CN116426580A - Agricultural polyglutamic acid fermentation broth and preparation method and application thereof - Google Patents
Agricultural polyglutamic acid fermentation broth and preparation method and application thereof Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 131
- 230000004151 fermentation Effects 0.000 title claims abstract description 131
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 67
- 108010020346 Polyglutamic Acid Proteins 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 66
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 32
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 31
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 31
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 27
- 239000004220 glutamic acid Substances 0.000 claims abstract description 27
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 235000013379 molasses Nutrition 0.000 claims description 36
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 14
- 239000001110 calcium chloride Substances 0.000 claims description 14
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 12
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 12
- 235000005822 corn Nutrition 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 8
- 239000003337 fertilizer Substances 0.000 claims description 4
- 241000209149 Zea Species 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 abstract description 11
- 235000012907 honey Nutrition 0.000 abstract description 9
- 238000000746 purification Methods 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 2
- 235000010633 broth Nutrition 0.000 description 32
- 239000002609 medium Substances 0.000 description 15
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- 238000011218 seed culture Methods 0.000 description 5
- 240000008067 Cucumis sativus Species 0.000 description 4
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000012807 shake-flask culturing Methods 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000005416 organic matter Substances 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
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- 235000019319 peptone Nutrition 0.000 description 2
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- 241000282414 Homo sapiens Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05C—NITROGENOUS FERTILISERS
- C05C11/00—Other nitrogenous fertilisers
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/10—Bacillus licheniformis
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Abstract
The invention belongs to the technical field of microorganisms, and discloses an agricultural polyglutamic acid fermentation broth, a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) preparation of mixed seed liquid: mixing bacillus natto seed solution and bacillus licheniformis seed solution to obtain mixed seed solution; (2) fermentation: inoculating the mixed seed liquid into a fermentation medium for fermentation culture to obtain the agricultural polyglutamic acid fermentation broth. According to the invention, bacillus natto FK001 and bacillus licheniformis FK002 are used as fermentation strains, free glutamic acid in the concentrated honey fermentation liquid can be polymerized into low-molecular-weight polyglutamic acid, the prepared fermentation liquid has high polyglutamic acid content, no purification is needed, and the concentrated honey fermentation liquid can be directly used and has low production cost.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to an agricultural polyglutamic acid fermentation broth, and a preparation method and application thereof.
Background
Gamma-polyglutamic acid (gamma-PGA for short) is a biopolymer material. The water-soluble and biodegradable food has the advantages of good water solubility, biodegradability, edibility, no toxicity to human beings and the environment, and the like, so that the water-soluble and biodegradable food has wide application prospect in the fields of environment, medicine, food, cosmetics, and the like.
The polyglutamic acid can be prepared by a plurality of methods including chemical synthesis methods, extraction methods, microbiological synthesis methods and the like. The microbial synthesis method has become the main method and the way for producing the gamma-polyglutamic acid at present because the production process is easy to control, the fermentation yield is stable, the extraction rate is high, the large-scale production is convenient to realize and the like.
CN107760732a discloses a production method of agricultural grade gamma-polyglutamic acid, comprising the following steps: activating strains: inoculating an original strain of bacillus licheniformis on a slant culture medium for culture and activation; preparing seed liquid; inoculating the activated slant seeds into a seed culture medium to obtain seed liquid of gamma-polyglutamic acid; inoculating the seed liquid into a fermentation medium for fermentation; and (3) after fermentation, removing thalli by centrifugation, precipitating polyglutamic acid by alcohol, and drying to prepare yellow dry powder agricultural polyglutamic acid.
CN110042129a discloses a preparation method of gamma-polyglutamic acid for agriculture, which comprises the following steps: (1) microbial fermentation: transferring the strain into a fermentation tank, maintaining the fermentation temperature at 35-45 ℃, and the ventilation rate at 1.0-2.0vvm, stirring at 150-250rpm, fermenting at pH of 6.5-7.0, and fermenting for 48-60h to obtain fermentation liquor; (2) membrane filtration: adding 2-4 times of volume of water into the obtained fermentation liquor, uniformly mixing, and filtering to obtain concentrated solution; (3) spray drying: the resulting concentrate was heated and spray dried.
However, the production cost of polyglutamic acid is relatively high, which results in relatively high price, and limits the application of polyglutamic acid in agriculture. How to reduce the production cost of polyglutamic acid, so that the polyglutamic acid can be better applied to agricultural production, and has important significance.
