Summary of the invention
The object of this invention is to provide a kind of Aspergillus niger strain, this bacterial classification can promote growing of crop; Invention also provides the preparation method of Aspergillus niger strain spore powder, simple, be applicable to suitability for industrialized production.
Aspergillus niger strain of the present invention is the aspergillus niger SDKYSW-2# in Aspergillus (Aspergillus), Aspergillus niger strain is on October 12nd, 2013, preservation is to China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, Classification And Nomenclature is aspergillus niger (Aspergillusniger), and preserving number is CGMCC No.8327.
Aspergillus niger strain from booth (Yiyuan) nursery soil, adopts soil dilution partition method to be separated obtain.
The screening method of Aspergillus niger strain of the present invention, comprises the following steps:
One, soil sample collection: sample 200g from upper soll layer to 15cm, the three unities adopts several sample, mixes scalping, gets 25g and is used as to be separated;
Two, the separation of aspergillus niger:
1, instrument:
High-pressure steam sterilizing pan, drying baker, incubator, electronic balance, enamelled cup, beaker, packing funnel, test tube, suction pipe (10ml), suction that ball, glass stick, cotton, cotton rope, newspaper, culture dish, triangular flask (100ml), granulated glass sphere, pipettor (1ml), suction nozzle, medicine spoon, inoculating needle, spirit lamp.
2, medicine:
Zulkovsky starch, potassium primary phosphate, magnesium sulfate, ferrous sulfate, agar, sucrose, aspergillus niger.
3, the preparation of aspergillus niger isolation medium:
Zulkovsky starch 10g, potassium primary phosphate 0.5g, magnesium sulfate 0.1g, ferrous sulfate 0.1g, agar 10g, sucrose 10g, water 500ml; Weighed up by reagent, add water and put into enamelled cup, heating for dissolving on electric furnace, inhale in substratum 10ml packing test tube with suction pipe (10ml), be stoppered tampon, paper using wraps sterilizing, for subsequent use.
Substratum makes: reagent → dissolving → correction pH value → packing → add tampon, wrapping → sterilizing (0.1P is incubated 30 minutes) → for subsequent use.
4, working method:
(1) preparation work:
To use instrument culture dish, triangular flask, suction nozzle (some), medicine spoon, substratum, 10ml test tube etc., with high-pressure sterilizing pot 0.1p pressurize 30 minutes, sterilizing was for subsequent use.
Triangular flask: load 100ml water and add granulated glass sphere (some).
10ml test tube: load 9ml water, carry out numbering, be stoppered tampon, 8, for subsequent use.
5, culture dish is one group, wraps with newspaper, 6 bags 30.
1ml suction nozzle, each independent newspaper is wrapped (some).
(2) dilution of bacterium sample is separated:
By bacterium sample to be separated aseptically, get 1-2 ring, put into triangular flask with inoculating needle, in bottle, bacterium liquid is about containing spore 50-100 ten thousand/ml, and shake well about 5 minutes, makes spore each and every one be separated mixing, for subsequent use.
By aseptic technique requirement in inoculation tank, draw bacterium liquid in 1ml triangular flask with pipettor and put into No. 1 pipe, inhale 1ml from No. 1 pipe again and put into No. 2 pipes, by that analogy, until be diluted to No. 6 or No. 7 in vitro, require often to inhale toward test tube once to change a suction nozzle, between about individual containing spore count 5-10 in every milliliter of diluent.
Get No. 5 No. 6 No. 7 in vitro bacterium liquid 0.5 milliliter, put into culture dish, each extent of dilution makees 10 flat boards, is poured in culture dish by substratum (temperature 50 C) and fully mixes with bacterium liquid, set level, cool.
Within about about 20 minutes, take out after culture medium solidifying in culture dish, be inverted in incubator (30 DEG C), cultivate after 4 days, can observe with each spore for concentric(al) circles and the bacterium colony that grows on plating medium, bacterium colony is large and spore density is thick screens excellent bacterial strain.
Precaution:
This operation must be carried out in sterilized aseptic inoculation box, and is strictly undertaken by aseptic technique requirement.
The culture dish specification used and the quantity of substratum added must be identical, so that compare the bacterium colony in different culture dish.
The temperature adding substratum is unsuitable overheated or excessively cold, and general control (touches non-scald on hand with hand) otherwise spore can be made to scald dead or occur the uneven phenomenon of media surface between 45-50 DEG C.
Often dilute a test tube, change a suction nozzle.
(3) sieve triangular flask culture medium again: joined in siccative by inorganic salt solution, until when the water content of batching is 53-55wt.%, stop adding inorganic salt solution; The mass percent of described siccative consists of: wheat bran 50%, soybean cake powder 30% and peanut straw powder 20%; Described inorganic salt solution is containing 2.0gKH in every premium on currency
2pO
4, 1.4g (NH
4)
2sO
4, 0.3g urea, 0.3gMgSO
47H
2o, 0.3g CaCl
2, 5.0mg FeSO
47H
2o, 1.56mg MnSO
4h
2o, 1.4mg ZnSO
47H
2o, 2.0mgCoCl
2.Load triangular flask, 500ml triangular flask loads the wet feed 40g of moisture 85%, and sterilizing, accesses the Aspergillus niger strain of above-mentioned separation, cultivates cryodrying after three days for 30 DEG C, analyzes the enzyme system produced, in table 1.
The activity analysis of the various enzyme of table 1
|
Analytical procedure |
Calculating vigor |
Saccharifying enzymic activity |
GB |
127u/g |
Cellulase activity |
GB |
7090u/g |
Xylanase activity |
GB |
915u/g |
Mannosans enzyme activity |
GB |
5379u/g |
An Aspergillus niger strain is chosen in comprehensive analysis, called after SDKYSW-2#.
