Fermented lactobacillus mucilaginosus with hypoglycemic effect and application of fermented lactobacillus mucilaginosus in fermented tea beverage

Abstract

The invention discloses lactobacillus mucus with a hypoglycemic effect and an application of the lactobacillus mucus in a fermented tea beverage, the lactobacillus mucus is preserved in China Center for Type Culture Collection on December 3, 2024, and the preservation number is CCTCC NO: M20242692. The lactobacillus mucilaginosus disclosed by the invention not only has a remarkable hypoglycemic effect, but also shows a good hypoglycemic effect in a product obtained by co-fermenting the lactobacillus mucilaginosus and the Kangpu tea. The innovative combination not only enriches the nutritional value of the Kangpu tea, but also significantly improves the health benefits of the Kangpu tea, so that the Kangpu tea becomes a preferred beverage for diabetics and people pursuing healthy life, and opens up a new possibility for the functional beverage market.

Description

Technical Field

[0001] The present invention belongs to the technical field of Lactobacillus, and particularly relates to a Lactobacillus mucosae fermentum with hypoglycemic effect and its application in fermented tea beverages. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by abnormally elevated blood glucose levels, which are usually caused by insufficient insulin secretion or reduced biological efficacy of insulin. With the rising global obesity rate and lifestyle changes, the incidence of type 2 diabetes has shown a sharp upward trend and has become a global public health problem. The long-term hyperglycemic state not only affects the health of patients but may also lead to a series of complications such as cardiovascular diseases, kidney diseases, neuropathy, and foot problems, seriously threatening the quality of life of patients.

[0003] In the traditional management of diabetes, drug treatment and lifestyle changes are usually relied on. However, recent studies have shown that certain food ingredients and supplements can be used as adjuvant therapies to help improve blood glucose control. Among many natural hypoglycemic substances, lactic acid bacteria have received extensive attention due to their potential in regulating intestinal health and metabolism. Lactic acid bacteria show their potential in adjuvant hypoglycemia by improving the balance of intestinal flora, enhancing intestinal barrier function, and regulating signal pathways related to host metabolism.

[0004] Fermented tea beverages, such as kombucha, are a typical example of the application of lactic acid bacteria. This beverage is made by symbiotic fermentation of lactic acid bacteria, yeasts, and acetic acid bacteria, and not only has a unique sour-sweet taste but also brings a series of health benefits. Studies have shown that the active ingredients in kombucha may help improve intestinal health, increase insulin sensitivity, and have a positive impact on controlling blood glucose levels.

[0005] Therefore, screening lactic acid bacteria with significant hypoglycemic effects and exploring their application in fermented tea beverages are of great significance for the development of new hypoglycemic foods. This can not only meet the market demand for healthy foods but may also provide a new adjuvant treatment option for diabetic patients. The present invention aims to provide a Lactobacillus mucosae fermentum with hypoglycemic effect and its application in fermented tea beverages, in order to provide a new strategy for the dietary management of diabetes. Summary of the Invention

[0006] Technical problem to be solved: The present invention provides a fermented Limosilactobacillus fermentum with hypoglycemic effect and its application in fermented tea beverages. This strain has a significant hypoglycemic effect and is applied to the fermentation process of kombucha to develop an innovative functional fermented tea beverage. This beverage aims to combine traditional tea drinks with modern biotechnology, not only meeting the growing market demand for healthy beverages, but also providing a natural adjuvant hypoglycemic diet therapy plan for diabetic patients and those in need of blood glucose management. In addition, the present invention also aims to enhance consumers' confidence in the health benefits of the new fermented tea beverage by scientifically verifying the hypoglycemic mechanism of this Lactobacillus strain and its effect during the kombucha fermentation process. By optimizing the production process and formula, this beverage will provide a safe and effective natural hypoglycemic alternative, helping to improve consumers' health and quality of life. At the same time, this invention is also expected to promote innovation in the field of functional foods and bring new growth points to the food industry.

[0007] Technical solution: A fermented Limosilactobacillus fermentum YZU-lbfel with hypoglycemic effect, which is deposited in the China Center for Type Culture Collection, with the deposit date of December 3, 2024, and the deposit number of CCTCC NO: M20242692.

[0008] The application of the above-mentioned fermented Limosilactobacillus fermentum in the preparation of tea beverages.

[0009] A method for preparing a kombucha beverage, comprising the following steps: S1. Preparation of tea soup water: Mix tea leaves with water, boil and extract for 10 - 20 minutes, filter out the tea leaves, add sucrose to a final concentration of 5 - 10% and cool; S2. Pretreatment of Limosilactobacillus fermentum: After resuscitating and amplifying the Limosilactobacillus fermentum described in claim 1 in MRS medium, centrifuge and wash to remove the medium, and resuspend the cells with black tea soup; S3. Compound fermentation: Mix the tea soup water in step S1 with kombucha pellicle, add the cell suspension prepared in step S2, and anaerobically ferment at 25 - 37°C for 7 days.

