Enterococcus faecalis, flavi bacteria combination, bacterial agent and use thereof, and method for cultivating bovine calculus

Abstract

The present application belongs to the field of development and utilization of bile-resistant bovine gallstone conversion bacteria resources, and relates to a combination of Enterococcus faecalis and yellow-producing bacteria, a bacterial agent, an application thereof, and a method for cultivating bovine gallstones. A new bacterial species, Enterococcus faecalis WLK2401JZ-07, is screened, which has advantages in cultivating bovine gallstones compared with other yellow-producing bacteria, especially in the aspects of beta-glucuronidase activity in bile, colony survival number, conversion capacity of conjugated bilirubin and conjugated cholic acid during the cultivation process. The combination of Enterococcus faecalis and other bile-resistant microorganisms (such as Escherichia coli, Bacillus, Lactobacillus, etc.) can stably achieve the preparation of high-quality in-vivo cultivated bovine gallstones, and has a synergistic effect. Compared with single-strain fermentation, the method can efficiently produce calcium bilirubinate while ensuring the health of the bovine body, and has a good application prospect in cultivating bovine gallstones.

Description

Technical Field

[0001] This invention belongs to the field of development and utilization of bovine bile-resistant bezoar transformant resources, specifically involving a combination of Enterococcus faecalis, flammogenic bacteria, bacterial agents and their uses, and a method for cultivating bezoar. Background Technology

[0002] Natural bezoar primarily refers to pathological gallstones formed in the gallbladder or bile ducts of cattle. Essentially, it is a gallstone with bilirubin as its main component, containing a mixture of bile acids, deoxycholic acid, cholesterol, ergot cholesterol, fatty acids, lecithin, mucoprotein, calcium, metal ions, and various amino acids. Bezoar is generally classified into four categories: naturally occurring bezoar from cattle, cultured bezoar grown within the cattle body, artificially synthesized bezoar, and in vitro cultured bezoar. The formation process of natural bezoar is complex, involving intricate biochemical and pathological changes, with the core being the abnormal deposition and mineralization of bile components. Due to the unique and pathological nature of this formation process, the incidence of natural bezoar in cattle is very low (usually less than 2%), and its quality and size vary greatly, making it extremely rare and precious. Artificially synthesized bezoar is not as effective as other types of bezoar; although in vitro culture yields more bezoar than natural bezoar, its efficacy cannot match that of natural bezoar; bezoar produced in vivo in the gallbladder of cattle by implanting flavonoid bacteria is the closest in efficacy to natural bezoar and is expected to solve the problem of the severe shortage of natural bezoar.

[0003] The core inducing factor for natural bezoar is cholecystitis or biliary obstruction. The most common cause of cholecystitis is bacterial infection (such as Escherichia coli). Bacterial inflammation disrupts the normal structure and function of the gallbladder wall, while the β-glucuronidase produced by bacteria hydrolyzes conjugated bilirubin in bile, converting it into free bilirubin. Bovine bile itself has a broad-spectrum antibacterial effect; most intestinal bacteria cannot survive in bovine bile, let alone possess the ability to convert bezoar. Literature reports some bezoar-producing bacterial species and their luteinizing mechanisms. Bile-resistant luteogenic bacteria, such as Escherichia coli and their produced β-glucuronidase (β-G enzyme), play an important role in bezoar formation. Furthermore, parasites (such as liver flukes), bile duct strictures, bile duct stones, or other causes of biliary obstruction impede bile flow, ultimately leading to bile stasis, creating conditions for the precipitation of components such as bilirubin calcium.

