CN106518389A

CN106518389A - Biofertilizer with edible fungus residue as raw material and preparation thereof

Info

Publication number: CN106518389A
Application number: CN201610983597.5A
Authority: CN
Inventors: 黄亮; 班立桐; 孙宁; 杨红澎; 王玉; 张怡; 王睿沁
Original assignee: Tianjin Agricultural University
Current assignee: Tianjin Agricultural University
Priority date: 2016-10-25
Filing date: 2016-10-25
Publication date: 2017-03-22

Abstract

The invention discloses a biofertilizer with edible fungus residue as the raw material and preparation thereof. The invention relates to a biofertilizer with edible fungus residue as the raw material, and the biofertilizer comprises the following components by weight: 20-40 parts of pretreated Hypsizygus marmoreus fungus residue, 1-3 parts of matrine, and 4-10 parts of fermented cow dung. The fertilizer provided by the invention can enhance the drought resistance of crops, is especially suitable for use in saline-alkali soil, can improve soil and improve soil physical properties, and is beneficial to improving soil fertility.

Description

Bio-feritlizer and its preparation of a kind of edible fungi residue for raw material

Technical field

The present invention relates to a kind of bio-feritlizer.

Background technology

Existing bio-feritlizer is monistic bio-feritlizer mostly.Such as：Azotobacteria fertilizer, mainly by with nitrogen fixation Antibacterial and funguses composition；And for example：Phosphobacteria, its function are to decompose the organophosphors in soil, are allowed to become feasible to be absorbed by plants Phosphorus element；For another example：Antibacterial, its function are to decompose the potassium compound in soil, make can be absorbed and used by plants quick-acting Potassium.These fertilizer can play each self-applying when being used alone, and partly meet plant to nitrogen, phosphorus, the needs of potassium three elements.It Will not produce soil compaction phenomenon caused by life-time service chemical fertilizer.But its deficiency is：Above-mentioned three class is used alone biological Fertilizer can only solve the one side of plant desired nutritional, and relatively costly, and consumption is big, meanwhile, three is used cooperatively, and because It is difficult to accomplish that ratio suitably causes the not enough of dose or wastes.The practice of the single or compound bio-feritlizer of life-time service Prove, they can only replacing fertilizer less than 30%.

Edible fungus culturing culture medium still can utilize material containing certain proportion after cultivation use, how efficient utilization Culture medium of edible fungus so as to be fully used for the development of edible fungus culturing industry is with important impetus, it is also right The protection and utilization of environment and resource is a positive factor. crab flavour mushroom and Pleurotus eryngii are the larger edible fungi product of two yield Kind, the culture medium prescription of crab flavour mushroom:40 kilograms of cotton seed hullss, 38 kilograms of wood flour, 10 kilograms of wheat bran, 10 kilograms of Semen Maydis powder, Calcium Carbonate Or 1 kilogram of calcium sulfate, 1 kilogram of Calx, pH value 7.5 8, water content 65%.A kind of cultivation that can improve sporophore calcium content Substrate, application number：201410403374.8, a kind of cultivation matrix that can improve sporophore calcium content of disclosure of the invention is related to plant Training matrix technology field, it is characterised in that be made up of the component of following parts by weight：Bone meal 8-12 parts, straw 20-40 parts, natural pond Slag 1-2 parts, ash wood 2-4 parts, lichen 1-2 parts, Exocarpium Litchi 1-2 parts, beanstalk ash 1-2 parts, Strobilus Pini 1-2 parts, matcha 1-2 parts, richness Selenium yeast 0.5-1 parts, fishbone powder 1-2 parts, Monas cuspurpureus Went 1-2 parts, pit mud 4-6 part, turfy soil 3-5 parts, sawdust 4-6 parts, bamboo powder 1-2 Part, lobster shell 1-2 parts, fish scale 1-2 parts and antibacterial 1-2 parts.The sporophore of substrate output of the present invention can be supplemented needed for body Multi-nutrition, adjusts gastrointestinal function, adjusts microcirculation, and enhance immunity is particularly suitable for osteoporosises, hyperosteogeny, easily fractures Personage, improve resource utilization, produce higher economic benefit.

Microorganism fertilizer is a kind of to cause crops to obtain the micro- of specific fertilizer effect with microbial life activity and its product Biological living product, it in culture fertility, improve chemical fertilizer utilization ratio, suppress the harmful substance such as crops heavy metal and pesticide The aspect such as absorption, purification and rehabilitating soil, the utilization of becoming thoroughly decomposed of promotion agricultural crop straw and municipal refuse, raising quality of agricultural product has Irreplaceable effect.The efficacy exertion of microbial manure is mainly by the potentiation to traditional fertilizer, fertilizer, right The improvement activation of soil, and the mode such as the physiological action of microorganism is realizing.

Therefore, microbial manure just has chemical fertilizer, fertilizer, various fertility of beneficial organism bacterium and effect, is activation fertilizer Material nutrient, raising effect of fertilizer, crop improvement quality, the preferable fertilizer for promoting agricultural production efficiency.Microbial manure is live body Fertilizer, a large amount of beneficial microorganism vital movements that its effect contains mainly by it are completing.Only when these beneficial micro- lifes In the case that thing is in vigorous breeding and metabolism, material conversion and beneficial metabolic product constantly could be formed.Therefore, it is micro- In bio-feritlizer beneficial microorganism species, vital movement it is whether vigorous be its effectiveness basis, and unlike other fertilizer are By in the form of the essential elements such as nitrogen, phosphorus, potassium and it is how many based on.Just because of microbial manure is preparation of living, thus its fertilizer efficiency with Number of viable, intensity and ambient environmental conditions are closely related, live in soil including temperature, moisture, acid-base value, nutritional condition and original Indigenous microorganism repulsive interaction in earth has certain impact.

