Lactobacillus paracasei BL19 and application of lactobacillus paracasei BL19 in preparation of products with functions of dispelling effects of alcohol and protecting liver

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus paracasei BL19 and application thereof to preparation of products with functions of dispelling effects of alcohol and protecting liver. The lactobacillus paracasei BL19 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the lactobacillus paracasei BL19 is CGMCC No.34368. The lactobacillus paracasei BL19 is named as Lactobacillus paracasei BL19. The lactobacillus paracasei BL19 provided by the invention has relatively strong alcohol tolerance, alcohol degradation capability and relatively high activity of promoting alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH); the lactobacillus paracasei BL19 has the advantages that the lactobacillus paracasei BL19 has good anti-alcohol and anti-liver effects and strong capacity of protecting alcoholic injury liver cells, so that the anti-alcohol and anti-liver effects can be comprehensively achieved from multiple dimensions, and in addition, the lactobacillus paracasei BL19 has strong acid resistance and bile salt resistance, also has strong tolerance to simulated gastrointestinal fluid and has important application value in the fields of anti-alcohol and anti-liver products and the like.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a strain of Lactobacillus paracasei BL19 and its application in the preparation of products with hangover relief and liver protection functions. Background Technology

[0002] my country is the birthplace of wine culture and has a long history of drinking. With the improvement of people's living standards and changes in dietary structure, alcoholism, alcohol poisoning, and diseases caused by alcohol abuse have become health issues of global concern.

[0003] The liver is the most important organ for metabolizing alcohol in the body. It is widely known that drinking alcohol damages the liver, and excessive drinking will inevitably cause serious damage to the liver. The main reason why alcohol damages the liver is to analyze how alcohol is metabolized in the body. After alcohol is ingested, it is absorbed through the digestive tract such as the stomach and intestines and enters the bloodstream. It is then transported to various tissues and organs for metabolism and utilization, with about 90%-98% being metabolized in the liver.

[0004] There are three main enzyme systems in the liver involved in alcohol metabolism: the alcohol dehydrogenase system in the hepatocyte cytoplasm, the ethanol oxidation system in the mitochondria, and the catalase system. Among these, the alcohol dehydrogenase system in the hepatocyte cytoplasm is the most important, including alcohol dehydrogenase and aldehyde dehydrogenase. The function of alcohol dehydrogenase is to break down ethanol into acetaldehyde. Acetaldehyde, under the action of aldehyde dehydrogenase, is converted into acetic acid. Acetic acid is further metabolized into acetyl-CoA and enters the tricarboxylic acid cycle, ultimately breaking down completely into carbon dioxide and water, releasing energy. However, prolonged heavy drinking or excessive drinking in a short period of time can overload the alcohol dehydrogenase system until it can no longer break down alcohol, leading to liver disease. Initially, it manifests as hepatocyte steatosis, which progresses to fatty liver, hepatitis, liver fibrosis, and cirrhosis. In severe cases, it can worsen into widespread hepatocyte necrosis and even liver cancer. Currently in my country, alcoholic liver disease has become the second most common liver disease after viral hepatitis.

[0005] Current treatment options for alcoholic liver disease primarily include abstinence from alcohol, medication, and other methods. While medication is the most common treatment, it carries the risk of side effects. Therefore, probiotics, as an adjunct therapy, have potential value in both prevention and treatment. Numerous studies both domestically and internationally have demonstrated that consuming probiotics can alleviate and even treat alcoholic liver disease. For example, probiotic intake can reduce malondialdehyde (MDA) and triglyceride (TG) levels in mouse liver tissue, while increasing glutathione (GSH) levels, significantly improving the degree of liver damage.

[0006] Currently, there are relatively few invention patents in my country regarding the screening of edible strains that have a strong promoting effect on alcohol dehydrogenase. Even if edible strains with a strong promoting effect on alcohol dehydrogenase are screened out, their promoting effect on acetaldehyde dehydrogenase (ALDH) activity is still at a low level.

[0007] In summary, the goal of this field is to develop a natural and safe strain of bacteria that exhibits strong tolerance to alcohol, possesses the ability to degrade alcohol, exhibits strong alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity, can comprehensively provide hangover relief and liver protection from multiple dimensions, and has good tolerance to gastric and intestinal fluids. Summary of the Invention

[0008] To address the aforementioned issues, this application provides a strain of Lactobacillus paracasei (… Lactobacillus paracasei BL19 is a natural strain that exhibits significant tolerance to alcohol, while also being able to degrade alcohol and strongly promote the activity of ADH and ALDH enzymes. It is also resistant to gastric acid and bile salts, and has good intestinal colonization ability, making it of significant application value in areas such as hangover relief and liver protection.

[0009] The Lactobacillus paracasei BL19 provided in this application has the Latin scientific name: Lactobacillus paracasei It is deposited at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: April 27, 2025, accession number: CGMCC No. 34368.

