CN116042454B - Composition, microbial fertilizer containing composition and application of microbial fertilizer - Google Patents
Composition, microbial fertilizer containing composition and application of microbial fertilizer Download PDFInfo
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- CN116042454B CN116042454B CN202211436739.8A CN202211436739A CN116042454B CN 116042454 B CN116042454 B CN 116042454B CN 202211436739 A CN202211436739 A CN 202211436739A CN 116042454 B CN116042454 B CN 116042454B
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Classifications
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- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05C—NITROGENOUS FERTILISERS
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12R2001/10—Bacillus licheniformis
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Abstract
The invention discloses a composition, a microbial fertilizer containing the same and application thereof, wherein the composition comprises triple coexisting fungus raw powder and triple coexisting fungus metabolite raw powder; the triple coexisting fungus raw powder contains three fungi including paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3, bacillus licheniformis (Bacillus licheniformis) BL-1106 and bacillus subtilis (Bacillus subtilis) BS-56. The beneficial bacteria in the composition are not antagonized by the beneficial bacteria and other two bacteria metabolites in the growth and propagation process, and can comprehensively play the effect of promoting the healthy growth of plants with the triple coexisting bacteria metabolite raw powder.
Description
Technical Field
The invention belongs to the technical field of microbial fertilizers, and particularly relates to a composition, a microbial fertilizer containing the composition and application of the microbial fertilizer.
Background
The technology for preparing the microbial fertilizer or the microbial organic fertilizer by utilizing natural microorganisms is a green and environment-friendly biotechnology which is rapidly developed in the international range at the end of the 60 th century, and by comprehensively utilizing multiple subjects, the mixed microbial community falls into a specific environment to decompose multiphase organic matters, so that organic solid waste is fermented and decomposed into humic acid for fertilizing and improving soil, and the technology has the characteristics of economy, practicability and no secondary pollution, and is widely concerned and developed. Meanwhile, as the microbial fertilizer or the microbial organic fertilizer is prepared to be seemingly simple, at present, in the process of producing the microbial fertilizer or the microbial organic fertilizer, microbial strains and raw materials are not screened and conditioned in a batch manner, and are directly piled up and fermented, the fermentation period is long, the production efficiency is low, the quantity of mixed bacteria is large, even a large number of harmful pathogenic bacteria are contained, the target beneficial bacteria are undefined, and the target beneficial bacteria are used or the survival rate of the shelf life is low; the quality of the product is seriously affected, and the use safety of users is seriously affected.
Disclosure of Invention
In order to solve the technical problems of long fermentation period, low production efficiency, large quantity of mixed bacteria and even large quantity of harmful pathogenic bacteria, undefined target beneficial bacteria and low target beneficial bacteria use or shelf life survival rate, the application provides a composition which comprises triple coexisting bacterial raw powder and triple coexisting bacterial metabolite raw powder. The beneficial bacteria in the composition are not antagonized by the beneficial bacteria and other two bacteria metabolites in the growth and propagation process, and can comprehensively play the effect of promoting the healthy growth of plants with the triple coexisting bacteria metabolite raw powder.
In order to solve the technical problems, the first aspect of the invention provides a composition, which comprises triple coexisting fungus raw powder and triple coexisting fungus metabolite raw powder;
the triple coexisting fungus raw powder contains paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3, bacillus licheniformis (Bacillus licheniformis) BL-1106 and bacillus subtilis (Bacillus subtilis) BS-56;
the preservation number of the paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3 is GDMCC No:62375;
the preservation number of the bacillus licheniformis (Bacillus licheniformis) BL-1106 is GDMCC No:62323;
the deposit number of the bacillus subtilis (Bacillus subtilis) BS-56 is GDMCC No:62324.
Optionally, the triple co-existing bacteria metabolite raw powder comprises one or more of amino acids, B vitamins and active biological enzymes.
Optionally, the amino acids include one or more of the following: aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine and tryptophan.
Optionally, the content of aspartic acid in the metabolite raw powder is more than or equal to 10000mg/kg;
The content of glutamic acid in the metabolite raw powder is more than or equal to 20000mg/kg;
serine content in the metabolite raw powder is more than or equal to 5000mg/kg;
the content of glycine in the metabolite raw powder is more than or equal to 15000mg/kg;
the content of histidine in the metabolite raw powder is more than or equal to 2500mg/kg;
the content of arginine in the metabolite raw powder is more than or equal to 4500mg/kg;
the content of threonine in the metabolite raw powder is more than or equal to 4000mg/kg;
The content of alanine in the metabolite raw powder is more than or equal to 8000mg/kg;
the content of proline in the metabolite raw powder is more than or equal to 12000mg/kg;
the content of tyrosine in the metabolite raw powder is more than or equal to 1800mg/kg;
the content of valine in the metabolite raw powder is more than or equal to 4500mg/kg;
the content of methionine in the metabolite raw powder is more than or equal to 1800mg/kg;
the content of isoleucine in the metabolite raw powder is more than or equal to 3000mg/kg;
The content of leucine in the metabolite raw powder is more than or equal to 6000mg/kg;
The content of phenylalanine in the metabolite raw powder is more than or equal to 5000mg/kg;
The content of lysine in the metabolite raw powder is more than or equal to 7000mg/kg;
The content of tryptophan in the metabolite raw powder is more than or equal to 1100mg/kg;
optionally, the B vitamins include one or more of the following: niacin, niacinamide, pantothenic acid, vitamin B2, vitamin B7 and vitamin B6;
optionally, the content of nicotinic acid in the metabolite raw powder is more than or equal to 400 mug/kg;
The content of nicotinamide in the metabolite raw powder is more than or equal to 100 mug/kg;
the content of pantothenic acid in the metabolite raw powder is more than or equal to 6500 mug/kg;
the content of vitamin B2 in the metabolite raw powder is more than or equal to 1200 mug/kg;
the content of vitamin B7 in the metabolite raw powder is more than or equal to 125 mug/kg;
The content of vitamin B6 in the metabolite raw powder is more than or equal to 400 mug/kg;
Optionally, the active biological enzyme comprises one or more of the following: silk proteases, lysozyme, chitinases, lipases, pectinases, proteases, cellulases, amylases;
optionally, the content of the silk protease in the metabolite raw powder is more than or equal to 100IU/L;
the content of lysozyme in the metabolite raw powder is more than or equal to 40IU/L;
The content of chitinase in the metabolite raw powder is more than or equal to 100IU/L;
the content of lipase in the metabolite raw powder is more than or equal to 200IU/L;
the content of pectase in the metabolite raw powder is more than or equal to 80IU/L;
the content of protease in the metabolite raw powder is more than or equal to 200IU/L;
the content of cellulase in the metabolite raw powder is more than or equal to 30IU/L;
the content of amylase in the metabolite raw powder is more than or equal to 3000IU/L.
Optionally, the mass ratio of the triple coexisting fungus raw powder to the triple coexisting fungus metabolite raw powder is (1-2) to (1-9).
Optionally, the viable bacteria ratio of the paecilomyces lilacinus PM0327-3, the bacillus licheniformis BL-1106 and the bacillus subtilis BS-56 in the triple coexisting microbial raw powder is (1-10): 1-10.
