CN105219692A - Solid state fermentation produces the conidial method of Verticillium lecanii - Google Patents
Solid state fermentation produces the conidial method of Verticillium lecanii Download PDFInfo
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Abstract
The invention discloses solid state fermentation and produce the conidial method of Verticillium lecanii.Solid state fermentation provided by the present invention produces the conidial method of Verticillium lecanii, comprising: a) be inoculated in fermention medium by Verticillium lecanii and cultivate, obtain Verticillium lecanii culture; B) collect Verticillium lecanii culture, obtain Verticillium lecanii conidium; Fermention medium is fermented substrate and the composition of nutritive substance of 25:3 by mass ratio; Fermented substrate by mass ratio be the wheat bran of 3:1:1:3, dregs of beans, rice husk and water forms; Nutritive substance by mass ratio be the glucose of 5:2:5, yeast powder and W-Gum form.Method of the present invention can scale operation Verticillium lecanii conidium and reduce production cost, shorten the production time.
Description
Technical field
The present invention relates to solid state fermentation in biological technical field and produce the conidial method of Verticillium lecanii.
Background technology
Verticillium lecanii (Verticilliumlecanii) can be used for the vegetable at protected field prevention and control of plant diseases, pest control; both the insects such as trialeurodes vaporariorum, aphid, thrips can be parasitized; the phytopathogens such as rust, damping-off germ, Powdery Mildew can also be parasitized; and genetic stability is better; all murder by poisoning is found no to people, crop, domestic animal, fowl; to natural enemy insect safety, therefore in control vegetable at protected field disease and pest, there are important potentiality.
As microbial pesticide, the effective constituent of Verticillium lecanii is conidium, conidium can only be produced at present by solid state fermentation, because solid state fermentation produces the lack of homogeneity such as thalli growth, the transmission of nutritive substance and secretion of metabolite in conidial process of this bacterium, be difficult to accurately detect and control fermentation parameter, the more difficult requirement reaching extensive industrialization.Past people often adopt liquid (patent of invention: a kind of Verticillium lecanii and cultural method thereof and application, the patent No.: ZL200410058017.9) produce, but product form is blastospore, be unfavorable for long-term preservation, seriously limit industrialization and produce.Solid fermentation in the past mainly with grain rice or millet as matrix (as patent of invention: Verticillium lecanii and cultural method thereof and application, the patent No.: ZL200410058016.4) produce Verticillium lecanii, fermentation period is long, and the solid fermentation matrix of producing is rice, not only production cost is high, and can grain be consumed, be also unfavorable for merchandized handling.
Summary of the invention
Technical problem to be solved by this invention is scale operation Verticillium lecanii (Verticilliumlecanii) conidium of how low cost.
For solving the problems of the technologies described above, the conidial method of solid state fermentation production Verticillium lecanii that it is matrix that the present invention provide firstly with cheap agricultural byproducts.
Solid state fermentation provided by the present invention produces the conidial method of Verticillium lecanii, comprise following a) and b):
A) Verticillium lecanii is inoculated in fermention medium cultivates, obtain Verticillium lecanii culture;
B) collect described Verticillium lecanii culture, obtain Verticillium lecanii conidium;
Described fermention medium is the aseptic culture medium by fermented substrate and composition of nutritive substance, and the mass ratio of fermented substrate and described nutritive substance described in described fermention medium is (20-30): 3; Described fermented substrate is made up of wheat bran, dregs of beans, rice husk and water, and the mass ratio of wheat bran described in described fermented substrate, described dregs of beans, described rice husk and water is 3:(0.5-2): (0.5-2): (2-4); Described nutritive substance is made up of glucose, yeast powder and W-Gum, and glucose described in described nutritive substance, described yeast powder and described W-Gum mass ratio are (4-6): 2:(4-6).
Described wheat bran can be Wheat bran.Described dregs of beans can be soybean meal.Described rice husk can be rice husk.Described glucose specifically can be Chemical Reagent Co., Ltd., Sinopharm Group's product.Described yeast powder specifically can be Beijing bispin microbiological culture media products factory product.
In aforesaid method, described in described fermention medium, the mass ratio of fermented substrate and described nutritive substance can be 25:3; The mass ratio of wheat bran described in described fermented substrate, described dregs of beans, described rice husk and water can be 3:1:1:3; Glucose described in described nutritive substance, described yeast powder and described W-Gum quality comparable are 5:2:5.
In aforesaid method, described Verticillium lecanii is inoculated in fermention medium can be by containing the conidial spore suspension of Verticillium lecanii spray be inoculated on described fermention medium.
In aforesaid method, the available aseptic atomizer of described injection inoculation carries out.
