CN113142378B - Modification method of whey protein - Google Patents

Abstract

The invention discloses a method for modifying whey protein, which belongs to the field of protein processing and comprises the following steps: preparing whey protein hydrate with water content of 45-55 wt%; carrying out cold extrusion on the whey protein hydrate to obtain a whey protein cold extrusion product; drying and crushing to obtain crushed extruded whey protein powder; preparing an extruded whey protein aqueous solution with the concentration of 3-7 wt%, and adding cysteine to the aqueous solution until the concentration is 12-18 mmol/L; and then reacting for 8-12min at the temperature of 55-65 ℃, and immediately transferring into an ice water bath to terminate the reaction to obtain the modified whey protein. The invention adopts a cold extrusion method to open the tertiary structure of the lactalbumin, and fully exposes internal hydrophobic groups and disulfide bonds; cys is added as a free sulfydryl source and performs a sulfydryl disulfide exchange reaction with disulfide bonds in protein to form a macromolecular cross-linked product, and finally a modified whey protein product with good emulsibility and rheological property is formed.

Description

Modification method of whey protein

Technical Field

The invention relates to the field of protein processing, in particular to a method for modifying whey protein.

Background

Whey protein is a protein obtained by separating whey, which is a byproduct of cheese production, through processes such as ultrafiltration, concentration, spray drying and the like, and contains 48% of beta-lactoglobulin (beta-lg), 19% of alpha-lactalbumin (alpha-la), 5% of Bovine Serum Albumin (BSA), lactoferrin (Lf) enzyme, bioactive factors and the like. The whey protein is easy to digest and has high metabolic efficiency, so the whey protein has high biological utilization value and can be widely applied to the production field of dairy products, meat products and baked foods as a nutritional ingredient.

However, since both β -lg and α -la in whey protein are globulin, hydrophobic groups are distributed inside the molecule and hydrophilic groups are distributed on the surface to form spherical molecules with dense structures, and both emulsifiability and rheological properties are poor. In order to increase the availability of whey protein, improvements in emulsifying properties and rheological properties are required. However, in the research on the extrusion modification of the whey protein at home and abroad, the high temperature mainly focuses on destroying the biological activity of the whey protein through the high-temperature extrusion of the whey protein; when Cys is used alone to modify whey protein, the reaction is carried out at a temperature of more than 70 ℃, so that the biological activity of the whey protein is destroyed, and the nutritional value of the whey protein is reduced.

Cysteine (cyse) molecules have highly reactive sulfhydryl groups that can reduce disulfide bonds located on the surface of a protein, exposing active-SH groups to inter-molecular SH/SS exchange reactions with active-SH groups of another protein, thereby producing covalent crosslinks. However, since whey protein hardly has a good crosslinking effect with SH of Cys due to the rigid spherical structure of α -lactalbumin and β -lactoglobulin, the disulfide bonds in lactoglobulin molecules are mostly located in the molecule, and the like, Cys is not suitable for direct crosslinking with native whey protein, and thus whey protein needs to be denatured to improve the crosslinking reaction with Cys.

The cold extrusion technology is a low-temperature extrusion technology with the temperature lower than 50 ℃, and the three-stage structure of the protein is unfolded through the physical action of high pressure and shearing in the extrusion process, so that hydrophobic groups in the protein molecules are exposed, and the non-covalent crosslinking between the protein molecules through hydrophobic interaction is promoted. However, physical modification alone does not disrupt disulfide bonds and promote extensive covalent cross-linking between protein molecules.

When the method is combined with Cys to treat the whey protein, firstly, the cold extrusion treatment enables the whey protein to expand a three-level mechanism under a low-temperature condition, so that an internal disulfide bond is exposed on the surface of a molecule; the second step is that: the active sulfydryl on the Cys molecule and the disulfide bond exposed by cold extrusion treatment have SH/SS exchange reaction at lower temperature to initiate the extensive covalent crosslinking among protein molecules. Therefore, the cold extrusion combined with the Cys treatment is a method combining physical action and low-temperature chemical action, enhances the non-covalent and covalent interactions among whey protein molecules on the basis of protecting the biological activity of the whey protein, improves the emulsibility and rheological property of the whey protein and improves the cell activity.

Disclosure of Invention

The present invention has been made in view of the above problems of the prior art, and an object of the present invention is to provide a method for modifying whey protein.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a method for modifying whey protein, which comprises the following steps:

(1) preparing whey protein hydrate with water content of 45-55 wt%;

(2) carrying out cold extrusion on the whey protein hydrate by using a double-screw extruder to obtain a whey protein cold extrusion product; the screw rotating speed of the double-screw extruder is 250-350r/min, and the feeding speed is 3-4 kg/h;

heating is not carried out in the cold extrusion process, and the temperature of a discharge port of the double-screw extruder is not higher than 50 ℃;

(3) drying and crushing the whey protein cold extrusion product to obtain an extruded whey protein powder crushed material;

(4) preparing the extruded whey protein powder crushed material into an extruded whey protein water solution with the concentration of 3-7 wt%; adding cysteine into the extruded whey protein aqueous solution to ensure that the concentration of the cysteine in the extruded whey protein aqueous solution is 12-18 mmol/L; and then reacting for 8-12min at the temperature of 55-65 ℃, and immediately transferring into an ice water bath to terminate the reaction to obtain the modified whey protein.

Further, in the step (1), the water content of the whey protein hydrate is 50 wt%.

Further, in the step (2), the feed port temperature of the twin-screw extruder was 22 ℃.

Further, in the step (2), the screw rotating speed of the double-screw extruder is 300r/min, and the feeding speed is 3.5 kg/h.

