CN107794240B - Bacillus megaterium, polypeptide agricultural microbial agent thereof, and preparation method and application thereof - Google Patents
Bacillus megaterium, polypeptide agricultural microbial agent thereof, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to bacillus megaterium, a polypeptide agricultural microbial agent thereof, a preparation method and application. The invention takes vinasse, soybean meal and straw powder with rich protein content as main raw materials, and corn flour, wheat bran, inorganic salt and a small amount of carbon source are added for reasonable compounding and then are steamed; the agricultural microbial agent which takes active peptides, amino acids, polysaccharides and the like as high-efficiency components is prepared by firstly utilizing high-yield protease bacillus subtilis and then utilizing bacillus megatherium to decompose different proteins in raw materials under specific fermentation conditions, the microbial agent contains a large number of living spores, 17 free amino acids, various active peptides and polysaccharides, is rich in nutrient content and high in thallus activity, has a shelf life of more than 2 years, and achieves good quality-improving and yield-increasing effects through field experiments.
Description
Technical Field
The invention relates to bacillus megaterium, a polypeptide agricultural microbial agent thereof, a preparation method and application, and belongs to the technical field of microbial fertilizers.
Background
Amino acid (amino acid) is a general name of a class of organic compounds containing amino groups and carboxyl groups, is a basic composition unit of biological functional macromolecular protein, and is an organic compound containing basic amino groups and acidic carboxyl groups. Amino acids confer a specific molecular structural morphology to a protein, rendering its molecules biochemically active. The L-amino acid is a substance which can be absorbed and utilized by plants, can supplement amino acid necessary for crops, stimulate and regulate the rapid growth of the plants, and promote the absorption of nutrient substances. The quality of crops is improved, and the disease resistance of the crops is improved; can also chelate medium trace elements and promote the absorption and transportation of the trace elements in the plant body. The adaptability of the plant to the adverse environment is improved, and the amino acid is respectively applied in the early stage, the middle stage and the later stage of the stress, so that the influence of the stresses such as high temperature, low humidity, frost, insect damage, hail, flooding and the like can be prevented and recovered. Some amino acid derivatives can regulate plant growth, or be used as pesticides, and the like. In recent years, amino acids have attracted much attention at home and abroad in the field of plant nutrition research, and have made many new advances.
Proteins are important active molecules in the body, including enzymes and enzymes that catalyze metabolism. More than 100 peptide bonds are generally referred to as proteins, and more than 2 and less than 100 peptide bonds are generally referred to as polypeptides. Long-term experimental research shows that: the polypeptide not only can provide organic nutrient substances for the growth of crops, but also plays an important role in the aspects of signal transduction, cell activity activation, crop yield increase, fertilizer utilization efficiency increase, quality improvement and the like in the growth and development of plants. The polypeptide peptide chain has a plurality of carboxyl groups, a plurality of complexing groups are arranged around the peptide chain, the polypeptide peptide chain has strong chelation with metal ions, can be combined with nutrients and can clamp the nutrients in the ring to enrich the nutrients for plants, thereby improving the utilization efficiency of fertilizers, improving the yield of crops, and chelating mineral elements which are difficult to move in plants, such as calcitonin, by active peptides and well absorbed by the plants. The polypeptide can enhance the immunity of crops, improve the stress resistance and has obvious disease and pest resistance effect.
In recent years, polypeptides and amino acids have been widely used in the aspects of saving fertilizer, improving fertilizer utilization rate and improving crop stress resistance in crop cultivation, and remarkable effects are achieved, for example, chinese patent document CN 103204735A (application No. 201310116458) discloses a polypeptide water-soluble fertilizer and a preparation method thereof, the invention is prepared by mixing polypeptides with organic and inorganic trace elements, and the selected polypeptides are obtained by hydrolyzing animal and plant proteins with acid, neutralizing, filtering, and spray-drying the filtrate. Chinese patent document CN101747093A (application number: 200910248459) provides a method for producing mycoprotein and polypeptide organic fertilizer by using vitamin C fermentation waste, in the method, gulonic acid mother liquor is added into waste mash formed by a vitamin C production process, the pH value is adjusted to 2.0-5.0, then polypeptide and amino acid are produced by mild hydrolysis, the pH value is adjusted and appropriately concentrated, and a proper amount of inorganic elements are added to prepare the polypeptide organic fertilizer.
The two polypeptide fertilizers are produced by hydrolyzing proteins in organic matters into amino acids and small molecule active peptides by an acid hydrolysis mode. The polypeptide fertilizer obtained by acid hydrolysis has the following disadvantages: 1) the method needs to be carried out in a pressure kettle under the conditions of high temperature and high pressure, and has high cost and poor safety; 2) a large amount of acid and alkali are needed in the production process, which is not beneficial to environmental protection; 3) the active peptide obtained by acid hydrolysis of protein has single form and limited biological activity, most of the amino acid obtained by hydrolysis has changed configuration, cannot be absorbed and utilized by crops, and has low fertilizer efficiency.