[0010] Preferably, the inoculation amount of the Limosilactobacillus fermentum in step S3 is 1% - 3% of the volume of the tea soup water, and the concentration of the cell suspension is 10 8 CFU / mL.

[0011] Preferably, the addition amount of the kombucha pellicle in step S3 is 5% of the mass of the tea soup water, and the tank mouth is covered with gauze during the fermentation process to maintain a microaerobic environment.

[0012] A kombucha beverage prepared by the above method.

[0013] The application of the above-mentioned fermented Limosilactobacillus fermentum in the preparation of functional foods for improving impaired glucose tolerance or insulin resistance.

[0014] The above functional food is a fermented tea beverage, a probiotic preparation or a dietary supplement.

[0015] A compound bacterium agent contains a mixture of the above-mentioned Lactobacillus mucosae fermentum and Kombucha pellicle.

[0016] Beneficial effects: Through the present invention, we have successfully compounded and fermented Lactobacillus mucosae fermentum and Kombucha. The experimental results show that: compared with traditional Kombucha, after adding Lactobacillus mucosae fermentum, the in vitro inhibition rates of α-amylase and α-glucosidase are significantly increased (see Figure 1 and Figure 2 ). In the gavage experiment on high-fat mice, we observed that adding Lactobacillus mucosae fermentum can significantly slow down the rising trend of blood glucose, showing the potential to improve impaired glucose tolerance (see Figure 3 and Figure 4 ). In addition, compared with the non-gavaged high-fat mouse group, the fasting insulin level of the mice gavaged with Lactobacillus mucosae fermentum is significantly reduced (see Figure 5 ). The insulin resistance - IR results show that: obvious insulin resistance occurred in the mice in the normal saline gavage group, while the mice gavaged with the co-fermented Kombucha of Lactobacillus mucosae fermentum showed an improvement in insulin resistance (see Figure 6 ). The determination results of total bile acids in serum and liver show that, compared with the non-compounded traditional Kombucha group, the levels of total bile acids in serum and liver of the mice in the Lactobacillus mucosae fermentum group are significantly increased (see Figure 7 and Figure 8 ). To sum up, the Lactobacillus mucosae fermentum of the present invention not only has a significant blood glucose lowering effect, but also the product co-fermented with Kombucha shows good blood glucose lowering efficacy. This innovative combination not only enriches the nutritional value of Kombucha, but also significantly improves its health benefits, making it an optimal drink for diabetic patients and people pursuing a healthy lifestyle, and opening up new possibilities for the functional beverage market. Description of the Drawings

[0017] Figure 1 It is a graph of the inhibition rate of α-amylase.

[0018] Figure 2 It is a graph of the inhibition rate of α-glucosidase.

[0019] Figure 3 It is a graph of the oral glucose tolerance of mice.

[0020] Figure 4 It is a graph of the area under the blood glucose curve of mice.

[0021] Figure 5 It is a graph of the fasting insulin content of mice.

[0022] Figure 6It is a graph of the insulin resistance index of mice.

[0023] Figure 7 It is a graph of the total bile acid content in the serum of mice.

[0024] Figure 8 It is a graph of the total bile acid in the liver of mice. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments. Limosilactobacillus fermentum YZU-Ibfel is preserved in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, Hubei Province, China, the preservation date being December 3, 2024, and the preservation number being CCTCC NO: M20242692.

[0026] The strain Limosilactobacillus fermentum YZU-lbfel of the present invention belongs to facultative anaerobes, which can not only grow in an aerobic environment but also reproduce normally in an anaerobic jar. After Gram staining, its colonies exhibit significant characteristics of Gram-positive bacteria.

[0027] When the strain is inoculated on MRS solid medium and cultured at 37°C for 36 hours, the formed colonies have the following characteristics: the surface is smooth and moist, the color is white, the size is uniform, the overall shape is regular circular, the edge is smooth and neat, and the diameter is mostly between 1-3 mm.

[0028] When it is placed in MRS liquid medium and cultured with shaking at 37°C for 24 hours, a large amount of white bacterial cell precipitation will appear. As a kind of lactic acid bacteria, this strain has the characteristics of producing gas and acid during metabolism.

