CN112251356A - Method for rapidly screening high-yield polyglutamic acid strains - Google Patents
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Abstract
The invention discloses a method for rapidly screening a high-yield polyglutamic acid strain. Preparing a semisolid fermentation culture medium, subpackaging the culture medium into sterile culture pore plates after sterilization, picking a single colony into a pore filled with the semisolid fermentation culture medium by using a toothpick after the semisolid fermentation culture medium is condensed, covering a pore plate cover, carrying out inverted culture on the pore plate, and observing whether the pore plate cover has a liquid product or not after culturing for 24-96 hours; if so, the strain inoculated into the hole is a polyglutamic acid high-producing strain. The method simplifies the traditional process of screening the high-yield gamma-polyglutamic acid strain and greatly lightens the workload of the screening process.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to a method for rapidly screening a high-yield polyglutamic acid strain.
Background
Gamma-polyglutamic acid (gamma-PGA), an extracellular polypeptide secreted mainly by Bacillus (Bacillus), is a major component of the capsule of these microorganisms, and is polymerized from glutamic acid monomers through gamma-glutamine bonds. The gamma-PGA is a non-toxic harmless green biological product without pollution to the environment, has the unique physical and chemical properties and biological characteristics of high water solubility, strong water absorption capacity, degradability, good biocompatibility, strong film forming property, high viscosity, fibrillinity and the like, and has wide application prospect in the industries of medicine, food, environment, daily chemicals, agriculture and the like.
Currently, many researchers are still screening strains of γ -PGA with high yield to reduce the production cost of γ -PGA. However, the method still adopts the steps that a sample is diluted and coated in a separation culture medium, and then a single colony on the separation culture medium is inoculated into a liquid fermentation culture medium for shake flask fermentation; after fermentation, measuring the yield of the gamma-PGA in the fermentation liquor; the method has large workload, and the shake flask fermentation needs a large amount of space, so that a large amount of single bacteria on the separation culture medium cannot be screened at the same time. Therefore, it is required to develop a method for screening strains with high yield of γ -PGA in batch.
Disclosure of Invention
Aiming at the defect that the prior art method can not screen a large number of strains with high yield of gamma-PGA at the same time, the invention provides a method for quickly screening the strains with high yield of polyglutamic acid, and the strains are screened in batches in a pore plate by a semi-solid fermentation culture medium, so that the method is simple and easy to implement and has low cost.
The method for rapidly screening the high-yield polyglutamic acid strain comprises the following steps of:
a. coating a sample to be screened on a separation culture medium after gradient dilution for culture;
b. preparing a corresponding semisolid fermentation culture medium according to the type of strains/flora in a sample to be screened, subpackaging the melted culture medium into sterile culture pore plates after sterilization, transferring a single colony on an isolated culture medium onto the coagulated semisolid fermentation culture medium in the pore, covering a pore plate cover, and inversely placing the pore plate into an incubator with the relative humidity maintained above 80% for culture; and (4) observing whether the pore plate cover has a liquid product, if so, determining that the strain inoculated into the pore is the high-yield polyglutamic acid strain.
Preferably, the semi-solid fermentation medium contains agar in an amount of 0.2 to 0.8 percent by mass; the culture medium is subpackaged, and the subpackaged amount is controlled in such a way that the distance between the liquid level of the culture medium and the well covered orifice plate cover is 2-15 mm.
Preferably, the culture well plate is a 96-well, 48-well, 24-well or 12-well culture plate.
Preferably, the separation culture medium is a nutrient agar culture medium.
Preferably, the strain/flora in the sample to be screened is bacillus licheniformis, and the semi-solid fermentation medium is as follows: contains 12g/L of citric acid, 80g/L, L g/L of glycerol, 20g/L of glutamic acid, 7g/L of ammonium chloride, 0.5g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 0.15g/L of calcium chloride dihydrate, 0.104g/L of manganese sulfate monohydrate, 0.01g/L of ferric chloride hexahydrate and 2g/L of agar, and the balance of water, and has the pH value of 6.5.
Preferably, the sample to be screened is natto, and the semisolid fermentation medium of the natto is as follows: contains soybean powder 40g/L, agar 8g/L, and water in balance, and has a pH of 6.5.