In order to solve the technical problem, a second aspect of the present invention provides a method for preparing the composition according to the first aspect of the present invention, the method comprising the following steps:
(1) Deep fermentation to obtain bacillus licheniformis fermentation liquid, and solid-liquid separation to obtain slurry sludge I and liquid I;
deep fermentation to obtain bacillus subtilis fermentation liquor, and solid-liquid separation to obtain slurry sludge II and liquid II;
deep fermentation to obtain Paecilomyces lilacinus fermentation liquid, and solid-liquid separation to obtain liquid III;
solid state fermentation to obtain Paecilomyces lilacinus solid state culture;
(2) Respectively drying and mixing the slurry sludge I, the slurry sludge II and the paecilomyces lilacinus solid culture to obtain triple coexisting fungus raw powder;
mixing the liquid I, the liquid II and the liquid III, and drying to obtain triple coexisting fungus metabolite raw powder;
Preferably, the volume ratio of the liquid I to the liquid II to the liquid III is (1-10): 1-10;
(3) And mixing the triple coexisting fungus raw powder and the triple coexisting fungus metabolite raw powder to obtain the composition.
In order to solve the technical problems, a third aspect of the present invention provides a microbial fertilizer, which contains the composition according to the first aspect of the present invention or the composition prepared by the preparation method according to the second aspect of the present invention.
Preferably, the microbial fertilizer further comprises an inorganic carrier, wherein the inorganic carrier comprises 80-99 parts by mass of diatomite, 0.1-5 parts by mass of potassium sulfate, 0.1-5 parts by mass of light calcium carbonate, 0.1-5 parts by mass of manganese sulfate and 0.1-5 parts by mass of magnesium sulfate.
In order to solve the technical problems, the fourth aspect of the invention provides the composition of the first aspect of the invention, the composition prepared by the preparation method of the second aspect of the invention and the application of the microbial fertilizer of the third aspect of the invention in promoting crop growth.
In order to solve the technical problems, the fifth aspect of the invention provides the composition of the first aspect of the invention, the composition prepared by the preparation method of the second aspect of the invention, and the application of the microbial fertilizer of the third aspect of the invention in preparing products for promoting crop growth.
The invention has the advantages that:
1. The paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3, the bacillus licheniformis (Bacillus licheniformis) BL-1106 and the bacillus subtilis (Bacillus subtilis) BS-56 in the triple coexisting fungus raw powder are obtained by screening under selective pressure tolerance (high-concentration self-metabolite tolerance and high-concentration metabolite tolerance of other strains of target mixed bacteria), have good tolerance to the triple coexisting fungus metabolite raw powder in the composition, and are not antagonized by metabolites in the growth and propagation processes of other two bacteria in the growth and propagation process when the triple coexisting fungus raw powder acts as a microbial inoculum or a bacterial fertilizer, so that the triple coexisting fungus raw powder can comprehensively play the role of promoting the healthy growth of plants.
2. The composition provided by the application contains primary and secondary metabolites with biological activity, which are generated in the submerged fermentation culture growth process of the paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3, the bacillus licheniformis (Bacillus licheniformis) BL-1106 and the bacillus subtilis (Bacillus subtilis) BS-56, and has good biological activity, thereby being beneficial to promoting plant growth.
Biological material preservation information
Penicillium lilacinum (Paecilomyces lilacinus) PM0327-3 was deposited at the microbiological bacterial collection center (the university of Guangdong microbiological institute) (GDMCC) at month 12 of 2022, at the deposit address: building 5 of Guangzhou City martyr of Guangdong province, no. 100 of the university of road 59, post code: 510070, accession number is: GDMCC No A62375, culture designation PM0327-3, classification designation Paecilomyces lilacinus (Paecilomyces lilacinus).
Bacillus licheniformis (Bacillus licheniformis) BL-1106 was deposited at the microorganism strain collection (institute of microorganisms, academy of sciences, guangdong province) (GDMCC) at 3/25 of 2022, accession number: building 5 of Guangzhou City martyr of Guangdong province, no. 100 of the university of road 59, post code: 510070, accession number is: GDMCC No:62323, cultures were designated BL-1106 and B.licheniformis (Bacillus licheniformis).
Bacillus subtilis (Bacillus subtilis) BS-56, deposited at the microorganism strain collection (institute of microorganisms, academy of sciences, guangdong province) (GDMCC) at 3/25 of 2022, accession number: building 5 of Guangzhou City martyr of Guangdong province, no. 100 of the university of road 59, post code: 510070, accession number is: GDMCC No:62324, cultures were named BS-56 and Bacillus subtilis (Bacillus subtilis).
Detailed Description
For the purposes of the present application, unless otherwise indicated, the terms used herein have the following meanings:
the term "submerged fermentation" refers to a fermentation mode in which a strain is inoculated into a liquid fermentation medium, continuously introduced with sterile air, and stirred.
The term "solid state fermentation" refers to a biological reaction process of one or more microorganisms in a solid substrate having a certain humidity in the absence or very small amount of free water.
The term "vitamin B6" is also known as pyridoxine, which includes pyridoxine, pyridoxal and pyridoxamine.
The term "microbial fertilizer" refers to an article containing a living body of a specific microorganism, which is applied to agricultural production, increases the supply amount of plant nutrients or promotes plant growth, increases yield, improves the quality of agricultural products and agricultural ecological environment by the vital activities of the microorganisms contained therein, and currently, microbial fertilizers include microbial inoculants (microbial inoculants), composite microbial fertilizers and bio-organic fertilizers.
Wherein the microbial inoculant (microbial inoculant) refers to a viable bacteria product prepared by directly using one or more target microorganisms after industrial production and proliferation or concentrating, drying or adsorbing by a carrier, and comprises the following components:
Single microbial inoculum: a microbial inoculant made from a microbial species;
Composite microbial inoculum: a microbial inoculant made of two or more species of microorganisms that are not antagonistic to each other;
Organic material decomposing inoculant: a microbial inoculant capable of accelerating decomposition and decomposition of various organic materials (including crop straws, livestock manure, household garbage, municipal sludge and the like);
Bioremediation agent: the microbial inoculant can reduce the concentration of harmful substances in the environment, reduce toxicity or be harmless through the growth and metabolism activities of microorganisms.
The compound microbial fertilizer is a living bacteria product formed by compounding target microorganisms with nutrient substances after industrial production and proliferation.
The biological organic fertilizer is a living bacterial product which is formed by compositing target microorganisms after industrial production and proliferation with organic materials which mainly take animal and plant residues (such as livestock and poultry manure, crop straws and the like) as sources and are subjected to innocent treatment.
The term "CFU" (colony forming units) refers to colony forming units, which refer to each colony formed after incubation on an agar plate at a certain temperature and time.