The microbial fermentation method is adopted to replace the traditional method for hydrolyzing protein by an enzyme method, so that the defects of acid hydrolysis can be overcome, such as: chinese patent document CN102079674A (application No. 201010558843.5) provides a microbial organic fertilizer, which is composed of straw, flour (corn flour or bean flour), lime, monopotassium phosphate, water and liquid mixed strains NEM, wherein the NEM mixed strains comprise bacillus subtilis, candida utilis, bacillus megaterium, bacillus mucilaginosus, lactobacillus plantarum, photosynthetic bacteria and Pantoea agglomerans which are fermented and cultured. Chinese patent document CN104671961A (application number: 201010558843.5) provides a microbial fermentation organic fertilizer for vegetables, which comprises the following components in parts by weight: 40-100 parts of straw, 10-20 parts of soybean meal, 10-20 parts of composite microbial agent, 30-50 parts of turf and 5-15 parts of inorganic additive, wherein the composite microbial agent is a mixture of bacillus subtilis, bacillus megaterium, bacillus cereus, bacillus mucilaginosus, trichoderma harzianum, lactobacillus plantarum and saccharomycetes. In the two methods, microorganisms with different culture conditions grow and reproduce in the same environment, and have competition or inhibition effects with each other, although individual strains generate polypeptide components in the organic matter fermentation process, the quantity of the polypeptide components cannot achieve the due effect, and the microorganisms have competition effects and lower quantity of viable bacteria.
For another example: chinese patent document CN 105272407A (application number: 201510791723.2) discloses a method for preparing a biological polypeptide organic fertilizer by microbial fermentation and application thereof, wherein furfural residues and edible fungi culture material residues are used as main raw materials, wheat bran, bean cakes and the like with rich protein content are added, and primary treatment is firstly carried out to dissolve out the contained protein; and then, decomposing different proteins in the raw materials by utilizing two microorganisms in sequence to form biological polypeptide components with various forms, and preparing the biological polypeptide organic fertilizer taking the polypeptides as high-efficiency components. Although the organic fertilizer, the biological organic fertilizer polypeptide and the organic matter produced by the method have high content, the production process needs additional treatment or enzymolysis, the production cost is increased, most of nutrients in the fertilizer are water-insoluble and are difficult to be absorbed and utilized by crops when being applied to soil, and in addition, the organic fertilizer obtained by the method has very low viable bacteria amount and large using amount and is not suitable for long-term application. For another example: chinese patent document CN 105884405a (application No. 201510836791.6) discloses a preparation method of an amino acid-polypeptide liquid fertilizer. Although the amino acid-polypeptide liquid fertilizer produced by the invention has high polypeptide content, the production process is complicated, the energy consumption of liquid fermentation is high, and the subsequent filtering process is added for solid-liquid separation and product extraction, active functional strains in the fermentation process are filtered out, and partial incompletely degraded organic nutrients are also filtered into solid filter residues, so that the active strains and the organic nutrients are greatly lost, and the fertilizer efficiency is reduced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a bacillus megaterium, a polypeptide agricultural microbial agent thereof, a preparation method and application.
Summary of the invention:
the invention takes vinasse, soybean meal and straw powder with rich protein content as main raw materials, and corn flour, wheat bran, inorganic salt and a small amount of carbon source are added for reasonable compounding and then are steamed; the agricultural microbial agent which takes active peptides, amino acids, polysaccharides and the like as high-efficiency components is prepared by firstly utilizing high-yield protease bacillus subtilis and then utilizing bacillus megatherium to decompose different proteins in raw materials under specific fermentation conditions, the microbial agent contains a large number of living spores, 17 free amino acids, various active peptides and polysaccharides, is rich in nutrient content and high in thallus activity, has a shelf life of more than 2 years, and achieves good quality-improving and yield-increasing effects through field experiments.
The technical scheme of the invention is as follows:
a Bacillus megaterium strain HB is preserved in China general microbiological culture Collection center in 2016, 1 month and 7 days, and addresses are as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the preservation numbers are as follows: CGMCC NO. 11961.
According to the invention, the preparation method of the polypeptide agricultural microbial agent comprises the following steps:
(1) preparation of functional bacteria bacillus subtilis seed liquid
A. Activating the bacillus subtilis strain by using an LB slant culture medium to obtain an activated bacillus subtilis strain;
B. inoculating the activated bacillus subtilis strain into a liquid seed culture medium, and culturing at 32-37 ℃ for 16-24h to obtain a bacillus subtilis seed liquid for later use;
(2) preparation of functional bacterium bacillus megaterium seed liquid
a. Activating the bacillus megaterium strain by using an LB slant culture medium to obtain an activated bacillus megaterium strain;
b. inoculating the activated bacillus megaterium strain into a liquid seed culture medium, and culturing at 32-37 ℃ for 16-24h to obtain a bacillus megaterium seed liquid for later use;
(3) preparation of the Pre-treated mix
Taking and uniformly mixing the bean pulp powder, the straw powder, the wheat bran, the vinasse and the corn flour according to the proportion, wherein the weight parts of the bean pulp powder, the straw powder, the wheat bran, the vinasse and the corn flour are as follows: 40-80 parts of soybean meal, 6-28 parts of straw powder, 5-22 parts of wheat bran, 5-20 parts of vinasse and 5-15 parts of corn flour; adding water to adjust the water content to 60-70wt%, standing at 20-35 deg.C for 8-24 hr, steaming at 115-121 deg.C for 20-40min, and cooling to obtain pretreated mixture;
(4) inoculating the pretreated mixture prepared in the step (3) into the bacillus subtilis seed liquid prepared in the step (1) according to the inoculation amount of 8-30wt%, uniformly mixing, fermenting for 24-30 hours at the temperature of 32-36 ℃, and uniformly stirring;
(5) then inoculating the bacillus megaterium seed solution prepared in the step (2) according to the inoculation amount of 12-18wt%, uniformly mixing, raising the temperature to 38-42 ℃, continuing to ferment for 24-48 hours, cooling to room temperature after fermentation is finished, drying and crushing to obtain the agricultural microbial agent rich in high-content live bacteria, compound amino acid and active peptide.
