CN114717147A

CN114717147A - Metazoan prepared from Lactobacillus rhamnosus and used for relieving fatty liver and obesity, and application thereof

Info

Publication number: CN114717147A
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: 2022-07-08
Anticipated expiration: 2042-03-23
Also published as: CN114717147B

Abstract

The invention discloses an anagen prepared from lactobacillus rhamnosus and used for relieving fatty liver and obesity and application thereof, belonging to the technical field of microorganisms and medicines. The metazoan prepared by the rhamnosus CCFM1219 provided by the invention has the effects of relieving fatty liver and obesity: (1) significantly relieving the weight gain of mice on high-fat diet; (2) significantly improving the blood glucose homeostasis of mice on high-fat diet; (3) significantly inhibit the liver fat accumulation and pathological changes of high fat diet mice; (4) the size of white fat cells is obviously reduced, the normal form of brown fat is maintained, and the browning of the white fat is promoted; (5) remarkably reducing the levels of TC, GGT and ALP in the serum of a high-fat diet mouse; (6) effectively reduces the weight of the liver and the abdominal white adipose tissues of the high-fat diet mice. Therefore, the metazoan prepared by the lactobacillus rhamnosus CCFM1219 has huge application prospect in preparing products for preventing and/or treating fatty liver and obesity and even preventing and/or treating obesity and metabolic disorder related diseases.

Description

Metazoan prepared from Lactobacillus rhamnosus and used for relieving fatty liver and obesity, and application thereof

Technical Field

The invention relates to an anagen prepared from lactobacillus rhamnosus and used for relieving fatty liver and obesity and application thereof, belonging to the technical field of microorganisms and medicines.

Background

Obesity is a chronic, complex, heterogeneous disease that can result in over 200 medical conditions affecting the entire organ system. Since 1980, the obesity rate of 73 countries doubled, high BMI (b:, the obesity rate of 73 countries>25kg/m²) Causing about 400 million deaths worldwide and creating a serious burden of cardiovascular disease. The need to treat obesity is becoming more urgent, as more and more obese patients, and the significant health risks associated with obesity-related complications add to the increased need for treatment of obesity. Current major weight loss modalities include exercise, diet control, use of weight loss medications, and the like. International mainstream weight-reducing medicines such as phentermine and orlistat have large adverse reactions and damage to the health of patients, for example, phentermine can cause rare primary pulmonary hypertension or severe reflux heart valve diseases, orlistat can cause poor absorption of fat-soluble vitamin A, D, E, K, and the like. Therefore, the market of weight-reducing products still needs to be innovative.

Fatty liver includes non-alcoholic fatty liver disease (NAFLD) and Alcoholic Fatty Liver Disease (AFLD), and is mainly characterized by excessive fat deposition in the liver, and hepatocellular inflammation, hepatic fibrosis and the like in clinical manifestations of patients. The total disease rates of AFLD and NAFLD are 6% and 25%, respectively, which are the most common chronic liver diseases in western countries and are the 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 features, ranging from benign simple steatosis to steatohepatitis, to more severe diseases including late fibrosis and cirrhosis, which may ultimately lead to hepatocellular carcinoma, liver failure, and death. In addition to the adjustment of life style, the existing clinical treatment of fatty liver mainly uses metformin, vitamin E, ursodeoxycholic acid and the like to carry out insulin resistance, antioxidant stress and cell protection to treat fatty liver. However, drugs are mainly detoxified and metabolized in the liver, and excessive or inappropriate drug intake increases the burden on the liver and hepatotoxicity, and thus, drugs should be carefully selected to treat fatty liver.

Therefore, there is a continuing need for a drug or treatment that can be used for the prevention and/or treatment of obesity and fatty liver without causing side effects to the patient, and that can be applied to various classes of patients with good tolerance to the patient.

Disclosure of Invention

[ problem ] to

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

[ solution ]

The invention provides a Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219, which is deposited in Guangdong province microorganism strain collection center in 23.1.2022, and the deposit number is GDMCC No: 62231, the preservation address is No. 59 of the Zhou Dazhong 100 Jie, Guangzhou city.

The Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 is obtained by separating healthy human excrement, the 16S rRNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is compared with the nucleic acid sequence in NCBI, and after the comparison result is obtained, the Lactobacillus rhamnosus is identified as the result.

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

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

The invention provides a composition which comprises the lactobacillus rhamnosus CCFM1219 and/or a metazoan prepared from the lactobacillus rhamnosus CCFM 1219. .

