Lactobacillus rhamnosus and application thereof in relieving aflatoxin B1 induced liver injury

Abstract

The invention provides lactobacillus rhamnosus and application thereof in relieving aflatoxin B1 induced liver injury, and particularly belongs to the technical field of microorganisms. The lactobacillus rhamnosus TY-63 is characterized in that the preservation number of the lactobacillus rhamnosus TY-63 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 20251683, and the lactobacillus rhamnosus TY-63 is a lactobacillus rhamnosus TY-63. The bacterial strain has bacteriostatic activity and better oxidation resistance, and can effectively reduce the level of proinflammatory factors in serum, down-regulate the relative expression of pro-apoptosis genes and reduce the apoptosis of liver cells, thereby effectively relieving the damage to the liver caused by exposure to aflatoxin B1.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus rhamnosus and its application in alleviating aflatoxin B1-induced liver damage. Background Technology

[0002] Aflatoxins (AFTs) are dihydrofuranocoumarin fungal toxins produced by *Aspergillus flavus* and *Aspergillus parasiticus*, belonging to the class of highly toxic secondary metabolites. The four main naturally occurring types are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). AFB1 is the most toxic, inducing growth inhibition, inflammatory responses, and oxidative stress damage, exhibiting strong toxicity to multiple organs including the liver, intestines, and kidneys. AFB1 is widely found in crops such as corn, peanuts, and soybeans, and it is difficult for humans and animals to completely avoid ingestion through their diet. Long-term low-dose exposure to AFB1 induces chronic organ damage. The liver is the target organ for AFB1 toxicity and the primary site of AFB1 metabolism. AFB1 induces hepatotoxicity and liver damage, including oxidative damage, inflammatory responses, and apoptosis.

[0003] Compared with commonly used nutrient interventions and chemical drug interventions, lactic acid bacteria can alleviate AFB1-induced damage through multiple mechanisms, such as enhancing the host's antioxidant capacity, regulating immune responses, and inhibiting excessive hepatocyte apoptosis, with almost no side effects on the host.

[0004] *Lactaseibacillus rhamnosus* is a Gram-positive, facultative anaerobic, short rod-shaped probiotic (0.8–1.0 × 2.0–4.0 μm in size), non-spore-forming, non-motile, forming round, white, raised colonies on MRS media. Taxonomically, it belonged to the genus *Lactobacillus*, but was reclassified to the genus *Lactobacillus* in 2020 based on whole-genome phylogenetic analysis, and its official nomenclature was updated by the National Health Commission of China in 2022. This bacterium exhibits excellent environmental adaptability: a growth temperature range of 2–53℃ (optimal 30–40℃), strong acid resistance (optimal pH 5.5–6.2, tolerating pH <5), and a DNA G+C content of 45–47%. *Lactaseibacillus rhamnosus* possesses various probiotic functions, including inhibiting pathogen adhesion and infection, regulating intestinal flora balance, anti-oxidation and anti-inflammation, and enhancing immune function. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to alleviate aflatoxin B1 (AFB1)-induced liver damage.

[0006] To solve the above-mentioned technical problems, the present invention first provides Lactobacillus rhamnosus, specifically Lactobacillus rhamnosus TY-63, which has the accession number CCTCC NO: M 20251683 at the China Center for Type Culture Collection.

[0007] The present invention also provides a composition comprising the aforementioned Lactobacillus rhamnosus.

[0008] In one specific embodiment of the present invention, the composition is a bacterial suspension prepared from Lactobacillus rhamnosus, and the preparation method of the bacterial suspension is as follows: take live bacterial cells and resuspend them in PBS buffer.

[0009] The active ingredient in the above composition may be the Lactobacillus rhamnosus or / and the metabolites of the Lactobacillus rhamnosus or / and the culture of the Lactobacillus rhamnosus.

[0010] In the above text, the culture can be the substance obtained by culturing the Lactobacillus rhamnosus in a microbial culture medium.

[0011] The metabolites can be obtained from the shake culture broth of *Lactobacillus rhamnosus*. The metabolites can be bacterial metabolites of *Lactobacillus rhamnosus*. Specifically, the bacterial metabolites of *Lactobacillus rhamnosus* can be prepared by culturing *Lactobacillus rhamnosus* in MRS (Man-Rogosa-Sharpe) broth and collecting the bacterial solution; this bacterial solution is the bacterial metabolite of *Lactobacillus rhamnosus*.

[0012] MRS broth medium (g / L) is prepared from the following ingredients: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 15.0 g agar powder, 1.0 g Tween 80, and the remainder is water, pH 6.5±0.2.

[0013] The substance may also be a fermentation product, such as a fermentation broth containing the Lactobacillus rhamnosus and the substance secreted into a liquid culture medium, or a solid fermentation product containing the Lactobacillus rhamnosus and the substance secreted into a solid culture medium.

[0014] The above composition can be a culture, which can be a substance obtained by culturing the Lactobacillus rhamnosus in a microbial culture medium. The culture can be a substance obtained by culturing the Lactobacillus rhamnosus in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the Lactobacillus rhamnosus and a substance secreted into a liquid culture medium, or a solid fermentation product containing the Lactobacillus rhamnosus and a substance secreted into a solid culture medium).

[0015] The composition may be a microbial agent containing *Lactobacillus rhamnosus* and / or its metabolites. The metabolites may be products obtained by removing *Lactobacillus rhamnosus* from the culture, such as culturing *Lactobacillus rhamnosus* in a liquid fermentation medium, collecting the fermentation broth (containing *Lactobacillus rhamnosus* and substances secreted into the liquid medium), removing the *Lactobacillus rhamnosus* from the fermentation broth, collecting the remaining components of the fermentation broth, and obtaining the metabolites of *Lactobacillus rhamnosus*. In one embodiment of the invention, the metabolites may be a sterile supernatant.

[0016] The composition may be a microbial agent.

[0017] The composition described above may have at least one of the following properties:

[0018] A1) Relieves liver damage,

[0019] A2) Inhibits liver cell apoptosis.

[0020] A3) Reduce serum levels of pro-inflammatory factors.