[0010] This application also provides a Lactobacillus paracasei BL19 inoculum agent, which includes Lactobacillus paracasei BL19 as described above.

[0011] In one embodiment, the Lactobacillus paracasei BL19 inoculum includes the fermentation broth of Lactobacillus paracasei BL19 and / or the powder of Lactobacillus paracasei BL19.

[0012] In one embodiment, the fermentation broth of Lactobacillus paracasei BL19 includes one or more combinations of fermentation supernatant, cell-free extract, and bacterial cell metabolites.

[0013] This application also provides the use of Lactobacillus paracasei BL19 as described above in the preparation of fermentation products with the ability to degrade alcohol.

[0014] In one embodiment, the fermentation product is a Lactobacillus paracasei BL19 fermentation broth or a Lactobacillus paracasei BL19 bacterial suspension; wherein, the Lactobacillus paracasei BL19 is inoculated into MRS liquid medium and cultured at 30-40°C for 24-36 hours to obtain the Lactobacillus paracasei BL19 fermentation broth; the Lactobacillus paracasei BL19 fermentation broth is separated into solid and liquid components, and the bacterial sludge is resuspended in physiological saline to obtain the Lactobacillus paracasei BL19 bacterial suspension.

[0015] This application also provides the use of Lactobacillus paracasei BL19 as described above in the preparation of fermentation products with the ability to promote ADH and ALDH enzyme activity.

[0016] In one embodiment, the fermentation product is a Lactobacillus paracasei BL19 fermentation broth or a Lactobacillus paracasei BL19 bacterial suspension; wherein, the Lactobacillus paracasei BL19 is inoculated into MRS liquid medium and cultured at 30-40°C for 24-36 hours to obtain the Lactobacillus paracasei BL19 fermentation broth; the Lactobacillus paracasei BL19 fermentation broth is separated into solid and liquid components, and the bacterial sludge is resuspended in physiological saline to obtain the Lactobacillus paracasei BL19 bacterial suspension.

[0017] This application also provides the use of Lactobacillus paracasei BL19 as described above in the preparation of hangover relief and liver protection products.

[0018] This application also provides the application of the Lactobacillus paracasei BL19 bacterial agent as described above in the preparation of hangover relief and liver protection products.

[0019] This application also provides a method for determining alcohol tolerance, wherein Lactobacillus paracasei BL19 is inoculated into MRS liquid medium and cultured at 30-40°C for 24-36 h, and the culture solution is taken and inoculated into 5%, 10%, 15%, and 20% ethanol-modified MRS liquid medium at a 30% inoculation amount, cultured for 12-16 h, and the culture solution is counted by pouring method to calculate the survival rate to assess alcohol tolerance.

[0020] Based on the above characteristics, the Lactobacillus paracasei BL19 provided in this application has the following beneficial effects: The *Lactobacillus paracasei* BL19 provided in this application is a natural strain with high food safety. It not only has significant alcohol tolerance activity but also the ability to degrade alcohol and promote the activity of ADH and ALDH enzymes. At the same time, it has a strong ability to protect hepatocytes from alcohol-induced damage. Therefore, it can comprehensively achieve the effects of relieving hangovers and protecting the liver from multiple dimensions. In addition, it is resistant to gastric acid and bile salts and has good intestinal colonization ability, which has important application value in the fields of hangover relief and liver protection products. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a colony morphology diagram of Lactobacillus paracasei BL19 from Example 1 of this application; Figure 2 This is a Gram-stained image of Lactobacillus paracasei BL19 from Example 1 of this application; Figure 3 Figure 1 shows the tolerance of Lactobacillus paracasei BL19 to 20% and 25% alcohol. Figure 4 Graphs showing the degradation of 20% and 25% alcohol by Lactobacillus paracasei BL19; Figure 5 This is a graph showing the enzymatic activity analysis of Lactobacillus paracasei BL19 against alcohol dehydrogenase. Figure 6 This is a graph showing the enzymatic activity analysis of Lactobacillus paracasei BL19 against acetaldehyde dehydrogenase. Figure 7 Figure showing the survival of Lactobacillus paracasei BL19 in simulated intestinal and gastric fluids; Figure 8 The image shows the results of drug susceptibility analysis for Lactobacillus paracasei BL19. Figure 9 Figure 1 shows the cytotoxicity analysis of Lactobacillus paracasei BL19 fermentation broth and its effect on the activities of three enzymes, AST, ALT, and LDH, in human liver cancer cells. Figure 10 Figures showing the activity distance, ADH content, and liver fat staining of zebrafish treated with Lactobacillus paracasei BL19. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] This application also provides the following operational examples and embodiments: The specific process of isolating and screening Lactobacillus paracasei BL19 from an oral sample of a healthy, long-lived elderly person in Zhao'an County, Zhangzhou City, Fujian Province is as follows: Oral samples were collected from a healthy elderly person in Zhao'an County, Zhangzhou City, Fujian Province. Using the spread plate method, 5 g of oral samples from the healthy elderly person were placed in a sterile homogenizing bag, labeled, and 45 mL of 0.85% physiological saline was added and then thoroughly mixed by tapping.

