CN111304103A - Surface active complex enzyme, bionic peat humic acid, and preparation method and application thereof - Google Patents

Surface active complex enzyme, bionic peat humic acid, and preparation method and application thereof Download PDF

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CN111304103A
CN111304103A CN201811517775.0A CN201811517775A CN111304103A CN 111304103 A CN111304103 A CN 111304103A CN 201811517775 A CN201811517775 A CN 201811517775A CN 111304103 A CN111304103 A CN 111304103A
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complex enzyme
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CN111304103B (en
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周霞萍
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Shanghai Zhenyan Biotechnology Co ltd
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Abstract

The invention discloses a surface active complex enzyme, bionic peat humic acid, a preparation method and application thereof. The preparation method of the surface active complex enzyme comprises the following steps: inoculating the mixed strain into a mixed culture medium for fermentation culture; the mixed strain comprises yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis; the mixed culture medium is a mixture of PDA culture medium and MRS culture medium. The surface active complex enzyme can be connected with a reaction interface under the condition of micro-water or hydrophily, so that harmful bacteria are removed, the purpose of reducing external supplied energy is achieved, and the straw lignin can be quickly oxidized or ectopically condensed into humic acid. The bionic peat humic acid can be used as a seedling and cultivation substrate for rice, vegetables and flowers, and can also be used for removing moss and spore coats in rice fields, humic acid bio-organic fertilizers, soil improvement and restoration agents, bio-stimulants, Chinese herbal medicines and other soilless culture plants, quick fermentation and field return and the like.

Description

Surface active complex enzyme, bionic peat humic acid, and preparation method and application thereof
Technical Field
The invention relates to a surface active complex enzyme, bionic peat humic acid, a preparation method and application thereof.
Background
Peat is a natural marsh land product (also called peat or peat) formed for thousands of years, is coal with the lowest coalification degree, is also the most original state of the coal, and has the advantages of sterility, no toxicity, no pollution and good ventilation performance. The organic fertilizer has the advantages of light weight, water retention, fertilizer retention, contribution to microbial activity, enhanced biological performance, rich nutrition, being a seedling culture medium, a good soil conditioner, and high organic matters, humic acid and nutrient components. However, peat is a non-renewable resource, and some mining behaviors are very harmful to the environment.
Peat humic acid is a term of peat geology, refers to organic mixed acid which can be extracted by dilute caustic solution in peat, is an important component in peat organic matter, and is a complex mixture composed of macromolecular hydroxy aromatic carboxylic acid. The humic acid content in the peat can generally reach 20-50%, and the content of the humic acid is mainly related to the organic matter content and different peat types.
Because the resources of non-renewable resources, namely peat, are limited, most of China is imported from Finland, Russia, Indonesia and the like for medium-high-grade peat used for flowers, vegetables, rice and the like. Besides the geological chemical coal-forming condition, the humic acid resources such as peat and the like can be formed, and the crop straws are converted into the bionic peat humic acid through the biotechnology, and the practice proves that the humic acid has similar effect. The bionic peat humic acid has the following excellent effects: (1) the organic matter of the soil can be improved, the growth of the plants can be promoted, and raw materials are provided for photosynthesis; (2) the soil is loosened and penetrated through the physical properties of the soil; (3) the method can adjust soil chemical conditions such as soil pH value, ion balance and the like, and increase cation saturation; (4) it can provide energy for the biological characters of soil organisms and microorganisms; (5) the fertilizer can balance physical and chemical properties with self-cleaning capability and improve the stress resistance of crops; (6) it can increase the nutrient components of plant and raise the utilization rate of N, P and K.
In the prior art, the biotechnology for converting crop straws into bionic peat humic acid mainly comprises the following two technologies: the first method is as follows: the livestock and poultry metabolite compost can be used for fermenting and producing straw humic acid fertilizer, but the fermentation time is long, if the equipment sealing performance is poor, the production environment has peculiar smell, and the metabolite contains more salt and more metal ions, and can not be fermented into a seedling culture product for producing peat substrate without treatment, which is in the way of preventing the soil from being comprehensively repaired. The second method comprises the following steps: the crop straws can also be changed into fly ash or biochar through a thermoelectric device, but more nutrient components are lost in the preparation process. For example, in the case of rice straw, 4% of nutrients such as crude protein, 1% of crude fat, and 35% of crude fiber in rice straw are lost. In addition, in the prior art, the raw materials such as mushroom residues, vinasse and the like are fermented by containing biological enzyme, and the raw materials can also contain biomass wastes such as tree branches and leaves; even if the useful enzyme alone is used, the overall fermentation rate is slow because of the difference in the performance of the enzyme. The above problems are urgently needed to be solved.
