CN114317670A - Screening culture medium and preparation method and application thereof - Google Patents

Abstract

The invention discloses a screening culture medium and a preparation method and application thereof. Wherein the screening culture medium comprises a concentrated fermentation broth containing a substrate factor, and the concentrated fermentation broth containing the substrate factor is obtained by filtering and concentrating fermentation broth obtained from the fermentation of a starting strain to the end of a production and growth stagnation stage of a secondary metabolite. When the strain obtained by screening the screening culture medium is applied to industrial fermentation production of zhongshengmycin, the industrial fermentation production level is improved by more than 50% compared with a control.

Description

A screening medium and its preparation method and application

technical field

The invention relates to the technical field of biological pesticides, in particular to a screening medium and a preparation method and application thereof.

Background technique

According to the statistics of the United Nations Food and Agriculture Organization (FAO), in the process of agricultural and forestry production in the world, the annual loss caused by pests and weeds is more than one third. There are approximately 3 million chemical pesticide poisoning incidents worldwide each year. In developing countries, there are about 1,650 collective poisoning incidents caused by the consumption of pesticide-contaminated food every year. Therefore, it has become an inevitable trend and a global consensus to vigorously develop biopesticides that are safe, low-toxic and non-toxic, low-accumulation and non-accumulative, and do not pollute the environment.

Zhongshengmycin (chemical name: 1-N glycosyl stridyl-2-amino L-lysine-2 deoxygulosamine; molecular formula: C ₁₉ H ₃₄ O ₈ N ₈ ; molecular weight: 502.0) is a A new type of agricultural antibiotic bio-pesticides, its application belongs to the category of fungicides, and is used to prevent and control crop diseases. Its producing bacteria are isolated from the soil of Hainan Island and named as Streptomyces lavendulae var. hainanensis ), the deposit number is CGMCC No.1026. As an agricultural antibiotic biopesticide, it has a strong inhibitory or killing effect on Gram-positive bacteria, Gram-negative bacteria and filamentous fungi. It inhibits the generation of peptide bonds of pathogenic bacterial proteins, and eventually leads to bacterial death; it can inhibit the growth of mycelium and the germination of spores, and play a role in preventing and controlling fungal diseases; , apple ring scab and apple leaf spot have good control effect, and low toxicity to humans and animals is safe, compatible with the environment.

Therefore, improving the fermentation level of zhongshengmycin industrialization, realizing energy saving and consumption reduction of zhongshengmycin industrialization, expanding the production capacity of zhongshengmycin original drug, and expanding the market share of zhongshengmycin in green food production materials, are the most beneficial to human beings. Contributing to the cause of food health has good social and economic benefits.

It has been 20 years since the industrial fermentation and production of zhongshengmycin, but the laboratory research and industrial fermentation production process are affected by the self-metabolites of the zhongshengmycin-producing bacteria in the fermentation production process, such as glucose effect, self secondary The single-factor and multi-factor repression and feedback inhibition regulation of the metabolite mesobiotics make it impossible to further improve the fermentation production level of mesobiotics. The content of bacteriocin parent drug products cannot be further increased, which limits the market supply and demand of bio-pesticides biocide parent drug and its preparations, as well as its extended use in the fields of comprehensive application of large-scale and large-area flight control.

Based on this, it is expected to start with mediosin-producing bacteria, to screen high-yielding strains of mediosin that are resistant to its own secondary metabolites, and its other metabolites, and apply them to industrial fermentation production, so as to improve green production materials. At the industrialized fermentation production level of mesobiotics, a large number of fully water-soluble high-content parent drugs of mesobiotics have been obtained to meet the needs of the rapidly developing green ecological agriculture.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a screening medium and its preparation method and application. When the strain screened by the screening medium of the present invention is applied to the industrial fermentation production of zhongshengmycin, the industrialized fermentation production level is increased by more than 50% compared with the control, reaching more than 18000 μ/ml, and the content of zhongshengmycin is obtained. A large number of fully water-soluble zhongshengmycin parent drug dry powder products reaching more than 500000μ/g greatly reduce the industrial production cost of zhongshengmycin, laying the foundation for the market demand of biological pesticide zhongshengmycin and meeting the requirements of green ecological Expanded use of large-scale and large-scale flight control drugs in agriculture.

For realizing the above-mentioned purpose of the invention, the present invention realizes through the following aspects:

In a first aspect, the present invention proposes a screening medium comprising a substrate factor-containing concentrated fermentation broth derived from fermentation of a starting strain to secondary metabolite production The fermentation broth obtained at the end of the growth stagnation stage is obtained by concentration.

Preferably, the screening medium is a gradient concentration medium of substrate factors.

Preferably, the starting strain is Streptomyces lavendulae, more preferably, the starting strain is Streptomyces lavendulae var. hainanensis, such as Streptomyces lavendulae var. hainanensis .hainanensis) CGMCC No. 1026, or Streptomyces lavendulae var. hainanensis CGMCC No. 61906.

Preferably, reducing sugar at the end of the production growth stagnation stage: 5-7 g/L, amino nitrogen: 0.6-0.9 mg/L, total phosphorus 8-12 mg/L, bacterial concentration 25-27%, pH 6.8- 7.0; and/or, the titer of mesobiotics in the final stage of the production growth stagnation stage is 10000-12000 μ/ml.

Preferably, the maximum biological titer of biotin in the gradient concentration medium is 20,000 to 50,000 μ/ml; for example, 30,000 μ/ml.

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

The fermentation temperature is 26℃-35℃;

Air ventilation 0.5V/VM-1.5V/VM;

Fermentation tank stirring speed 50rpm-200rpm;

The fermentation culture cycle is 3 days to 10 days;

The formula of the fermentation medium used in the fermentation includes: glucose 4.0%-15.0%, corn starch 1.0%-6.0%, corn flour 0.5%-7.5%, soybean flour 3.0%-12.0%, sodium chloride 0.2%- 1.6%, ammonium chloride 0.1%-1.8%, calcium carbonate 0.2%-1.6%, potassium dihydrogen phosphate 0.01%-0.13%, magnesium sulfate 0.01%-0.16%, pH6.0-7.0, the % is mass volume percentage.

In the second aspect, the present invention proposes a method for preparing the above-mentioned screening medium, the method comprising the following steps:

Step S1: respectively preparing a plate medium and a concentrated fermentation broth containing substrate factors;

Step S2: pouring the flat culture medium into a culture utensil such as a petri dish, and placing the culture utensil at an inclination to solidify the flat culture medium to form a lower-layer screening medium with a gradually thickening gradient;

Step S3: The concentrated fermentation broth containing the substrate factor is mixed with the plate medium and then poured into the horizontally placed culture vessel of step S2. The thickened gradient-type upper screening medium is obtained.

Preferably, the preparation of the substrate factor comprises the following steps:

Step S11: fermenting the starting strain to obtain the fermentation broth at the end of the production growth stagnation stage;

Step S12: after filtering the fermentation broth, a filtrate is obtained, wherein the filtrate is further concentrated to obtain a concentrated fermentation broth containing a substrate factor; the concentration is preferably reduced pressure concentration.

Preferably, in the step S12, the concentration under reduced pressure includes the following steps: placing the filtrate in a rotary evaporator equipped with a filter, at a water bath temperature <45°C, vacuum degree -0.098Mpa, cooling water Concentrate under reduced pressure at a temperature of 5°C-7°C to obtain a concentrated fermentation broth containing substrate factors; the volume ratio of the concentrated fermentation broth to the fermentation broth is 1:(5-10), for example, 1:5.

