Bacterium for in vitro culture of bovine gallstones and application thereof

Abstract

The application provides a yellowing bacterium capable of being used for in-vitro culture of bovine calculus and application thereof, relates to the technical field of microorganisms, and is obtained by being separated and cultured from natural yak bile. The yellowing bacterium not only has better yellowing capacity, but also has higher bile resistance. The yellowing bacterium is screened by using an optimized screening strategy. The screening strategy can obtain gram-positive yellowing bacterium, and expands the screening range of potential yellowing bacterium. The yellowing bacterium can directly use natural yak, yellow cattle, buffalo and other bovine bile as raw materials to culture bovine calculus in vitro, so that the product is closer to the formation process of natural bovine calculus, has the potential to be developed into a natural bovine calculus substitute, and has a better application prospect in the in-vitro culture of bovine calculus.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to flavonoids that can be used for in vitro culture of bezoar and their applications. Background Technology

[0002] Bezoar was first recorded in the "Shennong's Classic of Materia Medica" and was revered as an essential medicine for clearing heat and detoxifying, opening the orifices and resolving phlegm, and calming wind and stopping spasms. From the time Princess Wencheng of the Tang Dynasty entered Tibet, the cultures of the Central Plains and Tibet have continuously merged and collided.

[0003] The Chinese Pharmacopoeia includes bezoar ( Calculus Bovis is a bovine (Bovidae). Bos taurus domesticus Gmelin (dried gallstones). Since the 2009 "Gansu Provincial Local Medicinal Materials Standard", yak gallstones have also been included in the standard and are used locally as bezoar.

[0004] Since its initial recording, bezoar has been chemically complex, primarily composed of bilirubin compounds (mainly bilirubin and its calcium salts), bile acid compounds (such as cholic acid and deoxycholic acid), cholesterol, various amino acids, polypeptides, proteins, and inorganic elements such as calcium, iron, zinc, and copper. Its unique therapeutic effects, especially its core role in classic emergency formulas like "Angong Niuhuang Wan," give it extremely high clinical value. However, the natural formation rate of bezoar is extremely low (approximately 1-2%), and its acquisition is relatively passive. Coupled with the cyclical nature of current cattle farming methods in my country, production is even lower, leading to an exceptionally acute supply-demand imbalance and persistently high market prices. Against this backdrop, the development of synthetic bezoar is not only an urgent need to alleviate market pressure but also a strategic choice to promote the sustainable development of traditional Chinese medicine.

[0005] Existing technologies for the artificial synthesis of bezoar include in vivo culture, artificial bezoar, and in vitro culture. In vivo culture involves implanting luteinizing factors into the gallbladder of a cow to stimulate bezoar formation. This method suffers from drawbacks such as a long cultivation cycle, reliance on large live animals, and surgical procedures, making large-scale, low-cost industrial production impossible. Artificial bezoar is produced by mixing various raw materials based on the determined chemical composition of natural bezoar. It only roughly simulates natural bezoar from a chemical composition perspective. Due to the complex chemical composition of natural bezoar, artificial bezoar cannot be completely equivalent to the natural product in terms of dissolution characteristics, pharmacokinetics, and the final potency and breadth of its effects. In vitro culture of bezoar utilizes bovine bile, Escherichia coli, and other microorganisms and / or enzymes to simulate the formation process of natural bezoar in vitro. The resulting in vitro cultured bezoar is very similar to natural bezoar in terms of its main components, chemical structure, and pharmacological effects, and its efficacy is far greater than that of chemically synthesized artificial bezoar. Therefore, obtaining a substitute for natural bezoar through in vitro culture has become an important direction in the artificial synthesis of bezoar in recent years. It is generally believed that biliary tract inflammation and infection caused by microbial infection are important pathological conditions for the formation of bezoar stones. Therefore, finding one or more microorganisms with bezoar-transforming capabilities is the key technical point and challenge in in vitro culture of bezoar. Summary of the Invention

[0006] The purpose of this invention is to provide a flavonoid bacterium that can be used for in vitro culture of bezoar, using natural yak bile as raw material, and its application. This flavonoid bacterium is isolated and cultured from natural yak bile and not only has a good flavonoid production capacity, but also has a high tolerance to bile, showing good application prospects in in vitro culture of bezoar.

