CN112852676A - Method for separating, screening, detecting and identifying bacteriocin-producing strain - Google Patents

Abstract

The invention discloses a method for separating, screening, detecting and identifying a bacteriocin-producing strain, which comprises the following operation steps: screening of fermented meat samples of bacteriocin-producing strains: the fermented meat product is one of important sources of the bacterial strains for producing the bacteriocin, a meat product sample is cut up and placed in a meat grinder to be ground under the aseptic environment, the sample is added into a triangular flask of aseptic normal saline, the triangular flask is fully vibrated, and the suspension is taken and added into an MRS liquid culture medium after the triangular flask is kept stand. According to the method for separating, screening, detecting and identifying the bacteriocin-producing strain, the acquired sample is detected through a bacteriostatic experiment by a disc tube method, the sample is used as a target for screening the bacteriocin-producing strain and carrying out subsequent experiments on subsequent researches on bacteriocin, and screening is carried out from the macroscopic aspect, namely, the sample containing the bacteriocin-producing lactic acid bacteria is screened firstly, and then the bacteriocin-producing strain is separated, so that purposeful separation and screening can improve the efficiency, shorten the screening period of the strain and bring better use prospects.

Description

Method for separating, screening, detecting and identifying bacteriocin-producing strain

Technical Field

The invention relates to the field of bacteriocin-producing strain identification, in particular to a method for separating, screening, detecting and identifying a bacteriocin-producing strain.

Background

The bacterial strain for producing the bacteriocin is mainly present in various foods such as fermented meat products, dairy products and the like. Among them, fermented meat products are one of the important sources of bacteriocin-producing strains. The bacteriocin generally has the advantages of high safety, strong antibacterial specificity, high pH stability, high thermal stability and the like, and along with the continuous development of science and technology, people have higher and higher requirements on separation, screening, detection and identification methods of bacteriocin-producing strains.

The traditional method for screening the bacteriocin-producing lactic acid bacteria comprises the steps of screening lactic acid bacteria from a sample, and then judging whether the lactic acid bacteria have the bacteriocin-producing capacity or not by detecting the bacteriostatic activity of fermentation supernatant of the lactic acid bacteria, so that the working efficiency is reduced, the use of people is not facilitated, and certain adverse effects are brought to the use process of people.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a method for separating, screening, detecting and identifying a bacteriocin-producing strain, which comprises the steps of detecting a bacteriostasis experiment on an acquired sample by a tube-disc method, taking the sample as a bacteriocin-producing strain screening object and carrying out a subsequent experiment on a subsequent bacteriocin research object, and screening from a macroscopic aspect, namely screening a sample containing bacteriocin-producing lactic acid bacteria firstly, and then separating the bacteriocin-producing strain, so that purposeful separation and screening can improve the efficiency, shorten the strain screening period and effectively solve the problems in the background technology.

(II) technical scheme

In order to achieve the purpose, the invention adopts the technical scheme that: a method for separating, screening, detecting and identifying a bacteriocin-producing strain comprises the following operation steps:

s1: screening of fermented meat samples of bacteriocin-producing strains: the fermented meat product is one of important sources of bacteriocin-producing strains, a meat product sample is cut and placed in a meat grinder to be ground in an aseptic environment, a sample is added into a triangular flask of aseptic normal saline, the triangular flask is fully vibrated, a suspension is taken and added into an MRS liquid culture medium after standing, the MRS liquid culture medium is placed in a constant temperature incubator, the cultured bacterial liquid is centrifuged to remove precipitates, an L-1NaOH solution is used for adjusting the pH of a supernatant to about 6.0, and the presence or absence of an antibacterial effect of the supernatant is checked by a tube-disc antibacterial experiment;

s2: separation and purification of lactic acid bacteria in a sample of a bacteriocin-producing strain: chopping meat products containing bacteriocin-producing strains in a sterile environment, placing the meat products into a meat grinder for mincing, taking samples, adding the samples into a triangular flask containing sterile normal saline, fully oscillating the samples, standing the samples to obtain a suspension of YS01, taking 5 additional sterile water test tubes containing 9mL, sucking 1mL of the suspension diluted into YS01, adding the suspension into the sterile normal saline test tubes, oscillating the suspension to be fully mixed to obtain 10-2 diluent, sequentially diluting the diluent by 10 times to 10-4, 10-5, 10-6 and 10-7 by the same method, taking 1mL of the diluent with each gradient into a sterile flat dish, pouring a molten MRS solid culture medium into the flat dish, solidifying the culture medium, carrying out inversion constant-temperature culture, carrying out plate culture, and then selecting colonies with different apparent characteristics according to colony morphology, size, color, growth position in the culture medium, surface, interior and bottom by using an inoculating loop to inoculate in an MRS liquid culture medium, placing the strain in a constant temperature incubator for culture, diluting the cultured strain by 10-fold series gradient, selecting proper dilution, adding 1mL of bacterial liquid into a plate, pouring the plate by using MRS culture medium containing calcium carbonate, solidifying, inverting the plate for constant temperature culture, selecting bacterial colony with a calcium dissolving ring for inoculation culture, performing microscopic examination by using gram staining, recording and archiving, and storing the purified lactobacillus at-20 ℃ by using glycerol skim milk for subsequent experiments;

s3: primary screening of the bacteriocin-producing lactic acid bacteria: activating the lactobacillus strain separated in the step S2, inoculating the lactobacillus strain into an MRS liquid culture medium according to the inoculation amount of 1%, culturing for 12-16h in a constant-temperature incubator at 37 ℃, activating the second generation by the same method, then inoculating the third generation and culturing for 24h in an incubator at 37 ℃, centrifuging the cultured bacterium liquid, removing the bacterial precipitation, adjusting the pH of the supernatant to about 6.0 by using an L-1NaOH solution, and detecting whether the fermentation supernatant of the lactobacillus strain has bacteriostatic activity by using the tube-disc method in the step S1;

s4: rescreening of bacteriocin-producing lactic acid bacteria: in order to eliminate the possibility that the bacteriostatic substances are acid and H2O2 and determine that the bacteriostatic substances are protein, activating the effective strains in the step S3 by the same method to obtain fermentation supernatant, and then performing an acid action elimination experiment, an H2O2 action elimination experiment and a determination experiment that the bacteriostatic substances are protein;

