Isolated lactobacillus crispatus, composition containing same and application

Abstract

The present disclosure provides isolated Lactobacillus crispatus, a composition comprising the same, and uses thereof, which can be used to treat, prevent, alleviate or alleviate liver and kidney diseases, metabolic diseases, and the like.

Description

Technical Field

[0001] The present disclosure relates to the field of microorganisms, and more particularly to isolated Lactobacillus crispatus, compositions containing the same, and uses thereof. Background Art

[0002] Currently, the prevalence of diseases such as obesity, diabetes, hypertension, hyperlipidemia, liver and kidney diseases is increasing, and studies have also shown that these diseases are related to the ecological imbalance of microbial flora. In the prior art, a mixture of multiple microorganisms is usually used to treat metabolic and obesity-related diseases or disorders, such as probiotics or intestinal flora transplantation (FMT). However, the mixture of multiple microorganisms makes the mechanism more complicated, and there is no mature research on the mutual influence between the microorganisms, and the use of microbial mixtures usually disrupts the homeostasis of the intestinal flora. In addition, the use of microbial mixtures also requires additional consideration of whether the microorganisms will affect each other's activity. At the same time, it is also unclear whether it is the synergistic effect of the microbial mixture that makes it play a role in the treatment or prevention of the disease, or whether a single bacteria plays a role in the treatment or prevention of the disease.

[0003] Lactobacillus crispatus is closely related to reproductive tract health and plays an important role in maintaining the balance of female vaginal microecology. The reduction in the abundance of Lactobacillus crispatus may be associated with a variety of female reproductive tract diseases such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), and may even lead to adverse pregnancy outcomes such as infertility and embryo arrest. There are few studies on Lactobacillus crispatus in weight loss and blood sugar reduction. CN114540257B discloses that the IOB901 strain can be fed to mice to lower blood lipids and blood sugar. Chicken-derived Lactobacillus crispatus can reduce cholesterol and triglycerides in ileal epithelial cells of chickens and reduce abdominal fat accumulation (Lactobacillus johnsonii 3-1 and Lactobacillus crispatus 7-4 promote the growth performance and ileum development and participate in lipid metabolism of broilers). However, whether Lactobacillus crispatus can alleviate liver damage caused by a high-fat diet and visceral fat accumulation has not been studied.

[0004] The number of microbial resources is extremely large, and screening out new species or strains that can alleviate liver damage and visceral fat accumulation caused by a high-fat diet, and then further effectively treat or prevent diseases such as obesity, diabetes, hypertension, hyperlipidemia, liver and kidney diseases, etc. is a huge challenge, but it also represents a huge unmet need. Summary of the invention

[0005] The present invention discloses the isolation of a new Lactobacillus crispatus. The new strain disclosed in the present invention can reduce liver weight; reduce the ratio of liver weight to body weight; reduce hepatic fatty degeneration and / or hepatic lobular inflammation; alleviate hepatocyte ballooning; reduce non-alcoholic fatty liver disease activity score (NAS); reduce serum AST and ALT; reduce the body weight of mammals; reduce the weight gain of mammals; reduce at least one of the epididymal fat weight, perirenal fat weight, subcutaneous fat weight, and mesenteric fat weight of mammals; reduce the ratio of visceral fat to body weight; reduce fasting blood sugar; reduce the level of at least one of the following indicators in mammal serum: total cholesterol level, triglyceride level, high-density lipoprotein cholesterol level, and low-density lipoprotein cholesterol level; thereby achieving the purpose of treating, preventing or alleviating liver and kidney diseases and metabolic diseases.

[0006] In a first aspect, the present disclosure provides an isolated Lactobacillus crispatus ( Lactobacillus crispatus ), which has an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with the strain with a deposit number of GDMCC No: 65554 or GDMCC No: 65556; and / or, having a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0007] In some embodiments, the Lactobacillus crispatus ( Lactobacillus crispatus ) has an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with the strain MNH22076 with a deposit number of GDMCC No: 65554; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 3.

[0008] In some embodiments, the Lactobacillus crispatus ( Lactobacillus crispatus) has an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with the strain MNH45330 with a deposit number of GDMCC No: 65556; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 4.

[0009] In some embodiments, the Lactobacillus crispatus ( Lactobacillus crispatus ) having a 16S rRNA sequence that is at least 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:3, or SEQ ID NO:4.

[0010] In some embodiments, the Lactobacillus crispatus is a Lactobacillus crispatus species ( Lactobacillus crispatus ) were found to have two new strains.

[0011] In some embodiments, the name of the Lactobacillus crispatus is: Lactobacillus crispatus ( Lactobacillus crispatus )MNH22076, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65554, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Lactobacillus crispatus MNH22076.

[0012] In some embodiments, the name of the Lactobacillus crispatus is: Lactobacillus crispatus ( Lactobacillus crispatus )MNH45330, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65556, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Lactobacillus crispatus MNH45330.

[0013] In a second aspect, the present disclosure provides a culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus described in the first aspect of the present invention; Wherein, the culture comprises any one of the following A) to D): A) fermentation broth of the Lactobacillus crispatus; B) the supernatant of the fermentation broth of the Lactobacillus crispatus; C) an inactivated fermentation broth of the Lactobacillus crispatus; D) The concentrated or dried product of any one of A) to C) above.

[0014] In a third aspect, the present disclosure provides a composition comprising the Lactobacillus crispatus, a culture thereof, and / or a metabolite thereof according to the first aspect.

[0015] The present disclosure provides a composition comprising the Lactobacillus crispatus described in the first aspect, or the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus described in the second aspect.

[0016] In some embodiments, the culture of Lactobacillus crispatus includes a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof.

[0017] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained under anaerobic culture conditions using a liquid culture medium.

[0018] In some embodiments, the composition is provided in liquid form or in solid form.

[0019] In some embodiments, the composition comprises 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4 Up to 1×10 12 cfu / mg of live Lactobacillus crispatus.

[0020] In some embodiments, the composition comprises 1×10 5 Up to 1×10 11 cfu / mL or 1×10 5 Up to 1×10 11 cfu / mg of live Lactobacillus crispatus.

[0021] In some embodiments, the composition comprises 1×10 6 Up to 1×10 10 cfu / mL or 1×10 6 Up to 1×10 10 cfu / mg of live Lactobacillus crispatus.

[0022] In some embodiments, the composition comprises 1×10 7 Up to 1×10 9cfu / mL or 1×10 7 Up to 1×10 9 cfu / mg of live Lactobacillus crispatus.

[0023] In some embodiments, each gram of the composition comprises 1×10 3 Up to 1×10 17 colony forming units (CFU); for example, 1×10 4 Up to 1×10 12 pcs, 1×10 5 Up to 1×10 11 or 1×10 6 Up to 1×10 10 Colony forming units (CFU), 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 , 4×107 、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 、4×10 13 、5×10 13, 6×10 13 ,7×10 13 , 8×10 13 ,9×10 13 or any value in between of colony forming units (CFU).

[0024] In some embodiments, the concentration of the Lactobacillus crispatus as an active ingredient in the composition is 10 7 Up to 10 12 CFU / g.

[0025] In some embodiments, the Lactobacillus crispatus in the composition is a live bacterium, an attenuated bacterium, a freeze-dried bacterium, or an inactivated bacterium, for example, a heat-inactivated bacterium, preferably a pasteurized bacterium.

[0026] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0027] 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.

[0028] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0029] In some embodiments, the composition further comprises one or more pharmaceutically and / or food-acceptable excipients.

[0030] The pharmaceutically and / or food acceptable excipients are well known to those skilled in the art.

[0031] 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.

[0032] 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.

[0033] In some embodiments, the composition further comprises one or more other active agents for preventing or treating metabolic diseases and / or liver and kidney diseases.

