CN116064285A - Lactobacillus rhamnosus ZJUIDS07 capable of reducing blood sugar and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus ZJUISD07 with a blood sugar function and application thereof in the fields of foods, health products, pet foods, medicines and the like. The invention discloses lactobacillus rhamnosus ZJUIDS07 with a preservation number of CGMCC No.23996. The invention also discloses application of the compound in preparation of products with the function of reducing blood sugar.

Description

Lactobacillus rhamnosus ZJUIDS07 capable of reducing blood sugar and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus ZJUISD07 with a blood sugar function and application thereof in the fields of foods, health products, pet foods, medicines and the like.

Background

Currently, diabetes has become the most abundant metabolic disease. The high blood sugar level in serum is considered to be a major factor in metabolic diseases such as diabetes. Thus, lowering serum blood glucose levels is directly related to the health of the human body. To date, a great deal of experiments prove that the administration of part of lactobacillus and related products thereof has the effects of reducing blood serum glucose content of human body and reducing complications caused by diabetes. Lactic acid bacteria are important probiotics in human intestinal tracts, the quantity and the composition of the lactic acid bacteria play a vital role in maintaining the microecological balance of a host and improving the function of an immune system, and researches show that the lactic acid bacteria have the effects of reducing the activity of alpha-glucosidase, DPP-IV inhibition rate and the like in vitro, so that the effect of reducing blood sugar is achieved.

Lactobacillus rhamnosus is closely related to human life and is one of beneficial microorganisms widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. Lactobacillus rhamnosus is used as one of lactobacillus, and can improve the taste, texture and flavor of food by adding the lactobacillus rhamnosus to food. Lactobacillus rhamnosus can also be planted in human body to play a probiotic role, such as regulating intestinal flora, inhibiting growth of intestinal pathogenic bacteria, reducing blood sugar content in animal body, enhancing immunity, improving lactose digestion, resisting tumor and oxidation, etc.

The currently available Lactobacillus rhamnosus strain is used as follows:

the invention of CN114287552A, a lactobacillus rhamnosus strain separated from natto yak milk pimple and application thereof, informs that: lactobacillus rhamnosus NAQU Plateau LR-1 (Lactobacillus rhamnosus NAQU Plateau LR-1) can convert sugar in food raw materials into organic acid metabolites such as lactic acid and the like, so that the flavor and the nutritional value of fermented food are remarkably improved, and the shelf life of the food is prolonged. The lactobacillus rhamnosus in the patent has the main function of inhibiting the activity of mould, and does not relate to the functional activities of reducing blood sugar, resisting oxidation, resisting pathogenic bacteria and the like.

The invention of CN114621896A, namely Lactobacillus plantarum84-3 with the functions of reducing blood sugar and blood lipid and application thereof, informs Lactobacillus plantarum84-3 that the Lactobacillus plantarum84-3 has the blood sugar reducing potential; except for the effect of animals in evaluating blood sugar reducing, the strain only informs that the strain has acid resistance and cholate resistance.

The invention of CN113462613A (Lactobacillus plantarum ZJUIDS04 for reducing blood sugar) and application thereof discloses Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04 with a preservation number of CGMCC NO.22609. The lactobacillus plantarum has strong blood sugar reducing function and strong bile salt hydrolase activity; compared with other lactic acid bacteria, the strain has obvious advantages in acid resistance and bile salt resistance, is suitable for gastrointestinal environment and has proliferation capacity; no antibiotic resistance, and antibacterial activity; has strong antioxidation activity.

The invention of CN108410761A, a strain of lactobacillus rhamnosus with nitrite reduction and oxidation resistance and a screening and separating method, proves that lactobacillus rhamnosus (Lactobacillus rhamnosus) BLCC2-0077 has strong acid resistance and bile salt resistance, and can degrade high-concentration nitrite and strong oxidation resistance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a lactobacillus rhamnosus ZJUISD07 for reducing blood sugar of fermented food sources and application thereof.

In order to solve the technical problems, the invention provides lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07: the preservation number is CGMCC NO.23996.

Improvement of lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 as the present invention: the 16S rDNA full sequence of lactobacillus rhamnosus ZJUISD07 is SEQ ID No: 1.

The invention also provides an application of the lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 in preparing a product with the blood sugar reducing function.

The product with the blood sugar reducing function is food, medicine, health care product, feed and pet food with the blood sugar reducing function;

the food with blood sugar reducing function comprises fermented fruit and vegetable juice (such as fermented pumpkin and dragon fruit juice) with blood sugar reducing function; fermented sour meat with low blood sugar function; yogurt with hypoglycemia function; the medicine with blood sugar reducing function includes live bacteria preparation with blood sugar reducing function. The viable count of Lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 in the viable count is 1.0X10 ¹¹ ～1×10 ¹² CFU/g；

The lactobacillus rhamnosus ZJUISD07 can be used for preparing solid beverage, pet food and other products with the function of reducing blood sugar by fermenting alone or in combination with other strains.

Lactobacillus rhamnosus ZJUISD07 of the invention, deposit name: lactobacillus rhamnosus Lactobacillus rhamnosus; preservation unit: china general microbiological culture Collection center, preservation address: beijing city, chaoyang district, north Chenxi lu 1, 3, accession number: CGMCC No.23996, and the preservation time is 2021, 11 and 29.

The invention screens lactobacillus rhamnosus ZJUISD07 (Lactobacillus rhamnosus ZJUIDS 07) from traditional fermented dairy products, and the bacterium is identified by combining the morphological, physiological and culture characteristics of the bacterium, 16S rDNA sequencing and the like.

The invention not only proves that the strain has the effect of reducing blood sugar according to the activity of alpha-glucosidase, but also proves that the strain has the effect of reducing blood sugar from the aspect of another mechanism (DPP-IV inhibition rate). Meanwhile, the invention also verifies that the strain has good blood sugar reducing effect on an animal model.

Lactobacillus rhamnosus ZJUISD07 (Lactobacillus rhamnosus) of the present invention also has the following capabilities:

1. has stronger antioxidation activity and is obviously higher than the existing similar strains.

2. Can tolerate acid and bile salts and has certain proliferation capacity; sensitivity to common antibiotics; has antibacterial activity; namely, the composition can withstand the gastrointestinal environment, has no antibiotic resistance, and can inhibit the harmful pathogenic bacteria in the intestines.

In conclusion, lactobacillus rhamnosus with strong blood glucose reducing function is screened from probiotics obtained by separating traditional fermented dairy products. The strain has stronger bile salt hydrolase activity. The strain has obvious advantages in acid resistance and bile salt resistance compared with other lactic acid bacteria, is suitable for gastrointestinal environment and has proliferation capacity. No antibiotic resistance and antibacterial activity. The lactobacillus rhamnosus has strong antioxidant activity and is obviously superior to the conventional lactobacillus rhamnosus, so that the lactobacillus rhamnosus can be widely used for developing products with relevant probiotic functions for reducing blood sugar.

