CN114717147B

CN114717147B - Post-natal element prepared from lactobacillus rhamnosus and used for relieving fatty liver and obesity and application thereof

Info

Publication number: CN114717147B
Application number: CN202210294380.9A
Authority: CN
Inventors: 崔树茂; 潘正浩; 张秋香; 唐鑫; 毛丙永; 杨波; 赵建新; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-23
Filing date: 2022-03-23
Publication date: 2023-07-25
Anticipated expiration: 2042-03-23
Also published as: CN114717147A

Abstract

The invention discloses a metagen for relieving fatty liver and obesity prepared from lactobacillus rhamnosus and application thereof, and belongs to the technical field of microorganisms and medicines. The metagen prepared by the rhamnose bacillus CCFM1219 provided by the invention has the functions of relieving fatty liver and obesity: (1) significantly alleviating weight gain in high fat diet mice; (2) significantly improving glycemic homeostasis in high fat diet mice; (3) Significantly inhibiting liver fat accumulation and lesions in high fat diet mice; (4) Significantly reducing white fat cell size, maintaining normal morphology of brown fat, and promoting brown fat browning; (5) Significantly reducing the level of high fat diet mouse serum TC, GGT, ALP; (6) Effectively reduces the weight of the liver and the white adipose tissue of the abdomen of the high-fat diet mice. Therefore, the metagen prepared by lactobacillus rhamnosus CCFM1219 has great application prospect in preparing products for preventing and/or treating fatty liver.

Description

Post-natal element prepared from lactobacillus rhamnosus and used for relieving fatty liver and obesity and application thereof

Technical Field

The invention relates to a post-natal for relieving fatty liver and obesity prepared from lactobacillus rhamnosus and application thereof, and belongs to the technical field of microorganisms and medicines.

Background

Obesity is a chronic, complex, heterogeneous disease that can lead to over 200 medical conditions affecting the whole organ system. Since 1980, the obesity rate of 73 countries doubled, and the BMI was high>25kg/m ² ) Causing about 400 ten thousand deaths worldwide and causing serious cardiovascular disease burden. More and more patients with obesity, together with the huge hidden danger of health caused by obesity related complications, have an urgent need for treating obesity. The current major modes of weight loss include exercise, diet control, the use of weight loss drugs, and the like. The international mainstream weight-reducing medicines such as phentermine and orlistat have great adverse reactions, and damage is caused to the health of patients, for example, the phentermine may cause rare primary pulmonary arterial hypertension or serious reflux heart valve diseases, and the orlistat can cause malabsorption of fat-soluble vitamin A, D, E, K and the like. Therefore, the weight loss product market still needs innovation.

Fatty liver includes nonalcoholic fatty liver disease (NAFLD) and Alcoholic Fatty Liver Disease (AFLD), and is mainly characterized by excessive fat deposition in liver, and the clinical manifestations of patients are hepatocyte inflammation, liver fibrosis, etc. The total prevalence of AFLD and NAFLD is 6% and 25%, respectively, is the most common chronic liver disease in western countries and is a global health burden with increasing incidence. NAFLD is not only closely associated with hepatocellular carcinoma (HCC), liver failure, etc., but also significantly increases the incidence of metabolic and cardiovascular-related complications. NAFLD and AFLD have the same histopathological characteristics, ranging from benign simple steatosis to steatohepatitis to more severe diseases, including advanced fibrosis and cirrhosis, ultimately leading to the potential for hepatocellular carcinoma, liver failure and death. In addition to lifestyle adjustments, currently, clinically, fatty liver is treated mainly by using metformin, vitamin E, ursodeoxycholic acid and the like for insulin resistance, oxidative stress resistance and cytoprotection. However, drugs are mainly detoxified and metabolized in the liver, and excessive or improper drug intake may increase the burden and hepatotoxicity of the liver, so that drugs should be carefully selected to treat fatty liver.

Therefore, there is still a need to continue to find a drug or therapeutic way, which can be used for preventing and/or treating obesity and fatty liver, does not bring side effects to patients, and can also be applied to various types of patients, and has good tolerance to the patients.

Disclosure of Invention

[ technical problem ]

The invention aims to solve the technical problem of providing a metagen which can be prepared from lactobacillus rhamnosus (Lactobacillus rhamnosus) and can relieve fatty liver and obesity.

