Screening process of gibberellin production bacterial colony

Abstract

The invention provides a screening process of gibberellin production bacterial colonies. The screening process of the gibberellin production bacterial colonies comprises the following steps: step 1, strain activation: respectively transferring a strain S and a strain Y to a PDA (potato dextrose agar) plate for culturing; 2, liquid seed culture; mixing the bacterial cake with the bacterial strain S and the bacterial strain Y with sterile water, oscillating and inoculating into a conical flask; step 3, a strain fermentation culture method: respectively inoculating the liquid seed S and the liquid seed Y into a conical flask for culture; 4, processing the fermentation liquor in the conical flask in the step 3 to obtain liquid precipitate; 5, measuring the content of GA4: measuring the content of GA4 and the number of bacterial colonies in the liquid precipitate in the step 4, and drawing growth curves of the strain S and the strain Y; and 6, determining the dry weight of hyphae: taking the fermentation liquor in the conical flask in the step 3, and determining the dry weight of the hyphae of the strain S and the strain Y. The screening technology of the gibberellin production bacterial colony has the advantage that the microbial strain capable of improving the yield of GA4 can be screened out.

Description

Technical Field

[0001] The invention relates to the technical field of gibberellin production, in particular to a screening process for gibberellin production colonies. Background Art

[0002] Gibberellic acid is a widely used plant growth hormone, which plays an important role in agricultural and forestry production and has broad market prospects. The new gibberellin GA4 has attracted much attention due to its unique advantages. The new gibberellin GA4 can break plant dormancy, but does not affect the plant's resistance to lodging, nor does it cause the plant's peel to crack. It is very effective in regulating apple biennials and alleviating brown rust. Therefore, GA4 has better functions and broader application prospects.

[0003] Currently, GA4 on the market mainly comes from liquid fermentation of Fusarium fuciformis, but low yield and high cost have become the main bottlenecks of large-scale industrial production. Enterprises urgently need to increase GA4 production. The GA4 synthesis efficiency can be effectively improved by optimizing fermentation conditions, but the increase is relatively limited. Therefore, there is an urgent need to screen a microbial strain that can increase GA4 production.

[0004] Therefore, it is necessary to provide a new screening process for gibberellin production colonies to solve the above-mentioned technical problems. Summary of the invention

[0005] The technical problem solved by the invention is to provide a screening process for screening gibberellin production colonies of microbial strains capable of improving GA4 production.

[0006] The screening process for gibberellin production colonies provided by the invention comprises the following steps: step 1: strain activation: taking out a freezing tube from a -80 DEG C low-temperature refrigerator to store strain S and strain Y, wherein the strain S is Gibberella fujikura, model CGMCC 17793; the strain Y is Fusarium fujikura, model NRRL 13620; the strain S and the strain Y are respectively transferred to a PDA plate, cultured at a constant temperature of 28 DEG C for 7-8 days, obtaining a strain S matrix plate and a strain Y matrix plate, and putting them into a 4 DEG C refrigerator for storage for standby use;

[0007] Step 2: Liquid seed culture: Multiple 250mL conical flasks are filled with 30mL seed culture medium respectively, the inner layer of the conical flask is sealed with eight layers of gauze, the outer layer is covered with newspaper, and the high temperature steam sterilization is carried out at 115℃ for 30min. When the culture medium is cooled to room temperature; use a sterile puncher to punch multiple bacterial cakes with a diameter of 10mm on the strain S matrix and the strain Y matrix in step 1 respectively, and take 5 bacterial cakes from the strain S matrix and the strain Y matrix respectively, and put them into a test tube containing 5mL sterile water and glass beads for 10min to obtain S bacterial suspension and Y bacterial suspension, respectively put the S bacterial suspension and Y bacterial suspension into the conical flask, the volume percentage ratio of the seed culture medium to the bacterial suspension is: 96%:4%, put the inoculated conical flask into a constant temperature oscillator, and culture at a constant temperature of 33℃ and 220rpm for 48h to obtain liquid seeds S and liquid seeds Y;

[0008] Step 3: strain fermentation culture method: multiple 250 mL conical flasks are respectively filled with 100 mL of fermentation medium, and then liquid seeds S and liquid seeds Y are inoculated into multiple conical flasks respectively, and the volume percentage ratio of fermentation medium to liquid seeds is: 96%:4%, and the conical flask containing strain S or strain Y is placed in a constant temperature oscillator, and cultured at a constant temperature and oscillation at 33°C and 220 rpm;

