Lactobacillus muci genosus with hypoglycemic effect and application thereof in fermented tea beverage

Abstract

A fermented lactobacillus muciapparius with hypoglycemic effect and its application in fermented tea beverage, the strain is preserved in China Center for Type Culture Collection, the preservation date is December 3, 2024, and the preservation number is CCTCC NO:M20242692. The fermented lactobacillus muciapparius of the present application not only has significant hypoglycemic effect, but also the product co-fermented with kungpao tea also shows good hypoglycemic effect. This innovative combination not only enriches the nutritional value of kungpao tea, but also significantly improves its health benefits, making it the preferred drink for diabetic patients and people pursuing a healthy lifestyle, opening up new possibilities for the functional beverage market.

Description

Technical Field

[0001] This invention belongs to the field of lactobacillus technology, specifically relating to a strain of fermentable mucoid lactobacillus with hypoglycemic effects and its application in fermented tea beverages. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by abnormally high blood sugar levels, usually caused by insufficient insulin secretion or decreased insulin bioefficacy. With rising global obesity rates and changing lifestyles, the incidence of type 2 diabetes is increasing dramatically, becoming a global public health issue. Long-term hyperglycemia not only affects patients' health but can also lead to a range of complications such as cardiovascular disease, kidney disease, neuropathy, and foot problems, seriously threatening their quality of life.

[0003] Traditional diabetes management typically relies on medication and lifestyle modifications. However, recent studies have shown that certain food components and supplements can serve as adjunctive therapies, helping to improve glycemic control. Among numerous natural blood sugar-lowering substances, lactic acid bacteria have attracted widespread attention due to their potential in regulating gut health and metabolism. Lactic acid bacteria demonstrate their potential in adjunctive blood sugar lowering by improving gut microbiota balance, enhancing intestinal barrier function, and regulating signaling pathways related to host metabolism.

[0004] Fermented tea beverages, such as kombucha, are a prime example of the application of lactic acid bacteria. These drinks, made through the symbiotic fermentation of lactic acid bacteria, yeast, and acetic acid bacteria, not only offer a unique sweet and sour taste but also a range of health benefits. Studies have shown that the active ingredients in kombucha may help improve gut health, enhance insulin sensitivity, and have a positive impact on blood sugar control.

[0005] Therefore, screening lactic acid bacteria with significant hypoglycemic effects and exploring their application in fermented tea beverages is of great significance for developing novel hypoglycemic foods. This can not only meet the market demand for health foods, but also potentially provide a new adjunctive treatment option for diabetic patients. This invention aims to provide a fermented *Lactobacillus mucilaginosus* with hypoglycemic effects 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 Solved: This invention provides a strain of fermentable *Lactobacillus mucinus* with hypoglycemic effects and its application in fermented tea beverages. This strain exhibits significant hypoglycemic effects and is applied to the fermentation process of kombucha to develop an innovative functional fermented tea beverage. This beverage aims to combine traditional tea drinking with modern biotechnology, not only meeting the growing market demand for healthy drinks but also providing a natural, supplementary hypoglycemic dietary therapy for diabetic patients and those requiring blood sugar management. Furthermore, this invention aims to enhance consumer confidence in the health benefits of this novel fermented tea beverage by scientifically validating the hypoglycemic mechanism of this *Lactobacillus* strain and its effect in the kombucha fermentation process. By optimizing the production process and formulation, this beverage will provide a safe and effective natural hypoglycemic alternative, helping to improve consumers' health and quality of life. Simultaneously, this invention is expected to drive innovation in the functional food field, bringing new growth points to the food industry.

[0007] Technical solution: A strain of fermenting Lactobacillus fermentum YZU-lbfel with hypoglycemic effect is deposited at the China Center for Type Culture Collection on December 3, 2024, with accession number CCTCC NO:M20242692.

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

[0009] A method for preparing a kombucha beverage includes the following steps: S1, tea infusion preparation: 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 fermenting *Lactobacillus mucinus*: revive and amplify the *Lactobacillus mucinus* as described in claim 1 on MRS medium, centrifuge and wash to remove the medium, and resuspend the bacteria in black tea infusion; S3, compound fermentation: mix the tea infusion from step S1 with kombucha bacterial film, add the bacterial suspension prepared in step S2, and anaerobic ferment at 25-37℃ for 7 days.