Disclosure of Invention
The invention provides a surface active complex enzyme, a bionic peat humic acid and a preparation method and application thereof, and aims to overcome the defects of long compost fermentation time, peculiar smell generation, poor operating environment, influence of raw materials on products, high salinity, easiness in pollution and the like in the prior art under the conditions of less equipment investment, no sealing and the like. The surface active complex enzyme can be connected with a reaction interface under the condition of micro-water or hydrophily, so that harmful bacteria are removed, the external supplied energy is reduced, the straw lignin can be quickly oxidized or ectopically condensed to become humic acid, the biological fermentation time is shortened, and the surface active complex enzyme is safe and environment-friendly. The bionic peat humic acid can be used as a seedling and cultivation substrate for rice, vegetables and flowers, and can also be used for removing moss and spore coats in rice fields, humic acid bio-organic fertilizers, soil improvement and restoration agents, bio-stimulants, Chinese herbal medicines and other soilless culture plants, quick fermentation and field return and the like.
The invention solves the technical problems through the following technical scheme.
The invention provides a preparation method of a surface active complex enzyme, which comprises the following steps: inoculating the mixed strain into a mixed culture medium for fermentation culture;
wherein the mixed strain comprises Yeast (Yeast), Rhizobium (Rhizobium), Penicillium (Penicillium), Lactobacillus plantarum (Lactobacillus plantarum) and Bacillus subtilis (Bacillus subtilis); the mixed culture medium is a mixture of a PDA culture medium and an MRS culture medium.
Wherein, the mass ratio of the yeast, the rhizobium, the penicillium herbaceum, the lactic acid bacteria and the bacillus subtilis can be conventional in the field, and is preferably (10-30): (1-5): (20-30): (20-25): (15-40), more preferably (10-15): 5: (15-20): (20-25): (35-40).
Wherein the amount of the mixed strain inoculated into the mixed culture medium is preferably 0.01 to 0.07%, more preferably 0.03 to 0.06%, for example 0.05%.
The PDA culture Medium is a potato glucose Agar culture Medium (i.e. Potatodextrose Agar (Medium) in the field, and corresponds to potato, glucose and Agar in English in sequence.
The preparation method of the PDA culture medium can be conventional in the field and is generally prepared by the following steps: weighing potatoes, peeling, and cutting into strips; adding deionized water in equal proportion into a container device, cooking in an electromagnetic container at 100 ℃ for 40-60 min until the shredded potatoes are soft and rotten, cutting off the power, filtering to remove potato dregs, then adding 1/5 weight of glucose and 1/6 agar powder, starting stirring until the potato shreds are completely dissolved, diluting to 2 times, and then placing in an autoclave (121 ℃ for 30min) to obtain the potato vermicelli.
The MRS culture medium is a conventional lactic acid bacteria culture medium in the field, consists of a plurality of components, is suitable for the growth of lactic acid bacteria, and is a culture medium for separating and culturing the lactic acid bacteria.
The preparation method of the MRS medium can be conventional in the field and is generally prepared by the following steps: placing 1 part of beef extract, 2 parts of peptone, 1 part of agar, 1 part of sodium chloride and 1 part of fulvic acid in 200 parts of distilled water, heating and dissolving for 30min, stirring until all the components are dissolved, diluting to 2 times, and placing in an autoclave (121 ℃, 30 min).
Wherein, the mass ratio of the PDA culture medium to the MRS culture medium can be conventional in the field, and is preferably 1: (1-3), more preferably 1: (1.5-2.5), for example, 1: 2.
the operation and conditions of the fermentation culture may be conventional in the art, among others. The temperature of the fermentation culture is generally 0 to 50 ℃, preferably 20 to 45 ℃, for example 30 ℃, 35 ℃, 36 ℃ or 37 ℃. The fermentation time is preferably 24-120 h, more preferably 58-105 h, such as 65h, 72h, 80h, 90h or 96 h.
