Lactobacillus rhamnosus AFY04 and application thereof in relieving food allergy

Abstract

The invention belongs to the field of functional microorganisms, and particularly discloses lactobacillus rhamnosus AFY04 and application thereof in relieving food allergy, the lactobacillus rhamnosus AFY04 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation date is June 11, 2024, and the preservation number is CGMCC No.30982, and the lactobacillus rhamnosus AFY04 is preserved in the China General Microbiological Culture Collection Center (CGMCC). It is found that the lactobacillus rhamnosus AFY04 can effectively relieve migration from Th1 cells to Th2 cells in an allergic mouse body and adjust Th1 / Th2 immune balance; in addition, normal structures of spleen and intestine tissues can be maintained, the tissue morphologies of colon, jejunum and spleen are protected to be complete, the intestinal barrier function is enhanced, and allergy is relieved; potential food additives, feed additives and medicine preparation application are realized.

Description

Technical Field

[0001] The invention belongs to the field of functional microorganisms, and specifically discloses a Lactobacillus rhamnosus AFY04 and an application thereof in alleviating food allergies. Background Art

[0002] Food allergy is a major public health issue affecting both adults and children. It is a pathological response of the immune system triggered by the ingestion of food protein antigens. Its incidence has increased substantially in recent decades. Exposure to very small amounts of food antigens can trigger an allergic reaction. Milk is one of the earliest and most common sources of allergens in the world, and β-lactoglobulin is a major allergen protein. To date, treatment of food allergies has been ineffective due to a lack of clear understanding of the molecular mechanisms involved. Therefore, the best way to treat this disease is to avoid exposure to food allergens; however, accidental ingestion is difficult to completely avoid.

[0003] According to whether the allergic reaction is mediated by IgE (Immunoglobulin E, IgE), food allergies can be divided into IgE-mediated food allergic reactions, non-IgE-mediated food allergic reactions, and mixed food allergies mediated by both IgE and immune cells. Among them, IgE-mediated food allergic reactions are the most common. In the study of the pathogenesis of IgE-mediated type 1 allergic reactions, it is because the proteins in the food are regarded as antigenic substances. After passing through the intestinal mucosal barrier, they are absorbed by dendritic cells (DC) and taken to the local lymph nodes, degraded into peptides, and delivered to native CD4+T cells through the class II molecules MHCⅢI of the major histocompatibility complex (MHC) on the surface of DC, activating helper T cell 2 (Th2) cells to differentiate and proliferate, secreting a large amount of Th2 cytokines such as IL-4'Interlcukin-4, IL-4, which can induce B cells to proliferate and differentiate into plasma cells that can produce IgE. The Fc segment of IgE produced by plasma cells has a high affinity receptor Fce rcccptorI (FccR 1) for mast cells and basophils. After binding to mast cells or basophils, the body is in a sensitized state. When the same food allergen enters the body again, the IgE antibodies on the surface of sensitized mast cells or basophils can quickly recognize and bind to the corresponding antibodies, prompting mast cells or basophils to degranulate and release a large number of pro-inflammatory mediators and cytokines, such as IL-4, IL-5, etc. Mast cells stimulate eosinophils and other inflammatory cells to produce allergic reactions by releasing existing or newly synthesized IL-5, IL-13 and other factors.

[0004] Lactobacillus rhamnosus is an anaerobic bacterium that mainly exists in the intestines of humans and animals. It has the ability to resist the acidic environment of the gastrointestinal tract and bile salts, and also has the characteristics of tolerance to multiple antibiotics. It has high tolerance, high adhesion and strong colonization ability for the gastrointestinal environment. These characteristics help to enhance the intestinal mucosal barrier function, inhibit the adhesion of pathogens, produce antibacterial substances, competitively exclude pathogenic microorganisms, and improve the body's nonspecific immunity. Therefore, it has been one of the most widely studied probiotic strains and is used in various commercially available probiotic products. The beneficial effects of this strain have been widely studied in clinical trials and human intervention studies. Finding and screening new strains of Lactobacillus rhamnosus with better beneficial effects is also a hot topic of current research. Summary of the invention

[0005] In view of the above problems, the present invention discloses a Lactobacillus rhamnosus AFY04 and an application thereof in alleviating food allergies.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] A Lactobacillus rhamnosus strain, the Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain AFY04, is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of June 11, 2024 and a deposit number of CGMCC No.30982.

[0008] The invention also discloses a lactobacillus rhamnosus product, which contains the live bacteria of the lactobacillus rhamnosus AFY04 and / or fermentation liquid and / or metabolites.

[0009] The present invention also discloses the use of the Lactobacillus rhamnosus or Lactobacillus rhamnosus product in preparing a preparation for alleviating / treating food allergy.

[0010] Furthermore, in the above use, the food allergy is caused by β-lactoglobulin.

[0011] The invention also discloses a food additive containing the lactobacillus rhamnosus or the lactobacillus rhamnosus product.

[0012] The invention also discloses a feed additive containing the lactobacillus rhamnosus or the lactobacillus rhamnosus product.

