Plantarum CCFM1366 and its postbiotic capable of transforming various pueraria flavonoids

Abstract

The present application discloses a plant lactobacillus CCFM1366 capable of transforming various pueraria flavones and having the effects of alcoholism relieving and liver protecting, and a postbiotic prepared from the plant lactobacillus CCFM1366, and belongs to the technical field of microorganisms. The plant lactobacillus CCFM1366 and the postbiotic prepared from the plant lactobacillus CCFM1366 have at least one of the following effects: (1) improving alcohol metabolism enzyme activity and enhancing alcohol metabolism; (2) improving liver steatosis, lipid accumulation, oxidative stress and inflammatory response caused by long-term alcohol exposure; (3) reducing ROS synthesis and thereby reducing oxidative damage to liver cells, promoting the synthesis of antioxidants and thereby improving the protective effect on liver cells; and (4) reducing inflammatory response. The plant lactobacillus CCFM1366 and the postbiotic prepared from the plant lactobacillus CCFM1366 can be used for preparing functional food and / or dietary supplements with the potential of alcoholism relieving and liver protecting, and have great application prospect.

Description

Technical Field

[0001] This invention relates to a strain of *Lactobacillus plantarum* CCFM1366, which can convert various kudzu flavonoids and has the effects of relieving hangovers and protecting the liver, and its post-genes, belonging to the field of microbial technology. Background Technology

[0002] With socioeconomic development, the number of drinkers is increasing, and alcoholic liver disease (ALD) caused by alcohol abuse, alcohol dependence, and alcohol poisoning has become a common public health problem worldwide. ALD is mainly caused by long-term alcohol consumption exceeding a certain limit, and drinking can also aggravate other liver diseases. The pathogenesis of ALD initially begins with alcoholic fatty liver, characterized by hepatic steatosis, where phospholipids, cholesterol esters, and triglycerides accumulate in the liver to form fatty liver. Lipid droplets initially appear in hepatocytes around small veins, gradually appearing in hepatocytes in the midlobules and around the portal vein. In some patients, the condition continues to worsen, with liver inflammation and hepatocyte damage, eventually leading to alcoholic steatohepatitis (ASH). ASH progresses slowly, but persistent liver damage and inflammation can eventually lead to liver fibrosis and cirrhosis, and may even develop into liver cancer.

[0003] Traditional Chinese medicine (TCM) has gained increasing public attention in recent years due to its stable supply, long-term efficacy, and minimal side effects. More and more TCM products (including medicinal herbs and phytochemicals) are being used to treat chronic liver diseases. TCM is characterized by its multi-component, multi-target, and multi-mechanism synergistic effects, and can regulate and protect the liver through various mechanisms. Currently, TCM is being studied as a complementary and alternative therapy for ALD. The protective effects of herbs such as kudzu root, Japanese raisin tree fruit, licorice root, schisandra fruit, and salvia miltiorrhiza on alcoholic liver injury have been extensively studied. Other active ingredients in TCM, such as flavonoids, saponins, amino acids, phenols, and polysaccharides, have shown significant activity in liver protection. TCM compound formulas, extracts, and their active ingredients can scavenge free radicals, enhance the activity of antioxidant enzymes, improve the body's defense against free radicals, and inhibit liver inflammation and apoptosis, thus achieving the goal of preventing and treating ALD.

[0004] However, the bioactive components of traditional Chinese medicine (TCM) are complex, and some natural products of TCM cannot be directly absorbed by humans and animals. Furthermore, the content of most bioactive components in TCM is low, and some natural products are toxic to humans and animals. Microbial fermentation is one of the traditional processing techniques for TCM, which can be carried out under appropriate temperature and humidity conditions. TCM fermentation can improve efficacy, reduce toxicity, produce new chemical components, and reduce the use of wild TCM resources. Records of fermented TCM and its products are found in classic TCM texts such as *Qimin Yaoshu*, *Shennong Bencao Jing*, *Compendium of Materia Medica*, and the *Pharmacopoeia of the People's Republic of China*. In fact, some microbially fermented TCMs exhibit better pharmacological activity than unfermented ones. Probiotics are live microorganisms beneficial to human health. Some natural products of certain TCMs can act as prebiotics, promoting the proliferation of beneficial microorganisms in the host's gut. Therefore, the synergistic effect of probiotic fermentation of TCM has great potential to enhance the effectiveness of TCM. However, according to the literature "Recent advances in methods of puerarin biotransformation," research on microbial transformation and fermentation of puerarin flavonoids is relatively limited. Furthermore, the paper "Bioconversion of isoflavones during the fermentation of Samso-Eum with Lactobacillus strains" mentions that most strains cannot utilize puerarin, and even those strains with transformation ability have very low transformation rates (≤5%). The strain reported in "Expression and characterization of the human intestinal bacterial enzyme which cleaves the C-glycosidic bond in 3″-oxo-puerarin," while capable of completely transforming puerarin, is inedible and difficult to cultivate, thus lacking other additional value. The literature "Bioconversion of soy isoflavones daidzin and daidzein by Bifidobacterium strains" reports that Bifidobacterium has the ability to transform daidzein into daidzein, with a transformation rate of approximately 48%-65% / 7 days; however, the low transformation rate makes it unsuitable for industrial applications.

