CN112481188A - Preparation method of mixed bacterium agent for efficiently producing beta-glucuronidase - Google Patents
Preparation method of mixed bacterium agent for efficiently producing beta-glucuronidase Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- G—PHYSICS
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
The invention discloses a preparation method of a mixed microbial inoculum for efficiently producing beta-glucuronidase, which comprises the following steps of collecting a fresh animal gallbladder sample; step two, separating escherichia coli; step three, identifying escherichia coli; step four, survival screening of the escherichia coli bile; step five, directional induction culture of multiple concentration gradients of cholic acid; step six, bionic dynamic simulation cultivation of the cow gall bladder; seventhly, measuring the activity of the escherichia coli beta-G; step eight, mixed culture of bacterial liquid; step nine, animal toxicity test detection; the invention obtains the Escherichia coli strain with long survival time and high reproduction activity in the bile by domestication and orientation of the bile and cholic acid; obtaining an escherichia coli strain adapting to the change of a gallbladder environment through bionic dynamic simulation of the bovine gallbladder; obtaining an escherichia coli strain with high beta-G activity by PCR technology, bacterial liquid mixing and enzyme activity determination; and through toxin and toxicity combined screening, the escherichia coli strain with the minimum influence on the growth of animals is obtained.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method of a mixed microbial inoculum for efficiently producing beta-glucuronidase.
Background
The bezoar has high medicinal value, large medical demand and very short medicine source, and the bezoar begins to be cultivated in a large area in the gall bladder of the living cow in the later period of the 20 th century and 80 th century in China, but the problems of low bezoar yield, weak medicinal effect compared with natural bezoar and the like still exist in China;
the artificially cultivated bezoar is mainly prepared by injecting sufficient Escherichia coli with beta-G activity into ox gall bladder, wherein the beta-G can hydrolyze combined Bilirubin (BDG) in ox bile to convert into free bilirubin (UCB), and the free bilirubin is one of main components forming bezoar and is a main judgment standard of medicinal value;
most studies only separate a single strain to cause yellowing, but the problems of short survival time in gall bladder, poor adaptability to gall bladder environment, low reproductive activity, low beta-glucuronidase activity and the like generally exist; aiming at the defects, a preparation method for efficiently producing the beta-glucuronidase mixed bacterial agent is necessary.
Disclosure of Invention
The invention aims to provide a preparation method of a mixed microbial inoculum for efficiently producing beta-glucuronidase, which aims to solve the problems in the background technology.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a mixed microbial inoculum capable of efficiently producing beta-glucuronidase comprises the steps of collecting a fresh animal gall bladder sample; step two, separating escherichia coli; step three, identifying escherichia coli; step four, survival screening of the escherichia coli bile; step five, directional induction culture of multiple concentration gradients of cholic acid; step six, bionic dynamic simulation cultivation of the cow gall bladder; seventhly, measuring the activity of the escherichia coli beta-G; step eight, mixed culture of bacterial liquid; step nine, animal toxicity test detection;
in the first step, the gall bladder of different animals such as chicken, duck, pig, cattle and the like is picked up in an artificial and sterile manner;
wherein in the second step, the separation of Escherichia coli comprises the following steps:
1) collecting bile in gallbladder with disposable syringe under aseptic condition, inoculating 0.1mL into eosin methylene blue agar culture medium EMB, simultaneously inoculating 1mL bile into 10mL, and inoculating into LB liquid culture medium for enrichment;
2) culturing in a 37 deg.C constant temperature incubator and a 37 deg.C constant temperature shaking table respectively for 16-24h, and if EMB bacteria free colony grows and LB is clear and transparent, determining that the bacteria is sterile;
3) when the EMB has no deep purple red colonies with metallic luster but the LB is turbid, 0.1mL of the LB culture medium can be taken again, and the LB culture medium is transferred to the EMB to be cultured for 16-24h at 37 ℃; if round, moist, smooth, neat-edged and medium-sized deep purple red colonies with metallic luster appear on the EMB, the numbered single suspicious colonies are picked by using a sterile inoculating loop, and the single suspicious colonies are repeatedly streaked and separated on an EMB culture medium to obtain purified colonies;
