Lactobacillus plantarum WL05 capable of efficiently degrading ethanol and application of lactobacillus plantarum WL05

Abstract

The invention discloses lactobacillus plantarum WL05 capable of efficiently degrading ethanol and application of the lactobacillus plantarum WL05, and belongs to the technical field of biology. The bacterial strain is lactobacillus plantarum WL05, the bacterial strain is preserved in the China General Microbiological Culture Collection Center on July 8, 2024, and the preservation number is CGMCC No.31202. The lactobacillus plantarum WL05 has relatively high acid production capacity and activity, and the lactobacillus plantarum WL05 is preserved in the China General Microbiological Culture Collection Center on July 8, 2024. The acid resistance and cholate resistance are high; after the lactobacillus plantarum WL05 is fermented in an MRS culture medium containing 10% ethanol, the ethanol degradation rate can reach 42.5%, and the ethanol dehydrogenase activation rate is 43.53%; the fermentation liquor is inoculated into a lalang grass rhizome-reed rhizome matrix culture medium, and after fermentation, the measured ethanol degradation rate is 63.9%, and the activation rate of ethanol dehydrogenase in the fermentation liquor is 66.5%. The strain has strong reducing ability and antibacterial ability at the same time. The ethanol degradation rate of the beverage prepared based on the strain can reach 32%. The product can be used for preparing functional food for dispelling effects of alcohol and protecting liver, and has high application value in the field of functional food.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Lactobacillus plantarum WL05 capable of efficiently degrading ethanol and a use thereof. Background Art

[0002] In recent years, with the acceleration of industrialization and the improvement of people's living standards, alcohol consumption has increased significantly, and excessive intake of ethanol has posed a potential threat to human health. The liver is the main organ that alcohol is metabolized 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, and severe cases can lead to cirrhosis and liver cancer. It has been reported that when alcohol is abnormally metabolized by the liver, excessive free radicals will be 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 received widespread attention.

[0003] Lactic acid bacteria and their fermented products can prevent and protect against alcoholic liver damage. For example, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus fermentum can protect alcoholic mice from liver damage by regulating intestinal microorganisms.

[0004] At present, there are also some reports on the use of probiotic compositions for alcohol sobering and liver protection. For example, the invention patent with application number CN202311255212.X discloses a probiotic fermentation agent with alcohol sobering and liver protection effects and its application, which is carried out by mixing multiple bacterial agents; the invention patent with application number CN202311220048.9 discloses a fermented lactobacillus strain A21252 and its application for alcohol sobering, liver protection, anti-oxidation and reducing digestive tract damage. After 24 in a culture medium containing 15% ethanol concentration, the ethanol degradation rate of strain 21252 can reach 30% under in vitro conditions. The invention patent with application number CN202310635075.6 discloses a salivarius Lactobacillus and its application in the preparation of alcohol sobering products. After the salivarius Lactobacillus PLJK-0039 is fermented by high-density process development-constant pH method, the ethanol degradation rate can reach 35%.

[0005] However, most of the strains in these studies have problems such as insufficient degradation rate and slow degradation speed, which affects their effectiveness in practical applications. Therefore, finding more efficient microbial strains to improve the degradation rate of ethanol remains the focus of research in this field. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies of the above-mentioned prior art and to provide a strain of Lactobacillus plantarum WL05 that can sober up and protect the liver and its application in the preparation of products with sober up and liver protection functions.

[0007] The first object of the present invention is to provide a Lactobacillus plantarum WL05 (Lactobacillus plantarum), which has been deposited in the China Microorganism Culture Collection Center on July 8, 2024, with a deposit number of CGMCC No. 31202, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

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

[0009] Lactic acid bacteria are recognized as safe microorganisms and are part of the normal intestinal flora in the human intestine. They have the ability to sober up and protect the liver. Lactic acid bacteria with strong sobering and liver protecting abilities are screened and used to develop foods or health products with such effects, making lactic acid bacteria fermented foods a daily liver protection supplement.

[0010] The second object of the present invention is to provide a microbial agent, which contains the Lactobacillus plantarum WL05 or its culture solution, or its freeze-dried powder.

[0011] Furthermore, the content of 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 invention, the microbial agent is a freeze-dried powder; the freeze-dried powder is prepared by freezing Lactobacillus plantarum WL05 at -40°C to -60°C, drying to a water content of 3% to 5%, and then crushing it into powder.

