Leuconostoc mesenteroides subsp. mesenteroides and application thereof

Abstract

The application discloses a Leuconostoc mesenteroides subsp. mesenteroides strain for promoting the efficacy of puerarin and application thereof, and belongs to the technical field of microorganisms. The Leuconostoc mesenteroides subsp. mesenteroides strain CCFM1282 screened by the application can convert puerarin into various active small molecular substances containing daidzein; the cell proliferation ability and the antioxidant capacity of HepG2 alcoholic cell damage can be improved after fermentation; the ability of liver cells to cope with alcohol damage can be improved by the compound pretreatment of puerarin and the strain; and the Leuconostoc mesenteroides subsp. mesenteroides strain CCFM1282 can significantly improve the efficacy of puerarin for dispelling alcohol. The Leuconostoc mesenteroides subsp. mesenteroides strain CCFM1282 is used for converting puerarin and significantly improving the efficacy of dispelling alcohol, and has great application prospect.

Description

Technical Field

[0001] This invention relates to a strain of Leuconostoc mesenteroides that promotes the efficacy of puerarin and its application, belonging to the field of microbial technology. Background Technology

[0002] China boasts a long and rich history of alcohol culture. Throughout Chinese civilization, alcohol has permeated almost every aspect of education and social life. However, excessive drinking can lead to alcohol poisoning. Currently, Western medicine primarily treats acute alcohol poisoning with injections of 50% glucose, but lacks effective drugs for preventing and treating hangovers or for post-drinking health maintenance. Therefore, finding antidote drugs from traditional Chinese herbal medicines holds immense potential.

[0003] Kudzu root was first recorded in the Han Dynasty's *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), and has a long history of being used both as a medicine and food. Its main components include isoflavones, triterpenoids, saponins, and polysaccharides. Among these, kudzu isoflavones are the main pharmacologically active components, generally existing as free aglycones and bound glycosides. The former is mainly daidzein, while the latter is mainly puerarin (C...). 21 H 20 O9), daidzein, etc. Studies have shown that puerarin isoflavones have broad antioxidant activity and great potential in inhibiting alcohol absorption, accelerating alcohol metabolism and excretion. However, the absorption rate of glycoside isoflavones in the human body is very low; the other metabolites of isoflavones are the ones that truly exert their effects. Therefore, the complex metabolic genes of the gut microbiota are usually key to the conversion of puerarin, but the following problems still exist:

[0004] 1. Some people lack the gut microbiota that can convert puerarin, so the effects of consuming puerarin-containing foods are poor.

[0005] Second, there is a lack of bacterial strains in the food and pharmaceutical fields that can enhance the efficacy of puerarin.

[0006] Although various probiotic functions have been studied, none have been found to alleviate hepatocellular damage by converting puerarin and synergistically enhancing its effects. Therefore, exploring such strains and achieving synergistic effects between the strains and diets rich in puerarin has broad application prospects.

[0007] Currently, *Leuconostoc mesenteroides* (Gastrodia elata) obtained through isolation and screening methods are... Leuconostoc mesensteroides *Leuconostoc mesenteroides* is a plant epiphytic fungus commonly found in traditional fermented dairy products and pickles. Because it can ferment and produce lactic acid and diacetyl compounds, it is considered to make a significant contribution to the flavor of these traditional foods. In 2012, the Ministry of Health classified *Leuconostoc mesenteroides* subsp. *mesenteroides* as a subsp. *mesenteroides*. Leuconostoc mesensteroides subsp .mesenteroides The fermentation characteristics and special physiological activities of Leuconostoc mesenteroides subsp. enterica have been included in the list of microbial strains that can be used in food (Announcement No. 8 of 2012 by the Ministry of Health). Research on the fermentation characteristics and special physiological activities of Leuconostoc mesenteroides subsp. enterica has been gradually deepening.

