Application of a Pediococcus acidilactici CCFM1365 strain and its prepared postbiotics for alcohol detoxification and liver protection, and conversion of kudzu root flavonoids

Abstract

The present invention discloses a strain of Pediococcus acidilactici CCFM1365 and its prepared postbiotics for alcohol sobering and liver protection, as well as the application of kudzu root flavonoids for conversion, belonging to the field of microbial technology. The Pediococcus acidilactici CCFM1365 and its prepared synbiotic preparation, live bacteria, and postbiotics provided by the present invention have at least one of the following effects: (1) improving liver steatosis and lipid accumulation caused by long-term alcohol exposure; (2) enhancing alcohol metabolism; having anti-oxidative stress and anti-inflammatory effects; (3) reducing the expression level of mRNA of genes related to inflammatory mediators, oxidative stress, and alcohol metabolism in liver tissue; (4) reducing intestinal permeability and reducing alcohol absorption. The Pediococcus acidilactici CCFM1365 and its prepared synbiotic preparation, live bacteria, and postbiotics provided by the present invention can be used to prepare functional foods and / or dietary supplements with the potential to sober up and protect the liver, and have great application prospects.

Description

Technical Field

[0001] The invention relates to a Pediococcus acidilactici CCFM1365 strain and a postbiotic prepared therefrom for relieving alcohol and protecting the liver, and application of the postbiotic in converting kudzu root flavonoids, belonging to the technical field of microorganisms. Background Art

[0002] As lifestyles change, the number of alcoholics continues to grow, leading to a rising prevalence of alcoholic liver disease (ALD), posing a significant social burden. According to the World Health Organization's "World Report on Alcohol and Health 2018," excessive drinking contributes to a total of 3 million deaths worldwide. In China, alcohol-related deaths reach 700,000 annually, ranking first globally in terms of alcohol-related deaths. The liver is a vital organ responsible for metabolism and digestion. After alcohol enters the body through the mouth, 2% to 10% enters the respiratory tract, kidneys, and other organs, and is excreted through urine, sweat, and breathing. 90% to 98% enters the stomach, small intestine, esophagus, and duodenum via the esophagus. Of the alcohol absorbed through the gastrointestinal tract, a portion undergoes initial metabolism by alcohol dehydrogenase, while over 90% is absorbed by the gastrointestinal mucosa, enters the bloodstream, and circulates through the liver for metabolism. Therefore, protecting and repairing liver damage is crucial.

[0003] Traditional Chinese medicine (TCM) has a long history of use in the prevention and treatment of liver disease. Currently, TCM has been studied as a complementary and alternative treatment for alcoholic liver injury. However, active ingredients in TCM are a crucial component of TCM. The anti-inflammatory and antioxidant components contained in these herbs can modulate alcohol-induced oxidative stress and inflammation, demonstrating significant therapeutic benefits in the adjunctive treatment of alcoholic liver disease (ALD). These ingredients have become a research hotspot in the field of ALD. For example, puerarin and silymarin preparations have been clinically used as hepatoprotective agents. However, in reports on the use of TCM in the prevention and treatment of liver disease, drawbacks of TCM treatment have become increasingly apparent. First, the bioactive components of TCM are complex, and some natural products cannot be directly absorbed by humans and animals. Second, the content of most bioactive ingredients in TCM is low, and some natural products are toxic to humans and animals. To address these issues, researchers are currently interested in using fermentation of TCM herbs to enhance their bioactivity and reduce their toxicity, laying the foundation for the future development of hepatoprotective drugs.

[0004] A growing number of studies have found that fermentation of Chinese herbal medicines can enhance their bioactivity. Following microbial fermentation, the chemical composition and content of Chinese herbal medicines often change, affecting the pharmacological activity of fermentation metabolites and the final fermentation products. Furthermore, because the intestinal microbial composition and drug absorption capacity of each host vary significantly, in vitro fermentation can standardize Chinese herbal medicine products and enhance their clinical efficacy. Probiotics are living microorganisms that are beneficial to human health. my country's edible probiotic catalog includes 35 species, some of which have been used to ferment Chinese herbal medicines. For example, after fermentation with the Bifidobacterium breve strain CCRC 14061, the bound glycosides in the herbal medicines can be converted to free aglycones by the microbial β-glucosidase or β-galactosidase, thereby enhancing their bioavailability.

[0005] Flavonoids are important active ingredients in Pueraria root. Converting puerarin, daidzin, and genistin, among other flavonoids, into easily absorbed small molecules called daidzein is crucial for Pueraria root's pharmacological effects. However, prior research on the microbial conversion and fermentation of Pueraria root flavonoids is relatively limited, and most strains are unable to utilize puerarin. Even those that do possess the ability to convert puerarin have very low conversion rates, typically below 5%. While the strain reported in "Expression and characterization of the human intestinal bacterial enzyme which cleaves the C-glycosidic bond in 3″-oxo-puerarin" can fully convert puerarin, it is inedible and difficult to cultivate, thus lacking any additional value. The bifidobacterium reported in "Bioconversion of soyisoflavones daidzin and daidzein by Bifidobacterium strains" demonstrates the ability to convert daidzin to daidzein, with a conversion rate of approximately 48%-65% per 7 days. However, this conversion rate remains low, making it unsuitable for industrial application.

[0006] Pediococcus acidilactici is a Gram-positive bacterium belonging to the genus Pediococcus, primarily found in plants and dairy products. It is a bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. Although its various probiotic functions have been studied, no studies have shown that Pediococcus acidilactici can simultaneously utilize multiple flavonoids from kudzu root and significantly alleviate alcoholic liver injury. Therefore, isolating and screening such probiotics to achieve a synergistic effect with kudzu root flavonoid-rich diets and enhance the effectiveness of traditional Chinese medicines has broad application prospects. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a strain of Pediococcus acidilactici and the application of postbiotics prepared therefrom and transformed kudzu root flavonoids to enhance the alcohol-detoxifying and liver-protecting effects. This method can improve the alcohol-detoxifying effect of a diet rich in kudzu root flavonoids and improve the effectiveness of Chinese herbal medicines.

