Lactobacillus rhamnosus BN06 for improving hyperglycemia symptoms and application thereof

Abstract

The present invention discloses a rhamnosus lactobacillus BN06 for improving hyperglycemia symptoms and its application. The rhamnosus lactobacillus is rhamnosus lactobacillus (Lacticaseibacillus rhamnosus) BN06, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 22, 2023, with a deposit number of CGMCC No. 29103. Rhamnosus lactobacillus BN06 can inhibit the rapid absorption of glucose and promote insulin secretion, sustainably and efficiently protect the health of the pancreas, regulate blood sugar to normal levels, and fundamentally improve the symptoms of hyperglycemia such as frequent hunger, dry mouth, and frequent urination.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a Lactobacillus rhamnosus BN06 for improving hyperglycemia symptoms and an application thereof. Background Art

[0002] The sugar in the blood is called blood sugar, in which glucose is the main component. Most of the energy required for the activities of various tissues and cells in the human body comes from glucose, so blood sugar must be maintained at a certain level to maintain the needs of various organs and tissues in the body. However, when blood sugar exceeds a certain value (such as: fasting blood sugar ≥6.1mmol / L, blood sugar ≥7.8mmol / L two hours after a meal), it will cause harm to the human body, and symptoms such as increased hunger, extreme dry mouth and thirst, frequent urination, dry skin, dehydration, abdominal discomfort, weight loss, weakness, rapid heartbeat, slow and deep breathing, etc.

[0003] Most of the blood sugar regulating products on the market are insulin and other hormones, and Western medicine products, and their functions are mainly unilateral functions such as sugar blocking and insulin supplementation. Although they can lower blood sugar during the period of taking the medicine, the effect of lowering blood sugar disappears after stopping the medicine. In addition, unilateral blood sugar lowering is also prone to cause hypoglycemia and insufficient energy supply to the body due to uncontrolled reduction, making the human body feel uncomfortable, endangering human health, and failing to regulate blood sugar to normal levels.

[0004] The intestine is the main site for glucose absorption, and probiotics in it play an important role in regulating glucose metabolism. Different probiotics not only have different regulatory methods, but also different effects. It is necessary to screen a probiotic strain that can effectively improve hyperglycemia. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a Lactobacillus rhamnosus for improving the symptoms of hyperglycemia, which can slow down the absorption rate of glucose, inhibit the rapid absorption of glucose, and promote insulin secretion.

[0006] A second object of the present invention is to provide a product for improving symptoms of hyperglycemia.

[0007] The third object of the present invention is to provide a probiotic fermentation product.

[0008] A fourth object of the present invention is to provide a method for preparing a probiotic fermentation product.

[0009] One of the purposes of the present invention is achieved by the following technical solution:

[0010] A rhamnosus Lactobacillus for improving symptoms of hyperglycemia, wherein the rhamnosus Lactobacillus is rhamnosus Lactobacillus (Lacticaseibacillus rhamnosus) BN06, and the rhamnosus Lactobacillus (Lactobacillus acidophilus) BN06 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on November 22, 2023, with a deposit number of CGMCC No.29103.

[0011] As a preferred embodiment of the present invention, the Lactobacillus rhamnosus BN06 improves the symptoms of hyperglycemia through at least one of the following items (I) to (III):

[0012] (Ⅰ) Slow down the absorption rate of glucose and inhibit the rapid absorption of glucose;

[0013] (Ⅱ) Promote insulin secretion;

[0014] (III) Improve symptoms of frequent hunger, dry mouth, and frequent urination caused by high blood sugar.

[0015] The second object of the present invention is achieved by adopting the following technical solution:

[0016] A product for improving symptoms of hyperglycemia, wherein the active ingredients of the product include live bacteria and / or inactivated bacteria of Lactobacillus acidophilus BN06, and the preservation number of Lactobacillus acidophilus BN06 is CGMCC No.29103.

[0017] As a preferred embodiment of the present invention, the product for improving hyperglycemia symptoms includes probiotic freeze-dried powder, probiotic fermentation supernatant, probiotic lysate and probiotic fermentation product.

[0018] As a preferred embodiment of the present invention, the active ingredient of the product for improving hyperglycemia symptoms is the live bacteria of Lactobacillus acidophilus BN06, and the product is the fermentation supernatant of Lactobacillus acidophilus BN06.

[0019] The third object of the present invention is achieved by adopting the following technical solution:

[0020] A probiotic fermented product is prepared by fermenting a composition with Lactobacillus rhamnosus BN06. The composition comprises the following components in percentage by weight: 10-45 parts of carambola, 25-45 parts of lotus leaves, and 25-45 parts of tea tree flowers. The preservation number of Lactobacillus rhamnosus BN06 is CGMCC No.29103.

[0021] Star fruit is rich in nutrients, containing 7 essential amino acids, carbohydrates, proteins, fats, organic acids, various trace elements, and functional factors such as polyphenols, flavonoids, vitamin C, etc. required by the human body. It has high nutritional and medicinal value. According to the "Compendium of Materia Medica", star fruit has many functions such as removing wind-heat, promoting fluid production and quenching thirst, removing toxins and regenerating muscles. Modern pharmacological studies have found that star fruit also has a significant effect on blood sugar regulation. Its juice can lower blood sugar levels and improve pancreatic cell apoptosis.

