A probiotic composition for alleviating acute drunkenness, and its preparation method and application

Abstract

The present invention discloses a probiotic composition for relieving acute drunkenness, a preparation method thereof and an application thereof, and belongs to the technical field of probiotic application. A probiotic composition for relieving acute drunkenness is provided, characterized in that the probiotic composition comprises Lactobacillus paracasei STB Lactobacillus paracasei STB and Lactobacillus mucilaginosus Care360 Limosilactobacillus reuteri Care360; the Lactobacillus paracasei STB has a CCTCC NO: M2022881; the Lactobacillus mucilaginosus Care360 has a CCTCC NO: M2025682. The probiotic combination, which combines Lactobacillus paracasei STB and Lactobacillus mucilaginosus Care360, can effectively alleviate the symptoms of acute intoxication and has antioxidant and anti-inflammatory effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic applications, and in particular relates to a probiotic composition for alleviating acute drunkenness, a preparation method thereof, and an application thereof. Background Art

[0002] Alcohol consumption in my country consistently ranks high. Current research and surveys in the field indicate that the number of people suffering liver damage from excessive alcohol consumption is significant. Alcohol plays an integral role in the daily lives of most people today, leading to the urgent need to alleviate intoxication caused by short-term alcohol consumption. Furthermore, most people have a low metabolic rate for ethanol, which increases the risk of ethanol-induced health damage. Studies have shown that intoxication from short-term alcohol consumption not only causes a variety of discomfort but also increases the risk of developing underlying inflammatory conditions. In recent years, a large number of products have emerged on the market, primarily focused on treating or alleviating acute or chronic alcoholic liver damage, including antioxidants, anti-inflammatory drugs, liver-protective medications, and probiotics. However, these medications suffer from significant side effects, limited efficacy, and limited effectiveness.

[0003] In recent years, a number of patents in this field have mentioned the use of probiotics to alleviate alcoholic liver damage and symptoms of alcoholic discomfort. For example, the patent number CN117987307A, entitled "A strain of Lactobacillus acidophilus that can alleviate acute alcoholic liver damage and its application", mentions that Lactobacillus acidophilus ( Ligilactobacillus acidipiscis ) has significant ethanol tolerance and can alleviate alcoholic liver damage; Publication No. CN117801991A, entitled "Probiotics and postbiotics composition for alleviating symptoms of discomfort after drinking and its use" mentions the use of animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp.lactis ) ProSci-246's probiotic postbiotic composition is used to relieve the symptoms of discomfort after drinking; Publication No. CN114426942A, entitled "A recombinant Lactococcus lactis, microcapsule and its application", mentions the use of genetic engineering technology to construct recombinant Lactococcus lactis to express alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) to decompose alcohol and reduce alcohol damage to the liver and intestines; Publication No. CN116622559A, entitled "A Lactobacillus casei producing acetaldehyde dehydrogenase, alcohol-relieving probiotic composition and its preparation method", mentions the use of Lactobacillus casei producing acetaldehyde dehydrogenase ( Lactobacillus casei) (FPHC0700) is combined with other ingredients (such as mung beans, kudzu root, dendrobium officinale, etc.) to prepare a hangover probiotic composition, which has the effects of hangover and liver protection; the publication number is CN117801991A, and the name is "Probiotic postbiotic composition and use for alleviating symptoms of discomfort after drinking", which mentions the use of inactivated probiotics, bacterial components and their metabolites (postbiotics) to relieve symptoms of discomfort after drinking, such as prolonging alcohol tolerance time, shortening the time of drunkenness, etc.

