Weizmannia coagulans and applications thereof

Abstract

The present application relates to the field of microorganisms, and discloses a weizmannia coagulans and application thereof. The weizmannia coagulans provided by the present application has a preservation number of CGMCC No. 38119, can be used for fermenting tea products, and can improve the flavor, gamma-aminobutyric acid, tea polyphenol and tea catechin content of the tea products and reduce the free radical content of the tea products; can be used for fermenting dairy products and improving the acidity and flavor of the dairy products; in addition, the weizmannia coagulans provided by the present application can not only degrade inosine, guanosine, hypoxanthine, xanthine and uric acid, but also can degrade ethanol, inhibit pathogenic bacteria, degrade cholesterol, inhibit angiotensin converting enzyme activity and inhibit alpha-glucosidase activity, and has application prospects for preparing related drugs or exerting corresponding in-vitro effects.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of Weizmann's coagulans and its applications. Background Technology

[0002] Fermented tea products are traditional specialty beverages, encompassing multiple categories such as black tea, dark tea, oolong tea, and fermented flower tea. Through the metabolic action of microorganisms, the inherent components of tea leaves, such as tea polyphenols, caffeine, and amino acids, are transformed into substances with unique flavors and physiological activities, such as theaflavins, thearubigins, and γ-aminobutyric acid. This not only gives the products a mellow and sweet taste but also endows them with numerous health benefits, including antioxidant properties, regulation of intestinal flora, and lowering of blood lipids, making them highly popular among consumers.

[0003] In the production of fermented tea products, microorganisms are one of the core factors determining the quality of tea products, playing a crucial role in improving flavor, increasing the content of active ingredients, and enhancing the stability of tea products. However, microorganisms from different sources exhibit significant differences in physiological characteristics, and their adaptability, metabolic capacity, and functional expression in the fermented tea system vary. Moreover, not only do substances such as tea polyphenols originally present in tea leaves have a certain inhibitory effect on microorganisms, but the gradually decreasing pH value and changing nutrient composition during fermentation may also lead to microbial inactivation, resulting in problems such as abnormal flavor and insufficient content of active ingredients in tea products.

[0004] Therefore, there is an urgent need to provide a strain of bacteria that is tolerant to the tea fermentation environment and has good growth performance in order to improve the nutritional value of fermented tea products. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned problems existing in the prior art and to provide a strain of Weizmann's coagulans and its applications.

[0006] To achieve the above objectives, the first aspect of the present invention provides a strain of Weizmann's coagulans ( Weizmannia coagulans The preservation number of the *Weizmannii coagulans* is CGMCC No. 38119.

[0007] A second aspect of the present invention provides a microbial agent containing the *Weizmannii coagulans* described in the first aspect.

[0008] A third aspect of the present invention provides a method for fermenting tea, the method comprising: inoculating the *Weizmannii coagulans* strain described in the first aspect into tea leaves and / or tea extract for fermentation.

[0009] The fourth aspect of the present invention provides a fermented tea product, which is prepared by the method described in the third aspect.

[0010] The fifth aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the *Weizmannii coagulans* as described in the first aspect, or the bacterial agent as described in the second aspect, or the fermented tea product as described in the fourth aspect.

[0011] The sixth aspect of the present invention provides a method for increasing the content of γ-aminobutyric acid in fermented tea products, the method comprising: inoculating the *Weizmannii coagulans* described in the first aspect into tea leaves and / or tea extract for fermentation.

[0012] The seventh aspect of the present invention provides the use of the *Weizmannii coagulates* strain described in the first aspect in enhancing at least one of γ-aminobutyric acid, tea polyphenols, and tea catechins in fermented tea products.

[0013] The eighth aspect of the present invention provides the use of the *Weizmannii coagulans* described in the first aspect in enhancing the acidity and / or flavor of fermented dairy products.

[0014] The ninth aspect of the present invention provides the use of the *Weizmannii coagulans* described in the first aspect in the preparation of a medicament for lowering uric acid, degrading ethanol, inhibiting pathogens, lowering blood pressure, and lowering blood sugar.

[0015] The beneficial effects obtained by the present invention through the above technical solution include at least the following: (1) The Weizmann's coagulation bacteria with accession number CGMCC No.38119 provided by the present invention can be used to ferment tea products and improve the flavor, γ-aminobutyric acid, tea polyphenols and tea catechin content of tea products and reduce the free radical content of tea products. (2) The Weizmann's coagulans strain with accession number CGMCC No.38119 provided by this invention can be used to ferment dairy products and improve the acidity and flavor of dairy products; (3) The *Weizmannii coagulans* strain with accession number CGMCC No.38119 provided by this invention can not only degrade inosine, guanosine, hypoxanthine, xanthine and uric acid, but also degrade ethanol, inhibit pathogens, degrade cholesterol, inhibit angiotensin-converting enzyme activity and inhibit α-glucosidase activity, and has the application prospect of being prepared into related drugs.

[0016] Biological Preservation The strain provided by this invention is classified and named *Weizmannella coagulans*. Weizmannia coagulans It was deposited on March 31, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38119 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0017] Figure 1This is a microscopic image of the cells and spores of *Weizmannii coagulates*. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] The inventors of this invention isolated a gastric acid-resistant strain, CCNH166, from the intestines of infants, which was confirmed by research to be *Weizmannii coagulans*. Weizmannia coagulans Through further research, the inventors discovered that CCNH166 can be used directly in the tea fermentation process without being combined with other strains. This not only effectively improves the flavor and quality of tea products but also significantly increases the content of γ-aminobutyric acid, tea polyphenols, and tea catechins, while reducing the free radical content. Based on the above findings, the first aspect of this invention provides a strain of *Weizmannii coagulans* (…). Weizmannia coagulans The preservation number of the *Weizmannii coagulans* is CGMCC No. 38119 (i.e., the strain CCNH166 mentioned above).

[0020] A second aspect of the present invention provides a microbial agent containing *Weizmannii coagulans* as described in the first aspect and / or the metabolites of *Weizmannii coagulans* as described in the first aspect.

[0021] According to some specific embodiments of the present invention, the metabolites may include active substances produced by *Weizmannia coagulans* fermentation, including intracellular metabolites and / or extracellular metabolites. Specific forms of the metabolites may include, but are not limited to, fermentation supernatant (mainly containing intracellular metabolites, obtained by centrifugation to remove bacterial cells after fermentation) and strain lysis supernatant (including intracellular metabolites and / or extracellular metabolites, obtained by centrifugation after fermentation and cell lysis, which can simultaneously enrich intracellularly released active ingredients and extracellularly secreted active substances). The metabolites can be obtained from the *Weizmannia coagulans* fermentation system using conventional processes such as centrifugation, filtration, concentration, and freeze-drying.

[0022] According to some specific embodiments of the present invention, the fermentation supernatant may further include the supernatant obtained by centrifuging to remove the bacterial cells after the strain ferments.

[0023] According to some specific embodiments of the present invention, the viable count of *Weizmannii* coagulating in the bacterial agent can be 10. 2 -10 10CFU / mL. 10 2 -10 10 CFU / mL refers to a concentration of 10 viable *Weizmannii* coagulans in the bacterial agent. 2 -10 10 CFU / mL level, for example, 1×10 2 CFU / mL, 5×10 2 CFU / mL, 8×10 2 CFU / mL, 9.9×10 2 CFU / mL were all within 10 2 CFU / mL level. That is, in this invention, the effective viable count of *Weizmannii* coagulating in the bacterial agent is greater than or equal to 1 × 10⁻⁶. 2 CFU / mL to less than 1×10 11 CFU / mL.