[0022] Lotus leaves are rich in alkaloids such as aporphine, monobenzylisoquinoline, bisbenzylisoquinoline, and flavonoids such as quercetin and kaempferol. They can increase the level of glucose transporter, increase the uptake and utilization of glucose by adipose tissue, improve insulin resistance of fat cells, and help lower blood sugar.

[0023] Tea flowers contain a large amount of amino acids, proteins, saponins, tea polyphenols, tea polysaccharides and other functional ingredients. Among them, it contains 16 kinds of free amino acids. The content of theanine is significantly higher than that of fresh tea leaves. Theanine, as the most important amino acid in tea, has beneficial effects in anti-cancer, calming the nerves, protecting the liver, and improving immunity. The protein content is higher than that of fresh tea leaves, of which the water-soluble protein that can be directly absorbed and utilized accounts for about 3.5% of the total protein. Saponins can lower blood sugar by inhibiting the synthesis of pancreatic lipase, while tea polyphenols and tea polysaccharides play an important role in promoting the repair of pancreatic β cells, stimulating the regeneration of pancreatic β cells and thus lowering blood sugar.

[0024] The composition provided by the present invention is made of carefully selected natural plant raw materials, covering three major categories of fruits, herbs and woody plants, so that the types of nutritional components of the composition are more comprehensive and the quantity is richer. It can not only provide sufficient nutrition and protection for probiotics, promote their proliferation and produce short-chain fatty acids, amino acids, bacteriocins, polysaccharides and other probiotic factors, but also after in vitro fermentation of probiotics, the functional components in the composition are well released, and the bioavailability is greatly improved. When consumed by humans, they can obviously feel that the symptoms of high blood sugar such as frequent hunger, dry mouth, frequent urination, etc. are improved, and the blood sugar value is restored to a normal level, which is beneficial to protecting the health of the pancreas. From the aspects of the types and proportions of the composition (excluding probiotics), it is very novel and innovative.

[0025] As a preferred embodiment of the present invention, in the probiotic fermented product, the composition comprises the following components in weight percentage: 45 parts of carambola, 30 parts of lotus leaves, and 25 parts of tea tree flowers.

[0026] As a preferred embodiment of the present invention, the fermented product is spread on a plate, pre-frozen by liquid nitrogen, then vacuum freeze-dried, and then pulverized.

[0027] The probiotic fermented product provided by the present invention is pre-frozen by liquid nitrogen in a freeze-drying method, and the liquid nitrogen pre-freezing speed is faster than that of ordinary pre-freezing, so that the water inside the cells can quickly seep out of the cells, avoiding a large amount of water from accumulating and condensing in the bacterial cells for a long time and causing cell death. In addition, the pre-freezing is performed in a flat manner, which increases the surface area of ​​the liquid in contact with the liquid nitrogen, further shortens the pre-freezing time, and is conducive to reducing the attenuation of the strain during the freeze-drying process.

[0028] The fourth object of the present invention is achieved by adopting the following technical solution:

[0029] A method for preparing a probiotic fermented product as described in any one of the third purposes, comprising the following steps:

[0030] Beating: put the formulated amount of carambola, lotus leaf and tea tree flower into a juicer, add distilled water and pectinase, stir, and obtain base material A;

[0031] Enzymatic hydrolysis: adjust the pH of base material A with citric acid to obtain base material B;

[0032] Sterilization: Place base material B in a sterilizer and sterilize to obtain base material C;

[0033] Activation of bacterial strains: Activate Lactobacillus acidophilus BN06 and culture to obtain bacterial suspension D;

[0034] Fermentation: Place base material C in a fermentation tank, add bacterial suspension D, and perform anaerobic fermentation to obtain fermentation stock solution E;

[0035] Freeze drying: spread the fermentation solution E on a plate filled with liquid nitrogen for pre-freezing, and then freeze-dry it in a vacuum freeze dryer to obtain base material F;

[0036] Grinding: Grind the base material F and pass it through a sieve to obtain freeze-dried particles G;

[0037] Bagging: Put the freeze-dried particles G into a filter bag to obtain a fermented product.

[0038] As a preferred embodiment of the present invention, the method for preparing the probiotic fermentation product comprises the following steps:

[0039] Raw material selection: Choose fresh, ripe, disease-free carambolas, fresh, blooming, non-rotten lotus leaves and tea flowers;

[0040] Pretreatment: Wash the carambola with clean water to remove the dirt on the surface, drain the surface water, remove the core, cut into pieces, and weigh according to the formula amount; wash the lotus leaf with clean water to remove the dirt on the surface, drain the surface water, cut into small strips, and weigh according to the formula amount; wash the tea tree flower with clean water to remove the dirt on the surface, drain the surface water, and weigh according to the formula amount;

[0041] Beating: put the weighed carambolas, lotus leaves and tea flowers into a juicer, add 80-150 parts of distilled water and 0.01-0.05% pectinase, and stir at 12000-15000 r / min for 0.5-2 min to obtain base material A;

[0042] Enzymatic hydrolysis: adjust the pH of base material A to 3-4 with citric acid, and keep it at 40-50°C for 0.8-1.2h to obtain base material B;

[0043] Sterilization: Place base material B in an autoclave, sterilize at 120-123°C for 15-22 minutes, and then cool to 35-37°C to obtain base material C;

