Lactobacillus rhamnosus B12 with effects of resisting allergy, enhancing immunity, conditioning intestinal flora and fermenting and application of lactobacillus rhamnosus B12

Abstract

The invention relates to the technical field of microorganisms, and provides a lactobacillus rhamnosus B12 with the effects of resisting allergy, enhancing immunity, conditioning intestinal flora and fermenting and application of the lactobacillus rhamnosus B12. The preservation number of the strain is CGMCC No.34366. The strain is wide in carbon source growth adaptability, has good short-chain fatty acid secretion level, biofilm forming ability, DPPH free radical scavenging ability and gastric juice and pancreatic juice tolerance, can promote expression of various immune factors of zebra fish, reduce the immune globulin IgE level, can optimize the intestinal flora structure, and can improve the immunity of the zebra fish. A good bacteriostatic effect is achieved on harmful pathogenic bacteria; in addition, various plant bases can be fermented, the SOD activity, the substrate chromaticity and the flavor quality of a fermented product are improved, and the content of red wolfberry protein and total amino acid is remarkably increased. The strain can provide a new probiotic source for the development of anti-allergic products, immunity enhancing products, intestinal flora conditioning products, antioxidant products, antibacterial products and fermented products.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a strain of Lactobacillus rhamnosus B12 with anti-allergic, immune-enhancing, gut microbiota-regulating and fermentation effects, and its applications. Background Technology

[0002] Allergies are diseases caused by abnormal immune system reactions. Common allergic diseases include allergic rhinitis, allergic asthma, eczema, and urticaria. With environmental pollution and changes in modern lifestyles, allergies have become the sixth leading cause of chronic disease worldwide, affecting approximately 20-30% of the global population. Avoiding allergens, medication, immunotherapy, and probiotic regulation are existing methods for treating allergies. Probiotics can further alleviate allergic reactions by strengthening the connections between intestinal epithelial cells and boosting immunity.

[0003] The human immune system is a robust defense for health, responsible for resisting pathogen invasion and maintaining normal bodily functions. Once the immune system is impaired, the body faces numerous health risks: a significantly increased chance of pathogen infection, recurrent chronic inflammation, a markedly increased risk of tumors, difficulty achieving the desired effects of vaccination, slow wound healing, and even psychological distress, all of which severely impact quality of life. Therefore, ensuring the normal functioning of the immune system plays an irreplaceable and crucial role in preventing disease, promoting recovery, and maintaining health.

[0004] In the complex ecosystem of the human body, the gut microbiota plays a vital role. It is composed of beneficial, neutral, and harmful bacteria, with beneficial bacteria forming the cornerstone of overall health. When the gut microbiota becomes unbalanced, with a decrease in beneficial bacteria and an increase in harmful bacteria, a series of health problems arise: digestive system dysfunction, leading to various gastrointestinal diseases; immune system dysfunction, resulting in decreased bodily defenses; impaired intestinal barrier function, allowing harmful substances to easily invade the body; psychological well-being is also affected, potentially leading to mood swings, anxiety, and depression; and the risk of chronic diseases increases, making obesity and metabolic abnormalities more likely. Therefore, maintaining a balanced gut microbiota and actively supplementing it with beneficial bacteria is crucial for preventing and treating various diseases and safeguarding health.

[0005] Short-chain fatty acids (SCFAs) are key metabolites produced by gut microbiota during the fermentation of dietary fiber and oligosaccharides. They mainly include acetic acid, propionic acid, butyric acid, and valeric acid. These fatty acids play multiple important roles in human health: they provide essential energy to intestinal cells, supporting normal cell growth and function; they participate in regulating the composition and balance of the gut microbiota, promoting the proliferation of beneficial bacteria; they precisely regulate immune responses, enhancing the body's immune defense capabilities; they influence metabolic processes, maintaining the body's energy balance and nutrient metabolism; they promote intestinal peristalsis, preventing constipation and maintaining normal intestinal physiological function; and they strengthen the intestinal barrier, preventing harmful substances from entering the bloodstream through the intestines. SCFAs have demonstrated significant application potential and value in the prevention and treatment of various diseases, including constipation, inflammatory bowel disease, obesity, diabetes, and cardiovascular disease.

[0006] In recent years, ready-to-eat beverages based on medicinal and edible plants such as ginseng and goji berries have gradually gained market acceptance. However, these products generally suffer from low protein content. How to increase protein content is a pressing technical problem that needs to be solved in this field.

[0007] Based on this, there is a need to develop edible probiotics that can alleviate allergies, enhance immunity, produce short-chain fatty acids, provide antioxidants, regulate the gut, and increase the content of plant-based proteins from medicinal and edible plants such as wolfberry through fermentation, so as to provide new probiotic sources for the development of related products. Summary of the Invention

[0008] To address the shortcomings of the prior art mentioned in the background, this application provides *Lactobacillus rhamnosus* B12. It originates from the intestines of children and is classified as *Lactobacillus rhamnosus*, with the Latin scientific name: Lacticaseibacillus rhamnosus It was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34366.

[0009] This application also provides a microbial agent comprising Lactobacillus rhamnosus B12 as described above.

[0010] This application also provides a composition comprising Lactobacillus rhamnosus B12 as described above.

[0011] In some embodiments, the number of *Lactobacillus rhamnosus* B12 in the composition is ≥1 × 10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g.

[0012] In some embodiments, the composition comprises one or more combinations of uninactivated Lactobacillus rhamnosus B12, inactivated Lactobacillus rhamnosus B12, metabolites of Lactobacillus rhamnosus B12 strain, and freeze-dried Lactobacillus rhamnosus B12 strain.

[0013] This application also provides the use of Lactobacillus rhamnosus B12 as described above or the composition as described above in the preparation of fermented foods.

[0014] This application also provides a fermentation product obtained by fermentation of Lactobacillus rhamnosus B12 as described above.

[0015] In some embodiments, Lactobacillus rhamnosus B12 is used as a probiotic to ferment fruits, medicinal herbs, etc., to prepare fermented foods.