In one embodiment, the present application provides a composition comprising a triple co-existing bacterial raw powder and a triple co-existing bacterial metabolite raw powder;
The triple coexisting fungus raw powder contains three fungi including paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3, bacillus licheniformis (Bacillus licheniformis) BL-1106 and bacillus subtilis (Bacillus subtilis) BS-56;
the preservation number of the paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3 is GDMCC No:62375;
the preservation number of the bacillus licheniformis (Bacillus licheniformis) BL-1106 is GDMCC No:62323;
the deposit number of the bacillus subtilis (Bacillus subtilis) BS-56 is GDMCC No:62324.
In a specific embodiment, the live bacteria ratio of the paecilomyces lilacinus PM0327-3, the bacillus licheniformis BL-1106 and the bacillus subtilis BS-56 is (1-10): 1-10; further, the viable bacteria ratio of the paecilomyces lilacinus PM0327-3, the bacillus licheniformis BL-1106 and the bacillus subtilis BS-56 is 2-10:1-8:1-8; further, the ratio of the viable bacteria of the paecilomyces lilacinus PM0327-3, the bacillus licheniformis BL-1106 and the bacillus subtilis BS-56 is (4-10): 1-6; further, the ratio of the viable bacteria of the paecilomyces lilacinus PM0327-3, the bacillus licheniformis BL-1106 and the bacillus subtilis BS-56 is (6-10): 3-6; further, the viable bacteria ratio of the paecilomyces lilacinus PM0327-3, the bacillus licheniformis BL-1106 and the bacillus subtilis BS-56 is 1:1:1.
In a specific embodiment, the mass ratio of the triple coexisting fungus raw powder to the triple coexisting fungus metabolite raw powder is 1-2:1-9; further, the mass ratio of the triple coexisting fungus raw powder to the triple coexisting fungus metabolite raw powder is 1-2:4-9; further, the mass ratio of the triple coexisting fungus raw powder to the triple coexisting fungus metabolite raw powder is (1-2): (6-9), for example, the mass ratio of the triple coexisting fungus raw powder to the triple coexisting fungus metabolite raw powder is 1:6.
In a specific embodiment, the triple co-existing bacteria metabolite raw powder comprises one or more of amino acids, B vitamins and active biological enzymes.
In a specific embodiment, the amino group comprises one or more of the following: aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine and tryptophan.
In a specific embodiment, the content of the aspartic acid in the metabolite raw powder is more than or equal to 10000mg/kg; preferably not less than 12000mg/kg; preferably not less than 15000mg/kg; preferably not less than 18000mg/kg; preferably not less than 20000mg/kg; preferably not less than 25000mg/kg. For cost reasons, the aspartic acid may be present in the metabolite raw powder in an amount of, for example, 12000-50000 mg/kg.
The content of glutamic acid in the metabolite raw powder is more than or equal to 20000mg/kg; preferably not less than 25000mg/kg; preferably not less than 30000mg/kg; preferably not less than 40000mg/kg. For cost reasons, the glutamic acid may be present in the metabolite raw powder in an amount of, for example, 21000 to 8000mg/kg.
Serine content in the metabolite raw powder is more than or equal to 5000mg/kg; preferably more than or equal to 7000mg/kg; preferably not less than 9000mg/kg; preferably not less than 11000mg/kg; preferably not less than 11300mg/kg. For cost reasons, the serine may be present in the metabolite raw powder in an amount of, for example, 5000 to 22000mg/kg.
The content of glycine in the metabolite raw powder is more than or equal to 15000mg/kg; preferably not less than 20000mg/kg;25000mg/kg; preferably not less than 30000mg/kg. For cost reasons, the glycine may be present in the metabolite raw powder in an amount of, for example, 15000 to 63000mg/kg.
The content of histidine in the metabolite raw powder is more than or equal to 2500mg/kg; preferably not less than 3000mg/kg; preferably not less than 4000mg/kg; preferably greater than or equal to 5000mg/kg; for cost reasons, the histidine may be present in the metabolite raw powder in an amount of, for example, 2500-10000 mg/kg.
The content of arginine in the metabolite raw powder is more than or equal to 4500mg/kg; preferably greater than or equal to 5000mg/kg; preferably more than or equal to 6000mg/kg; preferably more than or equal to 7000mg/kg; preferably not less than 8000mg/kg; preferably not less than 9000mg/kg; for cost reasons, the arginine may be present in the metabolite raw powder in an amount of, for example, 4500-18000 mg/kg.
The content of threonine in the metabolite raw powder is more than or equal to 4000mg/kg; preferably greater than or equal to 5000mg/kg; preferably more than or equal to 6000mg/kg; preferably more than or equal to 7000mg/kg; for cost reasons, the threonine may be present in the metabolite raw powder in an amount of, for example, 4000 to 16000mg/kg.
The content of alanine in the metabolite raw powder is more than or equal to 8000mg/kg; preferably more than or equal to 10000mg/kg; preferably not less than 12000mg/kg; preferably not less than 14000mg/kg; preferably not less than 16000mg/kg; preferably not less than 17000mg/kg; for cost reasons, the content of alanine in the metabolite raw powder may be, for example, 8000 to 34000mg/kg.
The content of proline in the metabolite raw powder is more than or equal to 12000mg/kg; preferably not less than 15000mg/kg; preferably not less than 18000mg/kg; preferably not less than 21000mg/kg; preferably greater than or equal to 23000mg/kg; preferably not less than 26000mg/kg; for cost reasons, the content of proline in the metabolite raw powder may be, for example, 13000-52000 mg/kg.
The content of tyrosine in the metabolite raw powder is more than or equal to 1800mg/kg; preferably not less than 2000mg/kg; preferably not less than 2400mg/kg; preferably not less than 2800mg/kg; preferably not less than 3200mg/kg; preferably not less than 3600mg/kg; for cost reasons, the tyrosine may be present in the metabolite raw powder in an amount of, for example, 1800 to 7200mg/kg.
The content of valine in the metabolite raw powder is more than or equal to 4500mg/kg; preferably greater than or equal to 5000mg/kg; preferably more than or equal to 6000mg/kg; preferably more than or equal to 7000mg/kg; preferably not less than 8000mg/kg; preferably not less than 9000mg/kg; for cost reasons, the valine may be present in the metabolite raw powder in an amount of, for example, 5000 to 20000mg/kg.
The content of methionine in the metabolite raw powder is more than or equal to 1800mg/kg; preferably not less than 2000mg/kg; preferably not less than 2400mg/kg; preferably not less than 2900mg/kg; preferably not less than 35000mg/kg; preferably not less than 3800mg/kg; for cost reasons, the methionine may be present in the metabolite raw powder in an amount of, for example, 1900 to 8000mg/kg.
The content of isoleucine in the metabolite raw powder is more than or equal to 3000mg/kg; preferably not less than 3800mg/kg; preferably no less than 4200mg/kg; preferably greater than or equal to 5000mg/kg; preferably greater than or equal to 5800mg/kg; preferably more than or equal to 6200mg/kg; for cost reasons, the isoleucine may be present in the metabolite raw powder in an amount of, for example, 3000 to 12000mg/kg.
The content of leucine in the metabolite raw powder is more than or equal to 6000mg/kg; preferably not less than 8000mg/kg; preferably more than or equal to 10000mg/kg; preferably not less than 11000mg/kg; preferably not less than 12000mg/kg; for cost reasons, the leucine may be present in the metabolite raw powder in an amount of e.g. 6000-24000 mg/kg.