wherein in the third step, the identification of Escherichia coli comprises the following steps:
1) microscopic examination, namely aseptically picking a single purified suspicious colony, staining the single suspicious colony by using a gram stain solution, and observing the single suspicious colony under an optical microscope, wherein the colony subjected to microscopic examination is primarily judged as escherichia coli if the colony is in a red short rod shape;
2) performing PCR amplification, namely performing PCR amplification on the strains judged to be positive in the previous step, and detecting the PCR products by using 1% agarose gel electrophoresis;
wherein in the fourth step, the escherichia coli bile survival screening comprises the following steps:
1) sucking 8 mu L of the strains judged to be positive in the step three 2) by using a micropipette, and inoculating the strains into 1mL of fresh ox bile after aseptic treatment;
2) culturing at 37 deg.C for 30 days, spreading 8 μ L of the extract on eosin methylene blue agar plate, culturing at 37 deg.C, and observing whether there is colony growth, if so, determining that the strain can survive in ox bile;
in the fifth step, the directional induction culture of the cholic acid multi-concentration gradient comprises the following steps:
1) preparing 1mL of sterile broth with the cholic acid content of 12%, 15%, 18%, 21%, 24%, 27% and 30% by using No. 5 bile salt and the cholic acid content of 70% -85% and the fresh ox bile after sterile treatment;
2) selecting the bacterial colony judged to be positive in the step four 2) in 1mL of 12% cholic acid-broth culture medium by using an aseptic inoculating loop, and inoculating a loop of bacterial liquid on an eosin methylene blue agar culture medium;
3) if a green colony with metallic luster is observed, obtaining a first generation strain, then placing the first generation strain in a cholic acid-broth culture medium with the cholic acid content of 15%, repeating the steps, and acclimatizing the strain to survive and propagate in a sterile broth with the cholic acid content of more than 21%;
in the sixth step, the bionic dynamic simulation cultivation of the bovine gallbladder comprises the following steps:
1) simulating the change of the temperature of the cow gallbladder, wherein the set temperature change range is as follows: selecting the bacterial strain which is determined to be positive in the previous step by using an aseptic inoculating loop at 37 ℃, 38 ℃, 39 ℃, 40 ℃ and 41 ℃, inoculating the bacterial strain into 1mL of sterile fresh oxgall, culturing in a constant-temperature incubator at 37 ℃, adjusting the temperature up to 1 ℃ every 8h until the temperature reaches 41 ℃, taking a loop of bacterial liquid before each time of up-regulation, streaking the loop of bacterial liquid on an eosin methylene blue agar culture medium, culturing at the constant temperature of 37 ℃ for 24h, and detecting the survival activity of the bacterial strain;
2) simulating the change of bilirubin concentration of cow gallbladder TBr: preparing bilirubin solution with concentration range of 0.1755mg/mL, 0.2048mg/mL, 0.2340mg/mL, 0.2633mg/mL and 0.2925mg/mL, selecting the strains judged to be positive in the previous step by using an aseptic inoculating loop, inoculating the strains into the bilirubin-oxgall solution, and up-regulating the bilirubin concentration in the oxgall every 8 hours until the bilirubin concentration is regulated to 0.2925 mg/mL. Taking a ring of bacteria liquid before each time of up-regulation, streaking the ring of bacteria liquid on an eosin methylene blue agar culture medium, culturing at the constant temperature of 37 ℃ for 24 hours, and detecting the survival activity;
wherein in the seventh step, the activity determination of the escherichia coli beta-G comprises the following steps:
1) drawing a p-nitrophenol standard curve;
2) sample treatment: picking the best single colony grown in the last step by using a sterile inoculating loop, shaking and culturing the single colony in a 20ml LTSB culture medium for 6 hours, taking out the single colony, and adjusting the concentration of the bacterial liquid to be 1 multiplied by 108Centrifuging at 5000r/min and 4 deg.C for 10min, discarding supernatant, washing thallus with PBS for 2-3 times, centrifuging, discarding supernatant, adding 3-4mL PBS into thallus, crushing with ultrasonic cell crusher in ice bath for 30-45min at 400HZ frequency, centrifuging at 5000r/min for 10min, and collecting supernatant;