[0007] The embodiments of the present invention are implemented as follows: Flammogenic bacteria that can be used for in vitro culture of bezoar include at least one of strains FDW-04, FDW-08 and FDW-09. The strain FDW-04 is Macrococcus caseolyticus, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35885 and deposit date of September 12, 2025. The strain FDW-08 belongs to the genus Lysinibacillus sp., and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35886 and deposit date of September 12, 2025. The strain FDW-09 is Bacillus licheniformis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35887 and deposit date of September 12, 2025.

[0008] An antimicrobial agent comprising the aforementioned flammogenic bacteria.

[0009] A method for isolating the above-mentioned flavonoid bacteria, comprising: S1-1. Fresh bile from healthy yaks is mixed with sterile enrichment medium and cultured. S1-2. Observe the culture mixture and screen out samples that show turbidity or color change; S1-3. Inoculate the screened samples onto solid isolation medium, select morphologically uniform monoclonal colonies for subculture until a pure strain is obtained.

[0010] A method for in vitro culture of bezoar, comprising: S2-1. Inoculate the flavonoids into sterilized bovine bile and culture. S2-2. Centrifuge the culture obtained after cultivation and collect the precipitate; S2-3. Freeze-dry the precipitate.

[0011] An in vitro cultured bezoar product is prepared using the above-mentioned in vitro cultured bezoar method.

[0012] The application of one of the above-mentioned flammogenic bacteria or bacterial agents in in vitro culture of bezoar.

[0013] The beneficial effects of the embodiments of the present invention are: This invention provides a flavonoid bacterium, derived from natural yak bile, suitable for in vitro culture of bezoar and its application. This flavonoid bacterium, isolated and cultured from natural yak bile, not only possesses excellent flavonoid production capacity but also exhibits high tolerance to bile. The flavonoid bacterium was obtained through an optimized screening strategy, which yields Gram-positive flavonoids, thus expanding the screening range of potential flavonoids. This flavonoid bacterium can be directly used as raw material for in vitro bezoar culture from bovine animals such as yaks, cattle, and buffalo, resulting in a product that more closely resembles the formation process of natural bezoar. It has the potential to be developed as a substitute for natural bezoar and shows promising application prospects in the in vitro culture of bezoar. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a growth morphology diagram of strain FDW-04 provided in Example 1 of the present invention; Figure 2 This is a morphological diagram of the growth of strain FDW-08 provided in Example 1 of the present invention; Figure 3 This is a morphological diagram of the growth of strain FDW-09 provided in Example 1 of the present invention; Figure 4 This is a flowchart illustrating a method for in vitro culture of bezoar provided in Embodiment 2 of the present invention; Figure 5 This is a microscopic view of the freeze-dried product obtained by the in vitro culture method of bezoar provided in Embodiment 2 of the present invention. Figure 6 The bile salt tolerance test results of the flavonoids provided in Test Example 1 of this invention; Figure 7 This is a comparison diagram of the yellowing effect of the flammogenic bacteria provided in Experimental Example 2 of the present invention; Figure 8 This is a diagram showing the yellowing effect of the flammogenic bacteria provided in Experimental Example 3 of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0017] The following is a detailed description of the flavonoids that can be used for in vitro culture of bezoar according to embodiments of the present invention and their applications.

[0018] This invention provides flavonoids that can be used for in vitro culture of bezoar, including at least one of strains FDW-04, FDW-08, and FDW-09; The strain FDW-04 is Macrococcus caseolyticus, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35885 and deposit date of September 12, 2025. The strain FDW-08 belongs to the genus Lysinibacillus sp., and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35886 and deposit date of September 12, 2025. The strain FDW-09 is Bacillus licheniformis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35887 and deposit date of September 12, 2025.

