CN111286461A

CN111286461A - Method for obtaining strain ZFM54

Info

Publication number: CN111286461A
Application number: CN201911342130.2A
Authority: CN
Inventors: 顾青; 顾容铖
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-06-16

Abstract

The invention discloses a method for acquiring a strain ZFM54, which comprises the following steps: (A) separating lactic acid bacteria from the infant feces; (B) activating and preserving strains; (C) culturing indicator bacteria; (D) primary screening of lactic acid bacteria; (E) re-screening the bacteriocin-producing lactic acid bacteria; and (F) identification of strains, wherein the strains ZFM54 can be used as natural food preservatives and antibiotic substitutes.

Description

Method for obtaining strain ZFM54

Technical Field

The invention relates to the field of strain culture and screening, and further relates to a method for obtaining a strain ZFM 54.

Background

Contamination by microorganisms and food-borne pathogenic bacteria is a major cause of food spoilage and food poisoning, and the most common method currently used in the food industry to prevent and control microbial contamination is to add chemical preservatives thereto. However, if the chemical preservative is not used properly, certain side effects can be caused, and harm is caused to human health. Over the new century, drug residues in animal-derived foods have become a serious threat to human health due to abuse of antibiotics, causing allergic reactions in humans andthe flora in the body is unbalanced, thereby causing diarrhea, vitamin deficiency and the like. But the ensuing problem of bacterial antibiotic resistance also has a very important impact on public health and livestock production. Therefore, the search for safe, green and nontoxic chemical preservative substitutes and the solution of the problem of antibiotic resistance is a worldwide trend. Probiotics (Probiotics), also known as microecologics, ecological products, live bacterial preparations and the like, refer to a class of microorganisms which can regulate the intestinal microecology by being added into food or feed, thereby producing beneficial effects on human beings or animals. The concept of probiotics was originally derived from greek and meant "life-beneficial". In 2001, the united nations Food and Agriculture Organization (FAO) and World Health Organization (WHO) joint experts group made the following definitions for probiotics: live bacteria which exert an effective effect on the health of the consumer when ingested in an appropriate amount. Probiotics exist widely in nature, in humans and animals, and mainly include bifidobacteria, lactobacilli, gram-positive cocci and fungi. At present, researches on some bifidobacteria, lactobacilli, lactobacillus acidophilus, nonpathogenic streptococcus, enterococcus, actinomycetes, yeasts and the like mainly derived from animal bodies are relatively extensive. The probiotics have the effects of regulating the intestinal flora structure, improving the intestinal health, promoting the digestion and absorption of nutrient elements, inhibiting the growth of pathogenic microorganisms and regulating immune response. With the intensive recognition of the probiotic function, the research and application of probiotics are increasingly emphasized, and the demand of people on food processing is more and more shifted to concepts such as green and natural. In this case, probiotics replace chemical antiseptics to achieve food antisepsis and freshness keeping, and replace antibiotics to be used as feed additives to feed livestock and poultry^[7-9]To improve their growth performance and as a new means of disease treatment are gradually entering the human vision. Lactic Acid Bacteria (LAB) are Generally Recognized As Safe (GRAS) food-grade microorganisms, and are absolutely safe to human and livestock health, as well as probiotics recognized in microbiology. Due to the safety and probiotic characteristics of the lactic acid bacteria and the metabolites thereof, the application of the lactic acid bacteria in food and feed has good feasibility, and the growth of undesirable bacteria can be controlledFood grade and more natural ways.

Lactic acid bacteria are a generic term for a group of non-spore, gram-positive bacteria that ferment carbohydrates to produce lactic acid. Lactic acid bacteria are generally described as cocci or bacilli, facultative anaerobes. In the middle of the 19 th century, pasteur, a french scientist, first discovered lactic acid bacteria when investigating the cause of alcoholism. Lister subsequently isolated the first pure cultured lactic acid bacteria in 1873. Lactic acid bacteria are widely present in milk, meat, fermented vegetables and fruits, and intestinal tracts, oral cavities and other parts of human and animals are also places where the growth of lactic acid bacteria is vigorous. The lactobacillus has the beneficial effects of improving the intestinal function of a human body, enhancing the immunity of the organism, preventing diarrhea, reducing cholesterol and the like, improving the texture and flavor of food, improving the nutritional value of the food, and inhibiting the growth of pathogenic bacteria in the food and preventing the food from being corrupted by organic acid, hydrogen peroxide, bacteriocin and other natural substances generated by the metabolism of the lactobacillus.

Disclosure of Invention

An object of the present invention is to provide a method for obtaining a strain ZFM54, wherein the strain ZMF54 is a Lactobacillus paracasei which can be used as a natural food preservative and antibiotic substitute.

The invention aims to provide a method for obtaining a strain ZMF54, which is characterized in that lactic acid bacteria are separated from feces of newborn infants, a lactic acid bacteria dominant strain with broad-spectrum antibacterial action is obtained through primary screening, a secondary screening test for eliminating organic acid, hydrogen peroxide and enzyme hydrolysis is carried out to determine that bacteriostatic substances produced by the strain contain proteins or polypeptides such as bacteriocin, and the strain is subjected to species identification by physiological and biochemical identification and 16S rDNA sequence analysis.

An object of the invention is to provide a method for obtaining a strain ZMF54, wherein the strain ZFM54 has antibacterial effects on gram-positive bacteria such as micrococcus luteus 10209 and Listeria monocytogenes LM1 and gram-negative bacteria such as escherichia coli DH5 and Salmonella typhimurium CMCC50015, and has strong antibacterial activity.

An object of the present invention is to provide a method for obtaining a strain ZMF54, in which strain ZMF54 is obtained by designing a double sieveExperiment to eliminate organic acids and H₂O₂Identifying a class of proteins or polypeptides that produce a bacteriostatic effect.

An object of the invention is to provide a method for obtaining a strain ZFM54, wherein the bacteriostatic substance in the fermentation supernatant of the strain ZFM54 is very sensitive to trypsin and proteinase K, and the pepsin can also make the bacteriostatic substance lose part of the activity.

One purpose of the invention is to provide a method for obtaining a strain ZMF54, which combines morphological identification, physiological and biochemical identification and 16S rDNA homology analysis to identify and screen a ZFM54 strain.

An object of the present invention is to provide a method for obtaining strain ZFM54, wherein the strain ZFM54 produces bacteriocin.

One aspect of the present invention provides a method for obtaining a strain ZFM54, comprising:

(A) separating lactic acid bacteria from the infant feces;

(B) activating and preserving strains;

(C) culturing indicator bacteria;

(D) primary screening of lactic acid bacteria;

(E) re-screening the bacteriocin-producing lactic acid bacteria; and

(F) and (5) identifying strains.

