CN113862183A - Polypeptide microbial agent, preparation method thereof and application of polypeptide microbial agent in preparation of polypeptide microbial fertilizer - Google Patents
Polypeptide microbial agent, preparation method thereof and application of polypeptide microbial agent in preparation of polypeptide microbial fertilizer Download PDFInfo
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 130
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 130
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 130
- 230000000813 microbial effect Effects 0.000 title claims abstract description 111
- 239000003337 fertilizer Substances 0.000 title claims abstract description 101
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 60
- 230000004151 fermentation Effects 0.000 claims abstract description 60
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 23
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 19
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 19
- 241001052560 Thallis Species 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 23
- 102000016943 Muramidase Human genes 0.000 claims description 21
- 108010014251 Muramidase Proteins 0.000 claims description 21
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 21
- 235000010335 lysozyme Nutrition 0.000 claims description 21
- 239000004325 lysozyme Substances 0.000 claims description 21
- 229960000274 lysozyme Drugs 0.000 claims description 21
- 239000003895 organic fertilizer Substances 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 9
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical group C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000004021 humic acid Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims 4
- 238000011081 inoculation Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 28
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract description 21
- 239000004202 carbamide Substances 0.000 abstract description 21
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 14
- 150000001408 amides Chemical class 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 abstract description 3
- 108090000145 Bacillolysin Proteins 0.000 abstract description 3
- 102000035092 Neutral proteases Human genes 0.000 abstract description 3
- 108091005507 Neutral proteases Proteins 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 239000002207 metabolite Substances 0.000 abstract 1
- 150000007524 organic acids Chemical class 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000002068 microbial inoculum Substances 0.000 description 8
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- 240000008067 Cucumis sativus Species 0.000 description 5
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 235000021120 animal protein Nutrition 0.000 description 4
- 238000003287 bathing Methods 0.000 description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 4
- 235000019797 dipotassium phosphate Nutrition 0.000 description 4
- 210000003608 fece Anatomy 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 239000010871 livestock manure Substances 0.000 description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- 235000021118 plant-derived protein Nutrition 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 108010064851 Plant Proteins Proteins 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
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- 239000011368 organic material Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 229910010272 inorganic material Inorganic materials 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000000853 biopesticidal effect Effects 0.000 description 1
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 239000002686 phosphate fertilizer Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention relates to the field of microbial fertilizers, in particular to a polypeptide microbial agent, a preparation method thereof and application thereof in preparing polypeptide microbial fertilizers, wherein the preparation method of the polypeptide microbial agent comprises the following steps: inoculating bacillus subtilis to the first fermentation culture medium for culture, crushing thalli after the culture is finished to obtain a second fermentation culture medium containing mycoprotein, inoculating bacillus amyloliquefaciens to the second fermentation culture medium, and obtaining the polypeptide microbial agent after the culture is finished. According to the preparation method, the micro-molecular amide nitrogen is converted into the mycoprotein by the microorganism, the extremely volatile ammonia gas is fixed by metabolites such as organic acid and the like generated in the microbial proliferation process, and then the mycoprotein is hydrolyzed into the polypeptide by utilizing neutral protease generated by bacillus amyloliquefaciens, so that the polypeptide content in the fermentation liquor is improved, and the microbial fertilizer produced by the method can reduce the environmental pollution caused by the loss of low utilization rate of urea after application.
Description
Technical Field
The invention relates to the field of microbial fertilizers, in particular to a polypeptide microbial agent, a preparation method thereof and application thereof in preparation of a polypeptide microbial fertilizer.
Background
The polypeptide is a compound formed by dehydrating and condensing amino acid molecules, and can promote the growth and development of plants, improve the microbial community structure of soil, and improve the application effects of biofertilizer, biopesticide, organic materials and the like in agricultural production. At present, most of methods for preparing polypeptide microbial fertilizers are to mix polypeptides obtained by hydrolyzing animal or plant proteins purchased in the market with microbial inoculum and organic materials, and on one hand, the method has the defects that the whole fermentation process needs 24-48h of polypeptide hydrolysis, 24h of freeze drying, 48-72 h of microbial inoculum preparation and at least 96h of time consumption; on the other hand, the prices of animal and plant proteins purchased in the market are different from 2400-6000 yuan/ton, and the total price cost can only be controlled to be 3000 yuan/ton as low as possible by adding required conventional reagents.
