Enterococcus faecalis, xanthogenic bacterium composition, microbial inoculum, application of microbial inoculum and method for cultivating calculus bovis

Abstract

The invention belongs to the field of resource development and utilization of oxgall-resistant bezoar transformed bacteria, and relates to enterococcus faecalis, a xanthogenic bacteria combination, a microbial inoculum, application of the microbial inoculum and a method for cultivating bezoar. The new strain enterococcus faecalis WLK2401JZ-07 is obtained through screening, and compared with other yellow-causing bacteria, the new strain enterococcus faecalis WLK2401JZ-07 has the advantages in calculus bovis cultivation, especially in the aspects of beta-glucuronidase activity in bile in the cultivation process, the survival number of bacterial colonies, the capability of being converted and combined with bilirubin and cholic acid and the like. Enterococcus faecalis and other bile-resistant microorganisms (such as escherichia coli, bacillus, lactobacillus and the like) are combined for use, so that preparation of high-quality in-vivo cultivated calculus bovis can be stably realized, and a synergistic effect is generated. And compared with single strain fermentation, the calcium bilirubin can be efficiently produced, meanwhile, the health of cattle bodies can be guaranteed, and the method has a good application prospect in the aspect of culturing calculus bovis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of development and utilization of bovine gallstone-forming bacteria resources, and particularly relates to a combination of Enterococcus faecalis and gallstone-forming bacteria, a bacterial agent, an application thereof, and a method for cultivating bovine gallstones. BACKGROUND

[0002] Natural bovine gallstones mainly refer to pathological stones formed in the gallbladder or bile duct of a cow. The essence of the bovine gallstones is a mixture of bilirubin, cholic acid, deoxycholic acid, cholesterol, ergot cholesterols, fatty acids, lecithin, mucin, calcium, metal ions, and various amino acids. Generally, bovine gallstones are divided into four categories: natural bovine gallstones generated in the body of a cow, cultivated bovine gallstones generated in the body of a cow, artificially synthesized bovine gallstones, and in-vitro cultivated bovine gallstones. The formation process of natural bovine gallstones is complex and involves complicated biochemical and pathological changes, with the core being abnormal deposition and mineralization of bile components. Due to the particularity and pathology of the formation process, the incidence of natural bovine gallstones in a cow population is very low (usually less than 2%), and the quality and size of the natural bovine gallstones vary greatly, resulting in the rarity and preciousness of the natural bovine gallstones. The effect of artificially synthesized bovine gallstones is not as ideal as that of other bovine gallstones; the in-vitro cultivated bovine gallstones have a larger yield than the natural bovine gallstones, but their efficacy cannot be compared with that of the natural bovine gallstones; the in-vivo cultivated bovine gallstones generated by implanting gallstone-forming bacteria in the gallbladder of a cow have the closest efficacy to the natural bovine gallstones, and are expected to solve the problem of the shortage of natural bovine gallstones.

[0003] The core cause of natural bovine gallstones is inflammation of the gallbladder or obstruction of the bile duct. The most common cause of cholecystitis is bacterial infection (such as Escherichia coli). The inflammation caused by bacteria can destroy the normal structure and function of the gallbladder wall, and the β-glucuronidase produced by the bacteria can hydrolyze the conjugated bilirubin in the bile to convert it into free bilirubin. The bovine bile itself has a broad-spectrum antibacterial effect, and most intestinal bacteria cannot survive in the bovine bile, let alone have the ability to form bovine gallstones. Some bovine gallstone-forming bacteria and their yellow-producing mechanisms have been reported in the literature. Bile-resistant gallstone-forming bacteria such as Escherichia coli and the β-glucuronidase (β-G enzyme) produced by the bacteria play an important role in the formation of bovine gallstones. In addition, parasites (such as liver flukes), bile duct stenosis, bile duct stones, or other causes of bile duct obstruction cause poor bile outflow, ultimately leading to bile stasis, which creates conditions for the deposition of components such as bilirubin calcium.