[0008] The present invention provides a Pediococcus acidilactici CCFM1365, which was deposited in the Guangdong Provincial Microbial Culture Collection on November 9, 2023, with a deposit number of GDMCC No: 63997, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] In one embodiment, the Pediococcus acidilactici is isolated from healthy human feces and has the following characteristics:

[0010] The Pediococcus acidilactici CCFM1365 is round or oval after being cultured on MRS culture medium for 48 hours, has a smooth and dense surface, a diameter of about 1.0 to 2.5 mm, and is white in color.

[0011] The Pediococcus acidilactici CCFM1365 is a Gram-positive bacterium, facultative anaerobic, with an optimum growth temperature of 35-40° C. and an optimum growth pH of 6.0-7.0.

[0012] The Pediococcus acidilactici CCFM1365 can efficiently convert a variety of pueraria flavonoids (such as puerarin and daidzein) into active substances (such as daidzein).

[0013] The present invention provides a composition, which contains the Pediococcus acidilactici CCFM1365 or a postbiotic prepared from the Pediococcus acidilactici CCFM1365.

[0014] In one embodiment, the postbiotics include fermentation supernatant, bacterial lysate and / or fermentation broth.

[0015] In one embodiment, the postbiotics are obtained by inoculating the above-mentioned Pediococcus acidilactici CCFM1365 into MRS culture medium to obtain a bacterial liquid, and then performing heat treatment and lysis to obtain the postbiotics.

[0016] In one embodiment, the heat treatment is performed at 60-70° C. for 25-35 minutes.

[0017] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging the fermentation broth.

[0018] In one embodiment, the method for preparing the bacterial lysate is to homogenize the heat-treated fermentation broth under high pressure and centrifuge to obtain the bacterial lysate.

[0019] In one embodiment, the postbiotics can be dried into powder by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, etc., or used directly.

[0020] The present invention also provides a synbiotic preparation with alcohol-relieving and liver-protecting effects and its application, wherein the synbiotic preparation contains Pediococcus acidilactici CCFM1365 and Pueraria root extract; the content of Pediococcus acidilactici CCFM1365 is not less than 10 6 CFU / mL or 10 6 CFU / g, Pueraria root extract concentration ≥5mg / mL.

[0021] The present invention also provides a product, which contains the Pediococcus acidilactici CCFM1365 or the above-mentioned synbiotic preparation.

[0022] In one embodiment of the present invention, the product is food, medicine or health product.

[0023] In one embodiment of the present invention, the food is a dairy product, soy product or fruit and vegetable product produced using a starter containing the Pediococcus acidilactici CCFM1365; or the food is a beverage or snack containing the Pediococcus acidilactici CCFM1365 according to claim 1.

[0024] The present invention also provides food, health products, medicines or cosmetics containing the Pediococcus acidilactici CCFM1365 postbiotics and synbiotic preparations.

[0025] In one embodiment, the food comprises the above composition and conventional auxiliary materials.

[0026] In one embodiment, the health care product comprises the above composition and conventional excipients.

[0027] In one embodiment, the drug comprises the above composition, a drug carrier and / or a pharmaceutical excipient.

[0028] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0029] In one embodiment, the cosmetic comprises the above composition, matrix raw materials and / or conventional excipients.

[0030] In one embodiment, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powdery raw materials, colloid raw materials, coagulants, and surfactants.

[0031] In one embodiment, the conventional excipients include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0032] The invention also provides a method for calculating the conversion rate of kudzu root flavonoids.

[0033] In one embodiment of the present invention, the method comprises the following steps:

[0034] (1) Streak the above-mentioned Pediococcus acidilactici CCFM1365 on MRS solid medium and incubate the plate upside down at 37°C for 48 hours. Pick a single colony and inoculate it into 5 mL of MRS liquid medium and incubate it at 37°C for 24 hours.

[0035] (2) Add 5% (v / v) of the bacterial solution of Pediococcus acidilactici to the fermentation base rich in Pueraria root extract, and culture at a constant temperature of 37° C. to prepare a fermentation broth.

[0036] (3) The fermentation broth was collected and centrifuged to obtain the supernatant, which was extracted twice with equal proportions of ethyl acetate. After concentration by refrigerated centrifugation, methanol was added to dissolve the solution again. After filtration through a 0.22 μm microporous filter membrane, the changes in the content of kudzu root flavonoids were detected by HPLC.

[0037] In one embodiment of the present invention, the concentration of the kudzu root extract in the fermentation base material rich in kudzu root extract in step (2) is 0.5 mg / mL.

[0038] In one embodiment of the present invention, the amount of the bacterial liquid of Pediococcus acidilactici CCFM1365 in step (2) is ≥1×10 6 CFU / mL or 1×10 6 CFU / g.

[0039] In one embodiment of the present invention, the constant temperature fermentation culture temperature in step (2) is 30-37° C., and the time is 0-120 h.

[0040] The present invention also provides a method for transforming kudzu root flavonoids, which comprises culturing the Pediococcus acidilactici CCFM1365 in a culture medium containing kudzu root extract.

[0041] In one embodiment of the present invention, the pueraria flavonoids include puerarin, daidzin and genistin.

[0042] The present invention also provides the use of the above-mentioned Pediococcus acidilactici CCFM1365, or the composition, or the synbiotic preparation in the preparation of medicines for preventing and / or treating alcoholic fatty liver.

[0043] The present invention also provides the use of the above-mentioned Pediococcus acidilactici CCFM1365, or the composition, or the synbiotic preparation in the preparation of a health product having an auxiliary protective effect on chemical liver damage.

[0044] The present invention also provides the use of the above-mentioned Pediococcus acidilactici CCFM1365, or the composition, or the synbiotic preparation in preparing a product that can promote the absorption of puerarin, daidzin and / or genistin.

[0045] Beneficial effects:

[0046] The present invention screened and obtained a strain of Pediococcus acidilactici CCFM1365. The Pediococcus acidilactici CCFM1365 and the postbiotics, synbiotic preparations, and live bacteria prepared therefrom have the ability to enhance alcohol detoxification and liver protection, and have a synergistic effect with Pueraria root:

[0047] 1. The Pediococcus acidilactici CCFM1365 can convert pueraria flavonoids: the conversion rate of puerarin is 54.05±2.92% / 48h, the conversion rate of daidzein is 33.37±3.18% / 48h, and the conversion rate of genistin is 62.90±3.11% / 48h, and the increase of product daidzein is 197.7±18.23% / 48h.