[0025] Then, 100 μL of sample was taken and serially diluted 10-fold. 3 10 4 10 5 100 μL of the sample was spread on an MRS plate containing 2.5% CaCO3 and incubated upside down at 37°C for 48 h.

[0026] Select colonies with good growth and large calcium dissolution zones, and repeatedly isolate and purify them using the streak plate method until a single colony is obtained. The isolated strain is numbered and named BL19 and stored in a bacterial bank at -80℃ with glycerol.

[0027] Example 1: Screening of strains with strong alcohol tolerance (i.e., Lactobacillus paracasei BL19) 1. Determination of alcohol tolerance Eighty lactic acid bacteria strains frozen in glycerol tubes were taken from the bacterial bank and activated for two generations. They were then inoculated into MRS liquid medium at an inoculum rate of 3% (v / v) and cultured at 37°C for 24 hours. The fermentation broth was then inoculated into MRS liquid medium modified with different concentrations of alcohol at an inoculum rate of 3% (v / v), with alcohol volume concentrations of 5%, 10%, 15%, 20%, or 25% (v / v) and cultured in a constant temperature incubator at 37°C for 24 hours.

[0028] Different strains were inoculated with different concentrations of alcohol to modify MRS culture for 24 hours. The culture broth was then taken out and diluted 10-fold serially to form bacterial suspensions of different concentrations. The bacterial suspensions of different concentrations were counted using the pour method, and the survival rate was calculated to assess the alcohol tolerance of the strains.

[0029] The survival rate is calculated as follows: Survival rate (%) = (A1 / A0) * 100%; In the formula: A1 is the counting result after 24 hours of incubation, and A0 is the counting result at 0 hours.

[0030] The data obtained from the above experiments were processed by formula (1) to calculate the alcohol tolerance of each strain. The strain with high alcohol tolerance was selected from more than 80 strains of lactic acid bacteria in the company's bacterial bank. It is the lactic acid bacteria numbered BL19 that was extracted, isolated and cultured from oral samples of healthy long-lived elderly people. It was then activated and cultured in MRS liquid medium to obtain Lactobacillus paracasei BL19. The tests showed that, under the above fermentation process with an inoculum size of 3%, a fermentation temperature of 37℃, and a culture time of 24 hours, *Lactobacillus paracasei* BL19 exhibited high tolerance to alcohol. The viable count was 5.7997 ± 0.0616 CFU / mL at a 20% alcohol concentration and 4.2371 ± 0.0079 CFU / mL at a 25% alcohol concentration. Specific data are as follows: Figure 3 As shown.

[0031] Example 2: Identification of Lactobacillus paracasei BL19 (1) Identification of colony morphology Take one tube of glycerol bacteria (BL19) from the bacterial bank, inoculate it into MRS liquid medium at a 3% (v / v) inoculation rate, activate it, and incubate it in a constant temperature incubator at 37°C for 24 hours. Then streak it on an MRS solid medium plate and incubate it upside down in a constant temperature incubator at 37°C for 24 hours to obtain a single colony.

[0032] Its colony morphology is as follows Figure 1 As shown, it is round, milky white, with a smooth, raised surface, neat edges, and is opaque.

[0033] (2) Physiological and biochemical experiments Lactobacillus paracasei BL19, isolated and purified by the streak plating method, was used to select healthy single colonies for Gram staining. Its physiological and biochemical parameters were then measured. The experimental results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species. Stained Lactobacillus paracasei, such as Figure 2 As shown in the figure, the experiment showed that the Lactobacillus paracasei BL19 was Gram-positive, with purple staining and rod-shaped cells.

[0034] (3) Identification of 16S rRNA 1) Extract the bacterial DNA. The specific process for extracting bacterial DNA should be carried out according to the instructions of the bacterial genomic DNA extraction kit from Tiangen Biotech Co., Ltd.

[0035] 2) PCR amplification of the 16S rRNA gene was performed using the gene DNA as a template.

[0036] The amplification primers used were the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3').

[0037] The PCR reaction system consisted of: 1 μL DNA, 1 μL 27F, 1 μL 1492R, 10 μL Premix Ex Taq, and 7 μL ddH2O.

[0038] PCR reaction conditions: 95℃ pre-denaturation for 8 min; 95℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 1 min 30 s, 30 cycles; final extension at 72℃ for 5 min.

[0039] Then, the PCR amplification products were subjected to DNA sequencing (the sequencing company was Sangon Biotech Co., Ltd.).

[0040] The sequencing results were used to search for similar sequences in the NCBI database using the BLAST software. The sequenced *Lactobacillus paracasei* BL19 was compared with the 16S rRNA gene sequences of related species obtained from gene banks. The gene sequences are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'.