[0008] Existing studies have confirmed that *Leuconostoc mesenteroides* subsp. *mianospermum* can ferment sugars to produce lactic acid, exhibiting high acid production capacity, antioxidant capacity, and resistance to pathogenic bacteria. Currently, *Leuconostoc mesenteroides* subsp. *mianospermum* is widely used as a flavoring agent in the food industry. Although various probiotic functions have been studied, none have been found to promote the hepatocellular damage-relieving effects of puerarin. Therefore, exploring this strain and achieving synergistic effects between the strain and diets rich in puerarin has broad application prospects. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a *Leuconostoc mesenteroides* subsp. *miano* capable of enhancing the efficacy of puerarin and its applications. This method provides a *Leuconostoc mesenteroides* subsp. *miano* that can enhance the relief of alcoholic hepatocellular damage through high-level conversion of puerarin into active small molecules, and provides an application method to improve its hangover-relieving effects. This method successfully enhances the hangover-relieving effect.

[0010] The technical solution of the present invention is as follows:

[0011] This invention provides a strain of *Leuconostoc mesenteroides* subsp. *enteroides* CCFM1282 that can enhance the hangover-relieving effect of puerarin. It was deposited on October 14, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 62884), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The taxonomic name of this strain is *Leuconostoc mesenteroides* subsp. *enteroides* (CCFM1282). Leuconostoc mesensteroides subsp .mesenteroides The strain, named CCFM1282, exhibits round, convex, or lenticular colonies after 48 hours of culture on modified MRS medium. These colonies are slightly white, opaque, and have a smooth to mucous-like soft surface. It can highly convert puerarin into daidzein and enhance its hangover-relieving effects.

[0012] The present invention also provides a composition containing the Leuconostoc mesenteroides subsp. CCFM1282.

[0013] In one embodiment, the composition is a drug.

[0014] In one embodiment, the number of *Leuconostoc mesenteroides* subsp. *CCFM1282* in the composition is ≥1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0015] In one embodiment, the composition contains Leuconostoc mesenteroides subsp. CCFM1282 and kudzu root powder.

[0016] In one embodiment, the composition is a fermentation broth from *Leuconostoc mesenteroides* subsp. *enteroides* fermented in a fermentation substrate containing puerarin.

[0017] In one embodiment, the composition is prepared by culturing *Zootrum lucidum* subsp. *enteromorphum* CCFM1282 in MRS liquid medium for a period of time, then transferring it to a fermentation substrate containing puerarin, and fermenting at 35-40°C for 24-48 hours to obtain a bacterial concentration of 8 × 10⁻⁶. 8 Cell cultures with cfu / mL.

[0018] In one embodiment, the concentration of puerarin in the fermentation substrate is ≥0.1 mg / mL.

[0019] In one embodiment, the cell culture is subjected to high-pressure homogenization.

[0020] The present invention also provides the application of the *Leuconostoc mesenteroides* subsp. *CCFM1282* in the conversion of puerarin.

[0021] In one embodiment, the application involves culturing the Leuconostoc mesenteroides subsp. CCFM1282 in a culture medium containing puerarin for a period of time.

[0022] In one embodiment, the method includes the following steps:

[0023] (1) The aforementioned Leuconostoc mesenteroides subsp. mesenteroides was streaked on MRS solid medium and the plates were 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 48 h.

[0024] (2) Add 5% (v / v) of the bacterial solution of Leuconostoc mesenteroides to the fermentation substrate rich in puerarin, and ferment at a constant temperature of 37°C for 48 hours.

[0025] In one embodiment, the concentration of puerarin in the puerarin-rich MRS liquid culture medium in step (2) is 0.1~0.8 mg / mL.

[0026] The present invention also claims protection for the use of the Leuconostoc mesenteroides subsp. mesenteroides or the composition thereof, or the use of cell cultures of the Leuconostoc mesenteroides subsp. mesenteroides in the preparation of hangover remedies.

[0027] The present invention also claims protection for the use of the *Leuconostoc mesenteroides* subsp. *mesenteroides* or a cell culture of the *Leuconostoc mesenteroides* subsp. *mesenteroides* in the preparation of health products for adjuvant protection against chemically induced liver injury.