[0048] 2. The Pediococcus acidilactici CCFM1365 and the synbiotic preparation prepared therefrom can alleviate alcoholic fatty liver, specifically in the following aspects:

[0049] (1) Reduce serum lipid (AST, TG and LDL-C) levels;

[0050] (2) Improve liver tissue pathological damage and reduce liver damage and lipid deposition caused by long-term drinking;

[0051] (3) Enhance the activity of antioxidant enzymes (GSH and SOD) in liver tissue;

[0052] (4) Reduce MDA levels, reduce lipid peroxidation and alleviate alcohol-induced liver damage;

[0053] (5) exerting anti-oxidative stress and anti-inflammatory responses through the CYP2E1 / Nrf2 / HO-1 and NF-κB signaling pathways;

[0054] (6) Reduce serum LPS levels, inhibit LPS intestinal leakage, Kupffer cell activation, and LPS receptor expression;

[0055] (7) Reduce the expression level of inflammatory mediators (IL-1β, TNF-α, IL-6) gene mRNA in liver tissue;

[0056] (8) Reduce the expression level of oxidative stress (Nrf2, HO-1, COX-2) gene mRNA;

[0057] (9) Increase the activity of alcohol metabolizing enzymes (ADH and ALDH) and reduce the mRNA expression levels of CYP2E1 and CYP1A2 genes;

[0058] (10) Upregulates the expression of tight junction associated protein-1 (ZO-1) and occludin in the intestine, reduces intestinal permeability, and reduces alcohol absorption.

[0059] Therefore, Pediococcus acidilactici CCFM1365 and the postbiotics, synbiotic preparations and live bacteria prepared therefrom have great application prospects in the preparation of alcohol-relieving and liver-protecting products.

[0060] Biomaterial Deposit

[0061] A strain of Pediococcus acidilactici CCFM1365, taxonomically named Pediococcus acidilactici, was deposited in the Guangdong Provincial Microbiological Culture Collection on November 9, 2023, with the deposit number GDMCC No: 63997, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 :Effects of Pediococcus acidilactici CCFM1365 and its synbiotic preparation and live bacteria on body weight and liver indexes in mice exposed to chronic alcohol.

[0063] Figure 2 : Effects of Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations on liver histopathology in mice exposed to chronic alcohol.

[0064] Figure 3 : Effects of Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations on liver function in mice exposed to chronic alcohol.

[0065] Figure 4 : Effects of Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations on liver oxidative stress levels in mice with chronic alcohol exposure.

[0066] Figure 5 : Effects of Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations on alcohol metabolism in mice exposed to chronic alcohol.

[0067] Figure 6 :Effects of Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations on the mRNA expression levels of inflammatory factors in the liver of mice exposed to chronic alcohol.

[0068] Figure 7 : Effects of Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations on intestinal barrier function in mice exposed to chronic alcohol.

[0069] Figure 8 : The primary transformation metabolic pathway of flavonoids from Pueraria lobata.

[0070] Figure 9 : HPLC results of Pediococcus acidilactici CCFM1365 before and after fermentation.

[0071] Figure 10 : Changes of flavonoids content in Pueraria lobata before and after fermentation by Pediococcus acidilactici CCFM1365. DETAILED DESCRIPTION

[0072] Technical terms:

[0073] The "Pueraria flavonoids" in the present invention include puerarin, daidzein, and genistin.

[0074] The "utilization rate" or "conversion rate" in the present invention refers to the reduction in the amount of pueraria flavonoids in the substrate before and after fermentation of the strain, and is calculated as follows: conversion rate = 1 - content after fermentation / content before fermentation × 100%.

[0075] "Postbiotics" in this invention refer to the collection of bacterial culture medium after fermentation (without the addition of Pueraria root) and the collection of a portion of the supernatant (i.e., the supernatant of the fermentation broth) and the collection of a precipitate (i.e., the bacterial lysate) after high-pressure homogenization and centrifugation. For details, see Example 3, "Preparation of Postbiotics," herein.

[0076] The "synbiotic preparation" in the present invention refers to a preparation in which the Pueraria root extract and live bacteria are thoroughly mixed and then administered to the mice before gavage. This means that the Pueraria root extract and live bacteria are mixed and prepared immediately.

[0077] As used herein, "alcoholic fatty liver" refers to a condition in which fat accumulates within liver cells due to excessive alcohol consumption. Initially, there are no specific symptoms, but inflammation may occur. As the disease progresses, symptoms such as helplessness, fatigue, fever, nausea, and vomiting may appear. If not properly treated, the disease can progress to alcoholic cirrhosis, manifested by ascites, esophageal variceal bleeding, and hepatic coma.

[0078] "Treatment" in the present invention refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0079] In the present invention, "prevention" refers to avoiding or reducing the risk of disease occurrence through treatment before the onset of the disease, making it difficult for the disease to occur or develop.

[0080] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited by the implementing regulations.

[0081] SPF male C57bl / 6J mice (6 weeks old, 18 ± 2 g) in the following examples were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; the herbal kudzu root extract (40% kudzu root flavonoids) involved in the following examples was purchased from Sanyuan Longsheng Biotechnology Co., Ltd.; puerarin (product number: P816259, CAS: 3681-99-0), daidzein (product number: D807006, CAS: 552-66-9), genistin (product number: G810547, CAS: 529-59-9) and daidzein (product number: D807011, CAS: 486-66-8) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; Lieber-DeCarli liquid control diet (TP4030C) and Lieber-DeCarli liquid model diet (TP40 30B) was purchased from Nantong Trophi Feed Technology Co., Ltd. and prepared as required; serum aspartate aminotransferase (AST), triglyceride (TG), lactate dehydrogenase (LDH), low-density lipoprotein cholesterol (LDL-C), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute; mouse LPS enzyme-linked immunosorbent assay kit was purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; chloroform, isopropanol, ethanol, TRIzol, and DEPC-treated water were purchased from China National Pharmaceutical Reagent Co., Ltd.; grinding beads were purchased from Huzhou Xiongsheng Grinding Co., Ltd.; BCA protein concentration assay kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; reverse transcription kit and real-time fluorescence quantification kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; primers were purchased from Shanghai Shenggong Bioengineering Co., Ltd. The culture media involved in the following examples are as follows:

[0082] MRS liquid medium (g / L): peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, diammonium hydrogen citrate 2 g / L, K₂HPO₄·3H₂O 2.6 g / L, MgSO₄·7H₂O 0.58 g / L, MnSO₄·7H₂O 0.25 g / L, Tween-80 1 g / L, distilled water 1000 g / L. Autoclave at 115°C for 20 min.

