A nutritional composition and its use in the manufacture of a product for alleviating milk protein allergy

Abstract

The present application relates to the field of food technology, and specifically discloses a kind of nutritional composition and its application in preparation product for relieving milk protein allergy.The present application is prepared by scientific compounding of deep hydrolysis whey protein, vegetable fat powder, breast milk oligosaccharide, nucleotide and bifidobacterium longum infantis i18, and the nutritional composition is prepared.The components in the composition are mutually synergistic, complementary, not only can effectively relieve the clinical symptoms of infant cow milk protein allergy, but also can guarantee the weight gain of milk protein allergy infants, simultaneously intervene from intestinal microecology, immune balance multidimensional, efficiently promote the body to establish stable, long-acting cow milk protein allergy immune tolerance.The present application is suitable for preparing related products suitable for infants, with the effect of relieving milk protein allergy and / or assisting in establishing milk protein immune tolerance.

Description

Technical Field

[0001] This invention relates to the field of food technology, and specifically discloses a nutritional composition and its application in the preparation of products that alleviate milk protein allergy. Background Technology

[0002] Cow's milk protein allergy is one of the most common types of food allergies in infants and young children, with a high clinical incidence. The clinical manifestations of cow's milk protein allergy are complex and diverse, primarily affecting the skin, gastrointestinal tract, and respiratory system. Symptoms often include eczema, gastrointestinal discomfort, and abnormal coughing and wheezing. It not only severely impacts the child's daily feeding, sleep quality, and normal life, but also interferes with the body's nutrient absorption in the long term, posing potential adverse effects and safety risks to the infant's growth and development.

[0003] Currently, the mainstream clinical intervention for infant milk protein allergy primarily involves dietary avoidance, which means replacing regular milk powder with extensively hydrolyzed protein formula or amino acid-based formula. This approach, by degrading large milk protein molecules and reducing protein antigenicity, can reduce the occurrence of allergic reactions to some extent and alleviate acute allergic symptoms in children. However, clinical application has revealed that relying solely on formula-based dietary intervention still presents several challenges, including persistent allergic symptoms in some children, slow establishment of allergy tolerance, and a high long-term recurrence rate. The therapeutic efficacy and tolerance-inducing ability of existing intervention programs remain significantly insufficient, necessitating the development of more efficient and optimized intervention strategies that can promote long-term desensitization and tolerance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a nutritional composition and its application in the preparation of products for alleviating milk protein allergy. This invention combines extensively hydrolyzed whey protein, vegetable fat powder, human milk oligosaccharides, and Bifidobacterium longum to prepare a nutritional composition. This composition exhibits significant synergistic effects in alleviating milk protein allergy, increasing the body weight of individuals with milk protein allergy, and establishing milk protein tolerance.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a nutritional composition, the raw materials for which it is made include extensively hydrolyzed whey protein, Bifidobacterium longum subsp. infantis i18, vegetable fat powder, human milk oligosaccharides and nucleotides. The molecular weight distribution of the deeply hydrolyzed whey protein is as follows: ≤1000Da 80%~92%, 1000Da~10000Da 10%~25%.

[0006] Bifidobacterium longum subsp. infantis i18 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 25, 2024. The accession number is CGMCC No. 31411, and the Latin name is *Bifidobacterium longum* subsp. infantis. This strain was first disclosed in Chinese patent document CN202510937932.7.

[0007] This invention achieves multidimensional functional complementarity in a low-allergenic nutritional matrix, intestinal barrier repair, and immune homeostasis regulation by scientifically compounding extensively hydrolyzed whey protein, vegetable fat powder, human milk oligosaccharides, nucleotides, and Bifidobacterium longum subsp. infantis i18. Specifically, extensively hydrolyzed whey protein significantly reduces the allergenicity of cow's milk protein, decreasing the occurrence of allergic reactions; Bifidobacterium longum subsp. infantis i18 regulates allergic immune responses, repairs the intestinal mucosal barrier, and improves the intestinal microecology of infants. Furthermore, the added vegetable fat powder and human milk oligosaccharides significantly enhance the stress resistance of Bifidobacterium longum subsp. infantis i18, effectively strengthening its acid resistance, bile salt tolerance, and Caco-2 cell adhesion and colonization ability, ensuring the survival efficiency and colonization effect of probiotics in the infant's intestine; nucleotides significantly improve the integrity of the intestinal epithelial barrier in a cow's milk protein allergy model, playing a positive role in reshaping the immune microenvironment and promoting a shift from pro-inflammatory to tolerant microenvironment.

[0008] The nutritional composition provided by this invention features synergistic and complementary components that not only effectively alleviate the clinical symptoms of milk protein allergy in infants, but also intervene from multiple dimensions, including gut microbiota and immune balance, to efficiently promote the establishment of stable and long-lasting milk protein allergy tolerance. This nutritional composition not only effectively alleviates the clinical symptoms of milk protein allergy in infants, but also ensures the weight gain of infants with milk protein allergy.

[0009] Preferably, in addition to extensively hydrolyzed whey protein and Bifidobacterium longum subsp. infantis i18, the nutritional composition also includes vegetable fat powder, human milk oligosaccharides, and nucleotides.

