CN119842569A - Isolated lactobacillus crispatus, compositions comprising same and uses - Google Patents

Abstract

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

Description

Isolated lactobacillus crispatus, compositions comprising same and uses

Technical Field

The present disclosure relates to the field of microorganisms, more particularly to isolated lactobacillus crispatus, compositions comprising the same, and uses thereof.

Background

The prevalence of diseases such as obesity, diabetes, hypertension, hyperlipidemia, liver and kidney function diseases is increasing, and studies have also shown that these diseases are associated with dysbiosis of microbial flora. Mixtures of various microorganisms are commonly used in the art for the treatment of metabolic and obesity related diseases or disorders, such as probiotics or intestinal flora transplants (FMT). However, the mechanism is further complicated by a mixture of microorganisms, the interactions between the microorganisms have not been well studied, and the use of a mixture of microorganisms often breaks the homeostasis of the intestinal flora. Furthermore, the use of a mixture of microorganisms requires additional consideration of whether the individual microorganisms affect each other's activity, nor is it clear whether the synergistic effect of the mixture of microorganisms makes it useful in the treatment or prevention of a disease or whether a single species of bacteria plays a role in the treatment or prevention of a disease.

Lactobacillus crispatus is closely related to genital tract health and plays an important role in maintaining female vaginal microecological balance. The decrease in the abundance of Lactobacillus crispatus may be associated with various female genital tract diseases such as Bacterial Vaginosis (BV), vulvovaginal candidiasis (VVC), and even lead to bad pregnancy outcomes such as infertility, embryo arrest, and the like. Less researches on weight losing and blood glucose reducing of lactobacillus crispatus are carried out, and CN114540257B discloses that the IOB901 strain can be used for feeding mice to reduce blood fat and blood glucose. Chicken-derived lactobacillus crispatus is capable of reducing cholesterol and triglycerides of ileal epithelial cells of chickens, reducing 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 is capable of alleviating liver function damage caused by high fat diet, and visceral fat accumulation has not been studied.

The number of microbial resources is extremely large, and the screening of new species or strains from which to alleviate liver function damage caused by high fat diets, and visceral fat accumulation, and further to be effective in the treatment or prevention of diseases such as obesity, diabetes, hypertension, hyperlipidemia, liver and kidney function diseases, is a great challenge, but represents a great unmet need.

Disclosure of Invention

The present disclosure isolated novel Lactobacillus crispatus (Lactobacillus crispatus). The novel strain can reduce liver weight, reduce the ratio of the liver weight to the weight, reduce liver steatosis and/or liver lobular inflammation, reduce ballooning of liver cells, reduce the activity score (NAS) of nonalcoholic fatty liver diseases, reduce serum AST, ALT, reduce the weight of mammals, reduce the weight increment of the mammals, reduce at least one of epididymal fat weight, perirenal fat weight, subcutaneous fat weight and mesenteric fat weight of the mammals, reduce the ratio of visceral fat to the weight, reduce fasting blood glucose, reduce the level of at least one index of total cholesterol level, triglyceride level, high-density lipoprotein cholesterol level and low-density lipoprotein cholesterol level in serum of the mammals, and can achieve the aim of treating, preventing or relieving liver and kidney diseases and metabolic diseases.

In a first aspect, the present disclosure provides an isolated lactobacillus crispatus (Lactobacillus crispatus) having 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 a strain having deposit number GDMCC No:65554, or GDMCC No:65556, and/or a 16S rRNA sequence having at least 98.65% identity with a sequence as set forth in SEQ ID No. 3, or as set forth in SEQ ID No. 4.

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 strain MNH22076 having deposit number GDMCC No:65554, and/or has a 16S rRNA sequence at least 98.65% identical to the sequence set forth in SEQ ID NO. 3.

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 strain MNH45330 having deposit number GDMCC No:65556, and/or has a 16S rRNA sequence at least 98.65% identical to the sequence set forth in SEQ ID NO. 4.

In some embodiments, the lactobacillus crispatus (Lactobacillus crispatus) has 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 as SEQ ID No. 3, or as SEQ ID No. 4.

In some embodiments, the lactobacillus crispatus is two new strains of lactobacillus crispatus species (Lactobacillus crispatus).

In some embodiments, the Lactobacillus crispatus is designated Lactobacillus crispatus (Lactobacillus crispatus) MNH22076, deposited with the Guangdong university microbiological strain collection center (GDMCC) under accession number GDMCC No:65554 for 2024, 11 months and 28 days, at accession number 5 building, 30 th university, martyr, guangzhou, university, and national institute of microbiology, under accession number Lactobacillus crispatusMNH22076.

In some embodiments, the Lactobacillus crispatus is designated Lactobacillus crispatus (Lactobacillus crispatus) MNH45330, deposited with the Guangdong university microbiological strain collection center (GDMCC) under accession number GDMCC No:65556 for 2024, 11 months and 28 days, at accession number 5 building, 30 th university, martyr, guangzhou, university, and national institute of microbiology, under accession number Lactobacillus crispatusMNH45330.

In a second aspect, the present disclosure provides a culture, viable bacteria, lyophilized bacteria or inactivated bacteria of the lactobacillus crispatus of the first aspect of the invention;

wherein the culture comprises any one of the following A) to D):

A) A fermentation broth of the lactobacillus crispatus;

b) A fermentation broth supernatant of the lactobacillus crispatus;

c) A fermentation broth deactivation of the lactobacillus crispatus;

d) The concentrated or dried product of any of the a) -C).

In a third aspect, the present disclosure provides a composition comprising the lactobacillus crispatus of the first aspect, a culture thereof, and/or a metabolite thereof.

The present disclosure provides a composition comprising a lactobacillus crispatus according to the first aspect, or a culture, viable bacteria, lyophilized bacteria or inactivated bacteria of the lactobacillus crispatus according to the second aspect.

In some embodiments, the culture of lactobacillus crispatus comprises a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof of lactobacillus crispatus.

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 medium.

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

In some embodiments, the composition comprises 1x 10 ⁴ to 1x 10 ¹² cfu/mL or 1x 10 ⁴ to 1x 10 ¹² cfu/mg of the live lactobacillus crispatus.

In some embodiments, the composition comprises 1x 10 ⁵ to 1x 10 ¹¹ cfu/mL or 1x 10 ⁵ to 1x 10 ¹¹ cfu/mg of the live lactobacillus crispatus.

In some embodiments, the composition comprises 1x 10 ⁶ to 1x 10 ¹⁰ cfu/mL or 1x 10 ⁶ to 1x 10 ¹⁰ cfu/mg of the live lactobacillus crispatus.

In some embodiments, the composition comprises 1x 10 ⁷ to 1x 10 ⁹ cfu/mL or 1x 10 ⁷ to 1x 10 ⁹ cfu/mg of the live lactobacillus crispatus.

In some embodiments, 1X 10 ³ to 1X 10 ¹⁷ Colony Forming Units (CFU) of bacteria, e.g., 1X 10 ⁴ to 1X 10 ¹², 1X 10 ⁵ to 1X 10 ¹¹, or 1X 10 ⁶ to 1X 10 ¹⁰ Colony Forming Units (CFU) of bacteria, specifically, e.g., 1×10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴、1×10⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³、9×10¹³ or any value therebetween, are included per g of the composition.

In some embodiments, the concentration of the lactobacillus crispatus as an active ingredient in the composition is from 10 ⁷ to 10 ¹² CFU/g.

In some embodiments, the lactobacillus crispatus in the composition is a live, attenuated, lyophilized or inactivated bacterium, for example, may be a heat-inactivated bacterium, preferably pasteurized.

In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (e.g., lyophilized powder), or gel.

In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained release formulation, nano-formulation, or microencapsulated capsule.

In some embodiments, the composition is in the form of an oral or injectable formulation.

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

Such pharmaceutically, and/or food acceptable excipients are well known to those skilled in the art.

In some embodiments, the adjuvant may be at least one selected from the group consisting of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula, or sterile 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)), lyoprotectant, preservative, stabilizer, binder, compacter, lubricant, dispersion enhancer, disintegrant, antioxidant, flavoring, sweetener, and colorant.

In some embodiments, the composition further comprises one or more additional active agents for preventing or treating metabolic disorders and/or liver and kidney disorders.

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

In some embodiments, the liver disease comprises at least one of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver function impairment.

In some embodiments, the additional active agent has at least one of (a) suppressing appetite, (b) preventing metabolic disease, (c) treating metabolic disease, (d) preventing liver and kidney disease, and (e) treating liver and kidney disease.

In some embodiments, the suppressing appetite comprises reducing food intake and/or reducing appetite.