[0005] Lactiplantibacillus planturum is widely found in nature and is commonly used in food fermentation, animal feed, industrial lactic acid bacteria fermentation, and healthcare industries due to its probiotic properties and safety. Although its various probiotic functions have been studied, it has not yet been found that Lactiplantibacillus planturum can simultaneously and efficiently utilize multiple puerarin flavonoids (especially puerarin, daidzein, and genistein) and significantly alleviate alcoholic liver damage. Therefore, isolating and screening such probiotics to achieve the synergistic effect of probiotic fermentation of diets rich in puerarin flavonoids, thereby improving the effectiveness of traditional Chinese medicine, has broad application prospects. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a strain of Lactiplantibacillus plantarum and its preparation of post-biotics and the application of kudzu flavonoids to enhance the effects of alcohol detoxification and liver protection. This method can improve the alcohol detoxification effect of diets rich in kudzu flavonoids and enhance the effectiveness of traditional Chinese medicine.

[0007] This invention provides a strain of Lactiplantibacillus plantarum (CCFM1366) that can significantly enhance the hangover-relieving effect of kudzu flavonoids. It was deposited on November 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63998, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The Lactiplantibacillus plantarum mentioned above was isolated from the feces of healthy human beings and has the following characteristics:

[0009] The *Lactobacillus plantarum* CCFM1366, when cultured on MRS medium for 48 hours, appears as round, raised, smooth, fine-textured, white, occasionally light yellow or dark yellow.

[0010] The *Lactobacillus plantarum* CCFM1366 is a Gram-positive bacterium that is anaerobic or facultatively anaerobic. Its optimal growth temperature is 30–35°C, and its optimal growth pH is around 6.5.

[0011] The plant lactobacillus CCFM1366 can efficiently convert various puerarin flavonoids (such as puerarin, daidzein and genistein) into active substances (such as daidzein and genistein).

[0012] The present invention provides a composition containing *Lactobacillus plantarum* CCFM1366, or containing a metabiotic prepared from *Lactobacillus plantarum* CCFM1366.

[0013] In one embodiment, the metabiotic includes fermentation supernatant, cell lysate, and / or fermentation broth.

[0014] In one embodiment, the metabiotic is obtained by inoculating the above-mentioned *Lactobacillus plantarum* CCFM1366 into MRS medium, culturing the bacterial solution, and then subjecting it to heat treatment and lysis.

[0015] In one embodiment, the heat treatment is performed at 60–70°C for 25–35 minutes.

[0016] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging the above-mentioned fermentation broth.

[0017] In one embodiment, the method for preparing the cell lysate is to homogenize the heat-treated fermentation broth under high pressure and centrifuge it to obtain the cell lysate.

[0018] In one embodiment, the post-generic can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, and fluidized bed drying.

[0019] This invention provides the application of the above-mentioned Lactiplantibacillus plantarum CCFM1366 in the preparation of products for relieving hangovers and protecting the liver.

[0020] The present invention also provides food, health products, pharmaceuticals or cosmetics containing the said *Lactobacillus plantarum* CCFM1366 postbiotic.

[0021] In one embodiment, the food product includes the above-described composition and conventional excipients.

[0022] In one embodiment, the health product includes the above-described composition and conventional excipients.

[0023] In one embodiment, the pharmaceutical product comprises the above-described composition, a drug carrier, and / or pharmaceutical excipients.

[0024] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0025] In one embodiment, the cosmetic comprises the above-described composition, matrix ingredients, and / or conventional excipients.

[0026] In one embodiment, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.

[0027] In one embodiment, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0028] The present invention also provides a method for converting puerarin, daidzein and / or genistein using the aforementioned *Lactobacillus plantarum* CCFM1366, wherein the *Lactobacillus plantarum* CCFM1366 is added to a reaction system containing puerarin, daidzein and / or genistein for fermentation.

[0029] The present invention also provides the use of the aforementioned *Lactobacillus plantarum* CCFM1366, or the aforementioned composition, or the aforementioned product in the preparation of products that can promote the absorption of puerarin, daidzein, and / or genistein.

[0030] The present invention also provides the use of the aforementioned *Lactobacillus plantarum* CCFM1366, or the aforementioned composition, or the aforementioned product in the preparation of a medicine for relieving hangovers and protecting the liver.

[0031] The present invention also provides the use of the aforementioned *Lactobacillus plantarum* CCFM1366, or the aforementioned composition, or the aforementioned product in the preparation of health products that have an auxiliary protective effect against chemically induced liver injury.

