Hypoallergenic milk whey protein powder and preparation method thereof

Abstract

The invention provides hypoallergenic milk whey protein powder and a preparation method thereof. The hypoallergenic beta-LG protein powder comprises beta-lactoglobulin which is non-covalently bound with a small molecule substance, the small molecular substance is ascorbic acid, erythritol or beta-carotene. Non-covalent binding of small molecular substances and beta-LG is utilized, the sensitization of beta-LG is reduced by pertinently covering a beta-LG epitope and an IgE binding site, the hypoallergenic beta-LG is mixed with lactoferrin and alpha-lactalbumin, then part of immunoglobulin is added, and after freeze-drying, the hypoallergenic milk formula whey protein powder is prepared. The functional dairy product meets the national food safety standard, is suitable for multiple people in exercise and fitness, daily nutrition supplement and special physiological stages, and has wide application prospects in the functional dairy product processing industry.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, specifically to a low-allergenic milk whey protein powder and its preparation method. Background Technology

[0002] In the interdisciplinary field of public health and food science, and in dairy industry production practices, food safety assurance, especially allergy risk prevention and control, has always been a core focus. With the increasing proportion of dairy products in the global diet, health problems caused by food allergies have gradually become a global public health challenge, receiving widespread attention from health departments, the food industry, and research institutions worldwide. Food allergies are essentially abnormal immune responses of the body to specific components in food. When allergic individuals come into contact with the corresponding allergens, they may experience a series of local or systemic symptoms, such as hives and itching in the skin, abdominal pain, diarrhea, and nausea in the digestive system, and dizziness or even fainting in the nervous system. In severe cases, they can even directly threaten life. Among many allergenic foods, milk is one of the eight internationally recognized categories of allergenic foods, and its allergenicity is universal and consistent globally, with related problems being particularly prominent in dairy consumption scenarios.

[0003] Milk β - Lactoglobulin ( β -Lactoglobulin, abbreviated as β -LG is the core allergen; clinical data shows that approximately 82% of milk allergy sufferers will also experience allergic reactions. β -LG generates an immune response, and this data fully demonstrates β -LG's key role in the mechanism of milk allergy. Meanwhile, β -LG accounts for up to 50% of whey protein components, and whey protein, as an important food ingredient, is widely used in dairy products, functional foods, infant formula, and many other fields. In terms of market size, the global whey powder market exceeded US$10 billion in 2024 and is expected to maintain an average annual growth rate of 5%-7% in the coming years; among them, the demand for high-purity whey protein has surged due to the rapid expansion of the functional food market. Against this backdrop, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. β LG's research on allergen regulation technology is not only an important topic in food science and public health, but also directly related to product upgrading in the dairy industry and ensuring dietary safety for allergy sufferers, possessing significant practical application value. Currently, reducing... β - Lactoglobulin ( βThe technologies for addressing sensitization (LG) primarily include physical modification, enzymatic hydrolysis, fermentation engineering, genetic engineering, and chemical modification. Industrial applications often utilize a combination of enzymatic hydrolysis and fermentation, as seen in extensively hydrolyzed milk powder and fermented yogurt products. While gene editing technology holds promise, it has not yet been commercialized due to ethical controversies and regulatory restrictions. Emerging technologies (such as nanoencapsulation) remain in the laboratory stage, requiring solutions to cost and large-scale production challenges. Chemical modification commonly employs glycosylation reactions, but improper reaction conditions can lead to browning of the products.

[0004] Currently, in dairy processing and the production of related functional foods, the core challenge lies in how to effectively reduce the levels of certain substances in milk. β -LG's approach to allergen control balances product safety, compliance, and market applicability. On one hand, with the increasing market demand for whey protein-related products and the expanding consumer base, including a large number of people with milk allergies, existing whey protein products without allergen control cannot meet the consumption needs of this group, limiting market reach. On the other hand, if allergen control cannot be effectively implemented... β - The allergenicity of LG milk products poses health risks to individuals with allergies, impacting not only consumer health but also potential food safety disputes, negatively affecting the brand reputation of dairy companies and the industry's development. Furthermore, from a public health perspective, milk allergies have become a significant public health burden, particularly among children, where the incidence is high. There is an urgent need to address the allergenicity of dairy products through technological means to reduce related health risks and alleviate public health pressure.

[0005] In response to the above β The technical challenges of regulating LG sensitization have been addressed in existing research, with various attempts made to reduce sensitization through different technological approaches. β -LG's sensitizing properties. One important research direction is the use of plant-derived polyphenols and... β -LG combines through covalent or non-covalent bonding, altering... β -LG spatial structure, thereby reducing its sensitizing properties. Related studies have found that some plant-derived polyphenol compounds can interact with... β -LG forms a stable complex, making β - LG's antigenic epitopes are masked or destroyed, reducing the body's immune response and thus lowering sensitization. The advantage of this approach lies in the relatively wide availability of plant-derived polyphenol compounds, some of which possess certain antioxidant and other physiological activities. Theoretically, it has the potential to simultaneously reduce sensitization and enhance function, making it a current research hotspot in this field. Related research findings have already been validated to some extent at the laboratory level.