According to one embodiment, in the step (A), under aseptic conditions, the feces of the newborn baby is diluted by 1mL of sterile normal saline, the feces is shaken to be fully and uniformly mixed and then is kept still, 100 mu L of supernatant is taken and coated on an MRS solid medium plate added with 2% calcium carbonate, and the MRS solid medium plate is placed upside down in an incubator at 37 ℃ for anaerobic culture for 36-48 hours.

According to one embodiment, in the step (B), the single colony of lactobacillus is primarily determined, inoculated into 10mL of MRS liquid medium and cultured for 24h at 37 ℃, subcultured twice with the inoculum size of 1% (v/v), numbered and preserved after activation, and 700 μ L of bacterial liquid is taken and 300 μ L of sterilized glycerol is added to the strain preserved at-80 ℃.

According to an embodiment, in the step (C), each indicator strain preserved in a glycerin tube at-80 ℃ is streaked on an LB solid medium plate, and is cultured at the respective optimum temperature until a single colony appears, and the single colony is picked and inoculated in an LB liquid medium for shake culture to a logarithmic phase.

According to one embodiment, in the step (D), the activated lactobacillus strains are respectively inoculated in 10mL of MRS liquid culture medium according to the proportion of 1%, after static culture is carried out for 24h at the temperature of 37 ℃, centrifugation is carried out for 20min at 8000r/min at the temperature of 4 ℃ to obtain supernatant, and the fermentation supernatant is tested for bacteriostatic activity by adopting an oxford cup agar diffusion method.

The method for obtaining the strain ZFM54 according to one embodiment, wherein the step (E) comprises the steps of: eliminating the interference of organic acid, firstly measuring the pH value of ZFM54 fermentation supernatant, then adjusting the pH value of the fermentation supernatant to 5.0 by using 1M sodium hydroxide solution, carrying out an oxford cup agar diffusion method bacteriostasis test on Micrococcus luteus 10209 by using a sterile MRS culture medium which is adjusted to the same pH value by using lactic acid and acetic acid as a control, measuring the diameter of a bacteriostasis zone, and comparing the differences of bacteriostasis activities.

The method for obtaining the strain ZFM54 according to one embodiment, wherein the step (E) comprises the steps of: eliminating the interference of hydrogen peroxide, concentrating twice the fermentation supernatant, adjusting the pH value to the original pH value of the fermentation supernatant, preparing catalase working solution with the concentration of 5mg/mL, mixing the fermentation supernatant concentrated solution and the catalase working solution according to the volume ratio of 1:1, carrying out water bath treatment at 37 ℃ for 2h, and testing the bacteriostatic activity of the fermentation supernatant by adopting an oxford cup method; meanwhile, the fermentation supernatant concentrated solution mixed by 1:1 and PBS buffer solution with pH7.0 are used as blank control, and the diameter of the inhibition zone is respectively measured and compared.

The method for obtaining the strain ZFM54 according to one embodiment, wherein the step (F) comprises the steps of: determining bacteriocin substances, preparing 5mg/mL of enzyme solution of pepsin, trypsin and proteinase K, respectively adjusting the pH value of fermentation supernatant to the optimum pH value of each proteinase, then adding corresponding enzyme solution to make the final concentration of the enzyme solution be 1mg/mL, treating the enzyme solution in a water bath kettle at 37 ℃ for 2 hours, then adjusting the initial pH value of the fermentation supernatant, taking the fermentation supernatant which is not treated by the proteinase as a blank control, carrying out a bacteriostasis test by adopting an Oxford cup method, measuring the diameter of a bacteriostasis zone, and comparing the diameter and the difference.

The method for obtaining a bacterial species ZFM54 according to one embodiment, wherein the step (F) comprises the steps of: and (3) morphological identification, namely scribing on an MRS solid plate to separate the lactic acid bacteria, preliminarily judging the type of the strain according to the characteristics of the colony shape, color, transparency, whether the edge is neat, whether the surface is smooth and the like of a single colony growing on the plate, then selecting the colony which is in accordance with the colony shape of the lactic acid bacteria and has bacteriostatic activity to perform gram staining, observing the morphological characteristics of bacteria by using a microscope, and judging whether the bacteria belong to gram positive bacteria or negative bacteria according to the color.

The method for obtaining the strain ZFM54 according to one embodiment, wherein the step (F) comprises the steps of: physiological and biochemical identification, namely performing an esculin hydrolysis experiment, a starch decomposition experiment, a sugar alcohol fermentation experiment, a catalase experiment, a hydrogen sulfide experiment, a gelatin hydrolysis experiment and a bacteria V-P experiment respectively.

The method for obtaining a bacterial species ZFM54 according to one embodiment, wherein the step (F) comprises the steps of: 16S rDNA-based molecular biological identification.

Drawings

Fig. 1 is a block diagram of a method for obtaining a bacterial species ZFM54 according to a preferred embodiment of the present invention.

FIG. 2 shows the growth state of the lactic acid bacteria isolation medium strain according to an embodiment of the present invention

Fig. 3 is a test chart of bacteriostatic activity according to an example of the present invention, excluding the influence of organic acids on bacteriostatic activity.

FIG. 4A colony morphology of Strain ZFM54

FIG. 4B is the gram stain test results

FIG. 5 is an electrophoretogram of 16S rDNA amplification product of ZFM54

FIG. 6 phylogenetic tree of 16S rDNA gene sequences of Strain ZFM54

Lactobacillus paracasei ZFM54(Lactobacillus casei ZFM54) accession information:

the preservation date is as follows: 2016 (11 months) and 23 days

The preservation unit: china center for type culture Collection

Address: wuhan university No. 16 Lojia mountain street in Wuhan City, Hubei province of China

The preservation number is: CCTCC NO M2016667

Detailed Description

The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art. The basic principles of the invention, as defined in the following description, may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.

Referring to fig. 1, a schematic block diagram of a method for obtaining a strain ZFM54 according to an embodiment of the present invention is shown. The invention provides a method for obtaining a strain ZFM54, wherein the strain ZFM54 is a preserved strain and is named after classification: lactobacillus paracasei ZFM54(Lactobacillus casei ZFM 54).

Preservation information:

the preservation date is as follows: 2016 (11 months) and 23 days

The preservation unit: china Center for Type Culture Collection (CCTCC)

Address: wuhan university of Wuhan, China

The preservation number is: CCTCC NO M2016667

The method for obtaining the strain ZFM54 comprises the following steps:

s0: preparing raw materials; mainly comprises the following aspects:

(a) determination of the origin of the species

The strain screened in the embodiment is derived from feces of newborn infants in obstetrical and gynecological hospitals affiliated to medical colleges of Zhejiang university. The indicator bacteria used in the bacteriostasis experiment are Micrococcus luteus 10209(Micrococcus luteus 10209), Staphylococcus aureus D48(Staphylococcus aureus D48), Listeria monocytogenes LM1 (Listerionocytogenes LM1), Escherichia coli DH5(Escherichia coli DH5), Salmonella typhimurium CMCC50015 (Salmonella typhimurium CMCC 50015) deposited in the laboratory.