[0004] In the production of in vivo cultivation of bovine calculus, the general steps are to prepare a certain amount of bacterial solution in vitro, to implant the bacterial solution into the gallbladder of a cow, and to obtain in vivo cultivation of bovine calculus through long-term feeding. In fact, after the bacterial inoculation, firstly, due to the lack of bile nutrients compared with the culture medium, the bacteria need a certain time for adaptation, and the bacteria cannot rapidly proliferate in a short time; secondly, a small amount of bacteria is implanted into the gallbladder and is in a suspended state in a short time, and the bacteria are diluted by the bile, and since the gallbladder can discharge bile through physiological contraction and expansion, part of the bacteria implanted into the gallbladder are discharged from the gallbladder with the bile. Thirdly, the implantation surgery generally causes a wound, and if it is a traditional surgery, the wound is generally about 10 cm, and the immune response of the cow body will kill the bacteria to a great extent. The above factors will all cause the survival rate of the implanted bacteria to decrease, thereby causing the yield of in vivo cultivation of bovine calculus to be low, the quality to be poor, and even the implantation to fail.

[0005] Most studies on in vivo cultivation of bovine calculus are based on single inoculation of a single yellow-producing strain, and in vivo cultivation of bovine calculus is a complex mixture with multiple synergistic effects, which is the result of multiple factors. However, a single strain does not have the ability to produce multiple synergistic effects, nor does it have the ability to produce high-quality in vivo cultivation of bovine calculus. Single inoculation cannot guarantee the survival rate of the yellow-producing strain, nor can it guarantee the composition ratio of the surviving yellow-producing strain, and the implantation rate cannot be guaranteed, and the quality of the bovine calculus obtained cannot be guaranteed. In addition, the success rate of the traditional “one-size-fits-all” implantation method is greatly affected by individual differences in the cow body. If the implantation amount is too large, it may cause the cow body to be uncomfortable or the immune response to be too large, which affects the fattening effect. If the implantation amount is too small, it may cause the implantation to fail due to low enzyme content and low conversion and combination of cholic acid content.

[0006] Therefore, screening of excellent yellow-producing strains and / or bacterial flora and how to successfully apply them to the production of a large amount of in vivo cultivation of bovine calculus are the key points to obtain high-quality in vitro cultivation of bovine calculus. This has important significance for meeting the demand of the medical market for bovine calculus and improving the income of breeders, and large-scale promotion of the production of in vivo cultivation of bovine calculus. SUMMARY

[0007] In view of the defects of the prior art, the present application provides a Enterococcus faecalis, a yellow-producing bacterial combination, a bacterial agent and its use, and a method for cultivating bovine calculus, which aims to solve the problems of low single-strain colonization rate, low implantation success rate, and low quality of yellow production; and to solve the problems of low implantation success rate due to individual differences in the cow body, uncontrollable quality of yellow production, and uncertain benefits of breeders.

[0008] The present application provides a Enterococcus faecalis, which is preserved in the Guangdong Microbial Culture Collection Center, with a preservation date of December 13, 2024, and a preservation number of GDMCC NO: 65630.

[0009] The present application provides a yellowing bacteria combination, which comprises Enterococcus faecalis, Escherichia coli, and the quantity ratio is 0.2-1:0.2-1; the Enterococcus faecalis comprises 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 is composed of the following ratio of bacteria: The quantity ratio of Enterococcus faecalis:Escherichia coli:Bacillus sp.:Lactobacillus is 0.25-0.4:0.25-0.4:0.1-0.25:0.1-0.25; the Enterococcus faecalis comprises the Enterococcus faecalis as described above.

[0013] Preferably, the Bacillus sp. comprises Bacillus coagulans Bacillus coagulans , and the Lactobacillus comprises Lactobacillus crispatus Lactobacillus crispatus .

[0014] The present application provides a bacterial agent, which comprises the Enterococcus faecalis as described above, or the yellowing bacteria combination as described in any one of the above.

[0015] The present application provides the use of the Enterococcus faecalis as described above, the yellowing bacteria combination as described in any one of the above, or the bacterial agent as described above in cultivating bovine bezoar.

[0016] The present application provides a method for cultivating bovine bezoar, which comprises: mixing the Enterococcus faecalis as described above, the yellowing bacteria combination as described in any one of the above, or the bacterial agent as described above with bile, and fermenting.

