CN105505799A - Fermentation method and application of trichoderma viride mutant strains - Google Patents
Fermentation method and application of trichoderma viride mutant strains Download PDFInfo
- Publication number
- CN105505799A CN105505799A CN201610049068.8A CN201610049068A CN105505799A CN 105505799 A CN105505799 A CN 105505799A CN 201610049068 A CN201610049068 A CN 201610049068A CN 105505799 A CN105505799 A CN 105505799A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- viride
- powder
- mutant strain
- fermentation process
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention discloses a fermentation method and application of trichoderma viride mutant strains. The method comprises the steps that the strains are activated, a spore suspension of the activated strains is cultured, a solid fermentation cultivation base material is prepared, and solid fermentation is performed to obtain a fermentation product of the trichoderma viride mutant strains. The fermentation product can be used for preparing a medicament for preventing soil-borne disease. Chitin and crystallite carboxymethylcellulose are screened out to be used as an inductor for promoting fungus synthesis of chitinase and cellulase, graminaceous crop strawand beast and bird slaughtered blood are used as a nitrogen source base material for fermentation by means of the characteristics of the fungus of being addicted to cane sugar, crop disease resistance is achieved, growth is promoted, agriculture and animal husbandry waste can also be comprehensively utilized, environmental pollution is reduced, and coordinated development of economic, social and ecological benefits is facilitated.
Description
Technical field
The present invention relates to field of microbial culture technology, specifically a kind of fermentation process of viride mutant strain and application.
Background technology
Viride is a kind of imperfect fungi of ideal anti-soil-borne disease in Trichoderma, and not only effect is fast, and antibacterial broad spectrum is strong, also has the effect of certain short plant growth.Especially the damping-off in control soil and head blight are all had and acted on soon and significantly, when above-mentioned disease occurs, both can symptom management rapidly, also can admix seed when crop seed is sowed and play epidemic prevention effect.Good action can be played in addition to environmental factor adaptation aspects such as the nutritional utilization of crop, envrionment temperature and light.Therefore, on the basis of biological action understanding it, filter out and be suitable for it and grow and synthesize the product playing biology bacteriostatic action, be applied to minimizing crop pest, promotion plant growth, provide the food of safety and Health significant for people and animals.
The research institute Sun Yong of (2007 ~ 2014) Heilongjiang Forestry occupation in recent years qin studies biological characteristics and the control of generation to the harm of cultivating black fungus by viride, the people such as Wang Chun filter out from sclerotium blight of sunflower and sprout biological and ecological methods to prevent plant disease, pests, and erosion trichoderma viride strain about restraint to hyphal cluster germ growth and sclerotium disease thereof, more than 90%, more than 80% is reached respectively to the preventive effect about germ spore germination suppression and excised leaf, and finds that cultivating this biological and ecological methods to prevent plant disease, pests, and erosion trichoderma viride strain has the good production traits and biocontrol effect.Namely its mechanism to be the wild viride of institute's screening and separating through the second time purifying fungus of mutagenesis screening through further enlarged culturing (namely ferment); form a large amount of sporophyte or mycelium; suitably add additive again and make granule, pulvis; make it to be convenient to admix seed to be sown into together in soil; sprout when raining or soil humidity is suitable; by decomposing the soil organism (as crop material); its mycelia is wound around also secretory cell product along allos thalline and is killed by allos fungus; or suppress other varied bacteria growings, protection farm crop.
Summary of the invention
The object of the present invention is to provide that a kind of cost is low, efficiency is high, the fermentation process of free of contamination viride mutant strain and application.
