Lactobacillus plantarum VB165 and application thereof

Abstract

The invention provides a lactobacillus plantarum VB165, which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on September 28, 2022, and the preservation number is CGMCC NO.25839. The lactobacillus plantarum VB165 is named as VB165. The lactobacillus plantarum VB165 has the advantages that the lactobacillus plantarum VB165 is named as VB165; under the condition of simulating the intestinal environment, the strain can inhibit the activity of alpha-glucosidase, also has the activity of degrading cholate and inhibiting main intestinal pathogens, has good tolerance to gastric acid and choline, and can be applied to preparation of blood sugar control products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a plant lactobacillus and application thereof in a blood sugar-lowering product. Background Art

[0002] Lactobacillus plantarum is a type of lactic acid bacteria, with an optimal growth temperature of 30-35°C, anaerobic or facultative anaerobic, and belongs to homofermentative lactic acid bacteria. As a member of the genus Lactobacillus, Lactobacillus plantarum has been widely used in food production, including kimchi, winemaking, fermented yogurt, fermented juice, synbiotic preparations, microecological preparations, etc. At the same time, Lactobacillus plantarum is used in aquaculture, animal husbandry, etc. because of its high number of live bacteria and its ability to produce lactobacillus, a biological preservative.

[0003] Lactobacillus plantarum is widely distributed in nature, existing in the intestines of humans and insects, dairy products, fruits and vegetables, and has a strong ability to metabolize sugars. As a beneficial intestinal microorganism, Lactobacillus plantarum can occupy and colonize in the gastrointestinal tract through competitive inhibition, inhibit pathogenic bacteria from invading the gastrointestinal tract, regulate the balance of intestinal flora, and also has immunomodulatory functions, such as regulating the proliferation capacity of spleen cells and affecting the balance of Th1 / Th2 cells. Lactobacillus plantarum is widely distributed in the intestines of adults, and the human body has immune tolerance to it and will not cause immune rejection effects. The U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) have recognized that Lactobacillus plantarum is safe, and Chinese laws and regulations have also approved the use of Lactobacillus plantarum in food and health foods.

[0004] The incidence of diabetes has increased dramatically around the world. Most T2DM patients in China have elevated postprandial blood glucose (PPG). Elevated PPG is directly related to the development of diabetic complications. Controlling PPG is an important strategy to control the occurrence and development of diabetic complications. α-glucosidase inhibitors inhibit α-glucosidase on the intestinal mucosa, slow down the rate at which starch is broken down into glucose, and reduce the absorption of glucose in the small intestine. They have a very obvious effect on controlling PPG. Currently, the commonly used α-glucosidase inhibitors in clinical practice are mainly acarbose and voglibose. These drugs often cause gastrointestinal adverse reactions, such as abdominal discomfort, gastrointestinal flatulence, increased flatulence, and diarrhea.

[0005] Hyperlipidemia can easily cause cardiovascular and cerebrovascular diseases, such as myocardial infarction, coronary atherosclerotic heart disease, and can easily induce acute pancreatitis. Bile salts play an important role in fat digestion. They can reduce the surface tension of fat, emulsify fat into very fine chylomicrons, increase the contact area between fat and enzymes, and facilitate the decomposition and absorption of fat. At the same time, bile acid is mainly synthesized in the liver and is an important pathway for cholesterol metabolism. One-third of cholesterol catabolism is achieved through bile acid synthesis. Bile salts are sodium or potassium salts formed by the combination of bile acid and glycine or taurine. The consumption of bile salts will not only reduce the absorption of fat, but also promote the metabolism of cholesterol in the direction of bile acid, thereby decomposing more cholesterol. Summary of the invention

[0006] The present invention provides a Lactobacillus plantarum VB165 screened from adult feces, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 28, 2022, with a deposit number of CGMCC NO. 25839. With adult feces as the isolation source, the obtained strain has higher safety and is more suitable for the production of health products.

[0007] The present invention provides a plant lactobacillus capable of effectively inhibiting the activity of α-glucosidase. By utilizing the plant lactobacillus' intestinal colonization and strict anaerobic characteristics, the plant lactobacillus can inhibit the activity of α-glucosidase in the intestine, degrade bile salts, and inhibit major intestinal pathogens without causing adverse reactions. The plant lactobacillus can bring many medical and health benefits in terms of controlling the increase of host PPG, improving hyperlipidemia and cholesterol, and antagonizing intestinal infections.

