CN104774770B - Fengyang aerogenesis removing mildew - Google Patents

Abstract

The invention discloses a kind of Fengyang aerogenesis removing mildew, Fengyang aerogenesis removing mildew is the mould solid fermentation product of Fengyang aerogenesis；The preparation method of solid fermentation product is：Aerogenesis mould one grade fermemtation seed in Fengyang is inoculated on fermentation substrate and fermented, obtains solid fermentation product.Or Fengyang aerogenesis removing mildew be by solid fermentation product carry out nutrient solution coating and air-dry after gains；Preparation method is：By solid fermentation product according to 1g:0.9~1.1ml solid-to-liquid ratio is soaked in after sterilizing in nutrient solution, and soak time is 10~12 hours；It is 6%~10% that the solid fermentation product of gained after immersion, which is air-dried to moisture content,.During Fengyang aerogenesis removing mildew actual use of the present invention, 30~80 grams of Fengyang aerogenesis removing mildew is placed in every 10 liters of containers, the effect of preservation and antisepsis sterilization can be just produced to the material in container.

Description

Fengyang aerogenesis removing mildew

Technical field

The present invention relates to field of biological pesticide, is provided in particular in a kind of endogenetic fungus preparation, is to produce more precisely Fengyang aerogenesis removing mildew of raw bacteriostatic activity gas.

Background technology

Aerogenesis mould (Muscodor spp.) (also referred to as Moschus is mould now) is a kind of endogenetic fungus for belonging to Xylariaceae, most Early separating obtained from xylocinnamomum by Worapong etc. (2001), it, which is noteworthy characterized by, can produce VOC (volatile organic compounds, abbreviation VOCs), these VOCs have extensive bioactivity, can suppress or kill Many disease funguses, pathogenetic bacteria and some insects.Then, people utilize its distinctive morphology, physiology, biochemistry side The mould category endogenetic fungus of new aerogenesis, the increasing mould kind of aerogenesis are found and identified to the feature in face and the sequence of rRNA It is found.Due to the mould VOCs that can have extensive bacteriostatic activity of aerogenesis, scientists utilize the chemical analysis such as GC-MS means point From the mould caused VOCs compositions of aerogenesis are identified, it is found that VOCs compositions caused by different strains are not quite similar.

Very important link is produced by fermentation-scale metaplasia in the exploitation of biological agent.Fermentation be divided into solid fermentation and Liquid fermentation.Solid fermentation (solid-state fermentation, SSF) is to be not present in finger or almost do not have Free water Fermented under conditions of (matrix must have the growth and metabolism that enough humidity supports microorganism).(Pandey etc., 1992；1994；2000；2001).From the point of view of bioprocesses, solid state fermentation is the biology using gas phase as continuous phase Course of reaction, one can consider that solid fermentation is a kind of a kind of hair utilized on the basis of culture medium is solid carrier Ferment means (Mitchell etc., 2000).

Solid fermentation has the advantages of many notable compared with liquid fermentation：Low (the often agriculture waste of fermentation raw material cost Thing), energy resource consumption is low, simple to operate, and less waste water produces；It is relatively low to sterility requirements, it is not susceptible to the dirt of large area Dye；The yield of unit volume is high, can be fermented with less reactor；And it is environmentally friendly, because this technology solves Solid waste handling problems (Pandey etc., 2008；Matthew etc., 2009).SSF is similar to micro- life due to yeasting The natural custom of thing, for microorganism, SSF is very suitable for it and grows and can produce valuable byproduct.Solid is sent out Ferment has become a kind of applied to the potential of production microniological proudcts such as feed, fuel, industrial chemical and medical product Technology.

Development on solid fermentation can trace back to the 1950s.Steroids is converted using fungal culture, utilizes SSF Mycotoxin product is produced, ox feed of the solid fermentation production rich in protein (includes agroindustry waste residue during this Utilization, thus provide and the value-added Particular craft of inexpensive waste residue is developed, Singhania etc., 2009)；This is a series of solid The achievement of body fermentation causes interest of the researchers to SSF, and SSF achieves unprecedented development (Singhania etc., 2009). The different microniological proudcts of the development of basic research, bioreactor on SSF, modeling and production (such as food, feed, no Same basis and secondary metabolite and such as Bioleaching, bio-pulping, the bioprocess of biological prosthetic, biological ore dressing) Aspect generates numerous studies.

So far, have many people study the biochemistry for influenceing solid fermentation processes and engineering science factor (Mitchell etc., 2000a, 2000b；Pandey, 2003), although many ins and outs in SSF be not resolved yet (Deng 2004), But utilizations of the SSF in biotechnology it is still quite varied (Raimbault etc., 1998；Pandey etc., 2000a, 2001b).Poison Element, bacterial endotoxin, plant growth factor, antibiotic, immunosuppressive drug, alkaloid, these important bioactive substances Produced by wide coverage by SSF.Industrial enzyme, such as amylase, cellulase, zytase, protease, phytase, fat Fat enzyme etc., organic acid such as citric acid and lactic acid etc. also largely produces (Krishna etc., 2005) by SSF.

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of Fengyang aerogenesis removing mildew.

