CN115261376A - Efficient screening and mutagenesis optimization method for broad-spectrum antibacterial lactobacillus reuteri - Google Patents
Efficient screening and mutagenesis optimization method for broad-spectrum antibacterial lactobacillus reuteri Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to a high-efficiency screening and mutation optimization method of broad-spectrum antibacterial lactobacillus reuteri, which comprises the steps of preparing a sample to be tested, carrying out amplification culture by using an improved MRS culture medium, carrying out plate scribing and colony PCR amplification after screening a positive sample by using bacteria liquid PCR amplification, screening a positive lactobacillus reuteri colony, inoculating the positive colony to fermentation liquor in the improved MRS culture medium, and detecting the antibacterial effect of the fermentation liquor on an indicating strain by using escherichia coli and staphylococcus aureus as the indicating strain; the lactobacillus reuteri with the bacteriostatic effect on the indicating strain is selected, mutagenized and optimized, and inoculated with fermentation liquor, escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger and pseudomonas aeruginosa are used as the indicating strain, the bacteriostatic effect of the fermentation liquor on the indicating strain is detected, and the lactobacillus reuteri with the inhibitory effect on the indicating strain is screened, so that the broad-spectrum bacteriostatic lactobacillus reuteri is obtained.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a high-efficiency screening and mutagenesis optimization method of broad-spectrum antibacterial lactobacillus reuteri.
Background
Lactobacillus reuteri is present in the gastrointestinal tract of humans, pigs, poultry and other animals and is an indigenous strain of the intestinal tract. The lactobacillus reuteri can better tolerate the animal digestive tract environment, can be planted in the human and animal digestive tracts, and has various effects of regulating intestinal flora, preventing and treating diarrhea, preventing decayed teeth, treating various intestinal discomfort, improving immunity and the like.
Extracellular metabolites of lactobacillus reuteri mainly include lactic acid, acetic acid, ethanol and the like, and can metabolize glycerol to produce a non-protein broad-spectrum antibacterial substance called "reuterin". Roy's bacteriocin is a mixture of monomers, hydrates and cyclic dimers of 3-hydroxypropanal, and exists in dynamic equilibrium in aqueous solution. The Roy's bacteriocin can widely inhibit the growth of gram-positive bacteria, gram-negative bacteria, yeast, mold and pathogenic protozoa, etc. Can be used as bacteriostatic agent, anti-infection therapeutic agent, biological tissue fixing agent, etc. in food and medical industry.
The lactobacillus reuteri has great value in the aspects of probiotic property and bacteriostasis, most of the existing methods for separating the lactobacillus reuteri are obtained by randomly selecting a large number of samples, and the method has long test period and complicated process.
Disclosure of Invention
The invention aims to provide a method for efficiently screening and mutagenizing broad-spectrum antibacterial lactobacillus reuteri, and aims to solve the problem of long test period of the conventional method for separating lactobacillus reuteri.
In order to realize the aim, the invention provides a high-efficiency screening and mutagenesis optimization method of broad-spectrum antibacterial lactobacillus reuteri, which comprises the following steps:
preparing a plurality of samples to be tested from saliva and excrement of healthy people without gastrointestinal medical history;
amplifying each sample to be detected by improving an MRS culture medium, and amplifying the specificity of the sample to be detected by using a bacterial liquid PCR to screen out a positive sample containing lactobacillus reuteri;
carrying out plate streaking on the positive sample, and carrying out colony PCR amplification by using a specific primer to screen out a positive lactobacillus reuteri colony;
inoculating the positive bacterial colony into an improved MRS culture medium for culture, taking escherichia coli and staphylococcus aureus as indicator strains, and detecting the bacteriostatic effect of each fermentation liquid on the indicator strains by adopting an oxford cup bacteriostatic ring method;
carrying out mutagenesis optimization on the lactobacillus reuteri with the bacteriostatic effect of the indicator strain, inoculating a mutagenized bacterial colony to the fermentation liquor, taking escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger and pseudomonas aeruginosa as the indicator strain, adopting an oxford cup bacteriostasis ring method to detect each fermentation liquor, wherein the fermentation liquor is right for the bacteriostatic effect of the indicator strain, screening each lactobacillus reuteri with the inhibitory effect of the indicator strain, and obtaining broad-spectrum bacteriostatic lactobacillus reuteri.