[0016] In some embodiments, the fermentation product is obtained by inoculating the Lactobacillus rhamnosus B12 onto a medicinal and edible plant substrate and then fermenting it.

[0017] In some embodiments, the medicinal and edible plant base includes one or more combinations of ginseng, wolfberry, and white grape.

[0018] This application also provides the use of Lactobacillus rhamnosus B12 and / or the fermentation products described above in the preparation of functional products.

[0019] In some embodiments, the functions of the functional product include one or more of the following: anti-allergy, immune enhancement, gut microbiota regulation, and antioxidant properties.

[0020] In some embodiments, the functional product includes an antibacterial product.

[0021] Based on the above, compared with the prior art, the Lactobacillus rhamnosus B12 provided in this application has the following beneficial effects: The *Lactobacillus rhamnosus* B12 carbon source provided in this application exhibits broad growth adaptability, good short-chain fatty acid secretion levels, biofilm formation ability, DPPH free radical scavenging ability, and good tolerance to gastric and pancreatic juices. It can promote the expression of various immune factors in zebrafish, reduce immunoglobulin IgE levels, and optimize the intestinal flora structure. It also shows good antibacterial effects against *Streptococcus mutans* and *Staphylococcus aureus*. Furthermore, it can ferment plant-based products such as ginseng, goji berries, and white grapes, enhancing the SOD activity, matrix color, and flavor quality of the fermented products, and significantly increasing the protein and total amino acid content of red goji berries. Therefore, this bacterium can provide a new probiotic source for the development of anti-allergy products, immune-enhancing products, intestinal flora-regulating products, antioxidant products, antibacterial products, and fermented products, and has significant application value.

[0022] Other features and beneficial effects of this application will be set forth in the following description and will be apparent in part from the description or may be learned by practicing the application. The objectives and other beneficial effects of this application may be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A colony morphology diagram of Lactobacillus rhamnosus B12 provided in an embodiment of this application; Figure 2 Gram staining image of Lactobacillus rhamnosus B12 provided in the embodiments of this application; Figure 3 Scanning electron microscope image of Lactobacillus rhamnosus B12 provided in the embodiments of this application; Figure 4 Agarose gel electrophoresis image of the amplified 16S rDNA target fragment of Lactobacillus rhamnosus B12 provided in the embodiments of this application; Figure 5 Phylogenetic tree diagram of the 16S rDNA gene of Lactobacillus rhamnosus B12 provided in the embodiments of this application; Figure 6 A schematic diagram illustrating the growth of Lactobacillus rhamnosus B12 using oligosaccharides as the sole carbon source, as provided in an embodiment of this application. Figure 7 A graph showing the results of short-chain fatty acid production by Lactobacillus rhamnosus B12 using oligosaccharides, provided in an embodiment of this application. Figure 8 A graph illustrating the biofilm formation level of Lactobacillus rhamnosus B12 provided in this application embodiment; Figure 9 A photograph of the inhibitory level of Lactobacillus rhamnosus B12 against pathogenic bacteria provided in the embodiments of this application; Figure 10 A graph showing the effect of Lactobacillus rhamnosus B12 on the body length and weight of zebrafish, provided in an embodiment of this application. Figure 11 A graph showing the effect of Lactobacillus rhamnosus B12 on immune factors in zebrafish, as provided in an embodiment of this application. Figure 12 Venn diagram showing the influence of different treatments on the gut microbiota of zebrafish. Figure 13 A graph showing the colony diversity index analysis among different treatments in an experiment on factors influencing the gut microbiota of zebrafish. Figure 14 A graph showing the effect of feeding with *Lactobacillus rhamnosus* B12 on the genus level of gut microbiota in zebrafish, provided in an embodiment of this application. Figure 15 The images provided in this application are real-life photographs showing the color changes of ginseng, red goji berries, and white grape juice before and after fermentation with Lactobacillus rhamnosus B12. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The technical features designed in the different implementations of this application described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0026] In the description of this application, it should be noted that all terms used in this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and should not be construed as limiting this application; it should be further understood that the terms used in this application should be understood to have the same meaning as those in the context of this specification and the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this application.

[0027] This application provides a strain of Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus B12, obtained from the intestines of children using anaerobic culture in MRS medium, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34366.

[0028] Example 1: Screening and isolation of Lactobacillus rhamnosus B12 Take approximately 1g of intestinal fecal sample from a 2.5-month-old child and dilute it to 10 μL in physiological saline solution. -2 times, 10 -3 times, 10 -4 Take 0.1 mL of each diluted solution and place it in MRS medium for anaerobic culture. Select several colonies suspected to be lactic acid bacteria, purify them for three generations, and then test their oligosaccharide utilization, growth, and free radical scavenging effects. Select the strain with the best oligosaccharide utilization and free radical scavenging effects, preserve it, and name it B12.

[0029] Example 2: Identification of Lactobacillus rhamnosus B12 2.1 Morphological observation of Lactobacillus rhamnosus B12 The colony morphology of Lactobacillus rhamnosus B12 is as follows: Figure 1 As shown, the bacterial cell morphology is as follows Figure 2 and Figure 3 As shown. The main morphological characteristics of Lactobacillus rhamnosus B12 are as follows: The colonies are milky white, irregularly umbrella-shaped, with forked edges, Gram-positive, and rod-shaped, 0.45-0.8 μm wide and 2.7-6.9 μm long.

[0030] 2.2 Analysis of carbon source utilization of Lactobacillus rhamnosus B12 The biochemical experiments on lactic acid bacteria were conducted according to the standard method of GB4789.35, specifically as follows: Prepare a basal culture medium for lactic acid bacteria containing sorbitol, cellobiose, maltose, mannitol, salicin, sucrose, raffinose, inulin, lactose, and 1% (w / v) sodium hippurate. Inoculate *Lactobacillus rhamnosus* B12 into the medium at 1% (w / v).

[0031] In addition, after the culture is completed, 0.2 mL of ninhydrin solution (the amount of 1% sodium hippurate in this culture medium is 2 mL) should be slowly added along the wall of the test tube. Do not shake. After placing in a water bath at 36℃±1℃ for 10 min, the results should be interpreted. The interpretation results are detailed in Table 1.

