Ox-bile-tolerant in-vitro calculus bovis culture transformation strain and application thereof

Abstract

The invention discloses an oxgall-tolerant in-vitro calculus bovis culture transformation strain and application thereof, and belongs to the technical field of calculus bovis culture. The strain is obtained by separating and purifying fresh ox bile, is identified to belong to escherichia coli, has excellent ox bile tolerance, has the characteristics of beta-glucuronidase, high bezoar conversion rate and high safety, and can be used in the production of in-vitro artificial cultivation of bezoar. The strain is a brand new strain for in-vitro calculus bovis transformation, not only enriches a strain resource library of calculus bovis transformation bacteria, but also provides a new thought for deep research of subsequent in-vitro calculus bovis culture.

Description

Technical Field

[0001] This invention belongs to the field of bezoar cultivation technology, specifically relating to an in vitro bezoar cultivation transformation strain resistant to bovine bile and its application. Background Technology

[0002] Bezoar is a gallstone formed in the gallbladder, bile duct, and hepatic duct of bovine animals. As a precious traditional Chinese medicine, its natural occurrence rate is extremely low, with a single-unit yield typically only a few grams, far from meeting the needs of the pharmaceutical field. To address the shortage and high price of bezoar resources, various alternatives, such as cultured bezoar, in vitro cultured bezoar, and artificial bezoar, have been successfully developed.

[0003] Natural bezoar is a type of pathological gallstone in cattle. Microbial infection of the gallbladder is one of the pathological conditions for the formation of bezoar stones from gallbladder bile. However, bovine bile has broad-spectrum antibacterial properties, and most intestinal bacteria not only cannot survive in it, but also lack the ability to transform into bezoar. Domestic research shows that *Escherichia coli* and its produced β-glucuronidase (β-G enzyme) play an important role in the formation of bezoar. This enzyme can break down conjugated bilirubin (CB) into free bilirubin (UCB). Free bilirubin has poor water solubility and easily combines with metal ions such as calcium and magnesium to form calcium bilirubin salt precipitates, which then polymerize with proteoglycan complexes to form small bezoar particles.

[0004] Existing studies have shown that high concentrations of live bacteria produce large amounts of β-G enzymes. The free bilirubin generated under the action of this enzyme undergoes a series of changes with related components in bile, mainly manifesting in two forms: First, when free bilirubin is in a low-concentration supersaturated state, it promotes cholesterol crystallization, precipitating along with or successively with a large amount of cholesterol and other components to form gallstones with cholesterol as the main component. Second, when free bilirubin is in a high-concentration supersaturated state, it mainly exists in a self-aggregating form, precipitating together with a small amount of cholesterol and other components to form gallstones with bile pigment as the main component. Other reports indicate that bilirubin can disproportionately reduce bile lipids without affecting bile salt secretion, and that the rate of cholesterol crystallization accelerates with changes in the type of bile lecithin, thereby promoting gallstone formation.

[0005] In vitro cultured bezoar, as a raw material for traditional Chinese medicine, health products, and prepared Chinese medicinal herbs, has huge market demand and broad development prospects. The bacterial strain is the core element in the in vitro culture of bezoar; not all bacteria can transform bovine bile into bezoar. Furthermore, the broad-spectrum antibacterial effect of bovine bile inhibits the survival of most intestinal bacteria, and most bacteria cannot survive in bile for long periods. In particular, some highly efficient bezoar-transforming bacteria cannot survive in bile for more than 48 hours. This severely affects the production efficiency of bezoar transformation, directly determining production costs and indirectly impacting the market price of in vitro cultured bezoar.

[0006] The prior art CN117965358A discloses a flavin-producing bacterium that is tolerant to bile. This strain is derived from anal swabs of healthy calves and not only has excellent bovine bile tolerance but also produces high levels of β-glucuronidase, thus possessing the advantages of high bovine gall conversion rate and high safety.

