Soy proteins with various surface properties prepared by limited enzymatic hydrolysis and their potential on emulsion thickening and controlling lipolysis

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Food Hydrocolloids 156 (2024) 110274

Available online 7 June 2024

Soy proteins with various surface properties prepared by limited enzymatic

hydrolysis and their potential on emulsion thickening and

controlling lipolysis

Jinjin Wu

, Weiye Liu

, Min Zhong

, Mouming Zhao

, Qiangzhong Zhao

Feibai Zhou

School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China

Guangdong Food Green Processing and Nutrition Regulation Technology Research Center, Guangzhou, 510640, China

ARTICLE INFO

Keywords:

Soy protein

Enzymatic hydrolysis

Pickering particle

Protein aggregates

Emulsion thickens

Lipid digestion

ABSTRACT

In recent years, proteins with designed surface properties as interfacial stabilizers to obtain functional emulsions

with controlled lipolysis has received increasing attention. In this work, four types of soy proteins (I, II, III, IV)

with different surface properties were obtained by controlled enzymatic hydrolysis (Neutrase). Protein particle

size, morphology, surface hydrophobicity and interfacial wettability were analyzed along with changes in sub-

units composition and secondary structure, aiming to establish a certain relationship between protein surface

properties and its capacity in modulating emulsion properties. Specically, Type I (

’,

, β, A and B subunits) as

protein nanoparticles showed high surface hydrophobicity (+84%) and could form a thick interface to delay

lipolysis (k

-24.0%, k-24.2%) through Pickering effect. Type II (

, β, A and B subunits) as protein aggregates in

larger size showed high surface hydrophobicity (+45%) and roughness, which could enhance viscosity (equiv-

alent to that of φ0.3–0.4 oil fraction) and delay lipolysis (k

-24.0%) by bridge occulation. Further hydrolysis

caused partial precipitation (originated from β and B subunits) and the soluble portion (Type III and IV) with

decreased surface hydrophobicity and increased structural exibility showed poor emulsication capacity and

formed weak interface. It can be concluded that regulating subunit composition by enzymatic hydrolysis is an

effective way to obtain proteins with multi-surface properties, which plays a vital role in modulating emulsion

viscosity and lipolysis behavior. Results from the present study could provide a new strategy for the design of

low-calorie emulsions for weight management.

1. Introduction

High-fat diets can lead to obesity and subsequently increase the risk

of several chronic diseases, such as diabetes, cardiovascular disease and

certain cancers (Nordestgaard & Varbo, 2014). In processed food, most

fat or lipids are emulsied and found in emulsion systems where milk,

yogurt, cream, mayonnaise, cheese, and even some meat products are all

included. Therefore, researches on how to modulate lipid intake in

emulsion systems can provide certain guidance when addressing the

health problems caused by the overconsumption of lipid-containing

foods.

There are commonly two strategies for reducing fat intake in emul-

sion systems: (i) Directly decreasing the content of lipid. However, this

approach tends to decrease the viscosity and thereby reducing the

overall quality of the emulsion (McClements, 2015). To address this

problem, thickeners are commonly used but gradually cannot meet the

increased demand for clean label. It has been found that some interfacial

stabilizers are capable of inducing bridging or depletion occulation of

emulsion droplets and act as ller of continuous phase. This effect may

lead to an improvement in emulsion viscosity as the droplet movement is

slowed down (Farjami & Madadlou, 2019). (ii) Controlling the rate of

lipolysis to prolong satiety through activated ileal brake. Emulsions

lipolysis is typically an interfacial process and is triggered by the

adsorption of lipases onto the oil droplets once the interface was

replaced by bile salts. Therefore, creating an interfacial layer which is

mechanically strong or resistant to bile salt replacement may slowed

down the lipolysis (Wilde & Chu, 2011). As can be seen, both strategies

mentioned above can be achieved by interfacial engineering.

* Corresponding author.School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.

E-mail address: feibaizhou@scut.edu.cn (F. Zhou).