In some embodiments, the metabolic disease is a metabolic disease, metabolic disorder, or a disease caused by a metabolic disorder, including but not limited to at least one of liver disease, liver function disease, obesity and obesity related disease, cardiovascular disease, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), atherosclerosis, kidney disease.

In some embodiments, the liver and kidney disease includes liver disease, liver function injury-related disease, kidney injury, kidney dysfunction, and the like.

In some embodiments, liver-like disorders include, but are not limited to, at least one of fatty liver, NAFLD/NASH, liver dysfunction, extrahepatic cholestasis, hepatitis, liver injury, intrahepatic cholestasis, liver fibrosis, cirrhosis, and liver cell damage.

In some embodiments, the additional active agent is selected from the group consisting of GLP-1 receptor agonists, GLP-1 receptor and GCG receptor dual agonists, GLP-1 receptor, GIP receptor and GCG receptor triple agonists, AMPK agonists, or active agents that promote GLP-1 secretion.

In some embodiments, the additional active agent is selected from the group consisting of metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3-fatty acids, statins, ezetimibe, ursodeoxycholic acid, semaglutinin, liraglutide, exenatide, benalundin.

In some embodiments, the other active agent may be a probiotic, one or more of prebiotics, or a combination thereof.

In some embodiments, the probiotic is selected from one or more of lactic acid bacteria, lactobacillus, butyric acid bacteria.

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 grignard, lactobacillus helveticus, lactobacillus johnsonii, lactobacillus equi-like, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus fermentum, lactobacillus reuteri, lactobacillus plantarum, lactobacillus salivarius, lactobacillus curvatus, lactobacillus cantonensis, lactococcus lactis, lactococcus cremoris.

In some embodiments, the prebiotic is selected from inulin, mulberry leaf extract, berberine, ganoderma lucidum, green coffee bean extract, oat, pectin, potato or an extract thereof, citrus polyphenols, cassiterite, chromium, ergothioneine, astaxanthin, quercetin, curcumin, procyanidins, resistant dextrin, yeast beta-glucan, ginseng or an extract thereof, nutritional compounds, biotin, polydextrose, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, mannooligosaccharides (MOS), fructo-oligosaccharide-rich inulin, gluco-oligosaccharides, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS), and any combination thereof.

In some embodiments, the composition may be formulated as a frozen composition, for example, frozen composition made by quick freezing and drying, or freeze drying, for storage and/or transportation.

In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained via electrostatic spray drying.

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

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

In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric formulation. In some embodiments, the enteric formulation is a dosage form having an enteric coating. For example, the enteric formulation may 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 or soft capsule, or the capsule is a slow release capsule, a controlled release capsule, or an enteric capsule, or the capsule may be a microencapsulated capsule, or a microcapsule.

In some embodiments, the composition is a pharmaceutical, or a nutraceutical or food product.

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

In some embodiments, the composition is administered parenterally or parenterally.

In a fourth aspect, the present disclosure provides the use of a lactobacillus crispatus as described in the first aspect, a culture, viable bacteria, lyophilized bacteria or inactivated bacteria of the lactobacillus crispatus as described in the second aspect, or a composition of the third aspect, for the manufacture of a medicament, health product or food for treating, preventing, alleviating or alleviating a liver-kidney disease, metabolic disease.

The present disclosure provides use of the lactobacillus crispatus of the first aspect, the culture, viable bacteria, lyophilized bacteria or inactivated bacteria of the lactobacillus crispatus of the second aspect, or the composition of the third aspect, for the preparation of a medicament, health product or food for treating, preventing, alleviating, or alleviating liver diseases, diabetes, obesity, hypercholesterolemia, hyperlipidemia, cardiovascular and cerebrovascular diseases.

In some embodiments, the liver disease comprises at least one of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver function impairment.

In some embodiments, the metabolic disease is a metabolic disease, metabolic disorder, or a disease caused by a metabolic disorder, including but not limited to at least one of liver disease, liver function disease, obesity and obesity related disease, cardiovascular disease, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), atherosclerosis, kidney disease.

In some embodiments, the liver and kidney disease comprises at least one of liver disease, liver function injury-related disease, kidney injury, kidney dysfunction, and the like.

In some embodiments, the kidney disease includes, but is not limited to, kidney injury, renal dysfunction, and the like. The kidney disease includes, but is not limited to, primary glomerulonephritis, hypertensive glomerulonephritis, diabetic nephropathy, secondary glomerulonephritis, tubular interstitial lesions (e.g., chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic kidney disease, etc. In some embodiments, the predisposition to kidney disease comprises at least one of a high fat diet, a high sugar diet, a high cholesterol diet. In some embodiments, the kidney disease is manifested as elevated blood creatinine.

In some embodiments, liver-like disorders include, but are not limited to, at least one of fatty liver, NAFLD/NASH, liver dysfunction, extrahepatic cholestasis, hepatitis, liver injury, intrahepatic cholestasis, liver fibrosis, cirrhosis, and liver cell damage.

The composition comprises a consortium of an isolated and purified population of viable microorganisms to reduce ALT and/or AST levels by at least 2U/L,5U/L,10U/L,25U/L,30U/L, or 50U/L as compared to ALT and/or AST levels in a subject prior to administration of the isolated and purified consortium of a microorganism species.

As used herein, a microbiota generally refers to a microbiota consisting essentially of a single strain, species or genus, as may be the case when a population is cultured from isolated and purified sub-populations of such strain, species or genus. Thus, for a given population of microorganisms, if cultured from an isolated microorganism species or strain, such population will be referred to herein as purified or substantially pure. The resulting population may generally be at least 80% pure for the microorganism 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 microorganism species or strains within the particular population. Conversely, the level of undesired strains in any particular desired microorganism 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, such as other undesired microorganism strains or species, in the purified population of microorganisms may be proportioned to or less than the level described above for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. Where the composition comprises a consortium of a plurality of microbial populations, each population may be of the purity described above, either prior to its incorporation into the composition or when a polymerization measurement is performed on the consortium. For example, the impurity levels in the purified population of microorganisms, such as other undesirable strains or species of microorganisms, may be scaled to or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. Where the composition comprises a consortium of a plurality of microbial populations, each population may be of the purity described above, either prior to its incorporation into the composition or when a polymerization measurement is performed on the consortium. For example, the impurity levels in the purified population of microorganisms, such as other undesirable strains or species of microorganisms, may be scaled to or below the above levels for each desired population.

The compositions of the present disclosure may also include cellular components of the metabolism of lactobacillus crispatus, metabolites, secreted molecules and compounds, and the like. The isolated form of the component from Lactobacillus crispatus, or any mixture of one or more components from Lactobacillus crispatus, may correspond, for example, to the recovery of the supernatant of the Lactobacillus crispatus culture or by extracting a cellular component or cellular fraction, metabolite or secreted compound from the Lactobacillus crispatus culture.

In some embodiments, the Lactobacillus crispatus, cultures, live bacteria, lyophilized bacteria, or inactivated bacteria, or compositions of the present disclosure may be used to treat or prevent a disease associated with impaired liver function, including at least one of fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, liver cancer, or liver cell damage.

In some embodiments, the predisposition to liver-like disease includes, but is not limited to, at least one of a high fat diet, a high cholesterol diet, a high sugar diet, hyperlipidemia, hyperglycemia, or hypercholesterolemia.

In some embodiments, the liver disease comprises high fat diet induced, high cholesterol diet induced, high sugar diet induced, high fat high cholesterol induced, high fat high sugar induced, and/or high fat high cholesterol high sugar induced.

In some embodiments, the Lactobacillus crispatus, cultures, live bacteria, lyophilized bacteria, or inactivated bacteria, or compositions of the present disclosure can be used to treat, prevent, or ameliorate liver function damage-related diseases including at least one of fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, or liver cell damage.

In particular, the lactobacillus crispatus, cultures, live bacteria, lyophilized bacteria or inactivated bacteria of the present disclosure, or compositions, may be used to treat or prevent liver injury. The liver injury can be liver injury caused by long-time working tired, liver injury caused by long-time drinking, fatty liver, drug liver disease and liver injury caused by genetic metabolism factor.

In some embodiments, the lactobacillus crispatus, culture, viable bacteria, lyophilized bacteria, or inactivated bacteria of the present disclosure, or composition may reduce ALT and/or AST levels.

In some embodiments, the lactobacillus crispatus, cultures, live bacteria, lyophilized bacteria or inactivated bacteria of the present disclosure, or compositions may reduce liver weight.

In some embodiments, the kidney disease includes, but is not limited to, kidney injury, renal dysfunction, and the like. The kidney disease includes, but is not limited to, primary glomerulonephritis, hypertensive glomerulonephritis, diabetic nephropathy, secondary glomerulonephritis, tubular interstitial lesions (e.g., chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic kidney disease, etc. In some embodiments, the predisposition to kidney disease comprises at least one of a high fat diet, a high sugar diet, a high cholesterol diet. In some embodiments, the kidney disease is manifested as elevated blood creatinine.