[0013] The third object of the present invention is to provide a fermented beverage containing the plant lactobacillus WL05, wherein the beverage is prepared by adding the plant lactobacillus WL05 to a mixture of an extract of Imperata cylindrica and an extract of Phragmites australis and fermenting the mixture;

[0014] Furthermore, the inoculation ratio of Lactobacillus plantarum WL05 in the beverage is 3% to 9% of the mixed solution of Imperata cylindrica root extract and Phragmites australis root extract.

[0015] In some embodiments of the present invention, the preparation method of the Imperata root extract is as follows: the Imperata root is crushed and then extracted with deionized water, and then the solid-liquid separation is followed by sterilization;

[0016] In some embodiments of the present invention, the preparation method of the reed rhizome extract is as follows: the reed rhizome is crushed and then extracted with deionized water, and then the solid-liquid separation is followed by sterilization.

[0017] In some embodiments of the present invention, the preparation method of the Imperata root extract is as follows: crush the Imperata root, extract by ultrasonic at 85°C for 30 minutes according to a material-water ratio of 1:4, filter twice through an 80-mesh sieve, sterilize in a water bath at 95°C for 5 minutes, and then cool;

[0018] In some embodiments of the present invention, the method for preparing the reed root extract is: crush the reed root, ultrasonically extract at 85°C for 30 minutes according to a material-water ratio of 1:4; filter twice through an 80-mesh sieve, sterilize in a 95°C water bath for 5 minutes, and then cool.

[0019] Furthermore, the ratio of Imperata cylindrica root extract to Phragmites australis root extract in the mixed solution is 1:2-2:1;

[0020] Furthermore, the fermentation time of the mixed solution is 24-36 hours.

[0021] In some embodiments of the present invention, the method for preparing a fermented beverage of Lactobacillus plantarum WL05 comprises the following steps: (1) strain purification: applying Lactobacillus plantarum WL05 on MRS solid medium for purification and culture;

[0022] (2) Expansion culture: The strain was picked from the MRS solid medium and placed in MRS liquid medium supplemented with 10% ethanol and cultured at 37°C for 12-24 h;

[0023] (3) Preparing a culture medium of Imperata rhizome-Phragmites australis extract: extract the Imperata rhizome raw material at a material-water ratio of 1:4 at 85°C for 30 min, filter it twice through an 80-mesh sieve, sterilize it in a water bath at 95°C for 5 min, and cool it for later use; extract the Phragmites australis raw material at a material-water ratio of 1:4 at 85°C for 30 min, filter it twice through an 80-mesh sieve, sterilize it in a water bath at 95°C for 5 min, and cool it for later use; mix the Imperata rhizome extract and the Phragmites australis extract at a ratio of 1:2 for inoculation and culture;

[0024] (4) Fermentation of Imperata cylindrica-Phragmites australis extract: Inoculate the Imperata cylindrica-Phragmites australis extract medium with Lactobacillus plantarum WL05 at a ratio of 3% to 9% (v / v), and ferment at 37°C for 24 h to 36 h. After fermentation, sterilize at 95°C for 20 min and cool for later use.

[0025] The fourth object of the present invention is to provide a culture medium for Lactobacillus plantarum WL05, which is a Imperata root-Phragmites phalaris extract matrix culture medium; the preparation method is: crushing Imperata root, adding an appropriate amount of water to extract, separating the solid and liquid, and sterilizing the liquid phase for later use; taking Phragmites phalaris, crushing, adding an appropriate amount of water to extract, separating the solid and liquid, and sterilizing the liquid phase for later use; mixing Imperata root extract and Phragmites phalaris extract in a ratio of 1:2 for inoculating and culturing Lactobacillus plantarum WL05.

[0026] In one embodiment, the preparation method of the Imperata rhizome-Phragmites australis extract matrix culture medium is as follows: crush the Imperata rhizome, ultrasonically extract at 85°C for 30 minutes according to a material-water ratio of 1:4, filter twice through an 80-mesh sieve, sterilize at 95°C in a water bath for 5 minutes, and cool for use; take the Phragmites australis and crush it, ultrasonically extract at 85°C for 30 minutes according to a material-water ratio of 1:4; filter twice through an 80-mesh sieve, sterilize at 95°C in a water bath for 5 minutes, and cool for use; mix the Imperata rhizome extract and the Phragmites australis extract in a ratio of 1:2 for inoculating and culturing Lactobacillus plantarum WL05.

[0027] The fourth object of the present invention is to provide the use of the Lactobacillus plantarum WL05 or the microbial agent in the preparation of a functional product for degrading ethanol;

[0028] Furthermore, the degraded ethanol functional products include but are not limited to beverages, snacks, and dairy products.

