Probiotic and plant extract composition for reducing trimethylamine oxide

Abstract

The invention provides a composition. The composition is prepared from probiotics, a plant extract and acceptable auxiliary materials according to a specific content ratio. Wherein the probiotics comprise the following components: Weizmannia coagulans AUSA001, Lactobacillus plantarum AUSA002, Bifidobacterium longum subsp. Longum FL006, and the probiotics comprise the following components: the Weizmannia coagulans AUSA001, the Lactobacillus plantarum AUSA002, the Bifidobacterium longum subsp. Longum FL006, the Lactobacillus plantarum AUSA002 and the Bifidobacterium longum subsp. Longum FL006. The plant extract comprises a kudzuvine root extract, a grape seed extract and a turmeric extract. The composition has a synergistic effect, can reduce the content of human intestinal trimethylamine and serum trimethylamine oxide, and is used for prevention or adjuvant therapy of related diseases.

Description

Technical Field

[0001] This invention relates to a nutritional composition containing probiotics and plant extracts, used to reduce the levels of trimethylamine (TMA) in the human gut and trimethylamine oxide (TMAO) in serum. This invention belongs to the technical field of microorganisms and plant extracts. Background Technology

[0002] The gut microbiota resembles a dynamic endocrine system, producing numerous bioactive metabolites that play a vital role in human nutrition and metabolic health. Trimethylamine N-oxide (TMAO) is a metabolic product of the human gut microbiota, primarily derived from carnitine and choline, abundant in animal-based foods such as red meat, eggs, and shellfish. Carnitine and choline from animal-based foods are metabolized into trimethylamine (TMA) by the gut microbiota and various enzymes. TMA is absorbed into the liver, where it is catalyzed by flavin monooxygenase to generate TMAO. Excessive intake of seafood, red meat, eggs, dairy products, and other animal products; a long-term high-fat diet; excessive use of L-carnitine supplements; and exposure to highly toxic dioxin-like environmental pollutants can all lead to elevated blood TMAO levels.

[0003] Studies have demonstrated that TMAO is closely related to the development and progression of diseases such as diabetes, cardiovascular disease, obesity, chronic kidney disease, metabolic diseases, neurological disorders, and cancer. Therefore, TMAO, as an emerging detection indicator, is gaining prominence in clinical medicine and may become a potential biomarker for disease diagnosis, prognosis, and treatment intervention. For trimethylamine metabolism disorders, such as trimethylamineuria (also known as TMAU, TMA-induced uremia, or rotten fish syndrome), the liver cannot effectively convert TMA into TMAO due to enzyme gene deficiencies. Currently, the primary approach is to reduce TMA intake. Other abnormalities associated with elevated TMA levels include vaginal odor caused by TMA production by vaginal bacteria, body odor caused by TMA production by bodily bacteria, and halitosis caused by TMA production by oral bacteria. Furthermore, elevated blood TMAO during pregnancy (late pregnancy and postpartum) is associated with an increased risk of thrombosis; therefore, reducing TMA and TMAO levels has significant clinical implications.

[0004] Currently, the main methods for lowering TMAO levels include medication and dietary adjustments. Specifically, TMA-producing bacteria can be targeted and inhibited by taking TMA lysin inhibitors such as 3,3-dimethyl-1-butanol (DMB), iodomethylcholine (IMC), and flumethylcholine (FMC), thereby reducing the production of TMA and TMAO. Statins can also lower TMAO levels. However, there are currently no approved drugs specifically for lowering TMAO. For example, DMB, as a choline analog, significantly lowers TMAO in experimental animals but is not approved for human use, while statins have potential side effects such as liver damage and rhabdomyolysis. Exploring safe and effective strategies for lowering TMAO may provide new treatment avenues for related diseases.

[0005] Probiotics may be one way to combat TMAO. *Weizmannii coagulans* is a type of Gram-positive, facultative anaerobic to microaerobic bacterium, possessing facultative anaerobic and acid-tolerant characteristics, making it easier for it to survive in the low-oxygen environment of the human digestive system, especially the intestines. *Lactobacillus plantarum* is a type of lactic acid bacteria, a Gram-positive, short rod-shaped, microaerophilic, acid-resistant, non-spore-forming, non-respiratory, low-G+C content, heterofermentative lactobacillus group. *Bifidobacterium longum* is a microorganism of the genus *Bifidobacterium*, belonging to the Gram-positive bacillus group, capable of fermenting sugars to produce acid.