The end point of the fermentation can be conventional in the art, preferably the enzyme activity index of the surface active complex enzyme reaches 85-326U/g, more preferably the enzyme activity index of the surface active complex enzyme reaches 150-325U/g, such as 172U/g, 175U/g, 177U/g, 195U/g or 215U/g.
The invention also provides the surface active complex enzyme prepared by the preparation method.
Wherein the surface active complex enzyme contains mixed enzyme capable of generating surface active cellulase, hemicellulase, protease, hydrolase, oxidoreductase, esterase, alkalophilic enzyme, lipopeptide, phospholipid, lipopolysaccharide, fatty acid, polysaccharide protein complex enzyme and the like.
In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase can be (8-12): (3-7): (18-22): (28-32): (28-32): (13-17), preferably 10: 5: 20: 30: 30: 15.
wherein the enzyme activity index of the surface active complex enzyme is preferably 85-326U/g, more preferably 150-325U/g, such as 172U/g, 175U/g, 177U/g, 195U/g or 215U/g
The invention also provides an application of the surface active complex enzyme in preparation of bionic peat humic acid by taking crop straws as raw materials.
Wherein the crop stalks can be conventional crop stalks in the field, such as corn stalks, rice straw stalks, soybean stalks, peanut stalks, cotton stalks and the like.
The invention also provides a preparation method of the bionic peat humic acid, which comprises the following steps: carrying out enzymolysis on the crop straws containing the surface active complex enzyme.
Wherein, the mass percentage of the surface active complex enzyme in the crop straws is preferably 0.01-0.10%, more preferably 0.02-0.06%, for example 0.03%.
Wherein the crop stalks can be conventional crop stalks in the field, such as corn stalks, rice straw stalks, soybean stalks, peanut stalks, cotton stalks and the like.
Wherein, the operation and condition of the enzymolysis can be the conventional operation and condition in the field. The temperature of the enzymolysis is preferably-15 to 70 ℃, more preferably 0 to 50, for example 30 ℃. The time for the enzymatic hydrolysis is preferably 1 to 8 days, more preferably 2 to 5 days, for example 4 days.
The invention also provides the bionic peat humic acid prepared by the preparation method.
Preferably, the biomimetic peat humic acid has an organic matter content of 65-90% (e.g., 68.35%, 70.51%, 75%, 77.15%, 80.33%, 85.02%, 85.05%, 88.52%), a water content of 5-30% (5.56%, 10.10%, 10.69%, 12.33%, 15.9%, 20.7%, 23.10%, 25.83%), a total humic acid content of 25-35% (e.g., 25.06%, 30.05%, 30.28%, 31.7%, 32.68%, 32.11%, 33.02%, 33.29%), a fulvic acid content of 10-30% (e.g., 10.28%, 11.67%, 11.78%, 12.66%, 12.79%, 13.18%, 13.5%, 13.81%), and the percentages are mass percentages of the components relative to the biomimetic peat humic acid.
Wherein the particle size of the bionic peat humic acid is 1-10 mm.
Wherein, the pH value of the bionic peat humic acid is preferably 6.0-8.0.
Wherein, the decomposition stability grade of the bionic peat humic acid is preferably 5 at the temperature of 0-10 ℃.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The raw materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the surface active complex enzyme can be connected with a reaction interface under the condition of micro-water or hydrophily, so that harmful bacteria are removed, the purpose of reducing external supplied energy is achieved, and the straw lignin can be quickly oxidized or ectopically condensed into humic acid. The bionic peat humic acid can be used as a seedling and cultivation substrate for rice, vegetables and flowers, and can also be used for removing moss and spore coats in rice fields, humic acid bio-organic fertilizers, soil improvement and restoration agents, bio-stimulants, Chinese herbal medicines and other soilless culture plants, quick fermentation and field return and the like.
