A low-sensitization beta-lactoglobulin peptide, and a preparation method and application thereof

Abstract

The application provides a low-sensitization beta-lactoglobulin peptide and a preparation method and application thereof, and relates to the field of food processing technology.The method comprises the following steps: co-dissolving beta-lactoglobulin, apple chlorogenic acid and reducing sugar, performing low-temperature atmospheric pressure plasma pretreatment, and then freeze-drying; performing short-time glycosylation reaction under vacuum conditions; performing two-step protease step-by-step enzymolysis, dialysis purification, and then performing spray drying, so as to obtain the low-sensitization beta-lactoglobulin peptide.The peptide is compounded with maltodextrin, a nutrition enhancer and L-carnitine, so as to prepare a sports nutrition solid beverage which meets the GB 24154 standard.The application provides a new idea for the preparation of the low-sensitization, high-antioxidation and low-AGEs beta-lactoglobulin peptide and the development of sports nutrition food.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a low-allergenic β-lactoglobulin peptide, its preparation method, and its applications. Background Technology

[0002] Sports nutrition foods are foods specifically developed to meet the unique nutritional needs of athletes. According to GB 24154, the National Food Safety Standard for Sports Nutrition Foods, they can be divided into three main categories based on characteristic nutrients: protein supplements, energy supplements, and energy control products. Among these, protein supplements are characterized by high protein content and rapid digestion and absorption. Beta-lactoglobulin, a major component of whey protein, is rich in branched-chain amino acids, is rapidly absorbed, and effectively promotes muscle synthesis and repair, making it a high-quality protein source for sports nutrition foods. However, beta-lactoglobulin also has strong allergenic properties, easily triggering intolerance or mild allergic reactions in some adult consumers, affecting the universality of related products and the consumer experience.

[0003] Currently, three main methods are used to reduce the allergenicity of β-lactoglobulin: enzymatic hydrolysis, heat treatment, and glycosylation modification. Enzymatic hydrolysis, which breaks down β-lactoglobulin into small peptides using proteases, is a widely used desensitization method. However, it easily produces bitter peptides, which can damage the original functional properties and nutritional value of the protein, leading to decreased product solubility. Furthermore, single-method enzymatic hydrolysis is insufficient to completely eliminate all allergenic epitopes, resulting in limited allergenicity reduction. Heat treatment (such as high-temperature sterilization) can denature proteins to some extent, but its effect on reducing allergenicity is limited, and excessive heat treatment can impair the digestibility and absorption properties of proteins. Traditional glycosylation, while able to mask allergen epitopes to some extent by covalently binding reducing sugars to proteins, typically requires high temperatures and long reaction times. This can lead to severe browning of the product, affecting sensory quality, reducing protein digestibility and bioavailability, and promoting the formation of large amounts of advanced glycation end products (AGEs). These AGEs can damage human cells and tissues, accelerate aging, and contribute to various diseases such as diabetes and its complications, cardiovascular disease, and tumors.

[0004] In addition, athletes produce reactive oxygen species and reactive nitrogen species in their bodies after high-intensity training, which leads to increased oxidative stress, muscle fatigue, and enhanced inflammatory response, thereby affecting athletic performance and recovery speed.

[0005] Therefore, developing a protein ingredient that can maintain the high-quality nutritional and functional properties of β-lactoglobulin, significantly reduce its allergenicity, and also possess comprehensive properties such as antioxidant properties, high absorption rate, and good taste is of great practical significance. Summary of the Invention

[0006] The purpose of this invention is to provide a low-allergenic β-lactoglobulin peptide, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a method for preparing a low-allergenic β-lactoglobulin peptide, comprising the following steps: S1. Dissolve β-lactoglobulin, natural phenolic acids and reducing sugars together in pure water, adjust the pH to 7.5-8.5, and pretreat the resulting mixture with low-temperature atmospheric pressure plasma. S2. The solution pretreated in step S1 is pre-frozen and then freeze-dried under vacuum to obtain freeze-dried powder. S3. Place the lyophilized powder obtained in step S2 into a vacuum heating chamber for vacuum glycosylation reaction; S4. After dissolving the glycosylated product obtained in step S3, add the first complex protease and the second complex protease in sequence for stepwise enzymatic hydrolysis to inactivate the enzyme. S5. The enzymatic hydrolysate obtained in step S4 is purified and dried to obtain low-allergenic β-lactoglobulin peptide.