Preferably, the LB slant culture medium in the steps (1) and (2) comprises the following components in percentage by weight: tryptone 0.8-1.2%, yeast extract 0.4-0.6%, sodium chloride 0.4-0.6%, agar powder 1.5-2.0%, and water in balance, wherein the pH value is 7.2.
Preferably, in the step A and the step a, the single colony is picked and cultured on an LB culture medium slant culture medium at 37 ℃ for 18-24 h.
Preferably, the components and weight percentages of the liquid seed culture medium in the steps B and B are as follows: 2.0-3.0% of glucose, 1.1-1.8% of corn steep liquor dry powder, 1.5-4.0% of soybean meal, 0.1-0.2% of yeast extract, 0.01-0.03% of potassium dihydrogen phosphate, 0.01-0.02% of sodium chloride, 0.01-0.02% of magnesium sulfate and the balance of water, wherein the pH value is 7.0-7.3;
preferably, according to the invention, the inoculation amount of the bacillus subtilis seed solution in the step (4) is 10-20wt%, the fermentation temperature is 34-36 ℃, and the fermentation time is 26-30 hours;
most preferably, the inoculation amount of the bacillus subtilis seed solution is 10 wt%, the fermentation temperature is 35 ℃, and the fermentation time is 30 hours.
Preferably, according to the invention, the inoculation amount of the bacillus megaterium seed liquid in the step (5) is 14-18wt%, the fermentation temperature is 40-42 ℃, and the fermentation time is 40-48 hours;
most preferably, the inoculation amount of the bacillus megaterium seed liquid is 15 wt%, the fermentation temperature is 42 ℃, and the fermentation time is 48 hours;
preferably, according to the invention, the drying temperature in step (5) is from 50 to 110 ℃.
According to the invention, the straw powder is one or more of corn straw powder, wheat straw powder or rice straw powder which are optionally mixed.
According to the invention, the grain diameter of the straw powder and the vinasse is preferably less than or equal to 0.6cm, and the grain diameter of the wheat bran, the soybean meal and the corn flour is preferably 0.4-1 mm.
According to the invention, the number of viable bacteria in the bacillus subtilis seed liquid is preferably 5.0 x 109-1.5×1010CFU/ml, viable count in Bacillus megaterium seed liquid is 1.2 × 109-8.6×109CFU/ml。
The above-mentioned preparation processes are all according to the prior art and are not defined in detail.
According to the invention, the bacillus subtilis is preferably a bacillus subtilis strain p-1 which is purchased from China agricultural microbial culture collection management center and has the collection number of ACCC No. 11025.
The bacillus megaterium is plant growth-promoting rhizobacteria (PGPR), and has the characteristics of stronger capability of decomposing lipoprotein and phosphoprotein in raw materials, 110-DEG C high temperature resistance, high salt resistance, strong colonization capability and high propagation speed compared with other similar bacteria; the bacillus megatherium can be fast colonized at the rhizosphere of crops after fully utilizing protein applied to soil in the fermentation production process of microbial inoculum and the soil, fully utilizing amino acid resources such as protein nutrient and tryptophan, converting into indoleacetic acid (IAA) through aerobic fermentation, improving the micro-ecological environment of the rhizosphere soil, keeping the status of dominant bacteria, occupying the living space of harmful bacteria, degrading organophosphorus pesticide, converting invalid phosphorus into effective phosphorus, improving the absorption and utilization efficiency of the phosphorus in the soil, and recovering ecological balance.
The bacillus subtilis has the specific function of secreting a large amount of active enzymes such as alpha-amylase, protease, cellulase and the like in the growth and propagation processes, can produce a large amount of water-soluble extracellular polysaccharide in the fermentation process, converts organic waste in soil into small molecular substances which are easily absorbed and utilized by crops, can secrete polysaccharide substances in the growth and propagation processes of bacteria, and has the functions of preserving water, preserving fertilizer and improving soil fertility.
An agricultural microbial agent rich in high-content live bacteria, compound amino acids and active peptides is prepared by the method, wherein the effective live bacteria number in the microbial agent is 10-50 hundred million/g, the mass content of free amino acids is 4-12wt%, and the mass content of the active peptides is 11-22 wt%.
The application of the microbial agent of the invention is as follows: the fertilizer synergist is used for increasing the yield of vegetables, melons, fruit trees and grain and cotton crops or is compounded with a compound fertilizer and an organic-inorganic compound fertilizer for use.
Preferably, the application method is used for increasing the yield of vegetables, melons, fruit trees and grain and cotton crops, and comprises the following steps: uniformly spreading the microbial agent on the soil surface before planting crops, and ploughing into soil, or according to the mass ratio: mixing soil according to the proportion of 1:20, applying the mixture into planting holes or sowing holes, and then transplanting seedlings or sowing;
according to the invention, the amount of the microbial inoculum is preferably: 5-15 kg of vegetable crops per mu, 10-20 kg of fruit trees per mu, 10-20 kg of melon crops per mu and 5-15 kg of grain and cotton crops per mu;
the dosage of the microbial inoculum during hole application is as follows: 3-6 kg of vegetable crops per mu and 5-10 kg of melons per mu.