Contents lists available at ScienceDirect

Food Hydrocolloids

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

https://doi.org/10.1016/j.foodhyd.2024.110274

Received 23 December 2023; Received in revised form 4 May 2024; Accepted 6 June 2024

Food Hydrocolloids 156 (2024) 110274

Proteins are the most widely used interfacial stabilizers. However,

they are easy to be digested by proteases or displaced by bile salts, which

have less effect in retarding lipid digestion (Guo, Ye, Bellissimo, Singh,

& Rousseau, 2017; Maldonado-Valderrama et al., 2008). Therefore, to

achieve the goals as mentioned above, mixed or conjugated interfaces

are designed for protein-based materials where other biomaterials like

polysaccharides or polyphenols are usually involved (Liu, Ma, Gao, &

McClements, 2017; Nimaming, Sadeghpour, Murray, & Sarkar, 2023;

Sun, Zhang, et al., 2022). For instance, a mixed interface from proteins

and polysaccharides showed increased interaction sites among droplets,

which might facilitate the formation of a gel or gel-like structure and

improve the viscosity of emulsions (Luo et al., 2023; Lv et al., 2023;

Zhao et al., 2023). In addition, increased thickness of the interface might

reduce the contact of enzyme and bile salts towards the interface by

steric hindrance, thereby delaying lipid digestion (Araiza-Calahorra,

Glover, Akhtar, & Sarkar, 2020; Cofrades et al., 2023; Wulff-P´

erez,

G´

alvez-Ruíz, de Vicente, & Martín-Rodríguez, 2010). Compared to an-

imal proteins, plant-based proteins have gained increasing attention

within decades for their natural resistance to digestive enzymes (Bellesi,

Ruiz-Henestrosa, & Pilosof, 2014), showing promising potential in

retarding lipid digestion. Filippidi, Patel, Bouwens, Voudouris, and

Velikov (2014) successfully fabricated zein-based microcapsules by

antisolvent precipitation and found that particle with strong surface

hydrophobicity could tightly adhere to the interface, forming shell-like

interface with adjustable thickness to control lipid digestion. Tang and

Liu (2013) reported that SPI with improved surface hydrophobicity and

shielded charges could be obtained upon thermal treatment with

modulated ionic strength. These properties facilitated the bridging

occulation of emulsion droplets, creating a gel network structure with

enhanced the viscosity. Sun, Li, et al. (2022) recently found that soy

lipophilic protein, β-conglycinin, and globulin can form Pickering par-

ticles when treated by acid, and those with small size, high charges, and

better emulsication stability can form high internal phase emulsions

with gel-like network, which consequently delays lipid digestion. It can

be found that surface properties of proteins, specically, their size, hy-

drophobicity, rigidity, and morphology, etc. are multidimensional and

play an important role in determining the properties of emulsions

(Zhang, Hu, et al., 2021; Zhang, Fan, Liu, Huang, & Li, 2022). By

establishing a relationship between the protein surface properties and

emulsion properties, it may be feasible to discover interfacial stabilizers

that can meet both strategies proposed. To date, the relationship

mentioned above is still unclear, and the research to modulate protein

structure based on their surface properties is relatively limited.

Recently, we successfully obtained soy proteins with various surface

properties by enzymatic hydrolysis. For example, the rigidity and hy-

drophobicity of proteins can be altered by hydrolyzing A, B and part of

subunits (Cui, Zhao, Yuan, Zhang, & Ren, 2013). Size and surface charge

of proteins can be altered by changing the content of B subunit (Yuan,

Zhou, Niu, Shen, & Zhao, 2023). Morphology, size and hydrophobicity

of proteins can be regulated by hydrolyze β-conglycinin and A subunits

(Niu, Zhou, Yuan, & Zhao, 2024; Shen et al., 2020). It was proved that

proteins with relatively complete subunit composition can self-assemble

into small, rigid soy protein nanoparticles with high surface hydro-

phobicity, which can adsorb at the interface rapidly. They act as Pick-

ering stabilizers to delay lipid digestion in the intestinal tract (Zhao

et al., 2021). It suggested that protein with various surface properties

could be well obtained through subunit regulation by controlled enzy-

matic hydrolysis.

