CN113846034A

CN113846034A - Lysobacter enzymogenes L-43 and application thereof

Info

Publication number: CN113846034A
Application number: CN202111255172.XA
Authority: CN
Inventors: 王志新; 贾英民; 宁亚维; 贾紫伟; 刘洋; 封成玲
Original assignee: Hebei University of Science and Technology
Current assignee: Hebei University of Science and Technology
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2021-12-28
Anticipated expiration: 2041-10-27
Also published as: CN113846034B

Abstract

The invention discloses a lysobacter enzymogenes L-43 and application thereof, wherein the lysobacter enzymogenes L-43 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms No. 3 of Navy No. 1 Hospital No. 3 in the south of Chaozhou, Beijing, 1 month and 5 days in 2021, and the preservation number is CGMCC NO. 21593; the bacteria can produce antibacterial peptide by fermentation. The method is suitable for separating the lysobacter enzymogenes L-43, the lysobacter enzymogenes L-43 obtained by separation can generate the antibacterial peptide, and the generated antibacterial peptide can be further applied to the fields of food preservation, animal nonreactive breeding and the like.

Description

Lysobacter enzymogenes L-43 and application thereof

Technical Field

The invention belongs to the field of bioengineering, and relates to a strain, in particular to lysobacter enzymogenes L-43 and application thereof.

Background

The microbial antibacterial substance has the characteristics of low toxicity, high efficiency, stability, no residue, short production period, low cost and the like, and is gradually a research hotspot in the fields of food, agriculture, medicine and the like; along with the rapid development of biotechnology, more and more natural antibacterial substances are produced by taking microorganisms and metabolites thereof as raw materials and utilizing technologies such as fermentation, extraction and the like;

at present, antibacterial substances from microorganisms have bioactivity on bacteria, fungi, viruses and even tumor cells, and are widely applied to various fields of food, agriculture, medical treatment and the like due to the characteristics of high stability, difficult generation of drug resistance, good inhibition effect and the like, so the development and application prospects are good; the microorganisms have huge groups in nature, can be fully excavated, and various strains with antibacterial properties are screened out from the groups, so that strain resources are provided for the development of novel antibacterial substances;

the lysobacter is Proteobacteria (Proteobacteria), Gamma-Proteobacteria (Gamma-Proteobacteria), Xanthomonas (Xanthomonadales), Xanthomonas (Xanthoonaceae); currently, about 50 kinds of reported Lysobacter bacteria are known, and four typical species among them are Lysobacter enzymogenes (Lysobacter enzymogenes), Lysobacter antibioticus (Lysobacter antibioticus), Lysobacter collodionalis (Lysobacter gummosus), and Lysobacter palmatus (Lysobacter palmescens), have high antibacterial activity and plant disease control potential, and are a new type of plant disease control bacteria; the bacteria have obvious antagonistic action on plant pathogenic fungi, oomycetes, gram-negative bacteria, positive bacteria and nematodes, and the action mechanism is as follows: (1) producing small molecule antibacterial secondary metabolites; (2) secretion to produce large amounts of extracellular enzymes including chitinase, beta-1, 3-glucan, protease and cellulase; (3) inducing plants to generate disease resistance; (4) better colonization ability;

in conclusion, the novel lysobacter strain with the bacteriostatic activity is separated and identified, so that a high-quality resource can be provided for the microbial antibacterial substance, and the novel lysobacter strain has great potential for developing novel, efficient and nontoxic natural medicines.

Disclosure of Invention

The invention aims to provide lysobacter enzymogenes L-43 so as to achieve the purposes of high growth speed, good adaptability and low fermentation cost, and the fermentation product can effectively resist gram-positive bacteria;

another object of the present invention is to provide the use of the above described lysobacter enzymogenes L-43.

In order to achieve the purpose, the invention adopts the technical scheme that:

the zymogenic bacillus L-43 is preserved in the common microbe center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No. 1 of Beijing, sunward, the preservation date is 2021 year, 1 month and 5 days, the preservation number is CGMCC NO.21593, and the Latin literature name is Lysobacter enzymogens.