Further, in the step (3), the drying is carried out for 20-28h at 35-45 ℃ until the water content of the whey protein cold extrusion product is 5 wt%; the crushing is to be crushed to a particle size of less than 0.5 mm.

Further, in step (4), the concentration of the extruded whey protein aqueous solution is 5 wt%; the concentration of cysteine in the extruded whey protein aqueous solution is 15 mmol/L; the reaction is carried out for 10min at 60 ℃.

The invention also provides a modified whey protein obtained by the method for modifying whey protein, wherein the modified whey protein contains the following intermolecular disulfide bond cross-linked peptide segments:

WENDECAQK(6)-CEVFR(1)

LSFNPTQLEEQCHI(12)-CEVFR(1)

WENDECAQK(6)-ALCSEK(3)

LSFNPTQLEEQCHI(12)-ALCSEK(3)

LDQWLCEK(6)-LDQWLCEK(6)

LSFNPTQLEEQCHI(12)-LDQWLCEK(6)

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

the invention discloses the following technical effects:

cysteine (Cys) is a safe food additive with free sulfydryl, and the free sulfydryl in the Cys and a protein disulfide bond generate extensive sulfydryl/disulfide bond exchange reaction and sulfydryl oxidation reaction, so that the whey protein generates intermolecular crosslinking to generate a macromolecular crosslinking product, and the food additive has the advantages of safety, easiness in obtaining and wide crosslinking. The invention adopts a cold extrusion method to open the tertiary structure of the lactalbumin, and fully exposes internal hydrophobic groups and disulfide bonds; cys is added as a free sulfydryl source and generates sulfydryl disulfide exchange reaction with disulfide bonds in protein to form a macromolecular cross-linked product, and finally a modified whey protein product with good emulsifying property and rheological property is formed.

Compared with the untreated raw material WPI, the TWPI-Cys intermolecular disulfide crosslinking of the raw material TWPI-Cys treated by cold extrusion combined with Cys is more extensive:

in untreated WPI, a total of 5 intermolecular disulfide-crosslinked peptide fragments were identified, each

In the TWPI-Cys material treated by cold extrusion combined with Cys, the 13 intermolecular disulfide-crosslinked peptide fragments were identified.

Infrared chromatography: compared with the secondary structure of WPI, in the TWPI-Cys sample treated by cold extrusion and Cys, the ordered secondary structure alpha-helix and beta-fold are respectively reduced by 2.64 percent and 3.71 percent; disordered secondary structure β -turns and random curls increased by 1.77% and 4.58%, respectively.

Rheological properties: the gel onset temperature of WPI was about 74 ℃. However, cold pressing combined with Cys treatment significantly (P <0.05) reduced the gel onset temperature of the TWPI-Cys samples to 57 ℃. At 85 deg.C, the Cys-treated TWPI's G' and G 'were maximal, increasing 22.41-fold and 18.18-fold over WPI's G 'and G', respectively (P < 0.05).

No significant high molecular weight protein was detected in WPI as detected by exclusion chromatography, whereas a peak eluting at 24.21 minutes was detected in TWPI-Cys, these polymer molecules were larger in size than BSA (about 66KDa), indicating that high molecular weight cross-linked protein was formed between the whey protein molecules during the cold-extrusion Cys-binding treatment.

Cell activity: the survival rate of the cells treated by the TWPI-Cys with the concentration of 2.0mg/mL is 190.41 percent after being cultured for 24 hours, which is 26.81 percent higher than that of the WPI; after the cells treated by 2.0mg/mL TWPI-Cys are cultured for 48 hours, the survival rate is 137.20 percent, which is 1.65 percent higher than that of WPI;

emulsibility: the EAI and ESI of WPI were 60.27% and 42.07%, respectively, and the EAI and ESI of TWPI-Cys increased by 10.75% and 12.49%, respectively, after treatment with cold pressing in combination with Cys.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a LC/MS/MS analysis chart of TWPI-Cys; A. total Ion Current (TIC) plot, b. base peak plot, b, y ion match plot of LC/MS cross-linked peptide WENDECAQK (6) -CEVFR (1) extracted by c.plink software;

FIG. 2 is a Fourier infrared chromatogram and a quantitative analysis chart of a protein sample; A. fourier transform infrared spectroscopy of a protein sample in an amide I area, Fourier self-deconvolution chromatography of B.TWPI-Cys, alpha-helix content before and after crosslinking of C.WPI, OWPI and TWPI with Cys, beta-folding content before and after crosslinking of D.WPI, OWPI and TWPI with Cys, beta-turn content before and after crosslinking of E.WPI, OWPI and TWPI with Cys, and irregular convolution content before and after crosslinking of F.WPI, OWPI and TWPI with Cys;

FIG. 3 shows the results of temperature sweep test of protein sample dispersion; WPI, OWPI and TWPI, B.WPI-Cys, OWPI-Cys and TWPI-Cys;

FIG. 4 is a size exclusion chromatogram of a dispersion of a protein sample; WPI-Cys, OWPI-Cys, TWPI-Cys and standard Cys, B.WPI, OWPI and TWPI;

FIG. 5 is a graph of the effect of protein samples on cell viability of HUVEC; A. the culture time is 24h, and the culture time is 48 h;

FIG. 6 is the emulsification properties of protein samples; effect of cys treatment on EAI of TWPI, OWPI and WPI, b. influence of cys treatment on ESI values of TWPI, OWPI and WPI.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

Example 1

A method for modifying whey protein isolate by Cysteine combined with cold extrusion comprises the following steps:

(1) hydration of whey protein:

whey Protein Isolate (WPI) was dissolved in deionized water to form whey protein hydrate with a dry matter content of 50% (W/W) and stored at 4 ℃ for 4 hours to be sufficiently hydrated.