[0034] In some embodiments, the composition further comprises one or more other active agents for preventing or treating liver disease, diabetes, obesity, hypercholesterolemia, hyperlipidemia, or cardiovascular and cerebrovascular diseases.

[0035] In some embodiments, the liver disease includes at least one of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver function impairment.

[0036] In some embodiments, the other active agent has at least one of the following functions: (a) suppresses appetite, (b) prevents metabolic diseases, (c) treats metabolic diseases, (d) prevents liver and kidney diseases, (e) treats liver and kidney diseases.

[0037] In some embodiments, suppressing appetite comprises reducing food intake and / or reducing appetite.

[0038] In some embodiments, the metabolic disease is a metabolic disease, a metabolic disorder, or a disease caused by a metabolic disorder, including but not limited to: liver diseases, liver function diseases, obesity and obesity-related diseases, cardiovascular diseases, 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 fatty hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of kidney disease.

[0039] In some embodiments, the liver and kidney diseases include liver diseases, diseases related to liver damage, kidney damage, abnormal kidney function and other kidney diseases.

[0040] In some embodiments, liver diseases include, but are not limited to, at least one of fatty liver, NAFLD / NASH, abnormal liver function, extrahepatic cholestasis, hepatitis, liver injury, intrahepatic cholestasis, liver fibrosis, cirrhosis, and liver cell injury.

[0041] In some embodiments, the other active agent is selected from: a GLP-1 receptor agonist, a dual agonist of a GLP-1 receptor and a GCG receptor, a triple agonist of a GLP-1 receptor, a GIP receptor and a GCG receptor, an AMPK agonist or an active drug that promotes GLP-1 secretion.

[0042] In some embodiments, the other 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, ursodeoxycholic acid, semaglutide, liraglutide, exenatide, and benaglutide.

[0043] In some embodiments, the other active agent may be one or more of probiotics, prebiotics, or a combination thereof.

[0044] In some embodiments, the probiotics are selected from one or more of lactic acid bacteria, lactobacilli, and butyrate-producing bacteria.

[0045] In some embodiments, the probiotic is selected from at least one of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus krum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus creamer.

[0046] In some embodiments, the prebiotic is selected from 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, fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharide, manno-oligosaccharide (MOS), inulin rich in fructooligosaccharide, oligo-glucose, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS) and any combination thereof.

[0047] In some embodiments, the composition can be formulated as a frozen composition, such as a frozen composition prepared by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0048] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0049] 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 alive. In some embodiments, the strain in the composition is freeze-dried or spray-dried and can partially or completely colonize the intestine. In some embodiments, the strain is reconstituted before administration. In some cases, the reconstitution is carried out by using a diluent as described herein.

[0050] 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.

[0051] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric preparation. In some embodiments, the enteric preparation is a dosage form with an enteric coating. For example, the enteric preparation can be an enteric granule, an enteric tablet, or an enteric 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 capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

[0052] In some embodiments, the composition is a medicine, or a health product or a food.

[0053] In some embodiments, the composition is in an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0054] In some embodiments, the composition is in a parenteral dosage form.

[0055] In a fourth aspect, the present disclosure provides the use of the Lactobacillus crispatus described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus described in the second aspect, or the composition of the third aspect in the preparation of drugs, health products or foods for treating, preventing, alleviating or reducing liver and kidney diseases and metabolic diseases.

[0056] The present disclosure provides the use of the Lactobacillus crispatus described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus described in the second aspect, or the composition of the third aspect in the preparation of drugs, health products or foods for treating, preventing, alleviating or alleviating liver diseases, diabetes, obesity, hypercholesterolemia, hyperlipidemia, cardiovascular and cerebrovascular diseases.

[0057] In some embodiments, the liver disease includes at least one of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver function impairment.

[0058] In some embodiments, the metabolic disease is a metabolic disease, a metabolic disorder, or a disease caused by a metabolic disorder, including but not limited to: liver diseases, liver function diseases, obesity and obesity-related diseases, cardiovascular diseases, 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 fatty hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of kidney disease.

[0059] In some embodiments, the liver and kidney diseases include at least one of liver diseases, diseases related to liver damage, kidney diseases such as kidney damage and abnormal kidney function.

[0060] In some embodiments, the renal disease includes but is not limited to renal injury, abnormal renal function, etc. The renal disease includes but is not limited to primary glomerulonephritis, hypertensive arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial lesions (such as chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic nephropathy, etc. In some embodiments, the cause of the renal disease includes at least one of the following: high-fat diet, high-sugar diet, high-cholesterol diet. In some embodiments, the manifestation of the renal disease is increased blood creatinine.

[0061] In some embodiments, liver diseases include, but are not limited to, at least one of fatty liver, NAFLD / NASH, abnormal liver function, extrahepatic cholestasis, hepatitis, liver injury, intrahepatic cholestasis, liver fibrosis, cirrhosis, and liver cell injury.

[0062] The composition comprises a consortium of isolated and purified live microbial populations to reduce ALT and / or AST levels. One or more of aspartate aminotransferase (AST) and alanyl aminotransferase (ALT) in a subject is reduced by at least 2 U / L, 5 U / L, 10 U / L, 25 U / L, 30 U / L, or 50 U / L compared to the ALT and / or AST levels in the subject before administration of the isolated and purified consortium of microbial species.

[0063] As used herein, a microbial population generally refers to a microbial population consisting essentially of a single strain, species or genus, which may be the case when a population is cultured from a subpopulation of such strain, species or genus that is separated and purified. Thus, for a given microbial population, if cultured from an isolated microbial species or strain, such a population will be referred to herein as purified or substantially pure. The resulting population may typically be at least 80% pure for the microbial species or strain, 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 microbial species or strains within the particular population. In contrast, the level of non-desirable strains in any particular desired microbial population 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 in a purified population population, such as other undesirable microbial strains or species, may be proportional to or less than the above levels for each desired population. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case where the composition comprises a consortium of multiple microbial populations, each population can have the above-mentioned purities, either before it is incorporated into the composition, or when the consortium is measured in an aggregate. For example, the level of impurities in the purified population consortium, such as other undesirable microbial strains or species, can be proportional to the above-mentioned levels for each desired population or below the above-mentioned levels. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case where the composition comprises a consortium of multiple microbial populations, each population can have the above-mentioned purities, either before it is incorporated into the composition, or when the consortium is measured in an aggregate. For example, the level of impurities in the purified population consortium, such as other undesirable microbial strains or species, can be proportional to the above-mentioned levels for each desired population or below the above-mentioned levels.

[0064] The compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by Lactobacillus crispatus, and the like. Cellular components or cell fractions, metabolites or secreted compounds may be obtained, for example, by recovering the supernatant of a Lactobacillus crispatus culture or by extracting from a Lactobacillus crispatus culture; may correspond to components in isolated form from Lactobacillus crispatus, or any mixture of one or more components from Lactobacillus crispatus.

[0065] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition disclosed herein can be used to treat or prevent diseases related to liver function damage, wherein the diseases related to liver function damage include at least one of the following diseases: fatty liver, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, liver fibrosis, cirrhosis, liver cancer or liver cell damage.

[0066] In some embodiments, the causes of the liver disease include, but are not limited to, at least one of a high-fat diet, a high-cholesterol diet, a high-sugar diet, high blood lipids, high blood sugar, or high cholesterol.

[0067] In some embodiments, the liver disease includes that caused by a high-fat diet, caused by a high-cholesterol diet, caused by a high-sugar diet, caused by high fat and high cholesterol, caused by high fat and high sugar, and / or caused by high fat, high cholesterol and high sugar.

[0068] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition described in the present disclosure can be used to treat, prevent or alleviate diseases related to liver function damage, wherein the diseases related to liver function damage include at least one of the following: fatty liver, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, liver fibrosis, cirrhosis or liver cell damage.