[0006] Natural plant extracts may also be a means of combating TMAO. Kudzu root extract, a plant compound isolated from kudzu root, has anticholinergic, antioxidant, and free radical scavenging effects. Grape seed extract, a class of polyphenols extracted from grape seeds, mainly consists of proanthocyanidins, catechins, epicatechin, gallic acid, and epicatechin gallate, and has anti-aging, immune-boosting, skin-improving, and eye-fatigue-relieving effects. Turmeric extract, also an effective component extracted from turmeric, has anti-inflammatory, anticoagulant, and antiplatelet aggregation effects.

[0007] In our exploratory experiments, we found that the single application of probiotics or plant extracts had limited effects against TMAO. However, combining these two types of substances in different proportions significantly enhanced their effects on inhibiting trimethylamine (TMA)-producing bacteria, inhibiting the conversion of choline to trimethylamine TMA, and reducing plasma oxidized trimethylamine (TMAO) levels. Therefore, this invention, through optimization, provides a multi-component composition with synergistic effects, exhibiting superior efficacy compared to using any single component alone, thereby achieving the purpose of preventing or treating TMAO-related diseases. Summary of the Invention

[0008] To address the shortcomings of current trimethylamine oxide products, this invention provides a safe and effective composition.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A composition comprising:

[0011] a) probiotics; b) plant extracts; c) acceptable excipients or carriers, or mixtures thereof. The plant extracts are selected from one or more of kudzu root extract, grape seed extract, and turmeric extract, and the probiotics are selected from one or more strains of the following:

[0012] (1) *Weizmannii coagulans* AUSA001, deposited at Guangdong Provincial Microbial Culture Collection Center on March 15, 2024, with accession number GDMCC No: 64416, classified and named as follows: The depository of Weizmannia coagulans is located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0013] (2) *Lactobacillus plantarum* AUSA002, deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 15, 2024, with accession number GDMCC No: 64417, classified and named as follows: Lactiplantibacillus plantarum, deposited at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province;

[0014] (3) *Bifidobacterium longum* subspecies FL006, deposited at Guangdong Provincial Microbial Culture Collection Center on June 5, 2024, with accession number GDMCC No: 1.4842, and classified as follows: Bifidobacterium longum subsp.Longum, deposited at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0015] In this invention, the plant extract is obtained by solvent extraction, filtration, purification, concentration, and drying of the effective parts of the plant.

[0016] In this invention, the main components of kudzu root extract include daidzein, daidzin, puerarin, puerarin-7-xyloside, etc.

[0017] In this invention, the main components of grape seed extract include polyphenols, such as proanthocyanidins, catechins, epicatechin, gallic acid, epicatechin gallate, etc.

[0018] In this invention, the main components of turmeric extract include curcumin, curcuminone, curcuminol, etc.

[0019] In this invention, the mass ratio of probiotics to plant extracts is 1-10:1-10, preferably 1-3:1-3, and more preferably 1:1.

[0020] In this invention, the probiotic is Weizmannii coagulans AUSA001, and the plant extract is kudzu root extract. The ratio of Weizmannii coagulans AUSA001 to kudzu root extract by mass is 1:1.

[0021] Alternatively, the probiotics may be Lactobacillus plantarum AUSA002 and Bifidobacterium longum subsp. FL006, the plant extract may be grape seed extract, and the mass ratio of the probiotics to the plant extract may be 2:1.

[0022] Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, the plant extract may be turmeric extract, and the mass ratio of the probiotics to the plant extract may be 3:3.3.

[0023] Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, the plant extract may be turmeric extract, and the mass ratio of the probiotics to the plant extract may be 3:1.

[0024] Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, and the plant extracts may be kudzu root extract and turmeric extract, with the mass ratio of probiotics to plant extracts being 3:20.

[0025] In this invention, the probiotic is *Weizmannii coagulans* AUSA001, and the plant extract is kudzu root extract. The ratio of *Weizmannii coagulans* AUSA001 to kudzu root extract by mass is 1:1.