[0006] Although existing methods utilize plant-derived polyphenol compounds to regulateβ While research on the allergenicity of LG has made some progress, this technological approach still faces key challenges in practical industrial application, with a core deficiency being insufficient compliance. According to the current GB 2760-2024 "National Food Safety Standard for the Use of Food Additives," none of the plant-derived polyphenol compounds used in the research are included in the permitted category of food additives. This research is merely academic exploration and has no practical significance in guiding production. In food production and processing, the use of any additive must strictly adhere to the provisions of national food safety standards. Substances not included in the standard's scope cannot be used as food additives in food production; otherwise, the product will fail to meet food safety standards, cannot pass regulatory approval, and cannot enter the market. This problem directly prevents related technological achievements from moving from the laboratory to industrial application, making it difficult to truly transform them into marketable products and effectively solve the problems in the dairy industry. β -The actual need for LG sensitization regulation.

[0007] In summary, existing technical solutions face obstacles to industrial application due to their failure to meet national food safety standards, and thus cannot effectively solve current problems in dairy processing. β The core issue in regulating LG's allergenicity urgently requires the development of a method based on compliant food additives. β -LG's sensitization regulation technology aims to fill existing technological gaps and meet industry development and public health needs. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a low-allergenic milk whey protein powder and its preparation method, utilizing small molecule substances and... β -LG's non-covalent binding, through targeted masking β -LG antigen epitopes and IgE binding sites reduce its sensitization, thus reducing the hyposensitization level. β -LG is mixed with lactoferrin and α-lactalbumin, and then some immunoglobulins are added. After freeze-drying, it is prepared into a low-allergenic milk formula whey protein powder that meets national food safety standards. It is suitable for multiple groups of people who are engaged in sports and fitness, daily nutritional supplementation, and special physiological stages, and has broad application prospects in the functional dairy processing industry.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a low-sensitization method β -LG protein powder, including those that are non-covalently bound to small molecules. β - Lactoglobulin; the small molecule substances are ascorbic acid, erythritol, β - At least one of the carotene.

[0010] Furthermore, the aforementioned β- The mass ratio of added lactoglobulin to small molecules is (2~5):1.

[0011] This invention also provides a low-sensitivity β The preparation method of LG protein powder, with specific steps as follows: Will β - Lactoglobulin dissolved in phosphate buffer yields β -LG solution; Add small molecules β In a LG solution, after stirring at room temperature and allowing to stand, a non-covalent complex was obtained. The non-covalent complex was freeze-dried under vacuum to obtain a low-sensitivity product. β -LG protein powder.

[0012] Furthermore, the aforementioned β The final concentration of the -LG solution is 0.1 mg / mL to 0.5 mg / mL.

[0013] This invention also provides a hypoallergenic milk formula whey protein powder, the raw materials of which include lactoferrin powder, α-lactalbumin powder, immunoglobulins and hypoallergenic... β -LG protein powder, the hypoallergenic β -LG protein powder is a hypoallergenic product as described in any one of claims 1. β -LG protein powder.

[0014] Furthermore, the lactoferrin powder, α-lactalbumin powder, and hypoallergenic... β - The addition ratio of LG protein powder is 5:2:0.1 to 4:1:0.1.

[0015] Furthermore, the lactoferrin powder, α-lactalbumin powder, and hypoallergenic... β -LG protein powder contains 5% to 8% immunoglobulin by weight.

[0016] Furthermore, the raw materials also include calcium salts, citric acid, and flavoring agents. The calcium salts are calcium carbonate, tricalcium phosphate, or calcium citrate. The amount of calcium salt added per 100g of mixed protein powder 2 is 0.8-2.0g. Mixed protein powder 2 is lactoferrin powder, α-lactalbumin powder, or a hypoallergenic agent. β - A mixture of LG protein powder and immunoglobulins; citric acid is added at 0.15% of mixed protein powder 3, which consists of lactoferrin powder, α-lactalbumin powder, and hypoallergenic... β - A mixture of LG protein powder, immunoglobulins and calcium salts; the flavoring agent is vanillin or L-theanine, wherein the amount of vanillin added does not exceed 5 mg / 100 mL; and the amount of L-theanine added is 100 mg / kg-310 mg / kg.

[0017] This invention also provides a method for preparing hypoallergenic milk formula whey protein powder, the specific steps of which are as follows: Lactoferrin powder and α-lactalbumin powder were combined with hypoallergenic... β - LG protein powder is mixed at room temperature to obtain mixed protein powder 1.

[0018] Immunoglobulin was added to the mixed protein powder 1 and allowed to stand at room temperature to obtain hypoallergenic milk formula whey protein powder.

[0019] Furthermore, 0.07g of antioxidants are added to each gram of hypoallergenic milk formula whey protein powder.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a low-sensitivity β -LG protein powder's core advantage lies in completely resolving the pain point of insufficient compliance in existing hypoallergenic whey protein technologies, filling the gap in compliant hypoallergenic whey protein technology, and providing a new path for its industrial application. The core components of this protein powder are small molecule substances and... β - Lactoglobulin ( β The non-covalent complex of -LG, and the selected small molecules are limited to ascorbic acid, erythritol, β - At least one of the carotene components is a dairy additive permitted under GB 2760-2024 "National Food Safety Standard for the Use of Food Additives," ensuring its safety and compliance with clear national standards. Compared to commonly used plant-derived polyphenol compounds in existing research, the small molecule substances selected in this invention completely circumvent the industrialization obstacles caused by additives not being included in national standards, ensuring that the product meets food safety standards from the source and laying a compliant foundation for industrial promotion.

