In-vitro fermentation system for rapid targeted screening and regulation of generation of intestinal TMA, establishment method and application

Abstract

The invention relates to the technical field of intestinal microecology, and particularly discloses an in-vitro fermentation system for rapid targeted screening, regulation and control of intestinal TMA generation, an establishment method and application, and the in-vitro fermentation system comprises the following components in parts by weight: 2-3 parts of yeast extract, 0.8-1.2 parts of L-cysteine, 1.5-2.5 parts of heme, 0.8-1.0 part of sodium chloride, 0.008-0.012 part of calcium chloride hexahydrate and 0.4-0.5 part of dipotassium phosphate. The feed is prepared from the following raw materials in parts by weight: 0.08 to 0.1 part of magnesium sulfate heptahydrate, 0.15 to 0.25 part of vitamin mixture, 7 to 9 parts of starch, 0.01 to 0.8 part of choline and 0.8 to 1.2 parts of resazurin. The method has the characteristics that the large-scale and high-flux active substance screening requirement can be met, and the TMA generation and related substrate metabolism quantitative control can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of intestinal microecology, and particularly relates to a rapid targeted screening in vitro fermentation system for regulating intestinal TMA generation, a method for establishing the same and application. BACKGROUND

[0002] In recent years, cardiovascular disease (CVD) and cerebrovascular disease (CBVD) are still one of the major causes of death worldwide; although drug treatment and intervention means are constantly improving, the incidence, recurrence rate and all-cause mortality of CVD are still high. According to the statistics of the World Health Organization, more than 1760 million people die of CVD every year, accounting for one-third of the global total mortality rate; the traditional treatment strategy mainly focuses on blood lipid, blood pressure and blood glucose regulation, but cannot completely block the disease progression from the metabolic source, which makes the scientific community begin to pay attention to the relationship between intestinal microecology and cardiovascular metabolism. A large number of studies have shown that intestinal flora and its metabolites play a key role in the occurrence and development of CVD, especially intestinal microbial metabolite trimethylamine-N-oxide (TMAO), which is derived from the metabolism of intestinal flora to nutrients rich in choline, carnitine, betaine and phosphatidylcholine, etc. These substrates are metabolized by intestinal microorganisms to generate trimethylamine (TMA), and then TMA is absorbed into the liver, where it is oxidized to form TMAO under the action of flavin monooxygenase (mainly FMO3); a number of clinical and animal experiments have confirmed that the increase in TMAO concentration is closely related to atherosclerosis, vascular endothelial dysfunction, lipid metabolism disorder and inflammatory reaction activation, therefore, how to effectively reduce the generation of intestinal TMA or inhibit the formation of TMAO has become a new direction of CVD prevention and treatment research. However, existing research mainly relies on animal models or clinical intervention experiments, which have long cycle, high cost and complex variables, and are difficult to meet the needs of large-scale, high-throughput active substance screening; traditional in vivo models have significant limitations, on the one hand, TMAO generation involves a complex process of “substrate absorption-bacterial metabolism-liver oxidation”; on the other hand, the intestinal flora of different individuals differs greatly, making the results less reproducible; in view of these problems, researchers have proposed to use an in vitro simulated intestinal fermentation system to reconstruct the human post-intestinal metabolic environment, and to study the TMA generation process under controllable conditions to provide an efficient tool for active substance screening. Based on this demand, it is of great significance to develop a rapid targeted screening in vitro fermentation system for regulating intestinal TMA generation.

[0003] Meanwhile, the patent document with the publication number "CN119552770A" discloses a method for constructing an in-vitro simulation system of intestinal microorganisms, which takes the contents of the cecum, mixes them uniformly, inoculates them into an anaerobic fermentation tank for fermentation, and controls the addition speed of the culture medium; the pH is controlled at 6.0-7.0 during fermentation, and the fermentation is carried out for 1-7 days to obtain a fermentation liquor containing intestinal microorganisms. The method provided by the present application can restore the dominant flora of the original flora in the body to a certain extent, and can simulate the dynamic changes of intestinal microorganisms in vitro. However, this scheme lacks the ability to regulate specific metabolic pathways, cannot achieve quantitative control of TMA generation and related substrate metabolism, and does not consider the problems of redox balance and metabolic stability. SUMMARY

[0004] In order to solve the above technical problems existing in the present TMA generation process research, the present application provides a rapid targeted screening in-vitro fermentation system for regulating TMA generation in the intestinal tract, a method for establishing and application, which has the characteristics of being able to meet the demand of large-scale and high-throughput active substance screening, and being able to realize quantitative control of TMA generation and related substrate metabolism.

[0005] The first technical solution of the present invention is an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA production, comprising the following components by weight: 2-3 parts yeast extract, 0.8-1.2 parts L-cysteine, 1.5-2.5 parts heme, 0.8-1.0 parts sodium chloride, 0.008-0.012 parts calcium chloride hexahydrate, 0.4-0.5 parts dipotassium hydrogen phosphate, 0.08-0.1 parts magnesium sulfate heptahydrate, 0.15-0.25 parts vitamin mixture, 7-9 parts starch, 0.01-0.8 parts choline, and 0.8-1.2 parts resazurin. The yeast extract in this invention serves as a complex nitrogen source and growth factor source, providing amino acids, small peptides, nucleotides, vitamins, and trace elements to ensure rapid initiation of growth and metabolism in intestinal microorganisms. L-cysteine, as a reducing agent, consumes oxygen in the system and, in conjunction with resazurite, rapidly establishes and maintains a strictly anaerobic environment, providing essential sulfur for microorganisms and creating and maintaining anaerobic conditions crucial for colonic microbial survival, preventing excessive growth of aerobic microorganisms and ensuring the accuracy of experimental results. Heme is used to simulate the heme environment in the intestine, supporting the growth of heme-dependent bacteria that play a crucial role in the metabolism of choline, a precursor to TMA, thus making the fermentation system more realistically reflect the in vivo situation. The sodium chloride component in this invention is the main osmotic pressure regulator, maintaining an isotonic environment similar to human intestinal fluid, preventing microbial death due to osmotic imbalance. Calcium ions in calcium chloride hexahydrate are enzyme cofactors, participating in signal transduction and cell wall stability maintenance, providing a suitable physiological environment for microorganisms, ensuring their normal physiological functions and structural integrity. The system is characterized by: The presence of dipotassium hydrogen phosphate and acidic substances produced during fermentation, such as short-chain fatty acids, forming a phosphate buffer system that maintains relative pH stability. This system provides phosphorus, essential for microbial synthesis of nucleic acids, ATP, and phospholipids, as well as potassium, necessary for maintaining intracellular potential. It prevents a sharp drop in pH due to excessive acid production, thus avoiding inhibition of most intestinal microorganisms and ensuring a smooth fermentation process. Magnesium sulfate heptahydrate provides magnesium and sulfur ions, ensuring the basal metabolism and energy supply for microorganisms. The vitamin mixture in this invention provides a range of B vitamins, such as biotin, folic acid, and thiamine, supporting the growth of bacteria requiring exogenous vitamins and diversifying the microbial community. Starch, as the primary carbon and energy source, provides a continuous and stable energy source for the entire microbial community, supporting its long-term fermentation and metabolism. Choline, as a direct substrate for TMA generation, is added exogenously to provide raw materials for the TMA generation reaction, enabling direct and quantitative detection of TMA yield changes under different intervention conditions, achieving rapid targeted screening.As an anaerobic indicator, resazurin appears pink or purple under aerobic conditions and is reduced to colorless halogen under anaerobic conditions, providing experimenters with an intuitive and visual indication of the anaerobic environment. The disappearance of the pink color signifies that the system has reached an anaerobic state and fermentation experiments can begin. This invention precisely recreates the anaerobic, isotonic, and weakly acidic environment of the colon through inorganic salts, buffer pairs, reducing agents, and anaerobic indicators. Yeast extracts, vitamins, and heme provide comprehensive and balanced nutrition to support the growth of complex microbiota. The targeted approach, with the direct addition of the TMA precursor choline, makes the research objective highly focused. The addition of heme takes into account its potential impact on gut microbiota and metabolism, making the model more realistic. Using resazurin as an indicator significantly reduces operational difficulty and failure rate. Overall, this is a scientifically designed, structurally complete, and highly targeted in vitro fermentation system, highly rational, advanced, and practical for rapidly screening substances that regulate intestinal TMA production. Choline is a key substrate for the formation of trimethylamine (TMA); resazurin is a colorimetric indicator of the redox state in the system. Resazurin accurately reflects changes in the anaerobic environment through a reversible change from blue to pink to colorless, enabling visual monitoring of the reduction state of the fermentation system.

[0006] Preferably, the product comprises the following components by weight: 2.2–2.8 parts yeast extract, 0.9–1.1 parts L-cysteine, 1.8–2.2 parts heme, 0.85–0.95 parts sodium chloride, 0.009–0.011 parts calcium chloride hexahydrate, 0.42–0.48 parts dipotassium hydrogen phosphate, 0.09 parts magnesium sulfate heptahydrate, 0.18–0.22 parts vitamin mixture, 7.5–8.5 parts starch, 0.05–0.4 parts choline, and 0.9–1.1 parts resazurin.

[0007] Preferably, the product comprises the following components by weight: 2.5 parts yeast extract, 1 part L-cysteine, 2 parts heme, 0.9 parts sodium chloride, 0.01 parts calcium chloride hexahydrate, 0.45 parts dipotassium hydrogen phosphate, 0.09 parts magnesium sulfate heptahydrate, 0.2 parts vitamin mixture, 8 parts starch, 0.2 parts choline, and 1 part resazurin.

[0008] Preferably, L-cysteine ​​is prepared as an L-cysteine ​​solution. The preparation of the L-cysteine ​​solution includes the following steps: (A01) Analytical grade L-cysteine ​​is added to distilled water (with oxidation removed) under nitrogen protection and stirred to dissolve. Analytical grade ensures high purity and low impurities of the raw material, avoiding the introduction of unknown chemicals that may interfere with microbial growth or subsequent detection. Distilled water (with oxidation removed) is used because dissolved oxygen in water is a major enemy of L-cysteine; removing oxygen from the water beforehand reduces the risk of L-cysteine ​​oxidation during preparation. By creating an oxygen-free, inert nitrogen gas environment above the solution, contact between air, especially oxygen, and the solution is physically isolated, ensuring that L-cysteine ​​remains in a reduced state to the maximum extent during dissolution, resulting in a solution. A high-purity, high-reducing-activity L-cysteine ​​initial solution provides a reliable foundation for subsequent steps; (A02) The pH of the solution from step (A01) is adjusted to 6.8–7.2 using NaOH solution to ensure that the stock solution does not cause drastic pH fluctuations when added to the main fermentation medium. The pH of human colon and most intestinal microbial culture media is within this range. If the L-cysteine ​​solution is strongly acidic, direct addition may cause local over-acidity, potentially damaging microorganisms or affecting the buffer system. In preparation for filtration and sterilization, the filter membrane performs best and has the best compatibility at a specific pH. A near-neutral pH is usually a safe choice. A bottle of L-cysteine ​​solution with a pH that matches the final application environment is obtained, avoiding adverse effects on the system caused by pH shocks.

[0009] (A03) The solution from step (A02) is filtered through a PES microporous membrane, and the resulting filtrate is the L-cysteine ​​solution. The PES polyethersulfone microporous membrane can physically trap microorganisms such as bacteria and fungi, removing any insoluble particulate impurities that may be introduced during the preparation process. The PES membrane has low protein adsorption, high flow rate, and good chemical compatibility, ensuring that the effective components of L-cysteine ​​are minimized during filtration, resulting in a bottle of sterile, clear L-cysteine ​​stock solution. All steps serve to protect the reducing activity of L-cysteine ​​and ensure the cleanliness and sterility of the solution, anticipating all major risks that may be encountered from preparation to use. Oxidation risks are avoided through nitrogen protection and deoxygenated water, pH shock risks are avoided through pH pre-adjustment, and microbial contamination risks are avoided through sterile filtration. Specific pH parameters and designated PES filtration methods ensure the consistency of reagent quality between different batches.

[0010] Preferably, the pH of the solution in step (A01) is adjusted to 6.9–7.1. More preferably, the pH of the solution in step (A01) is adjusted to 7. Precise control of the pH value ensures perfect pH compatibility with the main fermentation system to the greatest extent possible, avoiding any minor pH fluctuations.

[0011] Preferably, the dissolution container in step (A01) is a double-walled glass reaction flask. The double-walled glass reaction flask enables precise temperature control, while also being structurally robust, well-sealed, and easy to connect to nitrogen pipelines and stirring devices. This provides an ideal hardware platform for precise temperature control in the next step and ensures that the entire dissolution process takes place in a closed, inert environment.

[0012] Preferably, the dissolution temperature in step (A01) is 23°C to 27°C. More preferably, the dissolution temperature in step (A01) is 24°C to 26°C. Even more preferably, the dissolution temperature in step (A01) is 25°C. Precise control of the dissolution temperature can effectively control the dissolution rate; it can reduce chemical degradation, as low temperature helps to slow down any potential chemical degradation reactions; temperature affects solution viscosity and filtration speed, and constant temperature facilitates standardized operation, minimizing thermodynamic instability while ensuring rapid dissolution, and ensuring completely consistent preparation conditions for each batch.

