CN115851497B - Ox bile-resistant bezoar transformation strain and application thereof - Google Patents

Abstract

The invention belongs to the field of development and utilization of bovine biliary-resistant bovine bezoar conversion bacteria resources, and particularly relates to a bovine bezoar conversion bacteria resistant to bovine bezoar and application thereof. The characteristic base sequence of the 16S rDNA of the bovine biliary-resistant calculus bovis transformation bacterium is shown as SEQ No.1, and the characteristic base site comprises: 265-A,275-C,280-G,282-A,537-A,581-A, a unique Citrobacter oxgall, strain accession number: CGMCC No.24893 is applied to the fermentation production of in vitro cultured bezoar, and the content of bilirubin and cholic acid is increased by more than 2 times compared with natural oxgall after fermentation for 16 days at 30 ℃. The strain NDZNM-01 can tolerate oxgall with broad-spectrum antibacterial effect, has survival time of more than 9 days in pure oxgall and is in a delayed period within 48 hours, and then enters a logarithmic growth phase to be generally cultured until the cell density reaches a peak value within 96 hours.

Description

Ox bile-resistant bezoar transformation strain and application thereof

Technical Field

The invention belongs to the field of development and utilization of bovine biliary-resistant bovine bezoar conversion bacteria resources, and particularly relates to a bovine bezoar conversion bacteria resistant to bovine bezoar and application thereof.

Background

Because the social demand of natural bezoar is very large and the contradiction between supply and demand is very prominent, in order to solve the contradiction, artificial bezoar is successfully developed in the 50 s of the 20 th century, and the artificial bezoar belongs to formula bezoar, and most cases cannot replace natural bezoar. For this reason, the teaching Cai Gongjiao of the 90 th century was first developed into in vitro cultured bezoar (CN 1022668C, CN1044402A, CN1041105 a) and incorporated into the chinese pharmacopoeia (2020 edition). The in vitro bezoar cultivation is carried out by simulating the formation process of bezoar under pathological conditions, and is formed by co-culturing and transforming ox gall and microorganism, the chemical components and clinical effects of the bezoar are similar to those of natural bezoar, and most of the cases can be equivalent to the use of natural bezoar in medicine and clinical application.

The in vitro cultured bezoar is used as a raw material of Chinese patent medicines and health care products and Chinese medicinal decoction pieces, and has huge market demand space and wide development prospect. At present, only the in vitro cultivation bezoar is produced by the Qingjinshen of Wuhan, and the in vitro cultivation of the production needs to meet the content requirement of the quality detection index of pharmacopoeia 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 the finished product is extremely expensive and exceeds one third of that of natural bezoar, and the biological conversion efficiency of bilirubin and bile acid of the produced strain is also reflected.

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

Disclosure of Invention

Aiming at the problems existing in-vitro cultivation of bezoar, the invention screens and obtains a strain of bezoar transformation bacteria resistant to ox bile, researches the bezoar transformation capability and obtains high bezoar transformation efficiency. Improves the production efficiency of in vitro bezoar cultivation, reduces the production cost, and can realize the aim that the rare traditional Chinese medicine bezoar is not expensive any more.

The technical scheme of the invention is as follows:

a strain of bezoar-resistant bacteria is prepared from Citrobacter bovisCitrobacter bovisNDZNM-01, samples of which were deposited at the China general microbiological culture Collection center, accession number: CGMCC No. 24893.

Preferably, the characteristic base sequence of the 16S rDNA of the bezoar transformation bacteria is shown as SEQ No. 1. Preferably, the amplification primer of the characteristic base sequence of the 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 steps: 5'-CGCAAGCCTGATGCAG-3';

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

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

The identification method of the ox gall-resistant bezoar transformation bacteria adopts the amplification primer and controls the characteristic base site.

Further, the bezoar transformation bacteria survive for more than 9 days in pure oxgall.

The invention also aims to protect the application of the ox bile-resistant bezoar transformation bacteria in cultivation production of in-vitro cultivated bezoar.

