Lactobacillus plantarum lpa2 having alcohol degradation function, formulation thereof and use thereof

Abstract

Provided are a Lactobacillus plantarum strain LpA2 having an alcohol degradation function and the use thereof. Particularly provided is the use of Lactobacillus plantarum LpA2 in alcohol degradation and the like. Further provided is a composition for treating or relieving intoxication, comprising a drug, etc.

Description

Lactobacillus plantarum LpA2 with alcohol degradation function and its preparation and application Technical Field

[0001] The product belongs to the fields of biopharmaceuticals and food / health products. Specifically, it relates to Lactobacillus plantarum LpA2 with alcohol-degrading properties, its preparations, and applications. Background Art

[0002] Alcohol intolerance refers to the physiological effects and negative behaviors caused by recently consumed alcohol. Mild alcohol intoxication is also called drunkenness. Symptoms of low-dose alcohol intoxication include mild sedative effects and poor coordination, while higher doses may cause slurred speech, difficulty walking, and vomiting.

[0003] The proportion is higher in East Asian populations, and it is often inherited due to reduced activity of acetaldehyde dehydrogenase (AldH2), which leads to the accumulation of acetaldehyde in the body, causing flushing, rapid heartbeat, nausea, blurred vision, shortness of breath, etc.; in addition, acetaldehyde is a known carcinogen and can increase the risk of cancers related to it, such as liver cancer, esophageal cancer and digestive tract cancer.

[0004] Currently, commercially available alcohol-degrading health products primarily consist of single or combined natural products. One category includes natural products containing large amounts of glucose and organic acids, such as curcumin and puerarin. Recently, some naturally ethanol-resistant probiotics, such as Pediococcus pentosaceus, have also emerged. However, the main drawback of these products is that they have not been tested in vivo on experimental animals, and only in vitro alcohol degradation experiments have been conducted, so their true effectiveness has not been verified experimentally. Furthermore, alcohol and microorganisms enter the body through a complex environmental system, characterized by low pH and hypoxia. Whether microorganisms can effectively contribute to this effect remains unknown.

[0005] Currently, there is no effective method or medicine for alleviating the symptoms of drunkenness and shortening the duration of drunkenness with little side effects in this field.

[0006] Therefore, this area is in the urgent need of developing a new method or medicine that has no toxic side effects and is used to prevent drunkenness and shorten the time of drunkenness and alleviate the symptoms of drunkenness.

[0007] Summary of the Invention

[0008] The present invention provides a novel method without toxic or side effects for preventing drunkenness, shortening the drunkenness time and alleviating the drunkenness symptoms and its application.

[0009] In a first aspect of the present invention, a Lactobacillus plantarum strain is provided. The Lactobacillus plantarum strain is Lactobacillus plantarum LpA2, and the Lactobacillus plantarum strain LpA2 has an alcohol-degrading effect.

[0010] In another preferred embodiment, the Lactobacillus plantarum preservation number is CCTCC NO.M 2023105.

[0011] In another preferred embodiment, the 16S rRNA gene sequence of the Lactobacillus plantarum is as shown in SEQ ID NO.: 1 or has at least 99.5% identity with SEQ ID NO.: 1.

[0012] In another preferred embodiment, the Lactobacillus plantarum is isolated from sauerkraut.

[0013] In a second aspect of the present invention, a composition for promoting alcohol degradation is provided, comprising: (a) a safe and effective amount of the Lactobacillus plantarum and / or its metabolites according to the first aspect of the present invention; and (b) a food or pharmaceutically acceptable carrier.

[0014] In another preferred embodiment, the composition is selected from the following group: a food composition, a health product composition, a pharmaceutical composition, a beverage composition, or a combination thereof.

[0015] In another preferred embodiment, the composition is an oral preparation.

[0016] In another preferred embodiment, the composition further contains an additional alcohol-degrading component.

[0017] The dosage form of the composition is: (i) liquid; (ii) solid preparation; (iii) semi-solid preparation.

[0018] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, powders, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0019] In another preferred embodiment, the food composition includes an emulsion product, a solution product, a powder product, or a suspension product.

[0020] In another preferred embodiment, the food composition includes dairy products, milk powder, or emulsion.

[0021] In another preferred embodiment, the liquid preparation is selected from the following group: a solution preparation or a suspension preparation.

[0022] In another preferred embodiment, the composition contains 1×10-1×10 20cfu / mL or cfu / g LpA2, preferably 1×10 4 -1×10 15 cfu / mL or cfu / g LpA2, based on the total volume or weight of the composition.

[0023] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt% of the Lactobacillus plantarum and / or its metabolites, based on the total weight of the composition.