[0004] Although probiotics have shown certain effects in the field of hangover relief, the effects of single strains in the prior art are limited. For example, the publication number CN116622559A, entitled "A Lactobacillus casei producing acetaldehyde dehydrogenase, a hangover relief probiotic composition and its preparation method", mentions that in the prior art, multiple probiotics or combinations with traditional Chinese medicine ingredients are usually required to achieve a good hangover relief effect, and the effect of a single strain has not been fully verified. Although existing hangover relief products can alleviate some of the discomfort symptoms after drinking, the hangover relief effect is still not significant enough, especially in the case of acute alcoholic liver damage. The existing probiotic composition may not be able to completely relieve the damage caused by alcohol to the liver. Alcoholics have serious intestinal flora disorders. Although existing probiotic products can regulate the intestinal flora, the effect is limited and cannot completely restore the balance of the intestinal flora. Especially in the case of long-term alcoholism; Publication No. CN117987307A, entitled "A strain of Lactobacillus acidophilus capable of alleviating acute alcoholic liver damage and its application" mentions that although existing drug treatments have certain anti-inflammatory and antioxidant effects, the intake of drugs may bring side effects and even increase the burden on the liver. Therefore, the development of a safe and effective method for sobering up is still a challenge; Publication No. CN114426942A, entitled "A recombinant Lactococcus lactis, microcapsule and its application" mentions that although existing sobering products can shorten the recovery time after drinking, the sobering speed is still slow, especially after drinking a lot of alcohol. Existing probiotic products cannot quickly decompose alcohol, resulting in a longer period of drunkenness. There is no detailed research on the existing probiotic sobering products in alleviating acute drunkenness, and there is an urgent need to develop a more effective and practical probiotic composition. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a probiotic composition for relieving acute drunkenness, a preparation method and an application thereof, wherein the probiotic composition is prepared by Lactobacillus paracasei STB. Lactobacillus paracasei STB and Lactobacillus mucilaginosus Care360 Limosilactobacillus reuteri The Care360 combination can effectively relieve the symptoms of acute drunkenness and has antioxidant and anti-inflammatory effects.

[0006] To achieve the above object, the present invention provides a probiotic composition for alleviating acute drunkenness, wherein the probiotic composition comprises Lactobacillus paracasei STB Lactobacillus paracasei STB and Lactobacillus mucilaginosus Care360 Limosilactobacillus reuteri Care360;

[0007] Lactobacillus paracasei STB Lactobacillus paracasei STB was deposited in the China Center for Type Culture Collection on June 14, 2022, at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M2022881.

[0008] Lactobacillus mucilaginosus Care360 Limosilactobacillus reuteri Care360 was deposited in the China Center for Type Culture Collection on April 2, 2025, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M2025682.

[0009] Preferably, the Lactobacillus paracasei STB in the probiotic composition Lactobacillus paracasei STB and Lactobacillus mucilaginosus Care360 Limosilactobacillus reuteri Care360 has a mass ratio of 1:1.

[0010] Preferably, the Lactobacillus paracasei STB Lactobacillus paracasei The effective viable count of STB is 2×10 8 ~4.5×10 8 CFU / mL, the Lactobacillus reuteri Care360 Limosilactobacillus reuteri The effective viable bacterial count of Care360 is 3×10 8 ~4.1×10 8 CFU / mL.

[0011] The present invention also provides a method for preparing the probiotic composition, comprising the following steps:

[0012] (1) Lactobacillus paracasei STB Lactobacillus paracasei STB was inoculated into MRS liquid culture medium and fermented to obtain Lactobacillus paracasei fermentation liquid;

[0013] (2) Lactobacillus reuteri Care360 Limosilactobacillus reuteri Care360 was inoculated into MRS liquid medium and fermented to obtain Lactobacillus reuteri fermentation broth;

[0014] (3) The Lactobacillus paracasei fermentation liquid obtained in step (1) is centrifuged to obtain a Lactobacillus paracasei bacterial sludge, and the Lactobacillus paracasei bacterial sludge is resuspended in physiological saline to obtain a Lactobacillus paracasei bacterial suspension;

[0015] (4) centrifuging the Lactobacillus reuteri fermentation broth obtained in step (2) to obtain Lactobacillus reuteri bacterial sludge, and resuspending the Lactobacillus reuteri bacterial sludge with physiological saline to obtain a Lactobacillus reuteri bacterial suspension;

[0016] (5) The Lactobacillus paracasei suspension obtained in step (3) and the Lactobacillus muciniphila suspension obtained in step (4) are mixed in a mass ratio of 1:1 to obtain a probiotic composition.

[0017] Preferably, the Lactobacillus paracasei STB in step (1) Lactobacillus paracasei The inoculum amount of STB is calculated as 2-6% of the volume of the MRS liquid culture medium; the fermentation temperature in step (1) is 37°C, and the fermentation time is 8-16 hours.

[0018] Preferably, the Lactobacillus reuteri Care360 in step (2) Limosilactobacillus reuteri The inoculum amount of Care360 is calculated as 2-6% of the volume of the MRS liquid culture medium; the fermentation temperature in step (2) is 37°C, and the fermentation time is 8-16 hours.

