Hypoallergenic eye-protecting milk tablet for children and preparation method of hypoallergenic eye-protecting milk tablet

Abstract

The invention provides hypoallergenic eye-protecting children milk tablets and a preparation method thereof, and the hypoallergenic eye-protecting children milk tablets comprise hypoallergenic formula whey protein powder, casein hydrolysate freeze-dried powder, eye-protecting composite active ingredients, a flavoring agent, an antioxidant and auxiliary materials, wherein the low-sensitization formula whey protein powder comprises a mixture of lactoferrin powder, alpha-lactalbumin powder and low-sensitization beta-LG protein powder, and the low-sensitization beta-LG protein powder comprises a non-covalent compound formed by combining a small molecule substance and cow milk beta-LG; the casein hydrolysate freeze-dried powder comprises bovine milk casein hydrolyzed by Alcalase protease and bacillus licheniformis protease. The selected small molecule substances are all dairy product available additives permitted by GB2760-2024 National Food Safety Standard Food Additives Usage Standards, and related reports of the food additives for beta-LG sensitization improvement do not exist in the existing research at present, so that the blank in the prior art is filled, and the eating safety of children is guaranteed.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, specifically to a hypoallergenic eye-protecting children's milk tablet and its preparation method. Background Technology

[0002] Milk allergy is an internationally recognized food safety and public health issue, and its sensitizing effect is prevalent and consistent globally. Milk is mainly composed of whey protein and casein, among which whey protein contains... β - Lactoglobulin ( β β-Lactoglobulin (Beta-lactoglobulin) is a core allergen in cow's milk. Clinical data shows that approximately 82% of people with milk allergies are allergic to cow's milk. β -LG exhibits an immune response, which highlights β -LG plays a central role in the mechanism of milk allergy. Therefore, developing drugs targeting milk allergies is crucial. β LG's improved technology has become an important research direction in the fields of food science and public health, aiming to reduce the risk of milk allergies and ensure dietary safety for allergy sufferers. Meanwhile, whey protein, as an important food ingredient, has wide applications in dairy products, functional foods, and other fields. According to relevant statistics, the global whey powder market exceeded US$10 billion in 2024 and is expected to continue growing at an average annual rate of 5%-7% in the coming years. The demand for high-purity whey protein is also rapidly increasing due to the expansion of the functional food market. Furthermore, α-lactone, the main component of cow's milk casein... s1 -casein and β Casein also has strong allergenic properties, and its allergenicity needs to be considered in dairy product processing.

[0003] Although milk and its products play an important role in the food industry, milk allergies limit their widespread use among allergy sufferers. Currently, improved technologies targeting milk allergies face two main challenges: firstly, how to effectively reduce the core allergens in milk. β - How to address the allergenicity of LG while maintaining the nutritional value and functional properties of milk; secondly, how to handle allergenic components in cow's casein, such as α-protein. s1 -casein and β - Casein, to reduce its potential risks to people with allergies. Especially in the application of whey protein, because... β -LG accounts for up to 50% of the whey protein component, and reducing its allergenicity has become key to technological improvements. Currently, reducing... β - Lactoglobulin ( βThe main technologies for reducing sensitization (LG) include physical modification, enzymatic hydrolysis, fermentation engineering, genetic engineering, and chemical modification. Industrial applications often utilize a combination of enzymatic hydrolysis and fermentation, such as in extensively hydrolyzed milk powder and fermented yogurt products. While gene editing technology shows 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 uses glycosylation reactions, but improper reaction conditions can lead to browning of the products. Furthermore, some existing methods for reducing sensitization, such as the use of plant-derived polyphenolic compounds, do not fall under the scope of GB2760-2024, the National Food Safety Standard for the Use of Food Additives, which limits their widespread application in the food industry.

[0004] Regarding milk allergies, existing technologies have implemented several measures to reduce the allergenicity of milk. β -LG, some studies have attempted to reduce the allergenicity of plant-derived polyphenols through covalent or non-covalent binding, but these methods are difficult to apply due to non-compliance with food safety standards. For cow's casein, the industry commonly employs extensive hydrolysis technology, which breaks down the large peptide chains of milk proteins into smaller peptide fragments and amino acids, thereby disrupting the antigenic epitopes of allergenic proteins and significantly reducing their allergenicity. However, while effective, extensive hydrolysis may reduce the nutritional value of milk and incur higher processing costs. Furthermore, for certain populations, such as infants and the elderly, extensively hydrolyzed products may not meet their nutritional needs.

[0005] Although existing technologies have reduced the allergenicity of milk to some extent, many problems still exist. Firstly, regarding... β - While LG (Liquid Oxygen Protein) reduces allergenicity, there is a lack of effective methods that meet both food safety standards and practical applications. Secondly, although extensive hydrolysis technology can significantly reduce the allergenicity of cow's casein, it may lead to a decline in the product's nutritional value and has high processing costs, limiting its widespread market application. Furthermore, different populations react differently to milk allergies, making it difficult for existing technologies to provide personalized solutions. Therefore, developing a new technology that can effectively reduce milk allergenicity while maintaining its nutritional value and functional properties, and meeting food safety standards, has become an urgent problem to be solved. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a hypoallergenic eye-protecting children's milk tablet and its preparation method, using nationally approved eye-protecting small molecule substances, non-covalently bonded. β -LG reduces the allergenicity of whey protein powder; casein is hydrolyzed by compound enzymes and then powdered after ultrafiltration; the protein components are mixed with excipients such as eye protectants and prebiotics and compressed into tablets to produce hypoallergenic eye-protecting children's milk tablets.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a hypoallergenic eye-protecting milk tablet for children, comprising hypoallergenic whey protein powder, casein hydrolysate lyophilized powder, eye-protecting compound active ingredients, flavoring agent, antioxidant, and excipients, wherein: the hypoallergenic whey protein powder comprises lactoferrin powder, α-lactalbumin powder, and hypoallergenic... β - A blend of LG protein powders, hypoallergenic β -LG protein powder contains small molecules and milk β -LG-bound non-covalent complexes, small molecules including ascorbic acid, β - At least one of carotene and erythritol; casein hydrolysate lyophilized powder including bovine casein hydrolyzed with Alcalase protease and Bacillus licheniformis protease; eye-protecting complex active ingredients including ascorbic acid, lutein and β - Carotene.