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

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

[0034] (1) The above-mentioned Lactiplantibacillus plantarum CCFM1366 was streaked on MRS solid medium and incubated upside down at 37°C for 48 h. A single colony was picked and inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 24 h.

[0035] (2) Add 5% (v / v) of the aforementioned Lactobacillus plantarum bacterial solution to the fermentation substrate rich in kudzu root extract, and ferment at a constant temperature of 37°C to prepare the fermentation broth.

[0036] (3) Collect the fermentation broth, centrifuge to obtain the supernatant, add ethyl acetate in equal proportion for extraction twice, concentrate by freezing centrifugation, add methanol to redissolve, filter through a 0.22μm microporous membrane and detect the change in pueraria flavonoid content by HPLC.

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

[0038] In one embodiment of the present invention, the number of *Lactobacillus plantarum* CCFM1366 bacterial suspension in step (2) is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

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

[0040] Beneficial effects:

[0041] This invention screened and obtained a strain of Lactiplantibacillus plantarum, CCFM1366. This strain of Lactiplantibacillus plantarum, CCFM1366, and its prepared metabiotics possess the ability to enhance the liver's ability to detoxify and protect against alcohol, and have a synergistic effect with kudzu root. Specifically, this is reflected in:

[0042] In one embodiment, the product includes at least one of the following functions:

[0043] 1. *Lactobacillus plantarum* CCFM1366 can efficiently utilize flavonoids in kudzu root. The conversion rate of puerarin was 49.22±3.76% / 48h; the conversion rate of daidzein was 96.57±2.45% / 48h; and the conversion rate of genistein was 90.10±3.13% / 48h. The concentration of daidzein after fermentation was 23.30±2.27 μg / mL, with an increase of 4909±440.3% / 48h.

[0044] 2. *Lactobacillus plantarum* CCFM1366 has the effect of relieving hangovers and protecting the liver:

[0045] (1) Reverse changes in biochemical parameters (ALT, AST, TG and HDL-C) and histopathological changes, and alleviate steatosis and liver damage caused by long-term alcohol consumption;

[0046] (2) Reduce MDA levels, enhance the activity of antioxidant enzymes (GSH, SOD and CAT) in liver tissue, enhance the body's antioxidant capacity, reduce lipid peroxidation and alleviate liver tissue damage caused by alcohol.

[0047] (3) Increase the activity of alcohol metabolism enzymes (ADH and ALDH), inhibit the secretion of CYP2E1 and CYP1A2, reduce ROS synthesis and thus reduce oxidative damage to hepatocytes, activate the expression of Nrf2 gene and its downstream gene sequences HO-1 and GCLC, promote the synthesis of antioxidants and thus enhance the protective effect on hepatocytes.

[0048] (4) Inhibit NF-κB activation and downregulate the expression of IL-1β, TNF-α and IL-10 mRNA in liver tissue, thereby reducing the inflammatory response.

[0049] Therefore, Lactiplantibacillus plantarum CCFM1366 and its prepared metabiotics have great application potential in the preparation of products for relieving hangovers and protecting the liver.

[0050] Preservation of biological materials

[0051] A strain of Lactiplantibacillus plantarum (CCFM1366), taxonomically named Lactiplantibacillus plantarum, was deposited on November 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63998), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0052] Figure 1 Preliminary metabolic pathways of kudzu flavonoids.

[0053] Figure 2 HPLC results of Lactobacillus plantarum CCFM1366 before and after fermentation.

[0054] Figure 3 Changes in the content of flavonoids in kudzu root before and after fermentation with Lactobacillus plantarum CCFM1366.

[0055] Figure 4 Effects of Lactobacillus plantarum CCFM1366 and its prepared metabiotic on body weight and liver index in mice with chronic alcohol exposure.

[0056] Figure 5 Effects of Lactobacillus plantarum CCFM1366 and its prepared metabiotic on liver histopathology in mice with chronic alcohol exposure.

[0057] Figure 6 Effects of Lactobacillus plantarum CCFM1366 and its prepared metabiotic on liver function in mice with chronic alcohol exposure.

[0058] Figure 7Effects of Lactobacillus plantarum CCFM1366 and its prepared metabiotic on liver oxidative stress levels in mice with chronic alcohol exposure.

[0059] Figure 8 Effects of Lactobacillus plantarum CCFM1366 and its prepared metabiotic on alcohol metabolism in mice with chronic alcohol exposure.

[0060] Figure 9 Effects of Lactobacillus plantarum CCFM1366 and its prepared metabiotic on the expression levels of liver inflammatory factors mRNA in mice with chronic alcohol exposure. Detailed Implementation

[0061] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the implementation regulations.