[0029] In some embodiments, the present invention also provides a functional supplement prepared using Lactobacillus plantarum WL05, which is prepared by freezing Lactobacillus plantarum WL05 at -40°C to -60°C, drying to a moisture content of 3% to 5%, and then crushing it into a powder. Milk powder, oligosaccharides, maltodextrin or flavoring agents can be added to the powdered Lactobacillus plantarum WL05 to prepare a powdered or flaky product for daily consumption.

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

[0031] (1) Strain activation: Lactobacillus plantarum WL05 was spread on MRS solid medium for activation culture.

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

[0033] (2) Fermentation to produce ethanol dehydrogenase: The activated Lactobacillus plantarum WL05 was transferred to the fermentation medium for cultivation.

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

[0035] Beneficial Effects

[0036] After the plant lactobacillus WL05 provided by the present invention is fermented in an MRS medium containing 10% ethanol concentration, the ethanol degradation rate can reach 42.5%, and the alcohol dehydrogenase activation rate is 43.53%;

[0037] The plant lactobacillus WL05 provided by the present invention has a high survival rate in a simulated human body environment and strong acid and bile salt resistance. After fermentation at 37°C for 24 hours in an MRS liquid medium, the pH value of the fermentation liquid is 3.59, and the number of viable bacteria is greater than 2.1×10 12 CFU / mL, with high acid production capacity and vitality; also has strong antioxidant activity and antibacterial advantages;

[0038] Using different substrates for fermentation, the alcohol dehydrogenase capacity of the strain was significantly improved compared with the original strain. After Lactobacillus plantarum WL05 was fermented in MRS medium containing 10% ethanol concentration, the fermentation broth was inoculated into the Imperata rhizome-Phragmites australis matrix medium, and the ethanol degradation rate reached 63.9%, the alcohol dehydrogenase activation rate in the fermentation broth was 66.5%, and the alcohol dehydrogenase enzyme activity reached 63U / mL.

[0039] The traditional medicinal and edible white imperata rhizome and reed rhizome fermented by Lactobacillus plantarum WL05 provided by the present invention shows good alcohol-resolving and liver-protecting abilities, and can be compounded with other lactic acid bacteria to develop alcohol-resolving and liver-protecting fermented milk products or lactic acid bacteria beverages, and the culture of the lactic acid bacteria can also be freeze-dried to produce functional supplements for alcohol-resolving and liver-protecting.

[0040] Biomaterial Deposit

[0041] Lactobacillus plantarum WL05 was deposited in the China Center for Microbiological Culture Collection on July 8, 2024, with the deposit number: CGMCC No.31202, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1It is the electrophoresis diagram of Lactobacillus plantarum WL05 of the present invention (spotting order: WL01, WL04, WL05, WL06, WL07, WL08, WL09, WL010, Marker). DETAILED DESCRIPTION

[0043] The technical scheme of the present invention is described clearly and completely below in conjunction with the accompanying drawings and implementation cases. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and do not limit the claims of the present invention in any way. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0044] Imperata root (also known as Imperata grass, Imperata grass, Imperata grass); purchased from: Ali Health Pharmacy;

[0045] Reed root (also known as: reed root, reed root, reed head, reed root); purchased from: Ali Health Pharmacy.

[0046] Related culture media and reagent formulas

[0047] MRS solid medium: peptone 10.0 g, sodium acetate trihydrate 3.0 g, beef extract 8.0 g, ammonium citrate trihydrate 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 hydrogen phosphate 2.0 g, agar 15 g, distilled water 1000 mL, adjust the pH to 6.4.

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

[0049] MRS fermentation medium: peptone 10.0g, sodium acetate trihydrate 3.0g, beef extract 8.0g, ammonium citrate trihydrate 2.0g, yeast extract 4.0g, magnesium sulfate heptahydrate 0.2g, glucose 10.0g, manganese sulfate monohydrate 0.04g, Tween-80 1.0mL, potassium hydrogen phosphate 2.0g, 10% ethanol, distilled water 1000mL. Adjust pH to 6.4, sterilize at 121℃ for 15min, and cool for use.

[0050] Imperata rhizome-Phragmites australis extract matrix culture medium: Imperata rhizome raw material, at a material-water ratio of 1:4, ultrasonically extract at 85°C for 30 min, filter through 80-mesh sieve twice, sterilize at 95°C water bath for 5 min, and cool for use; Imperata rhizome raw material, at a material-water ratio of 1:4, ultrasonically extract at 85°C for 30 min, filter through 80-mesh sieve twice, sterilize at 95°C water bath for 5 min, and cool for use; Imperata rhizome extract and Phragmites australis extract are mixed at a ratio of 1:2 for inoculation and culture.