[0008] Furthermore, the addition ratio of the hypoallergenic whey protein powder to the casein hydrolysate lyophilized powder is 1:4 to 1:3, and the amount of the eye-protecting compound active ingredient added is 1% to 3% of the total amount of the hypoallergenic whey protein powder and the casein hydrolysate lyophilized powder.

[0009] Furthermore, the lactoferrin powder, α-lactalbumin powder, and hypoallergenic... β - The mixing ratio of LG protein powder is 5:2:0.1 to 4:1:0.1; milk β - The mass ratio of LG to small molecules is 2:1 to 5:1.

[0010] Furthermore, the preparation of hypoallergenic whey protein powder is as follows: Small molecule substances are added to milk at concentrations of 0.1 mg / mL to 0.5 mg / mL. β A non-covalent complex was obtained by stirring at room temperature in an LG solution. The non-covalent complex was freeze-dried under vacuum to obtain a low-sensitivity compound. β -LG protein powder; Lactoferrin powder, α-lactalbumin powder and hypoallergenic β -LG protein powder is mixed at room temperature to obtain a hypoallergenic whey protein powder formula.

[0011] Furthermore, the ratio of Alcalase protease to Bacillus licheniformis protease is 0.5:0.4~0.7:0.5; the enzymatic hydrolysis conditions are pH=8~9, enzyme addition amount is 5.50%~7.5%, temperature is 60℃~63℃, hydrolysis time is 45~55min, and degree of hydrolysis is ≥60%.

[0012] Furthermore, the preparation of the casein hydrolysate lyophilized powder is as follows: Bovine milk casein was hydrolyzed using both Alcalase and Bacillus licheniformis protease to obtain the hydrolysate. The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the filtrate was collected, then freeze-dried to obtain casein hydrolysate lyophilized powder.

[0013] Furthermore, the ascorbic acid, lutein, β - The ratio of added carotene is 1:0.3:5 to 2:0.8:6.

[0014] This invention also provides a method for preparing hypoallergenic eye-protecting milk tablets for children, the specific steps of which are as follows: Hypoallergenic whey protein powder and casein hydrolysate lyophilized powder were mixed to obtain mixture 1; Add the eye-protecting complex active ingredients to mixture 1 in sequence, and mix to obtain mixture 2; Add the flavoring agent to mixture 2, and mix to obtain mixture 3; An antioxidant was added to mixture 3, and the mixture was then mixed to obtain mixture 4. Add excipients to mixture 4, mix, and compress into tablets to obtain hypoallergenic eye-protecting children's milk tablets.

[0015] Furthermore, 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 to 310 mg / kg.

[0016] Furthermore, the antioxidant is sodium D-isoascorbate, with 0.07g of antioxidant added per gram of mixture 3; the excipients include sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3, and the amount of excipients added accounts for 4% of the mass of mixture 4.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a hypoallergenic eye-protecting milk tablet for children, achieving precise matching of allergen control with nutritional and eye-protecting functions, thus solving the technical pain points of existing children's milk tablets with high allergy risk and limited functionality. The small molecule substances used in this invention (ascorbic acid, ... β - Carotene and erythritol are both permitted additives for dairy products under GB2760-2024 "National Food Safety Standard for the Use of Food Additives," and their safety is clearly supported by national standards. Furthermore, current research has not found any evidence of these food additives being used in dairy products. β The reports on LG's allergenicity improvement are highly innovative, filling a gap in existing technology while avoiding the safety hazards and compliance risks associated with the use of additives that have not been approved for dairy products, thus ensuring the safety of children's consumption.

[0018] Furthermore, this invention utilizes small molecule substances with milk β -LG forms non-covalent complexes, which can effectively mask [the underlying structure]. β -LG's antigenic epitopes and IgE binding sites reduce the allergenicity of milk protein at the source, specifically addressing the issue of children unable to consume dairy products due to milk protein allergies, thus expanding the product's target population. Simultaneously, the hypoallergenic whey protein powder utilizes lactoferrin, α-lactalbumin, and modified... β The -LG compound formula fully restores the core nutrients of milk whey protein, retains the immunomodulatory function of lactoferrin and the easily digestible properties of α-lactalbumin, and does not contain added lactose or fat, making it suitable for children's delicate digestive systems. It can supplement children with high-quality protein while avoiding discomfort caused by lactose intolerance and excessive fat intake, thus taking into account both nutrition and suitability.

