Hyposensitization functionalized whey-pea compound protein as well as preparation method and application thereof

Abstract

The invention discloses a low-sensitization functionalized whey-pea compound protein as well as a preparation method and application thereof, and belongs to the technical field of food processing. According to the invention, pH driving is combined with heat treatment to induce whey protein and pea protein to carry out structural recombination, and sensitization epitopes of the whey protein are effectively replaced and destroyed, so that the pea-whey compound protein with low sensitization and high technical function characteristics is prepared; meanwhile, the sensitization of the whey protein is reduced, and the technical functional characteristics of the pea protein are improved. The preparation method provided by the invention is green, simple, convenient and low in cost; the whey-pea compound protein provided by the invention can be used as a high-protein functional ingredient suitable for being eaten by whey allergy groups, and has a wide application prospect in the production of products such as infant formula milk powder, milk-containing beverages, yoghourt and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food processing, in particular to a low-allergy functional whey-pea composite protein and a preparation method and application thereof. BACKGROUND

[0002] Whey protein is a high-quality protein ingredient widely used in the food industry, but its main components (beta-lactoglobulin and alpha-lactalbumin) are allergenic, which can cause severe allergic reactions in certain consumer groups (such as infants), seriously threatening people's health. Therefore, how to effectively reduce the allergenicity of whey protein is a key problem that food processing practitioners need to solve.

[0003] Currently, the commonly used techniques to reduce the allergenicity of whey protein mainly include physical, chemical and enzymatic hydrolysis methods. Among them, simple physical processing cannot effectively reduce the allergenicity of whey protein; the commonly used chemical reaction method (such as glycosylation, polyphenol covalent modification) has the defects of complex process, product safety and high cost. The most widely used method is enzymatic hydrolysis, which usually needs to be deeply hydrolyzed to effectively destroy the allergenic epitopes (conformational epitopes, linear epitopes) of allergens and reduce their allergenicity. However, deep hydrolysis of proteins leads to an increase in bitter peptides and severe destruction of protein structure, resulting in a decrease in the technical functional properties of proteins.

[0004] Pea protein is a high-nutrition, low-allergy and sustainable plant protein. However, its low technical functional properties limit its application in the food field. Studies have shown that the structure of a single protein can be effectively modified through protein recombination technology, and the technical functional properties of a single protein can be improved. However, there is no report on the reduction of the allergenicity of whey protein through low-allergy protein structure recombination. SUMMARY

[0005] The purpose of the present application is to provide a low-allergy functional whey-pea composite protein and a preparation method and application thereof to solve the problems existing in the prior art. The present application uses pH-driven combined heat treatment to induce structural recombination of whey protein and pea protein, effectively replacing and destroying the allergenic epitopes of whey protein, thereby preparing a low-allergy pea-whey composite protein with high technical functional properties, and reducing the allergenicity of whey protein and improving the technical functional properties of pea protein.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides a preparation method of a low-allergy functional whey-pea composite protein, comprising the following steps:

[0008] Disperse whey protein isolate and pea protein isolate in water to obtain a protein solution, and adjust the pH of the protein solution;

[0009] The protein solution is subjected to heat treatment, and then cooled;

[0010] The pH of the cooled protein solution is adjusted to obtain a whey-pea composite protein solution;

[0011] The whey-pea composite protein solution is subjected to electrodialysis, vacuum concentration, and spray drying to obtain the whey-pea composite protein.

[0012] Preferably, the mass-volume ratio of the whey protein isolate and the pea protein isolate to the water is (0.5-5) g:100 mL.

[0013] Preferably, the mass ratio of the whey protein isolate to the pea protein isolate is 1:1.

[0014] Preferably, the pH of the protein solution is adjusted to 10-12.

[0015] Preferably, the heat treatment is performed at a temperature of 70-90℃ for 20 min.

[0016] Preferably, the pH of the cooled protein solution is adjusted to 7.

[0017] The application also provides a whey-pea composite protein prepared according to the preparation method.

[0018] The application also provides use of the whey-pea composite protein in preparation of a low-allergy protein food.

[0019] Optionally, the low-allergy protein food comprises milk powder, a milk-containing beverage, or yogurt.

