A method for cultivating high-quality bezoar using bacterial flora

Abstract

The invention discloses a method for cultivating high-quality bezoar by using bacterial flora, belonging to the technical field of in vitro cultivation of bezoar, comprising the steps of processing, bacteriostasis, fermentation and finished product of natural ox bile, in which the first bacterial liquid and the third bacterial liquid are added to the first mixed liquid mixed with vitamin C, rosemary and natural ox bile to obtain the second mixed liquid, the second mixed liquid is slowly shaken for 2-4 hours in a 37°C environment, and then the second bacterial liquid is added to obtain the third mixed liquid, and the third mixed liquid is slowly shaken for 8-10 hours in a 37°C environment; the mass ratio of the first bacterial liquid, the third bacterial liquid, the second bacterial liquid and the first mixed liquid is 0.8-1.2:0.7-0.9:0.4-0.6:90-110. The method enriches and balances the components of bezoar by allocating the types of bacterial liquids in ox bile to make them cooperate with each other and be as close as possible to the complex environment of stone formation in the bile of the ox body, thereby not only improving the utilization rate of ox bile, but also giving full play to the synergistic effect between the components, making it closer to the efficacy of natural bezoar.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro cultivation of bezoar, and particularly relates to a method for cultivating high-quality bezoar by utilizing bacterial flora. Background Art

[0002] Bezoar is a stone in the gallbladder, bile duct or hepatic duct of cattle or buffalo. Due to its functions of clearing away heat and detoxifying, clearing the heart and resolving phlegm, invigorating the orifices and cooling the liver, calming the nerves and reducing swelling, strengthening the heart and promoting bile secretion, it has become the main raw material for more than 600 kinds of Chinese patent medicines such as Angong Niuhuang Pills and Liushen Pills. The natural occurrence rate of natural bezoar is only about one in a thousand, making it extremely scarce and there is no substitute.

[0003] In order to ease the contradiction between supply and demand and promote the development of medicine and health, my country has included the cultivation of bezoar in the pharmacopoeia. At present, there are three main types of bezoar cultivation: in vivo bezoar cultivation, in vitro bezoar cultivation and chemical synthesis. Among them, in vivo bezoar cultivation uses a surgical method to implant a yellowing factor in the gallbladder of the cow, forming a bezoar-like substance in the gallbladder. The bezoar bed is removed and scraped during surgery or slaughter. Although the bezoar produced by this method is very close to natural bezoar, it cannot be mass-produced, the output is small and the cycle is long; chemical synthesis can achieve industrial production, but the effective ingredients are low and cannot replace natural bezoar, resulting in limited use, which has no obvious help in alleviating the imbalance between supply and demand of medical bezoar. Therefore, in vitro cultivation of bezoar has become the most feasible way.

[0004] The current in vitro bezoar cultivation technology can be roughly divided into two types. One is an invention patent with a publication number of CN102429927B, in which reactants of effective ingredients required by the pharmacopoeia are added to natural bovine bile to obtain treated bovine bile, and then the treated bovine bile is fermented with a bacterial solution, and the pH value of the fermentation is adjusted. The dynamic process of bile in the gallbladder is simulated by shaking to obtain bezoar. Although the main effective ingredients of the bezoar obtained by this method meet the requirements of the Chinese Pharmacopoeia, the process of artificial participation is increased, which to a certain extent destroys the balance of the components of bezoar. The other is an invention patent with a publication number of CN103316046B, in which enzymes are added to natural bovine bile, that is, enzymatic bovine bile reaction is used to obtain bezoar. Although this method reduces the degree of artificial participation and improves the quality of bezoar to a certain extent, it is still far from natural bezoar and has a high cost. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for cultivating high-quality bezoar by using bacterial flora, which can improve the quality and yield of bezoar under the premise of reducing costs.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: design a method for cultivating high-quality bezoar using bacterial flora, comprising the following steps:

[0007] S1 treatment: adding vitamin C and rosemary to the sterilized natural ox bile to obtain a first mixed solution;

[0008] S2 Preparation of the first bacterial solution: A single colony of Escherichia coli is inoculated into the sterilized first culture medium to form a first culture solution, and the first culture solution is cultured by shaking until the cell concentration in the first culture solution reaches 2×10 8 CFU / mL, then centrifuge at 5000g for 15 min at 4°C to obtain the supernatant to prepare the first bacterial culture. The culture temperature of the first culture was 37°C and the shaking speed was 180-200 rpm;

