New application of acetobacter pasteurianus CGMCC No.3089 and alcoholism-reducing and alcoholism-relieving fungicide

Abstract

The invention belongs to the technical field of microorganisms, and relates to new application of acetobacter pasteurianus CGMCC No.3089 and an alcoholism-reducing and alcoholism-relieving bacterial agent. The strain has strong alcohol tolerance and acid resistance, can survive well in a human gastric juice environment, and can degrade alcohols in the stomach. The strain can effectively degrade miscellaneous oleyl alcohol and ethanol in vivo, can effectively eliminate headache and drunkenness of drinkers, has remarkable alcoholism-reducing and alcoholism-relieving functions, and can be used for preparing alcoholism-relieving and liver-protecting products. The strain and the prepared hangover-alleviating and liver-protecting microbial agent can also be combined with other auxiliary materials or medicinal and edible food materials to prepare medicines, health-care products or foods and the like, and are applied to the field of hangover-alleviating and liver-protecting. The product is natural and safe, has a probiotic effect, and can reduce the damage of alcohol to the liver and improve the metabolic capability of a human body. The invention has great market potential and application prospect, and can effectively meet the requirements of hangover-alleviating and liver-protecting products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a new application of Acetobacter pasteurianus CGMCC No.3089 and an alcohol-reducing and alcohol-detoxifying bacterial agent. Background Art

[0002] Alcohol has been a part of Chinese civilization for 5,000 years. It plays an important role in people's lives and is an indispensable part of their daily lives and social life. Moderate drinking is good for health, but acute drinking can cause adverse symptoms and, in severe cases, damage to multiple organs in the body, especially the liver.

[0003] Acetic acid bacteria is an important microorganism widely used in vinegar fermentation. Its main function is to oxidize ethanol, carbohydrates and sugar alcohols to produce corresponding products, among which the most important is to oxidize ethanol to acetic acid. In addition, acetic acid bacteria also have the ability to promote stomach ethanol oxidation, regulate liver lipid metabolism, and regulate the body's oxidative stress, so it has significant potential in alcohol detoxification and liver protection. Existing patents such as CN116687978A, CN116751722A, and CN116144550A disclose the use of Acetobacter pasteurianus in alcohol detoxification agents.

[0004] The existing technology mainly uses acetic acid bacteria or acetic acid bacteria mixture to degrade ethanol to achieve the purpose of sobering up and protecting the liver. However, although these methods can degrade ethanol to a certain extent, they do not show ideal degradation effects on higher alcohols in alcoholic products, and it is difficult to effectively eliminate headaches and drunkenness in drinkers. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a new application of Acetobacter pasteurianus CGMCC No.3089. The strain has a certain alcohol tolerance, can effectively degrade miscellaneous oleyl alcohol and ethanol in the body, and can be used to prepare alcohol-detoxifying and liver-protecting products. The strain and the alcohol-detoxifying and liver-protecting bacterial agent prepared can also be used in combination with other auxiliary materials or medicinal and edible ingredients to prepare medicines, health products, foods, etc., which are natural and safe and have a probiotic effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the use of Acetobacter pasteurianus CGMCC No.3089 in degrading miscellaneous alcohols and ethanol.

[0007] Fusel alcohols, also known as higher alcohols in the alcohol and liquor industry, are byproducts of the winemaking process and are a general term for monohydric alcohols with more than three carbon atoms, of which isopentanol and isobutyl alcohol are the most toxic to the human body. Fusel oils oxidize more slowly in the body than ethanol and last longer. They are irritating to the eyes and nose, can cause congestion in the central nervous system, and can cause headaches, dizziness, nausea, vomiting, and diarrhea in drinkers.

[0008] The Acetobacter pasteurianus involved in the present invention ( Acetobacter pasteurianus ) CGMCC No.3089 has been published in "The Evolutionary Response of Alcohol Dehydrogenase and AldehydeDehydrogenases of Acetobacter pasteurianus CGMCC 3089 to Ethanol Adaptation" (Yu Zheng et al., Food Sci. Biotechnol. 24(1): 133-140 (2015)). Compared with other acetic bacteria, this strain has strong environmental adaptability, can tolerate 9% ethanol concentration, and can survive in human gastric juice. This characteristic ensures that it can play a role in sobering up in the stomach after entering the human body. At the same time, in addition to the ability of acetic bacteria to convert ethanol into acetic acid, this strain also has a variety of alcohol dehydrogenases and aldehyde dehydrogenases, which can use other higher alcohols as substrates and oxidize them into corresponding acid compounds. It has been reported that this strain can effectively reduce the content of miscellaneous alcohols in wine when fermented with brewer's yeast in sequence.

[0009] In a second aspect, the present invention also provides the use of Acetobacter pasteurianus CGMCC No.3089 in the preparation of a hangover-relieving and liver-protecting product; the active ingredient in the hangover-relieving product is the Acetobacter pasteurianus CGMCC No.3089 and / or a metabolite of Acetobacter pasteurianus CGMCC No.3089.

[0010] The products include but are not limited to medicines, health products, and food.

[0011] In a third aspect, the present invention further provides an alcohol-reducing and alcohol-detoxifying bacterial agent, comprising Acetobacter pasteurianus CGMCC No. 3089 and a biologically acceptable microbial agent carrier and / or auxiliary material.

[0012] The alcohol-reducing and alcohol-detoxifying bacterial agent provided by the present invention can degrade ethanol and miscellaneous alcohols in alcohol in the gastrointestinal tract of the human body, and at the same time increase the activity of liver ADH enzyme, prolong the time of drunkenness, shorten the sobering time and alleviate the discomfort after drinking. The time of taking can be any time before, during or after drinking.