[0007] In the cultivation of bezoar, the flavonoids used must meet several requirements: they must be able to survive and multiply in the gallbladder for a long time, be non-toxic to cattle, have a certain inflammatory effect, and possess strong β-glucuronidase production and bile hydrolysis capabilities. Due to the broad-spectrum antibacterial properties of bovine bile, the growth of most bacteria (including some highly effective flavonoid strains) is inhibited in bile, and some cannot even survive for long periods. This significantly affects the bezoar conversion efficiency and increases the production cost of live bezoar cultivation. Therefore, screening for bile-tolerant, highly effective bezoar-converting strains is a key and challenging aspect of the live bezoar cultivation process. Summary of the Invention

[0008] The purpose of this invention is to provide an in vitro bezoar culture transformation strain that is resistant to bovine bile. This strain is obtained by isolation and purification from fresh bovine bile. The strain has been identified as belonging to Escherichia coli and has excellent bovine bile resistance, possesses β-glucuronidase, and features high bezoar conversion rate and high safety. It can be used in the production of artificially cultured bezoar in vitro.

[0009] This invention is achieved through the following technical solution:

[0010] An in vitro bezoar-transformed strain resistant to bovine bile, wherein the in vitro bezoar-transformed strain is Escherichia coli BC01, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20251645 and deposit date of July 21, 2025.

[0011] Preferably, the in vitro bezoar culture transformant strain is derived from fresh bovine bile, and its 16S rDNA characteristic base sequence is shown in SEQ ID NO.1; the serotype of the transformant strain is ONT:H7.

[0012] An in vitro bezoar culture transformant agent resistant to bovine bile, said agent containing the aforementioned in vitro bezoar culture transformant strain.

[0013] The application of an in vitro bezoar-producing strain resistant to bovine bile in the production and cultivation of bezoar, wherein the production and cultivation refers to the enzymatic culture of the in vitro bezoar-producing strain resistant to bovine bile in bovine bile to obtain the finished bezoar product.

[0014] A method for in vitro production and cultivation of bezoar includes the following steps:

[0015] S1. Select the in vitro bezoar transformation strain that is resistant to bovine bile and inoculate it in the culture medium and culture it overnight to obtain the strain culture solution;

[0016] S2. Select the bacterial culture solution and inoculate it into a new culture medium for continued culture to obtain seed culture;

[0017] S3. Mix the seed liquid, fresh ox bile, antioxidant and stabilizer thoroughly and place them in a dark place for static incubation. After the incubation is completed, the fermentation liquid is obtained.

[0018] S4. After adding chitosan to the fermentation broth, the mixture is allowed to settle in an ice bath in the dark, and the supernatant is removed by a vacuum pump to obtain the sediment.

[0019] S5. The sediment is freeze-dried and ground to obtain the product.

[0020] Preferably, in step S1, 1-2% (V / V) of an in vitro bezoar-resistant transformed strain tolerant to bovine bile is placed in 100 ml of LB medium and cultured overnight at 35-37°C and 200-220 rpm to obtain a strain culture solution.

[0021] In step S2, 100 ml of the strain culture solution is inoculated into 900 ml of LB medium and cultured at 35–37°C and 200–220 rpm to obtain the seed culture.

[0022] Preferably, in step S3, the mixing ratio of the seed liquid, fresh ox bile, antioxidant, and stabilizer is 1L:4.5L:0.5g:200ml;

[0023] The antioxidant is ascorbic acid;

[0024] The stabilizer is calcium chloride;

[0025] The conditions for static incubation are: 40-45℃ for 30-36 hours.

[0026] Preferably, in step S4, 500 ml of chitosan with a mass concentration of 1% is added to the fermentation broth while stirring at 80-100 rpm. The 1% chitosan is prepared by dissolving it in 1% acetic acid.

[0027] The ice bath and light-protected settling time is 3.5 to 4 hours.

[0028] Preferably, in step S5, the total bilirubin content in the product accounts for 28-59% of the total bilirubin in fresh bovine bile.

[0029] Compared with the prior art, the present invention has at least the following technical effects:

[0030] (I) This invention provides an in vitro bezoar culture transformation strain that is resistant to bovine bile. The strain is obtained by isolation and purification from fresh bovine bile. The strain is identified as belonging to Escherichia coli. It has excellent bovine bile resistance and is a strain with β-glucuronidase, as well as high bezoar conversion rate and high safety. It can be used in the production of artificially cultured bezoar in vitro.

[0031] (II) The in vitro bezoar transformation strain resistant to bovine bile provided by this invention is a novel strain for in vitro bezoar transformation. It not only enriches the strain resource bank of bezoar transformation bacteria, but also provides new ideas for further in-depth research on in vitro culture of bezoar.

