A coagulant Weizmannella that efficiently metabolizes ethanol and its application

Abstract

The present application provides a strain of Weizmannella coagulans AlcoKer0031 that efficiently metabolizes ethanol, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.31222. The strain is isolated from kimchi, and the strain source is natural and safe. It can colonize in the intestine, efficiently metabolize ethanol and protect the liver. The combination of the strain with plant extracts such as Pueraria lobata and Hovenia dulcis can be used to prepare alcohol-detoxifying and liver-protecting products, which have good market application value.

Description

Technical Field

[0001] The present application belongs to the field of microorganisms. Specifically, the present application provides a Weizmannella coagulans that can efficiently metabolize ethanol and an application thereof. Background Art

[0002] With economic development and the accelerated pace of life, people's ethanol (alcohol) consumption in social activities continues to rise, and alcohol abuse has become an increasingly severe public health challenge. Excessive drinking has been shown to be strongly correlated with a variety of diseases, which may lead to health problems such as high blood pressure, indigestion, alcoholic cardiomyopathy, chronic gastrointestinal inflammation, and may cause damage to the nervous system, leading to brain dysfunction, neurasthenia, distraction, and memory loss. In severe cases, alcohol poisoning can lead to deep depression of the central nervous system, even coma and death. The liver is the main organ for alcohol metabolism, and long-term drinking may also cause fatty cirrhosis. Direct metabolism of ethanol can reduce the health risks of alcohol, and help treat alcohol dependence and improve recovery ability after alcohol intake.

[0003] The oxidative metabolism of alcohol in the human body is mainly carried out through the liver. After alcohol is ingested, a small part is metabolized in the stomach, and most of it is absorbed through the gastrointestinal tract, dispersed to various tissues throughout the body through the systemic circulation, and finally gathered in the liver for oxidative metabolism. In the liver, alcohol dehydrogenase (ADH) catalyzes the oxidation of ethanol to acetaldehyde, and acetaldehyde dehydrogenase (ALDH) further converts acetaldehyde into acetic acid, which eventually decomposes into CO2 and water. If ethanol is consumed excessively, it is easy to cause alcohol poisoning in the human body and cause damage to the nervous system. In this regard, many methods such as drug treatment (naloxone hydrochloride, aspirin) and nutritional supplements (vitamin B, vitamin C) are used to accelerate the metabolism of ethanol in the human body. However, the above methods have obvious side effects and ineffective effects. Improving the direct metabolic capacity of ethanol can reduce the production of acetaldehyde, thereby reducing or preventing these health risks.

[0004] Probiotics are increasingly used in accelerating alcohol metabolism and reducing alcoholic liver disease due to their low side effects and strong functionality. For example, patent CN 114107121A discloses the use of Bacillus coagulans in the treatment of alcoholic liver disease. The strain reduces the levels of alanine aminotransferase and aspartate aminotransferase in serum, reduces the expression of proinflammatory factors and triglycerides in liver tissue, and increases the expression of anti-inflammatory factors and intestinal junction proteins in liver tissue, thereby repairing the intestinal barrier and preventing and treating alcoholic liver disease. However, the strain has the problem of not being able to directly metabolize alcohol. Patent CN116731896A discloses a Bacillus coagulans capable of degrading ethanol and its application. The strain is mutagenized by ultraviolet light, guanidine nitrite, and normal pressure and room temperature plasma to improve the ability to degrade alcohol, which helps to reduce the alcohol content before human absorption. However, genetically modified strains may change their interactions with the human body or the environment, leading to unforeseen side effects. Summary of the invention

[0005] Based on the shortcomings of the above patents, the present invention proposes a Weizmannella coagulans AlcoKer0031 strain, a bacterial agent, a preparation method and further applications thereof that can efficiently metabolize ethanol. The Weizmannella coagulans is extracted from kimchi, has high safety, can directly metabolize ethanol efficiently, has high gastric acid resistance and choline resistance survival rate, and has the ability to increase the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase. Through the dual-pathway synergistic effect of the plant extract and the probiotic, the effect of rapid sobering up and protecting the liver can be achieved.