Drawings

FIG. 1 is a colony morphology of Lactobacillus rhamnosus ZJUISD 07.

FIG. 2 is a diagram showing the morphology of gram-stained cells of Lactobacillus rhamnosus ZJUISD 07.

FIG. 3 is an electrophoretically identified map of 16S rDNA of Lactobacillus rhamnosus ZJUISD 07;

the first band on the right is Maker and the second band is the electrophoresis band of ZJUIDS 07.

FIG. 4 shows the effect of ZJUIDS07 on total cholesterol, triglycerides, high density lipoproteins and low density lipoproteins in mouse blood.

FIG. 5 shows the effect of ZJUIDS07 on IL-8, IL-10, IL-1. Beta. And TNF-. Alpha.inflammatory factors in mouse blood.

FIG. 6 shows the effect of ZJUIDS07 on glycosylated hemoglobin content and glycogen in mice.

FIG. 7 shows the effect of ZJUIDS07 on insulin levels in mice.

FIG. 8 shows the effect of ZJUIDS07 on superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and Glutathione (GSH) in mouse liver.

Fig. 9 is the effect of ZJUIDS07 on short chain fatty acids (acetic acid, propionic acid, and butyric acid) in mouse feces.

FIG. 10 shows the effect of ZJUIDS07 on the intestinal flora of mice (bifidobacteria, S24-7_g, turicibacter).

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1, screening and identification of lactobacillus rhamnosus ZJUISD 07:

1. screening of lactic acid bacteria

1.1 sample Source

The strains used in the following cases are all obtained by separating fermented dairy products (taken as samples) such as fermented mare milk, cheese, milk skin, milk lumps and the like which are made by the inner Mongolian herdsman family. At least 30 samples are collected for subsequent separation, purification and identification.

1.2 isolation and purification of Strain

About 5g of the sample was collected by a sterile tube and immediately sent to a laboratory for strain isolation. Putting 1g of sample into 9mL of MRS liquid culture medium, and carrying out enrichment culture for 48h at 37 ℃ after vortex mixing uniformly; then 1mL of enrichment solution is sucked up in an ultra-clean bench, ten-fold gradient dilution is carried out by using sterile physiological saline, and 10 is selected ^-6 、10 ^-7 、10 ^-8 Three dilution gradients, 100. Mu.L of each of which was plated on MRS agar medium and incubated at 37℃for 48h. After the culture is finished, selecting a plate with 50-150 single colonies growing on the agar medium, picking typical colonies, carrying out repeated streak purification on the MRS agar plate until the colony morphology on the whole plate is consistent, and picking single colonies to the MRS liquid medium for enrichment culture. The obtained strains were all frozen and stored at-80℃in MRS liquid medium containing 40% glycerol.

A strain meeting the requirements of good growth and good genetic stability is selected and obtained, and is named ZJUIDS07.

2. Identification of lactobacillus rhamnosus ZJUISD07

2.1 colony characterization

After lactobacillus rhamnosus ZJUISD07 is cultured in MRS agar medium for 48h, the diameter is between 0.5 and 1.3mm, and the colony is round, and has neat edge, white color and moist and smooth surface, as shown in figure 1.

2.2 morphology under microscope:

lactobacillus rhamnosus ZJUISD07 colony smear: gram staining was positive, sporulation was absent, straight-ended, single, paired or short-chain, see figure 2.

2.3 16S rDNA identification

Extracting target strain genome DNA by using Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out PCR experiment of 16S rDNA by using bacterial universal primers 27F and 1492R, taking PCR products after PCR amplification, carrying out agarose gel detection and photographing, and the amplified fragment length is about 1.5kb, as shown in figure 3. The PCR product was sent to Beijing Liuhua big Gene (Wuhan) Inc. for sequencing, and the result was shown as SEQ ID NO:1, BLAST sequence alignment on NCBI website shows that the sequence has more than 99% homology with the 16S rDNA sequence of lactobacillus rhamnosus.

The sequence comparison result and the physiological and biochemical result of the strain ZJUIDS07 are combined, and the screened lactobacillus ZJUIDS07 is determined to be lactobacillus rhamnosus (Lactobacillus rhamnosus).

The strain ZJUIDS07 has the following preservation information: preservation name: lactobacillus rhamnosus Lactobacillus rhamnosus; preservation unit: china general microbiological culture Collection center, preservation address: beijing city, chaoyang district, north Chenxi lu 1, 3, accession number: CGMCC No.23996, and the preservation time is 2021, 11 and 29.

Example 2 confirmation of the in vitro hypoglycemic ability of lactobacillus rhamnosus ZJUISD07 (see methods described in CN113462613 a):

1. alpha-glucosidase inhibition rate

Preparation of Strain fermentation liquor

The strain ZJUISD07 stored in the glycerol tube is firstly streaked and activated on an MRS agar plate for 2 to 3 times, then single bacterial colony is selected and cultured in an MRS liquid culture medium for 18 hours at 37 ℃ in an enlarged way, and the concentration of bacterial liquid reaches 10 ⁷ About CFU/mL, as a bacterial suspension. Bacteria are inoculated withThe suspension was inoculated in an inoculum size of 2% (vol%) into MRS liquid medium having a glucose (water-soluble) concentration of 100. Mu.g/mL, and cultured at 37℃for 48 hours, to thereby obtain a strain fermentation broth. The above-mentioned cultivation was carried out at a rotation speed of 4000 r/min.

The strain fermentation broth is centrifuged for 20min at 12000rpm to obtain supernatant and thalli, part of thalli is crushed by a cell crusher (the power of the crusher is 40 percent, the crushing time is 8 minutes), and the supernatant obtained after centrifugation for 10min at 12000r/min is the content.

A total volume of 205 μl was set up, consisting of the following ingredients: to 50. Mu.L of PBS buffer solution (concentration 0.1mol/L, pH=6.8), 50. Mu.L of p-nitrophenol-. Alpha. -D-glucopyranoside (PNPG) solution (concentration 20 mmol/L) and 25. Mu.L of sample to be tested (supernatant/cell/content) were added, the mixture was incubated at 37℃for 10min, 30. Mu.L of alpha. -glucosidase solution (20U/mL) was added, and the reaction was continued for 20min, 50. Mu.L of Na (concentration 1 mol/L) was added ₂ CO ₃ As a reaction termination solution.

And measuring the light absorption value of the obtained reaction liquid at 405nm, wherein the light absorption value is in direct proportion to the free quantity of p-nitrophenol PNP, adopting a PBS solution with the pH value of 6.8 and the concentration of 0.1mol/L as a blank control of an alpha-glucosidase solution and a sample to be measured in the reaction system, and calculating the inhibition of lactobacillus on the alpha-glucosidase after the reaction.