Preferably, the strain/flora in the sample to be screened is bacillus subtilis, and the semi-solid fermentation medium is as follows: contains 60g/L glucose, 20g/L peptone, 1.5g/L dipotassium hydrogen phosphate, 0.05g/L magnesium sulfate heptahydrate, 0.1g/L calcium chloride dihydrate, 0.104g/L manganese sulfate monohydrate, 0.01g/L ferric chloride hexahydrate and 5g/L agar, and the balance of water, and has the pH value of 6.5.
Preferably, the sample to be screened is a saline-alkali soil sample, and the semi-solid fermentation medium is as follows: contains 100g/L glucose, 50g/L peptone, 100g/L sodium glutamate, 2g/L dipotassium hydrogen phosphate, 0.05g/L magnesium sulfate heptahydrate and 3g/L agar, and the balance water, and has a pH value of 6.5.
Preferably, the plating on the isolation medium in step a is performed at 37 ℃ for 24 hours.
Preferably, the plate of step b is placed upside down and cultured in an incubator with a relative humidity of 80% or more, and the plate is cultured at 37 ℃ for 24-96 h.
Has the advantages that:
the method provided by the invention provides a method for rapidly screening the strains with high yield of gamma-PGA, is simple and rapid, and can screen thousands of strains simultaneously. In the conventional method, each single bacterium is inoculated into liquid fermentation culture, then the liquid fermentation culture is shaken for several days, and finally, the yield of the gamma-PGA in each shake flask is measured. Compared with the traditional method, the method of the invention is simple, simplifies the traditional screening process, directly observes high-yield strains by naked eyes and reduces the screening workload.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
lyophilized powder of space-mutagenized Bacillus licheniformis ATCC9945a was dissolved in sterile physiological saline, and 100. mu.L of the solution was applied to nutrient agar plates and cultured at 37 ℃ for 24 hours.
Melting the semi-solid fermentation medium, adding 100 μ L of the melted medium into a 96-well culture plate, wherein the distance between the liquid surface of the melted medium and the cover of the plate is 5 mm.
The semi-solid fermentation medium comprises the following components: contains 12g/L of citric acid, 80g/L, L g/L of glycerol, 20g/L of glutamic acid, 7g/L of ammonium chloride, 0.5g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 0.15g/L of calcium chloride dihydrate, 0.104g/L of manganese sulfate monohydrate, 0.01g/L of ferric chloride hexahydrate and 2g/L of agar, and the balance of water, and the pH is adjusted to be 6.5; the preparation method comprises mixing the above materials, adjusting pH, and sterilizing.
After the culture medium in the pore plate is coagulated, single colony on the plate of nutrient agar is picked into the hole of a 96-hole culture plate filled with the semi-solid fermentation culture medium by a toothpick, and the cover of the pore plate is covered. The well plate was then placed upside down in an incubator maintained at a relative humidity of 80% or more and incubated at 37 ℃ for 96 h. γ -PGA is a product secreted to the outside of cells, and γ -PGA has a large viscosity. When the pore plate is inversely cultured, if the yield of gamma-PGA is large and the accumulation is large, the gravity of the pore plate is larger than the viscous force of the gamma-PGA, and the product under the action of gravity can drop onto the pore plate cover; if the γ -PGA yield is low and its own gravity is less than its viscosity, the product can only stick in the well plate. Therefore, whether the pore plate cover has the liquid product or not is observed by naked eyes, and the bacterial strain inoculated to the pore corresponding to the position of the pore plate cover with the liquid product is the high-yield polyglutamic acid bacterium.
768 single colonies are selected, and 24 strains of the production bacteria of the gamma-PGA with high yield are screened according to the visual observation and judgment method. Before mutagenesis, the yield of gamma-PGA of the emerging strain was 30.73 g/L. And performing shake flask fermentation on the screened 24 strains, wherein the average yield of the gamma-PGA is 55.52 g/L.
Example 2:
adding a commercially available natto sample into sterile normal saline, diluting in a gradient manner, taking 100 microliters, coating on a nutrient agar plate, and culturing at 37 ℃ for 24 hours.