[0006] On the one hand, the present application provides a strain of Weizmannella coagulans that efficiently metabolizes ethanol ( Weizmannia coagulans ) AlcoKer0031, the coagulant Weizmannella AlcoKer0031 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; deposit number: CGMCC No.31222, deposit date: July 8, 2024, classification name: Weizmannella coagulans Weizmannia coagulans .

[0007] On the other hand, the present application provides the use of the above-mentioned Weizmannella coagulans AlcoKer0031 in the preparation of health products or medicines for sobering up and protecting the liver.

[0008] Furthermore, the health care product or medicine is in the form of tablets or powder.

[0009] Furthermore, the health product or medicine contains coagulant Weizmannella AlcoKer0031 microcapsules.

[0010] Furthermore, the mass ratio of the Weizmannella AlcoKer0031 microcapsules to the health care product or medicine is 30-40%.

[0011] Furthermore, the health care product or medicine also includes kudzu root extract, Hovenia dulcis fruit extract, fructooligosaccharides, xylooligosaccharides, maltodextrin and corn oligopeptides.

[0012] Furthermore, the preparation process of the coagulated Weizmannella AlcoKer0031 microcapsules comprises:

[0013] The coagulant Weizmannella AlcoKer0031 is inoculated into LB medium for activation culture to obtain a bacterial seed liquid; the seed liquid is inoculated into an expansion culture medium for fermentation culture to obtain a fermentation liquid, and the fermentation liquid is freeze-dried to obtain a coagulant Weizmannella AlcoKer0031 starter;

[0014] By mass, 10-15 parts of sodium alginate were dissolved in 80-100 parts of water, 3-5 parts of Weizmannella coagulans AlcoKer0031 starter were added, and the mixture was stirred and mixed evenly. 15 parts of gelatin and 5 parts of calcium chloride solution were added, and the mixture was emulsified at 11000 r / min for 10 minutes. The microcapsules were obtained after freeze-drying.

[0015] Furthermore, the concentration of live bacteria in the coagulated Weizmannella AlcoKer0031 microcapsules is at least 5×10 9 cfu / g.

[0016] Furthermore, the expansion culture medium formula and preparation method are as follows: 5.0 g of glucose, 10.0 g of peptone, 5.0 g of yeast powder, 0.25 g of sodium chloride, and 0.15 g of anhydrous calcium chloride, add 1000 mL of distilled water, adjust the pH to 5.0-5.5 with 2 mol / L sodium hydroxide solution or 2 mol / L lactic acid solution, heat to dissolve, dispense into conical flasks, and sterilize under high pressure at 121°C for 15 min-20 min.

[0017] Furthermore, in the health product or medicine, the mass ratio of fructooligosaccharide to xylooligosaccharide is 1:1-1:3; the mass ratio of maltodextrin to corn oligopeptide is 1:1-1:5.

[0018] Furthermore, the preparation method of the Pueraria root extract and the Hovenia dulcis fruit extract is as follows: after the Pueraria root and the Hovenia dulcis fruit are crushed respectively, 5 to 10 times the mass of 60% to 95% ethanol is added at a mass ratio of 1:1 to 1:3, the ultrasonic power is 20 to 40 kHz, the water bath temperature is 50°C to 60°C, the water bath time is 2 to 3 hours, and the first filtrate is obtained after filtration; then 3 to 5 times the mass of 50% to 80% ethanol is added to the filter residue again, and the ultrasonic water bath is continued for 1 to 2 hours according to the above parameters and then filtered to obtain the second filtrate, which is mixed with the first filtrate and then the ethanol is removed by rotary evaporation, and freeze-dried to obtain the Pueraria root extract and the Hovenia dulcis fruit extract; the freeze-drying parameters are as follows: freezing at -25°C for 8 to 12 hours, cold trap temperature of -60°C to -80°C, vacuum degree of 50 to 150 Pa, and drying time of 20 to 24 hours.