[0010] Preferably, in step S3, the inoculum amount of *Lactobacillus mucinus* is 1%-3% of the tea infusion volume, and the bacterial suspension concentration is 10. 8 CFU / mL.

[0011] Preferably, the amount of kombucha film added in step S3 is 5% of the mass of the tea infusion, and the jar opening is covered with gauze during fermentation to maintain a micro-oxygen environment.

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

[0013] A compound microbial agent comprising a mixture of the aforementioned fermenting Lactobacillus mucilaginosus and Kombucha film.

[0014] Beneficial Effects: Through this invention, we successfully combined fermented *Lactobacillus myxoidis* with kombucha for fermentation. Experimental results show that compared with traditional kombucha, the addition of *Lactobacillus myxoidis* significantly increased the in vitro inhibition rates of α-amylase and α-glucosidase (see...). Figure 1 and Figure 2 In a gavage experiment on high-fat mice, we observed that the addition of fermented Lactobacillus mucin significantly slowed the rise in blood glucose, demonstrating its potential to improve impaired glucose tolerance (see [link to study]). Figure 3 and Figure 4 Furthermore, compared with the high-fat mouse group that did not receive gavage, mice gavaged with fermented Lactobacillus mucinus showed significantly lower fasting insulin levels (see [link]). Figure 5 Insulin resistance-IR results showed that mice administered saline by gavage developed significant insulin resistance, while mice administered kombucha co-fermented with *Lactobacillus mucinus* by gavage showed improved insulin resistance (see [link]). Figure 6 The results of serum and liver total bile acid measurements showed that, compared with the unblended conventional kombucha group, the levels of serum and liver total bile acids in mice in the fermented *Lactobacillus mucinus* group were significantly increased (see [link to study]). Figure 7 and Figure 8 In summary, the fermented *Lactobacillus mucinus* of this invention not only has a significant hypoglycemic effect, but its co-fermentation with kombucha also exhibits good hypoglycemic efficacy. This innovative combination not only enriches the nutritional value of kombucha but also significantly enhances its health benefits, making it a preferred beverage for diabetic patients and those pursuing a healthy lifestyle, thus opening up new possibilities for the functional beverage market. Attached Figure Description

[0015] Figure 1 This is a graph showing the inhibition rate of α-amylase.

[0016] Figure 2 This is a graph showing the inhibition rate of α-glucosidase.

[0017] Figure 3 This is a graph showing the oral glucose tolerance test in mice.

[0018] Figure 4 This is a graph showing the area under the blood glucose curve in mice.

[0019] Figure 5 This is a graph showing the fasting insulin levels in mice.

[0020] Figure 6 This is a graph of the insulin resistance index in mice.

[0021] Figure 7 This is a graph showing the total bile acid content in mouse serum.

[0022] Figure 8 This is a graph showing total bile acids in mouse liver. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. *Limosilactobacillus fermentum* YZU-Ibfel is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China, on December 3, 2024, with accession number CCTCC NO: M20242692.

[0024] The strain of this invention, *Lactobacillus mucinus* YZU-lbfel, is a facultative anaerobic bacterium that can grow not only in aerobic environments but also reproduce normally in anaerobic environments. After Gram staining, its colonies exhibit significant Gram-positive bacterial characteristics.

[0025] When the strain was inoculated onto MRS solid medium and cultured at 37°C for 36 hours, the resulting colonies exhibited the following characteristics: smooth and moist surface, white color, uniform size, regular circular shape, smooth and neat edges, and diameter mostly between 1 and 3 mm.

[0026] When placed in MRS liquid medium and cultured with shaking at 37°C for 24 hours, a large amount of white bacterial precipitate will appear. This strain, as a type of lactic acid bacteria, has the characteristics of gas production and acid production during metabolism.

[0027] Example 1

[0028] 1. Preparation of tea infusion

[0029] Take 1L of purified water, boil it, add 5g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Co., Ltd.), steep for 15 minutes, then filter out the tea leaves. Add 70g of cane sugar (Shanghai Sugar, Tobacco & Alcohol Co., Ltd.), stir to dissolve, and let stand to cool to obtain tea infusion;

[0030] 2. Fermentation

[0031] Add 800 ml of tea infusion to the fermentation tank. Add 40 g of kombucha spawn. Cover the tank opening with three layers of gauze. Incubate at 30°C for 7 days. After the above steps, we obtained the kombucha beverage (Group K).