[0024] In another preferred embodiment, the composition is in unit dosage form (a tablet, a capsule or a vial), and the mass of the composition in each unit dosage form is 0.05-5 g, preferably 0.1-1 g.

[0025] In another preferred embodiment, the composition further contains other probiotics and / or prebiotics.

[0026] In another preferred embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof.

[0027] In another preferred embodiment, the prebiotic is selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylooligosaccharides (XOS), lactofructooligosaccharides (LACT), soy oligosaccharides (SOS), inulin, or a combination thereof.

[0028] In another preferred embodiment, the composition is in the form of a solid powder.

[0029] In another preferred embodiment, the composition is in the form of a lyophilized powder.

[0030] In the third aspect of the present invention, there is provided a use of the Lactobacillus plantarum or its similar Lactobacillus described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, for preparing a medicament, wherein the medicament is used for one or more uses selected from the following groups: (a) preventing drunkenness; (b) shortening the duration of drunkenness; and / or (c) alleviating the symptoms of drunkenness.

[0031] In a fourth aspect of the present invention, there is provided a use of the Lactobacillus plantarum or a similar Lactobacillus plantarum described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, for preparing a medicament or preparation for (i) reducing the alcohol content in serum.

[0032] In a fifth aspect of the present invention, there is provided a method for preparing the composition according to the second aspect of the present invention, comprising the steps of:

[0033] The Lactobacillus plantarum or similar Lactobacillus plantarum and / or its metabolites described in the first aspect of the present invention are mixed with a food or pharmaceutically acceptable carrier to form the composition described in the second aspect of the present invention.

[0034] In a sixth aspect of the present invention, there is provided a production method comprising the steps of:

[0035] (a) culturing the Lactobacillus plantarum described in the first aspect of the present invention under conditions suitable for cultivation, thereby obtaining a culture;

[0036] (b) optionally, isolating Lactobacillus plantarum cells and / or metabolites thereof from the culture product; and

[0037] (c) Optionally, the Lactobacillus plantarum and / or other metabolites isolated and obtained in the previous step are mixed with a food or pharmaceutically acceptable carrier to prepare a composition.

[0038] In a seventh aspect of the present invention, a method for preparing the freeze-dried powder of Lactobacillus plantarum according to the first aspect of the present invention is provided, comprising the steps of:

[0039] (s1) Prepare a modified version of MRS broth;

[0040] (s2) inoculating the Lactobacillus plantarum LpA2 product into the MRS broth for cultivation;

[0041] (s3) washing and precipitating the Lactobacillus plantarum LpA2 twice by centrifugation to remove culture components, thereby obtaining a bacterial suspension;

[0042] (s4) centrifuging the bacterial suspension again to obtain precipitated bacterial sludge; and

[0043] (s5) The precipitated bacterial sludge is aseptically mixed with the auxiliary materials, shaken evenly, pre-frozen at -80°C for 1 hour, and freeze-dried in a vacuum to obtain a lyophilized powder.

[0044] In another preferred embodiment, the formula of the improved version of MRS broth is: soy peptone 10 g / L, yeast extract powder 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium citrate 2 g / L, sodium acetate 5 g / L, glucose 20 g / L, Tween 80 1 ml, magnesium sulfate heptahydrate 0.58 g / L, manganese sulfate tetrahydrate 0.25 g / L, and sterile water 1000 ml.

[0045] In another preferred embodiment, the method comprises the steps of: strain activation, strain fermentation culture, fermentation broth detection, fermentation broth centrifugation washing, freeze vacuum drying and bacterial powder detection.

[0046] In an eighth aspect of the present invention, a method for shortening the duration of drunkenness or alleviating the symptoms of drunkenness is provided, comprising administering the Lactobacillus plantarum described in the first aspect of the present invention or the composition described in the second aspect of the present invention to the subject.

[0047] In another preferred embodiment, the administration comprises oral administration.

[0048] In another preferred embodiment, the administration dosage is 0.01-5 g / 50 kg body weight / day, preferably, 0.1-2 g / 50 kg body weight / day.

[0049] In another preferred embodiment, the subject includes mammals, such as humans.

[0050] In another preferred embodiment, the method is non-therapeutic.

[0051] In a ninth aspect of the present invention, an in vitro method for reducing alcohol content is provided, wherein the Lactobacillus plantarum described in the first aspect of the present invention or the composition described in the second aspect of the present invention is co-cultured with an alcohol-containing sample.