[0021] This invention utilizes small molecule substances and β -LG's non-covalent bonding, efficient improvement β -LG allergenicity addresses the core concern of ensuring safe milk protein intake for people with milk allergies. ELISA in vitro testing has verified that three small molecules are... β -LG binding significantly reduces antigen content, with even more pronounced effects when used in combination. The mechanism involves small molecules modifying and masking the antigen through non-covalent binding. β -LG antigen epitopes reduce the probability of binding to human IgE antibodies, lowering the risk of allergies and providing a safe and compliant dairy protein supplement option for people with allergies. This binding method is gentle, requires no complex chemical modification processes, and does not damage... β -LG's physicochemical properties and nutritional activity are safer and easier to operate than traditional chemical modification, and there is no need to add toxic or harmful reagents, further ensuring food safety.

[0022] In addition, this protein powder has the advantage of multiple functions, with the small molecule substances themselves possessing multiple effects and β -LG, when combined, achieves synergistic effects. Ascorbic acid (vitamin C) can enhance the product's antioxidant activity and help boost immunity; β - Carotene can be converted into vitamin A, which also has eye-protecting effects; erythritol, as a prebiotic, can regulate the balance of intestinal flora and improve intestinal health. Compared with single hypoallergenic products, the protein powder of this invention has antioxidant, eye-protecting, and intestinal-regulating functions, making it suitable for a wider range of applications, including functional dairy products, health foods, and infant complementary foods, with significant market application value. In summary, this hypoallergenic... β -LG protein powder combines compliance, safety, functionality, and practicality, effectively solving existing technological bottlenecks and meeting the market demand for low-allergenic, multifunctional dairy proteins.

[0023] This invention clearly states β The mass ratio of added lactoglobulin to small molecules is (2~5):1. This ratio, verified through multiple experiments, represents the optimal range balancing low allergenicity, nutritional activity, and production costs. This ratio ensures that the small molecules and... β -LG binds fully, maximally masking antigenic epitopes. Experiments have verified that this ratio... β -LG antigen content reduction effect is significantly better than deviating from the range ratio, which can avoid poor sensitization improvement due to insufficient addition of small molecule substances, or waste of resources and increased costs due to excessive addition, thus achieving rational use of resources and effective cost control.

[0024] At the same time, this ratio can effectively preserve β - LG enhances the nutritional activity of small molecules, avoiding imbalances that could disrupt their synergistic effect. When the mass ratio is controlled at (2~5):1, the non-covalent bonding is optimal, preventing excessive small molecules from causing adverse reactions. β -LG has an unusual spatial conformation, and its additional functions will not be affected by insufficient addition. In addition, this formula meets the requirements of industrial cost control. The selected small molecule substances are inexpensive and readily available. The amount of raw materials can be reasonably controlled while ensuring product performance, thereby enhancing the advantages of industrial promotion and achieving a balance between economic and social benefits.

[0025] The low sensitization provided by this invention β The -LG protein powder preparation method addresses the shortcomings of existing methods, such as complex processes, easy damage to protein properties, and difficulty in large-scale production, constructing a simple, efficient, and industrially scalable preparation process. This method only includes... β The three core steps of -LG solution preparation, non-covalent complex preparation, and vacuum freeze-drying require no complex equipment or cumbersome operations. They are easy to control and can reduce equipment and labor costs, improve production efficiency, and break the dependence on high-end equipment and professional personnel.

[0026] This method prepares the complex by stirring and allowing it to stand at room temperature, without requiring high temperature, high pressure, or toxic reagents, and can retain the maximum amount of the complex. β -LG enhances the nutritional activity of small molecules, avoiding problems such as antigenic epitope exposure and small molecule degradation caused by traditional vigorous processing. It also reduces energy consumption, prevents flavor abnormalities and nutrient loss, and ensures product palatability and nutritional integrity. Vacuum freeze-drying technology offers significant advantages over conventional drying methods, removing moisture in a low-temperature vacuum environment and avoiding... β -LG denaturation and degradation of small molecules ensure product stability. After drying, the product is loose and porous with good solubility, making it easy to store and process. The production process is safe and environmentally friendly, in line with the concept of green production.

[0027] This invention clearly states β The final concentration of the LG solution is 0.1~0.5 mg / mL. This concentration is crucial for achieving the desired interaction between small molecules and... β -LG's high-efficiency combination is key to ensuring stable product quality. This concentration ensures... β -LG is uniformly dispersed in phosphate buffer to avoid excessively high concentrations that lead to molecular aggregation and decreased binding efficiency, or excessively low concentrations that result in increased energy consumption and costs, and reduced production efficiency. Experimental verification has shown that the complexes prepared within this concentration range... β -LG antigen content decreases significantly more and can maintain buffer pH stability, ensuring consistency between preparation process and product quality. It is compatible with subsequent freeze-drying processes, improving product processing performance and usability. No special adjustment equipment is required, further enhancing industrial feasibility.

