CN113897293B - Self-solution for new strain LE16 of lysobacter enzymolyticus and preparation method thereof - Google Patents

Self-solution for new strain LE16 of lysobacter enzymolyticus and preparation method thereof Download PDF

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CN113897293B
CN113897293B CN202110914917.2A CN202110914917A CN113897293B CN 113897293 B CN113897293 B CN 113897293B CN 202110914917 A CN202110914917 A CN 202110914917A CN 113897293 B CN113897293 B CN 113897293B
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CN113897293A (en
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黄纯杨
漆夏燕
李治模
董祥立
彭玉龙
温明霞
王小彦
杨红军
黄建国
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Zunyi Tobacco Co Of Guizhou Tobacco Corp
Southwest University
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Southwest University
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Abstract

The invention discloses a preparation method of an autolysate for a new strain LE16 of lysobacter enzymolyticus (Lysobacter enzymogenes); the self-solution of LE16 prepared by the method does not contain thalli, does not need filtration and sterilization, has good and stable effect of preventing and treating plant powdery mildew and gray mold, long storage time, little influence by environmental conditions, simple application method, strong practicability and easy popularization, and the preparation process and technology system has simple and universal equipment, less resource consumption and low industrial production requirements, and can meet the production requirements of common microbial preparation factories.

Description

Self-solution for new strain LE16 of lysobacter enzymolyticus and preparation method thereof
Technical Field
The invention relates to a microbial preparation, in particular to a self-solution for a new strain LE16 of lysobacter enzymolyticus and a preparation method thereof.
Background
Lysobacter enzymogenes (Lysobacter enzymogenes), a typical representative of the genus lysobacter, has the ability to secrete a variety of hydrolytic enzymes, which is also the cause of its name. At present, the produced and applied lysobacter enzymogenes comprises lysobacter enzymogenes C3 (L.enzymogenes C3), lysobacter enzymogenes 3.1T8 (L.enzymogenes 3.1T8), lysobacter enzymogenes N4-7 (L.enzymogenes N4-7), and lysobacter enzymogenes OH11 (L.enzymogenes OH 11) and the like, and play an important role in crop disease control. Has the functions of promoting growth and preventing diseasesThe novel strain L.enzymogenes LE16 (hereinafter referred to as LE 16) of the Bacillus dysenteriae. At present, the strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is No.14215, and the sequencing result is submitted to the national center for biotechnology gene (NCBI) and the accession number MK044898 is obtained. Through sequencing the whole genome of strain LE16, it was found that there are a plurality of synthetic genes of antibacterial substances which are difficult to synthesize artificially or have high cost of synthesizing artificially in the gene sequence of the strain, including a tetrapeptide Xenotetrapeptide BGC0001132 (C 21 H 38 N 4 O 4 ) The method comprises the steps of carrying out a first treatment on the surface of the Bicantaloupe A1 Bicorenutin A1 BGC0001135 (C) 34 H 71 N 19 O 5 ) The method comprises the steps of carrying out a first treatment on the surface of the Bicantaloupe A2 Bicorenutin A2 BGC0001135 (C) 38 H 70 N 18 O 5 ) The method comprises the steps of carrying out a first treatment on the surface of the 3, 5-dihydroxy-4-isopropyl stilbene BGC0001180 isopropylestilbene; grass ether Sodorifen BGC0001361 (C) 16 H 26 ) The method comprises the steps of carrying out a first treatment on the surface of the Huang Yuansu Xanthomonadin I BGC0000840 (C) 28 H 30 Br 2 O 3 A yellow pigment, which may protect the cells from light); myxoviridin A1 BGC0001104 (C) 35 H 61 NO 8 An antibiotic having broad-spectrum antagonistic activity against gram-negative bacteria, which is also highly adhesive and is often used for the treatment of dental plaque and gingivitis); doxorubicin teixobatin BGC0001207 (C 58 H 95 N 15 O 15 The novel antibiotic has strong antagonistic activity on staphylococcus aureus, methicillin-resistant staphylococcus aureus and the like; FR901464 (C) 27 H 41 NO 8 A natural product produced by pseudomonas having anti-tumor activity); talandamine Thailandamide BGC0000186 (C) 43 H 59 NO 8 A linear polyene natural product having strong and selective antimicrobial activity against gram-positive bacteria and gram-negative bacteria with reduced cell walls; talan damide Thailandamide lactone (C) 43 H 55 NO 8 A polyketide metabolite); lotion of mycin lotilibcin WAP-8294A2 (C) 73 H 111 N 17 O 21 High activity secondaryMetabolites, which antagonize the growth of general staphylococcus aureus and methicillin-resistant staphylococcus aureus, are 14 times more active than vancomycin and have been used in clinical phase II studies; elansolid A1 (C) 38 H 52 O 7 Has antibacterial activity against gram-positive bacteria); caliamantadine KalimantacinA BGC0001099 (C) 30 H 48 N 2 O 7 Is an antibiotic with strong antifungal activity); mupirocin (C) 26 H 44 O 9 ) The pseudomonas acid A is a natural broad-spectrum antibiotic produced by pseudomonas fluorescens, has high inhibition effect on a plurality of gram-positive cocci and gram-negative bacteria, and is not easy to produce drug resistance; antagonizing growth of methicillin-resistant staphylococcus aureus, having antagonistic activity incomparable with other antibiotics, and inhibiting growth of partial fungi such as candida albicans and trichophyton mentagrophytes, which are not found by the bacteria of the genus lysobacter at present; lysin lysobacin (C) 58 H 97 N 15 O 17 The high-activity antibiotics can inhibit the biosynthesis of cell membranes and cell walls, and have strong antagonistic activity on a plurality of gram-positive and gram-negative pathogenic bacteria; the lysin has strong antagonistic activity on methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci, and the drug effect of the lysin is 2-50 times of that of the vancomycin respectively. Among the above-mentioned frequency metabolites, BGC0001132, bilobalide A1 BGC0001135, bilobalide A2 BGC0001135, 3, 5-dihydroxy-4-isopropyl stilbene, oxyfluorfen, mucin, doxorubicin, mupirocin and the like are not produced in most of the known lysobacter enzymogenes (L.enzymogens C3, L.enzymogens M497-1 and the like). During the specific liquid culture, L.enzymogenes LE16 (hereinafter referred to as LE 16) autolyzes. The solution (microbial inoculum) contains the various antibacterial substances, has remarkable inhibition effect on various plant pathogenic bacteria such as powdery mildew and gray mold bacteria of crops, and can economically, simply and conveniently prevent and treat powdery mildew and gray mold of crops without pollution. LE16 autolysis has strict requirements, and the formula of the culture medium, the volume of the culture tank, the liquid loading amount, the culture temperature and the like are not prepared at home and abroadProcesses and application techniques for preparing the product from the solution.
Disclosure of Invention
In view of the above, the invention aims to provide a self-solution for a new strain LE16 of lysobacter enzymolyzing, and a preparation method thereof, which are free of thalli, free of filter sterilization, good and stable in effect of preventing and treating plant gray mold and powdery mildew, long in storage time, small in influence of conditions such as environment, simple in application method, strong in practicability, easy to popularize, simple in preparation technology system equipment, universal, low in resource consumption, low in industrial production requirement, and capable of meeting production in a common microbial preparation factory.
The self-solution for the new strain LE16 of the lysobacter enzymolyticus comprises the following raw materials in parts by weight: 10-20 parts of peptone, 5-15 parts of beef extract, 5-15 parts of NaCl, 0.5-3 parts of tryptophan betaine, 0.5-3 parts of asparagine, 0.05-0.5 part of compound vitamin (NH) 4 ) 2 SO 4 1-10 parts, 5-15 parts of glucose, KH 2 PO 4 1-5 parts of MgSO 4 0.5-3 parts;
further, the self-dissolving liquid raw material comprises the following components in parts by weight: 16 parts of peptone, 8 parts of beef extract, 8 parts of NaCl, 1 part of tryptophan betaine, 1 part of asparagine, 0.2 part of compound vitamin and (NH) 4 ) 2 SO 4 4. 8 parts of glucose and KH 2 PO 4 2 parts of MgSO 4 1 part.