[0051] Skim milk matrix culture medium: 120 g skim milk powder, 1000 mL distilled water.

[0052] Method for determining ethanol tolerance: In the blank group, 1 mL of seed solution was inoculated into 50 mL of MRS liquid medium, cultured at 37°C for 12-24 hours, and the absorbance value Ac of the bacterial solution at 600 nm was determined. The experimental group was operated in the same way as the blank group, but the seed solution was inoculated into 50 mL of MRS fermentation medium, and the absorbance value was recorded as Ai. By comparing the absorbance values, the ethanol tolerance of the strain was determined. Calculation formula: Ethanol tolerance (%) = (Ai / Ac) × 100%.

[0053] Example 1 Isolation and Identification of Lactobacillus plantarum WL05

[0054] 1. Isolation of lactic acid bacteria

[0055] The lactic acid bacteria were isolated from the feces of the longevity elderly in the longevity village in Guangxi. The collected samples were diluted in gradients and inoculated into MRS solid medium. They were cultured at 37°C for 48 hours. Typical colonies on the plate were picked and streaked to obtain pure colonies. The pure colonies on the plate were picked into MRS liquid medium. After anaerobic culture at 37°C for 24 hours, the strain concentration reached 2.1-2.6×10 12 CFU / mL, refrigerate at 4℃ for later use or freeze-dried for storage.

[0056] 2. Identification of lactic acid bacteria

[0057] 16s rDNA sequence determination for strain identification: The prepared lactic acid bacteria genomic DNA was used as a template for PCR amplification, and PCR amplification was performed using a 50μL reaction system. Template DNA 2 uL, 1.5μL of 10mmol upstream and downstream primers, 25μL of 2×Taq enzyme, supplemented to 50μL with ddH2O. PCR cycle parameters are: 94℃ pre-denaturation for 5min; 94℃ denaturation for 1min, 64℃ annealing for 45s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 8min. 5μL of PCR product was aspirated and mixed with 1mL Loading buffer, and electrophoresed with 1.0% agarose gel at a voltage of 80V. After the electrophoresis, it was stained with ethidium bromide EB for 20-30min and photographed. After electrophoresis detection of the amplified product, it was sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The sequence obtained by sequencing was compared with the sequence in the GenBank database, and the results showed that the homology between strain WL05 and Lactobacillus plantarum was 99.9%. The results showed that the strain WL05 of the present invention is Lactobacillus plantarum.

[0058] 3. Determination of acid production and ethanol tolerance of Lactobacillus plantarum WL05

[0059] Acid production capacity of lactic acid bacteria: Take the strains after refrigeration in Example 1 (live bacteria concentration 2.1-2.6×10 12 CFU / mL), inoculated into MRS liquid medium at a ratio of 3% (v / v), cultured at 37°C for 24h, and the pH of the fermentation broth was measured with a pH meter. Each strain was measured in parallel 3 times, and the results were averaged. The results showed that the pH value of the fermentation broth of Lactobacillus plantarum WL05 after 24h of fermentation in MRS matrix was 3.59, indicating that the strain had good acid production ability and good growth condition.

[0060] After the Lactobacillus plantarum WL05 was fermented in MRS medium for 24 hours, the pH value of the fermentation liquid was 3.59, and the number of viable bacteria was greater than 2.1×10 12 CFU / mL, it can be seen that Lactobacillus plantarum WL05 has higher acid production ability and vitality.

[0061] The strains after refrigeration in Example 1 were tested for their ethanol resistance: the results showed that the ethanol resistance of Lactobacillus plantarum WL05 was 80.5%, which showed a high ethanol tolerance.

[0062] 4. Determination of ethanol degradation rate

[0063] (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% and 1.0% (v / v) to 10 mL culture tubes respectively, add 1 mL of potassium dichromate-sulfuric acid solution to each test tube, and shake well; place the test tube in a boiling water bath for 10 min, and measure the absorbance of potassium dichromate-sulfuric acid solution at a wavelength of 610 nm; prepare the ethanol standard curve with ethanol volume concentration (v / v) as the horizontal axis and absorbance as the vertical axis.

[0064] Among them, ethanol standard solution: take an appropriate amount of anhydrous ethanol, add it into sterile water to prepare ethanol standard solutions of different concentrations (0.1%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (v / v)); potassium dichromate-sulfuric acid solution: take 1g of potassium dichromate and dissolve it in 25mL of water, and slowly add 4mL of concentrated sulfuric acid while stirring.

