CN107475162B

CN107475162B - Lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity and application thereof

Info

Publication number: CN107475162B
Application number: CN201710874502.0A
Authority: CN
Inventors: 刘鹭; 吕加平; 张书文; 逄晓阳; 芦晶; 王芬
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2017-09-25
Filing date: 2017-09-25
Publication date: 2020-10-23
Anticipated expiration: 2037-09-25
Also published as: CN107475162A

Abstract

The invention discloses lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity and application thereof, wherein the strain is deposited in China general microbiological culture Collection center with the following preservation numbers: CGMCC NO. 14571; the preservation date is as follows: 29/8/2017; the classification is named as: lactobacillus casei subsp rhamnosus. The inhibition rate of the screened strain on the dipeptidyl peptidase-IV reaches 27.113%, and on the basis, the screened strain is fully evaluated by simulating the digestion environment of a human body, and the result shows that the strain is sensitive to acid, has good tolerance on bile salt, has survival rates in simulated saliva, gastric juice and intestinal juice of 98.46%, 36.51% and 50.62% respectively, and can survive and play a role in the environment in the human body well. The lactobacillus rhamnosus ZNJ05 with high dipeptidyl peptidase-IV inhibition activity and excellent survival characteristics for human gastrointestinal transport can be used for preparing functional foods for reducing blood sugar, and has good market prospect.

Description

Lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity and application thereof.

Background

In recent years, diabetes has been widely prevalent throughout the world, and has gradually become a major disease that endangers human life and health, and various antidiabetic drugs have been continuously developed. The common main oral hypoglycemic agents include insulin secretion promoters, insulin sensitizers, alpha-glucosidase inhibitors and the like, but some drug defects inevitably occur.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP-1) are two kinds of endogenous insulinotropic hormones mainly existing in human bodies, and can promote the secretion of insulin and maintain the blood sugar balance. Dipeptidyl peptidase-IV (DPP-IV) is a membrane glycoprotein with serine exopeptidase activity that cleaves proline dipeptides from the N-terminus of the polypeptide. GLP-1 is rapidly degraded by DPP-IV after being released, so that the levels of GIP and GLP-1 can be improved by inhibiting DPP-IV by a dipeptidyl peptidase inhibitor, and the effects of improving insulin level and maintaining blood sugar balance are further achieved. Therefore, dipeptidyl peptidase inhibitors have become important targets for treating type II diabetes. Probiotics are widely used in food fermentation, medicine and other industrial fields. In recent years, it has been found that probiotics have a variety of physiological functions as a beneficial flora that colonizes the intestinal tract: relieving lactose intolerance, improving constipation and diarrhea, maintaining intestinal microecological balance and intestinal canal function, enhancing immunity and resisting tumor, influencing development of cardiovascular diseases, influencing diabetes and blood sugar metabolism, relieving anaphylaxis, etc. Numerous studies have shown that probiotics have the potential to reduce the incidence of diabetes, but few studies have been made on probiotics as a therapeutic agent for diabetes. Researches and screens out probiotics with high dipeptidyl peptidase-IV inhibitory activity, and has wide application prospect in pharmacy, health food, special diet and the like. The probiotic is taken by a human body daily, the intestinal health is improved, meanwhile, the abnormal blood sugar metabolism can be intervened as early as possible, and other side effects such as hypoglycemia or weight gain caused by drug control are avoided. Provides a novel functional ingredient for obese people, blood sugar disorder and diabetes patients to develop health-care functional food with the functions of regulating and intervening blood sugar and promoting body health, and lays a foundation.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Still another object of the present invention is to provide a lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity and uses thereof, wherein the lactobacillus rhamnosus has excellent capability of inhibiting dipeptidyl peptidase-IV activity in vitro; the lactobacillus rhamnosus is used for preparing the functional food for reducing blood sugar and has effective blood sugar reducing effect.

To achieve these objects and other advantages in accordance with the present invention, there is provided a lactobacillus rhamnosus strain with high dipeptidyl peptidase-IV inhibitory activity, which has been deposited with the general microbiological center of the chinese committee for culture collection of microorganisms under the accession number: CGMCC NO. 14571; the preservation date is as follows: 29/8/2017; the classification is named as: lactobacillus casei subsp rhamnosus; the preservation address is as follows: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The method for obtaining the lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity comprises the following steps:

step 1, obtaining a strain with highest inhibitory activity on dipeptidyl peptidase-IV by screening; the screening includes preparation of a sample and determination of dipeptidyl peptidase-IV inhibitory activity.