In order to solve the above-mentioned technical problem, the present invention provides a kind of Fengyang aerogenesis removing mildew, and Fengyang aerogenesis removing mildew is The mould solid fermentation product of Fengyang aerogenesis；The preparation method of the mould solid fermentation product of Fengyang aerogenesis is to comprise the following steps：

By the mould one grade fermemtation seed of Fengyang aerogenesis (i.e. liquid fermentation seed) according to 10~20ml:100g is (preferably 20ml:Amount ratio 100g), which is inoculated on fermentation substrate, to be fermented, and zymotechnique is：In 24.5~25.5 DEG C (preferably 25 DEG C) the quiet culture of dark, incubation time is 8~10 days；Obtain solid fermentation product.The solid fermentation product can produce directly as Fengyang Gas removing mildew.

Improvement as Fengyang aerogenesis removing mildew of the present invention：Fengyang aerogenesis removing mildew is to enter solid fermentation product Field headquarters nutrient solution be coated and air-dry after gains；Preparation method is：

Prepare nutrient solution, the nutrient solution for it is following any one：3%~5% aqueous trehalose solution, 3%~5% Sodium alginate aqueous solution, 0.1%~0.2% xanthan gum solution, water (preferable), above-mentioned % are quality %；

By solid fermentation product according to 1g:0.9~1.1ml (preferably 1g:Solid-to-liquid ratio 1ml) is soaked in nutrition after sterilizing In liquid, soak time is 10~12 hours (preferably 10 hours)；

Remarks explanation：By nutrient solution through conventional sterilant (in 121 DEG C, 1.1 atmospheric pressure sterilizing 20min), must sterilize rear batallion Nutrient solution；

By after immersion gained solid fermentation product air-dry (superclean bench air-dries) to moisture content be 6%~10% (compared with Good is that 8%, % is quality %)；Product is Fengyang aerogenesis removing mildew after air-drying.

Further improvement as Fengyang aerogenesis removing mildew of the present invention：Fengyang aerogenesis removing mildew is subjected to packing processes； The packing processes include vacuumizing and plastic packaging processing.

Further improvement as Fengyang aerogenesis removing mildew of the present invention：The fermentation substrate is wheat solid fermentation culture Base or low-cost fermentation culture medium；

The preparation method of the wheat solid fermentation culture medium is：Wheat is put into 7~9h of immersion in water, by institute after immersion The wheat obtained is sterilized, and (usual manner sterilizes 2 times, i.e. is specially：In 121 DEG C, 1.1 atmospheric pressure sterilizing 40min)；

The preparation method of the low-cost fermentation culture medium is：Preparation raw material, the raw material by following weight content into It is grouped into：40% wheat bran, 20% soya-bean cake, 30% iblet and 10% rice bran；By raw material and deionized water according to 1g：1ml ratio Being sterilized after example mixing, (usual manner sterilizes 2 times, i.e. is specially：In 121 DEG C, 1.1 atmospheric pressure sterilizing 40min).

Further improvement as Fengyang aerogenesis removing mildew of the present invention：Mould one grade fermemtation seed (the i.e. liquid of Fengyang aerogenesis Body fermentation seed) preparation method be：By ZJL024 bacterium in PDB nutrient solutions, 200r/min, dark 25 DEG C of cultures 6 days, one is obtained Level fermentation seed.

Further improvement as the preparation method of Fengyang aerogenesis removing mildew of the present invention：Fengyang aerogenesis removing mildew Storage temperature is 4 DEG C.

Mould Fengyang aerogenesis of the present invention is Muscodor sp.ZJLQ024 (also names： Muscodor.fengyangensis ZJLQ024), in the invention of Patent No. 200910153512.0《Muscodorsp. belong to Plant endogenesis epiphyte ZJLQ024 and application thereof and bactericide》In have and clearly inform, preserving number is CGMCC No.2863.

Aerogenesis is mould to suppress even killing effect, its conduct because VOCs caused by it has to most bacteriums, fungi and insect Biological control agent has very big application potential, at present by more and more section skilled workers in agricultural, medicine and commercial Application The attention of author.

The solid fermentation product of gained of the invention, can be used directly；The solid fermentation product of gained of the invention is only with going out After bacterium after nutrient solution immersion, also it can be used directly.Fengyang aerogenesis removing mildew after nutrient solution soaks and air-dried after sterilizing, before use Need：Sterile cotton is layered on aseptic plastic bottom of bottle portion, adds sterile water-soaked, adds above-mentioned Fengyang aerogenesis removing mildew (sterilizing rear batallion Nutrient solution soak and air-dry after Fengyang aerogenesis removing mildew), then with water spray wetting surface, lid lid.In 4 DEG C of preservations, in use, i.e. Open and use.

During Fengyang aerogenesis removing mildew actual use of the present invention, 30~80 grams of Fengyang aerogenesis is placed in every 10 liters of containers Removing mildew, the effect of preservation and antisepsis sterilization can be just produced to the material in container.

Fengyang aerogenesis removing mildew of the present invention is prepared using solid fermentating mode, have cost is relatively low, concise in technology, The advantages of preservation and antisepsis has been sterilized.

Brief description of the drawings

The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.

