CN115851497A - Bovine bile resistant type bezoar transformation bacterium and application thereof - Google Patents

Abstract

The invention belongs to the field of development and utilization of ox bile resistant bezoar transformation bacteria resources, and particularly relates to an ox bile resistant bezoar transformation bacteria and application thereof. The characteristic base sequence of the 16S rDNA of the ox bile-resistant bezoar transformation bacterium is shown in SEQ No.1, and the characteristic base sites comprise: 265-A,275-C,280-G,282-A,537-A,581-A, is a unique Citrobacter bovini strain with the accession number: CGMCC No.24893, is used in the fermentation production of in vitro cultured bezoar, and has bilirubin and cholic acid content increased by over 2 times compared with natural ox bile after fermentation at 30 deg.c for 16 days. The bacterial strain NDZNM-01 can tolerate oxgall with broad-spectrum bacteriostatic action, the survival time of the oxgall in pure oxgall is more than 9 days, a delay period is within 48 hours, a logarithmic growth period is started later, and the thallus density reaches a peak value after 96 hours of culture.

Description

Bovine bile resistant type bezoar transformation bacterium and application thereof

Technical Field

The invention belongs to the field of development and utilization of ox bile resistant bezoar transformation bacteria resources, and particularly relates to an ox bile resistant bezoar transformation bacteria and application thereof.

Background

Because the social demand of the natural bezoar is extremely large and the contradiction between supply and demand is very prominent, in order to solve the contradiction, the artificial synthetic bezoar is successfully developed in 50 years of the 20 th century, but the artificial bezoar belongs to the formula bezoar, and can not replace the natural bezoar in most cases. Therefore, in the last 90 th generation, professor Cai Gongjiao developed bezoar (CN 1022668C, CN1044402a, CN1041105 a) in vitro culture, and was recorded in chinese pharmacopoeia (2020 edition). The in vitro cultured bezoar is formed by simulating the forming process of gallstone of a cow under pathological conditions and applying coculture and transformation of oxgall and microorganisms, the chemical components and clinical efficacy of the bezoar are similar to those of natural bezoar, and the bezoar can be used as a medicine and clinically in most cases.

The in vitro cultured bezoar is used as the raw material of Chinese patent medicine, the raw material of health products and the Chinese herbal pieces, and has huge market demand space and wide development prospect. At present, only Wuhan Jianmin Dapeng exclusively produces in-vitro cultured bezoar, and from the patent literature, the in-vitro cultured bezoar produced by the method needs to meet the content requirement of quality detection indexes of pharmacopeia by additionally adding a large amount of compound bilirubin calcium salt and bile acid, so that the production cost is high, the market price of a finished product is extremely high and exceeds one third of that of natural bezoar, and the biotransformation efficiency of bilirubin and bile acid of a strain produced by the method is low.

The bacterial strain is the key for culturing bezoar to become yellow in vitro, and any bacteria can not convert the ox bile to form the bezoar; in addition, the ox bile has broad-spectrum antibacterial action, most intestinal bacteria cannot tolerate the bile and cannot survive in the bile for a long time, and particularly, some high-efficiency calculus bovis transformation bacteria are difficult to survive in the bile for more than 48 hours, so that the production efficiency of calculus bovis transformation is seriously influenced, the production cost is directly determined, and the market price of in-vitro cultured calculus bovis is indirectly influenced. Therefore, screening strains which can tolerate ox bile for a long time and can be efficiently converted into gallstones is the key for improving the production efficiency of in vitro cultured bezoar, reducing the production cost and realizing that the rare traditional Chinese medicine is not expensive and the common Hui Zhong is raw.

Disclosure of Invention

Aiming at the problems of in vitro calculus bovis cultivation, the invention screens and obtains a calculus bovis bile-resistant type calculus bovis converting bacterium, researches the calculus bovis converting capacity of the calculus bovis converting bacterium, and obtains high calculus bovis converting efficiency. Improves the production efficiency of in vitro cultured bezoar, reduces the production cost, is put on the market, and can realize the purpose that the precious traditional Chinese medicine bezoar is no longer expensive.

The technical scheme of the invention is as follows:

the ox bile resistant bezoar transformation bacterium is ox bile citric acid bacillusCitrobacter bovisNDZNM-01, samples of which were deposited at 12.5.2022 in the China general microbiological culture Collection center (CGMCC), accession number: CGMCC No. 24893.

