Use of a high-yield n-acetylglycine-producing lactobacillus reuteri in alleviating obesity-related metabolic diseases

Abstract

This invention discloses the application of a high-N-acetylglycine-producing *Lactobacillus reuteri* strain in alleviating obesity-related metabolic diseases, belonging to the field of microbial technology. The *Lactobacillus reuteri* NO1 strain of this invention (…) Limosilactobacillus reuteri (NO1) possesses the ability to produce N-acetylglycine in vitro. This strain can inhibit weight gain in obese mice without affecting food intake; improve glucose tolerance in obese mice while reducing blood glucose, serum insulin, and insulin resistance index; regulate liver triglycerides in obese mice, and alleviate liver tissue damage. The *Lactobacillus reuteri* strain described in this invention has very broad application prospects for preparing anti-obesity pharmaceutical compositions and fermented foods.

Description

Technical Field

[0001] This invention relates to the application of a high-N-acetylglycine-producing Lactobacillus reuteri in alleviating obesity-related metabolic diseases, and belongs to the field of microbial technology. Background Technology

[0002] Metabolic syndrome (MS) has become a major public health challenge worldwide, with clinical manifestations often encompassing multiple systemic abnormalities such as central obesity, abnormal glucose metabolism, decreased insulin sensitivity, dyslipidemia, and persistent hypertension. Recent studies have shown that gut microbiota imbalance caused by long-term high-fat / high-calorie diets (HFD) is a significant environmental factor driving obesity, insulin resistance, and other metabolic characteristics of MS. Consistently observed in both human and animal models, a decrease in the abundance of beneficial gut microbiota (such as Bifidobacteria and Lactobacillus) and a significant proliferation of pro-inflammatory and opportunistic pathogens (such as Enterobacteriaceae) are closely related to the activation of inflammatory states in adipose tissue, systemic low-grade inflammation, and the occurrence and development of various metabolic disorders.

[0003] The gut microbiota can participate in host metabolic regulation through multiple molecular pathways, such as maintaining intestinal barrier integrity, specifically metabolizing dietary components, and generating bioactive substances like short-chain fatty acids, thus playing a crucial role in alleviating obesity and related metabolic diseases. Currently, dietary intervention and probiotic supplementation are widely explored strategies for gut microbiota regulation, which can specifically reshape the gut microecological structure. However, existing probiotic preparations still face a series of technical bottlenecks in their development and application, including controlling the number of live bacteria and production stability, insufficient colonization efficiency of strains in the host gut, and inconsistent efficacy shown in preclinical animal experiments and human clinical trials.

[0004] Studies using animal experiments have shown that many species of Lactobacillus (Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, and Lactobacillus delbrueckii) have beneficial effects on improving host glucose tolerance. Lactobacillus reuteri is a dominant lactic acid bacterium naturally found in the intestines of humans and animals. It possesses probiotic functions that inhibit pathogenic intestinal bacteria and has been included in the "List of Microbial Strains that Can Be Used in Food" issued by the Ministry of Health. Lactobacillus reuteri has excellent colonization ability in the host intestine and can exert its probiotic functions after colonization, showing good efficacy in the treatment of various diseases.

[0005] Publication number CN 116077536 A describes a strain of Lactobacillus reuteri. Limosilactobacillus reuteri LR08The strain and its application in lowering blood lipids and blood sugar, and regulating inflammatory factors; Publication number CN 114344344B describes Lactobacillus reuteri J1 and its efficacy in improving obesity induced by a high-fat diet and the enhancement effect of lactic acid bacteria extracellular polysaccharides; Publication number CN 117618476 A describes the application of a strain of Lactobacillus reuteri combined with chlorogenic acid in the treatment of metabolic disorder-related diseases. Publication number CN 117899126 A describes a strain of *Lactobacillus reuteri* PRS-156 and its application in preventing and treating obesity, diabetes, hyperuricemia, gout caused by hyperuricemia, gout complications, or kidney damage; Publication number CN 116508993 B describes *Lactobacillus reuteri* LR08 and its application in adjusting blood lipid levels and correcting metabolic disorders in obese mice; Publication number CN 106794207 A describes the combined effects of *Lactobacillus reuteri* with *Bifidobacterium longum*, *Lactobacillus rhamnosus*, and *Bifidobacterium lactis* and its application in preventing or treating microbial dysbiosis. Therefore, most applications of the probiotic effects of *Lactobacillus reuteri* are limited to its ability to alleviate obesity or merely regulate the host's intestinal flora, while insulin resistance caused by obesity is closely related to intestinal flora dysbiosis.

