Probiotic composition for relieving acute drunkenness as well as preparation method and application thereof

Abstract

The invention discloses a probiotic composition for relieving acute drunkenness as well as a preparation method and application of the probiotic composition, belongs to the technical field of probiotic application, and provides a probiotic composition for relieving acute drunkenness, and is characterized in that the probiotic composition comprises lactobacillus paracasei STB and lactobacillus reuteri Cre360, the lactobacillus paracasei STB comprises lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus reuteri Cre360, lactobacillus paracasei, lactobacillus The preservation number of the lactobacillus paracasei STB is CCTCC (China Center For Type Culture Collection) NO: M2022881; the preservation number of the lactobacillus reuteri Crea360 is CCTCC (China Center for Type Culture Collection) NO: M2025682. The probiotic combination group is formed by combining lactobacillus paracasei STB and lactobacillus reuteri mucus Cre360, so that the symptom of acute drunkenness can be effectively relieved, and the probiotic combination group has the effects of resisting oxidation and resisting inflammation.

Description

Technical Field

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

[0002] The liquor consumption in China has always been at the forefront. According to current research and investigation reports in the field, the number of people suffering from liver damage caused by excessive drinking of ethanol cannot be ignored. At present, liquor plays an indispensable role in the daily life of most people. Based on this, there is a problem of how to solve the emergency of relieving drunkenness when a person ingests a large amount of liquor in a short time. In addition, most people have a low rate of ethanol catabolism, which increases the probability of damage to the body's health caused by ethanol. Research shows that short-term drinking of liquor and getting drunk will not only cause various uncomfortable symptoms in the body, but also increase the risk of inducing potential diseases such as inflammation. In recent years, a large number of products on the market mainly focus on treating or relieving acute or chronic alcoholic liver injury, including antioxidants, anti-inflammatory drugs, hepatoprotective drugs, and probiotics. However, these drugs have problems such as large side effects, limited efficacy, and limited effects.

[0003] In recent years, a number of patents in the field have mentioned the use of probiotics to relieve alcoholic liver injury and discomfort symptoms after drinking. For example, the patent with the publication number CN117987307A, titled "A Lactobacillus acidipiscis strain capable of relieving acute alcoholic liver injury and its application", mentions that Lactobacillus acidipiscis ( Ligilactobacillus acidipiscis ), isolated from the fermented grains and cellar mud of old cellars, has significant ethanol tolerance and can relieve alcoholic liver injury; the patent with the publication number CN117801991A, titled "Probiotic postbiotic composition for relieving discomfort symptoms after drinking and its use", mentions using a probiotic postbiotic composition of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ), ProSci-246, to relieve discomfort symptoms after drinking; the patent with the publication number CN114426942A, titled "A recombinant Lactococcus lactis, microcapsule and its application", mentions constructing recombinant Lactococcus lactis by genetic engineering technology to express alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) to decompose alcohol and reduce the damage of alcohol to the liver and intestine; the patent with the publication number CN116622559A, titled "A Lactobacillus casei strain producing aldehyde dehydrogenase, an anti-alcoholism probiotic composition and its preparation method", mentions using Lactobacillus casei ( Lactobacillus casei) (FPHC0700) is combined with other ingredients (such as mung bean, 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 effect of a single strain in the prior art is 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 a combination with 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 the existing hangover relief products can relieve 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 of alcohol to the liver. The intestinal flora of alcoholics is seriously disordered. Although the 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 that can relieve acute alcoholic liver damage and its application" mentioned 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" mentioned that although existing sobering up products can shorten the recovery time after drinking, the sobering up speed is still slow, especially after drinking a lot of alcohol, the existing probiotic products cannot quickly decompose alcohol, resulting in a longer drunken time. There is no detailed research on the existing probiotic sobering up products in relieving 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 relieving acute drunkenness, and the probiotic composition includes Lactobacillus paracasei STB Lactobacillus paracasei STB and Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360; The Lactobacillus paracasei STB Lactobacillus paracasei STB was deposited at the China Center for Type Culture Collection on June 14, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2022881; The Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360 was deposited at the China Center for Type Culture Collection on April 2, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025682.