[0065] (2) Determination of ethanol degradation ability: Lactobacillus plantarum activated twice was inoculated into MRS liquid enrichment medium for enrichment for 5-10 hours, then diluted and spread on solid screening medium. A single colony was picked from the screening medium and inoculated into MRS liquid medium for 18 hours, and the OD was measured. The bacterial solution was adjusted to 10 7 CFU / mL. Inoculate at 5% inoculation volume into MRS ethanol culture medium of different concentrations (5%, 10%, 15%), and use MRS ethanol culture medium without bacterial solution as control, and culture for 24h.

[0066] Centrifuge the 24h fermentation culture (10000rpm, 10min), take 0.1ml of the supernatant, add 1mL of potassium dichromate-sulfuric acid solution, and shake well; place the test tube in a boiling water bath for 10min and measure the absorbance of the potassium dichromate-sulfuric acid solution at a wavelength of 610nm.

[0067] The ethanol residual amount is calculated using the ethanol standard curve, and the quantitative size of the strain's alcohol sobering ability can be obtained by comparison.

[0068] Lactobacillus plantarum WL05 was fermented in MRS medium containing 10% ethanol concentration at 37°C for 19-24h, and the ethanol degradation rate was measured to be 42.5%. Subsequently, the fermentation liquid was inoculated into skim milk matrix medium and Imperata root-Phragmites australis matrix medium at a 6% inoculum amount, respectively. After fermentation in skim milk matrix medium at 37°C for 24h, the ethanol degradation rate was measured to be 35.8%, and after fermentation in Imperata root-Phragmites australis matrix medium at 37°C for 24h, the ethanol degradation rate was measured to be 63.9%.

[0069] Lactobacillus plantarum WL05 was fermented at 37°C for 19-24h in MRS medium containing 15% ethanol concentration, and the ethanol degradation rate was measured to be 40.2%. Subsequently, the fermentation liquid was inoculated into skim milk matrix medium and Imperata root-Phragmites australis matrix medium according to 6% inoculation amount, and the ethanol degradation rate was measured to be 32.5% after fermentation at 37°C in skim milk matrix medium for 24h, and the ethanol degradation rate was measured to be 60.4% after fermentation at 37°C in Imperata root-Phragmites australis matrix medium for 24h.

[0070] 5. Alcohol Dehydrogenase Activity Assay

[0071] Preparation of fermentation broth

[0072] (1) Preparation of bacterial solution: Inoculate the bacterial strain into MRS liquid culture medium and culture at 37°C for 12-24 h.

[0073] (2) Fermentation to produce ethanol dehydrogenase: Add the test bacterial solution to 10 mL of MRS medium, skimmed milk medium or Imperata cylindrica-Phragmites australis medium at a volume ratio of 6%. After culturing in a constant temperature incubator at 37°C for 24-60 hours, centrifuge at 4000 rpm for 10 minutes and collect the supernatant fermentation solution.

[0074] Determination of alcohol dehydrogenase in fermentation broth: Referring to Chai Zheng, the modified Waller-Hoch method was used to detect ADH activity. 1.5 mL of pH 8.8 sodium pyrophosphate solution was added to a stoppered test tube, followed by 1.0 mL of 0.027 mol / L oxidized coenzyme I (NAD + ), 0.5mL 11% ethanol solution and 0.1mL sample solution. Mix well and place in a 37℃ water bath for 5min. After the insulation, add 0.1mL ADH (0.25U / mL) and replace the 11.5% ethanol solution with 0.5mL distilled water for blank control zeroing. Then shake the test tube solution, set the 0μg / mL experimental group as the negative control, shake the solution and then measure the absorbance value A340 at a wavelength of 340nm. The change in absorbance here can represent the enzymatic reaction rate of ADH. Record once every 30s and continuously record for 5min, and calculate the average absorbance value per minute. The specific calculation is shown in formulas (1) and (2):

[0075]

[0076] Where: V is the total reaction volume; M is the sample amount; ΔA340 / min is the average absorbance per minute.

[0077] Activation rate / % = (U 1 -U 0 ) / U 0 ×100% (2)

[0078] Where: U 1 , U 0 The results were measured three times in parallel.

[0079] The alcohol dehydrogenase activity of Lactobacillus plantarum WL05 was detected to be over 15U / mL in the fermentation broth of MRS medium, over 10U / mL in the fermentation broth of skimmed milk matrix medium, and over 63U / mL in the fermentation broth of Imperata root-Phragmites australis matrix medium.

