CN114292774A

CN114292774A - Mycelium or thallus, application thereof and method for breeding bacterial strain by utilizing mycelium or thallus

Info

Publication number: CN114292774A
Application number: CN202111532064.2A
Authority: CN
Inventors: 陈俊升; 陈灼湖; 刘炳珠; 刘茂花; 林小敏; 郑毅伟
Original assignee: Fujian Kaili Bio Product Co ltd; Shanghai Jiaotong University
Current assignee: Fujian Kaili Bio Product Co ltd; Shanghai Jiaotong University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-04-08

Abstract

The invention discloses a mycelium or thallus, application thereof and a method for breeding strains by using the mycelium or thallus. The mycelium or the thallus is obtained from fermentation liquor at the end of a rapid growth and accumulation stage of production of secondary metabolites in the fermentation production process of the strains. When the mycelium or the thallus obtained by the invention is applied to the breeding of the streptomyces lavendulae strain with the resistance to the self metabolite, the high-yield bacterial strain of the zhongshengmycin is screened, the industrial fermentation production level of the high-yield bacterial strain can be greatly improved, the industrial production cost of the zhongshengmycin is greatly reduced, a foundation is laid for the market demand of the zhongshengmycin in biological pesticides, and the expanded use in the fields of large-scale flight control drugs in green ecological agriculture and the like is met.

Description

Mycelium or thallus, application thereof and method for breeding bacterial strain by utilizing mycelium or thallus

Technical Field

The invention relates to the technical field of biological pesticides, in particular to a mycelium or thallus, application thereof and a method for breeding a strain tolerant to a self metabolite.

Background

According to the statistics of the Food and Agriculture Organization (FAO) of the United nations, in the agriculture and forestry production process all over the world; the annual loss caused by diseases, insect pests and weeds is more than one third. Approximately 300 million chemical pesticide intoxication events occur worldwide each year. In developing countries, collective poisoning events caused by food contamination with pesticides occur around 1650 worldwide each year. Therefore, the vigorous development of biopesticides with safety, low toxicity and non-toxicity, low and no accumulation, and no environmental pollution has become an inevitable trend and global consensus.

Zhongshengmycin (chemical name: 1-N glycoside chain internal fixation-2-amino L-lysine-2 deoxy gulo sugar amine; molecular formula: C)₁₉H₃₄O₈N₈(ii) a Molecular weight: 502.0) is a novel agricultural antibiotic biological pesticide, the application belongs to the category of bactericide, and is used for preventing and treating crop diseases, the producing strain is separated from the soil of Hainan island, and is named as Streptomyces lavendulae Hainanensis variant (Streptomyces lavondulans. hainanensis), and the preservation number is CGMCC No. 1026. As an agricultural antibiotic biological pesticide, the compound has strong inhibiting or killing effects on gram-positive bacteria, gram-negative bacteria and filamentous fungi. It ultimately leads to bacterial death by inhibiting peptide bond formation of pathogenic bacterial proteins; can inhibit growth of hypha and sporeThe germination of the seed plays a role in preventing and treating fungal diseases; has good control effect on rice bacterial leaf blight, cabbage soft rot, apple ring spot, apple leaf spot and the like, is safe and low-toxic to human and livestock, and is compatible and friendly with the environment.

Therefore, the industrial fermentation level of the zhongshengmycin is improved, the energy conservation and the consumption reduction of the zhongshengmycin industrialization are realized, the productivity of the zhongshengmycin raw pesticide is expanded, the market share of the zhongshengmycin in green food production data is expanded, the brick is added for the food health industry of human, and the method has good social benefit and economic benefit.

The industrialized fermentation production of the zhongshengmycin has been carried out for 20 years so far, but the laboratory research and the industrialized fermentation production process are subjected to the repression and feedback inhibition regulation of various single factors and multi-factors such as the metabolism products of the zhongshengmycin producing bacteria, such as glucose effect, the zhongshengmycin of the secondary metabolism products of the zhongshengmycin and the like in the fermentation production process, so that the fermentation production level of the zhongshengmycin cannot be further improved, the biological potency is loin between 8000 mu/ml and 12000 mu/ml, the content of a zhongshengmycin mother medicine product cannot be further improved, and the market supply requirements of the zhongshengmycin mother medicine and the preparation thereof in the biological pesticide and the field of flight control of comprehensive application in a large range and large area are limited for extended use.

Based on the above, starting from the zhongshengmycin producing strain, the zhongshengmycin high-yield strain which can tolerate the zhongshengmycin of the self secondary metabolite and other self metabolites is expected to be screened and applied to industrial fermentation production, so that the industrial fermentation production level of the zhongshengmycin in green production data is improved, a large amount of water-soluble zhongshengmycin high-content mother medicines are obtained, and the requirement of rapidly developing green ecological agriculture is met.

Disclosure of Invention

In order to solve the technical problems, the invention provides a mycelium or thallus, application thereof and a method for breeding strains by using the mycelium or thallus. The technical scheme of the invention starts with the zhongshengmycin producing strain, screens the zhongshengmycin high-yield strain which can tolerate the self secondary metabolite (such as zhongshengmycin) and other self metabolites and applies the zhongshengmycin high-yield strain to the industrialized fermentation production, thereby realizing the improvement of the industrialized fermentation production level of the zhongshengmycin in green production data, obtaining a large amount of water-soluble high-content mother medicines of the zhongshengmycin and meeting the requirement of the rapidly developed green ecological agriculture.

In order to achieve the above object, the present invention is achieved by the following aspects:

in a first aspect, the present invention provides a mycelium or a thallus obtained from a fermentation broth at a last stage of a rapid growth and accumulation stage of production of a secondary metabolite in a fermentation production process of a strain.

In some preferred embodiments, the strain is Streptomyces lavendulae, preferably, a Streptomyces lavendulae varietal (Streptomyces lavendovasculus var. hainanensis); more preferably, the strain is the Hainan variant of the Streptomyces lavendulae with the preservation number of CGMCC NO.1026 or CGMCC 61906.

Preferably, the fermentation meets one or more of the following conditions:

the fermentation temperature is 26-35 ℃;

the air ventilation volume is 0.5V/VM-1.5V/VM;

the stirring speed of the fermentation tank is 50rpm-200 rpm;

the fermentation culture period is 3-10 days;

the formula of the fermentation medium used for fermentation comprises: 4.0-15.0% of glucose, 1.0-6.0% of corn starch, 0.5-7.5% of corn flour, 3.0-12.0% of soybean meal, 0.2-1.6% of sodium chloride, 0.1-1.8% of ammonium chloride, 0.2-1.6% of calcium carbonate, 0.01-0.13% of monopotassium phosphate, 0.01-0.16% of magnesium sulfate and pH6.0-7.0, wherein the percentages are mass volume percentages.

In a second aspect, the present invention provides a method for breeding a strain tolerant to self-metabolites, the method comprising the steps of:

step S1: fermenting the selected starting strain to obtain fermentation liquor at the end of a rapid growth and accumulation stage of secondary metabolite production and preparing thallus filtrate;

step S2: obtaining fermentation liquor at the end of growth stagnation stage of the same batch of secondary metabolite production, filtering to obtain filtrate, and concentrating, preferably concentrating under reduced pressure to obtain concentrated fermentation liquor containing substrate factors;

step S3: preparing a screening culture medium containing concentrated fermentation liquor;

step S4: and coating and culturing the thallus filtrate on the screening culture medium, and screening to obtain the bacterial strain which is tolerant to the self metabolite.

Preferably, the screening culture medium is a gradient concentration culture medium of a substrate factor, and the maximum value of the biological potency of the biotoxin in the gradient concentration culture medium is 20000-50000 mu/ml; for example 30000. mu.l/ml.

And/or 15-20 g/L, preferably 18-19 g/L, 1.2-1.5 mg/L, preferably 1.3-1.4 mg/L, 25-30 mg/L, preferably 28-29 mg/L, 27-30% of bacterial concentration, preferably 28-29% and pH 6.5-7.0 of reducing sugar at the end stage in the rapid growth accumulation stage of production;

and/or the titer of the zhongshengmycin in the terminal stage of the rapid growth accumulation stage of the production is 6000 to 10000, preferably 7000 to 8000 mu/ml.

