Bacillus coagulans and its application in sobering up, nourishing the stomach and protecting the liver

Abstract

The present invention discloses Bacillus coagulans and its application in sobering up, nourishing the stomach and protecting the liver, and relates to the technical field of microorganisms. Bacillus coagulans ), the deposit number of the Bacillus coagulans is CGMCC No. 25453. The present invention also discloses the use of the above-mentioned Bacillus coagulans in the preparation of products for preventing, treating or assisting in the treatment of alcohol poisoning. The Bacillus coagulans can significantly reduce the drunkenness rate and prolong the tolerance time by regulating the activity of alcohol metabolic enzymes in the liver, can effectively improve liver tissue cell damage and the changes in body weight and liver index caused by it, improve the expression of liver damage-related indicators and inflammation-related genes in serum, reduce alcohol-induced oxidative stress damage, can effectively prevent gastric mucosal damage caused by alcohol, and has good application prospects in the preparation of products for protecting against alcohol poisoning.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus coagulans and application thereof in sobering up, nourishing the stomach and protecting the liver. Background Art

[0002] Acute alcohol poisoning (AAI) is a central nervous system dysfunction phenomenon caused by the body's intake of large amounts of ethanol or ethanol-containing beverages in a short period of time. It is often manifested as abnormal behavior and consciousness, and often leads to dysfunction of multiple organs and systems, among which liver and stomach damage are particularly common. After alcohol is ingested by the human body, it is mainly metabolized in the liver, converted into acetaldehyde by the liver enzyme system, oxidized into acetic acid by the action of acetaldehyde dehydrogenase, and finally retained in the blood to participate in the tricarboxylic acid cycle to generate carbon dioxide and water. If it exceeds the metabolic capacity of the liver, excessive acetaldehyde will cause damage to multiple systems throughout the body.

[0003] There are many kinds of products for hangover relief, including Western medicine and Chinese herbal medicine, covering a wide range. The Western medicine naloxone is a drug used clinically to treat patients with acute alcohol poisoning. Naloxone is an opioid receptor antagonist. It stimulates the sympathetic adrenal medulla, improves the metabolism of adenosine triphosphate (ATP), stabilizes the cell membrane, and relieves the inhibition of circulation and respiration by β-endorphins, thereby increasing cardiopulmonary blood volume, stimulating the myocardium, and improving respiration. It can play a role in rapid awakening and restoring the normal function of the central nervous system. However, its therapeutic effect is mostly to relieve symptoms such as discomfort after drinking, and its effect on improving liver and stomach damage is not good. There are many types of Chinese herbal medicines with the function of sobering up, including Pueraria root, Pueraria flower, and Hovenia dulcis, but their taste is poor, and long-term use may have toxic side effects.

[0004] Bacillus coagulans ( Bacillus coagulans ) belongs to the genus Bacillus of the Firmicutes phylum and is one of the safer probiotics currently being studied and applied to animals and humans. Chinese patent CN117603868A discloses a strain of Bacillus coagulans that can be used to sober up, reduce uric acid, and improve intestinal flora. The Bacillus coagulans BC-Y09 of the invention has excellent alcohol tolerance and the ability to degrade ethanol and acetaldehyde; acid and bile salt resistance; efficient uric acid degradation and inhibition of uric acid production enzyme activity; the ability to inhibit pathogens, promote the growth of probiotics, and improve intestinal flora. The Bacillus coagulans BC-Y09 of the invention can be used to prepare products for sobering up and reducing uric acid.

[0005] A probiotic fermented functional food for sobering up and nourishing the liver and a preparation method thereof are disclosed in Chinese patent CN110521784A, wherein the probiotic fermented functional food is mixed in proportion after soybean, turmeric and Hovenia dulcis powder making, and is obtained by segmented solid-state fermentation using Bacillus coagulans, Pediococcus acidilactici, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus paracasei and Bacillus subtilis. While effectively improving the content of each probiotic in the product, the content and type of beneficial substances in the product can be improved, and some macromolecular substances can be converted into small molecular substances, which is beneficial to the absorption of human body, and the effect of gain functional food in sobering up and nourishing the liver, and it is made to have the effect of regulating intestinal health. In addition, the probiotic fermentation in the product can produce a large amount of lactic acid, which can effectively inhibit the growth of corrupt microorganisms, and is helpful to improve the shelf life of the product, without the need to add preservatives, and the quality of the product itself is not affected.

