Application of lactobacillus reuteri DSM17938 and product of lactobacillus reuteri DSM17938

Abstract

The invention discloses application of lactobacillus reuteri DSM17938 and a product of the lactobacillus reuteri DSM17938. Aiming at the technical problems that the existing chemical anti-alcohol medicine has side effects and the anti-alcohol effect of common probiotics is unstable, the key point of the technical scheme of the invention is to provide application of a specific strain DSM17938 in preparation of functional foods or medicaments, and the viable organism content of lactobacillus reuteri DSM17938 in the product is 1 * 10 < 9 >-5 * 10 < 10 > CFU / mL or CFU / g; the product can effectively improve the content of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the body, and further improve the total activity of the body, thereby prolonging the drunkenness latency of alcohol gavage mice and shortening the drunkenness sleep time of the mice. The invention also provides yoghurt which is prepared by fermenting the strain and has the effect of increasing the content of alcohol metabolic enzyme, and medicaments such as oral liquid and capsules containing the strain. The main purpose of the invention is to provide a safe, efficient and stable probiotic solution for relieving drunkenness symptoms, promoting alcohol metabolism and protecting the liver.

Description

Technical Field

[0001] This invention relates to the field of microbial application and product technology, specifically to the application of Lactobacillus reuteri DSM17938 and its products. Background Technology

[0002] Alcohol consumption is widespread globally, but excessive alcohol intake can lead to acute intoxication, liver damage, and other metabolic diseases. The metabolism of alcohol in the body primarily relies on the catalytic action of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the activities of which directly affect the rate of alcohol breakdown and the efficiency of acetaldehyde clearance. Acetaldehyde, as an intermediate product of alcohol metabolism, is highly toxic; its accumulation can cause discomfort such as facial flushing, nausea, and palpitations, and even increase long-term health risks. Therefore, increasing the levels of ADH and ALDH to enhance their overall activity is considered a key approach to alleviating alcohol-related problems.

[0003] Currently, several methods have been attempted to enhance alcohol metabolism through drugs or functional foods. For example, certain chemical drugs (such as metadoxine) or traditional probiotic preparations have been used to promote alcohol breakdown. However, these methods often have limitations: chemical drugs may be accompanied by side effects such as gastrointestinal discomfort or dependence, while ordinary probiotic products are inconsistent in their ability to increase the activity of specific metabolic enzymes and lack precise regulation of alcohol metabolism. Furthermore, current probiotic applications mostly focus on gut health regulation, with insufficient research on their direct enhancing effects on alcohol-metabolizing enzymes, especially the optimized application of specific strains, which has not yet been fully explored.

[0004] Lactobacillus reuteri, a common probiotic, has been extensively studied for its role in maintaining gut microbiota balance and immune regulation. However, its application in specifically enhancing the activity of alcohol-metabolizing enzymes (ADH and ALDH) is relatively rare. Furthermore, some existing probiotic hangover remedies suffer from insufficient live bacteria stability, low survival rates upon reaching the intestines, and insignificant or unstable hangover-relieving effects due to low strain specificity. Therefore, there is an urgent need in this field for a safe and effective approach based on specific probiotic strains that can reliably enhance ADH and ALDH activity in the body, thereby fundamentally improving alcohol metabolism efficiency and alleviating hangover-related symptoms.

[0005] The present invention aims to solve the above-mentioned technical problems by providing specific applications of Lactobacillus reuteri DSM17938, overcoming the shortcomings of the prior art, and achieving more efficient regulation of alcohol metabolism. Summary of the Invention

[0006] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0007] To achieve these and other advantages of the present invention, the use of Lactobacillus reuteri DSM17938 in the preparation of products for increasing the content of alcohol-metabolizing enzymes is now provided.

[0008] Specifically, the product of this invention enhances the total activity of the body by increasing the levels of ADH and ALDH, and can prolong the latency period of alcohol intoxication and / or shorten the duration of alcohol-induced sleep in mice administered alcohol via gavage. The strain used in this invention is *Lactobacillus reuteri* (…). Limosilactobacillusreuteri The strain DSM 17938 is publicly available from the German Society for the Preservation of Microbial Cultures (DSMZ). To meet the requirements of Chinese patent law, this strain was deposited on June 15, 2019, at the Shanghai Biotechnology Center for Collections, with accession number SHBCC D24679, address: No. 938 Shunda Road, Jiading District, Shanghai, 201802, China. The strain is classified as DSM17938, and the microorganism (strain) is *Lactobacillus reuteri*. Limosilactobacillusreuteri Its taxonomic units are: Bacteria; Bacillota; Bacilli; Lactobacillales; Lactobacillaceae; Limosilactobacillus ; reuteri .

[0009] Preferably, the product of the present invention is yogurt or a pharmaceutical preparation; the viable count of Lactobacillus reuteri DSM17938 in the product is 1×10⁻⁶. 9 Up to 5×10 10 CFU / mL or CFU / g.