When the bionic peat humic acid is used for removing moss coats in rice fields, the removal effect reaches 90-98%, the rate of inhibiting mixed bacteria is 80-90%, and the quick fermentation time is 2-5 days. When the bionic peat humic acid is used for vegetable seedling and flower substrates, the rate of inhibiting mixed bacteria is 70-85%, and the enzymolysis time of the straw into the bionic peat is 3-8 days. When the bionic peat humic acid is used for a bio-organic fertilizer, the fertilizer consumption can be reduced by 35-50%, and the crop yield can be increased by 12-35%. When the bionic peat humic acid is used as a soil improvement repairing agent and a biological stimulant, the crop quality can be improved, and the dosage of pesticides such as a growth regulator and the like is reduced by 30-70%. When the bionic peat humic acid is used for soilless culture planting of Chinese herbal medicines and the like, the effect is 105-150% of that of mineral humic acid biological stimulin.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples, the enzyme activity index is defined as: under experimental conditions, the enzyme amount for hydrolyzing a substrate to generate 1umol of reducing sugar per minute is 1 enzyme activity unit (U/mL), and the enzyme activity is measured by a DNS method: 100 μ L of 0.5% substrate solution (pH 7.5 Na)2HPO3And NaH2PO3Buffer solution preparation), adding the enzyme solution which is properly diluted by the buffer solution, fully and uniformly mixing, placing in a water bath at 50 ℃ for 10min, adding 100 mu L of DNS reagent, cooling by running water after boiling the water bath for 10min, measuring the absorbance value (OD value) at 550nn, taking the enzyme solution inactivated in advance at high temperature as a blank control (except for special specification, the substrate is guar gum), and referring specifically: GB/T36756 and 2018 tools enzyme activity determination general requirements. Each sample was measured 3 times and averaged.
In the following examples, the PDA culture Medium is a Potato Dextrose Agar culture Medium, referred to as "Potato Dextrose Agar" (Medium), which in turn corresponds to Potato Dextrose Agar, and English. The preparation method comprises the following steps: weighing potatoes, peeling, and cutting into strips; adding deionized water in equal proportion into a container device, cooking in an electromagnetic container at 100 ℃ for 40-60 min until the shredded potatoes are soft and rotten, cutting off the power, filtering to remove potato dregs, then adding 1/5 weight of glucose and 1/6 agar powder, starting stirring until the potato shreds are completely dissolved, diluting to 2 times, and then placing in an autoclave (121 ℃ for 30min) to obtain the potato vermicelli.
The MRS culture medium is prepared by the following steps: placing 1 part of beef extract, 2 parts of peptone, 1 part of agar, 1 part of sodium chloride and 1 part of fulvic acid in 200 parts of distilled water, heating and dissolving for 30min, stirring until all the components are dissolved, diluting to 2 times, and placing in an autoclave (121 ℃, 30 min).
In the following examples, the enzymatic activity index of the surface active complex enzyme was measured by UV-2600 UV spectrophotometer, Usneco Shanghai instruments Co.
The enzyme activity index of the surface active complex enzyme mentioned in the invention is the sum of the enzyme activity indexes of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase.
Example 1
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 15: 5: 20: 25: 40; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2; the inoculation amount of the mixed strain in the mixed culture medium is 0.05 percent; the fermentation time is 58h, the fermentation temperature is 36 ℃, and the enzyme activity index of the surface active complex enzyme is 215U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15 (obtained by separating each specific enzyme in the surface active complex enzyme, weighing respectively and calculating).
The surface tension of the surface active complex enzyme is measured by adopting a K100 surface tension meter of Germany Kruss company, and the surface tension is 5.86 mN/m.
The wetting angle of the surface active complex enzyme is measured by adopting a DSA 100 contact angle (wetting angle) instrument, and is 12.63 degrees.
2. Preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 4 days. The crop straw is corn straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.03%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; when the temperature is 0-10 ℃, the decomposition stability (decomposition degree) grade is 5; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 70.51 percent, and the content of water is 25.83 percent; the total humic acid content is 33.29%, the fulvic acid content is 12.66%, and the percentages are mass percentages of the components relative to the bionic peat humic acid.