Melting the semi-solid fermentation medium, adding 1mL of the melted medium into a 12-well culture plate, wherein the distance between the liquid level of the melted medium and the cover of the plate is 10 mm.
The semi-solid fermentation medium comprises the following components: 40g/L of crushed soybean meal, 8g/L of agar and the balance of water, and adjusting the pH value to 6.5; the preparation method comprises mixing the above materials, adjusting pH, and sterilizing.
After the culture medium in the pore plate is coagulated, picking a single colony on a plate of nutrient agar into a hole of a 12-hole culture plate filled with the semi-solid fermentation culture medium by using a toothpick, and covering the hole plate with a cover. The well plate was then placed upside down in an incubator maintained at a relative humidity of 80% or more and incubated at 37 ℃ for 24 h. And then observing whether the pore plate cover has a liquid product or not by naked eyes, wherein the bacterial strain inoculated into the pore corresponding to the position of the pore plate cover with the liquid product is the high-yield polyglutamic acid bacterium.
768 single colonies are selected in total, and 26 strains of the gamma-PGA producing bacteria with high yield are screened out according to the visual observation and judgment method. The screened 26 strains were subjected to solid fermentation, and the average yield of γ -PGA was 36.31 g/L.
Example 3:
adding sterile physiological saline into PGA-7 lyophilized powder of Bacillus subtilis (Bacillus subtilis) subjected to microgravity mutagenesis, performing gradient dilution, coating 100 μ L of the lyophilized powder on a nutrient agar plate, and culturing at 37 deg.C for 24 h. The Bacillus subtilis PGA-7 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number as follows: CCTCC NO: m206102, which is disclosed in patent No.: ZL 200610122640.5, invention name: gamma-polyglutamic acid producing strain and method for preparing gamma-polyglutamic acid by using the strain are disclosed.
Melting the semi-solid fermentation medium, adding 0.5mL of the melted medium into a 24-well culture plate, wherein the distance between the liquid level of the melted medium and the cover of the plate is 15 mm.
The components of the fermentation medium are as follows: contains 60g/L glucose, 20g/L peptone, 1.5g/L dipotassium hydrogen phosphate, 0.05g/L magnesium sulfate heptahydrate, 0.1g/L calcium chloride dihydrate, 0.104g/L manganese sulfate monohydrate, 0.01g/L ferric chloride hexahydrate and 5g/L agar, and the balance of water, and the pH is adjusted to 6.5; the preparation method comprises mixing the above materials, adjusting pH, and sterilizing.
After the culture medium in the pore plate is coagulated, picking a single colony on a plate of nutrient agar into a hole of a 24-hole culture plate filled with the semi-solid fermentation culture medium by using a toothpick, and covering the hole plate with a cover. The well plate was then placed upside down in an incubator maintained at a relative humidity of 80% or more and incubated at 37 ℃ for 48 h. And then observing whether the pore plate cover has a liquid product or not by naked eyes, wherein the bacterial strain inoculated into the pore corresponding to the position of the pore plate cover with the liquid product is the high-yield polyglutamic acid bacterium.
768 single colonies are selected in total, and 15 strains of the production bacteria of the gamma-PGA with high yield are screened out according to the visual observation and judgment method. Before mutagenesis, the yield of gamma-PGA of the emerging strain was 2.8 g/L. And performing shake flask fermentation on the screened 24 strains, wherein the average yield of the gamma-PGA is 4.08 g/L.
Example 4:
dissolving saline-alkali soil samples in sterile physiological saline, performing gradient dilution, taking 100 mu L of the diluted saline-alkali soil samples, coating the diluted saline-alkali soil samples on a nutrient agar plate, and culturing the nutrient agar plate for 24 hours at 37 ℃.
Melting the semi-solid fermentation medium, adding 300 μ L of the melted medium into a 48-well culture plate, wherein the distance between the liquid surface of the melted medium and the cover of the plate is 2 mm.