[0008] Optionally, the natural phenolic acid substance in step S1 is apple chlorogenic acid extract with a chlorogenic acid content ≥70%; the mass ratio of the natural phenolic acid substance to the β-lactoglobulin is (0.02-0.08):1.

[0009] Optionally, the reducing sugar in step S1 is at least one of glucose, isomaltooligosaccharide, or galactooligosaccharide; the mass ratio of the reducing sugar to the β-lactoglobulin is (0.8-1.2):1.

[0010] Optionally, the conditions for the low-temperature atmospheric pressure plasma pretreatment in step S1 are: voltage 10-50kV, frequency 20-30kHz, treatment time 4-10min, and atmosphere is air.

[0011] Optionally, the pH adjustment is performed using food-grade sodium bicarbonate.

[0012] Optionally, the pre-freezing temperature in step S2 is -80℃, and the pre-freezing time is 4-6 hours; the vacuum freeze-drying time is 48-72 hours.

[0013] Optionally, the conditions for the vacuum glycosylation reaction in step S3 are: absolute pressure ≤10kPa, temperature 95-105℃, and heating time 25-35min.

[0014] Optionally, in step S4, the first complex protease is bromelain and neutral protease; the bromelain and the neutral protease are compounded in a mass ratio of 1:(1-2); the conditions for adding the first complex protease for enzymatic hydrolysis are: pH 6.0-7.0, temperature 45-55℃, the ratio of the amount of the first complex protease added to the substrate is 1000-2000U:1mL, and the hydrolysis time is 1-1.5h.

[0015] Optionally, the second complex protease is papain and flavor protease; the papain and the flavor protease are compounded in a mass ratio of 1:(1-2); the conditions for adding the second complex protease for enzymatic hydrolysis are: pH 6.5-7.5, temperature 50-60℃, the ratio of the amount of the second complex protease added to the substrate is 2000-3000U:1mL, and the hydrolysis time is 0.5-1.5h.

[0016] Optionally, the enzyme inactivation conditions are: treatment at 80-100℃ for 10-15 minutes.

[0017] Optionally, the purification process in step S5 involves dialysis at 4°C for 36 hours using a dialysis bag with a molecular weight cutoff of 1000-3500 Da, with the solution changed every 12 hours; the drying process is spray drying, and the spray drying conditions are: inlet air temperature 130-160°C, outlet air temperature 65-75°C, feed solids content 15-20%, feed flow rate 10-20 mL / min, and atomization pressure 0.2-0.4 MPa.

[0018] Secondly, the present invention provides a low-allergenic β-lactoglobulin peptide prepared by any of the above-mentioned optional preparation methods.

