Lactobacillus casei with ethanol reducing capacity and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a lactobacillus casei strain with ethanol reducing capacity and application of the lactobacillus casei strain, the lactobacillus casei strain is lactobacillus casei Lc-PG-10, the classification name of the lactobacillus casei strain is Lacticaseibacillus casei, the preservation number is CGMCC No.31863, the preservation time is September 4, 2024, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC No.31863, the preservation number is CGMCC No.31863, the preservation number is CGMCC No.31863, the preservation number is CGMCC No.31863, the preservation number is CGMCC No.31863, and the preservation number is CGMCC No.31863. The address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is obtained by separating and purifying pickled Chinese cabbage fermentation liquor, the strain has high capacity of degrading ethanol, the ethanol inhibition rate of supernate of the strain is 57.55 + / -0.2%, and the strain has high capacity of resisting oxidation, acid, bile salt and artificial simulated gastrointestinal fluid and has the characteristics of probiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of *Lactobacillus casei* with the ability to reduce ethanol and its applications. Background Technology

[0002] Alcohol culture has a long history in my country and serves as an important vehicle for social interaction and folk etiquette. However, excessive drinking leads to the production of large amounts of reactive oxygen species and acetaldehyde metabolites during alcohol metabolism. Excessive reactive oxygen species cause oxidative stress, further damaging the liver; while acetaldehyde is highly toxic and directly damages liver cells. Continued liver cell damage activates hepatic stellate cells, causing them to synthesize large amounts of extracellular matrix, leading to liver fibrosis, alcoholic liver disease, alcoholic hepatitis, and in severe cases, liver failure, alcoholic cirrhosis, and even liver cancer, endangering life.

[0003] The main intervention for alcoholic liver disease is the use of related metabolic regulating drugs, such as disulfiram and metadoxine. However, these drugs have side effects such as nausea, vomiting, dizziness, abnormal liver function, and arrhythmia.

[0004] Existing research has shown that probiotics, which are beneficial to human health and have no obvious side effects, can degrade ethanol and alleviate or treat alcoholic liver disease. Probiotics that degrade ethanol can also regulate the balance of gut microbiota, reduce alcohol-induced intestinal barrier damage, decrease increased intestinal mucosal permeability, and reduce endotoxin entry into the bloodstream. Furthermore, probiotics can improve the gastrointestinal microenvironment and alleviate discomfort symptoms such as nausea, bloating, and indigestion caused by excessive alcohol intake.

[0005] Furthermore, long-term or excessive alcohol intake exacerbates oxidative stress in the body, generating a large number of free radicals. This not only further damages liver cells but also causes insulin resistance by affecting the insulin signaling pathway. Therefore, oxidative damage is considered an important cause of alcoholic liver injury and related metabolic disorders. Thus, the antioxidant activity of probiotics is also an important ability to help alleviate the harm of excessive alcohol consumption. However, currently reported probiotic ethanol degradation rates are low. Therefore, identifying probiotics with high ethanol degradation rates and good antioxidant capabilities is of great significance for leveraging the ethanol-degrading function of probiotics and alleviating alcoholic liver disease. Summary of the Invention

[0006] Therefore, the present invention aims to provide a strain of Lactobacillus casei that is acid-resistant, bile-salt-resistant, resistant to simulated gastrointestinal fluid, has strong antioxidant capacity, and has a high ethanol-reducing ability.

[0007] The present invention provides a strain of Lactobacillus casei with the ability to reduce ethanol, namely Lc-PG-10 (Lacticaseibacillus casei), which was deposited on September 4, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31863, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the Lactobacillus casei provided by this invention includes the following applications:

[0009] 1) Application in the degradation of ethanol;

[0010] 2) As a probiotic, its application in improving the gastrointestinal microenvironment;

[0011] 3) Used in the preparation of drugs that regulate intestinal flora balance, reduce alcohol-induced intestinal barrier damage, reduce increased intestinal mucosal permeability, and reduce endotoxin entry into the blood.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] The strain of this invention has strong resistance to acid, bile salts, simulated gastrointestinal fluid, and antioxidant capacity, and also has a high ability to reduce ethanol. Attached Figure Description

[0014] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0015] Figure 1 The colony morphology and Gram staining results of Lactobacillus casei Lc-PG-10 plate;

[0016] Figure 2 The results of the acid resistance test for Lactobacillus casei Lc-PG-10;

