Preparation method of lactobacillus rhamnosus leavening and application of lactobacillus rhamnosus leavening in body immune regulation, immune balance maintenance and allergic symptom and inflammatory response relieving

Abstract

The invention belongs to the technical field of probiotic fermentation, and particularly relates to application of lactobacillus rhamnosus and medicinal and edible leavening preparation in the aspects of body immune regulation, immune balance maintenance and allergic symptom and inflammatory response relieving. Lactic acid bacteria with anti-allergic activity and good fermentation performance are screened to serve as fermentation strains, fructo-oligosaccharide, lilies and poria cocos with immunoregulatory activity are fermented, and further, the produced fermentation product with the anti-allergic activity is compounded with bifidobacteria to form the function-enhanced anti-allergic composition. Results of embodiments show that the prepared medicinal and edible leavening not only can effectively improve allergy caused by Thl / Th2 imbalance, but also can effectively regulate the intestinal flora structure and maintain immune balance.

Description

Technical Field

[0001] This invention belongs to the field of probiotic fermentation technology, specifically relating to the preparation method of Lactobacillus rhamnosus ferment and its application in regulating the body's immune system, maintaining immune balance, relieving allergy symptoms, and reducing inflammatory responses. Background Technology

[0002] Th1 / Th2 imbalance refers to a disruption of the dynamic balance between the two major immune subsets, helper T cells 1 (Th1) and T cells 2 (Th2), leading to a state of immune system dysfunction. This imbalance can directly induce or aggravate various diseases. If Th1 cell function is excessive, it can easily trigger autoimmune diseases (such as rheumatoid arthritis and thyroiditis) or organ transplant rejection. Conversely, if Th2 cells have a dominant response, it often leads to allergic diseases (such as asthma, allergic rhinitis, and atopic dermatitis) and increased susceptibility to parasites. Long-term Th1 / Th2 imbalance can also weaken the body's immune surveillance capabilities and affect health homeostasis.

[0003] Both Bifidobacteria and Lactobacilli possess anti-allergic activity. Bifidobacteria primarily affect gastrointestinal allergic reactions through intestinal colonization, while Lactobacilli can directly inhibit mast cell degranulation, showing significant improvement in respiratory allergies. Furthermore, fermented products rich in short-chain fatty acids, polypeptides, polyphenols, polysaccharides, and other active small molecules have also been widely proven to have anti-allergic activity. However, Bifidobacteria are anaerobic bacteria with poor fermentation performance.

[0004] Chinese invention patent CN114848685 discloses an anti-allergy probiotic composition and its application, as well as a probiotic fermented traditional Chinese medicine product. Although the probiotic composition includes Lactobacillus and Bifidobacterium, the use of these strains together in the fermentation of the traditional Chinese medicine composition cannot ensure the survival of anaerobic Bifidobacterium in the fermentation product. Chinese invention patent CN103907927A discloses a diet for regulating special constitutions, which, although it adds plant (traditional Chinese medicine source) ingredients and extracts some plants, is then directly mixed with probiotics. However, its probiotic formula is complex, including Bifidobacteria (Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum) and Lactobacillus (Lactobacillus acidophilus powder, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus subsp. lactis, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus salivarius, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus johnsonii), which is an accumulation of most known probiotics, making it impossible to confirm or verify the rationality of its formulation.

[0005] Therefore, there is an urgent need to develop a well-matched, safe and effective probiotic composition to regulate the Th1 / Th2 imbalance in the body. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Lactobacillus rhamnosus ferment and its application in regulating the body's immune system, maintaining immune balance, relieving allergy symptoms, and reducing inflammatory responses. The Lactobacillus rhamnosus ferment provided by this invention has a significant effect on allergies caused by Th1 / Th2 imbalance, including allergic rhinitis and food allergies, and its effect is superior to that of a single traditional Chinese medicine composition, a single probiotic composition, or a probiotic fermentation product.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a Lactobacillus rhamnosus ferment, which uses Lactobacillus rhamnosus NKU ML1-2 as the fermentation strain, and the fermentation product obtained after fermentation is combined with Bifidobacterium NKU FB3-14 to prepare Lactobacillus rhamnosus ferment. The Lactobacillus rhamnosus NKU ML1-2 has the accession number GDMCC No. 66837; the Bifidobacterium NKUFB3-14 has the accession number CGMCC No. 25762.

[0008] Preferably, the fermentation process includes mixing and fermenting Lactobacillus rhamnosus NKU ML1-2 with millet protein peptides, fructooligosaccharides, lily powder and Poria cocos powder; The carbon-nitrogen mass ratio of the oligofructose is 1:5.

[0009] The present invention also provides a method for preparing the above-mentioned Lactobacillus rhamnosus ferment, comprising the following steps: S1. Add millet protein peptides, fructooligosaccharides, lily powder and poria powder to water in proportion to obtain a liquid mixture; S2. Inoculate Lactobacillus rhamnosus NKU ML1-2 into the liquid mixture, ferment at 37°C for 24 h, centrifuge, and collect the fermentation supernatant. S3. Inoculate the fermentation supernatant with live Bifidobacterium NKU FB3-14 to obtain Lactobacillus rhamnosus ferment.

[0010] Preferably, in step S1, the added masses of millet protein peptide, fructooligosaccharide, lily powder and poria powder are 10%, 2%, 1% and 1% of the water volume, respectively, based on the volume of water.

[0011] Preferably, in step S2, the inoculum size of *Lactobacillus rhamnosus* NKU ML1-2 is 10 g / L based on the volume of the liquid mixture. 8 CFU / mL.

[0012] Preferably, in step S3, the inoculum size of Bifidobacterium NKU FB3-14 is 10 g / L based on the volume of the fermentation supernatant. 8 CFU / mL.

