CN113293119A - High-density liquid fermentation medium for bacteria and fermentation method thereof - Google Patents
High-density liquid fermentation medium for bacteria and fermentation method thereof Download PDFInfo
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Abstract
The invention discloses a high-density liquid fermentation medium for bacteria and a fermentation method thereof. A bacterial high density liquid fermentation medium, said fermentation medium comprising: 1) carbon source: 1 to 1.5 percent of soluble starch, 0.3 to 2.0 percent of snowflake powder and 0 to 3.5 percent of corn flour; 2) nitrogen source: 2.0 to 2.8 percent of fish meal and 0.2 to 2 percent of acid hydrolysis amino acid; 3) the inorganic salt additive comprises: 0.1 to 0.3 percent of sodium chloride, 0.02 to 0.05 percent of monopotassium phosphate, 0.1 to 0.4 percent of dipotassium phosphate and 0.01 to 0.03 percent of magnesium sulfate heptahydrate; the balance being water. The fermentation conditions were: the inoculation amount is 2 percent, the liquid loading amount of the triangular flask is 40 percent, the temperature is 37 ℃, the rotating speed is 190r/min, the pH value is 7.0, and the fermentation time is 12 hours. The content of viable bacteria of Bacillus amyloliquefaciens SQR9 in the fermentation solution is more than or equal to 1 × 109CFU/mL。
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and particularly relates to a high-density liquid fermentation culture medium for bacteria and a fermentation method thereof.
Background
China is a large agricultural country with a large population, and agricultural production plays a significant role in national economy. Since the 19 th century, the wide use of chemical fertilizers has improved the yield and quality of crops, and has brought huge economic benefits to agricultural production. At present, the yield and the use amount of the chemical fertilizer in China are stable in the first place in the world, and although the agricultural production efficiency can be improved by using a large amount of chemical fertilizer, the conditions of land pollution and environmental deterioration are increasingly serious, so that the chemical fertilizer is a key factor for hindering the sustainable development of modern agriculture in the long term.
Microbial fertilizers have gradually entered the public vision as high-yield, high-efficiency, healthy fertilizers. At present, about one hundred thousand tons of microbial fertilizers are put into agricultural production in China every year, and mainly comprise three types of microbial agents, biological organic fertilizers and compound microbial fertilizers. The microbial fertilizer can produce friendly effect on the environment and mainly comprises the following two aspects: firstly, the growth and the propagation of pathogenic microorganisms are inhibited. After the microbial fertilizer is applied, the beneficial microorganisms can form dominant flora at plant rhizosphere, and occupy beneficial space sites through antagonistic action with pathogenic bacteria, so that the proliferation of the pathogenic bacteria is inhibited; and the second is the improvement effect on the soil. The traditional fertilizer is easy to cause the residue of elements such as nitrogen, phosphorus, potassium and the like, so that the soil is hardened and the fertility is reduced, and the microbial fertilizer can release insoluble phosphate and other trace elements in the soil, thereby improving the element utilization rate and enhancing the soil performance.
Gram-positive bacteria of the bacillus amyloliquefaciens, bacillus, widely exist in soil, air, water and animals and plants, and have the excellent characteristics of secreting a large amount of antibacterial substances, inducing host plants to generate resistance, secreting various hormones to promote plant growth and the like. Can effectively inhibit the growth of pathogenic bacteria such as fusarium oxysporum, aspergillus niger, botrytis cinerea and the like, and is one of biocontrol strains approved by the ministry of agriculture and applicable to agricultural production. The SQR9 strain is a strain of bacillus amyloliquefaciens identified and stored in a high-tech research key laboratory by resource utilization of solid organic wastes in Jiangsu province of Nanjing agricultural university, can effectively promote plant growth and prevent and control soil-borne diseases through tests, and is a beneficial bacterium capable of being widely produced and popularized. At present, the bacillus amyloliquefaciens is mainly prepared from expensive materials such as yeast powder, peptone and beef extract, the production cost is high, the fermentation density is low, and the production and the application of related products are not facilitated.
Disclosure of Invention
The invention aims to solve the problems that the raw materials of the high-density fermentation culture medium for bacteria in the prior art are expensive, the density content of thalli in the obtained fermentation liquor is low and the like, and provides the high-density liquid fermentation culture medium for bacteria with low cost.