Disclosure of Invention
In order to solve the technical problems, the invention provides an agricultural polyglutamic acid fermentation broth, a preparation method and application thereof, wherein concentrated molasses fermentation broth is used as a main raw material, bacillus natto FK001 and bacillus licheniformis FK002 are utilized for fermentation, free glutamic acid in the concentrated molasses fermentation broth is polymerized into low-molecular polyglutamic acid, the obtained fermentation broth has high polyglutamic acid content, can be directly used without purification, reduces production cost, and meets the agricultural market demand.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the preparation method of the agricultural polyglutamic acid fermentation broth comprises the following steps:
(1) Preparing mixed seed liquid: mixing bacillus natto seed solution and bacillus licheniformis seed solution to obtain mixed seed solution;
(2) Fermentation: inoculating the mixed seed liquid into a fermentation medium for fermentation culture to obtain the agricultural polyglutamic acid fermentation broth.
Preferably, the bacillus natto in the bacillus natto seed solution in the step (1) is bacillus natto FK001; the bacillus licheniformis in the bacillus licheniformis seed solution is bacillus licheniformis FK002.
Preferably, the concentration of the Bacillus natto seed solution and the Bacillus licheniformis seed solution in the step (1) is 1×10 7 CFU/ml。
Preferably, in the step (1), the volume ratio of the bacillus natto seed solution to the bacillus licheniformis seed solution is 3-8:1; further preferably 4-7:1; most preferably 5:1.
Preferably, the mixed seed solution in step (2) is inoculated in an amount of 8-25%.
Preferably, the fermentation medium in step (2) is: 10-30g/L of molasses, 100-250g/L of concentrated molasses fermentation liquid, 15-25g/L of glutamic acid, 25-50g/L of corn steep liquor and 1-5g/L of calcium chloride, and the pH value is 6.0-7.0.
Further preferably, the fermentation medium in step (2) is: 12-25g/L of molasses, 120-240g/L of concentrated molasses fermentation liquid, 18-22g/L of glutamic acid, 30-45g/L of corn steep liquor and 1.5-4.5g/L of calcium chloride, and the pH value is 6.0-7.0.
Most preferably, the fermentation medium in step (2) is: 20g/L of molasses, 200g/L of concentrated molasses fermentation liquid, 20g/L of glutamic acid, 35g/L of corn steep liquor and 2.5g/L of calcium chloride, and the pH value is 6.0-7.0.
Preferably, the conditions of the fermentation culture in step (2) are: the temperature is 32-40 ℃, the rotating speed is 160-280rpm, and the culture time is 20-36h.
Further preferably, the conditions of the fermentation culture in step (2) are: the temperature is 34-38 ℃, the rotating speed is 170-200rpm, and the culture time is 25-30h.
Most preferably, the conditions of the fermentation culture in step (2) are: the temperature is 35 ℃, the rotating speed is 180rpm, and the culture time is 25 hours.
According to the invention, bacillus natto FK001 and bacillus licheniformis FK002 are used as fermentation strains, free glutamic acid in the concentrated honey fermentation liquid can be polymerized into low-molecular-weight polyglutamic acid, the prepared fermentation liquid has high polyglutamic acid content (35-51.4 g/L), purification is not needed, and the concentrated honey fermentation liquid can be directly used, so that the agricultural production requirement is met.
The concentrated honey fermentation liquor is a concentrated molasses fermentation liquor of Weidan, is a byproduct of preparing glutamic acid by biological fermentation, and contains glutamic acid and a sugar source which is not digested and utilized by a strain; the concrete components are as follows: the content of free glutamic acid is 25g/L, the nitrogen and potassium content is more than or equal to 70g/L (40-0-30), and the organic matter content is more than or equal to 300g/L. The invention adopts the concentrated molasses fermentation liquid to replace part of glutamic acid and carbon source molasses, so that the production cost can be reduced.
The invention also provides the agricultural polyglutamic acid fermentation broth prepared by the preparation method.
The invention also provides application of the agricultural polyglutamic acid fermentation broth prepared by the preparation method in preparation of liquid fertilizer.