[0019] Furthermore, the preparation method of the medicine or health product also includes the steps of mixing the kudzu root extract, the Hovenia dulcis extract, oligofructose, oligoxylose, maltodextrin and corn oligopeptide and an optional tableting step.

[0020] In addition to the above ingredients, auxiliary materials including adhesives, fillers, coating agents, suspending agents, flavoring agents, and pH adjusters may be added to the health care products or medicines of the present application for dosage form or taste requirements. The selection and application of such auxiliary materials can be completed by those skilled in the art with conventional techniques and knowledge.

[0021] On the other hand, the present application provides the use of the above-mentioned Weizmannella coagulans AlcoKer0031 in the fermentation preparation of alcohol dehydrogenase.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The live bacteria of Weizmannella coagulans AlcoKer0031 provided by the present invention can colonize in the intestine, and experimental verification has shown that it has the function of efficiently metabolizing ethanol in vivo and protecting the liver.

[0024] (2) The Weizmannella coagulans AlcoKer0031 provided by the present invention can be cultured and produced by in vitro fermentation technology to prepare alcohol sobering and liver protecting products, which has strong market application value.

[0025] (3) The coagulant Weizmannella AlcoKer0031 provided by the present invention is isolated from kimchi, and the strain is natural and safe. Through the combined action with plant extracts (Kuanga Root, Hovenia Dulcis Fruit, etc.), the effects of metabolizing ethanol in the body and protecting the liver are significantly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0027] Figure 1 The effects of different embodiments on the pathological morphology of liver tissue in acutely intoxicated mice are shown. DETAILED DESCRIPTION

[0028] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0029] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention.

[0031] Example 1: Screening and domestication process of Weizmannella coagulans

[0032] Bacteria screening:

[0033] The strains are screened from the bacterial flora isolated from kimchi, and the specific steps are as follows:

[0034] (1) Preliminary enrichment: Add 2 mL of sample to 100 mL of MRS liquid culture medium and culture at 37°C for 16 to 22 hours.

[0035] (2) Gradient dilution: Take 0.5 mL of the enriched sample from step (1) and add it to 4.5 mL of sterile saline to prepare 10 -1 Then from 10 -1 Take 0.5 mL of the diluent and add it to another 4.5 mL of saline to prepare 10 -2 Repeat this process to prepare 10 -3 , 10 -4 , 10 -5 of dilution.

[0036] (3) Plate culture: Take 100 μL of each gradient dilution described in step (2) and spread it on MRS solid culture medium, setting up one plate for each gradient; culture at 37°C for 48 hours.

[0037] (4) Streak separation: Select colonies with typical characteristics based on their morphology, size, edge, and transparency. Use an inoculation loop to pick colonies from the coated plate and streak them on a selective culture medium. Then, culture them again at 37°C for 48 hours to obtain purified single colonies.

[0038] (5) Inoculate the single colony described in step (4) into 5 mL of MRS liquid culture medium and continue to culture at 37°C for 16 to 22 hours. Number each strain and further acclimate it.

[0039] (6) Stress culture: The strain described in step (5) was inoculated in MRS liquid and solid culture media with gradient ethanol concentrations (0%, 5%, 10%, 15%, 20% v / v) for initial stress culture. After culturing in liquid culture media for 18 h and in solid culture media for 36 h, strains with good growth were selected and transferred to MRS liquid and solid culture media with higher gradient ethanol concentrations (20%, 30%, 40%, 50%, 60% v / v) for further domestication. After culturing in liquid culture media for 18 h and in solid culture media for 36 h, strains with good growth were selected for high concentration ethanol (80% v / v) domestication. After gradient domestication, four strains that grew well on the high concentration ethanol plate were selected for further identification.

[0040] The MRS liquid culture medium in steps (1), (5) and (6) is prepared as follows: 15.0 g of glucose, 10.0 g of peptone, 4.0 g of yeast powder, 4.0 g of beef extract powder, 5.0 g of sodium acetate and 2.0 g of dipotassium hydrogen phosphate are added with 1000 mL of distilled water, the pH is adjusted to 5.5-6.0 with 2 mol / L sodium hydroxide solution or 2 mol / L lactic acid solution, the mixture is heated to dissolve, the mixture is dispensed into conical flasks and sterilized under high pressure at 121° C. for 15 min-20 min.