[0083] MRS solid medium (g / L): peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, diammonium hydrogen citrate 2 g / L, K₂HPO₄·3H₂O 2.6 g / L, MgSO₄·7H₂O 0.58 g / L, MnSO₄·7H₂O 0.25 g / L, Tween-80 1 g / L, agar 20 g / L, distilled water 1000 g / L. Autoclave at 115°C for 20 min.

[0084] Fermentation medium (0.5 g / L): 0.5 g / L Pueraria extract, 5 g / L yeast extract, 5 g / L glucose, 1000 g / L distilled water. Autoclave at 115°C for 20 min.

[0085] PBS buffer solution ( / L): 8.0 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, adjust the pH to 7.4. Autoclave at 115°C for 20 min.

[0086] Example 1: Screening, identification and preservation of strains

[0087] 1. Screening

[0088] The fecal samples from healthy people were used as samples and diluted 10-fold to 10 -6 , and then take 100 μL of each dilution factor of 10 -4 , 10 -5 , 10 -6 The dilution was plated on MRS solid culture medium and cultured at 37℃ for 48h. The colony morphology was observed and recorded. Colonies with different morphologies on the MRS solid culture medium were picked for streak separation. After culture at 37℃ for 48h, single colonies with different morphologies on the MRS solid culture medium were picked again for streak separation until pure single colonies with consistent morphology were obtained. Pure colonies on the MRS solid culture medium were picked and inoculated into 5mL of MRS liquid culture medium and cultured at 37℃ for 24h. 1mL of bacterial solution was taken into a sterile centrifuge tube, centrifuged at 8000r / min for 3min, and the upper culture medium was discarded. The obtained bacterial mud was freeze-dried.

[0089] 2. Identification

[0090] The isolated strain was subjected to PCR amplification of its 16S rDNA. The PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The resulting PCR product was then sequenced by a biotechnology company. The resulting sequence was searched and compared against GeneBank using BLAST to obtain species-level identification. A strain of Pediococcus acidilactici was obtained and named Pediococcus acidilactici CCFM1365. The strain's 16S rDNA sequence is shown in SEQ ID No. 1.

[0091] The primers used for 16S rDNA amplification are as follows:

[0092] 27F (forward): 5′-AGAGTTTGATCCTGGCCTCA-3′;

[0093] 1492R (reverse): 5'-GGTTACCTTGTTACGACTT-3'.

[0094] The 16S rDNA amplification procedure is as follows:

[0095] 94°C for 5 min; repeat 30 cycles (94°C for 30 s; 55°C for 30 s; 72°C for 2 min); 72°C for 10 min; 12°C for 2 min.

[0096] 3. Save

[0097] Pediococcus acidilactici CCFM1365 was inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 24 h. 1 mL of the bacterial solution was placed in a sterile centrifuge tube and centrifuged at 8000 r / min for 3 min. The upper layer of the culture medium was discarded, and the bacterial sludge was resuspended in 30% glycerol solution and stored at -80°C. The Pediococcus acidilactici CCFM1365 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with the deposit number GDMCC No: 63997.

[0098] Example 2: Conversion of Pueraria lobata flavonoids by Pediococcus acidilactici CCFM1365

[0099] 1. Fermentation of Pediococcus acidilactici CCFM1365

[0100] 1. Streak the Pediococcus acidilactici CCFM1365 in Example 1 on MRS solid medium and incubate the plate upside down at 37°C for 24-48 hours; pick a single colony and inoculate it into 5 mL of MRS liquid medium and incubate it at 37°C for 18-24 hours; after thorough mixing, take the bacterial solution and inoculate it into new MRS liquid medium at a 2% (v / v) inoculum size and incubate it under the same conditions. Repeat this step 3-5 times to obtain a seed solution.

[0101] 2. Add 5% (v / v) of the bacterial solution of Pediococcus acidilactici obtained in step 1 to the fermentation medium rich in kudzu flavonoids, and ferment at 37°C for 0 to 120 hours. The initial inoculation concentration of the bacterial solution is 1.2×10 9 CFU / mL.

[0102] 3. The fermentation broth at different time points was centrifuged and the supernatant was collected. An equal volume of ethyl acetate was added for extraction. After thorough mixing, the supernatant was allowed to stand for 5 minutes. After centrifugation (5000g, 5 minutes), the upper liquid was collected and concentrated into a dry powder in a 45°C refrigerated centrifuge. 200 μL of 100% chromatographic grade methanol was added to reconstitute the powder, and the powder was filtered through a 0.22 μm microporous organic filter membrane to obtain the Pueraria flavonoid fermentation product. The product was refrigerated and stored at 4°C for testing.

[0103] 2. Detection of Pueraria lobata flavonoids content before and after fermentation

[0104] The flavonoid content of Pueraria root before and after fermentation was determined by HPLC. The chromatographic conditions were as follows: chromatographic detection system: Waterse 2695; chromatographic column: Atlantis™ T3 (5 μm, 4.6×250 mm); flow rate: 1.0 mL / min; detection wavelength: 280 nm; injection volume: 5 μL; mobile phase: 0.2% acetic acid aqueous solution (A) - 100% methanol solution (B); column temperature: 35°C. Elution was performed according to the following gradient: 0-20 min, 30%-70% B; 20-21 min, 70%-30% B; 21-25 min, 30% B.

