阅读说明：本技术 一种替考游动放线菌的保藏方法 (Preservation method of actinoplanes teichoi ) 是由 王耀耀 张雪霞 朱研研 郝惠云 郄丽萍 王亚莉 于 2018-09-05 设计创作，主要内容包括：本发明提供一种用于促进替考游动放线菌工业生产菌株产生丰富的游动孢囊孢子的培养方法,菌种培养基配方中选用小麦胚芽粉作为孢子萌发的主要碳氮源,以天冬酰胺、硫酸铵作为补充氮源,微量元素补充铁、锰、钴、锌离子。培养温度采用分阶控制。并以甘油、硬葡聚糖作为复合冷冻保护剂进行-80℃或超低温液氮保藏制备菌种库,菌种库的保藏稳定性得到大幅提高。(The invention provides a culture method for promoting a test actinoplanes industrial production strain to produce rich zoosporangium spores. The culture temperature is controlled in a step mode. And glycerol and scleroglucan are used as composite cryoprotectants to be preserved in liquid nitrogen at the temperature of minus 80 ℃ or ultralow temperature to prepare the strain library, so that the preservation stability of the strain library is greatly improved.)

1. A preservation method for actinoplanes teichoensis comprising: (1) preparing a spore-producing culture medium; (2) inoculating the preserved strain to a spore-producing culture medium, wherein the culture temperature of the strain is controlled in a stepwise manner; (3) collecting mature sporangiospores by adopting a composite freezing protective agent of glycerol and scleroglucan, carrying out process cooling by a certain cooling program, and carrying out freezing preservation to prepare a multi-stage strain library; the method is characterized in that:

the spore-producing culture medium comprises the following components in percentage by weight: 15-20g of wheat germ powder, 0.1-0.2 g of asparagine, 2.0-3.0g of ammonium sulfate, 0.5-1.0 g of monopotassium phosphate, 2.0-4.0g of calcium carbonate, 0.02-0.03g of ferrous sulfate heptahydrate, 0.005-0.01g of manganese chloride tetrahydrate, 0.01-0.02g of zinc sulfate heptahydrate, 0.002-0.005g of cobalt chloride, 20g of agar powder and 1000mL of distilled water.

2. A preservation method according to claim 1, characterized in that: the strain culture temperature in the step (2) is controlled as follows: 0-144h 27-29 ℃, 144-192h 24-26 ℃.

3. A preservation method according to claim 1, characterized in that: the cryoprotectant in the step (3) comprises the following components in percentage by weight: glycerol 100-^-110-20 g.L of scleroglucan^-1。

4. A preservation method according to claim 1, characterized in that: the cooling procedure in the step (3) is as follows: the speed is reduced to-35 ℃ at 7 ℃/min, and then the mixture is rapidly preserved in liquid nitrogen or at-75 to-85 ℃.

5. A preservation method according to claim 1, characterized in that: the preparation of the strain library in the step (3) comprises the following steps: the preservation method of the strain stock seed bank is ultra-low temperature liquid nitrogen preservation, the main strain seed bank is frozen and preserved at-75 ℃ to-85 ℃, and the production seed bank is frozen and preserved at-75 ℃ to-85 ℃.

Technical Field

The invention relates to a preservation preparation method of microbial strains for antibiotic fermentation production, in particular to a culture method for producing a large amount of zoosporangium spores by using actinoplanes teichoensis and a strain preservation method.

Background

Teicoplanin is another drug-resistant bacteria antibiotic with important clinical curative effect after vancomycin and demethyl vancomycin, and the action mechanism of teicoplanin generates strong antibacterial activity to gram-positive bacteria by inhibiting bacterial cell wall synthesisThe other is the resistance to severe infection caused by multi-drug resistant bacteria staphylococcus aureus and enterococcus, and is another drug barrier for treating antibiotic resistance infection. The industrial production of teicoplanin is a microbial fermentation method by using rare actinomycetes-teicoplasma natans (Actinoplanes teichomyceticus) Performing pure fermentation production. In fermentation production, a multi-stage strain library is constructed by adopting a proper strain preservation means, and the expression of high-yield fermentation characteristics of strains is maintained. The stability of the strain stock and the strain preservation quality are directly related to key indexes such as production stability, production efficiency, final product quality and the like, and are important technical links of the whole production flow.