As a limitation, the 16SrRNA gene sequence of the lysobacter enzymogenes L-43 is as follows:

GGCTCAGAGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGCAGCACAGAGGAGCTTGCTCCTTGGGTGGCGAGTGGCGGACGGGTGAGGAATACGTCGGAATCTGCCTATTTGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGTGGGGGACCTTCGGGCCTCACGCAGATAGATGAGCCGACGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAAGCTTAGGGTTAATAACCCTGAGTCATGACGGTACCGGAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTTTGTTAAGTCTGATGTGAAAGCCCTGGGCTCAACCTGGGAATGGCATTGGAAACTGGCTTACTAGAGTGCGGTAGAGGGTAGCGGAATTCCCGGTGTAGCAGTGAAATGCGTAGATATCGGGAGGAACATCCGTGGCGAAGGCGGCTACCTGGACCAGCACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGGGCAACTTGGCCCTCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGAAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGCAGAACCTTACCTGGCCTTGACATGTCGAGAACTTGGCAGAGATGCCTTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGT。

the invention also provides an application of the lysobacter enzymogenes L-43, and the lysobacter enzymogenes L-43 is used for food preservation or animal nonreactive breeding.

The present invention also provides another use of the above lysobacter enzymogenes L-43 for producing an antibacterial peptide by fermentation.

As a limitation, the fermentation comprises the following raw materials of effective components of a fermentation medium per 100g by weight: 0.5-2.0 parts of carbon source, 0.5-1.5 parts of nitrogen source, 0.05-0.15 part of inorganic salt and the balance of water.

As a further limitation, the carbon source is at least one of maltose, fructose, and liquid sugar; the nitrogen source is tryptone; the inorganic salt is magnesium sulfate or magnesium chloride.

As another limitation, the initial pH value of the fermentation is 6.0-7.0, the temperature is 30-32 ℃, the rotation speed is 180-.

The invention also provides an application of the antibacterial peptide, wherein the antibacterial peptide inhibits the growth of bacteria and fungi;

the bacteria are staphylococcus aureus, bacillus subtilis, bacillus cereus, listeria monocytogenes and micrococcus luteus;

the fungi are Aspergillus niger and Rhodotorula mucilaginosa.

As a limitation, the antimicrobial peptides are used for food preservation or antibiotic-free animal farming.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

the lysobacter enzymogenes L-43 obtained by the invention has the beneficial effects of fast growth, good adaptability and low fermentation cost;

secondly, the zymogenic lysobacter L-43 obtained by the invention has simple and easy operation fermentation method and improves the yield of the antibacterial peptide; the antibacterial peptide obtained by fermentation has broad-spectrum antibacterial property, can inhibit the growth of various bacteria and fungi, particularly shows excellent gram-positive bacteria resisting activity, and can well inhibit the growth of gram-positive bacteria;

the zymogenic lysobacter L-43 obtained by the invention is fermented to obtain the antibacterial peptide which is used as a substitute of antibiotics, can be used as a food preservative or a feed additive, is widely applied to the fields of food preservation, animal nonreactive breeding and the like, and has the characteristics of high efficiency and safety.

The method is suitable for separating the lysobacter enzymogenes L-43, the separated lysobacter enzymogenes L-43 can produce the antibacterial peptide through fermentation, and the produced antibacterial peptide can be further applied to the fields of food preservation, animal nonreactive breeding and the like.

Drawings

The invention will be described in more detail with reference to the following figures and embodiments:

FIG. 1 is a colony morphology on a plate of the strain of example 1 of the present invention;

FIG. 2 is a gram stain micrograph of the strain of example 1 of the present invention;

FIG. 3 is a phylogenetic tree of the strains according to example 1 of the present invention;

FIG. 4 is a graph showing the thermal stability of the L.enzymogenesis L-43 antibacterial substance in example 3 of the present invention;

FIG. 5 is a graph showing the pH stability of the L.enzymogenesis L-43 antibacterial substance in example 3 of the present invention;

FIG. 6 is a graph showing the storage stability of the L.enzymogenes L-43 antibacterial substance in example 3 of the present invention;

FIG. 7 is a diagram showing the fermentation process of L.enzymogenesis L-43 in the 5L fermenter for producing antibacterial peptide of example 10 of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure.

