Lactobacillus plantarum WL05 capable of efficiently degrading ethanol and use thereof

Abstract

The present invention belongs to the technical field of biology. Provided are Lactobacillus plantarum WL05 capable of efficiently degrading ethanol and the use thereof. The strain is Lactobacillus plantarum WL05, which was deposited at the China General Microbiological Culture Collection Center on July 8, 2024, with the deposit number CGMCC No. 31202. Lactobacillus plantarum WL05 exhibits high acid production ability and viability and high acid and bile salt tolerance. After being fermented in an MRS medium containing 10% ethanol concentration, Lactobacillus plantarum WL05 can achieve an ethanol degradation rate of 42.5%, and an ethanol dehydrogenase activation rate of 43.53%. The fermentation broth is inoculated into a substrate medium containing Imperatae Rhizoma and Phragmitis Rhizoma, and after fermentation, it is determined that the ethanol degradation rate is 63.9%, and the ethanol dehydrogenase activation rate in the fermentation broth is 66.5%. Moreover, the strain exhibits strong reduction ability and bacteriostatic ability. The beverage prepared on the basis of the strain can achieve an ethanol degradation rate of 32%. The strain can be used for preparing functional foods for counteracting the effects of alcohol and protecting the liver.

Description

Lactobacillus plantarum WL05 with high efficiency of degrading ethanol and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to Lactobacillus plantarum WL05 with high efficiency of degrading ethanol and application thereof. BACKGROUND

[0002] In recent years, with the acceleration of industrialization and the improvement of people's living standards, the consumption of alcohol has increased significantly, and the excessive intake of ethanol has posed a potential threat to human health. The liver is the main organ for the metabolism of alcohol in the human body, and excessive drinking can cause damage to the liver. The development of alcoholic liver disease (ALD) involves many stages, from general fatty hepatitis, alcoholic hepatitis, liver fibrosis, to severe cirrhosis and liver cancer. It has been reported that when the metabolism of alcohol in the liver is abnormal, excessive free radicals are produced, causing oxidative stress and leading to liver damage. Therefore, the development of effective ethanol degradation biotechnology, especially the use of microorganisms for ethanol degradation, has attracted widespread attention.

[0003] Lactic acid bacteria and their fermented products can prevent and protect against alcoholic liver damage; for example, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus fermentum, etc. can protect alcohol poisoning mice from liver damage by regulating the intestinal microbiota.

[0004] There are also some related reports on the use of probiotic compositions for alcohol detoxification and liver protection. For example, the invention patent with application number CN202311255212.X discloses a probiotic fermenting agent with alcohol detoxification and liver protection functions and its application, which uses a mixture of multiple bacterial agents; the invention patent with application number CN202311220048.9 discloses a fermented Lactobacillus strain A21252 for alcohol detoxification, liver protection, antioxidant and digestive tract damage reduction and its application. After 24 hours of cultivation in a culture medium containing 15% ethanol, the ethanol degradation rate of strain 21252 under in vitro conditions can reach 30%. The invention patent with application number CN202310635075.6 discloses a Lactobacillus salivarius and its application in preparing an alcohol detoxification product. After fermentation by the high-density process-constant pH method, the ethanol degradation rate of Lactobacillus salivarius PLJK-0039 can reach 35%.

[0005] However, the strains in these studies mostly have problems such as insufficient degradation rate and slow degradation speed, which affect their effectiveness in practical applications. Therefore, finding more efficient microbial strains to improve the degradation rate of ethanol is still the focus of research in this field. SUMMARY

[0006] The present application aims at the deficiencies of the prior art, and provides a Lactobacillus plantarum WL05 capable of protecting liver and relieving alcoholism and application thereof in preparation of a liver-protecting and alcohol-relieving functional product.

[0007] The first object of the present application is to provide a Lactobacillus plantarum WL05, which has been preserved in the China General Microbiological Culture Collection Center on July 8, 2024, with a preservation number of CGMCC No.31202 and an address of 1 Qingdao West Road, No.3, Beijing Chaoyang District.

[0008] Further, the Lactobacillus plantarum WL05 is isolated from the feces of a centenarian in Guangxi Changshou Village, and has a nucleotide sequence of SEQ ID NO.1. The Lactobacillus plantarum WL05 is isolated from the feces of a centenarian in Guangxi Changshou Village, and can be cultured by using a common culture medium for culturing lactic acid bacteria, such as MRS culture medium.

[0009] As a recognized safe microorganism, lactic acid bacteria are normal intestinal flora in the human intestinal tract and have the ability to protect liver and relieve alcoholism. Screening of lactic acid bacteria with strong alcohol-relieving and liver-protecting ability can be used for development of food or health products with alcohol-relieving and liver-protecting effects, so as to make lactic acid bacteria fermented food a daily supplement for liver protection.

[0010] The second object of the present application is to provide a microbial agent containing the Lactobacillus plantarum WL05 or a culture solution or freeze-dried powder thereof.