[0013] Furthermore, the above-mentioned feed additive is used for feeding mice.

[0014] The invention also discloses a medicine for alleviating / treating food allergy, which contains the Lactobacillus rhamnosus or the Lactobacillus rhamnosus product as mentioned above.

[0015] Furthermore, in the above-mentioned medicine, the food allergy is caused by β-lactoglobulin.

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

[0017] The present invention utilizes beta-lactoglobulin intraperitoneal injection to construct a mouse allergy model, and beta-lactoglobulin (BLG) is the most immunogenic and highest content allergic milk protein in whey protein. Its pathogenesis is studied in IgE-mediated type 1 allergic reaction, because the protein in food is regarded as an antigenic substance, after passing through the intestinal mucosal barrier, a series of reactions induce B cell proliferation, and finally differentiate into plasma cells that can produce IgE. When the same food allergen enters the body again, the IgE antibodies on the surface of sensitized mast cells or basophils can quickly identify the corresponding antibodies and combine with them, prompting mast cells or basophils to undergo degranulation reactions, releasing a large amount of proinflammatory mediators and cytokines, causing intestinal barrier damage, imbalance of Th1 / Th2 immune response, and increased secretion of Th2 and Th17 type cytokines. The results of the embodiment of the present invention show that Lactobacillus rhamnosus AFY04 can protect the normal morphology of young mouse colon, jejunum, spleen tissue and cells in vivo, and can increase the relative expression of tight junction proteins and enhance intestinal barrier function, and alleviate allergies. In addition, Lactobacillus rhamnosus AFY04 can inhibit Th2 response by inducing the differentiation of Th1 cells and maintain the relative balance of Th1 / Th2 immune response.

[0018] In summary, Lactobacillus rhamnosus AFY04 is an important bacterium that can effectively alleviate the shift of Th1 cells to Th2 cells in allergic mice and regulate the Th1 / Th2 immune balance. In addition, it can maintain the normal structure of spleen and intestinal tissue, protect the integrity of colon, jejunum and spleen tissue morphology, and enhance intestinal barrier function; it has potential uses as a food additive, feed additive and pharmaceutical preparation.

[0019] The strain deposit information is as follows:

[0020] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;

[0021] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0022] Deposit date: June 11, 2024;

[0023] Deposit number: CGMCC No.30982;

[0024] Taxonomic name: Lacticaseibacillus rhamnosus. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Mouse weight changes;

[0026] Figure 2 Pictures of symptoms of food allergy in mice;

[0027] Figure 3 Allergy symptom scores of mice. The letters in the bar graphs indicated significant differences in the Ducan polynomial test (p < 0.05). AFY04 low-dose group: 1×10 8 CFU / kg.BW; AFY04 high-dose group: 1×10 9 CFU / kg.BW;

[0028] Figure 4 Immunoglobulin levels in mouse serum. The letters ad in the bar graphs show significant differences in the Ducan polynomial test (p < 0.05). AFY04 low-dose group: 1×10 8 CFU / kg.BW; AFY04 high-dose group: 1×10 9 CFU / kg.BW;

[0029] Figure 5 Cytokine levels in mouse serum. The letters ad in the bar graphs show significant differences in the Ducan polynomial test (p < 0.05). AFY04 low-dose group: 1×10 8 CFU / kg.BW; AFY04 high-dose group: 1×10 9 CFU / kg.BW;

[0030] Figure 6 Pathological observation of mouse colon H&E staining;

[0031] Figure 7 Pathological observation of mouse jejunum by H&E staining;

[0032] Figure 8 Pathological observation of mouse spleen by H&E staining;

[0033] Fig. 9 mRNA expression of intestinal function-related factors in mice. Different letters in the bar graphs indicated significant differences in the Ducan polynomial test (p < 0.05). AFY04 low-dose group: 1×10 8 CFU / kg.BW; AFY04 high-dose group: 1×10 9 CFU / kg.BW;

[0034] Fig.10mRNA expression of cytokines in mouse jejunum tissue. The letters ad in the bar graphs show significant differences in the Ducan polynomial test (p < 0.05). AFY04 low-dose group: 1×10 8 CFU / kg.BW; AFY04 high-dose group: 1×10 9 CFU / kg.BW. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0037] 1 Materials and reagents

[0038] Lactobacillus rhamnosus AFY04 was isolated from naturally fermented yogurt in herdsmen's homes in Kashgar, Xinjiang Uygur Autonomous Region by the Collaborative Innovation Center for Child Nutrition and Healthy Development. It is now preserved in the China General Microbiological Culture Collection Center with the collection number: CGMCC No.30982.