[0024] For example, the viable count of *Weizmannii* coagulating in the bacterial agent can be 1 × 10⁻⁶. 2 CFU / mL, 5×10 2 CFU / mL, 9×10 2 CFU / mL, 1×10 3 CFU / mL, 5×10 3 CFU / mL, 9×10 3 CFU / mL, 1×10 4 CFU / mL, 5×10 4 CFU / mL, 9×10 4 CFU / mL, 1×10 5 CFU / mL, 5×10 5 CFU / mL, 9×10 5 CFU / mL, 1×10 6 CFU / mL, 5×10 6 CFU / mL, 9×10 6 CFU / mL, 1×10 7 CFU / mL, 2×10 7 CFU / mL, 3×10 7 CFU / mL, 4×10 7 CFU / mL, 5×10 7 CFU / mL, 6×10 7 CFU / mL, 7×10 7 CFU / mL, 8×10 7 CFU / mL, 9×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 3×108 CFU / mL, 4×10 8 CFU / mL, 5×10 8 CFU / mL, 6×10 8 CFU / mL, 7×10 8 CFU / mL, 8×10 8 CFU / mL, 9×10 8 CFU / mL, 1×10 9 CFU / mL, 2×10 9 CFU / mL, 3×10 9 CFU / mL, 4×10 9 CFU / mL, 5×10 9 CFU / mL, 6×10 9 CFU / mL, 7×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL, 1×10 10 CFU / mL, 5×10 10 CFU / mL, 9×10 10 CFU / mL, 9.9×10 10 CFU / mL, or any value between the above values ​​or a range consisting of any two values.

[0025] According to some preferred embodiments of the present invention, the method for preparing the microbial agent or metabolite (or preparation containing the metabolite) may include at least one of the following: (1) Bacterial suspension: CCNH166 was inoculated into MRS liquid medium and cultured at 40-44℃ until the logarithmic or stationary growth phase. The viable cell concentration of the fermentation broth was adjusted to 10. 7 -10 9 CFU / mL is sufficient; Alternatively, the fermentation broth can be filtered or centrifuged to collect the cells, and then resuspended in phosphate buffer and / or sterile physiological saline to a viable cell concentration of 10⁻⁶. 7 -10 9 CFU / mL; (2) Supernatant: CCNH166 is inoculated into MRS liquid medium and cultured at 40-44℃ to the logarithmic or stable growth phase. The cells are removed by filtration or centrifugation, and the remaining liquid is the supernatant.

[0026] Preferably, the preparation method (2) above may further include sterilizing the collected supernatant. Sterilization is preferably performed by filtration (e.g., using a 0.22 μm filter membrane).

[0027] A third aspect of the present invention provides a method for fermenting tea, the method comprising: inoculating tea leaves and / or tea extract with the *Weizmannii coagulans* strain described in the first aspect or the microbial agent described in the second aspect for fermentation.

[0028] In this invention, the tea can be any common variety used in the art, including unfermented teas such as white tea, yellow tea, and green tea, as well as fermented teas (including semi-fermented teas) such as black tea and dark tea (e.g., Liubao tea). Fermented teas can undergo secondary fermentation to optimize flavor profiles, increase the content of active ingredients, or improve quality characteristics. Examples include oolong tea (a semi-fermented tea that can be further fermented to enhance its mellowness) and dark tea (which can be further fermented to enhance its aged aroma). It should be noted that green tea is an unfermented tea and is usually not fermented. The aforementioned examples of green tea do not contradict the statement that "fermented tea can be further fermented," and are merely examples of common tea varieties to which this invention applies.

[0029] In this invention, the fermentation can be carried out using methods commonly found in the art, such as solid-state fermentation (e.g., fermentation using tea leaves as a solid substrate) or liquid-state fermentation (e.g., fermentation of tea extract or tea powder into a liquid substrate).

[0030] In this invention, the tea extract can be prepared using conventional methods in the art. According to some specific embodiments of the invention, the preparation method of the tea extract may include: steeping tea leaves in a liquid (e.g., water) at a temperature higher than 40°C (preferably higher than 60°C, more preferably higher than 75°C). The steeping time can be 0.5 min to 60 min, for example, 0.5 min, 1 min, 5 min, 10 min, 12 min, 15 min, 20 min, 30 min, 60 min, or any value between these values ​​or any range of two of these values. The steeping time can be adjusted according to the type of tea and the requirements for the extract. According to some specific embodiments of the invention, the steeping time for fermented tea leaves (slightly fermented tea leaves, semi-fermented tea leaves, fully fermented tea leaves, post-fermented tea leaves; such as oolong tea, black tea, or dark tea) can be 10-60 min. According to other specific embodiments of the invention, the steeping time for non-fermented tea leaves (such as green tea) can be 0.5-30 min.

[0031] According to some specific embodiments of the present invention, the method for preparing tea extract may further include: a mass ratio of tea leaves to liquid of 1:(10-1000), for example, 1:10, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:1000, preferably 1:(10-200), and more preferably 1:(70-110). The mass ratio of tea leaves to liquid can be adjusted according to the type of tea and the requirements for the extract. According to some specific embodiments of the present invention, the volume ratio of oolong tea to liquid is 1:70-90. According to other specific embodiments of the present invention, the volume ratio of black tea or dark tea to liquid is 1:90-110. According to other specific embodiments of the present invention, the volume ratio of green tea to liquid is 1:30-50.

[0032] According to some preferred embodiments of the present invention, the method for preparing tea extract may further include: performing solid-liquid separation on the solid-liquid mixture obtained after soaking to obtain a liquid that does not contain solid matter (tea leaves).

[0033] It is understood that the CCNH166 provided by the present invention can be directly inoculated onto tea leaves, or onto tea extracts that do not contain tea leaves, or onto a mixture of tea leaves and tea extracts; all of the above inoculation methods can achieve tea fermentation, and can be reasonably selected according to actual fermentation conditions and product requirements, and all fall within the protection scope of the present invention.

[0034] According to some preferred embodiments of the present invention, a method for fermenting tea (liquid fermentation method) includes: Inoculate CCNH166 into tea extract (or a mixture of tea leaves and tea extract) and ferment at 30-45℃ for 10-100 hours.

[0035] According to some preferred embodiments of the present invention, the amount of CCNH166 inoculated into the tea extract (or a mixture of tea and tea extract) is such that the viable CCNH166 count in the inoculated liquid is 10-1. 1 -10 8 CFU / mL, for example, can be 1×10⁻⁶. 1 CFU / mL, 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 5×10 4 CFU / mL, 9×10 4 CFU / mL, 1×10 5 CFU / mL, 2×10 5 CFU / mL, 5×10 5CFU / mL, 9×10 5 CFU / mL, 1×10 6 CFU / mL, 3×10 6 CFU / mL, 5×10 6 CFU / mL, 9×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL, or any value between the above values ​​or a range of any two values, preferably 1×10⁻⁶. 5 -10 7 CFU / mL.

[0036] According to some preferred embodiments of the present invention, the fermentation temperature after inoculating CCNH166 into tea extract (or a mixture of tea and tea extract) can be 30°C, 31°C, 32°C, 33°C, 35°C, 36°C, 37°C, 38°C, 39°C, 41°C, 42°C, 43°C, 45°C, or any value between the above values ​​or any two of the above values, preferably 31-43°C, more preferably 35-37°C.