[0013] The present invention also provides the application of the above-mentioned Lactobacillus rhamnosus ferment or the Lactobacillus rhamnosus ferment prepared by the above preparation method in the preparation of products that regulate Th1 / Th2 imbalance.

[0014] The present invention also provides the application of the above-mentioned Lactobacillus rhamnosus ferment or the Lactobacillus rhamnosus ferment prepared by the above preparation method in the preparation of products that regulate the balance of intestinal flora.

[0015] This invention also provides the application of the above-mentioned Lactobacillus rhamnosus ferment or the Lactobacillus rhamnosus ferment prepared by the above-mentioned method in the preparation of products having one or more of the following functions: (1) Regulates the body's immunity; (2) Maintain immune balance; (3) Relieve allergy symptoms and inflammatory response.

[0016] The beneficial effects of this invention are: The Lactobacillus rhamnosus ferment prepared in this invention can effectively alleviate allergy symptoms and inflammatory responses by regulating the Th1 / Th2 immune balance and reducing the level of mast cell protease MCPT-1 in serum.

[0017] The Lactobacillus rhamnosus ferment prepared by this invention also achieved microbial community... Faecalibaculum , unclassified_Muribaculaceae , Lactobacillus , Odoribacter , Bacteroides and Alistipes It specifically enriches various beneficial bacteria, rebuilds intestinal homeostasis, maintains immune balance, and thus inhibits allergic reactions. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Diagram showing the construction of an OVA-sensitized mouse model under the intervention of fermentation composition; Figure 2 The graphs show the changes in body weight and body temperature of OVA-sensitized mice; where A is the graph showing the changes in mouse body weight and B is the graph showing the changes in mouse body temperature. Figure 3The graph shows the statistical levels of OVA-specific antibodies and MCPT-1 in mouse serum; where A represents the serum OVA-sIgG1 level; B represents the serum OVA-sIgG2 level; C represents the serum OVA-sIgG1 / OVA-sIgG2 ratio; D represents the serum OVA-sIgE level; and E represents the serum mast cell protease MCPT-1 level. Figure 4 The graph shows the statistical effects of the fermentation composition on the spleen index and cytokine levels in OVA-sensitized mice; where A represents the spleen index; B represents the spleen IL-4 level; C represents the spleen IL-5 level; D represents the spleen IL-10 level; E represents the spleen IL-12 level; F represents the spleen IFN-γ level; G represents the spleen IL-4 / IFN-γ ratio; H represents the spleen IL-4 / IL-12 ratio; and I represents the spleen IL-5 / IL-12 ratio. Figure 5 H&E staining pathological sections of lung tissue from OVA-sensitized mice in each treatment group, including NC, Alom, OVA, ML1-2, Mp, LM, LBM, LM+Bi, FB3-14 and H-LM+Bi groups; Figure 6 This is a statistical graph showing the changes in gut microbiota diversity and phylum-level species composition in OVA-sensitized mice; where AC represents the microbiota diversity index, specifically the Chao 1 index and the species richness index (Chao 1 index, phylum-level species richness ... Observed species )and shannon Index; D represents the phylum-level bacterial classification; E represents the Firmicutes phylum ( Bacillota ) / Bacteroidetes ( Bacteroidata The ratio; FL represents the relative abundance of bacterial groups that show significant differences at the phylum level, specifically Firmicutes (…). Bacillota Bacteroidetes ( Bacteroidota ), Iron-depleting bacteria ( Deferribacterota ) 、 Pseudomonas ( Pseudomonadota ), Actinobacteria ( Actinomycetota ), Patescibacteria and thermodesulfobacteria ( Thermodesulfobacteriota ); Figure 7 This is a statistical graph showing the changes in the species composition of the gut microbiota at the genus level in OVA-sensitized mice; where A represents the bacterial taxonomic composition at the genus level; BH represents the relative abundance statistics of the top 7 bacterial genera, namely, *Romebutzim* (…). Romboutsia ), Broutella ( Blautia ), unclassified_Muribaculaceae bacteria 、 Group NK4A136 of the family Trichophytonceae ( Lachnospiraceae _NK4A136_ group ), desulfuric vibrio ( Desulfovibrio) and Lactobacillus genus ( Ligilactobacillus ); Figure 8 A statistical chart showing the relative abundance changes of typical bacterial genera in the gut microbiota of OVA-sensitized mice: AI represents the relative abundance of bacterial groups with significant differences at the genus level, specifically Lactobacillus (…). Lactobacillus ), Coliform ( Colidextribacter ), Zurich bacillus ( Turicibacter ), Riken genus ( Rikenella ), Lachnoclostridium, Mucispirillum, Bacteroides ( Bacteroides ), Odorbacterium genus ( Odoribacter ) and other fungi ( Alistipes ). Detailed Implementation

[0020] This invention provides an anti-allergic Lactobacillus rhamnosus ferment that regulates the Th1 / Th2 imbalance in the body, as well as its preparation and application. The Lactobacillus rhamnosus ferment of this invention is prepared by fermenting the fermentation broth obtained from Lactobacillus rhamnosus NKU ML1-2 with a liquid mixture containing 10% millet protein peptide, 2% fructooligosaccharides (carbon-nitrogen mass ratio of 1:5), 1% lily powder, and 1% Poria cocos powder, followed by compounding with Bifidobacterium NKU FB3-14.