[0006] Currently, there are few studies on the use of Bacillus coagulans in the prevention and treatment of alcohol poisoning, and there is a need to provide a probiotic that can effectively alleviate alcohol poisoning. Summary of the invention

[0007] The invention aims to provide bacillus coagulans and application thereof in sobering up, nourishing the stomach and protecting the liver.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a Bacillus coagulans ( Bacillus coagulans ), the deposit number of the Bacillus coagulans is CGMCC No. 25453.

[0010] Specifically, the colony characteristics of the Bacillus coagulans on TSB culture medium are: the colony is light yellow, round, moist, opaque, and has neat edges. Microscopic examination shows that the bacteria are rod-shaped, 0.6-0.8 μm×1.7-2.5 μm, arranged individually or in groups, Gram-positive, the spores are oval, proximally grown, and the cysts are not swollen.

[0011] Specifically, the present invention provides a method for culturing Bacillus coagulans, which comprises inoculating Bacillus coagulans into a culture medium for culturing.

[0012] Furthermore, the culture medium includes but is not limited to MRS medium, TSB medium, M17 medium, PDA medium, LB medium, TB medium, SOB medium, TPY medium, improved TJA medium, improved MC medium, and YCFA medium.

[0013] Furthermore, the culture medium is MRS culture medium.

[0014] In the present invention, the Bacillus coagulans BC100 is the Bacillus coagulans with a deposit number of CGMCC No. 25453.

[0015] In another aspect, the present invention provides a preparation comprising the Bacillus coagulans, wherein the preparation is selected from one or more of the fermentation broth of Bacillus coagulans, the precipitate of the fermentation broth of Bacillus coagulans, the supernatant of the fermentation broth of Bacillus coagulans, the live bacteria of Bacillus coagulans, the dead bacteria of Bacillus coagulans, and the lyophilized powder of Bacillus coagulans.

[0016] Specifically, the fermentation liquid refers to the liquid obtained by inoculating the bacteria into the culture medium and culturing the culture medium.

[0017] More specifically, the fermentation broth supernatant refers to the clarified liquid on the upper layer of the fermentation broth after centrifugation; it contains abundant metabolites and some bacterial fragments during the bacterial growth and reproduction process.

[0018] More specifically, the fermentation liquid precipitation refers to the liquid precipitation after centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix.

[0019] More specifically, the dead bacteria refer to microorganisms that have lost their vitality and are unable to grow and reproduce, and the probiotics lose their vitality due to the growth process.

[0020] In yet another aspect, the present invention provides a bacterial agent, comprising the above-mentioned Bacillus coagulans or the above-mentioned preparation.

[0021] Specifically, the bacterial agent is a powder or a liquid preparation.

[0022] Specifically, the bacterial agent can be solid, liquid, semi-solid or any physical form that can be obtained by those skilled in the art through current or future technologies and contains the necessary active ingredients to achieve any application, function or effect described in the present invention.

[0023] In some embodiments, the bacterial agent is in solid form, and can be freeze-dried powder.

[0024] Specifically, the preparation method of the lyophilized powder can adopt the method currently conventionally used in the art, or other methods that may appear in the future to obtain a solid form product of a microorganism or microbial culture. It should be clarified that the preparation method of the lyophilized powder should not become a condition limiting the scope of protection of the present invention.

[0025] In another aspect, the present invention provides the use of the above-mentioned Bacillus coagulans or the above-mentioned preparation or the above-mentioned bacterial agent in the preparation of a product for preventing, treating or assisting in the treatment of alcoholism.

[0026] Specifically, the products include but are not limited to medicines, foods and health foods.

[0027] More specifically, the food includes but is not limited to compressed candies, yogurt, canned food, biscuits, chocolate, cakes, cream, cheese, sour cream, milk powder, ice cream, popsicles, jam, puree, candied fruit, preserved fruit, bread, egg rolls, protein drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee, and puffed food.