[0011] Further, the content of the Lactobacillus plantarum WL05 in the microbial agent is at least 2.1×10 12 CFU / mL or 2.1×10 12 CFU / g.

[0012] In some embodiments of the present application, the microbial agent is a freeze-dried powder; and the preparation method of the freeze-dried powder comprises the following steps: freezing the Lactobacillus plantarum WL05 at-40℃ to-60℃, drying to a water content of 3% to 5%, and crushing into powder.

[0013] The third object of the present application is to provide a fermented beverage containing the Lactobacillus plantarum WL05, wherein the beverage is prepared by fermenting a mixture of white flag root extract and reed root extract with the Lactobacillus plantarum WL05.

[0014] Further, the inoculation ratio of the Lactobacillus plantarum WL05 in the beverage is 3% to 9% of the mixture of white flag root extract and reed root extract.

[0015] In some embodiments of the present application, the preparation method of the Imperata cylindrical extract liquid is as follows: crushing the Imperata cylindrical, adding deionized water for extraction, and sterilizing after solid-liquid separation.

[0016] In some embodiments of the present application, the preparation method of the Phragmites communis Trin extract liquid is as follows: crushing the Phragmites communis Trin, adding deionized water for extraction, and sterilizing after solid-liquid separation.

[0017] In some embodiments of the present application, the preparation method of the Imperata cylindrical extract liquid is as follows: crushing the Imperata cylindrical, adding deionized water for extraction at a ratio of 1:4 at 85°C for 30 min, filtering twice through an 80-mesh sieve, and sterilizing in a 95°C water bath for 5 min, and then cooling.

[0018] In some embodiments of the present application, the preparation method of the Phragmites communis Trin extract liquid is as follows: crushing the Phragmites communis Trin, adding deionized water for extraction at a ratio of 1:4 at 85°C for 30 min, filtering twice through an 80-mesh sieve, and sterilizing in a 95°C water bath for 5 min, and then cooling.

[0019] Further, the ratio of the Imperata cylindrical extract liquid to the Phragmites communis Trin extract liquid in the mixed liquid is 1:2-2:1.

[0020] Further, the fermentation time of the mixed liquid is 24-36 h.

[0021] In some embodiments of the present application, the preparation method of the fermented drink of Lactobacillus plantarum WL05 includes the following steps: (1) strain purification: spreading Lactobacillus plantarum WL05 on MRS solid culture medium for purification culture;

[0022] (2) scale-up culture: picking the strain from the MRS solid culture medium and placing it in MRS liquid culture medium added with 10% ethanol, and culturing at 37°C for 12-24 h;

[0023] (3) preparation of Imperata cylindrical-Phragmites communis Trin extract liquid medium: crushing the Imperata cylindrical, adding deionized water for extraction at a ratio of 1:4 at 85°C for 30 min, filtering twice through an 80-mesh sieve, and sterilizing in a 95°C water bath for 5 min, and then cooling for standby; crushing the Phragmites communis Trin, adding deionized water for extraction at a ratio of 1:4 at 85°C for 30 min, filtering twice through an 80-mesh sieve, and sterilizing in a 95°C water bath for 5 min, and then cooling for standby; mixing the Imperata cylindrical extract liquid and the Phragmites communis Trin extract liquid at a ratio of 1:2 for inoculation culture;

[0024] (4) fermentation of Imperata cylindrical-Phragmites communis Trin extract liquid: inoculating Lactobacillus plantarum WL05 into the Imperata cylindrical-Phragmites communis Trin extract liquid medium at a ratio of 3%-9% (v / v), and fermenting at 37°C for 24 h-36 h; after fermentation, sterilizing at 95°C for 20 min, and then cooling for standby.

[0025] The fourth object of the present application is to provide a culture medium of Lactobacillus plantarum WL05, which is a Imperata cylindrica-Phragmites communis extraction liquid matrix culture medium; the preparation method is as follows: the Imperata cylindrica is crushed, and then extracted with an appropriate amount of water, followed by solid-liquid separation; the liquid phase is sterilized and then reserved; the Phragmites communis is crushed, and then extracted with an appropriate amount of water, followed by solid-liquid separation; the liquid phase is sterilized and then reserved; the Imperata cylindrica extraction liquid and the Phragmites communis extraction liquid are mixed in a ratio of 1:2, and used for inoculating and culturing Lactobacillus plantarum WL05.

[0026] In an embodiment, the preparation method of the Imperata cylindrica-Phragmites communis extraction liquid matrix culture medium is as follows: the Imperata cylindrica is crushed, and then extracted with water at a ratio of 1:4 at 85 DEG C for 30 min by ultrasonic extraction, filtered twice through an 80-mesh sieve, sterilized in a 95 DEG C water bath for 5 min, and then cooled for reservation; the Phragmites communis is crushed, and then extracted with water at a ratio of 1:4 at 85 DEG C for 30 min; filtered twice through an 80-mesh sieve, sterilized in a 95 DEG C water bath for 5 min, and then cooled for reservation; the Imperata cylindrica extraction liquid and the Phragmites communis extraction liquid are mixed in a ratio of 1:2, and used for inoculating and culturing Lactobacillus plantarum WL05.