[0069] In particular, the Lactobacillus crispatus, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition disclosed herein can be used to treat or prevent liver damage. The liver damage can be liver damage caused by long-term hard work; liver damage caused by long-term drinking; liver damage caused by fatty liver, drug-induced liver disease, and genetic metabolic factors.

[0070] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition of the present disclosure can reduce ALT and / or AST levels.

[0071] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition of the present disclosure can reduce liver weight.

[0072] In some embodiments, the renal disease includes but is not limited to renal injury, abnormal renal function, etc. The renal disease includes but is not limited to primary glomerulonephritis, hypertensive arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial lesions (such as chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic nephropathy, etc. In some embodiments, the cause of the renal disease includes at least one of the following: high-fat diet, high-sugar diet, high-cholesterol diet. In some embodiments, the manifestation of the renal disease is increased blood creatinine.

[0073] In some embodiments, the obesity and obesity-related diseases include, but are not limited to, overweight, obesity, metabolic syndrome, cardiovascular disease, cardiovascular and cerebrovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and fatty liver hepatitis.

[0074] 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.

[0075] In some embodiments, the obesity and obesity-related diseases include but are not limited to: obesity caused by a high-fat diet, obesity caused by high cholesterol, obesity caused by a high-sugar diet, obesity caused by high fat and high cholesterol, obesity caused by high fat and high sugar, obesity caused by high fat, high cholesterol and high sugar, or obesity in patients with NAFLD or NASH.

[0076] In some embodiments, the obesity and obesity-related diseases include at least one of the following: obesity, metabolic syndrome, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease and steatohepatitis.

[0077] In some embodiments, the obesity is peripheral obesity and / or central obesity. In some embodiments, the obesity is dietary obesity and / or metabolic obesity. In particular, the obesity is abdominal obesity or apple-shaped obesity, such as excess visceral fat.

[0078] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can reduce visceral fat and / or subcutaneous fat.

[0079] 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.

[0080] In some embodiments, the causes of diabetes include, but are not limited to, at least one of islet cell dysfunction, decreased insulin secretion, increased insulin resistance, a high-fat diet, a high-sugar diet, high cholesterol, hyperlipidemia, hyperglycemia, NAFLD, or NASH.

[0081] In some embodiments, the causes of diabetes include at least one of the following: caused by a high-fat diet, caused by a high-sugar diet, and caused by a high-cholesterol diet.

[0082] In some embodiments, the diabetes mellitus is manifested by hyperglycemia resulting from low levels of insulin and / or peripheral insulin resistance.

[0083] In some embodiments, the metabolic disorder includes but is not limited to: (1) diabetes caused by disordered glucose metabolism, or (2) diabetes caused by abnormal glucose tolerance or decreased glucose tolerance, or (3) diabetes caused by damaged insulin B cells, or (4) diabetes caused by insulin resistance.

[0084] 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.

[0085] 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 hypercholesterolemia.

[0086] In some embodiments, the drug, health product or food has at least one effect selected from the following: reducing liver weight; reducing the ratio of liver weight to body weight; reducing hepatic steatosis and / or hepatic lobular inflammation; alleviating hepatocyte ballooning; reducing non-alcoholic fatty liver disease activity score (NAS); reducing serum AST and ALT; reducing the body weight of a mammal; reducing the weight gain of a mammal; reducing at least one of epididymal fat weight, perirenal fat weight, subcutaneous fat weight, and mesenteric fat weight in a mammal; reducing the ratio of visceral fat to body weight; reducing fasting blood sugar; reducing the level of at least one of the following indicators in the serum of a mammal: total cholesterol level, triglyceride level, high-density lipoprotein cholesterol level, low-density lipoprotein cholesterol level; treat, prevent or alleviate liver function damage; treat, prevent or alleviate fatty liver; treat, prevent or alleviate NAFLD or NASH; treat, prevent or alleviate obesity; treat, prevent or alleviate metabolic syndrome; treat, prevent or alleviate local subcutaneous fat excess, epididymal fat excess, perirenal fat excess, and / or mesenteric fat excess; treat, prevent or alleviate coronary heart disease; treat, prevent or alleviate atherosclerosis; treat, prevent or alleviate hyperglycemia; treat, prevent or alleviate hyperlipidemia; treat, prevent or alleviate high cholesterol; treat, prevent or alleviate hypertension; treat, prevent or alleviate diabetes, preferably gestational diabetes or type 2 diabetes or diabetes mediated by HDAC activity.

[0087] The Lactobacillus crispatus isolated in the present invention can also synthesize a large amount of short-chain fatty acids, and thus can effectively treat or prevent metabolic diseases, reduce the subject's body weight, body fat, blood sugar, blood lipids, and cholesterol, and improve or alleviate non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0088] In some embodiments, the medicine, health product or food can reduce weight while improving liver damage.

[0089] In some embodiments, the medicine, health product or food can reduce blood lipids while reducing serum AST and / or ALT. The lipid reduction is to reduce visceral fat or subcutaneous fat.

[0090] In some embodiments, the medicine, health product or food can improve liver damage while improving diabetes.

[0091] In some embodiments, the medicine, health product or food can improve kidney damage while improving diabetes.

[0092] In some embodiments, the medicine, health product or food can improve liver damage while improving hypercholesterolemia, hyperlipidemia, and / or cardiovascular and cerebrovascular diseases.

[0093] In some embodiments, the medicine, health product or food can reduce lipids while improving hypercholesterolemia, hyperlipidemia, and / or cardiovascular and cerebrovascular diseases. The lipid reduction is to reduce visceral fat or subcutaneous fat.

[0094] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition described in the present disclosure has at least one of the effects of affecting or regulating fasting glucose homeostasis, affecting or regulating cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, and promoting local adipose tissue metabolism.

[0095] In some embodiments, the Lactobacillus crispatus, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition described in the present disclosure has the effect of reducing liver and / or kidney damage, wherein reducing liver damage is reducing one or more elevated liver damage or liver disease indicators of the subject, such as reducing the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and / or liver weight indicators of the subject; reducing kidney damage is reducing blood urea nitrogen (BUN) in the serum of the subject and / or reducing the CRE (creatinine) indicator in the serum.

[0096] A method for preventing, treating, improving or alleviating liver and kidney diseases and metabolic diseases, comprising administering an effective amount of the Lactobacillus crispatus described in the first aspect of the present disclosure, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus described in the second aspect, or the composition of the third aspect to a subject in need.

[0097] In some embodiments, the liver and kidney diseases and metabolic diseases are the liver and kidney diseases and metabolic diseases mentioned above. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] The embodiments will be described below in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0099] Figure 1 : shows the colony morphology photograph of strain MNH22076.

[0100] Figure 2 : Gram staining photographs of strain MNH22076 are shown.

[0101] Figure 3 : shows the electron micrograph of strain MNH22076.

[0102] Figure 4 : shows the colony morphology photograph of strain MNH45330.

[0103] Figure 5 : Gram staining photographs of strain MNH45330 are shown.

[0104] Figure 6 : shows the electron micrograph of strain MNH45330.

[0105] Figure 7 : Shows the results of the tolerance of strain MNH22076 to different pH.

[0106] Figure 8 : Shows the tolerance results of strain MNH22076 to different concentrations of NaCl.

[0107] Fig. 9 : Shows the tolerance results of strain MNH22076 to different concentrations of bile salts.

[0108] Fig.10 : Shows the results of the tolerance of strain MNH45330 to different pH.

[0109] Fig.11 : Shows the tolerance results of strain MNH45330 to different concentrations of NaCl.

[0110] Fig.12 : Shows the tolerance results of strain MNH45330 to different concentrations of bile salts.

[0111] Fig.13 : The phylogenetic tree of strain MNH22076 is shown.

[0112] Fig.14 : The phylogenetic tree of strain MNH45330 is shown.