The invention also discloses a preparation method of the new strain LE16 of the lysobacter enzymolyticus self-solution, which comprises the following steps:
a. seed pot medium: dissolving peptone, beef extract, naCl, tryptophan betaine, asparagine and vitamin B complex in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone, (NH) 4 ) 2 SO 4 Beef extract, glucose, KH 2 PO 4 、 NaCl、MgSO 4 The tryptophan betaine, the asparagine and the vitamin B complex are dissolved in distilled water, and the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: inoculating LE16 bacteria solution after sterilizing the seed tank culture solution, and culturing in dark until autolysis of the culture solution occurs;
d. seed pot culture: c, sterilizing the seed tank culture solution in the step a, inoculating LE16 bacterial solution, and culturing to obtain seed solution;
e. culturing in a fermentation tank: injecting a fermentation tank culture solution into a fermentation tank, sterilizing, inoculating a seed solution for culture, and then injecting an original self-solution for continuous culture to obtain an LE16 self-solution;
further, in the step c, sterilizing for 15-25min at 110-130 ℃ and 0.05-0.2Mpa, cooling to 30-35 ℃, inoculating LE16 bacterial liquid, and performing dark culture at 25-35 ℃ and 140-160 revolutions per minute until autolysis occurs in the culture liquid;
further, in step d, sterilizing the seed tank culture solution in step a at 110-130deg.C and vacuum degree of 0.05-0.2Mpa for 15-25min, cooling to 30-35deg.C, inoculating LE16 bacteria solution at 28-35deg.C, 140-160 rpm, and ventilation of 0.25M 3 Culturing for 34-38 hours at/min;
further, in step d, the inoculation amount of the LE16 bacterial liquid is 10 3 cell/mL；
In step e, the fermentation tank culture solution is injected into the fermentation tank, sterilized for 15-25min at 110-130 deg.C and 0.05-0.2Mpa, cooled to 30-35 deg.C, and the seed solution is introduced into the fermentation tank at aeration rate of 0.25M 3 And (2) culturing at the temperature of 28-35 ℃ for 115-125 hours at 140-160 rpm for 72-96 hours after injecting the primary self-solution, and preparing the LE16 self-solution.
The invention has the beneficial effects that: the prepared LE16 self-solution for the new strain LE16 of the lysobacter enzymolyzing and the preparation method thereof have the characteristics of high temperature resistance (30 minutes of high temperature treatment at 100 ℃), long storage time (no obvious reduction of the antibacterial effect) and sealed storage for 1 year at normal temperature (0-50 ℃), good confidentiality (the self-solution does not contain living bacteria and living cells thereof, so functional bacteria cannot be obtained from the self-solution and cannot be imitated), and various substances with high cost which are difficult to synthesize artificially or synthesize artificially, for example Huang Yuansu for protecting the bacteria from being damaged by light; mucinous toxins having broad spectrum antagonistic activity against gram negative bacteria; talandamine having strong and selective antibacterial activity against gram-positive bacteria and gram-negative bacteria with reduced cell walls; can antagonize the growth of general staphylococcus aureus and methicillin-resistant staphylococcus aureus, and is applied to the high-activity secondary metabolite-namely the lothecin for clinical phase II research at present; natural product FR901464 (C27H 41NO 8) with antitumor activity; calidamycin with strong antifungal activity, and the like. The bactericide does not contain thalli, does not need filtration and sterilization, has good and stable effect of preventing and treating plant gray mold and powdery mildew, long storage time, little influence by conditions such as environment, simple application method, strong practicability and easy popularization. The strain LE16 self-solution preparation process technical system has simple equipment, is universal, has less resource consumption and low industrial production requirement, and can meet the production requirement of a common microbial preparation factory.
Drawings
Fig. 1 is a graph showing the experimental effect of LE16 from solution in example one.
Detailed Description
Example 1
The self-solution for the new strain LE16 of the lysobacter enzymolyticus in the embodiment comprises the following raw materials in parts by weight: 10 parts of peptone, 5 parts of beef extract, 5 parts of NaCl, 0.5 part of tryptophan betaine, 0.5 part of asparagine, 0.05 part of compound vitamin (NH) 4 ) 2 SO 4 1 part, 5 parts of glucose, KH 2 PO 4 1 part of MgSO 4 0.5 part.