[0013] The active microorganism contained in the bacterial agent can be Acetobacter pasteurianus CGMCC No. 3089 or a probiotic composition containing Acetobacter pasteurianus CGMCC No. 3089. In the probiotic composition, in addition to Acetobacter pasteurianus CGMCC No. 3089, other probiotics can be selected from probiotics with alcohol sobering effects (e.g., Bifidobacterium bifidum, Lactobacillus plantarum) or probiotics that can assist Acetobacter pasteurianus CGMCC No. 3089 in sobering up (e.g., Lactobacillus plantarum, Lactobacillus helveticus).

[0014] The alcohol-reducing and hangover-relieving bacterial agent provided by the present invention has dosage forms including but not limited to liquid, powder, granule, tablet and capsule. By selecting suitable carriers and / or excipients to prepare bacterial agents in different dosage forms, the stability, activity and use effect of the bacterial agent can be improved to meet different use requirements.

[0015] Preferably, the degrading high-alcohol hangover relieving agent is solid bacterial powder, and the auxiliary material is a drying protective agent; the drying protective agent is one or more combinations of trehalose, Hovenia dulcis powder, Rosa roxburghii powder, wolfberry juice, maltodextrin, skim milk powder, mannitol and Pueraria root powder.

[0016] The solid bacterial powder can be easily carried and stored, and the solid bacterial powder can be easily used to prepare pills, tablets or other products.

[0017] Hovenia dulcis, Rosa roxburghii, Lycium barbarum, and Pueraria root are both medicinal and edible materials with natural safety. They can slow down the gastrointestinal absorption of ethanol and thus accelerate the metabolism of ethanol. The polysaccharide components they contain can also protect the bacteria. When added to the bacterial agent as a protective agent, they can sober up and protect the liver. They can also reduce the death of bacteria during drying processes, improve cell survival rate and strain activity, and prolong the survival time of acetic acid bacteria in gastric juice.

[0018] Preferably, Lactobacillus plantarum and / or Lactobacillus helveticus are also included.

[0019] Acetobacter pasteurianus can quickly oxidize alcohol into acetaldehyde, and then further oxidize it into acetic acid. Lactobacillus plantarum can metabolize organic acids such as acetic acid, and at the same time produce lactic acid in the body, reducing the accumulation of organic acids in the body and helping to maintain acid-base balance. These two strains have complementary functions in the metabolic process and can accelerate the metabolism of alcohol.

[0020] More preferably, the ratio of the viable counts of Acetobacter pasteurianus CGMCC No. 3089 to Lactobacillus plantarum is 1:10-100. Optionally, Lactobacillus plantarum is Lactobacillus plantarum CGMCC 21791.

[0021] When Lactobacillus helveticus is co-cultured with Acetobacter pasteurianus CGMCC No.3089, Lactobacillus helveticus can promote the growth of Acetobacter pasteurianus CGMCC No.3089 and stimulate the enzyme activity of Acetobacter pasteurianus, enabling it to decompose and utilize alcohol more effectively.

[0022] More preferably, the Lactobacillus helveticus is Lactobacillus helveticus CMCC 1.1877 or Lactobacillus helveticus CGMCC 12062.

[0023] Acetobacter pasteurianus CGMCC No.3089 and Lactobacillus plantarum and / or Lactobacillus helveticus can be separately cultured and then mixed to prepare the alcohol-lowering and alcohol-detoxifying bacterial agent, or can be prepared into the alcohol-lowering and alcohol-detoxifying bacterial agent by co-culturing.

[0024] More preferably, the Acetobacter pasteurianus CGMCC No.3089 and Lactobacillus plantarum are separately cultured in pure form and then mixed.

[0025] More preferably, the Acetobacter pasteurianus CGMCC No. 3089 is co-cultured with Lactobacillus helveticus.

[0026] Preferably, the Acetobacter pasteurianus CGMCC No.3089 is cultured in a culture medium containing 2.5%-15% lactic acid bacteria cell-free supernatant for 12-36 hours.

[0027] The lactic acid bacteria cell-free supernatant is a cell-free lactic acid supernatant obtained by separating the activated bacterial solution of lactic acid bacteria. The lactic acid bacteria fermentation solution can increase the number of live bacteria and enzyme activity of Acetobacter pasteurianus CGMCC No.3089 and accelerate its oxidation ability to alcohols.

[0028] Exemplarily, the present invention provides a method for preparing a lactic acid bacteria cell-free supernatant, comprising the following steps: The activated bacterial solution of lactic acid bacteria was centrifuged at 8000-10000 r / min for 10-15 min, and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a lactic acid bacteria cell-free supernatant.

[0029] More preferably, the Acetobacter pasteurianus CGMCC No.3089 is cultured in a culture medium containing 5%-15% lactic acid bacteria cell-free supernatant for 16-24 hours.

[0030] More preferably, the lactic acid bacteria is Lactobacillus helveticus.

[0031] Optionally, the Lactobacillus helveticus is Lactobacillus helveticus CMCC 1.1877 or Lactobacillus helveticus CGMCC 12062.

[0032] In a fourth aspect, the present invention also provides a method for preparing an alcohol-reducing and alcohol-detoxifying bacterial powder, the preparation method comprising mixing a live bacterial sludge containing Acetobacter pasteurianus CGMCC No.3089 with a drying protective agent and then performing a drying treatment, or preparing a bacterial suspension of the live bacterial sludge and mixing it with a drying protective agent and then performing a drying treatment to obtain an alcohol-reducing and alcohol-detoxifying bacterial powder containing Acetobacter pasteurianus.

[0033] Exemplarily, the present invention provides a method for preparing alcohol-reducing and alcohol-degrading bacterial powder, comprising the following steps: S1: Pick a single colony of Acetobacter pasteurianus CGMCC No.3089 and inoculate it into the seed culture medium, culture it at 30-37°C for 16-18h, subculture it 2-3 times, and obtain an activated bacterial solution.

