Use of an acidic ovine milk oligosaccharide composition in the manufacture of an anti-allergic medicament or food

Abstract

The present application relates to the application of an acidic goat milk oligosaccharide composition in the preparation of an anti-allergy drug or food, the composition comprising 24 acidic sugar chains 24SMAOs, among which, according to the classification of sialic acid structure, 12 Neu5Ac modified sialylation sugar chains, 10 Neu5Gc modified sialylation sugar chains and 2 sialylation sugar chains modified by both Neu5Ac and Neu5Gc, according to the classification of sialic acid connection mode, 11 alpha2,3 connected sialic acid sugar chains, 10 alpha2,6 connected sialic acid sugar chains and 3 sialic acid sugar chains connected by both alpha2,3 and alpha2,6. The composition belongs to a natural anti-allergy substance, has safety and effectiveness, and realizes excellent anti-allergy performance.

Description

Technical Field

[0001] The present invention belongs to the field of biological preparations, and in particular relates to the application of an acidic goat milk oligosaccharide composition in the preparation of anti-allergic drugs or foods. Background Art

[0002] Food allergy (FA) is an abnormal immune response characterized by the involvement of allergen-specific immunoglobulin E (IgE) and Th2 cells. Studies have shown that the development of immune cells and the cytokines (interleukin (IL)-4) and active mediators (β-Hex and histamine) they produce can lead to a series of allergic symptoms such as vomiting, diarrhea, anaphylactic shock, and even death, posing a significant threat to public health. Currently, treatments for food allergies (epinephrine, antihistamines, or glucocorticoids) do not fundamentally eliminate the patient's sensitivity to allergens and may lead to drug dependence or even adverse reactions. Therefore, it is crucial to find safe and effective natural anti-allergic substances to reduce the risk of food allergies. Summary of the Invention

[0003] In response to the above-mentioned prior art, the present invention proposes an application of an acidic sheep milk oligosaccharide composition in the preparation of anti-allergic drugs or foods, which solves the technical problem of the lack of natural anti-allergic substances in the prior art, can relieve intestinal damage and inflammation, and has excellent anti-allergic activity.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A use of an acidic goat milk oligosaccharide composition in the preparation of an anti-allergic drug or food, the composition comprising 24 acidic sugar chains 24SMAOS. The 24SMAOS, classified according to sialic acid structure, include 12 Neu5Ac-modified sialylated sugar chains, 10 Neu5Gc-modified sialylated sugar chains, and 2 sialylated sugar chains modified with both Neu5Ac and Neu5Gc; and classified according to the sialic acid linkage mode, include 11 α2,3-linked sialylated sugar chains, 10 α2,6-linked sialylated sugar chains, and 3 sialylated sugar chains with both α2,3 and α2,6 linkages.

[0006] Furthermore, the content of the 12 Neu5Ac-modified sialylated sugar chains was 52%, the content of the 10 Neu5Gc-modified sialylated sugar chains was 45%, and the content of the 2 sialylated sugar chains modified with both Neu5Ac and Neu5Gc was 3%.

[0007] Furthermore, the content of the 11 α2,3 linked sialic acid sugar chains was 28%, the content of the 10 α2,6 linked sialic acid sugar chains was 68%, and the content of the 3 sialic acid sugar chains with both α2,3 and α2,6 links was 4%.

[0008] Furthermore, the composition alleviates intestinal damage and inflammation.

[0009] Furthermore, the composition reduces sIgE, histamine, IgG2a, IFN-γ, and IL-4 in serum.

[0010] Furthermore, the composition induces Treg cell differentiation.

[0011] Furthermore, the composition not only regulates the relative abundance of Lachnospiraceae, Erysipelotrichaceae and Bacteroidaceae to return to normal levels, but also promotes the proliferation of beneficial bacteria Lactobacillaceae, Marinifilaceae and Clostridiaceaede, reduces the relative abundance of Desulfovibrionaceae, and promotes the relative abundance of Prevotellaceae, Marinifilaceae and Lactobacillaceae.

