CN114350547B - Bifidobacterium lactis strain B-622 and application thereof in preparation of medicines for treating diabetes - Google Patents
Bifidobacterium lactis strain B-622 and application thereof in preparation of medicines for treating diabetes Download PDFInfo
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- CN114350547B CN114350547B CN202111550440.0A CN202111550440A CN114350547B CN 114350547 B CN114350547 B CN 114350547B CN 202111550440 A CN202111550440 A CN 202111550440A CN 114350547 B CN114350547 B CN 114350547B
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Abstract
The invention discloses a bifidobacterium lactis strain B-622 and application thereof in preparing a medicament for treating diabetes. The preservation number of the strain B-622 is CCTCC NO: m20211355, deposited with the China center for type culture Collection, at 2021, 11 and 2. The strain screened by the invention plays the role of reducing blood sugar by promoting glucagon-peptide-1 (GLP-1) secretion, repairing insulin secretion function, regulating blood lipid metabolism, reducing chronic low-grade inflammation and inhibiting dipeptidyl peptidase-4 activity, and simultaneously can inhibit type 2 diabetes model mice and stimulate proliferation of islet beta cells of the type 2 diabetes model mice.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bifidobacterium lactis strain B-622 and application thereof in preparing a medicament for treating diabetes.
Background
Diabetes (diabetes) is an endocrine metabolic disease associated with abnormal insulin production and action and characterized mainly by hyperglycemia, and mainly includes type I and type II diabetes. The diabetes group in China is mainly type II diabetes, and accounts for more than 90% of the total number of diabetics. Type II diabetes mellitus, which is based on relative deficiency of insulin and insulin resistance, eventually leads to hyperglycemia, and chronic hyperglycemia symptoms can lead to a range of chronic complications such as foot disease (foot ulcers, infections and gangrene), kidney disease (renal failure, uremia), eye disease (retinopathy, blurry, blindness), brain disease (cerebrovascular disease), heart disease, skin disease, venereal disease, etc. Type II diabetes is a slowly progressive disease whose central links to its onset are insulin resistance and islet beta cell dysfunction. At present, the number of patients suffering from type II diabetes in China is the first place in the world, so that prevention and treatment measures for developing type II diabetes are not slow. Many studies have found that probiotics have good biological effects on improving type II diabetes, but the mechanism of action is not clarified, and strains, ferments and core technologies of functional probiotics are monopolized abroad.
Incretins (incretin) are a hormone secreted by small intestine endocrine cells that helps the body to produce a postprandial insulin response after eating carbohydrates, and their insulinotropic effect accounts for about 60% of the total postprandial insulin secretion. To date, 2 incretins have been isolated, namely glucose-dependent insulin release peptide (GIP) and glucagon-like peptide-1 (Glucagon like peptide-1, GLP-1). Clinical researches show that GLP-1 and analogues thereof have important application value in the treatment process of type 2 diabetes mellitus, and GLP-1 has physiological functions of promoting insulin secretion, promoting islet beta cell proliferation, inhibiting apoptosis, slowing gastric emptying, inhibiting postprandial glucagon secretion, reducing hepatic glycogen synthesis, improving insulin sensitivity, controlling appetite and the like. GLP-1 is therefore of increasing interest as a safe, effective insulinotropic secretion. However, GLP-1 is degraded by dipeptidyl peptidase-4 (Dipeptidyl peptidase-4, DPP-4) in vivo very rapidly, the half-life is only 1-2min, and the dose for exciting GLP-1 receptor cannot be accumulated under the condition of insufficient endogenous GLP-1 secretion, so that the GLP-1 receptor is difficult to be applied to clinic. DPP-4 inhibitor can inhibit degradation of DPP-4 to GLP-1, so that endogenous GLP-1 can be accumulated in vivo to achieve blood sugar reducing effect. The two medicines have the functions of protecting islet cells, reducing weight and the like, and simultaneously rarely cause hypoglycemia reaction, so that the clinical application is increasingly wide. The most common adverse reactions of the two medicines are gastrointestinal reactions such as nausea, vomiting and the like.