In some embodiments, the obesity and obesity-related disorders include, but are not limited to, overweight, obesity, metabolic syndrome, cardiovascular disease, cerebrovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, steatohepatitis.

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, hyperlipidemia, hyperglycemia, NAFLD, or NASH.

In some embodiments, the obesity and obesity-related disorders 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 and high cholesterol and high sugar, or obesity in NAFLD or NASH patients.

In some embodiments, the obesity and obesity-related disorders include at least one of obesity, metabolic syndrome, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

In some embodiments, the obesity is peripheral obesity, and/or central obesity. In some embodiments, the obesity is dietary obesity, and/or is metabolic obesity. In particular, the obesity is abdominal obesity or apple type obesity, such as visceral fat superscript.

In some embodiments, the lactobacillus crispatus, culture, viable bacteria, lyophilized bacteria, or inactivated bacteria, or composition of the present disclosure is capable of reducing visceral fat and/or subcutaneous fat.

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 mellitus caused by damage to islet B cells, diabetes mellitus caused by insulin resistance, diabetes mellitus caused by obesity.

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.

In some embodiments, the predisposition to diabetes comprises at least one of a high fat diet-induced, a high sugar diet-induced, a high cholesterol diet-induced.

In some embodiments, the diabetes is manifested as hyperglycemia due to low levels of insulin and/or peripheral insulin resistance.

In some embodiments, the metabolic disorder includes, but is not limited to, (1) diabetes caused by a disorder of glucose metabolism, or (2) diabetes caused by abnormal glucose tolerance or reduced glucose tolerance, or (3) diabetes caused by impaired insulin B cells, or (4) diabetes caused by insulin resistance.

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

In some embodiments, the predisposition to cardiovascular disease or cardiovascular disease includes, but is not limited to, at least one of atherosclerosis, NAFLD, NASH, hyperlipidemia, hyperglycemia, or hypercholesterolemia.

In some embodiments, the medicament or nutraceutical or food product has at least one effect selected from the group consisting of reducing liver weight; reducing the ratio of liver weight to body weight; reducing liver steatosis, and/or liver lobular inflammation; reducing liver cell ballooning, reducing non-alcoholic fatty liver disease activity score (NAS), reducing serum AST, ALT, reducing weight in a mammal, reducing weight gain in a mammal, reducing at least one of epididymal fat weight, perirenal fat weight, subcutaneous fat weight, mesenteric fat weight in a mammal, reducing the ratio of visceral fat to weight, reducing fasting blood glucose, reducing the level of at least one indicator in the mammal serum of total cholesterol level, triglyceride level, high density lipoprotein cholesterol level, low density lipoprotein cholesterol level, treating, preventing or alleviating liver function impairment, treating, preventing or alleviating fatty liver, treating, preventing or alleviating NAFLD or NASH, treating, preventing or alleviating obesity, treating, preventing or alleviating metabolic syndrome, treating, preventing or alleviating local subcutaneous fat excess, epididymal fat excess, perirenal fat excess, and/or mesenteric fat excess, treating, preventing or alleviating coronary heart disease, treating, preventing or alleviating atherosclerosis, treating, preventing or alleviating hyperglycemia, treating, preventing or alleviating high cholesterol level, treating, preventing or alleviating diabetes mellitus, or diabetes mellitus is a mediated by alleviating or a high blood lipid level.

The isolated lactobacillus crispatus of the present disclosure is also capable of synthesizing a large amount of short chain fatty acids, and thus is capable of effectively treating or preventing metabolic diseases, reducing body weight, body fat, blood sugar, blood lipid, cholesterol in a subject, improving or reducing non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH).

In some embodiments, the medicament or health product or food product is capable of reducing weight while ameliorating liver damage.

In some embodiments, the medicament or health product or food product is capable of reducing blood lipid while reducing serum AST and/or ALT. The lipid lowering is visceral fat or subcutaneous fat lowering.

In some embodiments, the medicament or nutraceutical or food improves diabetes while also improving liver damage.

In some embodiments, the medicament or nutraceutical or food improves diabetes while also improving kidney damage.

In some embodiments, the medicament or nutraceutical or food improves hypercholesterolemia, hyperlipidemia, and/or cardiovascular and cerebrovascular disease while also improving liver damage.

In some embodiments, the medicament or nutraceutical or food is capable of reducing blood lipid while ameliorating hypercholesterolemia, hyperlipidemia, and/or cardiovascular and cerebrovascular diseases. The lipid lowering is visceral fat or subcutaneous fat lowering.

In some embodiments, the lactobacillus crispatus, culture, live bacteria, lyophilized bacteria, or inactivated bacteria, or composition described in the present disclosure has an effect in at least one of affecting or regulating fasting glucose homeostasis, affecting or regulating cholesterol homeostasis, triglyceride homeostasis, repairing liver injury, kidney injury, promoting local adipose tissue metabolism.

In some embodiments, a lactobacillus crispatus, culture, viable bacteria, lyophilized bacteria, or inactivated bacteria, or composition of the present disclosure has an effect in reducing liver and/or kidney damage, which is reducing one or more elevated liver damage or liver disease indicators of the subject, such as reducing serum levels and/or liver weight indicators of aspartate Aminotransferase (AST), alanyl Aminotransferase (ALT) of the subject, and/or reducing urea nitrogen (BUN) in serum and/or reducing CRE (creatinine) indicators in serum of the subject.

A method of preventing, treating, ameliorating or reducing liver and kidney diseases, metabolic diseases, administering to a subject in need thereof an effective amount of a lactobacillus crispatus according to the first aspect of the present disclosure, a culture of lactobacillus crispatus according to the second aspect, a live, lyophilized or inactivated bacterial cell, or a composition of the third aspect.

In some embodiments, the liver and kidney disease, metabolic disease is the liver and kidney disease, metabolic disease described above.

Drawings

The foregoing and other aspects and advantages of the invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings.

FIG. 1 is a photograph showing colony morphology of strain MNH 22076.

FIG. 2 shows a gram of the strain MNH 22076.

FIG. 3 shows an electron micrograph of strain MNH 22076.

FIG. 4 is a photograph showing colony morphology of strain MNH 45330.

FIG. 5 shows a gram of the strain MNH 45330.

FIG. 6 shows an electron micrograph of strain MNH 45330.

FIG. 7 shows the results of tolerance of strain MNH22076 to different pH.

FIG. 8 shows the results of the tolerance of strain MNH22076 to different concentrations of NaCl.

FIG. 9 shows the results of the tolerance of strain MNH22076 to bile salts at different concentrations.

FIG. 10 shows the results of tolerance of strain MNH45330 to different pH.

FIG. 11 shows the results of the tolerance of strain MNH45330 to different concentrations of NaCl.

FIG. 12 shows the results of the tolerance of strain MNH45330 to bile salts at different concentrations.

FIG. 13 shows a phylogenetic tree of strain MNH 22076.

FIG. 14 shows a phylogenetic tree of strain MNH 45330.

FIG. 15a shows that strain MNH22076 can significantly reduce liver function index ALT. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used t-test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.01 compared to HFD-Control group.

FIG. 15b shows that strain MNH22076 can significantly reduce liver function index AST. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used t-test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.01 compared to HFD-Control group.

FIG. 16 shows that strain MNH22076 and MNH45330 significantly lower fasting plasma glucose in high-fat diet-induced diabetic mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used t-test (Student's t test) analysis method, p <0.05 compared to HFD-Control group.

FIG. 17a shows that strain MNH22076 and MNH45330 significantly reduced the body weight of high fat diet induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). The statistical analysis uses the t test (Student's t test) analysis method, 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, and p <0.0001 compared with the HFD-Control group.

FIG. 17b shows that strain MNH22076 and MNH45330 significantly reduced the weight change rate of high fat diet-induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). The statistical analysis uses the t test (Student's t test) analysis method, 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, and p <0.0001 compared with the HFD-Control group.

FIG. 18a shows that strain MNH22076 can significantly reduce total cholesterol in serum of high fat diet induced obese mice (TCHO). Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used the t-test (Student's t test) analysis method, p <0.01 compared to HFD-Control group, p <0.0001 compared to HFD-Control group.

FIG. 18b shows that strain MNH22076 can significantly reduce Triglyceride (TG) in serum of high-fat diet-induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used t-test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.01 compared to HFD-Control group.

FIG. 18C shows that strain MNH22076 can significantly reduce 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 t-test (Student's t test) analysis method, p <0.05 compared to the HFD-Control group.