[0006] Although studies have shown that *Lactobacillus reuteri* can alleviate metabolic disorders, the bioactive metabolites produced by *Lactobacillus reuteri* are important functional substances for alleviating obesity, and N-acetylglycine is one of the main metabolites produced by *Lactobacillus reuteri*. Currently, there are no reports demonstrating the ameliorative effect of *Lactobacillus reuteri* with high N-acetylglycine production on metabolic disorders.

[0007] Therefore, developing a strain of *Lactobacillus reuteri* that can produce high levels of N-acetylglycine and improve metabolic disorders is of great significance in the fields of medicine, food, and health products. Summary of the Invention

[0008] To alleviate gut microbiota dysbiosis, associated glucose metabolism disorders, and liver damage resulting from obesity-related metabolic diseases induced by a high-fat diet, this invention screened out a *Lactobacillus reuteri* strain that can alleviate obesity. It was demonstrated that this strain can improve obesity under different gut microbiota backgrounds and further combat accompanying metabolic disorders. This has significant implications and broad prospects for dietary intervention in alleviating obesity.

[0009] This invention provides a strain of *Lactobacillus reuteri* (… Limosilactobacillus reuteri Accession number N01 was deposited on December 30, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67579.

[0010] The Lactobacillus reuteri ( Limosilactobacillus reuteri N01 was isolated from the fermentation broth of fecal samples from normal individuals in Nanchang, Jiangxi Province. Sequencing analysis revealed its 16S rDNA sequence to be shown in SEQ ID NO. 1. The sequence was then compared with the nucleic acid sequence of *Lactobacillus reuteri* using NCBI, showing a 99.33% similarity. It was named *Lactobacillus reuteri*. Limosilactobacillus reuteri )N01.

[0011] In one embodiment of the present invention, the microbial agent contains *Lactobacillus reuteri* (… Limosilactobacillus reuteri The viable count of N01 is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

[0012] In one embodiment of the present invention, the product contains *Lactobacillus reuteri* (… Limosilactobacillus reuteri The viable count of N01 is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

[0013] In one embodiment of the present invention, the product is food, medicine, or health product.

[0014] In one embodiment of the present invention, the food includes beverages, dairy products, or other products containing the aforementioned *Lactobacillus reuteri* (…). Limosilactobacillus reuteri Food item No. 01.

[0015] In one embodiment of the present invention, the pharmaceutical product comprises *Lactobacillus reuteri* (… Limosilactobacillus reuteri ) N01, drug carriers and / or pharmaceutical excipients.

[0016] In one embodiment of the present invention, the dosage form of the medicine or health product includes granules, capsules, tablets, pills or oral liquids.

[0017] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0018] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, pH adjusters, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0019] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0020] In one embodiment of the present invention, the adhesive is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone. In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup. In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate and / or sodium bicarbonate.

[0021] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol. In one embodiment of the present invention, the flavoring agent is a simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid and / or sodium bicarbonate.

[0022] The present invention also provides a method for preparing N-acetylglycine, wherein the aforementioned *Lactobacillus reuteri* NO1 is inoculated into MRS medium for fermentation.

[0023] In one embodiment of the present invention, the culture medium comprises peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate, manganese sulfate, Tween, and glucose.

[0024] The present invention also provides the above-mentioned *Lactobacillus reuteri* ( Limosilactobacillus reuteri ) N01, or the above-mentioned microbial agents, in the preparation of medicines for the prevention and / or treatment of metabolic diseases.

[0025] In one embodiment of the invention, the metabolic disease includes insulin resistance or diabetes.

[0026] The present invention also provides the above-mentioned *Lactobacillus reuteri* ( Limosilactobacillus reuteri ) N01, or the above-mentioned microbial agents, in the preparation of health products that help stabilize blood sugar.

[0027] In one embodiment of the present invention, the product contains *Lactobacillus reuteri* (… Limosilactobacillus reuteri The viable count of N01 is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

[0028] In one embodiment of the present invention, the pharmaceutical product comprises *Lactobacillus reuteri* (… Limosilactobacillus reuteri ) N01, drug carriers and / or pharmaceutical excipients.

[0029] In one embodiment of the present invention, the dosage form of the medicine or health product includes granules, capsules, tablets, pills or oral liquids.