[0062] In the following examples, SPF-grade male C57bl / 6J mice (6 weeks old, 18±2g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; the herbal kudzu root extract (40% kudzu 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), genistein (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 were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. The body model feed (TP4030B) was purchased from Nantong Trofi Feed Technology Co., Ltd. and formulated according to requirements; the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute; 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.; the BCA protein concentration assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the reverse transcription kit and real-time fluorescence quantitative PCR kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; and primers were purchased from Shanghai Sangon Biotech Co., Ltd.

[0063] The culture media involved in the following examples are as follows:

[0064] MRS liquid culture medium (g / L): peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, glucose 20g / L, anhydrous sodium acetate 2g / L, diammonium citrate 2g / L, K₂HPO₄·3H₂O 2.6g / L, MgSO₄·7H₂O 0.58g / L, MnSO₄·7H₂O 0.25g / L, Tween-80 1g / L, distilled water 1000g / L. Autoclave at 115℃ for 20 min.

[0065] MRS solid culture medium (g / L): peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, glucose 20g / L, anhydrous sodium acetate 2g / L, diammonium citrate 2g / L, K₂HPO₄·3H₂O 2.6g / L, MgSO₄·7H₂O 0.58g / L, MnSO₄·7H₂O 0.25g / L, Tween-80 1g / L, agar 20g / L, distilled water 1000g / L. Autoclave at 115℃ for 20 min.

[0066] Fermentation medium (0.5 g / L): 0.5 g / L kudzu root extract, 5 g / L yeast extract, 5 g / L glucose, 1000 g / L distilled water. Autoclave at 115℃ for 20 min.

[0067] PBS buffer solution ( / L): Sodium chloride 8.0g, potassium chloride 0.2g, disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, adjust pH to 7.4. Autoclave at 115℃ for 20min.

[0068] Example 1: Screening, identification and preservation of strains

[0069] 1. Screening

[0070] Using fecal samples from healthy individuals as the sample, sterile physiological saline was used for 10-fold serial dilution to 10-1. -6 Then take 100 μL of each diluted 10. -4 10 -5 10 -6 The diluted solution was plated on MRS solid medium and incubated at 37°C for 48 h. The colony morphology was observed and recorded. Colonies of different morphologies were picked from the MRS solid medium and streaked for isolation. After incubation at 37°C for 48 h, single colonies of different morphologies were picked from the MRS solid medium again and streaked for isolation until pure single colonies with consistent morphology were obtained. Pure colonies from the MRS solid medium were inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 24 h. 1 mL of bacterial solution was taken into a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the upper medium was discarded. The obtained bacterial sludge was freeze-dried.

[0071] 2. Identification

[0072] The isolated strain underwent PCR amplification of 16S rDNA. The PCR products were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The resulting PCR products were then sent to a biotechnology company for sequencing. The obtained sequences were searched in GeneBank using BLAST and similarity comparisons to obtain strain identification results. Finally, one strain of *Lactiplantibacillus plantarum* was obtained and named *Lactiplantibacillus plantarum* CCFM1366. The spliced ​​sequence and identification results of the strain are shown in SEQ ID NO.1.

[0073] The primers used for 16S rDNA amplification are as follows:

[0074] 27F (positive): 5'-AGAGTTTGATCCTGGCCTCA-3';

[0075] 1492R (reverse): 5'-GGTTACCTTGTTACGACTT-3'.

[0076] The 16S rDNA amplification procedure is as follows:

[0077] 94℃ for 5 min; repeat for a total of 30 cycles (94℃ for 30 s; 55℃ for 30 s; 72℃ for 2 min); 72℃ for 10 min; 12℃ for 2 min.

[0078] 3. Save

[0079] Lactiplantibacillus plantarum CCFM1366 was inoculated into 5 mL of MRS liquid medium and cultured at 37 °C for 24 h. 1 mL of bacterial suspension was taken into a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, the upper culture medium was discarded, and the bacterial sludge was resuspended in 30% glycerol solution and stored at -80 °C.

[0080] The Lactiplantibacillus plantarum CCFM1366 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCC No: 63998.

[0081] Example 2: Preparation of postbiotics from *Lactobacillus plantarum* CCFM1366

[0082] Before activation, *Lactobacillus plantarum* CCFM1366 was stored in 30% glycerol at -80°C. A small amount of *Lactobacillus plantarum* CCFM1366 bacterial suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated aerobically at 37°C for 24–48 h. Single colonies were then picked and inoculated into MRS liquid medium and incubated at 37°C for 18–24 h. After thorough mixing, the bacterial suspension was inoculated into fresh MRS liquid medium at a 2% (v / v) inoculation rate and incubated under the same conditions. This process was repeated 3–5 times to obtain 3.8 × 10⁸ cells / mL. 8 CFU / mL bacterial suspension.