Probiotics (probiotics) are microecological preparations which have promoting effects on body health, mainly comprise lactobacillus and bifidobacterium, and are a type of microorganisms accepted as safe to eat. The probiotics have the effects of improving intestinal tract, enhancing immunity, resisting allergy, resisting oxidation, reducing cholesterol, lowering blood pressure, reducing weight, etc. In recent years, some lactic acid bacteria have been studied to have hypoglycemic effect. The mechanism of the blood glucose reducing effect mainly comprises: improving intestinal mucosa barrier function; enhancing immune modulating function; repairing oxidative damage of organism and improving antioxidation capability; promoting insulin secretion and improving insulin sensitivity; inhibit or delay glucose absorption by intestinal tract, promote glucose utilization by peripheral tissues and target organs, etc.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a bifidobacterium lactis strain B-622 and application thereof in preparing a medicament for treating diabetes, wherein the strain plays the roles of reducing blood sugar by promoting glucagon-1 (GLP-1) secretion, repairing insulin secretion function, regulating blood lipid metabolism, reducing chronic low-grade inflammation and inhibiting dipeptidyl peptidase-4 activity, and simultaneously can inhibit type 2 diabetes model mice and stimulate proliferation of type 2 diabetes model mouse islet beta cells.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a bifidobacterium lactis (Bifidobacterium animalis subsp. Lactis) strain B-622, wherein the preservation number of the strain B-622 is CCTCC NO: m20211355, deposited with the China center for type culture Collection, at 2021, 11 and 2.
Further, comprising the strain B-622, the crushed material or the culture of the strain B-622 according to claim 1 as an active ingredient.
Further, the hypoglycemic probiotic preparation is powder, tablets or capsules.
The application of the hypoglycemic probiotic preparation in preparing medicaments for preventing blood sugar from rising or assisting in reducing blood sugar.
The application of the strain B-622 in preparing hypoglycemic drugs.
The application of the strain B-622 in preparing a medicament for treating diabetes.
The application of the hypoglycemic probiotic preparation in preparing the medicament for treating diabetes is provided.
Further, strain B-622 or the hypoglycemic probiotic preparation can inhibit apoptosis of islet beta cells, and promote proliferation of islet beta cells.
Further, diabetes is type ii diabetes.
The invention has the beneficial effects that:
the invention takes lactobacillus separated and identified from faeces samples of 2 year old healthy infants which are breast-fed from Tibetan areas and are not contacted with antibiotics as a study object, and obtains a new lactobacillus strain named bifidobacterium lactis B-622 through a large number of experimental screening. The invention adopts high-fat feed and streptozotocin to build a diabetes model, and the bifidobacterium lactis B-622 is administrated by the stomach of a mouse for 28 continuous days, so as to discuss the treatment effect of the bifidobacterium lactis B-622 on the diabetes model mouse. The research result shows that the bifidobacterium lactis B-622 plays the role of reducing blood sugar by promoting the secretion of glucagon-like peptide-1 (GLP-1), repairing insulin secretion function, regulating blood fat metabolism, reducing chronic low-grade inflammation and inhibiting dipeptidyl peptidase-4 activity.
The bifidobacterium lactis B-622 strain has the effects of preventing and treating diabetes mellitus, so that the bifidobacterium lactis B-622 strain can be used for preparing foods, health-care products or medicines with the function of reducing blood sugar, and the like, and meanwhile, the bifidobacterium lactis B-622 strain provided by the invention can be used as functional probiotics for preparing foods, health-care products or medicines, is safe, has no toxic or side effect, and has a very wide application prospect. Screening of lactic acid bacteria with the function of reducing blood sugar is carried out, the audience population is wide, and the market space is huge.
Drawings
FIG. 1 is the effect of bifidobacterium lactis B-622 on blood glucose in type 2 diabetic mice;
FIG. 2 shows that bifidobacterium lactis B-622 inhibits apoptosis of islet beta cells in model mice with type 2 diabetes;
FIG. 3 shows the stimulation of proliferation of islet beta cells in mice model for type 2 diabetes by bifidobacterium lactis B-622.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
EXAMPLE 1 isolation screening and identification of bifidobacterium lactis B-622
1. Sample collection
Fecal samples were collected from 2 year old healthy infants breast fed in the Tibetan area who were not exposed to antibiotics.