The beneficial effects of the invention are as follows:
(1) The concentrated honey fermentation liquor is a concentrated molasses fermentation liquor of Weidan, is a byproduct of preparing glutamic acid by biological fermentation, and contains glutamic acid and sugar sources which are not digested and utilized by strains; the concrete components are as follows: the content of free glutamic acid is 25g/L, the nitrogen and potassium content is more than or equal to 70g/L (40-0-30), and the organic matter content is more than or equal to 300g/L. The invention adopts the concentrated molasses fermentation liquid to replace part of glutamic acid and carbon source molasses, thereby reducing the production cost;
(2) According to the invention, bacillus natto FK001 and bacillus licheniformis FK002 are firstly applied to the production of polyglutamic acid, free glutamic acid in concentrated honey fermentation liquor can be polymerized into polyglutamic acid with low molecular weight by utilizing the combined action of bacillus natto FK001 and bacillus licheniformis FK002, the content of polyglutamic acid in the prepared fermentation liquor is high (35-51.4 g/L), purification is not needed, and the concentrated honey fermentation liquor can be directly used and has low production cost.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The following examples, unless otherwise specified, do not contain other components not explicitly indicated except for unavoidable impurities. The raw materials used in each example were commercially available products. Wherein the concentrated honey fermentation liquor is a pellet concentrated molasses fermentation liquor, and the specific components are as follows: the content of free glutamic acid is 25g/L, the nitrogen and potassium content is more than or equal to 70g/L (40-0-30), and the organic matter content is more than or equal to 300g/L. The bacillus licheniformis FK002 and the bacillus natto FK001 are both derived from the Hongyuan biological fertilizer company.
EXAMPLE 1 preparation of Bacillus natto FK001 seed solution
(1) Activating strains: inoculating the bacillus natto on the inclined plane live freeze-dried tube to a nutrient agar culture plate, and culturing in a culture box at 38+/-2 ℃ for 12-16h.
(2) Preparing shake flask seed liquid: selecting colonies cultured by 1-2 rings of flat plates, and shake-culturing in shake flask culture medium at constant temperature of 38+ -2deg.C for 12-16 hr to obtain shake flask seed liquid;
the formula of the liquid shake flask culture medium is as follows: 20-50g/L of molasses, 50-100g/L of glutamic acid, 10-50g/L of peptone, 0.1-1g/L of ammonium sulfate and 0.1-1g/L of calcium chloride, and distilled water is added to 1000mL.
(3) Seed liquid preparation: inoculating the shake flask seed liquid into a primary seed culture medium at the temperature of 32-40 ℃ and the rotating speed of 80-200rpm for 6-12 hours according to the inoculation amount of 3-10%, and obtaining bacillus natto FK001 seed liquid;
the formula of the first-stage seed culture medium is as follows: 5-30g/L of molasses, 50-150g/L of concentrated molasses fermentation liquid, 10-20g/L of glutamic acid, 10-20g/L of corn steep liquor, 0.1-1g/L of calcium chloride and pH value of 6.0-7.0.
EXAMPLE 2 preparation of Bacillus licheniformis FK002 seed solution
(1) Activating strains: inoculating the bacillus licheniformis with the inclined plane live freeze-dried tube onto a nutrient agar culture plate, and culturing for 12-16h in a culture box at 38+/-2 ℃.
(2) Preparing shake flask seed liquid: selecting colonies cultured by 1-2 rings of flat plates, and shake-culturing in shake flask culture medium at constant temperature of 38+ -2deg.C for 12-16 hr to obtain shake flask seed liquid;
the formula of the liquid shake flask culture medium is as follows; 20-50g/L of molasses, 50-100g/L of glutamic acid, 10-50g/L of peptone, 0.1-1g/L of ammonium sulfate and 0.1-1g/L of calcium chloride, and distilled water is added to 1000mL.
(3) Seed liquid preparation: inoculating the shake flask seed liquid into a primary seed culture medium at the temperature of 32-40 ℃ and the rotating speed of 80-200rpm for 6-12 hours according to the inoculation amount of 3-10%, and obtaining bacillus licheniformis FK002 seed liquid;
the formula of the first-stage seed culture medium is as follows: 5-30g/L of molasses, 50-150g/L of concentrated molasses fermentation liquid, 10-20g/L of glutamic acid, 10-20g/L of corn steep liquor, 0.1-1g/L of calcium chloride and pH value of 6.0-7.0.
