Pediococcus acidilactici ccfm1364 and preparation of its alcohol-protecting probiotics and application of the probiotics in transforming pueraria flavones
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2024-04-25
- Publication Date
- 2026-07-14
Smart Images
Figure CN118360201B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a strain of *Pediococcus lactis* CCFM1364 and its preparation as a post-biotic for hangover relief and liver protection, as well as the application of converted kudzu root flavonoids, belonging to the field of microbial technology. Background Technology
[0002] Alcohol-related liver disease (ALD) refers to liver damage caused by chronic and abusive alcohol, encompassing a range of liver manifestations from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Various host and environmental factors and comorbidities have been shown to alter the development and progression of ALD, including age, sex, genetic factors, drinking patterns, obesity, and chronic viral hepatitis. Despite significant progress in identifying the pathogenesis and therapeutic targets of ALD, there are currently no effective treatments other than abstinence from alcohol. Currently, traditional Chinese medicine (TCM) is being studied as a complementary and alternative therapy for ALD due to its advantages of stable supply, long-term efficacy, and minimal side effects, as well as its multi-component, multi-target, and multi-mechanism synergistic effects. An increasing number of TCM products (including medicinal herbs and phytochemicals) are also being used to treat chronic liver diseases.
[0003] Kudzu root, the dried root of *Pueraria lobata* (Willd.) Ohwi, a legume, has been used to treat alcoholic liver injury for thousands of years. Its main pharmacologically active component is isoflavones, which can alleviate liver damage through anti-oxidative stress, regulation of lipid metabolism, and inhibition of LPS intestinal leakage. Studies have reported that ethanol can promote lipogenesis and inhibit lipolysis by affecting enzyme functions such as adenosine monophosphate-activated protein kinase and peroxisomes, leading to excessive lipid deposition in hepatocytes. To investigate this, researchers established an alcoholic fatty liver disease model by exposing zebrafish larvae to a 2% ethanol solution for 32 hours, and then administered kudzu root flavonoids and puerarin to the zebrafish. The results showed that kudzu root flavonoids and puerarin significantly reduced lipid accumulation and the levels of total cholesterol (TC) and triglycerides (TG) in the zebrafish larvae.
[0004] Although numerous studies have demonstrated the preventative and therapeutic effects of traditional Chinese medicine (TCM) on alcohol-induced liver injury, particularly its significant antioxidant and anti-inflammatory properties, laying the foundation for the future development of hepatoprotective drugs, the content of most bioactive components in TCM is low, and some natural products are toxic to humans and animals. In recent years, an increasing number of studies have employed microbial fermentation of TCM to enhance the content of bioactive components. The composition and function of microorganisms play a crucial role in TCM fermentation, influencing the pharmacological activity of fermentation metabolites and final fermentation products. Probiotics are live microorganisms beneficial to human health. my country's catalog of edible probiotics includes 35 species, some of which have been used in the fermentation of TCM. However, few microorganisms have been reported to have the ability to convert and ferment puerarin. Most strains cannot utilize puerarin, and even those with the ability to convert puerarin have very low conversion rates, typically below 5%. Edible strains capable of converting puerarin are even rarer. The literature "Bioconversion of soyisoflavones daidzin and daidzein by Bifidobacterium strains" reports a Bifidobacterium strain capable of converting daidzein to daidzein with a conversion rate of approximately 48%-65% / 7 days. However, due to the low conversion rate, it is not suitable for industrial application. Currently, there is a lack of edible strains capable of converting flavonoids such as puerarin and daidzein.
[0005] Lactic acid bacteria are commonly used probiotics in the fermentation of traditional Chinese medicine (TCM). They have been used to ferment ginseng, atractylodes macrocephala, Taiwan golden thread, iron broom, salvia miltiorrhiza, and other TCM herbs, as well as in TCM compound formulas such as Xiao Chai Hu Tang, Zi Yin Jiang Huo Tang, and Huang Lian Jie Du Tang. Given the vast number of probiotics or probiotic combinations existing in nature, screening for new probiotic strains capable of utilizing TCM herbs to produce active ingredients, achieving the synergistic effect of probiotic fermentation to enrich diets with kudzu flavonoids, promoting the development of TCM fermentation, and improving the efficacy of TCM herbs has broad application prospects. Summary of the Invention
[0006] The technical problem to be solved by this invention is to provide a strain of Pediococcus acidilactici 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 Pediococcus acidilactici CCFM1364, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCC No: 63996, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0008] The Pediococcus acidilactici strain was isolated from the feces of healthy human individuals and possesses the following characteristics:
[0009] The *Pediococcus lactis* CCFM1364 cultured on MRS medium for 48 hours was round or oval, with a smooth, fine surface, a diameter of about 1.0 to 2.5 mm, and a white color.