[0012] Furthermore, the composition increases the concentrations of acetic acid, propionic acid and isobutyric acid in the intestine.

[0013] Furthermore, the composition restores the expression of colon mucin O-glycans.

[0014] Furthermore, the composition is prepared by the following method: taking a certain amount of goat milk, thawing it at room temperature, and centrifuging it to remove fat; then adding ethanol twice the volume of the goat milk, fully mixing it, centrifuging it and collecting the supernatant, freeze-concentrating and drying it to obtain freeze-dried goat milk crude oligosaccharides; dissolving the freeze-dried goat milk crude oligosaccharides in double-distilled water, slowly adding it to a DEAE-52 cellulose chromatography column, eluting neutral goat milk oligosaccharides with double-distilled water, and continuing to add 0.25M NaCl solution to elute acidic goat milk oligosaccharides; using a graphite carbon column to wash and desalt the acidic goat milk oligosaccharides to obtain the composition.

[0015] Furthermore, the conditions of the two centrifugation processes include: 3-5°C, 8000-13000 rpm, 15-30 min.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] 1. The acidic goat milk oligosaccharide composition of the present invention is derived from goat milk and is a natural anti-allergic substance with safety and effectiveness;

[0018] 2. The acidic goat milk oligosaccharide composition of the present invention maximizes the retention of anti-allergic active ingredients in goat milk, thereby achieving excellent anti-allergic properties;

[0019] 3. The acidic goat milk oligosaccharide composition of the present invention can relieve intestinal damage and inflammation, reduce sIgE, histamine, IgG2a, IFN-γ, and IL-4 in serum, induce Treg cell differentiation and inhibit Th2 response, and not only regulate the relative abundance of Lachnospiraceae, Erysipelotrichaceae and Bacteroidaceae to normal levels, but also promote the proliferation of beneficial bacteria Lactobacillaceae, Marinifilaceae and Clostridiaceaede, reduce the relative abundance of Desulfovibrionaceae, promote the relative abundance of Prevotellaceae, Marinifilaceae and Lactobacillaceae, increase the concentration of acetic acid, propionic acid and isobutyric acid in the intestine, restore the expression of mucin O-sugar chains, and achieve anti-allergic function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Evaluation of the anti-allergic activity of Example 1 and Comparative Example 1 of the present invention;

[0021] Figure 2 The effects of Example 1 of the present invention and Comparative Example 1 on antibodies, allergic mediators and cytokines in mouse serum;

[0022] Figure 3 The effects of Example 1 of the present invention and Comparative Example 1 on Treg cell differentiation in mouse mesenteric lymph node cells are shown;

[0023] Figure 4 The effects of Example 1 and Comparative Example 1 on intestinal microorganisms in mice;

[0024] Figure 5 The effects of Example 1 of the present invention and Comparative Example 1 on short-chain fatty acids in mouse feces;

[0025] Figure 6 The effects of Example 1 of the present invention and Comparative Example 1 on the O-glycosylation level of colon mucin;

[0026] Figure 7 This is a qualitative and quantitative analysis of Example 1 and Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below in conjunction with the examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0028] Material:

[0029] Goat milk (Saanen goats, mature milk, 30 pooled samples) was provided by the dairy goat breeding base of Red Star Meiling Co., Ltd. Cow milk (Chinese Holstein cattle, mature milk, 30 pooled samples) was provided by Haiwen Ranch in Yanliang District, Xi'an, Shaanxi Province. Samples were transferred to a −80°C freezer within 2 hours for storage. Aniline, deuterated (d5-)aniline, activated carbon, ovalbumin (OVA), aluminum adjuvant, and protease inhibitor (PMSF) were purchased from Sigma-Aldrich, Inc.; loratadine (Lora) was purchased from Xi'an Janssen Pharmaceuticals Co., Ltd.; Sep-Pak C18 (200 mg / 3 mL) and porous graphitic carbon (PGC) (150 mg / 3 mL) solid-phase extraction cartridges were from Simon-Aldrich, Germany. DEAE-52 cellulose was purchased from Hefei Bomei Biotechnology Co., Ltd. All other reagents were of analytical grade. Water used in the experiments was purified using a Milli-Q ultrapure water system from Millipore, Inc.