[0004] The general procedure for producing in vivo cultured bezoar involves preparing a certain amount of bacterial inoculum solution externally, inoculating the bacteria into the bovine gallbladder, and then obtaining in vivo cultured bezoar through a long-term feeding process. However, after inoculation, several factors are encountered. First, because bile is less nutritious than the culture medium, the bacteria require an adaptation period and cannot multiply rapidly in a short time. Second, a small amount of bacteria introduced into the gallbladder remains suspended for a short period and is diluted by the bile. The gallbladder's physiological contraction and expansion releases bile, causing some of the inoculated bacteria to be expelled with it. Third, the implantation surgery typically creates an incision, usually around 10 cm in length, and the antibody immune response produced by the bovine body can kill a significant portion of the bacteria. All these factors contribute to a low survival rate of the implanted bacteria, resulting in low yield, poor quality, or even implantation failure of in vivo cultured bezoar.

[0005] Most studies exploring in vivo cultivation of bezoar rely on a single inoculation with a single luteogenic strain. However, in vivo bezoar cultivation involves a complex mixture of factors, resulting from multiple interactions. A single strain often lacks the ability to produce synergistic effects or generate high-quality in vivo bezoar. A single inoculation cannot guarantee the survival rate of the luteogenic strain, nor can it guarantee the composition ratio of the surviving strain, thus compromising the luteogenic rate and the quality of the resulting bezoar. Furthermore, the success rate of the traditional "one-size-fits-all" implantation method is greatly affected by individual differences in cattle. Excessive implantation may lead to discomfort or an overreaction in the cattle, affecting fattening results; insufficient implantation may result in failure due to low enzyme content and low ability to convert and bind bile acids.

[0006] Therefore, screening for high-performance jaundice-inducing strains and / or flora and successfully applying them to the large-scale production of in vivo cultured bezoar are key to obtaining high-quality in vitro cultured bezoar. This is of great significance for meeting the pharmaceutical market's demand for bezoar, increasing farmers' profits, and promoting the large-scale production of in vivo cultured bezoar. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a combination of Enterococcus faecalis, flavonoids, microbial agents, their uses, and a method for cultivating bezoar. The aim is to solve the problems of low colonization rate of single microbial strains, low success rate of bezoar cultivation, and poor quality of bezoar; as well as the problems of low success rate of bezoar cultivation and uncontrollable bezoar quality due to individual differences in cattle, which leads to uncertain benefits for farmers.

[0008] This invention provides a strain of Enterococcus faecalis, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 13, 2024, with accession number GDMCC NO: 65630.

[0009] The present invention provides a combination of flammable bacteria, comprising Enterococcus faecalis and Escherichia coli in a ratio of 0.2-1:0.2-1; wherein the Enterococcus faecalis includes the Enterococcus faecalis as described above.

[0010] Preferably, the quantity ratio is 0.2-0.8:0.2:-0.8.

[0011] Preferably, the quantity ratio is 0.2-0.5:0.5-0.8.

[0012] Preferably, it consists of the following proportions of microbial strains:

[0013] The ratio of Enterococcus faecalis: Escherichia coli: Bacillus: Lactobacillus is 0.25-0.4:0.25-0.4:0.1-0.25:0.1-0.25; the Enterococcus faecalis includes the Enterococcus faecalis as described above.

[0014] Preferably, the Bacillus includes Bacillus coagulans. Bacillus coagulans The lactobacilli include Lactobacillus curvatureii. Lactobacillus crispatus .

[0015] The present invention provides a microbial agent comprising Enterococcus faecalis as described above, or a combination of flammogenic bacteria as described in any of the preceding claims.

[0016] The present invention provides the use of Enterococcus faecalis as described above, the combination of flammogenic bacteria as described in any of the preceding claims, or the bacterial agent as described above in the cultivation of bezoar.

[0017] This invention provides a method for cultivating bezoar, comprising:

[0018] The Enterococcus faecalis, the combination of flammable bacteria as described above, or the bacterial agent as described above are mixed with bile and fermented.

[0019] Preferably, the cultivation is in vivo cultivation; the in vivo cultivation method includes:

[0020] Step 1: Mix the Enterococcus faecalis, the flammogenic bacteria combination, or the bacterial agent with bile to prepare a flammogenic liquid;

[0021] Step 2: The bezoar bed is implanted into the gallbladder of the cow;

[0022] Step 3: Introduce the yellowing liquid into the bezoar bed;

[0023] Step 4: Normal feeding;

[0024] The order of steps 1 and 2 can be changed arbitrarily.