[0010] The *Pediococcus lactis* CCFM1364 is a Gram-positive, facultatively anaerobic bacterium with an optimal growth temperature of 35–40°C and an optimal growth pH of 6.0–7.0.
[0011] The lactic acid cocci CCFM1364 can efficiently convert various puerarin flavonoids (such as puerarin and daidzein) into active substances (such as daidzein).
[0012] The present invention provides a composition containing the *Pediococcus lactis* CCFM1364, or containing a metabiotic prepared from the *Pediococcus lactis* CCFM1364.
[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 *Pediococcus lactis* CCFM1364 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 also provides a synbiotic preparation with hangover relief and liver protection effects, wherein the content of *Pediococcus lactis* CCFM1364 in the synbiotic preparation is not less than 10%. 6 CFU / mL or 10 6 CFU / g.
[0020] In one embodiment of the present invention, the synthetic preparation further contains kudzu root extract, wherein the concentration of the kudzu root extract is ≥5 mg / mL.
[0021] The present invention also provides a product containing the aforementioned *Pediococcus lactis* CCFM1364 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, medicine, or health product; or the food is a dairy product, soy product, or fruit and vegetable product produced using a fermentation agent containing the lactic acid cocci CCFM1364; or the food is a beverage or snack containing the lactic acid cocci CCFM1364 of claim 1.
[0024] The present invention also provides a method for converting kudzu flavonoids by culturing the *Pediococcus lactis* CCFM1364 in a culture medium containing kudzu extract.
[0025] In one embodiment of the present invention, the puerarin flavonoids include puerarin and daidzein.
[0026] This invention provides the application of the aforementioned Pediococcus acidilactici CCFM1364 or the aforementioned synbiotic preparation in the preparation of products for relieving hangovers and protecting the liver.
[0027] The present invention also provides food, health products, pharmaceuticals or cosmetics containing the lactic acid cocci CCFM1364 postbiotic and synbiotic preparation.
[0028] In one embodiment, the food product includes the above-described composition and conventional excipients.
[0029] In one embodiment, the health product includes the above-described composition and conventional excipients.
[0030] In one embodiment, the pharmaceutical product comprises the above-described composition, a drug carrier, and / or pharmaceutical excipients.
[0031] 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.
[0032] In one embodiment, the cosmetic comprises the above-described composition, matrix ingredients, and / or conventional excipients.
[0033] 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.
[0034] 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.
[0035] The present invention also provides the use of the aforementioned Pyrococcus lactis CCFM1365 or a preparation containing the aforementioned compound in the preparation of health products that have an auxiliary protective effect against chemically induced liver injury.
[0036] The present invention also provides the use of the aforementioned *Pediococcus lactis* CCFM1364, or the composition thereof, in the preparation of products that can promote the absorption of puerarin and / or daidzein.
[0037] This invention also provides a method for calculating the conversion rate of kudzu flavonoids.
[0038] In one embodiment of the present invention, the method includes the following steps:
[0039] (1) Pleococcus acidilactici (CCFM1364) 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.
[0040] (2) Add 5% (v / v) of the aforementioned Pyrrosia lingua 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.
[0041] (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.
[0042] 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.
[0043] In one embodiment of the present invention, the number of *Pediococcus lactis* CCFM1364 bacterial suspensions in step (2) is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.
[0044] 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.
[0045] Beneficial effects:
[0046] 1. This invention screened and obtained a strain of *Pediococcus acidilactici* CCFM1364, which has the ability to convert puerarin flavonoids: the conversion rate of puerarin is 69.39±2.60% / 48h; the conversion rate of daidzein is 39.65±3.18% / 48h; and the increase in daidzein product is 550.0±36.50% / 48h.