According to the invention, when the microbial agent is used as a fertilizer synergist, 60-120 kg of the microbial agent is preferably added to each ton of fertilizer.
The invention has the following excellent effects:
1. the microbial agent contains a large amount of high-activity spore dormancy bodies of bacillus megatherium and bacillus subtilis, and the number of effective live bacteria can reach as high as 50 hundred million/g
The microbial inoculum product prepared by the invention contains billions of functional spores per gram, wherein the bacillus megaterium spores with strong specific functions account for more than 80 percent, has strong stress resistance, can tolerate high-salt and high-temperature environments, has strong colonization capacity, can be quickly propagated into dominant floras when being applied to soil, decomposes organic phosphorus in the soil, converts ineffective phosphorus into effective phosphorus, and effectively improves the absorption efficiency of the phosphorus in the soil. The bacillus megaterium can also fully utilize protein nutrients, tryptophan and other amino acid resources in the fermentation production process of the microbial inoculum and soil, and can be converted into indoleacetic acid (IAA) through aerobic fermentation, wherein the indoleacetic acid is a natural plant growth promoter and can promote seed germination, crop growth and developed root system.
2. The microbial agent contains rich plant source free amino acids, active peptides and rich strain metabolites
The microbial agent is rich in a large number of living spores, 17 free amino acids (lysine, arginine, glutamic acid, leucine, isoleucine, valine, alanine, glycine, aspartic acid, threonine, serine, cystine, methionine, tyrosine, phenylalanine, histidine and proline), active peptides and polysaccharides with different molecular weights, is rich in nutrient content and high in thallus activity, has a shelf life of more than 2 years, and is easy to absorb by crops, so that the synthesis of dry substances is promoted, the product is thicker in color and luster, larger in size, higher in VC and sugar content, and the yield and quality of the crops are greatly improved. The amino acid and the active peptide can promote photosynthesis, improve the absorption and transportation efficiency of nutrient substances such as trace elements and the like in plants, stimulate the root systems of crops to be developed, strengthen the branches and the stems of the crops, promote the flowering and the fruit setting of the crops, enhance the cold resistance, the drought resistance, the disease resistance and the insect resistance of the crops, enable the crops to have fast fruit expansion and high fruit setting rate, and prolong the harvest time of the crops. The combination of polysaccharide and other substances in the microbial inoculum and nutrients in the fertilizer can reduce the loss of nitrogen, phosphorus and potassium nutrients of the chemical fertilizer and the fixation of phosphorus elements, promote the transport rate of the nutrient elements in crops, and improve the absorption and utilization efficiency of the crops on the fertilizer.
3. The preparation method of the invention has high efficiency and good comprehensive effect
The method uses two microorganisms to ferment the organic raw material rich in protein, firstly uses bacillus subtilis to decompose casein and silk protein in the raw material, and then uses bacillus megatherium to decompose lipoprotein and phosphoprotein in the raw material. The method reduces the mutual influence among microorganisms, improves the conversion efficiency of free amino acid and active peptide, and can play a good role in improving the utilization rate of fertilizer and increasing the yield of crops under the condition of using a plurality of kilograms in each mu of land. The field experiment result shows that compared with a blank control, the microbial inoculum increases the yield of 19.49%, 25.98%, 7% or more of the sugar-acid ratio and 11.41% of the color pepper in a greenhouse tomato planting experiment, advances the harvesting period by 3-7 days, and increases the content of vitamin C and soluble solids by 15% or more.
4. The preparation method has simple process and short production period
The invention uses wheat bran, soybean meal and the like as raw materials, does not need chemical process treatment and additional enzyme enzymolysis, is completely realized by microbial fermentation in the whole production process, does not need complicated and energy-consuming post-treatment processes such as separation, purification and the like, and also does not need additional living microorganisms, thereby greatly reducing the production cost of the compound amino acid and active peptide microbial inoculum. The bacillus subtilis and the bacillus megatherium are fermented in a combined way, the whole fermentation period is about 3 days, and a large amount of microbial spores are obtained while a large amount of metabolites such as free amino acids, active peptides, polysaccharides, indoleacetic acid and the like are obtained.
5. The fermentation and preparation method of the microbial inoculum of the invention has scientific and reasonable design
The invention carries out high-density fermentation on the bacterial strain through bacillus subtilis to obtain active peptide, free amino acid and polysaccharide, then adds the high-temperature resistant active functional bacterial strain bacillus megaterium to carry out propagation of the composite bacterial strain, stops fermentation when the spore production rate of the bacillus megaterium reaches more than 90 percent, and then dries and crushes the bacillus megaterium to obtain the composite amino acid and active peptide microbial inoculum which contains 17 kinds of free amino acid, polysaccharide, active peptide and living spore, has rich nutrient content and high activity of the bacteria. The fertilizer efficiency of the active peptide microbial inoculum is greatly improved by the synergistic effect of a plurality of organic nutrients and microorganisms, and a good effect is obtained through field experiments. The field experiments of the two crops show that compared with the treatment without the microbial inoculum, the microbial inoculum of the invention has the advantages that the yield of the strawberries in the greenhouse is increased by 7.2 to 12.5 percent, the Vc content is increased by 6.2 to 7.9 percent, the tomato yield is increased by 7.3 to 11.2 percent, and the Vc content is increased by 6.9 to 9.1 percent.