[0105] Accurately weigh approximately 10 mg of each reference substance, puerarin, daidzin, genistin, and daidzein, into a 100 mL volumetric flask. Dose to volume with analytical grade ethanol and ultrasonically extract for 30 min at 300 W, 50 Hz, and then add ethanol to compensate for the missing weight. Accurately pipette these solutions and dilute them to 2 mL of concentrations of 10.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, 75.0 μg / mL, and 100.0 μg / mL, respectively. Create a working curve using the mobile phase, obtain the results, and fit the curve. Based on the standard curves, calculate the contents of puerarin, daidzin, genistin, and daidzein in the fermentation medium and the pueraria flavonoid fermentation broth, respectively.

[0106] It is reported that microorganisms can transform kudzu root flavonoids into a variety of active small molecules through the enzyme system they produce ( Figure 8 ). HPLC test results showed that the contents of puerarin, daidzein and genistin in the pueraria flavonoid fermentation broth were reduced. This shows that pueraria flavonoids can be partially converted and utilized under appropriate conditions, and daidzein is one of the main metabolites ( Figure 9 ).

[0107] The reduction in the amount of pueraria flavonoids (conversion rate) and the increase in the product daidzein before and after fermentation by Pediococcus acidilactici CCFM1365 were compared. The calculation formula is: conversion rate = 1 - content after fermentation / content before fermentation × 100%; increase = (content after fermentation - content before fermentation) / content before fermentation × 100%. At 48 hours of fermentation, the conversion rate of puerarin in pueraria flavonoids by Pediococcus acidilactici was 54.05±2.92% / 48 hours; the conversion rate of daidzein was 33.37±3.18% / 48 hours; the conversion rate of genistin was 62.90±3.11% / 48 hours. The concentration of daidzein after fermentation was 1.24±0.21μg / mL, and the increase was 197.7±18.23% / 48 hours. The changes in the content of pueraria flavonoids at different time points are shown in Figure 2. Figure 10 The transformation ability of different strains to kudzu flavonoids is shown in Table 2.

[0108] Table 1 Summary of the transformation ability of different lactic acid bacteria to flavonoids from Pueraria lobata

[0109]

[0110] Example 3: Preparation of postbiotics, live bacteria and synbiotic preparations using Pediococcus acidilactici CCFM1365

[0111] 1. Preparation of postbiotics

[0112] Before activation, Pediococcus acidilactici CCFM1365 was stored in a -80°C freezer with 30% glycerol. A small amount of Pediococcus acidilactici CCFM1365 bacterial solution was streaked onto MRS solid medium using a sterile inoculating loop and incubated aerobically at 37°C for 24-48 hours. A single colony was picked and inoculated into MRS liquid medium and incubated at 37°C for 18-24 hours. After thorough mixing, the bacterial solution was inoculated into new MRS liquid medium at a 2% (v / v) inoculum and incubated under the same conditions. This step was repeated 3-5 times to obtain 4.6×10 8 CFU / mL of bacterial solution.

[0113] The bacterial solution was heat treated at 65°C for 30 minutes, centrifuged (8000 g, 4°C, 15 minutes), and the supernatant was collected and lyophilized to obtain a freeze-dried powder of Pediococcus acidilactici CCFM1365 supernatant (CCFM1365-S) for later use. Separate bacterial solution was heat treated and then homogenized under high pressure (800-1200 MPa, 3 times) to obtain a bacterial lysate. This was lyophilized to obtain a freeze-dried powder of Pediococcus acidilactici CCFM1365 bacterial lysate (CCFM1365-L) for later use.

[0114] 2. Preparation of live bacteria / synbiotic preparations

[0115] (1) Preparation of live bacteria of Pediococcus acidilactici CCFM1365

[0116] Dip an appropriate amount of lactic acid Pediococcus CCFM1365 bacterial liquid and streak it, and incubate it aerobically at 37°C for 24 to 48 hours; pick a single colony to MRS liquid medium, incubate it at 37°C for 18 to 24 hours, mix it thoroughly, and then take the bacterial liquid and inoculate it into a new MRS liquid medium at a 2% (v / v) inoculation volume and incubate it under the same environment. Repeat this step 3 to 5 times to obtain a seed liquid; inoculate the prepared seed liquid into MRS medium at a 2% (v / v) inoculation volume and incubate it for 18 to 36 hours, collect the bacterial sludge by centrifugation, rinse it thoroughly with PBS buffer solution 3 to 5 times, and resuspend it to 10 10 CFU / mL, and finally freeze-dried to obtain the freeze-dried powder of live bacteria of Pediococcus acidilactici CCFM1365 (CCFM1365-H). The content of Pediococcus acidilactici CCFM1365 in the bacterial powder is not less than 10 10 CFU / g.

[0117] The freeze-dried protective agent ingredients are 100 g / L skim milk powder, 30 mL / L glycerol, 100 g / L maltodextrin, and 150 g / L trehalose. These freeze-dried protective agent raw materials are mixed with purified water to fully dissolve and sterilized at 115° C. for 20 minutes.

[0118] (2) Preparation of synbiotic preparations

[0119] The powder of Pediococcus acidilactici CCFM1365 in step (1) is mixed with the kudzu root extract to obtain a synbiotic preparation (CCFM1365+PLE), wherein the kudzu root extract content in the synbiotic preparation is not less than 5 mg / mL, and the kudzu root extract content and the number of viable bacteria in the synbiotic preparation are adjusted according to the gavage dose.