The specific mode for preparing a plurality of samples to be detected from saliva and excrement of healthy people without gastrointestinal medical history is as follows:
inoculating saliva and feces samples of healthy people without gastrointestinal disease history into an improved MRS culture medium, standing at 37 ℃ and anaerobically culturing for 20-40h;
centrifuging 4ml of each culture solution at 5500rpm for 10min, washing the centrifuged thallus with sterile physiological saline with the pH value of 6.8, centrifuging again at 6000rpm for 8min, and repeating twice to obtain sterile pure water suspended thallus;
400uL of the sterile pure water was suspended in the cells, centrifuged in a boiling water bath for 10min at 10000rpm for 15min, and the supernatant was collected.
The modified MRS culture medium has the pH of 7.2 +/-0.2, and comprises 15.0g/L of peptone, 15.0g/L of beef extract, 10.0g/L of yeast powder, 30.0g/L of glucose, 15.0g/L of sucrose, 1.0g/L of magnesium sulfate, 5.0g/L of sodium acetate, 5.0g/L of triammonium citrate, 5.0g/L of dipotassium hydrogen phosphate, 0.1g/L of manganese sulfate and 801.0g/L of Tween.
Wherein the reaction system of the PCR amplification is as follows: 10 mu L of 2 xTaq PCR mix, 9 mu L of DDH2O, 0.4 mu L of upstream primer F, 0.4 mu L of downstream primer R and 0.2 mu L of sample template, evenly mixing in a vortex, then flashing off for 5S, placing on a PCR nucleic acid amplification instrument for reaction, wherein the reaction conditions of PCR amplification are pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10S, annealing at 55 ℃ for 30S, extension at 72 ℃ for 90S, 28 cycles of the whole system, and extension at 72 ℃ for 5min.
Wherein the mutagenesis optimization is to dilute the thallus to 10 by using sterile physiological salt5And (3) coating 200 mu L of CFU/ml on an improved MRS agar medium plate, performing ultraviolet mutagenesis for 5-20min under an ultraviolet germicidal lamp, performing static culture for 20-40h at 37 ℃ in the dark, and selecting a plate with no more than 200 colonies for screening.
Wherein, the specific primer sequence used by the PCR amplification of the bacterial liquid and the colony PCR amplification is SEQ ID NO. 1-2.
The invention relates to a high-efficiency screening and mutagenesis optimization method of broad-spectrum antibacterial lactobacillus reuteri, which is used for preparing a plurality of samples to be detected; amplifying each sample to be detected by improving an MRS culture medium, and screening out a positive sample containing lactobacillus reuteri by using the PCR amplification specificity of a bacterial liquid; carrying out plate streaking on the positive sample, and carrying out colony PCR amplification by using a specific primer to screen out a positive lactobacillus reuteri colony; inoculating the screened colonies into an improved MRS culture medium for culture, taking escherichia coli and staphylococcus aureus as indicator strains, and detecting the bacteriostatic effect of each fermentation liquor on the indicator strains by adopting an oxford cup bacteriostatic ring method; the lactobacillus reuteri with the bacteriostatic effect of the indicator strain is subjected to mutagenesis optimization, a mutagenized bacterial colony is inoculated to the fermentation liquor, escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger and pseudomonas aeruginosa are used as the indicator strain, the bacteriostatic effect of each fermentation liquor on the indicator strain is detected by adopting an oxford cup bacteriostatic circle method, the lactobacillus reuteri with the inhibitory effect on each indicator strain is screened, and the broad-spectrum bacteriostatic lactobacillus reuteri wndPL-14 is obtained.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the results of gel imaging of the specific PCR amplification product of the present invention.