[0028] More specifically, the food may further include auxiliary materials acceptable in food.

[0029] More specifically, the acceptable excipients in the food include, but are not limited to, sweeteners, flavoring agents, colorants, stabilizers, glidants, fillers, surfactants, carbohydrates, antioxidants, chelating agents, preservatives, carriers, wetting agents, isotonic agents, solvents, and emulsifiers.

[0030] Preferably, the health food contains 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g of Bacillus coagulans.

[0031] Further preferably, the health food further comprises conventional supplementary materials for health food, and the supplementary materials include but are not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids and nutritional enhancers.

[0032] More preferably, the health food includes emulsion products, solution products, powder products and solid products.

[0033] Specifically, the number of viable bacteria of Bacillus coagulans in the product is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g, such as 1×10 3 CFU / mL (CFU / g), 1×10 4 CFU / mL (CFU / g), 1×10 5 CFU / mL (CFU / g), 1×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 1×10 10CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), 1×10 13 CFU / mL (CFU / g), 1×10 14 CFU / mL (CFU / g), etc. Other point values ​​within this numerical range can be selected.

[0034] Furthermore, the viable count of Bacillus coagulans in the product is 2×10 8 -2×10 9 CFU / mL or 2×10 8 -2×10 9 CFU / g.

[0035] Furthermore, the viable count of Bacillus coagulans in the product is 2×10 9 CFU / mL or 2×10 9 CFU / g.

[0036] Specifically, when the product is a medicine, the medicine may further include a pharmaceutically acceptable carrier.

[0037] Further preferably, the pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

[0038] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose and mannitol.

[0039] Specifically, the stabilizer is selected from at least one of agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0040] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0041] Specifically, the binder is selected from at least one of ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinyl pyrrolidone.

[0042] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0043] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0044] Specifically, the dosage form of the drug is drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, solutions, pellets, pills, buccal tablets, lyophilized powder injections, gels, suppositories or aerosols.

[0045] In another aspect, the present invention provides the use of the above-mentioned Bacillus coagulans or the above-mentioned preparation or the above-mentioned bacterial agent in the preparation of a product for preventing, treating or assisting in the treatment of alcoholic liver injury.

[0046] In yet another aspect, the present invention provides a product for preventing, treating or assisting in the treatment of alcoholism, wherein the product comprises the above-mentioned Bacillus coagulans or the above-mentioned preparation or the above-mentioned bacterial agent.

[0047] Specifically, the number of viable bacteria of Bacillus coagulans in the product is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g.

[0048] Furthermore, the viable count of Bacillus coagulans in the product is 2×10 8 -2×10 9 CFU / mL or 2×10 8 -2×10 9 CFU / g.

[0049] Specifically, the products include but are not limited to medicines, foods and health foods.

[0050] The beneficial effects of the present invention are:

[0051] The invention discloses the application of Bacillus coagulans in preparing a product for treating alcohol poisoning. It is found through experiments that when Bacillus coagulans BC100 is applied to acute alcohol intoxication (AAI) mice, BC100 pretreatment can significantly reduce the drunkenness rate of AAI mice, prolong the tolerance time, especially shorten the sober time, by regulating the activity of alcohol metabolic enzymes in the liver, and can effectively improve the liver tissue cell damage of AAI mice and the changes in body weight and liver index caused by it, and improve the expression of liver damage-related indicators and inflammation-related genes in serum; by regulating the antioxidant enzyme indicators in the liver, the oxidative stress damage induced by alcohol is reduced; and the gastric mucosal damage caused by alcohol can be effectively prevented.

[0052] Collection Instructions

[0053] Strain name: BC100;

[0054] Deposit number: CGMCC No. 25453;

[0055] Classification name: Bacillus coagulans Bacillus coagulans ;

[0056] Deposit time: August 1, 2022;

[0057] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0058] Abbreviation of depository unit: CGMCC;

[0059] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The tolerance time and intoxication time of each group of mice after intragastric administration of alcohol. Different letters represent significant differences.

[0061] Figure 2 is the alcohol content in the serum of mice in each group. Different letters represent significant differences.