[0028] In one embodiment, the cell culture comprises a cell culture medium of Leuconostoc mesenteroides subsp. enterica CCFM1282 cultured in a culture medium for a period of time, or a product obtained by homogenizing and disrupting the cell culture medium.

[0029] Beneficial effects:

[0030] (1) This invention provides a strain of Leuconostoc mesenteroides that can enhance the alcohol-relieving effect of puerarin and its application. It can convert puerarin into a variety of active small molecules including daidzein, and the concentration of daidzein in the metabolites after fermentation is higher than 15.91 μg / mL (0.1 mg / mL puerarin).

[0031] (2) The fermentation broth prepared by transforming Leuconostoc mesenteroides into puerarin has the ability to alleviate alcoholic liver damage, can improve the cell proliferation and antioxidant capacity of HepG2 alcohol-induced cell damage, and has a strong alcohol detoxification effect.

[0032] Preservation of biological materials

[0033] Leuconostoc mesenteroides subsp. enterica ( Leuconostoc mesensteroides subsp. mesenteroides CCFM1282, taxonomically named Leuconostoc mesensteroides subsp. mesenteroides It was deposited on October 14, 2022 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 62884. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0034] Figure 1 The percentage of MDA content in each group.

[0035] Figure 2 The cell viability ratio for each group.

[0036] Figure 3 The ratio of aspartate aminotransferase (AST) activity in each group.

[0037] Figure 4 The ratio of alanine aminotransferase (ALT) activity in each group. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the implementation regulations.

[0039] The following examples used puerarin (product number: P816259, CAS: 3681-99-0) purchased from Maclean's; herbal pueraria powder purchased from Beijing Tongrentang; daidzein (product number: SD8010, CAS: 486-66-8) purchased from Solarbio Science & Technology Co., Ltd.; DMEM culture medium and fetal bovine serum purchased from Gibco; anhydrous ethanol purchased from Sinopharm Chemical Reagent Co., Ltd.; aspartate aminotransferase (AST) kit, alanine aminotransferase (ALT) kit, and malondialdehyde (MDA) kit all purchased from Nanjing Jiancheng Co., Ltd.; acetic acid was of analytical grade, and methanol was of chromatographic grade, both purchased from Sinopharm Chemical Reagent Co., Ltd.

[0040] The culture media involved in the following examples are as follows:

[0041] MRS liquid culture 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 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.

[0042] MRS solid culture 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 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.

[0043] Fermentation substrate containing puerarin (g / L): puerarin 0.1 mg / mL, peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, diamine 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.

[0044] Example 1: Screening, identification and preservation of Leuconostoc mesenteroides subsp. mesenteroides

[0045] 1. Screening

[0046] Using pickled vegetable samples from Chengdu, Sichuan as the sample, sterile physiological saline was used for 10-fold serial dilution to 10-1. -6Then take 100 μL of each diluted by a factor of 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, and the color of each colony was observed.

[0047] 2. Identification

[0048] The isolated strain was subjected to PCR amplification of 16S rDNA. The PCR product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the nucleic acid sequence in NCBI. Finally, a strain of Leuconostoc mesensteroides subsp. meseneroides was obtained and named Leuconostoc mesensteroides subsp. meseneroides CCFM1282.

[0049] 3. Save

[0050] Leuconostoc mesensteroides subsp. mesenteroides CCFM1282 was inoculated into 5 mL of MRS liquid medium and cultured at 37 °C for 24 h. 1 mL of the bacterial suspension was then transferred to a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the supernatant was discarded. The bacterial sludge was resuspended in 30% glycerol solution and stored at -80 °C.

[0051] Example 2: High-efficiency conversion of Leuconostoc mesenteroides subsp. mesenteroides into puerarin

[0052] 1. The enteromembranous subsp. enterospermum of Leuconostoc mesenteroides CCFM1282 from Example 1 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 48 h.