[0028] The hypoallergenic milk formula whey protein powder provided by this invention has low allergenicity. β Based on LG protein powder, this formula is compounded with lactoferrin, α-lactalbumin, and immunoglobulins to optimize nutrition and enhance function, addressing the weakness of existing products with limited nutritional content. This formula is also hypoallergenic. β - LG protein powder is the core ingredient, ensuring low allergenicity. The three blended proteins are key nutrients of whey protein, which can restore the natural composition of whey protein, meet the human body's need for high-quality protein, and achieve the dual goals of low allergenicity and high nutrition.

[0029] Lactoferrin can regulate immunity, has antibacterial and antiviral effects, and promotes iron absorption; α-lactalbumin is easily digestible and suitable for people with weak digestive function; immunoglobulins can supplement antibodies and strengthen immunity. All three are low in allergens. β -LG protein powder features a blend of ingredients to achieve nutritional complementarity, catering to the needs of a wider range of people. Furthermore, the formula is lactose-free and fat-free, making it suitable for those with lactose intolerance and those seeking weight loss or control, thus expanding its applicability and enhancing its market competitiveness.

[0030] This invention specifies the exact proportions and amounts of each component to ensure nutritional balance, functional stability, and good palatability. Lactoferrin, α-lactalbumin, and low-allergenicity are included. β -LG protein powder has a formulation ratio of 5:2:0.1 to 4:1:0.1, which can achieve nutritional complementarity and functional optimization; the addition of 5% to 8% immunoglobulin can strengthen immune function; the types and amounts of calcium salts, citric acid and flavoring agents strictly comply with national standards, which can supplement calcium, improve solubility and palatability, ensure product safety and compliance, and meet the needs of industrial quality control.

[0031] The protein powder preparation method provided by this invention employs a mild dry-mixing process, comprising only two mixing steps. This simple and efficient operation reduces equipment and labor costs, improves production efficiency, and is suitable for large-scale production. The dry-mixing process eliminates the need for water, avoiding protein denaturation and interactions between components, maximizing the retention of the nutritional activity of each protein. Room temperature operation further prevents the destruction of protein activity by high temperatures, ensuring stable product quality.

[0032] This method limits the addition of antioxidants to 0.07g per gram of product, effectively inhibiting protein oxidation and deterioration, extending shelf life, and preventing flavor and nutrient loss. The dosage complies with national standards, ensuring food safety. The preparation process involves no toxic reagents, making it safe and environmentally friendly. The product has stable quality, good solubility, and is easy to store and process. Compared to existing methods, it is simpler and safer, enabling large-scale industrial production and catering to the segmented needs of different consumer groups, demonstrating significant market application value. Attached Figure Description

[0033] Figure 1 For milk β -LG structure and its antigenic epitope distribution.

[0034] Figure 2 for β -LG complex protein antigen activity assay.

[0035] Figure 3 for β -LG complex protein structure analysis. (AC) UV-Vis absorption spectroscopy; (D) Fourier transform infrared spectroscopy; (EF) circular dichroism spectroscopy.

[0036] Figure 4 For small molecules and β -LG molecular docking model. β A three-dimensional schematic diagram (A) and a two-dimensional docking diagram (B) of molecular docking of -LG-AA. β Three-dimensional docking diagram (C) and two-dimensional docking diagram (D) of the -LG-CAR complex. β Three-dimensional docking diagram (E) and two-dimensional docking diagram (F) of the -LG-ERY complex.

[0037] Figure 5 for β - LG complex protein antioxidant activity analysis.

[0038] Figure 6 for β -LG complex protein emulsification analysis. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0040] This invention provides a low-sensitivity β -LG protein powder uses small molecule substances as food additives, specifically including: ascorbic acid, erythritol, or β - Carotene, the specific steps for its preparation are as follows: (1) β -LG is dissolved in phosphate buffer (pH 5-8, concentration 0.1 mol / L) to obtain β -LG solution, β The final concentration of the -LG solution is 0.1 mg / mL to 0.5 mg / mL.

[0041] (2) Ascorbic acid, erythritol, and β - Added carotene β -LG solution, β - The mass ratio of LG to small molecules was set to (2~5):1. After stirring at room temperature for 15~30 minutes, the mixture was allowed to stand at room temperature to obtain... β -LG-ascorbic acid ( β -LG-AA), β -LG-carotene ( β -LG-CAR) and β -LG-erythritol ( β (-LG-ERY) are three non-covalent complexes.

[0042] (3) The above three non-covalent complexes were freeze-dried under vacuum (-80℃~-70℃, 14~16h) to obtain low-sensitization compounds. β -LG protein powder.

[0043] This invention also provides a hypoallergenic milk formula whey protein powder, comprising lactoferrin powder, α-lactalbumin powder, immunoglobulin, calcium salt, citric acid, and the aforementioned hypoallergenic... β -LG protein powder is mixed to obtain the following specific preparation steps: (1) Combine lactoferrin powder and α-lactalbumin powder with the above-mentioned hypoallergenic β -LG protein powder is mixed at a certain ratio (5:2:0.1~4:1:0.1) at room temperature to obtain mixed protein powder 1.

[0044] (2) Add 5% to 8% of immunoglobulin to the mixed protein powder 1 and let it stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0045] (3) Crush the calcium salts (calcium carbonate, tricalcium phosphate, calcium citrate) to a particle size ≤50μm. The amount of calcium salt added to each 100g of mixed protein powder 2 is 0.8~2.0g. Let it stand at room temperature for 15~20 minutes to obtain mixed protein powder 3.