[0019] Furthermore, this invention incorporates eye-protecting compound active ingredients (ascorbic acid, lutein, ... β - Carotene is explicitly included in the product ingredients, constructing a scientific eye protection system. Among them, ascorbic acid can resist oxidation and promote collagen synthesis, while lutein can protect the retina and filter blue light. β - Carotene can be converted into Vitamin A and relieve eye fatigue. The synergistic effect of these three factors can specifically meet the eye care needs during children's growth and development, achieving a dual function of low allergen and eye protection. Compared with existing single-function children's milk tablets, this solution has more comprehensive product functions, addressing both allergy concerns and the special needs of children's vision protection, significantly improving its practicality and market competitiveness.

[0020] This invention further clarifies the specific ratio range of each core component and key preparation process parameters (the addition ratio of low-allergenic whey protein powder to casein hydrolysate lyophilized powder is 1:4 to 1:3, and the addition amount of eye-protecting compound active ingredients is 1% to 3% of the total amount of the two protein powders). Through precise ratio control, the synergistic optimization of allergen control, nutritional supply and eye protection effect is achieved, which not only ensures low allergenicity, but also avoids the safety risks caused by insufficient addition of eye-protecting ingredients leading to functional failure or excessive addition.

[0021] In whey protein blends and β - Regarding LG modification, lactoferrin powder, α-lactalbumin powder, and low-allergenicity products were identified. β - The mixing ratio of LG protein powder is 5:2:0.1 to 4:1:0.1, and the milk... βThe mass ratio of LG to small molecules is 2:1 to 5:1. This ratio range can fully restore the nutritional composition of milk whey protein while minimizing allergenicity, and can ensure the activity of lactoferrin and α-lactalbumin, giving full play to their advantages of immune regulation and easy digestibility. Meanwhile, this invention specifies that the ratio of Alcalase protease to Bacillus licheniformis protease is 0.5:0.4 to 0.7:0.5, and the enzymatic hydrolysis conditions are limited to pH=8 to 9, enzyme addition amount of 5.50% to 7.5%, temperature of 60℃ to 63℃, and degree of hydrolysis ≥60%. These process parameters can achieve deep hydrolysis of bovine milk casein, resulting in a very low proportion of highly allergenic intact casein molecules. The hydrolysis products are mainly small molecule peptides and free amino acids, which are unlikely to trigger an immune response in the body, further improving the low allergenicity of the product. Furthermore, the plan clarifies the preparation process of casein hydrolysate lyophilized powder, specifying the use of a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa for ultrafiltration of the enzymatic hydrolysate. This effectively retains incompletely hydrolyzed large-molecule allergenic proteins, collecting only small-molecule peptides and amino acids with a molecular weight less than 1 kDa, further reducing the risk of allergies, while improving the product's digestibility and absorption rate, making it suitable for children's digestive system. Simultaneously, the plan clarifies the ascorbic acid, lutein, and other active ingredients in the eye-protecting complex. β - The addition ratio of carotene is 1:0.3:5~2:0.8:6. This ratio allows the three eye-protecting ingredients to work synergistically to maximize the eye-protecting effect. Compared with a single eye-protecting ingredient, it can better meet the diverse needs of children's vision development, further enhance the product's functional advantages, provide precise process guidance for industrial production, and reduce quality fluctuations during the production process.

[0022] This invention provides a method for preparing hypoallergenic eye-protecting children's milk tablets. The method employs a segmented mixing process, first mixing hypoallergenic whey protein powder with freeze-dried casein hydrolysate powder, then sequentially adding eye-protecting compound active ingredients, flavoring agents, antioxidants, and excipients. This step-by-step mixing avoids interference between different components, prevents protein denaturation due to direct contact with other components, effectively preserves the nutritional activity and hypoallergenic properties of the protein, and ensures the activity of the eye-protecting ingredients is not destroyed, thus guaranteeing the product's functional stability. This invention specifies the exact types and amounts of flavoring agents and antioxidants, selecting vanillin or L-theanine as flavoring agents. 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. This meets national standards and imparts a pleasant aroma to the product, improving the taste of the children's milk tablets and increasing children's acceptance. Simultaneously, L-theanine can also relieve fatigue and help regulate mood, further enriching the product's nutritional functions. Sodium D-isoascorbate was selected as an antioxidant, and the amount added to each gram of mixture 3 was limited to 0.07g. This effectively inhibits the oxidative deterioration of proteins and eye-protecting ingredients in the product, extends the product's shelf life, and avoids the odor or safety hazards caused by using other antioxidants, thus balancing the product's stability and safety.

[0023] Furthermore, the plan specifies the exact composition and dosage of the excipients. The excipients consist of sucrose, maltodextrin, magnesium stearate, erythritol, and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3, with the dosage accounting for 4% of the mixture's mass. This excipient formulation effectively improves the tableting performance of the product, ensuring that the hardness and brittleness of the compressed milk tablets meet edible requirements, avoiding issues of easy breakage and difficulty in chewing, and is suitable for children's eating habits. Simultaneously, erythritol, as a prebiotic, can regulate the balance of intestinal flora, helping to promote children's intestinal health, achieving a multi-functional integration of low allergenicity, eye protection, and intestinal care. The entire preparation process requires no complex equipment, is simple and easy to operate, and has clearly defined parameters for each step, enabling large-scale, standardized production, reducing production costs, and improving production efficiency. This provides strong support for the industrial promotion of the product, combining practicality and economy. Attached Figure Description

[0024] Figure 1 For milk β -LG structure and its antigenic epitope distribution.

[0025] Figure 2 for β -LG complex protein antigen activity assay.

[0026] Figure 3 for β-LG complex protein structure analysis. (A~C) UV-Vis absorption spectroscopy; (D) Fourier transform infrared spectroscopy; (E~F) Circular dichroism spectroscopy.