[0053] 2. Add 5% (v / v) of *Leuconostoc mesenteroides* subsp. *enteroides* bacterial suspension to the fermentation substrate containing puerarin, so that the bacterial concentration after inoculation reaches 1×10⁻⁶. 7CFU / mL, fermented at 37℃ for 48 hours to achieve a cell concentration of 1×10⁻⁶ after fermentation. 9 CFU / mL.

[0054] 3. After fermentation, the fermentation broth was extracted with an equal volume of ethyl acetate. The supernatant was then subjected to rotary distillation at 50°C in a fume hood and finally evaporated to dryness in a water bath at 55°C to obtain puerarin fermentation product, which was then stored at 4°C.

[0055] 4. HPLC analysis of the puerarin fermentation product revealed that puerarin was no longer detectable. This indicates that puerarin can be completely converted under suitable conditions, but daidzein is only one of the metabolites.

[0056] Example 3: HPLC determination of daidzein content after puerarin fermentation

[0057] 1. Chromatographic conditions were as follows: Column: Waters HSS Peptide T3; Flow rate: 1.0 mL / min; Detection wavelength: 250 nm; Injection volume: 5 μL; Mobile phase: Methanol (A) - 0.2% acetic acid water (B); Column temperature: 35°C. Elution was performed according to the following gradient: 0–10 min, 0–30% A; 10–30 min, 30–70% A; 30–35 min, 70–0% A.

[0058] 2. Accurately weigh approximately 10 mg each of puerarin and daidzein reference standards into 100 mL volumetric flasks, dilute to volume with analytical grade ethanol solution, 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 10.0 g / L. g / mL, 25.0 g / mL, 50.0 g / mL, 75.0 g / mL, 100.0 Prepare a 2 mL solution with a concentration of g / mL. Plot a working curve under the mobile phase, obtain the results, and fit the curve.

[0059] 3. When all the raw materials have fermented, the concentration of the sample solution is 0.1 mg / mL, and the standard concentration is 10. The concentration of the analyte was calculated by using g / mL as the internal reference concentration.

[0060] 4. The sample to be tested is diluted from 100... Dilute to 10 g / ml After obtaining g / ml, take 10 mL of puerarin fermentation product (the sample to be tested) and use 0.45... After filtration through a membrane, the solution was diluted to 100 mL with analytical grade ethanol. The reference solution and the sample solution were injected, and the chromatographic conditions described above were followed. The daidzein content in the fermentation product was calculated using the external standard method. The result showed that the daidzein concentration in the fermentation broth of *Leuconostoc mesenteroides* subsp. *enteroides* was 15.91 mg / L. g / mL, the cell concentration in the fermentation broth is 1×10 9 CFU / mL.

[0061] Example 4: Verification of the hangover-relieving effect of the combination of strain and puerarin using HepG2 liver cancer cells.

[0062] Preparation of the fermentation broth of *Leuconostoc mesenteroides* subsp. *enteroides* CCFM1282 and puerarin: The activated *Leuconostoc mesenteroides* subsp. *enteroides* CCFM1282 from Example 1 was inoculated into MRS liquid medium and anaerobically cultured at 37℃ for 24 h. A 5% (v / v) inoculum was then added to a fermentation substrate containing puerarin, and fermented at 37℃ for 48 h, yielding a bacterial concentration of 8 × 10⁻⁶. 8 The concentration of cfu / mL was subjected to high-pressure homogenization and centrifugation at 8000 r / min for 20 min. The supernatant was collected and the pH was adjusted to 7.2-7.4. The mixture was then filtered through a 0.22 μm disposable filter for sterilization, aliquoted, and stored at -20℃ to obtain the combined fermentation broth of Enterococcus enterica subsp. enterica CCFM1282 and puerarin.

[0063] Verify the hangover relief effect by following these steps:

[0064] 1. HepG2 cells were cultured in DMEM containing 5% fetal bovine serum at a ratio of 1:3 in a 37°C, 5% CO2 incubator. After repeated passage and centrifugation, 1 ml of culture medium was added to prepare a cell suspension. 100 μL of the cell suspension was transferred to a small centrifuge tube, and 100 μL of culture medium was added simultaneously. Pour in 0.4% trypan blue solution and mix thoroughly. Take approximately 10 μL of the solution. Add the L mixture droplet to one side of the coverslip, let it stand for a period of time, and use an inverted microscope to count the total number of blue cells in the four squares.