Drawings
FIG. 1 is a photograph showing electrophoresis of fermented active peptide proteins in microbial agents prepared in examples 1 to 4 of the present invention, wherein the electrophoresis is shown in lane 1, example 1, lane 2, example 2, lane 3, example 3, lane 4, and example 4.
FIG. 2 is a chromatogram of 17 free amino acids in the microbial agent prepared in example 1 of the present invention.
FIG. 3 is a chromatogram of 17 free amino acids in the microbial inoculum prepared in example 2 of the present invention.
FIG. 4 is a chromatogram of 17 free amino acids in the microbial inoculum prepared in example 3 of the present invention.
FIG. 5 is a chromatogram of 17 free amino acids in the microbial inoculum prepared in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further clearly explained below with reference to specific examples, but is not to be construed as being limited in any way. The experimental methods in the following examples are all conventional methods unless otherwise specified; the percentages in the following examples are by mass unless otherwise specified.
The bacillus megaterium selects HB strain, and is preserved in the China general microbiological culture Collection center in 2016, 1, 7 and the preservation number is CGMCC NO. 11961.
The bacillus subtilis is ACCC NO.1102, purchased from China agricultural microorganism culture preservation management center.
Example 1: a preparation method of a polypeptide agricultural microbial agent comprises the following steps:
(1) preparation of functional bacteria bacillus subtilis seed liquid
A. Activating a bacillus subtilis strain p-1 stored at a low temperature on an LB plate culture medium, selecting a single strain, and culturing the single strain on an LB culture medium slant culture medium at 37 ℃ for 24 hours to obtain an activated strain;
B. scraping a ring of the activated strain in the step A by using a sterile inoculating ring, inoculating the bacteria into 50ml of liquid seed culture medium, and culturing at 35 ℃ for 18h to obtain the bacillus subtilis seed solution, wherein the number of viable bacteria in the seed solution is 3.8 x 109CFU/ml。
(2) Preparation of functional bacterium bacillus megaterium seed liquid
a. Activating a bacillus megaterium strain HB stored at a low temperature on an LB plate culture medium, selecting a single strain, and culturing the single strain on an LB slant culture medium at 37 ℃ for 24 hours to obtain an activated strain;
b. scraping a ring of the activated strain in the step A by using a sterile inoculating ring, inoculating the bacteria into 50ml of liquid seed culture medium, and culturing at 35 ℃ for 18h to obtain a bacillus megaterium seed solution, wherein the number of viable bacteria in the seed solution is 1.9 x 109CFU/ml。
(3) Preparation of the mix
Taking 40 parts of soybean meal, 10 parts of rice straw powder, 10 parts of wheat bran, 25 parts of vinasse and 5 parts of corn flour by mass, putting into a container, uniformly mixing, adding water to adjust the water content to 60%, and standing for 18 hours at the temperature of 30 ℃; steaming at 120 deg.C for 25min to obtain mixture, and cooling;
(4) preparation of the fermentation
According to the mass parts, the mixture prepared in the step (3) is inoculated into the bacillus subtilis seed liquid prepared in the step (1) according to the inoculum size of 30%, the mixture is uniformly mixed, the mixture is placed for 28 hours at the temperature of 35 ℃, then the mixture is uniformly stirred, the mixture is inoculated into the bacillus megaterium seed liquid prepared in the step (2) according to the inoculum size of 15%, the mixture is uniformly mixed, the temperature is increased to 38 ℃, the fermentation is continued for 35 hours, and the temperature is reduced to the room temperature after the fermentation is finished, so that a fermented product is obtained;
(5) preparation of microbial agent
And (4) drying the fermentation product in the step (4) at the temperature of 80 ℃, and crushing to obtain the agricultural microbial agent rich in high-content live bacteria, compound amino acid and active peptide.
The detection is carried out according to the method in the national standard GB/T2087-2006, and the result is as follows: the microbial agent contains effective viable bacteria 35 hundred million/g, free amino acid 4.9% and active peptide 11.5%.
As can be seen from FIGS. 1 and 2, the obtained microbial preparation contains 17 amino acids and multiple active peptides.
Example 2: a preparation method of a polypeptide agricultural microbial agent comprises the following steps:
(1) the functional bacteria Bacillus subtilis seed liquid is prepared in the same way as in example 1, and the number of viable bacteria in the seed liquid is 3.2 x 109CFU/ml。
(2) The functional bacteria Bacillus megaterium seed liquid is prepared in the same way as in example 2, and the number of viable bacteria in the seed liquid is 2.2 x 109CFU/ml。
(3) Preparation of the mix
Taking 80 parts of soybean meal, 5 parts of peanut straw powder, 5 parts of wheat bran, 5 parts of vinasse and 5 parts of corn flour by mass, putting the materials into a container, uniformly mixing, adding water to adjust the water content to 70%, and placing the mixture for 24 hours at the temperature of 28 ℃; steaming at 121 deg.C for 20min to obtain mixture, and cooling;
(4) preparation of the fermentation
Taking the mixture prepared in the step (3), inoculating the bacillus subtilis seed liquid prepared in the step (1) according to 15% of inoculation amount, uniformly mixing, placing for 30 hours at 34 ℃, then uniformly stirring, inoculating the bacillus megaterium seed liquid prepared in the step (2) according to 20% of inoculation amount, uniformly mixing, raising the temperature to 39 ℃, continuing to ferment for 38 hours, and cooling to room temperature after fermentation is finished to obtain a fermented product;
(5) preparation of microbial agent
And (4) drying the fermentation product in the step (4) at 90 ℃, and crushing to obtain the agricultural microbial agent rich in high-content live bacteria, compound amino acid and active peptide.