[0026] Alternatively, the probiotics are *Lactobacillus plantarum* AUSA002 and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract is grape seed extract. The ratio of *Weizmannii coagulans* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, to grape seed extract is 1:1:1 by mass.

[0027] Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract. The ratio of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, and turmeric extract by mass is 1:1:1:3.3.

[0028] Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract. The ratio of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, and turmeric extract by mass is 1:1:1:1.

[0029] Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extracts may be kudzu root extract and turmeric extract. The ratio of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, kudzu root extract, and turmeric extract, by mass, is 1:1:1:10:10.

[0030] In this invention, the composition contains 50-500 parts of probiotics and 50-500 parts of plant extracts; preferably, the composition contains 100-300 parts of probiotics and 100-300 parts of plant extracts.

[0031] In this invention, the probiotic is Weizmannii coagulans AUSA001, and the plant extract is kudzu root extract, wherein the probiotic is 100 parts and the plant extract is 100 parts.

[0032] Alternatively, the probiotics may be Lactobacillus plantarum AUSA002 and Bifidobacterium longum subsp. FL006, and the plant extract may be grape seed extract, wherein the probiotics are 200 parts and the plant extract is 100 parts.

[0033] Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, and the plant extract may be turmeric extract, wherein the probiotics are 30 parts and the plant extract is 33 parts.

[0034] Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, and the plant extract may be turmeric extract, wherein the probiotics are 300 parts and the plant extract is 100 parts.

[0035] Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, and the plant extracts may be kudzu root extract and turmeric extract, wherein the probiotics are 30 parts and the plant extracts are 200 parts.

[0036] In this invention, the probiotic is Weizmannii coagulans AUSA001, and the plant extract is kudzu root extract, wherein the amount of Weizmannii coagulans AUSA001 is 100 parts and the amount of kudzu root extract is 100 parts.

[0037] Alternatively, the probiotics may be *Lactobacillus plantarum* AUSA002 and *Bifidobacterium longum* subsp. FL006, and the plant extract may be grape seed extract, wherein *Lactobacillus plantarum* AUSA002 is 100 parts, *Bifidobacterium longum* subsp. FL006 is 100 parts, and grape seed extract is 100 parts.

[0038] Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, wherein *Weizmannii coagulans* AUSA001 comprises 10 parts, *Lactobacillus plantarum* AUSA002 comprises 10 parts, *Bifidobacterium longum* subsp. *longum* FL006 comprises 10 parts, and turmeric extract comprises 33 parts.

[0039] Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, wherein the amount of *Weizmannii coagulans* AUSA001 is 100 parts, the amount of *Lactobacillus plantarum* AUSA002 is 100 parts, the amount of *Bifidobacterium longum* subsp. *longum* FL006 is 100 parts, and the amount of turmeric extract is 100 parts.

[0040] Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extracts may be kudzu root extract and turmeric extract, wherein the amount of *Weizmannii coagulans* AUSA001 is 10 parts, the amount of *Lactobacillus plantarum* AUSA002 is 10 parts, the amount of *Bifidobacterium longum* subsp. *longum* FL006 is 10 parts, the amount of kudzu root extract is 100 parts, and the amount of turmeric extract is 100 parts.

[0041] In this invention, a portion can be a microgram, a milligram, a gram, a microliter, a milliliter, a deciliter, etc.

[0042] In this invention, the total number of live probiotics is 0.5 × 10⁻⁶. 8CFU / g ~ 2.0 × 10⁻⁶ 11 CFU / g; the total live bacteria count of probiotics is preferably 1×10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g; specifically, the total viable count of *Weizmannii* AUSA001 was 1 × 10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g, preferably 1×10 9 ~1×10 10 CFU / g; The total viable count of *Lactobacillus plantarum* AUSA002 was 1×10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g, preferably 1×10 9 ~1×10 10 CFU / g; The total viable count of Bifidobacterium longum subsp. FL006 was 1×10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g, preferably 1×10 9 ~1×10 10 CFU / g.

[0043] In this invention, acceptable excipients or carriers or mixtures thereof include one or more of sugars or oligosaccharides or functional sweeteners, fillers, wetting agents, binders, lubricants, and microbial powder cryoprotectants.