Technical scheme

The invention provides a lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219, wherein lactobacillus rhamnosus (Lactobacillus rhamnosus) is stored in the microorganism strain collection in Guangdong province for 1 month 23 days in 2022, and the storage number is GDMCC No:62231, the preservation address is Guangzhou Mr. first 100 college No. 59 building.

The lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 is separated from healthy human body excrement, the 16S rRNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is subjected to nucleic acid sequence comparison in NCBI, and after the comparison result is obtained, the lactobacillus rhamnosus is identified.

The colony of lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 on MRS solid culture medium is round, white and smooth.

The lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 is a gram-positive bacterium, is facultative anaerobic, has a temperature preference, has an optimal growth temperature of 35-40 ℃ and an optimal growth pH of 6.0-7.0.

The invention provides a composition comprising the lactobacillus rhamnosus CCFM1219 and/or the progeny cells prepared from the lactobacillus rhamnosus CCFM1219. .

In one embodiment, the metazoan comprises dead cells, fermentation supernatant, bacterial lysate, and/or fermentation broth.

In one embodiment, the preparation method of the fermentation broth comprises inoculating lactobacillus rhamnosus CCFM1219 into a fermentation medium to obtain a bacterial solution, and performing heat treatment on the bacterial solution to obtain the fermentation broth.

In one embodiment, the heat treatment is performed at 60 to 70℃for 25 to 35 minutes.

In one embodiment, the preparation method of the bacterial lysate comprises the steps of homogenizing the fermentation liquor under high pressure and centrifuging to obtain the bacterial lysate.

In one embodiment, the preparation method of the dead cells is to obtain the dead cells after heat treatment or freeze drying of bacterial sludge sediment obtained by centrifuging the bacterial liquid.

In one embodiment, the fermentation supernatant is obtained by centrifuging the bacterial liquid or the fermentation liquid.

In one embodiment, the metazoan is prepared as a powder or liquid formulation.

In one embodiment, the powder is a solid powder prepared by drying the metazoan described above.

In one embodiment, the drying means comprises spray drying, vacuum freeze drying, fluid bed drying or vacuum drying.

The invention provides application of lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 and/or in preparing a product for preventing and/or treating fatty liver and obesity-related diseases.

The invention provides application of the composition in preparing products for preventing and/or treating fatty liver and obesity related diseases.

In one embodiment of the invention, the obesity-related disorder comprises diabetes, musculoskeletal disorders, cardiovascular disorders, malignant tumors, hyperlipidemia or metabolic syndrome.

In one embodiment of the invention, the fatty liver-associated disease comprises alcoholic fatty liver, non-alcoholic fatty liver, liver inflammation, cirrhosis or liver cancer.

In one embodiment, the product comprises a food or pharmaceutical product.

In one embodiment of the invention, the pharmaceutical product comprises the above-described composition, a pharmaceutical carrier and/or a pharmaceutical adjuvant.

In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.

In one embodiment of the invention, the pharmaceutical excipients comprise solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retarders.

In one embodiment, the food product comprises the above composition and conventional excipients,

in one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional supplements.

The invention provides a product for preventing and/or treating fatty liver and obesity related diseases, which comprises the lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 and/or the composition.

In one embodiment, the product comprises at least one of the following effects:

(1) Significantly alleviating weight gain in high-fat diet mice;

(2) Significantly improving the glycemic homeostasis of high-fat diet mice;

(3) Significantly inhibiting liver fat accumulation and lesions in high fat diet mice;

(4) Significantly reducing white fat cell size, maintaining normal morphology of brown fat, and promoting brown fat browning;

(5) Significantly reducing the level of high fat diet mouse serum TC, GGT, ALP;

(6) Effectively reduces the weight of the liver and epididymis white adipose tissues of the high-fat diet mice.

In one embodiment of the invention, the dose of the offspring produced by lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 as described above is not less than 90mg/kg body weight in the product.

In one embodiment of the invention, the product comprises a food or pharmaceutical product.

In one embodiment of the invention, the pharmaceutical product comprises a composition, a pharmaceutical carrier and/or a pharmaceutical adjuvant.

In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

In one embodiment of the invention, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills or oral liquids.

[ advantageous effects ]

The invention screens and obtains a lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219, and the metagen prepared by the lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 has the effect of relieving fatty liver and obesity, and is specifically expressed in the following steps:

(1) Significantly alleviating weight gain in high-fat diet mice;

(2) Significantly improving the glycemic homeostasis of high-fat diet mice;

(5) Significantly reducing the level of high fat diet mouse serum TC, GGT, ALP;

(6) The weight of the liver and epididymis white adipose tissue of the high-fat diet mice is obviously reduced.