3) and (3) enzyme activity determination: sucking 80 mul of supernatant into 96 holes, adding an equivalent amount of PNPG substrate solution as a sample solution, and keeping the temperature at 36 ℃ for 1 h; sucking 80 mul, putting PBS in 96 holes, adding the same amount of PNPG substrate solution as a blank control, and keeping the temperature at 36 ℃ for 1 h; after the heat preservation of each sample is finished, taking out each hole, adding 36 mu L of NaOH solution with the concentration of 0.1mol/L to each hole to terminate the reaction, using a full-wavelength microplate reader to correct the zero point by using a blank under the wavelength of 405nm, measuring the absorbance of the sample solution, substituting the absorbance value into a standard curve equation to obtain the concentration of the p-nitrophenol, and then calculating the activity unit of the enzyme according to the following formula;
in the eighth step, the mixed culture of the bacterial liquid comprises the following steps:
1) screening out Escherichia coli combinations which are matched and mixed randomly in pairs at the concentration of 1:1 and have synergistic effect on enzyme activity, and performing concentration ratios of 1:1, 1: 2, 1: 4, 1: 8, 8: 1, 4: 1 and 2: 1 in the combinations respectively;
2) after mixed culture, measuring the enzyme activity of the mixed bacteria liquid, comparing the enzyme activity with the mixed bacteria liquid with the same combination in the concentration ratio of 1:1, and screening out the optimal pairing and concentration ratio of the mixed bacteria liquid capable of highly yielding beta-G;
wherein in the ninth step, the animal toxicity test comprises the following steps:
1) numbering the combinations of the screened mixed bacterial liquids capable of highly producing beta-G enzyme, taking 3-week-old adult mice, each group containing 6 mice, setting two groups of the mixed bacterial liquids which are respectively injected into the abdominal cavity with pathogenic escherichia coli or normal saline, and the other groups of the mixed bacterial liquids which are respectively injected into the abdominal cavity with the screened mixed bacterial liquids, each 1 × 109CFU/mL, test feeding for three weeks;
2) and (4) performing experimental feeding for three weeks, and observing whether the screened strains are non-pathogenic escherichia coli, wherein the result shows that the strains which are non-pathogenic escherichia coli are stored for later use.
According to the technical scheme, in the step one, in the collection of the fresh animal gallbladder sample, the used animal gallbladder is required to be fresh and complete, the cystic duct is ligated, and the content is not in contact with the outside.
According to the technical scheme, the shaking table in the second step 2) is 200 r/min.
According to the technical scheme, a primer is designed according to the uid and A gene which are published on Genebank and code beta-G in the step three 2).
According to the technical scheme, the step five 2) is carried out with constant temperature culture at 37 ℃ for 24 h.
According to the above technical means, in the step seven 3), β -G enzyme unit/L (U/L) is p-nitrophenol release amount (μmol)/incubation time (h)/test liquid amount (L).
Compared with the prior art, the invention has the following beneficial effects: the preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase is simple to operate, and the escherichia coli strain with long survival time and high propagation activity in bile is obtained through domestication and orientation of the bile and cholic acid; obtaining an escherichia coli strain adapting to the change of a gallbladder environment through bionic dynamic simulation of the bovine gallbladder; obtaining an escherichia coli strain with high beta-G activity by PCR technology, bacterial liquid mixing and enzyme activity determination; and through toxin and toxicity combined screening, the escherichia coli strain with the minimum influence on the growth of animals is obtained.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a morphological diagram of the strain of the invention under a light microscope;
FIG. 3 is a graph of the growth of a strain of the invention;
FIG. 4 is a kinetic diagram of the beta-G activity of the strain of the invention;
FIG. 5 is a diagram showing the results of PCR identification of the gallbladder strain of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-5, the present invention provides a technical solution: a preparation method of a mixed microbial inoculum capable of efficiently producing beta-glucuronidase comprises the steps of collecting a fresh animal gall bladder sample; step two, separating escherichia coli; step three, identifying escherichia coli; step four, survival screening of the escherichia coli bile; step five, directional induction culture of multiple concentration gradients of cholic acid; step six, bionic dynamic simulation cultivation of the cow gall bladder; seventhly, measuring the activity of the escherichia coli beta-G; step eight, mixed culture of bacterial liquid; step nine, animal toxicity test detection; the method is characterized in that:
in the first step, the gall bladder of different animals such as chickens, ducks, pigs, cows and the like is picked up manually and aseptically, and in the process of collecting a fresh animal gall bladder sample, the gall bladder of the used animal must be kept fresh and complete, the gall bladder duct is ligated, and the content is not contacted with the outside;
wherein in the second step, the separation of Escherichia coli comprises the following steps:
1) collecting bile in gallbladder with disposable syringe under aseptic condition, inoculating 0.1mL into eosin methylene blue agar culture medium EMB, simultaneously inoculating 1mL bile into 10mL, and inoculating into LB liquid culture medium for enrichment;
2) respectively culturing in a 37 deg.C constant temperature incubator and a 37 deg.C constant temperature shaking table at 200r/min for 16-24 hr, and if EMB bacteria colony grows and LB is clear and transparent, determining as bacteria free;
3) when the EMB has no deep purple red colonies with metallic luster but the LB is turbid, 0.1mL of the LB culture medium can be taken again, and the LB culture medium is transferred to the EMB to be cultured for 16-24h at 37 ℃; if round, moist, smooth, neat-edged and medium-sized deep purple red colonies with metallic luster appear on the EMB, the numbered single suspicious colonies are picked by using a sterile inoculating loop, and the single suspicious colonies are repeatedly streaked and separated on an EMB culture medium to obtain purified colonies;
wherein in the third step, the identification of Escherichia coli comprises the following steps:
1) microscopic examination, namely aseptically picking a single purified suspicious colony, staining the single suspicious colony by using a gram stain solution, and observing the single suspicious colony under an optical microscope, wherein the colony subjected to microscopic examination is primarily judged as escherichia coli if the colony is in a red short rod shape;
2) performing PCR amplification, namely performing PCR amplification on the strain judged to be positive in the previous step, designing a primer according to the uid and A genes which are disclosed on Genebank and encode beta-G, and detecting a PCR product by using 1% agarose gel electrophoresis;
wherein in the fourth step, the escherichia coli bile survival screening comprises the following steps:
1) sucking 8 mu L of the strains judged to be positive in the step three 2) by using a micropipette, and inoculating the strains into 1mL of fresh ox bile after aseptic treatment;
2) culturing at 37 deg.C for 30 days, spreading 8 μ L of the extract on eosin methylene blue agar plate, culturing at 37 deg.C, and observing whether there is colony growth, if so, determining that the strain can survive in ox bile;
in the fifth step, the directional induction culture of the cholic acid multi-concentration gradient comprises the following steps:
1) preparing 1mL of sterile broth with the cholic acid content of 12%, 15%, 18%, 21%, 24%, 27% and 30% by using No. 5 bile salt and the cholic acid content of 70% -85% and the fresh ox bile after sterile treatment;
2) selecting the bacterial colony judged to be positive in the step four 2) in 1mL of 12% cholic acid-broth culture medium by using an aseptic inoculating loop, culturing at the constant temperature of 37 ℃ for 24 hours, and inoculating a loop of bacterial liquid on an eosin methylene blue agar culture medium;
3) if a green colony with metallic luster is observed, obtaining a first generation strain, then placing the first generation strain in a cholic acid-broth culture medium with the cholic acid content of 15%, repeating the steps, and acclimatizing the strain to survive and propagate in a sterile broth with the cholic acid content of more than 21%;
in the sixth step, the bionic dynamic simulation cultivation of the bovine gallbladder comprises the following steps:
1) simulating the change of the temperature of the cow gallbladder, wherein the set temperature change range is as follows: selecting the bacterial strain which is determined to be positive in the previous step by using an aseptic inoculating loop at 37 ℃, 38 ℃, 39 ℃, 40 ℃ and 41 ℃, inoculating the bacterial strain into 1mL of sterile fresh oxgall, culturing in a constant-temperature incubator at 37 ℃, adjusting the temperature up to 1 ℃ every 8h until the temperature reaches 41 ℃, taking a loop of bacterial liquid before each time of up-regulation, streaking the loop of bacterial liquid on an eosin methylene blue agar culture medium, culturing at the constant temperature of 37 ℃ for 24h, and detecting the survival activity of the bacterial strain;