[0027] 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.

[0028] Figure 5 for β -Scanning electron micrograph of the complex formed by LG and small molecules (magnification 12000x), (A) β -LG,(B) β -LG-AA,(C) β -LG-CAR and (D) β -LG-ERY.

[0029] Figure 6 The components of bovine milk protein hydrolysate filtrate were analyzed using MALDI-TOF-MS mass spectrometry.

[0030] Figure 7 for β The -LG complex was co-cultured with H2O2-treated ARPE-19 cells, and the intracellular ROS level was measured. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] This invention provides a hypoallergenic eye-protecting milk tablet for children, the specific preparation steps of which are as follows: I. Preparation of Hypoallergenic Whey Protein Powder (1) Put the milk β -LG is dissolved in phosphate buffer (pH 5-8, concentration 0.1 mol / L) to obtain β -LG in milk with a final concentration of 0.1~0.5 mg / mL β -LG solution.

[0033] Small molecule substances selected as food additives include ascorbic acid, β - At least one of carotene and erythritol; The small molecule substances are respectively reacted with milk. β -LG solution was mixed, stirred at room temperature for 15-30 minutes, and then 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 A non-covalent complex of -LG-ERY, wherein: β - The mass ratio of LG to small molecule substances is set to 2:1 to 5:1.

[0034] (2) The above non-covalent complex was subjected to vacuum freeze drying (-80℃~-70℃, 14~16h) to obtain a low-sensitization compound. β -LG protein powder.

[0035] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powder is mixed in a certain ratio (5:2:0.1~4:1:0.1) at room temperature to prepare a hypoallergenic whey protein powder formula.

[0036] II. Hydrolyzed Bovine Casein (1) Bovine milk casein was hydrolyzed using Alcalase protease (Novozymes China) and Bacillus licheniformis protease (Sigma-Aldrich), with a complex enzyme ratio of E. Alcalase酶 E 地衣芽孢杆菌蛋白酶 =0.5:0.4~0.7:0.5. The enzymatic hydrolysis conditions are: pH=8~9, enzyme addition amount (enzyme / substrate ratio, E / S) 5.50%~7.5%, temperature 60℃~63℃, hydrolysis time 45~55min, degree of hydrolysis ≥60%, to obtain the enzymatic hydrolysate.

[0037] (2) The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was first washed with 0.1 mol / L NaOH solution, and then washed with pure water until neutral. During sample injection, the inlet pressure was ensured not to exceed 20 Pa and the outlet pressure was not to exceed 5 Pa. The enzymatic hydrolysate was allowed to pass through the 1 kDa polyethersulfone membrane. The filtrate was collected and freeze-dried at -55°C and a vacuum of 10 Pa to obtain casein hydrolysate lyophilized powder.

[0038] III. Preparation of Hypoallergenic Eye-Protecting Children's Milk Tablets (1) The low-allergenic whey protein powder prepared above is mixed with casein hydrolysate lyophilized powder in a ratio of 1:4 to 1:3 to obtain mixture 1.

[0039] (2) Add the following eye-protecting compound active ingredients to mixture 1 in sequence: ascorbic acid, lutein, β- Carotene, to obtain mixture 2; wherein, the eye-protecting complex active ingredients account for 1%~3% of the mass of mixture 1, ascorbic acid, lutein, β - The ratio of added carotene is 1:0.3:5 to 2:0.8:6.

[0040] (3) Add vanillin (<5mg / 100mL) or L-theanine (100–310mg / kg) to mixture 2 according to flavor requirements to obtain mixture 3.

[0041] (4) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of mixture 3 to obtain mixture 4.

[0042] (5) Add 4% of excipients (containing sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3) to mixture 4. Under aseptic conditions, use a single-punch tablet press to uniformly mix mixture 4 and excipients and then compress the mixture into tablets. Compress the tablets at room temperature for 3 seconds under a pressure of 50KN. The tablet weight is about 3.0g. Pack the tablets in aluminum foil bags at room temperature to obtain hypoallergenic eye-protecting children's milk tablets.

[0043] Example 1: This embodiment provides a method for preparing hypoallergenic eye-protecting milk tablets for children, the specific steps of which are as follows: (1) Put the milk 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 A non-covalent complex of -LG-AA, β - The mass ratio of LG to small molecules was set to 2:1. After stirring at room temperature for 15 minutes, the mixture was allowed to stand at room temperature.

[0044] (2) β -LG-ascorbic acid was freeze-dried at -80°C for 14 hours to obtain a low-sensitivity product. β -LG protein powder.

[0045] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powders were mixed in a certain ratio (5:2:0.1) at room temperature to prepare a hypoallergenic whey protein powder formula.

[0046] (4) Bovine milk casein was hydrolyzed using Alcalase and Bacillus licheniformis protease in a compound enzyme ratio of E. Alcalase酶 E 地衣芽孢杆菌蛋白酶=0.5:0.4. The enzymatic hydrolysis conditions were: pH=8, enzyme addition (E / S) 5.50%, temperature 60℃, hydrolysis time 45min, degree of hydrolysis ≥60%, to obtain the enzymatic hydrolysate.

[0047] (5) The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was first washed with 0.1 mol / L NaOH solution, and then washed with purified water until neutral. During sample injection, the inlet pressure was ensured not to exceed 20 Pa and the outlet pressure was not to exceed 5 Pa. The enzymatic hydrolysate was allowed to pass through the 1 kDa polyethersulfone membrane. The filtrate was collected and freeze-dried at -55°C and a vacuum of 10 Pa to obtain casein hydrolysate lyophilized powder.

