CN106389478B

CN106389478B - Application of bacteroides fragilis in treatment and/or prevention of obesity or diabetes

Info

Publication number: CN106389478B
Application number: CN201510459406.0A
Authority: CN
Inventors: 智发朝; 白杨; 王晔; 刘洋洋; 王从峰
Original assignee: Guangzhou Zhiyi Biotechnology Co Ltd
Current assignee: Guangzhou Zhiyi Biotechnology Co Ltd
Priority date: 2015-07-31
Filing date: 2015-07-31
Publication date: 2019-12-24
Anticipated expiration: 2035-07-31
Also published as: CN106389478A; WO2017020786A1

Abstract

The application of bacteroides fragilis in treatment and/or prevention of obesity or diabetes, in particular to the application of bacteroides fragilis in preparation of a pharmaceutical composition for treatment and/or prevention of obesity or diabetes, wherein the bacteroides fragilis is bacteroides fragilis ZY-312, the bacteroides fragilis ZY-312 is preserved in the China general microbiological culture collection center on 4-2 days 2015, and the preservation number is CGMCC No. 10685. The bacteroides fragilis has a good effect on treating and/or preventing obesity or diabetes, does not generate drug resistance, is safe and non-toxic, does not contain enterotoxin gene bft, has the probiotic characteristics of cholate resistance, gastric acid resistance and the like compared with the existing strains, and has wide application prospect.

Description

Application of bacteroides fragilis in treatment and/or prevention of obesity or diabetes

Technical Field

The invention relates to the technical field of microorganisms, medicines and daily chemical products, in particular to application of bacteroides fragilis in treatment and/or prevention of obesity or diabetes.

Background

Diabetes Mellitus (Diabetes mellitis) is a metabolic disease of multiple etiologies, and is a disease caused by a generalized metabolic disorder due to a defect in insulin secretion or action. Diabetes is a disease characterized by persistent chronic hyperglycemia, and also causes metabolic disorders such as protein, fat, water, electrolytes, etc. in the body.

The etiology of diabetes is currently divided into two aspects, genetic factors and environmental factors. Genetic factors: there is significant genetic heterogeneity in both type 1 and type 2 diabetes. Diabetes has a family incidence tendency, and 1/4-1/2 patients have a family history of diabetes. Clinically, at least 60 genetic syndromes may be accompanied by diabetes. Type 1 diabetes has multiple DNA sites involved in the pathogenesis, wherein the DQ site polymorphism in HLA antigen gene is the most closely related. A variety of well-defined genetic mutations have been found in type 2 diabetes, such as the insulin gene, the insulin receptor gene, the glucokinase gene, the mitochondrial gene, and the like. Environmental factors: obesity caused by excessive food intake and reduced physical activity is the most important environmental factor of type 2 diabetes, and makes individuals with genetic susceptibility to type 2 diabetes easily develop the disease. Patients with type 1 diabetes have abnormal immune systems, which cause autoimmune reactions after infection with certain viruses such as coxsackie virus, rubella virus, mumps virus, etc., and destroy insulin beta cells.

Diabetes has become the third largest non-infectious disease following cardiovascular disease and tumor, and the World Health Organization (WHO) predicts: in 2030, diabetics will be more than 3.6 billion worldwide, with type 2 diabetes accounting for more than 90%. Type 2 diabetes is non-insulin dependent diabetes mellitus and is a common disease with an increasing incidence of disease year by year.

Clinically diabetes is mainly divided into two categories:

the first type: insulin-dependent diabetes mellitus (IDDM), which has typically had an onset age below 30 years, has been referred to in the past as "juvenile onset diabetes" and may occur at virtually any age. Type i diabetes is an Autoimmune Disease (Autoimmune Disease) in which the beta cells of islets of langerhans (islets of langerhans) of the pancreas are destroyed by the Autoimmune system. The causes are related to genetic inheritance of individuals, viral infection in environmental factors, destruction of beta cells of pancreas by toxic substances, formation of antibodies against beta cells by autoimmunity, and attack of beta cells by cellular immune action. Finally, the pancreas of the patient cannot normally secrete insulin, so ketoacidosis is very easy to occur, and insulin needs to be injected for treatment.

The second type: non-insulin dependent diabetes mellitus (NIDDM) mostly occurs after the age of 40, patients are mostly obese, which are called adult type diabetes in the past, but it may also occur in young people, and the familial onset of the diabetes is more common, and the diabetes accounts for more than 95% of the total population of diabetes in Taiwan. This type of diabetes is caused by defective insulin secretion, and insulin resistance (insulin resistance); although some patients have reduced insulin secretion, most patients have sufficient insulin secretion capacity, and therefore, most patients rely on diet control and oral hypoglycemic drugs to control blood glucose without immediate insulin therapy. In addition, most patients are associated with symptoms of insulin resistance (insulin resistance). The formation of insulin resistance is mainly caused by excessive secretion of insulin (hyperinsulinamia) by beta cells of islets of langerhans of the pancreas, which results in a decrease in insulin sensitivity (insulin sensitivity) of peripheral tissues such as skeletal muscle, adipose tissue and liver. Thus reducing the utilization rate of glucose by tissues and causing the phenomenon of hyperglycemia. This type of disease progresses slowly and therefore, there are no typical symptoms of diabetes in the early stages and is not readily detectable. But often accompanied by chronic complications such as diabetic macroangiopathy (such as myocardial infarction and cerebral apoplexy) and small angiopathy (such as kidney, eye net membrane and neuropathy).