2. Isolation of strains
1g of sample is taken and put into 9mL of physiological saline to be vibrated and mixed uniformly, after the bacterial suspension is subjected to gradient dilution, 100 mu L of a proper gradient bacterial liquid is absorbed on a bifidobacterium separation culture medium agar plate added with mupirocin lithium salt, the mixture is uniformly coated, and after replacement air is oxygen-free air, the mixture is subjected to standing and constant temperature culture for 48 hours at 37 ℃. After colonies grow out, opaque milky white, round, glossy, regular in edge, raised and wet on the surface, repeated streak purification culture is performed on MRS solid culture medium to obtain 1 strain of infant fecal bacterial strain, and the strain is preserved at 4 ℃ for later use. Bifidobacterium isolation medium (g/L): beef extract 10, proteinPeptone 10, yeast extract 5, glucose 5, sodium acetate 5, diammonium hydrogen citrate 2, tween 80 1, K 2 HPO 4 2、MgSO 4 ·7H 2 O 0.58、MnSO 4 ·H 2 O 0.25、CaCO 3 20 Sterilizing at 121deg.C for 20min, adding 1.5% agar powder into MRS solid culture medium, stabilizing agar chemical structure, and solidifying agent in culture medium, which melts into liquid at 100deg.C, and cooling to below 45deg.C for re-solidification. The bacterial colony of the strain is opaque and milky white, round and glossy, and has neat edge, raised surface and wetness. The strain is sensitive to oxygen and is strict anaerobe, and air is replaced by anaerobic air during culture. The optimal growth temperature of the strain is 37 ℃, and the optimal initial pH value is 6.8.
3. Molecular biological identification of bifidobacterium lactis B-622
The primary strain was collected after purification culture, and genomic DNA was extracted using a genomic DNA extraction kit (TIANGEN Co.) using the upstream primer 27F: AGTTTGATCMTGGCTCAG (SEQ ID NO. 1) and a downstream primer 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO. 2) and amplifying the 16SrDNA sequence to obtain a PCR product. And sequencing the PCR product. Wherein the PCR reaction system comprises: 2*Super PfX MasterMix 25 mu L (CW 2965M, kangji, china) of each 2 mu L of the upstream and downstream primers, 2 mu L of the DNA template, ddH 2 O21. Mu.L. PCR reaction conditions: 95 ℃ for 10min;94 ℃ for 30s,55 ℃ for 30s,72 ℃ for 2min,35 cycles; and at 72℃for 10min. The PCR product was detected by gel electrophoresis and sent to Beijing engine company for sequencing. The measured gene sequence is submitted to NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis and comparison, and 16S rDNA gene sequence homology is more than or equal to 99% as an identification standard.
EXAMPLE 2 evaluation of Bifidobacterium lactis B-622 on tolerance to simulated artificial gastric fluid and artificial intestinal fluid
100 mu L of test bacterial liquid is inoculated into a nitrogen-filled anaerobic glass tube filled with 7mL of MRS liquid culture medium, and is subjected to stationary culture activation at a constant temperature of 37 ℃ for 2 times. And (3) taking 200 mu L of activated bifidobacterium lactis B-622 bacterial liquid, centrifuging, collecting bacterial bodies, adding 1mL of simulated artificial gastric fluid with the pH of 2.5 and simulated artificial intestinal fluid with the pH of 8.0 respectively, fully and uniformly mixing, digesting for 3.0h at the temperature of 37 ℃, sampling, detecting the number of viable bacteria, and calculating the survival rate, wherein the result is shown in Table 1. Wherein the artificial gastric juice and the artificial intestinal juice are prepared and used at present, and are placed at 4 ℃ for refrigeration for standby in a short time. Survival% = number of viable bacteria of post-digestion bacterial liquid/number of viable bacteria of pre-digestion bacterial liquid x 100%.
