CN113846034B

CN113846034B - Lysobacter enzymogenes L-43 and application thereof

Info

Publication number: CN113846034B
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: 2024-01-16
Anticipated expiration: 2041-10-27
Also published as: CN113846034A

Abstract

The invention discloses a lysobacter enzymogenes L-43 and application thereof, wherein the bacterium is preserved in China general microbiological culture Collection center (CGMCC No. 21593) of national institute of microbiological culture collection center No. 3 in North Chen West Lu No. 1 in the Korean area of Beijing in 2021, 1 month and 5 days; the bacteria can be used for producing the antibacterial peptide through fermentation. The invention is suitable for separating the lysobacter enzymogenes L-43, and the lysobacter enzymogenes L-43 obtained by separation can produce antibacterial peptide.

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 source antibacterial substance has the characteristics of low toxicity, high efficiency, stability, no residue, short production period, low cost and the like, and becomes a research hotspot in the fields of food, agriculture, medicine and the like; with the rapid development of biotechnology, the use of microorganisms and their metabolites as raw materials, and the use of fermentation, extraction and other technologies to produce natural antibacterial substances is increasing;

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

the lysobacter is Proteus (Proteus), gamma-Proteus (gamma-Proteus), xanthomonas (Xanthomonas adacae); at present, about 50 types of reported lysobacter bacteria exist, wherein four typical species are lysobacter enzymogenes (Lysobacter enzymogenes), lysobacter antibioticus (Lysobacter antibioticus), lysobacter gelatinosum (Lysobacter gummosus) and lysobacter palmi (Lysobacter brunescens), and the bacteria have high-efficiency antibacterial activity and plant disease biocontrol potential, and are novel plant disease biocontrol bacteria; the bacteria have remarkable antagonism to plant pathogenic fungi, oomycetes, gram-negative bacteria, positive bacteria and nematodes, and the action mechanism is as follows: (1) producing a small molecule antimicrobial secondary metabolite; (2) Secretion produces a large number of extracellular enzymes including chitinase, beta-1, 3-glucan, proteases and cellulases; (3) inducing disease resistance in plants; (4) better colonisation ability;

in conclusion, the novel lysobacter strain with antibacterial activity can be separated and identified, so that high-quality resources can be provided for microbial source antibacterial substances, and the novel and efficient nontoxic natural medicine has great potential for developing novel and nontoxic natural medicines.

Disclosure of Invention

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

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

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the lysobacter enzymogenes L-43 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), and has a preservation address of Hospital No. 3, north Chen West Lu No. 1, the North Korea of Beijing, a preservation date of 2021, a preservation number of CGMCC No.21593 and a Latin name of Lysobacter enzymogenes.

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

GGCTCAGAGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGCAGCACAGAGGAGCTTGCTCCTTGGGTGGCGAGTGGCGGACGGGTGAGGAATACGTCGGAATCTGCCTATTTGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGTGGGGGACCTTCGGGCCTCACGCAGATAGATGAGCCGACGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAAGCTTAGGGTTAATAACCCTGAGTCATGACGGTACCGGAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTTTGTTAAGTCTGATGTGAAAGCCCTGGGCTCAACCTGGGAATGGCATTGGAAACTGGCTTACTAGAGTGCGGTAGAGGGTAGCGGAATTCCCGGTGTAGCAGTGAAATGCGTAGATATCGGGAGGAACATCCGTGGCGAAGGCGGCTACCTGGACCAGCACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGGGCAACTTGGCCCTCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGAAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGCAGAACCTTACCTGGCCTTGACATGTCGAGAACTTGGCAGAGATGCCTTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGT。

the invention also provides another application of the lysobacter enzymogenes L-43, which is used for producing the antibacterial peptide through fermentation.