[0009] S3: preparing the second bacterial solution: inoculating the Enterococcus faecalis seed solution into the sterilized second culture medium at a 2% inoculation rate to form a second culture solution; culturing the second culture solution at 37°C for 24 hours, centrifuging at 3000 rpm for 2 minutes, and taking the supernatant as the second bacterial solution;

[0010] The second culture medium is composed of the following raw materials in parts by mass:

[0011] Tryptone 0.6-1 part

[0012] Soybean peptone 0.6-1 part

[0013] Yeast extract 0.4-0.6 parts

[0014] Sodium chloride 0.4-0.6 parts

[0015] 0.25-0.35 parts of potassium dihydrogen phosphate

[0016] 0.04-0.06 parts of magnesium chloride hexahydrate

[0017] Cysteine ​​hydrochloride 0.04-0.06 parts

[0018] 0.001 part of ferrous sulfate heptahydrate

[0019] Tween-80 0.08~0.12 parts

[0020] 2.5-3.5 parts of glucose

[0021] 90-110 parts of distilled water

[0022] S4 Preparation of the third bacterial solution: inoculate the P. acnes strain into the sterilized third culture medium to form a third culture solution, culture the third culture solution in an anaerobic environment at 37°C for 4 to 7 days, and dilute it with the third culture medium until the concentration of P. acnes reaches 1×10 8 CFU / mL, centrifuge at 3000 rpm for 10 min, and take the supernatant to prepare the third bacterial solution;

[0023] S5 fermentation: adding the first bacterial liquid of step S2 and the third bacterial liquid of step S4 to the first mixed liquid of step S1 to obtain a second mixed liquid, slowly shaking the second mixed liquid at 37° C. for 2 to 4 hours, then adding the second bacterial liquid of step S3 to obtain a third mixed liquid, and slowly shaking the third mixed liquid at 37° C. for 8 to 10 hours; the mass ratio of the first bacterial liquid, the third bacterial liquid, the second bacterial liquid and the first mixed liquid is 0.8 to 1.2:0.7 to 0.9:0.4 to 0.6:90 to 110;

[0024] S6 finished product: let the third mixed solution treated in step S5 stand, discard the supernatant, filter, and dry to obtain bezoar.

[0025] Furthermore, in step S2, the method for preparing a single colony of Escherichia coli is:

[0026] S21 was dipped into -80℃ frozen E. coli solution and evenly streaked on LB agar plate and cultured at 37℃ overnight;

[0027] S22 Dip a small amount of colonies from the LB agar plate, add it to 1mL phosphate buffer, mix it to make a bacterial stock solution, take 100μL of the stock solution and add it back to 900μL phosphate buffer to dilute the stock solution tenfold until 1mL of bacterial solution is obtained. Spread the bacterial solution evenly on the LB agar plate and culture it at 37℃ for 24h to obtain dispersed single colonies of Escherichia coli.

[0028] Furthermore, in step S2, the first culture medium is LB broth.

[0029] Furthermore, in step S3, the preparation method of Enterococcus faecalis seed solution is:

[0030] Enterococcus faecalis strains were inoculated into the second culture medium at a 2% inoculation rate and cultured at 37°C for 24 h. After three generations of continuous activation, the temperature was lowered to 4°C and the cells were collected by centrifugation at 3000 rpm for 20 min. The cells were washed three times by centrifugation with sterile saline and the cell concentration was adjusted to 10 6 CFU / mL, and made into Enterococcus faecalis seed solution.

[0031] Furthermore, the preparation method of the second culture medium is:

[0032] 0.6-1 parts by weight of tryptone, 0.6-1 parts by weight of soy peptone, 0.4-0.6 parts by weight of yeast extract, 0.4-0.6 parts by weight of sodium chloride, 0.25-0.35 parts by weight of potassium dihydrogen phosphate, 0.04-0.06 parts by weight of magnesium chloride hexahydrate, 0.04-0.06 parts by weight of cysteine ​​hydrochloride, 0.001 parts by weight of ferrous sulfate heptahydrate, 0.08-0.12 parts by weight of Tween-80 and 2.5-3.5 parts by weight of glucose are dissolved in 90-110 parts by weight of distilled water to form a first solution, the pH value of the first solution is adjusted to 6.5, and the first solution is sterilized at 121° C. for 15 minutes to obtain a second culture medium.