3. The bionic peat humic acid prepared in the step 2 is used as a rice field seedling raising substrate, the seedling emergence is neat, and the seedling emergence rate reaches 98%.
Compared with a control group (the control group is the northeast peat seedling substrate), the fertility of soil humic acid is increased by 25 percent (calculated from the chemical fertilizer reduction angle), and the planted rice is superior in grinding, appearance, cooking and nutritional quality.
Example 2
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 10: 5: 20: 25: 35; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2.5; the inoculation amount of the mixed strain in the mixed culture medium is 0.03 percent; the fermentation time is 65h, the fermentation temperature is 35 ℃, and the enzyme activity index of the surface active complex enzyme is 177U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 4 days. The crop straws are rice straws, the mass ratio of the surface active complex enzyme in the crop straws is 0.02%, and the dosage of the crop straws is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; when the temperature is 0-10 ℃, the decomposition stability grade is 5; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 75 percent, and the content of water is 20.70 percent; the total humic acid content is 32.11 percent, the fulvic acid content is 11.67 percent, and the percentages are mass percentages of the components relative to the bionic peat humic acid respectively.
3. And (2) using the bionic peat humic acid prepared in the step (2) as an organic fertilizer (wherein the content of organic matters is 75%) for corn planting, and compared with a control group (blank, namely no fertilizer application), increasing the quantity of soil bio-enzyme by 8.5% (measured by an enzyme-linked immunosorbent assay), increasing the fertility of soil humic acid by 32% (here, the indexes of soil organic matters and the like representing the fertilizer are increased by 32%), and enabling the planted corn to have bright yellow appearance, sweet in cooked glutinous rice and good nutritional taste.
Example 3
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 10: 5: 15: 25: 35; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 1.5; the inoculation amount of the mixed strain in the mixed culture medium is 0.06 percent; the fermentation time is 72h, the fermentation temperature is 37 ℃, and the enzyme activity index of the surface active complex enzyme is 172U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 4 days. The crop straw is peanut straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.01%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; the decomposition stability grade is 5 when the temperature is 0-10 ℃; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 85.02 percent, and the content of water is 10.69 percent; the total humic acid content is 30.28 percent, the fulvic acid content is 13.50 percent, and the percentages are mass percentages of the components relative to the bionic peat humic acid respectively.
3. And (3) using the bionic peat humic acid prepared in the step (2) as biological stimulants (containing trace saccharomycetes and other metabolites such as gibberellin, amino acid, potassium, sodium, iron, zinc, magnesium and the like) for planting Chinese herbal medicine astragalus (planted according to the application amount of 100 kilograms per mu) to increase the antioxidant dismutase index of the planted astragalus SOD by 20% compared with a control group (the control group is a blank control group, namely the bionic peat humic acid is not used as the biological stimulants).
The method for testing the antioxidant dismutase index of the astragalus SOD comprises the following steps: inoculating activated 10 strains of plant lactobacillus into MGS liquid culture medium at a ratio of 1%, standing and culturing in a constant temperature incubator at 35 deg.C for 46h, centrifuging the fermentation liquid, and collecting supernatant to measure SOD activity.
Example 4
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 15: 5: 20: 20: 40; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2; the inoculation amount of the mixed strain in the mixed culture medium is 0.01 percent; the fermentation time is 96h, the fermentation temperature is 30 ℃, and the enzyme activity index of the surface active complex enzyme is 205U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 4 days. The crop straw is corn straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.01%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; the decomposition stability grade is 5 when the temperature is 0-10 ℃; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 80.33%, the content of water is 15.90%, the content of total humic acid is 31.70%, and the content of fulvic acid is 13.81%, wherein the percentages are mass percentages of the components relative to the bionic peat humic acid.
3. When the bionic peat humic acid prepared in the step 2 is used for removing the moss coats of the rice field, the removal effect reaches 90-98%, the rate of inhibiting mixed bacteria is 80-90%, and the quick fermentation time is 4 days.