[0019] This invention also provides a bacterial agent comprising the aforementioned flavinogenic bacteria. This flavinogenic bacteria and the bacterial agent not only possess excellent flavin-producing ability but also exhibit high tolerance to bile.

[0020] Optionally, the microbial agent includes strains FDW-04, FDW-08, and FDW-09, with the ratio of strains FDW-04, FDW-08, and FDW-09 being (1~2):(1~2):(1~2). Using these three strains together can have a synergistic effect, achieving better yellowing efficiency.

[0021] This invention also provides a method for isolating the above-mentioned flavonoid bacteria, comprising: S1-1. Fresh bile from healthy yaks is mixed with sterile enrichment medium and cultured.

[0022] Furthermore, fresh bile is collected from healthy adult yaks. Specifically, several complete gallbladders are obtained from healthy adult yaks slaughtered at a slaughterhouse. During the gallbladder collection process, exogenous microbial contamination and bile leakage are minimized. Specifically, after slaughter, the yaks are immediately transferred to a pre-sterilized area, and the abdominal skin is disinfected by wiping with povidone-iodine. Then, the yak's abdomen is incised using sterilized instruments to expose the gallbladder. During gallbladder removal, the proximal and distal ends of the common bile duct are carefully clamped with sterile hemostatic forceps, and then double-ligated at the clamping points with sterile sutures. The common bile duct is cut between the ligatures to ensure gallbladder integrity and prevent bile leakage. The obtained complete gallbladders are then quickly placed in a pre-prepared sterile, sealed container and transferred to the laboratory at 2–8°C. Once in the laboratory, the complete yak gallbladders are removed under sterile conditions, and the surface is sterilized by wiping with 75% alcohol. The yak gallbladder was then opened using sterile surgical instruments, and the bile was poured into a sterile container for preservation. Optionally, each yak gallbladder contains approximately 50 mL of bile, which can be preserved in a 250 mL Erlenmeyer flask. Freshly extracted bile is a dark green, viscous, transparent liquid. It is then mixed with sterile enrichment medium and cultured.

[0023] Preferably, the enrichment medium of the present invention is BHI medium, i.e., brain and heart extract medium. In other embodiments, other equivalent nutrient enhancers may also be used. 10-20 ml of BHI medium is added to each 250 mL Erlenmeyer flask, and the flask is sealed with sterile sealing film after addition. Optionally, the culture conditions are 36-38°C and 100-200 rpm shaking for 48-96 hours. The shaking process is achieved using a shaker.

[0024] Furthermore, the method for isolating flavonoids provided in this embodiment of the invention further includes: S1-2. Observe the culture mixture and screen out samples that show turbidity or color change; S1-3. Inoculate the screened samples onto solid isolation medium, select morphologically uniform monoclonal colonies for subculture until a pure strain is obtained.

[0025] Furthermore, in steps S1-3, the solid separation medium is TSA medium, which is tryptone soybean agar medium. In other embodiments, other equivalent substrates may also be used.

[0026] Specifically, purification and separation were performed using the streak plating method at 36–38°C. The screened samples were inoculated onto solid isolation medium using a streak plater and incubated for 24 hours. After incubation, the morphology of the colonies on the plates was observed. Several morphologically consistent, independent bacterial single colonies, spaced far from other colonies, were picked using sterile cotton swabs and resuspended in 1 mL of physiological saline. After resuspending, the colonies were again inoculated onto solid isolation medium using a streak plater and incubated at 36–38°C for 24 hours. This process could be repeated multiple times until all colonies growing on the plate were morphologically consistent with the naked eye, thus obtaining the isolated bacterial strain.