[0062] Figure 3 Are the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of mice in each group. Different letters represent significant differences.

[0063] Figure 4 are the activities of superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) in the serum and liver of mice in each group. Different letters represent significant differences.

[0064] Figure 5 are the serum liver function indicators of mice in each group, and different letters represent significant differences.

[0065] Figure 6 HE staining of liver and stomach tissues of mice in each group.

[0066] Figure 7 A in the figure is the thickness of gastric mucosa layer of mice in each group. Figure 7 B in the figure represents the thickness of the gastric wall muscle layer, and different letters represent significant differences.

[0067] Figure 8 The effect of BC100 on the mRNA levels of TNF-α, IL-1β, Nrf-2, and NF-κB inflammatory genes in mouse liver. Different letters represent significant differences. DETAILED DESCRIPTION

[0068] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0069] Basic Example

[0070] Isolation, purification and identification of Bacillus coagulans BC100:

[0071] Culture medium: MRS liquid and solid culture medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), MC liquid and solid culture medium (purchased from Best Biotechnology Co., Ltd.), BHI liquid and solid culture medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.).

[0072] The isolated samples were collected from watermelon soybeans in Fuyang City, Anhui Province. 10g soybeans were added to a triangular flask. After shaking for 30 minutes, 1mL was drawn into 9mL dilution to obtain a 10-1 dilution. The dilution was graded and diluted 6 times in sequence, and then inoculated on MRS / MC / BHI agar medium in sequence, and then placed at 37℃ for anaerobic inversion culture for 72h. The plate was taken out after culture, and the colony morphology on the solid culture medium was observed, including shape, color, size, surface, edge, ridge, transparency, etc. Single bacteria with different colony morphology were selected for partition and streaking, and cultured at 37℃ for 48h. The bacterial colonies were examined under a microscope and photographed. Use a sterile toothpick to pick the purified colony and connect it to a liquid test tube, and place it at 37℃ for anaerobic culture for 48h. Take 1.6mL of the liquid after 48h of culture and dispense it into a glycerol tube, mark it and mix it evenly, and store it at -80℃. The bacterial genome was extracted using a bacterial genome extraction kit, and the 16s rRNA was amplified using 27F / 1492R primers and sent to Sangon Biotech Co., Ltd. for sequencing. After sequencing, it was compared with the NCBI database and identified as Bacillus coagulans.

[0073] The sequence is as follows (SEQ ID NO: 11):

[0074]

[0075] Example 1

[0076] 1 Materials and methods

[0077] 1.1 Materials and Equipment

[0078] Lactic acid bacteria culture medium (MRS culture medium, purchased from Beijing Solebow Technology Co., Ltd., Cat. No. M8540); primers (Nanjing Jiancheng Bioengineering Institute); RNA extraction kit (Beijing Solebow Technology Co., Ltd., Cat. No. 50preps-DP451); cDNA reverse transcription kit, purchased from Thermo Fisher Scientific (Cat. No. K1622); real-time fluorescence quantitative RT-PCR reagents (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., Cat. No. 11201ES08); all ELISA kits and biochemical kits were purchased from Beijing Solebow Technology Co., Ltd., alcohol dehydrogenase (ADH) detection kit (Cat. No. BC1085), acetaldehyde dehydrogenase (ALDH) detection kit (Cat. No. BC0755) ), superoxide dismutase (SOD) detection kit (catalog number BC5165), malondialdehyde (MDA) detection kit (catalog number BC0025), reduced glutathione (GSH) detection kit (catalog number BC1175), glutathione peroxidase (GSH-Px) detection kit (catalog number BC1195); alanine aminotransferase (ALT) detection kit (catalog number BC1555), aspartate aminotransferase (AST) detection kit (catalog number BC1565), γ-glutamyltransferase (GGT) detection kit (catalog number BC1225); Metadoxine was purchased from Roen Biotechnology Co., Ltd. (catalog number R058009-10g); other common reagents were of analytical grade.