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 liquid comprises the steps of inoculating the lactobacillus rhamnosus CCFM1219 into a fermentation medium to be cultured to obtain a bacterial liquid, and carrying out heat treatment on the bacterial liquid to obtain the fermentation liquid.

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

In one embodiment, the bacterial cell lysate is prepared by homogenizing the fermentation broth under high pressure and centrifuging.

In one embodiment, the dead cells are prepared by heat-treating or freeze-drying a bacterial sludge precipitate obtained by centrifuging the bacterial liquid.

In one embodiment, the fermentation supernatant is a supernatant 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 above mentioned metazoan.

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

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

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

In one embodiment of the invention, the disease associated with obesity comprises diabetes, a musculoskeletal disease, a cardiovascular disease, a malignancy, hyperlipidemia, or metabolic syndrome.

In one embodiment of the present invention, the fatty liver-related 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 present invention, the pharmaceutical product comprises the above composition, a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and an additive.

In one embodiment of the invention, the pharmaceutical excipients comprise solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

In one embodiment, the food comprises the composition and conventional auxiliary materials,

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

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 relieving the weight gain of mice on high-fat diet;

(2) significantly improving the blood glucose homeostasis of mice on high-fat diet;

(3) remarkably inhibiting liver fat accumulation and lesion of high-fat diet mice;

(4) the size of white fat cells is obviously reduced, the normal form of brown fat is maintained, and the browning of the white fat is promoted;

(5) remarkably reducing the serum TC, GGT and ALP levels of high fat diet mice;

(6) effectively reduces the weight of the liver and the white adipose tissue of the epididymis of mice with high-fat diet.

In one embodiment of the invention, the product is prepared by using the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 for the postnatal preparation at a dose of not less than 90mg/kg body weight.

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 excipient.

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

[ advantageous effects ]

The invention obtains a Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 strain through screening, the anagen prepared by the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 strain has the function of relieving fatty liver and obesity, and the specific expression is as follows:

(1) significantly relieving the weight gain of mice on high-fat diet;

(5) remarkably reducing the levels of TC, GGT and ALP in the serum of a high-fat diet mouse;

(6) obviously reduces the weight of the liver and the white adipose tissue of the epididymis of mice with high-fat diet.

Therefore, the metazoan 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 obesity and metabolic disorder related diseases.

Biological material preservation

Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1219, which is taxonomically named Lactobacillus rhamnous and is deposited in Guangdong province collection of microorganisms and strains in 23 months 1 in 2022 with the deposit number being GDMCC No: 62231, the preservation address is No. 59 of the Zhou Dazhong 100 Jie, Guangzhou city.

Drawings

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

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

FIG. 3: carrying out HE staining on tissue sections of livers of mice of different groups;

FIG. 4: liver oil red O staining tissue sections of mice of different groups;

FIG. 5: white fat staining tissue sections of epididymis of different groups of mice;

FIG. 6: brown adipose-stained tissue sections of mice of different groups;

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

FIG. 8: total weight gain and tissue weight in 12 weeks for different groups of mice;

FIG. 9: simulated gastric fluid and intestinal fluid tolerance of lactobacillus paracasei CCFM 1219.

"+" indicates significant difference from Model group (P <0.05), "+" indicates very significant difference from Model group (P < 0.01).

Detailed Description

The invention is further illustrated with reference to specific examples.

The C57BL/6N mice referred to in the examples below were purchased from Wintolite, Beijing.

The media involved in the following examples are as follows:

MRS liquid medium: 5.0g/L yeast powder, 10.0g/L beef extract, 10.0g/L peptone, 20.0g/L glucose, 2.0g/L anhydrous sodium acetate, 2.0g/L hydrogencitrate diamine, 2.6g/L dipotassium hydrogen phosphate, 0.25g/L manganese sulfate monohydrate, 0.5g/L magnesium sulfate heptahydrate and Tween-801 mL/L, and the pH value is 6.2-6.4.

MRS solid medium: 5.0g/L yeast powder, 10.0g/L beef extract, 10.0g/L peptone, 20.0g/L glucose, 2.0g/L anhydrous sodium acetate, 2.0g/L hydrogencitrate diamine, 2.6g/L dipotassium hydrogen phosphate, 0.25g/L manganese sulfate monohydrate, 0.5g/L magnesium sulfate heptahydrate, Tween-801 mL/L and 20.0g/L agar, wherein the pH value is 6.2-6.4.

