Breast milk oligosaccharide compound and application thereof in preparation of composition for improving intestinal homeostasis

Abstract

The invention provides a breast milk oligosaccharide compound and application of the breast milk oligosaccharide compound in preparation of a composition for improving intestinal homeostasis. The breast milk oligosaccharide compound comprises a bifidobacterium strain, one or more breast milk oligosaccharides, whey protein and aromatic amino acid, and the bifidobacterium strain is selected from bifidobacterium longum or bifidobacterium bifidum; the aromatic amino acid is selected from phenylalanine or tryptophan. The breast milk oligosaccharide compound promotes growth of specific probiotics and generation of aromatic amino acid metabolites, so that the establishment of the intestinal homeostasis of infants as early as possible is promoted, and the infection risk of the infants in the period is reduced.

Description

Technical Field

[0001] This invention relates to the field of infant nutrition. Specifically, this invention relates to a human milk oligosaccharide complex and its use in the preparation of compositions for improving intestinal homeostasis. Background Technology

[0002] The first 1000 days of life are a critical period for infants and young children to establish gut homeostasis and build their immune system. This is also a critical period for the development of various diseases. Once the gut homeostasis of an infant or young child is imbalanced, leading to immune system dysfunction, it can cause intestinal diseases such as diarrhea and abdominal pain, inflammatory bowel disease, and allergic diseases such as food allergies, asthma, and eczema. Dietary factors are one of the key factors affecting the gut homeostasis of infants and young children. Breast milk is recognized as the golden source of nutrition for infants and young children. Human milk oligosaccharides (HMOs) play various functional roles in the process of establishing the gut microbiota and immune system and achieving gut homeostasis balance, such as directly regulating intestinal immunity and acting as prebiotics.

[0003] Recent studies have shown that human milk oligosaccharides can promote the proliferation of certain Bifidobacteria, thereby driving their metabolism of aromatic amino acids to produce aromatic lactic acids, including indolelacic acid, phenyllactic acid, and hydroxyphenyllactic acid. These metabolites, as another important gut microbiota-host interaction regulatory molecule that has attracted widespread attention after short-chain fatty acids, can play a key regulatory role in early life gut immune development by activating signaling pathways such as the aryl hydrocarbon receptor (AhR) and hydroxycarboxylic acid receptor 3 (HCA3).

[0004] However, in existing milk-based nutritional compositions, the content and structural diversity of human milk oligosaccharides are generally low, which is considered one of the important factors contributing to the functional gap between them and breast milk. To bridge this gap, some studies have attempted to combine prebiotic oligosaccharides with probiotics in related products. However, current research still has significant limitations: the range of oligosaccharide types and strains used is relatively limited, mostly based on random selection, lacking a systematic and in-depth exploration of the synergistic effects and mechanisms of action between different strains and oligosaccharides with different structures, resulting in the actual application failing to meet expectations.

[0005] Therefore, there is an urgent need for an optimized human milk oligosaccharide composition that supports the establishment of intestinal homeostasis to overcome the problems of blind combination selection and insignificant effects in the prior art. Summary of the Invention

[0006] In view of this, the present invention provides a human milk oligosaccharide complex comprising Bifidobacterium animalis strains, one or more human milk oligosaccharides, whey protein, and aromatic amino acids.

[0007] Furthermore, another object of the present invention is to provide the use of the aforementioned human milk oligosaccharide complex in the preparation of a composition for improving intestinal homeostasis in infants and young children. The composition improves intestinal homeostasis in infants and young children by promoting the growth of specific probiotics in the infant's gut and promoting the production of aromatic amino acid metabolites—aromatic lactic acid.

[0008] On one hand, the present invention provides a human milk oligosaccharide complex, the human milk oligosaccharide complex comprising a strain of Bifidobacterium, one or more human milk oligosaccharides, whey protein and aromatic amino acids, wherein the strain of Bifidobacterium is selected from Bifidobacterium longum or Bifidobacterium bifidum; and the aromatic amino acid is selected from phenylalanine or tryptophan.

[0009] Optionally, the human milk oligosaccharide is selected from 2'-fucosylated lactose (2'-FL), 3'-sialylated lactose (3'-SL), 6'-sialylated lactose (6'-SL), lactose-N-neotetrasaccharide (LNnT), or lactose-N-tetrasaccharide (LNT).

[0010] Optionally, the *Bifidobacterium longum* is *Bifidobacterium longum* subsp. *longum* BB536, and the *Bifidobacterium bifidum* is *Bifidobacterium bifidum* R0071.

[0011] Optionally, the human milk oligosaccharide complex is human milk oligosaccharide complex 1 comprising Bifidobacterium longum subsp. BB536, lactose-N-tetrasaccharide, whey protein, and phenylalanine, or human milk oligosaccharide complex 2 comprising Bifidobacterium bifidum R0071, 2'-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lactose-N-neotetrasaccharide or lactose-N-tetrasaccharide, whey protein, and tryptophan.

[0012] Optionally, the weight ratio of human milk oligosaccharides, whey protein, and phenylalanine in the human milk oligosaccharide complex 1 is 10-85 : 30-380 : 2-11, and the content of the Bifidobacterium strain is 2 × 10⁻⁶. 6 CFU - 2×10 9 CFU; and, in the human milk oligosaccharide complex 2, the weight ratio of human milk oligosaccharides, whey protein and tryptophan is 10-560 : 60-750 : 3-24, and the content of the Bifidobacterium strain is 2×10 6 CFU - 2×10 9 CFU.

[0013] Optionally, the weight ratio of human milk oligosaccharides, whey protein, and phenylalanine in the human milk oligosaccharide complex 1 is 12-80 : 35-350 : 2-11, and the content of the Bifidobacterium strain is 2 × 10⁻⁶. 7 CFU - 2×10 9 CFU; and, in the human milk oligosaccharide complex 2, the weight ratio of human milk oligosaccharides, whey protein and tryptophan is 13-520 : 70-700 : 3-22, and the content of the Bifidobacterium strain is 2×10 7 CFU - 2×10 9 CFU.

[0014] Optionally, when multiple human milk oligosaccharides are added, the weight ratio of 2'-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lactose-N-neotetrasaccharide, and lactose-N-tetrasaccharide in the human milk oligosaccharide complex 2 is 45-55 : 5-10 : 2-6 : 3-7 : 10-15. Preferably, the addition ratio of 2'-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lactose-N-neotetrasaccharide, and lactose-N-tetrasaccharide can be 53 : 7 : 4 : 5 : 13.

[0015] On the other hand, the present invention provides the use of the aforementioned human milk oligosaccharide complex in the preparation of compositions for improving intestinal homeostasis.

[0016] Optionally, the human milk oligosaccharide complex is used to improve intestinal homeostasis problems caused by food allergies.

[0017] Optionally, the human milk oligosaccharide complex promotes intestinal homeostasis by alleviating pathological damage to jejunal tissue, inhibiting the secretion of pro-inflammatory factors and restoring the secretion of anti-inflammatory factors, and enriching intestinal probiotics.

[0018] The beneficial effects achieved by this invention include, but are not limited to:

[0019] This invention discloses a compound containing specific probiotics, human milk oligosaccharides, whey protein, and aromatic amino acids, which promotes the growth of specific probiotics in the gut and simultaneously promotes the production of aromatic amino acid metabolites. Through the optimal combination of this compound, the differences between cow / goat milk formula and breast milk are further reduced, promoting the early establishment of intestinal homeostasis in infants. This not only reduces the risk of infection and enhances immunity during infancy, but more importantly, it has a profound and far-reaching impact on various functions that influence a child's lifelong healthy development, such as the immune system and cognition. Attached Figure Description

[0020] Figure 1The growth curves of six Bifidobacterium strains utilizing human milk oligosaccharides are shown (Note: "MRS-glc" represents the removal of glucose from the culture medium; "MRS-glc+HMO" represents the removal of glc and the addition of human milk oligosaccharides as a substitute).