The autolysate for the new strain LE16 of the lysobacter enzymolyticus is used for preparing an LE16 autosolution, and comprises the following steps: a. seed pot medium: dissolving peptone (6 parts), beef extract (4 parts), naCl parts, tryptophan betaine (0.25 parts), asparagine (0.25 parts) and vitamin B complex (0.0025 parts) in distilled water, wherein the initial pH is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone (4 parts), (NH) 4 ) 2 SO 4 Beef extract (1 part), grapeSugar, KH 2 PO 4 、NaCl、MgSO 4 Tryptophan betaine (0.25 part), asparagine (0.25 part), vitamin B complex (0.0025 part) and distilled water, wherein the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: sterilizing the culture solution of the seed tank at 110deg.C under vacuum degree of 0.05Mpa for 15min, cooling to 30deg.C, inoculating LE16 bacteria solution, and dark culturing at 25deg.C at 140 r/min until autolysis occurs;
d. seed pot culture: sterilizing the seed tank culture solution in step a at 110deg.C under vacuum degree of 0.05Mpa for 15min, cooling to 30deg.C, inoculating LE16 bacterial solution, and sterilizing at 28deg.C for 140 r/min with ventilation amount of 0.25M 3 Culturing for 34 hr at/min to obtain seed solution with LE16 bacteria solution inoculation amount of 10 3 cell/mL；
e. Culturing in a fermentation tank: injecting fermentation tank culture solution into fermentation tank, sterilizing at 110deg.C under vacuum degree of 0.05Mpa for 15min, cooling to 30deg.C, and introducing seed solution at ventilation rate of 0.25M 3 And (3) culturing at 28 ℃ for 115 hours at 140 revolutions per minute, injecting the primary self-solution, continuously culturing for 72 hours, preparing the LE16 self-solution, aseptically filling and sealing.
Example two
The self-solution for the new strain LE16 of the lysobacter enzymolyticus in the embodiment comprises the following raw materials in parts by weight: 20 parts of peptone, 15 parts of beef extract, 15 parts of NaCl, 3 parts of tryptophan betaine, 3 parts of asparagine, 0.5 part of compound vitamin and (NH) 4 ) 2 SO 4 10 parts of glucose 15 parts of KH 2 PO 4 5 parts of MgSO 4 3 parts.
The autolysate for the new strain LE16 of the lysobacter enzymolyticus is used for preparing an LE16 autosolution, and comprises the following steps: a. seed pot medium: dissolving peptone (15 parts), beef extract (10 parts), naCl, tryptophan betaine (1.5 parts), asparagine (1.5 parts) and vitamin B complex (0.25 parts) in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b.fermentation broth: peptone (5 parts), (NH) 4 ) 2 SO 4 Beef extract (5 parts), glucose, KH 2 PO 4 、NaCl、MgSO 4 Tryptophan betaine (1.5 parts), asparagine (1.5 parts), vitamin B complex (0.25 parts) and distilled water, wherein the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: sterilizing the culture solution of the seed tank at 130deg.C under vacuum degree of 0.2Mpa for 25min, cooling to 35deg.C, inoculating LE16 bacteria solution, and dark culturing at 35deg.C at 60 r/min until autolysis occurs;
d. seed pot culture: sterilizing the seed tank culture solution in step a at 130deg.C under vacuum degree of 0.2Mpa for 25min, cooling to 35deg.C, inoculating LE16 bacterial solution, and sterilizing at 35deg.C under 160 r/min with ventilation amount of 0.25M 3 Culturing for 38 hr at/min to obtain seed solution with LE16 bacterial liquid inoculation amount of 10 3 cell/mL；
e. Culturing in a fermentation tank: injecting fermentation tank culture solution into fermentation tank, sterilizing at 130deg.C under 0.2Mpa for 25min, cooling to 35deg.C, and introducing seed solution at ventilation rate of 0.25M 3 And (3) culturing at 35 ℃ for 125 hours at 160 revolutions per minute, injecting the primary self-solution, continuously culturing for 96 hours, preparing the LE16 self-solution, aseptically filling and sealing.
Example III
The self-solution for the new strain LE16 of the lysobacter enzymolyticus in the embodiment comprises the following raw materials in parts by weight: 10 parts of peptone, 15 parts of beef extract, 5 parts of NaCl, 3 parts of tryptophan betaine, 0.5 part of asparagine, 0.5 part of compound vitamin and (NH) 4 ) 2 SO 4 1 part, 15 parts of glucose, KH 2 PO 4 1 part of MgSO 4 3 parts.