Example 5
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 15: 5: 20: 20: 40; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2; the inoculation amount of the mixed strain in the mixed culture medium is 0.03 percent; the fermentation time is 105h, the fermentation temperature is 50 ℃, and the enzyme activity index of the surface active complex enzyme is 215U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 4 days. The crop straw is corn straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.01%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; the decomposition stability grade is 5 when the temperature is 0-10 ℃; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 68.35 percent, and the content of water is 23.10 percent; the total humic acid content is 25.06 percent, the fulvic acid content is 10.28 percent, and the percentages are mass percentages of the components relative to the bionic peat humic acid respectively.
3. The bionic peat humic acid prepared in the step 2 is used for flower substrates, the rate of inhibiting mixed bacteria is 92% (calculated according to a colony technique method), and the enzymolysis time is 4 days.
Example 6
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 15: 5: 20: 20: 40; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2; the inoculation amount of the mixed strain in the mixed culture medium is 0.05 percent; the fermentation time is 90h, the fermentation temperature is 45 ℃, and the enzyme activity index of the surface active complex enzyme is 195U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 4 days. The crop straw is corn straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.01%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; the decomposition stability grade is 5 when the temperature is 0-10 ℃; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 88.52 percent, and the content of water is 5.56 percent; the total humic acid content is 30.05 percent, the fulvic acid content is 13.18 percent, and the percentages are mass percentages of the components relative to the bionic peat humic acid respectively.
3. When the bionic peat humic acid prepared in the step 2 is used for the bio-organic fertilizer, compared with a control group (blank, i.e. no fertilizer application), the fertilizer consumption can be reduced by 35%, and the crop yield can be increased by 20%.
Example 7
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 15: 5: 20: 20: 40; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2; the inoculation amount of the mixed strain in the mixed culture medium is 0.01 percent; the fermentation time is 80h, the fermentation temperature is 50 ℃, and the enzyme activity index of the surface active complex enzyme is 195U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 1 day. The crop straw is corn straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.06%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; the decomposition stability grade is 5 when the temperature is 0-10 ℃; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 77.15 percent, and the content of water is 12.33 percent; the total humic acid content is 32.68 percent, the fulvic acid content is 11.78 percent, and the percentages are mass percentages of the components relative to the bionic peat humic acid respectively.
3. When the bionic peat humic acid prepared in the step 2 is used as a soil improvement repairing agent and a biological stimulant, the crop quality can be improved, and the dosage of pesticides such as a growth regulator and the like can be reduced by 55% (calculated by a comparative experiment method-a material weight method).
Example 8
1. Preparation of surface active complex enzyme
Inoculating the mixed strain into a mixed culture medium for fermentation culture;
in the mixed strain, the proportion of yeast, rhizobium, penicillium herbarum, lactobacillus plantarum and bacillus subtilis is 15: 5: 20: 20: 40; in the mixed culture medium, the mass ratio of the PDA culture medium to the MRS culture medium is 1: 2; the inoculation amount of the mixed strain in the mixed culture medium is 0.03 percent; the fermentation time is 120h, the fermentation temperature is 20 ℃, and the enzyme activity index of the surface active complex enzyme is 175U/g. In the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is 10: 5: 20: 30: 30: 15.
2. preparation of bionic peat humic acid
Carrying out enzymolysis on crop straws containing the surface active complex enzyme and an aqueous medium. The enzymolysis temperature is 30 ℃, and the enzymolysis time is 2 days. The crop straw is corn straw, the mass ratio of the surface active complex enzyme in the crop straw is 0.03%, and the dosage of the crop straw is 1 ton.
The pH value of the obtained bionic peat humic acid is 6-8; the decomposition stability grade is 5 when the temperature is 0-10 ℃; the grain size of the bionic peat humic acid is 1-10 mm.
In the bionic peat humic acid, the content of organic matters is 85.05 percent, and the content of water is 10.10 percent; the total humic acid content is 33.02 percent, the fulvic acid content is 12.79 percent, and the percentages are mass percentages of the components relative to the bionic peat humic acid respectively.
3. When the bionic peat humic acid prepared in the step 2 is used for soilless culture planting of Chinese herbal medicines and the like, the effect is 120 percent (calculated by a comparative experiment method-material weight method) of the mineral source humic acid bio-stimulin.