[0019] Preferably, the centrifugal temperature in step (3) is 0-4°C, the centrifugal speed is 5000-7000 rpm, and the centrifugal time is 4-6 min; the effective viable bacteria count in the Lactobacillus paracasei suspension in step (3) is 2×10 8 ~4.5×10 8 CFU / mL.

[0020] Preferably, the centrifugation temperature in step (4) is 0-4°C, the centrifugation speed is 5000-7000 rpm, and the centrifugation time is 4-6 min; the effective viable count of Lactobacillus reuteri suspension in step (4) is 3×10 8 ~4.1×10 8 CFU / mL.

[0021] The present invention also provides the use of the probiotic composition in preparing a rapid alcohol sobering and liver protecting product.

[0022] The present invention also provides the use of the probiotic composition prepared by the preparation method in preparing a rapid alcohol sobering and liver protecting product.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] The present invention uses Lactobacillus reuteri Care360 for the first time Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB Lactobacillus paracaseiSTB is used as the core strain. Both strains have high gastric acid and bile salt resistance. They can survive in the intestine and produce a large amount of alcohol dehydrogenase and acetaldehyde dehydrogenase, thereby effectively decomposing alcohol and its metabolite acetaldehyde, thereby alleviating the discomfort symptoms caused by acute drunkenness and reducing the possibility of inflammation induced by it. Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB, which is highly resistant to gastric acid and bile salts Lactobacillus paracasei The STB combination has the potential to alleviate acute intoxication, synergistically relieving it and preventing inflammation and other damage caused by excessive drinking. This combination of bacteria is more realistic in alleviating acute intoxication, more realistically reflecting its hangover-relieving effects, and further enhancing the protective effect of the Lactobacillus combination against acute intoxication. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Lactobacillus paracasei STB Lactobacillus paracasei STB evolutionary tree diagram;

[0027] Figure 2 Lactobacillus reuteri Care360 Limosilactobacillus reuteri Care360 evolutionary tree diagram;

[0028] Figure 3 This is a heat map of alcohol dehydrogenase activity in different strains;

[0029] Figure 4 This is a heat map of acetaldehyde dehydrogenase activity in different strains;

[0030] Figure 5 Figure 2 is a graph showing the number of viable bacteria of different strains tolerant to ethanol;

[0031] Figure 6 The figure shows the number of viable bacteria of different strains resistant to acetaldehyde;

[0032] Figure 7 This is a graph showing the ethanol tolerance time of mice in different treatment groups;

[0033] Figure 8 Figure 2 is the blood ethanol level of mice in different treatment groups;

[0034] Figure 9 Figure 2 is the blood acetaldehyde level of mice in different treatment groups;

[0035] Figure 10 This is the graph of aspartate aminotransferase activity in mice under different treatment groups;

[0036] Figure 11 This is a graph showing the activity of alanine aminotransferase in mice in different treatment groups;

[0037] Figure 12 This is the graph of alcohol dehydrogenase activity in the liver of mice in different treatment groups;

[0038] Figure 13 This is the graph of acetaldehyde dehydrogenase activity in the liver of mice in different treatment groups;

[0039] Figure 14 Figure 2 shows the reduced glutathione content in the liver of mice in different treatment groups;

[0040] Figure 15 Figure 2 shows the activity of glutathione peroxidase in the liver of mice in different treatment groups;

[0041] Figure 16 Figure 2 is the malondialdehyde content in the liver of mice in different treatment groups;

[0042] Figure 17 Figure 2 is the catalase activity in the liver of mice in different treatment groups;

[0043] Figure 18 Figure 2 is the total antioxidant capacity of the liver of mice in different treatment groups;

[0044] Figure 19 The graph shows the total superoxide dismutase activity in the liver of mice in different treatment groups. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] Example 1

[0051] Lactobacillus paracasei STB Lactobacillus paracasei STB (Lactobacillus paracasei STB) and Lactobacillus mucilaginosus Care360 Limosilactobacillus reuteri Care360 (Lactobacillus reuteri Care360) classification and species identification.

[0052] like Figure 1 As shown, STB and Lactobacillus paracasei ATCC 25302 T In the same evolutionary branch, the branch node is 99%, indicating that STB and Lactobacillus paracasei The genetic relationship is very close. Therefore, the strain STB was finally determined to be Lactobacillus paracasei STB.