[0030] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0031] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, pH adjusters, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0032] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0033] In one embodiment of the present invention, the adhesive is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone. In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup. In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate and / or sodium bicarbonate.

[0034] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol. In one embodiment of the present invention, the flavoring agent is a simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid and / or sodium bicarbonate.

[0035] Beneficial effects 1. This invention screened out a strain of *Lactobacillus reuteri* that produces high levels of N-acetylglycine (…). Limosilactobacillus reuteri The *Lactobacillus reuteri* strain N01 can produce 76.64 μM of N-acetylglycine in an in vitro culture medium environment. The yield is 11.5 times that of the model strain *Lactobacillus reuteri* DSM 20016 (6.64 μM). Compared with other *Lactobacillus reuteri* strains screened at the same time (17.53~64.14 μM), the N-acetylglycine yield is increased by 1.2~4.3 times, which is a significant improvement.

[0036] 2. The *Lactobacillus reuteri* strain screened in this invention (… Limosilactobacillus reuteri NO1 has the effect of improving obesity, specifically in the following ways: 1) It can inhibit the weight gain of mice on a high-fat diet without affecting their food intake; 2) It can improve glucose tolerance in high-fat induced obese mice; 3) It can reduce blood glucose and serum insulin in high-fat induced obese mice, reduce the insulin resistance index (HOMA-IR), and improve glucose metabolism disorders in mice; 4) It can reduce the increase in liver weight in obese mice induced by a high-fat diet, lower liver triglyceride levels, and alleviate liver tissue damage; 3. *Lactobacillus reuteri* ( Limosilactobacillus reuteri Lactobacillus reuteri (Lactobacillus reuteri) is a type of probiotic and is currently included in the "List of Microbial Strains that Can Be Used in Food" issued by the Ministry of Health. Therefore, the Lactobacillus reuteri (Lactobacillus reuteri) screened in this invention... Limosilactobacillus reuteri NO1 has no side effects on the human body and can be used to prepare drug compositions and fermented foods that can improve obesity, with a very wide range of application prospects.

[0037] Preservation of biological materials A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri No. 01 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 30, 2025, and its taxonomic name is: Limosilactobacillus reuteri The accession number is GDMCC No: 67579, and the accession address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Provincial Center for Microbial Culture Collection. Attached Figure Description

[0038] Figure 1 Lactobacillus reuteri ( Limosilactobacillus reuteri The following graphs show the weight change trend of mice during the intervention of high-fat diet-induced obesity in mice and the food intake of mice at the end of week 8; where A is the weight change trend of mice in each group during the intervention period; and B is the average food intake during the 8-week experiment. Figure 2 Lactobacillus reuteri ( Limosilactobacillus reuteri The oral glucose tolerance test curves and area under the glucose tolerance test curves of N01 mice on a high-fat diet during the last week of intervention; where A is the glucose tolerance test curve; and B is the area under the glucose tolerance test curve.

[0039] Figure 3 Lactobacillus reuteri ( Limosilactobacillus reuteri N01 mice on a high-fat diet were tested for fasting blood glucose concentration, serum insulin concentration, and insulin resistance index in the last week after intervention. Where A is fasting insulin concentration, B is fasting blood glucose, and C is insulin resistance index.

[0040] Figure 4 Lactobacillus reuteri ( Limosilactobacillus reuteri) Changes in liver function indicators in mice after N01 intervention on a high-fat diet; where A is the level of alanine aminotransferase (ALT); B is the level of aspartate aminotransferase (AST); C is the level of liver triglycerides; and D is the liver weight after intervention.

[0041] Figure 5 Lactobacillus reuteri ( Limosilactobacillus reuteri Liver photographs, H&E staining results, and Oil Red O staining results of liver tissue in mice after N01 intervention on a high-fat diet.

[0042] "*" indicates a significant difference from the model group (M) (*: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001); Data results in some tables are expressed as averages. Data analysis was performed using SPSS 24 with one-way ANOVA and Tukey's HSD post-hoc test. Different letters "a, b, c, etc." in the same column indicate significant differences between groups. p <0.05). Detailed Implementation

[0043] The mice used in the following examples were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and were housed at 25±2℃, constant humidity 50±5%, with 12 hours of light (8:00-20:00), soundproofed, and with free access to food and water. Experiments began after two weeks of acclimatization. The insulin kit (purchased from Crystal Chem, catalog number: 90080), blood glucose test strips, and blood glucose meter used in the following examples were purchased from Roche; the triglyceride TG kit (A110-1-1) was purchased from Nanjing Jiancheng Bioengineering Institute. The fecal DNA extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd. (DP328); all culture medium components used in the following examples were purchased from Qingdao Haibo Co., Ltd. The high-fat diet used in the following examples was purchased from Research Diets, Inc. (D12492).