In aforesaid method, described spore suspension can be the liquid obtained that to be suspended by described Verticillium lecanii conidium sterilized water.
In aforesaid method, described Verticillium lecanii conidium can be and carried out activating the conidium obtained by described Verticillium lecanii.
In aforesaid method, be describedly inoculated in fermention medium by Verticillium lecanii, the ratio of described Verticillium lecanii conidium and described fermention medium can be (1-3) × 10
8cfu:0.1kg, as 2 × 10
8cfu:0.1kg.
In aforesaid method, the temperature of described cultivation is 20-25 DEG C; The humidity of described cultivation is 85-95%; The time of described cultivation is 9-11 days.
Described 20-25 DEG C specifically can be 23 DEG C.Described 85-95% specifically can be 90%.Within described 9-11 days, specifically can be 10 days.
In aforesaid method, described cultivation can be carried out in an aseptic environment.Described gnotobasis specifically can be aseptic proving room.The ventilation time of described aseptic proving room can be 15 minutes per hour.
In aforesaid method, described collection described Verticillium lecanii culture can comprise following 1) and 2):
1) described Verticillium lecanii culture is carried out drying, obtain dried culture;
2) obtain dried culture described in pulverizing, obtain containing the conidial spore powder of Verticillium lecanii.
In aforesaid method, described collection described Verticillium lecanii culture also can comprise described step of sifting out containing the conidial spore powder of Verticillium lecanii.
In aforesaid method, described sieve specifically can be 100 mesh sieve.
In aforesaid method, the temperature of described drying can be 28-32 DEG C; The time of described drying can be 36-60 hour.
In aforesaid method, described 28-32 DEG C specifically can be 30 DEG C.Within described 36-60 hour, specifically can be 48 hours.
In aforesaid method, described Verticillium lecanii specifically can be Verticillium lecanii (Verticilliumlecanii) VerleBj085-1 being numbered CGMCCNo.1187 at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
For solving the problems of the technologies described above, present invention also offers the application of described method in preparation controlling plant diseases and/or insect pest of the plant product.
In above-mentioned application, described Plant diseases specifically can be plant rust, damping-off or Powdery Mildew; Described insect pest of the plant specifically can be trialeurodes vaporariorum, aphid or thrips.
For solving the problems of the technologies described above, present invention also offers described fermention medium.
Experiment proves, solid state fermentation of the present invention is produced the conidial method of Verticillium lecanii and can be carried out scale operation Verticillium lecanii (Verticilliumlecanii) conidium and reduce production cost, shortening production time: solid state fermentation of the present invention is produced in the conidial method of Verticillium lecanii and adopted spray inoculation method, use and obtain by solid fermentation the inoculation liquid that conidium is directly prepared from, spore homoepitaxial can not only be made in fermented substrate, the transformation efficiency of abundant raising matrix biopolymers mass-energy, by 2 × 10
9the initial Verticillium lecanii conidium of cfu can obtain 5 × 10 after cultivating in 1kg solid-state fermentation culture medium
9cfu Verticillium lecanii conidium/gram spore powder, has significantly saved initial inoculum, reduces production cost and the production time of inoculation liquid; Solid state fermentation of the present invention produces the raw materials used convenience of drawing materials of the conidial method of Verticillium lecanii, and super quality and competitive price, fully ensure that the supply of carbon source, nitrogenous source, nutritional condition, and production process are without the discharge of waste gas, waste residue, waste water, environmental protection.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Verticillium lecanii (Verticilliumlecanii) in following embodiment is documented in Chinese patent CN1274807C.
Glucose in following embodiment is Chemical Reagent Co., Ltd., Sinopharm Group's product; Yeast powder is Beijing bispin microbiological culture media products factory product.
Atomizer in following embodiment is the atomizer of 100ml capacity.
Pulverizer in following embodiment is Beijing Huan Ya Tianyuan Mechanical Technology Inc. product, and model is HY-04B.
Embodiment 1, solid state fermentation produce Verticillium lecanii conidium
One, the preparation of fermention medium
The fermention medium that the present embodiment is used is by fermented substrate and composition of nutritive substance, and wherein the mass ratio of fermented substrate and nutritive substance is 25:3; Fermented substrate is made up of wheat bran, dregs of beans, rice husk and water, and in fermented substrate, the mass ratio of Wheat bran, soybean meal, rice husk and water is 3:1:1:3; Nutritive substance is made up of glucose, yeast powder and W-Gum, and in nutritive substance, glucose, yeast powder and W-Gum mass ratio are 5:2:5.Be dispensed into by fermention medium in high temperature resistant sterilizing bag, under 121 DEG C of conditions, moist heat sterilization obtains without bacteria fermentation culture medium for 20 minutes.In fermention medium, the quality of Wheat bran, soybean meal, rice husk, glucose, yeast powder and W-Gum is all with dry weight basis.
