Lactobacillus plantarum YYS-K3 and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to *Lactobacillus plantarum* YYS-K3 and its applications. This *Lactobacillus plantarum* YYS-K3 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27598. The *Lactobacillus plantarum* YYS-K3 provided by this invention exhibits strong acid and bile salt resistance, good tolerance in simulated gastric and intestinal fluids, and can successfully reach the human intestine. Furthermore, *Lactobacillus plantarum* YYS-K3 possesses the ability to produce ethyl carbamate-degrading enzymes and ureases. Due to its high ethyl carbamate degradation capacity and ability to degrade urea, it can be widely used in the production of fermented products, such as alcoholic beverages, soy sauce, and enzymes, significantly reducing the ethyl carbamate content in these products. It can also be used in conjunction with probiotics to reduce the health risks associated with alcoholic beverages.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a plant lactobacillus YYS-K3 and its applications. Background Art

[0002] Ethyl carbamate (C3H7NO2, EC), also known as urethane, is a natural compound widely found in fermented foods, especially in fermented alcoholic beverages (yellow wine, wine, baijiu, etc.). It is a carcinogen that seriously endangers human health. The formation of ethyl carbamate is mainly due to incomplete metabolism of nitrogenous substances during fermentation, producing substances such as urea, citrulline, cyanide, diethyl pyrophosphate, and carbamoyl phosphate. These precursors readily react with ethanol to form ethyl carbamate, and among these precursors, the reaction of urea with ethanol is the primary cause of EC formation.

[0003] As an important precursor in the synthesis of urethane, urea is also a harmful substance to the human body. The accumulation of large amounts of urea in the body can affect various systems and organs, easily causing dysfunction. Urea is the final product of protein breakdown in the human body and is generally excreted through the kidneys. Clinically, urea levels are often used as a biomarker reflecting kidney damage. Elevated urea levels in the body may lead to kidney damage such as nephritis, renal dysfunction, renal tuberculosis, and renal tumors. Furthermore, it can cause excessive protein breakdown, resulting in upper gastrointestinal bleeding, hyperthyroidism, and acute infectious diseases.

[0004] Currently, there are four main approaches to reduce EC content: directly removing EC using ethyl carbamate degrading enzymes, controlling the content of precursor substances, optimizing production processes, and breeding low-urea-producing engineered bacteria. Among these, optimizing production processes may affect the final flavor of the wine product, while engineered bacteria, due to genetic modification, have significant limitations and safety risks in their application in the food industry.

[0005] Therefore, using enzymatic methods to reduce the content of EC or its precursor urea in fermented wines is the most common approach. EC hydrolases can degrade EC into carbon dioxide, ethanol, and ammonia, while acid urease can degrade urea into carbon dioxide, water, and ammonia; all of these metabolites are harmless to the human body. Reducing the urea content in raw materials and during fermentation through microbial enzyme production not only helps reduce EC formation but also largely preserves the original brewing process. This is a safe, efficient, and highly feasible method that ensures food safety.

[0006] Chinese invention patent application number CN201510195626.7, published on 20150708, discloses a strain of *Lactobacillus plantarum* that does not produce amino acid decarboxylase but produces high urease. In the process of brewing rice wine, this strain is mixed with raw materials rice, yeast, and wheat koji for fermentation. The resulting rice wine has a 28.1% lower biogenic amine content and a 75.9% lower ethyl carbamate content compared to the control group rice wine (which does not contain this *Lactobacillus plantarum* for fermentation).

[0007] Chinese invention patent application number CN202111428903.6, published on February 24, 2023, discloses a strain of brewing yeast G2 capable of degrading urea and a strain of Lactobacillus fermentum d6 producing ethyl carbamate-degrading enzyme. When these two strains are added simultaneously to the fermentation process of rice wine, the final test results show that, compared to a control rice wine, urea and ethyl carbamate levels have decreased by 70.06% and 68.73%, respectively. This technology requires a combination of two strains with different degradation functions to achieve the effect of simultaneously degrading urea and ethyl carbamate.

[0008] Currently, most strains that degrade ethyl carbamate are Bacillus species, with few probiotics, and most reported strains can only degrade either urea or ethyl carbamate.

