A hypoallergenic milk powder and its use

Abstract

The application provides a low-sensitization milk powder and application thereof, and belongs to the technical field of dairy products. The milk powder simulates the protein proportion of breast milk, and can reduce the sensitization risk of the milk powder by using the natural advantage of low sensitization of sheep milk. The milk powder can reduce the levels of allergic reaction markers and inflammatory factors, reduce the damage degree of the intestinal barrier, promote the growth of intestinal probiotics, and maintain intestinal homeostasis.

Description

Technical Field

[0001] This invention belongs to the field of dairy product technology, and in particular relates to a hypoallergenic milk powder and its application. Background Technology

[0002] Milk contains various specific proteins that are common food allergens, easily triggering allergic reactions in humans, especially infants and young children whose digestive and immune systems are not yet fully developed. The incidence of milk protein allergy can reach 2% to 6%, making it the most common type of food allergy in infants and young children, seriously affecting their healthy development. Some allergic symptoms can even persist into adulthood, leading to long-term allergic diseases. At the same time, some adults with weakened immune systems and those with sensitive stomachs are also prone to intolerance and allergic symptoms such as bloating, diarrhea, rashes, and vomiting after consuming regular milk powder, greatly limiting the suitable population for regular milk powder.

[0003] Studies have shown that the main allergenic components in cow's milk are β-lactoglobulin, casein, and α-lactalbumin, with β-lactoglobulin being the most significant allergen, accounting for approximately 50% of total whey protein. Over 80% of people with cow's milk allergies experience allergic reactions to this protein. This protein contains multiple specific allergenic epitopes in its molecular structure. After ingestion, undigested intact protein molecules enter the intestinal mucosa, stimulating a specific immune response and triggering immediate or delayed-type allergic reactions. Compared to breast milk proteins, cow's milk proteins have a larger molecular weight, more allergenic epitopes, and higher structural stability, making them difficult for the human digestive system to digest and break down quickly. This is the core reason for the high allergenicity of regular infant formula.

[0004] To address the issue of milk powder allergies, the industry has developed various hypoallergenic milk powder preparation technologies, with mainstream solutions mainly falling into three categories: protein hydrolysis modification, physical modification, and raw material screening and optimization. However, existing hypoallergenic milk powder technologies still suffer from several unavoidable drawbacks, resulting in significant technological bottlenecks in the industry. Firstly, conventional enzymatic hydrolysis processes have poor controllability, making it difficult to precisely control the degree of hydrolysis. Over-hydrolysis leads to an excessively high proportion of short peptides and free amino acids in the milk powder, not only disrupting its original nutritional structure and reducing its nutritional value but also imparting a distinctly bitter and unpleasant taste, negatively impacting the consumer experience. Incomplete hydrolysis leaves behind a large amount of intact protein and large peptide fragments, with allergenic epitopes not being fully destroyed, resulting in limited allergy reduction and a still high risk of allergies, failing to meet the consumption needs of highly sensitive individuals. Secondly, physical modification methods such as high temperature and high pressure can only slightly change the spatial structure of proteins and cannot fundamentally eliminate protein sensitization sites. The desensitization effect is weak. Moreover, high temperature treatment can easily cause the loss of heat-sensitive nutrients in milk powder, destroy functional components such as vitamins and active proteins, and reduce the nutritional quality of milk powder.

[0005] Meanwhile, existing hypoallergenic infant formulas generally suffer from limited functionality and poor adaptability. Most hypoallergenic infant formulas on the market focus solely on reducing allergens, neglecting the compatibility with human digestion and absorption, as well as nutritional balance. Modified formulas often have amino acid ratios and fatty acid compositions that do not match human needs well, and long-term consumption may lead to insufficient nutrient absorption.

[0006] Therefore, providing a hypoallergenic milk powder to reduce the risk of allergies in infants and young children not only fills the gap in existing products and technologies, but also provides a new direction and theoretical basis for the research and development of hypoallergenic infant formula. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a low-allergenic milk powder that mimics the protein ratio of breast milk and utilizes the natural advantage of the low allergenicity of goat milk to reduce the risk of allergies in the milk powder.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a hypoallergenic milk powder, comprising, by weight, the following ingredients: 95-105 parts whole goat milk powder, 550-580 parts demineralized goat whey powder D90, 160-180 parts compound vegetable oil, 25-35 parts refined olive oil, 20-30 parts goat butter, 3-5 parts fructooligosaccharide powder, 70-80 parts galactooligosaccharide syrup, 0.5-1.5 parts of compound stage 1 milk powder vitamin fortifier B, and compound stage 1 milk powder mineral fortifier E. 0.5-1.5 parts, calcium carbonate 2.5-3.5 parts, dicalcium phosphate 4-6 parts, sodium citrate 3-5 parts, potassium citrate 1-2 parts, magnesium sulfate 1-1.5 parts, choline chloride 1-1.5 parts, L-carnitine 0.08-0.12 parts, potassium chloride 5-8 parts, phospholipids 3-4 parts, inositol 0.2-0.4 parts, mixed tocopherol concentrate 0.2-0.4 parts, 10% w / w docosahexaenoic acid powder 9-10 parts, 10% w / w arachidonic acid powder 10-12 parts, compound 0.5% w / w lutein nutritional fortifier 0.2-0.4 parts, tricalcium phosphate 0.5-1.5 parts, Bifidobacterium animalis Bb-12 0.2~0.4 parts, L-ascorbic acid sodium 1~1.5 parts, compound nucleotide nutritional fortifier 0.2~0.4 parts, lactoferrin 0.5~0.7 parts.