[0021] The Lactobacillus rhamnosus NKU ML1-2 described in this invention has the accession number GDMCC No. 66837 (disclosed in patent application number 202511982720.7); the Lactobacillus rhamnosus NKU ML1-2 is isolated from breast milk and has been biologically preserved (Lactobacillus rhamnosus NKU ML1-2 is preserved at Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, accession number: GDMCC No. 66837, preservation date: August 13, 2025), and experiments have proven that the Lactobacillus rhamnosus NKU ML1-2 is a safe strain and can be used in humans and animals. The accession number of Bifidobacterium longum subspecies infantis NKU FB3-14 is CGMCC No. 25762. Bifidobacterium longum NKU FB3-14 was isolated from the feces of breastfed infants and has been biopreserved. It was disclosed in Chinese Patent CN116286551A (Bifidobacterium longum subspecies infantis NKU FB3-14 is deposited at the China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 25762, accession date: September 21, 2022). Furthermore, experiments have proven that Bifidobacterium longum subspecies infantis NKU FB3-14 is a safe strain suitable for human and animal use.

[0022] The results of this invention's embodiments show that the *Lactobacillus rhamnosus* ferment can effectively alleviate allergic symptoms and inflammatory responses by regulating the Th1 / Th2 immune balance and reducing the level of mast cell protease MCPT-1 in serum; the *Lactobacillus rhamnosus* ferment also achieves the following in the bacterial community ( Faecalibaculum , unclassified_Muribaculaceae , Lactobacillus , Odoribacter , Bacteroides )and Alistipes It specifically enriches various beneficial bacteria, rebuilds intestinal homeostasis, maintains immune balance, and thus inhibits allergic reactions.

[0023] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] In this invention, fructooligosaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S11133; lily powder (product number: 10075429320342) and Poria cocos powder (product number: 10066867346272) were both food-grade raw materials. The plant was fed AIN 93G growth feed (purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.), and after the pre-season, AIN 93M maintenance feed (purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.) was introduced. Ovalbumin (OVA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S12015; adjuvant Al(OH)3 was purchased from InvivoGen, USA, product number vac-alu-50. The mouse IL-4 ELISA kit was purchased from Jiangsu Enzyme-Label Reagent Company, catalog number MB-3400A; the IL-5 ELISA kit was purchased from Jiangsu Enzyme-Label Reagent Company, catalog number MB-3401A; the IL-10 ELISA kit was purchased from Jiangsu Enzyme-Label Reagent Company, catalog number MB-2912A; the IFN-γ ELISA kit was purchased from Jiangsu Enzyme-Label Reagent Company, catalog number MB-2918A; and the IL-12 ELISA kit was purchased from Jiangsu Enzyme-Label Reagent Company, catalog number MB-3436A. The mouse mast cell protease MPCT-1 was purchased from Jiangsu Enzyme-Label Reagent Company, catalog number MB-66724A.

[0025] The millet protein peptides described in this invention were purchased from Fufeng Sinote Biotechnology Co., Ltd.

[0026] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0027] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0028] Example 1: Preparation of Lactobacillus rhamnosus ferment 1.1 Microbial culture Take out the cryopreserved culture (-80℃ NKU ML1-2 Lactobacillus rhamnosus facultative anaerobic), add 24 mL of MRS broth medium to the cryopreserved tube for complete activation, and activate and culture at 37℃ for 24 h; after 24 h of culture, take 1 mL of the activated bacterial solution and add it to 30 mL of MRS liquid medium for subculture.

[0029] Bifidobacterium NKU FB3-14 was strictly anaerobic cultured in an anaerobic workstation at 37°C using modified MRS broth medium supplemented with 0.05% (w / v) L-cysteine ​​hydrochloride.

[0030] 1.2 Preparation of Lactobacillus rhamnosus ferment broth In subsequent experiments, the Lactobacillus rhamnosus ferment can be described as: a combination of lactic acid bacteria fermentation product and Bifidobacterium NKU FB3-14 (LM+Bi group).

[0031] According to 10 8 At an inoculum size of CFU / mL, *Lactobacillus rhamnosus* NKU ML1-2 was inoculated into 20 mL of a liquid mixture containing 10% millet protein peptide, 2% fructooligosaccharides (C:N ratio 1:5), 1% lily powder, and 1% *Poria cocos* powder (the remainder being water). The mixture was fermented in a shaker at 37°C for 24 h, then centrifuged at 8000 rpm for 10 min. The fermentation supernatant was collected and processed according to a 10... 8 A CFU / mL fermentation system was used to add live Bifidobacterium NKU FB3-14 bacteria obtained from the fermentation product to prepare Lactobacillus rhamnosus ferment.

[0032] Example 2: Preparation of different fermentation compositions Add 10% millet protein peptides, 2% fructooligosaccharides (carbon-nitrogen mass ratio of 1:5), 1% lily powder, and 1% poria cocos powder (the remainder being water) to the liquid fermentation system. Set up different experimental groups to prepare for subsequent animal experiments.

[0033] (1) A live bacteria intervention group (ML1-2) of *Lactobacillus rhamnosus* NKU ML1-2 was set up: freeze-dried *Lactobacillus rhamnosus* NKUML1-2 bacterial powder was subcultured, and 30 mL of *Lactobacillus rhamnosus* NKU ML1-2 bacterial suspension was centrifuged at 6000 g for 10 min. The bacterial sludge was resuspended in 30 mL of a protective agent (10% (w / v) trehalose, 2% (w / v) glycine and 0.85% NaCl solution), transferred to a petri dish, pre-frozen at -80℃, and then freeze-dried for 24 h. After freeze-drying, the viable bacteria content in the freeze-dried bacterial powder was determined by the plate spreading method. The bacterial count was found to be 9 × 10⁻⁶ per 0.5 g of freeze-dried bacterial powder. 10 CFU, freeze-dried Lactobacillus rhamnosus NKU ML1-2 bacterial powder is a live bacterial intervention ML1-2.