[0046] (4) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain low-allergenic milk formula whey protein powder.

[0047] Furthermore, depending on flavor requirements, vanillin (<5mg / 100mL) or L-theanine (100–310mg / kg) can be added to prepare various flavored hypoallergenic milk formula whey protein powders according to known methods.

[0048] Furthermore, 0.07g of the antioxidant sodium D-isoascorbate is added to each gram of hypoallergenic milk formula whey protein powder, and it is packaged in aluminum foil bags at room temperature.

[0049] Example 1: 1. Preparation of hypoallergenic milk formula whey protein powder (1) β -LG is dissolved in phosphate buffer (pH=5, concentration 0.1 mol / L) to make... β -LG achieved a final concentration of 0.1 mg / mL. Preparation β -LG-ascorbic acid ( β -LG-AA) non-covalent complex, β - The mass ratio of LG to small molecules was set to 3:1. After stirring at room temperature for 15 minutes, the mixture was allowed to stand at room temperature.

[0050] (2) To β -LG-ascorbic acid was freeze-dried at -80°C for 14 hours to obtain a low-sensitivity product. β -LG protein powder.

[0051] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powders were mixed at a certain ratio (5:2:0.1) at room temperature to obtain mixed protein powder 1.

[0052] (4) Add 5% by weight of immunoglobulin to mixed protein powder 1, let stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0053] (5) Add an appropriate amount of calcium salt (calcium carbonate) to (4), and crush the calcium salt to a particle size ≤50μm. The amount of calcium salt added in each 100g of mixed protein powder 2 is 0.8g. Let it stand at room temperature for 15 minutes to obtain mixed protein powder 3.

[0054] (6) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain low-allergenic milk formula whey protein powder.

[0055] (7) Add vanillin (<5mg / 100mL) according to flavor requirements and prepare vanilla-flavored low-allergenic milk formula whey protein powder according to known methods.

[0056] (8) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of hypoallergenic milk formula whey protein powder, and package in aluminum foil bags at room temperature.

[0057] 2. Quality testing of hypoallergenic milk formula whey protein powder Detection and analysis are conducted in terms of sensory characteristics, microorganisms, and nutrients.

[0058] Table 1. Quality testing of hypoallergenic milk formula whey protein powder

[0059] 3. Functional testing of hypoallergenic milk formula whey protein powder Dissolve hypoallergenic milk formula whey protein powder at a ratio of 0.1 mg / 10 mL in 40 mL of water. o In water C, stir well, let stand at room temperature for 5 minutes, and then use cow... β - LG ELISA quantitative kit for detection: Use cattle β The LG ELISA Quantitative Kit measures the antigenicity of all samples (control and experimental groups) using a sandwich enzyme-linked immunosorbent assay (ELISA). Each well of a 96-well microtiter plate is pre-coated with capture antibody diluted in 0.05 mol / L phosphate buffer. After incubation overnight at 4°C, the plate is washed five times with ELISA wash buffer, and 350 μL of blocking buffer is added to each well to block any remaining free binding sites. After incubation at room temperature for 30 min, the blocking buffer is discarded, and the plate is washed five times to remove unbound antibodies. Immediately, 50 μL of standards and diluted samples under different conditions are added, followed by 100 μL of horseradish peroxidase (HRP)-labeled [sample / treatment] to each well. β-LG antibody detection: Seal the reaction wells with sealing film, incubate at 37℃ for 1 h, discard the liquid and pat dry on absorbent paper. Add 350 μL of ELISA washing buffer to each well, let stand for 1 min, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times. Then add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to each well, and incubate in the dark at 37℃ for 15 min. Finally, add stop solution (2 mol / L H2SO4, 50 μL) to each well to stop the reaction, and measure the absorbance at 450 nm using a microplate reader. According to the standard curve ( β -LG's linear correlation range is 6.25-200 μg / mL) to calculate the antigenicity of the complex, i.e., the equivalent micrograms of protein antigen per milliliter of sample.

[0060] Measured cattle β -LG antigen content decreased by 20%, and the antioxidant DPPH free radical scavenging ability was 68%.

[0061] Example 2: 1. Preparation of hypoallergenic milk formula whey protein powder (1) β -LG was dissolved in phosphate buffer (pH=6, concentration 0.1 mol / L) to make... β -LG achieved a final concentration of 0.2 mg / mL. Preparation β -LG-carotene ( β -LG-CAR) non-covalent complex, β - The mass ratio of LG to small molecules was set to 2:1. After stirring at room temperature for 20 minutes, the mixture was allowed to stand at room temperature.

[0062] (2) To β -LG-carotene was freeze-dried at -75°C for 15 hours to obtain a low-allergenicity product. β -LG protein powder.

[0063] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powders were mixed at a certain ratio (5:1:0.1) at room temperature to obtain mixed protein powder 1.

[0064] (4) Add 6% by weight of immunoglobulin to mixed protein powder 1, let stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0065] (5) Add an appropriate amount of calcium salt (tricalcium phosphate) to (4), and crush the calcium salt to a particle size ≤50μm. The amount of calcium salt added in each 100g of mixed protein powder 2 is 1.8g. Let it stand at room temperature for 20 minutes to obtain mixed protein powder 3.

[0066] (6) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain low-allergenic milk formula whey protein powder.