[0021] A4) Reduces the activity of alanine aminotransferase in serum.

[0022] A5) Reduces the activity of serum aspartate aminotransferase.

[0023] A6) Reduces the activity of alkaline phosphatase in serum.

[0024] A7) Reduces the activity of lactate dehydrogenase in serum.

[0025] A8) Reduces the level of malondialdehyde in the liver.

[0026] A9) Increases the level of glutathione in the liver.

[0027] A10) increases the activity of superoxide dismutase in the liver.

[0028] A11) regulates hepatocyte apoptosis-related pathways.

[0029] A12) Upregulates the relative expression of the anti-apoptotic gene BCL2.

[0030] A13) downregulates the relative expression of the pro-apoptotic gene BAX.

[0031] A14) Downregulates the relative expression of the pro-apoptotic gene CASP3.

[0032] A15) Treatment for weight loss in animals caused by aflatoxin B1

[0033] A16) Treatment for increased liver organ coefficient caused by aflatoxin B1

[0034] A17) antagonizes pathogenic bacteria.

[0035] In this invention, the term "treatment" generally refers to a method performed to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, narrowing of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease state, and remission (whether partial or complete), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment.

[0036] The active ingredients of the above composition may also contain other biological or non-biological components, and those skilled in the art can determine the other active ingredients of the above composition based on the effects of the composition.

[0037] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.

[0038] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0039] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0040] The present invention also provides a method for preparing the above composition, the method comprising the step of using the aforementioned Lactobacillus rhamnosus as a component of the composition.

[0041] The scope of protection of this invention also includes the use of the aforementioned Lactobacillus rhamnosus or its composition in the preparation of products.

[0042] The product may have at least one of the following properties:

[0043] B1) alleviates liver damage.

[0044] B2) Inhibits liver cell apoptosis.

[0045] B3) Reduces serum levels of pro-inflammatory factors.

[0046] B4) Reduces the activity of alanine aminotransferase in serum.

[0047] B5) Reduces the activity of serum aspartate aminotransferase.

[0048] B6) Reduces the activity of alkaline phosphatase in serum.

[0049] B7) Reduces the activity of lactate dehydrogenase in serum.

[0050] B8) Reduces the level of malondialdehyde in the liver.

[0051] B9) Increases the level of glutathione in the liver.

[0052] B10 increases the activity of superoxide dismutase in the liver.

[0053] B11 regulates hepatocyte apoptosis-related pathways.

[0054] B12) upregulates the relative expression of the anti-apoptotic gene BCL2.

[0055] B13) downregulates the relative expression of the pro-apoptotic gene BAX.

[0056] B14) downregulates the relative expression of the pro-apoptotic gene CASP3.

[0057] B15) was used to treat weight loss in animals caused by aflatoxin B1.

[0058] B16) is used to treat the increased liver organ coefficient caused by aflatoxin B1.

[0059] B17) antagonizes pathogenic bacteria.

[0060] In this invention, the pathogenic bacteria can be either pathogenic bacteria or pathogenic fungi. Specifically, the pathogenic bacteria may be *Escherichia coli* ATCC 11775, *Micrococcus luteus* ATCC 4698, *Staphylococcus aureus* ATCC 29213, *Pseudomonas aeruginosa* ATCC 27853, *Listeria monocytogenes* ATCC 51780, *Klebsiella pneumoniae* ATCC BAA-1898, or *Salmonella enterica subsp. enterica* ATCC 13076.

[0061] The specific pathogenic fungi mentioned may be Aspergillus flavus BNCC 142803, Penicillium expansum BNCC 146144, and Aspergillus niger ATCC 16888.

[0062] In one specific embodiment of the present invention, the liver cell apoptosis-related pathway includes the anti-apoptotic gene BCL2, the pro-apoptotic gene BAX, and CASP3.

[0063] In the above compositions or products, the pro-inflammatory factors may be IL-1β, IL-17, and NO, etc.

[0064] Specifically, the product may be a reagent, preparation, drug, pharmaceutical composition, or vaccine.

[0065] The present invention also provides a method for alleviating liver damage, the method comprising taking the aforementioned Lactobacillus rhamnosus or the composition thereof to alleviate liver damage.

[0066] In this invention, the liver damage is caused by aflatoxin B1.

[0067] The purpose of the above methods can be for disease prognosis and / or disease treatment; or it can be for non-disease prognosis and non-disease treatment purposes.

[0068] This invention isolates a strain of Lactobacillus rhamnosus TY-63 that is resistant to acid, bile salts, and the gastrointestinal environment from fecal samples of healthy infants. TY-63 has antibacterial activity and good antioxidant capacity, as well as certain self-aggregation ability and hydrophobicity, and has good safety.

[0069] This invention provides the application of *Lactobacillus rhamnosus* TY-63 in AFB1-induced liver injury. *Lactobacillus rhamnosus* TY-63 can effectively reduce the levels of pro-inflammatory factors such as IL-17, IFN-γ, and NO in serum; reduce the activities of AST, ALT, ALP, and LDH; reduce the content of MDA in the liver, increase the content of GSH, and increase SOD activity; inhibit hepatocyte apoptosis; upregulate the relative expression of the anti-apoptotic gene BCL2; and downregulate the relative expression of the pro-apoptotic genes BAX and CASP3, effectively alleviating liver damage caused by AFB1 exposure.

[0070] Preservation Instructions

[0071] Strain name: Lactobacillus rhamnosus

[0072] Latin name: Lacticaseibacillus rhamnosus

[0073] Strain number: TY-63

[0074] Preservation Institution: China Center for Type Culture Collection

[0075] Abbreviation for depository institution: CCTCC

[0076] Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province

[0077] Deposit date: July 23, 2025

[0078] Collection Center Registration Number: CCTCC NO: M 20251683 Attached Figure Description

[0079] Figure 1 The images show the morphology of strain TY-63. (A) shows the colony morphology of strain TY-63, and (B) shows an optical microscope image of strain TY-63. Detailed Implementation

[0080] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0082] The following examples used SPSS 27.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and a one-way ANOVA test was used. The lowercase letters after the numerical values ​​indicate the significance of the differences. Treatments with the same letter showed no significant difference at the 0.05 level, while treatments with different letters showed significant differences at the 0.05 level.

