Application of chitosan oligosaccharide in relieving cow milk allergy and regulating immunity

Abstract

The invention relates to the field of application of food functional factors, and particularly discloses a method for relieving cow milk allergy by using chitosan oligosaccharide and application. According to the method, chitosan oligosaccharide is given before sensitization, during sensitization or after sensitization; the chitosan oligosaccharide can be used for remarkably reducing the levels of serum IgE (Immunoglobulin E), IgG1 (Immunoglobulin G1), IL-4 (Interleukin-4), IL-10 (Interleukin-10), IL-17A (Interleukin-10), histamine and mast cell proteinase-1, and increasing the expression of IFN (Interferon)-gamma to regulate the immune balance of Th1 / Th2 / Th17; the alpha diversity and beta diversity of intestinal flora are improved, and the stable state of the flora is recovered; the allergy symptoms are relieved; the intestinal tract tissue injury is repaired. The chitosan oligosaccharide can be used for preparing functional foods or compositions for resisting cow milk allergy, and has application prospects in infant formula foods, solid beverages and dairy products.

Description

Technical Field

[0001] This invention relates to the field of food functional factors and their application in the prevention and treatment of allergies, and particularly to a method and its application of using chitosan oligosaccharides (COS) to regulate immune responses and gut microbiota composition to alleviate milk allergy. Background Technology

[0002] Cow's milk allergy (CMA) is one of the most common food allergies in infants and young children, mainly caused by an IgE-mediated immune response induced by milk protein. Existing avoidance, immunotherapy, and probiotic interventions still have problems such as long treatment courses, poor compliance, and unstable effects. Summary of the Invention

[0003] To address the problems of existing technologies, this invention provides an application of chitosan oligosaccharide in alleviating milk allergy and regulating immunity.

[0004] A method for alleviating milk allergy using chitosan oligosaccharide involves administering chitosan oligosaccharide (COS) to milk proteins at different stages before, during, or after sensitization to alleviate milk allergy (CMA).

[0005] Furthermore, the chitosan oligosaccharide is a water-soluble oligomer with a degree of deacetylation ≥90% and a molecular weight of 340–1630 Da.

[0006] Furthermore, chitosan oligosaccharide is administered at a dose of 50-600 mg / kg (preferably 200 mg / kg) per kilogram of body weight.

[0007] Furthermore, the chitosan oligosaccharide is a chitosan oligosaccharide solution with a concentration of 6-10 mg / mL, preferably 8 mg / mL.

[0008] Furthermore, the chitosan oligosaccharide was administered orally.

[0009] Furthermore, cow's milk allergy is a type of cow's milk protein allergy.

[0010] Furthermore, the method significantly alleviates allergy symptoms by regulating the immune balance of Th1, Th2, and Th17 (including reducing IgE, IgG1, IL-4, IL-10, IL-17A, histamine, and MCPt-1, and increasing IFN-γ) and restoring gut microbiota homeostasis (including reducing the Firmicutes / Bacteroidota (F / B) ratio, significantly reducing the abundance of Bacteroidetes, significantly increasing the abundance of Verrucomicrobia and Proteobacteria, increasing the abundance of Muribaculaceae and Lactobacillaceae, decreasing the abundance of Akkermansiaceae, decreasing the abundance of Akkermansia and Muribaculum, and increasing the abundance of Prevotella, Rikenella, Lactobacillus, Alistipes, and Parabacteroides).

[0011] The application of a chitosan oligosaccharide in the preparation of functional foods or compositions for relieving milk allergy.

[0012] Furthermore, the chitosan oligosaccharide dosage is equivalent to 50-600 mg / kg body weight in animal experiments, preferably 200 mg / kg body weight.

[0013] Furthermore, the functional food is infant formula, solid beverage, nutritional supplement, or dairy product.

[0014] Furthermore, the composition is a pharmaceutical composition.

[0015] Chitosan oligosaccharide (COS) is a water-soluble oligomer of chitosan that has the potential to modulate immunity, reduce inflammation, and improve the intestinal barrier. It has a relieving effect on CMA at different intervention stages.