The detection is carried out according to the method in the national standard GB/T2087-2006, and the result is as follows: the microbial agent contains 49 hundred million/g of effective live bacteria, 11.6 percent of free amino acid and 20.5 percent of active peptide.
As can be seen from FIGS. 1 and 3, the obtained microbial preparation contains 17 amino acids and multiple active peptides.
Example 3: a preparation method of a polypeptide agricultural microbial agent comprises the following steps:
(1) the functional bacteria Bacillus subtilis seed liquid is prepared in the same way as in example 1, and the number of viable bacteria in the seed liquid is 3.72 x 109CFU/ml。
(2) The functional bacteria Bacillus megaterium seed liquid is prepared in the same way as in example 2, and the number of viable bacteria in the seed liquid is 2.89 x 109CFU/ml。
(3) Preparation of the mix
Taking 65 parts of soybean meal, 15 parts of corn straw powder, 5 parts of wheat bran, 10 parts of vinasse and 5 parts of corn flour according to the parts by mass, putting the materials into a container, uniformly mixing, adding water to adjust the water content to 65%, and placing the mixture for 20 hours at the temperature of 30 ℃; steaming at 121 deg.C for 25min to obtain mixture, and cooling;
(4) preparation of the fermentation
According to the mass parts, the mixture prepared in the step (3) is inoculated into the bacillus subtilis seed liquid prepared in the step (1) according to the inoculum size of 25%, the mixture is uniformly mixed, the mixture is placed for 28 hours at the temperature of 35 ℃, then the mixture is uniformly stirred, the mixture is inoculated into the bacillus megaterium seed liquid prepared in the step (2) according to the inoculum size of 20%, the mixture is uniformly mixed, the temperature is increased to 40 ℃, the fermentation is continued for 38 hours, and the temperature is reduced to the room temperature after the fermentation is finished, so that a fermented product is obtained;
(5) preparation of microbial agent
And (4) drying the fermentation product in the step (4) at 100 ℃, and crushing to obtain the agricultural microbial agent rich in high-content live bacteria, compound amino acid and active peptide.
The detection is carried out according to the method in the national standard GB/T2087-2006, and the result is as follows: the microbial agent contains 28 hundred million/g of effective viable bacteria, 7.88 percent of free amino acid and 11.1 percent of active peptide.
As can be seen from FIGS. 1 and 4, the obtained microbial preparation contains 17 amino acids and multiple active peptides.
Example 4: a preparation method of a polypeptide agricultural microbial agent comprises the following steps:
(1) the functional bacteria Bacillus subtilis seed liquid is prepared in the same way as in example 1, and the viable count of the seed liquid is 3.89 x 109CFU/ml。
(2) The functional bacteria Bacillus megaterium seed liquid is prepared in the same way as in example 2, and the viable count of the seed liquid is 2.12 x 109CFU/ml。
(3) Preparation of the mix
Taking 60 parts of soybean meal, 15 parts of corn straw powder, 5 parts of wheat bran, 10 parts of vinasse and 10 parts of corn flour according to the parts by mass, putting the materials into a container, uniformly mixing, adding water to adjust the water content to 65%, and placing the mixture for 20 hours at the temperature of 30 ℃; steaming at 121 deg.C for 20min to obtain mixture, and cooling;
(4) preparation of the fermentation
According to the mass parts, inoculating the mixture prepared in the step (3) into the bacillus subtilis seed liquid prepared in the step (1) according to the inoculation amount of 20%, inoculating the bacillus megaterium seed liquid prepared in the step (2) according to the inoculation amount of 20%, uniformly mixing, standing at 35 ℃ for 28 hours, uniformly stirring, increasing the temperature to 40 ℃, continuing to ferment for 42 hours, and cooling to room temperature after fermentation is finished to obtain a fermented product;
(5) preparation of microbial agent
And (4) drying the fermentation product in the step (4) at 110 ℃, and crushing to obtain the agricultural microbial agent rich in high-content live bacteria, compound amino acid and active peptide.
The detection is carried out according to the method in the national standard GB/T2087-2006, and the result is as follows: the microbial agent contains 20 hundred million/g of effective viable bacteria, 6.1 percent of free amino acid and 11.59 percent of active peptide.
As can be seen from FIGS. 1 and 5, the obtained microbial preparation contains 17 amino acids and multiple active peptides.
Comparative example 1
A method of preparing an agricultural microbial inoculant, as described in example 1, except that: in the method, only the bacillus subtilis seed solution is inoculated for fermentation, and the fermentation conditions are the same as those in example 1.
Comparative example 2
A method of preparing an agricultural microbial inoculant, as described in example 1, except that: in the method, only the seed liquid of the bacillus megaterium is inoculated for fermentation, and the fermentation conditions are the same as those of the example 1.
Comparative example 3
A method of preparing an agricultural microbial inoculant, as described in example 1, except that: the method adopts the bacillus subtilis seed solution and the bacillus megaterium seed solution to be mixed and then inoculated, and the inoculation amount and the fermentation conditions are the same as those in the example 1.
Comparative example 4
A method of preparing an agricultural microbial inoculant, as described in example 1, except that: the bacillus megaterium adopted by the method is a common commercial bacillus megaterium which is purchased from China general microbiological culture collection center with the collection number: CGMCCNO.1.1870.