[0039] Table 1 Experimental materials and reagents

[0040]

[0041]

[0042] 2. Instruments and Equipment

[0043] Table 2 Instruments and equipment

[0044]

[0045]

[0046] 3 Experimental methods

[0047] 3.1 Animal Model

[0048] Twenty-eight four-week-old BALB / c mice were randomly divided into four groups: a high-dose AFY04 group (1×10 9 CFU / mL), low-dose AFY04 group (1×10 8CFU / mL), normal group, BLG induced group, 7 mice in each group. The normal group used AIN-95G standard feed throughout the experimental period and had free access to water. In addition to normal feeding of AIN-95G, free drinking water and oral gavage of the corresponding lactobacilli, the BLG induced group, the low-dose group of Lactobacillus rhamnosus AFY04, and the high-dose group of Lactobacillus rhamnosus AFY04 were sensitized by intraperitoneal injection of lactoglobulin on the 7th, 14th and 21st days. Each mouse was injected with 0.2ml of sensitization solution (20mg lactoglobulin, 10ml saline and 10ml Freund's adjuvant). On the 28th day, lactoglobulin stimulator was gavaged twice, and each mouse was gavaged with 1mg / g stimulating solution and lactoglobulin. The normal group was modeled and given saline as a control. The allergic symptoms of mice were evaluated, blood was collected from the eyeballs after anesthesia, and the mice were killed by dislocation. The thymus and spleen of the mice were weighed and the weight was recorded. About 5cm of colon and jejunum were cut, and 1 / 3 of the spleen was placed in 4% paraformaldehyde for fixation.

[0049] 3.2 Determination of mouse body weight and organ index

[0050] Body weight measurement: Place the beaker securely on the electronic scale, adjust to zero and confirm that the pointer is at 0 again, gently place the mouse in the groove, and quickly read the measurement results when the BALB / c mouse is in a static state and record the measurement data with an accuracy of 0.01. On the 28th day, dissect the mouse, weigh and record the weight of the thymus and spleen.

[0051] 3.3 Evaluation of allergic symptoms in mice

[0052] The food allergy symptom score ratios of mice are shown in Table 3.

[0053] Table 3 Food allergy symptom score table for mice

[0054]

[0055] 3.4 Determination of mouse immunoglobulin and cytokine content

[0056] Blood was collected from the retroorbital plexus on day 28 and then centrifuged at 4000 rpm for 20 min at 4°C; the serum was then stored at −80°C until use. The levels of immunoglobulins IgE and IgM, and cytokines IL-4, IL-5, IL-13, IL-17A, TNF-α, TGF-γ, and IFN-β in serum were measured by ELISA according to the manufacturer's instructions.

[0057] 3.5 H&E staining sections of mouse colon, jejunum, and spleen tissues

[0058] The colon, jejunum and spleen tissues were fixed, dehydrated, embedded and stained, and finally observed under a microscope and photographed.

[0059] 3.6 Determination of mRNA expression in mouse jejunum tissue

[0060] Total RNA was extracted from jejunal tissue and reverse transcribed into cDNA. SYBR, DDW, forward and reverse primers (Table 4) and template cDNA were added in sequence. The Ct value was obtained by 40 cycles of 95°C for 15s, 55°C for 30s, and 72°C for 35s. -ΔΔCt The expression level of target gene mRNA was calculated by this method.

[0061] Table 4 qPCR primer sequences (numbered in sequence as SEQ ID No. 1-28)

[0062]

[0063]

[0064] 3.7 Data Processing

[0065] SPSS 23.0 software was used to perform statistical analysis on the data of each group. The Duncan method was used to analyze the mean and variance of each group, and different letters ad indicate significant differences in the mean values ​​(p<0.05).

[0066] Example 1

[0067] Isolation and identification of Lactobacillus rhamnosus strain AFY04.

[0068] 1. Experimental Materials

[0069] Naturally fermented yak yogurt was collected from the Altay region of Xinjiang. 40 mL of naturally fermented yogurt was taken into a sterile centrifuge tube, placed in a food sampling box, and stored in a 4°C refrigerator in the laboratory for later use.

[0070] 2. Experimental Methods

[0071] 1. Isolation and identification of Lactobacillus

[0072] 1.1 Isolation and purification of Lactobacillus

[0073] Take 1 mL of naturally fermented yogurt sample and dilute it 10 times to 10 with sterile saline. -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of 3 gradient bacterial solutions were spread on the plate and cultured at 37℃ for 24-48h. The colony morphology was observed and recorded. Colonies with different morphologies on the plate were picked for streaking separation. After culturing at 37℃ for 48h, single colonies with different morphologies on the plate were picked for streaking separation again. This was repeated 2 to 3 times until pure single colonies with consistent morphology were obtained.

[0074] 1.2 Lactobacillus DNA extraction

[0075] Inoculate the purified suspected target strain into MRS broth, culture at 37℃ for 18-24h, and then use a bacterial genomic DNA extraction kit to extract DNA. Number the extracted DNA and store it in a -20℃ freezer for later use.

[0076] 1.4 PCR amplification of genomic DNA and agarose gel electrophoresis detection

[0077] The extracted DNA was subjected to PCR amplification, including 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTCAG-3'=SEQ ID No.29), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3'=SEQID No.30), 12.5 μL of 2×Taq plus Buffer, 1 μL of template DNA, and the system was supplemented to 25 μL with sterile dd H2O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94℃ 5min; 94℃ 30s, 55℃ 30s, 72℃ 1min, a total of 29 cycles, and finally 72℃ extension for 5min.