[0053]

[0054] like Figure 2 As shown in the figure, Care360 and Limosilactobacillus reuteri subsp. reuteri DSM20016 T In the same evolutionary branch, the branch node is 100%, indicating that Care360 and Limosilactobacillus reuteri subsp. reuteri The strain Care360 was thus identified as Lactobacillus reuteri Care360.

[0055]

[0056] Example 2

[0057] 1. Screening of lactobacilli producing high-activity intracellular alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0058] 1. Source of strain.

[0059] The strains used were all from the strain library of the Microbiology Laboratory of the College of Food Science and Technology of Nanjing Agricultural University.

[0060] 2. Preparation of culture medium.

[0061] The culture medium used was MRS liquid medium with the following ingredients: peptone 10.0 g, glucose 20.0 g, beef extract 10.0 g, yeast extract 5.0 g, anhydrous sodium acetate 5.0 g, triammonium citrate 2.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.58 g, MnSO4·4H2O 0.25 g, Tween-80 1.0 mL, distilled water 1000.0 mL, adjusted to pH 6.8, and sterilized at 121°C for 20 min.

[0062] 3. Determination of alcohol dehydrogenase and acetaldehyde dehydrogenase activity.

[0063] 1) Centrifuge the fermentation broth at 6000 rpm for 5 minutes at 4°C. Discard the supernatant and collect the cells. Resuspend in 1x PBS buffer and centrifuge again. Discard the supernatant and collect the cells. Weigh the fresh weight of the cells and resuspend in physiological saline (0.85% sodium chloride in water). Ultrasonicate the cells at 400 W for 30 minutes on ice, with a 1-second on / 2-second off cycle. Inspect the cells microscopically for complete cell disruption. Centrifuge at 8000 rpm at 4°C for 10 minutes. Collect the supernatant, which is the crude enzyme solution. Store in an ice box at 4°C and assay activity as soon as possible. Prepare the working solution according to the instructions for the alcohol dehydrogenase (ADH) test kit (item number: A083-2-1, Nanjing Jiancheng Bioengineering Institute) and add the sample to be tested as required. Start timing when adding the sample, mix thoroughly, and pipette 200 μL of the mixture into a 96-well plate after 15 seconds. Read the absorbance A1 at 340 nm. Quickly place the reaction solution in a 37°C water bath for 10 minutes, then take it out and read the absorbance A2 at 340 nm. Calculate Each test was repeated three times. A3 is the absorbance of the blank sample before reaction, and A4 is the absorbance of the blank sample after reaction.

[0064] ADH activity (U / g fresh weight) = {[(A2-A1)-(A4-A3)] / (6.22×0.5)}×3000.

[0065] 2) Centrifuge the fermentation broth at 4°C and 6000rpm for 5 minutes, discard the supernatant, collect the bacteria, add 1×PBS buffer to resuspend, centrifuge again, discard the supernatant to collect the bacteria; weigh the fresh weight of the bacteria, add the extract from the aldehyde dehydrogenase (ALDH) test kit (item number: A075-1-1, Nanjing Jiancheng Bioengineering Institute), and ultrasonically disrupt the cells in an ice bath at 400W power for 30 minutes. Work for 1 second and stop for 2 seconds, then check under a microscope to see if the cells are completely broken. Centrifuge at 8000rpm and 4°C for 10 minutes, collect the supernatant, which is the crude enzyme solution. Store it in a 4°C ice box temporarily and perform activity determination as soon as possible. Prepare the working solution according to the instructions for use of the kit, and add the sample to be tested as required. After the sample and working solution are evenly mixed, take 200μL to a 96-well plate, read the absorbance A1 at 340nm, incubate at 37°C for 5 minutes, read the absorbance A2 at 340nm, and calculate , three parallels in each group.

[0066] ;

[0067] In the formula, W is the fresh weight of the bacteria.

[0068] 2. In vitro ethanol and acetaldehyde resistance test.