[0009] Preferably, the mass ratio of whey protein to casein in the milk powder is 70~90:30~10.

[0010] Preferably, the mass ratio of whey protein to casein in the milk powder is 75~85:25~15.

[0011] Preferably, the mass ratio of whey protein to casein in the milk powder is 80:20.

[0012] Preferably, the milk source of the milk powder is goat milk.

[0013] The present invention also provides the application of the milk powder in the preparation of hypoallergenic products.

[0014] The present invention also provides the application of the milk powder in the preparation of products that regulate intestinal flora.

[0015] Preferably, the milk powder maintains the stability of the microbial community and promotes the growth of probiotics.

[0016] The present invention also provides the application of the milk powder in the preparation of products that maintain intestinal barrier function.

[0017] Preferably, the milk powder promotes the production of short-chain fatty acids.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a hypoallergenic infant formula. By adjusting the mass ratio of whey protein to casein to mimic the protein ratio in breast milk, the risk of allergic reactions can be reduced. The infant formula provided by this invention can lower the levels of allergy markers and inflammatory factors, reduce intestinal barrier damage, promote the growth of beneficial intestinal bacteria, and maintain intestinal homeostasis. Attached Figure Description

[0019] Figure 1 The results showed that symptom scores and rectal temperature changes after sensitization and challenge were statistically significant (P<0.05). A represents symptom scores after challenge, and B represents rectal temperature changes after challenge. Figure 2 The values ​​represent serum levels of specific antibodies IgE and IgG1; where (A) represents specific antibody IgE and (B) represents specific antibody IgG1; different letters indicate significant differences (P<0.05); Figure 3 The levels of histamine, β-Hex, TPS, and ACAN in serum are represented by (A) histamine (HIS); (B) β-aminohexosidase (β-Hex); (C) trypsin (TPS); and (D) proteoglycan (ACAN). Significant differences were found between the different letters (P<0.05). Figure 4 The values ​​represent the expression levels of intestinal permeability-related mRNAs; where A represents serum DAO content; B represents serum D-lactic acid content; and C represents the relative expression levels of intestinal barrier-related gene mRNAs. Significant differences were found between the different letters (P<0.05). Figure 5Venn diagram for identifying protein levels in the spleen; where Control represents the control group; 6B represents the 60% bovine whey protein group; 8B represents the 80% bovine whey protein group; 8C represents the 80% goat whey protein group; and HB represents the bovine hydrolyzed protein group. Figure 6 This is a principal component analysis diagram of proteomics; where A is the PCA cluster analysis of all samples; B is the cluster analysis of different whey-casein ratios from bovine milk with the control group; C is the cluster analysis of different whey-casein ratios from bovine and ovine milk with the control group; and D is the cluster analysis of intact and hydrolyzed proteins from bovine milk with the control group. Figure 7 Principal component analysis diagram of mouse gut microbiota; Figure 8 The results represent the gut microbiota diversity analysis; where A represents Chao1 α diversity; B represents Shannon α diversity; C represents β diversity; D represents the relative abundance of phylum-level gut microbiota; and E represents the relative abundance of class-level gut microbiota. Figure 9 This study analyzes the differential abundance of gut microbiota and its genus-level community structure. A represents hierarchical clustering based on Euclidean distance, plotting a heatmap of the relative abundance of top-level genera across all groups, with color levels indicating z-score-normalized relative abundance. B represents a T-test comparison of differentially abundant genera among selected group pairs. Figure 10 The content of short-chain fatty acids in feces; where A is acetic acid; B is propionic acid; C is butyric acid; D is valeric acid; E isovaleric acid; F is isobutyric acid; different letters indicate significant differences (P<0.05); Figure 11 The diagram shows the principal component analysis of metabolites; where A represents the cluster analysis of all six groups of metabolites; B represents the cluster analysis of metabolites from different milk sources and with different whey to casein ratios (6B, 8B, 8C, and control); and C represents the cluster analysis of metabolites from the hydrolyzed protein group and the intact protein group (6B, 8B, HB, and control group). Detailed Implementation