[0034] (2) Set up the unfermented composition intervention group (MP): Add 10% millet protein peptide, 2% fructooligosaccharide (carbon-nitrogen mass ratio of 1:5), 1% lily powder and 1% poria powder to 20 mL of water, mix thoroughly, but do not inoculate with Lactobacillus rhamnosus NKU ML1-2, culture in a shaker at 37℃ for 24 h, centrifuge at 8000 rpm for 10 min, collect the supernatant, and record it as the unfermented composition MP.

[0035] (3) Setting up the lactic acid bacteria fermentation composition intervention group (LM): Take out the frozen culture (-80℃ NKU ML1-2 Lactobacillus rhamnosus facultative anaerobic), add 24 mL of MRS broth medium to the cryovial for complete activation, and activate and culture at 37℃ for 24 h; after 24 h of culture, take 1 mL of the activated bacterial solution and add it to 30 mL of MRS liquid medium for subculture; according to 10 8 CFU / mL Lactobacillus rhamnosus NKU ML1-2 was inoculated into 20 mL of water containing 10% millet protein peptide, 2% fructooligosaccharide (carbon-nitrogen mass ratio of 1:5), 1% lily powder and 1% Poria cocos powder. After fermentation in a shaker at 37℃ for 24 h, the mixture was centrifuged at 8000 rpm for 10 min, and the fermentation supernatant was collected and denoted as lactic acid bacteria fermentation composition LM.

[0036] (4) An intervention group (LBM) was set up using a compound fermentation composition of *Lactobacillus rhamnosus* NKU ML1-2 and *Bifidobacterium* NKU FB3-14: *Bifidobacterium longum* infant NKU FB3-14 was subjected to strict anaerobic culture in a 37°C anaerobic workstation using modified MRS broth medium supplemented with 0.05% (w / v) L-cysteine ​​hydrochloride. Following a 10 8CFU / mL Lactobacillus rhamnosus NKU ML1-2 and 10 8 Bifidobacterium NKU FB3-14 was inoculated into 20 mL of water containing 10% millet protein peptide, 2% fructooligosaccharide (C:N mass ratio of 1:5), 1% lily powder and 1% Poria cocos powder. After fermentation at 37℃ for 24 h, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected and denoted as Lactobacillus and Bifidobacterium complex fermentation composition LBM.

[0037] (5) Bifidobacterium NKU FB3-14 live bacteria intervention group (FB3-14): Bifidobacterium longum subsp. infantile NKUFB3-14 was strictly anaerobic cultured in a 37℃ anaerobic workstation using modified MRS broth medium supplemented with 0.05% (w / v) L-cysteine ​​hydrochloride. After 24 h of culture, the bacterial sludge was collected by centrifugation at 25℃ and 6000 rpm for 10 min and resuspended in sterile PBS for subsequent animal experiments.

[0038] (6) Set up an intervention group (H-LM+Bi) of Lactobacillus rhamnosus NKU ML1-2 fermentation composition + Bifidobacterium NKU FB3-14 heat inactivation complex: according to 10 8 Lactobacillus rhamnosus NKU ML1-2 was inoculated into 20 mL of water containing 10% millet protein peptide (purchased from Fufeng Sinote Biotechnology Co., Ltd.), 2% fructooligosaccharides (C:N ratio 1:5), 1% lily powder, and 1% Poria cocos powder. Fermentation was carried out at 37℃ for 24 h, followed by centrifugation at 8000 rpm for 10 min. The fermentation supernatant was collected and processed according to a 10... 8 In a CFU / mL fermentation system, live Bifidobacterium FB3-14 bacteria obtained from the culture were added to the fermentation product. The resulting fermentation broth and Bifidobacterium complex were subjected to ultra-high temperature sterilization (135℃, 8 s). The resulting heat-inactivated complex intervention was denoted as fermentation broth + Bifidobacterium heat-sterilization complex intervention group H-LM+Bi.

[0039] Example 3 Animal Experiment The experimental animals were 3-week-old weaned female BALB / c mice of specific pathogen-free grade. The mice were placed in polycarbonate boxes under controlled conditions of 22±2℃, 65%±5% humidity, and a 12-hour light / dark cycle. They were pre-fed for 3 weeks, during which they were fed AIN 93G growth feed. After the pre-fed period, they were fed AIN 93M maintenance feed. Feed was prepared by the mice themselves (stored at -20℃ or 4℃) and changed twice weekly.

[0040] After pre-culture, mice were randomly divided into 10 groups as follows: blank control group (NC), OVA (ovalbumin) sensitization model group (OVA), adjuvant group Al(OH)3 (Alum), Lactobacillus rhamnosus NKU ML 1-2 bacterial culture intervention group (ML1-2), unfermented composition intervention group (MP), lactic acid bacteria fermentation composition intervention group (LM), lactic acid bacteria and bifidobacteria compound fermentation composition intervention group (LBM), lactic acid bacteria fermentation product + bifidobacteria live bacteria composition intervention group (LM+Bi), bifidobacteria NKUFB3-14 live bacteria intervention group (FB3-14), and lactic acid bacteria fermentation product + bifidobacteria heat inactivated composition intervention group (H-LM+Bi). From 7 to 27 days, the intervention groups received continuous gavage administration of the six interventions, with Lactobacillus rhamnosus NKU ML 1-2 (ML 1-2) and bifidobacteria NKU FB3-14 (FB3-14) administered at doses of 10 gavage. 8 CFU / animal / day, the gavage dose for the following compositions was 0.3 mL / animal / day: unfermented composition (MP), lactic acid bacteria fermented composition (LM), lactic acid bacteria and bifidobacteria complex fermented composition (LBM), lactic acid bacteria fermentation product + live bifidobacteria composition (LM+Bi), and lactic acid bacteria fermentation product + heat-inactivated bifidobacteria composition (H-LM+Bi).