In addition, abnormal lipid metabolism is often associated with type II diabetes patients, such as increased plasma Triglyceride (TG) concentration, decreased high density lipoprotein cholesterol (HDL-C) concentration, and increased low density lipoprotein cholesterol (LDL-C) concentration. These symptoms can lead to a risk of cardiovascular disease in patients with type II diabetes. According to the research, the liver blood lipid value clearing capability of serious diabetes patients is reduced. When triglyceride and low-density lipoprotein cholesterol in the liver are accumulated continuously, liver cells are diseased to form non-alcoholic fatty liver, and the liver function is seriously affected.

The current methods for treating diabetes are divided into non-drug therapy and drug therapy, in addition to the administration of insulin. In the non-drug treatment aspect, the treatment is mainly carried out by means of diet regulation and exercise. In terms of drug therapy, the main purposes are to increase insufficient insulin, regulate hyperglycemia after eating, improve insulin resistance and the like. The drugs currently used for the treatment of diabetes can be divided into:

(1) sulfonyl ureas (Sulfonylurea): the main action mechanism of the medicine is to promote the secretion of insulin in pancreas, in particular to strengthen the action of insulin released by pancreas beta cells to the stimulation of glucose; commonly used sulfonyl urea hypoglycemic agents are Youkang (glibenclamide, trade name: euglucon), pyrithia (glipizide, trade name: minitab) and daimikrolon (gliclazide, trade name: diamicron). However, in addition to the side effects such as rash and itching which have been found, the subjects to which such drugs are administered are limited, and such hypoglycemic drugs are not suitable for patients with severe liver and kidney dysfunction, pregnant women and mammals, and patients who are severely allergic to sulfonylurea drugs.

(2) Inhibitors of alpha-Glucosidase (alpha-Glucosidase): the main action mechanism of the medicines is to inhibit the activity of pancreatic alpha-amylase (alpha-amylase) and intestinal alpha-glucosidase (alpha-glucosidase), and further inhibit the decomposition and absorption of carbohydrate in the intestinal tract, and the medicines can effectively reduce the blood sugar and the insulin concentration after meals, but have side effects of abdominal distension or occasional diarrhea, abdominal pain and nausea.

(3) Thiazolidinedione (Thiazolidinedione) derivatives: the medicines mainly have the effects of increasing the activity of peroxisome proliferator-activated receptor-gamma (PPAR) -gamma in a cell nucleus, further strengthening the action of insulin, increasing glucose transfer proteins GLUT2 and GLUT4 in the cell and conveying glucose into the cell for utilization. Troglitazone (trade name: rezulin), rosiglitazone (trade name: avandia), pioglitazone (trade name: actos), and the like are commonly used clinically; however, it is noteworthy that troglitazone (troglitazone) caused fatal hepatotoxicity and was therefore banned two months after marketing in the uk. In addition, Thiazolidinedione (Thiazolidinedione) derivatives have also been extensively recovered and banned by the United states directive.

(4) Biguanides (Biguanides): biguanide drugs are derivatives of guanidine (guanidine), and currently, biguanide hypoglycemic drugs are mainly metoformin. The medicine does not stimulate the secretion of insulin, and the action mechanism of controlling blood sugar is as follows: a. suppressing appetite, so preferentially used in obese type II diabetics, reducing food intake and weight to improve insulin action, b delaying glucose absorption in intestinal tracts, c promoting anaerobic decomposition of glucose in intestinal tracts, further increasing utilization of glucose in intestinal tracts, but possibly generating excessive lactate (lactate) which is easy to cause lactic acidosis, d strengthening action of insulin in liver, thus suppressing glucose neogenesis of liver, reducing release of glucose from liver, e promoting glucose transfer protein GLUT4 stored in cells to participate in delivery work on cell surfaces, and obviously increasing the amount of glucose transfer protein on cell surfaces. In addition, side effects of the hypoglycemic drugs, such as gastrointestinal discomfort, anorexia, nausea, vomiting or diarrhea and the like, can occur after the hypoglycemic drugs are taken for the first time, skin rash can occur in a small number of people, and inactivation can occur after the hypoglycemic drugs are taken for a long time.

As described above, there have been many studies and efforts to develop drugs for treating or preventing obesity and diabetes to date, but the results have been unsatisfactory. Many of the above chemicals have been developed as drugs for treating obesity and diabetes, but all have side effects. These drugs cause loss of body fat along with valuable proteins. As a result, none of the drugs are capable of treating or inhibiting obesity and diabetes from the source alone.

Bacteroides fragilis (Bacteroides fragilis) is an obligate anaerobic bacterium which is gram-negative, rod-shaped, blunt and densely stained at both ends, capsular, spore-free and unpowered, and is classified into enterotoxigenic type and non-enterotoxigenic type. Bacteroides fragilis, as part of the normal flora of the human and animal intestinal tract, is mainly present in the colon, and furthermore, colonizes and grows in the respiratory, gastrointestinal and genitourinary tracts. Currently, bacteroides fragilis is widely studied as a conditional pathogen, when host mucosa is damaged, the bacteroides fragilis can invade submucosa to cause infection, and can cause suppurative infection of organs of a body such as intestinal tract, abdominal cavity, liver, lung and brain tissues and accompanied with abscess and symptoms such as acute and chronic diarrhea through blood flow; in addition, bacteroides fragilis also has promoting effect on the occurrence of colon cancer and rectal cancer.