[0019] Thirdly, the present invention provides the application of the above-mentioned low-allergenic β-lactoglobulin peptide or the low-allergenic β-lactoglobulin peptide prepared by any of the above-mentioned optional preparation methods in the preparation of sports nutrition foods.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The phenolic hydroxyl groups of malic chlorogenic acid can efficiently scavenge free radicals and covalently bind chlorogenic acid to β-lactoglobulin peptide, thus protecting its antioxidant activity during processing. At the same time, vacuum glycosylation can significantly inhibit the oxidation side reaction under high temperature and aerobic environment, avoid the excessive oxidation and destruction of antioxidant active ingredients, and still achieve appropriate grafting of reducing sugar and protein, ultimately achieving synergistic effect. (2) Plasma treatment loosens the protein structure, making it easier for the phenolic hydroxyl groups of malic chlorogenic acid to undergo nucleophilic addition with the lysine and arginine residues of β-lactoglobulin, preemptively occupying some allergic epitopes. At the same time, it induces a moderate change in the protein structure, exposing the deeply buried epitopes to the surface, which can further reduce the sensitization of the product. Vacuum glycosylation covalently modifies the remaining free amino groups with reducing sugars to form steric hindrance. Finally, the molecular weight is controlled at 1000-3500 Da by complex enzymatic hydrolysis, and residual linear epitopes are completely removed. (3) This invention innovatively conducts the glycosylation reaction in a vacuum environment with an absolute pressure not exceeding 10 kPa, reducing the reaction temperature to 95-105℃ and shortening the time to 25-35 min. This effectively inhibits severe browning and the formation of AGEs products, resulting in better sensory quality. Simultaneously, chlorogenic acid, by capturing glycosylation intermediates and scavenging free radicals, blocks the conversion pathway to AGEs. This ensures that the product will not induce chronic inflammation and metabolic disorders during long-term consumption, which is of great significance for the long-term health of athletes. (4) The present invention adopts a two-stage compound enzymatic hydrolysis scheme. Since the two compound enzymes have different action sites, such as bromelain can cleave the carboxyl terminus of basic amino acids such as lysine and arginine, and papain has broad specificity and can cleave a variety of peptide bonds. Stepwise enzymatic hydrolysis allows different enzymes to act sequentially under their respective optimal conditions, which not only makes the removal of allergic epitopes more thorough, but also makes the final product less bitter. The bitterness sensory score of the obtained product reaches 9.6 points (out of 10, the higher the score, the less bitter). (5) The preparation method provided by this invention precisely controls the molecular weight of the product within the range of 1000-3500 Da using a dialysis bag, which not only preserves the nutritional value of the protein but also has good absorption characteristics, allowing athletes to quickly obtain amino acid supply after training, promoting muscle protein synthesis and accelerating recovery. Low-allergenic β-lactoglobulin peptides can provide high-absorption, non-bitter high-quality protein, and when combined with L-carnitine to promote fatty acid oxidation, it can delay muscle fatigue; maltodextrin is a fast-acting carbohydrate that can immediately replenish glycogen consumed during exercise; calcium carbonate can strengthen bone health; the addition of zinc gluconate can enhance immune function; vitamin premix comprehensively supports energy metabolism and antioxidant defense; added collagen peptides nourish joints and tendons; branched-chain amino acids directly inhibit muscle breakdown and accelerate repair. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the preparation process of the hypoallergenic β-lactoglobulin peptide of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0023] This invention provides a method for preparing low-allergenic β-lactoglobulin peptide, comprising the following steps: S1. Dissolve β-lactoglobulin, natural phenolic acids and reducing sugars together in pure water, adjust the pH to 7.5-8.5, and pretreat the resulting mixture with low-temperature atmospheric pressure plasma. S2. The solution pretreated in step S1 is pre-frozen and then freeze-dried under vacuum to obtain freeze-dried powder. S3. Place the lyophilized powder obtained in step S2 into a vacuum heating chamber for vacuum glycosylation reaction; S4. After dissolving the glycosylated product obtained in step S3, add the first complex protease and the second complex protease in sequence for stepwise enzymatic hydrolysis to inactivate the enzyme. S5. The enzymatic hydrolysate obtained in step S4 is purified and dried to obtain low-allergenic β-lactoglobulin peptide.

[0024] In some embodiments, the natural phenolic acid used in step S1 is apple chlorogenic acid extract with a chlorogenic acid content ≥70%; the mass ratio of the natural phenolic acid used to β-lactoglobulin is (0.02-0.08):1.

[0025] In fact, malic chlorogenic acid plays a key "multi-effect" role in the system: First, its abundant phenolic hydroxyl structure, after plasma pretreatment, is more likely to undergo nucleophilic addition with the lysine and arginine residues exposed by β-lactoglobulin, preemptively occupying some allergic epitopes through covalent bonds, thus achieving a pre-reduction of sensitization; Second, the covalently bound chlorogenic acid stably anchors high antioxidant activity to the peptide chain, enabling the final product to achieve a DPPH free radical scavenging rate of over 65%, providing additional antioxidant protection for athletes whose oxidative stress levels increase after high-intensity exercise. Thirdly, in subsequent vacuum glycosylation reactions, chlorogenic acid can effectively capture Maillard reaction intermediates (such as dicarbonyl compounds) and scavenge free radicals, blocking the conversion pathway to harmful advanced glycation end products (AGEs). Compared with traditional aerobic glycosylation, it significantly reduces the carboxymethyl lysine content from over 21.53 μg / mL to 4.70-6.33 μg / mL. Fourthly, apple chlorogenic acid extract is derived from apple processing byproducts (such as apple pomace), which has been approved for use as a food ingredient, ensuring high safety and conforming to the concept of green processing. The introduction of apple chlorogenic acid enables this application to simultaneously achieve a triple functional gain of "pre-masking allergic epitopes, imparting antioxidant activity, and inhibiting AGEs formation".