[0113] Fig.15a:Shows that strain MNH22076 can significantly reduce liver function index ALT. Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group.

[0114] Fig.15b :Showed that strain MNH22076 can significantly reduce liver function index AST. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group.

[0115] Fig.16 :Shows that strains MNH22076 and MNH45330 can significantly reduce fasting blood glucose in high-fat diet-induced type 2 diabetic mice. Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group.

[0116] Fig.17a :Shows that strains MNH22076 and MNH45330 can significantly reduce the body weight of high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group; ***, p < 0.001 compared with the HFD-Control group; ****, p < 0.0001 compared with the HFD-Control group.

[0117] Fig.17b :Shows that strains MNH22076 and MNH45330 can significantly reduce the weight change ratio of high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t test); *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group; ***, p < 0.001 compared with the HFD-Control group; ****, p < 0.0001 compared with the HFD-Control group.

[0118] Fig.18a:Strain MNH22076 can significantly reduce total cholesterol (TCHO) in the serum of high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t test); **, p < 0.01 compared with the HFD-Control group; ****, p < 0.0001 compared with the HFD-Control group.

[0119] Fig.18b :Strain MNH22076 can significantly reduce serum triglycerides (TG) in high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group.

[0120] Fig.18c :Strain MNH22076 can significantly reduce the low-density lipoprotein cholesterol (LDL-C) in the serum of high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group.

[0121] Fig.19a :Shows that strains MNH22076 and MNH45330 can significantly reduce epididymal fat weight in high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group); **, p < 0.01 compared with the HFD-Control group; ****, p < 0.0001 compared with the HFD-Control group.

[0122] Fig.19b :Shows that strains MNH22076 and MNH45330 can significantly reduce the perirenal fat weight of high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t test); *, p < 0.05 compared with the HFD-Control group); **, p < 0.01 compared with the HFD-Control group; ****, p < 0.0001 compared with the HFD-Control group.

[0123] Fig.19c:Shows that strains MNH22076 and MNH45330 can significantly reduce mesenteric fat weight in high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group); ****, p < 0.0001 compared with the HFD-Control group.

[0124] Fig.19d :Shows that strains MNH22076 and MNH45330 can significantly reduce the subcutaneous fat weight of high-fat diet-induced obese mice. Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t test); *, p < 0.05 compared with the HFD-Control group); **, p < 0.01 compared with the HFD-Control group; ****, p < 0.0001 compared with the HFD-Control group.

[0125] Fig.19e :Shows that strains MNH22076 and MNH45330 can significantly reduce the percentage of visceral fat in high-fat diet-induced obese mice. Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the HFD-Control group); ****, p < 0.0001 compared with the HFD-Control group.

[0126] Deposit of strains

[0127] Lactobacillus crispatus strain ( Lactobacillus crispatus )MNH22076, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65554, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Lactobacillus crispatus MNH22076, taxonomic name: Lactobacillus crispatus .

[0128] Lactobacillus crispatus ( Lactobacillus crispatus )MNH45330, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65556, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Lactobacillus crispatus MNH45330, taxonomic name: Lactobacillus crispatus . DETAILED DESCRIPTION

[0129] The present invention separates two new strains of Lactobacillus crispatus, with the deposit numbers GDMCC No: 65554 and GDMCC No: 65556, respectively, and identifies them using traditional classification methods and molecular biology methods. The identification results show that the two strains are new strains of Lactobacillus crispatus. Furthermore, the present invention studies the biochemical properties and therapeutic uses of the two strains.

[0130] It is known in the art that bacterial species can be classified and identified by traditional classification methods and molecular biological methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biological methods include, but are not limited to, ribosomal RNA sequence determination methods, determination methods based on whole genome sequencing, etc.

[0131] 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).

[0132] 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.

[0133] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics or medical foods. In some cases, the composition is applied in the form of a pharmaceutical preparation. In some cases, the composition is applied in the form of a nutritional supplement. In some cases, the composition is applied in the form of a dietary supplement. In some cases, the composition is applied in the form of a medical food. In some cases, the composition is applied in the form of a medical probiotic. In some cases, the composition (e.g., dietary supplements, nutritional supplements, medical probiotics or medical foods) can be orally administered, for example as a capsule, pill or tablet.

[0134] 16S rRNA is a ribosomal RNA of prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is common to all bacteria, while the variable region has different degrees of difference between different bacteria. By comparing the 16S rRNA gene sequences of bacteria, according to the base number of sequence differences and their evolutionary distance, an evolutionary tree can be drawn. 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 between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary 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 the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), P23).

[0135] 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.

[0136] The 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 by methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value needs to reach more than 95% to be identified 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.).

[0137] Various existing mature ANI value calculation tools can be used, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0138] By using the above method, a person skilled in the art can determine whether an isolated strain belongs to the species of Lactobacillus crispatus identified by the inventors. For example, when compared with Lactobacillus crispatus with a deposit number of GDMCC No: 65554 or GDMCC No: 65556 ( Lactobacillus crispatus ) has an average nucleotide identity ANI value of at least 95%, for example at least 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%, 97.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.1%, 99.2%, 99.3%, 9 ... .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 they belong to the same species.

[0139] For another example, when its 16S rRNA sequence has at least 98.65% identity with the sequence shown in SEQ ID NO: 3 or 4, 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% identity, it can be determined to belong to the same bacterial species.

[0140] A "strain" refers to a member of a bacterial species that has a genetic characteristic that allows it to be distinguished from closely related members of the same bacterial species. The genetic characteristic can be the total or partial absence of at least one gene, the total or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosomal 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, ribosomal 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 the case of a strain that gains or loses antibiotic resistance (compared to another strain of the same species) or gains or loses a biosynthetic ability (e.g., an auxotrophic strain), the strains can be distinguished by selection or counter-selection using antibiotics or nutrients / metabolites.

[0141] "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.

[0142] The Lactobacillus crispatus described herein can be used to prepare a composition, for example, by using a pharmaceutically acceptable excipient. The pharmaceutical composition comprises a pharmaceutically effective amount of the Lactobacillus crispatus, for example, the Lactobacillus crispatus with a deposit number of GDMCC No: 65554 or GDMCC No: 65556. Similarly, the Lactobacillus crispatus with a deposit number of GDMCC No: 65554 or GDMCC No: 65556 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, which comprises a pharmaceutically effective amount of the Lactobacillus crispatus.

[0143] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0144] The composition herein can be formulated into any form suitable for enhancing the abundance of Lactobacillus crispatus in a subject. The composition can be administered orally (e.g., by oral gavage), intramuscular injection, inhalation, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, intragastrointestinal, bile infusion, cardiac infusion, anterior anal, rectal, spinal subcutaneous, sublingual, topical, intravaginal, transdermal, ureteral or urethral.

[0145] Examples of dosage forms suitable for the composition 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.

[0146] 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 food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder or a toothpaste, etc.

[0147] In some embodiments, administration can also be by inclusion in a subject's diet, such as inclusion in a functional food for humans or companion animals.

[0148] The compositions provided herein may include a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutically acceptable excipient, diluent or carrier is well known in the art.

[0149] In some embodiments, the Lactobacillus crispatus in the compositions of the present disclosure is freeze-dried. In some embodiments, the Lactobacillus crispatus in the compositions of the present disclosure is spray-dried. In some embodiments, the Lactobacillus crispatus in the compositions of the present disclosure is freeze-dried or spray-dried and is alive. In some embodiments, the Lactobacillus crispatus in the compositions of the present disclosure is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the freeze-dried Lactobacillus crispatus is reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent as described herein.

[0150] 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.

[0151] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0152] The composition disclosed herein includes medicines, health products or foods.