[0008] The plant lactobacillus provided by the present invention is in the form of living cells, for example, the strain is in the form of bacterial culture fluid.

[0009] The plant lactobacillus provided by the present invention is in the form of non-living cells, for example, the strain is in the form of freeze-dried powder.

[0010] The plant lactobacillus provided by the present invention can be used to prepare one or more of medicines, health products, foods, beverages, additives and feeds for controlling or lowering blood sugar, and preferably controls or lowers blood sugar by inhibiting α-glucosidase activity.

[0011] The plant lactobacillus provided by the invention can be used to prepare one or more of medicines, health products, foods, beverages, additives and feeds for degrading bile salts.

[0012] The plant lactobacillus provided by the invention can be used to prepare one or more of medicines, health products, foods, beverages, additives and feeds for inhibiting the activity of intestinal pathogens.

[0013] The present invention provides a composition comprising the above Lactobacillus plantarum. The composition may comprise other probiotic materials and may further comprise prebiotic materials.

[0014] The composition provided by the present invention further comprises an ingestible carrier, which can be a pharmaceutically acceptable carrier, such as a capsule, a tablet or a powder; or it can be a food, such as acidified milk, yogurt, frozen yogurt, milk powder, condensed milk, cheese sauce, condiment or beverage.

[0015] The plant lactobacillus provided by the present invention can be subjected to anaerobic fermentation in a nutrient medium containing an assimilable carbon source and / or nitrogen source.

[0016] In the preferred technical solution, the assimilable carbon source is selected from starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol or any combination thereof; preferably glucose.

[0017] In the preferred technical solution, the assimilable nitrogen source is selected from yeast extract, yeast powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor, soybean meal, peptone, urea, ammonium salt, or any combination thereof; preferably yeast extract.

[0018] In the preferred technical solution, the nutrient medium also includes an inorganic salt, which is selected from one of trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate, or any combination thereof; preferably dipotassium hydrogen phosphate and magnesium sulfate.

[0019] Beneficial effects:

[0020] The plant lactobacillus VB165 provided by the present invention has a strong inhibitory effect on α-glucosidase, and can control or reduce blood sugar through this approach; it can also degrade bile salts, and improve the state of hyperlipidemia and hypercholesterolemia through this approach; and it has strong abilities of inhibiting major intestinal pathogens, acid resistance and bile salt resistance, and can be used as a probiotic for the preparation of blood sugar lowering or blood sugar control products.

[0021] Collection information:

[0022] Strain name: Lactobacillus plantarum VB165

[0023] Date of deposit: September 28, 2022

[0024] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0025] Deposit number: CGMCC NO.25839 BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the inhibition test of α-glucosidase activity by acarbose, blank culture medium, Lactobacillus rhamnosus LGG and Lactobacillus plantarum VB165 provided by the present invention are shown. DETAILED DESCRIPTION

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0029] The MAGEN bacterial genomic DNA extraction kit used was purchased from Shanghai Maigen Biotechnology Co., Ltd. Phusion high-fidelity DNA polymerase was purchased from Thermo Scientific.

[0030] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.

[0031] In the culture medium used in the following examples, the percentage (%) used in the material ratio is the mass percentage unless otherwise specified.

[0032] Example 1 Bacterial source

[0033] Lactobacillus plantarum VB165 is isolated from adult feces. Take fresh adult feces and add sterile PBS buffer in a sterile container. After sufficient shaking, dilute and spread on MRS solid culture medium plates. After 48 hours of culture, pick single colonies of different sizes and shapes and streak on fresh plates. Then pick single colonies to the slant. After the slant is cultured, the colony morphology is examined under a microscope. After the microscopic examination, identification is performed after no miscellaneous bacteria are found. Using adult feces as the isolation source, the obtained strain has higher safety and is more suitable for the production of health products.

[0034] Example 2: Bacterial species identification

[0035] The experiment was conducted according to the relevant contents in the book "Molecular Cloning Experiment Guide". After the purified strains were cultured in MRS medium for 1 day, 1 mL of bacterial solution was centrifuged at 10,000 rpm for 1 min, the supernatant was discarded, the bacteria were collected, and DNA was extracted according to the operation steps of the MAGEN Bacterial Genomic DNA Extraction Kit. Using the extracted DNA as a template, 16S rDNA was amplified by PCR (polymerase chain reaction) using universal primers 337F (5′-GACTCCTACGGGAGGCWGCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). The PCR reaction system was 50 μL, and the reaction system and program were configured according to the instructions of Phusion high-fidelity DNA polymerase, with an annealing temperature of 55°C. The PCR product was sent to Shanghai Bioengineering for sequencing.