For achieving the above object, the invention provides following technical scheme:
A fermentation process for viride mutant strain, comprises the following steps:
(1) liquid fermenting, comprises the following steps:
11) activation of bacterial strain: the viride mutant strain through colchicine chemomorphosis is inoculated on PDA substratum cultivate 5 ~ 7 days, its fungus activate and adapt to fermentation new environment;
12) after activation, the spore suspension of bacterial strain is cultivated: get the bacterial strain after activation, inoculation culture is in showing spore maturation under the microscope, with the spore after the activation of 10ml aseptic water washing in the aseptic triangular flask of 150ml, then on constant-temperature table with 120r/min vibrate 20min, after spore ball is fully disperseed, with Hematocyte Counter counting, then with 1 × 10
8the inoculum size of individual/ml is inoculated in certain volume and the fermentation cylinder for fermentation 5 ~ 7 days of liquid nutrient medium is housed, and obtains the spore suspension activating rear bacterial strain; During the fermentation, regulate pH between 4.3 ~ 6.5 with 0.10 ~ 0.25molNaOH;
(2) solid fermentation, comprises the following steps:
21) solid fermentation culture base-material prepares: according to mass percent, be made up of carbon element 38%, nitrogen 4%, phosphate ore prime element 1%, stone carbon dust 22%, water 25% and fungus 10%; Described carbon source is from bagasse, crop material powder, wheat bran, corn husk powder, one or more the mixture of cracking rice in chaff of the equal < 10% of water ratio; Described nitrogen comprises inorganic nitrogen-sourced and organic nitrogen source, and inorganic nitrogen-sourced is urea, (NH
4) NO
3, NaNO
3, one or more mixture in Sodium Nitrite and ammonium sulfate, organic nitrogen source is one or more the mixture in peptone, fish meal and blood meal; Described phosphate ore prime element is KH
2pO
4, K
2hPO
4, Na
2hPO
4, NaH
2pO
4, (NH
4)
2hPO
4(NH
4) H
2pO
4in one or more mixture; Described fungus is derived from the spore suspension of bacterial strain after the activation in step (1); The preparation of described blood meal: purchase country fair slaughter after animal blood, pour in the boiling water of steam cooker, namely 3min pulls precipitation out, pulverize air-dry or dry break into powder again, be blood meal;
22) solid fermentation: carbon element, nitrogen, phosphate ore prime element, stone carbon dust are combined according to the above ratio, through fully stirring, the high-temperature sterilization 30min under 1.13 normal atmosphere in steam cooker or disinfection tank, discharging, after being chilled to room temperature, add the spore suspension of bacterial strain after activation in step (1) in proportion, after fully mixing thoroughly, proceed to large fermentation tank temperature controlled fermentation 5 ~ 7 days, leavening temperature is at 28 ± 0.5 DEG C, cold wind dries up packaging, obtains the tunning of viride mutant strain.
As the further scheme of the present invention: described step 12) in, also add the amount of 80 units of Penicillin 200 μ L according to often liter of bulk solution, add 80 units of Penicillin; Fermentor tank domestic demand continues through apparatus of oxygen supply and keeps oxygen supply, meanwhile, also needs the space keeping leaving 1/3 ~ 2/5 in fermentor tank.
As the further scheme of the present invention: described step 12) in, during the fermentation, regulate pH between 5.3 ~ 6.2 with 0.10 ~ 0.25molNaOH.
As the further scheme of the present invention: described step 12) in, also comprise and adopt the method for rotary evaporation or vacuum concentration that the spore suspension of bacterial strain after the activation obtained is concentrated into 3 ~ 5 times.
As the further scheme of the present invention: described crop material powder comprises sugarcane bar powder, milled powders of cornstalk, rice straw powder and beans straw powder.
As the further scheme of the present invention: described carbon element is the one in wheat bran+sugarcane bar powder, corn husk powder+wheat bran, sugarcane bar powder+corn husk powder, wheat bran+chaff of cracking rice, chaff of cracking rice+corn husk powder, corn husk powder.
As the further scheme of the present invention: described carbon element is corn husk powder+wheat bran, wheat bran+sugarcane bar powder, or chaff of cracking rice+corn husk powder; Described inorganic nitrogen-sourced be ammonium nitrate and urea; Described organic nitrogen source is peptone and blood meal; Described phosphate ore prime element is mass ratio is KH
2pO
4, or etc. the KH of quality
2pO
4+ (NH
4)
2hPO
4.
As the further scheme of the present invention: in described step (1), the preparation of liquid nutrient medium: peeled potatoes 600g, first thinly slice to pulverize again and boil 20min, filter with tail cloth and wring out waste and stay juice, add 45g wheat bran or corn husk powder or chaff of cracking rice as vitamin B group donor, boil half an hour more than, filtered while hot waste; With sucrose 60g, honey 90g, inject 800,000 units of Penicillin with syringe from fermentor tank well, yeast powder 15g, concentration is the colloidal state chitin 3ml of 10g/L, then adds water until the space of only remaining 2L in 5L fermentor tank, sterilizing; The chitinous preparation of described colloidal state: take chitin 10g, add 100ml in 85% phosphoric acid solution of 4 DEG C of precoolings, abundant mixing, and be placed in 4 DEG C of insulations 2 round the clock, add again excessive pH value be 6.0 phosphoric acid buffer adjustment chitin concentration be 10g/L, put into vial, add the granulated glass sphere of diameter 3mm, again on shaking table with the velocity fluctuation 2h of 180r/min, the colloidal state chitin that concentration is 10g/L can be obtained.