[0017] Figure 3 The results of the bile salt tolerance test for Lactobacillus casei Lc-PG-10;

[0018] Figure 4 The results of the test on the ability of Lactobacillus casei Lc-PG-10 to tolerate artificial simulated gastrointestinal fluid;

[0019] Figure 5 The results of the test on the inhibition of pathogenic bacteria by the supernatant of Lactobacillus casei Lc-PG-10 fermentation broth;

[0020] Figure 6The results of the hemolytic activity test for Lactobacillus casei Lc-PG-10;

[0021] Figure 7 A standard curve for determining the ethanol degradation rate of Lactobacillus casei Lc-PG-10;

[0022] Figure 8 The results of the Lc-PG-10 ethanol tolerance test for Lactobacillus casei;

[0023] Figure 9 The results of the test on the DPPH free radical scavenging ability of Lactobacillus casei Lc-PG-10;

[0024] Figure 10 The results of the test on the hydroxyl radical scavenging ability of Lc-PG-10 of Lactobacillus casei;

[0025] Figure 11 The results of the test on the ABTS+ free radical scavenging ability of Lactobacillus casei Lc-PG-10;

[0026] Figure 12 The results show the scavenging ability of Lactobacillus casei Lc-PG-10 against superoxide anion free radicals. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Screening and identification of Lacticaseibacillus casei Lc-PG-10

[0029] 1. Strains Isolation and Purification

[0030] Take 1 mL of sauerkraut fermentation broth, dilute it with physiological saline, and then take 100 μL of the diluted broth at different dilution gradients of 10 μL and 10 μL respectively. -2 ~10 -7 The fermentation broth was evenly spread onto MRS medium and incubated at 37°C for 48 h. Single colonies of different morphologies and sizes were picked from the plates and streaked multiple times on MRS agar for purification until a pure strain was identified.

[0031] 2. Microscopic examination

[0032] Single colonies were picked and inoculated into MRS broth medium. After culturing for 24 h, Gram staining was performed. The bacterial culture was smeared, Gram-stained, and the cell morphology and arrangement were observed under a light microscope. The observations were recorded and photographed for later use. Finally, Gram-positive cultures were identified as suspected lactic acid bacteria. 1.0 mL of the pure culture was transferred to a storage tube containing 50% glycerol for preservation and future use.

[0033] 3. Physiological and biochemical identification

[0034] The hydrogen peroxide test, indole test, and carbohydrate utilization test were used to identify and screen the bacterial strains. The isolated and purified strain was milky white, with a raised center, a smooth and moist surface, and a diameter of 1.15 mm; the strain was Gram-positive, and the cells grew in flocculent single or clumps. Figure 1 Its physicochemical characteristics include a negative hydrogen peroxide test; a positive indole reaction; a positive aescin hydrolysis test; and the ability to ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0035] 4. Molecular biological identification

[0036]

[0037] A comparison with the NCBI BLAST database revealed that the gene sequence of strain Lrn-PG-06 was 99.79% homologous to Lactobacillus casei. Combined with Gram staining results and physiological and biochemical characteristics, strain Lc-PG-10 was identified as Lactobacillus casei.

[0038] Example 2: Tolerance test of Lactobacillus casei Lc-PG-10

[0039] 1. Results of acid resistance test

[0040] After the strain was activated and passaged twice in MRS medium at 37℃, a bacterial suspension was prepared and resuspended in MRS medium with pH values ​​adjusted to 5.0, 4.0, 3.0, 2.0 and 1.0 respectively using 4.0 mol / L HCl. After incubation at 37℃ for 24 h, the bacterial suspension was collected. The absorbance of the bacterial suspension was measured at 600 nm wavelength, with 3 replicates. The absorbance of pH 6.0 MRS medium at 600 nm wavelength was used as a control. The survival rate was calculated.

[0041] Survival rate = N t / N0

[0042] Where: N t The absorbance values ​​are for MRS culture media with pH values ​​ranging from 1.0 to 5.0.

[0043] N0 represents the absorbance of the MRS medium at pH 6.0.

[0044] In healthy individuals, the pH of gastric juice on an empty stomach ranges from 0.9 to 1.8. After ingestion, the pH of gastric juice changes, fluctuating between approximately 1.8 and 5.0. Therefore, this experiment selected pH values ​​of 0, 1, 2, 3, 4, and 5 to test the acid tolerance of the bacterial strains. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that *Lactobacillus casei* Lc-PG-10 exhibits poor tolerance at pH 1.0, with a survival rate of only 4.30% after 24 hours of treatment; however, it demonstrates strong tolerance at pH 4.0, with a survival rate of 63.83% after 24 hours of treatment. This indicates that the bacterium has a strong ability to tolerate acidic conditions.