[0033] Furthermore, the raw materials of the second culture medium consist of 0.8 parts by mass of tryptone, 0.8 parts by mass of soy peptone, 0.5 parts by mass of yeast extract, 0.5 parts by mass of sodium chloride, 0.3 parts by mass of potassium dihydrogen phosphate, 0.05 parts by mass of magnesium chloride hexahydrate, 0.05 parts by mass of cysteine ​​hydrochloride, 0.001 parts by mass of ferrous sulfate heptahydrate, 0.1 parts by mass of Tween-80, 3 parts by mass of glucose and 100 parts by mass of distilled water.

[0034] Further, the preparation method of the third culture medium is:

[0035] The BHI powder and deionized water were mixed at a mass ratio of 1:10 to form a mixed solution, and the pH value of the mixed solution was adjusted to 7.0 to prepare a third culture medium.

[0036] Furthermore, in step S1, the weight ratio of natural ox bile, vitamin C and rosemary is 1000:4-7:1.8-2.2.

[0037] Furthermore, the weight ratio of natural ox bile, vitamin C and rosemary is 1000:5.5:2.

[0038] Furthermore, in step S5, the mass ratio of the first bacterial liquid, the third bacterial liquid, the second bacterial liquid and the first mixed liquid is 1:0.8:0.5:100.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The present invention enriches and balances the components of bezoar by adjusting the types of bacterial liquid in the bile of cattle to make them cooperate with each other and be as close as possible to the complex environment of stone formation in the bile of cattle, thereby not only improving the utilization rate of the bile of cattle, but also giving full play to the synergistic effect of the various components, making it closer to the efficacy of natural bezoar.

[0041] 2. The present invention can effectively maintain the original components and proportions of the natural cow bile and its original color by adding vitamin C and rosemary to the natural cow bile, thereby reducing the changes in its components caused by changes in environmental conditions.

[0042] 3. The present invention can not only realize the mass production of bezoar, but also approach the quality of natural bezoar to a greater extent, improve its medicinal value, and facilitate popularization and application. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific implementation modes. Embodiment 1

[0044] High-quality bezoar is obtained through the following steps:

[0045] S1 treatment: Add vitamin C and rosemary to the sterilized natural cow bile to obtain the first mixed solution, and the weight ratio of natural cow bile, vitamin C and rosemary is 1000:4:1.8. Vitamin C and rosemary can effectively maintain the original components and proportions of cow bile and its original color, thereby reducing the changes in its components caused by changes in environmental conditions, and laying a high-quality raw material foundation for improving the quality of bezoar.

[0046] S2 Preparation of the first bacterial solution: A single colony of Escherichia coli is inoculated into the sterilized first culture medium to form a first culture solution, and the first culture solution is cultured by shaking until the cell concentration in the first culture solution reaches 2×10 8 CFU / mL, and then centrifuged at 5000g for 15 minutes at 4°C to obtain the supernatant to prepare the first bacterial culture. The culture temperature of the first culture was 37°C and the shaking speed was 180rpm. The first culture medium used was LB broth.

[0047] The preparation method of the above-mentioned Escherichia coli single colony is as follows:

[0048] S21 was dipped into -80℃ frozen E. coli solution and evenly streaked on LB agar plates and cultured at 37℃ overnight.

[0049] S22 Take a small amount of colonies from the LB agar plate, add them to 1 mL of phosphate buffer, and mix them to make a bacterial stock solution. Take 100 μL of the stock solution and add it back to 900 μL of phosphate buffer to dilute the stock solution tenfold until 1 mL of bacterial solution is obtained. Spread the bacterial solution evenly on the LB agar plate and culture it at 37°C for 24 hours to obtain dispersed single colonies of E. coli.