[0009] In summary, how to develop natural probiotics with urea degradation and urethane degradation capabilities is precisely the technical problem that this field is committed to solving. Summary of the Invention

[0010] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus plantarum* YYS-K3 strain. This *Lactobacillus plantarum* YYS-K3 is a natural strain capable of simultaneously producing ethyl carbamate-degrading enzymes and ureases, exhibiting excellent degradation of ethyl carbamate. It can be widely applied in the production of fermented products, such as alcoholic beverages, soy sauce, and enzymes, significantly reducing the ethyl carbamate content in these products. It can also be used in conjunction with probiotics for detoxification to reduce the health hazards of alcoholic beverages.

[0011] The *Lactobacillus plantarum* YYS-K3 provided by this invention has the following Latin scientific name: Lactiplantibacillus plantarum The strain, *Lactobacillus plantarum* YYS-K3, was deposited on June 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27598. Isolated from rice wine, this strain was sequenced and, through BLAST sequence comparison, showed high homology with *Lactobacillus plantarum*, and was named *Lactobacillus plantarum* YYS-K3.

[0012] Among them, Lactobacillus plantarum YYS-K3 can produce ethyl carbamate degrading enzyme and urease, and it has the ability to degrade urea and ethyl carbamate.

[0013] The present invention also provides the application of *Lactobacillus plantarum* YYS-K3 as described above in the preparation of fermented products; wherein, fermented products include alcoholic beverages, soy sauce, enzymes and other fermented products.

[0014] When Lactobacillus plantarum YYS-K3 is used in the preparation of fermented products, it can significantly reduce the content of ethyl carbamate in the products.

[0015] In one embodiment, the alcoholic beverage product includes one or more of rice wine, fruit wine, grape wine, and health wine.

[0016] The present invention also provides the application of Lactobacillus plantarum YYS-K3 as described above in the preparation of hangover relief products.

[0017] The Lactobacillus plantarum YYS-K3 is used in hangover relief products. It can work together with hangover relief probiotics to relieve hangovers and reduce harmful components in alcohol, thereby reducing the harm of alcoholic products to human health.

[0018] The present invention also provides a method for producing ethyl carbamate degrading enzyme and / or urease, wherein *Lactobacillus plantarum* YYS-K3 as described above is inoculated into MRS liquid culture medium and cultured at 30-40°C for 24-36 h, the culture medium is separated into solid and liquid, the solid is resuspended and cell wall is broken to obtain an enzyme solution containing ethyl carbamate degrading enzyme and / or urease.

[0019] The present invention also provides a freeze-dried product, the components of which include Lactobacillus plantarum YYS-K3 as described above.

[0020] In one embodiment, the viable count of *Lactobacillus plantarum* YYS-K3 in the freeze-dried product is (2–8) × 10⁻⁶. 11 CFU / g.

[0021] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0022] 1) Preparation of Lactobacillus plantarum seed culture YYS-K3;

[0023] 2) Seed culture expansion;

[0024] 3) Seed culture fermentation to obtain fermentation broth;

[0025] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0026] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0027] 6) The emulsion is freeze-dried and pulverized to obtain plant lactobacillus YYS-K3 freeze-dried powder.

[0028] The present invention also provides a microbial agent, wherein the components of the microbial agent comprise the freeze-dried powder as described above, and / or the components of the microbial agent comprise *Lactobacillus plantarum* YYS-K3 as described above.

[0029] In one embodiment, the microbial agent further includes other ingredients, which are existing ingredients suitable for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, thickeners, and inclusion agents.

[0030] The present invention also provides the application of Lactobacillus plantarum YYS-K3 as described above in the preparation of probiotic products.

[0031] This invention also provides the application of *Lactobacillus plantarum* YYS-K3 as described above in the preparation of functional products, said functional products comprising at least one of the following functions:

[0032] (1) It has the ability to degrade urea;

[0033] (2) It has the ability to degrade urethane.

[0034] Based on the above characteristics, the *Lactobacillus plantarum* YYS-K3 provided by this invention has the following beneficial effects:

[0035] The *Lactobacillus plantarum* YYS-K3 provided by this invention has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can successfully reach the human intestine.

[0036] Lactobacillus plantarum YYS-K3 has the ability to produce ethyl carbamate degrading enzyme and urease, which can degrade urea and can be used in the development of kidney protection products.

[0037] It has a high ethyl carbamate degradation capacity and can be widely used in the production of fermented products (such as wine, soy sauce, enzymes, etc.). It can significantly reduce the ethyl carbamate content in the products and can also be used in combination with probiotics to reduce the harm of alcoholic products to human health. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The colorimetric reaction diagram of Lactobacillus plantarum YYS-K3 in the primary screening medium;

[0040] Figure 2 A standard curve of ammonium ions in the degradation of ethyl carbamate and urea by Lactobacillus plantarum YYS-K3 is shown.