And/or, said production of growth arrest stage end-stage reducing sugars: 5-7 g/L, amino nitrogen: 0.6-0.9 mg/L, 8-12 mg/L of total phosphorus, 25-27% of bacterial concentration and 6.8-7.0 of pH;

and/or the titer of the biotins in the terminal phase of the production growth stagnation stage is 10000-12000 mu/ml.

More preferably, in the step S1, the starting strain is Streptomyces lavendulae, preferably, a Streptomyces lavendulae Hainanensis variant (Streptomyces lavendulae var. hainanensis), more preferably, a Streptomyces lavendulae Hainanensis variant with a preservation number of CGMCC No.1026 or CGMCC 61906.

In some preferred embodiments, in the step S1, the fermentation satisfies one or more of the following conditions:

the fermentation temperature is 26-35 ℃;

the air ventilation volume is 0.5V/VM-1.5V/VM;

the stirring speed of the fermentation tank is 50rpm-200 rpm;

the fermentation culture period is 3-10 days;

In some preferred embodiments, in step S2, the thallus filtrate is obtained under aseptic conditions by: fully stripping and dispersing mycelium of the fermentation liquor and a culture medium of the fermentation liquor, and naturally filtering at normal temperature by using sterile cotton (preferably sterilized) as a filter medium to obtain thallus filtrate;

and/or, the concentrated fermentation broth containing the substrate factor is obtained under aseptic conditions by: fully stripping and dispersing mycelia of the fermentation liquor and a culture medium of the fermentation liquor, naturally filtering at normal temperature by using sterile cotton (preferably sterilized) as a filter medium through a Buchner funnel, and filtering the obtained filtrate through a sterile bacterial funnel to obtain concentrated fermentation liquor containing substrate factors; the concentration is preferably concentration under reduced pressure;

preferably, the concentration under reduced pressure comprises the following steps: putting the filtrate into a rotary evaporator provided with a filter, and concentrating under reduced pressure under the conditions that the water bath temperature is less than 45 ℃, the vacuum degree is-0.098 Mpa and the cooling water temperature is 5-7 ℃ to obtain concentrated fermentation liquor containing substrate factors; the volume ratio of the concentrated fermentation liquor to the fermentation liquor is preferably 1 (5-10), for example 1: 5.

In some preferred embodiments, in said step S2,

putting the fermentation liquor into a glass ball before filtering, and oscillating to fully strip the mycelium in the fermentation liquor from the culture medium;

preferably, 20-50, preferably 30-40 glass balls are arranged in every 500ml of fermentation liquor; and/or the oscillation speed is 220rpm, and the oscillation time is 60 min.

In some preferred embodiments, in step S3, the screening medium is a gradient concentration medium.

In some preferred embodiments, a step of mutagenizing the bacterial filtrate is further included before the step S4.

In some preferred embodiments, the mutagenesis is cumulative decreasing ultraviolet-light revival composite mutagenesis, wherein ultraviolet light and sunlight are irradiated for 2-10 times alternately, and the irradiation time is decreased gradually.

Preferably, the ultraviolet light is from a 30W ultraviolet lamp and the effective interval wavelength is

The sunlight comes from a 30W fluorescent lamp.

More preferably, the number of times of the alternating irradiation of the ultraviolet light and the sunlight is five, wherein,

the first time is as follows: turning on the ultraviolet lamp for 30s, and then turning off the ultraviolet lamp for 20 s; starting the fluorescent lamp for 20 s; turning off the fluorescent lamp for 20 s;

the second time is that: turning on the ultraviolet lamp for 25s, and then turning off the ultraviolet lamp for 20 s; starting the fluorescent lamp for 20 s; turning off the fluorescent lamp for 20 s;

the third time is that: turning on the ultraviolet lamp for 20s, and then turning off the ultraviolet lamp for 10 s; starting a fluorescent lamp for 10 s; turning off the fluorescent lamp for 10 s;

the fourth time is: turning on the ultraviolet lamp for 15s, and then turning off the ultraviolet lamp for 10 s; starting a fluorescent lamp for 10 s; turning off the fluorescent lamp for 10 s;

the fifth time is: turning on the ultraviolet lamp for 10s, and then turning off the ultraviolet lamp for 5 s; starting a fluorescent lamp for 5 s; the fluorescent lamp is turned off for 5 s.

In a third aspect, the invention provides the application of the mycelium or the thallus in breeding of the self-metabolite-resistant strain.

In the technical scheme, the inventor reasonably breeds the zhongshengmycin high-yield strain which is tolerant to the self metabolite from the source of the zhongshengmycin producing strain, applies to the zhongshengmycin industrial fermentation production and finally improves the zhongshengmycin industrial fermentation production level.

The specific process is as follows: the method comprises the steps of taking mutagenic bacteria (UV-3) of Hainan variant strain of Streptomyces lavendulae (with the preservation number of CGMCC No.1026) for industrial fermentation production of zhongshengmycin, selecting a fermentation tank batch with the industrial fermentation production fermentation level being more than or equal to 10000 mu/ml, taking mycelium in fermentation liquor at the last stage of fast growth and accumulation of the zhongshengmycin production in secondary metabolites in the fermentation process of the fermentation tank batch as a target starting strain for screening the tolerant self metabolites, enabling the target starting strain to obtain variation by an accumulation and decrement ultraviolet-light revival composite mutagenesis means, taking concentrated solution of filtrate of the fermentation liquor at the last stage of the slow growth and accumulation of the zhongshengmycin production in the secondary metabolites produced by industrial fermentation of the same batch as a substrate factor source for screening the zhongshengmycin producing bacteria of the tolerant self metabolites, taking concentrated solution containing the substrate factor as a source for preparing a gradient concentration culture medium, rationally breeding strains with higher concentration of metabolites of the zhongshengmycin producing strains. The target starting strain, the composite mutagenesis means and the screening substrate factor are combined and applied to select a single colony of the target strain with mutagenesis variation tolerance and higher concentration of self metabolite, and the high-yield zhongshengmycin strain is obtained through preliminary screening, secondary screening and stability tests. The fermentation level high-yield character of the zhongshengmycin high-yield strain is stable in the large-scale industrialized fermentation process.

The method for rationally breeding the zhongshengmycin high-yield strain tolerant to the self metabolite is a method for rationally synthesizing various screening factors to design, develop mutagenesis, screen and optimally detect the zhongshengmycin high-yield strain according to the problems that the zhongshengmycin producing strain plays a role in feedback inhibition or repression in the process of producing the zhongshengmycin to the zhongshengmycin producing strain by the zhongshengmycin producing strain in the whole fermentation growth production process, so that the industrialized fermentation level of the zhongshengmycin is stagnated and the fermentation level cannot be further improved.

The autogenous metabolites such as zhongshengmycin are produced by using inorganic salts such as carbon source, nitrogen source and phosphate in a fermentation medium in the fermentation production process of the zhongshengmycin producing bacteria, decomposing and converting the carbon source, nitrogen source and phosphate into energy such as ATP required by the zhongshengmycin producing bacteria for growth and production, amino acid, oligopeptide, polypeptide, monosaccharide, disaccharide and polysaccharide, enzyme substances with biological activity and vitamins, minerals and the like required by other zhongshengmycin producing bacteria for growth and reproduction so as to ensure the normal growth and reproduction of the zhongshengmycin producing bacteria, realize the transition from the reproduction growth period to the production period of antibiotic, complete the primary metabolic process of thallus growth and incremental reproduction of the zhongshengmycin producing bacteria in the fermentation process, and further transfer into the secondary metabolic process accumulated in the zhongshengmycin production; the growth and the propagation of the zhongshengmycin producing bacteria, the rapid growth and the accumulation of the zhongshengmycin production, the growth and the growth retardation of the zhongshengmycin production, the bacterial growth decay and even the production of various metabolites of the zhongshengmycin producing bacteria generated in the period process of resisting the production decline and the decomposition products of the zhongshengmycin producing bacteria for decomposing and utilizing the culture medium are completed.

The target starting strain for rationally breeding the zhongshengmycin high-yield strain tolerant to the self metabolite is selected according to the fact that mycelium grows and breeds when the zhongshengmycin producing strain undergoes primary metabolism in the fermentation growth and production process, and then is induced to be converted into the fermentation growth and production cycle process of the zhongshengmycin product produced by secondary metabolism, and mycelium at the end of the secondary metabolite fast growth and accumulation stage of the zhongshengmycin producing strain, which passes through the thallus growth and reproduction stage, the fast growth and accumulation stage of the zhongshengmycin production in the secondary metabolite, the growth and stagnation stage of the zhongshengmycin production in the secondary metabolite and the fast growth and accumulation stage of the thallus growth and death stage in the production process are used as the target starting strain for rationally breeding the zhongshengmycin high-yield strain.