[0032] Table 1. Carbohydrate utilization of Lactobacillus rhamnosus B12

[0033] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0034] Conclusion: According to the results in Table 1, Lactobacillus rhamnosus B12 can utilize sugars such as sorbitol, cellobiose, maltose, mannitol, salicin, sucrose, raffinose, inulin, and lactose, and has a wide substrate adaptability. However, it cannot utilize sodium hippurate.

[0035] 2.3 Molecular biological identification of Lactobacillus rhamnosus B12 ① Extraction of genomic DNA from Lactobacillus rhamnosus B12: Genomic DNA was extracted using a bacterial genomic DNA extraction kit from TIANGEN. ② PCR amplification of the 16S rDNA sequence: The primers used to amplify the 16S rDNA gene sequence are: F 9-27: 5'-GAGTTTGATCCTGGCTCAG-3'; R 1525-1542: 5'–AGAAAGGAGTGATCCAGCC-3'; PCR reaction system: 12.5 μL of 2×Mix, 1 μL each of primers and DNA, and 9.5 μL of ddH2O.

[0036] PCR amplification program: 93℃ pre-denaturation for 4 min. Then denature at 94℃ for 30 s, 55℃ (16S rDNA), 72℃ extension for 90 s, for a total of 30 cycles, and finally extend at 72℃ for 10 min, and store at 4℃.

[0037] ③ PCR product detection and sequencing analysis: 5 μL of PCR product was separated and examined by gel electrophoresis in 1.0% (w / v) agarose gel containing EB. The amplified 16S rDNA target fragment was 1395 bp in length (see agarose gel electrophoresis image of 16S rDNA target fragment amplification). Figure 4 ); ④ Phylogenetic analysis: Blast alignment analysis was performed on each 16S rRNA sequence in NCBI data to obtain the sequence and... Lacticaseibacillus rhamnosus The sequence homology was greater than 99.6%, and the developmental tree was constructed using the Neighbor-joining method in MEGA 4 (results are shown in [link to results]). Figure 5 ).

[0038] The 16S rDNA sequence determination results of bacteria B12 are as follows:

[0039] Conclusion: Based on morphological observation, lactic acid bacteria biochemical identification, and homology analysis in the DNA phylogenetic tree, *Lactobacillus rhamnosus* B12 was identified as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus ) bacterial strains.

[0040] The performance characterization of Lactobacillus rhamnosus B12 provided in this application is as follows: Example 3: Growth of Lactobacillus rhamnosus B12 using oligosaccharides Various oligosaccharide media were prepared by replacing glucose in MRS medium with an equal amount of oligosaccharides. Lactobacillus rhamnosus B12 yeast culture was inoculated at 1% (v / v) into MRS media containing different oligosaccharides and cultured at 37°C for 48 h. The bacterial OD was then measured. 600 value.

[0041] See results Figure 6 The results indicate that *Lactobacillus rhamnosus* B12 can grow in MRS media with isomaltooligosaccharide, galactooligosaccharide, inulin, fructooligosaccharide, and xylooligosaccharide as glucose substitutes. The OD values ​​for growth in isomaltooligosaccharide and inulin were 1.12 and 1.02, respectively, indicating that it can utilize the above polysaccharides as the sole carbon source for growth.

[0042] The preparation process of Lactobacillus rhamnosus B12 yeast culture is as follows: an appropriate amount of -80℃ freeze-dried strain B12 is inoculated into MRS+L-cysteine ​​liquid medium and cultured at 37℃ for 24-38 hours.

[0043] Example 4: Production of short-chain fatty acids by Lactobacillus rhamnosus B12 using oligosaccharides The short-chain fatty acid content of the culture medium after 48 hours of culture in Example 3 was determined by gas chromatography, specifically the short-chain fatty acids propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and valeric acid.

[0044] Figure 7 The results of the detection of propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and valeric acid produced from B12 showed that the yield of propionic acid ranged from 7.63 to 12.84 μg / mL, isobutyric acid from 3.45 to 7.71 μg / mL, n-butyric acid from 4.47 to 5.94 μg / mL, isovaleric acid from 1.28 to 3.33 μg / mL, and valeric acid from 1.94 to 2.15 μg / mL. Isomaltooligosaccharide, galactooligosaccharide, fructooligosaccharide, and xylooligosaccharide had a good promoting effect on the yield of the above short-chain fatty acids.

[0045] Example 5: Biofilm levels of Lactobacillus rhamnosus B12 MRS medium was prepared by adding various oligosaccharides at a concentration of 0.5 g / L to replace glucose. *Lactobacillus rhamnosus* B12 was inoculated into each medium at 1.0% (w / v) and fermented at 37°C for 48 h. Using the medium containing different oligosaccharides as dilutions, flow cytometry was used to adjust the bacterial count in each fermentation broth to (1.0 ± 0.1) * 102. 8 Take 200 μL of the adjusted fermentation dilution (cfu / mL) and place it in a 96-well plate. Incubate at 37℃ for 24 h. Discard the culture medium, add 0.2 mL of 1% crystal violet and react for 30 min. Rinse with distilled water and dissolve in 0.2 mL of 95% (v / v) ethanol. Measure the absorbance of the resulting colored solution at 590 nm. Repeat the experiment three times and record the average value as the ODX value. Use the uninoculated culture medium as a control and record the replicate value as the ODC.

[0046] When the ODX:ODC ratio is greater than 1, it indicates that the bacteria have the ability to form biofilms; the higher the ratio, the stronger the ability. Test results are shown below. Figure 8 The results show that the ODX:ODC values ​​of all treatments are greater than 2.78, indicating that Lactobacillus rhamnosus B12 has good biofilm formation ability. The addition of inulin, fructooligosaccharides and galactooligosaccharides further enhances the biofilm formation ability (ODX:ODC values ​​of 8.52, 6.14 and 5.20, respectively), which is higher than the 3.00 value on the MRS. This suggests that inulin, fructooligosaccharides and galactooligosaccharide isomaltoose help to further promote the formation of biofilm.