Another object of the present invention is to provide a bacterial high density liquid fermentation process.
The purpose of the invention can be realized by the following technical scheme:
a bacterial high density liquid fermentation medium, said fermentation medium comprising:
1) carbon source: 1 to 1.5 percent of soluble starch, 0.3 to 2.0 percent of snowflake powder and 0 to 3.5 percent of corn flour;
2) nitrogen source: 2.0 to 2.8 percent of fish meal and 0.2 to 2 percent of acid hydrolysis amino acid;
3) the inorganic salt additive comprises: 0.1 to 0.3 percent of sodium chloride, 0.02 to 0.05 percent of monopotassium phosphate, 0.1 to 0.4 percent of dipotassium phosphate and 0.01 to 0.03 percent of magnesium sulfate heptahydrate;
the balance of water, wherein the percent is the mass volume percentage and represents g/100 mL.
As a preferred aspect of the present invention, the fermentation medium composition comprises: 1 to 1.5 percent of soluble starch, 0.3 to 0.5 percent of snowflake powder, 3 to 3.5 percent of corn flour, 2 to 2.5 percent of fish meal, 1.5 to 2 percent of acid hydrolysis amino acid, 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the balance being water.
As a further preferred aspect of the present invention, the fermentation medium composition comprises: 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn flour, 2.4% of fish meal, 1.6% of acid hydrolysis amino acid, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.15% of dipotassium hydrogen phosphate and 0.02% of magnesium sulfate heptahydrate; the balance being water.
As another preferred aspect of the present invention, the fermentation medium composition comprises: 1.5 percent of soluble starch, 2.0 percent of snowflake powder, 2.5 percent of fish meal, 0.5 percent of acid hydrolysis amino acid (stock solution), 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the balance being water.
The content of free amino acid in the acidolysis amino acid (stock solution) is 15 percent, and the concentration of free hydrogen ions is 3M.
The preparation method of the acidolysis amino acid (stock solution) is shown in the patent issued by the applicant before: a liquid amino acid compound produced by utilizing protein of pigs died of illness and application thereof (ZL201410042218.3), the preparation method of the acid hydrolysis amino acid (stock solution) comprises the following steps: is prepared by adding sulfuric acid into lean meat for acidolysis; preferably lean meat of pig, cattle, sheep; in the acidolysis, sulfuric acid with the concentration of 5-7 mol/L is used for hydrolyzing the lean meat for 5-7 hours at 80-90 ℃, and the material ratio of the lean meat to the sulfuric acid with the concentration of 5-7 mol/L is 1: 1.5-2.5; preferably, the lean meat is hydrolyzed by using sulfuric acid with the concentration of 5mol/L for 6 hours at 80 ℃, and the material ratio of the lean meat to the sulfuric acid with the concentration of 5mol/L is 1: 1.5.
A method for fermenting bacteria by high-density liquid is characterized in that a bacterial seed solution is inoculated into the bacterial high-density liquid fermentation culture medium for fermentation; the fermentation conditions are that the temperature is 25-41 ℃, the inoculum size is 1-5%, the rotating speed is 150-.
The fermentation condition is preferably 37 ℃, the inoculum size is 2%, the rotating speed is 190r/min, the pH value is 7.0, the liquid loading amount is 40%, and the time is 12 h.
The invention relates to an application of a bacterial high-density liquid fermentation culture medium in bacterial high-density fermentation.
The bacteria are preferably Bacillus, Pseudomonas, and Acinetobacter.
The bacteria are preferably Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SQR9 with the preservation number of CGMCC No.5808, Bacillus (Bacillus) W19, Pseudomonas (Pseudomonas) PSE13 or Acinetobacter (Acinetobacter) Y40 with the preservation number of CGMCC No. 10241.
When the content of viable bacteria in the fermentation liquor is more than or equal to 1 multiplied by 109The culture was terminated at CFU/mL.