The formula of the artificial gastric juice comprises the following components: preparing 0.85% physiological saline, regulating pH value to 2.5 with dilute hydrochloric acid, adding 0.3% pepsin, dissolving completely, and filtering with 0.22 μm microporous membrane for sterilization.
The formula of the artificial intestinal juice comprises: preparing 0.85% physiological saline, adjusting pH to 8.0 with sodium hydroxide, adding 0.1% trypsin, dissolving completely, and filtering with 0.22 μm microporous membrane for sterilization.
TABLE 1 bifidobacterium lactis B-622 simulated digestive tract environment experimental data sheet
The results in Table 1 show that the bifidobacterium lactis B-622 has high survival rate in simulated artificial gastric fluid and artificial intestinal fluid, and shows that the bifidobacterium lactis B-622 can effectively resist the influence of gastrointestinal tract and still has high activity after passing through the gastrointestinal tract.
EXAMPLE 3 evaluation of Lactobacillus bifidus B-622 antibacterial Activity
100 mu L of test bacterial liquid is inoculated into a nitrogen-filled anaerobic glass tube filled with 7mL of MRS liquid culture medium, and the mixture is subjected to stationary culture and activation at a constant temperature of 37 ℃. 100 mu L of pathogenic indicator strain such as Escherichia coli, staphylococcus aureus and Salmonella typhi are respectively inoculated into 10mL of liquid LB culture medium, and cultured and activated at a constant temperature of 37 ℃ and a shaking table of 100 rpm. Diluting the activated pathogenic bacteria liquid to a concentration of 10 8 The CFU/mL bacterial suspension is added into 30mL MRS solid culture medium with proper temperature (about 50 ℃) to be evenly mixed (indicating the bacterial number of living bacteria 10) by 300 mu L of each pathogenic bacterial suspension 6 CFU/mL), pouring into a plate in which 4 sterile oxford cups are placed in advance, taking out the oxford cups with forceps after the culture medium is solidified, and adding 160 mu L of test bacterial liquid (the viable count is 10) into the holes left after the oxford cups are pulled out 8 CFU/mL), 3 parallels, 1 pairAnd (3) irradiating, wherein the control is replaced by MRS liquid culture medium. The plate was covered with a light cover and then was placed in a normal position, and after the replacement air was oxygen-free air, the plate was cultured in a constant temperature incubator at 37℃for a suitable period of time for observation, and the diameter of the inhibition zone was measured with a vernier caliper, and the results are shown in Table 2.LB medium: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride, 1000ml of distilled water, pH 7.0-7.2 and sterilizing at 121 ℃ for 15 min.
TABLE 2 data sheet of antagonistic ability of bifidobacterium lactis B-622 against pathogenic bacteria
The results in Table 2 show that the bifidobacterium lactis B-622 has a strong inhibition effect on common intestinal pathogenic bacteria such as escherichia coli, staphylococcus aureus and salmonella typhi.
EXAMPLE 4 evaluation of antibiotic susceptibility of Bifidobacterium lactis B-622
After marking and activating test bacteria on an MRS solid plate, selecting bacterial mud to prepare bacterial suspension in normal saline, and adjusting the concentration of the bacterial suspension to 10 8 CFU/mL, 100 mu L of bacterial suspension is added to an MRS solid flat plate poured in advance, bacterial liquid is uniformly coated on the flat plate by using a sterile cotton swab, an antibiotic drug sensitive sheet is attached, replacement air is oxygen-free air, then the culture is carried out at 37 ℃ in a normal position, after 24 hours, the diameter of the bacterial strain sensitive to the antibiotic is measured by using a vernier caliper, and the result is shown in Table 3.
TABLE 3 data sheet of sensitivity of bifidobacterium lactis B-622 to antibiotics
The results in Table 3 show that bifidobacterium lactis B-622 is not resistant to 17 common antibiotics, and the bifidobacterium lactis is proved to be safe in the application process.