[0010] Preferably, the molecular weight distribution of the deeply hydrolyzed whey protein is: ≤1000Da 80%~90%, 1000Da~3000Da 1%~20%, and 3000Da~10000Da 0~5%.

[0011] For example, the molecular weight distribution of the deeply hydrolyzed whey protein is: ≤1000Da 86.4%, 1000Da~3000Da 11.5%, 3000Da~10000Da 2.1%.

[0012] Preferably, the vegetable fat powder includes at least two of glucose syrup, edible vegetable blended oil, sodium octenyl succinate starch, mono- and diglycerides of fatty acids, or sodium ascorbate; the human milk oligosaccharide includes at least one of 2-fucoyl lactose or lactose-N-neotetrasaccharide.

[0013] For example, the edible vegetable blend oil includes sunflower seed oil, low erucic acid rapeseed oil, coconut oil, mixed tocopherol concentrate, and ascorbyl palmitate.

[0014] For example, the mass ratio of 2-fucoyl lactose or lactose-N-neotetrasaccharide is (0.6~1.0):(0.3~0.5).

[0015] More preferably, the viable count of the *Bifidobacterium longum* in the nutritional composition is 1 × 10⁻⁶. 6 CFU / g ~5×10 7 CFU / g.

[0016] More preferably, the mass ratio of the deeply hydrolyzed whey protein, vegetable fat powder, human milk oligosaccharides, nucleotides and Bifidobacterium longum is (14.5~16.5):(60~66):(1~1.5):(0.02~0.08):(0.003~0.4).

[0017] Preferably, the nutritional composition further includes at least one of the following: syrup, complex minerals, complex vitamins, long-chain polyunsaturated fatty acids, or amino acid composition.

[0018] For example, the composite minerals include sodium chloride, potassium chloride, potassium citrate, copper sulfate, magnesium carbonate, ferric pyrophosphate, zinc citrate, manganese sulfate, calcium carbonate, calcium hydrogen phosphate, potassium iodide, and sodium selenite in a mass ratio of (90~95):(460~480):(175~185):(345~355):(30~38):(8~12):(2~5):(0.1~0.3):(50~60):(450~480):(0.05~0.15):(0.01~0.02). The compound vitamins comprise, in a mass ratio of (530~550):(8~15):(530000~550000):(25~35):(440~460):(620~720):(400~420):(0.1~0.3):(5600~5800):(50~150):(3500~3700):(4800~5200):(10~30):(120000~140000), retinyl acetate, cholecalciferol, dl-α-tocopherol acetate, phytonabinone, thiamine nitrate, riboflavin, pyridoxine hydrochloride, cyanocobalamin, nicotinamide, folic acid, D-calcium pantothenate, sodium L-ascorbate, D-biotin, and choline tartrate; The long-chain polyunsaturated fatty acids include docosahexaenoic acid and arachidonic acid in a mass ratio of (0.5~1.5):(0.5~1.5); The amino acid composition comprises phenylalanine, tyrosine, histidine, and tryptophan in a mass ratio of (0.1~0.4):(0.1~0.3):(0.08~0.15):(0.02~0.1).

[0019] More preferably, the mass ratio of the deeply hydrolyzed whey protein, syrup, complex minerals, complex vitamins, long-chain polyunsaturated fatty acids and amino acid composition is (14.5~16.5):(10~15):(2~4):(0.5~1):(1~3):(0.5~2).

[0020] Secondly, the present invention provides the application of the above-mentioned nutritional composition in the preparation of products that alleviate milk protein allergy.

[0021] Thirdly, the present invention provides the use of the above-mentioned nutritional composition in the preparation of products that alleviate milk protein allergy and help establish milk protein tolerance.

[0022] Fourthly, the present invention provides a product for relieving milk protein allergy, the product comprising the nutritional composition provided in the first aspect.

[0023] Fifthly, the present invention provides a product that alleviates milk protein allergy and helps establish milk protein tolerance, said product comprising the nutritional composition provided in the first aspect.

[0024] This invention scientifically combines extensively hydrolyzed whey protein, vegetable fat powder, human milk oligosaccharides, nucleotides, and Bifidobacterium longum subsp. infantis i18 to prepare a nutritional composition. The components in this composition work synergistically and complement each other, effectively alleviating the clinical symptoms of milk protein allergy in infants and ensuring their weight gain. Simultaneously, it intervenes from multiple dimensions, including gut microbiota and immune balance, to efficiently promote the establishment of stable and long-lasting milk protein allergy immune tolerance. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The following is an example of the changes in body weight of mice in different groups under different intervention methods in Example 1 of the present invention; Figure 2 The above are the statistical results of allergy symptom scores of mice in different groups in Example 1 of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] The vegetable fat powder used in this invention was purchased from Qingdao Haizhiyuan Life Technology Co., Ltd.; each ton of vegetable fat powder is prepared by spray drying after mixing the following components: 661.3 kg of glucose syrup, 390 kg of edible vegetable blended oil (specifically including 234.19 kg of sunflower seed oil, 96.67 kg of low erucic acid rapeseed oil, 58.63 kg of coconut oil, 0.45 kg of mixed tocopherol concentrate, and 0.06 kg of ascorbate palmitate), 95 kg of sodium octenyl succinate starch, 16 kg of mono- and diglycerides of fatty acids, and 3 kg of sodium ascorbate.