The mycelium of the last stage in the secondary metabolite zhongshengmycin production rapid growth accumulation stage in the zhongshengmycin producing and metabolic process (the last stage in the production rapid growth accumulation stage refers to the period from the beginning to the post 2/3 time when the biological potency or concentration of the target secondary metabolite of the fermentation liquid is increased to the maximum value after the speed increase, and the maximum value can be determined by a person skilled in the art through real-time measurement or deduced through drawing a curve), is the mycelium which is converted from primary metabolism into secondary metabolism and has the highest growth and production capacity in the zhongshengmycin producing and bacterial fermentation production process, and the mycelium of the last stage in the secondary metabolite zhongshengmycin production rapid growth accumulation stage which grows in the secondary metabolite zhongshengmycin self fermentation growth production environment in industrial scale production is used as the target starting strain for screening, the screened strain is more suitable for the primary metabolite and the secondary metabolite in the self growth and production process of the industrial fermentation of the zhongshengmycin; the permeability of the cell wall of the mycelium in the fast growth and accumulation stage of the secondary metabolite zhongshengmycin production is better than that of the cell wall of bacterial colony spores, the composite mutagenesis of ultraviolet-light revival is easier to accept, the target strain which can tolerate the metabolite of the mycelium is easier to screen on the culture medium of the sexual breeding, and the capability of the bred target strain in adapting to the industrialized fermentation growth and production environment is stronger.

The selection of the screening substrate factor for rationally breeding the zhongshengmycin high-yield strain tolerant to the self metabolite is carried out according to the fermentation growth and production process of the zhongshengmycin producing strain, the zhongshengmycin producing strain undergoes primary metabolism thallus growth and propagation and is further induced and converted into the fermentation growth and production metabolic cycle process of a secondary metabolism produced zhongshengmycin product, and the concentrated solution of the filtrate of the fermentation liquor which is rich in the zhongshengmycin in the secondary metabolism product and other various metabolites produced in the secondary metabolism product zhongshengmycin producing strain and decomposition substances of a culture medium at the end of the growth and growth retardation stage in the growth and production process of the zhongshengmycin producing strain is used as the source of the screening substrate factor for rationally breeding the zhongshengmycin high-yield strain.

The production of the zhongshengmycin in the secondary metabolite of the zhongshengmycin is in the end of the production growth stagnation phase (the period of fermentation tank discharge). The zhongshengmycin is fermented to the end of the growth arrest stage of the zhongshengmycin production of the secondary metabolite, the fermentation liquor is rich in the zhongshengmycin of the secondary metabolite, other various metabolites produced by primary metabolism and secondary metabolism and culture medium resolvent, the substances can suppress or feedback inhibit the production of the zhongshengmycin, and the zhongshengmycin is slowly increased or not increased at the stage; if the fermentation culture is continued, the step of growth and death of the zhongshengmycin-produced metabolic thallus is carried out, substances such as active enzymes in metabolites of the zhongshengmycin in the fermentation liquor in the thallus growth and death step can even decompose the formed zhongshengmycin into other small molecular substances, so that the fermentation level of the zhongshengmycin is not increased any more, even falls down, the increase and waste of the fermentation time are caused, and the increase of the energy consumption cannot achieve the uneconomical effect of improving the fermentation level of the zhongshengmycin.

In the breeding of antibiotic self-product tolerant strains, the traditional method basically performs the breeding of single factors such as feedback inhibition of self secondary metabolites, release of glucose effect, phosphate repression and the like, so the effect is weaker.

Different from the prior art, the fermentation liquor of the secondary metabolite zhongshengmycin production growth retardation stage of the zhongshengmycin producing bacteria in the production and metabolism process is rich in other various complex self metabolites in the production and fermentation process of the zhongshengmycin besides the zhongshengmycin of the secondary metabolite zhongshengmycin. The concentrated solution of the fermentation liquor filtrate at the end of the production and growth retardation stage of the secondary metabolite zhongshengmycin is selected as a source of a screening substrate factor for screening a self metabolite tolerant strain of the zhongshengmycin, so that not only can the feedback inhibition of the zhongshengmycin of the self secondary metabolite be relieved, but also various carbon sources generated or remained by catabolism in a fermentation medium in the fermentation process of the zhongshengmycin producing bacteria, such as starch, polysaccharide, disaccharide, glucose, pentose, tetrose, triose, alpha-ketoglutaric acid, oxaloacetic acid and the like, can be relieved; nitrogen sources, such as various amino acids like beta-lysine of beta-amino acids, oligopeptides, short peptides, polypeptides and zhongshengmycin precursors, N-acetylglucosamine which can be converted into cell wall peptidoglycan of producer bacteria of the basic structural unit of aminoglycoside antibiotics, N-acetylmuramic acid, repression or feedback inhibition of intracellular and extracellular enzymes with various biological activities, such as oxidoreductases, dehydrogenases, etc. The method is used for relieving the repression and feedback inhibition of the self-product zhongshengmycin and the growth production metabolite thereof in the fermentation production process of the zhongshengmycin producing bacteria, realizing the tolerance to various repression or feedback inhibition single factors or multiple factors and multiple synergistic factors, and finally relieving or partially relieving the influence of various repression or feedback inhibition on the zhongshengmycin production, and is the root for improving the industrialized fermentation production level of the zhongshengmycin.

The mutagenesis method for rationally breeding the high-yield zhongshengmycin strain tolerant to the metabolite of the strain adopts an accumulative decrement ultraviolet-light reactivation composite mutagenesis method, which is characterized in that single-factor physical mutagenesis generated by a zhongshengmycin original strain through single ultraviolet irradiation is changed into the starting strain to generate mutagenesis through ultraviolet irradiation, and then a natural light daylight lamp is used for carrying out combined application of a light reactivation repair function on a mutation site generated by the zhongshengmycin producing strain to form the ultraviolet-light reactivation double-factor composite mutagenesis; furthermore, the ultraviolet-light revived compound mutagenesis is carried out for multiple times of decrement dosage to carry out cumulative decrement mutagenesis, so that the effect that the mutation site of thymine dimer between single strands or double strands of DNA formed as a result of the ultraviolet irradiation mutagenesis does not easily generate reversion repair after multiple times of dimer formation → dimer depolymerization → dimer formation → dimer depolymerization → dimer formation.

In the ultraviolet-light revival composite mutagenesis method, the ultraviolet is selected to be the wavelength in the interval most effective for the mutagenesis of the microbial strains

The sterilization and mutagenesis in this interval of ultraviolet wavelengths is best. Biological effect of ultraviolet ray can cause biological damage of DNA and thymine base pair is dimerized, so that formation of thymine dimer is the main path for changing DNA biological activity and causing thallus death or variation by ultraviolet ray, and has great effect on causing thallus variation. When DNA undergoes self-replication, the double strands become single-stranded first, and then each forms a complementary strand with a nearby base, however, the two strands of DNA are cross-linked due to thymine forming a dimer between the two strands, which prevents the double strands from separating and replicating. Adjacent thymines in the same strand form a diThe mer, too, may prevent normal base pairing. In normal conditions, thymine pairs with adenine and if two adjacent thymines form a dimer, the original pairing may be altered, disrupting the normal incorporation of adenine, at which point replication is abruptly halted, or performed incorrectly. If the replication is made erroneously and there is an altered base sequence on the newly formed strand, the DNA strand whose base sequence has been altered will still replicate as it is during the subsequent replication process, resulting in a molecule whose base sequence is wrong on both strands and thus causing a mutation; however, the effect of ultraviolet rays on the genetic activity of DNA can be recovered by visible light fluorescent lamps, which is known as a photoreaction. Because the substance damaged by ultraviolet ray is mainly dimer, the dimer is decomposed into monomer under the action of the photo-reactivating enzyme to restore the DNA to normal, so the strain is subjected to ultraviolet ray mutagenesis treatment and is prevented from being irradiated by visible light. Ultraviolet light revivification, which shows that ultraviolet mutagenesis is a complex process, the initial effect of ultraviolet light is to induce genetic materials to be in various types of metastable, and a certain time is needed for transition from the metastable to a stable mutation state, so the ultraviolet light and visible white light fluorescent lamp are used for repeatedly processing the mycelium by using the time accumulation and decrement ultraviolet light-revivification composite mutagenesis method, so that each site of the mycelium which has formed mutation errors is not easy to recover to an initial normal base sequence, and the mutation rate and the mutation amplitude of the mycelium can be improved.