[0041] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

[0042] The 16S rRNA gene sequence (SEQ ID NO.1) was determined as follows:

[0043] In summary, based on the comprehensive analysis of colony morphology, cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence of this *Lactobacillus paracasei* BL19, and referring to *Bergey's Manual of Systematic Bacteriology, 8th Edition*, this *Lactobacillus paracasei* BL19 was identified as *Lactobacillus paracasei*. Lactobacillus paracasei ).

[0044] Example 3: Determination of the alcohol degradation capacity of Lactobacillus paracasei BL19 Drinking alcohol is often unavoidable at meals; however, if ingested alcohol is not broken down or digested in the body for an extended period, it can cause discomfort such as dizziness, nausea, and vomiting. Therefore, rapidly breaking down ingested alcohol is crucial for alleviating these post-drinking symptoms.

[0045] The standard curve for alcohol concentration was determined by colorimetry. 0.1 mL of 0%, 5%, 10%, 15%, 20%, 25%, or 30% ethanol standard solution was added to test tubes, respectively. 2 mL of potassium dichromate-sulfuric acid solution (100 mL of 5% potassium dichromate, with 10 mL of concentrated sulfuric acid added) was added to each test tube, and the mixture was thoroughly shaken. The test tubes were placed in a boiling water bath for 10 min to ensure complete oxidation of the potassium dichromate-sulfuric acid solution. The test tubes were then removed, and the absorbance of the potassium dichromate-sulfuric acid solution was measured at a wavelength of 600 nm.

[0046] Lactobacillus paracasei BL19 was cultured to passage 2 and inoculated into MRS liquid medium at a 3% (v / v) inoculum. The culture was then incubated at 37°C for 24 h to obtain the fermentation broth. This broth was then inoculated into MRS liquid medium modified with different concentrations of alcohol (20% and 25%) at a 3% (v / v) inoculum and incubated at 37°C for 24 h. After incubation, 1 mL of the fermentation broth was centrifuged at 8000 rpm for 2 min. 100 μL of the supernatant was used to replace the ethanol solution in the standard curve determination and was subjected to the same sample loading and testing procedure.

[0047] like Figure 4 As shown, substituting the data obtained from the above experiments into the standard curve formula, we obtained that the alcohol degradation rate of Lactobacillus paracasei BL19 at a 20% alcohol concentration was 26.08±0.41%; and the alcohol degradation rate at a 25% alcohol concentration was 25.76±0.09%.

[0048] Example 4: Determination of the ability of Lactobacillus paracasei BL19 to promote ADH enzyme activity Prolonged or excessive alcohol consumption can damage the digestive, circulatory, and nervous systems, with the liver and nervous system being the most severely affected. The body contains alcohol dehydrogenase (ADH), an enzyme that catalyzes the oxidation of alcohol into acetaldehyde. Stronger ADH activity means that alcohol can be rapidly oxidized to acetaldehyde.

[0049] In this embodiment, the ability of Lactobacillus paracasei BL19 to promote ADH enzyme activity was determined by spectrophotometry: Add 100 μL of pH 8.8 sodium pyrophosphate solution to a stoppered test tube, followed by 75 μL of 0.027 mol / L oxidized coenzyme I (NAD+), 35 μL of 20% ethanol solution, and 10 μL of *Lactobacillus paracasei* BL19 resuspension. Mix well and incubate at 37°C for 5 min. After incubation, add 10 μL of ADH (2.5 U / mL), and use 0.5 mL of distilled water as a control instead of the bacterial suspension. Shake the test tube solution well and measure the absorbance (A) at 340 nm. 340 Measurements were taken every 1 minute for 5 consecutive minutes. The change in absorbance here represents the rate of the enzymatic reaction of ADH.

[0050] The preparation process of Lactobacillus paracasei BL19 resuspension is as follows: 1 mL of the 10 mL Lactobacillus paracasei BL19 fermentation broth obtained in Example 2 is centrifuged to separate the solid and liquid components, and the bacterial sludge is resuspended in 1 mL of physiological saline to obtain the solution.

[0051] The formula for calculating ADH enzyme activity is: ADH enzyme activity (U·mL) -1 )=ΔA 340 × (total reaction volume / enzyme amount) × 10 6 / (6.2×10 3 ); Where, ΔA 340 This represents the increase in sample absorbance every 60 seconds.

[0052] like Figure 5 As shown, substituting the data obtained from the above experiment into the standard curve formula, the ADH enzyme activity of Lactobacillus paracasei BL19 fermentation broth was found to be 95.52±0.93 U / mL.

[0053] Example 5: Determination of the ability of Lactobacillus paracasei BL19 to promote ALDH enzyme activity When alcohol is ingested, it is broken down into acetaldehyde by alcohol dehydrogenase. However, acetaldehyde is far more harmful to the human body than alcohol, and it is listed as a Group 2B carcinogen. Therefore, the activity of acetaldehyde dehydrogenase in the body is crucial, as it is necessary to oxidize the acetaldehyde obtained after alcohol oxidation by alcohol dehydrogenase into acetic acid, which is harmless to the human body.