[0045] 2. Results of bile salt tolerance test

[0046] After two generations of activation and subculturing, *Lactobacillus casei* strain Lc-PG-10 was inoculated at a 3% inoculum into MRS medium containing 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% bile salts, respectively. After incubation at 37°C for 24 h, the bacterial suspension was collected, and the absorbance of the bacterial suspension was measured at a wavelength of 600 nm. Three replicates were set up, with the absorbance of MRS medium without bile salts at a wavelength of 600 nm as a control. The survival rate of the strain was calculated.

[0047] Survival rate = N t / N0

[0048] Where: N t The number of viable bacteria in MRS culture media containing different concentrations of bile salts;

[0049] N0 represents the number of viable bacteria in MRS medium without bile salts.

[0050] The concentration of bile salts in the small intestine of most humans is around 0.3% (w / v). Probiotics need to tolerate the bile salts in the gut to exert their beneficial effects. This study selected five Lc-PG-10 strains with different mass fractions of bile salts (0.1%, 0.3%, 0.5%, 0.7%, and 0.9%) for 24 h (with strains cultured without bile salts serving as a control). The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the survival rate of the strain gradually decreases with increasing bile salt concentration. After 24 h of treatment with 0.3% bile salt, the survival rate was still 38.64%, indicating that Lactobacillus casei Lc-PG-10 has good bile salt tolerance.

[0051] 3. Results of simulated gastrointestinal fluid tolerance test

[0052] Lactobacillus casei Lc-PG-10 strain was activated and passaged twice in MRS medium at 37℃. A bacterial suspension was prepared, resuspended in simulated gastric fluid, and treated at 37℃ for 3 h. After centrifugation and discarding the supernatant, the bacterial sludge was collected and washed three times with sterile physiological saline. The sludge precipitate was collected aseptically and mixed with physiological saline at a ratio of 1:5. The mixture was then serially diluted before colony counting.

[0053] Take 1.0 g of bacterial sludge after 3 h of gastric fluid treatment, add simulated bile, and treat at 37℃ for 20 min. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge: physiological saline of 1:5, mix well, perform serial dilution, and count the colonies.

[0054] Take 1.0 g of bacterial sludge after treating the bile for 20 min, add simulated intestinal fluid, and treat at 37℃ for 4 h. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge: physiological saline of 1:5, mix well, perform serial dilution, and count the colonies.

[0055] Survival rate of artificial gastrointestinal fluid and bile = N t / N0

[0056] Where: N t To simulate the number of viable bacteria after treatment with gastric juice, bile, and intestinal fluid;

[0057] N0 represents the number of viable bacteria before treatment.

[0058] Gastric juice contains mucus, gastric acid, pepsin, etc. For probiotics to exert their beneficial effects, they must survive in the stomach, meaning they need to be able to tolerate the acidic environment and resist pepsin. Food typically stays in the stomach for 3 hours; therefore, this study used simulated gastric juice to treat *Lactobacillus casei* Lc-PG-10 bacterial culture for 3 hours. After digestion in the stomach, food enters the intestines. The small intestine has a weakly alkaline environment and contains trypsin and bile. The viable count of probiotics in the intestines reaches 10-10. 6 Probiotics need a concentration of at least CFU / mL to exert their probiotic function; therefore, they must be able to tolerate the weakly alkaline environment of the intestine and resist trypsin and bile. Food typically stays in the small intestine for 3–8 hours. Therefore, this experiment used simulated intestinal fluid to treat *Lactobacillus casei* Lc-PG-10 bacterial culture for 4 hours. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that after 3 hours of treatment with gastric juice, the survival rate of *Lactobacillus casei* Lc-PG-10 was 77.74%, with a viable count of 12.87 ± 0.06 lg (cfu / g). After 20 minutes of treatment with bile, the survival rate was 68.53%, with a viable count of 11.34 ± 0.30 lg (cfu / g). After 4 hours of treatment with intestinal juice, the survival rate was still 65.42%, with a viable count of 10.83 ± 0.05 lg (cfu / g). This demonstrates that *Lactobacillus casei* Lc-PG-10 has a strong ability to tolerate simulated gastric and intestinal juices and bile.