[0083] The bacterial suspension was heat-treated at 65℃ for 30 min, centrifuged (8000g, 4℃, 15 min), and the supernatant was collected. After freeze-drying, the supernatant of *Lactobacillus plantarum* CCFM1366 was obtained as freeze-dried powder (CCFM1366-S) for later use. The bacterial suspension was heat-treated by high pressure (800-1200MPa, 3 times) to obtain bacterial cell lysate, which was then freeze-dried to obtain *Lactobacillus plantarum* CCFM1366 bacterial cell lysate freeze-dried powder (CCFM1366-L) for later use.

[0084] Example 3: Establishment of a mouse model of alcoholic liver disease (ALD)

[0085] The mouse model of alcoholic liver injury was established based on the method described in the literature with slight modifications. First, all mice underwent a 7-day acclimatization period to a liquid diet, during which they were fed a Lieber-DeCarli liquid control diet. After acclimatization, all mice were randomly divided into 5 groups: a blank control group (Control), an alcohol model group (Model), a positive control group (150 mg / kg / d biphenyl diester by gavage), a bacterial supernatant group (CCFM1366-S, 100 mg / kg / d lyophilized bacterial supernatant powder by gavage), and a bacterial lysate group (CCFM1366-L, 100 mg / kg / d lyophilized bacterial lysate powder by gavage), with 6 mice in each group. The blank control group continued to receive the Lieber-DeCarli liquid control diet, while the other groups received the Lieber-DeCarli alcohol liquid model diet for 8 weeks. During weeks 2-8 of the feeding period, mice were administered gavage daily before 5 PM (the daily gavage dose was calculated based on the mouse's body weight), with identical gavage volumes in all groups. Mice were observed for 30 minutes after each gavage. After the final gavage, all mice were fasted but allowed free access to water for 12 hours, weighed, anesthetized, had their eyeballs enucleated for blood collection, and were euthanized by cervical dislocation. Serum and tissue samples were collected. Animal groupings and drug administration are shown in Table 1.

[0086] Table 1. Grouping and administration of experimental animals

[0087]

[0088] Example 4: Transformation of Pueraria lobata flavonoids by Lactobacillus plantarum CCFM1366

[0089] 1. Fermentation of *Lactobacillus plantarum* CCFM1366 in a fermentation medium rich in puerarin flavonoids:

[0090] The *Lactobacillus plantarum* CCFM1366 from Example 1 was streaked on MRS solid medium and incubated upside down at 37°C for 24–48 h. A single colony was picked and inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 18–24 h. After thorough mixing, the bacterial solution was inoculated into a new MRS liquid medium at an inoculation rate of 2% (v / v) and incubated under the same conditions. This step was repeated 3–5 times to obtain the seed culture.

[0091] In a fermentation medium rich in kudzu flavonoids, 5% (v / v) of the seed culture of *Lactobacillus plantarum* obtained above was added, and fermentation was carried out at a constant temperature of 37℃ for 0–120 h. The initial inoculum concentration was 4.2 × 10⁻⁶. 9 CFU / mL.

[0092] Fermentation broths at different time points were centrifuged, and the supernatant was collected. An equal volume of ethyl acetate was added for extraction. After thorough mixing, the mixture was allowed to stand for 5 minutes, centrifuged (5000g, 5 minutes), and the supernatant was collected and concentrated into a dry powder using a refrigerated centrifuge at 45℃. 200 μL of 100% chromatographic grade methanol was added for reconstitution, and the mixture was filtered through a 0.22 μm microporous organic filter membrane to obtain kudzu flavonoid fermentation product. The product was then stored at 4℃ for analysis.

[0093] 2. HPLC method for determining the flavonoid content of kudzu root before and after fermentation:

[0094] The chromatographic conditions were as follows: Chromatographic detection system: Waters e2695; 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℃. 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.

[0095] Obtaining the standard curve: Accurately weigh approximately 10 mg each of puerarin, daidzein, genistein, and daidzein reference standards into 100 mL volumetric flasks, dilute to volume with analytical grade ethanol, and sonicate at 300 W 50 Hz for 30 min. After cooling, replenish the missing weight with ethanol. Accurately pipette the above solution and dilute to 2 mL concentrations of 10.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, 75.0 μg / mL, and 100.0 μg / mL. Plot a working curve in the mobile phase, obtain the results, and fit the curve. Based on the standard curve, calculate the contents of puerarin, daidzein, genistein, and daidzein in the fermentation medium and pueraria flavonoid fermentation broth, respectively.

[0096] It has been reported that microorganisms can convert kudzu flavonoids into a variety of active small molecules through the enzyme system they produce. Figure 1 HPLC analysis showed that the contents of puerarin, daidzein, and genistein in the fermentation broth of kudzu flavonoids were significantly reduced. The contents of daidzein and genistein were almost zero after 48 hours of fermentation with *Lactobacillus plantarum* CCFM1366, indicating a conversion rate close to 100%. This demonstrates that kudzu flavonoids can be largely converted under suitable conditions, with daidzein being one of the main metabolites. Figure 2 ).

