CN-121975712-A - Method for screening high-efficiency cultivated bezoar strains

CN121975712ACN 121975712 ACN121975712 ACN 121975712ACN-121975712-A

Abstract

A method for screening the high-efficient cultured bezoar strain includes such steps as inoculating the low-pathogenicity Escherichia coli to culture medium, comparing, choosing out single bacterial colony with good growth vigor, subculturing, and culturing, and features that the single bacterial colony is recovered in LB solid culture medium, culturing in the first gradient liquid bile culture medium, measuring OD 600 , and if OD 600 is greater than 0.6, inoculating the bacterial strain to the next gradient liquid bile culture medium, and culturing. The invention has the advantages that the invention can obtain the escherichia coli strain with high beta-glucuronidase activity for efficiently cultivating bezoar, and the escherichia coli strain has bile resistance property through in vitro cultivation verification, can obtain bezoar analogues, and has similar physicochemical properties with natural bezoar.

Inventors

LIU ZISHAN
ZHANG ZEXIU
WANG XINGLONG
LUO ZHIYI
XU YULI
LI HUI
WANG RUIQING
Dai Pengxiu
ZHANG XINKE

Assignees

漳州片仔癀药业股份有限公司
西北农林科技大学

Dates

Publication Date: 20260505
Application Date: 20250904

Claims (7)