Figure 19a shows that strain MNH22076 and MNH45330 significantly reduced epididymal fat weight in high fat diet induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used the t test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.01 compared to HFD-Control group, and p <0.0001 compared to HFD-Control group.

FIG. 19b shows that strain MNH22076 and MNH45330 significantly reduced perirenal fat weight in high-fat diet-induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used the t test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.01 compared to HFD-Control group, and p <0.0001 compared to HFD-Control group.

Figure 19c shows that strain MNH22076 and MNH45330 significantly reduced the mesenteric fat weight of high fat diet induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used the t test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.0001 compared to HFD-Control group.

FIG. 19d shows that strain MNH22076 and MNH45330 significantly reduced the subcutaneous fat weight of high fat diet induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used the t test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.01 compared to HFD-Control group, and p <0.0001 compared to HFD-Control group.

FIG. 19e shows that strain MNH22076 and MNH45330 significantly reduced visceral fat ratio in high fat diet-induced obese mice. Data are shown as Mean ± standard deviation (Mean ± SD). Statistical analysis used the t test (Student's t test) analysis method, p <0.05 compared to HFD-Control group, p <0.0001 compared to HFD-Control group.

Preservation of strains

Lactobacillus crispatus (Lactobacillus crispatus) MNH22076 is deposited with the Guangdong province microorganism strain collection center (GDMCC) under the accession number GDMCC No:65554 under the accession number 2024, 11 months and 28 days, with the address being building 5 No. 59 of the university 100 in Guangzhou City martyr under the accession number Lactobacillus crispatusMNH22076 and the taxonomic name Lactobacillus crispatus.

Lactobacillus crispatus (Lactobacillus crispatus) MNH45330 is deposited with the Guangdong province microorganism strain collection center (GDMCC) under the accession number GDMCC No:65556 under the accession number 2024, 11 months and 28 days, with the address being building 5 No. 59 of the university 100 in Guangzhou City martyr under the accession number Lactobacillus crispatusMNH45330 and the taxonomic name Lactobacillus crispatus.

Detailed Description

The method is used for separating two new strains of the lactobacillus crispatus strains, the preservation numbers of the two new strains are GDMCC No:65554 and GDMCC No:65556, and the two new strains are identified by using a traditional classification method and a molecular biological method. The identification results show that the two strains belong to a new strain under the Lactobacillus crispatus strain. Further, the present disclosure investigated the biochemical properties and therapeutic uses of both strains.

It is known in the art that classification and identification of species by conventional classification methods and molecular biology methods can be employed. Traditional classification methods include, but are not limited to, for example, cell morphology observations, gram staining, flagella staining, various metabolic experiments, and the like. Molecular biological methods include, but are not limited to, ribosomal RNA sequencing, whole genome sequencing-based assay methods, and the like.

The term "prebiotic" as used herein may be a generic term referring to a chemical substance and/or component capable of affecting the growth and/or activity of a microorganism in a host (e.g., may allow for specific changes in the composition and/or activity of a microbiome).

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

The compositions or formulations of the present disclosure may be administered as a pharmaceutical formulation, therapeutic composition, dietary supplement, nutritional supplement, medical probiotic, or medical food. In some cases, the composition is administered in the form of a pharmaceutical formulation. In some cases, the composition is administered in the form of a nutritional supplement. In some cases, the composition is administered in the form of a dietary supplement. In some cases, the composition is administered in the form of a medical food. In certain instances, the composition is administered in the form of a medical probiotic. In some cases, the composition (e.g., dietary supplement, nutritional supplement, medical probiotic, or medical food) may be administered orally, for example, as a capsule, pill, or tablet.

16S rRNA is a ribosomal RNA of prokaryotes, the 16S rRNA gene consists of a variable region common to all bacteria and a conserved region which varies to a different extent from bacterium to bacterium. By comparing the 16S rRNA gene sequences of bacteria, a biological evolutionary tree can be drawn according to the sequence difference base and the evolutionary distance. When the identity between the 16S rRNA gene sequences of the two strains is less than 98.65%, they can be judged to belong to different species (see pages ,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).

Known computer algorithms may be used to determine "identity" between two nucleic acid molecule sequences, such as the "FASTA" program, GCG program package, BLASTN or FASTA. Commercially or publicly available programs may also be, for example, DNAStar "MegAlign" programs.

And a second generation sequencing technology can be adopted to carry out strain identification based on whole genome sequencing, so that the strain identification result is more accurate. Average nucleotide identity (Average nucleotide identity, ANI) of bacterial genomes refers to the similarity of homologous genes between the genomes of two bacteria. ANI values can be calculated by BLAST or the like. In the field of bacterial taxonomy, it is generally believed that ANI values of over 95% are required to identify the same species （Jain C, Rodriguez-R L M, Phillippy A M, et al.High throughput ANI analysis of 90K prokaryotic genomes reveals clear speciesboundaries[J]. Nature Communications, 2018, 9（1）: 5114.）.

The existing calculation tools of various mature ANI values, such as local calculation software Jspecies (/ jspecies) and Gegenees (/ document. Html), on-line calculation tools ANI caculator (http:/eveomics. Gatech. Edu /), ezGenome (/ ezgenome/ANI) and ANItools, can be used.

Using the methods described above, one of skill in the art can determine whether an isolated strain belongs to the species Lactobacillus crispatus identified by the present inventors. For example, it can be determined that the species belongs to the same species when the average nucleotide identity ANI value with Lactobacillus crispatus (Lactobacillus crispatus) deposited under accession numbers GDMCC No:65554, or GDMCC No:65556 is at least 95%, such as 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%、97.4%、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%.

As another example, a species can be identified as belonging to the same species when its 16S rRNA sequence has at least 98.65% identity, e.g., 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, to the sequence set forth in SEQ ID NO.3 or 4.

By "strain" is meant a member of a bacterial species having genetic characteristics such that it can be distinguished from closely related members of the same bacterial species. The genetic characteristic may be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence (cure of at least one natural plasmid), the presence of at least one recombinant gene, the presence of at least one mutant gene, the presence of at least one exogenous gene (gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one unnatural 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 acquires or loses antibiotic resistance or acquires or loses biosynthetic capacity (e.g., auxotrophic strain) as compared to another strain of the same species, the strain or nutrient/metabolite can be distinguished by selection or counter-selection using the antibiotic.

"Supernatant" or "supernatant" within the meaning herein refers to a culture supernatant of a 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.

The compositions can be prepared using the lactobacillus crispatus described herein, for example, by using pharmaceutically acceptable excipients. The pharmaceutical composition comprises a pharmaceutically effective amount of the Lactobacillus crispatus, e.g., lactobacillus crispatus having deposit number GDMCC No:65554, or GDMCC No: 65556. Likewise, lactobacillus crispatus having deposit numbers GDMCC No:65554, or GDMCC No:65556 may also be prepared as pharmaceutical compositions, for example by using pharmaceutically acceptable excipients, which comprise a pharmaceutically effective amount of the Lactobacillus crispatus.

Suitable pharmaceutically acceptable excipients which may be used are, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavoring agents.

The compositions herein may be formulated in any form suitable for enhancing the abundance of lactobacillus crispatus in a subject. The compositions may be administered by oral administration (e.g., by oral gavage), intramuscular injection, inhalation, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, intragastrointestinal, biliary infusion, cardiac infusion, anterior to the anus, rectal, subcutaneous to the spine, sublingual, topical, intravaginal, transdermal, ureteral, or urethral routes.

Examples of dosage forms for which the compositions herein are suitable include, but are not limited to, tablets, aerosols, chewing sticks, capsules containing coated particles, capsules containing extended release particles, concentrates.

In some embodiments, the composition is a sugar-coated tablet, gel capsule, gel, emulsion, tablet capsule, hydrogel, nanofiber gel, electrospun fiber, food bar, candy, fermented milk, fermented cheese, chewing gum, powder, or toothpaste, or the like.

In some embodiments, administration may also be by inclusion in the subject's diet, for example, in a functional food for a human or companion animal.

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

In some embodiments, the lactobacillus crispatus in the compositions of the disclosure is lyophilized. In some embodiments, the lactobacillus crispatus in the compositions of the disclosure is spray dried. In some embodiments, the lactobacillus crispatus in the compositions of the present disclosure is lyophilized or spray dried and is viable. In some embodiments, the lactobacillus crispatus in the compositions of the present disclosure is lyophilized or spray-dried and is capable of partially or fully colonising the intestine. In some embodiments, the lyophilized lactobacillus crispatus is reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent as described herein.

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 oral, lingual or sublingual administration.

In some embodiments, the composition is prepared by freeze drying, or spray drying.

The composition of the present disclosure includes a pharmaceutical product, a health product, or a food product.