[0037] According to some preferred embodiments of the present invention, the fermentation time after inoculating the tea extract (or a mixture of tea and tea extract) with CCNH166 can be 10h, 20h, 25h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 40h, 45h, 46h, 47h, 48h, 49h, 50h, 60h, 80h, 100h, or any value between the above values ​​or any two of the above values, preferably 30-60h, more preferably 45-55h.

[0038] According to some preferred embodiments of the present invention, the method for fermenting tea (e.g., Liubao tea) (solid-state fermentation) may further include: I. Conventional Fermentation (Pre-fermentation): The raw materials are evenly mixed according to the requirements, piled for fermentation, sieved, subjected to the first high-temperature instantaneous steam treatment (single steaming), and cooled. The inventors of this invention optimized the fermentation process of tea by adding a post-fermentation step, taking the fermentation of Liubao tea as an example. The optimized fermentation process resulted in a fermented tea product with a higher viable bacteria count, a uniform reddish-brown liquid color, a typical aroma, and a slightly acidic flavor imparted by the strain, creating a harmonious and pleasant taste with good palatability and a distinct fermented flavor. The post-fermentation process is as follows: II. Post-fermentation process: (1) Probiotic fortification: Weigh out the freeze-dried powder of Weizmann's coagulation, mix it with water at a ratio of 1:10-100, activate it at 25-35℃ for 10-40 minutes, and then evenly sprinkle it into the tea leaves and stir it evenly (the total amount of freeze-dried powder of Weizmann's coagulation added is 0.05-1%). (2) Secondary steaming: The probiotic-enhanced Liubao tea undergoes a second high-temperature instantaneous steam treatment at a temperature of 80-90℃ for 10-60 seconds. (3) Pressing: The tea leaves are softened by the above-mentioned high-temperature instantaneous steam treatment and then pressed into shape to form a tightness that is conducive to the colonization of probiotics. For example, the tea leaves can be pressed with a pressure of 1.0-2.0 MPa. (4) Secondary fermentation culture: After pressing and shaping, the tea leaves are cultured at 25-40℃ and 60-90% humidity for 20-60 hours. (5) Low temperature drying: The drying temperature is ≤50℃ and the air humidity is ≤30%, and the tea leaves are dried to a moisture content of ≤7%.

[0039] The fourth aspect of the present invention provides a fermented tea product, wherein the fermented tea is prepared by the method described in the third aspect.

[0040] The fifth aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the *Weizmannii coagulans* as described in the first aspect, or the bacterial agent as described in the second aspect, or the fermented tea product as described in the fourth aspect.

[0041] According to some preferred embodiments of the present invention, the pharmaceutical composition may further contain pharmaceutically acceptable excipients. Any excipient commonly used in pharmaceutical preparation in the art is suitable for the present invention, such as excipients, preservatives, stabilizers, buffers, etc.

[0042] According to some specific embodiments of the present invention, the pharmaceutical composition may be provided in any dosage form, including liquid, semi-solid, and solid.

[0043] According to some specific embodiments of the present invention, the formulation may be a liquid formulation or a solid formulation; specifically, the supernatant / lysis supernatant may be concentrated, lyophilized or spray-dried to form a dry powder; the bacterial suspension may be lyophilized in the presence of a protectant to form a live bacterial powder; the dry powder may be further prepared into solid formulations such as granules, pills, capsules, tablets, or formulated with a pharmaceutically acceptable carrier into ointments, gels, sprays or liquid formulations.

[0044] The inventors discovered in their research that, compared to conventional Weizmannia coagulans (such as Weizmannia coagulans with the designation CICC 21736), CCNH166 can increase the γ-aminobutyric acid (GABA) content by 20 times or more. Based on this, a sixth aspect of the present invention provides a method for increasing the GABA content in fermented tea products, the method comprising: inoculating the Weizmannia coagulans described in the first aspect into tea leaves and / or tea extract for fermentation.

[0045] According to some preferred embodiments of the present invention, the method for increasing the γ-aminobutyric acid (GABA) content in fermented tea products further includes adding monosodium glutamate (MSG) to tea leaves and / or tea extract. The amount of MSG added can be adjusted according to actual production needs. According to some preferred embodiments of the present invention, the amount of MSG added is 0.1-1 g / 100 mL of extract.

[0046] According to some preferred embodiments of the present invention, the conditions for the fermentation of γ-aminobutyric acid include: a temperature of 30-45°C, for example, 30°C, 31°C, 32°C, 33°C, 35°C, 37°C, 39°C, 41°C, 42°C, 43°C, 45°C, or any value between the above values ​​or a range consisting of any two of the above values, preferably 31-43°C, more preferably 35-37°C.

[0047] The seventh aspect of the present invention provides the use of the *Weizmannii coagulates* strain described in the first aspect in enhancing at least one of γ-aminobutyric acid, tea polyphenols, and tea catechins in fermented tea products.

[0048] The present invention further provides a method for increasing the content of tea polyphenols and / or tea catechins in fermented tea products, the method comprising: inoculating the *Weizmannii coagulans* described in the first aspect into tea leaves and / or tea extract for fermentation.

[0049] The present invention further provides a method for reducing the free radical content in fermented tea products, the method comprising: inoculating the *Weizmannii coagulans* strain described in the first aspect into tea leaves and / or tea extract for fermentation.

[0050] The methods for increasing the content of tea polyphenols and / or tea catechins in fermented tea products and for reducing the content of free radicals in fermented tea products provided by this invention are the same as the fermented tea methods provided in the third aspect, and will not be repeated here.

[0051] According to some preferred embodiments of the present invention, the free radical includes DPPH free radical.

[0052] The eighth aspect of the present invention provides the use of the *Weizmannii coagulans* described in the first aspect in enhancing the acidity and / or flavor of fermented dairy products.

[0053] According to some preferred embodiments of the present invention, the raw material for the fermented dairy product is whey protein.

[0054] According to some preferred embodiments of the present invention, the method for preparing the fermented dairy product may include: inoculating 150 mL of whey protein water with a culture of *Weizmannii coagulates* and fermenting it at 30-45°C for 2-20 h.

[0055] According to some preferred embodiments of the present invention, after inoculating the *Weizmannii coagulate* bacterial solution into 150 mL of whey protein water, fermentation is preferably carried out at 38-45°C for 2-10 h, more preferably at 41-43°C for 3-7 h.

[0056] According to some preferred embodiments of the present invention, whey protein can be provided in liquid form during fermentation of dairy products. In the liquid, the concentration of whey protein can be 0.5-20 wt%, for example, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 10 wt%, 20 wt%, or any value between these values ​​or a range consisting of any two of the above values, preferably 1-5 wt%, more preferably 1-2 wt%.

[0057] According to some preferred embodiments of the present invention, whey protein can be pasteurized at 90-98°C before fermenting dairy products.

[0058] In further research, the inventors also discovered that CCNH166 also has the effects of lowering uric acid, degrading ethanol, inhibiting pathogens, degrading cholesterol, inhibiting angiotensin-converting enzyme activity, and inhibiting α-glucosidase activity. Based on this, the ninth aspect of the present invention provides the use of the *Weizmannii coagulans* described in the first aspect in the preparation of a medicament for lowering uric acid, degrading ethanol, inhibiting pathogens, lowering blood pressure, and lowering blood sugar.