[0054] In this embodiment, the ability of Lactobacillus paracasei BL19 to promote ALDH enzyme activity was determined by spectrophotometry. Add 300 μL of pH 8.5, 1 mol / L Tris-HCl to a stoppered test tube, followed by 100 μL of 0.020 mol / L oxidized coenzyme I (NAD+), 50 μL of 100 mmol / L acetaldehyde solution, and 50 μL of Lactobacillus paracasei BL19 resuspension (or 50 μL of Lactobacillus paracasei BL19 fermentation broth prepared in Example 2 above). Mix well, then add 0.1 mL of 3 mol / L KCl, 0.1 mL of 0.5 mol / L EDTA, 0.03 mL of 1 mol / L mercaptoethanol (2-Me), and 0.02 mL of 10% pyrazole. Finally, add 2 mL of distilled water. Mix well and incubate at 37°C for 5 min. After incubation, 100 μL of ALDH (2.5 U / mL) was added, bringing the final reaction volume to 2.85 mL. 0.5 mL of distilled water was used as a control instead of the bacterial suspension. After shaking the solution, the absorbance (A) was measured at 340 nm. 340 Measurements were taken every 60 seconds for 5 minutes. The change in absorbance value here represents the rate of ALDH enzymatic reaction.

[0055] The preparation process of Lactobacillus paracasei BL19 resuspension is as follows: 1 mL of the 10 mL Lactobacillus paracasei BL19 fermentation broth obtained in Example 2 is centrifuged to separate the solid and liquid components, and the bacterial sludge is resuspended in 1 mL of physiological saline to obtain the solution.

[0056] The formula for calculating ALDH enzyme activity and activation rate is as follows: ALDH enzyme activity (U·mL) -1 )=ΔA 340 × (total reaction volume / enzyme amount) × 10 6 / (6.2×10 3 ); Where, ΔA 340 This represents the increase in sample absorbance every 60 seconds.

[0057] like Figure 6As shown, substituting the data obtained from the above experiments into the standard curve formula, the ALDH enzyme activity of Lactobacillus paracasei BL19 physiological saline resuspension was 32.89±2.38 U / mL; the ALDH enzyme activity of the strain fermentation broth was 42.10±2.41 U / mL.

[0058] Example 6: In vitro digestion resistance test of Lactobacillus paracasei BL19 in simulated gastric and intestinal juices Human food undergoes a process of breakdown by gastric juice and digestion by intestinal juice. Studies have shown that the pH value of normal human gastric juice is generally between 0.9 and 1.5, and the concentration of bile salts in intestinal juice is generally between 0.3 and 3 g / L. A simulated digestive solution was prepared to fully mimic the digestive environment of human gastric juice and intestines, and the tolerance of *Lactobacillus paracasei* BL19 was determined.

[0059] (1) Preparation of simulated gastric juice: Pepsin (p7000) was prepared into a 2 g / L pepsin solution using sterilized 0.85% physiological saline. The pH was adjusted to 3 with 4 mol / L hydrochloric acid solution, and then filtered through a 0.22 μm microporous membrane for sterilization before use.

[0060] (2) Preparation of simulated intestinal fluid: Prepare a 2 g / L solution of trypsin (USP(P7545)) using sterilized 0.85% physiological saline, then add ox bile salt to make the concentration 0.2%, adjust the pH to 8.0 with 1 mol / L sodium hydroxide, and then filter it through a 0.22 μm microporous membrane for sterilization before use.

[0061] (3) Tolerance test for artificial gastric juice: 1 mL of BL19 bacterial suspension from Example 1 was added to 9 mL of prepared artificial gastric fluid. After thorough mixing, 1 mL of the mixture was diluted tenfold to a specific gradient, and after shaking to mix, 100 μL was taken and the viable bacterial count was determined using the pour plate method. Simultaneously, the simulated artificial gastric fluid was incubated at 37°C for 2 hours. Then, 1 mL of the mixture was diluted tenfold to a specific gradient, and after shaking to mix, 100 μL was taken and the viable bacterial count was determined using the pour plate method. The survival rate was calculated using the following formula: Strain survival rate (%) = b / a × 100%; In the formula: a is the number of viable bacteria after 0 h of adding 1 mL of bacterial suspension to 9 mL of artificial gastric fluid, in CFU / mL; b is the number of viable bacteria after incubation in artificial gastric fluid for 2 h, in CFU / mL.

[0062] like Figure 7As shown in the experimental results, *Lactobacillus paracasei* BL19 grew well in simulated gastric fluid, with a survival rate of 98.70 ± 0.86%. This indicates that after *Lactobacillus paracasei* BL19 entered the gastric fluid environment, the lower pH of the gastric fluid caused fewer bacteria to die, while a considerable number of bacteria survived. This demonstrates that *Lactobacillus paracasei* BL19 has strong tolerance to both acid and pepsin and can survive well in simulated gastric fluid environment.