[0032] (III) In the process of cultivating in vitro bezoar production, the in vitro bezoar-tolerant strain uses fresh bovine bile that has not been subjected to high-temperature sterilization. This method not only ensures the variety of bezoar produced, but also reduces production costs.

[0033] (iv) Antioxidants and stabilizers are used in combination. Ascorbic acid mainly protects the system by resisting oxidation and regulating the reducing environment, and adapts to microbial activity; calcium chloride provides the physical and nutritional basis for fermentation by stabilizing the colloidal structure, providing essential ions, and mitigating bile toxicity. The ultimate goal is to ensure that the mixed system is stable and homogeneous during the "45℃, 36h incubation in the dark" process, while creating suitable conditions for the proliferation and metabolism of seed culture microorganisms.

[0034] (v) The in vitro bezoar culture transformation strain resistant to bovine bile was applied to the fermentation production of in vitro cultured bezoar. The bilirubin yield of the product after fermentation at 45℃ for 36 hours reached 28% to 59% of the total bilirubin in bovine bile. It can tolerate bovine bile with broad-spectrum antibacterial activity, survive in pure bile and use the nutrients in bile for growth. Attached Figure Description

[0035] Figure 1 Example 5 shows a schematic diagram of the products after sedimentation;

[0036] Figure 2 This is a schematic diagram of the settling process in Example 5;

[0037] Figure 3 This is a schematic diagram of the serotype prediction results. Detailed Implementation

[0038] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] Example 1: The process of isolating and purifying the in vitro bezoar-tolerant transformant strain from bovine bile.

[0040] 1. Screening culture medium

[0041] Table 1. Culture media used for screening bovine bile-resistant transforming bacteria.

[0042]

[0043] The following two solid cultures are sterilized, mixed, and then 0.2% neutral red is added. After being poured onto plates, they are used.

[0044]

[0045] The above culture media were autoclaved at 115°C for 30 minutes.

[0046] Mix solid lactose broth and solid bile lactose broth in the specified proportions, and add 0.002% Neutral Red to produce 0%, 30%, 60%, and 100% bile lactose broth plates, respectively. While still hot, pour 20 mL into each 90 mm bacterial culture dish and allow to cool and solidify.

[0047]

[0048] 2. Assay for β-glucuronidase activity

[0049] The 4-nitrophenyl mother liquor was serially diluted and added to an equal volume of 0.05M PBS and 10% of the volume of 0.1M sodium hydroxide. A standard curve was established by measuring the absorbance at a wavelength of 405nm.

[0050] The experimental group consisted of 150 μL of crude enzyme solution, 150 μL of 0.5 M PBS, and 6 mM 4-nitrophenyl β-D-glucuronide; the control group consisted of 150 μL of crude enzyme solution and 150 μL of 0.5 M PBS.

[0051] After incubating in the dark for 1 hour, remove the sample and place it on ice. Add 30 μL of 0.1 M sodium hydroxide to terminate the reaction. Centrifuge and aspirate 200 μL of the supernatant to measure the absorbance at 405 nm. Subtract the absorbance of the control group from the absorbance of the experimental group and substitute it into the standard curve to obtain the enzyme activity value.

[0052] 3. Methods for determining bilirubin content

[0053] The bilirubin content in bovine bile, fermentation products, and waste liquid was tested according to the Chinese Pharmacopoeia (2025 edition, Part I, Medicinal Materials and Processed Pieces, page 187).

[0054] 4. Isolation and purification process of in vitro bovine bile-tolerant transformed strains for bovine bile culture

[0055] Take 50 μL of bile sample and spread it on a 0% bile lactose broth plate overnight. Observe the colonies. If there are no colonies or the number is too small, place the bile sample on a shaker at 37°C overnight to enrich it before spreading. If the colonies are too numerous and clustered together, dilute with 0.8% sterile saline before spreading.

[0056] Pick red colonies and streak them onto 30% bile lactose broth agar plates;

[0057] Pick red colonies and streak them onto 60% bile lactose broth agar plates;

[0058] Pick red colonies and streak them onto 100% bile lactose broth plates.

[0059] Colonies were picked and inoculated into bile lactose broth (liquid) and incubated overnight at 37°C. Obvious turbidity was observed, indicating that the strain was tolerant to bile. The bacterial culture was then frozen with 15% glycerol.