[0059] According to some preferred embodiments of the present invention, the pathogenic bacteria include Pseudomonas aeruginosa (… Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa At least one of the following, Staphylococcus aureus ( Staphylococcus aureus ) and Salmonella ( Salmonella At least one of the following.

[0060] The *Weizmannii coagulans* (including its cells and / or metabolites, preferably the fermentation supernatant of the strain) provided by this invention has the ability to inhibit a variety of pathogens, including *Salmonella typhimurium*, *Salmonella enteritidis*, *Escherichia coli*, *Staphylococcus aureus*, and *Streptococcus mutans*. Therefore, reagents containing *Weizmannii coagulans* provided by this invention (such as bacterial agents, fermented tea, or pharmaceutical compositions) have the potential to inhibit pathogens in vitro.

[0061] The fermented tea product containing *Weizmannii coagulans* provided by this invention can inhibit *Pseudomonas aeruginosa*, *Escherichia coli*, and *Staphylococcus aureus*. Therefore, fermented tea products containing *Weizmannii coagulans* provided by this invention have the potential to inhibit pathogens in vitro.

[0062] According to some preferred embodiments of the present invention, the Salmonella preferably includes Salmonella Typhimurium (Salmonella Typhimurium). Salmonella typhimurium ) and Salmonella enteritidis ( Salmonella enteritidis ).

[0063] According to some preferred embodiments of the present invention, the Escherichia coli is numbered ATCC 25922 and / or CICC10421.

[0064] According to some preferred embodiments of the present invention, the Staphylococcus aureus is designated as CMCC(B)26001 and / or CMCC(B)26003.

[0065] According to some preferred embodiments of the present invention, the Salmonella Typhimurium is designated ATCC 14028.

[0066] According to some preferred embodiments of the present invention, the Salmonella enteritidis is designated CVCC 3378.

[0067] The present invention further provides a method for reducing uric acid content in vitro, the method comprising: fermenting the *Weizmannii coagulans* described in the first aspect in vitro with uric acid and / or uric acid precursors.

[0068] According to some preferred embodiments of the present invention, the uric acid precursor includes at least one of inosine, guanosine, hypoxanthine, and xanthine.

[0069] The present invention further provides a method for in vitro degradation of ethanol, the method comprising: contacting the *Weizmannii coagulans* described in the first aspect with ethanol in vitro for fermentation.

[0070] The present invention further provides a method for in vitro degradation of cholesterol, the method comprising: fermenting the *Weizmannii coagulans* described in the first aspect in vitro with cholesterol.

[0071] The present invention further provides a method for inhibiting angiotensin-converting enzyme activity, the method comprising: fermenting the *Weizmannii coagulans* strain described in the first aspect in vitro with angiotensin-converting enzyme.

[0072] The present invention further provides a method for inhibiting α-glucosidase activity, the method comprising: fermenting the *Weizmannii coagulans* described in the first aspect in vitro with α-glucosidase.

[0073] The present invention will be described in detail below through examples. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0074] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 0.5 g / L, agar 15 g / L, diluted with pure water to 1 L, sterilized at 121 °C for 15 min; In-situ culture medium for tea: 10 g / L Liubao tea and 15 g / L agar during the pile fermentation period. After filtering the tea, pure water was brought to a final volume of 1 L and sterilized at 115 ℃ for 10 min.

[0075] Escherichia coli ( Escherichia coli ATCC25922 was purchased from the American Center for Type Culture Collection; Escherichia coli CICC10421 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa CICC10419 was purchased from the China Industrial Microbial Culture Collection Center. Staphylococcus aureus ( Staphylococcus aureus CMCC(B)26001 and CMCC(B)26003 were both purchased from the China Medical Bacteriological Preservation and Management Center.

[0076] salmonella( Salmonella Salmonella typhimurium (Salmonella typhimurium) Salmonella typhimurium ATCC14028, purchased from the American Center for Type Culture Collection; Salmonella enteritidis (ATCC14028, purchased from the American Center for Type Culture Collection); Salmonella enteritidis (ATCC14028, purchased from the American Center for Type Culture Collection). Salmonella enteritidis CVCC3378, purchased from the China Veterinary Microbial Culture Collection Center; Streptococcus mutans ( Streptococcus mutans CGMCC 1.2499, purchased from China General Microbiological Culture Collection Center; Reference strain: *Weizmannii coagulans* CICC 21736, purchased from the China Industrial Microbial Culture Collection Center; Fermentation medium: 20 g / L glucose, 15 g / L yeast extract, 15 g / L peptone, 4 g / L magnesium sulfate, 0.3 g / L dipotassium hydrogen phosphate.

[0077] Example 1 The inventors of this invention isolated a gastric acid-resistant bacterial strain, CCNH166, from the gut microbiota of healthy volunteers. The morphology of its bacterial cells and spores under a microscope is as follows: Figure 1 As shown, the 16S rDNA sequence is as shown in SEQ ID NO: 1:

[0078] Identified as a coagulating bacterium (Wietzmannii) Weizmannia coagulans It has more than 99% homology.

[0079] Example 2 This embodiment is used to illustrate the fermentation effect of CCNH166 liquid tea provided by the present invention.

[0080] The bacterial strain was inoculated onto the fermentation medium and fermented at 45°C for 48 hours to obtain a bacterial culture. The bacterial culture was centrifuged to obtain a bacterial sludge. The bacterial sludge was resuspended in physiological saline (0.9% (w / v) NaCl aqueous solution, sterile) to obtain a viable count of 10. 8 CFU / g bacterial suspension.

[0081] Prepare liquid fermented tea according to the following method: Methods for preparing extracts and conditions for fermenting the extracts: Oolong tea: water mass ratio 1:80, 80℃ for 12 minutes, filter to obtain extract; during fermentation, add 8wt% fructose syrup, inoculate with 1% volume of bacterial suspension, fermentation temperature 37℃, fermentation time 36 hours. Black tea or dark tea: water mass ratio 1:100, 80℃, 12 minutes of soaking time, filter to obtain the extract; during fermentation, the amount of fructose syrup added is 8wt%, the volume of bacterial suspension inoculated is 3‰, the fermentation temperature is 37℃, and the fermentation time is 48 hours. Green tea: water mass ratio 1:40, 100℃ for 1 minute, filter to obtain extract; during fermentation, add 8wt% fructose syrup, 4% of bacterial suspension inoculum volume, fermentation temperature 37℃, fermentation time 48 hours.

[0082] Add CCNH166 or CICC 21736 bacterial suspension to the extract and ferment according to their respective conditions to obtain fermentation broth.

[0083] Dispense 9 mL of physiological saline into test tubes. Separately, place 45 mL of physiological saline into a 100 mL Erlenmeyer flask and sterilize at 121°C for 20 min. After sterilization, remove and cool to room temperature. Add 5 g of fermentation broth (from oolong tea, black tea, green tea, or dark tea) to 45 mL of sterile physiological saline and mix well to obtain a suspension. Pipette 1 mL of this suspension into a test tube to form 10... -1 Diluent. Take 10 μL. -1 Add 1 mL of diluent to the next test tube to form 10 -2 Diluent. Continue diluting to 10... -5 Or 10 -6100 μL of each serial dilution was plated, allowed to air dry at room temperature, and then incubated at 45°C for 48 h. The viable count was determined using the GB4789.35-2023 method.

