A whey protein hydrolysate and its use in the preparation of anti-inflammatory, antioxidant or allergy relief related products

Abstract

The application provides a whey protein hydrolysate and application thereof in preparation of anti-inflammatory, antioxidant or allergy relief related products. The preparation method has the advantages of high preparation efficiency, fast speed and sufficient hydrolysis. The whey protein hydrolysate obtained through the preparation method is of excellent quality, rich in nutrition, easy to be absorbed by the human body, and can improve the cyclooxygenase-2 inhibition rate, improve the free radical clearance rate, and relieve the kidney and liver damage caused by the body allergy, and thus has the effects of anti-inflammation, anti-oxidation and allergy relief.

Description

Technical Field

[0001] This invention relates to biotechnology, and more particularly to a whey protein hydrolysate and its application in the preparation of anti-inflammatory, antioxidant, or allergy-relieving products. Background Technology

[0002] With the continuous improvement of global consumers' health awareness, natural products that combine nutritional supply and specific physiological functions are increasingly favored by the market, and their application demand in functional foods, health foods, and special medical purpose formulations continues to grow. Whey protein, as an important by-product generated during dairy processing, is rich in high-quality essential amino acids, immunoglobulins, lactoferrin, lactoperoxidase, and other active ingredients, and its amino acid profile is highly compatible with human needs. Therefore, it has become one of the research hotspots and core application raw materials in the field of functional ingredients.

[0003] However, due to its large molecular weight and complex spatial structure, natural whey protein presents significant allergenicity issues, easily triggering adverse reactions such as gastrointestinal discomfort and skin allergies in some individuals (especially infants and those with allergies), greatly limiting its application in products for specific populations. To address this issue, the industry typically employs processing and modification methods to treat whey protein, resulting in products such as whey protein concentrate, whey protein isolate, and whey protein hydrolysate (WPH). Among these, whey protein hydrolysate is a product obtained by breaking down large whey protein molecules into smaller peptides and free amino acids through enzymatic and acidic hydrolysis. Compared to unhydrolyzed whey protein, it not only has a faster digestion and absorption rate and higher bioavailability but also significantly reduces allergenicity, giving it a unique advantage in the field of specialized nutrition.

[0004] However, in existing technologies, the bioactivity of whey protein hydrolysates is significantly affected by hydrolysis conditions (such as enzyme selection, hydrolysis time, and temperature). Most products suffer from low levels of active peptides and lack of functional specificity, making it difficult to meet the precise needs of specific scenarios such as anti-inflammation, anti-oxidation, or allergy relief. Furthermore, some hydrolysis processes suffer from poor product stability and insufficient adaptability to industrial production, limiting their large-scale application in related functional products. Therefore, developing a whey protein hydrolysate with clearly defined bioactivity, strong specificity, and stable processing, and expanding its application in anti-inflammatory, anti-oxidative, and allergy relief products, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to utilize a three-step targeted enzymatic hydrolysis process to enable three enzyme preparations to perform their respective functions and synergistically enhance each other, thereby maximizing the retention and enrichment of active peptide components and improving the biological functional activity of the resulting whey protein hydrolysate. This allows it to be used in the preparation of anti-inflammatory, antioxidant, and allergy-relieving products, thus helping to broaden the application scenarios of whey protein, promote the deep processing of dairy products and the development of functional foods, and provide new solutions for non-pharmacological interventions for chronic inflammation, oxidative damage, and allergic diseases.

[0006] This invention provides a method for preparing whey protein hydrolysate, comprising the following steps:

[0007] The whey protein raw material was subjected to a first enzymatic hydrolysis using glutaminase to obtain the first enzymatic hydrolysis product.

[0008] The first enzymatic hydrolysis product was subjected to a second enzymatic hydrolysis using alkaline protease to obtain the second enzymatic hydrolysis product.

[0009] The second enzymatic hydrolysate was subjected to a third enzymatic hydrolysis using a neutral protease to obtain whey protein hydrolysate.

[0010] In the preparation method described above, the amount of glutaminase added, based on whey protein raw material, is 0.6-1.2% (m / v); and / or,

[0011] Based on whey protein raw materials, the amount of alkaline protease added is 0.7-1.5 wt%; and / or,

[0012] Based on whey protein raw materials, the amount of neutral protease added is 0.7-1.5 wt%.

[0013] In the preparation method described above, the glutaminase activity is 500-700 U / g; and / or,

[0014] The enzyme activity of alkaline protease is 90,000-110,000 U / g; and / or,

[0015] The enzyme activity of neutral protease is 10,000-30,000 U / g.

[0016] The preparation method described above, wherein the conditions for the first enzymatic hydrolysis treatment include: a temperature of 45-55°C, a time of 25-35 min, and a pH value of 5.5-7.5; and / or,

[0017] The conditions for the second enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 30-50 min, and a pH of 6.0-8.0; and / or,

[0018] The conditions for the third enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 2-3 h, and a pH value of 6.0-7.5.

[0019] In the preparation method described above, the whey protein raw material is obtained by mixing whey protein powder and water at a mass ratio of 1:(5-10).

[0020] In the preparation method described above, the protein content of the whey protein powder is ≥80wt%.