[0027] Furthermore, embodiments of the present invention also provide a method for in vitro culture of bezoar, comprising: S2-1. Inoculate the above-mentioned flavonoids into sterilized bovine bile and culture. S2-2. Centrifuge the culture obtained after cultivation and collect the precipitate; S2-3. Freeze-dry the precipitate.

[0028] This culture method is not only effective for yak bile, but also has good results for the culture of bile from traditional bovine animals such as cattle and buffalo.

[0029] Optionally, prior to step S2-1, activation and amplification of the flavonoids are also included. Specifically, the flavonoids are cultured on tryptone soy agar (TSA) plates at 36–38°C for 24 hours. Several clones are picked from the plates, resuspended in 1 mL of physiological saline, and then the McFarland turbidity is adjusted to 0.5 with physiological saline to obtain a bacterial concentration of 1 × 10⁻⁶. 7 cfu / mL ~1×10 8 Bacterial suspension with cfu / mL.

[0030] Furthermore, the bovine bile is obtained from the gallbladder of any one of the following: cattle, yaks, or buffalo. Specifically, fresh bovine bile is collected and sterilized at 40–75°C for 1–3 hours. After the bovine bile cools, the aforementioned bacterial solution is added, resulting in an inoculum of flavogenic bacteria of 1 × 10⁻⁶. 5 cfu / mL ~1×10 6 CFU / mL. Specifically, bovine bile can be dispensed into 250mL Erlenmeyer flasks in 50mL increments, and 0.5mL of the above bacterial solution can be added. After addition, seal the flasks with sterile sealing film.

[0031] Optionally, the culture conditions are 36–38°C and 100–200 rpm shaking for 48–96 hours. Shaking can be achieved using a shaker. The progress of yellowing can be judged by observing the color change of the culture during the culture process.

[0032] In step S2-3, the freeze-drying of the precipitate involves first freezing it at -50℃ for 2-5 hours, then freezing it at -20℃ with a vacuum of ≤10Pa for 12-16 hours, and finally maintaining the vacuum while gradually raising the temperature to 20℃ and holding it there for 2-5 hours to obtain the freeze-dried product.

[0033] This invention also provides an in vitro cultured bezoar product, which is prepared using the above-described in vitro culture method. The raw material for this bezoar product is natural bovine bile, including bile from bovine animals such as yaks, cattle, and buffalo, which allows the product to more closely resemble the formation process of natural bezoar.

[0034] This invention also provides an application of the above-mentioned flavonoids or the above-mentioned bacterial agents in the in vitro culture of bezoar.

[0035] The features and performance of the present invention will be further described in detail below with reference to embodiments. Example 1

[0036] This embodiment provides a flavonoid bacterium that can be cultured in vitro for bezoar, and its isolation method is as follows: S1-1. To minimize exogenous microbial contamination and bile leakage, several intact yak gallbladders were collected from healthy adult yaks at the slaughterhouse and transferred to the laboratory at 2-8℃. Under aseptic conditions, the intact yak gallbladders were removed and their surfaces were sterilized by wiping with 75% alcohol. The gallbladders were then opened using sterile surgical instruments, and the bile was poured into sterile 250mL Erlenmeyer flasks. Each gallbladder contained approximately 50mL of yak bile, which was a dark green, viscous, transparent liquid. 10mL of sterile Brain Heart Extract (BHI) culture medium was then added to each flask, and the flasks were sealed with sterile sealing film. After the procedure, the flasks were placed on a shaker at 37℃ and 200rpm / min for at least 48 hours.

[0037] S1-2. After culturing the mixture of yak bile and brain and heart extract in BHI medium for 48 hours, remove the Erlenmeyer flasks and visually inspect the mixture for turbidity or color change. Remove any Erlenmeyer flasks that show turbidity or color change, and continue culturing the remaining flasks for another 48 hours.