[0027] The fourth object of the present application is to provide the use of the Lactobacillus plantarum WL05 or the microbial inoculant in the preparation of an ethanol-degrading functional product.

[0028] Further, the ethanol-degrading functional product includes but is not limited to beverages, snacks, and dairy products.

[0029] In some embodiments, the present application also provides a functional supplement prepared by using Lactobacillus plantarum WL05, which is frozen at-40 DEG C to-60 DEG C, dried to a water content of 3% to 5%, and then crushed into a powder; milk powder, oligosaccharides, malt dextrin, or flavoring agents can be added to the powdered Lactobacillus plantarum WL05 to prepare a powder or tablet product for daily consumption.

[0030] The present application also provides a method for degrading ethanol by using the Lactobacillus plantarum WL05, which mainly includes the following steps:

[0031] (1) strain activation: Lactobacillus plantarum WL05 is inoculated on a MRS solid culture medium for activation culture,

[0032] The formula of the MRS solid culture medium is as follows: 10.0 g of proteose peptone, 3.0 g of sodium acetate trihydrate, 8.0 g of beef extract, 2.0 g of ammonium citrate tribasic, 4.0 g of yeast extract, 0.2 g of magnesium sulfate heptahydrate, 10.0 g of glucose, 0.04 g of manganese sulfate monohydrate, 1.0 mL of Tween-80, 2.0 g of potassium phosphate dibasic, 15 g of agar, and 1000 mL of distilled water, with the pH being adjusted to 6.4; the activation culture temperature is 37 DEG C, and the culture time is 16 to 48 h;

[0033] (2) fermentation of ethanol dehydrogenase: the activated Lactobacillus plantarum WL05 is inoculated into a fermentation medium for culture,

[0034] The fermentation medium formula is: 10.0 g of proteose peptone, 3.0 g of sodium acetate trihydrate, 8.0 g of beef extract, 2.0 g of ammonium citrate, 4.0 g of yeast extract, 0.2 g of magnesium sulfate heptahydrate, 10.0 g of glucose, 0.04 g of manganese sulfate monohydrate, 1.0 mL of Tween-80, 2.0 g of potassium phosphate dibasic, 10% ethanol, and 1000 mL of distilled water; the fermentation culture temperature is 37 DEG C, and the culture time is 16-48 h. Beneficial effects

[0035] The ethanol degradation rate of the Lactobacillus plantarum WL05 provided in the application can reach 42.5% after fermentation in the MRS medium containing 10% ethanol concentration, and the ethanol dehydrogenase activation rate is 43.53%;

[0036] The Lactobacillus plantarum WL05 provided in the application has a high survival rate in a simulated human environment and has strong acid and bile salt resistance; after 37 DEG C fermentation in the MRS liquid medium for 24 h, the pH values of the fermentation liquor are 3.59, respectively, and the viable cell count is greater than 2.1*10 12 CFU / mL, has high acid production capacity and activity, and has strong antioxidant activity and bacteriostatic advantages;

[0037] Compared with the original strain, the ethanol dehydrogenase capacity is significantly improved by fermentation with different substrates. After the Lactobacillus plantarum WL05 is fermented in the MRS medium containing 10% ethanol concentration, the ethanol degradation rate can reach 63.9% when the fermentation liquor is inoculated into the Imperata cylindrical-Aesurus linearis substrate medium, the ethanol dehydrogenase activation rate in the fermentation liquor is 66.5%, and the ethanol dehydrogenase enzyme activity reaches 63 U / mL.

[0038] The Lactobacillus plantarum WL05 provided in the application shows good alcoholism relieving and liver protecting capacity when fermented with traditional medicine and food homologous Imperata cylindrical-Aesurus linearis, and can be compounded with other lactic acid bacteria to develop alcoholism relieving and liver protecting fermented milk products or lactic acid bacteria beverages, or the culture of the lactic acid bacteria can be freeze-dried for producing functional supplements for alcoholism relieving and liver protecting.

[0039] Biological material preservation

[0040] The Lactobacillus plantarum WL05 is preserved in the China General Microbiological Culture Collection Center on July 8, 2024, with a preservation number of CGMCC No.31202 and a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is an electropherogram of Lactobacillus plantarum WL05 of the present application (loading order: WL01, WL04, WL05, WL06, WL07, WL08, WL09, WL010, Marker). DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be described clearly and completely below in combination with the drawings and examples. Obviously, the described examples are only some of the examples of the present application, but not all the examples, which do not limit the claims of the present application. The experimental methods described in the following examples are all conventional methods, unless otherwise specified. The reagents and materials described, unless otherwise specified, can be obtained commercially.

[0043] Imperata cylindrica (alias: silk grass, grass, white grass); purchased from: Ali health pharmacy;

[0044] Phragmites australis (alias: reed root, reed, reed root, reed root); purchased from: Ali health pharmacy.