[0041] The preparation method of the MRS solid culture medium in steps (3) and (6) is as follows: 15.0 g of glucose, 10.0 g of peptone, 4.0 g of yeast powder, 4.0 g of beef extract powder, 15.0 g of agar, 5.0 g of sodium acetate, and 2.0 g of potassium dihydrogen phosphate are added to 1000 mL of distilled water, and the pH is adjusted to 5.5-6.0 with 2 mol / L sodium hydroxide solution or 2 mol / L lactic acid solution. Heat to dissolve, dispense into conical flasks, and sterilize under high pressure at 121°C for 15 min-20 min.

[0042] The typical characteristics of the bacterial colonies in step (4) are: moist, sticky, smooth, relatively transparent, easy to pick up, and uniform in texture.

[0043] Filter results:

[0044] The four isolated single colonies were continuously cultured and activated, and the strains with better growth, numbered AlcoKer0031, AlcoKer1044, AlcoKer1120, and AlcoKer2306, were selected for simulated gastrointestinal tolerance experiments.

[0045] (1) After each single strain was activated for three generations (18 h each time), the experiment was conducted. The OD value of the strain activated for three generations at A600nm wavelength was measured, and the amount of bacterial solution required for OD600nm=5 was calculated.

[0046] (2) Centrifuge the desired bacterial solution at 8000 × g for 10 min, discard the supernatant, resuspend in 1 mL of simulated gastric fluid, and incubate at 37°C for 2.5 h before counting the live bacteria on the plate.

[0047] (3) Take the required bacterial solution and centrifuge it at 8000 × g for 10 min. Discard the supernatant and resuspend it in an equal volume of simulated intestinal fluid. Incubate at 37°C for 4 h and then count the live bacteria on the plate.

[0048] (4) Centrifuge the bacterial solution treated with simulated gastric fluid at 8000 × g for 10 min, discard the supernatant, resuspend the solution in an equal volume of simulated intestinal fluid, and culture at 37°C for 5 h before counting the live bacteria on a plate.

[0049] The survival rate after tolerance to gastric juice (%) = (the number of live bacteria in the bacterial solution after tolerance to simulated gastric juice / the original number of live bacteria in the bacterial solution) × 100%.

[0050] The survival rate after tolerance to intestinal fluid (%) = (the number of live bacteria in the bacterial solution after tolerance to simulated intestinal fluid / the number of live bacteria in the bacterial solution after tolerance to simulated gastric fluid) × 100%.

[0051] The survival rate after tolerance to gastrointestinal fluid (%) = (the number of live bacteria in the bacterial solution after tolerance to simulated gastrointestinal fluid / the original number of live bacteria in the bacterial solution) × 100%.

[0052] Preparation method of simulated gastric fluid: Take 16.4 ml of dilute hydrochloric acid, add 800 ml of water and 10 g of pepsin, shake well and dilute with water to 1000 ml.

[0053] Preparation method of simulated intestinal fluid: take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve it, and adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take another 10g of pancreatic enzyme, add appropriate amount of water to dissolve it, after the two liquids are completely mixed, add water to dilute to 1000ml.

[0054] The Duncan method was used to perform significant difference analysis among multiple groups of samples. The results are shown in Table 1. The survival rates of strain AlcoKer0031 under three stress environments of simulated gastric fluid for 2.5 h, simulated intestinal fluid for 4 h, and simulated gastrointestinal fluid for 7.5 h were higher than those of other strains, indicating that it has a strong tolerance to gastrointestinal fluid.