[0084] After fermentation in oolong tea, black tea, green tea, and dark tea, the viable count of CICC 21736 was 1.2 × 10⁻⁶, respectively. 4 2×10 6 1.5×10 5 and 1.0×10 6 The CFU / mL and the viable count of CCNH166 were 2.1 × 10⁻⁶. 7 2×10 7 1.2×10 6 and 3.0×10 8 CFU / mL.

[0085] Liquid fermented black tea was prepared using the same method to test its ability to produce γ-aminobutyric acid (GABA), except that monosodium glutamate (MSG) was added to the tea extract before fermentation at a rate of 0.5 g / 100 mL extract.

[0086] High-performance liquid chromatography (HPLC) was used to determine the γ-aminobutyric acid (GABA) content in the fermentation broth of black tea extract. Before injection, the sample needed to be derivatized. The specific procedure was as follows: 100 μL of the sample was thoroughly mixed with 100 μL of phthalaldehyde solution (10 mg phthalaldehyde dissolved in 2.5 mL acetonitrile, followed by the addition of 20 μL of β-mercaptoethanol) and 500 μL of 0.4 M borate buffer (adjusted to pH 9.5 with sodium hydroxide). After standing for 2 min, the mixture was filtered through a 0.22 μm filter before HPLC detection. HPLC conditions: Column: Hypersil ODS C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile: 0.02 M sodium acetate = 23:77; Flow rate: 0.9 mL / min; Column temperature: 30 °C; Injection volume: 20 μL; Detection wavelength: 333 nm.

[0087] The γ-aminobutyric acid (GABA) content in the fermented liquid of CCNH166 dark tea is 0.911 g / L, while the γ-aminobutyric acid (GABA) content in the fermented liquid of CICC 21736 dark tea is only 0.045 g / L.

[0088] Example 3 This embodiment is used to illustrate the solid-state tea fermentation effect of CCNH166 provided by the present invention.

[0089] The freeze-dried powder preparation process is as follows: Original microbial strains are taken from a -80℃ microbial culture library, activated, and then inoculated into a sterile fermentation medium for large-scale amplification. After fermentation, the microbial sludge is obtained by centrifugation and then transferred to a freeze dryer for pre-freezing to form uniform ice crystals. Subsequently, sublimation drying and desorption drying are carried out sequentially under vacuum to completely remove moisture and retain the maximum viability of the microorganisms. The final product is a powder with a viable count of 1×10⁻⁶. 9 CFU / g.

[0090] Raw tea materials of different origins, grades, seasons or quality characteristics are mixed evenly, fermented in piles, sieved, subjected to a first high-temperature instantaneous steam treatment (single steaming), and cooled to obtain Liubao tea. For details, please refer to GB / T 32719.4-2016 "Dark Tea Part 4: Liubao Tea".

[0091] After obtaining Liubao tea, the following fermentation process is carried out: (1) Probiotic fortification: Weigh CCNH166 freeze-dried powder and add it to purified water at a ratio of 1 (freeze-dried powder, g): 30 (volume ratio, mL). After activating at 30℃ for 30 min, evenly sprinkle it into Liubao tea leaves and stir evenly. The total amount of freeze-dried powder added is 0.1 wt%. Let it stand at 30℃ for 1 h to obtain fortified Liubao tea for later use.

[0092] (2) Secondary steaming: The Liubao tea is subjected to a second high-temperature instantaneous steam treatment at a temperature of 85°C for 30 seconds.

[0093] (3) Pressing: After the tea leaves are steamed twice, they are softened and then pressed at 1.4 MPa for 30 seconds to set their shape; (4) Secondary fermentation culture: The pressed and shaped tea leaves are cultured in a culture room at 37℃ and 85% humidity for 48 hours; (5) Low temperature drying: After secondary fermentation, the tea leaves are dried at a temperature of ≤50℃ and an air humidity of ≤30% until the moisture content of the pressed tea leaves is ≤7%, thus obtaining fermented Liubao tea raw tea.

[0094] In addition, CICC21736 fermented Liubao tea (fermented tea) was prepared using the same method.

[0095] The viable count was tested according to the method in Example 2, except that 5g of fermentation liquid was replaced with 5g of fermented tea.

[0096] The results showed that after fermentation, the viable cell count of CICC21736 was 1×10⁻⁶. 6 CFU / g, CCNH166 viable count was 1.2 × 10⁻⁶. 8 CFU / g, or CCNH166, exhibits stronger adaptability and stability during the fermentation process of Liubao tea.

[0097] In addition, sensory evaluation of the suspension showed that the CCNH166 fermented tea liquid was reddish-brown with a uniform color, had the typical aroma of Liubao tea, and also had a slight acidity from probiotics, exhibiting a distinct fermented flavor that was harmonious, pleasant, mellow, and palatable.

[0098] Example 4 This embodiment is used to illustrate the content of tea polyphenols and catechins, antibacterial properties, and antioxidant properties of the solid-state fermented tea water produced by CCNH166 fermentation provided by the present invention.

[0099] Take 3g of the fermented tea obtained in Example 3 and steep it in 150mL of boiling water for 10 minutes. Filter the tea leaves and the resulting liquid is tea water.

[0100] (a) Tea polyphenol content Ferrous tartrate solution: 1g ferrous sulfate, 5g potassium sodium tartrate, dissolved in water to a volume of 1L.

[0101] Phosphate buffer: 85 mL of disodium hydrogen phosphate aqueous solution (disodium hydrogen phosphate concentration 23.877 g / L). Mix 15 mL of potassium dihydrogen phosphate aqueous solution (9.078 g / L) to obtain phosphate buffer (pH 7.5).

[0102] Accurately pipette 1 mL of the tea obtained in Example 3 into a 25 mL volumetric flask, add 4 mL of water and 5 mL of ferrous tartrate solution, mix thoroughly, and then dilute to the mark with phosphate buffer to obtain the test liquid; use water instead of tea as the reference solution. Measure the absorbance A of the test liquid at a wavelength of 540 nm, and calculate the tea polyphenol content according to the following formula.

[0103] Tea polyphenol content (mg / mL) = A × 1.957 × 2; The polyphenol content of fermented Liubao tea infusion using CCNH166 was 1.49 mg / mL. The polyphenol content of fermented Liubao tea infusion using CICC21736 was 0.88 mg / mL, indicating that CCNH166 provided by this invention can significantly increase the polyphenol content in fermented tea.

[0104] (ii) Content of tea catechins The tea water obtained in Example 3 was diluted 25 times and filtered through a 0.22 μm bacterial filter to obtain the test solution, and the content of tea catechins in it was tested according to the following high performance liquid chromatography method.

[0105] Preparation of mobile phases for liquid chromatography: Mobile phase A: Add 90 mL of acetonitrile, 20 mL of acetic acid, and 2 mL of EDTA to a 1000 mL volumetric flask, dilute to the mark with water, and mix well; Mobile phase B: Add 800 mL of acetonitrile, 20 mL of acetic acid, and 2 mL of EDTA to a 1000 mL volumetric flask, dilute to the mark with water, and mix well.

[0106] Chromatographic conditions: Mobile phase flow rate: 1 mL / min; Column temperature: 35℃; UV detector: Detection wavelength: 278 nm; Gradient conditions: 100% phase A held for 10 min, then switched from 100% phase A to 68% phase A and 32% phase B within 15 min, then held for 10 min with 68% phase A and 32% phase B, and finally eluted with 100% phase A.