As a limitation, the raw materials of the active ingredients of the fermentation medium include, per 100g by weight: 0.5-2.0 parts of carbon source, 0.5-1.5 parts of nitrogen source, 0.05-0.15 parts 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 fermentation is carried out at an initial pH value of 6.0-7.0, a temperature of 30-32 ℃, a rotating speed of 180-200r/min, an inoculum size of 2-4% and a fermentation time of 12-22h.

The invention also provides an application of the antibacterial peptide, which can inhibit the growth of bacteria and fungi;

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

the fungus is Aspergillus niger or Rhodotorula mucilaginosa.

By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:

(1) the lysobacter enzymogenes L-43 has the beneficial effects of fast growth, good adaptability and low fermentation cost;

(2) the zymolytic bacillus 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 has excellent gram-positive bacterium resisting activity, and can well inhibit the growth of gram-positive bacteria;

the invention is suitable for separating the lysobacter enzymogenes L-43, and the lysobacter enzymogenes L-43 obtained by separation can be used for producing antibacterial peptide by fermentation.

Drawings

The invention will be described in more detail below with reference to the attached drawings and specific examples:

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

FIG. 2 is a microscopic morphology of gram staining results of the strain of example 1 of the present invention;

FIG. 3 is a phylogenetic tree of the strain of example 1 of the present invention;

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

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

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

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

Detailed Description

The invention will now be described in further detail by way of specific examples, which are to be understood as illustrative only and not limiting.

Example 1 screening and identification of lysobacter enzymogenes L-43

1. Strain information

The lysobacter enzymogenes L-43 is separated and screened from soil, and the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 1 month 5 of 2021, wherein the preservation address is No. 1,3 of North Chen Silu No. 1 in the Korean region of Beijing, the preservation number is CGMCC No.21593, and the Latin name is Lysobacter enzymogenes.

2. Separation and screening method of lysobacter enzymogenes L-43

The separation and purification method sequentially comprises the following steps:

s1, collecting 40 soil samples from each region of two provinces of Hebei and Jiangsu respectively, performing preliminary screening on the strains by adopting an oxford cup agar diffusion method and respectively taking staphylococcus aureus and escherichia coli as indication strains and taking a bacteria inhibition zone as a screening basis on a nutrient agar medium, and obtaining 45 strains capable of inhibiting the growth of the indication strains, wherein the numbers of the strains are L-1 to L-45;

s2, culturing a primary screening strain in an LB culture medium by taking bacillus cereus ATCC 11778, listeria monocytogenes 10403s and salmonella typhimurium ATCC 14028 as indication strains, taking the antibacterial activity of fermentation supernatant as a screening basis, and respectively measuring the antibacterial effect of the primary screening strains L-1 to L-45 fermentation supernatant on the indication strains by adopting an oxford cup agar diffusion method; wherein 6 strains have good antibacterial effect; the serial numbers are L-1, L-18, L-22, L-42, L-43 and L-45;

s3, further carrying out a hemolytic experiment on the 6 strains and the fermentation supernatant thereof, observing hemolytic rings of the strains through a blood plate, and measuring hemolytic of the fermentation supernatant through a hemolytic rate, wherein none of the strains L-18, L-42 and L-43 is hemolytic, and the hemolytic property of the fermentation supernatant of the strain L-43 is lowest;

s4, obtaining a strain L-43 with high yield of antibacterial substances through screening, wherein the antibacterial substances produced by the strain have large antibacterial circle on indicator bacteria, particularly have the best inhibitory capacity on gram-positive bacteria, and have no hemolysis.