Example 5 animal model establishment and experiment
1. Packet processing
100 male C57BL/6J mice were randomly divided into 4 groups of 20 mice each, and the grouping was as follows:
1. blank control group: feeding with common feed from beginning to end of the test; at week 9, the mice were given a single intraperitoneal injection of 30mg/kg of physiological saline 12 hours after empty stomach; the administration is carried out from week 10 to week 13, and the administration dosage is as follows: 12mL of sterile PBS buffer was administered per kilogram of body weight.
2. Model group: feeding with high-fat feed from week 1 to week 8, and feeding with common feed from week 9 to the end of the test; at week 9, mice were given a single intraperitoneal injection of streptozotocin 30mg/kg 12 hours on an empty stomach; administration is performed from week 10 to week 13; the dosage is as follows: 12mL of sterile PBS buffer was administered per kilogram of body weight.
3. Positive control group: feeding with high-fat feed from week 1 to week 8, and feeding with common feed from week 9 to the end of the test; at week 9, mice were given a single intraperitoneal injection of streptozotocin 30mg/kg 12 hours on an empty stomach; administration is performed from week 10 to week 13; the dosage is as follows: a solution of metformin in PBS was administered at a rate of 0.1mg/ml per body weight of 12ml/kg, once daily.
Group b-622: feeding with high-fat feed from week 1 to week 8, and feeding with common feed from week 9 to the end of the test; at week 9, mice were given a single intraperitoneal injection of streptozotocin 30mg/kg 12 hours on an empty stomach; administration is performed from week 10 to week 13; the dosage is as follows: 12mL of bacterial liquid is administered each time per kilogram of body weight. The preparation method of the bacterial liquid comprises the following steps: suspending the bifidobacterium lactis B-622 with PBS buffer to obtain a bifidobacterium lactis B-622 concentration of 1.0X10 9 CFU/mL bacterial liquid.
Week 1 to week 8, test days 1 to 56, week 10 to week 13, test days 64 to 91, and so on. Intraperitoneal injection of streptozotocin or physiological saline was performed on day 57 of the test.
2. Judging and establishing diabetes model standard
On week 9 (day 63 of the test), the tail vein was collected and the Fasting Blood Glucose (FBG) of the mice was measured. Mice meeting the following criteria are model type 2 diabetes mice: FBG is measured twice continuously and is more than or equal to 11.1mmol/L. The results indicated that on trial day 57, each of the model, positive control and B-622 mice was a model type 2 diabetes.
3. Blood glucose detection
Mice were blood-sampled for fasting blood glucose at week 10, week 11, week 12, week 13, and week 14, respectively (daily test, mean value for this week was calculated). The sugar tolerance test was performed on test day 99.
Fasting blood glucose results are shown in FIG. 1. The fasting blood glucose values of mice in group B-622 were decreased compared to the model group. The fasting blood glucose values of the mice in the B-622 group are obviously reduced at the 12 th week, the 13 th week and the 14 th week, the blood glucose value is reduced to 12.35mmol/L at the 14 th week, and the fasting blood glucose values are obviously different from those of the mice in the model group (P is less than 0.01). The results show that the bifidobacterium lactis B-622 has a therapeutic effect on hyperglycemia of type 2 diabetes and can obviously reduce fasting blood glucose values.
4. Detection of blood lipid, insulin and inflammatory cytokines
At the end of the test (day 100 of the test), each group of mice was collected, blood was collected from the eyeballs, and centrifuged at 3000r/min for 15min after 60min at room temperature, and the supernatant (serum) was collected. Detecting the contents of total cholesterol, triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol, insulin, endotoxin, anti-tumor necrosis factor-alpha and interleukin-6 in serum. The results of the blood lipid related factor detection in serum are shown in Table 1. The serum of mice in group B-622 had decreased TC and TG levels compared to the model group. The result shows that the bifidobacterium lactis B-622 can obviously improve the hyperlipidemia symptom of the type 2 diabetes.
TABLE 1 influence of bifidobacterium lactis B-622 on blood lipid in type 2 diabetic mice
Note that: compared to the model group, there was significant difference (P < 0.05), and there was significant difference (P < 0.01).
TABLE 2 Effect of Bifidobacterium lactis B-622 on ET, TNF-alpha, IL-6, INS, GLP-1, DPP-4 in serum of mice model for type 2 diabetes
Note that: compared to the model group, there was significant difference (P < 0.05), and there was significant difference (P < 0.01).