[0083] The culture medium preparation method involved in the following examples is as follows:

[0084] MRS (Man-Rogosa-Sharpe) broth medium (g / L): Each liter of medium contains 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 15.0 g agar powder, 1.0 g Tween 80, and the remainder is water, pH 6.5±0.2.

[0085] MRS solid medium (g / L): Each liter of medium contains 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 14.0g agar, 1.0g Tween 80, 15.0g agar powder, and the remainder is water, pH 6.5±0.2.

[0086] Nutrient Broth (NB) medium (g / L): Each liter of medium contains 10.0 g peptone, 5.0 g sodium chloride, 3.0 g beef extract powder, and the remainder is water, pH 7.2±0.2.

[0087] Nutrient agar (NA) medium (g / L): Each liter of medium contains 10.0g peptone, 5.0g sodium chloride, 3.0g beef extract, 15.0g agar powder, and the remainder is water, pH 7.3±0.1.

[0088] Potato Dextrose Agar (PDA) medium: The finished potato dextrose agar medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd., product number 02-023.

[0089] Columbia Blood Agar Medium: The finished Columbia Blood Agar medium was purchased from Shanghai Lianshuo Biotechnology Co., Ltd., product number CP0160.

[0090] LB liquid medium: Each liter of medium contains 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, and the remainder is water, pH 7.0±0.1.

[0091] Toluidine Blue-DNase Assay Agar: The finished toluidine blue-DNase assay agar was purchased from Aili Biotechnology (Shanghai) Co., Ltd., product number M1134-01.

[0092] Nitroreductase activity assay medium (g / L): Each liter of medium contains 10.0 g peptone, 1.0 g potassium nitrate, and the remainder is water, pH 7.4.

[0093] Example 1: Isolation, purification and identification of TY-63 strain

[0094] 1. Isolation and purification of the bacterial strain. 1g of fecal sample from a healthy infant collected in Xinxiang City, Henan Province, was added to 9mL of sterile water and shaken thoroughly. After serial dilution of the sample, 100μL was spread onto MRS solid medium and incubated at 37℃ for 48h. Colonies that were milky white, opaque, relatively moist, smooth, with regular edges and obvious ridges were selected and repeatedly streaked on MRS solid medium for purification until the colony morphology of the strain was uniform. The purified lactic acid bacteria strain was mixed with 10% glycerol and inoculated into cryovials, thoroughly mixed, and stored at -80℃. The purified strain is hereby referred to as TY-63.

[0095] 2. Morphological characteristics of the strain. The TY-63 strain, isolated and purified in step 1 above and exhibiting stable colony size during the logarithmic growth phase, was observed as a single colony. After culturing on MRS agar for 48 hours, the colonies of TY-63 were milky white, opaque, relatively moist, smooth, with regular edges and distinct ridges. Under a light microscope, the cells were rod-shaped, did not produce spores, and were Gram-positive. Results are as follows: Figure 1 (A) is a colony morphology diagram of strain TY-63, and (B) is an optical microscope image of strain TY-63.

[0096] 3. Molecular genetic identification of the strain. Whole genome sequencing. DNA was extracted from the screening strain using the TGUIDE S96 magnetic bead DNA extraction toolkit. Genomic DNA concentration was detected using a TBS 380, and genomic DNA purity was detected using Nanodrop. After the samples passed the tests, library construction was performed, and sequencing experiments were conducted only after the libraries passed quality control. Single-molecule PacBio sequencing and Illumina sequencing revealed that the genome size of TY-63 was 3,024,689 bp, with a GC content of 46.70%. The 16S rDNA of strain TY-63 has the nucleotide sequence of SEQ ID NO:1 (1493 bp), as follows:

[0097]

[0098] Finally, through morphological analysis and whole-genome sequencing, the selected strain was identified as *Lactobacillus rhamnosus*, and named *Lactobacillus rhamnosus* TY-63, abbreviated as TY-63. *Lactobacillus rhamnosus* TY-63 was deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCC NO: M 20251683.

[0099] II. Preparation of Lactobacillus rhamnosus TY-63 bacterial suspension

[0100] After two subcultures of Lactobacillus rhamnosus TY-63, single colonies were picked and inoculated into MRS broth medium and cultured at a constant temperature with shaking for 24 h (37℃, 180 rpm). The fermentation broth was centrifuged for 5 min (4℃, 8000 g), and the supernatant was poured into a pre-prepared sterile Erlenmeyer flask and filtered twice through a 0.22 μm filter membrane to obtain a cell-free supernatant. The precipitated bacterial cells were washed twice with sterile PBS buffer, and then the bacterial cells were mixed thoroughly with a certain amount of sterile PBS buffer. The viable cell count in the bacterial suspension was calculated using a hemocytometer, and the viable cell count in the bacterial suspension was adjusted to 1 × 10⁻⁶ using sterile PBS buffer. 10 CFU / mL was used to obtain a suspension of Lactobacillus rhamnosus TY-63, hereinafter referred to as TY-63 suspension or suspension.

[0101] III. Tolerance to Lactobacillus rhamnosus TY-63.

[0102] 1. Acid resistance.

[0103] The pH of MRS broth medium was adjusted to 3.0, 4.0, and 5.0 using dilute hydrochloric acid. TY-63 bacterial suspension was inoculated into different pH media at a 2% inoculum size and anaerobically cultured at 37°C for 24 hours. The digestion solutions at 0 hours and 4 hours were used to detect viable cell counts and calculate the survival rate. The survival rate (%) was calculated as A / B × 100%. Where A represents the viable cell count (CFU / mL) at 4 hours, and B represents the viable cell count (CFU / mL) at 0 hours.

[0104] (2) Conclusion: In the acid tolerance test, the survival rate of TY-63 was 90.41% at pH 3.0, and exceeded 100% at pH 4.0 and 5.0. After 4 hours of incubation, the number of viable bacteria in TY-63 at pH 4.0 and 5.0 was significantly higher than that at 0 hours. These results demonstrate the excellent adaptability and survival ability of TY-63 in acidic environments.