[0020] The application discloses the following technical effects:

[0021] The application uses pH-driven combined heat treatment to induce structural recombination of whey protein and pea protein, effectively replaces and destroys the allergenic epitopes of whey protein, thereby preparing a pea-whey composite protein with low allergy and high technical functional characteristics, and simultaneously reducing the allergenicity of whey protein and improving the technical functional characteristics of pea protein.

[0022] The preparation method provided by the application is green, simple, and low-cost; and the whey-pea composite protein can be used as a high-protein functional ingredient suitable for whey allergy groups, and has a wide application prospect in production of infant formula milk powder, milk-containing beverages, yogurt, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only illustrate some of the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0024] Figure 1 Effect of pH shift combined with heat treatment on the solubility of complex protein

[0025] Figure 2 Effect of pH shift combined with heat treatment on the solubility of complex protein

[0026] Figure 3 Effect of pH shift combined with heat treatment on the emulsifying properties of complex protein

[0027] Figure 4 Effect of pH shift combined with heat treatment on the foaming properties of complex protein

[0028] Figure 5 Effect of pH shift combined with heat treatment on the LgE binding capacity of complex protein

[0029] Figure 6 Effect of pH shift combined with heat treatment on the LgG binding capacity of complex protein

[0030] In the above figures, whey or whey protein indicates single whey protein isolate, pea or pea protein indicates single pea protein isolate, pH shift indicates Comparative Example 1, 70°C indicates Comparative Example 2, pH shift + 70°C indicates Example 1, pH shift + 80°C indicates Example 2, and pH shift + 90°C indicates Example 3. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and should not be construed to limit the scope of the application. It should be understood that the detailed description and specific examples, while indicating certain embodiments of the application, are intended to be illustrative only and are not intended to limit the scope of the application.

[0032] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or stated range within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] The technical solution for preparing whey-pea complex protein according to the present invention includes the following steps:

[0037] Equal masses of whey protein isolate (WPI) and pea protein isolate (PPI) were dispersed in deionized water (0.5-5% W / V, i.e., 0.5g-5g of WPI and PPI per 100 mL of deionized water). The pH of the protein solution was adjusted to 10-12 to induce the dissociation of protein subunits to form fully dissolved "molten spheres".

[0038] The above-mentioned "molten globular" protein solution was heat-treated (70-90℃, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2℃ using an ice-water bath.

[0039] The pH of the cooled protein solution was adjusted to 7, and the protein that had been induced to unfold reassembled into a new whey-pea complex protein by non-covalent intermolecular forces.

[0040] The recombinant whey-pea protein solution was subjected to electrodialysis (temperature 10℃, desalted protein solution conductivity not exceeding 0.1 mS / cm), vacuum concentration (vacuum degree 5 kPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea protein powder. Specific research is illustrated in the following examples.

[0041] Example 1: Preparation of a hypoallergenic functionalized whey-pea complex protein

[0042] (1) Disperse equal masses of WPI and PPI in deionized water (0.5% W / V), adjust the pH of the protein solution to 12, and induce the protein subunits to dissociate to form fully dissolved "molten spheres".

[0043] (2) The above-mentioned "molten globular" protein solution was heat-treated (70°C, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2°C using an ice-water bath.

[0044] (3) Adjust the pH of the cooled protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0045] (4) The above-mentioned recombinant whey-pea complex protein solution was prepared into whey-pea complex protein powder by electrodialysis (temperature 10℃, desalted protein solution conductivity not exceeding 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃).

[0046] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 55.03%, which was 209.33% higher than that of the single PPI (17.79%); the EAI (emulsifying activity index) and ESI (emulsifying stability index) were 74.60 m. 2 / g and 72.47%, compared to a single PPI (EAI 33.82 m 2 / g, ESI 43.78%) increased by 120.58% and 65.53% respectively; FC (foaming capacity) and FS (foaming stability) were 356.05 cm 3 / g and 80.78%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS52.13%) increased by 68.19% and 55.00% respectively; the binding capacity of LgE and LgG decreased to 58.43% and 63.90% of WPI respectively.

[0047] (1) Solubility detection method: The protein solution was appropriately diluted with deionized water and then centrifuged at 4000 rpm for 25 min. The protein content in the supernatant and the original sample was determined by the BCA method, and the protein solubility was calculated according to Formula 1.

[0048] Solubility = Protein content in supernatant / Protein content before centrifugation × 100 (Formula 1).