HSY culture medium: 60g/L glucose, 20g/L casein peptone, 1g/L yeast extract powder, 0.35g/L MgSO₄·7H₂O、0.1g/L MnSO₄·H₂O、2.6g/L K₂HPO₄·3H₂O。

Example 1: screening and identification of lactobacillus rhamnosus

The method comprises the following specific steps:

1. screening

The sample is from healthy human excrement in Shanghai city, the sample is stored in a refrigerator at minus 80 ℃ in 20% glycerol after being pretreated, the sample is taken out and thawed, the sample is uniformly mixed and 0.5mL of the sample is absorbed to be added into 4.5mL of physiological saline, gradient dilution is carried out by the physiological saline containing 9g/L, proper gradient dilution liquid is selected to be coated on an MRS solid culture medium and cultured for 48 hours at 37 ℃, a typical bacterial colony of lactobacillus rhamnosus is selected to be streaked and purified on the MRS solid culture medium, a single bacterial colony is selected to be transferred to an MRS liquid culture medium for enrichment, and 30% glycerol is preserved to obtain a strain which is named as CCFM 1219; wherein, the typical colony of the lactobacillus rhamnosus is round, white and smooth.

2. Identification

The genome of the strain CCFM1219 is extracted, the 16S rDNA of the strain CCFM1219 is amplified and sequenced (by Jinzhi Biotech Co., Ltd., Suzhou, the 16S rDNA amplified nucleotide sequence of the strain CCFM1219 is shown in SEQ ID NO. 1), and the nucleotide sequence of the sequence is compared in NCBI, so that the strain is Lactobacillus rhamnosus and is named as Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1219.

Example 2: prebiotics prepared by lactobacillus rhamnosus CCFM1219

Culturing for 24-48h in 37 ℃ water-proof constant temperature incubator by using MRS solid culture medium to obtain single colony; selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and culturing at 37 ℃ for 12h to obtain a culture solution 1; inoculating the culture solution 1 into an MRS liquid culture medium in an inoculation amount of 1% (v/v), and culturing at 37 ℃ for 12h to obtain a culture solution 2; inoculating the culture solution 2 into a fermentation culture medium (HSY culture medium) of the prepared prebiotics in an inoculation amount of 1% (v/v) to obtain a seed solution, and culturing at 37 ℃ for 12 h; inoculating the seed solution into HSY culture medium at 3-5% (v/v), and culturing at 37 deg.C for 24-36h to obtain bacterial solution.

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

And (4) freeze-drying the HSY culture medium to obtain culture medium freeze-dried powder.

Example 3: effect of metazoans prepared from Lactobacillus rhamnosus CCFM1219 on weight gain of high-fat diet mice

60 healthy male C57BL/6N mice at 7 weeks of age were randomly assigned to 10 groups of 6 mice each, 10 groups were: blank group (Control), Model group (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 metagenome: CCFM1219_1 (low dose: 200mg/kg mouse body weight), CCFM1219_2 (high dose: 800mg/kg mouse body weight), Lactobacillus rhamnosus FTJDG metagenome: 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 took 13 weeks: after the mice are adapted for one week, a blank group is fed with low-fat and low-sugar feed, the other groups are fed with high-fat and low-sugar feed, from the second week, the mice are gavaged by the HSY culture medium freeze-dried powder (dissolved in normal saline at a corresponding dose) of the culture medium group in an amount of 0.2 mL/one/day, each metazoan group is gavaged by the metazoan freeze-dried powder (dissolved in normal saline at a corresponding dose) prepared by the corresponding strain in an amount of 0.2 mL/one/day, and the blank group and the model group are used as controls by the same amount of normal saline for gavage until the experiment is finished. All groups were free water and food intake.

Mice were weighed at

weeks

1 and 12 of the post-natal intervention, respectively, and the increase in body weight per mouse is shown in figure 1. As can be seen from fig. 1, the metazoan prepared by lactobacillus rhamnosus CCFM1219 significantly inhibited the weight gain of the high-fat diet mice when administered at a high dose, compared to the metazoan prepared by the model group, the HSY medium group, and other bacteria.

According to the experimental results, the metazoan prepared by the lactobacillus rhamnosus CCFM1219 has the effect of relieving the weight increase of the high-fat diet mice, and when the experiment is finished, compared with the average weight increase of a model group of 21.1g, the weight increase of the metazoan CCFM1219_2 group is only 12.26g, the weight increase of the metazoan CCFM1219_1 group is 18.2g, and the weight increase of other intervention groups is 19.14g with the least weight increase.