(b) Culture medium

(1) MRS culture medium: 10g of beef extract, 20g of glucose, 10g of peptone, 5g of yeast extract, 3g of triammonium citrate, 2g of dipotassium phosphate, 5g of anhydrous sodium acetate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate and 801 mL of Tween, and the components are dissolved in ultrapure water and the volume is fixed to 1L. Adjusting the pH of the culture medium to 6.5, and autoclaving at 121 deg.C for 15 min. On the basis of the solid culture medium, 1.5 to 2 percent (W/V) of agar is added. The culture medium used for screening the lactic acid bacteria is additionally added with 2 percent of calcium carbonate.

(2) LB culture medium: 10g of sodium chloride, 10g of peptone and 5g of yeast extract, dissolving with ultrapure water, metering to 1L, adjusting the pH value of the culture medium to 7, and carrying out autoclaving at 121 ℃ for 15 min. 1.5 to 2 percent (W/V) of agar is added into the solid culture medium on the basis, and 1 to 1.2 percent (W/V) of agar is added into the semi-solid culture medium on the basis.

(c) Primary reagent

The bacteria micro-biochemical identification tube is purchased from Qingdao Haibo biotechnology limited; ammonium oxalate crystal violet, iodine solution with Roche, calcium carbonate, agarose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, absolute ethyl alcohol, hydrochloric acid, sodium hydroxide, catalase, trypsin, pepsin, proteinase K and a bacterial genome extraction kit are purchased from Biotechnology engineering (Shanghai) GmbH.

(d) Main instrument

TABLE 1 main apparatus for experiment

Further, the method for obtaining the strain ZMF54 comprises the following steps:

(S1): separation of lactic acid bacteria from infant feces

Under the aseptic condition, diluting the feces of the newborn infant with 1mL of sterilized normal saline, shaking to fully mix the feces uniformly, standing, taking 100 mu L of supernatant, coating the supernatant on an MRS solid culture medium plate added with 2% calcium carbonate, and inversely placing the plate in an incubator at 37 ℃ for anaerobic culture for 36-48 h. And selecting single colonies with good growth and obvious calcium soluble rings, respectively inoculating the single colonies in an MRS liquid culture medium for culture and numbering. And (3) repeatedly carrying out plate streaking separation on the bacterial liquid for several times, selecting a single bacterial colony, and carrying out gram staining and catalase test, wherein the bacterial colony with positive gram staining and negative catalase is primarily judged as the lactic acid bacteria.

(S2): activation and preservation of strains

The single colony of lactic acid bacteria is initially judged, inoculated into 10mL of MRS liquid culture medium and cultured for 24h at 37 ℃, and subcultured twice with the inoculum size of 1% (v/v). After activation, the strains are numbered and preserved, and 700 mu L of bacterial liquid is taken and 300 mu L of sterilized glycerol is added to preserve the strains at the temperature of minus 80 ℃.

(S3): cultivation of indicator bacteria

Marking the strains of the indicator bacteria preserved in a glycerin tube at the temperature of-80 ℃ on an LB solid culture medium plate respectively, culturing at the respective optimal temperature until obvious single colonies appear, selecting the single colonies, inoculating the single colonies in an LB liquid culture medium, and performing shaking culture on a shaker until the logarithmic phase (about 12h) is reached. The bacterial cell concentration was adjusted to OD600 of 0.6 with sterile physiological saline, and stored at 4 ℃ for further use.

(S4): primary sieve for lactic acid bacteria

Respectively inoculating the activated lactobacillus strains into 10mL of MRS liquid culture medium according to the proportion of 1% (v/v), standing and culturing at 37 ℃ for 24h, centrifuging at 8000r/min at 4 ℃ for 20min to obtain supernatant, and testing the antibacterial activity of the fermented supernatant by adopting an oxford cup agar diffusion method. Selecting the strain with the best bacteriostatic effect, and carrying out classification and identification for subsequent research. The specific method of the Oxford cup method bacteriostasis test is as follows:

(1) heating the sterilized LB semisolid culture medium to fully melt the culture medium, slightly shaking and uniformly mixing the culture medium and placing the culture medium in a 55 ℃ water bath kettle to keep the constant temperature to prevent agar from solidifying;

(2) uniformly placing the sterilized oxford cups on a disposable culture dish at intervals;

(3) uniformly mixing the indicator bacteria in a vortex oscillator, adding the indicator bacteria suspension into 15mL of semisolid culture medium according to the inoculation amount of 1% (v/v) after the LB semisolid culture medium is cooled to about 45 ℃, pouring the mixture into a culture dish after the mixture is sufficiently and uniformly mixed, slightly rotating the culture dish to uniformly cover the surface of the culture dish, and taking care not to pour the culture medium into an Oxford cup;

(4) after the semi-solid culture medium is completely cooled and solidified, carefully pulling out the Oxford cup by using sterilized tweezers to prepare a bacterium-containing flat plate with a cylindrical hole;

(5) after 100 microliter of sample to be detected is added into each hole, the flat plate is placed in a refrigerator at 4 ℃ to fully diffuse the sample for 4 hours, and then the sample is cultured for 12 hours according to respective optimal culture conditions of different indicator bacteria;

(6) the diameter of the zone was measured with a vernier caliper and recorded. Three groups of tests for bacteriostasis are performed in parallel.

(S5): bacteriocin-producing lactic acid bacteria rescreening

Besides bacteriocin, antibacterial active substances generated by the metabolism of lactic acid bacteria also comprise substances such as organic acid, hydrogen peroxide and the like, only lactic acid bacteria capable of generating antibacterial substances can be screened out by the Oxford cup method, and which substance plays a role in bacteriostasis cannot be determined, so that the antibacterial interference effect of the organic acid and the hydrogen peroxide on the indicating bacteria needs to be eliminated.

The method specifically comprises the following steps:

(S51) Elimination of interference of organic acids

Firstly, the pH value of ZFM54 fermentation supernatant is measured, then the pH value of the fermentation supernatant is adjusted to 5.0 by using 1M sodium hydroxide solution, an oxford cup agar diffusion method bacteriostasis test is carried out on Micrococcus luteus 10209 by using a sterile MRS culture medium which is adjusted to the same pH value by using lactic acid and acetic acid as a control, the diameter of a bacteriostasis ring is measured, and the differences of the bacteriostasis activities are compared.