[0041] Figure 3 A standard curve diagram showing the degradation of urea by Lactobacillus plantarum YYS-K3;

[0042] Figure 4 A graph showing the serum urea levels in mice after feeding them with Lactobacillus plantarum YYS-K3. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] The solution of the present invention:

[0045] This invention provides a plant lactobacillus ( Lactiplantibacillus plantarum YYS-K3:

[0046] Lactobacillus plantarum ( Lactiplantibacillus plantarum YYS-K3 is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 27598;

[0047] Source: The *Lactobacillus plantarum* strain was isolated from rice wine. After sequencing analysis, the strain was found to be highly homologous to *Lactobacillus plantarum* by Blast sequence comparison, and was named *Lactobacillus plantarum* YYS-K3.

[0048] Colony morphology: In MRS solid medium, colonies are milky white, round, and have smooth and neat edges.

[0049] Functions: It has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can reach the human intestine smoothly; it has the ability to produce ethyl carbamate degrading enzyme and urease, and can degrade urea and ethyl carbamate.

[0050] The present invention also provides an operational example of a method for producing ethyl carbamate degrading enzyme and / or urease:

[0051] As described above, *Lactobacillus plantarum* YYS-K3 was inoculated into MRS liquid medium and cultured at (30-40) °C for 24-36 h. The culture solution was centrifuged at (2-6) °C, the supernatant was discarded, the bacterial cells were resuspended in citrate-sodium citrate buffer and ultrasonically disrupted to obtain an enzyme solution containing ethyl carbamate degrading enzyme and / or urease.

[0052] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0053] 1) Preparation of Lactobacillus plantarum seed culture YYS-K3;

[0054] 2) Seed culture expansion;

[0055] 3) Seed culture fermentation to obtain fermentation broth;

[0056] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0057] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0058] 6) The emulsion is freeze-dried and pulverized to obtain plant lactobacillus YYS-K3 freeze-dried powder.

[0059] This invention also provides an operational example of its application method in fermented rice wine:

[0060] Soak glutinous rice, steam it until cooked, and let it cool. Add Bacillus plantarum YYS-K3 bacterial powder and / or freeze-dried powder, as well as brewing yeast. Mix well and add it to a fermentation tank to ferment together. After fermentation is complete, filter out the liquid, sterilize it at high temperature, and seal it for storage to obtain rice wine.

[0061] This invention also provides an operational example of its application method in fermented rice wine:

[0062] This invention also provides a method for brewing wine using *Lactobacillus plantarum* YYS-K3: grapes are washed, dried, and crushed, and *Lactobacillus plantarum* YYS-K3 bacteria and / or freeze-dried powder and brewing yeast are added and mixed for fermentation. After fermentation, the wine liquid is filtered out, sterilized at high temperature, sealed and stored to obtain the wine.

[0063] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1: Isolation and identification of Lactobacillus plantarum YYS-K3

[0065] Separation:

[0066] Aseptic sampling was performed on rice wine using the plate spread method. 5 g of rice wine sample was placed in a sterile homogenizing bag, and 45 mL of 0.85% physiological saline was added. The mixture was then homogenized to obtain the sample. 100 μL of the sample was then serially diluted 10-fold. -2 10 -3 10 -4 100 μL of the sample was spread onto an MRS solid plate containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h. Colonies with good growth and large calcium dissolution zones were picked and repeatedly isolated and purified by streak plating until single colonies were obtained. The isolated strain was named YYS-K3 and stored in a bacterial bank at -80°C with glycerol.

[0067] The colony morphology of the isolated and purified *Lactobacillus plantarum* YYS-K3 was as follows: in MRS solid medium, the colonies were milky white, round, and had smooth and neat edges.

[0068] The formula for MRS liquid medium is as follows: 10.0 g beef extract, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 1.0 L deionized water, pH 6.5 (add 1.5% agar to make MRS solid medium).

[0069] Strain identification:

[0070] The screened and purified strain YYS-K3 was subjected to Gram staining and catalase tests, and its physiological and biochemical indicators were measured. The test results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species. Figure 1 As shown, the test results showed that the selected strain YYS-K3 stained purple with Gram stain, indicating a positive result. It is rod-shaped, catalase-negative, and does not form spores.