[0067] (7) Add L-theanine (100mg / kg) according to flavor requirements and prepare low-allergenic milk formula whey protein powder with green tea flavor according to known methods.

[0068] (8) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of hypoallergenic milk formula whey protein powder, and package in aluminum foil bags at room temperature.

[0069] 2. Quality testing of hypoallergenic milk formula whey protein powder Detection and analysis are conducted in terms of sensory characteristics, microorganisms, and nutrients.

[0070] Table 2 Quality Testing of Hypoallergenic Milk Formula Whey Protein Powder

[0071] 3. Functional testing of hypoallergenic milk formula whey protein powder Dissolve hypoallergenic milk formula whey protein powder at a ratio of 0.1 mg / 10 mL in 40 mL of water. o In water C, stir well, let stand at room temperature for 5 minutes, and then use cow... β -LG ELISA quantitative kit (same as Example 1) was used to measure bovine... β -LG antigen content decreased by 21%, and the antioxidant DPPH free radical scavenging ability was 45%.

[0072] Example 3: 1. Preparation of hypoallergenic milk formula whey protein powder (1) β -LG is dissolved in phosphate buffer (pH=8, concentration 0.1 mol / L) to make β -LG reached a final concentration of 0.5 mg / mL during preparation. β -LG-erythritol ( β -LG-ERY) non-covalent complex, β - The mass ratio of LG to small molecules was set to 5:1. After stirring at room temperature for 30 minutes, the mixture was allowed to stand at room temperature.

[0073] (2) To β -LG-erythritol was freeze-dried at -70°C for 16 hours to obtain a low-sensitivity product. β -LG protein powder.

[0074] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β-LG protein powders were mixed at a certain ratio (4:1:0.1) at room temperature to obtain mixed protein powder 1.

[0075] (4) Add 8% by weight of immunoglobulin to mixed protein powder 1, let stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0076] (5) Add an appropriate amount of calcium salt (calcium citrate) to (4), and crush the calcium salt to a particle size ≤50μm. The amount of calcium salt added to each 100g of mixed protein powder 2 is 2g. Let it stand at room temperature for 20 minutes to obtain mixed protein powder 3.

[0077] (6) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain low-allergenic milk formula whey protein powder.

[0078] (7) According to the flavor requirements, L-theanine (310 mg / kg) is added, and green tea flavored low-allergenic milk formula whey protein powder is prepared according to known methods.

[0079] (8) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of hypoallergenic milk formula whey protein powder, and package in aluminum foil bags at room temperature.

[0080] 2. Quality testing of hypoallergenic milk formula whey protein powder Detection and analysis are conducted in terms of sensory characteristics, microorganisms, and nutrients.

[0081] Table 3 Quality testing of hypoallergenic milk formula whey protein powder

[0082] 3. Functional testing of hypoallergenic milk formula whey protein powder Dissolve hypoallergenic milk formula whey protein powder at a ratio of 0.1 mg / 10 mL in 40 mL of water. o In water C, stir well, let stand at room temperature for 5 minutes, and then use cow... β -LG ELISA quantitative kit (same as Example 1) was used to measure bovine... β -LG antigen content decreased by 23%, and the antioxidant DPPH free radical scavenging ability was 51%.

[0083] Example 4: 1. Preparation of hypoallergenic milk formula whey protein powder (1) β -LG is dissolved in phosphate buffer (pH=8, concentration 0.1 mol / L) to make β -LG reached a final concentration of 0.5 mg / mL, β-LG is compounded non-covalently with erythritol, ascorbic acid, and carotene. β - The mass ratio of LG to small molecule substances is set to 5:1:1.7:2.5 ( β -LG:Ascorbic acid ratio is 3:1 β -LG: Carotene 2:1, β -LG:Erythritol 5:1), stir at room temperature for 30 minutes and then let stand at room temperature.

[0084] (2) To β -LG-erythritol was freeze-dried at -70°C for 16 hours to obtain a low-sensitivity product. β -LG protein powder.

[0085] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powders were mixed at a certain ratio (4:1:0.1) at room temperature to obtain mixed protein powder 1.

[0086] (4) Add 8% by weight of immunoglobulin to mixed protein powder 1, let stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0087] (5) Add an appropriate amount of calcium salt (calcium citrate) to (4), and crush the calcium salt to a particle size ≤50μm. The amount of calcium salt added to each 100g of mixed protein powder 2 is 2g. Let it stand at room temperature for 20 minutes to obtain mixed protein powder 3.

[0088] (6) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain low-allergenic milk formula whey protein powder.

[0089] (7) According to the flavor requirements, L-theanine (310 mg / kg) is added, and green tea flavored low-allergenic milk formula whey protein powder is prepared according to known methods.

[0090] (8) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of hypoallergenic milk formula whey protein powder, and package in aluminum foil bags at room temperature.

[0091] 2. Quality testing of hypoallergenic milk formula whey protein powder Detection and analysis are conducted in terms of sensory characteristics, microorganisms, and nutrients.

[0092] Table 4 Quality Testing of Hypoallergenic Milk Formula Whey Protein Powder

[0093] 3. Functional testing of milk formula whey protein powder Dissolve milk-based whey protein powder at a ratio of 0.1 mg / 10 mL in 40 mL of water.o In water C, stir well, let stand at room temperature for 5 minutes, and then use cow... β -LG ELISA quantitative kit (same as Example 1) was used to measure bovine... β -LG antigen content decreased by 24%. Compared with the previous case, the compound... β - The antigen content of the LG protein complex decreased more significantly than that of a single small molecule protein complex.