Experimental example: the application effects of examples 1 to 4 and comparative examples 1 to 4 are as follows:
experiment time: month 9 to 2016 and 8.
Longevity greenhouse tomato test: let 8 treatments for examples 1-4 and comparative examples 1-4. The area of each cell is 66 square meters, the dosage of the microbial inoculum products of the examples 1-4 and the comparative examples 1-4 is 5 kilograms per mu, and meanwhile, the microbial inoculum is not applied as a control example. The application method is that the tomato seedlings are planted in the planting holes before the tomatoes are transplanted, the tomato seedlings are mixed with soil, other fertilizers are applied according to the habit of farmers, and all management measures are consistent. The application effect is shown in tables 1 and 2.
TABLE 1 tomato yield, Vc content and soluble solids content test results of examples and comparative examples, and comparative examples
TABLE 2 tomato application Effect% of examples 1-4 compared to control%
2. The near-Yi open grape test: let 8 treatments for examples 1-4 and comparative examples 1-4. The area of each cell is 30 square meters, the dosage of the microbial inoculum products of the examples 1-4 and the comparative examples 1-4 is 8 kilograms per mu, and meanwhile, the microbial inoculum is not applied as a control example. The application method is that the grape seedlings are planted in the cultivation holes before the grape is transplanted and mixed with soil, and other fertilizers are applied according to the habit of farmers, and all management measures are consistent. The application effects are shown in tables 3 and 4.
TABLE 3 test results of glucose yield, sugar acid ratio and soluble solid content of examples, comparative examples and comparative examples
TABLE 4 application Effect% of grapes of examples 1-4 compared to control example%
3. The near-Yi greenhouse color pepper test: let 5 treatments for examples 1-4 and comparative examples 1-4. The area of each cell is 20 square meters, the dosage of the microbial inoculum products in the embodiments 1-4 is 8 kilograms per mu, and the comparison example is comparison without applying the microbial inoculum. The application method is that the microbial inoculum is used as a base fertilizer, is mixed with soil uniformly and then is applied and turned over, other fertilizers are applied according to the habit of farmers, and all management measures are consistent. The application effects are shown in tables 5 and 6.
TABLE 5 test results of color pepper yield and Vc content of examples, comparative examples and comparative examples
TABLE 6 increase in yield in color pepper application% of examples 1-4 compared to comparative example%
| Item | Yield increase compared to control example% | Compared with the comparative example, the Vc is improved |
| Example 1 | 10.09 | 12.6 |
| Example 2 | 11.41 | 18.27 |
| Example 3 | 9.82 | 18.9 |
| Example 4 | 8.57 | 17.64 |
In conclusion, the microbial agent contains a large number of spore dormant bodies of high-activity bacillus megaterium and bacillus subtilis, the number of effective viable bacteria can reach 50 hundred million/g, 17 free amino acids (lysine, arginine, glutamic acid, leucine, isoleucine, valine, alanine, glycine, aspartic acid, threonine, serine, cystine, methionine, tyrosine, phenylalanine, histidine and proline), active peptides and polysaccharides with different molecular weights, the nutrient content is rich, the activity of the bacteria is high, the transport rate of nutrient elements in crops is promoted, the absorption and utilization efficiency of the crops on fertilizers is improved, and the yield and quality of the crops are improved.
Claims (10)
1. A preparation method of a polypeptide agricultural microbial agent comprises the following steps:
(1) preparation of functional bacteria bacillus subtilis seed liquid
A. Activating the bacillus subtilis strain by using an LB slant culture medium to obtain an activated bacillus subtilis strain; the bacillus subtilis is a bacillus subtilis strain p-1 purchased from China agricultural microbial strain preservation management center and having a preservation number of ACCC No. 11025;
B. inoculating the activated bacillus subtilis strain into a liquid seed culture medium, and culturing at 32-37 ℃ for 16-24h to obtain a bacillus subtilis seed liquid for later use;
(2) preparation of functional bacterium bacillus megaterium seed liquid
a. Activating the bacillus megaterium strain by using an LB slant culture medium to obtain an activated bacillus megaterium strain; the Bacillus megaterium (A), (B) and (C)Bacillus megaterium) Strain HB, deposited in the china general microbiological culture collection center on 1 month 7 days 2016, address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the preservation numbers are as follows: CGMCC NO. 11961;
b. inoculating the activated bacillus megaterium strain into a liquid seed culture medium, and culturing at 32-37 ℃ for 16-24h to obtain a bacillus megaterium seed liquid for later use;
(3) preparation of the Pre-treated mix
Taking and uniformly mixing the soybean meal, the straw powder, the wheat bran, the vinasse and the corn flour according to the proportion, wherein the soybean meal, the straw powder, the wheat bran, the vinasse and the corn flour are in parts by weight as follows: 40-80 parts of soybean meal, 6-28 parts of straw powder, 5-22 parts of wheat bran, 5-20 parts of vinasse and 5-15 parts of corn flour; adding water to adjust the water content to 60-70wt%, standing at 20-35 deg.C for 8-24 hr, steaming at 115-121 deg.C for 20-40min, and cooling to obtain pretreated mixture;
(4) inoculating the pretreated mixture prepared in the step (3) into the bacillus subtilis seed liquid prepared in the step (1) according to the inoculation amount of 8-30wt%, uniformly mixing, fermenting for 24-30 hours at the temperature of 32-36 ℃, and uniformly stirring;
(5) then inoculating the bacillus megaterium seed solution prepared in the step (2) according to the inoculation amount of 12-18wt%, uniformly mixing, raising the temperature to 38-42 ℃, continuing to ferment for 24-48 hours, cooling to room temperature after fermentation is finished, drying and crushing to obtain the agricultural microbial agent rich in high-content live bacteria, compound amino acid and active peptide.