[0071] DNA was extracted from YYS-K3 according to the instructions of the bacterial DNA extraction kit and amplified by PCR. The amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The strain YYS-K3 was identified as Lactobacillus plantarum.

[0072] The gene sequence is as follows:

[0073]

[0074] Example 2 Preparation of Lyophilized Powder of Lactobacillus plantarum YYS-K3

[0075] The activated *Lactobacillus plantarum* YYS-K3 was inoculated at a rate of 3% (v / v) into a culture medium sterilized at 121℃ for 15 min, and cultured at 37℃ for 24 h. After centrifugation at 4℃ and 6000 r / min for 10 min, the supernatant was discarded to obtain bacterial sludge. This sludge was then emulsified with a cryoprotectant for 15 min to obtain a bacterial solution with a concentration of 5 × 10⁻⁶. 10 An emulsion with a cfu / mL concentration was pre-frozen at -40°C for 4 hours and then freeze-dried at -35°C for 35 hours to obtain a freeze-dried bacterial powder of active *Lactobacillus plantarum* YYS-K3 with a viable count of 510 billion cfu / g.

[0076] The culture medium consists of: 2.5% glucose, 2.5% soybean peptone, 1% yeast extract, 1% beef extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% Tween 80, and the balance being water. The protective agent consists of 50 g / L skim milk powder, 10 g / L sucrose, 10 g / L trehalose, and 10 g / L L-glutamate.

[0077] It should be noted that the protective agent can be an existing protective agent component or formulation, including but not limited to the above-described embodiments.

[0078] Example 3: Study on the acid and bile salt resistance of Lactobacillus plantarum YYS-K3

[0079] 1. Acid resistance

[0080] The *Lactobacillus plantarum* YYS-K3 bacterial powder obtained in Example 2 was inoculated at a rate of 1% (w / v) into MRS liquid medium with pH 6.0, pH 4.0 and pH 2.0, respectively. After incubation at 37°C for 3 h, viable bacteria were counted using the plate count method. The results are shown in Table 1.

[0081] Table 1. Study on acid resistance of Lactobacillus plantarum YYS-K3

[0082]

[0083] It can be seen that under pH 2.0 conditions, Lactobacillus plantarum YYS-K3 still has a sufficient number of live bacteria that can pass through the gastric environment after 3 hours.

[0084] The formulation of the MRS liquid culture medium is the same as that in Example 1.

[0085] 2. Bile salt tolerance

[0086] The *Lactobacillus plantarum* YYS-K3 bacterial powder obtained in Example 2 was inoculated at a rate of 1% (w / v) into MRS liquid medium with a bile salt concentration of 0% (w / v) and 0.3% (w / v). After incubation at 37°C for 3 h, viable bacteria were counted using the plate count method. The results are shown in Table 2.

[0087] Table 2. Study on bile salt tolerance of Lactobacillus plantarum YYS-K3

[0088]

[0089] It can be seen that at a bile salt concentration of 0.3%, Lactobacillus plantarum YYS-K3 has good bile salt tolerance.

[0090] 3. Tolerance in artificial gastric and intestinal fluids

[0091] A certain amount of pepsin was dissolved in a PBS buffer solution at pH 3.0, and its final concentration was adjusted to 3.0 g / L to obtain simulated artificial gastric fluid. A certain amount of trypsin was dissolved in a PBS buffer solution at pH 8.0, and its final concentration was adjusted to 1.0 g / L to obtain simulated artificial intestinal fluid. Both the simulated gastric and artificial intestinal fluids were filtered through a 0.22 μm filter membrane under sterile conditions.

[0092] The *Lactobacillus plantarum* YYS-K3 bacterial powder obtained in Example 2 was inoculated at 1% (w / v) in MRS liquid medium and artificial gastric fluid and cultured at 37°C for 3 h. Then, the fermentation broth in the artificial gastric fluid was inoculated into artificial intestinal fluid at 10% and cultured at 37°C for 3 h. Viable bacteria were counted using the plate count method. The results are shown in Table 3.

[0093] Table 3. Tolerance of *Lactobacillus plantarum* YYS-K3 in simulated gastric and intestinal fluids.

[0094]

[0095] It can be seen that after passing through artificial gastric fluid and then artificial intestinal fluid, Lactobacillus plantarum YYS-K3 has good tolerance and can successfully reach the intestine to colonize and exert its effects.