[0020] This invention provides a hypoallergenic milk powder, comprising, by weight, the following ingredients: 95-105 parts whole goat milk powder, 550-580 parts demineralized goat whey powder D90, 160-180 parts compound vegetable oil, 25-35 parts refined olive oil, 20-30 parts goat butter, 3-5 parts fructooligosaccharide powder, 70-80 parts galactooligosaccharide syrup, 0.5-1.5 parts of compound stage 1 milk powder vitamin fortifier B, and compound stage 1 milk powder mineral fortifier E. 0.5-1.5 parts, calcium carbonate 2.5-3.5 parts, dicalcium phosphate 4-6 parts, sodium citrate 3-5 parts, potassium citrate 1-2 parts, magnesium sulfate 1-1.5 parts, choline chloride 1-1.5 parts, L-carnitine 0.08-0.12 parts, potassium chloride 5-8 parts, phospholipids 3-4 parts, inositol 0.2-0.4 parts, mixed tocopherol concentrate 0.2-0.4 parts, 10% w / w docosahexaenoic acid powder 9-10 parts, 10% w / w arachidonic acid powder 10-12 parts, compound 0.5% w / w lutein nutritional fortifier 0.2-0.4 parts, tricalcium phosphate 0.5-1.5 parts, Bifidobacterium animalis Bb-12 The milk powder contains 0.2-0.4 parts of whey protein, 1-1.5 parts of sodium L-ascorbate, 0.2-0.4 parts of compound nucleotide fortifier, and 0.5-0.7 parts of lactoferrin. The preferred mass ratio of whey protein to casein is 70-90:30-10, more preferably 75-85:25-15, and even more preferably 80:20. The milk source of the milk powder is goat milk. In this invention, the purity of L-carnitine is 99.98%, and the purity of sodium L-ascorbate is 99.99%.

[0021] Goat milk proteins are structurally closer to breast milk than cow milk proteins. Goat milk has a lower αs1-casein content, accounting for only 5.6% of total casein (compared to approximately 34% in cow milk), and its fat globules are smaller. Amino acid sequence alignment shows that goat milk and cow milk share only 88% homology in αs1-casein, with significant differences in isoelectric point and stability. These characteristics contribute to the better digestibility and potentially lower allergenicity of goat milk. This invention uses goat milk as the milk source to provide a milk powder with low allergenicity, which can reduce intestinal barrier damage, promote the growth of beneficial intestinal bacteria, and maintain intestinal homeostasis.

[0022] This invention does not limit the preparation method of milk powder; conventional methods in the art can be used for preparation.

[0023] This invention also provides the application of the milk powder in the preparation of hypoallergenic products. The milk powder provided by this invention significantly reduces the immune response to common allergens and has a low risk of allergy, providing a new direction and theoretical basis for the research and development of hypoallergenic products.

[0024] The present invention also provides the application of the milk powder in the preparation of products that regulate intestinal flora, wherein the milk powder maintains the ecological stability of the flora and promotes the growth of probiotics.

[0025] The present invention also provides the application of the milk powder in the preparation of products that maintain intestinal barrier function, wherein the milk powder can promote the production of short-chain fatty acids, which helps to enhance the integrity of the intestinal mucosal barrier and improve intestinal health.

[0026] In a specific embodiment of the present invention, whole goat milk powder was purchased from OVIGANIC IBERICA SL; demineralized goat whey powder D90 was purchased from OVIGANIC IBERICA SL; compound vegetable oil was purchased from Qinhuangdao Jinhai Special Edible Oil Industry Co., Ltd.; refined olive oil was purchased from ACEITES ABRIL.SL; goat butter was purchased from OVIGANIC IBERICA.SL; compound stage 1 milk powder vitamin fortifier B (VR27350264) was purchased from DSM Vitamins (Shanghai) Co., Ltd.; compound stage 1 milk powder mineral fortifier E (VR25916264) was purchased from DSM Vitamins (Shanghai) Co., Ltd.; mixed tocopherol concentrate was purchased from Shaanxi Haisifu Bioengineering Co., Ltd.; compound 0.5% w / w lutein fortifier (dry method) was purchased from DSM Vitamins (Shanghai) Co., Ltd.; Bifidobacterium animalis Bb-12 was purchased from CHR HANSEN; and compound nucleotide fortifier was purchased from Nantong Qiuzhiyou Nutritional Food Co., Ltd.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 A hypoallergenic milk powder, by weight, comprises the following ingredients: 100 parts whole goat milk powder, 565 parts demineralized goat whey powder D90, 170 parts compound vegetable oil, 30 parts refined olive oil, 25 parts goat butter, 4 parts fructooligosaccharide powder, 75 parts galactooligosaccharide syrup, 1 part compound stage 1 milk powder vitamin fortifier B, 1 part compound stage 1 milk powder mineral fortifier E, 3 parts calcium carbonate, 5 parts dicalcium phosphate, 4 parts sodium citrate, 1.5 parts potassium citrate, 1.2 parts magnesium sulfate, 1.4 parts choline chloride, 0.1 parts L-carnitine (99.98%), 7 parts potassium chloride, 3.7 parts phospholipids, 0.3 parts inositol, 0.3 parts mixed tocopherol concentrate, 9.5 parts 10% w / w docosahexaenoic acid powder, 11.5 parts 10% w / w arachidonic acid powder, and 0.5% compound. w / w lutein fortifier 0.3 parts, tricalcium phosphate 1 part, Bifidobacterium animalis Bb-12 0.3 parts, L-ascorbic acid sodium (99.99%) 1.3 parts, compound nucleotide fortifier 0.3 parts, lactoferrin 0.6 parts.