[0034] S2: The activated bacterial solution in S1 is transferred to the culture medium at an inoculation rate of 1%-5% by weight for expansion culture to obtain a fermentation liquid; the conditions for the expansion culture are: 30-37°C, 160-200 rpm, and culture for 12-36 h.

[0035] S3: The fermentation broth in S2 was centrifuged at 4000-8000 rpm for 10-30 min at 4°C, and then washed twice with sterile 0.9% saline to collect the bacterial sludge.

[0036] S4: The bacterial sludge in S3 is made into a bacterial suspension, which is then evenly mixed with a drying protective agent and dried to obtain Acetobacter pasteurianus powder, i.e., alcohol-reducing and alcohol-degrading single bacteria powder.

[0037] Furthermore, the culture medium of S2 contains 2.5%-15% lactic acid bacteria cell-free supernatant; Furthermore, the lactic acid bacteria cell-free supernatant is Lactobacillus helveticus cell-free supernatant.

[0038] Furthermore, the conditions for the expanded culture in S2 are: 30° C., 180 rpm for 18 h.

[0039] Furthermore, the centrifugation condition in S3 is 6000 rpm at 4°C for 10 min.

[0040] The preparation method provided by the present invention further comprises mixing the probiotic live bacteria mud with the effect of sobering up and / or the probiotic live bacteria mud with the effect of assisting Acetobacter Pasteurellii CGMCC No.3089 in sobering up with a drying protective agent, performing drying treatment, and then mixing with the Acetobacter Pasteurellii powder.

[0041] Exemplarily, the present invention provides a method for preparing alcohol-reducing and alcohol-resolving mixed bacteria powder, comprising the following steps: S1: Pick a single colony of Acetobacter pasteurianus CGMCC No.3089 and inoculate it into a liquid culture medium, culture it at 30-37°C for 16-18 h, and subculture it 2-3 times to obtain an activated bacterial solution of Acetobacter pasteurianus; transfer the activated bacterial solution of Acetobacter pasteurianus to the culture medium at an inoculation rate of 1%-5% by weight, culture it at 30-37°C and 160-200 rpm for 12-36 h to obtain a fermentation solution of Acetobacter pasteurianus; centrifuge the fermentation solution of Acetobacter pasteurianus and collect the bacterial mud of Acetobacter pasteurianus CGMCC No.3089.

[0042] S2: Evenly mix the Acetobacter pasteurianus CGMCC No.3089 bacterial mud and the drying protective agent, and perform drying treatment to obtain Acetobacter pasteurianus CGMCC No.3089 bacterial powder.

[0043] S3: Pick a single colony of Lactobacillus plantarum and inoculate it into a liquid culture medium, culture it at 37° C. for 16-18 h, and subculture it 2-3 times to obtain an activated Lactobacillus plantarum bacterial solution; transfer the activated Lactobacillus plantarum bacterial solution to a liquid culture medium at an inoculation rate of 1%-5% by weight, culture it at 30-37° C. and 160-200 rpm for 16-18 h to obtain a Lactobacillus plantarum fermentation liquid; centrifuge the Lactobacillus plantarum fermentation liquid and collect the Lactobacillus plantarum bacterial mud.

[0044] S4: Evenly mix the Lactobacillus plantarum mud and the drying protective agent, and dry them to obtain the Lactobacillus plantarum powder.

[0045] S5: Mix the Acetobacter pasteurianus CGMCC No.3089 powder obtained in S2 with the Lactobacillus plantarum powder obtained in S4, wherein the ratio of live bacteria of Acetobacter pasteurianus CGMCC No.3089 to Lactobacillus plantarum is 1:10-100, to obtain alcohol-reducing and alcohol-detoxifying mixed bacteria powder.

[0046] Exemplarily, the present invention provides a method for preparing alcohol-reducing and alcohol-resolving mixed bacteria powder, comprising the following steps: S1: Pick a single colony of Acetobacter pasteurianus CGMCC No.3089 and inoculate it into liquid culture medium, culture it at 30-37℃ for 16-18 h, and subculture it 2-3 times to obtain activated bacterial liquid of Acetobacter pasteurianus.

[0047] S2: Pick a single colony of Lactobacillus helveticus and inoculate it into MRS liquid culture medium at 37°C, 180 rpm for 16-18 h, subculture 2-3 times, and obtain activated bacterial solution of Lactobacillus helveticus.

[0048] S3: The activated bacterial solution of Acetobacter pasteurianus and the activated bacterial solution of Lactobacillus helveticus are transferred to the culture medium at an inoculation rate of 1%-5% by weight, respectively, and cultured at 30-37° C. and 160-200 rpm for 16-18 h to obtain a mixed fermentation solution; the mixed fermentation solution is centrifuged to collect the mixed bacterial mud.

[0049] S4: Evenly mix the mixed bacteria mud and the drying protective agent, and perform drying treatment to obtain mixed bacteria powder containing Acetobacter pasteurianus, that is, the alcohol-reducing and alcohol-detoxifying mixed bacteria powder.

[0050] The drying treatment method can be selected according to the tolerance of the strains involved in the bacterial powder and the production requirements, taking into account factors such as cost, efficiency and bacterial activity. The optional methods include but are not limited to freeze drying, spray drying, and fluidized bed drying.

[0051] Preferably, the drying treatment method is spray drying, and the drying protective agent is one or more combinations of trehalose, Hovenia dulcis powder, Rosa roxburghii powder, wolfberry juice, maltodextrin, skim milk powder, mannitol and Pueraria root powder.

[0052] The drying protective agent can be directly added to the bacterial mud suspension to form a suspension system, or the drying protective agent can be dissolved in water and mixed with the bacterial mud or the bacterial mud suspension to form a suspension system, and then dried. The bacterial mud suspension can be prepared by mixing physiological saline with an equal volume of the mixed bacterial fermentation liquid.

[0053] More preferably, the final concentration of each component in the drying protective agent in the suspension is 2 wt %-10 wt %.