[0030] Example 1:

[0031] Take 2L of goat milk and thaw at room temperature, centrifuge and remove fat (8000rpm, 20min), then add 2 volumes of ethanol respectively, mix thoroughly, centrifuge (10000rpm, 30min), collect the supernatant, freeze concentrate and dry to obtain freeze-dried goat milk crude oligosaccharides. The freeze-dried goat milk crude oligosaccharides are dissolved in double distilled water and slowly added to a DEAE-52 cellulose chromatography column. Neutral goat milk oligosaccharides are eluted with 3 column volumes of double distilled water, and 3 column volumes of 0.25MNaCl solution are added to elute acidic goat milk oligosaccharides. The acidic goat milk oligosaccharides obtained above are washed and desalted with a graphite carbon column to obtain the acidic goat milk oligosaccharide composition of Example 1. The above-mentioned neutral goat milk oligosaccharides are loaded on an activated carbon column, lactose is removed with 10 column volumes of 8% ethanol, and then eluted with 3 column volumes of 70% ethanol to obtain the neutral goat milk oligosaccharide composition of Example 1 with lactose removed.

[0032] Comparative Example 1:

[0033] The difference from Example 1 is that the goat milk in Example 1 is replaced by cow milk to obtain the acidic cow milk oligosaccharide composition and the neutral cow milk oligosaccharide composition of Comparative Example 1.

[0034] Test experiment:

[0035] 1. Evaluation of Antiallergic Activity in Mice

[0036] Six-week-old, clean-grade female Balb / c mice (weighing 20 ± 2 g) were provided by the Animal Center of Xi'an Jiaotong University School of Medicine. Balb / c mice were housed in an SPF-grade environment at a standard temperature (21–23°C) and humidity (45–55%). Subsequent experiments were performed after one week of acclimatization. All animal experiments were approved by the Laboratory Animal Care and Ethics Department of Northwestern University (Ethics Approval Number: NWU-AWC-20210903M).

[0037] Mice were randomly divided into 7 groups (control group (blank group), OVA group (model group), Lora group (loratadine drug group), SGMOs group (acidic goat milk oligosaccharide composition of Example 1), NGMOs group (neutral goat milk oligosaccharide composition of Example 1), SBMOs group (acidic bovine milk oligosaccharide composition of Comparative Example 1), and NBMOs group (neutral bovine milk oligosaccharide composition of Comparative Example 1)), with 6 mice in each group. Except for the control group, all other groups were intraperitoneally injected with 200 μL of sensitization solution (OVA: aluminum adjuvant: PBS = 1:1:1 (v:v:v)) on days 0 and 14 to induce sensitization twice. From days 27 to 40, mice in the control and OVA groups were gavaged with PBS, mice in the Lora group were gavaged with loratadine (0.2 mg / kg), and mice in the other groups were gavaged with SGMOs, NGMOs, SBMOs, and NBMOs (100 mg / kg) daily. At the same time, on days 28, 30, 32, 34, 36, and 38, mice were injected with 50 mg of OVA solution for challenge (except the control group), and the allergic symptoms and body weight of the mice were recorded. On day 40 of the experiment, fresh fecal samples were collected for 16SrRNA sequencing. On day 41, the rectal temperature and organ weight of the mice were measured, and blood, colon, and fecal contents were collected. The jejunal tissue of each group of mice was collected, placed in 4% paraformaldehyde fixative, dehydrated in graded ethanol solutions, and embedded in paraffin. The tissue samples were then cut into slices and stained with hematoxylin and eosin (H&E) respectively. The sections were sealed with neutral gum, and the sections were observed under a microscope and images were collected.