[0021] Figure 2 Growth curves showing the selective utilization of human milk oligosaccharides by M-16V, BB536, and R0071 are displayed.

[0022] Figure 3 The growth curves show the selective utilization of human milk oligosaccharides by M-16V, BB536, and R0071 in the validation experiment.

[0023] Figure 4 The growth of R0071 is shown under HMO as the carbon source and under different nitrogen source conditions;

[0024] Figure 5 The graph shows a comparison of the growth of R0071 under different nitrogen source conditions, with glucose (left) or mixed HMOs (2'-FL, 3'-SL, 6'-SL, LNnT, LNT) (right) as the carbon source (note: "-" indicates deletion, "+" indicates addition).

[0025] Figure 6 The growth of BB536 under LNT as carbon source and under different nitrogen source conditions is shown;

[0026] Figure 7 The graph shows a comparison of the growth of BB536 under different nitrogen source conditions with glucose (left) or LNT (right) as the carbon source (note: "-" indicates deletion, "+" indicates addition).

[0027] Figure 8 The growth of M-16V is shown under LNT as the carbon source and under different nitrogen source conditions;

[0028] Figure 9 The growth of M-16V is shown in a comparison graph with glucose (left) or LNT (right) as the carbon source and under different nitrogen source conditions (Note: "-" indicates deletion, "+" indicates addition).

[0029] Figure 10 The results show the indolelactic acid content produced by BB536 under the conditions of LNT as the carbon source and experimental groups 8, 4 and 2 as the nitrogen source;

[0030] Figure 11 The indolelactic acid content produced by R0071 is shown under the conditions of 2'-FL, 3'-SL, 6'-SL, LNnT, LNT as carbon sources and different nitrogen sources in experimental groups 1, 3, 5, 6 and 7.

[0031] Figure 12 The diagram shows a Balb / c juvenile rat model of OVA sensitization and intervention with human milk oligosaccharide complex.

[0032] Figure 13 The percentage change in weight of young mice during the experiment is shown in (A) and the symptoms of food allergy is shown in (B), where n=6. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. ns indicates p < 0.0001; ns indicates p > 0.05;

[0033] Figure 14 The effects of human milk oligosaccharide complex on histopathological symptoms of jejunal tissue in young mice were shown (n=6).

[0034] Figure 15 The serum OVA-sIgE levels of young mice are shown, where n=6. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. ns indicates p < 0.0001; ns indicates p > 0.05;

[0035] Figure 16 The serum levels of IL-4 (A), IL-5 (B), IL-13 (C), IL-17 (D), TNF-α (E), and IL-10 (F) in young mice are shown, where n=6. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. ns indicates p < 0.0001; ns indicates p > 0.05;

[0036] Figure 17 Immunohistochemical sections of tight junction protein from the colon tissue of young mice in blank group, control group, Example A3 and Example B3 are shown (n=6).

[0037] Figure 18 The expression of (A)claudin-1, (B) occludin, and (C) ZO-1 in the colon of young mice is shown, where n=6. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This indicates that p < 0.0001;

[0038] Figure 19The alpha diversity index of the gut microbiota of young mice was displayed, where K was the blank group, G was the control group, A was Example A3, and B was Example B3; and This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This indicates that p < 0.0001;

[0039] Figure 20 The Beta diversity analysis of the gut microbiota of young mice is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3;

[0040] Figure 21 The community composition at the phylum level is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3. Specifically, Figure 19 The relative abundance of Firmicutes, Bacteroidota, Desulfobacterota, Actinobacteria, Patescibacteria, Verrucomicrobiota, Deferribacterota, Campilobacterota, unclassified bacteria (domain level), Cyanobacteria, and Proteobacteria was compared.

[0041] Figure 22 The significance test of the differences between the gate level groups is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3; This means p ≤ 0.05. This means p≤0.01. This means p ≤ 0.001. Specifically, Figure 22 The study compared the phyla Desulfobacteria, Patrobacteria, Verruciformis, Deferobacteria ILA, Campylobacteria, Cyanobacteria ILA, and Proteobacteria.

[0042] Figure 23 The community composition at the genus level is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3. Specifically, Figure 23The study compared genera *Lactobacillus*, *Alistipes*, *Desulfovibrio*, *Muribaculaceae* (unnamed), *Lachnospiraceae* (NK4A136 group), *Enterorhabdus*, *Candidatus* (candidate saccharimonas), and *Clostridia* (unnamed). UCG-014), *Odoribacter*, *Faecalibaculum*, *norank_f_Lachnospiraceae*, *Akkermansia*, *unclassified_f_Lachnospiraceae*, *norank_o_Clostridia_vadiniBB60_group*, *Bifidobacterium*, *Turiciba* *Clostridium*, *Lachnoclostridium*, *Erysipelotrichaceae*, *Clostridium sensustricto*, *RF39*, ​​*Eggertaceae* The relative abundance of *Gergerthellaceae*, *Roseburia*, *Rikenella*, *Eubacterium siraeum* group, *norank f. Oscillospiraceae*, *unclassified f. Oscillospiraceae*, *Eubacterium xylanophilum* group, and *Monoglobus* genus;

[0043] Figure 24The significance test of differences between the genus level groups is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3; This means p ≤ 0.05. This means p≤0.01. This means p ≤ 0.001. Specifically, Figure 24 The study compared genera *Alternaria*, *Desulfovibrio*, *Unnamed* Trichobacteriaceae, *Trichobacteriaceae* NK4A136, *Candidate* *Saccharomyces*, *Unnamed* Clostridium* UCG-014, *Femobacterium*, *Unnamed* Trichobacteriaceae, *Ackermania*, and *Bifidobacterium*.

[0044] Figure 25 The community composition at the species level is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3. Specifically, Figure 25The study compared *Lactobacillus johnsonii*, uncultured *bacterium g. Alistipes*, uncultured *bacterium g. Desulfovibrio*, *Lactobacillus reuteri*, *Lactobacillus murinus*, and uncultured candidate *Saccharomyces*. Candidatus Saccharimonas, uncultured bacteria of the family Muribaculaceae, uncultured bacteria of the genus Enterorhabdus, uncultured bacteria of the family Lachnospiraceae NK4A136 group, and uncultured bacteria of the family Mucocephalomycetes. (uncultured bacteroidales bacterium f. Muribaculaceae), uncultured bacterium g. Odoribacter, unclassified g. Lachnospiraceae NK4A136 group, Faecalibaculum rodentium, Akkermansia mucini The following are listed: phila, unclassified g norank o Clostridia UCG-014, unclassified f Lachnospiraceae, uncultured bacterium f Lachnospiraceae, and uncultured bacterium o Clostridia UCG-014.

[0045] Figure 26 The significance test of differences between groups at different levels is shown, where K is the blank group, G is the control group, A is Example A3, and B is Example B3; This means p ≤ 0.05. This means p≤0.01. This means p ≤ 0.001. Specifically, Figure 26 The study compared *Lactobacillus johnsonii*, uncultured *Alternaria*, uncultured *Desulfovibrio*, *Lactobacillus reuteri*, *Lactobacillus murineis*, uncultured *candidate* *Saccharomyces*, uncultured *Muribaculaceae*, uncultured *Trichophyton* NK4A136, uncultured *Bacteroidetes* Trichophyton, and *Bacillus faecalis*.