The inhibition rate calculation formula is as follows:

α -glucosidase inhibition = [1- (a-B)/(C-D) ] = [ 100%;

in the middle of

A is a measured absorbance value of a sample to be measured containing an alpha-glucosidase solution;

B is a measured absorbance value of the sample to be measured without the alpha-glucosidase solution;

c is the measured absorbance value containing the alpha-glucosidase solution but no sample;

d is the measured absorbance value of the sample to be measured without the alpha-glucosidase solution;

that is to say,

a corresponds to the above system;

changing 30 mu L of alpha-glucosidase solution into 30 mu L of PBS buffer solution, and obtaining a system B;

changing 25 μl of the sample to be tested into 25 μl of PBS buffer solution, wherein the obtained system is C;

30. Mu.L of the alpha-glucosidase solution was changed to 30. Mu.L of PBS buffer solution, and 25. Mu.L of the sample to be tested was changed to 25. Mu.L of PBS buffer solution, and the resulting system was D.

Each strain was run in 3 replicates, each set of experiments was repeated 3 times, while MRS broth was used as negative control (α -glucosidase inhibition was essentially 0) and lactobacillus rhamnosus ATCC53103 was used as positive control strain.

TABLE 1 alpha-glucosidase inhibition Rate (%)

abc represents significant difference, P <0.05

As shown in Table 1, the supernatant, thalli and crushed contents of lactobacillus rhamnosus ZJUISD07 obtained by screening according to the invention have a significantly higher alpha-glucosidase inhibition rate than that of the standard strain lactobacillus rhamnosus ATCC53103. This indicates that lactobacillus rhamnosus ZJUISD07 strain has excellent alpha-glucosidase inhibitory ability.

Description: the results obtained in CN113462613a with lactobacillus plantarum (Lactobacillus plantarum) zjuds 04 are also compared with the present invention in table 1 above, and the content of ZJUISD07 of the present invention has significantly higher α -glucosidase inhibition rate than zjuds 04, indicating that the blood glucose lowering effect of the broken ZJUISD07 cells is significantly higher than ZJUIDS04.

DPP-IV inhibition

The fermentation supernatant, the cells and the cell contents were prepared as described above.

The measurement method is as follows: in a 96-well microplate, 25. Mu.l glycine-p-nitroaniline (0.2 mM) and 25. Mu.l bacterial sample (supernatant, bacterial suspension, contents, PBS control) were added and pre-incubated for 10 min at 37 ℃. Thereafter, 50. Mu.l DPP-IV (0.01U/ml) was added and incubated at 37℃for 60 minutes; the reaction was stopped by adding 100. Mu.l of sodium acetate buffer (1M, pH 4.0) and the absorbance of the sample was measured at 405 nm.

The PBS solution with pH of 6.8 and 0.1mol/L is adopted as a blank control of the DPP-IV solution and the sample to be detected in the reaction system;

DPP-IV inhibition = [1- (a-B)/(C-D) ]%

Wherein:

a is a measured absorbance value of a sample to be measured containing DPP-IV solution;

b is a measured absorbance value of the sample to be measured without DPP-IV solution;

c is the measured absorbance value with DPP-IV solution but without sample;

D is the measured absorbance of the sample to be tested without DPP-IV solution.

Each strain was run in 3 replicates, each set of experiments was repeated 3 times, while MRS broth was used as negative control and lactobacillus rhamnosus ATCC53103 was used as positive control strain.

TABLE 2 DPP-IV inhibition by strain (%)

Description: the results obtained with Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04 in CN113462613A are also compared to the present invention in Table 2 above, where the DPP-IV inhibition of the present content is significantly higher than ZJUIDS04.

The lactobacillus rhamnosus ZJUISD07 has excellent DPP-IV inhibition effect and can be used for screening blood glucose reducing standard strains.

The two experiments show that lactobacillus rhamnosus ZJUISD07 has good effects on DPP-IV and alpha-glucosidase inhibition, has obvious advantages compared with the standard strain lactobacillus rhamnosus ATCC53103, and can be used for later research and product development.

Example 3 confirmation of the in vivo hypoglycemic Capacity of Lactobacillus rhamnosus ZJUISD07

1. Experimental animals: 40C 57BL/6 male mice purchased from Shanghai Laek laboratory animal center, company license number: SCXK 2017-0005, which is fed into laboratory animal center of Zhejiang university, SPF environment.

2. Experimental animal feeding

After C57BL/6 mice are subjected to adaptive feeding for one week in SPF-class animal laboratories, the C57BL/6 mice at 3 weeks of age are randomly divided into 4 groups of 10 mice each, which are respectively a conventional feed+normal saline group (control group); high fat diet group: high fat feed + physiological saline group (diabetes model group), high fat feed + standard strain group (lactobacillus rhamnosus ATCC 53103), high fat feed + lactobacillus rhamnosus ZJUIDS07 group (lactobacillus rhamnosus ZJUIDS07 group).

3. Diabetes model construction

All the mice described above received intraperitoneal injections 12 hours after fasting. Control fed mice received intraperitoneal injection of 50mmol/L citrate buffer (pH 4.5), and high fat diet fed mice received STZ (Sigma, st. Louis) dissolved in 50mmol/L citrate buffer at a dose of 100mg/kg Body Weight (BW) per mouse. After one week, tail blood glucose levels were measured with a glucometer. Mice with FBG levels of 11.1mmol/L or more were defined as mice with successful model construction of type II diabetes.

4. Probiotic stomach lavage

Preparation of Lactobacillus rhamnosus ATCC53103 and Lactobacillus rhamnosus ZJUIDS07 fermentation broths were prepared as in the above examples, and after preparation of the fermentation broths, the cells were collected by centrifugation and prepared into 10 with sterile PBS solution ¹⁰ CFU/mL was used for subsequent lavage.

All mice were subjected to intragastric administration, wherein the control group was intragastric sterile saline, the diabetes model group was also intragastric sterile saline, and the lactobacillus rhamnosus ATCC53103 standard strain group was intragastric administration by 0.1ml 10 ¹⁰ CFU/mL ATCC53103 bacterial liquid, lactobacillus rhamnosus ZJUIDS07 group lavage 0.1mL 10 ¹⁰ CFU/mL ZJUIDS07 bacterial liquid. A total of 6 weeks of gastric lavage test was performed. After the test is finished, samples such as blood liver, intestinal contents and the like are collected for analysis and measurement.

5. Index measurement

Reference is made to the instructions of the Nanjing build or Beijing pride kit, etc., specifically as follows:

5.1 detection of serum Biochemical indicators and inflammatory factors

After the end of the administration, the anesthetized mice were heart-sampled, centrifuged at 3000×g for 10 minutes at 4 ℃ overnight, and serum was isolated. ELISA method is used for detecting serum triglyceride, total cholesterol, high density lipoprotein, low density lipoprotein, IL-8, IL-10, IL-1β, TNF- α, glycosylated hemoglobin and insulin levels of mice, and the operation methods are all carried out according to the instruction of the kit. And (3) taking a repeated average value for 3 times, drawing a standard curve according to the standard liquid absorbance result and the standard liquid concentration, obtaining a conversion equation, and calculating each index level.