Advantageous effects
The invention adopts low-cost agricultural resources (such as snow pollen, fish meal, corn flour and the like) as available carbon-nitrogen sources, and shortens the fermentation period by further optimizing the components of the culture medium and the fermentation conditions, so that the content of viable bacteria in the final fermentation liquid reaches 130.3 multiplied by 107CFU/mL effectively overcomes the defects of low biomass, long fermentation period, high production cost and the like in the traditional fermentation process, is convenient for processing related products, and is beneficial to large-scale popularization and application. The bacillus amyloliquefaciens SQR9 microbial inoculum can be used as a biological fertilizer to be applied to agricultural production. The fertilizer is safe and nontoxic, has obvious biocontrol effect, and can achieve the purposes of preventing and treating soil-borne diseases, promoting crop growth and improving yield and quality by being applied alone or being matched with other agents.
Drawings
FIG. 1 Effect of different fermentation conditions on the amount of liquid fermentation of SQR9 Strain
FIG. 2 optimization of fermentation medium and application of fermentation method in bacterial high-density liquid fermentation
Detailed Description
EXAMPLE 1 preliminary acquisition of fermentation Medium
1.1 preparation method of seed liquid
(1) Inoculating bacillus amyloliquefaciens SQR9 on an LB solid culture medium, and culturing at 37 ℃ for 24 hours to obtain a single colony, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar and 1000mL of deionized water, and the pH is natural.
(2) Inoculating the single colony obtained in the step (1) into an LB liquid culture medium, and culturing at the temperature of 37 ℃, the liquid loading amount of 40 percent and the rotating speed of 170r/min to OD600Obtaining seed liquid when the seed liquid is 0.7; the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride and 1000mL of deionized water, and the pH value is natural. 1.2 carbon Source screening
0.2% ammonium sulfate was fixed as a nitrogen source, and an inorganic salt additive was added. Different types of carbon sources to be tested are respectively arranged in the following gradient to form a fermentation medium: 0.50 percent of soluble starch, 0.70 percent of soluble starch, 1.00 percent of soluble starch, 1.30 percent of soluble starch and 1.50 percent of soluble starch; 0.40 percent of cane sugar, 0.70 percent of cane sugar, 1.00 percent of cane sugar, 1.30 percent of cane sugar and 1.60 percent of cane sugar; 0.50 percent of snowflake powder, 0.70 percent of snowflake powder, 1.00 percent of snowflake powder, 1.30 percent of snowflake powder and 1.60 percent of snowflake powder; 1.50% of corn flour, 2.00%, 2.50%, 3.00% and 3.50%; 0.40%, 0.70%, 1.00%, 1.30%, 1.60% of lactose; glucose 0.40%, 0.70%, 1.00%, 1.30%, 1.60%; 0.02%, 0.04%, 0.06%, 0.08%, 0.10% of citric acid monohydrate and 0.10%, 0.50%, 1.00%, 1.50%, 2.00% of composite carbon-nitrogen source beef extract powder; peptone 0.50%, 1.00%, 1.50%, 2.00%, 2.50%; the balance of water. Inoculating the seed solution of 1.1 into fermentation medium at inoculation amount of 1%, and fermenting at rotation speed of 170r/min, temperature of 37 deg.C, liquid loading amount of 40%, and pH of 7.0 for 24h to obtain fermentation liquid. The results show that the new formulation, which uses soluble starch and snowflake powder as carbon sources respectively, is most suitable for the growth of SQR9 strain, and the effect of using corn flour, beef extract powder and peptone respectively is inferior. And (3) comprehensively considering factors such as production cost and the like, and finally selecting soluble starch, snowflake powder and corn flour as the raw materials of the composite carbon source.
The inorganic salt additive comprises 0.1% sodium chloride, 0.05% potassium dihydrogen phosphate, 0.15% dipotassium hydrogen phosphate and 0.02% magnesium sulfate heptahydrate.