[0016] This invention involves administering chitosan oligosaccharides before, during, or after sensitization to bovine milk proteins. The chitosan oligosaccharides significantly reduce serum levels of IgE, IgG1, IL-4, IL-10, IL-17A, histamine, and mast cell protease-1; increase IFN-γ expression to regulate Th1 / Th2 / Th17 immune balance; improve gut microbiota α and β diversity, restoring gut microbiota homeostasis; alleviate allergy symptoms; and repair intestinal tissue damage. The chitosan oligosaccharides can be used to prepare functional foods or pharmaceutical compositions for treating bovine milk allergy, showing promising applications in infant formula, solid beverages, and dairy products.

[0017] Compared with existing technologies, the present invention has the following characteristics: multi-stage applicability; synergistic effect of multiple immune targets; restoration of microecological homeostasis; and good safety and edibility. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0019] Figure 1 Scoring of allergic symptoms in each group of mice;

[0020] Figure 2 The change in body weight of mice in each group;

[0021] Figure 3 HE staining was performed on the jejunum and colon of mice in each group, with a scale bar of (100 μm). The mouse jejunum (AE) and colon (FJ) were stained with HE; A was the jejunum Con group, B was the jejunum CMA group, C was the jejunum Int group, D was the jejunum Pre group, E was the jejunum Tre group, F was the colon Con group, G was the colon CMA group, H was the colon Int group, I was the colon Pre group, and J was the colon Tre group.

[0022] Figure 4 The changes in interleukin-4 in each group of mice;

[0023] Figure 5 The changes in interleukin-17 in each group of mice;

[0024] Figure 6 Changes in histamine levels in each group of mice;

[0025] Figure 7 The changes in interferon-gamma in each group of mice;

[0026] Figure 8 The changes in interleukin-10 in each group of mice;

[0027] Figure 9 The changes in IgE in each group of mice;

[0028] Figure 10 Changes in IgG1 levels in each group of mice;

[0029] Figure 11 Changes in mast cell protease-1 in mice of different groups;

[0030] Figure 12 The α-diversity of the mouse microbiota in each group is represented by: A for Chao1 index analysis, B for Observed species index analysis, C for Shannon index analysis, and D for Simpson index analysis.

[0031] Figure 13 The β-diversity of the gut microbiota in each group of mice;

[0032] Figure 14This is an analysis of the phylum level of the intestinal flora in mice; where A is Bacteroidota, B is Firmicutes, B is Firmicutes, C is Actinobacteriota, D is Verrucomicrobiota, E is Proteobacteria, and F is the ratio of (F / B).

[0033] Figure 15 This study analyzed the gut microbiota at the family level in mice; where A represents Akkermansiaceae, B represents Bacteroidaceae, C represents Lactobacillaceae, D represents Lachnospiraceae, and E represents Muribaculaceae.

[0034] Figure 16 This study analyzed the gut microbiota of mice at the genus level, where A represents Prevotella, B represents Lactobacillus, C represents Akkermansia, and D represents Muribaculum.

[0035] Figure 17 Changes in various prebiotic cytokines during RAW264.7 cell experiments;

[0036] Figure 18 Changes in various prebiotic cytokines during RBL-2H3 cell experiments;

[0037] Figure 19 The changes in prebiotic histamine and mast cell protease-1 in RBL-2H3 cell experiments. Detailed Implementation

[0038] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0039] In the examples below, chitosan oligosaccharide is a water-soluble oligomer with a degree of deacetylation ≥90% and a molecular weight of 340-1630 Da, food grade, purchased from Qingdao Hehai Biotechnology Co., Ltd.; casein was purchased from Beijing Solarbio Technology Co., Ltd.; whey protein was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; fucoidan, galacto-oligosaccharide, inulin, stachyose, isomaltoose, fructooligosaccharide, and trehalose were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0040] (1) Model and grouping: BALB / c mice were used as the model and divided into five groups: Con, CMA, Pre, Int, and Tre.

[0041] (2) Administration and stimulation: COS solution 8 mg / mL, 200 mg / kg body weight, was administered by gavage; oral milk protein was administered daily from day 35 to 49 for stimulation;

[0042] (3) Detection: Record symptom scores and weight, and detect serum IgE, IgG1, IL-4, IL-10, IL-17A, IFN-γ, histamine, MCPt-1, and conduct 16S rRNA sequencing analysis to identify diversity, F / B, Akkermansia, Lactobacillus, and Prevotella and other related indicators.