[0063] (4) Tolerance test for artificial intestinal fluid: After incubating the BL19 bacterial culture in artificial gastric fluid for 2 hours, shake well and add 1 mL to 9 mL of prepared artificial intestinal fluid. Mix thoroughly and incubate at 37°C for 1 hour. Then, take 1 mL of the mixture and dilute it tenfold to a certain gradient. Shake well and take 100 μL to determine the viable bacterial count using the pour plate method. Calculate the survival rate using the following formula: Strain survival rate (%) = c / b × 100%; In the formula: b is the number of viable bacteria after incubation in artificial gastric fluid for 2 hours, in CFU / mL; c is the number of viable bacteria after incubation in artificial intestinal fluid for 1 hour, in CFU / mL.

[0064] like Figure 7 As shown in the experimental results, *Lactobacillus paracasei* BL19 showed good growth in simulated artificial intestinal fluid, with a survival rate of 81.10 ± 1.44%. This indicates that after *Lactobacillus paracasei* BL19 entered the intestinal fluid environment, the high bile salt concentration in the intestinal fluid caused a very small portion of the bacteria to die, while the majority of the remaining bacteria survived.

[0065] Example 7: Antimicrobial susceptibility testing of Lactobacillus paracasei BL19 To demonstrate that the screened Lactobacillus paracasei BL19 does not possess strong drug resistance, Example 8 tested its drug susceptibility using three antibiotics: tetracycline, azithromycin, and amoxicillin.

[0066] Lactobacillus paracasei BL19 was activated for two generations at an inoculum of 3% (v / v) and then inoculated into MRS liquid medium and cultured at 37°C for 24 hours. The bacterial suspension was serially diluted 10-fold with physiological saline. 100 μL of each diluted suspension was injected onto an MRS solid agar plate, spread evenly with a spreader, and allowed to air dry under an alcohol lamp. Once the plate surface was clear, a drug sensitivity test strip was placed flat on the MRS agar plate, and the plate was then inverted and incubated at 37°C for 24 hours. After incubation, the size of the inhibition zone was observed.

[0067] The results of the drug sensitivity test are as follows Figure 8 As shown: Figure 8Three inhibition zones were displayed, corresponding to tetracycline (TE), azithromycin (AZM), and amoxicillin (AML), respectively. The inhibition zones were compared to a 10mm outer diameter comparison disc; all three inhibition zones had an outer diameter greater than 10mm. Since inhibition zones smaller than 10mm indicate insensitivity, BL19 is sensitive to all three antibiotics (TE, AZM, and AML), proving it does not exhibit drug resistance.

[0068] Example 8: Determination of ALT and AST activities in human HepG2 liver cancer cells by Lactobacillus paracasei BL19 (1) First, cytotoxicity experiments were performed on Lactobacillus paracasei BL19, and cell viability was detected by CCK8 assay. HepG2 cells in logarithmic growth phase were digested with digestive solution, and then DMEM complete medium was added to terminate the digestion. Cells were collected by centrifugation and then processed at 2*10-1. 5 Cells were seeded per well in a 96-well plate. 12–16 h after seeding, the cells were inoculated with ethanol (0, 200, 400, 500, 600, 700, 800, 900, 1000 mM) or a sample (10 mM). 2 10 3 10 4 10 5 10 6 10 7 10 8 CFU probiotic samples were diluted to different concentrations with culture medium and incubated with cells for 24 hours. The CCK8 assay was then performed. After discarding the cell culture medium, 100 µL of 10% CCK8 reagent was added to each well and incubated at 37°C for about 30 min to 1 h. The absorbance was measured at 405 nm using an ELISA reader.

[0069] The probiotic sample was prepared by diluting the bacterial suspension obtained after cultivation with DMEM cell culture medium.

[0070] Cell viability is calculated using the following formula: Cell viability (%) = (A 样品 -A 空白组 ) / (A 对照组 -A 空白组 ) × 100%.

[0071] The blank represents the absence of bacterial samples and the detection reagents from the CCK-8 kit, while the control group represents the presence of the detection reagents from the CCK-8 kit but without bacterial samples.

[0072] (2) Methods for determining injury indicators: HepG2 cells in the logarithmic growth phase were digested and dispersed to prepare a single-cell suspension, and the cell concentration was adjusted to 5*102. 5 Inoculate 1 mL per well into 12-well plates and incubate for 24 h.

[0073] After inducing an alcoholic liver injury cell model by adding 400mM alcohol according to the experimental groups (except for the blank control group), different concentrations of non-toxic probiotic samples (10) were added. 5 and 10 7 CFU), each group was set with 3 replicates. After incubation for 24 hours, cells were extracted and various indicators were measured according to the method and steps of the kit (the content of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) was measured according to the kit purchased from Nanjing Jiancheng Bioengineering Institute). Among the hepatocyte indicators, the activities of AST, ALT and LDH were inversely proportional to the health of hepatocytes.