2) simulating the change of bilirubin concentration of cow gallbladder TBr: preparing bilirubin solution with concentration range of 0.1755mg/mL, 0.2048mg/mL, 0.2340mg/mL, 0.2633mg/mL and 0.2925mg/mL, selecting the strains judged to be positive in the previous step by using an aseptic inoculating loop, inoculating the strains into the bilirubin-oxgall solution, and up-regulating the bilirubin concentration in the oxgall every 8 hours until the bilirubin concentration is regulated to 0.2925 mg/mL. Taking a ring of bacteria liquid before each time of up-regulation, streaking the ring of bacteria liquid on an eosin methylene blue agar culture medium, culturing at the constant temperature of 37 ℃ for 24 hours, and detecting the survival activity;
wherein in the seventh step, the activity determination of the escherichia coli beta-G comprises the following steps:
1) drawing a p-nitrophenol standard curve;
2) sample treatment: picking the best single colony grown in the last step by using a sterile inoculating loop, shaking and culturing the single colony in a 20ml LTSB culture medium for 6 hours, taking out the single colony, and adjusting the concentration of the bacterial liquid to be 1 multiplied by 108Centrifuging at 5000r/min and 4 deg.C for 10min, discarding supernatant, washing thallus with PBS for 2-3 times, centrifuging, discarding supernatant, adding 3-4mL PBS into thallus, crushing with ultrasonic cell crusher in ice bath for 30-45min at 400HZ frequency, centrifuging at 5000r/min for 10min, and collecting supernatant;
3) and (3) enzyme activity determination: sucking 80 mul of supernatant into 96 holes, adding an equivalent amount of PNPG substrate solution as a sample solution, and keeping the temperature at 36 ℃ for 1 h; sucking 80 mul, putting PBS in 96 holes, adding the same amount of PNPG substrate solution as a blank control, and keeping the temperature at 36 ℃ for 1 h; after the heat preservation of each sample is finished, taking out each hole, adding 36 mu L of NaOH solution with the concentration of 0.1mol/L to terminate the reaction, using a full-wavelength microplate reader to correct the zero point by using a blank at the wavelength of 405nm, determining the absorbance of the sample solution, substituting the absorbance value into a standard curve equation to obtain the concentration of p-nitrophenol, and then calculating the activity unit of the enzyme according to the following formula, wherein beta-G enzyme unit/L (U/L) is p-nitrophenol release amount (mu mol)/heat preservation time (h)/liquid volume to be detected (L);
in the eighth step, the mixed culture of the bacterial liquid comprises the following steps:
1) screening out Escherichia coli combinations which are matched and mixed randomly in pairs at the concentration of 1:1 and have synergistic effect on enzyme activity, and performing concentration ratios of 1:1, 1: 2, 1: 4, 1: 8, 8: 1, 4: 1 and 2: 1 in the combinations respectively;
2) after mixed culture, measuring the enzyme activity of the mixed bacteria liquid, comparing the enzyme activity with the mixed bacteria liquid with the same combination in the concentration ratio of 1:1, and screening out the optimal pairing and concentration ratio of the mixed bacteria liquid capable of highly yielding beta-G;
wherein in the ninth step, the animal toxicity test comprises the following steps:
1) numbering the combinations of the screened mixed bacteria liquid capable of highly producing beta-G enzyme, taking 6 adult mice of 3 weeks old, setting two groups for respectively injecting pathogenic escherichia coli or physiological bacteria into abdominal cavitySaline, mixed bacterial liquid selected from the rest groups by intraperitoneal injection, each 1 × 109CFU/mL, test feeding for three weeks;
2) and (4) performing experimental feeding for three weeks, and observing whether the screened strains are non-pathogenic escherichia coli, wherein the result shows that the strains which are non-pathogenic escherichia coli are stored for later use.
The combination of E.coli strains which were mixed at a 1:1 concentration in random pairwise combinations and which were synergistic for the enzymatic activity is given in the following table:
the mixed bacterial liquid is mixed and cultured according to the concentration ratio of 1:1, 1: 2, 1: 4, 1: 8, 8: 1, 4: 1 and 2: 1, and the enzyme activity is compared with the mixed bacterial liquid with the concentration ratio of 1:1 of the same combination, and the results are shown in the table.