[0026] In some embodiments, the reducing sugar used in step S1 is at least one of glucose, isomaltooligosaccharide, or galactooligosaccharide; the mass ratio of the reducing sugar used to β-lactoglobulin is (0.8-1.2):1.

[0027] In some embodiments, the conditions for low-temperature atmospheric pressure plasma pretreatment used in step S1 are: voltage 10-50kV, frequency 20-30kHz, treatment time 4-10min, and atmosphere is air.

[0028] In practice, low-temperature atmospheric pressure plasma treatment involves treating a mixture of β-lactoglobulin, malic chlorogenic acid, and reducing sugars at a voltage of 10-50 kV and a frequency of 20-30 kHz in an air atmosphere for 4-10 minutes. This process utilizes high-energy active particles (such as free radicals, ions, and electrons) to bombard protein molecules, causing their higher-order structures to unfold and their spatial conformation to loosen. This exposes previously buried hypersensitive epitopes on the surface, creating more accessible sites for subsequent reactions. Simultaneously, plasma treatment introduces active functional groups (such as carbonyl and hydroxyl groups) onto the protein surface, promoting nucleophilic addition reactions between chlorogenic acid phenolic hydroxyl groups and protein lysine and arginine residues, resulting in more stable covalent binding of natural antioxidants to the peptide chain. Furthermore, this treatment is conducted entirely at low temperatures, avoiding the damage to the nutritional value and functional properties of proteins caused by traditional high-temperature pretreatment. Plasma pretreatment significantly improves the desensitization efficiency of subsequent glycosylation and enzymatic hydrolysis, laying the foundation for the high antioxidant activity of the product.

[0029] In some embodiments, pH is adjusted using food-grade sodium bicarbonate.

[0030] In some embodiments, the pre-freezing temperature used in step S2 is -80°C, and the pre-freezing time is 4-6 hours; the vacuum freeze-drying time used is 48-72 hours.

[0031] In some embodiments, the conditions for the vacuum glycosylation reaction used in step S3 are: absolute pressure ≤10kPa, temperature 95-105℃, and heating time 25-35min.

[0032] In fact, vacuum glycosylation, by placing the lyophilized powder in a low-pressure environment with an absolute pressure not exceeding 10 kPa and heating it briefly at 95-105℃ for 25-35 minutes, can significantly inhibit the excessive Maillard reaction compared to traditional aerobic high-temperature long-term glycosylation. It controls the content of carboxymethyl lysine (CML), a representative marker of advanced glycation end products (AGEs), at a low level of 4.70-6.33 μg / mL. At the same time, it effectively avoids severe browning and the formation of bitter substances in the product. While achieving covalent modification of free amino groups of proteins by reducing sugars and forming steric hindrance to mask allergic epitopes, it maximizes the preservation of the product's sensory quality, digestibility and absorption characteristics, and nutritional safety.

[0033] In some embodiments, the first complex protease used in step S4 is bromelain and neutral protease; bromelain and neutral protease are compounded in a mass ratio of 1:(1-2); the conditions for adding the first complex protease for enzymatic hydrolysis are: pH 6.0-7.0, temperature 45-55℃, the ratio of the amount of the first complex protease added to the substrate is 1000-2000U:1mL, and the hydrolysis time is 1-1.5h.

[0034] In some embodiments, the second complex protease used is papain and flavor protease; the papain and the flavor protease are compounded in a mass ratio of 1:(1-2); the conditions for adding the second complex protease for enzymatic hydrolysis are: pH 6.5-7.5, temperature 50-60℃, the ratio of the amount of the second complex protease added to the substrate is 2000-3000U:1mL, and the hydrolysis time is 0.5-1.5h.