Therefore, the metagen prepared by lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 has great application prospect in preparing products for preventing and/or treating fatty liver and obesity and even preventing and/or treating diseases related to obesity and metabolic disorder.

Preservation of biological materials

Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219, taxonomic designation Lactobacillus rhamnosus, was deposited at the collection of microorganism strains, accession No. GDMCC No:62231, the preservation address is Guangzhou Mr. first 100 college No. 59 building.

Drawings

Fig. 1: weight gain of mice of different groups;

fig. 2: oral glucose tolerance in mice of different groups;

fig. 3: liver HE stained tissue sections of different groups of mice;

fig. 4: liver oil red O stained tissue sections of different groups of mice;

fig. 5: white adipose stained tissue sections of epididymis of mice of different groups;

fig. 6: brown fat stained tissue sections of different groups of mice;

fig. 7: comparing serum biochemical indexes of different groups of mice;

fig. 8: total 12-week weight gain and tissue weight in different groups of mice;

fig. 9: lactobacillus paracasei CCFM1219 mimics gastric juice, intestinal juice tolerance.

", indicates significant differences from the Model group (P < 0.05), and", indicates extremely significant differences from the Model group (P < 0.01).

Detailed Description

The invention is further illustrated below in conjunction with specific examples.

The C57BL/6N mice referred to in the examples below were purchased from Beijing vitamin Toril LiHua Co.

The following examples relate to the following media:

MRS liquid medium: 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L of magnesium sulfate heptahydrate and 1mL/L of tween-80, and the pH value is 6.2-6.4.

MRS solid medium: 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L of magnesium sulfate heptahydrate, 20.0g/L of tween-80 and agar, and pH value of 6.2-6.4.

HSY medium: 60g/L glucose, 20g/L casein peptone, 1g/L yeast extract, 0.35g/L MgSO ₄ ·7H ₂ O、0.1g/L MnSO ₄ ·H ₂ O、2.6g/L K ₂ HPO ₄ ·3H ₂ O。

Example 1: screening and identification of lactobacillus rhamnosus

The method comprises the following specific steps:

1. screening

The sample is derived from healthy human body excrement in Shanghai city, the sample is pretreated and then stored in a refrigerator at the temperature of minus 80 ℃ in 20 percent of glycerol, after being taken out and thawed, the sample is evenly mixed and absorbed into 4.5mL of physiological saline, the physiological saline is added into the sample to carry out gradient dilution, a proper gradient diluent is selected to be coated on an MRS solid culture medium, the sample is cultured for 48 hours at the temperature of 37 ℃, typical bacterial colonies of lactobacillus rhamnosus are picked up to be streaked and purified on the MRS solid culture medium, single bacterial colonies are picked up and transferred to the MRS liquid culture medium for enrichment, 30 percent of glycerol is stored, and the bacterial strain is obtained and named as CCFM1219; among them, typical colony of lactobacillus rhamnosus is round, white and smooth.

2. Authentication

Extracting genome of the strain CCFM1219, amplifying and sequencing 16S rDNA of the strain CCFM1219 (the nucleotide sequence of the amplified 16S rDNA of the strain CCFM1219 is shown as SEQ ID NO.1 by Jin Weizhi biotechnology Co., ltd. In Suzhou), and comparing the sequence with nucleic acid sequence in NCBI, so that the strain is lactobacillus rhamnosus and named as lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219.

Example 2: preparation of post-metazoan by lactobacillus rhamnosus CCFM1219

Culturing in a 37 ℃ water-proof constant temperature incubator for 24-48 hours by using an MRS solid culture medium to obtain single colonies; single colony is selected and inoculated into MRS liquid culture medium, and cultured for 12 hours at 37 ℃ to obtain culture solution 1; inoculating the culture solution 1 into MRS liquid culture medium with an inoculum size of 1% (v/v), and culturing at 37 ℃ for 12h to obtain a culture solution 2; inoculating the culture solution 2 into a fermentation medium (HSY medium) for preparing metazoan at an inoculum size of 1% (v/v) to obtain a seed solution, and culturing at 37 ℃ for 12h; inoculating the seed solution into HSY culture medium at 3-5% (v/v), and culturing at 37deg.C for 24-36 hr to obtain bacterial solution.

