A Lactobacillus strain for preventing obesity, its product, composition and application

Abstract

The present disclosure provides a strain of the family Lactobacillaceae and its products, compositions and applications. In a first aspect of the present disclosure, a strain of the family Lactobacillaceae ( Lactobacillaceae ) strain, the Lactobacillaceae strain may include strain MNH08891 with a deposit number of GDMCC No. 65551, or strain MNH22076 with a deposit number of GDMCC No. 65554, or a strain having at least 98.65% identity to the 16S rRNA sequence of strain MNH08891 or strain MNH22076, or a strain having at least 98.65% identity to the 16S rRNA sequence set forth in SEQ ID NO: 1. The strains disclosed in the embodiments of the present disclosure or products based on the strains have excellent effects in treating, preventing, or alleviating metabolic diseases, as well as treating, preventing, or ameliorating metabolic disorders after discontinuation of drugs used to treat metabolic diseases.

Description

Technical Field

[0001] The present disclosure generally relates to the field of microbial technology. More specifically, the present disclosure relates to a strain of the family Lactobacillus that prevents obesity, and its products, compositions, and applications. Background Art

[0002] Obesity has become a growing public health problem worldwide. To address this challenge, the medical community is committed to developing various treatments, one of which is based on incretin-like peptide-1 (GLP-1) receptor agonists. Growing evidence shows that by mimicking insulin secretion, these drugs can significantly reduce patient weight, helping to suppress appetite and reduce food intake. While these drugs have demonstrated significant weight loss efficacy, a significant number of patients discontinue their use for various reasons.

[0003] Although the weight loss effect of GLP-1 receptor agonists such as liraglutide and semaglutide is indeed significant in the short term, studies have found that the incidence of four gastrointestinal adverse reactions induced by GLP-1 agonists semaglutide and liraglutide is higher than that of other weight loss drugs, including biliary tract disease, pancreatitis, intestinal obstruction, and gastric spasm. In addition, GLP-1 receptor agonists are prone to rebound after discontinuation. Clinical trials have shown that participants who received semaglutide treatment regained more than 50% of their lost weight one year after discontinuation of the drug. In other words, persistent use of semaglutide can indeed ensure weight loss; but once the intervention is stopped, the possibility of rebound is probably even higher.

[0004] A population-based study found that from enrollment to one year after discontinuation, the liraglutide group regained a total of 8.7 kg, the most among all intervention groups. The combined liraglutide and moderate-to-high-intensity exercise group regained the least weight, a full 5.1 kg less (P=0.04). Researchers believe that one reason for the increased risk of weight regain after discontinuation of liraglutide is that liraglutide suppresses appetite and delays gastric emptying, effectively forcing us to control our appetite. However, once the drug is discontinued, we may be unable to cope with the return of appetite, ultimately leading to weight regain. (Jensen SBK, Blond MB, Sandsdal RM, et al. Healthy weight loss maintenance with exercise, GLP-1 receptor agonist, or both combined followed by one year without treatment: a post-treatment analysis of a randomized placebo-controlled trial[J]. eClinicalMedicine, 2024.)

[0005] That is, due to artificial intervention (i.e., an increase in exogenous GLP-1), the body detects higher levels of "fake" GLP-1, and the synthesis of endogenous GLP-1 decreases accordingly. After abruptly stopping the drug, the body's previously high levels of GLP-1 drop sharply, and hunger and appetite "return with a vengeance," leading to a rapid rebound in weight, blood sugar, blood lipids, and body fat.

[0006] As can be seen, most existing weight loss products on the market only treat symptoms, not the root cause. Some even compromise overall health, disrupting intestinal flora balance, disrupting metabolism, and easily causing rebound or dependency, or even serious side effects. Most people experience weight regain after stopping medication, and health indicators such as blood pressure, blood sugar, and cholesterol levels deteriorate. Given the significant weight rebound issue, preventing weight regain and maintaining blood sugar balance after discontinuing GLP-1 agonists have become pressing issues.

[0007] Probiotics have shown potential in treating metabolic disorders, including obesity, diabetes, and metabolic-associated fatty liver disease (MAFLD). However, since appetite tends to return with a vengeance after discontinuation of weight-loss drugs, weight loss becomes more difficult. Therefore, screening out new species or strains of probiotics that can be effectively used to prevent weight regain remains a huge challenge, but also represents a huge unmet need. Summary of the Invention

[0008] In order to at least solve one or more of the above-mentioned technical problems, the present disclosure provides a lactobacillus and its products, compositions and uses in multiple aspects.

[0009] The present invention discloses the isolation of a new Lactobacillus family ( Lactobacillaceae ) strains MNH08891 and MNH22076.

[0010] The strain disclosed herein can effectively prevent weight regain and maintain healthy weight, blood sugar, and body fat after weight loss. Specifically, the strain's effects in preventing weight regain include: 1. improving weight regain after drug discontinuation and maintaining weight loss; 2. improving blood sugar homeostasis imbalance after drug discontinuation and maintaining blood sugar balance; and 3. improving localized fat accumulation after drug discontinuation and maintaining the localized fat loss effect.

[0011] The new strain disclosed herein is tolerant to salt and choline, making it easy to colonize. It can utilize a variety of carbon sources to produce acid and is easy to culture. It is sensitive to most types of antibiotics, with a low risk of antibiotic resistance. It is safe and non-toxic. It can reduce weight gain and the rate of weight gain after discontinuation of semaglutide treatment; improve the imbalance of blood glucose homeostasis and blood glucose elevation after discontinuation of semaglutide treatment; and significantly reduce fat accumulation after discontinuation of semaglutide treatment. It has the potential to prevent elevated blood glucose and fat accumulation in the body, prevent weight gain again, and maintain blood glucose balance.

[0012] According to a first aspect of the present disclosure, the present disclosure provides an isolated Lactobacillusaceae ( Lactobacillaceae ) strain, the average nucleotide identity (ANI) values ​​between it and the strain deposited in Guangdong Provincial Microbiological Culture Collection with the deposit number of GDMCC No: 65551 or the deposit number of GDMCC No: 65554 are 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 97.1%, 97.2%, 97. 7.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

[0013] In some embodiments, the Lactobacillaceae ( Lactobacillaceae) strain and the strain with a deposit number of GDMCC No: 65551 or a deposit number of GDMCC No: 65554 have an alignment score (AF) of at least 90%, at least 90.5%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100%.

[0014] In some embodiments, the Lactobacillaceae ( Lactobacillaceae ) strain and the strain with a deposit number of GDMCC No: 65551 or a deposit number of GDMCC No: 65554 have a 16S rRNA sequence that is at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to the sequence shown in SEQ ID NO. 1 or 2.

[0015] In some embodiments, the lactobacillus is Lactobacillus paracasei ( Lacticaseibacillus paracasei )MNH08891, deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), the deposit number is GDMCC No: 65551, and the deposit date is November 28, 2024.

[0016] In some embodiments, the lactobacillus is Lactobacillus crispatus ( Lactobacillus crispatus )MNH22076, deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), the deposit number is GDMCC No: 65554, and the deposit date is November 28, 2024.

[0017] In some embodiments, the Lactobacillus paracasei MNH08891 has a gene cluster that produces primary metabolites that convert pyruvate into acetate and formate.

[0018] In some embodiments, the Lactobacillus crispatus MNH22076 has a gene cluster that can produce a primary metabolite that converts arginine into bicarbonate.

[0019] According to a second aspect of the present disclosure, provided is a culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillaceae strain described in the first aspect.

[0020] In some embodiments, the culture includes a fermentation broth of the Lactobacillusaceae strain or a concentrated or dried product of the fermentation broth.

[0021] In some embodiments, the culture comprises a solid culture, a fermentation culture, or a supernatant of a fermentation culture of a Lactobacillaceae strain.

[0022] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained using a liquid culture medium under anaerobic culture conditions.

[0023] According to a third aspect of the present disclosure, a composition for preventing obesity is provided, comprising an effective amount of a culture, live bacteria, freeze-dried bacteria, or inactivated bacteria of the Lactobacillaceae strain of the first aspect or the Lactobacillaceae strain of the second aspect; wherein the culture comprises any one of the following A) to D):

[0024] A) fermentation broth of the strain;

[0025] B) the supernatant of the fermentation broth of the strain;

[0026] C) an inactivated fermentation broth of the strain;

[0027] D) The concentrated or dried product of any one of A) to C) above.