[0107] After the flow rate and column temperature stabilize, perform a blank run. Accurately pipette 10 μL of the mixed standard series of tea catechin working solution and inject it into the high-performance liquid chromatograph to establish a standard curve for tea catechin content. Under the same chromatographic conditions, inject 10 μL of the test solution and substitute the peak area of ​​the test solution into the standard curve to obtain the tea catechin content.

[0108] The catechin content of fermented Liubao tea by CCNH166 was 1.06 mg / mL, and the catechin content of fermented Liubao tea by CICC 21736 was 0.45 mg / mL, indicating that the *Weizmannii coagulans* provided by this invention can increase the catechin content in fermented tea.

[0109] (III) Antibacterial effect Staphylococcus aureus CMCC(B)26001, Escherichia coli ATCC25922, and Pseudomonas aeruginosa CICC10419 were streaked on LB medium, and single colonies were picked and cultured in LB liquid medium at 37°C and 180 rpm to obtain indicator bacterial seed culture.

[0110] 2% agar was poured into a Petri dish to form a 3mm base. After solidification, sterile Oxford cups were placed evenly spaced into the dish. Indicator bacteria were added at 0.5% (v / v) to LB solid medium containing 0.75% agar at 50℃, shaken well, and poured into the Petri dish containing the Oxford cups, to a thickness of half the cups. After solidification, the Oxford cups were removed, and 200μL of tea was added to each well. The tea was used to replace the sterile water as a control group. The diameter of the clear zone (in mm) was measured to evaluate the antibacterial effect. The inhibition zone results of CCNH166 and CICC 21736 against different bacteria are shown in Table 1. The inhibition zone of the control group was 0mm.

[0111] Table 1

[0112] The inhibition zone test results in Table 1 show that the fermented Liubao tea made with *Weizmannii coagulates* CCNH166 has an inhibitory effect on pathogenic bacteria such as *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa* after brewing, and the inhibition zone is larger than that of CICC 21736, indicating that fermented tea made with CCNH166 has the potential to inhibit harmful microorganisms and regulate intestinal flora.

[0113] (iv) Antioxidant effect Prepare a DPPH solution with a concentration of 0.1 mg / mL using anhydrous ethanol; prepare a DPPH solution with a concentration of 12.5 mg / mL using 50% ethanol as a positive control. Store refrigerated and protected from light.

[0114] 100 μL each of tea and positive control VC glucoside were added to a 96-well plate, followed by 100 μL of DPPH solution to obtain the experimental group and positive control group. A blank group was also set up, in which 100 μL of anhydrous ethanol was replaced with tea. The sample background group consisted of 100 μL of tea sample + 100 μL of anhydrous ethanol to subtract the color interference from the tea itself. Similarly, the positive control background group consisted of 100 μL of VC glucoside solution + 100 μL of anhydrous ethanol.

[0115] After reacting in the dark for 30 minutes, the absorbance was measured at 517 nm using an ELISA reader. The absorbance of the reagent blank group was A0, the absorbance of the sample background group was A0', the absorbance of the experimental group was A1, and the absorbance of the positive control group was A1'. The scavenging ability against DPPH free radicals was calculated using the following formula.

[0116] DPPH clearance rate (%) = 1 - (A1 - A1') / (A0 - A0') × 100%.

[0117] The test results showed that the DPPH free radical scavenging rate of CCNH166 fermented Liubao tea was 90.33%, while that of CICC21736 fermented Liubao tea was 55.34%, indicating that the CCNH166 fermented tea provided by this invention has better antioxidant capacity.

[0118] Example 5 This embodiment is used to illustrate the acid-producing effect of CCNH166 provided by the present invention using whey protein as raw material.

[0119] Preparation of bacterial culture: The bacterial strain was inoculated into the fermentation medium and fermented at 45°C for 48 hours to obtain bacterial culture. The bacterial culture was centrifuged to obtain bacterial sludge. The bacterial sludge was resuspended in physiological saline (0.9% (w / v) NaCl aqueous solution, sterile) to obtain a viable count of 10. 8 CFU / g bacterial suspension.

[0120] Both *Wietzmannii* CCNH166 and CICC 21736 were activated and prepared to a concentration of 1×10⁻⁶. 8 For each CFU / g bacterial culture, 1 mL of the culture was inoculated into 150 mL of whey protein water (whey protein concentration 1.22 wt%, pasteurized at 95°C for 5 min and cooled to room temperature before use), shaken to mix, covered and left to ferment at 42°C for 4 h and 6 h respectively.

[0121] The acidity (in °T) and pH of the fermentation at different times were tested using GB 19302—2025, and the flavor was tested by sensory evaluation. The results are shown in Table 2.

[0122] Table 2

[0123] CCNH166 has a stronger acid-producing capacity than CICC 21736, indicating that CCNH166 provided by this invention has stronger fermentation performance and can impart a better flavor to whey protein water.

[0124] Example 6 This embodiment is used to illustrate the degradation capabilities of inosine, guanosine, hypoxanthine, and xanthine in CCNH166 provided by the present invention.

[0125] The bacterial cultures of CCNH166 and CICC 21736 prepared in Example 5 were centrifuged and resuspended in sterile physiological saline to obtain a viable count of 1×10⁻⁶. 9 A bacterial suspension of CFU / mL was washed twice with sterile physiological saline and resuspended in equal volumes. One mL of the suspension was centrifuged at 5000 rpm for 5 min to collect the bacterial sludge. 0.9 mL of each of the following were added: inosine (Solarbio, catalog number SI8430) and guanosine (Solarbio, catalog number SG9310), both at a concentration of 0.5 g / L; hypoxanthine (Solarbio, catalog number SH8900) and xanthine (Solarbio, catalog number X8030), both at a concentration of 50 mg / L. After mixing, the solutions were incubated at 37℃ and 180 rpm for 10 min, 30 min, and 1 h. The concentrations of inosine, guanosine, xanthine, and xanthine were determined by HPLC. A control group without the added bacterial strains was included. The HPLC conditions were as follows: mobile phase: methanol:water = 1:9, retention time 15 min, C18 column, 4.6 × 250 mm, 4.6 μm; column temperature 30℃, injection volume 20 μL, flow rate 0.7 mL / min, detection wavelength 254 nm, and UV detector. The degradation rates of inosine, guanosine, xanthine, and xanthine by CCNH166 and CICC 21736 at 1 h, and the degradation rates of inosine and guanosine at 10 min, 30 min, and 1 h were calculated using the following formula.

[0126] Degradation rate (%) = 1 - (C) t / C0)×100%; Degradation rate (mg / min) = (C0 - C) t ) / T; Wherein, C0 is the content of the control group substance (g), T is the incubation time, and Ct The substance content at time T.

[0127] The results are shown in Table 3. The degradation rate and degradation rate of CCNH166 under incubation conditions of 10 min and 30 min were significantly higher than those of the reference strain CICC 21736. p <0.01).

[0128] Table 3

[0129] Example 7 This embodiment is used to illustrate the uric acid degradation ability of CCNH166 provided by the present invention.

[0130] Initial screening plate: 4 g / L uric acid, 0.5 g / L NaCl, 0.5 g / L MgSO4, 2 g / L K2HPO4, 0.5 g / L KH2PO4, 1.5% agar, pH=6.