And (3) carrying out heat treatment (65 ℃ for 30 min) on the bacterial liquid to obtain a metazoan fermentation liquid, and carrying out freeze-drying to obtain metazoan freeze-dried powder for later use.

Lyophilizing the HSY culture medium to obtain lyophilized culture medium powder.

Example 3: effect of Lactobacillus rhamnosus CCFM 1219-prepared metazoan on weight gain in high-fat diet mice

60 healthy male C57BL/6N mice of 7 weeks of age are randomly divided into 10 groups of 6 mice each, and the 10 groups are respectively: blank (Control), model (Model), HSY medium group: m_1 (low dose: 200mg/kg mouse body weight), M_2 (high dose: 800mg/kg mouse body weight), lactobacillus rhamnosus CCFM1219 metatuple: CCFM1219_1 (low dose: 200mg/kg mouse body weight), CCFM1219_2 (high dose: 800mg/kg mouse body weight), lactobacillus rhamnosus FTJDG metatuple: ftjdg_1 (low dose: 200mg/kg mouse body weight), ftjdg_2 (high dose: 800mg/kg mouse body weight), lactobacillus paracasei CQYY metagenome: CQYY_1 (low dose: 200mg/kg mouse body weight), CQYY_2 (high dose: 800mg/kg mouse body weight).

The experiment was performed for 13 weeks: after mice were acclimatized for one week, the blank group was fed with low-fat low-sugar feed, the remaining groups were fed with high-fat low-sugar feed, and from the second week onwards, mice were perfused with medium group HSY medium lyophilized powder (dissolved in physiological saline at the corresponding dose) at an amount of 0.2 mL/day, each metagroup was perfused with metagroup lyophilized powder prepared with the corresponding strain (dissolved in physiological saline at the corresponding dose) at an amount of 0.2 mL/day, and the blank group and model group were perfused with the same amount of physiological saline as the control until the end of the experiment. All groups were free drinking and ingestion.

Mice were weighed at week 1 and week 12 of metazoan intervention, respectively, and the weight gain per mouse was as shown in figure 1. As can be seen from fig. 1, the metazoans prepared from lactobacillus rhamnosus CCFM1219 significantly inhibited weight gain in high-fat diet mice when administered at high doses, compared to the model group, HSY medium group and metazoans prepared from other bacteria.

As shown by experimental results, the post-natal cell prepared by the lactobacillus rhamnosus CCFM1219 has the effect of relieving the weight gain of the high-fat diet mice, and at the end of the experiment, compared with the average weight gain of a model group, the weight gain of the post-natal cell CCFM1219_2 group is only 12.26g, the weight gain of the post-natal cell CCFM1219_1 group is 18.2g, and the weight gain of other intervention groups is 19.14g.

Table 1 experimental animal groups

Example 4: the effect of the metazoan prepared by lactobacillus rhamnosus CCFM1219 on the blood glucose homeostasis of high-fat diet mice is obtained by randomly dividing 60 healthy male C57BL/6N mice of 7 weeks of age into 10 groups of 6 mice each, wherein the 10 groups are respectively: blank (Control), model (Model), HSY medium group: m_1 (low dose: 200mg/kg mouse body weight), M_2 (high dose: 800mg/kg mouse body weight), lactobacillus rhamnosus CCFM1219 metatuple: CCFM1219_1 (low dose: 200mg/kg mouse body weight), CCFM1219_2 (high dose: 800mg/kg mouse body weight), lactobacillus rhamnosus FTJDG metatuple: ftjdg_1 (low dose: 200mg/kg mouse body weight), ftjdg_2 (high dose: 800mg/kg mouse body weight), lactobacillus paracasei CQYY metagenome: CQYY_1 (low dose: 200mg/kg mouse body weight), CQYY_2 (high dose: 800mg/kg mouse body weight).

The experiment was performed for 13 weeks: after mice were acclimatized for one week, the blank group was fed with low-fat low-sugar feed, the remaining groups were fed with high-fat low-sugar feed, and from the second week onwards, the mice were perfused with the lyophilized powder of the medium HSY (dissolved in physiological saline at the corresponding dose) at an amount of 0.2 mL/day, the metazoans prepared with the corresponding strain (dissolved in physiological saline at the corresponding dose) were perfused with the same amount of physiological saline as the model group, and the blank group and the model group were perfused with the same amount of physiological saline until the end of the experiment. All groups were free drinking and ingestion.