1. A method for screening high-efficiency cultured bezoar strains is characterized by comprising the following steps: inoculating low-pathogenicity escherichia coli into an LB solid culture medium and a bile LB solid culture medium, culturing for 20 hours at a temperature of 37 ℃, comparing and observing, picking single bacterial colonies with good growth vigor, placing the single bacterial colonies into a liquid LB culture medium for subculture, placing a strain four-area line after subculture into a bile LB solid culture plate with increased bile concentration for culture, comparing and screening strains with consistent bacterial colony size, large bacterial colony and high growth speed, picking single bacterial colonies, placing the single bacterial colonies into an antibiotic-free liquid LB culture medium, shaking the strain overnight at the temperature of 37 ℃, and then preserving the strains; And secondly, taking a primary screening deposited strain to resuscitate in a four-section line of an LB solid culture medium, placing a single colony in 10ml of a first gradient liquid bile culture medium, measuring OD 600 after culturing for 10-16 hours, placing the strain in the next gradient liquid bile culture medium if OD 600 is more than 0.6, and taking three gradient liquid bile culture mediums which are the first gradient liquid bile culture medium, the second gradient liquid bile culture medium and the third gradient liquid bile culture medium respectively, wherein the strain which can grow in the three gradients can be inoculated in fresh oxgall for culturing for 14-18 hours, measuring OD 600 , and considering the bile resistance degree to be 100% if OD 600 is close to 0.6 or more than 0.6.
2. The method for efficiently culturing bovine bacterial strain according to claim 1, wherein the preserving bacterial strain in the first step is prepared by placing bacterial solution of glycerol=1:1 in a 1.5mlEP tube and preserving at-80 ℃ for secondary screening.
3. The method for efficiently culturing the bezoar strain according to claim 1, wherein the LB solid medium in the first step is prepared by weighing 0.5g of yeast extract, 1g of tryptone, 1g of sodium chloride and 1.5g of agar powder, adding 100mL of deionized water, sterilizing at 121 ℃ for 30min under high pressure, and pouring 10mL into a 70mm dish when cooling to 45-60 ℃.
4. The method for efficiently culturing bovine bezoar strain according to claim 1, wherein the bile LB solid culture operation with increasing bile concentration in the first step comprises three gradient bile LB solid culture media, wherein the proportion of the first gradient bile LB solid culture media is 50mL LB liquid culture media+50 mL fresh oxgall+1.5 g agar powder, the proportion of the second gradient bile LB solid culture media is 40mL LB liquid culture media+60 mL fresh oxgall+1.5 g agar powder, the proportion of the third gradient bile LB solid culture media is 30mL LB liquid culture media+70 mL fresh oxgall+1.5 g agar powder, the three gradient bile LB solid culture media are sterilized under high pressure for 30min at the temperature of 121 ℃ after being prepared, and the mixture is poured into a plate for standby after being cooled to 45-60 ℃ and poured into a culture dish with the volume of 10mL to 70 mm.
5. The method of claim 1, wherein the fresh oxgall is filtered by a bacterial filter with a diameter of 0.22 μm.
6. The method for screening high-efficiency cultured bezoar bacteria according to claim 1, wherein: the first gradient liquid bile culture medium comprises yeast extract 0.75g, tryptone 1.5g, sodium chloride 1.5g and fresh oxgall 150mL; The second gradient liquid bile culture medium comprises yeast extract 0.375g, tryptone 0.75g, sodium chloride 0.75g and fresh oxgall 150mL; the third gradient liquid bile culture medium comprises yeast extract 0.1875g, tryptone 0.375g, sodium chloride 0.375g and fresh oxgall 150mL; Preparing a gradient liquid bile culture medium according to the formula, respectively autoclaving at 121 ℃ for 30min, and cooling to room temperature for later use.
7. The method for efficiently cultivating bezoar strain according to claim 1, wherein the step of screening the strain for the highly active beta-glucuronidase comprises the steps of: Step 1, establishing a standard curve of pNP colorimetric method Preparing 1mMpNP mother solution by using 1 XPBS as a solvent, preparing standard working solution with the concentration range of 10-480 mu M by adopting a 10-fold gradient dilution method, preparing a system comprising 100 mu L of standard working solution and 900 mu LPBS, uniformly mixing, taking 200 mu L of system solution, respectively adding the system solution into 20 mu L of 1MNA 2 CO 3 enzyme-labeled holes, measuring the OD 405 of the system to obtain an OD 405 -pNP concentration scatter diagram, linearly regressing and fitting a standard curve, and obtaining an OD 405 -pNP concentration linear regression equation, and obtaining the slope C of the equation; Step 2, determination of bacterial liquid beta-glucuronidase activity Taking the deposited strain in the second step, carrying out three-section line on a Maiconkai culture plate, culturing for 12 hours at the temperature of 37 ℃ and the rotating speed of 180rpm, picking up a monoclonal with good shape in the plate, placing in 10ml of non-antibiotic LB, carrying out shaking culture for 3-6 hours at the temperature of 37 ℃ and the rotating speed of 180rpm until the strain liquid OD 600 =0.6-1, measuring OD 600 and recording, centrifuging for 15 minutes at the rotating speed of 6000rpm, discarding the supernatant to obtain bacterial precipitate, re-suspending the precipitate by 1ml of PBS, centrifuging for 5 minutes at the rotating speed of 6000rpm, repeating the steps for 3 times, concentrating the bacterial liquid to 1ml, carrying out ultrasonic crushing on ice until the liquid is clear and transparent, centrifuging at the rotating speed of 12000rpm to separate cell fragments, and preparing an enzymatic reaction system comprising 100 mu L ultrasonic crushing supernatant +100. Mu.L 10g/LpNPG +800. Mu. LPBS, a set of blank controls: 100 mu LPBS +100 mu L of 10g/LpNPG +800 mu LPBS, and taking out the liquid of the reaction system 20 Mu L+180 mu L1M sodium carbonate, at this time, as 0 point time measuring OD 405 , with the reaction occurrence of visual solution color yellowing, according to different reaction speed selecting 3-4 time points in 3h, repeating the above steps, obtaining time-OD 405 scatter diagram, performing scatter diagram fitting, obtaining time-absorbance curve, calculating slope k of the curve, calculating enzyme activity formula of GUS generated by strain as follows: Where k is the slope of the OD 405 -time scatter plot fit curve for the enzymatic reaction, which can reflect the reaction rate over the time of measurement. C is the slope of an OD 405 -pNP concentration standard curve, V 1 is the total volume of an enzymatic reaction system, V 2 is the volume of enzyme solution used in the enzymatic reaction, V 3 is the dilution multiple of the enzyme solution in absorbance measurement, and OD 600 is the absorbance of the bacterial solution before ultrasonic disruption, and the concentration of the bacterial solution is reflected.