[0007] Preferably, in the probiotic composition, the mass ratio of the Lactobacillus paracasei STB Lactobacillus paracasei STB to the Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360 is 1:1.

[0008] Preferably, the effective viable count of the Lactobacillus paracasei STB Lactobacillus paracasei STB is 2×10 8 ~4.5×10 8 CFU / mL, and the effective viable count of the Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360 is 3×10 8 ~4.1×10 8 CFU / mL.

[0009] The present invention also provides a preparation method of the probiotic composition, including the following steps: (1) Inoculate Lactobacillus paracasei STB Lactobacillus paracasei STB into MRS liquid medium and ferment to obtain a Lactobacillus paracasei fermentation broth; (2) Inoculate Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360 into MRS liquid medium and ferment to obtain a Lactobacillus mucosae fermentation broth; (3) Centrifuge the Lactobacillus paracasei fermentation broth obtained in step (1) to obtain a Lactobacillus paracasei bacterial sludge, and resuspend the Lactobacillus paracasei bacterial sludge with physiological saline to obtain a Lactobacillus paracasei bacterial suspension; (4) Centrifuge the Lactobacillus mucosae fermentation broth obtained in step (2) to obtain a Lactobacillus mucosae bacterial sludge, and resuspend the Lactobacillus mucosae bacterial sludge with physiological saline to obtain a Lactobacillus mucosae bacterial suspension; (5) The Lactobacillus paracasei bacterial suspension obtained in step (3) and the Lactobacillus mucosae Care360 bacterial suspension obtained in step (4) are mixed in a mass ratio of 1:1 to obtain the probiotic composition.

[0010] Preferably, the Lactobacillus paracasei STB in step (1) Lactobacillus paracasei The inoculation amount of STB is calculated according to 2-6% of the volume of the MRS liquid medium; the fermentation temperature in step (1) is 37 °C, and the fermentation time is 8-16 h.

[0011] Preferably, the Lactobacillus mucosae Care360 in step (2) Limosilactobacillus reuteri The inoculation amount of Care360 is calculated according to 2-6% of the volume of the MRS liquid medium; the fermentation temperature in step (2) is 37 °C, and the fermentation time is 8-16 h.

[0012] Preferably, the centrifugation temperature in step (3) is 0-4 °C, the centrifugation speed is 5000-7000 rpm, and the centrifugation time is 4-6 min; the effective viable count in the Lactobacillus paracasei bacterial suspension in step (3) is 2×10 8 ~4.5×10 8 CFU / mL.

[0013] 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 in the Lactobacillus mucosae bacterial suspension in step (4) is 3×10 8 ~4.1×10 8 CFU / mL.

[0014] The present invention also provides the application of the probiotic composition in the preparation of a product for quickly relieving alcohol and protecting the liver.

