CN105524863B - Method for producing bacillus amyloliquefaciens in high density by combining liquid and semisolid fermentation processes - Google Patents
Method for producing bacillus amyloliquefaciens in high density by combining liquid and semisolid fermentation processes Download PDFInfo
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Abstract
The invention discloses a method for producing bacillus amyloliquefaciens in high density by combining liquid and semisolid fermentation processes, which comprises the following steps: inoculating the seed liquid of the bacillus amyloliquefaciens into a liquid fermentation culture medium, wherein the inoculation amount is 1-5%, and culturing for 20-26h at the temperature of 35-38 ℃, the air inlet amount of 25-35L/min and the pressure of 0.02-0.04Mpa to obtain a liquid fermentation product; semi-solid fermentation: inoculating the liquid fermentation product to a semisolid fermentation culture medium containing coconut bran powder and cassava residue powder, uniformly mixing, wherein the inoculation amount is 18-22%, and culturing for 4-6 days under the conditions that the temperature is 35-38 ℃ and the humidity is 50-70% to obtain a semisolid fermentation product; the invention combines the liquid and semi-solid fermentation processes and optimizes the process parameters thereof to obtain the bacillus amyloliquefaciens final product with the thallus content of 3.21 multiplied by 1012cfu/g, the spore rate is as high as 93 percent.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for producing bacillus amyloliquefaciens in a high density mode by combining a liquid fermentation process and a semisolid fermentation process.
Background
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a bacterium with broad-spectrum antibacterial activity, has strong secondary metabolite production capacity, and can inhibit various plant pathogenic fungi such as fusarium oxysporum and the like.
Currently, the optimization of fermentation of bacillus amyloliquefaciens at home and abroad mainly focuses on producing enzyme substances such as antibacterial lipopeptide, α -amylase, protease and the like, and the optimization of the fermentation process of the bacillus amyloliquefaciens quantity is less researched, and in the research of the optimization of the fermentation of the bacillus amyloliquefaciens quantity, most research aims are to provide a bacillus amyloliquefaciens liquid submerged fermentation technology and a bacillus amyloliquefaciens high-density fermentation technology.
Currently, industrial production modes of bacillus products are mainly divided into liquid submerged fermentation and solid fermentation, and the liquid submerged fermentation has the advantages that the fermentation process is easy to control, the automation degree is high, the product purity is high, but the requirements on equipment are high, the investment is large, the energy consumption is high, the environmental pollution is serious, adsorption drying is needed after fermentation, and the number of process procedures is large; the solid fermentation has the advantages of high fermentation yield, no adsorption after fermentation, direct drying and crushing for use, simple process, low production cost and difficult control of the fermentation process and the mixed bacteria pollution. Semisolid fermentation is a type of solid fermentation, which refers to a microbial fermentation process that is carried out in a state where the medium is in a solid state, although rich in water, but is free or substantially free of free-flowing water.
Chinese patent ZL201110428027.7 discloses a high-density fermentation method of bacillus coagulans for livestock and poultry, a preparation prepared by the method and application of the bacillus coagulansPulverizing and sieving to obtain high-density bacillus coagulans final product with thallus number of 115.5 × 108cfu.g-1, the spore rate can reach 77.2%. But has the problems of low bacterial count, low spore rate and the like of the final product.
Disclosure of Invention
In view of the above, the invention provides a method for producing bacillus amyloliquefaciens in high density by combining liquid and semisolid fermentation processes, and solves the problems of low bacterial quantity and low spore rate in industrial production of bacillus amyloliquefaciens.