[0054] Optionally, the drying protective agent is a combination of wolfberry juice, maltodextrin, skim milk powder and mannitol. The final concentration of wolfberry juice in the suspension is 2wt%; the final concentration of maltodextrin in the suspension is 5wt%; the final concentration of skim milk powder in the suspension is 5wt%; and the final concentration of mannitol in the suspension is 2.5wt%.

[0055] Optionally, the drying protective agent is a combination of trehalose, Hovenia dulcis powder and Rosa roxburghii powder. The final concentration of trehalose in the suspension is 5wt%, the final concentration of Hovenia dulcis powder in the suspension is 2.5wt%, and the final concentration of Rosa roxburghii powder in the suspension is 2.5wt%.

[0056] Optionally, the drying protective agent is a combination of trehalose, roxburghii powder and kudzu root powder. The final concentration of trehalose in the suspension is 5wt%; the final concentration of roxburghii powder in the suspension is 3wt%; and the final concentration of kudzu root powder in the suspension is 3wt%. The drying protective agent can be used to protect Lactobacillus plantarum.

[0057] In a fifth aspect, the present invention further provides a hangover-relieving and liver-protecting drug, comprising Acetobacter pasteurianus CGMCC No.3089, a probiotic combination comprising Acetobacter pasteurianus CGMCC No.3089, the above-mentioned alcohol-reducing and alcohol-relieving bacterial agent, or the alcohol-reducing and alcohol-relieving bacterial agent prepared by the above-mentioned preparation method.

[0058] The alcohol-detoxifying and liver-protecting drug can also be added with suitable carriers or excipients to prepare different preparation forms such as granules, tablets and capsules acceptable to humans. The above-provided bacterial agent can be used to prepare an oral preparation to prolong the survival time of Acetobacter pasteurianus CGMCC No.3089 in gastric juice, facilitate carrying and improve compliance.

[0059] Illustratively, the present invention provides a hangover and liver protection tablet, comprising the above-mentioned alcohol-reducing and hangover-resolving bacteria agent or the alcohol-reducing and hangover-resolving bacteria agent prepared by the above-mentioned preparation method and auxiliary materials; the auxiliary materials are one or more combinations of fillers, binders, disintegrants, lubricants and flavoring agents.

[0060] In a sixth aspect, the present invention further provides a hangover-degrading and liver-protecting composite food, which comprises Acetobacter pasteurianus CGMCC No.3089, a probiotic combination comprising Acetobacter pasteurianus CGMCC No.3089, the above-mentioned alcohol-degrading bacterial agent for degrading higher alcohol or the alcohol-degrading bacterial agent for degrading alcohol prepared by the above-mentioned preparation method, and food ingredients having the same origin as medicine.

[0061] Food and medicine ingredients are not only safe for consumption but also have certain medicinal effects. They can be used together with Acetobacter pasteurianus CGMCC No.3089 to make alcohol-detoxifying and liver-protecting foods, which can fill the gap in alcohol-detoxifying and liver-protecting foods on the market.

[0062] Preferably, the food and medicine ingredients are food and medicine ingredients with alcohol sobering and liver protecting effects, including but not limited to Pueraria root, Hovenia dulcis fruit and turmeric.

[0063] The hangover-relieving and liver-protecting composite food provided by the present invention may also include prebiotics or postbiotics, such as isomaltooligosaccharide, fructooligosaccharide, stachyose, galacto-oligosaccharide and mannose oligosaccharide. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0065] The strains used in the following examples of the present invention are derived from the following: Acetobacter pasteurianus ( Acetobacter pasteurianus ) CGMCC No.3089, provided by China Microbiological Culture Collection Center (CGMCC), this strain has been used in "The Evolutionary Response of AlcoholDehydrogenase and Aldehyde Dehydrogenases of Acetobacter pasteurianus CGMCC3089 to Ethanol Adaptation” (Yu Zheng et al., Food Sci. Biotechnol. 24(1): 133-140(2015)).

[0066] Acetobacter pasteurianus ( Acetobacter pasteurianus )AS 1.41, purchased from Shanghai Collection Biotechnology Center.

[0067] Acetobacter pasteurianus ( Acetobacter pasteurianus ) CICC 20001, purchased from China Industrial Microbiological Culture Collection Center. This strain has been disclosed in the patent application publication number CN 109401908A, entitled "A Youth Anti-fatigue Functional Fruit Vinegar Beverage and Its Preparation Method".

[0068] Acetobacter pasteurianus ( Acetobacter pasteurianus ) BNCC 335801, purchased from Beijing Beina Chuanglian Biotechnology Research Institute. This strain has been disclosed in the patent application publication number CN 116687978 A, entitled "An alcohol detoxification agent, its preparation method and application".

[0069] Lactobacillus helveticus ( Lactobacillus helveticus ) CGMCC 12062, provided by China Microbiological Culture Collection Center. This strain has been used in “Interaction of acetic acid bacteria and lactic acid bacteria in multispecies solid-state fermentation of traditional Chinese cereal vinegar” (Menglei Xia et al., Frontiers in Microbiology. (2022.9.29)).

[0070] Lactobacillus helveticus ( Lactobacillus helveticus ) CGMCC 1.1877, provided by China Microbial Culture Collection Center. This strain has been used in "Study on the Interaction between Lactic Acid Bacteria and Acetic Acid Bacteria in the Acetic Acid Fermentation Stage of Shanxi Old Vinegar" (Mu Jun et al., Master's thesis . (2018.6)).

[0071] Lactobacillus plantarum ( Lactiplantibacillus plantarum ) CICC 21791, provided by China Microbiological Culture Collection Center. This strain has been disclosed in the patent application publication number CN 110669632 A, entitled "A kind of alcohol-detoxifying vinegar rich in probiotics and its preparation method and use".