[0046] Figure 27 The levels of indolelactone in the feces of young mice in the blank group, control group, intervention group A, and intervention group B are shown. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. ns indicates p < 0.0001; ns indicates p > 0.05;

[0047] Figure 28 Spearman correlation analysis showed the relationship between the levels of two metabolites and food allergy phenotypes. This represents 0.4. <r<0.6, Indicates 0.6 <r<0.8, This represents 0.8 <r<1.0。 Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the following specific embodiments. Obviously, the described specific embodiments are only a part of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0049] Example

[0050] The present invention will be further illustrated below with reference to the embodiments. The embodiments of the present invention are merely illustrative descriptions of the technical solutions of the present invention, and do not imply that the technical solutions of the present invention are limited to these specific embodiments.

[0051] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods.

[0052] Unless otherwise specified, all reagents and materials used in this invention are prepared using conventional methods or obtained commercially.

[0053] Example 1. Screening of strains utilizing human milk oligosaccharides

[0054] This invention screens human milk oligosaccharide-utilizing strains from six Bifidobacterium strains (Bifidobacterium longum subsp. BB536, Bifidobacterium breve M-16V, Bifidobacterium bifidum R0071, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium animalis subsp. lactis CP-9, and Bifidobacterium animalis subsp. lactis HN019).

[0055] Specific filtering methods:

[0056] 1. Purification and activation of Bifidobacteria:

[0057] Weigh out 20 g / L glucose, 10 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 1 g / mL Tween-80, 2 g / L sodium dihydrogen phosphate, 5 g / L anhydrous sodium acetate, 2 g / L triammonium citrate, 0.02 g / L manganese sulfate, 0.1 g / L magnesium sulfate, 1 g / L cysteine ​​hydrochloride, 1000 mL distilled water, and 15 g / L agar. Adjust the pH to 6.5 and sterilize at 115℃ for 20 min to prepare solid MRS medium.

[0058] Pour the prepared solid MRS medium into plates. After the medium cools and solidifies, place the plates in an anaerobic workstation overnight for deoxygenation. The next day, use an inoculation loop to collect six Bifidobacterium strains from the glycerol storage container, streak the plates, and place them back in the anaerobic workstation for anaerobic incubation at 37°C for 16–24 hours. Collect the colonies of each strain after incubation and inoculate them into liquid MRS medium, then anaerobically incubate at 37°C until the logarithmic growth phase of each strain.

[0059] 2. Screening for strains that utilize human milk oligosaccharides:

[0060] Take 4% of the logarithmic growth phase bacterial culture of each strain and inoculate it into the culture medium shown in Table 1 below. The culture medium for negative control 1 is MRS medium with glucose (glc) removed (MRS-glc); the culture medium for positive control 1 is MRS medium with glucose (glc) as the sugar source (MRS); and the culture medium for experimental group a is MRS medium with glucose (glc) removed and replaced with a mixture of human milk oligosaccharides (HMOs) of equal concentration (MRS-glc+HMOs). The mixed human milk oligosaccharides were prepared according to the concentration ratio of each HMO in breast milk: 0.53% 2'-FL, 0.07% 6'-SL, 0.04% 3'-SL, 0.05% LNnT, 0.13% LNT and 0.18% ultrapure water, and mixed thoroughly.

[0061] Each bacterial culture was placed in an anaerobic workstation and anaerobic incubated at 37°C for 72 hours. The 72-hour growth curve of each group of bacterial cultures was measured using a fully automated growth curve measuring instrument (Shanghai Weizai Technology Co., Ltd.).

[0062] Table 1. Screening experiment for human milk oligosaccharides using strains

[0063]

[0064] 3. Results

[0065] like Figure 1 It was found that under conditions where mixed human milk oligosaccharides were the sole sugar source, the three animal Bifidobacterium subsp. lactis BB12, CP-9, and HN019 could not grow at all. However, Bifidobacterium longum subsp. longum BB536, Bifidobacterium bifidum R0071, and Bifidobacterium breve M-16V showed a significant growth advantage under these conditions. In particular, Bifidobacterium bifidum R0071 showed the greatest growth advantage, almost consistent with the growth advantage under conditions where glc was used as the sugar source.

[0066] Therefore, the mixed human milk oligosaccharide-utilizing strains selected from the six Bifidobacterium strains were Bifidobacterium longum subsp. BB536, Bifidobacterium breve M-16V, and Bifidobacterium bifidum R0071.

[0067] Example 2. Effects of different types of human milk oligosaccharides on strains that utilize human milk oligosaccharides

[0068] 1. Screening Experiment

[0069] According to the purification and activation method in Example 1, Bifidobacterium longum subsp. BB536, Bifidobacterium breve M-16V and Bifidobacterium bifidum R0071 were cultured to the second-generation logarithmic phase.

[0070] Four percent of the bacterial suspension in the logarithmic growth phase of each strain was inoculated into the culture media shown in Table 2 below. Positive control 2 used five mixed human milk oligosaccharides as the sole sugar source, negative control 2 had no sugar source, and experimental groups A-E used four mixed human milk oligosaccharides (each with one different type of human milk oligosaccharide removed) as the sugar source. Under the above culture medium composition and anaerobic conditions at 37℃, the growth curves of each strain were measured for 72 hours.

[0071] Table 2. Screening experiment for optimal human milk oligosaccharides by strains utilizing mixed human milk oligosaccharides

[0072]

[0073] The results are as follows Figure 2As shown, the growth of M-16V and BB536 was maximally restricted after removing LNT from the mixed human milk oligosaccharides; the growth of R0071 was maximally restricted after removing 2'-FL from the mixed human milk oligosaccharides. Therefore, it can be preliminarily determined that the optimal human milk oligosaccharides for M-16V, BB536, and R0071 are likely LNT, LNT, and 2'-FL, respectively. However, this conclusion requires further verification experiments to confirm the final results.

[0074] 2. Verification Experiment

[0075] Four percent of the logarithmic growth phase bacterial suspension of each strain was inoculated into the culture media shown in Table 3 below. The concentration of each human milk oligosaccharide in the culture medium was kept consistent, and it was used as the sole sugar source. Mixed human milk oligosaccharide and sugar-free MRS were used as positive control 3 and negative control 3, respectively. The growth curves of each strain were measured for 72 hours under the specified culture medium composition and anaerobic conditions at 37°C.

[0076] Table 3. Validation experiment for screening optimal human milk oligosaccharides from strains utilizing human milk oligosaccharides

[0077]

[0078] The results are as follows Figure 3 As shown, the growth curves of M-16V and BB536 correspond to the results of the screening experiments above, further demonstrating that M-16V and BB536 have the greatest growth advantage under the condition that LNT is the only sugar source.

[0079] However, the growth curve of R0071 showed no significant difference in growth advantage among the various human milk oligosaccharides. Therefore, the reason why the growth curve of R0071 showed the greatest limiting effect after removing 2'-FL from the mixed human milk oligosaccharides in the screening experiment is because 2'-FL had the highest concentration; therefore, the reduction in total sugar source concentration after removal led to the growth restriction of R0071.

[0080] Based on the experimental data above, the optimal utilization of human milk oligosaccharides for Bifidobacterium longum subsp. BB536, Bifidobacterium breve M-16V, and Bifidobacterium bifidum R0071 are LNT, LNT, and mixed human milk oligosaccharides, respectively.