[0065] 2. With 10 5 Cells were seeded at a density of [number] cells / mL into 24-well plates, with 2 mL of cell suspension added to each well. Anhydrous ethanol at five concentration gradients (0, 250, 500, 750, 1000, and 1250 mM) was added to each well, with four replicates per concentration. After 24 h of incubation, 25 mL of CCK-8 solution was added to each well. L, incubated for 1 h, absorbance (A value) was measured at 450 nm, and cell proliferation activity was calculated.

[0066] 3. A hepatocyte injury model was established using 750 mM ethanol. After cell counting of the cell suspension, culture medium was added to dilute the suspension to a final cell density of 102. 5 Cells / mL, using a 96-well plate, add 200 mL to each well. L cell suspension was cultured for 6 hours and then the well plate was removed.

[0067] 4. The experiment was conducted in three groups: an experimental group, a model group, and a control group. In the experimental group, 9g of [unspecified substance] was added to each well. L of anhydrous ethanol, according to four concentration gradients (based on the concentration of the composite fermentation broth, 10 L... g / mL, 20 g / mL, 40 g / mL, 80 Add (g / mL) the fermentation broth of *Zootrum mucosa* subsp. *enteroides* CCFM1282 and puerarin; the model group only added 9 g / mL. The control group received anhydrous ethanol and cell suspension; the control group received only cell suspension. The 96-well plates were returned to the incubator and cultured for another 12 hours.

[0068] 5. Cell proliferation activity was determined using the CCK-8 assay. Experiments were conducted according to the operating instructions of the malondialdehyde (MDA), aspartate aminotransferase (AST / GOT), and alanine aminotransferase (ALT / GPT) test kits. The final values ​​of MDA, GOT, and GPT were obtained. Results are as follows: Figures 1 - 4 As shown, the combined fermentation of *Zootrum lucidum* subsp. *enteroides* CCFM1282 and puerarin (CCFM1282 metabolized to puerarin group in the figure) significantly improved cell proliferation activity by more than 12% compared to the model group at different concentration gradients; reduced intracellular MDA content by more than 40% compared to the model group at different concentration gradients; reduced AST activity by more than 15% compared to the model group at different concentration gradients; reduced ALT activity by more than 15% compared to the model group at different concentration gradients; enhanced antioxidant effects; and further improved hangover relief efficacy.

[0069] Example 5: Experimental verification of the effect of combining strain with herbal kudzu root powder on enhancing hangover relief using HepG2 liver cancer cells.

[0070] Preparation of a compound solution of *Leuconostoc mesenteroides* subsp. *enteroides* CCFM1282 and kudzu root powder (CCFM1282 compound kudzu root powder): The activated *Leuconostoc mesenteroides* subsp. *enteroides* CCFM1282 from Example 1 was inoculated into MRS liquid medium and anaerobically cultured at 37℃ for 24 h. The bacterial sludge was collected by centrifugation at 8000 r / min for 20 min, then freeze-dried under vacuum to obtain powder (ensuring the bacterial activity of the powder was 1×10⁻⁶). 11The CFU / g of the herbal kudzu root powder was mixed with an equal proportion and dissolved in sterile water to make the concentration of the herbal kudzu root powder 0.1 mg / ml and the bacterial concentration 8 × 10⁻⁶. 8 The concentration of CFU / mL was adjusted to pH 7.2-7.4; after aliquoting, the solution was stored at -20℃ to obtain a compound solution of Enterococcus membranaceus subsp. enterica CCFM1282 and herbal kudzu root powder.

