Breeding method for improving strain production capacity of antibiotics

Abstract

The invention relates to the technical field of microbial breeding and biology, and provides a breeding method for improving the strain production capacity of antibiotics. The method comprises the following steps: burying production strain seeds in soil, influencing for 3-6 months, separating single colonies, inoculating with an inclined plane, carrying out shake flask titer verification on inclined plane spores, and carrying out strain screening. According to the method disclosed by the invention, the situation that most antibiotic production strains are obtained from natural soil is considered, the strains are returned to the main nutrient of the soil, and a hotbed effect is generated through breath exchange of the strains, the soil environment and growth factors in the soil for a period of time, so that the strains have the effect of stimulating the activity under the influence of a mild external environment condition.

Description

Technical Field

[0001] The present invention belongs to the fields of microbial breeding and biotechnology, and particularly relates to a method for breeding strains for activating and enhancing the strain ability in the production of antibiotics by Streptomyces and bacteria. Background Art

[0002] Microbial secondary metabolites have rich structures and diverse biological activities, and are widely used in the pharmaceutical field. They are an important source of natural medicine lead compounds. Since the lead compounds derived from wild strains have low content and complex components, which seriously affect their industrial application, the research on how to effectively improve the antibiotic production ability has always been one of the research hotspots in industrial microbial genetic breeding. So far, in industrial microbial breeding, each enterprise still uses physical mutagenesis, chemical mutagenesis or a combination of both to mutate strains, hoping to improve the growth level, but its efficiency and accuracy are relatively poor;

[0003] From the perspective of the traditional concept of optimizing strains, these methods for mutating and transforming strains are a kind of damage to microbial individuals. After being damaged, the first thing for the microorganisms is to revive, and then it is the repair after mutation. It is actually very difficult to obtain excellent individuals after repair. In view of this background, the following method is invented. Summary of the Invention

[0004] In order to solve the problems of difficult strain optimization and slow production progress, the present invention provides a method for optimizing strains of antibiotic-producing bacteria.

[0005] The technical solution adopted by the present invention is as follows: A breeding method for improving the ability of antibiotic-producing strains, comprising the following steps: burying the seeds of the production bacteria in the soil for 3 - 6 months, separating single colonies, inoculating slants, verifying the flask titer of the slant spores, and screening the strains.

[0006] A further technical solution lies in that it specifically includes the following steps:

[0007] A. Take the soil in the experimental field;

[0008] B. Mix the seeds of the production bacteria into the sterilized soil, sub-pack them into culture dishes, and conduct gas exchange with the outside world;

[0009] C. Place the culture dishes containing the soil in step B in different environments for influence;

[0010] D. After 3 - 6 months, take back the culture dishes containing the soil in step C to the laboratory;

[0011] E. Dilute the soil in step D with water under sterile conditions and separate it into single colonies;

[0012] F. Transfer the slants after the single colonies mature;

[0013] G. After the inclined plane matures, inoculate the inoculation bottle and transfer it to the fermentation bottle to verify the titer.

[0014] H. Select the strains with a titer higher than the control for preservation.

[0015] A further technical solution is that the soil in step A is the soil below 20 cm in depth on the ground surface that has not been polluted by chemical fertilizers and pesticides and has been sterilized twice at 121 °C.

[0016] A further technical solution is that the production bacteria in step B are the spores or cells of any randomly selected strain.

[0017] A further technical solution is that in step B, the culture dish is fixed crosswise with a 1 cm wide tape, so that the soil in the culture dish can exchange air with the outside world.

[0018] A further technical solution is that in step C, the different environments for storage are the working laboratory, the excavation site in the experimental field, and the grassland at the same depth as the excavation by the lake in the park.

[0019] A further technical solution is that the inclined plane culture medium, the seed bottle culture medium, and the fermentation culture medium are the conventional culture media used respectively.

[0020] A further technical solution is that the culture conditions of the inclined plane, the seed bottle, and the fermentation bottle are the respective conventional culture temperatures and humidities.