3. Identification of the lysobacter enzymogenes L-43 Strain

a1. Morphological identification:

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

the gram staining results are shown in figure 2;

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

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

a2. molecular identification:

the molecular identification of the strain L-43 is carried out by the Shanghai bioengineering Co., ltd. Through the analysis of the homology of the 16SrDNA sequence of the strain, the full length of the 16SrDNA sequence of the strain L-43 is 1481bp, and the specific sequence is as follows:

GGCTCAGAGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGCAGCACAGAGGAGCTTGCTCCTTGGGTGGCGAGTGGCGGACGGGTGAGGAATACGTCGGAATCTGCCTATTTGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGTGGGGGACCTTCGGGCCTCACGCAGATAGATGAGCCGACGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAAGCTTAGGGTTAATAACCCTGAGTCATGACGGTACCGGAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTTTGTTAAGTCTGATGTGAAAGCCCTGGGCTCAACCTGGGAATGGCATTGGAAACTGGCTTACTAGAGTGCGGTAGAGGGTAGCGGAATTCCCGGTGTAGCAGTGAAATGCGTAGATATCGGGAGGAACATCCGTGGCGAAGGCGGCTACCTGGACCAGCACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGGGCAACTTGGCCCTCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGAAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGCAGAACCTTACCTGGCCTTGACATGTCGAGAACTTGGCAGAGATGCCTTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGT

BLAST analysis and comparison are carried out by utilizing NCBI, the result is shown in Table 1, the homology of the screened strain L-43 with Lysobacter enzymogenes M497-1 and Lysobacter sp.OC7 is the highest, and the homology is 99.46%, and the 16S rDNA sequence of the strain L-43 is uploaded to GeneBank to obtain the sequence number MT229166;

TABLE 1 16S 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 figure 3, and the strain L-43 has the closest relationship with lysobacteria genes 521; the 16SrDNA sequence of the strain was aligned with a standard strain of lysobacter enzymogenes, and the results showed that the strain was aligned with Lysobacter enzymogenes DSM2043 ^T (AJ 298291) homology of 99.19%, further confirming that the strain L-43 belongs to the lysobacter enzymogenes, designated Lysobacter enzymogenes L-43;

the strain is identified as zymolytic bacillus by combining the result of the sequence comparison, the physiological and biochemical characteristics and the morphological characteristics of the strain 16SrDNA, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation address of North Chen West Lu No. 1 and No. 3 in the Korean region of Beijing, the preservation date is 2021, 1 and 5 days, the preservation number is CGMCC No.21593, and the Latin name is Lysobacter enzymogenes.

Example 2 antibacterial peptide produced by lysobacter enzymogenes L-43 antibacterial effect verification

The embodiment is a bacteriostasis effect verification experiment of antibacterial peptide produced by fermentation of lysobacter enzymogenes L-43, and the specific method is as follows: the bacteriostasis spectrum of the lysobacter enzymogenes antibacterial peptide is measured by an agar diffusion method, and the indicator bacteria mainly comprise common pathogenic bacteria and putrefying fungi, wherein the indicator bacteria comprise 5 positive bacteria and 2 fungi, and the specific indicator bacteria and the bacteriostasis effect are shown in table 2.

TABLE 2 antibacterial Effect of antibacterial peptides produced by lysobacter enzymogenes L-43

Indicating bacteria name	Diameter/mm of inhibition zone
		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 shown in Table 2, the antibacterial peptide produced by the lysobacter enzymogenes L-43 can inhibit gram-positive bacteria and fungi, and has a wide antibacterial range and a good antibacterial effect.

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

The present examples 3 to 8 are fermentation methods of lysobacter enzymogenes L-43, respectively, specifically: weighing carbon source, nitrogen source, inorganic salt and distilled water according to table 4, mixing, adjusting pH value, sterilizing at 121deg.C for 20min to obtain fermentation culture medium; inoculating the lysobacter enzymogenes L-43 into a corresponding fermentation culture medium, and fermenting for 12-22 hours under the conditions that the temperature is 28-32 ℃, the rotating speed is 180-200r/min and the inoculum size is 2% -4%; they differ only in the raw materials, amounts, initial pH and process parameters, as detailed in table 3.