[0043] Example 1: Establishment and sampling of a mouse model of bovine milk allergy

[0044] 1) Animal grouping: 50 mice were randomly divided into five groups of ten each: control group (Con group); sensitization group (CMA group); COS intervention group (Int group); COS prevention group (Pre group); and COS treatment group (Tre group).

[0045] (1) Control group: From day 8 to day 49, sterile saline was administered by gavage three times a week, with a volume of 0.5 mL / 20 g.

[0046] (2) COS prevention group: Chitosan oligosaccharide was prepared into a solution of 8 mg / mL with sterile saline and administered by gavage at a concentration of 200 mg / kg body weight. The gavage was performed once every morning from day 8 to day 22, with a volume of 0.5 mL / 20 g. The preventive administration was carried out for two consecutive weeks.

[0047] (3) Animal models of allergy induced by bovine milk protein (sensitization group, COS prevention group, COS intervention group, COS treatment group): Bovine milk protein (mass ratio of casein:whey protein = 8:2) was prepared into a solution with sterile physiological saline and mixed with Freund's adjuvant (Sigma, USA) at a volume ratio of 1:1 to prepare a solution with a protein concentration of 2 mg / ml. The injection concentration was 20 mg / kg body weight. The model was established by intraperitoneal injection on the afternoon of day 8 and the afternoon of day 22, with an administration volume of 0.1 mL / 10 g.

[0048] (4) COS intervention group: COS (chitosan oligosaccharide prepared with sterile saline to an 8 mg / mL solution) was administered by gavage three times every morning for six weeks from day 8 to day 49, with an gavage concentration of 200 mg / kg body weight and a volume of 0.5 mL / 20 g. From day 8 to day 22, except for the COS prevention group, other groups were treated with the same dose of sterile saline. From day 23 to day 34, all other groups were treated with the same dose of sterile saline. From day 35 to day 49, except for the COS treatment group, all other groups were treated with the same dose of sterile saline.

[0049] (5) COS treatment group: COS (chitosan oligosaccharide was prepared into a solution of 8 mg / mL with sterile saline) was administered by gavage once every morning for two weeks from day 35 to day 49. The gavage concentration was 200 mg / kg body weight and the administration volume was 0.5 mL / 20 g.

[0050] 2) From day 35 to day 49, every two days, the COS intervention group, COS prevention group, COS treatment group and sensitization group were given oral antigen challenge. Skim milk powder (Beijing Sanyuan Foods Co., Ltd.) was prepared into a solution with a concentration of 100 mg / mL using sterile deionized water, and the gavage volume was 0.5 mL / 20 g.

[0051] 3) On the afternoons of days 6, 23, and 34, feces were collected from mice in all groups except the control group. On the morning of day 50, mice were orally challenged before sacrifice, and allergy scores were measured. Feces were collected from all groups. The collected feces were flash-frozen in liquid nitrogen and stored at -80°C.

[0052] Example 2: Symptom assessment in milk-allergic mice

[0053] 1. Allergic symptoms were evaluated using a scoring system (Table 1) 1 hour after oral challenge on day 50. Allergy scores were obtained as follows: Figure 1 The allergy symptom score of the sensitized group was 2.29, which was significantly higher than that of the control group (P<0.0001). COS intervention, prevention and treatment all played a certain role in inhibiting allergic reactions, among which COS intervention (1.43) and treatment (1.29) had more obvious effects.

[0054] Table 1 Allergy Symptom Scoring Table

[0055]