The content of phenylalanine in the metabolite raw powder is more than or equal to 5000mg/kg; preferably more than or equal to 7000mg/kg; preferably not less than 8000mg/kg; preferably not less than 9000mg/kg; preferably more than or equal to 10000mg/kg; preferably not less than 11000mg/kg; for cost reasons, the phenylalanine may be present in the metabolite raw powder in an amount of, for example, 5000 to 22000mg/kg.
The content of lysine in the metabolite raw powder is more than or equal to 7000mg/kg; preferably not less than 9000mg/kg; preferably not less than 11000mg/kg; preferably not less than 12000mg/kg; preferably not less than 13000mg/kg; preferably not less than 14000mg/kg; for cost reasons, the lysine content in the metabolite raw meal may be, for example, 7000 to 30000mg/kg.
The content of tryptophan in the metabolite raw powder is more than or equal to 1100mg/kg; preferably not less than 1300mg/kg; preferably more than or equal to 1500mg/kg; preferably more than or equal to 1700mg/kg; preferably not less than 1900mg/kg; preferably more than or equal to 2200mg/kg; for cost reasons, the tryptophan may be present in the metabolite raw meal in an amount of, for example, 1100 to 5000mg/kg.
Optionally, the B vitamins include one or more of the following: niacin, niacinamide, pantothenic acid, vitamin B2, vitamin B7 and vitamin B6;
Optionally, the content of nicotinic acid in the metabolite raw powder is more than or equal to 400 mug/kg; preferably not less than 500. Mu.g/kg; preferably not less than 600. Mu.g/kg; preferably not less than 700. Mu.g/kg; for cost reasons, the niacin may be present in the metabolite raw powder in an amount of, for example, 400-1600 μg/kg.
The content of nicotinamide in the metabolite raw powder is more than or equal to 100 mug/kg; preferably not less than 150. Mu.g/kg; preferably not less than 200. Mu.g/kg; preferably not less than 250. Mu.g/kg; for cost reasons, the nicotinamide may be present in the metabolite raw powder in an amount of, for example, 150 to 600. Mu.g/kg.
The content of pantothenic acid in the metabolite raw powder is more than or equal to 6500 mug/kg; preferably not less than 8000. Mu.g/kg; preferably greater than or equal to 10000 mug/kg; preferably not less than 11000. Mu.g/kg; preferably not less than 12000. Mu.g/kg; preferably not less than 13000. Mu.g/kg; for cost reasons, the pantothenic acid can be present in the metabolite raw powder in an amount of, for example, 6500 to 28000. Mu.g/kg.
The content of vitamin B2 in the metabolite raw powder is more than or equal to 1200 mug/kg; preferably not less than 1500. Mu.g/kg; preferably not less than 2000. Mu.g/kg; preferably greater than or equal to 2500 mug/kg; for cost reasons, the vitamin B2 may be present in the metabolite raw powder in an amount of, for example, 1200-4800. Mu.g/kg.
The content of vitamin B7 in the metabolite raw powder is more than or equal to 125 mug/kg; preferably not less than 150. Mu.g/kg; preferably not less than 200. Mu.g/kg; preferably not less than 240. Mu.g/kg; for cost reasons, the vitamin B7 may be present in the metabolite raw powder in an amount of, for example, 125-500. Mu.g/kg.
The content of vitamin B6 in the metabolite raw powder is more than or equal to 400 mug/kg; preferably not less than 600. Mu.g/kg; preferably not less than 700. Mu.g/kg; preferably not less than 800. Mu.g/kg; for cost reasons, the vitamin B6 may be present in the metabolite raw powder in an amount of, for example, 400 to 1800. Mu.g/kg.
Optionally, the active biological enzyme comprises one or more of the following: silk proteases, lysozyme, chitinases, lipases, pectinases, proteases, cellulases, amylases;
optionally, the content of the silk protease in the metabolite raw powder is more than or equal to 100IU/L; preferably not less than 150IU/L; preferably not less than 180IU/L; preferably not less than 200IU/L; for cost reasons, the content in the metabolite raw powder may be, for example, 100 to 400IU/L.
The content of lysozyme in the metabolite raw powder is more than or equal to 40IU/L; preferably not less than 50IU/L; preferably not less than 60IU/L; preferably not less than 70IU/L; for cost reasons, the lysozyme may be present in the metabolite raw powder in an amount of, for example, 40 to 1600IU/L.
The content of chitinase in the metabolite raw powder is more than or equal to 100IU/L; preferably not less than 120IU/L; preferably not less than 150IU/L; preferably not less than 180IU/L; preferably not less than 190IU/L; for cost reasons, the chitinase may be present in the metabolite raw powder in an amount of, for example, 100-400 IU/L.
The content of lipase in the metabolite raw powder is more than or equal to 200IU/L; preferably not less than 250IU/L; preferably not less than 300IU/L; preferably not less than 350IU/L; preferably not less than 390IU/L; for cost reasons, the lipase may be present in the metabolite raw powder in an amount of, for example, 200-800 IU/L.
The content of pectase in the metabolite raw powder is more than or equal to 80IU/L; preferably not less than 100IU/L; preferably not less than 120IU/L; preferably not less than 140IU/L; preferably not less than 160IU/L; for cost reasons, the pectinase may be present in the metabolite raw powder in an amount of, for example, 80-320 IU/L.
The content of protease in the metabolite raw powder is more than or equal to 200IU/L; preferably not less than 250IU/L; preferably not less than 300IU/L; preferably not less than 350IU/L; preferably not less than 390IU/L; for cost reasons, the protease may be present in the metabolite raw powder in an amount of, for example, 200-800 IU/L.
The content of cellulase in the metabolite raw powder is more than or equal to 30IU/L; preferably not less than 40IU/L; preferably not less than 50IU/L; preferably not less than 60IU/L; for cost reasons, the cellulase may be present in the metabolite raw powder in an amount of, for example, 30-120 IU/L.
The content of amylase in the metabolite raw powder is more than or equal to 3000IU/L. Preferably not less than 4000IU/L; preferably not less than 50000IU/L; preferably not less than 5500IU/L; preferably greater than or equal to 5800IU/L; for cost reasons, the amylase may be present in the metabolite raw powder in an amount of, for example, 3000 to 12000IU/L.
According to an aspect of the present application, there is provided a method of preparing a composition as described above, the method comprising the steps of:
deep fermentation to obtain bacillus licheniformis fermentation liquid, and solid-liquid separation to obtain slurry sludge I and liquid I;
deep fermentation to obtain bacillus subtilis fermentation liquor, and solid-liquid separation to obtain slurry sludge II and liquid II;
deep fermentation to obtain Paecilomyces lilacinus fermentation liquid, and solid-liquid separation to obtain liquid III;
solid state fermentation to obtain Paecilomyces lilacinus solid state culture;
respectively drying and mixing the slurry sludge I, the slurry sludge II and the paecilomyces lilacinus solid culture to obtain triple coexisting fungus raw powder;
Mixing the liquid I, the liquid II and the liquid III, and drying to obtain triple coexisting fungus metabolite raw powder; preferably, the volume ratio of the liquid I to the liquid II to the liquid III is (1-10): 1-10; more preferably, the volume ratio of the liquid I, the liquid II and the liquid III is 1:1:1;
and mixing the triple coexisting fungus raw powder and the triple coexisting fungus metabolite raw powder to obtain the composition.