[0021] This invention provides a whey protein hydrolysate, wherein the whey protein hydrolysate is prepared by the above-described preparation method.

[0022] The whey protein hydrolysate as described above, wherein the whey protein hydrolysate has a moisture content ≤4wt%, an ash content ≤5wt%, a fat content ≤0.5wt%, and a lactose content ≤1.2wt%; and / or,

[0023] The whey protein hydrolysate contains ≥80wt% protein and ≥65wt% peptides.

[0024] The whey protein hydrolysate as described above, wherein the content of peptides with a molecular weight <500 u in the whey protein hydrolysate is ≥60 wt%.

[0025] The present invention also provides a whey protein hydrolysate prepared by the above preparation method and / or the application of the above whey protein hydrolysate in the preparation of anti-inflammatory, antioxidant or allergy relief related products.

[0026] This invention provides a method for preparing whey protein hydrolysate, which has the following advantages:

[0027] The preparation method provided by this invention has the advantages of high efficiency, fast speed, and complete hydrolysis. The whey protein hydrolysate obtained by this method is of excellent quality, rich in nutrients, and easily absorbed by the human body. Furthermore, it can improve the inhibition rate of cyclooxygenase-2, increase the DPPH free radical scavenging rate, and enhance ABST... + It has a high free radical scavenging rate and can alleviate kidney and liver damage caused by allergies, thus possessing anti-inflammatory, antioxidant, and allergy-relieving effects. Attached Figure Description

[0028] Figure 1 Thermogravimetric and differential thermogravimetric results of whey protein powder in Test Example 2 of this invention are shown in the figure.

[0029] Figure 2 Thermogravimetric and differential thermogravimetric results of whey protein hydrolysate in Test Example 2 of this invention are shown in the figure.

[0030] Figure 3 This is a differential scanning calorimetry curve of whey protein powder in Test Example 2 of the present invention;

[0031] Figure 4This is a differential scanning calorimetry (DSC) curve of whey protein hydrolysate in Test Example 2 of the present invention;

[0032] Figure 5 This is a graph showing the COX-2 inhibition rate of whey protein hydrolysate in Test Example 3 of the present invention;

[0033] Figure 6 This is a graph showing the DPPH scavenging rate of whey protein hydrolysate in Test Example 4 of the present invention;

[0034] Figure 7 ABTS of whey protein hydrolysate in Test Example 5 of this invention + Clearance rate results graph;

[0035] Figure 8 HE staining images of the kidneys and livers of BN rats in Test Example 6 of this invention. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. 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.

[0037] To develop a whey protein hydrolysate that can be used for anti-inflammatory, antioxidant, and allergy relief purposes, the first aspect of this invention provides a method for preparing a whey protein hydrolysate, comprising the following steps:

[0038] The whey protein raw material was subjected to a first enzymatic hydrolysis using glutaminase to obtain the first enzymatic hydrolysis product.

[0039] The first enzymatic hydrolysis product was subjected to a second enzymatic hydrolysis using alkaline protease to obtain the second enzymatic hydrolysis product.

[0040] The second enzymatic hydrolysate was subjected to a third enzymatic hydrolysis using a neutral protease to obtain whey protein hydrolysate.

[0041] This invention employs a three-step targeted enzymatic hydrolysis process, enabling three enzyme preparations to perform their respective functions while synergistically enhancing their effects. This maximizes the retention and enrichment of active peptide components, thereby improving the biological functional activity of the resulting whey protein hydrolysate. This allows it to be used in anti-inflammatory, antioxidant, and allergy relief applications, helping to broaden the application scenarios of whey protein, promote the deep processing of dairy products and the development of functional foods, and provide new solutions for non-pharmacological interventions for chronic inflammation, oxidative damage, and allergic diseases.

[0042] Specifically, in the first enzymatic hydrolysis, glutaminase specifically hydrolyzes glutamine residues in whey protein raw materials to generate glutamic acid and its derivatives. Simultaneously, it disrupts the dense spatial structure of whey protein macromolecules, breaks down inter-peptide cross-links, and provides more hydrolysis sites for the subsequent two proteases, laying the foundation for the generation of small peptides and initially reducing the allergenicity of the raw materials. In the second enzymatic hydrolysis, alkaline protease preferentially breaks down hydrophobic amino acid residues in the protein peptide chains, efficiently degrading the protein fragments from the first enzymatic hydrolysis into small and medium-sized peptides. This further disrupts the allergenic epitopes of whey protein, reducing the allergenicity of the product. Furthermore, this protease has high hydrolysis efficiency, produces mild products, and does not generate off-flavors that affect the quality of the finished product. In the third enzymatic hydrolysis process, the hydrolysis specificity of neutral protease is mild, which can avoid excessive hydrolysis that would degrade the active peptides into non-functional amino acids. At the same time, it further refines the product of the second step into small molecule peptides with molecular weights concentrated below 500 u. These peptides are easily absorbed by the human body and have extremely low sensitization. In addition, its mild hydrolysis characteristics can retain the active groups in the peptide chain, and synergistically enhance the anti-inflammatory, antioxidant and allergy-relieving functions of the product with the first two enzyme preparations, ensuring the stability of the final product's bioactivity.