[0081] Example 3. Effects of milk protein and its enzymatic hydrolysates / aromatic amino acid types on various human milk oligosaccharide-utilizing strains

[0082] 1. Experimental Preparation

[0083] Considering that milk proteins and their enzymatic hydrolysates / aromatic amino acids cannot enter the large intestine intact, in vitro simulation of static digestion of milk proteins and their enzymatic hydrolysates / aromatic amino acids by infants was conducted before screening.

[0084] Table 4. Preparation of 1.25× Electrolyte Stock Solution

[0085]

[0086] Note: ① When preparing electrolyte stock solutions simulating gastric and small intestinal digestive fluids, adjust the pH to 5.3 and 6.6 respectively using HCl solution; ② Due to Ca 2+ It is prone to precipitation in electrolyte stock solutions, so CaCl2 should be added separately before the digestion test.

[0087] Table 5. Preparation of sample solutions

[0088]

[0089] (1) Gastric digestion: Mix the sample with SGF at a ratio of 63:37, adjust the pH to 5.3, and then add pepsin (268 U / mL). Mix and react simultaneously (1 h, 37 °C, 50 rpm). Add an appropriate amount of 1 mol / L NaHCO3 to the sample to adjust the pH to 7-8 to inactivate the pepsin and terminate the reaction.

[0090] (2) Intestinal digestion: Gastric digestive juice and SIF were mixed at a ratio of 62:38, and pancreatic enzyme (42.1 U / mL) and bile salt (3.1 mmol / L) were added. The pH was adjusted to 6.6, and the mixture was reacted while mixing (1 h, 37 °C, 50 rpm). After that, the sample was heated in a boiling water bath for 5 min to inactivate the enzyme and terminate the reaction.

[0091] (3) Freeze-drying: The digested sample was pre-frozen at -80℃ overnight, and then freeze-dried for 48 hours to obtain the final sample.

[0092] 2. Effects of milk protein and its enzymatic hydrolysates / aromatic amino acid types on strains utilizing human milk oligosaccharides

[0093] To determine the effects of milk proteins and their enzymatic hydrolysates / aromatic amino acids on the above-mentioned human milk oligosaccharide-utilizing strains (M-16V, BB536, R0071), the growth and metabolism of each strain were measured under the conditions of using the optimal human milk oligosaccharide (LNT / mixed human milk oligosaccharides) as the carbon source and eight different nitrogen sources (Table 6) to conduct preliminary screening for metabolomics assays. The experimental group with glucose as the sole carbon source was used as a control.

[0094] The specific screening method is as follows:

[0095] (1) Preparation of chemically limited basal culture medium (bCDM):

[0096] Weigh out 0.04g sodium chloride, 0.016g potassium dihydrogen phosphate, 0.016g dipotassium hydrogen phosphate, 0.004g magnesium sulfate heptahydrate, 0.004g calcium chloride hexahydrate, 0.8g sodium bicarbonate, 0.02% L-cysteine, 0.2g bile salts, 4μL vitamin K, 0.8mL Tween 80, and 0.002g heme. Then add 160mL of water, mix thoroughly, adjust the pH to 7, and aliquot into 40 15mL centrifuge tubes (4mL per tube) (Note: Calcium chloride + 40mL distilled water is prepared and sterilized separately).

[0097] Adding a carbon source:

[0098] Forty centrifuge tubes containing bCDM culture medium were divided into two groups of 20 tubes each. In one group, 0.1 g of glucose was added to each tube, and in the other group, 0.1 g of LNT / mixed human milk oligosaccharides was added to each tube.

[0099] Adding a nitrogen source:

[0100] Weigh out two portions of each of the eight digested nitrogen sources listed in Table 5 above, 0.02 g each, and add them to the corresponding bCDM culture medium test tubes. Adjust the final pH of the bCDM containing the nitrogen source to 7. Then, sterilize the mixture together with the calcium chloride solution at 110°C for 10 min using high-temperature steam sterilization.

[0101] Addition of calcium chloride solution:

[0102] Add 1 mL of separately sterilized calcium chloride solution to each of the 40 sterilized culture tubes to complete the final culture medium preparation.

[0103] (2) Inoculation and instrumental determination of nitrogen source screening experiment of M-16V / BB536 / R0071:

[0104] 4% of the logarithmic growth phase bacterial suspensions of strains M-16V, BB536, and R0071 were inoculated into the culture media shown in Table 6 below. Specifically, a culture medium with 3% glucose as the sole carbon source and eight different nitrogen sources (glucose group) was used as a control, and the growth and metabolism of each strain were observed in a culture medium with the optimal utilization of human milk oligosaccharides (LNT / mixed human milk oligosaccharides) as the sole carbon source and eight different nitrogen sources, as shown in Table 6 below.

[0105] After thoroughly mixing each culture medium using a vortex mixer, transfer 200 μL of each medium to a Bioscreen culture plate in an automated growth curve analyzer. Place the sample solution and culture plate together in an anaerobic workstation. After deoxygenating the sample solution in the Bioscreen culture plate for 20-30 minutes, take 100 μL of paraffin oil to seal the bacterial solution, and then measure its 72-hour growth curve using the instrument.

[0106] Table 6. Nitrogen source screening experiment

[0107]

[0108] Note: "bCDM-glc+HMO" means that glc is removed from bCDM medium and human milk oligosaccharides are added to replace it.

[0109] The results are as follows Figures 4-9 As shown, the growth advantage of strains supplemented with nitrogen was greater than that of the negative control group without nitrogen supplementation. Furthermore, the growth advantage of the bCDM-glc+HMO group, which optimally utilizes human milk oligosaccharides as the sole carbon source, was greater than that of the bCDM group using glucose as the carbon source in the groups supplemented with BB536 and R0071 strains. However, although the growth efficiency of the groups supplemented with whey protein and its enzymatic hydrolysates was greater than that of the negative control group, the growth efficiency of the groups supplemented with M-16V strain was greater than that of the bCDM-glc+HMO group in the bCDM group using glucose as the carbon source.

[0110] (3) Determination of aromatic lactic acid metabolites

[0111] Pretreatment of bacterial culture and qualitative and quantitative determination of aromatic lactic acid metabolites:

[0112] Mix 1 mL of culture supernatant with 4 mL of methanol, incubate at room temperature for 5 minutes, then centrifuge at 13,000 g for 5 minutes. Transfer 1 mL of the supernatant to a new test tube and evaporate to dryness using a nitrogen evaporator. Add 1 mL of resuspension solvent (0.1% formic acid (v / v) aqueous solution), vortex for approximately 30 seconds to dissolve the compound, and clarify the sample by filtration through a 0.22 μm sterile membrane. Then, perform qualitative and quantitative analysis of aromatic lactic acid using high-performance liquid chromatography-mass spectrometry (HPLC-MS).

[0113] like Figure 10 As shown, for BB536, in all four groups of different nitrogen sources, including the negative control group, indolelacic acid was produced to varying degrees. Compared with other nitrogen source groups, experimental group 8 (digested bovine whey protein hydrolysate + phenylalanine) produced the highest indolelacic acid content.

[0114] like Figure 11 As shown, for strain R0071, in all six selected nitrogen source groups, including the negative control group, indolelacic acid was produced to varying degrees. Compared with other nitrogen source groups and the BB536 group, the indolelacic acid content produced by experimental group 7 (digested bovine whey protein hydrolysate + tryptophan) of strain R0071 was significantly higher than that of other groups.