[0044] Activation of bacterial strains: Take out the freeze-dried tube of Lactobacillus rhamnosus BN06 stored at -80°C, use a pipette to measure 0.3-0.4 mL of sterile saline and add it dropwise to the freeze-dried tube, shake well, use a pipette to draw 150-200 μL, inject it onto the plate of solid culture medium, and use a coating rod to make it evenly distributed, and then culture it in a 37°C constant temperature incubator for 45-50 hours to obtain the first-generation bacterial strain; use an inoculation loop to pick up independent colonies of the first-generation bacterial strain and put them into the liquid culture medium, culture them in a 37°C shaker at 120-180 rpm for 45-48 hours, centrifuge at 7500-8500 r / min for 8-12 minutes, take the precipitate to obtain the activated second-generation bacterial strain, wash it with saline for 2-3 times, and then resuspend and dilute it with saline to adjust its viable bacterial count to 1.0×10 10 CFU / mL, bacterial suspension D was obtained;

[0045] Fermentation: Place base material C in a fermentation tank, add 3-6% bacterial suspension D, and perform anaerobically fermentation at 37°C and 90-110 r / min for 45-48 hours to obtain fermentation stock solution E;

[0046] Freeze drying: spread the fermentation stock solution E on a plate filled with liquid nitrogen for 8 to 10 minutes to a thickness of 4 to 5 mm, and then freeze dry it in a vacuum freeze dryer. Keep the shelf temperature at -50°C for 25 to 30 minutes, raise the temperature to -10 to -8°C within 60 minutes, keep it for 9 to 11 hours, and then raise the temperature to 8 to 12°C and keep it for 1.8 to 2.2 hours to obtain base material F. The vacuum degree of the whole process is 0.4 to 0.6 Pa, and the cold trap temperature is -60 to -55°C.

[0047] Grinding: Grind the base material F and pass it through a 10-mesh sieve to obtain freeze-dried particles G;

[0048] Bag filling: put the freeze-dried particles G into 300-mesh filter bags, 15 g per bag, to obtain the fermented product.

[0049] The probiotic fermented product provided by the present invention innovatively adopts live bacteria tea bags. The filter bag specification is carefully selected to be 300 mesh and the particle size is 8.47 μm, which is much larger than the cell diameter of Lactobacillus rhamnosus. During the brewing process of the fermented product, Lactobacillus rhamnosus can enter the water through the filter membrane. In addition, the efficacy factors can also enter the water with the release of live bacteria. After soaking in warm water at 37°C for 5 minutes, a cup of live bacteria beverage can be obtained. When the human body consumes this live bacteria beverage, the Lactobacillus rhamnosus and the efficacy factors can smoothly reach the human intestine under the transportation of water, and play the effect of improving the symptoms of hyperglycemia, and the high efficiency and sustainability are fully reflected.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The Lactobacillus rhamnosus for improving hyperglycemia symptoms provided by the present invention was screened out by the applicant through a large number of experiments. It can inhibit the rapid absorption of glucose, promote insulin secretion, sustainably and efficiently protect the health of the pancreas, regulate blood sugar to normal levels, and fundamentally improve the symptoms of hyperglycemia such as frequent hunger, dry mouth, and frequent urination.

[0052] (2) The probiotic fermented product provided by the present invention contains active Lactobacillus rhamnosus BN06, which can not only allow Lactobacillus rhamnosus BN06 to colonize the intestine, but also more effectively improve symptoms of hyperglycemia such as frequent hunger, dry mouth, and frequent urination.

[0053] Biological material preservation information: Lactobacillus rhamnosus BN06, the preservation number is CGMCCNo.29103, the classification name is: Lacticaseibacillus rhamnosus, it was deposited on November 22, 2023 in the General Microbiology Center of China Culture Collection Administration (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101), and the abbreviation of the depository unit is CGMCC. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following embodiments can be purchased through commercial channels unless otherwise specified.

[0055] The Lactobacillus rhamnosus BN06 for improving hyperglycemia symptoms provided in the embodiment of the present invention is a probiotic strain obtained by screening through cell experiments and animal experiments.

[0056] System 1: Screening of bacteria that inhibit rapid glucose absorption

[0057] 1. Experimental strains: Lactobacillus acidophilus, Lactobacillus fermentans, Lactobacillus plantarum, Streptococcus salivarius subsp. thermophilus, Lactobacillus rhamnosus, numbered A to E.

[0058] 2. Experimental cells: Caco-2 human colorectal adenocarcinoma cells.

[0059] 3. Reagent preparation:

[0060] (1) Culture medium A: 20 g glucose, 10 g beef extract, 5 g yeast extract, 10 g tryptone, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80. Add 800 mL of distilled water and stir to dissolve. Adjust the pH to 6.5 and make up to 1000 mL. Sterilize at 121°C for 20 min.

[0061] (2) Medium B: 20 ​​g glucose, 10 g beef extract, 5 g yeast extract, 10 g tryptone, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80, 20 g agar, add 800 mL distilled water and stir to dissolve, adjust the pH to 6.5, and make up to 1000 mL. Sterilize at 121°C for 20 min.

[0062] (3) Culture medium C: DMEM culture medium, 20% fetal bovine serum, and 1% penicillin-streptomycin.

[0063] (4) HEPES buffer: 20 mM / mL HEPES, 137 mM / mL NaCl, 4.7 mM / mL KCl, 1.8 mM / mL CaCl2, and 1.2 mM / mL MgSO4. The solvent is water. Sterilize at 121°C for 20 min.