As the further scheme of the present invention: in described step (2), the component of solid fermentation used medium and proportioning are: peeled potatoes 150 ~ 200g/L; Sucrose 101.5g/L; Sodium phosphate 3.5-5.0%; FeSO
40.1 ~ 0.3g; KH
2pO
40.2 ~ 2.0g; MgSO
40.5 ~ 0.35g; CaSO
40.5 ~ 0.10g; ZnSO
40.1 ~ 0.2g; Redistilled water 1.0L; PH state of nature.
The application of tunning in the medicament of preparation control soil-borne disease of the viride mutant strain that described fermentation process obtains.
Compared with prior art, the invention has the beneficial effects as follows:
Viride mutant strain of the present invention is the mutant strain that separation and purification wild strain out obtains the class soil-borne diseases such as Effective Anti wheat scab through chemomorphosis from residual rotten timber, it is a kind of mutant strain having hereditary defect, only need suitable substratum, it just better can grow and synthesize comparatively wild strain higher output yield and the good bioactive product of germ resistance, to improve the use value of target product, reduce production cost, reach good economic benefit.The antibacterial target product chitinase of this viride mutant strain, the activity of cellulase comparatively wild-type are increased to 710U by 340U respectively, 14U rises to 58U, may be used for the harmful cause of disease fungus prevented in soil, on the one hand, its underproduction caused that harms the crops can be avoided, on the other hand, it can also be avoided to affect human and livestock health by food chain.
The present invention is by filtering out chitin and crystallite carboxymethyl cellulose and carry out promoting this fungi to the synthesis of chitinase and cellulase as inductor and according to the characteristic addicted to sucrose of this bacterium, with gramineous crop stalk, slaughtering blood for nitrogenous source for base-material ferments, reach not only to crop disease-resistant but also growth promoting effects, also can realize fully slaughtering waste to husbandry to fully utilize, reduce environmental pollution, promote economic, social, ecological three benefit cooperative developments.The inventive method cost is low, efficiency is high, pollution-free.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of fermentation process of viride mutant strain, comprises the following steps:
(1) liquid fermenting, comprises the following steps:
11) activation of bacterial strain: the viride mutant strain through separation screening mutagenesis is inoculated on PDA substratum cultivate 5 ~ 7 days, its fungus activate and adapt to fermentation new environment;
Wherein the mutagenic processes of viride mutant strain is: first sample from the aqueous solution of the residual body of field plant decay, be inoculated on PDA solid medium in 26 DEG C and cultivate 3 ~ 5 days, contrasting national microbial bacteria storehouse announces after pattern M1, M2 carry out Screening and Identification, through two generation cultured continuously go out purifying trichoderma viride strain; By purifying trichoderma viride strain, through liquid PDA substratum, (this liquid PDA substratum is formed by following material configuration according to weight percent: potato 2 ~ 3% again; Fish protein powder 0.5 ~ 3.3%; FeSO
40.01 ~ 0.03%; KH
2pO
40.02 ~ 0.20%; MgSO
40.05 ~ 0.35%; Na
2b
2o
710H
2o0.025 ~ 0.05%; Na
2seO
30.025 ~ 0.05%, ZnSO
40.01 ~ 0.02%, surplus is water; PH value is adjusted to 5.8 ~ 6.5, culture temperature is 27 ± 1.0 DEG C, incubation time is 5 days) middle enlarged culturing, centrifugally remove clear liquid (retain 4 DEG C, refrigerator), measure active, use secondary pure water again, with the centrifugal 15min of 8000r/min, get and precipitate and repeat an enlarged culturing, centrifugally remove clear liquid (retain 4 DEG C, refrigerator), measure active, use secondary pure water again, with the centrifugal 15min of 8000r/min, get after precipitation dries up and be stored in refrigerator dark place, less to ensure that fungus excellent characteristic changes; Clear liquid after centrifugal is put into PDA substratum, with chitin or crystallized carboxylic acid's sodium cellulosate, for reaction substrate, (wherein chitinous addition adds in fermenting container according to fermented product volume capacity preparation (10g/L) separately, adding of crystallized carboxylic acid's sodium cellulosate is also add in fermenting container according to fermented product volume capacity preparation (10g/L) separately), using chemical mutagen---colchicine is as mutagenic compound, carry out mutagenesis, wherein the concentration of mutagenic compound is 0.005 ~ 0.08wt%; Mutation time is 6 ~ 120h, and mutagenesis through centrifuging and taking throw out, is viride mutant strain, is placed in dark place and stablizes 4 ~ 6h after stopping, and avoids meeting light and recovers sudden change and lead to the failure.