[0096] Example 4: Study on the degradation of urea by Lactobacillus plantarum YYS-K3

[0097] 1. Initial screening

[0098] The *Lactobacillus plantarum* YYS-K3 strain was activated to the second generation, streaked into solid screening medium, and incubated at 37°C for 48 h. The color change of the medium was observed; a change from yellow to purple indicated that it had the function of degrading urea.

[0099] Screening medium: MRS solid medium (formulation consistent with Example 1) with 5 g / L urea and 6 mg / L bromocresol purple added, and pH adjusted to 4.5.

[0100] 2. Determination of urease (i.e., urease) activity

[0101] Enzyme activity is defined as the amount of enzyme that produces 1 μmol of ammonia per minute by breaking down urea at 37°C and pH 4.5.

[0102] Enzyme activity assay method – Berthelot reaction: The enzyme activity of urea-degrading enzyme was determined by colorimetry. Take 1 mL of enzyme solution, add 1 mL of 3% (w / v) urea solution, react at 37℃ for 20 min, add 1 mL of stop agent, then add 1 mL of colorimetric reagent 1 and 1 mL of colorimetric reagent 2, shake well, and incubate at 37℃ for 20 min. Make up to 10 mL with ultrapure water, and measure the absorbance at 625 nm.

[0103] The terminator is 10% trichloroacetic acid. Colorimetric reagent I: Weigh 60 g of phenol and 3.0 g of sodium nitroferricyanide, add pure water to a final volume of 1 L, and store at 4℃. Colorimetric reagent II: Weigh 52.5 g of sodium hydroxide and 30 mL of sodium hypochlorite, add pure water to a final volume of 1 L, and store at 4℃.

[0104] The enzyme solution acquisition process in this embodiment is as follows: Plantlet Lactobacillus plantarum YYS-K3 is inoculated into MRS liquid medium (formula is the same as in Example 1), cultured at 37°C for 36 h, the culture solution is centrifuged at 4°C, the supernatant is discarded, the bacterial cells are resuspended with citrate-sodium citrate buffer and ultrasonically disrupted, and the resulting crude enzyme solution is obtained; wherein, the inoculation amount of Plantlet Lactobacillus plantarum YYS-K3 is 3% (v / v) of the culture medium.

[0105] Result calculation method:

[0106] The formula for enzyme activity determination is: Enzyme activity = OD 625 ×n×1 / k×1 / 20;

[0107] In the formula, n is the dilution factor for enzyme solution testing, k is the slope of the fitted standard curve, and 20 is the enzyme-catalyzed reaction time.

[0108] The standard curve was prepared by using ammonium chloride to prepare standard solutions of different concentrations (0 mmol / L, 0.2 mmol / L, 0.4 mmol / L, 0.6 mmol / L, 0.8 mmol / L, 1.0 mmol / L, 1.2 mmol / L, and 1.4 mmol / L), and measuring the OD using the Berthelot reaction method for enzyme activity assay. 625Absorbance, in NH4 + Plot a linear standard curve with concentration on the x-axis and OD value on the y-axis (see [reference]). Figure 2 )

[0109] The results showed that the urea-degrading enzyme activity of Lactobacillus plantarum YYS-K3 was 0.25 ± 0.06 U / mL.

[0110] 4.3 Urea Degradation Test by Lactobacillus plantarum YYS-K3

[0111] Lactobacillus plantarum YYS-K3 was activated to generation 2 and inoculated at a rate of 3% (v / v) into MRS liquid medium containing 5 g / L urea (formula consistent with Example 1). The medium was then incubated at 37°C for 24 h to obtain the test solution.

[0112] The urea content was determined using the diacetyl-oxime method: 2 mL of the test solution was added to a 10 mL colorimetric tube, along with 0.5 mL of diacetyl-oxime-thiamide solution and 5 mL of iron-phosphate solution. The mixture was thoroughly mixed and incubated in a water bath at 100°C for 10 min. After the water bath, the colorimetric tube was quickly placed in ice water to cool for 2 min, and the absorbance at 530 nm was measured.

[0113] The diacetylmonoxime-thiamide solution was prepared by weighing 30 mg of thiamide and 600 mg of diacetylmonoxime, then diluting with pure water to a final volume of 100 mL and storing in a brown volumetric flask. The iron-phosphoric acid solution was prepared by weighing 600 mg of ferric ammonium sulfate, dissolving it in concentrated phosphoric acid, and diluting to a final volume of 100 mL, then storing at room temperature. Results were calculated as follows:

[0114] The formula for determining urea degradation rate is: Urea degradation rate = (initial urea concentration - remaining urea concentration) / initial urea concentration × 100%;

[0115] Urea standard curve: Prepare a 1.0 g / L urea solution, and dilute it with pure water to concentrations of 0 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and 64 mg / L, respectively. Take 2 mL of each dilution and place it in a 10 mL colorimetric tube. Measure the absorbance at 530 nm using the diacetyl monooxime method. Plot a standard curve with urea concentration on the x-axis and absorbance on the y-axis (see [reference]). Figure 3 ).