[0010] Preferably, the pharmaceutical preparation of the present invention is in the form of capsules, tablets, oral liquids, or granules; the pharmaceutical preparation contains *Lactobacillus reuteri* DSM17938 and a pharmaceutically acceptable carrier, with a viable bacterial count of 1 × 10⁻⁶. 9 Up to 5×10 10 CFU / mL or CFU / g; wherein, when the drug is an oral liquid, Lactobacillus reuteri DSM17938 is microencapsulated and suspended in a stable matrix containing a lyophilization protectant; when the drug is a granule, Lactobacillus reuteri DSM17938 is mixed with excipients and then granulated by a dry granulation process.

[0011] Preferably, the yogurt of the present invention, which has an increased content of alcohol-metabolizing enzymes, is fermented with Lactobacillus reuteri DSM17938, and wherein the viable count of Lactobacillus reuteri DSM17938 is ≥1×10⁻⁶. 9CFU / g or CFU / mL; the yogurt is prepared by a method comprising the following steps: fermentation of milk substrate inoculated with Lactobacillus reuteri DSM17938 at 37 ℃ for 8-10 hours to obtain a fermentation product; the fermentation product is cooled to 4 ℃ at a rate of 0.5-1 ℃ / min and refrigerated, wherein the viable count of Lactobacillus reuteri DSM17938 is ≥1×10⁻⁶. 9 CFU / g.

[0012] A method for preparing yogurt with increased alcohol-metabolizing enzyme content includes: mixing Lactobacillus reuteri DSM17938 bacterial powder with milk at a mass ratio of 1:4, fermenting at 37 ℃ for 8-10 hours to obtain a fermentation product; cooling the fermentation product to below 4 ℃ at a rate of 0.5-1 ℃ / min and storing it in the dark to obtain yogurt with increased alcohol-metabolizing enzyme content.

[0013] Preferably, when the product is cooled to 10-15 °C, microencapsulated Lactobacillus reuteri DSM17938 bacterial powder is added and mixed at a stirring speed of 150-200 rpm, and then the temperature is further reduced to 4 °C; wherein the amount of microencapsulated bacterial powder added increases the total viable count of Lactobacillus reuteri DSM17938 in the final yogurt by no less than 1 × 10⁻⁶. 9 CFU / g.

[0014] Preferably, the method for preparing yogurt with increased alcohol-metabolizing enzyme content further includes: after obtaining the fermentation product, transferring it to a temperature of 15-20 °C and maintaining it for 15-30 minutes, then cooling it to below 4 °C at a rate of 0.5-1 °C / min, and storing it in the dark at this temperature to obtain yogurt with increased alcohol-metabolizing enzyme content.

[0015] The present invention provides a pharmaceutical preparation for increasing the content of alcohol-metabolizing enzymes. When the preparation is an oral liquid, the stable matrix containing the lyophilization protectant is composed of trehalose, skim milk, and glycerol in a mass ratio of 5-10:1-3:0.5-2. The Lactobacillus reuteri DSM17938 microcapsule powder is mixed with the stable matrix in a mass ratio of 1:2-5, and the mixture is stirred at 200-400 rpm for 30-60 minutes at 15-25 °C to form a homogeneous suspension, which is the oral liquid for increasing the content of alcohol-metabolizing enzymes.

[0016] The present invention provides a preparation of a pharmaceutical agent that increases the content of alcohol-metabolizing enzymes, comprising: dissolving trehalose, skim milk, and glycerol in purified water at a mass ratio of 7.5:2:1, stirring until completely dissolved to form a homogeneous and stable aqueous matrix, thereby obtaining a stable matrix; mixing microencapsulated Lactobacillus reuteri DSM17938 bacterial powder with the stable matrix at a mass ratio of 1:3.5, and continuously stirring at 300 rpm for 45 minutes at 20±2 °C to form a homogeneous suspension; aseptically filling the resulting suspension into 10 mL brown oral liquid glass bottles, and immediately sealing with nitrogen to obtain an oral liquid that increases the content of alcohol-metabolizing enzymes; wherein the obtained oral liquid product should be stored below 25 °C under light-protected conditions, and its shelf life is 24 months; during the shelf life, the viable bacterial survival rate is not less than 80%.

[0017] The present invention has at least the following beneficial effects: 1. This invention uses *Lactobacillus reuteri* DSM17938 within a specific live bacteria content range to prepare a product that effectively increases the levels of ADH and ALDH in the body, thereby significantly increasing the total content of these two enzymes. This prolongs the latency period of intoxication and shortens the duration of intoxication-related sleep. This solves the problem of insufficient probiotic regulation of alcohol metabolism in existing technologies, achieving a safe and efficient effect in promoting alcohol decomposition and alleviating intoxication symptoms. Simultaneously, the clearly defined live bacteria content range ensures the consistency and reliability of product function, providing users with quantifiable health benefits and avoiding efficacy fluctuations caused by insufficient live bacteria.

[0018] 2. By specifying the product as either yogurt or a pharmaceutical preparation, multiple application forms are provided to meet the needs of different scenarios. Yogurt, as an everyday food, is easily accepted, while the pharmaceutical preparation is suitable for medical use. This solves the problem of the limited product form of existing technologies, achieving the effect of enhancing user compliance and expanding the scope of application. This flexibility allows the product to be adapted to a wider range of people, such as everyday consumers or patients who need specific dosage forms, thereby enhancing practicality and market potential.

