CN113293119B - Bacterial high-density liquid fermentation medium and fermentation method thereof - Google Patents
Bacterial high-density liquid fermentation medium and fermentation method thereof Download PDFInfo
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Abstract
The invention discloses a bacterial high-density liquid fermentation medium and a fermentation method thereof. A bacterial high density liquid fermentation medium, the fermentation medium comprising: 1) Carbon source: 1-1.5% of soluble starch, 0.3-2.0% of snowflake powder and 0-3.5% of corn powder; 2) Nitrogen source: 2.0 to 2.8 percent of fish meal and 0.2 to 2 percent of acidolysis amino acid; 3) The inorganic salt additive includes: sodium chloride 0.1-0.3%, potassium dihydrogen phosphate 0.02-0.05%, dipotassium hydrogen phosphate 0.1-0.4%, magnesium sulfate heptahydrate 0.01-0.03%; the balance being water. The fermentation conditions are as follows: the inoculation amount is 2%, the liquid amount in the triangular flask is 40%, the temperature is 37 ℃, the rotating speed is 190r/min, the pH is 7.0, and the fermentation time is 12h. The content of the viable bacteria of the bacillus amyloliquefaciens SQR9 in the fermentation liquid is more than or equal to 1 multiplied by 10 9 CFU/mL。
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and particularly relates to a bacterial high-density liquid fermentation medium and a fermentation method thereof.
Background
China is a large agricultural country with a large population, and agricultural production occupies a significant position in national economy. The wide use of fertilizers has improved crop yield and quality since the 19 th century, bringing great economic benefits to agricultural production. At present, the yield and the usage amount of chemical fertilizers in China stably stay in the first place in the world, and the use of a large amount of chemical fertilizers can improve the agricultural production efficiency, but also make the land pollution and the environmental deterioration increasingly serious, which is a key factor for preventing the sustainable development of modern agriculture in the long term.
Microbial fertilizers have gradually entered the public as high-yield and efficient healthy fertilizers. At present, about one hundred thousand tons of microbial fertilizer are put into agricultural production in China every year, and the microbial fertilizer mainly comprises three types of microbial agents, biological organic fertilizers and compound microbial fertilizers. Microbial fertilizers can produce friendly effects on the environment, and mainly comprise the following two aspects: first, inhibit the growth and reproduction of pathogenic microorganisms. When the microbial fertilizer is applied, beneficial microorganisms can form dominant bacterial groups on plant rhizosphere, and occupy favorable space sites through antagonism with pathogenic bacteria, so that proliferation of the pathogenic bacteria is inhibited; secondly, the soil is improved. The traditional fertilizer is easy to cause the residue of nitrogen, phosphorus, potassium and other elements, so that the soil is hardened and the fertility is reduced, and the microbial fertilizer can release indissolvable phosphate and other trace elements in the soil, so that the element utilization rate is improved, and the soil performance is improved.
The bacillus amyloliquefaciens is a gram positive bacterium, and the bacillus is widely existing in soil, air, water and animals and plants, and has 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 agricultural department and used for agricultural production. The SQR9 strain is a bacillus amyloliquefaciens identified and stored in a key laboratory by recycling solid organic wastes in Jiangsu province of Nanjing agricultural university, and can effectively promote plant growth and prevent and control soil-borne diseases through experiments, and is a beneficial bacterium capable of being produced and popularized in a large scale. At present, materials with expensive price such as yeast powder, peptone and beef extract are mainly used for culturing bacillus amyloliquefaciens, the production cost is higher, the fermentation density is lower, and the production and the application of related products are not facilitated, so that materials with low price and sufficient nutrient content are searched for fermentation production, the number of functional bacteria in a fermentation product is continuously improved, and the method is a research hot spot at present.
Disclosure of Invention
The invention aims to solve the problems of high price of raw materials of a bacterial high-density fermentation medium, low density content of thalli in the obtained fermentation broth and the like in the prior art, and provides a bacterial high-density liquid fermentation medium with low cost.
It is another object of the present invention to provide a bacterial high density liquid fermentation process.