[0047] 2. The *Pediococcus lactis* CCFM1364 described in this invention, along with its prepared post-biotics, synergistic agents, and live bacteria, possess the ability to enhance alcohol detoxification and liver protection, and exhibit synergistic effects with kudzu root. Specifically, this is reflected in:
[0048] (1) Lower blood lipid levels (ALT, AST, TC, TG and LDL-C);
[0049] (2) Improves pathological damage to liver tissue and reduces fatty degeneration and liver damage caused by long-term alcohol consumption;
[0050] (3) Enhance the activity of antioxidant enzymes (GSH, SOD, CAT) in liver tissue;
[0051] (4) Reduce MDA content and alleviate the damage of free radicals and lipid peroxides to hepatocytes;
[0052] (5) Downregulates the mRNA expression of IL-1β, IL-6, TNF-α, and IL-10 genes in liver tissue;
[0053] (6) Upregulates the mRNA expression of Nrf2, HO-1, and COX-2 genes in liver tissue;
[0054] (7) Increase the activity of alcohol-metabolizing enzymes (ADH and ALDH), inhibit the upregulation of CYP2E1 and CYP1A2 in the liver induced by alcohol and the activation of the NF-κB pathway to prevent alcoholic liver injury.
[0055] Therefore, Pediococcus acidilactici CCFM1364 and its prepared metabiotics, synergistic preparations, and live bacteria have great application potential in the preparation of products for relieving hangovers and protecting the liver.
[0056] Preservation of biological materials
[0057] A strain of *Pediococcus acidilactici*, CCFM1364, taxonomically named *Pediococcus acidilactici*, was deposited on November 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63996), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description
[0058] Figure 1 Preliminary metabolic pathways of kudzu flavonoids.
[0059] Figure 2 HPLC results of Pietrococcus lactis CCFM1364 before and after fermentation.
[0060] Figure 3 Changes in the content of kudzu flavonoids before and after fermentation with Lactococcus lactis CCFM1364.
[0061] Figure 4 Effects of Pyrococcus lactis CCFM1364 and its prepared postbiotic and synbiotic formulations on body weight and liver index in mice with chronic alcohol exposure.
[0062] Figure 5 Effects of Pediococcus lactis CCFM1364 and its prepared postbiotics and synbiotics on liver histopathology in mice with chronic alcohol exposure.
[0063] Figure 6 Effects of Pyrococcus lactis CCFM1364 and its prepared postbiotics and synbiotics on liver function in mice with chronic alcohol exposure.
[0064] Figure 7 Effects of Pyrococcus lactis CCFM1364 and its prepared postbiotics and synbiotics on liver oxidative stress levels in mice with chronic alcohol exposure.
[0065] Figure 8 Effects of Pyrococcus lactis CCFM1364 and its prepared postbiotics and synbiotics on alcohol metabolism in mice with chronic alcohol exposure.
[0066] Figure 9 Effects of Pediococcus lactis CCFM1364 and its prepared postbiotic and synbiotic formulations on the expression levels of liver inflammatory factor mRNA in mice with chronic alcohol exposure. Detailed Implementation
[0067] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the implementation regulations.
[0068] 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), 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 (TP4030B) were purchased from Nantong Teruo. The following reagents were purchased from Fei Feed Technology Co., Ltd., and prepared according to requirements: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-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.; BCA protein concentration assay kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; reverse transcription kits and real-time fluorescence quantitative kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; primers were purchased from Shanghai Sangon Biotech Co., Ltd.
[0069] The culture media involved in the following examples are as follows:
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] Example 1: Screening, identification and preservation of strains
[0075] 1. Screening
[0076] 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.
[0077] 2. Identification
[0078] The isolated strain underwent PCR amplification of its 16S rDNA. The PCR products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The resulting PCR products were then sequenced by a biotechnology company. The obtained sequences were searched in GeneBank using BLAST and compared for similarity to obtain strain identification results. Ultimately, one strain of *Pediococcus acidilactici* was obtained and named *Pediococcus acidilactici* CCFM1364. The 16S rDNA sequence of this strain is shown in SEQ ID No. 1.
[0079] The primers used for 16S rDNA amplification are as follows:
[0080] 27F (positive): 5'-AGAGTTTGATCCTGGCCTCA-3';
[0081] 1492R (reverse): 5'-GGTTACCTTGTTACGACTT-3'.
[0082] The 16S rDNA amplification procedure is as follows:
[0083] 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.
[0084] 3. Save
[0085] Pediococcus acidilactici (CCFM1364) 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 rpm for 3 min, and the supernatant was discarded. The bacterial sludge was resuspended in 30% glycerol solution and stored at -80 °C. The Pediococcus acidilactici (CCFM1364) was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 9, 2023, with accession number GDMCC No: 63996.