[0079] Multifunctional microplate reader (purchased from Thermo Fisher Scientific, Thermo); micro-spectrophotometer (Nano-300, Hangzhou Aosheng Instrument Co., Ltd.); syringe (BD); fluorescence quantitative PCR instrument (SepOne Plus, purchased from Thermo Fisher Scientific, Thermo); upright optical microscope (BX43, Olympus Corporation, Japan); centrifuge (Baiyang Medical Instrument Co., Ltd., BY-160C); biological sample homogenizer (Bioprep-24, Hangzhou Aosheng Instrument Co., Ltd.); constant temperature incubator (BI-150A, Shidukai Instrument Equipment (Shanghai) Co., Ltd.); clean bench (SW-CJ-2FD, Suzhou Antai Air Technology Co., Ltd.); high-pressure steam sterilizer (LDZM-80KCS-II, Shanghai Shen'an Medical Instrument Factory).

[0080] 1.2 Preparation of bacterial culture

[0081] Bacillus coagulans BC100 was obtained from Jiangsu Shan En Kang. The bacteria were activated using MRS medium and cultured at 37°C under anaerobic conditions for 48 h until the bacterial logarithmic growth phase. The number of colony forming units (CFU) was standardized by measuring the optical density (wavelength of 600 nm). After centrifugation, the number of colony forming units (CFU) was calculated according to 2×10 9 CFU / kg and 2×10 8 CFU / kg, and prepare bacterial suspension with PBS for later use.

[0082] 2 Experimental methods

[0083] 2.1 Animal grouping

[0084] Thirty SPF C57 mice were selected, male, aged 8-10 weeks, and weighing 30-35 g. The experimental protocol was approved by the Ethics Committee of the Collaborative Innovation Center for Child Nutrition and Healthy Development of Chongqing Second Normal University (approval number: 2023032702B), and the operations were carried out in accordance with the requirements of the Experimental Animal Ethics Committee. The experiment was divided into 5 groups, with 6 mice in each group: normal control group (NC group), acute alcohol intoxication model group (AAI group), gavage Metadoxine positive control group (Metadoxine group), gavage high concentration Bacillus coagulans BC100 experimental group (BC-H group), and gavage low concentration Bacillus coagulans BC100 experimental group (BC-L group).

[0085] 2.2 AAI model establishment and intervention

[0086] At 9:00 a.m. every day after the experiment, the mice in the metadoxine group were given metadoxine at a concentration of 20 g / kg by gavage, and the mice in the BC-H group and BC-L group were given metadoxine at a concentration of 10 g / kg. 9 CFU / (kg m b d) and 10 8 CFU / (kg m b ·d) were gavaged with Bacillus coagulans at a dose of 1.447 mg / kg body weight / day. The mice in the blank group and the model group were gavaged with the same volume of PBS, 0.2 ml / mouse / day for all mice. After one week of continuous administration, 30 minutes after the last administration, except for the blank group which was gavaged with normal saline, the mice in other groups were gavaged with liquor (56%, v / v) (5.6 g / kg body weight / day) for three consecutive days. The time of disappearance of righting reflex, recovery time and sleep time of the mice were observed and recorded.

[0087] 2.3 Observation indicators and detection methods

[0088] 2.3.1 Righting reflex test

[0089] After mice were gavaged with 56% alcohol, they were observed for about 20 minutes. When the mice were swaying or slow in walking, they were turned over so that their backs were facing down. If the mice could not turn over to their backs up on their own within 30 seconds and their righting reflex disappeared, they were considered drunk. When the mice could turn over to their backs up on their own and their righting reflex appeared, they were considered sober. The number of mice with lost righting reflex was observed and recorded. Mice that did not appear drunk were recorded as 0 minutes, and mice that were not sober after 7 hours were recorded as 420 minutes.

[0090] 2.3.2 Serum collection and tissue homogenization

[0091] At the end of the experiment, peripheral blood of mice was collected by eye removal in a 1.5 ml sterile centrifuge tube. After standing at room temperature for 2 hours and then placed at 4°C overnight, the blood was centrifuged at 3500 r / min for 10 minutes. The upper serum was separated and collected and frozen at -80°C for testing. An appropriate amount of liver tissue was cut into pieces, and a pH 7.4 tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) solution was added at a ratio of 1:9 (W:V). After the tissue was homogenized, it was centrifuged at 3500 r / min for 10 minutes. The precipitate was discarded and the supernatant was taken to obtain the liver tissue homogenate.