The technical means adopted by the invention are as follows: a method for producing Bacillus amyloliquefaciens by combining a liquid fermentation process and a semi-solid fermentation process with high-density fermentation, comprising the following steps of:
① liquid fermentation
Inoculating the seed liquid of the bacillus amyloliquefaciens into a liquid fermentation culture medium, wherein the inoculation amount is 1-5%, and culturing for 20-26h at the temperature of 35-38 ℃, the air inlet amount of 25-35L/min and the pressure of 0.02-0.04Mpa to obtain a liquid fermentation product;
the liquid fermentation culture medium comprises 18-22g/L sucrose, 8-12g/L soybean meal, 0.8-1.2g/L yeast powder, 1.0-1.4g/L K2HPO4And 0.8-1.2g/L MgCL2；
② semi-solid fermentation
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium, uniformly mixing, wherein the inoculation amount is 18-22%, and culturing for 4-6 days under the conditions that the temperature is 35-38 ℃ and the humidity is 50-70% to obtain a semi-solid fermentation product;
the semi-solid culture medium comprises, by weight, 80-100 parts of corn flour, 8-12 parts of rice flour, 35-50 parts of coconut bran powder, 14-17 parts of cassava slag powder and 160-200 parts of water.
Preferably, the seed solution concentration of Bacillus amyloliquefaciens is 1.2X 1010-5.6×1010cfu/mL。
Preferably, a method for producing bacillus amyloliquefaciens by a combination of liquid and semi-solid fermentation processes and high-density fermentation comprises the following steps:
① liquid fermentation
Inoculating the seed liquid of the bacillus amyloliquefaciens into a liquid fermentation culture medium, wherein the inoculation amount is 3%, and culturing for 24 hours at the temperature of 37 ℃, the air inlet amount of 30L/min and the pressure of 0.03Mpa to obtain a liquid fermentation product;
the seed liquid of Bacillus amyloliquefaciens has a concentration of 4.0 × 1010cfu/mL Bacillus amyloliquefaciens;
the liquid fermentation culture medium comprises 20g/L sucrose, 10g/L soybean meal, 1.0g/L yeast powder and 1.2g/LK2HPO4And 1.0g/L MgCL2；
② semi-solid fermentation
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium, uniformly mixing, wherein the inoculation amount is 20%, and then culturing for 5 days under the conditions that the temperature is 37 ℃ and the humidity is 60% to obtain a semi-solid fermentation product;
the semi-solid culture medium comprises 90 parts by weight of corn flour, 10 parts by weight of rice flour, 40 parts by weight of coconut bran powder, 15 parts by weight of cassava slag powder and 200 parts by weight of water.
Preferably, the seed solution of bacillus amyloliquefaciens is obtained by culturing the following methods: inoculating bacillus amyloliquefaciens to an LB culture medium for culture for 32-48 h, and then inoculating to a liquid seed culture medium for culture to obtain a seed solution, wherein the liquid seed culture medium comprises 20g/L of sucrose and 10g/L, K of yeast powder2HPO41.2g/L、MgCL21.0g/L and 0.2mL of antifoaming agent.
The invention combines the liquid and semi-solid fermentation processes and optimizes the process parameters thereof to obtain the bacillus amyloliquefaciens final product with the thallus content of 3.21 multiplied by 1012cfu/g, the spore rate is as high as 93 percent.
The invention provides a method for producing bacillus amyloliquefaciens in high density by combining a liquid fermentation process and a semisolid fermentation process, which combines the liquid fermentation production with the semisolid fermentation production, firstly utilizes liquid to culture fast-growing spore thalli, and then transfers the spore thalli to a semisolid nutrient substrate containing coconut bran powder and cassava residue powder to generate a large amount of spores, thereby realizing mass production on the basis of improving the yield and the spore rate of the thalli.
Furthermore, the method provided by the invention has simple production process, obviously improves the yield of the spores, reduces the pollution probability in the culture process and does not need complex equipment.
In the method provided by the invention, the raw materials of the culture medium mainly comprise tropical special processing byproducts and common agricultural and sideline products. The coconut husk powder and the cassava residue powder are special processing byproducts in tropical regions, the coconut husk powder is fiber powder of coconut shells, and is a natural degradable and purely natural organic matter medium which falls off from the processing process of the coconut shell fibers. The coconut bran powder has good air permeability and water retention property in semisolid fermentation and can provide trace elements required by microbial fermentation. The cassava residue powder is a byproduct after starch is extracted from cassava, the main indexes comprise crude fiber, a small amount of starch and crude protein, and the cassava residue powder contains rich mineral substances (comprising nitrogen, phosphorus, potassium, carbon, calcium, magnesium, sulfur, zinc, manganese, copper, iron and sodium). The rice flour and the corn flour have wide and sufficient sources, low cost, easy regulation and control and high success rate.