[0019] 3. This invention, through a specific fermentation process and programmed cooling steps, ensures a high viable count and functional activity of *Lactobacillus reuteri* DSM17938 in yogurt. This overcomes the shortcomings of existing yogurt products in terms of probiotic activity and efficacy, reliably increasing the content of alcohol-metabolizing enzymes and providing long-term stability. Programmed cooling reduces damage to the bacteria from sudden temperature changes, and refrigeration further maintains the viable count, enabling yogurt not only to be consumed as an everyday food but also to provide sustained functional benefits.

[0020] 4. This invention optimizes the growth and preservation conditions of *Lactobacillus reuteri* DSM17938 by controlling the mixing ratio of the starter culture and milk, fermentation temperature, and cooling rate. This solves the problems of low viable culture yield and poor stability in existing methods, achieving efficient preparation of yogurt with high viable culture content while ensuring its function of enhancing alcohol metabolism enzyme content. Light-protected storage further prevents photosensitive degradation, allowing the product to maintain its efficacy during storage, thus improving commercial viability and user satisfaction.

[0021] 5. This invention reduces the loss of live bacteria during the addition process by adding microencapsulated bacterial powder when the temperature is cooled to 10-15°C and controlling the stirring speed, thus ensuring an increase in the total number of live bacteria. This solves the problem of unstable live bacteria replenishment in existing methods, achieving a significant increase in the probiotic content and functionality of yogurt. Microencapsulation protects the bacteria from environmental stress, and uniform stirring ensures consistent distribution, thereby enhancing the overall quality and reliability of the product.

[0022] 6. This invention reduces the risk of heat shock by introducing a 15-20°C maintenance phase after fermentation, allowing the microorganisms to gradually adapt to temperature changes before programmed cooling. This solves the negative impact of existing cooling methods on the activity of live bacteria, achieving better preservation of live bacteria function and extending product shelf life. This step optimizes the metabolic regulation of the microorganisms, ensuring that the yogurt maintains a high enzyme content and its ability to enhance enzyme activity during storage and use, thus improving product consistency and durability.

[0023] 7. This invention, through a specific ratio of freeze-drying protectants and optimized mixing conditions, forms a homogeneous suspension that effectively protects the activity of the microencapsulated bacterial powder. This solves the problem of poor stability of live bacteria in oral liquids, achieving the effects of improving product uniformity, shelf life, and post-consumption bioavailability. The combination of protectants, such as trehalose, skim milk, and glycerin, works synergistically to prevent bacterial inactivation, while controlling the stirring environment and speed ensures the homogeneity of the suspension, making the product easy to take and reliably effective.

[0024] 8. This invention ensures a high survival rate and long-term stability of live bacteria in the oral liquid by optimizing the stable matrix ratio, mixing parameters, and packaging conditions. This solves the problems of easy product spoilage and significant loss of live bacteria in existing methods, achieving the effect of extending shelf life and maintaining the increased content of highly efficient alcohol-metabolizing enzymes. Nitrogen-filled sealing and light-protected storage further reduce the risk of oxidation and photodegradation, ensuring that the live bacteria survival rate of the product is not less than 80% within 24 months, guaranteeing the effectiveness of commercial transportation and user use. Attached Figure Description

[0025] Figure 1 This is the phylogenetic tree of strain LR DSM17938 based on the 16S rDNA gene sequence of the present invention; Figure 2The effect of LR DSM17938 administration in this invention on serum AST and ALT levels in acutely intoxicated mice; Figure 3 The effect of LR DSM17938 administration in this invention on serum ADH and ALDH levels in acutely intoxicated mice; Figure 4 This invention investigates the effects of LR DSM17938 administration on liver MDA content, SOD activity, and GSH levels in acutely intoxicated mouse models. Figure 5 This invention produces yogurt samples fermented for 14 h at 37 ℃ with different ratios (bacterial starter: milk); Figure 6 This invention relates to the effect of LRFM administration on serum AST and ALT levels in mice with acute alcohol intoxication. Figure 7 This invention relates to the effect of LRFM administration on serum ADH and ALDH levels in acutely intoxicated mice. Figure 8 This invention investigates the effects of LRFM administration on liver MDA content, SOD activity, and GSH levels in mice with acute alcohol intoxication. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0027] Example 1 Application of Lactobacillus reuteri DSM17938 in the preparation of yogurt products for increasing the content of alcohol-metabolizing enzymes.

[0028] Step 1: On a sterile work surface, weigh 200 mg of Lactobacillus reuteri DSM17938 starter culture powder and mix it with 800 g of pasteurized whole milk, ensuring thorough mixing. Dispense the mixture into aseptic yogurt bottles and place them in a 37°C incubator for fermentation. Fermentation continues for 8-10 hours, until the fermentation product coagulates and exhibits a typical yogurt texture. Sampling and testing at this stage showed that the viable Lactobacillus reuteri DSM17938 content in the fermentation product reached 1 × 10⁻⁶. 9 CFU / g.