[0035] In fact, the stepwise complex enzymatic hydrolysis involves two stages: "bromelain + neutral protease" and "papain + flavor protease." Utilizing the specific differences in peptide bond cleavage sites among different proteases, it achieves synergistic clearance of β-lactoglobulin hypersensitivity epitopes: The first complex enzyme (bromelain and neutral protease, mass ratio 1:1-2) preferentially cleaves the carboxyl termini of basic amino acid residues such as lysine and arginine, as well as various internal peptide bonds, under conditions of pH 6.0-7.0 and 45-55℃, efficiently cleaving the intact protein into medium-molecular-weight polypeptide fragments and fully exposing residual linear hypersensitivity epitopes. The second complex enzyme (papain and flavor protease, mass ratio 1:1-2) further acts on a wide range of specific peptide bonds under conditions of pH 6.5-7.5 and 50-60℃. Simultaneously, the flavor protease, through its unique exonuclease activity, directionally cleaves hydrophobic amino acid residues (such as phenylalanine, leucine, and valine), eliminating the formation of bitter peptides at the source.

[0036] Compared to single or simultaneous enzymatic hydrolysis, this stepwise strategy allows the two complex enzyme systems to exert their maximum activity sequentially under their respective optimal pH and temperature conditions. This avoids the problem of incomplete desensitization caused by mutual inhibition of enzyme activity or conflict of optimal conditions. Ultimately, it achieves a synergistic effect with an antigenicity reduction rate of over 85% and a bitterness sensory score of 9.6 out of 10. At the same time, it precisely controls the molecular weight of the product within the small peptide range of 1000-3500 Da, with an in vitro digestibility of over 95%. It combines low allergenicity, good taste, and high absorption and utilization rate.

[0037] In some embodiments, the enzyme inactivation conditions used are: treatment at 80-100°C for 10-15 minutes.

[0038] In some embodiments, the purification process in step S5 involves dialysis at 4°C for 36 hours using a dialysis bag with a molecular weight cutoff of 1000-3500 Da, with the solution changed every 12 hours. The drying process is spray drying, with the following conditions: inlet air temperature 130-160°C, outlet air temperature 65-75°C, feed solids content 15-20% (w / v), feed flow rate 10-20 mL / min, and atomization pressure 0.2-0.4 MPa.

[0039] The present invention also provides a low-allergenic β-lactoglobulin peptide prepared by the preparation method in any of the above embodiments.

[0040] The present invention also provides the application of the above-mentioned low-allergenic β-lactoglobulin peptide or the low-allergenic β-lactoglobulin peptide prepared by the preparation method in any of the above embodiments in the preparation of sports nutrition foods.

[0041] Specifically, the obtained low-allergenic β-lactoglobulin peptide is used to prepare a sports nutrition solid beverage, comprising 15-35 parts of low-allergenic β-lactoglobulin peptide, 40-60 parts of maltodextrin, 0.5-1.5 parts of calcium carbonate, 0.1-0.3 parts of zinc gluconate, 0.5-1.5 parts of vitamin premix, 5-15 parts of L-carnitine, 10-20 parts of collagen peptide, and / or 5-15 parts of branched-chain amino acids. The resulting solid beverage meets the technical requirements for protein-supplemented sports nutrition foods in GB 24154.

[0042] Example 1

[0043] This embodiment 1 provides a method for preparing low-allergenic β-lactoglobulin peptide, comprising the following steps: S1. Raw material mixing and plasma pretreatment: Dissolve 100g of β-lactoglobulin powder (purity ≥90%), 2g of apple chlorogenic acid extract (chlorogenic acid content ≥70%), and 100g of glucose in 2000mL of pure water. Adjust the pH of the system to 8.0 with food-grade sodium bicarbonate. Perform dielectric barrier discharge plasma treatment with the following parameters: voltage 35kV, frequency 25kHz, air atmosphere, and treatment time 6min.

[0044] S2. Freeze-drying: The pretreated solution is dispensed into freeze-drying trays and pre-frozen at -80℃ for 5 hours, followed by vacuum freeze-drying for 60 hours to obtain freeze-dried powder.