[0078] Then 5 μL of the amplified product was taken for agarose gel electrophoresis detection, the agarose concentration was 1.5%, the electrophoresis conditions were 110V, 45min. The successfully detected PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequenced sequence was compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0079] 1.5 Lactobacillus in vitro resistance screening

[0080] 1.5.1 Ability of Lactobacillus to Tolerate 0.3% Bile Salts

[0081] Porcine bile salt was added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make the concentration 0.3%, and sterilized at 121℃ for 15min. 5mL of activated bacteria were inoculated into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt at a 2% (v / v) inoculation volume, respectively. Blank medium (MRS-THIO medium without bacteria) was used as control. After culturing at 37℃ for 24h, the OD values ​​of the above different concentrations of culture medium were measured respectively. 600nm The value was used to calculate the tolerance of the strain to bile salts according to formula (1):

[0082]

[0083] 1.5.2 Artificial gastric juice tolerance test

[0084] Preparation of artificial gastric juice: Artificial gastric juice is composed of 0.2% NaCl and 0.35% pepsin. The NaCl and pepsin required for the test are weighed according to the corresponding mass volume ratio for preparation. The pH of the prepared artificial gastric juice is adjusted to 3.0 with 1 mol / L HCl, and then filtered with a 0.22 μm filter membrane for sterilization and set aside.

[0085] In the clean bench, 5 mL of the cultured bacterial culture was taken into a 10 mL sterile centrifuge tube, centrifuged at 3000 r / min for 10 min, the upper culture medium was discarded and the bacteria were collected, and an equal volume (5 mL) of sterile physiological saline was added and mixed to make a bacterial suspension, and then 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric juice of pH 3.0, and 1 mL of the above mixture was taken as the sample of artificial gastric juice treatment 0 h, and the remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37 ° C, 150 r / min) and cultured for 3 h. The samples of 0 h and 3 h were diluted by 10 times gradient, and the number of viable bacteria was determined by plate coating method with appropriate gradients, and cultured on MRS solid medium at 37 ° C for 48 h, and the survival rate (%) was calculated according to formula 2.

[0086]

[0087] 2. Results of determination of Lactobacillus in samples

[0088] 2.1.1 Sequencing of Lactobacillus PCR amplification products

[0089] The sequence of AFY04 is as follows: SEQ ID No. 31 =

[0090]

[0091] 2.2.3 Gene Bank analysis of Lactobacillus species

[0092] BLAST program comparison analysis showed that the homology between AFY04 and known Lactobacillus rhamnosus in the Gene Bank database reached 99%.

[0093] Table 5 16S rDNA sequence analysis results of AFY04

[0094]

[0095]

[0096] 2.2.4 Results of in vitro resistance of Lactobacillus

[0097] As shown in Table 6, Lactobacillus rhamnosus AFY04 has a higher survival rate in artificial gastric juice and a stronger ability to tolerate bile salts.

[0098] Table 6 Survival rate of Lactobacillus rhamnosus AFY04 in pH 3.0 artificial gastric juice and 0.3% bile salt

[0099]

[0100] Example 2

[0101] 1 Effects of food allergy on body weight and organ parameters of mice

[0102] Food allergy modeling can cause weight loss in mice. The mice were weighed on the 0th and 28th days of the experiment. On the 28th day, mice other than the normal group were gavaged with lactoglobulin stimulator twice, with each mouse gavaged with 1 mg / g lactoglobulin stimulator. After the stimulation, the mice were fasted for 3 hours and weighed again. Figure 1 As shown in the figure, before the challenge, the weight of mice in the normal group decreased slightly due to fasting, and the weight of mice in the BLG-induced group decreased significantly. The low-dose AFY04 group and the high-dose AFY04 group alleviated the weight loss of mice caused by food allergy modeling, and the relief effect of the high-dose AFY04 group was better than that of the low-dose AFY04 group. The weight of mice after the challenge showed the same trend as before the challenge, and decreased relative to the weight before the challenge.

[0103] Table 7 Thymus and spleen indicators of mice (unit: %)

[0104]

[0105] In Table 7, the thymus index and spleen index were ranked from large to small as follows: BLG induction group, AFY04 low-dose group, AFY04 high-dose group and normal group (p < 0.05).

[0106] 2 Evaluation of allergic symptoms in mice

[0107] On the 28th day, each group of mice was gavaged with lactoglobulin stimulator, and the symptoms of the mice were observed during the fasting process. The general symptoms of food allergy include scratching, messy hair, weight loss, reduced spontaneous activity, diarrhea, purple lips, no response to stimulation, etc. ( Figure 2 ). Real-time observation of the mice after stimulation showed repeated scratching of their ears and mouths, scratching of the ear canals with their hind legs, reduced activity, swelling around the eyes and mouth, and even swelling of the feet.