[0105] Table 1 L. rhamnosus TY - Survival rate of 63 in acidic environment

[0106]

[0107] 2. Bile salt tolerance

[0108] (1) Method: Bovine bile salts were weighed and added to MRS broth medium to achieve a bile salt concentration of 0.3% (3 g / L). The medium was autoclaved at 121℃ for 20 min. A 2% inoculum of TY-63 bacterial suspension was inoculated into MRS broth medium with a bile salt concentration of 0.3% (w / v). The medium was incubated at 37℃. Three parallel experiments were conducted, with samples taken at 0 h, 1 h, 2 h, 3 h, and 4 h of incubation. The samples were serially diluted with sterile PBS buffer, plated onto MRS solid medium, and viable cell counts were performed. The average value of the three parallel experiments was calculated. The control group consisted of MRS broth medium without added bile salts. The formula for calculating the bile salt tolerance of the strain was: Bile salt tolerance (%) = Nx / N × 100%. In the formula: Nx represents the number of viable bacteria (CFU / mL) in the culture medium containing 0.3% bile salts at 1, 2, 3 and 4 h; N represents the number of viable bacteria (CFU / mL) in the MRS broth culture medium without added bile salts at the corresponding time point.

[0109] (2) Conclusion:

[0110] Table 2 L. rhamnosus TY - Survival rate of 63 in a 0.3% bile salt environment

[0111]

[0112] 3. Tolerance to artificial gastrointestinal fluids

[0113] (1) Methods: Preparation of artificial gastric fluid: 0.35g of pepsin was diluted in 0.9% sterile physiological saline, the pH was adjusted to 2.5 with dilute hydrochloric acid, and filtered through a 0.22μm filter membrane to obtain simulated gastric fluid with a pepsin mass fraction of 0.2%. Preparation of artificial intestinal fluid: 0.1g of trypsin, 1.8g of ox bile salts, 1.1g of sodium carbonate and 0.2g of sodium chloride were added to 100mL of sterile water, the pH was adjusted to 8.0 with sodium hydroxide, and filtered through a 0.22μm filter membrane to obtain simulated intestinal fluid with a trypsin mass fraction of 0.1%. 20mL of TY-63 bacterial suspension was added to 20mL of simulated gastric fluid and cultured at 37℃ for 3h. Then, 20mL of simulated intestinal fluid was added to the co-culture medium and cultured at 37℃ for 4h to simulate the digestive process of the stomach and small intestine. Samples were taken at 0h, 1.5h, 3h, 5h and 7h of culture, and the viable bacterial count was determined by dilution plating method.

[0114] (2) Conclusion: Under artificial gastrointestinal fluid conditions, the survival rates of TY-63 strain after 1.5h, 3h, 5h, and 7h of culture were 72.23%, 39.94%, 9.27%, and 10.53%, respectively. TY-63 was in the artificial gastric fluid digestion stage from 0 to 3h. The survival rate of TY-63 was relatively high from 0 to 1.5h. As the digestion time increased, the survival rate of TY-63 decreased, but it began to rise in the later stages of artificial intestinal fluid digestion, suggesting that TY-63 gradually adapted to and proliferated in the artificial intestinal fluid environment.

[0115] Table 3 L. rhamnosus TY - Survival rate of 63 in an artificial gastrointestinal environment

[0116]

[0117] IV. Lactobacillus rhamnosus TY - 63 has beneficial properties.

[0118] The indicator bacteria are all ATCC products, specifically: Escherichia coli ATCC 11775, Micrococcus luteus ATCC 4698, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Listeria monocytogenes ATCC 51780, Klebsiella pneumoniae ATCC BAA-1898, and Salmonella enterica subsp. enterica ATCC 13076.

[0119] Indicator fungi: Aspergillus flavus BNCC 142803, Penicillium ex pansum BNCC 146144 and Aspergillus niger ATCC 16888.

[0120] 1. Ability to inhibit pathogenic bacteria.

[0121] (1) Method: The ability to inhibit pathogenic bacteria was tested by the hole punching method.

[0122] After two subcultures of Lactobacillus rhamnosus TY-63, a single colony was picked and inoculated into MRS broth medium and cultured at a constant temperature with shaking for 24 h (37℃, 180 rpm). The fermentation broth was centrifuged for 5 min (4℃, 8000 g), and the supernatant was poured into a pre-prepared sterile Erlenmeyer flask and filtered twice through a 0.22 μm filter membrane to obtain the cell-free supernatant, a metabolite of TY-63.

[0123] The pathogenic bacterial strains *Escherichia coli* ATCC 11775, *Micrococcus luteus* ATCC 4698, *Staphylococcus aureus* ATCC 29213, *Pseudomonas aeruginosa* ATCC 27853, *Listeria monocytogenes* ATCC 51780, *Klebsiella pneumoniae* ATCC BAA-1898, and *Salmonella enterica subsp. enterica* ATCC 13076 were activated and subcultured twice. Single colonies were then picked and inoculated onto NB medium and cultured at a constant temperature with shaking for 12 hours (37℃, 180 rpm). The optical density of the fermentation broth at 600 nm was measured using a UV spectrophotometer. Density (OD) value, determined by using sterile water to measure the OD value of the fermentation broth. 600 Adjust to 1.0. In a clean bench, add the indicator bacteria fermentation broth to sterile NA medium cooled to approximately 50°C at a ratio of 2% (v / v), pour evenly into disposable plastic petri dishes, and allow to cool and solidify. Use a sterilized punch (10 mm diameter) to make three wells on the plate, add 200 μL of the prepared cell-free supernatant of TY-63 to each well, and use sterile MRS broth medium as a control. Incubate at 37°C for 48 h, observe whether there is a transparent inhibition zone around the well, and measure the diameter (mm) of the inhibition zone using calipers to evaluate the antibacterial activity of TY-63 metabolites.

