CN102352317B - Micro-culture method of pleuromutilin producing bacteria and high-throughput screening method of high-yield bacteria of pleuromutilin - Google Patents

Abstract

The invention provides a micro-cultivation method for pleuromutilin-producing bacteria, the steps are as follows: isolate single colonies after culturing the pleuromutilin-producing bacteria; prepare a solid fermentation medium and add it to each well of an enzyme-labeled plate after autoclaving , inoculate the mycelium of a single colony of pleuromutilin-producing bacteria on an enzyme plate and culture it for 7-9 days. The present invention also provides a high-throughput screening method for high-yielding pleuromutilin-producing bacteria. The steps are as follows: add extracts to the wells of microplate microplates of pleuromutilin-producing bacteria for extraction, and absorb the extracts in each well to transfer the plate. , adding a chromogen to develop the color, using a microplate reader to measure and analyze the results, and the screened pleuromutilin high-producing bacteria were obtained. Due to the adoption of solid fermentation, the step of seed cultivation is simplified, and the fermentation time is greatly shortened. Compared with the common screening method, the high-throughput screening method of the present invention has a good correlation coefficient, which shows that the high-throughput screening method of the present invention can be applied to the screening of high-yield pleuromutilin bacteria.

Description

Microculture method for pleuromutilin-producing bacteria and high-throughput screening method for high-yielding bacteria

technical field

The invention relates to a micro-cultivation method for pleuromutilin-producing bacteria and a high-throughput screening method for high-yielding bacteria.

Background technique

Pleuromutilin derivatives include warnimulin, tiamulin, etc., which are mainly added to animal feed as a premix, which not only prevents swine malaria and pneumonia, but also increases the output of pigs. The role of pleuromutilin antibiotics in production practice has become increasingly prominent, and its fermentation research has gradually become a research hotspot. In order to increase fermentation yield, breeding high-yielding strains is a breakthrough point for the application of pleuromutilin antibiotics.

At present, high-throughput microbial micro-cultivation and detection methods are gradually becoming the main method for strain screening, but the micro-culture methods of various microorganisms are different, and there is no micro-culture method for a class of higher basidiomycete fungi that produce pleuromutilin. It has been well established; the rapid detection technology is mainly based on the optical detection of high-throughput microplate reader, but the effective, fast and accurate trace detection method for diterpene substances such as pleuromutilin antibiotics has not been reported .

The key to applying microplate reader detection technology to high-throughput, high-yield bacterial strain screening lies in the stable characteristic absorption peak of the product and the efficient transfer of the product to the plate. Only the combination of micro-culture and microplate reader detection can solve this problem well, and the current micro-culture and high-throughput detection technologies are not suitable for higher basidiomycete fungi such as pleuromutilin-producing bacteria, and Its special diterpene structure.

Contents of the invention

The invention provides a micro-culture method for pleuromutilin-producing bacteria, and also provides a high-throughput screening method for high-yield pleuromutilin-producing bacteria.

In order to achieve the above object, the present invention provides a micro-cultivation method of pleuromutilin-producing bacteria, the steps of the micro-culture method are as follows:

1) Dilute the pleuromutilin-producing bacteria into a bacterial suspension, spread it on a plate, and isolate a single colony after culturing;

2) Preparation of solid fermentation medium: the following are mass percentages: glucose 5.7%, corn steep liquor 4%, soybean oil 0.4%, MgSO ₄ 0.04%, soybean peptone 1%, agar powder 1.5%, and the rest is water;

3) After autoclaving the solid fermentation medium in step 2), add it to the microtiter plate in a sterile environment, and the amount added to each well is 1/3-2/3 of the well volume;

4) Inoculate the single colony mycelium of the pleuromutilin-producing bacteria formed in step 1) on the microplate plate prepared in step 3), and culture at 25°C for 7-9 days to complete the production of pleuromutilin Microculture of bacteria.

Further, in step 3), the amount of the solid fermentation medium added to each well is 1/2 of the volume of the well.