[0013] Preferably, the stirring speed in step (A01) is 180 r / min to 200 r / min. More preferably, the stirring speed in step (A01) is 185 r / min to 195 r / min. Even more preferably, the stirring speed in step (A01) is 190 r / min. Precise control of the stirring speed ensures rapid and uniform dissolution of L-cysteine, avoiding excessively high or low local concentrations; it also allows for control of gas mass transfer. Under nitrogen protection, a moderate stirring speed promotes dissolution without generating excessive vortices due to vigorous stirring, thus avoiding increased contact between the solution and potentially residual oxygen. This results in a solution with uniform concentration and allows the mixing process to be completed in a controllable and repeatable manner.

[0014] Preferably, the concentration of the NaOH solution in step (A02) is 1 mol / L. Specifying a NaOH solution concentration of 1 mol / L allows for pH adjustment using a higher concentration of alkali solution; only a small volume needs to be added, without significantly altering the final volume and concentration of the L-cysteine ​​solution. A medium concentration of alkali solution facilitates finer pH adjustments, avoiding the problem of over-adjustment when using excessively concentrated alkali solutions, ensuring the accuracy of the working concentration of L-cysteine, and facilitating precise pH adjustment.

[0015] Preferably, the filtrate from step (A03) is stored in a brown sterile bottle for later use. The brown bottle avoids light exposure, especially ultraviolet-catalyzed oxidation reactions, and its connection with the filtration and sterilization step ensures that the storage container is also sterile.

[0016] Preferably, the pore size of the PES microporous filter membrane in step (A03) is 0.2 μm to 0.24 μm. More preferably, the pore size of the PES microporous filter membrane in step (A03) is 0.21 μm to 0.23 μm. Even more preferably, the pore size of the PES microporous filter membrane in step (A03) is 0.22 μm. Specifying the pore size of the PES filter membrane ensures the retention of all bacteria and fungi, providing absolute sterility assurance.

[0017] Preferably, the L-cysteine ​​solution in the brown sterile bottle is stored at -22°C to -18°C. More preferably, it is stored at -21°C to -19°C. Even more preferably, it is stored at -20°C. Low-temperature freezing can greatly inhibit all chemical and enzymatic reactions, maintain the stability of the reagent for a long time, and provide long-term and stable storage conditions for the prepared highly active reagent, making it convenient to prepare once and use it multiple times.

[0018] Preferably, before use, the frozen L-cysteine ​​solution is thawed naturally to room temperature, and an equal volume of FeCl3 solution is added to the L-cysteine ​​solution sample. The color change of the solution is observed. If a light green complex color is produced immediately, it indicates that L-cysteine ​​has been oxidized and the L-cysteine ​​solution needs to be reconstituted. If a light green complex color is not produced immediately, it indicates that L-cysteine ​​has not been oxidized and can continue to be used.

[0019] Preferably, the concentration of the FeCl3 solution is 0.01 mol / L. Reducing L-cysteine ​​can undergo a complexation reaction with ferric ions to form a light purple or light green complex. If L-cysteine ​​has been oxidized to cystine, it loses this reactivity. This emphasizes the immediacy of the color change, eliminating interference from other slow reactions. It provides a rapid, simple, and low-cost method to verify whether the reducing activity of the L-cysteine ​​solution remains effective before each use.

[0020] Preferably, the preparation of heme includes the following steps: (B01) taking defatted pig liver, cutting it into small pieces, adding it to a mixed solvent of ethanol and acetone, adjusting the pH to 1.8-2.2, stirring and extracting to obtain the extract; pig liver is a rich natural source of heme, and pre-defatting can remove most of the fat impurities, simplifying the subsequent purification steps and improving the extraction efficiency; the mixed solvent formed by ethanol and acetone can effectively destroy cell membranes and protein structures, dissociating and dissolving heme from the hemoglobin bound to it; adjusting the pH to strong acidity, under strong acidity conditions, can ensure that the iron in the heme molecule is in a high-iron state and makes it more stable to dissolve in organic solvents, while promoting the complete denaturation and decomposition of hemoglobin; thus achieving the effective release and preliminary dissolution of heme from liver tissue; (B02) taking the step After vacuum filtration of the extract from step (B01), the filtrate is collected. Physical separation is performed to remove insoluble tissue residues, denatured protein precipitates, and other solid impurities, resulting in a clear organic solution containing heme and other soluble components. (B03) The filtrate from step (B02) is distilled under reduced pressure under a nitrogen stream to remove the organic solvent, yielding a crude heme precipitate. Reduced pressure distillation lowers the boiling point, allowing ethanol and acetone to evaporate rapidly at a lower temperature, avoiding prolonged heating at high temperatures that could degrade heme. During the evaporation process, inert nitrogen is continuously introduced to prevent heme from being oxidized by oxygen in the air, while also helping to remove solvent vapors, thus recovering the crude heme and removing a large amount of organic solvent. (B04) The crude heme precipitate from step (B03) is dissolved in NaOH solution, and the pH is adjusted to 7.1–7.3. After standing, centrifuge and collect the supernatant; under weakly alkaline conditions, heme can form soluble ferric heme, thus separating it from lipids and other nonpolar impurities insoluble in alkaline water. A near-neutral, slightly alkaline environment is the suitable pH for heme dissolution, while avoiding damage to the heme porphyrin ring due to excessive alkalinity. Centrifugation further removes trace impurities that remain insoluble in alkaline solutions, achieving preliminary purification and separating heme from a large number of lipid-soluble impurities; (B05) Purify the supernatant from step (B04) through a Sephadex G-25 gel filtration column, using the fraction with the main peak at 398 nm as the pure product; Sephadex The G-25 gel filter column can completely separate heme with larger molecular weights from impurities such as salts, small peptides, and amino acids. 398nm is the characteristic absorption peak of the porphyrin ring of heme. By monitoring the absorbance at this wavelength, the fraction with the highest heme content can be accurately collected, ensuring the purity and concentration of the product and obtaining a high-purity heme solution. (B06) The purified product from step (B05) is purged with nitrogen to remove dissolved oxygen and then dispensed into brown glass bottles to obtain purified heme. Nitrogen purging removes dissolved oxygen from the purified aqueous solution, maximizing the delay of heme oxidation during storage. Brown bottles are stored away from light to prevent photocatalytic oxidation reactions. Dispensing facilitates each use and avoids product deterioration caused by repeated freeze-thaw cycles or opening of the main packaging. This provides long-term stable storage conditions for high-purity heme products, ensuring that they maintain high activity during use. The overall process followed the classic separation and purification logic of extraction → crude purification → refined purification → preservation. It comprehensively utilized various techniques, including solvent extraction, acid-base dissolution, centrifugation, vacuum distillation, and column chromatography. Each technique specifically addressed a particular purification problem. The 398nm absorption peak was used as the collection standard to ensure the chemical purity and consistency of the final product. Throughout the process, the oxidation of heme was carefully monitored, and its long-term effectiveness was guaranteed through nitrogen protection and light-proof storage. Continuous nitrogen purging prevented the self-oxidation of choline in air to form trimethylamine oxide (TMAO).

[0021] Preferably, the mass-to-volume ratio of defatted pork liver to the mixed solvent in step (B01) is 1:5 to 10. More preferably, the mass-to-volume ratio of defatted pork liver to the mixed solvent in step (B01) is 1:6 to 9. Even more preferably, the mass-to-volume ratio of defatted pork liver to the mixed solvent in step (B01) is 1:7 to 8. This specific feed-to-liquid ratio optimizes mass transfer efficiency. Sufficient solvent volume ensures that tissue fragments are fully submerged and permeated, providing a sufficient concentration gradient driving force for the diffusion of heme from the tissue to the solvent. Too low a ratio may lead to incomplete extraction, while too high a ratio wastes solvent and increases the burden on subsequent concentration steps.

[0022] Preferably, the volume ratio of ethanol to acetone in step (B01) is 1 to 2:1. Optimizing solvent polarity, acetone has strong polarity, which is beneficial for penetrating cell membranes and dehydration, while ethanol has slightly weaker polarity and better solubility for heme. The mixture of the two can produce a synergistic effect, resulting in higher extraction efficiency than a single solvent, and achieving the best balance between cell-damaging ability and heme-dissolving ability.

[0023] Preferably, the pH is adjusted to 1.9–2.1 in step (B01). More preferably, the pH is adjusted to 2 in step (B01). Precise pH control ensures the optimization of the reaction environment, which is crucial for ensuring complete denaturation of hemoglobin, stable heme iron in the Fe3+ state, and maximizing its solubility in the organic phase. Even slight pH fluctuations can affect the extraction rate. Precise pH control ensures that each batch of experiments achieves maximum and consistent extraction efficiency.

[0024] Preferably, the stirring in step (B01) is magnetic stirring. Magnetic stirring provides a gentle and uniform mixing, avoiding the excessive shear force or heat generation problems that may occur with mechanical stirring.

[0025] Preferably, the extraction temperature in step (B01) is 2°C to 4°C. More preferably, the extraction temperature in step (B01) is 2.5°C to 3.5°C. Even more preferably, the extraction temperature in step (B01) is 3°C. Low-temperature extraction can greatly inhibit the degradation of impurities, the growth of microorganisms, and the chemical degradation of heme itself during the extraction process. While ensuring thorough mixing, it maximizes the preservation of the natural structure and stability of heme and reduces the dissolution of impurities.

[0026] Preferably, the extraction time in step (B01) is 1 to 2 hours. More preferably, the extraction time in step (B01) is 1.5 hours. Precise control of the extraction time can balance the extraction rate and the dissolution of impurities. If the time is too short, the extraction will be insufficient; if the time is too long, it will increase the dissolution of impurities and increase the risk of heme degradation. The optimal balance point between extraction rate and product purity has been found.

[0027] Preferably, the vacuum filtration in step (B02) is performed within a Buchner funnel. The Buchner funnel, combined with filter paper, is a standard laboratory instrument for achieving rapid, large-scale solid-liquid separation. Vacuum filtration significantly improves filtration speed and efficiently separates large amounts of extract from tissue residue.

[0028] Preferably, the concentration of the NaOH solution in step (B04) is 0.1 mol / L. This concentration is appropriate, sufficient to dissolve heme to form ferrous heme, without causing damage due to excessive alkalinity.

[0029] Preferably, the pH is adjusted to 7.2 in step (B04). Precise control at a slightly alkaline pH is the optimal pH window for heme solubility.

[0030] Preferably, the settling time in step (B04) is 20 min to 30 min. More preferably, the settling time in step (B04) is 22 min to 28 min. Even more preferably, the settling time in step (B04) is 25 min. Sufficient time is given to allow the heme to completely dissolve and for insoluble impurities to fully aggregate and precipitate.

[0031] Preferably, the centrifugal speed in step (B04) is 6000 r / min to 8000 r / min. More preferably, the centrifugal speed in step (B04) is 6500 r / min to 7500 r / min. Even more preferably, the centrifugal speed in step (B04) is 7000 r / min. Providing sufficient centrifugal force and time ensures that tiny insoluble impurity particles are completely settled to the bottom of the tube.

[0032] Preferably, the centrifugation time in step (B04) is 8 to 10 minutes. More preferably, the centrifugation time in step (B04) is 9 minutes. This series of precise parameters works together to ensure maximum recovery of heme from the crude product and efficient removal of insoluble impurities, preparing high-quality samples for the next column chromatography step.

[0033] Preferably, the nitrogen purging time in step (B06) is 3 to 5 minutes. More preferably, the nitrogen purging time in step (B06) is 4 minutes. The precise control of the nitrogen purging time is achieved by physically purging dissolved oxygen in the water by blowing nitrogen into the liquid. If the time is too short, the deoxygenation will be incomplete; if the time is too long, the solution may evaporate and concentrate or generate aerosols.

[0034] Preferably, the vitamin mixture comprises biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine, wherein the mass ratio of biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine is 1–2:1–2:1–2:1–2:1–2. Biotin, as a coenzyme for carboxylases, participates in energy metabolism, fatty acid synthesis, and gluconeogenesis; cobalamin participates in methyl transfer, DNA synthesis, and erythropoiesis; para-aminobenzoic acid is part of the folic acid molecule and is a precursor for folic acid synthesis in some microorganisms; folic acid participates in carbon unit transfer and nucleic acid synthesis; pyridoxine is a coenzyme for transaminases and decarboxylases, participating in amino acid metabolism. This mixture covers multiple key pathways of basic microbial metabolism, including energy, amino acid, nucleic acid, and fatty acid metabolism, providing comprehensive support for various gut microbes with specific vitamin requirements. The defined mass ratio ensures that the various components provide sufficient and balanced amounts of vitamins in the culture medium, avoiding the limitation of single vitamin deficiency on microbial growth.