The selection of the culture medium for rationally selecting and breeding the high-yield zhongshengmycin strain tolerant to the self metabolite is a gradient concentration culture medium for the high-yield zhongshengmycin strain tolerant to the self growth to produce the metabolite, and the concentrated solution of the filtrate of the fermentation liquor rich in the zhongshengmycin and the self metabolite at the end of the secondary metabolite zhongshengmycin production growth retardation stage in the industrial fermentation process of the zhongshengmycin is used as a source of a screening substrate factor for rationally selecting the high-yield zhongshengmycin strain. The concentrated solution rich in the zhongshengmycin and other primary metabolites, secondary metabolites and culture medium decomposition products generated in the zhongshengmycin fermentation growth production process is used as a main component of the screening culture medium, and the gradual concentration increase of the concentration of the self metabolites from low to high is formed on the culture medium of the screening plate, so that the targeted high-yield strain which is tolerant to the self metabolites is bred reasonably and rapidly in a targeted manner. The mycelium which is mutagenized by the accumulation and decrement ultraviolet-light revival composite mutagenesis method to generate mutation can be expressed and shown rapidly on a screening substrate factor gradient culture medium for rational breeding, and the working intensity is greatly reduced.

The gradient concentration culture medium of the zhongshengmycin producing bacteria tolerant to the self metabolites is characterized in that a layer of screening culture medium is paved on the bottom layer of a sterilized double-dish plate with the diameter of 90mm, the culture medium is obliquely placed, the culture dish is horizontally placed after the screening culture medium on the bottom layer is solidified, then the screening culture medium which is rich in the concentrated solution of the filtrate of the fermentation liquor of the zhongshengmycin, other primary metabolites, secondary metabolites and culture medium resolvent generated in the fermentation growth production process of the zhongshengmycin producing bacteria is added into the culture dish, and the screening culture medium is solidified, so that the screening culture medium for the zhongshengmycin producing bacteria rational breeding with gradually increased concentration of screening substrate factors of various substances of the zhongshengmycin and the self metabolites of the zhongshengmycin producing bacteria is formed from low to high.

The method for rationally breeding the zhongshengmycin high-yield strain tolerant to the self metabolite selects mycelium with high-activity zhongshengmycin production capacity at the tail section in the rapid growth and accumulation stage of the zhongshengmycin production of the secondary metabolite in the process of producing the zhongshengmycin by fermenting the zhongshengmycin producing strain as a target starting strain; selecting high-unit zhongshengmycin in fermentation liquor at the end of the growth retardation stage of the zhongshengmycin production of secondary metabolites produced by the fermentation of zhongshengmycin producing bacteria, and a large amount of carbon sources, nitrogen sources, phosphates, precursor substances, bioactive enzymes and the like generated in the processes of primary metabolism and secondary metabolism as tolerant screening substrate factors; a compound mutagenesis method of cumulative decrement of time and dosage and ultraviolet-light revival is used as a mutagenesis factor; the culture dish of the culture medium with gradient concentration, wherein the concentration of the self metabolite of the culture medium is gradually increased from low to high, and the culture dish is represented by the zhongshengmycin and is used as a detection tool for screening and detecting target strains which realize mutagenic mutation.

The combined screening method of screening culture medium by using mycelium or thallus at the end of the fast growth and accumulation stage of the production of the zhongshengmycin in the secondary metabolite as a target starting strain, using the concentrated solution of the filter liquor of the fermentation liquor at the end of the growth and stagnation stage of the production of the zhongshengmycin in the secondary metabolite as a screening substrate factor source, using cumulative diminishing ultraviolet ray-light reviving composite mutagenesis as a mutagenesis method, and using a culture dish with gradually increased concentration of the self metabolite formed on the culture medium of a screening plate from low to high as a detection tool to screen the culture medium is adopted to breed the high-yield zhongshengmycin strain resistant to the self metabolite, screen a zhongshengmycin producing strain, and apply the high-yield zhongshengmycin producing strain to 40m after stability verification³The fermentation tank carries out industrialized fermentation mass production, the fermentation level of the selected bacterial strain is improved by 52.5 percent under the same fermentation process and cycle conditions as the industrialized fermentation of a reference bacterial strain, and the fermentation unit can reach 18050 mu/ml, thereby the fermentation level of the obtained bacterial strain for producing the zhongshengmycin is greatly improved compared with the prior art.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

when the mycelium or the thallus obtained by the invention is applied to breeding of the streptomyces lavendulae strain with self-metabolite resistance, a zhongshengmycin high-yield strain is screened, and when the high-yield strain is used for industrially producing zhongshengmycin, the industrial fermentation production level of the zhongshengmycin can be greatly improved by more than 50 percent, and the highest industrial fermentation production level can reach more than 18000 mu/ml. Therefore, a large amount of water-soluble zhongshengmycin mother medicine dry powder products with the zhongshengmycin content of more than 500000 mu/g are obtained, the industrialized production cost of the zhongshengmycin is greatly reduced, a foundation is laid for the market demand of the zhongshengmycin in biological pesticides, and the expanded use of the fields of large-scale flight control medicines in green ecological agriculture and the like in a large scale is met.

Biological material preservation information

The Streptomyces lavendulae Hainanensis variant (Streptomyces lavendianensis var. hainanensis) UV-11 is preserved in Guangdong province microorganism culture collection (GDMCC) at 31/8/2021, and the preservation address is as follows: the institute of microbiology (Guangdong province center for microbiological analysis and detection) of Guangdong province, building 59 of Mieli Zhou 100, Guangzhou city. And E, postcode: 510070, accession number: GDMCC No.61906, culture name UV-11, taxonomic designation Streptomyces sp.

Drawings

FIG. 1 shows the HPLC test results of the control strain UV-3;

FIG. 2 is the HPLC test result of the UV-11 strain of the present invention;

FIG. 3 illustrates the structure of the screening medium of the present invention.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Wherein, the formula of the culture medium related to fermentation and the fermentation culture conditions are as follows:

the seed culture medium formula (the components are in mass volume percentage): 1.0 percent of glucose, 1.5 percent of corn starch, 1.0 percent of corn flour, 2.0 percent of soybean meal, 0.3 percent of sodium chloride, 0.3 percent of ammonium chloride and 0.3 percent of calcium carbonate, and the pH value is adjusted to 6.6.

The fermentation medium formula (the components are in mass volume percentage): 5.0 percent of glucose, 2.0 percent of corn starch, 2.0 percent of corn flour, 4.0 percent of soybean flour, 0.6 percent of sodium chloride, 0.8 percent of ammonium chloride, 0.7 percent of calcium carbonate, 0.02 percent of monopotassium phosphate and 0.05 percent of magnesium sulfate, and the pH value is adjusted to be 6.6.

The fermentation culture conditions are as follows: the fermentation temperature is 26-35 ℃; the air ventilation volume is 0.5V/VM-1.5V/VM; the stirring speed of the fermentation tank is 50rpm-200 rpm.

Wherein, the related inclined plane and plate culture medium formula is as follows:

slant and plate culture medium (components are mass volume percent): 1.05% of soluble starch, 0.06% of peptone, 0.07% of magnesium sulfate, 0.25% of sodium nitrate, 0.05% of potassium chloride, 0.02% of ferrous sulfate, 0.12% of potassium dihydrogen phosphate and 2.05% of agar, and adjusting the pH value to 6.6.

Example 1A mutagenized strain (UV-3) of Hainan variant strain of Streptomyces lavendulae (preservation number CGMCC No.1026) as an industrial production strain for the industrial fermentation production of zhongshengmycin

The strain UV-3 is a mutant strain of Hainan variant of Streptomyces lavendulae, which is obtained by mutagenesis of a Hainan variant strain of Streptomyces lavendulae with the preservation number of CGMCC No.1026 and is applied to industrial fermentation production of zhongshengmycin.

UV-3 is set at 40m³Carrying out industrialized fermentation in the fermentation tank (according to the formula of the culture medium and the fermentation culture conditions of the fermentation, and the fermentation culture period is 5 days), and collecting fermentation liquor at different stages in the fermentation process.