[0080] In MRS medium containing 10% ethanol, after fermentation at 37℃ for 19-24 hours, the activation rate of alcohol dehydrogenase in the fermentation broth of Lactobacillus plantarum WL05 was 43.53%. Subsequently, the fermentation broth was inoculated into skim milk matrix medium and Imperata cylindrica-Phragmites australis matrix medium, respectively. After fermentation at 37℃ for 24 hours in skim milk matrix medium, the activation rate of alcohol dehydrogenase in the fermentation broth was 32.26%; while after fermentation at 37℃ for 24 hours in Imperata cylindrica-Phragmites australis matrix medium, the activation rate of alcohol dehydrogenase in the fermentation broth was 66.5%.

[0081] In MRS medium containing 15% ethanol, Lactobacillus plantarum WL05 was fermented at 37°C for 19-24 hours, and the activation rate of alcohol dehydrogenase in the fermentation broth was 41.2%. Subsequently, the fermentation broth was inoculated into skim milk matrix medium and Imperata cylindrica-Phragmites australis matrix medium, respectively. After fermentation at 37°C for 24 hours in skim milk matrix medium, the activation rate of alcohol dehydrogenase was 30.6%, while after fermentation at 37°C for 24 hours in Imperata cylindrica-Phragmites australis matrix medium, the activation rate of alcohol dehydrogenase in the fermentation broth was 62.6%.

[0082] 6. Determination of acid and bile resistance of Lactobacillus plantarum WL05

[0083] The Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture, and then the strain was picked from the plate culture medium and placed in MRS liquid culture medium, and cultured at 37°C for 12-24h;

[0084] The Lactobacillus plantarum strain WL05 culture was centrifuged at 10000×g for 5 min to collect the cells, and washed twice with PBS, resuspended with physiological saline, and 1 mL of the bacterial suspension was inoculated into 9 mL of simulated gastric fluid at pH 3.0, shaken evenly, and cultured in a 37°C incubator. Samples were taken at 0 h and 3 h to plate, and the number of viable cells was determined, and the survival rate was calculated;

[0085] The culture of Lactobacillus plantarum strain WL05 was centrifuged at 10000×g for 5 min to collect the cells, washed twice with PBS, resuspended with physiological saline, and 1 mL of the bacterial suspension was inoculated into MRS liquid culture medium containing 0%, 0.1%, 0.3% and 0.5% (w / v) bile salts, and cultured at 37°C for 3 h. Samples were taken and plated at 0 h and 3 h to determine the number of viable cells and calculate the survival rate.

[0086] The results showed that the survival rate of Lactobacillus plantarum WL05 in artificial gastric juice with a pH of 3.0 was 69.2%, and the survival rates after culturing for 3 hours in MRS medium with bile salt concentrations of 0.10%, 0.30%, and 0.50% were 74.5%, 43.7%, and 15.5%, respectively.

[0087] 7. Determination of Antioxidant Capacity of Lactobacillus plantarum WL05

[0088] (1) DPPH clearance determination

[0089] The Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture, and then the strain was picked from the plate culture medium and placed in MRS liquid culture medium, and cultured at 37° C. for 12-24 hours; the Lactobacillus plantarum strain WL05 culture was centrifuged at 10000×g for 5 minutes, and the supernatant was taken as a sample.

[0090] Accurately measure 0.5 mL of sample solution and add it to a stoppered test tube. Then add 0.2 mL of DPPH solution (0.2 mmol / LDPPH dissolved in 95% ethanol solution). Mix well and react at 40°C in the dark for 20 minutes. Measure the absorbance at a wavelength of 517 nm and record it as A. i Blank group: Accurately measure 0.5 ml of sample solution and add it to a stoppered test tube. Replace the DPPH solution with 0.2 ml of 95% ethanol solution. After mixing, measure the absorbance at a wavelength of 517 nm and record it as A. j ; Control group: Add 2.0mL DPPH solution and 2mL 95% ethanol solution to a stoppered test tube, and measure the absorbance at a wavelength of 517nm, which is recorded as A c .

[0091] The total reaction volume of the above experimental groups was 4 mL, which was supplemented with 95% ethanol solution. The DPPH free radical scavenging rate was calculated according to the following formula: Scavenging rate (%): = [1-(A i -A j )] / A c *100

[0092] The results showed that the DPPH scavenging rate of Lactobacillus plantarum WL05 was 78.5%.

[0093] (2) Determination of reducing ability

[0094] The Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture, and then the strain was picked from the plate culture medium and placed in MRS liquid culture medium, and cultured at 37° C. for 12-24 hours; the Lactobacillus plantarum strain WL05 culture was centrifuged at 10000×g for 5 minutes, and the supernatant was taken as a sample.

