CN113303395B

CN113303395B - Preparation method of modified whey protein

Info

Publication number: CN113303395B
Application number: CN202110656786.2A
Authority: CN
Inventors: 侯俊财; 姜瞻梅; 李金鹏; 马佳歌; 李金哲
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-12-24
Anticipated expiration: 2041-06-11
Also published as: CN113303395A

Abstract

The invention discloses a preparation method of modified whey protein, and belongs to the technical field of protein processing. The method comprises the following steps: pretreating whey protein by extrusion to obtain extrudates with different extrusion temperatures; and carrying out catalytic crosslinking reaction on the extrudate and transglutaminase to obtain the modified whey protein. Specifically, the extrusion technology is combined with transglutaminase crosslinking reaction, so that the structure of the protein can be unfolded by high pressure and high shear force generated in the extrusion process, thereby exposing a site crosslinked with TGase, and particularly, the improvement of the crosslinking degree of catalyzing WPI by TGase is facilitated by extrusion at low temperature (50 ℃), the improvement of the water retention property, the emulsifying activity, the stability and other functions of the whey protein is realized, and the modified whey protein prepared by the method is favorably applied to different fields.

Description

Preparation method of modified whey protein

Technical Field

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

Background

The whey protein is a general term for soluble proteins present in whey after the casein is precipitated, and accounts for about 18 to 20% of the total amount of milk proteins. The main components of the milk protein powder comprise alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), immunoglobulin and bovine serum albumin, which respectively account for 22%, 60%, 9% and 6% of the total amount of the whey protein. In addition, other compounds such as lactoferrin, lactoperoxidase, lysozyme and the like are also present. Whey protein has high nutritional value and diversified functional properties, and is widely used in food processing as a nutritional supplement, an emulsifier, a thickener, a gelling agent, and the like. With the development of food processing technology, the original characteristics of whey protein have not been able to meet various needs of the food industry, and thus, whey protein needs to be modified to enhance its functional characteristics.

Currently, whey proteins are often modified using a single modification process, such as by physical, chemical or enzymatic means. Extrusion technology has been applied in the food field as a means of physical modification. The technology utilizes the synergistic effect of high temperature, high pressure and high shearing force on the material in the extrusion process to change the structure of protein, thereby leading the functional characteristics of the protein to be changed to a certain extent. In addition, studies have shown that transglutaminase (TGase) is an enzyme capable of catalyzing acyl transfer reactions, crosslinking reactions, and deamidation reactions, and can improve functional properties of proteins such as emulsification and foaming by forming epsilon- (gamma-glutamine) -lysine isopeptide bonds within or between molecules of proteins using epsilon-amino groups of lysine residues in proteins as acyl acceptors to cause crosslinking reactions of protein molecules.

Although there are many reports that the effect of extrusion technology or TGase cross-linking on proteins was studied separately, there is currently no report that extrusion technology can increase the accessibility of TGase cross-linking. Therefore, the whey protein is treated by combining the extrusion technology with TGase crosslinking, and the change of the structure and the functional characteristics of the whey protein is researched, so that the functional characteristics of the whey protein are improved, and the application field of the whey protein is widened.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and provides a preparation method of modified whey protein, which realizes the modification of whey protein by extrusion combined with TGase crosslinking, improves the functions of the whey protein such as emulsifying activity, stability and the like, and can be applied to wider fields.

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

the invention provides a preparation method of modified whey protein, which comprises the following steps:

pretreating whey protein by extrusion to obtain extrudates with different extrusion temperatures;

and carrying out catalytic crosslinking reaction on the extrudate and transglutaminase to obtain the modified whey protein.

Preferably, the extrusion pretreatment is carried out by a parallel twin-screw extruder, and the extrusion parameters of the parallel twin-screw extruder comprise: the feeding speed is 10-13g/min, the water content of the material is 40-50% (w/w), the water inlet speed is 5.0-6.0mL/min, and the screw rotating speed is 200-.

Preferably, the parallel twin-screw extruder comprises 4 functional zones (8 independent heating zones in total), the extrusion temperatures of the different functional zones being set: a feeding area (the first area is 20-30 ℃); a mixing zone (second zone 30-40 ℃, third zone 40-50 ℃); a cooking zone (fourth zone-sixth zone 50-90 ℃); a discharging area (a seventh area is 40-50 ℃, and an eighth area is 20-30 ℃).

Preferably, the extrusion temperature of the cooking zone (fourth zone-sixth zone) is set at 45-55 ℃ at the same time.

Preferably, the addition amount of the transglutaminase is 25-35U/g, and the catalytic crosslinking reaction time is 3-5 h.

