CN116035111A - Modified whey protein and preparation method thereof - Google Patents

Abstract

The invention relates to the field of protein processing, in particular to modified whey protein and a preparation method thereof. The modification of whey protein is achieved by cold extrusion of whey protein and cysteine together to allow the cysteine to enter the protein molecule. According to the invention, cys can enter protein molecules rapidly through coextrusion of WPI and Cys, so that the three-level structure of the protein is unfolded, and disulfide cross-linking is formed through sulfhydryl oxidation or SH/S-S exchange reaction. The invention adopts the method of Cys and WPI co-cold extrusion to modify whey protein molecules, simplifies the two-step reaction into one step, has simple method, is suitable for industrial production, provides a quick and effective method for developing WPI functional components for food industry, improves the functional characteristics and digestion characteristics of whey protein molecules, and widens the application field of whey protein.

Description

A modified whey protein and preparation method thereof

Technical Field

The invention relates to the field of protein processing, in particular to a modified whey protein and a preparation method thereof.

Background Art

Whey protein isolate (WPI) has excellent nutritional value and is widely used as a raw material for food processing in the food industry. WPI molecules have a spherical structure with hydrophilicity on the outside and hydrophobicity on the inside, and have good solubility, but their functional properties such as emulsification and gelation cannot meet the requirements of the food industry, and WPI needs to be modified.

Extrusion technology is a physical processing operation, which includes cold extrusion and hot extrusion through a combination of different steps such as mixing, molding, kneading, cooking and molding. Generally speaking, hot extrusion is commonly used to process proteins in domestic and foreign research, but the high temperature of hot extrusion will cause many bioactive substances in the protein to be inactivated, and the aggregation of protein molecules will greatly reduce the solubility of the protein. In order to avoid these situations, cold extrusion (≤50℃) is a good choice for processing WPI.

Cysteine (Cys) is a sulfur-containing α-amino acid. The addition of Cys is an important means to change the molecular structure and intermolecular interactions of WPI. Theoretically, in the presence of exogenous Cys, its free sulfhydryl groups will undergo SH/S-S exchange reactions or sulfhydryl oxidation reactions with protein molecules. Previous studies have shown that Cys can induce disulfide cross-linking and improve some properties of protein products, but Cys is not suitable for direct cross-linking with natural whey protein, and some modification of whey protein is required.

Summary of the invention

Based on the above content, the present invention provides a modified whey protein and a preparation method thereof, wherein the whey protein molecules are modified by a Cys and WPI co-cold extrusion method, thereby improving the emulsification properties and in vitro digestibility of the whey protein and broadening the application field of the whey protein.

To achieve the above object, the present invention provides the following solutions:

One of the technical solutions of the present invention is a method for modifying whey protein, which achieves the modification of whey protein by cold-extruding whey protein and cysteine together to allow Cys to enter the protein molecule.

Further, the method comprises the following steps: adding whey protein into a twin-screw extruder through a feed screw, and simultaneously adding a cysteine aqueous solution into the twin-screw extruder at the same feeding speed as the whey protein for co-extrusion to obtain a whey protein cold extrusion product;

The whey protein cold extrusion product is dried to obtain modified whey protein.

Furthermore, the feeding rate of the whey protein is 3-4 kg/h.

Furthermore, the concentration of the cysteine aqueous solution is 20-100 mmol/L; preferably, 60-100 mmol/L.

Furthermore, the water content of the whey protein cold extrusion product is 45-55wt%.

Furthermore, the drying treatment is drying or freeze drying; the drying is specifically: drying at 35-45°C for 20-28h; and the drying treatment also includes the steps of crushing and screening.

The drying specifically includes drying until the moisture content is about 5 wt %; and the pulverizing specifically includes pulverizing until the particle size is less than 0.5 mm.

Furthermore, no heating is performed during the co-extrusion process; the temperature of the feeding zone of the twin-screw extruder is set to 25°C, the temperature of the mixing zone is set to 30°C, the temperature of the cooking zone is set to 35°C, and the temperature of the discharge zone is not higher than 50°C.

Furthermore, the screw diameter of the twin-screw extruder is 25 mm, the aspect ratio is 24:1, and the screw speed is 250-350 r/min.

In the present invention, the factors affecting the protein extrusion effect are mainly the screw configuration and the operating conditions, wherein the operating conditions mainly refer to the feed rate, the moisture content, the screw speed and the barrel temperature.

Temperature: The present invention uses a cold extrusion method to process protein, and the extruder temperature is controlled to be ≤50° C. When the temperature is too high, the high temperature of hot extrusion will cause a large degree of aggregation and polymerization of whey protein, and cause its solubility to drop significantly.

Moisture content: The moisture content of the cold extruded extrudate in the present invention is 45-55wt%. If the moisture content of the extrudate is too low, the viscosity of the material will be too high, which may easily cause the main machine to overload; if the moisture content is too high, the material will not be easy to shape.

Feeding speed and screw speed: During the extrusion process of the twin-screw extruder, in order to ensure stable extrusion, the feeding speed of the feeding device and the speed of the extruder screw are usually linked to ensure the balance between the feeding amount and the twin-screw extrusion amount. In the present invention, the feeding speed of whey protein is 3-4kg/h, and the screw speed is 250-350r/min. When the feeding speed and screw speed continue to increase, the shearing and friction effects on the material are enhanced, which will result in the inability to prepare a uniform and well-stirred extrudate.

The second technical solution of the present invention is the modified whey protein prepared according to the above-mentioned whey protein modification method.

Furthermore, the modified whey protein contains the following intermolecular disulfide bond cross-linked peptide segments:

CEVFR(1)-CEVFR(1)

LDQWLCEKL(6)-LDQWLCEK(6)

LDQWLCEK(6)-LDQWLCEK(6)

LDQWLCEK(6)-LDQWLCEK(6)

WENGECAQK(6)-CEVFR(1)

WENDECAQK(6)-WENGECAQK(6)

LSFNPTQLEEQCHI(12)-CEVFR(1)

LSFNPTQLEEQCHI(12)-ALCSEK(3)

LSFNPTQLEEQCHI(12)-LDQWLCEKL(6)

The third technical solution of the present invention is an application of the above-mentioned modified whey protein in the food industry.

The present invention discloses the following technical effects:

Extrusion is an operation that uses shear force, heating and mechanical pressure to change the structure and composition of food raw materials. Extrusion technology can be applied to the modification of soy protein, pea protein and whey protein isolate. Cysteine (Cys) is a reducing agent with free thiol groups. In the presence of exogenous Cys, the free thiol groups on the Cys molecule will undergo SH/S-S exchange reaction or thiol oxidation reaction with the disulfide bonds and free thiol groups of the protein molecule. Generally speaking, it is cumbersome to modify WPI with Cys alone or by cold extrusion followed by Cys treatment, which is not suitable for industrial production. Co-extrusion of WPI and Cys is a faster and more direct modification method that can form macromolecular cross-linked products, and ultimately form modified whey protein products with good functional properties and digestibility.

The co-extrusion of WPI and Cys can make Cys quickly enter the protein molecule, which is beneficial to the unfolding of the tertiary structure of the protein, and disulfide crosslinking is formed through sulfhydryl oxidation or SH/S-S exchange reaction. The present invention adopts the method of co-cold extrusion of Cys and WPI to modify the whey protein molecule, simplifies the two-step reaction into one step, and the method is simple and suitable for industrial production. A fast and effective method is provided for the food industry to develop WPI functional ingredients, improve the functional properties and digestion properties of whey protein molecules, and broaden the application field of whey protein.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a total ion current (TIC) diagram (A) and a base peak diagram (B) of T+C ₁₀₀ of the present invention; a matching diagram of b and y ions of the disulfide cross-linked peptide segment WENGECAQKK(6)-WENGECAQK(6) (C);

FIG2 is a graph showing the molecular weight distribution of non-reduced (A) and reduced (B) co-extruded WPI-Cys samples of the present invention;

FIG3 is a non-reduced (A) and reduced (B) SDS-PAGE electrophoretogram of the co-extruded WPI-Cys sample of the present invention;

FIG4 shows the particle size (A), Zeta potential (B) and median particle size (D ₅₀ ) (C) of the co-extruded WPI-Cys sample of the present invention;

FIG5 shows the intrinsic fluorescence (A) and surface hydrophobicity (B) of the co-extruded WPI-Cys sample of the present invention;

FIG6 shows the emulsification activity (A) and emulsification stability (B) of the co-extruded WPI-Cys sample of the present invention;