[0056] 2. Weigh the mice weekly. Observe the weight changes in the control group, sensitized group, COS intervention group, COS prevention group, and COS treatment group. Weight is one of the indicators of mouse health; rapid weight loss is usually accompanied by metabolic disorders and the occurrence of diseases, such as diabetes, hyperthyroidism, tuberculosis, and cancer. This experiment also observed the phenomenon of weight loss in mice sensitized by cow's milk, such as... Figure 2As shown, compared with the control group (19.70±0.95g), the sensitized group (17.87±0.72g) showed a significant decrease in body weight (P<0.0001). Starting from week 2, the body weight of mice in both the COS intervention and COS prevention groups began to increase. By week 4, the body weight of mice in the COS intervention group exceeded that of the control group, while the body weight of mice in the COS prevention group began to approach that of the control group. After the sensitization period ended, the body weight of mice in both the sensitized and COS treatment groups began to increase. By week 5, the body weight of mice in the sensitized group began to decrease, while the body weight of mice in the COS treatment group continued to increase. This indicates that COS treatment improved the weight loss symptoms caused by milk allergy. By week 7, the body weight of mice in the COS intervention group, COS prevention group, and COS treatment group was significantly increased compared with the sensitized group. The COS intervention group (20.58±0.92g) had the largest increase, followed by the COS prevention group (19.49±1.21g) and the COS treatment group (19.11±0.97g).

[0057] 3. On day 50, the colon and jejunum of the experimental mice were removed and fixed in 4% paraformaldehyde. Intestinal sections were stained with hematoxylin-eosin to observe intestinal morphology. Figure 3 HE staining of the jejunum of each group of mice is shown below. Figure 3 As shown in the AE, the intestinal barrier in the control group was intact, and the intestinal villi were relatively complete and normally arranged. In contrast, the sensitized group showed broken and defective intestinal villi, accompanied by aggregation and infiltration of eosinophils stained red, and lacked complete intestinal villi arrangement. Compared to the sensitized group, the COS intervention group, COS prevention group, and COS treatment group showed intact intestinal villi and intestinal mucosal barrier. Although inflammatory cell infiltration was also present, the inflammatory response was improved to some extent, with the intervention and treatment groups showing the most significant effects.

[0058] Colon HE staining Figure 3 As shown in Figure FJ, the crypts in the control group were relatively intact and contained numerous goblet cells. Compared to the control group, the sensitized group showed severe damage to the crypt structure and mucosal epithelium, a significantly reduced number of goblet cells, and marked inflammatory cell infiltration. In contrast, the COS intervention, COS prevention, and COS treatment groups showed no significant intestinal mucosal damage; the crypt structure was restored compared to the sensitized group, and inflammatory cell infiltration was reduced. This indicates that COS can alleviate the inflammatory response in the intestines of milk protein-sensitized mice.

[0059] Example 3: Determination of cytokines and immunoglobulins in milk-allergic mice

[0060] Serum samples were collected to determine cytokines and immunoglobulins. Mice were euthanized 1 hour after milk challenge, and blood samples were collected from the eye. Serum was obtained by centrifugation at 4000 rpm for 20 min at 4°C and then stored at -80°C before assay. The concentrations of serum IL-4, interleukin-17A (IL-17A), histamine, interferon-gamma (IFN-γ), interleukin-10 (IL-10), total IgE, IgG1, and mast cell protease (MCPt-1) were determined using an ELISA kit according to the product instructions. Figure 4-11 As shown.

[0061] IL-4 is an important inflammatory cytokine secreted by Th2 cells, and allergic reactions are often accompanied by elevated IL-4 levels. Studies have observed significantly elevated IL-10 levels in infants with cow's milk protein allergy, and high levels of IL-10 may play a pro-inflammatory role in allergic diseases. The effects of COS on IL-4 and IL-10 in cow's milk protein-induced allergic mice are as follows: Figure 4 and Figure 8 As shown, compared with the control group, the concentrations of IL-4 and IL-10 in the sensitized group were significantly increased (P<0.05). After COS treatment, the concentrations of IL-4 in the COS prevention and treatment groups, and the concentrations of IL-10 in the COS intervention and prevention groups were significantly decreased compared with the sensitized group (P<0.05). Overall, the concentrations of IL-4 and IL-10 in all three groups showed a decreasing trend after COS treatment, which is consistent with the IgE detection results, proving that COS can inhibit the increase of IL-4 and IL-10 caused by sensitization.

[0062] IL-17A is primarily secreted by Th17 cells and is a major inflammatory mediator within the IL-17 family. Under normal conditions, IL-17 secreted by Th17 cells maintains intestinal homeostasis by producing barrier-protective cytokines. However, immune imbalances can lead to autoimmune diseases and inflammatory conditions such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Changes in IL-17A levels in mice are shown below. Figure 5 As shown, the IL-17A level in the sensitized group was significantly higher than that in the control group (P<0.0001), while the IL-17A levels in the COS intervention group, COS prevention group, and COS treatment group were significantly lower than those in the sensitized group (P<0.001). This indicates that COS can inhibit the secretion of IL-17A by Th17 cells. This suggests that COS may alleviate allergic reactions by inhibiting Th2 and Th17 cell-induced allergic responses and promoting Th1 cell responses.