And agricultural microbial agent in the market is mainly single microorganism fungus kind, it is difficult to reduce Tiny ecosystem knot Structure, while the synergism of common strain and crops is weak, it is unfavorable to absorb as improved stress resistance of plant and root system nutrition, such as it is public Cloth number for CN102888356A patent application, disclose a kind of utilization bacillus subtilises prepare microbial manure method and Its application.Although having occurred some complex micro organism fungicides in the market, itself or single function, or its using method Be restricted, the crop yield effect after use can't reach it is satisfactory, so needing exist for exploitation suitable for various Route of administration, complex micro organism fungicide or fertilizer with comprehensive function and good effect of increasing production.

Put into as China's research in terms of bio-feritlizer for a long time lacks so that the bio-feritlizer industry of China is still present Integral level is not high, technological innovation is not enough, product quality and application effect show understable problem.

The content of the invention

The technical problem to be solved is to overcome the deficiencies in the prior art, there is provided a kind of edible fungi residue food With the bio-feritlizer that bacterium bacteria residue is raw material, the parts by weight are consisted of:

Pretreatment crab flavour mushroom bacteria residue 20-40, matrine 1-3, fermented cow dung 4-10；

The preparation method of pretreatment crab flavour mushroom bacteria residue is as follows：

Add crushing maize core powder and powder of straw after crab flavour mushroom bacteria residue is crushed after cultivation, moisture is controlled in 10-35%, Addition aspergillus awamori culture, control temperature keep 2-5 little in 26-35 DEG C of aerlbic culture 10-20 hours adjustment temperature 5-12 DEG C When after adjust temperature to 20-28 DEG C, spray yeast liquid culture 10-20 hours obtain final product.

The preparation method of fermented cow dung is as follows：Comprise the steps：

Cattle manure is added aspergillus niger culture regulation moisture and is built after proportionally being mixed with crushing straw and the Herba Sophorae alopecuroidiss crushed Heap ferments, and temperature keeps 3-5 hours, holding to turn at intervals of every 3-4 hours once after reaching 65-70 DEG C, treat that temperature reaches 20- Terminate fermentation process after 32 DEG C of addition bacillus subtilises culture fermentation 20-25 hours；

The cattle manure, crushing straw, Herba Sophorae alopecuroidiss and aspergillus niger culture parts by weight composition:Cattle manure 10-20 parts, crushing straw Stalk 5-10 parts, Herba Sophorae alopecuroidiss 2-5；Aspergillus niger culture 3-8 part；

1-2% of the bacillus subtilises culture addition for cattle manure quality；

The moisture of fermented cow dung should be controlled 40～65%.

The preparation method of yeast liquid：Yeast seeds access fluid medium culture 15-25 hours addition L cysteine After be obtained；

It is prepared by the bacillus subtilises culture：From inclined-plane switching culture bacillus subtilises, the kind after spreading cultivation step by step Sub- liquid is transferred in fermentation tank, and fermentation finishes fermentation liquid and bacillus subtilises culture is obtained after plate-and-frame filtration, drying.

It is prepared by aspergillus awamori culture：Spawn culture, solid fermentation culture：Spore liquid is inoculated into the training of aspergillus oryzae solid fermentation In nutriment, cultivate to mycelia for 26-33 DEG C and cover with compost, low temperature fluidized bed dry, pulverize dried object.

Bacillus subtilises (Bacillus subtilis subsp) CGMCC 7926

Aspergillus awamori (Aspergillus awamori) CGMCC3.6484

The depositary institution of above-mentioned strain is China General Microbiological culture presevation administrative center.Address：Zhongguancun, Beijing City Northern No. 13 institutes of microbiology of the Chinese Academy of Sciences；Postcode：100080.

Beneficial effect

The fertilizer of the present invention can strengthen the drought-resistant ability of crop, be particularly suitable for the fertilizer using the present invention in salt-soda soil, Being capable of improved soil.In fertilizer, beneficial microbe can produce glutathion and plant mucilage, and mineral idiosome and organic colloid are combined Together, soil aggregate can be improved, strengthens the loss of the physical property and reduction soil particle of soil, in certain bar Under part, moreover it is possible to participate in humus and formed.So applying microbial manure can improve soil physical property, be conducive to improving soil fertilizer Power.

This product using method is simple, and per mu of ground usage amount 0.3-1 kilogram, cost are only 80-100 units/mu, seed dressing or life Earth's surface sprinkling before pouring water for a long time.

The conversion of geobiont enzyme, reduce agriculture production cost, reduce chemical fertilizer use, recover soil ecology soil fertility and Effectively improve the aspects such as crop yield and can play obvious action, give full play to the organic matter efficiency of soil.