[0045] S3. Vacuum glycosylation reaction: Place the lyophilized powder in a vacuum heating chamber, evacuate to an absolute pressure ≤10kPa, heat to 100℃, heat for 30min, and then cool naturally.

[0046] S4. Stepwise complex enzymatic hydrolysis: Redissolve the glycosylated product in pure water to 5% (w / v), adjust the pH to 6.5 with food-grade sodium bicarbonate, add the first complex protease (bromelain and flavor protease, mass ratio 1:1) at a rate of 1500 U per mL of reaction solution (based on total activity), and hydrolyze in a 50°C water bath with shaking for 1.25 h. After the first enzymatic hydrolysis, adjust the pH to 7.0 with food-grade sodium bicarbonate, add the second complex protease (neutral protease and papain, mass ratio 1:1) at a rate of 2000 U per mL of reaction solution (based on total activity), and continue enzymatic hydrolysis in a 55°C water bath with shaking for 1 h. After enzymatic hydrolysis, heat at 95°C for 10 min to inactivate the enzyme.

[0047] S5. Purification and Drying: After cooling, the enzymatic hydrolysate was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in pure water at 4℃ for 36 hours, with the solution changed every 12 hours. The liquid in the bag was collected, and the feed solids content was adjusted to 15-20% (w / v). Spray drying was carried out at a feed flow rate of 15 mL / min and an atomization pressure of 0.3 MPa. The inlet air temperature for spray drying was set to 140℃ and the outlet air temperature to 70℃, yielding low-allergenic β-lactoglobulin peptide powder. This is denoted as peptide A1.

[0048] Example 2 (Adjusting the amount of apple chlorogenic acid) The only difference between Example 2 and Example 1 is that the amount of apple chlorogenic acid extract used in S1 is changed to 5g. The resulting product is denoted as peptide A2.

[0049] Example 3 (Adjusting Vacuum Glycosylation Parameters) The difference between Example 3 and Example 1 is that the vacuum glycosylation reaction conditions in S3 are changed to heating to 95°C for 40 minutes. The resulting product is denoted as peptide A3.

[0050] Example 4 (Adjusting the ratio of complex protease) The difference between Example 4 and Example 1 is that the mass ratio of the first complex protease (bromelain and neutral protease) in S4 is changed to 1:2, and the mass ratio of the second complex protease (neutral protease and papain) is changed to 1:2. The resulting product is denoted as peptide A4.

[0051] Example 5

[0052] This embodiment 5 provides a method for preparing a sports nutrition solid beverage using peptide A1 and compounded collagen peptides, including: Formula (per 1000g): Peptide A1 250g, maltodextrin (DE10-15) 580g, calcium carbonate 10g, zinc gluconate 2g, vitamin premix (containing vitamin A 0.5g, vitamin D 0.01g, vitamin B1 0.5g, vitamin B2 0.5g, vitamin B6 0.5g, vitamin B12 0.001g, vitamin C 10g, with the remainder being maltodextrin filler) 10g, L-carnitine 80g, collagen peptide (fish source, 2000Da) 120g.

[0053] Preparation method: Add the above raw materials into a three-dimensional mixer in sequence and mix for 40 minutes until uniform. Package them individually in aluminum foil bags (25g per bag), seal with nitrogen, and the sports nutrition solid beverage is obtained.

[0054] Example 6

[0055] This embodiment 6 provides a method for preparing sports nutrition solid beverages using peptide A3 and compounded branched-chain amino acids, including: Formula (per 1000g): Peptide A3 350g, maltodextrin 480g, calcium carbonate 10g, zinc gluconate 2g, vitamin premix 10g, L-carnitine 80g, branched-chain amino acids (leucine:isoleucine:valine = 2:1:1) 100g.

[0056] Preparation method: The mixing method is the same as in Example 5, to obtain a sports nutrition solid beverage.

[0057] Comparative Example 1 (without malic chlorogenic acid) The only difference between Comparative Example 1 and Example 1 is that malic chlorogenic acid in Example 1 is omitted, and the resulting product is denoted as Comparative Peptide B.