Two, the preparation of proving room
Volume is 20m
2the small-sized fermentation workshop of (floorage) × 3m (height), this proving room throughput is every batch of 30 kilograms of fermented products.
The sterilization of proving room: the potassium permanganate weighed up is put into container, then injects formaldehyde solution, close door immediately, stifling office work of being allowed for access after 24 hours, later every day carries out disinfection by ultraviolet light half an hour.Every cubic metre of space formaldehyde solution 10ml, potassium permanganate 5g.
The temperature of proving room: 23 DEG C ± 1 DEG C.
The humid control of proving room: 90% ± 2%.
The ventilation of proving room controls ventilation per hour 15 minutes, is regulated by proving room top portion ventilation fan.
Three, the preparation of conidial suspension
By original strain Verticillium lecanii (Verticilliumlecanii) VerleBj085-1CGMCCNo.1187) the line of PDA substratum 23 DEG C, back-off cultivates 7-10 days under the condition of relative humidity 90%, collect conidium, by the sectional growing spore suspension that obtains in sterilized water, obtain conidial suspension, the concentration of conidial suspension is 1 × 10
8cfu conidium/ml conidial suspension.
Four, solid state fermentation produces Verticillium lecanii conidium
To hollow out aseptic plastic case (being highly 20cm) in add step one without bacteria fermentation culture medium, each plastic box 1kg, without bacteria fermentation culture medium, is about 2cm without bacteria fermentation culture medium thickness, obtains the inoculation tank without bacteria fermentation culture medium is housed.The conidial suspension of step 3 is filled in aseptic atomizer, and with aseptic atomizer by conidial suspension spray be inoculated in inoculation tank without in bacteria fermentation culture medium, every kilogram without bacteria fermentation culture medium inoculation 20mL conidial suspension, to stir a little without bacteria fermentation culture medium after inoculation, to spray in the Hu Zui distance inoculation tank of atomizer during inoculation without the height of bacteria fermentation culture medium lower than 20cm, inoculate after terminating and obtain being connected to the conidial inoculation tank of Verticillium lecanii.To be connected to the conidial inoculation tank of Verticillium lecanii inserts according in the ready proving room of step 2, cultivates 10 days, obtains the inoculation tank containing Verticillium lecanii culture.
Inoculation tank containing Verticillium lecanii culture is placed in baking oven drying treatment 48 hours, obtains dried culture; Dried culture is crumbed in sterile seal bag, obtain the dried culture of bulk processing, the dried culture of bulk processing is pulverized in pulverizer and within 1 minute, obtains meal, meal is crossed 100 mesh sieve, obtain containing the conidial spore powder of Verticillium lecanii (abbreviation spore powder).Spore powder can be contained in sealing bag and be stored in 0 DEG C of-10 DEG C of dry environment.
The conidial detection of Verticillium lecanii in spore powder, concrete grammar is as follows: take 5g spore powder, add and fill in 100ml sterilized water triangular flask, add 0.01% tween-80, put into 5 granulated glass sphere concussion 3-5min, fully spore is separated from matrix, then filter with two-layer gauze, the filtrate obtained is placed on blood counting chamber, counting conidium.Spore output unit: the spore count in every gram of spore powder.Result shows, containing 5.0 × 10 in every gram of spore powder
9± 0.2 × 10
9cfu Verticillium lecanii conidium.
Above-mentioned solid state fermentation is produced in the conidial method of Verticillium lecanii, by 2 × 10
9the initial Verticillium lecanii conidium of cfu can obtain 5.0 × 10 after bacteria fermentation culture medium cultivates 10 days
9± 0.2 × 10
9cfu Verticillium lecanii conidium/g spore powder.
In Chinese invention patent CN1274808C, describe a kind of Verticillium lecanii (Verticilliumlecanii) VerleBj085-1CGMCCNo.1187) cultural method: by strain inoculation in liquid nutrient medium, cultivate at 23-25 DEG C and obtain Verticillium lecanii product, this liquid nutrient medium contains the sucrose of 1-3% weight, the starch of 2-4% weight, the cereuisiae fermentum of 2-4% weight, the calcium carbonate of 0.1-0.3% weight, surplus is water.The Verticillium lecanii product form obtained by the method is blastospore, is unfavorable for long-term preservation, seriously limits industrialization and produces.