[0048] (6) The low-allergenic whey protein powder prepared above is mixed with casein hydrolysate lyophilized powder in a ratio of 1:4 to obtain mixture 1.

[0049] (7) Add the eye-protecting complex active ingredients ascorbic acid, lutein, and... to mixture 1 in sequence. β - A compound system of carotene in a ratio of 1:0.3:5, accounting for 1% of the mass of mixture 1, yields mixture 2.

[0050] (8) Add vanillin (<5mg / 100mL) to mixture 2 according to flavor requirements to obtain mixture 3.

[0051] (9) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of mixture 3 to obtain mixture 4.

[0052] (10) Add 4% of excipients (containing sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3) to mixture 4. Under sterile conditions, use a single-punch tablet press to uniformly mix mixture 4 and then compress the mixture into tablets. Compress the tablets at room temperature for 3 seconds under a pressure of 50KN. The tablet weight is about 3.0g. Pack the tablets in aluminum foil bags at room temperature to obtain hypoallergenic eye-protecting children's milk tablets.

[0053] Example 2: This embodiment provides a method for preparing hypoallergenic eye-protecting milk tablets for children, the specific steps of which are as follows: (1) Put the milk β -LG is dissolved in phosphate buffer (pH 6, concentration 0.1 mol / L) to make β - The final concentration of LG reached 0.2 mg / mL. Preparation β -LG-carotene ( β A non-covalent complex of -LG-CAR, β- The mass ratio of LG to small molecules was set to 3:1. After stirring at room temperature for 20 minutes, the mixture was allowed to stand at room temperature.

[0054] (2) β -LG-carotene was freeze-dried at -75°C for 15 hours to obtain a low-allergenicity product. β -LG protein powder.

[0055] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powders were mixed in a certain ratio (5:1:0.1) at room temperature to prepare a hypoallergenic whey protein powder formula.

[0056] (4) Bovine milk casein was hydrolyzed using Alcalase and Bacillus licheniformis protease in a compound enzyme ratio of E. Alcalase酶 E 地衣芽孢杆菌蛋白酶 =0.62:0.45. The enzymatic hydrolysis conditions were: pH 8, enzyme addition (E / S) 6.5%, temperature 62℃, hydrolysis time 50 min, degree of hydrolysis ≥65%, to obtain the enzymatic hydrolysate.

[0057] (5) The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was first washed with 0.1 mol / L NaOH solution, and then washed with purified water until neutral. During sample injection, the inlet pressure was ensured not to exceed 20 Pa and the outlet pressure was not to exceed 5 Pa. The enzymatic hydrolysate was allowed to pass through the 1 kDa polyethersulfone membrane. The filtrate was collected and freeze-dried at -55°C and a vacuum of 10 Pa to obtain casein hydrolysate lyophilized powder.

[0058] (6) The low-allergenic whey protein powder prepared above is mixed with the lyophilized casein hydrolysate powder at a ratio of 1:3.5 to obtain mixture 1.

[0059] (7) Add the eye-protecting complex active ingredients ascorbic acid, lutein, and... to mixture 1 in sequence. β - A compound system of carotene in a ratio of 1:0.8:6, accounting for 2% of the total protein powder mass, yielded mixture 2.

[0060] (8) Add vanillin (<5mg / 100mL) to mixture 2 according to flavor requirements to obtain mixture 3.

[0061] (9) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of mixture 3 to obtain mixture 4.

[0062] (10) Add 4% of excipients (containing sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3) to mixture 4. Under sterile conditions, use a single-punch tablet press to uniformly mix mixture 4 and then compress the mixture into tablets. Compress the tablets at room temperature for 3 seconds under a pressure of 50KN. The tablet weight is about 3.0g. Pack the tablets in aluminum foil bags at room temperature to obtain hypoallergenic eye-protecting children's milk tablets.

[0063] Example 3: This embodiment provides a method for preparing hypoallergenic eye-protecting milk tablets for children, the specific steps of which are as follows: (1) Put the milk β -LG is dissolved in phosphate buffer (pH 7, concentration 0.1 mol / L) to make β - The final concentration of LG reached 0.4 mg / mL. Preparation β -LG-erythritol ( β A non-covalent complex of -LG-ERY, β - The mass ratio of LG to small molecules was set to 4:1. After stirring at room temperature for 30 minutes, the mixture was allowed to stand at room temperature.

[0064] (2) β -LG-erythritol was freeze-dried at -70°C for 15 hours to obtain a low-sensitivity product. β -LG protein powder.

[0065] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powder was mixed at a certain ratio (4:1.6:0.1) at room temperature to prepare a hypoallergenic whey protein powder formula.

[0066] (4) Bovine milk casein was hydrolyzed using Alcalase and Bacillus licheniformis protease in a compound enzyme ratio of E. Alcalase酶 E 地衣芽孢杆菌蛋白酶 =0.63:0.55. The enzymatic hydrolysis conditions were: pH=9, enzyme addition (E / S) 6.9%, temperature 63℃, hydrolysis time 52min, degree of hydrolysis ≥63%, to obtain the enzymatic hydrolysate.

[0067] (5) The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was first washed with 0.1 mol / L NaOH solution, and then washed with purified water until neutral. During sample injection, the inlet pressure was ensured not to exceed 20 Pa and the outlet pressure was not to exceed 5 Pa. The enzymatic hydrolysate was allowed to pass through the 1 kDa polyethersulfone membrane. The filtrate was collected and freeze-dried at -55°C and a vacuum of 10 Pa to obtain casein hydrolysate lyophilized powder.