[0050] S3 prepares the second bacterial solution: inoculate the Enterococcus faecalis seed solution into the sterilized second culture medium at a 2% inoculation rate to form a second culture solution, culture the second culture solution at 37°C for 24 hours, centrifuge at 3000 rpm for 2 minutes, and take the supernatant as the second bacterial solution. The preparation method of the Enterococcus faecalis seed solution is as follows:

[0051] Enterococcus faecalis strains were inoculated into the second culture medium at a 2% inoculation rate and cultured at 37°C for 24 h. After three generations of continuous activation, the temperature was lowered to 4°C and the cells were collected by centrifugation at 3000 rpm for 20 min. The cells were washed three times by centrifugation with sterile saline and the cell concentration was adjusted to 1×10 6 CFU / mL, and the seed solution of Enterococcus faecalis was prepared.

[0052] The preparation method of the second culture medium used is:

[0053] 0.6 parts by mass of tryptone, 0.6 parts by mass of soy peptone, 0.4 parts by mass of yeast extract, 0.4 parts by mass of sodium chloride, 0.25 parts by mass of potassium dihydrogen phosphate, 0.04 parts by mass of magnesium chloride hexahydrate, 0.04 parts by mass of cysteine ​​hydrochloride, 0.001 parts by mass of ferrous sulfate heptahydrate, 0.08 parts by mass of Tween-80 and 2.5 parts by mass of glucose were dissolved in 90 parts by mass of distilled water to form a first solution, the pH value of the first solution was adjusted to 6.5, and the first solution was sterilized at 121° C. for 15 min to prepare a second culture medium.

[0054] S4 Preparation of the third bacterial solution: inoculate the P. acnes strain into the sterilized third culture medium to form a third culture solution, culture the third culture solution in an anaerobic environment at 37°C for 4 days, and dilute it with the third culture medium until the concentration of P. acnes reaches 1×10 8 CFU / mL, centrifuge at 3000rpm for 10min, and take the supernatant to prepare the third bacterial solution. The preparation method of the third culture medium used is:

[0055] BHI powder and deionized water were mixed in a mass ratio of 1:10 to form a mixed solution, and the pH value of the mixed solution was adjusted to 7.0 to prepare a third culture medium.

[0056] S5 fermentation: the first bacterial liquid of step S2 and the third bacterial liquid of step S4 are added to the first mixed liquid of step S1 to obtain a second mixed liquid, the second mixed liquid is slowly shaken in an environment of 37°C for 2 hours, and then the second bacterial liquid of step S3 is added to obtain a third mixed liquid, and the third mixed liquid is slowly shaken in an environment of 37°C for 8 hours; the mass ratio of the first bacterial liquid, the third bacterial liquid, the second bacterial liquid and the first mixed liquid is 0.8:0.7:0.4:90.

[0057] S6 finished product: let the third mixed solution treated in step S5 stand, discard the supernatant, filter, and dry to obtain bezoar.

[0058] The above-mentioned Escherichia coli cryopreservation solution, LB agar plate, LB broth, Enterococcus faecalis strains and Propionibacterium acnes strains are all purchased items. Among them, the Escherichia coli cryopreservation solution was purchased from Shanghai Weidi Biotechnology Co., Ltd., and its number is BL21 (DE3). The LB agar plate, LB broth and phosphate buffer were all purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. The LB agar plate is HBPM0129, the LB broth is HB0128, and the phosphate buffer is HBXBHP001. The Enterococcus faecalis strain was purchased from the China Center for Type Culture Collection, and its preservation number is CCTCC M 2022312, and the preservation time is March 25, 2022. The Propionibacterium acnes strain was purchased from the Shanghai Collection Biotechnology Center, and its number is NCTC 737. Embodiment 2

[0059] The difference between this embodiment and the first embodiment is that:

[0060] In step S1, the weight ratio of natural ox bile, vitamin C and rosemary is 1000:7:2.2.

[0061] In step S2, the oscillation speed is 200 rpm.

[0062] In step S3, the first solution is composed of 1 part by mass of tryptone, 1 part by mass of soy peptone, 0.6 parts by mass of yeast extract, 0.6 parts by mass of sodium chloride, 0.35 parts by mass of potassium dihydrogen phosphate, 0.06 parts by mass of magnesium chloride hexahydrate, 0.06 parts by mass of cysteine ​​hydrochloride, 0.001 parts by mass of ferrous sulfate heptahydrate, 0.12 parts by mass of Tween-80, 3.5 parts by mass of glucose and 110 parts by mass of distilled water.

[0063] In step S4, the static culture time of the third culture solution is 7 days.