Specific embodiment

Unless stated otherwise, technological means used in the present invention are method known in those skilled in the art.Separately Outward, embodiment is interpreted as illustrative, and unrestricted the scope of the present invention, and the spirit and scope of the invention only will by right Book is asked to be limited.To those skilled in the art, on the premise of without departing substantially from spirit and scope of the present invention, to these enforcements The various changes or change that reaction condition, separation and Extraction condition in scheme is carried out fall within protection scope of the present invention.

The Wine brewing yeast strain deposit number is that CGMCC No.12789 are specially saccharomyces cerevisiae (Saccharomyces Cerevisiae) tlj2016, the bacterial strain is in being preserved in the China Committee for Culture Collection of Microorganisms of on July 15th, 2016 Common micro-organisms center, deposit number be CGMCC No.12789, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Number, Institute of Microorganism, Academia Sinica, postcode 100101.

The most suitable growth pH of the saccharomyces cerevisiae tlj2016 is 6.0-6.5, and optimum growth temperature is 28-35 DEG C；

The saccharomyces cerevisiae tlj2016 is isolated below saccharomyces cerevisiae starting strain Jing from the orchard of one plant of Ningxia Step is obtained：

The original strain that sets out → test tube activation → dithyl sulfate (DES) mutation → hypertonic plate screening → nitrosoguanidine (NTG) secondary screening (producing GSH abilities) → mitotic stability test is screened → fermented to mutagenesis screening → hypertonic flat board primary dcreening operation → shaking flask.

1st, saccharomyces cerevisiae provided by the present invention reaches 300g/L to the tolerance of glucose, beneficial to which in high concentration Portugal GSH is produced under the conditions of grape sugar；

2nd, saccharomyces cerevisiae provided by the present invention reaches 3308mg/L in 5L fermentation cylinder for fermentation production GSH final concentrations；

3rd, the ability of saccharomyces cerevisiae tolerance L-Cysteine provided by the present invention is far above starting strain, in 5mmol/L Slow growth is remained under L-Cysteine effect, remains to keep GSH to synthesize in a large number under the effect of 40mmol/LL- cysteine；

4th, saccharomyces cerevisiae salt resistance ability provided by the present invention reaches 18%, is conducive to extending its application.

GSH capacity experimentals are produced in tlj2016 fermentations under the conditions of high sugar

(1) shake-flask culture

One ring of tlj2016 slant strains is taken, is accessed and 150rpm in the 250mL shaking flasks of 30mL Shake flask mediums is housed, 30 DEG C Culture 30h obtains seed liquor；

Shake flask medium (g/L)：(NH₄)₂SO₄6th, glucose 35, K₂HPO₄·3H₂O 3、KH₂PO₄0.5th, yeast powder 11, MnSO₄ 0.1、KCL 0.1、FeSO4 0.1、MgSO₄·7H₂O 0.1, pH6.0；

(2) 5L fermentor cultivation

Seed liquor is pressed into 10% inoculum concentration, is accessed in the fermentation tank equipped with 3L fermentation medium, 30 DEG C, ventilation 6L/ Min, tank pressure 0.03MPa, 500rpm carry out fermentation culture under the conditions of permanent pH6.0, when fermenting to 30h, disposably add final concentration For the L-Cysteine of 25mmol/L, total fermentation time is 50h；

Fermentation medium (g/L)：(NH₄)₂SO₄10th, glucose 100, K₂HPO₄·3H₂O 8、KH₂PO₄0.5th, yeast powder 11、MnSO₄ 0.1、KCL 0.1、FeSO4 0.1、MgSO₄·7H₂O 0.1,pH6.0；

After fermentation ends, the content for determining GSH in fermentation liquid is 3308mg/L.

L-Cysteine tolerance is tested

By starting strain and each ring of tlj2016 slant strains, the 250mL equipped with 30mL Shake flask mediums is respectively connected to 150rpm in shaking flask, 30 DEG C are cultivated, and the half Guang ammonia of L- of different final concentrations when culture is to 12h, is added in shaking flask Acid, is further cultured for 10h, determines dry cell weight, as a result table 2,3；

Shake flask medium (g/L)：(NH₄)₂SO₄6th, glucose 20, K₂HPO₄·3H₂O 3、KH₂PO₄0.5th, yeast powder 11, MnSO₄ 0.1、KCL 0.1、FeSO4 0.1、MgSO₄·7H₂O 0.1, pH6.0；

Table 2：Starting strain L-Cysteine tolerance

L-Cysteine concentration mmol/L	0	5	10	15	20	40
							Starting strain dry weight g/L	22.6	15.7	10.2	4.3	2.2	0.8
GSH concentration (mg/L)	35.6	46.7	43.2	40.7	37.9	25.3

Table 3tlj2016L- cysteine tolerances

L-Cysteine concentration mmol/L	0	5	10	15	20	40
							Tlj2016 dry weights g/L	25.7	28.5	23.6	21.2	20.6	18.7
GSH concentration (mg/L)	73.2	98.3	113.5	121.7	127.5	135.8

From the results shown in Table 2, for starting strain, in culture medium, add L-Cysteine, cell stops growing, And start self-dissolving, cause GSH rate of increase to reduce with the rising of L-Cysteine concentration；And low concentration L-Cysteine Under, tlj2016 still is able to slow growth, with the raising of L-Cysteine concentration, under the dry cell weight of tlj2016 bacterial strains is slow Drop, and GSH concentration sustainable growths, this result pass through to add half Guang ammonia of precusor amino acids-L- in being beneficial to GSH production processes Acid proposes the production for promoting GSH.