(2) Whey protein extrusion:

adding the whey protein isolate hydrate into a double-screw extruder for cold extrusion. Setting the rotating speed of the screw rod to be 300r/min and the feeding speed to be 3.5 kg/h; the temperature of the feed inlet is 22 ℃ at room temperature, the mechanical energy is converted into heat energy under the combined action of pressure and shearing force without heating in the extrusion process, so that the temperature is gradually increased until the temperature of the discharge outlet is increased to about 48 ℃ (not higher than 50 ℃).

(3) Drying of the extruded whey protein: the whey protein cold extrusion product is dried in a blast type constant temperature drying oven for 24 hours at the constant temperature of 40 ℃ until the water content is about 5 percent.

(4) Crushing of the extruded whey protein: cutting the dried extruded whey protein into pieces having a volume of about 0.5cm³The small blocks enter a cyclone mill to be further processedPulverizing, and sieving with 0.5mm mesh sieve.

(5) Cys crosslinking cold extrusion: and re-dissolving the extruded whey protein powder fragments in deionized water to prepare an extruded whey protein aqueous solution with the concentration of 5% (W/W), adding Cys, adjusting the concentration to be 15mmol/L, carrying out water bath at 60 ℃ for 10min, and immediately transferring to an ice water bath for 15min to obtain the modified whey protein TWPI-Cys.

Example 2

A method for modifying whey protein isolate by Cysteine combined with cold extrusion comprises the following steps:

(1) hydration of whey protein:

whey Protein Isolate (WPI) was dissolved in deionized water to form a whey protein hydrate having a dry matter content of 45% (W/W) and stored at 4 ℃ for 4 hours to be sufficiently hydrated.

(2) Whey protein extrusion:

adding the whey protein isolate hydrate into a double-screw extruder for cold extrusion. Setting the rotating speed of a screw rod to be 350r/min and the feeding speed to be 3 kg/h; the temperature of the feed inlet is 22 ℃ at room temperature, the mechanical energy is converted into heat energy under the combined action of pressure and shearing force without heating in the extrusion process, so that the temperature is gradually increased until the temperature of the discharge outlet is increased to about 48 ℃ (not higher than 50 ℃).

(3) Drying of the extruded whey protein: the whey protein cold extrusion product is dried in a blast type constant temperature drying oven for 28 hours at the constant temperature of 35 ℃ until the water content is about 5 percent.

(4) Crushing of the extruded whey protein: cutting the dried extruded whey protein into pieces having a volume of about 0.5cm³The small blocks enter a cyclone mill for further crushing, and pass through a screen with the aperture of 0.5 mm.

(5) Cys crosslinking cold extrusion: and re-dissolving the extruded whey protein powder fragments in deionized water to prepare an extruded whey protein aqueous solution with the concentration of 3% (W/W), adding Cys, adjusting the concentration to be 18mmol/L, carrying out water bath at 55 ℃ for 12min, and immediately transferring to an ice water bath for 10min to obtain the modified whey protein TWPI-Cys.

Example 3

A method for modifying whey protein isolate by Cysteine combined with cold extrusion comprises the following steps:

(1) hydration of whey protein:

whey Protein Isolate (WPI) was dissolved in deionized water to give a whey protein hydrate having a dry matter content of 55% (W/W) and stored at 4 ℃ for 4 hours to be sufficiently hydrated.

(2) Whey protein extrusion:

adding the whey protein isolate hydrate into a double-screw extruder for cold extrusion. Setting the rotating speed of the screw rod to be 250r/min and the feeding speed to be 4 kg/h; the temperature of the feed inlet is 22 ℃ at room temperature, the mechanical energy is converted into heat energy under the combined action of pressure and shearing force without heating in the extrusion process, so that the temperature is gradually increased until the temperature of the discharge outlet is increased to about 48 ℃ (not higher than 50 ℃).

(3) Drying of the extruded whey protein: the whey protein cold extrusion product is dried in a blowing type constant temperature drying oven at the constant temperature of 45 ℃ for 20 hours until the water content is about 5 percent.

(4) Crushing of the extruded whey protein: cutting the dried extruded whey protein into pieces having a volume of about 0.5cm³The small blocks enter a cyclone mill for further crushing, and pass through a screen with the aperture of 0.5 mm.

(5) Cys crosslinking cold extrusion: and re-dissolving the extruded whey protein powder fragments in deionized water to prepare an extruded whey protein aqueous solution with the concentration of 7% (W/W), adding Cys, adjusting the concentration to be 12mmol/L, carrying out water bath at 65 ℃ for 8min, and immediately transferring to an ice water bath for 18min to obtain the modified whey protein TWPI-Cys.

Analysis of experimental cold extrusion combined with Cysteine modified whey protein product

1. Determination of disulfide bond Change

And (3) carrying out trypsin hydrolysis: whey Protein Isolate (WPI), Oxidized Whey Protein Isolate (OWPI), cold pressed whey protein isolate (TWPI), cysteine-treated whey protein isolate (WPI-Cys), cysteine-treated oxidized whey protein isolate (OWPI-Cys) and cysteine-treated cold pressed whey protein isolate (TWPI-Cys) samples were each diluted to a concentration of 1.0 mg/mL. Alkylation was first carried out with 30mM acrylamide at room temperature for 30 min. Then trypsin was added to the protein solution at a ratio of 1:20(W/W) and adjusted to pH6.8 with 0.1M NaOH solution, and the mixture was hydrolyzed at 37 ℃ overnight.

LC/MS/MS analysis: the protein samples were analyzed for peptide fragments cross-linked by disulfide bonds using the LC/MS/MS method. Mass spectrometry was performed by a Q-exact Plus mass spectrometer (Thermo Scientific, Calif., USA) consisting of a hybrid quadrupole rod-track structure, Easy-nLC 1200 ultra high performance liquid chromatography (Thermo-Dionex Sunnyvale, Calif.) system and a reversed phase C18 column (100mm x 3mm, 2.7 μm, Agilent) connected in-line to the mass spectrometer by a nanospray ion source.