5.2 liver index determination

100mg of liver is accurately weighed, and the lysate is added in a proportion of 1mg of liver to 20 mu L of lysate. The tissue is crushed by a full-automatic sample rapid grinding instrument, then the tissue is kept stand for 10min, a proper amount of supernatant is transferred into a 1.5mL centrifuge tube, the liver SOD, GSH-Px, MDA, GSH and liver glycogen levels of the mice are detected by adopting an ELISA method, and the operation method is carried out according to the instruction of the kit. And (3) taking a repeated average value for 3 times, drawing a standard curve according to the standard liquid absorbance result and the standard liquid concentration, obtaining a conversion equation, and calculating each index level.

5.3 short chain fatty acid assay

Extruding the segmented colon slice with sterile forceps, taking out the content, and storing in a-80deg.C low-temperature storage tube. The colon contents were diluted five times with ultrapure water and vortexed for 3 minutes. Next, the suspension was allowed to stand for 5 minutes, and then centrifuged at 5000 Xg for 20 minutes at 4 ℃. One milliliter of the supernatant was mixed with 20. Mu.L of chromatographic grade phosphoric acid, and the mixture was injected into a chromatographic bottle through a 0.45 μm membrane filter for gas chromatography. The gas chromatograph consisted of an AOC-20S autosampler and GC-2010 equipped with a flame ionization detector. Nitrogen was used as a carrier gas at a flow rate of 3ml/min. An SH stable wax high-polarity column is arranged on the gas chromatograph, the sample injection quantity is 0.2 mu L, the split injection ratio is 50, and the injection temperature is 200 ℃. Ethyl acetate was injected as a blank solvent between each sample to eliminate any memory effect. The initial column temperature was set at 80℃and held for 1 minute, then increased to 170℃at a rate of 8℃per minute, then immediately increased to 220℃at a rate of 20℃per minute and held for 4 minutes. The total time was 18.75 minutes. Finally, the content calculated by the SCFAs is calibrated by an external standard method according to the SCFAs standard curve.

5.4 analysis of the flora in the intestinal tract

Samples of the colon contents were collected for total DNA isolation and 16s rRNA high throughput sequencing techniques by the hangzhou Ming family organism. 16s rRNA was amplified in the V3-V4 region, the amplicon was purified using QIA rapid PCR purification kit, sequencing was performed by the Illumina Novaseq platform (PE 300), and the original sequence was quality controlled by UPARSE. The Operational Taxon (OTU) was constructed by binding sequences into clusters with sequence similarity greater than 97% using QIIME.

6. Analysis of experimental results

The results of fig. 4 demonstrate that the liver triglyceride, total cholesterol, and low density cholesterol levels were significantly higher in the hyperglycemic model group than in the control group, and that the high density cholesterol levels were significantly lower than in the control group, indicating that the lipid metabolism and cholesterol metabolism of the hyperglycemic model mice were affected. Compared with the hyperglycemia model group, the triglyceride, total cholesterol and low-density cholesterol levels of the liver of the lactobacillus rhamnosus ZJUISD07 group are obviously lower than those of the model group, and the high-density cholesterol is obviously higher than that of the control group. It was demonstrated that lactobacillus rhamnosus ZJUISD07 was able to reduce accumulation of lipids and cholesterol with better effect than the standard strain.

The results in FIG. 5 demonstrate that the serum IL-8, IL-1. Beta. And TNF-a levels were significantly higher in the hyperglycemic model group than in the control group, while IL-10 levels were significantly lower than in the control group, indicating an increased inflammatory level in the hyperglycemic model mice. The serum IL-8, IL-1. Beta. And TNF-. Alpha.were significantly reduced in the Lactobacillus rhamnosus ZJUISD07 group, while IL-10 levels were significantly increased compared to the hyperglycemia model group. The lactobacillus rhamnosus ZJUISD07 can reduce the inflammation level of the hyperglycemia mice, and the effect is superior to that of the standard strain.

The results in FIG. 6 show that Lactobacillus rhamnosus ZJUISD07 has hypoglycemic effect. Compared with the control group, the glycosylated hemoglobin and liver glycogen of the hyperglycemia model group are obviously higher than those of the control group, the dry prognosis of lactobacillus rhamnosus ZJUISD07 is obviously reduced, and the effect is better than that of a standard strain.

The results of fig. 7 demonstrate that lactobacillus rhamnosus ZJUISD07 is able to reduce serum fasting insulin levels in mice. Compared with the control group, the fasting serum insulin level of the hyperglycemia model group is obviously increased, and the fasting insulin level of the mice is obviously reduced after the lactobacillus rhamnosus ZJUISD07 is dried. It was demonstrated that lactobacillus rhamnosus ZJUISD07 was able to lower blood glucose in mice, thereby reducing the stimulatory effect on insulin.

The result of fig. 8 shows that lactobacillus rhamnosus ZJUISD07 has stronger antioxidant capacity. Normally, antioxidant enzymes in the natural antioxidant defense system of organisms act synergistically with antioxidants in diets or drugs to scavenge peroxides. Superoxide dismutase (SOD) is an antioxidant enzyme which can catalyze superoxide anions to perform disproportionation reaction in the metabolic process of human bodies, and the SOD activity is positively correlated with the antioxidant capacity of organisms. Glutathione peroxidase (GSH-Px) can protect cells, cell membranes and the like from peroxidation damage, and has important physiological activity in human bodies. On the other hand, the level of MDA, a lipid peroxidation product, was examined to indirectly determine the severity of the free radical attack on the cells. Reduced Glutathione (GSH) is a non-enzymatic antioxidant that is significant in maintaining normal operation of the body, consisting primarily of glycine residues, cysteine, and glutamate, and plays an important role in the primary biochemical metabolism within the human body. Compared with the control group, the SOD, GSH-Px and GSH of the hyperglycemia model group are obviously lower than the control group, and the MDA is obviously higher than the control group. Compared with the hyperglycemia model group, the performance of serum SOD and GSH-Px, GSH, MDA of the lactobacillus rhamnosus ZJUISD07 group is obviously improved, which indicates that the lactobacillus rhamnosus ZJUISD07 can enhance the antioxidation capability of the hyperglycemia mice.

The results in FIG. 9 show that Lactobacillus rhamnosus ZJUISD07 has the ability to promote intestinal short chain fatty acid synthesis, and that SCFA is the main metabolite produced by intestinal microbial fermentation. SCFA act as their metabolic end products, maintaining redox equivalents in the aerobically environment of the gut. SCFA are saturated fatty acids with 1-6 carbon atoms, with the most abundant SCFA (-95%) being acetic acid (C2), propionic acid (C3) and butyric acid (C4). The results show that the intervention of lactobacillus rhamnosus ZJUISD07 significantly increases the contents of acetic acid, propionic acid and butyric acid in intestinal tracts; the lactobacillus rhamnosus ZJUISD07 can improve intestinal flora and has obvious promotion effect on balance of intestinal microecology.