Salt resistance ability is tested

Take tlj2016 bacterium solutions 1mL inoculation strain in containing different NaCl concentrations (concentration gradients are 0%, 2%, 5%, 10%th, 15%, 10mL YPD fluid mediums (pH=6.5) 18%), is placed at 30 DEG C and cultivates 24h respectively, and each processes 3 Individual repetition.Respectively take 1ml samples bacterium solution to mix in 9ml normal saline, prepare dilution factor solution, take 0.1ml diluents solid in YPD It is coated with body flat board, culture 36 hours (each dilution factor do 3 parallel) record is inverted in 30 DEG C of biochemical cultivation cases and calculates flat Bacterium number number on plate.The results are shown in Table 4, it is known that the resistance to salinity of the bacterium is 18%, illustrates that tlj2016 not only can be in conventional ring Survive in border, still there is under high salt conditions vigor, can be applicable to consumption sugar in the high salt food processing process such as soy sauce, curing food Produce glutathion.

4 salt resistance ability of table detection (× 10⁷cfu/ml)

NaCl contents

0%

2%

5%

10%

15%

18%

Original strain

5.16±0.42

4.38±0.42

2.15±0.21

0.12±0.11

0

tlj2016

5.33±0.28

5.10±0.71

4.83±0.42

3.98±0.33

2.57±0.48

0.83±0.15

Bacillus subtilises (Bacillus subtilis) Li-2013-02, the bacterial strain is in the preservation of July 15 in 2013 In China Committee for Culture Collection of Microorganisms's common micro-organisms center, (abbreviation CGMCC, address is：City of BeiJing, China is exposed to the sun The institute 3 of area North Star West Road 1), preserving number is CGMCC No.7926.

The strain characteristic is that the enzyme activity of product Thermostable α-Amylase is high, and heat-resisting, acid resistance is strong.

Thermostable α-Amylase enzyme activity prepared by the bacterial strain is 30000-35000u/ml；Applicable temperature scope is 105-115 DEG C, 110 DEG C of optimal reactive temperature, in 110 DEG C of enzyme activity complete stabilities；Applicable pH value in reaction scope is 3.0-7.0, Enzyme activity complete stability when pH value is 3.0, optimal reaction pH value is 4.2.

The bacterial strain feature is as follows：

Bacterial strain colony colour on solid plate is milky, and dry tack free is opaque, and neat in edge is with fortune The aerobic bacteria of dynamic property.Microscopy is elongated rod shape, and Gram’s staining is positive.The bacterium can utilize citrate, nitrate reductase, V-P realities Test into the positive.

Bacillus subtilises (Bacillus subtilis) Li-2013-02 is by one plant of product Thermostable α-Amylase Bacillus subtilises Li-2013 Jing UV-LiCls-dithyl sulfate Mutation screening is obtained, and concrete screening step is such as Under：

(1) preparation of bacteria suspension

The Li-2013 single bacterium colonies grown after plate streaking is separated are accessed in seed culture medium, 100r/min, 40 DEG C of trainings After foster 12h, take after 1mL medium centrifugals with brine twice, and it is resuspended with 9mL normal saline in.

(2) UV-LiCl-dithyl sulfate complex mutation

Bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirring irradiation 100s under the uviol lamp of power 15w.Will Lithium chloride flat board is coated Jing after gradient dilution through the bacterium solution irradiated, and flat board is applied with the bacterium solution dilution without ultra-vioket radiation and done Control.By above-mentioned coating uniform flat board, wrapped with the cloth or newspaper of black, put 40 DEG C of culture 48h, growing the flat board of bacterium colony On filter out hydrolysis circle with colony diameter ratio the maximum choose to inclined-plane preserve, be configured to bacteria suspension, Jing gradient dilutions after purification Being sufficiently mixed with dithyl sulfate stock solution afterwards, and 40min being processed in 40 DEG C of concussions, the bacterium solution for processing is applied Jing after gradient dilution It is distributed in lithium chloride flat board.

(3) primary dcreening operation of high-yield strains

By above-mentioned coating uniform flat board, 40 DEG C of culture 48h are put, primary dcreening operation goes out hydrolysis circle and bacterium on the flat board for grow bacterium colony The diameter ratio that falls the greater is chosen to inclined-plane and is preserved, and obtains three plants of bacterium Li-2013-01, Li-2013-02, Li-2013- after purification 03。

(4) shake flask fermentation secondary screening

The three plants of bacterium Li-2013-01 that will be obtained, Li-2013-02, Li-2013-03 are containing 30mL fermentation medium Shake flask fermentation, seed inoculum concentration 10% (V/V), 40 DEG C, 100r/min culture 72h, in centrifuging and taking fermentation are carried out in 250mL shaking flasks Clear liquid is obtained crude enzyme liquid.

(5) enzyme activity determination

The definition of enzyme-activity unit：1mL crude enzyme liquids, under the conditions of 105 DEG C, pH 4.2,1min liquefaction 1mg soluble starches, As 1 enzyme activity unit, is represented with U/mL.

Jing is determined, and bacterial strain Li-2013-02 is stable most superior strain, and enzyme activity reaches 30000U/mL.