[0047] Example 6: Analysis of the scavenging ability of Lactobacillus rhamnosus B12 against 2,2-biphenyl-1-picrylhydrazyl (DPPH) free radicals Preparation of Lactobacillus rhamnosus B12 fermentation broth: Take 2 ml of fermentation broth (cultured in MRS medium at 37℃ for 48 h), centrifuge at 3000 r / min for 10 min, the supernatant is the extracellular enzyme, and the intracellular enzyme is the bacterial cells plus 2 ml PBS, sonicate at 40% room temperature for 5 min, centrifuge at 3000 r / min for 10 min and take the supernatant. The extracellular enzyme is compared with the uninoculated MRS medium as the control, and the intracellular enzyme is compared with the PBS control. The absorbance at 515 nm of each treatment and control sample is measured according to the instructions of the DPPH free radical scavenging ability test kit (Solarbio).

[0048] Based on the absorbance at 515 nm, the DPPH free radical scavenging rate of the extracellular enzyme of *Lactobacillus rhamnosus* B12 was calculated to be 47.95 ± 0.38%, and the intracellular enzyme free radical scavenging rate of *Lactobacillus rhamnosus* B12 was 20.39 ± 0.20%.

[0049] Example 7: Analysis of the antibacterial activity of Lactobacillus rhamnosus B12 The antibacterial ability of Lactobacillus rhamnosus B12 against pathogenic bacteria was tested. Streptococcus mutans ATCC25175 was spread on brain heart infusion medium, and then holes were made with a punch. The fermentation broth of Lactobacillus rhamnosus B12, which had been fermented for 24 h (24 h at 37℃ on MRS medium), was injected into the holes and aerobically cultured at 37℃ for 24 h.

[0050] like Figure 9 As shown, the inhibition zone of *Lactobacillus rhamnosus* B12 against *Streptococcus mutans* ATCC25175 was 1.42 ± 0.08 cm, and the inhibition zone against *Staphylococcus aureus* was 1.37 ± 0.15 cm, indicating that *Lactobacillus rhamnosus* B12 has good antibacterial activity against these two pathogenic bacteria. Example 8: Survival analysis of Lactobacillus rhamnosus B12 in a simulated artificial gastric juice environment (1) The survival rate test process is as follows: Fermentation broth of *Lactobacillus rhamnosus* B12, fermented for 24 h, was centrifuged at 12000 r / min for 5 min to collect bacterial cells. An equal volume of 0.85% (w / v) physiological saline was added and mixed thoroughly to obtain a bacterial suspension. Artificial gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3, and 3 g / L pepsin) was prepared, and the pH was adjusted to 3.0. The suspension was then filtered through a 0.22 μM microporous membrane for later use. 1 mL of the bacterial suspension was added to 9 mL of the artificial gastric fluid and incubated at 37℃ for 1, 2, 3, 4, and 5 h. Samples were treated at each time (1, 2, 3, 4, and 5 h). 0.9 mL of the treated sample was taken each time, and 0.1 mL of 20 μg / mL propidium iodide (PI) was added. After staining at 37℃ for 10 min, 0.1 mL of the treated sample was added to 0.9 mL of the pepsin solution. In mL of ultrapure water, the total bacterial count P1 and the number of dead bacteria P2 were detected by flow cytometry, and the survival rate / % at different time periods was calculated. The survival rate in the bacterial suspension without artificial gastric juice treatment was used as a 100% control to calculate the gastrointestinal tolerance of each treatment. The formula for calculating bacterial survival rate at different treatment times is as follows: Survival rate (%) = [(P1) 处理组 -P2 处理组 ) / P1 处理组 ] / [(P1 对照 -P2 对照 ) / P1 对照 ]; Among them, P1 处理组 The total number of bacterial particles in the treatment group, P2 处理组 This refers to the number of dead bacterial particles in the treatment group, P1 对照 This refers to the total number of bacterial particles in the control group, P2. 对照 This refers to the number of dead bacterial particles in the control group.

[0051] (2) The survival rate of Lactobacillus rhamnosus B12 in different gastric fluid environments is shown in Table 6. According to the data, the following can be seen: The survival rate of *Lactobacillus rhamnosus* B12 after 1 hour of treatment in a simulated gastric fluid environment was 80.01%, and the survival rate remained stable between 72.92% and 79.87% after 2-5 hours of treatment, indicating that *Lactobacillus rhamnosus* B12 has good tolerance in a simulated gastric fluid environment.

[0052] Table 2 Survival rate (%) of Lactobacillus rhamnosus B12 in an artificial simulated gastric juice environment

[0053] Example 9: Survival analysis of Lactobacillus rhamnosus B12 in a simulated artificial pancreatic juice environment (1) Take the fermentation broth of Lactobacillus rhamnosus B12 fermented for 24 h, centrifuge at 12000 r / min for 5 min to collect the bacterial cells, add the same volume of 0.85% (w / v) physiological saline and mix well to prepare a bacterial suspension for later use; prepare protein pancreatic juice [0.1% (w / v) secretin, 0.15% (w / v) bovine bile], adjust the pH value to 7.5, filter through a 0.22 μM microporous membrane for later use, take 1 mL of the above bacterial suspension in 9 mL of protein pancreatic juice with different pH values, and incubate at 37℃. After 3 h of treatment, take a sample, take 0.9 mL of the treated sample, add 0.1 mL of PI dilution solution, stain at 37℃ for 10 min, and detect the total bacterial count P1 and the number of dead bacteria P2 by flow cytometry, and calculate the survival rate at different time points. The survival rate of bacteria is calculated with the survival number of untreated bacterial suspension as a control. The formula for calculating bacterial survival rate is: Survival rate (%) = [(P1) 处理组 -P2 处理组 ) / P1 处理组 ] / [(P1 对照 -P2 对照 ) / P1 对照 ]; Among them, P1 处理组 This refers to the total number of bacterial particles in the treatment group, P2 处理组 This refers to the number of dead bacterial particles in the treatment group, P1 对照 This refers to the total number of bacterial particles in the control group, P2. 对照 This refers to the number of dead bacterial particles in the control group. The bacterial survival rate of *Lactobacillus rhamnosus* B12 is shown in Table 7. Based on the data, we can see that: The test results showed that after being treated in a pancreatic juice environment at pH 7.5 for 3 hours, the survival rate of Lactobacillus rhamnosus B12 was 74.14 ± 0.20%, indicating that it has good tolerance to artificially simulated pancreatic juice.