A great deal of research has been conducted on Bacteroides fragilis in the art. For example, a bacteroides strain BF839 is isolated from the intestinal tract of a well-developed infant or a young animal, and can increase the growth and development of children after being prepared into a live bacterial preparation, and has a good curative effect on prevention and treatment of acute and chronic enteritis, dysbacteriosis, upper respiratory infection, neurosis and the like (see the Chinese patent application with the application number of 90102847.9 and the name of 'a beneficial strain and application thereof'; Zhang Ji et al. clinical application research of bacteroides fragilis (BF839) bacterial solution, journal of Chinese biological sciences, 1995, volume 8, phase 2, pages 63-65)).

As another example, application No. 201310095126.7 entitled "bacteroides fragilis with probiotic properties"; application No. 201310085744.3 entitled "use of bacteroides fragilis for the preparation of a composition for the treatment of acute radiation enteritis"; the bacteroides fragilis disclosed in the Chinese invention patent application with the application number of '201310085716.1' and the name of 'application of bacteroides fragilis in preparation of a composition for treating inflammatory bowel disease' is a bacteroides fragilis strain (with the preservation number of CGMCC NO.7280) with probiotic properties separated from infant feces in 2012 and can be used for treating inflammatory bowel disease, diarrhea and the like. In addition, the bacterial morphology, culture characteristics and physiological and biochemical reaction results of the strain (Bd312) are similar to those of Bacteroides fragilis through further identification of the strain, the homology of the isolated strain and the standard strain ATCC25285 of Bacteroides fragilis reaches 99% through BLASTN sequence alignment, and drug sensitive experiments indicate that the strain Bd312 is insensitive to cefradine, amoxicillin, gentamicin, sulfamethoxazole and trimethoprim, and acute and chronic toxicity tests indicate no toxicity (Liuyangyang, and the like. the isolation and identification of nontoxic Bacteroides fragilis in healthy infants. China medical journal, 2014, volume 94, stage 30, page 2372-2374). However, there is currently no literature on the use of bacteroides fragilis for the treatment of obesity and diabetes and its complications.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a bacteroides fragilis strain and application thereof in treating and/or preventing obesity or diabetes.

In order to achieve the above object, the present invention provides the use of bacteroides fragilis for the treatment and/or prevention of obesity or diabetes.

The application of the Bacteroides fragilis is that the Bacteroides fragilis ZY-312 has a preservation number of CGMCC No. 10685.

In order to better achieve the above objects, the present invention also provides a pharmaceutical composition for treating and/or preventing obesity or diabetes, wherein the pharmaceutical composition comprises a pharmaceutically effective dose of bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and a pharmaceutically acceptable carrier.

The pharmaceutical composition is a tablet, a capsule, an oral liquid or a freeze-dried powder.

The pharmaceutical composition of the above, wherein the pharmaceutically acceptable carrier is one or more of skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

The invention has the technical effects that:

experiments prove that the bacteroides fragilis ZY-312 has a good effect on treating and/or preventing obesity or diabetes, does not generate drug resistance, is safe and non-toxic, and provides a new choice for treating and/or preventing obesity or diabetes. In addition, the bacteroides fragilis ZY-312 is a new bacteroides fragilis strain which is obtained by separating and purifying the feces of well-developed infants and has probiotic characteristics, and experiments show that the separated bacteroides fragilis ZY-312 does not contain enterotoxin gene bft (bacteroides fragiliss toxin), and compared with the existing strains, the bacteroides fragilis ZY-312 has the probiotic characteristics of bile salt resistance, gastric acid resistance and the like, and has wide application prospects.

The invention is described in detail below with reference to the drawings and specific examples, but the invention is not limited thereto.

Drawings

FIG. 1 is a diagram showing the morphology of a colony of Bacteroides fragilis ZY-312 of the present invention after anaerobic culture;

FIG. 2 is a gram-stained microscopic image (1000X) of Bacteroides fragilis ZY-312 of the present invention.

FIG. 3 is a scanning electron micrograph of Bacteroides fragilis ZY-312 of the present invention (30000X).

FIG. 4 is a comparison of the results of gel electrophoresis of PCR products of the present invention;

FIG. 5 is a comparison of the results of gel electrophoresis of PCR products of the present invention;

FIG. 6 is a phylogenetic tree constructed from whole genome sequence comparisons;

FIG. 7 is a graph comparing the mean blood glucose changes of mice during the test of the present invention;

FIG. 8 is a graph comparing the 1 st week glucose tolerance test of the present invention;

FIG. 9 is a graph comparing the 7 th week sugar tolerance test of the present invention;

FIG. 10 is a graph comparing the change in body weight of mice during the test of the present invention.

The Bacteroides fragilis (Bacteroides fragilis) is named as ZY-312, is preserved in the China general microbiological culture collection center (CGMCC) 4 and 2 days 2015, has the preservation number of CGMCC No.10685, and has the preservation address of No. 3 Hospital No.1 of North Chen West Lu of the sunward area in Beijing.