The industrialized fermentation liquor is sampled and collected from '0' hour after inoculation of the fermentation tank according to the requirement of aseptic sampling, and the fermentation liquor sample is sampled and collected once every 12 hours. The specific method for sterile sampling comprises the following steps: sterilizing the sampling port of the Zhongshengmycin fermentation tank for 30 minutes by using live steam of which the pressure is not less than 0.3Mpa, quickly closing a steam valve of the sampling port of the fermentation tank under the protection of a flame ring, opening a discharging valve of the sampling port of the fermentation tank, and allowing fermentation liquor to flow out of the sampling port; under the protection of flame, rapidly pulling out the cotton plug at the sampling port while the sterilized glass triangular bottle with the cotton plug is under the protection of flame, rapidly acquiring fermentation liquid by aligning the bottle mouth with the fermentation liquid flowing out of the sampling port, rapidly covering the cotton plug on the triangular bottle, closing a discharge valve of the sampling port, opening a steam valve to sterilize the sampling port for 10 minutes, and closing the steam valve. And immediately putting the collected fermentation liquor sample into a refrigerator at 4 ℃ for cold storage after each collection and sampling until the batch of industrial fermentation is put into a tank. At the same time, after sampling, the sterility of the bevel, broth and the microscope were carried out immediatelyThe hypha morphology is observed by microscopic examination, and only fermentation liquor which is not infected by other mixed bacteria can be used as a sample for measuring relevant parameters of the fermentation liquor sample by slant surface, broth sterility test and microscopic observation, wherein the values of the end of the growth rapid growth accumulation stage and the end of the production growth stagnation stage are shown in Table 1. It should be noted that the biological potency increase rate v can be calculated by the following formula, wherein u₁Is a previous time t₁Biological potency of u₂Is a later time t₂Biological potency of u₁And u₂The interval time of (a) may be defined according to the specific conditions of each embodiment, in this embodiment u₁And u₂At intervals of 12 hours:

table 1.

It should be noted that the test method mentioned in table 1 is performed by the following method:

1.1 determination of the biological potency

The biological potency is determined by the method of detecting antibiotic microorganism in Chinese pharmacopoeia (2015 edition). The reagents and solutions used were: phosphate buffer (pH 7.8); a culture medium I; bacillus subtilis suspension (Bacillus subtilis number: CMCC 63501); volumetric flask (100ml, 50 ml).

1.2 determination of microbial metabolism of reducing sugars

The method for measuring the microbial metabolic reducing sugar adopts a Fehling reagent colorimetric method, and the preparation of the Fehling reagent is as follows:

1. preparing a Fehling reagent A solution: 120 grams of copper sulfate hydrate (CuSO) are accurately weighed₄·5H₂O), analytically pure, content > 99.0%), completely dissolved with 2000ml of distilled water.

2. Preparing a Fehling reagent B solution: at an open positionThe mouth glass container was filled with 2000ml of distilled water. Accurately weighing potassium sodium tartrate (C)₄H₄KNaO₆·4H₂O, analytically pure, content > 99.0%) 375 g was added thereto and stirred well until dissolved. 250 g of sodium hydroxide (analytically pure) is accurately weighed, and the solution is slowly added into the solution in several times while stirring.

3. Preparing a Fehling reagent solution C: 300 g of potassium iodide (analytically pure, content > 98.0%) are weighed out accurately, dissolved in distilled water and diluted to 1000 ml.

4. Preparing a Fehling reagent: the feilin reagent A, B, C solution was sequentially contained in a 10000ml brown reagent bottle via a large funnel and carefully shaken vigorously to homogenize, i.e., 5000ml of the feilin reagent was prepared.

5.0.1mol/L sodium thiosulfate solution preparation: 26g of sodium thiosulfate and 0.2g of anhydrous sodium carbonate are weighed, dissolved in 1000ml of water, slowly boiled for 10min and cooled. After standing for two weeks, filtration was carried out. And after filtration, calibrating according to GB/T601-2002.

6.1% preparation of starch indicator: weighing 1g of starch, adding a small amount of water to prepare a starch solution, adding boiling distilled water to 100ml, and boiling for 2 minutes to finish the preparation.

Operating procedure

And comprehensively judging the expected range of the total residual sugar concentration according to the grade and the culture time of the microbial culture medium. Centrifuging the culture solution filtrate or culture medium supernatant with the concentration of more than 10g/100ml, sucking 1ml, adding 4ml of distilled water for dilution, and sucking 1ml of the solution for determination after uniform mixing; for the filtrate or supernatant with total residual sugar concentration less than or equal to 10g/100ml and greater than 5g/100ml, 0.5ml is extracted for determination, and for the filtrate or supernatant with total sugar concentration less than or equal to 5g/100ml, 1ml is extracted for determination.

Accurately sucking 1ml of filtrate or supernatant, placing the filtrate or supernatant in a 150ml triangular flask, directly adding 20ml of Fehling reagent, uniformly heating the mixture on an electric hot plate, and starting timing after boiling. And timing for two minutes, taking the triangular flask down, and placing the triangular flask in an operating disk filled with cooling water for cooling. In the operation process, the exhaust fan is in an opening state.

After cooling, 15ml of a 2mol/L sulfuric acid solution is added, rapidly added with 0.09 mol/L98mol/L Na₂S₂SO₃The solution was titrated to a light yellow color and about 1ml of starch indicator was added by titration and titration continued until the blue color disappeared. Note the volume of sodium thiosulfate solution consumed: v_{Sample (A)}。

Blank experiment: accurately measuring 20ml of the Fehling reagent, placing the Fehling reagent in a 150ml triangular flask, adding no to-be-detected solution, and adding 1ml of distilled water to replace the sample. The procedure was followed for total sugar determination. The blank bottle should be boiled with the sample bottle at the same time to minimize accidental errors. After the titration was complete, the volume of sodium thiosulfate solution consumed was recorded as V_{Air conditioner}。

ΔV＝V_{Air conditioner}-V_{Sample (A)}. And (3) checking a sugar table according to the delta V value, and multiplying the checked sugar value by the dilution multiple to obtain the total sugar or reducing sugar concentration of the sample, wherein the unit is as follows: g/100 ml.

1.3 measurement of amino Nitrogen in microbial metabolism

Reagent preparation

0.1% methyl red indicator formulation: 0.1g of methyl red is accurately weighed into a 250ml beaker, 100ml of 75% alcohol is added, and the methyl red is completely dissolved by fully stirring with a clean glass rod. After the reagent is prepared, the reagent is filled into about 2/3-drop bottles for use.

Preparation of 0.015mol/L sulfuric acid solution: 75ml of distilled water was accurately measured in a 1000ml beaker. Accurately measuring a 2mol/L sulfuric acid solution by using a 100ml measuring cylinder, draining by using an inclined glass rod, slowly adding into a beaker, and uniformly stirring while adding.

0.02858mol/L sodium hydroxide solution preparation: 1.1432g of sodium hydroxide (analytically pure) was dissolved in distilled water and the solution was taken up to a volume of 1000ml in a volumetric flask. And (6) calibrating.

Preparation of 18% neutral formaldehyde solution: 50ml of 37-40% formaldehyde is taken and evenly mixed with 50ml of distilled water to prepare 18% formaldehyde solution.

Preparation of 1% phenolphthalein indicator: accurately weighing 1g of phenolphthalein into a 250ml beaker, adding 100ml of 75% ethanol, and stirring thoroughly with a clean glass rod until phenolphthalein is completely dissolved.

Operating procedure

2ml of the culture filtrate or the supernatant after centrifugation was accurately aspirated into a 200ml Erlenmeyer flask, and 50ml of distilled water was added.

1 drop of 1% methyl red indicator was added and titrated to reddish with 0.015mol/L sulfuric acid solution.

The solution was added dropwise to orange yellow with 0.02858mol/L NaOH solution.

Adding 2ml of 18 percent neutral formaldehyde solution, shaking evenly, and standing for 10 minutes

2-8 drops of 1% phenolphthalein indicator are added, and the solution is titrated to reddish with 0.02858mol/L sodium hydroxide solution, and the number of milliliters of the current titration of 0.02858mol/L standard sodium hydroxide solution is recorded.