[0064] In step S5, the shaking time of the second mixed solution is 4 hours, and the shaking time of the third mixed solution is 10 hours. The mass ratio of the first bacterial solution, the third bacterial solution, the second bacterial solution and the first mixed solution is 1.2:0.9:0.6:110.

[0065] The rest is the same as in Example 1. Embodiment 3

[0066] The difference between this embodiment and the first embodiment is that:

[0067] In step S1, the weight ratio of natural ox bile, vitamin C and rosemary is 1000:5.5:2

[0068] In step S2, the oscillation speed is 190 rpm.

[0069] In step S3, the first solution is composed of 0.8 parts by mass of tryptone, 0.8 parts by mass of soy peptone, 0.5 parts by mass of yeast extract, 0.5 parts by mass of sodium chloride, 0.3 parts by mass of potassium dihydrogen phosphate, 0.05 parts by mass of magnesium chloride hexahydrate, 0.05 parts by mass of cysteine ​​hydrochloride, 0.001 parts by mass of ferrous sulfate heptahydrate, 0.1 parts by mass of Tween-80, 3 parts by mass of glucose and 100 parts by mass of distilled water.

[0070] In step S4, the static culture time of the third culture solution is 6 days.

[0071] In step S5, the shaking time of the second mixed solution is 3 hours, and the shaking time of the third mixed solution is 9 hours. The mass ratio of the first bacterial solution, the third bacterial solution, the second bacterial solution and the first mixed solution is 1:0.8:0.5:100.

[0072] The rest is the same as in Example 1. Embodiment 4

[0073] The difference between this embodiment and the first embodiment is that:

[0074] In step S1, the weight ratio of natural ox bile, vitamin C and rosemary is 1000:7:1.8.

[0075] In step S2, the oscillation speed is 200 rpm.

[0076] In step S3, the first solution is composed of 0.6 parts by mass of tryptone, 1 part by mass of soy peptone, 0.4 parts by mass of yeast extract, 0.6 parts by mass of sodium chloride, 0.25 parts by mass of potassium dihydrogen phosphate, 0.06 parts by mass of magnesium chloride hexahydrate, 0.04 parts by mass of cysteine ​​hydrochloride, 0.001 parts by mass of ferrous sulfate heptahydrate, 0.12 parts by mass of Tween-80, 2.5 parts by mass of glucose and 110 parts by mass of distilled water.

[0077] In step S4, the static culture time of the third culture solution is 5 days.

[0078] In step S5, the shaking time of the second mixed solution is 4 hours, and the shaking time of the third mixed solution is 9 hours. The mass ratio of the first bacterial solution, the third bacterial solution, the second bacterial solution and the first mixed solution is 0.8:0.9:0.4:110;

[0079] The rest is the same as in Example 1. Embodiment 5

[0080] The difference between this embodiment and the first embodiment is that:

[0081] In step S1, the weight ratio of natural ox bile, vitamin C and rosemary is 1000:4:2.2.

[0082] In step S2, the oscillation speed is 180 rpm.

[0083] In step S3, the first solution is composed of 1 part by mass of tryptone, 0.6 part by mass of soy peptone, 0.6 part by mass of yeast extract, 0.4 part by mass of sodium chloride, 0.35 part by mass of potassium dihydrogen phosphate, 0.04 part by mass of magnesium chloride hexahydrate, 0.06 part by mass of cysteine ​​hydrochloride, 0.001 part by mass of ferrous sulfate heptahydrate, 0.08 part by mass of Tween-80, 3.5 parts by mass of glucose and 90 parts by mass of distilled water.

[0084] In step S4, the static culture time of the third culture solution is 7 days.

[0085] In step S5, the shaking time of the second mixed solution is 2 hours, and the shaking time of the third mixed solution is 10 hours. The mass ratio of the first bacterial solution, the third bacterial solution, the second bacterial solution and the first mixed solution is 1.2:0.7:0.6:90.

[0086] The rest is the same as in Example 1.

[0087] Comparative Example 1

[0088] In this comparative example, steps S3 and S4 are removed on the basis of Example 1, that is, the second bacterial liquid and the third bacterial liquid are not added. On this basis, the fermentation step is adjusted as follows: the first bacterial liquid of step S2 is added to the first mixed liquid of step S1 to obtain a second mixed liquid, the second mixed liquid is slowly shaken at 37° C. for 2 h, the mass ratio of the first bacterial liquid to the first mixed liquid is 0.8:90, the second mixed liquid is allowed to stand, the supernatant is discarded, filtered, and dried to obtain bezoar.