[0124] (2) Conclusion:

[0125] The inhibition zone diameter of the cell-free supernatant metabolite of TY-63 against Listeria monocytogenes ATCC 51780 was 21.13±0.33 mm; against Staphylococcus aureus ATCC 29213 was 21.40±0.25 mm; against Micrococcus luteus ATCC 4698 was 25.80±0.75 mm; against Escherichia coli ATCC 11775 was 14.88±0.58 mm; against Pseudomonas aeruginosa ATCC 27853 was 15.49±0.13 mm; and against Klebsiella pneumoniae ATCC 51780 was 21.13±0.33 mm. The inhibition zone diameter of BAA-1898 was 15.40±0.51 mm, and the inhibition zone diameter of Salmonella enterica subsp. enterica ATCC 13076 was 16.94±0.43 mm.

[0126] Table 4. Inhibitory effect of Lactobacillus rhamnosus TY-63 on pathogenic bacteria.

[0127] Note: 1. The values ​​are the diameter (mm) of the inhibition zone of lactic acid bacteria against pathogenic bacteria, and are the average of three parallel experiments. 2. The size of the inhibition zone includes the diameter of the punch (10mm).

[0128] 2. Ability to inhibit pathogenic fungi

[0129] (1) Methods: After two subcultures of Aspergillus flavus BNCC 142803, Penicillium expansum BNCC 146144, and Aspergillus niger ATCC 16888, spores on the plates were collected with sterile physiological saline and filtered through sterile four-layer gauze to obtain spore suspensions of the three pathogenic fungi. The spores were counted using a hemocytometer, and the concentration of the spore suspensions was adjusted to 1×10⁻⁶. 6spores / mL. The inhibitory effect of TY-63 on pathogenic fungi was evaluated using the double-layer plate method. 15 mL of MRS solid medium was poured into a disposable plastic petri dish as the lower layer. After solidification, a single colony of the activated strain TY-63 was picked up with a sterile toothpick and streaked with two parallel lines 2 cm long, 1 cm apart, on the lower layer. The dish was then anaerobically incubated at 37°C for 36 h. A spore suspension of the three pathogenic fungi was added at a ratio of 2% (v / v) to unsolidified sterile PDA medium (T < 50°C). 5 mL of the unsolidified PDA medium containing the spore suspension was poured onto the lower layer, and the mixture was gently shaken to distribute the upper layer evenly across the entire plate. After complete solidification, the dish was incubated at 28°C for 4 days. The inhibition zone around the lactic acid bacteria was examined to evaluate the inhibitory effect.

[0130] (2) Conclusion: TY-63 has a strong inhibitory effect on Aspergillus flavus and Penicillium expansum, with the area of ​​the inhibition zone around each lactic acid bacteria line accounting for more than 8% of the total plate area; it has a weak inhibitory effect on Aspergillus niger, with the area of ​​the inhibition zone around each lactic acid bacteria line accounting for 0.1-3% of the total plate area.

[0131] Table 5 L. rhamnosus TY - 63. Inhibitory effect on pathogenic fungi

[0132]

[0133] Note: "–" indicates no inhibitory effect; "+" indicates that TY-63 has a weak inhibitory effect on pathogenic fungi, with the area of ​​the inhibition zone around each line accounting for 0.1-3% of the total plate area; "++" indicates that TY-63 has a moderate inhibitory effect on pathogenic fungi, with the area of ​​the inhibition zone around each line accounting for 3-8% of the total plate area; "+++" indicates that TY-63 has a strong inhibitory effect on pathogenic fungi, with the area of ​​the inhibition zone around each line accounting for more than 8% of the total plate area.

[0134] 3. Self-aggregation ability.

[0135] (1) Method: Take 15 mL of TY-63 bacterial suspension, mix it evenly on a vortex mixer, and measure its absorbance (A0) at 600 nm using a spectrophotometer. Take 3 mL of TY-63 bacterial suspension and let it stand at 37℃ for 2 h, 4 h, and 24 h. Measure the absorbance (A1) of the upper layer liquid at 600 nm at three time points. Perform three repeated experiments. The formula for calculating the self-aggregation ability of the strain is: Self-aggregation rate (%) = (1 - A1 / A0) × 100%.

[0136] (2) Conclusion: The self-aggregation rate of TY-63 was 13.24% at 2h, 15.85% at 4h, and 46.22% at 24h.

[0137] Table 6 L. rhamnosus TY - Self-aggregation ability of 63

[0138]

[0139] 4. Hydrophobic.

[0140] (1) Method: The surface hydrophobicity of the strain was determined by the method of microbial adhesion to hydrocarbons. 3 mL of xylene and ethyl acetate were respectively mixed with 9 mL of TY-63 bacterial suspension, vortexed for 1 min, and allowed to stand at 37℃ for 40 min. The organic phase was removed, and the absorbance (At) of the aqueous phase at 600 nm was measured. The experiment was repeated three times. The formula for calculating the surface hydrophobicity of the strain was: Surface hydrophobicity (%) = (1 - At / A0) × 100%, where At is the absorbance after standing treatment, and A0 is the absorbance before standing treatment.

[0141] (2) Conclusion: TY-63 has a hydrophobicity of 56.95% for xylene and 48.35% for ethyl acetate.

[0142] Table 7 L. rhamnosus TY - Hydrophobicity of 63

[0143]

[0144] 5. Antioxidant capacity.

[0145] (1) Method: Heat-inactivated bacterial suspension: The bacterial suspension of TY-63 was autoclaved at 121℃ for 15 min to obtain a heat-inactivated bacterial suspension. Cell lysis buffer: According to the viable cell count (102...) 4 CFU / mL): The extraction solution volume (mL) was 1000:1. The extraction solution provided in the kit was added to the bacterial suspension, and the mixture was ultrasonically broken up in an ice bath (power 200W, sonication for 5s, interval 5s, total time 30min) to obtain the lysate of TY-63. The antioxidant capacity of the live bacterial suspension, heat-inactivated bacterial suspension, and lysate of XX1-2 was determined using a total antioxidant capacity assay kit (DPPH method, Shanghai Maclean Biochemical Technology Co., Ltd., catalog number T931095). Samples were added to EP tubes according to the instructions, mixed thoroughly, and reacted at room temperature in the dark for 20min. The absorbance was measured at 515nm in a 1mL glass cuvette. The absorbance of the blank tube was recorded as A-blank, and the absorbance of the assay tube was recorded as A-measurement. The results are expressed as DPPH free radical scavenging rate (%). The formula for calculating the DPPH free radical scavenging rate (%) is: DPPH free radical scavenging rate (%) = (A-blank - A-measurement) ÷ A-blank × 100%.