[0086] Example 2: Preparation of probiotics, live bacteria, and synergistic agents from P. lactis CCFM1364
[0087] 1. Preparation of post-genetic agents
[0088] Before activation, *Pediococcus lactis* CCFM1364 was stored in 30% glycerol at -80°C. A small amount of *Pediococcus lactis* CCFM1364 culture 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 inoculated into MRS liquid medium and incubated at 37°C for 18–24 h. After thorough mixing, the culture was inoculated at a rate of 2% (v / v) into fresh MRS liquid medium and incubated under the same conditions. This process was repeated 3–5 times to obtain 9.4 × 10⁹ cells / mL. 7 CFU / mL bacterial suspension.
[0089] The bacterial suspension was heat-treated at 65℃ for 30 min, centrifuged (8000g, 4℃, 15 min), and the supernatant was collected. After lyophilization, the supernatant of *Pediococcus lactis* CCFM1364 was obtained as lyophilized powder (CCFM1364-S) for later use. Separately, the bacterial suspension was heat-treated, then homogenized under high pressure (800–1200 MPa, 3 times) to obtain bacterial cell lysates. After lyophilization, the bacterial cell lysate of *Pediococcus lactis* CCFM1364 was obtained as lyophilized powder (CCFM1364-L) for later use.
[0090] 2. Preparation of live bacteria / synthetic preparations
[0091] (1) Preparation of live Pseudococcus lactis CCFM1364
[0092] Streaking an appropriate amount of *Pediococcus lactis* CCFM1364 bacterial suspension and incubating at 37°C in an aerobic, inverted manner for 24–48 h; picking single colonies and transferring them to MRS liquid medium, incubating at 37°C for 18–24 h, then thoroughly mixing, and then inoculating the bacterial suspension at a 2% (v / v) inoculation rate into fresh MRS liquid medium and incubating under the same conditions, repeating this step 3–5 times to obtain the seed culture; inoculating the prepared seed culture into MRS medium at a 2% (v / v) inoculation rate and incubating for 18–36 h, centrifuging to collect the bacterial sludge, rinsing thoroughly with PBS buffer 3–5 times, and resuspending with lyophilization protectant to 10... 10 CFU / mL, and finally lyophilized to obtain live lyophilized powder of *Pediococcus lactis* CCFM1364 (CCFM1364-H). The content of *Pediococcus lactis* CCFM1364 in the powder is not less than 10 CFU / mL. 10 CFU / g.
[0093] The freeze-drying protectant consists of 100g / L skim milk powder, 30mL / L glycerol, 100g / L maltodextrin, and 150g / L trehalose. These freeze-drying protectant raw materials are mixed with purified water to fully dissolve them and then sterilized at 115℃ for 20 minutes.
[0094] (2) Preparation of synbiotic preparations
[0095] The synbiotic preparation (CCFM1364+PLE) is prepared by mixing the P. 1364 lactic acid cocci powder with kudzu root extract in step (1). The content of kudzu root extract in the synbiotic preparation is not less than 5 mg / mL. The content of kudzu root extract and the number of live bacteria in the synbiotic preparation are adjusted according to the gavage dose.
[0096] Example 3: Establishment of a mouse model of alcoholic liver disease (ALD)
[0097] 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 7 groups: a blank control group (Control), an alcohol model group (Model), a positive control group (Biphenyl diester administered by gavage), and a group containing *Pediococcus lactis* CCFM1364 combined with kudzu root extract (CCFM1364+PLE, administered by gavage at 100 mg / kg / d of kudzu root extract and 0.1 mL of 5×10⁻⁶ PLE). 9 CFU / mL / d live bacteria), live bacteria group (CCFM1364-H, 0.1mL orally, 5×10⁻⁶ cells / day) 9Mice were divided into three groups: live bacteria (CFU / mL / d), supernatant group (CCFM1364-S, lyophilized supernatant of 100 mg / kg / d via gavage), and lysate group (CCFM1364-L, lyophilized lysate of 100 mg / kg / d via gavage), with 6 mice in each group. The blank control group continued to be given Lieber-DeCarli liquid control diet, while the other groups of mice were given Lieber-DeCarli alcohol liquid model diet until week 8. During the feeding period from week 2 to week 8, mice were gavaged daily before 5 pm (the daily gavage dose was calculated based on the mouse's body weight), with the same gavage volume in each group. Mice were observed for 30 minutes after each gavage. After the last gavage, all mice were fasted but allowed free access to water for 12 hours, weighed, anesthetized, had their eyes enucleated to collect blood, and were euthanized by cervical dislocation. Serum and tissue samples were collected from the mice. The experimental animal groups and drug administration are shown in Table 1.