In a specific embodiment, in order to solve the problems of long fermentation period, low production efficiency, large quantity of mixed bacteria, even containing a large quantity of harmful pathogenic bacteria, undefined target beneficial bacteria and low survival rate of target beneficial bacteria in shelf life in the prior art, the invention provides a composition, wherein three target beneficial bacteria are subjected to directional fermentation culture by using a biotechnological culture method of pure fermentation value-added target beneficial bacteria, and the obtained target beneficial bacteria have high content of target beneficial bacteria, small quantity of mixed bacteria, no harmful pathogenic bacteria and high survival rate of recovery of each beneficial bacteria in the shelf life; meanwhile, primary and secondary metabolites with biological activity generated in the submerged fermentation culture growth process of the three beneficial bacteria are concentrated and extracted, so that the three beneficial bacteria growth production metabolites with biological activity are obtained.
In a specific embodiment, a microbial fertilizer is provided, which contains the composition described above or a composition prepared according to the preparation method described above.
Preferably, the microbial fertilizer further comprises an inorganic carrier, wherein the inorganic carrier comprises 80-99 parts by mass of diatomite, 0.1-5 parts by mass of potassium sulfate, 0.1-5 parts by mass of light calcium carbonate, 0.1-5 parts by mass of manganese sulfate and 0.1-5 parts by mass of magnesium sulfate.
In a specific embodiment, the microbial fertilizer further comprises an inorganic carrier, wherein the inorganic carrier is mainly calcined diatomite, and is compounded with spore germination agent, moisture absorption drying agent, acid-base regulator manganese sulfate, potassium sulfate, magnesium sulfate and light calcium carbonate as carriers; the microbial fertilizer rich in directional target beneficial bacteria including Paecilomyces lilacinus, bacillus licheniformis and Bacillus subtilis triple coexisting bacteria living bacteria and metabolites thereof is formed by preparing each beneficial bacteria product, each beneficial bacteria metabolite with physiological activity and a compound carrier according to different proportions.
In a specific embodiment, the mass ratio of the triple coexisting fungus raw powder to the triple coexisting fungus metabolite raw powder to the inorganic carrier is (1-2): 1-9): 90-98.
In a specific embodiment, the inorganic carrier comprises 80-99 parts by mass of diatomite, 0.1-5 parts by mass of potassium sulfate, 0.1-5 parts by mass of light calcium carbonate, 0.1-5 parts by mass of manganese sulfate and 0.1-5 parts by mass of magnesium sulfate.
Example 1 Breeding of Paecilomyces lilacinus PM0327-3, bacillus licheniformis BL-1106, bacillus subtilis BS-56
Preparation of the mixed fermentation concentrate
1. Preparation of Paecilomyces lilacinus submerged fermentation concentrate:
Deep fermenting Penicillium lilacinum (Paecilomyces lilacinus) CICC 40276 to obtain fermentation broth with viable spore number of 30 hundred million CFU/ml, ceramic membrane filtering, concentrating the filtrate 6 times, and preserving the fermentation concentrate in refrigerator at 4-6deg.C.
2. Preparation of bacillus licheniformis submerged fermentation concentrated solution:
The bacillus licheniformis (Bacillus licheniformis) CICC 10037 submerged fermentation liquid has viable spore count of 120 hundred million CFU/ml, the fermentation liquid is filtered by a ceramic membrane, the filtrate is concentrated by 6 times, and the fermentation concentrate is preserved in a refrigerator at 4-6 ℃.
3. Preparation of bacillus subtilis submerged fermentation concentrate:
Deeply fermenting bacillus subtilis (Bacillus subtilis) CMCC 63501 to 150 hundred million CFU/ml of viable spores of the fermentation broth; filtering the fermentation liquor by a ceramic membrane, concentrating the filtrate for 6 times, and preserving the fermentation concentrate in a refrigerator at the temperature of 4-6 ℃.
4. Mixing the three fermentation concentrated solutions in equal proportion uniformly to obtain mixed solution of fermentation concentrated solutions rich in three bacteria fermentation metabolites, and filtering the mixed solution by a sterilizing filter under aseptic operation conditions to obtain mixed fermentation concentrated solution.
Breeding of bacillus subtilis BS-56
Scraping and washing lawn on a mature inclined plane of bacillus subtilis (Bacillus subtilis) CMCC63501 by using 5ml of sterile mixed fermentation concentrated solution, diluting the mixed fermentation concentrated solution, placing the diluted mixed fermentation concentrated solution in a constant-temperature water bath box at 70 ℃ for 25min, coating the diluted mixed fermentation concentrated solution on a double-dish flat plate for culture at 35 ℃ for 36h, and then sequentially culturing the diluted mixed fermentation concentrated solution by an acid-resistant flat plate (peptone 10g, beef extract 3g, sodium chloride 5g, soluble powder 10g, agar 18g, distilled water 1000ml, pH4.5 adjustment and sterilization at 121 ℃ for 30 min to prepare a separated double-dish flat plate) and an alkali-resistant flat plate (peptone 10g, beef extract 3g, sodium chloride 5g, soluble powder 10g, agar 18g, distilled water 1000ml, pH9.5 adjustment and sterilization at 121 ℃ for 30 min to prepare a separated double-dish flat plate).
The obtained bacillus subtilis BS-56 strain which is resistant to self, bacillus licheniformis and paecilomyces lilacinus metabolites, acid, alkali and high temperature is sent to the Guangdong province microbiological strain collection center (the institute of microbiology, academy of sciences, guangdong province) to be preserved, and is bacillus subtilis (Bacillus subtilis) GDMCC No:62324.
Breeding of paecilomyces lilacinus strain PM0327-3
Scraping and washing lawn on mature inclined plane of Paecilomyces lilacinus (Paecilomyces lilacinus) CICC 40276 with 5ml of sterile mixed fermentation concentrated solution, diluting with the mixed fermentation concentrated solution, placing into a constant temperature water bath box at 35 ℃ for 20min, coating on a double-dish plate for culturing at 29 ℃ for 180h, and culturing by acid-resistant plates (PDA culture medium is used for adjusting pH to 4.5 to prepare a separated double-dish plate) and alkali-resistant plates (PDA culture medium is used for adjusting pH to 9.5 to prepare a separated double-dish plate).
Obtaining paecilomyces lilacinus strain PM0327-3 which is resistant to self, bacillus licheniformis and bacillus subtilis metabolites, acid, alkali and high temperature, and is delivered to the Guangdong province microorganism strain collection center (the university of Guangdong university microbiological institute) for preservation, wherein the preservation number is: GDMCC No:62375.