[0043] Experiments have verified that the preparation method of the present invention has the following advantages:

[0044] (1) The preparation method of the present invention can improve the quality of the obtained whey protein hydrolysate, reduce the moisture, ash, fat and lactose content, and effectively increase the peptide content.

[0045] (2) The preparation method of the present invention can effectively improve the degree of hydrolysis of the obtained whey protein hydrolysate and reduce its molecular weight, thereby helping to improve its digestibility and absorption and promote its rapid digestion and absorption by the gastrointestinal tract.

[0046] (3) The whey protein hydrolysate obtained by the preparation method of the present invention has anti-inflammatory effects, specifically: it can increase the inhibition rate of cyclooxygenase-2 (COX-2). COX-2 can catalyze the conversion of arachidonic acid into inflammatory prostaglandins, thereby triggering typical inflammatory symptoms such as vasodilation, increased permeability, pain sensitivity, and elevated body temperature. Therefore, inhibiting COX-2 can precisely block its activity, reduce the generation of inflammatory mediators from the source, and achieve anti-inflammatory effects.

[0047] (4) The whey protein hydrolysate obtained by the preparation method of the present invention has antioxidant effects, specifically: it can improve the DPPH free radical scavenging rate and ABST +Free radical scavenging rate. Among them, 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical is not a naturally occurring free radical in organisms, but a synthetically produced, stable nitrogen-centered free radical. Its scavenging rate can evaluate the antioxidant activity of a substance. 2,2'-Bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is a water-soluble nitrogen heterocyclic compound, which is colorless in itself. Under the action of oxidants, it is oxidized to a stable blue-green cationic free radical (ABTS). + Therefore, ABST + Free radical scavenging rate can be used as an indicator for evaluating in vitro antioxidant activity.

[0048] (5) The whey protein hydrolysate obtained by the preparation method of the present invention has the effect of allergy relief, specifically: it can relieve kidney and liver damage caused by allergies, and the effect is similar to that of the allergy drug loratadine.

[0049] (6) The preparation method provided by the present invention has the advantages of high preparation efficiency, fast speed and full hydrolysis. The whey protein hydrolysate obtained by the preparation method can achieve the effects of being nutritious and easily absorbed by the human body. It is easily accepted by people with poor digestion or who need to quickly supplement protein and can be used as a nutritional functional ingredient.

[0050] In the above technical solutions, the amount of enzyme added can regulate the enzymatic hydrolysis rate and affect the degree of hydrolysis of whey protein raw materials. Higher enzyme additions usually accelerate the hydrolysis process, resulting in a higher degree of hydrolysis, thereby affecting the molecular weight distribution and functional properties of whey protein hydrolysates.

[0051] Experiments have shown that, based on whey protein raw materials, adding 0.6-1.2% (m / v) of glutaminase, 0.7-1.5 wt% of alkaline protease, or 0.7-1.5 wt% of neutral protease helps to further promote a good match between the three enzyme preparations and the whey protein raw materials, thereby promoting the enzymatic hydrolysis process towards the production of whey protein hydrolysates with anti-inflammatory, antioxidant, and allergy-relieving effects.

[0052] In some embodiments, the whey protein raw material is in the form of a solution of whey protein powder and water; therefore, the amount of glutaminase added is 0.6-1.2% (m / v) based on the volume of the whey protein raw material in solution form; the amount of alkaline protease added is 0.7-1.5 wt% based on the mass of the whey protein powder; and the amount of neutral protease added is 0.7-1.5 wt% based on the mass of the whey protein powder.

[0053] In the above technical solutions, enzyme activity may affect the degree of hydrolysis of whey protein raw materials. By limiting enzyme activity, it is possible to precisely control the degree of degradation of whey protein, thereby obtaining whey protein hydrolysates with specific molecular weight distribution and functional properties.

[0054] Experiments have shown that when the enzyme activity of glutaminase is 500-700 U / g, the enzyme activity of alkaline protease is 90,000-110,000 U / g, or the enzyme activity of neutral protease is 10,000-30,000 U / g, it helps to promote a good matching effect between glutaminase, alkaline protease, neutral protease, and whey protein raw materials, thereby exerting a good catalytic effect and promoting the enzymatic hydrolysis process towards producing whey protein hydrolysates with anti-inflammatory, antioxidant, and allergy-relieving effects.

[0055] In the above technical solution, regulating the enzymatic hydrolysis temperature, time, and pH value helps to accurately control the degree of hydrolysis of whey protein raw materials, thereby promoting the enzymatic hydrolysis process to obtain whey protein hydrolysate with anti-inflammatory, antioxidant, and allergy-relieving effects.

[0056] The conditions for the first enzymatic hydrolysis treatment may include: a temperature of 45-55℃, a time of 25-35 min, and a pH of 5.5-7.5; the conditions for the second enzymatic hydrolysis treatment may include: a temperature of 45-55℃, a time of 30-50 min, and a pH of 6.0-8.0; and the conditions for the third enzymatic hydrolysis treatment may include: a temperature of 45-55℃, a time of 2-3 h, and a pH of 6.0-7.5.