FIG. 2 shows the results of experiments on screening Lactobacillus reuteri inhibiting Escherichia coli by the Oxford cup bacteriostatic ring method.
FIG. 3 shows the results of the experiment of screening Lactobacillus reuteri inhibiting Staphylococcus aureus by the Oxford cup bacteriostasis ring method.
FIG. 4 shows the results of the experiments on the inhibition of Escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger and Pseudomonas aeruginosa by Lactobacillus reuteri wndPL-14 of the present invention.
FIG. 5 is a flow chart of the efficient screening and mutagenesis optimization method for broad-spectrum antibacterial Lactobacillus reuteri provided by the present invention.
Detailed Description
Referring to fig. 1 to 5, the present invention provides a method for efficient screening and mutagenesis optimization of broad-spectrum antibacterial lactobacillus reuteri, comprising the following steps:
s1, preparing a plurality of samples to be detected;
specifically, S11, all saliva and excrement samples from healthy people without gastrointestinal disease history are inoculated in an improved MRS culture medium and subjected to standing anaerobic culture at 37 ℃ for 20-40h;
s12, respectively taking 4ml of each culture solution, centrifuging at 5500rpm for 10min, washing the centrifuged thallus with sterile physiological saline with the pH value of 6.8, centrifuging again after washing at 6000rpm for 8min, and repeating twice to obtain sterile pure water suspended thallus;
s13, 400uL of the sterile pure water is used for suspending thalli, a boiling water bath is used for 10min,10000rpm is used for centrifuging for 15min, and supernatant is collected.
S2, amplifying each sample to be tested by improving an MRS culture medium, and amplifying the specificity of the sample to be tested by using a bacterial liquid PCR to screen out a positive sample containing the lactobacillus reuteri;
specifically, the sample to be tested is prepared, each sample to be tested is subjected to amplification culture through a modified MRS culture medium, and the positive sample containing lactobacillus reuteri is screened out through bacterial liquid PCR amplification specificity, wherein the pH of the modified MRS culture medium is 7.2 +/-0.2, the modified MRS culture medium comprises 15.0g/L of peptone, 15.0g/L of beef extract, 10.0g/L of yeast powder, 30.0g/L of glucose, 15.0g/L of sucrose, 1.0g/L of magnesium sulfate, 5.0g/L of sodium acetate, 5.0g/L of triammonium citrate, 5.0g/L of dipotassium hydrogen phosphate, 0.1g/L of manganese sulfate and 801.0g/L of Tween, 15g/L of agar is added on the basis of a liquid culture medium for solid culture, and the PCR amplification reaction system is as follows: 2 × Taq PCR mix 10 μ L, DDH2O9 mu L, upstream primer F1.4 mu L, downstream primer R1.4 mu L and sample template 0.2 mu L, vortex mixing uniformly, then flash separating for 5s, placing on a PCR nucleic acid amplification instrument for reaction, wherein the reaction conditions of the PCR amplification reaction are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10S, annealing at 55 ℃ for 30S, extension at 72 ℃ for 90S, performing 28 cycles on the whole system, extension at 72 ℃ for 5min, and performing gel gelation on PCR productsAnd (4) performing electrophoresis detection, photographing and detecting under a gel imaging system, and determining the sample as a positive sample if a band of about 1400bp is obvious.
