High-throughput screening of lactobacillus plantarum with ethanol degradation capability and application of lactobacillus plantarum

Abstract

The invention discloses high-throughput screening of lactobacillus plantarum with ethanol degradation capacity and application of the lactobacillus plantarum, and belongs to the technical field of microorganisms. According to the method for high-throughput screening of the ethanol degradation strains, 8340 strains are successfully and rapidly screened, the strains are evaluated in vitro, and the degradation rates of A1H11 strains in vitro for 6 hours are 33.7% and 24.2% respectively under the alcohol concentrations of 10% and 20% (v / v); the resistance to gastric juice is shown, proliferation to a certain degree can be achieved, and the survival rate reaches 106%. And finally obtaining the lactobacillus plantarum which is relatively good in ethanol degradation capability and resistant to gastric acid.

Description

Technical Field

[0001] The invention relates to high-throughput screening of plant lactobacillus with ethanol degradation ability and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Moderate drinking helps reduce the risk of chronic cardiovascular disease and is beneficial to the human body. However, when the human body drinks a lot of alcohol and the amount of alcohol metabolized is less than the amount of alcohol intake, alcohol will accumulate in the body in a short period of time. The water solubility of ethanol allows it to freely enter and exit cells, causing toxic effects on cells. Its metabolite acetaldehyde can cause permanent damage to the DNA in cells and inhibit the DNA repair process. Long-term excessive drinking can lead to intestinal dysbiosis, anemia, pancreatitis, cancer, brain diseases, etc.

[0003] Ethanol is mainly eliminated by oxidation in the liver with the help of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). Microorganisms carry ADH and ALDH, which can be delivered to the intestines to help the body quickly break down alcohol. Considering that genetic engineering may involve environmental risks, biosafety risks, and ethical issues, it may be safer to screen wild strains in the environment that have the effect of sobering up. At present, there are not many studies on the screening of sobering up strains, and there are problems such as low screening efficiency, reliance on traditional plate separation methods, difficulty in quickly screening a large number of strains, and limited screening range.

[0004] The ethanol oxidation process is closely related to the NADH content. With the participation of NAD+, ethanol is first oxidized to acetaldehyde with the help of ADH, and acetaldehyde is oxidized to acetic acid with the help of ALDH. Acetic acid enters the tricarboxylic acid cycle and is eventually oxidized to carbon dioxide and water. The cofactor NAD+ is reduced to NADH in both steps. Therefore, by quantifying the NADH content produced under the action of ethanol, the ethanol oxidase activity of the strain can be reflected from the side. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a high-throughput screening of Lactobacillus plantarum with ethanol degradation ability and its use, aiming at quickly screening alcohol-degrading strains in the environment by means of a color development reaction.

[0006] The first technical solution provided by the present invention is a high-throughput screening method for Lactobacillus plantarum with ethanol degradation ability. The method comprises the following steps: a plurality of single colonies are separated from a sample, and the plurality of single colonies are respectively inoculated into a screening medium containing ethanol and WST-8 for anaerobic culture. The ethanol degradation amount is characterized by the absorbance value at a dual wavelength of 450 / 600 nm, thereby screening out Lactobacillus plantarum with ethanol degradation ability.

[0007] In certain embodiments, the sample is derived from sauerkraut, kimchi, kimchi mother water, enzyme, aged rice wine, etc.

[0008] In certain embodiments, the sample is derived from milk curd, cheese, yogurt, etc.

[0009] In certain embodiments, the sample is derived from fermented grains, lees, etc.

[0010] In certain embodiments, the sample is derived from the intestine, feces, skin, etc.

[0011] In certain embodiments, the sample originates from soil, water source, factory floor, plant, etc.

[0012] In some embodiments, the specific steps of sample preparation are: diluting the sample 10-fold with 0.85% (w / v) saline, selecting a suitable dilution for coating, culturing at 37°C for 24 hours, and selecting plates with colony counts between 30-300 as samples for high-throughput screening.

[0013] In certain embodiments, the specific steps of the screening are as follows: using an automatic dispensing workstation to dispense the test culture medium into a 96-well plate, 200 μL per well. Using a QPix 420 instrument, pick a single colony on the plate into the test culture medium in the 96-well plate, place the inoculated 96-well plate in an anaerobic culture at 37°C for 4 to 12 hours, and use an ELISA reader to detect the absorbance (450 / 600nm dual wavelength). After 12 hours, the strain grows appropriately, and the bacterial solution in the well with the highest absorbance is taken for coating and culture. After a single colony grows, it is separated by streaking to obtain plant lactobacillus with ethanol degradation ability.