[0038] Construction of OVA-sensitized mouse model Figure 1 As shown in A, Balb / c mice were challenged with OVA (50 mg / kg) for 6 times and fed with oligosaccharide samples for 2 weeks, and then the allergic symptoms of the mice were analyzed ( Figure 1 B- Figure 1 I). Compared with the control group, the OVA group mice showed obvious loose stools in the colon ( Figure 1 B), severe allergic symptoms such as scratching the ears and cheeks and rash on the tail ( Figure 1C), body weight of mice ( Figure 1 D) and a significant increase in rectal temperature ( Figure 1 E)(p<0.05), indicating that the allergic model was successfully constructed. Compared with the OVA group, oral administration of oligosaccharide samples and loratadine (Lora) positive drugs can restore the fecal morphology of mice to normal and significantly reduce the allergic symptom score (p<0.05). In addition, SGMOs, NGMOs and Lora significantly alleviated the weight loss of mice (p<0.05), and SGMOs, NGMOs, SBMOs and Lora significantly increased the rectal temperature of mice, among which the rectal temperature changes of mice in the SGMOs and Lora groups were significantly higher than those in the NGMOs, SBMOs and NBMOs groups (p<0.05). Compared with the control group, the thymus index of mice in the OVA group ( Figure 1 F) and spleen index ( Figure 1 G) was significantly increased. Compared with the OVA group, the thymus index of mice in the oligosaccharide treatment groups and the Lora group was significantly reduced (p < 0.05), among which the thymus index of the SGMOs group was significantly lower than that of the NBMOs group (p < 0.05). Although OVA, Lora and oligosaccharides had no effect on the length of the mouse colon ( Figure 1 H), but the results of jejunal tissue sections showed that oligosaccharides could alleviate intestinal damage and inflammation caused by OVA ( Figure 1 I).

[0039] 2. Effects of Antibodies, Allergic Mediators, and Cytokines in Mouse Serum

[0040] like Figure 2 As shown in the results, by detecting the expression of specific antibodies, allergic mediators and cytokines in the serum of mice, it was found that compared with the control group, the levels of sIgE, IgG2a, histamine, IFN-γ and IL-4 in the serum of mice in the OVA group were significantly increased (p < 0.05), indicating that the OVA-induced food allergy model of Balb / c mice was successfully established. Compared with the OVA group, the sIgE ( Figure 2 A) and histamine ( Figure 2 The level of Th1 cytokine IgG2a (B) was significantly reduced (p < 0.05), proving that oligosaccharides have anti-allergic activity. Food allergy is a type I allergic disease, which is associated with the balance between Th1 and Th2 tilting towards Th2. Compared with the OVA group, the Lora group and oligosaccharide group significantly reduced the expression of Th1 cytokine IgG2a ( Figure 2 C) and IFN-γ( Figure 2 D) production (p < 0.05), among which the expression level of IFN-γ in the SGMOs group was significantly higher than that in the SBMOs and NBMOs groups, indicating that SGMOs induced a stronger Th1 response than BMOs. Compared with the OVA group, the Lora group and the oligosaccharide group significantly reduced the expression of Th2 cytokine IL-4 ( Figure 2 E) production (p < 0.05), among which the expression level of IL-4 in the SGMOs group was significantly lower than that in the NGMOs group, indicating that SGMOs inhibited Th2 response more effectively than NGMOs. Compared with the OVA group, the anti-inflammatory factor IL-10 ( Figure 2 The levels of F) in milk oligosaccharides were significantly increased (p < 0.05). In summary, milk oligosaccharides can alleviate allergic symptoms by reducing Th2 cytokines, inhibiting Th2 responses, and reducing the production of sIgE and histamine. In addition, compared with neutral NGMOs, acidic SGMOs significantly inhibited Th2 responses; compared with bovine milk SBMOs, goat milk SGMOs significantly promoted the production of Th1 cytokines.