[0108] The food allergy symptoms induced by β-lactoglobulin were evaluated and the allergy symptoms of mice were scored according to the above scoring table ( Figure 3 ), the normal group mice had smooth and shiny hair, normal weight, and no reduction in activity. The BLG-induced group showed severe symptoms of food allergy (anaphylactic reaction, 2.71±0.45 points), with erect hair, scratching of the head and ears, reduced activity, and edema around the eyes and mouth. The mice in the low-dose AFY04 group did not show edema, but still had messy hair and scratching. The mice in the high-dose AFY04 group were close to the normal group, with no significant changes (1.14±0.35 and 0.29±0.25 points, respectively). Supplementation of Lactobacillus rhamnosus AFY04 significantly inhibited BLG-induced food allergy, and the high dose was better than the low dose.

[0109] 3 Effect of Lactobacillus rhamnosus AFY04 on the level of immunoglobulin in mouse serum

[0110] IgE-mediated food allergy is an immune response that induces B cells to produce IgE antibodies when the body comes into contact with food allergens. Therefore, the severity of mouse allergies can be assessed by detecting the content of specific IgE antibodies in serum. Figure 4 As shown, according to the determination of IgE and IgM in mouse serum, the low-dose and high-dose AFY04 groups significantly reduced the levels of immunoglobulins IgE and IgM in mouse serum compared with the BLG-induced group.

[0111] 4. Effect of Lactobacillus rhamnosus AFY04 on cytokine levels in mouse serum

[0112] Figure 5The results are as follows: The contents of cytokines in mouse serum. The contents of IL-4, IL-5, IL-13, IL-17A and TNF-α in the serum of mice in the BLG-sensitized group were significantly increased compared with those in the normal group. The contents of IL-4, IL-5, IL-13, IL-17A and TNF-α in the serum of mice in the AFY04 group were significantly decreased compared with those in the BLG-sensitized group (p < 0.05), but there were still significant differences compared with the normal group (p < 0.05). In addition, compared with the normal group, the contents of TGF-β and IFN-γ in the BLG-sensitized group were significantly decreased (p < 0.05). After AFY04 intervention, the contents of TGF-β and IFN-γ were significantly increased compared with those in the BLG-sensitized group (p < 0.05). The trends of the AFY04 low-dose group and the AFY04 high-dose group were consistent, but there were still significant differences compared with the normal group (p < 0.05).

[0113] 5. Observation of Colon Histopathology in Food Allergic Mice

[0114] The colon tissue structure of mice in the normal group was intact, the number and structure of goblet cells were normal, the glandular structure was neatly arranged, and there was no inflammatory cell infiltration. After sensitization with β-lactoglobulin, compared with the normal group, a large number of inflammatory cell infiltrations, erosion of epithelial cells, damage to the mucosa and submucosa were found in the colon sections of the BLG-induced group, and the colon glandular structure and goblet cells disappeared to varying degrees. The colon sections of the AFY04 low-dose group showed improved glandular structure and a small amount of inflammatory cell infiltration. The normality of cells in the AFY04 high-dose group was significantly close to that of the normal group. The intestinal glandular cells were arranged regularly, the goblet cells tended to be normal, and there were still a small number of inflammatory cells ( Figure 6 ).

[0115] 6. Observation of Jejunum Histopathology in Food Allergic Mice

[0116] The results of H&E staining of mouse jejunum are as follows Figure 4-8 As shown in the figure, the integrity of the intestinal mucosa and the structure of the villi in the BLG-induced group were destroyed, and the submucosal layer was obviously edematous, with immune cell infiltration. The injury symptoms in the low-dose AFY04 group were alleviated, the swelling of the villi was relieved to a certain extent, and the infiltration of inflammatory cells was reduced. The injury symptoms of the jejunal tissue in the high-dose AFY04 group were further alleviated, the villi tended to be normal, and there was still a small amount of inflammatory cell infiltration ( Figure 7 ).

[0117] 7. Histopathological observation of spleen in food allergic mice

[0118] The spleen is the largest immune organ in the body and one of the important tissues for the body to produce antigen-specific T cell responses and B cells to produce antibodies. Figure 8In the middle, the pink part at the edge of the specimen is the capsule, and the inside is the spleen parenchyma. In the BLG-induced group, the capsule structure was severely damaged, the red and white pulp boundaries were unclear, the splenic white pulp lymphocytes decreased, the splenic nodules were smaller, and a large number of macrophages proliferated around the nodules. In comparison, the red and white pulp boundaries in the AFY04 low-dose group were acceptable, and the apoptotic lymphocytes decreased. In the AFY04 high-dose group, the capsule was slightly damaged, the splenic red and white pulp boundaries were clear, the number of lymphocytes was large, and the overall structural integrity was close to that of the normal group.