Step 2, evaluating the probiotic characteristics of the screened strains; the probiotic property evaluation includes an acid resistance test, a bile salt resistance test, adhesion to HT-29 cells, resistance to simulated gastrointestinal tract, and a digestion test that simulates probiotics in vitro.

And 3, determining a strain through 16SrDNA gene sequencing analysis to obtain the lactobacillus rhamnosus with the inhibition effect on the dipeptidyl peptidase-IV.

Preferably, the preparation of said sample in step 1 comprises preparation of a cellular metabolite; the specific process for preparing the cell metabolite comprises the following steps: standing and culturing probiotic in MRS culture medium at 37 deg.C for 16-20 hr, centrifuging to collect thallus, and treating with sterile phosphorusWashing with acid salt buffer solution for 2-4 times, re-suspending in phosphate buffer solution, and adjusting the concentration of bacterial liquid to 1 × 10⁶-1×10¹⁰cfu/mL, mixing, incubating, centrifuging, collecting supernatant, and filtering to obtain cell metabolite.

Preferably, the preparation of the sample in step 1 comprises preparation of the cell contents; the specific process for preparing the cell content comprises the following steps: standing probiotic bacteria in MRS culture medium at 37 deg.C for 16-20 hr, centrifuging to collect thallus, washing with sterile phosphate buffer solution for 2-4 times, suspending in phosphate buffer solution, and regulating bacteria liquid concentration to 1 × 10⁶-1×10¹⁰cfu/mL, mixing, ultrasonication, centrifugation at 4 deg.C to collect supernatant, and filtration to obtain cell content.

Preferably, the conditions of the ultrasonication are: crushing with 200W power in 3-5s pulse under ice bath condition for 10-14 min.

Preferably, the determination of dipeptidyl peptidase-IV inhibitory activity in step 1 consists essentially of the following steps:

step A, taking an enzyme label plate, adding 20-30 mu L of cell metabolite or cell content, 45-55 mu L of buffer solution and 0.5-1.5 mu L of enzyme into a reaction hole by using a micropipette, culturing for 8-12min at 37 ℃ in a dark place, adding 45-55 mu L of buffer solution and 1-3 mu L of substance, reacting for 1-30min at 37 ℃ by using an enzyme label instrument, and measuring the light absorption value of the reaction solution at 405nm every minute.

And step B, taking sitagliptin as a positive control, and taking 0.1M phosphate buffer solution with pH of 6.8 as a blank control.

And C, calculating the relative inhibition rate to obtain the strain with the highest inhibitory activity on the dipeptidyl peptidase-IV.

The application of the lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibiting activity in preparing hypoglycemic functional food.

The invention at least comprises the following beneficial effects:

the invention adopts an in vitro test method to screen out the bacterial strain with the strongest inhibition ability to the dipeptidyl peptidase-IV, the bacterial strain is sensitive to acid, but has good tolerance to bile salt, can well survive in the internal environment of a human body, can be used for preparing the hypoglycemic functional food, and has good market prospect. The food containing the lactobacillus rhamnosus for inhibiting the dipeptidyl peptidase-IV has the effects of assisting in reducing blood sugar and also has the advantages of probiotics, and is suitable for developing foods for obese people, insulin resistance and diabetic patients.

The food containing the lactobacillus rhamnosus provided by the invention has no adverse effect and toxic or side effect, and is high in safety.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a process flow chart of the Lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity obtained by the present invention;

FIG. 2 is a bar graph of probiotic properties of Lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity according to the present invention;

FIG. 3 is an agarose gel electrophoresis image according to the invention;

FIG. 4 is a graph showing the inhibitory effect of Lactobacillus rhamnosus ZNJ05 on dipeptidyl kininase from different sources in experiment 1 of the present invention;

FIG. 5 is a graph of fasting plasma glucose values for each group after 13 weeks of feeding in experiment 2 according to the invention;

FIG. 6 is a graph showing glycosylated hemoglobin values of each group after 13 weeks of feeding in experiment 2 according to the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

As shown in figure 1, the invention provides lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity, and the strains have the following preservation numbers in China general microbiological culture Collection center: CGMCC NO. 14571; the preservation date is as follows: 29/8/2017; the classification is named as: lactobacillus casei subsp Lactobacillus rhamnosus.