[0038] S1-3. For conical flasks showing turbidity or color change, inoculate onto trypto-soy agar (TSA) plates using a streak plating apparatus and incubate at 37°C for 24 hours. After incubation, observe the morphology of the colonies on the plates. Use sterile cotton swabs to pick several morphologically identical, independent bacterial single colonies that are far apart from other colonies, resuspend them in 1 mL of physiological saline, and then inoculate them again onto trypto-soy agar (TSA) plates using a streak plating apparatus and incubate at 37°C for 24 hours. This process can be repeated multiple times until all colonies growing on the plates are morphologically identical bacterial single colonies, thus obtaining the isolated bacterial strain. The strain should then be numbered and preserved in the laboratory.

[0039] Three strains with good tolerance to yak bile and luteinizing ability were isolated using this method: FDW-04, FDW-08, and FDW-09. (The details are as follows...) Figures 1-3 As shown.

[0040] Genomic DNA was extracted from three strains, FDW-04, FDW-08, and FDW-09, and fragmented into approximately 300bp fragments using a Covaris M220 instrument. Based on this, libraries were constructed and paired-end sequencing was performed using a next-generation sequencer.

[0041] The obtained sequencing results were used for genome assembly, and species identification was performed using KmerFinder 3.2. The results are shown in Table 1: Table 1. Results of strain identification FDW-04 Chlorococcus lysate Macrococcus caseolyticus Macrococcoides caseolyticum corrig. 69966 FDW-08 Lysine Bacillus Lysinibacillus spp Lysinibacillus sp. YS11 2072025 FDW-09 Bacillus licheniformis Bacillus licheniformis Bacillus licheniformis 1402 According to the identification results, strain FDW-04 is Macrococcus caseolyticus, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35885 and deposit date of September 12, 2025; The strain FDW-08 belongs to the genus Lysinibacillus sp., and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35886 and deposit date of September 12, 2025. The strain FDW-09 is Bacillus licheniformis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35887 and deposit date of September 12, 2025.

[0042] It is important to note that all three strains are Gram-positive bacteria. However, current research on artificial bezoar flavonoids has largely focused on Gram-negative bacteria, primarily *Escherichia coli*. One possible reason for this is that most bacteria, especially Gram-positive bacteria, are intolerant to bile, making them difficult to screen using traditional methods—a limitation of previous research conditions.

[0043] The separation method of the present invention can isolate Gram-positive bacteria that are tolerant to bile and have the ability to transform bezoar from yak bile, thus expanding the range of strains to be screened and providing more ideas and directions for discovering the mechanism and process of natural bezoar formation. Example 2

[0044] This embodiment provides a method for in vitro culture of bezoar, which includes: S2-1. The flavonoid bacteria from Example 1 were cultured on tryptone soybean agar (TSA) plates at 37°C for 24 hours. Several clones were picked from the plates and resuspended in 1 mL of physiological saline. The McFarland turbidity was then adjusted to 0.5 with physiological saline, at which point the bacterial concentration was approximately 5 × 10⁻⁶. 7 cfu / mL.

[0045] Fresh yak bile was collected and sterilized at 72℃ for 1 hour. After sterilization, 50 mL was dispensed into 250 mL Erlenmeyer flasks, and 0.5 mL of bacterial dilution solution adjusted to McFarland turbidity of 0.5 was added. The Erlenmeyer flasks were sealed with sterile sealing film and incubated at 37℃ and 200 rpm for 72 hours on a shaker.

[0046] S2-2. Add the culture obtained after incubation to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 20 min. Discard the supernatant after centrifugation. Resuspend the precipitate after centrifugation in 1 mL of sterile ultrapure water and transfer it to a vial.

[0047] S2-3. Freeze-dry the precipitate. First, freeze at -50℃ for 3 hours, then freeze at -20℃ under a vacuum of 10Pa for 12 hours. Finally, while maintaining the vacuum, gradually raise the temperature to 20℃ and continue to maintain it for 3 hours to obtain the freeze-dried product.

[0048] The operation process is as follows Figure 4 As shown, the freeze-dried product is a yellow / golden yellow solid.