[0009] Step 4: Every 24 hours, take an equal amount of samples and select the fermentation liquid of the production strain S and the fermentation liquid of the wild strain Y in the conical flask in step 3 with good growth status at 3d, 6d, and 8d, respectively, and put them into a centrifuge, centrifuge at 1000rpm and 4℃ for 10min, discard the supernatant, obtain the cell precipitate, add 3mL of 1xPBS prepared with enzyme-free water to suspend the cell precipitate, centrifuge at 200rpm for 5min, wash twice, and discard the supernatant; transfer the precipitate to a threaded freezing tube that can withstand -192℃ low temperature, and freeze the fermentation liquid precipitates of strain S and strain Y in liquid nitrogen for 1h and then store them at -80℃ for later use;

[0010] Step 5: Determine the GA4 content: Determine the GA4 content and colony count in the liquid precipitate of step 4, and draw the growth curves of strain S and strain Y;

[0011] Step 6: Determine the dry weight of mycelium: Take 5 mL of fermentation liquid from the conical flask in step 3 every 24 hours, centrifuge to remove the supernatant, add appropriate amount of 0.9% NaCl solution to wash twice, place the precipitate in a culture dish, dry at 80°C to constant weight, and weigh and record the dry weight of mycelium of strain S and strain Y respectively.

[0012] Preferably, the raw material ratio of the PDA culture medium in step 1 is: 200g potato, 20g glucose, 20g agar powder, 1L distilled water, and natural pH.

[0013] Preferably, the raw material ratio of the seed culture medium in step 2 is: sucrose 30g, soybean meal 12g, NH4Cl 3g, NaNO3 3g, KH2PO4 1.5g, MgSO4·7H2O 0.2g, K2SO4 0.2g, distilled water 1L, pH 6.8, and high-pressure steam sterilization at 121°C for 30min.

[0014] Preferably, the raw material ratio of the fermentation medium in step three is: lactose 140g, soybean meal powder 10g, corn syrup powder 10g, corn gluten powder 5g, K2SO4 2g, ZnSO4·7H2O 0.005g, MgSO4·7H2O 1g, CoCl2·6H2O 0.005g, urea 1.25g, distilled water 1L, pH 7.0, 121°C high pressure steam sterilization for 30min. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 are the growth curves of strain S and strain Y;

[0016] Figure 2 is the GA4 synthesis curve of strain S and strain Y;

[0017] Figure 3 This is the GA4 synthesis curve of strain S and strain Y per unit mycelium dry weight.

[0018] Compared with the related art, the screening process for gibberellin-producing bacterial colonies provided by the present invention has the following beneficial effects:

[0019] like Figure 1 As shown in the figure, the growth curves of strain S and strain Y. The results showed that the growth curves of strain S and strain Y were mainly divided into lag phase (0-1d), logarithmic phase (1-3d), and stable phase (3-10d). From the second day of fermentation, the growth rate of strain S was significantly greater than that of strain Y, and the final biomass was significantly higher than that of strain Y. Figure 2 As shown in the figure, strain S began to accumulate GA4 on the second day, and strain Y began to accumulate GA4 on the third day, one day later. During the stable period, the mycelial dry weight of strains S and Y tended to be stable, but the GA4 production showed an upward trend, reaching the highest value on the ninth day, which was 513.0 mg / L and 177.8 mg / L, respectively. At this time, the GA4 production of strain S was 2.9 times that of strain Y; as shown in the attached Figure 3As shown, the GA4 yield per unit mycelium dry weight of strain S and strain Y reached the highest value on the 9th day, which was 1.6g / gDCW (Dry Cell Weight) and 0.6g / gDCW, respectively. At this time, the GA4 yield per unit mycelium dry weight of strain S was 2.7 times that of strain Y. By comparing the growth curve, GA4 synthesis curve and GA4 synthesis curve of the strains, strain S was significantly better than strain Y in the process of producing GA4, and the GA4 yield could be significantly increased by using strain S for fermentation. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0021] Please refer to Figure 1 , Figure 2 and Figure 3 ;

[0022] A screening process for gibberellin production bacterial colonies comprises the following steps:

[0023] Step 1: Activation of strains: Take out the cryovials from the -80°C low-temperature refrigerator to preserve strain S and strain Y, strain S is Fusarium fujikura, model CGMCC 17793; strain Y is Fusarium fujikura, model NRRL 13620; strain S and strain Y are transferred to PDA plates respectively, cultured at 28°C for 7-8 days, and strain S matrix plates and strain Y matrix plates are obtained, and stored in a 4°C refrigerator for later use;

[0024] Step 2: Liquid seed culture: Multiple 250mL conical flasks are filled with 30mL seed culture medium respectively, the inner layer of the conical flask is sealed with eight layers of gauze, the outer layer is covered with newspaper, and the high temperature steam sterilization is carried out at 115℃ for 30min. When the culture medium is cooled to room temperature; use a sterile puncher to punch multiple bacterial cakes with a diameter of 10mm on the strain S matrix and the strain Y matrix in step 1 respectively, and take 5 bacterial cakes from the strain S matrix and the strain Y matrix respectively, and put them into a test tube containing 5mL sterile water and glass beads for 10min to obtain S bacterial suspension and Y bacterial suspension, respectively put the S bacterial suspension and Y bacterial suspension into the conical flask, the volume percentage ratio of the seed culture medium to the bacterial suspension is: 96%:4%, put the inoculated conical flask into a constant temperature oscillator, and culture at a constant temperature of 33℃ and 220rpm for 48h to obtain liquid seeds S and liquid seeds Y;

[0025] Step 3: strain fermentation culture method: multiple 250 mL conical flasks are respectively filled with 100 mL of fermentation medium, and then liquid seeds S and liquid seeds Y are inoculated into multiple conical flasks respectively, and the volume percentage ratio of fermentation medium to liquid seeds is: 96%:4%, and the conical flask containing strain S or strain Y is placed in a constant temperature oscillator, and cultured at a constant temperature and oscillation at 33°C and 220 rpm;

[0026] Step 4: Every 24 hours, take an equal amount of samples and select the fermentation liquid of the production strain S and the fermentation liquid of the wild strain Y in the conical flask in step 3 with good growth status at 3d, 6d, and 8d, respectively, and put them into a centrifuge, centrifuge at 1000rpm and 4℃ for 10min, discard the supernatant, obtain the cell precipitate, add 3mL of 1xPBS prepared with enzyme-free water to suspend the cell precipitate, centrifuge at 200rpm for 5min, wash twice, and discard the supernatant; transfer the precipitate to a threaded freezing tube that can withstand -192℃ low temperature, and freeze the fermentation liquid precipitates of strain S and strain Y in liquid nitrogen for 1h and then store them at -80℃ for later use;

[0027] Step 5: Determine the GA4 content: Determine the GA4 content and colony count in the liquid precipitate of step 4, and draw the growth curves of strain S and strain Y;

[0028] Step 6: Determine the dry weight of mycelium: Take 5 mL of fermentation liquid from the conical flask in step 3 every 24 hours, centrifuge to remove the supernatant, add appropriate amount of 0.9% NaCl solution to wash twice, place the precipitate in a culture dish, dry at 80°C to constant weight, and weigh and record the dry weight of mycelium of strain S and strain Y respectively.

[0029] Specifically, the raw material ratio of the PDA culture medium in step 1 is: 200g potato, 20g glucose, 20g agar powder, 1L distilled water, and natural pH.

[0030] Specifically, the raw material ratio of the seed culture medium in step 2 is: sucrose 30g, soybean meal powder 12g, NH4Cl 3g, NaNO3 3g, KH2PO4 1.5g, MgSO4·7H2O 0.2g, K2SO4 0.2g, distilled water 1L, pH 6.8, 121°C high pressure steam sterilization for 30min.

[0031] Specifically, the raw material ratio of the fermentation medium in step three is: lactose 140g, soybean meal powder 10g, corn syrup powder 10g, corn gluten powder 5g, K2SO4 2g, ZnSO4·7H2O 0.005g, MgSO4·7H2O 1g, CoCl2·6H2O 0.005g, urea 1.25g, distilled water 1L, pH 7.0, 121°C high pressure steam sterilization for 30min.

[0032] As the instruction manual Figure 1 , Instruction Manual Figure 2 And the instruction manual Figure 3 As shown, in the process of producing GA4, strain S is significantly better than strain Y, and the GA4 yield can be significantly increased by using strain S for fermentation.