s5: RAPD analysis of bacteriocin-producing strains: extracting DNA of the bacterial strain producing the bacteriocin, activating the screened bacterial strain producing the bacteriocin, extracting the DNA after partial condition optimization based on a traditional method, and screening suitable primers from 7 random primers existing in a laboratory: p1252, P1284, OPA-18, OPL-16, OPM-05, OPA-03 and OPV-07, primers with poor amplification effect and fuzzy bands are eliminated, primers with more than 5 amplified total bands and clear bands are used for secondary screening, and primers with good polymorphism and amplification repeatability, clear and more bands are selected from an electrophoretogram after amplification and are used for RAPD analysis of a bacteriocin-producing strain DNA sample;

s6: morphological and physiological and biochemical identification of the bacteriocin-producing strain: mainly comprises culture characteristic identification, morphological characteristic identification and physiological and biochemical characteristic identification;

s7: 16S rDNA identification of bacteriocin-producing strains: carrying out 16S rDNA PCR amplification on the target strain genome DNA extracted in the step S5, using a primer used in a 16S rDNA PCR amplification reaction as a universal primer, adopting PCR to amplify a target fragment, simultaneously setting a blank control group, namely replacing a DNA template in an amplification system with ddH2O, storing a PCR amplification product at 4 ℃ after the PCR is finished, uniformly mixing the PCR amplification product with bromophenol blue, detecting the PCR amplification result by using 1% agarose gel electrophoresis, determining that a single strip appears, carrying out gel cutting, recovering and purifying, sequencing, carrying out BLAST comparison on a sequencing result on NCBI, and constructing a system development tree by using the obtained result;

s8: virulence detection of bacteriocin-producing strains: in order to evaluate the potential pathogenicity of the bacteria and detect whether the bacteria contain some virulence genes, the method is to carry out PCR on the virulence genes, the virulence genes to be detected comprise asa1, namely aggregates and ace, namely collagen adhesins and esp, namely enterococcus surface protein and efaAfm, namely cell wall adhesins and cylA/B, namely activation and expression of cell lysins, wherein the annealing temperatures of different primers are different, PCR programs are set according to the annealing temperatures of the respective primers in the experiment, PCR amplification products are stored at 4 ℃ after the PCR is finished, the PCR amplification products are uniformly mixed with bromophenol blue, 1.5% agarose gel electrophoresis is used for detecting the PCR amplification results, and the results are observed and photographed and recorded in an automatic gel imaging instrument.

As a preferred technical scheme, the preparation of the common culture medium mainly comprises an MRS culture medium and a BHI culture medium.

As a preferred technical solution, the common reagents and buffers mainly comprise physiological saline: 0.85% NaCl solution, glycerolipid demulsification: glycerol was diluted to 20% -30% with 12% sterile skim milk, Tris-SDS: 6.06g Tris-base was mixed with 2.1mL concentrated HCl, then 40mL distilled water and 40mL 0.5M EDTA were added, then cooled to room temperature, pH adjusted to 9.0, and finally made to 250mL, 50mL 10% SDS, phenol/chloroform/isoamyl alcohol, anhydrous ethanol, isopropanol, chloroform, bromophenol blue, EDTA, TE saturated phenol, EB, sodium acetate, agarose, etc. were added.

As a preferred technical scheme, the experimental sample is screened in three aspects of shelf life, fermentation and heat treatment intensity, products with long shelf life and low fermentation and heat treatment intensity are preferentially selected, and different types of fermented meat products are finally determined and stored at 4 ℃ for later use.

As a preferred technical scheme, the determination of the antibacterial activity of the supernatant in the step S1 generally adopts a double-layer agar culture tube-disk method, the water agar is poured into a plate in an aseptic environment, the plate is kept still, after the plate is solidified, the indicator bacterium listeria monocytogenes is sucked and added into the molten BHI solid culture medium, the plate is fully and uniformly mixed on a vortex oscillator and poured into the plate while the plate is hot, after the plate is solidified, a plate cover is opened in a super clean bench, the water is volatilized for 20min in the aseptic environment, then an oxford cup is lightly placed on the solidified culture medium, the fermentation supernatant is added into the oxford cup, the plate is placed in a 4 ℃ refrigerator for 6-12h, the constant temperature culture is carried out, the culture temperature is 37 ℃, the culture time is 12-16h, whether an antibacterial ring appears or not is detected, and the diameter size of the antibacterial ring is recorded.

As a preferred technical solution, in the step S4, an experiment is performed:

acid exclusion experiment: adjusting the pH of the fermentation supernatant to be about neutral, taking the fermentation supernatant without the adjusted pH as a reference, and measuring the bacteriostatic activity of the fermentation supernatant by using a pipe-disc method;

H2O2 action exclusion experiment: after the experiment of eliminating the acid action, adjusting the pH of the fermentation supernatant of the effective strain to 7.0, adding H2O2 enzyme to make the final concentration of the enzyme be 1 mg/mL-1, treating for 2H at 37 ℃, and measuring the bacteriostatic activity of the fermentation supernatant by a pipe-disc method by taking the fermentation supernatant which is not treated by the H2O2 enzyme as a control;

determination of the bacteriostatic substance as protein: after the screening of the two experiments, the pH of the effective fermentation supernatant is adjusted to 7.0, trypsin is added to make the final concentration of the effective fermentation supernatant to be 1 mg/mL-1, the effective fermentation supernatant is treated for 2 hours at 37 ℃, and the antibacterial activity of the effective fermentation supernatant is measured by a tube-disc method by taking the fermentation supernatant which is not treated by the trypsin as a control.