The results of fig. 10 show that lactobacillus rhamnosus ZJUISD07 promotes the content of probiotics bifidobacteria and S24-7_g in the intestinal tract, and the two bacteria are closely related to the balance and health of the intestinal tract, the health of reducing blood sugar, blood pressure and the like; at the same time, the content of harmful bacteria Turicibacter is reduced, and the strain has obvious influence on the blood sugar elevation. The results show that lactobacillus rhamnosus ZJUISD07 can significantly improve intestinal health.

Comparison: lactobacillus rhamnosus NAQU Plateau LR-1, lactobacillus plantarum (Lactobacillus plantarum) zjuds 04, lactobacillus rhamnosus (lactobacillus rhamnosus) BLCC2-0077 were subjected to the analysis of the flora in the intestinal tract by the above method according to the present invention, and the results were: the content of probiotic bifidobacteria and S24-7_g in the intestinal tracts corresponding to the 3 strains is not as good as that of ATCC53103 standard strain group.

Example 4 confirmation of antioxidant capacity of lactobacillus rhamnosus ZJUISD07 (see method described in CN113462613 a):

1. total antioxidant capacity (FRAP method)

The method for measuring the total antioxidant capacity was slightly modified according to the method of Giuberti et al. To the ELISA plate, 150. Mu.L of TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, mixed at V: V=10:1:1, as-prepared) and 20. Mu.L of sample were added, mixed with shaking, reacted at 37℃for 10min, and the absorbance of the solution at 593nm was measured. Substituting the absorbance measured by the sample into a ferrous sulfate standard curve, wherein the antioxidant capacity of the sample is expressed as ferrous sulfate equivalent (mu mol FeSO) ₄ /mL sample). Each sample was repeated 3 times and averaged.

Ferrous sulfate standard curve: ferrous sulfate solutions with different mass concentrations (0 mu M, 50 mu M, 100 mu M, 200 mu M, 400 mu M, 600 mu M and 800 mu M) were prepared, the ferrous sulfate solutions with different molar concentrations, 10mM TPTZ buffer and 0.3M acetate buffer were mixed according to V: V=1:1:10, 170 mu L of the mixed solution was added to an ELISA plate, the reaction was carried out at 37℃for 10min, and the absorbance of the solution at 593nm was measured. And drawing a standard curve by taking absorbance as an ordinate and ferrous sulfate mass concentration as an abscissa, and measuring.

2. Reducing ability

Determination of reducing ability reference is made to the method of Lin et al with some modifications. 1mL of the sample was placed in a centrifuge tube, and 1mL of each of a PBS buffer solution and 1% (w/v) potassium ferricyanide solution, 0.2M, pH6.6, and the mixture was mixed. Water bath at 50 ℃ for 20min, and ice bath cooling. Then, 1mL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 600 r/min for 5min, 1mL of the supernatant was collected, 1mL of 0.1% (w/v) ferric trichloride and 1mL of distilled water were added, and the mixture was mixed uniformly, allowed to stand for 10min, and absorbance was measured at 700 nm. The samples were replaced with PBS buffer or MRS broth as blank. Each sample was repeated 3 times and averaged.

Reduction capacity (%) = [ (As-Ab)/Ab ]. 100; wherein: as—absorbance of the sample set; ab—blank absorbance.

3. DPPH free radical scavenging ability

The method for measuring the DPPH radical scavenging ability was described with reference to Shimada et al and with some modifications. Preparing 1000mg/ml VC standard solution, and diluting to different concentration gradients (0-30 mu g/ml). Adding 100 mu L of a sample to be detected (or VC standard solution) and 100 mu L of 0.2mM DPPH ethanol solution (prepared by absolute ethanol, stored at 4 ℃ in a dark place and used at present) into an ELISA plate, shaking uniformly, keeping away from light at room temperature for 30min, and measuring the absorbance of the solution at 517 nm; 100 mu L of absolute ethyl alcohol is used to replace 100 mu L of DPPH ethanol solution to form a blank group; 100. Mu.L of PBS buffer (0.2M, pH6.6 PBS or MRS broth) was used as a control instead of 100. Mu.L of the sample to be tested, and the control was zeroed with 100. Mu.L of a mixture of PBS buffer (or MRS broth) and absolute ethanol. Each sample was repeated 3 times and averaged.

DPPH radical scavenging ability (%) = [1- (As-Ab)/Ac ] ×100; wherein: as—absorbance of the sample set; ab—blank absorbance; ac—control absorbance;

TABLE 3 antioxidant Activity of Lactobacillus rhamnosus ZJUISD07

* Representing significant differences, P <0.05; * Representing significant differences, P <0.01.

As shown in Table 3, the total antioxidant capacity, reducing capacity and DPPH free radical of lactobacillus rhamnosus ZJUISD07 fermentation supernatant and bacterial suspension obtained by screening according to the invention are higher than those of the standard strain ATCC53103, in particular to the reducing capacity. This demonstrates that lactobacillus rhamnosus ZJUISD07 fermentation supernatant and bacterial suspension have high antioxidant capacity.

Example 5 confirmation of bile salt hydrolase activity of lactobacillus rhamnosus ZJUISD07 (see method described in CN113462613 a):

1. qualitative determination of bile salt hydrolase produced by lactobacillus rhamnosus ZJUISD07

Adding 0.3% (m/v), i.e., 3g/1000 ml), sodium deoxytaurocholate, 0.2% (m/v) sodium thioglycolate, 0.37g/L CaCl to freshly prepared MRS agar medium ₂ It was completely dissolved. Sterilizing at 121deg.C for 15min, pouring into sterile flat plate, placing sterile filter paper sheet into flat plate after solidification, and dripping 10 μl lactobacillus rhamnosus ZJUISD07 bacterial suspension (about 10 ⁸ CFU/mL) was added dropwise with 10 μl of sterile phosphate buffer as a blank. Plates were anaerobically incubated in an anaerobic jar (OXOID) at 37℃for 72h. A white precipitate around the filter paper sheet is considered to have bile salt hydrolase activity.