The lithium chloride flat board：Starch 1%, peptone 1%, (NH)₂SO₄0.4%, K₂HPO₄0.8%, CaCl₂0.2%, lithium chloride 0.9%, agar 2%.

Described seed culture medium：Yeast powder 0.5%, peptone 1%, soluble starch 1%, NaCl 1%.

Described fermentation medium：Semen Maydis powder 5%-15%, soybean cake powder 4%-10%, (NH)₂SO₄0.4%, K₂HPO₄ 0.8%, CaCl₂0.2%.

Described shake flask culture conditions：The bacterium in the 250mL shaking flasks containing 30mL fermentation medium, inoculum concentration 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.

The high-temperature resistant alpha-amylase, its zymologic property are as follows：

(1) the enzyme temperature adaptation wider range, optimum temperature is between 100-110 DEG C, and preserves below 110 DEG C , temperature stability is preferable, and more than 110 DEG C preservation long-time temperature stabilities are poor.

(2) the enzyme optimal reaction pH value is 4.2.There is high enzyme vigor between pH value 3.0-7.0, be 3.0 in pH value When enzyme activity complete stability.

(3) enzymatic activity：By mutant Li-2013-02 provided by the present invention, the Thermostable α-Amylase enzyme activity of preparation For 30000-35000U/ml.

The preparation method of aspergillus awamori microbial inoculum：

Slant strains activation culture：Aspergillus awamori slant strains are transferred on slant medium, 27 DEG C are cultivated 3 days.

Solid first order seed culture：Picking aspergillus awamori slant strains access 500 milliliters of triangles equipped with 100 grams of culture medium Seed culture is carried out in bottle, 30 DEG C are cultivated 3 days.

Solid secondary seed culture：Above-mentioned cultured solid first order seed stirring is equipped with into 1000 grams to add after fragment Seed culture, condition of culture are carried out in 5000 milliliters of triangular flasks of culture medium：30 DEG C are cultivated 3 days.

Solid fermentation culture：Second-level shake flask seed is crushed, is mixed in adding the fermentation vat equipped with sterilising medium or pallet Close it is uniform after cultivate, the control of zymogenic cultivation temperature at 26-35 DEG C, humidity 80-90%, during culture every 10 hours stirrings once Between 5-7 days；The culture of solid zymogenic is using conventional zymogenic culture technique；Treat that compost terminates culture, culture by covering with mycelia The advance Jing high temperature steamings sterilization treatment of base, sterilising conditions 121 DEG C of temperature of control, 1 hour time.

Drying and crushing：Fermentation ends compost is dried on fluid bed or other drying equipments, baking temperature control At 60 DEG C, be dried to moisture below 10%, then solid culture medium crushed, crushing material aperture 60 mesh with On.

Culture medium is constituted：Solid material：Wheat bran 80, soybean cake powder 10%, corn starch 10% add equivalent tap water；Just Beginning pH nature.

The preparation method of bacillus subtilises culture：

The acquisition of fermentation liquid：Spread cultivation step by step acquisition bacillus subtilis fermentation liquor using slant strains；

(1) first order seed culture：Bacillus subtilises slant strains are accessed in 500 milliliters of shaking flasks, culture medium loading amount 100 Milliliter, 180 revs/min of rotary shaker, 30 DEG C of cultivation temperature, incubation time 24 hours；

(2) secondary seed culture：First order seed is accessed in 500 milliliters of secondary seed shaking flasks according to 10% inoculum concentration, Condition of culture is identical with first order seed；

(3) three-level seed culture：Secondary seed is accessed in 5000 milliliters of three-level seed flasks with 10% inoculum concentration, culture 1000 milliliters of base loading amount, 100 revs/min of rotary shaker, 30 DEG C of cultivation temperature, incubation time 24 hours；

(4) first class seed pot culture：Three-level seed is accessed into first class seed pot of the total measurement (volume) as 150L with 10% inoculum concentration, Fermentation medium loading amount 100L, 28 DEG C of cultivation temperature, 100 revs/min of mixing speed, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours；

(5) fermentation culture：First class seed pot strain is accessed into total measurement (volume) as 1.5 tons of secondary seed tanks with 10% inoculum concentration, 1 ton of fermentation medium loading amount, 28 DEG C of condition of culture cultivation temperature, 100 revs/min of mixing speed, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours.Fermentation finishes fermentation liquid and microbial inoculum containing bacillus subtilis is obtained after plate-and-frame filtration, drying In interior bacillus subtilises culture.

Culture medium is constituted：Glucose 6%, yeast extract 1%, peptone 0.2%, CaCO₃1%, pH6.8.

Embodiment 1：

The technical problem to be solved is to overcome the deficiencies in the prior art, there is provided a kind of high-efficiency composite biological is organic Fertilizer, the parts by weight are consisted of:

Pretreatment crab flavour mushroom bacteria residue 30, matrine 2, fermented cow dung 8；

Add crushing maize core powder and powder of straw after crab flavour mushroom bacteria residue is crushed after cultivation, control moisture adds 18% Plus aspergillus awamori culture, control temperature adjustment temperature after 31 DEG C of aerlbic cultures, 8 DEG C of adjustment temperature holding 3 hours in 15 hours and arrive 25 DEG C, sprinkling yeast liquid culture is obtained final product for 15 hours.