As a preferred technical solution, in the step S5, DNA is extracted: taking 1mL to 2mL of a centrifugal tube of a bacterial liquid under an aseptic condition, centrifuging, discarding a supernatant, adding 1mL of PBS, sufficiently and uniformly mixing by a vortex oscillator, centrifuging for 5min at 4 ℃ at a centrifugal acceleration of 20627 Xg, discarding the supernatant, adding 0.3g of glass beads, 300. mu.L of Tris-SDS, 500. mu.L of TE saturated phenol, homogenizing in a homogenizer for 30s after balancing, balancing and centrifuging again, transferring 400. mu.L of a centrifuged supernatant suspension into a new 2.0mL centrifugal tube, adding 400. mu.L of phenol/chloroform/isoamylol, shaking by hand, uniformly mixing, centrifuging, taking 250. mu.L of a suspended supernatant, transferring into a new 1.5mL centrifugal tube, adding 25. mu.L of 3M sodium acetate, uniformly mixing with 300. mu.L of isopropanol, discarding after centrifuging, adding 500. mu.L of 70% ethanol, uniformly mixing, centrifuging, discarding the supernatant, throwing a liquid for a centrifugal tube to the bottom, sucking out an excessive liquid by using a gun head, drying at room temperature, adding 100. mu.L of TE buffer solution, adding 5. mu.L of LRNA enzyme, mixing uniformly, standing at 37 ℃ for 10min, and detecting the extracted DNA by electrophoresis: preparing 1% m/V agarose gel, uniformly mixing 5 mu L DNA sample with 1 mu L bromophenol blue, loading, performing electrophoresis for 40min at 120V voltage, and observing, photographing and recording in an automatic gel imager.

As a preferred technical solution, the identification step in the step S6:

the culture characteristics are as follows: streak-culturing the bacterial strain producing the bacteriocin in an MRS solid culture medium, carrying out inversion constant-temperature culture at the culture temperature of 37 ℃ for 48h, observing the colony morphology and size, the surface dry-wet condition, the uplift or flat condition, the rough or smooth condition, whether the edge is neat, the colony transparent state, the loose or tight color and texture condition and the like, and photographing and recording the result;

morphological characteristics: inoculating and culturing the bacterial strain producing the bacteriocin in an MRS liquid culture medium, culturing at a constant temperature of 37 ℃ for 12-16h, then carrying out gram staining on the bacterial strain, observing the form of the bacterial strain, and taking a picture to record the result, wherein the negative or positive bacterial strain is negative;

physiological and biochemical characteristic identification: the physiological and biochemical characteristics of the bacteriocin-producing strain are identified according to a common bacteria system identification manual and a lactic acid bacteria classification identification and experimental method, wherein the physiological and biochemical characteristics comprise growth conditions under the conditions of 10 ℃ and 45 ℃, growth conditions under the conditions of 6.5% NaCl culture solution and pH 9.6, catalase tests, H2S production tests, V-P tests, motility tests and sugar and alcohol fermentation tests.

(III) advantageous effects

Compared with the prior art, the invention provides a method for separating, screening, detecting and identifying a bacteriocin-producing strain, which has the following beneficial effects: according to the method for separating, screening, detecting and identifying the bacteriocin-producing strain, a collected sample is subjected to detection of a bacteriostatic experiment by a tube-disc method, the sample is used as a bacteriocin-producing strain screening object and a subsequent experiment is carried out on a subsequent bacteriocin research object, and screening is carried out from a macroscopic aspect, namely, a sample containing bacteriocin-producing lactic acid bacteria is firstly screened, and then the bacteriocin-producing strain is separated, so that purposeful separation and screening can improve the efficiency and shorten the strain screening period.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

A method for separating, screening, detecting and identifying a bacteriocin-producing strain comprises the following operation steps:

s1: screening of fermented meat samples of bacteriocin-producing strains: the fermented meat product is one of important sources of bacteriocin-producing strains, a meat product sample is cut and placed in a meat grinder to be ground in an aseptic environment, a sample is added into a triangular flask of aseptic normal saline, the triangular flask is fully vibrated, a suspension is taken and added into an MRS liquid culture medium after standing, the MRS liquid culture medium is placed in a constant temperature incubator, the cultured bacterial liquid is centrifuged to remove precipitates, an L-1NaOH solution is used for adjusting the pH of a supernatant to about 6.0, and the presence or absence of an antibacterial effect of the supernatant is checked by a tube-disc antibacterial experiment;

s2: separation and purification of lactic acid bacteria in a sample of a bacteriocin-producing strain: chopping meat products containing bacteriocin-producing strains in a sterile environment, placing the meat products into a meat grinder for mincing, taking samples, adding the samples into a triangular flask containing sterile normal saline, fully oscillating the samples, standing the samples to obtain a suspension of YS01, taking 5 additional sterile water test tubes containing 9mL, sucking 1mL of the suspension diluted into YS01, adding the suspension into the sterile normal saline test tubes, oscillating the suspension to be fully mixed to obtain 10-2 diluent, sequentially diluting the diluent by 10 times to 10-4, 10-5, 10-6 and 10-7 by the same method, taking 1mL of the diluent with each gradient into a sterile flat dish, pouring a molten MRS solid culture medium into the flat dish, solidifying the culture medium, carrying out inversion constant-temperature culture, carrying out plate culture, and then selecting colonies with different apparent characteristics according to colony morphology, size, color, growth position in the culture medium, surface, interior and bottom by using an inoculating loop to inoculate in an MRS liquid culture medium, placing the strain in a constant temperature incubator for culture, diluting the cultured strain by 10-fold series gradient, selecting proper dilution, adding 1mL of bacterial liquid into a plate, pouring the plate by using MRS culture medium containing calcium carbonate, solidifying, inverting the plate for constant temperature culture, selecting bacterial colony with a calcium dissolving ring for inoculation culture, performing microscopic examination by using gram staining, recording and archiving, and storing the purified lactobacillus at-20 ℃ by using glycerol skim milk for subsequent experiments;

s3: primary screening of the bacteriocin-producing lactic acid bacteria: activating the lactobacillus strain separated in the step S2, inoculating the lactobacillus strain into an MRS liquid culture medium according to the inoculation amount of 1%, culturing for 12-16h in a constant-temperature incubator at 37 ℃, activating the second generation by the same method, then inoculating the third generation and culturing for 24h in an incubator at 37 ℃, centrifuging the cultured bacterium liquid, removing the bacterial precipitation, adjusting the pH of the supernatant to about 6.0 by using an L-1NaOH solution, and detecting whether the fermentation supernatant of the lactobacillus strain has bacteriostatic activity by using a tube-disc method in the step S1;