The autolysate for the new strain LE16 of the lysobacter enzymolyticus is used for preparing an LE16 autosolution, and comprises the following steps: a. seed pot medium: dissolving peptone (7 parts), beef extract (11 parts), naCl, tryptophan betaine (1.5 parts), asparagine (0.25 parts) and vitamin B complex (0.25 parts) in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone (3 parts), (NH) 4 ) 2 SO 4 Beef extract (4 parts), glucose, KH 2 PO 4 、NaCl、MgSO 4 Tryptophan betaine (1.5 parts), asparagine (0.25 parts), vitamin B complex (0.25 parts) and distilled water, wherein the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: sterilizing the culture solution of the seed tank at 110deg.C under vacuum degree of 0.2Mpa for 15min, cooling to 35deg.C, inoculating LE16 bacteria solution, and dark culturing at 25deg.C at 160 r/min until autolysis occurs;
d. seed pot culture: sterilizing the seed tank culture solution in step a at 110deg.C under vacuum degree of 0.2Mpa for 15min, cooling to 35deg.C, inoculating LE16 bacterial solution, and sterilizing at 28-35deg.C for 140 r/min with ventilation of 0.25M 3 Culturing for 38 hr at/min to obtain seed solution with LE16 bacterial liquid inoculation amount of 10 3 cell/mL；
e. Culturing in a fermentation tank: injecting fermentation tank culture solution into fermentation tank, sterilizing at 110deg.C under vacuum degree of 0.2Mpa for 15min, cooling to 35deg.C, and introducing seed solution at ventilation rate of 0.25M 3 And (3) culturing at 28 ℃ for 115 hours at 160 revolutions per minute, injecting the primary self-solution, continuously culturing for 96 hours, preparing the LE16 self-solution, aseptically filling and sealing.
Example IV
The self-solution for the new strain LE16 of the lysobacter enzymolyticus in the embodiment comprises the following raw materials in parts by weight: 20 parts of peptone, 5 parts of beef extract, 15 parts of NaCl, 0.5 part of tryptophan betaine, 3 parts of asparagine, 0.05 part of compound vitamin (NH) 4 ) 2 SO 4 10 parts of glucose 5 parts of KH 2 PO 4 5 parts of MgSO 4 0.5 part.
The autolysate for the new strain LE16 of the lysobacter enzymolyticus is used for preparing an LE16 autosolution, and comprises the following steps: a. seed pot medium: dissolving peptone (14 parts), beef extract (3 parts), naCl, tryptophan betaine (0.25 parts), asparagine (1.5 parts) and vitamin B complex (0.025 parts) in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone (6 parts), (NH) 4 ) 2 SO 4 Beef extract (2 parts), glucose, KH 2 PO 4 、NaCl、MgSO 4 Tryptophan betaine (0.25 part), asparagine (1.5 part), vitamin B complex (0.025 part) and distilled water, wherein the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: sterilizing the culture solution of the seed tank at 130deg.C under vacuum degree of 0.05-Mpa for 25min, cooling to 30deg.C, inoculating LE16 bacteria solution, and dark culturing at 35deg.C at 140 r/min until autolysis occurs;
d. seed pot culture: sterilizing the seed tank culture solution in step a at 130deg.C under vacuum degree of 0.05Mpa for 25min, cooling to 30deg.C, inoculating LE16 bacterial solution, and sterilizing at 35deg.C for 140 r/min with ventilation amount of 0.25M 3 Culturing for 38 hr at/min to obtain seed solution with LE16 bacterial liquid inoculation amount of 10 3 cell/mL；
e. Culturing in a fermentation tank: injecting fermentation tank culture solution into fermentation tank, sterilizing at 130deg.C under 0.05Mpa for 25min, cooling to 30deg.C, and introducing seed solution at ventilation rate of 0.25M 3 And (3) culturing at 35 ℃ for 125 hours at 140 revolutions per minute, injecting the primary self-solution, continuously culturing for 72 hours, preparing the LE16 self-solution, aseptically filling and sealing.