[0033] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A screening process for gibberellin-producing bacterial colonies, characterized in that: The following steps are involved: Step 1: Activation of strains: Take out the cryovials from the -80°C low-temperature refrigerator to preserve strain S and strain Y, strain S is Fusarium fujikura, model CGMCC 17793; strain Y is Fusarium fujikura, model NRRL 13620; strain S and strain Y are transferred to PDA plates respectively, cultured at 28°C for 7-8 days, and strain S matrix plates and strain Y matrix plates are obtained, and stored in a 4°C refrigerator for later use; Step 2: Liquid seed culture: Multiple 250mL conical flasks are filled with 30mL seed culture medium respectively, the inner layer of the conical flask is sealed with eight layers of gauze, the outer layer is covered with newspaper, and the high temperature steam sterilization is carried out at 115℃ for 30min. When the culture medium is cooled to room temperature; use a sterile puncher to punch multiple bacterial cakes with a diameter of 10mm on the strain S matrix and the strain Y matrix in step 1 respectively, and take 5 bacterial cakes from the strain S matrix and the strain Y matrix respectively, and put them into a test tube containing 5mL sterile water and glass beads for 10min to obtain S bacterial suspension and Y bacterial suspension, respectively put the S bacterial suspension and Y bacterial suspension into the conical flask, the volume percentage ratio of the seed culture medium to the bacterial suspension is: 96%:4%, put the inoculated conical flask into a constant temperature oscillator, and culture at a constant temperature of 33℃ and 220rpm for 48h to obtain liquid seeds S and liquid seeds Y; Step 3: strain fermentation culture method: multiple 250 mL conical flasks are respectively filled with 100 mL of fermentation medium, and then liquid seeds S and liquid seeds Y are inoculated into multiple conical flasks respectively, and the volume percentage ratio of fermentation medium to liquid seeds is: 96%:4%, and the conical flask containing strain S or strain Y is placed in a constant temperature oscillator, and cultured at a constant temperature and oscillation at 33°C and 220 rpm; Step 4: Every 24 hours, take an equal amount of samples and select the fermentation liquid of the production strain S and the fermentation liquid of the wild strain Y in the conical flask in step 3 with good growth status at 3d, 6d, and 8d, respectively, and put them into a centrifuge, centrifuge at 1000rpm and 4℃ for 10min, discard the supernatant, obtain the cell precipitate, add 3mL of 1xPBS prepared with enzyme-free water to suspend the cell precipitate, centrifuge at 200rpm for 5min, wash twice, and discard the supernatant; transfer the precipitate to a threaded freezing tube that can withstand -192℃ low temperature, and freeze the fermentation liquid precipitates of strain S and strain Y in liquid nitrogen for 1h and then store them at -80℃ for later use; Step 5: Determine the GA4 content: Determine the GA4 content and colony count in the liquid precipitate of step 4, and draw the growth curves of strain S and strain Y; Step 6: Determine the dry weight of mycelium: Take 5 mL of fermentation liquid from the conical flask in step 3 every 24 hours, centrifuge to remove the supernatant, add appropriate amount of 0.9% NaCl solution to wash twice, place the precipitate in a culture dish, dry at 80°C to constant weight, and weigh and record the dry weight of mycelium of strain S and strain Y respectively.

2. The screening process for gibberellin production bacterium colonies according to claim 1, characterized in that: The raw material ratio of the PDA culture medium in step 1 is: 200g potato, 20g glucose, 20g agar powder, 1L distilled water, and natural pH.

3. The screening process for gibberellin production bacterium colonies according to claim 1, characterized in that: The raw material ratio of the seed culture medium in step 2 is: sucrose 30g, soybean meal 12g, NH4Cl 3g, NaNO3 3g, KH2PO4 1.5g, MgSO4·7H2O0.2g, K2SO4 0.2g, distilled water 1L, pH 6.8, 121℃ high pressure steam sterilization for 30min.

4. The screening process for gibberellin production bacterium colonies according to claim 1, characterized in that: The raw material ratio of the fermentation medium in step three is: lactose 140g, soybean meal powder 10g, corn syrup powder 10g, corn gluten powder 5g, K2SO4 2g, ZnSO4·7H2O 0.005g, MgSO4·7H2O 1g, CoCl2·6H2O 0.005g, urea 1.25g, distilled water 1L, pH7.0, 121°C high-pressure steam sterilization for 30min.

Images