Mobile phase a consisted of 0.1% (v/v) Formic Acid (FA) ultrapure aqueous solution. Mobile phase B comprises 20% of H₂O and 80% acetonitrile, and contains 0.1% FA. The sample was eluted with mobile phase B under a linear gradient. First, the concentration of mobile phase B increased from 3% to 8% between 0-5min, from 5-63min to 28%, then from 63-75min to 38%, and finally from 75-80min to 100%, for 10 min. The total run time was 90min and the column flow was maintained at 300 nL/min. The electrospray ion source operating conditions were as follows: the capillary voltage was set to 1.8kV and the temperature of the ion transfer tube was set to 300 ℃. In the positive ion mode, mass scanning was performed at a resolution of 70000 with a maximum ion implantation time of 50min ranging from 350-2000 mass-to-nuclear ratio (m/z). The MS/MS scanning is extended from 200m/z to 2000m/z, the resolution is 17500, the maximum ion injection time of the measurement scanning is 50min, and the MS/MS scanning is 45 min. The first 20 most abundant precursor ions were fragmented by collision dissociation with a normalized collision energy of 27 eV.

Identification of disulfide-bond cross-linking peptides: identification of cross-linked peptides and disulfide cross-linking site localization in samples was achieved by comparing the mass list in the experimental results with a database containing theoretical masses. Analysis was performed by 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). Data was searched under a "bovine" database downloaded from UniProt (Swissprot and treEMBL; https:// www.uniprot.org /).

The results are shown in table 1, table 2 and fig. 1.

TABLE 1 Overall results for the quality and protein type of cross-linking peptides in WPI, OWPI and TWPI

Note: a naturally occurring disulfide bond; recombinant disulfide bond

TABLE 2 comprehensive results of cross-linking peptides in WPI-Cys, OWPI-Cys and TWPI-Cys and their masses and protein types

Note: a naturally occurring disulfide bond; recombinant disulfide bond

The results show that: a total of 17 intermolecular disulfide cross-links were identified in the cysteine-treated protein samples (TWPI-Cys, OWPI-Cys and WPI-Cys), but 12 intermolecular disulfide cross-links were present in the non-cysteine-treated protein samples (TWPI, OWPI and WPI). The number of protein intermolecular disulfide-crosslinked peptides was higher in the Cys-treated protein sample than in the untreated sample, indicating that cysteine facilitates protein crosslinking through intermolecular disulfide bonds. Generally, cysteine molecules contain highly reactive sulfhydryl groups that reduce intramolecular disulfide bonds in the native protein structure, exposing reactive-SH groups to react with reactive-SH groups of another protein, forming intermolecular aggregation and polymerization of proteins by covalent crosslinking.

6 identical intermolecular disulfide-bond cross-linking peptide fragments were identified in TWPI-Cys, OWPI-Cys and WPI-Cys:

WENDECAQK(6)-CEVFR(1)

LSFNPTQLEEQCHI(12)-CEVFR(1)

WENDECAQK(6)-ALCSEK(3)

LSFNPTQLEEQCHI(12)-ALCSEK(3)

LDQWLCEK(6)-LDQWLCEK(6)

LSFNPTQLEEQCHI(12)-LDQWLCEK(6)

five of the peptides are cross-linked peptides between α -la and β -lg via intermolecular disulfide bonds, for example, the β -lg (160) site forms intermolecular disulfide bonds with α -la (6), (111) (120) sites, and the β -lg (66) site forms intermolecular disulfide bonds with α -la (6), (111) sites, only one cross-linking occurs

Between the sites.

In the TWPI, OWPI and WPI samples, 3 identical intermolecular disulfide cross-linked peptide fragments were identified. The two intermolecular disulfide bond crosslinks of alpha-la and beta-lg occur separately

A site and

between sites, disulfide cross-linking of a peptide segment occurs

Between the sites. This means that a broad interaction between α -la and β -lg occurs in Cys treated and untreated protein samples, rather than between the corresponding homologous α -la and α -la or β -lg and β -lg. Cysteines at the β -lg (66,160) and α -la (6,111,120) sites are active participants in the formation of disulfide bonds between proteins. This confirms that β -lg (160) is the most active participant in the "thiol-disulfide" interaction in heated whey protein (85 ℃, 10 min).

The reactivity of a cysteine in a protein molecule to participate in a thiol-disulfide interaction depends on its position in the original tertiary structure. The higher reactivity of alpha-la (6), alpha-la (111) and alpha-la (120) is due to

And

are located on the surface of the a-la molecule. In addition, bovine serum albumin cross-linked to α -la only in the OWPI-Cys and TWPI-Cys samples. For example, in TWPI-Cys samples BSA (447) and BSA (460) crosslinked with α -la (6), respectively, whereas in OWPI-Cys samples BSA (264) crosslinked with α -la (120). Similarly, in sample TWPI BSA (447) and BSA (199) crosslinked only with α -la (6), in sample OWPI BSA (460) crosslinked with α -la (111). BSA contains three domains (I,II and III), each domain is composed of two subdomains (a and B), with a common basic structural pattern. In addition, BSA (119,264) and BSA (460,447) are located in IIA and IIIA domains. Disulfide bond recombination in these regions indicates that the IIA and IIIA domains are exposed during cold extrusion and oxidation. During cold extrusion, the shear force generated by the screw unfolded the native tertiary structure of BSA and the disulfide bonds were exposed. The disulfide bonds then more readily access and react with cysteines, forming new intermolecular crosslinks. This crosslinking reaction can be induced by oxidation of the thiol groups during mechanical mixing.