[0119] 8. Effects of Lactobacillus rhamnosus AFY04 on intestinal function in food allergic mice

[0120] Probiotics can alleviate the damage to the intestinal barrier caused by food allergies. Lactobacillus rhamnosus LGG can upregulate the mRNA expression of tight junction proteins Claudin1, Occludin and Tjp1 in the intestine of allergic mice, enhance intestinal barrier function and reduce intestinal permeability. The tight junction proteins Claudin1, Occludin, Tjp1, JAM1, TSLP, IL-25 and IL-33 in the jejunum were measured. Fig. 9 As shown in the figure, compared with the normal group, the expression of Claudin1, Occludin, Tjp1 and JAM1 in the BLG sensitized group was reduced, while the expression levels of Claudin1, Occludin and Tjp1 showed a recovery trend after supplementation with Lactobacillus rhamnosus AFY04, but were still lower than those in the normal group. Compared with the low-dose AFY04 group, the expression levels in the high-dose AFY04 group were increased. In addition, TSLP, IL-25 and IL-33 were highly expressed in the BLG induced group, with the lowest content in the normal group. The TSLP, IL-25 and IL-33 contents in the low-dose AFY04 group were higher than those in the high-dose AFY04 group, and the difference was statistically significant (p < 0.05).

[0121] 9. Effects of Lactobacillus rhamnosus AFY04 on cytokines in jejunum of mice with food allergy

[0122] Food allergy can cause an imbalance in the Th1 / Th2 immune response, and the secretion of Th2 and Th17 cytokines increases. The mediators of β-lactoglobulin-induced intestinal allergies include cytokines secreted by Th2 cells, mast cells, and various inflammatory cells. To explore the effects of probiotic strains on immune response, the levels of Th2 cytokines IL-4, IL-5, IL-6, IL-13, Th17 cytokine IL-17F, Th1-related cytokine IFN-γ, and Treg cytokine TGF-β in jejunal tissue were measured ( Fig.10). After intervention with Lactobacillus rhamnosus AFY04, the expression levels of IL-4, IL-5, IL-6, IL-13, and IL-17F in the jejunal tissue of allergic mice were significantly reduced, and the expression of IFN-γ and TGF-β in the high-dose AFY04 group was significantly increased, by about 7 and 6 times, respectively (p < 0.05).

[0123] 10. Analysis and discussion:

[0124] Food allergy is a pathological response of the immune system triggered by the ingestion of food protein antigens. [1] In order to study the alleviating effect of Lactobacillus rhamnosus on food allergy induced by β-lactoglobulin in young mice, this experiment used β-lactoglobulin solution to induce the allergy model. Milk is one of the earliest and most common sources of allergens in the world, and β-lactoglobulin is a major allergen protein. [2] During the development of food allergies, the colon mucosa of mice will be damaged. The jejunum is the site where the structural changes of food allergies are most obvious and is one of the evaluation indicators of food allergies. The spleen is an important immune organ, the center of cellular immunity and humoral immunity, and the largest immune organ in the body. [3-5] The results of this experiment showed that the colon and jejunum of mice in the BLG-induced group had large areas of deep ulcers, infiltration of inflammatory cells and immune cells, and damage to the mucosal structure under the microscope. The spleen sections showed large areas of structural damage, unclear boundaries between the red and white pulps of the spleen, decreased lymphocytes, and increased macrophages. The colon and jejunum sections of the group that were gavaged with Lactobacillus rhamnosus AFY04 showed reduced inflammatory infiltration under the microscope, neatly arranged glands, tightly arranged intestinal villi, and few cases of shedding or breaking. The boundaries between the red and white pulps in the spleen sections were acceptable, with more lymphocytes and tending to normal morphology. Therefore, Lactobacillus rhamnosus AFY04 may have the effect of restoring intestinal tissue morphology and protecting the spleen.

[0125] Exposure to very small amounts of food antigens can trigger an allergic reaction, with typical symptoms including gastrointestinal reactions, urticaria, and respiratory inflammation, ranging in severity from mild to life-threatening. It can also be inferred that food allergy modeling can cause weight loss in mice [6] According to the experimental results, the weight of each group of mice was 24.18±0.38g on day 0. The mice were weighed before and after the stimulation on day 28. The weight of the BLG-induced group decreased by 9.32% and 22.56%, respectively. The weight of the AFY04 low-dose group and the AFY04 high-dose group increased significantly, and the effect of the AFY04 high-dose group was better than that of the AFY04 low-dose group. In addition, the β-lactoglobulin-induced food allergy in mice also damages the health of the mice's organs. The experiment found that the thymus and spleen of the BLG-induced group were enlarged. After intervention with AFY04, the thymus and spleen indexes of the mice were significantly reduced, but still higher than those of the normal group.

[0126] At the same time, the allergy symptom score can also reflect the severity of food allergy in mice to a certain extent, and oral administration of AFY04 has a certain allergic effect. After sensitization and stimulation, the allergy symptoms such as scratching, convulsions, and decreased activity of mice treated with Lactobacillus rhamnosus AFY04 were alleviated compared with the BLG-induced group.