Preferably, the extrudate also comprises freeze drying, grinding and dissolving processes before the enzymatic transglutaminase crosslinking reaction, wherein the concentration of the whey protein after dissolving is 30-45 mg/mL.

Preferably, the whey protein solution is adjusted to pH7.0 and then cross-linked with transglutaminase under heating in a water bath at 45-55 deg.C.

Preferably, the extrudate is further subjected to enzyme inactivation, cooling, freeze drying and grinding after the enzymatic crosslinking reaction is catalyzed by the transglutaminase.

The invention also provides the modified whey protein prepared by the preparation method.

The invention discloses the following technical effects:

the invention carries out extrusion pretreatment on whey protein, and then carries out cross-linking reaction with TGase. Experiments show that the high pressure and high shear force generated in the low-temperature extrusion (50 ℃) process can expand the structure of the protein, thereby exposing the sites for crosslinking with the TGase and being more beneficial to promoting the crosslinking degree of the WPI catalyzed by the TGase. Meanwhile, the characteristics of water retention, emulsification and the like of the whey protein are improved, and experiments prove that compared with the WPI-TGase (WPI subjected to TGase crosslinking) which is not treated by the preparation method, the emulsifying activity and the emulsifying stability of the WPI-TGase extruded at 50 ℃ are respectively improved by 63.78 percent and 9.99 percent, namely the emulsifying activity is increased from 41.47 percent to 67.92 percent, and the emulsifying activity is increased from 29.82 percent to 32.80 percent; the water holding capacity is increased by 97.73%, i.e. from 0.44g/g to 0.87 g/g. Therefore, the invention combines extrusion with TGase crosslinking, not only realizes whey protein modification, but also improves the function of the modified whey protein, and is more beneficial to applying the modified whey protein to different fields.

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 graph of the effect of different extrusion temperatures on WPI free amino content after cross-linking of WPI and TGase;

FIG. 2 is a graph of the effect of different extrusion temperatures on WPI molecular weight distribution;

FIG. 3 is a graph of the effect of different extrusion temperatures on the molecular weight distribution of WPI after TGase cross-linking;

FIG. 4 is a graph of the effect of different extrusion temperatures on WPI molecular weight distribution (treated with SDS and beta-mercaptoethanol);

FIG. 5 shows the effect of different extrusion temperatures on the molecular weight distribution of WPI after TGase cross-linking (treatment with SDS and. beta. -mercaptoethanol);

figure 6 is a graph of the effect of different extrusion temperatures on WPI particle size distribution;

FIG. 7 shows the effect of different extrusion temperatures on the WPI particle size distribution after TGase cross-linking;

FIG. 8 is a graph showing the effect of different extrusion temperatures on the average WPI particle size after cross-linking of WPI and TGase;

FIG. 9 is a graph of the effect of different extrusion temperatures on WPI free thiol content after cross-linking of WPI and TGase;

FIG. 10 is a graph of the effect of different extrusion temperatures on WPI surface hydrophobicity after cross-linking of WPI and TGase;

FIG. 11 is a graph of the effect of different extrusion temperatures on WPI emulsifying activity after cross-linking of WPI and TGase;

figure 12 is a graph of the effect of different extrusion temperatures on WPI emulsion stability after cross-linking of WPI and TGas;

figure 13 is a graph of the effect of different extrusion temperatures on WPI water retention after WPI and TGas cross-linking;

figure 14 is a graph of the effect of different extrusion temperatures on WPI secondary structure;

FIG. 15 shows the effect of different extrusion temperatures on the WPI secondary structure after TGase cross-linking;

figure 16 is a micrograph of an uncompressed WPI (magnification 1000);

figure 17 is a 50 ℃ extrusion WPI microstructure diagram (1000 x magnification);

figure 18 is a 90 ℃ extrusion WPI microstructure diagram (1000 x magnification);

figure 19 is a 130 ℃ extrusion WPI microstructure diagram (1000 x magnification);

FIG. 20 is a micrograph of an uncompressed WPI after TGase cross-linking (magnification 1000);

FIG. 21 is a 50 ℃ extrusion WPI microstructure diagram (1000 times magnification) after TGase cross-linking;

FIG. 22 is a view of a 90 ℃ extrusion WPI microstructure after TGase cross-linking (magnification 1000 times);

FIG. 23 is a view showing a microscopic structure of a 130 ℃ extrusion WPI after TGase crosslinking (magnification: 1000 times).