Meanwhile, the peptide fragments that β -lg (66) and β -lg (106 or 119 or 121) cross-linked by intramolecular disulfide recombination were found only in TWPI-Cys and TWPI samples. Due to the loss of the α -helix and β -sheet structure during cold extrusion, β -lg (106,119,121) embedded in the hydrophobic pocket was exposed and then cross-linked with β -lg (66), indicating that hydrophobic interactions favoured the disulfide cross-linking reaction. In addition, peptide fragments cross-linked by disulfide bonds between β -lg (106 or 119 or 121) and β -lg (66) were identified in the heated fibrinolysin hydrolysate of β -lg.

However, in both Cys-treated and untreated protein samples, the recombination of disulfide bonds within the protein molecule occurs

And

the above. This is due to the fact that α -la (111 and 120) is at the c-terminus of the peptide chain, at

And

after cleavage of the native disulfide bond between them, the peptide chain can freely extend from the molecule.

CEVFR(1)-CEVFR(1)

WENDECAQK(6)-CEVFR(1)

LSFNPTQLEEQCHI(12)-CEVFR(1)

2. Fourier infrared chromatography

Fourier Infrared chromatography of protein samples FTIR Spectroscopy (Nicolet, Madison, USA) at full wavelength (4000-^-1) The range is scanned. A 2mg protein sample was mixed with 200mg kbr and the mixture was ground to a uniform powder using an agate mortar. Infrared spectrum at 4cm^-1Resolution, recording was performed by 32 scans. Secondary structural elements were quantified by deconvolution and multi-band fitting methods using peakfit 4 software. Peaks in the second derivative spectrum are identified according to the following intervals: alpha-helix (-1660 cm)^-1) Beta-sheet (1640-1630 and 1620-1610 cm)^-1) Beta-turn (1690) 1670cm^-1) And random crimp (1650 cm)^-1)。

The results show that: the IR spectrum of the TWPI-Cys, OWPI-Cys and WPI-Cys samples in the amide I region is shown in FIG. 2A. Since the amide I region is a conformationally sensitive region, changes in the secondary structural elements of the protein are analyzed by infrared spectroscopy on the percentage of area of the component to which each band of the amide I region is fitted. The Fourier self-deconvolution chromatogram of TWPI-Cys is shown in FIG. 2B. In the amide I region, the absorption peaks of Cys-treated TWPI, OWPI and WPI are different from the untreated absorption peaks due to stretching vibration of carbon-oxygen double bonds and carbon-nitrogen bonds. By deconvolution of the amide I region of TWPI-Cys, 8 bands were identified as 1689,1678,1668,1658,1647,1637,1627 and 1616cm, respectively^-1. 1689 and 1616cm^-lBands can be assigned to intermolecular beta-folds. 1689 and 1616cm^-lBands can be assigned to intermolecular beta-turns and are present in the spectra of all whey proteins. 1637cm^-1The bands are due to beta-sheet within the alpha-la molecule. 1626cm^-1Bands have been extensively attributed to antiparallel beta-structures. 1658cm^-1Bands can be assigned to alpha-helices. 1647cm^-1The bands were assigned random crimp.

The ordered secondary structure of proteins is characterized by α -helices and β -sheets, and is related to the local sequence of amino acids and the interaction of protein molecules. The alpha-helix, beta-sheet, beta-turn and random coil for TWPI-Cys, OWPI-Cys and WPI-Cys samples are shown in FIGS. 2C, D, E, F. The ordered structures of TWPI-Cys, OWPI-Cys and WPI-Cys (alpha-helix and beta-sheet) decreased by 3.93%, 3.41% and 4.13%, respectively (P <0.05), compared to TWPI, OWPI and WPI. In all six samples, TWPI-Cys possessed 12.55% alpha helix and 34.38% beta sheet, being the lowest content of ordered structure. Cys is introduced to reduce the disulfide bond of protein and break the ordered secondary structure of protein molecule. Furthermore, adjacent protein molecules are cross-linked by intermolecular disulfide bonds, resulting in irreversible destruction of secondary structures. The total unordered secondary structure increased correspondingly in the TWPI-Cys, OWPI-Cys and WPI-Cys samples, indicating that the addition of Cys loosens the secondary structure of TWPI, OWPI and WPI. It is demonstrated that heat treatment of WPI in the presence of 50mM cysteine (above 65 ℃) resulted in extensive disruption of ordered secondary structure. In general, the α -helix is predominantly from α -la, whereas β -lg is predominantly a β -sheet protein. Thus, the reduction of the alpha-helix in TWPI-Cys, OWPI-Cys and WPI-Cys may be linked to the intermolecular disulfide bond of alpha-la to another alpha-la and beta-lG molecule, as evidenced by the intermolecular disulfide-linked peptide stretches identified by LC/MS/MS. WPI has a high level of ordered structure, with the molecule consisting of 15.20% alpha-helix and 38.09% beta-sheet. After cold extrusion and oxidation, ordered α -helices and β -sheets in TWPI and OWPI decreased by 2.43% and 0.88%, respectively (P < 0.05). This indicates that the internal structural unfolding of the β -lg molecule causes a loss in the a-helix and β -sheet structure. Furthermore, the disorder structures (β -turn and random coil) in TWPI and OWPI decreased by 2.43% and 0.79%, respectively. The increase in conformational entropy and the increase in random coil at the protein surface during protein denaturation leads to thermodynamically driven irreversible aggregation between proteins.