[0069] The eight lactic acid bacteria strains screened out in "I. Screening of Lactobacillus Producing High-Activity Intracellular Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase" were inoculated into MRS liquid culture medium, cultured at 37°C, and activated and passaged twice (the frozen lactobacilli were inoculated at a volume ratio of 4% and inoculated into MRS liquid culture medium, cultured at 37°C for 24 h, the activated bacterial solution was inoculated at a volume ratio of 4%, and again inoculated into new activation culture medium, cultured at 37°C for 12 h to obtain the secondary activated fermentation broth of lactobacilli). The inoculation volume of 4% was then inoculated into MRS liquid culture medium with 15%, 10%, 5%, and 0% ethanol concentrations and 100× (42 μL of 0.4% acetaldehyde solution was added to 100 mL), 50×, 10×, and 0× acetaldehyde concentrations, respectively. After culture for 3 h, 1 mL of the activated sample was aseptically taken and added to 9 mL of 0.85% sterile saline to dilute to 10 -1 Dilution of concentration, take 1mL from the dilution and add it into a test tube containing 9mL of 0.85% sterile saline to dilute it to 10 -2 Concentration of dilution. So dilute in sequence to get 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Multiple dilution. Select 10 -5 , 10 -6 , 10 -7 , 10-8 0.2 mL of each dilution of the concentration was taken and evenly spread on MRS solid culture medium plates, and cultured upside down in a 37°C incubator for 48 h. The ethanol and acetaldehyde resistance of each strain was compared.

[0070] 3. Preparation of Lactobacillus combination.

[0071] Probiotic fermentation: The activated liquids of Lactobacillus paracasei STB and Lactobacillus reuteri Care360 that have been secondary activated (the frozen lactobacilli are inoculated at an inoculum size of 4% by volume and inoculated into MRS liquid culture medium, cultured at 37°C for 24 hours, the activated bacterial liquid is taken out and inoculated at an inoculum size of 4% by volume and inoculated again into new activation culture medium, cultured at 37°C for 12 hours to obtain secondary activated fermentation liquid of lactobacilli) are inoculated into MRS liquid culture medium at an inoculum size of 4% of the total volume of the MRS liquid culture medium, respectively, and fermented at 37°C for 12 hours to obtain fermentation liquid.

[0072] Normal saline: Add 0.85g sodium chloride to 100mL water, sterilize, and cool for use.

[0073] Preparation of the composition: The fermentation broth of the two probiotics was centrifuged at 4°C and 6000 rpm for 5 min, the supernatant was discarded, and the slurry was mixed with physiological saline and resuspended in the slurry. The slurry was centrifuged at 4°C and 6000 rpm for 5 min, and repeated twice. The slurry was then mixed with physiological saline to prepare a bacterial suspension. Finally, the two probiotics were mixed in a volume ratio of 1:1 (the effective viable count of Lactobacillus paracasei STB was 3.25×10 8 CFU / mL, the effective viable count of Lactobacillus reuteri Care360 is 3.55×10 8 CFU / mL).

[0074] 4. Verification of the effectiveness of the lactobacillus combination in relieving acute drunkenness in animals.

[0075] 1) Determination of the intoxication protocol: Red Star Erguotou (light-flavor liquor, 56% vol) purchased from a supermarket was selected for the mouse intoxication experiment.

[0076] To establish a safe and stable model of acute intoxication, 9-week-old male BALB / c mice weighing 25±2g were randomly divided into three groups (n=8 per group) and administered 4 mg / g BW, 6 mg / g BW, and 8 mg / g BW, respectively. The righting reflex was used as a criterion for determining the degree of intoxication. Briefly, mice were placed on their backs on the ground with their abdomen and limbs facing upward. If the mice failed to turn over within 30 seconds, they were considered to have lost their normal righting reflex. The point of loss of the righting reflex was defined as the point of intoxication, and the time between the first alcohol intake and intoxication was defined as the alcohol tolerance time. The alcohol tolerance time for all three groups of mice was less than 20 minutes. To eliminate the influence of individual variability, mice that did not lose their righting reflex within 1 hour were considered to have not experienced acute intoxication. Considering the intoxication rate and safety, the 6 mg / g BW dose was selected for subsequent experiments (Table 1).

[0077] Table 1 Establishment of acute intoxication model

[0078] ;

[0079] 2) Animal Experiment: Nine-week-old male BALB / c mice weighing 25±2 g were randomly divided into eight treatment groups (n=8 in each treatment group), including blank control group, Lactobacillus plantarum STB9b liquid group, Lactobacillus paracasei STB liquid group, Lactobacillus mucilaginosus reuteri Care360 liquid group, Lactobacillus plantarum STB9b liquid + Lactobacillus paracasei STB liquid group, Lactobacillus plantarum STB9b liquid + Lactobacillus mucilaginosus reuteri Care360 liquid group, Lactobacillus paracasei STB liquid + Lactobacillus mucilaginosus reuteri Care360 liquid group, and Lactobacillus plantarum STB9b liquid + Lactobacillus paracasei STB liquid + Lactobacillus mucilaginosus reuteri Care360 liquid group, with eight mice in each group.