Preferably, the characteristic base sequence of 16S rDNA of the bezoar transformation bacterium is shown as SEQ No. 1. Preferably, the amplification primer of the characteristic base sequence of 16S rDNA of the bezoar transformation bacterium is:

the base sequence of the forward F primer is shown as SEQ No. 2; the method comprises the following specific steps: 5'-CGCAAGCCTGATGCAG-3';

the base sequence of the reverse R primer is shown as SEQ No. 3; the method specifically comprises the following steps: 5'-CTTCGCGTTGCATCGA-3'.

Preferably, the characteristic base site of the 16S rDNA characteristic base sequence of the bezoar bovis bacteria comprises: 265-A,275-C,280-G,282-A,537-A,581-A.

The method for identifying the ox bile-resistant bezoar transformation bacteria adopts the amplification primer and contrasts the characteristic base site.

Further, the survival time of the bezoar transformation bacteria in the pure oxgall is more than 9 days.

The invention also aims to protect the application of the ox bile-resistant bezoar transformation bacterium, which is applied to the cultivation and production of in vitro cultivated bezoar.

Further, the fermentation culture conditions of the bezoar transformation bacteria applied to the culture production of in vitro cultured bezoar are as follows: the pH value of the fermentation liquor is 6.5-7.5, and the fermentation temperature is 20-35 ℃.

Further, the culture medium raw materials of the bezoar transformation bacteria applied to the strain scale culture in the culture production of in vitro cultured bezoar comprise: tryptone, beef extract, sodium chloride; according to the adding amount of the agar powder, a semi-solid culture medium and a solid culture medium are prepared.

The invention has the beneficial effects that:

1. bilirubin and cholic acid as main medicinal components for efficiently converting ox bile to form in-vitro cultured bezoar

Strains of the present applicationCitrobacter bovisWhen NDZNM-01 is cultured in oxgall at the culture temperature of 30 ℃, the contents of bilirubin and cholic acid in 16d fermentation respectively reach 2.01 per mill and 6.42 percent, the bilirubin content is increased by 2.35 times compared with the natural oxgall, and the cholic acid content is increased by 2.13 times.

2. Long survival time in ox bile

As the oxgall has broad-spectrum antibacterial action, most intestinal bacteria hardly survive in the oxgall for more than 48 hours, the bacterial strain of the applicationCitrobacter bovisNDZNM-01, through the growth curve determination, the survival time in pure oxgall is more than 9 days, when the fermentation temperature in oxgall is 30 ℃, the culture is in a lag phase within 48h, then the logarithmic growth period of the rapid increase of the thallus density is entered, the thallus density is maximum when the culture is carried out for 96h, and then the culture is continued for 4 days, and the thallus density begins to slowly decrease.

3. Transformed bovine bezoar bacterium of the present applicationCitrobacter bovisNDZNM-01 is a protein derived from oxgallSpecific bovine bile Citrobacter

To date, there is no literature reporting that Citrobacter can survive in oxgall, and even more, there is no literature reporting that the metabolic activity of Citrobacter can increase the conversion efficiency of bilirubin and cholic acid in oxgall.

Strain preservation information:

preservation time: 12/05/2022;

the preservation unit: china general microbiological culture Collection center;

the preservation number is: CGMCC No. 24893;

the address of the depository: the microbial research institute of the national academy of sciences, no.3, west way, no.1, north chen, chaoyang, zip code: 100101;

and (3) classification and naming: bovine bile CitrobacterCitrobacter bovis。

Drawings

FIG. 1 is a morphological observation of an oxgall-resistant strain NDZNM-01 and its bezoar conversion performance; a, morphological characteristics (magnification 10 multiplied by 40, scale 50 μm) of the oxgall-resistant strain NDZNM-01; b, fermenting oxgall to form turbid liquid of bezoar particles by an oxgall-resistant strain NDZNM-01; c, a process of polymerizing bezoar particles into bezoar stones caused by fermenting the oxgall through the oxgall-resistant strain NDZNM-01; d, a process of fermenting ox bile by the ox bile-resistant strain NDZNM-01 to initiate the polymerization of bezoar particles to form bezoar;

FIG. 2 shows the sequencing result of the 16S rDNA amplified fragment of bovine bile resistant bezoar transformed strain NDZNM-01;