[0029] Example 1

[0030] 1. Preparation of tea soup water

[0031] Take 1 L of pure water, boil it and add 5 g of tea leaves (purchased from Yiming Ecological Tea Industry Co., Ltd., Wuyishan, Xiamen), filter out the tea leaves after extraction for 15 min. Add 70 g of sucrose (Shanghai Sugar, Tobacco and Alcohol Co., Ltd.), stir and dissolve it, and then let it stand and cool to obtain the tea soup water;

[0032] 2. Fermentation

[0033] Add 800 ml of tea soup water to the fermentation tank. Add 40 g of kombucha culture. Cover the tank mouth with three layers of gauze. Culture it in a constant temperature incubator at 30°C for 7 days. Through the above steps, we obtained kombucha beverage (Group K).

[0034] Example 2

[0035] 1. Preparation of tea soup water

[0036] Take 1 L of pure water, boil it, add 5 g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Industry Co., Ltd.), filter the tea leaves after extraction for 15 min. Add 70 g of sucrose (Shanghai Sugar, Tobacco and Alcohol Co., Ltd.), stir to dissolve and then let it stand and cool to obtain the tea soup water.

[0037] 2. Resuscitation and culture of Lactobacillus plantarum

[0038] After taking out the Lactobacillus plantarum strain from the cryopreservation tube, inoculate it into MRS liquid medium at an inoculation amount of 2%, and anaerobically culture it at 37 °C for 24 h. Streak the cultured Lactobacillus plantarum on MRS solid medium, pick single colonies after culturing at 37 °C for 24 h. Inoculate the selected colonies into MRS liquid medium and culture them again at 37 °C for 24 hours. Use the dilution coating plate method to count Lactobacillus plantarum, and store the bacterial solution in a 4 °C refrigerator. Take 8 mL of the bacterial solution, centrifuge it at 5000 rpm for 3 min to remove the supernatant medium, add black tea soup and mix well, then centrifuge again, and repeat the operation 3 times to wash the medium. After the last centrifugation is completed, add 8 mL of black tea soup to the precipitate, mix well and set aside.

[0039] 3. Compound fermentation

[0040] Take 800 mL of tea soup water in a fermentation tank, inoculate 40 g of Kombucha bacterial membrane, and inoculate 8 mL of Lactobacillus plantarum (10 8 CFU / mL). Cover the tank mouth with 3 layers of gauze and culture it in a constant temperature incubator at 30 °C for 7 days to obtain Kombucha beverage (Group A).

[0041] Example 3

[0042] 1. Tea soup water preparation

[0043] Take 1 L of pure water, boil it, add 5 g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Industry Co., Ltd.), filter the tea leaves after extraction for 15 min. Add 70 g of sucrose (Shanghai Sugar, Tobacco and Alcohol Co., Ltd.), stir to dissolve and then let it stand and cool to obtain the tea soup water.

[0044] 2. Resuscitation and culture of Lactobacillus mucosae

[0045] After taking out the Lactobacillus mucosae fermentum strain from the cryotube, inoculate it into MRS liquid medium at an inoculation amount of 2%, and culture it anaerobically at 37°C for 24 h. Streak the cultured Lactobacillus mucosae fermentum on MRS solid medium, and pick single colonies after culturing at 37°C for 24 h. Inoculate the selected colonies into MRS liquid medium and culture again at 37°C for 24 hours. Use the dilution coating plate method to count Lactobacillus mucosae fermentum, and store the bacterial liquid in a 4°C refrigerator. Take 8 mL of the bacterial liquid, centrifuge it at 5000 rpm for 3 min to remove the supernatant medium, add black tea soup and mix well, then centrifuge again. Repeat the operation 3 times to wash the medium. After the last centrifugation is completed, add 8 mL of black tea soup to the precipitate, mix well and set aside.

[0046] 3. Compound fermentation

[0047] Take 800 mL of tea soup water in a fermentation tank, inoculate 40 g of kombucha biofilm, and inoculate 8 mL of Lactobacillus mucosae fermentum (10 8 CFU / mL). Cover the tank mouth with 3 layers of gauze and culture it in a constant temperature incubator at 30°C for 7 days to obtain kombucha beverage (Group B).

[0048] Example 4

[0049] 1. Preparation of tea soup water

[0050] Take 1 L of pure water, add 5 g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Industry Co., Ltd.) after boiling, filter the tea leaves after extraction for 15 min. Add 70 g of sucrose (Shanghai Sugar, Tobacco and Wine Co., Ltd.), stir to dissolve and let it stand and cool to obtain tea soup water.

[0051] 2. Resuscitation and culture of Lactobacillus acidophilus

[0052] After taking out the Lactobacillus acidophilus strain from the cryotube, inoculate it into MRS liquid medium at an inoculation amount of 2%, and culture it anaerobically at 37°C for 24 h. Streak the cultured Lactobacillus acidophilus on MRS solid medium, and pick single colonies after culturing at 37°C for 24 h. Inoculate the selected colonies into MRS liquid medium and culture again at 37°C for 24 hours. Use the dilution coating plate method to count Lactobacillus acidophilus, and store the bacterial liquid in a 4°C refrigerator. Take 8 mL of the bacterial liquid, centrifuge it at 5000 rpm for 3 min to remove the supernatant medium, add black tea soup and mix well, then centrifuge again. Repeat the operation 3 times to wash the medium. After the last centrifugation is completed, add 8 mL of black tea soup to the precipitate, mix well and set aside.