Detailed Description

The invention will be described in detail with reference to the following drawings, which are provided for illustration purposes and the like:

embodiments of the invention include: the invention screens a large amount of feces from healthy infants to screen a large amount of bacteroides fragilis strains, discovers a new bacteroides fragilis (bacteroides fragilis) through physicochemical experimental identification, is named ZY-312, is proved to be free of enterotoxin gene bft (bacteroides fragilis toxin), is an avirulent strain, and is discovered through fermentation culture, staining microscopy, physiological and biochemical characteristic analysis and animal experiments: compared with the existing bacteroides fragilis strains, the bacteroides fragilis strains have outstanding probiotic characteristics such as bile salt resistance and gastric acid resistance, can make up for some defects of the original probiotics, still maintain higher biological activity under the conditions of bile salt content and acidity of the digestive tract, can effectively overcome the defects that the existing bacteroides fragilis is easy to inactivate in the digestive tract and the like, can effectively treat and/or prevent obesity or diabetes, and are preferred strains of probiotic products. The Bacteroides fragilis (Bacteroides fragilis) ZY-312 is preserved in China general microbiological culture Collection center (CGMCC) 4.22015, the preservation number is CGMCC No.10685, and the preservation address is Beijing Shangyang Beichen Xilu No.1 Hospital No. 3.

The Bacteroides fragilis ZY-312 can be prepared into pharmaceutical composition. The pharmaceutical composition comprises pharmaceutically effective dose of Bacteroides fragilis ZY-312 and pharmaceutically acceptable carrier, wherein the preservation number of the Bacteroides fragilis ZY-312 is CGMCC No. 10685. The pharmaceutical composition is a tablet, a capsule, an oral liquid or a freeze-dried powder. The pharmaceutically acceptable carrier is one or more of skimmed milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

The present invention will be further described with reference to the following specific examples. It is to be noted that all dosage forms within the scope of the present invention have been tested, and hereinafter, only for illustration, only a few of which are described in the examples, but should not be construed as limiting the present invention, since bacteroides fragilis for treating and/or preventing obesity or diabetes or a pharmaceutical composition comprising bacteroides fragilis of the present invention can be applied to the indications and exhibit the functions described above after administration to a subject.

Unless otherwise specified, the reagents used in the present invention are commercially available.

Example 1

Isolation and purification of Bacteroides fragilis ZY-312

Reagent and apparatus

(1) Culture medium A: an improved formula is added on the basis of Bacteroides-bile-esculin (BBE) agar (Qingdao Haibo Biotech limited, Cat: HB7028), and the specific components are as follows:

TABLE 1 culture and Components

(2) And (3) a culture medium B: the improved formula is added on the basis of Bacteroides-bile-esculin (BBE) agar (Qingdao Haibo Biotech limited, Cat: HB7028), and the specific components are as follows:

TABLE 2 culture and Components

(3) And (3) a culture medium C: fetal bovine serum was added to Brookfield broth (Qingdao Haibo Biotechnology Co., Ltd., product No. HB0241) in an amount of 5% (v/v) (Zhejiang Hangzhou Biotechnology Co., Ltd., brand: Sijiqing, product No. HB 0205).

(4) Laboratory apparatus

2.5L sealed culture pot (Mitsubishi gas chemical Co., Ltd., C-31)

Constant temperature incubator (Shanghai-Heng scientific instruments Co., Ltd., type: DHP-9082)

Microscope (Nikon instruments (Shanghai) Co., Ltd., model: E100)

PCR instrument (Saimer Feishale science and technology company, model: Applied)PCR System 9700)

Electrophoresis apparatus (model: DYCP-32B of six instruments of Beijing City)

(5) Reagent

Anaerobic gas bag (Mitsubishi gas chemical company, Commodity number: C-1)

Bacterial DNA extraction Kit (Bacterial DNA Kit) OMEGA, cat # D3350-01)

Taq enzyme (Bio-engineering (Dalian) Ltd., Cat. No.: DR100A)

Agarose (Brand: Biowest, cat # 91622)

Sulfamethoxazole (Sigma, cat # S7507-10G)

Trimethoprim (Sigma, cat # T7883-5G)

Vitamin K1 (Qingdao Nishui Biotech Co., Ltd., product number: 21005)

DL1000 DNA Marker (bioengineering (Dalian) Co., Ltd., product number: D526A)

Bacteroides fragilis enterotoxigenic strain (provided by Sunyong teacher of digestive department of southern Hospital, isolated from patients with clinical diarrhea)

Bacteroides fragilis standard strain ATCC25285 (purchased from Guangdong institute for microorganisms)

Bacteroides fragilis strain Bd312 (preservation number CGMCC No.7280, provided by Guangzhou photobioscience Co., Ltd.)

BF839 strain (isolated from totem probiotic).

(6) Media preparation

Preparation of a culture medium A: weighing 61.5 g of BBE culture medium, heating and dissolving in 1000mL of distilled water, autoclaving at 121 ℃ for 15 minutes, cooling to about 50 ℃, adding 1g of sulfamethoxazole, 4g of trimethoprim and 50mL of sterile defibrinated goat blood, mixing uniformly, and pouring into a sterile plate for later use.