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 by extrusion combined with transglutaminase treatment, comprising the steps of:

(1) preparation of extruded whey protein: whey Protein Isolate (WPI) was used as a raw material and subjected to extrusion pretreatment using a parallel twin-screw extruder. The extrusion parameters are set as follows: the feeding speed is 10g/min, the water content of the material is 40% (w/w), the water inlet speed is 5.0mL/min, and the screw rotating speed is 200 rpm. The extruder was divided into 4 functional zones (total of 8 independent heating zones, including 7 built-in heating zones, 1 external die heating zone), the temperature of each functional zone was kept constant: a feeding zone (first zone 20 ℃); a mixing zone (second zone 30 ℃, third zone 40 ℃); a cooking zone (fourth zone 50 ℃, fifth zone 50 ℃, sixth zone 50 ℃); a discharge zone (a seventh zone at 40 ℃ C., an eighth zone (die heating zone) at 20 ℃ C.). After the extrusion is finished, the obtained extrudates with different temperatures are freeze-dried and ground to obtain the extruded whey protein powder.

(2) Preparation of extruded whey protein solution: dissolving the extruded whey protein powder in deionized water, and magnetically stirring for 2 hours at room temperature to obtain 30mg/mL whey protein solution;

(3) preparation of TGase cross-linked extruded whey protein: the whey protein solution was adjusted to pH7.0 with 1mol/L sodium hydroxide solution, and then 25U/g TGase was added to the solution and heated in a water bath at 45 ℃ for 3 hours to catalyze the crosslinking reaction. After the crosslinking reaction is finished, inactivating enzyme of the crosslinking solution at 75 ℃ for 15min, and cooling at room temperature. And finally, freeze-drying and grinding a sample of the TGase cross-linked WPI extrudate to obtain the modified whey protein powder.

Example 2 a method of modifying whey protein by extrusion in combination with transglutaminase treatment, comprising the steps of:

(1) preparation of extruded whey protein: whey Protein Isolate (WPI) was used as a raw material and subjected to extrusion pretreatment using a parallel twin-screw extruder. The extrusion parameters are set as follows: the feeding speed is 10g/min, the water content of the material is 40% (w/w), the water inlet speed is 5.5mL/min, and the screw rotating speed is 240 rpm. The extruder was divided into 4 functional zones (total of 8 independent heating zones, including 7 built-in heating zones, 1 external die heating zone), the temperature of each functional zone was kept constant: a feeding zone (first zone 25 ℃); a mixing zone (second zone 35 ℃, third zone 45 ℃); a cooking zone (fourth zone 50 ℃, fifth zone 50 ℃, sixth zone 50 ℃); the discharge zone (seventh zone 45 ℃ C., eighth zone (die heating zone) 25 ℃ C.). After the extrusion is finished, the obtained extrudates with different temperatures are freeze-dried and ground to obtain the extruded whey protein powder.

(2) Preparation of extruded whey protein solution: dissolving the extruded whey protein powder in deionized water, and magnetically stirring for 2 hours at room temperature to obtain a whey protein solution of 40 mg/mL;

(3) preparation of TGase cross-linked extruded whey protein: the whey protein solution was adjusted to pH7.0 with 1mol/L sodium hydroxide solution, and then 30U/g TGase was added to the solution and heated in a water bath at 50 ℃ for 4 hours to catalyze the crosslinking reaction. After the crosslinking reaction is finished, inactivating enzyme of the crosslinking solution at 75 ℃ for 15min, and cooling at room temperature. And finally, freeze-drying and grinding a sample of the TGase cross-linked WPI extrudate to obtain the modified whey protein powder.

Example 3 a method of modifying whey protein by extrusion in combination with transglutaminase treatment, comprising the steps of:

(1) preparation of extruded whey protein: whey Protein Isolate (WPI) was used as a raw material and subjected to extrusion pretreatment using a parallel twin-screw extruder. The extrusion parameters are set as follows: the feeding speed is 13g/min, the water content of the material is 50% (w/w), the water inlet speed is 6.0mL/min, and the screw rotating speed is 250 rpm. The extruder was divided into 4 functional zones (8 total independent heating zones, including 7 built-in heating zones, 1 external die heating zone), the temperature of each functional zone was kept constant: a feeding zone (first zone 30 ℃); a mixing zone (second zone 40 ℃, third zone 50 ℃); a cooking zone (fourth zone 90 ℃, fifth zone 90 ℃, sixth zone 90 ℃); the discharge zone (seventh zone 50 ℃, eighth zone (die heating zone) 30 ℃). After the extrusion is finished, the obtained extrudates with different temperatures are freeze-dried and ground to obtain the extruded whey protein powder.