TABLE 3 list of parameters of the fermentation process of antibacterial peptide-producing lysobacter enzymogenes

After the fermentation is finished, fermentation liquid delta 1-delta 6 is obtained, the fermentation liquid delta 1-delta 6 is centrifuged for 20min at the rotating speed of 8000r/min, supernatant fluid is collected, so that fermentation supernatant fluid epsilon 1-epsilon 6 is obtained, staphylococcus aureus ATCC 25923 is used as indicator bacteria, and the yield of antibacterial peptide in the fermentation supernatant fluid epsilon 1-epsilon 6 is measured by a double dilution method, wherein the antibacterial titer is 364.5-4271.4AU/mL.

Example 9 characterization of antibacterial peptides produced by lysobacter enzymogenes L-43

The experiment for identifying the chemical property of the antibacterial peptide produced by the lysobacter enzymogenes L-43 is specifically carried out as follows:

b1. enzyme Activity assay

Principle of enzyme Activity measurement: most of lysobacilli can secrete extracellular hydrolases such as lysozyme, protease, chitinase, cellulase and the like, and the extracellular hydrolases achieve a bacteriostatic effect by degrading the cell wall of the indicator bacteria;

according to bacteriostasis spectrum experiments, antibacterial substances produced by L.enzymes L-43 mainly inhibit bacteria, and based on the antibacterial substances, enzyme activities such as lysozyme and the like hydrolyzing cell walls of bacteria are mainly examined to analyze whether the L.enzymes L-43 produce hydrolases such as lysozyme and protease;

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

b2. protease treatment

Mixing L.enzymogenes L-43 fermentation supernatant, wherein the fermentation supernatant epsilon 1 and protease liquid are mixed in a volume ratio of 1:1, the final concentration of protease is 10mg/mL, the mixture is placed in a water bath kettle at 37 ℃ for reaction for 2 hours, boiling water is treated for 5 minutes to terminate enzyme reaction, deionized water is used as a blank control, and the antibacterial activity recovery rate is measured, wherein the result is shown in Table 4;

wherein trypsin, proteinase K and proteinase E are dissolved in 0.1mol/L Tris-HCl buffer with pH 7.5; pepsin was dissolved in 0.1mol/L, pH KCl-HCl buffer at 2.0; the catalase is dissolved by using phosphate buffer solution with the concentration of 0.2mol/L, pH of 7.0;

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

Enzyme name	Recovery rate of bacteriostatic activity/%
		Trypsin, trypsin and its preparation method	79.00±2.07
Pepsin	77.05±2.65
		Proteinase K	78.74±0.45
Proteinase E	73.43±1.13
		Catalase enzyme	100±0.96

As shown in Table 4, the activity of the fermentation supernatant epsilon 1 is 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 loss of activity after catalase treatment indicates that the fermentation supernatant does not contain hydrogen peroxide; indicating that the chemical nature of the antibacterial substances in the fermentation supernatant epsilon 1 is mainly protein or polypeptide;

b3. heat resistance treatment

Respectively placing fermentation supernatant epsilon 1 in water baths at 40 ℃, 60 ℃, 80 ℃ and 100 ℃ for heat treatment for 30min; treating the fermentation supernatant with a high-pressure steam sterilizing pot at 121deg.C for 20min; respectively measuring antibacterial activity, and taking fermentation supernatant epsilon 1 without heat treatment as a control;

the recovery rate of the antibacterial activity of the fermentation supernatant epsilon 1 after being treated at different temperatures is shown in a figure 4, and the antibacterial substance produced by L.zymogenic esL-43 has good thermal stability, the antibacterial activity of the fermentation supernatant epsilon 1 still remains more than 98% after being treated for 30min at 100 ℃, and the recovery rate of the antibacterial activity is 87.77% after being treated for 20min at 121 ℃; it is known that the antibacterial substance is a polypeptide with low possibility of macromolecular protein and good heat resistance;

b4. acid-base stabilization treatment

Taking 100ml of fermentation supernatant epsilon 2, equally dividing into 10 equal parts, respectively marking 10ml of each part as alpha 1-alpha 10, respectively adjusting the pH value 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 solution, carrying out water bath for 2 hours at 37 ℃, then adjusting the solution back to the initial pH value of the fermentation broth, namely 8.8 by using HCl or NaOH, finally fixing the volume to 30ml, and measuring the antibacterial activity of the fermentation broth to investigate the stability of antibacterial substances to acid and alkali, wherein the result is shown in figure 5;