[0029] The multivitamins were purchased from DSM FineMeyers Nutrition (Shanghai) Co., Ltd.; each 1 kg of multivitamins contains the following vitamins by weight: retinyl acetate 546 μg, cholecalciferol 13 μg, dl-α-tocopherol acetate 546 mg, phytonabinone 32 μg, thiamine nitrate 447 μg, riboflavin 696.7 μg, pyridoxine hydrochloride 405 μg, cyanocobalamin 0.2 μg, nicotinamide 5723 μg, folic acid 100 μg, D-calcium pantothenate 3601 μg, L-sodium ascorbate 5 mg, D-biotin 15 μg, and choline tartrate 129.2 mg / kg; The compound minerals were purchased from DSM FineMeyers Nutrition (Shanghai) Co., Ltd.; each kg of compound minerals contains the following raw materials by weight: sodium chloride 93.753 mg, potassium chloride 468.98 mg, potassium citrate 180.67 mg, copper sulfate 351.600 mg, magnesium carbonate 34.700 mg, ferric pyrophosphate 10.817 mg, zinc citrate 3.9 mg, manganese sulfate 121 μg, calcium carbonate 54.698 mg, calcium hydrogen phosphate 464.585 mg, potassium iodide 99 μg, and sodium selenite 12.6 μg. Human milk oligosaccharides, purchased from Glycom Manufacturing A / S; Extensively hydrolyzed whey protein was purchased from Dato Dairy Products (Shanghai) Co., Ltd. Its molecular weight distribution is as follows: ≤1000Da 86.4%, 1000Da~3000Da 11.5%, 3000Da~10000Da 2.1%.

[0030] Example 1 This invention provides a nutritional composition comprising the following ingredients by weight: 15.2g of extensively hydrolyzed whey protein, 63.5g of vegetable fat powder, 1.2g of human milk oligosaccharides, 0.05g of nucleotides, and 33mg of Bifidobacterium longum subsp. infantis i18 bacterial powder. The live bacteria count of Bifidobacterium longum infantis subsp. i18 powder was 3×10⁻⁶. 11 CFU / g; The human milk oligosaccharide consists of 0.8 g of 2-fucoyllactose and 0.4 g of lactose-N-neotetrasaccharide.

[0031] Example 2 This invention provides a nutritional composition comprising the following ingredients by weight: 14.5g of extensively hydrolyzed whey protein, 66g of vegetable fat powder, 1g of human milk oligosaccharides, 0.02g of nucleotides, and 0.03g of Bifidobacterium longum subsp. infantis i18 bacterial powder. The live bacteria count of Bifidobacterium longum infantis subsp. i18 powder was 3×10⁻⁶. 10 CFU / g; The human milk oligosaccharide consists of 0.6 g of 2-fucoyllactose and 0.4 g of lactose-N-neotetrasaccharide.

[0032] Example 3 This invention provides a nutritional composition comprising the following ingredients by weight: 16.5g of extensively hydrolyzed whey protein, 60g of vegetable fat powder, 1.5g of human milk oligosaccharides, 0.08g of nucleotides, and 0.4g of Bifidobacterium longum subsp. infantis i18 bacterial powder; The live bacteria count of Bifidobacterium longum subsp. infantis i18 powder was 3×10⁻⁶. 9 CFU / g; The human milk oligosaccharide consists of 1g of 2-fucoyllactose and 0.5g of lactose-N-neotetrasaccharide.

[0033] Example 4 This invention provides a nutritional composition comprising the following ingredients by weight: 15.2g of extensively hydrolyzed whey protein, 63.5g of vegetable fat powder, 14.30g of solid corn syrup, 2.5g of complex minerals, 0.75g of complex vitamins, 0.9g of docosahexaenoic acid, 1g of arachidonic acid, 0.6g of an amino acid composition, 1.2g of human milk oligosaccharides, 0.05g of nucleotides, and 33mg of Bifidobacterium longum subsp. infantis i18 bacterial powder; The live bacteria count of Bifidobacterium longum subsp. infantis i18 powder was 3×10⁻⁶. 11 CFU / g; The human milk oligosaccharide is composed of 0.8 g of 2-fucoyllactose and 0.4 g of lactose-N-neotetrasaccharide; The amino acid composition consists of 0.29 g of L-phenylalanine, 0.14 g of L-tyrosine, 0.11 g of L-histidine, and 0.06 g of L-tryptophan.

[0034] Example 5 This invention provides a nutritional composition comprising the following ingredients by weight: 15.0g of extensively hydrolyzed whey protein, 63.7g of vegetable fat powder, 13.53g of solid corn syrup, 3g of complex minerals, 1g of complex vitamins, 0.5g of docosahexaenoic acid, 0.5g of arachidonic acid, 1.5g of an amino acid composition, 1.2g of human milk oligosaccharides, 0.05g of nucleotides, and 0.02g of Bifidobacterium longum subsp. infantis i18 bacterial powder; The live bacteria count of Bifidobacterium longum subsp. infantis i18 powder was 1×10⁻⁶. 10 CFU / g; The human milk oligosaccharide is composed of 0.8 g of 2-fucoyllactose and 0.4 g of lactose-N-neotetrasaccharide; The amino acid composition consists of 0.72g of L-phenylalanine, 0.35g of L-tyrosine, 0.28g of L-histidine, and 0.15g of L-tryptophan.