The Chinese patent with the application number of 201310638649.1 discloses that the polypeptide source in the fertilizer containing the polypeptide is obtained by hydrolyzing plant or animal protein with protease, animal and plant protein is mixed with sulfuric acid aqueous solution, the pH value is adjusted after 24-48h of hydrolysis to obtain filtrate, the filtrate is concentrated, frozen and dried and then mixed with microbial inoculum and organic and inorganic materials, the polypeptide source in the method is animal and plant raw material, the price is low, the yield of crops can be greatly increased, but the polypeptide hydrolysis step is complicated, and the time is long.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a polypeptide microbial agent, a preparation method thereof and a use thereof in preparing a polypeptide microbial fertilizer, which are used for solving the problems in the prior art.
In order to achieve the above objects and other related objects, the present invention provides a method for preparing a polypeptide microbial agent, comprising the steps of: inoculating bacillus subtilis to the first fermentation culture medium for culture, crushing thalli after the culture is finished to obtain a second fermentation culture medium containing mycoprotein, inoculating bacillus amyloliquefaciens to the second fermentation culture medium, and obtaining the polypeptide microbial agent after the culture is finished.
The invention also provides the polypeptide microbial agent obtained by the preparation method of the polypeptide microbial agent.
The invention also provides application of the polypeptide microbial agent in preparation of a polypeptide microbial fertilizer.
The invention provides a polypeptide microbial fertilizer which comprises a polypeptide microbial agent.
The invention also provides a preparation method of the polypeptide microbial fertilizer, which comprises the step of mixing the polypeptide microbial agent with a solid fertilizer and/or a liquid fertilizer.
As mentioned above, the polypeptide microbial agent, the preparation method and the application thereof in preparing the polypeptide microbial fertilizer have the following beneficial effects:
1. the polypeptide hydrolysis process and the microbial inoculum preparation process are combined, the total fermentation time is only 74 hours, and the fermentation time is saved by 23 percent.
2. The price cost of the required fermentation reagent and the lysozyme is about 1000 yuan/ton, and the cost is reduced by 2/3 compared with the prior art.
3. The polypeptide microbial inoculum obtained by fermentation is mixed with solid fertilizer or humic acid water-soluble fertilizer, so that the microbial quantity and activity in the microbial inoculum are ensured, the nitrogen nutrient content is also ensured, urea is converted into organic nitrogen by taking the urea as a nitrogen source in the fermentation process, the utilization rate of the urea is improved, the loss of the urea in the application process is reduced, the emission of ammonia is reduced, the environmental pollution is reduced, the polypeptide content is greatly increased, the polypeptide can promote the growth and development of plants, the crop quality is improved from the aspects of improving the stress resistance of crops, promoting the growth of root systems, improving the absorption and utilization efficiency of the fertilizer and the like, the disease resistance of the crops is improved, and meanwhile, the effects of improving the microbial community structure of soil and the like are achieved.
Detailed Description
The invention provides a preparation method of a polypeptide microbial agent, which comprises the following steps: inoculating bacillus subtilis to the first fermentation culture medium for culture, crushing thalli after the culture is finished to obtain a second fermentation culture medium containing mycoprotein, inoculating bacillus amyloliquefaciens to the second fermentation culture medium, and obtaining the polypeptide microbial agent after the culture is finished.
In one embodiment, the Bacillus subtilis is selected from Bacillus subtilis with the strain number of hlj158, which is deposited in the general microbiological center of the China Committee for culture Collection of microorganisms with the collection number of CGMCC No.13026, the collection date of 2016, 09, 21 days, and the collection unit address of institute of microbiology, China institute of sciences, No. 3, North Cheng West Lu 1, the south China area, Beijing. The Bacillus subtilis with the strain number of hlj158 has the function of fixing nitrogen.