[0057] It is understood that those skilled in the art can select the actual enzymatic hydrolysis temperature, time, and pH value within the above range according to the actual situation. For example, the temperatures for the first, second, and third enzymatic hydrolysis treatments can be 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, or any value between any two of the above ranges. The time for the first enzymatic hydrolysis treatment can be 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, or any value between any two of the above ranges; the time for the second enzymatic hydrolysis treatment can be 30 min, 35 min, 40 min, 45 min, 50 min, or any value between any two of the above ranges; and the time for the third enzymatic hydrolysis treatment can be 2 h, 2.5 h, 3 h, or any value between any two of the above ranges. The pH value for the first enzymatic hydrolysis treatment can be 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or any value between any two of the above ranges; the pH value for the second enzymatic hydrolysis treatment can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6. The pH values ​​for the third enzymatic hydrolysis treatment can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any value between any two of the above ranges;

[0058] In the above technical solution, the whey protein raw material is obtained by mixing whey protein powder and water at a mass ratio of 1:(5-10). Compared with solid whey protein powder, whey protein raw material has a certain degree of fluidity, a larger surface area, and is more conducive to subsequent enzymatic hydrolysis, which helps to obtain target whey protein hydrolysates with low bitterness and good digestibility. If too little water is added, the whey protein raw material will have poor fluidity, which is not conducive to the action of enzyme preparations and will easily lead to a decrease in enzymatic hydrolysis efficiency; if too much water is added, the reaction volume during enzymatic hydrolysis will be too large, which may increase the load of subsequent operations and the processing cost will also increase accordingly.

[0059] Furthermore, the whey protein powder has a protein content of ≥80wt%, which helps to match with other enzymatic hydrolysis operations to obtain whey protein hydrolysates with anti-inflammatory, antioxidant and allergy-relieving effects.

[0060] In one embodiment of the present invention, 90% whey protein isolate (WPI) can be selected as whey protein powder. WPI90 is a high-protein product obtained by purifying whey protein through ultrafiltration, ion exchange, and other refining processes, removing most of the lactose, fat, and ash, making it a high-quality raw material for preparing highly active whey protein hydrolysates.

[0061] In addition, after adding water to WPI90 at a mass ratio of 1:5-10, it can be stirred in a water bath at 40-60℃ to fully hydrate it, and finally obtain a whey protein solution (i.e. whey protein raw material).

[0062] In practice, after the third enzymatic hydrolysis, the hydrolysate can be subjected to enzyme inactivation treatment at 90-100℃ for 10-20 minutes to avoid the inactivation of active peptides due to over-hydrolysis. Next, it can be concentrated at 70-80℃ to 10-30 Baume degrees to increase the concentration of whey protein hydrolysate and improve its shelf life. Finally, it can be spray-dried at an inlet air temperature of 150-170℃ and an outlet air temperature of 100-130℃ to obtain powdered whey protein hydrolysate, which is convenient for storage and transportation.

[0063] Based on the above preparation method, a second aspect of the present invention provides a whey protein hydrolysate, which is prepared by the above preparation method.

[0064] The whey protein hydrolysate contains ≤4wt% moisture, ≤5wt% ash, ≤0.5wt% fat, ≤1.2wt% lactose, ≥80wt% protein, and ≥65wt% peptide. Therefore, the whey protein hydrolysate of this invention has fewer impurities, and most of the protein exists in the form of small molecule peptides.

[0065] Furthermore, the whey protein hydrolysate contains ≥60 wt% peptides with a molecular weight <500 u. This demonstrates that the whey protein hydrolysate of the present invention has a low molecular weight. Low molecular weight peptides are more easily absorbed and utilized by the body, resulting in good digestibility and high bioavailability. Moreover, small molecular weight peptides generally have lower allergenicity and higher stability than large protein molecules, thus the whey protein hydrolysate also possesses the advantages of low immunogenicity and high stability.

[0066] Based on the above research results, the third aspect of the present invention provides a whey protein hydrolysate prepared by the above preparation method and / or the application of the above whey protein hydrolysate in the preparation of anti-inflammatory, antioxidant or allergy relief related products.

[0067] Furthermore, the related products may include at least one of food, functional food, health food, and medicine. It is understood that the related products of this invention can be applied to infants, children, adolescents, adults, and the elderly.

[0068] When the relevant products include food, they can be at least one of snacks, complementary foods, food additives, dietary supplements, and nutritional fortifiers.

[0069] When the relevant products include functional foods or nutritional products, they can be at least one of the following: special dietary foods, special medical purpose foods, infant formula milk powder, children's formula milk powder, adolescent formula milk powder, adult formula milk powder, functional powders, functional granules, functional capsules, functional beverages, and sports nutrition foods.

[0070] When the relevant product includes health food, it can be at least one of the following: anti-inflammatory, antioxidant, or health food that improves or alleviates allergic reactions.

[0071] When the relevant product includes a drug, it may be at least one of the following: a drug for preventing or treating inflammation, a drug for preventing or treating oxidative stress damage, or a drug for preventing or treating allergic reactions.

[0072] Furthermore, the aforementioned products may be in the form of at least one of the following: oral liquid, tablets, granules, capsules, pills, aqueous solutions, powders, soft capsules, and films.