Fig. 1：The mould ZJLQ024 of Fengyang aerogenesis with PDB shaking table culture time changes biomass；

Fig. 2：The biomass of the mould ZJLQ024 differences initial inoculum fermentation different time of Fengyang aerogenesis；

Fig. 3：Aerogenesis mould ZJLQ024 initial inoculums in Fengyang are 20ml fermented product figure；

Fig. 4：The bacteriostatic activity of the mould ZJLQ024 solid fermentation products of aerogenesis and bacterial strain on PDA；

In Fig. 4, each column from left to right represents ck, 5mm Fengyang aerogenesis mould cake respectively and one grown Fengyang production Face-off growth photo of the mould wheat of gas to various pathogenic bacteria；

Fig. 5：The mould ZJLQ0246d of Fengyang aerogenesis nucleic acid A values are cultivated on the culture medium of different substrates；

Fig. 6：Influence of each factor level to nucleic acid A values；

Fig. 7：Aerogenesis mould ZJLQ024 biological agents in Fengyang preserve scheme；

Fig. 8：Initial water content (moisture content) 8% respectively handles the survival rates of 4 DEG C of preservation different times；

Fig. 9：Initial water content 8% respectively handles the survival rates of 10 DEG C of preservation different times；

Figure 10：Initial water content 8% respectively handles the survival rates of 25 DEG C of preservation different times；

Figure 11：Initial water content 6% respectively handles the survival rates of 25 DEG C of preservation different times；

Figure 12：Initial water content 10% respectively handles the survival rates of 25 DEG C of preservation different times；

Figure 13：The survival rate of 6%4 DEG C of preservation different times of initial water content；

Figure 14：The survival rate of 10%4 DEG C of preservation different times of initial water content；

Figure 15：Preserve the preparation active constituent content of 3 months；

In Figure 15, every group is followed successively by from left to right：0.1% xanthan gum solution, 0.2% xanthan gum solution, 3% marine alga Acid sodium aqueous solution, 5% sodium alginate aqueous solution, 3% aqueous trehalose solution, 5% aqueous trehalose solution, water；

Figure 16：The mould ZJLQ024 fumigation controls green mold of apple of Fengyang aerogenesis.

Embodiment

Material：

Strains tested is the mould Muscodor.fengyangensis ZJLQ024 of Fengyang aerogenesis.Fengyang aerogenesis is mould Inventions of the Muscodor.fengyangensis ZJLQ024 in Patent No. 200910153512.0《Muscodorsp. category is planted Thing endogenetic fungus ZJLQ024 and application thereof and bactericide》In have and clearly inform, preserving number is CGMCC No.2863.

Cause of disease indicator bacteria includes：KH8 Penicillium notatum Penicillium expansum；MH10 strawberry anthrax-bacilus Colletotrichum fragariae；TZ01 peach brown rot fungus Monilinia fructicola；WJ0101 botrytis cinereas Botrytis cinerea；ZAU05 Rhizoctonia solani Kuhn Rhizoctonia solani；ZAU22 Pythium ultimum bacterium Pythium ultimum。

Reagent include trehalose, sodium alginate, xanthans, 5% trichloroacetic acid etc..

Instrument involved in the present invention includes superclean bench, constant incubator, electronic balance, pH meter, baking oven, electromagnetism Stove, micro-wave oven, refrigerator, vacuum drying chamber, GRT-20D type 50L solid-state fermentation tanks, ultrasonoscope, Agilent 6890N-5975B Type GC-MS instrument, MLS3750 types high-pressure sterilizing pot, BS233S type analysis balance, headspace solid-phase microextraction device etc..

The present invention relates to five kinds of culture mediums, respectively PDA culture medium, PDB culture mediums, Czapek's medium, freezen protective training Base and wheat solid fermentation culture medium are supported, its compound method difference is as follows：

PDA culture medium (potato dextrose agar)：Potato 200g, glucose 20g, 1.5% agar is (i.e., Agar 15g), running water 1000mL.Take quantitative potato to be cut into uniform fritter, add boiling to boil to the stamp of potato glass bar one and break up, 8 Layer filtered through gauze, 20g glucose is added, cooling, adds running water to be settled to 1000mL, the agar of triangle bottled 1.5%, adds liquid Body, encapsulation, 121 DEG C, 15min autoclavings.

PDB culture mediums (potato dextrose medium)：Potato 200g, glucose 20g, running water 1000mL.Take quantitative Potato is cut into uniform fritter, adds boiling to boil to the stamp of potato glass bar one and breaks up, 8 layers of filtered through gauze, adds 20g glucose, cold But, add running water to be settled to 1000mL, encapsulate, 121 DEG C, 15min autoclavings.

Czapek's medium：Sodium nitrate 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate (MgSO4.7H2O) 0.5g, potassium chloride 0.5g, sulphur Acid ferrous 0.01g, sucrose 30g, agar 20g, distilled water 1000mL.By ionic compound sodium nitrate, dipotassium hydrogen phosphate, sulfuric acid Magnesium, potassium chloride and ferrous sulfate add ionized water to be made into mother liquor, with sterilizing filter filtered fluid.Remaining is such as sucrose, agar, distillation Water is bottled in proportion, 121 DEG C, 15min autoclavings.Before being poured on culture dish after being dissolved, inorganic ionic compound is mixed Liquid mixes with culture medium in proportion.