In a preferred embodiment, the preparation of said sample in step 1 comprises the preparation of a cellular metabolite; the specific process for preparing the cell metabolite comprises the following steps: standing probiotic bacteria in MRS culture medium at 37 deg.C for 16-20 hr, centrifuging to collect thallus, washing with sterile phosphate buffer solution for 2-4 times, suspending in phosphate buffer solution, and regulating bacteria liquid concentration to 1 × 10⁶-1×10¹⁰cfu/mL, mixing, incubating, centrifuging, collecting supernatant, and filtering to obtain cell metabolite.

In a preferred embodiment, the preparation of the sample in step 1 comprises preparation of the cell contents; the specific process for preparing the cell content comprises the following steps: standing probiotic bacteria in MRS culture medium at 37 deg.C for 16-20 hr, centrifuging to collect thallus, washing with sterile phosphate buffer solution for 2-4 times, suspending in phosphate buffer solution, and regulating bacteria liquid concentration to 1 × 10⁶-1×10¹⁰cfu/mL, mixing, ultrasonication, centrifugation at 4 deg.C to collect supernatant, and filtration to obtain cell content.

In a preferred embodiment, the conditions of the ultrasonication are as follows: crushing with 200W power in 3-5s pulse under ice bath condition for 10-14 min.

In a preferred embodiment, the dipeptidyl peptidase-IV inhibitory activity assay of step 1 consists essentially of the steps of:

The lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity is applied to preparing hypoglycemic functional foods or medicines.

In the scheme, the bacterial strain with the strongest inhibition capacity on the dipeptidyl peptidase-IV is screened out by adopting an in-vitro test method, is sensitive to acid, has good tolerance on bile salt, can well survive in the internal environment of a human body, can be used for preparing the hypoglycemic functional food, and has good market prospect. The food containing the lactobacillus rhamnosus for inhibiting the dipeptidyl peptidase-IV has the effects of assisting in reducing blood sugar and also has the advantages of probiotics, and is suitable for developing foods for obese people, insulin resistance and diabetic patients. The food containing the lactobacillus rhamnosus provided by the invention has no adverse effect and toxic or side effect, and is high in safety.

The specific acquisition method of the lactobacillus rhamnosus with the high dipeptidyl peptidase-IV inhibitory activity comprises the following steps:

screening of probiotics having dipeptidyl peptidase-IV (DPP-IV) inhibitory action:

1. preparation of samples

(1)CFS(cell-free excretory supernatant)

The probiotics is statically cultured in MRS culture medium at 37 ℃ for 18h, and then is centrifuged at 12000r/min at 4 ℃ for 15min to collect thalli. The collected cells were washed 3 times with sterile 0.1M PBS (pH6.8), resuspended in PBS, and the concentration of the cells was adjusted to 1X 10⁹cfu/mL, mix well. Incubating at 37 deg.C for 12h, centrifuging at 4 deg.C for 15min at 12000r/min, collecting supernatant, filtering with 0.22 μm water system microfiltration membrane to obtain CFS, and storing at-80 deg.C.

(2)CFE(cell-free intracellular extract)

The probiotics is statically cultured in MRS culture medium at 37 ℃ for 18h, and then is centrifuged at 12000r/min at 4 ℃ for 15min to collect thalli. The collected cells were washed 3 times with sterile 0.1M PBS (pH6.8), resuspended in PBS, and the concentration of the cells was adjusted to 1X 10⁹cfu/mL, mixed well and sonicated. The conditions of ultrasonication were: the disruption was carried out with a pulse of 3-5s (3 s at work, 5s at 200W) for 12min under ice bath conditions. Centrifuging the liquid obtained after ultrasonic treatment at 12000r/min at 4 deg.C for 15min, collecting supernatant, filtering with 0.22 μm water system microfiltration membrane to obtain CFE, and storing at-80 deg.C.