[0029] The mass ratio of whey protein to casein is 80:20.

[0030] Comparative Example 1 A hypoallergenic milk powder, by weight, contains the following ingredients: 800 parts raw milk, 40 parts demineralized whey powder D905, 25 parts whey protein powder (WPC80), 170 parts compound vegetable oil, 40 parts anhydrous butter, 10 parts refined olive oil, 4 parts fructooligosaccharide powder, 75 parts galactooligosaccharide syrup, 1 part vitamin B fortifier for stage 1 milk powder, and 1 part mineral fortifier E for stage 1 milk powder. 1 part, magnesium sulfate 2 parts, calcium carbonate 4.5 parts, sodium citrate 4 parts, calcium hydrogen phosphate 5.5 parts, choline chloride 1.2 parts, compound taurine nutritional fortifier (wet method) 0.4 parts, L-carnitine (99.98%) (wet method) 0.1 parts, potassium chloride 5 parts, phospholipids 3.7 parts, inositol (wet method) 0.6 parts, mixed tocopherol concentrate 0.3 parts, sodium L-ascorbate (99.99%) (dry method A) 1.3 parts, tricalcium phosphate 1 part, compound lutein (0.5%) nutritional fortifier 0.3 parts, docosahexaenoic acid powder (10%) 9 parts, arachidonic acid powder (10%) 11 parts, lactoferrin 0.6 parts, compound nucleotide nutritional fortifier 0.4 parts, Bifidobacterium animalis Bb-12 0.3 parts.

[0031] The mass ratio of whey protein to casein is 80:20, and the milk source is cow's milk.

[0032] Comparative Example 2 A hypoallergenic milk powder differs from Comparative Example 1 in that the mass ratio of whey protein to casein is 60:40, and the milk source is cow's milk.

[0033] Example 5 1. Construct a mouse sensitization model.

[0034] BALB / c litter mice (one mother and her 4-5 day old pups) were housed in an IVC environment, provided with standard feed and water. The ambient temperature was maintained at 23±2℃, with a 12-hour light-dark cycle. Female offspring were selected for subsequent experiments. To ensure consistent growth conditions for pups raised by different mothers, each mother housed 6 female pups, and pups from the same litter were randomly assigned to different experimental groups.

[0035] Seventy-two female offspring were selected and randomly divided into six groups. The specific grouping and processing are as follows: The control group (n=12) was given saline by gavage on days 9, 14, 19, 24 and 29, and saline was given to stimulate gavage on day 34. The adjuvant control group (CT, n=12) was administered 0.5 μg / g bw Vibrio cholerae toxin by gavage on days 9, 14, 19, 24 and 29, and challenged by gavage with physiological saline on day 34. The sensitization group with 80% goat milk whey (8C, n=12): 200 μg / g.bw goat milk mixed with 0.5 μg / g.bw CT adjuvant was administered by gavage on days 9, 14, 19, 24, and 29, and 1 mg / g.bw goat milk-based stimulation was administered by gavage on day 34. The sensitization group with 80% bovine milk whey (8B, n=12) was sensitized by gavage on days 9, 14, 19, 24 and 29 with 200 μg / g.bw bovine milk whey mixed with 0.5 μg / g.bw CT adjuvant, and challenged by gavage with 1 mg / g.bw bovine milk whey (80%) on day 34. The sensitization group with 60% bovine milk whey (6B, n=12) was sensitized by gavage on days 9, 14, 19, 24 and 29 with 200 μg / g.bw bovine milk whey mixed with 0.5 μg / g.bw CT adjuvant, and challenged by gavage with 1 mg / g.bw bovine milk whey (60%) on day 34. Hydrolyzed milk sensitization group (HB, n=12): Hydrolyzed milk 200 μg / g.bw mixed with CT adjuvant 0.5 μg / g.bw was administered by gavage on days 9, 14, 19, 24 and 29, and hydrolyzed milk 1 mg / g.bw was administered by gavage on day 34.

[0036] Among them, 80% of the whey was from sheep milk and was the milk powder provided in Example 1; 80% of the whey was from cow milk and was the milk powder provided in Comparative Example 1; 60% of the whey was from cow milk and was the milk powder provided in Comparative Example 2; and the hydrolyzed cow milk was Lacprodan IF-3070 produced by Arla Foods Ingredients in Denmark.

[0037] Mice were anesthetized with isoflurane after stimulation, and whole blood was collected by enucleation. After euthanasia by cervical dislocation, fresh feces from the spleen, mesenteric lymph nodes, jejunum, and colon were collected.

[0038] 2. Score of allergic symptoms and changes in rectal temperature in mice after challenge.

[0039] Allergic symptoms and rectal temperature in mice were assessed within 30 minutes after challenge. The symptom scoring table is shown in Table 1. Table 1 Scoring table for allergic symptoms in mice after provocation

[0040] After six consecutive oral sensitizations, on day 34, mice were induced to exhibit allergic symptoms by administering a large dose of the sensitizing protein via oral gavage. The symptoms and changes in rectal temperature 30 minutes after stimulation were assessed. Figure 1As shown in A, the allergy symptom score results are: 6B group > 8B group > 8C group ≈ HB group ≈ CT group ≈ Control group.