Cattle manure is added aspergillus niger culture regulation moisture and is built after proportionally being mixed with crushing straw and the Herba Sophorae alopecuroidiss crushed Heap ferments, and temperature is kept for 4 hours after reaching 66 DEG C, and holding is turned at intervals of once, treating that per 3.5 hours temperature reaches 41 DEG C of additions Bacillus subtilises culture terminates fermentation process after fermenting 23 hours；

The cattle manure, crushing straw, Herba Sophorae alopecuroidiss and aspergillus niger culture parts by weight composition:15 parts of cattle manure, crushing straw 8 Part, Herba Sophorae alopecuroidiss 4；5 parts of aspergillus niger culture；

The bacillus subtilises culture addition for cattle manure quality 1%；

The moisture of fermented cow dung should be controlled in 50-55%.

Matrine preparation technology is as follows:

1st, smash：Herba Sophorae alopecuroidiss are smashed to 40 mesh of fineness degree.

2nd, soak：By solid-liquid ratio 1:10(m：V weight) soaks 3h, immersion temperature in the mixed solution of sodium carbonate and ethanol Degree control is at 65 DEG C；

3rd, extract：Soaked Herba Sophorae alopecuroidiss mixture is placed in ultrasonic extraction tank and extracts matrine, ultrasonic frequency 60-80kHz, extraction time 35min, extraction time 2 times, temperature control is at 65 DEG C；

4th, extracting solution filtering and concentrating：

The Radix Sophorae Flavescentiss alkali extracting solution Jing vacuum concentration equipments for having extracted are concentrated into into Brix15-16, PH6.5-7.0.Matrine Content 7.6%；The extraction rate reached more than 93% of matrine.

The bio-fertilizer preparation method is as follows：Various microbial inoculums are mixed according to aforementioned proportion, or pelletized after mixing.

Bacillus subtilises (Bacillus subtilis subsp) CGMCC 7926

Aspergillus awamori (Aspergillus awamori) CGMCC3.6484

Prepared by yeast fermentation liquor culture, adopt Wine brewing yeast strain deposit number for CGMCC No.12789.

(1) shake-flask culture

(2) 5L fermentor cultivation

Seed liquor is pressed into 10% inoculum concentration, is accessed in the fermentation tank equipped with 3L fermentation medium, 30 DEG C, ventilation 6L/ Min, tank pressure 0.03MPa, 500rpm carry out fermentation culture under the conditions of permanent pH6.0, when fermenting to 14-15h, disposable addition is eventually Concentration obtains yeast fermentation liquor for the L-Cysteine of 16mmol/L；

Fermentation medium (g/L)：(NH₄)₂SO₄10th, glucose 50, K₂HPO₄·3H₂O 8、KH₂PO₄0.5th, yeast powder 11、MnSO₄ 0.1、KCL 0.1、FeSO4 0.1、MgSO₄·7H₂O 0.1,pH6.0；

Embodiment 2

A kind of high-efficiency composite biological organic fertilizer, the parts by weight are consisted of:

Pretreatment crab flavour mushroom bacteria residue 20, matrine, fermented cow dung 4；

Add crushing maize core powder and powder of straw after crab flavour mushroom bacteria residue is crushed after cultivation, control moisture adds 30% Plus aspergillus awamori culture, control temperature adjustment temperature after 35 DEG C of aerlbic cultures, 5 DEG C of adjustment temperature holding 5 hours in 10 hours and arrive 20 DEG C, sprinkling yeast liquid culture is obtained final product for 10 hours.

Cattle manure is added aspergillus niger culture regulation moisture and is built after proportionally being mixed with crushing straw and the Herba Sophorae alopecuroidiss crushed Heap ferments, and temperature is kept for 4 hours after reaching 65-70 DEG C, and holding is turned at intervals of every 3-4 hours once, treats that temperature reaches 25 DEG C Terminate fermentation process after addition bacillus subtilises culture fermentation 20-25 hours；

The cattle manure, crushing straw and matrine parts by weight composition:20 parts of cattle manure, 5 parts of crushing straw, Herba Sophorae alopecuroidiss 2；It is black 8 parts of aspergillosis culture；

The bacillus subtilises culture addition for cattle manure quality 2%；

The moisture of fermented cow dung should be controlled 65%.

The preparation method of yeast liquid：With example 1.

Matrine preparation technology is as follows:

Smash：Herba Sophorae alopecuroidiss are smashed to 40 mesh of fineness degree.

Immersion：By solid-liquid ratio 1:12(m：V weight) soaks 4h, soaking temperature in the mixed solution of sodium carbonate and ethanol Control is at 60 DEG C；The mass ratio of the sodium carbonate and ethanol is 1：6.

Extract：Soaked Herba Sophorae alopecuroidiss mixture is placed in ultrasonic extraction tank and extracts matrine, ultrasonic frequency 60- 80kHz, extraction time 40min, extraction time 1 time, temperature control is at 60-70 DEG C；

Extracting solution is concentrated：The Radix Sophorae Flavescentiss alkali extracting solution Jing vacuum concentration equipments for having extracted are concentrated into into Brix16, PH6.5- 7.0。

Matrine content 7.6%；The extraction rate reached more than 93% of matrine.