[0044] In this invention, the cryoprotectant for the bacterial powder includes one or more of inulin, maltodextrin, and fructooligosaccharides.

[0045] In this invention, the dosage forms of the composition include, but are not limited to, solid beverages (powders, granules, microcapsules), tablets (ordinary tablets, bilayer tablets, multilayer tablets, chewable tablets, lozenges, compressed candies), lyophilized powders, oral liquids, injections, pills, syrups, semi-solid preparations (gels, gel candies, soft candies), capsules (hard capsules, soft capsules, crystal balls), candies, liquid preparations, and other dosage forms.

[0046] In this invention, the composition is used in the preparation of a product for reducing serum trimethylamine oxide.

[0047] In this invention, the composition is used in the preparation of a product for reducing trimethylamine.

[0048] In this invention, the composition is used in the preparation of products for treating diseases associated with elevated serum trimethylamine oxide.

[0049] In this invention, the composition is used in the preparation of products for treating trimethylamine metabolism disorders, including trimethylamineuria.

[0050] The beneficial effects of this invention are as follows: The probiotic strains used in this invention were all isolated from healthy human subjects by the inventors and have been deposited at the Guangdong Provincial Microbial Culture Collection Center. This invention mixes the selected probiotics and plant extracts in a specific form and proportion, and processes them according to a specific process to formulate a composition product that reduces trimethylamine oxide (TMA). The combined supplementation of each component produces an unexpected synergistic effect, which can regulate intestinal flora-related metabolites, reduce intestinal TMA-producing microorganisms, thereby reducing the levels of trimethylamine and serum TMA in the human body, achieving the purpose of preventing and adjuvant treatment of diseases. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments, but this is not intended to limit the present invention. Any equivalent substitutions made in the art in accordance with the content of the present invention shall fall within the protection scope of the present invention.

[0052] Example 1: In vitro inhibition experiment of trimethylamine (TMA)-producing bacteria

[0053] I. Methods

[0054] Preparation of plant extracts: Kudzu root and turmeric were selected. The raw materials were freeze-dried and ground into powder, or they were mixed and crushed, and then extracted by rotary evaporation or soaking in methanol and heating under reflux. After filtration and concentration, the paste was obtained and then freeze-dried or vacuum-dried to obtain solid powder, which was used as plant extract for later use.

[0055] Preparation of bacterial suspension and bacterial fragments: Three probiotic strains were selected: *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *flavovirens* FL006. They were cultured according to their respective culture media and conditions. The preparation method of the bacterial agent was as follows: *Lactobacillus plantarum* AUSA002 was cultured on MRS agar medium at 35–37°C under facultative anaerobic conditions for 24–40 h until the strain concentration reached 1 × 10⁻⁶. 9 CFU / ml; *Bifidobacterium longum* subsp. *longum* FL006 was cultured on MRS agar medium supplemented with cysteine ​​hydrochloride under strictly anaerobic conditions at 35–37°C for 40–48 h until the strain concentration reached 1×10⁻⁶. 9 CFU / ml; *Weizmannii coagulans* AUSA001 was cultured in a dedicated culture medium for *Bacillus coagulans* at 40℃ under facultative anaerobic conditions for 24–40 h until the strain concentration reached 1×10⁻⁶. 9CFU / ml. Taking *Lactobacillus plantarum* AUSA002 as an example (other strains are treated similarly), inoculate 2% into 500 mL of the corresponding liquid culture medium and culture. After culture, centrifuge at 4°C, 6000 rpm for 10 min. Collect the supernatant and bacterial cells. Store the bacterial culture for later use or freeze at -80°C. Resuspend the bacterial cells in 50 mL of sterile water or 1×PBS buffer and centrifuge twice. Resuspend the washed bacterial cells again and sonicate at 25 kHz and 450 W for 20 min. Mix with lysozyme at a 1:1 ratio and incubate at 37°C for 30 min before centrifugation. Finally, store for later use or freeze at -80°C.