[0063] Histamine is an inflammatory mediator released by mast cells that can dilate capillaries and increase their permeability, making it a key factor in vasodilation and localized skin edema. In allergic reactions, histamine can cause symptoms such as rash, asthma, abdominal pain, diarrhea, decreased blood pressure, and even severe shock. Studies have shown that COS (co-osmotic pressure) has an anti-allergic effect on food allergies induced by shrimp tropomyosin. The results showed that COS treatment significantly reduced serum histamine levels in mice compared to the model group, demonstrating a significant desensitization effect. Histamine levels in each group of mice are shown below. Figure 6 As shown, the histamine content in the sensitized group (22.72±5.60 ng / mL) was significantly higher than that in the control group (14.61±1.76 ng / mL) (P<0.01). The histamine content in the COS intervention group, COS prevention group, and COS treatment group was lower than that in the sensitized group, with the intervention group showing the most significant decrease (14.98±4.76 ng / mL) (P<0.01). In conclusion, COS can reduce the secretion of MCPt-1 and histamine.

[0064] IFN-γ is a cytokine primarily secreted by Th1 cells, but it can also be secreted by cytotoxic T cells (CD8+ T cells), natural killer cells (NK cells), and natural killer T cells (NKT cells). IFN-γ possesses antiviral, antitumor, antimicrobial, and immunomodulatory functions, and it can inhibit the differentiation of Th0 cells into Th2 cells and the production of Th2 cytokines. The regulation of IFN-γ in mouse serum by COS is as follows... Figure 7 As shown, compared with the control group, the IFN-γ content in the sensitized group was significantly reduced (P<0.05), while after COS treatment, the serum IFN-γ levels in the COS intervention group, COS prevention group and COS treatment group were significantly increased compared with the sensitized group (P<0.001).

[0065] Elevated serum IgE and IgG1 levels are typical symptoms of IgE-mediated allergic reactions. IgE can bind to sensitizing proteins to form antibodies, inducing degranulation of basophils and mast cells, releasing histamine and causing inflammation. The concentrations of total IgE and IgG1 in mouse serum are as follows: Figure 9 and Figure 10As shown, compared with the control group, the total IgE and IgG1 levels in the sensitized group were significantly increased (P<0.05). The IgE levels in the COS intervention group (21.48±3.74 U / mL), COS prevention group, and COS treatment group were significantly lower than those in the sensitized group (P<0.01). The IgG1 levels in the COS intervention and COS prevention groups were somewhat lower than those in the sensitized group, with the most significant decrease in the COS intervention group (P<0.05). However, the IgG1 level in the COS treatment group was significantly higher than that in the sensitized group (P<0.01). The trends in allergy symptom scores and serum total IgE were basically consistent. After the last oral challenge, mice in the sensitized group exhibited symptoms such as nasal itching and scratching, facial redness and swelling, reduced activity, and even immobility. The COS intervention, COS prevention, and COS treatment groups all reduced the occurrence of these allergy symptoms. This indicates that COS has a significant inhibitory effect on milk protein-induced allergic reactions.