(IV) breeding bacillus licheniformis BL-1106:
scraping and washing lawn on a mature inclined plane of CICC 10037 of bacillus licheniformis (Bacillus licheniformis) by using 5ml of sterile mixed fermentation concentrated solution, diluting the mixed fermentation concentrated solution, placing the diluted mixed fermentation concentrated solution in a constant-temperature water bath box at 70 ℃ for 30min, coating the diluted mixed fermentation concentrated solution on a double-dish flat plate for culture at 35 ℃ for 36h, then sequentially culturing the diluted mixed fermentation concentrated solution by using acid-resistant flat plates (peptone 10g, beef extract 3g, sodium chloride 5g, soluble powder 10g, agar 15-20 g, distilled water 1000ml, pH4.5 adjustment, sterilization at 121 ℃ for 30min, split charging the diluted mixed fermentation concentrated solution on 90mm double-dish flat plate) and alkali-resistant flat plates (peptone 10g, beef extract 3g, sodium chloride 5g, soluble powder 10g, agar 15-20 g, distilled water 1000ml, pH9.5 adjustment, sterilization at 121 ℃ for 30min and split charging the diluted mixed fermentation concentrated solution on 90mm double-dish flat plate).
Obtaining the bacillus licheniformis (Bacillus licheniformis) BL-1106 strain which is resistant to self and paecilomyces lilacinus and bacillus subtilis metabolites, acid, alkali and high temperature, and delivering the bacillus licheniformis BL-1106 strain to the Guangdong province microorganism strain collection center (the university of Guangdong province microbiological institute) for preservation, wherein the preservation number is: GDMCC No:62323.
EXAMPLE 2 production of Bacillus subtilis (Bacillus subtilis) GDMCC No:62324 by deep submerged fermentation and centrifugal spray drying
1. Production strain: bacillus subtilis (Bacillus subtilis) GDMCC No:62324.
2. The formula of the culture medium comprises:
2.1, plate and slant culture medium: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 10g of soluble starch, 15g of agar and 1000mL of distilled water, and regulating pH to 7.2.
2.2, Shake flask and seed culture medium (wt%): 3.0 parts of soybean meal, 2.0 parts of soybean peptone, 0.3 parts of sodium chloride, 2.0 parts of glucose, 1.0 part of corn starch and adjusting the pH value to be 7.2.
2.3, Fermentation medium (wt%): glucose 2, soybean peptone 1.5, angel yeast powder 2.0, soybean meal 2.0, beef extract 2.0, corn starch 2.0, sodium chloride 0.3, potassium dihydrogen phosphate 0.2, magnesium sulfate 0.05, ammonium sulfate 0.15, manganese sulfate 0.03, light calcium carbonate 0.3, and pH7.2.
3. Culture conditions:
3.1, plate and slant culture: picking up mature monospore, drawing lines on a flat plate or a test tube inclined plane, and placing at 35 ℃ for 36h of culture time.
3.2, Shake flask and seed culture: the inclined spore blocks are dug and cultured in shake flask at 35 deg.c and 200r/min for 36 hr, and the shake flask ferments about 40 hundred million CFU/ml of viable spores.
3.3, Fermentation conditions of a fermentation tank: the inoculation amount is 5%, the temperature is 32 ℃, the rotating speed is 250rpm, the ventilation amount of sterile air is 1:1v/vm, the dissolved oxygen in the culture medium is controlled to be 60%, the pH is controlled to be 7, the fed-batch is fed in 15-70 hours of fermentation, the reducing sugar is ensured to be 1.1+/-0.2%, the fermentation period is 84 hours, and the colony number of the fermentation liquid placed in a tank is about 150 hundred million CFU/ml.
4. Membrane filtration separation and centrifugal spray drying:
4.1, the fermentation liquor is subjected to solid-liquid separation by a ceramic membrane filter liquor, and a pasty sludge part (pasty sludge I) containing bacillus subtilis thalli and a ceramic membrane permeate (liquid I) are collected for standby.
4.2, Spray drying the slurry sludge I, wherein the colony number of the obtained bacillus subtilis product is about 2200 hundred million CFU/g, and the moisture content is less than or equal to 3.5%.
5. Packaging and storing: the product is vacuum packed in aluminum plastic bags with 1000g each, and stored under the conditions of temperature less than or equal to 20 ℃, relative humidity less than or equal to 65% and no direct sunlight.
EXAMPLE 3 production of Bacillus licheniformis (Bacillus licheniformis) GDMCC No:62323 pure submerged fermentation and centrifugal spray drying
1. Production strain: bacillus licheniformis (Bacillus licheniformis) GDMCC No:62323
2. The formula of the culture medium comprises:
2.1, plate and slant culture medium: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 10g of soluble starch, 15g of agar and 1000mL of distilled water, and regulating pH to 7.2.
2.2, Shake flask and seed culture medium (wt%): 3.0 parts of soybean meal, 2.0 parts of soybean peptone, 0.3 parts of sodium chloride, 2.0 parts of glucose, 1.0 part of corn starch and adjusting the pH value to be 7.2.
2.3, Fermentation medium (wt%): glucose 2, corn steep liquor 1.0, yeast extract 0.5, soybean meal 3.0, beef extract 0.5, corn starch 2.0, sodium chloride 0.3, monopotassium phosphate 0.2, dipotassium phosphate 0.3, magnesium sulfate 0.05, ammonium sulfate 0.15, manganese sulfate 0.03 and pH value adjustment of 7.2.
3. Culture conditions
3.1, Plate and slant culture: picking up mature monospore, drawing lines on a flat plate or a test tube inclined plane, and culturing for 36h at 34+/-1 ℃.
3.2, Shake flask and seed culture: digging inclined spore blocks in shake flask, and shake culturing at 34+ -1deg.C and 200r/min for 36 hr to obtain liquid shake flask fermentation broth with viable spore count of about 30 hundred million CFU/ml.
3.3, Fermenting and culturing in a fermentation tank: the inoculation amount is 5%, the temperature is controlled at 32 ℃, the period is 84 hours, the feeding is fed in after 15 hours of fermentation to 12 hours before tank discharge, the reducing sugar is ensured to be between 1.1+/-0.2%, the rotating speed is 500rpm, the ventilation amount is 1.1v/vm, the dissolved oxygen in the fermentation tank is controlled to be between 60+/-5%, the pH value is controlled to be 7.5+/-1.0, and the viable spore count of the fermentation liquid is measured to be about 120 hundred million CFU/ml by tank discharge.
4. Membrane filtration separation and spray drying:
4.1, the fermentation liquor is subjected to solid-liquid separation by a ceramic membrane filter liquor, and a pasty sludge part (pasty sludge II) containing bacillus licheniformis thalli and ceramic membrane permeate (liquid II) are collected for standby.
4.2, Spray drying the slurry sludge II, wherein the colony count of the obtained bacillus licheniformis product is about 1300 hundred million CFU/g, and the moisture content is less than or equal to 3.5%.
5. Packaging and storing: the product is vacuum packed in aluminum plastic bags with 1000g each, and stored under the conditions of temperature less than or equal to 20 ℃, relative humidity less than or equal to 65% and no direct sunlight.