In conclusion, the culture method of the bacillus amyloliquefaciens provided by the invention has the advantages of economical and practical production process, high production efficiency, less investment, small occupied area, realization of large-scale production without the three wastes, suitability for large-scale popularization and application and huge economic benefit.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The selected Bacillus amyloliquefaciens HC200 has the registration number of CGMCC No.10371 in the common microorganism center of China Committee for culture Collection of microorganisms, and the strain is disclosed in the application patent CN 201510653843.6. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5, which is deposited at the institute of environmental and plant protection, national academy of tropical agricultural sciences in China.
The bacillus amyloliquefaciens fermented product produced by the method is dried into powder and can be directly used for preventing and controlling soil-borne diseases such as banana wilt, tomato bacterial wilt, sweet melon wilt, potato blight, apple red rot, scab and the like and overground diseases such as mango anthracnose, melon powdery mildew, grape powdery mildew and the like.
The first embodiment is as follows:
the production method of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:
1. strain culture
The bacillus amyloliquefaciens HC200 is inoculated on an LB medium plate and cultured for 32 hours at 37 ℃ until the bacteria grow full of the plate.
LB culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 17g of agar, and the volume is adjusted to 1000ml by water, and the pH value is 7.0.
2. Liquid seed culture
Placing 400ml liquid seed culture medium into 1000ml triangular flask, inoculating strain obtained in step 1, inoculating 1/6 petri dish plate strain (petri dish size is 90mm) into each triangular flask, culturing at 37 deg.C and 180rpm for 24 hr to obtain seed solution with strain concentration of 1.2 × 1010cfu/mL。
Liquid seed culture medium: taking 20g of sucrose, 10g of yeast powder and K2HPO41.2g and MgCL21g, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
3. Liquid fermentation production
A100 liter mechanically stirred fermenter was charged with 60 liters of liquid fermentation medium and then inoculated with 1% of the seed solution obtained in step 2 (i.e., 99 volumes of liquid fermentation medium per 1 volume of seed solution).
The culture conditions are as follows: introducing air at 35 deg.C with ventilation rate of 25L/min, maintaining pressure at 0.02Mpa, and culturing for 20 hr.
After the above culture was completed, all the substances in the fermenter were used as liquid fermentation products.
A large amount of spore thallus appears in the liquid fermentation product through microscope observation, and the fermentation liquid is turbid.
Liquid fermentation medium: taking 18g of cane sugar, 12g of soybean meal powder, 0.8g of yeast powder and K2HPO41.0g and MgCL20.8g0.2mL of defoaming agent is dissolved in water and the volume is fixed to 1000mL by water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
4. Semi-solid fermentation production
A culture rack: the four-upright-post rack with nylon gauze interlayer in the cavity has the length of 3.2m, the width of 0.7m and the height of 2.6m, is separated into 7 layers by a watertight nylon gauze, the layer spacing is 30cm, and the four upright posts are made of carbon steel sprayed plastics.
The semi-solid fermentation medium comprises the following components in parts by weight: uniformly mixing 100 parts of corn flour, 8 parts of rice flour, 50 parts of coconut bran powder, 14 parts of cassava slag powder and 170 parts of water; natural pH; steam sterilization was carried out at 121 ℃ for 60 minutes.
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium in a fermentation chamber subjected to fumigation sterilization by formaldehyde and potassium permanganate, uniformly mixing, wherein the inoculation amount is 18% (namely, 18 volume parts of the liquid fermentation product are inoculated to 82 volume parts of the semi-solid fermentation culture medium), then placing the mixture on a nylon gauze of a culture rack in the fermentation chamber, and culturing for 4 days under the conditions that the temperature is 35 ℃ and the humidity is 50%. During the cultivation, the material is frequently turned over.
After the above incubation was complete, all the material in the container was designated as semi-solid fermentation product.