[0049] Observe under a microscope (40x) Take an appropriate amount of the product powder onto a glass slide, add a small amount of chloral hydrate to prepare a slide, and observe it under a microscope: the results show obvious yellow granular substances ( Figure 5 (When left to stand for a short time, the pigment dissolves quickly and turns a bright golden yellow, but turns green after a long time.)

[0050] Take a small amount of the product and apply it to your nails to dye them yellow, and the color will not fade easily.

[0051] Experimental Example 1 This experiment used the flavonoids FDW-04, FDW-08, and FDW-09 isolated in Example 1 to test their bile salt tolerance. The specific test method is as follows: The isolated strains FDW-04, FDW-08, and FDW-09 were cultured on tryptone soy agar (TSA) plates at 37°C for 24 hours. Following strain numbering, several clones from each plate were picked and resuspended in 1 mL of physiological saline. The McFarland turbidity was then adjusted to 0.5 with physiological saline, at which point the bacterial concentration was approximately 5 × 10⁻⁶. 7 cfu / mL.

[0052] Fresh yak bile was collected and sterilized at 72℃ for 1 hour. After sterilization, it was divided into two portions. One portion was added with 20% of the sterilized bile volume of brain and heart extract BHI medium and labeled as yak bile + BHI. The other portion was left untreated and labeled as yak bile. Several 250mL Erlenmeyer flasks were prepared, and 50mL of either yak bile + BHI or yak bile was added to each. Then, 0.5mL of different numbered bacterial dilutions were added to each flask, adjusted to McFarland turbidity of 0.5. At this point, the bacterial concentration in either the yak bile + BHI or yak bile was approximately 5 × 10⁻⁶. 5 cfu / mL.

[0053] After sealing the conical flask with sterile sealing film, incubate at 37°C on a shaker at 200 rpm for 72 h, and take samples at 0 h, 24 h, 48 h and 72 h.

[0054] After sampling, the bacterial concentration in yak bile + BHI or yak bile was counted. The method was as follows: 1 mL of yak bile + BHI or yak bile was diluted 1000-fold and 10000-fold with physiological saline, respectively. Then, 100 μL of the diluted solution was plated on tryptone soybean agar (TSA) plates and incubated at 37°C for 24 h. After incubation, the number of clones on the plates was counted. The bacterial count at 0 hours was set as 1. The counting results are shown below. Figure 6 As shown.

[0055] Depend on Figure 6 It can be seen that FDW-04, FDW-08, and FDW-09 not only survive in sterilized yak bile, but also utilize the nutrients within it for growth and proliferation. After 24 hours, they achieve approximately a 3-5 fold increase in growth, and after 48 hours, the increase reaches 5-10 times. Adding 20% ​​BHI (brain and heart extract) culture medium to the bile enhances bacterial proliferation due to the increased nutrient supply, achieving a 10-15 fold increase after 24 hours, and exceeding 30-fold growth after 48 hours. Therefore, the flavonoids isolated using the method of this invention exhibit excellent tolerance to yak bile.

[0056] Experimental Example 2 In this experiment, the flammogenic bacteria isolated in Example 1 were used to culture bezoar in vitro using the method in Example 2, and their bezoar production capacity was tested.

[0057] The tests were conducted using three single groups of FDW-04, FDW-08, and FDW-09, as well as a mixed group of FDW-04+FDW-08+FDW-09 (1:1:1), all at a ratio of 5×10⁻⁶. 5 Inoculation and culture were performed at a concentration of CFU / mL, and the culture results were as follows: Figure 7 As shown.