Claims (10)

1. A preparation method of a surface active complex enzyme is characterized by comprising the following steps: inoculating the mixed strain into a mixed culture medium for fermentation culture;
wherein the mixed strain comprises Yeast (Yeast), Rhizobium (Rhizobium), Penicillium (Penicillium), Lactobacillus plantarum (Lactobacillus plantarum) and Bacillus subtilis (Bacillus subtilis); the mixed culture medium is a mixture of a PDA culture medium and an MRS culture medium.
2. The method for preparing the surface active complex enzyme according to claim 1, wherein the mass ratio of the yeast, the rhizobium, the penicillium herbaceum, the lactic acid bacteria and the bacillus subtilis is (10-30): (1-5): (20-30): (20-25): (15-40), preferably (10-15): 5: (15-20): (20-25): (35-40);
and/or the mixed strain is inoculated in the mixed culture medium in an amount of 0.01-0.07%, preferably 0.03-0.06%, for example 0.05%.
3. The method for preparing surface active complex enzyme according to claim 1, wherein the mass ratio of the PDA culture medium to the MRS culture medium is 1: (1-3), preferably 1: (1.5-2.5), preferably 1: 2.
4. the preparation method of the surface active complex enzyme as claimed in claim 1, wherein the temperature of the fermentation culture is 20-45 ℃, preferably 30 ℃, 35 ℃, 36 ℃ or 37 ℃;
and/or the fermentation culture time is 24-120 h, preferably 58-105 h, more preferably 65h, 72h, 80h, 90h or 96 h;
and/or the end point of the fermentation is that the enzyme activity index of the surface active complex enzyme is 85-326U/g, preferably the enzyme activity index of the surface active complex enzyme is 150-325U/g, and more preferably the enzyme activity index of the surface active complex enzyme is 172U/g, 175U/g, 177U/g, 195U/g or 215U/g.
5. A surface active complex enzyme prepared by the method for preparing the surface active complex enzyme as claimed in any one of claims 1-4.
6. The surface active complex enzyme according to claim 5, wherein in the surface active complex enzyme, the mass ratio of cellulase, hemicellulase, protease, hydrolase, oxidoreductase and esterase is (8-12): (3-7): (18-22): (28-32): (28-32): (13-17), preferably 10: 5: 20: 30: 30: 15;
and/or the enzyme activity index of the surface active complex enzyme is 85-326U/g, preferably 150-325U/g, more preferably 172U/g, 175U/g, 177U/g, 195U/g or 215U/g.
7. The use of the surface active complex enzyme as claimed in claim 5 or 6 in the preparation of bionic peat humic acid from crop straw.
8. A preparation method of bionic peat humic acid comprises the following steps: carrying out enzymolysis on crop straws containing the surface active complex enzyme as claimed in claim 5 or 6.
9. The method for preparing bionic peat humic acid according to claim 8, wherein the mass percentage of the surface active complex enzyme in the crop straw is 0.01-0.10%, preferably 0.02-0.06%, more preferably 0.03%;
and/or the crop straws are one or more of corn straws, rice straw straws, soybean stalks, peanut straws and cotton straws;
and/or the temperature of the enzymolysis is-15 to 70 ℃, preferably 0 to 50, and more preferably 30 ℃;
and/or the enzymolysis time is 1-8 days, preferably 2-5 days, and more preferably 4 days.
10. A biomimetic peat humic acid prepared by the method for preparing the biomimetic peat humic acid according to claim 8 or 9.
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CN106957188A (en) * 2017-04-19 2017-07-18 淮海工学院 A kind of method for preparing organic fertilizer by composite fermentation by substrate of straw
CN108315275A (en) * 2017-12-31 2018-07-24 三门峡龙飞生物工程有限公司 A kind of high yield " three plain enzymes " straw decomposing inoculant and preparation method thereof
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Publication number Priority date Publication date Assignee Title
JP2001199782A (en) * 2000-01-12 2001-07-24 Marutomo:Kk Method for producing fertilizer and compost having crumb structure
CN102048025A (en) * 2010-11-10 2011-05-11 天津科建科技发展有限公司 Composite leavening agent combining xylanase with multiple strains and method for fermenting straw feed
CN102277349A (en) * 2011-07-24 2011-12-14 李绩 Production method of biological compound enzyme
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