[0035] Preferably, the preparation of the vitamin mixture includes the following steps: (C01) Take appropriate amounts of biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine, add them to a mixed solvent of anhydrous ethanol and distilled water, and stir to dissolve under nitrogen protection. These five vitamins have different polarities; pure water cannot effectively dissolve hydrophobic vitamins like biotin, while pure ethanol may reduce the solubility of folic acid. Using a mixed solvent of ethanol and water provides a wider polarity range, ensuring that all components are fully dissolved. Folic acid and pyridoxine are very sensitive to oxygen and are easily oxidized and deactivated. Introducing nitrogen during the dissolution stage is a key measure to prevent oxidative degradation from the source, resulting in a clear, homogeneous initial vitamin solution with unoxidized active ingredients. (C02) Adjust the pH of the solution from step (C01) to 6.6–7, and add an appropriate amount of ascorbic acid before mixing. Strong acid or strong alkaline conditions will accelerate the dissolution of most vitamins. The hydrolysis and chemical degradation of vitamins, maintaining them within a certain pH range, is a safe choice to preserve their chemical stability and is also compatible with the pH environment of the fermentation system. Ascorbic acid, a powerful antioxidant, plays a "guardian" role, preferentially reacting with trace amounts of residual oxygen in the solution, thereby protecting other more vulnerable vitamins such as folic acid and pyridoxine from oxidation. This creates a chemically mild solution environment with antioxidant buffering capacity, greatly enhancing the overall stability of the vitamin mixture. Ascorbic acid acts as an antioxidant stabilizer to delay the oxidation of folic acid and pyridoxine. (CO3) The mixture from step (CO2) is filtered through a PES microporous membrane, and the resulting filtrate is the vitamin mixture. The PES membrane is used to remove bacteria and fungi, preventing the vitamin solution from deteriorating during storage and preventing contamination of the main fermentation system. This ensures a completely clear and sterile solution, provides sterility assurance, extends shelf life, and ensures product clarity. The five selected vitamins form a basic vitamin package that supports the growth of a wide range of microorganisms; a triple stabilization system consisting of a nitrogen-inert environment, a mild chemical environment, and ascorbic acid chemical protection is constructed to comprehensively combat the main factors that cause vitamin deterioration—oxidation and hydrolysis; and sterile filtration ensures that pure growth factors are introduced, rather than sources of contamination.

[0036] Preferably, in step (C01), the volume ratio of anhydrous ethanol to distilled water is 1:1 to 2. This solvent volume ratio creates a moderately polar environment that can effectively dissolve both hydrophilic vitamins, such as cobalamin, and hydrophobic vitamins, such as biotin, ensuring that all components are completely dissolved and preventing any precipitation. This results in a clear, homogeneous vitamin solution, laying the foundation for subsequent precise addition and filtration.

[0037] Preferably, the mass-to-volume ratio of the five active ingredients to the mixed solvent in step (C01) is 0.15–0.25:100. More preferably, the mass-to-volume ratio of the five active ingredients to the mixed solvent in step (C01) is 0.18–0.22:100. Even more preferably, the mass-to-volume ratio of the five active ingredients to the mixed solvent in step (C01) is 0.2:100. This solid-liquid ratio ensures that the vitamin mixture has a suitable working concentration, avoiding the need to add excessive volumes that would affect the fermentation system due to excessively low concentrations, and also avoiding the increased risk of precipitation during storage or at low temperatures due to excessively high concentrations, thus providing a stable, easy-to-use, and calculable stock solution concentration.

[0038] Preferably, the stirring speed in step (C01) is 150 r / min to 200 r / min. More preferably, the stirring speed in step (C01) is 160 r / min to 190 r / min. Even more preferably, the stirring speed in step (C01) is 170 r / min to 180 r / min. The stirring speed is sufficient to allow dissolution to proceed quickly without generating excessive foam or causing solution splashing due to excessive vigor, thus reducing contact with air.

[0039] Preferably, the dissolution time in step (C01) is 10 min to 15 min. More preferably, the dissolution time in step (C01) is 11 min to 14 min. Even more preferably, the dissolution time in step (C01) is 12 min to 13 min. Precise time control ensures that the degree of dissolution of each batch of product is completely consistent, avoiding batch-to-batch differences caused by intuition, achieving repeatability and maximizing efficiency in the dissolution process, while minimizing the risks of mechanical shearing and oxidation.

[0040] Preferably, in step (CO2), the pH of the solution from step (CO1) is adjusted to 6.7–6.9. More preferably, in step (CO2), the pH of the solution from step (CO1) is adjusted to 6.8. Precise pH control minimizes exposure to strong acids or bases, protecting all five vitamins and the ascorbic acid antioxidant, minimizing their hydrolysis and degradation rates, and providing a stable chemical environment for the entire vitamin mixture.

[0041] Preferably, the concentration of ascorbic acid in step (CO2) is 0.1%. Specifying the ascorbic acid concentration provides sufficient but not excessive antioxidant protection, limiting the concentration to effectively remove dissolved oxygen and any subsequent trace amounts of oxygen that may invade the solution, providing strong sacrificial protection for other vitamins, establishing a robust reducing environment, and significantly extending the shelf life of the vitamin mixture.

[0042] Preferably, the filtrate from step (C03) is packaged into brown glass ampoules for later use. Brown glass provides the highest level of light protection, preventing photocatalytic oxidation reactions. Unlike ordinary screw-top bottles, ampoules form a completely airtight and liquid-tight seal after fusion sealing, completely isolating oxygen and providing the best physical barrier. Repackaging into ampoules facilitates single use, avoids solution contamination and repeated oxygen intrusion caused by repeated opening and use, and provides the best long-term storage stability conditions currently achievable.

[0043] Preferably, the pore size of the PES microporous filter membrane in step (C03) is 0.2 μm to 0.24 μm. More preferably, the pore size of the PES microporous filter membrane in step (C03) is 0.21 μm to 0.23 μm. Even more preferably, the pore size of the PES microporous filter membrane in step (C03) is 0.22 μm; specifying the pore size of the PES filter membrane ensures sterility without any risk.

[0044] Preferably, the preparation of choline includes the following steps: (D01) using analytical grade choline chloride C5H 14 C l NO, dissolved in deoxidized distilled water, magnetically stirred under nitrogen protection until clear; analytical grade choline chloride, specifying a clear chemical substance to ensure the use of high-purity raw materials and avoid interference from impurities. Choline chloride is a stable salt form of choline, easier to store and use than free choline; deoxidized distilled water, although choline itself is not easily oxidized, using deoxygenated water is standard practice in the preparation of reagents for the entire fermentation system, aiming to maintain the consistency of the inert environment of the entire preparation system and prevent the introduction of any oxidizing substances that may affect other sensitive components, such as subsequently added vitamins or microorganisms; nitrogen protection provides an inert environment for the dissolution process, preventing slight pH fluctuations caused by the dissolution of carbon dioxide in the air, and further eliminating any potential oxidation risks; magnetically stirred until clear. Clarification provides a gentle and effective mixing process, ensuring complete dissolution and yielding a homogeneous solution, resulting in a pure, clear, and anaerobic choline chloride stock solution; (D02) Adjust the pH of the solution from step (D01) to 6.9–7.1 and filter it through a PES microfiltration membrane. The resulting filtrate is the choline solution; the choline chloride solution is slightly acidic. Precisely adjusting its pH to a neutral range that is essentially consistent with the human colon and fermentation medium avoids pH shock when added to the main fermentation system, thus protecting pH-sensitive intestinal microorganisms; filtration through a PES microfiltration membrane removes bacteria, fungi, and other microorganisms that may have been introduced during preparation, preventing contamination of the nutrient-rich fermentation system, resulting in a bottle of pH-compatible, sterile choline working solution that can be directly and safely used in fermentation experiments.

[0045] Preferably, the magnetic stirring speed in step (D01) is 200 r / min to 250 r / min. More preferably, the magnetic stirring speed in step (D01) is 210 r / min to 240 r / min. Even more preferably, the magnetic stirring speed in step (D01) is 220 r / min to 230 r / min. Precise control of the magnetic stirring speed optimizes dissolution efficiency. Choline chloride is readily soluble, and a moderate stirring speed ensures rapid and uniform dissolution, avoiding the formation of concentration gradients or powder floating. This speed range ensures efficiency while also preventing excessive vortices and bubbles generated by vigorous stirring, thereby reducing the contact area with potentially residual oxygen and achieving a rapid, uniform, and controlled dissolution process.

[0046] Preferably, the stirring temperature in step (D01) is 19°C to 21°C. More preferably, the stirring temperature in step (D01) is 20°C. Precise control of the stirring temperature can eliminate the potential impact of temperature fluctuations on the dissolution rate and any trace degradation reactions, making the dissolution process a completely standardized step and ensuring high consistency between batches.

[0047] Preferably, the filtrate from step (D02) is stored in a brown glass bottle for later use. Using brown glass bottles for storage, protecting the solution from light, maintains consistency with the packaging materials of the entire reagent preparation system, such as L-cysteine ​​and vitamin mixtures, avoids confusion, prevents any potential or unknown photocatalytic reactions, provides an extra layer of protection, and achieves packaging standardization.

[0048] Preferably, in step (D02), a 0.1 mol / L NaOH solution is used to adjust the pH. Specifying the use of a 0.1 mol / L NaOH solution for pH adjustment achieves precise and controllable pH control. Using this moderately concentrated alkali solution avoids the problem of over-adjusting the pH due to excessively high concentrations, facilitating fine-tuning and easily and accurately adjusting the pH to the target value.

[0049] Preferably, in step (D02), the pH of the solution from step (D01) is adjusted to 7.

[0050] Preferably, the pore size of the PES microporous filter membrane in step (D02) is 0.2 μm to 0.24 μm. More preferably, the pore size of the PES microporous filter membrane in step (D02) is 0.21 μm to 0.23 μm. Even more preferably, the pore size of the PES microporous filter membrane in step (D02) is 0.22 μm. Specifying the pore size of the PES filter membrane ensures sterility without any risk.

[0051] Preferably, before use, the resamarinzane is compared with a 0.01 mol / L Na₂S₂O₄ standard reducing agent for colorimetric sensitivity calibration. Resamarinzane is blue or pink in its oxidized state and becomes colorless in its completely reduced state. Sodium dithionite is a powerful reducing agent that can instantly and completely reduce resamarinzane to a colorless state. The calibration method involves taking a small amount of resamarinzane stock solution, adding sufficient Na₂S₂O₄ standard solution, and observing whether it immediately and completely fades. This confirms that the batch of resamarinzane reagent used is effective and can be reduced. If the fading is incomplete or slow, it indicates that the reagent may have degraded and become ineffective. This provides researchers with an absolute colorless endpoint visual standard for determining whether the fermentation system has reached a completely anaerobic state.

[0052] The second technical solution of the present invention: a method for establishing an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA generation, comprising the following steps,

[0053] (S01) Take appropriate amounts of yeast extract, L-cysteine, heme, sodium chloride, calcium chloride hexahydrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, vitamin mixture, starch, choline, and resazurite, and mix them. Deoxygenate under nitrogen protection to achieve an anaerobic state. Combine all carefully designed and prepared nutrients, substrates, and indicators to form the basic fermentation medium. Before entering the anaerobic workstation, remove dissolved oxygen from the medium solution by purging nitrogen, and use the reducing effect of L-cysteine ​​to initially establish a reducing environment. This greatly reduces the workload of the subsequent anaerobic workstation and can achieve a strictly anaerobic state more quickly, thus preparing a complete medium with preliminarily established anaerobic conditions. (S0) 2) A mixture of nitrogen and carbon dioxide gas is introduced into the anaerobic workstation to establish a simulated intestinal anaerobic environment. The mixture from step (S01) is placed in the anaerobic workstation to form a closed system, maintaining constant temperature and pressure. The nitrogen gas is used to maintain the anaerobic environment, while the CO2 content is typically 5%–10% to maintain the pH stability of the culture medium, form a carbonate buffer pair, and simulate the high CO2 environment (approximately 10%) produced by microbial metabolism and blood diffusion in the colon. Constant temperature and pressure are maintained; the temperature is typically 37°C, simulating the core temperature of the human body, which is the optimal growth temperature for microorganisms. Maintaining a slight positive pressure prevents external oxygen-containing air from infiltrating the workstation, creating a stable environment where physical and chemical conditions highly mimic those of the human colon. A stable and controllable in vitro environment; (S03) Collect fecal samples from patients with cardiovascular diseases, anaerobic homogenize and centrifuge to remove impurities, and obtain fecal inoculum; Collect fecal samples from patients with cardiovascular diseases. The composition of the intestinal flora and the ability to generate TMA in patients with cardiovascular diseases are different from those in healthy people. Using the flora of a specific population can more effectively screen for substances that regulate TMA generation under pathological conditions; Anaerobic homogenization is to uniformly suspend the fecal sample in a buffer solution under anaerobic conditions to ensure the survival of all microorganisms. Centrifugation to remove impurities is usually done at low speed to remove undigested food residues and large particles, while retaining the supernatant rich in microorganisms, resulting in an inoculum that represents the intestinal microbial community of a specific population, is active, and has been free of physical impurities; (S04) The fecal inoculum from step (S03) was added to the mixture from step (S02) for anaerobic culture to form an in vitro TMA fermentation system. The active microbial community from step (S03) was introduced into the simulated gut environment from step (S02) to initiate the fermentation process, thus establishing an operable fermentation system containing a complete microbial community and a simulated gut environment. (S05) Samples of the TMA in vitro fermentation system from step (S04) were periodically taken to detect the concentrations of TMA and short-chain fatty acids in the samples, and the color change of resazurin was monitored in real time to verify the anaerobic stability and metabolic activity of the TMA in vitro fermentation system. TMA is the primary readout data for the screening work, and the effect of any intervention is evaluated by changes in TMA concentration.SCFAs serve as key indicators of system health and function. They are the main beneficial metabolites produced by gut microbiota fermentation of carbohydrates. Monitoring SCFAs can verify whether the overall metabolic activity of the microbial community is normal, avoiding false positive results caused by a decrease in TMA due to system imbalance. Real-time monitoring of the color change of resplendent zygosperm and continuous visual monitoring of anaerobic stability ensure that the system remains in a strictly anaerobic state throughout the entire culture period. This enables multiple, real-time validations of the fermentation system, confirming the core target of TMA generation, assessing the overall metabolic health of the microbial community (SCFAs), and continuously monitoring environmental stability, thus forming a robust quality control closed loop. From culture medium preparation, environmental simulation, and microbial inoculation to final efficacy verification, a seamless workflow was established. Each step revolved around the core objective of rapid, targeted, and in vitro screening of substances that regulate TMA generation. Multiple indicators—rezazulen and SCFAs—were used to validate the system, ensuring the reliability and validity of the experimental data. The selection of feces from specific disease populations as inoculum significantly improved the efficiency and targeting of the screening. The establishment method of this in vitro fermentation system is highly rational, scientific, and highly operable. It successfully integrates all the meticulous component design and preparation processes into a powerful research tool, providing an ideal platform for the rapid and reliable screening of substances that regulate TMA generation, such as drugs, prebiotics, and functional foods.