12) after activation, the spore suspension of bacterial strain is cultivated: get the bacterial strain after activation, inoculation culture is in showing spore maturation under the microscope, with the spore after the activation of 10ml aseptic water washing in the aseptic triangular flask of 150ml, then on constant-temperature table with 120r/min vibrate 20min, after spore ball is fully disperseed, with Hematocyte Counter counting, then with 1 × 10
8the inoculum size of individual/ml is inoculated in certain volume and is equipped with in the fermentation unit tank (V:30L-5 ton) of liquid nutrient medium ferments 5 ~ 7 days, obtains the spore suspension activating rear bacterial strain;
Wherein being set as of fermentor tank effective volume: the fermentation of such fungi is aerobic fermentation, for ensureing fermentation validity, certain apparatus of oxygen supply need be continued through and keep oxygen supply, except not stopping to stir except oxygenation, also make in fermentor tank, to keep 1/3 ~ 2/5 space absence of liquid substratum to be stained with at the bottom of wall and tank, prevent living contaminants.
The antiforeign bacteria of fermentation system pollutes: though the pathogenic agent of the anti-soil-borne disease of viride, and still affect germ resistance by similar different strains pollutes during the fermentation, during the fermentation, often liter of bulk solution need add 80 units of Penicillin 200 μ L.
In fermentor tank, reaction system pH controls: the startup of fermentation, continuity and surrounding environment pH are closely related, the crucial output height starting rear perdurability and product, timely detection, regulates pH between 4.3 ~ 6.5 with 0.10 ~ 0.25mol or 0.10 ~ 0.25molNaOH, better with 5.3 ~ 6.2.
The vitality test of fermented product primary product: the mensuration of vigor represents the validity of the height of quality product, anti-microbial effect.With product chitinase and cellulase activity size most important, the former measures by DN5 method, and (Li Jiang is far, Jiang Like; Wood mould on level ground with effect preliminary study [J] on rye grass) Practaculture Science, 2008,23 (1): 103-105); The latter measures [Kong Lei etc., filter paper sugar-DNA colorimetric method for determining cellulose enzyme vigor [J] print and dye, 1999,25 (1): 36 ~ 38] by filter paper sugar-DNA method.
Concentrated and the storage of fermented liquid, the active size of liquid fermentation production is relevant to fermented liquid working substance content, finite concentration need be reached by concentrated for keeping greater activity, need under general normal temperature to be concentrated into 3 ~ 5 times, after can reaching 6 months, the activity (namely enzyme is lived) of concentrated solution active substance is not less than less than 50% of former activity.Therefore normal employing rotary evaporation or vacuum concentration, the former is more satisfactory.
(2) solid fermentation, comprises the following steps:
21) solid fermentation culture base-material prepares:
The carbohydrate base-material of solid fermentation: the bagasse (or corn stalk, straw, beans stalk etc.) collected after making squeezing dries and is ground into 0.10 ~ 0.50mm, through natural air drying or infrared drying, makes water content < 10%, stores; Wheat bran, corn husk and chaff of cracking rice (dry equally); Ordinary tap water;
Other non-carbohydrate base-materials of solid fermentation: purchase country fair slaughter after animal blood, pour in the boiling water of steam cooker, namely 3min pulls precipitation out, pulverize air-dry or dry break into powder again, be blood meal, store with bottled.
Inorganic elements needed for solid fermentation: KH
2pO
4; Na
2hPO
4; NaH
2pO
4; (NH
4) NO
3, urea, (NH
4)
2sO
4, MgSO
4.
Fermentation inducement thing: because the bacterial strain of fermentation is addicted to chitin defective type, needs supply chitin maybe can supply containing chitinous intermediate, promotes that bacterial strain is bred, the chitin of the antibacterial enzyme of synthesis high-yield and high-efficiency chitin, glucose, sucrose, maltose, peptone.
Solid fermentation base-material proportioning combines:
1, additional carbon
A. title: wheat bran, sugarcane bar powder, milled powders of cornstalk, chaff of cracking rice, corn husk powder.
B. carbon source combination: 1. wheat bran+sugarcane bar powder; 2. corn husk powder+wheat bran; 3. sugarcane bar powder+corn husk powder; 4. wheat bran+chaff of cracking rice; 5. to crack rice chaff+corn husk powder; 6. corn husk powder.