The present invention also provides a high-throughput screening method for high-yielding pleuromutilin bacteria. The steps of the high-throughput screening method are as follows:

1) Dilute the pleuromutilin-producing bacteria into a bacterial suspension, spread it on a plate, and isolate a single colony after culturing;

2) Preparation of solid fermentation medium: the following are mass percentages: glucose 5.7%, corn steep liquor 4%, soybean oil 0.4%, MgSO ₄ 0.04%, soybean peptone 1%, agar powder 1.5%, and the rest is water;

3) After autoclaving the solid fermentation medium in step 2), add it to the microplate in a sterile environment, and the amount added to each well is 1/3-2/3 of the well volume;

4) Inoculate the single colony mycelia of the pleuromutilin-producing bacteria formed in step 1) on the microplate prepared in step 3), and culture at 25°C for 7-9 days;

5) Add 50-100 μl methanol or ethanol to each well in step 4) for extraction;

6) Take a certain amount of extract from each well and transfer to another microplate;

7) Add concentrated sulfuric acid to each well for color development, the volume of the concentrated sulfuric acid is greater than or equal to the volume of the extract;

8) Dilute each well to the same multiple, transfer the diluted solution to another microplate plate, measure the ultraviolet absorption of the microplate reader at a wavelength of 450nm-460nm, analyze the measurement results, and obtain the screened pleuromutilin High-yielding bacteria.

Further, in step 3), the amount of the solid fermentation medium added to each well is 1/2 of the volume of the well.

Further, the volume of the concentrated sulfuric acid in step 7) is the same as the volume of the extraction solution. At this time, it can be ensured that the amount of concentrated sulfuric acid is minimized while the color development of pleuromutilin is sufficient.

Further, the dilution factor in step 8) is 10-100 times. At this time, it can be ensured that the concentration of the diluted extract is within the detection range of the microplate reader.

Further, in step 8), the ultraviolet absorption measurement with a microplate reader is carried out at a wavelength of 455 nm.

Further, the extraction time in step 5) is 5-20 minutes.

Further, the extraction time in step 5) is 10 minutes.

The technical effects of the technical solution of the present invention are as follows:

1. A micro-culture method for pleuromutilin-producing bacteria is provided. Due to the high-throughput single colony transfer culture, there is no cross-effect between each well, and the colony growth environment is highly consistent, and the nutritional conditions are restricted. In this case, the yield depends entirely on the genetics of the strain itself.

2. Due to the adoption of solid fermentation technology, the fermentation process is simplified, and there is no need to adjust the medium and other conditions too much during the static culture process, so that the culture conditions between each single colony are consistent throughout the fermentation process, reducing the artificial operation and other processing. error. When the amount of solid fermentation medium added to each well is 1/3-2/3 of the volume of the well, it can not only ensure the sufficient growth nutrient needs of the bacteria, but also leave enough space to ensure the oxygen supply.

3. Due to the use of multi-well plate culture, the original laboratory culture scale has been greatly increased. Using 96-well microplate culture, one person can transfer more than 1,000 strains, and the efficiency of each round of screening has increased by more than 100 times, and can guarantee The consistency of the culture conditions of all once-transferred strains.

4. Due to the use of solid fermentation, the step of seed cultivation is simplified and the fermentation is carried out directly. The fermentation time is greatly shortened, and the traditional fermentation cycle is shortened by half. Four rounds of screening can be carried out every month, and the cultivation conditions are easier to control.

5. Since there is only one subculture in the whole process, for microorganisms whose genetic background is easily affected by the environment, the chance of genetic background changes in multiple subcultures is reduced, and high-yield strains can be quickly separated and better maintained.

6. Pleuromutilin is easy to extract and can be extracted with only one reagent: pleuromutilin is highly soluble in methanol or ethanol. And in a trace environment, excessive extractant will also destroy the cell wall structure, so that the extraction of pleuromutilin is more sufficient, and better reflects the level of yield.

7. The extraction time is short, and the pleuromutilin in the microplate can be extracted into the solvent in 5 minutes, and the trace amount avoids incomplete extraction, and the result is more reliable.

8. The extract solution is transferred to the plate to quickly separate pleuromutilin from the culture conditions, the culture time is the same, and the detection environment is unified; the color reaction is carried out at the same time, the color development time is short, and the excess color reagent makes all pleuromutilin fully develop color , therefore, the pleuromutilin content detected by using a microplate reader is the pleuromutilin content in the pleuromutilin-producing bacteria obtained by fermentation.

9. The extracted pleuromutilin undergoes the color reaction of concentrated sulfuric acid to generate colored substances, which avoids the influence of other interfering substances on the determination of the yield of pleuromutilin, and the color development time is short, and the color development can be completed in 20s-60s , when the volume of concentrated sulfuric acid is greater than or equal to the volume of the extract, sufficient color development can be ensured. Concentrated sulfuric acid is a specific color-developing substance of terpenoids. Using concentrated sulfuric acid for color development makes the generated product have a stable characteristic absorption peak, which further increases the credibility of the results.