[0014] In certain embodiments, the assay medium contains 5% (v / v) ethanol and 7.5% (v / v) WST-8.

[0015] In certain embodiments, the detection culture medium further contains 6.8 g / L disodium hydrogen phosphate, 3.0 g / L dipotassium hydrogen phosphate, 1.0 g / L ammonium chloride, 0.5 g / L sodium chloride, and 5.0 g / L glucose.

[0016] The second technical solution provided by the present invention is a strain of Lactobacillus plantarum A1H11, which has been deposited in the China Center for Type Microorganism Collection on January 2, 2025, and the strain preservation number is CCTCC NO: M2025003.

[0017] The third technical solution provided by the present invention is a microbial agent containing Lactobacillus plantarum A1H11 described in the second technical solution.

[0018] The present invention provides a fourth technical solution, a method for degrading ethanol, wherein the method comprises introducing the plant lactobacillus A1H11 described in the second technical solution or the microbial agent described in the third technical solution into an environment containing ethanol to degrade the ethanol.

[0019] The fifth technical solution provided by the present invention is the use of Lactobacillus plantarum A1H11 described in the second technical solution or the microbial agent described in the third technical solution in degrading ethanol.

[0020] The technical effects of the present invention are as follows:

[0021] The high-throughput screening method for ethanol-degrading strains of the present invention successfully screened 8340 strains rapidly and evaluated the strains in vitro. At 10% and 20% (v / v) alcohol concentrations, the in vitro 6-hour degradation rates of the A1H11 strain were 33.7% and 24.2%, respectively; thereby a strain with good ethanol degradation ability was obtained.

[0022] Biological deposit materials

[0023] Lactobacillus plantarum A1H11, taxonomic name Lactobacillus plantarum, was deposited in the China Center for Type Microorganism Collection on January 2, 2025. The deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2025003.

[0024] Lactobacillus plantarum I3D12, taxonomic name Lactobacillus plantarum, was deposited in the China Center for Type Microorganism Collection on January 2, 2025. The deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2025004. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 High-throughput screening process.

[0026] Figure 2 It is the absorbance value of 8340 strains detected at dual wavelengths of 450nm and 600nm.

[0027] Figure 3 Degradation rates of 10% (v / v) and 20% (v / v) ethanol by the strains.

[0028] Figure 4 It is the self-agglutination ability of the strain. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0030] Test method:

[0031] Ethanol content was detected by GC-MS:

[0032] Add 10% (v / v) and 20% (v / v) ethanol to the bacterial solution and culture at 37°C for 6h. After mixing, take 1ml of the mixed solution and centrifuge at 4000rpm for 5min. Take the supernatant to detect the remaining ethanol content. Gas chromatography conditions: incubate at 70°C for 5min, stirring speed is 500rpm. Chromatographic column: TG-WAXMS (30mx0.25umx0.25mm); injection port temperature is 220°C, carrier gas is high-purity helium, flow rate is 1.0mL / min, split injection, split ratio 10:1; heating program: starting temperature 35°C, hold for 4min, then increase to 240°C at a rate of 40°C / min, hold for 2min. Mass spectrometry conditions: ionization mode: E1; emission current: 50 A; electron energy: 70 eV; ion source temperature: 250 °C; transfer line temperature: 230 °C; ion scanning range: 29, 44, 43, 31, 45, 46 m / z.

[0033] Example 1 High-throughput screening sample processing

[0034] 1. Processing of sauerkraut and kimchi samples

[0035] Add 5g kimchi to a centrifuge tube containing 45ml 0.85% (w / v) saline, vortex for 30s; take 1mL sample solution and add it to 9mL 0.85% saline, dilute 10 times, take 100μL of 10-5, 10-6, 10-7 dilutions and spread them on MRS solid plates, 3 parallels for each gradient, and culture anaerobically at 37℃ for 24h. Select samples with colony counts between 30-300 as samples for high-throughput bacterial selection. Store samples in a refrigerator at 4℃.

[0036] 2. Enzyme Sample Processing

[0037] 0.1 mL of sample solution was added to 0.9 mL of 0.85% (w / v) physiological saline, and diluted 10 times in a gradient. 100 μL of each dilution of 10-5, 10-6, and 10-7 was spread on the MRS solid plate, with 3 parallels for each gradient. After culturing at 37°C for 24 hours, the colony count between 30 and 300 was selected and stored as a sample for high-throughput bacterial selection. The sample was stored in a refrigerator at 4°C.