[0153] The subject of the present disclosure may be a human or an animal, and the animal includes but is not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0154] The medicine disclosed herein can be used to treat, prevent or alleviate metabolic diseases, metabolic disorders, or diseases caused by metabolic disorders.

[0155] In some embodiments, the metabolic disease, metabolic disorder, or disease caused by metabolic disorder includes but is not limited to: liver disease, liver function disease, obesity and obesity-related diseases, liver and kidney function disease, 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 fatty hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of kidney disease.

[0156] diabetes Diabetes includes type 1 diabetes (T1D), type 2 diabetes (T2D) and gestational diabetes mellitus (GDM). Type 1 diabetes is caused by autoimmune damage or idiopathic reasons. It is characterized by absolute destruction of pancreatic islet function. It mostly occurs in children and adolescents. It must be treated with insulin to achieve satisfactory results, otherwise it will be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / fat metabolism, usually including hyperglycemia, hypertension and abnormal cholesterol. Type 2 diabetes is caused by the inability of insulin to work effectively (low binding to receptors). Therefore, it is necessary not only to check fasting blood sugar, but also to observe blood sugar 2 hours after a meal, especially to do pancreatic islet function tests. There are two types of diabetes during pregnancy. One is that the patient has been diagnosed with diabetes before pregnancy, which is called "diabetes complicated with pregnancy"; the other is that the patient has normal glucose metabolism or potential glucose tolerance before pregnancy, and the diabetes that appears or is diagnosed during pregnancy, which is also called "gestational diabetes (GDM)". More than 80% of pregnant women with diabetes have GDM.

[0157] Four metabolic disease-related models, the high-fat diet (HFD)-induced mouse obesity model, the high-fat, high-sugar, and high-cholesterol-induced mouse NASH model, the high-fat diet combined with streptozotocin (HFD-STZ)-induced mouse type II diabetes model, and the leptin receptor gene-deficient mouse model (db / db), are all commonly used metabolic disease mouse models. The model mice are usually accompanied by metabolic diseases such as obesity, insulin resistance, hyperglycemia, hyperlipidemia, hypercholesterolemia, NAFLD / NASH, etc.

[0158] Insulin resistance refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons. The body compensatory secretion of excessive insulin produces hyperinsulinemia to maintain blood sugar stability. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes.

[0159] Liver function disorders Liver function diseases are abnormal liver function or liver function damage. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver function diseases. When the body's liver function is abnormal (for example, liver damage, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), ALT and / or AST in the blood will increase significantly. In general, ALT is more sensitive than AST in reflecting acute liver damage. Continuous increase in ALT indicates chronic liver damage. In cases of chronic hepatitis, cirrhosis, liver cancer, etc., AST increases significantly and can exceed ALT. AST levels indicate the chronicity, extensiveness and severity of liver lesions, and even indicate the prognosis of chronic liver disease.

[0160] Common liver diseases with elevated ALT and / or AST include, but are not limited to: acute viral hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E); Epstein-Barr virus, cytomegalovirus infection; chronic hepatitis B or chronic hepatitis C; autoimmune liver disease; alcoholic liver disease (ALD); non-alcoholic fatty liver disease (NAFLD or NASH); drug-induced / toxic liver injury; cirrhosis; liver cancer; Wilson's disease; α1-antitrypsin deficiency; hemochromatosis, etc.

[0161] In addition, fat accumulation in the liver is an important factor in the development of non-alcoholic fatty liver disease (NAFLD or NASH). Therefore, when ALT and / or AST levels decrease and liver weight decreases after drug intervention, it can show that the drug has a certain therapeutic improvement effect.

[0162] Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of excess fat in the liver in the form of triglycerides (TG) (fatty degeneration). Some patients with NAFLD have liver cell damage and inflammation (steatohepatitis) in addition to excess fat, namely non-alcoholic steatohepatitis (NASH). NASH is widely considered to be the liver manifestation of metabolic syndrome, such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglycerides) and hypertension.

[0163] Liver and kidney disease Liver and kidney function diseases refer to functional acute renal failure that occurs in severe liver disease and decompensated cirrhosis. Due to insufficient effective circulating blood volume and decreased prostaglandins, hepatorenal syndrome may occur.

[0164] Cardiovascular disease TG (triglycerides) are mainly involved in energy metabolism in the human body and generate heat energy. Excessive TG content in the blood can lead to blood viscosity, causing lipids to deposit on the blood vessel walls, gradually forming small plaques, namely atherosclerosis. Increased LDL-C is the main and independent risk factor for the occurrence and development of atherosclerosis; the increased level of LDL-C is also an indicator for measuring coronary heart disease. Since HDL-C can transport cholesterol in the blood vessel wall to the liver for decomposition and metabolism (i.e., reverse cholesterol transport), it can reduce the deposition of cholesterol in the blood vessel wall and play an anti-atherosclerotic role.

[0165] obesity Obesity refers to a certain degree of obvious overweight and excessive fat layer. It is a state caused by excessive accumulation of body fat, especially triglycerides. It is an abnormal or excessive accumulation of fat that poses a risk to health. Excessive accumulation of body fat due to excessive food intake or changes in body metabolism causes excessive weight gain and causes pathological, physiological changes or lurking in the human body. A body mass index of more than 25 is considered overweight, and more than 30 is considered obese. Obesity increases the risk of many physical and mental diseases. It is mainly related to metabolic syndrome, a combination of diseases including type 2 diabetes, hypertension, hypercholesterolemia and hypertriglyceridemia. In general, the effects of obesity on health can be divided into two categories: diseases that can be attributed to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases that increase the number of fat cells (diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver or non-alcoholic steatohepatitis, etc.). 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, atherosclerosis, 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.

[0166] 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.

[0167] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature of the art or in accordance with the product or instrument instructions are used. All reagents or instruments are indicated by the manufacturer and can be purchased commercially.

[0168] Example

[0169] The anaerobic blood agar plate involved in the embodiment was purchased from Huankai Microorganisms, with a formula of 10 g / L casein pancreatic digest, 3 g / L heart 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.

[0170] The PYG liquid culture medium involved in the embodiment, each 1 L PYG liquid culture medium contains: Trypticase peptone (trypticase peptone) 5.0 g, Peptone (peptone) 5.0 g, Yeast extract (yeast extract powder) 10.0 g, Beef extract (beef extract powder) 5.0 g, Glucose (glucose) 5.0 g, K2HPO4 2.0 g, Tween 80 (Tween 80) 1.0 ml, Resazurin (resazurin) 1.0 mg, 40 ml Salt solution (salt solution) 40 ml (its components are as follows: CaCl2·2H2O 0.01 g, MgSO4·7H2O 0.02 g, K2HPO4 0.04 g, NaHCO3 0.4 g, NaCl 0.08 g, balance water), balance water, pH 7.2±0.2 (25°C).

[0171] The MRS plate medium involved in the embodiment, each 1LMRS plate medium contains: peptone 10.0g, beef extract powder 10.0g, yeast extract powder 5.0g, glucose 20.0g, Tween-80 1.0mL, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, triammonium citrate 2.0g, magnesium sulfate (MgSO4·7H2O) 0.1g, manganese sulfate (MnSO4·4H2O) 0.05g, agar 15.0g, and water balance.

[0172] The MRS liquid culture medium involved in the embodiment, each 1LMRS liquid culture medium contains: peptone 10.0g, beef extract powder 10.0g, yeast extract powder 5.0g, glucose 20.0g, Tween-80 1.0mL, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, triammonium citrate 2.0g, magnesium sulfate (MgSO4·7H2O) 0.1g, manganese sulfate (MnSO4·4H2O) 0.05g, and water balance.