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

The pharmaceutical products of the present disclosure are useful for treating, preventing, or alleviating metabolic diseases, metabolic disorders, or diseases caused by metabolic disorders.

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

Diabetes mellitus

Diabetes includes type I diabetes (T1D), type II diabetes (T2D), and Gestational Diabetes (GDM). Type I diabetes is caused by autoimmune damage or idiopathic causes, and is characterized by absolute destruction of islet function, and occurs in children and adolescents, where insulin therapy is necessary to achieve satisfactory efficacy, otherwise life threatening. Type II diabetes is a multifactorial syndrome characterized by abnormal carbohydrate/fat metabolism, generally involving hyperglycemia, hypertension, and cholesterol abnormalities. Type II diabetes is caused by the inability of insulin to function effectively (low level of binding to the receptor), and therefore, it is required to examine not only fasting blood glucose but also blood glucose 2 hours after meal, and it is particularly desirable to examine islet function. Diabetes during pregnancy is characterized by two conditions, one is diabetes which is diagnosed before pregnancy, called "diabetes mellitus combined pregnancy", the other is diabetes which is normal in sugar metabolism or has potential impaired glucose tolerance before pregnancy and occurs or is diagnosed during pregnancy, also called "Gestational Diabetes Mellitus (GDM)", and more than 80% of pregnant women with diabetes mellitus are GDM.

The 4 metabolic disease related models, namely a High Fat Diet (HFD) induced mouse obesity model, a high fat, high sugar and high cholesterol induced mouse NASH model, a high fat diet combined with streptozotocin (Streptozotocin) (HFD-STZ) induced mouse type II diabetes model and a leptin receptor gene deficiency mouse model (db/db), are all common metabolic disease type mouse models, and the model mice are usually accompanied with obesity, insulin resistance, hyperglycemia, hyperlipidemia, high cholesterol, NAFLD/NASH and other metabolic diseases.

Insulin resistance refers to the decrease of the efficiency of insulin to promote glucose uptake and utilization for various reasons, and the compensatory hypersecretion of insulin by the body produces hyperinsulinemia to maintain the stability of blood sugar, and insulin resistance is liable to cause metabolic syndrome and type II diabetes.

Liver function diseases

Liver function disorders are liver function abnormalities or liver function injuries. Glutamic-pyruvic transaminase (ALT) and/or glutamic-oxaloacetic transaminase (AST) are sensitive markers of liver function diseases. ALT and/or AST are significantly elevated in the blood in the event of abnormal liver function in the body, such as liver injury, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In general, ALT reflects a higher sensitivity to acute liver injury than AST. Sustained elevation of ALT is suggested to be chronic liver injury. In cases of chronic hepatitis, cirrhosis, liver cancer, etc., AST is elevated significantly, and ALT can be exceeded. AST levels mark the chronicity, prevalence and severity of liver lesions and even suggest prognosis of chronic liver disease.

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), EB 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/mid-toxic liver injury, cirrhosis, liver cancer, hepatolenticular degeneration, alpha 1-antitrypsin deficiency, hemochromatosis, and the like.

In addition, fat accumulation in liver is an important factor for the development of nonalcoholic fatty liver disease (NAFLD or NASH), so that the decrease of ALT and/or AST levels and the decrease of liver weight can show that the drug has a certain treatment improving effect when the drug is dry.

Nonalcoholic fatty liver disease (NAFLD) refers to excessive fat accumulating in the liver in the form of Triglycerides (TG) (steatosis). Some patients with NAFLD have liver cell damage and inflammation (steatohepatitis), i.e., nonalcoholic steatohepatitis (NASH), in addition to excess fat. NASH is widely recognized as a liver manifestation of metabolic syndrome such as type II diabetes, insulin resistance, central obesity, hyperlipidemia (low high density lipoprotein cholesterol, hypertriglyceridemia) and hypertension.

Liver and kidney function diseases

Liver and kidney function diseases refer to functional acute renal failure occurring in severe liver diseases, decompensated liver cirrhosis, and liver and kidney syndrome may occur due to insufficient blood volume of effective circulation, prostaglandin decrease, and the like.

Cardiovascular diseases

TG (triglyceride) is mainly involved in energy metabolism in the human body, producing heat energy. Too high a TG content in the blood can cause the blood to be viscous, causing lipid to deposit on the vessel wall and gradually form small plaques, i.e. atherosclerosis. Increased LDL-C is a major, independent risk factor for the development and progression of atherosclerosis, and increased levels of LDL-C are also an indicator of coronary heart disease. Because HDL-C can transport cholesterol in the vessel wall to the liver for catabolism (i.e. reverse cholesterol transport), the deposition of cholesterol on the vessel wall can be reduced, and the anti-atherosclerosis effect can be achieved.

Obesity and obesity control method

Obesity refers to a state where a degree of apparent overweight and fat layers are too thick, and is a condition resulting from excessive accumulation of fat, particularly triglycerides, in the body, and is abnormal or excessive accumulation of fat that constitutes a risk to health. Excessive body fat accumulation due to excessive food intake or altered body metabolism results in excessive weight gain and causes pathological, physiological changes or latency in the human body. Body mass index exceeding 25 is considered overweight and exceeding 30 is considered obese. Obesity increases the risk of developing many physical and mental disorders. It is mainly associated with metabolic syndrome, a combination of diseases including type 2 diabetes, hypertension, hypercholesterolemia and hypertriglyceridemia. In general, the effects of obesity on health are divided into two major categories, diseases attributable to increased body fat (e.g., osteoarthritis, obstructive sleep apnea, etc.) and diseases with increased numbers of adipocytes (diabetes, dyslipidemia, cancer, cardiovascular disease, nonalcoholic fatty liver, nonalcoholic steatohepatitis, etc.). The "obesity-related diseases" may be selected from the group consisting of overeating (overeating), binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentrations, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, atherosclerosis, hypercholesterolemia, hyperuricemia, lower back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, nonalcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as those subjects having these obesity-related diseases including those desiring weight loss.

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

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are described in the literature in the art or are carried out in accordance with the instructions for the product or the apparatus. All reagents or instruments are commercially available to the manufacturer.

Examples

The anaerobic blood agar plates related in the examples are purchased from a circular Kai microorganism, and the formula comprises 10g/L of casein pancreatin digest, 3g/L of cardiopancreatin digest, 1g/L of corn starch, 5g/L of meat gastric enzyme digest, 5g/L of yeast extract powder, 5g/L of sodium chloride, 15g/L of agar, 50-100 mL/L of sterile defibrinated sheep blood and pH of 7.3+/-0.2.

The PYG liquid medium referred to in the examples, per 1LPYG liquid medium, contained TRYPTICASE PEPTONE (tryptone) 5.0g, pepone (peptone) 5.0g,Yeast extract (yeast extract) 10.0g,Beef extract (beef extract) 5.0g, glucose 5.0g, K ₂HPO₄ 2.0.0 g, tween 80 (Tween 80) 1.0ml, resazurin (Resazurin) 1.0mg,40mlSalt solution (salt solution) 40ml (its composition: :CaCl₂·2H₂O0.01g,MgSO₄·7H₂O 0.02g,K₂HPO₄0.04g,NaHCO₃0.4g,NaCl 0.08g, water balance, pH 7.2.+ -. 0.2 (25 ℃).

The MRS plate medium in the example comprises 10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 1.0mL of Tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of tri-ammonium citrate, 0.1g of magnesium sulfate (MgSO ₄·7H₂ O), 0.05g of manganese sulfate (MnSO ₄·4H₂ O), 15.0g of agar and the balance of water.

The MRS liquid culture medium in the example comprises 10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 1.0mL of Tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of tri-ammonium citrate, 0.1g of magnesium sulfate (MgSO ₄·7H₂ O), 0.05g of manganese sulfate (MnSO ₄·4H₂ O) and the balance of water per 1LMRS liquid culture medium.

The above-described culture medium can be prepared by conventional preparation methods and sterilization methods.

Example 1 isolation and identification of strains

1.1 Isolation and purification of Strain MNH22076 and MNH45330

Both intestinal strains lactobacillus crispatus (Lactobacillus crispatus MNH22076 and Lactobacillus crispatus MNH 45330) were isolated from fecal samples of healthy volunteers. The separation method adopts a conventional strain separation method, utilizes gradient dilution, then adopts separation culture to pick single colony, and adopts strain purification and anaerobic culture at 37 ℃. The pure culture strain is prepared into 20% glycerol/water-bacterial solution, and the pure culture strain is preserved at a low temperature of-80 ℃.

Specifically, the method for separating and purifying the strain is as follows:

The donor takes fresh excrement 2-5 g from himself, puts it into sample collecting and preserving tube, shakes and homogenizes the excrement sample, puts it into ice box, and sends it to laboratory for strain separation within 24 hours.