[0058] Depend on Figure 7As can be seen, after 72 hours of cultivation, FDW-08 (labeled 8 in the figure) transformed the sterile yak bile, which was a dark green, transparent, viscous liquid, into a deep yellow, turbid culture through growth and reproduction in sterile yak bile without any additives. Compared to the FDW-08 yak bile culture, the yak bile cultures of FDW-04 (labeled 4 in the figure) and FDW09 (labeled 9 in the figure) were darker in color, appearing as dark brown or dark green turbid substances. This result further confirms that strains FDW-04, FDW-08, and FDW-09 can not only survive in yak bile but can also utilize the nutrients within it for further reproduction. From the perspective of culture color, intuitively, strain FDW-08 exhibits a better yellow-producing ability than strains FDW-04 and FDW-09. Furthermore, it can be clearly seen that the experimental group (labeled 489 in the figure) with the addition of three strains, FDW-04, FDW-08 and FDW-09, produced a golden-yellow yak bile culture. This color was significantly more yellow than the sterilized yak bile culture using only the three strains. This visually indicates that the three strains exhibited a synergistic effect during the culture process, enhancing the yellowing capacity.

[0059] The precipitate was further separated to prepare a lyophilized product. The contents of free bilirubin, bilirubin, and bile acid in the lyophilized product were determined separately using the following methods: 1) Methods for determining bilirubin Accurately weigh an appropriate amount of bilirubin reference standard and prepare a solution containing 40 μg per ml using dichloromethane. Accurately weigh approximately 50–200 mg of lyophilized powder (finely ground with a glass rod) (passed through a No. 6 sieve), place it in a stoppered conical flask, add 10 ml of 10% oxalic acid solution, seal tightly, vortex to mix, accurately add 20–50 ml of water-saturated dichloromethane, seal tightly, weigh, shake thoroughly, vortex to mix, sonicate (500 W, 53 kHz, 25–35 °C) for 40 minutes, cool, weigh again, replenish the lost weight with water-saturated dichloromethane, shake well, centrifuge (4000 rpm), separate the dichloromethane solution, filter, and use the filtrate as the test sample.

[0060] The liquid chromatograph used octadecylsilane-bonded silica gel as the stationary phase and acetonitrile-1% glacial acetic acid solution (95:5) as the mobile phase; the detection wavelength was 450 nm. Accurately pipette 5 μl each of the reference solution and the test solution and inject them into the liquid chromatograph for determination.

[0061] 2) Determination method of free bilirubin Take an appropriate amount of bilirubin reference standard, accurately weigh it, and add dichloromethane to prepare a solution containing 6.87 μg per ml, which is used as the reference standard.

[0062] Take approximately 10mg~100mg of lyophilized powder (passed through a No. 6 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 10~25ml of dichloromethane, seal tightly, weigh it, shake to mix, sonicate in an ice bath (power 500W, frequency 53kHz) for 40 minutes, weigh it again, replenish the lost weight with dichloromethane, shake well, centrifuge (speed 4000 rpm), separate the dichloromethane solution, filter it, and take the filtrate as the sample to be tested.

[0063] The HPLC system used octadecylsilane-bonded silica gel as the stationary phase and acetonitrile-1% glacial acetic acid solution (95:5) as the mobile phase. The detection wavelength was 450 nm. 5 μl of each of the reference solution and the test solution were precisely injected into the HPLC system for determination.

[0064] 3) Methods for determining bile acids Take an appropriate amount of bile acid reference standard, accurately weigh it, add an appropriate amount of 60% glacial acetic acid to dissolve and dilute to 0.5 mg / mL, and use it as the reference standard.

[0065] Accurately weigh approximately 10-100 mg of lyophilized powder (passed through a No. 6 sieve), place it in a stoppered conical flask, accurately add 10-25 ml of water, seal tightly, weigh, shake to mix, and sonicate in an ice bath (500 W, 53 kHz) for 15 minutes. Weigh again, add 1 mL of dilute hydrochloric acid, shake well, and then extract five times with 15 ml of chloroform each time. Combine the chloroform extracts, evaporate to dryness in a water bath, and dilute to 10 ml in a volumetric flask with methanol to obtain the sample for testing.

[0066] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile-1% glacial acetic acid solution (95:5) was used as the mobile phase; 20 μl of the reference solution and the test solution were accurately pipetted separately and determined by HPLC-ELSD.