[0028] In some embodiments, the composition is provided in liquid form or solid form.

[0029] In some embodiments, the composition comprises 1×10 4 to 1×10 12 cfu / mL or 1×10 4 to 1×10 12 cfu / mg of the live Lactobacillus bacteria.

[0030] In some embodiments, the composition comprises 1×10 5 to 1×10 11 cfu / mL or 1×10 5 to 1×10 11 cfu / mg of the live Lactobacillus bacteria.

[0031] In some embodiments, the composition comprises 1×10 6 to 1×10 10 cfu / mL or 1×10 6 to 1×10 10 cfu / mg of the live Lactobacillus bacteria.

[0032] In some embodiments, the composition comprises 1×107 to 1×10 9 cfu / mL or 1×10 7 to 1×10 9 cfu / mg of the live Lactobacillus bacteria.

[0033] In some embodiments, each gram of the composition contains 1×10 3 to 1×10 17 colony-forming units (CFU) of bacteria; for example, 1×10 4 to 1×10 12 1×10 5 to 1×10 11 or 1×10 6 to 1×10 10 colony forming units (CFU), specifically, for example, 1 × 10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、3×10 13 、4×1013 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 In some embodiments, the lactobacillus in the composition is an attenuated bacterium, a killed bacterium, a freeze-dried bacterium, or an irradiated bacterium, for example, a heat-inactivated bacterium, preferably a pasteurized bacterium.

[0034] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0035] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid preparation, sustained-release preparation, nanoformulation, or microencapsulated capsule.

[0036] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0037] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers, active agents, excipients and / or adjuvants.

[0038] In some embodiments, the pharmaceutically acceptable excipients are well known to those skilled in the art.

[0039] In some embodiments, the auxiliary material may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

[0040] In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0041] In some embodiments, the active agent further comprises one or more agents for preventing or treating cardiovascular and cerebrovascular diseases, metabolic diseases, lowering lipids, and improving intestinal flora.

[0042] The active agent has at least one of the following functions: (a) suppressing appetite, (b) preventing metabolic diseases, and (c) treating metabolic diseases.

[0043] In some embodiments, the active agent is selected from: a GLP-1 receptor agonist, a dual agonist of the GLP-1 receptor and the GCG receptor, a triple agonist of the GLP-1 receptor, the GIP receptor and the GCG receptor, an AMPK agonist or an active drug that promotes GLP-1 secretion.

[0044] In some embodiments, the active agent is selected from the group consisting of metformin, sulfonylureas, meglitinide, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3 fatty acids, statins, ezetimibe, and ursodeoxycholic acid.

[0045] In some embodiments, the GLP-1 receptor agonist includes one or more of the following: Exenatide, Liraglutide, Lixisenatide, Dulaglutide, Albiglutide, Semaglutide, Betaglutide, Losenapeptide, and Somalutide.

[0046] In some embodiments, the active agent is one or more of probiotics, prebiotics, or a combination thereof.

[0047] In some embodiments, the probiotics are selected from one or more of lactic acid bacteria, lactobacilli, lactococci, butyrate-producing bacteria, bifidobacteria, thermophilic Streptococcus, faecal Streptococcus, and mesenteric Leuconostoc.

[0048] In some embodiments, the prebiotic is selected from the group consisting of inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in fructooligosaccharides, oligo-glucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS), and any combination thereof.

[0049] In some embodiments, the composition can be formulated as a frozen composition, for example, by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0050] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0051] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using a diluent as described herein.

[0052] In some embodiments, the composition can be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0053] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric-coated formulation. In some embodiments, the enteric-coated formulation is a dosage form having an enteric coating. For example, the enteric-coated formulation can be an enteric granule, an enteric-coated tablet, or an enteric-coated capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained-release capsule, a controlled-release capsule, or an enteric-coated capsule, or the capsule can be a microencapsulated capsule or a microcapsule.

[0054] In some embodiments, the composition is a medicament.

[0055] In some embodiments, the composition is in an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0056] In some embodiments, the composition is in a parenteral or enteral dosage form.

[0057] According to a fourth aspect of the present disclosure, there is provided the Lactobacillaceae ( Lactobacillaceae ) strain, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillaceae strain described in the second aspect or the composition described in the third aspect in the preparation of a medicament for preventing weight regain or maintaining weight after weight loss.

[0058] In some embodiments, the drug has at least one selected from the group consisting of:

[0059] (1) maintain weight after discontinuation of weight-loss medication;

[0060] (2) prevent blood sugar increases and / or maintain blood sugar balance after discontinuation of weight loss medication;

[0061] (3) Prevent body fat accumulation after discontinuation of weight loss drugs.

[0062] In some embodiments, the body fat includes one or more of subcutaneous fat, epididymal fat, white fat, and visceral fat.

[0063] In some embodiments, the weight loss drug is a GLP-1 receptor agonist.

[0064] In some embodiments, the subject of the application is a subject receiving a GLP-1 receptor agonist for weight loss.

[0065] In some embodiments, the GLP-1 receptor agonist includes one or more of the following: liraglutide or semaglutide.

[0066] The compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by the lactobacilli, and the like. This can be obtained, for example, by recovering the supernatant of a lactobacillus culture or by extracting cellular components or cell fractions, metabolites or secreted compounds from a lactobacillus culture; this can correspond to components from the lactobacillus in isolated form, or any mixture of one or more components from the lactobacillus.

[0067] According to a fifth aspect of the present disclosure, there is provided the Lactobacillaceae ( Lactobacillaceae ) strain, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus family strain described in the second aspect or the composition described in the third aspect in the preparation of a drug for preventing blood sugar from rising or maintaining blood sugar balance after stopping weight loss medication.

[0068] According to a sixth aspect of the present disclosure, there is provided the Lactobacillaceae ( Lactobacillaceae ) strain, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillaceae strain described in the second aspect or the composition described in the third aspect in the preparation of a drug for preventing, treating, alleviating or reducing metabolic diseases, metabolic disorders, or diseases caused by metabolic disorders, liver and kidney diseases, cardiovascular and cerebrovascular diseases in subjects in need.

[0069] In some embodiments, the metabolic disease includes at least one of obesity and obesity-related diseases, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.

[0070] As used herein, a microbial population generally refers to a population of microorganisms consisting essentially of a single strain, species, or genus, as may be the case when the population is cultured from an isolated and purified subpopulation of such a strain, species, or genus. Thus, for a given population of microorganisms, if such population is cultured from an isolated species or strain of microorganisms, such population will be referred to herein as purified or substantially pure. The resulting population will typically be at least 80% pure with respect to the species or strain of microorganism, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure relative to other species or strains of microorganisms within that particular population. Conversely, the level of undesirable strains in any particular desired population of microorganisms will be less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%. For example, the level of impurities, such as other undesirable strains or species, in a purified population of microorganisms can be at or below the aforementioned levels for each desired population, proportionally. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case of a composition comprising a consortium of multiple populations of microorganisms, each population may have the above-mentioned purities, either prior to its incorporation into the composition, or when measured in aggregate for the consortium. For example, the level of impurities, such as other undesirable strains or species of microorganisms, in the purified population consortium may be proportional to or below the above-mentioned levels for each desired population. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case of a composition comprising a consortium of multiple populations of microorganisms, each population may have the above-mentioned purities, either prior to its incorporation into the composition, or when measured in aggregate for the consortium. For example, the level of impurities, such as other undesirable strains or species of microorganisms, in the purified population consortium may be proportional to or below the above-mentioned levels for each desired population.

[0071] In some embodiments, the causes of obesity and obesity-related diseases include, but are not limited to, at least one of a high-fat diet, a high-sugar diet, high cholesterol, high blood lipids, high blood sugar, NAFLD, or NASH.

[0072] In some embodiments, the diabetes includes but is not limited to type I diabetes, type II diabetes, gestational diabetes, diabetes mediated by HDAC activity, diabetic nephropathy, diabetic neuropathy, diabetic eye disease, diabetic retinopathy, diabetic foot, diabetes caused by damage to pancreatic B cells, diabetes caused by insulin resistance, and diabetes caused by obesity.