[0095] Take 1.0mL of sample in a centrifuge tube, add 2.5mL of 0.2mol / L phosphate buffer solution (pH6.6) and 1% potassium ferricyanide solution, mix thoroughly, keep warm in a water bath at 50℃ for 20min, quickly cool to room temperature, add 2.5mL of 10% trichloroacetic acid solution, centrifuge at 3500r / min for 10min, accurately pipette 2.5ml of supernatant into a stoppered test tube, add 2.5mL of distilled water and 0.5mL of 0.1% ferric chloride solution in sequence, mix thoroughly, and measure the absorbance of the solution at a wavelength of 200nm. VC is used as the control sample.

[0096] The results showed that the reducing capacity of Lactobacillus plantarum WL05 reached 0.85, compared with the reducing capacity of VC (1.20), indicating that Lactobacillus plantarum WL05 has high antioxidant activity.

[0097] 8. Determination of antibacterial ability of Lactobacillus plantarum WL05

[0098] Lactobacillus plantarum WL05 was inoculated into the culture medium and cultured at 37°C for 24 hours. The antibacterial ability of lactic acid bacteria against Escherichia coli, Staphylococcus aureus, Salmonella, and Bacillus subtilis was determined by the Oxford cup method. The bacterial suspensions of Escherichia coli, Staphylococcus aureus, Salmonella, and Bacillus subtilis were spread into LB culture medium, 200 μL of lactic acid bacteria fermentation liquid was added to the Oxford cup, and cultured at a constant temperature for 48 hours, and the inhibition zone was observed.

[0099] The results showed that Lactobacillus plantarum WL05 had high antibacterial ability against Escherichia coli, Staphylococcus aureus, Salmonella and Bacillus subtilis, and the diameters of the inhibition zones were 22.50 mm, 18.35 mm, 17.22 mm and 16.50 mm, respectively.

[0100] Example 2 Preparation of fermented Imperata cylindrica-Phragmites australis beverage containing Lactobacillus plantarum WL05;

[0101] (1) Strain activation: Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture.

[0102] (2) Expansion culture: Lactobacillus plantarum WL05 was inoculated into the optimized MRS medium at a 6% inoculum amount and cultured at 37°C for 24-48 hours.

[0103] (3) Preparation of Imperata cylindrica / Phragmites australis extract:

[0104] The Imperata root raw material was ultrasonically extracted at 85°C for 30 minutes at a material-water ratio of 1:4, filtered through an 80-mesh sieve twice, sterilized in a 95°C water bath for 5 minutes, and the Imperata root extract was obtained, which was cooled for later use;

[0105] The reed root raw material was ultrasonically extracted at 85°C for 30 minutes at a material-water ratio of 1:4; filtered through an 80-mesh sieve twice, sterilized in a 95°C water bath for 5 minutes, and the reed root extract was obtained and cooled for later use;

[0106] (4) Preparation of Imperata cylindrica-Phragmites australis culture medium;

[0107] The Imperata root extract and the Phragmites australis extract are mixed in a certain proportion to obtain the Imperata root-Phragmites australis culture medium, and the mixed Imperata root-Phragmites australis extract is inoculated with bacteria, the inoculation amount is 3%-9% (v / v), and the fermentation time is 24h-36h at 37℃.

[0108] After fermentation, dilute the Imperata cylindrica-Phragmites australis fermented liquid by 1 time with sterilized and cooled water, add appropriate amount of sweetener and acidifier to make a lactic acid bacteria beverage that can be drunk directly.

[0109] The fermentation conditions were respectively followed in Table 1 to obtain Imperata cylindrica-Phragmites australis beverage, and then the ethanol degradation rate was tested (the testing method was the same as in Example 1). The fermentation conditions and ethanol degradation rate are shown in Table 1.

[0110] Table 1 Fermented Imperata cylindrica-Phragmites australis beverages with Lactobacillus plantarum WL05

[0111] Imperata cylindrica-Phragmites australis ratio Inoculation amount Fermentation time Ethanol degradation rate (10%) 1：1 3％ 24h 23％ 1：2 3％ 24h 29％ 2：1 3％ 24h 27％ 1：2 6％ 24h 30％ 1：2 9％ 24h 28％ 1：2 6％ 28h 32％ 1：2 6％ 32h 26％ 1：2 6％ 36h 23％

[0112] From the data in Table 1, it can be seen that the fermented Imperata cylindrica-Phragmites australis 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%.

[0113] Example 3 Preparation method of functional supplement containing Lactobacillus plantarum WL05

[0114] (1) Strain activation: Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture.