FIG. 3 is a rootless phylogenetic tree of 16S rRNA gene fragment sequences of bovine bile resistant transformed bezoar bacterial strain NDZNM-01 and its kindred bacterial strains constructed based on Fast minium Evolution method;

FIG. 4 shows the bezoar conversion rate of bezoar transformed strain NDZNM-01 resistant to ox bile at different pH values of fermented bile;

FIG. 5 is a graph showing the effect of the culture temperature on the bile acid-converting ability of bovine bile resistant bezoar transformed strain NDZNM-01;

FIG. 6 is a graph showing the effect of culture temperature on the bilirubin conversion ability of bovine bile resistant bezoar transforming strain NDZNM-01;

FIG. 7 is a graph showing the growth of bovine bile resistant transformed bezoar bovis NDZNM-01.

Detailed Description

Example 1 Process for isolating and purifying ox bile-resistant bezoar bovis Seu Bubali transformant bacteria from ox bile with yellow gallbladder

1. Screening Medium

The culture medium formula for screening the bezoar transformation bacteria in the ox bile comprises the following components: 1.00g of tryptone, 0.30g of beef extract and 0.50g of sodium chloride, adding deionized water to a constant volume of 100ml, heating to dissolve, cooling, adjusting the pH value to 7.2, boiling for 10 min, cooling, filtering, subpackaging and autoclaving for 30 min. 1.5g of agar powder is added when preparing a solid culture medium; when preparing the semi-solid culture medium, 0.3g of agar powder is added.

2. Method for measuring content of bilirubin and cholic acid

According to Chinese pharmacopoeia (2020 edition I, crude drugs and decoction pieces, page 181), the contents of bilirubin and cholic acid in ox bile, a culture medium and in vitro cultured bezoar are detected.

3. Process for separating and purifying ox bile-tolerant bezoar transformation bacteria

Taking fresh ox bile with yellow gall bladder, streaking and culturing on the solid culture medium, culturing at 37 ℃ for 24 h, selecting single bacterial colonies with good separation degree, respectively inoculating the single bacterial colonies in the semisolid culture medium, numbering, culturing for 24 h, respectively adding 100 mu L of bacterial liquid into 50 ml sterilized ox bile, culturing for 96h, respectively taking 10 mu L of bacterial liquid, coating the bacterial liquid on the solid culture medium, culturing for 72 h, counting the number of bacterial colonies, and designating the bacterial colonies with the number of bacterial colonies more than 1000 as the ox bile tolerant strain. And (3) measuring the content of bilirubin and cholic acid in the oxgall of each culture tolerant strain, and designating bacterial colonies with the content of bilirubin and cholic acid in the fermented oxgall more than 2 times of that in the natural oxgall as bezoar transformation bacteria.

Example 2 morphological Observation and physiological and Biochemical identification of bovine bile resistant bovine bezoar-converting bacteria

Example 1 separation and get 46 single colony, with the liquid medium containing 70% oxgall cultured, every 1d get culture solution to count and bilirubin and cholic acid content measure, choose a bile-resistant strain with high bilirubin and cholic acid comprehensive conversion rate named NDZNM-01, inoculate the strain on the solid medium of example 1, after culturing 24 h at 37 ℃, record its colony morphological characteristics, carry on gram staining, carry on morphological observation.

The purified NDZNM-01 strain is inoculated in the semi-solid culture medium of example 1, shaking culture is carried out at 37 ℃ and 160 rpm for 24 h, 10 mu L of bacterial liquid is inoculated in a bacterial micro biochemical reaction tube, culture is carried out at 37 ℃ for 24 h-48 h, and physiological and biochemical identification tests are carried out according to Berger's bacteria identification handbook, wherein the physiological and biochemical identification tests comprise gram staining, glucose and sucrose fermentation tests, citrate and nitrate utilization tests, acetyl methyl methanol tests (V.P tests), starch hydrolysis tests and methyl red tests (M.R tests), and extracellular enzyme activity tests (catalase, oxidase, lysine decarboxylase and phenylalanine deaminase) are carried out. The results are shown in FIG. 1, from which it can be seen that: the strain NDZNM-01 forms a gray round colony which is wet, low-convex, smooth in surface, neat in edge and semitransparent on a solid culture medium, is gram-negative, is in a short rod shape under a light mirror, is blunt-rounded on two sides, is provided with peripheral flagella and aseptic hair, and the estimated size of the bacterial body is about 2.5 microns multiplied by 3.5 microns.