5. Drying of the fermentation product
Drying the semi-solid fermentation product in a fermentation chamber with an air conditioner and a dehumidifier, and pulverizing to obtain the final product (with water content of 6.0% by detection).
And calculating the thallus content and the spore rate of the bacillus amyloliquefaciens HC200 in the final product by adopting plate counting.
Example two:
the production method of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:
1. strain culture
The bacillus amyloliquefaciens HC200 is inoculated on an LB medium plate and cultured for 40 hours at 37 ℃ until the bacteria grow full of the plate.
LB culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 17g of agar, and the volume is adjusted to 1000ml by water, and the pH value is 7.0.
2. Liquid seed culture
Placing 400ml liquid seed culture medium into 1000ml triangular flask, inoculating strain obtained in step 1, inoculating 1/6 petri dish plate strain (petri dish size is 90mm) into each triangular flask, culturing at 37 deg.C and 180rpm for 24 hr to obtain seed solution with strain concentration of 4.0 × 1010cfu/mL。
Liquid seed culture medium: taking 20g of sucrose, 10g of yeast powder and K2HPO41.2g and MgCL21g, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
3. Liquid fermentation production
A100 liter mechanically stirred fermenter was charged with 60 liters of liquid fermentation medium and then inoculated with 3% of the seed liquid obtained in step 2 (i.e., 97 volumes of liquid fermentation medium per 3 volumes of seed liquid).
The culture conditions are as follows: introducing air at 37 deg.C with ventilation rate of 30L/min, maintaining pressure at 0.03MPa, and culturing for 24 hr.
After the above culture was completed, all the substances in the fermenter were used as liquid fermentation products.
A large amount of spore thallus appears in the liquid fermentation product through microscope observation, and the fermentation liquid is turbid.
Liquid fermentation medium: taking 20g of cane sugar, 10g of soybean meal powder, 10g of yeast powder and K2HPO41.2g and MgCL21.0g of defoaming agent and 0.2mL of defoaming agent, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
4. Semi-solid fermentation production
A culture rack: the four-upright-post rack with nylon gauze interlayer in the cavity has the length of 3.2m, the width of 0.7m and the height of 2.6m, is separated into 7 layers by a watertight nylon gauze, the layer spacing is 30cm, and the four upright posts are made of carbon steel sprayed plastics.
The semi-solid fermentation medium comprises the following components in parts by weight: uniformly mixing 90 parts of corn flour, 10 parts of rice flour, 40 parts of coconut bran powder, 15 parts of cassava slag powder and 180 parts of water; natural pH; steam sterilization was carried out at 121 ℃ for 60 minutes.
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium in a fermentation chamber subjected to fumigation sterilization by formaldehyde and potassium permanganate, uniformly mixing, wherein the inoculation amount is 20% (namely 20 parts by volume of the liquid fermentation product is inoculated to 80 parts by volume of the semi-solid fermentation culture medium), then placing the mixture on a nylon gauze of a culture rack in the fermentation chamber, and culturing for 5 days under the conditions that the temperature is 37 ℃ and the humidity is 60%. During the cultivation, the material is frequently turned over.
After the above incubation was complete, all the material in the container was designated as semi-solid fermentation product.
5. Drying of the fermentation product
Drying the semi-solid fermentation product in a fermentation chamber with an air conditioner and a dehumidifier, and pulverizing to obtain the final product (with water content of 6.0% by detection).
And calculating the content and the spore rate of the bacillus amyloliquefaciens HC200 in the final product by adopting plate counting.
Example three:
the production method of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:
1. strain culture
The bacillus amyloliquefaciens HC200 is inoculated on an LB medium plate and cultured for 48 hours at 37 ℃ until the bacteria grow full of the plate.
LB culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 17g of agar, and the volume is adjusted to 1000ml by water, and the pH value is 7.0.