[0072] The culture medium used in the following examples of the present invention is as follows: GY medium (g / L): 20 g glucose, 15 g yeast extract, distilled water to 1 L, pH natural. For solid medium, 20 g agar powder should be added, but for liquid medium, no agar powder should be added, and sterilized at 121°C for 20 min.

[0073] MRS medium (g / L): 20 g glucose, 10 g peptone, 10 g beef extract, 5 g yeast extract, 5 g anhydrous sodium acetate, 2 g ammonium citrate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1 mL Tween 80, 1000 mL distilled water, pH adjusted to 6.2-6.8. For solid medium, 20 g agar powder should be added, but not for liquid medium, and sterilized at 121°C for 20 min.

[0074] Seed culture medium, expansion culture medium (g / L): 10 g wolfberry juice, 10 g glucose, 15 g yeast extract, distilled water to 1 L, natural pH. Solid culture medium needs to add 20 g agar powder on this basis, but liquid culture medium does not need to add it.

[0075] Detection method for the number of live bacteria in the bacterial agent: adopt the national standard "GB4789.2-2022 National Food Safety Standard Food Microbiology Inspection Total Colony Count Determination".

[0076] PBS: 8.0 g sodium chloride, 0.2 g potassium chloride, 1.42 g sodium dihydrogen phosphate, 0.27 g potassium dihydrogen phosphate, dissolved in deionized water and adjusted to 1 L, pH adjusted to 7.4.

[0077] Artificial gastric juice: sterile PBS, 0.35% pepsin, adjusted to pH 3.0 with hydrochloric acid, and sterilized by filtration with a 0.22 µm filter membrane.

[0078] Example 1 Single colonies of Acetobacter pasteurianus CGMCC No.3089, Acetobacter pasteurianus AS 1.41, Acetobacter pasteurianus CICC 20001, and Acetobacter pasteurianus BNCC 335801 were inoculated into GY liquid medium at 30°C, 180 rpm / min for 18 h, and subcultured at an inoculum size of 2%. The subculture was repeated twice to obtain activated bacterial solution 1, activated bacterial solution 2, activated bacterial solution 3, and activated bacterial solution 4, respectively.

[0079] 1. Acetobacter pasteurianus ( Acetobacter pasteurianus )CGMCC No.3089 has the ability to degrade higher alcohols and ethanol efficiently The bacterial concentration of activated bacterial solution 1 was adjusted to 1.0×10 8 CFU / mL, 5.0×108 CFU / mL, 1.0×10 9 CFU / mL, 5.0×10 9 CFU / mL, 1.0×10 10 CFU / m. Take 5 mL of the activated bacterial solution 1 with different bacterial concentrations and add it to the mixture of artificial gastric juice and liquor, and record the content of ethanol, isoamyl alcohol and isobutanol in the simulated gastric juice after 1 hour. The results are shown in Table 1.

[0080] Table 1 Comparison of the ability of Acetobacter pasteurianus to degrade ethanol and miscellaneous alcohols

[0081] It can be seen that the Acetobacter pasteurianus CGMCC No. 3089 of the present invention can effectively reduce the content of harmful alcohols such as ethanol, isoamyl alcohol and isobutanol in the mixed solution, and as the number of live bacteria of Acetobacter pasteurianus CGMCC No. 3089 increases, the content of the three alcohol substances shows a downward trend. This shows that Acetobacter pasteurianus CGMCC No. 3089 has the ability to significantly degrade ethanol and miscellaneous alcohols, and is dependent on the bacterial amount. When the bacterial concentration is 1.0×10 10 CFU / mL has the best alcohol-lowering effect.

[0082] 2. Comparison of the ability of Acetobacter pasteurianus to degrade higher alcohols and ethanol Adjust the bacterial concentration of activated bacterial solution 1-4 to 1.0×10 10 CFU / mL. Take 100 mL of artificial gastric juice and 50 mL of bulk liquor with an alcohol content of 56 degrees and mix them well. Then add 5 mL of activated bacterial solution 1-4. After mixing well, let it stand at room temperature. Record the survival rate of Acetobacter pasteurianus, ADH specific enzyme activity, ALDH specific enzyme activity, and ethanol, isoamyl alcohol and isobutanol content after 1 hour. And use an equal dose of PBS buffer as a blank control. The results are shown in Table 2.

[0083] Table 2 Comparison of the ability of Acetobacter pasteurianus to degrade ethanol and miscellaneous alcohols

[0084] From the data in Table 2, it can be seen that Acetobacter pasteurianus CGMCC No.3089 has better ethanol tolerance and gastric acid tolerance than other Acetobacters, shows excellent survival rate in the mixture of artificial gastric juice and white wine, and can significantly reduce the content of ethanol, isopentanol and isobutanol in the mixture.

[0085] 3. Effect of Lactobacillus helveticus fermentation broth on the alcohol-degrading ability of Acetobacter pasteurianus CGMCC No.3089 A single colony of Lactobacillus helveticus CGMCC 12062 was selected and inoculated into MRS liquid medium at 37°C and 180 rpm for 18 h, and then subcultured at an inoculation rate of 2%. The subculture was repeated twice to obtain an activated bacterial solution. The bacterial solution was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken and filtered through a 0.22 μm sterile filter membrane to obtain a cell-free lactic acid supernatant. The activated bacterial solution 1 was inoculated into GY liquid medium containing 2.5%, 5%, 10%, and 15% cell-free lactic acid supernatant at an inoculation rate of 2%, and cultured at 30°C and 180 rpm for 18 h until the early stage of the stable phase to obtain activated bacterial solution 5, activated bacterial solution 6, activated bacterial solution 7, and activated bacterial solution 8.

[0086] The number of live bacteria in activated bacterial solution 1 and activated bacterial solution 5-8 was detected, and then 5 mL of activated bacterial solution 1 and activated bacterial solution 5-8 were added to the mixture of artificial gastric juice and liquor, and the ADH specific enzyme activity, ALDH specific enzyme activity, and ethanol, isopentanol, and isobutanol contents in the simulated gastric juice were recorded 1 hour later. The results are shown in Table 3.