The aim of the invention can be achieved by the following technical scheme:
a bacterial high density liquid fermentation medium, the fermentation medium comprising:
1) Carbon source: 1-1.5% of soluble starch, 0.3-2.0% of snowflake powder and 0-3.5% of corn powder;
2) Nitrogen source: 2.0 to 2.8 percent of fish meal and 0.2 to 2 percent of acidolysis amino acid;
3) The inorganic salt additive includes: sodium chloride 0.1-0.3%, potassium dihydrogen phosphate 0.02-0.05%, dipotassium hydrogen phosphate 0.1-0.4%, magnesium sulfate heptahydrate 0.01-0.03%;
the balance being water, the above% being mass volume percent representing g/100mL.
As a preferred aspect of the present invention, the fermentation medium composition includes: 1 to 1.5 percent of soluble starch, 0.3 to 0.5 percent of snowflake powder, 3 to 3.5 percent of corn powder, 2 to 2.5 percent of fish meal, 1.5 to 2 percent of acidolysis 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.
As a further preferred aspect of the present invention, the fermentation medium composition includes: 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn powder, 2.4% of fish meal, 1.6% of acidolysis amino acid, 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the balance being water.
As another preferred aspect of the present invention, the fermentation medium composition includes: 1.5% of soluble starch, 2.0% of snowflake powder, 2.5% of fish meal, 0.5% of acidolysis amino acid (stock solution), 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the balance being water.
The content of free amino acid in acidolysis amino acid (stock solution) is 15%, and the concentration of free hydrogen ions is 3M.
The preparation method of the acidolysis amino acid (stock solution) is disclosed in the patent of the invention which is previously authorized by the applicant: a liquid amino acid compound produced by using pig protein died of illness and application thereof (ZL 201410042218.3), wherein the preparation method of acidolysis amino acid (stock solution) comprises the following steps: is prepared by acidolysis of lean meat with sulfuric acid; lean meat of pig, cattle and sheep is preferred; the acidolysis is to hydrolyze lean meat for 5-7 hours at 80-90 ℃ by using sulfuric acid with the concentration of 5-7 mol/L, 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, lean meat is hydrolyzed at 80 ℃ for 6 hours by using sulfuric acid with the concentration of 5mol/L, and the material ratio of lean meat to the 5mol/L sulfuric acid is 1:1.5.
A method for fermenting bacteria in a high-density liquid comprises the steps of inoculating bacterial seed liquid into a bacterial high-density liquid fermentation medium for fermentation; the fermentation condition is that the temperature is 25-41 ℃, the inoculation amount is 1-5%, the rotating speed is 150-230r/min, the pH is 5.5-8.0, the liquid loading amount is 10-50%, and the time is 9-28h.
The fermentation condition is preferably at 37 ℃, the inoculation amount is 2%, the rotating speed is 190r/min, the pH value is 7.0, the liquid loading amount is 40%, and the time is 12h.
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 bacteria, pseudomonas bacteria, acinetobacter bacteria.
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 10 9 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 and nitrogen sources, shortens the fermentation period by further optimizing the components of the culture medium and the fermentation conditions, and ensures that the viable bacteria content in the final fermentation liquid reaches 130.3 multiplied by 10 7 CFU/mL, effectively solved the biomass that exists in the traditional fermentation process and low, fermentation cycle is long, manufacturing cost is high grade shortcoming, is convenient for relevant product processing, is favorable to extensive popularization and application. The bacillus amyloliquefaciens SQR9 microbial inoculum can be used as a biological fertilizer to be applied to agricultural production. The pesticide is safe and nontoxic, has obvious biocontrol effect, and can achieve the purposes of preventing and controlling soil-borne diseases, promoting crop growth and improving yield and quality by being singly applied or being matched with other medicaments.
Drawings
FIG. 1 Effect of different fermentation conditions on the number of liquid fermentations of SQR9 Strain
FIG. 2 optimized 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 solution
(1) Bacillus amyloliquefaciens SQR9 is inoculated on an LB solid culture medium, and is cultured for 24 hours at 37 ℃ to obtain single colonies, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar, 1000mL of deionized water and natural pH.