In one embodiment, the first fermentation medium comprises, based on the volume of the first fermentation medium:
in one embodiment, the pH of the first fermentation medium is from 7.2 to 7.5.
In one embodiment, the Bacillus subtilis is inoculated at a ratio of 1% to 3% by volume of the first fermentation medium.
In one embodiment, the culture conditions of the bacillus subtilis are selected from any one or more of the following: the culture temperature is 26-30 ℃, the rotating speed is 180-220 r/min, and the culture time is 18-36 h.
In one embodiment, after the bacillus subtilis is cultured, the bacteria are crushed by adding lysozyme and ultrasonic, and the lysozyme is heated and denatured to inactivate the lysozyme, so that the second fermentation medium containing mycoprotein is obtained.
In one embodiment, the lysozyme is present at a concentration of 5mg/L to 50 mg/L. The action concentration refers to the initial concentration of lysozyme after being added into the system.
In one embodiment, the lysozyme is added and then heated at 35-45 ℃ for 45-75 min.
The lysozyme can selectively decompose microbial cells without damaging other tissues, and is nontoxic and harmless per se.
In one embodiment, the sonication conditions are 100-500w for 2-4min at 3s intervals per minute.
In one embodiment, the Bacillus amyloliquefaciens is selected from Bacillus amyloliquefaciens with strain number ahsz909, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.13028, the preservation date of 2016, 9, 21 days, and the preservation unit address of institute of microbiology, China institute of sciences, institute of microbiology, No. 3, North Chen Lu No.1, of the sunward area, Beijing.
In one embodiment, the bacillus amyloliquefaciens is inoculated in an amount of 1% to 3% by volume of the second fermentation medium.
In one embodiment, the culture conditions of the bacillus amyloliquefaciens are selected from any one or more of the following: the culture temperature is 30-38 ℃, the rotating speed is 180-220 r/min, and the culture time is 18-72 h.
In one embodiment, the method further comprises concentrating and/or drying the fermentation liquid after the culture of the bacillus amyloliquefaciens is finished.
According to the preparation method of the polypeptide microbial agent, urea in a first fermentation culture medium is used as a unique nitrogen source, after bacillus subtilis is inoculated, the bacillus subtilis ferments amide nitrogen in the urea in a mass propagation process to convert the amide nitrogen into mycoprotein, and then lysozyme is used for crushing bacillus subtilis to release the mycoprotein, so that a second fermentation culture medium containing a large amount of mycoprotein is obtained. After inoculating the bacillus amyloliquefaciens, the bacillus amyloliquefaciens generates neutral protease to hydrolyze the mycoprotein in the second fermentation medium into polypeptide which can be absorbed by plants, and the polypeptide microbial agent containing the polypeptide and the bacillus amyloliquefaciens is obtained. The polypeptide chain has a large number of carboxyl groups, and rich complexing groups around the chain and metal ions form chelation, so that nutrients are enriched, the utilization rate of the fertilizer can be improved by preparing the polypeptide microbial fertilizer by using the polypeptide microbial agent, and the crop yield is improved at the same time.
By the safe and efficient method, the content of the polypeptide is increased while the content of the nitrogen nutrients and the number of microorganisms are ensured, the utilization rate of the urea is improved, the loss of the urea in the application process is reduced, the environmental pollution is reduced, the fermentation time is shortened, the stress resistance of crops can be improved by the polypeptide, the growth of root systems is promoted, the absorption and utilization efficiency of fertilizers is improved, and the like.
The invention also provides the polypeptide microbial agent obtained by the preparation method of the polypeptide microbial agent.
The polypeptide microbial agent is solid or liquid. Specifically, a solid polypeptide microbial agent is obtained by concentrating and drying fermentation liquor after the culture of the bacillus amyloliquefaciens is finished; and concentrating the fermentation liquor after the culture of the bacillus amyloliquefaciens is finished to obtain the liquid polypeptide microbial agent. In one embodiment, the solid polypeptide microbial agent is a powder.