[0056] In vitro inhibition of TMA bacteria experiment: The probiotics represented by the compound strain composed of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. FL006 mentioned above, with a mass ratio of 1:1:1; the plant extracts represented by the compound plant extract composed of kudzu root extract and turmeric extract mentioned above, with a mass ratio of 1:1. The experiment was divided into 6 groups: 5 experimental groups and 1 control group. The experimental groups included probiotic culture supernatant, probiotic cell fragments, and plant extracts, or combinations thereof (in a 1:1 volume ratio), i.e., a combination of probiotic supernatant and plant extracts, or a combination of probiotic cell fragments and plant extracts. The control group used sterile water or 1×PBS buffer. The cultures were then co-cultured at 35–37°C for 24 hours with bacteria containing TMA lyase (CntA or cutC / D gene clusters), such as *Proteus mirabilis*, *Clostridium saccharolyticum*, or *Escherichia coli*, in a 1:1 volume ratio. The OD of the culture medium was measured using a microplate reader. 600 Monitor the growth of TMA-producing bacteria and determine the OD value after 24 hours. 600 The size of the sample determines the inhibitory effect of probiotics, plant extracts, or a combination of both on TMA-producing intestinal bacteria.

[0057] II. Results

[0058] As shown in Table 1, negative values ​​represent the percentage reduction compared to the blank control; that is, the larger the value after the negative sign, the better the antibacterial effect. Compared with the blank control (water) group, the OD values ​​of the probiotic culture supernatant group, probiotic cell fragment group, and plant extract group were significantly higher. 600 Both groups showed a significant decrease (P < 0.05), with the OD values ​​of the probiotic culture supernatant + plant extract group and the probiotic cell fragments + plant extract group being significantly lower. 600All values ​​were further reduced (P < 0.01), indicating that the combination of the probiotic strains consisting of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *followii* FL006 with plant extracts consisting of kudzu root extract and turmeric extract produced a highly significant reduction in OD. 600 The effect of in vitro inhibition of trimethylamine (TMA)-producing bacteria is significant.

[0059] Table 1. Inhibitory effect of the composition of the present invention on TMA-producing intestinal bacteria.

[0060]

[0061] Note: Compared with the blank control group, a P<0.05, aa P<0.01.

[0062] Example 2: In vitro experiment on inhibition of choline to trimethylamine (TMA) conversion

[0063] I. Methods

[0064] Preparation of plant extracts: Kudzu root and turmeric were selected. The raw materials were freeze-dried and ground into powder, or they were mixed and crushed, and then extracted by rotary evaporation or soaking in methanol and heating under reflux. After filtration and concentration, the paste was obtained and then freeze-dried or vacuum-dried to obtain solid powder, which was used as plant extract for later use.

[0065] Preparation of bacterial suspension and bacterial fragments: Three probiotic strains were selected: *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *flavovirens* FL006. They were cultured according to their respective culture media and conditions. The preparation method of the bacterial agent was as follows: *Lactobacillus plantarum* AUSA002 was cultured on MRS agar medium at 35–37°C under facultative anaerobic conditions for 24–40 h until the strain concentration reached 1 × 10⁻⁶. 9 CFU / ml; *Bifidobacterium longum* subsp. *longum* FL006 was cultured on MRS agar medium supplemented with cysteine ​​hydrochloride under strictly anaerobic conditions at 35–37°C for 40–48 h until the strain concentration reached 1×10⁻⁶. 9 CFU / ml; *Weizmannii coagulans* AUSA001 was cultured in a dedicated culture medium for *Bacillus coagulans* at 40℃ under facultative anaerobic conditions for 24–40 h until the strain concentration reached 1×10⁻⁶. 9CFU / ml. Taking *Lactobacillus plantarum* AUSA002 as an example (other strains are treated similarly), inoculate 2% into 500 mL of the corresponding liquid culture medium and culture. After culture, centrifuge at 4°C, 6000 rpm for 10 min. Collect the supernatant and bacterial cells. Store the bacterial culture for later use or freeze at -80°C. Resuspend the bacterial cells in 50 mL of sterile water or 1×PBS buffer and centrifuge twice. Resuspend the washed bacterial cells again and sonicate at 25 kHz and 450 W for 20 min. Mix with lysozyme at a 1:1 ratio and incubate at 37°C for 30 min before centrifugation. Finally, store for later use or freeze at -80°C.