Preparation of a culture medium B: weighing 61.5 g of BBE culture medium, heating and dissolving in 1000mL of distilled water, autoclaving at 121 ℃ for 15 minutes, cooling to about 50 ℃, adding 1g of sulfamethoxazole and 4g of trimethoprim which are subjected to filtration sterilization, mixing uniformly, and pouring into a sterile plate for later use.

Preparation of a culture medium C: weighing 28.1g of Brookfield broth, heating and stirring to dissolve in 1000mL of distilled water, subpackaging in a triangular flask, and autoclaving at 121 ℃ for 15 minutes for later use. Before use, 5% fetal bovine serum was added.

Preparation of Brucella broth: weighing 28.1g of Brookfield broth, heating and stirring to dissolve in 1000mL of distilled water, subpackaging in a triangular flask, and autoclaving at 121 ℃ for 15 minutes for later use.

The method comprises the following steps:

1. separating and purifying

0.5g of fresh baby feces was taken and placed in a flask containing 4.5mL of Brookfield broth, and shaken for 1 minute. 0.1mL of the culture broth was dropped onto a Brookfield culture medium, streaked, and then placed in an anaerobic jar, and cultured at 37 ℃ for 48 hours. Typical colonies were picked on liquid medium for 24h and gram stained. Observing the shape under a microscope, selecting a gram-negative bacterium solution, streaking and inoculating the gram-negative bacterium solution on a blood plate, and carrying out anaerobic culture for 48 hours. And judging whether the bacteria are purified or not according to the morphological characteristics of the colonies on the plate and the staining characteristics, size, club shape and distribution condition of the bacteria observed under a mirror. If the bacteria are not pure, the steps are continued, and the separation and the passage are repeated for a plurality of times until the purified bacterial strain is obtained.

2. Characteristics of bacterial colony

After the bacteroides fragilis ZY-312 is cultured on a blood plate for 48 hours, the bacteroides fragilis ZY-312 presents a round and slightly convex shape, is semitransparent and white, has a smooth surface and is not hemolyzed, and the diameter of a colony is 1-3mm, as shown in figure 1.

3. Microscopic form

Gram-stained bacteroides fragilis ZY-312 was used as gram-negative bacteria, and was typically rod-shaped, with blunt and densely stained ends, and non-staining areas in the middle of the cells, such as vacuoles, as shown in FIG. 2.

4. Morphology under electron microscope

Fixing the fixing solution, and observing by a scanning electron microscope. Under the microscope, the size of the bacteroides fragilis ZY-312 is 0.5-0.8 multiplied by 1-4.5 μm, and the bacteroides fragilis ZY-312 has no flagella and spores, and is shown in figure 3.

5. Biochemical identification

The results of biochemical identification are shown in Table 3 below (in the table, + represents positive, -represents negative)

TABLE 3 Biochemical identification results

The results of the physiological and biochemical reactions of API20A (Biochemical reaction assay plate, Meirieo GmbH, a biological organism of France) showed that: bacteroides fragilis ZY-312 can ferment glucose, lactose, sucrose, maltose, xylose, esculetin, mannose, and raffinose, and is in accordance with the characteristics of Bacteroides fragilis.

Example 2

Identification of Bacteroides fragilis ZY-312

The Polymerase Chain Reaction (PCR) primers (synthesized by England Weiji (Shanghai) trade, Inc.) were of the following sequence:

primer pair 1:

a forward primer: 5'-ACGCTTGCACCCTCCGTATTA-3'

Reverse primer: 5'-GCTTAGAGTTTGATCCTGGCTCAG-3'

And (3) primer pair 2:

a forward primer: 5'-TGGGTGGTTGCTGCCTGGACACA-3'

Reverse primer: 5'-CATCCGGGTATGGATATGAA-3'

And (3) primer pair:

a forward primer: 5'-GATGCTCCAGTTACAGCTTCCATTG-3'

Reverse primer: 5'-CGCCCAGTATATGACCTAGTTCGTG-3'

bft gene primer set:

a forward primer: 5'-GACGGTGTATGTGATTTGTCTGAGAGA-3'

Reverse primer: 5'-ATCCCTAAGATTTATTATCCCAAGTA-3'

1. PCR identification (PCR, i.e., polymerase chain reaction, is a commonly used method for rapid amplification of genes)

(1)16S rRNA sequencing

Inoculating the strain to culture medium A, and anaerobically culturing at 37 deg.C for 48 hr. Inoculating single strain into liquid culture medium, and anaerobically culturing at 37 deg.C for 48 hr. The DNA extraction kit extracts bacterial DNA (Tiangen Biochemical technology (Beijing) Ltd., product number: DP302-02) as PCR template DNA.

Amplification of 16S rRNA gene sequence: the size of the amplified fragment of the primer pair 1 is about 531 bp; the size of the amplified fragment of the primer pair 2 is 518 bp; the amplified fragment of primer pair 3 is about 970bp in size.

20 μ L of PCR reaction system was used: 10 mu L of Taq enzyme, 2 mu L of template DNA, 1 mu L of forward and reverse primers respectively and 6 mu L of sterile deionized water.

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, and extension at 72 ℃ for 10 min.