EXAMPLE 4 Effect of extrusion temperature on the content of free amino groups of TGase Cross-Linked WPI (WPI-TGase)

1) Determination of the content of free amino groups

The protein sample was diluted with deionized water to a final concentration of 5 mg/mL. And respectively adding 3mL of an o-phthalaldehyde reagent into 100 mu L of the diluted protein sample, quickly mixing, standing for 5min in a dark place at room temperature, recording an absorbance value at a wavelength of 340nm, and calculating the free amino content of the sample according to the absorbance value. Deionized water (100 μ L) was used as a reagent blank instead of the protein sample. Standard curve lineObtained by using L-leucine (0.1-0.5 mg/mL) as a standard: y 1.362x-0.0094, R²＝0.9996。

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the content of free amino groups of the TGase cross-linked WPI was studied, and the specific test results are shown in FIG. 1.

As can be seen from FIG. 1, the low temperature extrusion pretreatment (50 ℃ C. and 70 ℃ C.) increased the free amino group content of the protein samples (P < 0.05). After TGase is added, the cross-linking reaction in WPI molecules and among WPI molecules can be catalyzed, and epsilon- (gamma-glutamine) lysine isopeptide bond formation is induced, so that the free amino group content of three groups of protein samples, namely non-extruded protein samples, 50 ℃ extruded protein samples and 70 ℃ extruded protein samples is reduced (P is less than 0.05). Although the free amino content of the 50 ℃ pressing WPI-TGase and the 70 ℃ pressing WPI-TGase is not significantly different from that of the WPI-TGase (P <0.05), the free amino content of the 50 ℃ and 70 ℃ pressing protein sample groups is reduced more than that of the non-pressing WPI, indicating that the free amino content consumed by the 50 ℃ and 70 ℃ pressing protein samples is larger by the TGase crosslinking, i.e. the protein after the low temperature pressing (50 ℃ and 70 ℃) is likely to be crosslinked with the TGase to a larger extent.

With the further increase of the extrusion temperature (90-130 ℃), the free amino content of the extruded WPI and the extruded WPI-TGase has no significant difference (P is greater than 0.05), namely the crosslinking degree of the WPI and the TGase after high-temperature extrusion is not large.

Example 5 Effect of extrusion temperature on molecular weight distribution of TGase Cross-Linked WPI

1) Determination of size exclusion chromatography

The distribution of protein molecular weights was studied by size exclusion chromatography. The HPLC was equipped with a TSK G2000-SW analytical column (7.5 mm. times.60 cm, 1mm) and a TSK guard column (7.5 mm. times.7.5 cm). Protein samples were first diluted with deionized water to a final concentration of 5mg/mL and then filtered using a 0.45 μm filter. The samples were divided into two groups for measurement, one of which was diluted with a solution containing 87.5% Sodium Dodecyl Sulfate (SDS) and 4.17% β -mercaptoethanol to disrupt non-covalent bonds and disulfide bonds in the samples; the other group is not processed. The column was equilibrated and eluted with a 30% acetonitrile solution containing 0.1% trifluoroacetic acid as a mobile phase at an elution flow rate of 0.5 mL/min. The detection wavelength of the ultraviolet absorption detector was set to 280 nm.

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the molecular weight distribution of the TGase cross-linked WPI was studied, and the specific test results are shown in FIGS. 2-3.

As shown in FIG. 2, the extruded WPI showed the first peak at 23.74min, indicating the formation of the macromolecular polymer. The peak height gradually decreased with increasing extrusion temperature. This is because the insoluble aggregates of macromolecules produced by WPI after high temperature pressing (90 deg.C, 11 deg.C 0 and 130 deg.C pressing) are filtered and cannot be detected because they need to be filtered through a 0.45 μm filter before detection. The gradual decrease in the peak height of the second peak (. beta. -LG) also indicates that the consumption of. beta. -LG is caused by the formation of macromolecular substances. As shown in FIG. 3, the peak heights of the 50 ℃ pressing WPI-TGase and the 70 ℃ pressing WPI-TGase were higher than those of the corresponding peaks in FIG. 2. It was shown that in the extruded WPI after TGase cross-linking, 50 ℃ and 70 ℃ extrudates are more favorable for cross-linking with TGase.

SDS and beta-mercaptoethanol were added to eliminate the effect of disulfide bonds on the results, which are shown in FIGS. 4 and 5.

As shown in fig. 4, the protein aggregates formed by the pressing treatment disappeared. Indicating that the macromolecular species formed during extrusion may be due to disulfide bonds. As can be seen from the enlarged portion of FIG. 5, the peak heights gradually decreased with increasing extrusion temperature, and the peak heights of the 50 ℃ and 70 ℃ extrusion WPI-TGases were slightly higher than that of the WPI-TGase. This indicates that more aggregates can be formed with the degree of crosslinking of the extrudate with TGase at 50 ℃ and 70 ℃ being greater than the degree of crosslinking of WPI with TGase. Furthermore, the 50 ℃ extrusion temperature is more favorable than the 70 ℃ extrusion temperature for exposing the sites of TGase cross-linking by the protein.