[0074] Specific data is as follows Figure 9 As shown, in the above experiment, the BL19 effect on CCK8 cell viability was as follows: when the viable cell count was 10... 5 The cell viability was 109.79% and the viable bacterial count was 10. 7 The cell viability was 121.3%, indicating that the *Lactobacillus paracasei* BL19 strain in this application had weak cytotoxicity. The AST, ALT, and LDH results were 9.4631 ± 1.24 U / gprot (10... 5 ), 13.0656±0.76 U / gprot (10 7 ) and 0.4436±0.01 U / gprot (10 5 ).

[0075] The results showed that, compared with the model group (Mods), the activities of alanine aminotransferase (AST), aspartate aminotransferase (ALT), and lactate dehydrogenase (LDH) in human liver cancer cells HepG2 in the experimental group were reduced, indicating that Lactobacillus paracasei BL19 has a strong ability to protect against alcohol-induced liver damage, thus helping to improve liver cell health.

[0076] Example 9: Evaluation of the effects of Lactobacillus paracasei BL19 on the alcohol metabolism behavior of zebrafish First, zebrafish were placed in aquaculture water containing alcohol and treated at 28°C for 1 hour to induce a "drunken" state, exhibiting behaviors similar to those of humans who are intoxicated, such as decreased activity and loss of balance.

[0077] Following alcohol exposure, zebrafish were rapidly transferred to a facility containing 2×10⁻⁶ mmol / L. 8The zebrafish were administered CFU / mL of BL19 bacterial suspension and positive control drug solution (RU21, concentration of 100 μg / mL) in aquaculture water via immersion. The subsequent behavioral changes and related physiological indicators of zebrafish in different treatment groups were observed.

[0078] The results are as follows Figure 10 As shown in Figure a, behavioral observations were conducted on the hangover relief of zebrafish after treatment. The results showed that Lactobacillus paracasei BL19 has a hangover relief effect. Specifically, compared with the model group (Mods), the addition of Lactobacillus paracasei BL19 increased the total movement distance of zebrafish.

[0079] RU-21 is a well-known over-the-counter hangover remedy. It is not a drug approved by the FDA in China or the United States, but rather a dietary supplement.

[0080] Example 10: Evaluation of the hepatoprotective effect of Lactobacillus paracasei BL19 on zebrafish First, zebrafish were placed in aquaculture water containing alcohol and treated at 28°C for 1 hour to induce a "drunken" state, exhibiting behaviors similar to those of humans who are intoxicated, such as decreased activity and loss of balance.

[0081] Following alcohol exposure, zebrafish were rapidly transferred to a facility containing 2*10 8 The zebrafish were administered CFU / mL of BL19 bacterial suspension and positive control drug solution (RU21, concentration of 100 μg / mL) in aquaculture water via immersion. The subsequent behavioral changes and related physiological indicators of zebrafish in different treatment groups were observed.

[0082] The results are as follows Figure 10 b、 Figure 10 As shown in Figure c, behavioral observations of zebrafish after treatment revealed that Lactobacillus paracasei BL19 has a liver-protective effect in relieving alcohol consumption, specifically by increasing ADH levels and reducing the intensity of liver fat staining.

[0083] Example 11: Preparation of Lactobacillus paracasei BL19 inoculum Lactobacillus paracasei BL19 was activated for two generations at an inoculum of 3% (v / v), then inoculated into MRS liquid medium and cultured at 37℃ for 24 h. After centrifugation at 8000 r / min for 5 min at 4℃, the supernatant was discarded, and the bacterial sludge was retained. The bacterial sludge was mixed with a freeze-drying protectant at a mass ratio of 1:(1-2) and then embedded. The resulting embedded bacterial sludge was stored at -80℃ overnight. The pre-frozen embedded bacterial sludge was then freeze-dried in a vacuum freeze dryer for 48 h to obtain Lactobacillus paracasei BL19 freeze-dried bacterial powder with a viable count of 200 billion CFU / g. The freeze-drying protectant was prepared as follows: 8 g skim milk powder, 2 g sucrose, and 100 g distilled water were added, stirred to dissolve, sterilized at 115℃ for 15 min, and used after cooling.

[0084] It should be noted that the freeze-drying protectant can be composed of other protectants or formulations, including but not limited to the above-described embodiments.