[0052] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows (A) a schematic diagram of the separation and screening plate, the screening medium components: 1% yeast extract, 1% glucose, 1% calcium carbonate, 4% anhydrous ethanol, 2% agar powder; (B) a photograph of the plate with the colony morphology of the strain LpA2; (C) a photograph of the purified strain LpA2 observed under a microscope (1000X).

[0054] Figure 2 shows the Neighbor-Joining phylogenetic tree of strain LpA2 constructed based on the 16S rRNA gene sequence alignment results with Oenococcus oeni NBRC 100497 (AB681195) as the outer branch.

[0055] Figure 3 shows a heat map generated by the whole genome of strain LpA2 calculated using OAT software.

[0056] FIG4 shows the long-term stability of the lyophilized powder of strain LpA2.

[0057] Figure 5 shows the distribution of ethanol metabolism in mice.

[0058] FIG6A shows the in vitro degradation of ethanol in a reaction system with an ethanol concentration of 2 g / L (c. blank control, Pp. Pediococcus pentosaceus, BS. Bacillus subtilis, Lp-8. Lactobacillus plantarum).

[0059] FIG6B shows the in vitro degradation of ethanol in a reaction system with an ethanol concentration of 4 g / L (c. blank control, Pp. Pediococcus pentosaceus, BS. Bacillus subtilis, Lp-8 and Lp-12. Lactobacillus plantarum).

[0060] FIG. 7 shows the in vitro ethanol degradation by strain LpA2 (c. blank control, Lp-8. Lactobacillus plantarum). DETAILED DESCRIPTION

[0061] After extensive and intensive research and experimentation, and through extensive screening, the inventors unexpectedly obtained a strain of Lactobacillus plantarum LpA2, or Lactobacillus plantarum LpA2, that has an alcohol (ethanol)-degrading effect. Experiments have shown that the strain Lactobacillus plantarum LpA2 has significant efficacy in preventing drunkenness, sobering up, and promoting soberness. Administration of an active composition containing strain LpA2 to experimental subjects can alleviate adverse reactions caused by alcohol intolerance after drinking, significantly shorten the duration of drunkenness, reduce serum ethanol content, and improve drunkenness. The present invention was completed on this basis.

[0062] Lactobacillus plantarum and its application

[0063] Lactobacillus plantarum was formerly known as "Lactobacillus plantarum".

[0064] As used herein, the terms "Lactobacillus plantarum LpA2 of the present invention", "Lactobacillus plantarum LpA2 of the present invention", "Lactobacillus plantarum LpA2 of the present invention", "Lactobacillus plantarum LpA2 of the present invention", "strain LpA2 of the present invention" and "LpA2 of the present invention" are used interchangeably and all refer to Lactobacillus plantarum LpA2 of the present invention, which has the accession number CCTCC NO.M 2023105.

[0065] The Lactobacillus plantarum strain LpA2 of the present invention is isolated from sauerkraut. The physiological characteristics of the LpA2 strain are as follows: after culturing on MRS medium for 24 hours, the LpA2 strain produces white, opaque colonies with raised, moist, smooth, and shiny edges, and a diameter of 0.8 to 1.0 mm. Its optimal growth temperature is 37°C, and it can grow and reproduce at a pH greater than 3.

[0066] The present invention provides the use of strain LpA2 for preventing and alleviating symptoms of alcohol intoxication. According to a preferred embodiment of the present invention, strain LpA2 co-cultured with mouse contents exhibits a certain ability to degrade ethanol. Therefore, the strain can be used to prevent and / or alleviate symptoms of alcohol intoxication.

[0067] As used herein, "ethanol" and "alcohol" are used interchangeably and have the same meaning.

[0068] sauerkraut

[0069] Sauerkraut, also known as pickled cabbage, has a salty and sour taste, a crisp texture, and a bright color. It is appetizing, refreshing, and helps sober up and remove greasiness. Pickling sauerkraut primarily involves converting plant sugars into organic acids, which produce acidic substances in the vegetables. In everyday cooking, pickled cabbage is often added with some white wine to enhance its flavor. Once the sauerkraut matures, the alcohol is consumed.

[0070] The strain of the invention is selected from sauerkraut food, is a natural strain, is not subjected to genetic engineering or mutagenesis treatment, and is safe and stable.

[0071] Composition and application thereof

[0072] The present invention also provides a composition, preferably a pharmaceutical composition. The composition comprises an effective amount of Lactobacillus plantarum LpA2, and in a preferred embodiment, the composition further comprises a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof; and / or a prebiotic selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylooligosaccharides (XOS), lactofructooligosaccharides (LACT), soy oligosaccharides (SOS), inulin, or a combination thereof.