TABLE 1 influence of different kinds and concentrations of carbon sources on the content of viable bacteria in the fermentation broth of the strain SQR9
1.3 Nitrogen Source selection
Fixing 1% of sucrose as carbon source, and adding inorganic salt additive. Different types of nitrogen sources to be tested are respectively arranged in the following gradient to form a fermentation medium: 1.00 percent of fish meal, 1.50 percent of fish meal, 2.00 percent of fish meal, 2.50 percent of fish meal and 3.00 percent of fish meal; 1.00 percent of meat and bone meal, 1.50 percent, 2.00 percent, 2.50 percent and 3.00 percent; 0.20 percent of ammonium chloride, 0.50 percent of ammonium chloride, 0.70 percent of ammonium chloride, 1.00 percent of ammonium chloride and 1.20 percent of ammonium chloride; 0.20 percent of urea, 0.50 percent of urea, 0.70 percent of urea, 1.00 percent of urea and 1.20 percent of urea; 0.20 percent of ammonium sulfate, 0.50 percent of ammonium sulfate, 0.70 percent of ammonium sulfate, 1.00 percent of ammonium sulfate and 1.20 percent of ammonium sulfate; 0.2 percent, 0.5 percent, 1.0 percent, 1.5 percent and 2.0 percent of acidolysis amino acid and 0.10 percent, 0.50 percent, 1.00 percent, 1.50 percent and 2.00 percent of composite carbon nitrogen source beef extract powder; peptone 0.50%, 1.00%, 1.50%, 2.00%, 2.50%; the balance being water. The pH was adjusted to 7.00 with 1mol/L NaOH. Inoculating the seed solution of 1.1 into fermentation medium at inoculation amount of 1%, and fermenting at rotation speed of 170r/min, temperature of 37 deg.C, liquid loading amount of 40%, and pH of 7.0 for 24h to obtain fermentation liquid. The results show that the culture effect of peptone and beef extract powder is the best, and the culture effect of acid hydrolysis amino acid and fish powder is the second best. And (3) comprehensively producing cost and the like, and finally selecting fish meal and acid hydrolysis amino acid as a compound nitrogen source.
The inorganic salt additive comprises 0.1% sodium chloride, 0.05% potassium dihydrogen phosphate, 0.15% dipotassium hydrogen phosphate and 0.02% magnesium sulfate heptahydrate.
TABLE 2 influence of different kinds and concentrations of nitrogen sources on the content of viable bacteria in the fermentation broth of the strain SQR9
1.3 Complex carbon-nitrogen sources
And (3) combining the screened 3 carbon sources and 2 nitrogen sources into a composite carbon-nitrogen source, setting four horizontal gradients, generating a 5-factor-4-level orthogonal test table, combining 16 tests, and repeating each test for 3 times. The result shows that the formula 9 has the most obvious effect, and the highest viable bacteria amount reaches 52.2 multiplied by 107CFU/mL. The No. 9 test medium consists of: 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn flour, 2.4% of fish meal, 1.6% of acid hydrolysis amino acid, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.15% of dipotassium hydrogen phosphate and 0.02% of magnesium sulfate heptahydrate. The fermentation conditions were: the inoculation amount is 1 percent, the temperature is 37 ℃, the rotating speed is 170r/min, the pH value is 7.0, the liquid loading amount is 40 percent, and the time is 24 hours.
TABLE 3L16(45) Level of orthogonality test factor
TABLE 4L16(45) Orthogonal test design and result analysis
Example 2 fermentation condition optimization
On the basis of a fermentation medium, the following gradients are respectively set for different fermentation conditions: the inoculation amount is 1%, 2%, 3%, 4% and 5%; pH5.5, 6.0, 6.5, 7.0, 7.5, 8.0; the rotating speeds are 150r/min, 170r/min, 190r/min, 210r/min and 230 r/min; the temperature is 25 ℃, 29 ℃, 33 ℃, 37 ℃ and 41 ℃; the liquid loading amount is 10%, 20%, 30%, 40% or 50%. The results show that the effect is best when the inoculation amount is 2 percent, the pH value is 7.0, the liquid loading amount is 40 percent, the rotating speed is 190r/min, the temperature is 37 ℃, and the content of viable bacteria respectively reaches 97 multiplied by 107CFU/mL、113.78×107CFU/mL、78×107CFU/mL、97×107CFU/mL and 95.78X 107CFU/mL. Under the optimal conditions, the time gradients are set to be 9h, 12h, 16h, 20h,The results of 24h and 28h show that the content of the viable bacteria is highest at 12h, and reaches 94.78 multiplied by 107CFU/mL. The fermentation medium comprises 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn flour, 2.4% of fish meal, 1.6% of acidolysis amino acid, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.15% of dipotassium hydrogen phosphate and 0.02% of magnesium sulfate heptahydrate.