Description

Method for screening high-efficiency cultivated bezoar strains Technical Field The invention belongs to the field of bezoar strain screening, and in particular relates to a method for screening and efficiently cultivating bezoar strains. Background Bezoar is dry gall-stone of cattle, and is used in 650 kinds of 4500 kinds of traditional Chinese medicines in Shennong herbal Jing recorded in Shennong herbal Jing for more than two thousands of years at the earliest, has the effects of clearing heat and detoxicating, tranquilizing and allaying excitement and the like, is used for treating diseases such as high fever, convulsion and the like, and is one of precious medicinal materials of traditional Chinese medicine. The natural incidence rate of bovine cholelithiasis is extremely low, natural bezoar is not easy to obtain, the medicine source is short, the supply source is far behind the actual demand, and the demand of the medicine industry can not be met, so that the research and development of cultivated bezoar and substitutes are urgent demands in the field of traditional Chinese medicine. It has been shown that the formation of natural calculus bovis involves complex regulation of bilirubin metabolism, in which beta-Glucuronidase (GUS) is a key enzyme for the hydrolysis of bilirubin glucuronide, and GUS releases free bilirubin (UCB) by catalyzing the hydrolysis of bound bilirubin (CB), which is bound to calcium ions to form an insoluble bilirubin calcium salt complex, constituting the core component of calculus bovis, and this process is accomplished under specific bile conditions (e.g., pH, ion concentration) within the gallbladder and under the synergistic action of microorganisms. In addition, the expression level of GUS is directly related to the crystal form of bilirubin calcium salt, and the covalent binding proportion of the bilirubin calcium salt in natural bezoar is obviously higher than that of in-vitro cultivated products, which is probably a key factor with better biological activity. At present, the technology of in vitro culturing bezoar has been industrialized, for example, the publication number is CN119842527A, the name is a composite microbial inoculum for producing beta-glucuronidase and the application of the composite microbial inoculum in bezoar production are industrialized, but the core technology still depends on chemical synthesis of bilirubin calcium salt compound, and the problems of low biological activity of the product, more impurity residues, obvious difference between crystal morphology and natural bezoar and the like exist. Disclosure of Invention The invention provides a method for screening and efficiently cultivating bezoar strains, which is used for solving the defects in the prior art. The invention is realized by the following technical scheme: a method for screening high-efficiency cultured bezoar strains comprises the following steps: inoculating low-pathogenicity escherichia coli into an LB solid culture medium and a bile LB solid culture medium, culturing for 20 hours at a temperature of 37 ℃, comparing and observing, picking single bacterial colonies with good growth vigor, placing the single bacterial colonies into a liquid LB culture medium for subculture, placing a strain four-area line after subculture into a bile LB solid culture plate with increased bile concentration for culture, comparing and screening strains with consistent bacterial colony size, large bacterial colony and high growth speed, picking single bacterial colonies, placing the single bacterial colonies into an antibiotic-free liquid LB culture medium, shaking the strain overnight at the temperature of 37 ℃, and then preserving the strains; Recovering the strain in LB solid culture medium, placing single colony in 10ml first gradient liquid bile culture medium, culturing for 10-16 hr, measuring OD 600, if The strain can be placed in the next gradient liquid bile culture medium with OD 600 being more than 0.6, three gradients of liquid bile culture medium are provided, namely a first gradient liquid bile culture medium, a second gradient liquid bile culture medium and a third gradient liquid bile culture medium, the strain which can grow in all three gradients can be inoculated in fresh oxgall for culture for 14-18 hours, OD 600 is measured, and if OD 600 is close to 0.6 or more than 0.6, the bile resistance degree is considered to be 100%. And (3) preserving the strain with the bile resistance degree of 100% for measuring and screening the bacterial liquid beta-glucuronidase activity. The method for efficiently culturing the bezoar strain is characterized in that the preserved strain in the first step and the second step is prepared by placing bacterial liquid, glycerol=1:1, in a 1.5mlEP tube, and preserving at-80 ℃ for secondary screening. The preparation process of the LB solid culture medium in the first step includes weighing yeast extract 0.5g, tryptone 1g, sodium chloride 1g and agar pow