[0045] Related culture medium and reagent formula

[0046] MRS solid medium: proteose peptone 10.0 g, sodium acetate trihydrate 3.0 g, beef extract 8.0 g, triammonium citrate 2.0 g, yeast extract 4.0 g, magnesium sulfate heptahydrate 0.2 g, glucose 10.0 g, manganese sulfate monohydrate 0.04 g, Tween-80 1.0 mL, potassium phosphate dibasic 2.0 g, agar 15 g, distilled water 1000 mL, adjust pH to 6.4.

[0047] MRS liquid medium: proteose peptone 10.0 g, sodium acetate trihydrate 3.0 g, beef extract 8.0 g, triammonium citrate 2.0 g, yeast extract 4.0 g, magnesium sulfate heptahydrate 0.2 g, glucose 10.0 g, manganese sulfate monohydrate 0.04 g, Tween-80 1.0 mL, potassium phosphate dibasic 2.0 g, distilled water 1000 mL. Adjust pH to 6.4, sterilize at 121℃ for 15 min, cool for standby.

[0048] MRS fermentation medium: proteose peptone 10.0 g, sodium acetate trihydrate 3.0 g, beef extract 8.0 g, triammonium citrate 2.0 g, yeast extract 4.0 g, magnesium sulfate heptahydrate 0.2 g, glucose 10.0 g, manganese sulfate monohydrate 0.04 g, Tween-80 1.0 mL, potassium phosphate dibasic 2.0 g, 10% ethanol, distilled water 1000 mL. Adjust pH to 6.4, sterilize at 121℃ for 15 min, cool for standby.

[0049] Imbibition liquid of Imperata cylindraea and Phragmites communis substrate culture medium: Imperata cylindraea raw material, according to the ratio of 1:4, 85 ℃ ultrasonic extraction for 30 min, 80 mesh filter 2 times, 95 ℃ water bath sterilization for 5 min, and then cool standby; Phragmites communis raw material, according to the ratio of 1:4, 85 ℃ ultrasonic extraction for 30 min; 80 mesh filter 2 times, 95 ℃ water bath sterilization for 5 min, and then cool standby; Imperata cylindraea extraction liquid and Phragmites communis extraction liquid were mixed according to the ratio of 1:2, used for inoculation culture.

[0050] Skim milk substrate culture medium: skim milk powder 120 g, distilled water 1000 mL.

[0051] Ethanol tolerance determination method: the blank group took 1 mL of seed liquid inoculated in 50 mL of MRS liquid medium, 37 ℃ culture for 12-24 h, the absorbance value of the bacterial liquid at 600 nm was measured Ac. The experimental group and the blank group were operated in the same way, but the seed liquid was inoculated in 50 mL of MRS fermentation medium, and the absorbance value was recorded as Ai. By comparing the absorbance value, the ethanol tolerance of the strain was determined. The calculation formula: ethanol tolerance (%) = (Ai / Ac) x 100%.

[0052] Example 1 Isolation and identification of Lactobacillus plantarum WL05

[0053] 1. Isolation of lactic acid bacteria

[0054] The lactic acid bacteria were isolated from the feces of the Longevity Village in Guangxi, and the collected samples were gradient diluted and inoculated in MRS solid culture medium, 37 ℃ culture for 48 h, typical colonies on the plate were picked and streaked for isolation and culture to obtain pure colonies. The pure colonies on the plate were picked in MRS liquid culture medium, 37 ℃ anaerobic culture for 24 h, the strain concentration reached 2.1-2.6 x 10 12 CFU / mL, 4 ℃ refrigerator cold storage or freeze-dried preservation.

[0055] 2. Identification of lactic acid bacteria

[0056] 16s rDNA sequence determination for strain identification: the prepared lactic acid bacteria genomic DNA was used as a template for PCR amplification, and a 50 μL reaction system was used for PCR amplification. 2 uL of template DNA, 1.5 μL of 10 mmol upper and lower primers, 25 μL of 2x Taq enzyme, and ddH2O was added to 50 μL. The PCR cycle parameters are: 94°C pre-denaturation for 5 min; 94°C denaturation for 1 min, 64°C annealing for 45 s, 72°C extension for 1 min, 35 cycles; 72°C extension for 8 min. 5 μL of PCR product was mixed with 1 mL Loading buffer, and 1.0% agarose gel electrophoresis was used, and the voltage was 80V. After electrophoresis, EB was used for staining for 20-30 min, and the picture was taken. After electrophoresis detection of the amplification product, it was sent to Shanghai Sunway Biotech Co., Ltd. for sequencing. The sequence obtained by sequencing was compared and analyzed with the sequence in the GenBank database, and the results showed that the homology of strain WL05 and Lactobacillus plantarum was 99.9%. The results show that the strain WL05 of the application is Lactobacillus plantarum.