[0064] (5) PBS buffer: 0.27 g potassium dihydrogen phosphate, 1.42 g disodium hydrogen phosphate, 8 g sodium chloride, 0.2 g potassium chloride, add about 800 mL of distilled water and stir thoroughly to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.4, finally make up to 1 L, and sterilize at 121°C for 20 min.

[0065] 4. Preparation of fermentation supernatant:

[0066] (1) Activation of bacterial strains: Take out the freeze-dried tube of bacterial strains stored at -80°C, use a pipette to measure 0.4 mL of sterile saline and add it dropwise to the freeze-dried tube, shake well, use a pipette to draw 200 μL, inject it onto the plate of culture medium B, and use a coating rod to evenly distribute it, then culture it in a 37°C constant temperature incubator for 48 hours to obtain the first-generation bacterial strains. Use an inoculation loop to pick up independent colonies of the first-generation bacterial strains and add them to culture medium A, culture them in a shaker at 37°C at 120-180 rpm for 48 hours, centrifuge at 8000 r / min for 10 minutes, take the precipitate, obtain the activated second-generation bacterial strains, wash them twice with saline, then resuspend and dilute them with saline to adjust the number of viable bacteria to 1.5×10 8 CFU / mL, and obtain bacterial suspension.

[0067] (2) Fermentation: 2 mL of bacterial suspension was inoculated into 80 mL of culture medium A. After culturing at 37°C for 48 h, the mixture was centrifuged at 8000 rpm for 10 min and the supernatant was obtained.

[0068] 5. Experimental methods:

[0069] (1) Establishment of monolayer cell model: Caco-2 cells were digested into single free cells and each well (1.12 cm 2 )3.0×105 cells were seeded in a 12-well Transwell chamber (pore size 0.4 μm, polycarbonate membrane), and the culture medium was replaced every two days.

[0070] (2) Incubation: The blank group and the control group were incubated with 0.4 mg / mL culture medium C for 60 min, and the experimental group was incubated with 0.4 mg / mL fermentation supernatant for 60 min. The experimental groups were Groups A to E, and the corresponding fermentation supernatants used were the fermentation supernatants of the strains numbered A to E. After that, the cell culture medium and fermentation supernatant were removed, and the cells were washed twice with 1 mL of HEPES buffer. Then, 0.5 mL and 1.5 mL of HEPES buffer were added to the intestinal side (AP) and basolateral side (BL) of the Transwell chamber, respectively. After incubation for 30 min, the buffer was removed.

[0071] (3) Testing:

[0072] 1) In the experimental group and the control group, 0.5 mL of HEPES buffer containing 2 mg / mL D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. In the blank group, HEPES buffer without D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. The basolateral buffer was collected after 15 minutes.

[0073] 2) In the experimental group and the control group, 0.5 mL of HEPES buffer containing 2 mg / mL D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. In the blank group, HEPES buffer without D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. The basolateral buffer was collected after 30 minutes.

[0074] 3) In the experimental group and the control group, 0.5 mL of HEPES buffer containing 2 mg / mL D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. In the blank group, HEPES buffer without D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. The basolateral buffer was collected after 60 minutes.

[0075] 4) In the experimental group and the control group, 0.5 mL of HEPES buffer containing 2 mg / mL D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. In the blank group, HEPES buffer without D-glucose was added to the intestinal lumen side, and 1.5 mL of HEPES buffer was added to the basolateral side. The basolateral buffer was collected after 90 minutes.

[0076] (4) The polycarbonate membrane in the Transwell chamber was washed twice with 4°C precooled PBS buffer to stop the Caco-2 cells on the polycarbonate membrane from continuing to uptake glucose.

[0077] (5) 500 μL of NaOH solution (0.1 M) was added to each Transwell well to dissolve the cells on the polycarbonate membrane. The reaction was carried out at 37° C. for 30 min. The glucose content in the cells was determined using a glucose oxidase kit. The results are shown in Table 1.

[0078] 6. Results and analysis:

[0079] Table 1 Glucose content in cells of each group at different time periods (mmol / L)

[0080] Group 15min 30min 60min 90min Blank Group 0.165±0.020 0.164±0.014 0.162±0.018 0.160±0.015 Control group 0.597±0.021 0.683±0.017 0.724±0.016 0.751±0.034 Group A 0.308±0.035 0.345±0.036 0.457±0.033 0.564±0.029 Group B 0.390±0.004 0.437±0.019 0.529±0.024 0.647±0.006 Group C 0.363±0.010 0.391±0.030 0.486±0.019 0.595±0.003 Group D 0.376±0.008 0.413±0.022 0.504±0.025 0.602±0.018 Group E 0.184±0.013 0.218±0.011 0.295±0.010 0.393±0.026

[0081] Intestinal epithelial cells are the last barrier for glucose to enter the blood circulation. The structure of Caco-2 cells is similar to that of intestinal epithelial cells. Therefore, Caco-2 cells can be used instead of intestinal epithelial cells for related research on sugar absorption. As shown in Table 1, compared with the blank group, the intracellular glucose content of the control group and the experimental group increased, indicating that the glucose in the solution can be absorbed by the cells; compared with the control group, the intracellular glucose content of the experimental groups (groups A to E) decreased to varying degrees at different time periods, indicating that the fermentation supernatant of the experimental group can slow down the rate of glucose absorption by the cells, among which the effect of group E was the best, that is, the fermentation supernatant of group E had the best effect in inhibiting the rapid absorption of glucose. Therefore, Lactobacillus rhamnosus was selected as the best strain to inhibit the rapid absorption of glucose.