[0025] Preferably, in step 3, the yellowing solution and polyacrylamide hydrogel are introduced into the bezoar bed separately and / or simultaneously;

[0026] And / or, in step 4, after normal feeding for 7-15 days, the Enterococcus faecalis or the combination of flammogenic bacteria are implanted a second time, and the animals are fed for another 9-12 months.

[0027] This invention screened and obtained a novel bacterial strain, *Enterococcus faecalis* WLK2401JZ-07, which has advantages in cultivating bezoar compared to other flammogenic bacteria, especially in terms of β-glucuronidase activity in bile, colony survival rate, and ability to convert conjugated bilirubin and bile acids during the cultivation process. Combining *Enterococcus faecalis* with other bile-resistant microorganisms (such as *Escherichia coli*, *Bacillus*, and *Lactobacillus*) can stably achieve the preparation of high-quality in vivo cultured bezoar. Furthermore, compared to single-strain fermentation, it can efficiently produce bilirubin calcium while ensuring the health of the cattle. Moreover, the microorganisms described in this invention are all common symbiotic and / or probiotic microorganisms in the gastrointestinal tract. In the preparation of bacterial solutions and bezoar production, they offer advantages such as a mild and simple production process, low production cost, no use of flammable or explosive solvents, reduced environmental pollution, safe production process, emission reduction and energy saving, and safe product quality when applied to pharmaceutical products, resulting in significant economic and social benefits.

[0028] The flavin-inducing bacteria combination provided by this invention, with Enterococcus faecalis as its main component, has great potential in the cultivation of bezoar. Enterococcus faecalis' excellent mucosal barrier-forming function has a positive impact on postoperative inflammation control and recovery in cattle. Enterococcus faecalis also has a strong colonization ability on the mucosa, ensuring the concentration of bacteria and the amount of β-G enzyme produced in the gallbladder, thus guaranteeing the abundance of the combined bacteria after implantation and ensuring the quality of the bezoar produced. The flavin-inducing bacteria and their combination of this invention all have excellent β-glucuronidase production capabilities, especially Escherichia coli, which has high β-glucuronidase activity and strong ability, resulting in a high direct bilirubin conversion rate, which can accelerate the conversion of bile into bezoar. It is safe to use and can be applied to the efficient in vitro production of bezoar and the live culture of bezoar from cattle. The combined bacteria provided by this invention contain lactic acid bacteria, which can regulate the bile microenvironment. Under acidic conditions, free bilirubin and cholesterol co-precipitate, making it easier for conjugated bilirubin and calcium ions in the bile fermentation broth to become free, increasing the probability of bilirubin calcium formation and thus increasing bezoar yield. Compared with single-strain fermentation, using the flavin-inducing bacterial composition of this invention for in vivo bezoar cultivation allows for synergistic effects between bacterial strains, promoting the colonization of the flavin-inducing bacterial composition in the gallbladder. This results in high-quality in vivo cultured bezoar in a shorter time, significantly reducing the in vivo bezoar cultivation time.

[0029] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0030] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0031] Instructions for the Preservation of Biological Samples

[0032] Enterococcus faecalis WLK2401JZ-07 was deposited on December 13, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC: 65630. Its Latin scientific name is... Enterococcus faecalis . Detailed Implementation

[0033] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0034] TSB medium (Tryptic Soy Broth), also known as tryptic soybean broth or tryptic soybean soup, contains: tryptic peptone 16.5-17.0 g / L, soybean papain hydrolysate 2.87-3.02 g / L, sodium chloride 4.96-5.02 g / L, glucose 2.46-2.63 g / L, dipotassium hydrogen phosphate 2.43-2.56 g / L, pH 7.3±0.2.