1.4 microbial concentration assay

Shaking up the fermentation liquor;

weigh the centrifuge tube by electronic scale₁Pouring about 10ml of fermentation liquid into the centrifuge tube, and weighing the centrifuge tube again₂(ii) a Placing the mixture in a centrifuge, and centrifuging the mixture for 15 minutes at 3000 r/min;

the supernatant was poured off, and the remaining part was weighed to give a weight m₃；

As a result: concentration of fungus (m)₃-m₁)/(m₂-m₁)×100％。

1.5 detection of Total phosphorus by microorganisms

Instrument detection method for total phosphorus detection

The instrument used for the total phosphorus is a multi-parameter water quality tester (5B-3B (V11)) manufactured by Yongxing science and technology development limited company of Beijing Lihua, and all reagents used by the instrument are manufactured by manufacturers.

EXAMPLE 2 preparation of starting strains of interest

1. According to the determination result of the biological titer of the fermentation liquid sample, the fermentation liquid sample collected from the fermentation period of example 1 to 48 hours (end period in the rapid growth accumulation period) is selected.

2. Taking out a triangular flask which is stored in a refrigerator at 4 ℃ and is filled with a zhongshengmycin fermentation liquid sample which is not polluted by other microorganisms, carrying out ultraviolet irradiation disinfection on the outer surface of the triangular flask, carrying out omnibearing wiping disinfection on the outer wall of a bottle body of the triangular flask by 75% alcohol cotton, then moving the triangular flask into a super-clean workbench of an aseptic operation room, and carrying out aseptic operation in the whole process;

3. in a sterile room, uniformly shaking the zhongshengmycin fermentation liquor at normal temperature, sucking 100ml of the zhongshengmycin fermentation liquor into a 250ml triangular flask with 20 glass balls inside (the triangular flask and contents are sterilized), and shaking on a shaking table at 220rpm at normal temperature for 60min to ensure that mycelia of the fermentation liquor and a culture medium of the fermentation liquor are fully peeled and dispersed;

4. and (2) in a sterile room, under the sterile operation condition, carrying out natural filtration at normal temperature on the fermentation liquor which is subjected to sufficient vibration and enables mycelia and a culture medium to be fully peeled and dispersed, using sterilized sterile cotton as a Buchner funnel of a filter medium, obtaining target thallus starting strain filtrate, preserving the target thallus starting strain filtrate in a refrigerator at 4 ℃, carrying out infection-free other microorganism tests (called sterile tests) by means of inoculation, and using the strain filtrate as starting strain filtrate only when the sterile tests are negative.

Example 3 preparation of tolerant substrate factor

1. According to the determination result of the biological titer of the fermentation liquid sample, the fermentation liquid sample collected from the fermentation of example 1 for 120 hours (end of growth stagnation stage) is selected.

2. Taking out a triangular flask which is stored in a refrigerator at 4 ℃ and is filled with a zhongshengmycin fermentation liquid sample which is not polluted by other microorganisms, sterilizing the outer surface of the triangular flask, wiping and sterilizing the outer wall of a triangular flask body in an all-round way by 75% alcohol cotton, transferring the triangular flask into a super-clean workbench of a sterile operation room, and carrying out aseptic operation in the whole process;

3. shaking the zhongshengmycin fermentation liquor uniformly, sucking 500ml into a 1000ml triangular flask with 50 glass balls inside (the triangular flask and the content are sterilized), and placing on a shaking table at 220rpm for shaking for 60min to ensure that the mycelium of the fermentation liquor and the culture medium of the fermentation liquor are fully peeled and dispersed;

4. the sterile fermentation liquor which is fully shaken to fully peel off and disperse the mycelium and the culture medium is naturally filtered by a Buchner funnel which takes sterilized and disinfected sterile cotton as a filter medium, the obtained filtrate is filtered by a sterile bacterial funnel to obtain 400ml of target filtrate with the mycelium removed, and the target filtrate is preserved in a refrigerator at 4 ℃;

5. in a sterile room, 400ml of the target filtrate without mycelium obtained above is placed in a sterilized rotary evaporator by a sterile operation method, and is subjected to low-temperature reduced pressure concentration to the residual 80ml under the conditions of the temperature being less than 45 ℃, the vacuum degree being-0.098 Mpa and the cooling water temperature being 5-7 ℃, so as to obtain a sterile filtrate concentrated solution of the target filtrate without the mycelium of the zhongshengmycin and the like (the sterile filtrate concentrated solution is a concentrated solution containing the substrate factor), and the measured data of the concentrated solution are shown in the following table 2:

table 2.

Example 4 preparation of screening Medium

1. Plate medium preparation of concentrate without target filtrate: preparing a culture medium according to the formula of the slant culture medium and the flat culture medium, performing steam damp-heat sterilization on the culture medium at the temperature of 121 ℃ for 30 minutes, cooling, and storing in a water bath at the constant temperature of 48-50 ℃ for later use.

2. Preparation of plate medium containing concentrate of target filtrate: the amount of the concentrate of the objective filtrate of example 3 used was calculated to give a zhongshengmycin bio-titer of 30000. mu./ml as same as 3.0%; the other components were the same as the above "slant and plate medium formulation". The culture medium is subjected to steam moist heat sterilization and cooling at 121 ℃ for 30 minutes, and then is stored in a water bath at a constant temperature of 48-50 ℃ for later use.

The specific operation is as follows:

2.1. according to the biotoxin biological potency of the target filtrate sterile concentrated solution in the example 3 of 53286 mu/ml, the volume of the target filtrate sterile concentrated solution in the example 3 required for converting the content of the biotoxin into 3 percent is 56.6ml, and the sterile concentrated solution is measured for standby by a sterile operation amount of 56.6 ml.

2.2. Preparing other components of a target filtrate concentrated solution plate culture medium: the other medium components excluding the sterile concentrated solution of the objective filtrate were prepared in a total of 43.4 ml. The medium was weighed according to the "slant and plate medium recipe" described above, based on the total volume of the medium, which was 100 ml. The 43.3ml culture medium is sterilized by steam damp heat at 121 ℃ for 30min, cooled to 48-50 ℃, and then placed in a water bath at 48-50 ℃ for constant temperature storage for later use.

2.3. 56.6ml of the target filtrate sterile concentrated solution with the biotoxin biological titer of 53286 mu/ml measured by the sterile operation is heated to 45 ℃ by the sterile operation, the sterile concentrated solution is placed in a 45 ℃ water bath for constant temperature storage for standby application, the shorter the 45 ℃ heat preservation time is, the better the time is, the more 10 minutes are not needed to exceed, and the inactivation of the biological activity of the self metabolite in the concentrated solution can be reduced.

2.4. Rapidly and uniformly mixing 43.4ml of the sterilized culture medium which is preserved in a water bath at the constant temperature of 48-50 ℃ and is subtracted from other culture medium components except the target filtrate sterile concentrated solution with 56.6ml of the sterilized concentrated solution of the target filtrate preserved in a water bath at the constant temperature of 45 ℃ to obtain a plate culture medium containing 3% of self-metabolites such as zhongshengmycin and the like and no zhongshengmycin thallus, and immediately preparing a gradient concentration culture medium after uniformly mixing the culture medium containing 3% of the self-metabolites such as zhongshengmycin and the like.

3. The preparation of the gradient concentration culture medium is as follows:

3.1. under the aseptic operating condition of a sterile room, taking 10ml of flat culture medium stored in a water bath at constant temperature of 48-50 ℃, placing the flat culture medium in a 90mm culture dish, quickly leaning against one side of the culture dish on a cross bar and placing the cross bar obliquely, so that the bottom surface of the culture dish leaning against the high position of the cross bar just touches the culture medium to form the culture dish with the culture medium being thin at the top and thick at the bottom, placing the culture dish after the culture medium is solidified and placing the culture dish flat, wherein the culture dish forms a culture medium inclined plane which gradually thickens from one end to the other end, the back of the culture dish is marked with an arrow mark from the thickest part of the culture medium to the thinnest part, and the head part of the arrow mark [ < i > is the thinnest part of the culture medium;

3.2. under the aseptic operation condition of a sterile room, after the culture dish of the plate culture medium on which the solid inclined plane is formed is given the mark arrow as the thinnest position, the culture dish is horizontally placed; taking 10ml of a just prepared culture medium containing 3% of self-metabolites such as zhongshengmycin and the like, quickly placing the culture medium into a culture dish of the plate culture medium with a solid inclined plane, quickly shaking up and spreading horizontally, placing the culture medium containing the self-metabolites such as zhongshengmycin and the like for solidification, and obtaining the culture dish containing the culture medium with the gradient concentration of the self-metabolites such as zhongshengmycin and the like, wherein the content of the self-metabolites such as zhongshengmycin and the like in the culture dish is gradually increased along the direction of an arrow, the content of the zhongshengmycin at the head of the arrow "< below > is 3%, and the concentration of other self-metabolites in the culture medium is also highest at the head of the arrow" < below >;

3.3. under the aseptic operating condition of a sterile room, simultaneously preparing 6 culture dishes containing the culture medium with the gradient concentration of the metabolites of the Zhongshengmycin and the like according to the method; the content of zhongshengmycin at the head of an arrow at the bottom of the culture dish is 3%, and the concentration of other self products in the culture medium is highest at the head of an arrow "↓" in the same way.