[0041] Sensitization phase: After pre-feeding, except for the NC and Alum groups, mice in other groups were intraperitoneally injected with OVA (ovalbumin) solution on days 1, 7, 14, and 21. 1.5 mg of OVA was dissolved in 3 mL of sterile PBS solution, then mixed with an equal volume of adjuvant Al(OH)3. Each mouse was given 200 μL of the OVA-Alum mixture intraperitoneally, meaning each mouse received 50 μg of OVA for sensitization. Simultaneously, the NC group received an equal volume of sterile PBS solution intraperitoneally, and the Alum group received an equal volume of the PBS-Alum mixture intraperitoneally.

[0042] Challenge phase: Challenge was performed on days 23, 26, and 27. Mice in the OVA, ML1-2, MP, LM, LBM, LM+Bi, FB3-14, and H-LM+Bi groups were intranasally administered 20 µL of OVA solution (i.e., 0.5 mg / 20 µL). Under isoflurane inhalation anesthesia, the OVA solution was administered intranasally to both nostrils in multiple doses of 2 μL every 30 seconds using a micropipette. At the same time, mice in the NC and Alum groups were challenged with an equal volume of PBS.

[0043] The specific experimental procedure is as follows: Figure 1As shown, during the experiment, the body weight of the mice was measured on days 1, 7, 14, 21, and 28, and the rectal temperature of the mice was measured on days 1, 7, and 27. The mice were fasted on the afternoon of day 27, and were euthanized by cervical dislocation on day 28. Serum, spleen, lungs, intestinal tissue, and cecal contents were collected for further analysis.

[0044] Example 4 The effects of the fermentation composition on the changes in body weight and body temperature of allergic mice in the animal experiment of Example 3 were analyzed.

[0045] During the experiment, the mice's weight was measured weekly, and the results were as follows: Figure 2 As shown in (A), the results revealed an overall trend of weight gain in the adjuvant group and the blank control group. However, the OVA and ML1-2, MP, LM, LBM, LM+Bi, FB3-14, and H-LM+Bi intervention groups showed a significant trend of initial weight gain followed by a decrease, with a marked weight loss trend after 14 days. It is speculated that the mice experienced a significant allergic reaction after two intraperitoneal sensitizations, leading to the weight loss trend. Observations showed that the LM+Bi and H-LM+Bi intervention groups exhibited the most significant weight gain, and their weight status after the intervention was closest to that of the blank control group. Furthermore, there was no significant difference between the LM+Bi and H-LM+Bi groups.

[0046] One of the typical pathological features of food allergies is abnormal fluid distribution caused by vasodilation, which may reduce blood flow to the thermoregulatory center, thus causing a drop in body temperature. Therefore, decreased body temperature is often considered an important clinical manifestation of food allergies. Figure 2 As shown in (B), compared with the NC group and the Alum group, the OVA-sensitized mice showed a significant decrease in body temperature after challenge. The decrease in rectal temperature in all six intervention groups was less than that in the OVA group. The body temperature changes in the ML1-2, LM+Bi, and H-LM+Bi intervention groups were statistically different from those in the OVA group (P<0.05, P<0.001, P<0.001). Among them, the decrease in body temperature in the LM+Bi and H-LM+Bi groups was smaller, with no significant difference between the two groups, and was closest to the blank control group.

[0047] Example 5 The effects of the fermentation composition in Example 3 on serum immunoglobulin and MCPT-1 levels in allergic mice were analyzed.

[0048] After the intervention in Example 3, mouse serum was collected, and the levels of IgG1, IgG2a, IgE, and mast cell protease MCPT-1 in the serum were measured. The method for measuring the level of OVA-specific antibody in mouse serum was as follows: OVA protein was diluted to 5 μg / mL with carbonate buffer, and 100 μL was added to each well of the ELISA plate. The plate was placed in a refrigerator at 4°C overnight, then removed and placed at room temperature for 30 min. After returning to room temperature, 100 μL of PBST was used to wash the plate 4 times, and the washing buffer was patted dry. Then, 150 μL of 1% BSA blocking buffer was added to each well, and the plate was incubated at 37°C for 2 h. After washing 4 times, the plate was patted dry, and then 100 μL of antibody dilution buffer (standard or sample + dilution buffer) was added. The plate was incubated at 37°C for 2 h, and then washed 4 times. Finally, 100 μL of diluted goat anti-rabbit HRP-IgG was added to each well, and the plate was incubated at 37°C for 1 h. The plate was washed thoroughly (6 times, 1 min each time). After washing, add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 15 min. When blue is visible to the naked eye, remove the well and quickly add 50 μL of sulfuric acid stop solution to each well. Measure the OD value at 450 nm.

[0049] OVA-sIgE is a core indicator for measuring the specific immune response induced by OVA sensitization. Elevated levels of OVA-sIgE indicate an enhanced Th2 immune response and are positively correlated with the severity of allergic reactions.