[0044] The normal feed used in the following examples was purchased from Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd. (mice maintenance feed, fed during the adaptation period).

[0045] The following examples involve culture media: Preparation of activation medium (g / L): The components include carbon sources: pectin 0.047, xylan 0.047, arabinogalactan, amylopectin 0.04, soluble starch 0.392; nitrogen sources: bacterial peptone 24, tryptone 24; inorganic salts: magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.5, sodium chloride 4.5, calcium chloride dihydrate 0.45, ferric sulfate heptahydrate 0.005; bile salts 0.4, cysteine ​​hydrochloride 0.2, and acid-base buffer (MES) 19.52. First, the above components were prepared, and the pH was adjusted to 6 before deoxygenation and sterilization (121℃, 15 min). After sterilization, the culture medium was transferred to an anaerobic glove box. 1 μg of heat-sensitive heme, 1 μg of vitamin K3 (VK3), and 0.1 mL of a vitamin mixture (Wolfe's Vitamin Solution) were added to 1 L of the culture medium and filtered through a 0.22 μm filter membrane. The medium was then deoxygenated overnight in the anaerobic glove box to obtain the activated liquid culture medium.

[0046] Preparation of enrichment medium: Each liter of enrichment medium is prepared by mixing 350 mL of solution A, 150 mL of solution B, 500 mL of solution C, 1 mL of solution D, and 0.08 mL of Wolfe's Vitamin Solution. The formula (g / L) includes: Solution A: bacterial peptone 68.57, tryptone 68.57, bile salts 1.14, anaerobic agent cysteine ​​hydrochloride 1.43, magnesium sulfate 1.14, potassium monohydrogen phosphate 5.48, sodium chloride 12.86, calcium chloride 0.97, ferric sulfate heptahydrate 0.014; Solution B: acid-base buffer (MES) 130; Solution C: heme 10 mg, vitamin K3 (VK3) 8 mg. First, the autoclaved components (solutions A and B) are prepared, the pH is adjusted to 6, and deoxygenation is performed, followed by sterilization (121℃, 15 min). After sterilization, the culture medium was transferred to an anaerobic glove box and left overnight. Finally, solution C and Wolfe's Vitamin Solution (filtered through a 0.22 μm membrane) were added to the culture medium in the specified proportions to obtain the enrichment medium.

[0047] MRS liquid culture medium (g / L): peptone 10.0, beef extract 8.0, yeast extract 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 1.0, dissolved in 1L of distilled water, and cysteine ​​hydrochloride 0.5-1 g / L was added. The mixture was mixed thoroughly, and the pH was adjusted to 6.6-7.0. After sterilization at 121℃ for 15 min, the MRS liquid culture medium was obtained.

[0048] Preparation of MRS solid medium: Add 1.5-2% agar to the MRS liquid medium. Mix well, then adjust the pH to 6.6-7.0, and sterilize at 121℃ for 15 min to obtain the MRS solid medium.

[0049] The detection methods involved in the following embodiments are as follows: Liver function assay in mice: After week 8 of the experiment, mice were anesthetized and sacrificed. Blood was collected from the orbital sinus of the mice, centrifuged at 3000 rpm for 15 min, and the resulting serum was obtained. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which reflect liver function, in the mouse serum were measured using a blood biochemistry analyzer.

[0050] Weight determination of mouse liver tissue: After the 8th week of the experiment, the mice were anesthetized and sacrificed, the complete livers of the mice were separated, weighed and recorded.

[0051] Histological observation of mouse liver: (1) H&E staining: The tissue was embedded in paraffin, frozen and cut into sections. Before staining, the paraffin sections were dewaxed and stained with hematoxylin and eosin respectively. After dehydration, the sections were mounted and observed under a microscope. (2) Oil Red O staining: The tissue was embedded in paraffin, frozen and cut into sections. The sections were then immersed in Oil Red staining solution and counterstained with hematoxylin. After mounting with glycerol gelatin, the sections were observed under a microscope.