Freezen protective culture medium (liquid)：Glucose 10g, yeast extract 1g, acid hydrolyzed casein 0.5g, glycerine (glycerine) 180mL.With distilled water by after glucose, yeast extract, acid hydrolyzed casein dissolving, add glycerine, finally use Distilled water is settled to 1000mL, 121 DEG C, sterilizing 20min.

Wheat solid fermentation culture medium：A certain amount of wheat is taken to add running water soaked overnight, soak time 7-9h or so.Outwell The unnecessary moisture of wheat, 100g wheats are filled to 1L triangular flask, in 121 DEG C, the continuous autoclaving 40min of 1.1 atmospheric pressure.

Embodiment 1, the preservation of bacterial strain and activation culture (belonging to routine techniques)

1st, the normal temperature or Cord blood of bacterial strain

In superclean bench, preserve pipe in 2ml sterile cryos and add the PDA culture medium no more than 1.5ml thawings, cover lid, Slant setting.After to be solidified, the bacterial strain (the mould Muscodor.fengyangensis ZJLQ024 of Fengyang aerogenesis) of preservation is inoculated with To slant medium, after a couple of days, mycelia is successfully colonized in inclined-plane, it is seen that fresh mycelia grows, and adds through the sterilizing of 3 sub-high pressures Paraffin floods mycelia, lid lid, and under normal temperature or 10 DEG C of refrigerators preserve.In use, with sterile toothpick picking hyphostroma, it is inoculated in Activated in flat board PDA culture medium.

2nd, the freezen protective of bacterial strain

In superclean bench, the bacterium colony on PDA takes 4-5 ferfas blocks, is placed in 2ml sterile cryos and preserves pipe, adds high The freezen protective liquid of sterilizing is pressed, fungus block is flooded.Lid lid, after freezen protective pipe is placed into 1h at 4 DEG C and -20 DEG C successively, turn Move in -80 DEG C of ultra low temperature freezers and preserved for a long time.In use, the naturally to thaw in room temperature, picking fungus block inoculation therein Activated in flat board PDA culture medium.

3rd, the activation of bacterial strain

Fungus block is obtained from the freezen protective pipe for preserving bacterial strain with toothpick, is inoculated into PDA culture medium, sealed membrane sealing In 25 DEG C, dark culturing case culture.

Embodiment 2, preparation fermentation process optimization

1st, one grade fermemtation seed inoculation kind age optimization

The present invention is used to ferment using one grade fermemtation seed to be inoculated with, and preferably shake flask fermentation is inoculated with, and is inoculated with kind of age certain journey Degree determines the height of fermentation efficiency, the present invention first optimization inoculation kind age：3 pieces of 5mm ZJL024 bacteria cakes are cultivated in 100ml PDB In liquid, 200r/min, dark 25 DEG C of cultures.Fix time and take the mycelia liquid of fermented and cultured to filter, dried to constant weight in baking oven (60 DEG C), Weigh record data, draws growth curve.It was found that in shaking table 200r/min liquid fermentations Fengyang mould ZJLQ 024 of aerogenesis, culture 6d fermentation seeds activity is most strong (Fig. 1).Hyphae length is slowly increased to 7d and reaches plateau after 6d.

Therefore, with ZJL024 bacteria cakes in PDB nutrient solutions, 200r/min, dark 25 DEG C of cultures, 6 days gained, as one-level Fermentation seed.

2nd, volume and fermentation time optimization are inoculated with

5ml, 6ml, 8ml are inoculated with respectively, and 10ml, 15ml, 20ml, 25ml one grade fermemtations seed is to after equipped with 100g sterilizings In wheat (that is, wheat solid fermentation culture medium) triangular flask (capacity 1L), observation result of fixing time, CFU is surveyed (CFU, Colony-Forming Units), choose optimal inoculation volume.

CFU (CFU, Colony-Forming Units) refers to the viable bacteria number in unit volume.Trained in viable bacteria When supporting counting, formed colony is bred in cultured on solid medium by single thalline or the pockets of multiple thalline of aggregation, is claimed For CFU, the quantity of viable bacteria is expressed with it.

Survey CFU：The solid fermentation product of different volumes inoculation liquid is taken into 20 (uniform in size) in superclean bench The mortar of sterilizing adds 20ml sterilized waters, and pestle is ground into homogeneous solution, takes 100ul solution to add 900ul water, is taken after well mixed 100ul applies PDA plate, double-deck Parafilm sealed membranes sealing, 25 DEG C of dark static gas wave refrigerators, treats that bacterium colony grows counting.

The either culture of bacterial strain plate or fermented and cultured, bacterial strain can all reach the stage of aging, and the present invention needs Tunning is used for subsequent experimental, is more likely to the tunning for selecting number of viable most.Optimal incubation time is The tunning activity that arrives is strong, quantity is more.The present embodiment obtains the optimal optimization time by CFU.