[0054] Example 10: Effects of Lactobacillus rhamnosus B12 on growth and immune factors in zebrafish Two- to three-month-old AB / TU strain blue zebrafish were acclimatized for one week and then divided into four groups (Con, B12L, B12M, and B12H), with 45 fish in each group and 15 fish per tank. After fasting for one day and drying off their body surface, the weight and length of each zebrafish were measured and recorded. Following this, a feeding experiment with *Lactobacillus rhamnosus* B12 was conducted. The control group (Con) was fed chilled small red worms without B12, while the B12L, B12M, and B12H groups were fed chilled small red worms supplemented with astaxanthin (B12 bacterial suspension added to small red worms to a final concentration of 10). 7 10 8 10 9 Feed the treated red worms at a dose of 1.3 g per group, twice a day, morning and evening. (±0.1 CFU / g).

[0055] Four weeks after feeding, the weight and length of each zebrafish were weighed and recorded again using the same method. Changes in weight and length before and after feeding were statistically analyzed. Then, zebrafish from each treatment group were harvested, and the liver, foregut, and spleen were removed. The tissues were rinsed with pre-cooled phosphate buffer (0.01 mol / L, pH=7.4) to remove residual plasma. After weighing, the tissues were minced. The minced tissues were added to a glass homogenizer with the corresponding volume of phosphate buffer (1:9 weight-to-volume ratio) and homogenized thoroughly on ice until complete lysis. The homogenate was centrifuged at 5000 r / min for 10 min, and the supernatant was used to detect the immune factors TNF-α, IL-1β, and IL-10 using a kit.

[0056] (1) Effects of Lactobacillus rhamnosus B12 on zebrafish growth like Figure 10 As shown, after feeding, the body length of zebrafish in both the Con control group and the Lactobacillus rhamnosus B12 group increased significantly, from 2.41-2.48 cm before feeding to 2.67-2.79 cm. However, there was no significant difference in body length between the Con control group and the Lactobacillus rhamnosus B12 (low, medium, and high) groups before and after feeding. The body weight of zebrafish in both the Con control group and the Lactobacillus rhamnosus B12 group increased significantly from 0.112-0.120 g before feeding to 0.152-0.169 g after feeding. However, there was no significant difference in body weight between the Con control group and the Lactobacillus rhamnosus B12 (low, medium, and high) groups before and after feeding. This indicates that feeding with Lactobacillus rhamnosus B12 did not have a significant effect on the body length and body weight of zebrafish.

[0057] (2) Effects of Lactobacillus rhamnosus B12 on immune factors in zebrafish Interleukin-10 (IL-10), as a pleiotropic cytokine, plays a crucial role in the field of immune regulation. Tumor necrosis factor-α (TNF-α) is an important inflammatory factor in the body. It can not only directly kill tumor cells or inhibit their growth, but also promote the phagocytic function, proliferation, and differentiation of neutrophils, playing an indispensable role in maintaining physiological homeostasis and autoimmune regulation.

[0058] Interleukin-1β (IL-1β) is also an important regulator of the occurrence and development of inflammatory immune responses. It can activate immune cells, promote the proliferation of T cells, participate in the antibody production process, and thus promote the inflammatory response.

[0059] like Figure 11 As shown, the effects of *Lactobacillus rhamnosus* B12 on immune factors in zebrafish were analyzed. The results showed that the levels of IL-10 in the low, medium, and high dose groups (B12L, B12M, and B12H) of *Lactobacillus rhamnosus* B12 were significantly higher than those in the control group (32.19 ng / L), reaching 43.46, 39.83, and 40.61 ng / L, respectively. The levels of TNF-α in the low, medium, and high dose groups were 117.54, 171.28, and 209.26 ng / L, respectively, significantly higher than the 84.40 ng / L in the control group (Con). The levels of IL-1β in the medium and high dose groups were 21.99 and 25.60 ng / L, respectively, significantly higher than the 11.18 ng / L in the control group (Control group). These results indicate that feeding zebrafish with *Lactobacillus rhamnosus* B12 at all doses significantly increased the levels of IL-10 and TNF-α, while medium and high doses increased the level of IL-1β.

[0060] Example 11: Anti-allergic effect of Lactobacillus rhamnosus B12 on zebrafish and its influence on gut microbiota. AB / TU strain blue zebrafish aged 7-8 months were acclimatized for one week and then divided into two groups (Con and B12), with 45 fish in each group and 15 fish per tank. A feeding experiment using *Lactobacillus rhamnosus* B12 was conducted. The control group (Con) was fed fresh red worms without *Lactobacillus rhamnosus* B12, while the group with *Lactobacillus rhamnosus* B12 was fed fresh red worms containing a *Lactobacillus rhamnosus* B12 suspension (the final concentration of the *Lactobacillus rhamnosus* B12 suspension added to the red worms was (10...) 7 ~10 9 The treated red worms were fed at a dose of 1.3g per group, twice a day, morning and evening, with a concentration of ±0.1 CFU / g. After 4 weeks of feeding, the intestines were rinsed three times with sterile saline, and then the intestines were harvested under sterile conditions, quickly frozen in liquid nitrogen, and sent to Nomi Metabolism on dry ice for microbial community diversity analysis. The remaining viscera were tested for anti-allergy effects using fish immunoglobulin IgE.

[0061] (1) Effect of Lactobacillus rhamnosus B12 on zebrafish immunoglobulin IgE Allergen induction leads to the secretion of specific IgE by plasma cells, etc. Therefore, detecting IgE levels in animals using immunoassay methods such as ELISA can measure the state of allergic reactions. Zebrafish visceral tissue (intestinal portion removed) was collected, and IgE levels were measured using an ELISA kit. The results are shown in Table 3. It can be seen that after feeding with *Lactobacillus rhamnosus* B12 (B12 suspension with small red worms added to a final concentration of 10), the IgE levels were significantly higher. 7 The IgE level in zebrafish decreased from 0.53 μg / mL in the control group (Con group) to 0.07 μg / mL, with a significant reduction effect (P≤0.05) and a reduction rate of 86.00%. This indicates that consuming Lactobacillus rhamnosus B12 can significantly reduce the allergy level in zebrafish and has a significant anti-allergic effect.