[0049] (2) Emulsification test: The prepared sample solution was diluted with deionized water to a protein concentration of 5 mg / mL and stirred at 450 rpm for 30 min. The diluted sample was mixed with soybean oil at an oil-water volume ratio of 1:4 and then homogenized at 20,000 rpm for 1 min to prepare an emulsion.

[0050] Immediately aspirate 50 μL of the emulsion from the bottom and add it to 5 mL of 0.1% sodium dodecyl sulfate (SDS) solution. Immediately read the absorbance value A0 at 500 nm using a UV spectrophotometer. After allowing the emulsion to stand for 10 min, repeat the sampling procedure to measure the absorbance value A0. 10 The formulas for calculating the Emulsification Activity Index (EAI) and Emulsification Stability Index (ESI) are shown in Formulas 2 and 3.

[0051] (Formula 2);

[0052] (Formula 3).

[0053] Where DF is the dilution factor (100), C is the protein concentration (g / mL), φ is the oil phase volume fraction (0.20), and A0 and A 10 The values ​​represent the absorbance at 500 nm at 0 min and 10 min, respectively.

[0054] (3) Foaming property test: The sample was diluted with deionized water to a protein concentration of 5 mg / mL, and then the solution was homogenized at 20,000 rpm for 1 min, and the foam volume V0 was recorded. After standing for 30 min, the foam volume V0 was read again. 30 The formulas for calculating foaming capacity (FC) and foaming stability (FS) are shown in Formulas 4 and 5.

[0055] (Formula 4);

[0056] (Formula 5);

[0057] Where m is the mass of the sample protein, V0 is the volume after homogenization, and V 30 This is the volume after standing for 30 minutes.

[0058] (4) Indirect ELISA analysis of protein sensitization: The sample was diluted to 5 μg / mL with 0.05 mol / L sodium bicarbonate buffer (pH 9.6), and 100 μL of sample was added to a 96-well plate and incubated overnight at 4°C. After removing the sample coating solution, 250 μL of gelatin solution (3%, w / v) was added and incubated at 37°C for 1 h to block residual binding sites. Goat anti-human IgE-HRP conjugate or goat anti-human IgG-HRP conjugate was added respectively, and incubated at 37°C for 1 h. The absorbance of each well at 450 nm was read using a microplate reader to determine the binding capacity of IgE and IgG respectively. The absorbance of whey protein was taken as 100%, and the antibody binding capacity of other samples was expressed as the percentage of its absorbance to whey protein.

[0059] Example 2: Preparation of a hypoallergenic functionalized whey-pea complex protein

[0060] (1) Disperse equal masses of WPI and PPI in deionized water (0.5% W / V), adjust the pH of the protein solution to 12, and induce the protein subunits to dissociate to form fully dissolved "molten spheres".

[0061] (2) The above-mentioned "molten globular" protein solution was heat-treated (80℃, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2℃ using an ice-water bath.

[0062] (3) Adjust the pH of the cooled protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0063] (4) The above-mentioned recombinant whey-pea complex protein solution was subjected to electrodialysis (temperature 10℃, the conductivity of the desalted protein solution does not exceed 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea complex protein powder.

[0064] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 63.17%, which was 255.09% higher than that of the single PPI (17.79%); the EAI and ESI were 85.89 m. 2 / g and 78.37%, respectively, representing increases of 153.96% and 79% compared to single PPI (EAI 33.82m2 / g, ESI 43.78%); FC and FS were 364.89 cm 3 / g and 84.68%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 72.36% and 62.44% respectively; the binding capacity of LgE and LgG decreased to 54.40% and 62.61% of WPI respectively.

[0065] Example 3: Preparation of a hypoallergenic functionalized whey-pea complex protein

[0066] (1) Disperse equal masses of WPI and PPI in deionized water (0.5% W / V), adjust the pH of the protein solution to 12, and induce the protein subunits to dissociate to form fully dissolved "molten spheres".

[0067] (2) The above-mentioned "molten globular" protein solution was heat-treated (90℃, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2℃ using an ice-water bath.

[0068] (3) Adjust the pH of the cooled protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0069] (4) The above-mentioned recombinant whey-pea complex protein solution was subjected to electrodialysis (temperature 10℃, the conductivity of the desalted protein solution does not exceed 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea complex protein powder.