[0066] In vitro inhibition of choline to TMA conversion experiment: The probiotic represented by the aforementioned compound strain consisting of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *flavovirens* FL006, with a mass ratio of 1:1:1. The plant extract represented by the aforementioned compound plant extract consisting of kudzu root extract and turmeric extract, with a mass ratio of 1:1. The experiment was divided into six groups: five experimental groups and one control group. The experimental groups consisted of probiotic culture supernatant, probiotic cell fragments, and plant extracts, or combinations thereof (in a 1:1 volume ratio), i.e., a combination of probiotic supernatant and plant extracts, or a combination of probiotic cell fragments and plant extracts. The control group used sterile water or 1×PBS buffer. Each group was then co-cultured with bacteria containing TMA lysin (CntA or cutC / D gene clusters), such as *Proteus mirabilis*, *Clostridium saccharolyticum*, or *Escherichia coli*, at a 1:1 volume ratio at 35–37°C for 24 hours. Choline chloride (CC) (1M stock solution) was added to the culture medium to achieve a final concentration of 0.5 mM. After cultivation, 600 μL of a mixed solution of acetonitrile:methanol:water (V:V:V, 40:40:20) was added to each sample to precipitate proteins and other substances. Simultaneously, d9-TMA at a final concentration of 10 μM was added as an internal standard. The mixture was shaken and incubated at -80℃ for 2 h. After centrifugation at 12000×g for 15 min at 4℃, the supernatant was transferred to a sample vial and stored at -80℃. TMA concentration was determined using HPLC-MS / MS. The concentration of TMA produced during cultivation was determined by constructing regression curves using the response (peak area) versus concentration for each standard, using TMA and d9-TMA calibration standards.

[0067] II. Results

[0068] As shown in Table 2, compared with the blank control (water) group, the TMA content in the probiotic culture supernatant group, the probiotic cell fragment group, and the plant extract group was significantly reduced (P < 0.05). The TMA content in the probiotic culture supernatant + plant extract group and the probiotic cell fragment + plant extract group was further reduced (P < 0.01). This indicates that the combination of the compound probiotic strain composed of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. FL006 with the plant extract composed of kudzu root extract and turmeric extract produced a very significant effect in reducing TMA content, that is, a significant effect in inhibiting the conversion of choline to trimethylamine (TMA) in vitro.

[0069] Table 2. Effect of the composition of the present invention on in vitro inhibition of choline to trimethylamine (TMA) conversion.

[0070]

[0071] Note: Compared with the blank control group, a P<0.05, aa P<0.01.

[0072] Example 3: Effects of the composition of the present invention on TMAO in rats

[0073] I. Methods

[0074] Experimental animals and grouping: SD rats, half male and half female, weighing 200±20g, were purchased from Guangdong Provincial Medical Laboratory Animal Center. They were housed in an environment with room temperature of 18–28℃ and relative humidity of 40%–70%, with free access to food and water. After one week of acclimatization feeding with standard diet, they were randomly divided into groups as shown in Table 3 below, with 10 rats in each group.

[0075] Model construction and drug administration: Except for the model control group, rats in other groups were administered the corresponding test samples by gavage at the doses specified in Table 3. The viable bacterial count in the samples was 1.0 × 10⁻⁶. 10 Rats in the model control group were administered an equal volume of phosphate-buffered saline by gavage daily. Simultaneously, all rats were administered 53 mg / kg choline chloride by gavage daily, and all rats had free access to a high-fat diet (purchased from the Guangdong Provincial Medical Laboratory Animal Center). On day 14 of the experiment, plasma was collected from all rats via ocular vein sampling (after a 12-hour fast). All plasma samples were then centrifuged at 3000g for 20 minutes at 4°C, and serum was collected. The concentration of trimethylamine oxide (TMAO) was determined using liquid chromatography-mass spectrometry.