Above-mentioned 6 groups all suitable, wherein with 1. and 5. better, both containing the sugar that viride is had a liking for, have again metabolism synthesize needed for VITAMIN and mineral substance.
2, additional nitrogenous source
Its more suitable nitrogenous source: inorganic nitrogen-sourced (urea, (NH
4) NO
3, NaNO
3, Sodium Nitrite, ammonium sulfate); Organic nitrogen source (peptone, fish meal, blood meal).Wherein better with ammonium nitrate and urea in inorganic nitrogen, source easily and extensively, easy handling; Better with peptone and blood meal in organonitrogen, easily, cost is low in source, and fermentation is fast.
Phosphate ore prime element: KH
2pO
4, K
2hPO
4, Na
2hPO
4, (NH
4) H
2pO
4, wherein with 1: 1 KH
2pO
4+ (NH
4)
2hPO
4or independent KH
2pO
4as well, consumption is respectively 1wt%.
Solid medium combine: mass ratio is the corn husk powder+wheat bran of 1: 4, or mass ratio is the wheat bran+sugarcane bar powder of 3: 2, and carbon element is set to A, and nitrogen is set to B, and phosphate ore prime element is C, and stone carbon dust is set to D, and water is E, and fungus is F, institute become compare be A
38b
4c
1d
22e
25f
10each alphabetical lower right corner is quality hundred number of fermentation system.
22) solid fermentation: A, B, C, D are combined according to the above ratio, through fully stirring, in steam cooker or disinfection tank high-temperature sterilization (under 1.13 normal atmosphere) sterilization 30min, discharging, after being chilled to room temperature, adds the spore suspension of bacterial strain after activation in step (1) according to setting share, after fully mixing thoroughly, proceed to large fermentation tank temperature control (28 ± 0.5 DEG C) fermentation 5 ~ 7 days, cold wind dries up packaging, obtains the tunning of viride mutant strain.Timing (6 ~ 8h) sampling Detection pH is needed in fermentation.
Particularly, in the embodiment of the present invention, by solid-liquid two kinds of product design weight ratios, preparation substratum:
Solid production of hybrid seeds substratum: peeled potatoes 150 ~ 200g/L; Sucrose 101.5g/L; Sodium phosphate 3.5-5.0%; FeSO
40.1 ~ 0.3g; KH
2pO
40.2 ~ 2.0g; MgSO
40.5 ~ 0.35g; CaSO
40.5 ~ 0.10g; ZnSO
40.1 ~ 0.2g; Redistilled water 1.0L; PH state of nature;
The preparation of induction indemnity: take inductor chitin (also known as chitin) (if this locality have agent company can from local acquisition) 10g, add 100ml in 85% phosphoric acid solution of 4 DEG C of precoolings, abundant mixing, and be placed in 4 DEG C of insulations 2 round the clock; Add again excessive pH value be 6.0 phosphoric acid buffer adjustment chitin concentration be 10g/L, put into heavy-walled glass bottle, add the granulated glass sphere of diameter 3mm, again on shaking table with the velocity fluctuation 2h of 180r/min, the colloidal state chitin that concentration is 10g/L can be obtained, produce chitinase and the cellulase of Anti-bacterium for triggering hole type viride mutant strain, improve disease resistance effect.If without above-mentioned inductor chitin, then can refer to Jiang Li section method self-control (ZL200510039128.X).
The chitinous method of Jiang Like system: by the clean oven dry such as shell, crab shell, dried small shrimp after rejecting remnants (remaining) slough, be ground into powder, 0.2molcp grade hydrochloric acid lixiviate 1 round the clock, do not stop to stir (or stirring 80 times/min with hertz oscilltor), filter or centrifugal gained throw out tap water extremely neutrality, chlorion is washed away again, drying and crushing with distilled water.
Liquid nutrient medium: peeled potatoes 600g, first thinly slice to pulverize again and boil 20min, filter with tail cloth and wring out waste (giving over to feed) and stay juice, add 45g wheat bran (or etc. heavy corn husk powder, or etc. heavy chaff of cracking rice) as vitamin B group donor, boil half an hour more than, filtered while hot waste; With sucrose 60g, honey 90g, inject 800,000 units of Penicillin with syringe from fermentor tank well, yeast powder 15g, concentration is the colloidal state chitin 3ml of 10g/L, then adds water until be equivalent to the volume of 3L in 5L fermentor tank, sterilizing.