Based on the above, the preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase has the advantages that the operation is simple, and the escherichia coli strains with long survival time and high propagation activity in bile are directionally obtained through the domestication of the bile and the cholic acid; obtaining an escherichia coli strain adapting to the change of a gallbladder environment through bionic dynamic simulation of the bovine gallbladder; obtaining an escherichia coli strain with high beta-G activity by PCR technology, bacterial liquid mixing and enzyme activity determination; and through toxin and toxicity combined screening, the escherichia coli strain with the minimum influence on the growth of animals is obtained.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A preparation method of a mixed microbial inoculum capable of efficiently producing beta-glucuronidase comprises the steps of collecting a fresh animal gall bladder sample; step two, separating escherichia coli; step three, identifying escherichia coli; step four, survival screening of the escherichia coli bile; step five, directional induction culture of multiple concentration gradients of cholic acid; step six, bionic dynamic simulation cultivation of the cow gall bladder; seventhly, measuring the activity of the escherichia coli beta-G; step eight, mixed culture of bacterial liquid; step nine, animal toxicity test detection; the method is characterized in that:
in the first step, the gall bladder of different animals such as chicken, duck, pig, cattle and the like is picked up in an artificial and sterile manner;
wherein in the second step, the separation of Escherichia coli comprises the following steps:
1) collecting bile in gallbladder with disposable syringe under aseptic condition, inoculating 0.1mL into eosin methylene blue agar culture medium EMB, simultaneously inoculating 1mL bile into 10mL, and inoculating into LB liquid culture medium for enrichment;
2) culturing in a 37 deg.C constant temperature incubator and a 37 deg.C constant temperature shaking table respectively for 16-24h, and if EMB bacteria free colony grows and LB is clear and transparent, determining that the bacteria is sterile;
3) when the EMB has no deep purple red colonies with metallic luster but the LB is turbid, 0.1mL of the LB culture medium can be taken again, and the LB culture medium is transferred to the EMB to be cultured for 16-24h at 37 ℃; if round, moist, smooth, neat-edged and medium-sized deep purple red colonies with metallic luster appear on the EMB, the numbered single suspicious colonies are picked by using a sterile inoculating loop, and the single suspicious colonies are repeatedly streaked and separated on an EMB culture medium to obtain purified colonies;
wherein in the third step, the identification of Escherichia coli comprises the following steps:
1) microscopic examination, namely aseptically picking a single purified suspicious colony, staining the single suspicious colony by using a gram stain solution, and observing the single suspicious colony under an optical microscope, wherein the colony subjected to microscopic examination is primarily judged as escherichia coli if the colony is in a red short rod shape;
2) performing PCR amplification, namely performing PCR amplification on the strains judged to be positive in the previous step, and detecting the PCR products by using 1% agarose gel electrophoresis;
wherein in the fourth step, the escherichia coli bile survival screening comprises the following steps:
1) sucking 8 mu L of the strains judged to be positive in the step three 2) by using a micropipette, and inoculating the strains into 1mL of fresh ox bile after aseptic treatment;
2) culturing at 37 deg.C for 30 days, spreading 8 μ L of the extract on eosin methylene blue agar plate, culturing at 37 deg.C, and observing whether there is colony growth, if so, determining that the strain can survive in ox bile;
in the fifth step, the directional induction culture of the cholic acid multi-concentration gradient comprises the following steps:
1) preparing 1mL of sterile broth with the cholic acid content of 12%, 15%, 18%, 21%, 24%, 27% and 30% by using No. 5 bile salt and the cholic acid content of 70% -85% and the fresh ox bile after sterile treatment;
2) selecting the bacterial colony judged to be positive in the step four 2) in 1mL of 12% cholic acid-broth culture medium by using an aseptic inoculating loop, and inoculating a loop of bacterial liquid on an eosin methylene blue agar culture medium;
3) if a green colony with metallic luster is observed, obtaining a first generation strain, then placing the first generation strain in a cholic acid-broth culture medium with the cholic acid content of 15%, repeating the steps, and acclimatizing the strain to survive and propagate in a sterile broth with the cholic acid content of more than 21%;
in the sixth step, the bionic dynamic simulation cultivation of the bovine gallbladder comprises the following steps:
1) simulating the change of the temperature of the cow gallbladder, wherein the set temperature change range is as follows: selecting the bacterial strain which is determined to be positive in the previous step by using an aseptic inoculating loop at 37 ℃, 38 ℃, 39 ℃, 40 ℃ and 41 ℃, inoculating the bacterial strain into 1mL of sterile fresh oxgall, culturing in a constant-temperature incubator at 37 ℃, adjusting the temperature up to 1 ℃ every 8h until the temperature reaches 41 ℃, taking a loop of bacterial liquid before each time of up-regulation, streaking the loop of bacterial liquid on an eosin methylene blue agar culture medium, culturing at the constant temperature of 37 ℃ for 24h, and detecting the survival activity of the bacterial strain;