FIG7 shows the solubility (A) and free thiol groups (B) of the coextruded WPI-Cys sample of the present invention;

FIG8 FTIR spectrum of the co-extruded WPI-Cys sample of the present invention in the amide I region (A), deconvoluted FTIR spectrum of T+C100 in the amide I region (B), and the contents of α-helix (C), β-sheet (D), β-turn (E), and random coil (F) of the sample;

FIG9 is a transverse relaxation spectrum of the co-extruded WPI-Cys sample gel of the present invention;

FIG10 Simulated digestibility of the co-extruded WPI-Cys sample in the stomach (A) and intestine (B);

FIG11 OD ₇₀₀ nm of simulated digests of the co-extruded WPI-Cys sample of the present invention in the stomach (A) and intestine (B);

FIG12 α-glucosidase activity inhibition rate of gastric (A) and intestinal (B) digesta of the co-extruded WPI-Cys sample of the present invention;

FIG13 Inhibition rate of xanthine oxidase activity in gastric (A) and intestinal (B) digesta of the co-extruded WPI-Cys sample of the present invention;

FIG14 is a graph showing the correlation between the structure and functional properties of the co-extruded WPI-Cys sample of the present invention;

Fig. 15 is a schematic diagram of the co-extrusion process of the present invention; in the figure, 1-feeding zone; 2-mixing zone; 3, 4, 5, 6-cooking zones; 7-discharging zone.

DETAILED DESCRIPTION

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

The schematic diagram of the co-extrusion process of the present invention is shown in FIG15 ; in the figure, 1-feeding zone; 2-mixing zone; 3, 4, 5, 6-cooking zone; 7-discharging zone.

Example 1

A method for co-cold extrusion of WPI and Cys to modify whey protein isolate comprises the following steps:

(1) Co-extrusion of WPI and Cys: whey protein was extruded using a twin-screw extruder, with the screw speed set at 300 r/min and the feeding rate at 3.5 kg/h; at the same time, different concentrations of Cys (0, 20, 40, 60, 80, 100 mmol/L, the corresponding co-extruded WPI-Cys samples were marked as T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ ) aqueous solutions were added at the same speed through an automatic valve, and the moisture content of the extrudate was controlled to be 50 wt%. The temperatures of the feeding zone (T ₁ ), mixing zone (T ₂ ) and cooking zone (T ₃ ) were set to 25° C., 30° C. and 35° C., respectively. No heating was performed during the extrusion process, and the temperature of the discharge zone (T ₄ ) was ensured to be about 50° C. (not higher than 50° C.). A whey protein cold extrusion product was obtained.

(2) Drying of extruded whey protein: The cold extruded whey protein product is dried in a constant temperature drying oven at 40°C (35-45°C is equivalent to 40°C in terms of technical effect) for 24 hours (20-28 hours is equivalent to 24 hours in terms of technical effect) to a water content of about 5 wt %, thereby obtaining an extruded whey protein dry product.

(3) Crushing of extruded whey protein: The dried extruded whey protein was cut into small pieces with a volume of about 0.5 ^cm3 , put into a cyclone mill for further crushing, and passed through a 0.5 mm pore size sieve to obtain a WPI and Cys co-extruded modified whey protein product (co-extruded WPI-Cys sample).

Effect verification example

1. Identification and analysis of disulfide cross-linking sites

Trypsin hydrolysis:

The samples (T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C 80 and T+C ₁₀₀ ) co-extruded with different concentrations of Cys (0, 20, 40, 60, ₈₀ , 100 mmol/L) were diluted to a concentration of 1.0 mg/mL. The protein solution was first alkylated with 30 mmol/L acrylamide at room temperature for 30 min, and then the protein solution was mixed with trypsin at a ratio of 1:20 (W/W), and the pH was adjusted to 6.8 with 0.1 M NaOH, and the mixture was hydrolyzed at 37°C overnight.

LC/MS/MS analysis:

The LC/MS/MS method was used to analyze the sites of action of disulfide cross-linking in protein samples. The Easy-nLC1200 ultra-high performance liquid chromatography (Thermo-Dionex Sunnyvale, California, USA) system and a reversed phase C18 column (100 mm × 3 mm, 2.7 μm, Agilent) were connected to the mass spectrometer online through a nanospray ion source. The mass spectrometric analysis was performed by a Q-Exactive Plus mass spectrometer (Thermo Scientific, California, USA).

Liquid chromatography conditions:

Mobile phase A was prepared as 0.1% (v/v) formic acid (FA) ultrapure aqueous solution, and mobile phase B was prepared as a mixture of 20% H ₂ O and 80% acetonitrile. Linear gradient elution was performed using mobile phases A and B: first, the concentration of mobile phase B increased from 3% to 8% between 0 and 5 min, and increased to 28% between 5 and 63 min; then, it continued to increase to 38% between 63 and 75 min; finally, it was linearly increased to 100% between 75 and 80 min and maintained for 10 min. The total running time was 90 min, and the column flow rate was maintained at 300 nL/min.

Mass spectrometry conditions:

Ionization was performed using an electrospray ion source, with the capillary voltage set to 1.8 kV and the ion transfer tube temperature set to 300 °C. Primary mass spectrometry: In positive ion mode, molecular weight scans were performed with a resolving power of 70,000, a maximum ion injection time of 50 min, and a range of 350 to 2000 mass-to-nuclear ratio (m/z). Secondary mass spectrometry: MS/MS scans were extended from 200 to 2000 m/z, with a resolution of 17,500, a maximum ion injection time of 50 min for the measurement scan, and a MS/MS scan of 45 min. The 20 most abundant precursor ions were fragmented by collision dissociation, with a normalized collision energy of 27 eV.

Identification of disulfide cross-linked peptides:

Disulfide-cross-linked peptides in the samples were identified and located using Proteome Discoverer 2.2 software (version 2.2; Thermo Fisher Scientific) and pLink software (version 2.3.5, http://pfind.ict.ac.cn/software/pLink/2014/pLink-SS.htmL) by comparing the molecular weight lists in the experimental results with the theoretical molecular weights contained in the “cow” database downloaded from UniProt (Swissprot and trEMBL; https://www.uniprot.org/).

The results are as follows:

(1) Intermolecular cross-linking

Table 1 Disulfide cross-linked peptides in the co-extruded WPI-Cys samples and their molecular weights and cross-linking sites

和

可以看出，α-La(6)和α-La(120)是α-La分子间二硫键交联反应最活跃的位点，这证实了

在α-La的四个分子内二硫键中稳定性最低。同样，在β-Lg(β-乳球蛋白)分子的两个天然二硫键中，

的稳定性最低，是分子间二硫键交联反应最活跃的位点。并且在共有的五种分子间二硫键交联肽中，

和

的二硫键交联发生在α-La(α-乳白蛋白)和β-Lg分子之间，

和

的二硫键交联发生在同源α-La之间，说明α-La比β-Lg更易于发生分子间二硫交联。The total ion current (TIC) and base peak diagrams of T+C ₁₀₀ , and the matching diagrams of b and y ions of the disulfide cross-linked peptide WENGECAQKK(6)-WENGECAQK(6) are shown in Figures 1(A), (B), and (C). A total of 29 intermolecular disulfide cross-linked peptides were detected in the WPI, T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ , and T+C ₁₀₀ samples, and the results are shown in Table 1. In WPI, no intermolecular disulfide cross-linked peptides were detected. However, five common intermolecular disulfide cross-linked peptides were identified in the extruded WPI sample (T+C ₀ ), namely,

and

It can be seen that α-La(6) and α-La(120) are the most active sites for disulfide cross-linking reaction between α-La molecules, which confirms that

It has the lowest stability among the four intramolecular disulfide bonds of α-La. Similarly, among the two native disulfide bonds of β-Lg (β-lactoglobulin) molecule,

The stability is the lowest and it is the most active site for intermolecular disulfide cross-linking reaction. And among the five intermolecular disulfide cross-linked peptides,

and

The disulfide cross-linking occurs between α-La (α-lactalbumin) and β-Lg molecules.

and

The disulfide cross-linking occurs between homologous α-La, indicating that α-La is more prone to intermolecular disulfide cross-linking than β-Lg.