[0017] Preferably, the cultivation is in vivo; the in vivo cultivation method comprises: Step 1, mixing the Enterococcus faecalis, the yellowing bacteria combination, or the bacterial agent with bile to prepare a yellowing liquid; Step 2, implanting a bovine bezoar bed into the gallbladder of a bovine; Step 3, introducing the yellowing liquid into the bovine bezoar bed; Step 4, normal feeding; The order of steps 1 and 2 can be changed arbitrarily.

[0018] Preferably, in step 3, the yellowing liquid and polyacrylamide hydrogel are introduced into the bovine bezoar bed respectively and / or simultaneously; and / or, in step 4, after normal feeding for 7-15 days, the Enterococcus faecalis or the yellowing bacteria combination is implanted again, and then the bovine is fed for 9-12 months.

[0019] The present application screens a new strain of Enterococcus faecalis WLK2401JZ-07, which has advantages in cultivating bovine calculus compared with other yellow-producing bacteria, especially in the activity of beta-glucuronidase in bile, the number of colony survival, the ability of transforming and combining bilirubin and cholic acid during cultivation. 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 calculus, and compared with single-strain fermentation, it can efficiently produce calcium bilirubinate while ensuring the health of the bovine body. Moreover, the microorganisms described in the present application are all common gastrointestinal symbiotic microorganisms and / or probiotic microorganisms, which have the advantages of mild and simple production process, low production cost, no use of flammable and explosive solvent vehicles, reduction of environmental pollution, safe production process, environmental protection, and quality and safety of products applied in medical products, and have good economic and social benefits.

[0020] The combination of yellow-producing bacteria provided by the present application mainly uses Enterococcus faecalis as an important component, which has great potential in cultivating bovine calculus. The excellent mucosal barrier function of Enterococcus faecalis has a positive effect on inflammation control and postoperative recovery of the bovine body; Enterococcus faecalis also has strong colonization ability on the mucosa, which ensures the concentration of bacterial strains in the gallbladder and the amount of beta-G enzyme produced, so that the abundance of the combined bacterial strains after implantation is guaranteed, and the quality of the produced bovine calculus is also guaranteed; the yellow-producing bacteria and their combinations of the present application all have excellent beta-glucuronidase production ability, especially Escherichia coli, which has high beta-glucuronidase activity, strong ability, and high direct bilirubin conversion rate, which can accelerate the conversion of bile to bovine calculus. It is safe to use and can be used for in-vitro bovine calculus production and bovine in-vivo cultivation of bovine calculus. The combination bacteria provided by the present application contain lactic acid bacteria, which can adjust the bile microenvironment. Under acidic conditions, free bilirubin and cholesterol co-precipitate, making it easier for bound bilirubin and calcium ions in the bile fermentation broth to be free, increasing the probability and the probability of calcium bilirubinate formation, thereby increasing the yield of bovine calculus. Compared with single-strain fermentation, the use of the yellow-producing bacteria combination of the present application for in-vivo bovine calculus cultivation can produce a synergistic effect between the bacterial strains, promote the colonization of the yellow-producing bacteria combination in the gallbladder, and obtain high-quality in-vivo cultivated bovine calculus in a shorter time, greatly shortening the cultivation time of in-vivo bovine calculus.

[0021] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above-mentioned basic technical ideas of the present application.

[0022] The above content of the present application will be further described in detail through the specific embodiments below. However, it should not be understood that the scope of the above-mentioned subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application.

[0023] Biological sample preservation instructions Enterococcus faecalis WLK2401JZ-07 has been preserved in Guangdong Microbial Culture Collection Center on December 13, 2024, the preservation address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, the preservation number is GDMCC: 65630, and the Latin name is Enterococcus faecium . DETAILED DESCRIPTION

[0024] In the following examples and experimental examples, the reagents and materials not specifically stated are commercially available.

[0025] TSB medium (Tryptic Soy Broth), also known as Tryptic Soy Broth or Tryptic Soy Broth, composition: Tryptic Soy Broth 16.5-17.0 g / L, soybean papain protease hydrolysate 2.87-3.02 g / L, sodium chloride 4.96-5.02 g / L, glucose 2.46-2.63 g / L, potassium phosphate 2.43-2.56 g / L, pH 7.3±0.2.