2. Liquid seed culture
Placing 400ml liquid seed culture medium into 1000ml triangular flask, inoculating strain obtained in step 1, inoculating 1/6 petri dish plate strain (petri dish size is 90mm) into each triangular flask, culturing at 37 deg.C and 180rpm for 24 hr to obtain seed solution with strain concentration of 5.0 × 1010cfu/mL。
Liquid seed culture medium: taking 20g of sucrose, 10g of yeast powder and K2HPO41.2g and MgCL21g, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; high-pressure extinguishing at 121 DEG CThe bacteria were allowed to cool to 40 ℃ for 20 minutes before inoculation.
3. Liquid fermentation production
A100 liter mechanically stirred fermenter was charged with 60 liters of liquid fermentation medium and then inoculated with 5% of the seed liquid obtained in step 2 (i.e., 95 volumes of liquid fermentation medium per 5 volumes of seed liquid).
The culture conditions are as follows: introducing air at 38 deg.C with ventilation rate of 35L/min, maintaining pressure at 0.04MPa, and culturing for 26 hr.
After the above culture was completed, all the substances in the fermenter were used as liquid fermentation products.
A large amount of spore thallus appears in the liquid fermentation product through microscope observation, and the fermentation liquid is turbid.
Liquid fermentation medium: taking 22g of sucrose, 8g of soybean meal, 1.2g of yeast powder and K2HPO41.4g and MgCL21.2g of defoaming agent and 0.2mL of defoaming agent, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
4. Semi-solid fermentation production
A culture rack: the four-upright-post rack with nylon gauze interlayer in the cavity has the length of 3.2m, the width of 0.7m and the height of 2.6m, is separated into 7 layers by a watertight nylon gauze, the layer spacing is 30cm, and the four upright posts are made of carbon steel sprayed plastics.
Semi-solid fermentation medium: uniformly mixing 80 parts by mass of corn flour, 12 parts by mass of rice flour, 35 parts by mass of coconut bran powder, 17 parts by mass of cassava slag powder and 200 parts by mass of water; natural pH; steam sterilization was carried out at 121 ℃ for 60 minutes.
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium in a fermentation chamber subjected to fumigation sterilization by formaldehyde and potassium permanganate, uniformly mixing, wherein the inoculation amount is 22% (namely, 22 volume parts of the liquid fermentation product are inoculated to 78 volume parts of the semi-solid fermentation culture medium), then placing the mixture on a nylon gauze of a culture rack in the fermentation chamber, and culturing for 6 days under the conditions that the temperature is 38 ℃ and the humidity is 70%. During the cultivation, the material is frequently turned over.
After the above incubation was complete, all the material in the container was designated as semi-solid fermentation product.
5. Drying of the fermentation product
Drying the semi-solid fermentation product in a fermentation chamber with an air conditioner and a dehumidifier, and pulverizing to obtain the final product (with water content of 6.0% by detection).
And calculating the thallus content and the spore rate of the bacillus amyloliquefaciens HC200 in the final product by adopting plate counting.
Example four:
the production method of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:
1. strain culture
The bacillus amyloliquefaciens HC200 is inoculated on an LB medium plate and cultured for 48 hours at 37 ℃ until the bacteria grow full of the plate.
LB culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 17g of agar, and the volume is adjusted to 1000ml by water, and the pH value is 7.0.
2. Liquid seed culture
Placing 400ml liquid seed culture medium into 1000ml triangular flask, inoculating strain obtained in step 1, inoculating 1/6 petri dish plate strain (petri dish size is 90mm) into each triangular flask, culturing at 37 deg.C and 180rpm for 24 hr to obtain seed solution with strain concentration of 5.6 × 1010cfu/mL。
Liquid seed culture medium: taking 20g of sucrose, 10g of yeast powder and K2HPO41.2g and MgCL21g, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
3. Liquid fermentation production
A100 liter mechanically stirred fermenter was charged with 60 liters of liquid fermentation medium and then inoculated with 4% of the seed liquid obtained in step 2 (i.e., 96 volumes of liquid fermentation medium per 4 volumes of seed liquid).
The culture conditions are as follows: introducing air at 36 deg.C with aeration rate of 32L/min, maintaining pressure at 0.03MPa, and culturing for 25 hr.