(S52): eliminating interference of hydrogen peroxide

The fermentation supernatant was concentrated twice and then the pH was adjusted to the original pH to prepare a catalase working solution (solvent PBS buffer solution of pH 7.0) at a concentration of 5 mg/mL. Mixing the fermentation supernatant concentrated solution and catalase working solution according to the volume ratio of 1:1, carrying out water bath treatment at 37 ℃ for 2h, and testing the antibacterial activity of the mixture by adopting an Oxford cup method; meanwhile, the fermentation supernatant concentrated solution mixed by 1:1 and PBS buffer solution with pH7.0 are used as blank control, and the diameter of the inhibition zone is respectively measured and compared.

(S53): determination of bacteriocin-like substances (polypeptides)

Preparing 5mg/mL of enzyme solutions of pepsin, trypsin and proteinase K, respectively adjusting the pH values of the fermentation supernatants to the optimum pH values of the proteases (pepsin pH2.0, trypsin pH5.4 and proteinase K pH7.6), adding the corresponding enzyme solutions to make the final concentration be 1mg/mL, treating in a water bath kettle at 37 ℃ for 2h, and adjusting the initial pH values of the fermentation supernatants back. And taking the fermentation supernatant without protease treatment as a blank control, performing an antimicrobial test by adopting an Oxford cup method, measuring the diameter of an antimicrobial zone, and comparing for difference.

The method for obtaining the strain ZFM54 further comprises the following steps:

(S6): strain identification

According to the classification identification and experimental method of lactobacillus bacteria and Bojie's manual of systematic bacteriology, the selected strains with optimal antibacterial activity are subjected to colony thalli morphological identification and physiological and biochemical identification. Meanwhile, molecular biological identification is carried out based on the 16S rDNA gene.

(S61): morphological identification

The method comprises the steps of separating lactic acid bacteria on an MRS solid plate by scribing, preliminarily judging the type of a strain according to the characteristics of colony shape, color, transparency, neat edge, smooth surface and the like of a single colony growing on the plate, then selecting the colony which accords with the colony form of the lactic acid bacteria and has bacteriostatic activity to perform gram staining, observing the morphological characteristics of bacteria by using a microscope, and judging whether the bacteria belong to gram positive bacteria or negative bacteria according to the color.

Gram staining and microscopic examination:

(1) tabletting: dropping a drop of physiological saline on a clean glass slide, picking out a single bacterial colony by using an inoculating loop, uniformly mixing the single bacterial colony with the physiological saline, smearing the mixture into a thin layer with the diameter of about 1cm, and then smearing the mixture into a smear;

(2) fixing: heating and fixing the glass slide by passing the glass slide above the flame of the alcohol lamp for one to two times;

(3) dyeing: dropping a drop of ammonium oxalate crystal violet on the smear area, staining for 1min, and carefully washing with sterile water;

(4) mordant dyeing: adding one drop of Luoge iodine solution, washing with sterile water after 1min, and drying with a piece of lens wiping paper;

(5) and (3) decoloring: the glass slide is inclined, 95% ethanol is added into the glass slide for decolorization by a dropper flow until no purple fall off, and then the glass slide is washed by sterile water;

(6) counterdyeing: 0.5% safranine is dripped to be dyed for one drop, and after dyeing is carried out for 30s, washing is carried out;

(7) microscopic examination: the water was blotted with a paper lens and observed with an oil lens.

(S62): physiological and biochemical identification

(1) Hydrolysis test of esculin

Some bacteria can decompose and utilize esculin to produce escin, which can react with ferrous ions in the medium to form black compounds. The experiment was carried out using bacterial micro biochemical reaction tubes, and the same colony that had been purified and cultured was picked from the plate with an inoculating loop and inoculated into the biochemical tube of the esculin assay. After inoculation, the mixture is statically placed in a constant temperature box at 37 ℃ for 24 hours (the mixture is sealed by a sealing film, the sealing film is wiped by 75% alcohol for sterilization), if black appears, the esculin is hydrolyzed, and the positive reaction is obtained, otherwise, the negative reaction is obtained.

(2) Starch decomposition test

Taking out the strain from-80 ℃, streaking and activating the strain on a MRS solid culture medium plate, picking out a single colony by using an inoculating loop, inoculating the single colony in an MRS liquid culture medium containing 0.5% of starch, and standing in a 37 ℃ incubator for culturing for 24 hours. Then, a few drops of iodine solution were added to the culture solution and shaken up to observe whether the color changed. If the color does not change, the starch is completely hydrolyzed, and the reaction is positive, and if the color changes to blue or bluish purple, the starch is not completely hydrolyzed, and the reaction is negative.

(3) Sugar alcohol fermentation test

The experiment was performed using a micro biochemical reaction tube for each sugar. Inoculating single colony from the plate with inoculating loop into glucose trace biochemical reaction tube, fructose trace biochemical reaction tube, cellobiose trace biochemical reaction tube, maltose trace biochemical reaction tube, sucrose trace biochemical reaction tube, melibiose trace biochemical reaction tube, raffinose trace biochemical reaction tube, lactose trace biochemical reaction tube, mannitol trace biochemical reaction tube and sorbitol trace biochemical reaction tube, standing in 37 deg.C incubator for 24h (sealing with sealing film, and wiping sealing film with 75% alcohol for sterilization), and observing the result. If the color of the reaction tube is yellow, acid production is indicated, and the reaction tube is a positive reaction, otherwise, the reaction tube is a negative reaction.

(4) Catalase assay

The strain is taken out from minus 80 ℃, streaked and activated on a MRS solid culture medium plate, and placed at 37 ℃ for static culture for 24 hours. A single colony was then picked and smeared onto a glass slide and a few drops of 5% hydrogen peroxide solution were added dropwise to the smear area. If the bacteria can produce catalase, the hydrogen peroxide is decomposed by the enzyme to generate bubbles, and the reaction is positive, otherwise, the reaction is negative.

(5) Hydrogen sulfide test

Some bacteria are capable of decomposing sulfur-containing amino acids such as cystine, cysteine, etc. and producing hydrogen sulfide. The hydrogen sulfide can react with the ferric salt in the culture medium to generate black ferric sulfide precipitate. And selecting a single colony from the flat plate by using an inoculating loop, inoculating the single colony into a trace biochemical reaction tube for a hydrogen sulfide test, standing in a constant temperature box at 37 ℃ for culturing for 24h (sealing by using a sealing film, wiping and sterilizing the sealing film by using 75% alcohol), observing the result, and judging the result as a positive reaction if black is shown, or else, judging the result as a negative reaction.

(6) Gelatin hydrolysis test

This experiment was used to determine whether the bacteria could produce gelatinase to hydrolyze gelatin into polypeptides. The experiment was performed using a micro biochemical reaction tube. A single colony is picked from a flat plate by using an inoculating loop and inoculated into a trace biochemical reaction tube for a gelatin hydrolysis test, the micro biochemical reaction tube is statically placed in a constant temperature box at 37 ℃ for culturing for 24 hours (the opening of the micro biochemical reaction tube is sealed by a sealing film, and the sealing film is wiped and sterilized by 75% alcohol), and the gelatin hydrolysis condition is observed.