Example 6 Effect of extrusion temperature on particle size distribution of TGase Cross-Linked WPI

1) Measurement of particle diameter

The protein sample concentration was diluted to 1mg/mL with phosphate buffer (PBS buffer, pH7.0, concentration 0.01mol/L) and then measured with a particle size analyzer.

2) On the basis of example 2, the extrusion temperature conditions in the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the particle size distribution of the TGase cross-linked WPI was studied, and the specific test results are shown in FIGS. 6-8.

As can be seen from FIG. 6, the particle size distribution gradually shifted to the right with increasing extrusion temperature (90 to 130 ℃), i.e., the average particle size gradually increased (as shown in FIG. 8). After TGase cross-linking, the particle size distribution of all protein samples shifted to the right overall (as shown in figure 7). This indicates that the protein sample is cross-linked with TGase, resulting in a change in the particle size distribution.

Since the data of the two groups of 110 ℃ extrusion and 130 ℃ extrusion are much larger than those of the three groups of non-extrusion, 50 ℃ extrusion and 70 ℃ extrusion, the difference of the three groups of data is not significant, and therefore, the four groups of data of non-extrusion, 50 ℃ extrusion, 70 ℃ extrusion and 90 ℃ extrusion are independently subjected to significance analysis, as shown in the enlarged diagram of fig. 8. The average particle size of the extruded WPI at 50 ℃ and 70 ℃ was not significantly different from that of the non-extruded WPI (P >0.05), but after TGase cross-linking, the average particle size of the extruded WPI-TGase at 50 ℃ and 70 ℃ was significantly larger than that of the non-extruded WPI-TGase (P < 0.05). This indicates that the low temperature extrusion (50 ℃ C., 70 ℃ C.) of WPI cross-linked with TGase produced more polymer than the non-extruded WPI, i.e., the degree of cross-linking was greater than the non-extruded WPI. And the average particle size of the WPI after high-temperature extrusion (90-130 ℃) is not significantly different from that of the WPI-TGase crosslinked and extruded through the TGase, which shows that the protein sample after high-temperature extrusion is not beneficial to crosslinking with the TGase.

Example 7 Effect of extrusion temperature on the content of free thiol groups in the TGase Cross-Linked WPI

1) Determination of the content of free mercapto groups

The protein sample was diluted to a final concentration of 2mg/mL with a standard buffer (0.086mol/L Tris, 0.09mol/L glycine and 4mmol/L Na2EDTA) at pH8.0, and then centrifuged at 3000g for 10min, and the supernatant was taken for assay. To 3mL of supernatant was added 30. mu.L of Ellman's reagent solution (4mg DTNB/mL standard buffer) and mixed rapidlyAfter mixing, the mixture was left standing for 15min at room temperature in the dark and the absorbance was recorded at 412 nm. The blank was prepared without protein. The molar extinction coefficient of the Ellman's reagent was 1.36X 10⁴. The free thiol (SH) content is expressed as:

wherein A is₄₁₂Represents the absorbance value of the sample at a wavelength of 412 nm; d represents dilution factor; c represents the concentration of the sample (mg/mL).

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the content of free thiol groups of the TGase cross-linked WPI was studied, and the specific test results are shown in FIG. 9.

As can be seen from FIG. 9, the free thiol content of both the extruded WPI and the extruded WPI-TGase decreased with increasing extrusion temperature, and was less than that of the non-extruded WPI and WPI-TGase (P < 0.05). The extruded WPI had minimal free thiol content at an extrusion temperature of 130 ℃. This is because proteins form disulfide bonds by consuming a large amount of free thiol groups during high temperature extrusion, and then form high molecular weight protein-protein and/or protein-non-protein polymers by disulfide cross-linking. After TGase cross-linking, free thiols form disulfide bonds by oxidation reactions during TGase catalysis. And it can be seen that the crosslinking with TGase after low temperature extrusion (50 ℃ and 70 ℃) can obviously reduce the content of free sulfhydryl (P <0.05), so that the low temperature extrusion can indirectly indicate that the crosslinking with TGase is more favorable.