[0041] All mice in the four milk powder groups showed varying degrees of allergic symptoms. Group 6B exhibited the most severe allergic symptoms, with most mice displaying typical allergic symptoms (scoring 4 or higher), remaining motionless for more than one minute, and showing reduced sensitivity to whisker stimulation. Group 8B also showed similar allergic symptoms, primarily reduced activity, self-isolation, and periorbital edema. In contrast, only some mice in groups 8C and HB exhibited symptoms such as scratching and rubbing around their noses and heads.

[0042] Sensitized mice exhibited a brief drop in body temperature after exposure to a large amount of allergen. Therefore, the rectal temperature difference was measured 60 minutes before and 30 minutes after challenge. Figure 1 As shown in B in the figure. The range of rectal temperature changes in each group was as follows: 6B group ≈ 8B group > CT group ≈ Control group ≈ 8C group ≈ HB group. Neither the CT nor Control group showed any symptoms, and their rectal temperatures remained unchanged, indicating that the influence of adjuvants on the experiment could be largely ruled out. Compared with the Control group, the rectal temperatures in the 6B and 8B groups decreased significantly after stimulation, reflecting a stronger allergic reaction. This temperature change is consistent with the study by Shin et al. (SHIN HS, SEE HJ, JUNG SY, et al. Turmeric (Curcuma longa) attenuates food allergy symptoms by regulating type 1 / type 2 helper T cells (Th1 / Th2) balance in amouse model of food allergy [J]. Journal of Ethnopharmacology, 2015, 175: 21-9.). There was no significant difference in the changes between the 8C and HB groups.

[0043] 3. Measurement of serum-specific IgE and IgG1.

[0044] 3000g of collected whole blood was centrifuged at 4℃ for 10 min, and the serum in the supernatant was collected. The levels of specific IgE and IgG1 in the serum of the 6B / 8B / 8C / HB groups were detected by enzyme-linked immunosorbent assay (ELISA).

[0045] IgE is a key marker reflecting the body's allergic reaction to foreign proteins and is usually closely related to acute allergic symptoms; IgG1 reflects the degree of activation of the Th2 immune response and is closely related to humoral immunity levels. This invention assesses the severity of allergies in mice by measuring the levels of IgE and IgG1 antibodies, markers of allergic reactions, in serum. Figure 2 As shown in (A) and (B), the IgE levels were: 6B > 8B ≈ 8C ≈ HB ≈ CT ≈ Control; the IgG1 levels were: 6B > CT ≈ Control ≈ 8C ≥ 8B ≈ HB. Mice in group 6B produced large amounts of specific antibodies IgE and IgG1. The IgE and IgG1 antibody levels in groups 8B, 8C, and HB were similar to those in the Control group, possibly because groups 8B, 8C, and HB had lower levels of allergy and did not produce large amounts of IgE and IgG1.

[0046] 4. Measurement of serum histamine, β-aminohexosidase and cytokine levels.

[0047] According to the instructions provided by Shanghai Enzyme-Link Biotechnology Co., Ltd., the levels of histamine (HIS), β-aminohexosidase (β-Hex), trypsin (TPS), proteoglycan (ACAN), IL-4, IL-5, IL-6, IL-10, IL-12, IFN-γ, DAO, and D-lactic acid in the serum of young mice after sensitization and challenge were measured.

[0048] The results are as follows Figure 3 As shown, the HIS levels were: 6B≈8B>8C≈HB≥CT≥Control; β-Hex levels were: 6B≈8B>8C≈HB≈CT>Control; TPS levels were: 6B≈8B>8C≈HB≈CT>Control; and ACAN levels were: 6B>8B≈HB≥8C≥CT>Control. The release levels of HIS, β-Hex, TPS, and ACAN in groups 6B and 8B were significantly higher than those in groups Control and CT. Furthermore, the release levels of HIS, β-Hex, TPS, and ACAN in groups 8C and HB were significantly lower than those in groups 6B and 8B, and there was no significant difference compared to the adjuvant groups. This indicates that the degranulation of mast cells in mice in groups 6B and 8B was the greatest, causing the most significant inflammation, while the degranulation of mast cells in groups 8C and HB was less severe.

[0049] 5. Measurement of intestinal permeability-related mRNA expression levels.

[0050] (1) Tissue lysis: Take 20 mg of tissue into a 1.5 mL enzyme-free centrifuge tube, add 500 μL of Buffer RL lysis buffer and 4 zirconium beads, and grind at 60 Hz for 60 s.

[0051] (2) RNA extraction: The lysis buffer was centrifuged at 12,000 rpm for 30 seconds using FastPure gDNA-Filter Column III, and the filtrate was collected. 0.5 times the volume of anhydrous ethanol was added and mixed well. The mixture was then loaded into FastPure RNA Column III and centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded. The sample was washed sequentially with 700 μL Buffer RW1 and 700 μL Buffer RW2, and centrifuged at 12,000 rpm for 30 seconds after each wash, and the filtrate was discarded. 500 μL Buffer RW2 was added, and the sample was centrifuged at 12,000 rpm for 2 minutes, and the collection tube was discarded. 90 μL of enzyme-free water was added, and the RNA was eluted by centrifugation at 12,000 rpm for 1 minute.