[0115] Based on the above results, the optimal in vitro compound for improving intestinal homeostasis in infants and young children is Bifidobacterium longum subsp. BB536+LNT+bovine whey protein hydrolysate+phenylalanine and Bifidobacterium bifidum R0071+mixed human milk oligosaccharides+bovine whey protein hydrolysate+tryptophan.

[0116] Example 4. Effects of human milk oligosaccharide complex on intestinal allergy

[0117] 1. Solution preparation

[0118] (1) Preparation of strains

[0119] Preparation of experimental strains: The glycerol culture tubes of BB536 and R0071 were removed and streaked onto MRS solid medium. They were then placed in an anaerobic incubator and incubated at 37°C for 72 h. Single colonies were picked and activated in liquid MRS medium. After thorough mixing, the bacterial suspensions were aliquoted and stored in glycerol at -80°C. Aliquots of the glycerol-preserved strains were re-inoculated into liquid MRS medium at a 4% inoculum and anaerobically cultured at 37°C for 24 h. 1 mL of the bacterial suspension was then diluted 1–8 times with physiological saline. 100 μL of each dilution was plated onto MRS plates and anaerobically cultured at 37°C for 48 h for counting. The experiment was repeated three times to obtain strains that reached a growth rate of 1 × 10⁻⁶ after 24 h of culture. 8 CFU / mL, meaning that one day before the experiment, 4% BB536 and R0071 were initially aliquoted into equal volumes of glycerol-preserved strains. After culturing for the same duration using the same method, 2 mL of the culture medium (1×10⁻⁶) was taken. 8 After washing and resuspending (CFU / mL), the daily gavage dose for each juvenile mouse is 2 × 10⁻⁶ CFU / mL. 8 CFU vs. 2×10 9 CFU, each baby mouse weighs approximately 20g.

[0120] (2) Preparation of LNT solution: The gavage dosage is shown in Table 8 below. The weight of the young mice is measured every three days. If the gavage dosage of LNT is 400 mg / kg BW per young mouse per day, taking a young mouse weight of 20g as an example, then each young mouse needs to be gavaged with 8mg LNT per day.

[0121] (3) Preparation of multiple human milk oligosaccharide solutions (HMO solutions): Weigh 0.424g 2'-FL, 0.032g 3'-SL, 0.056g 6'-SL, 0.04g LNnT and 0.104g LNT, and mix them evenly in 0.144g deionized water to obtain multiple human milk oligosaccharide solutions (HMO solutions). The gavage dosage is shown in Table 8 below. The weight of the pups was measured every three days. If the gavage dosage of HMO solution is 400 mg / kg BW per pup per day, taking a pup weighing 20g as an example, then each pup needs to be gavaged with 8 mg of HMO solution per day.

[0122] (4) Preparation of bovine whey hydrolyzed protein (BWPH): Based on the daily intake data provided by Ausnutria Dairy (China) Co., Ltd., the BWPH intake dose for young mice was converted as shown in Table 8 below.

[0123] (5) Preparation of tryptophan solution (Trp solution): Based on the daily intake data provided by Ausnutria Dairy (China) Co., Ltd., the Trp intake dose for young mice was converted as shown in Table 8 below.

[0124] (6) Preparation of phenylalanine solution (Phe solution): Based on the daily intake data provided by Ausnutria Dairy (China) Co., Ltd., the Phe intake dose for young mice was converted as shown in Table 8 below.

[0125] (7) Human milk oligosaccharide complex: According to the specific contents shown in Table 8, LNT, BWPH, Phe solution and BB536 strain were mixed to obtain the first group of human milk oligosaccharide complex (intervention group A): LNT+BB536+BWPH+Phe; the second group of human milk oligosaccharide complex (intervention group B) was obtained by the same method: multiple HMOs+R0071+BWPH+Trp.

[0126] (8) Preparation of sensitizing solution

[0127] 1) OVA and aluminum adjuvant suspension: Weigh 2.5 mg OVA and 100 mg aluminum hydroxide powder, dissolve in 10 mL of physiological saline, vortex to allow the aluminum hydroxide to fully adsorb the OVA. After mixing, inject intraperitoneally immediately (prepare and use immediately). Each mouse should be injected with 0.2 mL containing 50 μg OVA and 2 mg aluminum hydroxide.

[0128] 2) High-concentration OVA solution: Weigh 2.25g of OVA and dissolve it in 9mL of physiological saline to prepare a 250mg / mL OVA solution. Mix thoroughly and use immediately before gavage for challenge. Inject each mouse with 0.2mL containing 50mg of OVA.

[0129] 2. Animal experimental protocol

[0130] All animal experiments were approved by the Experimental Animal Welfare and Ethics Review Committee of Hunan Yuantai Biotechnology Co., Ltd. (Approval No.: 202409121535000403133).

[0131] This invention uses 3-week-old, SPF-grade female BALB / C mice (provided by Hunan Slack Jingda Experimental Animal Co., Ltd.) and housed them in the barrier environment animal laboratory of Hunan Yuantai Biotechnology Co., Ltd. (temperature 23±2℃, relative humidity 60±20%, 12 hours of light and 12 hours of darkness per day. The mice can eat and drink freely).

[0132] Table 7. Grouping of Animal Experiments

[0133]

[0134] Note: BWPH represents hydrolyzed bovine whey protein; Phe represents phenylalanine; Trp represents tryptophan.

[0135] Table 8. Intake of components from human milk oligosaccharide complexes

[0136]

[0137] Animal experimental procedures such as Figure 12 As shown, BALB / C pups were acclimatized for 3 days and then divided into 4 groups according to their weight. There was no significant difference in average weight among the groups (13-16g). The control group, intervention group A, and intervention group B received intraperitoneal injections of 0.2 mL of OVA and aluminum hydroxide suspension on day 0 (the time of the first intraperitoneal injection sensitization was recorded as day 0) and day 14. From day 28 to day 40, 0.2 mL of high-concentration OVA solution was administered by gavage every other day to establish a food allergy model. Intervention groups A and B chose 10:00 AM as the administration time for the intervention. From day 17 to day 40, 0.2 mL of human milk oligosaccharide compound solution was administered by gavage once consecutively. If OVA gavage challenge was required, the human milk oligosaccharide compound solution was administered 4 hours before the challenge. In the blank control group, both the injected and gavage contents were replaced with an equal volume of physiological saline / deionized water. The treatment methods and intake amounts for each group are shown in Tables 7 and 8 above.

[0138] 2.1 Monitoring of body weight and clinical symptoms

[0139] (1) Regular weighing records: The young mice were weighed every 3 days in the early stage and every week in the later stage. The weight was expressed as a percentage (weight% = current weight / initial weight (day 0) × 100%).

[0140] (2) Clinical symptoms: The food weight of each group of young mice was controlled at 50g. The remaining food weight was weighed every 2-3 days to record the food intake of each group of young mice. After the young mice were sensitized, their clinical symptoms were observed within 2 hours. The scoring criteria are shown in Table 9.

[0141] Table 9. Clinical Symptom Scoring Criteria for Food Allergies in Young Mice

[0142]

[0143] 2.2 Histopathological examination of jejunum tissue

[0144] After the young mice were sacrificed, the jejunum (about 1 cm) was dissected and collected, fixed with 4% paraformaldehyde solution, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and scanned and imaged using a 3DHISTECH scanner (CaseViewer, Pannoramic SCAN). Finally, histopathological changes such as hemorrhage, edema, necrosis, and inflammatory changes were observed in detail.