[0055]

[0056] The single bacterial strain with the best gastrointestinal tolerance, numbered AlcoKer0031, was selected, DNA was extracted, and the 16S rRNA gene of the single bacterial strain AlcoKer0031 was amplified by PCR using universal primers 27 F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R (SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3'). The obtained PCR product was sent to Meige Gene Sequencing Co., Ltd. for sequencing, and the sequencing result was Weizmannella coagulans ( Weizmannia coagulans ), the sequencing results are as follows:

[0057] SEQ ID NO.3:

[0058]

[0059] This strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 8, 2024, with the deposit number: CGMCC No.31222.

[0060] The coagulant Weizmannella AlcoKer0031 was inoculated into LB medium for activation culture to obtain a bacterial seed liquid; the seed liquid was inoculated into an expansion culture medium for fermentation culture (the formula and preparation method are as follows: 5.0 g of glucose, 10.0 g of peptone, 5.0 g of yeast powder, 0.25 g of sodium chloride, 0.15 g of anhydrous calcium chloride, 1000 mL of distilled water was added, and the pH was adjusted to 5.0-5.5 with 2 mol / L sodium hydroxide solution or 2 mol / L lactic acid solution, heated to dissolve, and divided into conical bottles, and high-pressure sieved at 121°C. Sterilize for 15min-20min), obtain fermentation liquid, freeze-dry to obtain coagulated Weizmannella AlcoKer0031 fermentation agent; by mass, dissolve 10-15 parts of sodium alginate in 80-100 parts of water, add 3-5 parts of coagulated Weizmannella AlcoKer0031 fermentation agent, stir and mix evenly, add 15 parts of gelatin and 5 parts of calcium chloride solution, emulsify at 11000r / min for 10min, and freeze-dry to obtain coagulated Weizmannella AlcoKer0031 microcapsules (live bacteria concentration is 5×10 9 cfu / g).

[0061] Example 2: Detection of the properties of coagulating Weizmannella

[0062] 1. Colonization ability:

[0063] Three strains with similar functional effects to Weizmannella coagulans AlcoKer0031 were selected from the market and numbered as strain 1, strain 2, and strain 3. Among them, strain 1 and strain 2 are other Weizmannella coagulans, and strain 3 is a certain Lactobacillus plantarum. The strains that grow well on high-concentration ethanol plates are continuously activated and cultured to conduct gastrointestinal tolerance comparison experiments.

[0064] Each single strain was activated for three generations (18 h each time) before the experiment. The OD value of the strain activated for three generations at A600nm wavelength was measured, and the amount of bacterial solution required for OD600nm=5 was calculated.

[0065] Take the required bacterial solution and centrifuge it at 8000×g for 10 min, discard the supernatant, resuspend it in 1 mL of simulated gastric fluid, and culture it at 37℃ for 2.5 h before counting the live bacteria on the plate.

[0066] The required bacterial solution was centrifuged at 8000×g for 10 min, the supernatant was discarded, and the solution was resuspended in an equal volume of simulated intestinal fluid. After incubation at 37°C for 4 h, the live bacteria were counted on the plate.

[0067] The bacterial solution treated with simulated gastric fluid was centrifuged at 8000×g for 10 min, the supernatant was discarded, and the solution was resuspended in an equal volume of simulated intestinal fluid. After incubation at 37°C for 5 h, the live bacteria were counted on the plate.

[0068] The survival rate after tolerance to gastric juice (%) = (the number of live bacteria in the bacterial solution after tolerance to simulated gastric juice / the original number of live bacteria in the bacterial solution) × 100%.

[0069] The survival rate after tolerance to intestinal fluid (%) = (the number of live bacteria in the bacterial solution after tolerance to simulated intestinal fluid / the number of live bacteria in the bacterial solution after tolerance to simulated gastric fluid) × 100%.

[0070] The survival rate after tolerance to gastrointestinal fluid (%) = (the number of live bacteria in the bacterial solution after tolerance to simulated gastrointestinal fluid / the original number of live bacteria in the bacterial solution) × 100%.

[0071]

[0072] The results showed that compared with other strains, the survival rate of Weizmannella coagulans AlcoKer0031 in gastric juice, intestinal juice and gastrointestinal juice was significantly higher.