Description: ZJUIDS07 cells obtained in the above example were resuspended in PBS buffer (0.2M, pH 7.0) to obtain ZJUIDS07 cell suspension (about 10) ⁸ CFU/mL)。

2. Quantitative determination of Lactobacillus rhamnosus ZJUISD07 bile salt hydrolase activity

Preparation of cells and bacterial suspensions, etc. referring to the above examples, 0.1mL of a cell disruption supernatant solution of Lactobacillus rhamnosus ZJUISD07 was taken, and 1.8mL of 0.1mol/L PBS buffer (0.2M, pH 7.0) and 0.1mL of 6mmol/L sodium taurocholate were added thereto. These mixtures were incubated at 37℃for 30min, after which 0.5mL of 15% trichloroacetic acid was added to terminate the enzymatic reaction. This was centrifuged, and 0.5mL of the supernatant was added to 1mL of ninhydrin color solution. Mixing the above materials under vortex vibration, and boiling for 35min. After cooling, the absorbance at 570nm was measured. One bile salt hydrolase activity unit is defined as the amount of enzyme required to release 1. Mu. MoL taurine per minute from the substrate.

3. The above-mentioned determination of bile salt hydrolase activity was performed using lactobacillus rhamnosus (Lactobacillus rhamnosus) ATCC53103 as a positive control. The determination of protein concentration uses bovine serum albumin as a standard and all experiments were repeated 3 times.

4. Table 4 shows quantitative measurement results of bile salt hydrolase activities of two strains. As can be seen from Table 4, the bile salt hydrolase activities of both strains were 1.0U/mg or more. Experimental results show that lactobacillus rhamnosus ZJUISD07 has higher bile salt hydrolase activity.

TABLE 4 results of bile salt hydrolase assay of strains

Note that: + indicates that a precipitation ring is produced, -indicates that no precipitation ring is produced.

5. Bile salt hydrolase can hydrolyze in vivo combined bile salt into free bile salt, and the free bile salt does not participate in liver and intestine circulation and is discharged out of the body along with feces, so that the activity of the bile salt hydrolase is a key factor for reducing blood sugar in the body. The lactobacillus rhamnosus ZJUISD07 provided by the invention has stronger bile salt hydrolase activity.

Example 6 confirmation of acid resistance and bile salt resistance of lactobacillus rhamnosus ZJUISD07 (see method described in CN113462613 a):

1. acid resistance test

Lactobacillus rhamnosus ZJUISD07 single colony is picked up and cultured for 18 hours at 37 ℃ in an MRS liquid culture medium, and the amplified bacterial suspension is inoculated into the MRS liquid culture medium in an amount of 1 percent and cultured for 18 hours at 37 ℃. The culture broth was centrifuged at 8000r/min for 5min at 4℃to collect the cells, which were washed 2 times with PBS buffer (pH 6.8,0.1 mol/L). The cells were suspended in MRS liquid medium with pH adjusted to 3.0 in advance, and the initial viable count was adjusted to about 10 ⁸ CFU/mL, cultured at 37℃for 3h. Counting living bacteria in 0h and 3h samples by adopting a pouring plate method, culturing the poured plates for 48h at 37 ℃, and measuring the survival rate, wherein the calculation formula of the survival rate is as follows:

in the above, N ₀ Viable count (CFU/mL) for 0h of test strain; n (N) _t The strain was tested for viable count (CFU/mL) for 3h.

2. Experiment of bile salt resistance

Inoculating activated lactobacillus rhamnosus ZJUISD07 suspension into MRS liquid culture medium at 1%, culturing at 37deg.C for 18 hr, mixing with vortex, and correcting initial viable count to about 10 ⁹ CFU/mL. The cells were inoculated in an amount of 10% into MRS liquid medium containing 0.3% (m/v) of bovine bile salt (control MRS liquid medium containing no bovine bile salt) and cultured at 37℃for 3 hours. The number of viable bacteria in the sample was then counted using the pour plate method. The poured plate was incubated at 37℃for 48h. The bile salt tolerance of the strain is expressed as the logarithm of the difference between the number of viable bacteria per milliliter of bile salt-containing medium at 3 hours and the number of viable bacteria in the non-bile salt-containing medium (log CFU/mL).

3. The acid and bile salt resistance was measured using lactobacillus rhamnosus (Lactobacillus acidophilus) ATCC53103 as a control.

4. As shown in Table 5, the acid and bile salt resistance of Lactobacillus rhamnosus ZJUISD07 is significantly better than that of the control strain ATCC53103. Experiments prove that lactobacillus rhamnosus ZJUISD07 has higher gastrointestinal tract viability.

TABLE 5 results of acid and bile salt tolerance by strains

Strain	Acid resistance (%)	Bile salt tolerance (DeltaLog CFU/mL)
			Lactobacillus rhamnosus ZJUISD07	71.54±0.36	1.24±0.13
Lactobacillus rhamnosus ATCC53103	62.12±0.18	1.03±0.23

5. Probiotics must be able to withstand a range of adverse conditions in the gastrointestinal tract such as gastric acid and bile to survive their probiotic action. The lactobacillus rhamnosus ZJUISD07 provided by the invention can grow and proliferate under the condition of pH 3.0, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. Meanwhile, lactobacillus rhamnosus ZJUISD07 can resist bile salts, can survive in intestinal tracts, and further can effectively improve intestinal flora and further play a role in reducing blood sugar.

Example 7 confirmation of the hydrophobic ability of lactobacillus rhamnosus ZJUISD07 (see method described in CN113462613 a):

1. measurement of hydrophobicity

The lactic acid bacteria pellet was washed twice with clean PBS buffer (0.1 mol/L, pH 6.8) and resuspended to OD ₆₁₀ The absorbance of the obtained solution is about 0.5, and the lactobacillus suspension is obtained.

Thoroughly mixing 2ml lactobacillus suspension and 2ml xylene, shaking in 37 deg.C water bath for 5min, and measuring OD of water phase after 0 hr and 2 hr respectively ₆₁₀ Absorbance values.

A ₀ Absorbance value=0h, a _t Absorbance of =th.

The results obtained are shown in Table 6 below.

TABLE 6 surface hydrophobicity of different strains (%)

2. Analysis of results

The hydrophobicity of lactobacillus rhamnosus ZJUISD07 is measured to be 42.36 percent, which is obviously higher than that of a control standard strain. The strain has strong adhesion capability, can adhere to human intestinal tracts, and improves the health of intestinal flora.

Example 8 confirmation of antibiotic susceptibility of lactobacillus rhamnosus ZJUISD07 (see method described in CN113462613 a):

culturing for 18h at a concentration of about 10 ⁷ CFU/mL lactobacillus rhamnosus ZJUISD07 bacterial suspension is added into a sterilized MRS agar culture medium cooled to about 45 ℃ according to the amount of 1%, fully mixed and quantitatively added into 15 mL/dish. After solidification, the drug sensitive paper is taken by forceps and placed on the culture medium. The dishes were placed right side up in a 37℃incubator for 24h. Paper sheets without antibiotics were used as blank. And measuring the diameter of the inhibition zone. Each was repeated three times.