[0073] In a preferred embodiment, the composition is a liquid preparation, a solid preparation, or a semi-solid preparation.

[0074] In a preferred embodiment, the liquid preparation is selected from the following group: a solution preparation or a suspension preparation.

[0075] In a preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, powders, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0076] The pharmaceutical composition of the present invention can be administered in the form of tablets, injections or capsules. The pharmaceutical preparation includes excipients, pharmaceutically acceptable media and carriers, which can be selected according to the route of administration. The pharmaceutical preparation of the present invention can further include auxiliary active ingredients.

[0077] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil can be used as carriers, excipients or diluents of the pharmaceutical composition of the present invention.

[0078] In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical composition of the present invention can be produced as an enteric coating formulation by various well-known methods so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0079] Alternatively, the microorganisms of the present invention can be used in the form of capsules prepared by conventional methods. For example, standard excipients can be mixed with the freeze-dried microorganisms of the present invention to form pellets, which can then be filled into gelatin capsules. Furthermore, the microorganisms of the present invention can be mixed with pharmaceutically acceptable excipients such as liquid glue, cellulose, silicates, or mineral oil to form suspensions or dispersions, which can be filled into soft gelatin capsules.

[0080] The pharmaceutical composition of the present invention can be prepared as enteric-coated tablets for oral administration. The term "enteric coating" as used herein includes all conventional pharmaceutical coatings that are not degraded by gastric acid but fully decompose in the small intestine to rapidly release the microorganisms of the present invention. The enteric coating of the present invention can be maintained in synthetic gastric acid, such as a pH 1 HCl solution, at 36-38°C for more than 2 hours and preferably decomposes within 1.0 hour in synthetic intestinal fluid, such as a pH 7.0 buffer.

[0081] The enteric coating of the present invention is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The thickness of the enteric coating of the present invention is 5-100 μm, and ideally 20-80 μm. The enteric coating ingredients are selected from conventional polymers known in the art.

[0082] The preferred enteric coating of the present invention is prepared from a copolymer of cellulose acetate phthalate polymer or trimellitate polymer and methacrylic acid (for example, a copolymer of methacrylic acid containing more than 40% and methylcellulose hydroxypropyl phthalate or its ester derivatives).

[0083] The cellulose acetate phthalate used in the casing of the present invention has a viscosity of approximately 45-90 cp, an acetyl content of 17-26%, and a phthalic acid content of 30-40%. The cellulose acetate trimellitate used in the casing has a viscosity of approximately 5-21 cs and an acetylphthalate content of 17-26%. Cellulose acetate trimellitate is manufactured by Eastman Coda and can be used as the casing material of the present invention.

[0084] The hydroxypropyl methylcellulose phthalate used in the enteric coating of the present invention generally has a molecular weight of 20,000-130,000 Daltons, an ideal molecular weight of 80,000-100,000 Daltons, a hydroxypropyl content of 5-10%, a methoxy content of 18-24%, and a phthaloyl content of 21-35%.

[0085] The hydroxypropyl methylcellulose phthalate used in the enteric coating of the present invention is HP50, manufactured by Shin-Etsu Chemidnl Co. Ltd. of Japan. HP50 contains 6-10% hydroxypropyl groups, 20-24% methoxy groups, 21-27% propyl groups, and has a molecular weight of 84,000 Daltons. Another enteric coating material is HP55, which contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy groups, 27-35% phthalic acid, and has a molecular weight of 78,000 Daltons.

[0086] The enteric coating of the present invention is prepared as follows: an enteric coating solution is sprayed onto the cores using conventional methods. In this enteric coating method, all solvents are alcohols (e.g., ethanol), ketones (e.g., acetone), halogenated hydrocarbon compounds (e.g., dichloromethane), or combinations thereof. A softening agent, such as di-n-butyl phthalate and triacetin, is added to the enteric coating solution in a ratio of 1 part coating to about 0.05 or about 0.3 parts softening agent. The spraying process is preferably performed continuously, and the amount of material sprayed can be controlled according to the coating conditions employed. The spray pressure can be adjusted as desired; generally, ideal results are achieved at an average pressure of 1-1.5 bar.

[0087] In the specification, "pharmaceutically effective amount" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals. For example, in the present invention, a drug containing 1×10-1×10 20 cfu / ml or cfu / g (especially, can contain 1×10 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 cfu / ml or cfu / g) of Lactobacillus plantarum LpA2 and / or its metabolites.