Example 8 Effect of extrusion temperature on surface hydrophobicity of TGase Cross-Linked WPI

1) Determination of surface hydrophobicity

The protein sample is diluted to 0.2-1.0 mg/mL by PBS buffer (pH7.0, concentration is 0.01mol/L), 20 μ L of ANS solution (concentration is 8mmol/L) is added to 4mL of diluted protein sample, and the mixture is shaken, mixed and reacted in the dark for 15 min. The excitation wavelength was set at 390nm, the emission wavelength at 470nm, and the slit width at 5 nm. And performing linear regression analysis by taking the measured fluorescence intensity as a vertical coordinate and the protein concentration as a horizontal coordinate, and taking the obtained initial slope as the surface hydrophobicity of the protein sample.

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the surface hydrophobicity of the TGase cross-linked WPI was studied, and the specific test results are shown in FIG. 10.

As can be seen from fig. 10, the surface hydrophobicity of the extruded WPI at 50 ℃ is significantly higher than that of the non-extruded WPI (P < 0.05). This may be due to exposure of partially hydrophobic amino acids inside the protein under high pressure and high shear during extrusion, resulting in increased hydrophobicity of the protein surface. But the surface hydrophobicity of the extruded WPI gradually decreases with increasing extrusion temperature. This is probably due to the high temperature during extrusion causing aggregation of the protein, thereby confining most of the hydrophobic sites to the interior of the aggregates.

After addition of TGase, the surface hydrophobicity of the pressed WPI-TGase at 50 ℃ and 70 ℃ was significantly increased (P <0.05) compared to the non-pressed WPI-TGase by 18.58% and 4.91%, respectively. This is probably because the low temperature extrusion increases the degree of TGase cross-linking, exposing more hydrophobic amino acids and increasing the accessibility to ANS binding sites, thus increasing its surface hydrophobicity. Compared with the high-temperature extrusion WPI without TGase crosslinking, the surface hydrophobicity of the WPI subjected to TGase crosslinking (110 ℃ and 130 ℃) has no significant difference (P is more than 0.05), which indicates that the high-temperature extrusion is not beneficial to the TGase crosslinking, and the result is consistent with the conclusion obtained by indexes such as size exclusion chromatography and the like.

Example 9 Effect of extrusion temperature on emulsification Properties of TGase Cross-Linked WPI

1) Determination of the emulsification Properties

First, 3mL of 0.5mg/mL protein sample solution and 1mL of soybean oil were mixed together, followed by homogeneous emulsification at 10000rpm for 2min using a high-speed emulsifier. Then, 50. mu.L of the emulsion was aspirated from the bottom of the tube, left for 0min and 10min, respectively, and immediately mixed with 5mL of a 0.1% SDS solution, and the absorbance was recorded at 500 nm. 0.1% SDS solution was used as a reagent blank. The emulsifying activity (EAI, m2/g) and the emulsifying stability (ESI,%) were calculated using the following formulas:

wherein A is₀The absorbance measured after the emulsion was left for 0min was indicated, D was the dilution factor of 100,. phi.is the oil phase volume fraction of the emulsion of 0.25, C is the concentration of the protein solution before emulsification (mg/mL), and T is the turbidity of 2.303.

Wherein A is₀Denotes the absorbance, A, measured after the emulsion has been left to stand for 0min₁₀The absorbance measured after the emulsion was left for 10min is shown.

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the emulsification characteristics of the TGase cross-linked WPI was studied, and the specific test results are shown in FIGS. 11 and 12.

As can be seen from fig. 11, the emulsification activity of the WPI subjected to the extrusion pre-treatment was significantly increased (P <0.05) compared to the WPI without the extrusion pre-treatment, and the emulsification activity of the WPI extruded at 50 ℃ was increased by 62.29% compared to the WPI without the extrusion pre-treatment. Mozafarpor et al also found that the emulsifying activity of the extruded protein was significantly higher than that of the untreated soy protein by extruding the soy protein. In addition, the emulsifying activity of the extruded WPI gradually decreased with increasing extrusion temperature. This is probably due to the fact that as the extrusion temperature increases, it leads to the formation of aggregates, thus some exposed hydrophobic groups are buried inside the protein, changing the hydrophobic and hydrophilic amino acid distribution of the extruded WPI surface, and thus changing the interfacial properties of the protein. Compared with non-extruded WPI-TGase, the emulsifying activity of the extruded WPI-TGase (extruded at 50-90 ℃) is obviously increased (P < 0.05). The emulsifying activity of the 50 ℃ squeezed WPI-TGase was increased 63.78%, i.e., from 41.47% to 67.92%, compared to the non-squeezed WPI-TGase. However, the emulsifying activity of the TGase-treated WPI-TGase and extruded WPI-TGase was significantly reduced (P <0.05) compared to WPI and extruded WPI without TGase cross-linking. The reason for this may be that the high molar mass and extended protein structure slows down the interfacial adsorption capacity of the protein, promotes its aggregation in the interfacial region and, therefore, leads to a reduced emulsifying activity of the cross-linked WPI and extruded WPI.