as can be seen from FIG. 5, the activity recovery rate was maintained above 80% at pH 2-10, wherein the activity recovery rate was 100% at pH 9, because pH 9 was closest to the initial pH of the broth of 8.8; when the pH exceeds 11, the activity recovery rate is obviously reduced, and when the pH is 12, the activity recovery rate is only 55.7 percent, and almost half of the activity recovery rate is lost, so that compared with an alkaline environment, the antibacterial substance produced by L.enzymogenes L-43 is more stable in an acidic environment and a neutral environment;

b5. storage stability

100ml of fermentation supernatant ε 6 was taken and its bacteriostatic activity was measured after 30d, 60d, 90d, 120d, 160d and 180d storage at 4deg.C, and the results are shown in FIG. 6;

as shown in FIG. 6, the recovery rate of activity in the fermentation broth in storage 150d was kept at 90% or more, and the recovery rate was reduced to about 87.32% after storage to 180d, whereby it was found that the antibacterial substance had good storage stability.

EXAMPLE 10 fermentation tank experiment of lysobacter enzymogenes L-43

The result of the amplification experiment of antibacterial peptide by the lysobacter enzymogenes L-43 in the 5L fermenter is shown in FIG. 7;

as shown in FIG. 7, in the process of producing the antibacterial peptide by fermentation in a 5L fermenter, the antibacterial peptide content rapidly increases from 4 hours later to the peak value of 3570.21AU/mL at 12 hours, and the fermentation time is greatly shortened compared with shaking flask fermentation (the fermentation medium is inoculated with the lysobacter enzymogenes L-43 in an inoculum size of 2%, and the fermentation time is increased by 180r/min at 32 ℃ and the peptide production time is increased by 22 hours).

EXAMPLE 11 Effect of antibacterial peptides produced by lysobacter enzymogenes L-43 on preservation of foods

The embodiment is an application effect experiment of antibacterial peptide produced by lysobacter enzymogenes L-43 in enema corrosion prevention, comprising the following steps in sequence:

s1, self-making a preservative-free enema:

taking 600g of fresh minced pork, adding 18g of refined salt, 6g of white sugar and 90mL of water, uniformly stirring, then placing in a refrigerator for pickling for two hours (adding the 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 clysis beta 1-beta 4, wherein beta 1 is a blank control group without adding the antibacterial peptide, beta 2 is a positive control group with adding potassium sorbate (according to the addition amount of 1 mg/g), beta 3 is a low-dose experimental group with adding 240AU/mL of the antibacterial peptide of the lysobacter enzymogenes, and beta 4 is a high-dose experimental group with adding 600AU/mL of the antibacterial peptide;

s2, storing the clysis beta 1-beta 4 in a sealed bag, wherein the storage temperature is 25 ℃, randomly sampling and observing on days 0, 1, 2, 3, 4 and 5 respectively, and measuring relevant indexes according to the steps c1-c 4;

c1. determination of colony count

Removing sausage casing from beta 1-beta 4, putting into a homogenizing bag, kneading, adding 0.85% physiological saline, beating and homogenizing for 2min at 4000r/min to thoroughly mix the sample with the physiological saline, preparing a sample theta 1-theta 4 for measuring the total number of bacterial colonies, and measuring the total number of bacterial colonies in theta 1-theta 4 according to the standard of GB 4789.2-2016;

c2. determination of fat oxidation number

Clysis beta 1-beta 4, removing casing, grinding, adding 7.5% trichloroacetic acid (containing 0.1% ethylenediamine tetraacetic acid), oscillating for 40min, suction filtering, adding 0.02 mol/L2-thiobarbituric acid solution, boiling water bath at 100deg.C for 40min, cooling, centrifuging, collecting supernatant, adding chloroform, layering, collecting supernatant, colorizing at absorbance of 532nm and 600nm, and recording absorbance;

the fat oxidation value is calculated as follows:

TBA＝(A532-A600)/155×1/10×72.6×100

wherein:

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

absorbance of samples at a 532-532 nm;

absorbance of the sample at 600nm to 600 nm;

c3.pH determination

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

c4. enema sensory evaluation

10 panelists were invited to perform sensory evaluation on clysis β1- β4, and the evaluation criteria are shown in table 5;

table 5 enema sensory evaluation

The results were as follows:

c1. determination of colony count

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

c2. determination of fat oxidation number

Fat oxidation number is an important indicator for assessing freshness of meat products. The greater the fat oxidation number, the greater the degree of fat oxidation and the greater the degree of enema spoilage. Day 1, group 1 had a fat oxidation number of 0.316mg/100g, group 2 had a fat oxidation number of 0.098mg/100g, group 3 had a fat oxidation number of 0.161mg/100g, and group 4 had a fat oxidation number of 0.141mg/100g; on day 5, there was no significant difference in fat oxidation values for group 2, group 3 and group 4, but all were significantly lower than group 1, 3-4 times lower than group 1. It can be seen that the addition of the antibacterial peptide or potassium sorbate effectively inhibits the spoilage of the enema;

c3.pH determination

After 5 days of measurement, the pH value of the experimental group added with the antibacterial peptide is lower than that of the control group in the whole situation. The pH of groups 1-4 was around 6 on day 0, the pH of group 1 was highest on day 5, and the pH of group 4 was lowest, 6.19, and it was seen that an appropriate amount of the antimicrobial peptide inhibited the growth of microorganisms;

c4. enema sensory evaluation

The sensory evaluation of 3 days shows the difference that the color index of the antibacterial peptide experimental group is uniform in color and little in difference, the score is 7.5, and the color index score of the group 1 and the group 2 is 5.5; the antimicrobial peptide experimental group was slightly lower in odor than groups 1 and 2; in the aspects of elasticity and viscosity, the group 1 is poor in elasticity, is not easy to recover and sticky after being pressed, and is 2 groups, the group 2 and the antibacterial peptide experimental group are good in elasticity, can recover after being pressed and slightly sticky, and the overall score is 7 groups;

the results show that the addition of the lysobacter enzymogenes antibacterial peptide and the potassium sorbate in the enema can have the effect of inhibiting the growth and the reproduction of microorganisms, and the degree of fat oxidation can have influence on sensory evaluation.

SEQUENCE LISTING

<110> university of Hebei technology

<120> a lysobacter enzymogenes L-43 and use thereof

<130> 1027

<160> 1

<170> PatentIn version 3.3

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<211> 1481

<212> DNA

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

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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. An enzyme-producing lysobacter L-43, characterized in that: it is preserved in China general microbiological culture Collection center (CGMCC) with the preservation address of No. 1,3, and the preservation date of No. 1, 5, 2021 and No.21593, latin nameLysobacter enzymogenes。

2. Use of a lysobacter enzymogenes L-43 according to claim 1, wherein: the lysobacter enzymogenes L-43 produces the antibacterial peptide by fermentation.

3. Use of a lysobacter enzymogenes L-43 according to claim 2, 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 parts of inorganic salt and the balance of water.

4. Use of a lysobacter enzymogenes L-43 as claimed in claim 3, 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.

5. Use of a lysobacter enzymogenes L-43 according to claim 2, wherein: the initial pH value is 6.0-7.0, the temperature is 30-32 ℃, the rotating speed is 180-200r/min, the inoculation amount is 2-4%, and the fermentation time is 12-22h.

6. Use of an antimicrobial peptide according to any one of claims 3 to 5, characterized in that: the antimicrobial peptides inhibit bacterial and fungal growth;

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

the fungus is Aspergillus niger or Rhodotorula mucilaginosa.