[0073] In some embodiments, the causes of diabetes include but are not limited to at least one of: pancreatic islet cell dysfunction, decreased insulin secretion, increased insulin resistance, a high-fat diet, a high-sugar diet, high cholesterol, hyperlipidemia, hyperglycemia, NAFLD, or NASH.

[0074] In some embodiments, the causes of diabetes include at least one of the following: a high-fat diet, a high-sugar diet, and a high-cholesterol diet.

[0075] In some embodiments, the diabetes mellitus is manifested as hyperglycemia resulting from low levels of insulin and / or peripheral insulin resistance.

[0076] In some embodiments, the metabolic disorder includes but is not limited to: (1) diabetes caused by impaired glucose metabolism, or (2) diabetes caused by impaired glucose tolerance or decreased glucose tolerance, or (3) diabetes caused by damaged insulin B cells, or (4) diabetes caused by insulin resistance.

[0077] In some embodiments, the liver and kidney diseases include at least one of liver diseases, diseases related to liver damage, and kidney diseases.

[0078] In some embodiments, the cardiovascular and cerebrovascular diseases include atherosclerosis, coronary heart disease, hypertension, cardiovascular diseases in patients with NAFLD or NASH, cardiovascular and cerebrovascular diseases in patients with NAFLD or NASH, and high cholesterol diseases.

[0079] In some embodiments, the liver disease comprises at least one of fatty liver, NAFLD, NASH, abnormal liver function, extrahepatic cholestasis, hepatitis, liver injury, intrahepatic cholestasis, liver fibrosis, cirrhosis and liver cell damage.

[0080] In some embodiments, the renal disease comprises at least one of renal injury, abnormal renal function, primary glomerulonephritis, hypertensive arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial disease, and ischemic nephropathy.

[0081] In some embodiments, the cardiovascular disease or cardiovascular and cerebrovascular disease includes but is not limited to: atherosclerosis, coronary heart disease, hypertension, cardiovascular disease in patients with NAFLD or NASH, cardiovascular and cerebrovascular disease in patients with NAFLD or NASH, and high cholesterol disease.

[0082] In some embodiments, the causes of the cardiovascular disease or cardiovascular and cerebrovascular disease include, but are not limited to, at least one of atherosclerosis, NAFLD, NASH, hyperlipidemia, hyperglycemia, or high cholesterol.

[0083] In some embodiments, the drug can treat, prevent, alleviate or improve at least one of the following: (1) weight loss, weight control or delayed weight gain; (2) improvement of blood glucose homeostasis imbalance, improvement of blood glucose elevation; (3) prevention, treatment, improvement or alleviation of diabetes; (4) reduction of fat accumulation or reduction of local fat; (5) reduction of blood lipid and / or cholesterol levels; (6) prevention, treatment and improvement of hypertension and atherosclerosis; (7) improvement of intestinal flora; (8) prevention of weight gain.

[0084] In some embodiments, the local fat includes one or more of subcutaneous fat, epididymal fat, white fat, visceral fat, inguinal fat, perirenal fat, intermuscular fat, retroperitoneal fat, pericardial fat, and omental fat.

[0085] In addition, the present application also provides the Lactobacillaceae ( Lactobacillaceae ) strain, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus family strain described in the second aspect or the composition described in the third aspect are used in the preparation of health products that help control body fat and maintain healthy blood sugar levels after stopping weight loss drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an illustrative and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0087] Figure 1 Shown is a picture of the colony morphology of MNH08891.

[0088] Figure 2 Shown is a Gram-stained microscopic morphological photograph of MNH08891.

[0089] Figure 3 Shown: Electron micrograph of MNH08891.

[0090] Figure 4 The colony morphology of MNH22076 is shown.

[0091] Figure 5 Shown are Gram-stained microscopic photographs of strain MNH22076.

[0092] Figure 6 An electron micrograph of MNH22076 is shown.

[0093] Figure 7 The results of the strains' tolerance to different pH are shown.

[0094] Figure 8The results show the tolerance of the strains to different concentrations of NaCl.

[0095] Figure 9 The results of the strains' tolerance to different concentrations of bile salts are shown.

[0096] Figure 10 The results show the tolerance of the strains to different pH values.

[0097] Figure 11 The results show the tolerance of the strains to different concentrations of NaCl.

[0098] Figure 12 The results of the strains' tolerance to different concentrations of bile salts are shown.

[0099] Figure 13 The phylogenetic tree of strain MNH08891 is shown.

[0100] Figure 14 The phylogenetic tree of strain MNH22076 is shown.

[0101] Figure 15 The figure shows the body weight at the end of the trial after discontinuation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group.

[0102] Figure 16 The figure shows the weight gain at the end of the study after discontinuation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the HFD-Sema group.

[0103] Figure 17 Figure 2 shows the body weight gain after cessation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the HFD-Sema group.

[0104] Figure 18 Figure 2 shows blood glucose levels 2 hours after discontinuation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group.

[0105] Figure 19Figure 2 shows fasting blood glucose levels after discontinuation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group.

[0106] Figure 20 Subcutaneous fat weights were shown after MNH08891 / MNH22076 administration following semaglutide discontinuation. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group; ***, p < 0.001 compared with the HFD-Sema group.

[0107] Figure 21 Figure 2 shows epididymal fat weights after semaglutide discontinuation and administration of MNH08891 / MNH22076. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group; ***, p < 0.001 compared with the HFD-Sema group.

[0108] Figure 22 White fat was measured after MNH08891 / MNH22076 administration after semaglutide discontinuation. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group; ***, p < 0.001 compared with the HFD-Sema group.

[0109] Figure 23 The white fat mass ratios of MNH08891 / MNH22076 after semaglutide discontinuation were shown. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group; ***, p < 0.001 compared with the HFD-Sema group.

[0110] Figure 24Figure 2 shows visceral fat in patients receiving MNH08891 / MNH22076 after discontinuation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group; ***, p < 0.001 compared with the HFD-Sema group.

[0111] Figure 25 The visceral fat to body weight ratio is shown following administration of MNH08891 / MNH22076 after discontinuation of semaglutide treatment. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Sema group; **, p < 0.01 compared with the HFD-Sema group; ***, p < 0.001 compared with the HFD-Sema group.

[0112] Collection information:

[0113] Lactobacillus paracasei MNH08891 was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on November 28, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number GDMCC NO.65551. The test result is survival, and the taxonomic name is Lacticaseibacillus paracasei .

[0114] Lactobacillus crispatus MNH22076 was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on November 28, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number GDMCC NO.65554. The test result is survival, and the taxonomic name is Lactobacillus crispatus . DETAILED DESCRIPTION

[0115] The following will clearly and completely describe the technical solutions in the embodiments of this disclosure in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of this disclosure, not all of them. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this disclosure.

[0116] The present invention has isolated two new strains of Lactobacillus: MNH08891, with a deposit number of GDMCC NO.65551, belonging to the family Lactobacillaceae, Lactobacillus paracasei ( Lacticaseibacillus paracasei ); and MNH22076, deposited with GDMCC NO.65554, belonging to Lactobacillus crispatus of the family Lactobacillaceae ( Lactobacillus crispatus ). Furthermore, the present invention studies the biochemical properties and therapeutic uses of the strain.

[0117] As is known in the art, bacterial species can be classified and identified using traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagellar staining, and various metabolic assays. Molecular biology methods include, but are not limited to, ribosomal RNA sequencing and whole genome sequencing-based methods.

[0118] As used herein, the term "prebiotic" may be a general term referring to chemicals and / or ingredients that can affect the growth and / or activity of microorganisms in a host (e.g., can allow for specific changes in the composition and / or activity of a microbiome).

[0119] The terms "subject," "subject," "individual," "host," and "patient" are used interchangeably herein to refer to any animal subject, including humans, mammals, laboratory animals, livestock, and domestic pets.

[0120] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, or medical probiotics. In some cases, the composition is used in the form of a pharmaceutical preparation. In some cases, the composition is used in the form of a medical probiotic. In some cases, the composition (e.g., medical probiotics) can be administered orally, for example, as a capsule, pill, or tablet.