As shown in Table 2, the INS content in the serum of the mice in each administration group was increased to a different extent during the treatment period, and the INS content in the serum of the mice in the positive control group and the B-622 group was extremely significantly increased (P < 0.01) and the ET level in the serum of the mice in the B-622 group was significantly decreased (P < 0.01) compared with the serum of the mice in the model group. B-622 can obviously reduce the content of TNF-alpha and IL-6 in the serum of a type 2 diabetes mouse, and effectively relieve the chronic inflammation of the organism. The secretion amount of GLP-1 in the group B-622 is obviously increased, and DPP-IV enzyme activity is obviously inhibited.
5. Effect of bifidobacterium lactis B-622 on islet beta cells in model mice with type 2 diabetes
All mice are killed by cervical vertebra removal after blood collection on the 100 th day, pancreas specimens are taken, 4% formalin is used for fixation, then dehydrated and transparent to prepare wax blocks, the slice thickness is 5um, and subsequent staining is carried out. Verification of Bifidobacterium lactis B-622 inhibition of apoptosis of islet beta cells of type 2 diabetes model mice and stimulation of proliferation of islet beta cells of type 2 diabetes model mice
1. Bifidobacterium lactis B-622 inhibits apoptosis of islet beta cells in model mice with diabetes mellitus 2
The apoptosis test TUNEL apoptosis kit (cat No. 11684817910, rogowski) completed staining according to the kit instructions to develop green fluorescence, the primary antibody was polyclonal guinea pig anti-insulin (cat No. a0564, DAKO), the secondary antibody was goat anti-guinea pig Texas Red stained with Red fluorescence. It was demonstrated that B-622 can inhibit apoptosis of islet beta cells.
2. Bifidobacterium lactis B-622 stimulates proliferation of islet beta cells in model mice with diabetes mellitus 2
Experiments on bifidobacterium lactis B-622 to stimulate proliferation of islet beta cells in mice model for type 2 diabetes we used double staining of insulin and PCNA, labelling of islet beta cells: the primary antibody was polyclonal guinea pig anti-insulin (cat No. a0564, DAKO) and the secondary antibody was Red fluorescent (solid line delineated area) stained for goat anti-guinea pig Texas Red. Labeling of proliferating cells: the primary antibody is a mouse monoclonal antibody [ PC10] to PCNA (product number: ab29, abcam), and the secondary antibody is goat anti-mouse FITC stained with green fluorescence (marked by gray arrow). In FIG. 3, it can be seen that the B-622 group and the positive control group showed significantly higher green fluorescence expression than the model group, indicating that B-622 can stimulate islet beta cell proliferation.
Claims (9)
1. Lactobacillus bifidusBifidobacterium animalis subsp.Lactis) The strain B-622 is characterized in that the preservation number of the strain B-622 is CCTCC NO: m20211355, deposited with the China center for type culture Collection, at 2021, 11 and 2.
2. A hypoglycemic probiotic preparation comprising, as active ingredient, the strain B-622, a crushed product or culture of strain B-622 according to claim 1.
3. The hypoglycemic probiotic preparation according to claim 2, wherein the hypoglycemic probiotic preparation is a powder, a tablet or a capsule.
4. Use of a hypoglycemic probiotic preparation according to claim 2 or 3, for the preparation of a medicament for preventing blood glucose elevation, or for assisting in blood glucose lowering.
5. Use of strain B-622 of claim 1 in the preparation of a hypoglycemic agent.
6. Use of strain B-622 of claim 1 in the preparation of a medicament for the treatment of diabetes.
7. Use of a hypoglycemic probiotic preparation according to any one of claims 2 or 3 for the preparation of a medicament for the treatment of diabetes.
8. The use according to claim 6 or 7, wherein the strain B-622 or hypoglycemic probiotic preparation inhibits apoptosis of islet β cells and at the same time promotes proliferation of islet β cells.
9. The use according to claim 6 or 7, wherein the diabetes is type ii diabetes.
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