Example five
The self-solution for the new strain LE16 of the lysobacter enzymolyticus in the embodiment comprises the following raw materials in parts by weight: 15 parts of peptone, 8 parts of beef extract, 13 parts of NaCl, 2 parts of tryptophan betaine, 2 parts of asparagine, 0.3 part of compound vitamin and (NH) 4 ) 2 SO 4 1 part, 15 parts of glucose, KH 2 PO 4 1 part of MgSO 4 2 parts.
The autolysate for the new strain LE16 of the lysobacter enzymolyticus is used for preparing an LE16 autosolution, and comprises the following steps: a. seed pot medium: dissolving peptone (8 parts), beef extract (6 parts), naCl, tryptophan betaine (1 part), asparagine (1 part) and vitamin B complex (0.15 part) in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone (7 parts), (NH) 4 ) 2 SO 4 Beef extract (2 parts), glucose, KH 2 PO 4 、NaCl、MgSO 4 Tryptophan betaine (1 part), asparagine (1 part), vitamin B complex (0.15 part) and distilled water, wherein the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: sterilizing the culture solution of the seed tank at 120deg.C under vacuum degree of 0.1Mpa for 20min, cooling to 32deg.C, inoculating LE16 bacteria solution, and dark culturing at 28deg.C at 150 r/min until autolysis occurs;
d. seed pot culture: sterilizing the seed tank culture solution in step a at 115deg.C under vacuum degree of 0.1Mpa for 18min, cooling to 33deg.C, inoculating LE16 bacterial solution, and sterilizing at 32deg.C for 155 r/min with ventilation amount of 0.25M 3 Culturing for 36 hr at/min to obtain seed solution with LE16 bacteria solution inoculation amount of 10 3 cell/mL；
e. Culturing in a fermentation tank: injecting fermentation tank culture solution into fermentation tank, sterilizing at 125deg.C under vacuum degree of 0.08Mpa for 18min, cooling to 31deg.C, introducing seed solution at ventilation rate of 0.25M 3 And (3) culturing at 30 ℃ for 122 hours at 155 revolutions per minute, injecting the primary self-solution, continuously culturing for 90 hours, preparing the LE16 self-solution, aseptically filling and sealing.
Example six
The self-solution for the new strain LE16 of the lysobacter enzymolyticus in the embodiment comprises the following raw materials in parts by weight: 16 parts of peptone, 8 parts of beef extract, 8 parts of NaCl, 1 part of tryptophan betaine, 1 part of asparagine, 0.2 part of compound vitamin and (NH) 4 ) 2 SO 4 4 parts of glucose 8 parts of KH 2 PO 4 2 parts of MgSO 4 1. Parts by weight.
The autolysate for the new strain LE16 of the lysobacter enzymolyticus is used for preparing an LE16 autosolution, and comprises the following steps: a. seed pot medium: dissolving peptone (10 parts), beef extract (6 parts), naCl, tryptophan betaine (0.5 parts), asparagine (0.5 parts) and vitamin B complex (0.1 parts) in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone (6 parts), (NH) 4 ) 2 SO 4 Beef extract (2 parts), glucose, KH 2 PO 4 、NaCl、MgSO 4 Tryptophan betaine (0.5 part), asparagine (0.5 part), vitamin B complex (0.1 part) and distilled water, wherein the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: sterilizing the culture solution of the seed tank at 120deg.C under vacuum degree of 0.1Mpa for 20min, cooling to 32deg.C, inoculating LE16 bacteria solution, and dark culturing at 30deg.C at 150 r/min until autolysis occurs;
d. seed pot culture: sterilizing the seed tank culture solution in step a at 120deg.C under vacuum degree of 0.12Mpa for 20min, cooling to 33deg.C, inoculating LE16 bacterial solution, and sterilizing at 33deg.C for 150 r/min with ventilation amount of 0.25M 3 Culturing for 36 hr at/min to obtain seed solution with LE16 bacteria solution inoculation amount of 10 3 cell/mL；
e. Culturing in a fermentation tank: injecting fermentation tank culture solution into fermentation tank, sterilizing at 120deg.C under vacuum degree of 0.1Mpa for 20min, cooling to 31deg.C, and introducing seed solution at aeration rate of 0.25M 3 And (3) culturing at 32 ℃ for 120 hours at 150 revolutions per minute, injecting the primary self-solution, continuously culturing for 85 hours, preparing the LE16 self-solution, aseptically filling and sealing.