[0088] When used to prepare a pharmaceutical composition, the effective dosage of Lactobacillus plantarum LpA2 or its metabolites may vary depending on the mode of administration and the severity of the disease to be treated. The dosage form suitable for oral administration comprises about 1×10-1×10 20 cfu / ml or cfu / g (especially, can contain 1×10 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 cfu / ml or cfu / g) of active Lactobacillus plantarum LpA2 or the active ingredient produced by fermentation. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, depending on the exigencies of the therapeutic condition, several divided doses can be given daily, or the dosage can be reduced proportionally.

[0089] The plant lactobacillus LpA2 or its metabolites can be administered orally or in combination. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include culture media, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), as long as they are suitable for the characteristics of the plant lactobacillus LpA2 or its metabolites and the required specific administration mode. Commonly used adjuvants in the preparation of pharmaceutical compositions can also be advantageously included, such as flavorings, pigments, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0090] From the standpoint of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

[0091] The composition of the present invention is administered to the individual once or more per day. The dosage unit represents a dosage that can be divided in form and is suitable for humans or all other mammalian individuals. Each unit contains a pharmaceutically acceptable carrier and an effective therapeutic amount of the microorganism of the present invention. The dosage varies with the patient's weight and severity of obesity, the supplementary active ingredients included and the microorganisms used. In addition, if possible, the administration can be separated and, if necessary, continuous. Therefore, the dosage does not limit the present invention. In addition, the "composition" in the present invention not only means a medicine but also means a functional food and a health supplement food. In a preferred embodiment, the composition includes: beverages, food, medicines, animal feed, etc.

[0092] In a preferred embodiment of the present invention, a food composition is also provided, which contains an effective amount of Lactobacillus plantarum LpA2 and / or its metabolites, and the remainder is a food-acceptable carrier, and the dosage form of the food composition is selected from solid, dairy, solution products, powder products, or suspension products.

[0093] In a preferred embodiment, the composition is formulated as follows:

[0094] 1×10-1×10 20 cfu / mL of Lactobacillus plantarum LpA2 and / or its metabolites; and a food or pharmaceutically acceptable carrier, and / or excipient.

[0095] In another preferred embodiment, the composition is formulated as follows:

[0096] 1×10 6 -1×10 11 cfu / mL of Lactobacillus plantarum LpA2 and / or its metabolites; and a food or pharmaceutically acceptable carrier, and / or excipient.

[0097] Production method of Lactobacillus plantarum LpA2

[0098] Generally, Lactobacillus plantarum LpA2 can be prepared by conventional methods.

[0099] In the present invention, a method for large-scale production of Lactobacillus plantarum LpA2 is provided, specifically comprising the following steps:

[0100] (a) culturing the Lactobacillus plantarum LpA2 under conditions suitable for cultivation, thereby obtaining a culture product;

[0101] (b) optionally, isolating Lactobacillus plantarum LpA2 cells and / or metabolites thereof from the culture product; and

[0102] (c) Optionally, the Lactobacillus plantarum LpA2 cells and / or their metabolites obtained by separation in the previous step are mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier to prepare a composition.

[0103] Methods to prevent and / or shorten the duration of intoxication

[0104] In another preferred embodiment, the method comprises: taking the pharmaceutical composition, food composition, beverage composition, or a combination thereof of the present invention. The experimental subject is a human.

[0105] In another preferred embodiment, the method comprises: taking the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof. The experimental subject is an animal, preferably a rodent or a rabbit.

[0106] The main advantages of the present invention are:

[0107] (a) The Lactobacillus plantarum LpA2 of the present invention can significantly degrade alcohol in alcohol co-culture, shorten the time of intoxication, and can significantly degrade alcohol in vitro co-culture in the mouse intestine.

[0108] (b) The present invention relates to a freeze-dried powder of Lactobacillus plantarum LpA2 and a preparation method thereof. The freeze-dried powder preparation can be stored dry and cool (20° C.) for 2 months. The bacterial powder has strong stability and high activity, which can save a large part of transportation and storage costs.

[0109] (c) The strain LpA2 of the present invention was screened from sauerkraut food and is a natural strain without genetic engineering or mutagenesis treatment, so it is safe and stable.

[0110] (d) The alcohol-degrading effect of the strain of the present invention can indirectly protect the liver.

[0111] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0112] Example 1. Screening and identification of LpA2 strains

[0113] 1.1 Strain Source

[0114] The method collects pickled cabbage and kimchi from residents' homes, takes 0.5 g of the sample and adds it to 4.5 mL of sterile physiological saline, fully shakes and disperses the sample, and takes 100 μL of the sample for gradient dilution.

[0115] Select a suitable gradient and evenly spread it on a medium containing CaCO3 and screening, as shown in Figure 1A, and culture it under anaerobic conditions at 37°C for 36 to 48 hours.