[0060] The strain was inoculated into lactose broth medium and cultured at 37°C and 220 rpm for 18 hours, and the OD600 was measured.

[0061] Centrifuge 1 mL of the solution and resuspend in 0.5 M PBS, washing three times. Sonicate at 200 W for 3 seconds, then repeat 3 seconds * 20 times (crude enzyme solution).

[0062] The activity of β-glucuronidase was measured in the crude enzyme solution. Strains with enzyme activity values ​​higher than those in the blank control group were selected as the chosen strains. The selected strains were inoculated into bile for fermentation. When the bilirubin content in the fermentation product was higher than that in the control group without inoculation, it was designated as the bezoar-transformed strain.

[0063] Example 2: Morphological observation and biochemical identification of bovine bile-resistant in vitro transformed strains of bovine bezoar

[0064] Four strains were isolated in Example 1, inoculated on LB solid plates, and cultured upside down at 37°C for 16 hours. Morphological characteristics were recorded, and Gram staining was performed for microscopic examination.

[0065] Four purified bacterial strains were inoculated onto LB agar plates and incubated upside down at 37°C for 16 hours. A suitable amount of bacterial cells was scraped and placed in sterile 0.8% physiological saline. Following the instructions in the "Biochemical Identification Tubes for Enterobacteriaceae" (Hangwei), the cells were inoculated into micro-biochemical tubes and incubated at 37°C. Positive reactions were observed and recorded as required. The results are shown in Table 2.

[0066] Table 2

[0067]

[0068]

[0069] Note: * indicates that gas production was observed in the tube; - indicates that the reaction was negative; -* indicates that the reaction was negative and gas production was observed; +* indicates that the reaction was positive and gas production was observed; + indicates that the reaction was positive.

[0070] The strain was not found by referring to the Enterobacter biochemical coding book, indicating that the strain is uncommon or has not been reported.

[0071] Combination Figure 2 It can be seen that the colonies of this type of strain are round, with neat edges, smooth and translucent surfaces, and appear as short rods when Gram-negative bacteria are examined under a microscope.

[0072] Example 3: Molecular identification of bovine bile-resistant in vitro bovine gallstone-transformed strains

[0073] 3.1 Crude genome extraction of the strain

[0074] (1) The bacterial cells were lysed using 0.2M NaOH and 1% SDS (sodium dodecyl sulfate) and diluted with sterile pure water. An appropriate amount of bacterial cells was picked up from the tip of a single-colony pipette, pipetted several times in 10 μL of lysis buffer, and then diluted with 150 μL of sterile water.

[0075] 3.2 PCR amplification of 16S rDNA from four strains

[0076] The 16S rDNA of the bacterial strain was amplified using the commonly used universal primers 27F and 1492R.

[0077] The base sequence of the forward F primer is Eubac 27F: AGAGTTTGATCMTGGCTCAG, as shown in SEQ ID NO.5.

[0078] The reverse R primer has the base sequence Eubac 1492R: ggttaccttgttacgactt, as shown in SEQ ID NO. 6.

[0079] 3.3 PCR reaction system for amplifying 16S rDNA of four strains

[0080] PCR reaction system (50 μL): DNA template 2 μL, forward and reverse primers 2 μL each, sterile pure water 19 μL, 2× PCR Master Mix 25 μL. Pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 90 s, repeated 30 times; final extension at 72℃ for 5 min, cooled at 4℃. Electrophoresis on a 0.01% Sybr Green TAE 1% agarose gel at 120V for 30 min, then sent to BGI Genomics for sequencing.

[0081] 3.4 BLAST data comparison using 16S rDNA of the strain

[0082] The sequencing sequences were compared with the Core_nt and Sequences from type material libraries to obtain information on similar strains.

[0083] 3.5 The 16S rDNA sequences of these four strains are as follows:

[0084] BC01, as shown in SEQ ID NO.1;

[0085] BC02, as shown in SEQ ID NO.2;

[0086] BC03, as shown in SEQ ID NO.3;

[0087] BC04, as shown in SEQ ID NO.4.

[0088] Based on the peak plot, a reliable portion was extracted and compared with the Core_nt and Sequences from type material libraries. Multiple strains showed a high degree of similarity to the 16S rDNA of this strain. Considering that strains of the Shigella and Escherichia genera have high 16S rDNA similarity, further data support is needed to determine the genus and species.