In this work, a series of proteins with different subunit compositions

were fabricated by controlled enzymatic hydrolysis, and their surface

properties were evaluated in a more systematic manner in terms of size,

surface hydrophobicity, and interfacial wettability, etc. Their potential

as interfacial stabilizer to improve emulsion viscosity and retarding

lipolysis were both explored, aiming to establish a certain relationship

between them. Results from the present work could provide certain

guidance for further design of low-calorie food.

2. Materials and methods

2.1. Materials

Low-heat defatted soy meal were supplied by Yuwang Industrial and

Commercial Co., Ltd. (Shandong, China). Neutrase (0.8 Au/g) was

supplied by Novozymes Co. (Bagsvaerd, Denmark). L-serine, o-phtha-

laldehyde (OPA), bovine bile salt (#B875069, bile acid of ≥75.0%), and

sodium 8-anilino-1-naphthalenesulfonate (ANS-Na) were purchased

from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). DL-

dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) were supplied by

Genview Scientic Inc., USA. Soybean oil was purchased at a local su-

permarket (Guangzhou, China). Bromophenol blue, Nile red, Nile blue,

Florisil, porcine pepsin (#P7000: ≥250 units/mg solid), pancreatin

(#P7545: 8 ×USP specications, trypsin of ≥200 units/mg and lipase of

≥16 units/mg), and lipase (#L3126: 100–500 units/mg protein) were

obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All

other reagents used were of analytical grade.

2.2. Soy protein isolates (SPI) preparation and hydrolysis

SPI was prepared from low-heat defatted soy meal by the method of

alkali-soluble acid precipitation according to the previous work (Wan,

Wang, Wang, Yuan, & Yang, 2014). Briey, milled and sieved soy meal

was suspended in deionized water (1:10, w/w), followed by alkaline

extraction at pH 8.0 (2 h) and centrifuged (10,000×g, 10 min, 25 ◦C).

The supernatants were then precipitated at pH 4.5 (30 min) and

centrifuged. The precipitates were washed twice, resuspended in

deionized water by adjusting the pH to 7.0, then dialyzed (4 ◦C, 48 h, 14,

000 Da) and freeze-dried before use. The protein content of SPI powder

determined by Kjeldahi method (N =6.25) was 92.33 ±0.23%.

The freeze-dried SPI was resuspended in deionized water (4%, w/w)

and subjected to Neutrase (pH 7.0, 50 ◦C) treatment with an enzyme:

substrate (E/S) ratio of 1:100. Samples were boiled to inactivate en-

zymes and centrifuged (10,000×g, 10 min, 25 ◦C) to collect the super-

natants before subjected to SDS-PAGE analysis (Fig. S1). Hydrolysates

with different representative subunit compositions (1h, 4h, 8h and 12h)

were collected and freeze-dried for further use, and labeled as I, II, III,

and IV.

2.3. SPI hydrolysates characterization

2.3.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE)

To monitor the representative subunit compositions changing by

enzymatic hydrolysis, SDS-PAGE was conducted under both non-

reducing and reducing (containing β-mercaptoethanol, β-ME) condi-

tions, according to the previous method with some modications

(Laemmli, 1970). Sample suspensions were prepared from freeze-dried

powders and diluted with the loading buffer solution to reach a pro-

tein concentration of 2 mg/mL. Particularly, the sample solutions with

β-ME were boiled before centrifugation (10,000×g, 10 min). The marker

and the sample solutions (10

L) were loaded onto the gel (consisting of

a 5% stacking gel and a 12% running gel) and subjected to electropho-

resis at a constant voltage of 80 V, using a Mini-PROTEIN 3 Cell appa-

ratus (Bio-Rad Laboratories, USA). Then, gels were stained with 0.25%

(w/v) Coomassie Brilliant Blue (R-250) and destained in a solution

containing 10% acetic acid and 30% methanol.

2.3.2. Degree of hydrolysis (DH)

The degree of hydrolysis (DH) was determined using a derivatization

protocol with OPA described by Nielsen, Petersen, and Dambmann

(2001). Briey, each sample (400

L) was mixed with OPA reagent (3

mL) thoroughly. After 2 min reaction, the absorbance value (340 nm)

was measured using a UV spectrophotometer (UV-1800, Shimadzu Co.,

Japan). Deionized water and serine solution (0.9516 meqv/L) were set

J. Wu et al.

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