[0035] Comparative Example 1 The present invention provides a nutritional composition comprising the following ingredients by weight: 15.2g of extensively hydrolyzed whey protein, 63.5g of vegetable fat powder, 1.2g of human milk oligosaccharides, 0.05g of nucleotides, and 33mg of Bifidobacterium longum subsp. infantis i19 bacterial powder. The live bacteria count of Bifidobacterium longum infantis subsp. i19 powder was 3×10⁻⁶. 11CFU / g; Bifidobacterium longum subsp. infantis i19 is deposited at Junlebao Culture Collection Center and first disclosed in Chinese Invention Patent CN202511193067.6; The human milk oligosaccharide consists of 0.8 g of 2-fucoyllactose and 0.4 g of lactose-N-neotetrasaccharide.

[0036] Comparative Example 2 The present invention provides a nutritional composition comprising the following ingredients by weight: 15.2g of extensively hydrolyzed whey protein, 63.5g of vegetable fat powder, 1.2g of human milk oligosaccharides, and 0.05g of nucleotides; The human milk oligosaccharide is composed of 0.8g of 2-fucoyllactose and 0.4g of lactose-N-neotetrasaccharide.

[0037] Comparative Example 3 The present invention provides a nutritional composition comprising the following ingredients by weight: 15.2g of extensively hydrolyzed whey protein, 63.5g of vegetable fat powder, 1.2g of human milk oligosaccharides, and 33mg of Bifidobacterium longum subsp. infantis i18 powder. The live bacteria count of Bifidobacterium longum subsp. infantis i18 powder was 3×10⁻⁶. 11 CFU / g; The human milk oligosaccharide consists of 0.8 g of 2-fucoyllactose and 0.4 g of lactose-N-neotetrasaccharide.

[0038] Comparative Example 4 The present invention provides a nutritional composition in a comparative example, the types and amounts of raw materials of which are basically the same as those in Example 4, the only difference being that Bifidobacterium longum subsp. infantis i18 powder is not added.

[0039] Example 1 This invention provides the effect of the nutritional composition provided in Example 4 on the body weight of sensitized model mice, the details of which are as follows: I. Mouse experimental grouping and construction of allergy model 1. Four-week-old SPF-grade female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of acclimatization, all mice were randomly divided into four groups of 10 mice each, as follows: Normal control group (CON): No sensitization was performed from day 0 to 28. From day 29 to 57, 0.2 mL of PBS buffer was administered by gavage once a day. Allergy model group (MOD): Allergy model was established from 0 to 28 days. From 29 to 57 days, 0.2 mL of PBS buffer was administered by gavage once a day. Comparative Example 4 Intervention Group (referred to as EHF group): An allergy model was established from 0 to 28 days. From 29 to 57 days, 0.2 mL of the nutritional composition provided in Comparative Example 4 and a solution prepared with PBS buffer were administered by gavage once a day. The gavage dose of the nutritional composition was 1 g / kg / day. Example 4 Intervention Group (referred to as EHF+BB group): An allergy model was established from 0 to 28 days. From 29 to 57 days, 0.2 mL of the nutritional composition provided in Example 4 and a solution prepared with PBS buffer were administered by gavage once a day. The gavage dose of the nutritional composition was 1 g / kg / day.

[0040] 2. Methods for constructing allergy models The sensitization method was intraperitoneal injection. Except for the CON group, mice in other groups were sensitized by intraperitoneal injection of 0.2 mL of sensitizing agent (0.1 mg of bovine milk β-lactoglobulin dissolved in 100 μL of sterile physiological saline, and then emulsified with 100 μL of complete Freund's adjuvant in a 1:1 ratio) on days 1, 7, 21 and 28 of the experiment.

[0041] The CON group was injected with an equal volume of sterile saline at the same time point. After the last sensitization (day 28) confirmed successful model establishment, mice in each group began a 4-week intervention, once a day, until the end of the experiment.

[0042] All mice were housed in an SPF environment with a constant temperature (25 ± 2℃) and relative humidity (50 ± 5%), with a 12-hour light / dark cycle, and had free access to standard sterilized basic feed (Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd., XTI01FZ-011) and drinking water.

[0043] II. Sample Collection and Processing Procedure During the experiment, the mice were weighed and their weight data were recorded weekly. The symptoms of the mice in each group were observed during the experimental period, and the allergic symptoms of the mice in different groups were scored.

[0044] III. Experimental Results 1. Weight The changes in body weight of mice in each group under different intervention methods are as follows: Figure 1 As shown.

[0045] Depend on Figure 1 It can be seen that during the 28-day intervention period, the body weight of mice in the CON group, EHF group, and EHF+BB group all showed a steady upward trend; while the body weight gain of mice in the MOD group was significantly lagging behind and remained at a low level. This indicates that the allergic state interfered with the normal growth process of mice, which may be related to the energy consumption of immune activation caused by allergies and the impact of gastrointestinal dysfunction on nutrient absorption.