EXAMPLE 4 production of Mucor lilacinus (Paecilomyces lilacinus) GDMCC No:62375 pure strain "deep layer+solid state" fermentation, dry air Normal temperature air flow drying
1. Production strain: penicillium lilacinum (Paecilomyces lilacinus) GDMCC No:62375.
2. The formula of the culture medium comprises:
2.1, plate and slant culture medium: PDA medium was used.
2.2, Shake flask and seed culture medium (wt%): 15 parts of potato, 5 parts of corn steep liquor, 5 parts of soybean meal, 3 parts of glucose and natural pH.
2.3, Fermentation medium (wt%): 8 parts of potato, 4 parts of corn steep liquor, 3 parts of soybean meal, 2 parts of glucose, 1 part of corn meal, 2 parts of corn starch and 6.5 parts of pH value.
2.4, Solid state fermentation Medium (wt%): 58.0 parts of rice, 5.0 parts of bran, 6.0 parts of rice bran, 3.0 parts of soybean powder and 28.0 parts of water. Soaking rice in hot water heated to 60deg.C for 1 hr, taking out, air drying, adding testa Tritici, semen glycines powder, testa oryzae, and water, mixing, and stirring. And (5) sterilizing.
3. Culture conditions:
3.1, plate and slant culture: mature monospore is picked up and drawn on the slant of the test tube, and cultured for 7d at 29 ℃.
3.2, Shake flask fermentation and seed culture: digging the spore blocks on the inclined plane into a shake flask. Shaking on a shaking table with a rotation speed of 180r/min, and culturing at 28+ -1deg.C for 4-5 days.
3.3, Fermenting and culturing in a fermentation tank: the ventilation rate is 1:1v/vm, the rotating speed is 200rpm, the temperature is 28 ℃ +/-1 ℃, the inoculation amount is 8%, the feed is fed in 24 hours before the fermentation is carried out for 10 hours to the tank discharge, the reducing sugar is ensured to be 1.1+/-0.2%, the culture period is 148 hours, the pH is controlled to be 7.1, and the viable spore count of the fermentation liquid is measured to be about 35 hundred million CFU/ml by the tank discharge.
3.4, Solid state fermentation culture: and (3) performing solid-liquid separation on the fermentation liquor through a ceramic membrane filtrate, collecting a pasty sludge part containing the paecilomyces lilacinus bodies and a ceramic membrane permeate (liquid III), and collecting for later use. Taking the slurry sludge containing the thalli according to the inoculation amount of 10 percent by mass, uniformly mixing the slurry sludge with a culture medium, culturing for 5 days at the temperature of 28+/-1 ℃ and the relative humidity of 90+/-2%, adjusting the temperature to 33+/-1 ℃, continuously culturing for 5 days, and turning most of mycelia into light purple.
4. And (3) drying: sieving the solid culture with 30 mesh sieve to obtain spore powder with water content less than or equal to 3.5% and bacterial colony number of about 120 hundred million CFU/g.
5. Packaging and storing: the product is vacuum packed in aluminum plastic bags with 1000g each, and stored under the conditions of temperature less than or equal to 20 ℃, relative humidity less than or equal to 65% and no direct sunlight.
EXAMPLE 5 preparation of the metabolic and decomposition products of submerged fermentation of three bacteria
And respectively collecting spare liquid I, liquid II and liquid III, uniformly mixing the spare liquid I, liquid II and liquid III in equal volumes, obtaining mixed liquid, shrinking liquid, spray drying, and obtaining three bacterial metabolite raw powder products (the moisture content is less than 3.5%), wherein the metabolite raw powder products are vacuum-packed by aluminum plastic, and the products are rich in various amino acids, B vitamins, pectinase, protease, amylase, lipase, cellulase, chitinase, lysozyme and various active enzymes of silk protease (see table 1-table 3), and the moisture content is less than or equal to 3.5%.
TABLE 1 amino acids contained in three bacterial metabolite raw powders and the contents thereof
| Sequence number | Name of the name | Content (mg/kg) | Sequence number | Name of the name | Content (mg/kg) |
| 1 | Aspartic acid | 26181.59 | 10 | Tyrosine | 3645.94 |
| 2 | Glutamic acid | 42489.72 | 11 | Valine (valine) | 9526.91 |
| 3 | Serine (serine) | 11316.48 | 12 | Methionine | 3870.76 |
| 4 | Glycine (Gly) | 31563.46 | 13 | Isoleucine (Ile) | 6214.35 |
| 5 | Histidine | 5053.96 | 14 | Leucine (leucine) | 12414.89 |
| 6 | Arginine (Arg) | 9184.65 | 15 | Phenylalanine (Phe) | 11041.88 |
| 7 | Threonine (Thr) | 7927.48 | 16 | Lysine | 14907.82 |
| 8 | Alanine (Ala) | 17667.01 | 17 | Tryptophan | 2294.34 |
| 9 | Proline (proline) | 26241.97 |
Table 2B vitamins contained in the three bacterial metabolite raw powders and the contents thereof
| Sequence number | Name of the name | Content (μg/kg) |
| 1 | Nicotinic acid | 798.39 |
| 2 | Nicotinamide | 273.41 |
| 3 | Pantothenic acid | 13765.82 |
| 4 | Vitamin B2 | 2520.66 |
| 5 | Vitamin B7 | 249.10 |
| 6 | Vitamin B6 | 852.43 |
Table 3 bioactive enzymes contained in the three bacterial metabolite raw powders and their contents
Example 6 preparation of microbial Fertilizer containing Tri-co-located bacteria and its metabolite
1. Preparing raw powder of paecilomyces lilacinus, bacillus licheniformis and bacillus subtilis triple coexisting bacteria:
Weighing 6 parts of bacillus subtilis raw powder (about 2200 hundred million CFU/g), 9 parts of bacillus licheniformis raw powder (about 1300 hundred million CFU/g) and 90 parts of paecilomyces lilacinus raw powder (about 120 hundred million CFU/g) by mass, and uniformly mixing to obtain triple coexisting fungus raw powder.
2. Preparing a microbial fertilizer carrier: 0.3 parts of potassium sulfate, 0.5 parts of light calcium carbonate, 0.5 parts of magnesium sulfate, 0.2 parts of manganese sulfate and 98.5 parts of calcined diatomite are prepared into the inorganic carrier of the microbial fertilizer.
3. And (3) preparing a microbial fertilizer: and taking 93 parts of the prepared microbial fertilizer carrier, 6 parts of the triple coexisting fungus metabolite and 1 part of the triple coexisting fungus raw powder by mass, and uniformly mixing to obtain a microbial fertilizer product.
EXAMPLE 7 microbial fertilizer containing Triplex and its metabolite for cultivation of tomato and cucumber
1. 6 Blocks of land for cultivating tomatoes and cucumbers in the Changtai of Minnan are selected, and each block of land is 2 meters multiplied by 6 meters (0.018 mu). In the sunny weather of 10 late days, the temperature is 31 ℃, and simultaneously, the soil is ploughed to a depth of 25cm, and the ploughed soil is insolated for 3 days in the sun and ploughed once every day;
2. Taking 1000g (the total number of bacterial colony measured by a flat plate is 2.95 hundred million CFU/g, and 1kg of the bacterial manure is measured per mu) of microbial fertilizer products of aluminum-plastic vacuum packed triple coexisting bacteria and metabolites thereof stored for 6 months under the conditions that the temperature is less than or equal to 20 ℃ and the relative humidity is less than or equal to 65 percent and the conditions that the sunlight is not direct irradiation, and uniformly mixing the microbial fertilizer products with mineral source potassium fulvate and fine silt according to the proportion of 1:50:50, thereby being used as the hole-applied bacterial manure.