[0082] System 2: Screening of strains that inhibit rapid glucose absorption

[0083] 1. Experimental strains: Lactobacillus rhamnosus BN06, Lactobacillus rhamnosus CICC 22173 (preservation number: CICC22173), Lactobacillus rhamnosus CICC 22151 (preservation number: CICC 22151), numbered F to H, respectively.

[0084] Among them, Lactobacillus rhamnosus CICC 22173 (preservation number: CICC 22173) and Lactobacillus rhamnosus CICC22151 (preservation number: CICC 22151) were purchased from the China Industrial Microbiological Culture Collection Center.

[0085] 2. Experimental method: The glucose absorption of the experimental strain was measured according to the method of system one.

[0086] 3. Experimental results: The results are shown in Table 2.

[0087] Table 2 Glucose content in cells of each group at different times (mmol / L)

[0088] Group 15min 30min 60min 90min Blank Group 0.164±0.005 0.163±0.010 0.163±0.015 0.162±0.013 Control group 0.595±0.020 0.685±0.016 0.721±0.014 0.750±0.011 Group F 0.180±0.013 0.211±0.015 0.289±0.011 0.378±0.017 Group G 0.190±0.016 0.236±0.026 0.305±0.021 0.399±0.019 Group H 0.189±0.010 0.227±0.018 0.313±0.020 0.385±0.008

[0089] As shown in Table 2, compared with the control group, the intracellular glucose content of the experimental groups (F to H groups) at different time periods decreased to varying degrees, indicating that the fermentation supernatant of the experimental groups can slow down the rate at which cells absorb glucose. There are few differences between the F to H groups, indicating that Lactobacillus rhamnosus has a significant effect in inhibiting the rapid absorption of glucose. Among them, the intracellular glucose content of the F group was the lowest at different times, indicating that the strain of the F group, namely Lactobacillus rhamnosus BN06, has the best effect in inhibiting the rapid absorption of glucose.

[0090] System 3: Screening of strains that promote insulin secretion

[0091] 1. Experimental strains: Lactobacillus rhamnosus BN06, Lactobacillus rhamnosus CICC 22173 (preservation number: CICC22173), Lactobacillus rhamnosus CICC 22151 (preservation number: CICC 22151).

[0092] 2. Experimental animals: Male SD rats, weighing 250 g.

[0093] 3. Reagent preparation:

[0094] (1) Culture medium A: 20 g glucose, 10 g beef extract, 5 g yeast extract, 10 g tryptone, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80. Add 800 mL of distilled water and stir to dissolve. Adjust the pH to 6.5 and make up to 1000 mL. Sterilize at 121°C for 20 min.

[0095] (2) Medium B: 20 ​​g glucose, 10 g beef extract, 5 g yeast extract, 10 g tryptone, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80, 20 g agar, add 800 mL distilled water and stir to dissolve, adjust the pH to 6.5, and make up to 1000 mL. Sterilize at 121°C for 20 min.

[0096] (3) 1640 medium: Weigh 10.4 g of 1640 medium powder, add 900 mL of distilled water, stir to dissolve, add 2 g of sodium bicarbonate, stir to dissolve, add distilled water to make up to 1000 mL, and sterilize at 121°C for 20 min.

[0097] (4) Hanks buffer:

[0098] 1) Liquid A: 0.06 g disodium hydrogen phosphate, 0.06 g anhydrous potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 1 g glucose, 8 g sodium chloride, 750 mL distilled water.

[0099] 2) Solution B: 0.14 g calcium chloride, 100 mL distilled water.

[0100] 3) Dissolve 0.35 g of sodium bicarbonate in 100 mL of distilled water at 37°C, add 0.02 g of phenol red, stir to dissolve, and obtain mixed solution A. Add solution B to solution A, then add mixed solution A, make up to 1000 mL with distilled water, mix well, store in a refrigerator at 4°C overnight, sterilize at 121°C for 15 min, and cool to obtain.

[0101] (5) KRBH buffer: NaCl 128.8mmol / L, KCl 4.8mmol / L, CaCl22.5mmol / L, KH2PO41.2mmol / L, MgSO41.2mmol / L, NaHCO35mmol / L, HEPES10mmol / L, albumin 2%, pH 7.4.

[0102] 4. Preparation of fermentation supernatant:

[0103] (1) Activation of bacterial strains: Take out the freeze-dried tube of bacterial strains stored at -80°C, use a pipette to measure 0.4 mL of sterile saline and add it dropwise to the freeze-dried tube, shake well, use a pipette to draw 200 μL, inject it onto the plate of medium B, and use a coating rod to evenly distribute it, then culture it in a 37°C constant temperature incubator for 48 hours to obtain the first-generation bacterial strains. Use an inoculation loop to pick up independent colonies of the first-generation bacterial strains and add them to medium A, culture them in a shaker at 37°C at 120-180 rpm for 48 hours, centrifuge at 8000 r / min for 10 minutes, take the precipitate, obtain the activated second-generation bacterial strains, wash them twice with saline, then resuspend and dilute them with saline, adjust the viable count to 1.5×108 CFU / mL, and obtain a bacterial suspension.