[0015] The present invention also provides the application of the probiotic composition prepared by the preparation method in the preparation of a product for quickly relieving alcohol and protecting the liver.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention uses Lactobacillus mucosae Care360 for the first time Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB Lactobacillus paracaseiAs the core strain combination, both of these two strains have the characteristics of high gastric acid tolerance and bile salt tolerance, and can survive in the intestine and produce a large amount of alcohol dehydrogenase and aldehyde dehydrogenase, so as to effectively decompose alcohol and its metabolite acetaldehyde, and then relieve the discomfort symptoms caused by acute drunkenness and reduce the possibility of inflammation induced thereby. By using Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360 with high alcohol dehydrogenase and aldehyde dehydrogenase production and Lactobacillus paracasei STB with high gastric acid tolerance and bile salt tolerance Lactobacillus paracasei STB combination, this combination has the effect of relieving acute drunkenness, and can synergistically relieve acute drunkenness and prevent damage such as body inflammation caused by excessive drinking. This strain combination for relieving acute drunkenness is more in line with the actual use scenario, can more truly reflect its hangover effect, and further improve the protective effect of the lactobacillus combination on acute drunkenness. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is Lactobacillus paracasei STB Lactobacillus paracasei Phylogenetic tree diagram of STB; Figure 2 It is Lactobacillus mucosae Care360 Limosilactobacillus reuteri Phylogenetic tree diagram of Care360; Figure 3 It is the heat map of alcohol dehydrogenase activity of different strains; Figure 4 It is the heat map of aldehyde dehydrogenase activity of different strains; Figure 5 It is the viable cell number map of different strains tolerant to ethanol; Figure 6 It is the viable cell number map of different strains tolerant to acetaldehyde; Figure 7 It is the ethanol tolerance time map of mice in different treatment groups; Figure 8 It is the blood ethanol level map of mice in different treatment groups; Figure 9 It is the blood acetaldehyde level map of mice in different treatment groups; Figure 10 It is the glutamic oxaloacetic transaminase activity map of mice in different treatment groups; Figure 11 It is the glutamic pyruvic transaminase activity map of mice in different treatment groups; Figure 12 The figure shows the ethanol dehydrogenase activity in the livers of mice in different treatment groups; Figure 13 The figure shows the acetaldehyde dehydrogenase activity in the livers of mice in different treatment groups; Figure 14 The figure shows the reduced glutathione content in the livers of mice in different treatment groups; Figure 15 The figure shows the glutathione peroxidase activity in the livers of mice in different treatment groups; Figure 16 The figure shows the malondialdehyde content in the livers of mice in different treatment groups; Figure 17 The figure shows the catalase activity in the livers of mice in different treatment groups; Figure 18 The figure shows the total antioxidant capacity in the livers of mice in different treatment groups; Figure 19 The figure shows the total superoxide dismutase activity in the livers of mice in different treatment groups. Detailed implementation manners

[0019] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0022] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0023] As used herein, terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0024] Example 1 Lactobacillus paracasei STB Lactobacillus paracasei STB (Lactobacillus paracasei STB) and Lactobacillus mucosae Care360 Limosilactobacillus reuteri Classification and species identification of Care360 (Lactobacillus mucosae Care360).

[0025] As Figure 1 shown, STB and Lactobacillus paracasei ATCC 25302 T are in the same evolutionary branch, and the node of the branch is 99%, indicating that STB and Lactobacillus paracasei have a very close genetic relationship. Therefore, the strain STB was finally determined to be Lactobacillus paracasei STB.

[0026]

[0027] As shown Figure 2 in the figure, Care360 and Limosilactobacillus reuteri subsp. reuteri DSM20016 T are on the same evolutionary branch, and the node of the branch is 100%, indicating that Care360 and Limosilactobacillus reuteri subsp. reuteri are closely related. Therefore, the strain Care360 was finally determined to be Limosilactobacillus reuteri Care360.

[0028]

[0029] Example 2 I. Screening of Lactobacillus with high - activity intracellular alcohol dehydrogenase and aldehyde dehydrogenase.

[0030] 1. Strain source.

[0031] All the strains used were from the strain bank reserved in the Microbiology Laboratory of the College of Food Science and Technology, Nanjing Agricultural University.

[0032] 2. Preparation of the culture medium.

[0033] The culture medium used was MRS liquid medium, and the components were as follows: peptone 10.0 g, glucose 20.0 g, beef extract 10.0 g, yeast extract 5.0 g, anhydrous sodium acetate 5.0 g, ammonium 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. Adjust the pH to 6.8 and sterilize at 121℃ for 20 min.

[0034] 3. Determination of the activities of alcohol dehydrogenase and aldehyde dehydrogenase.