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

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

The invention has the beneficial effects that:

1. high-efficiency conversion of ox bile to form bilirubin and cholic acid as main medicinal components for in vitro culturing bezoar

Strains of the present applicationCitrobacter bovisWhen NDZNM-01 is cultured in oxgall at 30deg.C, the bilirubin and cholic acid content of fermentation 16d respectively reach 2.01%o and 6.42%, bilirubin content is increased by 2.35 times than natural oxgall, and cholic acid content is increased by 2.13 times.

2. Long survival time in oxgall

Because oxgall has broad-spectrum antibacterial effect, most intestinal bacteria are difficult to survive for more than 48 hours in oxgall, and the strain of the applicationCitrobacter bovisNDZNM-01, as measured by growth curve, survives for more than 9 days in pure oxgall, and is a stagnation period within 48 hours when cultured at a fermentation temperature of 30 ℃ in oxgall, then enters a logarithmic growth phase with rapidly increased cell density, and the cell density is maximum when cultured for 96 hours, and then continues to culture for 4 days, and the cell density begins to slowly decrease.

3. The bezoar transformation bacterium of the present applicationCitrobacter bovisNDZNM-01 is a special Citrobacter bovis Seu Bubali-derived from fel bovis Seu Bubali

To date, no document reports that Citrobacter can survive in oxgall, and no document reports that the metabolic activity of Citrobacter can increase the conversion efficiency of bilirubin and cholic acid in oxgall.

Strain preservation information:

preservation time: 2022, 05, 12;

preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);

preservation number: CGMCC No. 24893;

deposit unit address: the institute of microbiology, national institute of sciences, no.3, north chen west way 1, region of korea, beijing city, postal code: 100101;

classification naming: citrobacter oxgallCitrobacter bovis。

Drawings

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

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

FIG. 3 is a root system-free phylogenetic tree of 16S rRNA gene segment sequences of bovine biliary powder-resistant transformed strain NDZNM-01 and related strains constructed based on Fast Minimum Evolution method;

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

FIG. 5 shows the effect of culture temperature on the bile-resistant bovine bezoar transformation strain NDZNM-01 cholic acid transformation capacity;

FIG. 6 shows the effect of culture temperature on the ability of bovine biliary-tolerant bovine bezoar transformant strain NDZNM-01 to convert bilirubin;

FIG. 7 shows the growth curve of bovine biliary-resistant transformed strain NDZNM-01.

Detailed Description

EXAMPLE 1 Process for isolation and purification of bovine biliary-tolerant transformed bacteria from bovine bile yellow in the gallbladder

1. Screening media

The formula of the culture medium for screening bezoar transformation bacteria in the oxgall is as follows: 1.00g of tryptone, 0.30g of beef extract and 0.50g of sodium chloride, adding deionized water to a volume of 100ml, heating for dissolving, cooling, adjusting the pH to 7.2, boiling for 10 min, cooling, filtering, packaging, and sterilizing under high pressure for 30 min. 1.5g of agar powder is added when preparing a solid culture medium; 0.3g of agar powder was added when preparing the semisolid culture medium.

2. Bilirubin and cholic acid content determination method

Bilirubin and cholic acid content in oxgall, culture medium and in vitro cultured calculus bovis are detected according to Chinese pharmacopoeia (2020 edition one, medicinal materials and decoction pieces, 181 pages).

3. Process for separating and purifying bezoar transformation bacteria tolerant to oxgall

Fresh oxgall is taken to yellow oxgall, streak culture is carried out on the solid culture medium, after the culture is carried out at the constant temperature of 37 ℃ for 24 h, single colonies with good separation degree are selected and respectively inoculated into the semisolid culture medium, serial numbers are made, after 24 h is cultivated, 100 mu L of bacterial liquid is respectively taken and added into 50 ml sterilized oxgall, after 96h is cultivated, 10 mu L of bacterial liquid is respectively taken and coated on the solid culture medium, after 72 h is cultivated, the number of the colonies is counted, and the colonies with the number of the colonies being more than 1000 are designated as oxgall-resistant strains. The bilirubin and cholic acid content in the oxgall of each cultured tolerant strain was measured, and colonies having bilirubin and cholic acid content in the fermented oxgall of more than 2 times that in the natural oxgall were designated as bezoar transformants.