Preferably, in the step S12, a sterile bacterial funnel is used for filtration;

Preferably, glass beads are put into the fermentation liquid before filtration, and the mycelium in the fermentation liquid is fully peeled off from the culture medium by shaking;

Preferably, 20-50 glass balls, preferably 30-40 glass balls are built into every 500ml of fermentation broth; and/or, the shaking speed is 220 rpm, and the shaking time is 60 min.

Preferably, in the step S3, the added amount of the concentrated fermentation broth is 40-80% of the volume percentage of the upper screening medium.

In the third aspect, the present invention proposes the application of the above-mentioned screening medium in culturing and/or screening strains resistant to self-metabolites.

In the above technical solution, the inventors of the present case started from the source of the mesobiotics-producing bacteria, rationally selected high-yielding mesobiotics strains resistant to their own metabolites, and applied them to the industrialized fermentation production of mesbiotics. Production level of biotin industrial fermentation.

The specific process is as follows: use the mutant strain (UV-3) of Streptomyces lilacinus Hainan variant strain (the preservation number is CGMCC No. 1026) to produce mesobiotics by industrial fermentation, and select the industrial fermentation to produce the fermentation level ≥ 10000μ/ml fermenter batch, starting with the mycelium in the fermentation broth in the middle and last stage of the rapid growth and accumulation stage of secondary metabolites in the fermentation process of the fermenter batch as the target for screening self-tolerant metabolites Strains, the target starting strains were mutated by means of cumulative decreasing UV-photoreactivation compound mutagenesis, and the same batch of zhongshengmycin was used to produce secondary metabolites by industrial fermentation. The concentrated solution is used as the source of substrate factor for screening mediosin-producing bacteria resistant to its own metabolites, and the concentrated solution containing the substrate factor is used as the source to prepare gradient concentration medium, and rational selection is carried out to tolerate the production of mediosinin. strains with higher concentrations of their own metabolites. The above target starting strains, compound mutagenesis methods, and screening substrate factors are used in combination to select the target strains whose mutagenesis variation tolerates higher concentrations of their own metabolites for single colony culture. Biocine high-producing strains. In the large-scale industrial fermentation process, the high-yield and high-yield properties of the mesobiotics high-yielding strain are stable.

The method for rationally selecting and breeding high-producing strains of zhongshengmycin that is resistant to self-metabolites is based on the effect of zhongshengmycin and other self-metabolites produced by the zhongshengmycin-producing bacteria during the entire fermentation growth and production process on the zhongshengmycin. The producing bacteria play the role of feedback inhibition or repression in the process of producing zhongshengmycin, resulting in the stagnation of the industrialized fermentation level of zhongshengmycin and the inability to further improve the fermentation level. Starting from the selection of target starting strains, the selection of screening substrate factors, the selection of mutagenesis methods, and the selection of targeted screening and breeding media, the above-mentioned various screening factors are rationally integrated to design and induce the neutrophil-producing bacteria. A method for mutation, screening, and optimization of detection to obtain high-producing strains of mesobiotics.

The self-metabolites such as the mesobiocin are the carbon source, nitrogen source, phosphate and other inorganic salts in the fermentation medium used by the mesobiocin-producing bacteria in the fermentation production process and the carbon source, nitrogen source, phosphate and the like. Decomposition and conversion of phosphates, etc. into ATP and other energy required for the growth and production of mesobiotics, amino acids, oligopeptides, polypeptides, monosaccharides, disaccharides, polysaccharides, biologically active enzymes, and other mesobiotics The vitamins and minerals necessary for the growth and reproduction of the bacteria are produced to ensure the normal growth and reproduction of the mesobiotics-producing bacteria, and the transition from the reproductive growth period to the anti-production period is realized, and the bacteria of the mesobiotics-producing bacteria in the fermentation process are completed. The primary metabolic process of body growth and incremental reproduction, and then transferred to the secondary metabolic process of mesobiotic production and accumulation; complete the growth and reproduction of mesobiotics-producing bacteria, the rapid growth and accumulation of mesobiotics production, and the completion of mesobiotics production and accumulation. During the cycle of stagnant growth of cytosine production, decline of bacterial growth and even decline of production of antibiotics, various metabolites of melanobiocin-producing bacteria and the decomposition products of melanobiocin-producing bacteria that decompose and utilize the medium.

The selection of the target starting strain for the rational selection of high-producing strains of zhongshengxin that is resistant to its own metabolites is based on the fermentation growth and production process of the zhongshengmycin-producing bacteria when the bacteria grow and reproduce when they undergo primary metabolism, and then induce conversion into In the process of fermentation growth and production of secondary metabolites produced by mesobiotics, the growth of mesobiocin-producing bacteria, the growth of bacteria in the production process, the rapid growth and accumulation of secondary metabolites Stage, secondary metabolite mesobiotics production growth stagnation stage, cell growth and decline stage in different stages of secondary metabolite mesobiotics production in different stages of rapid growth and accumulation stage Mycelium in the middle and last stages of the accumulation stage as a rational selection The target starting strain of the high-producing strain of biocine.

The middle and last stage of the rapid growth and accumulation stage of the secondary metabolite in the process of growth and production of the mesobiotics producing bacteria (the middle and last stage of the rapid growth and accumulation stage of the production refers to the target secondary stage of the fermentation broth. The biological potency or concentration of the metabolite increases from the beginning to the second 2/3 of the time period after the growth rate reaches the maximum value, and the maximum value can be determined by those skilled in the art according to the real-time determination or obtained by drawing a curve. Mycelium is the mycelium with the most vigorous growth and production capacity in the process of fermentative growth and production of mesobiocin-producing bacteria, which has been converted from primary metabolism to secondary metabolism, so that it can be used in industrial scale production. Self-fermentative growth and production environment of biocine, the secondary metabolite of mesobiotics growing in the production environment, the mycelium in the middle and last stage of the accumulation stage is used as the target starting strain for screening, and the screened strains are more suitable for mesobiotics The primary metabolites and secondary metabolites in the growth and production process of industrial fermentation; the permeability of the cell wall of the mycelium in the rapid growth and accumulation stage of the secondary metabolite production is higher than that of the colony spores. It is better and easier to accept the compound mutagenesis of UV-light revival, and it is easier to screen out the target strains that are resistant to their own metabolites on the rationally selected medium, and the selected target strains are adapted to the industrial fermentation growth and production environment more capable.

The selection of the screening substrate factor for the rational selection of the high-producing strains of zhongshengmycin that is resistant to its own metabolites is based on the fermentation growth and production process of the zhongshengmycin-producing bacteria, after the growth and reproduction of the primary metabolizing bacteria, and then the induction conversion. In the process of fermentation growth and production of secondary metabolism to produce mesobiotics products, the secondary metabolites during the growth and production process of mesobiotics are rich in secondary metabolites at the end of the growth stagnation stage. The concentrated solution of the filtrate of the fermentation broth of zhongshengmycin and other various metabolites and the decomposed substances of the medium produced during the growth and metabolism of zhongshengmycin-producing bacteria is used as the screening base for rational selection of zhongshengmycin high-producing strains source of biological factors.