[0035] TSA (Tryptic Soy Broth), also known as tryptic soybean solid medium or tryptic soybean solid medium, consists of: tryptic peptone 16.5-17.0 g / L, soybean papain hydrolysate 2.87-3.02 g / L, sodium chloride 4.96-5.02 g / L, glucose 2.46-2.63 g / L, dipotassium hydrogen phosphate 2.43-2.56 g / L, 12 g / L agar, 5-10% defibrinated sheep blood, and pH 7.3±0.2.

[0036] The jaundice-inducing bacteria strain used in this invention was derived from the bezoar bile produced in the gallbladder of a 5-year-old (multiparous) Simontal cow from a cattle farm in Ningxia.

[0037] The fresh bile used in this invention comes from a slaughterhouse in Sichuan Province, and all fresh bile is collected and used immediately.

[0038] Thermosensitive gel for in vivo transplantation is sterilized and ready for use (please refer to patent CN118716281A for details).

[0039] The conventional plate coating method was used to screen jaundice-causing bacteria in bovine bile. The obtained single strains were sent to a professional microbial identification institution for identification, and some of them were sent to the Guangdong Provincial Microbial Culture Collection Center for preservation.

[0040] All other strains used in this invention are commercially available.

[0041] E. coli Escherichia coli GDMCC NO. 801268;

[0042] Bacillus coagulans Bacillus coagulans GDMCC NO. 1.420;

[0043] Lactobacillus curvature Lactobacillus crispatus GDMCC NO. 1.4925.

[0044] Example 1: Screening of Flavogenic Bacteria

[0045] The first flammable bacteria (Enterococcus) were obtained by screening using the following method:

[0046] (1) Initial screening

[0047] Bovine bile was extracted from the bovine gallbladder and serially diluted with sterile PBS (10⁻¹⁰). -1 Up to 10 -5 Take 100 μL of stock solution (or diluted solution) and spread it evenly on the bile-resistant Enterococcus faecalis selection medium. Invert the plate in a biochemical incubator and incubate at 37°C for 24-48 hours.

[0048] The primary screening medium for bile-resistant enterococci (Bile BAE) consists of the following components: 17.5 g tryptone, 3 g soybean peptone, 2.5 g glucose, 40 g oxalate, 2.5 g dipotassium hydrogen phosphate (K₂HPO₄), 0.25 g sodium azide (NaN₃), 1 g aescin, 0.5 g ferric ammonium citrate, 15 g agar, pH 7.1 ± 0.2. (Dissolve in 0.5 L of water, sterilize at 121 °C and 0.1 MPa for 15 min, and add 0.5 L of preheated sterile bile to 50 °C when cooled to 50 °C).

[0049] Black colonies were obtained by screening on bile BEA.

[0050] (2) Secondary screening

[0051] The secondary screening was performed using bile-resistant enterococci secondary screening medium (Bile Slanetz-Bartley), with the following components:

[0052] 15.0 g tryptone, 5.0 g yeast extract, 2.0 g glucose, 0.5 g dipotassium hydrogen phosphate (K₂HPO₄), 15.0 g agar, 0.4 g sodium azide (NaN₃), and 0.1 g triphenyltetrazolium chloride (TTC). (Dissolve the above substances in 0.5 L of water, sterilize at 121 °C and 0.1 MPa for 15 min, and cool to 50 °C. Mix with 0.5 L of preheated sterile bile containing 0.4 g sodium azide and 0.1 g TTC, pour aseptically into petri dishes, and store in the dark after solidification (TTC is light-sensitive).)

[0053] Black colonies from the bile BEA were streaked onto modified bile Slanetz-Bartley agar plates and incubated at 37°C for 24 hours. Red colonies were candidate bacteria of the Enterococcus spp.

[0054] (3) Proliferation culture

[0055] Red to dark red colonies (1–2 mm in diameter, round and raised) were selected from bile on Slanetz-Bartley plates for liquid fermentation, while 16S rRNA sequencing and biological preservation were performed simultaneously.