[0035] 1) Take the fermentation broth, centrifuge at 4℃ and 6000 rpm for 5 min, discard the supernatant, collect the thalli, resuspend with 1×PBS buffer, centrifuge again, discard the supernatant and collect the thalli. Weigh the fresh weight of the thalli, resuspend the thalli with normal saline (sodium chloride aqueous solution with a mass concentration of 0.85%), and ultrasonically disrupt at a power of 400 W in an ice bath for 30 min. After working for 1 s and stopping for 2 s, check under the microscope whether the cells are completely disrupted. Centrifuge at 8000 rpm and 4℃ for 10 min, collect the supernatant, which is the crude enzyme solution. Place it in an ice box at 4℃ for temporary storage and perform the activity determination as soon as possible. Prepare the working solution according to the instruction manual of the alcohol dehydrogenase (ADH) test kit (product 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 well, and at 15 s, pipette 200 μL of the mixed solution into a 96 - well plate, read the absorbance A1 at 340 nm. Quickly place the reaction solution in a water bath at 37℃ for 10 min and then take it out, read the absorbance A2 at 340 nm, and calculate , and each group of experiments is parallel three times. Among them, A3 is the absorbance of the blank sample before the reaction, and A4 is the absorbance of the blank sample after the reaction.

[0036] ADH activity (U / g fresh weight) = {[(A2 - A1) - (A4 - A3)] / (6.22×0.5)}×3000.

[0037] 2) Centrifuge the fermentation broth at 4°C and 6000 rpm for 5 min, discard the supernatant, collect the bacterial cells, resuspend them in 1×PBS buffer, centrifuge again, and discard the supernatant to collect the bacterial cells; weigh the fresh weight of the bacterial cells, add the extract in the aldehyde dehydrogenase (ALDH) test kit (product item number: A075-1-1, Nanjing Jiancheng Bioengineering Institute), and ultrasonically disrupt at a power of 400 W in an ice bath for 30 min. After working for 1 s and stopping for 2 s, check under a microscope whether the cells are completely disrupted. Centrifuge at 8000 rpm and 4°C for 10 min, collect the supernatant, which is the crude enzyme solution. Store it temporarily in an ice box at 4°C and perform the activity assay as soon as possible. Prepare the working solution according to the kit instructions and add the test sample as required. After mixing the sample and the working solution evenly, take 200 μL and transfer it to a 96-well plate, read the absorbance A1 at 340 nm, incubate at 37°C for 5 min, read the absorbance A2 at 340 nm, and calculate , with three parallels in each group.

[0038] ; In the formula, W is the fresh weight of the bacterial cells.

[0039] II. In vitro ethanol and acetaldehyde tolerance tests.

[0040] Respectively inoculate the 8 strains of lactic acid bacteria screened in "I. Screening of Lactobacillus with High-Vitality Intracellular Ethanol Dehydrogenase and Aldehyde Dehydrogenase" into MRS liquid medium, culture at 37°C, and activate and passage twice (calculate the inoculation amount of the cryopreserved Lactobacillus as 4% by volume, inoculate it into MRS liquid medium, culture at 37°C for 24 h, take the activated bacterial liquid, calculate the inoculation amount as 4% by volume, and inoculate it into a new activated medium again, culture at 37°C for 12 h to obtain the secondary activated fermentation broth of Lactobacillus), and then inoculate them into MRS liquid medium with ethanol concentrations of 15%, 10%, 5%, 0% and acetaldehyde concentrations of 100× (add 42 μL of 0.4% acetaldehyde solution to 100 mL), 50×, 10×, 0× at an inoculation amount of 4%. After culturing for 3 h, take 1 mL of the activated sample under sterile conditions and add it to 9 mL of 0.85% sterile physiological saline to dilute it to 10 -1 dilution of concentration, pipette 1 mL from the dilution and add it to a test tube containing 9 mL of 0.85% sterile physiological saline to dilute it to 10 -2 dilution of concentration. Dilute in this way successively to obtain dilutions of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 times dilutions. Select 10 -5 , 10 -6 , 10 -7 , 10 -8Diluted solutions of various concentrations were each taken at 0.2 mL and evenly spread on MRS solid medium plates, which were then incubated upside down in an incubator at 37 °C for 48 h to compare the ethanol tolerance and acetaldehyde tolerance of each strain.