Viride mutant strain of the present invention is the mutant strain that separation and purification wild strain out obtains the class soil-borne diseases such as Effective Anti wheat scab through chemomorphosis from residual rotten timber, it is a kind of mutant strain having hereditary defect, only need suitable substratum, it just better can grow and synthesize comparatively wild strain higher output yield and the good bioactive product of germ resistance, to improve the use value of target product, reduce production cost, reach good economic benefit.The antibacterial target product chitinase of this viride mutant strain, the activity of cellulase comparatively wild-type are increased to 710U by 340U respectively, 14U rises to 58U, may be used for the harmful cause of disease fungus prevented in soil, on the one hand, its underproduction caused that harms the crops can be avoided, on the other hand, it can also be avoided to affect human and livestock health by food chain.
The present invention is by filtering out chitin and crystallite carboxymethyl cellulose and carry out promoting this fungi to the synthesis of chitinase and cellulase as inductor and according to the characteristic addicted to sucrose of this bacterium, with gramineous crop stalk, slaughtering blood for nitrogenous source for base-material ferments, reach not only to crop disease-resistant but also growth promoting effects, also can realize fully slaughtering waste to husbandry to fully utilize, reduce environmental pollution, promote economic, social, ecological three benefit cooperative developments.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (10)
1. a fermentation process for viride mutant strain, is characterized in that, comprises the following steps:
(1) liquid fermenting, comprises the following steps:
11) activation of bacterial strain: the viride mutant strain through colchicine chemomorphosis is inoculated on PDA substratum cultivate 5 ~ 7 days, its fungus activate and adapt to fermentation new environment;
12) after activation, the spore suspension of bacterial strain is cultivated: get the bacterial strain after activation, inoculation culture is in showing spore maturation under the microscope, with the spore after the activation of 10ml aseptic water washing in the aseptic triangular flask of 150ml, then on constant-temperature table with 120r/min vibrate 20min, after spore ball is fully disperseed, with Hematocyte Counter counting, then with 1 × 10
8the inoculum size of individual/ml is inoculated in certain volume and the fermentation cylinder for fermentation 5 ~ 7 days of liquid nutrient medium is housed, and obtains the spore suspension activating rear bacterial strain; During the fermentation, regulate pH between 4.3 ~ 6.5 with 0.10 ~ 0.25molNaOH;
(2) solid fermentation, comprises the following steps:
21) solid fermentation culture base-material prepares: according to mass percent, be made up of carbon element 38%, nitrogen 4%, phosphate ore prime element 1%, stone carbon dust 22%, water 25% and fungus 10%; Described carbon source is from bagasse, crop material powder, wheat bran, corn husk powder, one or more the mixture of cracking rice in chaff of the equal < 10% of water ratio; Described nitrogen comprises inorganic nitrogen-sourced and organic nitrogen source, and inorganic nitrogen-sourced is urea, (NH
4) NO
3, NaNO
3, one or more mixture in Sodium Nitrite and ammonium sulfate, organic nitrogen source is one or more the mixture in peptone, fish meal and blood meal; Described phosphate ore prime element is KH
2pO
4, K
2hPO
4, Na
2hPO
4, NaH
2pO
4, (NH
4)
2hPO
4(NH
4) H
2pO
4in one or more mixture; Described fungus is derived from the spore suspension of bacterial strain after the activation in step (1); The preparation of described blood meal: purchase country fair slaughter after animal blood, pour in the boiling water of steam cooker, namely 3min pulls precipitation out, pulverize air-dry or dry break into powder again, be blood meal;
22) solid fermentation: carbon element, nitrogen, phosphate ore prime element, stone carbon dust are combined according to the above ratio, through fully stirring, the high-temperature sterilization 30min under 1.13 normal atmosphere in steam cooker or disinfection tank, discharging, after being chilled to room temperature, add the spore suspension of bacterial strain after activation in step (1) in proportion, after fully mixing thoroughly, proceed to large fermentation tank temperature controlled fermentation 5 ~ 7 days, leavening temperature is at 28 ± 0.5 DEG C, cold wind dries up packaging, obtains the tunning of viride mutant strain.
2. the fermentation process of viride mutant strain according to claim 1, is characterized in that, described step 12) in, also add the amount of 80 units of Penicillin 200 μ L according to often liter of bulk solution, add 80 units of Penicillin; Fermentor tank domestic demand continues through apparatus of oxygen supply and keeps oxygen supply, meanwhile, also needs the space keeping leaving 1/3 ~ 2/5 in fermentor tank.