[0052] Oral glucose tolerance test (OGTT): Before the end of week 8, mice in each group were fasted for 6 hours and then administered glucose solution (1.5 g / kg) by gavage. Blood glucose levels were measured at 0, 15, 30, 60, and 90 minutes after gavage using a blood glucose meter and matching test strips collected from the tail vein of each mouse. The area under the oral glucose tolerance curve (AUC) was calculated using the following formula: Among them, BG0, BG 15 BG 30 BG 60 and BG 90 The blood glucose values ​​were measured at 0 min, 15 min, 30 min, 60 min, and 90 min.

[0053] Fasting blood glucose (FBG) determination: After fasting for 12 hours in week 8, blood glucose levels in each group of mice were measured by blood collection from the tail vein using a blood glucose meter and matching test strips.

[0054] Serum insulin determination: After the 8th week of the experiment, the mice were anesthetized and sacrificed, and serum was obtained. The insulin content in the serum was determined according to the kit instructions.

[0055] Methods for calculating the HOMA-IR (Homo da Génifique Insulin Resistance Index): ; Determination of triglycerides in the liver: Triglyceride assay kit was used.

[0056] Example 1: Lactobacillus reuteri ( Limosilactobacillus reuteri Separation and screening of N01 1. Sample collection Fecal samples were collected from normal individuals in Nanchang, Jiangxi Province. The samples were placed in preservation tubes and 5 times their weight of protective solution were added (preparation of protective solution: weigh 1 g / L cysteine ​​hydrochloride and 200-300 g / L glycerol, dissolve them evenly in PBS (1×), and sterilize at 115-121℃ for 15-20 min). The samples were stored in an insulated box containing dry ice and brought back to the laboratory. They were then quickly placed in a -80 °C freezer for separation and screening.

[0057] 2. Accumulation of fecal bacteria The above fecal microbiota solution was removed from the -80 ℃ freezer, thawed, and centrifuged at low speed (500 g, 5 min, 4 ℃) to obtain the supernatant. The supernatant was then filtered through a 100 μm filter to remove impurities. The supernatant fecal microbiota solution was inoculated into activation medium (fecal microbiota solution: activation medium = 1:9, (v / v)) and incubated at 37 ℃ and 140 rpm for 16 h. Then, it was inoculated into enrichment medium at an inoculation ratio of 10% (v / v) and incubated at 37 ℃ and 140 rpm for 24 h. The enriched fecal microbiota solution was obtained. All the above operations were performed in a sterile anaerobic environment.

[0058] 3. Isolation and purification of *Lactobacillus reuteri* (1) Gradual dilution of fecal microbial solution: In a sterile anaerobic environment, take the above-enriched fecal microbial solution and add it to 9 mL of physiological saline to obtain the first gradient dilution. Take 1 mL of the first gradient dilution and add it to 9 mL of physiological saline to obtain the second gradient dilution. And so on, to prepare a total of 5 gradient dilutions. (2) Spreading culture: Take 100 μL of each of the above gradient dilutions and place them on MRS fixation medium. After spreading, incubate under anaerobic conditions at 37°C for 48 h to obtain diluted spread plates. (3) Purification culture: Select pure single colonies with neat edges, slightly white, opaque, moist and smooth surfaces, and uniform morphology from the solid culture medium and inoculate them into 5 mL of liquid MRS medium. The initial inoculation amount is 10. 5 The culture medium was incubated at CFU / mL under anaerobic conditions at 37°C for 24 h to obtain a purified culture medium.

[0059] 4. Preservation and Identification of Microbial Strains The purified culture medium with the best growth obtained in step 3 was centrifuged at 8000 r / min for 10 min, and the supernatant was discarded to obtain bacterial cells. PCR was performed using bacterial 16S rDNA PCR-specific primers (see Table 1). After confirmation by nucleic acid electrophoresis analysis, the amplified product was sent to the company for sequencing. Its 16S rDNA sequence is shown in SEQ ID NO.1. The sequencing results were compared with sequences in the NCBI database; the results showed that the nucleic acid sequence similarity with *Lactobacillus reuteri* was as high as 99.33%, and it was named *Lactobacillus reuteri*. Limosilactobacillus reuteri )N01.