After the above culture was completed, all the substances in the fermenter were used as liquid fermentation products.
A large amount of spore thallus appears in the liquid fermentation product through microscope observation, and the fermentation liquid is turbid.
Liquid fermentation medium: taking 21g of sucrose, 9g of soybean meal, 0.9g of yeast powder and K2HPO41.2g and MgCL21.2g of defoaming agent and 0.2mL of defoaming agent, dissolving in water and fixing the volume to 1000mL by using water; pH 6.5; autoclaved at 121 ℃ for 20 minutes, cooled to 40 ℃ and used for inoculation.
4. Semi-solid fermentation production
A culture rack: the four-upright-post rack with nylon gauze interlayer in the cavity has the length of 3.2m, the width of 0.7m and the height of 2.6m, is separated into 7 layers by a watertight nylon gauze, the layer spacing is 30cm, and the four upright posts are made of carbon steel sprayed plastics.
Semi-solid fermentation medium: uniformly mixing 100 parts by mass of corn flour, 9 parts by mass of rice flour, 45 parts by mass of coconut bran powder, 170 parts by mass of cassava slag powder and 170 parts by mass of water; natural pH; steam sterilization was carried out at 121 ℃ for 60 minutes.
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium in a fermentation chamber subjected to fumigation sterilization by formaldehyde and potassium permanganate, uniformly mixing, wherein the inoculation amount is 21% (namely 21 volume parts of the liquid fermentation product are inoculated to 79 volume parts of the solid fermentation culture medium), then placing the mixture on a nylon gauze of a culture rack in the fermentation chamber, and culturing for 5 days under the conditions that the temperature is 36 ℃ and the humidity is 65%. During the cultivation, the material is frequently turned over.
After the above incubation was complete, all the material in the container was designated as semi-solid fermentation product.
5. Drying of the fermentation product
Drying the semi-solid fermentation product in a fermentation chamber with an air conditioner and a dehumidifier, and pulverizing to obtain the final product (with water content of 6.0% by detection).
The number of Bacillus amyloliquefaciens HC200 in the final product was calculated by plate counting to be 1.58X 1012And (4) obtaining the final product.
Comparative example one:
inoculating Bacillus amyloliquefaciens HC200 on sterilized solid culture medium (the solid culture medium comprises, by mass, beef extract 0.3%, peptone 0.6%, agar 1.5%, and water in balance, and has pH of 7.0, 121 deg.C, and 0.11Mpa sterilized for 20min.) by aseptic streaking, and culturing at 37 deg.C for 4 days.
After washing the bacterial colony on the solid culture medium with sterile normal saline, inoculating the bacterial colony in a sterile culture medium of a 1000ml shake flask (the culture medium comprises, by mass, 0.4% of glucose, 0.3% of beef extract, 1.0% of peptone, 0.5% of yeast extract, 0.1% of dipotassium hydrogen phosphate, 0.2% of ammonium sulfate, 0.5% of sodium chloride, 0.15% of an antifoaming agent, and the balance of water, and is sterilized at pH7.0, 121 ℃ and 0.11MPa for 20min), wherein the loading amount of the culture medium is 200ml, and the shake culture conditions are as follows: the culture was carried out at 37 ℃ and 220rpm for 16 hours.