5ml, 6ml, 8ml, 10ml one grade fermemtation seed are first inoculated with respectively to 100g wheat solid fermentation culture medium (1L triangles Bottle), course of fermentation is observed every three days.Wherein 10ml one grade fermemtations seed can see that bacterium successfully colonizes.Further expand inoculation Optimal inoculation volume is screened in volume to 10ml, 15ml, 20ml, 25ml first order seed fermentation, is seen every three days in fermentation process Examine.As a result find that the bacterium 3-4d of 10ml, 15ml and 20ml inoculation successfully colonizes wheat, but 25ml bacterium needs 5-6d to determine Grow.By taking the tunning corresponding to 0.2g 10ml, 15ml and 20ml to carry out CFU measure every three days, aerogenesis is found to The growth balance period of trichoderma strain, about 8 days, it is inoculated with clump count in 20ml culture medium and at most (CFU contents highest), therefore, is inoculated with Volume is 20ml (Fig. 2, Fig. 3), i.e., the inoculum concentration of liquid fermentation seed is the 20% ideal of fermentation substrate；Simultaneously by not With inoculum concentration tunning, record the CFU of different times, draw growth curve, from Fig. 2 growth curve it will be evident that Between 8-10d, peak of the single inoculum concentration in different growing stage can be reached, therefore can determine that optimal fermented incubation time It is 8-10d (as shown in Figure 2).

3rd, solid fermentation product/bacterial strain plate determination of activity

Take above-mentioned optimal culture condition (20% inoculum concentration (be inoculated with 20ml first order seeds), the quiet culture 8 of 25 DEG C of dark My god) obtained by solid fermentation product (including wheat culture) 1/Fengyang aerogenesis trichoderma strain 5mm (embodiment 1 gained) to two lattice A wherein lattice for PDA plating mediums, with double-deck Parafilm ParafilmTMs culture dish, 25 DEG C of dark static gas wave refrigerators.After 4 days By 5mm sizes indicator bacteria (Rhizoctonia solani Kuhn R.solani, ZAU22 Pythium ultimum bacterium P.ultimum, the KH8 Penicillium notatums of ZAU 05 P.expansum, TZ01 peach brown rot fungus M.fructicola, MH10 strawberry anthrax-bacilus C.fragariae, WJ0101 botrytis cinerea B.cinerea) bacteria cake accesses another lattice in two lattice PDA plating mediums.Not meet ZJLQ024 as control, 3 repetitions, 25 DEG C Dark quiet culture, the colony diameter for the treatment of group is taken pictures and recorded when control group will cover with the half of plate, wherein, ZAU 05 Rhizoctonia solani Kuhn R.solani face-offs growth 1 day, ZAU22 Pythium ultimum bacterium P.ultimum face-offs growth 1 day, KH8 Penicillium notatums P.expansum face-offs growth 3 days, TZ01 peach brown rot fungus M.fructicola face-offs growth 3 days, MH10 strawberry anthrax-bacilus C.fragariae face-offs growth 3 days, WJ0101 botrytis cinereas B.cinerea face-offs growth 2 days.

Remarks explanation：In Fig. 4, left-to-right is CK respectively, and Fengyang aerogenesis of 5mm pure culture biscuits involvng inoculations is mould, and wheat culture (has been given birth to It is mould to have grown Fengyang aerogenesis) one, whether the same the mould bacteriostatic activity of Fengyang aerogenesis for seeing under different condition of culture is, and conclusion is nothing Mould by growth Fengyang aerogenesis where, fungistatic effect is all good, and all pathogens are all suppressed to grow.

It is specific as follows：

Brown rot germ (TZ01), ash arrhizus bacteria (WJ0101), anthrax bacteria (MH10), Pythium ultimum (ZAU22), vertical withered silk Pyrenomycetes (ZAU05) and mould germ (KH8) in the PDA plate normal growths without aerogenesis mould, and mould containing aerogenesis (including 5mm pure culture biscuits involvng inoculations and come from solid fermentation product wheat inoculation Fengyang aerogenesis it is mould) PDA plates do not grow (Fig. 4), Therefore aerogenesis mould is to the antibacterial of brown rot germ, ash arrhizus bacteria, anthrax bacteria, Pythium ultimum, Rhizoctonia solani Kuhn and mould germ Rate is 100%.As a result show, the aerogenesis mould wheat culture (solid fermentation product) of present invention gained has strong resist Bacterium bioactivity, it is consistent with its active ingredient bacterial strain ZJLQ024 bacteriostatic activity.

Embodiment 3, low-cost fermentation medium optimization

1) medium matrix single factor experiment

Culture medium prescription：5g medium matrixs (wheat bran, iblet, soya-bean cake and rice bran), 5ml deionized waters；Encapsulation, 121 DEG C 40min autoclavings.It is cooled to room temperature, inoculation 1ml mycelia liquid (that is, the one grade fermemtation obtained by the step 1 of above-described embodiment 2 Seed) in culture medium, 25 DEG C of quiet cultures of dark.Nucleic acid is surveyed after 6d.

Culture 6d different culture media matrix is taken to carry out nucleic acid determination, as a result display contains core containing chaffy medium matrix Sour A values highest, ZJLQ 024 growth (Fig. 5) is more suitable for compared with rice bran, corn and soya-bean cake etc..

2) screening of medium matrix best of breed

Using wheat bran as main matrix, iblet, soya-bean cake and rice bran are factor, set orthogonal array.According to orthogonal array Formulated in combination 10g (5g medium matrix+5ml deionized waters) system culture medium, 121 DEG C of 40min autoclavings, be cooled to room Temperature.It is quiet in culture medium, 25 DEG C of dark to be inoculated with mycelia liquid (that is, the one grade fermemtation seed obtained by the step 1 of above-described embodiment 2) 1ml Culture.Constant weight is dried to after 10d, nucleic acid content is surveyed and assesses fermentation efficiency.