Split charging physiological saline, 9 mL/tube in a biosafety cabinet, preparing strain separation culture medium anaerobic blood agar plates, transferring the strain separation culture medium anaerobic blood agar plates into an anaerobic workstation in advance for 24 hours, and marking sample information, culture medium type, separation date and the like.

Placing fresh fecal samples into an anaerobic workstation, oscillating 1 min by using a vortex oscillator, uniformly mixing, sucking 1 mL samples into 9 mL physiological saline, uniformly mixing to obtain 10 ^-1 diluent, and then carrying out gradient dilution to obtain 10 ^-6 diluent for later use.

Dripping 10 ^-6 dilution liquid into an anaerobic blood agar plate, dripping the liquid into 100 mu L/dish, uniformly coating, and culturing for 3-5 days at 37 ℃ after the surface of the plate is dried.

The growth of the strain on the isolated medium (anaerobic blood agar plate) was observed, single colonies were picked up with sterilized toothpicks, strain purification was performed, and the purified strain was placed at 37 ℃ for anaerobic culture. The pure culture strain is prepared into 20% glycerol/water-bacterial solution, and the pure culture strain is preserved at a low temperature of-80 ℃.

1.2 Morphological characteristics of Strain MNH22076 and MNH45330

1.2.1 Morphological characteristics of Strain MNH22076

After bacterial strain MNH22076 is inoculated in MRS plate culture medium and subjected to anaerobic culture at 37 ℃ for 48: 48h, visible bacterial colonies are formed on the MRS plate culture medium, the bacterial colonies are round, have regular and smooth edges and have a diameter of about 1-2 mm, are white and opaque, are gram-positive, and have no flagella, no movement and rod shape and have a size of about 0.5-1 mu m multiplied by 1.5-4 mu m. The colony morphology photograph of strain MNH22076 in MRS plate culture 48h is shown in fig. 1. Gram staining photographs of strain MNH22076 are shown in FIG. 2 and electron micrographs are shown in FIG. 3.

1.2.2 Morphological characteristics of Strain MNH45330

After the strain MNH45330 is inoculated in an MRS flat plate culture medium and subjected to anaerobic culture at 37 ℃ for 48: 48h, visible colonies are formed on the MRS flat plate culture medium, the colonies are round, have regular and smooth edges and have the diameter of about 0.5-2mm, are white and opaque, the strain is gram-positive, and have no flagella, no movement and rod shape and the size of about 0.5-1 mu m multiplied by 1.5-4 mu m. The colony morphology photograph of strain MNH45330 cultured in MRS plate for 48h is shown in FIG. 4. Gram staining photographs of strain MNH45330 are shown in FIG. 5 and electron microscopy photographs are shown in FIG. 6.

1.3 Physiological and biochemical characteristics of Strain 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, the optimal growth pH is 8.0 (the result of the tolerance of the strain to different pH is shown in figure 7), the strain MNH22076 can still grow in a culture medium with NaCl content exceeding 6% (the result of the tolerance of the strain to NaCl with different concentrations is shown in figure 8), the strain MNH22076 can grow in a living state in the range of 0% -0.1% of bile salt concentration, and the strain cannot grow when the bile salt concentration is more than or equal to 0.15% (the result of the tolerance of the strain to bile salt with different concentrations is shown in figure 9).

1.3.2 Physiological and Biochemical characteristics of Strain MNH45330

The strain MNH45330 can grow in the range of pH 4.0 to 10.0, the optimal growth pH is 8.0 (the tolerance result of the strain to different pH is shown in figure 10), the strain can still grow on the culture medium with NaCl content exceeding 6% (the tolerance result of the strain to different concentration NaCl is shown in figure 11), and the strain MNH45330 can not grow in the culture medium with bile salt concentration above 0.1% (the tolerance result of the strain to different concentration bile salt is shown in figure 12).

1.4 Biochemical identification results of Strain MNH22076 and MNH45330

1.4.1 Results of biochemical characterization of Strain MNH22076 via API 50CHL

Using API 50CHL (Mei Liai, CN 5041010), specific experimental procedures are directed to conventional API reagent procedures. Culture conditions were 37℃and anaerobic. The experimental results are shown in Table 1.

MNH22076 can be fermented to produce acid using galactose GAL, glucose GLU, fructose FRU, mannose MNE, alpha-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, escin, liu Chun SAL, cellobiose CEL, maltose MAL, lactose LAC, MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN. Thus galactose GAL, glucose GLU, fructose FRU, mannose mno, alpha-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, escin, liu Chun SAL, cellobiose CEL, maltose MAL lactose LAC, MEL, sucrose SAC, trehalose TRE, INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN, and derivatives thereof may be used as carbon sources during fermentation or cultivation of strain MNH 22076.

1.4.2 Results of biochemical characterization of Strain MNH45330 via API 50CHL

Using API 50CHL (Mei Liai, CN 5041010), specific experimental procedures are directed to conventional API reagent procedures. Culture conditions were 37℃and anaerobic. The experimental results are shown in Table 2.

MNH45330 can be fermented to produce acid using galactose GAL, glucose GLU, fructose FRU, mannose MNE, mannitol MAN, alpha-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, escin, liu Chun SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN. Thus galactose GAL, glucose GLU, fructose FRU, mannose mno, mannitol MAN, alpha-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, escin, liu Chun SAL, cellobiose CEL, maltose MAL, lactose LAC, MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN, and derivatives thereof may be used as carbon sources during fermentation or cultivation of strain MNH 45330.

1.5 Antibiotic minimum inhibitory concentration test of Strain MNH22076 and MNH45330

1.5.1 Antibiotic minimum inhibitory concentration test of Strain MNH22076

The minimum antibiotic inhibitory concentration of strain MNH22076 was determined using E-test (purchased from Liofilchem) paper sheets and the test results are shown in Table 3.

The results show that MNH22076 is 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 that prolonged administration of MNH22076 results in a lower risk of developing antibiotic resistance in the subject.

1.5.2 Antibiotic minimum inhibitory concentration test of Strain MNH45330

The minimum antibiotic inhibitory concentration of strain MNH45330 was determined using E-test (purchased from Liofilchem) paper sheets and the test results are shown in Table 4.

The results show that MNH45330 is sensitive to imipenem, ampicillin sulbactam, dopopenem, ertapenem, meropenem, piperacillin-tazobactam, ceftriaxone. It can be seen that MNH45330 is sensitive to most types of antibiotics and that prolonged administration of MNH45330 results in a lower risk of developing antibiotic resistance in the subject.

1.6 Identification of Strain MNH22076 and MNH45330

1.6.1 16S rRNA Gene amplification of Strain MNH22076 and MNH45330

Fresh cultures of strain MNH22076 and MNH45330 were taken and the genomic DNA of the strain was extracted. The 16S rRNA gene amplification was performed using the extracted genomic DNA of the strain as a template.

The primer pairs used for PCR of the 16S rRNA gene were:

27F:5’-AGAGTTTGATCMTGGCTCAG-3’(SEQ ID NO:1);

1492R:5’-TACGGYTACCTTGTTACGACTT-3’(SEQ ID NO:2)。

The PCR reaction procedure was as follows:

Pre-denaturation at 94℃4 min, (denaturation at 94℃50 sec, annealing at 52℃40 sec; extension at 72℃70 sec), 36 cycles, final extension at 72℃10 min.

1.6.2 16S rRNA Gene sequencing of Strain MNH22076 and MNH45330

The PCR product was purified and the 16S rRNA gene was sequenced by the Productivity Co, to obtain the 16S rRNA gene.

Wherein the 16S rRNA gene sequence of the strain MNH22076 is shown as SEQ ID NO. 3 （AGTCGAGCGAGCGGAACTAACAGATTTACTTCGGTAATGACGTTAGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTTAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAAAGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCCATTTGTAGAGATACAAAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCTGCAATGCCCAAAGCCGGTGGCCTAACC）;

The 16S rRNA gene sequence of the strain MNH 45330 is shown as SEQ ID NO. 4 （AAGTCGAGCGAGCGGAACTAACAGATTTACTTCGGTAATGACGTTAGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTTAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAAAGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCCATTTGTAGAGATACAAAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGGA）.

1.6.3 Identification results of Strain MNH22076 and MNH45330

The 16S rRNA gene sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 were analyzed by NCBI Basic Local ALIGNMENT SEARCH Tool to confirm strain classification information.