Inoculating the seed solution obtained in the step into a sterilization culture medium of a 100L seed fermentation tank (the culture medium comprises, by mass, 0.4% of glucose, 0.3% of beef extract, 1.0% of peptone, 0.5% of yeast extract, 0.1% of dipotassium hydrogen phosphate, 0.2% of ammonium sulfate, 0.02% of magnesium sulfate, 0.005% of manganese sulfate, 0.5% of sodium chloride, 0.15% of a defoaming agent, and the balance of water, sterilizing at pH7.0, 121 ℃ and 0.11MPa for 20min), wherein the culture medium loading is 70L, the inoculation amount is 5% (v/v), and the fermentation conditions are as follows: the temperature is 37 ℃, the tank pressure is 0.05Mpa, the aeration ratio is 1:0.6(v/v) at the beginning, the aeration ratio is increased to 1:1(v/v) after 6 hours of fermentation, the initial stirring revolution is 180rpm, the stirring revolution is adjusted to 220rpm after 6 hours of fermentation, and the fermentation culture is carried out for 10 hours, so as to obtain the seed bacterial liquid used by the secondary tank;
inoculating the seed solution cultured in the step into a sterilized culture medium of a 1000L fermentation tank (the culture medium comprises, by mass, 0.4% of glucose, 2% of corn starch, 0.1% of corn micropowder, 0.1% of dipotassium phosphate, 0.2% of ammonium sulfate, 0.01% of magnesium sulfate, 0.2% of sodium chloride, 0.15% of an antifoaming agent, and the balance of water, sterilizing at a pH of 7.0, 121 ℃ and 0.11MPa for 20-30min), wherein the culture medium has a loading capacity of 700L, and the fermentation conditions are as follows: the temperature was 37 ℃ and the pot pressure was 0.05MPa, and the aeration ratio was 1:0.6(v/v) at the initial aeration ratio, and after 6 hours of fermentation, the aeration ratio was increased to 1:1(v/v) at the initial stirring speed of 180rpm, and after 6 hours of fermentation, the stirring speed was adjusted to 220rpm at the inoculation rate of 10% (v/v), and the fermentation was carried out for 36 hours. Collecting fermentation culture solution to obtain bacillusA liquid product. Viable count of 9.6X 10 on nutrient agar plate9cfu/ml, spore rate 75%.
The experimental conditions and results of examples one to three, comparative example are summarized in table 1.
TABLE 1
Example five:
the difference between the fifth embodiment and the first embodiment is that: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5 is used for replacing the Bacillus amyloliquefaciens HC200 for fermentation production.
Example six:
the difference between the sixth embodiment and the second embodiment is that: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5 is used for replacing the Bacillus amyloliquefaciens HC200 for fermentation production.
Example six:
the difference between the seventh embodiment and the third embodiment is that: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5 is used for replacing the Bacillus amyloliquefaciens HC200 for fermentation production.
Example eight:
the difference between the eighth example and the fourth example is that: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5 is used for replacing the Bacillus amyloliquefaciens HC200 for fermentation production.
Comparative example two:
comparative example two differs from comparative example one in that: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5 is used for replacing the Bacillus amyloliquefaciens HC200 for fermentation production.
The experimental conditions and results of examples five to eight, comparative example two are summarized in table 2.
TABLE 2
From the above experimental results, the liquid fermentation conditions and the semi-solid fermentation conditions have a great influence on the yield and the spore rate of the bacillus amyloliquefaciens, especially the ratio of the nutrient components of the semi-solid culture medium to water, the inoculation amount, the fermentation temperature and the like. The invention combines the liquid and semi-solid fermentation processes and optimizes the process parameters thereof to obtain the bacillus amyloliquefaciens final product with the thallus content of 3.21 multiplied by 1012cfu/g, the spore rate is as high as 93 percent.
The invention provides a method for producing bacillus amyloliquefaciens with high density by combining liquid and semisolid fermentation processes.
The method provided by the invention has simple production process, obviously improves the yield of the spores, reduces the pollution probability in the culture process and does not need complex equipment.
In the method provided by the invention, the raw materials of the culture medium mainly comprise tropical specific processing byproducts and common agricultural and sideline products. The coconut husk powder and the cassava residue powder are special processing byproducts in tropical regions, the coconut husk powder is fiber powder of coconut shells, and is a natural degradable and purely natural organic matter medium which falls off from the processing process of the coconut shell fibers. The coconut bran powder has good air permeability and water retention property in semisolid fermentation and can provide trace elements required by microbial fermentation. The cassava residue powder is a byproduct after starch is extracted from cassava, the main indexes comprise crude fiber, a small amount of starch and crude protein, and the cassava residue powder contains rich mineral substances (comprising nitrogen, phosphorus, potassium, carbon, calcium, magnesium, sulfur, zinc, manganese, copper, iron and sodium). The rice flour and the corn flour have wide and sufficient sources, low cost, easy regulation and control and high success rate.