The resulting gradient concentration medium is the screening medium for rational screening, the structure of which is shown in FIG. 3.

As shown in FIG. 3, the side of the tail portion of the arrow "×" is 0mm, and the gradient concentration of the metabolite gradually increases in the direction of the arrow. In FIG. 3, I shows a plate medium of the concentrate containing no objective filtrate at the time of preparation, and II shows a plate medium of the concentrate containing objective filtrate at the time of preparation.

EXAMPLE 5 preparation of Complex mutagenic liquid of target cell filtrate

1. The bacterial body filtrate of the target starting strain obtained in example 2 and stored in a refrigerator at 4 ℃ was taken out, and the culture dishes containing the culture medium with the gradient concentration of the metabolites of the strains such as zhongshengmycin and the like, which had been prepared in example 4, were placed together in a sterile dark room with a red light.

2. Cumulative degressive ultraviolet-light revival composite mutagenesis:

2.1. in a sterile dark room with a red light, a 30w fluorescent lamp and a 30w wavelength of

The ultraviolet lamp and the fluorescent lamp in the mutagenesis box of the ultraviolet lamp are both simultaneously started for preheating and stabilizing for 15 minutes, and the ultraviolet lamp and the fluorescent lamp are closed (the mutagenesis box is covered by a red and black cloth cover);

2.2. 10ml of the bacterial filtrate of the target starting strain is sucked into a 90mm double-disc sterile culture dish, a cover on the upper layer of the culture dish is removed in a sterile dark room with a red light, and the culture dish is placed on a rotating disk 20cm below the double lights in a mutagenesis box provided with a 30w fluorescent lamp and a 30w ultraviolet lamp and slowly rotated.

2.3. The UV-light revival mutagenesis was performed 5 times in succession:

2.3.1. firstly, turning on an ultraviolet lamp for irradiating for 30s, and then turning off the ultraviolet lamp for 20 s; starting the fluorescent lamp for 20 s; turning off the fluorescent lamp for 20 s;

2.3.2. then turning on the ultraviolet lamp for irradiating for 25s, and then turning off the ultraviolet lamp for 20 s; starting the fluorescent lamp for 20 s; turning off the fluorescent lamp for 20 s;

2.3.3. then the ultraviolet lamp is turned on for irradiating for 20s, and then the ultraviolet lamp is turned off for 10 s; starting a fluorescent lamp for 10 s; turning off the fluorescent lamp for 10 s;

2.3.4. then the ultraviolet lamp is turned on for irradiating for 15s, and then the ultraviolet lamp is turned off for 10 s; starting a fluorescent lamp for 10 s; turning off the fluorescent lamp for 10 s;

2.3.5. then the ultraviolet lamp is turned on for irradiating for 10s, and then the ultraviolet lamp is turned off for 5 s; starting a fluorescent lamp for 5 s; turning off the fluorescent lamp for 5 s;

2.4. after 5 times of ultraviolet-light revival dosage accumulation degressive composite mutagenesis, the composite mutagenic liquid of the target starting strain thallus filtrate is obtained.

Example 6 rational screening

1. Coating, culturing and screening the composite mutagenic liquid of the bacterial filtrate of the target starting bacterial strain obtained by 5 times of ultraviolet-light reviving and dose accumulation decrement composite mutagenesis, and specifically comprises the following steps:

1.1. coating: 6 culture dishes containing a medium with a gradient concentration of a self-metabolite such as zhongshengmycin are placed in a red-light sterile dark room, and the same operation is carried out on 5 culture dishes under sterile operating conditions: sucking 1ml of the composite mutation liquid of the bacterial filtrate of the target starting strain obtained in example 5, and uniformly coating the composite mutation liquid on a 90mm double-dish culture dish containing 1 culture medium with the gradient concentration of the self metabolites such as zhongshengmycin and the like; the remaining 1 dish was coated with the remaining cell filtrate of the target starting strain that was not subjected to UV-light rejuvenation mutagenesis, and used as a control dish.

1.2. Culturing: in a sterile darkroom, the temperature and the humidity of the darkroom are regulated to be constant as follows: the temperature is 28.5-29.5 ℃, and the relative humidity is 55-65%; and (3) covering the control culture dish and 5 culture dishes coated with the same bacterium solution subjected to compound mutagenesis by using a cover, wherein the covers are upward, and after one day of culture, inverting the whole culture dish by using the cover as the bottom, and culturing for 7-10 days to avoid the drying of the culture medium.

1.3. Selecting a single colony strain which is tolerant to high-concentration self metabolites:

1.3.1. a sparse colony grows at the tail part marked by an arrow on a culture dish of a control, the colony is sparse along with the extension in the direction of the arrow, only one colony is at the position of 22mm at most, the colony is pink, the concentration of the zhongshengmycin resistant colony of 22mm is about 7300 mu/ml according to the zhongshengmycin gradient concentration, and the colony is marked as d 22;

1.3.2. on 5 culture dishes tested, the colonies marked at the tail part of the arrow are sparsely grown, the colonies are almost not grown along with the extension in the direction of the arrow, but 3 culture dishes respectively have one colony at the highest position of each of 52mm, 58mm and 63mm, and the concentration of the resistant zhongshengmycin of the colonies of 52mm, 58mm and 63mm is respectively about 17000 mu/ml, 19000 mu/ml and 21000 mu/ml according to the zhongshengmycin gradient concentration; the 52mm and 63mm bacterial colonies are small and compact, the bacterial colonies are full of spore bacterial colonies in a flat steamed bun shape, and the spores are pink; the colony of 58mm is small and compact, presents a colony with spores covered with steamed bread with higher middle bulge, but the spores are more white and not obvious pink; colonies of 52mm, 58mm, 63mm on the three test plates were labeled n52, n58, n 63.

1.3.3. Selecting the colonies of d22, n52, n58 and n63, inoculating to an inclined plane, culturing at 29 ℃ for 7 days, wherein the bacterial lawn on the inclined plane of d22 is thicker and is pink, and the back of the inclined plane of the test tube is dark pink; the bacterial tongue fur on the inclined surfaces of n52 and n63 is thick and pink, and the back of the inclined surface of the test tube is dark pink; the thick lawn on the n58 slant surface appears gray and slightly light pink, and the back of the slant surface of the test tube appears light pink.

1.3.4 fermentation test: the screened resistant strains n52, n58, n63 which are resistant to zhongshengmycin and self metabolites are subjected to 5L fermentation tank fermentation test (according to the formula of the fermentation medium and the fermentation culture conditions, and the fermentation culture period is 7 days) together with the control strain d22 and the strain UV-3 used in industrial production, 3 times of test are repeated, and the fermentation level of the zhongshengmycin is examined, and the related result data are shown in the following table 3:

table 3.

As can be seen from Table 3, the zhongshengmycin titers obtained by n52, n58 and n63 are all much higher than 11389 mu/ml of the starting strain UV-3, and it can be seen that more high-producing strains can be obtained by the screening method of the present invention. The n58 with the highest yield is preserved, namely the preservation number is: streptomyces lavendulae Hainan variant UV-11 of GDMCC No. 61906.

Example 7 Strain conservation and stability verification

1. The 5 bevels of UV-3, d22, n52, n58 and n63 in the above example 6 were sealed with sterile liquid paraffin and stored in a refrigerator at-80 ℃ for 3 months.

2. Taking out the sterile liquid paraffin to seal and store the slant strains of UV-3, d22, n52, n58 and n63 which are preserved by freezing, carrying out plate single colony separation and culturing for 7-10 days to observe the growth condition of single colonies, wherein the observation results are shown in the following table 4:

table 4.