[0121] 16S rRNA is a type of ribosomal RNA in prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is shared by all bacteria, while the variable region varies to varying degrees among different bacteria. By comparing bacterial 16S rRNA gene sequences and basing the number of sequence differences and their evolutionary distance, an evolutionary tree can be constructed. When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence betweenaverage nucleotide identity and 16S rRNA gene sequence similarity for speciesdemarcation of prokaryotes. International Journal of Systematic andEvolutionary Microbiology, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini,R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening thetaxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), p23).

[0122] The "identity" between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the "FASTA" program, the GCG program package, BLASTN or FASTA. Commercial or publicly available programs may also be, for example, the DNAStar "MegAlign" program.

[0123] Second-generation sequencing technology can also be used to identify bacterial species based on whole-genome sequencing, making the identification results more accurate. The average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. The ANI value can be calculated using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value must reach above 95% to be considered as belonging to the same species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nature Communications, 2018, 9(1):5114.).

[0124] Various existing mature ANI value calculation tools can be used, such as the local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0125] Using the above method, a person skilled in the art can determine whether an isolated strain belongs to the Lactobacillus species identified by the inventors. For example, when the average nucleotide identity ANI value with MNH08891 is at least 95%, such as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9 ... When the expression level is 7.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that it belongs to the same strain as MNH08891. species; when the average nucleotide identity ANI value with MNH22076 is at least 95%, for example 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 97.1%, 97.2%, 97.3%, 97.9%, 97.1 ...2%, 97.3%, 97.9%, 97.1%, 97.1%, 97.2%, 97.3%, 97.9%, 97.1%, 97.1%, 97.2%, 97.3%, 97.9%, 97.1%, 97.1%, 97.2%, 97.3%, 97.9%, 97.1%, 97.1%, 97.2%, 97.3%, 97.9%, 97.1%, 97.1%, .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that it belongs to the same species as MNH22076.

[0126] For another example, when its 16S rRNA sequence is at least 98.65%, for example, at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the sequence shown in SEQ ID NO. 1, it can be determined to belong to the same species as MNH08891; or

[0127] When its 16S rRNA sequence is at least 98.65% identical to the sequence shown in SEQ ID NO: 2, for example, at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical, it can be determined to belong to the same species as MNH22076.

[0128] A "strain" is a member of a bacterial species that has genetic characteristics that allow it to be distinguished from closely related members of the same bacterial species. A genetic characteristic can be the complete or partial absence of at least one gene, the complete or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence ("cure") of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In cases where one strain (compared to another strain of the same species) acquires or loses antibiotic resistance or a biosynthetic capacity (e.g., an auxotrophic strain), the strains can be distinguished by selection or counterselection using antibiotics or nutrients / metabolites.

[0129] "Supernatant" or "supernatant" within the meaning herein refers to the culture supernatant of the bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0130] Compositions can be prepared using MNH08891 or MNH22076 described herein, for example, by using pharmaceutically acceptable excipients. The pharmaceutical compositions comprise a pharmaceutically effective amount of the lactobacillus, such as Lactobacillus paracasei with a deposit number of GDMCC NO. 65551 or Lactobacillus crispatus of the Lactobacillaceae family with a deposit number of GDMCC NO. 65554.

[0131] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavors.

[0132] The compositions herein can be formulated into any form suitable for enhancing the abundance of lactobacilli in a subject. The compositions can be administered orally (e.g., by oral gavage), intramuscularly, by inhalation, intracranially, intralymphatically, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrointestinally, by bile infusion, by cardiac infusion, pre-anally, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, or by ureteral or urethral routes.

[0133] Examples of dosage forms suitable for the compositions herein include, but are not limited to, tablets, aerosols, chewable sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0134] In some embodiments, the composition is a sugar-coated tablet, a gel capsule, a gel, an emulsion, a tablet, a sheet capsule, a hydrogel, a nanofiber gel, an electrospun fiber, a powder, a toothpaste, or the like.

[0135] The compositions provided herein may comprise a pharmaceutically acceptable excipient, diluent or carrier. Such pharmaceutically acceptable excipients, diluents or carriers are well known in the art.

[0136] In some embodiments, the MNH08891 or MNH22076 in the composition of the present disclosure is lyophilized. In some embodiments, the MNH08891 or MNH22076 in the composition of the present disclosure is spray-dried. In some embodiments, the MNH08891 or MNH22076 in the composition of the present disclosure is lyophilized or spray-dried and is alive. In some embodiments, the MNH08891 or MNH22076 in the composition of the present disclosure is lyophilized or spray-dried and can partially or completely colonize in the intestine. In some embodiments, the lyophilized MNH08891 or MNH22076 is reconstituted before administration. In some embodiments, the reconstitution is carried out using a diluent as described herein.

[0137] In some embodiments, the compositions of the present disclosure are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

[0138] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0139] The compositions of the present disclosure include pharmaceutical compositions.

[0140] The subject of the present disclosure may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0141] The pharmaceutical composition disclosed herein can be used to treat, prevent or alleviate metabolic diseases or diseases caused by metabolic disorders.

[0142] In some embodiments, the metabolic disease, metabolic disorder, or disease caused by metabolic disorder includes but is not limited to at least one of obesity and obesity-related diseases, cardiovascular disease, diabetes, dyslipidemia, cardiovascular and cerebrovascular disease, glucose intolerance, atherosclerosis, coronary heart disease or hypertension, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, hypoglycemia, thrombotic disease, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and atherosclerosis.

[0143] diabetes

[0144] Diabetes includes type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes mellitus (GDM). Type 1 diabetes, caused by autoimmune or idiopathic factors, is characterized by the complete destruction of pancreatic islet function. It primarily occurs in children and adolescents and requires insulin therapy for satisfactory results; otherwise, it can be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / fat metabolism, typically including hyperglycemia, hypertension, and abnormal cholesterol levels. Type 2 diabetes is caused by ineffective insulin action (low insulin receptor binding), so it is important to monitor not only fasting blood sugar levels but also two hours after meals, with particular emphasis on pancreatic islet function tests. There are two types of diabetes during pregnancy: one is diagnosed with diabetes before pregnancy, known as "diabetes complicated by pregnancy"; the other is normal glucose metabolism or potential impaired glucose tolerance before pregnancy, which develops or is diagnosed during pregnancy. This is also known as "gestational diabetes mellitus (GDM)." Over 80% of pregnant women with diabetes have GDM.

[0145] cardiovascular disease

[0146] Triglycerides (TG) are primarily involved in energy metabolism and heat generation in the human body. Excessive TG levels in the blood can lead to blood viscosity, causing lipids to deposit on blood vessel walls, gradually forming small plaques known as atherosclerosis. Elevated LDL-C is a major independent risk factor for the development and progression of atherosclerosis; elevated LDL-C levels are also a marker for coronary heart disease. Because HDL-C transports cholesterol from the blood vessel wall to the liver for breakdown (reverse cholesterol transport), it reduces cholesterol deposition in the vessel wall and plays an anti-atherosclerotic role.

[0147] obesity

[0148] Obesity refers to a certain degree of significant overweight and an excessive fat layer. It is a condition caused by excessive accumulation of body fat, particularly triglycerides. It is an abnormal or excessive accumulation of fat that poses a health risk. Excessive body fat accumulation, caused by excessive food intake or altered metabolism, leads to excessive weight gain and can cause pathological or physiological changes or potential health problems. A body mass index (BMI) over 25 is considered overweight, and over 30 is considered obese. Obesity increases the risk of many physical and mental illnesses. It is primarily associated with metabolic syndrome, a combination of conditions including type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia. Generally speaking, the health effects of obesity fall into two main categories: conditions attributable to increased body fat (such as osteoarthritis and obstructive sleep apnea) and conditions attributed to an increased number of fat cells (such as diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis). The "obesity-related diseases" can be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, low back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as subjects with these obesity-related diseases including those who wish to lose weight.

[0149] The obesity-related diseases in the present disclosure include at least one of the following diseases: obesity, metabolic syndrome, cardiovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

[0150] The pharmaceutical composition disclosed herein can be used to treat, prevent or improve metabolic disorders after discontinuation of GLP-1 receptor agonists.

[0151] In some embodiments, the metabolic disease includes metabolic diseases, metabolic disorders, or diseases caused by metabolic disorders, including but not limited to: obesity and obesity-related diseases, cardiovascular disease, cardiovascular and cerebrovascular diseases, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), at least one of atherosclerosis.