[0071] Verify the hangover relief effect by following these steps:

[0072] The specific implementation method is the same as in Example 4, except that the experiment is divided into an experimental group, a model group, and a blank group. 9g of [unspecified substance] was added to each well in the experimental group. L of anhydrous ethanol was added to a compound solution of *Zootrum lucidum* subsp. *enteroides* CCFM1282 and *Pueraria lobata* powder at four concentration gradients, so that the final concentration of the compound solution in each well was 10. g / mL, 20 g / mL, 40 g / mL, 80 g / mL; only 9 g / mL was added to the model group. The control group received anhydrous ethanol and cell suspension; the control group received only cell suspension. The 96-well plates were returned to the incubator and cultured for another 12 hours.

[0073] Cell proliferation activity was determined using the CCK-8 assay. Experiments were conducted according to the operating instructions of the malondialdehyde (MDA), aspartate aminotransferase (AST / GOT), and alanine aminotransferase (ALT / GPT) test kits. The final values ​​of MDA, GOT, and GPT were obtained. Results are as follows: Figures 1 - 4 As shown, the combined fermentation of *Zootrum lucidum* subsp. *enteroides* CCFM1282 and puerarin significantly improved cell proliferation activity, increasing it by more than 12% compared to the model group at different concentration gradients; it reduced intracellular MDA content, decreasing it by more than 40% compared to the model group at different concentration gradients; it reduced the aspartate aminotransferase (AST) activity ratio, decreasing it by more than 15% compared to the model group at different concentration gradients; it reduced the alanine aminotransferase (ALT) activity ratio, decreasing it by more than 15% compared to the model group at different concentration gradients; it enhanced antioxidant effects and further improved hangover relief efficacy.

[0074] Comparative Example 1: HepG2 cell experiments verify the hangover-relieving effect of puerarin alone.

[0075] The specific implementation method is the same as in Example 4, except that the experiment is divided into an experimental group, a model group, and a blank group. 9g of [unspecified substance] was added to each well in the experimental group. L of anhydrous ethanol, based on the final concentration, add 10 g / mL, 20 g / mL, 40 g / mL, 80 g / mL single puerarin; the model group only added 9 g / mL puerarin. The control group received anhydrous ethanol and cell suspension; the control group received only cell suspension. The 96-well plates were returned to the incubator and cultured for another 12 hours.

[0076] Cell proliferation activity was determined using the CCK-8 assay. Experiments were conducted according to the operating instructions of the malondialdehyde (MDA), aspartate aminotransferase (AST / GOT), and alanine aminotransferase (ALT / GPT) test kits. The final values ​​of MDA, GOT, and GPT were obtained. Results are as follows: Figures 1 - 4 As shown, although puerarin alone can improve cell proliferation, reduce intracellular MDA content, aspartate aminotransferase and alanine aminotransferase content, and enhance antioxidant effects, its effect is far lower than that of the fermentation transformation product of Leuconostoc mesenteroides subsp. enterica CCFM1282.

[0077] Comparative Example 2: HepG2 cell experiments verify the alcohol-relieving efficacy of a single strain.

[0078] The specific implementation method is the same as in Example 4, except that *Zootrum lucidum* subsp. *enteromorphum* CCFM1282 is inoculated into MRS liquid medium and cultured anaerobically at 37°C until the bacterial concentration reaches 8 × 10⁻⁶. 8 The sample was homogenized under high pressure at cfu / mL, centrifuged at 8000 rpm for 20 min, and the supernatant was collected. The pH was adjusted to 7.2–7.4. The mixture was then filtered through a 0.22 μm disposable filter for sterilization, aliquoted, and stored at -20°C to obtain the autofermentation lysate of *Zootrum mucosae* subsp. *enterostomum* CCFM1282. The experiment was conducted in three groups: experimental group, model group, and control group. For the experimental group, 9 μL of cfu / mL ... L of anhydrous ethanol was added to the self-fermentation lysis broth at four concentration gradients to achieve a concentration of 10 L / well in each well. g / mL, 20 g / mL, 40 g / mL, 80 g / mL; only 9 g / mL was added to the model group. The control group received anhydrous ethanol and cell suspension; the control group received only cell suspension. The 96-well plates were returned to the incubator and cultured for another 12 hours.