[0057] 3. Acid production capacity and ethanol tolerance of Lactobacillus plantarum WL05

[0058] Lactic acid bacteria acid production capacity: the strain (viable bacteria concentration 2.1-2.6x10 12 CFU / mL) after cold storage in Example 1 was inoculated into MRS liquid medium at a ratio of 3% (v / v), and cultured at 37°C for 24h, and the pH of the fermentation broth was measured by pH meter, and each strain was measured in triplicate, and the average value was taken. The results show that the pH value of the fermentation broth of Lactobacillus plantarum WL05 after 24h fermentation in MRS medium is 3.59, indicating that the strain has good acid production capacity and good growth condition.

[0059] The pH value of the fermentation broth of the Lactobacillus plantarum WL05 after 24h fermentation in MRS medium is 3.59, and the viable bacteria count is greater than 2.1x10 12 CFU / mL, so the Lactobacillus plantarum WL05 has high acid production capacity and activity.

[0060] The ethanol tolerance of the strain after cold storage in Example 1 was tested, and the results show that the ethanol tolerance of Lactobacillus plantarum WL05 is 80.5%, and it has high ethanol tolerance.

[0061] 4. Ethanol degradation rate determination

[0062] (1) Preparation of ethanol standard curve: add 0.1 mL of ethanol standard solution with volume concentration of 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0% (v / v) respectively into 10 mL culture tubes, add 1 mL of potassium dichromate-sulfuric acid solution into each test tube, shake well; place the test tubes in boiling water bath for 10 min, measure the absorbance of potassium dichromate-sulfuric acid solution at wavelength 610 nm; prepare ethanol standard curve with volume concentration (v / v) of ethanol as abscissa and absorbance as ordinate.

[0063] Wherein, ethanol standard solution: take an appropriate amount of anhydrous ethanol, add sterile water to prepare ethanol standard solution with different concentrations (0.1%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (v / v)); potassium dichromate-sulfuric acid solution: take 1 g of potassium dichromate, dissolve in 25 mL of water, slowly add 4 mL of concentrated sulfuric acid while stirring.

[0064] (2) Ethanol degradation capacity determination: take Lactobacillus plantarum activated twice, inoculate in MRS liquid enrichment medium for enrichment for 5-10 h, then dilute, spread on solid screening medium. Take single colony from the screening medium, inoculate in MRS liquid medium and culture for 18 h, measure OD, adjust the bacterial liquid to 10 7 CFU / mL. Inoculate in MRS ethanol culture medium with different concentrations (5%, 10%, 15%) at inoculation amount of 5%, take MRS ethanol culture medium without bacterial liquid as control, culture for 24 h.

[0065] Take 24 h fermentation culture, centrifuge (10000 rpm, 10 min), take 0.1 mL of supernatant, add 1 mL of potassium dichromate-sulfuric acid solution, shake well; place the test tubes in boiling water bath for 10 min, measure the absorbance of potassium dichromate-sulfuric acid solution at wavelength 610 nm.

[0066] Use ethanol standard curve to calculate ethanol residual amount, compare to obtain the quantitative size of alcohol tolerance of the strain.

[0067] Lactobacillus plantarum WL05 was fermented in MRS medium containing 10% ethanol concentration at 37°C for 19-24 h, the measured ethanol degradation rate was 42.5%, then the fermentation liquid was inoculated into skim milk medium and white flag grass-Phragmites australis medium at inoculation amount of 6%, after fermentation at 37°C for 24 h in the skim milk medium, the measured ethanol degradation rate was 35.8%, and after fermentation at 37°C for 24 h in the white flag grass-Phragmites australis medium, the measured ethanol degradation rate was 63.9%.

[0068] The Lactobacillus plantarum WL05 was fermented in MRS medium containing 15% ethanol at 37°C for 19-24h, and the ethanol degradation rate was 40.2%. Then, the fermentation liquor was inoculated into skim milk medium and white flag grass-reed medium at a ratio of 6%, and the ethanol degradation rate was 32.5% after fermentation in the skim milk medium at 37°C for 24h, and the ethanol degradation rate was 60.4% after fermentation in the white flag grass-reed medium at 37°C for 24h.

[0069] 5. Ethanol dehydrogenase activity determination

[0070] Preparation of fermentation liquor

[0071] (1) Preparation of bacterial solution: the bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 12-24h.

[0072] (2) Fermentation of ethanol dehydrogenase: the test bacterial solution was added into 10mL MRS medium, skim milk medium or white flag grass-reed medium at a ratio of 6%. After incubation at 37°C for 24-60h, the fermentation liquor was obtained by centrifugation at 4000rpm for 10min.

[0073] Determination of ethanol dehydrogenase in fermentation liquor: according to the method of Chai Zheng, 1.5mL of sodium pyrophosphate solution (pH 8.8) was added into a test tube with a stopper, followed by the addition of 1.0mL of 0.027mol / L oxidized coenzyme I (NAD + ), 0.5mL of 11% ethanol solution and 0.1mL of sample solution. After mixing, the test tube was placed in a 37°C water bath for 5min. After incubation, 0.1mL of ADH (0.25U / mL) was added, and 0.5mL of distilled water was used as a blank control instead of 11.5% ethanol solution. The test tube solution was shaken well, and the experimental group with 0μg / mL was used as a negative control. The solution was shaken and then the absorbance value A340 was determined at a wavelength of 340nm. The absorbance value change represented the enzymatic reaction rate of ADH. The absorbance value was recorded every 30s for 5min, and the average absorbance value per minute was calculated according to the following formulas (1) and (2):

[0074] In the formula, V is the total reaction volume, M is the sample amount, and ΔA340 / min is the average absorbance value per minute.