[0056] Enterococcal bile proliferation medium (1L): Bovine brain extract: 200 g, bovine heart extract: 250 g, peptone: 10 g, glucose: 2 g, sodium chloride (NaCl): 5 g, disodium hydrogen phosphate (Na2HPO4): 2.5 g, pH: 7.4 ± 0.2. (Dissolve the above substances in 0-0.1L of water, sterilize at 121℃, 0.1MPa for 15 min, cool to room temperature, and then add 0.9-1L of sterile bile.)

[0057] Place the inoculated bile-resistant enterococci at 10°C–45°C (preferably 37°C) and incubate statically or with gentle shaking (preferably 100–150 rpm) for 8–30 h (preferably 18–24 h).

[0058] The OD600 value, β-glucuronidase activity, and bile salt hydrolase (BSH) activity of the proliferation culture medium were measured.

[0059] When OD600≥1.2, β-glucuronidase activity≥500U / L, and bile salt hydrolase (BSH) activity≥15U / mg protein, it is judged as a complete primary flammatory bacterium culture.

[0060] And / or,

[0061] After serial dilution, the samples were plated onto BHI agar, and the CFU / mL count, β-glucuronidase activity, and bile salt hydrolase (BSH) activity were calculated.

[0062] When CFU / mL is ≥1.0 10 12 Bacteria are classified as flammogenic bacteria when β-glucuronidase activity is ≥500 U / L and bile salt hydrolase (BSH) activity is ≥15 U / mg.

[0063] After screening using the above methods, the jaundice-causing bacteria strain was obtained and identified by a professional microbial identification institution in Sichuan Province as: Enterococcus faecalis (…). Enterococcus faecalis WLK2401JZ-07 was deposited on December 13, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC: 65630.

[0064] Example 2 Enterococcus faecalis ( Enterococcus faecalis WLK2401JZ-07 is used for in vivo culture of bezoar.

[0065] The Enterococcus faecalis WLK2401JZ-07 strain obtained in Example 1 was used to culture bezoar in vivo. The steps are as follows:

[0066] Step S1: Prepare sterile bile.

[0067] Fresh bile is collected, filtered through eight layers of gauze to remove impurities, and then placed in a sterilization bag for irradiation sterilization to remove any possible microorganisms and viruses.

[0068] The sterilization method is CO2 60 Irradiation sterilization (dosage can be selected from 10-20KGy), preferably 15KGy.

[0069] Step S2: Prepare concentrated flavonoid agent.

[0070] Enterococcus faecalis WLK2401JZ-07 was fermented and propagated using conventional fermentation methods. The resulting fermentation broth was centrifuged at 4°C and 5000 rpm for 10 min, and then resuspended in 5 mL of sterile bile to achieve a bacterial concentration of 10. 13 Concentrated flammogenic bacteria solution at CFU / mL.

[0071] Step S3: First in vivo culture.

[0072] The first implantation of the bezoar bed, thermosensitive polyacrylamide hydrogel, and bacterial strain (for details, please refer to patent CN118716281A) is as follows:

[0073] Step 1: The bezoar bed is implanted into the gallbladder of a Simmental cow using minimally invasive surgery;

[0074] Step 2: Mix the prepared thermosensitive polyacrylamide hydrogel with 20 mL of concentrated flavogenic bacteria agent and inject it into the bezoar bed;

[0075] Alternatively, the prepared thermosensitive polyacrylamide hydrogel can be injected into the bezoar bed first, and then 20 mL of concentrated flavonoid agent can be injected into the bezoar bed.

[0076] Step S4: Normal feeding.

[0077] Step S5: Perform a routine blood test on the cow within 7-15 days after the operation, and carefully observe and record the cow's postoperative diet, defecation, sleep, weight gain and other conditions.

[0078] Once the indicators described in step S5 return to preoperative levels, bile is extracted for testing of bilirubin, bile acids, β-glucuronidase, and bacterial count.

[0079] Step S6: Implant the jaundice-inducing agent for the second time. The composition and amount of the jaundice-inducing agent should be the same as those used for the first implantation.