[0070] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 71.40%, which was 301.35% higher than that of the single PPI (17.79%); the EAI and ESI were 89.28 m. 2 / g and 81.76%, respectively, representing increases of 153.96% and 79% compared to single PPI (EAI 33.82m² / g, ESI 43.78%); FC and FS were 379.14 cm⁻¹. 3 / g and 86.48%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 72.36% and 62.44% respectively; the binding capacity of LgE and LgG decreased to 47.78% and 56.79% of WPI respectively.

[0071] Example 4: Preparation of a hypoallergenic functionalized whey-pea complex protein

[0072] (1) Disperse equal masses of WPI and PPI in deionized water (0.5% W / V), adjust the pH of the protein solution to 10, and induce the protein subunits to dissociate to form fully dissolved "molten spheres".

[0073] (2) The above-mentioned "molten globular" protein solution was heat-treated (90℃, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2℃ using an ice-water bath.

[0074] (3) Adjust the pH of the cooled protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0075] (4) The above-mentioned recombinant whey-pea complex protein solution was subjected to electrodialysis (temperature 10℃, the conductivity of the desalted protein solution does not exceed 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea complex protein powder.

[0076] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 53.61%, which was 201.35% higher than that of the single PPI (17.79%); the EAI and ESI were 58.53 m. 2 / g and 64.39%, respectively, representing increases of 73.06% and 47.08% compared to single PPI (EAI 33.82m2 / g, ESI 43.78%); FC and FS were 294.57.14 cm 3 / g and 68.48%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 39.15% and 31.36% respectively; the binding capacity of LgE and LgG of the complex protein decreased to 63.51% and 71.09% of WPI respectively.

[0077] Example 5: Preparation of a hypoallergenic functionalized whey-pea complex protein

[0078] (1) Disperse equal masses of WPI and PPI in deionized water (5% W / V), adjust the pH of the protein solution to 12, and induce the protein subunits to dissociate to form fully dissolved "molten spheres".

[0079] (2) The above-mentioned "molten globular" protein solution was heat-treated (90℃, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2℃ using an ice-water bath.

[0080] (3) Adjust the pH of the cooled protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0081] (4) The above-mentioned recombinant whey-pea complex protein solution was subjected to electrodialysis (temperature 10℃, the conductivity of the desalted protein solution does not exceed 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea complex protein powder.

[0082] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 55.93%, which was 214.39% higher than that of the single PPI (17.79%); the EAI and ESI were 60.89 m. 2 / g and 66.30%, compared to a single PPI (EAI 33.82m) 2 / g, ESI 43.78%) increased by 80.04% and 51.43% respectively; FC and FS were 303.46 cm 3 / g and 71.28%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 43.34% and 36.74% respectively; the binding capacity of LgE and LgG decreased to 65.73% and 73.68% of WPI respectively.

[0083] Comparative Example 1: Preparation of a whey-pea complex protein

[0084] The difference from Example 1 is that only pH shifting is performed, and the heat treatment operation is omitted. The specific steps are as follows:

[0085] (1) Disperse equal masses of WPI and PPI in deionized water (0.5% W / V), adjust the pH of the protein solution to 12, and induce the protein subunits to dissociate to form fully dissolved "molten spheres".

[0086] (2) The above “melted ball” protein solution was treated at room temperature (25±2℃) for 20 min.

[0087] (3) Adjust the pH of the protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0088] (4) The above-mentioned recombinant whey-pea complex protein solution was subjected to electrodialysis (temperature 10℃, the conductivity of the desalted protein solution does not exceed 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea complex protein powder.

[0089] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 35.9%, which was 101.80% higher than that of the single PPI (17.79%); the EAI and ESI were 55.99 m. 2 / g and 50.79%, compared to a single PPI (EAI 33.82 m 2 / g, ESI 43.78%) increased by 65.55% and 16.01% respectively; FC and FS were 299.86 cm 3 / g and 65.55%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 41.64% and 25.74% respectively; the binding capacity of LgE and LgG decreased to 73.05% and 76.83% of WPI respectively.

[0090] Comparative Example 2

[0091] The difference from Example 1 is that only heat treatment is performed, and the pH shifting operation is omitted. The specific steps are as follows:

[0092] (1) Disperse equal masses of WPI and PPI in deionized water (0.5% W / V) to form a mixed protein solution.