The PCR product was electrophoresed in 2% agarose gel under 100V for 15 min.

The result of gel electrophoresis of the PCR product is shown in FIG. 4, wherein lanes 1 and 2 are the amplification products of primer pair 1 and primer pair 2, respectively; 4. lanes 5 are primer pair 1 and primer pair 2 amplification products (results of repeated PCR), respectively; 3. lane 6 is the amplification product of primer pair 3; lane 7 is a DNA molecular weight standard (DL1000 DNA marker). The size of the product of the separated strain DNA after the PCR amplification by the primer pair 1 is 531bp, the size of the product after the PCR amplification by the primer pair 2 is 518bp, the size of the product after the PCR amplification by the primer pair 3 is 970bp, and the separated strain is Bacteroides fragilis according to the expectation.

The PCR products were subjected to nucleotide sequence determination (determined by Shenzhen Huada Gene science and technology Co., Ltd.). Sequencing results BLAST alignments (http:// www.ncbi.nlm.nih.gov/BLAST /) were performed on Genbank (DNA sequence database established by the national center for Biotechnology information, USA), see Table 4.

The result shows that the bacteroides fragilis is separated.

TABLE 416S rRNA sequences BLAST alignment results (partial)

(2) PCR detection of bft Gene

The strains screened by sequencing were inoculated into medium C and anaerobically cultured at 37 ℃ for 48 hours. 2mL of the culture solution is taken, and DNA is extracted by using a bacterial DNA extraction kit to be used as PCR template DNA. The amplification of the bft gene adopts bft gene primers, and the size of an amplified fragment is 294 bp.

20 μ L of PCR reaction system was used: 10 mu L of Taq enzyme, 2 mu L of template DNA, 1 mu L of upper primer and lower primer respectively and 6 mu L of sterile deionized water.

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45s, for 30 cycles, and extension at 72 ℃ for 10 min. The PCR product was subjected to 2% agarose gel electrophoresis under 100V for 15 min.

The results are shown in FIG. 5, in which lanes 1, 2, 3 and 4 are the electrophoresis results of ZY-312 isolate; 5. 6 and 7 are electrophoresis results of enterotoxigenic bacteroides fragilis; lane 8 is DL1000 DNA marker. 4. Lane 5 is the amplification product of the bft gene primer pair; 1. lane 7 is the amplification product of primer pair 2; 2. 6 is the amplification product of the primer pair 1; lane 3 is the amplification product of primer pair 3.

The result shows that ZY-312 is a bacteroides fragilis, does not contain enterotoxin bft gene, and is a new avirulent strain.

2. Whole genome sequencing analysis and identification

The Bacteroides fragilis ZY-312 was subjected to whole genome sequencing (Shenzhen Huada Gene science and technology Co., Ltd.), the sequencing results were compared with the published strain sequences, and an NJ-tree was constructed by using a neighbor joining method using treebest software (http:// treeboft. svn. sourceform. net/viewrc/treeboft/. can be freely downloaded on a sourceform website) or a maximum likelihood tree was constructed by using software PhyML using a maximum likelihood method. Phylogenetic tree shows (see FIG. 6) that Bacteroides fragilis ZY-312 is in the same branch as Bacteroides fragilis standard strain ATCC25285 (i.e. NCTC9343), indicating that Bacteroides fragilis ZY-312 is a new strain of Bacteroides fragilis, homologous to ATCC 25285.

And carrying out virulence gene analysis on the whole genome sequencing result to verify whether the whole genome sequencing result contains the toxigenic bft gene. The result shows that the bacteroides fragilis ZY-312 does not contain bft gene in the whole genome and is a new bacteroides fragilis which does not produce enterotoxin.

Example 3

Tolerance of bacteroides fragilis ZY-312 to gastric acid

1. Artificial gastric juice preparation (according to 2010 'Chinese pharmacopoeia' artificial gastric juice preparation method)

23.4mL of concentrated HCl was dissolved in 100mL of purified water to obtain dilute hydrochloric acid. Taking 8.2mL of diluted hydrochloric acid, adding 400mL of purified water and 5g of pepsin (Porcine source, 1: 15000) The volume is up to 500 mL. Magnetically stirring overnight at 37 deg.C to obtain artificial gastric juice.

2. Preparation of cells

Collecting bacteria liquid, centrifuging, discarding supernatant, resuspending with normal saline, centrifuging again, discarding supernatant, and keeping thallus for use.

3. Adding artificial gastric juice to determine viable count

Adding artificial gastric juice into the thallus, resuspending, and measuring viable count for 0, 1, 2 and 3h respectively.

Viable bacteria count adopts a 10-time serial dilution method: mu.L of the broth was added to 900. mu.L of Brookfield broth and gradually diluted in gradient to the appropriate concentration. There were 4 concentration gradients per plate spot, and 3 replicates of each gradient were spotted, 20 μ L per spot. Anaerobic culture was carried out at 37 ℃ for 48 hours, and the number of colonies was counted (counting by taking a concentration gradient of 3-30).