The polypeptide content of the polypeptide microbial agent is more than 6 mg/ml.
The total nitrogen content of the polypeptide microbial agent is more than 5.6%. The total nitrogen content is determined according to the method in the NY/T798-2015 standard of the compound microbial fertilizer.
The invention also provides application of the polypeptide microbial agent in preparation of a polypeptide microbial fertilizer.
The invention provides a polypeptide microbial fertilizer which comprises a polypeptide microbial agent.
In one embodiment, the polypeptide microbial fertilizer comprises the polypeptide microbial agent and an organic fertilizer.
The organic fertilizer is a fertilizer prepared by decomposing or fermenting natural organic matters through microorganisms, and the industrial standard NY525-2002 established by the ministry of agriculture is suitable for the organic fertilizer.
The organic fertilizer can directly supply nutrient elements necessary for the growth and development of crops and is rich in organic substances. The organic fertilizer is selected from green manure, human excrement, stable manure, compost, biogas manure or waste fertilizer, and further comprises mud manure, smoked soil, pit soil or dregs.
In one embodiment, the polypeptide microbial fertilizer comprises the polypeptide microbial agent and an inorganic fertilizer.
The inorganic fertilizer refers to inorganic salt fertilizer prepared by extraction, mechanical crushing, chemical synthesis and other processes, and is also called mineral fertilizer and mineral fertilizer. Since most of chemical fertilizers are inorganic fertilizers, the inorganic fertilizers are also called chemical fertilizers, which are called chemical fertilizers for short.
The inorganic fertilizer mainly contains nitrogen, phosphorus, potassium and other nutrient elements which exist in the form of inorganic compounds, and most of the inorganic fertilizers are produced by chemical industry.
In one embodiment, the inorganic fertilizer is selected from a nitrogen fertilizer, a phosphate fertilizer or a complex fertilizer.
In one embodiment, the polypeptide microbial fertilizer comprises the polypeptide microbial agent, an organic fertilizer and an inorganic fertilizer.
The polypeptide microbial fertilizer is solid or liquid. When the polypeptide microbial fertilizer is liquid, the organic fertilizer or inorganic fertilizer is selected from water-soluble fertilizers. In one embodiment, the water soluble fertilizer is a humic acid water soluble fertilizer.
When the polypeptide microbial fertilizer is solid, the organic fertilizer or the inorganic fertilizer is a solid organic fertilizer or a solid inorganic fertilizer respectively.
The effective viable count in the solid polypeptide microbial fertilizer is more than 5 hundred million/g. The effective viable count in the liquid polypeptide microbial fertilizer is more than 10 hundred million/mL.
The invention also provides a preparation method of the polypeptide microbial fertilizer, which comprises the step of mixing the polypeptide microbial agent with an organic fertilizer and/or an inorganic fertilizer.
Specifically, the preparation method of the solid polypeptide microbial fertilizer comprises the step of mixing the solid polypeptide microbial agent with a solid organic fertilizer and/or a solid inorganic fertilizer.
The preparation method of the liquid polypeptide microbial fertilizer comprises the step of mixing the liquid polypeptide microbial agent with a water-soluble fertilizer.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1
A method for increasing the polypeptide content in a microbial fertilizer, comprising the steps of:
10g of glucose, 0.5g of starch, 2.5g of dipotassium phosphate, 2.5g of monopotassium phosphate, 0.3g of magnesium sulfate heptahydrate, 2g of calcium carbonate and 1L of water are prepared into a 1L first fermentation culture medium, the pH value is adjusted to 7.2-7.5, 50mL of the first fermentation culture medium is subpackaged into 250mL triangular flasks per flask, and the first fermentation culture medium is cooled after being sterilized at the high temperature and the high pressure of 115 ℃ for 20 min. The urea is prepared into 35g/100mL mother liquor, sterilized by a 0.22 mu m filter membrane, and 2mL urea mother liquor is added into each bottle. Hlj158 inoculating the seed liquid (viable count 10)8cfu/mL, the viable count of the seed liquid in the following examples is the same as that in the examples), culturing for 24h at 28 ℃, 200r/min, adding 100mg/mL lysozyme 200 mu L, carrying out water bath at 40 ℃ for 1h, carrying out ultrasonic crushing for 200w and 3min after the water bath is finished, carrying out time interval of 3s, heating to 80 ℃ for water bath to denature and inactivate the lysozyme, cooling, and inoculating ahsz909 seed liquid (viable count of 10)8cfu/mL, the viable count of the seed liquid in the following examples is the same as that in the present example) 2%, culturing at 37 ℃ at 200r/min for 24h to obtain the polypeptide microbial agent, and measuring the content of the polypeptide.