2. Measurement of DPP-IV inhibitory Activity

(1) Reaction system

Taking an enzyme label plate, adding 25 mu L of CFS or CFE, 49 mu L of buffer and 1 mu L of Lenzyme into a reaction hole by using a micropipette, culturing for 10min at 37 ℃ in a dark place, adding 49 mu L of buffer and 2 mu L of substance, reacting for 1-30min at 37 ℃ by using an enzyme label instrument, and measuring the light absorption value of the reaction solution at 405nm every minute. Sitagliptin was used as a positive control and 0.1M PBS pH6.8 was used as a blank control.

(2) The calculation formula of the inhibition rate is as follows:

for each assay, a Sample Blank was made, and the slope of the Sample set was subtracted from the slope of the Blank set. The corrected values were used to calculate the relative inhibition ratios.

% relative inhibition

Slope ═ FLU₂-FLU₁)/(T₂-T₁)

Slope SM: slope of Sample Inhibitor

Slope EC: slope of Enzyme Control

TABLE 1 screening results for DPP-IV inhibitors

Note: in the table ND means that no inhibition was detected (Dot detected)

As can be seen from Table 1, 14 probiotics all show inhibition effect on DPP-IV activity, and the inhibition rate can reach 5.669-27.113%. Wherein the positive control group sitagliptin has the highest inhibition rate, and the half inhibition rate is 44.195%; only CFE of the strain 1.6970 has DPP-IV inhibition effect, and the inhibition rate is high and is 17.789%; the inhibition rate of 10 probiotics reaches more than 10 percent, the inhibition rates of ZNJ05, ST-2 and 1.1881 of the strains are respectively 27.113 percent, 21.525 percent and 14.632 percent, and the inhibition rate can reach 33 percent of the half inhibition rate of the sitagliptin of a positive control group. Finally, according to the inhibition rate results in table 1, probiotic characteristics of the strain ZNJ05 with the highest DPP-IV inhibitory activity were evaluated.

Evaluation of probiotic Properties

By evaluating beneficial characteristics, the survival capability of the strain ZNJ05 in the gastrointestinal tract is discussed, and a reference is provided for whether the probiotic can play a health promotion role in the human digestive tract environment.

1. Acid resistance test

Inoculating probiotic bacteria into MRS broth at an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. Resuspending the collected thallus in MRS broth with pH of 2.0, 3.0, 7.0, and adjusting the concentration of the bacteria solution to 1 × 10⁹cfu/mL. Incubate at 37 ℃ for 3h, collect the bacterial solution, count viable bacteria according to GB 4789.2-2010. The tolerance is calculated as follows:

Survival rate(％)＝(logN₁/logN₀)×100 (2)

N₁: viable count under the condition of enduring treatment

N₀: pH 6.4 (Normal) MRS Broth Medium

2. Bile salt resistance test

Inoculating probiotic bacteria into MRS broth at an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. Resuspending the collected thallus in sterile deionized water with pH of 8.0 and containing 2% bile salt, and adjusting the concentration of the bacteria solution to 1 × 10⁹cfu/mL. Incubate at 37 ℃ for 24h, collect the bacterial solution, count the viable bacteria according to GB 4789.2-2010. The same calculation as in equation (2).

3. Adhesion to HT-29 cells

(1) Fluorescent labeling of probiotics by cFDA-SE

A stock solution of 1mmol/L cFDA-SE (5.0 mg cFDA-SE is dissolved in 8.969mL of DMSO reagent) was prepared in Dimethyl sulfoxide (DMSO), sterilized by filtration through a 0.22 μm aqueous microfiltration membrane, and stored at-20 ℃ until use.

Inoculating the screened probiotics into MRS broth culture medium at an inoculum size of 4% (v/v), standing and culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 5min at 3000 Xg, discarding supernatant, and collecting thallus. Washing the collected thallus with sterile PBS for 3 times, suspending in PBS solution, adjusting the concentration of the thallus to 1 × 10⁹cfu/mL. Adding cFDA-SE stock solution (1mmol/L) to a final concentration of 20. mu. mol/L, standing at 37 deg.C in the dark for 20min, centrifuging at 4 deg.C (5000 Xg, 10min), discarding supernatant, and collecting thallus. The cells were washed 3 times with PBS, resuspended in PBS solution, and subjected to flow cytometry at 488nm excitation wavelength to analyze the labeling rate of cFDA-SE.