[0093] (2) The above mixed protein solution was heat-treated (70℃, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2℃ using an ice water bath.

[0094] (3) Prepare whey-pea protein powder by spray drying the above whey-pea protein solution (inlet air temperature 150℃, outlet air temperature 60℃).

[0095] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 28.79%, which was 61.83% higher than that of the single PPI (17.79%); the EAI and ESI were 43.91 m. 2 / g and 45.63%, compared to a single PPI (EAI 33.82m) 2 / g, ESI 43.78%) increased by 29.83% and 4.23% respectively; FC and FS were 269.17 cm 3 / g and 56.55%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 27.15% and 8.48%, respectively; the binding capacity of LgE and LgG decreased to 76.65% and 78.39% of WPI, respectively.

[0096] Comparative Example 3

[0097] The difference from Example 1 is that the parameters in step (1) are adjusted, and the specific steps are as follows:

[0098] (1) Disperse WPI and PPI in deionized water at a mass ratio of 2:1 (6% W / V), adjust the pH of the protein solution to 9, and induce the protein subunits to dissociate to form dissolved "molten spheres".

[0099] (2) The above-mentioned "molten globular" protein solution was heat-treated (70°C, 20 min) to further denature and expand the protein subunits, and then cooled to 25±2°C using an ice-water bath.

[0100] (3) Adjust the pH of the cooled protein solution to 7, and the protein that has been induced to unfold will reassemble into a new whey-pea complex protein by non-covalent intermolecular forces.

[0101] (4) The above-mentioned recombinant whey-pea complex protein solution was subjected to electrodialysis (temperature 10℃, the conductivity of the desalted protein solution does not exceed 0.1 mS / cm), vacuum concentration (vacuum degree 5 KPa, temperature 37℃), and spray drying (inlet air temperature 150℃, outlet air temperature 60℃) to prepare whey-pea complex protein powder.

[0102] Testing showed that the solubility of the whey-pea protein complex prepared under the above conditions was 51.64%, which was 190.27% higher than that of the single PPI (17.79%); the EAI and ESI were 54.65 m. 2 / g and 58.71%, respectively, representing increases of 61.59% and 34.10% compared to single PPI (EAI 33.82m2 / g, ESI 43.78%); FC and FS were 286.51 cm 3 / g and 61.85%, compared to a single PPI (FC 211.70 cm⁻¹). 3 / g, FS 52.13%) increased by 35.34% and 18.65% respectively; the binding capacity of LgE and LgG decreased to 78.08% and 80.29% of WPI respectively.

[0103] Experimental effect test

[0104] (1) The hydration particle size of the whey-pea complex protein prepared in Examples 1-3 and Comparative Examples 1-2 was tested, with single whey protein and single pea protein as controls.

[0105] Hydration particle size determination: The protein solution was diluted with deionized water to 1 mg / mL, and the particle size and polydispersity index (PDI) of the sample were determined using a Malvern laser particle size analyzer (NANO ZS90).

[0106] The results are as follows Figure 1 As shown, the particle size and PDI of the composite protein prepared by pH shift treatment are between those of whey protein and pea protein. The particle size and PDI of the composite protein prepared by pH shift combined with heat treatment are further reduced compared to the pH shift-treated composite protein, and are both smaller than those of whey protein. Furthermore, the particle size and PDI show a decreasing trend with increasing heat treatment temperature. This indicates that pH shift combined with heat treatment can induce pea protein and whey protein to assemble into a stable composite protein with reduced size.

[0107] (2) The solubility of whey-pea complex proteins prepared in Examples 1-3 and Comparative Examples 1-2 was compared, with single whey protein and single pea protein as controls. The results are as follows: Figure 2 As shown, the solubility of the composite protein prepared by pH shift treatment is between that of whey protein and pea protein. The solubility of the composite protein prepared by pH shift combined with heat treatment is further improved compared to the pH-shifted composite protein, and the solubility shows an increasing trend with increasing heat treatment temperature (the solubility at temperatures above 80℃ is comparable to that of whey protein). This indicates that pH shift combined with heat treatment can induce pea protein and whey protein to assemble into a composite protein with significantly improved solubility.