[0060] Table 1 Primer Names

[0061] 5. Targeted metabolism assay to determine the ability of 20 strains of *Lactobacillus reuteri* to produce N-acetylglycine. The purified culture medium with the best growth obtained in step 3 was centrifuged at 12000 rpm for 10 min, and the supernatant was collected to remove the bacterial cells. 100 μL of the supernatant was added to 400 μL of 50% acetonitrile water, vortexed, centrifuged at 12000 rpm for 15 min, and then filtered through a membrane. N-acetylglycine was detected using QTRAP 6500 HPLC-MS / MS. The experimental results are shown in Table 2. The N-acetylglycine production range of the 20 strains of *Lactobacillus reuteri* was 17.53–76.63 μM, among which *Lactobacillus reuteri* (… Limosilactobacillus reuteri N01 had the highest N-acetylglycine production at 76.64 μM, significantly higher than the *Lactobacillus reuteri* model strain. Limosilactobacillus reuteri DSM 20016.

[0062] Table 2. N-acetylglycine production capacity of 20 strains of *Lactobacillus reuteri*.

[0063] Example 2: Lactobacillus reuteri ( Limosilactobacillus reuteri Effects of N01 on body weight and diet in obese mice on a high-fat diet The specific steps are as follows: 1. Preparation of cryopreservation medium for *Lactobacillus reuteri*: (1) Cultivation method: In a sterile anaerobic environment, streak Lactobacillus reuteri on MRS solid medium and culture under anaerobic conditions for 48 h. After single colonies are formed, pick single colonies and inoculate them into MRS liquid medium. Culture under anaerobic conditions at 37℃ for 16-24 h to reach the stationary phase. At this time, the OD value is 1.0~1.4, and the seed liquid is prepared.

[0064] (2) Preparation of protective agent: Weigh 1 g / L cysteine ​​hydrochloride and 200-300 g / L glycerol, dissolve them evenly in distilled water, and sterilize at 115-121℃ for 15-20 min.

[0065] (3) Preparation of cryoprotectant: After centrifuging the Lactobacillus reuteri seed culture cultured to the stable period in step (1) (8000 rpm, 10 min, 4℃), wash it 1-2 times with sterile phosphate buffer (pH 7.2), and then resuspend the bacterial culture with the protectant prepared in step (2) to obtain Lactobacillus reuteri cryoprotectant, which is stored at -80℃ for later use.

[0066] 2. Preparation of *Lactobacillus reuteri* preparation: (1) Activation of strains: The cryopreservation agent of Lactobacillus reuteri prepared in step 1 was streaked on MRS solid medium and cultured under anaerobic conditions for 48 h. After single colonies were formed, the single colonies were inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 16-24 h to reach the stationary phase (OD value: 1.0-1.4).

[0067] (2) Preparation of bacterial agent: Take 100 μL of the culture medium obtained in step (1) at different dilution ratios and spread it on MRS solid medium. Count the number of colonies on the MRS solid plate and calculate the number of viable bacteria in the liquid medium of step (1). After washing 1-2 times with sterile phosphate buffer (pH 7.2), prepare the bacterial solution to a concentration of 1×10⁻⁶. 10 For formulations with a concentration of CFU / mL, the gavage volume is 0.2 mL.

[0068] 3. Experimental methods: This invention uses a high-fat diet to induce obesity in mice. Sixteen healthy male C57BL / 6J mice aged 6 weeks were randomly divided into two groups (n=8 per group): a high-fat diet obesity group (model group, denoted by M), and a high-fat diet plus *Lactobacillus reuteri* (model group). Limosilactobacillus reuteri Group N01 (Lactobacillus reuteri Group N01, denoted by L).

[0069] The experimental procedure is shown in Table 3. After a two-week adaptation period; Intervention process: From week 0 to week 8, mice in the model group (M) and the Lactobacillus reuteri N01 group (L) were fed a high-fat diet. After 8 weeks of high-fat diet, fasting blood glucose and fasting serum insulin levels were measured in each high-fat diet group, and the area under the oral glucose tolerance curve and insulin sensitivity index were calculated. The results showed that the above indicators were significantly higher in the model group than in the Lactobacillus reuteri intervention group (n=6 in each group); specific indicators are shown in Table 6. Intervention experiment process: Model group (M): During the intervention period (weeks 0-8), the group was fed a high-fat diet, and 0.2 mL of sterile phosphate buffer was administered by gavage once a day. Water was available freely. Lactobacillus reuteri group (L): During the intervention period (weeks 0-8), the diet remained high-fat, and 0.2 mL of Lactobacillus reuteri bacterial suspension (concentration of 1×10⁻⁶) was administered by gavage once daily. 10 (CFU / mL), free access to water; During weeks 0-8, mice in each group had free access to food and water, and their condition was monitored and recorded every 3 days.