[0120] Example 4: Pediococcus acidilactici CCFM1365 and its synbiotic preparation reduce body weight and liver index in mice

[0121] Establishment of Alcoholic Liver Disease (ALD) Mouse Model:

[0122] The mouse model of alcoholic liver injury was established according to the method described in the literature with slight modifications. First, all mice underwent a 7-day liquid diet adaptation period, during which they were fed a Lieber-DeCarli liquid control diet. After the liquid diet adaptation period, all mice were randomly divided into 7 groups: blank control group (Control), alcohol model group (Model), positive control group (Positive, gavage 150 mg / kg / d bifendate), Pediococcus acidilactici CCFM1365 combined with Pueraria root extract group (CCFM1365+PLE, gavage 100 mg / kg / d Pueraria root extract and 0.1 mL 5×10 9 CFU / mL live bacteria), live bacteria group (CCFM1365-H, oral administration 0.1mL 5×10 9 Mice were divided into three groups: a CCFM1365-S group (live bacteria, CFU / mL / day), a CCFM1365-S group (100 mg / kg / day of lyophilized CCFM1365 supernatant powder administered orally), and a CCFM1365-L group (100 mg / kg / day of lyophilized CCFM1365 lysate powder administered orally), with six mice in each group. The blank control group continued to receive the Lieber-DeCarli liquid control diet, while all other groups received the Lieber-DeCarli alcohol model diet until week 8. During weeks 2-8 of this feeding period, mice were gavaged daily before 5:00 PM (daily gavage dose calculated based on mouse body weight), with the same gavage volume in each group. Mice were observed for 30 minutes after each gavage. After the final gavage, all mice were fasted for 12 hours, weighed, anesthetized, and their eyes enucleated for blood collection. The mice were then sacrificed by cervical dislocation, and serum and tissue samples were collected. Animal grouping and dosing are shown in Table 1.

[0123] Table 2 Experimental animal groups and drug administration

[0124]

[0125] During the ALD membrane formation period, the mental state of the mice was observed regularly every day and the weight of the mice was recorded. During the dissection, the intact liver tissue was sampled and washed with PBS buffer solution. The wet weight was measured after drying on filter paper. The mouse liver index = liver weight / mouse body weight. Figure 1As shown, compared with the blank control group (control, weight 28.00±0.85g, liver index 3.61±0.14%), the body weight of mice in the alcohol model group (Model) was significantly reduced (23.92±0.29g), and the liver index (4.90±0.09%) was significantly increased (P<0.001). Compared with the model group, the liver index of mice in the synbiotic preparation group (CCFM1365+PLE, 4.50±0.04%) and the live bacteria group (CCFM1365-H, 4.24±0.12%) was significantly reduced (P<0.001), indicating that the synbiotic preparation prepared from Pediococcus acidilactici CCFM1365 and the live bacteria can effectively inhibit liver swelling in mice induced by chronic alcohol exposure.

[0126] Example 5: Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations alleviate alcohol-induced liver pathological changes in mice

[0127] The specific experimental setup was as described in Example 4. The liver tissue was fixed in 4% paraformaldehyde solution for 24 h and then stained with hematoxylin-eosin (H&E) to assess the degree of liver steatosis and inflammation. Frozen sections of fresh liver tissue embedded in OCT were cut and stained with Oil Red O to observe the accumulation of lipids in the liver. The stained tissue sections were observed under an optical microscope. The section results are shown in Figure 4. Figure 2 As shown, the liver tissue structure of mice in the Control group was normal, with no obvious fat droplet vacuoles or steatosis. The livers of mice in the Model group showed severe steatosis and lipid droplet accumulation, with numerous round vacuoles of varying sizes visible in the cytoplasm, indicating that the 7-week chronic ALD model was successfully established. Compared with the Model group, the liver lobules and hepatocyte cord structures of mice in the synbiotic preparation group (CCFM1365+PLE), the live bacteria group (CCFM1365-H), and the bacterial lysate group (CCFM1365-L) were basically normal, with significantly reduced steatosis. A small amount of fat droplet particles and inflammatory cell infiltration were visible in the tissues, and hepatocyte edema was lower than that in the Model group. This indicates that postbiotics prepared from Pediococcus acidilactici CCFM1365, synbiotic preparations, and live bacteria can effectively alleviate alcohol-induced liver pathological changes in mice.

[0128] Example 6: Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations improve alcohol-induced lipid accumulation in mice

[0129] The specific experimental settings were as shown in Example 4. Serum AST, TG and LDL-C levels were measured using the corresponding detection kits according to the instructions. Figure 3As shown in the results, compared with the control group, the serum AST, TG, and LDL-C levels of mice in the Model group were significantly increased (P < 0.001), indicating that long-term alcohol intake causes lipid accumulation in mice. Compared with the Model group, the serum AST, TG, and LDL-C levels of mice treated with the synbiotic preparation, live bacteria, bacterial supernatant, and bacterial lysate decreased to varying degrees (P < 0.05). Among them, the combination of Pediococcus acidilactici CCFM1365 and Pueraria root extract reduced serum AST, TG, and LDL-C levels by 53.14±4.80%, 28.75±3.30%, and 39.48±6.89%, respectively (P<0.001). The combination of Pediococcus acidilactici CCFM1365 and live bacteria reduced serum AST, TG, and LDL-C levels by 32.85±2.91%, 16.74±4.89%, and 57.27±4.04%, respectively (P<0.001). Both the combination and live bacteria achieved regulatory levels comparable to those in the positive control group. Furthermore, lipid accumulation in mice treated with the bacterial supernatant and bacterial lysate groups was alleviated to varying degrees. The results showed that postbiotics, synbiotic preparations and live bacteria prepared from Pediococcus acidilactici CCFM1365 had good lipid-lowering and liver-protecting effects on alcohol-induced liver damage in mice, especially the synbiotic preparation, which significantly reduced the degree of liver damage and fatty lesions.

[0130] Example 7: Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations inhibit alcohol-induced increase in oxidative stress levels in mouse liver

[0131] The specific experimental setup was as described in Example 4. Part of the frozen liver tissue was weighed and added to PBS (pH 7.4, 4°C) at a ratio of 1:9 (m / V). The mixture was homogenized and centrifuged at 4°C and 12,000×g for 30 min. The supernatant was collected and the levels of MDA, GSH, and SOD were determined according to the instructions of the corresponding kit. Total RNA was extracted from the mouse liver tissue using an animal RNA extraction kit, and then the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Real-time fluorescence quantitative PCR was performed using the cDNA as a template using a fluorescent dye intercalation method, and mouse GAPDH was used as an internal reference gene. -ΔΔCt The mRNA expression levels of the target genes COX-2, Nrf2, and HO-1 were calculated using the PCR method. The expression levels of the target genes in each group are expressed relative to the blank control group (set to 1.0). Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; primer sequences are shown in Table 3.