[0131] The strain was activated and inoculated into MRS medium for culture at 42°C for 24-48 hours. The cells were collected by centrifugation, washed three times with phosphate buffer (100 mM, pH 7.0), and the viable count was adjusted to 10⁻⁶ cells / mL using phosphate buffer. 8 A CFU / mL resuspended bacterial solution was taken, and 10 μL of the resuspended bacterial solution was spotted onto a primary screening plate and incubated at 42℃ for 2-3 days. The size of the clear zone produced by uric acid degradation was observed. The clear zone diameter of *Weizmannii coagulating* CCNH166 reached 10 mm, while the clear zone diameter of the reference strain *Weizmannii coagulating* CICC 21736 was 0 mm. It was preliminarily determined that *Weizmannii coagulating* CCNH166 has the ability to degrade uric acid. Bacterial cells were collected by centrifugation and then inoculated into sterile MRS medium containing 1.0 g / L uric acid. The OD was adjusted to 1, and the culture was carried out at 42℃ and 180 rpm for 72 h. The supernatant was then centrifuged, and the residual uric acid concentration was measured to determine its uric acid degradation ability. Uric acid concentration was detected using a uric acid detection kit from Wuhan Shengzhiyuan Biotechnology Co., Ltd. The uric acid degradation rate of CCNH166 reached 93.48%, while that of CICC 21736 was 75.23%, indicating that *Weizmannii coagulans* CCNH166 has a better uric acid-lowering ability.

[0132] Example 8 This embodiment is used to illustrate the ethanol degradation ability of CCNH166 provided by the present invention.

[0133] Add 0.1 mL of ethanol standard solutions with volume concentrations of 0%, 2%, 4%, 6%, 8%, and 10% to glass test tubes, respectively. Add 1 mL of potassium dichromate-sulfuric acid solution to each test tube and shake thoroughly. Place the test tubes in a boiling water bath for 10 minutes to allow the ethanol to fully evaporate and be oxidized by the potassium dichromate-sulfuric acid solution. Remove the test tubes and measure the absorbance of the potassium dichromate-sulfuric acid solution at a wavelength of 610 nm. Plot a standard curve of ethanol content (in volume%) with ethanol volume concentration (volume%) on the x-axis and absorbance on the y-axis (y = 0.1581x + 0.0058, R0). 2 =0.9946).

[0134] Take 5 mL of 20% ethanol culture medium and 200 µL of culture medium with a viable count of 10 6 CFU / mL bacterial culture was added to a test tube, mixed well, and incubated at 42℃ and 180 rpm for 48 h to obtain the test sample. An equal volume of MRS culture medium was used as the control sample. In a 25 mL volumetric flask, 2 mL of 4wt% potassium dichromate and 1 mL of 98% concentrated sulfuric acid were added, shaken well, and cooled to 25℃. Then, 1 mL of either the test sample or the control sample was added. After reacting for 10 min, the volume was adjusted to a final volume, and the OD of the control sample was measured. 600 As the initial ethanol OD 600 Measure the OD of the sample to be tested 600 OD of ethanol after the reaction 600 According to the standard curve equation, the initial ethanol OD was obtained by... 600 and the OD of ethanol after the reaction 600 Calculate the initial ethanol content and the ethanol content after the reaction, and calculate the ethanol degradation rate according to the following formula.

[0135] Ethanol degradation rate (%) = (Initial ethanol content - Post-reaction ethanol content) / Initial ethanol content × 100% CCNH166 showed an ethanol degradation rate of 23.44%, while CICC21736 showed a degradation rate of 10.3%. *Weizmannii coagulans* CCNH166 exhibits good ethanol-reducing ability and has potential alcohol-degrading capabilities.

[0136] Example 9 This embodiment is used to illustrate the antibacterial ability of the CCNH166 supernatant provided by the present invention.

[0137] Indicator strains: Salmonella Typhimurium ATCC14028, Escherichia coli CICC10421, Staphylococcus aureus CMCC26001, Streptococcus mutans CGMCC 1.2499.

[0138] The indicator strains were inoculated into LB liquid medium and activated overnight at 37°C. They were then transferred to fresh LB liquid medium and cultured at 37°C until OD500.600 =1, diluted to 10 with fresh LB liquid medium. 5 CFU / mL was used as the indicator bacterial solution.

[0139] Glycerol tubes of CCNH166 and CICC21736 were inoculated into fresh, sterile LB liquid medium and cultured overnight at 42 °C for 18 h. Then, 2% (v / v) of the inoculum was added to fresh LB liquid medium, and the mixture was incubated statically at 42 °C for 18 h. The supernatant was collected by centrifugation and sterilized by membrane filtration. LB liquid medium was used as a negative control. Three experimental groups were set up: S1: 100 μL supernatant, 100 μL indicator bacterial solution; S2: 50 μL supernatant, 150 μL indicator bacterial solution; S3: 25 μL supernatant, 175 μL indicator bacterial solution. All were incubated at 42 °C for 18 h, and OD was measured after incubation. 600 Additionally, a negative control group was set up by replacing the supernatant with an equal volume of MRS liquid culture medium, and the inhibition rate was calculated according to the following formula.

[0140] Antibacterial rate (%) = ×100% Where A0 is the OD of the negative control sample after culture. 600 - OD before cultivation 600 A represents the OD of the experimental group after culture. 600 -Pre-culture OD 600 value.

[0141] Table 4

[0142] Example 10 This embodiment is used to verify the ability of CCNH166 to lower blood pressure by inhibiting angiotensin-converting enzyme activity.

[0143] The activated bacterial cultures of CCNH166 and CICC21736 were inoculated into MRS liquid medium at a volume percentage of 1% and incubated at 37°C for 18 hours. The resulting culture was centrifuged at 8000 rpm for 10 minutes and filtered through a 0.22 μm filter membrane to obtain sterile supernatant as the sample to be tested.

[0144] Assay for angiotensin-converting enzyme (ACE) inhibitory activity: 40 μL of the test sample, 50 μL of FAPPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine) substrate, and 10 μL of angiotensin-converting enzyme were added sequentially to a 96-well plate to obtain the experimental group. A control group was prepared by replacing the test sample with (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (HEPES) buffer at pH 8.3. After mixing, the absorbance A1 and B1 of the experimental and control groups at 340 nm were immediately measured. The reaction was then carried out at 37℃ for 30 min, and the absorbance A2 and B2 of the experimental and control groups at 340 nm were measured. The ACE inhibition rate was calculated as follows: ACE inhibition rate = [1 - (A1 - A2) / (B1 - B2)] × 100%.

[0145] The results showed that CICC21736 inhibited angiotensin-converting enzyme (ACE) activity by approximately 30.89 ± 3.54%, while strain CCNH166 inhibited ACE activity by approximately 86.64 ± 4.91%. This indicates that CCNH166 provided by this invention can significantly inhibit ACE activity, continuously converting angiotensin I to angiotensin II, thereby lowering blood pressure.

[0146] Example 11 This embodiment is used to verify the ability of CCNH166 to exert a hypoglycemic effect by inhibiting α-glucosidase activity.

[0147] Prepare the following solutions: A 1 mg / mL acarbose positive control solution was prepared using distilled water. 49 mL of 0.2 mol / L disodium hydrogen phosphate was mixed with 51 mL of 0.2 mol / L sodium dihydrogen phosphate and brought to a final volume of 200 mL with sterile ultrapure water. The pH was adjusted to 6.8 and the solution was sterilized at 121 °C to obtain 0.1 mol / L PBS buffer. A 20 mM 4-nitrophenyl-β-D-galactopyranoside (PNPG) substrate solution was prepared using the PBS buffer (aliported and stored at -20 °C protected from light). The lyophilized α-glucosidase powder was dissolved in PBS to prepare a 0.2 U / mL enzyme working solution. 0.1 mol / L sodium carbonate stop solution.