Example 2
10g of glucose, 0.5g of starch, 2.5g of dipotassium phosphate, 2.5g of monopotassium phosphate, 0.3g of magnesium sulfate heptahydrate, 2g of calcium carbonate and 1L of water are prepared into a 1L first fermentation culture medium, the pH value is adjusted to 7.2-7.5, 50mL of the first fermentation culture medium is subpackaged into 250mL triangular flasks per flask, and the first fermentation culture medium is cooled after being sterilized at the high temperature and the high pressure of 115 ℃ for 20 min. Urea was prepared as a 45g/100mL stock solution, sterilized through a 0.22 μm filter and 2mL urea stock solution was added per bottle. Inoculating hlj158 seed liquid 2%, culturing at 28 deg.C and 200r/min for 24h, adding 100mg/mL lysozyme 200 μ L, water bathing at 40 deg.C for 1h, ultrasonicating for 200w and 3min, heating to 80 deg.C for water bathing to denature and inactivate lysozyme, cooling, inoculating ahsz909 seed liquid 2%, culturing at 37 deg.C and 200r/min for 48h to obtain polypeptide microbial preparation, and determining polypeptide content.
Example 3
A method for increasing the polypeptide content in a microbial fertilizer, comprising the steps of:
10g of glucose, 0.5g of starch, 2.5g of dipotassium phosphate, 2.5g of monopotassium phosphate, 0.3g of magnesium sulfate heptahydrate, 2g of calcium carbonate and 1L of water are prepared into a 1L first fermentation culture medium, the pH value is adjusted to 7.2-7.5, 50mL of the first fermentation culture medium is subpackaged into 250mL triangular flasks per flask, and the first fermentation culture medium is cooled after being sterilized at the high temperature and the high pressure of 115 ℃ for 20 min. Urea was prepared as a 45g/100mL stock solution, sterilized through a 0.22 μm filter and 2mL urea stock solution was added per bottle. Inoculating hlj158 seed liquid 2%, culturing at 28 deg.C and 200r/min for 24h, adding 100mg/mL lysozyme 300 μ L, water bathing at 40 deg.C for 1h, ultrasonicating for 300w and 3min, heating to 80 deg.C for water bathing to denature and inactivate lysozyme, cooling, inoculating ahsz909 seed liquid 2%, culturing at 37 deg.C and 200r/min for 24h to obtain polypeptide microbial preparation, and determining polypeptide content.
Example 4
10g of glucose, 0.5g of starch, 2.5g of dipotassium phosphate, 2.5g of monopotassium phosphate, 0.3g of magnesium sulfate heptahydrate, 2g of calcium carbonate and 1L of water are prepared into a 1L first fermentation culture medium, the pH value is adjusted to 7.2-7.5, 50mL of the first fermentation culture medium is subpackaged into 250mL triangular flasks per flask, and the first fermentation culture medium is cooled after being sterilized at the high temperature and the high pressure of 115 ℃ for 20 min. Preparing 35g/100mL of urea mother solution, filtering with a 0.22 mu m filter membrane for sterilization, adding 2mL of urea mother solution into each bottle, inoculating hlj158 seed solution 2%, culturing at 28 ℃ and 200r/min for 24h, adding 100mg/mL of lysozyme 300 mu L, carrying out water bath at 40 ℃ for 1h, carrying out ultrasonic crushing for 300w and 3min, carrying out time interval of 3s, heating to 80 ℃ for water bath to denature and inactivate the lysozyme, cooling, adding ahsz909 seed solution 2%, culturing at 37 ℃ and 200r/min for 48h, obtaining the polypeptide microbial inoculum, and determining the content of the polypeptide.