In Chinese invention patent CN1274807C, describe a kind of Verticillium lecanii (Verticilliumlecanii) VerleBj085-1CGMCCNo.1187) cultural method: first rice is parboiled, be loaded in the high pressure resistant plastics bag of 30 × 20cm after air-dry, often packed 200 grams, sterilizing 0.5 hour under 100 DEG C and 15 pounds of pressure conditions.Verticillium lecanii (Verticilliumlecanii) VerleBj085-1CGMCCNo.1187 by after activation) to be diluted to concentration with sterilized water be 10
7spore/milliliter spore suspension, by the rice of 150 milliliters of inoculation of suspension liquid after sterilising treatment under aseptic condition, 23-25 DEG C, relative humidity cultivates 20 days under being the condition of 85-90%.The fermentation period that the method produces Verticillium lecanii is long, and for meter Zuo Wei fermented substrate, not only production cost is high, and can consume grain, is also unfavorable for merchandized handling.
The solid state fermentation being matrix with cheap agricultural byproducts provided by the invention produces the conidial method of Verticillium lecanii fast, significantly reduces production cost, is more conducive to merchandized handling.
Claims (10)
1. solid state fermentation produces Verticillium lecanii conidial method, comprise following a) and b):
A) Verticillium lecanii is inoculated in fermention medium cultivates, obtain Verticillium lecanii culture;
B) collect described Verticillium lecanii culture, obtain Verticillium lecanii conidium;
Described fermention medium is by fermented substrate and composition of nutritive substance, and the mass ratio of fermented substrate and described nutritive substance described in described fermention medium is (20-30): 3; Described fermented substrate is made up of wheat bran, dregs of beans, rice husk and water, and the mass ratio of wheat bran described in described fermented substrate, described dregs of beans, described rice husk and water is 3:(0.5-2): (0.5-2): (2-4); Described nutritive substance is made up of glucose, yeast powder and W-Gum, and glucose described in described nutritive substance, described yeast powder and described W-Gum mass ratio are (4-6): 2:(4-6).
2. method according to claim 1, is characterized in that: the mass ratio of fermented substrate and described nutritive substance described in described fermention medium is 25:3; The mass ratio of wheat bran described in described fermented substrate, described dregs of beans, described rice husk and water is 3:1:1:3; Glucose described in described nutritive substance, described yeast powder and described W-Gum mass ratio are 5:2:5.
3. according to the method for claim 1 or 2, it is characterized in that: described being inoculated in by Verticillium lecanii in fermention medium is inoculated on described fermention medium for spraying containing the conidial spore suspension of Verticillium lecanii.
4. method according to claim 3, is characterized in that: be describedly inoculated in fermention medium by Verticillium lecanii, and the ratio of described Verticillium lecanii conidium and described fermention medium is (1-3) × 10
8cfu:0.1kg.
5., according to described method arbitrary in claim 1-4, it is characterized in that: the temperature of described cultivation is 20-25 DEG C; The humidity of described cultivation is 85-95%; The time of described cultivation is 9-11 days.
6., according to described method arbitrary in claim 1-5, it is characterized in that: described collection described Verticillium lecanii culture comprises following 1) and 2):
1) described Verticillium lecanii culture is carried out drying, obtain dried fermented product;
2) obtain dried fermented product described in pulverizing, obtain containing the conidial spore powder of Verticillium lecanii.
7. method according to claim 6, is characterized in that: the temperature of described drying is 28-32 DEG C; The time of described drying is 36-60 hour.
8., according to described method arbitrary in claim 1-7, it is characterized in that: described Verticillium lecanii is Verticillium lecanii (Verticilliumlecanii) VerleBj085-1CGMCCNo.1187.
9. the application of arbitrary described method in preparation controlling plant diseases and/or insect pest of the plant product in claim 1-8.
10. arbitrary described fermention medium in claim 1 or 2.
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| CN101248799A (en) * | 2008-03-04 | 2008-08-27 | 云南农业大学 | Verticillium lecanii pesticides and uses thereof |
| CN102796672A (en) * | 2012-08-14 | 2012-11-28 | 黑龙江省农业科学院植物保护研究所 | Verticillium lecanii solid fermentation medium, preparation method and application |
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2015
- 2015-11-04 CN CN201510741208.3A patent/CN105219692A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587376A (en) * | 2004-08-06 | 2005-03-02 | 中国农业大学 | Lecanium verticillin and cultivating method and use |
| CN1864491A (en) * | 2006-06-20 | 2006-11-22 | 徐向群 | A method for preparing biological control agent of Verticillium spp. |
| CN101248799A (en) * | 2008-03-04 | 2008-08-27 | 云南农业大学 | Verticillium lecanii pesticides and uses thereof |
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