10. Immediately after the reaction, measure the ultraviolet absorption of the microplate reader. The measurement time of each 96-well microplate plate is 2 seconds, and a large number of microplate plates can be measured in a short time. At present, the high-performance liquid phase measurement is 12 minutes at a time. For samples, the detection efficiency of the microplate reader assay method of the present invention is 34560 times that of the high performance liquid phase detection.

11. The whole process is high-throughput and time-compressed in each link, and the screening efficiency is greatly improved under the condition of one person's operation: the high-throughput fermentation time is half of the original fermentation time; one person can operate 10 96-well microwell plates A total of 960 strains were fermented, and conventional shake flask fermentation can operate up to 12 strains at a time, and the efficiency of the fermentation process has increased by 2×960/12=160; the efficiency of the measurement process is 34560 times that of the original, and the screening efficiency is 34560×160= of the original screening efficiency 5529600 times, and can guarantee the accuracy of the results.

Description of drawings

Fig. 1 is a data comparison diagram of the application of the high-throughput screening method of the present invention and the application of common screening methods (shaking flask fermentation screening, high performance liquid chromatography method). Among them, the abscissa is the strain number of the selected high-yielding pleuromutilin bacteria, the ordinate is the pleuromutilin concentration (μg/ml), and the dark column represents the strain obtained by applying the high-throughput screening method of the present invention. The pleuromutilin concentration of the pleuromutilin in the light-colored column represents the pleuromutilin concentration obtained by applying the common screening method.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the implementation manners of the present invention will be further described in detail below.

The reagents used in the present invention are as follows:

Pleuromutilin producing strain: produced in Bulgaria, model HK-101;

Every 100 grams of solid fermentation medium contains: glucose 5.7g, corn steep liquor 4g, soybean oil 0.4g, MgSO ₄ 0.04g, soybean peptone 1g, agar powder 1.5g, water 87.36g;

96-well ELISA plate.

Example 1

The microculture steps of pleuromutilin-producing bacteria are as follows:

(1) Dilute the pleuromutilin-producing bacteria 10 times with sterile water to make a bacterial suspension, spread it on a plate, incubate at 25°C for 3-5 days, and isolate a single colony;

(2) Prepare a 96-well microtiter plate containing 1/2 of the solid fermentation medium for later use: Autoclave the solid fermentation medium at 121°C for 15 minutes, and add it to the 96-well microtiter plate in an ultra-clean bench aseptically. The amount added to the well is 150 μl;

(3) Pick up the single colony mycelium of the pleuromutilin-producing bacteria formed in step (1) with a bamboo stick, inoculate it in a 96-well microplate containing solid fermentation medium, repeat the plate once, and leave Used for identification of shake flasks;

(4) Incubate the inoculated 96-well ELISA plate at 25°C for 7 days to complete the microculture of pleuromutilin-producing bacteria.

The high-throughput screening method of pleuromutilin high-producing bacteria is as follows:

(1) Add 100 μl methanol to each well of the microplate well containing the pleuromutilin-producing bacteria after micro-cultivation, and extract for 10 minutes;

(2) After the extraction, absorb 50 μl of the extraction solution from each well, and transfer the plate to another 96-well microtiter plate;

(3) Add 50 μl of concentrated sulfuric acid to each well to fully develop the color reaction. The color development time is 20 s. After the reaction is completed, dilute each well 10 times, transfer 200 μl of the diluted solution to another 96-well microplate plate , carry out the ultraviolet absorption measurement of microplate reader at 455nm place;

(4) The OD values measured by ultraviolet absorption are shown in Table 1. The higher the OD value, the greater the concentration of pleuromutilin. Select 11 wells with high OD values. These 11 wells correspond to The strain is the screened pleuromutilin high-yielding bacteria. The position numbers of the pleuromutilin high-yielding bacteria in the 96-well microtiter plate are in order of OD value: B1O, C11, C8, H6, H7, E9, C9, B9 ,, H5, C10, G7. Number them from 1-11.

Table 1

 。

.