[0050] Research results are as follows Figure 3As shown in (AD), compared with the NC and Alum groups, the serum OVA-sIgE level in the OVA group was significantly increased (P<0.0001, P<0.001), while the serum OVA-sIgE level in the six intervention groups was lower than that in the OVA group. Except for the ML1-2 and FB3-14 intervention groups, the other intervention groups showed statistically significant differences. Among them, the LM+Bi and H-LM+Bi groups had the lowest OVA-sIgE levels, with no significant difference between the two groups, essentially returning to the NC group level. IgG1 is an immunoglobulin secreted by B cells under the stimulation of Th2 cytokines, and its elevated level is usually considered a marker of enhanced Th2 immune response. IgG2a is an immunoglobulin produced by Th1 cytokines (such as IFN-γ), and its elevated level is usually associated with enhanced Th1 immune response. ELISA results showed that OVA sensitization significantly increased the serum OVA-sIgG1 level in mice (P<0.0001), while the serum OVA-sIgG1 level in the ML1-2, MP, LM, LBM, LM+Bi, FB3-14 and H-LM+Bi intervention groups was significantly lower than that in the OVA group, with the LM+Bi and H-LM+Bi groups showing the most significant reduction. On the other hand, ML1-2, LM+Bi and H-LM+Bi interventions all increased the serum OVA-sIgG2a level in allergic mice, and the OVA-sIgG2a levels in the two intervention groups were statistically different from those in the OVA group (P<0.05, P<0.01, P<0.01). In addition, the serum OVA-sIgG1 / OVA-sIgG2a ratios in the ML1-2, LM+Bi, and H-LM+Bi groups were significantly lower than those in the OVA group (P<0.01, P<0.001, P<0.001), while the LM, LBM, and FB3-14 groups showed a decrease compared to the OVA group, but there was no statistically significant difference between the two groups.

[0051] In addition, the level of mast cell protease MCPT-1 in mouse serum was measured using an ELISA kit, and the results are as follows: Figure 3As shown in (E), the results indicated that, compared with the OVA group, the serum mast cell protease MCPT-1 levels in the six intervention groups were significantly reduced, decreasing from 39.28±0.2710 pg / mL (OVA) to 33.23±1.658 pg / mL (ML1-2), 35.35±0.6829 pg / mL (MP), 33.21±0.8981 pg / mL (LM), 35.35±0.6829 pg / mL (LBM), 31.36±0.6680 pg / mL (LM+Bi), 33.23±1.037 pg / mL (FB3-14), and 30.76±0.2842 pg / mL (H-LM+Bi).

[0052] In summary, the fermentation composition effectively alleviated OVA-induced allergic reactions in mice, reduced serum levels of OVA-sIgE and OVA-sIgG1, and suppressed the Th2 immune response in mice; simultaneously, it increased serum levels of OVA-sIgG2a, enhancing the Th1 immune response in mice. Furthermore, the fermentation composition significantly reduced serum levels of mast cell protease MCPT-1, effectively alleviating allergic symptoms and inflammatory responses. Overall, among the multiple intervention groups, the LM+Bi group and the heat-inactivated H-LM+Bi group showed the best intervention effects, with no significant difference between the two groups. The intervention effects of the remaining groups were, in descending order: LM, LBM, ML1-2, FB3-14, and MP.

[0053] Example 6 The effects of the fermentation composition on the spleen immune response of OVA-sensitized mice in the animal experiment of Example 3 were analyzed.

[0054] After the animal experiment in Example 3, mouse spleen tissue was taken to observe its morphology, photographed, weighed and recorded, and the spleen index was calculated. The results are as follows: Figure 4 As shown in (A). Changes in the spleen index can reflect the strength of the body's immune response and the degree of allergic inflammation to a certain extent. Compared with the NC group, OVA-sensitized mice showed significant splenomegaly and a significantly increased spleen index (P<0.0001), indicating that OVA sensitization triggered a significant proliferative response of immune organs. Except for the MP group, the interventions in the LM, LBM, ML1-2, LM+Bi, FB3-14, and H-LM+Bi groups significantly alleviated splenomegaly and the abnormal increase in the spleen index, and to a certain extent had immunomodulatory and protective effects on OVA-sensitized mice (P<0.05, P<0.05, P<0.05, P<0.001, P<0.05, P<0.001). Among them, the interventions in the LM+Bi and H-LM+Bi groups showed the best effects, superior to the other intervention groups.

[0055] To further assess changes in Th1 / Th2 immune balance, ELISA was used to detect the levels of relevant cytokines in the spleen of mice. The results are as follows: Figure 4 As shown in (BF), the results indicated that compared with the NC group, the OVA group showed significantly increased levels of Th2 cytokines IL-4 and IL-5 (P<0.0001, P<0.05), indicating activation of the Th2 immune response. LM, LM+Bi, and H-LM+Bi interventions significantly reduced IL-4 and IL-5 levels in the mouse spleen. Simultaneously, compared with the NC group, the OVA group and the ML1-2, MP, LM, LBM, LM+Bi, FB3-14, and H-LM+Bi intervention groups showed significantly increased serum levels of Th1 cytokines. Among the multiple intervention groups, only the LM+Bi and H-LM+Bi groups showed statistically significant differences compared with the OVA model group, with significantly increased levels of Th1 cytokines IL-12 and IFN-γ (P<0.01, P<0.05). The serum level of Tregs-related factor IL-10 in the OVA group was significantly increased compared with that in the NC group (P<0.01). Specifically, the IL-10 levels in the LM, LM+Bi, and H-LM+Bi groups were similar to those in the NC group, but significantly lower than those in the OVA group (P<0.05, P<0.01, P<0.01). The elevated IL-10 level in the spleen of OVA-sensitized mice may be a negative feedback regulation mechanism initiated by the body in response to Th2-type inflammatory responses, aiming to suppress excessive inflammation, reduce tissue damage, and restore immune homeostasis. On the other hand, IL-10 is a key pleiotropic cytokine that can both promote and inhibit Th2-dependent allergic responses. Previous studies have shown that IL-10 cytokines can promote mast cell activation and enhance the allergic effects of IgE.