[0146] (2) Conclusion: The DPPH free radical scavenging rates of TY-63 live bacterial suspension, heat-inactivated bacterial suspension and lysis buffer were 55.78%, 46.53% and 52.03%, respectively.

[0147] Table 8 L. rhamnosus TY - Antioxidant capacity of 63

[0148]

[0149] V. Lactobacillus rhamnosus TY - 63% security.

[0150] 1. Drug sensitivity test

[0151] (1) Method: The paper disc diffusion method (KB method) was used to test the antibiotic susceptibility of TY-63. 100 μL of bacterial suspension (1×10⁻²) was taken separately. 10 The antibiotic susceptibility test strips (CFU / mL) were spread onto MRS solid medium. The strips were placed on the medium with sterile forceps and anaerobically incubated at 37°C for 48 hours. The diameter of the inhibition zone was measured with calipers. Three replicate tests were conducted for each antibiotic.

[0152] (2) Conclusion: TY-63 is sensitive to erythromycin, penicillin, chloramphenicol, tetracycline, rifampin, gentamicin, doxycycline and ampicillin, but resistant to streptomycin, vancomycin and kanamycin.

[0153] Table 9 L.rhamnosus TY - 63. Susceptibility to antibiotics

[0154]

[0155]

[0156] 2. Hemolytic activity

[0157] (1) Method: Single colonies were picked and inoculated onto Columbia blood agar plates and incubated at 37°C for 48 hours. Hemolysis was judged based on the color and morphology around the colonies. Result determination: Staphylococcus aureus ATCC 29213 was used as a positive control strain. A clear transparent zone around the colony was considered positive.

[0158] (2) Conclusion: Using Staphylococcus aureus as a positive control, a transparent area was observed around the colony during its growth on blood agar plates. This is because Staphylococcus aureus has β-hemolytic activity. Under the same experimental conditions, no transparent area was observed around TY-63, which indicates that this strain does not have hemolytic activity.

[0159] 3. Gelatin hydrolase activity

[0160] (1) Methods: Preparation of modified LB medium: 75 g / L gelatin was added to LB liquid medium. TY-63 bacterial suspension was inoculated into modified LB medium at a 2% inoculum (v / v) and cultured at 37℃ for 48 h (37℃, 180 rpm), then refrigerated at 4℃ for 2 h. Result determination: Staphylococcus aureus ATCC 29213 was used as a positive control. If the medium did not solidify after cooling, it was considered to have positive gelatin hydrolase activity, indicating that the strain had the ability to produce gelatin hydrolase.

[0161] (2) Conclusion: Using Staphylococcus aureus as a positive control, it was observed that the modified LB medium did not solidify after cooling, which is because Staphylococcus aureus has gelatin hydrolase activity; under the same experimental conditions, the modified LB medium inoculated with TY-63 solidified, indicating that TY-63 does not have gelatin hydrolase activity.

[0162] 4. DNA enzyme activity

[0163] (1) Method: Wells were punched on toluidine blue-DNase test agar plates. 200 μL of TY-63 bacterial suspension was added to each well, and the plates were anaerobically cultured at 37℃ for 48 h. The color change around the wells was observed. Result determination: Staphylococcus aureus ATCC29213 was used as a positive control strain. A distinct pinkish-purple area around the well was considered a positive DNase activity.

[0164] (2) Conclusion: Using Staphylococcus aureus as a positive control, a distinct pinkish-purple color was observed around the wells where Staphylococcus aureus was added. This is because Staphylococcus aureus has DNase activity. Under the same experimental conditions, no pinkish-purple color appeared around the wells where TY-63 bacterial suspension was added, indicating that TY-63 does not have DNase activity.

[0165] 5. Nitroreductase activity

[0166] (1) Method: Single colonies of TY-63 were picked and inoculated into MRS broth medium and anaerobically cultured at 37℃ for 24 h. Then, 3% (v / v) of the fermentation broth of TY-63 was inoculated into nitroreductase activity detection medium and anaerobically cultured at 37℃ for 48 h. Appropriate amounts of α-naphthylamine solution and p-aminobenzosulfonic acid solution were added sequentially, mixed thoroughly, and allowed to stand at room temperature. The color of the medium was then observed. Result determination: Using Escherichia coli ATCC 11775 as a positive control, if the medium changed from colorless to brownish-yellow, it indicated that the strain possessed nitroreductase activity.

[0167] (2) Conclusion: Using Escherichia coli as a positive control, the color of the medium for detecting nitroreductase activity inoculated with Escherichia coli turned brownish-yellow, which is because Escherichia coli has nitroreductase activity; under the same experimental conditions, the color of the medium inoculated with TY-63 did not change, indicating that TY-63 does not have nitroreductase activity.

[0168] Table 10 L. rhamnosus TY - 63. Hemolytic activity, gelatin hydrolase, DNase and nitroreductase activity

[0169] strain hemolytic activity Gelatin hydrolase activity DNA enzyme activity Nitroreductase activity <![CDATA[L.rhamnosus TY - 63]]> - - - -

[0170] Note: + indicates hemolytic activity, as well as gelatin hydrolase, DNase, and nitroreductase activity; - indicates no hemolytic activity, as well as gelatin hydrolase, DNase, and nitroreductase activity.

[0171] Example 2: Effect of Lactobacillus rhamnosus TY-63 on aflatoxin B1 (AFB1)-induced liver damage.

[0172] Reagents: AFB1 standard was purchased from Northern Weiye Metrology Group Co., Ltd. Silymarin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0173] 1. Grouping and administration of experimental animals.