[0116] 1.2 Isolation and purification of strains

[0117] Pick a single colony that produces a clear transparent ring. Pick the colony obtained in Figure 1A and streak it on MRS solid medium. Repeat the purification until no other bacteria are found.

[0118] After culturing LpA2 on MRS medium for 24 h, the colonies were white, opaque, raised, moist, with smooth edges and a glossy appearance, and the colony diameter was 0.8 to 1.0 mm, as shown in Figures 1B-1C.

[0119] The preparation method of MRS solid medium is as follows: peptone 10g, beef extract powder 5g, Tween 80 1mL, potassium hydrogen phosphate 2g, yeast extract powder 4g, magnesium sulfate 0.2g, ammonium citrate 2g, glucose 20g, manganese sulfate 0.05g, sodium acetate 5g, agar 16g, distilled water 1L, pH 6.2±0.2, sterilize at 121℃ for 25min and set aside.

[0120] 1.3 16S rRNA gene identification of strains

[0121] The lactic acid bacteria genome was extracted using a rapid bacterial genomic DNA extraction kit and used as a template for PCR amplification. Successful PCR products were sequenced and assembled to obtain DNA sequences, which were then compared for sequence homology using the NCBI BLAST search system.

[0122] Select the known strain with the highest similarity and analyze its similarity.

[0123] A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA6 software. As shown in Figure 2 , the phylogenetic tree suggests that LpA2 is a newly identified strain of Lactobacillus plantarum, and its closest relative is Lactobacillus plantarum ATCC14917, with a homology of 65%.

[0124] After sequencing, the gene sequence of 16S rRNA of the LpA2 strain is shown as SEQ ID NO: 1.

[0125] Example 2 Physiological and biochemical properties of strains

[0126] Each batch of LPA2 product is prepared from glycerol cryovials of the working strain bank.

[0127] Thaw the glycerol cryovials and inoculate them into MRS broth (without animal protein) in a biosafety cabinet. Culture conditions: 37°C, 200 rpm, 18-20 h. After culture, analyze the OD values. 600 The bacterial concentration was assessed by using the values ​​obtained from the assay. The samples were inoculated into different physical and chemical test kits and cultured for observation.

[0128] MRS broth (does not contain animal protein) formula: soy peptone 10g / L, yeast extract powder 5g / L, dipotassium hydrogen phosphate 2g / L, diammonium citrate 2g / L, sodium acetate 5g / L, glucose 20g / L, Tween 80 1ml, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate tetrahydrate 0.25g / L, sterile water 1000ml.

[0129] The results of the physiological and biochemical characteristics test of strain LpA2 are shown in Table 1-2.

[0130] Table 1 Physiological and biochemical characteristics of strain LpA2 – enzyme activity, carbon source oxidation Note: +: positive reaction; -: negative reaction.

[0131] Table 2 Physiological and biochemical characteristics of strain LpA2 – Acid production using carbon sources Note: +: positive reaction; -: negative reaction.

[0132] As shown in Table 1-2, the strain can produce β-galactosidase, can ferment glucose and mannitol, and can use glucose, fructose, galactose and the like to produce acidic substances.

[0133] Example 3. Whole genome sequencing of strain LpA2

[0134] The lactic acid bacteria strain genome was extracted using a bacterial genomic DNA rapid extraction kit and then sequenced for comparison. The results are shown in Figure 3: The ANI value of the strain LpA2 was 97.21% when compared with the full genome data of the model strain Lactiplantibacillus plantarum (ATCC14579). This ANI value is greater than the threshold of 95-96% for identification of the same species.

[0135] Strain identification results: Combined with the microbial morphological characteristics, physiological and biochemical indicators, 16SrRNA gene sequence and whole genome comparison results, the LpA2 strain was determined to be a new strain of Lactobacillus plantarum LpA2 (Lactiplantibacillus plantarum LpA2) in the genus Lactobacillus.

[0136] Example 4. Preparation of LpA2 preparation (lyophilized powder)

[0137] Modified MRS broth was chosen because the original culture medium formula contained animal protein.

[0138] Formula of modified medium MRS: soy peptone 10 g / L, yeast extract powder 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium citrate 2 g / L, sodium acetate 5 g / L, glucose 20 g / L, Tween 80 1 ml, magnesium sulfate heptahydrate 0.58 g / L, manganese sulfate tetrahydrate 0.25 g / L, and sterile water 1000 ml.

[0139] Each batch of LPA2 product will be prepared from glycerol cryovials of the working strain bank.