[0052] (3) RNA reverse transcription: Nanodrop was used to detect and standardize RNA concentration; the reaction system was prepared as follows: 7 μL of enzyme-free water, 4 μL of 4×gDNAwiper Mix, and 5 μL of RNA template, with a total volume of 16 μL. The mixture was incubated at 42℃ for 2 min; 4 μL of 5×HiScriptⅢ qRT SuperMix was added and mixed well to complete reverse transcription. The cDNA was stored at -20℃.

[0053] (4) cDNA quantification Table 2 Primers for related gene mRNAs

[0054] qPCR quantitative reaction system (10μL): 5μL SYBR Green, 0.2μL each of forward and reverse primers, 4.1μL enzyme-free water, and 0.5μL cDNA; LightCycle-96 instrument was used, program: 95℃ for 10s, 60℃ for 10s, 40 cycles, for relative quantification of mRNA.

[0055] The results are as follows Figure 4 As shown in A and B, the serum DAO and D-lactic acid levels in mice in groups 6B and 8B were significantly higher than those in the control group, indicating impaired intestinal barrier function and abnormal permeability. However, there were no significant differences between groups 8C and HB and the control group, indicating that the intestinal barrier was intact.

[0056] like Figure 4As shown in C, compared to the Control group, the expression of Occludin, Claudin-1, Claudin-2, ZO-1, and ZO-2 was downregulated in groups 6B and 8B, while the expression of Claudin-2 in the pore-forming TJ was upregulated. Claudin-2 promotes fluid flow from the serosal side to the intestinal lumen side, leading to fluid leakage to clear pathogens, and is part of the innate immune system (Fanning AS, Jameson BJ, Jesitis LA, et al. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton[J]. Journal of Biological Chemistry, 1998, 273(45): 29745-53. Itoh M, Nagafuchi A, Moroi S, et al. Involvement of ZO-1 in cadherin-based celladhesion through its direct binding to catenin and actin filaments [J]. Journal of Cell Biology, 1997, 138(1): 181-92. Monaco A, Ovryn B, Axis J, et al. The Epithelial Cell Leak Pathway [J]. International Journal of Molecular Sciences, 2021, 22(14).). These results indicate that the intestinal barrier was severely damaged in groups 6B and 8B, leading to an immune response in the small intestine. Compared to the Control group, there were no significant differences in the expression of Occludin, Claudin-1, Claudin-2, ZO-1, and ZO-2 between groups 8C and HB. The connectivity of gut-associated proteins remained intact, and the intestinal barrier in groups 8C and HB was not significantly disrupted. In conclusion, the intestinal barrier was severely damaged in groups 6B and 8B, while the damage to the intestinal barrier in groups 8C and HB was relatively mild.

[0057] 6. Spleen proteomics.

[0058] (1) Protein extraction: Take approximately 60 mg of sample, freeze-dry in liquid nitrogen until powdered, add 9 times the volume of extraction buffer (90% acetone, 8% water, 2% saturated NaCl, 1 mM DTT), grind at -20℃ for 4 min until no lumps remain; centrifuge at 4℃, 10000g for 5 min, discard the supernatant. Add 1 mL of washing buffer (80% acetone, 18% water, 2% saturated NaCl), grind at low temperature for 4 min, centrifuge under the same conditions and discard the supernatant. Add 1 mL of stock solution (100 mM HEPES, 1% SDC), grind at low temperature for 4 min to reconstitute, sonicate in a water bath at room temperature for 2 min, cool for 2 min, and sonicate again for 2 min; centrifuge at 4℃, 12000g for 10 min, and determine the protein concentration using the BCA method.

[0059] (2) Proteolysis and desalting: The sample was mixed with TCEP / CAA at a ratio of 20:1 and centrifuged. 0.5 μg / μL trypsin was prepared and added at a protein:enzyme ratio of 50:1. After vortexing, the mixture was digested at 42°C for 2–4 h and then temporarily stored at 4°C. The digested product was desalted using a C18 column, and the peptides were freeze-dried for later use.

[0060] (3) LC-MS / MS analysis: A Q-Exactive Plus mass spectrometer coupled with an EASY-nanoLC120 was used. 3 μL of sample was loaded onto an Acclaim PepMap C18 analytical column (75 μm × 25 cm); column temperature 40 °C, flow rate 300 nL / min, electrospray voltage 2 kV; nonlinear gradient separation was performed for 60 min: the B phase increased from 2% to 35% in 47 min, then to 100% in 1 min and maintained for 12 min. Mass spectrometry was performed in data-dependent acquisition mode: MS scan range m / z 200-2000, resolution 70000, AGC target 3e6, maximum injection time 50 ms; HCD-MS / MS resolution 17500, AGC target 1e5, maximum injection time 45 ms, collision energy 28%, dynamic exclusion 30 s.