The end stage of the growth stagnation stage of the mesobiotics fermentation secondary metabolite (the last stage of the production growth stagnation stage is the time period during which the fermentation is put into the tank). At the end of the stagnation stage of the production of stagnant from the fermentation of mesobiotics to the secondary metabolites, the fermentation broth is rich in secondary metabolites mesbiotics and various other metabolites produced by primary and secondary metabolism. The decomposition products of the base, these substances play a role in repressing or feedback inhibition of the production of neutrophils. At this stage, the growth of neutrophils is slow or no longer increases; if the fermentation culture is continued, it will enter the growth of neutrophils to produce metabolic bacteria. In the decay stage, the growth and decay stage of the bacterial cells, some active enzymes and other substances in the metabolites of zhongshengxin in the fermentation broth will even decompose the formed zhongshengxin into other small molecular substances, resulting in the fermentation level of zhongshengxin no longer. If it increases, it will even decrease, resulting in the increase of fermentation time and waste, and the increase of energy consumption, but the uneconomical effect of improving the fermentation level of mesobiotics cannot be achieved.

In the breeding of antibiotic self-product-tolerant strains, traditional methods are basically the feedback inhibition of their own secondary metabolites, the release of glucose effect, and the breeding of single-factor tolerance such as phosphate repression, so the effect is relatively weak. .

What is different from the prior art is that the fermentation broth at the end of the growth stagnation stage of the secondary metabolite of the mesobiotics-producing bacteria involved in this case in the production and metabolism process is not rich in mesocidins. In addition to its own secondary metabolite mesobiotics, it is also rich in other complex self-metabolites in the process of growth, production and fermentation of mesbiotics. The fermentation broth filtrate concentrate at the end of the growth stagnation stage of the secondary metabolite mesobiotics was selected as the source of the screening substrate factor for screening the mesobiotic metabolite-tolerant strains. The feedback inhibition of biocine can also relieve the various carbon sources produced or left by catabolism in the fermentation medium during the fermentation of mesobiocin-producing bacteria, such as starch, polysaccharide, disaccharide, glucose, pentose, tetose, Triose, α-ketoglutaric acid, oxaloacetic acid, etc.; various nitrogen sources, such as various amino acids such as β-amino acid and β-lysine, oligopeptides, short peptides, polypeptides, and neutrophil precursor substances , N-acetylglucosamine, N-acetylmuramic acid, intracellular and extracellular enzymes such as redox, which can be converted into the basic structural unit of aminoglycoside antibiotics, the cell wall peptidoglycan of the bacteria Repression or feedback inhibition of enzymes, dehydrogenases, etc. Relieve the inhibition and feedback inhibition of the self-product zhongshengxin and its growth production metabolites in the fermentation production process of the zhongshengxin-producing bacteria, and realize the resistance to various inhibition or feedback inhibition of single or multiple factors and multiple synergistic factors Therefore, it is the fundamental to improve the industrial fermentation production level of zhongshengmycin to finally realize or partially release the influence of various repression or feedback inhibition on the production of zhongshengmycin.

The selection of the mutagenesis method for rationally selecting and cultivating high-yielding strains of neutrophils resistant to self-metabolites is to adopt the cumulative decreasing ultraviolet-photoreactivation compound mutagenesis method, which is to combine simple ultraviolet irradiation on the neutrophils. The single-factor physical mutagenesis produced by the strain becomes the original strain that is irradiated with ultraviolet light to produce mutagenesis, and then uses the natural light fluorescent lamp to carry out photorejuvenation and repair of the mutation site produced by the neutropenin-producing bacteria. Two-factor compound mutagenesis; further, this ultraviolet-photoreactivation compound mutagenesis is carried out with multiple decreasing doses for cumulative decreasing mutagenesis, so as to achieve the DNA single-strand or double-strand DNA formed as a result of ultraviolet radiation mutagenesis. The variation site of thymine dimer undergoes multiple formation of dimer→dimer depolymerization→formation of dimer→dimer depolymerization→formation of dimer, and the effect of restoration and repair is no longer easy to occur.

这一区间紫外线波长的杀菌和诱变作用最好。紫外线的生物学效应能引起DNA的生物损伤与胸腺嘧啶碱基对被二聚化，所以胸腺嘧啶二聚体的形成是紫外线改变DNA生物活性、造成菌体死亡或变异的主要途径，对引起菌体变异也起很大作用。当DNA进行自身复制时，双链先变成单链，然后各自再与附近的碱基形成互补链，然而由于两链间的胸腺嘧啶形成了二聚体，使DNA两条链交联，这样就阻碍了双链的分开和复制。同一链上的相邻胸腺嘧啶形成了二聚体，也会阻碍碱基的正常配对。在正常情况下，胸腺嘧啶与腺嘌呤配对，如果两个相邻的胸腺嘧啶形成了二聚体，就可能改变原来的配对情况，破坏了腺嘌呤的正常掺入，复制就在这一点上突然停止，或错误地进行。如果错误地进行复制，且在新形成的链上有一个改变了的碱基次序，则在随后的复制过程中，碱基次序已改变的DNA链仍照自身进行复制，产生了一个在二条链上碱基次序都是错误的分子因而引起突变；然而，紫外线损伤DNA遗传活性的作用，能被可见光日光灯恢复，这种现象是众所周知的光复活作用。由于被紫外线损伤的物质主要是二聚体，故在光复活酶作用下，二聚体就重新分解成为单体，使DNA恢复正常，所以菌株经紫外线诱变处理后，要避免可见光的照射。紫外线光复活作用，说明紫外线诱变是一个复杂的过程，紫外线的初始效应是诱发遗传物质处于各种类型的亚稳定，从亚稳定过渡到稳定的突变态需要一定的时间，故用时间上累积递减紫外线-光复活复合诱变方法，使用紫外线与可见白光日光灯多次反复处理菌丝体，使该菌丝体已经形成突变出错的各个位点不易回复恢复到初始正常的碱基序列，将能提高菌丝体的变异率和扩大变异幅度。In the ultraviolet-photoreactivation compound mutagenesis method, ultraviolet light is the most effective interval wavelength for microbial strain mutagenesis.

This range of ultraviolet wavelengths has the best bactericidal and mutagenic effects. The biological effect of ultraviolet rays can cause DNA biological damage and thymine base pairs to be dimerized, so the formation of thymine dimer is the main way for ultraviolet rays to change DNA biological activity and cause bacterial death or mutation. Somatic variation also plays a big role. When DNA replicates itself, the double strands first become single strands, and then each forms complementary strands with nearby bases. However, due to the dimerization of thymine between the two strands, the two strands of DNA are cross-linked, so that This hinders the separation and replication of the double strands. Adjacent thymines on the same strand form dimers that also prevent the normal pairing of bases. Under normal conditions, thymine pairs with adenine, and if two adjacent thymines form a dimer, it may change the original pairing situation and disrupt the normal incorporation of adenine, and replication suddenly occurs at this point stop, or proceed incorrectly. If the replication is performed by mistake, and there is an altered base order on the newly formed strand, then during subsequent replication, the DNA strand with the altered base order still replicates itself, resulting in a second strand Mutations are caused by molecules with the wrong base sequence; however, the effects of UV-induced damage to DNA's genetic activity can be restored by visible-light fluorescent lamps, a phenomenon known as photoreactivation. Since the substances damaged by ultraviolet rays are mainly dimers, under the action of photoreactivation enzymes, the dimers will be decomposed into monomers again, so that the DNA will return to normal. The photoreactivation effect of ultraviolet light shows that ultraviolet mutagenesis is a complex process. The initial effect of ultraviolet light is to induce the genetic material to be in various types of metastable states. It takes a certain amount of time to transition from metastable to stable mutant state, so it takes time to accumulate The method of decreasing ultraviolet-photoreactivation compound mutagenesis uses ultraviolet and visible white light fluorescent lamps to repeatedly treat the mycelium, so that the various sites where the mycelium has formed a mutation and error is not easy to restore to the original normal base sequence. Increase the mutation rate of mycelium and expand the variation range.