[0140] Thaw the glycerol cryovials and inoculate them into MRS broth (without animal protein) in a biosafety cabinet. Culture conditions: 37°C, 200 rpm, 18-20 h. After culture, analyze the OD values. 600 The bacterial concentration was assessed by centrifugation at 10,000 r / min, and then the precipitated bacteria were washed twice with an equal amount of normal saline (NS) to remove the culture medium components. The bacterial density of the bacterial suspension was calculated using OD 600 value to measure.

[0141] Centrifuge the bacterial suspension again at 10,000 rpm, weigh the wet weight of the precipitated bacterial sludge, and add the following excipients: sucrose: soluble starch: bacterial sludge: sterile water = 3:1:20:10. Mix aseptically and shake until evenly combined. Prefreeze at -80°C for 1 hour, freeze-dry under vacuum, and prepare a lyophilized powder. Store in a cool, dry place. Samples should be collected and viable bacterial counts should be performed using a gradient dilution method to determine the viable bacterial concentration of the lyophilized powder.

[0142] The long-term stability of the freeze-dried powder of strain LpA2 prepared by the preparation method of the present invention is shown in FIG4 .

[0143] The freeze-dried powder prepared from the strain can be stored at room temperature of 25°C for more than 40 days, and the decrease in the concentration of live bacteria is maintained within one order of magnitude.

[0144] Example 5. Alcohol metabolism test of ALDH2 (acetaldehyde dehydrogenase)- / - mice

[0145] 1 hour after mice were gavaged with 100 μL of ethanol (equivalent to an adult male ingesting 75-85 ml of ethanol), the ethanol concentration distribution in each organ was as shown in Figure 5. The ethanol concentration in blood, liver, and intestine was 2-3 g / L.

[0146] Therefore, the co-culture system was selected to have an ethanol concentration of 2-4 g / L in subsequent experiments.

[0147] Example 6. In vitro co-cultivation of LpA2 strains for ethanol degradation

[0148] 6.1 Experimental methods

[0149] Each batch of LPA2 product will be prepared from glycerol cryovials of the working strain bank.

[0150] Thaw the glycerol cryovials and inoculate them into MRS broth (without animal protein) in a biosafety cabinet. Culture conditions: 37°C, 200 rpm, 18-20 h. After culture, analyze the OD values. 600 The bacterial concentration was assessed by centrifugation at 10000 r / min, and then the precipitated bacteria were washed twice with an equal amount of normal saline (NS) to remove the culture medium components and adjust the bacterial solution concentration to 10 10 CFU / mL, stored at 4°C for future use; ethanol was added to prepare reaction systems with final concentrations of 2 g / L and 4 g / L ethanol, and anaerobically cultured at 37°C for 4 hours. The ethanol content in the co-culture solution was detected by HPLC.

[0151] 6.2 Experimental Results

[0152] As shown in Figure 6A, Figure 6B and Table 3.

[0153] Table 2 Ethanol content of each group after 4 hours in 2g / L and 4g / L ethanol reaction systems Note: C: control; Pp, BS and Lp-8 are all alcohol-relieving probiotics on the market; LpA12 is another strain of Lactobacillus plantarum screened out by the present invention.

[0154] As can be seen, the LpA2 strain, when treated with 2g / L and 4g / L ethanol, could degrade ethanol to concentrations of 16.25% and 8.3%, respectively, demonstrating a robust alcohol degradation capacity, particularly at low alcohol concentrations. This performance compares favorably to commercially available alcohol-degrading probiotics (Pp, BS, and Lp-8). Furthermore, LpA12, another strain of Lactobacillus plantarum selected by the present invention, showed no significant ethanol degradation effect, as shown in Table 3 and Figures 6A and 6B.

[0155] Example 7. In vitro co-culture of strain LpA2 with mouse intestinal contents

[0156] 7.1 Experimental Methods

[0157] Each batch of LPA2 product will be prepared from glycerol cryovials of the working strain bank.

[0158] Thaw the glycerol cryovials and inoculate them into MRS broth (without animal protein) in a biosafety cabinet. Culture conditions: 37°C, 200 rpm, 18-20 h. After culture, analyze the OD values. 600 The bacterial concentration was assessed by centrifugation at 10000 r / min, and then the precipitated bacteria were washed twice with an equal amount of normal saline (NS) to remove the culture medium components and adjust the bacterial solution concentration to 10 10 CFU / mL, stored at 4℃ for later use.