[0092] 2.3.3 Detection of alcohol concentration in mouse serum and metabolic enzymes in liver

[0093] Biochemical kits were used to detect the alcohol content in serum (the kit was purchased from Nanjing Jiancheng Bioengineering Institute, item number E036-1-1), the activity of alcohol metabolism enzymes ADH and ALDH in liver tissue homogenate, and the activity of antioxidants SOD, MDA, GSH and GSH-Px in serum and liver; in addition, the activity of ALT, AST and GGT in liver tissue homogenate was detected to verify the level of liver damage. According to the steps in the manual, the blank well, zero well and parallel control were set, and the absorbance (A) value was measured at a wavelength of 450 nm. The content of the measured index was calculated by drawing a standard curve.

[0094] 2.3.4 Histological observation

[0095] After blood was collected from mice, they were killed by cervical dislocation. Fresh liver, stomach, small intestine and colon tissues were fixed in 4% paraformaldehyde for more than 12-24 hours until further processing. After routine tissue dehydration, they were dried, transparent, paraffin-embedded, sliced ​​to a thickness of 4-5 μm, stained with HE, and observed for pathological changes under an optical microscope.

[0096] 2.4 RT-qPCR detection of gene expression

[0097] Immediately after sampling, the fresh mouse liver tissue was frozen at -80℃ for storage. When extracting tissue RNA, a certain amount of tissue was taken out and cut to extract the whole genome RNA according to the instructions of the RNA extraction and reverse transcription kit. After measuring the concentration, the RNA was reverse transcribed into cDNA. After testing the purity of the cDNA, it was used as a sample for later use. The RT-qPCR process and parameters were carried out according to the reagent instructions (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product number 11201ES08). After adding the premixed solution for real-time quantitative PCR amplification (Hieff® qPCR SYBR Green Master Mix (No Rox)) and the template, it was run on the machine. β -actin is the internal reference gene, and the primer sequences of all detected genes are shown in the following table:

[0098] Table 1 Primer sequences for RT-qPCR gene detection

[0099]

[0100] Note: IL-1β, interleukin-1β; NF-κB, nuclear factor κB; TNF-α, tumor necrosis factor α; Nrf-2, nuclear factor 2-related factor 2.

[0101] 2.5 Statistical analysis

[0102] The experimental data were expressed as mean ± standard deviation (x ± s). All statistical analyses and graphs were drawn using GraphPad Prism 8. The completely randomized one-way ANOVA statistical analysis was used. When the variances of the groups were homogeneous, the pairwise comparisons between the means of multiple samples were performed using Tukey's multiple comparisons test. If the variances were unequal, the Tamhane's T2 method was used. The test level was set at two-sided α = 0.05, and P < 0.05 was considered statistically significant.

[0103] 3 Experimental Results

[0104] 3.1 Effects of BC100 on body weight, liver index, and intoxication rate of AAI mice

[0105] As shown in Table 2, there was no significant difference in body weight among the five groups after one week of intervention and three days of intragastric alcohol administration (Table 2). At the same time, the liver index (considered to be one of the indicators of alcoholic liver disease) increased to varying degrees due to continuous alcohol intake, but the liver index of the metadoxine and BC-H groups increased the least, and the effect of the BC-L group was slightly worse than that of the BC-H group. The average three-day drunkenness rate was the same for the BC-H group, the BC-L group, and the AAI group, all at 88.89%, and the metadoxine group at 77.78%.

[0106] Table 2 Statistics of body weight, liver index and drunkenness rate of mice in each group after intragastric alcohol administration for 3 days

[0107]

[0108] 3.2 Effects of BC100 on tolerance time and intoxication time of AAI mice

[0109] The experimental results are as follows Figure 1 As shown in the figure, it can be seen that the tolerance time and sleep time of mice in the AAI model group were 12±5.8min and 317±56.98min, respectively. Compared with the AAI model group, the mice in the metadoxine group significantly prolonged the tolerance time (P<0.05), and the BC-H and BC-L groups had no significant effect on the tolerance time, but the three intervention groups all significantly shortened the sleep time (P<0.05), with metadoxine having the best effect, followed by the BC-H group. The results showed that BC100 could reduce the drunkenness rate of AAI mice to a certain extent, prolong the tolerance time, and especially shorten the sober time, and there was a significant dose-effect relationship with BC100.