[0152] In some embodiments, the metabolic disorder comprises weight gain, imbalanced blood glucose homeostasis, and fat accumulation.

[0153] Disturbance of blood sugar homeostasis

[0154] These include abnormal blood sugar metabolism, insulin resistance, abnormal postprandial blood sugar levels, or abnormal fasting blood sugar levels.

[0155] Fat accumulation

[0156] Fat accumulation includes subcutaneous fat, epididymal fat, white fat, visceral fat, inguinal fat, perirenal fat, intermuscular fat, retroperitoneal fat, pericardial fat, and omental fat.

[0157] GLP-1 receptor agonists are compounds that have agonist activity at the glucagon-like peptide-1 receptor. Their ability to increase insulin secretion by binding to the GLP-1 receptor has been discovered.

[0158] In some embodiments, the GLP-1 receptor agonist includes one or more of the following: Exenatide, Liraglutide, Lixisenatide, Dulaglutide, Albiglutide, Semaglutide, Betaglutide, Losenapeptide, and Somalutide.

[0159] The present disclosure provides the Lactobacillus family ( Lactobacillaceae ) strain, its culture, live bacteria, freeze-dried bacteria or inactivated bacteria or the composition thereof in the preparation of a medicament for preventing weight regain or maintaining weight after weight loss.

[0160] In some embodiments, the drug has at least one selected from the group consisting of:

[0161] (1) maintain weight after discontinuation of weight-loss medication;

[0162] (2) prevent blood sugar increases and / or maintain blood sugar balance after discontinuation of weight loss medication;

[0163] (3) Prevent body fat accumulation after discontinuation of weight loss drugs.

[0164] In some embodiments, the body fat includes one or more of subcutaneous fat, epididymal fat, white fat, and visceral fat.

[0165] In some embodiments, the weight loss drug is a GLP-1 receptor agonist.

[0166] In some embodiments, the subject of the application is a subject receiving a GLP-1 receptor agonist for weight loss.

[0167] In some embodiments, the GLP-1 receptor agonist includes one or more of the following: liraglutide or semaglutide.

[0168] The present disclosure also provides the Lactobacillaceae ( Lactobacillaceae ) strain, its culture, live bacteria, freeze-dried bacteria or inactivated bacteria or the composition thereof in the preparation of a health product that helps control body fat and maintain healthy blood sugar levels after discontinuation of weight loss drugs.

[0169] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature of the art or in accordance with the product or instrument specifications are used. All reagents or instruments are commercially available if their manufacturers are specified. Example

[0170] The following are non-limiting examples for practicing the present disclosure. The following examples are provided merely to illustrate embodiments of the present disclosure and should not be construed as limiting the present disclosure in any way.

[0171] The culture medium involved in the embodiment includes:

[0172] Anaerobic blood agar plates: purchased from Huankai Microorganisms, formula: 10 g / L casein pancreatic digest, 3 g / L cardiac pancreatic digest, 1 g / L corn starch, 5 g / L meat gastric digest, 5 g / L yeast extract powder, 5 g / L sodium chloride, 15 g / L agar, 50-100 mL / L sterile defibrinated sheep blood, pH 7.3 ± 0.2.

[0173] MRS medium: peptone 10.0 g, beef extract powder 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, Tween-80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, triammonium citrate 2.0 g, magnesium sulfate (MgSO4·7H2O) 0.1 g, manganese sulfate (MnSO4·4H2O) 0.05 g.

[0174] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0175] Example 1. Isolation and identification of strains

[0176] 1.1 Isolation of strains MNH08891 and MNH22076

[0177] The two intestinal lactobacilli strains, Lacticaseibacillus paracasei MNH08891 and Lactobacillus crispatus MNH22076, screened in the present invention were both isolated from fecal samples of healthy volunteers.

[0178] Specifically, the strain isolation method is as follows:

[0179] The donor collected 2-5 g of fresh feces and placed it in a sample collection and preservation tube. After shaking and homogenization, the processed fecal sample was placed in an ice box and sent to the laboratory for strain isolation within 24 hours.

[0180] Aliquot physiological saline solution in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation and transfer them to the anaerobic workstation 24 h in advance. Label the plates with sample information, culture medium type, isolation date, etc.

[0181] Take fresh fecal sample and place it in anaerobic workstation and shake it with vortex shaker for 1 min to mix it. Pipette 1 mL of sample into 9 mL of normal saline and mix it for 10 min. -1 dilution, and then serially diluted to 10 -6 Dilution solution, set aside.

[0182] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a rate of 100 μL / plate. Spread evenly. After the plate surface is dry, invert the plate and culture at 37°C for 3-5 days.

[0183] 1.2 Strain purification

[0184] Observe the growth of the strain and pick a single colony with a sterilized toothpick for strain purification. The purified strain was placed at 37°C for anaerobically culture.

[0185] The pure culture strain was prepared into 20% glycerol / water-bacteria solution and stored at -80℃.

[0186] 1.3 Strain preservation

[0187] Lactobacillus paracasei MNH08891 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on November 28, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number GDMCC NO.65551. The test result was survival, and the taxonomic name is Lacticaseibacillusparacasei.

[0188] Lactobacillus crispatus MNH22076 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on November 28, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number GDMCC NO.65554. The test result was survival, and the taxonomic name is Lactobacillus crispatus.

[0189] 1.4 Morphological characteristics of strains

[0190] Morphological characteristics of strain MNH08891

[0191] Strain MNH08891 was inoculated into MRS medium and incubated anaerobically at 37°C for 48 hours. Visible colonies formed on the MRS plate. These colonies were round, with regular, smooth edges, approximately 1-3 mm in diameter, and white, opaque. The strain was Gram-positive. Microscopic observation revealed that the strain lacked flagella, was nonmotile, and had a rod-shaped shape, approximately 0.5-1 µm × 1-2 µm in size. A photograph of the colony morphology of strain MNH08891 after 48 hours of MRS plate culture is shown in the accompanying image. Figure 1 Gram staining microscopic morphology of strain MNH08891 is shown in Figure 2 , Gram staining positive. Electron micrographs are shown in Figure 3 .

[0192] Morphological characteristics of strain MNH 22076

[0193] Strain MNH22076 was inoculated into MRS medium and incubated anaerobically at 37°C for 48 hours. Visible colonies formed on the MRS plate. These colonies were round, with regular, smooth edges, approximately 1-2 mm in diameter, and white, opaque. The strain was Gram-positive. Microscopic observation revealed that the strain lacked flagella, was nonmotile, and had a rod-shaped shape, approximately 0.5-1 µm × 1.5-4 µm in size. A photograph of the colony morphology of strain MNH 22076 after 48 hours of culture on an MRS plate is shown in the accompanying image. Figure 4 Gram staining microscopic morphology of strain MNH22076 is shown in Figure 5 , Gram staining positive. Electron micrographs are shown in Figure 6 .

[0194] 1.5. Physiological and biochemical characteristics of strains

[0195] The strain MNH08891 can grow in the pH range of 4.0 to 10.0, with the optimal growth pH being 6.0 to 9.0 (see the results of the strain's tolerance to different pH values). Figure 7 ); it does not grow on a culture medium containing more than 5% w / w NaCl (the results of the strain's tolerance to different concentrations of NaCl are shown in Figure 8 ); strain MNH08891 can survive and grow in the range of bile salt concentrations from 0% to 0.25%, but cannot grow when the bile salt concentration is greater than or equal to 0.3% (the results of the strain's tolerance to different concentrations of bile salts are shown in Figure 9 ). This indicates that strain MNH08891 is tolerant to salt concentrations less than 5% and choline concentrations less than 0.3%.

[0196] Physiological characteristics of strain MNH22076

[0197] The strain MNH22076 can grow in the pH range of 5.0 to 10.0, with the optimal growth pH being 8.0 (see the results of the strain's tolerance to different pH values ​​for details). Figure 10 ); it can still grow in a culture medium with a NaCl content of more than 6% (the results of the strain's tolerance to different concentrations of NaCl are shown in Figure 11 ); strain MNH22076 can survive and grow in the bile salt concentration range of 0% to 0.1%, and cannot grow when the bile salt concentration is greater than or equal to 0.15% (the results of the strain's tolerance to different concentrations of bile salts are shown in Figure 12 ). This indicates that strain MNH22076 is tolerant to salt concentrations less than 6% and choline concentrations less than 0.15%.