[0159] Adult wild mice (C57BL / 6J, male, 8 weeks old, purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) were selected and gavaged with 200 μL of normal saline and 10 11 CFU / mL of fresh LpA2 and Lp-8 bacterial liquid, 0.5h after the mouse ileum contents were collected, 1:1 (mass) of PBS solution (phosphate buffer solution) with 4g / L ethanol was added, and after anaerobically incubated at 37℃ for 3h, the ethanol content in the co-culture was detected by HPLC.

[0160] 7.2 Experimental Results

[0161] The experimental results are shown in Figure 7 and Tables 4-5.

[0162] Table 4 In vitro experiments on intestinal contents Note: 0: PBS containing 2 g / L ethanol; C: Probiotics not administered orally. LpA2 experimental strain, Lp-8 commercial strain

[0163] Table 5. Grouping of intestinal contents of LpA2 strains in vitro

[0164] From the data in the table and the figure, it can be seen that the co-culture of LpA2 and mouse contents has a certain ability to degrade ethanol, which is similar to the efficacy of alcohol-degrading probiotics on the market.

[0165] discuss

[0166] The existing preparation method and auxiliary material formula of freeze-dried powder of late fermentation of Lactobacillus plantarum are stored at 4°C or -20°C. This storage method has great restrictions and costs for transportation and storage. In addition, the temperature change caused by transportation will cause the concentration of live bacteria in the powder to drop rapidly, resulting in poor stability.

[0167] The post-fermentation freeze-drying process and auxiliary material formula of this strain of bacteria can enable the freeze-dried powder of this strain to be stored in a cool and dry place (25°C) for 2 months, with a decrease of less than 1 order of magnitude. The bacterial powder has strong stability and high activity, which can save a large part of the transportation and storage costs and ensure the stability of the effect.

[0168] strain preservation

[0169] The plant lactobacillus LpA2 (Lactobacillus plantarum LpA2) of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) (Wuhan, China) on February 10, 2023, with the deposit number: CCTCC NO.M 2023105.

[0170] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A plant lactobacillus, characterized in that The plant lactobacillus is plant lactobacillus LpA2 (Lactobacillus plantarum LpA2), and the plant lactobacillus LpA2 has an alcohol degradation effect.

2. The plant lactobacillus according to claim 1, characterized in that The deposit number of the Lactobacillus plantarum is CCTCC NO.M 2023105.

3. A composition for promoting alcohol degradation, characterized in that: The composition comprises: (a) a safe and effective amount of the plant lactobacillus and / or its metabolites according to claim 1; and (b) a food or pharmaceutically acceptable carrier.

4. The composition according to claim 3, characterized in that The composition is selected from the group consisting of a food composition, a health product composition, a pharmaceutical composition, a beverage composition, or a combination thereof.

5. The composition according to claim 3, characterized in that The composition also contains an additional alcohol degrading ingredient.

6. The composition according to claim 3, characterized in that The dosage form of the composition is: (i) liquid; (ii) solid preparation; (iii) semi-solid preparation.

7. A use of the Lactobacillus plantarum or its similar Lactobacillus plantarum according to claim 1, or the composition according to claim 3, characterized in that: Used for preparing a medicament for one or more uses selected from the following groups: (a) preventing drunkenness; (b) shortening the duration of drunkenness; and / or (c) alleviating symptoms of drunkenness.

8. A method for preparing the composition according to claim 3, characterized in that: Includes steps: The Lactobacillus plantarum or its similar Lactobacillus plantarum and / or its metabolites as claimed in claim 1 are mixed with a food or pharmaceutically acceptable carrier to form the composition as claimed in claim 3.

9. A production method, characterized in that: Includes steps: (a) culturing the plant lactobacillus according to claim 1 under conditions suitable for cultivation, thereby obtaining a culture; (b) optionally, isolating Lactobacillus plantarum cells and / or metabolites thereof from the culture product; and (c) Optionally, the Lactobacillus plantarum and / or other metabolites separated in the previous step are mixed with a food or pharmaceutically acceptable carrier to prepare a composition.

10. A method for preparing the freeze-dried powder of Lactobacillus plantarum according to claim 1, characterized in that: Includes steps: (s1) Prepare a modified version of MRS broth; (s2) inoculating the Lactobacillus plantarum LpA2 product into the MRS broth for culturing; (s3) washing and precipitating the Lactobacillus plantarum LpA2 twice by centrifugation to remove culture components, thereby obtaining a bacterial suspension; (s4) centrifuging the bacterial suspension again to obtain precipitated bacterial sludge; and (s5) The precipitated bacterial sludge is aseptically mixed with auxiliary materials, shaken evenly, pre-frozen at -80°C for 1 hour, and freeze-dried in a vacuum to obtain a lyophilized powder.

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