s4: rescreening of bacteriocin-producing lactic acid bacteria: in order to eliminate the possibility that the bacteriostatic substances are acid and H2O2 and determine that the bacteriostatic substances are protein, activating the effective strains in the step S3 by the same method to obtain fermentation supernatant, and then performing an acid action elimination experiment, an H2O2 action elimination experiment and a determination experiment that the bacteriostatic substances are protein;

s5: RAPD analysis of bacteriocin-producing strains: extracting DNA of the bacterial strain producing the bacteriocin, activating the screened bacterial strain producing the bacteriocin, extracting the DNA after partial condition optimization based on a traditional method, and screening suitable primers from 7 random primers existing in a laboratory: p1252, P1284, OPA-18, OPL-16, OPM-05, OPA-03 and OPV-07, primers with poor amplification effect and fuzzy bands are eliminated, primers with more than 5 amplified total bands and clear bands are used for secondary screening, and primers with good polymorphism and amplification repeatability, clear and more bands are selected from an electrophoretogram after amplification and are used for RAPD analysis of a bacteriocin-producing strain DNA sample;

s6: morphological and physiological and biochemical identification of the bacteriocin-producing strain: mainly comprises culture characteristic identification, morphological characteristic identification and physiological and biochemical characteristic identification;

s7: 16S rDNA identification of bacteriocin-producing strains: carrying out 16S rDNA PCR amplification on the target strain genome DNA extracted in the step S5, using a primer used in a 16S rDNA PCR amplification reaction as a universal primer, adopting PCR to amplify a target fragment, simultaneously setting a blank control group, namely replacing a DNA template in an amplification system with ddH2O, storing a PCR amplification product at 4 ℃ after the PCR is finished, uniformly mixing the PCR amplification product with bromophenol blue, detecting the PCR amplification result by using 1% agarose gel electrophoresis, determining that a single strip appears, carrying out gel cutting, recovering and purifying, sequencing, carrying out BLAST comparison on a sequencing result on NCBI, and constructing a system development tree by using the obtained result;

s8: virulence detection of bacteriocin-producing strains: in order to evaluate the potential pathogenicity of the bacteria and detect whether the bacteria contain some virulence genes, the method is to carry out PCR on the virulence genes, the virulence genes to be detected comprise asa1, namely aggregates and ace, namely collagen adhesins and esp, namely enterococcus surface protein and efaAfm, namely cell wall adhesins and cylA/B, namely activation and expression of cell lysins, wherein the annealing temperatures of different primers are different, PCR programs are set according to the annealing temperatures of the respective primers in the experiment, PCR amplification products are stored at 4 ℃ after the PCR is finished, the PCR amplification products are uniformly mixed with bromophenol blue, 1.5% agarose gel electrophoresis is used for detecting the PCR amplification results, and the results are observed and photographed and recorded in an automatic gel imaging instrument.

Furthermore, the preparation of the common culture medium mainly comprises MRS culture medium and BHI culture medium.

Further, the common reagents and buffers mainly include physiological saline: 0.85% NaCl solution, glycerolipid demulsification: glycerol was diluted to 20% -30% with 12% sterile skim milk, Tris-SDS: 6.06g Tris-base was mixed with 2.1mL concentrated HCl, then 40mL distilled water and 40mL 0.5M EDTA were added, then cooled to room temperature, pH adjusted to 9.0, and finally made to 250mL, 50mL 10% SDS, phenol/chloroform/isoamyl alcohol, anhydrous ethanol, isopropanol, chloroform, bromophenol blue, EDTA, TE saturated phenol, EB, sodium acetate, agarose, etc. were added.

Furthermore, the experimental samples are screened according to the quality guarantee period, whether the experimental samples are fermented and the heat treatment intensity, products with long quality guarantee period and low fermentation and heat treatment intensity are preferentially selected, and different types of fermented meat products are finally determined and stored at 4 ℃ for later use.

Further, the determination of the antibacterial activity of the supernatant in the step S1 generally adopts a double-layer agar culture tube-disk method, the water agar is poured into a plate in a sterile environment, the plate is kept still, after the plate is solidified, the indicating bacterium listeria monocytogenes is absorbed and added into the molten BHI solid culture medium, the BHI solid culture medium is fully and uniformly mixed on a vortex oscillator, the plate is poured into the plate while the plate is hot, after the plate is solidified, a plate cover is opened in a super clean bench, the water is volatilized for 20min in the sterile environment, then an oxford cup is lightly placed on the solidified culture medium, the fermentation supernatant is added into the oxford cup, the plate is placed in a refrigerator at 4 ℃ for 6-12h, the plate is vertically placed for constant-temperature culture, the culture temperature is 37 ℃, the culture time is 12-16h, whether an antibacterial ring appears or not is detected, and the diameter of the antibacterial ring is recorded.

Further, an experiment was performed in step S4:

acid exclusion experiment: adjusting the pH of the fermentation supernatant to be about neutral, taking the fermentation supernatant without the adjusted pH as a reference, and measuring the bacteriostatic activity of the fermentation supernatant by using a pipe-disc method;

H2O2 action exclusion experiment: after the experiment of eliminating the acid action, adjusting the pH of the fermentation supernatant of the effective strain to 7.0, adding H2O2 enzyme to make the final concentration of the enzyme be 1 mg/mL-1, treating for 2H at 37 ℃, and measuring the bacteriostatic activity of the fermentation supernatant by a pipe-disc method by taking the fermentation supernatant which is not treated by the H2O2 enzyme as a control;

determination of the bacteriostatic substance as protein: after the screening of the two experiments, the pH of the effective fermentation supernatant is adjusted to 7.0, trypsin is added to make the final concentration of the effective fermentation supernatant to be 1 mg/mL-1, the effective fermentation supernatant is treated for 2 hours at 37 ℃, and the antibacterial activity of the effective fermentation supernatant is measured by a tube-disc method by taking the fermentation supernatant which is not treated by the trypsin as a control.