[0076] To verify the scientific validity of the pharmaceutical composition provided by this invention, and to demonstrate that the combination of multiple components in the pharmaceutical composition is reasonable and that their synergistic effect is not simply the sum of pharmacological effects, the Jin Zhengjun Q-value method is introduced for analysis. The Jin Zhengjun Q-value method, also known as the probability addition method, calculates the pharmacological effects of two drugs used in combination and two drugs used alone within the dose-response curve region using the following formula: Q = E A+B / (E A +E B -E A *E B In the formula, the numerator represents the "measured combined effect," and the denominator represents the "expected combined effect." (To satisfy the analysis of the pharmacological relationship between components and compositions, their pharmacological effects are transformed into effects that can intuitively reflect the strength of pharmacological effects. The calculation formula is: E...) i =1-P i / P 模型组 P i For the pharmacological indicators of each component, P 模型组 (These are pharmacological indicators for the model group), where Q is the ratio of the two drugs: when Q is less than 0.85, the combined use of the two drugs is considered antagonistic; when Q is less than 1.15 but greater than 0.85, it is considered additive; and when Q is greater than or equal to 1.15, it is considered synergistic. Based on the calculation formula for the combined use of two drugs, the pharmacological effects of the combined use of three drugs and the pharmacological effects of the three drugs used alone are calculated using the following formula:

[0077] Q = E A+B+C / (E A +E B +E C -E A *E B -E A *E C -E B *E C -E A *E B *E C ).

[0078] II. Results

[0079] As shown in Table 3, compared with the model control group, there were no significant differences in plasma TMAO levels in the single-drug group, the low (high) dose AUSA002+FL006 group, the low dose AUSA001+AUSA002+FL006 group, the low dose AUSA001+pueraria extract group, and the low dose AUSA002+FL006+grape seed extract group (P>0.05). However, the plasma TMAO levels in the high dose AUSA001+AUSA002+FL006 group, the high dose AUSA001+pueraria extract group, and the low dose AUSA001+AUSA002+FL006+turmeric extract group were all significantly lower (P<0.05). The high dose AUSA002+FL006+grape seed extract group, the high dose AUSA001+AUSA002+FL006+turmeric extract group, and the low dose AUSA001+...

[0080] The plasma TMAO content in rats in the AUSA002+FL006+high-dose kudzu root extract+turmeric extract group was significantly reduced (P<0.01), indicating that the above-mentioned combination has the effect of significantly reducing plasma TMAO content and can degrade TMA in vivo.

[0081] Meanwhile, the Q values ​​of the low (high) dose AUSA001 + kudzu root extract group, the low (high) dose AUSA002 + FL006 + grape seed extract group, the low (high) dose AUSA001 + AUSA002 + FL006 + turmeric extract group, and the low dose AUSA001 + AUSA002 + FL006 + high dose kudzu root extract + turmeric extract group in reducing plasma TMAO content were all >1.15, indicating that the components of the composition of the present invention have a synergistic effect in reducing TMA generation and lowering plasma TMAO content.

[0082] Table 3 Effects of the composition on TMAO levels in rat plasma ( n = 9 to 10)

[0083]

[0084] Note: Compared with the model control group, a P<0.05, aa P<0.01.

Claims

1. A composition comprising: a) Probiotics; b) Plant extracts; c) An acceptable excipient or carrier or a mixture thereof; wherein the plant extract is selected from one or more of kudzu root extract, grape seed extract, and turmeric extract, and the probiotics are selected from one or more of the following strains: (1) Weizmann's coagulation bacterium AUSA001, deposited at Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCCNo: 64416; (2) Lactobacillus plantarum AUSA002, deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 64417; (3) Bifidobacterium longum subspecies FL006, deposited at Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCCNo: 1.4842.

2. The composition according to claim 1, characterized in that, The mass ratio of the probiotics to the plant extract is 1-10:1-10, preferably 1-3:1-3, and more preferably 1:

1.

3. The composition according to claim 2, characterized in that, The probiotic is Weizmannii coagulans AUSA001, the plant extract is kudzu root extract, and the mass ratio of the probiotic to the plant extract is 1:

1. Alternatively, the probiotics may be *Lactobacillus plantarum* AUSA002 and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be grape seed extract, with the mass ratio of the probiotics to the plant extract being 2:1; preferably, the mass ratio of *Weizmannii coagulans* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006 to grape seed extract is 1:1:

1. Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, with the mass ratio of the probiotics to the plant extract being 3:3.3; preferably, the mass ratio of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, and turmeric extract is 1:1:1:3.

3. Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, with the mass ratio of the probiotics to the plant extract being 3:1; preferably, the mass ratio of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, and turmeric extract is 1:1:1:

1. Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extracts may be kudzu root extract and turmeric extract, with the mass ratio of the probiotics to the plant extracts being 3:20; preferably, by mass, the ratio of *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, *Bifidobacterium longum* subsp. *longum* FL006, kudzu root extract, and turmeric extract is 1:1:1:10:

10.

4. The composition according to claim 1, characterized in that, The composition contains 50 to 500 parts of probiotics and 50 to 500 parts of plant extract; preferably, the composition contains 100 to 300 parts of probiotics and 100 to 300 parts of plant extract.

5. The composition according to claim 4, characterized in that, The probiotic is Weizmannii coagulans AUSA001, and the plant extract is kudzu root extract, wherein the probiotic is 100 parts and the plant extract is 100 parts. Alternatively, the probiotics may be *Lactobacillus plantarum* AUSA002 and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be grape seed extract, wherein the probiotics comprise 200 parts and the plant extract comprises 100 parts; or the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, wherein the probiotics comprise 30 parts and the plant extract comprises 33 parts. Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, and the plant extract may be turmeric extract, wherein the probiotics are 300 parts and the plant extract is 100 parts. Alternatively, the probiotics may be Weizmannii coagulans AUSA001, Lactobacillus plantarum AUSA002, and Bifidobacterium longum subsp. FL006, and the plant extracts may be kudzu root extract and turmeric extract, wherein the probiotics are 30 parts and the plant extracts are 200 parts.

6. The composition according to claim 5, characterized in that: The probiotics are *Lactobacillus plantarum* AUSA002 and *Bifidobacterium longum* subsp. FL006, and the plant extract is grape seed extract, wherein *Lactobacillus plantarum* AUSA002 is 100 parts, *Bifidobacterium longum* subsp. FL006 is 100 parts, and grape seed extract is 100 parts. Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, wherein *Weizmannii coagulans* AUSA001 comprises 10 parts, *Lactobacillus plantarum* AUSA002 comprises 10 parts, *Bifidobacterium longum* subsp. *longum* FL006 comprises 10 parts, and turmeric extract comprises 33 parts. Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extract may be turmeric extract, wherein the amount of *Weizmannii coagulans* AUSA001 is 100 parts, the amount of *Lactobacillus plantarum* AUSA002 is 100 parts, the amount of *Bifidobacterium longum* subsp. *longum* FL006 is 100 parts, and the amount of turmeric extract is 100 parts. Alternatively, the probiotics may be *Weizmannii coagulans* AUSA001, *Lactobacillus plantarum* AUSA002, and *Bifidobacterium longum* subsp. *longum* FL006, and the plant extracts may be kudzu root extract and turmeric extract, wherein the amount of *Weizmannii coagulans* AUSA001 is 10 parts, the amount of *Lactobacillus plantarum* AUSA002 is 10 parts, the amount of *Bifidobacterium longum* subsp. *longum* FL006 is 10 parts, the amount of kudzu root extract is 100 parts, and the amount of turmeric extract is 100 parts.

7. The composition according to claim 1, characterized in that, The total number of live bacteria in the probiotics is 0.5 × 10⁻⁶. 8 CFU / g ~ 2.0 × 10⁻⁶ 11 CFU / g, preferably 1×10 8 CFU / g ~ 1.5 × 10 11 CFU / g.

8. The composition according to claim 7, characterized in that, The total viable count of the *Weizmannii coagulans* AUSA001 was 1 × 10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g, preferably 1×10 9 ~1×10 10 CFU / g; The total viable count of the *Lactobacillus plantarum* AUSA002 is 1×10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g, preferably 1×10 9 ~1×10 10 CFU / g; The total viable count of the *Bifidobacterium longum* subsp. *FL006* is 1×10⁻⁶. 8 CFU / g ~ 1.5 × 10 11 CFU / g, preferably 1×10 9 ~1×10 10 CFU / g.

9. Use of the composition according to any one of claims 1 to 8 in the preparation of products for reducing serum trimethylamine oxide or treating diseases related to elevated serum trimethylamine oxide.

10. Use of the composition according to any one of claims 1 to 8 in the preparation of a product for reducing trimethylamine or treating trimethylamineuria.

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