[0046] Furthermore, it can be seen that intervention using only the nutritional composition provided in Comparative Example 4 has a certain effect on improving weight gain, but the combined use of Bifidobacterium longum subsp. infantis i18 (i.e., Example 4) on the basis of the nutritional composition provided in Comparative Example 4 has a more prominent effect. On day 28, the weight of mice in the EHF+BB group was significantly higher than that in the MOD group and the EHF group, and the weight level was close to that of the control group.

[0047] The above results demonstrate that the nutritional composition provided in Example 4 of this invention can not only regulate the disordered immune response under allergic conditions, but also help restore the body's metabolic and nutrient absorption homeostasis, and improve the growth and development restriction problems associated with allergies.

[0048] 2. Allergy Symptom Score This invention investigated the effects of different intervention methods on allergy symptom scores in mice. The allergy symptom scoring criteria are shown in Table 1 below.

[0049] Table 1 The statistical results of allergy symptom scores of mice in different groups are as follows: Figure 2 As shown.

[0050] Depend on Figure 2 It was found that the CON group mice did not exhibit any allergic symptoms, with a symptom score of 0; while the MOD group mice showed a significant increase in allergic symptom scores, exhibiting obvious allergic reactions such as rapid breathing and decreased activity. After intervention, the allergic symptom scores of the EHF and EHF+BB groups were significantly lower than those of the MOD group, indicating that both treatments could alleviate allergic symptoms to some extent; among them, the EHF+BB group had the lowest score, approaching the level of the normal control group, and its intervention effect was the most prominent.

[0051] The above results indicate that the nutritional composition provided by this invention has a positive effect on allergic symptoms in allergic mice. Extensively hydrolyzed whey protein may reduce the risk of allergic triggering by breaking down sensitizing proteins, while Bifidobacterium longum subsp. infantis i18 may alleviate allergic reactions by regulating gut microbiota and enhancing immune tolerance; when used synergistically, the effect of regulating allergic symptoms is more significant, demonstrating better improvement potential.

[0052] Example 2 Bifidobacterium longum subsp. infantis is an important species involved in the early formation of the infant gut microbiota. Whether this strain can reach the intestine and colonize in live bacterial form is a key condition for its aforementioned allergy-improving effects. Adhesion of the strain to the intestinal epithelium is an important prerequisite for its retention in the intestine, thereby exerting immune regulation and intestinal barrier protection functions; the better the adhesion, the higher the strain's retention potential in the intestine, and the more sustainably it can mediate immune regulation and barrier repair effects. Therefore, developing compositions that can enhance the gastric acid and bile salt tolerance of Bifidobacterium longum subsp. infantis and improve its intestinal colonization performance is of great significance for improving various related symptoms in individuals with milk protein allergy.

[0053] 1. Acid and bile salt resistance test methods This invention investigated the acid and bile salt resistance of Bifidobacterium longum subsp. infanti i18 and compared its performance with that of Bifidobacterium longum subsp. infanti i19 under the same conditions.

[0054] Take 1 mL of activated third-generation *Bifidobacterium longum* subsp. *infantii* i18 or i19 bacterial culture, centrifuge at 10000 r / min for 2 min to collect the cells, wash three times with PBS buffer, add 1 mL of physiological saline solution containing 1% compound functional components, and then inoculate into modified MRS liquid medium at pH 2.5 or 5 g / L bile salts, respectively. Incubate anaerobically at 37℃ for 4 h. Measure the viable cell count at 2 h and 4 h using the plate count method, and evaluate the strain's tolerance using the survival rate relative to 0 h. The modified MRS liquid medium is MRS liquid medium supplemented with 0.05% cysteine.

[0055] 2. Cell adhesion assay method This invention investigated the adhesion ability of Bifidobacterium longum infant subspecies i18 to Caco-2 cells and compared it with the adhesion performance of Bifidobacterium longum infant subspecies i19 under the same conditions.

[0056] Bifidobacterium longum subsp. infantis i18 or i19 was cultured at 37°C for 20 h and activated three times. After centrifugation, the bacterial cells were washed twice with PBS and resuspended in PBS. After centrifugation again, the cells were resuspended in 100 mL of DMEM medium containing 1 mL of 10% (w / v) physiological saline solution of the compound functional component, which is serum-free, antibiotic-free, and free of antibiotics.

[0057] A sterile round coverslip is heated by moving it back and forth near an alcohol lamp flame. The heated coverslip is then placed into a 6-well plate (sterile coverslips are added directly to the 6-well plate). The counted Caco-2 cell suspension (10 μL) is then aspirated. 5Add 300 μL of Caco-2 cells / mL to a coverslip, place the 6-well plate in a CO2 incubator, and incubate for 30-60 min. Remove the plate, add approximately 2.5 mL of DMEM complete culture medium, and place it back in the CO2 incubator. Observe until the cells have completely covered the coverslip. Wash twice with PBS to remove excess unattached Caco-2 cells.

[0058] Add 2 mL of the prepared bacterial suspension (adjusting the concentration of Bifidobacterium longum subsp. infantis i18 to 10). 7 Add either CFU / mL or an equal volume of DMEM medium (as a control) to the above-mentioned 6-well plates containing Caco-2 cells, and incubate at 37°C for 2 hours in a CO2 incubator. Wash three times with sterile PBS to remove unbound Bifidobacteria, fix with methanol for 30 minutes, Gram stain (0.5% crystal violet), and randomly observe 20 fields under an oil immersion microscope (1000×) to calculate the total number of Bifidobacteria adhering to Caco-2 cells.