The bio-fertilizer preparation method is as follows：According to granulation after the mixing of above-mentioned substance ratio.

Bacillus subtilises (Bacillus subtilis subsp) CGMCC 7926

Aspergillus awamori (Aspergillus awamori) CGMCC3.6484

Embodiment 3

Product effect experimental

Selection experimental field and EXPERIMENTAL DESIGN：Test in the August of 2016 on the 27th April in 2015 30 days in Jinghai county Salt-soda soil is carried out.

Experimental plot reaches 5 mu of field maize planting, uses 0.4 kilogram per mu of product of the present invention in plantation respectively, emerges 1 Or so moon uses 0.4 kilogram of invention product, matched group to use commercially available organic fertilizer by loose ground mode.

Invention product reaches 500 kilograms using milpa corn yield, and test group is sent out seedling stage diseases and pests and significantly reduced, parasite killing Medicine is the 40% of matched group；Matched group reaches 260 kilograms；The plot is reached in the 2nd year plant spring wheat, spring wheat production 420 kilograms, 60% is improve than matched group per unit area yield.And experimental plot Soil structure is good, without hardened.

Claims

1. bio-feritlizer of a kind of edible fungi residue edible fungi residue for raw material, the bio-feritlizer parts by weight consist of:In advance Process crab flavour mushroom bacteria residue 20-40, matrine 1-3, fermented cow dung 4-10.

2. edible fungi residue edible fungi residue according to claim 1 is the bio-feritlizer of raw material, it is characterised in that described The preparation method of pretreatment crab flavour mushroom bacteria residue is as follows：

Add crushing maize core powder and powder of straw after crab flavour mushroom bacteria residue is crushed after cultivation, control moisture is in 10-35%, addition Aspergillus awamori culture, control temperature is after 26-35 DEG C of aerlbic culture 10-20 hours adjustment temperature 5-12 DEG C keeps 2-5 hours Adjustment temperature to 20-28 DEG C, sprinkling yeast liquid culture 10-20 hours are obtained final product.

3. edible fungi residue edible fungi residue according to claim 1 is the bio-feritlizer of raw material, it is characterised in that described The preparation method of fermented cow dung is as follows：

Cattle manure is added aspergillus niger culture regulation moisture and builds heap after proportionally being mixed with crushing straw and the Herba Sophorae alopecuroidiss crushed Ferment, temperature keep 3-5 hours, holding to turn at intervals of every 3-4 hours once after reaching 65-70 DEG C, treat that temperature reaches 20-32 DEG C Terminate fermentation process after addition bacillus subtilises culture fermentation 20-25 hours；

The cattle manure, crushing straw and Herba Sophorae alopecuroidiss parts by weight composition:Cattle manure 10-20 parts, crushing straw 5-10 parts, Herba Sophorae alopecuroidiss 2- 5；Aspergillus niger culture 3-8 part；

1-2% of the bacillus subtilises culture addition for cattle manure quality；

The moisture of fermented cow dung should be controlled 40～65%.

4. edible fungi residue edible fungi residue according to claim 1 is the bio-feritlizer of raw material, it is characterised in that described

The preparation method of yeast liquid：Yeast seeds are made after accessing fluid medium culture 15-25 hours addition L cysteine .

5. edible fungi residue edible fungi residue according to claim 1 is the bio-feritlizer of raw material, it is characterised in that described The yeast strain deposit number is CGMCC No.12789.

6. edible fungi residue edible fungi residue according to claim 1 is the bio-feritlizer of raw material, it is characterised in that described Bacillus subtilises (Bacillus subtilis subsp) CGMCC 7926, aspergillus awamori (Aspergillus awamori) CGMCC3.6484。

7. according to the arbitrary edible fungi residue edible fungi residue of claim 1-6 for raw material bio-feritlizer preparation method,

Pretreatment crab flavour mushroom bacteria residue, matrine, fermented cow dung are proportionally mixed,

Add crushing maize core powder and powder of straw after crab flavour mushroom bacteria residue is crushed after cultivation, control moisture 30%, steep by addition Aspergillosis culture is contained, control temperature adjusts temperature to 25 after 31 DEG C of aerlbic cultures, 8 DEG C of adjustment temperature holding 3 hours in 15 hours DEG C, sprinkling yeast liquid culture is obtained final product for 15 hours；

The preparation method of fermented cow dung is as follows：Comprise the steps：The Herba Sophorae alopecuroidiss of cattle manure and crushing straw and crushing are proportionally After mixing add aspergillus niger culture adjust moisture build heap fermentation, temperature reach 66 DEG C after keep 4 hours, holding turn at intervals of Per 3.5 hours once, reach after 41 DEG C of addition bacillus subtilises cultures ferment 23 hours after temperature and terminate fermentation process；Institute State cattle manure, crushing straw, Herba Sophorae alopecuroidiss and aspergillus niger culture parts by weight composition:15 parts of cattle manure, 8 parts of crushing straw, Herba Sophorae alopecuroidiss 4；5 parts of aspergillus niger culture；The bacillus subtilises culture addition for cattle manure quality 1%；The moisture of fermented cow dung Content should be controlled in 50-55%；