[0089] Comparative Example 2

[0090] In this comparative example, step S4 is removed on the basis of Example 1, that is, the third bacterial liquid is not added. On this basis, the fermentation steps are adjusted as follows: the first bacterial liquid of step S2 is added to the first mixed liquid of step S1 to obtain a second mixed liquid, the second mixed liquid is slowly shaken in an environment of 37° C. for 2 h, and then the second bacterial liquid of step S3 is added to obtain a third mixed liquid, and the third mixed liquid is slowly shaken in an environment of 37° C. for 8 h; the mass ratio of the first bacterial liquid, the second bacterial liquid and the first mixed liquid is 0.8:0.4:90.

[0091] Comparative Example 3

[0092] In this comparative example, step S2 is removed on the basis of Example 1, that is, the second bacterial liquid is not added. On this basis, the fermentation step is adjusted as follows: the first bacterial liquid of S2 and the third bacterial liquid of step S4 are added to the first mixed liquid of step S1 to obtain a second mixed liquid, the second mixed liquid is slowly shaken in an environment of 37° C. for 2 hours, the mass ratio of the first bacterial liquid, the third bacterial liquid and the first mixed liquid is 0.8:0.7:90, and the second mixed liquid is allowed to stand, the supernatant is discarded, filtered, and dried to obtain bezoar.

[0093] According to the method described in the 2020 edition of the Chinese Pharmacopoeia, the contents of bile acid and bilirubin in the bezoar prepared in the above examples and comparative examples were determined, and the yield of each bezoar was calculated, yield = mass of prepared bezoar / mass of natural ox bile × 1000%. The results are shown in the following table:

[0094]

[0095] It can be seen that the present invention not only significantly improves the yield of bezoar, but also relatively increases the content of bile acid, while avoiding the toxic and side effects caused by excessive bilirubin, making its ratio closer to that of natural bezoar and improving its medicinal value.

[0096] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for cultivating high-quality bezoar using bacterial flora, comprising the following steps: S1 treatment: adding vitamin C and rosemary to the sterilized natural cow bile to obtain a first mixed solution, wherein the weight ratio of natural cow bile, vitamin C and rosemary is 1000:4-7:1.8-2.2; S2 Preparation of the first bacterial solution: A single colony of Escherichia coli is inoculated into the sterilized first culture medium to form a first culture solution, and the first culture solution is cultured by shaking until the cell concentration in the first culture solution reaches 2×10 8 CFU / mL, then centrifuge at 5000g for 15 min at 4°C to obtain the supernatant to prepare the first bacterial solution. The culture temperature of the first culture solution is 37°C, the shaking speed is 180-200 rpm, and the first culture medium is LB broth; S3: preparing the second bacterial solution: inoculating the Enterococcus faecalis seed solution into the sterilized second culture medium at a 2% inoculation rate to form a second culture solution; culturing the second culture solution at 37°C for 24 hours, centrifuging at 3000 rpm for 2 minutes, and taking the supernatant as the second bacterial solution; The second culture medium is composed of the following raw materials in parts by mass: Tryptone 0.6-1 part Soybean peptone 0.6-1 part Yeast extract 0.4-0.6 parts Sodium chloride 0.4-0.6 parts 0.25-0.35 parts of potassium dihydrogen phosphate 0.04-0.06 parts of magnesium chloride hexahydrate Cysteine ​​hydrochloride 0.04-0.06 parts 0.001 part of ferrous sulfate heptahydrate Tween-80 0.08~0.12 parts 2.5-3.5 parts of glucose 90-110 parts of distilled water S4 Preparation of the third bacterial solution: inoculate the P. acnes strain into the sterilized third culture medium to form a third culture solution, culture the third culture solution in an anaerobic environment at 37°C for 4 to 7 days, and dilute it with the third culture medium until the concentration of P. acnes reaches 1×10 8 CFU / mL, centrifuge at 3000 rpm for 10 min, and take the supernatant to prepare the third bacterial solution; S5 fermentation: adding the first bacterial liquid of step S2 and the third bacterial liquid of step S4 to the first mixed liquid of step S1 to obtain a second mixed liquid, slowly shaking the second mixed liquid at 37° C. for 2 to 4 hours, then adding the second bacterial liquid of step S3 to obtain a third mixed liquid, and slowly shaking the third mixed liquid at 37° C. for 8 to 10 hours; the mass ratio of the first bacterial liquid, the third bacterial liquid, the second bacterial liquid and the first mixed liquid is 0.8 to 1.2:0.7 to 0.9:0.4 to 0.6:90 to 110; S6 finished product: let the third mixed solution treated in step S5 stand, discard the supernatant, filter, and dry to obtain bezoar.