[0061] Venn diagrams of each sample group and the control group ( Figure 5 The results showed that group 8C shared the most proteins with group Control (9355 types), exhibiting the highest similarity; while groups 6B and 8B shared slightly fewer proteins with group Control, but still maintained a high degree of consistency (approximately 99%). In terms of the number of specific proteins expressed, groups HB and Control each had the most specific proteins (HB had 35 types, and Control had 67 types), indicating that hydrolyzed peptides and intact proteins have different effects on protein expression in the spleen.

[0062] PCA analysis compared the differences in spleen protein expression profiles among different groups. Figure 6The results showed that group 8B was closest to the control group, indicating high proteomic similarity; while group 6B was farther from the control group, indicating lower similarity. Further comparison of different protein sources revealed that group 8C was closest to the control group in protein expression, while group 6B showed the lowest similarity. In the comparison between hydrolyzed and intact proteins, group HB showed the highest similarity to the control group, followed by groups 8B and 6B. Comprehensive analysis indicates that the proteomic expression of groups 8C and HB was closest to the control group, while the similarity between groups 8B and 6B was relatively low, suggesting that allergies induced by groups 8B and 6B can lead to changes in the protein composition of the spleen.

[0063] 7. Gut microbiota.

[0064] Total fecal DNA was extracted using the TIANamp Stool DNA kit. After passing Nanodrop assay, 20–30 ng of DNA was used as a template to amplify the 16S rRNA V3–V4 region using primers 341F / 806R. The amplified DNA was purified with magnetic beads and sequenced using Illumina MiSeq / Novaseq. Clustering was performed using VSEARCH (1.9.6) with 97% similarity. OTU species annotation was completed using RDPClassifier based on the Silva 138 database. Community composition and relative abundance of taxa were analyzed. Alpha diversity and heatmaps were analyzed online using MicrobiomeAnalyst, and beta diversity was calculated using R3.5.0 software.

[0065] The results showed that the overall distribution of the bacterial communities at the phylum, class, order, family, and genus levels was relatively similar, with no significant differences. Figure 7 However, at the species level, significant differences existed between the groups. The bovine milk protein treatment groups 6B and 8B showed significantly different gut microbiota structures compared to the Control group; while the goat milk protein group 8C was closer to the Control group, indicating a more similar gut microbiota composition.

[0066] Results of gut microbiota diversity analysis ( Figure 8 The results showed that group 8C exhibited high similarity to the control and CT groups in both α-diversity indices (Chao1 and Shannon) and β-diversity indices (Bray-Curtis), indicating a stable gut microbiota structure, stable ecological balance, and good intestinal compatibility. In contrast, groups 6B and 8B showed significant differences from the control group in both microbiota richness and structural composition.

[0067] At the genus level ( Figure 9In groups A and 6B, the relative abundance of *Mucispirillum* and *Negativibacillus* was increased, suggesting their association with intestinal barrier damage and inflammatory responses; while in group 8B, these two types of bacteria were less abundant, indicating less disturbance in the gut microbiota. Compared to 8B, group 8C was enriched with more probiotics such as *Blautia* and *Bacteroides*, showing a more stable microbiota structure and reflecting its good gut ecological regulation ability. Group 8B was relatively enriched with inflammatory bacteria *Enterococcus*, the increase of which may be related to increased IgE levels and mucosal immune activation. Overall, groups 8C, Control, and CT showed high clustering in the distribution of microbiota at the genus level, with consistent distribution of probiotics such as *Bacteroides*, *Akkermansia*, and *Lactobacillus*, indicating that their microbiota ecological stability was better than other treatment groups.

[0068] T-test analysis results ( Figure 9 As shown in B), the relative abundance of Alistipes, Monoglobus, and Harryflintia was significantly higher in group 8C than in group 8B. These genera are mostly anaerobic fermenting bacteria with a certain ability to synthesize short-chain fatty acids (such as propionic acid and butyric acid), suggesting that goat milk protein is more conducive to promoting the growth of probiotics and maintaining intestinal homeostasis.

[0069] 8. Short-chain fatty acids.

[0070] Weigh 0.2g of feces, add acetonitrile and 2-ethylbutyric acid internal standard, grind, extract at 4℃, centrifuge, and filter the supernatant through a 0.22μm filter membrane for analysis; prepare a series of acetic acid standard solutions and add an equal amount of internal standard; use a Thermo TRACE 1310 / ISQ7000 GC-MS system with a Nukoll capillary column, set the injection port, transfer line and ion source temperature, optimize the helium flow rate and column temperature program, EI ionization (70 eV), and use full scan qualitative and SIM mode quantitative analysis for butyric acid.