Matrine preparation technology is as follows:Smash：Herba Sophorae alopecuroidiss are smashed to 40 mesh of fineness degree；Immersion：By solid-liquid ratio 1:10(m：V) Weight soaks 3h in the mixed solution of sodium carbonate and ethanol, and soaking temperature is controlled at 65 DEG C；Extract：By soaked Herba Sophorae alopecuroidiss Mixture is placed in ultrasonic extraction tank and extracts matrine, ultrasonic frequency 60-80kHz, extraction time 35min, extraction time 2 Secondary, temperature control is at 65 DEG C；Extracting solution filtering and concentrating：The Radix Sophorae Flavescentiss alkali extracting solution Jing vacuum concentration equipments for having extracted are concentrated into Brix15-16, PH6.5-7.0；Matrine content 7.6%；The extraction rate reached more than 93% of matrine；

It is prepared by the bacillus subtilises culture：From inclined-plane switching culture bacillus subtilises, the seed liquor after spreading cultivation step by step Transfer in fermentation tank, fermentation finishes fermentation liquid and bacillus subtilises culture is obtained after plate-and-frame filtration, drying；

It is prepared by aspergillus awamori culture：Spawn culture, solid fermentation culture：Spore liquid is inoculated into aspergillus oryzae solid state fermentation culture material In, to cultivate to mycelia for 26-33 DEG C and cover with compost, low temperature fluidized bed dry, pulverize dried object；Bacillus subtilises (Bacillus subtilis subsp) is CGMCC 7926, aspergillus awamori (Aspergillus awamori) CGMCC3.6484；

It is prepared by yeast fermentation liquor culture：

(1) shake-flask culture

One ring of tlj2016 slant strains is taken, 150rpm in the 250mL shaking flasks equipped with 30mL Shake flask mediums, 30 DEG C of cultures are accessed 30h obtains seed liquor；

(2) 5L fermentor cultivation

Seed liquor is pressed into 10% inoculum concentration, is accessed in the fermentation tank equipped with 3L fermentation medium, 30 DEG C, ventilation 6L/min, tank Pressure 0.03MPa, 500rpm, carry out fermentation culture, when fermenting to 14-15h, disposably add final concentration of under the conditions of permanent pH6.0 The L-Cysteine of 16mmol/L obtains yeast fermentation liquor；

Fermentation medium (g/L)：(NH₄)₂SO₄10th, glucose 50, K₂HPO₄·3H₂O 8、KH₂PO₄0.5th, yeast powder 11, MnSO₄ 0.1、KCL 0.1、FeSO4 0.1、MgSO₄·7H₂O 0.1,pH6.0。

8. according to the arbitrary edible fungi residue edible fungi residue of claim 1-6 for raw material bio-feritlizer preparation method, The parts by weight are consisted of:Pretreatment crab flavour mushroom bacteria residue 20, matrine 1, fermented cow dung 4；

The preparation method of pretreatment crab flavour mushroom bacteria residue is as follows：Add crushing maize core powder and straw after crab flavour mushroom bacteria residue is crushed after cultivation Stalk powder, control moisture add aspergillus awamori culture 16%, and control temperature is in 35 DEG C of aerlbic cultures, 10 hours adjustment temperature 5 DEG C of degree adjusts temperature to 20 DEG C after being kept for 5 hours, sprinkling yeast liquid culture is obtained final product for 10 hours；

The preparation method of fermented cow dung is as follows：Comprise the steps：The Herba Sophorae alopecuroidiss of cattle manure and crushing straw and crushing are proportionally Add aspergillus niger culture regulation moisture after mixing and build heap fermentation, temperature is kept for 4 hours after reaching 65-70 DEG C, between holding is turned It is divided into every 3-4 hours once, terminates fermentation after temperature reaches 25 DEG C of addition bacillus subtilises culture fermentation 20-25 hours Process；The cattle manure, crushing straw and Herba Sophorae alopecuroidiss parts by weight composition:20 parts of cattle manure, 5 parts of crushing straw, Herba Sophorae alopecuroidiss 2；Black fermented preparation 8 parts of mould culture；The bacillus subtilises culture addition for cattle manure quality 2%；The moisture of fermented cow dung should Control 65%；

Matrine preparation technology is as follows:

Smash：Herba Sophorae alopecuroidiss are smashed to 40 mesh of fineness degree；

Immersion：By solid-liquid ratio 1:12(m：V weight) soaks 4h, soaking temperature control in the mixed solution of sodium carbonate and ethanol At 60 DEG C；Extract：Soaked Herba Sophorae alopecuroidiss mixture is placed in ultrasonic extraction tank and extracts matrine, ultrasonic frequency 60- 80kHz, extraction time 40min, extraction time 1 time, temperature control is at 60-70 DEG C；

Extracting solution is concentrated：The Radix Sophorae Flavescentiss alkali extracting solution Jing vacuum concentration equipments for having extracted are concentrated into into Brix16, PH6.5-7.0；

Matrine content 7.6%；The extraction rate reached more than 93% of matrine；

It is prepared by aspergillus awamori culture：Spawn culture, solid fermentation culture：Spore liquid is inoculated into aspergillus oryzae solid state fermentation culture material In, to cultivate to mycelia for 26-33 DEG C and cover with compost, low temperature fluidized bed dry, pulverize dried object；Bacillus subtilises (Bacillus subtilis subsp) is CGMCC 7926, and aspergillus awamori (Aspergillus awamori) is CGMCC3.6484。