3. the fermentation process of viride mutant strain according to claim 1, is characterized in that, described step 12) in, during the fermentation, regulate pH between 5.3 ~ 6.2 with 0.10 ~ 0.25molNaOH.
4. the fermentation process of viride mutant strain according to claim 1, is characterized in that, described step 12) in, also comprise and adopt the method for rotary evaporation or vacuum concentration that the spore suspension of bacterial strain after the activation obtained is concentrated into 3 ~ 5 times.
5. the fermentation process of viride mutant strain according to claim 1, is characterized in that, described crop material powder comprises sugarcane bar powder, milled powders of cornstalk, rice straw powder and beans straw powder.
6. the fermentation process of viride mutant strain according to claim 5, it is characterized in that, described carbon element is the one in wheat bran+sugarcane bar powder, corn husk powder+wheat bran, sugarcane bar powder+corn husk powder, wheat bran+chaff of cracking rice, chaff of cracking rice+corn husk powder, corn husk powder.
7. the fermentation process of viride mutant strain according to claim 6, is characterized in that, described carbon element is corn husk powder+wheat bran, wheat bran+sugarcane bar powder, or chaff of cracking rice+corn husk powder; Described inorganic nitrogen-sourced be ammonium nitrate and urea; Described organic nitrogen source is peptone and blood meal; Described phosphate ore prime element is mass ratio is KH
2pO
4, or etc. the KH of quality
2pO
4+ (NH
4)
2hPO
4.
8. the fermentation process of viride mutant strain according to claim 1, it is characterized in that, in described step (1), the preparation of liquid nutrient medium: peeled potatoes 600g, first thinly slice to pulverize again and boil 20min, filter with tail cloth and wring out waste and stay juice, add 45g wheat bran or corn husk powder or chaff of cracking rice as vitamin B group donor, boil half an hour more than, filtered while hot waste; With sucrose 60g, honey 90g, inject 800,000 units of Penicillin with syringe from fermentor tank well, yeast powder 15g, concentration is the colloidal state chitin 3ml of 10g/L, then adds water until the space of only remaining 2L in 5L fermentor tank, sterilizing; The chitinous preparation of described colloidal state: take chitin 10g, add 100ml in 85% phosphoric acid solution of 4 DEG C of precoolings, abundant mixing, and be placed in 4 DEG C of insulations 2 round the clock, add again excessive pH value be 6.0 phosphoric acid buffer adjustment chitin concentration be 10g/L, put into vial, add the granulated glass sphere of diameter 3mm, again on shaking table with the velocity fluctuation 2h of 180r/min, the colloidal state chitin that concentration is 10g/L can be obtained.
9. the fermentation process of viride mutant strain according to claim 1, is characterized in that, in described step (2), the component of solid fermentation used medium and proportioning are: peeled potatoes 150 ~ 200g/L; Sucrose 101.5g/L; Sodium phosphate 3.5-5.0%; FeSO
40.1 ~ 0.3g; KH
2pO
40.2 ~ 2.0g; MgSO
40.5 ~ 0.35g; CaSO
40.5 ~ 0.10g; ZnSO
40.1 ~ 0.2g; Redistilled water 1.0L; PH state of nature.