[0115] (2) Expanded culture: Lactobacillus plantarum WL05 was inoculated into the optimized MRS medium at a 5% inoculum amount and cultured at 37°C for 24-48 hours.

[0116] (3) Preparation of freeze-dried powder: freeze Lactobacillus plantarum WL05 to -40°C to -60°C, dry to a moisture content of 3% to 5%, and crush into powder.

[0117] The powdered product obtained in Example 3 can be added with milk powder, oligosaccharides, maltodextrin, flavoring agents, etc. to prepare a powdered or flaky product for daily consumption.

[0118] After the plant lactobacillus WL05 provided by the present invention is fermented in MRS liquid culture medium at 37°C for 24h, the pH value of the fermentation liquid is 3.59, and the number of viable bacteria is greater than 2.1×1012CFU / mL, which shows that the plant lactobacillus WL05 has a high acid production ability and vitality. The survival rate in artificial gastric juice with a pH of 3.0 is greater than 69.2%, and the survival rate in MRS culture medium with bile salt concentrations of 0.10%, 0.30%, and 0.50% for 3h is greater than 74.5%, 43.7%, and 15.5%, respectively. After the plant lactobacillus WL05 is fermented in MRS culture medium containing 10% ethanol concentration, the ethanol degradation rate can reach 42.5%, and the alcohol dehydrogenase activation rate is 43.53%; subsequently, the fermentation liquid is inoculated into the Imperata cylindrica-Phragmites australis matrix culture medium, and after fermentation, the ethanol degradation rate is measured to be 63.9%, and the alcohol dehydrogenase activation rate in the fermentation liquid is 66.5%. The DPPH removal rate of the plant lactobacillus WL05 is 78.5%, and it also has strong antibacterial ability. The ethanol degradation rate of beverages prepared based on this strain can reach 32%. It has high application value in the field of functional foods.

[0119] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Lactobacillus plantarum WL05 (Lactobacillus plantarum), which has been deposited in the China Microorganism Culture Collection Center on July 8, 2024, with a deposit number of CGMCC No.31202, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. The plant lactobacillus WL05 according to claim 1, characterized in that The Lactobacillus plantarum WL05 is isolated from the feces of centenarians in a longevity village in Guangxi, and its nucleotide sequence is SEQ ID NO.

1.

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

4. The microbial agent according to claim 3, characterized in that: The content of Lactobacillus plantarum WL05 in the microbial agent is at least 2.1×10 12 CFU / mL or 2.1×10 12 CFU / g.

5. The microbial agent according to claim 2, characterized in that: The preparation method of the freeze-dried powder is as follows: freezing the plant lactobacillus WL05 at -40°C to -60°C, drying to a water content of 3% to 5%, and then crushing into powder.

6. A fermented beverage containing Lactobacillus plantarum WL05, characterized in that: The beverage is prepared by adding the Lactobacillus plantarum WL05 described in any one of claims 1 to 2 and fermenting the mixture with Imperata cylindrica extract and Phragmites australis extract; The inoculation ratio of Lactobacillus plantarum WL05 is 3% to 9% of the mixed solution.

7. The fermented beverage containing Lactobacillus plantarum WL05 according to claim 6, characterized in that: The preparation method of the Imperata root extract comprises the following steps: crushing the Imperata root, adding deionized water for extraction, and sterilizing after solid-liquid separation; The preparation method of the reed root extract is as follows: the reed root is crushed, deionized water is added for extraction, and the solid-liquid separation is followed by sterilization.

8. The fermented beverage containing Lactobacillus plantarum WL05 according to claim 7, characterized in that: The ratio of Imperata cylindrica root extract to Phragmites australis root extract in the mixed solution is 1:2-2:1; The fermentation time is 24-36h.

9. A culture medium for Lactobacillus plantarum WL05, characterized in that: The culture medium is a Imperata rhizome-Phragmites australis extract matrix culture medium; the preparation method is: crush Imperata rhizome, add appropriate amount of water to extract, separate solid and liquid, sterilize in liquid phase and set aside; take Phragmites australis and crush it, add appropriate amount of water to extract, separate solid and liquid, sterilize in liquid phase and set aside; mix Imperata rhizome extract and Phragmites australis extract in a ratio of 1:2, and use them to inoculate and culture Lactobacillus plantarum WL05.

10. Use of the Lactobacillus plantarum WL05 according to any one of claims 1 to 2 or the microbial agent according to any one of claims 3 to 5 in the preparation of a functional product for degrading ethanol; The degraded ethanol functional products include but are not limited to beverages, snacks, and dairy products.

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