[0108] (3) The emulsifying properties of the whey-pea complex proteins prepared in Examples 1-3 and Comparative Examples 1-2 were compared, with single whey protein and single pea protein as controls. The results are as follows: Figure 3 As shown, the emulsifying properties of the composite protein prepared by pH shift treatment were significantly higher than those of pea protein and approached those of whey protein. The emulsifying properties of the composite protein prepared by pH shift combined with heat treatment were further improved compared to the pH-shifted composite protein, and the emulsifying properties increased with increasing heat treatment temperature. This indicates that pH shift combined with heat treatment can induce pea protein and whey protein to assemble into a composite protein with significantly enhanced emulsifying properties.

[0109] (4) The foaming properties of the whey-pea complex proteins prepared in Examples 1-3 and Comparative Examples 1-2 were compared, with single whey protein and single pea protein as controls. The results are as follows: Figure 4 As shown, the foaming properties of the composite protein prepared by pH shift treatment are between those of whey protein and pea protein. The foaming properties of the composite protein prepared by pH shift combined with heat treatment are further improved compared to the pH shift-treated composite protein (and both are higher than those of whey protein), and the foaming properties increase with increasing heat treatment temperature. This indicates that pH shift combined with heat treatment can induce pea protein and whey protein to assemble into a composite protein with significantly improved foaming properties.

[0110] (5) The allergenicity of whey-pea complex proteins prepared in Examples 1-3 and Comparative Examples 1-2 was compared, with single whey protein and single pea protein as controls. The results are as follows:Figures 5-6 As shown, the binding capacity of IgE and IgG in the composite protein prepared by pH shift treatment is between that of whey protein and pea protein. The binding capacity of IgE and IgG in the composite protein prepared by pH shift combined with heat treatment is further reduced compared to the pH-shifted composite protein, and the binding capacity decreases with increasing heat treatment temperature. This indicates that pH shift combined with heat treatment can induce pea protein and whey protein to assemble into a composite protein with significantly reduced allergenicity.

[0111] Depend on Figures 1-6 It was found that the particle size of the mixed protein prepared by heating alone was larger than that of the composite protein prepared by pH shifting alone. Its functional properties (solubility, emulsifying properties, and foaming properties) were worse than those of the composite protein prepared by pH shifting alone, while its sensitization (IgE and IgG binding capacity) was stronger. Its functionality was weaker than that of the composite protein prepared by a combination of pH shifting and heat treatment, and its sensitization was stronger. Therefore, it is inferred that there is a synergistic effect in the process of pH shifting combined with heat treatment inducing the assembly of whey and pea proteins into a highly functional, low-sensitization composite protein.

[0112] Based on the above experimental results, this invention uses pH-driven combined heat treatment to induce structural recombination of whey protein and pea protein, effectively replacing and destroying the sensitizing epitopes of whey protein, thereby preparing a pea-whey complex protein with low sensitivity and high technical functional properties, while simultaneously reducing the sensitivity of whey protein and improving the technical functional properties of pea protein.

[0113] The preparation method provided by this invention is green, simple and low cost; the whey-pea complex protein provided can be used as a high-protein functional ingredient suitable for people with whey allergies, and has broad application prospects in the production of infant formula, milk-containing beverages, yogurt and other products.

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a hypoallergenic functionalized whey-pea complex protein, characterized in that, Includes the following steps: Disperse whey protein isolate and pea protein isolate in water to obtain a protein solution, and adjust the pH of the protein solution; The protein solution was heat-treated and then cooled. Adjust the pH of the cooled protein solution to obtain a whey-pea complex protein solution; The whey-pea complex protein solution is obtained by electrodialysis, vacuum concentration, and spray drying.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the whey protein isolate and pea protein isolate to the water is (0.5-5) g: 100 mL.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the whey protein isolate to the pea protein isolate is 1:

1.

4. The preparation method according to claim 1, characterized in that, The pH of the protein solution is adjusted to 10-12.

5. The preparation method according to claim 1, characterized in that, The heat treatment conditions are 70-90℃ and 20 min.

6. The preparation method according to claim 1, characterized in that, The pH of the cooled protein solution is adjusted to 7.

7. The whey-pea complex protein prepared by the preparation method according to any one of claims 1-6.

8. The application of the whey-pea complex protein as described in claim 7 in the preparation of low-allergenic protein foods.

9. The application as described in claim 8, characterized in that, The low-allergenic protein foods include milk powder, milk-containing beverages, or yogurt.

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