When powdery mildew of flue-cured tobacco and gray mold of tomato occur, LE16 of the above example is sprayed from solution until the leaf surface and stem surface are wetted and water is dropped.
The LE16 of the first embodiment is sprayed with rape leaves in an amount of 10mL each, and the effect of preventing and treating gray mold of rape is 62.38%, which is equivalent to the chemical pesticide carbendazim. The pesticide composition is used for spraying flue-cured tobacco leaves, 10mL of each plant is used for preventing and treating powdery mildew, the effect is 81.52 percent, and the pesticide composition is equivalent to triazolone. The effect is shown in figure 1, wherein A represents the morbidity of tobacco, B represents the morbidity of rape, C and D represent the prevention and treatment effects of the tobacco and the rape (compared with triazolone) sprayed with LE16 self-solution.
While the LE16 autolyzed effect of examples 2-6 was significantly better than that of example one. And the effect of example 6 is relatively excellent.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims (5)

1. A method for preparing a self-solution for a new strain LE16 of lysobacter enzymolyticus (Lysobacter enzymogenes), which is characterized by comprising the steps of: the method comprises the following steps:
a. seed pot medium: dissolving peptone, beef extract, naCl, tryptophan betaine, asparagine and vitamin B complex in distilled water, wherein the initial pH value is 7.0-7.2; preparing a seed tank culture solution;
b. fermentation broth: peptone, (NH) 4 ) 2 SO 4 Beef extract, glucose, KH 2 PO 4 、NaCl、MgSO 4 The tryptophan betaine, the asparagine and the vitamin B complex are dissolved in distilled water, and the initial pH value is 7.0-7.2; preparing a fermentation tank culture solution;
c. preparing a primary self-solution: inoculating LE16 bacteria solution after sterilizing the seed tank culture solution, and culturing in dark until autolysis of the culture solution occurs;
d. seed pot culture: c, sterilizing the seed tank culture solution in the step a, inoculating LE16 bacterial solution, and culturing to obtain seed solution;
e. culturing in a fermentation tank: injecting a fermentation tank culture solution into a fermentation tank, sterilizing, inoculating a seed solution for culture, and then injecting an original self-solution for continuous culture to obtain an LE16 self-solution; wherein the new strain LE16 of the lysobacter enzymolyticus is preserved in the China general microbiological culture Collection center with the preservation number of No.14215.
2. The method for preparing a novel strain LE16 of lysobacter enzymolyticus (Lysobacter enzymogenes) from a solution according to claim 1, wherein the method comprises the steps of: in the step c, sterilizing for 15-25min at 110-130 ℃ and 0.05-0.2Mpa, cooling to 30-35 ℃, inoculating LE16 bacterial liquid, and dark culturing at 25-35 ℃ and 140-160 rpm until autolysis occurs in the culture liquid.
3. The method for preparing a novel strain LE16 of lysobacter enzymolyticus (Lysobacter enzymogenes) from a solution according to claim 1, wherein the method comprises the steps of: in step d, sterilizing the seed tank culture solution in step a at 110-130deg.C and vacuum degree of 0.05-0.2Mpa for 15-25min, cooling to 30-35deg.C, inoculating LE16 bacteria solution at 28-35deg.C, 140-160 rpm, and ventilation of 0.25M 3 Culturing for 34-38 hours at/min.
4. A method for preparing a novel strain LE16 of lysobacter enzymolyticus (Lysobacter enzymogenes) as claimed in claim 3 from solution, wherein: in step d, the inoculation amount of the LE16 bacterial liquid is 10 3 cell/mL。
5. The method for preparing a novel strain LE16 of lysobacter enzymolyticus (Lysobacter enzymogenes) from a solution according to claim 1, wherein the method comprises the steps of: in step e, the fermentation tank culture solution is injected into the fermentation tank, sterilized for 15-25min at 110-130 ℃ and 0.05-0.2Mpa, cooled to 30-35 ℃, and the seed solution is introduced into the fermentation tank at ventilation of 0.25M 3 And (2) culturing at the temperature of 28-35 ℃ for 115-125 hours at 140-160 rpm for 72-96 hours after injecting the primary self-solution, and preparing the LE16 self-solution.
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