[0073] Furthermore, the aforementioned products may also include a carrier and / or physiologically acceptable excipients. The carrier includes at least one of microcapsules, microspheres, nanoparticles, and liposomes. The excipients include at least one of fillers, flavoring agents, diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, and isotonic or isotropic regulators.

[0074] The technical solution of this application will be further explained below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or obtained through public channels.

[0075] Example 1

[0076] This embodiment provides a method for preparing whey protein hydrolysate, including the following steps:

[0077] Add water to 90% whey protein isolate (WPI) at a mass ratio of 1:5, and then stir in a 50°C water bath to fully hydrate it, thus obtaining a whey protein solution.

[0078] Glutaminase was added to a whey protein solution for the first enzymatic hydrolysis to obtain the first hydrolysis product. The amount of glutaminase added was 1% (m / v) (based on the whey protein solution), the enzyme activity of glutaminase was 600 U / g, the first hydrolysis temperature was 50℃, the first hydrolysis time was 30 min, and the first hydrolysis pH was 6.5.

[0079] The pH of the first enzymatic hydrolysis product was adjusted to 7.5 using NaOH or HCl, and then alkaline protease was added to it for a second enzymatic hydrolysis to obtain the second enzymatic hydrolysis product. The amount of alkaline protease added was 1 wt% (based on WPI 90%), the enzyme activity of alkaline protease was 10 w U / g, the second enzymatic hydrolysis temperature was 50℃, the second enzymatic hydrolysis time was 50 min, and the second enzymatic hydrolysis pH was 7.5.

[0080] The pH of the second enzymatic hydrolysis product was adjusted to 7.0 using NaOH or HCl, and then a neutral protease was added for a third enzymatic hydrolysis to obtain the third enzymatic hydrolysis product. The amount of neutral protease added was 1 wt% (based on WPI 90%), the enzyme activity of the neutral protease was 18000 U / g, the third enzymatic hydrolysis temperature was 50℃, the third enzymatic hydrolysis time was 3 h, and the third enzymatic hydrolysis pH was 7.0.

[0081] After the third enzymatic hydrolysis, the product was subjected to enzyme inactivation treatment at 98°C for 15 min, then concentrated to 20 Baume degrees at 73°C, and then spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 110°C to obtain deeply hydrolyzed whey protein hydrolysates (WPH).

[0082] Test Example 1: Characteristic Index Detection

[0083] The moisture content, ash content, protein content, fat content, lactose content, molecular weight distribution, peptide content, and degree of hydrolysis of the above-mentioned whey protein hydrolysates were determined using the following methods:

[0084] Moisture content was determined according to GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food"; ash content was determined according to GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Food"; protein content was determined according to GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Food"; fat content was determined according to GB 5009.6-2016 "National Food Safety Standard - Determination of Fat in Food"; lactose content was determined according to GB 5009.6-2016 "National Food Safety Standard - Determination of Lactose in Food"; molecular weight distribution was determined according to GB / T 22729-2008 "Marine Fish Oligopeptide Powder"; peptide content was determined according to GB / T 22492-2008 "Soybean Peptide Powder"; and the degree of hydrolysis was determined using the o-phthalaldehyde (OPA) method. The results are shown in Tables 1 and 2.

[0085] Table 1

[0086]

[0087] Table 2

[0088]

[0089] Table 1 shows the moisture content, ash content, protein content, fat content, lactose content, peptide content, and degree of hydrolysis of whey protein hydrolysates; Table 2 shows the molecular weight distribution of whey protein hydrolysates. The test results indicate that whey protein hydrolysates are mainly composed of protein and peptides, and the molecular weight distribution is concentrated in the range below 500 u.

[0090] Test Example 2: Thermal Stability Test

[0091] Thermal stability analyses of the above-mentioned WPI 90% (WP) and whey protein hydrolysate (WPH) were performed using a simultaneous thermogravimetric / differential calorimetry (TG) analyzer, employing differential scanning calorimetry (DSC) and thermogravimetric analysis (TG) under nitrogen atmosphere at temperatures ranging from 35 to 800 °C and a heating rate of 10 °C / min. The TG and differential thermogravimetric (DTG) results for WP are as follows: Figure 1 As shown, the TG and DTG results for WPH are as follows: Figure 2 As shown, the DSC curve of WP is as follows: Figure 3 As shown, the DSC curve of WPH is as follows: Figure 4 As shown.

[0092] Figure 1 and Figure 2The results showed that the thermal decomposition process of both WP and WPH exhibited three weight loss stages: the weight loss ranges for WP were room temperature - 154℃ and 154℃ - ​​600℃, while the weight loss range for WPH shifted towards lower temperatures to room temperature - 131℃ and 131℃ - 600℃. In the first weight loss stage, characteristic endothermic peaks appeared in the DTG curves of both WP and WPH. This may be due to the evaporation of free and bound water adsorbed on the samples, or the release of water accompanying the breaking of hydrogen bonds between short peptide chains or intramolecular condensation reactions. Since both samples were dried and sealed for storage, the mass loss in the first stage reflects their hygroscopic properties. WP and WPH experienced relatively low mass losses in this temperature range, with loss rates of 7.11% and 3.71%, respectively, indicating that WP is more deliquescent in air. In the second weight loss stage, the decomposition rate of the samples increased significantly, at which point the chemical bonds of the polysaccharide peptides were broken. The weight loss rates for WP and WPH were 19.89% and 10.16%, respectively. The third stage is high-temperature decomposition, with weight loss rates of 49.34% for WP and 63.64% for WPH. This weight loss is related to the dehydration of hydroxyl groups and the oxidative decomposition of carbon atoms in the structure. This difference indicates that enzymatic hydrolysis weakens the stability of hydrogen bonds and hydrophobic interactions between peptide chains by disrupting the protein's native folding structure, making WPH more susceptible to thermally induced degradation and resulting in a greater weight loss percentage. DTG curves further show that the maximum thermal weight loss rate of WP corresponds to a temperature of 314.26℃, while that of WPH decreases to 294.08℃.