Further, in step S5, DNA is extracted: taking 1mL to 2mL of a centrifugal tube of a bacterial liquid under an aseptic condition, centrifuging, discarding a supernatant, adding 1mL of PBS, sufficiently and uniformly mixing by a vortex oscillator, centrifuging for 5min at 4 ℃ at a centrifugal acceleration of 20627 Xg, discarding the supernatant, adding 0.3g of glass beads, 300. mu.L of Tris-SDS, 500. mu.L of TE saturated phenol, homogenizing in a homogenizer for 30s after balancing, balancing and centrifuging again, transferring 400. mu.L of a centrifuged supernatant suspension into a new 2.0mL centrifugal tube, adding 400. mu.L of phenol/chloroform/isoamylol, shaking by hand, uniformly mixing, centrifuging, taking 250. mu.L of a suspended supernatant, transferring into a new 1.5mL centrifugal tube, adding 25. mu.L of 3M sodium acetate, uniformly mixing with 300. mu.L of isopropanol, discarding after centrifuging, adding 500. mu.L of 70% ethanol, uniformly mixing, centrifuging, discarding the supernatant, throwing a liquid for a centrifugal tube to the bottom, sucking out an excessive liquid by using a gun head, drying at room temperature, adding 100. mu.L of TE buffer solution, adding 5. mu.L of LRNA enzyme, mixing uniformly, standing at 37 ℃ for 10min, and detecting the extracted DNA by electrophoresis: preparing 1% m/V agarose gel, uniformly mixing 5 mu L DNA sample with 1 mu L bromophenol blue, loading, performing electrophoresis for 40min at 120V voltage, and observing, photographing and recording in an automatic gel imager.

Further, the identification step in the step S6:

the culture characteristics are as follows: streak-culturing the bacterial strain producing the bacteriocin in an MRS solid culture medium, carrying out inversion constant-temperature culture at the culture temperature of 37 ℃ for 48h, observing the colony morphology and size, the surface dry-wet condition, the uplift or flat condition, the rough or smooth condition, whether the edge is neat, the colony transparent state, the loose or tight color and texture condition and the like, and photographing and recording the result;

morphological characteristics: inoculating and culturing the bacterial strain producing the bacteriocin in an MRS liquid culture medium, culturing at a constant temperature of 37 ℃ for 12-16h, then carrying out gram staining on the bacterial strain, observing the form of the bacterial strain, and taking a picture to record the result, wherein the negative or positive bacterial strain is negative;

physiological and biochemical characteristic identification: the physiological and biochemical characteristics of the bacteriocin-producing strain are identified according to a common bacteria system identification manual and a lactic acid bacteria classification identification and experimental method, wherein the physiological and biochemical characteristics comprise growth conditions under the conditions of 10 ℃ and 45 ℃, growth conditions under the conditions of 6.5% NaCl culture solution and pH 9.6, catalase tests, H2S production tests, V-P tests, motility tests and sugar and alcohol fermentation tests.