(2) Inoculating the single colony obtained in the step (1) into LB liquid culture medium, and culturing at 37 ℃ and 40% of liquid loading amount and 170r/min until OD 600 =0.7, giving seed solution; the LB liquid medium comprises 10g/L peptone, 5g/L yeast powder, 5g/L sodium chloride, 1000mL deionized water and has natural pH. 1.2 screening of carbon sources
Fixing 0.2% ammonium sulfate as nitrogen source, and adding inorganic salt additive. The following gradient composition fermentation media are respectively arranged for different types of carbon sources to be tested: soluble starch 0.50%, 0.70%, 1.00%, 1.30%, 1.50%; sucrose 0.40%, 0.70%, 1.00%, 1.30%, 1.60%; 0.50%, 0.70%, 1.00%, 1.30%, 1.60% of snow pollen; corn flour 1.50%, 2.00%, 2.50%, 3.00%, 3.50%; lactose 0.40%, 0.70%, 1.00%, 1.30%, 1.60%; glucose 0.40%, 0.70%, 1.00%, 1.30%, 1.60%; 0.02%, 0.04%, 0.06%, 0.08%, 0.10% citric acid monohydrate, 0.10%, 0.50%, 1.00%, 1.50%, 2.00% beef extract powder of a composite carbon and nitrogen source; peptone 0.50%, 1.00%, 1.50%, 2.00%, 2.50%; the balance being water. Inoculating the seed solution of 1.1 into fermentation medium at 1% inoculation amount, and fermenting at rotation speed of 170r/min, temperature of 37deg.C, liquid loading amount of 40% and pH7.0 for 24 hr to obtain fermentation broth. The results show that the new formulation composed of soluble starch and snow pollen as carbon sources is most suitable for SQR9 strain growth, and the effects of corn flour, beef extract and peptone are inferior. And finally, selecting soluble starch, snow pollen and corn powder as raw materials of the composite carbon source by combining factors such as production cost and the like.
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 Effect of different types and concentrations of carbon sources on the effective viable count in SQR9 fermentation broths
1.3 Nitrogen Source screening
Fixing 1% sucrose as carbon source, and adding inorganic salt additive. The following gradients are respectively arranged on different types of nitrogen sources to be tested to form a fermentation culture medium: 1.00%, 1.50%, 2.00%, 2.50% and 3.00% of fish meal; meat and bone powder 1.00%, 1.50%, 2.00%, 2.50%, 3.00%; ammonium chloride 0.20%, 0.50%, 0.70%, 1.00%, 1.20%; urea 0.20%, 0.50%, 0.70%, 1.00%, 1.20%; ammonium sulfate 0.20%, 0.50%, 0.70%, 1.00%, 1.20%; acidolysis amino acid 0.2%, 0.5%, 1.0%, 1.5%, 2.0% and composite carbon nitrogen source beef extract 0.10%, 0.50%, 1.00%, 1.50%, 2.00%; 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 1% inoculation amount, and fermenting at rotation speed of 170r/min, temperature of 37deg.C, liquid loading amount of 40% and pH7.0 for 24 hr to obtain fermentation broth. The results showed that peptone and beef extract had the best culture effect, and acidolysis of amino acids and fish meal were inferior. And (3) combining factors such as production cost and the like, and finally selecting fish meal and acidolysis 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 Effect of different species and concentrations of Nitrogen sources on the effective viable bacteria content in SQR9 fermentation broths
1.3 Complex carbon Nitrogen Source
The 3 carbon sources and 2 nitrogen sources are combined into a composite carbon-nitrogen source, four horizontal gradients are set, a 5-factor 4-level orthogonal test table is generated, 16 test combinations are formed, and each test is repeated for 3 times. The result shows that the test effect of the No. 9 formula is most obvious, and the highest viable count reaches 52.2 multiplied by 10 7 CFU/mL. The No. 9 test culture medium comprises the following components: 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn powder, 2.4% of fish meal, 1.6% of acidolysis amino acid, 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate. The fermentation conditions are as follows: the inoculation amount is 1%, the temperature is 37 ℃, the rotating speed is 170r/min, the pH is 7.0, the liquid loading amount is 40%, and the time is 24h.