和

可以得出结论，当Cys的浓度增加到60mmol/L以上时，BSA的IIA结构域展开。

和

三个分子内二硫键都暴露出来，与β-Lg(160)发生巯基氧化或SH/S-S交换反应，生成分子间二硫键交联。相对于β-Lg，BSA对SH/S-S交换反应具有更高的活性。在T+C₆₀、T+C₈₀和T+C₁₀₀样品中，检测到3个β-Lg分子间二硫键交联肽，分别是

和

这表明，随着Cys浓度增加到60mmol/L以上，SH/S-S交换反应更加活跃，使β-Lg中位于

和

的二硫键都发生断裂，释放出游离巯基，产生分子间二硫键交联。在T+C₁₀₀样品中，位于不同α-La分子相同位点的二硫交联肽段

被鉴定出来。这可能是由于两个α-La分子中的

和

二硫键分别与Cys发生了SH/S-S交换反应，导致释放出的两个游离巯基发生巯基氧化反应。或者，一个α-la分子的

或

断裂生成一个游离巯基，与另一个α-la分子发生SH/S-S交换反应，产生分子间二硫键交联。In addition, nine intermolecular disulfide cross-linked peptides were detected for the first time in T+C ₆₀ , T+C ₈₀ or T+C ₁₀₀ samples, among which four disulfide cross-links occurred between β-Lg and BSA molecules, namely

and

It can be concluded that when the concentration of Cys increases to above 60 mmol/L, the IIA domain of BSA unfolds.

and

The three intramolecular disulfide bonds were exposed and underwent sulfhydryl oxidation or SH/SS exchange reaction with β-Lg (160) to generate intermolecular disulfide crosslinks. Compared with β-Lg, BSA had higher activity in SH/SS exchange reaction. Three β-Lg intermolecular disulfide crosslinked peptides were detected in the T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples, which were

and

This indicates that as the Cys concentration increases above 60 mmol/L, the SH/SS exchange reaction becomes more active, making the

and

The disulfide bonds of the peptides are broken, releasing free sulfhydryl groups _and generating intermolecular disulfide crosslinks.

This may be due to the fact that

and

The disulfide bonds undergo SH/SS exchange reactions with Cys, resulting in the release of two free sulfhydryl groups that undergo sulfhydryl oxidation reactions.

or

The cleavage generates a free thiol group, which undergoes SH/SS exchange reaction with another α-la molecule to produce intermolecular disulfide cross-linking.

(2) Intramolecular cross-linking

Table 2 Intramolecular disulfide cross-linked peptides and their molecular weights and cross-linking sites in co-extruded WPI-Cys samples

Note: * indicates disulfide bonds within natural molecules.

和

这可能是由于β-Lg分子中的空间位阻较大，使得分子内二硫键重组难以发生，更多地参与了分子间的二硫键交换反应。表1中的结果也证实了这一点。相对而言，仅在T+C₆₀、T+C₈₀和T+C₁₀₀样品中发现了二硫键重组的BSA肽。其中

和

位于BSA的结构域IA中，

和

位于BSA的结构域IB中，

位于BSA的结构域IIIB中。这表明共挤压过程中，只有当Cys浓度高于60mmol/L时，BSA的结构域I和结构域III才能打开，发生分子内二硫键重组。In the co-extruded WPI-Cys samples, a total of 7 intramolecular native disulfide bonds and 16 intramolecular recombinant disulfide cross-linked peptides were identified (Table 2). In the T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples, the four intramolecular native disulfide bonds of α-La were involved in the reorganization of intramolecular disulfide bonds. α-La(61, 73, 77) were cross-linked with α-La(111), α-La(120) and α-La(91) through disulfide bonds, respectively. In addition, intramolecular disulfide bond reorganization reactions occurred at any two sites between α-La(91), α-La(111) and α-La(120). This indicates that the addition of exogenous Cys during cold extrusion promoted the reorganization of extensive disulfide bond reactions within the α-La molecule and prevented the renaturation of α-La. However, only two intramolecular recombinant disulfide peptides were identified in the β-Lg molecule.

and

This may be due to the large steric hindrance in the β-Lg molecule, which makes it difficult for the intramolecular disulfide bond recombination to occur, and more disulfide bond exchange reactions are involved. The results in Table 1 also confirm this. Relatively speaking, disulfide bond reorganized BSA peptides were only found in T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples.

and

Located in domain IA of BSA,

and

Located in domain IB of BSA,

Located in domain IIIB of BSA. This indicates that during co-extrusion, only when the Cys concentration is higher than 60mmol/L can domain I and domain III of BSA be opened and intramolecular disulfide bond reorganization occur.

(3) Cyclic disulfide crosslinks

Table 3 Cyclic disulfide cross-linked peptides of co-extruded WPI-Cys samples and their molecular weights and cross-linking sites

肽段。此外，在T+C₀和T+C₂₀中也发现了

表明α-La(73)是分子内环状二硫键交联的活性位点。T+C₀、T+C₂₀和T+C₄₀样品中位于β-Lg(121)的游离巯基分别与邻近的β-Lg(119)和β-Lg(106)形成环状交联，表明在Cys浓度≤40mmol/L的样品中，β-Lg(121)不能与邻近的蛋白质分子发生聚合反应。然而，在Cys浓度≥60mmol/L时，β-Lg(121)能够与邻近的β-Lg发生分子间二硫键交联原因可能是高浓度的Cys有利于扩大β-Lg的三级结构，减少空间位阻，促进游离巯基与相邻蛋白质分子发生二硫键交联反应。类似地，在较低的Cys浓度下(≤40mmol/L)，BSA分子的结构域II和III中仅发生分子内环状二硫键交联，而在较高的Cys浓度下(≥60mmol/L)，BSA才发生分子间二硫键交联。The cyclic disulfide cross-linking results of the co-extruded WPI-Cys samples are shown in Table 3. In the T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples,

In addition, T+C ₀ and T+C ₂₀ were also found

The results showed that α-La(73) was the active site for intramolecular cyclic disulfide crosslinking. The free thiol group at β-Lg(121) in samples T+C ₀ , T+C ₂₀ and T+C ₄₀ formed cyclic crosslinks with adjacent β-Lg(119) and β-Lg(106), respectively, indicating that in samples with Cys concentration ≤40mmol/L, β-Lg(121) could not undergo polymerization reaction with adjacent protein molecules. However, when Cys concentration was ≥60mmol/L, β-Lg(121) could undergo intermolecular disulfide crosslinking with adjacent β-Lg. The reason may be that high concentration of Cys is conducive to expanding the tertiary structure of β-Lg, reducing steric hindrance, and promoting disulfide crosslinking reaction between free thiol groups and adjacent protein molecules. Similarly, at lower Cys concentrations (≤40 mmol/L), only intramolecular cyclic disulfide cross-linking occurred in domains II and III of the BSA molecule, whereas at higher Cys concentrations (≥60 mmol/L), intermolecular disulfide cross-linking occurred in BSA.

(4) Single-chain disulfide crosslinks

Table 4 Single-chain disulfide cross-linked peptides of co-extruded WPI-Cys samples and their molecular weights and cross-linking sites

The results of single-chain disulfide crosslinking of coextruded WPI-Cys samples are shown in Table 4. Single-chain disulfide crosslinking of α-La(91) was identified in samples T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ , indicating that α-La(91) is not easily crosslinked with other protein molecules after sulfhydryl exchange reaction with free sulfhydryl groups in Cys. Fourteen single-chain disulfide crosslinked peptides were identified in sample T+C ₂₀ , including α-La(91), α-La(61), α-La(120), β-Lg(66), β-Lg(106), β-Lg(119), β-Lg(160), BSA(75), BSA(123), BSA(288), BSA(315) and BSA(447). The results showed that when the Cys concentration was 20mmol/L, protein molecules were most likely to undergo single-chain disulfide cross-linking, and when the Cys concentration was higher (≥60mmol/L), the number of single-chain disulfide cross-linked peptides decreased significantly. The reason may be that high concentrations of Cys may make the structure of the protein looser, exposing the disulfide bonds within the molecule on the surface, promoting the continued reaction of single-chain disulfide cross-linked peptides with free thiol groups on adjacent protein molecules, resulting in a significant reduction in the number of single-chain disulfide cross-linked peptides identified.

2. Size Exclusion Chromatography (SEC)

The molecular weight distribution of protein samples was determined using a Waters 2695/2487 combination system (Waters, Etten-Leur, the Netherlands) by connecting a TSK guard column (7.5 mm × 7.5 mm) and a TSK G2000sw (7.5 mm × 60 cm, 1 μm, TosoHaas, Montgomeryville, PA, USA) column in series. Non-reducing exclusion chromatography: The column was equilibrated and eluted at a flow rate of 0.5 mL/min using a 30% acetonitrile aqueous solution containing 0.1% TFA. After filtering the sample (5 mg/mL) through a 0.45 μm filter membrane, 15 μL was injected for analysis. The UV absorption detector wavelength was set to 280 nm and calibrated with β-lactoglobulin (β-Lg), α-lactalbumin (α-La), bovine serum albumin (BSA), and Cys as standards, respectively. Reduction exclusion chromatography: First, the protein was dissolved in 0.1 M sodium phosphate buffer (pH 6.8) containing 5% SDS and 1% β-mercaptoethanol, then heated at 85°C for 15 min, immediately removed and cooled to 4°C in an ice water bath before measurement. The other steps were the same as non-reduction exclusion spectroscopy.