[0132] Table 3 Real-time fluorescence quantitative PCR detection primer sequences

[0133]

[0134]

[0135] The results are as follows Figure 4 As shown, compared with the control group, the activities of GSH and SOD in the liver of mice in the Model group were significantly reduced (P < 0.001), indicating that the liver of mice in the Model group had oxidative damage. Consistent with the intervention effect of bifendate in the positive control group (Positive), alcohol-induced GSH depletion and decreased SOD activity were significantly improved after intervention with CCFM1365+PLE, CCFM1365-H, and CCFM1365-L (P < 0.01). Among them, after CCFM1365+PLE intervention, the GSH content and SOD level in the liver of mice increased by 210.5±22.64% and 223.2±13.97%, respectively (P < 0.001), reaching a regulatory level comparable to that of the positive control group. Furthermore, the Model group mice had the highest MDA content in their livers. Compared to the Model group, the CCFM1365+PLE group had a 57.72±2.32% decrease in MDA content (P<0.001). The CCFM1365-H and CCFM1365-L groups also experienced varying degrees of reduction (P<0.001), returning to levels comparable to those in the control group. This suggests that the antioxidant capacity of the liver tissues of mice in the postbiotic, synbiotic, and live bacteria groups was enhanced.

[0136] In addition, compared with the control group, COX-2 gene expression in liver tissues of mice in the Model group was significantly increased (P < 0.001), while Nrf2 and HO-1 gene expression was significantly decreased (P < 0.001). Compared with the Model group, COX-2 gene expression in liver tissues of mice in the Positive group and CCFM1365 + PLE group was significantly decreased, while Nrf2 and HO-1 gene expression was significantly increased (P < 0.001). Specifically, COX-2 gene expression in liver tissues of mice in the CCFM1365 + PLE group decreased by 54.99 ± 5.24% (P < 0.001), while Nrf2 and HO-1 gene expression increased by 437.0 ± 29.44% and 235.6 ± 24.52%, respectively (P < 0.001), restoring the expression of each gene to levels comparable to those in the control group. These results indicate that a synergistic combination of Pediococcus acidilactici CCFM1365 and Pueraria root extract effectively inhibited the elevated liver oxidative stress levels in mice induced by long-term alcohol exposure. Furthermore, the synergistic combination significantly improved the repair of alcohol-induced liver damage in mice compared to mice treated with live Pediococcus acidilactici CCFM1365 alone.

[0137] Example 8: Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations regulate alcohol metabolism in mice

[0138] The specific experimental setup refers to Example 4. The metabolism of ethanol mainly occurs in the liver, and ethanol is metabolized into carbon dioxide and water and excreted from the body through several different metabolic pathways. The cytochrome P450 enzyme system includes many types, of which CYP4502E1 is only one subclass, and CYP2E1 is a metabolic enzyme subfamily metabolic pathway with significant polymorphism. Other metabolic pathways also include alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), etc. Therefore, it is important to assess the degree of liver damage by detecting the activity and expression levels of enzymes related to alcohol metabolism in liver tissue. The qRT-PCR method was used to detect the mRNA expression levels of Cyp2e1 and CYP1A2 in mouse liver tissue. The primer sequences are shown in Table 4.

[0139] Table 4 Real-time fluorescence quantitative PCR detection primer sequences

[0140]

[0141] The results are as follows Figure 5 As shown in the results, compared with the Control group, the activities of ADH and ALDH in the liver tissues of mice in the Model group were significantly decreased (P < 0.001), and the expression levels of Cyp2e1 and CYP1A2 genes were significantly increased (P < 0.001). Compared with the Model group, the activities of ADH and ALDH in the liver tissues of mice in the CCFM1365+PLE group were increased by 190.76±38.24% and 125.49±6.04%, respectively (P < 0.001), and the expression levels of Cyp2e1 and CYP1A2 genes were decreased by 71.57±11.31% and 64.37±7.06%, respectively (P < 0.001), reaching levels comparable to those of the Control group. These results indicate that the protective effects of a synergistic formulation of Pediococcus acidilactici CCFM1365 and Pueraria root extract on mice induced by chronic alcohol exposure include: regulating the activity of alcohol-metabolizing enzymes, promoting the activity of ADH and ALDH, inhibiting the activity of Cyp2e1 and CYP1A2, and downregulating gene expression, thereby alleviating liver damage caused by alcohol metabolites. Furthermore, compared with the model group, liver ADH and ALDH activities were increased to varying degrees in the live bacteria (CCFM1365-H) and bacterial supernatant (CCFM1365-S) groups, while Cyp2e1 and CYP1A2 expression levels were decreased (P < 0.05).

[0142] Example 9: Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations alleviate alcohol-induced liver inflammation and damage in mice

[0143] The specific experimental setup was as described in Example 4. Part of the frozen liver tissue was weighed and added to PBS (pH 7.4, 4°C) at a ratio of 1:9 (m / V). The mixture was homogenized and centrifuged at 4°C and 12,000×g for 30 min. The supernatant was collected and the LPS level in the liver was detected according to the relevant operating instructions of the ELISA kit. Total RNA was extracted from the mouse liver tissue using an animal RNA extraction kit, and then the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Real-time fluorescence quantitative PCR was performed using cDNA as a template using a fluorescent dye intercalation method, and mouse GAPDH was used as an internal reference gene. -ΔΔCt The mRNA expression levels of the target genes TNF-α, IL-6, and IL-1β were calculated using the ELISA. The expression levels of the target genes in each group are expressed relative to the blank control group (set to 1.0). Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; primer sequences are shown in Table 5.