[0097] The reduction (conversion rate) of puerarin, the substrate, and the increase of daidzein, the product, were compared before and after fermentation with *Lactobacillus plantarum* CCFM1366. The calculation formulas were: Conversion rate = 1 - (content after fermentation / content before fermentation) × 100%; Increase = (content after fermentation - content before fermentation) / content before fermentation × 100%. After 48 hours of fermentation, the conversion rates of puerarin in puerarin from *Lactobacillus plantarum* were 49.22 ± 3.76% / 48h; for daidzein, 96.57 ± 2.45% / 48h; and for genistein, 90.10 ± 3.13% / 48h. The concentration of daidzein after fermentation was 23.30 ± 2.27 μg / mL, with an increase of 4909 ± 440.3% / 48h. The changes in puerarin content at different time points are shown in [Figure showing the changes]. Figure 3 .

[0098] Example 5: Lactobacillus plantarum CCFM1366 and its prepared postbiotic reduced body weight and liver index in mice.

[0099] The specific experimental setup is as described in Example 3. During ALD membrane formation, the mice's mental state was observed and their weight was recorded daily. For dissection, intact liver tissue was sampled, washed with PBS buffer, blotted dry on filter paper, and its wet weight was measured. The mouse liver index was calculated as liver weight / mouse body weight. Results are as follows: Figure 4As shown, compared with the blank control group (Control), the body weight of mice in the alcohol model group (Model) was significantly reduced, 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 bacterial supernatant group (CCFM1366-S, 4.51±0.20%) and the bacterial lysate group (CCFM1366-L, 4.34±0.10%) was significantly reduced (P<0.01), suggesting that the metabiotic prepared from *Lactobacillus plantarum* CCFM1366 can effectively inhibit liver swelling in mice caused by chronic alcohol exposure.

[0100] Example 6: Lactobacillus plantarum CCFM1366 and its prepared postbiotic alleviate alcohol-induced liver pathological changes in mice.

[0101] The specific experimental setup was as described in Example 3. Liver tissue was fixed in 4% paraformaldehyde solution for 24 hours, followed by hematoxylin and eosin (H&E) staining to assess the degree of hepatic steatosis and inflammation. Frozen sections of fresh liver tissue embedded in OCT were cut and stained with Oil Red O to observe hepatic lipid accumulation. The stained tissue sections were observed using an optical microscope. The section results are as follows: Figure 5 As shown, the liver tissue structure of mice in the Control group was normal, with no obvious fat droplets, vacuoles, or steatosis. Mice in the Model group showed severe steatosis and lipid droplet accumulation in their livers, with numerous round vacuoles of varying sizes visible in the cytoplasm, indicating successful establishment of a 7-week chronic ALD model. Compared to the Model group, mice in the bacterial lysate group (CCFM1366-L) showed largely normal liver lobules and hepatocyte cord structures, significantly reduced steatosis, and a small number of fat droplets and inflammatory cell infiltration in the tissues, with lower hepatocyte edema compared to the Model group. This indicates that the metabiotic prepared from *Lactobacillus plantarum* CCFM1366 can effectively alleviate alcohol-induced liver pathological changes in mice.

[0102] Example 7: Lactobacillus plantarum CCFM1366 and its prepared postbiotic improve alcohol-induced lipid accumulation in mice

[0103] The specific experimental setup is as described in Example 3. Serum ALT, AST, TG, and HDL-C levels were measured using the corresponding detection kits according to the instructions. Results are as follows: Figure 6As shown, compared with the control group, the serum ALT, AST, and TG levels in the model group mice were significantly increased, while the HDL-C level was significantly decreased (P < 0.001), indicating that long-term alcohol intake caused lipid accumulation in the mice. Compared with the model group mice, after intervention with bacterial supernatant, serum ALT and AST levels decreased by 49.67 ± 5.67% and 29.38 ± 1.17%, respectively; after intervention with bacterial lysate, serum ALT and AST levels decreased by 44.22 ± 2.72% and 32.19 ± 4.67%, respectively; and serum HDL-C levels increased by 113.9 ± 1.12% and 110.7 ± 1.17%, respectively. The results indicate that the metabiotic prepared from *Lactobacillus plantarum* CCFM1366 has a good lipid-lowering and liver-protective effect on alcohol-induced liver damage in mice, especially the bacterial lysate group (CCFM1366-L), which significantly reduced the degree of liver damage and fatty lesions.

[0104] Example 8: Lactobacillus plantarum CCFM1366 and its prepared metabiotic inhibited the increase in alcohol-induced oxidative stress in mouse liver.