[0054] Preferably, the culture temperature in step (S04) is 36°C to 38°C. More preferably, the culture temperature in step (S04) is 36.5°C to 37.5°C. Even more preferably, the culture temperature in step (S04) is 37°C.

[0055] Preferably, the culture time in step (S04) is 12h to 36h. More preferably, the culture time in step (S04) is 18h to 30h. Even more preferably, the culture time in step (S04) is 20h to 24h.

[0056] Preferably, step (S01) is as follows: (S01-1) Under aseptic conditions, distilled water (after removing oxides) is added to appropriate amounts of yeast extract, L-cysteine, heme, sodium chloride, calcium chloride hexahydrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, a vitamin mixture, starch, choline, and resazurite, and the mixture is stirred and mixed; (S01-2) Immediately under nitrogen protection, nitrogen gas is continuously passed through the solution after stirring and mixing in step (S01-1) to remove dissolved oxygen from the system. This achieves the separation and optimization of physical mixing and chemical deoxygenation. All components are first dissolved and homogenized under aseptic conditions, and then deoxygenation is performed centrally. This avoids operations that may introduce oxygen, such as adding solids during deoxygenation, making the entire process logic clearer, the goal of each step more singular, and facilitating operation and quality control.

[0057] Preferably, the mixing vessel in step (S01-1) is a double-walled constant-temperature reactor. Using a double-walled constant-temperature reactor as the mixing vessel, connected to a circulating water bath, can keep the temperature during the mixing process constant within an ideal range, avoiding the impact of heat generated by stirring or fluctuations in ambient temperature on heat-sensitive components such as vitamins and heme. The reactor itself is designed for chemical reactions, has good sealing properties, and is easy to connect to nitrogen pipelines and stirring devices, making it an ideal platform for such precision operations. It provides a stable, professional, and functionally integrated operating platform for the preparation of culture media.

[0058] Preferably, the stirring speed in step (S01-1) is 180 r / min to 200 r / min. More preferably, the stirring speed in step (S01-1) is 185 r / min to 195 r / min. Even more preferably, the stirring speed in step (S01-1) is 190 r / min. Precise control of the stirring speed ensures that all components, especially powdered yeast extract, starch, etc., can be quickly and uniformly dissolved and dispersed to form a homogeneous solution; a moderate speed can provide effective mixing without generating excessive foam and vortex, obtaining a culture medium solution with uniform component concentration, and creating favorable conditions for the next step of deoxygenation.

[0059] Preferably, the continuous nitrogen aeration time in step (S01-2) is 10 min to 30 min. More preferably, the continuous nitrogen aeration time in step (S01-2) is 15 min to 25 min. Even more preferably, the continuous nitrogen aeration time in step (S01-2) is 20 min. Precise control of the continuous nitrogen aeration time ensures thorough deoxygenation. The nitrogen aeration time needs to be long enough to effectively displace the dissolved oxygen in the solution through physical bubbling. The defined time range is a time required to stably reduce dissolved oxygen to an extremely low level, determined by measuring dissolved oxygen levels. This ensures that the culture medium is in a highly anaerobic state before entering the anaerobic workstation.

[0060] Preferably, in step (S01-2), the change of resplenium from blue to light pink is used as a visual indicator for establishing the anaerobic state. Using the color change of resplenium as a visual indicator, from blue to light pink, provides a real-time and intuitive standard for judging the anaerobic state. Resplenium is blue in the oxidized state, and as the oxygen partial pressure decreases, it gradually turns pink and finally becomes colorless when there is no oxygen. Light pink is the endpoint. Pursuing complete colorlessness may require an extremely long deoxygenation time, while light pink indicates that the oxygen partial pressure in the solution has dropped to a very low level, which is sufficient to meet the needs of most strict anaerobic bacteria. At the same time, it is also an easily identifiable and repeatable visual node, providing the operator with a clear and reliable endpoint judgment basis, making the deoxygenation process standardized rather than based on intuition, and greatly improving the standardization and success rate of the operation. By specifying the container, stirring speed, and deoxygenation time, the consistency of the operation process was ensured. The introduction of the objective visual indicator of the color change of azure cyanide transformed a complex anaerobic chemical state into a simple and intuitive qualitative judgment, greatly reducing the difficulty of operation and human error. The choice of light pink instead of completely colorless as the endpoint optimized the operation time while ensuring the anaerobic effect, reflecting the principle of efficiency.

[0061] Preferably, step (S02) involves transferring the deoxygenation system from step (S01) into an anaerobic workstation, and continuously purging the reaction space of the anaerobic workstation with a mixture of nitrogen and carbon dioxide gas to maintain constant total pressure and temperature. Continuous purging with the mixed gas and maintaining constant pressure and temperature allows the dynamic airflow to continuously remove trace amounts of oxygen that may seep in from the workstation's seals or operating ports, and to carry away gases produced by microbial respiration, such as hydrogen. This maintains the anaerobic purity of the environment and a positive pressure slightly higher than standard atmospheric pressure, which is the most effective physical barrier to prevent external air infiltration. This provides the optimal growth temperature for microorganisms and creates a stable, controllable, and continuously renewing anaerobic environment.

[0062] Preferably, the volume ratio of nitrogen to carbon dioxide is 6-8:2. More preferably, the volume ratio is 6.5-7.5:2. Even more preferably, the volume ratio is 7:2. This nitrogen-to-carbon dioxide volume ratio simulates the gaseous environment of the colon. The gases in the human colon are mainly composed of N2, CO2, H2, and CH4, with CO2 accounting for more than 10%. High concentrations of CO2 dissolved in the culture medium form a carbonate / bicarbonate buffer pair, which is the most effective and physiologically sound buffer system for maintaining the pH of the culture medium within the neutral range. This effectively neutralizes the large amounts of short-chain fatty acids produced by microbial metabolism, preventing excessive acidification of the system. It provides both an anaerobic environment and a powerful, self-balancing pH buffer system.

[0063] Preferably, the total pressure is maintained at standard atmospheric pressure. Maintaining the total pressure at standard atmospheric pressure provides a stable physical environment without additional pressure. A slight positive pressure, such as 5% to 10% above atmospheric pressure, is used to prevent leakage, but the overall pressure is maintained at a normal level. This avoids unknown stress on microbial cells caused by high or low pressure, and provides a standard and safe culture pressure condition.

[0064] Preferably, the maintained temperature is between 36°C and 38°C. More preferably, the maintained temperature is between 36.5°C and 37.5°C. Even more preferably, the maintained temperature is 37°C. This precisely simulates the core temperature of the human body, which is the optimal growth temperature for gut microbiota derived from the human body, ensuring that the microorganisms can grow and metabolize at the highest efficiency.

[0065] Preferably, the deoxygenation system is added to a pressure-resistant glass fermentation flask, and the flask is sealed after controlling the gas-liquid ratio within the flask to be 1.5–2:1. Controlling the gas-liquid ratio within the pressure-resistant glass fermentation flask provides sufficient gas space. Sufficient gas phase space allows gas accumulation without causing a rapid increase in internal pressure, preventing flask explosion or seal failure. An appropriate gas-liquid ratio ensures that the culture medium has sufficient surface area for gas exchange with the gas phase, maintaining dynamic equilibrium in the system. This provides space for the gases produced by microorganisms during fermentation, avoiding liquid overflow or pressure runaway due to gas production. Within a single fermentation cycle, this maintains the physical stability within the fermentation flask, ensuring experimental safety and promoting equilibrium between the gas and liquid phases.

[0066] Preferably, the gas-liquid ratio inside the pressure-resistant glass fermentation flask is 1.6–1.9:1. More preferably, the gas-liquid ratio inside the pressure-resistant glass fermentation flask is 1.7–1.8:1. The combination of physiological simulation and engineering control not only considers the simulated gut but also the engineering requirements of the in vitro system. Through continuous purging and setting the gas-liquid ratio, a dynamic equilibrium system capable of responding to microbial metabolic activities, acid production, and gas production, is established, rather than a static system. The set gas-liquid ratio ensures the safe and stable operation of the long-term culture process.

[0067] Preferably, step (S03) involves: (S03-1) taking a fecal sample from a cardiovascular disease patient and mixing it with pre-deoxygenated sterile saline under anaerobic conditions in an anaerobic workbench; (S03-2) homogenizing the mixture from step (S03-1) and centrifuging to remove residue to obtain the fecal inoculation solution. This process optimization, by first initially mixing the feces and saline under anaerobic conditions to facilitate processing, followed by mechanical homogenization, avoids harsh mechanical processing in a dry state, making the operation smoother and potentially reducing mechanical damage to microorganisms.

[0068] Preferably, the fecal sample in step (S03-1) is processed within 1 to 2 hours. More preferably, the fecal sample in step (S03-1) is processed within 1.5 hours. This maximizes the preservation of the activity and composition of the microbial community. After feces leave the human body, drastic environmental changes such as temperature, hypoxia, and pH fluctuations rapidly kill oxygen-sensitive strict anaerobic bacteria and alter the metabolic activity of the microbial community. Shortening the processing time is crucial to ensuring the authenticity of the microbial community, ensuring that the microbial community inoculated into the fermentation system represents its original state in the host's intestine to the greatest extent possible, and reducing bias caused by sample processing delays.

[0069] Preferably, the mass-to-volume ratio of fecal sample to sterile saline in step (S03-1) is 1:4-5. More preferably, the mass-to-volume ratio of fecal sample to sterile saline in step (S03-1) is 1:4.5. This mass-to-volume ratio of feces to sterile saline aims to prepare a fecal suspension of suitable concentration. If the ratio is too low, the suspension will be too viscous, which is not conducive to homogenization and subsequent centrifugation to remove impurities, and may also lead to an excessively high inoculum concentration. If the ratio is too high, the suspension will be too diluted, which may lead to an excessively low microbial concentration, affecting the start-up speed of subsequent fermentation. The goal is to obtain a fecal suspension with suitable viscosity, standardized microbial concentration, and ease of subsequent operation.

[0070] Preferably, the homogenization time in step (S03-2) is 2 to 3 minutes. More preferably, the centrifugation time in step (S03-2) is 1 to 2 minutes. The homogenization time ensures that the fecal clumps are fully broken up, and the microorganisms are effectively released into the solution to form a uniform suspension. Too short a time results in inhomogeneity, while too long a time may damage the microorganisms due to heat generation or mechanical shear force. The purpose of limiting the centrifugation time is to gently settle large molecular impurities such as food residues and undigested particles, while retaining the desired bacterial cells in the supernatant. This yields a clear inoculum solution rich in active microorganisms and free of physical impurities, preventing impurities from becoming uncontrollable carbon sources or interfering with subsequent detection in the fermentation system.

[0071] Preferably, step (S04) is as follows: (S04-1) the mixture system from step (S02) is loaded into a pressure-resistant glass fermentation bottle and inoculated with the fecal inoculum from step (S03); (S04-2) immediately after inoculation in step (S04-1), nitrogen is used to replace the space inside the bottle to an anaerobic state.

[0072] (S04-3) Place the bottle from step (S04-2) in a constant temperature anaerobic incubator for culture to form a TMA in vitro fermentation system. Step (S04) is broken down into inoculation, replacement, and culture to achieve clarity and optimization of the process. The inoculation operation is separated from the final deoxygenation of the bottle space, ensuring that any trace oxygen that may be introduced during the brief process of opening the bottle for inoculation can be immediately removed. Then, closed culture is carried out, forming a rigorous closed loop of preparation-inoculation-deoxygenation-culture, which maximizes the protection of the anaerobic state at the start of the culture.

[0073] Preferably, the concentration of the fecal inoculum in step (S04-1) is 10%. A sufficiently high inoculum ensures rapid fermentation initiation, allowing microorganisms to quickly occupy their ecological niche, inhibiting the growth of potential contaminants, and ensuring sufficient microbial biomass to produce detectable metabolites. Excessive inoculum, such as 20%, may introduce too much fecal impurity or make the initial microbial community too close to its original state, hindering the observation of dynamic changes in the microbial community. This provides a standardized starting point for achieving a balance between fermentation initiation speed, metabolite yield, and experimental reproducibility.

[0074] Preferably, the time for purging the space inside the bottle with nitrogen in step (S04-2) is 2 to 3 minutes. This eliminates the oxygen introduced during the inoculation process. During the brief period of adding the inoculum by opening the bottle, the anaerobic environment inside the bottle can be invaded by air. Quickly purging the space at the bottle opening with a nitrogen stream for 2 to 3 minutes can effectively replace this oxygen, restoring a strictly anaerobic environment. This ensures that the headspace of the fermentation bottle is anaerobic at the beginning of the culture, protecting the newly inoculated, oxygen-sensitive intestinal microorganisms.