[0174] Animal experiments were conducted at the Experimental Animal Center of Zhengzhou University (hereinafter referred to as the Experimental Animal Center) and were approved by the Experimental Animal Ethics Committee of Zhengzhou University (hereinafter referred to as the Ethics Committee), with the approval number ZZU-LAC20231229

[25] . All procedures complied with the guidelines of the Ethics Committee and were subject to supervision and inspection by the Ethics Committee and the Experimental Animal Center.

[0175] Forty-five 4-week-old SPF-grade male Wistar rats, weighing 100±5g, were randomly divided into 7 groups of 9 rats each, with 3 rats per cage. They were housed in an environment with a temperature of 20-25℃, relative humidity of 50-60%, and a 12-hour diurnal light cycle. All rats had free access to food and water. Standard commercial rodent feed and water were provided by the experimental animal center, with feed and water changed daily. After a 5-day acclimatization period, the weights of the 7 groups were adjusted. Data analysis showed no significant differences in weight among the groups, and the experimental period lasted 21 days.

[0176] Animal grouping and experimental treatment are shown in Table 11. AFB1 standard was dissolved in 10% DMSO (dimethyl sulfoxide) aqueous solution to obtain AFB1 stock solution. The AFB1 stock solution was diluted with sterile PBS buffer to prepare the required AFB1 challenge solution. Silymarin was dissolved in 10% DMSO aqueous solution to prepare silymarin solution.

[0177] In the model group (AFB1 group), each rat was administered 1 mL of AFB1 challenge solution by gavage to make the AFB1 dosage 80 μg AFB1 / kg bw, and then 1 mL of sterile PBS buffer was administered by gavage.

[0178] The positive control group (AFB1 + silymarin group) was given 80 μg AFB1 / kg bw by gavage, followed by 50 mg silymarin / kg bw by gavage.

[0179] Experimental group (AFB1+TY-63 group): Each rat was administered 1 mL of AFB1 challenge solution by gavage to achieve an AFB1 dose of 80 μg AFB1 / kg bw, followed by gavage administration of the TY-63 bacterial suspension from step two. The dose of Lactobacillus rhamnosus TY-63 was 4 × 10⁻⁶. 10 CFU / each.

[0180] The concentrations of AFB1 solution and silymarin solution were adjusted according to the rat's body weight to ensure that each rat received a 1 mL dose via gavage. The viable count of the bacterial suspension was kept constant at 4 × 10⁻⁶. 10 CFU / d, with each gavage administration of 1 mL of bacterial suspension.

[0181] Control group: Each rat was administered 2 mL of sterile PBS buffer by gavage.

[0182] Solvent control group (DMSO group): Each rat was administered 2 mL of 10% DMSO sterile PBS solution by gavage.

[0183] TY-63TY-632. Indicator Testing and Results

[0184] (1) Weight gain: Throughout the experiment, the animals were observed daily for any signs of toxicity, and the weight of each group of rats was recorded every two days before gavage. The weight gain of the rats was calculated at the end of the experiment.

[0185] result:

[0186] Table 11 Weight gain in rats

[0187] Group Name Weight gain (g) Control group (Contro group) 158.57a Solvent control group (DMSO group) 160.08a <![CDATA[Model group (AFB1 group)]]> 127.87c <![CDATA[Positive control group (AFB1 + silymarin group)]]> 149.56ab <![CDATA[Experimental group (AFB1 + TY-63 group)]]> 147.10ab

[0188] (2) Liver organ coefficient:

[0189] The formula for calculating the liver organ coefficient is: liver weight at the end of the experiment (g) / body weight at the end of the experiment (100g). The results are shown in Table 12.

[0190] Table 12 Liver organ coefficients in rats

[0191]

[0192]

[0193] (3) Detection of serum inflammatory factors: The levels of interleukin-1β (IL-1β) and interleukin-17 (IL-17) in the serum of rats in each group were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Boyan Biotechnology Co., Ltd.), and the serum nitric oxide (NO) content was detected by a biochemical method (nitric oxide test kit, Nanjing Jiancheng Bioengineering Institute). The procedures were performed according to the kit instructions. The results are shown in Table 13.

[0194] Table 13 Serum levels of inflammatory factors

[0195]

[0196] (4) Detection of liver function indicators: The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in rat serum were measured using a kit (Shenzhen Raydu Life Science Co., Ltd.) and a fully automated biochemical analyzer. The results are shown in Table 14.

[0197] Table 14 Serum ALT, AST, ALP and LDH activities

[0198] Group Name ALT(U / L) AST(U / L) ALP(U / L) LDH(U / L) Control group (Contro group) 53.93b 65.80c 180.71cd 109.32d Solvent control group (DMSO group) 53.55b 81.06bc 167.36d 89.33d <![CDATA[Model group (AFB1 group)]]> 76.63a 183.03a 315.96a 443.67a <![CDATA[Positive control group (AFB1 + silymarin group)]]> 60.03b 103.675bc 222.06bc 235.37c <![CDATA[Experimental group (AFB1 + TY-63 group)]]> 46.14b 120.33b 238.81b 323.45b

[0199] (5) Effects of TY-63 on oxidative stress in rat liver. Liver tissue homogenate supernatant was prepared, and protein concentration was determined using a BCA protein quantification kit (Wuhan Saive Biotechnology Co., Ltd.). The contents of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue homogenate supernatant were determined using a kit (Wuhan Saive Biotechnology Co., Ltd.), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined using a kit (Wuhan Saive Biotechnology Co., Ltd.), following the kit instructions. The results are shown in Table 15.

[0200] Table 15. MDA and GSH content, SOD, CAT and GSH-Px activities in liver tissue.