[0029] Step 2: After fermentation, transfer the yogurt bottles to a programmable thermostatic incubator. Start the cooling program, setting the target temperature to 4°C and the cooling rate to 0.8°C / min, ensuring the entire process is conducted in the dark. Once the product reaches 4°C, the incubator will automatically switch to keep-warm mode. At this temperature, continue storage in the dark for at least 12 hours to complete the post-ripening process of the yogurt, resulting in yogurt with increased alcohol-metabolizing enzyme content.

[0030] Example 2 Application of Lactobacillus reuteri DSM17938 in the preparation of yogurt products for increasing the content of alcohol-metabolizing enzymes.

[0031] The difference from Example 1 is that step two is replaced with: Step 2: When the temperature sensor in the incubator shows that the temperature has dropped to 12 ℃, pause the cooling process. Under aseptic conditions, quickly add 2% by weight of microencapsulated Lactobacillus reuteri DSM17938 starter culture to each bottle of yogurt. Then, stir at 180 rpm to ensure that the starter culture is evenly dispersed throughout the fermentation product without disrupting its gel structure.

[0032] After stirring, restart the cooling program and continue cooling the product to 4°C at a rate of 0.8°C / min. Once the product reaches 4°C, the incubator automatically switches to the heat preservation mode. At this temperature, continue to store in the dark for at least 12 hours to complete the post-ripening process of the yogurt, thus obtaining yogurt with increased alcohol metabolism enzyme content.

[0033] Example 3 Application of Lactobacillus reuteri DSM17938 in the preparation of yogurt products for increasing the content of alcohol-metabolizing enzymes.

[0034] The difference from Example 1 is that step two is replaced with: Step 2: After fermentation, transfer the yogurt bottles to a programmable thermostat incubator at 18°C ​​and maintain this temperature for 20 minutes. Then, start the cooling program, setting the target temperature to 4°C and the cooling rate to 0.8°C / min, ensuring the entire process is conducted in the dark. Once the product reaches 4°C, the incubator will automatically switch to keep-warm mode. At this temperature, continue storing in the dark for at least 12 hours to complete the post-ripening process of the yogurt, resulting in yogurt with increased alcohol-metabolizing enzyme content.

[0035] Example 4 Application of Lactobacillus reuteri DSM17938 in the preparation of oral solutions for increasing the content of alcohol-metabolizing enzymes.

[0036] Step 1: Weigh out 375 g of trehalose, 100 g of skim milk powder, and 50 g of glycerin. Add them to a clean container containing 2 L of purified water. Stir at 200 rpm while slowly heating to 45-50°C until all components are completely dissolved, forming a clear, transparent solution. Stop heating, add purified water to bring the total volume to 3.5 L, and continue stirring for 15 minutes at 20-25°C to ensure a homogeneous and stable aqueous matrix, i.e., a stable matrix. Set aside.

[0037] Step 2: Centrifuge the *Lactobacillus reuteri* DSM17938 fermentation broth at 4 ℃ and 8000 rpm for 15 minutes, discard the supernatant, and collect the bacterial sludge. Resuspend the sludge in sterile physiological saline and wash it again by centrifugation to remove fermentation metabolites. Then resuspend the sludge in sterile physiological saline to prepare a high-concentration bacterial suspension (viable cell concentration ≥ 5.0 × 10¹). 0 (CFU / mL), place in an ice bath at 4 ℃ until ready for use.

[0038] Sodium alginate was dissolved in purified water to prepare a 3% sodium alginate solution, which was then sterilized at high temperature and cooled to room temperature. The high-concentration bacterial suspension prepared above was mixed with the 3% sodium alginate solution at a volume ratio of 1:4, and homogenized at 5000 rpm for 2 minutes using a high-speed shear disperser to form a homogeneous bacterial-sodium alginate mixture. The mixture was then injected dropwise into a 4% calcium chloride solution using a peristaltic pump through a 0.4 mm orifice needle. The calcium chloride solution was stirred at a constant speed of 150 rpm. The bacterial-sodium alginate droplets immediately underwent an ionic cross-linking reaction in the calcium chloride solution, forming spherical gel microbeads. The microbeads were then allowed to solidify in a coagulation bath for 30 minutes to allow them to fully harden.

[0039] The solidified sodium calcium alginate microbeads were removed using a sterile sieve and gently rinsed with purified water to remove residual calcium chloride from the surface. They were then transferred to a 0.5% chitosan-acetic acid solution (prepared with 1% acetic acid) and stirred slowly at 80 rpm for 20 minutes at room temperature. During this process, positively charged chitosan molecules adsorbed onto the negatively charged sodium calcium alginate microbeads through electrostatic interactions, forming a dense protective outer membrane. After the reaction was complete, the microcapsules were again removed using a sieve and rinsed with purified water to obtain intact, moist Lactobacillus reuteri microcapsules.

[0040] The wet microcapsules were mixed with a pre-prepared lyophilization protectant at a mass ratio of 1:1 and gently stirred to ensure full contact. The mixture was then spread evenly in a lyophilization tray and dried in a freeze dryer to obtain microencapsulated Lactobacillus reuteri DSM17938 bacterial powder.