Actinoplanes teichou (A)Actinoplanes teichomyceticus）Belongs to actinoplanes, no aerial hyphae are formed in the hypha development, and the dormant form is zoospores which are embedded on sporocyst stalks and are wrapped in sporocysts. The adoption of dormant bodies of the strains for preservation as much as possible is a basic strategy for strain preservation. Previous studies have demonstrated the special culture characteristics and impact requirements of numerous natural wild-type actinoplanes. Italian scholars F, PARENT et al^[1]A actinoplanes capable of producing teicoplanin was originally isolated, and the culture characteristics thereof showed that the strains can form zoosporangia on various culture media such as ISP2, ISP3 and ISP 7. Joachim M. Wink et al 2006^[2]The modern microorganism classification identification technology is utilized to name the teicoplanin producing bacteria AB8317 as teicoplanin actinoplanes (A)Actinoplanes teichomyceticus) The new species, the morphological feature analysis of which produced a large number of zoosporangia on ISP 3. Chinese scholar 'Tianmin' and the like^[3]A separated teicoplanin producing bacterium teicoplasma motinum variant (Actinoplanes teichomyceticusvar.jianensis) It is suitable for the production of cysts on glucose asparagine medium, whereas no cysts are produced on ISP3, ISP 4. With the progress of generating the physiological breeding by teicoplanin in recent years, Jinshihua and the like^[4]Breeding obtained valine hydroxamic acid resistance high-yield mutant strain and Wu military camp and the like^[5]The bred high-yield sodium acetate resistant mutant strains show the characteristics of reduced bacterial types and compact basic filaments. Go toThe step study shows that the spore of the high-yield strain is reduced or eliminated under the original slant culture condition, and the preservation stability of the strain is obviously reduced. From Carlo Taurino et al^[6]Optimization research of teicoplanin fermentation process published in recent years and Beltrametti F et al^[7]The strain preservation means adopted in the published research on the self-resistance mechanism of the teicoplanin producing strain all adopt shake flask hypha amplification and then carry out freezing preservation, and the report of adopting zoosporangium spores to carry out strain preservation is not seen.

Disclosure of Invention

The invention aims at the strain produced by teicoplanin industry (Actinoplanes teichomyceticus) The problem of reduced preservation efficiency caused by less or no sporangiospores in freezing preservation provides a strain preservation method for culturing zoophobous teichonii, obtaining abundant zoosporangium spores and preparing a multi-stage strain bank for producing seeds.

A preservation method for actinoplanes teichoensis comprising: (1) preparing a spore-producing culture medium; (2) inoculating the preserved strain to a spore-producing culture medium, wherein the culture temperature of the strain is controlled in a stepwise manner; (3) collecting mature sporangiospores by adopting a composite freezing protective agent of glycerol and scleroglucan, carrying out process cooling by a certain cooling program, and carrying out freezing preservation to prepare a multi-stage strain library; the method is characterized in that:

the spore-producing culture medium comprises the following components in percentage by weight: 15-20g of wheat germ powder, 0.1-0.2 g of asparagine, 2.0-3.0g of ammonium sulfate, 0.5-1.0 g of monopotassium phosphate, 2.0-4.0g of calcium carbonate, 0.02-0.03g of ferrous sulfate heptahydrate, 0.005-0.01g of manganese chloride tetrahydrate, 0.01-0.02g of zinc sulfate heptahydrate, 0.002-0.005g of cobalt chloride, 20g of agar powder and 1000mL of distilled water.

The technology of the strain preservation method is as follows:

1. spore production medium:

in the formula, the natural raw material wheat germ powder rich in various amino acids is selected as a main carbon nitrogen source for spore germination, asparagine and ammonium sulfate are used as supplementary nitrogen sources, and trace elements supplement iron, manganese, cobalt and zinc ions.

2. The concrete proportion is as follows: 15-20g of wheat germ powder, 0.1-0.2 g of asparagine, 2.0-3.0g of ammonium sulfate, 0.5-1.0 g of monopotassium phosphate, 2.0-4.0g of calcium carbonate, 0.02-0.03g of ferrous sulfate heptahydrate, 0.005-0.01g of manganese chloride tetrahydrate, 0.01-0.02g of zinc sulfate heptahydrate, 0.002-0.005g of cobalt chloride, 20g of agar powder and 1000mL of distilled water.

3. Inoculation and culture

The teicoplanin producing strain (Actinoplanes teichomyceticus) Inoculating spore-producing culture medium under aseptic condition, and controlling culture temperature by stages at 0-144h 27-29 deg.C and 144-192h 24-26 deg.C.

4. Freezing preservation method

The cryoprotectant adopted by the invention is a glycerol and scleroglucan composite formula, and the preparation method comprises the following steps:

solution 1: weighing 2-4g of scleroglucan, suspending in 100mL of distilled water, and swelling for 1 h.

Solution 2: 20-40g of glycerol was weighed and dissolved in 100mL of distilled water.

Solution 1 and solution 2 were mixed according to the ratio of 1: 1 volume ratio and sterilized at 121 ℃ for 30 minutes.

Adding the sterile cryoprotectant solution into a spore culture under sterile conditions, culturing by the culture method 3 to prepare a spore suspension, shaking for 10min at 220r/min, filtering by medium-speed filter paper, transferring the spore solution into a sterile freezing tube after filtering, reducing the temperature rate to-35 ℃ at 7 ℃/min, and rapidly storing in liquid nitrogen at-80 ℃ or ultralow temperature.

When the multi-stage strain library is prepared for production and seed supply, a teicoplanin production seed stock library, a production seed library main strain library and a production seed library are respectively prepared according to the strain culture and cryopreservation methods.

Through comparative experiments, it can be known that teicoplanin produces bacterial species: (Actinoplanes teichomyceticus) The improved spore producing strain culture medium has rich sporocyst, obviously superior to the reference culture medium, and sporocyst live count up to 8.0X 10⁶The above. The verification proves that the survival rate of the strains in the teicoplanin production strain three-level strain stock is over 84 percent after 1 year.

Drawings

FIG. 1 teicoplanin producing strain (Actinoplanes teichomyceticus) Hyphal cyst morphology on improved spore production medium.

FIG. 2 teicoplanin producing species (Actinoplanes teichomyceticus) Mycelial cyst morphology on control ISP7 medium.

Detailed Description