[0198] 1.6. Biochemical identification of strain MNH08891 using API 50CHL

[0199] Strain MNH08891 was biochemically identified using the Lactobacillus assay kit API 50CHL (Mérieux, CN5041010) according to the manufacturer's instructions. The carbon source for acid production was determined using anaerobic culture at 37°C. The results are shown in Table 1.

[0200] As shown in Table 1, strain MNH08891 can utilize ribose, galactose, glucose, fructose, mannose, dulose, mannitol, sorbitol, methyl-D-glucose, N-acetylglucosamine, starch, arabitol, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, melezitose, gentiobiose, turanose, and tagatose to ferment and produce acid. Therefore, these substances and their derivatives can be used as carbon sources during the fermentation or cultivation of strain MNH08891.

[0201] Table 1. Test results of strain MNH08891

[0202] 0 1 2 3 4 5 6 7 8 9 Control CTRL Glycerol GLY Erythritol ERY D-arabinose DARA L-arabinose LARA Ribosomal B-ribose D-Xylose DXYL L-Xylose LXYL ADO β-Methyl-D-xylosylgoside MDX - - - - - + - - - - 10 11 12 13 14 15 16 17 18 19 Galactose GAL Glucose GLU Fructose Mannose MNE Sorbitol SBE Rhamnose RHA Dulcitol DUL Inositol INO Mannitol MAN Sorbitol SOR + + + + - - + - + + 20 21 22 23 24 25 26 27 28 29 α-Methyl-D-mannoside MDM α-Methyl-D-glucoside MDG N-acetyl-glucosamine NAG Amygdalin Arbutin ARB Seven Leaf Spirit ESC Salicylate SAL Cellobiose CEL Maltose MAL Lactose LAC - + + + + + + + + + 30 31 32 33 34 35 36 37 38 39 Melibiose MEL Sucrose SAC Trehalose TRE Inulin INU Melezitose MLZ Raffinose RAF Starch AMD Glycogen GLYG Xylitol XLT Gentianobiose GEN - + + - + - - - - + 40 41 42 43 44 45 46 47 48 49 D-turanose TUR D-Lyxose LYX D-Tag D-rock sugar DFUC L-rock sugar LFUC D-arabinitol DARL L-arabinitol LARL Gluconate GNT 2-Keto-gluconate 2KG 5-Keto-gluconate 5KG + - + - - - - - - -

[0203] As shown in Table 1, strain MNH08891 can utilize ribose, galactose, glucose, fructose, mannose, dulose, mannitol, sorbitol, methyl-D-glucose, N-acetylglucosamine, starch, arabitol, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, melezitose, gentiobiose, turanose, and tagatose to ferment and produce acid. Therefore, these substances and their derivatives can be used as carbon sources during the fermentation or cultivation of strain MNH08891.

[0204] 1.7 Results of biochemical identification of strain MNH 22076 using API 50CHL.

[0205] The experimental results of strain MNH 22076 are shown in Table 2.

[0206] As shown in Table 2, strain MNH22076 can utilize galactose, glucose, fructose, mannose, methyl-D-glucose, N-acetylglucosamine, starch, arabitol, esculin, salicin, cellobiose, maltose, lactose melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, and gentiobiose to ferment and produce acid. Therefore, these substances and their derivatives can be used as carbon sources during the fermentation or cultivation of strain MNH22076.

[0207] Table 2. Test results of strain MNH22076

[0208] 0 1 2 3 4 5 6 7 8 9 Control CTRL Glycerol GLY Erythritol ERY D-arabinose DARA L-arabinoseLARA Ribosomal B-ribose D-Xylose DXYL L-Xylose LXYL ADO β-Methyl-D-xylosylgoside MDX - - - - - - - - - - 10 11 12 13 14 15 16 17 18 19 Galactose GAL Glucose GLU Fructose Mannose MNE Sorbitol SBE Rhamnose RHA Dulcitol DUL Inositol INO Mannitol MAN Sorbitol SOR + + + + - - - - - - 20 21 22 23 24 25 26 27 28 29 α-Methyl-D-mannoside MDM α-Methyl-D-glucoside MDG N-acetyl-glucosamine NAG Amygdalin Arbutin ARB Seven Leaf Spirit ESC Salicylate SAL Cellobiose CEL Maltose MAL Lactose LAC - + + + + + + + + + 30 31 32 33 34 35 36 37 38 39 Melibiose MEL Sucrose SAC Trehalose TRE Inulin INU Melezitose MLZ Raffinose RAF Starch AMD Glycogen GLYG Xylitol XLT Gentianobiose GEN + + + + + + + - - + 40 41 42 43 44 45 46 47 48 49 D-turanose TUR D-Lyxose LYX D-Tag D-rock sugar DFUC L-rock sugar LFUC D-arabinitol DARL L-arabinitol LARL Gluconate GNT 2-Keto-gluconate 2KG 5-Keto-gluconate 5KG - - - - - - - - - -

[0209] As shown in Table 2, strain MNH22076 can utilize galactose, glucose, fructose, mannose, methyl-D-glucose, N-acetylglucosamine, starch, arabitol, esculin, salicin, cellobiose, maltose, lactose melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, and gentiobiose to ferment and produce acid. Therefore, these substances and their derivatives can be used as carbon sources during the fermentation or cultivation of strain MNH22076.

[0210] 1.7. Minimum inhibitory concentration test of bacterial strain antibiotics

[0211] Minimum inhibitory concentration test of antibiotics for strain MNH08891

[0212] The minimum inhibitory concentration of antibiotics for MNH08891 was determined using E-test (Liofilchem) paper strips according to the instructions. The test results are shown in Table 3.

[0213] Table 3. Minimum inhibitory concentration test results of antibiotics for strain MNH08891

[0214] antibiotic Minimum inhibitory concentration (mg / L) Moxifloxacin (MXF) 0.38 Ampicillin (AMP) 8.00 Chloramphenicol (C) 4.00 Clindamycin (CD) 0.50 Amoxicillin (AMC) 0.70 Rifampicin (RD) 1.25 Penicillin (P) 0.50 Cefquinome (CZX) 18 Tetracycline (TE) 0.50

[0215] The results are shown in Table 3. Strain MNH08891 is sensitive to most types of antibiotics, and the risk of subjects developing antibiotic resistance due to long-term use of MNH19250 is low.

[0216] 1.8 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH22076

[0217] The minimum inhibitory concentration of antibiotics for MNH22076 was determined using E-test (Liofilchem) paper strips. The test results are shown in Table 4.

[0218] Table 4 Minimum inhibitory concentration test results of antibiotics for strain MNH22076

[0219] antibiotic Minimum inhibitory concentration (mg / L) Ampicillin (AMP) 3.00 Chloramphenicol (C) 3.00 Clindamycin (CD) 6.00 Amoxicillin (AMC) 0.25 Rifampicin (RD) 0.38 Imipenem (IMI) 64.00 Penicillin (P) 0.19 Cefquinome (CZX) 1.00 Tetracycline (TE) 1.50

[0220] The results are shown in Table 4. The strain MNH22076 is sensitive to most types of antibiotics, and the risk of subjects developing antibiotic resistance due to long-term use of MNH22076 is low.

[0221] 1.9 Identification of strains

[0222] 1.9.1. 16S rRNA gene amplification

[0223] Fresh cultures of strains MNH08891 and MNH22076 were used to extract genomic DNA, which was then used as a template for 16S rRNA gene amplification.

[0224] The primer pairs used in the 16S rRNA gene PCR of the present invention are:

[0225] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No: 3)

[0226] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID No: 4).

[0227] The PCR reaction procedure is as follows:

[0228] Initial denaturation: 94°C, 4 min; denaturation: 94°C, 50 sec; annealing: 52°C, 40 sec; extension: 72°C, 70 sec; final extension: 72°C, 10 min (36 cycles).

[0229] 1.9.2. 16S rRNA gene sequencing

[0230] After PCR amplification, the PCR product was purified and sent to Sangon for 16S rRNA gene sequencing to obtain the 16S rRNA gene sequence.