[0041] III. Preparation of Lactobacillus combinations.

[0042] Probiotic fermentation: The activated solutions of Lactobacillus paracasei STB and Lactobacillus reuteri Care360 after secondary activation (the frozen-preserved Lactobacillus was inoculated at an inoculation amount of 4% by volume into MRS liquid medium and cultured at 37 °C for 24 h. The activated bacterial liquid was taken and inoculated again into a new activated medium at an inoculation amount of 4% by volume and cultured at 37 °C for 12 h to obtain the secondary activated fermentation liquid of Lactobacillus) were each inoculated into MRS liquid medium at an inoculation amount of 4% of the total volume of MRS liquid medium and fermented at 37 °C for 12 h to obtain fermentation broths.

[0043] Normal saline: 0.85 g of sodium chloride was added to 100 mL of water, sterilized, and cooled for later use.

[0044] Composition preparation: The two probiotic fermentation broths were each centrifuged at 4 °C and 6000 rpm for 5 min, the supernatant was poured off, and then the bacterial sludge was resuspended and mixed with normal saline and centrifuged at 4 °C and 6000 rpm for 5 min, and this was repeated twice. Then, the bacterial suspension was prepared by mixing with normal saline again. Finally, the two probiotics were mixed at a volume ratio of 1:1 (the effective viable count of Lactobacillus paracasei STB was 3.25×10 8 CFU / mL, and the effective viable count of Lactobacillus reuteri Care360 was 3.55×10 8 CFU / mL).

[0045] IV. Verification of the effect of the Lactobacillus combination in alleviating acute drunkenness in animals.

[0046] 1) Determination of the drunkenness protocol: Red Star Erguotou (light-flavor liquor, 56% vol) purchased from the supermarket was selected for the mouse drunkenness experiment.

[0047] To establish a safe and stable acute intoxication model, 9-week-old male BALB / c mice weighing 25±2g were randomly divided into three groups (n=8 in each group) and given 4mg / g BW, 6mg / g BW, and 8mg / g BW, respectively. The righting reflex was used as the criterion for determining the degree of intoxication. Briefly, the mouse was placed on its back with its abdomen and limbs facing upward. If the mouse could not turn over within 30s, it was considered to have lost its normal righting reflex. The time point when the righting reflex disappeared was defined as the intoxication point, and the time point between the first drinking and intoxication was defined as the alcohol tolerance time. The alcohol tolerance time of the three groups of mice was less than 20min. In order to exclude the influence of individual differences, we defined that mice that did not lose the righting reflex within 1h were considered to be not acutely intoxicated. Considering the intoxication rate and safety, a dose of 6mg / gBW was selected for subsequent experiments (Table 1).

[0048] Table 1 Establishment of acute intoxication model ;

[0049] 2) Animal experiment: 9-week-old male BALB / c mice weighing 25±2g were randomly divided into 8 treatment groups (n=8 in each treatment group), including blank control group, Lactobacillus plantarum STB9b liquid group, Lactobacillus paracasei STB liquid group, Lactobacillus muciniphila Care360 liquid group, Lactobacillus plantarum STB9b liquid + Lactobacillus paracasei STB liquid group, Lactobacillus plantarum STB9b liquid + Lactobacillus muciniphila Care360 liquid group, Lactobacillus paracasei STB + Lactobacillus muciniphila Care360 liquid group and Lactobacillus plantarum STB9b liquid + Lactobacillus paracasei STB liquid group + Lactobacillus muciniphila Care360 liquid group, with 8 mice in each group.