(7) Bacterial V-P assay

This experiment was used to test the ability of bacteria to produce acetomethyl methanol. Certain bacteria are capable of breaking down glucose to ultimately produce acetomethyl carbinol. Under alkaline conditions, the acetyl methyl methanol is oxidized into diacetyl, and then combined with guanidino substances such as arginine in the culture medium to form red compounds, namely, the V-P test is positive. The experiment was performed using a micro biochemical reaction tube, and a single colony was picked from the plate with an inoculating loop and inoculated into the micro biochemical reaction tube. After inoculation, the mixture is stood in a thermostat at 37 ℃ for 24 hours (the mixture is sealed by a sealing film, the sealing film is wiped by 75% alcohol for sterilization), then VP A solution and VP B solution are dripped, and the red color is positive reaction, otherwise, the red color is negative reaction.

(S63): 16S rDNA-based molecular biology identification

(1) Extraction of lactic acid bacteria genomic DNA

Extracting the whole bacterial genome by using an Ezup column type bacterial genome DNA extraction kit. Before the experiment is started, adding a corresponding amount of isopropanol into PW Solution according to requirements, uniformly mixing, adding a corresponding amount of absolute ethyl alcohol into Wash Solution, uniformly mixing, making a label on a bottle body, and sealing and storing at room temperature. Before use, the Buffer diagnostic was checked for precipitation and, if so, dissolved at 56 ℃ before use.

1) Taking the strains stored at minus 80 ℃ out of a MRS solid culture medium plate, streaking and activating, selecting a single colony after the colony grows out, inoculating the single colony into 10mL of MRS liquid culture medium, and culturing in a constant temperature incubator at 37 ℃ for 24 hours;

2) shaking the cultured bacterial liquid on a vortex oscillator to fully and uniformly mix the bacteria, sucking 1mL of bacterial liquid into a 1.5mLEP tube, centrifuging at 8000r/min for 1min, and discarding the supernatant. mu.L of lysozyme solution (prepared as a 20mg/mL lysozyme solution with the corresponding lytic enzyme added to the enzyme lysis buffer before use) was added to the suspension and the solution was allowed to stand for one hour at 37 ℃. Add 20. mu.L of protease K solution, shake and mix well. Carrying out water bath at 56 ℃ for 30min until the cells are completely lysed;

3) adding 200 mu L of Buffer BD, fully reversing and uniformly mixing;

4) adding 200 μ L of anhydrous ethanol, fully reversing and mixing;

5) putting an adsorption column into a collecting pipe, sucking all the solution into the adsorption column by using a liquid transfer device, standing for 2min, centrifuging at 12000r/min for 1min, and pouring waste liquid in the collecting pipe;

6) putting the adsorption column back into the collection tube, adding 500 μ L PW Solution, centrifuging at 10000r/min for 30s, and pouring off the filtrate in the collection tube;

7) the adsorption column is put back into the collection tube, 500 mu L of Wash Solution is added, 10000r/min is centrifuged for 30s, and the filtrate in the collection tube is poured out.

8) The column was returned to the collection tube and centrifuged at 12000r/min for 2min to remove the remaining Washsolution. Opening the cover of the adsorption column, and standing at room temperature for several minutes to thoroughly dry the Washsolution remained in the adsorption material so as to avoid influencing the yield of the genome DNA and subsequent experiments;

9) the adsorption column was taken out and placed in a new 1.5mL EP tube, 100. mu.L of CE Buffer was added and left to stand for 3min, and centrifuged at 12000r/min for 2min, and the DNA solution was collected and stored at-20 ℃ for further experiments.

(2) Agarose gel electrophoresis

1) Preparing glue: weighing 0.2g of agarose, adding 40mL of 1 XTAE solution, mixing, heating for 40s until the agarose is completely dissolved, cooling to about 60 ℃ at room temperature, adding 1uL of ethidium bromide, fully mixing, pouring into a gel preparation tank, inserting a comb, cooling completely, solidifying, and carefully pulling out the comb.

2) Loading: the prepared gel was placed in an electrophoresis tank such that the 1 × TAE solution in the tank was submerged through the entire gel. 1uL 10 XLoading buffer is taken on a plastic film, 5uL of the extracted DNA sample is taken to be evenly mixed with the 10 Xloading buffer, the sample is carefully driven into a gel hole, and 3uL 10000bp DNA Marker is added into a groove hole on the edge. The glue is punctured by a careful gun head during sample loading, taking care not to have air bubbles.

3) Electrophoresis and imaging: electrophoresis is carried out for about 45min under the constant voltage of 80V, and the electrophoresis is finished when the blue strip runs to two thirds of the whole gel. The gel was removed and photographed in a gel imager to view the results.

(3) PCR amplification

The PCR amplification is carried out by using a universal primer of lactobacillus 16S rDNA and synthesizing by biological engineering (Shanghai) company Limited.

27F(5′-AGAGTTTGATCCTGGCTCAG-3′)

1492R(5′-GGTTACCTTGTTACGACTT-3′)

The PCR amplification reaction system is shown in Table 2 below:

TABLE 2 PCR amplification reaction System

TABLE 3 PCR cycling conditions

The products after PCR amplification are recovered by using a SanPrep column type DNA glue recovery kit, and the synthesis of lactobacillus 16S rDNA universal primers and the sequencing of PCR product sequences are all completed by the company of Biotechnology engineering (Shanghai).

(4)16S rDNA homology analysis and construction of phylogenetic tree

The sequence obtained by sequencing is submitted to a GenBank database of NCBI for BLAST analysis, strains with the highest homology with the sequence of the tested strain 16S rDNA are screened, the sequences of the strains are extracted, a phylogenetic tree is constructed by bioinformatics software MEGA6.0, and phylogenetic analysis is carried out to determine the species relationship of the separated lactobacillus strains.

(S7): analysis of results

Separating and primary screening of bacteriocin-producing lactic acid bacteria

The feces of newborn infants in maternal and child care hospitals in Hangzhou city, Zhejiang province are used as samples, and lactic acid bacteria capable of producing calcium lysozyme are obtained by screening, as shown in figure 2. 4 single colonies with obvious calcium ring and single colony morphology are selected and inoculated in an MRS liquid culture medium and are respectively numbered as S1, S2, S3 and S4.

The bacteriostatic activity of the 4 lactic acid bacteria on indicator bacteria such as escherichia coli DH5 α and micrococcus luteus 10209 is detected by an oxford cup agar diffusion method, and the result is shown in table 4.