EXAMPLE 2 morphological observation and physiological Biochemical identification of bovine bile-tolerant transformed bovine bacteria

Example 1 46 single colonies were isolated, cultured in a liquid medium containing 70% oxgall, the culture medium was taken every 1d for cell count and bilirubin and cholic acid content detection, a bile-resistant strain with high comprehensive conversion rate of bilirubin and cholic acid was selected and designated as NDZNM-01, the strain was inoculated on the solid medium of example 1, cultured at 37 ℃ for 24 h, and then the colony morphology was recorded for gram staining and morphological observation.

The purified NDZNM-01 strain was inoculated in the semi-solid medium of example 1, shaking culture was performed at 37℃and 160 rpm for 24 h, 10. Mu.L of the bacterial liquid was inoculated in a bacterial micro biochemical reaction tube, and culture was performed at 37℃for 24. 24 h.about.48. 48h, and physiological biochemical identification tests including gram staining, glucose and sucrose fermentation tests, citrate and nitrate utilization tests, acetylmethyl methanol tests (V.P tests), starch hydrolysis tests, methyl red tests (M.R tests) and extracellular enzyme activity tests (contact enzyme, oxidase, lysine decarboxylase, phenylalanine deaminase) were performed with reference to "Berger's bacteria identification handbook". The results are shown in FIG. 1, and can be seen from FIG. 1: the strain NDZNM-01 forms a gray round colony which is moist, low-convex, smooth in surface, neat in edge and semitransparent on a solid culture medium, is gram-negative, takes a short rod shape under a light microscope, has round two sides, and is periflagellum and sterile hair, and the estimated thallus size is about 2.5 mu m multiplied by 3.5 mu m.

The strain NDZNM-01 is inoculated in a biochemical identification tube for physiological and biochemical identification test, the result is shown in table 1, and gram staining, V.P experiment, starch hydrolysis, sucrose fermentation experiment and the like of the strain NDZNM-01 are negative, glucose, citrate and nitrate are positive by experiment, M.R experiment, contact enzyme detection and the like, and oxidase, lysine decarboxylase, phenylalanine deaminase activity detection and the like are negative. Comparing the detection result with the ' Berger's bacteria identification Manual ', and primarily judging that the strain NDZNM-01 is the bacteria of the genus Citrobacter.

TABLE 1 physiological and biochemical Properties of Strain NDZNM-1

Note that: "+": positive or reactive; "-": negative or no reaction

EXAMPLE 3 molecular identification of bovine bile-tolerant bovine bezoar transformant 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 amplified 16S rDNA of strain NDZNM-01

The BLASTN program is used for carrying out multiple comparison on the enterobacter 16S rDNA sequences recorded in the nr database of GenBank, and a conserved consensus sequence is selected as a PCR amplification primer according to the comparison result.

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 System (25. Mu.L): 1.0. Mu.L of DNA template, 3.0. Mu.L of 5 XBuffer, 0.5. Mu.L of each of F primer (10. Mu.M) and R primer (10. Mu.M), 1. Mu.L of dNTP (2.50 mM), and ddH ₂ O was fixed to a volume of 25. Mu.L. PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; 95. denaturation at 30S, annealing at 50℃for 30S, elongation at 72℃for 90S, and after 30 cycles, elongation at 72℃for 12min. The amplified product was subjected to agarose gel electrophoresis, and purified and recovered using a TIANGEN (DP 209) agarose gel DNA recovery kit to complete sequencing.

4. 16S rDNA sequence creation system evolution tree using strain NDZNM-01

The BLASTN program was used to search the nr database of GenBank, and homology alignment was performed on the 16S rDNA sequences thus obtained. A phylogenetic tree is constructed by adopting a Fast Minimum Evolution method, and the value of the tree Max Seq Difference is set to be 0.1. Sequence statistical analysis was performed using the edit seq subroutine of DNAStar.