TABLE 1 groups of experimental animals

Example 4: influence of metazoan prepared by lactobacillus rhamnosus CCFM1219 on blood glucose homeostasis of high-fat diet mice 60 7-week-old healthy male C57BL/6N mice were randomly divided into 10 groups of 6 mice each, and the 10 groups were: blank group (Control), Model group (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 metagenome: CCFM1219_1 (low dose: 200mg/kg mouse body weight), CCFM1219_2 (high dose: 800mg/kg mouse body weight), Lactobacillus rhamnosus FTJDG metagenome: 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 took 13 weeks: after the mice are adapted for one week, a blank group is fed with low-fat and low-sugar feed, the other groups are fed with high-fat and low-sugar feed, the mice are gavaged by using the HSY culture medium freeze-dried powder (dissolved in normal saline at a corresponding dose) in an amount of 0.2 mL/one/day in the culture medium group from the second week, the mice are gavaged by using the metazoan freeze-dried powder (dissolved in normal saline at a corresponding dose) prepared by using corresponding strains in an amount of 0.2 mL/one/day in the metazoan group, and the blank group and the model group are used as controls by using normal saline with the same amount as the gavage of the blank group and the model group until the experiment is finished. All groups were free water and food intake.

The steady state of blood glucose in mice was evaluated by Oral Glucose Tolerance Test (OGTT) before the end of the experiment at week 13, after fasting for 12h, the mice were gavaged with glucose at a dose of 2mg/g body weight, blood was collected in the tail vein before gavage (0min) and 15, 30, 60, 90, 120min after gavage, respectively, and blood glucose was measured using Accu-Chek active blood glucose test strips.

As shown in FIG. 2, it is understood from FIG. 2 that the fasting glucose levels of the postnatal high dose administration group (fasting glucose values of 6.38mmol/L, respectively) prepared from Lactobacillus rhamnosus CCFM1219 were lower and the blood glucose was reduced to the fasting level at 2h compared with the model group (fasting glucose value of 9mmol/L), the medium group and the other postnatal groups (fasting glucose value of at least 8.1 mmol/L).

According to experimental results, the metazoan prepared by the lactobacillus rhamnosus CCFM1219 has the effects of improving the blood sugar stability of high-fat diet mice and increasing the oral glucose tolerance.

Example 5: effect of metazoans prepared from Lactobacillus rhamnosus CCFM1219 on liver fat accumulation and lesions of mice with high-fat diet

After the experiment, blood was taken and the mice were sacrificed, and liver tissues of the mice were taken for HE staining and oil red O staining, and the staining results are shown in fig. 3 and fig. 4.

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

Experimental results show that the metazoan prepared by the lactobacillus rhamnosus CCFM1219 effectively inhibits the liver fat accumulation and lesion of high-fat diet mice

Example 6: effect of metazoans prepared from Lactobacillus rhamnosus CCFM1219 on scapular brown fat and epididymal white fat in high-fat diet mice

The experiment took 13 weeks: after the mice are adapted for one week, a blank group is fed with low-fat and low-sugar feed, the other groups are fed with high-fat and low-sugar feed, from the second week, the mice are gavaged by using the HSY culture medium freeze-dried powder (dissolved in normal saline in a corresponding dose) in an amount of 0.2 mL/body/day in the culture medium group, the mice are gavaged by using the metazoan freeze-dried powder (dissolved in normal saline in a corresponding dose) prepared by using corresponding strains in an amount of 0.2 mL/body/day in the metazoan group, and the blank group and the model group are gavaged with equal amount of normal saline as a control until the experiment is finished. All groups were free water and food intake.

After the experiment, blood was taken and the mice were sacrificed, and scapular brown fat and epididymal white adipose tissue of the mice were taken for HE staining, and the staining results are shown in fig. 5 and 6.

As can be seen from FIGS. 5 and 6, when compared with the model group, the medium group and the other metazoan intervention group, the metazoan prepared by Lactobacillus rhamnosus CCFM1219 increases the proportion of small fat cells (diameter less than or equal to 50 μm) and decreases the proportion of large fat cells (diameter greater than 50 μm) when administered at a high dose, thereby effectively reducing the fat accumulation in the fat cells. The high-fat diet can change the shape and function of fat cells from brown to white, and the metaplast prepared by the lactobacillus rhamnosus CCFM1219 obviously improves the white adipose tissue lipid accumulation caused by the high-fat diet, reduces the lipid infiltration and invasion, and maintains the normal shape and function of the brown adipose tissue.