[0144] Table 5 Real-time fluorescence quantitative PCR detection primer sequences

[0145]

[0146]

[0147] The results are as follows Figure 6 As shown in the results, compared with the Control group, the LPS level in the liver tissue of mice in the Model group was significantly increased (P < 0.001), and the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in the liver tissue of mice were significantly increased (P < 0.001), indicating that long-term ethanol intake induced an inflammatory response in the liver. However, consistent with the trend in the Positive group, the LPS level in the liver of alcohol-treated mice and the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the liver tissue of mice were improved to varying degrees after intervention with the synbiotic preparation, live bacteria, and bacterial lysate (P < 0.05). Among them, after intervention with the synbiotic preparation, the level of LPS in the mouse liver decreased by 43.01±2.52% (P<0.001), and the mRNA expression levels of TNF-α, IL-6, and IL-1β decreased by 93.01±2.47%, 45.11±13.28%, 85.65±2.97%, 92.17±7.60%, and 63.84±7.89%, respectively (P<0.001). This indicates that the synbiotic preparation prepared by Pediococcus acidilactici CCFM1365 and Pueraria root extract can significantly inhibit the expression of inflammatory factors in liver cells induced by alcohol. Compared with the group treated with live Pediococcus acidilactici CCFM1365 alone, it has a better repair effect on alcohol-induced liver inflammation and damage.

[0148] Example 10: Pediococcus acidilactici CCFM1365 and its postbiotic and synbiotic preparations improve alcohol-induced intestinal barrier function impairment in mice

[0149] The specific experimental settings refer to Example 4. It has been reported that alcohol and its metabolites can destroy the intestinal barrier function, causing excessive enteric endotoxin LPS to enter the blood and liver, thereby activating liver immune cells and triggering a series of inflammatory responses, ultimately leading to liver damage. According to the aforementioned studies, live bacteria of Pediococcus acidilactici CCFM1365 and postbiotics and synbiotic preparations prepared therefrom can improve liver fatty degeneration and lipid accumulation caused by long-term alcohol exposure to varying degrees, and reduce the damage of free radicals and lipid peroxides to liver cells. Therefore, it is necessary to continue to pay attention to the effects of live bacteria of Pediococcus acidilactici CCFM1365, postbiotics and synbiotic preparations on intestinal barrier function. The expression levels of tight junction proteins Z0-1 and Occludin in the mouse ileum were detected by immunofluorescence staining ( Figure 7 Observation results showed that the fluorescence intensity of Z0-1 (red) and occludin (green) in the CCFM1365+PLE group was significantly higher than that in the Model group, indicating that the expression levels of intestinal barrier-related functional proteins in the CCFM1365+PLE intervention group were increased, and intestinal barrier function was improved. Further semi-quantitative analysis of the expression of tight junction proteins Z0-1 and occludin in the mouse ileum was performed by measuring the mean fluorescence intensity (mean fluorescence intensity) (sum of fluorescence intensity in the region / area of ​​the region). The results showed that the mean fluorescence intensities of ZO-1 and occludin in the CCFM1365+PLE intervention group (mean fluorescence intensities of ZO-1 and occludin were 43.85±1.71 and 53.72±3.11, respectively) and the CCFM1365-H intervention group (mean fluorescence intensities of ZO-1 and occludin were 42.49±2.40 and 50.26±2.99, respectively) were significantly higher than those in the Model group (mean fluorescence intensities of ZO-1 and occludin were 38.11±1.48 and 44.61±2.67, respectively) (P<0.05). This suggests that the expression levels of intestinal barrier-related functional proteins in the CCFM1365+PLE and CCFM1365-H intervention groups were increased, and intestinal barrier function was improved.

[0150] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Pediococcus acidilactici ( Pediococcus acidilactici ) CCFM1365 was deposited in Guangdong Provincial Microbiological Culture Collection on November 9, 2023, with the deposit number GDMCC No: 63997.

2. A composition comprising the Pediococcus acidilactici CCFM1365 according to claim 1.

3. The composition according to claim 2, characterized in that The composition contains Pediococcus acidilactici CCFM1365 strain, or contains postbiotics prepared from Pediococcus acidilactici CCFM1365.

4. The composition according to claim 3, characterized in that The Pediococcus acidilactici CCFM1365 strain includes a live strain and / or a dried strain of Pediococcus acidilactici CCFM1365; the postbiotics include a fermentation supernatant, a bacterial lysate and / or a fermentation liquid of Pediococcus acidilactici CCFM1365.

5. A synbiotic preparation comprising the Pediococcus acidilactici CCFM1365 according to claim 1, or the composition according to any one of claims 2 to 4.

6. The synbiotic preparation according to claim 5, characterized in that In the synbiotic preparation, the content of Pediococcus acidilactici CCFM1365 is not less than 10 6 CFU / mL or 10 6 CFU / g.

7. The synbiotic preparation according to claim 5 or 6, characterized in that The synbiotic preparation further contains kudzu root extract, and the content of the kudzu root extract is ≥5 mg / mL.

8. A product comprising the Pediococcus acidilactici CCFM1365 according to claim 1, or the composition according to any one of claims 2 to 4, or the synbiotic preparation according to any one of claims 5 to 7, characterized in that: The products include food, medicine or health products.

9. The product according to claim 8, characterized in that The food is a dairy product, soy product or fruit and vegetable product produced using a starter containing the Pediococcus acidilactici CCFM1365; or the food is a beverage or snack containing the Pediococcus acidilactici CCFM1365 according to claim 1.

10. A method for converting kudzu root flavonoids, characterized in that: The Pediococcus acidilactici CCFM1365 according to claim 1 is added to a culture medium containing kudzu root extract and cultured.

11. The method according to claim 10, characterized in that The pueraria flavonoids include puerarin, daidzein and genistin.

12. Use of the Pediococcus acidilactici CCFM1365 according to claim 1, or the composition according to any one of claims 2 to 4, or the synbiotic preparation according to any one of claims 5 to 7 in the preparation of a medicament for treating alcoholic fatty liver disease.

13. Use of the Pediococcus acidilactici CCFM1365 according to claim 1, or the composition according to any one of claims 2 to 4, or the synbiotic preparation according to any one of claims 5 to 7 in the preparation of a health product having an auxiliary protective effect against chemical liver damage.

14. Use of the Pediococcus acidilactici CCFM1365 according to claim 1, or the composition according to any one of claims 2 to 4, or the synbiotic preparation according to any one of claims 5 to 7 in the preparation of a product capable of promoting the absorption of puerarin, daidzin and / or genistin.

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