Results: The non-reduced and reduced SEC results of the co-extruded WPI-Cys sample are shown in Figure 2 (A) and (B). The non-reduced SEC results (Figure 2 (A)) showed that the retention times of BSA, β-Lg and α-La in WPI were 24.69, 28.62 and 30.62 min, respectively. In T+C ₀ , high molecular weight substances greater than 66 KDa were eluted at 24.60 min and mixed with BSA. Compared with the BSA monomer, its peak area increased by 22.98% (P < 0.05). The results showed that after extrusion, protein molecules aggregated to form large molecular weight substances. At the same time, with the increase of Cys addition, the amount of large molecular weight polymers gradually increased (P < 0.05). Compared with WPI, the peak areas of high molecular weight substances in T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ increased by 50.05%, 50.23%, 76.01%, 139.68% and 164.94% respectively (P<0.05). In addition, the retention time of high molecular weight substances gradually decreased, and the retention time of polymers in T+C ₁₀₀ sample was the smallest (24.55min), indicating that the molecular weight of polymers contained in T+C ₁₀₀ sample was the largest. This indicates that coextrusion promotes intermolecular aggregation or polymerization of proteins. In addition, the peak areas of β-Lg and α-La decreased significantly in T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples, indicating that β-Lg and α-La participated in intermolecular aggregation and polymerization. Interestingly, in T+C ₀ , more β-Lg participated in intermolecular polymerization, while in the co-extruded samples, α-La was the main reactant for intermolecular aggregation and polymerization, and the content of α-La participating in the reaction gradually increased with the increase of Cys concentration. Compared with WPI, the consumption of α-La gradually increased by 10.24%, 11.44%, 24.65%, 25.79% and 35.96% in T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples, respectively (P < 0.05).

Since α-La (4) has more disulfide bonds than β-Lg (2), the amount of intermolecular aggregation and polymerization reaction of α-La during co-extrusion is significantly higher than that of β-Lg. In addition, Cys was not eluted in all protein samples, indicating that all Cys participated in sulfhydryl oxidation reaction or SH/S-S exchange reaction. From the reduction results of SEC (Figure 2 (B)), it can be seen that the retention time of BSA, β-Lg and α-La increased, which may be because SDS destroyed the non-covalent bonds in the protein molecules, making them linear structures and enhancing the adsorption capacity in the chromatographic column. In addition, in all protein samples, no substances with molecular weight higher than BSA were eluted, and there was no significant difference in the content of various proteins.

3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

The molecular weight distribution of co-extruded whey protein was analyzed by SDS-PAGE electrophoresis. The concentration of acrylamide gel was 20% and the concentration of protein sample was 5 mg/mL. 40 μL of sample was mixed with 10 μL of SDS buffer (pH 8.0) containing 10 mmol/LTris-HCl, 1 mmol/LEDTA, 2.5% SDS, 0.01% bromophenol blue and 5.0% β-mercaptoethanol, and then quickly cooled to 4 ° C after 5 minutes of boiling water bath. The electrophoresis voltage was set to 120 V, the molecular weight marker range was 14-200 kDa, and the sample volume was 10 μL. The gel was stained with Coomassie brilliant blue for 15 minutes, then decolorized in a solution containing 30% methanol and 10% acetic acid, and photographed and analyzed with a gel imager after the bands were clear.

Results: The results of non-reducing and reducing SDS-PAGE electrophoresis of the co-extruded WPI-Cys samples are shown in Figure 3 (A) and (B). In WPI and T+C ₀ , the optical density of the material with a molecular weight higher than 200 kDa in the top comb of the lane was low, while in the co-extruded samples, the optical density of the band in the top comb of the lane was high, indicating the presence of undissolved macromolecular protein polymers in the sample. Soluble macromolecular polymer bands appeared in all lanes near 200 kDa in the gel, and the optical density of the bands gradually increased as the Cys concentration increased from 20 to 100 mmol/L. This indicates a gradual increase in the content of soluble polymers in the sample. The band at a molecular weight of 66 KDa is BSA, and there is no significant difference in the optical density of the bands of all samples. The reason may be that the electrophoresis method is not as sensitive as LC/MS/MS, and the slight decrease in the BSA content was not detected. The bands near the molecular weights of 18.4 and 14.2 KDa are β-Lg and α-La, respectively. Compared with the WPI sample, the optical density of the β-Lg and α-La bands decreased slightly as the Cys concentration increased from 0 to 40 mmol/L. However, when the Cys concentration was ≥ 60 mmol/L, the optical density of the α-La band decreased significantly, proving that α-La was the main intermolecular polymerization reaction in the co-extruded samples, which was consistent with the results of non-reduced SEC (Figure 2 (A)). In addition, in the co-extruded samples T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ , a band with a strong optical density appeared at a molecular weight of 37 KDa, which may be a dimer formed by β-Lg.

From the reduction electrophoresis diagram (Figure 3 (B)), after all protein samples were treated with reducing agents, the precipitate in the comb-like part at the top of the lane disappeared. This indicates that the insoluble macromolecular polymers are mainly polymerized through intermolecular disulfide bonds and dissociated under the action of the reducing agent. The soluble aggregate band at a molecular weight of 200 kDa was also not detected, indicating that the soluble macromolecular proteins were also aggregated through disulfide bonds. Similarly, in the co-extruded samples (T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ ), no band with a strong optical density of 37 KDa was detected, indicating that the dimer formed by β-Lg was mainly connected by covalent bonds. The results show that co-extrusion promotes the cross-linking of WPI through intermolecular disulfide bonds to form a large molecular weight polymer.

4. Particle size potential

The Zetasizer Nano-ZS 90 nanometer particle size potential instrument was used to measure the nanometer particle size and potential. The protein sample was dissolved in 0.01 mol/L PBS (pH 7.0) at a concentration of 1 mg/mL, and the Zeta-potential was measured; and the protein particle size was calculated by the scattered light intensity. The HYL-1076 laser particle size distribution analyzer (DanDong Hylology Technology co., LTD, China) was used to measure the micron-scale particle size of the Cys synchronous cold extrusion WPI sample.

Results: The particle size, Zeta-potential and median particle size (D ₅₀ ) of the coextruded WPI-Cys samples are shown in Figure 4 (A), (B) and (C), respectively. As can be seen from Figure 4 (A) and (C), the particle sizes of WPI and T+C ₀ are both less than 10 μm, and their D ₅₀ are approximately 1.11 and 1.28 μm, respectively. This indicates that no aggregation and polymer of macromolecular proteins were detected during the cold extrusion process. However, as the Cys concentration gradually increased from 20 to 100 mmol/L, the particle size distribution of the coextruded WPI-Cys sample further broadened and shifted to the right, and its D ₅₀ also increased significantly compared with WPI and T+C _0. The D ₅₀ value of T+C ₁₀₀ was the largest, which was approximately 29.41 times that of WPI (P < 0.05). The reason may be that the addition of Cys promotes the breaking of disulfide bonds and the exposure of hydrophobic groups inside WPI, which leads to the occurrence of hydrophobic interactions and the formation of extensive molecular aggregation.

As can be seen from Figure 4 (B), there is no significant difference in the absolute value of the Zeta potential between WPI and T+C _0. However, with the increase in the amount of Cys added, the absolute value of the Zeta potential of the co-extruded WPI-Cys sample gradually increases. When the Cys addition amount reaches 100mmol/L, the absolute value of the Zeta potential is the largest (P<0.05). This result may be due to the SH/SS exchange reaction that causes the disulfide bonds inside the protein to break, and the free thiol groups are exposed to the protein surface, making the protein surface carry more negative charges, resulting in an increase in the absolute value of the Zeta potential.

5. Intrinsic fluorescence

The endogenous fluorescence spectrum was measured by a fluorescence spectrophotometer. The protein sample was diluted to a concentration of 0.5 mg/mL using 0.01 mol/L PBS (pH 7.0). The excitation wavelength was set to 280 nm, the emission wavelength was set to 290-420 nm, the slit width was set to 5 nm, and the operating speed was set to 240 nm/s to scan the endogenous fluorescence spectrum.