(2) Culture of HT-29 cells

After the HT-29 cells are recovered, the cells are subcultured according to the ratio of 1:3, transferred to a new culture flask, and the growth state of the cells is observed. Changing the culture solution every 1-2 days until the cell confluence degree is about 90%, removing the culture solution by suction, washing with PBS for 3 times, adding 1mL of 0.25% pancreatin, digesting in a constant temperature incubator at 37 deg.C and 5% CO2 for 2min, and suckingDiscarding pancreatin, adding culture solution to terminate digestion, counting cells, and adjusting cell number to 1 × 10⁵cells/mL, plating, observing the cell growth state, and performing a bacterial adhesion test when the confluency of cells in the culture plate is 90-100%.

(3) Adhesion assay for HT-29 cells

Washing HT-29 cells cultured in a 24-well plate for 3 times by using PBS, and respectively adding 500 mu L of PBS and cFDA-SE labeled probiotic bacterial suspension; the control group was blank wells + 500. mu.L cFDA-SE labeled probiotic suspension. Incubate at 37 ℃ for 2h in a 5% CO2 incubator and wash 3 times with sterile PBS. Adding 350 μ L of 0.25% pancreatin into each well, digesting in a CO2 incubator for 10min until the cells completely fall off from the bottom of the culture plate, adding 150 μ L of cell culture solution to stop digestion, mixing, taking out 100 μ L of suspension, transferring into a 96-well plate, wrapping with tinfoil paper, and measuring the fluorescence intensity. Fluorescence detection conditions: the excitation wavelength is 485nm, and the emission wavelength is 530 nm. The formula for calculating the adhesion rate is as follows:

(A-A) indicating the cell adhesion rate%₀)/(A₁-A₀)×100％ (3)

In the formula: a: HT-29 cells + cFDA-SE labeled bacterial suspension;

A₀: HT-29 cells + PBS;

A₁: cFDA-SE labeled bacterial suspensions.

4. Tolerance to simulated gastrointestinal tract

The simulated digestive juice is prepared by referring to Versantvorort and the like, and the digestive environment in the oral cavity and the gastrointestinal tract of a human body is fully simulated.

Inoculating probiotic bacteria into MRS broth at an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. The collected bacteria were resuspended in artificial simulated saliva (pH 7.0), gastric juice (pH 2.0), and intestinal fluid (pH 8.0) respectively, and the concentration of the bacteria solution was adjusted to 1 × 10⁹cfu/mL. Incubating for 5min, 3h and 24h at 37 ℃ in each environment respectively, collecting bacterial liquid, and counting viable bacteria according to GB 4789.2-2010. The same calculation as in equation (2).

5. Digestion test for in vitro simulated probiotics

Inoculating probiotic bacteria into MRS broth at an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. The collected bacterial cells were resuspended in simulated saliva at pH 7.0, and the bacterial liquid concentration was adjusted to 1X 10⁹cfu/mL. Incubating at 37 deg.C for 5min, and at 4 deg.C for 6000r/min, centrifuging for 10min, and collecting thallus; suspending the collected thallus in simulated gastric fluid with pH of 2.0, incubating at 37 deg.C for 3h, incubating at 4 deg.C for 6000r/min, centrifuging for 10min, and collecting thallus; finally, the collected thalli are resuspended in simulated intestinal fluid with the pH value of 8.0, incubated for 24 hours at 37 ℃, and bacterial fluid is collected and viable bacteria count is carried out according to GB 4789.2-2010. The same calculation as in equation (2).

The probiotic properties of strain ZNJ05 are shown in figure 2.