According to experimental results, the metazoan prepared by the lactobacillus rhamnosus CCFM1219 effectively reduces fat accumulation, maintains the normal form of brown fat and promotes browning of white fat.

Example 7: influence of metazoan prepared from Lactobacillus rhamnosus CCFM1219 on biochemical indexes of serum of high-fat diet mouse

60 healthy male C57BL/6N mice at 7 weeks of age were randomly assigned to 10 groups of 6 mice each, 10 groups were: blank group (Control), Model group (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 metagenome: CCFM1219_1 (low dose: 200mg/kg mouse body weight), CCFM1219_2 (high dose: 800mg/kg mouse body weight), Lactobacillus rhamnosus FTJDG metazoan group: 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).

Blood is got and the mouse is sacrificed after the experiment, with mouse whole blood centrifugation serum of getting, adopts full-automatic biochemical analyzer serum relevant index to detect, relevant index includes: TG (triglyceride), TC (total cholesterol), GGT (glutamyltranspeptidase), AST (aspartate aminotransferase), ALP (alkaline phosphatase). The results are shown in FIG. 7.

As can be seen from fig. 7, when compared with the model group, when the postnatal prepared from lactobacillus rhamnosus CCFM1219 is administered at a high dose, serum TC of the high-fat diet mice is reduced more significantly than that of the culture group and other postnatal intervention groups (20.05% lower than that of the model group and 6.68% lower than that of the culture group and other postnatal intervention groups at the highest), GGT (34% lower than that of the model group and 14% lower than that of the model group and that of the culture group and other postnatal intervention groups at the highest), ALP (36.45% lower than that of the model group and 13.63% lower than that of the culture group and other postnatal intervention groups at the highest), and TG, AST and TG content are also reduced.

According to experimental results, the metazoan prepared by the lactobacillus rhamnosus CCFM1219 effectively reduces the serum biochemical index level of a high-fat diet mouse related to obesity and fatty liver, and is beneficial to relieving the obesity and the fatty liver.

Example 8: the effect of each component of metazoan on the weight and tissue weight of mice on high-fat diet.

Cell lysate group: and (3) homogenizing the metazoan fermentation liquor obtained in the example 2 under high pressure, centrifuging to obtain a thallus lysate, and freeze-drying for later use.

And (3) fermentation liquor group: after the bacterial liquid of the embodiment 2 is subjected to heat treatment (65 ℃, 30min), the post-biogenic fermentation liquid is obtained, and the fermentation liquid is taken and freeze-dried to obtain post-biogenic powder for later use.

And (3) dead bacteria group: and (3) centrifuging the bacterial liquid obtained in the embodiment 2 to obtain bacterial sludge precipitate, 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 further use.

30 healthy male C57BL/6N mice at the age of 7 weeks were randomly divided into 6 groups of 5 mice each, 6 groups were: blank group (Control), Model group (Model), dead bacteria group, thallus lysate group, fermentation supernatant group and fermentation liquor group.

The experiment took 13 weeks: after the mice are adapted for one week, a blank group is fed with low-fat and low-sugar feed, the other groups are fed with high-fat and low-sugar feed, from the second week, a dried group is respectively filled with dead bacteria (the dose is 24mg/kg of the weight of the mice), thallus lysate (the dose is 24mg/kg of the weight of the mice), fermentation supernatant freeze-dried powder (the dose is 800mg/kg of the weight of the mice) and fermentation broth freeze-dried powder (the dose is 800mg/kg of the weight of the mice) in an amount of 0.2 mL/mouse/day, and physiological saline with the same amount as the stomach filling of the blank group and the model group is used as a control until the experiment is finished. All groups were free water and food intake.

Mice were weighed at the end of the acclimation period and before sacrifice, and after sacrifice, their livers and abdominal white fats were weighed, and the total increase in body weight and tissue weight for 12 weeks are shown in fig. 8. As can be seen from FIG. 8, compared to the model group, the metazoans prepared by various forms of Lactobacillus rhamnosus CCFM1219 were effective in inhibiting weight gain in high-fat diet mice, reducing the weight of white fat in liver and abdomen, wherein the fermentation supernatant was slightly less effective.