[0041] 3. Mouse Mesenteric Lymph Node CD4 + Foxp3 + Effects on Treg cell differentiation

[0042] Regulatory T cells (Treg) are immunosuppressive CD4 + T cells, CD4 + CD25 + Foxp3 + It is the main phenotype of Treg cells, which mainly secrete cytokines such as IL-10 in the body to inhibit the activation of T cells and thus play an immunosuppressive role. Figure 3 As shown, flow cytometry was used to detect Treg cell-specific antibodies (CD4 + Foxp3 + ), we can see that, if Figure 3 As shown in A, the cells in the R3 region are CD4+Foxp3+Treg cells. The ratio of Treg cells to CD4+T cells is as follows. Figure 3 As shown in B. Compared with the control group, the level of Treg cells in the mesenteric lymph node cells of mice in the OVA group was significantly reduced by 0.5 times. Compared with the OVA group, the proportion of Treg cells in all oligosaccharide groups was significantly increased (p < 0.01), among which the proportion of Treg cells in the SGMOs group increased by 3.8 times, which was significantly higher than that in the NGMOs group, SBMOs group, and NBMOs group (p < 0.05). The above research results show that compared with neutral / acidic bovine milk oligosaccharides and neutral goat milk oligosaccharides, SGMOs have significant advantages in inducing Treg cell differentiation, inhibiting Th2 response, and alleviating OVA-induced food allergy symptoms.

[0043] 4. Effects of Mouse Intestinal Microorganisms

[0044] like Figure 4As shown in the results, 16S rRNA sequencing showed that compared with the control group, at the phylum level, the relative abundance of Bacteroidota and Proteobacteria in the intestinal flora of OVA group mice increased significantly (p < 0.05), and the relative abundance of Firmicutes decreased significantly (p < 0.05) ( Figure 4 A), these results are consistent with those reported in the literature. The study found that some strains of symbiotic bacteria such as Bacteroidota and Proteobacteria are associated with the occurrence of intestinal inflammation. Compared with the OVA group, the relative abundance of intestinal flora returned to normal levels after SGMOs treatment, among which the relative proportion of Firmicutes was significantly higher than that of the Control group (p<0.05). Firmicutes is the main bacterial phylum in the healthy human intestine. Dietary fiber can increase the abundance of certain firmicutes in the intestine and alleviate related diseases by changing intestinal permeability, inflammation, sugar metabolism, fatty acid oxidation / synthesis, and energy consumption. At the family level, compared with the control group, the relative abundance of Lachnospiraceae and Oscillospiraceae beneficial bacteria in the OVA group was significantly reduced (p<0.05), and the relative abundance of Erysipelotrichaceae harmful bacteria and Bacteroidaceae symbiotic bacteria was significantly increased (p<0.05) ( Figure 4B) OVA-induced changes in the intestinal microbiota have been shown to be consistent with those seen in patients with food allergies. Studies have found that a decrease in the Lachnospiraceae family is associated with allergic diseases in children. Pectin and tea polysaccharides can promote the proliferation of Lachnospiraceae, potentially reducing intestinal inflammation. Oscillospiraceae can produce short-chain fatty acids such as butyrate and propionate, stimulating the increase in goblet cells and mucus production, maintaining the integrity of the intestinal epithelium and thus reducing damage and inflammation in colonic tissue. Studies have shown that oligosaccharides (FOS, FUC, GOS, HMOs) can increase the abundance of Oscillospiraceae. Recent reports have found that Erysipelotrichaceae has the activity of converting primary bile acids into secondary bile acids, affecting host metabolism and thus triggering intestinal inflammation. Compared with the OVA group, SGMOs treatment not only restored the relative abundance of Lachnospiraceae, Erysipelotrichaceae, and Bacteroidaceae to normal levels, but also promoted the proliferation of beneficial bacteria Lactobacillaceae, Marinifilaceae, and Clostridiaceae, while reducing the relative abundance of Desulfovibrionaceae. Furthermore, compared with the control group, SGMOs treatment significantly increased the relative abundance of Prevotellaceae, Marinifilaceae, and Lactobacillaceae (p < 0.05). Prevotellaceae and Lactobacillaceae can reduce the incidence of infant allergies.

