Fermentation process of bacillus velezensis

Abstract

The invention discloses a bacillus velezensis fermentation process, which comprises the steps of bacillus velezensis slant strain preparation, seed solution preparation, fermentation broth preparation and the like, the titer of the bacillus velezensis fermentation broth prepared by the method is 100-160CFU / mL, the spore content of the fermentation broth is obviously improved, and the bacillus velezensis fermentation broth has the advantages of high yield, high yield and the like. Therefore, the biocontrol effect of the obtained fermentation liquor is improved; and on the other hand, the preparation method maximizes the utilization rate of the raw materials, shortens the fermentation period, is suitable for batch production, and improves the economic benefit.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a fermentation process for Bacillus belye. Background Technology

[0002] Rice is one of my country's most important food crops, and diseases cause enormous losses to rice production, seriously threatening my country's food security. The occurrence and damage of rice bakanae disease are becoming increasingly serious; however, current basic research on rice bakanae disease is weak, control levels are low, and control effects are unsatisfactory, failing to meet production needs. Currently, the most common and effective methods for controlling plant diseases are utilizing varietal resistance, chemical control, and biological control.

[0003] Rice bakanae disease, also known as excessive growth disease, occurs in all rice-growing areas of China. Infected grains often fail to germinate or emerge from the soil after sowing. During the seedling stage, infected seedlings are thinner and taller than healthy seedlings, with slender leaf sheaths, pale yellow leaves, and poor root development. Some infected seedlings die before transplanting. A pale red or white powdery mold, the conidia of the pathogen, is present on the dead seedlings. Under high humidity, the surface of dead diseased plants becomes covered with a pale brown or white powdery mold, later developing into small black dots, the fungal cysts. Mildly infected plants exhibit premature heading, but the panicles are small and empty. Grains can also be affected during the heading stage; severely infected grains turn brown and fail to set grains. Pale red mold grows in the crevices of the glumes. Mild cases may not show symptoms, but mycelium may already be latent inside.

[0004] Bacillus velezensis is a new species of Bacillus that promotes plant growth and resists pathogenic microorganisms in agricultural production, making it a microorganism with great potential for developing biopharmaceuticals. Widely distributed in nature, Bacillus velezensis exhibits rapid growth and stability, is easy to isolate and culture, is harmless to humans and animals, does not pollute the environment, and has abundant metabolites with broad-spectrum antibacterial activity and strong stress resistance. Summary of the Invention

[0005] The purpose of this invention is to provide a fermentation process for Bacillus vesicularis, which has an inhibitory effect on rice bakanae disease, shortens the fermentation cycle, is suitable for mass production, and improves economic benefits.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a fermentation process for Bacillus belye, comprising the following steps: 1) Preparation of Bacillus belysin slant culture: Bacillus belysin culture was inoculated into eggplant flask slant culture and cultured at 30-35℃ for 3-6 days. The slant culture medium formula was: beef extract 5g / L, peptone 10g / L, glucose 5g / L, agar 15g / L, and pH 7.0-7.5 before sterilization. 2) Preparation of Bacillus belysin seed culture: Bacillus belysin seed culture was inoculated on eggplant slant agar plates and cultured at 28-36℃ for 12-20h. The seed culture medium formula was: 5g / L beef extract, 10g / L peptone, and 5g / L glucose. The pH before sterilization was 7.0-7.5. 3) Preparation of Bacillus vesiculosus fermentation broth: Inoculate the fermentation broth with Bacillus vesiculosus seed liquid at an inoculation rate of 2%-5%, ferment at a temperature of 28-36℃, and ferment for 2-3 days. The fermentation broth culture medium formula is as follows: rice flour 5-20g / L, yeast powder 5-20g / L, fish peptone 5-20g / L, wheat flour 20-50g / L, potassium dihydrogen phosphate 0.2-1.5g / L, magnesium sulfate 2-10g / L, calcium carbonate 2-10g / L, manganese sulfate 0.01-0.2g / L, sodium chloride 2-10g / L, sodium bicarbonate 0.1-1.5g / L, and potassium chloride 0.2-1.5g / L.