[0104] (2) Fermentation: 2 mL of bacterial suspension was inoculated into 80 mL of culture medium A. After culturing at 37°C for 48 h, the mixture was centrifuged at 8000 rpm for 10 min and the supernatant was obtained.

[0105] 5. Experimental methods:

[0106] (1) Isolation and culture of islets: Anesthetize and kill rats, open the abdominal cavity, find the intersection of the common bile duct and pancreatic duct at the intestinal end, clamp it with hemostats, and under a stereomicroscope, peel off the common bile duct and cut a small incision. Use a syringe to slowly inject 10 mL (1 mg / mL) of prepared collagenase P into the pancreas through the cut small incision. After the pancreas swells, peel it off and put it into a 50 mL centrifuge tube. Let it stand for digestion at 38°C for 11 minutes, then add 15 mL of 1640 culture medium and Hanks buffer at 4°C containing 10% fetal bovine serum, centrifuge at 1200 r / min for 3 minutes, discard the supernatant, add 10 mL of histopaque-1077 separation solution, blow it evenly, and then use a syringe to slowly inject 10 mL of 1640 culture medium into the tube along the tube wall. Centrifuge at 3200 r / min for 25 minutes. The islets seen between the culture medium and the separation solution are pancreatic islets.

[0107] (2) Add 1 mL of KRBH buffer containing 2.8 mmol / L glucose to the EP tube, add the islets to the EP tube, place it in an incubator at 37°C for 30 min, then remove it and discard the supernatant. The EP tubes containing the islets are divided into groups. The treatment methods for each group are as follows:

[0108] Group A: islets were incubated in KRBH solution for 30 min;

[0109] Group B: islets were incubated in KRBH solution containing 2.8 mmol / L glucose for 30 min;

[0110] Group C: islets were placed in KRBH solution containing 2.8 mmol / L glucose and 0.5 g / L fermentation supernatant of Lactobacillus rhamnosus BN06 and incubated for 30 min;

[0111] Group D: islets were incubated in KRBH solution containing 2.8 mmol / L glucose and 0.5 g / L fermentation supernatant of Lactobacillus rhamnosus CICC 22173 for 30 min;

[0112] Group E: islets were incubated in KRBH solution containing 2.8 mmol / L glucose and 0.5 g / L fermentation supernatant of Lactobacillus rhamnosus CICC 22151 for 30 min;

[0113] (3) After the incubation, the supernatant of each group was aspirated and stored at -20°C. The insulin content in the supernatant was determined by radioimmunoassay. The results are shown in Table 3.

[0114] 6. Results and analysis:

[0115] Table 3 Insulin content in the supernatant of each group

[0116] Group Insulin content (μU / mL) Group A 1.35±0.81 Group B 6.74±1.06 Group C 8.96±0.58 Group D 7.03±0.62 Group E 7.10±0.75

[0117] As shown in Table 3, compared with group A, the insulin content in the supernatant of groups B to E increased significantly, indicating that under the stimulation of glucose, the pancreatic islets will secrete insulin; compared with group B, the insulin content in the supernatant of groups C to E increased to varying degrees, indicating that the fermented supernatant of the strains of groups C to E can promote the secretion of insulin by the pancreatic islets, among which the insulin content in the supernatant of group C is the highest, indicating that the strains of group C have the best effect in promoting insulin secretion, that is, after Lactobacillus rhamnosus BN06 colonizes in the intestine, the metabolites produced during its proliferation can promote the secretion of insulin by the pancreatic islets, which is beneficial to improve the blood sugar level of hyperglycemia, and then improve the symptoms of hyperglycemia. Therefore, Lactobacillus rhamnosus BN06 was selected as the best strain to promote insulin secretion.

[0118] System 4: Screening of composition formula of fermented products

[0119] 1. Sample preparation

[0120] Table 4 Composition formula of fermented products

[0121]

[0122]

[0123] 1. Raw material selection: Choose fresh, ripe, disease-free carambolas (bitter melons), fresh, blooming, non-rotten lotus leaves and tea flowers;

[0124] 2. Pretreatment: Wash the surface dirt of carambola (bitter melon) with clean water, drain the surface water, remove the core, cut into pieces, and weigh according to the weight parts in Table 4; wash the surface dirt of lotus leaf with clean water, drain the surface water, cut into small strips, and weigh according to the weight parts in Table 4; wash the surface dirt of tea tree flower with clean water, drain the surface water, and weigh according to the weight parts in Table 4;

[0125] 3. Pulping: Put carambola (bitter melon), lotus leaves and tea tree flowers in a juicer, add 100 parts of distilled water and 0.03% pectinase, and stir at 15000r / min for 1min to obtain base material A;

[0126] 4. Enzymatic hydrolysis: adjust the pH of base material A to 3.5 with citric acid and keep it at 45°C for 1 hour to obtain base material B;

[0127] 5. Sterilization: Place base material B in a sterilizer, sterilize at 121°C for 20 minutes, and then cool to 37°C to obtain base material C;

[0128] 6. Culture medium preparation:

[0129] (1) Culture medium A: 20 g glucose, 10 g beef extract, 5 g yeast extract, 10 g tryptone, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80, add 800 mL distilled water and stir to dissolve, adjust the pH to 6.5, and make up to 1000 mL, sterilize at 121°C for 20 min;