Results: The intrinsic fluorescence spectra of the co-extruded WPI-Cys sample are shown in Figure 5 (A). As can be seen from the figure, compared with the WPI sample, the maximum absorption wavelengths of T+C ₀ and co-extruded WPI-Cys did not change significantly, indicating that there was no red shift or blue shift. This result shows that neither single extrusion nor co-extrusion can change the polarity of the microenvironment of tryptophan residues. In addition, as the amount of Cys added increased from 0 to 100 mmol/L, the fluorescence intensity of the sample gradually increased, and the fluorescence intensity of T+C ₁₀₀ reached the maximum. Compared with the single cold extrusion sample, the fluorescence intensity of the T+C ₁₀₀ sample increased by 92.54% (P < 0.05). The possible reason is that single extrusion causes the tryptophan (Trp) in the hydrophobic group inside the protein to be exposed, and the fluorescence intensity increases. The co-extrusion treatment can destroy the disulfide bonds of the protein molecules through the SH/SS exchange reaction, which further promotes the exposure of Trp inside the protein, resulting in a greater increase in fluorescence intensity.

6. Surface hydrophobicity

The surface hydrophobicity of proteins was determined using 8-aniline-1-naphthalenesulfonic acid (ANS) as a fluorescent probe. Protein samples with concentrations of 5, 10, 15, 20 and 25 mg/mL were prepared using 0.01 mol/L PBS buffer (pH 7.0). 20 μL of ANS (8 mmol/L PBS buffer) was added to 4 mL of protein solution and reacted in the dark at room temperature for 15 min. A Hitachi F4500 fluorescence spectrometer (Hitachi, Science Systems, Ibaraki, Japan) was used to determine the fluorescence intensity at excitation and emission wavelengths of 390 nm and 470 nm with a slit width of 5 nm. The surface hydrophobicity (H ₀ ) was expressed as the initial slope of fluorescence intensity versus protein concentration.

Results: The H ₀ of the co-extruded WPI-Cys sample is shown in Figure 5 (B). It can be seen that the H ₀ of WPI and T+C ₀ are very small. However, as the Cys concentration increases from 20 to 100 mmol/L, the H ₀ of the co-extruded WPI-Cys sample shows a significant increase trend, and its H ₀ increases by 157.88%, 186.75%, 193.05%, 198.71% and 205.07% compared with WPI, respectively. It may be due to the shear force, pressure and heat during the co-extrusion process that destroyed the tertiary structure of WPI, resulting in the exposure of the hydrophobic area hidden in the spherical structure and the increase of hydrophobic binding sites on the molecular surface.

7. Emulsification properties

3 mL of protein solution (5 mg/mL) and 1 mL of soybean oil were mixed and homogenized at 12000 g for 2 min using a high-speed homogenizer (IKA, T18 digital Ultra-turrax, Germany). The newly prepared emulsion (50 μL) was kept for 0 and 10 min, respectively, and then immediately dispersed into 5 mL of 1 mg/mL SDS solution. Then, the absorbance of the emulsion at 500 nm was recorded.

The emulsifying activity index (EAI) (m ² /g) is defined as:

表示油的体积分数。Where: _A0 represents the initial absorbance measured at 500nm, C represents the concentration of the protein solution (g/mL),

Indicates the volume fraction of oil.

The Emulsion Stability Index (ESI) (%) is defined as:

Where: _A0 represents the initial absorbance measured at 500 nm, and _A10 is the absorbance measured after the emulsion has been kept for 10 min.

Results: The effect of co-extrusion on the emulsification properties of WPI-Cys samples is shown in Figure 6 (A) and (B). As can be seen from Figure 6 (A), the EAI of T+C ₀ increased by 32.37% (P < 0.05) compared with WPI. This is because cold extrusion enlarged the total surface area of WPI and improved its fluidity and adsorption capacity at the oil-water interface. When the Cys concentration varied in the range of 20-100 mmol/L, the EAI of the co-extruded samples increased by 37.39%, 46.13%, 49.48%, 60.19% and 77.51% (P < 0.05) compared with WPI, respectively. This may be because co-extrusion further promoted the change of the tertiary structure of protein molecules and the increase of the total surface area, while increasing the flexibility of the molecules.

As can be seen from Figure 6 (B), the ESI of T+C ₀ increased by 82.15% compared with WPI. However, as the amount of Cys added increased from 20 to 100 mmol/L, the ESI of the co-extruded WPI-Cys samples increased by 136.41%, 157.66%, 187.74%, 193.00% and 193.95% (P < 0.05), respectively, and the ESI of T+C ₁₀₀ reached the maximum value. The reason for this result is that the addition of Cys promotes the polymerization of protein molecules and the formation of viscoelastic films, and as the amount of Cys added increases, the content of intermolecular disulfide bonds increases, increasing the viscoelasticity of the interfacial film, thereby making the formed emulsion more stable. In addition, the addition of Cys promotes the increase of the free thiol content, increases the absolute value of the surface net negative charge and Zeta-potential, enhances the electrostatic repulsion between protein molecules, inhibits the aggregation of oil droplets and promotes the formation of a stable emulsion, so T+C ₁₀₀ has the highest ESI.

8. Solubility

A protein sample with a concentration of 10 mg/mL was centrifuged at 8000 g for 15 min at room temperature, and the protein content in the supernatant was determined using the biuret method. The protein content in the supernatant was expressed as a percentage of the total protein content in the solution to indicate the sample solubility.

Results: The solubility of the co-extruded WPI-Cys sample is shown in Figure 7(A). It can be seen that both single extrusion and co-extrusion resulted in a decrease in the solubility of the sample. When the concentration of Cys increased from 0 to 100 mmol/L, the solubility of the WPI-Cys sample decreased by 15.39%, 19.22%, 26.68%, 25.38%, 29.86% and 32.18% (P < 0.05) compared with WPI, respectively. This is because during the co-extrusion process, as the tertiary structure of the protein unfolds, Cys immediately undergoes SH/S-S exchange reaction with the exposed disulfide bonds, resulting in aggregation and polymerization of protein molecules and preventing the renaturation of WPI. As the Cys concentration increases, the covalent cross-linking of WPI gradually increases, and the solubility decreases more significantly.

9. Free thiol:

The free thiol groups of the sample were determined using Ellman's (DTNB) reagent. The protein sample was diluted to a concentration of 2 mg/mL with Tris-Gly buffer at pH 8.0. 30 μL of DTNB (40 mg of DTNB/10 mL of Tris-Gly buffer) was added to 3 mL of the protein solution. After the mixture was incubated at ambient temperature for 15 min, the absorbance was measured at 412 nm. The free thiol content (μmol/g protein) in the sample was calculated according to the following formula.

Where: D-dilution factor; C-protein concentration (mg/mL).

Results: The three major whey proteins (β-Lg, α-La and BSA) contained different numbers of Cys residues per mole of whey protein (5 mol in β-Lg, 8 mol in α-La and 35 mol in BSA). The results of free thiol groups are shown in Figure 7 (B). The free thiol groups of WPI were 20.41 μmol/g, and after single extrusion, the free thiol groups decreased by 8.67%. This may be due to the fact that cold extrusion promoted the thiol oxidation reaction of β-Lg molecules and reduced the thiol content on the protein surface by forming intermolecular disulfide bonds. The free thiol contents of the co-extruded samples T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ were 2.05, 2.88, 2.89, 5.02 and 6.25 times that of WPI, respectively (P < 0.05). This may be because when WPI is co-extruded with Cys, as the tertiary structure of the protein unfolds, the small molecule Cys can quickly undergo SH/SS exchange reactions with the exposed intramolecular disulfide bonds. This causes the free thiol groups located inside the molecule to move to the surface, thereby increasing the number of free thiol groups. In addition, as the Cys concentration increases, the rate of SH/SS exchange reactions increases. The T+C ₁₀₀ sample has the highest free thiol content of 147.95 μmol/g.

10. Secondary structure

The protein samples were scanned over the full wavelength range (4000-400 cm-1) using a Fourier transform infrared spectrometer (FTIR) analyzer (Nicolet, Madison, USA). 2 mg of protein sample was mixed with 200 mg of KBr, ground into a uniform powder using an agate mortar, and then pressed into thin sheets. The resolution was set to 4 cm ^-1 and 32 scans were performed. The secondary structure elements were quantified by deconvolution and multi-band fitting using peakfit 4 software. The chromatographic peaks in the second-order derivative spectrum were identified according to the following intervals: α-helix (~1660 cm ^-1 ), β-sheet (1640-1630 and 1620-1610 cm ^-1 ), β-turn (1690-1670 ^cm-1 ) and random coil (~1650 cm ^-1 ).