[0098] Table 1. Grouping and administration of experimental animals
[0099]
[0100]
[0101] Example 4: Transformation of Pueraria Flavonoids by Pleurotus ostreatus CCFM1364
[0102] 1. Fermentation with Pyrococcus lactis CCFM1364
[0103] The *Pediococcus lactis* CCFM1364 from Example 1 was streaked onto 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 culture was inoculated into 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.
[0104] In a fermentation medium rich in pueraria flavonoids, 5% (v / v) of the seed culture of *Pediococcus lactis* obtained in step 1 was added, and fermentation was carried out at a constant temperature of 37℃ for 0–120 h, with a bacterial concentration of 3.4 × 10⁻⁶. 9 CFU / mL.
[0105] Fermentation broths at different time points were centrifuged and the supernatant was collected. An equal volume of ethyl acetate was added for extraction. The supernatant was collected and concentrated into a dry powder using a refrigerated centrifuge at 45°C. 200 μL of methanol was added to reconstitute the powder. The mixture was then filtered through a 0.22 μm microporous organic filter membrane to obtain kudzu flavonoid fermentation product, which was stored at 4°C for later analysis.
[0106] 2. HPLC method for determining the content of puerarin flavonoids before and after fermentation.
[0107] 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.
[0108] Accurately weigh approximately 10 mg each of puerarin, daidzein, 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 under mobile phase, obtain the results, and fit the curve. Calculate the contents of puerarin, daidzein, and daidzein in the fermentation medium and pueraria flavonoid fermentation broth according to the standard curve.
[0109] 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 and daidzein in the fermentation broth of kudzu flavonoids were significantly reduced. This indicates that kudzu flavonoids can be partially converted under suitable conditions, with daidzein being one of the main metabolites. Figure 2 ).
[0110] The reduction (conversion rate) of puerarin, the substrate, and the increase of daidzein, the product, were compared before and after fermentation with *Pediococcus lactis* CCFM1364. 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 rate of puerarin in puerarin from *Pediococcus lactis* was 69.39 ± 2.60% / 48h; the conversion rate of daidzein was 39.65 ± 3.18% / 48h. After 48 hours of fermentation, the concentration of daidzein was 5.45 ± 1.60 μg / mL, with an increase of 550.0 ± 36.50% / 48h. The changes in puerarin content at different time points are shown in [reference needed]. Figure 3 The conversion ability of different strains of kudzu flavonoids is shown in Table 1.
[0111] Table 2 Summary of the conversion capacity of different lactic acid cocci for pueraria flavonoids
[0112]
[0113] Example 5: Pyrococcus lactis CCFM1364 and its prepared postbiotics and synergistic agents reduced body weight and liver index in mice.
[0114] 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 phosphate-buffered saline (PBS), 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 4 As 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 was significantly increased (P < 0.001). Compared with the Model group, the liver index of mice in the synbiotic preparation group (CCFM1364+PLE) and the bacterial supernatant group (CCFM1364-S) was significantly reduced (P < 0.01), and the liver index of the synbiotic preparation group recovered to a level comparable to that of the control group, suggesting that the post-biotic prepared from *Pediococcus lactis* CCFM1364 and the synbiotic preparation can effectively inhibit liver swelling in mice caused by chronic alcohol exposure.
[0115] Example 6: Pediococcus lactis CCFM1364 and its prepared post-biotic and synergistic preparations alleviate alcohol-induced liver pathological changes in mice.
[0116] 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 synbiotic preparation group (CCFM1364+PLE) and the bacterial lysate group (CCFM1364-L) showed basically normal liver lobules and hepatocyte cord structures, significantly reduced steatosis, and a small number of fat droplets and inflammatory cell infiltration were visible in the tissues. Hepatocyte edema was also lower than in the Model group. This indicates that both the post-biotic and synbiotic preparations made from *Pediococcus lactis* CCFM1364 can effectively alleviate alcohol-induced liver pathological changes in mice.
[0117] Example 7: Pietrococcus lactis CCFM1364 and its prepared postbiotics and live bacteria improve alcohol-induced lipid accumulation in mice.