[0093] Figure 3 and Figure 4 The results showed that WP exhibited a significant endothermic peak at 240.67℃, corresponding to an enthalpy of 6.80 J / g. This phenomenon can be attributed to an endothermic decomposition process triggered by the breakage of the protein backbone or the destruction of its ordered structure. In contrast, the endothermic peak temperature of WPH shifted significantly forward to 164.40℃, and the enthalpy decreased to 0.38 J / g.

[0094] Thermal stability testing is of significant practical importance for the development of liquid beverage products. Industrial production of liquid beverages requires high-temperature sterilization. Thermal stability testing effectively assesses the product's and its active ingredients' tolerance to this processing stage, mitigating issues such as ingredient inactivation and decreased product stability caused by high-temperature sterilization. The typical temperature range for high-temperature sterilization of liquid beverages is 100-140℃. Therefore, although the temperature corresponding to the maximum weight loss rate of whey protein hydrolysate (WPH) is lower than that of whey protein (WP), this thermal characteristic still fully meets the requirements of industrial production of liquid beverages. Furthermore, in the temperature ranges of the first and second stages of heat loss, which involve the most frequent applications in product use, the heat loss rate of WPH is significantly lower than that of WP, indicating that it exhibits superior thermal stability within the core temperature range of high-temperature processing for liquid beverages and is more suitable for the industrial production requirements of liquid dosage forms.

[0095] Test Example 3: Detection of COX-2 Inhibition Rate

[0096] Cyclooxygenase-2 (COX-2) is a key inducible enzyme mediating inflammatory responses, and its inhibitors can effectively treat inflammation. Under normal physiological conditions, COX-2 is present in extremely low levels in body tissues. However, when stimulated by inflammatory factors, mechanical damage, or other stimuli, macrophages and endothelial cells rapidly induce its high expression. COX-2 catalyzes the conversion of arachidonic acid into inflammatory prostaglandins, thereby triggering typical inflammatory symptoms such as vasodilation, increased permeability, heightened pain sensitivity, and elevated body temperature. Therefore, inhibiting COX-2 precisely blocks its activity, reducing the production of inflammatory mediators at the source to achieve an anti-inflammatory effect.

[0097] To detect the COX-2 inhibition rate of whey protein hydrolysate (WPH), the following experiment was conducted: WPH was prepared at concentrations of 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL, respectively. Following the instructions of the cyclooxygenase-2 (COX-2) inhibitor screening kit, the COX-2 inhibitory activity of WPH was detected. The results are shown below. Figure 5 .

[0098] Figure 5 The results showed that the inhibition rate of COX-2 increased in a concentration-dependent manner with increasing WPH concentration, reaching a maximum inhibition rate of 72.47±1.41% at 80 mg / mL. Celecene, the positive control, is a COX-2 inhibitor whose core mechanism of action is the specific inhibition of COX-2 activity, blocking the conversion of arachidonic acid into inflammatory prostaglandins such as PGE2, thereby alleviating symptoms at the site of inflammation. Its IC50 value is 40 nM. It has been reported that celecene's inhibition rate at 40 nM is approximately 60%. Using celecene as a positive control for WPH, experiments showed that 100 nM celecene had an inhibition rate of 74.58±4.21% for COX-2, similar to and without significant difference from the inhibition rate at 80 mg / mL WPH. This suggests that WPH can achieve its anti-inflammatory effect by inhibiting COX-2.

[0099] Test Example 4: DPPH Free Radical Scavenging Rate Detection

[0100] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable organic free radical that appears deep purple in organic solvents, with a characteristic absorption peak at 517 nm. When an antioxidant is added to the system, the antioxidant reacts with the DPPH free radical by donating hydrogen atoms or electrons, reducing it to a stable, colorless product (1,1-diphenyl-2-trinitrophenylhydrazine), causing the purple color of the solution to fade. Therefore, the scavenging ability of the substance against DPPH free radicals can be calculated by measuring the change in absorbance at 517 nm before and after the reaction. The DPPH free radical scavenging rate can be used to evaluate the antioxidant activity of a substance.