[0094] Comparative Example 1 This comparative example provides a milk-based whey protein powder, and the specific preparation steps are as follows: 1. Preparation of milk-based whey protein powder (1) β -LG is dissolved in phosphate buffer (pH=8, concentration 0.1 mol / L) to make β -LG reached a final concentration of 0.5 mg / mL during preparation. β -LG-Mogroside ( β -LG-LHK) non-covalent complex, β - The mass ratio of LG to small molecules was set to 6:1. After stirring at room temperature for 30 minutes, the mixture was allowed to stand at room temperature.

[0095] (2) To β -LG-mogrosides were freeze-dried (-75℃, 16h) to obtain β -LG protein powder.

[0096] (3) Mix lactoferrin powder and α-lactalbumin powder with β -LG protein powders were mixed at a certain ratio (4:1:0.1) at room temperature to obtain mixed protein powder 1.

[0097] (4) Add 8% by weight of immunoglobulin to mixed protein powder 1, let stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0098] (5) Add an appropriate amount of calcium salt (calcium citrate) to (4), and crush the calcium salt to a particle size ≤50μm. The amount of calcium salt added to each 100g of mixed protein powder 2 is 2g. Let it stand at room temperature for 20 minutes to obtain mixed protein powder 3.

[0099] (6) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain milk formula whey protein powder.

[0100] (7) Add L-theanine (310 mg / kg) according to flavor requirements and prepare green tea flavored milk formula whey protein powder according to known methods.

[0101] (8) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of milk formula whey protein powder and package in aluminum foil bags at room temperature.

[0102] 2. Quality testing of milk formula whey protein powder Detection and analysis are conducted in terms of sensory characteristics, microorganisms, and nutrients.

[0103] Table 5 Quality Inspection of Milk Formula Whey Protein Powder

[0104] 3. Functional testing of milk formula whey protein powder Dissolve milk-based whey protein powder at a ratio of 0.1 mg / 10 mL in 40 mL of water. o In water C, stir well, let stand at room temperature for 5 minutes, and then use cow... β -LG ELISA quantitative kit (same as Example 1) was used to measure bovine... β - The LG antigen content did not decrease significantly (the difference did not reach the level required for statistical significance). p<0.05 ), namely mogrosides and β -LG compound cannot reduce its allergenicity. This shows that not all food additives used in dairy products have an allergenicity-reducing effect.

[0105] Comparative Example 2 This comparative example provides a milk-based whey protein powder, and the specific preparation steps are as follows: 1. Preparation of milk-based whey protein powder (1) β -LG is dissolved in phosphate buffer (pH=8, concentration 0.1 mol / L) to make β -LG reached a final concentration of 0.5 mg / mL, with the addition of mogrosides and erythritol, making β -LG forms non-covalent complexes with mogrosides and erythritol. β The mass ratio of LG to small molecules was set to 6:1:1.2. After stirring at room temperature for 30 minutes, the mixture was allowed to stand at room temperature to obtain a complex protein.

[0106] (2) The composite protein was freeze-dried under vacuum (-75℃, 16h) to obtain β -LG protein powder.

[0107] (3) Mix lactoferrin powder and α-lactalbumin powder with β -LG protein powders were mixed at a certain ratio (4:1:0.1) at room temperature to obtain mixed protein powder 1.

[0108] (4) Add 8% by weight of immunoglobulin to mixed protein powder 1, let stand at room temperature for 3 minutes to obtain mixed protein powder 2.

[0109] (5) Add an appropriate amount of calcium salt (calcium citrate) to (4), and crush the calcium salt to a particle size ≤50μm. The amount of calcium salt added to each 100g of mixed protein powder 2 is 2g. Let it stand at room temperature for 20 minutes to obtain mixed protein powder 3.

[0110] (6) Crush the citric acid to a particle size ≤50μm, add 0.15% citric acid to the mixed protein powder 3, mix for 20 minutes to obtain milk formula whey protein powder.

[0111] (7) Add L-theanine (310 mg / kg) according to flavor requirements and prepare green tea flavored milk formula whey protein powder according to known methods.

[0112] (8) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of milk formula whey protein powder and package in aluminum foil bags at room temperature.

[0113] 2. Quality testing of milk formula whey protein powder Detection and analysis are conducted in terms of sensory characteristics, microorganisms, and nutrients.

[0114] Table 6 Quality Inspection of Milk Formula Whey Protein Powder

[0115] 3. Functional testing of milk formula whey protein powder Dissolve milk-based whey protein powder at a ratio of 0.1 mg / 10 mL in 40 mL of water. o In water C, stir well, let stand at room temperature for 5 minutes, and then use cow... β -LG ELISA quantitative kit (same as Example 1) was used to measure bovine... β -LG antigen content decreased by 14.5%, indicating that mogrosides and β -LG complex influences erythritol and β The proportion of -LG in the combination leads to a smaller decrease in the antigen content when the two are combined, resulting in a weaker effect, which is not as good as the effect of erythritol alone.

[0116] Figure 1 For milk β -LG structure and its antigenic epitope distribution.