The data from GenBank were analyzed by BLAST and the comparison showed that the strain with highest similarity to MNH22076 was Lactobacillus crispatus, the similarity was 100%, the whole genome ANI analysis showed that the strain with highest similarity to Lactobacillus crispatus (GCF 002088015.1) was 97.63%, the comparison score (AF) was 86%, and thus strain MNH22076 was Lactobacillus crispatus, the strain with highest similarity to MNH45330 was Lactobacillus crispatus, the similarity was 100%, and the whole genome ANI analysis showed that the strain with highest similarity to Lactobacillus crispatus (GCF 002088015.1) was 97.4%, the comparison score (AF) was 86%, and thus strain MNH45330 was Lactobacillus crispatus.

MNH22076 and MNH45330 were compared with the 16S rRNA gene sequences of Lactobacillus crispatus bacteria related strains called from GenBank database, etc., and phylogenetic tree was constructed.

And (3) carrying out multi-sequence comparison on sequences of the strain with high sequence similarity of the 16S rRNA genes in MNH22076, MNH45330 and NCBI databases, and then constructing a phylogenetic tree by using software MEGA 5 (constructing the phylogenetic tree by adopting a maximum likelihood method), wherein the phylogenetic tree nodes in figures 13 and 14 only show that the Bootstrap value is larger than 50%.

From the phylogenetic tree (FIGS. 13, 14) it can be seen that the strain MNH22076, together with Lactobacillus and Lactobacillus crispatus, is a new strain under Lactobacillus crispatus. The strain MNH45330 and Lactobacillus are together with Lactobacillus crispatus, and are novel strains under Lactobacillus crispatus.

1.7 Genome analysis of Strain MNH22076 and MNH45330

1.7.1 Genomic analysis of Strain MNH22076

The genome of the strain MNH22076 is subjected to sequence fragmentation by an ultrasonic method, the fragmentation length ranges from 350bp, and then an Illumina sequencing library is constructed by using a standard DNA library-building kit (NEB UltraTM). The constructed sequencing library was subjected to double-ended 150bp sequencing using NovaSeq (Illumina). Sequencing gave 1.28Gbp data with a Q20 ratio of 95.28%.

Genomic raw sequencing data was data filtered using fastp (version: 0.20.0), filter parameters: "-poly_g_min_len10-poly_x_min_len10-q 15-u 40-n 5-l 50". The filtered raw data was genome assembled using SPAdes (version: v3.14.0), assembly parameters "- -isolate- -cov-cutoff 10". Genome assembly gave a total length of 2.28mbp, an N50 length of 33.0kbp and a GC content of 36.59%.

Genomic genes genomic gene prediction analysis was performed using the prokaryotic analysis software genome annotation procedure prokka (version: 1.14.5), parameters "- -gcode 11- -evalue e-09". In total 2257 CDS sequences were predicted, of which the average CDS sequence length was 890bp.

Potential antibiotic resistance genes in the genome were analyzed using RGI protocol (version 4.2.2), where the database of antibiotic resistance genes was CARD (version 3.0.0, https:// card.mcmaster.ca/analyze/RGI). There was no resistance gene after comparison.

The analysis of potential virulence factors and related genes in the genome used NCBI blastp (version: 2.7.1+) versus virulence factor database VFDB (virulence factor database, http:// www.mgc.ac.cn/cgi-bin/VFs/v5/main. Cgi, update date 2019, 9, 19 days). The detailed comparison results are shown in Table 5.

The analysis of potential primary metabolic gene clusters in the genome used gutSMASH (version: 1.0.0). The detailed comparison results are shown in Table 6.

1.7.2 Genomic analysis of Strain MNH45330

The genome of the strain MNH45330 is subjected to sequence fragmentation by an ultrasonic method, the fragmentation length ranges from 350bp, and then an Illumina sequencing library is constructed by using a standard DNA library-building kit (NEB UltraTM). The constructed sequencing library was subjected to double-ended 150bp sequencing using NovaSeq (Illumina). Sequencing gave 1.43Gbp data with a Q20 ratio of 95.33%.

Genomic raw sequencing data was data filtered using fastp (version: 0.20.0), filter parameters: "-poly_g_min_len10-poly_x_min_len10-q 15-u 40-n 5-l 50". The filtered raw data was genome assembled using SPAdes (version: v3.14.0), assembly parameters "- -isolate- -cov-cutoff 10". Genome assembly gave a total length of 2.45mbp, an N50 length of 43.3kbp and a GC content of 36.51%.

Genomic genes genomic gene prediction analysis was performed using the prokaryotic analysis software genome annotation procedure prokka (version: 1.14.5), parameters "- -gcode 11- -evalue e-09". A total of 2468 CDS sequences were predicted, with an average CDS sequence length of 870bp.

Potential antibiotic resistance genes in the genome were analyzed using RGI protocol (version 4.2.2), where the database of antibiotic resistance genes was CARD (version 3.0.0, https:// card.mcmaster.ca/analyze/RGI). The comparison shows no resistance gene.

The analysis of potential virulence factors and related genes in the genome used NCBI blastp (version: 2.7.1+) versus virulence factor database VFDB (virulence factor database, http:// www.mgc.ac.cn/cgi-bin/VFs/v5/main. Cgi, update date 2019, 9, 19 days). The detailed comparison results are shown in Table 7.

The analysis of potential primary metabolic gene clusters in the genome used gutSMASH (version: 1.0.0). The detailed comparison results are shown in Table 8.

Example 2 improvement of liver function and related diseases by Strain MNH22076 and MNH45330 in high fat diet induced liver injury mouse model

The improvement of the strain MNH22076 and MNH45330 on liver and kidney functions and related diseases is studied by using a high-fat diet liver and kidney injury mouse model. The protocol has been reviewed ethically by the Mu En committee for biological laboratory animal management and use.

Liver function disorders are liver function abnormalities or liver function injuries. Glutamic-pyruvic transaminase (ALT) and/or glutamic-oxaloacetic transaminase (AST) are sensitive markers of liver function diseases. ALT and/or AST are significantly elevated in the blood in the event of abnormal liver function in the body, such as liver injury, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In general, ALT reflects a higher sensitivity to acute liver injury than AST. Sustained elevation of ALT is suggested to be chronic liver injury. In cases of chronic hepatitis, cirrhosis, liver cancer, etc., AST is elevated significantly, and ALT can be exceeded. AST levels mark the chronicity, prevalence and severity of liver lesions and even suggest prognosis of chronic liver disease.

2.1 Experimental methods

1) Experimental animals the experimental mice were C57BL/6J mice, 5-6 weeks old, purchased from Guangdong Kangshen Biotechnology Co.

2) The preparation of test substances of strains MNH22076 and MNH45330 comprises the steps of melting strain glycerol freezing tubes of strains MNH22076 and MNH45330 at 37 ℃, inoculating the strain glycerol freezing tubes on an MRS flat plate medium for activation in an anaerobic workstation, inoculating the activated strain in the MRS liquid medium, carrying out anaerobic culture to obtain a sufficient number of cultures, centrifuging and concentrating the cultured bacterial liquid, and then re-suspending the bacterial liquid by using PBS containing 0.05% of cysteine hydrochloride (L-Cys HCl) to obtain the test substance with the purity and viable count (2X 10 ⁹ CFU/mL) meeting the requirements of animal experiments.

3) Negative control PBS containing 0.05% L-Cys HCl was used as the negative control.

4) Positive control, namely thawing a glycerol freezing tube of a bifidobacterium animalis subspecies B420 lactic acid strain (B420 is disclosed in patent CN 109069553A) at 37 ℃, inoculating the strain into a PYG culture medium for activation in an anaerobic workstation, inoculating the activated strain into the PYG liquid culture medium, carrying out anaerobic culture to obtain a culture with sufficient quantity, centrifuging and concentrating the cultured bacterial liquid, and then using PBS containing 0.05% L-Cys HCl to suspend the bacterial liquid, thereby obtaining the positive control with purity and viable count (2X 10 ⁹ CFU/mL) meeting the requirements of animal experiments.

5) The experimental process is that 40 mice with the weight range of 18g-22g are selected after the quarantine period is finished, and the mice are randomly layered and grouped according to the weight, 8 mice/group are divided into 5 groups, namely NCD-Control group, HFD-Control group, MNH22076 group, MNH45330 group and B420 group. Wherein NCD-Control group is fed with maintenance feed (product number: D12451B, derivative of derivative), and the other four groups are fed with 60% high-fat feed (product number: D12492, derivative of derivative). After the grouping (D1), the negative Control substances are given to the NCD-Control group and the HFD-Control group, the positive Control substance is given to the B420 group, the MNH22076 group is given to the MNH22076 and the MNH45330 group is given to the MNH45330, 1 time per day, 200 mu L each time is given for 56 days, and the mice drink and eat freely during the experiment, and the 12h/12h day and night circulation is adopted. During the trial, 1 general clinical observation was performed after each dosing was completed. The test end point is the next day (D57) after the drug administration, the test end point performs dissection sampling according to a scheme, and the data is summarized to analyze each anatomical data result and serum detection data result. All data are expressed in mean±sd format, plotted using GRAPHPAD PRISM software and statistically analyzed. For the pairwise comparison, a t-test (Student's t test) analysis method was used. No significance is indicated and difference significance is indicated by p <0.05, p <0.01, p <0.001, p <0.0001.