[0080] Step S7: A second jaundice-inducing bacterial culture is implanted using an epidural puncture procedure.

[0081] Step S8: Feed the implanted cattle normally.

[0082] Step S9: Slaughter the cattle 9-12 months later, remove the gallbladder, filter the bile, and extract the bezoar.

[0083] Step S10: The obtained in vivo cultured bezoar was analyzed according to the Pharmacopoeia, and the results are shown in Table 1.

[0084] Table 1

[0085]

[0086] Example 3: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0087] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0088] Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus curvature = 0.4:0.4:0.1:0.1.

[0089] The method of Example 2 was used to cultivate bezoar in vivo, except that concentrated flavonoid agents of the above four strains were prepared separately, and then mixed in the ratio of Enterococcus faecalis: Escherichia coli: Bacillus: Lactobacillus = 0.4:0.4:0.1:0.1 to obtain a mixed bacterial solution. The total volume of the mixed bacterial solution used for the first in vivo culture was 20 mL.

[0090] Example 4: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0091] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0092] Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus curvature = 0.3:0.3:0.2:0.2.

[0093] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0094] Example 5: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0095] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0096] Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus curvature = 0.25: 0.25: 0.25: 0.25.

[0097] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0098] Example 6: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0099] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0100] Enterococcus faecalis: Escherichia coli = 0.5:0.5.

[0101] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0102] Example 7: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0103] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0104] Enterococcus faecalis: Escherichia coli = 0.4:0.6.

[0105] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0106] Example 8: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0107] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0108] Enterococcus faecalis: Escherichia coli = 0.6:0.4.

[0109] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0110] Example 9: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0111] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0112] Enterococcus faecalis: Escherichia coli = 0.2:0.8.

[0113] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0114] Example 10: A combination of flavonoid bacteria and its application in in vivo culture of bezoar

[0115] The flammatory bacteria combination in this embodiment consists of the following bacterial species in the following proportions:

[0116] Enterococcus faecalis: Escherichia coli = 0.8:0.2.

[0117] The method of Example 3 was used to cultivate bezoar in vivo, with the only difference being the ratio of flavonoid bacteria.

[0118] The technical solution of the present invention will be further explained through experiments below. The Enterococcus faecalis used in the following experimental examples was obtained through screening in Example 1, and Escherichia coli was used... Escherichia coli GDMCC NO. 801268, Bacillus coagulans was used Bacillus coagulans GDMCC NO. 1.420, Lactobacillus curvatureis was used Lactobacillus curly GDMCC NO. 1.4925.

[0119] Experimental Example 1: Screening Experiment of Flammogenic Bacterial Combinations

[0120] I. Experimental Methods

[0121] 1. Prepare concentrated flavonoid agent.

[0122] According to conventional fermentation methods, Enterococcus faecalis ( Enterococcus faecalis WLK2401JZ-07, Escherichia coli ( Escherichia coli ), Lactobacillus curvature ( Lactobacillus crispatus ), Bacillus coagulans ( Bacillus coagulans The fermentation broth was subjected to fermentation and propagation. The resulting fermentation broth was centrifuged at 4°C and 5000 rpm for 10 min, and then resuspended in 5 mL of sterile bile to form four bacterial strains at a concentration of 10. 13 Concentrated flammogenic bacteria solution at CFU / mL.

[0123] 2. Experimental Grouping

[0124] This experiment was divided into three groups: single flammable bacteria group, Enterococcus faecalis + Escherichia coli group, and quadruple flammable bacteria group. The single flammatory bacteria group was inoculated with *Enterococcus faecalis*, *Escherichia coli*, *Bacillus coagulans*, and *Lactobacillus curvatureii*, respectively. The *Enterococcus faecalis* + *Escherichia coli* group was inoculated with mixed bacterial solutions of *Enterococcus faecalis* and *Escherichia coli* in ratios of 0.5:0.5, 0.4:0.6, 0.6:0.4, 0.2:0.8, and 0.8:0.2, respectively. The quadruple flammatory bacteria group was inoculated with mixed bacterial solutions of *Enterococcus faecalis*, *Escherichia coli*, *Bacillus coagulans*, and *Lactobacillus curvatureii* in ratios of 0.25:0.25:0.25:0.25, 0.4:0.4:0.1:0.1, 0.1:0.1:0.4:0.4, 0.3:0.3:0.2:0.2, and 0.2:0.2:0.3:0.3, respectively. The volume of flammatory bacteria solution introduced into the biomimetic fermentation system for each group was 20 mL.