[0050] Before the experiment, mice were fasted but not allowed to drink for 3 h, and then fasted but not allowed to drink for 1 h. Mice in the blank control group were given an equal volume of saline (0.01 mL / g BW) and then given liquor 2 h later. Mice in the Lactobacillus plantarum 9b group were given a bacterial liquid with a bacterial count of 1×10 9 cfu / mL(OD 600 =1.0) (0.01 mL / g BW) of physiological saline suspension and then administrated with liquor 2 h later; the number of bacteria in each oral gavage of the mice in the STB group was 1×10 9 cfu / mL(OD 600 =1.0) (0.01 mL / g BW) of physiological saline bacterial suspension and then administrated with liquor 2 h later; the number of bacteria per gavage was 1×10 9 cfu / mL(OD 600After administering a saline bacterial suspension of (0.01 mL / g BW) with an optical density (OD 9 ) of 1.0, the mice were given white wine 2 hours later. For the group of Lactiplantibacillus plantarum 9b bacterial liquid + Lactobacillus paracasei STB bacterial liquid, the number of bacteria administered to each mouse by gavage was 1×10 9 cfu / mL (OD 600 After administering a saline bacterial suspension of (0.01 mL / g BW) with an optical density (OD 9 ) of 1.0, the mice were given white wine 2 hours later. For the group of Lactiplantibacillus plantarum 9b + Limosilactobacillus reuteri Care360 bacterial liquid, the number of bacteria administered to each mouse by gavage was 1×10 9 cfu / mL (OD600 = 1.0). After administering a saline bacterial suspension of (0.01 mL / g BW) 2 hours later, the mice were given white wine. For the group of Lactobacillus paracasei STB + Limosilactobacillus reuteri Care360 bacterial liquid, the number of bacteria administered to each mouse by gavage was 1×10 9 cfu / mL (OD 600 After administering a saline bacterial suspension of (0.01 mL / g BW) with an optical density (OD 9 ) of 1.0, the mice were given white wine 2 hours later. For the group of Lactiplantibacillus plantarum 9b bacterial liquid + Lactobacillus paracasei STB bacterial liquid + Limosilactobacillus reuteri Care360 bacterial liquid, the number of bacteria administered to each mouse by gavage was 1×10 9 cfu / mL (OD 600 After administering a saline bacterial suspension of (0.01 mL / g BW) with an optical density (OD 600 ) of 1.0, the mice were given white wine 2 hours later.

[0051] 3) Hangover effect test.

[0052] Observe the activities of the mice. Use the dragging of the hind abdomen, unsteady crawling, and lazy movement with closed eyes of the mice as the indicators of sleep (drunkenness), and use the flexible limbs, free movement, and mental recovery as the indicators of awakening (sobering up). Record the time for the mice to fall asleep and get drunk.

[0053] Effects on the concentrations of ethanol, acetaldehyde, etc. in the blood of acutely drunk mice: 2 hours after gavage treatment, draw blood from the orbital sinus of the mice, collect the blood, centrifuge the blood at 3000×g for 15 minutes at room temperature to obtain serum. Use an ethanol content detection kit (product number: BC6030, Beijing Solarbio Science & Technology Co., Ltd.), an acetaldehyde content determination kit (product number: BL1839B, LanJieK Co., Ltd.) to measure the ethanol and acetaldehyde contents in the mouse serum, and use the corresponding aspartate aminotransferase (AST / GOT) test kit (product number: C010-2-1, Nanjing Jiancheng Bioengineering Institute), alanine aminotransferase (ALT / GPT) test kit (product number: C009-2-1, Nanjing Jiancheng Bioengineering Institute) to detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum.

[0054] Effects on the activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), etc. in the livers of acutely intoxicated mice: After blood collection by eye enucleation in each group, the mice were sacrificed by cervical dislocation, immediately dissected, the livers were quickly removed, rinsed with pre-cooled physiological saline and blotted dry with filter paper. A small amount of liver was cut and a 10% liver homogenate was prepared using physiological saline as the homogenization medium. After centrifugation at 3000×g for 15 min, the supernatant was taken. According to the instructions of the ADH detection kit (Nanjing Jiancheng Bioengineering Institute), ALDH detection kit (Nanjing Jiancheng Bioengineering Institute), etc., the activities of ADH and ALDH in the mouse liver homogenate and the contents of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC) (antioxidant indicators), etc. were measured.