3. The potassium fulvate ore source and the fine silt are uniformly mixed according to the proportion of 50:51 to be used as a control group A.
4. The microbial fertilizer containing the triple bacteria and the metabolites thereof with the same viable count is prepared by using paecilomyces lilacinus (Paecilomyces lilacinus) CICC 40276, bacillus licheniformis (Bacillus licheniformis) CICC 10037 and bacillus subtilis (Bacillus subtilis) CMCC 63501 as production strains by the same preparation method as the microbial fertilizer containing the triple coexisting bacteria and the metabolites thereof, and the control microbial fertilizer containing the triple bacteria and the metabolites thereof with the same viable count is uniformly mixed with the mineral source potassium fulvate and the fine silt according to the proportion of 1:50:50 to be used as a control group B.
5. Making holes with a hole spacing of 80cm for 3 days, respectively adding 150g of each group of fertilizer into the bottom layer of each land block hole, covering the hole surface with fine soil of about 10cm, and covering with plastic film mulching film; the next day, tomato and cucumber are transplanted and cultivated according to the conventional cultivation method, and the colony survival number represented by the paecilomyces lilacinus and the plant height and fruit harvest condition in 2 weeks are observed:
5.1, growing conditions of tomato and cucumber cultivation for 2 weeks:
5 plants were sampled by diagonal crossing and observed for 2 weeks of tomato growth, and the average plant heights are shown in Table 4.
Table 4 growth of tomatoes in each group
5 Strains were sampled by diagonal crossing method, and cucumber growth was observed for 2 weeks, and the average strain heights are shown in Table 5.
Table 5 cucumber growth conditions of each group
As can be seen from tables 4 and 5, the microbial fertilizer containing the triple coexisting bacteria and the metabolites thereof of the experimental group can more effectively promote the growth of tomatoes and cucumbers.
Survival rate of paecilomyces lilacinus in test group, control group B: the number of surviving colonies of Mucor lilacinus was measured as represented by Mucor lilacinus having poor resistance to adverse environment.
The sampling method comprises the following steps: sampling every 1 month, sampling the depth of 15cm-20cm under the soil surface layer of 5 point application sites by a diagonal crossing method, uniformly mixing 20g-30g of soil samples of each point application site, and bagging and preserving; from the 5-point storage bags, a total of 10g of soil samples were weighed and mixed uniformly, and the total number of colonies was counted in a Paecilomyces lilacinus plate medium, and the results are shown in tables 6 and 7.
TABLE 6 statistics of the colony count of Paecilomyces lilacinus for each tomato plot
TABLE 7 statistics of the colony count of Paecilomyces lilacinus in cucumber plots
As can be seen from tables 6 and 7, the bacterial colony number of Paecilomyces lilacinus in the test group is higher, which indicates that the Paecilomyces lilacinus in the test group has better stability, is not antagonized by metabolites in the growth and propagation processes of other two bacteria in the growth and propagation process, and can comprehensively play the effect of promoting the healthy growth of plants with the triple coexisting bacteria metabolite raw powder.
5.3. Yield of tomato and cucumber cultivation plots:
Yield comparison of tomato test group and control group a is shown in table 8, land mass yield increase of test group is 16.16%:
TABLE 8 tomato yield conditions
Yield of cucumber test group and yield of control group A are compared, and yield of test group land block is increased by 20.47%:
TABLE 9 cucumber yield conditions
From tables 8 and 9, it can be seen that the microbial fertilizer containing the triple coexisting bacteria and the metabolites thereof in the experimental group can effectively improve the yield of tomatoes and cucumbers.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, but rather, the invention is defined by the appended claims and description.
Claims (8)
1. A composition, characterized in that the composition comprises a triple co-existence bacteria raw powder and a triple co-existence bacteria metabolite raw powder;
The triple coexisting fungus raw powder consists of paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3, bacillus licheniformis (Bacillus licheniformis) BL-1106 and bacillus subtilis (Bacillus subtilis) BS-56;
The triple coexisting fungus metabolite raw powder is obtained by mixing and drying liquid obtained by solid-liquid separation of fermentation liquor of the paecilomyces lilacinus, the bacillus licheniformis and the bacillus subtilis;
The preservation number of the paecilomyces lilacinus (Paecilomyces lilacinus) PM0327-3 is GDMCC No:62375;
The preservation number of the bacillus licheniformis (Bacillus licheniformis) BL-1106 is GDMCC No:62323;
The deposit number of the bacillus subtilis (Bacillus subtilis) BS-56 is GDMCC No:62324.
2. The composition of claim 1, wherein the mass ratio of the co-viable bacteria powder to the co-viable bacteria metabolite powder is (1-2): 1-9.
3. The composition of claim 1, wherein the live bacteria ratio of paecilomyces lilacinus PM0327-3, bacillus licheniformis BL-1106 and bacillus subtilis BS-56 in the triple coexisting microbial raw powder is (1-10): 1-10.
4. A process for the preparation of a composition as claimed in any one of claims 1 to 3, comprising the steps of:
(1) Deep fermentation to obtain bacillus subtilis fermentation liquor, and solid-liquid separation to obtain slurry sludge I and liquid I;
Deep fermentation to obtain bacillus licheniformis fermentation liquid, and solid-liquid separation to obtain slurry sludge II and liquid II;
deep fermentation to obtain Paecilomyces lilacinus fermentation liquid, and solid-liquid separation to obtain liquid III;
solid state fermentation to obtain Paecilomyces lilacinus solid state culture;
(2) Respectively drying and mixing the slurry sludge I, the slurry sludge II and the paecilomyces lilacinus solid culture to obtain triple coexisting fungus raw powder;
mixing the liquid I, the liquid II and the liquid III, and drying to obtain triple coexisting fungus metabolite raw powder;
(3) And mixing the triple coexisting fungus raw powder and the triple coexisting fungus metabolite raw powder to obtain the composition.
5. The method of claim 4, wherein the volume ratio of liquid I, liquid II and liquid III is (1-10): 1-10.
6. A microbial fertilizer characterized in that it contains the composition according to any one of claims 1 to 3 or the composition prepared by the preparation method according to claim 4 or 5.
7. Use of a composition according to any one of claims 1-3, a composition prepared according to the preparation method of claim 4 or 5, a microbial fertilizer according to claim 6 for promoting crop growth; the crop is tomato or cucumber.
8. Use of a composition according to any one of claims 1-3, a composition prepared according to the preparation method of claim 4 or 5, a microbial fertilizer according to claim 6 for the preparation of a product for promoting crop growth; the crop is tomato or cucumber.
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