[0059] Bifidobacterium longum subspecies i19 is deposited at the Junlebao Culture Collection Center and was first disclosed in Chinese Invention Patent CN202511193067.6.

[0060] The composition and mass percentage of the different composite functional components are shown in Table 2 below. The content of each component listed in the table represents the amount of the corresponding raw material used in the composition per 1 kg or 1 t of product.

[0061] Table 2 3. Experimental Results The effects of culture media containing different composite functional components on the gastric acid resistance, bile salt resistance, and Caco2 cell adhesion ability of Bifidobacterium longum subsp. i18 and Bifidobacterium longum subsp. i19 are shown in Table 3.

[0062] Table 3 * indicates that, under the same experimental conditions, the groups with different added compound functional ingredients were compared with those without added compound functional ingredients, and P < 0.05; # indicates that under the same experimental conditions, the i18 group and the i19 group were compared, and P < 0.05.

[0063] As shown in Table 3, under the same compound functional ingredient and dosage, *Bifidobacterium longum* subsp. infantis i18 exhibits better resistance to gastric acid and bile salts compared to *Bifidobacterium longum* subsp. infantis i19. Furthermore, compared to other compound functional ingredients, plant fat powder helps *Bifidobacterium longum* subsp. infantis i18 better tolerate gastric acid and bile salt stimulation, while human milk oligosaccharides enhance the cell adhesion ability of *Bifidobacterium longum* subsp. infantis i18, thereby helping it colonize the intestines and exert its effects.

[0064] Example 3 This invention further investigated the effects of different nutritional compositions on the intestinal barrier and immune microenvironment, and evaluated the role of different nutritional compositions in establishing immune tolerance in a milk protein allergy cell model. The specific details are as follows: I. Preparation of cells, culture medium, bacterial suspension, and experimental grouping 1. Experimental cells: Caco-2 cell line.

[0065] 2. DMEM complete medium: DMEM medium is supplemented with 2mM L-glutamine, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0066] 3. Preparation of bacterial suspension: Bifidobacterium longum subsp. infantis i18 and i19 bacterial suspensions were resuspended in DMEM medium, and the bacterial suspension concentration was adjusted to 10. 7 CFU / mL, for later use; Preparation of nucleotide solution: Take nucleotides and dilute them with DMEM medium to a concentration of 0.1 mg / mL for later use.

[0067] 4. Experimental grouping: The test composition group was intervened with the nutrient composition provided in the embodiments or comparative examples of the present invention (nutrient composition + Caco-2 cell slurry); at the same time, zeroing wells (DMEM medium without Caco-2 cells), blank control group (containing only Caco-2 cell slurry), strain control group (Caco-2 cell slurry + i18 or Caco-2 cell slurry + i19) were set up, and the cell culture conditions of each group were kept consistent.

[0068] II. Cell Plating and Model Construction 1. Construction of an intestinal epithelial cell differentiation model First, the Transwell culture plates were pre-equilibrated: DMEM complete medium was added to the multi-well plate and the Transwell nest, and the culture plates were placed in a cell culture incubator at 37°C for 1 hour to complete the pre-equilibration of the system.

[0069] Caco-2 cells in the logarithmic growth phase were passaged and cultured. After cell counting, they were inoculated at a rate of 5.3 × 10⁻⁶ cells / year. 5The cells were seeded at a density of cells / mL in 6-well Transwell chambers (pore size 0.4 μm; membrane area 4.67 cm²). 2 (Corning), in which 1 mL of cell suspension and 0.5 mL of DMEM complete medium are added to the upper chamber, and 2 mL of DMEM complete medium is added to the lower chamber. The cells are then incubated at 37°C in a 5% CO2 incubator. The medium is changed every other day after inoculation, and then every other day thereafter, continuing the culture until the cells differentiate and mature.

[0070] The cell plating setup is shown in Table 4 below, and a cell-free blank zeroing well (Ctrl well) is also included for subsequent resistance baseline correction. In this invention, two 6-well Transwell culture plates are set up in this manner.

[0071] Table 4 Transmembrane resistance was measured every 2 days using a cell resistance meter to dynamically monitor the integrity of the Caco-2 cell monolayer barrier. The resistance of the cell-free blank wells (Ctrl wells) was measured first, followed by the resistance of each sample well sequentially. Each group was measured three times, and the average value was calculated. Cells were continuously cultured until day 10 after plating, when the transmembrane resistance of the monolayer cell layer stabilized at 700 Ω·cm. 2 When the values ​​fluctuate slightly and show only a slight downward trend, and the overall condition is relatively stable, the intestinal epithelial barrier model is considered to be mature and ready for subsequent experiments.

[0072] The resistance detection process includes the following steps: Before measuring the resistance value, immerse the test electrode in 75% alcohol for 15 minutes. During measurement, insert one end of the electrode into the cell culture medium in the lower chamber of the Transwell, and the other end into the upper chamber, taking care to prevent convection of the culture medium between the two chambers to avoid short circuits. Read and record the value displayed on the instrument screen. The formula for calculating the resistance value is as follows: Transmembrane resistance TEER = (R 样品孔 -R 空白孔 ) × 4.67 In the formula, the unit of transmembrane resistance is Ω·cm. 2 R 样品孔 R represents the resistance value of the orifice in the experimental group. 空白孔 The resistance value of the blank control group well is 4.67, which is the effective membrane area of ​​the 6-well Transwell culture plate, and the fixed parameters of this type of chamber are 4.67.