The FTIR spectrum results of the co-extruded WPI-Cys sample in the amide I region are shown in Figure 8 (A). In particular, the deconvoluted FTIR spectrum of the T+C ₁₀₀ sample in the amide I region is shown in Figure 8 (B). The contents of α-helix, β-sheet, β-turn and random coil in all protein samples are shown in Figure 8 (C), (D), (E) and (F), respectively. The stretching vibration of the carbon-nitrogen bond and the carbon-oxygen double bond leads to significant differences in the absorption peaks of the protein samples in the amide I region. By deconvoluting the amide I region sample T+C ₁₀₀ and fitting it using the Gaussian area method, 9 peaks were fitted, namely 1614, 1624, 1633, 1643, 1687, 1652, 1661, 1679 and 1670 cm ^-1 . The α-helix content in the WPI sample is 17.61%. Compared with WPI, the α-helix content of T+C ₀ decreased by 1.57%. The α-helix of T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ decreased by 2.38%, 3.09%, 3.21%, 4.01% and 4.46% respectively (P＜0.05). This shows that the α-helix content decreases sharply with the increase of Cys addition. Among whey protein molecules, α-La is the main α-helix protein. It may be that the free sulfhydryl group in Cys opens the intramolecular disulfide bond of α-La through SH/SS exchange reaction, thereby destroying the ordered secondary structure of α-La. The β-fold content in WPI is 43.62%. Compared with WPI, the β-fold content of T+C ₀ and T+C ₂₀ decreased by 4.42% and 4.44% respectively, and the difference in the degree of reduction of β-fold protein content between the two was not significant. With the increase of Cys concentration, the β-fold of T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples decreased by 6.10%, 7.56%, 8.15% and 9.55% respectively compared with WPI (P＜0.05). β-Lg is the main β-fold protein in whey protein. The high concentration of Cys may destroy the disulfide bonds in the β-Lg molecule, resulting in the change of β-Lg secondary structure and the reduction of β-fold content.

The sum of the α-helix and β-sheet contents is the ordered secondary structure content of the protein, and the ordered secondary structure of the WPI sample accounts for 61.23%. The ordered structure content of T+C ₀ decreased by 5.99%, and the ordered structure contents of T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ decreased by 6.82%, 9.19%, 10.76%, 12.15% and 14.00% respectively (P＜0.05). The reason may be that the addition of Cys leads to a reduction in protein disulfide bonds, significantly reduces the content of α-helix and β-sheet, and breaks the ordered secondary structure. With the increase of Cys concentration, the ordered structure of the protein gradually decreases. The content of disordered structure (β-turn and random coil) in the WPI sample accounts for 38.77%. Compared with WPI, the disordered structure content of T+C ₀ increased by 5.99%, and the disordered structure contents of T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ increased by 6.82%, 9.19%, 10.76%, 12.15% and 14.00% respectively (P＜0.05). Whether it is single extrusion or co-extrusion, the secondary structure of WPI gradually changes from highly ordered to disordered structure. Moreover, with the increase of Cys addition, the disordered structure content increases.

11. Gel moisture distribution

Table 5 _T2 relaxation time and _P2 peak area percentage of water in co-extruded WPI-Cys sample gel

8 mL of protein sample solution (12%) was placed in a 10 mL beaker, incubated in a 90°C water bath for 10 min, cooled in an ice water bath for 15 min, and stored at 4°C overnight for testing. Relaxation measurements of protein gels were performed at 25°C using a low-field nuclear magnetic resonance imaging analyzer (Meso MR 23-060H-I).

Relaxation time measurement: Set the magnetic field strength to 0.5T, the operating frequency to 23MHz, and use the CPMG sequence (pulse interval time is 1ms, repetition time between scans is 2s) to measure, obtain 3000 echo data, and perform 16 scan repetitions. Use MultiExp InvAnalysis software to invert the data and obtain the corresponding relaxation time in the decay curve. Among them, T ₂₁ , T ₂₂ and T ₂₃ represent the transverse relaxation time of bound water, immobile water and free water, respectively, and the peak areas P ₂₁ , P ₂₂ and P ₂₃ represent the corresponding content percentages of various water.

Results: The transverse relaxation spectra of water in the co-extruded WPI-Cys sample gel and the corresponding transverse relaxation time and content are shown in Figure 9 and Table 5. As can be seen from Figure 9, compared with WPI, the T ₂₂ of the co-extruded WPI-Cys sample is significantly reduced. As can be seen from Table 5. As the Cys concentration increases from 20 to 100 mmol/L, the transverse relaxation time T ₂₂ of the immobile water decreases by 4.21, 4.22, 11.75 and 15.28 ms, respectively, while the peak area P ₂₂ increases by 0.40, 0.48, 1.34 and 1.16%, respectively (P < 0.05). This shows that co-extrusion increases the immobile water content in the protein gel, and as the Cys concentration increases, the binding force of the protein molecules on the immobile water gradually increases.

As can be seen from Table 5, in the co-extruded samples, as the Cys concentration increases, the content of free water P ₂₃ in the gel gradually decreases. When the Cys concentration increases from 20 to 100 mmol/L, T ₂₃ decreases from 1162.32 ms to 714.94 ms, the free water content P ₂₃ decreases from 2.01 to 0.58%, and the water molecules in the gel lose their mobility. These results indicate that co-extrusion induces the formation of hydrogen bonds between peptide chains and water molecules in protein gels, which increases the binding of water molecules, thereby significantly improving the water retention of protein gels.

12. Simulated gastric and intestinal digestibility in vitro

Simulated gastric fluid (SGF) digestion: The protein sample solution (3 mg/mL) was adjusted to pH 1.5 with 1.0 M HCl, and a pepsin stock solution (5 mg/mL 0.01 M HCl) was added, and the mixture was shaken at 37°C for 30 min. Subsequently, the gastric digestion product was adjusted to pH 6 with 1.0 M NaOH to inactivate pepsin and interrupt the digestion process to obtain a gastric digest.

Simulated intestinal fluid (SIF) digestion: The gastric digest was adjusted to pH 7.8 with 1.0 M NaOH solution. Trypsin stock solution (5 mg/mL PBS buffer) was added to the pepsin pre-digest and shaken at 40°C for 60 min. Subsequently, 150 mmol/L Na ₂ CO ₃ solution was added to terminate the reaction and obtain the intestinal digest.

Determination of digestibility: The protein content in the digestive juice was determined by the biuret method, and a standard curve was prepared using BSA. The in vitro digestibility (%) was calculated using the following formula:

Results: The gastric and intestinal simulated digestibility results of the co-extruded WPI-Cys samples are shown in Figure 10 (A) and (B). As can be seen from the figure, during gastric digestion, the digestibility of the co-extruded WPI-Cys sample was significantly higher than that of WPI and T+C ₀ , and the increase in the digestibility of the co-extruded sample depended on the concentration of Cys. Specifically, when the Cys concentration increased from 0 to 60mmol/L, the gastric digestibility of WPI-Cys increased by 24.82%, 32.63%, 37.38% and 61.99% compared with WPI, respectively. (P < 0.05). This is because co-extrusion causes the tertiary structure of the protein molecule to unfold, and the free sulfhydryl groups in the Cys molecules quickly undergo SH/SS exchange reactions with the exposed intramolecular disulfide bonds, resulting in irreversible conformational changes in the molecular structure, exposing the hydrophobic amino acids that interact with pepsin, and increasing the gastric digestibility. However, when the Cys concentration exceeds the critical point (60mmol/L), the gastric digestibility will not continue to increase. This may be due to the formation of extensive covalent cross-links between protein molecules, which hinder the entry of pepsin. Similarly, in simulated intestinal digestion, as the Cys concentration increased from 0 to 40 mmol/L, the intestinal digestibility gradually increased.

13. Simulate the antioxidant properties of gastric and intestinal digestive matter

Take 1 mL of the protein digestion product (3 mg/mL) and mix it with 1 mL of 0.2 M PBS (pH 6.6) and 1 mL of 1% potassium ferricyanide (K ₃ Fe(CN ₆ )). The mixture is reacted in a 50°C water bath for 20 min, and 1 mL of 10% trichloroacetic acid is added. Then, centrifuge at 750 g for 10 min at 25°C, and the obtained supernatant (1 mL) is treated with 1 mL of distilled water and 200 μL of 0.1% FeCl ₃ , and the absorbance of the reaction mixture is measured at 700 nm. The OD ₇₀₀ value is used as a standard for measuring reducing power.