[0036] After the 16S rDNA sequence of the strain was collated, a homologous sequence BLAST comparison was performed with the sequences of related species and genera in the GenBank database to determine the taxonomic status of the strain. After comparison, it was found that its homology with Lactobacillus plantarum strain 1942 was as high as 99.93%, and its homology with Lactobacillus plantarum strain 1809 was as high as 99.86%, so the strain was identified as a Lactobacillus plantarum strain. The internal number of this strain is VB165, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 28, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.25839.

[0037] 16S sequencing sequence:

[0038]

[0039] Example 3: Bacteria fermentation

[0040] 1. Bacteria activation

[0041] Take the glycerol tube of the original strain of VB165, thaw it at room temperature, draw 0.1 ml of the bacterial suspension and inoculate it onto the MRS solid plate, spread it evenly, put the plate into an anaerobic bag (Haibo Biotechnology), and culture it in a 37°C incubator for 48 hours to obtain activated single colonies.

[0042] 2. Seed preparation

[0043] Take a single colony that has been activated, scrape a loop of colonies with an inoculation loop, and then inoculate it into a 500ml triangular flask containing 100ml MRS liquid culture medium, wrap it up and put it into an anaerobic bag, shake and culture it on a shaker at 37℃ and 70rpm for 48h, and control the OD value of the seed liquid to be ≥2.0. Adjust the pH of the MRS liquid culture medium to 7.0±0.2 before disinfection, and the disinfection conditions are 121~123℃ and 30min.

[0044] 3. Fermentation preparation

[0045] The cultured seed liquid is inoculated into a 500ml Erlenmeyer flask containing 100ml liquid culture medium at a ratio of 3-5% (V / W). The culture medium is MRS, or artificial intestinal fluid (containing enzymes, plus BD MRS, Coolaber), or artificial intestinal fluid plus 1% soluble starch. The culture is wrapped and placed in an anaerobic bag. The culture is shaken on a shaker at 37°C and 70rpm for 24-48h, and the OD value of the fermentation liquid is controlled to be ≥3.0. The pH of the MRS liquid culture medium is adjusted to 7.0±0.2 before disinfection, and the disinfection conditions are 121-123°C and 30min.

[0046] MRS liquid medium: yeast extract 0.5%, peptone 1.0%, beef extract 1.0%, glucose 2.0%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005% (the above are mass percentages), Tween 80 0.1% (volume / mass ratio).

[0047] MRS solid medium: Add 1.5% agar powder to MRS liquid medium.

[0048] Artificial intestinal fluid: contains enzymes, BD MRS, Coolaber.

[0049] Artificial intestinal fluid + starch: artificial intestinal fluid, 1% soluble starch.

[0050] Example 4: Detection of the inhibitory ability of α-glucosidase activity

[0051] In order to detect the ability of strains to produce AGI in different environments, especially intestinal environments, artificial simulated small intestinal fluid was used to simulate the intestinal state, and artificial small intestinal fluid with 1% soluble starch added was used to simulate the state of carbohydrate intake. The inhibitory activity of the fermentation supernatant of VB165 and Lactobacillus rhamnosus LGG (ATCC53103) on α-glucosidase was compared in these environments.

[0052] The culture medium was centrifuged after 48 hours of cultivation to obtain the fermentation supernatant. The reaction system of the α-glucosidase activity experiment was 100 μL. First, 25 μL of the substrate 4-nitrophenol-α-D-pyranoglucoside (PNPG, 10 mmol / L, Solebao) and 25 μL of the sample were mixed and incubated at 37°C for 10 min, and then 50 μL of α-glucosidase (40 U / L, Sigma) was added and mixed and reacted at 37°C for 30 min. Subsequently, 100 μL of Na2CO3 (0.1 mol / L) was quickly added to terminate the reaction, and the absorbance at 405 nm was measured with an enzyme reader. Three parallel experiments were set for each group of experiments. The absorbance of the sample was calibrated with the absorbance of the sample blank control (50 μL PBS was used instead of α-glucosidase in the system). The negative control (no sample inhibiting α-glucosidase in the system) used 25 μL PBS instead of the sample, and the negative blank control (no α-glucosidase activity in the system) used 25 μL PBS instead of the sample and 50 μL PBS instead of α-glucosidase.