Example 3 fermentation Medium further optimization
In order to further save the production cost and reduce the solid matter content in the fermentation liquor, the fermentation culture medium is optimized again. The acid hydrolysis amino acid is replaced by the acid hydrolysis amino acid (stock solution) which is cheaper, and the following concentration gradients are respectively set for 2 carbon sources and 2 nitrogen sources: 0.5 percent of soluble starch, 1.0 percent of soluble starch and 1.5 percent of soluble starch; 0.7% of snowflake powder, 1.3% of snowflake powder and 2.0% of snowflake powder; 2.0%, 2.5% and 3.0% of fish meal; 0.2 percent, 0.5 percent and 1.0 percent of acidolysis amino acid (stock solution). An orthogonal test table with 4-factor 3 level is generated, 9 test combinations are combined, and the test results and data analysis show that the best effect is achieved when the soluble starch is 1.5 percent, the snowflake powder is 2.0 percent, the fish meal is 2.5 percent and the acid hydrolysis amino acid (stock solution) is 0.5 percent, and the content of viable bacteria can reach 130.3 multiplied by 10 to the maximum7CFU/mL。
TABLE 5L9(34) Level of orthogonality test factor
TABLE 6L9(34) Orthogonal test design and result analysis
Example 4 comparison of fermentation SQR9 strains for different fermentation Processes
The strain SQR9 is used as a strain, the strain SQR9 is inoculated in a sterile environment, the strain is streaked in an LB solid culture medium, and the single colony is obtained after culturing for 24 hours at the temperature of 37 ℃, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar and 1000mL of deionized water, and the pH is natural.
Inoculating the obtained single colony in LB liquid culture medium, culturing at 37 deg.C to OD at 170r/min in 40% liquid loading600And (3) obtaining a seed liquid, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, and the pH value is natural.
Inoculating the seed liquid into an LB liquid culture medium (original LB) with the inoculation amount of 1%, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, the fermentation condition is that the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 h. The viable bacteria content of the fermentation liquor after the culture is finished is 8.67 multiplied by 107CFU/mL。
Inoculating the seed liquid into a fermentation culture medium (optimal formula) and an LB liquid culture medium (optimal LB) according to the inoculation amount of 2%, wherein the fermentation culture medium comprises the following components: 1.5 percent of soluble starch, 2.0 percent of snowflake powder, 2.5 percent of fish meal, 0.5 percent of acid hydrolysis amino acid (stock solution), 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the LB liquid medium comprises 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride. The fermentation conditions were: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH value is 7.0, and the time is 12 h. After the culture is finished, the content of viable bacteria in the fermentation medium (optimal formula) is 130.3 multiplied by 107CFU/mL, LB liquid medium (excellent LB) viable bacteria content of 17X 107CFU/mL。
Example 5 comparison of Bacillus fermentum W19 strains for different fermentation processes
A bacillus identified and stored in a high-tech research key laboratory by resource utilization of solid organic wastes of Jiangsu province of Nanjing agriculture university is classified and named as: bacillus (Bacillus) W19, see published articles Effects of novel biological inductor produced by Bacillus amyloliquefaciens W19 on antimicrobial of fungi with salt of bacteria and Fertility of soil, 2013,49,435 and 446. Inoculating a W19 preserved strain with a bacillus W19 strain in a sterile environment, streaking the strain in an LB solid culture medium, and culturing at 37 ℃ for 24 hours to obtain a single colony, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar and 1000mL of deionized water, and the pH value is natural.
Inoculating the obtained single colony in LB liquid culture medium, culturing at 37 deg.C to OD at 170r/min in 40% liquid loading600And (3) obtaining a seed liquid, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, and the pH value is natural.