[0066] Mast cells (MCPt-1) are effector cells in food allergy responses. When allergens bind to IgE on the surface of mast cells, they stimulate degranulation, releasing inflammatory factors such as histamine, leukotrienes, and mast cell proteases, triggering allergic reactions and inflammatory symptoms. Therefore, measuring MCPt-1 and histamine levels helps determine the inhibitory effect of COS on allergic reactions and inflammatory symptoms caused by cow's milk allergy. MCPt-1 levels in mice are shown below. Figure 11 As shown, compared with the control group (0.60±0.33 ng / mL), the level of MCPt-1 in the sensitized group (1.04±0.43 ng / mL) was significantly increased (P<0.05). After COS treatment, the level of MCPt-1 in the COS treatment group (0.57±0.25 ng / mL) was significantly lower than that in the sensitized group. Both the COS intervention group and the COS prevention group also showed a decreasing trend, but the difference was not significant (P>0.05). Example 4: Intestinal flora analysis of milk-allergic mice Cecal contents were collected from five rats in each group, flash-frozen in liquid nitrogen, and sent to Shanghai Paiseno Biotechnology Co., Ltd. for microbial community diversity profile analysis. DNA was extracted from the samples, and paired-end sequencing was performed on the community DNA fragments using a sequencing platform. Characteristic sequences (ASVs) were obtained using the QIIME2 (2019.4) software with DADA2 methods, including primer removal, quality filtering, denoising, splicing, and chimera removal. The sequences were annotated using the Greengenes database, and a phylogenetic tree was constructed using the FastTree method within the Maximum Likelihood framework. The ASV abundance table was flattened using a rarefaction method to facilitate subsequent analysis.

[0069] Ecologists use alpha diversity and beta diversity indices to characterize species diversity within and between habitats, respectively, to comprehensively assess their overall diversity. We assessed the alpha diversity of gut microbiota in mouse feces (…). Figure 12 ) and β diversity ( Figure 13 ). Figure 12 The Chao 1 and Observed Species indices were used to represent richness, while the Shannon index was used to represent diversity. Compared with the control group, the Chao 1, Observed Species, and Shannon indices were significantly decreased in the sensitized group (P<0.05). After COS treatment, the Chao 1, Observed Species, and Shannon indices were all increased in the COS intervention group, COS prevention group, and COS treatment group compared with the sensitized group, with the COS intervention group showing the most significant increase (P<0.01). There was no significant difference between the COS treatment group and the sensitized group. This indicates that bovine milk protein sensitization causes a decrease in the richness and diversity of the gut microbiota in mice, and this situation is improved after COS treatment.

[0070] The study of the β diversity index uses the WeightedUnifrac algorithm to characterize the relationships between groups. Principal coordinates analysis (PCoA) is a classic unconstrained ordination method. It expands the sample distance matrix in a low-dimensional space after projection, preserving the original sample distance relationships to the greatest extent possible. PCoA considers sample distances as a whole, which is more consistent with the characteristics of ecological data than principal component analysis (PCA), thus making it a suitable ordination tool. Figure 13 As shown, the control group and the sensitized group, as well as the COS intervention group and the sensitized group, are completely separated into two distance matrices, indicating significant differences in species abundance between the control group and the sensitized group, and between the COS intervention group and the sensitized group. The COS intervention group is closest to the control group, demonstrating the smallest difference between the two groups. The COS treatment group and the sensitized group have a large overlap, indicating a small difference in species abundance between the COS treatment group and the sensitized group.

[0071] phylum-level analysis of gut microbiota ( Figure 14 The coccidial matrix (CMA) is mainly composed of Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia, and Proteobacteria. Compared with the control group, the abundance of Bacteroidetes was significantly reduced in the CMA group, while the abundance of Verrucomicrobia and Proteobacteria was significantly increased, and the Firmicutes / Bacteroidetes (F / B) ratio was significantly increased. Both the COS intervention group and the COS prevention group significantly increased the abundance of Bacteroidetes and decreased the abundance of Verrucomicrobia, with the F / B ratio in the COS prevention group being closest to that of the control group.

[0072] In terms of scientific level ( Figure 15 Compared with the control group, the abundance of Akkermansiaceae and Staphylococcaceae increased in the CMA group, while the abundance of Rikenellaceae, Muribaculaceae, Bacteroidaceae, and Lactobacillaceae decreased. The COS intervention group, COS prevention group, and COS treatment group all increased the abundance of Muribaculaceae and Lactobacillaceae and decreased the abundance of Akkermansiaceae, with the COS intervention group showing a significant increase in Lactobacillaceae (P<0.05).

[0073] From a horizontal perspective ( Figure 16In the CMA group, the abundance of Akkermansia and Muribaculum increased, while the abundance of Prevotella, Cryptobacteroides, and Alistipes decreased. After COS treatment, the abundance of Akkermansia and Muribaculum decreased, while the abundance of Prevotella, Rikenella, Lactobacillus, Alistipes, and Parabacteroides increased. Increased Lactobacillus was observed in all COS intervention, COS prevention, and COS treatment groups.