[0038] 3. Sample processing of aged rice wine

[0039] The main microorganisms in rancid yellow wine are difficult to culture in conventional culture media. Take 1 ml of wine sample, dilute it 10 times, select the appropriate dilution gradient, take 50 μL of the dilution solution and spread it on rancid yellow wine solid culture medium (in MRS solid culture medium, add 0.14% (w / v) L-cysteine ​​(L-Cys), adjust the pH value to 5.4, and add 9% (v / v) anhydrous ethanol before use), and culture it in an anaerobic incubator for 3-4 days. Take an obvious single colony and streak it for purification.

[0040] Example 2: High Throughput Screening

[0041] refer to Figures 1-2 High-throughput screening was performed. The test culture medium was dispensed into 96-well plates using a high-throughput fully automated strain screening workstation (Freedom EVO-2200BASE, Tecan, Switzerland), with 200 μL per well. A high-throughput fully automated microbial colony picking workstation (QPix 420, American Molecular Instruments, USA) was used to screen 8340 strains ( Figure 2 a) Take photos of the colonies on the plate and automatically identify them, pick a single colony on the plate and place it in the test medium in the 96-well plate. The 96-well plate after inoculation is incubated anaerobically at 37°C for 4 hours ( Figure 2 b) 8h( Figure 2 c) 12h( Figure 2 d), and the absorbance was detected by an ELISA reader (EPOCH2, BioTek, USA) (dual wavelength Lm1 = 450, Lm2 = 600 nm). After 12 h, the strain was properly grown, and 100 μL of the bacterial solution in the well with higher absorbance was spread on the MRS solid plate, and cultured anaerobically at 37°C. After a single colony grew, it was streaked and purified, and stored at -80°C using the glycerol method.

[0042] By setting specific absorbance thresholds—absorbance value ≥ 0.6 at 4 hours, absorbance value ≥ 1.0 at 8 hours, and absorbance value ≥ 1.5 at 12 hours, 34 strains were successfully screened and preserved. Specific information is shown in Table 1.

[0043] Table 1 Detailed information of 34 strains obtained by high-throughput screening

[0044]

[0045]

[0046] Example 3: Strain Verification

[0047] 1. Ethanol degradation capability verification

[0048] The bacterial suspension of the 34 strains screened in Example 2 was added with 10% (v / v) and 20% (v / v) ethanol and cultured at 37°C for 6 hours. After mixing, 1 ml of the mixture was centrifuged at 4000 rpm for 5 minutes, and the supernatant was taken to detect the residual ethanol content.

[0049] like Figure 3 A1H11 showed strong degradation ability for 10% (v / v) ethanol, and the degradation ability was 33.7% after 6 hours of anaerobic culture. Further tests showed that A1H11 also had strong degradation ability for 20% (v / v) ethanol, and the degradation ability was 24.2% after 6 hours of anaerobic culture.

[0050] 2. Gastric acid resistance verification

[0051] Referring to the group standard T / CNHFA 435-2024 "Test Method for Gastric Juice Tolerance of Probiotics" issued by the China Nutrition and Health Food Association, simulated gastric juice was prepared and the gastric juice tolerance of the strain was evaluated.

[0052] Electrolyte A solution: weigh 0.064 g potassium chloride, 0.015 g potassium dihydrogen phosphate, 0.263 g sodium bicarbonate, 0.345 g sodium chloride, 0.003 g magnesium chloride hexahydrate, 0.006 g ammonium carbonate, 1.5 g tryptone, and 0.05 g L-cysteine ​​hydrochloride monohydrate, add 95 mL of distilled water to fully dissolve, adjust the pH to 3.0 with concentrated hydrochloric acid or 1 mol / L sodium hydroxide solution, make up to 100 mL, and sterilize by high pressure at 121°C for 15 min.

[0053] Electrolyte B solution: Weigh 0.022 g of calcium chloride dihydrate, dissolve it in water and make up to 100 mL, and sterilize it by high pressure at 121°C for 15 min.

[0054] Take 8.0mL of electrolyte A solution and 1.0mL of electrolyte B solution in a beaker, add pepsin equivalent to 4000U, adjust the pH value (2.0 for fasting, 4.0 for full stomach) with 1mol / L hydrochloric acid solution or 1mol / L sodium hydroxide solution, make the volume to 10mL, mix well and filter through a 0.22μm sterile filter membrane to prepare simulated gastric fluid, which is prepared and used immediately.