The result shows that after the strain ZNJ05 is cultured for 3 hours under the acidic condition of pH3.0, the survival rate is 97.61 percent and is more than 95 percent; after the culture is carried out for 3 hours under the condition that the pH value is 2.0, all the cells die, which indicates that the cells are sensitive to acid; after being cultured in deionized water containing 2 percent of bile salt for 24 hours, the culture medium has good survivability and high survival rate of 70.15 percent and has good tolerance to the bile salt; the strain ZNJ05 is marked by cFDA-SE with the final concentration of 20 mu mol/L, and the marking rate is detected by a flow cytometer, so that the marking rate is high and is 89.44 percent; however, the adhesion rate was very low, only 1.32%, and not more than 2.0%. In a simulated gastrointestinal environment, the survival rate of the strain ZNJ05 in simulated saliva, gastric juice and intestinal juice is 98.46%, 36.51% and 50.62%, respectively, and after digestion from oral cavity to gastrointestinal tract, the survival rate is 89.61%, so that the strain can well survive in the simulated in vivo environment.

16S-rDNA Gene sequencing

Strain ZNJ05 was inoculated at 4% (v/v) into MRS broth and cultured at 37 ℃ for 18 h. And 2mL of bacterial liquid is taken to extract a genome according to the bacterial genome DNA extraction kit. The purity and concentration of the DNA are detected and analyzed by agarose gel electrophoresis and ultraviolet spectrophotometry, and PCR amplification is carried out by bacterial 16S rDNA amplification universal primers 27F and 1492R. The base sequences of the universal primers are shown in Table 1. And detecting the amplified product by an agarose gel electrophoresis method. Finally, the amplified product is exchanged with Beijing Hua large gene research center to complete sequencing.

TABLE 1 bacterial 16SrDNA amplification Universal primers 27F and 1492R

(1) Amplification reaction system

(2) Reaction conditions

35 cycles, final extension at 72 ℃ for 10min, agarose gel electrophoresis of the PCR amplification products and results are shown in FIG. 3. (Marker band compositions from bottom to top are respectively 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, electrophoresis direction from top to bottom.)

The results showed that strain ZNJ05 was lactobacillus rhamnosus.

Example 1: sour soybean milk prepared from Lactobacillus rhamnosus ZNJ05 and having diabetes preventing effect

Adding 6% of sucrose into raw material soybean milk, sterilizing for 15min at 121 ℃, adding 7% of lactobacillus rhamnosus ZNJ05, fermenting at 37 ℃ until the titration acidity is 0.7-0.8% (calculated by lactic acid), refrigerating to 4 ℃, and refrigerating for storage to obtain the sour soybean milk with the function of preventing diabetes.

Example 2: lactobacillus rhamnosus ZNJ 05-containing lactobacillus beverage for preventing diabetes

Sterilizing raw milk (skimmed milk, fresh milk, reconstituted milk, etc.) at 100 deg.C for 10min, cooling to 4 deg.C, adding Lactobacillus rhamnosus ZNJ05 of the present invention to make its concentration reach 10⁶And (5) storing at the temperature of 4 ℃ for more than cfu/ml to obtain the viable bacteria milk beverage.

Example 3: yoghourt with function of preventing diabetes mellitus prepared from lactobacillus rhamnosus ZNJ05

Sterilizing raw milk (skim milk, fresh milk, reconstituted milk and the like) at 100 ℃ for 10min, cooling to 4 ℃, adding 3-5% of lactobacillus rhamnosus ZNJ05 and commensally-available commercial leavening agents for preparing the yoghourt such as lactobacillus bulgaricus and streptococcus thermophilus, performing mixed fermentation at 37 ℃ until the titration acidity is 0.6-0.7% (calculated by lactic acid), refrigerating to 4 ℃, and refrigerating for storage to obtain the yoghourt with the function of preventing diabetes.

Experiment 1, the specific results of inhibition experiments of dipeptidyl peptidase-IV from different sources by using lactobacillus rhamnosus ZNJ05 are shown in fig. 4.

Wherein the extraction method and the determination method of the dipeptidyl peptidase-IV are as follows:

1. determination of dipeptidyl peptidase-IV activity in mouse intestinal tract extraction enzyme

Taking an enzyme label plate, 25 mu L of 1.5mM Gly-Pro-pNA, 25 mu L CFS (0.1M PBS with pH6.8 as a blank control), culturing for 10min at a constant temperature of a metal bath at 37 ℃, adding 50 mu L (mouse intestinal tract) of dipeptidyl peptidase-IV (50 mu L), reacting for 60min at 37 ℃, adding 100 mu L of 1M sodium acetate buffer solution to terminate the reaction, and measuring the light absorption value of a sample at 405nm by using an enzyme label reader. Repeat 3 times.