[0118] The specific experimental setup is as described in Example 3. Serum ALT, AST, TC, TG, and LDL-C levels were measured using the corresponding detection kits according to the instructions. Results are as follows: Figure 6 As shown, compared with the Control group, the serum ALT, AST, TC, TG, and LDL-C levels in the Model group mice were significantly increased (P < 0.001), indicating that long-term alcohol intake caused lipid accumulation in the mice. Compared with the Model group mice, the serum ALT, AST, TC, TG, and LDL-C levels in mice were reduced to varying degrees after intervention with the probiotic, live bacteria, bacterial supernatant, and bacterial lysate (P < 0.05). Among them, after intervention with live *Pediococcus lactis* CCFM1364, the serum ALT, AST, TC, TG, and LDL-C levels in mice decreased by 47.61 ± 5.82%, 37.72 ± 3.71%, 30.72 ± 2.21%, 36.65 ± 5.72%, and 50.43 ± 4.41%, respectively (P < 0.001), which was similar to the degree of regulation in the positive control group. In addition, lipid accumulation in mice in the bacterial supernatant group and bacterial lysate group was also significantly alleviated. The results showed that the post-biotic and live bacteria prepared from Pleurotus ostreatus CCFM1364 had good lipid-lowering and liver-protecting effects on alcohol-induced liver damage in mice, especially the live bacteria group, which significantly reduced the degree of liver damage and fatty lesions.
[0119] Example 8: Pyrococcus lactis CCFM1364 and its prepared post-biotic and synergistic preparations inhibited the increase in alcohol-induced oxidative stress levels in mouse livers.
[0120] 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, and HO-1 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 3.
[0121] Table 3 Primer sequences for real-time quantitative PCR detection
[0122]
[0123] The results are as follows Figure 7 As shown, compared with the control group, the activities of GSH, SOD, and CAT in the liver of mice in the model group were significantly reduced (P < 0.001), indicating oxidative damage in the liver of mice in the model group. Consistent with the intervention of biphenyl diester in the positive control group, alcohol-induced GSH depletion and the reduction in SOD and CAT activities were significantly improved after intervention with CCFM1364+PLE, CCFM1364-H, CCFM1364-L, and CCFM1364-S (P < 0.01). Specifically, after CCFM1364+PLE intervention, the GSH content, SOD, and CAT levels in mouse liver increased by 224.5±22.49%, 172.6±19.46%, and 131.7±5.95%, respectively (P<0.001); after CCFM1364 live bacteria intervention, the GSH content, SOD, and CAT levels in mouse liver increased by 254.75±10.27%, 213.88±3.52%, and 137.00±2.91%, respectively. Furthermore, the Model group mice had the highest liver MDA content. Compared to the Model group, the CCFM1364+PLE group mice showed a 47.39±1.70% decrease in liver MDA content (P<0.001); the CCFM1364-H group mice showed a 55.68±4.27% decrease in liver MDA content; and the CCFM1364-L and CCFM1364-S groups mice also showed varying degrees of decrease in liver MDA content (P<0.001). This indicates that the antioxidant capacity of the liver tissue of mice treated with postbiotics, synbiotics, and live bacteria was improved.
[0124] 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 and HO-1 genes was significantly decreased (P < 0.001). Compared with the Model group, the expression levels of COX-2 gene in the liver tissue of mice in the Positive group and the CCFM1364+PLE group were significantly decreased, while the expression levels of Nrf2 and HO-1 genes were significantly increased (P < 0.001). Specifically, the expression level of COX-2 gene in the liver tissue of mice in the CCFM1364+PLE group decreased by 82.98 ± 5.28% (P < 0.001), while the expression levels of Nrf2 and HO-1 genes increased by 363.0 ± 68.53%, 193.7 ± 40.94%, and 143.2 ± 25.11%, respectively (P < 0.001), recovering to levels comparable to those in the control group. The above results indicate that the synergistic preparation of *Pediococcus lactis* CCFM1364 and kudzu root extract effectively inhibited the increase in oxidative stress levels in the liver of mice induced by long-term alcohol exposure. Furthermore, the synergistic preparation group showed better repair effects on alcohol-induced liver damage than the group treated with live *Pediococcus lactis* CCFM1364 via gavage alone.
[0125] Example 9: Regulation of alcohol metabolism in mice by *Pediococcus lactis* CCFM1364 and its prepared post-biotic and synergistic preparations.