Results: The antioxidant properties of simulated gastric and intestinal digesta of coextruded WPI-Cys samples were expressed as iron reducing capacity, and the results are shown in Figure 11 (A) and (B). After 30 min of gastric digestion, the ability of coextruded WPI-Cys digest to reduce Fe ³⁺ was significantly higher than that of WPI and T+C _0. Specifically, as the Cys concentration increased from 0 to 100 mmol/L, the iron reducing power in gastric digesta increased by 26.35%, 40.63%, 48.57%, 51.43%, 53.65% and 54.6% (P < 0.05) compared with WPI, respectively. During 60 min of intestinal digestion, as the Cys concentration increased from 0 to 100 mmol/L, the iron reducing capacity of coextruded WPI-Cys samples increased by 12.92, 34.83, 37.64, 38.76, 42.32 and 44.57%, respectively, compared with WPI. The possible reason is that the reducing ability of iron is related to the electron donating ability of peptides. As the Cys concentration increases, the digestibility increases, and more amino acid residues such as Cys, methionine (Met), Trp, and tyrosine (Tyr) are produced in the digestion products, which can donate electrons and play a reducing role.

14. Inhibition rate of α-glucosidase activity in simulated gastric and intestinal digesta

100 μL of α-glucosidase (0.075u/mL) was premixed with the sample (50 μL), and 3 mmol/L p-nitrophenyl-β-D-pyranoglucoside (pNPG) was added to the mixture as a PBS substrate and incubated at 37°C for 30 min. 2 mL of 0.1 M Na ₂ CO ₃ was added to terminate the reaction, and the α-glucosidase activity was characterized by measuring the absorbance of p-nitrophenol released by pNPG at 400 nm. The α-glucosidase inhibitory activity was calculated according to the following formula:

Where: A _control is the absorbance value when no sample is added, and A _sample is the absorbance value of the sample.

Results: The α-glucosidase inhibition rates of simulated gastric and intestinal digesta of the co-extruded WPI-Cys samples are shown in Figure 12 (A) and (B). As can be seen from Figure 12, the α-glucosidase inhibition rates of simulated gastric and intestinal digesta of the co-extruded samples were significantly increased compared with WPI and T+C ₀ (P < 0.05). In addition, as the Cys concentration increased from 0 to 80 mmol/L, the α-glucosidase inhibition rates of gastric digesta increased by 14.56%, 24.60%, 24.80%, 25.24% and 29.92% respectively compared with WPI, and the α-glucosidase inhibition rates of intestinal digesta increased by 9.97%, 17.57% and 19.68%, 23.35% and 25.25% respectively (P < 0.05). However, when the Cys concentration exceeded 80 mmol/L, there was no significant difference in the α-glucosidase inhibition rate (P> 0.05). The peptide can bind to the active site of the α-glucosidase inhibitory rate through hydrophobic interactions, thereby delaying the absorption of glucose and acting as a competitive inhibitor. Co-extrusion treatment exposes more hydrophobic side chains of WPI molecules, which facilitates the interaction between the peptide and α-glucosidase, thereby inhibiting the activity of α-glucosidase.

15. Xanthine oxidase (XOD) inhibition rate of simulated gastric and intestinal digesta

Take 100 μL of XOD solution (0.2u/mL 0.2M PBS pH7.5) and add it to 100 μL of sample to mix. Add 200 μL of substrate xanthine solution (0.04mmol/L) to the mixture to initiate the reaction and incubate at 37°C for 15 min. Add 200 μL of 1M HCl to the reaction system to terminate the reaction and measure its absorbance at 290nm. XOD inhibitory activity is calculated according to the following formula:

Where: XOD _blank is the absorbance value of the blank group; XOD _sample is the absorbance value of the sample group.

Results: The XOD inhibition rates of simulated gastric and intestinal digesta of co-extruded WPI-Cys samples are shown in Figure 13 (A) and (B). As can be seen from the figure, as the concentration of Cys gradually increased from 0 to 100mmol/L, the XOD inhibition rate in the stomach and intestine gradually increased (P < 0.05). Trp can inhibit XOD and is a non-competitive inhibitor of XOD. Co-extrusion promotes the unfolding of the WPI molecular structure, thereby promoting the exposure of Trp and enhancing the inhibitory activity of XOD.

16. Correlation between structural and functional properties of co-cold extruded whey protein

The results of this study are expressed as mean ± standard deviation (three replicates, n = 3). SPSS software 17.0 (SPSS, Inc., Chicago, IL, USA) was used to perform a one-way analysis of variance (ANOVA) and Duncan's multiple comparison method (P < 0.05), and the differences were marked with different letters. The Pearson correlation coefficient method was used to analyze the correlation between the structure and functional properties of cold-extruded and modified whey protein. Origin 2021Pro software was used for drawing.

Results: The Pearson linear correlation coefficient (γ) between the structure and functional properties of co-extruded WPI-Cys is shown in Figure 14. It can be seen that the emulsifying activity of co-extruded whey protein is strongly positively correlated with surface hydrophobicity, free thiol (γ＝0.91, P＜0.05), β-turn and random coil, and is strongly negatively correlated with solubility, potential (γ＝－0.81, P＜0.05), α-helix and β-fold. The emulsification stability is strongly positively correlated with surface hydrophobicity (γ＝0.94, P＜0.05), free thiol, β-turn and random coil, and is strongly negatively correlated with solubility, potential, α-helix and β-fold. This may be because co-extrusion unfolds the tertiary structure of whey protein, enhances surface hydrophobicity, promotes the disulfide bonds of protein molecules through SH/S-S exchange reaction, releases free thiol, reduces Zeta-potential, and enhances intermolecular electrostatic repulsion, thereby improving emulsification activity and emulsification stability. The immobile water content was strongly positively correlated with surface hydrophobicity (γ＝0.94, P＜0.05), free thiol (γ＝0.88, P＜0.05), β-turn and random coil, and was strongly negatively correlated with solubility, potential, α-helix and β-sheet. The correlation between free water content and co-extruded WPI-Cys structure was opposite to the correlation between immobile water content and co-extruded WPI-Cys structure. This may be because co-extruded WPI-Cys promoted the release of free thiol groups from whey protein molecules, strengthened the hydrogen bonding between protein side chains and water molecules, and strengthened the binding of water molecules, thereby increasing the immobile water content and reducing the free water content.

In summary, the following contents are achieved by using the modification method of the present invention:

Disulfide cross-linking: A total of 52 peptides cross-linked by disulfide bonds were detected in WPI, T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ samples. Among them, 29 were intermolecular disulfide cross-linked peptides, 12 were intramolecular recombinant disulfide cross-linked peptides, 4 were intramolecular original or recombinant disulfide cross-linked peptides, and 7 were natural intramolecular original disulfide cross-linked peptides. There were 5 common intermolecular disulfide cross-linked peptides in all extruded samples, of which 3 intermolecular disulfide cross-links occurred between α-La and β-Lg molecules, and 2 intermolecular disulfide cross-links occurred between α-La and α-La, indicating that α-La is more prone to intermolecular disulfide cross-linking than β-Lg.

Size exclusion chromatography: No substances with molecular weight higher than BSA (about 66 kDa) were eluted in the WPI sample, while the peak areas of high molecular weight substances in T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ increased by 50.05%, 50.23%, 76.01%, 139.68% and 164.94% (P < 0.05) respectively compared with WPI, indicating that co-extrusion promoted intermolecular aggregation or polymerization of proteins.

SDS-PAGE electrophoresis: After all protein samples were treated with reducing agents, the precipitates at the top comb part of the lane disappeared, indicating that the insoluble macromolecular polymers were mainly polymerized through intermolecular disulfide bonds. In the co-extruded samples, the band with a strong optical density of 37 kDa molecular weight was not detected, indicating that the dimer formed by β-Lg was mainly linked by covalent bonds.

Particle size potential: With the increase of Cys concentration, the particle size of protein samples gradually increases. With the increase of Cys addition, the absolute value of Zeta potential also gradually increases. When the Cys concentration increases to 100mmol/L, the absolute value of Zeta potential is the largest. This shows that during the co-extrusion process, Cys reduces the disulfide bonds in the protein molecules, releases free thiol groups in the molecules, promotes intermolecular hydrophobic interactions and SH/S-S exchange reactions, and thus causes extensive aggregation and polymerization of protein molecules, which significantly increases the protein particle size. At the same time, free thiol groups are exposed to the WPI surface, making the surface of the protein carry more negative charges, resulting in an increase in the absolute value of Zeta potential.