[0062] Table 3. Effects of Lactobacillus rhamnosus B12 on immunoglobulin IgE in zebrafish.

[0063] (2) Venn diagram analysis of the differences in gut microbiota of zebrafish by Lactobacillus rhamnosus B12 Group B12 was conducted (small red worms were added to the B12 suspension to a final concentration of 10). 9 ±0.1 CFU / g) and Wayne analysis of the Con group, as shown Figure 12 As shown, common and unique OTUs were obtained among the samples after feeding with B12 bacteria. Among them, 60 OTUs were unique to Con, 64 OTUs were unique to the B12 group, and 271 were common.

[0064] (3) Alpha diversity analysis Sample diversity analysis (Alpha diversity) can reflect the abundance and diversity of microbial communities, such as... Figure 13 As shown, the bacterial community observation values ​​(Observed), bacterial community abundance indices (Chao1 and Ace), bacterial community uniformity index (Pielou), and bacterial community diversity indices (Shannon and Simpson) were tested and statistically analyzed. It was found that the bacterial community diversity indices (Shannon and Simpson) and bacterial community uniformity index (Pielou) of the Lactobacillus rhamnosus B12 feeding group were higher than those of the control group, but the abundance of the bacterial community observation values ​​(Observed), bacterial community abundance indices (Chao1 and Ace) was lower than that of the control group (Con). This indicates that feeding Lactobacillus rhamnosus B12 has a certain improvement on the diversity and uniformity of zebrafish, but it has not reached a significant level (p=0.2~1).

[0065] (4) Analysis of the abundance of gut microbial material among samples like Figure 14 As shown, an analysis of the effects of *Lactobacillus rhamnosus* B12 on the gut microbiota of zebrafish at the genus level revealed that feeding with *Lactobacillus rhamnosus* B12 reduced the number of *Lactococcus* species (…). Lactococcus The relative abundance of Cetacea ( ) increased the abundance of Cetacea ( ) Cetobacterium ) and Lactobacillus genus ( Lactobacillus The relative abundance of ).

[0066] Lactococcus typically plays a role in the intestinal environment, but excessive abundance is not beneficial to the host. Feeding with *Lactobacillus rhamnosus* B12 significantly reduced the relative abundance of *Lactococcus* and significantly increased the abundance of *Lactobacillus* through competitive inhibition. This suggests that the increased *Lactobacillus* is most likely *Lactobacillus rhamnosus* B12, indicating that *Lactobacillus rhamnosus* B12 can successfully enter the zebrafish intestine and exert its effects of enhancing immunity, inhibiting bacteria, and combating allergies. Cetacea is an important vitamin-synthesizing bacterium in the gut, especially vitamin B12. Vitamin B12 is crucial for the development of the nervous system, erythrocyte production, and DNA synthesis in zebrafish. An increase in Cetacea helps meet the zebrafish's need for vitamin B12 and maintain its normal physiological functions.

[0067] Example 12: Effects of Lactobacillus rhamnosus B12 on the fermentation of ginseng, wolfberry and white grapes Lactobacillus rhamnosus B12 was inoculated into MRS medium and fermented for 24-28 hours to obtain fermentation broth. Ginseng, red goji berries, black goji berries and grapes were taken separately. Ginseng, red goji berries and black goji berries were mixed with 15 times water and pulped, 12% sucrose was added and then sterilized. White grapes were directly pulped and sterilized. Fermentation was carried out with 0.1% B12 inoculum for 96 hours.

[0068] The results are shown in Table 4 and Figure 15 As shown, after fermentation, the pH values ​​of each sample decreased significantly. The pH value of ginseng decreased from 5.38 in the control to 3.48, that of red goji berries decreased from 4.96 in the control to 3.57, that of black goji berries decreased from 4.99 to 3.58, and that of grape juice decreased from 4.26 to 3.53. This indicates that Lactobacillus rhamnosus B12 can ferment at least a variety of medicinal and edible products and fruits. The SOD values ​​of each sample before and after fermentation were detected using a superoxide dismutase (SOD) kit (Nanjing Jiancheng). It was found that the SOD values ​​of ginseng, red goji berries, black goji berries, and white grapes increased from 19.96, 22.72, 23.00, and 20.56 (U / mL) before fermentation to 41.29, 24.57, 28.86, and 23.36 (U / mL) after fermentation. After 4 days of fermentation, the viable cell count reached 8.3–9.59 log CFU / mL. The color of each sample was brightened after fermentation with B12 bacteria, all indicating that fermentation can enhance the antioxidant capacity of the fermentation medium.

[0069] Table 4. Effects of B12 fermentation on the quality of ginseng, red goji berries, and white grapes.

[0070] * indicates a significant difference between B12 fermentation and CK unfermented (p≤0.05).

[0071] Furthermore, the flavor of the fermented samples was evaluated. After high-pressure sterilization, the above plant-based samples exhibited different degrees of cooking flavor characteristics. After fermentation with B12, the cooking flavor of each sample was significantly removed and the flavor characteristics were significantly improved. Among them, ginseng exhibited fermented aroma, sour aroma and fresh aroma, red goji berries and black goji berries exhibited sour aroma and aromatic aroma, and grapes exhibited sour aroma and floral aroma, indicating that fermentation can significantly improve the flavor quality of the above samples.

[0072] Example 13: Effects of Lactobacillus rhamnosus B12 on protein and amino acids in red goji berries Red goji berry + Bacterium B12 fermentation broth was prepared according to Example 12 above. The amino acid content was determined according to the determination of amino acids in food (GB5009.124-2016), and the protein content was determined according to the determination of protein in food (GB 5009.2-2016).