[0007] The Bacillus berberis fermentation broth prepared by the method of the present invention has a potency of 100-160 CFU / mL, which significantly increases the spore content of the fermentation broth and thus improves the biocontrol effect of the obtained fermentation broth. On the other hand, the preparation method of the present invention also maximizes the utilization rate of raw materials, shortens the fermentation cycle, is suitable for mass production, and improves economic benefits. Detailed Implementation

[0008] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0009] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0010] The fermentation process of Bacillus belye described in this invention includes the following steps: 1) Pour the prepared slant culture medium evenly into 50-80 mL eggplant-shaped flasks, sealing each flask with a stopper, wrapping it with newspaper, and tying it tightly with string. 2) Pour the prepared seed culture medium evenly into 500 mL Erlenmeyer flasks, filling each flask with 20-70 mL, sealing each flask with gauze and newspaper, and tying it tightly with string. 3) Pour the prepared fermentation broth culture medium evenly into 500 mL Erlenmeyer flasks, filling each flask with 20-70 mL, wrapping each flask with gauze and newspaper. Seal the contents and tie them tightly with a rope. Place the sealed eggplant bottle slant, seed liquid Erlenmeyer flask, and fermentation liquid Erlenmeyer flask in a high-pressure steam sterilizer for 30 minutes at a temperature of 121℃. After sterilization, place the eggplant bottle slant on a small wooden stick and let it solidify. After solidification, place it in a constant temperature incubator at 33-38℃ for 1-2 days and then remove it for later use. Dry the sterilized seed liquid and fermentation liquid Erlenmeyer flasks with the residual heat in the sterilizer. After the gauze and newspaper are dry, remove them for later use.

[0011] 2) Inoculate the Bacillus belysin strain onto the slant of the eggplant bottle on a sterile operating table. After inoculation, place the slant of the eggplant bottle in a constant temperature incubator at 30-35℃ for 3-6 days, then remove and place in a refrigerator at 4℃ for later use.

[0012] 3) On a sterile operating table, inoculate the eggplant slant culture from the refrigerator into the seed culture flask. Place the seed culture flask in a constant temperature shaker at 28-36℃ and 160-240rpm for 12-20 hours. Take the fermentation broth in the logarithmic growth phase and adjust the OD of the fermentation broth. 600 The seed solution was prepared by adjusting the concentration to 0.8-1.2.

[0013] 4) On a sterile operating table, inoculate the Bacillus belye seed culture into the fermentation broth Erlenmeyer flask at an inoculation rate of 2%-5%. Place the fermentation broth Erlenmeyer flask in a constant temperature shaker at 28-36℃ and 160-240rpm for 48-72h to prepare the Bacillus belye fermentation broth.

[0014] In step 1) above, the slant culture medium formula is: 5g / L beef extract, 10g / L peptone, 5g / L glucose, 15g / L agar, with a pH of 7.0-7.5 before sterilization; the seed culture medium formula is: 5g / L beef extract, 10g / L peptone, 5g / L glucose, with a pH of 7.0-7.5 before sterilization; the fermentation broth culture medium formula is: 5-20g / L rice flour, 5-20g / L yeast powder, 5-20g / L fish peptone, 20-50g / L wheat flour, 0.2-1.5g / L potassium dihydrogen phosphate, 2-10g / L magnesium sulfate, 2-10g / L calcium carbonate, 0.01-0.2g / L manganese sulfate, 2-10g / L sodium chloride, 0.1-1.5g / L sodium bicarbonate, and 0.2-1.5g / L potassium chloride. Example

[0015] In the fermentation process of Bacillus belyssus provided in this embodiment, the slant culture medium formula is as follows: beef extract 5g / L, peptone 10g / L, glucose 5g / L, agar 15g / L, pH 7.0 before sterilization; the seed culture medium formula is as follows: beef extract 5g / L, peptone 10g / L, glucose 5g / L, pH 7.0 before sterilization; the fermentation broth culture medium formula is as follows: rice flour 5g / L, yeast powder 5g / L, fish peptone 5g / L, flour 20g / L, potassium dihydrogen phosphate 0.2g / L, magnesium sulfate 2g / L, calcium carbonate 2g / L, manganese sulfate 0.01g / L, sodium chloride 2g / L, sodium bicarbonate 0.1g / L, potassium chloride 0.2g / L. Example