Endogenous fluorescence spectrum: When the Cys concentration increased to 100mmol/L, the fluorescence intensity of the sample was the highest. Compared with the single cold extrusion sample, the fluorescence intensity of the T+C ₁₀₀ sample increased by 92.54% (P<0.05). This shows that the cold extrusion with Cys can promote the SS rupture of protein molecules, exposing more hydrophobic amino acids (tryptophan) than the single cold extrusion, thereby increasing the fluorescence intensity of the sample.

Surface hydrophobicity (H ₀ ): WPI had the smallest H ₀ , and after single extrusion, WPI's H ₀ increased by 10.07%; in the co-extruded whey protein samples T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ , H ₀ gradually increased by 157.88%, 186.75%, 193.05%, 198.71% and 205.07% (P < 0.05). This indicates that the shear force, pressure and heat during co-extrusion fully destroyed the tertiary structure of WPI, resulting in the exposure of the hydrophobic region buried in the globular structure and the increase of hydrophobic binding sites on the molecular surface. With the increase of Cys concentration, the hydrophobic region was exposed more and the surface hydrophobicity was further increased.

Emulsification performance: When the Cys concentration increased from 0 to 100 mmol/L, the EAI of the WPI sample increased by 32.37%, 37.39%, 46.13%, 49.48%, 60.19% and 77.51% compared with WPI, respectively. This indicates that the co-extrusion of WPI and Cys further promotes the change of the tertiary structure of the protein molecule and the increase of the total surface area. At the same time, the increase in surface hydrophobicity leads to higher surface activity of the protein molecule, thereby increasing the EAI of the protein molecule. Similarly, with the increase in the amount of Cys added, the ESI of the co-extruded WPI-Cys showed an upward trend, especially the ESI of T+C ₁₀₀ was 193.95% higher than that of WPI (P<0.05). The reason for this result is that with the increase in the amount of Cys added during the co-extrusion process, the content of disulfide bonds between molecules increases, which can promote the increase of the viscoelasticity of the interfacial film, thereby making the formed emulsion more stable. Furthermore, the addition of Cys promoted the increase of free thiol content, increased the net negative charge on the surface and the absolute value of zeta potential, which enhanced the electrostatic repulsion between protein molecules, inhibited the aggregation of oil droplets and promoted the formation of stable emulsions. Therefore, coextrusion improved the EAI and ESI of WPI molecules.

Solubility: When the concentration of Cys increased from 0 to 100 mmol/L, the solubility of WPI-Cys samples decreased by 15.39%, 19.22%, 26.68%, 25.38%, 29.86% and 32.18%, respectively, compared with WPI (P < 0.05).

Free thiol: The free thiol of WPI is 20.41 μmol/g, and after single extrusion, the free thiol decreased by 8.67%. The free thiol content of co-extruded samples T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ are 2.05, 2.88, 2.89, 5.02 and 6.25 times that of WPI, respectively (P < 0.05), and the free thiol content of T+C ₁₀₀ sample is the highest.

Secondary structure: Compared with WPI, the α-helix content of T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ decreased by 1.57%, 2.38%, 3.09%, 3.21%, 4.01% and 4.46%, respectively (P<0.05). The β-sheet content in WPI was 43.62%, and compared with WPI, the β-sheet content of T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ decreased by 4.42%, 4.44%, 6.10%, 7.56%, 8.15% and 9.55%, respectively (P<0.05). Compared with WPI, the disordered structure contents of T+C ₀ , T+C ₂₀ , T+C ₄₀ , T+C ₆₀ , T+C ₈₀ and T+C ₁₀₀ increased by 5.99%, 6.82%, 9.19%, 10.76%, 12.15% and 14.00% respectively (P＜0.05). Whether extruded alone or co-extruded, the secondary structure of WPI gradually changed from highly ordered to disordered structure. Moreover, with the increase of Cys addition, the disordered structure content increased.

Gel water retention: As the Cys concentration increased from 20 to 100 mmol/L, the transverse relaxation time T ₂₂ of immobile water decreased by 4.21, 4.22, 11.75 and 15.28 ms, respectively, while the peak area P ₂₂ increased by 0.40, 0.48, 1.34 and 1.16% (P < 0.05), respectively. This indicates that the content of immobile water in the co-extruded WPI gel increased, and as the Cys concentration increased, the binding force of protein molecules on immobile water gradually increased. Therefore, co-extrusion can induce the formation of hydrogen bonds between peptide chains and water molecules in protein gels, increase the binding of water molecules, and thus significantly improve the water retention of protein gels.

Simulated gastric and intestinal digestibility in vitro: The digestibility of the co-extruded WPI-Cys samples was significantly higher than that of WPI or EWPI during simulated gastric and intestinal digestion. Specifically, when the Cys concentration increased from 0 to 60 mmol/L, the gastric digestibility of WPI-Cys increased by 24.82%, 32.63%, 37.38%, and 61.99% (P < 0.05) compared with WPI, respectively. Similarly, in simulated intestinal digestion, as the Cys concentration increased from 0 to 40 mmol/L, the intestinal digestibility gradually increased.

Antioxidant activity of simulated gastric and intestinal digesta: After 30 min of gastric digestion, the ability of co-extruded WPI-Cys digesta to reduce Fe ³⁺ was significantly higher than that of WPI and T+C _0. Specifically, as the Cys concentration increased from 0 to 100 mmol/L, the iron reducing power in gastric digesta increased by 26.35%, 40.63%, 48.57%, 51.43%, 53.65% and 54.6% (P < 0.05) compared with WPI, respectively. Moreover, during 60 min of intestinal digestion, as the Cys concentration increased from 0 to 100 mmol/L, the iron reducing power of co-extruded WPI-Cys samples increased by 12.92, 34.83, 37.64, 38.76, 42.32 and 44.57%, respectively, compared with WPI.

α-glucosidase activity inhibition rate of simulated gastric and intestinal digesta: As the Cys concentration increased from 0 to 80mmol/L, the α-glucosidase inhibition rate of gastric digesta increased by 14.56%, 24.60%, 24.80%, 25.24% and 29.92% respectively compared with WPI, while the α-glucosidase inhibition rate of intestinal digesta increased by 9.97%, 17.57% and 19.68%, 23.35% and 25.25% respectively (P<0.05). However, when the Cys concentration exceeded 80mmol/L, there was no significant difference in the α-glucosidase inhibition rate (P>0.05).

Xanthine oxidase inhibition rate in simulated gastric and intestinal digesta: As the concentration of Cys increased from 0 to 100 mmol/L, the XOD inhibition rate in the stomach and intestines gradually increased (P < 0.05).

The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (10)

1. A method for modifying whey protein, characterized in that the whey protein and cysteine are cold-extruded together to allow cysteine to enter the protein molecule, thereby achieving the modification of the whey protein. 2. The method for modifying whey protein according to claim 1, characterized in that it comprises the following steps: adding the whey protein to a twin-screw extruder through a feed screw, and simultaneously adding a cysteine aqueous solution to the twin-screw extruder at the same feeding speed as the whey protein for co-extrusion to obtain a whey protein cold extrusion product; The whey protein cold extrusion product is dried to obtain modified whey protein. 3. The method for modifying whey protein according to claim 2, wherein the feeding rate of the whey protein is 3-4 kg/h. 4. The method for modifying whey protein according to claim 2, characterized in that the concentration of the cysteine aqueous solution is 20-100 mmol/L. 5. The method for modifying whey protein according to claim 2, characterized in that the water content of the whey protein cold extrusion product is 45-55wt%. 6. The method for modifying whey protein according to claim 2, characterized in that the drying treatment is drying or freeze drying; and the drying treatment further comprises the steps of crushing and screening. 7. The method for modifying whey protein according to claim 2 is characterized in that no heating is performed during the co-extrusion process; the temperature of the feeding zone of the twin-screw extruder is set to 25°C, the temperature of the mixing zone is set to 30°C, the temperature of the cooking zone is set to 35°C, and the temperature of the discharge zone is not higher than 50°C. 8. The method for modifying whey protein according to claim 2, characterized in that the screw diameter of the twin-screw extruder is 25 mm, the aspect ratio is 24:1, and the screw speed is 250-350 r/min. 9. Modified whey protein prepared according to the method for modifying whey protein according to any one of claims 1 to 8. 10. Use of the modified whey protein according to claim 9 in the food industry.

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