Using wheat bran as main matrix, table (table 1) is designed according to the horizontal quadrature of 4 factor 3 and carries out medium matrix soya-bean cake, corn The formulated in combination culture medium of grain, rice bran and wheat bran adds the mycelia liquid of inoculation, fermented and cultured 10d, determines nucleic acid A values.Will be all Obtained A values carry out range analysis, and the sequence for obtaining factor influence is soya-bean cake ＞ rice bran ＞ iblets, and preferable combination For：20% soya-bean cake, 30% iblet, 10% rice bran.Therefore 1g soya-bean cakes, 1.5g iblets, 0.5g rice brans, 2g brans in 10g systems Skin is optimal medium matrix combination.It is not notable that the results of analysis of variance finds that three factors influence, and only soya-bean cake contribution rate is 15.55%, remaining two factor is negative value, overall error 0.96.Specifically as described in 2~table of table 4.

Table 1, L9 (3^4) factor level table

Sequence number	Factor	Level 1	Level 2	Level 3
					1	Soya-bean cake	10%	20%	30%
2	Iblet	10%	20%	30%
					3	Rice bran	10%	20%	30%
4	Blank	Blank	Blank	Blank

Table 2, Orthogonal Experiment and Design experimental result

Table 3, range analysis result

Table 4, the analysis of medium matrix intraclass variance

Therefore, draw the low-cost culture medium after optimization be 40% wheat bran, 20% soya-bean cake, 30% iblet and 10% meter Chaff.

Embodiment 4, preparation prepare and preserved optimization

By in embodiment 2 20% inoculum concentration (be inoculated with 20ml first order seeds), using wheat solid fermentation culture medium as The solid fermentation product of the quiet 8 days gained of culture of fermentation substrate, 25 DEG C of dark is tested as follows：

1st, preparation preserves

The above-mentioned solid fermentation product containing wheat is dried to constant weight in vacuum desiccator, takes quantitatively be loaded on parcel respectively Pack, it is stored in 4 DEG C, 25 DEG C of incubators and normal temperature (10 DEG C).Survival rate and inhibiting rate, the meter of survival rate are surveyed at regular intervals Calculation method is to take out quantitative solid fermentation product to be inoculated in PDA, and the quantity of survival, survival rate (%)=survival are counted after 20d Quantity/inoculation quantity × 100%.It was found that the solid fermentation product survival rates of 4 DEG C of preservation 0.5-5 months are up to more than 80%, while institute There is the inhibiting rate of the solid fermentation product of survival up to 100% (table 5)；But the first quarter moon that is only capable of preserving of 25 DEG C and room temperature preservation (is deposited Motility rate 63.3%, inhibiting rate 100%；Survival rate 36.7%, inhibiting rate 100%), it is then all dead (table 5).It is final to determine 4 DEG C it is most suitable storage temperature.

The survival rate and inhibiting rate for the solid fermentation product difference holding time that the different temperatures of table 5 preserves

2nd, the selection of drying equipment：

In order to determine the optimum drying mode of the mould solid fermentation product of Fengyang aerogenesis, this experiment is to superclean bench, 25 DEG C baking oven (the mould optimum growth temp of Fengyang aerogenesis) and 35 DEG C of 3 kinds of vacuum desiccators (the instrument minimum set temperature is 35 DEG C) are dry Dry mode is screened.PDA plate, observation bacterium colony size, life will be inoculated in after the solid fermentation product rehydration of all dryings Long speed is to determine the influence of drying process ZJLQ024 survival rates mould to Fengyang aerogenesis.As a result find that 3 kinds of drying modes are being inoculated with Solid fermentation product afterwards can all grow bacterium colony；Bacterium colony is can be seen in 3 days in 25 DEG C of baking ovens and superclean bench, but 35 DEG C of vacuum are done The solid fermentation product of dry device processing could grow bacterium colony on PDA after 5 days, the wheat (solid fermentation product) after oven It is full flat inconsistent, differed greatly in the growth rate of PDA plate.Therefore, the last selection superclean bench natural wind of the present invention Dry mode is dehydrated to solid fermentation product.

3rd, the preparation shelf-life optimizes：

The basic ideas of preparation such as Fig. 7, solid fermentation product is handled, and processing mode is coated for nutrient solution, ventilation Cupboard air-dries, 4 DEG C, 10 DEG C, and plastic packaging preserves at a temperature of 25 DEG C three kinds, obtains optimal preservation scheme.

Comprise the following steps that：

The solid fermentation product (white aerogenesis fungicidin grows the culture of wheat) of culture 8 days, by 1g:1ml ratio leaching (7 kinds of nutrient solutions are respectively 3% aqueous trehalose solution, and 5% aqueous trehalose solution, 3% is extra large in the 7 kinds of nutrient solutions of bubble after sterilization Alginic acid sodium water solution, 5% sodium alginate aqueous solution, 0.1% xanthan gum solution, 0.2% xanthan gum solution and water, above-mentioned % It is quality %), overnight (about 10 hours).Spread out, air-dried in superclean bench, obtain Fengyang aerogenesis removing mildew.Specifically such as Under：Air-dry to water content 10% or so and carry out first time preservation.Storage temperature：4 DEG C, 10 DEG C and 25 DEG C；Water content 8% or so is entered Second of preservation of row, storage temperature：4 DEG C, 10 DEG C and 25 DEG C；Water content 6% or so carries out third time preservation, storage temperature：4 DEG C, 10 DEG C and 25 DEG C.After preserving certain time, take out quantitative solid fermentation product (Fengyang aerogenesis removing mildew) and be inoculated in PDA, Count survival volume.Water content=(quality before air-dried-quality after air-drying)/air-dry preceding quality.