[0053] The calculation formula of α-glucosidase inhibition rate is:

[0054] α-glucosidase inhibition rate = (1-(AB) / (CD)) × 100%

[0055] Wherein: A is the absorbance of the sample at 405nm; B is the absorbance of the sample blank control at 405nm; C is the absorbance of the negative control at 405nm; D is the absorbance of the negative blank control at 405nm.

[0056] See attached for the results Figure 1, the inhibition rate of acarbose 10μM on α-glucosidase was 92.3% (MRS), 97.0% (artificial intestinal fluid), 97.0% (artificial intestinal fluid + starch); the inhibition rate of blank medium was 1.4% (MRS), 4.4% (artificial intestinal fluid), 3.8% (artificial intestinal fluid + starch); the inhibition rate of rhamnosus casei LGG was 28.1% (MRS), 8.9% (artificial intestinal fluid), 13.1% (artificial intestinal fluid + starch); the inhibition rate of Lactobacillus plantarum VB165 was 56.6% (MRS), 63.6% (artificial intestinal fluid), 68.7% (artificial intestinal fluid + starch). The results showed that under the conditions of simulating artificial intestinal fluid, including with and without the addition of starch, the fermentation supernatant of Lactobacillus plantarum VB165 had a strong inhibitory effect on α-glucosidase, and was significantly higher than that of rhamnosus casei LGG.

[0057] Example 5 Bile Salt Degradation Ability Detection

[0058] In order to detect whether the strain has the ability to degrade bile salts, the following six major bile salts (source leaf organisms) were added to the Lactobacillus MRS culture medium: sodium taurocholate (TAC), sodium taurochenodeoxycholate (TCDC), sodium taurodeoxycholate (TDC), sodium glycocholate (GC) 4mmol / L, sodium glycochenodeoxycholate (GCDC), sodium glycodeoxycholate (GDC) 2mmol / L, and the bile salt degradation was evaluated according to the consumption rate after cultivation. MRS containing bile salts was added to a 48-well plate at 2ml per well, and then the revived strain was added at 1% of the volume, placed in an anaerobic bag, set the temperature to 37℃, 90rpm, and cultured anaerobically for 24h. Sample processing: Take 1ml of the well plate liquid, centrifuge, take the supernatant, and perform HPLC analysis to determine the bile salt degradation ability.

[0059] The analysis conditions were as follows: chromatographic column: Ultimate AQ-C18 (5um, 4.6×250mm); flow rate: 1.0ml / min; detection wavelength: 204nm; column temperature: 45°C; mobile phase A: phosphate buffer, mobile phase B: acetonitrile, A:B=56:44.

[0060] The results showed that Lactobacillus plantarum VB165 can degrade five bile salts, namely sodium taurocholate (TAC), sodium taurochenodeoxycholate (TCDC), sodium taurodeoxycholate (TDC), sodium glycocholate (GC), and sodium glycodeoxycholate (GDC). However, Lactobacillus rhamnosus LGG can only degrade sodium glycochenodeoxycholate (GCDC) and sodium glycodeoxycholate (GDC). Therefore, Lactobacillus plantarum VB165 has a stronger ability to degrade bile salts.

[0061] Table 1 Results of the test on the ability of strains to degrade bile salts

[0062]

[0063] Example 6 Antagonistic Pathogen Ability Test

[0064] The antibacterial ability of the strains was evaluated by double-layer plate culture method.

[0065] Preparation of the lower layer probiotics: After thawing the activated frozen tubes of different single bacteria, spot them on the MRS solid plate, put them in an anaerobic bag, and culture them anaerobically at 37°C for 48 hours. The inoculation amount of the bacteria is 2ul / spot.

[0066] Preparation of the upper pathogenic bacteria layer: The high temperature sterilized upper culture medium is cooled to 40-50°C, and different pathogens are added as required (the final concentration of pathogens in the culture medium is about 10 6 / ml) and mix well, add 7ml / dish of the medium containing pathogenic bacteria to the above-mentioned probiotic single bacterial plates that have grown, and after solidification, culture them according to the growth conditions of different pathogenic bacteria (Table 2). After the upper plate is grown, observe whether there is an antibacterial circle around the probiotics to determine whether it has antibacterial ability. All pathogenic bacteria strains were purchased from Qingdao Haibo Biological.

[0067] Table 2 Types of pathogenic strains and culture conditions

[0068]

[0069]

[0070] As shown in Table 3, Lactobacillus plantarum VB165 can inhibit all 7 common pathogens included in Table 2 and has the ability to antagonize pathogens.