S3, performing plate streaking on the positive sample, and performing colony PCR amplification by using a specific primer to screen out a positive lactobacillus reuteri colony;
s4, inoculating the positive bacterial colonies into an improved MRS culture medium for culture, taking escherichia coli and staphylococcus aureus as indicator strains, and detecting the bacteriostatic effect of each fermentation liquid on the indicator strains by adopting an oxford cup bacteriostatic ring method;
s5, carrying out mutagenesis optimization on the lactobacillus reuteri with the bacteriostatic effect of the indicator strain, inoculating a mutagenized bacterial colony to the fermentation liquor, taking escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger and pseudomonas aeruginosa as the indicator strain, adopting an oxford cup bacteriostasis ring method to detect each fermentation liquor, namely the fermentation liquor, the bacteriostatic effect of the indicator strain, screening each lactobacillus reuteri with the inhibitory effect of the indicator strain, and obtaining broad-spectrum bacteriostatic lactobacillus reuteri.
Specifically, the mutagenesis optimization is to dilute the thallus to 10 by using sterile physiological salt5And coating 200 mu L of the lactobacillus reuteri wndPL-14 on an improved MRS agar medium plate, performing ultraviolet mutagenesis for 5-20min under an ultraviolet germicidal lamp, performing static culture for 20-40h at 37 ℃ in a dark place, selecting the plate with not more than 200 bacterial colonies for screening, wherein the lactobacillus reuteri wndPL-14 can be applied to regulating intestinal flora, preventing and treating diarrhea and other intestinal discomfort, preventing decayed teeth, improving immunity and the like, and the fermentation product can also be applied to preparing bacteriostatic agents, anti-infection therapeutic agents, biological tissue fixing agents and the like, and is used in food and medical industries.
The present invention also provides the following embodiments:
the examples relate to nucleotide sequence information:
the sequence information of SEQ ID NO.1 is the base sequence cagtgtgcct aatacatgca agtcg of the upstream primer F1.
The sequence information of SEQ ID NO.2 is the base sequence cgttacaaac tcccatggtg tgacg of the downstream primer R1.
Example 1 Lactobacillus reuteri specific PCR amplification screening experiment
And (3) subpackaging the sterilized improved MRS liquid culture medium into 24-deep-hole plates according to 4.5mL per hole, inoculating saliva and excrement samples from healthy people without gastrointestinal disease history according to one hole, and standing at 37 ℃ for anaerobic culture for 20-40h. Centrifuging each culture solution 4ml respectively, 5500rpm centrifugating for 10min, washing thallus with sterile physiological saline with pH of 6.8, 6600rpm centrifugating for 8min, repeating for 2 times. 400uL of sterile pure water was suspended in the cells, centrifuged at 10min in boiling water bath and 10000rpm for 15min, and the supernatant was collected. Configuring a PCR amplification reaction system (2 XTaq PCR mix 10. Mu.L, DDH2O9 mu L, upstream primer 0.4 mu L, downstream primer 0.4 mu L and sample template 0.2 mu L, vortex mixing, flash separating 5S, placing on a PCR nucleic acid amplification instrument for reaction (the reaction conditions of the PCR amplification reaction are: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10S, annealing at 55 ℃ for 30S, extension at 72 ℃ for 90S, 28 cycles of the whole system, and extension at 72 ℃ for 5 min). And (3) carrying out gel electrophoresis detection on the PCR product, and photographing and detecting under a gel imaging system, wherein a band of about 1400bp is obvious and is determined as a positive sample. Streaking on an improved MRS agar culture medium plate, standing at 37 ℃ for anaerobic culture for 20-40h, inoculating one colony per hole into a 24-deep-hole plate containing 4.5mL of the improved MRS liquid culture medium per hole, carrying out anaerobic culture at 37 ℃ for 20-40h, repeating the steps of the specific PCR amplification screening experiment, carrying out repeated streaking on the plate and the specific PCR amplification screening experiment on the positive colony obtained by screening for 3 times, and determining the positive colony as a Lactobacillus reuteri strain if the 3 repeated experiments are positive.