[0087] Table 3 Effects of Lactobacillus helveticus fermentation supernatant on Acetobacter pasteurianus CGMCC No.3089

[0088] From the data in Table 3, it can be seen that different concentrations of Lactobacillus helveticus supernatant have a certain promoting effect on the growth and alcohol-reducing ability of acetic acid bacteria. With the increase of the amount of supernatant added, its promoting effect first increases and then weakens. When the amount of lactic acid bacteria added is 5%, its promoting effect is most obvious.

[0089] Example 2 This embodiment provides a monosaccharide powder for reducing alcohol and relieving alcohol, which is prepared by the following steps: (1) Pick a single colony of Acetobacter pasteurianus CGMCC No.3089 and inoculate it into the seed culture medium. Cultivate it at 180 rpm and 30°C for 18 h. Subculture it twice to obtain an activated bacterial solution.

[0090] (2) The activated bacterial liquid was transferred to the expansion medium at an inoculum rate of 2% by weight, and cultured at 30°C and 180 rpm for 20 h to obtain the fermentation liquid; the fermentation liquid was then centrifuged at 6000 rpm for 10 min at 4°C, washed twice with sterile 0.9% saline, and the bacterial sludge was collected.

[0091] (3) Using a suspension of bacterial slurry with a volume of wolfberry juice equal to that of the fermentation liquid, a drying protective agent was added and mixed thoroughly to form a suspension; the drying protective agent was a combination of wolfberry juice, maltodextrin, skim milk powder and mannitol, and the final concentration of wolfberry juice in the suspension was 2wt%, the final concentration of maltodextrin in the suspension was 5wt%, the final concentration of skim milk powder in the suspension was 5wt%, and the final concentration of mannitol in the suspension was 2.5wt%.

[0092] (4) The suspension was spray dried. The spray drying parameters were as follows: inlet temperature: 110 °C, outlet temperature: 60 °C, fan frequency: 60 Hz, striker interval: 5 s, execution time: 0.3 s, and feed rate: 500 mL / h.

[0093] The viable count of Acetobacter pasteurianus CGMCC No.3089 in the alcohol-reducing and alcohol-detoxifying bacteria powder was 4.49×10 10 CFU / g.

[0094] Example 3 This embodiment provides a monosaccharide powder for reducing alcohol and relieving alcohol, which is prepared by the following steps: (1) Pick a single colony of Acetobacter pasteurianus CGMCC No.3089 and inoculate it into GY liquid medium. Cultivate it at 160 rpm and 30℃ for 16 h. Subculture it three times to obtain an activated bacterial solution.

[0095] (2) The activated bacterial liquid was transferred to GY liquid culture medium at an inoculum rate of 2% by weight, and cultured at 30°C and 160 rpm for 18 h to obtain fermentation liquid; the fermentation liquid was then centrifuged at 6000 rpm for 10 min at 4°C, washed twice with sterile 0.9% saline, and the bacterial sludge was collected.

[0096] (3) Suspending bacterial sludge with physiological saline of an equal volume to the fermentation liquid to obtain a bacterial suspension; suspending trehalose, Hovenia dulcis powder, and Rosa roxburghii powder in water to obtain a drying protection solution; and uniformly mixing the bacterial suspension with the drying protection solution to obtain a suspension, wherein the final concentration of trehalose in the suspension is 5 wt %, the final concentration of Hovenia dulcis powder is 2.5 wt %, and the final concentration of Rosa roxburghii powder is 2.5 wt %.

[0097] (4) The suspension was spray dried. The spray drying parameters were as follows: inlet temperature: 130 °C, outlet temperature: 65 °C, fan frequency: 60 Hz, striker interval: 5 s, execution time: 0.3 s, and feed rate: 250 mL / h.

[0098] The number of viable bacteria of Acetobacter pasteurianus CGMCC No.3089 in the alcohol-reducing and alcohol-detoxifying bacteria powder was 5.23×10 10 CFU / g.

[0099] Example 4 This embodiment provides a monosaccharide powder for reducing alcohol and detoxifying alcohol. Compared with Embodiment 3, the difference is that in step 2, the activated bacterial liquid is transferred to a GY liquid culture medium containing 5% Lactobacillus helveticus cell-free supernatant at an inoculum rate of 2% by weight.

[0100] Preparation method of Lactobacillus helveticus cell-free supernatant: Pick a single colony of Lactobacillus helveticus and inoculate it into MRS liquid culture medium, culture it at 37°C and 180 rpm for 18 h to obtain an activated bacterial solution. Subculture it at an inoculation volume of 2%, repeat the subculture twice, and obtain an activated bacterial solution. Transfer the activated bacterial solution to MRS liquid culture medium at an inoculation volume of 2% by weight, culture it at 37°C and 180 rpm for 18 h, and repeat the subculture twice. Centrifuge the bacterial solution at 10,000 rpm for 10 min, and take the supernatant to pass through a 0.22 μm sterile filter membrane to obtain Lactobacillus helveticus cell-free supernatant.

[0101] The viable count of Acetobacter pasteurianus CGMCC No.3089 in the alcohol-reducing and alcohol-detoxifying bacteria powder was 6.67×10 10 CFU / g.

[0102] Example 5 The present embodiment provides a mixed bacterial powder for reducing alcohol and relieving alcohol, which is prepared by mixing single bacterial powder of Acetobacter pasteurianus CGMCC No.3089 and single bacterial powder of Lactobacillus plantarum in a mass ratio of 1:1.

[0103] The preparation method of single bacterial powder of Acetobacter pasteurianus CGMCC No.3089 is the same as that in Example 3.