[0075] Activation rate / %=(U1-U0) / U0x100% (2)

[0076] In the formula, U1 and U0 are the enzyme activities of the experimental group and the control group, respectively. The results were determined in triplicate.

[0077] Lactobacillus plantarum WL05 was detected more than 15 U / mL of alcohol dehydrogenase enzyme activity in MRS medium fermentation broth, more than 10 U / mL of alcohol dehydrogenase enzyme activity in skim milk substrate medium fermentation broth, more than 63 U / mL of alcohol dehydrogenase enzyme activity in Imperata-Carex substrate medium fermentation broth.

[0078] In MRS medium containing 10% ethanol concentration, Lactobacillus plantarum WL05 was fermented at 37℃ for 19-24 hours, the activation rate of alcohol dehydrogenase in fermentation broth was 43.53%. Then, the fermentation broth was inoculated into skim milk substrate medium and Imperata-Carex substrate medium respectively, the activation rate of alcohol dehydrogenase in fermentation broth was 32.26% after 24 hours fermentation at 37℃ in skim milk substrate medium, while the activation rate of alcohol dehydrogenase in fermentation broth was 66.5% after 24 hours fermentation at 37℃ in Imperata-Carex substrate medium.

[0079] In MRS medium containing 15% ethanol concentration, Lactobacillus plantarum WL05 was fermented at 37℃ for 19-24 hours, the activation rate of alcohol dehydrogenase in fermentation broth was 41.2%. Then, the fermentation broth was inoculated into skim milk substrate medium and Imperata-Carex substrate medium respectively, the activation rate of alcohol dehydrogenase in fermentation broth was 30.6% after 24 hours fermentation at 37℃ in skim milk substrate medium, while the activation rate of alcohol dehydrogenase in fermentation broth was 62.6% after 24 hours fermentation at 37℃ in Imperata-Carex substrate medium.

[0080] 6. Determination of acid tolerance and bile salt tolerance of Lactobacillus plantarum WL05

[0081] Lactobacillus plantarum WL05 was activated by spreading on plate medium, and then the strain was picked from plate medium and inoculated into MRS liquid medium, and incubated at 37℃ for 12-24 hours.

[0082] Lactobacillus plantarum strain WL05 culture was centrifuged at 10000 x g for 5 min to collect the bacterial cells, and then washed twice with PBS, and resuspended with physiological saline. 1 mL of bacterial suspension was inoculated into 9 mL of simulated gastric juice at pH 3.0, and incubated in a 37℃ incubator. Samples were taken at 0h and 3h for plate counting to determine the number of viable bacteria and calculate the survival rate.

[0083] Lactobacillus plantarum strain WL05 culture was centrifuged at 10000 x g for 5 min to collect the bacterial cells, and then washed twice with PBS, and resuspended with physiological saline. 1 mL of bacterial suspension was inoculated into MRS liquid medium containing 0%, 0.1%, 0.3% and 0.5% (w / v) bile salts, and incubated at 37℃ for 3h. Samples were taken at 0h and 3h for plate counting to determine the number of viable bacteria and calculate the survival rate.

[0084] The results show that the survival rate of Lactobacillus plantarum WL05 in artificial gastric juice with pH of 3.0 is 69.2%, and the survival rates in MRS culture medium with 0.10%, 0.30% and 0.50% of bile salt concentration for 3 hours are 74.5%, 43.7% and 15.5% respectively.

[0085] 7. Determination of antioxidant capacity of Lactobacillus plantarum WL05

[0086] (1) DPPH scavenging rate determination

[0087] Lactobacillus plantarum WL05 is activated and cultured on a flat plate medium, and then the strain is picked from the flat plate medium and placed in MRS liquid medium for 12-24 hours of culture at 37°C; the culture of Lactobacillus plantarum strain WL05 is centrifuged at 10000xg for 5 minutes, and the supernatant is taken as a sample.

[0088] 0.5mL of the sample solution is accurately measured and added into a test tube with a stopper, and then 0.2mL of DPPH solution (0.2mmol / L DPPH dissolved in 95% ethanol solution) is added, and after mixing, the reaction is carried out at 40°C for 20 minutes in the dark, and the absorbance is measured at a wavelength of 517nm, which is recorded as A i ; a blank group: 0.5ml of the sample solution is accurately measured and added into a test tube with a stopper, and then 0.2mL of 95% ethanol solution is used instead of DPPH solution, and after mixing, the absorbance is measured at a wavelength of 517nm, which is recorded as A j ; a control group: in a test tube with a stopper, 2.0mL of DPPH solution and 2mL of 95% ethanol solution are added respectively, and the absorbance is measured at a wavelength of 517nm, which is recorded as A c .