[0073] 2. Ability of Weizmannella coagulans AlcoKer0031 to metabolize alcohol dehydrogenase in vitro

[0074] Weizmannella coagulans AlcoKer0031 and strain 1, strain 2, and strain 3 were inoculated into MRS liquid medium (added with 52% ethanol, v / v), respectively, and cultured at 37°C for 16 h. The cells were then centrifuged at 8000 rpm, washed with PBS buffer, and then resuspended in fermentation broth. After low-temperature ultrasonic disruption, the supernatant was centrifuged at 4000 rpm, and the alcohol dehydrogenase (ADH) activity was determined by ELISA. The results are shown in the table.

[0075]

[0076] The results of enzyme activity detection showed that the alcohol dehydrogenase activity was from high to low, in the order of AlcoKer0031, strain 2, strain 3, and strain 1, and the difference between AlcoKer0031 and strains 1 and 3 reached a very significant level. The alcohol dehydrogenase activity of AlcoKer0031 was 2.9 times, 1.3 times, and 2.1 times that of strains 1, 2, and 3, respectively.

[0077] Example 3: Preparation method of probiotic composition tablet candy for hangover relief and liver protection

[0078] This embodiment provides a probiotic composition tablet candy for sobering up and protecting the liver and a preparation method thereof. The formula and preparation process of the tablet candy are as follows: Specification: 6 g / pack, 6 tablets per pack.

[0079] The formula is shown in Table 4.

[0080]

[0081] Preparation method:

[0082] (1) Take raw materials: Take 1.0g of Pueraria root extract and 1.0g of Hovenia dulcis extract, and add excipients including 0.5g of oligofructose, 0.5g of oligoxylose, 0.6g of maltodextrin, and 0.6g of corn oligopeptide. Use a water activity meter to test the water activity of the above raw materials and excipients (the water activity of the raw materials and excipients is required to be kept below 5%). If they do not meet the requirements, put the above materials into an oven and dry them at a low temperature of 45°C with circulating air. Take part of the raw materials and excipients for water activity testing every 1 hour until the water activity is lower than 5%;

[0083] (2) Put the Pueraria extract with a water activity of less than 5% obtained in step (1), the Hovenia dulcis extract, fructooligosaccharides, xylooligosaccharides, maltodextrin and corn oligopeptides into a stirrer, and stir at a speed of 200 r / min for 5 minutes to fully mix the raw materials and auxiliary materials to obtain a mixed material for standby use;

[0084] (3) Take 1.8 g of coagulated Weizmannella AlcoKer0031 microcapsules (live bacteria concentration 5×10 9 cfu / g) was added to the mixture obtained in step 2), and then stirred at a speed of 200 r / min for 5 minutes to make the microcapsules, raw materials and auxiliary materials evenly mixed to obtain a solid powder of the alcohol-detoxifying probiotic composition.

[0085] (4) The solid powder of the hangover-relieving probiotic bacteria described in step (3) is tableted, the ambient temperature is set to 25° C., the ambient humidity is set to 35%, the tableting punch is triangular, the pressure is set to a positive pressure greater than 200 N, and the side pressure greater than 150 N. After the solid powder in (3) is filled and adjusted to be stable, the tablet press is turned on and the tablets are collected in a clean container to obtain tablet candies.

[0086] The preparation methods of Pueraria root extract and Hovenia dulcis fruit extract are as follows: after crushing Pueraria root and Hovenia dulcis fruit respectively, 5 to 10 times the mass of 60% to 95% ethanol is added at a mass ratio of 1:1 to 1:3, the ultrasonic power is 20 to 40KHz, the water bath temperature is 50°C to 60°C, the water bath time is 2 to 3h, and the first filtrate is obtained after filtration; then 3 to 5 times the mass of 50% to 80% ethanol is added to the filter residue again, and the ultrasonic water bath is continued for 1 to 2h according to the above parameters and then filtered to obtain the second filtrate, which is mixed with the first filtrate and then the ethanol is removed by rotary evaporation, and freeze-dried to obtain the Pueraria root extract and the Hovenia dulcis fruit extract; the freeze-drying parameters are as follows: freezing at -25°C for 8 to 12 h, cold trap temperature of -60°C to -80°C, vacuum degree of 50 to 150Pa, and drying time of 20 to 24h.