[0026] TSA medium (Tryptic Soy Broth), also known as Tryptic Soy Broth or Tryptic Soy Broth, composition: Tryptic Soy Broth 16.5-17.0 g / L, soybean papain protease hydrolysate 2.87-3.02 g / L, sodium chloride 4.96-5.02 g / L, glucose 2.46-2.63 g / L, potassium phosphate 2.43-2.56 g / L, 12 g / L agar, 5-10% defibrillated sheep blood, pH 7.3±0.2.

[0027] The gall-producing bacteria used in the present application are derived from the gall-producing bacteria in the gallbladder of a 5-year-old Simmental cow (multiparous) in a certain cattle farm in Ningxia.

[0028] The fresh bile used in the present application is from a certain slaughterhouse in Sichuan Province, and all the fresh bile is used immediately after collection.

[0029] The temperature-sensitive gel for in vivo transplantation is sterilized for standby use (for details, please refer to patent CN118716281A).

[0030] The gall-producing bacteria in the gallbladder are screened by using the conventional plate coating method, the obtained single strain is sent to a professional strain identification institution for identification, and part of it is sent to the Guangdong Microbial Culture Collection Center for preservation.

[0031] The other strains of the present application are commercially available: Escherichia coli Escherichia coli GDMCC NO. 801268; Bacillus coagulans Bacillus coagulansGDMCC NO. 1.420; Lactobacillus crispatus Lactobacillus crispatus GDMCC NO. 1.4925.

[0032] Example 1 Screening of yellowing bacteria The first yellowing bacteria liquid (Enterococcus) was screened according to the following method: (1) Preliminary screening The bovine gallbladder produced bovine gallbladder bile, which was diluted by gradient (10 -1 to 10 -5 ) with sterile PBS, and 100 μL of the original solution (or dilution) was uniformly coated on the bile-resistant Enterococcus screening medium. The coated plate was inverted in a biochemical incubator and cultured at 37°C for 24-48 hours.

[0033] The bile-resistant Enterococcus preliminary screening medium (Bile BAE) has the following components: Tryptone 17.5 g, soybean peptone 3 g, glucose 2.5 g, oxgall 40 g, K2HPO4 2.5 g, NaN3 0.25 g, esculin 1 g, ferric ammonium citrate 0.5 g, agar 15 g, pH 7.1 ± 0.2. (When preparing, 0.5 L of water is used to dissolve, sterilized at 121°C, 0.1 MPa for 15 min, cooled to 50°C, and then 0.5 L of sterile bile preheated to 50°C is added).

[0034] Black colonies were screened on bile BEA.

[0035] (2) Rescreening The bile-resistant Enterococcus rescreening medium (Bile Slanetz-Bartley) was used for rescreening, and had the following components: Tryptone 15.0 g, yeast extract 5.0 g, glucose 2.0 g, K2HPO4 0.5 g, agar 15.0 g, sodium azide (NaN3) 0.4 g, 2,3,5-chlorinated triphenyl tetrazolium (TTC) 0.1 g. (When preparing, 0.5 L of water is used to dissolve the above-mentioned substances, sterilized at 121°C, 0.1 MPa for 15 min, and cooled to 50°C. Mix with 0.5 L of sterile bile preheated to 50°C with 0.4 g of sodium azide and 0.1 g of TTC in advance, pour into sterile culture dishes under sterile conditions, and store in the dark after solidification (TTC is sensitive to light).

[0036] 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.

[0037] (3) Proliferation culture Red to dark red colonies (1–2 mm in diameter, round and raised) were selected from bile Slanetz-Bartley plates for liquid fermentation, while 16S rRNA sequencing and biological preservation were performed.

[0038] 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.) 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).

[0039] The OD600 value, β-glucuronidase activity, and bile salt hydrolase (BSH) activity of the proliferation culture medium were measured. 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. And / or, 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. 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.

[0040] 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 faecium 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.

[0041] Example 2 Enterococcus faecalis ( Enterococcus faecium) WLK2401JZ-07 for in vivo cultivation of bovine gallstones The Enterococcus faecalis WLK2401JZ-07 screened in Example 1 is applied to in vivo cultivation of bovine gallstones, with the following steps: Step S1: Preparation of sterile bile.