3. Selecting characteristic single colony in the single colony separation plate, placing the transfer inclined plane in an aseptic room with the temperature of 28.5-29.5 ℃ and the relative humidity of 55-65%, and culturing for 7-10 days; further observation of the slant lawn morphology: the inclined surface lawn of the UV-3 is thicker and pink, the inclined surface lawn of the d22 is thicker and pink, and the back of the inclined surface of the test tube is dark pink; n52 the bacterial tongue coating on the inclined plane is pink, and the back of the inclined plane of the test tube is dark pink; n58 the bacterial tongue coating on the inclined plane is pink, and the back of the inclined plane of the test tube is dark pink; n63 the bacterial tongue coating on the inclined plane is pink, and the back of the inclined plane of the test tube is dark pink;

4. fermentation test: the liquid paraffin slant strain preserved at-80 deg.C is subjected to single colony separation, and then transferred to slant strains UV-3, d22, n52, n58, and n63 to perform 5L fermentation tank test (according to the above-mentioned "fermentation medium formula and fermentation culture conditions", fermentation culture period is 7 days), and repeating the test for 3 times.

The fermentation level of zhongshengmycin was examined, and the results are shown in the following table 5:

table 5.

As can be seen from Table 5, the fermentation level of the biotins in 3 strains (i.e. n52, n58 and n63) which are resistant to higher doses of the self metabolites such as the biotins is improved compared with the existing strains, and is also greatly improved compared with the control group (UV-3, d22), especially the maximum improvement of n58 is over 55%.

Example 8 verification of industrialized fermentation production

UV-3 and UV-11 were applied to 40m³And (3) carrying out industrialized fermentation production verification on the fermentation tank (according to the formula of the fermentation medium and the fermentation culture conditions, and the fermentation culture period is 5 days), and obtaining various parameters of the fermentation liquor of the zhongshengmycin in the tank. The fermentation broth of UV-11 has the following parameters:

total sugar: 1.306g/100ml, reducing sugar: 0.6359g/100ml, amino nitrogen: 88.58mg/100ml, total phosphorus 10.25mg/L, bacterial concentration 27.2%, pH6.95, biological potency 18050 u/ml.

Under the same fermentation conditions and fermentation period, the fermentation level of the zhongshengmycin in the UV-3 strain tank batch is only 11936 mu/ml, while the fermentation unit of the zhongshengmycin in the UV-11 strain tank batch reaches 18050 mu/ml, namely, the industrialized fermentation level of the UV-11 strain is improved by 52.5 percent compared with that of the UV-3 strain as a control strain. The components of the fermentation liquid in the UV-11 fermentation discharge liquid were further measured by high performance liquid chromatography, and the results are shown in FIGS. 1 and 2. Wherein, FIG. 1 is the HPLC test result of the control strain UV-3; FIG. 2 shows the results of HPLC test of the UV-11 strain.

As can be seen from FIGS. 1 and 2, the retention time of each component in the UV-11 fermentation effluent was in one interval with the retention time of the control strain UV-3, and the specific data are shown in Table 6 below.

Table 6.

The conditions employed in HPLC were as follows:

model: waters e2695

Mobile phase: the gradient elution conditions are shown in table 7 below:

table 7.

Time/min	Acetonitrile/% (V/V)	Mixed salt solution/% (V/V)
			0	15	85
2	15	85
			10	22	78
20	22	78
			22	15	85
30	15	85

Flow rate: 1.0 mL/min.

Column temperature: 30 ℃ plus or minus 2 ℃.

Detection wavelength: 200 nm.

Sample introduction volume: 10 μ L.

Column: water SymmetryShield^TMRP18 5μm

Example 9 preparation of a preparation comprising a Zhongshengmycin

1. Acidifying the fermentation liquor: an acidified solution obtained by adjusting the pH of 18050 μ/ml fermentation broth obtained in example 8 to 3.5;

2. solid-liquid separation of acidified liquid: pumping the acidified liquid of the fermentation liquor into a ceramic membrane with the aperture of 0.20 mu m for solid-liquid separation to obtain a permeate, and pumping the permeate into the ceramic membrane with the aperture of 0.05 mu m for liquid-solid separation to obtain a secondary permeate of the fermentation liquor;

3. concentrating secondary permeate of fermentation liquor: concentrating the secondary permeate through a nanofiltration membrane with the molecular weight of 400Da to obtain a concentrated solution;

4. spray drying: directly spray-drying the concentrated solution to obtain Zhongshengmycin full-water-soluble mother medicine dry powder, and measuring the biological value of the mother medicine dry powder to be 510025 mu/g.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, so that any changes or modifications made in the claims and the specification of the present invention should fall within the scope of the present invention.

Claims

1. A mycelium or thallus, wherein the mycelium or thallus is obtained from a fermentation liquid at the end of a rapid growth and accumulation stage of production of a secondary metabolite in a strain fermentation production process.

2. Mycelium or thallus according to claim 1, characterised in that the strain is Streptomyces lavendulae, preferably Streptomyces lavendulae Hainanensis variant (Streptomyces lavendulae var. hainanensis).

3. The mycelium or the bacterial body according to claim 2, wherein the fermentation satisfies one or more of the following conditions:

the fermentation temperature is 26-35 ℃;

the air ventilation volume is 0.5V/VM-1.5V/VM;

the stirring speed of the fermentation tank is 50rpm-200 rpm;

the fermentation culture period is 3-10 days;

4. A method for breeding a strain tolerant to a self-metabolite, the method comprising the steps of:

5. The method as claimed in claim 4, wherein the screening medium is a gradient concentration medium of substrate factors, and the maximum value of the biological potency of the biotoxin in the gradient concentration medium is 20000-50000 mu/ml; for example 30000. mu.l/ml; and/or the presence of a gas in the gas,

15-20 g/L, preferably 18-19 g/L, of reducing sugar at the last stage in the rapid growth accumulation stage of production; 1.2-1.5 mg/L of amino nitrogen, preferably 1.3-1.4 mg/L; 25-30 mg/L of total phosphorus, preferably 28-29 mg/L; the bacterial concentration is 27-30%, and the preferable concentration is 28-29%; the pH value is 6.5-7.0;

and/or the titer of the zhongshengmycin in the tail section in the rapid growth and accumulation stage of production is 6000-10000 mu/ml, preferably 7000-8000 mu/ml; and/or the presence of a gas in the gas,

5-7 g/L of reducing sugar at the end of the production growth stagnation stage, 0.6-0.9 mg/L of amino nitrogen, 8-12 mg/L of total phosphorus, 25-27% of microbial concentration and 6.8-7.0 of pH;

6. The method according to claim 4, wherein, in the step S2,

the thallus filtrate is obtained by the following method under the aseptic condition: peeling and dispersing the mycelium of the fermentation liquor and the culture medium of the fermentation liquor, and naturally filtering at normal temperature by using a Buchner funnel with sterile cotton as a filtering medium to obtain thallus filtrate;

and/or, the concentrated fermentation broth containing the substrate factor is obtained under aseptic conditions by: the mycelium of the fermentation liquor is peeled and dispersed from the culture medium of the fermentation liquor, the natural filtration is carried out at normal temperature by using a Buchner funnel with sterile cotton as a filter medium, and the obtained filtrate is filtered by using a sterile bacterial funnel to obtain the concentrated fermentation liquor containing the substrate factors.

7. The method of claim 6, wherein the concentrating under reduced pressure comprises the steps of: putting the filtrate into a rotary evaporator provided with a filter, and concentrating under reduced pressure under the conditions that the water bath temperature is less than 45 ℃, the vacuum degree is-0.098 Mpa and the cooling water temperature is 5-7 ℃ to obtain concentrated fermentation liquor containing substrate factors; the volume ratio of the concentrated fermentation liquor to the fermentation liquor 2 is preferably 1 (5-10), for example 1: 5.

8. The method according to any one of claims 4 to 7, further comprising a step of subjecting the bacterial cell filtrate to mutagenesis before step S4.

9. The method according to claim 8, wherein the mutagenesis is cumulative decreasing ultraviolet-light revival composite mutagenesis, ultraviolet light and sunlight are alternately irradiated for 2-10 times, and the irradiation time is sequentially decreased;

The sunlight comes from a 30W fluorescent lamp;

10. Use of the mycelium or the mycelia according to any one of claims 1 to 3 for the selective breeding of a self-metabolite resistant strain.