The selection of the targeted screening and breeding medium for rationally selecting high-producing strains of zhongshengmycin that tolerates its own metabolites is a gradient concentration medium for the production of metabolites that are tolerant to self-growth by the zhongshengmycin-producing bacteria. The secondary metabolite in the industrial fermentation process of zhongshengmycin The concentrated solution of the filtrate of the fermentation broth rich in zhongshengmycin and its own metabolites at the end of the growth stagnation stage is used as a rational selection for zhongshengxin Sources of screening substrate factors for high-yielding strains of bacteriocin. The concentrated solution rich in zhongshengxin and its other primary metabolites, secondary metabolites, and medium decomposition products produced in the process of fermentation growth and production of zhongshengmycin was used as a main component of the screening medium. The concentration of self-metabolites is gradually increased from low to high on the culture medium, and the targeted high-yielding strains that are resistant to self-metabolites can be selectively and rapidly selected geographically. The mycelia mutated by the cumulative decreasing UV-photoreactivation compound mutagenesis method can express rapidly on the gradient medium of the screening substrate factor of rational selection, which greatly reduces the work intensity.

The gradient concentration culture medium of the mesobiocin-producing bacteria tolerating self-metabolites is to spread a layer of screening medium on the bottom of a sterilized double-disc plate with a diameter of 90 mm, and place it at an incline. After the solidification, the petri dish is placed horizontally, and then the fermentation broth rich in zhongshengmycin and its other primary metabolites, secondary metabolites, and medium decomposition products produced in the process of fermentative growth and production of zhongshengmycin-producing bacteria is added. The screening medium of the concentrated solution of the filtrate is added to the petri dish, and it is solidified, thereby forming a screening substrate factor concentration of various substances containing zhongshengmycin and its own metabolites of zhongshengmycin-producing bacteria from low to high. A selection medium for rational selection of neutrophil-producing bacteria at progressively higher concentrations.

The method for rationally selecting high-producing mesobiotic strains resistant to self-metabolites is to select the secondary metabolite mesbiotics in the process of fermenting and producing mesbiotics in the stage of rapid growth and accumulation of secondary metabolites. The mycelium with high vigor and ability to produce mesobiotics in the middle and last stage was used as the target starting strain; the fermentation broth in the last stage of the growth stagnation stage of mesobiotics production was selected by fermentation of the mesobiotics-producing bacteria to produce secondary metabolites. High units of neutrophils, a large number of carbon sources, nitrogen sources, phosphates, precursors, biologically active enzymes, etc. produced in the process of primary metabolism and secondary metabolism are used as screening substrate factors for tolerance; time and dose The compound mutagenesis method of cumulative decreasing UV-photoreactivation was used as the mutagenic factor; the culture dish of the gradient concentration medium with the gradually increasing concentration of self-metabolites represented by neutrophils from low to high concentration was used as the screening method. The detection tool to achieve the target strain of mutagenic variation is to screen and detect the obtained strain.

The above-mentioned mycelium in the middle and last stage of the rapid growth and accumulation stage of secondary metabolite mesobiocin production in the fermentation production process of the same industrial fermentation production tank batch is used as the target starting strain, secondary metabolites The concentrated solution of the filtrate of the fermentation broth at the end of the growth stagnation stage of biotin production was used as the source of the screening substrate factor, the cumulative decrease UV-photoreactivation compound mutagenesis was used as the mutagenesis method, and the concentration of self-metabolites formed on the medium of the screening plate From low to high gradually increasing concentrations of petri dishes as a detection tool, a combined screening method for screening media, to select high-producing strains of mesobiotics that are resistant to self-metabolites, and screen a strain of mesobiotics. The strain was applied to a 40m ³ fermentor for industrial fermentation and large-scale production after the stability verification. Under the same fermentation process and cycle conditions as the industrial fermentation of the control strain, the fermentation level of the selected strain was increased by 52.5%, and the fermentation level was increased by 52.5%. The unit can reach 18050μ/ml, which shows that the fermentation level of the mesobiocin-producing bacteria strain obtained in this case has been greatly improved compared with the prior art.

On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

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

The positive progressive effect of the present invention is:

When the strains screened by the screening medium of the present invention are applied to the industrial fermentation production of mesobiotics, the industrial fermentation production level is increased by more than 50% compared with the control, and the highest can reach more than 18000 μ/ml, and the mesobiotics are obtained. A large number of fully water-soluble zhongshengmycin parent drug dry powder products with a content of more than 500000μ/g greatly reduce the industrial production cost of zhongshengmycin, laying a foundation for the market demand of biological pesticide zhongshengmycin and satisfying The expanded use of large-scale and large-scale flying control drugs in green ecological agriculture.

Biomaterial deposit information

The Streptomyces lavendulae var.hainanensis UV-11 of the present invention has been deposited in the Guangdong Provincial Microorganism Culture Collection Center (GDMCC) on August 31, 2021, and the deposit address is: Guangdong Academy of Sciences Microorganisms Research Institute (Guangdong Provincial Microbiological Analysis and Testing Center) Bacteria Room, Building 59, No. 100 Xianlie Middle Road, Guangzhou City. Postcode: 510070, deposit number: GDMCC No. 61906, culture name is UV-11, classification name is Streptomyces sp.

Description of drawings

Fig. 1 is the HPLC test result of control strain UV-3;

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

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

Detailed ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.

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

Seed medium formula (components are mass and volume percentages): glucose 1.0%, corn starch 1.5%, corn flour 1.0%, soybean flour 2.0%, sodium chloride 0.3%, ammonium chloride 0.3%, calcium carbonate 0.3%, adjusted to pH 6.6.

Fermentation medium formula (components are mass and volume percentage): glucose 5.0%, corn starch 2.0%, corn flour 2.0%, soybean flour 4.0%, sodium chloride 0.6%, ammonium chloride 0.8%, calcium carbonate 0.7%, phosphoric acid Potassium dihydrogen 0.02%, magnesium sulfate 0.05%, adjusted to pH 6.6.

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

Among them, the involved slant and plate medium formulations are as follows:

Slant and plate medium (components are mass and volume percentage): soluble starch 1.05%, peptone 0.06%, magnesium sulfate 0.07%, sodium nitrate 0.25%, potassium chloride 0.05%, ferrous sulfate 0.02%, potassium dihydrogen phosphate 0.12 %, agar 2.05%, adjusted to pH 6.6.

Example 1 The mutant strain (UV-3) of Streptomyces lilacinus Hainan variety strain (preservation number is CGMCC No. 1026) was used as an industrialized production strain to carry out industrialized fermentation production of mesobiotics

Strain UV-3 is a Hainan varietal of Streptomyces lilacinus that has been obtained by mutagenesis with the applicant's preservation number as CGMCC No. 1026 of Streptomyces lilacinus cv. mutant strains.

The UV-3 was fermented industrially in a 40m ³ fermenter (according to the aforementioned "fermentation medium formula and fermentation culture conditions", the fermentation culture period was 5 days) and the fermentation broth at different stages in the fermentation process was collected.