The method for measuring the content of the polypeptide is a biuret method.
TABLE 1 measurement results
| Name of process | Total nitrogen% | Polypeptide mg/mL |
| Example 1 | 5.6 | 6.04 |
| Example 2 | 5.8 | 6.58 |
| Example 3 | 5.9 | 6.97 |
| Example 4 | 5.8 | 7.12 |
Concentrating and drying the obtained polypeptide microbial agent, and mixing with super-Yio (brand of Shanghai agricultural product, biological products, Ltd.) organic fertilizer and compound fertilizer (15-15-15) according to the mass percentage of 5%: 50%: 45 percent of the mixture is mixed to prepare the polypeptide microbial fertilizer, and each index of the polypeptide microbial fertilizer is measured according to a method in a composite microbial fertilizer (NY/T798-2015) standard, and the result is as follows: n: 7.8-9.7%, P2O5：6.8-7.4％，K2O: 6.9-7.5%, organic matter: not less than 20%, effective viable count (cfu): 5 hundred million/g.
The fermentation liquor and the humic acid water-soluble fertilizer (macroelement type) are mixed according to the volume percentage of 10%: 90 percent of the mixture is mixed to prepare a polypeptide microbial liquid fertilizer, and each index of the polypeptide microbial fertilizer is measured according to a method in a compound microbial fertilizer (NY/T798-2015) standard, and the result is as follows: n: 6.6-9.3%, P2O5：5.1-5.7％，K2O: 5.4-5.9%, organic matter: not less than 20%, effective viable count (cfu): 10 hundred million/mL.
The polypeptide has the effects of improving the quality of crops, improving the utilization rate of the fertilizer, improving the yield of the crops and resisting diseases, and whether the polypeptide microbial fertilizer prepared by mixing the polypeptide microbial fertilizer with the organic fertilizer can provide nutrients for the growth of the crops and has the effect of the polypeptide or not is verified by the following examples.
Example 5 promoting Effect of polypeptide microbial Fertilizer on cucumber growth
The experimental site: shanghai Nongle biological product GmbH No.1 test shed
And (3) verifying by taking an organic fertilizer as a reference, applying the organic fertilizer and the polypeptide microbial fertilizer prepared in the example 3 into soil, turning over the soil, planting cucumber seedlings with two leaves and one core, and additionally applying a humic acid water-soluble fertilizer and a polypeptide microbial liquid fertilizer every 10-15 days in the middle.
TABLE 2 test treatments and numbering
TABLE 3 growth promoting effect of polypeptide microbial fertilizer on cucumber
By applying the polypeptide microbial fertilizer, under the condition of reducing the dosage of 1/3 base fertilizer, the yield of each plant of cucumber is still obviously increased (note: LSD multiple comparison method, different lower case letters such as a, b and c in different treatment rooms indicate that the difference reaches an obvious level (P is less than 0.05), and the same lower case letter in different treatment rooms indicates that the difference is not obvious), and the plant in seedling stage is higher than the control, which shows that the polypeptide added in the fertilizer has the effects of improving quality and increasing yield and improving the utilization rate of the fertilizer. The quantity of soil bacteria is increased, the quantity of pathogenic bacteria is reduced, the soil microbial community structure is improved, and the disease risk of cucumber fungal diseases is reduced.