In conclusion, the production method of the bacillus amyloliquefaciens provided by the invention has the advantages of economical and practical production process, high production efficiency, less investment, small occupied area, realization of large-scale production without the three wastes, suitability for large-scale popularization and application and huge economic benefit.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. A method for producing Bacillus amyloliquefaciens in high density by combining liquid and semisolid fermentation processes is characterized by comprising the following steps:
① liquid fermentation
Inoculating the seed liquid of the bacillus amyloliquefaciens into a liquid fermentation culture medium, wherein the inoculation amount is 1-5%, culturing for 20-26h at the temperature of 35-38 ℃, the air amount of 25-35L/min and the pressure of 0.02-0.04Mpa to obtain a liquid fermentation product, and a 100L mechanical stirring fermentation tank is used in the liquid fermentation process;
the liquid fermentation culture medium comprises 18-22g/L of sucrose, 8-12g/L of soybean meal, 0.8-1.2g/L of yeast powder and 1.0-1.4g/L K2HPO4And 0.8-1.2g/L MgCl2；
② semi-solid fermentation
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium, uniformly mixing, wherein the inoculation amount is 18-22%, and culturing for 4-6 days under the conditions that the temperature is 35-38 ℃ and the humidity is 50-70% to obtain a semi-solid fermentation product;
the semi-solid fermentation medium comprises, by weight, 80-100 parts of corn flour, 8-12 parts of rice flour, 35-50 parts of coconut bran powder, 14-17 parts of cassava slag powder and 160-200 parts of water.
2. The method for producing Bacillus amyloliquefaciens in high density by combining liquid and semi-solid fermentation processes according to claim 1, wherein the seed liquor concentration of the Bacillus amyloliquefaciens is 1.2 x 1010-5.6×1010cfu/mL。
3. The method for producing bacillus amyloliquefaciens in high density by combining liquid and semi-solid fermentation processes according to claim 2, comprising the steps of:
① liquid fermentation
Inoculating the seed liquid of the bacillus amyloliquefaciens into a liquid fermentation culture medium, wherein the inoculation amount is 3%, culturing for 24h at the temperature of 37 ℃, the air inlet amount of 30L/min and the pressure of 0.03Mpa to obtain a liquid fermentation product, and a 100L mechanical stirring fermentation tank is used in the liquid fermentation process;
the concentration of the seed liquid of the bacillus amyloliquefaciens is 4.0 multiplied by 1010cfu/mL；
The liquid fermentation culture medium comprises 20g/L of sucrose, 10g/L of soybean meal powder, 1.0g/L of yeast powder and 1.2g/L K2HPO4And 1.0g/L MgCl2；
② semi-solid fermentation
Inoculating the liquid fermentation product to a semi-solid fermentation culture medium, uniformly mixing, wherein the inoculation amount is 20%, and then culturing for 5 days under the conditions that the temperature is 37 ℃ and the humidity is 60% to obtain a semi-solid fermentation product;
the semi-solid fermentation medium comprises 90 parts of corn flour, 10 parts of rice flour, 40 parts of coconut bran powder, 15 parts of cassava residue powder and 200 parts of water in parts by weight.
4. The method for producing Bacillus amyloliquefaciens in high density by combining liquid and semi-solid fermentation processes according to any one of claims 1 to 3, wherein the seed liquid of the Bacillus amyloliquefaciens is obtained by culturing: inoculating bacillus amyloliquefaciens to an LB culture medium for culture for 32-48 h, and then inoculating to a liquid seed culture medium for culture to obtain a seed solution, wherein the liquid seed culture medium comprises 20g/L of sucrose and 10g/L, K of yeast powder2HPO41.2 g/L、MgCl21.0g/L and 0.2 mL/L of antifoaming agent.
5. The method for producing Bacillus amyloliquefaciens in high density by combining liquid and semi-solid fermentation processes according to any one of claims 1-3, further comprising drying and pulverizing the semi-solid fermentation product in a fermentation chamber to obtain the final product.
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