The above-mentioned industrial fermentation broth is sampled to collect fermentation broth samples from "0" hours after the inoculation of the fermenter according to the requirements of aseptic sampling, and the fermentation broth samples are collected every 12 hours. The specific method of aseptic sampling is as follows: the sampling port of the mesobiocin fermenter is sterilized with live steam of ≥0.3Mpa for 30 minutes, and under the protection of the flame circle, the steam valve of the sampling port of the fermenter is quickly closed, and the sampling port of the fermenter is opened. The fermented liquid flows out from the sampling port; put the sterilized glass triangular flask with the cotton plug under the protection of flame, and quickly remove the cotton plug at the sampling port (the pulled cotton plug is under the protection of the flame), and the bottle mouth The fermentation broth flowing out of the quasi-sampling port was quickly obtained, the triangular flask was quickly covered with a cotton plug, the outlet valve of the sampling port was closed, the steam valve was opened to sterilize the sampling port for 10 minutes, and the steam valve was closed. The collected fermentation broth samples were placed in a 4°C refrigerator immediately after each collection and sampling for refrigerated storage until the batch of industrial fermentation was put into the tank. At the same time, immediately after sampling, the sterility test of the slant and broth, and the microscopic examination of the hyphae were carried out. Only the fermented liquid of the slant, the sterility of the broth and the microscopic observation without being infected by other miscellaneous bacteria can be used as a sample. The relevant parameters of the fermentation broth samples were measured using the values in Table 1 for the values in the middle and end of the accumulation stage of rapid growth and the end of the stagnant stage of production growth. It should be noted that the biological titer growth rate v can be calculated by the following formula, where u ₁ refers to the biological titer at the previous time t ₁ , u ₂ refers to the biological titer at the next time t ₂ , and u ₁ and u The interval time of ₂ can be limited according to the specific conditions of each embodiment. In this embodiment, the interval between u ₁ and u ₂ is 12 hours:

Table 1.

It should be noted that the test methods involved in Table 1 are tested by the following methods:

1.1 Determination of biological potency

The determination method of the biological potency adopts the antibiotic microbiological assay method of "Chinese Pharmacopoeia (2015 Edition)". The reagents and solutions used were: phosphate buffer (pH 7.8); medium I; Bacillus subtilis bacterial suspension (Bacillus subtilis serial number: CMCC63501); volumetric flasks (100ml, 50ml).

1.2 Determination of microbial metabolism of reducing sugars

The determination of microbial metabolism of reducing sugars is determined by Fehling's reagent colorimetric method. The preparation of Fehling's reagent is as follows:

1. Preparation of Fehling's reagent A solution: Accurately weigh 120 grams of copper sulfate hydrate (CuSO ₄ ·5H ₂ O), analytically pure, with a content greater than 99.0%), and dissolve it completely with 2000 ml of distilled water.

2. Preparation of Fehling's reagent B solution: put 2000ml of distilled water in an open glass container. Accurately weigh 375 grams of potassium sodium tartrate (C ₄ H ₄ KNaO ₆ ·4H ₂ O, analytically pure, with a content greater than 99.0%), add it, and stir well until dissolved. Then accurately weigh 250 grams of sodium hydroxide (analytical grade), slowly add the above solution in stages, and stir while adding.

3. Preparation of Fehling's reagent C solution: Accurately weigh 300 grams of potassium iodide (analytical purity, content greater than 98.0%), dissolve and dilute to 1000 ml with distilled water.

4. Preparation of Fehling's reagent: Put Fehling's reagent A, B, and C solution into a 10000ml brown reagent bottle through a large funnel in turn, and shake it carefully and vigorously to prepare 5000ml of Fehling's reagent.

5. Preparation of 0.1 mol/L sodium thiosulfate solution: Weigh 26 g of sodium thiosulfate, add 0.2 g of anhydrous sodium carbonate, dissolve in 1000 ml of water, slowly boil for 10 minutes, and cool. Filter after two weeks. After filtering, calibrate according to GB/T601-2002.

6. Preparation of 1% starch indicator: Weigh 1 gram of starch and add a small amount of water to prepare a starch solution, add boiling distilled water to 100ml, and boil for 2 minutes to complete the preparation.

operating procedure

The expected range of the total residual sugar concentration is comprehensively determined according to the grade of the microbial medium and the cultivation time. For the culture filtrate or the centrifugation supernatant of the culture medium with a concentration of more than 10g/100ml, draw 1ml, add 4ml of distilled water to dilute, and after mixing evenly, draw 1ml of it for determination; for the total residual sugar concentration less than or equal to 10g/100ml, more than 5g /100ml of filtrate or supernatant, draw 0.5ml for measurement, and for filtrate or supernatant with total sugar concentration less than or equal to 5g/100ml, draw 1ml for measurement.

Accurately draw 1ml of filtrate or supernatant and place it in a 150ml conical flask, directly add 20ml of Fehling's reagent to heat evenly on an electric hot plate, and start timing after boiling. Timing for two minutes, remove the triangular flask and place it in a surgical tray filled with cooling water to cool. During operation, the exhaust fan is on.

After cooling, accurately add 15ml of 2mol/L sulfuric acid solution, quickly titrate with 0.0998mol/L Na ₂ S ₂ SO ₃ solution to light yellow, add about 1ml of starch indicator by titration, and continue to titrate until the blue color disappears. Note the volume of sodium thiosulfate solution consumed: V _sample .

Blank experiment: accurately measure 20ml of Fehling's reagent and place it in a 150ml conical flask, without adding the liquid to be tested, add 1ml of distilled water instead of the sample. Afterwards, the procedure is operated according to the determination of total sugar. The blank vial should be heated and boiled at the same time as the sample vial to minimize accidental errors. After the titration is completed, note down the volume of sodium thiosulfate solution consumed, which is denoted as V _empty .

ΔV=Vnull _{- Vsample} . Check the sugar table according to the ΔV value, and multiply the detected sugar value by the dilution factor, which is the total sugar or reducing sugar concentration of the sample, unit: g/100ml.

1.3 Determination of microbial metabolism of amino nitrogen

Reagent preparation

Preparation of 0.1% methyl red indicator: Accurately weigh 0.1 g of methyl red into a 250 ml beaker, add 100 ml of 75% alcohol, stir well with a clean glass rod, and the methyl red is completely dissolved. After the preparation of the reagent is completed, it can be used in about 2/3 of the dropper bottle.

Preparation of 0.015mol/L sulfuric acid solution: accurately measure 75ml of distilled water in a 1000ml beaker. Accurately measure 2mol/L sulfuric acid solution with a 100ml measuring cylinder, drain with an inclined glass rod, slowly add it to the beaker, and stir evenly while adding.

Preparation of 0.02858mol/L sodium hydroxide solution: dissolve 1.1432g sodium hydroxide (analytical grade) in distilled water, and set the volume in a 1000ml volumetric flask. Calibration is performed.

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

Preparation of 1% phenolphthalein indicator: Accurately weigh 1g of phenolphthalein into a 250ml beaker, add 100ml of 75% alcohol, stir well with a clean glass rod, and the phenolphthalein is completely dissolved.

operating procedure

Accurately draw the filtrate of the culture solution or add 2ml of the supernatant after centrifugation into a 200ml conical flask, and add 50ml of distilled water.

Add 1 drop of 1% methyl red indicator, and titrate to reddish with 0.015mol/L sulfuric acid solution.

Use 0.02858mol/L sodium hydroxide solution dropwise to orange-yellow.

Add 2ml of 18% neutral formaldehyde solution, shake gently and let stand for 10 minutes

Add 2-8 drops of 1% phenolphthalein indicator, use 0.02858mol/L sodium hydroxide solution, titrate to reddish, and record the titration ml of 0.02858mol/L standard sodium hydroxide solution.