Survival rate (%)=amount of survival/inoculation quantity × 100%.

Remarks explanation：By nutrient solution through conventional sterilant (in 121 DEG C, 1.1 atmospheric pressure sterilizing 20min), must sterilize rear batallion Nutrient solution.

Acquired results are as shown in Fig. 8~Figure 14.

After preserving December, the water content of the coated preparation drying to 8% of 7 kinds of Different Nutrition liquid is after 4 DEG C of survivals preserved Rate is kept at more than 90% (Fig. 8), but the preparation of same water content was stored in 10 DEG C and 25 DEG C of survival rate from 3 months Start slowly decline, and be stored in 25 DEG C since 6 months survival rate be gradually reduced to 0 (Fig. 9, Figure 10).

Therefore, initial water content is more suitable for preserving for a long time 8% or so.

At 25 DEG C, moisture content is that the preparation of 6% and 10% 3 first quarter moons of preservation starts death, but moisture content 8% is until 6 Month is just gradually dead (Figure 10,11,12).Different solvent Cotton seeds solid fermentation products, obtained data and disunity, 80%-100% water content is nearly all maintained in each processing of 4 DEG C of preservations, and the place for air-drying wheat is soaked with sterilized water Reason, preservation effect is more stable, even if the initial aqueous rate preserved is different, but survival rate is between 90-100%, in institute There is survival rate scope fluctuation in processing minimum.Therefore generally speaking, initial water content 8%, 4 DEG C of temperature, sterile water process are protected It is best to deposit effect, preservation can be stablized and surpass 1 year.

Embodiment 5, a kind of preparation method of Fengyang aerogenesis removing mildew, successively following steps：

1) the mould solid fermentation product of Fengyang aerogenesis, is prepared：

3 pieces of 5mm ZJL024 bacteria cakes are in 100ml PDB nutrient solutions, 200r/min, and dark 25 DEG C are cultivated 6 days；Obtain one-level Fermentation seed.

The mould one grade fermemtation seed of Fengyang aerogenesis (i.e. liquid fermentation seed) is inoculated in fermentation according to 20% inoculum concentration Fermented in matrix, in 25 DEG C of dark static gas wave refrigerators 8 days, obtain solid fermentation product.

Fermentation substrate is wheat solid fermentation culture medium or low-cost fermentation culture medium.

The preparation method of wheat solid fermentation culture medium is：Wheat is put into 7~9h of immersion in water, by gained after immersion Wheat is sterilized, and (usual manner sterilizes 2 times, i.e. is specially：In 121 DEG C, 1.1 atmospheric pressure sterilizing 40min)；

The preparation method of the low-cost fermentation culture medium is：Preparation raw material, the raw material by following weight content into It is grouped into：40% wheat bran, 20% soya-bean cake, 30% iblet and 10% rice bran, by raw material and deionized water according to 1g：1ml ratio Being sterilized after example mixing, (usual manner sterilizes 2 times, i.e. is specially：In 121 DEG C, 1.1 atmospheric pressure sterilizing 40min).

Above-mentioned 20% inoculum concentration, i.e.,：One grade fermemtation seed (i.e. liquid fermentation seed)：Fermentation substrate=20ml:100g.

2), solid fermentation product carries out nutrient solution coating and air-dried：

The nutrient solution is following any；

3% aqueous trehalose solution, 5% aqueous trehalose solution；3% sodium alginate aqueous solution, 5% sodium alginate aqueous solution, 0.1% xanthan gum solution, 0.2% xanthan gum solution and water, above-mentioned % are quality %；

By nutrient solution through conventional sterilant (in 121 DEG C, 1.1 atmospheric pressure sterilizing 20min), nutrient solution after must sterilizing.

By solid fermentation product according to 1g:1ml mass ratio is soaked in after sterilizing in nutrient solution, and soak time is 10 small When；

It is 8% that the solid fermentation product of gained after immersion is air-dried into (superclean bench air-dries) to moisture content；After must air-drying Product -- Fengyang aerogenesis removing mildew；

3), rear product must will be air-dried obtained by step 2) -- the conventional packing processes of Fengyang aerogenesis removing mildew progress (including take out Vacuum and plastic packaging processing).

Remarks explanation：

When fermentation substrate is wheat solid fermentation culture medium, Fengyang aerogenesis removing mildew I is obtained；

When fermentation substrate is low-cost fermentation culture medium, Fengyang aerogenesis removing mildew II is obtained.

The solid fermentation product (fermentation substrate is used as using wheat solid fermentation culture medium) in step 1) directly air-dry to 8% moisture content, obtain Fengyang aerogenesis removing mildew III (as a comparison case).