As can be seen from FIG. 12, the protein after high temperature extrusion (90 deg.C, 110 deg.C, 130 deg.C) had the highest emulsion stability, increased by 27.34%, 39.06%, 35.16%, respectively, as compared to the non-extruded WPI. The increase of the large molecular weight aggregates may cause deterioration of flexibility of the protein, decrease of adsorption capacity of the protein at the oil/water interface, and prevent re-aggregation of oil droplets, thereby improving emulsion stability of the protein. After TGase crosslinking treatment, the emulsification stability of WPI-TGase, WPI-TGase extruded at 50 ℃ and WPI-TGase extruded at 70 ℃ is obviously increased (P is less than 0.05), and the emulsification stability of crosslinked samples after high-temperature extrusion (90-130 ℃) is not obvious (P is more than 0.05). This also indicates that low temperature extrusion (50 ℃ and 70 ℃) favours TGase cross-linking to form a more rigid structure, resulting in an increase in the emulsion stability of the cross-linked protein.

Example 10 Effect of extrusion temperature on the Water binding Capacity of TGase Cross-Linked WPI

1) Measurement of Water holding Property

Weighing about 0.2g (m)₀) Protein samples were placed in 10mL centrifuge tubes and weighed (m)₁) 5mL of deionized water was added, vortex mixed for 30s, and then allowed to stand at room temperature for 24 h. Subsequently, centrifugation is carried out for 15min at 3000g, the supernatant is aspirated and weighed (m)₂). Each sample was repeated 3 times.

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the water holding capacity of the TGase cross-linked WPI was studied, and the specific test results are shown in FIG. 13.

As can be seen from fig. 13, the water binding capacity of the extruded WPI increased with increasing extrusion temperature (P <0.05) compared to WPI without extrusion pretreatment. The water retention of the extruded WPI at 130 ℃ was significantly increased 1695.29% over WPI without extrusion pretreatment. This indicates that increasing the temperature of the extrusion can significantly improve the water holding capacity of the WPI. The high temperature extrusion can make the original space structure of the protein become more compact and have small porosity (see fig. 16-23), so that the compact protein network traps water, and the water holding capacity is increased. After TGase crosslinking, the water binding capacity of the extruded WPI-TGase is remarkably increased (P <0.05) compared with that of the extruded WPI in the temperature range of 50-90 ℃, the water binding capacities of the extruded WPI-TGase are higher than those of the WPI-TGase which is not subjected to extrusion pretreatment, and the water binding capacities of the extruded WPI-TGase at 110 ℃ and 130 ℃ are not remarkably changed (P > 0.05). The water binding capacity of the extruded WPI-TGase was increased by 97.73%, i.e., from 0.44g/g to 0.87g/g, at 50 ℃ compared to the non-extruded WPI-TGase. This indicates that WPI treated by low temperature extrusion is more favorable to form a continuous network structure with TGase cross-linking to entrap moisture, compared to WPI treated by high temperature extrusion.

Example 11 Effect of extrusion temperature on the Secondary Structure of TGase Cross-Linked WPI

1) Determination of Secondary Structure

Performing full wave number (4000-400 cm) on a protein sample at room temperature by using an FTIR spectrometer^-1) Spectral scanning of (2). Approximately 2mg of the lyophilized protein sample was mixed with 200mg of KBr in a mortar and ground to a uniform powder and pressed into a thin sheet. The spectra were then recorded in 32 scans with a resolution of 4cm^-1. For amide I band (1700-1600 cm) in protein sample^-1) The secondary structural changes (alpha-helix, beta-sheet, beta-turn and random coil) were quantified.

2) On the basis of example 2, the extrusion temperature conditions of the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the secondary structure of the TGase cross-linked WPI was studied, and the specific test results are shown in FIGS. 14 and 15.

As can be seen from fig. 14, the content of alpha-helices was significantly reduced after the extrusion pretreatment (P < 0.05). This may be due to a drastic change in the secondary structure of the protein at higher extrusion temperatures, thereby reducing the helicity of the protein. Tomczynska-Mleko et al found that WPI was denatured during heating, thereby reducing the content of alpha-helical structures. Compared with the WPI without extrusion pretreatment, the beta-sheet content of the WPI extruded at 50-90 ℃ is remarkably increased, and the beta-turn content of the WPI extruded at 90-130 ℃ is remarkably increased (P < 0.05). According to the Beck et al studies, it was found that the increase in the content of β -turn structures in pea protein after extrusion is due to the formation of aggregates consisting of intermolecular hydrogen bonds of the β -turn structures. Thus, the increase in the proportion of β -turn structure in the WPI after the extrusion treatment indicates the formation of intermolecular aggregates. In addition, the content of beta-sheet increases after extrusion treatment, which indicates that the molecular structure of the protein is unfolded from folding and the molecular structure is changed from order to disorder, thereby promoting the exposure of hydrophobic groups buried in the protein.