Viable count (CFU/mL) three spotted colony totals/3 × 50 × dilution

TABLE 5 gastric acid tolerance test results for different strains (data are log values of viable bacteria concentration, h is hour)

Probiotics must enter the gastrointestinal tract of the body to reach a certain concentration before they can exert their function. From the mouth to the intestine, the probiotic bacteria must first pass through the stomach in a viable state before entry into the intestine is possible. The time for food (especially fluid) to pass through the stomach is typically 1-2 hours. According to different dietary structures, the pH value of gastric juice in human body greatly fluctuates, usually about pH3.0, and can reach pH1.5 in the case of empty stomach or acidic food, and can reach pH 4-5 in the case of alkaline food, and the acidic environment of gastric juice can activate pepsinogen, thereby killing bacteria entering stomach along with food. Probiotics must have a certain resistance to acids and pepsin if they are to exert their probiotic effect in the human body.

The results show (Table 5), compared with other strains of Bacteroides fragilis, the viable bacteria concentration of the Bacteroides fragilis ZY-312 is still higher after 3h, and the viable bacteria concentration of other strains is reduced rapidly along with time, which indicates that ZY-312 has good gastric acid tolerance, and has good probiotic potential and application prospect.

Example 4

Bacteroides fragilis ZY-312 test for tolerance to bile salts

1. Experimental Material

Tryptone soy broth (TSB for short, brand: OXOID, cat # CM0129B)

Tryptone soy agar (TSA, brand: OXOID, cat # CM0131B)

Ox gall powder (bioengineering Shanghai (stock) Co., Ltd., product number: ON1210)

Fetal bovine serum (U.S. MP Biomedicals corporation, cat # 2916754)

2. Preparation of strains and reagents

And (3) bile powder solution: the three final concentrations, 10g/L (1% of ox gall powder), 20g/L (2% of ox gall powder) and 40g/L (4% of ox gall powder), were set by adding ox gall powder to TSB. After sterilization, serum (final concentration 50mL/L) was added for use. Meanwhile, TSB without bile powder was used as a control.

Culturing and collecting strains: the strains (ZY-312, Bd312, BF839 and ATCC25285) were subjected to anaerobic liquid static culture at 37 ℃ until late logarithmic growth (about 14 to 16 hours), and then they were dispensed into centrifuge tubes, each of which was filled with 3ml of a bacterial solution, and centrifuged at 4000rpm at room temperature for 5 minutes. The cells were washed with 0.01M PBS 1 time (centrifuged at 4000rpm for 5 minutes at room temperature), the supernatant was discarded and the pellet was used.

3. Culturing in artificial bile powder culture medium

Resuspending the washed bacteria with the above bile powder solution, and adding bile powderAdjusting the initial bacterial liquid concentration to 1 × 10⁸CFU/mL. And anaerobically cultured at 37 ℃ for 1, 2, 4 hours, plated to count the change of viable bacteria number, and the bacteria number at 0 hour is used as a control. The experiment was done 3 times in parallel.

4. Calculating the tolerant condition of the bacteria

And (3) comparing the plate coating results of the three time points with the corresponding 0 hour results to obtain the results of the bacterial strains which can tolerate the bile powder after acting in the artificial bile powder solution for different time, wherein the results are described by mean +/-standard deviation and statistical results.

TABLE 6 result of gallbladder powder-resistant experiment of SK08 strain (n ═ 3)

The results are shown in Table 6, and the observation of 0-4h shows that ZY-312 can normally grow in the bile powder with the concentration of 1%, 2% and 4%, and the viable count of ZY-312 is obviously higher than that of other bacterial strain groups as the concentration of the bile powder is increased. The results show that ZY-312 is tolerant to bile salts and is significantly superior to other strains.

Bile salt is sodium salt or potassium salt formed by combining bile acid secreted by liver cells with glycine or taurine, and is a main component of bile participating in digestion and absorption. After bile salts are excreted to small intestine, most of the bile salts are absorbed into blood by small intestine mucous membrane and then enter liver to form bile. The mass concentration of bile salts in the small intestine of a human body fluctuates within the range of 0.03-0.3 g/100 mL.

In the case of living cells, bile salts can disrupt the cell membrane, and thus tolerance to bile salts is one of the important indicators for the evaluation of probiotics. The probiotic bacteria produce bile salt hydrolase which catalyzes the hydrolysis of glycine and taurine-bound bile salts to amino acid residues and free bile salts. The strain with bile salt dissociation capability can reduce serum cholesterol level of hypercholesterolemia population and prevent hypercholesterolemia of normal people. The concentration of bile salts in the digestive tract is not constant, the mass concentration of bile salts is 15-20 g/L at the beginning of 1h of food intake digestion, and then the mass concentration is reduced to about 3 g/L. The probiotic bacteria must survive the passage through the gastrointestinal tract at normal bile salt concentrations, and must be resistant to inhibition by bile salts, e.g., for colonization in the small intestine. Therefore, compared with other strains of Bacteroides fragilis, ZY-312 has better application prospect.