[0089] Example 4: Bacterial identification and genotyping based on second-generation sequencing results.

[0090] 4.1 Bacterial cell collection

[0091] Streak the inoculated strain on LB agar plates. Pick a single colony and incubate overnight on LB liquid medium. Inoculate 1% onto LB and culture until OD 0.6. Collect bacterial cells by centrifugation at 8000 rpm for 5 min. Resuspend in 0.8% physiological saline (sterile) and wash three times. Centrifuge to remove supernatant, flash freeze in liquid nitrogen, and mail on dry ice. Send for second-generation assay.

[0092] 4.2 Bacterial identification based on second-generation sequencing results

[0093] From the second-generation sequencing reads, 2000 high-quality reads were randomly selected after filtering and compared with the NT library using Blast software. The results were then combined with the annotation information of this species in the NT library and the comparison results.

[0094]

[0095] The strain was identified as [bacteria](taxid:562)Escherichia coli, belonging to the genus Escherichia.

[0096] 4.3 MLST typing based on second-generation sequencing results

[0097] Upload the data to the MLST prediction website. https: / / cge.food.dtu.dk / services / MLST-2.0 / Obtain the MLST genotype results.

[0098] BC01:Organism:Escherichia coli#1

[0099] Sequence Type: 2005

[0100] adk_6, fumC_29, gyrB_14, icd_16, mdh_9, purA_8, recA_14

[0101] Organism: Escherichia coli #2

[0102] Sequence Type: Unknown

[0103] Nearest ST:952

[0104] dinB_32, icdA_160*, pabB_4, polB_10, putP_26*, trpA_8, trpB_2, uidA_2

[0105] Notes:*alleles with less than 100% identity found

[0106] *putP:Novel allele,ST may indicate nearest ST.

[0107] *icdA:Novel allele,ST may indicate nearest ST.

[0108] 4.4 Serological analysis based on second-generation sequencing results (draft)

[0109] Upload the data to the MLST prediction website. https: / / cge.food.dtu.dk / services / SerotypeFinder / Obtain serotype prediction results.

[0110] The serotype of BC01 is ONT:H7.

[0111] After the above verification, this application selected Escherichia coli BC01, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 20251645 and deposit date of July 21, 2025.

[0112] Example 5: In vitro culture of bezoar from transformant strains resistant to bovine bile

[0113] Experimental Group 1:

[0114] 1% Escherichia coli BC01 was inoculated into 100 mL LB medium and incubated overnight at 37°C and 220 rpm to obtain the culture medium;

[0115] Seed culture was obtained by inoculating 10% of the culture medium into 900 mL of LB medium (100 mL of bacterial culture + 900 mL of medium) at 37℃, 220 rpm, for 6 h.

[0116] Add 0.5g ascorbic acid, 200mL 50% calcium chloride, and 1L seed culture to 4.5L of fresh ox bile and mix thoroughly. Incubate at 45℃ in the dark for 36 hours to obtain the fermentation broth.

[0117] Add 500 mL of 1% chitosan (dissolved in 1% acetic acid) to the fermentation broth while stirring at 100 rpm. Let it settle in an ice bath in the dark for 4 hours, then remove the supernatant using a vacuum pump.

[0118] The remaining sediment volume was approximately 1L. After freeze-drying and grinding, a total of 127.17g of product was obtained, containing 0.0659g of bilirubin.

[0119] The bile sample was tested and calculated to contain 0.1764g of bilirubin in 4.5L of bile sample, with a bilirubin conversion rate of approximately 37.29% (the proportion of total bilirubin in the sediment to the total bilirubin in the raw bile).

[0120] Experimental group 2:

[0121] 1% Escherichia coli BC01 was inoculated into 20 mL LB medium and incubated overnight at 37°C and 220 rpm to obtain the culture medium;

[0122] Seed culture was obtained by inoculating 10% of the culture medium into 180 mL of LB medium (20 mL of bacterial culture + 180 mL of medium) at 37℃, 220 rpm, for 6 h.

[0123] Add 0.5g ascorbic acid, 10mL 40% calcium chloride, and 0.1L seed culture to 4.5L of fresh ox bile and mix thoroughly. Incubate at 45℃ in the dark for 36 hours to obtain the fermentation broth.