[0105] The specific experimental setup is as described in Example 3. A portion of frozen liver tissue was weighed and added to PBS (pH 7.4, 4℃) at a ratio of 1:9 (m / V). The mixture was homogenized and centrifuged at 12,000×g for 30 min at 4℃. The supernatant was collected, and the levels of MDA, GSH, SOD, and CAT were determined according to the corresponding kit instructions. Total RNA was extracted from mouse liver tissue using an animal RNA extraction kit, and then the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Using cDNA as a template, real-time quantitative PCR was performed using a fluorescent dye intercalation method, with mouse GAPDH used as an internal reference gene. The results were obtained through 2... -ΔΔCt The mRNA expression levels of the target genes COX-2, Nrf2, HO-1, and Gclc were calculated using a 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 Biotech Co., Ltd., and their sequences are shown in Table 2.

[0106] Table 2 Primer sequences for real-time quantitative PCR detection

[0107]

[0108]

[0109] The results are as follows Figure 7As shown, compared with the control group, the activities of GSH (8.38±0.66 μmol / gprot), SOD (99.93±7.55 U / mgprot), and CAT (21.79±0.54 U / mgprot) in the liver of the model group mice were significantly reduced (P<0.001), while the content of MDA (10.99±0.45 nmol / mgprot) was significantly increased, indicating that there was oxidative damage in the liver of the model group mice. Consistent with the intervention of biphenyl diester in the positive control group, alcohol-induced GSH depletion and decreased SOD and CAT activities were significantly improved after intervention in CCFM1366-L (14.46±0.85 μmol / gprot, 149.18±4.79 U / mgprot, 25.83±0.65 U / mgprot, respectively) and CCFM1366-S (12.57±0.95 μmol / gprot, 112.01±5.11 U / mgprot, 24.18±0.47 U / mgprot, respectively) (P<0.05). Furthermore, the liver MDA content of mice in the CCFM1366-L group (7.68±0.12 nmol / mgprot) and the CCFM1366-S group (8.28±0.48 nmol / mgprot) was also reduced to varying degrees (P<0.001). This indicates that the antioxidant capacity of the liver tissue of mice in the post-genetic group was improved.

[0110] Furthermore, compared with the Control group, the expression of COX-2 gene in the liver tissue of mice in the Model group was significantly increased (P < 0.001), while the expression of Nrf2, HO-1, and Gclc genes was significantly decreased (P < 0.001). Compared with the Model group, the expression level of COX-2 gene in the liver tissue of mice in the Positive group, the bacterial supernatant group (CCFM1366-S), and the bacterial lysate group (CCFM1366-L) was significantly decreased, while the expression levels of Nrf2, HO-1, and Gclc genes were significantly increased (P < 0.01). Among them, the relative expression levels of COX-2, Nrf2, HO-1, and Gclc genes in the bacterial lysate group (CCFM1366-L) were 4.05 ± 0.91, 0.99 ± 0.10, 1.26 ± 0.27, and 1.09 ± 0.05, respectively. The above results indicate that the post-biotic intervention prepared from *Lactobacillus plantarum* CCFM1366 effectively inhibited the increase in oxidative stress levels in the liver of mice induced by long-term alcohol exposure. Furthermore, the repair effect of the bacterial lysate group (CCFM1366-L) on alcohol-induced liver injury was superior to that of the bacterial supernatant group (CCFM1366-S).

[0111] Example 9: Lactobacillus plantarum CCFM1366 and its prepared postbiotics regulate alcohol metabolism in mice

[0112] The specific experimental setup is described in Example 3. Ethanol metabolism primarily occurs in the liver, where it is metabolized into carbon dioxide and water and excreted through several different metabolic pathways. The cytochrome P450 enzyme system comprises many types, with CYP4502E1 being just one subclass. CYP2E1 is a metabolic pathway exhibiting significant polymorphism within this subfamily of enzymes. Other metabolic pathways include alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Therefore, detecting the activity and expression levels of alcohol metabolism-related enzymes in liver tissue is crucial for assessing the degree of liver damage. The mRNA expression levels of Cyp2e1 and CYP1A2 in mouse liver tissue were detected using qRT-PCR; primer sequences are shown in Table 3.

[0113] Table 3 Primer sequences for real-time quantitative PCR detection

[0114]

[0115]

[0116] The results are as follows Figure 8 As shown, compared with the Control group, the activities of ADH (7.09±0.97 U / mgprot) and ALDH (13.60±0.53 U / mgprot) in the liver tissue of Model group mice were significantly decreased (P<0.001), while the relative expression levels of Cyp2e1 (3.36±0.46) and CYP1A2 (1.56±0.11) genes were significantly increased (P<0.05). Compared with the Model group, the activities of ADH (10.95±0.50 U / mgprot) and ALDH (15.30±1.18 U / mgprot) in the liver tissue of bacterial lysate group (CCFM1366-L) mice were significantly increased, while the relative expression levels of Cyp2e1 (1.83±0.54) and CYP1A2 (1.27±0.20) genes were significantly decreased (P<0.05). The above results indicate that the metabiotic prepared from *Lactobacillus plantarum* CCFM1366 has a protective effect against long-term alcohol exposure-induced liver damage in mice by: regulating the activity of alcohol-metabolizing enzymes, promoting ADH and ALDH activity, inhibiting Cyp2e1 and CYP1A2 activity, and downregulating gene expression, thereby reducing liver damage caused by alcohol metabolites. Example 10: *Lactobacillus plantarum* CCFM1366 and its prepared metabiotic alleviate alcohol-induced liver inflammation and damage in mice.