The working principle is as follows: chopping a meat product sample in a sterile environment, mincing the meat product sample in a meat mincer, adding 25g of the sample into a triangular flask containing 225mL of sterile physiological saline, fully oscillating the sample, standing the mixture for 20min, adding 1mL of suspension into 100mL of a liquid culture medium, placing the mixture into a constant-temperature incubator at the culture temperature of 37 ℃ for 16-24h, centrifuging the cultured bacterium solution (1927 Xg, 15min) to remove precipitates, adjusting the pH of a supernatant to about 6.0 by using a 3 mol. L-1NaOH solution, and testing whether the supernatant has an antibacterial effect by using a tube-disc antibacterial experiment, wherein the antibacterial activity of the supernatant is generally determined by using a tube-disc method of double-layer agar culture: pouring 7 mL1.5% (W/V) of water agar into a plate in an aseptic environment, standing, sucking 100 mu L of indicator bacterium Listeria monocytogenes into 10mL of molten BHI solid culture medium (the number of bacteria is about 107 cfu. mL < -1 >), fully mixing on a vortex oscillator, pouring the mixture into the plate while the mixture is hot, opening a plate cover in a super clean bench after the mixture is solidified, volatilizing water in the aseptic environment for 20min, slightly putting an Oxford cup on the solidified culture medium, adding 100 mu L of fermentation supernatant into an Oxford cup, putting the plate into a refrigerator at 4 ℃ for 6-12h, culturing at the constant temperature of 37 ℃ for 12-16h, detecting whether an antibacterial ring appears or not, recording the diameter of the antibacterial ring, separating and purifying lactobacillus in a sample of a bacteriocin strain, under the aseptic environment, mincing meat products containing bacteriocin-producing strains and placing the minced meat products in a meat mincer, adding 25g of samples into a triangular flask containing 225mL of sterile normal saline, fully shaking the mixture, standing the mixture for 20min to obtain a suspension of YS01, taking 5 sterile water test tubes containing 9mL, sucking 1mL of the suspension diluted into YS01, adding the suspension into the sterile normal saline test tubes, shaking the suspension to fully mix the suspension uniformly to obtain 10-2 diluents, sequentially diluting the diluents by 10 times to 10-4, 10-5, 10-6 and 10-7, taking 1mL of each gradient diluent into a sterile flat dish, pouring the molten MRS solid culture medium into the flat dish, solidifying the culture medium, inverting the culture medium for constant temperature culture at 37 ℃ for 48h, after flat plate culture, selecting different apparent characteristics of bacterial colonies according to the shape, size and color of the bacterial colonies, and the growth positions (surface, interior and bottom) in the culture medium by using inoculating loops to inoculate the bacterial colonies in an MRS liquid culture medium, culturing in a constant temperature incubator at 37 deg.C for 12-16h, diluting the cultured strains in 10-fold series gradient, selecting appropriate dilution, adding 1mL of bacterial liquid into a plate, pouring the plate with calcium carbonate-containing MRS culture medium, solidifying, inverting, culturing at 37 deg.C for 48h, selecting bacterial colony with calcium-dissolving ring, inoculating and culturing, performing microscopic examination with gram stain, recording, storing in-20 deg.C with glycerol skim milk, performing primary screening for bacteriocin-producing lactobacillus, activating the separated lactobacillus strains, inoculating in 10 mM MRS liquid culture medium according to 1%, culturing in 37 deg.C constant temperature incubator for 12-16h, activating the second generation with the same method, inoculating the third generation, and culturing in 37 deg.C incubator for 24h, centrifuging the cultured bacterial liquid (1927 Xg, 15min), removing thallus precipitate, adjusting the pH of the supernatant to about 6.0 by using 3 mol. L-1NaOH solution, detecting whether the fermentation supernatant of the lactic acid bacterial strain has antibacterial activity by using a pipe-disk method, re-screening the bacteriocin-producing lactic acid bacteria to eliminate the possibility that the antibacterial substances are acid and H2O2 and determine that the antibacterial substances are protein, and activating the effective bacterial strain by the same method to obtain the fermentation supernatant, and performing the following three experiments, namely an acid action elimination experiment: adjusting the pH of the fermentation supernatant to be about neutral, taking the fermentation supernatant without the adjusted pH as a reference, and measuring the bacteriostatic activity of the fermentation supernatant by using a pipe-disc method; H2O2 action exclusion experiment: after the experiment of eliminating the acid action, the fermentation supernatant of the effective strain is adjusted to pH7.0, H2O2 enzyme is added to make the final concentration of the enzyme 1 mg/mL-1, the fermentation supernatant is treated for 2H at 37 ℃, and the bacteriostatic activity of the fermentation supernatant is measured by a pipe-disc method by taking the fermentation supernatant which is not treated by the H2O2 enzyme as a control; determination of the bacteriostatic substance as protein: adjusting pH of the fermentation supernatant to 7.0 after screening the effective effect of the two experiments, adding trypsin to make the final concentration 1 mg. mL-1, treating at 37 ℃ for 2h, using the fermentation supernatant without trypsin treatment as a control, measuring the antibacterial activity by a tube-disk method, performing RAPD analysis on the bacteriocin-producing strain, activating the screened bacteriocin-producing strain, extracting DNA after partial condition optimization based on the conventional method [46], taking 1mL to 2mL of the bacterial liquid in a centrifuge tube under aseptic condition, centrifuging (centrifugal acceleration 20627 Xg, 5min, 4 ℃), discarding the supernatant, adding 1mLPBS, fully shaking and mixing uniformly by a vortex shaker, centrifuging at a centrifugal acceleration of 20627 Xg for 5min at 4 ℃, discarding the supernatant, adding 0.3g of glass beads, 300 mu L of Tris-SDS, 500 mu L of TE saturated phenol, balancing, homogenizing in a homogenizer for 30s, re-balancing and centrifuging (centrifugal acceleration 20627 Xg, 5min, 4 ℃), transferring 400. mu.L of centrifuged supernatant into a new 2.0mL centrifuge tube, adding 400. mu.L of phenol/chloroform/isoamylol (25: 24: 1), shaking by hand, mixing, centrifuging (centrifugal acceleration 20627 Xg, 5min, 4 ℃), transferring 250. mu.L of suspended supernatant into a new 1.5mL centrifuge tube, adding 25. mu.L of 3M sodium acetate (pH5.3) and 300. mu.L of isopropanol, mixing, centrifuging (centrifugal acceleration 20627 Xg, 5min, 4 ℃), discarding supernatant, adding 500. mu.L of 70% ethanol, mixing, centrifuging (centrifugal acceleration 20627 Xg, 5min, 4 ℃), discarding supernatant, pouring off the liquid for centrifuge tube to the bottom, sucking out the liquid with a gun head, drying at room temperature, adding 100. mu. of LTE buffer solution, adding 5. mu. NA enzyme, mixing, standing at 37 ℃ for 10min, electrophoresis detection of extracted DNA: preparing 1% m/V agarose gel, taking 5 mu L DNA sample, mixing with 1 mu L bromophenol blue, loading, performing electrophoresis for 40min under the voltage of 120V, observing in an automatic gel imager, taking pictures and recording, and screening suitable primers from 7 random primers in a laboratory: p1252, P1284, OPA-18, OPL-16, OPM-05, OPA-03 and OPV-07, eliminating primers with poor amplification effect and unclear bands, using primers with more than 5 total amplification bands and clear bands for secondary screening, selecting primers with good polymorphism and amplification repeatability, clear bands and more bands from an electrophoretogram after amplification for RAPD analysis of a DNA sample of a strain producing bacteriocin, uniformly mixing a PCR amplification product with bromophenol blue, using agarose gel to carry out electrophoresis for 90min under electrophoresis buffer solution l × TAE and voltage of 120V, taking out the gel when the bromophenol blue runs to 2/3 of the whole gel, dyeing (the dyeing solution is 300mL1 × TAE +20 μ LEB), rinsing with clear water, finally placing in an automatic gel imager for observation and recording, screening 26 strains of lactic acid bacteria capable of inhibiting the growth of Listeria monocytogenes from Italyticum ham according to the traditional method of lactic acid bacteria separation and purification, and further reverse plate purification is carried out, 26 strains of lactic acid bacteria are screened out by gram staining, 26 strains of lactic acid bacteria are selected out for activation, when the third generation is carried out, the culture is carried out for 24h at the constant temperature of 37 ℃, the supernatant is centrifuged to measure the bacteriostatic activity, so that 14 strains of lactic acid bacteria have no bacteriostatic activity, 9 strains of 12 strains of lactic acid bacteria have the bacteriostatic activity, the bacteriostatic action of the lactic acid bacteria is probably due to the generation of one or more active substances such as organic acids (lactic acid, acetic acid, formic acid, propionic acid, butyric acid and the like), the reduction of the pH of the culture medium to enhance the action of the active substances, and the generation of other substances such as hydrogen peroxide, bacteriocins (nisin, pediocin, nisin, enterocin, and the like) [53], therefore, when the bacteriocin is screened by detecting the bacteriostatic activity of the fermentation supernatant, generally, the influence of acid and hydrogen peroxide is eliminated, and the supernatant is treated by protease to show that the bacteriostatic substance is the essence of protein, the 9 strains of bacteria obtained by primary screening are respectively subjected to an acid action elimination experiment, an H2O2 action elimination experiment and a verification experiment that the bacteriostatic substance is protein, the experimental results show that after acid neutralization, the bacteriostatic activity of the 9 strains of bacteria is not influenced, and after H2O2 enzyme is added, the bacteriostatic activity of the 9 strains of bacteria is not influenced, which indicates that the bacteriostatic activity of the 9 strains of bacteria is not caused by the action of acid and H2O 2; after protease is added, the bacteriostatic activity disappears, which indicates that the substance which enables the 9 strains to show bacteriostatic action is a protein substance, namely bacteriocin.