[0145] 2.3 Immunohistochemistry

[0146] After the young mice were sacrificed, their colons were dissected, collected, and separated, and fixed with 4% paraformaldehyde solution. For each slide, randomly selected fields of view were photographed (using a biological microscope, model ML51-N, Guangzhou Mingmei Optoelectronic Technology Co., Ltd.), ensuring the tissue filled the entire field of view and that the background lighting was consistent for each photograph. Image-Pro Plus 6.0 software was used to analyze the positive optical density (IOD) values ​​of each sample, and the average IOD value was used as the criterion for positive expression of colonic tight junction proteins.

[0147] 2.4 Measurement of serum OVA-specific IgE and immune factors (IL4, IL5, IL13, IL17, TNF-α, IL10)

[0148] Young mice were given a final gavage challenge, and after 3 hours without feeding, they were sacrificed, and blood was collected. Blood samples were allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm / min, 4°C for 15 minutes, and serum samples were collected in centrifuge tubes. Serum cytokine assays were performed according to the instructions of the ELISA kit.

[0149] 2.4 DNA extraction and 16S amplicon sequencing analysis of cecal contents

[0150] After euthanizing young mice, their cecal contents were collected and cryovials were flash-frozen in liquid nitrogen and stored at -80°C. DNA was extracted from each sample, and the integrity and purity of the extracted genomic DNA were detected by 1% agarose gel electrophoresis. Specific sequencing regions were selected, specific primers were synthesized, and low-cycle amplification was performed to obtain PCR products of appropriate concentrations. The PCR products were detected and quantified using the QuantiFluor™-ST blue fluorescence quantitative system, and then mixed in appropriate proportions according to the sequencing volume requirements of the samples. After library construction, the samples were ready for sequencing.

[0151] Bioinformatics analysis first involves assembling the PE reads obtained from sequencing and performing quality control on the sequences. Then, OTU clustering analysis and species classification analysis are performed. Based on OTU clustering and taxonomic analysis, diversity index analysis, species composition analysis, community structure analysis, and multivariate analysis and significance tests of community composition for each sample can be conducted.

[0152] 2.5 Determination of fecal aromatic lactic acid

[0153] Fresh fecal samples were collected one day before the young mice were euthanized, immediately flash-frozen in liquid nitrogen, and stored at -80°C. The samples were analyzed using LC-MS / MS according to the following procedure:

[0154] (1) Extraction of metabolites: After the sample is slowly thawed at 4°C, an appropriate amount of sample is added to a pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortexed, sonicated at low temperature, centrifuged, and the supernatant is vacuum dried. After reconstitution with acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v), the sample can be loaded for analysis.

[0155] (2) Preparation of standard solutions: Aromatic lactic acid standard (Shanghai McLean Biochemical Technology Co., Ltd.) was used to prepare standard stock solutions. The solutions were then serially diluted to obtain a series of standard solutions of different concentrations, which were then tested on the instrument.

[0156] (3) Chromatographic conditions: The chromatographic instrument was an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC, Thermo Fisher Scientific (China) Co., Ltd.); the chromatographic columns were HILIC and C18 columns; the column temperature was 35℃; the flow rate was 0.3 mL / min; the injection volume was 2 μL; mobile phase A was 90% water + 2 mM ammonium formate + 10% acetonitrile, and mobile phase B was acetonitrile + 0.4% formic acid; the gradient elution program is shown in Table 10 below:

[0157] Table 10. Chromatographic Flow Conditions

[0158]

[0159] (4) Mass spectrometry acquisition conditions: Mass spectrometer AB 6500+ QTRAP mass spectrometer (AB SCIEX); ion source is electrospray ionization (ESI); ion source temperature 580℃; spray gas (GS1) 45; spray gas (GS2) 60; curtain gas (Psi) 35; ionization voltage: 4500V (positive) / -4500V (negative).

[0160] 3. Results:

[0161] 3.1 Weight and Clinical Symptoms

[0162] Food allergies induced by OVA as an allergen can lead to weight loss; therefore, regular monitoring of weight changes in young mice can reflect their health status. Results showed ( Figure 13 Compared with the control group, the OVA attack caused a significant decrease in the weight gain of the young mice, while the young mice in Examples A3 and B3 were able to recover their weight gain to some extent.

[0163] Food allergies in young mice typically manifest as ruffled fur, scratching of the ears or cheeks, reduced spontaneous activity, decreased food intake, increased fecal water content, and cyanosis of the lips. This study observed and measured the symptoms in young mice within two hours after gavage administration of a high-concentration OVA solution, and quantified the symptoms using a symptom scoring system. The results showed ( Figure 13 In the control group, young mice exhibited rough fur, significantly reduced activity, frequent scratching of their ears and cheeks, decreased food intake, and increased fecal water content. In contrast, the control group showed normal behavior, with smooth fur, stable food intake, and dry, formed feces. Notably, intervention with the two human milk oligosaccharide complexes significantly alleviated the symptoms in young mice after OVA challenge, manifested as improved fur smoothness, increased activity, and restored food intake.

[0164] 3.2 Effects of human milk oligosaccharide complex on jejunal tissue damage in OVA-induced food-allergic young rats

[0165] HE staining results of jejunal tissue from young mice are as follows: Figure 14 The results showed that the intestinal tissue structure in the blank group was basically normal and intact; the intestinal tissue structure in the control group showed moderate abnormalities, with localized areas showing erosion and shedding of villous epithelial cells and exposure of the lamina propria (indicated by red arrows), and significant dilation of some villous spaces (indicated by blue arrows). In contrast, the intestinal tissue structure in Examples A3 and B3 showed only mild abnormalities, with intact and abundant intestinal villi, and localized areas showing erosion and shedding of villous epithelial cells and exposure of the lamina propria (indicated by red arrows). These results indicate that the human milk oligosaccharide complex of the present invention has a certain alleviating effect on the pathological damage of jejunal tissue caused by OVA allergy.

[0166] 3.3 Regulation of serum OVA-sIgE by human milk oligosaccharide complex in food-allergic young mice

[0167] The measurement results are as follows Figure 15 As shown, the serum OVA-sIgE level in the control group was significantly higher than that in the blank group, indicating that the OVA-induced food allergy model in young mice was successfully established. Compared with the control group, both intervention groups A (Examples A1-A5) and B (Examples B1-B5) significantly inhibited the secretion of OVA-sIgE, indicating that the human milk oligosaccharide complex of the present invention has a relieving effect on OVA-induced food allergy in young mice, and there was no significant difference in the relieving effect between the two groups (p>0.05).

[0168] Furthermore, according to comparative examples A1-A2, when the intake of the human milk oligosaccharide complex of the present invention is 2 × 10⁻⁶ BB536, it is found to be 2 × 10⁻⁶. 7 CFU / d ~ 2×10 9 When the CFU / d content of a single human milk oligosaccharide is higher than 800 mg / kg BW or lower than 120 mg / kg BW, the content of whey hydrolyzed protein is higher than 3500 mg / kg BW or lower than 350 mg / kg BW, and the content of aromatic amino acids is higher than 110 mg / kg BW or lower than 20 mg / kg BW, the inhibitory effect of this human milk oligosaccharide complex on OVA-sIgE secretion is significantly worse. According to comparative examples B1-B2, when the intake of the human milk oligosaccharide complex of this invention is 2 × 10⁻⁶, R0071 is 2 × 10⁻⁶. 7 CFU / d ~ 2×10 9 When the CFU / d content of various human milk oligosaccharides is higher than 2600 mg / kg BW or lower than 65 mg / kg BW, the content of whey hydrolyzed protein is higher than 3500 mg / kg BW or lower than 350 mg / kg BW, and the content of aromatic amino acids is higher than 110 mg / kg BW or lower than 15 mg / kg BW, the inhibitory effect of this human milk oligosaccharide complex on OVA-sIgE secretion is significantly worse.