The bacterial strain NDZNM-01 is inoculated in a biochemical identification tube for carrying out physiological and biochemical identification tests, the results are shown in table 1, and the gram staining, V.P experiment, starch hydrolysis, sucrose fermentation experiment and other negatives of the bacterial strain NDZNM-01, glucose, citrate and nitrate utilization experiment, M.R experiment, catalase detection and other negatives, oxidase, lysine decarboxylase, phenylalanine deaminase activity detection and other negatives can be seen. And comparing the detection result with a Bergey bacteria identification manual, and preliminarily judging that the strain NDZNM-01 is the Citrobacter bacteria.

TABLE 1 physiological and biochemical Properties of Strain NDZNM-1

Note: "+": positive or responsive; "-": negative or no reaction

Example 3 molecular characterization of bovine bile resistant transformed bezoar bacterium NDZNM-01

1. Genomic DNA extraction of strain NDZNM-01

The genomic DNA of strain NDZNM-01 was extracted using TIANGEN bacterial genomic DNA extraction kit (DP 302) according to the kit instructions.

2 PCR amplification of 16S rDNA of strain NDZNM-01

Multiple alignments of enterobacter 16S rDNA sequences recorded in nr database of GenBank were performed using BLASTn program, and conserved consensus sequences were selected as PCR amplification primers according to the alignment results.

Forward F primer (16 bp): 5'-CGCAAGCCTGATGCAG-3';

reverse R primer (16 bp): 5'-CTTCGCGTTGCATCGA-3'

3. PCR reaction system for amplifying 16S rDNA of strain NDZNM-01

PCR reaction (25. Mu.L): DNA template 1.0. Mu.L, 5 XBuffer 3.0. Mu.L, F primer (10. Mu.M) and R primer (10. Mu.M) each 0.5. Mu.L, dNTP (2.50 mM) 1. Mu.L, ddH ₂ O is added to a constant volume of 25 mu L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; 95. denaturation at 30S, annealing at 50 ℃ for 30S, extension at 72 ℃ for 90S, and extension at 72 ℃ for 12min after 30 cycles. And (3) carrying out agarose gel electrophoresis on the amplification product, and purifying and recovering the amplification product by using a TIANGEN (DP 209) agarose gel DNA recovery kit to complete sequencing.

4. Method for creating phylogenetic tree by using 16S rDNA sequence of strain NDZNM-01

The determined 16S rDNA sequences were aligned for homology using the BLASTn program to search the nr database of GenBank. A phylogenetic tree is constructed by adopting a Fast Minimum Evolution method, and the Max Seq Difference value of the constructed tree is set to be 0.1. Sequence statistics analysis was performed using the EditSeq subroutine of DNAStar.

5. Results and conclusions of the present example

The sequencing results are shown in FIG. 2. The total length of the sequence is 598bp, and the content of C + G is 55.69%. The sequence is as follows: CGCAAGCCTG ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT TCAGCGAGGA GGAAGGTGTT GTGGTTAATA ACCRCAGCAA TTGACGTTAC TCGCAGAAGA AGCACCGGCT AACTCCGTGC CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG CGTAAAGCGC ACGCAGGCGG TCTGTCAAGT CGGATGTGAA ATCCCCGGGC TCAACCTGGG AACTGCATCC GAAAATGGCA GGCTCGAGTG TAGTAGAGGG GGGTAGAATT CCAGGTGTAG CGGTGAAATG CGTAGAGATC TGGAGGAATA CCGGTGGCGA AGGCGGCCCC CTGGACAAAG ACTGACGCTC AGGTGCGAAA GCGTGGGGAG CAAACAGGAT TAGATACCCT GGTAGTCCAC GCCGTAAACG ATGTCGACTT GGAGGTTGTG CCCTTGAGGC GTGGCTTCCG GAGCTAACGC GTTAAGTCGA CCGCCTGGGG AGTACGGCCG CAAGGTTAAA ACTCAAATGA ATTGACGGGG GCCCGCACAA GCGGTGGAGC ATGTGGTTTA ATTCGATGCA ACGCGAAG.