[0127] IgE-mediated food allergy is an immune response that induces B cells to produce IgE antibodies when the body comes into contact with food allergens. Therefore, the severity of mouse allergies can be assessed by detecting the content of specific IgE antibodies in serum. [7] . According to the overall trend of serum IgE content in each group of mice during the sensitization stage, sensitization will cause the IgE of mice to increase. The IgE and IgM levels of mice in the BLG-induced group were the highest, and rhamnose AFY04 had a certain inhibitory effect on them. By studying the pathogenesis of IgE-mediated allergic reactions, it was found that allergic reactions are mainly caused by the rapid recognition and binding of IgE antibodies on the surface of sensitized mast cells or basophils to the corresponding antibodies, which prompts the mast cells or basophils to undergo degranulation reactions and release a large number of pro-inflammatory mediators and cytokines. Among them, inflammatory mediators such as tumor necrosis factor-α (TNF-α), IL-5, and IL-13 also play an important role in its pathogenesis. [8] .

[0128] T helper 2 cell (Th2) is a T cell subset that can secrete Th2-type cytokines (such as interleukins IL-4, IL-5 and IL-13, etc.), and belongs to CD4+T cells. These cytokines can promote the proliferation of Th2 cells and inhibit the proliferation of Th1 cells, while assisting the activation of B cells and playing a role in humoral immunity. B cell activation requires two signals, antigen stimulation signals (first signals) and co-stimulatory signals (second signals). Among them, co-stimulatory signals are generated by the interaction of multiple pairs of co-stimulatory molecules on the surfaces of Th2 cells and B cells. The most important co-stimulatory signal is CD40 / CD40L. CD40 is expressed on the surface of B cells, while CD40L is expressed on the surface of Th2 cells. On the other hand, cytokines secreted by Th2 cells (IL-4, IL-5, etc.) can act on a variety of cytokine receptors on the surface of B cells, causing B cells to proliferate in large numbers and further differentiate and produce antibodies. [9], effectively relieve allergic symptoms. The cytokines IL-4, IL-5, IL-13, IL-17A, TNF-α, IgM, TGF-β and IFN-γ were measured. The experimental results showed that compared with the BLG-induced group, the levels of IL-4, IL-5, IL-13, IL-17A and TNF-α in the serum of mice in the AFY04 group were significantly reduced, and the levels of TGF-β and IFN-γ increased, and the differences were significant (p<0.05). This shows that Lactobacillus rhamnosus AFY04 can reduce the levels of Th2 cytokines (IL-4, IL-5 and IL-13) and Th17 (IL-17A) in mice sensitized with β-lactoglobulin, and increase the levels of Th1 cytokines Treg cells (TGF-β) and Th1 cytokines (IFN-γ).

[0129] Tight junction (TJ) proteins provide intercellular adhesion in enterocytes and play a key role in regulating barrier permeability, which varies in different segments of the intestine. [10-11] Three types of transmembrane proteins are common to all TJs: claudins, MARVEL domain proteins, and junctional adhesion molecules (JAMs). Of these transmembrane proteins, occludin (OCLN) and claudins (CLDNs) form a barrier on the apical-lateral membrane of the cell, while peripheral membrane proteins, such as zona occludens (ZO), serve as links between transmembrane TJ proteins and the cytoskeleton

[41] . Claudins form selective pores for paracellular transport and are therefore the most important proteins involved in intestinal barrier permeability. [12-13] Immunofluorescence staining showed that Claudin-1 was mainly expressed in the colon

[14] , decreased claudin-1 expression levels may lead to tight junction dysfunction

[15] , and is considered to be a gastrointestinal

[16] Key mechanisms of allergic diseases. Tight junction proteins (such as Tjp1 and Occludin) form a barrier at the apical end of the adjacent epithelial cell membranes, preventing the transport of paracellular molecules between cells; together with adhesion junction proteins, they form the apical junction complex to regulate the integrity of the intestinal barrier.

[17] Studies have shown that the mRNA expression of Claudin-1, JAM, and Tjp1 in the proximal small intestine of mice was significantly downregulated after β-lactoglobulin sensitization, which may be a compensatory effect, indicating that the integrity of the intestinal epithelial cell layer is altered.

[18] The experimental results showed that after AFY04 supplementation, the expression of Claudin-1, JAM and Tjp1 in the jejunum increased, indicating that Lactobacillus rhamnosus AFY04 may affect intestinal permeability by regulating genes related to cell junction structure, thereby alleviating food allergy in mice.

[0130] Epithelial cells are essential for the initiation and coordination of immune responses, leading to sensitization to allergens. This includes the secretion of pro-allergenic cytokines such as IL-33, IL-25, and TSLP. [19-20] In this study, we found that the expression of IL-33, IL-25, and TSLP increased in the jejunal tissue of allergic mice after oral administration of Lactobacillus rhamnosus AFY04. The role of food proteases in the development of Th2 immune responses is associated with increased IL-25 and TSLP gene expression

[21] TSLP is an important intestinal epithelial cell factor that can activate Th2 cells.