1.4 Determination of microbial concentration

Shake the fermentation broth first;

Weigh the weight m ₁ of the centrifuge tube with an electronic scale, pour about 10ml of fermentation broth into the centrifuge tube, and weigh the weight m ₂ of the centrifuge tube again; put it in a centrifuge and centrifuge at 3000 r/min for 15 minutes;

The supernatant was poured out, and the weight m ₃ of the remaining part was weighed;

Result: bacterial concentration=(m ₃ -m ₁ )/(m ₂ -m ₁ )×100%.

1.5 Detection of microbial total phosphorus

The total phosphorus detection adopts the instrument detection method

The instrument used for total phosphorus is a multi-parameter water quality analyzer (5B-3B (V11)) manufactured by Beijing Lianhua Yongxing Technology Development Co., Ltd. The reagents used are all produced by the manufacturer.

The preparation of embodiment 2 target departure strain

1. According to the measurement results of the biological titer of the fermentation broth samples, select the fermentation broth samples collected in Example 1 from fermentation to 48 hours (the middle and end of the rapid growth accumulation stage).

2. Take out the triangular flask containing the neutrophil fermentation broth samples that have not been contaminated by other microorganisms stored in the refrigerator at 4°C, disinfect the outer surface of the triangular flask by ultraviolet irradiation, and sterilize the outer wall of the triangular flask by 75% alcohol cotton. After wiping and sterilizing in all directions, move it into the ultra-clean workbench of the sterile operating room for the whole process of aseptic operation;

3. In a sterile room, shake the above-mentioned mesobiocin fermentation liquid evenly at room temperature, and draw 100ml into a 250ml triangular flask with 20 glass balls built in (the triangular flask and its contents have been sterilized), at room temperature Shake for 60min on a shaking table at 220rpm, so that the mycelium of the fermentation broth and the culture medium of the fermentation broth are fully stripped and dispersed;

4. In a sterile room, under the conditions of aseptic operation, the above-mentioned fermentation broth that has been sufficiently shaken to make the mycelium and the culture medium fully peeled off and dispersed is carried out with the sterile cotton after sterilization as the Buchner funnel of the filter medium. Natural filtration at room temperature to obtain the filtrate of the starting strain of the target bacterial cell and store it in a refrigerator at 4°C, and at the same time, carry out a non-infection other microorganism test (called a sterility test).

Example 3 Preparation of Tolerant Substrate Factors

1. According to the measurement results of the biological titer of the fermentation broth samples, select the fermentation broth samples collected in Example 1 from fermentation to 120 hours (the end of the growth stagnation stage).

2. Take out the triangular flask containing the samples of zhongshengmycin fermentation broth that has not been contaminated by other microorganisms stored in the refrigerator at 4°C, disinfect the outer surface of the triangular flask and clean the outer wall of the triangular flask with 75% alcohol cotton. After wiping and sterilizing the position, move it into the ultra-clean workbench in the sterile operating room to carry out the whole process of aseptic operation;

3. Shake the above-mentioned mesobiocin fermentation liquid evenly, draw 500ml into a 1000ml triangular flask with 50 glass balls built in (the triangular flask and its contents have been sterilized), place on a 220rpm shaker and shake for 60min, The mycelium of the fermentation broth and the culture medium of the fermentation broth are fully stripped and dispersed;

4. The above-mentioned aseptic fermentation liquid that the mycelium and the culture medium are fully stripped and dispersed after being sufficiently shaken is naturally filtered through the Buchner funnel of the filter medium through sterilized aseptic cotton, and the obtained filtrate is then filtered through an aseptic filter. Bacterial funnel filtration to obtain 400ml of the target filtrate to remove mycelium, and store in a 4°C refrigerator;

5. In a sterile room, with aseptic operation method, place 400ml of the above-obtained target filtrate for removing mycelium in a sterilized rotary evaporator, at a temperature <45°C, vacuum degree-0.098Mpa, cooling water Under the condition of temperature 5 ℃-7 ℃, low temperature and reduced pressure are concentrated to the remaining 80ml, to obtain the sterile filtrate concentrate (the sterile filtrate containing the target filtrate such as neutropenin and other metabolites and without neutrophil mycelium). The concentrated solution is the concentrated solution containing the substrate factor), and the data of this concentrated solution is measured as shown in Table 2 below:

Table 2.

The preparation of embodiment 4 screening medium

1. Preparation of the plate medium without the concentrated solution of the target filtrate: prepare the medium according to the aforementioned "slope and plate medium formula", the medium is sterilized by steam moist heat at 121°C for 30 minutes, and then cooled at 48°C-50°C. Store at a constant temperature in a ℃ water bath until use.

2. Preparation of the plate medium containing the concentrated solution of the target filtrate: the usage amount of the concentrated solution of the target filtrate of Example 3 is calculated as 3.0% with the biological titer of 30,000 μ/ml of neutropenin; other components are the same as the aforementioned " Slant and Plate Medium Recipes". The culture medium was sterilized and cooled by steam moist heat at 121°C for 30 minutes, and then stored at a constant temperature of 48°C-50°C in a water bath until use. The specific operations are as follows:

2.1. According to the biological titer 53286 μ/ml of the target filtrate aseptic concentrate of embodiment 3, the volume of the target filtrate aseptic concentrate of the desired embodiment 3 of conversion content 3% is 56.6 ml, take 56.6ml of the sterile concentrate for use by aseptic operation.

2.2. Preparation of other components of the target filtrate concentrated liquid plate medium: the other medium components after deducting the target filtrate sterile concentrated liquid were formulated into a total of 43.4 ml. The medium was weighed according to the aforementioned "Slope and Plate Medium Recipe" with a total volume of 100 ml of medium. The 43.3ml culture medium was sterilized by steam moist heat at 121°C for 30min, cooled to 48°C-50°C, and then placed in a water bath at 48°C-50°C for constant temperature preservation for later use.

2.3. 56.6ml of the target filtrate aseptic concentrate with a biological potency of 53286μ/ml of zhongshengmycin measured by the above-mentioned aseptic operation, heated to 45 ℃ by aseptic operation, placed in a 45 ℃ water bath and kept at a constant temperature for use, and kept at 45 ℃ The shorter the time, the better it should not exceed 10 minutes, which will reduce the inactivation of the biological activity of the self-metabolites in the concentrate.

2.4. A total of 43.4ml of the culture medium except the sterile concentrate of the target filtrate that has been sterilized and stored at a constant temperature in a 48°C-50°C water bath and the target filtrate stored at a constant temperature in a 45°C water bath. 56.6ml of the concentrated solution was quickly mixed evenly to obtain a plate medium containing 3% zhongshengmycin and other self metabolites and no zhongshengmycin cells. , the preparation of the gradient concentration medium was carried out immediately.