[0041] Step 3: Accurately weigh 100 g of microencapsulated Lactobacillus reuteri DSM17938 bacterial powder and 350 g of the prepared stabilizing matrix. Transfer the stabilizing matrix to a clean and sterilized mixing tank. Maintain the system temperature at 20±2℃. While continuously stirring at 300 rpm, slowly and evenly sprinkle 100 g of bacterial powder into the stabilizing matrix. After all the bacterial powder has been added, maintain the stirring speed at 300 rpm and continue stirring for 45 minutes until a homogeneous suspension without visible particles is formed. Aseptically fill the resulting suspension to obtain an oral liquid that increases the content of alcohol-metabolizing enzymes. The oral liquid product should be stored below 25℃ and protected from light.

[0042] Experiments and Analysis Experiment 1: Effects of Lactobacillus reuteri DSM17938 intervention on acutely intoxicated mice I. Culture and Identification of Lactobacillus reuteri DSM17938 Lactobacillus reuteri DSM17938 bacterial powder was plated on MRS plates, and its colony morphology was observed. 16S rDNA gene amplification was performed using PCR, and the amplified products were sequenced by Guangzhou Aiji Biotechnology Co., Ltd. After sequencing the successfully amplified PCR products, the DNA sequences were assembled, and sequence homology was compared and analyzed using the NCBI BLAST search system. The known strains with the highest similarity were selected for similarity analysis. A phylogenetic tree was constructed using Neighbor-Joining (NJ) in MEGA11 software. Lactobacillus reuteri DSM17938 was purchased from Shanghai Preservation Biotechnology Center; MRS plates were purchased from Qingdao Haibo Biotechnology Co., Ltd. Results are as follows: Figure 1 As shown.

[0043] II. Effects of Lactobacillus reuteri DSM17938 intervention on acutely intoxicated mice Experimental animals: 60 healthy SPF-grade Kunming mice, half male and half female, 6-8 weeks old, weighing 18-22 g.

[0044] Reagents and chemicals: 52° Red Star Erguotou (a type of Chinese liquor), metadoxine, alanine aminotransferase (ALT) kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.), aspartate aminotransferase (AST) kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.), alcohol dehydrogenase (ADH) test kit (purchased from Nanjing Jiancheng Biotechnology Institute), acetaldehyde dehydrogenase (ALDH) test kit (purchased from Nanjing Jiancheng Biotechnology Institute), malondialdehyde (MDA) production test kit (purchased from Nanjing Jiancheng Biotechnology Institute), superoxide dismutase (SOD) activity test kit (purchased from Nanjing Jiancheng Biotechnology Institute), liver glutathione (GSH) content test kit (purchased from Nanjing Jiancheng Biotechnology Institute), sterile PBS buffer.

[0045] Experimental equipment: electronic balance, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader, gavage syringe.

[0046] Experimental methods: 1. After expanding the culture of Lactobacillus reuteri DSM17938, the bacterial sludge was collected by centrifugation and resuspended in sterile PBS buffer to prepare high, medium, and low concentration bacterial suspensions: 1×10 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 CFU / mL, store at 4℃ for later use.

[0047] 2. After one week of acclimatization feeding, the experimental mice were randomly divided into 6 groups, with 10 mice in each group. The groups included a blank control group (physiological saline), a model group (52° Red Star Erguotou liquor), and a high-dose LR group (1×10⁻⁶ oz). 9 CFU / mL), LR medium-dose group (1×10) 8 CFU / mL), LR low-dose group (1×10) 7 CFU / mL), positive group (metadone).

[0048] 3. Thirty minutes before administration of alcohol, based on body weight, the high, medium, and low dose groups of the LR were administered 1×10 mg / L via gavage. 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 CFU / mL LR bacterial suspension was administered to the positive group via gavage at 300 mg / kg metadoxine, while the blank control group and model group were administered the same dose of sterile PBS buffer via gavage. After 30 minutes, the blank control group was administered sterile PBS buffer via gavage, while the other groups were administered 0.16 mL / 10 g of 52° Red Star Erguotou (a type of Chinese liquor) via gavage to establish an acute alcohol intoxication mouse model. The latency time to intoxication (from the start of alcohol administration to the disappearance of the righting reflex), the duration of intoxication (from the disappearance of the righting reflex to its recovery), the intoxication rate, and the mortality rate were observed and recorded within 8 hours after alcohol administration (if the mice were not awake within 8 hours, the time was recorded as 8 hours). The results are shown in Table 1.

[0049] 4. After 8 hours of behavioral observation, blood was collected from the orbital vein into EP tubes, centrifuged at 3800 r / min for 15 min at 4 ℃, and the serum was separated and placed in a 1.5 mL centrifuge tube, stored at -20 ℃ for later testing. Mice were euthanized by cervical dislocation and dissected. The liver was collected and stored at -80 ℃ for liver homogenate preparation. Liver homogenate preparation: 0.1 g of liver tissue from the left lobe of the mouse liver was added to 900 uL of pre-cooled physiological saline, homogenized under ice-water bath conditions, and prepared into a 10% (w / v) tissue homogenate. The homogenate was centrifuged at 3500 r / min for 10 min, and the supernatant was collected for testing. The activities of ALT and AST, ADH, ALDH, MDA, GSH, and SOD in serum were measured strictly according to the instructions of the commercially available kit. Since the contents of ADH and ALDH are positively correlated with their catalytic activities, their content can effectively reflect the body's ability to metabolize alcohol. Results are as follows. Figures 2-4 .