2. The method for cultivating high-quality bezoar by utilizing bacterial flora according to claim 1, characterized in that: In step S2, the method for preparing a single colony of Escherichia coli is: S21 was dipped into -80℃ frozen E. coli solution and evenly streaked on LB agar plate and cultured at 37℃ overnight; S22 Dip a small amount of colonies from the LB agar plate, add it to 1mL phosphate buffer, mix it to make a bacterial stock solution, take 100μL of the stock solution and add it back to 900μL phosphate buffer to dilute the stock solution tenfold until 1mL of bacterial solution is obtained. Spread the bacterial solution evenly on the LB agar plate and culture it at 37℃ for 24h to obtain dispersed single colonies of Escherichia coli.

3. The method for cultivating high-quality bezoar by utilizing bacterial flora according to claim 1, characterized in that: In step S3, the preparation method of Enterococcus faecalis seed solution is: Enterococcus faecalis strains were inoculated into the second culture medium at a 2% inoculation rate and cultured at 37°C for 24 h. After three generations of continuous activation, the temperature was lowered to 4°C and the cells were collected by centrifugation at 3000 rpm for 20 min. The cells were washed three times by centrifugation with sterile saline and the cell concentration was adjusted to 10 6 CFU / mL, and made into Enterococcus faecalis seed solution.

4. The method for cultivating high-quality bezoar by utilizing bacterial flora according to claim 3, characterized in that: The preparation method of the second culture medium is as follows: 0.6-1 parts by weight of tryptone, 0.6-1 parts by weight of soy peptone, 0.4-0.6 parts by weight of yeast extract, 0.4-0.6 parts by weight of sodium chloride, 0.25-0.35 parts by weight of potassium dihydrogen phosphate, 0.04-0.06 parts by weight of magnesium chloride hexahydrate, 0.04-0.06 parts by weight of cysteine ​​hydrochloride, 0.001 parts by weight of ferrous sulfate heptahydrate, 0.08-0.12 parts by weight of Tween-80 and 2.5-3.5 parts by weight of glucose are dissolved in 90-110 parts by weight of distilled water to form a first solution, the pH value of the first solution is adjusted to 6.5, and the first solution is sterilized at 121° C. for 15 minutes to obtain a second culture medium.

5. The method for cultivating high-quality bezoar by utilizing bacterial flora according to claim 4, characterized in that: The raw materials of the second medium consisted of 0.8 parts by mass of tryptone, 0.8 parts by mass of soy peptone, 0.5 parts by mass of yeast extract, 0.5 parts by mass of sodium chloride, 0.3 parts by mass of potassium dihydrogen phosphate, 0.05 parts by mass of magnesium chloride hexahydrate, 0.05 parts by mass of cysteine ​​hydrochloride, 0.001 parts by mass of ferrous sulfate heptahydrate, 0.1 parts by mass of Tween-80, 3 parts by mass of glucose and 100 parts by mass of distilled water.

6. The method for cultivating high-quality bezoar by utilizing bacterial flora according to any one of claims 1 to 5, characterized in that: The preparation method of the third culture medium is: The BHI powder and deionized water were mixed at a mass ratio of 1:10 to form a mixed solution, and the pH value of the mixed solution was adjusted to 7.0 to prepare a third culture medium.

7. The method for cultivating high-quality bezoar by utilizing bacterial flora according to any one of claims 1 to 5, characterized in that: The weight ratio of natural ox bile, vitamin C and rosemary is 1000:5.5:

2.

8. The method for cultivating high-quality bezoar by using bacterial flora according to any one of claims 1 to 5, characterized in that: In step S5, the mass ratio of the first bacterial liquid, the third bacterial liquid, the second bacterial liquid and the first mixed liquid is 1:0.8:0.5:100.

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