Example 1 screening and identification of lysobacter enzymogenes L-43

First, strain information

The Lysobacter enzymogenes L-43 is separated and screened from soil, and its strain is preserved in China general microbiological culture Collection center at 1 month and 5 days of 2021, the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, the preservation number is CGMCC NO.21593, and the Latin name is Lysobacter enzymogenes.

Second, separation and screening method of lysobacter enzymogenes L-43

The separation and purification method is sequentially carried out according to the following steps:

s1, respectively collecting 40 soil samples from regions of Hebei and Jiangsu provinces, performing primary strain screening on a nutrient agar culture medium by adopting an Oxford cup agar diffusion method, respectively taking staphylococcus aureus and escherichia coli as indicator strains, taking the generated inhibition zone as a screening basis, and screening to obtain 45 strains capable of inhibiting the growth of the indicator strains, wherein the strains are respectively numbered from L-1 to L-45;

s2, culturing the primary screened strain in an LB (lysogeny broth) culture medium by taking bacillus cereus ATCC 11778, Listeria monocytogenes 10403s and Salmonella typhimurium ATCC 14028 as indicating strains, and respectively determining the bacteriostatic action of the primary screened strain L-1-L-45 fermentation supernatant on the indicating strains by adopting an oxford cup agar diffusion method by taking the bacteriostatic activity of the fermentation supernatant as a screening basis; 6 strains of the bacteria have good bacteriostatic effect; the serial numbers of the compounds are L-1, L-18, L-22, L-42, L-43 and L-45;

s3, further carrying out hemolytic experiment on the 6 screened bacteria and the fermentation supernatant thereof, observing hemolytic rings of the bacteria through a blood plate, and measuring hemolytic property of the fermentation supernatant through hemolytic rate, wherein the bacteria L-18, L-42 and L-43 are not hemolytic, and the hemolytic property of the fermentation supernatant of the bacteria L-43 is lowest;

s4, obtaining a bacterial strain L-43 with high antibacterial substance yield through screening, wherein the bacterial strain has a large inhibition zone on the indicator bacteria, particularly has the best inhibition capacity on gram-positive bacteria, and does not have hemolytic property.

Third, the identification of lysobacter enzymogenes L-43 strain

a1. And (3) morphological identification:

inoculating the strain L-43 screened in S3 to a nutrient agar plate, carrying out streak inversion culture in an incubator at 37 ℃, observing the edge characteristics, transparency, bulge shape, glossiness, color, morphology and size of a bacterial colony, carrying out gram staining, and observing the state under a microscope, wherein the morphology of the bacterial colony is shown in figure 1, wherein figure 1a is the morphology of the bacterial colony after 24h of culture, figure 1b is the morphology of the bacterial colony after 36h of culture, and figure 1c is the morphology of the bacterial colony after 48h of culture;

the gram stain results are shown in figure 2;

as can be seen from fig. 1: the bacterial colony of the strain L-43 on the nutrient agar plate has a large shape, the bacterial colony is in a round white sphere shape when cultured for 24 hours, the bacterial colony is in a yellowish color when cultured for 36 hours, which indicates that pigment is generated in the growth process, and the edge of the bacterial colony has a clear diffusion edge and a sliding movement phenomenon when cultured for 48 hours;

as can be seen from FIG. 2, the bacterial colony is purple after gram staining, and the bacterial strain L-43 is determined to be gram-negative bacteria, the thallus is in a fine rod shape and no spore is generated;

a2. and (3) molecular identification:

the molecular identification of the strain L-43 is entrusted to Shanghai bioengineering GmbH, and the 16SrDNA sequence of the strain L-43 has a full length of 1481bp through 16SrDNA sequence homology analysis of the strain, and the specific sequence is as follows:

GGCTCAGAGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGCAGCACAGAGGAGCTTGCTCCTTGGGTGGCGAGTGGCGGACGGGTGAGGAATACGTCGGAATCTGCCTATTTGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGTGGGGGACCTTCGGGCCTCACGCAGATAGATGAGCCGACGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAAGCTTAGGGTTAATAACCCTGAGTCATGACGGTACCGGAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTTTGTTAAGTCTGATGTGAAAGCCCTGGGCTCAACCTGGGAATGGCATTGGAAACTGGCTTACTAGAGTGCGGTAGAGGGTAGCGGAATTCCCGGTGTAGCAGTGAAATGCGTAGATATCGGGAGGAACATCCGTGGCGAAGGCGGCTACCTGGACCAGCACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGGGCAACTTGGCCCTCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGAAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGCAGAACCTTACCTGGCCTTGACATGTCGAGAACTTGGCAGAGATGCCTTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGT

when the results of BLAST analyses and comparisons were performed using NCB I, as shown in Table 1, the selected strain L-43 showed the highest homology of 99.46% with Lysobacter enzymogenes M497-1 and Lysobacter sp.OC7, and the 16S rDNA sequence of the strain L-43 was uploaded to GeneBank to obtain the sequence number MT 229166;

table 116S rDNA sequence homology identification results

Constructing a growth and development tree of the strain L-43 according to the result, wherein the result is shown in FIG. 3, and the strain L-43 has the closest relationship with Lysobacter enzymogenes 521;the 16SrDNA sequence of the strain is compared with the standard strain of Lysobacter enzymogenes, and the result shows that the strain is compared with Lysobacter enzymogenes DSM2043^T(AJ298291) having a homology of 99.19%, it was further confirmed that the strain L-43 belongs to Lysobacter enzymogenes, which was named Lysobacter enzymogenes L-43;

the strain is identified as Lysobacter enzymogenes by integrating the 16SrDNA sequence comparison result, physiological and biochemical characteristics and morphological characteristics of the strain, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No. 1 of Beijing Korean district, the preservation date is 2021 year, 1 month and 5 days, the preservation number is CGMCC No.21593, and the Latin is named as Lysobacter enzymogenes.

Example 2 verification of the antibacterial Effect of the antibacterial peptide produced by lysobacter enzymogenes L-43

The embodiment is a bacteriostatic effect verification experiment of antibacterial peptide produced by fermenting lysobacter enzymogenes L-43, and the specific method comprises the following steps: the antibacterial spectrum of the lysobacter enzymogenes antibacterial peptide is determined by adopting an agar diffusion method, the indicator bacteria mainly comprise common pathogenic bacteria and putrefactive fungi, the indicator bacteria comprise 5 strains of positive bacteria and 2 strains of fungi, and the specific indicator bacteria and the antibacterial effect are shown in table 2.

TABLE 2 bacteriostatic effect of antibacterial peptide produced by lysobacter enzymogenes L-43

Name of indicator bacterium	Diameter/mm of bacteriostatic circle
		Staphylococcus aureus ATCC 25923	17.45±0.55
Bacillus cereus ATCC 11778	14.55±0.30
		Bacillus subtilis ATCC 6051	21.30±0.26
Listeria monocytogenes 10403s	16.65±0.38
		Micrococcus luteus ATCC 10240	18.93±0.78
Aspergillus niger ATCC 16404	13.20±0.13
		Rhodotorula mucilaginosa	14.15±0.13

As can be seen from Table 2, the antibacterial peptide produced by lysobacter enzymogenes L-43 can inhibit gram-positive bacteria and fungi, and has a wide antibacterial range and a good antibacterial effect.

EXAMPLE 3-8 fermentation method of lysobacter enzymogenes L-43

The embodiments 3 to 8 are fermentation methods of lysobacter enzymogenes L-43, respectively, and specifically include: weighing carbon source, nitrogen source, inorganic salt and distilled water according to the table 4, mixing uniformly, adjusting pH value, sterilizing at 121 ℃ for 20min to prepare a fermentation medium; inoculating lysobacter enzymogenes L-43 into a corresponding fermentation culture medium, and fermenting for 12-22h under the conditions that the temperature is 28-32 ℃, the rotating speed is 180-; they differ only in the raw materials, amounts, initial pH and process parameters, as detailed in table 3.

TABLE 3 fermentation Process parameters List for lysobacter enzymogenes producing antimicrobial peptides

In each embodiment, after fermentation is finished, fermentation liquor delta 1-delta 6 is obtained, the fermentation liquor delta 1-delta 6 is respectively centrifuged for 20min under the condition that the rotating speed is 8000r/min, supernate is collected, fermentation supernate epsilon 1-epsilon 6 is obtained, staphylococcus aureus ATCC 25923 is used as indicator bacteria, the yield of antibacterial peptide in the fermentation supernate epsilon 1-epsilon 6 is measured by a double dilution method, and the antibacterial titer is 364.5-4271.4 AU/mL.