[0126] The specific experimental setup is as described in Example 3. Ethanol metabolism mainly 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 contains many types, with CYP4502E1 being just one subclass. CYP2E1 is a metabolic pathway within this subfamily exhibiting significant polymorphism. 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 4.
[0127] Table 4 Primer sequences for real-time quantitative PCR detection
[0128]
[0129] The results are as follows Figure 8As shown, compared with the control group, the activity of ADH and ALDH in the liver tissue of the model group mice was significantly reduced (P<0.001), and the expression levels of Cyp2e1 and CYP1A2 genes were significantly increased (P<0.05). Compared with the Model group, the activities of ADH and ALDH in the liver tissue of mice in the CCFM1364+PLE group increased by 187.41±17.29% and 147.15±9.02%, respectively (P<0.001), while the expression levels of Cyp2e1 and CYP1A2 genes decreased by 46.65±4.07% and 48.48±4.18%, respectively (P<0.001). In the CCFM1364-H group, the activities of ADH and ALDH in the liver tissue of mice increased by 177.95±5.81% and 156.23±1.99%, respectively, while the expression levels of Cyp2e1 and CYP1A2 genes decreased by 35.61±8.92% and 37.95±6.47%, respectively, returning to levels comparable to the control group. The above results indicate that the synthetic preparation of *Pediococcus lactis* CCFM1364 and *Pueraria lobata* extract 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 alleviating liver damage caused by alcohol metabolites. Furthermore, compared to the Model group, the liver ADH and ALDH activities of mice in the live bacteria (CCFM1364-H) and bacterial lysate (CCFM1364-L) groups were increased to varying degrees, while the expression levels of Cyp2e1 and CYP1A2 were decreased (P < 0.05).
[0130] Example 10: Pediococcus lactis CCFM1364 and its prepared post-biotic and synergistic preparations alleviate alcohol-induced liver inflammation and damage in mice.
[0131] 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 5.
[0132] Table 5 Primer sequences for real-time quantitative PCR detection
[0133]
[0134] The results are as follows Figure 9 As shown, compared with the Control group, the mRNA expression levels of inflammatory factors TNF-α, IL-6, IL-10, and IL-1β in the liver tissue of 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 mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, IL-10, and IL-1β in the liver tissue of alcohol-treated mice were improved to varying degrees after intervention with the combined preparation, live bacteria, bacterial lysate, and bacterial supernatant (P < 0.05). Among them, the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, IL-10, and IL-1β in the liver of CCFM1364+PLE group mice decreased by 92.85±1.41%, 83.71±2.37%, 97.56±1.58%, and 62.41±8.68%, respectively, after intervention with the combined preparation (P < 0.001). The results indicate that the synergistic preparation of Pseudococcus lactis CCFM1364 and Pueraria lobata extract can significantly inhibit the expression level of inflammatory factors in liver cells induced by alcohol.
[0135] 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 Pediococcus lactis ( Pediococcus acidilactici CCFM1364 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCC No: 63996.
2. A composition containing the *Pediococcus lactis* CCFM1364 of claim 1.
3. The composition according to claim 2, characterized in that, The composition contains Pediococcus lactis CCFM1364, or contains a metagene prepared from Pediococcus lactis CCFM1364; the metagene includes fermentation supernatant, cell lysate and / or fermentation broth.
4. A synergistic preparation containing the *Pediococcus lactis* CCFM1364 as described in claim 1, characterized in that, The compound preparation also contains kudzu root extract.
5. A method for converting kudzu root flavonoids, characterized in that, The *Pediococcus lactis* CCFM1364 of claim 1 was cultured in a medium containing kudzu root extract.
6. The method according to claim 5, characterized in that, The puerarin flavonoids include puerarin and daidzein.
7. The use of the Lactococcus lactis CCFM1364 of claim 1, or the composition of claim 2 or 3, or the synbiotic preparation of claim 4 in the preparation of a drug for relieving hangovers and protecting the liver.
8. The use of the Lactococcus lactis CCFM1364 of claim 1, or the composition of claim 2 or 3, or the synbiotic preparation of claim 4 in the preparation of health products with adjuvant protective effects against chemically induced liver injury.
9. The application of the *Pediococcus lactis* CCFM1364 according to claim 1 in the preparation of fermented products, characterized in that, The products include food or medicines for treating alcoholic liver damage.