5. Results and conclusions of implementation of the present embodiment

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 rRNA/ITS databases were searched using the BLASTN program (https:// blast. NCBI. Lm. Nih. Gov/blast. Cgi) provided by NCBI (http:// www.ncbi.nlm.nih.gov), and the results showed that the 16S rRNA gene amplified fragment of strain NDZNM-01 had a similarity of greater 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. The phylogenetic tree was constructed using Fast Minimum Evolution method, and the value of the tree Max Seq Difference was set to 0.7. As can be seen from FIG. 3, the strain NDZNM-01 forms a single line with the other three bacteria of the genus Citrobacter in one branch.

The bacterial strain NDZNM-01 belongs to an intestinal tract bacterium of the genus Citrobacter from oxgall, and is primarily named as the bacterial strain NDZNM-01 of oxgallCitrobacter bovis NDZNM-01 Wang Houwei）。

EXAMPLE 4 detection of bezoar conversion ability of bovine bile-tolerant bovine bezoar conversion bacterium NDZNM-01

1. The method for detecting the bezoar transformation capacity of the bezoar transformation bacteria NDZNM-01 which tolerates the oxgall

According to Chinese pharmacopoeia (2020 edition 1 part), the bezoar conversion ability of the strain NDZNM-01 is expressed in terms of the content of bilirubin and bile acid in fermented oxgall. Bacterial strain NDZNM-01 is inoculated into fresh oxgall respectively, continuous shake culture (120 rpm) is carried out at 37 ℃ for 14d, the same batch of oxgall without inoculating bacterial strain is set as a blank control, the light absorption value of the culture is detected at 560 nm every 1 day, and the content of bilirubin and bile acid is detected.

2. Influence of pH value of oxgall fermentation liquor on bezoar conversion capability of bezoar conversion bacteria NDZNM-01 tolerant to oxgall

Bacterial strain NDZNM-01 is inoculated into semi-solid culture medium of example 1 containing 60% oxgall, cultured for 3d, pH value of oxgall is regulated by citric acid and sodium hydroxide solution, bacterial strain culture solution 5.0 mL is respectively inoculated into 95.0 mL oxgall with pH value of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, shake culture (120 rpm) is carried out at 37 ℃, after 7 d is cultured, light absorption values of 560 nm are respectively detected, and bilirubin and cholate content in oxgall is detected according to 1.3.1 method.

The results are shown in FIG. 4, where strain NDZNM-01 ferments oxgall at pH7.5 with a bilirubin content of 16.75% (mg.%), with a cholic acid content of 5.16 (g.%) at pH6.5, and unfermented oxgall with a bilirubin content of only 7.14 (mg.%) and a cholic acid content of 2.42 (g.%). Thus, the content of cholic acid and bilirubin in the oxgall fermented by the strain NDZNM-01 is 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 an inconsistent effect on the conversion rate of cholic acid and bilirubin of the strain NDZNM-01, the conversion rate of bilirubin is high under the slightly alkaline condition, and the conversion rate of cholic acid is high under the slightly acidic condition. The cholic acid content is far beyond the standards of Chinese pharmacopoeia, so the pH value of the fermented oxgall is the principle of conversion of bilirubin. The bezoar transformation efficiency of the strain NDZNM-01 is better that the pH value of fermented bile is 7.2, and too high and too low pH values are not beneficial to bezoar transformation.

3. Influence of oxgall fermentation temperature on the conversion capability of the bezoar conversion strain NDZNM-01 tolerant to oxgall

The strain NDZNM-01 cultured in the semisolid culture medium of example 1 containing 60% bile was inoculated into 95.0 mL fresh oxgall, and cultured under shaking (120 r/min) at 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively for 16d, and the cell concentration was detected at 560 nm to detect the bilirubin and bile acid content in oxgall.