[0124] Add 500 mL of 1% chitosan (dissolved in 1% acetic acid) to the fermentation broth while stirring at 100 rpm. Let it settle in an ice bath in the dark for 4 hours, then remove the supernatant using a vacuum pump.

[0125] The remaining sediment volume was approximately 0.2 L. After freeze-drying and grinding, a total of 13.59 g of product was obtained, containing 0.0138 g of bilirubin.

[0126] The sample was tested and calculated to contain 0.0412g of bilirubin in 0.45L of bile, with a bilirubin conversion rate of approximately 33.50% (the proportion of total bilirubin in the sediment to the total bilirubin in the raw bile).

[0127] The sample contained 4.75g of bile acid. According to the analysis, the total bile acid content of 0.45L of bile sample was 15.95g, and the bile acid conversion rate was about 29% (the proportion of total bile acid in the sediment to the total bile acid in the raw bile).

[0128] like Figure 1 The diagram shown is a schematic of the sedimentation products in Example 5.

[0129] like Figure 2 The diagram shown is a schematic representation of the settling process in Example 5.

[0130] like Figure 3 The image shown is a schematic diagram of the serotype prediction results.

[0131] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. 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 bovine bile-resistant in vitro transformed strain of bovine bezoar, characterized in that, The in vitro bezoar culture transformation strain is Escherichia coli BC01, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251645 and deposit date of July 21, 2025.

2. The in vitro bovine bile-resistant transformed strain according to claim 1, characterized in that, The in vitro bezoar culture transformant strain was derived from fresh bovine bile, and its 16S rDNA characteristic base sequence is shown in SEQ ID NO.1; the serotype of the transformant strain is ONT:H7.

3. An in vitro bovine bile-resistant transformant for bezoar culture, characterized in that, The bacterial agent contains the in vitro bezoar culture transformation strain as described in claim 1.

4. The application of a bovine bile-resistant in vitro bezoar culture transformant strain in the in vitro bezoar production and cultivation, characterized in that, The production and cultivation refers to the process of enzymatically culturing the in vitro bezoar-resistant strain described in claim 1 in bovine bile to obtain the finished bezoar product.

5. A method for in vitro production and cultivation of bezoar, characterized in that, Includes the following steps: S1. Select the in vitro bezoar culture transformation strain that is resistant to bovine bile as described in claim 1, inoculate it in the culture medium and culture it overnight to obtain the strain culture solution; S2. Select the bacterial culture solution and inoculate it into a new culture medium for continued culture to obtain seed culture; S3. Mix the seed liquid, fresh ox bile, antioxidant and stabilizer thoroughly and place them in a dark place for static incubation. After the incubation is completed, the fermentation liquid is obtained. S4. After adding chitosan to the fermentation broth, the mixture is allowed to settle in an ice bath in the dark, and the supernatant is removed by a vacuum pump to obtain the sediment. S5. The sediment is freeze-dried and ground to obtain the product.

6. The method for in vitro production and cultivation of bezoar according to claim 5, characterized in that, In step S1, 1-2% (V / V) of bovine bile-tolerant in vitro bovine gall culture transformant strains are placed in 100 ml LB medium and cultured overnight at 35-37°C and 200-220 rpm to obtain the strain culture solution. In step S2, 100 ml of the strain culture solution is inoculated into 900 ml of LB medium and cultured at 35–37°C and 200–220 rpm to obtain the seed culture.

7. The method for in vitro production and cultivation of bezoar according to claim 5, characterized in that, In step S3, the mixing ratio of the seed liquid, fresh ox bile, antioxidant, and stabilizer is 1L:4.5L:0.5g:200ml; The antioxidant is ascorbic acid; The stabilizer is calcium chloride; The conditions for static incubation are: 40-45℃ for 30-36 hours.

8. The method for in vitro production and cultivation of bezoar according to claim 5, characterized in that, In step S4, 500 ml of chitosan with a mass concentration of 1% is added to the fermentation broth while stirring at 80-100 rpm. The 1% chitosan is prepared by dissolving it in 1% acetic acid. The ice bath and light-protected settling time is 3.5 to 4 hours.

9. The method for in vitro production and cultivation of bezoar according to claim 5, characterized in that, In step S5, the total bilirubin content in the product accounts for 28-59% of the total bilirubin in fresh bovine bile.

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