[0059] Example 3: Antibacterial activity test of Lactobacillus casei Lc-PG-10

[0060] Antibacterial test results

[0061] After activating the indicator bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella, Listeria monocytogenes, and Pseudomonas aeruginosa, they were inoculated into LB liquid medium and incubated at 37°C for 24 h. The resulting bacterial suspensions were then prepared for use. Lactobacillus casei Lc-PG-10 was activated and inoculated into liquid MRS medium and incubated at 37°C for 24 h. The supernatant was collected after centrifugation at 8000×g for 5 min. Using the Oxford cup agar diffusion method, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella, Listeria monocytogenes, and Pseudomonas aeruginosa were spread on plates. Sterile physiological saline served as a blank control. Four Oxford cups were evenly placed on each plate. 200 μL of fermentation supernatant was added to three Oxford cups, and 200 μL of sterile physiological saline was added to one Oxford cup. After incubation at 37°C for 24 h, the presence of inhibition zones around the bottom of the Oxford cups was observed, and the diameter of the inhibition zones was measured and averaged.

[0062] From Table 1 and Figure 5 As can be seen, inhibition zones appeared in all the petri dishes, and the diameter of the inhibition zones was greater than 15.0 mm. This indicates that Lactobacillus casei Lc-PG-10 has an inhibitory effect on the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella. The diameters of the inhibition zones were 21.47±0.42 mm, 20.22±0.50 mm, 17.23±0.44 mm, 15.88±0.49 mm, 19.38±0.33 mm, and 23.12±0.26 mm, respectively, showing that Lactobacillus casei Lc-PG-10 has a significant antibacterial effect and strong antibacterial ability.

[0063] Table 1. Experimental data on the antibacterial activity of Lactobacillus casei Lc-PG-10

[0064] Table 1

[0065] indicator bacteria Diameter of the inhibition zone (mm) Staphylococcus aureus 21.47±0.42 Bacillus subtilis 20.22±0.50 E. coli 17.23±0.44 Pseudomonas aeruginosa 15.88±0.49 Listeria monocytogenes 19.38±0.33 salmonella 23.12±0.26

[0066] Note: The diameter of the Oxford cup is 7.833 ± 0.172.

[0067] Example 4 Hemolysis test of Lactobacillus casei Lc-PG-10 strain

[0068] Lactobacillus casei Lc-PG-10 bacterial suspension was inoculated onto Columbia blood agar medium and incubated at 37°C for 24 h. Hemolysis was then observed around the colonies. A translucent hemolytic zone indicated α-hemolysis, a clearly defined, completely transparent hemolytic zone indicated β-hemolysis, and the absence of a hemolytic zone indicated γ-hemolysis (i.e., no hemolysis).

[0069] The test results for Lactobacillus casei Lc-PG-10 are as follows: Figure 6As shown, with Escherichia coli as the control strain (left image), Lactobacillus casei Lc-PG-10 (right image) did not hemolyze and was therefore a safe strain.

[0070] Example 5: Test on the ethanol-reducing ability of Lactobacillus casei Lc-PG-10

[0071] 1. Results of the ethanol-reducing capacity test

[0072] After activating and subculturing the Lactobacillus casei Lc-PG-10 strain in MRS medium at 37 ℃ for two generations, the supernatant was collected by centrifugation, and 0.3 mL of each of the 0%, 2%, 4%, 6%, 8%, and 10% ethanol standard solutions were prepared for use. Potassium dichromate-sulfuric acid solution was also prepared for use.

[0073] Take 0.3 mL of ethanol standard solutions of different mass concentrations (0%, 2%, 4%, 6%, 8%, 10%) and add them to the corresponding test tubes. Then add 3 mL of potassium dichromate-sulfuric acid solution to each test tube and mix thoroughly. Measure the absorbance of each mixture at a wavelength of 610 nm. Plot the ethanol standard curve with the absorbance value on the vertical axis and the ethanol mass concentration on the horizontal axis and obtain the regression equation.

[0074] The isolated Lactobacillus casei Lc-PG-10 was inoculated into MRS ethanol medium with a v / v concentration of 20% and 40%, respectively, and cultured under suitable conditions. At the same time, a blank control was set up, that is, the same concentration of MRS ethanol medium without inoculation was used and cultured under the same conditions.