[0101] To detect the DPPH radical scavenging rate of whey protein hydrolysate (WPH), the following experiment was conducted: WPH was prepared at concentrations of 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL to obtain WPH solutions of different concentrations. 100 μL of 0.1 mmol / L 1,1-diphenyl-2-picrylhydrazyl (DPPH) anhydrous ethanol solution and 100 μL of the above-mentioned WPH solutions of different concentrations were added sequentially to a 96-well plate. The plates were incubated at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm, recorded as Ax. Following the same experimental procedure, 100 μL of anhydrous ethanol was used instead of 100 μL of 0.1 mmol / L 1,1-diphenyl-2-picrylhydrazyl (DPPH) anhydrous ethanol solution, and the resulting absorbance was recorded as A0. Following the same experimental procedure, 100 μL of distilled water was used instead of 100 μL of 0.1 mmol / L distilled water. The absorbance values ​​obtained from μL of WPH solutions of different concentrations are denoted as A1; the formula for calculating the DPPH free radical scavenging rate is as follows:

[0102]

[0103] The results of DPPH free radical scavenging rate are shown below. Figure 6 . Figure 6 The results showed that the DPPH free radical scavenging capacity of WPH increased continuously with increasing concentration, reaching a scavenging rate of 92.65±4.90% at 80 mg / mL. Compared with most reported research papers focusing on the antioxidant function of peptides (around 90%), the scavenging rate of WPH at 80 mg / mL in this invention is at a relatively high level, indicating that WPH inhibits DPPH, thereby achieving an antioxidant effect.

[0104] Test Example 5: ABST + Free radical scavenging rate detection

[0105] ABTS, short for 2,2'-adiazonobis(3-ethylbenzothiazoline-6-sulfonic acid), is a synthetically produced, water-soluble nitrogen heterocyclic compound and one of the most widely used chromogenic reagents for evaluating in vitro antioxidant activity. ABTS itself is a colorless compound; under the action of an oxidizing agent, it is oxidized to a stable blue-green cationic free radical (ABTS). + This free radical exhibits characteristic absorption peaks at 414 nm, 645 nm, and 734 nm, with 734 nm being the most commonly used detection wavelength. When an antioxidant is added to the system, it will release the blue-green ABTS by donating electrons or hydrogen atoms. + Reduction to the colorless ABTS parent compound leads to a decrease in the absorbance of the solution. The degree of absorbance decrease is positively correlated with the concentration and activity of the antioxidant, from which ABST can be calculated. + Free radical scavenging rate.

[0106] Therefore, ABTS is used to detect whey protein hydrolysate (WPH). + To assess free radical scavenging rate, the following experiment was conducted: WPH was prepared in phosphate-buffered saline (PBS) solutions at concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL, and the pH was adjusted to 7.4 to obtain WPH solutions of different concentrations. Equal volumes of 7 mM 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution and 2.45 mM potassium persulfate solution were mixed and allowed to stand at room temperature in the dark for 16 h to obtain the ABTS working stock solution. Before use, the ABTS working stock solution was diluted with PBS to prepare the ABTS working solution. The absorbance of the ABTS working solution was measured at the corresponding wavelengths. After subtracting the absorbance value of the PBS blank control, the absorbance at 734 nm was required to meet the detection requirements of 0.7 ± 0.05 and 1.4 at 405 nm. A blank control group, a sample group, and a sample control group were set up, with three replicates for each group. The specific groupings were as follows: 10 μL of solution A and 190 μL of solution B were added to the wells of a 96-well plate, and the reaction was carried out at room temperature in the dark for 6 min. The absorbance was measured at 734 nm. In the blank control group, solution A was PBS and solution B was ABTS working solution; in the sample group, solution A consisted of different concentrations of WPH solution and solution B was ABTS working solution; in the sample control group, solution A consisted of different concentrations of WPH solution and solution B was PBS. + The formula for calculating the free radical scavenging rate is as follows:

[0107]

[0108] In the formula, B is the absorbance value corresponding to the sample group, C is the absorbance value corresponding to the sample control group, and D is the absorbance value corresponding to the blank control group.

[0109] ABTS + The results of free radical scavenging rate are visible Figure 7 . Figure 7 The results showed that within the WPH concentration range of 0.1-0.8 mg / mL, ABTS + The free radical scavenging rate increased proportionally to concentration, and there were significant differences in scavenging rates among different concentrations (P < 0.5). At a concentration of 0.8 mg / mL, WPH showed significant ABTS... + The scavenging rate reached 71.42±0.58%, indicating that WPH has a significant ABTS free radical scavenging ability.