[0080] Before the experiment, mice were fasted for 3 h and then fasted for 1 h. Mice in the blank control group were given an equal volume of normal saline (0.01 mL / g BW) and then given white wine 2 h later. Mice in the Lactobacillus plantarum 9b group were given 1×10 9 cfu / mL (OD 600 =1.0) in normal saline (0.01 mL / g BW) and then administrated with liquor 2 h later; the number of bacteria in each oral gavage of the mice in the Lactobacillus paracasei STB group was 1×10 9 cfu / mL (OD 600 =1.0) in saline (0.01 mL / g BW) and then administrated with liquor 2 h later; the mice in the Lactobacillus reuteri Care360 group received 1×10 9 cfu / mL (OD 600=1.0) (0.01 mL / g BW) of normal saline suspension and then 2 h later, white wine was administered; the number of bacteria per oral administration in the group of Lactobacillus plantarum 9b liquid + Lactobacillus paracasei STB liquid was 1×10 9 cfu / mL (OD 600 =1.0) in normal saline (0.01 mL / g BW) and then administrated with liquor 2 h later; the mice in the Lactobacillus plantarum 9b + Lactobacillus reuteri Care360 bacterial suspension group received 1×10 9 cfu / mL (OD600=1.0) of normal saline bacterial suspension (0.01mL / g BW) and then 2 hours later, white wine was administered orally; the number of bacteria in each oral administration of mice in the Lactobacillus paracasei STB+Lactobacillus reuteri Care360 bacterial suspension group was 1×10 9 cfu / mL (OD 600 =1.0) in normal saline (0.01 mL / g BW) and then 2 h later administered with white wine; the number of bacteria per oral administration was 1×10 9 cfu / mL (OD 600 =1.0) (0.01mL / g BW) of normal saline bacterial suspension and then white wine 2h later.

[0081] 3) Alcohol detoxification effect test.

[0082] Observe the mice's activity. Drag-the-back dragging, unsteady crawling, and closed eyes and laziness are indicators of sleep (drunkenness). Flexible limbs, free movement, and restored spirits are indicators of awakening (soberness). Record the time the mice fall asleep or become drunk.

[0083] Effects on the concentrations of ethanol, acetaldehyde, etc. in the blood of acutely intoxicated mice: 2 hours after gavage treatment, the eyeballs were removed and blood was collected. The blood was centrifuged at 3000×g for 15 minutes at room temperature to obtain serum. The ethanol content detection kit (item number: BC6030, Beijing Solebaugh Technology Co., Ltd.) and the acetaldehyde content determination kit (item number: BL1839B, Lanjieke Technology Co., Ltd.) were used to determine the ethanol and acetaldehyde levels in the mouse serum. The corresponding aspartate aminotransferase (AST / GOT) test kit (item number: C010-2-1, Nanjing Jiancheng Bioengineering Institute) and alanine aminotransferase (ALT / GPT) test kit (item number: C009-2-1, Nanjing Jiancheng Bioengineering Institute) were used to detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum.

[0084] Effects on the activities of alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH) and other enzymes in the liver of acutely drunk mice: After the eyeballs were removed and blood was collected, the mice were killed by cervical dislocation and immediately dissected. The liver was quickly removed, rinsed with pre-cooled saline and dried with filter paper. A small amount of liver was cut and 10% liver homogenate was prepared with saline as the homogenization medium. The supernatant was collected by centrifugation at 3000×g for 15 min, and the ADH and ALDH activities and the contents of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC) (antioxidant index) in the mouse liver homogenate were measured according to the instructions of the ADH detection kit (Nanjing Jiancheng Bioengineering Institute) and the ALDH detection kit (Nanjing Jiancheng Bioengineering Institute).