[0055] IV. Results.

[0056] 1) In in vitro experiments, as Figure 3 , Figure 4 , Figure 5 and Figure 6 showed, strains with higher activities of alcohol dehydrogenase and aldehyde dehydrogenase were screened, and 8 Lactobacillus strains with relatively excellent activities of related enzymes were obtained. Furthermore, the in vitro ethanol and acetaldehyde tolerance abilities of the above 8 Lactobacillus strains were continuously measured, and finally two Lactobacillus strains with higher activities of alcohol dehydrogenase and aldehyde dehydrogenase and stronger ethanol and acetaldehyde tolerance abilities were obtained.

[0057] 2) Combining the actual daily drinking scenarios of most people, the two obtained strains and a strain with strong acid tolerance and gastrointestinal digestion ability were combined to further explore their effects on alleviating mouse drunkenness. In terms of the most intuitive data, as Figure 7 showed, based on the tolerance time of mice from after gavage with alcohol to complete drunkenness, when the 3 strains were used alone for gavage in mice, it was found that the treatment group with Lactiplantibacillus plantarum 9b had the longest tolerance time. However, when the strains were combined and gavaged to mice, the treatment group with Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB in combination had the longest tolerance time. Next, 2 h after the gavage treatment of mice, blood was collected by eye enucleation and the mice were dissected, and the relevant indicators of mouse serum and liver were measured. Comparing with the group of mice that were completely untreated and not gavaged with alcohol and the group of mice that were gavaged with physiological saline and then gavaged with alcohol, as Figure 8 and Figure 9 showed, it was found that the contents of ethanol and acetaldehyde in the mouse serum also generally conformed to the trend of the tolerance time results. Among the single-strain treatments, the content of Lactiplantibacillus plantarum 9b was relatively lower, and the content of the combination of Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB was relatively the lowest; as Figure 10 and Figure 11As shown, the activities of AST and ALT in mouse serum are indicators reflecting the degree of liver damage in mice, and roughly similar results were obtained. In addition, some indicators in the mouse liver were also detected in this experiment, such as Figure 12 and Figure 13 As shown, in terms of the results of the activities of alcohol dehydrogenase and aldehyde dehydrogenase in the liver, which are the most relevant, the content of Lactiplantibacillus plantarum 9b was relatively lower in the single-strain treatment, and the content in the combined treatment of Limosilactobacillus reuteri Care360 and Lactobacillus paracasei was relatively the lowest. This may be because the alcohol dehydrogenase and aldehyde dehydrogenase of the orally administered Lactobacillus degraded the ethanol and acetaldehyde ingested by the mice and played a compensatory role.

[0058] Such as Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 and Figure 19 As shown, then the antioxidant indexes such as SOD activity, GSH content, CAT activity and GSH-PX activity in the liver were measured, and the results roughly conforming to the above trend were also obtained. All in all, the present invention discovers a combined treatment of Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB that has an excellent effect on alleviating acute drunkenness, and gives more application-scenario examples and references for the more common drinking needs in the population.

[0059] The results of animal experiments show that the combined composition of Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB significantly prolongs the time from sobriety to drunkenness in mice compared with mice that did not take probiotics. The combined bacteria of Limosilactobacillus reuteri Care360 and Lactobacillus paracasei STB in the present invention are selected from the List of Strains That Can Be Used in Foods, so there are no side effects like existing anti-hangover drugs on the market. The probiotic composition of the present invention can effectively relieve the symptoms of acute drunkenness, reduce the levels of ALT and AST in serum, and thus reduce the histopathological damage of liver tissue through specific probiotic strains and their metabolites. 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.

[0060] This Lactobacillus showed significant ability or effect in degrading ethanol and acetaldehyde in in vitro and in vivo experiments, and its activity is superior to most existing Lactobacillus.