[0231] The 16S rRNA gene sequence of strain MNH08891 is shown in SEQ ID No: 1:

[0232]

[0233] The 16S rRNA gene sequence of strain MNH 22076 is shown in SEQ ID No: 2:

[0234]

[0235] 1.9.3. Strain identification results

[0236] The strain 16S rRNA gene sequence returned by sequencing was submitted to the NCBI Basic Local Alignment Search Tool for strain 16S rRNA gene analysis to confirm the strain classification information.

[0237] The measured sequences were analyzed with the data in GenBank by BLAST. The alignment results showed that the strain with the highest similarity to MNH08891 was Lacticaseibacillus paracasei, with a similarity of 100%. Whole-genome ANI analysis showed a similarity of 98.14% with Lacticaseibacillus paracasei (GCF_000829035.1), with an alignment score (AF) of 89%. Therefore, strain MNH08891 is a species of Lacticaseibacillus paracasei. The strain with the highest similarity to MNH22076 was Lactobacillus crispatus, with a similarity of 100%. Whole-genome ANI analysis showed a similarity of 97.63% with Lactobacillus crispatus (GCF 002088015.1), with an alignment score (AF) of 86%. Therefore, strain MNH22076 is a species of Lactobacillus crispatus.

[0238] The 16S rRNA gene sequences of MNH08891 and MNH22076 were compared with those of related strains of Lacticaseibacillus retrieved from databases such as GenBank, and phylogenetic trees were constructed.

[0239] A multiple sequence alignment was performed between MNH08891 and MNH22076 and the sequences of model strains with high 16S rRNA gene sequence similarity in the NCBI database. A phylogenetic tree was then constructed using the software MEGA 5 (maximum likelihood method was used). Only nodes with bootstrap values ​​greater than 50% were displayed in the figure.

[0240] From the phylogenetic tree ( Figure 13 ) It can be seen that strain MNH08891 is clustered with Lacticaseibacillus paracasei and is a new strain of Lacticaseibacillus paracasei.

[0241] From the phylogenetic tree ( Figure 14 ) It can be seen that strain MNH22076 is clustered with Lactobacillus crispatus and is a new strain of Lactobacillus crispatus.

[0242] 1.10 Strain genome analysis

[0243] The genome of the original strain MNH08891 was fragmented using ultrasound to fragments ranging from ~350 bp in length. An Illumina sequencing library was then constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) with paired-end 150-bp sequencing. The sequencing yielded 1.12 Gbp of data, of which Q20 accounted for 96.19%.

[0244] The raw sequencing data were filtered using fastp (version 0.20.0) with the following parameters: "--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50". The filtered raw data were assembled using SPAdes (version 3.14.0) with the following parameters: "--isolate --cov-cutoff 10". The assembled genome yielded a total gene length of 3.02 Mbp, an N50 length of 98.7 kbp, and a GC content of 46.26%.

[0245] Genomic gene prediction analysis was performed using the prokaryotic analysis software Genome Annotation Pipeline prokka (version: 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 2911 CDS sequences were predicted, with an average CDS sequence length of 881 bp.

[0246] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), using the CARD antibiotic resistance gene database (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found in the comparison information.

[0247] The genome was analyzed for potential virulence factors and related genes using NCBI blastp (version 2.7.1+) against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated September 19, 2019). AntiSMASH6 (version 6.0.1) was used to analyze potential secondary metabolism gene clusters.

[0248] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 5.

[0249] Table 5. List of potential toxic genes of MNH08891

[0250] strain genes VFDB gene Gene name Comparison consistency (%) MNH08891_00051 VFG000080 clpE 64.364 MNH08891_00132 VFG048830 gnd 67.308 MNH08891_00231 VFG002190 cpsA 63.306 MNH08891_00640 VFG037100 pilB 60.145 MNH08891_01031 VFG002165 efaA 60.993 MNH08891_01118 VFG000079 clpC 61.063 MNH08891_01555 VFG000077 clpP 69.231 MNH08891_02078 VFG006717 lap 61.124 MNH08891_02583 VFG000964 hasC 70.134

[0251] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 6.

[0252] Table 6. List of potential primary metabolic gene clusters in MNH08891

[0253]

[0254] The results in Tables 5 and 6 show that a gene cluster capable of producing primary metabolites that convert pyruvate into acetate and formate was identified in strain MNH08891.

[0255] 1.11 Genome analysis of strain MNH 22076

[0256] The genome of the original strain MNH22076 was fragmented using ultrasound to fragments of approximately 350 bp. An Illumina sequencing library was then constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) with paired-end 150-bp sequencing. The sequencing yielded 1.28 Gbp of data, of which Q20 accounted for 95.28%.

[0257] The raw sequencing data were filtered using fastp (version 0.20.0) with the following parameters: "--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50". The filtered raw data were then assembled using SPAdes (version v3.14.0) with the following parameters: "--isolate --cov-cutoff 10". The assembled genome yielded a total gene length of 2.28 Mbp, an N50 length of 33.0 kbp, and a GC content of 36.59%.

[0258] Genomic gene prediction analysis was performed using the prokaryotic analysis software Genome Annotation Pipeline prokka (version 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 2257 CDS sequences were predicted, with an average CDS sequence length of 890 bp.

[0259] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), using the CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi) database for antibiotic resistance genes. No resistance genes were found after comparison.

[0260] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). Detailed comparison results are shown in Table 7.

[0261] Table 7. List of potential toxic genes of MNH22076

[0262] strain genes VFDB gene Gene name Comparison consistency (%) strain genes VFDB gene MNH22076_00022 VFG037100 pilB 61.268 MNH22076_00022 VFG037100 MNH22076_00126 VFG000077 clpP 69.634 MNH22076_00126 VFG000077 MNH22076_00736 VFG002182 cpsI 63.243 MNH22076_00736 VFG002182 MNH22076_00739 VFG000964 hasC 69.388 MNH22076_00739 VFG000964 MNH22076_01903 VFG000964 hasC 67.458 MNH22076_01903 VFG000964

[0263] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 8.

[0264] Table 8. List of potential primary metabolic gene clusters in MNH22076

[0265] Gene cluster range type from arrive Most similar known gene cluster abbreviation Similarity Region 24.1 Arginine2_Hcarbonate 1438 23972 Arginine to hydrogencarbonate P. aeruginosa ARG 100%

[0266] The results in Tables 7 and 8 show that a gene cluster capable of producing a primary metabolite that converts arginine into bicarbonate was identified in strain MNH22076.

[0267] Example 2. Metabolic Experiment

[0268] This example conducted an animal experiment on the effects of MNH08891 and MNH22076 on preventing weight relapse in an obese mouse model after cessation of semaglutide treatment.

[0269] Mice were fed a high-fat diet for 10 weeks, induced to obesity, and then treated with semaglutide for 4 weeks before withdrawal. Following withdrawal of semaglutide, MNH08891 and MNH22076 were administered to investigate their effects on preventing weight relapse after withdrawal of semaglutide. This invention has been reviewed by the Animal Ethics Committee of Muen Biotech.

[0270] The feed involved in this embodiment includes:

[0271] The basic feed product number is D12451B, which was purchased from Parker Biotechnology Co., Ltd.

[0272] High-fat feed was purchased from Parker Biotechnology Co., Ltd. with the product number D12492.

[0273] 1. Experimental Methods

[0274] 1) Experimental animals: C57BL / 6J mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0275] 2) Test strains: MNH08891 and MNH22076 strains were thawed in glycerol cryovials at 37°C and activated in MRS medium in a biosafety cabinet. The activated strains were inoculated into MRS liquid medium to obtain a sufficient number of cultures. The cultured liquid was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable count (2 × 10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0276] 3) Negative control: Use PBS containing 25% glycerol and 0.05% L-Cys HCl as a negative control

[0277] 4) Experimental Procedure: After the quarantine period, 8 C57BL / 6J male mice aged 5-6 weeks were randomly selected to be fed a basal diet. The remaining mice were all fed a high-fat diet for 10 weeks. After 10 weeks, all mice fed the basal diet were transferred to the non-congested diet group. For the high-fat diet group, 32 mice weighing 36-44 g were randomly assigned to stratify according to body weight, with 8 mice per group. Four groups were divided into: the high-fat diet group, the high-fat diet group (HFD), the high-fat diet group (HFD+Sema), the MNH08891 group, and the MNH22076 group. The NCD group continued to be fed a basal diet, while the other four groups continued to be fed a high-fat diet. Dosing began after grouping (D1). The NCD and HFD groups received a negative control, while the remaining three groups received semaglutide (30 nmol / kg once daily). Semaglutide administration was discontinued after 4 weeks. The HFD+Sema group began receiving the negative control, while the MNH08891 and MNH22076 groups began receiving MNH08891 and MNH22076 by gavage. The NCD and HFD groups continued to receive the negative control once daily, 200 μl each time, for a total of 28 days. During the experiment, mice had free access to water and food, and were maintained on a 12-hour / 12-hour day / night cycle. General clinical observations were conducted after each dose. The endpoint of the study was the day after the end of dosing (D57). General clinical observations were conducted after each dose during the study.