[0050] Table 1: Effects of LR DSM17938 on behavioral parameters in mice with acute alcohol intoxication In the blank control group, the mice were in good spirits, active, and had shiny fur. After being administered large amounts of alcohol by gavage, the mice in the model group and the groups treated with different doses of LR all showed symptoms of slow movement, lethargy, drowsiness, snoring, and intermittent tremors of the limbs.

[0051] According to the data in Table 1, the latency time to intoxication in the model group mice was 58.90 min, and the duration of intoxication was 408.60 min. After gavage administration of high, medium, and low doses of LR, the latency time to intoxication in mice was significantly prolonged, and the duration of intoxication was significantly shortened. Compared with the model group, the latency time to intoxication in the high-dose, medium-dose, and low-dose groups was prolonged by 281.2%, 257.4%, and 215.8%, respectively, while the duration of intoxication was shortened by 53.7%, 47.5%, and 34.4%, respectively. This indicates that after mice suffer from acute alcohol poisoning due to excessive alcohol consumption, high, medium, and low doses of LR can significantly prolong the latency time to intoxication, shorten the duration of intoxication, alleviate symptoms caused by acute alcohol poisoning, and reduce the rate of intoxication and mortality, thus exhibiting a certain anti-alcohol effect.

[0052] according to Figure 2 Compared with the blank control group, the serum AST and ALT activities of mice in the model group were significantly increased. Compared with the model group, the AST and ALT activities in the high, medium, and low dose LR groups were significantly decreased. These results indicate that LR can alleviate alcohol-induced liver damage in mice.

[0053] according to Figure 3Compared with the blank control group, the serum ADH and ALDH activities of mice in the model group were significantly reduced. Compared with the model group, the ADH and ALDH levels in the high, medium, and low dose LR groups were significantly increased. These results indicate that LR treatment can increase the content of alcohol-metabolizing enzymes, accelerate the metabolism of ethanol and acetaldehyde, and has a certain effect on relieving hangovers.

[0054] according to Figure 4 Compared with the blank control group, the model group mice showed significantly increased MDA levels and significantly decreased GSH and SOD levels in serum. Compared with the model group, the high, medium, and low dose LR groups showed significantly decreased MDA levels and significantly increased GSH and SOD levels. These results indicate that LR can reduce MDA, the end product of lipid peroxidation, reduce free radical damage to cell membranes, increase the levels of antioxidants GSH and SOD, effectively scavenge initial free radicals, and inhibit the source of damage. LR may exert its detoxifying effect through an antioxidant mechanism.

[0055] Experiment 2: Effects of Lactobacillus reuteri DSM17938 yogurt product intervention on acutely intoxicated mice I. Preparation of Yogurt Products Using Lactobacillus reuteri DSM17938 Referring to GB 19302-2025, the national food safety standard for fermented milk, the sensory requirements for fermented milk are as follows: uniform color, milky white or slightly yellow, possessing the characteristic taste and aroma of fermented milk, fine and uniform texture, with a small amount of whey separation permitted. The acidity of fermented milk should be ≥ 60 °T, i.e., pH approximately 4.1-4.3. The conventional requirements for fermented milk production processes are: pH 4-4.6, fermentation time 6-14 h, fermentation temperature 10-40 °C, and inoculum count ≥ 10... 6 CFU / g.

[0056] Samples were fermented for 14 hours at inoculum amounts for wells 1 (no starter culture), 2 (starter culture: milk = 1:2), 3 (starter culture: milk = 1:3), 4 (starter culture: milk = 1:4), 5 (starter culture: milk = 1:5), and 6 (starter culture: milk = 1:6). Figure 5 As shown in Table 2, the product with a 1:4 ratio (bacterial starter: milk) exhibited a uniform, milky-white color, a distinctive yogurt aroma, a fine and homogeneous texture, and a small amount of whey separation. The pH was measured every 2 hours after fermentation at the 1:4 ratio for 14 hours, and the results are shown in Table 2.

[0057] Table 2: pH values ​​of yogurt products prepared with starter culture and milk in a 1:4 ratio at different fermentation times. According to the data in Table 2, the pH of fermented milk after 10 and 12 hours is within the range of 4-4.6, which meets the standard for fermented milk.

[0058] II. Effects of Lactobacillus reuteri DSM17938 yogurt product intervention on acutely intoxicated mice Experimental animals: 60 healthy SPF-grade Kunming mice, half male and half female, 6-8 weeks old, weighing 18-22g.