[0068] (6) The low-allergenic whey protein powder prepared above is mixed with casein hydrolysate lyophilized powder at a ratio of 1:3.8 to obtain mixture 1.

[0069] (7) Add the eye-protecting complex active ingredients ascorbic acid, lutein, and... to mixture 1 in sequence. β - A compound system of carotene in a ratio of 2:0.5:6, accounting for 2.8% of the total protein powder mass, yielded mixture 2.

[0070] (8) According to the flavor requirements, L-theanine (210 mg / kg) was added to mixture 2 to obtain mixture 3.

[0071] (9) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of mixture 3 to obtain mixture 4.

[0072] (10) Add 4% of excipients (containing sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3) to mixture 4. Under sterile conditions, use a single-punch tablet press to uniformly mix mixture 4 and then compress the mixture into tablets. Compress the tablets at room temperature for 3 seconds under a pressure of 50KN. The tablet weight is about 3.0g. Pack the tablets in aluminum foil bags at room temperature to obtain hypoallergenic eye-protecting children's milk tablets.

[0073] Example 4: This embodiment provides a method for preparing hypoallergenic eye-protecting milk tablets for children, the specific steps of which are as follows: (1) Put the milk β -LG is dissolved in phosphate buffer (pH 8, concentration 0.1 mol / L) to make β -LG achieved a final concentration of 0.5 mg / mL. Preparation β -A non-covalent complex of LG with ascorbic acid, carotene and erythritol 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.

[0074] (2) The complex was freeze-dried under vacuum (-70℃, 16h) to obtain a low-sensitization compound. β -LG protein powder.

[0075] (3) Combine lactoferrin powder and α-lactalbumin powder with low allergenicity β -LG protein powders were mixed in a certain ratio (4:1:0.1) at room temperature to prepare a hypoallergenic whey protein powder formula.

[0076] (4) Bovine milk casein was hydrolyzed using Alcalase and Bacillus licheniformis protease in a compound enzyme ratio of E. Alcalase酶 E 地衣芽孢杆菌蛋白酶 =0.7:0.5. The enzymatic hydrolysis conditions were: pH 9, enzyme addition (E / S) 7.5%, temperature 63℃, hydrolysis time 55 min, degree of hydrolysis ≥65%, to obtain the enzymatic hydrolysate.

[0077] (5) The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was first washed with 0.1 mol / L NaOH solution, and then washed with purified water until neutral. During sample injection, the inlet pressure was ensured not to exceed 20 Pa and the outlet pressure was not to exceed 5 Pa. The enzymatic hydrolysate was allowed to pass through the 1 kDa polyethersulfone membrane. The filtrate was collected and freeze-dried at -55°C and a vacuum of 10 Pa.

[0078] (6) The hypoallergenic whey protein powder prepared above is mixed with casein hydrolysate lyophilized powder in a ratio of 1:3 to obtain mixture 1.

[0079] (7) Add the eye-protecting complex active ingredients, vitamin C, lutein, and... to mixture 1 in sequence. β - A compound system of carotene in a ratio of 2:0.8:6, accounting for 3% of the total protein powder mass, yielded mixture 2.

[0080] (8) According to the flavor requirements, L-theanine (310 mg / kg) was added to mixture 2 to obtain mixture 3.

[0081] (9) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of mixture 3 to obtain mixture 4.

[0082] (10) Add 4% of excipients (containing sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3) to mixture 4. Under sterile conditions, use a single-punch tablet press to uniformly mix mixture 3 and excipients and then compress the mixture into tablets. Compress the tablets at room temperature for 3 seconds under a pressure of 50KN. The tablet weight is about 3.0g. Pack the tablets in aluminum foil bags at room temperature to obtain hypoallergenic eye-protecting children's milk tablets.

[0083] Comparative Example 1: This comparative example provides a method for preparing eye-protecting children's milk tablets, the specific steps of which are as follows: (1) Put the milk β -LG is dissolved in phosphate buffer (pH 8, concentration 0.1 mol / L) to make β -LG achieved a final concentration of 0.5 mg / mL. Preparation β-LG-Mogroside ( β A non-covalent complex of -LG-LHK, 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.

[0084] (2) β -LG-mogrosides were freeze-dried (-70℃, 16h) to obtain β -LG protein powder.

[0085] (3) Mix lactoferrin powder and α-lactalbumin powder with β -LG protein powders are mixed in a certain ratio (4:1:0.1) at room temperature to prepare formulated whey protein powder.

[0086] (4) Bovine milk casein was hydrolyzed using Alcalase and Bacillus licheniformis protease in a compound enzyme ratio of E. Alcalase酶 E 地衣芽孢杆菌蛋白酶 =0.7:0.5. The enzymatic hydrolysis conditions were: pH 9, enzyme addition (E / S) 7.5%, temperature 63℃, hydrolysis time 55 min, degree of hydrolysis ≥65%, to obtain the enzymatic hydrolysate.

[0087] (5) The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was first washed with 0.1 mol / L NaOH solution, and then washed with purified water until neutral. During sample injection, the inlet pressure was ensured not to exceed 20 Pa and the outlet pressure was not to exceed 5 Pa. The enzymatic hydrolysate was allowed to pass through the 1 kDa polyethersulfone membrane. The filtrate was collected and freeze-dried at -55°C and a vacuum of 10 Pa.