[0089] The total volume of the reaction of the above experimental groups is 4mL, which is supplemented with 95% ethanol solution, and the DPPH free radical scavenging rate is calculated according to the following formula: scavenging rate (%) = [1-(A i -A j )] / A c *100

[0090] The results show that the DPPH scavenging rate of Lactobacillus plantarum WL05 reaches 78.5%.

[0091] (2) Determination of reducing capacity

[0092] Lactobacillus plantarum WL05 is activated and cultured on a flat plate medium, and then the strain is picked from the flat plate medium and placed in MRS liquid medium for 12-24 hours of culture at 37°C; the culture of Lactobacillus plantarum strain WL05 is centrifuged at 10000xg for 5 minutes, and the supernatant is taken as a sample.

[0093] Take 1.0 mL sample in centrifugal tube, then add 2.5 mL 0.2 mol / L phosphate buffer solution (pH 6.6) and 1% potassium ferricyanide solution respectively, mix well, incubate at 50℃ for 20 min, quickly cool to room temperature, then add 2.5 mL 10% trichloroacetic acid solution, centrifuge at 3500 r / min for 10 min, accurately take 2.5 ml supernatant in a test tube with stopper, then add 2.5 mL distilled water, 0.5 mL 0.1% ferric chloride solution in turn, mix well, and measure the absorbance of the solution at a wavelength of 200 nm. VC is used as a control sample.

[0094] The results show that the reducing capacity of the plant lactobacillus WL05 is 0.85, and the reducing capacity of VC is 1.20. It is shown that the plant lactobacillus WL05 has high antioxidant activity.

[0095] 8. Determination of the bacteriostatic ability of plant lactobacillus WL05

[0096] The plant lactobacillus WL05 is inoculated in the culture medium and cultured at 37℃ for 24h, and the bacteriostatic ability of the lactic acid bacteria to escherichia coli, staphylococcus aureus, salmonella and bacillus subtilis is determined by the oxford cup method. The bacterial suspension of escherichia coli, staphylococcus aureus, salmonella and bacillus subtilis is coated on the LB culture medium, 200μL of lactic acid bacteria fermentation liquor is added to the oxford cup, and incubated at constant temperature for 48h, and the bacteriostatic ring is observed.

[0097] The results show that the plant lactobacillus WL05 has high bacteriostatic ability to escherichia coli, staphylococcus aureus, salmonella and bacillus subtilis, and the diameters of the bacteriostatic rings are 22.50mm, 18.35mm, 17.22mm and 16.50mm respectively.

[0098] Example 2: Preparation of fermented white flag root-reed root beverage containing plant lactobacillus WL05

[0099] (1) Strain activation: the plant lactobacillus WL05 is coated on the flat plate culture medium for activation culture.

[0100] (2) Expansion culture: the plant lactobacillus WL05 is inoculated in the optimized MRS culture medium at a inoculation amount of 6%, and cultured at 37℃ for 24-48h.

[0101] (3) Preparation of white flag root / reed root extract:

[0102] The white flag root raw material is ultrasonically extracted at 85℃ for 30 min at a material-water ratio of 1:4, filtered twice through an 80 mesh sieve, and sterilized at 95℃ for 5 min to obtain the white flag root extract, which is cooled for standby use;

[0103] Phragmites communis raw material, according to the ratio of 1:4, 85 ℃ ultrasonic extraction 30 min; 80 mesh filter 2 times, 95 ℃ water bath sterilization 5 min, Phragmites communis extract, cooling ready for use;

[0104] (4)Imperatae-Phragmites culture medium preparation;

[0105] Imperatae extract and Phragmites extract mixed according to a certain proportion to obtain Imperatae-Phragmites culture medium, the mixed Imperatae-Phragmites extract is inoculated with bacteria, the inoculation amount is 3%-9%(v / v), and the fermentation time is 24h-36h at 37℃.

[0106] After fermentation, the Imperatae-Phragmites fermentation liquor is diluted 1 times with sterilized and cooled water, and appropriate sweeteners and sour agents are added to prepare a directly drinkable lactic acid bacteria beverage.

[0107] After fermentation according to the conditions in Table 1, the Imperatae-Phragmites beverage is prepared, and the ethanol degradation rate is tested (the test method is the same as in Example 1): the fermentation conditions and the ethanol degradation rate are shown in Table 1.

[0108] Table 1 Fermented Imperatae-Phragmites beverage of Lactobacillus plantarum WL05

[0109] As shown in Table 1, the fermented Imperatae-Phragmites beverage containing Lactobacillus plantarum WL05 has a high ethanol degradation rate. When the inoculation amount is 6% and the fermentation time is 28h, the ethanol degradation rate can reach 32%.