[0130] (2) Medium B: 20 ​​g glucose, 10 g beef extract, 5 g yeast extract, 10 g tryptone, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80, 20 g agar, add 800 mL distilled water and stir to dissolve, adjust the pH to 6.5, and make up to 1000 mL, sterilize at 121°C for 20 min;

[0131] 7. Activation of strains: Take out the freeze-dried tube of Lactobacillus rhamnosus BN06 stored at -80°C, use a pipette to measure 0.4mL of sterile saline and add it dropwise to the freeze-dried tube, shake well, use a pipette to draw 200μL, inject it onto the plate of medium B, and use a coating rod to evenly distribute it, then culture it in a 37°C constant temperature incubator for 48h to obtain the first-generation strains. Use an inoculation loop to pick up independent colonies of the first-generation strains and add them to medium A, culture them in a 37°C shaker at 120-180rpm for 48h, centrifuge at 8000r / min for 10min, take the precipitate, obtain the activated second-generation strains, wash them twice with saline, then resuspend and dilute them with saline to adjust the number of viable bacteria to 1.0×10 10 CFU / mL, bacterial suspension D was obtained;

[0132] 8. Fermentation: Place base material C in a fermentation tank, add 5% bacterial suspension D, and perform anaerobically fermentation at 37°C and 100 r / min for 48 hours to obtain fermentation stock solution E;

[0133] 9. Freeze drying: Spread the stock solution E in a plate containing liquid nitrogen for 10 minutes to a thickness of 5 mm, and then freeze dry it in a vacuum freeze dryer. Keep the shelf temperature at -50°C for 30 minutes, raise the temperature to -10°C within 60 minutes, keep it for 10 hours, and then raise the temperature to 10°C and keep it for 2 hours to obtain base material F. The vacuum degree of the whole process is 0.4 Pa, and the cold trap temperature is -60°C;

[0134] 10. Grinding: Grind the base material F and pass it through a 10-mesh sieve to obtain freeze-dried particles G;

[0135] 11. Bag filling: put base material G into 300 mesh filter bags, 15 g per bag, to obtain fermented products.

[0136] Among them, since Example 7 does not have a fermentation substrate, the fermentation product steps of Example 7 only retain steps 5 to 11, and the base material B is replaced with an equal amount of milk.

[0137] 2. Crowd Testing

[0138] 1. Subjects: People with high blood sugar levels, fasting blood sugar > 8.6mmol / L, 2-hour postprandial blood sugar > 9.8mmol / L, aged 25 to 60 years old, 55 people, with a male to female ratio of 1:1.

[0139] The following situations are excluded:

[0140] (1) Patients with type Ⅰ diabetes;

[0141] (2) Patients with serious diseases of the heart, liver, kidney and hematopoietic system, mental illness, or those taking glucocorticoids or other drugs that affect blood sugar;

[0142] (3) Those who cannot cooperate with dietary control and thus affect the observation results;

[0143] (4) Patients with diabetic ketoacidosis, ketoacidosis or infection in the past 3 months.

[0144] 2. Test methods:

[0145] (1) The population was randomly divided into 6 groups, 5 people in each group, numbered as Group A, Group B, Group C, Group D, Group E, Group F, Group G, Group H, Group I, Group J, and Group K, and the products of Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, Example 9, Example 10, and Example 11 were consumed respectively according to the consumption method;

[0146] (2) Consumption method: Consume half an hour after meals, 3 times a day, one bag each time. Soak individual bags of fermented products in 37°C warm water for 5 min before drinking. Do this for 21 consecutive days. On the 0th, 7th, 14th, and 21st days, rate the degree of improvement in hyperglycemia symptoms such as hunger and dry mouth. Calculate the average score for each group. The scoring criteria are shown in Table 5. The results are shown in Table 6.

[0147] Table 5 Scoring criteria for improvement of hyperglycemia symptoms in the subjects (score: X)

[0148] Very bad Poor No change better very good 0≤X≤2 2＜X≤4 4＜X≤6 6＜X≤8 8＜X≤10

[0149] 3. Results and analysis:

[0150] Table 6 Score table of improvement of hyperglycemia symptoms in the subjects (points)

[0151]

[0152] As shown in Table 6, compared with the 0th day, the improvement of hyperglycemia symptoms in different groups has different degrees of difference, and as the consumption time increases, the score is also constantly improving, indicating that the fermented products of different embodiments have different formulations, but they all have a promoting effect on the improvement of hyperglycemia symptoms. Among them, the improvement scores of hunger and dry mouth in Group E are the highest, indicating that the fermented product of E has the best effect on improving hyperglycemia symptoms. After the high blood sugar population in the test ate it for 21 days, their own blood sugar disorder was benignly improved, and hyperglycemia symptoms such as hunger and dry mouth were also greatly improved. Therefore, the fermented product of E, that is, Example 5, was selected as the best formulation of the fermented product composition.

[0153] The probiotic fermented product provided in the embodiment of the present invention is processed and produced by a fermentation composition of Lactobacillus rhamnosus BN06. As an active probiotic, the probiotic factor metabolites produced by the fermentation of Lactobacillus rhamnosus BN06 can not only inhibit the rapid absorption of glucose, but also regulate the absorption frequency of glucose, thereby avoiding insufficient insulin supply and pancreatic islet overload operation, liver and other internal organs caused by a sharp rise in blood sugar, which is beneficial to protecting the health of the pancreas and continuously improving the blood sugar level of hyperglycemia, but also can promote insulin secretion and accelerate blood sugar regulation of hyperglycemia, thereby improving the symptoms of hyperglycemia.