Table 3L 16 (4 5 ) Level of orthogonal test factors
Table 4L 16 (4 5 ) Orthogonal test design and result analysis
Example 2 fermentation condition optimization
Based on the fermentation medium, the following gradients are set for different fermentation conditions: 1%, 2%, 3%, 4%, 5% of inoculum size; 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 230r/min; the temperature was 25 ℃, 29 ℃, 33 ℃,37 ℃, 41 ℃; the liquid loading amount is 10%, 20%, 30%, 40% and 50%. The results show that the inoculation amount is 2%, the pH7.0, the liquid loading amount is 40%, the rotating speed is 190r/min, the effect is best, and the viable bacteria content is 97 multiplied by 10 7 CFU/mL、113.78×10 7 CFU/mL、78×10 7 CFU/mL、97×10 7 CFU/mL and 95.78X10 7 CFU/mL. Under the optimal conditions, the time gradients are set to be 9h, 12h, 16h, 20h, 24h and 28h, and the result shows that the content of viable bacteria is highest in 12h and reaches 94.78 multiplied by 10 7 CFU/mL. The fermentation medium comprises 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn powder, 2.4% of fish meal, 1.6% of acidolysis amino acid, 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate.
Example 3 further optimization of fermentation Medium
In order to further save the production cost and reduce the solid matter content in the fermentation broth, the fermentation medium is now optimized again. The acidolysis amino acid (stock solution) with lower price is used for replacing the acidolysis amino acid, and 2 carbon sources and 2 nitrogen sources are respectively provided with the following concentration gradients: 0.5%, 1.0% and 1.5% of soluble starch; 0.7 percent of snow pollen, 1.3 percent of snow pollen and 2.0 percent of snow pollen; 2.0 percent of fish meal, 2.5 percent of fish meal and 3.0 percent of fish meal; acidolysis amino acid (stock solution) 0.2%, 0.5% and 1.0%. Generating orthogonal test table of 4 factors and 3 levels, 9 test combinations, test results and numbersAnalysis shows that the effect is best when the soluble starch is 1.5%, the snowflake powder is 2.0%, the fish meal is 2.5%, and the acidolysis amino acid (stock solution) is 0.5%, and the content of viable bacteria can reach 130.3 multiplied by 10 at most 7 CFU/mL。
Table 5L 9 (3 4 ) Level of orthogonal test factors
Table 6L 9 (3 4 ) Orthogonal test design and result analysis
Example 4 comparison of different fermentation Processes fermented SQR9 Strain
SQR9 is used as a strain, SQR9 preserved strains are inoculated in a sterile environment, streaked in LB solid culture medium, and cultured for 24 hours at 37 ℃ to obtain single bacterial colonies, wherein the LB solid culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2% of agar, 1000mL of deionized water and natural pH.
Inoculating the obtained single colony into LB liquid medium, culturing at 37r/min, 40% liquid loading, and culturing at 37deg.C to OD 600 And (2) obtaining seed liquid, wherein the composition of the LB liquid culture medium is 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride and natural pH.
The seed liquid is inoculated into LB liquid culture medium (original LB) with 1 percent of inoculation amount, wherein the LB liquid culture medium consists of 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 percent, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 hours. The viable bacteria content of the fermentation broth after the culture is 8.67 multiplied by 10 7 CFU/mL。
Inoculating 2% seed solution into fermentation medium (optimal formula) and LB solutionIn the body culture medium (excellent LB), the fermentation medium composition comprises: 1.5% of soluble starch, 2.0% of snowflake powder, 2.5% of fish meal, 0.5% of acidolysis amino acid (stock solution), 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the LB liquid medium comprises 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride. The fermentation conditions are as follows: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH is 7.0, and the time is 12h. After the cultivation is finished, the viable bacteria content in the fermentation medium (optimal formula) is 130.3X10 7 CFU/mL, LB liquid medium (excellent LB) viable bacteria content of 17×10 7 CFU/mL。
Example 5 comparison of Bacillus fermenticus W19 Strain from different fermentation Processes
A bacillus strain identified and stored by a high-technology research key laboratory for recycling solid organic wastes in Jiangsu province of Nanjing agricultural university is named as follows: bacillus W19, see published article Effects of novel bioorganic fertilizer produced by Bacillus amyloliquefaciens W19 on antagonism of Fusarium wilt of banana. Biology and Fertility of Soils,2013,49,435-446. Inoculating a W19 preservation strain by using a bacillus W19 strain in a sterile environment, streaking 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, 1000mL of deionized water and natural pH.