[0045] 5. Effects of Short-Chain Fatty Acids in Mouse Feces

[0046] SCFAs are the most abundant metabolites of intestinal flora. They play a vital role as an important source of nutrients for intestinal epithelial cells and affect intestinal morphology and function. Figure 4 As shown in Figure 2, the SCFA content in cecal contents was determined by GC method ( Figure 4 C) Compared with the control group, the concentrations of acetate, propionate, butyrate, isobutyrate, valeric acid, and isovaleric acid in the OVA group were significantly decreased (p < 0.05). SGMO treatment significantly increased the concentrations of acetate, propionate, and isobutyrate compared with the OVA group. Acetate, propionate, and butyrate can ameliorate the development of allergic intestinal inflammation, thereby helping to restore intestinal mucosal and barrier function.

[0047] 6. Effect of colonic mucin O-glycosylation levels

[0048] The intestinal mucosa is the largest immune site in the body. Figure 5 As shown in Figure 2, qualitative and quantitative analysis of colon mucin O-glycans in mice from the Control, OVA, and SGMOs groups revealed that the types of colon mucin O-glycans in mice from different groups were similar, but the glycosylation levels were significantly different ( Figure 5 Compared with the Control group, the total O-glycosylation level of colon mucin in the OVA group mice was significantly reduced by 0.4 times (p < 0.05), of which the expression of total neutral O-glycans was reduced by 0.7 times, and the expression of total acidic O-glycans was reduced by 0.4 times; compared with the OVA group, the total O-glycosylation level of colon mucin in the SGMOs group mice was significantly increased by 0.7 times (p < 0.05) ( Figure 5 A), in which the expression of total neutral O-glycans increased significantly by 2.2 times (p < 0.05) ( Figure 5 B), the expression of total acidic O-glycans increased significantly by 0.6 times (p < 0.05) ( Figure 5 C). The study found that differences in intestinal glycosylation levels of sialylated and fucosylated modifications were associated with the severity of inflammation. Neutral O-glycans can be divided into fucosylated and non-fucosylated. Compared with the control group, the expression of fucosylated O-glycans in the OVA group was significantly reduced by 0.8 times ( Figure 5 D), there was no significant difference in the expression of non-fucosylated O-glycans. Compared with the expression of fucosylated neutral O-glycans in the OVA group, the expression of O-glycans in the SGMOs-treated group was significantly increased by 3 times (p < 0.05). The study found that the fucose at the end of the colonic mucin O-glycan can be utilized by intestinal flora, affecting the expression of bacterial metabolic pathways and reducing the expression of bacterial virulence genes. In addition, this fucose can also inhibit the expression of virulence genes in colitis-related intestinal epithelial cells. When intestinal fucosylation is lacking, pathogenic bacteria increase and beneficial bacteria decrease, leading to microbial translocation, destruction of the epithelial barrier and intestinal inflammation. Acidic sugars are modified by sialic acid, sulfate, and co-modification of sialic acid and sulfate. Compared with the Control group, the expression of sialylated O-glycans in the OVA group decreased by 0.3 times ( Figure 5 E), the expression of sulfated O-glycans decreased by 0.4-fold, while the expression of O-glycans modified with both sulfate and sialic acid showed no significant difference. Compared with the acidic O-glycans in the OVA group, the expression of sialylated O-glycans (0.7-fold) and sulfated O-glycans (0.5-fold) in the SGMOs-treated group was significantly increased (p < 0.05). Studies have shown that mucin sulfation can strengthen the mucus barrier and protect against pathogenic infections and intestinal inflammation.