[0110] Test Example 6: Animal experiments to verify the allergy-relieving effect

[0111] The experimental animals involved below, BN rats (SPF grade, 30 rats, 7-8 weeks old), were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed under standard conditions of ambient temperature (22±2℃), humidity 50%±5%, and 12 h light / dark cycle, and were provided with purified water. After one week of acclimatization, BN rats were randomly divided into 6 groups: blank control group (CON), ovalbumin model group (OVA), low-concentration WPH intervention group (WPH-L), medium-concentration WPH intervention group (WPH-M), high-concentration WPH intervention group (WPH-H), and positive control group (LT). The blank control group received no intervention. The ovalbumin model group received intraperitoneal injection of OVA mixture. The low-concentration WPH intervention group received intraperitoneal injection of OVA mixture and gavage 200 mg / kg / day WPH. The medium-concentration WPH intervention group received intraperitoneal injection of OVA mixture and gavage 500 mg / kg / day WPH. The high-concentration WPH intervention group received intraperitoneal injection of OVA mixture and gavage 1000 mg / kg / day WPH. The positive control group received intraperitoneal injection of OVA mixture and gavage 1000 mg / kg / day WPH. Loratadine was administered at a dose of mg / kg / d. The OVA mixture mentioned above refers to an equal volume mixture of OVA and Freund's complete adjuvant for the first injection, and an equal volume mixture of OVA and Freund's incomplete adjuvant for subsequent injections, with a total volume of 1 mL and a final OVA concentration of 0.02 mg / mL. Intraperitoneal injection of the OVA mixture was performed on days 0, 7, 14, 21, 28, and 35. After preliminary confirmation of successful model establishment by measuring allergic antibodies in rat serum on day 14, continuous daily gavage administration of WPH or loratadine was initiated until day 42. On day 42, a final challenge was performed using 1 mL of the OVA mixture with a final OVA concentration of 0.1 mg / mL.

[0112] Rats were sacrificed on day 42, and their livers and kidneys were harvested. These organs were fixed in 10% paraformaldehyde and trimmed. The livers and kidneys were then dehydrated with ethanol at different concentrations, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Histopathological changes were observed under a microscope and photographs were taken. Toluidine blue staining was used to observe mast cell degranulation. Detailed results can be found [link to results]. Figure 8 In the figure, A represents the HE staining results of the kidneys of BN rats (scale bar = 20 μm, n = 3), and B represents the HE staining results of the livers of BN rats (scale bar = 50 μm, n = 3).

[0113] Figure 8 Results A showed that in the CON and LT groups, the glomeruli had normal morphology and significant glomerular space; the glomerulus structure was regular, the basement membrane was intact, and the juxtaglomerular cells and macula densa were clearly distinguishable. In the OVA group, the glomeruli had abnormal morphology, and the glomerular space was almost completely lost; the glomeruli were significantly swollen and enlarged, and the glomerulus was significantly reduced or even blocked due to compression; mesangial cell proliferation led to thickening of the basement membrane, accompanied by inflammatory cell infiltration. After WPH intervention, the glomerular morphology returned to normal, and the glomerular structure and basement membrane were relatively intact, especially in the WPH-M and WPH-H groups. Figure 8 The results showed that the liver tissue morphology in the CON group was normal; the rats in the OVA group showed abnormal hepatocyte fusion and arrangement, enlarged intercellular spaces, and mild abnormal cell nuclei and hemocyte infiltration; while the intervention of WPH significantly alleviated liver damage caused by allergic reaction and reduced the infiltration of inflammatory cells.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing whey protein hydrolysate, characterized in that, Includes the following steps: The whey protein raw material was subjected to a first enzymatic hydrolysis using glutaminase to obtain the first enzymatic hydrolysis product. The first enzymatic hydrolysis product was subjected to a second enzymatic hydrolysis using alkaline protease to obtain a second enzymatic hydrolysis product. The second enzymatic hydrolysate was subjected to a third enzymatic hydrolysis using a neutral protease to obtain the whey protein hydrolysate.

2. The preparation method according to claim 1, characterized in that, Based on the whey protein raw material, the amount of glutaminase added is 0.6-1.2% (m / v); and / or, Based on the whey protein raw material, the amount of alkaline protease added is 0.7-1.5 wt%; and / or, Based on the whey protein raw material, the amount of neutral protease added is 0.7-1.5 wt%.

3. The preparation method according to claim 1 or 2, characterized in that, The glutaminase activity is 500-700 U / g; and / or, The alkaline protease has an enzyme activity of 90,000-110,000 U / g; and / or, The activity of the neutral protease is 10,000-30,000 U / g.

4. The preparation method according to any one of claims 1-3, characterized in that, The conditions for the first enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 25-35 min, and a pH value of 5.5-7.5; and / or, The conditions for the second enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 30-50 min, and a pH value of 6.0-8.0; and / or, The conditions for the third enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 2-3 h, and a pH value of 6.0-7.

5.

5. The preparation method according to any one of claims 1-4, characterized in that, The whey protein raw material is obtained by mixing whey protein powder and water at a mass ratio of 1:(5-10).

6. The preparation method according to claim 5, characterized in that, The whey protein powder contains ≥80wt% protein.

7. A whey protein hydrolysate, characterized in that, The whey protein hydrolysate was prepared by the preparation method according to any one of claims 1-6.

8. The whey protein hydrolysate according to claim 7, characterized in that, The whey protein hydrolysate has the following properties: moisture content ≤4 wt%, ash content ≤5 wt%, fat content ≤0.5 wt%, and lactose content ≤1.2 wt%; and / or, The whey protein hydrolysate contains ≥80wt% protein and ≥65wt% peptides.

9. The whey protein hydrolysate according to claim 7 or 8, characterized in that, The whey protein hydrolysate contains ≥60wt% peptides with a molecular weight <500 u.

10. The use of a whey protein hydrolysate prepared by any one of claims 1-6 and / or the whey protein hydrolysate prepared by any one of claims 7-8 in the preparation of anti-inflammatory, antioxidant or allergy relief related products.

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