The glycemic homeostasis of mice was assessed by the Oral Glucose Tolerance Test (OGTT) before the end of the 13 th week experiment, and after 12h of fasting, the mice were perfused with glucose at a dose of 2mg/g body weight, blood was collected at the tail veins before (0 min), 15, 30, 60, 90, 120min after the intragastric administration, respectively, and blood glucose was measured using the Accu-Chek viable blood glucose test paper.

As a result, as shown in FIG. 2, it is understood from FIG. 2 that the fasting blood glucose of the high-dose administered group (fasting blood glucose values of 6.38mmol/L, respectively) prepared from Lactobacillus rhamnosus CCFM1219 was relatively low compared with the model group (fasting blood glucose values of 9 mmol/L), the medium group and the other metazoan groups (fasting blood glucose values of 8.1mmol/L, respectively), and that blood glucose could be reduced to fasting level within 2 hours.

The experimental result shows that the metagen prepared by lactobacillus rhamnosus CCFM1219 has the effects of improving the blood glucose steady state of high-fat diet mice and increasing the oral glucose tolerance.

Example 5: effect of the offspring of Lactobacillus rhamnosus CCFM1219 on liver fat accumulation and lesions in high fat diet mice

After the experiment is finished, the mice are taken out and killed, the liver tissues of the mice are taken for HE staining and oil red O staining, and the staining results are shown in figures 3 and 4.

As can be seen from fig. 3 and 4, the liver tissue of mice in the high-dose administration group of the metazoan prepared from the blank group and lactobacillus rhamnosus CCFM1219 is uniformly colored, the fat accumulation is less, and the hepatocytes are compact and uniform and take on a regular shape. The liver tissues of the other groups of mice have more fat accumulation, obvious pathological changes, a large number of fat vacuoles around cells, loose connection of liver cells, outflow of cell contents, swelling of cells and damage of cell integrity.

As shown by experimental results, the metagen prepared from lactobacillus rhamnosus CCFM1219 effectively inhibits liver fat accumulation and lesions of high-fat diet mice

Example 6: effect of the offspring of Lactobacillus rhamnosus CCFM1219 on the brown fat and epididymal white fat of high fat diet mice

After the experiment is finished, the mice are taken out and killed, the brown fat of the shoulder blade and the white adipose tissue of the epididymis of the mice are taken for HE staining, and the staining results are shown in figures 5 and 6.

From FIGS. 5 and 6, it can be seen that the metazoans prepared by Lactobacillus rhamnosus CCFM1219, when administered at high doses, increased the proportion of small adipocytes (diameter. Ltoreq.50 μm), decreased the proportion of large adipocytes (diameter >50 μm) and effectively reduced fat accumulation in adipocytes, compared to the model group, the medium group and other metazoan intervention groups. The morphology and the function of fat cells are changed from brown to white by high-fat diet, and the metagen prepared by lactobacillus rhamnosus CCFM1219 remarkably improves lipid accumulation of white adipose tissues caused by high-fat diet, reduces lipid infiltration and invasion, and maintains the normal morphology and the function of brown adipose tissues.

Experimental results show that the metagen prepared by lactobacillus rhamnosus CCFM1219 effectively reduces fat accumulation, maintains the normal form of brown fat and promotes brown color of white fat.

Example 7: influence of the metazoan prepared by Lactobacillus rhamnosus CCFM1219 on serum biochemical indicators of high-fat diet mice

After the experiment is finished, taking blood and dying the mice, centrifuging whole blood of the mice to obtain serum, and detecting serum related indexes by adopting a full-automatic biochemical analyzer, wherein the related indexes comprise: TG (triglyceride), TC (total cholesterol), GGT (glutamyl transpeptidase), AST (aspartic acid aminotransferase), ALP (alkaline phosphatase). The results are shown in FIG. 7.

As can be seen from fig. 7, the post-natal prepared from lactobacillus rhamnosus CCFM1219 significantly reduced the serum TC of the high fat diet mice (20.05% lower in comparison to the control group and the other post-natal intervention groups, respectively), GGT (34% lower in comparison to the control group and the other post-natal intervention groups, 14% lower in comparison to the control group, respectively), ALP (36.45% lower in comparison to the control group and 13.63% lower in comparison to the control group and the other post-natal intervention groups, respectively) and TG, AST, content were also reduced when administered at high doses.