[0278] Fasting blood glucose and oral glucose tolerance tests in mice: Oral glucose tolerance test (OGTT): During the last week of dosing, an OGTT was performed after a 12-hour fast (e.g., fasting from 8:30 PM to 8:30 AM the following day). Weigh the mice and administer glucose orally based on their fasting weight at a dose of 2 g / kg (g glucose / kg fasting weight). Measure fasting blood glucose and 15, 30, 60, 90, and 120 minutes after glucose administration. Strictly time each mouse, and accurately measure blood glucose at six time points.

[0279] Subcutaneous fat, epididymal fat, perirenal fat, and mesenteric fat were directly dissected and weighed for measurement. Visceral fat was the sum of epididymal fat, perirenal fat, and mesenteric fat, and white fat was the sum of all fat.

[0280] At the end of the study, autopsies were performed according to the protocol. Data were summarized and analyzed for each autopsy and serum test results. All data were expressed as mean ± SD and analyzed using GraphPad Prism software. Pairwise comparisons were analyzed using the Student's t test. Significant differences are indicated with *, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0281] 1.1 MNH08891 and MNH22076 can improve weight regain after semaglutide treatment

[0282] Figure 15-17 The results showed that MNH08891 and MNH22076 can reduce weight gain and weight gain rate after discontinuation of semaglutide treatment, indicating that MNH08891 and MNH22076 can improve weight regain after discontinuation of semaglutide treatment and have the purpose of preventing weight regain.

[0283] 1.2 MNH08891 / MNH22076 can improve blood glucose elevation after cessation of semaglutide treatment

[0284] Figure 18-19 The results showed that MNH08891 and MNH22076 could reduce AUC-OGTT after discontinuation of semaglutide treatment, significantly reduce blood glucose 2 hours after a meal, and significantly reduce fasting blood glucose at the test endpoint, indicating that MNH08891 and MNH22076 could improve the imbalance of blood glucose homeostasis after discontinuation of semaglutide treatment, improve blood glucose elevation, and have the purpose of preventing blood glucose elevation and maintaining blood glucose balance.

[0285] 1.3 MNH08891 / MNH22076 can improve fat accumulation after semaglutide treatment

[0286] Figures 20-25 The results showed that MNH08891 and MNH22076 can significantly reduce the weight of subcutaneous fat, epididymal fat, white fat, and visceral fat after discontinuation of semaglutide treatment, and significantly reduce the proportion of white fat and visceral fat. This shows that MNH08891 and MNH22076 can improve fat accumulation after discontinuation of semaglutide treatment, and have the purpose of preventing body fat accumulation and weight gain.

[0287] In summary, the aforementioned examples demonstrate that the novel Lactobacillus species MNH08891 and MNH22076, isolated from fecal samples of healthy volunteers, are Gram-positive anaerobic bacteria that are tolerant to salt and choline and readily colonize. Lactobacillus species MNH08891 and MNH22076 can utilize a variety of carbon sources to produce acid, are easy to culture, and are sensitive to most antibiotics, with a low risk of antibiotic resistance.

[0288] Lactobacillus MNH08891 and MNH22076 can reduce weight gain and weight gain rate after discontinuation of semaglutide treatment; improve blood sugar homeostasis imbalance after discontinuation of semaglutide treatment and improve blood sugar elevation; significantly reduce fat accumulation after discontinuation of semaglutide treatment, and have the purpose of preventing the body's blood sugar from rising and fat accumulation, preventing weight regain, and maintaining blood sugar balance.

[0289] Although a plurality of embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art may conceive of many modifications, changes, and alternatives without departing from the ideas and spirit of the present disclosure. It should be understood that in practicing the present disclosure, various alternatives to the embodiments of the present disclosure described herein may be adopted. The appended claims are intended to define the scope of protection of the present disclosure and therefore cover equivalents or alternatives within the scope of these claims.

Claims

1. A Lactobacillus crispatus that prevents weight gain ( Lactobacillus crispatus ) strain, characterized in that It is deposited in Guangdong Provincial Microbiological Culture Collection with the deposit number GDMCC No: 65554.

2. A Lactobacillus paracasei that prevents weight gain ( Lacticaseibacillus paracasei ) strain, which was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65551.

3. The culture, live bacteria or freeze-dried bacteria of the strain according to claim 1 or 2, characterized in that: The culture is any one of the following A) to D): A) fermentation broth of the strain; B) fermentation broth and fermentation broth supernatant of the strain; C) fermentation broth and inactivated fermentation broth of the strain; D) The concentrated or dried product of any one of A) to C) above.

4. A composition for preventing weight regain, characterized in that: It comprises an effective amount of a culture, live bacteria or freeze-dried bacteria of the strain according to claim 1 or 2 or the strain according to claim 3; wherein the culture is any one of the following A) to D): A) fermentation broth of the strain; B) fermentation broth and fermentation broth supernatant of the strain; C) fermentation broth and inactivated fermentation broth of the strain; D) The concentrated or dried product of any one of A) to C) above.

5. The composition according to claim 4, wherein The composition further comprises one or more pharmaceutically acceptable excipients.

6. The composition according to claim 5, wherein The one or more pharmaceutically acceptable excipients include one or more of the following: a pharmaceutically acceptable carrier, an active agent, and an excipient.

7. The composition according to claim 6, wherein The active agent is one or more of probiotics and prebiotics.

8. The composition according to claim 4, wherein The composition is a medicine.

9. Use of the strain according to claim 1 or 2, the culture, live bacteria or lyophilized bacteria of the strain according to claim 3, or the composition according to any one of claims 4 to 8 in a medicament for preventing weight regain after discontinuation of a GLP-1 receptor agonist.

10. The use according to claim 9, characterized in that The drug is used for at least one selected from the following: (1) Maintaining body weight after discontinuation of GLP-1 receptor agonists; (2) Preventing body fat accumulation after discontinuation of GLP-1 receptor agonists.

11. The use according to claim 10, characterized in that The body fat includes one or more of subcutaneous fat, epididymal fat, white fat, and visceral fat.

12. The use according to claim 10 or 11, characterized in that The subject of the application is a subject who receives a GLP-1 receptor agonist for weight loss.

13. The use according to claim 12, characterized in that The GLP-1 receptor agonist includes one or more of the following: liraglutide or semaglutide.

14. Use of the strain according to claim 1 or 2, the culture, live bacteria or lyophilized bacteria of the strain according to claim 3, or the composition according to any one of claims 4 to 8 in the preparation of a medicament for maintaining weight after discontinuation of a GLP-1 receptor agonist.

15. Use of the strain according to claim 1 or 2, the culture, live bacteria or lyophilized bacteria of the strain according to claim 3, or the composition according to any one of claims 4 to 8 in the preparation of a medicament for preventing hyperglycemia after discontinuation of a GLP-1 receptor agonist.

16. Use of the strain according to claim 1 or 2, the culture, live bacteria or lyophilized bacteria of the strain according to claim 3, or the composition according to any one of claims 4 to 8 in the preparation of a medicament for maintaining blood sugar balance after discontinuation of a GLP-1 receptor agonist.

17. Use of the strain according to claim 1 or 2, the culture, live bacteria or lyophilized bacteria of the strain according to claim 3, or the composition according to any one of claims 4 to 8 in the preparation of a health product that helps control body fat or maintain healthy blood sugar levels after discontinuation of a GLP-1 receptor agonist.

18. The use according to any one of claims 14 to 17, characterized in that: The GLP-1 receptor agonist includes one or more of the following: liraglutide or semaglutide.

Images