[0085] 4. Results

[0086] 1) In in vitro experiments, such as Figure 3 、 Figure 4 、 Figure 5 and Figure 6 As shown, strains with high alcohol dehydrogenase and acetaldehyde dehydrogenase activities were screened to obtain eight lactobacilli with excellent activities of the relevant enzymes. Furthermore, the in vitro tolerance of ethanol and acetaldehyde of the eight lactobacilli was further measured, and finally two lactobacilli with high alcohol dehydrogenase and acetaldehyde dehydrogenase activities and strong tolerance to ethanol and acetaldehyde were obtained.

[0087] 2) Based on the actual drinking scenes of most people in their daily lives, the two strains obtained above were combined with a strain that has strong acid resistance and gastrointestinal digestion to further explore their effect on alleviating drunkenness in mice. Figure 7 As shown in the figure, based on the time it took for mice to tolerate alcohol until they were completely drunk, the group treated with Lactobacillus plantarum 9b using the three strains alone was the longest. However, the combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB was the longest. Next, 2 hours after the mice were treated with oral gavage, blood was collected from the eyeballs and the mice were dissected to measure the relevant indicators of the mouse serum and liver. The results were compared with the group of mice that were completely untreated and not given alcohol and the group that was given normal saline and then given alcohol. Figure 8 and Figure 9 As shown in the results, the ethanol and acetaldehyde levels in the mouse serum were roughly consistent with the trend of the tolerance time results. The content of Lactobacillus plantarum 9b was relatively lower in the single strain treatment, and the content of the combination treatment of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB was relatively lowest. Figure 10 and Figure 11As shown in the figure, the activity of AST and ALT in mouse serum is an indicator of the degree of liver damage in mice, and similar results were obtained. In addition, this experiment also tested some indicators in mouse liver, such as Figure 12 and Figure 13 As shown in the data, as for the most relevant results of alcohol dehydrogenase and acetaldehyde dehydrogenase activity in the liver, the content of Lactobacillus plantarum 9b was relatively lower in the single strain treatment, and the content of the combination treatment of Lactobacillus reuteri Care360 and Lactobacillus paracasei was relatively lowest. This may be because the alcohol dehydrogenase and acetaldehyde dehydrogenase of the lactobacillus itself in the oral administration play a compensatory role in the degradation of ethanol and acetaldehyde ingested by mice.

[0088] like Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 and Figure 19 As shown, antioxidant indicators such as SOD activity, GSH content, CAT activity, and GSH-PX activity in the liver were then measured, and results generally consistent with the above trends were obtained. In summary, the present invention has discovered a combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB that is highly effective in alleviating acute intoxication, providing a more practical example and reference for addressing the more common drinking needs of the population.

[0089] Animal experiment results show that a combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB significantly prolonged the time it took mice to go from sober to drunken compared to mice that did not take probiotics. The combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB in the present invention is selected from the "List of Bacteria Suitable for Food Use" and therefore does not have the side effects of existing hangover medications on the market. The probiotic composition of the present invention, through specific probiotic strains and their metabolites, can effectively alleviate the symptoms of acute drunkenness, reduce serum ALT and AST levels, and thus reduce liver tissue pathological damage. The probiotic composition of the present invention has significant antioxidant and anti-inflammatory effects, and can reduce oxidative stress and inflammatory responses caused by excessive drinking.

[0090] The lactobacillus exhibited significant ability or effect in degrading ethanol and acetaldehyde in both in vitro and in vivo experiments, and its activity was superior to that of most existing lactobacilli.

[0091] The combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB described herein rapidly alleviates inflammation following acute alcohol intoxication by inhibiting the production of reactive oxygen species (ROS) and promoting the activity of antioxidant enzymes (such as SOD and GSH-PX). The strain culture is simple and employs mild conditions, making it suitable for large-scale industrial production. The isolation method is environmentally friendly, efficient, and simple, requiring no extensive processing equipment. This combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB can be used to alleviate not only the symptoms of acute alcohol intoxication but also the inflammatory damage caused by alcohol consumption, offering broad application prospects. In in vitro testing, this combination of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB demonstrated high alcohol dehydrogenase and acetaldehyde dehydrogenase activities, as well as excellent tolerance to ethanol and acetaldehyde. This combination can significantly alleviate the symptoms of acute alcohol intoxication in animals and suppress inflammation, potentially improving users' ability to avoid the discomfort associated with acute alcohol intoxication. After mice took the combination, serum ethanol and acetaldehyde concentrations significantly decreased, and AST and ALT activity levels, reflecting liver damage from excessive alcohol consumption, improved. The combination was highly effective, with liver acetaldehyde dehydrogenase and acetaldehyde dehydrogenase activity remaining relatively normal after a single brief dose, reducing the metabolic burden on the liver caused by excessive ethanol and acetaldehyde concentrations following intoxication. In mouse experiments, the combination demonstrated significant inhibitory effects on inflammation, as measured by antioxidant markers (SOD, GSH, GSH-PX, etc.) in the mouse liver. This effect was evident after just one dose, potentially reducing the frequency of use and overall liver risk and burden after alcohol consumption. This combination offers new insights for alleviating acute intoxication and lays the foundation for the subsequent development of probiotics more tailored to practical applications, such as compound and sustained-release formulations.