[0117] Figure 2 for β -LG complex protein antigen activity assay. β -LG-ascorbic acid ( β -LG-AA), β LG-carotene ( β -LG-CAR) β -LG-Mogroside (β -LG-LHK) β -LG-erythritol ( β -LG-ERY), β -LG-ERY / LHK and β -LG-ERY / CAR / AA. As shown in the figure, except for mogroside, other complexes were significantly reduced. β -LG antigen concentration, i.e., reducing its sensitizing effect. Monk fruit glycosides cannot modify or improve... β -LG antigen concentration shows that not all food additives used in dairy products have the effect of reducing allergenicity.

[0118] Figure 3 for β -LG complex protein structure analysis. (AC) UV-Vis absorption spectroscopy; (D) Fourier transform infrared spectroscopy; (EF) circular dichroism spectroscopy. β The linear and conformational epitopes of the -LG protein complex were altered.

[0119] Figure 4 For small molecules and β -LG molecular docking model. β A three-dimensional schematic diagram (A) and a two-dimensional docking diagram (B) of molecular docking of -LG-AA. β Three-dimensional docking diagram (C) and two-dimensional docking diagram (D) of the -LG-CAR complex. β 3D docking diagram (E) and 2D docking diagram (F) of the -LG-ERY complex. Three small molecules and... β -LG complexes mask the antigenic epitopes, and the interaction sites fall within the antigenic epitope distribution area.

[0120] Figure 5 for β Antioxidant activity analysis of the LG complex proteins. Evaluation of DPPH and ABTS free radical scavenging capacity and iron ion reducing capacity. The antioxidant activity of all three complexes was altered to varying degrees.

[0121] Figure 6 for β - LG complex protein emulsification analysis. Emulsifying activity index (EAI) and emulsifying stability index (ESI). β -LG-CAR complex has the best emulsifying properties.

Claims

1. A hypoallergenic β - a LG protein powder characterized in that, comprising non-covalent binding to small molecule substances β - lactoglobulin; the small molecule substance is ascorbic acid, erythritol, β - at least one of the carotenes.

2. A hypoallergenic composition according to claim 1, wherein the composition is a food product. β - LG protein powder, characterized in that, The β - the mass ratio of lactoglobulin to small molecule substance addition is (2-5):

1.

3. A hypoallergenic composition according to claim 1 or 2. β A method for producing an LG protein powder, characterized by, The specific steps are as follows: The β - Lactoglobulin was dissolved in phosphate buffer to obtain β - LG solution; The small molecule substance is added β - The LG solution is stirred at room temperature and left to stand to obtain a non-covalent complex. The non-covalent complex was vacuum freeze-dried to obtain a hypoallergenic β -LG protein powder.

4. A hypoallergenic composition according to claim 3, wherein the protein is a recombinant protein. β A method for producing an LG protein powder, characterized by, The β The final concentration of the LG solution is 0.1 mg / mL ~ 0.5 mg / mL.

5. A hypoallergenic milk formula whey protein powder, characterized in that, The raw materials include lactoferrin powder, α-lactalbumin powder, immunoglobulin and hypoallergenic β -LG protein powder, the hypoallergenic β -LG protein powder is the hypoallergenic β -LG protein powder of any one of claims 1.

6. A hypoallergenic milk formula whey protein powder according to claim 5, characterized in that, The lactoferrin powder, alpha-lactalbumin powder and hypoallergenic β The addition ratio of the LG protein powder is 5:2:0.1~4:1:0.

1.

7. A hypoallergenic milk formula whey protein powder according to claim 5, characterized in that, The lactoferrin powder, the alpha-lactalbumin powder and the hypoallergenic β In the mixture of the LG protein powder, 5%-8% of immunoglobulin by mass is added.

8. A hypoallergenic milk formula whey protein powder according to claim 5, characterized in that, The raw materials further include calcium salt, citric acid and flavoring agent, wherein the calcium salt is calcium carbonate, tricalcium phosphate or calcium citrate, the addition amount of the calcium salt in 100 g of mixed protein powder 2 is 0.8-2.0 g, the mixed protein powder 2 is lactoferrin powder, α-lactalbumin powder, low-sensitization β -LG protein powder and immunoglobulin mixture; the addition amount of citric acid is 0.15% of mixed protein powder 3, the mixed protein powder 3 is lactoferrin powder, α-lactalbumin powder, low-sensitization β -LG protein powder, immunoglobulin and calcium salt mixture; the flavoring agent is vanillin or L-theanine, the addition amount of the vanillin is not more than 5 mg / 100 mL; the addition amount of L-theanine is 100 mg / kg-310 mg / kg.

9. A process for the preparation of a hypoallergenic milk formula whey protein powder as claimed in any one of claims 5 to 8, characterised in that, The specific steps are as follows: Mixing lactoferrin powder and alpha-lactalbumin powder with low allergenic β - The LG protein powder is mixed at room temperature to obtain mixed protein powder 1; The immunoglobulin is added into the mixed protein powder 1, and the mixture is left at room temperature to obtain a hypoallergenic milk formula whey protein powder.

10. A process for the preparation of a hypoallergenic milk formula whey protein powder as claimed in claim 9, characterized in that, 0.07 g of antioxidant is added into each gram of the hypoallergenic milk formula whey protein powder.

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