[0073] The results are shown in Table 5. The results indicate that after fermentation with B12, the total protein content in the fermented red goji berry broth reached 7.31±0.07 mg / g, which was significantly higher than that of the unfermented group (6.02±0.05 mg / g, p≤0.01), representing an increase of 21.53%. The total amino acid content after fermentation was 3.423±0.063 mg / g, which was significantly higher than that of the unfermented group (2.99±0.013 mg / g, p≤0.05), representing an increase of 14.55%. This indicates that B12 fermentation can significantly increase the protein and amino acid content of red goji berries.

[0074] Table 5. Effect of B12 fermentation on amino acid content of red goji berries (mg / g)

[0075] This application also provides the following application examples of Lactobacillus rhamnosus B12: Example 14: Preparation of probiotic agent from Lactobacillus rhamnosus B12 Lactobacillus rhamnosus B12 was inoculated into a culture medium, such as MRS medium, and cultured at 0-40°C for more than 15 hours. The bacterial cells were collected by centrifugation and resuspended in, for example, physiological saline or PBS buffer to prepare a liquid bacterial preparation containing Lactobacillus rhamnosus B12.

[0076] Optionally, somatic cells of Lactobacillus rhamnosus B12 are resuspended in a cell protectant and a carrier, and then freeze-dried to obtain a solid bacterial powder formulation containing Lactobacillus rhamnosus B12.

[0077] Optionally, Lactobacillus rhamnosus B12 can be used as a raw material component in anti-allergy, immune-enhancing, gut microbiota-regulating, and antioxidant products. Lactobacillus rhamnosus B12 can be present in the products in liquid or solid formulation form.

[0078] Example 15: Preparation of fermented foods using Lactobacillus rhamnosus B12 To prepare Lactobacillus rhamnosus B12 yeast broth, various fruits, medicinal and edible herbs, grain raw materials, and various sugars are used as auxiliary materials. Lactobacillus rhamnosus B12 is inoculated and fermented for a certain period of time under certain temperature conditions (0-40℃) to prepare fermented products. The fermented products are either inactivated or not inactivated. After the original liquid or different proportions are diluted, common beverage auxiliary materials are added to prepare fermented food.

[0079] Based on the results of the above embodiments, the Lactobacillus rhamnosus B12 provided in this application has the following properties and effects: Based on the results of the above embodiments, the Lactobacillus rhamnosus B12 provided in this application has the following properties and effects: (1) It can utilize sugars such as sorbitol, cellobiose, maltose, mannitol, salicin, sucrose, raffinose, inulin, and lactose, and has the characteristic of broad substrate adaptability; (2) It can grow well in MRS medium with isomaltooligosaccharide, galactooligosaccharide, inulin and fructooligosaccharide instead of glucose. It can produce high levels of short-chain fatty acids such as propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and valeric acid by isomaltooligosaccharide, galactooligosaccharide, fructooligosaccharide and xylooligosaccharide. (3) It has good biofilm formation ability, and the biofilm formation ability (ODX: ODC value) after using inulin, fructooligosaccharide and galactooligosaccharide isomaltosaccharide can reach 8.52, 6.14 and 5.20 respectively; (4) It has good free radical scavenging ability, with the DPPH free radical scavenging rate of the extracellular enzyme of bacterial B12 being 47.95% and the DPPH intracellular enzyme free radical scavenging rate being 20.39%; (5) It has a certain inhibitory effect on Streptococcus mutans and Staphylococcus aureus, with inhibition zone diameters of 1.42cm and 1.37cm, respectively; (6) It can survive for at least 5 hours in an artificial simulated gastric juice environment, with a survival rate of up to 80.1%; (7) After being treated in an artificial pancreatic juice environment for 3 hours, the survival rate reached 74.14%; (8) Bacterial B12 has a significant effect on enhancing the immunity of zebrafish. It can increase IL-10 from 32.19 ng / L in the Con group to a maximum of 43.46 ng / L, TNF-α from 84.40 ng / L in the Con group to a maximum of 209.26 ng / L, and IL-1β from 11.18 ng / L in the Con group to a maximum of 25.60 ng / L; (9) Bacterial B12 can significantly reduce zebrafish immunoglobulin IgE, with a reduction rate of 86.00%, and has a significant anti-allergic effect; (10) Significantly modulates the gut microbiota of zebrafish, changing the main gut microbiota of zebrafish from Lactococcus to Lactobacillus and Cetacea, thus optimizing the gut microbiota structure; (11) It can ferment ginseng, red goji berries, black goji berries and white grapes, significantly reduce the pH value of the fermentation substrate, and significantly increase the SOD value and color of the fermentation substrate. The number of viable bacteria of B12 in the substrate after 4 days of fermentation is as high as 8.3 to 9.59 log CFU / mL. B12 fermentation can significantly increase the protein content of red goji berries (increase by 21.53%) and the total amino acid content (increase by 14.55%).

[0080] Lactobacillus rhamnosus B12 was isolated from the intestines of healthy human children. It is highly safe for consumption and can be used in functional products such as anti-allergy products, immune-boosting products, products for regulating intestinal flora, and antioxidant products, showing broad application prospects.

[0081] In summary, compared with the prior art, the Lactobacillus rhamnosus B12 provided in this application has the following beneficial effects: The *Lactobacillus rhamnosus* B12 carbon source provided in this application exhibits broad growth adaptability, good short-chain fatty acid secretion levels, biofilm formation ability, DPPH free radical scavenging ability, and good tolerance to gastric and pancreatic juices. It can promote the expression of various immune factors in zebrafish, reduce immunoglobulin IgE levels, and optimize the intestinal flora structure. It also shows good antibacterial effects against *Streptococcus mutans* and *Staphylococcus aureus*. Furthermore, it can ferment plant-based products such as ginseng, goji berries, and white grapes, enhancing the SOD activity, matrix color, and flavor quality of the fermented products, and significantly increasing the protein and total amino acid content of red goji berries. Therefore, this bacterium can provide a new probiotic source for the development of anti-allergy products, immune-enhancing products, intestinal flora-regulating products, antioxidant products, antibacterial products, and fermented products, and has significant application value.