[0053] 3. Compound fermentation

[0054] Take 800 mL of tea soup water in a fermentation tank, inoculate 40 g of kombucha biofilm, and inoculate 8 mL of Lactobacillus acidophilus (10 8(CFU / mL). Cover the mouth of the can with three layers of gauze and culture in a constant temperature incubator at 30 °C for 7 days to obtain the kombucha beverage (Group C).

[0055] After functional testing of the four examples, we found that Example 3 (Group B) was particularly prominent in terms of the in vitro α-amylase inhibition rate, and its inhibitory effect was significantly better than the other three examples. Compared with the drug control group, the inhibition rate increased by more than 80%. In addition, the α-glucosidase inhibition rate of Group B was also significantly better than the other three examples, further demonstrating its advantage in hypoglycemic potential.

[0056] The results of the oral glucose tolerance test in mice fed a high-fat diet showed that the blood glucose levels of Group B mice at each time were lower than those of Group K mice without compounding. Statistical analysis of the area under the blood glucose curve showed that the area under the curve of mice that ingested the kombucha of Group B was significantly lower than that of high-fat mice in Group O that were gavaged with normal saline, showing a trend of improving impaired glucose tolerance. The fasting insulin level of the mice showed that Group B was significantly lower than Group K. The insulin resistance-IR test showed that obvious insulin resistance occurred in the mice in Group O gavaged with normal saline, and Group B could improve the insulin resistance induced by a high-fat diet. The detection of total bile acids in the serum of mice showed that the total bile acids in the serum of Example 3 were significantly higher than those of the other three examples; the detection of total bile acids in the liver of mice showed that the total bile acids in the liver of Example 3 were also significantly higher than those of the other three examples, indicating that Lactobacillus mucosae fermentum may reduce blood glucose by affecting bile acid metabolism.

[0057] In summary, Example 3 (Group B), that is, the kombucha beverage using Lactobacillus mucosae fermentum, not only shows excellent hypoglycemic potential in in vivo and in vitro experiments, but also improves the health value of the beverage, especially in terms of reducing blood glucose, which is particularly prominent.

[0058] The above embodiments describe the preferred embodiments of the present invention and do not limit the present invention. Without departing from the spirit or scope of the present invention, technical improvements and equivalent substitutions made by relevant technical personnel to the present invention are within the protection scope of the present invention.

Claims

1. A strain of fermented Lactobacillus mucilaginosus with hypoglycemic effect ( Limosilactobacillus fermentum )YZU-lbfel, deposited in China Center for Type Culture Collection, the preservation date is December 3, 2024, and the preservation number is CCTCCNO: M20242692.

2. Use of the fermented mucus lactobacillus according to claim 1 in preparing tea beverages.

3. A method for preparing a kombucha beverage, characterized in that: The following steps are involved: S1. Preparation of tea soup: Mix tea leaves with water, boil and soak for 10-20 minutes, filter out the tea leaves, add sucrose to a final concentration of 5-10% and cool; S2. Pretreatment of fermented mucus lactobacillus: resuscitating and amplifying the fermented mucus lactobacillus according to claim 1 with MRS culture medium, washing by centrifugation to remove the culture medium, and resuspending the bacteria with black tea soup; S3, compound fermentation: the tea soup of step S1 is mixed with the kombucha bacterial film, and the bacterial suspension prepared in step S2 is added, and anaerobically fermented at 25-37° C. for 7 days.

4. The preparation method according to claim 3, characterized in that: The inoculation amount of the fermented mucus lactobacillus in step S3 is 1%-3% of the volume of the tea soup, and the concentration of the bacterial suspension is 10 8 CFU / mL.

5. The preparation method according to claim 3, characterized in that: The amount of kombucha bacterial film added in step S3 is 5% of the mass of the tea soup, and the mouth of the tank is covered with gauze during the fermentation process to maintain a micro-oxygen environment.

6. A kombucha beverage, characterized in that Prepared by the method according to any one of claims 3 to 5.

7. Use of the fermented mucus lactobacillus according to claim 1 in preparing functional foods for improving impaired glucose tolerance or insulin resistance.

8. The use according to claim 7, characterized in that: The functional food is a fermented tea beverage, a probiotic preparation or a dietary supplement.

9. A composite bacterial agent, characterized in that: A mixture comprising the fermented mucus lactobacillus and kombucha biofilm according to claim 1.

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