It is noted that, herein, relational terms such as first and second (a, b, etc.) 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. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (8)

1. A method for separating, screening, detecting and identifying a bacteriocin-producing strain is characterized by comprising the following steps: the method comprises the following operation steps:

s1: screening of fermented meat samples of bacteriocin-producing strains: the fermented meat product is one of important sources of bacteriocin-producing strains, a meat product sample is cut and placed in a meat grinder to be ground in an aseptic environment, a sample is added into a triangular flask of aseptic normal saline, the triangular flask is fully vibrated, a suspension is taken and added into an MRS liquid culture medium after standing, the MRS liquid culture medium is placed in a constant temperature incubator, the cultured bacterial liquid is centrifuged to remove precipitates, an L-1NaOH solution is used for adjusting the pH of a supernatant to about 6.0, and the presence or absence of an antibacterial effect of the supernatant is checked by a tube-disc antibacterial experiment;

s2: separation and purification of lactic acid bacteria in a sample of a bacteriocin-producing strain: chopping meat products containing bacteriocin-producing strains in a sterile environment, placing the meat products into a meat grinder for mincing, taking samples, adding the samples into a triangular flask containing sterile normal saline, fully oscillating the samples, standing the samples to obtain a suspension of YS01, taking 5 additional sterile water test tubes containing 9mL, sucking 1mL of the suspension diluted into YS01, adding the suspension into the sterile normal saline test tubes, oscillating the suspension to be fully mixed to obtain 10-2 diluent, sequentially diluting the diluent by 10 times to 10-4, 10-5, 10-6 and 10-7 by the same method, taking 1mL of the diluent with each gradient into a sterile flat dish, pouring a molten MRS solid culture medium into the flat dish, solidifying the culture medium, carrying out inversion constant-temperature culture, carrying out plate culture, and then selecting colonies with different apparent characteristics according to colony morphology, size, color, growth position in the culture medium, surface, interior and bottom by using an inoculating loop to inoculate in an MRS liquid culture medium, placing the strain in a constant temperature incubator for culture, diluting the cultured strain by 10-fold series gradient, selecting proper dilution, adding 1mL of bacterial liquid into a plate, pouring the plate by using MRS culture medium containing calcium carbonate, solidifying, inverting the plate for constant temperature culture, selecting bacterial colony with a calcium dissolving ring for inoculation culture, performing microscopic examination by using gram staining, recording and archiving, and storing the purified lactobacillus at-20 ℃ by using glycerol skim milk for subsequent experiments;

s3: primary screening of the bacteriocin-producing lactic acid bacteria: activating the lactobacillus strain separated in the step S2, inoculating the lactobacillus strain into an MRS liquid culture medium according to the inoculation amount of 1%, culturing for 12-16h in a constant-temperature incubator at 37 ℃, activating the second generation by the same method, then inoculating the third generation and culturing for 24h in an incubator at 37 ℃, centrifuging the cultured bacterium liquid, removing the bacterial precipitation, adjusting the pH of the supernatant to about 6.0 by using an L-1NaOH solution, and detecting whether the fermentation supernatant of the lactobacillus strain has bacteriostatic activity by using the tube-disc method in the step S1;

s4: rescreening of bacteriocin-producing lactic acid bacteria: in order to eliminate the possibility that the bacteriostatic substances are acid and H2O2 and determine that the bacteriostatic substances are protein, activating the effective strains in the step S3 by the same method to obtain fermentation supernatant, and then performing an acid action elimination experiment, an H2O2 action elimination experiment and a determination experiment that the bacteriostatic substances are protein;

s5: RAPD analysis of bacteriocin-producing strains: extracting DNA of the bacterial strain producing the bacteriocin, activating the screened bacterial strain producing the bacteriocin, extracting the DNA after partial condition optimization based on a traditional method, and screening suitable primers from 7 random primers existing in a laboratory: p1252, P1284, OPA-18, OPL-16, OPM-05, OPA-03 and OPV-07, primers with poor amplification effect and fuzzy bands are eliminated, primers with more than 5 amplified total bands and clear bands are used for secondary screening, and primers with good polymorphism and amplification repeatability, clear and more bands are selected from an electrophoretogram after amplification and are used for RAPD analysis of a bacteriocin-producing strain DNA sample;

s6: morphological and physiological and biochemical identification of the bacteriocin-producing strain: mainly comprises culture characteristic identification, morphological characteristic identification and physiological and biochemical characteristic identification;

s7: 16S rDNA identification of bacteriocin-producing strains: carrying out 16S rDNA PCR amplification on the target strain genome DNA extracted in the step S5, using a primer used in a 16S rDNA PCR amplification reaction as a universal primer, adopting PCR to amplify a target fragment, simultaneously setting a blank control group, namely replacing a DNA template in an amplification system with ddH2O, storing a PCR amplification product at 4 ℃ after the PCR is finished, uniformly mixing the PCR amplification product with bromophenol blue, detecting the PCR amplification result by using 1% agarose gel electrophoresis, determining that a single strip appears, carrying out gel cutting, recovering and purifying, sequencing, carrying out BLAST comparison on a sequencing result on NCBI, and constructing a system development tree by using the obtained result;

s8: virulence detection of bacteriocin-producing strains: in order to evaluate the potential pathogenicity of the bacteria and detect whether the bacteria contain some virulence genes, the method is to carry out PCR on the virulence genes, the virulence genes to be detected comprise asa1, namely aggregates and ace, namely collagen adhesins and esp, namely enterococcus surface protein and efaAfm, namely cell wall adhesins and cylA/B, namely activation and expression of cell lysins, wherein the annealing temperatures of different primers are different, PCR programs are set according to the annealing temperatures of the respective primers in the experiment, PCR amplification products are stored at 4 ℃ after the PCR is finished, the PCR amplification products are uniformly mixed with bromophenol blue, 1.5% agarose gel electrophoresis is used for detecting the PCR amplification results, and the results are observed and photographed and recorded in an automatic gel imaging instrument.

2. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: the preparation of the common culture medium mainly comprises an MRS culture medium and a BHI culture medium.

3. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: the common reagents and buffers mainly comprise physiological saline: 0.85% NaCl solution, glycerolipid demulsification: glycerol was diluted to 20% -30% with 12% sterile skim milk, Tris-SDS: 6.06g Tris-base was mixed with 2.1mL concentrated HCl, then 40mL distilled water and 40mL 0.5M EDTA were added, then cooled to room temperature, pH adjusted to 9.0, and finally made to 250mL, 50mL 10% SDS, phenol/chloroform/isoamyl alcohol, anhydrous ethanol, isopropanol, chloroform, bromophenol blue, EDTA, TE saturated phenol, EB, sodium acetate, agarose, etc. were added.

4. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: the experimental samples are screened according to the quality guarantee period, whether the experimental samples are fermented and the heat treatment intensity, products with long quality guarantee period and low fermentation and heat treatment intensity are preferentially selected, and different types of fermented meat products are finally determined and stored at 4 ℃ for later use.

5. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: the determination of the antibacterial activity of the supernatant in the step S1 generally adopts a double-layer agar culture tube-disk method, water agar is poured into a plate in an aseptic environment, the plate is kept stand, after the plate is solidified, the indicating bacterium listeria monocytogenes is absorbed and added into a molten BHI solid culture medium, the liquid is fully and uniformly mixed on a vortex oscillator and poured into the plate while the plate is hot, after the plate is solidified, a plate cover is opened in a super clean bench, water is volatilized for 20min in the aseptic environment, an Oxford cup is lightly placed on the solidified culture medium, the fermentation supernatant is taken and added into the Oxford cup, the plate is placed in a refrigerator at 4 ℃ for 6-12h, the plate is cultured at the constant temperature, the culture temperature is 37 ℃, the culture time is 12-16h, whether an antibacterial ring appears or not is detected, and the diameter of the antibacterial ring is recorded.

6. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: the experiment in step S4 is performed:

acid exclusion experiment: adjusting the pH of the fermentation supernatant to be about neutral, taking the fermentation supernatant without the adjusted pH as a reference, and measuring the bacteriostatic activity of the fermentation supernatant by using a pipe-disc method;

H2O2 action exclusion experiment: after the experiment of eliminating the acid action, adjusting the pH of the fermentation supernatant of the effective strain to 7.0, adding H2O2 enzyme to make the final concentration of the enzyme be 1 mg/mL-1, treating for 2H at 37 ℃, and measuring the bacteriostatic activity of the fermentation supernatant by a pipe-disc method by taking the fermentation supernatant which is not treated by the H2O2 enzyme as a control;

determination of the bacteriostatic substance as protein: after the screening of the two experiments, the pH of the effective fermentation supernatant is adjusted to 7.0, trypsin is added to make the final concentration of the effective fermentation supernatant to be 1 mg/mL-1, the effective fermentation supernatant is treated for 2 hours at 37 ℃, and the antibacterial activity of the effective fermentation supernatant is measured by a tube-disc method by taking the fermentation supernatant which is not treated by the trypsin as a control.

7. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: extracting DNA in the step S5: taking 1mL to 2mL of a centrifugal tube of a bacterial liquid under an aseptic condition, centrifuging, discarding a supernatant, adding 1mL of PBS, sufficiently and uniformly mixing by a vortex oscillator, centrifuging for 5min at 4 ℃ at a centrifugal acceleration of 20627 Xg, discarding the supernatant, adding 0.3g of glass beads, 300. mu.L of Tris-SDS, 500. mu.L of TE saturated phenol, homogenizing in a homogenizer for 30s after balancing, balancing and centrifuging again, transferring 400. mu.L of a centrifuged supernatant suspension into a new 2.0mL centrifugal tube, adding 400. mu.L of phenol/chloroform/isoamylol, shaking by hand, uniformly mixing, centrifuging, taking 250. mu.L of a suspended supernatant, transferring into a new 1.5mL centrifugal tube, adding 25. mu.L of 3M sodium acetate, uniformly mixing with 300. mu.L of isopropanol, discarding after centrifuging, adding 500. mu.L of 70% ethanol, uniformly mixing, centrifuging, discarding the supernatant, throwing a liquid for a centrifugal tube to the bottom, sucking out an excessive liquid by using a gun head, drying at room temperature, adding 100. mu.L of TE buffer solution, adding 5. mu.L of LRNA enzyme, mixing uniformly, standing at 37 ℃ for 10min, and detecting the extracted DNA by electrophoresis: preparing 1% m/V agarose gel, uniformly mixing 5 mu L DNA sample with 1 mu L bromophenol blue, loading, performing electrophoresis for 40min at 120V voltage, and observing, photographing and recording in an automatic gel imager.

8. The method for the isolation, screening, detection and identification of a bacteriocin-producing strain according to claim 1, wherein: the identification step in the step S6:

the culture characteristics are as follows: streak-culturing the bacterial strain producing the bacteriocin in an MRS solid culture medium, carrying out inversion constant-temperature culture at the culture temperature of 37 ℃ for 48h, observing the colony morphology and size, the surface dry-wet condition, the uplift or flat condition, the rough or smooth condition, whether the edge is neat, the colony transparent state, the loose or tight color and texture condition and the like, and photographing and recording the result;

morphological characteristics: inoculating and culturing the bacterial strain producing the bacteriocin in an MRS liquid culture medium, culturing at a constant temperature of 37 ℃ for 12-16h, then carrying out gram staining on the bacterial strain, observing the form of the bacterial strain, and taking a picture to record the result, wherein the negative or positive bacterial strain is negative;

physiological and biochemical characteristic identification: the physiological and biochemical characteristics of the bacteriocin-producing strain are identified according to a common bacteria system identification manual and a lactic acid bacteria classification identification and experimental method, wherein the physiological and biochemical characteristics comprise growth conditions under the conditions of 10 ℃ and 45 ℃, growth conditions under the conditions of 6.5% NaCl culture solution and pH 9.6, catalase tests, H2S production tests, V-P tests, motility tests and sugar and alcohol fermentation tests.