[0075] After cultivation, the culture media from the experimental group (MRS ethanol medium inoculated with the bacterial strain) and the blank control group (MRS ethanol medium without the bacterial strain) were taken, and the total ethanol content was determined using the total ethanol content assay method. Figure 7 As shown, 0.3 mL of culture medium was added to each test tube, followed by 3 mL of potassium dichromate-sulfuric acid solution. After mixing, the absorbance at 610 nm was measured. Based on the regression equation of the ethanol standard curve, the ethanol concentration (v / v) A0 of the blank control group and the ethanol concentration (v / v) A1 of the experimental group were calculated. The ethanol degradation rate was calculated as follows:

[0076]

[0077] In the formula: A0 is the ethanol concentration (v / v) of the control group; A1 is the ethanol concentration (v / v) of the sample.

[0078] Table 2. Experimental data on the ethanol-reducing ability of the strains.

[0079] Table 2

[0080] strain name Ethanol degradation rate (%) Lactobacillus casei Lc-PG-10 57.55±0.2

[0081] At an ethanol concentration (v / v) of 20%, Lactobacillus casei Lc-PG-10 achieved an ethanol degradation rate of 57.55% ± 0.2%, which is outstanding compared to other probiotic strains. Therefore, Lactobacillus casei Lc-PG-10 has a strong ability to reduce ethanol.

[0082] 2. Comparison of ethanol degradation rate with common strains

[0083] Table 3 Comparison of ethanol-reducing abilities with other probiotic strains

[0084] Table 3

[0085] strain name Degradation rate (%) References Bacillus subtilis fmb8 33% LU J, ZHU X, ZHANG C, et al. Co-expression of alcoholdehydrogenase and aldehyde dehydrogenase in Bacillussubtilis for alcohol detoxification [J]. Food and ChemicalToxicology, 2020, 135: 110890. Pediococcus acidilactici RH2712 11.3% Song Jia, Yu Ping, Lin Xinmei, et al. Screening, identification and evaluation of the hangover relief and anti-drunkenness effects of ethanol-degrading lactic acid bacteria [J]. China Brewing, 2024, 43(02):106-112. Fermented Lactobacillus mucinus DACN611 30.93%±2.80% ZHANG L, ZHANG Y, LIU S, et al. Degradation effects and mechanisms of Limosilactobacillus fermentum on ethanol [J]. Food & Function, 2024. Lactobacillus helveticus, Streptococcus thermophilus 36.87±1.58%33.64±1.90% Liu Weiliang, Mao Ruixia, Wang Xuefeng, et al. Screening of ethanol-degrading bacteria and evaluation of their hangover-relieving effects in fermented milk products [J]. Food Science, 2020, 41(02):107-113.

[0086] Example 6: Ethanol Tolerance Inhibition Test of Lactobacillus casei Lc-PG-10

[0087] Results of Lactobacillus casei Lc-PG-10 ethanol tolerance test

[0088] After two generations of activation and subculturing of Lactobacillus casei strain Lc-PG-10 in MRS medium at 37℃, the supernatant was collected by centrifugation and inoculated into MRS liquid medium containing 12% ethanol at an inoculum of 2%. Ethanol intervention was carried out at 37℃ for 0, 2, 4, 6, 8, and 10 h, respectively. Meanwhile, the culture group in MRS liquid medium without ethanol was used as a negative control. After intervention, the OD value was measured at a wavelength of 610 nm using a UV spectrophotometer, and the survival rate was calculated according to the formula.

[0089]

[0090] In the formula: A1 represents the values ​​of ethanol-containing MRS medium after culturing for 0, 2, 4, 6, 8, and 10 hours. Value; A0 represents the values ​​after 0, 2, 4, 6, 8, and 10 hours of culture in MRS medium without ethanol. value.

[0091] The results of the Lc-PG-10 ethanol tolerance test for Lactobacillus casei were obtained from Figure 8 It can be seen that the survival rate of Lactobacillus casei Lc-PG-10 gradually decreased with the increase of culture time. At 2 h, the survival rate of Lactobacillus casei Lc-PG-10 was 76.33%; at 4 h, the survival rate of Lactobacillus casei Lc-PG-10 could still reach 58.0%, indicating that Lactobacillus casei Lc-PG-10 has good ethanol tolerance and can tolerate ethanol with a volume fraction of 12%.