Experimental results show that the metagen prepared by lactobacillus rhamnosus CCFM1219 effectively reduces serum biochemical index levels related to obesity and fatty liver of a high-fat diet mouse, and is beneficial to relieving the obesity and fatty liver.

Example 8: effect of metazoan components on weight and tissue weight of high fat diet mice.

Cell lysate group: homogenizing the metafermentation broth of example 2 under high pressure, centrifuging to obtain thallus lysate, and lyophilizing.

Fermentation liquor group: the bacterial liquid in example 2 is subjected to heat treatment (65 ℃ for 30 min) to obtain a metazoan fermentation liquid, and the fermentation liquid is taken and freeze-dried to obtain metazoan powder for later use.

Dead bacterial group: centrifuging the bacterial liquid in the embodiment 2 to obtain bacterial sludge sediment, and freeze-drying to obtain dead bacteria for later use.

Fermentation supernatant group: the bacterial liquid of example 2 was centrifuged to obtain a fermentation supernatant, which was lyophilized for use.

30 healthy male C57BL/6N mice of 7 weeks of age are randomly divided into 6 groups of 5 mice each, and the 6 groups are respectively: blank (Control), model (Model), dead bacteria, cell lysate, fermentation supernatant, and broth.

The experiment was performed for 13 weeks: after mice were acclimatized for one week, the blank group was fed low-fat low-sugar feed, the remaining groups were fed high-fat low-sugar feed, and from the second week onwards, the intervention group was perfused with normal saline in an amount of 0.2 mL/day with dead bacteria (dose of 24mg/kg mouse body weight), bacterial lysate (dose of 24mg/kg mouse body weight), fermented supernatant lyophilized powder (dose of 800mg/kg mouse body weight), fermented liquid lyophilized powder (dose of 800mg/kg mouse body weight), and the blank group and model group were perfused with normal saline in an amount equivalent to the stomach, respectively, as a control, until the experiment was completed. All groups were free drinking and ingestion.

Mice were weighed at the end of the conditioning period and prior to sacrifice, and after sacrifice, their liver, abdomen white fat were weighed and total 12 week body weight gain and tissue weight are shown in figure 8. As can be seen from fig. 8, the metazoans prepared from various forms of lactobacillus rhamnosus CCFM1219 effectively inhibited weight gain in high-fat diet mice and reduced liver and abdomen white fat weight in comparison with the model group, in which the fermentation supernatant was slightly weak.

Experimental results show that the components of the metagen prepared by lactobacillus rhamnosus CCFM1219 have the functions of losing weight and reducing liver fat accumulation.

Table 2 experimental animal groups

Example 9: acid and bile salt resistance of lactobacillus rhamnosus CCFM1219

Preparation of simulated gastric fluid: pepsin (1:10000) was dissolved in sterilized normal saline (pH 3) to a final concentration of 3g/L, and filtered through a 0.22 μm sterile filter membrane, and prepared for use.

Preparation of simulated intestinal juice: trypsin (1:250) was dissolved in sterilized normal saline (pH 8) to a final concentration of 1g/L, and bile salt was added to a final concentration of 0.30% (w/v), and the mixture was filtered through a 0.22 μm sterile filter membrane, and the mixture was ready to use.

The bacterial suspension at the end of the index of the lactobacillus rhamnosus CCFM1219 after equal amount of mixing is collected in 7mLEP pipes, the centrifugation is carried out for 10min at 8000 Xg, and the supernatant is discarded, so as to collect the bacterial body. Tube 1 was resuspended in 5mL of physiological saline (ph=7) and counted prior to treatment. 3, the bacterial strain is resuspended in simulated gastric juice with the same volume, and after being evenly mixed, the bacterial strain is placed at 37 ℃ for culturing for 1h, 2h and 4h, and then is counted. 3, the tube strain is resuspended in an equal volume of simulated intestinal fluid, and after being evenly mixed, the tube strain is placed at 37 ℃ for culturing for 1h, 2h and 4h, and then is counted.

Strain survival (%) = (N) _t /N ₀ ) 100%; in the test: n (N) ₀ Number of viable bacteria before 0h of treatment, N _t -viable count after 1h, 2h, 4h treatment. The results are shown in FIG. 9.