[0058] Comparative Example 1.1 The only difference between Comparative Example 1.1 and Example 5 is that peptide A1 was replaced with comparative peptide B to prepare the comparative beverage.

[0059] Comparative Example 2 (without plasma treatment) The only difference between Comparative Example 2 and Example 1 is that the plasma treatment in Example 1 is omitted, and the resulting product is denoted as Comparative Peptide C.

[0060] Comparative Example 2.1 The only difference between Comparative Example 2.1 and Example 5 is that peptide A1 was replaced with comparative peptide C to prepare the comparative beverage.

[0061] Comparative Example 3 (without vacuum glycosylation) The only difference between Comparative Example 3 and Example 1 is that the S3 vacuum glycosylation step in Example 1 is omitted, and the resulting product is denoted as Comparative Peptide D.

[0062] Comparative Example 3.1 The only difference between Comparative Example 3.1 and Example 5 is that peptide A1 was replaced with comparative peptide D to prepare the comparative beverage.

[0063] Comparative Example 4 (enzymatic hydrolysis only) The only difference between Comparative Example 4 and Example 1 is that steps S1, S2, and S3 are omitted, and untreated β-lactoglobulin is directly hydrolyzed without any pretreatment or glycosylation. The resulting product is denoted as Comparative Peptide E.

[0064] Comparative Example 4.1 The only difference between Comparative Example 4.1 and Example 5 is that peptide A1 was replaced with comparative peptide E to prepare the comparative beverage.

[0065] Performance testing 1. Determination of antigenicity reduction rate The IgE binding capacity of the samples was determined by indirect competitive ELISA, and the antigenicity reduction rate was calculated. The results are shown in Tables 1 and 2.

[0066] 2. DPPH free radical scavenging rate determination The scavenging ability of the samples for DPPH radicals was determined by the degree of attenuation of the characteristic absorption of DPPH radicals at 517 nm. The results are shown in Tables 1 and 2.

[0067] 3. Bitterness sensory score Ten trained sensory evaluators scored the samples using a 10-point scale (10 points for no bitterness, 1 point for extremely bitterness). The results are shown in Tables 1 and 2.

[0068] 4. In vitro protein digestibility determination The in vitro digestibility of the samples was determined using a two-step digestion method involving pepsin and trypsin. The results are shown in Table 1.

[0069] 5. Determination of carboxymethyl lysine (CML) content The CML content in the samples was determined using a competitive ELISA kit. The peptides were dissolved to a concentration of 20 mg / mL to assess the level of AGEs formation. The results are shown in Table 1.

[0070] Table 1: Effects of different treatments on the properties of β-lactoglobulin peptide

[0071] According to the data in Table 1, compared with peptide A1, peptide B (chlorogenic acid deficient) showed a significant decrease in DPPH clearance rate, indicating that chlorogenic acid is the key component for conferring antioxidant activity; peptide C (plasma pretreatment deficient) showed a significant decrease in antigenicity, indicating that plasma pretreatment can expose reaction sites and enhance the efficiency of subsequent modification; peptide D (vacuum glycosylation deficient) also showed a significant decrease in bitterness score, antigenicity reduction rate and digestibility, confirming that vacuum glycosylation can both mask allergic epitopes and improve the state of enzymatic hydrolysis substrates, and can also remove most bitter substances; peptide E (enzymatic hydrolysis only) showed a comprehensive deterioration in all four indicators, highlighting that single enzymatic hydrolysis cannot achieve the synergistic goal of low allergenicity, high antioxidant, no bitterness and high digestibility.

[0072] Table 2: Performance Indicators of Samples from Different Processes

[0073] The method for preparing low-allergenic β-lactoglobulin peptides provided by this invention organically integrates four technologies: malic chlorogenic acid pre-modification, low-temperature atmospheric pressure plasma pretreatment, vacuum short-time glycosylation reaction, and compound stepwise enzymatic hydrolysis. This synergistic approach achieves an antigenicity reduction rate >85%, significant inhibition of AGEs content, DPPH scavenging rate >65%, bitterness score >9, and digestibility >95%. The obtained peptides are then compounded with maltodextrin, nutritional fortifiers, and L-carnitine to produce a sports nutrition solid beverage conforming to GB 24154 standards. This invention provides a new approach for the preparation of low-allergenic, high-antioxidant, and low-AGEs β-lactoglobulin peptides and the development of sports nutrition foods.