[0049] like Figure 6As shown in Figure 2, further analysis of the effects of SGMOs on each type of O-glycan chain of colon mucin showed that colon mucin O-glycan chains are divided into acidic ( Figure 6 A) and neutral O-glycans ( Figure 6 B) Two types. Compared with the control group, the OVA group significantly reduced the levels of seven acidic O-glycans, including four sulfated modifications (S1F2H2N2, S1H1N1, S1F1H3N3, and S1H2N2), two sialylated modifications (A1F2H3N3 and A1F1H2N2), and one O-glycan with both sulfate and sialylation modifications (S1A1F1H2N2). The level of the O-glycan with the glycoform composition A1H2N4 was significantly increased by 1.7-fold. In addition, the levels of three neutral fucosylated O-glycans with the glycoform compositions F1H1N3, F1H2N2, and F2H2N2 were significantly reduced. Compared with the OVA group, the SGMOs group significantly increased the levels of two acidic O-glycans with glycoform compositions of S1F2H2N2 (0.8-fold) and A1F1H2N2 (1-fold), and significantly decreased the level of O-glycans with glycoform composition of A1H2N4 by 0.4-fold, indicating that the three acidic O-glycans with glycoform compositions of S1F2H2N2, A1F1H2N2 and A1H2N4 may play a key role in SGMOs restoring the expression of colonic mucin O-glycans; in addition, the levels of neutral O-glycans with glycoform compositions of F1H2N2, F2H2N3, F1H1N2 and F2H2N2 were significantly increased.

[0050] OVA sensitization leads to a general decrease in the levels of fucosylation, sialylation, and sulfated O-glycosylation of colonic mucin in mice. SGMO treatment significantly alters mucin O-glycosylation expression, restoring the expression of fucosylated (F1H2N2 and F2H2N2), sialic acid, and sulfated (A1F1H2N2 and S1F2H2N2) O-glycochains on colonic mucin in mice. The restoration of OVA-induced food allergy-induced intestinal mucosal damage by SGMOs is associated with significant changes in mucin O-glycoproteins.

[0051] 7. Qualitative and quantitative analysis of acidic goat milk / cow milk oligosaccharide composition

[0052] like Figure 7 As shown, the structure of sialylated oligosaccharides of SGMOs of Example 1 and SBMOs of Comparative Example 1 was analyzed using the "sugar queue" analysis strategy developed by our laboratory ( Figure 7A, 7B). Goat milk has a total of 24 sialylated glycans, classified by the number of sialic acids: 18 monosialylated and 6 disialylated. Cow milk has a total of 10 sialylated glycans: 8 monosialylated and 2 disialylated. Compared to SGMOs, SBMOs have a 58% reduction in the number of sialylated glycans. According to the classification of sialic acid structure, there are 12 types of Neu5Ac-modified sialylated sugar chains in SGMOs, 10 types of Neu5Gc-modified sialylated sugar chains, and 2 types of sialylated sugar chains modified by both Neu5Ac and Neu5Gc; there are 8 types of Neu5Ac-modified sialylated sugar chains in SBMOs, and 2 types of Neu5Gc-modified sialylated sugar chains; according to the classification of sialic acid connection mode, there are 11 types of α2,3-linked sialylated sugar chains in SGMOs, 10 types of α2,6-linked sialylated sugar chains, and 3 types of α2,3 and α2,6-linked sialylated sugar chains; there are 6 types of α2,3-linked sialylated sugar chains in SBMOs, 3 types of α2,6-linked sialylated sugar chains, and 1 type of sialylated sugar chains with both α2,3 and α2,6 connections. Figure 7 As shown in Figure C, further quantitative comparative analysis of acidic goat milk and acidic bovine milk oligosaccharides was conducted. The relative proportion of Neu5Ac-modified sialylated sugar chains in bovine milk (95%) was 1.8 times that of goat milk (52%), and the relative proportion of Neu5Gc-modified sialylated sugar chains in goat milk (45%) was 56 times that of bovine milk (0.8%). In addition, the α2,6-linked sialylated sugar chains in goat milk (68%) were 1.9 times that of bovine milk (36%), and the α2,3-linked sialylated sugar chains in bovine milk (60%) were 2.1 times that of goat milk (28%). Therefore, SBMOs are mainly composed of α2,3-linked sialylated sugar chains, while SGMOs are mainly composed of α2,6-linked sialylated sugar chains.