[0154] In addition, the new fermented product made from rhamnosus Lactobacillus BN06 not only contains active BN06, but is also rich in various functional factors such as short-chain fatty acids, alkaloids, flavonoids, polyphenols, amino acids, vitamins, etc. converted by BN06. The consumption process only requires soaking in warm water for 5 minutes to obtain a live bacteria drink. After human consumption, it can not only achieve live bacteria colonization in the intestines and continuously improve the symptoms of hyperglycemia, but also effectively improve the symptoms of hyperglycemia from the molecular level.

[0155] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A probiotic fermentation product, characterized in that: The fermented product is prepared by fermenting a composition with Lacticaseibacillus rhamnosus BN06, and the preservation number of the Lacticaseibacillus rhamnosus BN06 is CGMCC No.29103; the Lacticaseibacillus rhamnosus BN06 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on November 22, 2023; the composition includes the following components in weight percentage: 45 parts of carambola, 30 parts of lotus leaves, and 25 parts of tea tree flowers.

2. The probiotic fermented product according to claim 1, characterized in that: The fermented product is pre-frozen with liquid nitrogen, then vacuum-freeze-dried, and then pulverized.

3. A method for preparing a probiotic fermented product according to any one of claims 1 to 2, characterized in that: The following steps are involved: Beating: put the formulated amount of carambola, lotus leaf and tea tree flower into a juicer, add distilled water and pectinase, stir, and obtain base material A; Enzymatic hydrolysis: adjust the pH of base material A with citric acid to obtain base material B; Sterilization: Place base material B in an autoclave and sterilize to obtain base material C; Activation of bacterial strains: Activate Lacticaseibacillus rhamnosus BN06 and culture to obtain bacterial suspension D; Fermentation: Place base material C in a fermentation tank, add bacterial suspension D, and perform anaerobic fermentation to obtain fermentation stock solution E; Freeze drying: spread the fermentation solution E on a plate filled with liquid nitrogen for pre-freezing, and then freeze-dry it in a vacuum freeze dryer to obtain base material F; Grinding: Grind the base material F and pass it through a sieve to obtain freeze-dried particles G; Bagging: Put the freeze-dried particles G into a filter bag to obtain a fermented product.

4. The method for preparing a probiotic fermented product according to claim 3, characterized in that: The following steps are involved: Raw material selection: Choose fresh, ripe, disease-free carambolas, fresh, blooming, non-rotten lotus leaves and tea flowers; Pretreatment: Wash the carambola with clean water to remove the dirt on the surface, drain the surface water, remove the core, cut into pieces, and weigh according to the recipe; Wash the lotus leaves with clean water to remove dirt from the surface, drain the surface water, cut into small strips, and weigh according to the formula; wash the tea flowers with clean water to remove dirt from the surface, drain the surface water, and weigh according to the formula; Beating: put the weighed carambolas, lotus leaves and tea flowers into a juicer, add 80-150 parts of distilled water and 0.01-0.05% pectinase, and stir at 12000-15000 r / min for 0.5-2 min to obtain base material A; Enzymatic hydrolysis: adjust the pH of base material A to 3-4 with citric acid, and keep it at 40-50°C for 0.8-1.2h to obtain base material B; Sterilization: Place base material B in an autoclave, sterilize at 120-123°C for 15-22 minutes, and then cool to 35-37°C to obtain base material C; Activation of bacterial strains: Take out the freeze-dried tube of Lactobacillus rhamnosus BN06 stored at -80°C, use a pipette to measure 0.3~0.4mL of sterile saline and add it dropwise to the freeze-dried tube, shake well, use a pipette to draw 150~200μL, inject it onto the plate of solid culture medium, and use a coating rod to make it evenly distributed, and then culture it in a 37°C constant temperature incubator for 45~50h to obtain the first-generation bacterial strain; use an inoculation loop to pick up independent colonies of the first-generation bacterial strain and put them into liquid culture medium, culture them in a 37°C shaker at 120~180 rpm for 45~48h, centrifuge at 7500~8500r / min for 8~12min, take the precipitate to obtain the activated second-generation bacterial strain, wash it with saline for 2~3 times, and then resuspend and dilute it with saline to adjust its viable bacterial count to 1.0×10 10 CFU / mL, bacterial suspension D was obtained; Fermentation: Place base material C in a fermentation tank, add 3-6% bacterial suspension D, and perform anaerobically fermentation at 37°C and 90-110 r / min for 45-48 hours to obtain fermentation stock solution E; Freeze drying: spread the fermentation stock solution E on a plate filled with liquid nitrogen for 8-10 minutes to a thickness of 4-5 mm, and then freeze dry it in a vacuum freeze dryer. Keep the shelf temperature at -50°C for 25-30 minutes, raise the temperature to -10--8°C within 60 minutes, keep it for 9-11 hours, and then raise the temperature to 8-12°C and keep it for 1.8-2.2 hours to obtain base material F. The vacuum degree of the whole process is 0.4-0.6 Pa, and the cold trap temperature is -60--55°C. Grinding: Grind the base material F and pass it through a 10-20 mesh sieve to obtain freeze-dried particles G; Bag filling: put the freeze-dried particles G into 200-300 mesh filter bags, 10-20 g per bag, to obtain the fermented product.

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