3. Rheological analysis:

dynamic rheology of protein solutions (5%, W/W) during thermal gelation was measured using a Discovery Hybrid rheometer (HR-1, TA instruments, USA) in a small amplitude oscillatory shear mode. Shear was performed at a rate of 1rad/s and 1% strain. The temperature was increased from 50 ℃ to 85 ℃ at a rate of 1 ℃/min. The measurements were carried out using parallel plates (diameter 60mm) with a gap of 0.3 mm. The storage modulus G' and loss modulus G "recorded by the TA software were used to describe the dynamic rheological properties of the protein samples.

As a result: FIG. 3 shows the results of temperature sweep tests for dispersions (5%, w/w) containing TWPI-Cys, OWPI-Cys and WPI-Cys. In particular, WPI has a gel onset temperature of about 74 ℃. However, addition of Cys significantly (P <0.05) reduced the gel onset temperature of the TWPI-Cys samples to 57 ℃. Similar trends were also observed in the OWPI-Cys and WPI-Cys samples, which decreased the gel temperature to 62 ℃ and 65 ℃ respectively. This indicates that the addition of Cys significantly reduces the energy required for denaturation of the whey protein molecules. This can be attributed to the cysteine containing a highly reactive thiol group, which can break the disulfide bonds in native whey protein even at lower temperatures and unfold the molecular structure of the protein. Thus, the enhancement of intermolecular and intramolecular interactions in proteins leads to the formation of molecular aggregates, ultimately allowing gelation to occur at relatively low temperatures.

FIG. 3A shows the results of temperature sweep tests with TWPI, OWPI and WPI dispersions (5%, w/w). In addition, the storage modulus (G ') and loss modulus (G') of TWPI and OWPI increase dramatically at 72 ℃ indicating that the protein is converted from a sol to a gel. This also shows that cold extrusion and oxidation slightly lowers the gel temperature of the WPI. During the temperature programmed heat treatment, G 'and G' of TWPI, OWPI and WPI were at 10 deg.C as the temperature increased from 50 deg.C to 70 deg.C^-2pa fluctuates around with little change. Then, G 'and G' increase sharply when the temperature of the protein sample is raised from 70 ℃ to 75 ℃. Finally, as the temperature of the protein sample rose from 75 ℃ to 85 ℃, G' and G "rose steadily and then reached a maximum at 85 ℃. Accordingly, the number of the first and second electrodes,the viscosity and elasticity of the molecular network formed between the native protein and the denatured protein increases with the increase in denatured protein molecules. Furthermore, due to the higher proportion of denatured protein molecules in TWPI, the thickest network of molecules is formed, and thus the viscosity and elasticity of TWPI are greatest.

At 85 ℃, G' and G "were maximal for Cys-treated TWPI, 19.11 and 25.86 times greater than TWPI, respectively. However, at 85 ℃, there was no significant difference between Cys-treated OWPI or WPI and untreated OWPI or WPI in G' and G ″. Indicating that the viscoelastic modulus is related to the degree of cross-linking plasticization. The dramatic increase in viscoelastic modulus of Cys-treated TWPI is due to thickening of the protein network, as more of the deployed protein is attached through disulfide bonds and hydrophobic interactions.

4. Size exclusion chromatography:

molecular size distribution of protein samples was determined using a Waters 2695/2487 combination system (Waters, Etten-Leur, the Netherlands), a tandem TSK guard column (7.5 mm. times.7.5 mm) and TSK G2000sw (7.5 mm. times.60 cm, 10 μm, TosoHaas, Montgomeryville, Pa., USA). The column was equilibrated and eluted at a flow rate of 0.5mL/min using 30% acetonitrile containing 0.1% trifluoroacetic acid (TFA). After filtering the sample through a 0.45 μm filter, 15 μ L of the sample (5mg/mL) was injected for analysis. The uv detector wavelength was set to 280 nm. The calibration standard proteins are beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA), Bovine Serum Albumin (BSA) and cysteine, respectively.

As a result: the molecular weight distributions of Cys-treated and untreated TWPI, OWPI and WPI are shown in figures 4A and 4B, respectively. A peak eluting at 24.21 minutes was detected only in TWPI-Cys, indicating the formation of high molecular weight cross-linked protein. These polymers in TWPI-Cys have a higher molecular weight than BSA (about 66 kDa). In contrast, no cross-linked proteins with high molecular weight were found in OWPI-Cys and WPI-Cys. However, the peak intensity of α -la in TWPI-Cys decreased significantly, indicating that α -la is widely involved in protein intermolecular cross-linking of TWPI-Cys. Generally, without cysteine addition, whey protein aggregation is generally considered to be driven by β -lg, since its concentration is higher than other whey proteins. Beta-lg mainly controls the aggregation of whey proteins induced by heat treatment. Alpha-la has high thermal stability, due in part to the fact that alpha-la does not contain any free-SH groups, other than four disulfide bonds. Highly reactive cysteine-SH groups lead to the reduction of intramolecular disulfide bonds. The a-la molecule may be more sensitive to the reduction of the cysteine molecule because it contains four disulfide bonds in its structure, but only two disulfide bonds in the β -lg. Thus, the TWPI-Cys molecular weight is increased by aggregation through thiol-disulfide exchange reaction polymerization and hydrophobic interactions. In addition, the peak eluting at 44.90min in FIG. 4A indicates the standard cysteine. No peaks eluted at 44.90min in the TWPI-Cys, OWPI-Cys and WPI-Cys samples, revealing that cysteine was depleted in the chemical reactions with all three protein samples. As shown in FIG. 4B, three peaks of 25.79min (BSA), 28.34min (. beta. -LG) and 30.41min (. alpha. -LA) were detected in TWPI, OWPI and WPI, respectively. However, the strength of β -lg and α -la decreased slightly, indicating that β -lg and α -la aggregated slightly. Furthermore, there were no significant high molecular weight proteins in TWPI, OWPI, WPI. It is possible that the detector cannot detect the small intein polymers of β -lg and α -la due to sensitivity limitations. SEC analysis may underestimate the distribution of the cross-linked protein component due to poor solubility of the protein polymer, particularly high molecular weight cross-linked products. Meanwhile, in the present invention, size exclusion chromatography can only separate and identify soluble proteins in a high performance liquid chromatography solvent (acetonitrile).