Example 3 preparation method of agricultural polyglutamic acid fermentation broth
(1) Preparing mixed seed liquid: according to the volume ratio of 3:1, bacillus natto FK001 seed liquid (1 multiplied by 10 7 CFU/ml) and Bacillus licheniformis FK002 seed solution (1X 10) 7 CFU/ml) to obtain mixed seed liquid;
(2) Fermentation: inoculating the mixed seed solution into a fermentation medium according to the inoculation amount of 25% for fermentation culture to obtain the agricultural polyglutamic acid fermentation broth;
the fermentation medium is as follows: 10g/L of molasses, 250g/L of concentrated molasses fermentation liquid, 15g/L of glutamic acid, 25g/L of corn steep liquor and 1g/L of calcium chloride, and the pH value is 6.0-7.0;
the conditions of fermentation culture are as follows: the temperature is 32 ℃, the rotating speed is 160rpm, and the culture time is 36 hours.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained in the example is detected to be 40.8g/L.
Example 4 preparation method of agricultural polyglutamic acid fermentation broth
(1) Preparing mixed seed liquid: according to the volume ratio of 8:1, bacillus natto FK001 seed liquid (1 multiplied by 10 7 CFU/ml) and Bacillus licheniformis FK002 seed solution (1X 10) 7 CFU/ml) to obtain mixed seed liquid;
(2) Fermentation: inoculating the mixed seed solution into a fermentation medium according to the inoculation amount of 8% to perform fermentation culture to obtain the agricultural polyglutamic acid fermentation broth;
the fermentation medium is as follows: 30g/L of molasses, 100g/L of concentrated molasses fermentation liquid, 25g/L of glutamic acid, 50g/L of corn steep liquor and 5g/L of calcium chloride, and the pH value is 6.0-7.0;
the conditions of fermentation culture are as follows: the temperature is 40 ℃, the rotating speed is 280rpm, and the culture time is 20 hours.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained in the example is detected to be 35.0g/L.
Example 5 preparation method of agricultural polyglutamic acid fermentation broth
(1) Preparing mixed seed liquid: according to the volume ratio of 7:1, bacillus natto FK001 seed liquid (1 multiplied by 10 7 CFU/ml) and Bacillus licheniformis FK002 seed solution (1X 10) 7 CFU/ml) to obtain mixed seed liquid;
(2) Fermentation: inoculating the mixed seed solution into a fermentation medium according to the inoculation amount of 10 percent for fermentation culture to obtain the agricultural polyglutamic acid fermentation broth;
the fermentation medium is as follows: 12g/L of molasses, 240g/L of concentrated molasses fermentation liquid, 18g/L of glutamic acid, 30g/L of corn steep liquor and 1.5g/L of calcium chloride, and the pH value is 6.0-7.0;
the conditions of fermentation culture are as follows: the temperature was 34℃and the rotational speed was 170rpm, and the culture time was 30 hours.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained in the example is detected to be 44.9g/L.
Example 6 preparation method of agricultural polyglutamic acid fermentation broth
(1) Preparing mixed seed liquid: according to the volume ratio of 4:1, bacillus natto FK001 seed liquid (1 multiplied by 10 7 CFU/ml) and Bacillus licheniformis FK002 seed solution (1X 10) 7 CFU/ml) to obtain mixed seed liquid;
(2) Fermentation: inoculating the mixed seed solution into a fermentation medium according to the inoculation amount of 20% for fermentation culture to obtain the agricultural polyglutamic acid fermentation broth;
the fermentation medium is as follows: 25g/L of molasses, 120g/L of concentrated molasses fermentation liquid, 22g/L of glutamic acid, 45g/L of corn steep liquor and 4.5g/L of calcium chloride, and the pH value is 6.0-7.0;
the conditions of fermentation culture are as follows: the temperature is 38 ℃, the rotating speed is 200rpm, and the culture time is 25 hours.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained in the example is detected to be 42.1g/L.
Example 7 preparation method of agricultural polyglutamic acid fermentation broth
(1) Preparing mixed seed liquid: according to the volume ratio of 5:1, bacillus natto FK001 seed liquid (1 multiplied by 10 7 CFU/ml) and Bacillus licheniformis FK002 seed solution (1X 10) 7 CFU/ml) to obtain mixed seed liquid;
(2) Fermentation: inoculating the mixed seed solution into a fermentation medium according to the inoculation amount of 15% to perform fermentation culture to obtain the agricultural polyglutamic acid fermentation broth;
the fermentation medium is as follows: 20g/L of molasses, 200g/L of concentrated molasses fermentation liquid, 20g/L of glutamic acid, 35g/L of corn steep liquor and 2.5g/L of calcium chloride, and the pH value is 6.0-7.0;
the conditions of fermentation culture are as follows: the temperature is 35 ℃, the rotating speed is 180rpm, and the culture time is 25 hours.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained in the example is detected to be 51.4g/L.