EXAMPLE 9 characterization of the antimicrobial peptides produced by lysobacter enzymogenes L-43

This example is an experiment for identifying the chemical properties of the antibacterial peptide produced by lysobacter enzymogenes L-43, and the specific operations are as follows:

b1. enzyme Activity assay

Principle of enzyme activity assay: most lysobacters can secrete extracellular hydrolases, such as lysozyme, protease, chitinase, cellulase and the like, and the extracellular hydrolases achieve the bacteriostatic action by degrading the cell walls of indicator bacteria;

as can be known from an antibacterial spectrum experiment, antibacterial substances produced by the L.enzymogenes L-43 mainly inhibit bacteria, and on the basis, enzyme activities such as lysozyme and the like for hydrolyzing bacterial cell walls are mainly considered so as to analyze whether the L.enzymogenes L-43 produces hydrolase such as lysozyme, protease and the like;

through lysozyme activity determination and protease activity determination, the L.enzymogenes L-43 fermentation supernatant is found, namely lysozyme does not exist in the fermentation supernatant epsilon 1, and the protease activity is 199.73U/mL;

b2. protease treatment

Mixing L.enzymogenesis L-43 fermentation supernatant, the fermentation supernatant epsilon 1 and protease liquid in a volume ratio of 1:1, wherein the final concentration of protease is 10mg/mL, placing the mixture in a water bath kettle at 37 ℃ for reaction for 2h, treating the mixture with boiling water for 5min to terminate the enzyme reaction, and determining the recovery rate of the bacteriostatic activity by taking deionized water as a blank control, wherein the results are shown in Table 4;

wherein trypsin, proteinase K and proteinase E are solubilized with 0.1mol/L Tris-HCl buffer pH 7.5; dissolving pepsin with KCl-HCl buffer solution of 0.1mol/L, pH of 2.0; the catalase was dissolved in a phosphate buffer (0.2 mol/L, pH: 7.0);

TABLE 4 recovery of bacteriostatic activity of the fermentation supernatant ε 1 after enzyme treatment

Enzyme name	Percent recovery of bacteriostatic activity/%)
		Trypsin	79.00±2.07
Pepsin	77.05±2.65
		Proteinase K	78.74±0.45
Protease E	73.43±1.13
		Catalase enzyme	100±0.96

As shown in table 4, the activities of the fermentation supernatant epsilon 1 are lost after being treated by trypsin, pepsin, proteinase K and proteinase E, which indicates that the antibacterial substance produced by l.enzymogenes L-43 is protein or polypeptide substance; no activity loss after catalase treatment, indicating that the fermentation supernatant contains no hydrogen peroxide; the chemical nature of the antibacterial substance in the fermentation supernatant epsilon 1 is mainly protein or polypeptide;

b3. heat resistance treatment

Heat treating the fermentation supernatant epsilon 1 in water bath pots at 40 deg.C, 60 deg.C, 80 deg.C and 100 deg.C for 30 min; treating the fermented supernatant with high pressure steam sterilizing pot at 121 deg.C for 20 min; respectively measuring the antibacterial activity, and taking the fermentation supernatant epsilon 1 which is not subjected to heat treatment as a reference;

the antibacterial activity recovery rate of the fermentation supernatant epsilon 1 after treatment at different temperatures is shown in fig. 4, and it can be seen that the antibacterial substance produced by L.enzymogenes L-43 has good thermal stability, more than 98% of antibacterial activity is still retained after treatment at 100 ℃ for 30min, and the activity recovery rate is 87.77% after treatment at 121 ℃ for 20 min; therefore, the antibacterial substance is a macromolecular protein, is less likely to be a polypeptide with good heat resistance;