[0075] Preferably, the culture temperature in the anaerobic incubator in step (S04-3) is 36℃~38℃. More preferably, the culture temperature in the anaerobic incubator in step (S04-3) is 36.5℃~37.5℃. Even more preferably, the culture temperature in the anaerobic incubator in step (S04-3) is 37℃. This precisely simulates the core temperature of the human body, providing the optimal growth and metabolic temperature for human-derived gut microbiota, ensuring that microbial metabolic activities proceed at physiologically relevant rates, and making the rate and level of TMA generation biologically significant.

[0076] Preferably, the culture time in the constant temperature anaerobic incubator in step (S04-3) is 12h to 36h; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 18h to 30h; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 20h to 24h; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 22h. The limitation of isothermal incubation time is intended to capture the active metabolic phase, specifically the period from the end of the rapid logarithmic growth phase to the early stationary phase of microorganisms. During this time, TMA production should be at a high and easily detectable level to avoid over-fermentation. If the incubation time is too long, the nutrients in the culture medium may be depleted, metabolic waste may accumulate, and the pH may change significantly, leading to the decline of the microbial community or a change in metabolic pattern. TMA may be further transformed or degraded, complicating the results. A cycle of about one day is very suitable for high-throughput preliminary screening of drugs or functional components. Fermentation is terminated and samples are taken within an optimized time window, and the TMA concentration measured at this time can sensitively reflect the differences between different treatment groups.

[0077] Preferably, step (S05) is as follows: (S05-1) After fermentation in step (S04) for 12h to 36h, take a sample, centrifuge, and collect the supernatant.

[0078] (S05-2) The concentrations of choline, TMA, and short-chain fatty acids in the supernatant of step (S05-1) were detected using LC-MS / MS technology. If the color change of resazurin remained between light pink and colorless, it indicated that the system was in a stable anaerobic state. Optimizing the sampling time point is crucial to capturing the kinetic peak or plateau phase of TMA generation. Microbial metabolism is a dynamic process; the concentration of TMA initially increases over time, then stabilizes after reaching a peak or is further metabolized. Sampling too early may result in insufficient TMA accumulation before the microorganisms have entered their active metabolic phase, leading to insignificant concentration differences and difficulty in distinguishing the effects of different treatment groups. Sampling too late may result in the microorganisms entering their decline phase, depleting nutrients, and altering their metabolic patterns, potentially allowing other microorganisms to utilize TMA, leading to a decrease in concentration, which is also unfavorable for screening. Setting a sampling time window within the core culture period within a defined time range can capture the moment when TMA accumulation is most sufficient and the differences are most obvious, thus most sensitively reflecting the effect of the regulatory substances. LC-MS / MS technology can simultaneously and accurately detect and... This method quantifies multiple target compounds, such as choline, TMA, and various short-chain fatty acids, that may be present at very low concentrations in complex biological matrices. Combining liquid chromatography (LC) separation with mass spectrometry (MS) for qualitative and quantitative analysis effectively distinguishes structural analogs, avoiding interference from other metabolites and providing the most reliable and authoritative endpoint data for the entire screening experiment. Using resazurin color as a continuous indicator of anaerobic stability provides visualized quality control throughout the entire process. This not only determines the anaerobic state at the beginning but also continuously monitors the integrity of the system throughout the cultivation process. If the resazurin color remains between light pink and colorless throughout the cultivation period, it proves that the anaerobic environment is stable and the experimental data is valid. If a sample bottle shows blue or dark pink, it indicates that the system may be leaking, and the data should be discarded. This adds an important filter to the reliability of the data.

[0079] Preferably, after fermentation for 18-30 hours in step (S05-1), samples are taken and centrifuged. More preferably, after fermentation for 20-24 hours in step (S05-1), samples are taken and centrifuged.

[0080] Preferably, the sampling volume in step (S05-1) is 1 mL to 2 mL. This sampling volume balances detection requirements with system stability, meeting the detection criteria. LC-MS / MS technology has extremely high sensitivity, and 1 mL to 2 mL of sample, after appropriate processing, is sufficient for accurate quantification. Excessive sampling volume would significantly alter the total volume of the fermentation system, affecting nutrient concentration and total microbial count, and interfering with subsequent fermentation kinetics. 1 mL to 2 mL is a non-destructive or minimally destructive sampling volume with negligible impact on the system. While ensuring the reliability of data analysis, it maximizes the preservation of the original state of the fermentation system, allowing it to continue culturing or for subsequent sampling.

[0081] Preferably, the centrifugation time in step (S05-1) is 8 to 10 minutes. This centrifugation time allows for thorough solid-liquid separation, removing microbial cells and other insoluble substances from the sample, resulting in a clear supernatant for analysis. Sufficiently long centrifugation time ensures all bacterial cells are firmly deposited at the bottom of the tube, yielding a completely clear supernatant. This prevents cells or particulate matter from entering the sophisticated analytical instrument, clogging the tubing or column, and ensures that the concentration of extracellular metabolites is detected. This completes the rapid targeted screening process. Optimized sampling time points ensure the capture of the most biologically significant and statistically resolvable data. The use of cutting-edge LC-MS / MS technology guarantees the quality of the endpoint data, making targeted screening truly effective. The continuous indication from the LC-MS / MS transforms the abstract concept of environmental control into a simple, continuous monitoring indicator, greatly enhancing the reliability of the experimental results.

[0082] The third technical solution of the present invention: the application of an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA generation, wherein the application is any one of the following: (1) for functional screening studies of food-medicine homologous components and Chinese herbal extracts; the system simplifies the complex in vivo environment into a controllable in vitro system, which can simultaneously test dozens or even hundreds of different food-medicine homologous components or Chinese herbal extracts; combined with rapid culture and precise detection by LC-MS / MS, quantitative data on the inhibition of TMA generation by candidate substances can be obtained in a very short time, and its effectiveness can be quickly judged; the core readout index of the system is the TMA concentration, which directly serves the clear target of regulating TMA generation, avoiding the problem of target ambiguity in broad screening; compared with expensive animal experiments or lengthy human trials, the system is low in cost and has no ethical restrictions, making it very suitable for large-scale, preliminary functional screening; it can quickly, economically and efficiently select lead compounds or extracts with the potential to inhibit TMA generation from a large amount of natural product resources, providing a basis for the subsequent in-depth development of functional foods or drugs. Provides a robust candidate library; (2) Used to verify the specific regulatory effect of the screened active ingredients on the structure and function of the gut microbiota; By extracting microbial DNA after fermentation and performing 16S rRNA sequencing, it is possible to analyze whether the active ingredient broadly inhibits all microbiota or specifically enriches beneficial bacteria and inhibits known TMA-producing bacteria; By metagenomic sequencing or targeted metabolomics, it is possible to analyze its effect on the gene function of the entire microbiota or detect changes in other key metabolites besides TMA, such as SCFAs and secondary bile acids; If a component reduces TMA but also significantly reduces all SCFAs and causes a sharp decline in microbiota diversity, then its effect may be that of a non-specific antimicrobial agent rather than an ideal regulator. This application can identify and exclude such substances; Provides multidimensional mechanistic evidence for the screened active ingredients, elucidates the specificity and safety of their effects, and thus selects high-quality candidates that can precisely reshape the microbiota and directionally regulate metabolism, i.e., reduce TMA without affecting beneficial SCFAs. The two applications form a complementary and progressively in-depth research strategy. Application (1) is responsible for quickly scanning a wide area to discover targets, while application (2) is responsible for conducting in-depth analysis of the discovered targets, confirming their value, and formulating specific strategies.

[0083] Preferably, the functional screening study for food-medicine homology components and traditional Chinese medicine extracts involves (E01) adding candidate active substances, including astragaloside A, ginsenosides, codonopsis polysaccharides, lentinan, and / or auricularia polysaccharides, to an in vitro fermentation system and co-fermenting them with fecal inoculum from cardiovascular disease patients. The selected candidate substances cover the two major categories of food-medicine homology components: saponins such as astragaloside A and ginsenosides, and polysaccharides such as codonopsis polysaccharides, lentinan, and auricularia polysaccharides. These two categories of substances have different chemical structures and potential mechanisms of action, resulting in a broad and comparable screening outcome. These components are all derived from well-known tonic Chinese medicines or edible fungi, traditionally used to enhance bodily functions. Modern research shows that most of them are difficult for the human body to absorb directly, and one of their important mechanisms of action is the regulation of intestinal flora. Therefore, selecting them as screening targets has a solid theoretical and practical foundation; it allows researchers to test single or combined components according to actual conditions, increasing the flexibility of experimental design; it provides a list of candidate substances with high relevance and scientific value, making the screening research objectives clear and significant; it simulates the interaction between these active substances and gut microbiota in specific patient populations. Since the microbiota of cardiovascular disease patients may have specific TMA generation characteristics, the effective substances screened in this context are more likely to be directly applied to the intervention of this population, with higher translational medical value, greatly enhancing the clinical predictive value and specificity of the screening results; (E02) Detection step (E01) changes in the concentration of TMA and precursor choline in the fermentation broth to determine the regulatory effect of different active substance components on gut TMA generation. TMA concentration is the ultimate indicator for assessing the regulatory effect, while choline concentration is a crucial indicator of mechanism. If a substance causes a decrease in TMA while an increase in choline concentration, it strongly suggests that its mechanism of action is to inhibit the metabolic pathway by which microorganisms convert choline into TMA. If TMA and choline decrease simultaneously, it may mean that the substance inhibits the absorption of choline by microorganisms or promotes the conversion of choline to other metabolic pathways. This not only answers whether it is effective but also provides preliminary and crucial clues as to how it works.

[0084] Preferably, the fermentation time in step (E01) is 20h to 24h. More preferably, the fermentation time in step (E01) is 21h to 23h. Even more preferably, the fermentation time in step (E01) is 22h. Maintaining consistency with the core parameters of the entire system ensures the standardization and reproducibility of the screening conditions. The defined fermentation time is a point in time that can sensitively capture differences in TMA generation, ensuring that all candidate substances are compared within the same culture time, resulting in fair and reliable results.

[0085] Preferably, the method for verifying the specific regulatory effect of the screened active ingredients on the structure and function of the gut microbiota is as follows: (F01) After determining that a certain active substance can significantly reduce TMA generation, the active substance is added to the in vitro fermentation system; this ensures that the subsequent in-depth mechanism research, which requires a large amount of resources and effort, is based on a clear and reproducible phenotype, avoiding blindly exploring the mechanism of ineffective components, and concentrating research resources on the most promising candidates, thereby improving the efficiency of the entire research plan; (F02) By using 16S rRNA sequencing, metabolomics, and short-chain fatty acid analysis, the specific regulatory effect of the active ingredients on the structure and function of the gut microbiota is obtained. Step (F01) Observe the microbial community structure and metabolic characteristics in the system with added active substance; (F03) Obtain the microbial community structure and metabolic characteristics in the original in vitro fermentation system without added active substance through 16S rRNA sequencing, metabolomics, and short-chain fatty acid analysis; Establish a group with added active substance and a group without added active substance. By setting up an identical control system without added active substance, a baseline can be provided for all observed changes. Any differences observed in microbial community structure or metabolic characteristics can be confidently attributed to the addition of the active substance, rather than the culture system itself or accidental factors; 16S rRNA sequencing can precisely reveal which bacteria are significantly enriched and which are significantly suppressed at the phylum, family, and genus levels, specifically identifying key genera associated with reduced TMA phenotypes. Non-targeted metabolomics allows for broad exploration, uncovering other unknown and affected metabolic pathways besides TMA, potentially revealing novel mechanisms of action. Targeted SCFA analysis provides in-depth validation, focusing on metabolites crucial to host health, such as acetic acid, propionic acid, and butyric acid. It confirms that the reduction in TMA does not come at the expense of overall gut microbiota metabolic activity; if SCFAs also decrease significantly, it indicates that the substance possesses broad-spectrum antibacterial activity rather than specific regulation. It can be found that the active substance can reduce TMA while promoting the production of beneficial metabolites, revealing its multiple benefits; (F04) Compare the differences between the bacterial community structure and metabolic characteristics in step (F02) and step (F03) to obtain the target bacterial genus and metabolic pathway of the active substance; It can construct a hypothetical mechanism model, for example: the active substance X specifically inhibits the growth of bacterial genera A and B, thereby cutting off the metabolic pathway that converts choline precursor Y into TMA, while promoting the proliferation of bacterial genus C, resulting in an increase in butyrate production, providing clear targets and theoretical basis for precision nutritional intervention, probiotic development or drug design.