[0110] 3.3 Effect of BC100 on blood ethanol concentration in AAI mice

[0111] Blood alcohol concentration (BAC) can reflect the severity of alcohol intoxication. Figure 2 As shown, compared with the NC control group, the blood alcohol concentration of mice in the AAI alcohol-treated model group was significantly increased to 74.73 mmol / L (P<0.05); compared with the AAI model group, the blood alcohol concentration of mice in the metadoxine experimental group, BC-H group and BC-L group was significantly reduced (P<0.05), and there was no difference between the BC-H group and the metadoxine group, and the BC-H group was better than the BC-L group. This result shows that high concentrations of Bacillus coagulans can significantly reduce the blood alcohol content in mice with acute alcohol poisoning.

[0112] 3.4 Effect of BC100 on the activities of ADH and ALDH alcohol metabolism enzymes in the liver of AAI mice

[0113] Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are important indicators of how fast the liver metabolizes alcohol in the body. Figure 3 As shown in the figure, compared with the blank control group, the ADH and ADH activity in the liver of mice in the AAI model group increased significantly due to alcohol intake (P<0.05), and the ADH activity in the metadoxine group and the BC-H treatment group was slightly higher than that in the model group (P>0.05). It was proved that BC100 pretreatment can enhance ADH activity, and the effect of the high concentration group was more obvious. The opposite trend was observed in ALDH activity. The activity of the metadoxine group was similar to that of the AAI model group, but the ALDH activity of the BC-H treatment group decreased (P>0.05), which may be related to its protection of the gastric mucosa and inhibition of alcohol absorption.

[0114] 3.5 Effect of BC100 on the activity of antioxidant-related factors in serum and liver of AAI mice

[0115] The results are as follows Figure 4 As shown in the figure, compared with NC, the levels of GSH, GSH-px and SOD in serum and liver tissue of AAI mice were significantly reduced (P<0.05). The three intervention groups could increase the levels of GSH, GSH-px and SOD in serum and liver. Metadoxine was better than BC-H and BC-L, but it showed better effects on GSH-px, BC-H and BC-L in liver tissue. In addition, compared with the NC normal group, the MDA in serum and liver tissue of AAI model group was significantly increased (P<0.05), while the three intervention groups could inhibit the MDA level in serum and there was no significant difference between the groups (P>0.05), and the BC-L group had no significant effect on liver MDA.

[0116] 3.6 Effects of BC100 on liver damage indicators in serum of AAI mice

[0117] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important transaminases that produce and degrade amino acids in liver cells. Liver cell death will lead to increased enzyme activities of ALT and AST in serum. Together with alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT), they are important indicators of liver cell damage. Figure 5 It can be seen that compared with the NC control group, alcohol treatment significantly enhanced the activities of AST, ALT, GGT and ALP in the serum of mice in the AAI group (P<0.05), while the AST in the serum of mice pretreated with metadoxine and high concentration BC100 was significantly reduced to normal levels. Although the activities of ALT, ALP and GGT decreased in the three treatment groups, there was no statistical difference with AAI, and the effect of BC-H was better than that of BC-L group. The results show that alcohol gavage causes damage to hepatocytes, and high concentration BC100 pretreatment can prevent and alleviate liver damage caused by alcoholism to a certain extent.