b4. acid-base stability treatment

Taking 100ml of fermentation supernatant epsilon 2, dividing into 10 equal parts, respectively marking each 10ml as alpha 1-alpha 10, respectively adjusting the pH values to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 by using 0.1mol/L HCl and 0.1mol/L NaOH solutions respectively, carrying out water bath at 37 ℃ for 2h, then adjusting the pH values of the solutions back to the initial pH value of the fermentation liquor, namely 8.8, finally fixing the volume to 30ml, and measuring the bacteriostatic activity of the solution to investigate the stability of an antibacterial substance to acid and alkali, wherein the result is shown in figure 5;

as can be seen from FIG. 5, the activity recoveries were all maintained above 80% at pH 2-10, with 100% at pH 9, due analytically to pH 9 being closest to the initial broth pH of 8.8; when the pH value exceeds 11, the activity recovery rate is remarkably reduced, when the pH value is 12, the activity recovery rate is only 55.7 percent, and about half of the loss is achieved, so that the antibacterial substance produced by the L.enzymes L-43 is more stable in acidic and neutral environments compared with the alkaline environment;

b5. storage stability

Taking 100ml of fermentation supernatant epsilon 6, and measuring the bacteriostatic activity of the fermentation supernatant epsilon 6 after the fermentation supernatant epsilon 6 is stored for 30d, 60d, 90d, 120d, 160d and 180d at the temperature of 4 ℃, wherein the result is shown in figure 6;

as can be seen from fig. 6, the recovery rate of activity was maintained at 90% or more in 150d storage of the fermentation broth, and the recovery rate was decreased to about 87.32% after 180d storage, which indicates that the antibacterial substance had good storage stability.

EXAMPLE 10 fermentation tank experiment of lysobacter enzymogenes L-43

In this example, the results of an amplification experiment of antibacterial peptide by lysobacter enzymogenes L-43 in a 5L fermenter are shown in FIG. 7;

as can be seen from FIG. 7, in the process of producing the antimicrobial peptide by fermentation in the 5L fermentor, the content of the antimicrobial peptide is rapidly increased from 4h to reach the peak value of 3570.21AU/mL when reaching 12h, compared with the shake flask fermentation (2% of the inoculum size of lysobacter enzymogenes L-43 is inoculated in the fermentation medium, the fermentation is carried out at 32 ℃, 180r/min, and the maximum value of the peptide production is reached in 22 h), the fermentation time is greatly shortened.

EXAMPLE 11 Effect of antimicrobial peptides produced by lysobacter enzymogenes L-43 in food preservation

This example is an experiment of the effect of antibacterial peptide produced by lysobacter enzymogenes L-43 in enema preservation, and includes the following steps:

s1, self-made sausage without preservative:

taking 600g of fresh pork powder, adding 18g of refined salt, 6g of white sugar and 90mL of water, uniformly stirring, placing in a refrigerator for pickling for two hours (adding antibacterial peptide before pickling), adding 60g of corn starch, 30g of soybean protein powder, 1.8g of monosodium glutamate and 120mL of water after pickling, uniformly stirring, filling into four cellulose casings, steaming for 30 minutes, and then placing at room temperature for 30 minutes to obtain enema beta 1-beta 4, wherein beta 1 is a blank control group without the antibacterial peptide, beta 2 is a positive control group (according to the addition amount of 1 mg/g) with potassium sorbate, beta 3 is a low-dose experiment group with 240AU/mL of zymogen antibacterial peptide, and beta 4 is a high-dose experiment group with 600AU/mL of antibacterial peptide;

s2, storing the clysters beta 1-beta 4 in a sealed bag at the storage temperature of 25 ℃, randomly sampling and observing on

days

0, 1, 2, 3, 4 and 5 respectively, and determining related indexes according to steps c1-c 4;

c1. determination of the Total number of colonies

Removing casings of the enemas beta 1-beta 4, putting the enemas beta 1-beta 4 into a homogenizing bag, kneading and crushing the enemas beta 1-beta 4, adding 0.85% of normal saline, beating and homogenizing for 2min under the condition that the rotating speed is 4000r/min, fully mixing the sample with the normal saline, preparing a sample theta 1-theta 4 for determining the total number of bacterial colonies, and determining the total number of the bacterial colonies in the theta 1-theta 4 according to the standard of GB 4789.2-2016;

c2. determination of fat Oxidation number

Filling a sausage beta 1-beta 4, removing casings, grinding, adding 7.5% trichloroacetic acid (containing 0.1% ethylene diamine tetraacetic acid), oscillating for 40min, performing suction filtration, adding 0.02 mol/L2-thiobarbituric acid solution, performing boiling water bath at 100 ℃ for 40min, cooling, centrifuging, taking supernate, adding chloroform, layering, taking supernate, performing colorimetry at the absorbance of 532nm and 600nm respectively, and recording the light absorption value;

the fat oxidation value is calculated as follows:

TBA＝(A532-A600)/155×1/10×72.6×100

in the formula:

TBA value-fat oxidation number in mg/100 g;

a532-absorbance of sample at 532 nm;

a600-absorbance of sample at 600 nm;

c3. determination of pH

Grinding the sausage with casing removed, adding distilled water, and measuring pH value with pH meter;

c4. sensory evaluation of sausage

Inviting 10 experts to perform sensory evaluation on the enema beta 1-beta 4, wherein the evaluation standard is shown in the table 5;

TABLE 5 sensory evaluation of enema

The results of the measurement are as follows:

c1. determination of the Total number of colonies

After 5 days of determination, the total number of colonies of the group 1 and the group 2 control group is not obviously different, while the total number of colonies of the experimental group is lower than that of the control group, wherein the antibacterial effect of the antibacterial peptide (group 4) of the high-dose group is the best, and the total number of colonies is 1 Lg value lower than that of the group 1 and the group 2. Therefore, a certain amount of lysobacter enzymogenes antibacterial peptide can inhibit the growth of bacteria in pork, and the inhibition effect is more obvious when the addition amount of the antibacterial peptide is more;

c2. determination of fat Oxidation number

Fat oxidation number is an important indicator for assessing the freshness of meat products. The greater the fat oxidation number, the greater the degree of fat oxidation and the greater the degree of putrefaction of the enema. On day 1, the fat oxidation number was 0.316mg/100g for group 1, 0.098mg/100g for group 2, 0.161mg/100g for group 3, and 0.141mg/100g for group 4; on day 5, the fat oxidation values of

groups

2, 3 and 4 were not significantly different, but were all significantly lower than group 1, 3-4 fold lower than group 1. Therefore, the putrefaction of the enema is effectively inhibited by adding the antibacterial peptide or the potassium sorbate;

c3. determination of pH

After 5 days of measurement, the pH value of the experimental group added with the antimicrobial peptide was lower than that of the control group as a whole. On day 0, the pH of groups 1-4 was around 6, on day 5, the pH of group 1 was the highest and the pH of group 4 was the lowest, 6.19, indicating that the appropriate amount of antimicrobial peptide inhibited microbial growth;

c4. sensory evaluation of sausage

The differences are shown on sensory evaluation of 3 days, wherein on color indexes, the antibacterial peptide experimental groups have uniform and small differences, and the color index of the antibacterial peptide experimental groups is 7.5, and the color index of the

groups

1 and 2 is 5.5; in terms of odor, the antimicrobial peptide test group was slightly lower than those of

groups

1 and 2; in the aspects of elasticity and viscosity, the group 1 has poor elasticity, is not easy to recover and sticks to hands after being pressed, and has 2 points, the group 2 and the antibacterial peptide experimental group have good elasticity, can recover after being pressed, slightly sticks to hands, and has a total score of 7 points;

the result shows that the adding of the lysobacter enzymogenes antibacterial peptide and the potassium sorbate in the enema can inhibit the growth and the propagation of microorganisms, and the oxidation degree of fat can influence the sensory evaluation.

SEQUENCE LISTING

<110> university of Hebei science and technology

<120> lysobacter enzymogenes L-43 and application thereof

<130> 1027

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1481

<212> DNA

<213> Lysobacter enzymogenes L-43 (Lysobacter enzymogenes)