[0061] The combination composition of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB described in the present invention can rapidly relieve the inflammation after acute drunkenness by inhibiting ROS (reactive oxygen species) and promoting the activity of antioxidant enzymes (such as SOD, GSH-PX). The strain culture method is simple and the culture conditions are mild, which is suitable for large-scale industrial production. The strain isolation method is green, efficient and simple, without excessive processing equipment. The combination composition of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB can not only be used to relieve the symptoms of acute drunkenness, but also to relieve the inflammatory damage of the body caused by drinking, and has broad application prospects. In in vitro experimental tests, the combination composition of Lactobacillus reuteri Care360 and Lactobacillus paracasei STB showed good alcohol dehydrogenase and aldehyde dehydrogenase activities, and also had excellent tolerance to ethanol and acetaldehyde. It can significantly relieve the symptoms of acute drunkenness in animals and inhibit the potential of inflammation, and improve the discomfort symptoms of users from acute drunkenness. After mice took this composition, the concentrations of ethanol and acetaldehyde in the serum decreased significantly, and the degree of liver damage reflected by the activity levels of AST and ALT was improved. The composition is highly efficient. After mice took this composition once briefly, the activities of alcohol dehydrogenase and aldehyde dehydrogenase in the liver were relatively normal, reducing the burden on liver metabolism caused by excessive ethanol and acetaldehyde concentrations after drunkenness. In the mouse experiment, by measuring the antioxidant indexes (such as SOD, GSH, GSH-PX, etc.) in the mouse liver, it was found that the composition had a better inhibitory effect on the occurrence of inflammation, and showed a better effect after using the composition once, which can reduce the consumption frequency of users, thereby reducing the overall risk and burden of the liver after drinking. The combination provides a new idea for relieving acute drunkenness and lays a foundation for the subsequent development of probiotics that are more suitable for actual application scenarios (such as compound preparations, sustained-release preparations).

[0062] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

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

2. The probiotic composition according to claim 1, wherein The Lactobacillus paracasei STB in the probiotic composition Lactobacillus paracasei STB and the Limosilactobacillus reuteri Care360 Limosilactobacillus reuteri The mass ratio of Care360 is 1:

1.

3. The probiotic composition according to claim 1, wherein The Lactobacillus paracasei STB Lactobacillus paracasei The viable count of STB is 2×10 8 ~4.5×10 8 CFU / mL, and the Lactobacillus mucosae Care360 Limosilactobacillus reuteri The viable count of Care360 is 3×10 8 ~4.1×10 8 CFU / mL.

4. The preparation method of the probiotic composition according to any one of claims 1 to 3, characterized in that, The following steps are involved: (1)Lactobacillus paracasei STB Lactobacillus paracasei STB was inoculated into MRS liquid medium and fermented to obtain the Lactobacillus paracasei fermentation broth; (2)Lactobacillus mucosae Care360 Limosilactobacillus reuteri Care360 was inoculated into MRS liquid medium and fermented to obtain the fermentation broth of Lactobacillus mucosae Care360; (3) The Lactobacillus paracasei fermentation liquid obtained in step (1) is centrifuged to obtain 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.

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

6. The preparation method according to claim 4, characterized in that, Lactobacillus reuteri Care360 described in step (2) Limosilactobacillus reuteri The inoculum amount of Care360 is calculated according to 2-6% of the volume of MRS liquid medium; the fermentation temperature in step (2) is 37 °C, and the fermentation time is 8-16 h.

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

8. The preparation method according to claim 4, wherein In step (4), the temperature of centrifugation is 0~4°C, the rotation speed of centrifugation is 5000~7000 rpm, and the time of centrifugation is 4~6 min; the number of effective viable bacteria in the Lactobacillus reuteri mucilage suspension in step (4) is 3×10 8 ~4.1×10 8 CFU / mL.

9. Use of the probiotic composition according to any one of claims 1 to 3 in the preparation of a rapid alcohol sobering and liver protecting product.

10. Use of the probiotic composition prepared by the preparation method according to any one of claims 4 to 8 in preparing a quick alcohol sobering and liver protecting product.

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