[0125] According to the grouping, 50 mL of the above bacterial solution was taken and inoculated into the biomimetic fermentation system (for details, please refer to patent CN119736143A), and the changes in bile bilirubin, bile acid, β-glucuronidase content and colony count were detected.

[0126] 3. Methods for determining bilirubin content

[0127] Chinese Pharmacopoeia 2025 Edition, Part I, Calculus Bovis, Methods for Bilirubin Detection

[0128] 4. Methods for determining free bilirubin content

[0129] Chinese Pharmacopoeia 2025 Edition, Part I, Calculus Bovis Section, Method for Determination of Free Bilirubin

[0130] 5. Methods for determining bile acid content

[0131] The 2025 edition of the Chinese Pharmacopoeia, Part I, Bezoar section, combined with bile acid detection methods.

[0132] 6. Methods for determining the content of free bile acids

[0133] Chinese Pharmacopoeia 2025 Edition, Part I, Calculus Bovis Section, Method for Detection of Free Bile Acids

[0134] 7. Methods for determining β-glucuronidase activity

[0135] Use the enzyme-linked glucuronidase assay kit to perform the test according to the instructions.

[0136] 8. Methods for determining colony count

[0137] Viable bacteria were counted using the dilution plating method.

[0138] II. Experimental Results

[0139] The effects of the species and proportion of flavonoid strains on β-glucuronidase and colony count in bovine bile are shown in Table 2. The effects of the species and proportion of flavonoid strains on bilirubin and bile acids in bovine bile are shown in Table 3.

[0140] Table 2. Effects of different bacterial strains and their different implantation ratios on β-glucuronidase and colony count.

[0141]

[0142] Table 3. Effects of different bacterial strains and their different implantation ratios on bilirubin and bile acids.

[0143]

[0144] For the single flavin strain of Enterococcus faecalis, after the inoculation of flavin strain, the β-glucuronidase activity was significantly increased, the colony survival rate was high, and the ability to convert conjugated bilirubin and conjugated bile acids was strong, indicating that Enterococcus faecalis WLK2401JZ-07 can be used for in vivo culture of bezoar.

[0145] In the *Enterococcus faecalis* + *Escherichia coli* group, β-glucuronidase activity was significantly increased after implantation of the flavogenic bacteria, with a greater effect than any single flavogenic bacteria group at the same implantation dose, especially the mixed bacteria groups at ratios of 0.2:0.8, 0.5:0.5, and 0.4:0.6. Simultaneously, the colony survival rate was high, and the ability to convert conjugated bilirubin and bile acids was strong, with the conversion of conjugated bile acids being more effective than any single flavogenic bacteria group at the same implantation dose. This indicates that *Enterococcus faecalis* WLK2401JZ-07 and *Escherichia coli* at a ratio of 0.2-0.8:0.2:-0.8 produced a synergistic effect.

[0146] For the quadruple flavin-causing bacteria group, β-glucuronidase activity was increased after implantation of flavin-causing bacteria, especially at ratios of 0.4:0.4:0.1:0.1, 0.3:0.3:0.2:0.2, and 0.25:0.25:0.25:0.25. The improvement effect was significantly better than that of any single flavin-causing bacteria group with the same implantation dose, and the Enterococcus faecalis + Escherichia coli group with the same implantation dose at a ratio of 0.5:0.5. This indicates that the four flavin-causing bacteria screened in this invention produced a synergistic effect at ratios of 0.4:0.4:0.1:0.1, 0.3:0.3:0.2:0.2, and 0.25:0.25:0.25:0.25.