[0110] Example 3 Method for preparing functional supplement containing Lactobacillus plantarum WL05

[0111] (1) Strain activation: Lactobacillus plantarum WL05 is inoculated on a flat plate culture medium for activation culture,

[0112] (2) Expansion culture: Lactobacillus plantarum WL05 is inoculated into the optimized MRS culture medium at an inoculation amount of 5%, and cultured at 37℃ for 24-48h.

[0113] (3) Freeze-dried powder preparation: Lactobacillus plantarum WL05 is frozen to-40℃ to-60℃, dried to a water content of 3%-5%, and crushed into powder.

[0114] The powder product prepared in Example 3 can be added to milk powder, oligosaccharides, malt dextrin, flavoring agents, etc. to prepare powder or tablet products for daily consumption.

[0115] The Lactobacillus plantarum WL05 provided by the application has a pH value of 3.59 and a viable cell count of greater than 2.1*1012 CFU / mL after fermentation at 37 DEG C for 24 h in MRS liquid culture medium, so the Lactobacillus plantarum WL05 has high acid production capacity and activity. The survival rate of the Lactobacillus plantarum WL05 in artificial gastric juice with a pH of 3.0 is greater than 69.2%, and the survival rates in MRS culture medium with a bile salt concentration of 0.10%, 0.30% and 0.50% are greater than 74.5%, 43.7% and 15.5% respectively after 3 h of culture. The ethanol degradation rate of the Lactobacillus plantarum WL05 after fermentation in MRS culture medium containing 10% ethanol concentration is 42.5%, and the ethanol dehydrogenase activation rate is 43.53%; then, the fermentation liquor is inoculated into Imperata-Carex substrate culture medium, and after fermentation, the ethanol degradation rate is 63.9% and the ethanol dehydrogenase activation rate in the fermentation liquor is 66.5%. The DPPH clearance rate of the Lactobacillus plantarum WL05 is 78.5%, and the Lactobacillus plantarum WL05 has strong antibacterial ability. The ethanol degradation rate of a beverage prepared based on the strain is 32%, so the Lactobacillus plantarum WL05 has high application value in the field of functional food.

[0116] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, so the protection scope of the present application should be defined by the claims.

Claims

1. A Lactobacillus plantarum WL05, which has been deposited with the China General Microbiological Culture Collection Center on July 8, 2024, and has the accession number of CGMCC No.31202, and is located at No.3, Xibaheyi, Beijing, China.

2. The Lactobacillus plantarum WL05 according to claim 1, characterized in that, The Lactobacillus plantarum WL05 is isolated from the feces of a centenarian in Guangxi's Longevity Village, and has the nucleotide sequence of SEQ ID NO.

1.

3. A microbial inoculant, characterized in that, The microbial agent contains the Lactobacillus plantarum WL05 of claim 1 or its culture solution, or its freeze-dried powder.

4. The microbial inoculant of claim 3, wherein, The content of Lactobacillus plantarum WL05 in the microbial inoculant is at least 2.1 x 10 12 CFU / mL or 2.1 x 10 12 CFU / g.

5. The microbial inoculant of claim 2, wherein, The freeze-dried powder is prepared by freezing the Lactobacillus plantarum WL05 at-40℃ to-60℃, drying to a water content of 3% to 5%, and then crushing into powder.

6. A fermented drink comprising Lactobacillus plantarum WL05, characterized in that, The beverage is prepared by mixing the Iris lactea extract and the Phragmites australis extract, and then adding the Lactobacillus plantarum WL05 of any one of claims 1-2 for fermentation. The inoculation ratio of the Lactobacillus plantarum WL05 is 3% to 9% of the mixed solution.

7. The fermented drink containing Lactobacillus plantarum WL05 according to claim 6, characterized by, The Iris lactea extract is prepared by crushing the Iris lactea, adding deionized water for extraction, and sterilizing after solid-liquid separation. The Phragmites australis extract is prepared by crushing the Phragmites australis, adding deionized water for extraction, and sterilizing after solid-liquid separation. 8.The fermented beverage containing the Lactobacillus plantarum WL05 of claim 7, wherein The ratio of the Iris lactea extract to the Phragmites australis extract in the mixed solution is 1:2-2:

1. The fermentation time is 24-36h.

9. A culture medium of Lactobacillus plantarum WL05, characterized in that, The culture medium is Iris lactea-Phragmites australis extract medium, which is prepared by crushing the Iris lactea, adding an appropriate amount of water for extraction, solid-liquid separation, sterilizing the liquid phase, and then using; crushing the Phragmites australis, adding an appropriate amount of water for extraction, solid-liquid separation, sterilizing the liquid phase, and then using; mixing the Iris lactea extract and the Phragmites australis extract in a ratio of 1:2 for inoculating and culturing the Lactobacillus plantarum WL05. 10.Use of the Lactobacillus plantarum WL05 of any one of claims 1-2 or the microbial agent of any one of claims 3-5 in the preparation of an ethanol-degrading functional product. The ethanol-degrading functional product includes but is not limited to beverages, snacks, and dairy products.

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