In fig. 9, lactobacillus rhamnosus CCFM1219 is shown to have a survival rate up to 88.72% after treatment for 1 hour in a simulated gastric fluid environment at ph=3, a survival rate of 78.43% after 2 hours of treatment, and a survival rate of 66.32% after 4 hours; after the lactobacillus rhamnosus CCFM1219 is treated by simulated intestinal juice for 1h, the survival rate is up to 90.25%, the survival rate after 2h treatment is 80.37%, and the survival rate after 4h treatment is higher than 65%, which indicates that the lactobacillus rhamnosus CCFM1219 has good acid and bile salt resistance.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> a fatty liver and obesity relieving metazoan prepared from Lactobacillus rhamnosus and its application

<130> BAA220234A

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1443

<212> DNA

<213> rhamnose bacterium

<400> 1

tgcaagtcga acgagttctg attattgaaa ggtgcttgca tcttgattta attttgaacg 60

agtggcggac gggtgagtaa cacgtgggta acctgccctt aagtggggga taacatttgg 120

aaacagatgc taataccgca taaatccaag aaccgcatgg ttcttggctg aaagatggcg 180

taagctatcg cttttggatg gacccgcggc gtattagcta gttggtgagg taacggctca 240

ccaaggcaat gatacgtagc cgaactgaga ggttgatcgg ccacattggg actgagacac 300

ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg caagtctgat 360

ggagcaacgc cgcgtgagtg aagaaggctt tcgggtcgta aaactctgtt gttggagaag 420

aatggtcggc agagtaactg ttgtcggcgt gacggtatcc aaccagaaag ccacggctaa 480

ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat ttattgggcg 540

taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccctcggctt aaccgaggaa 600

gtgcatcgga aactggaaaa cttgagtgca gaagaggaca gtggaactcc atgtgtagcg 660

gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac 720

tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc 780

cgtaaacgat gaatgctagg tgttggaggg tttccgccct tcagtgccgc agctaacgca 840

ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa ttgacggggg 900

cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960

cttgacatct tttgatcacc tgagagatca ggtttcccct tcgggggcaa aatgacaggt 1020

ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080

aacccttatg actagttgcc agcatttagt tgggcactct agtaagactg ccggtgacaa 1140

accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200

gtgctacaat ggatggtaca acgagttgcg agaccgcgag gtcaagctaa tctcttaaag 1260

ccattctcag ttcggactgt aggctgcaac tcgcctacac gaagtcggaa tcgctagtaa 1320

tcgcggatca gcacgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380

ccatgagagt ttgtaacacc cgaagccggt ggcgtaaccc ttttagggag cgagccgtct 1440

aag 1443

Claims

1. Lactobacillus rhamnosus strainLactobacillus rhamnosus) CCFM1219, deposited at the microorganism seed collection of Guangdong province at day 1 and 23 of 2022Numbered GDMCC No:62231, the preservation address is Guangzhou Mr. first 100 college No. 59 building.

2. A composition comprising the lactobacillus rhamnosus CCFM1219 of claim 1 and/or the progeny cells produced by the lactobacillus rhamnosus CCFM1219 of claim 1;

the metazoan includes dead cells, fermentation supernatant, bacterial lysate and/or fermentation broth.

3. The composition according to claim 2, wherein the fermentation broth is prepared by inoculating lactobacillus rhamnosus CCFM1219 of claim 1 into a fermentation medium to obtain a broth, and performing heat treatment on the broth to obtain a fermentation broth; the preparation method of the thallus lysate comprises the steps of homogenizing the fermentation liquor under high pressure and centrifuging to obtain the thallus lysate; the preparation method of the dead cells comprises the steps of carrying out heat treatment or freeze-drying on bacterial sludge sediment obtained by centrifuging bacterial liquid to obtain the dead cells; the fermentation supernatant is obtained by centrifuging the bacterial liquid or the fermentation liquid.

4. The composition of claim 2, wherein the metazoan is prepared as a powder or liquid formulation.

5. Use of the composition according to any one of claims 2 to 4 in the preparation of a medicament for preventing and/or treating fatty liver.

6. A medicament for preventing and/or treating fatty liver, characterized in that the medicament comprises lactobacillus rhamnosus CCFM1219 of claim 1 or the composition of any of claims 2 to 4.

7. The medicament according to claim 6, characterized by comprising at least one of the following actions:

(1) Significantly alleviating weight gain in high-fat diet mice;

(2) Significantly improving the glycemic homeostasis of high-fat diet mice;

(5) Significantly reducing the level of high fat diet mouse serum TC, GGT, ALP;