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

3.1. Under the sterile operating conditions of the sterile room, take 10ml of the plate culture medium kept in a water bath at a constant temperature of 48°C-50°C, place it in a 90mm petri dish, and place the petri dish on a horizontal bar quickly and tilt it to make the culture The dish rests on the bottom surface of the high position of the horizontal bar and just touches the medium, forming a petri dish with a thin medium on top and a thick medium on the bottom. After the medium is solidified, the petri dish is placed flat, and the petri dish forms a gradually thickening from one end to the other end. Medium slope, mark the arrow mark from the thickest medium to the thinnest point on the back of the culture dish, and the head of the "↑" arrow is the thinnest point of the medium;

3.2. Under the aseptic operation conditions of the sterile room, place the petri dish of the above-mentioned flat medium that has formed a solid slant on the thinnest part of the marked arrow, and then place it horizontally; 10 ml of the medium for self-metabolites such as neutropenia was quickly placed in the petri dish of the above-mentioned flat plate medium with a solid slope formed, and the medium was quickly shaken to spread horizontally. The petri dish of the medium with gradient concentration of its own metabolites such as zhongshengmycin, the content of its own metabolites such as zhongshengmycin in the petri dish gradually increases in the direction of the arrow, and the content of zhongshengmycin at the head of the arrow "↑" is 3%, and the concentration of other self-metabolites in the same medium is also the highest at the head of the arrow "↑";

3.3. Under the sterile operating conditions of the sterile room, simultaneously prepare 6 petri dishes containing medium with gradient concentrations of self-metabolites such as zhongshengmycin according to the above method; the content of zhongshengmycin at the arrow head at the bottom of the culture dish is 3%, and the concentration of other self-products in the same medium is also the highest at the head of the arrow "↑".

The gradient concentration medium thus finally formed is the screening medium for rational screening, and the structure of the screening medium is shown in FIG. 3 .

As shown in Figure 3, the side where the tail of the arrow "↑" is located is 0 mm, and the gradient concentration of autometabolites gradually increases along the direction of the arrow. In Figure 3, I shows the plate medium prepared without the concentrate of the target filtrate, and II shows the plate medium containing the concentrate of the target filtrate at the time of preparation.

The preparation of compound mutagenesis liquid of embodiment 5 target thalline filtrate

1. Take out the target starting bacterial strain filtrate stored in the refrigerator at 4°C obtained in Example 2, and place the prepared culture dish containing the gradient concentration medium of self-metabolites such as zhongshengmycin in Example 4 together. A sterile dark room with red light.

2. Cumulative decreasing UV-photoreactivation compound mutagenesis:

的紫外灯的诱变箱内的日光灯与紫外灯都同时开启预热稳定15分钟，关闭紫外灯与日光灯(该诱变箱外用红黑布罩罩着)；2.1. In a sterile dark room with red lights, a 30w fluorescent lamp and a 30w wavelength of

The fluorescent lamp and ultraviolet lamp in the mutagenesis box of the ultraviolet lamp are turned on at the same time to preheat and stabilize for 15 minutes, and the ultraviolet lamp and fluorescent lamp are turned off (the outside of the mutagenesis box is covered with red and black cloth);

2.2. Draw 10ml of the target starting strain bacterial filtrate into a 90mm double-disc sterile petri dish, remove the upper cover of the petri dish in a sterile dark room with red light, and put it into a mutagenesis with a 30w fluorescent lamp and a 30w ultraviolet lamp. On the rotating plate 20cm below the double lamp in the box, rotate slowly.

2.3. Perform 5 consecutive UV-photoreactivation mutagenesis:

2.3.1. First turn on the UV light for 30s, then turn off the UV light for 20s; turn on the fluorescent light for 20s; turn off the fluorescent light for 20s;

2.3.2. Turn on the UV light again for 25s, then turn off the UV light for 20s; turn on the fluorescent light for 20s; turn off the fluorescent light for 20s;

2.3.3. Turn on the UV light for 20s, then turn off the UV light for 10s; turn on the fluorescent light for 10s; turn off the fluorescent light for 10s;

2.3.4. Turn on the UV light again for 15s, then turn off the UV light for 10s; turn on the fluorescent light for 10s; turn off the fluorescent light for 10s;

2.3.5. Turn on the UV light for 10s, then turn off the UV light for 5s; turn on the fluorescent light for 5s; turn off the fluorescent light for 5s;

2.4. After 5 times of UV-light revival dose-accumulating and decreasing compound mutagenesis, a compound mutagenesis solution of the bacterial filtrate of the target starting strain was obtained.

Example 6 Rational Screening

1. Coating, cultivating and screening the above-mentioned composite mutagenesis liquid of the target departure strain thalline filtrate obtained by the cumulative reduction composite mutagenesis of the above-mentioned 5 times of ultraviolet-light revival, specifically comprising the following steps:

1.1. Coating: Place 6 pre-prepared petri dishes containing gradient concentration medium of self-metabolites such as zhongshengmycin in a red light sterile dark room, under aseptic operation conditions, apply all 5 petri dishes to the medium. Carry out the same operation: draw 1 ml of the composite mutagenesis liquid of the target starting strain cell filtrate obtained in Example 5 and spread it evenly on a 90mm double-disc petri dish containing a medium containing gradient concentrations of self-metabolites such as neutropenia. On; the remaining 1 petri dish was coated with the remaining filtrate of the target starting strain without UV-photoreactivation mutagenesis, which was used as a control petri dish.

1.2. Cultivation: In a sterile dark room, adjust the dark room temperature and humidity to be constant: temperature 28.5°C-29.5°C, relative humidity 55%-65%; the control culture dish and 5 same bacterial liquids subjected to compound mutagenesis were used The coated petri dishes were covered with lids facing upwards. After one day of culture, turn the whole set of petri dishes upside down with the lid as the bottom and cultivate for 7-10 days to avoid drying of the medium.

1.3. Select single colony strains that tolerate high concentrations of their own metabolites:

1.3.1. There are sparse colonies at the tail of the arrow marked on the control petri dish. With the extension in the direction of the arrow, the colonies are sparse. There is only one colony at the highest point of 22mm, and the colonies are all pink. According to the calculation of the concentration of the neutrophil gradient, the 22mm colony is resistant to the concentration of mesobiotics at about 7300μ/ml, and the colony is marked as d22;

1.3.2. There are sparse colonies at the tail of the arrows on the 5 petri dishes in the test, and there are almost no colonies as they extend in the direction of the arrows, but there are 3 petri dishes that extend up to 52mm, 58mm and 63mm have one colony respectively. According to the gradient concentration of zhongshengxin, the 52mm, 58mm and 63mm colonies can tolerate zhongshengxin concentration of about 17000μ/ml, 19000μ/ml and 21000μ/ml respectively; 52mm , 63mm colonies are small and dense, showing a flat steamed bread-like colony covered with spores, and the spores are pink; 58mm colonies are small and dense, showing a steamed bread-shaped colony full of spores with a higher bulge in the middle, but the spores are more white, The pink color is not obvious; the colonies of 52mm, 58mm, and 63mm on the three test dishes are labeled as n52, n58, and n63.

1.3.3. Select the above-mentioned colonies of d22, n52, n58, and n63 to transfer the slanted surface, and cultivate at 29 °C for 7 days. The slanted surface of d22 is thicker and pink, and the back of the test tube slope is dark pink; n52, n63 slanted bacterial moss Thickness is pink, and the back of the bevel of the test tube is dark pink; the thickness of the moss on the n58 bevel is gray and white and slightly pale pink, and the back of the bevel of the test tube is light pink.

1.3.4 Fermentation test: The screened tolerant strains n52, n58, n63 that are resistant to mesobiotics and their own metabolites, together with the control strain d22 and the strain UV-3 used for industrial production, were subjected to a 5L fermenter fermentation test (according to aforesaid "the culture medium formula of fermentation and fermentation culture condition", the fermentation culture cycle is 7 days), repeat 3 tests, investigate the fermentation level of mesobiotics, and relevant result data are as shown in table 3 below:

table 3.

As can be seen from Table 3, the titers of zhongshengmycin obtained by n52, n58 and n63 are all much higher than the 11389 μ/ml of the initial strain UV-3. It can be seen that the screening method of the present invention can obtain more high-yielding strains. Among them, the n58 with the highest yield is preserved, namely the Streptomyces lilacinus Hainan variety UV-11 with the deposit number: GDMCC No.61906.