[0092] Example 7: In vitro antioxidant capacity test of Lactobacillus casei Lc-PG-10

[0093] 1. Results of DPPH free radical scavenging ability

[0094] Prepare a 0.1 mmol / L DPPH solution using anhydrous ethanol, store it protected from light, and adjust the absorbance at 517 nm to 1.25 ± 0.05 with anhydrous ethanol before use. Take 2.0 mL of bacterial culture into a 10 mL test tube, add 2.0 mL of DPPH solution, and incubate at room temperature in the dark for 20 min. Measure the absorbance at 517 nm and record it as A1. Record the absorbance when ethanol is used instead of DPPH as A2. Record the absorbance when ethanol is used instead of the sample as A0. Calculate the DPPH free radical scavenging rate using the formula.

[0095]

[0096] like Figure 9 As shown, the DPPH free radical scavenging rate of Lactobacillus casei Lc-PG-10 was 89.41±5.17%, which was significantly higher than that of LGG (79.34±7.04%).

[0097] 2. Results of scavenging ability against hydroxyl radicals

[0098] Take 1 mL of bacterial culture and place it in a 10 mL test tube. Then, add 1 mL of 9 mmol / L ferrous sulfate solution, 1 mL of 9 mmol / L salicylic acid ethanol solution, and 1 mL of 8.8 mmol / L H₂O₂ solution sequentially. Shake well and incubate the test tube at 37℃ for 30 min. After cooling to room temperature, measure the absorbance at 510 nm (A1). The absorbance of H₂O instead of H₂O₂ is A2. The absorbance of H₂O instead of the sample is A0. Calculate the hydroxyl radical scavenging rate using the formula.

[0099]

[0100] like Figure 10 The hydroxyl radical scavenging rate of Lactobacillus casei Lc-PG-10 was 95.46±2.14%, which was not significantly different from that of VC and LGG (96.83±1.09%).

[0101] 3. Regarding ABTS + Free radical scavenging ability results

[0102] First, prepare a 7 mmol / L ABTS solution and a 2.45 mmol / L K₂S₂O₈ solution, mix them in equal proportions, and react at 4°C in the dark for 12–16 h. Before use, dilute with anhydrous ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Take 0.5 mL of bacterial culture in a test tube, add 4.5 mL of the diluted ABTS stock solution, react at 30°C for 10 min, and measure the absorbance at 734 nm as A1. The absorbance at 734 nm using H₂O instead of ABTS is A2. The absorbance at 734 nm using H₂O instead of the sample is A0. Calculate the absorbance of ABTS using the formula. + Free radical scavenging rate.

[0103]

[0104] like Figure 11 The ABTS of Lactobacillus casei Lc-PG-10 shown + Although the free radical scavenging rate was lower than that of LGG, it was higher than 70%, and the ABTS of Lactobacillus casei Lc-PG-10 was... + The free radical scavenging rate was 74.37 ± 6.32%.

[0105] 4. Results of superoxide anion free radical scavenging ability

[0106] Take 1 mL of bacterial culture in a test tube, add 1 mL of 10 mmol / L pyrogallol solution and 5 mL of 50 mmol / L Tris-HCl (pH 8.2), react at 25℃ for 4 min, then add 1 mL of 8 mol / L HCl to terminate the reaction. Measure the absorbance at 320 nm (A1), the absorbance at 320 nm for H2O instead of pyrogallol (A2), and the absorbance at 320 nm for H2O instead of the sample (A0). Calculate the superoxide anion radical scavenging rate according to the formula.

[0107]

[0108] like Figure 12 Although the superoxide anion radical scavenging rate of L. casei Lc-PG-10 was significantly lower than that of LGG, it was still higher than 50%, with a superoxide anion radical scavenging rate of 52.19 ± 3.42%.

[0109] In summary, the results show that Lactobacillus casei Lc-PG-10 has high antioxidant capacity, and studies have shown that strains with antioxidant properties can play a positive role in the ethanol reduction reaction.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A strain of *Lactobacillus casei* with the ability to reduce ethanol, characterized in that, The specimen is Lactobacillus casei Lc-PG-10, which was deposited on September 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31863, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The *Lactobacillus casei* with ethanol-reducing ability according to claim 1, characterized in that, Including the following applications: 1) Application in the degradation of ethanol; 2) As a probiotic, its application in improving the gastrointestinal microenvironment; 3) Used in the preparation of drugs that regulate intestinal flora balance, reduce alcohol-induced intestinal barrier damage, reduce increased intestinal mucosal permeability, and reduce endotoxin entry into the blood.

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