The pleuromutilin high-producing bacteria will be verified by shaking flask fermentation screening and high-performance liquid chromatography as follows:

(1) Take the double plate of the preserved pleuromutilin-producing bacteria, and divide 11 high-yielding pleuromutilin strains according to the strain positions shown in Table 1 (that is, the position numbers are B1O, C11, C8, H6, H7, E9, C9, B9,, H5, C10, G7 strains) were picked out, made into bacterial liquid with 5ml of sterile water, and spread on the slant medium. The composition of the slant medium is: glucose 5.7g, corn steep liquor 4g, soybean oil 0.4g, MgSO ₄ 0.04g, soybean peptone 1g, agar powder 1.5g, water 87.36g;

(2) When the colonies are all over the slope, use an inoculation shovel to scoop out about 2cm ² of bacterial blocks, use 5ml of sterile water to make a bacterial solution and inoculate the seed medium for shake flask culture. Seed medium formula: soybean powder 7g, Ca(NO ₃ ) ₂ 0.05g, CaCO ₃ 0.05g, water 92.9g. The seed culture condition is 25°C, the rotation speed is 220rmp, and the culture time is three days;

(3) After the seeds are cultivated, the seed solution is added to the fermentation medium at a volume ratio of 10%. The fermentation medium contains the following components per 100 g: 5.7 g of glucose, 4 g of corn steep liquor, 0.4 g of soybean oil, 0.04 g of MgSO ₄ , 1 g of soybean peptone, and 88.86 g of water. The fermentation culture temperature is 25°C, the rotation speed is 220rmp, and the culture time is 9 days;

(4) After the fermentation is over, take 2.5ml of fermentation broth from each bottle, dilute it to a 25ml volumetric flask with methanol, extract it with ultrasonic waves for 30 minutes, filter, and perform high performance liquid chromatography on the filtrate. The results are shown in Figure 1.

As can be seen from Fig. 1: the correlation coefficient r=0.8656, p ＜0.01, wherein r is Peel Sen correlation coefficient, p is the probability of significant difference. The correlation between the two is good. Therefore, the high-throughput screening method of the present invention can replace common screening methods for screening high-producing bacteria of pleuromutilin.

Example 2

The microculture steps of pleuromutilin-producing bacteria are as follows:

(1) Dilute the pleuromutilin-producing bacteria to 100 times with sterile water to make a bacterial suspension, spread it on a plate, culture at 25°C for 3-5 days, and isolate a single colony;

(2) Prepare a 96-well microtiter plate containing 1/3 of the solid fermentation medium for later use: Autoclave the solid fermentation medium at 121°C for 15 minutes, and add it to the 96-well microtiter plate in an ultra-clean bench aseptically. The amount added to the well is 100 μl;

(3) Use an inoculation loop to pick up the single colony mycelium of the pleuromutilin-producing bacteria formed in step (1), and inoculate it in a 96-well microtiter plate containing a solid fermentation medium;

(4) Incubate the inoculated 96-well ELISA plate at 25°C for 8 days to complete the microculture of pleuromutilin-producing bacteria.

The high-throughput screening method of pleuromutilin high-producing bacteria is as follows:

(1) Add 100 μl of ethanol to each well of the microplate well containing the pleuromutilin-producing bacteria after micro-cultivation, and extract for 5 minutes;

(2) After the extraction, absorb 40 μl of the extraction solution from each well, and transfer the plate to another 96-well microtiter plate;

(3) Add 80 μl of concentrated sulfuric acid to each well to fully develop the color reaction. The color development time is 40 s. After the reaction, dilute each well 50 times, transfer 200 μl of the diluted solution to another 96-well microplate plate , at 450nm, carry out the ultraviolet absorption measurement of microplate reader, and the strain corresponding to the hole with high OD value is the screened pleuromutilin high-producing bacteria.

Example 3

The microculture steps of pleuromutilin-producing bacteria are as follows:

(1) Dilute the pleuromutilin-producing bacteria to 1000 times with sterile water to make a bacterial suspension, spread it on a plate, incubate at 25°C for 3-5 days, and isolate a single colony;

(2) Prepare a 96-well microtiter plate containing 2/3 of the solid fermentation medium for later use: Autoclave the solid fermentation medium at 121°C for 15 minutes, and add it to the 96-well microtiter plate aseptically in an ultra-clean bench. The amount added to the well is 200 μl;

(3) Pick up the single-colony mycelia of the pleuromutilin-producing bacteria formed in step (1) with an inoculation needle, and inoculate it in a 96-well microtiter plate containing a solid fermentation medium;

(4) Incubate the inoculated 96-well ELISA plate at 25°C for 9 days to complete the microculture of pleuromutilin-producing bacteria.