Since the WPI after low-temperature extrusion is beneficial to crosslinking with TGase, the contents of alpha-helix and beta-sheet of the WPI-TGase extruded at 50 ℃ and 70 ℃ are obviously reduced compared with the WPI extruded without crosslinking, the content of beta-turn is obviously increased (P <0.05), and the change of the secondary structure of the WPI-TGase extruded at 90-130 ℃ is not obvious in difference compared with the change of the secondary structure of the WPI-TGase extruded without TGase crosslinking (as shown in fig. 14 and 15). Meanwhile, Zhang et al also found that when 0.1% of TGase was added, the α -helix structure was changed to the β -turn structure to promote the extension of the protein molecular chain.

Example 12 Effect of extrusion temperature on the microstructure of TGase Cross-Linked WPI

1) Measurement by scanning Electron microscope

The change in microstructure of the protein sample was observed using a tungsten filament scanning electron microscope at an accelerating voltage of 5 kV. Prior to observation, the protein samples were gold plated using an ion sputter. The image was then taken 1000 times.

2) On the basis of example 2, the extrusion temperature conditions in the cooking zone were changed to 50 ℃, 70 ℃, 90 ℃, 110 ℃ and 130 ℃, and the influence of the extrusion temperature on the microstructure of the TGase cross-linked WPI was studied, and the specific test results are shown in FIGS. 16-23.

As shown in fig. 16 to 23, WPI without the extrusion pretreatment exhibited a fragmented state, compared to the samples subjected to the extrusion pretreatment. Whereas the extrusion pre-treated samples, the extruded WPI gradually formed large aggregates as the extrusion temperature increased. This is because the proteins undergo aggregation and cross-linking during high temperature extrusion (90 ℃, 110 ℃, 130 ℃), resulting in the formation of large molecular weight protein-protein and/or protein-non-protein polymers. At low temperature extrusion (50 ℃), the extruded WPI did not form large polymers but rather were sheared into smaller pieces and some coiled structures were formed on the surface of the protein, which made it possible to expose some TGase cross-linking sites. After the protein is subjected to TGase crosslinking, compared with the WPI-TGase which is not subjected to extrusion pretreatment, the WPI-TGase extruded at 50 ℃ has better crosslinking effect, and original protein fragments are crosslinked into a whole. For the 90 ℃ pressed WPI-TGase samples, it can be seen that there are cases where large aggregates are not cross-linked. While the WPI-TGase sample was crosslinked only slightly at 130 ℃.

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

1. A method for modifying whey protein by extrusion combined with transglutaminase treatment, comprising the steps of:

(1) preparation of extruded whey protein: carrying out extrusion pretreatment on Whey Protein Isolate (WPI) serving as a raw material by using a parallel double-screw extruder; the extrusion parameters are set as follows: the feeding speed is 10g/min, the water content of the material is 40% (w/w), the water inlet speed is 5.5mL/min, and the screw rotating speed is 240 rpm; the extruder is divided into 4 functional zones, the 4 functional zones have 8 independent heating zones in total, the extruder comprises 7 built-in heating zones and 1 external die heating zone, the temperature of each functional zone is kept constant, and the feeding zone comprises: first zone 25 ℃, mixing zone: the second zone is 35 ℃, the third zone is 45 ℃, and the cooking zone: the fourth zone is 50 ℃, the fifth zone is 50 ℃, the sixth zone is 50 ℃, the discharging zone is: seventh zone 45 ℃, die heating zone: an eighth area is 25 ℃; after extrusion, freeze-drying and grinding the obtained extrudates with different temperatures to obtain extruded whey protein powder;

(3) preparation of TGase cross-linked extruded whey protein: adjusting the pH value of the whey protein solution to 7.0 by using 1mol/L sodium hydroxide solution, then adding 30U/g TGase into the solution, and heating in a water bath at 50 ℃ for 4h to catalyze a crosslinking reaction; after the crosslinking reaction is finished, inactivating enzyme of the crosslinking solution at 75 ℃ for 15min, and cooling at room temperature; and finally, freeze-drying and grinding a sample of the TGase cross-linked WPI extrudate to obtain the modified whey protein powder.

2. A modified whey protein prepared by the method of claim 1.