[0088] (6) The prepared whey protein powder and casein hydrolysate lyophilized powder were mixed in a ratio of 1:3 to obtain mixture 1.

[0089] (7) Add the eye-protecting complex active ingredients ascorbic acid, lutein, and... to mixture 1 in sequence. β - A compound system of carotene in a ratio of 2:0.8:6, accounting for 3% of the total protein powder mass, yielded mixture 2.

[0090] (8) According to the flavor requirements, L-theanine (310 mg / kg) was added to mixture 2 to obtain mixture 3.

[0091] (9) Add 0.07g of antioxidant sodium D-isoascorbate to each gram of mixture 3 to obtain mixture 4.

[0092] (10) Add 4% of excipients (containing sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3) to mixture 4. Under sterile conditions, use a single-punch tablet press to uniformly mix mixture 4 and excipients and then compress the mixture into tablets. Compress the tablets at room temperature for 3 seconds under a pressure of 50KN. The tablet weight is about 3.0g. Pack the tablets in aluminum foil bags at room temperature to obtain eye-protecting children's milk tablets.

[0093] The products obtained in Examples 1-4 and Comparative Example 1 were tested, and the results are as follows: Table 1 lists the products. β - LG antigen concentration reduction rate, hardness and friability test results

[0094] As shown in Table 1, in terms of sensitization control effect, Examples 1-4... β -LG antigen concentration decreased by 15%~19%, showing a statistically significant reduction. p <0.05); while the decrease rate of control group 1 was only 2%, which did not reach a significant level. This result confirms that ascorbic acid, β -Carotene and erythritol can be modified through non-covalent binding. β -LG antigenic epitopes effectively reduce allergenicity, while mogrosides do not possess this modification ability, revealing the selective differences in allergenicity regulation by food additives. Example 4 is a combination of three small molecules ( β - Carotene, ascorbic acid, erythritol) and β -LG forms a complex, and compared with Examples 1-3, the complex shows a better effect in reducing sensitization than the single small molecule. β -LG complex.

[0095] Regarding textural stability, the hardness (4.35~4.63 kgf) and friability (0.41%~0.5%) of all formulations were within a stable range, with no significant differences between groups. This indicates that the formulation optimization for allergens in this invention did not negatively impact the physical processing characteristics and edible quality of the product, achieving synergistic protection of allergen control and textural stability.

[0096] Oxidative stress (ROS) levels showed that the ROS reduction rates in Example 1 (16.8%) and Example 2 (16.3%) were significantly higher than those in Control Example 1 (12.1%), mainly due to the combination of ascorbic acid and... β - The strong antioxidant activity of carotene; the relatively low ROS reduction rate (11.8%) in Example 3 also confirms the weak antioxidant activity of erythritol. These results indicate that ascorbic acid and βThe introduction of carotene can effectively compensate for the lack of antioxidant capacity of erythritol and enhance the physiological activity value of the product.

[0097] In summary, the composite formula developed in this invention achieves low allergenicity while also taking into account the product's textural stability and antioxidant activity, providing experimental evidence for the hypoallergenic and functional design of children's dairy products.

[0098] Sensory evaluations were conducted on the products obtained in Examples 1-4 and Comparative Example 1. The specific sensory index scoring criteria are shown in Table 2, and the scoring results are shown in Table 3.

[0099] Table 2 Sensory Indicator Scoring Criteria

[0100] Table 3 Sensory rating results

[0101] As shown in Tables 2 and 3, the sensory evaluation scores of the milk tablets obtained by the present invention are 87-95 points, indicating that the milk tablets obtained by the present invention have good texture, color, aroma, taste and reconstitution properties.

[0102] Figure 1 For milk β -LG structure and its antigenic epitope distribution.

[0103] 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) and β -LG- compound. As shown in the figure, except for mogroside, the other three complexes and the compound all showed significant reduction. 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 allergens.

[0104] Figure 3 for β -LG complex protein structure analysis. (A~C) UV-Vis absorption spectroscopy; (D) Fourier transform infrared spectroscopy; (E~F) Circular dichroism spectroscopy. βThe linear and conformational epitopes of the -LG protein complex were altered.

[0105] 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.

[0106] Figure 5 for β -Scanning electron micrograph of the complex formed by LG and small molecules (magnification 12000x), (A) β -LG,(B) β -LG-AA,(C) β -LG-CAR and (D) β -LG-ERY.

[0107] Figure 6 The components of the bovine milk protein hydrolysate filtrate were analyzed using MALDI-TOF-MS mass spectrometry. As shown in the figure, the m / z of each component is mainly small peptides with a molecular weight of less than 1000 Da, which proves that the complex enzyme has a high degree of hydrolysis.

[0108] Figure 7 for β The -LG complex was co-cultured with H2O2-treated ARPE-19 cells, and intracellular ROS levels were measured. Compared with the H2O2-treated ARPE-19 cell damage model, β -LG-AA and β The LG-CAR complex and its compound complex both showed a significant decrease in ROS levels, but erythritol and mogroside did not significantly reduce ROS levels. This demonstrates that not all food additives can alleviate H2O2-induced oxidative stress damage and protect retinal pigment epithelial cells by reducing intracellular ROS levels in ARPE-19 cells.

[0109] Enzyme-linked immunosorbent assay (ELISA) for determining the antigenicity of complexes: Use cattle βThe 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 LISA washing buffer to each well, let stand for 1 min, discard the washing buffer, pat dry on absorbent paper, 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 6.25~200μg / mL) to calculate the antigenicity of the complex, that is, the equivalent micrograms of protein antigen per milliliter of sample.