According to experimental results, each component of the metazoan prepared by the lactobacillus rhamnosus CCFM1219 has the effects of losing weight and relieving liver fat accumulation.

TABLE 2 groups of experimental animals

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

Preparing simulated gastric juice: pepsin (1: 10000) was dissolved in sterile physiological saline (pH3) to a final concentration of 3g/L, and the solution was filtered through a 0.22 μm sterile filter and used as it was.

Preparing simulated intestinal juice: trypsin (1: 250) was dissolved in sterile physiological saline (pH8) to a final concentration of 1g/L, and bile salt was added to a final concentration of 0.30% (w/v), and the solution was filtered through a 0.22 μm sterile filter and ready to use.

Collecting the equal and uniform bacterial suspension at the end of the index of the lactobacillus rhamnosus CCFM1219 in 7-mLEP tubes, centrifuging at 8000 Xg for 10min, removing the supernatant, and collecting the thallus. 1 tube of the cells was resuspended in 5mL of physiological saline (pH 7) and counted before treatment. And 3 tubes of the thallus are resuspended in simulated gastric juice with the same volume, mixed uniformly, cultured at 37 ℃ for 1h, 2h and 4h, and then counted. And 3 tubes of the thallus are resuspended in simulated intestinal fluid with the same volume, mixed uniformly, cultured at 37 ℃ for 1 hour, 2 hours and 4 hours, and then counted.

Strain survival rate (%) ═ (N)_t/N₀) 100% of the total weight; in the test: n is a radical of₀Number of viable bacteria before treatment of-0 h, N_tAnd the viable count after 1h, 2h and 4h of treatment. The results are shown in FIG. 9.

In fig. 9, it is shown that the survival rate of lactobacillus rhamnosus CCFM1219 is as high as 88.72% after treatment in a simulated gastric fluid environment with pH3 for 1h, 78.43% after treatment for 2h and 66.32% after 4 h; after the lactobacillus rhamnosus CCFM1219 is treated for 1 hour by simulated intestinal fluid, the survival rate is as high as 90.25%, the survival rate after 2 hours is 80.37%, and the survival rate after 4 hours is still higher than 65%, which indicates that the lactobacillus rhamnosus CCFM1219 has good acid and bile salt resistance.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 south of the Yangtze river

<120> metazoan for relieving fatty liver and obesity prepared by lactobacillus rhamnosus and application thereof

<130> BAA220234A

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1443

<212> DNA

<213> rhamnosus 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. A strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1219 is deposited at 23.1.2022 in Guangdong province of microorganism culture collection with the deposit number of GDMCC No.62231 and the deposit address of No. 59 great institute of Michelia Tokorea 100 in Guangzhou.

2. A composition comprising lactobacillus rhamnosus CCFM1219 according to claim 1 and/or an metazoan prepared from lactobacillus rhamnosus CCFM1219 according to claim 1.

3. The composition of claim 2, wherein the metazoan comprises dead cells, fermentation supernatant, bacterial lysate and/or fermentation broth.

4. The composition according to claim 3, wherein the fermentation broth is prepared by inoculating Lactobacillus rhamnosus CCFM1219 according to claim 1 into a fermentation medium, culturing to obtain a bacterial liquid, and performing heat treatment on the bacterial liquid to obtain a fermentation broth; the preparation method of the thallus lysate comprises the steps of homogenizing the fermentation liquor at 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 the bacterial liquid to obtain the dead cells; the fermentation supernatant is obtained by centrifuging the bacterial liquid or the fermentation liquid.

5. The composition of claim 2 or 3, wherein the metazoan is prepared as a powder or liquid formulation.

6. Use of the composition according to any one of claims 2 to 5 for the preparation of a product for the prevention and/or treatment of fatty liver and obesity related diseases.

7. The use according to claim 6, wherein the obesity-related disease comprises diabetes, musculoskeletal diseases, cardiovascular diseases, malignancies, hyperlipidemia, or metabolic syndrome; the fatty liver-related disease comprises alcoholic fatty liver, non-alcoholic fatty liver, liver inflammation, liver cirrhosis or liver cancer.

8. Use according to claim 6, wherein the product comprises a food or a pharmaceutical product.

9. A product for preventing and/or treating fatty liver and obesity related diseases, which comprises lactobacillus rhamnosus CCFM1219 of claim 1 or a composition of any of claims 2 to 5.

10. The product of claim 9, wherein the product comprises at least one of the following:

(1) significantly relieving the weight gain of mice on high-fat diet;