The results indicated that the mouse intestinal enzyme reacted with the substrate in a colored manner, indicating that dipeptidyl peptidase-IV was present.

Substrate	enzyme/PBS	Absorbance value
			Gly-Pro-pNA	PBS	0.1486±0.0026
Gly-Pro-pNA	Mouse intestinal tract extracting enzyme	0.2066±0.0164

Extraction of enzymes from mouse intestinal tract

A clean-grade male Balb/c mouse is killed by decapitation after fasting for 12h, the upper segment of the small intestine is taken for 30cm, the small intestine is washed by sterile PBS at 4 ℃, cut into pieces and placed in a glass homogenizer. Add about 4 volumes of PBS, grind, homogenize in ice bath, pour into centrifuge tube. Centrifuging at 4 deg.C and 4000r/min for 20min, collecting supernatant, packaging, and storing at-20 deg.C.

2. Extraction of enzymes from Caco-2 cells

Plates of Caco-2 cells cultured to 21d were placed on ice, the cells were washed 2 times with PBS, lysis buffer (100KIU/mL aprotinin and Triton X-100 in PBS) was added for 5min, the cells were gently blown and transferred into a centrifuge tube for centrifugation 2 times: centrifuging at 4 deg.C for 10min at 1000g for the first time to remove cell residue; centrifuging at 20000g for 30min at 4 deg.C for the second time, collecting supernatant, and storing at-80 deg.C.

Determination of protein content of extracted enzyme in Caco-2 cell

The protein content of the extracted enzyme in Caco-2 cells was 1.256mg/mL as determined by the BCA kit instructions.

Determination of dipeptidyl peptidase-IV Activity in Caco-2 cell extraction enzyme

25 mu L of 1.5mM Gly-Pro-p-nitroanilide and 25 mu L of CFS (0.1M PBS with pH6.8 as a blank control) are taken for an enzyme label plate, the plate is cultured for 10min at the constant temperature of 37 ℃ in a metal bath, 50 mu L of dipeptidyl peptidase-IV (Caco-2 cells) are added for reaction for 60min at the temperature of 37 ℃, 100 mu L of 1M sodium acetate buffer solution is added for stopping the reaction, and the light absorption value of a sample is measured at 405nm by an enzyme label instrument. Repeat 3 times.

The results showed that the enzyme extracted from Caco-2 cells reacted with the substrate in a colored manner, indicating that dipeptidyl peptidase-IV was contained therein.

Substrate	enzyme/PBS	Absorbance value
			Gly-Pro-pNA	PBS	0.1336±0.0388
Gly-Pro-pNA	Caco-2 cell extraction enzyme	0.7566±0.1902

3. Determination of dipeptidyl peptidase-IV Activity in porcine intestinal extract enzyme

Taking an enzyme label plate, 25 mu L of 1.5mM Gly-Pro-pNA, 25 mu L CFS (0.1M PBS with pH6.8 as a blank control), culturing for 10min at a constant temperature of a metal bath at 37 ℃, adding 50 mu L (pig small intestine) of dipeptidyl peptidase-IV, reacting for 60min at 37 ℃, adding 100 mu L of 1M sodium acetate buffer solution to terminate the reaction, and measuring the light absorption value of a sample at 405nm by using an enzyme label instrument. Repeat 3 times.

The results showed that the enzyme extracted from the pig small intestine reacted with the substrate with a color after 30-fold dilution, indicating that it contained dipeptidyl peptidase-IV.

Substrate	enzyme/PBS	Absorbance value
			Gly-Pro-pNA	PBS	0.0920±0.0131
Gly-Pro-pNA	Enzyme extracted from small intestine of pig	0.4753±0.0091

Experiment 2, animal experiment:

in vivo diabetes prevention experiments, a type 2 diabetes model was developed by feeding high fat diet plus injected Streptozotocin (STZ).

Balb/c mice, male, 4-5 weeks old weekly, purchased from Beijing Wittingle, Inc., certification number: SCXK (Jing) 2013-. The animal breeding stock is bred in SPF animal house of pharmaceutical plant institute of Chinese medical science institute, and the license number is as follows: SYXK (Kyoto) 2013-0023 at 22. + -. 2 ℃ and 60% RH relative humidity. The mouse cage is regularly washed by 12 hours of illumination and 12 hours of darkness and is freely drunk by drinking water, padding is regularly replaced, and the mouse cage is regularly washed.