[0042] Fresh bile is taken, filtered through 8 layers of gauze to remove impurities, and placed in a sterilized bag for irradiation sterilization to remove possible microorganisms and viruses.

[0043] The sterilization method is CO 60 Irradiation sterilization (dose can be selected as 10-20 KGy), preferably 15 KGy.

[0044] Step S2: Preparation of concentrated Enterococcus faecalis WLK2401JZ-07.

[0045] The Enterococcus faecalis WLK2401JZ-07 is fermented and proliferated according to the conventional fermentation method, and the obtained fermentation broth is centrifuged at 4°C and 5000 rpm for 10 min, and 5 mL of sterile bile is added to resuspend to form a concentrated Enterococcus faecalis WLK2401JZ-07 bacterial solution with a concentration of 10 13 CFU / mL.

[0046] Step S3: First in vivo cultivation.

[0047] The first implantation of bovine gallstone bed, temperature-sensitive polyacrylamide hydrogel and bacteria (for details, please refer to patent CN118716281A) is as follows: Step one: Minimally invasive surgery is used to implant the bovine gallstone bed into the gallbladder of a cow (Simmental cow); Step two: The prepared temperature-sensitive polyacrylamide hydrogel is mixed with 20 mL of concentrated Enterococcus faecalis WLK2401JZ-07 bacterial agent and injected into the bovine gallstone bed; Or first inject the prepared temperature-sensitive polyacrylamide hydrogel into the bovine gallstone bed, and then inject 20 mL of concentrated Enterococcus faecalis WLK2401JZ-07 bacterial agent into the bovine gallstone bed.

[0048] Step S4: Normal feeding.

[0049] Step S5: Within 7-15 days after the operation, the blood routine of the cow is detected, and the postoperative diet, defecation, sleep, and weight gain of the cow are carefully observed and recorded.

[0050] When the indicators in step S5 return to the preoperative level, the bile is extracted for detection of bilirubin, cholic acid, β-glucuronidase, and the number of bacteria.

[0051] Step S6: Second implantation of Enterococcus faecalis WLK2401JZ-07, with the same composition and implantation amount as the first implantation.

[0052] Step S7: Enterococcus faecalis WLK2401JZ-07 is implanted again using epidural puncture surgery.

[0053] Step S8: normal feeding of the implanted bovine.

[0054] Step S9: after 9-12 months, the bovine is slaughtered, the gallbladder is removed, the bovine bile is filtered, and the bovine gallstone is taken out.

[0055] Step S10: the components of the obtained in-vivo cultivated bovine gallstone are detected according to the Pharmacopoeia, and the results are shown in Table 1.

[0056] Table 1 Example 3: A combination of gallstone-inducing bacteria and its application in in-vivo cultivation of bovine gallstone The combination of gallstone-inducing bacteria of this example is composed of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus crispatus = 0.4:0.4:0.1:0.1.

[0057] The bovine gallstone is cultivated in-vivo according to the method of Example 2, with the difference that the concentrated gallstone-inducing bacterial agent of the above four bacterial species is prepared respectively, then mixed according to the ratio of Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus = 0.4:0.4:0.1:0.1, to obtain the mixed bacterial solution, and the total volume of the mixed bacterial solution used in the first in-vivo cultivation is 20 mL.

[0058] Example 4: A combination of gallstone-inducing bacteria and its application in in-vivo cultivation of bovine gallstone The combination of gallstone-inducing bacteria of this example is composed of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus crispatus = 0.3:0.3:0.2:0.2.

[0059] The bovine gallstone is cultivated in-vivo according to the method of Example 3, with the difference that the ratio of the combination of gallstone-inducing bacteria is different.

[0060] Example 5: A combination of gallstone-inducing bacteria and its application in in-vivo cultivation of bovine gallstone The combination of gallstone-inducing bacteria of this example is composed of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli: Bacillus coagulans: Lactobacillus crispatus = 0.25:0.25:0.25:0.25.

[0061] The bovine gallstone is cultivated in-vivo according to the method of Example 3, with the difference that the ratio of the combination of gallstone-inducing bacteria is different.