[0147] In addition, for the tetragenous flavonoids group, the colony survival rate and the ability to convert conjugated bilirubin and bile acids were high when the ratios were 0.4:0.4:0.1:0.1, 0.3:0.3:0.2:0.2, and 0.25:0.25:0.25.

[0148] In summary, Enterococcus faecalis WLK2401JZ-07 can be used for in vivo culture of bezoar, and its jaundice-inducing effect is superior to that of Bacillus coagulans and Lactobacillus curvature. Enterococcus faecalis WLK2401JZ-07 and Escherichia coli exhibit a synergistic effect in a combination of 0.2-0.8:0.2:-0.8. Enterococcus faecalis WLK2401JZ-07, Escherichia coli, Lactobacillus curvature, and Bacillus coagulans, four jaundice-inducing bacteria, exhibited a synergistic effect in ratios of 0.4:0.4:0.1:0.1, 0.3:0.3:0.2:0.2, and 0.25:0.25:0.25:0.25.

[0149] As can be seen from the above embodiments and experimental examples, the present invention screened a new strain of Enterococcus faecalis WLK2401JZ-07, which has advantages in the cultivation of bezoar compared with other flammogenic bacteria, especially in terms of β-glucuronidase activity in bile, colony survival count, and ability to convert conjugated bilirubin and bile acids during the cultivation process. Combining Enterococcus faecalis with other bile-resistant microorganisms (such as Escherichia coli, Bacillus, Lactobacillus, etc.) can stably achieve the preparation of high-quality in vivo cultured bezoar, and compared with single-strain fermentation, it can efficiently produce bilirubin calcium while ensuring the health of the cattle. Moreover, the microorganisms described in this invention are all common symbiotic microorganisms and / or probiotic microorganisms in the gastrointestinal tract. In the preparation of bacterial solutions and bezoar production, they have advantages such as mild and simple production processes, low production costs, no use of flammable and explosive solvents, reduced environmental pollution, safe production processes, emission reduction and energy saving, and safe product quality when applied to pharmaceutical products, resulting in good economic and social benefits.

Claims

1. Enterococcus faecalis (E. faecalis) characterized in that: Enterococcus faecalis It is preserved in Guangdong Microbial Culture Collection Center, the preservation date is December 13, 2024, and the preservation number is GDMCC NO: 65630. ​ 2. A yellowing bacteria combination, characterized by, It includes Enterococcus faecalis and Escherichia coli, and the quantity ratio is 0.2-1:0.2-1; the Enterococcus faecalis includes the Enterococcus faecalis of claim 1.

3. The xanthogenic bacterial combination according to claim 2, characterized in that: The quantity ratio is 0.2-0.8:0.2-0.

8.

4. The xanthogenic bacterial combination according to claim 2, characterized in that, It is composed of the following proportions of strains: The quantity ratio of Enterococcus faecalis, Escherichia coli, Bacillus and Lactobacillus is 0.25-0.4:0.25-0.4:0.1-0.25:0.1-0.25; the Enterococcus faecalis includes the Enterococcus faecalis of claim 1.

5. The xanthogenic bacterial combination according to claim 4, characterized in that: The Bacillus includes Bacillus coagulans (ATCC® 7053) Bacillus coagulans The Lactobacillus includes Lactobacillus crispatus (ATCC® 33820) Lactobacillus crispatus .

6. An inoculant characterized in that: The bacterial agent contains the Enterococcus faecalis of claim 1 or the Flavobacterium combination of any one of claims 2-4.

7. Use of the Enterococcus faecalis of claim 1, the Flavobacterium combination of any one of claims 2-5 or the bacterial agent of claim 6 in cultivating bovine calculus.

8. A method for cultivating bovine gallstones, characterized by, It includes: Mixing the Enterococcus faecalis of claim 1, the Flavobacterium combination of any one of claims 2-5 or the bacterial agent of claim 6 with bile and fermenting.

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