TABLE 4 bacteriostatic action of Lactobacillus fermentation broth on indicator bacteria

Note: the diameter of the oxford cup is 8.0 mm; "-" indicates no zone of inhibition

Rescreening of bacteriocin-producing lactic acid bacteria

A lactobacillus strain ZFM54 with a wider bacteriostatic spectrum is obtained through preliminary screening, in order to avoid interference of substances such as organic acids, hydrogen peroxide and the like generated by lactobacillus metabolism on bacteriostatic effect, the bacteriostatic action of bacteriocin substances is determined, and the interference factors are eliminated one by one through secondary screening experiments.

Eliminating the interference of organic acid on the bacteriostatic activity

Adjusting the pH of the fermentation supernatant of lactobacillus ZFM54 to 5.0 with 1M sodium hydroxide solution, adjusting the pH of the uninoculated MRS liquid culture medium to 5.0 with lactic acid and acetic acid, and performing bacteriostatic activity test with the fermentation supernatant (pH3.78) without pH adjustment as a control, wherein the indicator is Micrococcus luteus 10209, and the result is shown in FIG. 3, wherein 1 in FIG. 3 is ZFM54 fermentation supernatant; 2 is the fermentation supernatant at ph 5.0; 3 is lactic acid MRS at ph 5.0; 4 is MRS acetate at pH 5.0. The results of bacteriostatic experiments show that after the pH of the fermentation supernatant is adjusted to 5.0, the bacteriostatic zone has lower transparency and smaller bacteriostatic zone than that of the fermentation stock solution, but still has good bacteriostatic activity. While the lactic acid MRS and acetic acid MRS with the pH value adjusted to 5.0 have no bacteriostatic circle, and the experimental result shows that other non-acidic substances in the fermentation supernatant liquid play a bacteriostatic role.

Exclusion of H₂O₂Interference on bacteriostatic activity of lactic acid bacteria

Treating the fermentation supernatant of ZFM54 with catalase, and comparing with the fermentation stock solution, wherein the antibacterial effect of the catalase and ZFM54 on Micrococcus luteus is not significantly changed, and the size and transparency of the antibacterial zone are almost the same, thereby eliminating H₂O₂The bacteriostasis further indicates that other bacteriostatic active substances exist in the fermentation supernatant.

Effect of protease treatment on bacteriostatic Activity of fermentation supernatant

The bacteriostatic activity of the fermented supernatant of the strain ZFM54 after being treated by pepsin, trypsin and proteinase K is shown in Table 5. The observation shows that the bacteriostatic substance of the strain ZFM54 has different sensibility to different proteases, and the diameter of the bacteriostatic ring is obviously reduced and most of bacteriostatic activity is lost after the treatment of trypsin and proteinase K; after being treated by pepsin, the diameter of a bacteriostatic zone of the antibacterial fiber is slightly reduced, the transparency is reduced, and a small part of bacteriostatic activity is lost. The results preliminarily judge that the main antibacterial substance in the fermentation supernatant of the strain ZFM54 is a protein, polypeptide substance or not a single substance.

Table 5 effect of different protease treatments on the bacteriostatic activity of strain ZFM54

Note: the diameter of the oxford cup is 8.00 mm; the same letters in the same column indicate no significant difference (P > 0.05), and the different letters in the same column indicate significant difference (P < 0.05).

(S8) identification of bacteriocin-producing lactic acid bacteria species

(S81) morphological identification of bacterial colonies

The strain ZFM54 can be seen to grow well on an MRS solid medium plate through plate scribing separation, bacterial colonies are in a circular convex shape and have regular edges, the size of the bacterial colonies is 0.5-2.0 mm, the bacterial colonies are milky white, the surface of the bacterial colonies is smooth and opaque, and the bacterial colonies have typical growth characteristics of lactic acid bacteria (such as the bacterial colonies of the strain ZFM54 shown in figure 4A); the gram staining result is positive; the shape of the bacteria observed under an optical microscope is rod-shaped, without flagella and spores (as shown in FIG. 4B, gram stain result), and the colony shape and the gram stain microscopic result are consistent with the characteristics of lactobacillus.

(S82) physiological and biochemical identification of bacteria

Bacteria can perform various complex metabolic reactions in the process of growth and propagation, and enzyme catalysis is required in the reaction process. Different bacterial metabolic pathways generate different enzymes to catalyze the reaction, so that the metabolic types and products are different, and the bacterial species can be identified by biochemical detection. The results of physiological and biochemical tests of strain ZFM54 are shown in table 6.

TABLE 6 physiological and biochemical characteristics of Strain ZFM54

Note: "+" represents positive; "-" represents negativity

Strain ZFM54 warp H₂O₂The results of the enzyme test, the starch hydrolysis test, the hydrogen sulfide production test and the gelatin liquefaction test are negative, which indicates that the bacterium can not secrete amylase to degrade macromolecular substances and can not produce hydrogen sulfide and hydrogen peroxide in the growth and metabolism processes. In the decomposition experiments of various saccharides, ZFM54 can ferment various sugar alcohols such as glucose, fructose, maltose, mannitol, sorbitol and the like, and the result is positive; but acid production such as melibiose, raffinose and arabinose can not be fermented, and the result is negative. In addition, the bacterium can decompose esculin and the V-P reaction is negative, which substantially corresponds to the characteristics of the lactic acid bacteria population. By combining the morphological, physiological and biochemical characteristics, the strain ZFM54 is preliminarily identified as lactobacillus paracasei by referring to the lactic acid bacteria classification identification and experimental method and the common bacteria system identification handbook. However, because the Lactobacillus paracasei has close relativity with certain subspecies of Lactobacillus casei and Lactobacillus rhamnosus, and is difficult to distinguish by using the traditional fermentation characteristics, the identification needs to be further combined with a 16S rDNA method.

(S83): molecular biological identification of lactic acid bacteria

(1) Lactobacillus ZFM54 genome extraction and 16S rDNA PCR amplification

Extracting total DNA of lactobacillus ZFM54 by using an Ezup column type bacterial genome DNA extraction kit, performing PCR amplification by using the total DNA of lactobacillus ZFM54 as a template and using a lactobacillus 16S rDNA universal primer, and performing electrophoresis on an amplification product under 1% agarose gel to obtain a strip of about 1500bp, wherein the electrophoresis result is shown in figure 5. The 16S rDNA of lactic acid bacterium ZFM54 was sequenced by bio-engineering (shanghai) ltd.

(2) BLAST homology comparison and construction of phylogenetic Tree

The 16S rDNA sequence of lactobacillus ZFM54 obtained by sequencing was submitted to the GenBank database of NCBI for BLAST alignment, 16S rDNA gene sequences of 10 strains were selected, and MEGA6.0 software was used for multiple sequence alignment and phylogenetic tree construction, see fig. 6. In a phylogenetic tree, a strain ZFM54 and Lactobacillus paracasei are in the same branch, and the strain is identified as Lactobacillus paracasei by combining the morphological, physiological and biochemical identification of bacteria, and is named as Lactobacillus casei ZFM54, which is called ZFM54 for short.