The semi-solid fermentation medium comprises the following components: contains 100g/L glucose, 50g/L peptone, 100g/L sodium glutamate, 2g/L dipotassium hydrogen phosphate, 0.05g/L magnesium sulfate heptahydrate and 3g/L agar, and the balance of water, and the pH is adjusted to 6.5; the preparation method comprises mixing the above materials, adjusting pH, and sterilizing.
After the culture medium in the pore plate is coagulated, single colony on the plate of nutrient agar is picked into the hole of a 48-hole culture plate filled with the semi-solid fermentation culture medium by a toothpick, and the cover of the pore plate is covered. The well plate was then placed upside down in an incubator maintained at a relative humidity of 80% or more and incubated at 37 ℃ for 72 hours. And then observing whether the pore plate cover has a liquid product or not by naked eyes, wherein the bacterial strain inoculated into the pore corresponding to the position of the pore plate cover with the liquid product is the high-yield polyglutamic acid bacterium.
480 single colonies are picked out in total, and 2 strains of the production bacteria of the gamma-PGA with high yield are screened out according to the visual observation judgment method. The yield of the gamma-PGA is 38.15g/L and 35.55g/L respectively when the shake flask fermentation is carried out.
Claims (10)
1. A method for rapidly screening a high-yield polyglutamic acid strain is characterized by comprising the following steps of:
a. coating a sample to be screened on a separation culture medium after gradient dilution for culture;
b. preparing a corresponding semisolid fermentation culture medium according to the type of strains/flora in a sample to be screened, subpackaging the melted culture medium into sterile culture pore plates after sterilization, transferring a single colony on an isolated culture medium onto the coagulated semisolid fermentation culture medium in the pore, covering a pore plate cover, and inversely placing the pore plate into an incubator with the relative humidity maintained above 80% for culture; and (4) observing whether the pore plate cover has a liquid product, if so, determining that the strain inoculated into the pore is the high-yield polyglutamic acid strain.
2. The method of claim 1, wherein the semi-solid fermentation medium comprises agar in an amount of 0.2% to 0.8% by weight.
3. The method of claim 1, wherein the media is dispensed at a volume of 2-15mm from the well plate lid after the well plate lid is closed.
4. The method of claim 1, wherein the culture well plate is a 96-well, 48-well, 24-well or 12-well culture plate and the isolation medium is a nutrient agar medium.
5. The method of claim 1, wherein the strain/flora in the sample to be screened is bacillus licheniformis and the semi-solid fermentation medium is: contains 12g/L of citric acid, 80g/L, L g/L of glycerol, 20g/L of glutamic acid, 7g/L of ammonium chloride, 0.5g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 0.15g/L of calcium chloride dihydrate, 0.104g/L of manganese sulfate monohydrate, 0.01g/L of ferric chloride hexahydrate and 2g/L of agar, and the balance of water, and has the pH value of 6.5.
6. The method according to claim 1, wherein the sample to be screened is natto, and the semi-solid fermentation medium thereof is: contains soybean powder 40g/L, agar 8g/L, and water in balance, and has a pH of 6.5.
7. The method according to claim 1, wherein the strain/colony in the sample to be screened is bacillus subtilis, and the semi-solid fermentation medium is: contains 60g/L glucose, 20g/L peptone, 1.5g/L dipotassium hydrogen phosphate, 0.05g/L magnesium sulfate heptahydrate, 0.1g/L calcium chloride dihydrate, 0.104g/L manganese sulfate monohydrate, 0.01g/L ferric chloride hexahydrate and 5g/L agar, and the balance of water, and has the pH value of 6.5.
8. The method according to claim 1, wherein the sample to be screened is a saline-alkali soil sample, and the semi-solid fermentation medium is: contains 100g/L glucose, 50g/L peptone, 100g/L sodium glutamate, 2g/L dipotassium hydrogen phosphate, 0.05g/L magnesium sulfate heptahydrate and 3g/L agar, and the balance water, and has a pH value of 6.5.
9. The method of claim 1, wherein the plating on the isolation medium in step a is performed at 37 ℃ for 24 hours.
10. The method of claim 1, wherein the step b of inverting the well plate and incubating the plate in an incubator with a relative humidity above 80% is performed at 37 ℃ for 24-96 h.
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