[0082] For example: (1) Lactobacillus rhamnosus B12 can be used as a raw material component of the composition to prepare a composition with the above-mentioned anti-allergy, immune enhancement, intestinal flora regulation, antioxidant and antibacterial functions.

[0083] The bacterial strain present in the composition includes, but is not limited to, one or more combinations of *Lactobacillus rhamnosus* B12 (non-inactivated), *Lactobacillus rhamnosus* B12 (inactivated), metabolites of *Lactobacillus rhamnosus* B12 strain, and freeze-dried *Lactobacillus rhamnosus* B12 strain. Preferably, the number of *Lactobacillus rhamnosus* B12 in the composition is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. More preferably, the number of *Lactobacillus rhamnosus* B12 is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0084] (2) Various plants can be used as raw materials, combined with various ingredients, and inoculated with Lactobacillus rhamnosus B12 for fermentation to prepare fermented products. The fermented products have increased SOD content and amino acid content, and can be used to prepare products with antioxidant properties. The fermentation raw materials can be various commonly used plant fermentation raw materials, and the auxiliary materials can also be existing conventional auxiliary materials, including but not limited to the above-mentioned scheme selection.

[0085] In summary, Lactobacillus rhamnosus B12 and / or its ferments, based on their characteristics, can be used in functional products that include at least one of the following functions: (1) Good carbon source utilization level; (2) Good level of short-chain fatty acid production; (3) Good biofilm formation ability; (4) Excellent DPPH free radical scavenging ability; (5) Good tolerance to gastric and pancreatic juices; (6) Significantly enhances immunity; (7) It can significantly reduce IgE levels and has anti-allergic effects; (8) Good intestinal flora regulation effect; (9) It has good antibacterial ability against Streptococcus mutans and Staphylococcus aureus; (10) It can ferment plant-based materials such as ginseng, wolfberry and white grape, and improve their SOD value and flavor quality. The number of live bacteria in the above substrates after 4 days of fermentation is greater than 8.0 log CFU / mL, which significantly increases the content of protein and total amino acids in red wolfberry.

[0086] Among them, products with the above (1)-(10) functions include, but are not limited to, anti-allergy, immune enhancement, intestinal flora regulation, antioxidant and other manifest effects; and can also play other manifest effects that are beneficial to health based on the correlation between the functions of (1)-(10) and health care, including but not limited to, anti-allergy, immune enhancement, intestinal flora regulation, antioxidant and other manifest effects.

[0087] It should be noted that: (1) Definition: The term "food" as used in this article is used in a broad sense, including human food and drink.

[0088] The Lactobacillus rhamnosus B12 exists in the composition in forms including but not limited to non-inactivated bacteria, inactivated bacteria, metabolites, freeze-dried strains, etc. It is anticipated that the Lactobacillus rhamnosus B12 may also exist in the composition in other forms.

[0089] (2) The relevant prior art means or prior art terms involved in this application: "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. x "OD" is the optical density value measured when the wavelength is set to X nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0090] The determination of total bacterial count P1 and dead bacterial count P2 using flow cytometry is an existing technology, and its principles and methods will not be elaborated here.

[0091] Carbon source utilization analysis was performed using biochemical identification strips for lactic acid bacteria. This is an existing technology, and its principles and methods will not be elaborated here.

[0092] (3) The formulations of the culture media used in the examples are as follows: MRS medium (g / L): 10g casein peptone, 10g beef extract, 5g yeast extract, 5g glucose, 5g sodium acetate, 2g K2HPO4, 2g diammonium citrate, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 1mL Tween 80, pH=6.2. For solid medium, add 2% agar and 2% CaCO3 to the above and sterilize at 121℃ for 15min.

[0093] Brain and heart extract culture medium (g / L): brain extract powder 5.0, heart extract powder 12.5, peptone 5.0, tryptone 10.0, sodium chloride 5.0, glucose 2.0, disodium hydrogen phosphate 2.5, agar 20.0 (not included in liquid culture medium), pH 7.4±0.2, sterilized at 121 ℃ for 15 min.

[0094] LB medium (g / L): tryptone 10.0, sodium chloride 10.0, yeast extract 5.0, pH 7.5; (solid medium with 20.0g agar powder), sterilized at 121℃ for 15min.

[0095] MRS+L-cysteine ​​liquid medium: 0.5% (w / v) of L-cysteine ​​was added to the above MRS medium to obtain MRS+L-cysteine ​​liquid medium.

[0096] Unless otherwise specified, the experimental procedures involved in the embodiments of this application are conventional experimental procedures in the field, and the reagents or instruments involved can be obtained from legitimate channels.

[0097] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of this application can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus B12, characterized in that: Its accession number is CGMCC No. 34366.

2. A microbial agent, characterized in that: It contains Lactobacillus rhamnosus B12 as described in claim 1.

3. A composition, characterized in that: It contains Lactobacillus rhamnosus B12 as described in claim 1.

4. The composition according to claim 3, characterized in that: The composition comprises one or more combinations of Lactobacillus rhamnosus B12 non-inactivated bacteria, Lactobacillus rhamnosus B12 inactivated bacteria, metabolites of Lactobacillus rhamnosus B12 strain, and freeze-dried Lactobacillus rhamnosus B12 strain.

5. The use of Lactobacillus rhamnosus B12 as described in claim 1 or the composition as described in any one of claims 3-4 in the preparation of fermented foods.

6. A fermentation product, characterized in that: The ferment is obtained by fermentation of Lactobacillus rhamnosus B12 as described in claim 1.

7. The fermented product as described in claim 6, characterized in that: It was obtained by inoculating the Lactobacillus rhamnosus B12 onto a medicinal and edible plant substrate and then fermenting it.

8. The fermented product as described in claim 7, characterized in that: The medicinal and edible plant base includes one or more of ginseng, wolfberry, and white grape.

9. The use of Lactobacillus rhamnosus B12 as described in claim 1 and / or the fermentation product as described in claim 6 in the preparation of functional products.

10. The application as described in claim 9, characterized in that: The functions of the product include one or more of the following: anti-allergy, immune enhancement, gut microbiota regulation, and antioxidant properties.

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