[0021] Beneficial effects:

[0022] Based on the theory of life science, that is, biology, by studying the laws of life activities, development laws, and the mutual relationship between organisms and the environment, considering that most antibiotic-producing strains are obtained from the soil in nature, through years of research, it is found that when the strains return to the soil base camp, after a period of gas exchange between the strains, the soil environment, and the growth factors in the soil, a hotbed effect will occur, which has a unique impact on the stimulation of the strains. The present invention is applicable to Streptomyces, bacteria, etc. Under the influence of mild external environmental conditions, the strains obtain the effect of activating their vitality. This method plays a promoting and driving role in excavating the vitality of the strains and promoting the production level, and some varieties have been successfully applied to production. Description of the drawings

[0023] Figure 1 It is a flow schematic diagram of the present invention. Detailed implementation manners

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will refer to the accompanying drawings required in the embodiments or the description of the prior art. Figure 1For a brief introduction, obviously, the attached drawings in the following description Figure 1 are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] A breeding method for improving the ability of antibiotic-producing strains. Streptomyces spores or bacterial cells are buried in the soil for a certain period of time, single colonies are isolated, inoculated on slants, and then inoculated into shake flasks for ability verification, thereby screening the strains.

[0026] Preferably, the breeding method for improving the ability of antibiotic-producing strains specifically includes the following steps:

[0027] A. Take the soil in the experimental field;

[0028] B. Mix the seeds of the production bacteria into the sterilized soil, divide them into culture dishes, and conduct gas exchange with the outside world;

[0029] C. Place them in different environments for influence;

[0030] D. After a period of time, retrieve the bacteria-carrying soil in different environments back to the laboratory;

[0031] E. Dilute with water under sterile conditions to isolate single colonies;

[0032] F. Transfer the slants when the single colonies mature;

[0033] G. Inoculate the flasks after the slants mature, inoculate the fermentation flasks, and verify the titer;

[0034] H. Select the strains with a titer higher than the control for preservation.

[0035] Preferably, for step A of taking the soil in the experimental field, it is the soil below 20 cm deep without being polluted by chemical fertilizers and pesticides, and sterilized at 121 °C for ≥2 times;

[0036] Preferably, the strains in step B include Streptomyces or bacteria, etc., and are mixed with the sterilized soil;

[0037] Preferably, the mixed spore soil is divided into glass petri dishes, not sealed but fixed to prevent the petri dishes from being opened and enable gas exchange;

[0038] Preferably, the petri dishes are placed in different environments to be influenced and stimulated, and the locations include the working laboratory (construction site), the excavation site (original site), and the lawn near the lake (lake site);

[0039] Preferably, after a period of time, retrieve them back to the laboratory when the stimulation lasts for 6 months;

[0040] Preferably, use an inoculation spoon to scoop about 0.1 g of soil after environmental stimulation, add it to 10 mL of sterile water, and dilute it step by step;

[0041] Preferably, aspirate diluents of different gradients and spread them on petri dishes. The petri dishes are cultured using their respective conventional culture media and culture conditions;

[0042] Preferably, after a single colony grows to maturity, inoculate it onto its respective large test tube slant. The large test tube slant is cultured using its respective conventional culture media and culture conditions;

[0043] Preferably, after the slant matures, inoculate it into its respective seed bottle. The seed bottle is cultured using its respective conventional culture media and culture conditions;

[0044] Preferably, after the seed bottle matures, inoculate it into its respective fermentation bottle medium. The fermentation bottle is cultured using its respective conventional culture media and culture conditions;

[0045] Preferably, after the fermentation bottle reaches the cycle, perform potency determination. The determination method is its respective conventional potency detection method;

[0046] Preferably, select the corresponding slant spores or bacteria according to the test results for strain preservation.

[0047] Example 1

[0048] 1. Using Streptomyces viridochromogenes that produces avilamycin as the experimental strain, according to Figure 1 Mix the slant spores of Streptomyces viridochromogenes and the soil, and dispense them into 3 sterile petri dishes;

[0049] 2. Place the 3 petri dishes in the locker of the working laboratory, underground at the place where the soil was dug, and on the lawn by the lake in a park with elegant conditions for 6 months

[0050] The conventional culture media and culture conditions used are as follows:

[0051] The composition and ratio of the slant culture medium are (g / L): corn starch 20.0, KNO 3 1.0, K 2 HPO 4 0.5, CaCl 2 2.0, agar 30.0; the pH of the culture medium is 7.0 - 7.2, and the culture conditions are: culture temperature 28.0 °C, natural humidity, and culture cycle 7 days.