[0074] In summary, COS can improve the intestinal flora imbalance in milk protein sensitized mice, mainly by increasing the abundance of beneficial bacteria such as Bacteroidetes, Muribacaceae, and Lactobacillus, and reducing the abnormal proliferation of bacteria such as Akkermansia, thereby restoring intestinal flora homeostasis.

[0075] Example 5: Advantages of chitosan oligosaccharide compared to other prebiotics

[0076] To further clarify the unique advantages of chitosan oligosaccharide (COS) in alleviating allergic reactions, this embodiment designed a cell experiment to compare it with other common prebiotics (such as fructooligosaccharides, galactooligosaccharides, and inulin). RAW264.7 macrophages and RBL-2H3 mast cells were used as in vitro models to evaluate the effects of different prebiotics on immune regulation and allergy relief by detecting cytokine levels and the release of inflammatory mediators.

[0077] 1) RAW264.7 cell experiments

[0078] RAW264.7 cells were cultured in 96-well plates using standard methods. Before modeling, different prebiotics (chitosan oligosaccharide, COS; fucoidan oligosaccharide, AOS; galactooligosaccharide, GOS; inulin, Inu) were added to a modified Terra-Bio medium (Beijing Solarbio Science & Technology Co., Ltd.) to a final prebiotic concentration of 0.2 g / mL. These were then added to the cell culture system to a final prebiotic concentration of 300 ng / mL, and incubated for 6 h to obtain a cell suspension. Subsequently, cells containing 200 μL of the cell suspension (2*10⁻⁶ cells per well) were cultured. 5 Lipopolysaccharide (LPS, Sigma-Aldrich, USA) was added to 96-well plates (wells per cell) to a final concentration of 2 μg / mL, and incubated overnight to establish an inflammation model. The experiment was conducted in the following groups:

[0079] Control group (Con): Only modified Taiwanese liquid was added, without prebiotics and LPS, and no modeling was performed.

[0080] Model group (MOD): Only modified Taiwanese liquid is added, without prebiotics, but LPS is added for modeling.

[0081] Prebiotic intervention group: COS, AOS, GOS or Inu (0.2g / mL) were added before modeling according to the above method, followed by LPS for modeling.

[0082] Next, cell supernatant was collected for cytokine experiments. The levels of immune-related factors such as IL-10 and IFN-γ were detected using ELISA. The results are as follows: Figure 17 As shown, compared with the model group, the COS group exhibited a more significant immunomodulatory effect, with a significant decrease in IL-10 levels (P<0.05) and effective inhibition of IFN-γ levels, indicating that COS can alleviate the inflammatory response induced by sensitizing factors and reduce the release of macrophage-mediated pro-inflammatory signals. Compared with other prebiotic groups, COS had a more prominent effect in reducing inflammatory factors.

[0083] 2) RBL-2H3 cell experiment

[0084] RBL-2H3 cells are a commonly used effector cell model for allergic reactions, and their degranulation reaction can directly reflect the allergic process. RBL-2H3 cell modeling was performed when the cells were in optimal condition. First, anti-dinitrophenyl antibody (Anti-DNPIgE, Sigma-Aldrich, USA) was diluted to 2 mg / mL with a modified Trussard solution. Then, it was added to a 200 μL cell suspension (2 x 10⁻⁶ cells / mL). 5 In a 96-well plate culture medium containing cells (cells / well), the final concentration of anti-dinitrophenyl antibody was 0.5 mg / mL, and the plate was incubated at 37°C for 20 h. After incubation, the serum-containing medium was discarded, and the plate was washed three times with modified Trussard medium. Serum-free medium was then added to obtain serum-free medium containing cells.

[0085] Subsequently, different prebiotic solutions prepared with modified TrussorFlow (including chitosan oligosaccharide (COS), alginate oligosaccharide (AOS), galacto-oligosaccharide (GOS), inulin (Inu), stachyose (STA), isomaltooligosaccharide (IMO), fructooligosaccharide (FOS), and trehalose (TRE), with a prebiotic concentration of 0.2 g / mL) were added to the serum-free culture medium containing cells to achieve a final prebiotic concentration of 156.25 mg / mL. The culture was then incubated at 37°C for 4 h. Afterward, 40 μL of 2,4-dinitrophenyl-conjugated bovine serum albumin (DNP-BSA, AbMole, USA) was added to each well to achieve a final concentration of 400 ng / mL, and the culture was continued for another 4 h.