As can be seen from the above examples 3-7, the invention uses the concentrated molasses fermentation liquid of Weidan as the main raw material, uses Bacillus natto FK001 and Bacillus licheniformis FK002 as the fermentation strains, and the prepared fermentation liquid has high content of gamma-polyglutamic acid (35-51.4 g/L), can be directly used without purification, has low production cost, and meets the agricultural market demand.
Meanwhile, in order to further demonstrate the technical effects and novelty of the present invention, the following experiments were conducted to verify the synergy between strains.
Comparative example 1
This comparative example differs from example 4 only in that: in the preparation process of the mixed seed liquid, equal amount of bacillus natto FK001 seed liquid is used for replacing bacillus licheniformis FK002 seed liquid, and the final mixed seed liquid is only bacillus natto FK001 seed liquid.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained by the comparative example is 19.3g/L.
Comparative example 2
This comparative example differs from example 4 only in that: in the preparation process of the mixed seed solution, the equivalent amount of bacillus licheniformis FK002 seed solution is used for replacing bacillus natto FK001 seed solution; the final mixed seed solution was only bacillus licheniformis FK002 seed solution.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained by the comparative example is detected to be 12.7g/L.
Comparative example 3
This comparative example differs from example 4 only in that: in the preparation process of the mixed seed solution, the volume ratio of the bacillus natto FK001 seed solution to the bacillus licheniformis FK002 seed solution is 1:1.
The content of gamma-polyglutamic acid in the agricultural polyglutamic acid fermentation broth obtained by the comparative example is 17.2g/L.
Compared with single strain fermentation, the two strains can be compounded to obviously improve the content of gamma-polyglutamic acid in fermentation broth, and a synergistic effect is generated.
Evaluation of application Effect
Selecting 32 trays for cucumber seedling cultivation, thinning after cucumber seedlings emerge, selecting seedlings with consistent size and good growth, and transplanting 32 seedlings per tray.
The agricultural polyglutamic acid fermentation broths obtained in examples 3-7 and comparative examples 1-3 were diluted by 1000 times, respectively, and clear water was set as a control. Each disc was flushed 500mL, treated once every 3 days, and treated three times in total. After the test, the plant height, root length, SPAD value and leaf area were measured and the results are shown in table 1.
TABLE 1 Effect of agricultural polyglutamic acid fermentation broth on cucumber seedlings
From the above, the polyglutamic acid fermentation broth prepared by fermentation can be directly used as a water flush fertilizer after being diluted, and has the effects of promoting the growth and height of cucumber seedlings, promoting root system development, extending leaves and increasing chlorophyll content.
The invention has been further described above in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Claims (9)
1. The preparation method of the agricultural polyglutamic acid fermentation broth is characterized by comprising the following steps of:
(1) Preparing mixed seed liquid: mixing bacillus natto seed solution and bacillus licheniformis seed solution to obtain mixed seed solution;
(2) Fermentation: inoculating the mixed seed solution into a fermentation medium for fermentation culture to obtain the agricultural polyglutamic acid fermentation broth;
the bacillus natto in the bacillus natto seed liquid in the step (1) is bacillus natto FK001; the bacillus licheniformis in the bacillus licheniformis seed solution is bacillus licheniformis FK002.
2. The preparation method according to claim 1, wherein the volume ratio of the bacillus natto seed solution to the bacillus licheniformis seed solution in the step (1) is 3-8:1.
3. The method of claim 1, wherein the fermentation medium in step (2) is: 10-30g/L of molasses, 100-250g/L of concentrated molasses fermentation liquid, 15-25g/L of glutamic acid, 25-50g/L of corn steep liquor and 1-5g/L of calcium chloride, and the pH value is 6.0-7.0.
4. A method according to claim 3, wherein the fermentation medium in step (2) is: 12-25g/L of molasses, 120-240g/L of concentrated molasses fermentation liquid, 18-22g/L of glutamic acid, 30-45g/L of corn steep liquor and 1.5-4.5g/L of calcium chloride, and the pH value is 6.0-7.0.
5. The method according to claim 1, wherein the mixed seed liquid in the step (2) is inoculated in an amount of 8 to 25%.
6. The method according to claim 1, wherein the conditions of the fermentation culture in step (2) are: the temperature is 32-40 ℃, the rotating speed is 160-280rpm, and the culture time is 20-36h.
7. The method according to claim 6, wherein the conditions for the fermentation culture in the step (2) are: the temperature is 34-38 ℃, the rotating speed is 170-200rpm, and the culture time is 25-30h.