[0116] The test results showed that the degradation rate of urea by Lactobacillus plantarum YYS-K3 was 22.17% ± 1.4%.

[0117] Example 5: Application of *Lactobacillus plantarum* YYS-K3 powder in reducing blood urea levels in mice.

[0118] 1. Test materials

[0119] Experimental animals: 60 male mice, 3 weeks old, about 20 g.

[0120] Test subject: Lyophilized powder of Lactobacillus plantarum YYS-K3 obtained in Example 2.

[0121] 2. Animal grouping, modeling, and gavage methods

[0122] Mice were acclimatized to a basal diet for one week. From week two onwards, they were fed a diet high in urea. The control group continued to be fed the basal diet. After three weeks, the urea levels in the blood of both types of mice were measured. Mice with significantly higher urea levels than the control group (P < 0.05) were designated as high urea model mice. Mice with successfully induced urea model were selected and administered the following gavage protocol:

[0123] Control group: 0.2 mL of normal saline was administered by gavage daily for 7 consecutive days.

[0124] Experimental group: 0.2 mL of a 2% (w / v) solution of *Lactobacillus plantarum* YYS-K3 bacterial powder was administered by gavage daily for 7 consecutive days. The *Lactobacillus plantarum* YYS-K3 bacterial powder used was the lyophilized powder prepared in Example 2.

[0125] After the mice were administered the medication via gavage, the urea content in their serum was measured. The results showed that the urea content in the serum of the experimental group mice was significantly lower than that of the control group by 38.72 ± 2.3% (see details). Figure 4 ).

[0126] Example 6: Study on the degradation of ethyl carbamate by Lactobacillus plantarum YYS-K3

[0127] 1. Initial screening

[0128] The *Lactobacillus plantarum* YYS-K3 strain was activated to the second generation, streaked into solid screening medium, and incubated at 37°C for 48 h. The color change of the medium was observed; a change from yellow to purple indicated that it had the function of degrading ethyl carbamate.

[0129] Screening medium: MRS solid medium (formulation consistent with Example 1) was supplemented with 5 g / L ethyl carbamate and 6 mg / L bromocresol purple, and the pH was adjusted to 4.5.

[0130] 2. Assay of ethyl carbamate degrading enzyme activity

[0131] Enzyme activity is defined as the amount of enzyme that can decompose ethyl carbamate to produce 1 μmol of ammonia per minute at 37°C and pH 4.5. One enzyme activity unit (U) is defined as the amount of enzyme that can decompose ethyl carbamate to produce 1 μmol of ammonia per minute.

[0132] Enzyme activity assay method – Berthelot reaction: Take 1 mL of enzyme solution, add 1 mL of 3% ethyl carbamate solution, react at 37℃ for 20 min, add 1 mL of stop agent, then add 1 mL of colorimetric reagent 1 and 1 mL of colorimetric reagent 2, shake well, and incubate at 37℃ for 20 min. Make up to 10 mL with ultrapure water, and measure the absorbance at 625 nm. The enzyme activity calculation method and standard curve are as described in Example 4.

[0133] The enzyme solution acquisition process in this embodiment is the same as in Embodiment 4;

[0134] The test results showed that the ethyl carbamate degrading enzyme activity of Lactobacillus plantarum YYS-K3 was 0.21±0.04 U / mL.

[0135] 3. Degradation of ethyl carbamate by Lactobacillus plantarum YYS-K3

[0136] Lactobacillus plantarum YYS-K3 was activated to generation 2 and inoculated at a rate of 3% (v / v) into MRS solid medium containing 5 g / L ethyl carbamate (formula consistent with Example 1). The medium was incubated at 37°C for 24 h, and the remaining content of ethyl carbamate was determined.

[0137] The above-mentioned samples were sent to CTI Certification & Testing Group Co., Ltd. for GC-MS testing. The results showed that the degradation rate of ethyl carbamate by Lactobacillus plantarum YYS-K3 was 94.4%±2.5%.