[0173] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0174] Example 1. Isolation and identification of strains

[0175] 1.1 Isolation and purification of strains MNH22076 and MNH45330 Two intestinal strains of Lactobacillus crispatus ( Lactobacillus crispatus MNH22076 and Lactobacillus crispatusMNH45330) were isolated from fecal samples of healthy volunteers. The isolation method adopted the conventional strain isolation method, using gradient dilution, and then picking a single colony for isolation and culture, and then purifying the strain and anaerobic culture at 37°C. The pure culture strain was prepared into 20% glycerol / water-bacterial solution and stored at -80°C.

[0176] Specifically, the separation and purification methods of the strains are as follows: The donor takes 2-5 g of fresh feces and puts it into a sample collection and storage tube. After shaking and homogenization, the processed feces sample is placed in an ice box and sent to the laboratory for strain isolation within 24 hours.

[0177] Dispense physiological saline in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation culture medium, and transfer them to the anaerobic workstation 24 hours in advance, marking the sample information, culture medium type, isolation date, etc.

[0178] Take a fresh stool sample and place it in an anaerobic workstation and shake it on a vortex shaker for 1 min to mix it. Pipette 1 mL of the sample into 9 mL of normal saline and mix it for 10 min. -1 dilution, and then serially diluted to 10 -6 Dilution solution for later use.

[0179] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a drop size of 100 μL / plate. Spread evenly. After the surface of the plate is dry, invert the plate and culture at 37°C for 3-5 days.

[0180] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick a single colony with a sterilized toothpick for strain purification. The purified strain is placed at 37°C for anaerobically cultured. The pure culture strain is prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

[0181] 1.2 Morphological characteristics of strains MNH22076 and MNH45330 1.2.1 Morphological characteristics of strain MNH22076 The strain MNH22076 was inoculated on an MRS plate medium and cultured anaerobically at 37°C for 48 h. Visible colonies were formed on the MRS plate medium. The colonies were round, with regular and smooth edges, about 1-2 mm in diameter, white, and opaque. The strain was Gram-positive. Microscopic morphological observation revealed that it had no flagella, was non-motile, rod-shaped, and about 0.5-1µm×1.5-4 µm in size. The colony morphology of strain MNH 22076 cultured on an MRS plate for 48 h is shown in Figure 1 The Gram staining photos of strain MNH22076 are shown in Figure 2 See the electron microscope photos for Figure 3 .

[0182] 1.2.2 Morphological characteristics of strain MNH45330 The strain MNH45330 was inoculated on an MRS plate medium and cultured anaerobically at 37°C for 48 h. Visible colonies were formed on the MRS plate medium. The colonies were round, with regular and smooth edges, about 0.5-2 mm in diameter, white, and opaque. The strain was Gram-positive. Microscopic morphological observation revealed that it had no flagella, was non-motile, rod-shaped, and about 0.5-1 µm × 1.5-4 µm in size. The colony morphology of the strain MNH 45330 cultured on an MRS plate for 48 h is shown in Figure 4 The Gram staining photos of strain MNH45330 are shown in Figure 5 See the electron microscope photos for Figure 6 .

[0183] 1.3 Physiological and biochemical characteristics of strains MNH22076 and MNH45330 1.3.1 Physiological and biochemical characteristics of strain MNH22076 The strain MNH22076 can grow in the range of pH 5.0 to 10.0, with the optimal growth pH being 8.0 (see the results of strain tolerance to different pH values). Figure 7 ); it can still grow in a 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 8 ); strain MNH22076 can survive and grow in the bile salt concentration range of 0%-0.1%, and cannot grow when the bile salt concentration is greater than or equal to 0.15% (the strain's tolerance to different concentrations of bile salts is shown in Fig. 9 ).

[0184] 1.3.2 Physiological and biochemical characteristics of strain MNH45330 The strain MNH45330 can grow in the pH range of 4.0 to 10.0, with the optimal growth pH being 8.0 (see the results of the strain's tolerance to different pH values). Fig.10 ); it can still grow on medium with NaCl content exceeding 6% (the results of strain tolerance to different concentrations of NaCl are shown in Fig.11 ); strain MNH45330 could not grow in medium with bile salt concentration above 0.1% (the results of strain tolerance to different concentrations of bile salts are shown in Fig.12 ).

[0185] 1.4 Biochemical identification results of strains MNH22076 and MNH45330 1.4.1 Results of biochemical identification of strain MNH22076 using API 50CHL API 50CHL (Mérieux, CN5041010) was used. The specific experimental operation was as per the conventional API reagent operation guide. Culture conditions: 37°C, anaerobic. The experimental results are shown in Table 1.

[0186] MNH22076 can utilize galactose GAL, glucose GLU, fructose FRU, mannose MNE, α-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, and gentiobiose GEN to ferment and produce acid. Therefore, in the process of fermentation or cultivation of strain MNH22076, galactose GAL, glucose GLU, fructose FRU, mannose MNE, α-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN and their derivatives can be used as carbon sources.

[0187]

[0188] 1.4.2 Results of biochemical identification of strain MNH45330 using API 50CHL API 50CHL (Mérieux, CN5041010) was used. For specific experimental operations, please refer to the conventional API reagent operation guide. Culture conditions: 37°C, anaerobic. The experimental results are shown in Table 2.

[0189] MNH45330 can utilize galactose GAL, glucose GLU, fructose FRU, mannose MNE, Mannitol MAN, α-Methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN are fermented to produce acid. Therefore, in the process of fermentation or cultivation of strain MNH45330, galactose GAL, glucose GLU, fructose FRU, mannose MNE, Mannitol MAN,α-Methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN, and their derivatives can be used as carbon sources.

[0190]

[0191] 1.5 Antibiotic Minimum Inhibitory Concentration Test for Strains MNH22076 and MNH45330 1.5.1 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH22076 The minimum inhibitory concentration of antibiotics for strain MNH22076 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 3.

[0192]

[0193] The results showed that MNH22076 was sensitive to ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, penicillin, cefquinome, and tetracycline. It can be seen that MNH22076 is sensitive to most types of antibiotics, and the risk of long-term use of MNH22076 leading to antibiotic resistance in subjects is low.

[0194] 1.5.2 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH45330 The minimum inhibitory concentration of antibiotics for strain MNH45330 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 4.

[0195]

[0196] The results showed that MNH45330 was sensitive to imipenem, ampicillin-sulbactam, dopenem, ertapenem, meropenem, piperacillin-tazobactam, and ceftriaxone. It can be seen that MNH45330 is sensitive to most types of antibiotics, and the risk of long-term use of MNH45330 leading to antibiotic resistance in subjects is low.

[0197] 1.6 Identification of strains MNH22076 and MNH45330 1.6.1 Amplification of 16S rRNA genes of strains MNH22076 and MNH45330 Fresh cultures of strains MNH22076 and MNH45330 were taken to extract genomic DNA of the strains, and the extracted genomic DNA of the strains was used as a template for 16S rRNA gene amplification.

[0198] The primer pairs used for PCR of 16S rRNA gene are: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1); 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0199] The PCR reaction procedure is as follows: Pre-denaturation: 94℃, 4 min; (denaturation: 94℃, 50 sec, annealing: 52℃, 40 sec; extension: 72℃, 70sec), 36 cycles; final extension: 72℃, 10 min.

[0200] 1.6.2 16S rRNA gene sequencing of strains MNH22076 and MNH45330 The PCR product was purified and sequenced by Bioengineering Company to obtain the 16S rRNA gene.

[0201]

[0202] 1.6.3 Identification results of strains MNH22076 and MNH45330 The 16S rRNA gene sequences measured above, such as those shown in SEQ ID NO: 3 and SEQ ID NO: 4, were used to perform strain 16S rRNA gene analysis using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0203] The measured sequences were compared with the data in GenBank by BLAST analysis. The results showed that the strain with the highest similarity to MNH22076 was Lactobacillus crispatus The similarity was 100%. The whole genome ANI analysis showed that the similarity with Lactobacillus crispatus (GCF 002088015.1) was 97.63%, and the alignment score (AF) was 86%. Therefore, strain MNH22076 was Lactobacillus crispatus strains planted; the strain with the highest similarity to MNH45330 is Lactobacillus crispatus The similarity was 100%. The whole genome ANI analysis showed that the similarity with Lactobacilluscrispatus (GCF 002088015.1) was 97.4%, and the alignment score (AF) was 86%. Therefore, strain MNH45330 was Lactobacillus crispatus Planted strains.