[0052] The composition and ratio of the seed bottle culture medium are (g / L): soybean cake powder 15.0, yeast powder 25.0, glucose 5.0, dextrin 20.0, CaCl 2 2.0, CaCO 31.0, pH 7.0 - 7.2. The shaking table amplitude is 5 cm, the rotation speed is 200 - 220 rpm, the temperature is 28.0 °C, natural humidity, and the periodic culture is 25 h.

[0053] The components and ratios of the fermentation medium are (g / L): corn starch 40.0, glucose 20.0, soybean cake powder 10.0, CaCl 2 2.0, CaCO 3 4.0, NaCl 1.0, pH 7.0 - 7.2; the shaking table amplitude is 5 cm, the rotation speed is 200 - 220 h, the culture temperature is 28.0 °C, natural humidity, and the culture period is 96 h.

[0054] 3. Verify the titer of avila. The control titer is specified as "1", and the influence result is displayed in multiples compared with the control. The results are shown in the following table

[0055]

[0056] Strain preservation: The colony 9 buried in the original place for 6 months was preserved in the strain center, preservation number: SD24 - 24095. It has been applied to production.

[0057] Example 2

[0058] 1. Using Streptomyces albus that produces polylysine as the experimental strain, according to Figure 1 Mix the slant spores of Streptomyces albus and the soil, and divide them into 3 sterile petri dishes;

[0059] 2. The 3 petri dishes are respectively placed in the storage cabinet of the working laboratory, underground at the place where the soil is dug, and on the lawn by the lake in the elegant park, and the placement time is 6 months

[0060] The conventional medium and culture conditions used are as follows:

[0061] The components of the slant medium used are (g / L): glucose 10.0, peptone 1.0, yeast powder 1.5, beef extract 1.0, agar 20.0; the medium pH is 7.0 - 7.2, and the culture conditions: the culture temperature is 30.0 °C, natural humidity, and the culture period is 6 days.

[0062] The components and ratios of the seed bottle medium are (g / L): glucose 50.0, yeast powder 5.0, potassium dihydrogen phosphate 1.0, dipotassium hydrogen phosphate 0.8, ammonium sulfate 2.0, magnesium sulfate 0.5, pH 7.0 - 7.2. The shaking table amplitude is 5 cm, the rotation speed is 220 - 250 rpm, the temperature is 3.0 °C, natural humidity, and the periodic culture is 20 h

[0063] The components and ratios of the fermentation medium are (g / L): corn starch 20.0, glucose 20.0, yeast powder 5.0, potassium dihydrogen phosphate 1.0, dipotassium hydrogen phosphate 0.8, ammonium sulfate 10.0, magnesium sulfate 0.5, ferrous sulfate 0.03, pH 7.0 - 7.2; shaker amplitude 5 cm, rotation speed 220 - 250 h, culture temperature 27.0°C - 29.0°C, natural humidity, culture period 48 h

[0064] 3. Verify the titer of polylysine. The specified control titer is "1", and the influencing results are shown in multiples compared with the control. The results are shown in the following table

[0065]

[0066] Strain preservation: Colony 8 buried by the lake for 6 months was preserved in the strain center, preservation number: SD41 - 24021. It has been applied to production currently.

[0067] Example 3

[0068] 1. Using Bacillus polymyxa var. colistimus as the experimental strain, according to Figure 1 Mix the Bacillus polymyxa cells and the soil, and dispense them into 3 sterile petri dishes;

[0069] 2. Place the 3 petri dishes in the locker of the working laboratory, underground at the place where the soil was dug, and on the lawn by the elegant park lake respectively, for 6 months

[0070] The conventional culture medium and culture conditions used are as follows:

[0071] The components of the slant medium used are (g / L): glucose 1.0, yeast powder 1.0, beef extract 0.1, sodium chloride 0.5, light calcium carbonate 0.2, agar 2.0; the pH of the slant medium is 6.0 - 7.2. Culture temperature 31.0°C, culture period 48 h.