[0067] The test results are shown in Table 2.

[0068] Table 2. Test results of freeze-dried products FDW-04 0.90 0.001 8.6 FDW-08 0.88 0.001 8.5 FDW-09 0.80 0.001 8.7 FDW-04,08,09 1.10 0.001 8.4 Sterilized yak bile 0.00 0.000 5.7 As shown in Table 2, bilirubin and free bilirubin were not detected in sterilized yak bile. However, after culturing with the strains described in this invention, bilirubin and free bilirubin were produced in all samples. Among them, the mixed group of FDW-04+FDW-08+FDW-09 showed the best jaundice-producing effect, with a bilirubin content of 1.1% in the product. This indicates that the combined use of the three strains can produce a synergistic effect and further improve the jaundice-producing capacity.

[0069] Experimental Example 3 This experimental example follows the method of Experimental Example 2, but replaces the yak bile in the raw materials with an equal amount of yellow cattle bile, using a mixture of FDW-04+FDW-08+FDW-09 (1:1:1), all at a ratio of 5×10. 5 Inoculation and culture were performed at a concentration of CFU / mL, and the culture results were as follows: Figure 8 As shown.

[0070] Depend on Figure 8 It can be seen that, although quantitative conclusions have not yet been obtained after culturing the strains of the present invention, qualitative analysis based on the color changes shows that the strains of the present invention can produce similar effects on the bile of other cattle besides yak, proving that the strains screened by the present invention have good universality.

[0071] In summary, this invention provides a flavonoid bacterium for in vitro culture of bezoar and its application. This flavonoid bacterium, isolated and cultured from natural yak bile, not only possesses excellent flavonoid-producing ability but also exhibits high tolerance to bile. The flavonoid bacterium was obtained through an optimized screening strategy, which yields Gram-positive flavonoids, thus expanding the screening range of potential flavonoids. This flavonoid bacterium can be directly used as raw material for in vitro bezoar culture from bovine animals such as yaks, cattle, and buffalo, making the product more closely resemble the formation process of natural bezoar and potentially developing it as a substitute for natural bezoar. It shows promising application prospects in the in vitro culture of bezoar.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A flavonoid bacterium that can be used for in vitro culture of bezoar, characterized in that, The strain includes at least one of strains FDW-04, FDW-08, and FDW-09; strain FDW-04 is *Macrococcus caseolyticus*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35885 and deposit date of September 12, 2025; strain FDW-08 is *Lysinibacillus* sp., deposited at the CGMCC with accession number CGMCC No. 35886 and deposit date of September 12, 2025; strain FDW-09 is *Bacillus licheniformis*, deposited at the CGMCC with accession number CGMCC No. 35887 and deposit date of September 12, 2025.

2. A microbial agent, characterized in that, Includes the flavonoids described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The ratio of strains FDW-04, FDW-08 and FDW-09 is (1~2):(1~2):(1~2).

4. A method for in vitro culture of bezoar, characterized in that, include: S2-1. Inoculate the flavonoid bacteria of claim 1 into sterilized bovine bile and culture; S2-2. Centrifuge the culture obtained after culture and collect the precipitate; S2-3. Freeze-dry the precipitate.

5. The method according to claim 4, characterized in that, In step S2-1, the bovine bile is collected from the gallbladder of any one of the following: yak, cattle, or buffalo. The inoculum amount of flavonoids in the bovine bile is 1 × 10⁻⁶. 5 cfu / mL ~1×10 6 cfu / mL; culture conditions were 36-38℃, 100-200 rpm shaking for 48-96 h.

6. An in vitro cultured bezoar product, characterized in that, It is prepared using the in vitro culture method for bezoar as described in claim 4 or 5.

7. The use of a flavonoid as described in claim 1 or a fungal agent as described in any one of claims 2 to 3 in the in vitro culture of bezoar.

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