[0073] 2. Cell treatment Cells were randomly divided into four main groups, with the probiotic or nutritional combination intervention group containing seven subgroups. Each group was intervened in strict accordance with the following time sequence: Blank control group: Normal culture medium was added to the above non-Ctrl wells and cultured for a total of 28 hours.

[0074] Allergy model group: The inducing agent sodium dextran sulfate (DSS) was first added to the non-Ctrl well chambers to a final concentration of 1% w / v, and the pretreatment was carried out for 4 h after adding the inducing agent; then β-lactoglobulin was added to a final concentration of 1 mg / mL and incubated for a total of 24 h.

[0075] Positive control group: The non-Ctrl well chambers were pretreated with anti-inflammatory drug (dexamethasone at a final concentration of 1 μM) for 4 h; then the inducing agent DSS (at a final concentration of 1% w / v) was added and pretreated for 4 h; then β-lactoglobulin (at a final concentration of 1 mg / mL) was added and incubated for a total of 24 h.

[0076] Probiotic or nutritional composition intervention group (7 groups in total): Probiotics or nutritional composition were first added to the non-Ctrl well chambers for 4 hours of pretreatment; then DSS inducer was added (to a final concentration of 1% w / v) and pretreated for 4 hours; then β-lactoglobulin was added (to a final concentration of 1 mg / mL) and incubated for a total of 24 hours.

[0077] The probiotic or nutritional composition intervention group comprised seven subgroups, designated as i18 single strain group, i19 single strain group, Example 1 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and nucleotide group. The only difference between Example 1 group and Comparative Example 3 group was that Comparative Example 3 group did not contain nucleotides; the types and concentrations of the remaining components were identical. The final concentrations of the bacterial agents or main active ingredients in each subgroup are shown in Table 5.

[0078] Table 5 III. Indicator Testing This invention uses a resistance meter to measure the transmembrane resistance (TEER) of each pore 4 h after DSS induction and before β-lactoglobulin stimulation, 12 h after β-lactoglobulin stimulation and 24 h after β-lactoglobulin stimulation to assess the integrity of the intestinal epithelial barrier. In addition, cell culture supernatant was collected 24 hours after β-lactoglobulin stimulation. After centrifugation at 3000 rpm for 10 min at 4°C to remove cell debris, the concentrations of TSLP, IL-33, IL-10 and TGF-β in the supernatant were detected by ELISA. Immune microenvironment factors in each group were also detected.

[0079] All experiments in this invention were independently repeated at least three times. One-way ANOVA was used for comparisons among multiple groups, and P < 0.05 was considered statistically significant.

[0080] IV. Experimental Results 1. Assessment of intestinal epithelial barrier integrity Table 6 shows the transmembrane resistance (TEER) measurements of different groups 4 hours after DSS induction and before, 12 hours after, and 24 hours after β-lactoglobulin stimulation.

[0081] Table 6 Note: Different letter subscripts after the data in the same column indicate significant differences (P<0.05).

[0082] The repair rate is used to assess the integrity of the intestinal epithelial barrier, and its determination formula is as follows: Repair rate = [(TEER 实验组刺激24h后 -TEER 过敏模型组刺激24h后 ) / TEER 过敏模型组刺激24h后 ×100%. The repair rates in Table 6 are all compared with the allergy model group.

[0083] As shown in Table 6, from 4 hours after DSS induction and before β-lactoglobulin stimulation to 24 hours after β-lactoglobulin stimulation, the TEER value of cells in the allergy model group increased from the initial 712.8 Ω·cm. 2 It dropped sharply to 345.3 Ω·cm 2 (P<0.001) indicates that the tight junction structure of the intestinal epithelium was severely damaged and its permeability was significantly increased.

[0084] While adding Bifidobacterium longum subsp. infantis i18 or i19 alone could alleviate the decline in TEER values ​​to some extent, its repair capacity was limited compared to the allergy model group. However, the nutritional composition of Example 1 showed a significant protective effect, with the TEER value recovering to 549.2 Ω·cm 24 hours after DSS induction and β-lactoglobulin stimulation following the nutritional composition intervention. 2 The levels were significantly higher than those of the single-strain group. Notably, the repair effect of the nutritional composition provided in Example 1 of this invention was not statistically different from that of the dexamethasone positive control group. This result suggests that the combination of Bifidobacterium longum subsp. infantis with extensively hydrolyzed whey protein, HMOs, and nucleotides may promote the reassembly of tight junction proteins through a synergistic mechanism, thereby physically rebuilding the intestinal barrier.

[0085] Furthermore, a comparison of Example 1, Comparative Example 3, and the nucleotide group also shows that nucleotides may significantly promote the improvement of intestinal barrier function by probiotics and deeply hydrolyzed whey protein.

[0086] 2. Detection of immune microenvironment factors Table 7 shows the results of the determination of immune factors such as TSLP, IL-33, IL-10 and TGF-β in the supernatant of different groups 24 hours after allergen stimulation.