[0110] Hardness and friability: Hardness was measured using a hardness tester, and friability was measured using a friability tester.

[0111] Detecting intracellular reactive oxygen species (ROS) levels: Human retinal pigment epithelial cells (ARPE-19) were used and placed in DMEM / F12 (containing HEPES) complete medium containing 10% fetal bovine serum at 37°C. o ARPE-19 cells were cultured at C60°C. Pretreated with H2O2 was used as a model group, and intracellular ROS levels were detected using a sodium acetoacetate (DCFH-DA) fluorescent probe. Following the kit instructions, ARPE cells were incubated with DCFH-DA for 30 min, then collected, and 500 μL of 1 μmol / L DCFH-DA working solution was added and mixed thoroughly. The cells were incubated at 37 °C for 30 min in the dark. The probe was discarded, and the cells were washed three times with PBS. Fluorescence microscopy was used for observation, and fluorescence images were acquired simultaneously. The fluorescence intensity of the sample images was semi-quantitatively analyzed using ImageJ software V1.4.

Claims

1. A hypoallergenic eye-protecting milk tablet for children, characterized in that, This includes hypoallergenic whey protein powder, casein hydrolysate lyophilized powder, eye-protecting complex active ingredients, flavoring agents, antioxidants, and excipients. The hypoallergenic whey protein powder includes lactoferrin powder, α-lactalbumin powder, and hypoallergenic... β - A blend of LG protein powders, hypoallergenic β -LG protein powder contains small molecules and milk β -LG-bound non-covalent complexes, small molecules including ascorbic acid, β - At least one of carotene and erythritol; casein hydrolysate lyophilized powder including bovine casein hydrolyzed with Alcalase protease and Bacillus licheniformis protease; eye-protecting complex active ingredients including ascorbic acid, lutein and β - Carotene.

2. The hypoallergenic eye-protecting children's milk tablet according to claim 1, characterized in that, The addition ratio of the hypoallergenic whey protein powder to the casein hydrolysate lyophilized powder is 1:4 to 1:3, and the amount of eye-protecting compound active ingredients added is 1% to 3% of the total amount of the hypoallergenic whey protein powder and the casein hydrolysate lyophilized powder.

3. The hypoallergenic eye-protecting children's milk tablet according to claim 1, characterized in that, The lactoferrin powder, α-lactalbumin powder, and hypoallergenic β - The mixing ratio of LG protein powder is 5:2:0.1 to 4:1:0.1; milk β - The mass ratio of LG to small molecules is 2:1 to 5:

1.

4. The hypoallergenic eye-protecting children's milk tablet according to claim 1, characterized in that, The preparation of hypoallergenic whey protein powder is as follows: Small molecule substances are added to milk at concentrations of 0.1 mg / mL to 0.5 mg / mL. β A non-covalent complex was obtained by stirring at room temperature in an LG solution. The non-covalent complex was freeze-dried under vacuum to obtain a low-sensitivity compound. β -LG protein powder; Lactoferrin powder, α-lactalbumin powder and hypoallergenic β -LG protein powder is mixed at room temperature to obtain a hypoallergenic whey protein powder formula.

5. The hypoallergenic eye-protecting children's milk tablet according to claim 1, characterized in that, The ratio of Alcalase protease to Bacillus licheniformis protease is 0.5:0.4~0.7:0.5; the enzymatic hydrolysis conditions are pH=8~9, enzyme addition amount is 5.50%~7.5%, temperature is 60℃~63℃, hydrolysis time is 45~55min, and degree of hydrolysis is ≥60%.

6. The hypoallergenic eye-protecting children's milk tablet according to claim 1, characterized in that, The specific preparation method for casein hydrolysate lyophilized powder is as follows: Bovine milk casein was hydrolyzed using both Alcalase and Bacillus licheniformis protease to obtain the hydrolysate. The enzymatic hydrolysate was ultrafiltered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the filtrate was collected, then freeze-dried to obtain casein hydrolysate lyophilized powder.

7. The hypoallergenic eye-protecting children's milk tablet according to claim 1, characterized in that, The ascorbic acid, lutein, β - The ratio of added carotene is 1:0.3:5 to 2:0.8:

6.

8. A method for preparing a hypoallergenic eye-protecting children's milk tablet according to any one of claims 1 to 7, characterized in that, The specific steps are as follows: Hypoallergenic whey protein powder and casein hydrolysate lyophilized powder were mixed to obtain mixture 1; Add the eye-protecting complex active ingredients to mixture 1 in sequence, and mix to obtain mixture 2; Add the flavoring agent to mixture 2, and mix to obtain mixture 3; An antioxidant was added to mixture 3, and the mixture was then mixed to obtain mixture 4. Add excipients to mixture 4, mix, and compress into tablets to obtain hypoallergenic eye-protecting children's milk tablets.

9. The method for preparing a hypoallergenic eye-protecting children's milk tablet according to claim 8, characterized in that, 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 to 310 mg / kg.

10. The method for preparing a hypoallergenic eye-protecting children's milk tablet according to claim 8, characterized in that, The antioxidant is sodium D-isoascorbate, with 0.07g of antioxidant added per gram of mixture 3; the excipients include sucrose, maltodextrin, magnesium stearate, erythritol and sodium citrate in a mass ratio of 6:3:1.8:2.8:0.3, and the amount of excipients added accounts for 4% of the mass of mixture 4.

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