[0032] Example 2

[0033] 1. Preparation of tea infusion

[0034] Take 1L of purified water, boil it, add 5g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Co., Ltd.), steep for 15 minutes, then filter out the tea leaves. Add 70g of cane sugar (Shanghai Sugar, Tobacco & Alcohol Co., Ltd.), stir to dissolve, and let stand to cool to obtain tea infusion.

[0035] 2. Resuscitation and Culture of Lactobacillus plantarum

[0036] After removing the *Lactobacillus plantarum* strain from the cryopreservation tube, inoculate it into MRS liquid medium at a 2% inoculum and incubate anaerobically at 37°C for 24 hours. Streak the cultured *Lactobacillus plantarum* onto MRS solid medium, incubate at 37°C for 24 hours, and then pick single colonies. Inoculate the selected colonies into MRS liquid medium and incubate again at 37°C for 24 hours. Count the *Lactobacillus plantarum* using the dilution plating method and store the bacterial suspension at 4°C. Take 8 mL of the bacterial suspension, centrifuge at 5000 rpm for 3 min, remove the supernatant, add black tea infusion, mix well, and centrifuge again. Repeat this process three times to wash away the culture medium. After the final centrifugation, add 8 mL of black tea infusion to the precipitate, mix well, and set aside.

[0037] 3. Compound fermentation

[0038] Take 800mL of tea infusion into a fermentation tank, inoculate with 40g of kombucha spawn film, and inoculate with 8mL of Lactobacillus plantarum (10). 8 CFU / mL). Cover the mouth of the can with 3 layers of gauze and incubate in a constant temperature incubator at 30℃ for 7 days to obtain kombucha beverage (Group A).

[0039] Example 3

[0040] 1. Preparation of tea infusion

[0041] Take 1L of purified water, boil it, add 5g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Co., Ltd.), steep for 15 minutes, then filter out the tea leaves. Add 70g of cane sugar (Shanghai Sugar, Tobacco & Alcohol Co., Ltd.), stir to dissolve, and let stand to cool to obtain tea infusion.

[0042] 2. Recovery and culture of fermented Lactobacillus mucinus

[0043] After removing the *Lactobacillus fermentatus* strain from the cryopreservation tube, inoculate it into MRS liquid medium at a 2% inoculum and anaerobic culture at 37°C for 24 h. Streak the cultured *Lactobacillus fermentatus* onto MRS solid medium, incubate at 37°C for 24 h, and then pick single colonies. Inoculate the selected colonies into MRS liquid medium and incubate again at 37°C for 24 h. Count the *Lactobacillus fermentatus* using the dilution plating method and store the bacterial culture at 4°C. Take 8 mL of the bacterial culture, centrifuge at 5000 rpm for 3 min, remove the supernatant, add black tea infusion, mix well, and centrifuge again. Repeat this process 3 times to wash away the culture medium. After the final centrifugation, add 8 mL of black tea infusion to the precipitate, mix well, and set aside.

[0044] 3. Compound fermentation

[0045] Take 800mL of tea infusion into a fermentation tank, inoculate with 40g of kombucha spawn film, and inoculate with 8mL of fermenting lactobacillus (10... 8 (CFU / mL). Cover the can opening with three layers of gauze and incubate at 30℃ for 7 days to obtain kombucha beverage (Group B).

[0046] Example 4

[0047] 1. Preparation of tea infusion

[0048] Take 1L of purified water, boil it, add 5g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Co., Ltd.), steep for 15 minutes, then filter out the tea leaves. Add 70g of cane sugar (Shanghai Sugar, Tobacco & Alcohol Co., Ltd.), stir to dissolve, and let stand to cool to obtain tea infusion.

[0049] 2. Resuscitation and culture of Lactobacillus acidophilus

[0050] After removing the *Lactobacillus acidophilus* strain from the cryopreservation tube, inoculate it into MRS liquid medium at a 2% inoculum and incubate anaerobically at 37°C for 24 hours. Streak the cultured *Lactobacillus acidophilus* onto MRS solid medium, incubate at 37°C for 24 hours, and then pick single colonies. Inoculate the selected colonies into MRS liquid medium and incubate again at 37°C for 24 hours. Count the *Lactobacillus acidophilus* using the dilution plating method and store the bacterial suspension at 4°C. Take 8 mL of the bacterial suspension, centrifuge at 5000 rpm for 3 min, remove the supernatant, add black tea infusion, mix well, and centrifuge again. Repeat this process three times to wash away the medium. After the final centrifugation, add 8 mL of black tea infusion to the precipitate, mix well, and set aside.