[0016] In the fermentation process of Bacillus belyssus provided in this embodiment, the slant culture medium formula is as follows: beef extract 5g / L, peptone 10g / L, glucose 5g / L, agar 15g / L, pH 7.2 before sterilization; the seed culture medium formula is as follows: beef extract 5g / L, peptone 10g / L, glucose 5g / L, pH 7.2 before sterilization; the fermentation broth culture medium formula is as follows: rice flour 10g / L, yeast powder 10g / L, fish peptone 10g / L, wheat flour 30g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 5g / L, calcium carbonate 5g / L, manganese sulfate 0.05g / L, sodium chloride 5g / L, sodium bicarbonate 0.5g / L, potassium chloride 0.5g / L. Example

[0017] This embodiment provides a fermentation process for Bacillus belyssus. The slant culture medium formula is as follows: 5 g / L beef extract, 10 g / L peptone, 5 g / L glucose, 15 g / L agar, with a pH of 7.2 before sterilization; the seed culture medium formula is as follows: 5 g / L beef extract, 10 g / L peptone, 5 g / L glucose, with a pH of 7.5 before sterilization; the fermentation broth culture medium formula is as follows: 15 g / L rice flour, 15 g / L yeast powder, 15 g / L fish peptone, 40 g / L wheat flour, 1.0 g / L potassium dihydrogen phosphate, 8 g / L magnesium sulfate, 8 g / L calcium carbonate, 0.1 g / L manganese sulfate, 8 g / L sodium chloride, 1.0 g / L sodium bicarbonate, and 1.0 g / L potassium chloride. Example

[0018] This embodiment provides a fermentation process for Bacillus belyssus. The slant culture medium formula is as follows: 5 g / L beef extract, 10 g / L peptone, 5 g / L glucose, 15 g / L agar, with a pH of 7.5 before sterilization; the seed culture medium formula is as follows: 5 g / L beef extract, 10 g / L peptone, 5 g / L glucose, with a pH of 7.2 before sterilization; the fermentation broth culture medium formula is as follows: 20 g / L rice flour, 20 g / L yeast powder, 20 g / L fish peptone, 50 g / L wheat flour, 1.5 g / L potassium dihydrogen phosphate, 10 g / L magnesium sulfate, 10 g / L calcium carbonate, 0.2 g / L manganese sulfate, 10 g / L sodium chloride, 1.5 g / L sodium bicarbonate, and 1.5 g / L potassium chloride.

Claims

1. A fermentation process for Bacillus belye, characterized in that: Includes the following steps: 1) Preparation of Bacillus belysin slant culture: Bacillus belysin culture was inoculated into eggplant flask slant culture and cultured at 30-35℃ for 3-6 days. The slant culture medium formula was: beef extract 5g / L, peptone 10g / L, glucose 5g / L, agar 15g / L, and pH 7.0-7.5 before sterilization. 2) Preparation of Bacillus belysin seed culture: Bacillus belysin seed culture was inoculated on eggplant slant agar plates and cultured at 28-36℃ for 12-20h. The seed culture medium formula was: 5g / L beef extract, 10g / L peptone, and 5g / L glucose. The pH before sterilization was 7.0-7.

5. 3) Preparation of Bacillus vesiculosus fermentation broth: Inoculate the fermentation broth with Bacillus vesiculosus seed liquid at an inoculation rate of 2%-5%, ferment at a temperature of 28-36℃, and ferment for 2-3 days. The fermentation broth culture medium formula is as follows: rice flour 5-20g / L, yeast powder 5-20g / L, fish peptone 5-20g / L, wheat flour 20-50g / L, potassium dihydrogen phosphate 0.2-1.5g / L, magnesium sulfate 2-10g / L, calcium carbonate 2-10g / L, manganese sulfate 0.01-0.2g / L, sodium chloride 2-10g / L, sodium bicarbonate 0.1-1.5g / L, and potassium chloride 0.2-1.5g / L.

2. The fermentation process of Bacillus belye according to claim 1, characterized in that: Step 2) Preparation of Bacillus belye seed culture was obtained by shaking culture in a constant temperature shaker at 160-240 rpm.

3. The fermentation process of Bacillus belye according to claim 1, characterized in that: Step 3) Preparation of Bacillus vesiculus fermentation broth: The broth was prepared by shaking culture in a constant temperature shaker at 160-240 rpm.