Inoculating the seed liquid into an LB liquid culture medium (original LB) with the inoculation amount of 1%, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, the fermentation condition is that the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 h. The viable bacteria content of the fermentation liquor after the culture is finished is 31.67 multiplied by 107CFU/mL。
Inoculating the seed liquid into a fermentation culture medium (optimal formula) and an LB liquid culture medium (optimal LB) according to the inoculation amount of 2%, wherein the fermentation culture medium comprises the following components: 1.5 percent of soluble starch, 2.0 percent of snowflake powder, 2.5 percent of fish meal, 0.5 percent of acid hydrolysis amino acid (stock solution), 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the LB liquid medium comprises 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride. The fermentation conditions were: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH value is 7.0, and the time is 12 h. After the culture is finished, the content of viable bacteria in the fermentation medium (optimal formula) is 100 multiplied by 107CFU/mL, LB liquid medium (excellent LB) viable bacteria content is 41X 107CFU/mL。
Example 6 comparison of different fermentation Processes for the fermentation of Pseudomonas PSE13 Strain
A strain of pseudomonas identified and stored by a high-tech research key laboratory through resource utilization of solid organic wastes of Jiangsu province of Nanjing agriculture university is classified and named as: pseudomonas (Pseudomonas) PSE13, see published article Bio-organic filters additives microorganisms to enhance plant disease delivery. Microbiome,2020,8: 137. The method comprises the steps of taking pseudomonas PSE13 as a strain, inoculating a PSE13 preservation strain in a sterile environment, streaking the strain in an LB solid culture medium, and culturing for 24 hours at 37 ℃ to obtain a single colony, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar and 1000mL of deionized water, and the pH is natural.
Inoculating the obtained single colony in LB liquid culture medium, culturing at 37 deg.C to OD at 170r/min in 40% liquid loading600And (3) obtaining a seed liquid, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, and the pH value is natural.
Inoculating the seed liquid into an LB liquid culture medium (original LB) with the inoculation amount of 1%, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, the fermentation condition is that the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 h. The viable bacteria content of the fermentation liquor after the culture is finished is 1 multiplied by 107CFU/mL。
Inoculating the seed liquid into a fermentation culture medium (optimal formula) and an LB liquid culture medium (optimal LB) according to the inoculation amount of 2%, wherein the fermentation culture medium comprises the following components: 1.5 percent of soluble starch, 2.0 percent of snowflake powder, 2.5 percent of fish meal, 0.5 percent of acid hydrolysis amino acid (stock solution), 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the LB liquid culture medium is 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride. The fermentation conditions were: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH value is 7.0, and the time is 12 h. After the culture is finished, the content of viable bacteria in the fermentation medium (optimal formula) is 10.1 multiplied by 107CFU/mL, LB liquid medium (excellent LB) viable bacteria content is 8.89X 107CFU/mL。
Example 7 comparison of Acinetobacter strain Y40 fermented by different fermentation Processes
An acinetobacter identified and stored in a high-tech research key laboratory by resource utilization of solid organic wastes of Jiangsu province of Nanjing agriculture university, which is classified and named as: acinetobacter (Acinetobacter) Y40, see issued patent: an acinetobacter strain of phosphate-solubilizing growth-promoting bacteria Y40 and its application (grant No.: ZL201510399215. x). The method comprises the steps of inoculating a Y40 preserved strain to an acinetobacter Y40 strain in a sterile environment, streaking the strain in an LB solid culture medium, and culturing the strain at 37 ℃ for 24 hours to obtain a single colony, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar and 1000mL of deionized water, and the pH is natural.
Inoculating the obtained single colony in LB liquid culture medium, culturing at 37 deg.C to OD at 170r/min in 40% liquid loading600And (3) obtaining a seed liquid, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, and the pH value is natural.
Inoculating the seed liquid into an LB liquid culture medium (original LB) with the inoculation amount of 1%, wherein the LB liquid culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride, the fermentation condition is that the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 h. The viable bacteria content of the fermentation liquor after the culture is finished is 124 multiplied by 107CFU/mL。
Inoculating the seed liquid into a fermentation culture medium (optimal formula) and an LB liquid culture medium (optimal LB) according to the inoculation amount of 2%, wherein the fermentation culture medium comprises the following components: 1.5 percent of soluble starch, 2.0 percent of snowflake powder, 2.5 percent of fish meal, 0.5 percent of acid hydrolysis amino acid (stock solution), 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the LB liquid culture medium is 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride. The fermentation conditions were: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH value is 7.0, and the time is 12 h. After the culture is finished, the content of viable bacteria in the fermentation medium (optimal formula) is 458.3 multiplied by 107CFU/mL, LB liquid medium (excellent LB) viable bacteria content of 169.3X 107CFU/mL。
Claims (10)
1. A bacterial high density liquid fermentation medium, characterized in that said fermentation medium comprises:
1) carbon source: 1 to 1.5 percent of soluble starch, 0.3 to 2.0 percent of snowflake powder and 0 to 3.5 percent of corn flour;
2) nitrogen source: 2.0 to 2.8 percent of fish meal and 0.2 to 2 percent of acid hydrolysis amino acid;
3) the inorganic salt additive comprises: 0.1 to 0.3 percent of sodium chloride, 0.02 to 0.05 percent of monopotassium phosphate, 0.1 to 0.4 percent of dipotassium phosphate and 0.01 to 0.03 percent of magnesium sulfate heptahydrate;
the balance of water, wherein the percent is the mass volume percentage and represents g/100 mL.