[0204] MNH22076 and MNH45330 were compared with the Lactobacillus crispatus The 16S rRNA gene sequences of related strains of the genus were compared to construct a phylogenetic tree.

[0205] The sequences of MNH22076 and MNH45330 were aligned with those of the model strains with high 16S rRNA gene sequence similarity in the NCBI database, and then a phylogenetic tree was constructed using the software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method). Fig.13 , 14 Only the Bootstrap values ​​greater than 50% are displayed in the developmental tree nodes.

[0206] From the phylogenetic tree ( Fig.13 , 14 ) It can be seen that strain MNH22076 and Lactobacillus Bacteria and Lactobacillus crispatus Get together for Lactobacillus crispatus The strain MNH45330 and Lactobacillus Bacteria and Lactobacillus crispatusGet together for Lactobacillus crispatus New strains planted.

[0207] 1.7 Genome analysis of strains MNH22076 and MNH45330 1.7.1 Genome analysis of strain MNH22076 The genome of strain MNH22076 was fragmented by ultrasound, with a fragment length range of ~350bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150bp. 1.28Gbp of data was obtained by sequencing, of which Q20 accounted for 95.28%.

[0208] The raw sequencing data of the genome were filtered using fastp (version: 0.20.0) with filtering 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: v3.14.0) with assembly parameters "--isolate --cov-cutoff 10". The total gene length of the genome assembly was 2.28 Mbp, the N50 length was 33.0 kbp, and the GC content was 36.59%.

[0209] The genomic genes were predicted and analyzed 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.

[0210] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), and the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found after comparison.

[0211] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare the virulence factor database VFDB (virulence factor database, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). The detailed comparison results are shown in Table 5.

[0212]

[0213] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 6.

[0214]

[0215] 1.7.2 Genome analysis of strain MNH45330 The genome of strain MNH45330 was fragmented by ultrasound, with a fragment length range of ~350bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150bp. 1.43Gbp of data was obtained by sequencing, of which Q20 accounted for 95.33%.

[0216] The raw sequencing data of the genome were filtered using fastp (version: 0.20.0) with filtering 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: v3.14.0) with assembly parameters "--isolate --cov-cutoff 10". The total gene length of the genome assembly was 2.45 Mbp, the N50 length was 43.3 kbp, and the GC content was 36.51%.

[0217] The genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline prokka (version: 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 2468 CDS sequences were predicted, with an average CDS sequence length of 870 bp.

[0218] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), and the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found in the comparison.

[0219] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare the virulence factor database VFDB (virulence factor database, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). The detailed comparison results are shown in Table 7.

[0220]

[0221] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 8.

[0222]

[0223] Example 2: Strains MNH22076 and MNH45330 improve liver function and related diseases in a high-fat diet-induced liver injury mouse model The high-fat diet liver and kidney injury mouse model was used to study the improvement of liver and kidney function and related diseases by strains MNH22076 and MNH45330. This experimental protocol has been ethically reviewed by the Experimental Animal Care and Use Committee of Muen Biotechnology.

[0224] Liver function diseases are abnormal liver function or liver function damage. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver function diseases. When the body's liver function is abnormal (for example, liver damage, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), ALT and / or AST in the blood will increase significantly. In general, ALT is more sensitive than AST in reflecting acute liver damage. Continuous increase in ALT indicates chronic liver damage. In cases of chronic hepatitis, cirrhosis, liver cancer, etc., AST increases significantly and can exceed ALT. AST levels indicate the chronicity, extensiveness and severity of liver lesions, and even indicate the prognosis of chronic liver disease.

[0225] 2.1 Experimental methods 1) Experimental animals: C57BL / 6J mice, 5-6 weeks old, were purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0226] 2) Preparation of test materials of strains MNH22076 and MNH45330: After thawing the glycerol cryovials of strains MNH22076 and MNH45330 at 37°C, they were inoculated on MRS plate medium in an anaerobic workstation for activation. The activated strains were inoculated in MRS liquid medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 0.05% cysteine ​​hydrochloride (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.

[0227] 3) Negative control: Use PBS containing 0.05% L-Cys HCl as the negative control.

[0228] 4) Positive control: After thawing the glycerol cryopreservation tube of animal Bifidobacterium lactis subsp. B420 (B420 has been disclosed in patent CN109069553A) at 37°C, inoculate it into PYG medium in an anaerobic workstation for activation, inoculate the activated strain into PYG liquid medium, and culture it anaerobically to obtain a sufficient amount of culture. After centrifugation and concentration of the cultured bacterial solution, resuspend the bacterial strain in PBS containing 0.05% L-Cys HCl to obtain the purity and viable count (2×10 9 CFU / mL) is a positive control substance that meets the requirements of animal experiments.

[0229] 5) Experimental process: After the quarantine period, 40 C57BL / 6J male mice weighing 18g-22g were selected from 5-6 weeks old male mice. They were randomly divided into 5 groups, 8 mice / group, including NCD-Control group, HFD-Control group, MNH22076 group, MNH45330 group, and B420 group. The NCD-Control group was fed with maintenance feed (Cat. No.: D12451B, Parker Biotech), and the other four groups were fed with 60% high-fat feed (Cat. No.: D12492, Parker Biotech). Drug administration began after grouping (D1). The NCD-Control group and the HFD-Control group were given negative control products, the B420 group was gavaged with positive control products, the MNH22076 group was gavaged with MNH22076, and the MNH45330 group was gavaged with MNH45330. The drugs were administered once a day, 200 μL each time, for a total of 56 days. During the experiment, mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the experiment, a general clinical observation was performed once after each administration. The endpoint of this experiment was the day after the end of administration (D57). The end point of the experiment was anatomical sampling according to the protocol, and the results of each anatomical data and serum test data were summarized and analyzed. All data were expressed in the form of Mean±SD, and GraphPad Prism software was used for drawing and statistical analysis. For pairwise comparisons, the t test (Student's t test) analysis method was used. No significance is indicated; significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0230] 2.2 Experimental Results See also Figure 15a-15b , showing that strain MNH22076 can significantly reduce liver function indicators. Specifically, Figure 15a-15b The results showed that strain MNH22076 can significantly reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, so strain MNH22076 can improve abnormal liver function caused by a high-fat diet and has the use of treating or preventing liver function damage.

[0231] Example 3: Use of strains MNH22076 and MNH45330 for preventing or treating diabetes

[0232] 3.1 Experimental methods 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 5-6 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0233] 2) Preparation of test materials of strains MNH22076 and MNH45330: After thawing the glycerol cryovials of MNH22076 and MNH45330 strains at 37°C, inoculate them into MRS plate medium in an anaerobic workstation for activation. Inoculate the activated strains into MRS liquid medium and culture them anaerobically to obtain a sufficient amount of culture. After centrifugation and concentration of the cultured bacterial solution, resuspend the bacterial cells in PBS containing 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.

[0234] 3) Negative control: Use PBS containing 0.05% L-Cys HCl as negative control.

[0235] 4) Positive control: After thawing the glycerol cryopreserved tube of B420 strain at 37°C, inoculate it into PYG medium in an anaerobic workstation for activation. Inoculate the activated strain into PYG liquid medium and culture it anaerobically to obtain a sufficient amount of culture. After centrifugation and concentration of the cultured bacterial solution, resuspend the bacterial strain in PBS containing 0.05% L-Cys HCl to obtain the purity and viable count (2×10 9 CFU / mL) is a positive control substance that meets the requirements of animal experiments.