[0086] The present invention has the following beneficial effects: (1) Yeast extract, as a complex nitrogen source and growth factor source, can provide amino acids, small peptides, nucleotides, vitamins and trace elements, ensuring that intestinal microorganisms can quickly start growth and metabolism; L-cysteine, as a reducing agent, consumes oxygen in the system, and in combination with razor violet, quickly establishes and maintains a strict anaerobic environment, provides essential sulfur elements for microorganisms, creates and maintains anaerobic conditions that are crucial for the survival of colonic microorganisms, prevents excessive growth of aerobic microorganisms, and ensures the accuracy of experimental results; Heme is used to simulate the heme environment in the intestine, supporting the growth of heme-dependent bacteria, which can play an important role in the metabolism of TMA precursor choline, thereby making the fermentation system more realistically reflect (2) Sodium chloride is the main osmotic regulator, maintaining an isotonic environment similar to human intestinal fluid, preventing microorganisms from dying due to osmotic imbalance. Calcium ions in calcium chloride hexahydrate are enzyme cofactors, participating in signal transduction and cell wall stability maintenance, providing a suitable physiological environment for microorganisms, ensuring their normal physiological functions and structural integrity. Dipotassium hydrogen phosphate and acidic substances produced during fermentation, such as short-chain fatty acids, constitute a phosphate buffer system, maintaining the relative stability of the system's pH value. It can provide the phosphorus element necessary for microorganisms to synthesize nucleic acids, ATP, and phospholipids, as well as the potassium element necessary to maintain intracellular potential, preventing the system from experiencing a sharp drop in pH due to excessive acid production, avoiding inhibition of the growth of most intestinal microorganisms, and ensuring fermentation. The process proceeds smoothly; magnesium sulfate heptahydrate can provide magnesium ions and sulfur elements, ensuring the basic metabolism and energy supply of microorganisms; (3) vitamin mixture can provide a series of B vitamins such as biotin, folic acid, and thiamine, supporting the growth of bacteria that require exogenous vitamins, making the microbial community structure more diverse; starch, as the main carbon source and energy source, provides a continuous and stable energy source for the entire microbial community, supporting its long-term fermentation and metabolism; choline, as the direct substrate for TMA generation, provides raw materials for the TMA generation reaction by adding choline exogenously, thereby enabling direct and quantitative detection of changes in TMA production under different intervention conditions, achieving the purpose of rapid targeted screening; as an anaerobic indicator, razor cyanide, under aerobic conditions, razor cyanide It is pink or purple, and under anaerobic conditions, it is reduced to colorless halogen, providing an intuitive and visual indicator of the anaerobic environment for the experimental operator. The disappearance of pink means that the system has reached the anaerobic state and the fermentation experiment can begin; (4) Through inorganic salts, buffer pairs, reducing agents and anaerobic indicators, the anaerobic, isotonic and weakly acidic environment of the colon is accurately recreated; Through yeast extract, vitamins, heme, etc., comprehensive and balanced nutrition is provided to support the growth of complex flora; The target is clear, and the TMA precursor choline is added directly, making the research target very focused. The addition of heme takes into account its potential impact on intestinal flora and metabolism, making the model closer to reality. The use of razor violet as an indicator greatly reduces the difficulty of operation and the failure rate;The overall system is a scientifically designed, structurally complete, and highly targeted in vitro fermentation system, highly rational, advanced, and practical for rapidly screening substances that regulate intestinal TMA production. Choline is a key substrate for trimethylamine TMA formation; resazurin is a colorimetric indicator of the redox state in the system. Resazurin accurately reflects changes in the anaerobic environment through a reversible change from blue to pink to colorless, allowing for visual monitoring of the reduction state of the fermentation system. Attached Figure Description

[0087] Figure 1 This is a schematic flowchart of the method for establishing the in vitro fermentation system of the present invention; Figure 2 The diagram shows the effects of Examples 1-5 of the present invention on intestinal fermentation. Figure 3 The graph shows the residual choline content in the fermentation broth after fermentation in Examples 1-5 of this invention. Figure 4 The graph shows the effect of different active components on the generation of TMA by in vitro fermentation of gut microbiota in cardiovascular patients, using the in vitro fermentation system of Example 5 as an example. Figure 5 The following figure illustrates the animal experiment's verification of the effects of adding astragaloside A on high-fat induced obesity and metabolic abnormalities, using the in vitro fermentation system of Example 5 as an example. Figure 6 The figure shows the effect of adding astragaloside A on TMAO content in animal experiments, using the in vitro fermentation system of Example 5 as an example. Detailed Implementation

[0088] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0089] Example 1: A rapid targeted screening in vitro fermentation system for regulating intestinal TMA production, comprising the following components by weight: 2 parts yeast extract, 0.8 parts L-cysteine, 1.5 parts heme, 0.8 parts sodium chloride, 0.008 parts calcium chloride hexahydrate, 0.4 parts dipotassium hydrogen phosphate, 0.08 parts magnesium sulfate heptahydrate, 0.15 parts vitamin mixture, 7 parts starch, 0.01 parts choline, and 0.8 parts resazurin.

[0090] L-cysteine ​​was prepared into an L-cysteine ​​solution. The preparation of the L-cysteine ​​solution included the following steps: (A01) Analytical grade L-cysteine ​​was added to distilled water (after removing oxidizing substances) under nitrogen protection and stirred to dissolve; the dissolving container in step (A01) was a double-walled glass reaction flask; the dissolving temperature in step (A01) was 23℃; the stirring speed in step (A01) was 180 r / min; (A02) The pH of the solution in step (A01) was adjusted to 6.8 using NaOH solution; the concentration of the NaOH solution in step (A02) was 1 mol / L; (A03) The solution in step (A02) was filtered through a PES microporous membrane, and the resulting filtrate was L-cysteine. Solution; the filtrate from step (A03) is placed in a brown sterile bottle for later use; the pore size of the PES microporous filter membrane in step (A03) is 0.2 μm; the L-cysteine ​​solution in the brown sterile bottle is frozen at -22℃; before use, the frozen L-cysteine ​​solution is thawed naturally to room temperature, and an equal volume of FeCl3 solution is added to the L-cysteine ​​solution sample to be used. The color change of the solution is observed. If a light green complex color is produced immediately, it indicates that L-cysteine ​​has been oxidized and the L-cysteine ​​solution needs to be reconstituted. If a light green complex color is not produced immediately, it indicates that L-cysteine ​​has not been oxidized and can continue to be used; the concentration of FeCl3 solution is 0.01 mol / L.

[0091] The preparation of heme includes the following steps: (B01) Defatted pork liver is chopped and added to a mixed solvent of ethanol and acetone. After adjusting the pH to 1.8, the mixture is stirred and extracted to obtain an extract. In step (B01), the mass-to-volume ratio of defatted pork liver to the mixed solvent is 1:5; the volume ratio of ethanol to acetone is 1:1; the pH is adjusted to 1.9; the stirring in step (B01) is magnetic stirring; the extraction temperature in step (B01) is 2℃; and the extraction time in step (B01) is 2 hours. (B02) The extract from step (B01) is vacuum filtered, and the filtrate is collected. The vacuum filtration in step (B02) is completed in a Buchner funnel. (B03) The filtrate from step (B02) is distilled under reduced pressure under a nitrogen stream to remove the organic solvent and obtain crude heme. Precipitate; (B04) Dissolve the crude heme precipitate from step (B03) in NaOH solution, adjust the pH to 7.1, let stand, centrifuge, and take the supernatant; the concentration of NaOH solution in step (B04) is 0.1 mol / L; the standing time in step (B04) is 30 min; the centrifugation speed in step (B04) is 6000 r / min; the centrifugation time in step (B04) is 10 min; (B05) Purify the supernatant from step (B04) through a Sephadex G-25 gel filter column, and use the fraction with the main peak at 398 nm as the pure product; (B06) Purge the pure product from step (B05) with nitrogen to remove dissolved oxygen, and then dispense it into brown glass bottles to obtain refined heme; the nitrogen purging time in step (B06) is 5 min.

[0092] The vitamin mixture includes biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine, with a mass ratio of 1:1:1:1:1. The preparation of the vitamin mixture includes the following steps: (C01) Take appropriate amounts of the five active ingredients—biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine—and add them to a mixed solvent of anhydrous ethanol and distilled water, stirring to dissolve under nitrogen protection; in step (C01), the volume ratio of anhydrous ethanol to distilled water is 1:1; in step (C01), the mass ratio of the five active ingredients to the volume ratio of the mixed solvent is 0.15:100; in step (C01), the stirring speed is 150 r / min; in step (… (C01) The dissolution time is 15 min; (C02) Adjust the pH of the solution in step (C01) to 6.6, and add an appropriate amount of ascorbic acid and mix; the concentration of ascorbic acid in step (C02) is 0.1%; (C03) Filter the mixture in step (C02) through a PES microporous membrane, and the resulting filtrate is the vitamin mixture; store the filtrate in step (C03) in a brown glass ampoule for later use; the pore size of the PES microporous membrane in step (C03) is 0.2 μm.

[0093] The preparation of choline includes the following steps: (D01) Analytical grade choline chloride C5H is selected. 14 C l NO was dissolved in distilled water to remove oxides and stirred magnetically under nitrogen protection until clear; the magnetic stirring speed in step (D01) was 200 r / min; the stirring temperature in step (D01) was 19℃; (D02) the pH of the solution in step (D01) was adjusted to 6.9 and filtered through a PES microfiltration membrane, the resulting filtrate being the choline solution; the filtrate in step (D02) was stored in a brown glass bottle for later use; the pH was adjusted in step (D02) using 0.1 mol / L NaOH solution; the pore size of the PES microporous membrane in step (D02) was 0.2 μm. Before use, resazurin was compared with a 0.01 mol / L Na2S2O4 standard reducing agent for colorimetric sensitivity calibration; resazurin is a colorimetric indicator of the redox state in the system. Resazurin accurately reflects changes in the anaerobic environment through a reversible change from blue to pink to colorless, allowing for visual monitoring of the reduction state of the fermentation system.

[0094] like Figure 1 The method for establishing a rapid targeted screening and regulation in vitro fermentation system for intestinal TMA generation includes the following steps: (S01) Take appropriate amounts of yeast extract, L-cysteine, heme, sodium chloride, calcium chloride hexahydrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, vitamin mixture, starch, choline, and resazurite, mix them, and deoxygenate them under nitrogen protection to achieve an anaerobic state; Step (S01) is as follows: (S01-1) Under aseptic conditions, take distilled water to remove oxides, add appropriate amounts of yeast extract, L-cysteine, heme, sodium chloride, calcium chloride hexahydrate, and dipotassium hydrogen phosphate. Dipotassium sulfate, magnesium sulfate heptahydrate, vitamin mixture, starch, choline, and resazurite are stirred and mixed; the stirring and mixing container in step (S01-1) is a double-layered constant temperature reactor; the stirring speed in step (S01-1) is 180 r / min; (S01-2) immediately under nitrogen protection, nitrogen gas is continuously purged into the solution after stirring and mixing in step (S01-1) to remove dissolved oxygen from the system; the continuous nitrogen purging time in step (S01-2) is 10 min; in step (S01-2), the change of resazurite from blue to light pink is used as a visual indicator of the establishment of anaerobic state;

[0095] (S02) A mixture of nitrogen and carbon dioxide gas is introduced into the anaerobic workstation to establish a simulated intestinal anaerobic environment. The mixture from step (S01) is placed in the anaerobic workstation to form a closed system, maintaining constant temperature and pressure. Step (S02) involves transferring the deoxygenation system from step (S01) into the anaerobic workstation, continuously purging the reaction space of the anaerobic workstation with a mixture of nitrogen and carbon dioxide gas to maintain constant total pressure and temperature; the volume ratio of nitrogen to carbon dioxide is 6:2; the total pressure is maintained at standard atmospheric pressure; the temperature is maintained at 36°C; the deoxygenation system is added to a pressure-resistant glass fermentation bottle, and the gas-liquid ratio in the pressure-resistant glass fermentation bottle is controlled at 1.5:1 before the bottle mouth is sealed; (S03) Heart samples are collected. Fecal samples from patients with vascular diseases are anaerobically homogenized and centrifuged to remove impurities, yielding a fecal inoculum. Step (S03) involves: (S03-1) taking fecal samples from patients with cardiovascular diseases and mixing them with pre-deoxygenated sterile saline under anaerobic conditions in an anaerobic workbench; processing the fecal samples from step (S03-1) within 1 hour; the mass-to-volume ratio of fecal samples to sterile saline in step (S03-1) is 1:4; (S03-2) homogenizing the mixture from step (S03-1) and centrifuging to remove residue yields the fecal inoculum; the homogenization time in step (S03-2) is 2 minutes; (S04) adding the fecal inoculum from step (S03) to the mixture from step (S02). The mixture was anaerobically cultured in the system to form a TMA in vitro fermentation system; the culture temperature in step (S04) was 36℃; the culture time in step (S04) was 36h; step (S04) consisted of: (S04-1) the mixture from step (S02) was placed into a pressure-resistant glass fermentation bottle and inoculated with the fecal inoculum from step (S03); the concentration of the fecal inoculum in step (S04-1) was 10%; (S04-2) immediately after inoculation in step (S04-1), the space inside the bottle was purged with nitrogen to an anaerobic state; the time for purging the space inside the bottle with nitrogen in step (S04-2) was 2min; (S04-3) the bottle from step (S04-2) was placed in a constant temperature anaerobic incubator for cultivation. A TMA in vitro fermentation system is formed; the culture temperature in the constant temperature anaerobic incubator in step (S04-3) is 36℃; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 36h; (S05) the TMA in vitro fermentation system in step (S04) is sampled periodically to detect the concentration of TMA and short-chain fatty acids in the sample, and the color change of azuran is monitored in real time, so as to verify the anaerobic stability and metabolic activity of the TMA in vitro fermentation system; step (S05) is as follows: (S05-1) after fermentation in step (S04) for 12h, the sample is centrifuged and the supernatant is collected; the sample volume in step (S05-1) is 1mL; the centrifugation time in step (S05-1) is 8min.(S05-2) The concentrations of choline, TMA and short-chain fatty acids in the supernatant of step (S05-1) were detected using LC-MS / MS technology. If the color change of resazurin remained between light pink and colorless, it indicated that the system was in a stable anaerobic state.