2. The bacterial high density liquid fermentation medium of claim 1, characterized in that the fermentation medium composition comprises: 1 to 1.5 percent of soluble starch, 0.3 to 0.5 percent of snowflake powder, 3 to 3.5 percent of corn flour, 2 to 2.5 percent of fish meal, 1.5 to 2 percent of acid hydrolysis amino acid, 0.1 percent of sodium chloride, 0.05 percent of potassium dihydrogen phosphate, 0.15 percent of dipotassium hydrogen phosphate and 0.02 percent of magnesium sulfate heptahydrate; the balance being water.
3. The bacterial high-density liquid fermentation medium of claim 2, wherein said fermentation medium consists of: 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn flour, 2.4% of fish meal, 1.6% of acid hydrolysis amino acid, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.15% of dipotassium hydrogen phosphate and 0.02% of magnesium sulfate heptahydrate; the balance being water.
4. The bacterial high-density liquid fermentation medium of claim 1, wherein the fermentation medium consists of: 1.5 percent of soluble starch, 2.0 percent of snowflake powder, 2.5 percent of fish meal, 0.5 percent of acid hydrolysis amino acid, 0.1 percent of sodium chloride, 0.05 percent of monopotassium phosphate, 0.15 percent of dipotassium phosphate and 0.02 percent of magnesium sulfate heptahydrate; the balance being water.
5. Use of the bacterial high density liquid fermentation medium of claim 4 in bacterial high density fermentation.
6. Use according to claim 5, characterized in that said bacteria are selected from the group consisting of bacteria of the genus Bacillus, bacteria of the genus Pseudomonas, bacteria of the genus Acinetobacter.
7. The use according to claim 6, wherein the bacteria are selected from the group consisting of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SQR9 with the collection number CGMCC No.5808, Acinetobacter (Acinetobacter) Y40 with the collection number CGMCC No.10241, Bacillus (Bacillus) W19, and Pseudomonas (Pseudomonas) PSE 13.
8. A method for bacterial high-density liquid fermentation, characterized in that bacterial seed liquid is inoculated into a bacterial high-density liquid fermentation medium of any one of claims 1 to 4 for fermentation; the fermentation conditions are that the temperature is 25-41 ℃, the inoculum size is 1-5%, the rotating speed is 150-.
9. The method according to claim 8, wherein the fermentation conditions are 37 ℃ temperature, 2% inoculum size, 190r/min rotation speed, pH7.0, 40% liquid loading and 12h time.
10. The method of claim 9, wherein the viable bacteria content of the fermentation broth is 1 x 10 or more9The culture was terminated at CFU/mL.
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| CN109306330A (en) * | 2018-07-09 | 2019-02-05 | 东北农业大学 | The fermentation culture method of biocontrol bacteria strain bacillus amyloliquefaciens |
| CN109852562A (en) * | 2019-02-25 | 2019-06-07 | 山东京青农业科技有限公司 | A method of with water-soluble raw material culture bacillus amyloliquefaciens |
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| CN103614323A (en) * | 2013-11-27 | 2014-03-05 | 江苏苏滨生物农化有限公司 | Culture medium of bacillus amyloliquefaciens and application |
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| CN103804032A (en) * | 2014-01-28 | 2014-05-21 | 南京农业大学 | Liquid amino acid compound produced by dead porcine proteins and application thereof |
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