[0117] The specific experimental setup is as described in Example 3. Total RNA was extracted from mouse liver tissue using an animal RNA extraction kit, and then the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the cDNA as a template, real-time quantitative PCR was performed using a fluorescent dye intercalation method, with mouse GAPDH used as an internal reference gene. -ΔΔCt The mRNA expression levels of the target genes TNF-α, IL-6, IL-10, and IL-1β were calculated using a 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 Biotech Co., Ltd., and their sequences are shown in Table 4.

[0118] Table 4 Primer sequences for real-time quantitative PCR detection

[0119]

[0120] The results are as follows Figure 9 As shown, compared with the Control group, the relative mRNA expression levels of inflammatory factors TNF-α (3.46±0.92), IL-10 (2.62±0.70), and IL-1β (2.82±0.39) in the liver tissue of the Model group 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 relative mRNA expression levels of pro-inflammatory factors TNF-α, IL-10, and IL-1β in the liver tissue of alcohol-treated mice were improved to varying degrees after intervention with bacterial lysate (CCFM1366-L, 1.13±0.19, 1.61±0.28, and 1.08±0.50, respectively) and bacterial supernatant (CCFM1366-S, 2.52±0.28, 2.00±0.29, and 2.92±0.58, respectively) (P<0.05). This indicates that the postbiotic prepared from *Lactobacillus plantarum* CCFM1366 can inhibit the expression levels of inflammatory factors in liver cells induced by alcohol. Comparative Example 1: Comparison of the conversion abilities of *Lactobacillus plantarum* CCFM1283 and *Lactobacillus plantarum* CCFM1366 on kudzu root flavonoids.

[0121] The specific implementation method is described in Example 4, with the difference being that *Lactobacillus plantarum* CCFM1275 (disclosed in patent CN117143769A) and *Lactobacillus plantarum* CCFM1283 (disclosed in patent CN117625456A) were used to replace *Lactobacillus plantarum* CCFM1366, respectively. The results showed that *Lactobacillus plantarum* CCFM1275 could only convert daidzein in puerarin, but could not convert puerarin and genistein. Furthermore, the yield of daidzein was 4.4 ug / mL, which was 5.30 times lower than the yield of daidzein obtained by *Lactobacillus plantarum* CCFM1366 in this invention. Conversely, *Lactobacillus plantarum* CCFM1283 could only convert puerarin in puerarin, but could not convert daidzein and genistein. Furthermore, the yield of daidzein was 13.49 ug / mL, which was 3.07 times lower than the yield of daidzein obtained by *Lactobacillus plantarum* CCFM1366 in this invention (Table 5). Therefore, compared with *Lactobacillus plantarum* CCFM1283, the *Lactobacillus plantarum* CCFM1366 provided in this patent has more significant advantages in both the biotransformation of kudzu flavonoids and the enhancement of hangover relief effects by compounding kudzu root extract. Its application in the preparation of products with the potential to relieve hangovers and protect the liver has greater application prospects.

[0122] Table 5 Summary of the conversion capabilities of different plant lactobacilli to kudzu flavonoids

[0123]

[0124] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactiplantibacillus plantarum (CCFM1366), characterized in that, The *Lactobacillus plantarum* CCFM1366 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCCNo: 63998.

2. A composition containing the *Lactobacillus plantarum* CCFM1366 as described in claim 1.

3. A composition, characterized in that, The composition contains *Lactobacillus plantarum* CCFM1366, or contains a metagene prepared from *Lactobacillus plantarum* CCFM1366; the metagene includes fermentation supernatant and / or cell lysate, wherein the fermentation supernatant is obtained by centrifugation after heat treatment of the fermentation broth of *Lactobacillus plantarum* CCFM1366; the cell lysate is obtained by high-pressure homogenization and centrifugation after heat treatment of the fermentation broth of *Lactobacillus plantarum* CCFM1366; *Lactobacillus plantarum* CCFM1366 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCCNo: 63998.

4. A pharmaceutical product comprising the composition of any one of claims 2 to 3.

5. The method for converting puerarin, daidzein, and / or genistein using *Lactobacillus plantarum* CCFM1366 as described in claim 1, characterized in that, The *Lactobacillus plantarum* CCFM1366 described in claim 1 was added to a reaction system containing puerarin, daidzein, and / or genistein for fermentation.

6. The use of the *Lactobacillus plantarum* CCFM1366 of claim 1, or the composition of any one of claims 2 to 3, or the drug of claim 4 in the preparation of a drug for relieving hangovers and protecting the liver.

Images