[0087] Example 4: Preparation method of probiotic composition solid beverage for hangover relief and liver protection

[0088] This embodiment provides a probiotic composition solid beverage for sobering up and protecting the liver and a preparation method thereof. The formula and preparation process of the solid beverage are as follows: Specification: 6 g / bag.

[0089] The formula is shown in Table 5.

[0090]

[0091] Preparation method:

[0092] (1) Take raw materials: Take 0.7g of Pueraria root extract and 0.7g of Hovenia dulcis extract, and add 0.3g of oligofructose, 0.4g of oligoxylose, 0.6g of maltodextrin, 0.6g of corn oligopeptide and 0.3g of citric acid, and then use a dryer to perform heat drying at 90°C for 30min, and use a water activity meter to test the water activity of the above raw materials and auxiliary materials respectively (the water activity of the raw materials and auxiliary materials is required to be kept below 5%). If they do not meet the requirements, put the above materials into an oven and dry them at 45°C with circulating air at low temperature. Take part of the raw materials and auxiliary materials for water activity testing every 1h until the water activity is less than 5%;

[0093] (2) Put the Pueraria extract with a water activity of less than 5% obtained in step (1), the Hovenia dulcis extract, fructooligosaccharides, xylooligosaccharides, maltodextrin, corn oligopeptides and citric acid into a blender, and then stir at a speed of 200 r / min for 5 minutes to fully mix the raw materials and auxiliary materials to obtain a mixed material for standby use;

[0094] (3) Take 2.4 g of coagulated Weizmannella AlcoKer0031 microcapsules (live bacteria concentration 5×10 9cfu / g) was added to the mixture obtained in step (2), and then stirred at a speed of 200 r / min for 5 minutes to ensure that the probiotic powder, raw materials and auxiliary materials were evenly mixed to obtain a hangover probiotic composition solid beverage.

[0095] The preparation methods of Pueraria root extract and Hovenia dulcis fruit extract are as follows: after crushing Pueraria root and Hovenia dulcis fruit respectively, 5 to 10 times the mass of 60% to 95% ethanol is added at a mass ratio of 1:1 to 1:3, the ultrasonic power is 20 to 40KHz, the water bath temperature is 50°C to 60°C, the water bath time is 2 to 3h, and the first filtrate is obtained after filtration; then 3 to 5 times the mass of 50% to 80% ethanol is added to the filter residue again, and the ultrasonic water bath is continued for 1 to 2h according to the above parameters and then filtered to obtain the second filtrate, which is mixed with the first filtrate and then the ethanol is removed by rotary evaporation, and freeze-dried to obtain the Pueraria root extract and the Hovenia dulcis fruit extract; the freeze-drying parameters are as follows: freezing at -25°C for 8 to 12 h, cold trap temperature of -60°C to -80°C, vacuum degree of 50 to 150Pa, and drying time of 20 to 24h.

[0096] Example 5: Effect test of alcohol sobering and liver protecting products

[0097] The hangover-relieving and liver-protecting effects of the hangover-relieving and liver-protecting products in Examples 3 and 4 were tested.

[0098] 110 mice weighing 17-22 g were adaptively fed for 7 days (relative humidity of the feeding environment was 40% to 60%, temperature was 20 to 25°C), and randomly divided into blank control group, model group, and low, medium and high dose experimental groups after stratification by body weight, with 10 mice in each group. Mice in the blank control group were not treated: mice in the model group were gavaged with 52% vol liquor (10 mL / kg): the low, medium and high dose experimental groups were crushed the tablet candy obtained in Example 3 and the solid beverage obtained in Example 4, added water and prepared according to 65 g / L, 130 g / L and 195 g / L, and centrifuged at 8000 r / min at 4°C, and the supernatant was gavaged.