[0051] 3. Compound fermentation

[0052] Take 800mL of tea infusion into a fermentation tank, inoculate with 40g of kombucha spawn film, and inoculate with 8mL of Lactobacillus acidophilus (10). 8 (CFU / mL). Cover the mouth of the can with three layers of gauze and incubate at 30℃ for 7 days to obtain kombucha beverage (Group C).

[0053] After functional testing of the four embodiments, we found that Embodiment 3 (Group B) was particularly outstanding in terms of in vitro α-amylase inhibition rate, with its inhibition effect significantly exceeding that of the other three embodiments, and the inhibition rate was increased by more than 80% compared with the drug control group. In addition, the α-glucosidase inhibition rate of Group B was also significantly better than that of the other three embodiments, further demonstrating its advantage in hypoglycemic potential.

[0054] Oral glucose tolerance tests conducted on mice fed a high-fat diet showed that the blood glucose levels of group B mice were lower than those of the untreated group K mice at all time points. Statistical analysis of the area under the blood glucose curve (AUC) revealed that the AUC of group B mice, which ingested kombucha, was significantly lower than that of the high-fat group O mice administered saline by gavage, indicating a trend towards improved glucose tolerance. Fasting insulin levels in mice showed that group B was significantly lower than group K. Insulin resistance-IR tests indicated that group O mice administered saline by gavage developed significant insulin resistance, while group B was able to improve insulin resistance induced by a high-fat diet. Serum total bile acid assays in mice showed that the serum total bile acid levels in Example 3 were significantly higher than those in the other three examples; liver total bile acid assays in mice showed that the liver total bile acid levels in Example 3 were also significantly higher than those in the other three examples, suggesting that *Lactobacillus fermentum* may lower blood glucose by influencing bile acid metabolism.

[0055] In summary, Example 3 (Group B), namely the kombucha beverage made from fermented Lactobacillus mucinus, not only showed excellent blood sugar lowering potential in both in vitro and in vivo experiments, but also enhanced the health value of the beverage, especially in its remarkable effect on lowering blood sugar.

[0056] The above embodiments are preferred embodiments of the present invention and are not intended to limit the present invention. Any technical improvements and equivalent substitutions made to the present invention by those skilled in the art without departing from the spirit or scope of the present invention are within the protection scope of the present invention.

Claims

1. A strain of fermenting *Lactobacillus mucilaginosus* with hypoglycemic effects ( Limosilactobacillus fermentum YZU-lbfel, deposited at the China Center for Type Culture Collection, on December 3, 2024, with accession number CCTCCNO:M20242692.

2. The application of the fermented Lactobacillus mucilaginosus according to claim 1 in the preparation of tea beverages.

3. A method for preparing a kombucha beverage, characterized in that, Includes the following steps: S1. Preparation of tea infusion: Mix tea leaves with water, boil and steep for 10-20 minutes, filter out the tea leaves, add sucrose to a final concentration of 5-10% and cool. S2. Pretreatment of fermenting *Lactobacillus mucinus*: After the fermenting *Lactobacillus mucinus* of claim 1 is revived and amplified on MRS medium, the medium is removed by centrifugation and washing, and the bacteria are resuspended in black tea infusion; S3. Compound fermentation: The tea infusion of step S1 is mixed with the kombucha mycelium film, and the bacterial suspension prepared in step S2 is added, and the mixture is anaerobic fermented at 25-37℃ for 7 days.

4. The preparation method according to claim 3, characterized in that, In step S3, the inoculation amount of *Lactobacillus mucinus* is 1%-3% of the tea infusion volume, and the bacterial suspension concentration is 10. 8 CFU / mL.

5. The preparation method according to claim 3, characterized in that, In step S3, the amount of kombucha film added is 5% of the mass of the tea infusion, and the jar opening is covered with gauze during fermentation to maintain a micro-oxygen environment.

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

7. A compound microbial agent, characterized in that, A mixture comprising the fermented Lactobacillus mucilaginosus and Kombucha film as described in claim 1.

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