[0056] To more intuitively reflect the changing trends of the Th1 / Th2 immune balance, the proportions of key cytokines in each group were further analyzed. The results are as follows: Figure 4 As shown in (GI), compared with the NC group, the IL-4 / IFN-γ and IL-4 / IL-12 ratios in the OVA group mice were significantly increased (P<0.0001), indicating enhanced Th2 immune response and suppressed Th1 immune response. LM, LM+Bi, and H-LM+Bi interventions all significantly reduced the IL-4 / IFN-γ and IL-4 / IL-12 ratios in the spleen of mice (P<0.01, P<0.0001, P<0.0001), bringing them close to the levels of the NC group. There was no significant difference in the reduction effect between the LM+Bi and H-LM+Bi groups.

[0057] Meanwhile, compared with the NC group and the Alum group, the IL-5 / IL-12 ratio in the spleen of mice in the OVA group was slightly increased, but there was no statistical difference. Compared with mice in the OVA group, LM, LM+Bi and H-LM+Bi interventions significantly inhibited the increase in the IL-5 / IL-12 ratio (P<0.05, P<0.0001, P<0.0001).

[0058] The results in summary indicate that the intervention of fermentation composition can effectively restore the Th1 / Th2 immune balance. Among them, the intervention effect of lactic acid bacteria fermentation product + Bifidobacterium live bacteria composition (LM+Bi) is the best. The intervention effect of heat-inactivated H-LM+Bi is not significantly different from that of LM+Bi. The next best effects are LM, LBM, ML1-2, FB3-14 and MP.

[0059] Example 7 Analysis of Example 3 animal experiments showed that the fermented composition alleviated the pulmonary inflammatory response in OVA-sensitized mice.

[0060] Example 3: After the animal experiment, right middle lobe lung tissue was taken from mice. The tissue was placed in tissue fixative and embedded in paraffin after 24 h. Sections (4 μm) were placed on glass slides and stained with H&E. The pathological changes in the lung tissue of each group of mice in the H&E sections were observed under an optical microscope.

[0061] The results are as follows Figure 5 As shown, histological examination and H&E staining results revealed that intraperitoneal injection of OVA caused abnormal lung tissue structure in mice, including alveolar atrophy and collapse, thickening of the pulmonary septa, and severe infiltration of inflammatory cells around the bronchi and blood vessels. Different interventions, ML1-2, MP, LM, LBM, LM+Bi, FB3-14, and H-LM+Bi, could significantly reduce the invasion of OVA-induced inflammatory cells into lung tissue. In particular, the lung tissue structure and morphology of the LM+Bi intervention group were most similar to those of the NC group, and its alleviating effect on the OVA-induced lung inflammatory response was significantly better than that of the LM and LBM groups, and there was no significant difference compared with the H-LM+Bi group.

[0062] Example 8 The effect of the fermentation composition on improving the dysbiosis of the intestinal flora in OVA-sensitized mice was analyzed in the animal experiment of Example 3.

[0063] The species diversity and composition of the gut microbiota in OVA-sensitized mice were analyzed using 16S rRNA gene sequencing technology.

[0064] The results of α-diversity analysis of the microbial community are as follows: Figure 6As shown in (AC), the diversity indices of Chao1, Observed species, and Shannon in the OVA group were significantly lower than those in the NC and Alum groups. The interventions of LM, MP, LBM, and LM+Bi effectively improved the species diversity decline caused by OVA sensitization. In particular, the diversity index of the intervention group with lactic acid bacteria fermentation product + Bifidobacterium live bacteria combination (LM+Bi) was the highest, restoring it to the level of the blank control group.

[0065] Changes at the phylum level of the gut microbiota can provide a basis for the identification of potential biomarkers of food allergies. Therefore, the changes at the typical phylum level in the gut microbiota of OVA-sensitized mice were analyzed, and the results are as follows: Figure 6 As shown in (DL), the allergic OVA model group exhibited significant gut microbiota dysbiosis: among which Bacteroidetes ( Bacteroidota ) and rare bacteria ( Deferribacterota , Patescibacteria and Thermodesulfobacteriota The abundance of ) was significantly increased, while Firmicutes ( Bacillota ), Actinobacteria ( Actinomycetota ) and Pseudomonas ( Pseudomonadota The abundance of bacterial phyla was significantly reduced. All five intervention groups (ML1-2, MP, LM, LBM, and LM+Bi) effectively reversed this disorder, with the LM+Bi group showing the best effect, reducing the abundance of Bacteroidetes and rare bacteria to near the levels of the normal control NC group, while restoring the abundance of Firmicutes, Actinobacteria, and Pseudomonas. Furthermore, we found that OVA sensitization significantly reduced the ratio of Firmicutes to Bacteroidetes in the gut microbiota, indicating that OVA sensitization disrupted the gut microbiota balance and caused dysbiosis. The LM+Bi intervention significantly reversed the abnormal decrease in the Firmicutes-Bacteroidetes ratio (P<0.0001), maintaining gut microbiota balance.

[0066] Next, the composition of the gut microbiota in allergic mice was analyzed at the genus level, and the relative abundance of 16 typical gut microbiota genera was quantitatively analyzed. The results are as follows: Figure 7 (AH) and Figure 8 As shown in (AI), compared with the NC group, the bacterial flora of the OVA group mice contained Broutella spp. ( Blautia ), Desulfuric Vibrio spp. Desulfovibrio ), Mucispirillum and C. coli ( Colidextribacter The relative abundance increased significantly.