[0059] Reagents and chemicals: 52° Red Star Erguotou (a type of Chinese liquor), Metadoxine, and Lactobacillus reuteri DSM17938 bacterial powder were purchased from Shanxi Xinno Biotechnology Co., Ltd.; ALT (alanine aminotransferase) kit and AST (aspartate aminotransferase) kit were purchased from Nanjing Jiancheng Bioengineering Co., Ltd.; ADH (alcohol dehydrogenase) test kit, ALDH (acetaldehyde dehydrogenase) test kit, MDA (malondialdehyde) production test kit, SOD (superoxide dismutase) activity test kit, and liver glutathione (GSH) content test kit were purchased from Nanjing Jiancheng Bioengineering Co., Ltd.; sterile PBS buffer.

[0060] Experimental equipment: electronic balance, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader, gavage syringe.

[0061] Experimental methods: 1. After one week of acclimatization feeding, the experimental mice were randomly divided into 6 groups, with 10 mice in each group. The groups included a blank control group (physiological saline), a model group (52° Red Star Erguotou liquor), and a high-dose LRFM group (1×10⁻⁶). 9 CFU / mL), LRFM medium-dose group (1×10) 8 CFU / mL), LRFM low-dose group (1×10) 7 CFU / mL), positive group (metadone).

[0062] 2. Fasting was allowed for 12 hours prior to the experiment, but water was permitted. 30 minutes before alcohol administration, the high, medium, and low dose groups of LRFM were administered via gavage, with a bacterial count of 1×10⁻⁶ based on body weight. 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7CFU / mL LRFM was administered to the positive group via gavage, along with 300 mg / kg metadoxine. The blank control group and the model group were administered the same dose of sterile PBS buffer via gavage. After 30 minutes, the blank control group was administered sterile PBS buffer via gavage, while the other groups were administered 0.16 mL / 10 g of 52° Red Star Erguotou (a type of Chinese liquor) via gavage to establish an acute alcohol intoxication mouse model. The latency time to intoxication (from the start of alcohol administration to the disappearance of the righting reflex), the duration of intoxication (from the disappearance of the righting reflex to the recovery of the righting reflex), the intoxication rate, and the mortality rate were observed and recorded within 8 hours after alcohol administration (if the mice did not wake up within 8 hours, the data was recorded as 8 hours). The results are shown in Table 3.

[0063] 3. After 8 hours of behavioral observation, blood was collected from the orbital vein into EP tubes, centrifuged at 3800 r / min for 15 min at 4 ℃, and the serum was collected and placed in a 1.5 mL centrifuge tube, stored at -20 ℃ for later testing. Mice were euthanized by cervical dislocation and dissected. The liver was collected and stored at -80 ℃ for liver homogenate preparation. Liver homogenate preparation: 0.1 g of liver tissue from the left lobe of the mouse liver was added to 900 uL of pre-cooled physiological saline, homogenized under ice-water bath conditions, and prepared as a 10% (w / v) tissue homogenate. The homogenate was centrifuged at 3500 r / min for 10 min, and the supernatant was collected for testing. The activities of ALT and AST, ADH, ALDH, MDA, GSH, and SOD in serum were measured strictly according to the instructions of the commercially available kit. Results are as follows: Figures 6-8 The ratio of prolonged sleep time was calculated as follows: (Late latency of experimental group - Latency of blank model group) / Latency of blank model group × 100%; the ratio of shortened sleep time was calculated as follows: (Sleep time of blank model group - Sleep time of experimental group) / Sleep time of blank model group × 100%.

[0064] Table 3: Effects of Lactobacillus reuteri DSM17938 yogurt products on behavioral parameters in mice with acute alcohol intoxication. In the blank control group, the mice were in good spirits, active, and had shiny fur. After being administered large amounts of alcohol by gavage, the mice in the model group and the groups treated with different doses of LRFM all showed symptoms of slow movement, lethargy, drowsiness, snoring, and intermittent tremors of the limbs.

[0065] According to the data in Table 3, the latency time to intoxication in the model group mice was 45.90 min, and the duration of intoxication was 422.40 min. After gavage with high, medium, and low doses of LRFM, the latency time to intoxication in mice was significantly prolonged, and the duration of intoxication was significantly shortened. Compared with the model group, the latency time to intoxication in the high-dose, medium-dose, and low-dose groups was prolonged by 426.58%, 201.96%, and 129.19%, respectively, while the duration of intoxication was shortened by 45.38%, 20.57%, and 15.84%, respectively. This indicates that after mice suffer from acute alcohol poisoning due to excessive alcohol consumption, high, medium, and low doses of LRFM can significantly prolong the latency time to intoxication, shorten the duration of intoxication, alleviate symptoms caused by acute alcohol poisoning, and reduce the rate of intoxication and mortality, thus exhibiting a certain anti-alcohol effect.

[0066] according to Figure 6 Compared with the blank control group, the serum AST and ALT activities of mice in the model group were significantly increased. Compared with the model group, the AST and ALT activities of the high, medium and low dose LRFM groups were significantly decreased. These results indicate that LR can alleviate the liver damage caused by alcohol in mice.

[0067] according to Figure 7 Compared with the blank control group, the serum ADH and ALDH activities of mice in the model group were significantly reduced. Compared with the model group, the ADH and ALDH activities of the high, medium, and low dose LRFM groups were significantly increased. These results indicate that LRFM can increase the content of alcohol-metabolizing enzymes, accelerate the metabolism of ethanol and acetaldehyde, and has a certain effect on relieving hangovers.