[0104] The plant lactobacillus single bacterial powder is prepared by the following steps: (1) Pick a single colony of Lactobacillus plantarum CGMCC 21791 and inoculate it into MRS liquid culture medium. Culture it at 37°C for 18 h and subculture it three times to obtain an activated bacterial solution.

[0105] (2) Transfer the activated bacterial liquid to MRS liquid culture medium at a 2% inoculum volume and culture at 30°C and 180 rpm for 18 h until the early stage of the stabilizer. Centrifuge at 6000 rpm for 10 min at 4°C to collect the bacterial sludge and wash it twice with 0.9% saline.

[0106] (3) Suspending bacterial sludge with physiological saline of an equal volume to the fermentation liquid to obtain a bacterial suspension; suspending trehalose, roxburghii powder, and kudzu root powder in water to obtain a drying protection solution; and uniformly mixing the bacterial suspension with the drying protection solution to obtain a suspension, wherein the final concentration of trehalose in the suspension is 5wt%, the final concentration of roxburghii powder is 3wt%, and the final concentration of kudzu root powder is 3wt%.

[0107] (4) The suspension was spray dried. The spray drying parameters were as follows: inlet temperature of 120 °C, outlet temperature of 60 °C, fan frequency of 60 Hz, striker interval of 5 s, execution time of 0.3 s, and feed rate of 250 mL / h to obtain Lactobacillus plantarum single bacterial powder.

[0108] The number of viable Lactobacillus plantarum CGMCC 21791 in the Lactobacillus plantarum single bacterial powder was tested to be 4.86×10 12 CFU / g.

[0109] Example 6 This embodiment provides an alcohol-reducing and alcohol-resolving mixed bacteria powder. Compared with Example 3, the difference is that the activated bacterial liquid of Acetobacter pasteurianus CGMCC No.3089 and the activated bacterial liquid of Lactobacillus helveticus CMCC 1.187 are both transferred to the GY liquid culture medium at an inoculation rate of 2% by weight for co-cultivation.

[0110] Preparation method of activated bacterial solution of Lactobacillus helveticus CMCC 1.187: pick a single colony of Lactobacillus helveticus CMCC 1.187 and inoculate it into MRS liquid culture medium at 37°C and 180 rpm for 18 h, subculture it at an inoculation rate of 2%, repeat the subculture twice, and obtain an activated bacterial solution.

[0111] The number of viable bacteria of Acetobacter pasteurianus CGMCC No.3089 in the alcohol-reducing and alcohol-detoxifying mixed bacteria powder was detected to be 6.50×10 10 CFU / g, the viable count of Lactobacillus helveticus CMCC 1.1879 was 3.22×10 8 CFU / g.

[0112] Example 7 This embodiment provides a monosaccharide powder for reducing alcohol and detoxifying alcohol. Compared with Embodiment 3, the difference is that the drying protective agent used in step (3) is trehalose; and the final concentration of trehalose in the suspension is 10 wt%.

[0113] The viable count of Acetobacter pasteurianus CGMCC No.3089 in the alcohol-reducing and alcohol-detoxifying bacteria powder was 2.35×10 10 CFU / g.

[0114] Comparative Example 1 This comparative example provides a single bacterial powder of Lactobacillus plantarum, and the preparation method is the same as that of Example 5.

[0115] Verification Example 1 100 mL of artificial gastric juice was mixed with 50 mL of bulk liquor with an alcohol content of 56%, and 2 g of the bacterial powder obtained in Examples 3, 5 and Comparative Examples 1-2 were added thereto. The survival rate, ethanol, isoamyl alcohol and isobutanol content of Acetobacter pasteurianus CGMCC No. 3089 or Lactobacillus plantarum in the simulated gastric juice were recorded after 1 hour.

[0116] Table 4 Comparison of the ability of Acetobacter pasteurianus to degrade ethanol and miscellaneous alcohols

[0117] As shown in the data of Table 4, by comparing the results of Example 3 with those of Example 7, it can be seen that the medicinal and edible substances as protective agents can not only improve the freeze-dried survival rate of the strains, but also improve the survival rate of the bacteria in gastric juice. In addition, it can be seen from the results of Example 3, Example 5 and Comparative Example 1 that after the plant lactobacillus powder is mixed with the pasteurian acetobacter powder, the hangover effect is significantly improved, and the survival rate of pasteurian acetobacter CGMCC No.3089 in the artificial gastric juice and liquor mixture is also greatly improved.

[0118] Example 8 This embodiment provides a hangover-relief and liver-protecting tablet, which is composed of 25 g alcohol-reducing and alcohol-relief bacteria powder, 200 g microcrystalline cellulose, 10 g mannitol, 7.5 g low-substituted hydroxypropyl cellulose, 5 g magnesium stearate, 2.5 g aspartame, and 22.5 g starch slurry. The alcohol-reducing and alcohol-relief bacteria powder used is provided by Example 4. The specific preparation method is as follows: (1) Grind the alcohol-reducing and alcohol-detoxifying bacteria powder, microcrystalline cellulose, mannitol, low-substituted hydroxypropyl cellulose, magnesium stearate, aspartame, etc. separately with a grinder, and pass through an 80-mesh sieve for later use.

[0119] (2) Preparation of soft material: After evenly mixing the alcohol-reducing and alcohol-detoxifying bacteria powder with auxiliary materials (microcrystalline cellulose, mannitol), add starch slurry to prepare the soft material. The soft material should be in a state of "forming a ball when kneaded and falling apart when touched".

[0120] (3) Granulation: The soft material is granulated on a granulator with a 40-mesh screen.

[0121] (4) Drying: The wet granules are quickly placed in a drying oven at 75°C for drying. In order to fully avoid the wet granules formed by excessive moisture inside the drying process from agglomerating due to heat or deforming due to thermal pressure, the temperature should be maintained during the drying process. The moisture content after drying should be controlled below 3%.