[0087] Table 7 Note: Different letter subscripts after the data in the same column indicate significant differences (P<0.05).

[0088] Intestinal epithelial cells are not only a physical barrier but also a sensor for immune regulation. As shown in Table 7, the levels of TSLP and IL-33 epithelial warning in the cell supernatant of the allergy model group soared to 210.8 pg / mL and 165.5 pg / mL, respectively, accompanied by the depletion of secretion of anti-inflammatory factors IL-10 and TGF-β.

[0089] Unlike the single-strain group, which only partially inhibited TSLP secretion, the nutritional composition provided in Example 1 significantly inhibited TSLP and IL-33 levels to 69.1 pg / mL and 62.1 pg / mL, respectively, demonstrating a significantly better inhibitory effect than the single-strain group. More importantly, the nutritional composition provided in Example 1 exhibited unique advantages in immune tolerance indicators: its induced IL-10 secretion was significantly higher than that of the allergy model group; the composition provided in Example 1 induced TGF-β secretion not only significantly higher than that of the allergy model group but also slightly higher than that of the blank control group. This indicates that the composition not only passively inhibited inflammation but also actively induced the secretion of tolerance cytokines by intestinal epithelial cells. The nutritional composition provided by this invention intervenes in milk protein allergy and establishes a tolerance mechanism through a dual mechanism of anti-inflammatory and tolerance-promoting action.

[0090] A comparison of Example 1, Comparative Example 3, and the nucleotide group also shows that nucleotides can work with probiotics and deeply hydrolyzed proteins to reduce allergic reactions.

[0091] In summary, the nutritional composition provided by this invention can effectively improve clinical symptoms related to cow's milk protein allergy in infants and young children, while ensuring normal growth and weight gain, and avoiding growth retardation caused by cow's milk protein allergy. The components in this composition work synergistically to target and intervene from multiple dimensions, including repairing the intestinal barrier, regulating the intestinal microecology, and reshaping the body's immune balance. This effectively improves immune disorders in allergic states, helps infants and young children establish stable and long-lasting immune tolerance to cow's milk protein allergy, and fundamentally improves their allergic constitution.

[0092] The nutritional composition provided by this invention is highly safe and adapted to the physiological development characteristics of infants and young children. It can simultaneously alleviate allergy symptoms, ensure growth and development, and build immune tolerance. It can effectively solve the pain points of intervention for infant milk protein allergy, provide nutritional and immune protection for the healthy growth of allergic infants and young children, and has broad market application prospects and important social value. It can be widely used to prepare related products suitable for infants and young children that have the effect of relieving milk protein allergy and / or assisting in the establishment of milk protein immune tolerance.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nutritional composition, characterized in that, The raw materials used to make it include extensively hydrolyzed whey protein, Bifidobacterium longum subsp. infantis i18, vegetable fat powder, human milk oligosaccharides, and nucleotides; The molecular weight distribution of the deeply hydrolyzed whey protein is as follows: ≤1000Da 80%~92%, 1000Da~10000Da 10%~25%; The accession number of the Bifidobacterium longum subspecies i18 is CGMCC No. 31411.

2. The nutritional composition according to claim 1, characterized in that, The molecular weight distribution of the deeply hydrolyzed whey protein is as follows: ≤1000Da 80%~90%, 1000Da~3000Da 1%~20%, 3000Da~10000Da 0~5%.

3. The nutritional composition according to claim 2, characterized in that, The vegetable fat powder comprises at least two of glucose syrup, blended edible vegetable oil, sodium octenyl succinate starch, mono- and diglycerides of fatty acids, or sodium ascorbate; and / or The human milk oligosaccharide includes at least one of 2-fucoyllactose or lactose-N-neotetrasaccharide.

4. The nutritional composition according to claim 3, characterized in that, The viable count of the *Bifidobacterium longum* in the nutritional composition is 1 × 10⁻⁶. 6 CFU / g ~5×10 7 CFU / g; and / or The mass ratio of the deeply hydrolyzed whey protein, vegetable fat powder, human milk oligosaccharides, nucleotides and Bifidobacterium longum is (14.5~16.5):(60~66):(1~1.5):(0.02~0.08):(0.003~0.4).

5. The nutritional composition according to any one of claims 1 to 4, characterized in that, The nutritional composition also includes at least one of the following: syrup, complex minerals, complex vitamins, long-chain polyunsaturated fatty acids, or amino acid composition.

6. The nutritional composition according to claim 5, characterized in that, The mass ratio of the deeply hydrolyzed whey protein, syrup, complex minerals, complex vitamins, long-chain polyunsaturated fatty acids and amino acid composition is (14.5~16.5):(10~15):(2~4):(0.5~1):(1~3):(0.5~2).

7. The use of the nutritional composition according to any one of claims 1 to 6 in the preparation of a product for relieving milk protein allergy.

8. The use of the nutritional composition according to any one of claims 1 to 6 in the preparation of products that alleviate milk protein allergy and help establish milk protein tolerance.

9. A product for relieving milk protein allergy, characterized in that: The product contains the nutritional composition according to any one of claims 1 to 6.

10. A product that alleviates milk protein allergy and helps establish milk protein tolerance, characterized in that: The product contains the nutritional composition according to any one of claims 1 to 6.

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