Lactic acid bacteria are important probiotics in human intestinal tracts, and the quantity and the composition of the lactic acid bacteria play a vital role in maintaining the microecological balance of hosts and improving the functions of immune systems. Since the growth environment of exogenous probiotics is far from the environment of the human gastrointestinal tract, many scholars consider that the ideal probiotics are preferably from the human gastrointestinal tract itself. Researches show that the feces of infants contain probiotics, and the probiotics can effectively regulate intestinal flora and increase the content of short-chain fatty acid in intestinal tracts of mice and feces of human bodies.

In the embodiment of the invention, 4 lactic acid bacteria are separated from newborn infant excrement through calcium solution ring, colony morphology observation and gram staining experiments, the 4 lactic acid bacteria are preliminarily screened by respectively testing the antibacterial activity by an Oxford cup agar diffusion method, and an advantageous strain ZFMM 54 which has antibacterial effect and strongest antibacterial activity on gram-positive bacteria such as micrococcus luteus 10209, Listeria monocytogenes LM1 and the like and gram-negative bacteria such as Escherichia coli DH5 α, Salmonella typhimurium CMCC50015 and the like is screened from the 4 lactic acid bacteria₂O₂The substances cannot be shown to play a bacteriostatic action by the bacteriocin substances only through an Oxford cup method bacteriostatic test, so a re-screening test needs to be designed to eliminate organic acids and H₂O₂The interference of (2). The bacterial inhibition effect of the strain ZFM54 is not determined by organic acid and H through exclusion test₂O₂But causes other bacteriostatic substances to act. Through the stomachProtease, trypsin and proteinase K treatment of strain ZFM54 fermentation supernatant found: the bacteriostatic substance in the ZFM54 fermented supernatant is very sensitive to trypsin and proteinase K, and the pepsin can also make the bacteriostatic substance lose partial activity, so the substance which generates bacteriostatic effect in ZFM54 is preliminarily identified to be a protein or polypeptide.

Different microorganisms have different abilities to decompose and utilize carbohydrate, fat and protein substances, so the physiological and biochemical reactions of bacteria are important bases for strain identification. In the embodiment of the invention, the screened ZFM54 strain is identified to be Lactobacillus paracasei by combining morphological identification, physiological and biochemical identification and 16S rDNA homology analysis, and is named as Lactobacillus paracasei ZFM54(Lactobacillus casei ZFM 54).

It will be appreciated by persons skilled in the art that the embodiments of the invention described above and shown in the drawings are given by way of example only and are not limiting of the invention. The objects of the invention have been fully and effectively accomplished. The functional and structural principles of the present invention have been shown and described in the examples, and any variations or modifications of the embodiments of the present invention may be made without departing from the principles.

Claims

1. A method for obtaining a strain ZFM54 is characterized by comprising the following steps:

(A) separating lactic acid bacteria from the infant feces;

(B) activating and preserving strains;

(C) culturing indicator bacteria;

(D) primary screening of lactic acid bacteria;

(E) re-screening the bacteriocin-producing lactic acid bacteria; and

(F) and (5) identifying strains.

2. The method for obtaining bacterial strain ZFM54, according to claim 1, wherein in step (A), under aseptic conditions, the feces of newborn baby is diluted with 1mL of sterilized normal saline, shaken to be fully mixed, then kept still, 100 μ L of supernatant is spread on MRS solid medium plate added with 2% calcium carbonate, and the plate is placed upside down in a 37 ℃ incubator for anaerobic culture for 36-48 h.

3. The method for obtaining strain ZFM54, according to claim 1, wherein in step (B), single colony of lactic acid bacteria is initially determined, inoculated into 10mL MRS liquid medium and cultured at 37 ℃ for 24h, subcultured twice with 1% (v/v) inoculum size, numbered for conservation after activation, 700. mu.L of bacterial liquid is added with 300. mu.L of sterilized glycerol, and the strain is preserved at-80 ℃.

4. The method for obtaining strain ZFM54, according to claim 3, wherein MRS medium: 10g of beef extract, 20g of glucose, 10g of peptone, 5g of yeast extract, 3g of triammonium citrate, 2g of dipotassium phosphate, 5g of anhydrous sodium acetate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate and 801 mL of Tween, dissolving in ultrapure water, fixing the volume to 1L, adjusting the pH value of a culture medium to 6.5, and carrying out autoclaving at 121 ℃ for 15 min.

5. The method for obtaining bacterial species ZFM54, according to claim 3, wherein in step (C), the indicator bacterial species preserved in-80 ℃ glycerin tube are streaked on LB solid medium plate, cultured at respective optimum temperature until obvious single colony appears, and the single colony is picked and inoculated in LB liquid medium for shaking culture to logarithmic phase.

6. The method for obtaining strain ZFM54, according to claim 5, wherein the ratio of LB medium: 10g of sodium chloride, 10g of peptone and 5g of yeast extract, dissolving with ultrapure water, metering to 1L, adjusting the pH value of the culture medium to 7, and carrying out autoclaving at 121 ℃ for 15 min.

7. The method for obtaining bacterial strain ZFM54, according to claim 1, wherein in step (D), the activated lactobacillus strain is inoculated into 10mL MRS liquid culture medium according to 1% ratio, after static culture at 37 ℃ for 24h, the activated lactobacillus strain is centrifuged at 8000r/min at 4 ℃ for 20min to obtain supernatant, and the fermented supernatant is tested for bacteriostatic activity by Oxford cup agar diffusion method.

8. The method for obtaining strain ZFM54 according to claim 7, wherein the Oxford cup agar diffusion test method comprises the steps of: (1) heating the sterilized LB semisolid culture medium to fully melt the culture medium, slightly shaking and uniformly mixing the culture medium and placing the culture medium in a 55 ℃ water bath kettle to keep the constant temperature to prevent agar from solidifying; (2) uniformly placing the sterilized oxford cups on a disposable culture dish at intervals; (3) uniformly mixing the indicator bacteria in a vortex oscillator, adding the indicator bacteria suspension into 15mL of semisolid culture medium according to the inoculation amount of 1% (v/v) after the LB semisolid culture medium is cooled to about 45 ℃, pouring the mixture into a culture dish after the mixture is sufficiently and uniformly mixed, slightly rotating the culture dish to uniformly cover the surface of the culture dish, and taking care not to pour the culture medium into an Oxford cup; (4) after the semi-solid culture medium is completely cooled and solidified, carefully pulling out the Oxford cup by using sterilized tweezers to prepare a bacterium-containing flat plate with a cylindrical hole; (5) after 100 microliter of sample to be detected is added into each hole, the flat plate is placed in a refrigerator at 4 ℃ to fully diffuse the sample for 4 hours, and then the sample is cultured for 12 hours according to respective optimal culture conditions of different indicator bacteria; (6) the diameter of the zone was measured with a vernier caliper and recorded.