[0074] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing a low-allergenic β-lactoglobulin peptide, characterized in that, Includes the following steps: S1. Dissolve β-lactoglobulin, natural phenolic acids and reducing sugars together in pure water, adjust the pH to 7.5-8.5, and pretreat the resulting mixture with low-temperature atmospheric pressure plasma. S2. The solution pretreated in step S1 is pre-frozen and then freeze-dried under vacuum to obtain freeze-dried powder. S3. Place the lyophilized powder obtained in step S2 into a vacuum heating chamber for vacuum glycosylation reaction; S4. After dissolving the glycosylated product obtained in step S3, add the first complex protease and the second complex protease in sequence for stepwise enzymatic hydrolysis to inactivate the enzyme. S5. The enzymatic hydrolysate obtained in step S4 is purified and dried to obtain low-allergenic β-lactoglobulin peptide.

2. The preparation method according to claim 1, characterized in that, The natural phenolic acid substance mentioned in step S1 is apple chlorogenic acid extract with a chlorogenic acid content ≥70%; the mass ratio of the natural phenolic acid substance to the β-lactoglobulin is (0.02-0.08):

1.

3. The preparation method according to claim 1, characterized in that, The reducing sugar in step S1 is at least one of glucose, isomaltooligosaccharide, or galactooligosaccharide; the mass ratio of the reducing sugar to the β-lactoglobulin is (0.8-1.2):

1.

4. The preparation method according to claim 1, characterized in that, The conditions for the low-temperature atmospheric pressure plasma pretreatment in step S1 are: voltage 10-50kV, frequency 20-30kHz, treatment time 4-10min, and atmosphere is air; and / or, the pH adjustment is performed using food-grade sodium bicarbonate.

5. The preparation method according to claim 1, characterized in that, The pre-freezing temperature in step S2 is -80℃, and the pre-freezing time is 4-6 hours; the vacuum freeze-drying time is 48-72 hours.

6. The preparation method according to claim 1, characterized in that, The conditions for the vacuum glycosylation reaction in step S3 are: absolute pressure ≤10kPa, temperature 95-105℃, and heating time 25-35min.

7. The preparation method according to claim 1, characterized in that, In step S4, the first complex protease is bromelain and neutral protease; the bromelain and neutral protease are combined in a mass ratio of 1:(1-2); the conditions for adding the first complex protease for enzymatic hydrolysis are: pH 6.0-7.0, temperature 45-55℃, the ratio of the amount of the first complex protease added to the substrate is 1000-2000U:1mL, and the hydrolysis time is 1-1.5h; and / or, the second complex protease is papain and flavor protease; the papain and flavor protease are combined in a mass ratio of 1:(1-2); the conditions for adding the second complex protease for enzymatic hydrolysis are: pH 6.5-7.5, temperature 50-60℃, the ratio of the amount of the second complex protease added to the substrate is 2000-3000U:1mL, and the hydrolysis time is 0.5-1.5h; and / or, the conditions for enzyme inactivation are: treatment at 80-100℃ for 10-15min.

8. The preparation method according to claim 1, characterized in that, The purification process in step S5 involves dialysis at 4°C for 36 hours using a dialysis bag with a molecular weight cutoff of 1000-3500 Da, with the solution changed every 12 hours. The drying process is spray drying, and the spray drying conditions are as follows: inlet air temperature 130-160°C, outlet air temperature 65-75°C, feed solids content 15-20%, feed flow rate 10-20 mL / min, and atomization pressure 0.2-0.4 MPa.

9. A low-allergenic β-lactoglobulin peptide prepared by the preparation method according to any one of claims 1-8.

10. The use of a low-allergenic β-lactoglobulin peptide as described in claim 9 or a low-allergenic β-lactoglobulin peptide prepared by any one of the preparation methods described in claims 1-8 in the preparation of sports nutrition foods.

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