5. Cytotoxicity assays for WPI-Cys conjugates

To study the cytotoxicity of protein samples in vitro, Human Umbilical Vein Endothelial Cells (HUVEC) were selected as target cells and cell viability was assessed using the cell counting kit CCK8 as the basis for the colorimetric assay. Initially, 100. mu.L of a cell suspension (5X 10)⁴/mL) was added to a 96-well cell culture plate. Cells were cultured in humidified incubator for 24h (5% CO)₂At 37 ℃ C. After removal of the cell culture medium, 100. mu.L of protein suspension (0.5mg/mL, 1.0mg/mL, 2.0mg/mL) was added and incubated for 24h and 48h, respectively. Optical Density (OD) was measured at 450nm with a model 550 fully automatic quantitative plotter microplate reader (Bio-Rad, Hercules, Calif., USA).The cell viability was calculated as follows:

cell survival (%) ═ a₄₅₀(sample)/A₄₅₀(blank) × 100

Wherein A is₄₅₀(sample) is the absorbance at 450nm of the protein sample, A₄₅₀(blank) is the absorbance in the absence of sample.

The effect of cysteine-treated TWPI, OWPI and WPI (0.5,1.0 and 2.0mg/mL) on cell viability of HUVEC cultured for 24h is shown in FIG. 5A. The cell survival rates of TWPI-Cys, OWPI-Cys, WPI-Cyst, TWPI and WPI treatments were all above 100%, indicating that these proteins promote HUVEC proliferation. At 0.5mg/mL (p <0.05), cell survival rates were significantly higher for TWPI-Cys, OWPI-Cys, and WPI-Cys samples than for TWPI, OWPI, and WPI samples, indicating that cysteine treatment increased HUVEC survival. Furthermore, the cell viability for the 2.0mg/mL TWPI-Cys treatment was 190.41%, the highest among all samples.

Different protein aggregation morphologies of the same parent protein exhibit different cytotoxicity. The morphology of protein fibers is critical to cause cell death. Cys-treated proteins form more flexible fibers due to the reduction of the portion of S-S bonds in the protein molecule, further reducing damage to cell membrane integrity and cell damage. Meanwhile, the survival rate of WPI cells is higher than TWPI and OWPI (P < 0.05). During the extrusion and oxidation, whey protein molecules form aggregates. Aggregates in TWPI and OWPI produce cytotoxicity. In addition, the toxicity of these aggregates is caused by oxidative stress, ultimately leading to apoptosis or necrotic cell death. Thus, when the TWPI-Cys, WPI-Cys and WPI concentrations were increased from 0.5,1.0 to 2.0mg/mL, their survival rates were dose-dependently increased (p < 0.05).

The effect of Cys-treated TWPI, OWPI and WPI (0.5,1.0 and 2.0mg/mL) on HUVEC survival at 48h in culture is shown in FIG. 5B. The cell viability of all protein samples gradually decreased as the culture time increased from 24h to 48 h. Similarly, cell survival at 0.5,1.0 and 2mg/mL for TWPI-Cys and OWPI-Cys was significantly higher than for TWPI and OWPI (p <0.05), whereas cell survival at 2mg/mL for WPI-Cys was greater than for WPI, whereas cell survival at 0.5mg/mL and 1.0mg/mL for WPI-Cys and WPI was not significantly different. Furthermore, WPI has better cell viability compared to TWPI and OWPI, which is the same trend (p < 0.05). Cell viability increased significantly (p <0.05) as TWPI-Cys, OWPI-Cys, TWPI and OWPI concentrations varied from 0.5 to 1.0 mg/mL. Furthermore, as WPI-Cys increased from 1.0 to 2.0mg/mL, cell viability increased by 11.15% (P < 0.05).

Cell viability after treatment with 2.0mg/mL TWPI-Cys was 137.20%, reaching a maximum in all samples. Thus, the cell viability was greater than 85% for all Cys treated and untreated TWPI, OWPI and WPI at each concentration, indicating that the modified whey protein was non-toxic to HUVEC and met food production requirements.

6. Emulsifying property

The emulsifying properties were investigated using the modified turbidity method. 3mL of the protein dispersion (5mg/mL) and 1mL of soybean oil were mixed and homogenized with a high-speed homogenizer at 12000g for 120 seconds to prepare an emulsion. The freshly prepared emulsion (50. mu.L) was left to stand for 0 and 10min, respectively, and then immediately dispersed into 5mL of a 1mg/mL SDS solution. The absorbance of the emulsion was recorded at 500 nm.

Emulsification Activity Index (EAI) (M)²The/g) is defined as:

wherein: a. the₀The absorbance thereof, c the concentration of the protein solution (g/mL),

the volume fraction of the oil phase is shown, and dilution is a dilution factor.

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

wherein A is₁₀And A₀Are respectively shown inAbsorbance measured at 10min and 0 min.