The search of the rRNA/ITS databases using the BLASTn program (https:// blast. NCBI. Nlm. Nih. Gov/blast. Cgi) provided by NCBI (http:// www.ncbi.nlm.nih.gov) showed that the 16S rRNA gene amplified fragment of strain NDZNM-01 has a similarity of more than 98% to the corresponding sequence of Citrobacter, and that the characteristic base sites included: 265-A,275-C,280-G,282-A,537-A,581-A. A phylogenetic tree is constructed by applying a Fast Minimum Evolution method, and the Max Seq Difference value of the constructed tree is set to be 0.7. As can be seen from FIG. 3, the strain NDZNM-01 was clustered with three other Citrobacter bacteria to form a single line.

By combining morphological observation, physiological and biochemical identification and molecular identification results of the strain NDZNM-01, the strain NDZNM-01 can be judged to belong to an enterobacteria of the genus Citrobacter derived from oxgall, and is initially named as oxgall Citrobacter NDZNM-01 strain (Citrobacter bovis NDZNM-01 Wang Houwei）。

Example 4 examination of calculus bovis converting ability of calculus bovis-converting bacterium NDZNM-01 resistant to bovine bile

1. The method for detecting the bezoar transformation capability of the bezoar transformation bacteria NDZNM-01 which can tolerate oxgall

According to the Chinese pharmacopoeia (2020 edition 1 part), the bezoar conversion ability of the bacterial strain NDZNM-01 is expressed by the content of bilirubin and bile acid in the fermented ox bile. Respectively inoculating the bacterial strain NDZNM-01 into fresh oxgall, continuously shaking and culturing at 37 ℃ (120 rpm) for 14d, setting the same batch of oxgall without inoculated strains as a blank control, detecting the light absorption value of a culture 560 nm every 1 day, and detecting the content of bilirubin and cholic acid.

2. The influence of pH value of ox bile fermentation liquid on bezoar transformation capability of ox bile-tolerant bezoar transformation bacterium NDZNM-01

Inoculating the strain NDZNM-01 into the semisolid culture medium containing 60% oxgall in the embodiment 1, culturing for 3d, adjusting the pH value of the oxgall by using citric acid and sodium hydroxide solution, taking a strain culture solution 5.0 mL, respectively inoculating the strain culture solution into 95.0 mL oxgall with the pH values of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, performing shake culture (120 rpm) at the temperature of 37 ℃, respectively detecting 560 nm light absorption values after culturing 7 d, and detecting the contents of bilirubin and cholic acid in the oxgall according to a 1.3.1 method.

The results are shown in fig. 4, the strain NDZNM-01 had the highest bilirubin content in fermented oxgall of 16.75% (mg.%), at ph7.5, the highest cholic acid content of 5.16 (g.%), at ph6.5, whereas the bilirubin content in unfermented oxgall was only 7.14 (mg.%), and the cholic acid content was 2.42 (g.%). Therefore, the contents of cholic acid and bilirubin in the oxgall fermented by the strain NDZNM-01 are remarkably improved, wherein the content of bilirubin is increased by 2.35 times, and the content of cholic acid is increased by 2.13 times.

As can be seen from FIG. 4, the pH value of oxgall has inconsistent effect on the conversion rate of cholic acid and bilirubin of the strain NDZNM-01, the conversion rate of bilirubin is high under a slightly alkaline condition, and the conversion rate of cholic acid is high under a slightly acidic environment. Wherein the cholic acid content far exceeds the standard of Chinese pharmacopoeia, so the pH value of the fermented oxgall meets the conversion principle of bilirubin preferentially. In combination of two factors, the bezoar conversion efficiency of the bacterial strain NDZNM-01 is preferably that the pH value of the fermented bile is 7.2, and the bezoar conversion is not facilitated by too high and too low pH values.

3. Influence of ox bile fermentation temperature on bezoar transformation capability of bezoar transformation strain NDZNM-01 for resisting oxgall

The bacterial strain NDZNM-01 culture solution 5.0 mL cultured in the semisolid culture medium of example 1 containing 60% bile is inoculated into 95.0 mL fresh oxgall, 16d is cultured under the conditions of 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ in a shaking mode (120 r/min), the bacterial concentration is detected in 560 nm, and the content of bilirubin and cholic acid in the oxgall is detected.