[0138] Example 7 Application of Lactobacillus plantarum YYS-K3 in the production of ethyl carbamate degrading enzyme / urease

[0139] Lactobacillus plantarum YYS-K3 was inoculated into MRS liquid medium (formula consistent with Example 1) and cultured at 37°C for 24-36 h. The culture was centrifuged at 4°C, the supernatant was discarded, the cells were resuspended in citrate-sodium citrate buffer and ultrasonically disrupted to obtain crude enzyme solution.

[0140] The crude enzyme solution contained ethyl carbamate degrading enzyme and urease. The presence of urease and ethyl carbamate degrading enzyme could be identified using the enzyme activity assay methods shown in Examples 4 and 6. The enzyme solution prepared in these examples was confirmed to contain ethyl carbamate degrading enzyme and urease.

[0141] Example 8: Application of *Lactobacillus plantarum* YYS-K3 bacterial powder in the preparation of solid beverages

[0142] A probiotic solid beverage is obtained by mixing 10% of Lactobacillus plantarum YYS-K3 bacterial powder, 10% of Lactobacillus paracasei YYS-K1, 10% of erythritol, 20% of skim milk powder, 15% of fermented pineapple powder, 15% of maltodextrin, 10% of fructooligosaccharides, and 10% of galactooligosaccharides (wherein the percentages are by mass).

[0143] Example 9: Application of Lactobacillus plantarum YYS-K3 bacterial powder in fermented beverages

[0144] Mix 1.0 kg of water, 0.2 kg of concentrated blueberry juice, and 0.2 kg of concentrated apple juice. Inoculate with 0.4 g of *Lactobacillus plantarum* YYS-K3 powder and 0.4 g of *Lactobacillus paracasei* YYS-K1 obtained in Example 2. Ferment at 30°C for 48 h. The final pH of the fermentation is 3.56, thus obtaining a fermented fruit and vegetable juice beverage.

[0145] Example 10: Application of Lactobacillus plantarum YYS-K3 in fermented rice wine

[0146] Commercially available glutinous rice was selected as the raw material. The glutinous rice was soaked, steamed, and cooled. Without changing the brewing process, 1% (w / v) of the freeze-dried Lactobacillus plantarum YYS-K3 powder obtained in Example 2 and brewing yeast were added and mixed together for fermentation at room temperature for 10 days. The rice wine fermented without the addition of Lactobacillus plantarum YYS-K3 was used as a control. The content of ethyl carbamate in the finished rice wine was determined (the content determination was carried out according to Example 6, using the GC-MS detection method).

[0147] The results showed that the ethyl carbamate content of the rice wine fermented with added Lactobacillus plantarum YYS-K3 decreased by 80.3% ± 3.8% compared with the control rice wine.

[0148] Example 11 Application of Lactobacillus plantarum YYS-K3 in fermented wine

[0149] Commercially available Kyoho grapes were used as raw materials. After washing with clean water and drying, the grapes were crushed and mixed with 1% (w / v) of *Lactobacillus plantarum* YYS-K3 freeze-dried powder obtained in Example 2 and brewing yeast. The mixture was stirred and added to a fermentation tank for 7 days of fermentation. After the first fermentation, the upper layer of grape pomace was removed, and fermentation continued for another 7 days. The fermentation temperature was controlled at around 30°C throughout the process. After the fermentation was completed, the wine liquid was filtered out. The content of ethyl carbamate in the finished wine was determined using wine fermented without *Lactobacillus plantarum* YYS-K3 as a control (the content determination was performed according to Example 6, using the GC-MS detection method).

[0150] The results showed that the ethyl carbamate content of the wine fermented with added Lactobacillus plantarum YYS-K3 decreased by 51.5% ± 6.2% compared with the control wine.

[0151] Example 12: The combined use of Lactobacillus plantarum YYS-K3 and probiotics in a hangover remedy product:

[0152] When combined with probiotics for relieving hangovers, Lactobacillus plantarum YYS-K3 can produce ethyl carbamate degrading enzymes and ureases, which have a good degradation effect on both ethyl carbamate and urea. When used in combination with probiotics for relieving hangovers, it can help relieve hangovers and reduce harmful components in alcohol, thereby reducing the harm of alcoholic products to human health.

[0153] Among them, the probiotics for relieving hangovers include, but are not limited to, Lactobacillus paracasei YYS-K1 (accession number: CGMCC No. 26405).