[0096] Example 2: This example is basically the same as Example 1, except that the in vitro fermentation system for rapidly targeting and regulating intestinal TMA production includes the following components by weight: 3 parts yeast extract, 1.2 parts L-cysteine, 2.5 parts heme, 1.0 part sodium chloride, 0.012 parts calcium chloride hexahydrate, 0.5 parts dipotassium hydrogen phosphate, 0.1 parts magnesium sulfate heptahydrate, 0.25 parts vitamin mixture, 9 parts starch, 0.8 parts choline, and 1.2 parts resazurin. The dissolution temperature in step (A01) is 27℃; the stirring speed in step (A01) is 200 r / min; (A02) the pH of the solution in step (A01) is adjusted to 7.2 using NaOH solution; the pore size of the PES microporous membrane in step (A03) is 0.24 μm; the L-cysteine ​​solution in the brown sterile bottle is frozen at -18℃; (B01) defatted pork liver is chopped and added to a mixed solvent of ethanol and acetone, the pH is adjusted to 2.2, and then stirred and extracted to obtain the extract; the mass-volume ratio of defatted pork liver to the mixed solvent in step (B01) is 1:10; step ( In step (B01), the volume ratio of ethanol to acetone is 2:1; the pH is adjusted to 2.1; the extraction temperature is 4℃; the extraction time is 1 h; in step (B04), the crude heme precipitate from step (B03) is dissolved in NaOH solution, the pH is adjusted to 7.3, centrifuged after standing, and the supernatant is collected; the standing time is 30 min; the centrifugation speed is 8000 r / min; the centrifugation time is 10 min; and the nitrogen purging time is 5 min. The vitamin mixture includes biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine, with a mass ratio of 2:1:2:1:2. The preparation of the vitamin mixture includes the following steps: In step (C01), the volume ratio of anhydrous ethanol to distilled water is 1:2; in step (C01), the mass ratio of the five active ingredients to the volume ratio of the mixed solvent is 0.25:100; in step (C01), the stirring speed is 200 r / min; in step (C01), the dissolution time is 15 min; in step (C02), the pH of the solution in step (C01) is adjusted to 7, and an appropriate amount of ascorbic acid is added and mixed; in step (C03), the pore size of the PES microporous filter membrane is 0.24 μm; in step (D01), the magnetic stirring speed is 250 r / min; in step (D01), the stirring temperature is 21℃; in step (D02), the pH of the solution in step (D01) is adjusted to 7.1, and filtered through the PES microfiltration membrane, the resulting filtrate is the choline solution; in step (D02), the pore size of the PES microfiltration membrane is 0.24 μm.

[0097] A method for establishing an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA generation includes the following steps: Step (S01-1) involves stirring at 200 r / min; Step (S01-2) involves continuous nitrogen purging for 30 min; the volume ratio of nitrogen to carbon dioxide is 8:2; the temperature is maintained at 38℃; the deoxygenated system is added to a pressure-resistant glass fermentation flask, and the flask is sealed after controlling the gas-liquid ratio to 2:1; the fecal sample in Step (S03-1) is processed within 2 h; the mass-to-volume ratio of fecal sample to sterile saline in Step (S03-1) is 1:5; Step (… The homogenization time in step (S03-2) is 3 min; the culture temperature in step (S04) is 38℃; the culture time in step (S04) is 12 h; the time for purging the space inside the bottle with nitrogen in step (S04-2) is 3 min; the culture temperature in the constant temperature anaerobic incubator in step (S04-3) is 38℃; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 12; (S05-1) after fermentation for 36 h in step (S04), take a sample and centrifuge to collect the supernatant; the sample volume in step (S05-1) is 2 mL; the centrifugation time in step (S05-1) is 10 min.

[0098] Example 3: This example is basically the same as Example 1, except that the in vitro fermentation system for rapidly targeting and regulating intestinal TMA production includes the following components by weight: 2.2 parts yeast extract, 0.9 parts L-cysteine, 1.8 parts heme, 0.85 parts sodium chloride, 0.009 parts calcium chloride hexahydrate, 0.42 parts dipotassium hydrogen phosphate, 0.09 parts magnesium sulfate heptahydrate, 0.18 parts vitamin mixture, 7.5 parts starch, 0.05 parts choline, and 0.9 parts resazurin. The dissolution temperature in step (A01) is 24℃; the stirring speed in step (A01) is 185 r / min; the pH of the solution in step (A01) is adjusted to 6.9; the pore size of the PES microporous membrane in step (A03) is 0.21 μm; the L-cysteine ​​solution in the brown sterile bottle is frozen at -21℃; (B01) defatted pork liver is chopped and added to a mixed solvent of ethanol and acetone, the pH is adjusted to 1.9, and after stirring and extraction, the extract is obtained; the mass-volume ratio of defatted pork liver to the mixed solvent in step (B01) is 1:6; the ethanol in step (B01) The volume ratio of biotin to acetone was 1:1; in step (B01), the pH was adjusted to 1.9; the extraction temperature in step (B01) was 2.5℃; the extraction time in step (B01) was 1 h; (B04) the crude heme precipitate from step (B03) was dissolved in NaOH solution, the pH was adjusted to 7.1, centrifuged after standing, and the supernatant was collected; the standing time in step (B04) was 22 min; the centrifugation speed in step (B04) was 6500 r / min; the centrifugation time in step (B04) was 8 min; the nitrogen purging time in step (B06) was 3 min. The vitamin mixture included biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine, with a mass ratio of biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine of 2:1:2:1:2. In step (C01), the volume ratio of anhydrous ethanol to distilled water is 1:1; the mass ratio of the five active ingredients to the volume ratio of the mixed solvent is 0.18:100; the stirring speed in step (C01) is 160 r / min; the dissolution time in step (C01) is 11 min; in step (C02), the pH of the solution from step (C01) is adjusted to 6.7; in step (C03), the pore size of the PES microporous membrane is 0.21 μm. In step (D01), the magnetic stirring speed is 210 r / min; the stirring temperature in step (D01) is 19℃; in step (D02), the pH of the solution from step (D01) is adjusted to 6.9, and the solution is filtered through a PES microfiltration membrane to obtain a choline solution; the pore size of the PES microfiltration membrane in step (D02) is 0.21 μm.

[0099] A method for establishing an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA generation includes the following steps: Step (S01-1) involves stirring at 185 r / min; Step (S01-2) involves continuous nitrogen purging for 15 min; the nitrogen to carbon dioxide volume ratio is 6.5:2; the temperature is maintained at 36.5℃; the gas-liquid ratio in the pressure-resistant glass fermentation flask is 1.6:1; the fecal sample in Step (S03-1) is processed within 1.5 h; the mass-to-volume ratio of the fecal sample to sterile physiological saline in Step (S03-1) is 1:4.5; Step (S03-2)... The centrifugation time in step (S04) is 1 min; the culture temperature in step (S04) is 36.5℃; the culture time in step (S04) is 18 h; the time for replacing the space inside the bottle with nitrogen in step (S04-2) is 2.5 min; the culture temperature in the constant temperature anaerobic incubator in step (S04-3) is 36.5℃; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 30 h; after fermentation for 18 h in step (S05-1), samples are taken and centrifuged; the sample volume in step (S05-1) is 2 mL; the centrifugation time in step (S05-1) is 10 min.

[0100] Example 4: This example is basically the same as Example 1, except that the in vitro fermentation system for rapidly targeting and regulating intestinal TMA production includes the following components by weight: 2.8 parts yeast extract, 1.1 parts L-cysteine, 2.2 parts heme, 0.95 parts sodium chloride, 0.011 parts calcium chloride hexahydrate, 0.48 parts dipotassium hydrogen phosphate, 0.09 parts magnesium sulfate heptahydrate, 0.22 parts vitamin mixture, 8.5 parts starch, 0.4 parts choline, and 1.1 parts resazurin. The dissolution temperature in step (A01) is 26℃; the stirring speed in step (A01) is 195 r / min; the pH of the solution in step (A01) is adjusted to 7.1; the pore size of the PES microporous membrane in step (A03) is 0.23 μm; it is stored at -19℃; the mass-to-volume ratio of defatted pork liver to mixed solvent in step (B01) is 1:9; the volume ratio of ethanol to acetone in step (B01) is 1-2:1; the pH is adjusted to 2.1 in step (B01); the extraction temperature in step (B01) is 3.5℃; the extraction time in step (B01) is 1.5 h; the pH is adjusted to 7.2 in step (B04); the settling time in step (B04) is 28 min; the centrifugation speed in step (B04) is 7500 r / min; the centrifugation time in step (B04) is 9 min; the nitrogen purging time in step (B06) is 4 min. The vitamin mixture includes biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine, with a mass ratio of 1:1:2:2:1. In step (C01), the volume ratio of anhydrous ethanol to distilled water is 1:2; the mass ratio of the five active ingredients to the mixed solvent in step (C01) is 0.22:100; the stirring speed in step (C01) is 190 r / min; the dissolution time in step (C01) is 14 min; in step (C02), the pH of the solution from step (C01) is adjusted to 6.9; and in step (C03), the pore size of the PES microporous membrane is 0.23 μm. The magnetic stirring speed in step (D01) is 240 r / min; the stirring temperature in step (D01) is 20℃; the pH of the solution in step (D02) is adjusted to 7; the pore size of the PES microporous filter membrane in step (D02) is 0.23 μm;

[0101] A method for establishing an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA generation includes the following steps: Step (S01-1) involves a stirring speed of 195 r / min; Step (S01-2) involves continuous nitrogen purging for 25 min; the nitrogen to carbon dioxide volume ratio is 7.5:2; the temperature is maintained at 37.5℃; the gas-liquid ratio in the pressure-resistant glass fermentation flask is 1.9:1; the fecal sample in Step (S03-1) is processed within 1.5 h; the mass-to-volume ratio of fecal sample to sterile physiological saline in Step (S03-1) is 1:4.5; and the homogenization time in Step (S03-2) is 3 min. n; the centrifugation time in step (S03-2) is 2 min; the culture temperature in step (S04) is 37.5℃; the culture time in step (S04) is 30 h; the time for purging the space inside the bottle with nitrogen in step (S04-2) is 2.5 min; the culture temperature in the constant temperature anaerobic incubator in step (S04-3) is 37.5℃; the culture time in the constant temperature anaerobic incubator in step (S04-3) is 30 h; after fermentation for 30 h in step (S05-1), take a sample and centrifuge; the sample volume in step (S05-1) is 1 mL; the centrifugation time in step (S05-1) is 10 min.

[0102] Example 5: This example is basically the same as Example 1, except that the in vitro fermentation system for rapidly targeting and regulating intestinal TMA production includes the following components by weight: 2.5 parts yeast extract, 1 part L-cysteine, 2 parts heme, 0.9 parts sodium chloride, 0.01 parts calcium chloride hexahydrate, 0.45 parts dipotassium hydrogen phosphate, 0.09 parts magnesium sulfate heptahydrate, 0.2 parts vitamin mixture, 8 parts starch, 0.2 parts choline, and 1 part resazurin. The dissolution temperature in step (A01) is 25℃; the stirring speed in step (A01) is 190 r / min; the pH of the solution in step (A01) is adjusted to 7; the pore size of the PES microporous membrane in step (A03) is 0.22 μm; it is stored at -20℃; the mass-to-volume ratio of defatted pork liver to mixed solvent in step (B01) is 1:7.5; the volume ratio of ethanol to acetone in step (B01) is 1.5:1; the pH is adjusted to 2 in step (B01); the extraction temperature in step (B01) is 3℃; the extraction time in step (B01) is 1.5 h; the pH is adjusted to 7.2 in step (B04); the settling time in step (B04) is 25 min; the centrifugation speed in step (B04) is 7000 r / min; the centrifugation time in step (B04) is 9 min; the nitrogen purging time in step (B06) is 4 min. The mass ratio of biotin, cobalamin, para-aminobenzoic acid, folic acid, and pyridoxine is 2:2:2:2:1. In step (C01), the volume ratio of anhydrous ethanol to distilled water is 1:1.5; the mass ratio of the five active ingredients to the mixed solvent is 0.2:100; the stirring speed in step (C01) is 180 r / min; the dissolution time in step (C01) is 13 min; in step (C02), the pH of the solution from step (C01) is adjusted to 6.8; the pore size of the PES microporous membrane in step (C03) is 0.22 μm. In step (D01), the magnetic stirring speed is 230 r / min; the stirring temperature in step (D01) is 20 °C; the pH of the solution from step (D01) is adjusted to 7 in step (D02); the pore size of the PES microporous membrane in step (D02) is 0.22 μm.

[0103] A method for establishing an in vitro fermentation system for rapid targeted screening and regulation of intestinal TMA generation includes the following steps: Step (S01-1) involves stirring at 190 r / min; Step (S01-2) involves continuous nitrogen purging for 20 min; the nitrogen to carbon dioxide volume ratio is 7:2; the temperature is maintained at 37℃; the gas-liquid ratio in the pressure-resistant glass fermentation flask is 1.8:1; the fecal sample in Step (S03-1) is processed within 1.5 h; the mass-to-volume ratio of the fecal sample to sterile physiological saline in Step (S03-1) is 1:4.5; Step (S03-2)... The centrifugation time was 1.5 min; the culture temperature in step (S04) was 37℃; the culture time in step (S04) was 22 h; the time for replacing the space inside the bottle with nitrogen in step (S04-2) was 2.5 min; the culture temperature in the constant temperature anaerobic incubator in step (S04-3) was 37℃; the culture time in the constant temperature anaerobic incubator in step (S04-3) was 22 h; after fermentation for 24 h in step (S05-1), samples were taken and centrifuged; the sample volume in step (S05-1) was 1.5 mL; the centrifugation time in step (S05-1) was 9 min.