2) simulating the change of bilirubin concentration of cow gallbladder TBr: preparing bilirubin solution with concentration range of 0.1755mg/mL, 0.2048mg/mL, 0.2340mg/mL, 0.2633mg/mL and 0.2925mg/mL, selecting the strains judged to be positive in the previous step by using an aseptic inoculating loop, inoculating the strains into the bilirubin-oxgall solution, and up-regulating the bilirubin concentration in the oxgall every 8 hours until the bilirubin concentration is regulated to 0.2925 mg/mL. Taking a ring of bacteria liquid before each time of up-regulation, streaking the ring of bacteria liquid on an eosin methylene blue agar culture medium, culturing at the constant temperature of 37 ℃ for 24 hours, and detecting the survival activity;
wherein in the seventh step, the activity determination of the escherichia coli beta-G comprises the following steps:
1) drawing a p-nitrophenol standard curve;
2) sample treatment: picking the best single colony grown in the last step by using a sterile inoculating loop, shaking and culturing the single colony in a 20ml LTSB culture medium for 6 hours, taking out the single colony, and adjusting the concentration of the bacterial liquid to be 1 multiplied by 108Centrifuging at 5000r/min and 4 deg.C for 10min, discarding supernatant, washing thallus with PBS for 2-3 times, centrifuging, discarding supernatant, adding 3-4mL PBS into thallus, crushing with ultrasonic cell crusher in ice bath for 30-45min at 400HZ frequency, centrifuging at 5000r/min for 10min, and collecting supernatant;
3) and (3) enzyme activity determination: sucking 80 mul of supernatant into 96 holes, adding an equivalent amount of PNPG substrate solution as a sample solution, and keeping the temperature at 36 ℃ for 1 h; sucking 80 mul, putting PBS in 96 holes, adding the same amount of PNPG substrate solution as a blank control, and keeping the temperature at 36 ℃ for 1 h; after the heat preservation of each sample is finished, taking out each hole, adding 36 mu L of NaOH solution with the concentration of 0.1mol/L to each hole to terminate the reaction, using a full-wavelength microplate reader to correct the zero point by using a blank under the wavelength of 405nm, measuring the absorbance of the sample solution, substituting the absorbance value into a standard curve equation to obtain the concentration of the p-nitrophenol, and then calculating the activity unit of the enzyme according to the following formula; in the eighth step, the mixed culture of the bacterial liquid comprises the following steps:
1) screening out Escherichia coli combinations which are matched and mixed randomly in pairs at the concentration of 1:1 and have synergistic effect on enzyme activity, and performing concentration ratios of 1:1, 1: 2, 1: 4, 1: 8, 8: 1, 4: 1 and 2: 1 in the combinations respectively;
2) after mixed culture, measuring the enzyme activity of the mixed bacteria liquid, comparing the enzyme activity with the mixed bacteria liquid with the same combination in the concentration ratio of 1:1, and screening out the optimal pairing and concentration ratio of the mixed bacteria liquid capable of highly yielding beta-G;
wherein in the ninth step, the animal toxicity test comprises the following steps:
1) numbering the combinations of the screened mixed bacterial liquids capable of highly producing beta-G enzyme, taking 3-week-old adult mice, each group containing 6 mice, setting two groups of the mixed bacterial liquids which are respectively injected into the abdominal cavity with pathogenic escherichia coli or normal saline, and the other groups of the mixed bacterial liquids which are respectively injected into the abdominal cavity with the screened mixed bacterial liquids, each 1 × 109CFU/mL, test feeding for three weeks;
2) and (4) performing experimental feeding for three weeks, and observing whether the screened strains are non-pathogenic escherichia coli, wherein the result shows that the strains which are non-pathogenic escherichia coli are stored for later use.
2. The preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase according to claim 1, which is characterized by comprising the following steps: in the step one, during the collection of a fresh animal gallbladder sample, the used animal gallbladder must be kept fresh and complete, the cystic duct is ligated and the content is not in contact with the outside.
3. The preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase according to claim 1, which is characterized by comprising the following steps: the shaking table in the second step 2) is 200 r/min.
4. The preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase according to claim 1, which is characterized by comprising the following steps: in the step three 2), a primer is required to be designed according to the uid and A gene which are published on Genebank and code beta-G.
5. The preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase according to claim 1, which is characterized by comprising the following steps: the step five 2) is cultured for 24 hours at the constant temperature of 37 ℃.
6. The preparation method of the mixed microbial inoculum for efficiently producing the beta-glucuronidase according to claim 1, which is characterized by comprising the following steps: and (c) in the step seven 3), wherein the beta-G enzyme unit/L (U/L) is p-nitrophenol release amount (mu mol)/incubation time (h)/liquid volume to be detected (L).
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