[0068] according to Figure 8 Compared with the blank control group, the model group mice showed significantly increased MDA levels and significantly decreased GSH and SOD levels in serum. Compared with the model group, the high, medium, and low dose LRFM groups showed significantly decreased MDA levels and significantly increased GSH and SOD levels. These results indicate that LRFM can reduce MDA, the end product of lipid peroxidation, reduce free radical damage to cell membranes, increase the levels of antioxidants GSH and SOD, effectively scavenge initial free radicals, and inhibit the source of damage. LRFM exerts its hangover-relieving effect through an antioxidant mechanism.

[0069] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. Use of Lactobacillus reuteri DSM 17938, characterized in that, A product for increasing the content of alcohol metabolism enzymes.

2. Use according to claim 1, wherein The product is a yoghurt or a medicament; the viable cell content of Lactobacillus reuteri DSM 17938 in the product is from 1 x 10 9 to 5 x 10 10 CFU / mL or CFU / g.

3. Use according to any one of claims 1 or 2, wherein the compound is ###0001### The medicament is a capsule, a tablet, an oral solution or granules; the medicament comprises Lactobacillus reuteri DSM 17938 and a pharmaceutically acceptable carrier, in an amount of 1 x 10 9 to 5 x 10 10 CFU / mL or CFU / g of viable bacteria. When the medicament is an oral liquid, the Lactobacillus reuteri DSM17938 is microencapsulated and embedded, and is suspended in a stable matrix containing a freeze-drying protective agent; When the medicament is a granule, the Lactobacillus reuteri DSM17938 is mixed with excipients and then granulated by a dry granulation process.

4. A yoghurt having an increased content of alcohol metabolizing enzymes, fermented from Lactobacillus reuteri DSM 17938, and wherein the viable count of Lactobacillus reuteri DSM 17938 is > 1 x 10 9 CFU / g; said yoghurt being prepared by a process comprising the steps of inoculating Lactobacillus reuteri DSM 17938 starter culture into milk, fermenting at 37 °C for 8-10 hours to obtain a fermentation product; and cooling the fermentation product to 4 °C at a rate of 0.5-1 °C / minute and storing at 4 °C.

5. The method for preparing the yogurt having increased content of alcohol metabolism enzyme according to claim 4, characterized in that, It comprises: Lactobacillus reuteri DSM17938 bacterial powder is mixed with milk at a mass ratio of 1:4, and is fermented at 37°C for 8-10 hours to obtain a fermentation product; The fermentation product is cooled to below 4°C at a rate of 0.5-1°C / min, and is stored in the dark, to obtain yogurt with increased content of alcohol metabolism enzymes.

6. The method of claim 5, wherein, When cooled to 10-15°C, Lactobacillus reuteri DSM17938 bacterial powder microencapsulated and embedded is added to the fermentation product, and is mixed at a stirring speed of 150-200 rpm, and then is further cooled to 4°C; The microencapsulated bacteria powder is added in an amount such that the total viable count of Lactobacillus reuteri DSM 17938 in the final yogurt is increased by not less than 1 x 10 9 CFU / g.

7. The method of claim 5, wherein, It also comprises: After obtaining the fermentation product, it is transferred to a condition of 15-20°C for 15-30 minutes, and then is cooled to below 4°C at a rate of 0.5-1°C / min, and is stored in the dark at this temperature, to obtain yogurt with increased content of alcohol metabolism enzymes.

8. A medicament having an effect of increasing the content of alcohol metabolism enzymes, characterized by, When the medicament is an oral liquid, the stable matrix containing a freeze-drying protective agent is composed of trehalose, skim milk and glycerol at a mass ratio of 5-10:1-3:0.5-2; The mixed mass ratio of Lactobacillus reuteri DSM17938 microencapsulated bacterial powder and the stable matrix is 1:2-5, and they are mixed at a stirring speed of 200-400 rpm for 30-60 minutes in an environment of 15-25°C to form a homogeneous suspension, which is the oral liquid with increased content of alcohol metabolism enzymes.

9. The preparation for increasing the content of alcohol metabolism enzyme according to claim 8, wherein It comprises: Trehalose, skim milk and glycerol are dissolved in purified water at a mass ratio of 7.5:2:1, and are stirred until completely dissolved to form a homogeneous and stable aqueous phase matrix, to obtain the stable matrix; Lactobacillus reuteri DSM17938 bacterial powder microencapsulated and embedded is mixed with the stable matrix at a mass ratio of 1:3.5, and is continuously stirred at a stirring speed of 300 rpm for 45 minutes in an environment of 20±2°C to form a homogeneous suspension; The obtained suspension is filled into 10 mL brown oral liquid glass bottles under sterile conditions, and is immediately sealed by nitrogen, to obtain the oral liquid with increased content of alcohol metabolism enzymes; The obtained oral liquid product should be stored in the dark at below 25°C, and has a shelf life of 24 months; during the shelf life, the survival rate of live bacteria is not less than 80%.

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