[0092] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A probiotic composition for relieving acute drunkenness, characterized in that: The probiotic composition is composed of Lactobacillus paracasei ( Lacticaseibacillus paracasei )STB and Lactobacillus reuteri ( Limosilactobacillus reuteri ) Care360 composition; The Lactobacillus paracasei STB was deposited in the China Center for Type Culture Collection on June 14, 2022, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M2022881; The Lactobacillus reuteri Care360 was deposited on April 2, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M2025682. The mass ratio of the Lactobacillus paracasei STB to the Lactobacillus reuteri Care360 in the probiotic composition is 1:

1.

2. The probiotic composition according to claim 1, characterized in that The effective viable count of the Lactobacillus paracasei STB is 2×10 8 ~4.5×10 8 CFU / mL, the effective viable count of Lactobacillus reuteri Care360 is 3×10 8 ~4.1×10 8 CFU / mL.

3. The method for preparing the probiotic composition according to claim 1 or 2, wherein: The following steps are involved: (1) Lactobacillus paracasei STB is inoculated into MRS liquid culture medium and fermented to obtain Lactobacillus paracasei fermentation liquid; (2) Lactobacillus reuteri Care360 was inoculated into MRS liquid culture medium and fermented to obtain Lactobacillus reuteri fermentation liquid; (3) The Lactobacillus paracasei fermentation liquid obtained in step (1) is centrifuged to obtain a Lactobacillus paracasei bacterial sludge, and the Lactobacillus paracasei bacterial sludge is resuspended in physiological saline to obtain a Lactobacillus paracasei bacterial suspension; (4) centrifuging the Lactobacillus reuteri fermentation broth obtained in step (2) to obtain Lactobacillus reuteri bacterial sludge, and resuspending the Lactobacillus reuteri bacterial sludge with physiological saline to obtain a Lactobacillus reuteri bacterial suspension; (5) The Lactobacillus paracasei suspension obtained in step (3) and the Lactobacillus muciniphila suspension obtained in step (4) are mixed in a mass ratio of 1:1 to obtain a probiotic composition.

4. The preparation method according to claim 3, characterized in that The inoculation amount of Lactobacillus paracasei STB in step (1) is calculated as 2-6% of the volume of the MRS liquid culture medium; the fermentation temperature in step (1) is 37° C., and the fermentation time is 8-16 h.

5. The preparation method according to claim 3, characterized in that: The inoculation amount of Lactobacillus reuteri Care360 in step (2) is calculated as 2-6% of the volume of the MRS liquid culture medium; the fermentation temperature in step (2) is 37° C., and the fermentation time is 8-16 h.

6. The preparation method according to claim 3, characterized in that: The centrifugal temperature in step (3) is 0-4°C, the centrifugal speed is 5000-7000 rpm, and the centrifugal time is 4-6 min; the effective viable bacteria count in the Lactobacillus paracasei suspension in step (3) is 2×10 8 ~4.5×10 8 CFU / mL.

7. The preparation method according to claim 3, characterized in that: The centrifugation temperature in step (4) is 0-4°C, the centrifugation speed is 5000-7000 rpm, and the centrifugation time is 4-6 min; the effective viable count of Lactobacillus reuteri suspension in step (4) is 3×10 8 ~4.1×10 8 CFU / mL.

8. Use of the probiotic composition according to claim 1 or 2 in preparing a rapid alcohol sobering and liver protecting product, characterized in that: The product described is a pharmaceutical product.

9. Use of the probiotic composition prepared by the preparation method according to any one of claims 3 to 7 in preparing a fast alcohol sobering and liver protecting product, characterized in that: The product described is a pharmaceutical product.

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