et al., 2021). Soy powder was combined with the

deionized water at a ratio of 1:10 (w/v) for 2 h (Maintaining pH 7.5).

The obtained mixture was centrifuged at 4 ◦C for 30 min at 15800×g

using a centrifuge (CF16RXII high-speed refrigerated centrifuge, Hita-

chi, Japan). Subsequently, the slurry was centrifuged at 10000×g for 20

min after the pH of the supernatant was adjusted to 4.5 using 1 M HCl.

The precipitate was gathered, redissolved and adjusted to pH 7 with 1 M

NaOH, then lyophilized and stored in plastic tubes at −20 ◦C before use.

The protein content of the resultant powder was determined using the

Kjeldahl approach, suggesting a protein concentration of 93.7 ±0.7%.

2.3. Preparation of SP nanoparticles by disulde bond cleavage combined

with critical pH-driven method

SP powder was dissolved in distilled water and centrifuged at

10000×g for 30 min to get rid of impurities. Na

was also dissolved

in distilled water and used freshly. Then, an equal volume of Na

solution (8 mM) was added dropwise to the SP solution and agitated for

15 min. The resultant solution was freeze-dried after dialysis (dialysis

membrane molecular weight cut off: 3.5 kDa) to obtain SP powder

treated with Na

. The obtained powder was redissolved and the pH

of the solution was brought to 10.5 with 1 M NaOH (Sodium ionic

strength increased from 0.008 M to 0.0476 M), then held at this pH for

30 min to induce further protein unfolding, labeled S

. Note: The se-

lection of critical pH was based on previous research (Wang et al., 2024).

In addition, while other experimental conditions remained unchanged,

the untreated SP, the SP treated with Na

alone, and the SP treated

with NaOH alone were used as controls, labeled as S

, S

, and S

respectively.

2.4. Structural characterization of SP nanoparticles

Fluorescence spectroscopy (FL). The excitation wavelength was

selected at 290 nm, and the emission spectrum was collected between

300 and 500 nm. The excitation and emission slits were both 5 nm, and

the scanning speed was 240 nm/min, which was measured at 25 ◦C

using a uorescence spectrophotometer (F-2700, HITACHI, Japan) (He

et al., 2021; Kwaambwa & Maikokera, 2008).

Circular dichroism (CD). The CD spectra were captured at a scanning

speed of 50 nm/min on a solution containing 0.1 mg/mL protein. The

spectra of 190~260 nm were measured using the circular dichroism

spectrometer (J-1500, JASCO, Japan). The secondary structure was

computed using the CDSSTR program (Wang et al., 2022).

Dynamic light scattering (DLS). A 3DLS Dynamic and Static Laser Light

Scatterer (LS Instruments, Switzerland) was used to evaluate the mate-

rial, which had a concentration of 1 mg/mL and was ltered using an

aqueous membrane with a pore size of 0.22

M (Microporous aqueous

Y. Wang et al.

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