[0169] 3.4 Regulation of serum cytokines in OVA-induced food allergy-inducing young rats by human milk oligosaccharide complex

[0170] After ingestion of the allergen OVA, it can cross the intestinal mucosal barrier, induce a skewed Th2 and Th17 immune response, promote the secretion of Th2 and Th17 immune factors, thereby triggering intestinal inflammation and exacerbating clinical symptoms of food allergy. To investigate the alleviating effect of human milk oligosaccharide complex on OVA-induced food allergy, this invention measured the concentrations of relevant immune factors in the serum of young mice. Figure 16 As shown, compared with the blank group, the serum levels of pro-inflammatory factors IL-4, IL-5, IL-13, IL-17 and TNF-α in the control group were significantly increased, while the secretion of these pro-inflammatory factors was significantly inhibited in both groups in Examples A3 and B3.

[0171] Furthermore, OVA attack significantly reduced the level of the anti-inflammatory factor IL-10, while intervention with the two human milk oligosaccharide complexes significantly restored IL-10 expression. Elevated IL-10 levels are one of the important mechanisms for alleviating food allergies, as they can block type 2 immune responses and promote Th1 cell differentiation. These results indicate that the human milk oligosaccharide complexes of this invention can reduce the body's susceptibility to allergens by regulating gut microbiota composition, mediating T cell homeostasis, and inhibiting Th2 and Th17 immune responses.

[0172] 3.5 Regulation of colonic tight junction protein expression by human milk oligosaccharide complex

[0173] Increased intestinal barrier permeability, especially that caused by disruption of tight junctions in the intestinal epithelium, significantly increases the risk of food allergies. To assess the effects of human milk oligosaccharide complexes on intestinal barrier function, this invention examined the expression levels of tight junction proteins (Claudin-1, Occuldin, ZO-1) in the colon tissue of young mice. Figures 17-18 As shown, the expression of Claudin-1, Occuldin, and ZO-1 was significantly reduced in the control group, indicating that OVA attack disrupted the integrity of the colonic epithelium. However, in Examples A3 and B3, the expression of tight junction proteins was significantly increased after intervention with the human milk oligosaccharide complex, and there was no significant difference between the two groups.

[0174] These results indicate that the anti-allergic effect of the human milk oligosaccharide complex of the present invention does not depend on a single component, but can regulate gut microbiota metabolism and affect gut immune homeostasis through the synergistic effect of oligosaccharides, probiotics and their metabolites in the gut.

[0175] 3.6 Effects of human milk oligosaccharide complexes on gut microbiota

[0176] 3.6.1 Effects of human milk oligosaccharide complexes on gut microbiota diversity

[0177] To investigate the effects of human milk oligosaccharide complexes on gut microbiota diversity in OVA-induced food allergic pups, this invention performed 16S rRNA amplicon sequencing on the cecal contents of four groups of pups and analyzed their alpha and beta diversity. Alpha diversity was assessed using the Chao1 index, Ace index, and Sobs index.

[0178] The results show that ( Figure 19 There was no significant difference between the control group and Example A3, while the gut microbiota diversity of the young mice in Example B3 was significantly lower than that of the other three groups (p<0.05). In this invention, intervention group B provides five structurally diverse human milk oligosaccharides (HMOs), which can act as specific prebiotics to selectively promote the proliferation of strains such as Bifidobacterium and Lactobacillus that can utilize HMOs, while inhibiting the niche of other bacterial genera. This is similar to the mechanism of low-diversity gut microbiota formation in early infants; while intervention group A, because it only provides a single type of HMO, may have its growth and metabolic effects easily diluted by other carbon sources in the diet.

[0179] Beta diversity reflects the degree of difference in microbial communities by analyzing species composition and abundance information among samples. This invention uses principal component analysis (PCA) and principal coordinate analysis (PCoA) to assess the differences in microbial communities among samples, and the results are as follows: Figure 20 As shown, the gut microbiota composition of the blank group and the control group showed significant clustering and separation (p = 0.001). The intervention with the human milk oligosaccharide complex in Examples A3 and B3 significantly altered the OVA-induced microbiota structure, indicating that the human milk oligosaccharide complex of this invention has a regulatory effect on the gut microbiota structure of food-allergic young mice.

[0180] 3.6.2 Effects of human milk oligosaccharide complex on gut microbiota composition in food-allergic young mice

[0181] (1) Phylogenetic composition analysis: In order to investigate the effect of human milk oligosaccharide complex on the gut microbiota of food-allergic young mice, phylogenetic analysis was performed on the gut microbiota of four groups of young mice. Figures 21-22 Analysis revealed that 11 bacterial phyla were particularly important in all samples, with Bacteroidetes and Firmicutes being the dominant phyla in the intestines of young mice. Compared to the control group, OVA treatment significantly increased the abundance of Patescibacteria and significantly decreased the abundance of Desulfobacterota and Verrucomicrobia. However, the intervention with the human milk oligosaccharide complex in Examples A3 and B3 significantly decreased the abundance of Patescibacteria and significantly increased the abundance of Desulfobacterota and Verrucomicrobia.

[0182] (2) Genus-level species composition analysis: To further analyze the differences in the composition of the gut microbiota among the groups of young mice, a genus-level species composition and difference analysis was performed on the gut microbiota of the four groups of young mice. Figures 23-24 The results showed that *Lactobacillus* was the dominant genus of bacteria in the intestines of young mice. Similar to the phylum-level analysis, OVA treatment significantly reduced the abundance of *Desulfovibrio*, while the intervention with the human milk oligosaccharide complex in Examples A3 and B3 significantly increased the abundance of *Desulfovibrio*.

[0183] Compared with the control group, intervention with the human milk oligosaccharide complex significantly reduced the relative abundance of *Alistipes* and *Candidatus Saccharimonas*. Analysis showed that the enrichment of *Alistipes* was positively correlated with food allergy, while excessive proliferation of *Candidatus Saccharimonas* may exacerbate immune inflammation by disrupting the intestinal mucus barrier and was abnormally enriched in the oral cavity and intestines of patients with immune-related diseases such as food allergy. Notably, intervention with the human milk oligosaccharide complex significantly increased the abundance of *Akkermansia*, a genus crucial for maintaining intestinal barrier function. Previous literature has reported that children with food allergies have lower abundance of *Akkermansia* in their fecal microbiota compared to healthy children.

[0184] (4) Species composition analysis at the species level

[0185] To further clarify the characteristic bacterial communities of each group, this invention analyzed the species composition and inter-group differences at the species level. The results showed that ( Figures 25-26 In Examples A3 and B3, the relative abundance of *Lactobacillus johnsonii* (P<0.01) and *Lactobacillus reuteri* (p<0.05) was significantly higher than that in the blank group and the control group. Previous studies have shown that these two lactobacilli play an important role in alleviating food allergies and maintaining intestinal immune homeostasis.

[0186] It is evident that the human milk oligosaccharide complex of the present invention can regulate intestinal immunity by enriching specific beneficial bacteria (such as Lactobacillus johnsonii and Lactobacillus reuteri), thereby exerting a therapeutic effect.