[0071] The production of short-chain fatty acids (SCFAs) depends on the fermentation of dietary fiber by gut microbiota and plays a crucial role in the self-repair of the intestinal mucosa and in maintaining the compactness and integrity of intestinal epithelial cells. Disorders of gut microbiota commonly found in allergy models may lead to reduced SCFA synthesis. Figure 10As shown in Figures A through F, compared to the Control group, the levels of various short-chain fatty acids, including histidine, butyric acid, valerate, and isovaleric acid, in the feces of group 6B were significantly lower than in other groups, especially compared to groups 8B and Control. This suggests that bovine milk protein may affect the intestinal barrier in mice and inhibit the production of short-chain fatty acids. In contrast, groups 8C and HB had higher levels of short-chain fatty acids, indicating that goat milk protein and hydrolysis treatment may be more beneficial for maintaining intestinal metabolic function and have relatively lower sensitization potential. Overall, the results suggest that the interference of bovine milk protein with the intestinal microecology may be related to its strong sensitization potential.

[0072] 9. Fecal metabolites.

[0073] Fecal samples were weighed at -80℃, and internal standard and 20% acetonitrile methanol solution were added and vortexed. The samples were then centrifuged at 12000 rpm for 10 min at 4℃. Extraction buffer was added again, and the samples were allowed to stand at -20℃ for 30 min, followed by centrifugation under the same conditions for 3 min. The supernatant was used for LC-MS / MS metabolomics analysis. A Vanquish UPLC system with an ACQUITY UPLC HSS T3 column was used at 40℃, a flow rate of 0.3 mL / min, and an injection volume of 2 μL. Gradient elution was performed using the corresponding mobile phase in both positive and negative ion modes. Mass spectrometry was performed using Q Exactive Focus with electrospray ionization (ESI) in both positive and negative ion modes, with parameters such as sheath gas and auxiliary gas flow rates and injection voltage set.

[0074] Principal component analysis of intestinal metabolites (PCA) showed that ( Figure 11 Different protein sources have a significant impact on the composition of intestinal metabolites in mice. Figure 11 The results in B show that, among the different milk source and whey casein ratio treatment groups, the 8C group clustered most closely with the Control group, indicating that its metabolites were similar to those of the control group; while the 6B and 8B groups were far from the Control group, indicating that they had a certain impact on the metabolites. Figure 11 Further comparison of the C group in the study revealed differences between hydrolyzed and intact proteins. The results showed that the HB group clustered closely with the Control group, indicating that the HB group was similar to the control group and did not induce sensitization or changes in metabolites. However, the 6B and 8B groups showed significant deviations from the Control group in their metabolite profiles, suggesting that the allergic reactions induced by these two groups would induce significant changes in intestinal metabolites. In conclusion, the goat milk protein 8C group and the hydrolyzed milk protein HB group showed greater advantages in regulating intestinal metabolic homeostasis.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hypoallergenic milk powder, characterized in that, By weight, the product includes the following ingredients: 95-105 parts whole goat milk powder, 550-580 parts demineralized goat whey powder D90, 160-180 parts compound vegetable oil, 25-35 parts refined olive oil, 20-30 parts goat butter, 3-5 parts fructooligosaccharide powder, 70-80 parts galactooligosaccharide syrup, 0.5-1.5 parts vitamin B fortifier for stage 1 milk powder, and 0.5-1.5 parts mineral E fortifier for stage 1 milk powder. 0.5-1.5 parts, calcium carbonate 2.5-3.5 parts, dicalcium phosphate 4-6 parts, sodium citrate 3-5 parts, potassium citrate 1-2 parts, magnesium sulfate 1-1.5 parts, choline chloride 1-1.5 parts, L-carnitine 0.08-0.12 parts, potassium chloride 5-8 parts, phospholipids 3-4 parts, inositol 0.2-0.4 parts, mixed tocopherol concentrate 0.2-0.4 parts, 10% w / w docosahexaenoic acid powder 9-10 parts, 10% w / w arachidonic acid powder 10-12 parts, compound 0.5% w / w lutein nutritional fortifier 0.2-0.4 parts, tricalcium phosphate 0.5-1.5 parts, Bifidobacterium animalis Bb-12 0.2~0.4 parts, L-ascorbic acid sodium 1~1.5 parts, compound nucleotide nutritional fortifier 0.2~0.4 parts, lactoferrin 0.5~0.7 parts.

2. The milk powder according to claim 1, characterized in that, The mass ratio of whey protein to casein in the milk powder is 70~90:30~10.

3. The milk powder according to claim 1, characterized in that, The mass ratio of whey protein to casein in the milk powder is 75~85:25~15.

4. The milk powder according to claim 1, characterized in that, The mass ratio of whey protein to casein in the milk powder is 80:

20.

5. The milk powder according to any one of claims 1 to 4, characterized in that, The milk powder is made from goat milk.

6. The use of the milk powder according to any one of claims 1 to 5 in the preparation of hypoallergenic products.

7. The use of the milk powder according to any one of claims 1 to 5 in the preparation of products that regulate intestinal flora.

8. The application according to claim 7, characterized in that, The milk powder maintains the stability of the gut microbiota and promotes the growth of probiotics.

9. The use of the milk powder according to any one of claims 1 to 5 in the preparation of products that maintain intestinal barrier function.

10. The application according to claim 9, characterized in that, The milk powder promotes the production of short-chain fatty acids.

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