Inoculating the obtained single colony into LB liquid medium, culturing at 37r/min, 40% liquid loading, and culturing at 37deg.C to OD 600 And (2) obtaining seed liquid, wherein the composition of the LB liquid culture medium is 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride and natural pH.
The seed liquid is inoculated into LB liquid culture medium (original LB) with 1 percent of inoculation amount, wherein the LB liquid culture medium consists of 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 percent, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 hours. The living bacteria content of the fermentation broth after the culture is 31.67 multiplied by 10 7 CFU/mL。
Inoculating 2% seed solution into fermentationIn the culture medium (optimal formula) and the LB liquid culture medium (optimal LB), the fermentation culture medium comprises the following components: 1.5% of soluble starch, 2.0% of snowflake powder, 2.5% of fish meal, 0.5% of acidolysis amino acid (stock solution), 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the LB liquid medium comprises 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride. The fermentation conditions are as follows: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH is 7.0, and the time is 12h. After the cultivation is finished, the viable bacteria content in the fermentation medium (optimal formula) is 100 multiplied by 10 7 CFU/mL, LB liquid medium (excellent LB) viable bacteria content of 41×10 7 CFU/mL。
Example 6 comparison of Pseudomonas fermentum PSE13 Strain from different fermentation Processes
A Pseudomonas strain identified and stored by a high-technology research key laboratory for recycling solid organic wastes in Jiangsu province of Nanjing agricultural university is named as follows: pseudomonas (Pseudomonas) PSE13, see published paper Bio-organic fertilizers stimulate indigenous soil Pseudomonas populations to enhance plant disease support. Microbiol, 2020,8:137. Pseudomonas PSE13 is used as a strain, a PSE13 preservation strain is inoculated in a sterile environment, streaked in an LB solid culture medium, and cultured 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, 1000mL of deionized water and natural pH.
Inoculating the obtained single colony into LB liquid medium, culturing at 37r/min, 40% liquid loading, and culturing at 37deg.C to OD 600 And (2) obtaining seed liquid, wherein the composition of the LB liquid culture medium is 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride and natural pH.
The seed liquid is inoculated into LB liquid culture medium (original LB) with 1 percent of inoculation amount, wherein the LB liquid culture medium consists of 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 percent, the rotating speed is 170r/min, the pH value is 7.0, and the time is 24 hours. The living bacteria content of the fermentation broth after the culture is 1 multiplied by 10 7 CFU/mL。
Inoculating 2% seed solution into fermentation cultureIn a culture medium (optimal formula) and an LB liquid culture medium (optimal LB), the fermentation culture medium comprises the following components: 1.5% of soluble starch, 2.0% of snowflake powder, 2.5% of fish meal, 0.5% of acidolysis amino acid (stock solution), 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the LB liquid medium is peptone 10g/L, yeast powder 5g/L and sodium chloride 5g/L. The fermentation conditions are as follows: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH is 7.0, and the time is 12h. After the cultivation is finished, the viable bacteria content in the fermentation medium (optimal formula) is 10.1X10% 7 CFU/mL, LB liquid medium (excellent LB) viable bacteria content of 8.89×10 7 CFU/mL。
EXAMPLE 7 comparison of Acinetobacter fermentum Y40 Strain with different fermentation Processes
The Acinetobacter strain identified and stored by a key laboratory of high-technology research on recycling utilization of solid organic wastes in Jiangsu province of Nanjing agricultural university is named as follows in taxonomy: acinetobacter (Acinetobacter) Y40, see issued patent: acinetobacter phosphate-solubilizing and growth-promoting bacteria Y40 and application thereof (authorized number: ZL2015199215. X). Inoculating a Y40 preservation strain with an Acinetobacter Y40 strain in a sterile environment, streaking 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, 1000mL of deionized water and natural pH.
Inoculating the obtained single colony into LB liquid medium, culturing at 37r/min, 40% liquid loading, and culturing at 37deg.C to OD 600 And (2) obtaining seed liquid, wherein the composition of the LB liquid culture medium is 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride and natural pH.