2. The method for preparing the polypeptide agricultural microbial agent as claimed in claim 1, wherein the components and weight percentages of the LB slant culture medium in the steps (1) and (2) are as follows: tryptone 0.8-1.2%, yeast extract 0.4-0.6%, sodium chloride 0.4-0.6%, agar powder 1.5-2.0%, and water in balance, wherein the pH value is 7.2.
3. The method for preparing a polypeptide agricultural microbial inoculum according to claim 1, wherein in the step A and the step a, the activation is that single colonies are picked and cultured on an LB culture medium slant culture medium for 18-24h at 37 ℃; the components and weight percentages of the liquid seed culture medium in the step B and the step B are as follows: 2.0-3.0% of glucose, 1.1-1.8% of corn steep liquor dry powder, 1.5-4.0% of soybean powder, 0.1-0.2% of yeast extract, 0.01-0.03% of potassium dihydrogen phosphate, 0.01-0.02% of sodium chloride, 0.01-0.02% of magnesium sulfate and the balance of water, wherein the pH value is 7.0-7.3.
4. The method for preparing a polypeptide agricultural microbial inoculant according to claim 1, wherein the inoculation amount of the bacillus subtilis seed liquid in the step (4) is 10-20wt%, the fermentation temperature is 34-36 ℃, and the fermentation time is 26-30 hours.
5. The method for preparing a polypeptide agricultural microbial inoculant according to claim 1, wherein the inoculation amount of the bacillus megaterium seed liquid in the step (5) is 14-18wt%, the fermentation temperature is 40-42 ℃, and the fermentation time is 40-48 hours.
6. The preparation method of the polypeptide agricultural microbial agent as claimed in claim 1, wherein the drying temperature in step (5) is 50-110 ℃, the straw powder in step (3) is one or a mixture of more than two of corn straw powder, wheat straw powder or rice straw powder, the grain size of the straw powder and the vinasse is less than or equal to 0.6cm, and the grain size of the wheat bran, the soybean meal and the corn meal is 0.4-1 mm.
7. The method for preparing a polypeptide agricultural microbial inoculant according to claim 1, wherein the viable count of the bacillus subtilis seed solution prepared in the step (1) is 5.0 x 109-1.5×1010CFU/ml, the viable count in the bacillus megaterium seed liquid prepared in the step (2) is 1.2 multiplied by 109 -8.6×109CFU/ml。
8. An agricultural microbial agent rich in high-content live bacteria, compound amino acids and active peptides is prepared by the method of claim 2, wherein the microbial agent contains 10-50 hundred million/g of effective live bacteria, 4-12wt% of free amino acids and 11-22wt% of active peptides.
9. The application of the agricultural microbial agent rich in high-content live bacteria, compound amino acids and active peptides in claim 8: the application method is used for increasing the yield of vegetables, melons, fruit trees and grain and cotton crops and comprises the following steps: uniformly spreading the agricultural microbial agent on the soil surface before planting crops, and ploughing and burying the agricultural microbial agent into soil, wherein the dosage of the microbial agent in spreading is as follows: 5-15 kg of vegetable crops per mu, 10-20 kg of fruit trees per mu, 10-20 kg of melon crops per mu and 5-15 kg of grain and cotton crops per mu;
or the agricultural microbial agent and the planting soil are mixed according to the mass ratio: 1:20, mixing with soil, applying to planting holes or sowing holes, transplanting seedlings or sowing, wherein the amount of the microbial inoculum in hole application is as follows: 3-6 kg of vegetable crops per mu and 5-10 kg of melons per mu.
10. The application of the agricultural microbial agent rich in high-content live bacteria, compound amino acids and active peptides in claim 8:
the microbial agent is compounded with compound fertilizers and organic-inorganic compound fertilizers to be used as a fertilizer synergist, and 60-120 kg of the microbial agent is added into each ton of the fertilizer.
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| CN108640719A (en) * | 2018-06-27 | 2018-10-12 | 安徽瑞然生物药肥科技有限公司 | A kind of preparation method of amino acid foliage fertilizer |
| CN111592994A (en) * | 2019-02-21 | 2020-08-28 | 中国科学院南京土壤研究所 | Bacillus subtilis microbial inoculum with growth promoting function and preparation method thereof |
| CN110301182B (en) * | 2019-05-29 | 2021-05-25 | 金华市艾力生物科技有限公司 | Preparation method of seed initiator |
| CN110526760A (en) * | 2019-10-09 | 2019-12-03 | 吉林省农业科学院 | A kind of compound amino acid fertilizer and its application method improving nursery stage rice plant cold resistance |
| CN111302853A (en) * | 2020-02-16 | 2020-06-19 | 金葵子环保生物科技(国际)有限公司 | Microbial phosphorus-potassium fertilizer and preparation method thereof |
| CN111466413B (en) * | 2020-04-03 | 2021-05-18 | 上海辰山植物园 | Quercus plant root-promoting microbial microcapsule microbial inoculant and preparation method thereof |
| CN111621433B (en) * | 2020-04-10 | 2022-04-08 | 四川轻化工大学 | Bacillus megaterium for producing cellulase, preparation method and application |
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