The result is shown in FIG. 5, and when the strain NDZNM-01 is cultured at 30 ℃, the content of the cholic acid is the highest until 16d, and reaches 6.42%; fermenting to 14d until the cholic acid conversion efficiency enters a slow growth period when culturing at 25 ℃ and 30 ℃; fermenting to 12d until the cholic acid conversion rate enters a slow growth period when the culture is carried out at 35 ℃ and 40 ℃; at a bile fermentation temperature of 10 ℃, 15 ℃ and 20 ℃, the cholic acid content is positively correlated with the fermentation time, and no slow growth period occurs.

From FIG. 6, it is seen that when the strain NDZNM-01 is cultured at a fermentation temperature of 30 ℃ in oxgall, the bilirubin content reaches 2.01%o up to 16d, and the bilirubin conversion rate enters a slow increasing period when the strain is fermented to 14 d; fermenting to 12d bilirubin conversion rate to slow growth period at 35 ℃ and 40 ℃; at a fermentation temperature of 10 ℃, 15 ℃, 20 ℃, 25 ℃, the bilirubin content is positively correlated with the fermentation time, and no slow growth phase occurs.

Determination of the growth curve of bovine bile-tolerant bovine transformed bacteria NDZNM-01 described in example 5

Inoculating single colony of strain NDZNM-01 into 5 ml semisolid culture medium, shake culturing at 37deg.C for 6 hr, inoculating 1ml of bacterial liquid into 100ml liquid culture medium, shake culturing, and measuring cell density (OD) every 1 day ₅₆₀ ) At OD ₅₆₀ Values are on the ordinate and incubation time is on the abscissa to make a growth curve. As shown in FIG. 7, the survival time of the bezoar transformed bacterium NDZNM-01 in the pure oxgall is longer than 9 days, the stagnation period of the bacterial strain NDZNM-01 is within 48 hours, the bacterial strain NDZNM-01 enters a logarithmic growth phase with rapidly-increased bacterial density, the bacterial density is maximum when the bacterial density is cultured for 96 hours, and the bacterial density begins to slowly decrease after the bacterial density is continuously cultured for 4 days.

Claims (9)

1. A strain of bezoar transformation bacteria resistant to ox bile is characterized in that the bezoar transformation bacteria resistant to ox bile is Citrobacter aurantiacaCitrobacter bovis) The sample is preserved in China general microbiological culture Collection center, with the preservation number: CGMCC No. 24893.

2. The bovine biliary tree transformation-resistant bacterium according to claim 1, wherein the characteristic base sequence of 16S rDNA of the bovine tree transformation-resistant bacterium is shown in SEQ ID No. 1.

3. The bovine biliary tree-tolerant transformed bacterium according to claim 1, wherein the amplification primer of the base sequence characteristic of 16S rDNA of the bovine tree-tolerant transformed bacterium is:

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

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

4. The bovine biliary tree-tolerant transformed bacterium according to claim 2, wherein the characteristic base site of the 16S rDNA characteristic base sequence of the bovine tree-tolerant transformed bacterium is: 265-A,275-C,280-G,282-A,537-A,581-A.

5. The bezoar transformation bacteria of claim 1, wherein the bezoar transformation bacteria survive for a period of time greater than 9d in pure bezoar.

6. The method for identifying bovine biliary powder-resistant transformed bacteria according to any one of claims 1 to 4, wherein the amplification primer according to claim 3 is used to control the characteristic base site according to claim 4.

7. The use of the bovine biliary-resistant bovine bezoar transformant according to claim 1 for culturing bezoar in vitro.

8. The use according to claim 7, wherein the culturing conditions for culturing bezoar outside the bezoar transformed bacteria are: the pH of the fermentation liquid is 6.5-7.5, and the fermentation temperature is 20-35 ℃.

9. The use according to claim 7 or 8, wherein the culture medium materials for the expanded culture of the bacterial species for culturing bezoar in vitro of the bezoar transformation bacteria comprise: tryptone, beef extract, sodium chloride; the semisolid culture medium and the solid culture medium are prepared according to the adding amount of the agar powder.