[0131] Since both Th1 cells and Th2 cells can secrete cytokines to promote their own proliferation and inhibit the proliferation of the other, under normal circumstances, Th1 cells and Th2 cells in the body are in a relatively balanced state. However, when the body has dysfunctional functions, the balance often tends to one side, which is called "Th1 / Th2 drift". Once the balance between Th1 cells and Th2 cells is broken, it is likely to cause the dynamic balance of the human cytokine network to be destroyed, which in turn causes the occurrence and development of many diseases.

[22] For example, an imbalance in Th1 and Th2 cytokine levels is closely associated with the development of allergic reactions.

[23] Lactobacillus rhamnosus can regulate the balance of Th cells and related cytokine levels in a strain-specific manner. A large amount of evidence shows that Lactobacillus rhamnosus plays an important role in restoring the balance of Th1 / Th2 immune responses. IL-4 is a typical Th2 cytokine that plays an important role in allergic reactions and is involved in the proliferation and activation of mast cells.

[24] In addition, cytokines such as IL-5 and IL-13 secreted by Th2 cells are involved in intestinal mucus secretion, eosinophil and mast cell proliferation, and the production of allergen-specific IgE.

[25] . Lactobacillus rhamnosus AFY04 can significantly reduce the levels of Th2-related cytokines (IL-4, IL-5, IL-13) in mice after BLG induction and enhance the levels of Th1-related cytokines (IFN-γ) and Treg cytokines (TGF-β), thereby alleviating the allergic symptoms of mice. From the experimental results, it can be observed that Lactobacillus rhamnosus can effectively alleviate the levels of Th2 and Th17 cytokines in the jejunum of food-allergic mice, alleviate the shift of Th1 cells to Th2 cells, and play a better role in regulating the Th1 / Th2 immune balance.

[0132] 11. Conclusion

[0133] The present invention utilizes beta-lactoglobulin intraperitoneal injection to construct a mouse allergy model, and beta-lactoglobulin (BLG) is the most immunogenic and highest content allergic milk protein in whey protein. Its pathogenesis is studied in IgE-mediated type 1 allergic reaction, because the protein in food is regarded as an antigenic substance, after passing through the intestinal mucosal barrier, a series of reactions induce B cell proliferation, and finally differentiate into plasma cells that can produce IgE. When the same food allergen enters the body again, the IgE antibodies on the surface of sensitized mast cells or basophils can quickly identify the corresponding antibodies and combine with them, prompting mast cells or basophils to undergo degranulation reactions, releasing a large amount of proinflammatory mediators and cytokines, causing intestinal barrier damage, imbalance of Th1 / Th2 immune response, and increased secretion of Th2 and Th17 type cytokines. The results of the embodiment of the present invention show that Lactobacillus rhamnosus AFY04 can protect the normal morphology of young mouse colon, jejunum, spleen tissue and cells in vivo, and can increase the relative expression of tight junction proteins and enhance intestinal barrier function, and alleviate allergies. In addition, Lactobacillus rhamnosus AFY04 can inhibit Th2 response by inducing the differentiation of Th1 cells and maintain the relative balance of Th1 / Th2 immune response.

[0134] In summary, Lactobacillus rhamnosus AFY04 is an important intestinal bacterium that can effectively alleviate the shift of Th1 cells to Th2 cells in allergic mice and regulate the Th1 / Th2 immune balance. In addition, it can maintain the normal structure of spleen and intestinal tissue, protect the integrity of colon, jejunum and spleen tissue morphology, and enhance intestinal barrier function.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the specification and drawings of the present invention, directly or indirectly applying the above technical solutions to other related technical fields are all included in the protection scope of the patent of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus, characterized in that The Lactobacillus rhamnosus strain AFY04 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit date of June 11, 2024 and a deposit number of CGMCC No.30982.

2. A Lactobacillus rhamnosus product, characterized in that Contains the live bacteria of Lactobacillus rhamnosus AFY04 and / or fermentation liquid and / or metabolites as claimed in claim 1.

3. Use of Lactobacillus rhamnosus as claimed in claim 1 in preparing a preparation for alleviating / treating food allergies.

4. Use of the Lactobacillus rhamnosus product according to claim 2 in the preparation of a preparation for alleviating / treating food allergies.

5. The use according to claim 3 or 4, characterized in that: The food allergy is caused by beta-lactoglobulin.

6. A food additive, characterized in that: Containing the Lactobacillus rhamnosus according to claim 1 or the Lactobacillus rhamnosus product according to claim 2.

7. A feed additive, characterized in that: Containing the Lactobacillus rhamnosus according to claim 1 or the Lactobacillus rhamnosus product according to claim 2.

8. The feed additive according to claim 7, characterized in that The feed is used for feeding mice.

9. A drug for alleviating / treating food allergies, characterized in that: Containing the Lactobacillus rhamnosus according to claim 1 or the Lactobacillus rhamnosus product according to claim 2.

10. The drug according to claim 9, characterized in that The food allergy is caused by beta-lactoglobulin.

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