[0070] At the end of week 8 of the experiment, mice were anesthetized and euthanized. Liver was harvested, and blood was collected from the orbital rims. The blood was centrifuged at 3000 rpm for 15 min to obtain serum. Serum and liver were stored... 80℃ is used for subsequent analysis.

[0071] Table 3 Experimental Procedure

[0072] 4. Effects of *Lactobacillus reuteri* on body weight, body fat, and diet in insulin-resistant mice on a high-fat diet. The specific experimental procedure is the same as steps 1-3, except that during the 8-week high-fat induction period, the mice in each group were weighed every 7 days; after the intervention, the mice were anesthetized and sacrificed.

[0073] I. *Lactobacillus reuteri* ( Limosilactobacillus reuteri Effects of N01 on body weight and diet in obese mice on a high-fat diet (1) Experimental results are as follows Figure 1 As shown, after 8 weeks of intervention, the weight trends of mice in each group were as follows: Figure 1 As shown in Figure A: Compared with the model group (M), the weight gain of mice in the *Lactobacillus reuteri* N01 group (L) was inhibited, and the weight of mice in the L group was significantly reduced from 44.80 g to 38.02 g compared with the M group. p <0.001).

[0074] (2) Changes in the diet of mice during the intervention period (weeks 0-8) are shown in Table 4. From the time of gavage administration of *Lactobacillus reuteri*, there was no significant difference in food intake among the high-fat diet groups. p The value >0.05 indicates that the effect of *Lactobacillus reuteri* on mouse body weight is not caused by affecting the amount of food consumed by the mice.

[0075] Table 4. Feed intake (g / day / mice) of mice in each group during the intervention period (weeks 0-8)

[0076] The above results indicate that the *Lactobacillus reuteri* of this invention can inhibit the weight gain of high-fat induced obese mice, and this weight control is not caused by reducing the amount of food consumed by the mice.

[0077] Example 3: Lactobacillus reuteri ( Limosilactobacillus reuteri Effects of N01 on glucose tolerance in high-fat induced obese mice The specific steps are as follows: The specific experimental method was the same as in Example 2, except that at the end of week 7, mice in each group underwent an oral glucose tolerance test (OGTT), and after the experiment ended in week 8, the mice were anesthetized and sacrificed. The results are as follows... Figure 2 As shown in A~B.

[0078] The results showed that, Figure 2 As shown in A and Table 5, blood glucose levels in all groups of mice rose rapidly after gavage with glucose solution. At 15 min after gavage, the blood glucose level in the model group (M) (24.3 mmol / L) was significantly higher than that in the Lactobacillus reuteri group (L) (19.8 mmol / L).

[0079] like Figure 2 As shown in B, the area under the curve (AUC) of the oral glucose tolerance test (OGTT) was used to evaluate the ability of each group of mice to regulate blood glucose. The AUC of the model group (M) (31.27) was significantly higher than that of the Lactobacillus reuteri group (L) (25.81). p <0.05).

[0080] The above results indicate that the *Lactobacillus reuteri* of this invention can improve glucose tolerance in obese mice and restore their ability to regulate blood sugar.

[0081] Table 5 Results of the oral glucose tolerance test (OGTT) in each group of mice

[0082] Example 4: Effects of *Lactobacillus reuteri* on fasting blood glucose, serum insulin, and insulin resistance index in high-fat induced insulin-resistant mice The specific steps are as follows: The specific experimental method is the same as in Example 2, except that at the end of week 8, fasting blood glucose (FBG) was measured in each group of mice. After the experiment ended in week 8, the mice were anesthetized and sacrificed. After the mice were sacrificed, the insulin level in the serum of each group of mice was measured, and the insulin resistance index (HOMA-IR) was calculated by using FBG and serum insulin level.

[0083] Table 6. Fasting blood glucose, fasting insulin, and insulin resistance index of mice in each group.

[0084] The results showed that, Figure 3 As shown in Tables A and 6, the serum insulin level in the model group (M) mice (2.35 ng / mL) was significantly higher than that in the Lactobacillus reuteri N01 group (L) (1.38 ng / mL). p <0.05), indicating that the model group (M) mice had significant insulin resistance. After intervention with *Lactobacillus reuteri* N01, the serum insulin level of mice in the *Lactobacillus reuteri* N01 group (L) was significantly reduced.

[0085] like Figure 3 As shown in B and Table 6, the serum FBG level in the model group (M) (10.82 mmol / L) was significantly higher than that in the *Lactobacillus reuteri* N01 group (L) (8.06 mmol / L). p <0.01).