9. The method for obtaining a bacterial species ZFM54 as claimed in claim 1, wherein said step (E) comprises the steps of: eliminating the interference of organic acid, firstly measuring the pH value of ZFM54 fermentation supernatant, then adjusting the pH value of the fermentation supernatant to 5.0 by using 1M sodium hydroxide solution, carrying out an oxford cup agar diffusion method bacteriostasis test on Micrococcus luteus 10209 by using a sterile MRS culture medium which is adjusted to the same pH value by using lactic acid and acetic acid as a control, measuring the diameter of a bacteriostasis zone, and comparing the differences of bacteriostasis activities.

10. The method for obtaining a bacterial species ZFM54 as claimed in claim 1, wherein said step (E) comprises the steps of: eliminating the interference of hydrogen peroxide, concentrating twice the fermentation supernatant, adjusting the pH value to the original pH value of the fermentation supernatant, preparing catalase working solution with the concentration of 5mg/mL, mixing the fermentation supernatant concentrated solution and the catalase working solution according to the volume ratio of 1:1, carrying out water bath treatment at 37 ℃ for 2h, and testing the bacteriostatic activity of the fermentation supernatant by adopting an oxford cup method; meanwhile, a 1:1 mixed fermentation supernatant concentrated solution and a PBS buffer solution with the pH value of 7.0 are used as blank controls, and the diameter of the inhibition zone is respectively measured and compared.

11. The method for obtaining a bacterial species ZFM54 as claimed in claim 1, wherein said step (F) comprises the steps of: determining bacteriocin substances, preparing 5mg/mL of enzyme solution of pepsin, trypsin and proteinase K, respectively adjusting the pH value of fermentation supernatant to the optimum pH value of each proteinase, then adding corresponding enzyme solution to make the final concentration of the enzyme solution be 1mg/mL, treating the enzyme solution in a water bath kettle at 37 ℃ for 2 hours, then adjusting the initial pH value of the fermentation supernatant, taking the fermentation supernatant which is not treated by the proteinase as a blank control, carrying out a bacteriostasis test by adopting an Oxford cup method, measuring the diameter of a bacteriostasis zone, and comparing the diameter and the difference.

12. The method for obtaining a bacterial species ZFM54, as claimed in claim 1, wherein said step (F) comprises the steps of: and (3) morphological identification, namely scribing on an MRS solid plate to separate the lactic acid bacteria, preliminarily judging the type of the strain according to the characteristics of the colony shape, color, transparency, whether the edge is neat, whether the surface is smooth and the like of a single colony growing on the plate, then selecting the colony which is in accordance with the colony shape of the lactic acid bacteria and has bacteriostatic activity to perform gram staining, observing the morphological characteristics of bacteria by using a microscope, and judging whether the bacteria belong to gram positive bacteria or negative bacteria according to the color.

13. The method for obtaining a bacterial species ZFM54 as claimed in claim 11, wherein said step (F) comprises the steps of: physiological and biochemical identification, namely performing an esculin hydrolysis experiment, a starch decomposition experiment, a sugar alcohol fermentation experiment, a catalase experiment, a hydrogen sulfide experiment, a gelatin hydrolysis experiment and a bacteria V-P experiment respectively.

14. The method for obtaining strain ZFM54, according to claim 13, wherein the aesculin hydrolysis assay method is: the experiment is carried out by using a bacterial micro biochemical reaction tube, the same bacterial colony which is purified and cultured is selected from a flat plate by using an inoculating loop to be inoculated into the biochemical tube of the esculin test, the inoculated bacterial colony is placed in a constant temperature box at 37 ℃ for culturing for 24 hours after being inoculated, if black appears, the esculin is hydrolyzed, the reaction is a positive reaction, and otherwise, the reaction is a negative reaction.

15. The method for obtaining strain ZFM54, according to claim 13, wherein the amylolysis assay is: taking out the strain from-80 ℃, streaking and activating the strain on a MRS solid culture medium plate, picking out a single colony by using an inoculating loop, inoculating the single colony in an MRS liquid culture medium containing 0.5% of starch, and standing in a 37 ℃ incubator for culturing for 24 hours. And adding a few drops of iodine solution into the culture solution, shaking uniformly, observing whether the color is changed or not, wherein if the color is not changed, the starch is completely hydrolyzed, and the reaction is positive, and if the color is changed to blue or bluish purple, the starch is not completely hydrolyzed, and the reaction is negative.

16. The method for obtaining strain ZFM54, according to claim 13, wherein the sugar alcohol fermentation test method is: inoculating single colony from the plate with inoculating loop to glucose micro biochemical reaction tube, fructose micro biochemical reaction tube, cellobiose micro biochemical reaction tube, maltose micro biochemical reaction tube, sucrose micro biochemical reaction tube, melibiose micro biochemical reaction tube, raffinose micro biochemical reaction tube, lactose micro biochemical reaction tube, mannitol micro biochemical reaction tube and sorbitol micro biochemical reaction tube, standing in 37 deg.C incubator, culturing for 24 hr, and observing result. If the color of the reaction tube is yellow, acid production is indicated, and the reaction tube is a positive reaction, otherwise, the reaction tube is a negative reaction.

17. The method for obtaining strain ZFM54, as claimed in claim 13, wherein the catalase test method is: the strain is taken out from minus 80 ℃, streaked and activated on a MRS solid culture medium plate, and placed at 37 ℃ for static culture for 24 hours. Then picking a single colony and smearing the single colony on a glass slide, dripping a few drops of 5% hydrogen peroxide solution on the smear area, if bacteria can produce catalase, decomposing the hydrogen peroxide by enzyme to generate bubbles, and performing positive reaction, otherwise performing negative reaction.

18. The method for obtaining a bacterial species ZFM54, as claimed in claim 12, wherein said step (F) comprises the steps of: 16S rDNA-based molecular biological identification.

19. The method for obtaining strain ZFM54, according to claim 18, wherein the 16S rDNA based molecular biology characterization comprises: (1) extracting lactobacillus genome DNA; (2) performing agarose gel electrophoresis; (3) PCR amplification; (4)16S rDNA homology analysis and construction of phylogenetic tree.

20. The method for obtaining strain ZFM54 as claimed in any one of claims 1 to 19, wherein said strain ZFM54 has bacteriostatic effect against micrococcus luteus 10209, listeria monocytogenes LM1 gram-positive bacteria and escherichia coli DH5, salmonella typhimurium CMCC50015 gram-negative bacteria.

21. The method for obtaining the strain ZFM54, according to any of claims 1-19, wherein the strain ZFM54 has a bacteriostatic effect on a protein or polypeptide.