10. the application of tunning in the medicament of preparation control soil-borne disease of the viride mutant strain that the fermentation process as described in as arbitrary in claim 1 ~ 9 obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610049068.8A CN105505799A (en) | 2016-01-26 | 2016-01-26 | Fermentation method and application of trichoderma viride mutant strains |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610049068.8A CN105505799A (en) | 2016-01-26 | 2016-01-26 | Fermentation method and application of trichoderma viride mutant strains |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105505799A true CN105505799A (en) | 2016-04-20 |
Family
ID=55714101
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610049068.8A Pending CN105505799A (en) | 2016-01-26 | 2016-01-26 | Fermentation method and application of trichoderma viride mutant strains |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105505799A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107418944A (en) * | 2017-05-22 | 2017-12-01 | 河池学院 | The method of Trichoderma viride production cellulase and the application of institute's cellulase-producing |
| CN109414024A (en) * | 2016-04-28 | 2019-03-01 | 组合化学工业株式会社 | Pesticidal preparations composition, preparation method and the application method of trichoderma bacterium are used |
| CN114807006A (en) * | 2022-04-26 | 2022-07-29 | 广西大学 | Extraction method of sugarcane pollen spores |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771809A (en) * | 2005-10-14 | 2006-05-17 | 田连生 | Prepn process of corn stalk trichoderma as biological antiseptic |
| CN101260371A (en) * | 2008-04-18 | 2008-09-10 | 吉林农业大学 | Genome rearrangement trichoderma and its preparation and application |
-
2016
- 2016-01-26 CN CN201610049068.8A patent/CN105505799A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771809A (en) * | 2005-10-14 | 2006-05-17 | 田连生 | Prepn process of corn stalk trichoderma as biological antiseptic |
| CN101260371A (en) * | 2008-04-18 | 2008-09-10 | 吉林农业大学 | Genome rearrangement trichoderma and its preparation and application |
Non-Patent Citations (3)
| Title |
|---|
| 葛文中 等: "绿色木霉应用的研究进展", 《黑龙江八一农垦大学学报》 * |
| 袁铸 等: "秋水仙碱对木霉F10纤维素酶系的诱变处理研究", 《四川大学学报（自然科学版）》 * |
| 邵喜霞 等: "绿色木霉合成纤维素酶部分培养条件的优化", 《安徽农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109414024A (en) * | 2016-04-28 | 2019-03-01 | 组合化学工业株式会社 | Pesticidal preparations composition, preparation method and the application method of trichoderma bacterium are used |
| CN109414024B (en) * | 2016-04-28 | 2021-10-19 | 组合化学工业株式会社 | Pesticide preparation composition using trichoderma, preparation method and application method thereof |
| CN107418944A (en) * | 2017-05-22 | 2017-12-01 | 河池学院 | The method of Trichoderma viride production cellulase and the application of institute's cellulase-producing |
| CN114807006A (en) * | 2022-04-26 | 2022-07-29 | 广西大学 | Extraction method of sugarcane pollen spores |
| CN114807006B (en) * | 2022-04-26 | 2023-10-13 | 广西大学 | Extraction method of sugarcane pollen spores |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102531766B (en) | Microbial decomposing agent and production method thereof | |
| CN101875571B (en) | Method for preparing enhanced liquid microbial organic fertilizer | |
| CN109618944B (en) | Hypersensitive protein complex enzyme preparation as well as preparation method and application thereof | |
| CN100451105C (en) | Bacillus laterosporus and soil inoculation agent prepared from the strain | |
| CN107857689A (en) | The technique that a kind of bioanalysis prepares bio-organic fertilizer using chicken manure | |
| CN102491802A (en) | Tea seed cake biological organic fertilizer and production method thereof | |
| CN104560045B (en) | Microbial bacterial agent and chemical fertilizer excessively use growth-promoting secondary saline and alkaline and prone soil improved application | |
| CN101775359A (en) | Special microorganism composite bacterial agent for directly decomposing and fermenting crops straws to generate marsh gas and application method thereof | |
| CN102442849A (en) | Disease-preventing bio-organic fertilizer prepared from straw and its preparation method | |
| CN104193516A (en) | Environment-friendly fertilizer for strawberries and preparation method thereof | |
| CN102835251B (en) | Submerged fermentation culturing method for medicinal hericium erinaceus mycelium liquid | |
| CN107840762A (en) | A kind of method that biological organic fertilizer is made using cow dung | |
| CN104017739B (en) | Complex micro organism fungicide, its preparation method and its application in high protein potato dreg fodder is produced | |
| CN103588522B (en) | A kind of preparation method of quick-effective mixed fertilizer of organic active liquid | |
| CN107827608A (en) | A kind of method that biological organic fertilizer is made using sheep manure | |
| CN107711891A (en) | A kind of method prevented and treated matrimony vine root rot and extremely set | |
| CN106305487A (en) | Biological active padding material of fermentation bed for poultry, preparation method and application | |
| CN103387428A (en) | Preparation method for organic material decomposition agent | |
| CN109511465A (en) | A kind of oyster mushroom cultivating in bag method based on corncob | |
| CN105087421B (en) | The method that yeast fusion bacterium mixes microbial inoculum and preparation method thereof and production organic fertilizer | |
| CN104261980A (en) | Bio-organic fertilizer for planting lotus root | |
| CN105948831B (en) | A method of biological and ecological methods to prevent plant disease, pests, and erosion fertilizer is produced using brewex's grains | |
| CN109105271A (en) | A kind of padding and preparation method thereof of vaccary cowshed | |
| CN103098980A (en) | Microorganism straw feed fermenting agent | |
| CN107954759A (en) | A kind of method that biological organic fertilizer is made using methane slag |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160420 |
|
| RJ01 | Rejection of invention patent application after publication |