2.2 Experimental results

Referring to FIGS. 15a-15b, it is shown that strain MNH22076 can significantly reduce liver function index. In particular, FIGS. 15a-15b show that strain MNH22076 can significantly reduce alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in serum, and thus strain MNH22076 can improve liver dysfunction caused by high-fat diet, and has the application of treating or preventing liver function injury.

EXAMPLE 3 Strain MNH22076 and MNH45330 for the prevention or treatment of diabetes

3.1 Experimental methods

1) Experimental animals the experimental mice were C57BL/6J mice, 5-6 weeks old, purchased from Guangdong Kangshen Biotechnology Co.

2) The preparation of test substances of strains MNH22076 and MNH45330 comprises the steps of melting MNH22076 and MNH45330 strain glycerol freezing tubes at 37 ℃, inoculating the strain in an MRS flat plate culture medium for activation in an anaerobic workstation, inoculating the activated strain in the MRS liquid culture medium, carrying out anaerobic culture to obtain a culture with sufficient quantity, centrifuging and concentrating the cultured bacterial liquid, and then using PBS containing 0.05% L-Cys HCl to re-suspend the bacterial liquid, thereby obtaining the test substance with the purity and the viable count (2X 10 ⁹ CFU/mL) meeting the requirements of animal experiments.

3) Negative control PBS containing 0.05% L-Cys HCl was used as the negative control.

4) And (3) a positive control, namely melting a glycerol cryopreservation tube of the B420 strain at 37 ℃, inoculating the glycerol cryopreservation tube into a PYG culture medium in an anaerobic workstation for activation, inoculating the activated strain into the PYG liquid culture medium, performing anaerobic culture to obtain a sufficient number of cultures, centrifuging and concentrating the cultured bacterial liquid, and then re-suspending the bacterial liquid by using PBS containing 0.05% L-Cys HCl to obtain the positive control with purity and viable count (2X 10 ⁹ CFU/mL) meeting the requirements of animal experiments.

5) The experimental process is that 40 mice with the weight range of 18g-22g are selected after the quarantine period is finished, and the mice are randomly layered and grouped according to the weight, 8 mice/group are divided into 5 groups, namely NCD-Control group, HFD-Control group, MNH22076 group, MNH45330 group and B420 group. Wherein NCD-Control group is fed with maintenance feed, and the other four groups are fed with 60% high-fat feed. After the grouping (D1), the negative Control was given to NCD-Control group and HFD-Control group, the positive Control was given to B420 group, MNH22076 was given to MNH22076 group, MNH45330 was given to MNH45330 group, and the administration was performed by gastric lavage 1 time a day, 200. Mu.L each time, for 56 days, and the mice were allowed to drink and eat freely during the experiment, and the 12h/12h day-and-night cycle was adopted. During the trial, 1 general clinical observation was performed after each dosing was completed. Fasting blood glucose was measured once within the last 2 weeks of dosing and the test endpoint measured fasting blood glucose. All data are expressed in mean±sd format, plotted using GRAPHPAD PRISM software and statistically analyzed. For the pairwise comparison, a t-test (Student's t test) analysis method was used. No significance is indicated and difference significance is indicated by p <0.05, p <0.01, p <0.001, p <0.0001.

3.2 Experimental results

Referring to fig. 16, it can be seen that the strains MNH22076 and MNH45330 can significantly reduce fasting blood glucose in high-fat diet-induced type two diabetic mice, and thus the strains MNH22076 and MNH45330 have the use of preventing or treating diabetes.

EXAMPLE 4 use of strains MNH22076 and MNH45330 for the treatment or prevention of obesity and related diseases

4.1 Experimental method:

1) Experimental animals the experimental mice were C57BL/6J mice, 5-6 weeks old, purchased from Guangdong Kangshen Biotechnology Co.

2) The preparation of test substances of strains MNH22076 and MNH45330 comprises the steps of melting MNH22076 and MNH45330 strain glycerol freezing tubes at 37 ℃, inoculating the strain in an MRS flat plate culture medium for activation in an anaerobic workstation, inoculating the activated strain in the MRS liquid culture medium, carrying out anaerobic culture to obtain a culture with sufficient quantity, centrifuging and concentrating the cultured bacterial liquid, and then using PBS containing 0.05% L-Cys HCl to re-suspend the bacterial liquid, thereby obtaining the test substance with the purity and the viable count (2X 10 ⁹ CFU/mL) meeting the requirements of animal experiments.

3) Negative control PBS containing 0.05% L-Cys HCl (cysteine hydrochloride) as negative control

4) And (3) a positive control, namely melting a B420 strain glycerol cryopreservation tube at 37 ℃, inoculating the tube into a PYG culture medium in an anaerobic workstation for activation, inoculating the activated strain into the PYG liquid culture medium, performing anaerobic culture to obtain a sufficient number of cultures, centrifuging and concentrating the cultured bacterial liquid, and then re-suspending the bacterial liquid by using PBS containing 0.05% L-Cys HCl to obtain the positive control with purity and viable count (2X 10 ⁹ CFU/mL) meeting the requirements of animal experiments.

5) The experimental process is that 40 mice with the weight range of 18g-22g are selected after the quarantine period is finished, and the mice are randomly layered and grouped according to the weight, 8 mice/group are divided into 5 groups, namely NCD-Control group, HFD-Control group, MNH22076 group, MNH45330 group and B420 group. Wherein NCD-Control group is fed with maintenance feed, and the other four groups are fed with 60% high-fat feed. After the grouping (D1), the negative Control was given to NCD-Control group and HFD-Control group, the positive Control was given to B420 group, MNH22076 was given to MNH22076 group, MNH45330 was given to MNH45330 group, and the administration was performed by gastric lavage 1 time a day, 200. Mu.L each time, for 56 days, and the mice were allowed to drink and eat freely during the experiment, and the 12h/12h day-and-night cycle was adopted. During the trial, 1 general clinical observation was performed after each dosing was completed. Animal weights were measured twice weekly during the dosing period and were measured prior to the end of the test dissection. The test endpoint is the next day (D57) after the end of administration, the test endpoint takes anatomical materials according to the scheme, and the data is summarized to analyze the weight and the weight change percentage, each anatomical data result and serum detection data result. All data are expressed in mean±sd format, plotted using GRAPHPAD PRISM software and statistically analyzed. For the pairwise comparison, a t-test (Student's t test) analysis method was used. No significance is indicated and difference significance is indicated by p <0.05, p <0.01, p <0.001, p <0.0001.

4.2 Experimental results

Referring to fig. 17a-17b, it can be seen that strains MNH22076 and MNH45330 significantly reduced the weight and weight gain of high fat diet induced obese mice, and that after eight weeks of gastric lavage intervention, the weight was reduced by more than 10% compared to HFD-Control group, indicating that strains MNH22076 and MNH45330 had significant weight loss effects.

Referring to fig. 18a-18C, the results show that strain MNH22076 was able to significantly reduce the concentration of Total Cholesterol (TCHO), triglycerides (TG), low density lipoprotein cholesterol (LDL-C) in serum of high fat diet induced obese mice. The data show that the strain MNH22076 and MNH45330 have the effects of treating/preventing hypercholesterolemia, hyperlipidemia and/or cardiovascular and cerebrovascular diseases.

Referring to fig. 19a-19e, the results show that the strains MNH22076 and MNH45330 can significantly reduce the fat weight of epididymal fat, perirenal fat, subcutaneous fat and/or mesenteric fat in high-fat diet induced obese mice, significantly reduce the ratio of visceral fat to body weight, and demonstrate that the strains MNH22076 and MNH45330 have the effects of treating/preventing visceral fat percentage from being high and treating/preventing obesity.

Although the invention has been disclosed with reference to certain embodiments, it will be apparent that modifications and variations can be made without departing from the spirit and scope of the invention as disclosed herein and as provided in the appended claims. Furthermore, it should be understood that while all of the examples in the disclosure illustrate embodiments of the invention, they are provided as non-limiting examples only and therefore should not be considered as limiting the various aspects of the invention described thereby. The invention is intended to have the full scope defined by the disclosure, the language of the following claims, and any equivalents thereof. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not as restrictive.

Claims (10)

1. An isolated Lactobacillus crispatus, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of 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; 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. 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, lyophilized 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. According to the use of claim 8, the liver disease includes 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.