Example 5

Bacteroides fragilis ZY-312 action on obesity and diabetes mice

Experimental Material

db/db mouse (Nanjing Elmait Biotech Co., Ltd.), glucose (1.6g/Kg), SPF-grade pellet feed (Guangdong province center for laboratory animals), glucose meter and test paper (Sannuo biosensing Co., Ltd.).

db/db mice are derived from the C57BL/KsJ inbred strain autosomal recessive inheritance, and belong to the type II diabetes mellitus model. Animals begin to bulimia and become obese one month later, and consequently produce hyperglycemia, hyperinsulinemia, and elevated glucagon. Death typically occurs within 10 months. db/db (C57BL/KsJ) mice are characterized in that the Leptin receptor point mutation causes Leptin signal pathway disorder, thereby causing the mice to have symptoms of obesity, insulin resistance, hyperglycemia, fatty liver and the like. The db/db mouse can obviously increase obesity and fasting blood sugar 6 weeks after birth, increase water intake and urine intake, and is most obvious at 8-12 weeks, and can also have complications such as diabetic nephropathy. db/db mice are often used as research models for obesity, type 2 diabetes, and fatty liver.

B, B.fragilis ZY-312 fermentation culture: activated ZY-312 was inoculated into trypticase soy broth and cultured anaerobically at 37 ℃ for 12 h. The application method of the Bacteroides fragilis ZY-312 comprises the following steps: freeze-drying and pulverizing the fermented thalli to obtain bacterial powder, dissolving the bacterial powder in PBS according to the amount before intragastric administration, and intragastric administration to mice.

Experiment grouping

The db/db mice were randomly divided into 4 groups of 10 mice each, 4 groups being a control group, a low dose group, a medium dose group, and a high dose group, respectively.

Experimental procedures and results

The stomach of db/db mice is irrigated for 2 times in the morning and at the evening, the mice are irrigated for 7 weeks continuously, and the food intake and the body weight are measured every day. The gavage dose design for each group is shown in table 7.

TABLE 7 gavage dose design

Mean blood glucose Change in mice during the test

The blood sugar of the mice is measured after intragastric administration every week, tail vein blood sampling is carried out on the mice, and the blood sugar value is measured by a glucometer and blood sugar test paper.

Referring to fig. 7, the results in fig. 7 show that the blood sugar of the mice in the control group is gradually increased during the test period, the symptoms of diabetes are aggravated, and the blood sugar of each group of bacteroides fragilis ZY-312 is not continuously increased, and the blood sugar of the middle and low dose groups is obviously reduced and has a significant difference with the control group. The Bacteroides fragilis ZY-312 is shown to reduce blood sugar and improve diabetes symptoms.

(2) Sugar tolerance test

The glucose tolerance test was performed on the weekdays of weeks 1 and 7: fasting is carried out at night on saturday without water supply for 14h, fasting blood glucose of the mice is measured in the morning on sunday, gastric administration is carried out, glucose is filled after 30 minutes and is recorded as 0min, tail vein blood sampling is carried out on the mice respectively at 10min, 20min, 30min, 60min and 120min, and blood glucose value is measured by a glucometer and blood glucose test paper.

Referring to fig. 8, the results of fig. 8 show that the blood glucose values of the groups of the sugar load experiment are not obviously different after the mice are continuously gavaged for 1 week, and the blood glucose value of the high-dose group is obviously lower than that of the control group at 120 min.

Referring to fig. 9, the results of fig. 9 show that after 7 weeks of continuous gavage, the fasting blood glucose values of each group of the bacteroides fragilis ZY-312 are significantly lower than those of the control group, and the fasting blood glucose values of the high-dose group are recovered to be normal. In the process of the glucose tolerance experiment, the blood glucose value of each group of the bacteroides fragilis ZY-312 is lower than that of the control group, and the bacteroides fragilis ZY-312 has significant difference.

(3) Body weight changes of mice during the test

TABLE 8 mouse weight gain Rate

The results in FIG. 10 and Table 8 show that after 7 weeks of the experiment, the body weight of mice in each group of Bacteroides fragilis ZY-312 is obviously lower than that of the control group, the body weight increase rate of the diabetic mice is reduced, and the body weight increase rate is lower than that of the control group, which indicates that the obesity symptom is reduced.

The invention has good effect on treating and/or preventing obesity or diabetes, does not generate drug resistance, is safe and nontoxic, and provides a new choice for treating and/or preventing obesity or diabetes. Experiments show that the bacteroides fragilis ZY-312 does not contain enterotoxin gene bft (bacteroides fragilis toxin), and compared with the existing strains, the bacteroides fragilis ZY-312 has the probiotic characteristics of bile salt resistance, gastric acid resistance and the like, and has wide application prospects.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of the bacteroides fragilis in preparing the pharmaceutical composition for treating and/or preventing obesity or diabetes is characterized in that the bacteroides fragilis ZY-312 is bacteroides fragilis with the preservation number of CGMCC No.10685, and the bacteroides fragilis ZY-312 does not contain enterotoxin gene bft.

2. A pharmaceutical composition for treating and/or preventing obesity or diabetes, which comprises a pharmaceutically effective dose of Bacteroides fragilis ZY-312 and a pharmaceutically acceptable carrier, wherein the Bacteroides fragilis ZY-312 does not contain enterotoxin gene bft, and the preservation number of the Bacteroides fragilis ZY-312 is CGMCC No. 10685.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is a tablet, capsule, oral liquid, or lyophilized powder.

4. The pharmaceutical composition of claim 2 or 3, wherein the pharmaceutically acceptable carrier is one or more of skim milk, lactose, dextrose, sucrose, sorbitol, mannose, trehalose, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.

5. The pharmaceutical composition of claim 4, wherein the cellulose is microcrystalline cellulose.