[0062] Example 6: A combination of gallstone-inducing bacteria and its application in in-vivo cultivation of bovine gallstone The combination of gallstone-inducing bacteria of this example is composed of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli = 0.5:0.5.

[0063] The gallstones were cultivated in vivo according to the method of Example 3, with the exception that the proportions of the gallstone-inducing bacterial combination were different.

[0064] Example 7 A gallstone-inducing bacterial combination and its use in cultivating gallstones in vivo The gallstone-inducing bacterial combination of this example consists of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli = 0.4:0.6.

[0065] The gallstones were cultivated in vivo according to the method of Example 3, with the exception that the proportions of the gallstone-inducing bacterial combination were different.

[0066] Example 8 A gallstone-inducing bacterial combination and its use in cultivating gallstones in vivo The gallstone-inducing bacterial combination of this example consists of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli = 0.6:0.4.

[0067] The gallstones were cultivated in vivo according to the method of Example 3, with the exception that the proportions of the gallstone-inducing bacterial combination were different.

[0068] Example 9 A gallstone-inducing bacterial combination and its use in cultivating gallstones in vivo The gallstone-inducing bacterial combination of this example consists of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli = 0.2:0.8.

[0069] The gallstones were cultivated in vivo according to the method of Example 3, with the exception that the proportions of the gallstone-inducing bacterial combination were different.

[0070] Example 10 A gallstone-inducing bacterial combination and its use in cultivating gallstones in vivo The gallstone-inducing bacterial combination of this example consists of the following number ratio of bacterial species: Enterococcus faecalis: Escherichia coli = 0.8:0.2.

[0071] The gallstones were cultivated in vivo according to the method of Example 3, with the exception that the proportions of the gallstone-inducing bacterial combination were different.

[0072] The technical solutions of the present application are further described below through experiments. The Enterococcus faecalis used in the following experimental examples was screened from Example 1, the Escherichia coli used was Escherichia coli GDMCC NO. 801268, the Bacillus coagulans used was Bacillus coagulans GDMCC NO. 1.420, and the Lactobacillus crispatus used was Lactobacillus crispatus GDMCC NO. 1.4925.

[0073] Experimental Example 1: Screening Experiment of Flammogenic Bacterial Combinations I. Experimental Methods 1. Prepare concentrated flavonoid agent.

[0074] According to conventional fermentation methods, Enterococcus faecalis ( Enterococcus faecium 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.

[0075] 2. Experimental Grouping 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.

[0076] 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.

[0077] 3. Methods for determining bilirubin content Chinese Pharmacopoeia 2025 Edition, Part I, Calculus Bovis, Methods for Bilirubin Detection 4. Methods for determining free bilirubin content Chinese Pharmacopoeia 2025 Edition, Part I, Calculus Bovis Section, Method for Determination of Free Bilirubin 5. Methods for determining bile acid content The 2025 edition of the Chinese Pharmacopoeia, Part I, Bezoar section, combined with bile acid detection methods. 6. Methods for determining the content of free bile acids Method for detecting free cholic acid in the part of Calculus Bovis in the first volume of Chinese Pharmacopoeia 2025 edition 7. Method for detecting β-glucuronidase activity The enzyme-linked glucuronidase content detection kit was used to detect according to the method in the instruction.

[0078] 8. Method for detecting the number of bacteria The dilution coating plate method was used to count the number of living bacteria.

[0079] II. Experimental results The results of the influence of the type and proportion of yellow-producing strains on the β-glucuronidase and the number of bacteria in the bovine bile are shown in Table 2. The results of the influence of the type and proportion of yellow-producing strains on the bilirubin and cholic acid in the bovine bile are shown in Table 3.

[0080] Table 2. Results of the influence of different strains and their different implantation proportions on β-glucuronidase and the number of bacteria Table 3. Results of the influence of different strains and their different implantation proportions on bilirubin and cholic acid For the single yellow-producing strain group of Enterococcus faecalis, after implanting the yellow-producing strain, the β-glucuronidase activity was significantly improved, the survival rate of the number of bacteria was high, and the ability to transform and bind bilirubin and cholic acid was strong, indicating that Enterococcus faecalis WLK2401JZ-07 can be used for cultivating bovine calculus in vivo.