40 were randomly divided into 4 groups by weight: normal group, model group, positive drug group and probiotic feeding group (except normal group, all are fed with high fat feed, 10 per group).

Two weeks after prophylactic gavage administration, the patient was fasted one day in advance and kept water for 12 hours, and 100mg/kg streptozotocin (STZ, SIGMA) was intraperitoneally injected, and the injections were repeated every 3 days. The normal group was fed with basal diet (crude protein content 19.2%, crude fat content 4.6%, crude fiber 4.0%, crude ash content 6.3%, moisture 8.8%, nitrogen-free extract 55.9%); the model group and the positive drug group (sitagliptin phosphate tablet (tenofovir)) test group were fed with high fat diet (crude protein content 17.5%, crude fat content 17.9%, crude fiber 3.1%, crude ash content 4.5%, moisture 8.5%, nitrogen-free extract 48.5%); test group gastric lavage of lactic acid bacteria 1X 10 daily¹⁰CFU/mouse. One week after the STZ induction molding, the mice of each group are fasted for 12 hours without water prohibition, and the blood glucose value of the tail vein is measured by a glucometer to be the fasting blood glucose value. Model setThe fasting blood glucose level is 11.3mmoL/L, and the normal blood glucose level is 3.7 mmoL/L. The blood sugar value of the blood sugar group of the model group exceeds that of the normal group by more than 50 percent, which is the success of model building of the type II diabetes.

After the molding is successful, the positive drug group is gavaged with 10mg/kg every day, and the probiotic group is gavaged with 250 mu L of probiotic samples every day until the end of the 13-week experiment. Blood was sampled in the fasting state, and the fasting blood glucose level (FIG. 5) and the glycosylated hemoglobin level (FIG. 6) were measured. After 13 weeks, the fasting blood glucose value of the model group is 12.74mmoL/L, the fasting blood glucose value of the positive drug group is 8.084mmoL/L, and the fasting blood glucose value of the probiotic group is 6.74 mmoL/L. Compared with the model group, the fasting blood glucose value of the mice of the probiotic ZNJ05 group is reduced, and the differences are significant (P is less than 0.05); meanwhile, compared with the fasting blood glucose value of the mice in the normal group, the fasting blood glucose of the probiotic ZNJ05 group is higher than that of the mice in the normal group, but has no significant difference; compared with the fasting blood glucose value of the positive drug group (sitagliptin phosphate tablets (tenofovir)), the blood glucose reducing effect of the probiotic ZNJ05 group is not significantly different. The results show that the blood sugar regulating effect of the probiotics ZNJ05 has no significant difference with the positive medicament effect, and has significant blood sugar reducing effect.

The effect on glycosylated hemoglobin was further investigated in this experiment. Glycosylated hemoglobin is a gold index among diabetes evaluation indices and is used to evaluate the therapeutic effect of long-term blood glucose regulation. The results show that: compared with the model group, the glycosylated hemoglobin value of the ZNJ05 probiotic group is reduced, but no significant difference exists; meanwhile, compared with the glycosylated hemoglobin of the positive drug group, the glycosylated hemoglobin has no significant difference, which indicates that the regulating capacity of the probiotics ZNJ05 on the glycosylated hemoglobin is between that of the positive drug and that of the control group. (P is less than 0.05), and the glycosylated hemoglobin has no significant difference with the glycosylated hemoglobin of the positive drug group. The probiotics are proved to have certain hypoglycemic effect on the type II diabetes.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. The lactobacillus rhamnosus with high dipeptidyl peptidase-IV inhibitory activity is characterized in that the strain is deposited in China general microbiological culture Collection center with the following deposition numbers: CGMCC NO. 14571; the preservation date is as follows: 9 month 8 in 2017; the classification is named as: lactobacillus rhamnosus (A), (B), (C)Lactobacillus rhamnosus） ZNJ05。

2. Use of lactobacillus rhamnosus having a high dipeptidyl peptidase-IV inhibitory activity according to claim 1 for the preparation of a hypoglycemic functional food.