[0086] Control group (Con): No Anti-DNP IgE and DNP-BSA were added, only an equal volume of modified benchtop solution was added.

[0087] Model group (MOD): Add Anti-DNP IgE and DNP-BSA, but no prebiotics, only an equal volume of modified Bento liquid.

[0088] Prebiotic intervention group: Anti-DNP IgE and DNP-BSA were added according to the above method, and COS, AOS, GOS, Inu, STA, IMO, FOS or TRE were added for treatment respectively.

[0089] Cell supernatant was collected after incubation to detect cytokine levels. Its regulatory effects on histamine release, MCPt-1 levels, and inflammatory factors such as IL-4 and IL-17A were examined. Experimental results are as follows: Figure 18 and Figure 19 As shown, the COS group significantly reduced the release levels of histamine and MCPt-1, effectively inhibited the increase of Th2 / Th17-related cytokines (IL-4 and IL-17A), and promoted the recovery of IFN-γ, with significant differences compared to the model group (P<0.01). In contrast, although other prebiotic treatment groups also showed some improvement, the effects were not as significant as those of COS.

[0090] 3) Comprehensive Analysis

[0091] Dual validation using RAW264.7 and RBL-2H3 cells demonstrates that COS is superior to traditional prebiotics such as fructooligosaccharides, galacto-oligosaccharides, and inulin in regulating immune homeostasis, inhibiting the release of inflammatory factors, and alleviating allergic reactions. Its advantages are reflected in:

[0092] 1. It has a more significant inhibitory effect on Th2 / Th17 inflammatory factors;

[0093] 2. The intervention effect on mast cell mediator release (histamine, MCPt-1) is the most significant;

[0094] 3. It can simultaneously regulate both pro-inflammatory and anti-inflammatory factors, exhibiting a more balanced immune regulatory effect.

[0095] In summary, this embodiment demonstrates that chitosan oligosaccharide exhibits the best anti-allergy potential among many prebiotics, further highlighting its application value as an active ingredient in functional foods or pharmaceutical compositions.

Claims

1. A method for alleviating milk allergy using chitosan oligosaccharides, characterized in that: Chitosan oligosaccharide can be administered before, during, or after sensitization to milk protein to alleviate allergy symptoms.

2. The method according to claim 1, characterized in that: The chitosan oligosaccharide is a water-soluble oligomer with a degree of deacetylation ≥90% and a molecular weight of 340–1630 Da.

3. The method according to claim 1, characterized in that: Administer chitosan oligosaccharide at a dose of 50-600 mg / kg body weight.

4. The method according to claim 1, characterized in that: The chitosan oligosaccharide is a chitosan oligosaccharide solution with a concentration of 6-10 mg / mL.

5. The method according to claim 1, characterized in that: By regulating the immune balance of Th1, Th2, and Th17, and restoring gut microbiota homeostasis.

6. The method according to claim 4, characterized in that: Restoring the gut microbiota included a decrease in the Firmicutes / Bacteroidota ratio, a decrease in Bacteroidetes abundance, an increase in Verrucomicrobia and Proteobacteria abundance, an increase in Muribaculaceae and Lactobacillaceae abundance, a decrease in Akkermansiaceae abundance, a decrease in Akkermansia and Muribaculum abundance, and an increase in Prevotella, Rikenella, Lactobacillus, Alistipes, and Parabacteroides abundance.

7. The method according to claim 4, characterized in that: Immune regulation includes reducing IgE, IgG1, IL-4, IL-10, and IL-17A, increasing IFN-γ, and decreasing histamine and MCPt-1 levels.

8. The use of a chitosan oligosaccharide in the preparation of functional foods or compositions for relieving milk allergy.

9. The application according to claim 8, characterized in that: The dosage of chitosan oligosaccharide is 50-600 mg / kg body weight.

10. The application according to claim 8, characterized in that: The functional food is an infant formula, solid beverage, nutritional supplement, or dairy product; the composition is a pharmaceutical composition.

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