Example 2 screening of Lactobacillus reuteri strains inhibiting Escherichia coli and Staphylococcus aureus
And (3) inoculating each positive colony in the example 1 to an improved MRS liquid culture medium, standing at 37 ℃ for anaerobic culture for 20-40h, and preparing fermentation liquor to be tested. The sterilized nutrient broth agar medium is heated to completely melt, poured into a petri dish, 15-18ml per plate, and solidified. Diluting Escherichia coli and Staphylococcus aureus culture solution with sterile physiological salt to 10%8CFU/ml, respectively sucking 100 μ L of diluted bacterial liquid of Escherichia coli and Staphylococcus aureus onto the surface of the plate, coating with a coating rod to uniformly coat the bacterial liquid, and preparing Escherichia coli indicator bacteriumThe plate and a staphylococcus aureus indicator bacterium flat plate are vertically provided with an oxford cup on the surface of the culture medium, and the oxford cup is slightly pressurized to be in contact with the culture medium without a gap. 200. Mu.L of each fermentation broth was added to the cup on a cup-by-cup basis. Culturing at 37 deg.C for 18-24 h. Measuring the diameter of the inhibition zone by a millimeter ruler, and screening the lactobacillus reuteri with the diameter of the inhibition zone larger than 8ml for the lactobacillus reuteri strains of escherichia coli and staphylococcus aureus.
Example 3 screening of Lactobacillus reuteri strains inhibiting Escherichia coli and Staphylococcus aureus
Selecting Lactobacillus reuteri with antibacterial effect on Escherichia coli and Staphylococcus aureus, performing mutagenesis optimization, and diluting thallus with sterile physiological salt to 10%5CFU/ml, coating 200 mul on an improved MRS agar medium plate, ultraviolet mutagenesis for 5-20min under an ultraviolet germicidal lamp, standing and culturing for 20-40h at 37 ℃ in a dark place, selecting a plate with not more than 200 colonies for screening, inoculating the mutagenized colonies in an improved MRS medium to prepare fermentation liquor, taking escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger, pseudomonas aeruginosa and the like as indicator strains, and adopting an Oxford cup bacteriostasis ring method to detect the bacteriostasis effect of each fermentation liquor on the escherichia coli, the staphylococcus aureus, the candida albicans, the aspergillus niger and the pseudomonas aeruginosa, screening the lactobacillus reuteri with the inhibiting effect on the five indicator strains, and finally obtaining the broad-spectrum bacteriostasis lactobacillus reuteri dwndPL-14.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A high-efficiency screening and mutagenesis optimization method of broad-spectrum antibacterial Lactobacillus reuteri is characterized by comprising the following steps:
preparing a plurality of samples to be detected from saliva and excrement of healthy people without gastrointestinal medical history;
amplifying each sample to be detected by improving an MRS culture medium, and amplifying the specificity of the sample to be detected by using a bacterial liquid PCR to screen out a positive sample containing lactobacillus reuteri;
carrying out plate streaking on the positive sample, and carrying out colony PCR amplification by using a specific primer to screen out a positive lactobacillus reuteri colony;
inoculating the positive bacterial colony into an improved MRS culture medium for culture, taking escherichia coli and staphylococcus aureus as indicator strains, and detecting the bacteriostatic effect of each fermentation liquor on the indicator strains by adopting an oxford cup bacteriostatic ring method;
carrying out mutagenesis optimization on the lactobacillus reuteri with the bacteriostatic effect of the indicator strain, inoculating a mutagenized bacterial colony to the fermentation liquor, taking escherichia coli, staphylococcus aureus, candida albicans, aspergillus niger and pseudomonas aeruginosa as the indicator strain, adopting an oxford cup bacteriostasis ring method to detect each fermentation liquor, wherein the fermentation liquor is right for the bacteriostatic effect of the indicator strain, screening each lactobacillus reuteri with the inhibitory effect of the indicator strain, and obtaining broad-spectrum bacteriostatic lactobacillus reuteri.