[0154] Based on the results of the above embodiments, the *Lactobacillus plantarum* YYS-K3 provided by the present invention has the following properties and effects:

[0155] 1. It has strong resistance to acid and bile salts, and is well tolerated in artificial gastric and intestinal fluids, allowing it to reach the human intestine smoothly;

[0156] 2. Ethyl carbamate has a high degradation level, with a degradation rate as high as 94.4% in culture media, 80.3% in rice wine, and 51.5% in wine.

[0157] 3. It can simultaneously produce ethyl carbamate degrading enzyme and urease, and has a good degradation effect on both ethyl carbamate and urea.

[0158] 4. It can be used in conjunction with hangover remedies to help relieve hangovers and reduce the harm of alcoholic beverages to human health;

[0159] 5. The strain is derived from rice wine and can be used as a fermenting agent in the preparation of rice wine, fruit wine, grape wine, health wine, etc.

[0160] 6. This strain has a strong scavenging effect on urea accumulation in animals and humans, and can be applied to the development of kidney protection products.

[0161] In summary, compared with the prior art, the *Lactobacillus plantarum* YYS-K3 provided by this invention has the following beneficial effects:

[0162] The *Lactobacillus plantarum* YYS-K3 provided by this invention has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can successfully reach the human intestine.

[0163] Lactobacillus plantarum YYS-K3 has the ability to produce ethyl carbamate degrading enzyme and urease, and can degrade urea, which can be applied to the development of kidney protection products;

[0164] This strain also possesses a high ethyl carbamate degradation capacity, making it widely applicable in the production of fermented products such as alcoholic beverages, soy sauce, and enzymes. It can significantly reduce the ethyl carbamate content in these products and can also be used in conjunction with probiotics to reduce the health risks associated with alcoholic beverages.

[0165] 1. It can be applied to the fermentation of alcoholic beverages;

[0166] 2. Applicable to functional products that include at least one of the following functions (e.g., for the development of kidney protection products):

[0167] (1) It has the ability to degrade urea;

[0168] (2) It has the ability to degrade urethane;

[0169] Among them, products with the above (1)-(2) functions include, but are not limited to, the effects of clearing urea to protect the kidneys, etc., and can also be products with other effects based on the function of degrading urea or urethane.

[0170] It should be noted that:

[0171] (1) Definition:

[0172] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0173] (2) The relevant prior art means or prior art terms involved in this application:

[0174] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. 625 "OD" is the optical density value measured when the wavelength is set to 625nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0175] Ethyl carbamate, with the structural formula C3H7NO2, is abbreviated as EC.

[0176] (3) Application of strains:

[0177] The examples illustrate that Lactobacillus plantarum YYS-K3 can be applied to the fermentation of alcoholic beverages. According to the above design concept, this strain can be applied to various fermented alcoholic beverages containing ethanol, including but not limited to rice wine and wine in the examples.

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum YYS-K3, characterized in that, Its accession number is CGMCC No. 27598.

2. A method for producing ethyl carbamate-degrading enzyme and / or urease, characterized in that: Lactobacillus plantarum YYS-K3 was inoculated into MRS liquid medium and cultured at 30-40℃ for 24-36 h. The culture medium was then separated into solid and liquid components. The solid was resuspended and cell wall was broken to obtain an enzyme solution containing ethyl carbamate degrading enzyme and / or urease. The *Lactobacillus plantarum* YYS-K3 mentioned herein is the *Lactobacillus plantarum* YYS-K3 as described in claim 1.

3. The application of *Lactobacillus plantarum* YYS-K3 in the preparation of fermented products, characterized by: in, The fermented products include alcoholic beverages, soy sauce, or enzymes; The plant lactobacillus YYS-K3 used is the plant lactobacillus YYS-K3 as described in claim 1.

4. A freeze-dried product, characterized in that: Its components include *Lactobacillus plantarum* YYS-K3 as described in claim 1.

5. A microbial agent, characterized in that: The components of the microbial agent include the freeze-dried product as described in claim 4, or the components of the microbial agent include *Lactobacillus plantarum* YYS-K3 as described in claim 1.

6. The application of *Lactobacillus plantarum* YYS-K3 in the preparation of probiotic products, characterized by: The plant lactobacillus YYS-K3 used is the plant lactobacillus YYS-K3 as described in claim 1.

7. The use of Lactobacillus plantarum YYS-K3 as described in claim 1 in the preparation of functional products for degrading ethyl carbamate.

8. The use of Lactobacillus plantarum YYS-K3 as described in claim 1 in the degradation of urea for purposes other than disease diagnosis or treatment.

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