By above-mentioned Fengyang aerogenesis removing mildew I, Fengyang aerogenesis removing mildew II, aerogenesis removing mildew III (as a comparison case) in 4 DEG C Preserved, survival rate detection is carried out according to mode described in above-described embodiment 4, gained survival rate is containing tool after the different holding times Body is as follows：

Table 6, gained survival rate after the different holding times

Active constituent content measuring：

This experiment determines the active constituent content of preparation by surveying CFU (CFU).

Fengyang aerogenesis removing mildew that 3 months are preserved under the different temperatures of the gained of embodiment 5 is weighed (with the training of wheat solid fermentation Base is supported as fermentation substrate) about 0.2g, with 20mL PDB nutrient solution soaked overnights, outwells unnecessary PDB nutrient solutions, sample is put In 25 DEG C of incubator static gas wave refrigerator 4d, taking-up is put into sterile mortar, adds 20mL sterilized waters to grind granule, draws 100 μ L and hangs Turbid counts clump count in applying flat board in PDA culture medium after 25 DEG C of culture 4-5d.

The content W of active ingredient is calculated by formula (1) in sample：

W=200A/m ... ... ... ... ... ... ... ... (1)

In formula：A-bacterium colony number, CFU；

The sample weighting amount of m-sample, g；

W-active constituent content, CFU/g.

It is W=3.1 × 10 to calculate active constituent content according to the formula of active constituent content⁵。

The CFU data of Fengyang aerogenesis removing mildew after being preserved 3 months in 4 DEG C, 10 DEG C and 25 DEG C are as described in Figure 15.Nutrient solution For water when, relative efficacy is optimal.

Embodiment 6, as fumigant fruit postharvest diseases application

Choose the essentially identical apple of size, quality to be implemented, 6 repetitions of every group of setting.It is specific as follows：

The solid fermentation product (being used as fermentation substrate using wheat solid fermentation culture medium) of the gained of embodiment 5 is loaded into little Bai In bottle.

Mould spore suspension is dipped in sterile needle, stab inoculation apple, is placed in the container of closing, in container simultaneously Equipped with the above-mentioned little Bai bottles for opening lid, as experimental group (fumigant)；Above-mentioned little Bai bottles are not placed with the container of closing, made For control group (ck).

Experimental group, control group are placed naturally in indoor (about 18 DEG C), observation experiment result after 10 days.After control group 10d 2/3 apple morbidity, and the apple of experimental group only 1/6 has the generation of penicilliosis；Therefore, after aerogenesis removing mildew in Fengyang is adopted to apple Penicilliosis have certain prevention effect.

Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.

Claims (5)

1. Fengyang aerogenesis removing mildew, it is characterized in that：Fengyang aerogenesis removing mildew is that aerogenesis mould solid fermentation product in Fengyang is entered into field headquarters Nutrient solution be coated and air-dry after gains；

The preparation method of the mould solid fermentation product of Fengyang aerogenesis is to comprise the following steps：By the mould one grade fermemtation of Fengyang aerogenesis Seed is according to 10 ~ 20ml:100g amount ratio, which is inoculated on fermentation substrate, to be fermented, and zymotechnique is：In 24.5 ~ 25.5 DEG C Dark quiet culture, incubation time are 8~10 days；Obtain the mould solid fermentation product of Fengyang aerogenesis；

The nutrient solution for it is following any one：The % of the % of 3 %~5 aqueous trehalose solution, 3 %~5 sodium alginate aqueous solution, The % xanthan gum solutions of 0.1 %~0.2, water, above-mentioned % is quality %；

By the mould solid fermentation product of Fengyang aerogenesis according to 1g:0.9 ~ 1.1ml solid-to-liquid ratio is soaked in after sterilizing in nutrient solution, immersion Time is 10 ~ 12 hours；

It is 8% that the mould solid fermentation product of Fengyang aerogenesis of gained after immersion, which is air-dried to moisture content, and product is Fengyang aerogenesis after air-drying Removing mildew.

2. aerogenesis removing mildew in Fengyang according to claim 1, it is characterized in that：Fengyang aerogenesis removing mildew is carried out at packaging Reason；The packing processes include vacuumizing and plastic packaging processing.

3. aerogenesis removing mildew in Fengyang according to claim 1 or 2, it is characterized in that：The fermentation substrate is sent out for wheat solid Ferment culture medium or low-cost fermentation culture medium；

The preparation method of the wheat solid fermentation culture medium is：Wheat is put into 7~9h of immersion in water, by gained after immersion Wheat is sterilized；

The preparation method of the low-cost fermentation culture medium is：Preparation raw material, the raw material is by following weight content into packet Into：

40% wheat bran, 20% soya-bean cake, 30% iblet and 10% rice bran；By raw material and deionized water according to 1g：After 1ml ratio mixing Sterilized.

4. aerogenesis removing mildew in Fengyang according to claim 3, it is characterized in that：

The preparation method of the mould one grade fermemtation seed of Fengyang aerogenesis is：By preserving number be CGMCC No.2863 ZJLQ024 bacterium in In PDB nutrient solutions, 200 r/min, dark 25 DEG C of cultures 6 days, one grade fermemtation seed is obtained.

5. aerogenesis removing mildew in Fengyang according to claim 4, it is characterized in that：The storage temperature of Fengyang aerogenesis removing mildew For 4 DEG C.