[0072] The components of the seed flask medium are (g / L): yeast powder 30.0, malt extract 30.0, sodium chloride 5, medium pH 7.0 - 7.2. Activation culture conditions: rotation speed 200 - 220 rpm, culture temperature 31.0°C, culture period 16 h.

[0073] The components of the fermentation flask medium are (g / L): starch 90.0, soybean cake powder 20.0, calcium carbonate 14.0, ammonium sulfate 20.0, potassium dihydrogen phosphate 1.2, amylase 0.05, soybean oil 10.0, pH 7.2 - 7.4. Fermentation culture conditions: rotation speed 200 - 220 rpm, temperature 31.0°C, periodic culture 96 h.

[0074] 3. Verify the titer of colistin. The specified control titer is "1", and the influence result is shown in multiples compared with the control. The results are shown in the following table

[0075]

[0076] Strain preservation: This variety preserves the No. 4 strain in the lake area, preservation number: SD03-24939, which is currently to be applied to production;

[0077] As can be seen from the above examples, in Example 1, the green-producing Streptomyces viridifaciens of avilamycin has its strain vitality improved under the technical method of this invention. In Example 2, the white Streptomyces producing polylysine has its strain vitality improved under the technical method of this invention. In Example 3, Bacillus polymyxa has its strain vitality improved under the technical method of this invention. It can be seen that this method is applicable to Streptomyces, bacteria, etc. The strains have their vitality stimulated under the influence of mild external environmental conditions. This method plays a promoting and driving role in exploring the strain vitality to promote the production level, and some varieties have been successfully applied to production.

[0078] As described above, although the present invention randomly selects three varieties to show examples, and the stimulation time is only 6 months, the protection scope of the present invention is not limited thereto. Any person skilled in the art in the technical field of the present invention can understand that various changes, modifications, substitutions and variations can be made to these examples without departing from the principle and spirit of the present invention, and all should be covered within the protection scope of the present invention. The present invention provides a method for breeding antibiotic-producing strains. The flow chart of the invention is shown in the appendix Figure 1 .

Claims

1. A method for breeding bacteria to improve their ability to produce antibiotics, characterized in that: The method comprises the following steps: burying the production bacteria seeds in the soil for 3-6 months, isolating single colonies, inoculating the slant, performing shake flask titer verification on the slant spores, and screening the strains.

2. A method for breeding bacteria to improve the ability of producing antibiotics according to claim 1, characterized in that: The specific steps include: A. Take soil from the experimental field; B. Mix the seeds of the production bacteria into the sterilized soil, and dispense them into culture dishes to exchange air with the outside world; C. Place the culture dishes containing soil in step B in different environments for influence; D. After 3-6 months, take the culture dish containing soil in step C back to the laboratory; E. Dilute the soil in step D with water under sterile conditions and separate into single colonies; F, single colony matures and then spreads on a slant; G. After the slant matures, inoculate the bottle, connect it to the fermentation bottle, and verify the titer; H. Select strains that are higher than the control for preservation.

3. A method for breeding bacteria to improve the ability of producing antibiotics according to claim 1 or 2, characterized in that: The soil in step A is soil that is not polluted by fertilizers and pesticides and is sterilized twice at 121° C. at a depth of 20 cm below the surface.

4. A method for breeding bacteria to improve the ability of producing antibiotics according to claim 2, characterized in that: The production bacteria in step B are spores or cells of any randomly selected bacterial species.

5. A method for breeding bacteria to improve the ability of producing antibiotics according to claim 2, characterized in that: In step B, the culture dish is fixed with a 1 cm wide tape in a cross shape, so that the soil in the culture dish can exchange air with the outside world.

6. A method for breeding bacteria to improve the ability of producing antibiotics according to claim 2, characterized in that: The different environments in step C are storage environments, namely, a working laboratory, an excavation site in an experimental field, and a grassland at the same depth as the excavation site by a lake in a park.

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