[0104] Experimental Example: Feces from 3 cardiovascular patients were collected for in vitro fermentation. Based on parameters such as total gas production, short-chain fatty acid production, and gut microbiota structure, the effects of Examples 1-5 on intestinal fermentation were studied. Results are as follows: Figure 2 As shown, the propionic acid yield in the fermentation system of Example 5 was higher than that in Example 2, while the total acid and total gas production were higher than those in Examples 1 and 3, respectively. The total number of bacteria in Examples 1-5 was basically the same, indicating that Examples 1-5 all had good in vitro fermentation effects, with Example 5 showing the best in vitro fermentation effect.

[0105] To further determine the effects of Examples 1-5 on TMA production in intestinal bacteria, LC-MS / MS was used to detect choline and TMA, the main substrates for TMA production in the fermentation broths of different examples. The results are as follows: Figure 3 As shown: the residual choline in the fermentation broth was highest in Example 1, followed by Example 3, then Example 5, then Example 4, and finally Example 2. The residual choline in Example 4 was higher than in Example 1, indicating that the gut microbiota of cardiovascular patients can metabolize choline in the system. There was no difference in residual choline among Examples 1, 3, and 5. Furthermore, the TMA content in the fermentation broth was not different among Examples 1, 3, and 5. However, there was a trend of increasing TMA in Examples 4 and 2, indicating that the stability of gut microbial metabolism was slightly affected in the systems of Examples 4 and 2. In conclusion, the above results further demonstrate that Example 5 exhibited the best in vitro fermentation effect.

[0106] A rapid targeted screening in vitro fermentation system for regulating intestinal TMA production was developed for functional screening studies of food-medicine homologous components and extracts of traditional Chinese medicine.

[0107] The functional screening study of medicinal and edible components and extracts of traditional Chinese medicine involved the following steps: (E01) Candidate active substances, including astragaloside A, ginsenosides, codonopsis polysaccharides, lentinan, and / or auricularia polysaccharides, were added to an in vitro fermentation system and co-fermented with fecal inoculum from patients with cardiovascular diseases. The fermentation time in step (E01) was 20-24 hours; in step (E02), it was 21-23 hours; and in step (E03), it was 22 hours. (E02) The changes in the concentrations of TMA and precursor choline in the fermentation broth from step (E01) were detected to determine the regulatory effects of different active components on intestinal TMA production. Five traditional Chinese medicines and medicinal and edible active components with reported potential for reducing TMAO were selected, including astragaloside A, ginsenosides, codonopsis polysaccharides, lentinan, and auricularia polysaccharides. The rapid screening system developed in this invention was used to screen and verify the bioactive substances that can reduce TMA production. Specifically, the system of Example 5, which showed the best effect, was used as a representative for verification. First, fecal samples from 20 cardiovascular disease patients collected anaerobically were injected into the system of Example 5. Simultaneously, appropriate concentrations of the aforementioned traditional Chinese medicines and their food-medicine homologous active components—astragaloside A, ginsenosides, codonopsis polysaccharides, lentinan, and auricularia polysaccharides—were added to the system. After co-fermentation for 24 hours, the concentration of TMA was measured. The results are as follows: Figure 4 As shown, compared with the blank group, the addition of ginsenosides or astragaloside A significantly reduced the content of TMA in the fermentation system (p<0.05), the addition of lentinan significantly increased the content of TMA in the fermentation system (p<0.05), while the addition of Codonopsis pilosula polysaccharide or Auricularia auricula polysaccharide had no significant effect on the content of TMA in the fermentation system (p>0.05). The results indicate that different bioactive substances have different responses to the in vitro intestinal TMA generation and detection system for cardiovascular patients created in this invention. This system can be used as a platform for rapid screening to reduce TMA generation, but the screening results need further verification.

[0108] A rapid targeted screening in vitro fermentation system for regulating intestinal TMA production was used to verify the specific regulatory effects of the screened active ingredients on the structure and function of the intestinal flora. The verification process involved: (F01) After determining that a certain active substance could significantly reduce TMA production, the active substance was added to the in vitro fermentation system; (F02) The structure and metabolic characteristics of the flora in the system with the added active substance in step (F01) were obtained using 16S rRNA sequencing, metabolomics, and short-chain fatty acid analysis; (F03) The structure and metabolic characteristics of the flora in the original in vitro fermentation system without the added active substance were obtained using 16S rRNA sequencing, metabolomics, and short-chain fatty acid analysis; (F04) The differences between the flora structure and metabolic characteristics in step (F02) and step (F03) were compared to identify the target bacterial species and metabolic pathways of the active substance. To verify the regulatory effect of the above screening system on TMA generation, further mouse experiments were conducted. C57 mice were selected and an obese mouse model was established by feeding them a high-fat diet (D12492). Subsequently, astragaloside A (100 mg / kg / d) was administered by gavage for 8 consecutive weeks. After euthanasia with ether, the effects on body weight, fat deposition, glucose and lipid metabolism, inflammatory factor expression, serum TMAO levels, and gut microbiota structure were examined to verify the screening results of in vitro fermentation. The results are as follows: Figure 5 As shown, compared with the control group, the high-fat diet successfully established an obese mouse model. Furthermore, compared with the model group, gavage administration of astragaloside A significantly reduced the body weight, fat deposition index, serum triglyceride, and inflammatory factor levels in obese mice (p<0.05), suggesting that astragaloside A has the potential to improve cardiovascular disease. Furthermore, as... Figure 6 As shown, quantitative detection of serum TMAO revealed that the TMAO level in the model group was significantly higher than that in the control group (p < 0.05), while the serum TMAO level in the gavage group of mice was lower than that in the model group. Due to the complexity of metabolism and TMAO generation processes, the results of this study suggest that astragaloside A may have a potential effect of reducing TMAO in animals, and its regulatory role in TMA and TMAO metabolism needs further investigation. In summary, the results of this study indicate that astragaloside A, an active component that targets and reduces intestinal TMA in cardiovascular patients, obtained using this invention, has shown the potential to reduce TMA / TMAO and improve cardiovascular disease in both in vitro and in vivo experiments, confirming that the system constructed in this invention can be applied to the screening of active substances targeting intestinal TMA generation.

Claims

1. A rapid targeted screening in vitro fermentation system modulating TMA production in the gut, characterized in that: by weight, comprising 2-3 parts of yeast extract, 0.8-1.2 parts of L-cysteine, 1.5-2.5 parts of hematin, 0.8-1.0 parts of sodium chloride, 0.008-0.012 parts of calcium chloride hexahydrate, 0.4-0.5 parts of dipotassium hydrogen phosphate, 0.08-0.1 parts of magnesium sulfate heptahydrate, 0.15-0.25 parts of vitamin mixture, 7-9 parts of starch, 0.01-0.8 parts of choline, and 0.8-1.2 parts of resazurin.

2. The rapid targeted screening in vitro fermentation system modulating gut TMA production according to claim 1, characterized in that: The L-cysteine is configured into an L-cysteine solution, and the configuration of the L-cysteine solution comprises the following steps, (A01) Take analytical pure L-cysteine, and stir and dissolve in distilled water under nitrogen protection after adding an oxide-removing substance; (A02) Adjust the pH of the solution in step (A01) to 6.8-7.2 using a NaOH solution; (A03) Filter the solution in step (A02) through a PES microporous filter membrane, and the obtained filtrate is the L-cysteine solution.

3. The rapid targeted screening in vitro fermentation system modulating gut TMA production according to claim 1, characterized in that: The preparation of the hematin comprises the following steps, (B01) Take defatted pig liver, cut it, and then add it into a mixed solvent of ethanol and acetone, adjust the pH to 1.8-2.2, and then perform stirring extraction to obtain an extraction liquid; (B02) Perform vacuum filtration on the extraction liquid in step (B01), and take the filtrate; (B03) Perform vacuum distillation on the filtrate in step (B02) under a nitrogen flow, remove the organic solvent, and obtain a crude hematin precipitate; (B04) Dissolve the crude hematin precipitate in step (B03) in a NaOH solution, adjust the pH to 7.1-7.3, and then perform centrifugation after standing, and take the supernatant; (B05) Purify the supernatant in step (B04) through a Sephadex G-25 gel filtration column, and take the main peak fraction with an absorption peak of 398 nm as the pure product; (B06) Purify the pure product in step (B05) by blowing nitrogen gas thereon to remove dissolved oxygen, and then perform dispensing in brown glass bottles, and obtain refined hematin.

4. The rapid targeted screening in vitro fermentation system modulating gut TMA production according to claim 1, characterized in that: The vitamin mixture comprises biotin, cobalamin, p-aminobenzoic acid, folic acid, and pyridoxamine, and the mass ratio of the biotin, cobalamin, p-aminobenzoic acid, folic acid, and pyridoxamine is 1-2:1-2:1-2:1-2:1-2.

5. The rapid targeted screening in vitro fermentation system modulating gut TMA production according to claim 4, characterized by: The preparation of the vitamin mixture comprises the following steps, (C01) Take appropriate parts of biotin, cobalamin, p-aminobenzoic acid, folic acid, and pyridoxamine as five active ingredients, and stir and dissolve in a mixed solvent of anhydrous ethanol and distilled water under nitrogen protection; (C02) Adjust the pH of the dissolved solution in step (C01) to 6.6-7, and then mix after adding an appropriate amount of ascorbic acid; (C03) Filter the mixed solution in step (C02) through a PES microporous filter membrane, and the obtained filtrate is the vitamin mixture.

6. The rapid targeted screening in vitro fermentation system modulating gut TMA production according to claim 1, characterized by: The preparation of the choline comprises the following steps, (D01) Analytical pure choline chloride C5H 14 C l NO, dissolved in distilled water, removed of oxides, under magnetic stirring under nitrogen until clear; (D02) Adjust the pH of the solution in step (D01) to 6.9-7.1, and then filter through a PES microfilter membrane, and the obtained filtrate is the choline solution.

7. A method for establishing a rapid targeted screening system for regulating TMA producers in the intestinal tract in vitro fermentation system, characterized in that: comprises the following steps, ​ (S01) Take the appropriate amount of yeast extract, L-cysteine, hematin, sodium chloride, calcium chloride hexahydrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, vitamin mixture, starch, choline and resazurin are mixed, and deoxidized under nitrogen protection to reach anaerobic state; (S02) In the anaerobic workstation, nitrogen and carbon dioxide mixed gas is introduced to establish a simulated intestinal anaerobic environment, and the mixed system of step (S01) is placed in the anaerobic workstation to form a closed system, and the temperature and pressure are kept constant; (S03) Collect the fecal sample of cardiovascular disease patients, anaerobically homogenize and centrifuge to remove impurities, and obtain the fecal inoculum; (S04) The fecal inoculum of step (S03) is added to the mixed system of step (S02) for anaerobic culture to form a TMA in vitro fermentation system; (S05) The TMA in vitro fermentation system in step (S04) is sampled regularly, and the concentrations of TMA and short-chain fatty acids in the sample are detected, and the color change of resazurin is monitored in real time, thereby verifying the anaerobic stability and metabolic activity of the TMA in vitro fermentation system.

8. The method of claim 7, wherein the method is characterized by: The step (S01) is, (S01-1) Under sterile conditions, remove the oxidant from the distilled water, and add the appropriate amount of yeast extract, L-cysteine, hematin, sodium chloride, calcium chloride hexahydrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, vitamin mixture, starch, choline and resazurin for stirring and mixing; (S01-2) Immediately under nitrogen protection, continuously introduce nitrogen into the solution of step (S01-1) to remove dissolved oxygen in the system; The step (S02) is, The deoxidized system of step (S01) is transferred into the anaerobic workstation, and nitrogen and carbon dioxide mixed gas is continuously purged into the reaction space of the anaerobic workstation to maintain constant total pressure and temperature; The step (S03) is, (S03-1) Take the fecal sample of cardiovascular disease patients, mix with pre-deoxidized sterile normal saline under anaerobic conditions in the anaerobic workstation; (S03-2) Homogenize the mixture of step (S03-1) and centrifuge to remove residues to obtain the fecal inoculum; The step (S04) is, (S04-1) The mixed system of step (S02) is loaded into a pressure-resistant glass fermentation bottle, and the fecal inoculum of step (S03) is inoculated; (S04-2) Immediately after inoculation in step (S04-1), replace the space in the bottle with nitrogen to an oxygen-free state; (S04-3) The bottle of step (S04-2) is placed in a constant-temperature anaerobic incubator for culture to form a TMA in vitro fermentation system; The step (S05) is, (S05-1) After step (S04) is fermented for 12-36 hours, sample centrifugation, and take the supernatant; (S05-2) The concentrations of choline, TMA and short-chain fatty acids in the supernatant of step (S05-1) are detected by LC-MS / MS technology, and if the color change of resazurin is maintained between light pink and colorless, it indicates that the system is in a stable anaerobic state.

9. Use of a rapid targeted screening in vitro fermentation system modulating the production of TMA in the gut, characterized in that: The application is any of the following, (1) used for functional screening research of ingredients of food and medicine and Chinese herbal medicine extracts; (2) To verify the specific regulation of the active ingredients screened on the structure and function of intestinal flora.

10. 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