[0085] In summary: The Lactobacillus paracasei BL19 strain provided in this application was isolated and screened from oral samples of a healthy, long-lived elderly person in Zhao'an County, Zhangzhou City, Fujian Province. It is a natural strain and has high food safety. Lactobacillus paracasei BL19 not only exhibits significant alcohol tolerance but also possesses the ability to degrade alcohol and promote the activity of ADH and ALDH enzymes. Simultaneously, it can maximize the protection of alcohol-damaged hepatocytes, thus providing a comprehensive multi-dimensional effect in detoxifying and protecting the liver. Furthermore, it demonstrates a certain degree of resistance to gastric acid and bile salts, and exhibits good intestinal colonization capabilities. Its beneficial properties further enhance its application value in the field of products for detoxifying and protecting the liver. For example: (1) Prepare a microbial agent with the above functions using Lactobacillus paracasei as the raw material component of the microbial agent. (2) Lactobacillus paracasei is used to obtain fermented products with the above functions through fermentation treatment; (3) Lactobacillus paracasei is fermented and resuspended to obtain a resuspension, thus obtaining a fermented product with the above-mentioned functions; The aforementioned Lactobacillus paracasei BL19 can be widely used in the preparation of anti-hangover or anti-drinking agents, specifically in its ability to degrade alcohol and its promoting effect on the activity of various alcohol-degrading enzymes. It is universally applicable to people who are alcoholics, drink alcohol regularly, or have severe symptoms of alcohol-related discomfort.

[0086] It should be noted that: The culture medium involved in the embodiments of this application is specifically: MRS liquid medium: glucose 20.0 g, tryptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, magnesium sulfate 0.5 g, manganese sulfate monohydrate 0.25 g, deionized water 1 L, pH 6.5 (adding 1.5% agar powder makes it MRS solid medium); The method for preparing the alcohol-modified MRS liquid culture medium in this paper is as follows: 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of alcohol are added to 9.5 mL, 9 mL, 8.5 mL, 8 mL, and 7.5 mL of MRS liquid culture medium, respectively, so that the volume concentration of alcohol in the MRS liquid culture medium is 5%, 10%, 15%, 20%, and 25%.

[0087] Unless otherwise specified, the experimental procedures involved in the embodiments of this application are conventional experimental procedures in the field, and the reagents or instruments involved can be obtained from legitimate channels.

[0088] In addition, the *Lactobacillus paracasei* BL19 of this application can be used as a raw material component for probiotic products, fermented products, functional products, etc. Based on its characteristics, *Lactobacillus paracasei* BL19 can be applied to functional products that include at least one of the following functions: (1) Has alcohol tolerance; (2) It has the ability to degrade alcohol; (3) It has the ability to promote ALDH enzyme activity; (4) It has the ability to promote ADH enzyme activity; (5) It has the ability to protect hepatocytes from alcohol-induced damage; Among them, products with the above-mentioned (1)-(5) properties are not limited to hangover relief and liver protection products; they can also have other obvious effects that are beneficial to health based on the correlation between the effects of (1)-(5) and health care, including but not limited to hangover relief and liver protection.

[0089] In addition, in this paper, "Con" is the abbreviation for the control group and "Mods" is the abbreviation for the model group.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A strain of Lactobacillus paracasei ( Lactobacillus paracasei BL19, characterized in that, Its accession number is CGMCC No.34368.

2. A Lactobacillus paracasei BL19 inoculum, characterized in that: Includes Lactobacillus paracasei BL19 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that: This includes the fermentation broth of Lactobacillus paracasei BL19 and / or the bacterial powder of Lactobacillus paracasei BL19.

4. The application of Lactobacillus paracasei BL19 as described in claim 1 in the preparation of fermentation products with the ability to degrade alcohol.

5. The application according to claim 4, characterized in that: The fermentation product is Lactobacillus paracasei BL19 fermentation broth or Lactobacillus paracasei BL19 bacterial suspension; The Lactobacillus paracasei BL19 was inoculated into MRS liquid medium and cultured at 30-40°C for 24-36 h to obtain Lactobacillus paracasei BL19 fermentation broth; The fermentation broth of Lactobacillus paracasei BL19 was separated into solid and liquid components, and the bacterial sludge was resuspended in physiological saline to obtain a Lactobacillus paracasei BL19 bacterial suspension.

6. The application of Lactobacillus paracasei BL19 as described in claim 1 in the preparation of fermentation products with the ability to promote ADH and ALDH enzyme activity.

7. The application according to claim 6, characterized in that: The fermentation product is Lactobacillus paracasei BL19 fermentation broth or Lactobacillus paracasei BL19 bacterial suspension; The Lactobacillus paracasei BL19 was inoculated into MRS liquid medium and cultured at 30-40°C for 24-36 h to obtain Lactobacillus paracasei BL19 fermentation broth; The fermentation broth of Lactobacillus paracasei BL19 was separated into solid and liquid components, and the bacterial sludge was resuspended in physiological saline to obtain a Lactobacillus paracasei BL19 bacterial suspension.

8. The use of Lactobacillus paracasei BL19 as described in claim 1 or Lactobacillus paracasei BL19 agent as described in any one of claims 2-3 in the preparation of hangover relief and liver protection products.

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