2. The efficient screening and mutagenesis optimization method for broad-spectrum antibacterial Lactobacillus reuteri according to claim 1,
the specific mode for preparing a plurality of samples to be detected from saliva and excrement of healthy people without gastrointestinal history is as follows:
inoculating saliva and feces samples of healthy people without gastrointestinal disease history into an improved MRS culture medium, standing at 37 ℃ and anaerobically culturing for 20-40h;
centrifuging 4ml of each culture solution at 5500rpm for 10min, washing the centrifuged thallus with sterile physiological saline with the pH value of 6.8, centrifuging again after washing at 6000rpm for 8min, and repeating twice to obtain sterile pure water suspended thallus;
400uL of the sterile pure water was suspended in the cells, centrifuged in boiling water bath for 10min and 10000rpm for 15min, and the supernatant was collected.
3. The efficient screening and mutagenesis optimization method for broad-spectrum antibacterial Lactobacillus reuteri according to claim 1,
the improved MRS culture medium has the pH of 7.2 +/-0.2, and comprises 15.0g/L of peptone, 15.0g/L of beef extract, 10.0g/L of yeast powder, 30.0g/L of glucose, 15.0g/L of cane sugar, 1.0g/L of magnesium sulfate, 5.0g/L of sodium acetate, 5.0g/L of triammonium citrate, 5.0g/L of dipotassium hydrogen phosphate, 0.1g/L of manganese sulfate and 1.0g/L of Tween 80.
4. The efficient screening and mutagenesis optimization method for broad-spectrum antibacterial Lactobacillus reuteri according to claim 1,
the reaction system of the PCR amplification is as follows: 2 XTAQUART PCR mix 10. Mu.L, DDH2And 3, carrying out vortex mixing on 9 mu L of O, 10.4 mu L of an upstream primer F, 10.4 mu L of a downstream primer R and 0.2 mu L of a sample template, then carrying out flash separation for 5S, and placing the mixture on a PCR nucleic acid amplification instrument for reaction, wherein the reaction conditions of PCR amplification are pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10S, annealing at 55 ℃ for 30S and extension at 72 ℃ for 90S, the whole system is subjected to 28 cycles, and the extension is carried out at 72 ℃ for 5min.
5. The efficient screening and mutagenesis optimization method for broad-spectrum antibacterial Lactobacillus reuteri according to claim 1,
the mutagenesis optimization is to dilute the thallus to 10 by using sterile physiological salt5And (3) coating 200 mu L of CFU/ml on an improved MRS agar medium plate, performing ultraviolet mutagenesis for 5-20min under an ultraviolet germicidal lamp, performing static culture for 20-40h at 37 ℃ in the dark, and selecting a plate with no more than 200 colonies for screening.
6. The efficient screening and mutagenesis optimization method of broad-spectrum antibacterial Lactobacillus reuteri according to claim 1,
the specific primer sequence used for PCR amplification of the bacterial liquid and PCR amplification of the bacterial colony is SEQ ID NO. 1-2.
7. The method for efficient screening and mutagenesis optimization of broad-spectrum antibacterial lactobacillus reuteri according to claim 1, wherein the obtained broad-spectrum antibacterial lactobacillus reuteri wndPL-14 can be applied to not only regulation of intestinal flora, prevention and treatment of various intestinal discomfort such as diarrhea, prevention of dental caries, improvement of immunity and the like, but also preparation of antibacterial agents, anti-infection therapeutic agents, biological tissue fixing agents and the like for food and medical industry.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116606784A (en) * | 2023-07-18 | 2023-08-18 | 华南农业大学 | Application of novel Lactobacillus reuteri anti-freezing protective agent in vacuum freeze drying process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116606784A (en) * | 2023-07-18 | 2023-08-18 | 华南农业大学 | Application of novel Lactobacillus reuteri anti-freezing protective agent in vacuum freeze drying process |
| CN116606784B (en) * | 2023-07-18 | 2023-10-20 | 华南农业大学 | Application of novel Lactobacillus reuteri anti-freezing protective agent in vacuum freeze drying process |
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