[0081] For the Enterococcus faecalis + Escherichia coli group, after implanting the yellow-producing strain, the β-glucuronidase activity was significantly improved, and the improvement effect was better than that of any single yellow-producing strain group with the same implantation dose, especially the mixed bacteria group with the proportions of 0.2:0.8, 0.5:0.5 and 0.4:0.6. At the same time, the survival rate of the number of bacteria was high, and the ability to transform and bind bilirubin and cholic acid was strong, and the effect of transforming and binding cholic acid was stronger than that of any single yellow-producing strain group with the same implantation dose. It is shown that Enterococcus faecalis WLK2401JZ-07 and Escherichia coli have a synergistic effect in the combination of 0.2-0.8:0.2:-0.8.

[0082] For the four yellowing bacteria group, after implanting the yellowing bacteria, the β-glucuronidase activity is improved, especially when the ratio is 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 is significantly better than that of any single yellowing bacteria group with the same implantation dose, and the Enterococcus faecalis + Escherichia coli group with the ratio of 0.5:0.5, indicating that the four yellowing bacteria screened in the application have a synergistic effect at the ratio 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.

[0083] In addition, for the four yellowing bacteria group, the survival rate of the colony number and the ability to transform and bind bilirubin and cholic acid are high when the ratio is 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.

[0084] In summary, the Enterococcus faecalis WLK2401JZ-07 can be used for in vivo cultivation of bovine calculus, and the yellowing effect is better than that of Bacillus coagulans and Lactobacillus crispatus; the Enterococcus faecalis WLK2401JZ-07 and Escherichia coli have a synergistic effect at the ratio of 0.2-0.8:0.2:-0.8; the Enterococcus faecalis WLK2401JZ-07, Escherichia coli, Lactobacillus crispatus and Bacillus coagulans have a synergistic effect at the ratio 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.

[0085] As can be seen from the above examples and experimental examples, the Enterococcus faecalis WLK2401JZ-07 screened in the application has advantages in cultivating bovine calculus compared with other yellowing bacteria, especially in terms of β-glucuronidase activity in bile, colony survival number, ability to transform and bind bilirubin and cholic acid, etc. 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 calculus, and compared with single-strain fermentation, it can efficiently produce calcium bilirubinate while ensuring the health of the bovine body. Moreover, the microorganisms described in the application are common gastrointestinal symbiotic microorganisms and / or probiotic microorganisms, and have the advantages of mild and simple production process, low production cost, no use of flammable and explosive solvent vehicles, reduction of environmental pollution, safe production process, environmental protection, and quality and safety of products applied in medical products, etc., which have good economic and social benefits.

Claims

1. An Enterococcus faecalis, characterized in that: 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 comprises Enterococcus faecalis and Escherichia coli, and the quantity ratio is 0.2-1:0.2-1; the Enterococcus faecalis comprises 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 strains in the following proportions: 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 comprises the Enterococcus faecalis of claim 1.

5. The xanthogenic bacterial combination according to claim 4, characterized in that: The Bacillus includes Bacillus coagulans Bacillus coagulans The Lactobacillus includes Lactobacillus crispatus Lactobacillus crispatus .

6. An inoculant characterized in that: The bacterial agent comprises 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 bezoar.

8. A method for cultivating bovine gallstones, characterized by, It comprises: 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.

9. The method of cultivating calculus bovis according to claim 8, characterized by: The cultivation is in vivo cultivation; the in vivo cultivation method comprises: Step 1, 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 to obtain a yellowing liquid; Step 2, implanting a bovine bezoar bed into the gallbladder of a bovine; Step 3, introducing the yellowing liquid into the bovine bezoar bed; Step 4, normal feeding; The order of steps 1 and 2 can be arbitrarily changed.

10. The method for cultivating bovine bezoar according to claim 9, characterized in that: In step 3, the yellowing liquid and polyacrylamide hydrogel are introduced into the bovine bezoar bed separately and / or simultaneously; And / or, in step 4, after normal feeding for 7-15 days, the Enterococcus faecalis or the Flavobacterium combination is implanted again, and then fed for 9-12 months.

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