[0236] 5) Experimental process: After the quarantine period, 40 C57BL / 6J male mice with a body weight range of 18g-22g were selected for 5-6 weeks old mice. They were randomly stratified and divided into 5 groups according to their body weight, 8 mice / group, namely NCD-Control group, HFD-Control group, MNH22076 group, MNH45330 group, and B420 group. The NCD-Control group was fed with maintenance feed, and the other four groups were fed with 60% high-fat feed. After grouping (D1), drug administration began. The NCD-Control group and the HFD-Control group were given negative control products, the B420 group was given positive control products, the MNH22076 group was given MNH22076, and the MNH45330 group was given MNH45330. The drugs were administered orally once a day, 200μL each time, for a total of 56 days. During the experiment, the mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the trial, general clinical observation was performed once after each administration. Fasting blood glucose was measured once within the last 2 weeks of administration, and fasting blood glucose was measured at the end of the trial. All data were expressed in Mean±SD format, and GraphPad Prism software was used for plotting and statistical analysis. For pairwise comparisons, the t test (Student's t test) analysis method was used. No significance is indicated; significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0237] 3.2 Experimental Results See also Fig.16 It can be seen that strains MNH22076 and MNH45330 can significantly reduce the fasting blood glucose of type 2 diabetic mice induced by a high-fat diet. Therefore, strains MNH22076 and MNH45330 are useful for preventing or treating diabetes.

[0238] Example 4: Use of strains MNH22076 and MNH45330 in the treatment or prevention of obesity and related diseases

[0239] 4.1 Experimental methods: 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 5-6 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0240] 2) Preparation of test materials of strains MNH22076 and MNH45330: After thawing the glycerol cryopreserved tubes of MNH22076 and MNH45330 strains at 37°C, inoculate them into MRS plate medium in an anaerobic workstation for activation. The activated strains were inoculated into MRS liquid medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 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.

[0241] 3) Negative control: Use PBS containing 0.05% L-Cys HCl (cysteine ​​hydrochloride) as a negative control 4) Positive control: After thawing the glycerol cryopreserved tube of B420 strain at 37°C, inoculate it into PYG medium in an anaerobic workstation for activation. Inoculate the activated strain into PYG liquid medium and culture it anaerobically to obtain a sufficient amount of culture. After centrifugation and concentration of the cultured bacterial solution, resuspend the bacterial strain in PBS containing 0.05% L-Cys HCl to obtain the purity and viable count (2×10 9 CFU / mL) is a positive control substance that meets the requirements of animal experiments.

[0242] 5) Experimental process: After the quarantine period, 40 C57BL / 6J male mice with a body weight range of 18g-22g were selected for 5-6 weeks old mice. They were randomly stratified and divided into 5 groups according to their body weight, 8 mice / group, namely NCD-Control group, HFD-Control group, MNH22076 group, MNH45330 group, and B420 group. The NCD-Control group was fed with maintenance feed, and the other four groups were fed with 60% high-fat feed. After grouping (D1), drug administration began. The NCD-Control group and the HFD-Control group were given negative control products, the B420 group was given positive control products, the MNH22076 group was given MNH22076, and the MNH45330 group was given MNH45330. The drugs were administered orally once a day, 200μL each time, for a total of 56 days. During the experiment, the mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the trial, general clinical observation was performed once after each administration. The animals were weighed twice a week during the administration period; the animals were weighed before dissection at the end of the trial. The end point of this trial was the day after the end of administration (D57). The end point of the trial was dissected according to the protocol, and the body weight and body weight change percentage, various dissection data results and serum test data results were summarized and analyzed. All data were expressed in the form of Mean±SD, and GraphPad Prism software was used for drawing and statistical analysis. For pairwise comparisons, the t test (Student's t test) analysis method was used. No significance is not indicated; significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0243] 4.2 Experimental Results See also Figure 17a-17b It can be seen that strains MNH22076 and MNH45330 can significantly reduce the body weight and weight gain of obese mice induced by high-fat diet, and after eight weeks of gavage intervention, the body weight was reduced by more than 10% compared with the HFD-Control group, indicating that strains MNH22076 and MNH45330 have significant weight loss effects.

[0244] See also Figures 18a-18c The results showed that strain MNH22076 could significantly reduce the concentrations of total cholesterol (TCHO), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the serum of mice with high-fat diet-induced obesity. The above data indicate that strains MNH22076 and MNH45330 have the effect of treating / preventing hypercholesterolemia, hyperlipidemia, and / or cardiovascular and cerebrovascular diseases.

[0245] See also Figures 19a-19eThe results showed that strains MNH22076 and MNH45330 could significantly reduce the fat weight of epididymal fat, perirenal fat, subcutaneous fat and / or mesenteric fat in mice induced by high-fat diet, and significantly reduce the ratio of visceral fat to body weight, indicating that strains MNH22076 and MNH45330 have the effect of treating / preventing high visceral fat rate and treating / preventing obesity.

[0246] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations may be made without departing from the spirit and scope of the present invention as disclosed herein and as provided in the appended claims. In addition, it should be understood that although all examples disclosed illustrate embodiments of the present invention, they are provided only as non-limiting examples and therefore should not be considered as limiting the various aspects of the present invention thus described. The present invention is intended to have the full scope defined by the language of the present disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed description should be considered illustrative rather than restrictive.

Claims

1. An isolated Lactobacillus crispatus ( Lactobacillus crispatus ), which is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 65554 or GDMCC No: 65556.

2. The culture, live bacteria, freeze-dried bacteria or inactivated bacteria of Lactobacillus crispatus according to claim 1; in, The culture comprises any one of the following A) to D): A) fermentation broth of the Lactobacillus crispatus; B) the supernatant of the fermentation broth of the Lactobacillus crispatus; C) an inactivated fermentation broth of the Lactobacillus crispatus; D) The concentrated or dried product of any one of A) to C) above.

3. A composition comprising the Lactobacillus crispatus according to claim 1, or the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus according to claim 2.

4. The composition according to claim 3, further comprising one or more pharmaceutically and / or food-acceptable excipients; and / or The composition may further comprise one or more other active agents for preventing or treating liver disease, diabetes, obesity, hypercholesterolemia, hyperlipidemia, or cardiovascular and cerebrovascular diseases.

5. The composition of claim 4, wherein the other active agent comprises one or more of a probiotic, a prebiotic, a GLP-1 receptor agonist, an AMPK agonist or an active drug that promotes GLP-1 secretion.

6. The composition according to any one of claims 3 to 5, which is any one of a medicine, a health product or a food.

7. The composition according to claim 6, wherein the health product or food is any one of a nutritional composition, a food, a candy, a food bar, a food additive, a drink additive, and a dietary supplement.

8. Use of the Lactobacillus crispatus according to claim 1, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the Lactobacillus crispatus according to claim 2, or the composition according to any one of claims 3 to 7 in the preparation of medicines, health products or foods for treating, preventing, alleviating or alleviating liver diseases, diabetes, obesity, hypercholesterolemia, hyperlipidemia, cardiovascular and cerebrovascular diseases.

9. The use according to claim 8, wherein the liver disease comprises at least one of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver function damage.

10. The use according to any one of claims 8 to 9, wherein the medicine, health product or food is used for: 1) Prevent, treat, improve or alleviate liver damage, NAFLD or NASH; 2) Lose weight or control weight; 3) reducing the subcutaneous fat and / or visceral fat of the subject; 4) reducing the subject's blood lipid and / or cholesterol levels; 5) Prevent, treat, improve or alleviate hypercholesterolemia, hyperlipidemia, and / or cardiovascular and cerebrovascular diseases; 6) Prevent, treat, improve or alleviate diabetes.

Images