The high-throughput screening method of pleuromutilin high-producing bacteria is as follows:

(1) Add 100 μl methanol to each well of the microplate well containing the pleuromutilin-producing bacteria after micro-cultivation, and extract for 20 minutes;

(2) After the extraction, absorb 60 μl of the extraction solution from each well, and transfer the plate to another 96-well microtiter plate;

(3) Add 80 μl of concentrated sulfuric acid to each well to fully develop the color reaction. The color development time is 1 min. After the reaction is completed, each well is diluted 100 times, and 200 μl of the diluted solution is transferred to another 96-well ELISA plate. , at 460nm, carry out the ultraviolet absorption measurement of microplate reader, and the strain corresponding to the well with high OD value is the high-yielding pleuromutilin strain selected.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (9)

1. a microculture method of pleuromutilin producing bacteria, characterized in that: the steps of the microculture method are as follows: 1) Dilute the pleuromutilin-producing bacteria into a bacterial suspension, spread it on a plate, and isolate a single colony after culturing; 2) Preparation of solid fermentation medium: the following are mass percentages: glucose 5.7%, corn steep liquor 4%, soybean oil 0.4%, MgSO ₄ 0.04%, soybean peptone 1%, agar powder 1.5%, and the rest is water; 3) After autoclaving the solid fermentation medium in step 2), add it to the microtiter plate in a sterile environment, and the amount added to each well is 1/3-2/3 of the well volume; 4) Inoculate the single colony mycelium of the pleuromutilin-producing bacteria formed in step 1) on the microplate plate prepared in step 3), and culture at 25°C for 7-9 days to complete the production of pleuromutilin Microculture of bacteria. 2. The micro-cultivation method according to claim 1, characterized in that the amount of the solid fermentation medium added to each well in step 3) is 1/2 of the volume of the well. 3. A high-throughput screening method for pleuromutilin high-yielding bacteria, characterized in that: the steps of the high-throughput screening method are as follows: 1) Dilute the pleuromutilin-producing bacteria into a bacterial suspension, spread it on a plate, and isolate a single colony after culturing; 2) Preparation of solid fermentation medium: the following are mass percentages: glucose 5.7%, corn steep liquor 4%, soybean oil 0.4%, MgSO ₄ 0.04%, soybean peptone 1%, agar powder 1.5%, and the rest is water; 3) After autoclaving the solid fermentation medium in step 2), add it to the microplate in a sterile environment, and the amount added to each well is 1/3-2/3 of the well volume; 4) Inoculate the single colony mycelia of the pleuromutilin-producing bacteria formed in step 1) on the microplate prepared in step 3), and culture at 25°C for 7-9 days; 5) Add 50-100 μl methanol or ethanol to each well in step 4) for extraction; 6) Take a certain amount of extract from each well and transfer to another microplate; 7) Add concentrated sulfuric acid to each well for color development, the volume of the concentrated sulfuric acid is greater than or equal to the volume of the extract; 8) Dilute each well to the same multiple, transfer the diluted solution to another microplate plate, measure the ultraviolet absorption of the microplate reader at a wavelength of 450nm-460nm, analyze the measurement results, and the wells with high OD values correspond to The bacterial strain is a screened high-yielding pleuromutilin strain, and the screened high-yielding pleuromutilin strain is obtained. 4. The high-throughput screening method for high-producing pleuromutilin bacteria according to claim 3, characterized in that the amount of the solid fermentation medium added to each well in step 3) is 1/2 of the volume of the well. 5. The high-throughput screening method for high-producing pleuromutilin bacteria according to claim 3, characterized in that the volume of the concentrated sulfuric acid in step 7) is the same as the volume of the extract. 6. The high-throughput screening method for high-producing pleuromutilin bacteria according to claim 3, characterized in that: the dilution factor in step 8) is 10-100 times. 7. The high-throughput screening method for high-producing pleuromutilin bacteria according to claim 3, characterized in that: in step 8), the ultraviolet absorption measurement is performed with a microplate reader at a wavelength of 455 nm. 8. The high-throughput screening method for high-producing pleuromutilin bacteria according to claim 3, characterized in that the extraction time in step 5) is 5-20 minutes. 9. The high-throughput screening method for high-producing pleuromutilin bacteria according to claim 8, wherein the extraction time in step 5) is 10 minutes.