[0079] Cell proliferation activity was determined using the CCK-8 assay. Experiments were conducted according to the operating instructions of the malondialdehyde (MDA), aspartate aminotransferase (AST / GOT), and alanine aminotransferase (ALT / GPT) test kits. The final values ​​of MDA, GOT, and GPT were obtained. Results are as follows: Figures 1 - 4 As shown, although a single strain can improve cell proliferation, reduce intracellular MDA content, aspartate aminotransferase and alanine aminotransferase content, and enhance antioxidant effects, the effect is far lower than that of the fermentation transformation product of Leuconostoc mesenteroides subsp. enterica CCFM1282.

[0080] Comparative Example 3: HepG2 cell experiments verify the hangover-relieving effects of other probiotic fermentation processes.

[0081] The specific implementation method is the same as in Example 4, except that CCFM1282 is replaced with Lactobacillus plantarum with accession number CGMCCNo.6077. Lactobacillus plantarum Bifidobacterium animalis (CCFM8610) (described in the text of patent application publication number CN102586148 A), or accession number GDMCC No: 61495. Bifidobacterium animalis CCFM1155 (described in the patent text with publication number CN 113088473 A).

[0082] Cell proliferation activity was determined using the CCK-8 assay. Experiments were conducted according to the operating instructions of the malondialdehyde (MDA), aspartate aminotransferase (AST / GOT), and alanine aminotransferase (ALT / GPT) test kits. The final values ​​of MDA, GOT, and GPT were obtained. Results are as follows: Figures 1 - 4 As shown: Lactobacillus plantarum ( Lactobacillus plantarum CCFM8610 and Bifidobacterium animalis ( Bifidobacterium animalis Although the combined fermentation of CCFM1155 and puerarin can improve cell proliferation, reduce intracellular MDA content, aspartate aminotransferase and alanine aminotransferase content, and enhance antioxidant effects, the effect is far lower than that of the fermentation transformation product of Leuconostoc mesenteroides subsp. enterica CCFM1282.

[0083] Comparative Example 4: Other fermentation conversion technologies for puerarin and their effects in the prior art

[0084] 1. The article "Recent Advances in Methods of Puerarin Biotransformation" published in the journal "Mini Reviews in Medicinal Chemistry" mentions relatively few studies on the microbial transformation and fermentation of puerarin.

[0085] 2. Although the strain described in the article "Biotransformation of C-Glucosylisoflavone Puerarin to Estrogenic (3S)-Equolin Co-culture of Two Human Intestinal Bacteria" published in the journal "BIOLOGICAL & PHARMACEUTICAL BULLETIN" can completely transform puerarin, the strain is inedible and difficult to cultivate, so it does not have any other additional value.

[0086] 3. In the article "Bioconversion of Isoflavones during the Fermentation of Samso-Eum with Lactobacillus Strains" published in the journal "Biotechnology and Bioprocess Engineering", most strains were unable to convert puerarin, and even those strains that were able to convert had a very low conversion rate of only 5%.

[0087] 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 Leuconostoc mesenteroides subsp. enterica ( Leuconostoc mesensteroides subsp .mesenteroides CCFM1282 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 14, 2022, with accession number GDMCC No.62884.

2. A composition comprising Leuconostoc mesenteroides subsp. CCFM1282 as described in claim 1 and puerarin.

3. The composition according to claim 2, characterized in that, The composition is a drug.

4. The composition according to claim 2 or 3, characterized in that, The number of Leuconostoc mesenteroides subsp. CCFM1282 was ≥1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

5. The composition according to claim 2, characterized in that, The composition is the fermentation broth of Leuconostoc mesenteroides subsp. enterica CCFM1282 fermented in a fermentation substrate containing puerarin.

6. The use of Leuconostoc mesenteroides subsp. CCFM1282 as described in claim 1 in the conversion of puerarin to daidzein.

7. The use of the composition according to any one of claims 2 to 5 in the preparation of a hangover remedy.

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