[0187] Furthermore, this invention found that Bifidobacteria did not form a dominant flora in the mouse gut after intervention with human milk oligosaccharide complexes. It is noteworthy that the ability of Bifidobacteria to utilize human milk oligosaccharides is not the only determining factor for their dominant colonization in the gut of breastfed infants; the symbiotic relationships among gut microbiota are also key to understanding the role of various HMOs in regulating gut microbiota. In vitro experiments of this invention confirmed that Bifidobacteria capable of growing by utilizing multiple HMOs did not become a dominant flora in vivo, which may be related to microbiota interactions. Bifidobacteria, through their glycoside hydrolase system, degrade simple sugars such as galactose and fucose from human milk oligosaccharides, providing growth substrates for Lactobacillus. For example, the free fucose released by the degradation of fucoidylated human milk oligosaccharides by Bifidobacterium breve can promote the colonization of Lactobacillus reuteri; similarly, the metabolites of 2'-FL decomposed by Bifidobacterium IPLA 20048 can drive the proliferation of Lactobacillus gasseri. This division of labor, between primary decomposers (Bifidobacteria) and secondary users (Lactobacilli), further confirms the paradoxical phenomenon that exogenous Bifidobacterium intervention actually promotes Lactobacillus enrichment. Therefore, Lactobacillus enrichment can be a concrete manifestation of how human milk oligosaccharides regulate microbial interactions through a multi-level metabolic network.

[0188] 3.7 Effects of human milk oligosaccharide complex on aromatic lactic acid in the intestines of OVA-induced food-allergic young rats

[0189] In vitro studies of this invention have demonstrated that strains R0071 and BB536, under the promotion of their respective main utilized human milk oligosaccharides (multiple human milk oligosaccharides and LNTs), can efficiently metabolize aromatic lactic acid using BWPH+Trp and BWPH+Phe, respectively. To investigate the effects of the human milk oligosaccharide complex of this invention on aromatic lactic acid, a metabolite in the intestinal flora of FA infant mice, and its impact on intestinal health, fresh feces from four groups of infant mice were analyzed one day before their sacrifice.

[0190] 3.7.1 Effect of human milk oligosaccharide complex on aromatic lactic acid content in OVA-induced food allergic young rats

[0191] This invention uses targeted metabolomics to analyze the content of aromatic lactic acid in the feces of young mice in each group after approximately 3 weeks of intervention with a human milk oligosaccharide complex. The results showed that ( Figure 27Compared with the control group, Examples A1-A5 and Examples B1-B5 significantly increased the content of ILA (indolelacic acid) (p<0.05). Although a certain amount of OH-PLA (hydroxyphenyllactic acid) was detected in the young mice after intervention with various HMO complexes, its level was not significantly different from that of the blank group and the control group. Notably, PLA (phenyllactic acid) was not detected in the young mice of any group. Therefore, although Bifidobacterium did not become the dominant species, the significant increase in ILA after intervention with the human milk oligosaccharide complex of this invention is related to the enrichment of Lactobacillus johnsonii and Lactobacillus reuteri. The lack of PLA may be attributed to the lack of strains in the intestine capable of metabolizing phenylalanine.

[0192] 3.8 Correlation analysis of aromatic lactic acid and OVA-induced food allergy phenotype in young mice

[0193] Based on the beneficial effects of aromatic lactic acid on host gut health and immune regulation, this invention further analyzed the correlation between aromatic lactic acid and OVA-induced food allergy pathological indicators after intervention with human milk oligosaccharide complexes, to confirm whether human milk oligosaccharide complexes exert their beneficial effects through the metabolism of aromatic lactic acid. Figure 28 As shown, ILA was significantly negatively correlated with pro-inflammatory factors IL-4, IL-5, IL-13, IL-17 and the food allergy marker IgE, while it was significantly positively correlated with the anti-inflammatory factor IL-10; OH-PLA had a weaker correlation with pathological indicators of food allergy.

[0194] In summary, the two human milk oligosaccharide complexes LNT+BB536+BWPH+Phe and multiple HMOs+R0071+BWPH+Trp in this invention can promote ILA production by regulating the abundance of Lactobacillus johnsonii and Lactobacillus reuteri in the intestine. ILA alleviates OVA-induced food allergy symptoms in young mice by regulating intestinal immune homeostasis.

Claims

1. A human milk oligosaccharide complex, wherein, The human milk oligosaccharide complex contains Bifidobacterium strains, one or more human milk oligosaccharides, whey protein, and aromatic amino acids. The Bifidobacterium strain is selected from Bifidobacterium longum or Bifidobacterium bifidum; the aromatic amino acid is selected from phenylalanine or tryptophan.

2. The human milk oligosaccharide complex according to claim 1, wherein, The human milk oligosaccharides are selected from 2'-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lactose-N-neotetrasaccharide, or lactose-N-tetrasaccharide.

3. The human milk oligosaccharide complex according to claim 1 or 2, wherein, The *Bifidobacterium longum* is *Bifidobacterium longum* subsp. *longum* BB536, and the *Bifidobacterium bifidum* is *Bifidobacterium bifidum* R0071.

4. The human milk oligosaccharide complex according to any one of claims 1-3, wherein, The human milk oligosaccharide complex is either human milk oligosaccharide complex 1, which includes Bifidobacterium longum subsp. BB536, lactose-N-tetrasaccharide, whey protein, and phenylalanine, or human milk oligosaccharide complex 2, which includes Bifidobacterium bifidum R0071, 2'-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lactose-N-neotetrasaccharide, lactose-N-tetrasaccharide, whey protein, and tryptophan.

5. The human milk oligosaccharide complex according to claim 4, wherein, The weight ratio of human milk oligosaccharides, whey protein, and phenylalanine in the human milk oligosaccharide complex 1 is 10-85 : 30-380 : 2-11, and the content of the Bifidobacterium strain is 2 × 10⁻⁶. 6 CFU - 2×10 9 CFU; and, in the human milk oligosaccharide complex 2, the weight ratio of human milk oligosaccharides, whey protein and tryptophan is 10-560 : 60-750 : 3-24, and the content of the Bifidobacterium strain is 2×10 6 CFU - 2×10 9 CFU.

6. The human milk oligosaccharide complex according to claim 4, wherein, The weight ratio of human milk oligosaccharides, whey protein, and phenylalanine in the human milk oligosaccharide complex 1 is 12-80 : 35-350 : 2-11, and the content of the Bifidobacterium strain is 2 × 10⁻⁶. 7 CFU - 2×10 9 CFU; and, in the human milk oligosaccharide complex 2, the weight ratio of human milk oligosaccharides, whey protein and tryptophan is 13-520 : 70-700 : 3-22, and the content of the Bifidobacterium strain is 2×10 7 CFU - 2×10 9 CFU.

7. The human milk oligosaccharide complex according to claim 4, wherein, The weight ratio of 2'-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lactose-N-neotetrasaccharide, and lactose-N-tetrasaccharide in the human milk oligosaccharide compound 2 is 45-55: 5-10: 2-6: 3-7: 10-15.

8. Use of a human milk oligosaccharide complex according to any one of claims 1-7 in the preparation of a composition for improving intestinal homeostasis.

9. The application according to claim 8, wherein, The human milk oligosaccharide complex is used to improve intestinal homeostasis problems caused by food allergies.

10. The application according to claim 8 or 9, wherein, The human milk oligosaccharide complex promotes intestinal homeostasis by alleviating pathological damage to jejunal tissue, inhibiting the secretion of pro-inflammatory factors and restoring the secretion of anti-inflammatory factors, and enriching intestinal probiotics.

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