[0099] The mice in the low-dose experimental group were gavaged with the low-dose experimental group solution (65 g / L, 10 mL / kg) for 30 minutes, and then they were gavaged with 52% vol liquor (10 mL / kg); the mice in the medium-dose experimental group were gavaged with the medium-dose experimental group solution (130 g / L, 10 mL / kg) for 30 minutes, and then they were gavaged with 52% vol liquor (10 ml / kg); the mice in the high-dose experimental group were gavaged with the high-dose experimental group solution (195 g / L, 10 mL / kg) for 30 minutes, and then they were gavaged with 52% vol liquor (10 ml / kg).

[0100] After each group of mice was gavaged with liquor, the timing was immediately started and the righting reflex test was performed to determine whether the mice were drunk (observe whether the mice could keep their backs down for 30 seconds, if they could, they were drunk, otherwise they were not drunk), and the drunkenness time (last gavage time-righting reflex disappearance time) and sobering time (righting reflex disappearance time-righting reflex recovery time) were recorded. The results are shown in Table 6. Compared with the model group, Examples 3 and 4 significantly reduced the drunkenness and sobering time of the mice.

[0101] Each group of mice was gavaged once a day for 7 days, and the mice were killed by spinal dislocation 30 minutes and 4 hours after the last gavage. After blood was collected from the eyeball, the blood was added with anticoagulant sodium heparin and centrifuged at 4°C and 3000 r / min for 15 minutes, and the supernatant was taken and stored at -80°C for inspection. The serum ethanol content, alcohol dehydrogenase (ADH) content and acetaldehyde dehydrogenase (ALDH) content were detected at 30 minutes and 4 hours according to the ELISA kit operation steps. The results are shown in Tables 7 and 8. Compared with the model group, Examples 3 and 4 significantly reduced the alcohol content in the serum of mice 0.5 h and 4 h after alcohol gavage; compared with the model group, Examples 3 and 4 significantly increased the content of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the serum of mice after alcohol gavage, indicating that the alcohol-detoxifying probiotic composition of the present invention has a significant effect of accelerating ethanol metabolism.

[0102] The liver of the mouse sacrificed 4 hours later was taken, rinsed with ice saline, wiped dry, weighed, and photographed. Some of the liver tissues were fixed with paraformaldehyde (4%), embedded in paraffin, and sliced ​​(5 μm). The slices were stained with hematoxylin-eosin (HE), and the liver lesions were observed under a light microscope ( Figure 1 The degree of liver tissue damage was measured by referring to the naked eye observation method and the fatty degeneration scoring standard. Figure 1 As shown, compared with the model group, the degree of fatty degeneration in Example 3 and Example 4 was significantly lower than that in the model group, indicating that the alcohol-detoxifying probiotic composition of the present invention has the effect of significantly reducing alcoholic liver damage.

[0103] The data results were expressed as “mean + standard deviation”, and SPSS 26.0 software was used to perform one-way analysis of variance (ANOVA) on the data. p <0.05 was used as the basis to judge whether the difference was statistically significant.

Claims

1. Weizmannella coagulans ( Weizmannia coagulans ) A novel use of AlcoKer0031 in the fermentation preparation of alcohol dehydrogenase, characterized in that: The coagulant Weizmannella AlcoKer0031 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.31222, and the deposit date: July 8, 2024; Weizmannella coagulans AlcoKer0031 was inoculated into an MRS liquid culture medium containing 52% ethanol and cultured at 37°C for 16 h; the preparation method of the MRS liquid culture medium was as follows: 15.0 g of glucose, 10.0 g of peptone, 4.0 g of yeast powder, 4.0 g of beef extract powder, 5.0 g of sodium acetate, and 2.0 g of dipotassium hydrogen phosphate, 1000 mL of distilled water was added, the pH was adjusted to 5.5-6.0 with 2 mol / L sodium hydroxide solution or 2 mol / L lactic acid solution, heated to dissolve, dispensed into conical flasks, and sterilized under high pressure at 121°C for 15 min-20 min.

Images