[0067] Previous studies have shown that *Broutella* spp. Blautia The accumulation of Brutella (Brutella) was negatively correlated with lipid metabolism. OVA sensitization disrupted the lipid metabolism balance in mice, leading to increased lipid metabolism in Brutella. Blautia ) accumulates in the intestine. In addition, desulfovibrio ( Desulfovibrio), Mucispirillum and Coliforms ( Colidextribacter All of these are opportunistic pathogens. Their accumulation in allergic mice exacerbates immune imbalance, disrupts intestinal homeostasis, and intensifies inflammatory responses. Furthermore, compared to the blank control group, the OVA group mice showed a higher concentration of *Romebutzella* (*Romebutzella*) in their gut microbiota. Romboutsia ), NK4A136 group of Trichophyceae ( Lachnospiraceae _NK4A136_ group ), Lactobacillus spp. ( Ligilactobacillus ), Zurich bacillus ( Turicibacter ), Riken genus ( Rikenella ), Lachnoclostridium and Bacteroides ( Bacteroides The relative abundance of many beneficial bacteria decreased significantly. These bacteria have a variety of physiological functions, such as immune regulation, protection of the intestinal barrier, promotion of short-chain fatty acid production, and regulation of metabolism.

[0068] The results showed that Lactobacillus rhamnosus ML1-2, unfermented composition (MP), lactic acid bacteria fermentation composition (LM), lactic acid bacteria and bifidobacteria complex fermentation composition (LBM), and lactic acid bacteria fermentation product + live bifidobacteria composition (LM+Bi) could all effectively inhibit the abnormal accumulation of harmful bacteria and the reduction of beneficial bacteria in allergic mice. Among them, the lactic acid bacteria fermentation product + live bifidobacteria composition (LM+Bi) had the best intervention effect, followed by LM, LBM, ML1-2 and MP.

[0069] In addition to restoring gut microbiota to normal levels, LM+Bi intervention also significantly enriched a variety of novel beneficial bacteria, such as *Faecalibacterium*. Faecalibaculum ), unclassified_Muribaculaceae Lactobacillus ( Lactobacillus ), Odorbacterium genus ( Odoribacter Bacteroides ( Bacteroides ) and other fungi ( Alistipes These bacteria play an important role in protecting the intestinal barrier, promoting the production of short-chain fatty acids, and regulating immune responses.

[0070] In summary, the results indicate that OVA sensitization significantly reduces the species diversity and richness of the gut microbiota in mice, while simultaneously promoting the abnormal accumulation of harmful bacteria and the reduction of beneficial bacteria. All five different intervention groups effectively inhibited OVA-induced gut microbiota dysbiosis and harmful bacteria accumulation, with the Lactobacillus fermentation product + Bifidobacterium live bacteria combination (LM+Bi) showing the best effect, followed by LM and LBM. Furthermore, the LM+Bi intervention also achieved [a certain effect] in the gut microbiota. Faecalibaculum , unclassified_Muribaculaceae、Lactobacillus、Odoribacter、Bacteroides )and Alistipes It specifically enriches various beneficial bacteria, rebuilds intestinal homeostasis, maintains immune balance, and thus inhibits allergic reactions.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation product of Lactobacillus rhamnosus, characterized in that, The fermentation product obtained by using Lactobacillus rhamnosus NKU ML1-2 as the fermentation strain was combined with Bifidobacterium NKU FB3-14 to prepare the product. The Lactobacillus rhamnosus NKU ML1-2 has the accession number GDMCC No. 66837; the Bifidobacterium NKU FB3-14 has the accession number CGMCC No. 25762.

2. The Lactobacillus rhamnosus ferment according to claim 1, characterized in that, The fermentation process includes mixing Lactobacillus rhamnosus NKU ML1-2 with millet protein peptides, fructooligosaccharides, lily powder, and Poria cocos powder for fermentation. The carbon-nitrogen mass ratio of the oligofructose is 1:

5.

3. The method for preparing the Lactobacillus rhamnosus ferment as described in claim 1, characterized in that, Includes the following steps: S1. Add millet protein peptides, fructooligosaccharides, lily powder and poria powder to water in proportion to obtain a liquid mixture; S2. Inoculate Lactobacillus rhamnosus NKU ML1-2 into the liquid mixture, ferment at 37°C for 24 h, centrifuge, and collect the fermentation supernatant. S3. Inoculate the fermentation supernatant with live Bifidobacterium NKU FB3-14 to obtain Lactobacillus rhamnosus ferment.

4. The preparation method according to claim 3, characterized in that, In step S1, the added masses of millet protein peptide, fructooligosaccharide, lily powder and poria powder are 10%, 2%, 1% and 1% of the water volume, respectively, based on the volume of water.

5. The preparation method according to claim 3, characterized in that, In step S2, the inoculum size of *Lactobacillus rhamnosus* NKU ML1-2 is 10 g / L based on the volume of the liquid mixture. 8 CFU / mL.

6. The preparation method according to claim 3, characterized in that, In step S3, the inoculum size of Bifidobacterium NKU FB3-14 is 10 g / L based on the volume of the fermentation supernatant. 8 CFU / mL.

7. The use of the Lactobacillus rhamnosus ferment as described in claim 1 or the Lactobacillus rhamnosus ferment prepared by any one of claims 3-6 in the preparation of products that regulate Th1 / Th2 imbalance.

8. The use of the Lactobacillus rhamnosus ferment as described in claim 1 or the Lactobacillus rhamnosus ferment prepared by any one of claims 3-6 in the preparation of products that regulate the balance of intestinal flora.

9. The use of the *Lactobacillus rhamnosus* ferment as described in claim 1 or the *Lactobacillus rhamnosus* ferment prepared by the method according to any one of claims 3-6 in the preparation of products having one or more of the following functions: (1) Regulates the body's immunity; (2) Maintain immune balance; (3) Relieve allergy symptoms and inflammatory response.

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