[0055] The strain A1H11 obtained by screening in Example 1 and the strain I3D12 (as a control strain) were cultured in MRS liquid medium at 37°C for 24 hours, centrifuged at 3000rpm for 5 minutes, and the supernatant was discarded. The suspension was resuspended with 20mL 0.85% saline, and the total number of viable bacteria in the bacterial solution was determined. 2.0mL simulated gastric fluid and 2.0mL sample bacterial solution were taken into a 5mL centrifuge tube, vortexed and mixed, and the centrifuge tube was placed in a 37°C constant temperature water bath for culture (fasting for 0.5h, full for 3.0h), and the total number of viable bacteria was determined. The results are shown in Table 2 and Figure 4 shown.

[0056] Table 2 Resistance of strains to gastric juice

[0057]

[0058] 2. Self-agglutination ability test

[0059] Self-agglutination ability: Take 4 ml of the lactic acid bacteria suspension after adjusting the concentration, mix thoroughly, and incubate at room temperature for 5 hours. Pipette 200 μL of the surface bacterial solution, do not blow it, and keep the bacterial solution still. Use PBS buffer as a control to measure the OD value at 600nm, recorded as At. Repeat the experiment three times. Self-agglutination rate (%) = 1-At / A0×100%, A0 is the initial absorbance.

[0060] As shown in Table 2, A1H11 showed resistance to gastric juice and was able to achieve a certain degree of proliferation (survival rate reached 106%). In contrast, the number of I3D12 decreased in the satiety state. Figure 4 The self-aggregation ability of A1H11 increased first and then weakened within 1 to 4 hours, while the self-aggregation ability of I3D12 gradually increased over time. Among these strains, A1H11 had the strongest self-aggregation ability, with the self-aggregation rates reaching 17.88±1.59% and 14.45±2.24% at 3 hours and 4 hours, respectively.

[0061] 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 high-throughput screening method for Lactobacillus plantarum with ethanol degradation ability, characterized in that: The method comprises the following steps: separating a plurality of single colonies from a sample, respectively inoculating the plurality of single colonies into a screening medium containing ethanol and WST-8 for anaerobic culture, characterizing the ethanol degradation amount by the absorbance value of a dual wavelength of 450 / 600 nm, and further screening out plant lactobacillus with ethanol degradation ability.

2. The method according to claim 1, characterized in that The sample is derived from fermented food, intestinal tract, feces, skin, soil, water source, factory floor or plant; Optionally, the fermented food includes sauerkraut, kimchi, kimchi mother water, enzyme, chentanhuang, milk lumps, cheese, yogurt, fermented grains and distiller's grains.

3. The method according to claim 1, characterized in that The specific steps of sample preparation are: diluting the sample 10 times with 0.85% (w / v) physiological saline, spreading and culturing, and selecting plates with 30-300 colony counts as samples for high-throughput screening.

4. The method according to claim 1, characterized in that: The specific steps of the screening are as follows: using an automatic dispensing workstation to dispense the test culture medium into a 96-well plate, using a QPix 420 instrument to pick up a single colony on the plate and put it into the test culture medium in the 96-well plate, placing the inoculated 96-well plate at 37° C. for anaerobic culture for 4 h to 12 h, using an ELISA reader to detect the absorbance at a dual wavelength of 450 / 600 nm, after 12 h, the strain has grown appropriately, taking the bacterial solution in the well with the highest absorbance, applying it for culture, growing a single colony and separating it by streaking, and obtaining plant lactobacillus with ethanol degradation ability.

5. The method according to claim 4, characterized in that The assay medium contained 5% (v / v) ethanol and 7.5% (v / v) WST-8.

6. The method according to claim 5, characterized in that The detection culture medium also contains 6.8 g / L disodium hydrogen phosphate, 3.0 g / L dipotassium hydrogen phosphate, 1.0 g / L ammonium chloride, 0.5 g / L sodium chloride, and 5.0 g / L glucose.

7. A strain of Lactobacillus plantarum A1H11, characterized in that: It was deposited in the China Center for Type Microorganism Collection on January 2, 2025, and the strain deposition number is CCTCC NO: M 2025003.

8. A microbial agent containing the Lactobacillus plantarum A1H11 according to claim 7.

9. A method for degrading ethanol, characterized in that: The method comprises introducing the plant lactobacillus A1H11 described in claim 7 or the microbial agent described in claim 8 into an environment containing ethanol to degrade the ethanol.

10. Use of the Lactobacillus plantarum A1H11 according to claim 7 or the microbial agent according to claim 8 in degrading ethanol.

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