[0086] like Figure 3 As shown in Table C and Table 6, the insulin resistance index (HOMA-IR) can reflect the degree of insulin resistance in each group of mice. The insulin HOMA-IR of mice in the Lactobacillus reuteri N01 group (L) (10.94) was significantly lower than that of the M group (23.94), a decrease of 45.70%.

[0087] The above results indicate that the Lactobacillus reuteri NO1 of this invention can reduce blood glucose and serum insulin levels in insulin-resistant mice, decrease HOMA-IR, and improve glucose metabolism disorders in insulin-resistant mice induced by a high-fat diet.

[0088] Example 5: Effects of *Lactobacillus reuteri* N01 on liver damage in high-fat induced insulin-resistant mice The specific experimental method was the same as in Example 2. After the experiment ended (week 8), mouse serum was collected, and the levels of ALT and AST in the mouse serum were measured. The mouse livers were weighed, photographed, and stained to observe the morphological changes in the livers of each group. The results are as follows: Figures 4 - 5 As shown.

[0089] like Figure 4 As shown in A-4B, insulin resistance induced by a high-fat diet increased serum ALT and AST levels in the model group (M) mice (335.03 U / L and 279.69 U / L, respectively). After intervention with *Lactobacillus reuteri* N01, compared with the model group (M), serum ALT and AST levels in the *Lactobacillus reuteri* N01 group (L) mice (103.50 U / L and 209.87 U / L, respectively) decreased by 69.10% and 24.9%, respectively.

[0090] like Figure 4As shown in Figure C, the model group (M) mice had the highest liver TG level at 1.69 mmol / gprot, while the Lactobacillus reuteri N01 group (L) mice had a liver TG level of 1.22 mmol / gprot. Compared with the model group (M), the Lactobacillus reuteri group significantly reduced the liver TG level in insulin-resistant mice after intervention. p <0.001), a decrease of 27.8%.

[0091] like Figure 4 As shown in Figure D, the results indicated that, compared to the model group (M), the liver weight of mice in the *Lactobacillus reuteri* N01 group (L) (0.98 g) was significantly lower than that in the model group (M) (1.42 g). p The value <0.01 indicates that intervention with *Lactobacillus reuteri* can reduce the increase in liver weight in mice induced by a high-fat diet.

[0092] like Figure 5 As shown, the livers of mice in the L01 group (L) of *Lactobacillus reuteri* were bright in color and smooth in surface; while the livers of mice in the model group (M) were dull in color and significantly enlarged in volume. From the sections, the livers of the model group (M) showed obvious diffuse steatosis (vacuolization) and fibrosis. After treatment with *Lactobacillus reuteri*, the livers showed significant improvement compared with the model group (M).

[0093] The above results indicate that the Lactobacillus reuteri NO1 of this invention can significantly reduce liver weight, lower liver triglycerides, and improve liver tissue structure and morphology in insulin-resistant mice, thus alleviating liver damage in these mice.

[0094] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri Accession number N01 was deposited on December 30, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67579.

2. A microbial inoculant, characterized in that, Contains Lactobacillus reuteri NO1 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, In the microbial agent, the number of *Lactobacillus reuteri* NO1 cells is not less than 1 × 10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

4. A product characterized in that, The product contains Lactobacillus reuteri NO1 as described in claim 1, or contains the microbial agent as described in claim 2 or 3.

5. The product as described in claim 4, characterized in that, The dosage forms of the product include granules, capsules, tablets, pills, or oral liquids.

6. The product as described in claim 4 or 5, characterized in that, The product is food, medicine, or health product; the food includes beverages, dairy products, or other foods containing *Lactobacillus reuteri* NO1 as described in claim 1.

7. The product as described in claim 6, characterized in that, The drug contains Lactobacillus reuteri N01, and also contains a drug carrier and / or pharmaceutical excipients.

8. A method for preparing N-acetylglycine, characterized in that, The Lactobacillus reuteri NO1 of claim 1 is inoculated into a culture medium for fermentation.

9. The method as described in claim 8, characterized in that, The culture medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate, manganese sulfate, Tween, and glucose.

10. The use of *Lactobacillus reuteri* NO1 as described in claim 1, or the microbial agent as described in claim 2 or 3, in the preparation of products for the prevention and / or treatment of metabolic diseases, characterized in that... The product is a pharmaceutical or health supplement; the metabolic disease includes insulin resistance or diabetes.

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