The diameter of the antibiotic susceptibility zone of lactobacillus rhamnosus ZJUISD07 is shown in table 7. Reference to CLSI (2017) drug sensitivity test criteria can be obtained that lactobacillus rhamnosus ZJUISD07 exhibits sensitivity to penicillin G, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, compound neonomine and chloramphenicol. Intermediation is presented for ciprofloxacin. Experimental results show that lactobacillus rhamnosus ZJUISD07 is sensitive to common antibiotics.

TABLE 7 results of sensitivity of Lactobacillus rhamnosus ZJUISD07 to antibiotics

Note that: s, sensitivity; i, intermediation; r, drug resistance

With the wide application of antibiotics in clinical treatment, the drug resistance of lactobacillus is also more and more serious, and long-term intake of the drug-resistant lactobacillus can bring great difficulty to clinical treatment. The lactobacillus rhamnosus ZJUISD07 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.

Example 9 confirmation of pathogenic bacteria inhibitory capacity of lactobacillus rhamnosus ZJUISD07 (method described with reference to CN113462613 a):

the antibacterial activity of the lactic acid bacteria is measured by an international agar diffusion method. 10mL of LB agar medium was poured into a sterile dish and cooled to obtain a lower medium. Culturing for 18h to a concentration of about 10 ⁷ CFU/mL indicator fungus suspension is added into the sterilized LB agar medium cooled to about 45 ℃ according to the amount of 1%, and the mixture is fully mixed and quantitatively added into 10 mL/dish. Placing the sterilized oxford cup on the cup. After the upper culture medium is condensed, the oxford cup is gently pulled out. Samples of lactobacillus rhamnosus ZJUISD07 fermentation supernatant were dosed at 100 μl/well and PBS buffer (0.1 mol/L, pH 6.8) was used as a control. The strains with obvious inhibition zones around the small holes are selected, the diameters of the inhibition zones are measured, and each is repeated three times.

As shown in Table 8, the metabolites of Lactobacillus rhamnosus ZJUISD07 have certain inhibition effects on pathogenic bacteria such as staphylococcus aureus, escherichia coli, salmonella enteritidis, listeria monocytogenes and the like, and are superior to the antibacterial effect of ATCC 53103. The metabolite of the strain has antibacterial property.

TABLE 8 results of the inhibition ability of strains against pathogenic bacteria

Staphylococcus aureus is the most common pathogen in suppurative infection of humans, some escherichia coli can cause severe diarrhea and septicemia, and some salmonella species can also cause food poisoning in humans. Bacteriocin, organic acid, hydrogen peroxide and other antibacterial substances generated by lactic acid bacteria metabolism can inhibit the growth of pathogenic bacteria singly or jointly. The metabolite of lactobacillus rhamnosus ZJUISD07 provided by the invention has a certain antagonism to the three pathogenic bacteria, plays an important role in maintaining intestinal microecological balance and has a health promoting effect.

Example 10 preparation of functional fermented yogurt Using Lactobacillus rhamnosus ZJUISD07

1. The processing process flow of the yoghourt comprises the following steps:

raw material preheating, homogenizing, blending, sterilizing, cooling, preparing and inoculating, fermenting, after-ripening and refrigerating

2. Key points of operation

(1) Raw materials: 2L whole UHT sterilized milk or fresh cow milk;

(2) Preheating: placing the mixture into a container and heating to 63 ℃;

(3) Homogenizing: homogenizing in a homogenizer under 15-25 MPa), pouring the mixed solution into an iron tank, adding 100g white sugar, and sterilizing in water bath at 90deg.C for 10min;

(4) And (3) blending: adding ingredients into cow milk, and dissolving;

(5) Sterilizing: sterilizing the sweetened milk in water bath at 90deg.C for 10min;

(6) And (3) cooling: cooling sterilized cow milk to 40-50deg.C for use;

(7) Preparing a starter: lactobacillus rhamnosus ZJUISD07 strain was inoculated in a tube containing sterilized skim milk (12%, w/v) under aseptic conditions and cultured at 37 ℃ for 20 hours. The inoculation amount for each passage is 2-4% (v/v), the passage is carried out for 2-3 times to restore activity, and the culture medium is placed in a refrigerator at 4 ℃ for preservation;

(8) Inoculating and fermenting: under aseptic condition, activated lactobacillus rhamnosus ZJUISD07 is inoculated with an inoculum size of 2-4% (v/v). Fermenting at 42 deg.C for 6-10 hr;

(9) Post-ripening: after fermentation, placing the mixture in a refrigerator at the temperature of 4 ℃ for after-ripening for 12-24 hours;

(10) Filling and refrigerating: after finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.

Example 11 preparation of functional fermented fruit and vegetable juice Using Lactobacillus rhamnosus ZJUISD07

1. Process flow of fermented fruit and vegetable juice

Raw material cleaning, flash evaporation, pulping, blending, homogenizing, sterilizing, cooling, inoculating, sealing fermentation, after-ripening, filling and refrigerating

2. Key points of operation

(1) Raw materials: fresh pumpkin and dragon fruit are selected.

(2) Cleaning and cutting into blocks: cleaning, peeling (removing pulp of fructus Cucurbitae Moschatae), and cutting into small pieces.

(3) Flash evaporation: the enzyme is deactivated by flash evaporation, 0.5 to 1min is treated at 121 ℃, and the gas is rapidly exhausted.

(4) Pulping: according to the proportion of pumpkin to water (weight ratio) =1:1, a proper amount of pumpkin and water are gradually put into a colloid mill for grinding, and coarse grinding and fine grinding are carried out once. Pulping the dragon fruits by a pulping machine until pulp is uniform and has no lumps.

(5) Blending and homogenizing: 15% of pumpkin juice, 30% of dragon fruit juice, regulating the content of soluble solids to 10 degrees Brix by using sucrose, adding 0.2% of stabilizer CMC, uniformly mixing, and adopting a two-stage homogenization method, wherein the diameter and the grain size of the melon pulp are 2-3 mu m by adopting a low pressure (15 MPa) and a high pressure (25 MPa) firstly.

(6) Sterilizing and cooling: preserving the heat of the prepared compound fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃.

(7) Inoculating and fermenting: under aseptic condition, activated lactobacillus rhamnosus ZJUISD07 is inoculated, and the initial bacterial count is controlled at 10 ⁷ CFU/mL. Fermenting at 37deg.C for 24 hr.

(8) Post-ripening: after fermentation, the mixture is put into a refrigerator at 4 ℃ for 3 hours.

(9) Filling and refrigerating: after finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.

EXAMPLE 12 preparation of functional fermented Carnis Sus Domestica with Lactobacillus rhamnosus ZJUISD07

1. The processing technology flow of the fermented sour meat comprises the following steps:

raw material, slicing, mixing glutinous rice flour, inoculating, fermenting, pickling, packaging and obtaining finished products

2. Key points of operation

(1) Slicing: commercially available fresh streaky pork was cut into 3cm square pieces.

(2) Mixing glutinous rice flour: 1000g of raw meat and 250g of glutinous rice flour are mixed uniformly, and 1% of glucose is added.