According to the invention, a series of biochemical reactions in the microbial fermentation process are utilized, bacillus amyloliquefaciens which can produce neutral protease is utilized to hydrolyze bacterial protein obtained by adding lysozyme after bacillus subtilis is fermented, so that the polypeptide content in fermentation liquor is greatly improved, the microbial inoculum containing polypeptide and bacillus amyloliquefaciens can be directly obtained after the fermentation liquor is concentrated, the fermentation time is shortened, and the fermentation steps are saved. According to the invention, urea is used as the only nitrogen source for fermentation, amide nitrogen in the urea is converted into organic nitrogen, so that the pollution of the urea to the environment is reduced, and the utilization rate of the urea is improved. The bacillus amyloliquefaciens has various excellent effects of promoting growth and resisting diseases, improving and prolonging fertilizer efficiency and the like, and the polypeptide microbial fertilizer can be obtained by mixing a solid polypeptide microbial agent obtained by concentrating and drying fermentation liquor with a solid fertilizer or directly mixing a liquid polypeptide microbial agent obtained by concentrating the fermentation liquor with a water-soluble fertilizer.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (17)
1. A preparation method of a polypeptide microbial agent is characterized by comprising the following steps: inoculating bacillus subtilis to the first fermentation culture medium for culture, crushing thalli after the culture is finished to obtain a second fermentation culture medium containing mycoprotein, inoculating bacillus amyloliquefaciens to the second fermentation culture medium, and obtaining the polypeptide microbial agent after the culture is finished.
2. The method according to claim 1, wherein the Bacillus subtilis is Bacillus subtilis with a collection number of CGMCC No. 13026.
3. The method of claim 1, wherein the first fermentation medium comprises, based on the volume of the first fermentation medium:
4. the method according to claim 1, wherein the pH of the first fermentation medium is 7.2 to 7.5, and/or the inoculation ratio of Bacillus subtilis is 1 to 3% by volume of the first fermentation medium.
5. The production method according to claim 1, wherein the culture conditions of Bacillus subtilis are selected from any one or more of the following: the culture temperature is 26-30 ℃; rotating at a speed of 180-220 r/min; the culture time is 18-36 h.
6. The preparation method according to claim 1, wherein after the bacillus subtilis is cultured, the bacteria are crushed by adding lysozyme and ultrasonic, and the lysozyme is heated and denatured to inactivate the lysozyme, so that a second fermentation medium containing mycoprotein is obtained.
7. The preparation method according to claim 1, wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens with a preservation number of CGMCC No. 13028.
8. The process according to claim 1, wherein the amount of Bacillus amyloliquefaciens inoculated is 1 to 3% by volume of the second fermentation medium.
9. The method according to claim 1, wherein the Bacillus amyloliquefaciens is cultured under conditions selected from any one or more of the following: the culture temperature is 30-38 ℃, the rotating speed is 180-220 r/min, and the culture time is 18-72 h.
10. The method according to claim 1, further comprising concentrating and/or drying the fermentation broth after completion of the culture of Bacillus amyloliquefaciens.
11. The polypeptide microbial agent obtained by the preparation method according to any one of claims 1 to 10.
12. The polypeptide microbial inoculant according to claim 11, wherein the polypeptide microbial inoculant is solid or liquid and/or the polypeptide content of the polypeptide microbial inoculant is above 6 mg/ml.
13. Use of the polypeptide microbial inoculant of any one of claims 11-12 in the preparation of a polypeptide microbial fertilizer.
14. A polypeptide microbial fertilizer, which comprises either or both of an organic fertilizer and an inorganic fertilizer, and the polypeptide microbial agent according to any one of claims 11 to 12.
15. The polypeptide microbial fertilizer according to claim 14, wherein said polypeptide microbial fertilizer is solid or liquid; preferably, when the polypeptide microbial fertilizer is liquid, the organic fertilizer or inorganic fertilizer is selected from water-soluble fertilizers.
16. The polypeptide microbial fertilizer according to claim 15, wherein the water-soluble fertilizer is a humic acid water-soluble fertilizer.
17. The process for producing the polypeptide microbial fertilizer according to any one of claims 14 to 16, which comprises mixing the polypeptide microbial agent according to any one of claims 11 to 12 with an organic fertilizer and/or an inorganic fertilizer.
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