[0118] 3.7 Effects of BC100 on the morphology of liver and stomach tissues in AAI mice

[0119] The liver is the main organ that metabolizes alcohol. Most of the alcohol absorbed by the body is metabolized in the liver. Figure 6 It can be found that the liver cells of mice in the NC normal group are arranged neatly, forming liver plates in radial stripes, with complete liver cell structure, clear nuclear structure and clear boundaries. In contrast, the liver cells in the AAI model group are arranged irregularly, the liver cells are swollen, the intercellular space becomes larger (black arrows), some liver cell nuclei are atrophied and deformed or even disappear, the cell boundaries are blurred, large areas of necrosis can be seen (in the black frame), and a large number of inflammatory cells are infiltrated in the edges of blood vessels and surrounding tissues (in the black circle); while the inflammatory infiltration of the liver of mice pretreated with metadoxine and BC100 is reduced, and there is no large amount of cell necrosis in the model group. The morphological structure and arrangement of liver cells tend to be normal, and the high concentration group has better effects than the low concentration group. The results show that alcohol causes serious damage to the liver, and high concentration BC100 can play a better preventive and alleviating role.

[0120] From the structure of gastric tissue sections, we can see that compared with the NC normal group, AAI alcohol modeling caused significant damage to the gastric wall, thinning of the gastric mucosal layer, damage and loss of upper layer cells, edema separation distance between the mucosal layer and the submucosal layer increased, inflammatory cells infiltrated, and the muscular layer thickened; the gastric wall morphology of the metadoxine and BC100-H treatment groups was less damaged, which can significantly prevent and alleviate the thinning and damage of the gastric mucosa caused by alcohol ( Figure 7 A) and inflammatory fibrotic lesions of the gastric wall muscularis ( Figure 7 B in the figure).

[0121] 3.8 Effect of BC100 on the expression of inflammatory genes in the liver of AAI mice

[0122] The mRNA expression levels of TNF-α, IL-1β, Nrf-2, and NF-κB were detected to assess the degree of inflammatory response induced by AAI alcohol. Figure 8 As shown in the data, the mRNA expression levels of TNF-α, IL-1β and NF-κB in mice in the AAI acute alcohol model group were significantly increased (P<0.05), and the gene expression level trends in the three treatment groups were opposite to those in the AAI model group, demonstrating that BC100 has the same inhibitory effect on the expression of liver inflammatory genes as metadoxine and is positively correlated with the concentration of bacteria; in contrast, the mRNA expression level of Nrf-2 in the AAI model group was decreased (P<0.05), but BC-H showed the best effect in inhibiting the decrease in the expression level of Nrf-2 mRNA.

[0123] The above results show that Bacillus coagulans BC100 has good anti-inflammatory and antioxidant activities, can exert its alcohol-resolving function through the gastrointestinal tract and has the effect of protecting the liver and gastric mucosa.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus coagulans ( Bacillus coagulans ), characterized in that, The deposit number of the Bacillus coagulans is CGMCC No. 25453.

2. A preparation comprising the Bacillus coagulans according to claim 1, characterized in that The preparation is selected from one or more of the following: fermentation broth of Bacillus coagulans, precipitate of fermentation broth of Bacillus coagulans, supernatant of fermentation broth of Bacillus coagulans, live bacteria of Bacillus coagulans, dead bacteria of Bacillus coagulans, and freeze-dried powder of Bacillus coagulans.

3. A bacterial agent, characterized in that: The bacterial agent comprises the Bacillus coagulans according to claim 1 or the preparation according to claim 2.

4. Use of the Bacillus coagulans according to claim 1, the preparation according to claim 2, or the bacterial agent according to claim 3 in the preparation of a product for preventing, treating or assisting in the treatment of alcoholism.

5. The use according to claim 4, characterized in that: The viable count of Bacillus coagulans in the product is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g.

6. The use according to claim 5, characterized in that: The viable count of Bacillus coagulans in the product is 2×10 8 -2×10 9 CFU / mL or 2×10 8 -2×10 9 CFU / g.

7. The use according to claim 4, characterized in that: When the product is a medicine, the medicine also includes a pharmaceutically acceptable carrier.

8. Use of the Bacillus coagulans according to claim 1, the preparation according to claim 2, or the bacterial agent according to claim 3 in the preparation of a product for preventing, treating or assisting in the treatment of alcoholic liver damage.

9. A product for preventing, treating or assisting in the treatment of alcoholism, characterized in that: The product contains the Bacillus coagulans according to claim 1, the preparation according to claim 2, or the bacterial agent according to claim 3.

10. The product according to claim 9, characterized in that The viable count of Bacillus coagulans in the product is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g.

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