[0053] Compared with the prior art, the above specific implementation has the following beneficial effects:

[0054] 1. The acidic goat milk oligosaccharide composition of the present invention is derived from goat milk and is a natural anti-allergic substance with safety and effectiveness;

[0055] 2. The acidic goat milk oligosaccharide composition of the present invention maximizes the retention of anti-allergic active ingredients in goat milk, thereby achieving excellent anti-allergic properties;

[0056] 3. The acidic goat milk oligosaccharide composition of the present invention can relieve intestinal damage and inflammation, reduce sIgE, histamine, IgG2a, IFN-γ, and IL-4 in serum, induce Treg cell differentiation and inhibit Th2 response, and not only regulate the relative abundance of Lachnospiraceae, Erysipelotrichaceae and Bacteroidaceae to normal levels, but also promote the proliferation of beneficial bacteria Lactobacillaceae, Marinifilaceae and Clostridiaceaede, reduce the relative abundance of Desulfovibrionaceae, promote the relative abundance of Prevotellaceae, Marinifilaceae and Lactobacillaceae, increase the concentration of acetic acid, propionic acid and isobutyric acid in the intestine, restore the expression of mucin O-sugar chains, and achieve anti-allergic function.

[0057] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. An application of an acidic goat milk oligosaccharide composition in the preparation of an anti-allergic drug, characterized in that: The composition includes 24 acidic sugar chains, wherein the 24 acidic sugar chains are classified according to sialic acid structure, including 12 Neu5Ac-modified sialylated sugar chains, 10 Neu5Gc-modified sialylated sugar chains, and 2 sialylated sugar chains modified with both Neu5Ac and Neu5Gc; and according to the sialic acid linkage mode, the composition includes 11 α2,3-linked sialylated sugar chains, 10 α2,6-linked sialylated sugar chains, and 3 sialylated sugar chains with both α2,3 and α2,6 linkages; The composition is prepared by the following method: taking goat milk, thawing it at room temperature, and centrifuging it to remove fat; then adding ethanol with a volume twice that of the goat milk, fully mixing it, centrifuging it, collecting the supernatant, and freeze-concentrating and drying it to obtain freeze-dried goat milk crude oligosaccharides; dissolving the freeze-dried goat milk crude oligosaccharides in double-distilled water, and slowly adding it to a DEAE-52 cellulose chromatography column, eluting neutral goat milk oligosaccharides with double-distilled water, and continuously adding a 0.25M NaCl solution to elute acidic goat milk oligosaccharides; and washing the acidic goat milk oligosaccharides with water using a graphite carbon column to remove salts to obtain the composition.

2. The use according to claim 1, characterized in that The content of the 12 Neu5Ac-modified sialylated sugar chains was 52%, the content of the 10 Neu5Gc-modified sialylated sugar chains was 45%, and the content of the 2 sialylated sugar chains modified with both Neu5Ac and Neu5Gc was 3%.

3. The use according to claim 1, characterized in that The content of the 11 α2,3 linked sialic acid sugar chains was 28%, the content of the 10 α2,6 linked sialic acid sugar chains was 68%, and the content of the 3 sialic acid sugar chains with both α2,3 and α2,6 links was 4%.

4. The use according to claim 1, characterized in that The composition alleviates intestinal damage and inflammation.

5. The use according to claim 1, characterized in that The composition reduces sIgE, histamine, IgG2a, IFN-γ, and IL-4 in serum.

6. The use according to claim 1, characterized in that The composition induces Treg cell differentiation.

7. The use according to claim 1, characterized in that The composition not only regulates Lachnospiraceae 、 Erysipelotrichaceae and Bacteroidaceae The relative abundance of beneficial bacteria was restored to normal levels and Lactobacillaceae 、 Marinifilaceae and Clostridiaceaede The proliferation of Desulfovibrionaceae The relative abundance of Prevotellaceae 、 Marinifilaceae and Lactobacillaceae relative abundance.

8. The use according to claim 1, characterized in that The composition increases the concentration of acetic acid, propionic acid and isobutyric acid in the intestine.

9. The use according to claim 1, characterized in that The composition restores colonic mucin O -Expression of sugar chains.

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