8. The agricultural polyglutamic acid fermentation broth prepared by the preparation method of any one of claims 1-7.
9. The use of the agricultural polyglutamic acid fermentation broth prepared by the preparation method of any one of claims 1-7 in preparing liquid fertilizer.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999756A (en) * | 2006-12-18 | 2007-07-18 | 浙江大学 | Process of preparing gamma-polyglutamic acid by bacillus subtilis and glutamic acid bacillus mixed cultivating system |
| CN101109010A (en) * | 2007-07-17 | 2008-01-23 | 秦皇岛领先科技发展有限公司 | Mycopremna generating gamma- polyglutamic acid and culturing method thereof |
| KR20110080699A (en) * | 2010-01-07 | 2011-07-13 | 박귀조 | Manufacturing method of fermented soybean curd |
| CN102342371A (en) * | 2011-09-01 | 2012-02-08 | 安徽金农惠民生物技术有限公司 | Process for preparing compound feed additive from bacillus subtilis natto and bacillus licheniformis |
| CN103898022A (en) * | 2014-04-01 | 2014-07-02 | 湛江恒兴养殖技术服务有限公司 | Novel formula of photosynthetic bacteria culture medium and preparation method |
| CN108841882A (en) * | 2018-07-26 | 2018-11-20 | 武琳慧 | A method of thallus fermenting and producing polyglutamic acid is discarded using glutamic acid fermentation |
| CN109415751A (en) * | 2016-05-26 | 2019-03-01 | 密执安大学评议会 | Composition and method for microbial co culture |
| CN110862950A (en) * | 2019-12-24 | 2020-03-06 | 烟台富康生物科技有限公司 | Bacillus licheniformis and application thereof |
| CN110951798A (en) * | 2020-01-07 | 2020-04-03 | 广东丸美生物技术股份有限公司 | Biological fermentation method of gamma-polyglutamic acid and application thereof |
| CN111019861A (en) * | 2019-12-25 | 2020-04-17 | 烟台富康生物科技有限公司 | Bacillus natto and application thereof |
-
2023
- 2023-06-14 CN CN202310699528.1A patent/CN116426580B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999756A (en) * | 2006-12-18 | 2007-07-18 | 浙江大学 | Process of preparing gamma-polyglutamic acid by bacillus subtilis and glutamic acid bacillus mixed cultivating system |
| CN101109010A (en) * | 2007-07-17 | 2008-01-23 | 秦皇岛领先科技发展有限公司 | Mycopremna generating gamma- polyglutamic acid and culturing method thereof |
| KR20110080699A (en) * | 2010-01-07 | 2011-07-13 | 박귀조 | Manufacturing method of fermented soybean curd |
| CN102342371A (en) * | 2011-09-01 | 2012-02-08 | 安徽金农惠民生物技术有限公司 | Process for preparing compound feed additive from bacillus subtilis natto and bacillus licheniformis |
| CN103898022A (en) * | 2014-04-01 | 2014-07-02 | 湛江恒兴养殖技术服务有限公司 | Novel formula of photosynthetic bacteria culture medium and preparation method |
| CN109415751A (en) * | 2016-05-26 | 2019-03-01 | 密执安大学评议会 | Composition and method for microbial co culture |
| CN108841882A (en) * | 2018-07-26 | 2018-11-20 | 武琳慧 | A method of thallus fermenting and producing polyglutamic acid is discarded using glutamic acid fermentation |
| CN110862950A (en) * | 2019-12-24 | 2020-03-06 | 烟台富康生物科技有限公司 | Bacillus licheniformis and application thereof |
| CN111019861A (en) * | 2019-12-25 | 2020-04-17 | 烟台富康生物科技有限公司 | Bacillus natto and application thereof |
| CN110951798A (en) * | 2020-01-07 | 2020-04-03 | 广东丸美生物技术股份有限公司 | Biological fermentation method of gamma-polyglutamic acid and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| CHYI-HOW LAY等: "Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation", INTERNATIONAL JOURNAL OF HYDROGEN ENERGY, vol. 35, pages 13445 - 13451, XP027457106, DOI: 10.1016/j.ijhydene.2009.11.128 * |
| 房俊楠;雷娟;许力山;姬高升;刘杨;闫志英;: "微生物发酵生产γ-聚谷氨酸研究进展", 应用与环境生物学报, vol. 24, no. 05, pages 1041 - 1049 * |
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