[0071] Table 3 Antibacterial effect of Lactobacillus plantarum VB165

[0072] Pathogens Inhibition zone size (cm) Staphylococcus aureus <1 Salmonella Typhimurium 1-3 Listeria monocytogenes <1 Escherichia coli 1-3 Candida albicans 1-3 Clostridium perfringens 1-3 Shigella fulvei 1-3

[0073] Example 7 Acid and bile salt resistance

[0074] Take 1mL of 24h bacterial solution, centrifuge to remove the supernatant, add artificial gastric juice (Coolaber, pH3.0), or 0.1% ox bile salt (Coolaber, PBS preparation) to resuspend, and treat at 37℃ for 2h. Gradient dilution and colony count on the plate to calculate the survival rate of the tested bacteria. The results showed that the survival rate of Lactobacillus plantarum VB165 in artificial gastric juice with pH3.0 was 99.50% for 2h and 98.30% for 4h; the survival rate of Lactobacillus plantarum VB165 in 0.1% ox bile salt was 98.70% for 2h and 95.60% for 4h, which has strong acid and bile salt resistance.

[0075] Table 4 Acid and bile resistance of Lactobacillus plantarum VB165

[0076]

Claims

1. A Lactobacillus plantarum VB165, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.25839 and a deposit date of September 28, 2022.

2. The plant lactobacillus according to claim 1, wherein The Lactobacillus plantarum is in the form of living cells.

3. The plant lactobacillus according to claim 2, wherein The Lactobacillus plantarum is in the form of bacterial culture solution.

4. The plant lactobacillus according to claim 1, wherein The Lactobacillus plantarum is in the form of non-living cells.

5. The plant lactobacillus according to claim 4, wherein The Lactobacillus plantarum is in the form of freeze-dried powder.

6. A use of Lactobacillus plantarum according to any one of claims 1 to 5, characterized in that: Used to prepare one or more of medicines, health products, foods, beverages, additives, and feeds for controlling or lowering blood sugar.

7. The use according to claim 6, characterized in that Control or lower blood sugar by inhibiting α-glucosidase activity.

8. A use of Lactobacillus plantarum according to any one of claims 1 to 5, characterized in that: Used for preparing one or more of medicines, health products, foods, beverages, additives, and feeds for degrading bile salts.

9. A use of Lactobacillus plantarum according to any one of claims 1 to 5, characterized in that: Used to prepare one or more of medicines, health products, foods, beverages, additives, and feeds that inhibit the activity of intestinal pathogens.

10. A composition, characterized in that The composition comprises the Lactobacillus plantarum according to any one of claims 1 to 5.

11. The composition according to claim 10, characterized in that The composition may also comprise additional probiotic material.

12. The composition according to claim 10, characterized in that The composition also comprises a prebiotic material.

13. The composition according to any one of claims 10 to 12, characterized in that The composition also includes an ingestible carrier.

14. The composition according to claim 13, characterized in that The ingestible carrier is a pharmaceutically acceptable carrier.

15. The composition according to claim 13, characterized in that The ingestible carrier is a capsule, tablet or powder.

16. The composition according to claim 13, characterized in that The ingestible carrier is a food.

17. The composition according to claim 16, characterized in that The food is acidified milk, yogurt, frozen yogurt, milk powder, concentrated milk, cheese sauce, condiment or beverage.

18. A fermentation method of Lactobacillus plantarum according to any one of claims 1 to 5, characterized in that: The fermentation method comprises carrying out anaerobic fermentation in a nutrient medium containing an assimilable carbon source and / or nitrogen source.

19. The fermentation method according to claim 18, characterized in that The assimilable carbon source is selected from starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol or any combination thereof.

20. The fermentation method according to claim 18, characterized in that The assimilable carbon source is glucose.

21. The fermentation method according to claim 18, characterized in that The assimilable nitrogen source is selected from yeast extract, yeast powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor, soybean meal, peptone, urea, ammonium salt or any combination thereof.

22. The fermentation method according to claim 18, characterized in that The assimilable nitrogen source is yeast extract.

23. The fermentation method according to claim 18, characterized in that The nutrient medium also includes an inorganic salt, which is selected from one of trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate, or any combination thereof.

24. The fermentation method according to claim 18, characterized in that The nutrient medium also includes dipotassium hydrogen phosphate and magnesium sulfate.

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