[0122] (5) Granulation: The dried granules are sieved through a 40-mesh sieve to remove the granules that are stuck together into clumps or strips and set aside.

[0123] (6) Mixing: Lubricant magnesium stearate, disintegrant low-substituted cellulose and flavoring agent aspartame are added to the whole granules and mixed well.

[0124] (7) Tablet pressing: Use an automatic tablet press for tablet pressing, and the die specification is φ12 mm.

[0125] Verification Example 2: Animal Intoxication Experiment (1) Experimental subjects: Male Kunming mice, 8 weeks old, weighing 28±2.5 g. The mice were randomly divided into three groups: model group, positive control group, and experimental group, with 10 mice in each group. They were allowed to eat and drink freely. They were fasted but not watered for 4 h before oral gavage.

[0126] (2) Dosage: The model group was gavaged with 15 mL / kg of normal saline, the positive group was gavaged with 0.4 g / kg of Haiwang Jinzun alcohol-relieving tablets (crushed and dissolved in normal saline), and the experimental group was gavaged with 0.4 g / kg of alcohol-relieving and liver-protecting tablets (provided in Example 8, crushed and dissolved in normal saline). After 30 minutes, each group was gavaged with 15 mL / kg of 56-proof bulk liquor to create a drunkenness model. (The " / kg" refers to the weight of the mice) (3) Experimental content: The time required for mice to become drunk was recorded, and the serum ethanol concentration and liver ADH activity were measured using a biochemical colorimetric method 10 hours after the administration of white wine.

[0127] (4) Experimental results: Table 5 Mouse drunkenness experiment

[0128] In the experiment, compared with the control group, the time from drinking to drunkenness was significantly prolonged, the sobering time was shortened, and the ADH activity content in the liver was increased in the experimental group of mice.

[0129] In summary, the Acetobacter pasteurianus CGMCC No. 3089 in the present invention can effectively decompose fusel alcohol and ethanol in the body, significantly relieve headache and drunkenness after drinking, has a good alcohol-reducing and alcohol-relieving effect, and is suitable for preparing alcohol-relieving and liver-protecting products.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Acetobacter pasteurianus CGMCC No.3089 in the degradation of miscellaneous alcohols and ethanol in vivo.

2. The application of Acetobacter pasteurianus CGMCC No.3089 in the preparation of hangover relieving products, characterized in that: The active ingredient in the hangover relieving product is the Acetobacter pasteurianus CGMCC No.3089 and / or the metabolites of Acetobacter pasteurianus CGMCC No.3089.

3. An alcohol-reducing and alcohol-detoxifying bacterial agent, characterized in that: The invention comprises Acetobacter pasteurianus CGMCC No.3089 and biologically acceptable microbial agent carriers and / or auxiliary materials.

4. The alcohol-reducing and alcohol-detoxifying bacterial agent according to claim 3, characterized in that The high-alcohol alcohol degrading and hangover relieving agent is solid bacterial powder, and the auxiliary material is a drying protective agent; the drying protective agent is one or more combinations of trehalose, Hovenia dulcis powder, Rosa roxburghii powder, wolfberry juice, maltodextrin, skim milk powder, mannitol and kudzu root powder.

5. The alcohol-reducing and alcohol-detoxifying bacterial agent according to claim 3, characterized in that: It also includes Lactobacillus plantarum and / or Lactobacillus helveticus; preferably, Acetobacter pasteurianus CGMCC No.3089 and Lactobacillus plantarum are pure cultured and then mixed.

6. The alcohol-reducing and alcohol-detoxifying bacterial agent according to claim 3, characterized in that: The Acetobacter pasteurianus CGMCC No.3089 is cultured in a culture medium containing 2.5%-15% lactic acid bacteria cell-free supernatant or co-cultured with lactic acid bacteria for 12-36 hours; preferably, the Acetobacter pasteurianus CGMCC No.3089 is cultured in a culture medium containing 5%-15% lactic acid bacteria cell-free supernatant for 16-24 hours.

7. The alcohol-reducing and alcohol-detoxifying bacterial agent according to claim 6, characterized in that: The lactic acid bacteria is Lactobacillus helveticus.

8. A method for preparing alcohol-reducing and alcohol-detoxifying bacteria powder, characterized in that: The preparation method comprises the steps of mixing live bacterial sludge containing Acetobacter pasteurianus CGMCC No. 3089 with a drying protective agent and then performing a drying treatment, or preparing the live bacterial sludge into a suspension and mixing it with a drying protective agent and then performing a drying treatment, so as to obtain alcohol-reducing and alcohol-degrading bacterial powder containing Acetobacter pasteurianus.

9. The method for preparing alcohol-reducing and alcohol-detoxifying bacteria powder according to claim 8, characterized in that: The drying treatment method is spray drying, and the drying protective agent is one or more combinations of trehalose, Hovenia dulcis powder, Rosa roxburghii powder, wolfberry juice, maltodextrin, skim milk powder, mannitol and Pueraria root powder.

10. A drug for sobering up and protecting the liver, characterized in that: The invention comprises Acetobacter pasteurianus CGMCC No.3089, a probiotic combination comprising Acetobacter pasteurianus CGMCC No.3089, the alcohol-reducing and alcohol-detoxifying bacterial agent according to any one of claims 3 to 7, or the alcohol-reducing and alcohol-detoxifying bacterial agent prepared by the preparation method according to claim 8 or 9.

11. A composite food for sobering up and protecting the liver, characterized in that: The alcohol-detoxifying and liver-protecting composite food comprises Acetobacter pasteurianus CGMCC No.3089, a probiotic combination comprising Acetobacter pasteurianus CGMCC No.3089, the alcohol-reducing and alcohol-detoxifying bacterial agent according to any one of claims 3 to 7 or the alcohol-reducing and alcohol-detoxifying bacterial agent prepared by the preparation method according to claim 8 or 9, and food and medicine ingredients.

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