Example 7 strain preservation and stability verification

1. After the five slants of UV-3, d22, n52, n58, and n63 involved in the above Example 6 were sealed with sterile liquid paraffin, the slants were sealed in liquid paraffin, and then stored in a -80°C refrigerator for 3 months.

2. Take out the slanted strains of UV-3, d22, n52, n58, n63 that are sealed and stored in the above-mentioned sterile liquid paraffin, carry out plate single colony isolation and cultivate for 7 days to 10 days to observe the growth of single colony, and the observation results are shown in the following table 4:

Table 4.

3. Select the characteristic single colonies in the single colony isolation plate, place the transfer slope in a sterile room with a temperature of 28.5°C-29.5°C, a relative humidity of 55%-65%, and cultivate for 7 days to 10 days; further observe the morphology of the slope bacteria: UV-3 has a thicker slanted moss and is pink, d22 has a thicker slanted moss and is pink, and the back of the bevel of the test tube is dark pink; n52 has a thick slanted moss, and the back of the test tube is dark pink; n58 has a thick slanted moss. Pink, the back of the bevel of the test tube is dark pink; the thickness of the bacterial moss on the n63 bevel is pink, and the back of the bevel of the test tube is dark pink;

4. Fermentation test: the above-mentioned liquid paraffin slant strains through -80 ℃ of cryopreservation, through single colony separation, and then transferred to slanted slant strains UV-3, d22, n52, n58 and n63 to carry out a 5L fermenter test together (according to the aforementioned "Fermentation medium formula and fermentation culture conditions", the fermentation culture period is 7 days), and the experiment was repeated 3 times.

Investigate the fermentation level of mesobiotics, and the result data are as follows in Table 5:

table 5.

As can be seen from Table 5, 3 strains (i.e. n52, n58 and n63) that tolerate higher doses of self-metabolites such as mesobiotics, wherein the fermentation level of biotin is not only somewhat higher than that of the existing strains. Compared with the control group (UV-3, d22), it also has a relatively large increase, especially n58, whose highest increase is more than 55%.

Example 8 Industrialization Fermentation Production Verification

Apply UV- ³ and UV-11 to a 40m fermentor to carry out industrialized fermentation production verification (according to the aforementioned "fermented medium formula and fermentation culture conditions", the fermentation culture period is 5 days), and the zhongshengmycin is put into the tank parameters of the fermentation broth. The parameters of the fermentation broth of UV-11 are as follows:

Total sugar: 1.306g/100ml, reducing sugar: 0.6359g/100ml, amino nitrogen: 88.58mg/100ml, total phosphorus 10.25mg/L, bacterial concentration 27.2%, pH 6.95, biological potency 18050μ/ml.

Under the same fermentation conditions and fermentation cycle, the fermentation level of mesobiotics in the UV-3 strain tank batch was only 11936 μ/ml, while the mesobiotic fermentation unit of the UV-11 strain tank batch reached 18050 μ/ml. That is, the industrialized fermentation level of UV-11 strain was 52.5% higher than that of control strain UV-3. The components of the fermentation liquid in the UV-11 fermentation tank liquid were further determined by high performance liquid chromatography, and the results are shown in Figure 1 and Figure 2. Wherein, Figure 1 is the HPLC test result of the control strain UV-3; Figure 2 is the HPLC test result of the UV-11 strain.

It can be seen from Figure 1 and Figure 2 that the retention time of each component in the UV-11 fermentation tank liquid is in the same interval as the retention time of the control strain UV-3, and the specific data are shown in Table 6 below.

Table 6.

It should be noted that the conditions used by HPLC are as follows:

Model: waters e2695

Mobile phase: 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 twenty two 78 20 twenty two 78 twenty two 15 85 30 15 85

Flow rate: 1.0 mL/min.

Column temperature: 30℃±2℃.

Detection wavelength: 200nm.

Injection volume: 10 μL.

Column: water SymmetryShield ^TM RP18 5μm

Example 9 Preparation of Preparations Containing Zhongshengmycin

1. Acidification of the fermentation broth: the fermentation broth with a biological titer of 18,050 μ/ml obtained in Example 8 was adjusted to an acidified solution with a pH of 3.5;

2. Solid-liquid separation of acidified liquid: The acidified liquid of the fermentation broth is injected into a ceramic membrane with a pore size of 0.20 μm for solid-liquid separation to obtain a permeate, which is then injected into a ceramic membrane with a pore size of 0.05 μm for liquid-solid separation to obtain fermentation. Liquid secondary permeate;

3. Secondary permeate concentration of fermentation broth: the secondary permeate is concentrated through a nanofiltration membrane with a molecular weight of 400Da to obtain a concentrated solution;

4. Spray drying: The concentrated liquid is directly spray-dried to obtain a dry powder of the fully water-soluble parent drug of Zhongshengmycin. The biological potency of the dry powder of the parent drug is determined to be 510025 μ/g.

The above are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Therefore, any changes or modifications made according to the claims and description of the present invention shall be covered by the patent of the present invention. within the range.

Claims (10)

1. A screening medium, which is characterized by comprising a concentrated fermentation broth containing a substrate factor, wherein the concentrated fermentation broth containing the substrate factor is obtained by concentrating a fermentation broth obtained from fermentation of a starting strain to the end of a growth arrest phase for production of a secondary metabolite.

2. The screening medium of claim 1, which is a gradient concentration medium of substrate factors.

3. The screening medium according to claim 1, wherein the starting strain is Streptomyces lavendulae, preferably the starting strain is Streptomyces lavendulae Hainanensis variant (Streptomyces lavendale var. hainanensis).

4. The selection medium according to claim 3, wherein said production of growth arrest stage end 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 biological potency of the zhongshengmycin in the end of the production growth stagnation stage is 10000-12000 mu/ml.

5. The screening medium of claim 3, wherein the biological potency of the zhongshengmycin in the gradient medium is 20000-50000 μ/ml at the maximum; for example 30000. mu.l/ml.

6. The screening media of claim 3, wherein the conditions of the fermentation meet 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.

7. A method for preparing a screening medium according to any one of claims 1 to 6, comprising the steps of:

step S1: respectively preparing a plate culture medium and concentrated fermentation liquor containing substrate factors;

step S2: pouring the plate culture medium into a culture utensil such as a culture dish, and placing the culture utensil obliquely to solidify the plate culture medium to form a lower screening culture medium with a gradually thickened gradient;

step S3: and mixing the concentrated fermentation liquor containing the substrate factors with a plate culture medium, pouring the mixture into a horizontally placed culture apparatus in the step S2, and forming a gradually thickened gradient upper-layer screening culture medium in the opposite direction to the lower-layer screening culture medium in the culture apparatus after the mixture is solidified to obtain the culture medium.

8. The method of claim 7, wherein preparing the concentrated fermentation broth comprising the substrate factor comprises the steps of:

step S11: fermenting the starting strain to obtain fermentation liquor at the end of the growth stagnation stage;

step S12: filtering the fermentation liquor to obtain filtrate, wherein the filtrate is concentrated to obtain concentrated fermentation liquor containing substrate factors; the concentration is preferably concentration under reduced pressure.

9. The method according to claim 8, wherein in the step S12, the reduced-pressure concentration includes 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 is 1 (5-10), for example 1: 5.

10. Use of a screening medium according to any one of claims 1 to 6 for the cultivation and/or screening of a strain resistant to self-metabolites.

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