As a result: the emulsification activity index is related to the surface area per weight of protein stabilized. EAI for Cys-treated TWPI, OWPI and WPI are shown in fig. 6A. The EAI of the Cys-treated TWPI, OWPI and WPI were all higher than TWPI, OWPI and WPI (P)<0.05). After addition of Cys, EAI increased 2.18, 2.97 and 4.37% for TWPI, OWPI and WPI, respectively. The EAI values were greatest for Cys-treated TWPI compared to the other 5 protein samples. Cys breaks the disulfide bond of the protein, making the structure of the protein easily opened. In addition, the protein with more flexible structure (high random coil) is quickly adsorbed on the oil-water interface, and the EAI value of the protein is higher. In addition, Cys-treated TWPI, OWPI and WPI undergo structural rearrangement, resulting in a significant increase in their EAI. And WPI (60.27 m)²/g) increased by 0.50% compared to the EAI of the OWPI as oxidation control, indicating that there was no significant difference between the EAI of WPI and the EAI of OWPI (P > 0.05). The EAI of TWPI was increased by 10.59% (P) over WPI after cold extrusion treatment<0.05). During cold extrusion, the hydrophobic groups buried in β -lg are exposed as the globular structure expands. The increased hydrophobic interaction and molecular flexibility between adjacent protein molecules enhances the affinity of the protein for the oil and water phase interface. Because protein molecules are closely packed on an oil-water interface, the concentration of the interface protein is enhanced. The higher interface protein concentration is beneficial to forming a stronger viscoelastic film around the fat sphere, and the effective adsorption of denatured protein molecules on an oil-water interface is accelerated. However, the emulsion systems formed with WPI are not sufficient to achieve complete coverage of the droplet surface.

Emulsion stability: FIG. 6B shows the effect of Cys treatment on ESI values for TWPI, OWPI and WPI. With Cys addition, the ESIs of TWPI-Cys, OWPI-Cys and WPI-Cys increased significantly by 12.96,19.56 and 21.23%, respectively (P < 0.05). Free SH groups are introduced in the form of Cys, and the molecular structure of alpha-la is unfolded by destroying disulfide bonds in the alpha-la molecule, resulting in irreversible denaturation of alpha-la. Highly reactive SH groups facilitate the interaction of α -la molecules with β -la. The intermolecular interaction of proteins is enhanced by the covalent reaction of SH/S-S exchange, resulting in higher relative adsorption of proteins to the oil droplet surface. The ESI of TWPI increased significantly to 54.56% after Cys treatment. As a control, the ESI values of Cys-treated WPI and OWPI were 51.00% and 51.29%, respectively (P < 0.05).

TWPI has a significantly higher viscosity than WPI and OWPI, and forms a thick, rigid film at the oil-water interface due to hydrophobic interactions between protein molecules at the interface. In addition, the higher surface charge provides sufficient electrostatic repulsion between oil droplets, retarding the movement of oil droplets and retarding the formation of oil droplet coalescence.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (3)

1. A method for modifying whey protein, comprising the steps of:

(1) preparing whey protein hydrate with water content of 50 wt%;

(2) carrying out cold extrusion on the whey protein hydrate by using a double-screw extruder to obtain a whey protein cold extrusion product; the screw rotating speed of the double-screw extruder is 300r/min, and the feeding speed is 3.5 kg/h;

heating is not carried out in the cold extrusion process, and the temperature of a discharge port of the double-screw extruder is not higher than 50 ℃;

(3) drying and crushing the whey protein cold extrusion product to obtain an extruded whey protein powder crushed material;

(4) preparing the extruded whey protein powder crushed material into an extruded whey protein water solution with the concentration of 5 wt%; adding cysteine into the extruded whey protein aqueous solution to ensure that the concentration of the cysteine in the extruded whey protein aqueous solution is 15 mmol/L; then reacting for 10min at the temperature of 60 ℃, immediately transferring to an ice water bath to terminate the reaction, and obtaining the modified whey protein;

in the step (3), the drying is carried out for 20-28h at 35-45 ℃ until the water content of the whey protein cold extrusion product is 5 wt%; the crushing is to be crushed to a particle size of less than 0.5 mm.

2. A modified whey protein obtained by the method for modifying whey protein according to claim 1, characterized in that the modified whey protein contains the following intermolecular disulfide-bond-cross-linked peptide fragments:

WENDECAQK(6)-CEVFR(1) β-lg (66) ↔α-la (6)

LSFNPTQLEEQCHI(12)-CEVFR(1) β-lg (160) ↔α-la (6)

WENDECAQK(6)-ALCSEK(3) β-lg (66) ↔α-la (111)

LSFNPTQLEEQCHI(12)-ALCSEK(3) β-lg (160) ↔α-la (111)

LDQWLCEK(6)-LDQWLCEK(6) α-la (120) ↔α-la (120)

LSFNPTQLEEQCHI(12)-LDQWLCEK(6) β-lg (160) ↔α-la (120)。

3. the modified whey protein of claim 2, comprising the following intermolecular disulfide-linked peptide fragments:

CEVFR(1)-CEVFR(1) α-la (6) ↔α-la (6)

WENGECAQK(6)-CEVFR(1)β-lg (66) ↔α-la (6)

WENDECAQK(6)-CEVFR(1)β-lg (66) ↔α-la (6)

LSFNPTQLEEQCHI(12)-CEVFR(1)β-lg (160) ↔α-la (6)

MPCTEDYLSLILNR(3)-CEVFR(1) BSA (447) ↔ α-la (6)

LCVLHEK(2)-CEVFR(1) BSA (460) ↔α-la (6)

ALCSEK(3)-ALCSEK(3)α-la (111) ↔α-la (111)

WENGECAQK(6)-ALCSEK(3)β-lg (66) ↔α-la (111)

WENDECAQK(6)-ALCSEK(3)β-lg (66) ↔α-la (111)

LSFNPTQLEEQCHI(12)-ALCSEK(3)β-lg (160) ↔α-la (111)

LDQWLCEK(6)-LDQWLCEK(6)α-la (120) ↔α-la (120)

WENDECAQK(6)-LDQWLCEK(6)β-lg (66) ↔ α-la (120)

LSFNPTQLEEQCHI(12)-LDQWLCEK(6)β-lg (160) ↔α-la (120)。

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