The seed liquid is inoculated into an LB liquid culture medium (original LB) with the inoculation amount of 1%, wherein the composition of the LB liquid culture medium is 10g/L peptone, 5g/L yeast powder and 5g/L 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 is 7.0, and the time is 24 hours. The viable bacteria content of the fermentation broth after the culture is 124 multiplied by 10 7 CFU/mL。
Inoculating the seed solution into fermentation medium (optimal formula) and LB liquid medium (optimal LB) at an inoculum size of 2%, wherein the fermentation mediumThe nutrient medium comprises the following components: 1.5% of soluble starch, 2.0% of snowflake powder, 2.5% of fish meal, 0.5% of acidolysis amino acid (stock solution), 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the LB liquid medium is peptone 10g/L, yeast powder 5g/L and sodium chloride 5g/L. The fermentation conditions are as follows: the temperature is 37 ℃, the liquid loading amount is 40%, the rotating speed is 190r/min, the pH is 7.0, and the time is 12h. After the cultivation is finished, the viable bacteria content in the fermentation medium (optimal formula) is 458.3X10 7 CFU/mL, LB liquid medium (excellent LB) viable bacteria content is 169.3X10 7 CFU/mL。
Claims (9)
1. A bacterial high-density liquid fermentation medium, characterized in that the fermentation medium comprises:
1) Carbon source: 1-1.5% of soluble starch, 0.3-0.5% of snowflake powder and 3-3.5% of corn powder;
2) Nitrogen source: 2-2.5% of fish meal and 1.5-2% of acidolysis amino acid, wherein the acidolysis amino acid is prepared by acidolysis of lean meat with sulfuric acid; the acidolysis is to hydrolyze lean meat for 5-7 hours at 80-90 ℃ by using sulfuric acid with the concentration of 5-7 mol/L, 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;
3) The inorganic salt additive includes: sodium chloride 0.1%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.15%, magnesium sulfate heptahydrate 0.02%;
the balance being water, the above% being mass volume percent representing g/100mL.
2. Bacterial high-density liquid fermentation medium according to claim 1, characterized in that the fermentation medium composition comprises: 1% of soluble starch, 0.3% of snowflake powder, 3.2% of corn powder, 2.4% of fish meal, 1.6% of acidolysis amino acid, 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the balance being water.
3. Bacterial high-density liquid fermentation medium according to claim 1, characterized in that the fermentation medium composition comprises: 1.5% of soluble starch, 2.0% of snowflake powder, 2.5% of fish meal, 0.5% of acidolysis amino acid, 0.1% of sodium chloride, 0.05% of monopotassium phosphate, 0.15% of dipotassium phosphate and 0.02% of magnesium sulfate heptahydrate; the balance being water.
4. Use of the bacterial high-density liquid fermentation medium of any one of claims 1-3 in bacterial high-density fermentation.
5. The method according to claim 4, wherein the bacteria are selected from the group consisting of Bacillus bacteria, pseudomonas bacteria, acinetobacter bacteria.
6. The use according to claim 4, wherein the bacterium is selected from the group consisting of Bacillus amyloliquefaciens with a preservation number of CGMCC No.5808Bacillus amyloliquefaciens) SQR9 and bacillusBacillus) W19 and PseudomonasPseudomonas) PSE13 or AcinetobacterAcinetobacter）Y40。
7. A method of high-density liquid fermentation of bacteria, characterized in that bacterial seed liquid is inoculated into the bacterial high-density liquid fermentation medium of any one of claims 1 to 3 for fermentation; the fermentation condition is that the temperature is 35-37 ℃, the inoculation amount is 4-5%, the rotating speed is 170-190 r/min, the pH is 7.0, the liquid loading amount is 40% of the liquid loading amount of the triangular flask, and the time is 12-15h.
8. The method according to claim 7, wherein the fermentation condition is 37 ℃ temperature, inoculum size 4%, rotational speed 170r/min, pH7.0, liquid loading in triangular flask 40% and time 12h.
9. The method according to claim 7 or 8, wherein the bacterium is selected from the group consisting of Bacillus amyloliquefaciens with a preservation number of CGMCC No.5808Bacillus amyloliquefaciens) SQR9 and bacillusBacillus) W19 and PseudomonasPseudomonas) PSE13 or AcinetobacterAcinetobacter）Y40。
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| CN103642729A (en) * | 2013-12-03 | 2014-03-19 | 湖北工业大学 | Method for producing Bacillus subtilis for feeds by fermenting high-salt-content amino acid wastewater |
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| 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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