[0148] 25 μL of PBS buffer, 25 μL of PNPG solution, and 25 μL of the test sample prepared in Example 10 were added sequentially to the sample wells of a 96-well plate to obtain a sample group. After incubation at 37°C for 10 minutes, 50 μL of enzyme working solution was added, and the reaction was continued at 37°C for 15 minutes. Finally, 100 μL of sodium carbonate solution was added to terminate the reaction. A blank well, a blank control well, a sample control well, and a positive control well in which the test sample was replaced with a positive control solution were set up. Immediately after the reaction was terminated, the absorbance values ​​of the sample group, blank well, blank control well, and sample control well were measured at a wavelength of 405 nm, and the α-glucosidase inhibition rate was calculated according to the following formula.

[0149] Sample group: Add 25 μL PBS, 25 μL PNPG solution and 25 μL sample to be tested to a 96-well plate, incubate at 37℃ for 10 min, add 50 μL enzyme working solution and react for 15 min, add 100 μL sodium carbonate to stop the reaction, and measure the absorbance at 405 nm and record it as sample A.

[0150] Blank well (corresponding to blank A): Replace the sample with 25 μL PBS, and perform the other operations as with the sample group. Measure the absorbance at 405 nm and record it as blank A. This is used to eliminate interference from the absorbance of the system itself.

[0151] Blank control wells (corresponding to blank control A): Replace the test sample with 25 μL PBS and the enzyme working solution with 50 μL PBS. Perform the same operation as the sample group. Measure the absorbance at 405 nm and record it as blank control A. This is used to eliminate background interference from the enzyme-free system.

[0152] Sample control well (corresponding to sample A control): Replace the enzyme working solution with 50 μL PBS, and perform the other operations the same as the sample group. Measure the absorbance at 405 nm and record it as sample A control. This is used to exclude interference from the sample itself and non-enzymatic reactions.

[0153] Positive control wells: Replace the test sample with 25 μL of 1 mg / mL acarbose solution, and perform the same operation as the sample group. Measure the absorbance at 405 nm to compare the inhibition effect of the sample.

[0154] The α-glucosidase inhibition rate (%) was calculated as [1 - (Sample A - Sample A control) / (Blank A - Blank A control)] × 100%.

[0155] The experimental results showed that the supernatant of CICC21736 inhibited α-glucosidase by 25.11%, and the supernatant of CCNH166 inhibited α-glucosidase by 82.13%, which was slightly lower than the effect of the positive control acarbose (inhibition rate 98.89%). This confirmed that the strain can effectively inhibit α-glucosidase activity and exert a hypoglycemic effect.

[0156] Example 12 This embodiment is used to verify the ability of CCNH166 to degrade cholesterol and thus exert a lipid-lowering effect.

[0157] Mix 0.1 g cholesterol, 0.2 g taurine, 0.11 g sucrose ester, 1 mL Tween 80, and 5 mL anhydrous ethanol in a 15 mL centrifuge tube. Heat the mixture in a 90-100°C water bath, then sonicate at 80 W for 15-25 minutes (vortexing every 4-8 minutes) until the cholesterol is completely dissolved. Filter the resulting solution while hot to remove bacteria. Immediately add 360 μL of this hot cholesterol solution to 30 mL of MRS liquid culture medium pre-placed in a sterile rotor. Stir the mixture at 700-800 rpm for 10-15 minutes on a magnetic stirrer to ensure thorough mixing, thus preparing the cholesterol-containing assay medium.

[0158] Glyceryl tubes of CCNH166 and CICC21736 were inoculated into MRS liquid medium and cultured to the logarithmic growth phase. The OD value of the bacterial culture was adjusted and normalized to obtain the test bacterial culture. This culture was then transferred to the above-mentioned test medium at a 2% (v / v) inoculation rate. An uninoculated test medium was used as a blank control. The culture was incubated at 37°C for 8 hours. After incubation, the culture medium was centrifuged to collect the supernatant. The supernatant was then measured using a cholesterol detection kit (TC-W48-N, catalog number 1031): Test tubes (containing 20 μL of supernatant, i.e., sample A), blank tubes (containing 20 μL of isopropanol, i.e., blank A), and standard tubes (containing 20 μL of standard) were prepared. Each tube contained 180 μL of the working solution used in the kit. After mixing, the mixture was incubated at 37°C for 15 minutes. The absorbance values ​​of the blank tube and the test tube (blank A and sample A) were measured at 500 nm. The cholesterol degradation rate was calculated using the following formula.

[0159] Cholesterol degradation rate = (A blank - A sample) / A blank × 100%.

[0160] Experimental results showed that CICC21736 had a cholesterol degradation rate of 29.68%, while CCNH166 had a degradation rate of 95.6%, demonstrating significant cholesterol assimilation capabilities. It can effectively remove cholesterol through degradation via mechanisms such as direct assimilation or bile salt metabolism, and has the potential to be a cholesterol-lowering probiotic.

[0161] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Weizmann's coagulans ( Weizmannia coagulans ), characterized in that, The preservation number of the *Weizmannii coagulans* is CGMCC No. 38119.

2. A microbial agent, characterized in that, The bacterial agent contains *Weizmannii coagulans* as described in claim 1.

3. A method for fermenting tea, characterized in that, The method includes: inoculating the *Weizmannii coagulans* of claim 1 into tea leaves and / or tea extract for fermentation.

4. A fermented tea product, characterized in that, The fermented tea product is obtained by the following method: Inoculate the tea extract or a mixture of tea and tea extract with the *Weizmannii coagulans* as described in claim 1, and ferment at 30-45°C for 10-100 h; Alternatively, the method is as follows:

1. Conventional Fermentation: The raw tea leaves are blended evenly according to requirements, piled for fermentation, sieved, subjected to the first high-temperature instantaneous steam treatment, and then cooled. II. Post-fermentation process: (1) Probiotic fortification: Weigh the freeze-dried powder of Weizmann's coagulans as described in claim 1, mix it with water at a ratio of g:mL = 1:10-100, activate it at 25-35℃ for 10-40 min, and then evenly sprinkle it into the tea leaves and stir it evenly. (2) Secondary steaming: The probiotic-enhanced Liubao tea undergoes a second high-temperature instantaneous steam treatment at a temperature of 80-90℃ for 10-60 seconds. (3) Pressing: The tea leaves are softened by the above-mentioned high-temperature instantaneous steam treatment and then pressed into shape; (4) Secondary fermentation culture: After pressing and shaping, the tea leaves are cultured at 25-40℃ and 60-90% humidity for 20-60 hours. (5) Low temperature drying: The drying temperature is ≤50℃ and the air humidity is ≤30%, and the tea leaves are dried to a moisture content of ≤7%.

5. A method for increasing the γ-aminobutyric acid (GABA) content in fermented tea products, characterized in that, The method includes: inoculating the *Weizmannii coagulans* of claim 1 into tea leaves and / or tea extract for fermentation.

6. The use of the *Weizmannii coagulates* strain according to claim 1 in enhancing at least one of γ-aminobutyric acid, tea polyphenols, and tea catechins in fermented tea products.

7. The use of the *Weizmannii coagulans* strain according to claim 1 in enhancing the acidity and / or flavor of fermented dairy products.

8. The application according to claim 7, wherein, The raw material for the fermented dairy product is whey protein.

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