(3) Inoculating and fermenting:under aseptic condition, activated lactobacillus rhamnosus ZJUISD07 is inoculated, and the initial bacterial count is controlled at 10 ⁷ CFU/mL. Fermenting at 37deg.C for 18 hr.

(4) Pickling: adding 2% salt, and pickling at 25deg.C for 20d.

EXAMPLE 13 preparation of hypoglycemic powder Using Lactobacillus rhamnosus ZJUISD07

1. Preparation of lactobacillus rhamnosus ZJUISD07 bacterial mud

Lactobacillus rhamnosus ZJUISD07 single colony is selected and inoculated into 50mL of MRS liquid culture medium, and the culture is carried out in a 37 ℃ incubator for 18 hours. Activated again in 250mL MRS liquid culture medium according to the inoculum size of 5%, and placed in a 37 ℃ incubator for 24 hours. Finally, the activated lactobacillus rhamnosus ZJUISD07 is subjected to high-density anaerobic culture in a 10L fermentation tank at an inoculum size of 5%, and is cultured for 18 hours at 37 ℃ and pH of 6.8. Then centrifuging at 8000r/min and 4deg.C for 15min, discarding supernatant, collecting bacterial precipitate, and rinsing bacterial cells with sterile phosphate buffer solution (pH 7.0) for 2 times. Obtaining lactobacillus rhamnosus ZJUISD07 bacterial mud.

2. Preparation of protective agent

The lyoprotectant comprises 15% of skim milk powder, 5% of trehalose, 3% of sodium glutamate, 1% of glycerol and 0.5% of cysteine hydrochloride. Water was used as solvent. Sterilizing at 110deg.C.

3. Preparation of lactobacillus rhamnosus ZJUISD07 bacterial powder

And fully and uniformly mixing the lactobacillus rhamnosus ZJUISD07 bacterial precipitate prepared by the method with a protective agent solution according to the proportion of 1:5. Pre-freezing for 5 hours at the temperature of minus 40 ℃ to uniformly freeze the lactobacillus rhamnosus ZJUISD07 bacterial powder on the inner wall of the container, and then performing vacuum freeze drying for 18-20 hours. After rehydration with normal saline, washing twice, the number of viable bacteria in lactobacillus rhamnosus ZJUISD07 bacterial powder is 1.0X10 ¹¹ ～1×10 ¹² CFU/g。

EXAMPLE 14 preparation of probiotic solid drink Using Lactobacillus rhamnosus ZJUISD07

The preparation method of the probiotic solid drink by lactobacillus rhamnosus ZJUISD07 comprises the following steps:

(1) Preparation of probiotic bacteria powder: the lactobacillus rhamnosus ZJUISD07 probiotic powder is prepared by the above examples;

(2) The solid beverage comprises the following formula: edible corn starch, white granulated sugar (10%), fructo-oligosaccharide (6%), isomaltooligosaccharide (9%), citric acid, edible essence and probiotic bacteria powder (2-5%);

(3) Added according to the formula and evenly mixed, ensures that lactobacillus rhamnosus ZJUISD07 is between 10 ⁹ CFU/g or more.

(4) Packaging according to 10/20g of each small bag, and packaging 10-20 small bags into a large bag.

Preparation of a composite probiotic solid beverage: other powders (or purchased) were prepared with reference to the above examples of the invention, such as lactobacillus plantarum, lactobacillus paracasei, according to lactobacillus rhamnosus ZJUISD07: lactobacillus plantarum: mixing lactobacillus paracasei at a ratio of 1:1:1, and blending and packaging according to the solid beverage formula to obtain the composite probiotic solid beverage. The acid resistance test was performed by referring to the above examples for the compounded bacteria, and the results are shown in table 9: after the lactobacillus rhamnosus ZJUISD07 is compounded with the two strains, the acid resistance is obviously improved, so that the strain can better resist gastric acid and reach intestinal tracts.

TABLE 9 acid resistance of the strains (%)

Example 15 preparation of silage Using Lactobacillus rhamnosus ZJUISD07

The procedure for preparing alfalfa silage by lactobacillus rhamnosus ZJUISD07 is as follows:

(1) Raw material preparation: after the alfalfa is harvested, the alfalfa is ensured to be clean and free from spoilage, and is cut off to 1-2 cm.

(2) Modulation of silage: the water content is controlled at 60-65% when the alfalfa is silaged, 200g alfalfa raw materials are weighed and put into a vacuum packaging bag, lactobacillus rhamnosus ZJUISD07 bacterial powder is inoculated into the raw materials according to 0.02 per mill inoculum size, and the raw materials are subjected to vacuum packaging by a vacuum packaging machine and then are subjected to fermentation in a storage room.

(3) Storage conditions: the temperature is 15-45 ℃ and the time is 30-60 d.

Example 16 preparation of probiotic milk powder for pets Using Lactobacillus rhamnosus ZJUISD07

1. Preparation of lactobacillus rhamnosus ZJUISD07 bacterial powder

Referring to the above cases, lactobacillus rhamnosus ZJUISD07 bacterial powder freeze-dried bacterial powder is prepared, wherein the number of viable bacteria in the bacterial powder is 1.0X10 ¹¹ ～1×10 ¹² CFU/g。

2. Preparation of pet formula powder

Primary selection of raw materials: milk powder, fish meal, bone meal, cereal and vegetable oil, additives: vitamins, trace elements, functional factors and others;

automatic batching: throwing the obtained material raw materials into a material bin according to a formula;

crushing: crushing the weighed materials by a crusher;

mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniform mixing;

puffing: making the mixed materials into granular materials through a bulking machine;

and (3) drying: drying the mixed materials by a dryer, wherein the temperature is controlled to be 65-70 ℃;

and (3) classifying and screening: the stream was passed through a classifying screen with the particles controlled at 2.5-5 mm.

3. Preparation of probiotic formula powder for pets

The bacterial powder prepared in the step 1 and the pet feed prepared in the step 2 are mixed according to the following steps: 100, and the viable bacteria leaving the factory in the final product is 10 ⁸ CFU/g or more. After the product is filled, the product is put in storage for sale.

Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.

Claims (5)

1. Lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07, characterized by: the preservation number is CGMCC NO.23996.

2. Lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 according to claim 1, characterized in that: the 16S rDNA complete sequence of lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 is shown in SEQ ID No: 1.

3. Use of lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 as claimed in claim 1 or 2 for the manufacture of a product with hypoglycemic activity.

4. A use according to claim 3, characterized in that: the product with the blood sugar reducing function comprises a live bacterial preparation of lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS 07.

5. The use according to claim 4, characterized in that: in the viable bacteria preparation, lactobacillus rhamnosus (Lactobacillus rhamnosus) ZJUIDS07 has viable bacteria number of 1.0X10 ¹¹ ～1×10 ¹² CFU/g。

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