[0201]

[0202] (6) Effect of TY-63 on the expression levels of apoptosis-related gene mRNAs in rat liver tissue. The relative expression levels of jejunal apoptosis regulatory genes (BCL2, BAX, and CASP3) mRNAs were detected by qRT-PCR. GAPDH was used as an internal reference gene for normalization. Total RNA was extracted from liver tissue and reverse transcribed using a kit (Nanjing Novizan Biotechnology Co., Ltd.). The primer sequences for BCL2 were: F: 5′-CACAGAGGGGCTACGAGT-3′; R: 5′-GGCTGGAAGGAGAAGATG-3′. The primer sequences for BAX were: F: 5′-GAGGATGATTGCTCATGTG-3′; R: 5′-CAGTTGAAGTTGCCGTCT-3′. The primer sequences for CASP3 were: F: 5′-TGACCCAGATTATGTTTGA-3′; R: 5′-ATAGATAGGCACGTTGTGA-3′. The GAPDH primer sequences were: F: 5′-GACATGCCGCCTGGAGAAAC-3′; R: 5′-AGCCCAGGATGCCCTTTAGT-3′. The results are shown in Table 16.

[0203] Table 16. Relative mRNA expression levels of apoptosis-regulating genes

[0204] Group Name BCL2 BAX CASP3 Control group (Contro group) 1ab 1c 1c Solvent control group (DMSO group) 0.87b 0.76c 1.09c <![CDATA[Model group (AFB1 group)]]> 0.60c 2.83a 2.23a <![CDATA[Positive control group (AFB1 + silymarin group)]]> 0.87b 1.49b 2.07b <![CDATA[Experimental group (AFB1 + TY-63 group)]]> 1.19a 1.06c 1.51c

[0205] (6) Effect of TY-63 on apoptosis of rat liver cells. TUNEL staining was used to detect liver cell apoptosis. Three fields of view were randomly selected from each slice, and Image-Pro Plus 6.0 software was used to analyze liver cell apoptosis. Positive cells were defined as those with corresponding fluorescence channels around the blue cell nucleus. The number of positive cells and the total number of cells in each image were measured. The apoptosis rate was calculated as: apoptosis rate (%) = number of positive cells / total number of cells × 100%. The results are shown in Table 17.

[0206] Table 17 Apoptosis rate of rat liver cells

[0207]

[0208]

[0209] 3. Summary

[0210] This invention isolates a strain of Lactobacillus rhamnosus TY-63 that is resistant to acid, bile salts, and the gastrointestinal environment from fecal samples of healthy infants. TY-63 has antibacterial activity and good antioxidant capacity, as well as certain self-aggregation ability and hydrophobicity, and has good safety.

[0211] This invention provides the application of Lactobacillus rhamnosus TY-63 in AFB1-induced liver injury. Lactobacillus rhamnosus TY-63 can effectively reduce the levels of pro-inflammatory factors such as IL-17, IFN-γ, and NO in serum; reduce the activities of AST, ALT, ALP, and LDH; reduce the content of MDA in the liver, increase the content of GSH, and increase the activity of SOD; inhibit hepatocyte apoptosis, upregulate the relative expression of the anti-apoptotic gene BCL2, and downregulate the relative expression of the pro-apoptotic genes BAX and CASP3, effectively alleviating liver damage caused by AFB1 exposure.

[0212] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Lactobacillus rhamnosus, characterized in that, The Lactobacillus rhamnosus mentioned is Lactobacillus rhamnosus TY-63, whose accession number at the China Center for Type Culture Collection is CCTCC NO: M20251683.

2. The composition, characterized in that, The composition contains Lactobacillus rhamnosus as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is a culture, which is a substance obtained by culturing the Lactobacillus rhamnosus in a microbial culture medium.

4. The composition according to claim 2, characterized in that, The composition is a microbial agent.

5. The composition according to any one of claims 2-4, characterized in that, The composition has at least one of the following properties: A1) Relieves liver damage, A2) Inhibits liver cell apoptosis. A3) Reduce serum levels of pro-inflammatory factors. A4) Reduces the activity of alanine aminotransferase in serum. A5) Reduces the activity of serum aspartate aminotransferase. A6) Reduces the activity of alkaline phosphatase in serum. A7) Reduces the activity of lactate dehydrogenase in serum. A8) Reduces the level of malondialdehyde in the liver. A9) Increases the level of glutathione in the liver. A10) increases the activity of superoxide dismutase in the liver. A11) regulates hepatocyte apoptosis-related pathways. A12) Upregulates the relative expression of the anti-apoptotic gene BCL2. A13) downregulates the relative expression of the pro-apoptotic gene BAX. A14) Downregulates the relative expression of the pro-apoptotic gene CASP3. A15) Treatment for weight loss in animals caused by aflatoxin B1 A16) Treatment for increased liver organ coefficient caused by aflatoxin B1 A17) antagonizes pathogenic bacteria.

6. The composition according to claim 5, characterized in that, The liver damage was caused by aflatoxin B1.

7. A method for preparing the composition, characterized in that, The composition is the composition according to any one of claims 2-6, and the method includes the step of using Lactobacillus rhamnosus as a component of the composition according to claim 1.

8. The use of the Lactobacillus rhamnosus of claim 1 or the composition of claims 2-6 in the preparation of a product.

9. The product according to claim 8, characterized in that, The product has at least one of the following properties: B1) alleviates liver damage. B2) Inhibits liver cell apoptosis. B3) Reduces serum levels of pro-inflammatory factors. B4) Reduces the activity of alanine aminotransferase in serum. B5) Reduces the activity of serum aspartate aminotransferase. B6) Reduces the activity of alkaline phosphatase in serum. B7) Reduces the activity of lactate dehydrogenase in serum. B8) Reduces the level of malondialdehyde in the liver. B9) Increases the level of glutathione in the liver. B10 increases the activity of superoxide dismutase in the liver. B11 regulates hepatocyte apoptosis-related pathways. B12) upregulates the relative expression of the anti-apoptotic gene BCL2. B13) downregulates the relative expression of the pro-apoptotic gene BAX. B14) downregulates the relative expression of the pro-apoptotic gene CASP3. B15) was used to treat weight loss in animals caused by aflatoxin B1. B16) is used to treat the increased liver organ coefficient caused by aflatoxin B1. B17) antagonizes pathogenic bacteria.

10. The composition according to claim 5 or the product according to claim 8, characterized in that, The liver damage was caused by aflatoxin B1.

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