<400> 1

ggctcagagt gaacgctggc ggcaggccta acacatgcaa gtcgaacggc agcacagagg 60

agcttgctcc ttgggtggcg agtggcggac gggtgaggaa tacgtcggaa tctgcctatt 120

tgtgggggat aacgtaggga aacttacgct aataccgcat acgacctacg ggtgaaagtg 180

ggggaccttc gggcctcacg cagatagatg agccgacgtc ggattagcta gttggcgggg 240

taaaggccca ccaaggcgac gatccgtagc tggtctgaga ggatgatcag ccacactgga 300

actgagacac ggtccagact cctacgggag gcagcagtgg ggaatattgg acaatgggcg 360

caagcctgat ccagccatgc cgcgtgtgtg aagaaggcct tcgggttgta aagcactttt 420

gtccggaaag aaaagcttag ggttaataac cctgagtcat gacggtaccg gaagaataag 480

caccggctaa cttcgtgcca gcagccgcgg taatacgaag ggtgcaagcg ttactcggaa 540

ttactgggcg taaagcgtgc gtaggtggtt tgttaagtct gatgtgaaag ccctgggctc 600

aacctgggaa tggcattgga aactggctta ctagagtgcg gtagagggta gcggaattcc 660

cggtgtagca gtgaaatgcg tagatatcgg gaggaacatc cgtggcgaag gcggctacct 720

ggaccagcac tgacactgag gcacgaaagc gtggggagca aacaggatta gataccctgg 780

tagtccacgc cctaaacgat gcgaactgga tgttgggggc aacttggccc tcagtatcga 840

agctaacgcg ttaagttcgc cgcctgggaa gtacggtcgc aagactgaaa ctcaaaggaa 900

ttgacggggg cccgcacaag cggtggagta tgtggtttaa ttcgatgcaa cgcgcagaac 960

cttacctggc cttgacatgt cgagaacttg gcagagatgc cttggtgcct tcgggaactc 1020

gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080

caacgagcgc aacccttgtc cttagttgcc agcacgtaat ggtgggaact ctaaggagac 1140

cgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatggc ccttacggcc 1200

agggctacac acgtactaca atggtaggga cagagggctg caaacccgcg agggcaagcc 1260

aatcccagaa accctatctc agtccggatt ggagtctgca actcgactcc atgaagtcgg 1320

aatcgctagt aatcgcagat cagcattgct gcggtgaata cgttcccggg ccttgtacac 1380

accgcccgtc acaccatggg agtttgttgc accagaagca ggtagcttaa ccttcgggag 1440

ggcgcttgcc acggtgtggc cgatgactgg ggtgaagtcg t 1481

Claims

1. Lysobacter enzymogenes L-43, characterized in that: it is preserved in China general microbiological culture Collection center, the preservation address is No. 3 Xilu No. 1 Beijing, Chaoyang, the preservation date is 2021 year, 1 month and 5 days, the preservation number is CGMCC NO.21593, the Latin name isLysobacter enzymogenes。

2. The lysobacter enzymogenes L-43 according to claim 1, wherein: the 16SrRNA gene sequence is as follows:

3. the use of lysobacter enzymogenes L-43 according to claim 1 or 2, wherein: the lysobacter enzymogenes L-43 is used for food preservation or animal nonreactive breeding.

4. The use of lysobacter enzymogenes L-43 according to claim 1 or 2, wherein: the lysobacter enzymogenes L-43 is used for producing the antibacterial peptide by fermentation.

5. The use of lysobacter enzymogenes L-43 according to claim 4, wherein: the fermentation medium comprises the following raw materials in parts by weight per 100 g: 0.5-2.0 parts of carbon source, 0.5-1.5 parts of nitrogen source, 0.05-0.15 part of inorganic salt and the balance of water.

6. The use of lysobacter enzymogenes L-43 according to claim 5, wherein: the carbon source is at least one of maltose, fructose and liquid sugar;

the nitrogen source is tryptone; the inorganic salt is magnesium sulfate or magnesium chloride.

7. The use of lysobacter enzymogenes L-43 according to claim 4, wherein: the initial pH value of the fermentation is 6.0-7.0, the temperature is 30-32 ℃, the rotating speed is 180-.

8. A use of the antimicrobial peptide according to any one of claims 5 to 7, wherein: the antimicrobial peptides inhibit bacterial and fungal growth;

the fungi are Aspergillus niger and Rhodotorula mucilaginosa.

9. The use of an antimicrobial peptide according to claim 8, wherein: the antibacterial peptide is used for food preservation or animal nonreactive breeding.