The result is shown in figure 5, when the bacterial strain NDZNM-01 is cultured at 30 ℃, the cholic acid content is highest after fermentation until 16d reaches 6.42%; when the culture is carried out at 25 ℃ and 30 ℃, fermentation is carried out until 14d of cholic acid conversion efficiency enters a slow growth period; when the culture is carried out at 35 ℃ and 40 ℃, fermentation is carried out until 12d of cholic acid conversion rate enters a slow increasing period; when the fermentation temperature of the bile is 10 ℃, 15 ℃ and 20 ℃, the cholic acid content is positively correlated with the fermentation time, and the slow growth period does not appear.

As can be seen from figure 6, when the fermentation temperature of the bacterial strain NDZNM-01 in ox bile is 30 ℃ for culture, the content of bilirubin is highest after fermentation until 16d, and reaches 2.01 per mill, and the conversion rate of bilirubin enters a slow increasing period when the content of bilirubin is fermented until 14 d; when the fermentation temperature is 35 ℃ and 40 ℃, the conversion rate of the bilirubin is slowly increased after the fermentation is carried out for 12 d; when the fermentation temperature is 10 ℃, 15 ℃, 20 ℃ and 25 ℃, the bilirubin content is positively correlated with the fermentation time, and the slow growth period does not appear.

Example 5 determination of growth curves of bovine bile resistant transformed bezoar bovis NDZNM-01

Inoculating single colony of strain NDZNM-01 into 5 ml semisolid culture medium, shake culturing at 37 deg.C for 6 hr, inoculating 1ml of bacterial liquid into 100ml of liquid culture medium, shake culturing, and measuring thallus density (OD) every 1 day ₅₆₀ ) In OD ₅₆₀ The values are plotted on the ordinate and the incubation time is plotted on the abscissa to prepare a growth curve. As can be seen from FIG. 7, the survival time of the bezoar transformation bacterium NDZNM-01 in pure oxgall is more than 9 days, the lag phase of the bacterial strain NDZNM-01 is within 48h, then the logarithmic growth period of the bacterial density rapid growth is entered, the bacterial density is maximum when the bacterial density is cultured for 96h, and then the bacterial density begins to slowly decrease after the bacterial density is continuously cultured for 4 days.

Claims (9)

1. The bovine bile resistant bezoar transformation bacterium is characterized in that the bovine bile resistant bezoar transformation bacterium is bovine bile citrobacterCitrobacter bovisThe sample is preserved in China general microbiological culture Collection center on 12 th 5 th 2022 with the preservation number: CGMCC No. 24893.

2. The bovine bile resistant transformed bezoar having a nucleotide sequence characteristic of 16S rDNA of the transformed bezoar according to claim 1, wherein the nucleotide sequence is represented by SEQ No. 1.

3. The bovine bile resistant transformed bezoar bovis bacteria according to claim 1, wherein the primers for amplifying the characteristic base sequence of 16S rDNA of said bezoar bovis bacteria are:

the base sequence of the forward F primer is shown as SEQ No. 2; the method specifically comprises the following steps: 5'-CGCAAGCCTGATGCAG-3';

the base sequence of the reverse R primer is shown as SEQ No. 3; the method specifically comprises the following steps: 5'-CTTCGCGTTGCATCGA-3'.

4. The bovine bile resistant transformed bezoar bovis bacteria according to claim 2, wherein the characteristic base site of the 16S rDNA characteristic base sequence of said bezoar bovis bacteria comprises: 265-A,275-C,280-G,282-A,537-A,581-A.

5. The method for identifying bezoar bovis Seu Bubali-resistant transformant bacteria according to any one of claims 1 to 4, wherein the amplification primer according to claim 3 is used in comparison with the characteristic base site according to claim 4.

6. The bovine bile resistant bezoar transformation bacteria of claim 1, wherein said bezoar transformation bacteria survive in pure oxgall for a time greater than 9 days.

7. The use of the bovine bile resistant transformed bezoar according to claim 1, wherein the use is in the cultivation and production of in vitro cultivated bezoar.

8. The use according to claim 7, wherein the culture conditions of said transformed bovine bezoar bacteria for the in vitro cultured production of bovine bezoar are: the pH value of the fermentation liquor is 6.5-7.5, and the fermentation temperature is 20-35 ℃.

9. The use of the transformed bovine bezoar according to claim 7 or 8, wherein the culture medium raw material for the strain expansion culture in the culture production of in vitro cultured bovine bezoar comprises: tryptone, beef extract, sodium chloride; according to the addition amount of agar powder, a semisolid culture medium and a solid culture medium are prepared.

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