Bacterial classification feature: dibbling is on PDA substratum, and black-koji mould filament diameter 15 ~ 20pm, is about 1 ~ 3mm, wall thickness and smooth.Globe-roof capsule is formed on top, and it covers one deck metulae and one deck stigma comprehensively, and long on stigma have bunchiness memnonious spherical, diameter 2.5 ~ 4.0 μm.Conidial head is spherical, diameter 700 ~ 800 μm, brown-black.Spreading rapidly, be just white, after become aureus until black heavy fleece shape, the colourless or central authorities' slightly tawny in the back side.Hydroxyl pregnant sterone also can be converted into androstene by some bacterial strains.Mycelia is chocolate, and top capsule is spherical greatly, and stigma is double-deck, and conidium is spherical, darkly, chocolate, level and smooth or coarse.Strong to the patience of ultraviolet and ozone.Mycelia is flourishing, and multi-branched, has the many cells fungi of multinuclear.The heavy wall of conidiophore by specialization and the hyphal cell (podocyte) expanded vertically bears; Conidium, head is as " chrysanthemum ".Mycelia, the spore of aspergillus niger present shades of colour usually, as: black, brown, green, yellow, orange, brown etc., bacterial classification is different, and color is also different.
The preparation method of Aspergillus niger strain spore powder of the present invention, step is as follows:
(1) actication of culture: Aspergillus niger strain is seeded on slant medium and cultivates,
(2) cultivation of seed liquor: the bacterial classification activated is joined cultivation in seed culture medium and obtain seed liquor,
(3) ferment: the batching after seed liquor and sterilizing stirs, heat-preservation fermentation, and spore is sent out in temperature adjustment;
(4) aftertreatment: the low-temperature material drying that step (3) obtains is obtained work in-process, dry, pulverize to Standard mesh after adding anticorrosion antiultraviolet material, and spore is collected, and rechecks, packing and get final product.
Slant medium described in step (1) is wheat bran juice slant medium, and culture temperature is 28-30 DEG C, and incubation time is 70-74 hour.
The basic recipe of the wheat bran juice substratum described in step (1): Zulkovsky starch 2%, KH
2pO
40.1%, MgSO
40.03%, (NH
4)
2sO
40.05%, agar 2%, sugar 1%, wheat bran juice 500ml.
The volume ratio of the bacterial classification that the activation described in step (2) is good and seed culture medium is 1:8-12.
Culture temperature described in step (2) is 33-36 DEG C, and incubation time is 45-48 hour.
In the middle of when cultivating in step (2) every 1 hour delivering oxygen once.
The preparation method of the seed culture medium described in step (2) is as follows:
By the ratio mixing of wheat germ, wheat bran 1:9-11 in mass ratio, repeatedly rinse with 45-50 DEG C of warm water, by the scavenging solution that obtains at 125-130 DEG C, sterilizing 40-50min under 0.15-0.2Mpa, be cooled to 33-36 DEG C, make seed culture medium.
The mass ratio of the seed liquor described in step (3) and batching is 1:10-12.
Heat-preservation fermentation temperature described in step (3) is 32-36 DEG C, and the heat-preservation fermentation time is 12-15h; It is 32-38 DEG C that spore temperature is sent out in temperature adjustment, and it is 48-50h that the spore time is sent out in temperature adjustment.
Preparation of batch method described in step (3) is as follows:
Inorganic salt solution is joined in siccative, until when the water content of batching is 53-55wt.%, stops adding inorganic salt solution;
The mass percent of described siccative consists of: wheat bran 40-50%, soybean cake powder 30-40% and peanut straw powder 20-30%;
Described inorganic salt solution is containing 2.0gKH in every premium on currency
2pO
4, 1.4g (NH
4)
2sO
4, 0.3g urea, 0.3gMgSO
47H
2o, 0.3g CaCl
2, 5.0mg FeSO
47H
2o, 1.56mg MnSO
4h
2o, 1.4mg ZnSO
47H
2o, 2.0mgCoCl
2.
In step (3), the fermentation finished product of gained can directly use.
Anticorrosion antiultraviolet material described in step (4) is zinc oxide, and anticorrosion antiultraviolet material and half-finished mass ratio are 1-1.2:10000.
Cryodrying described in step (4) be spring and autumn winter dryness temperature be 32-34 DEG C, time of drying is 24-36h; Summer, drying temperature was 34-38 DEG C, and time of drying is 36-48h.
Half-finished moisture≤10% described in step (4).
Standard mesh described in step (4) is 120 sieve meshes.
Reinspection in step (4) is according to Q/SHN--2009 testing product quality.
Primary dcreening operation gating is excessively to the plant residue sample taking from different root system of plant soil and rot, with dilution-plate method, carry out single bacterium separation, by the multiple Aspergillus niger strains obtained, select growth and breeding fast, do product enzyme further and sieve culture experiment again, by measuring the vigor producing enzyme, select the energetic bacterial strain of cytohydrolist to preserve, by technical process, produce aspergillus niger spore powder field test, after three months, same sample plot sampling is separated flora, carry out bacterium dependency experiment turn out to be former experimental bacteria determine grow, and constantly improve experiment, in bacterial strain, promote plant growth, improve Soil structure, strengthen soil fertility, constant product quality, to people and animals, the target of crop and environmental safety.
In the present invention, bacterial strain screening samples in soil and rotten plant residue, but it is high to endanger serious soil and plant residual body screening probability with pathogenic bacteria, to contain multiple instruction culture medium for primary dcreening operation substratum, chooses active height and accepts or rejects bacterial strain; Multiple sieve is then considered the characteristic of bacterial strain and changes fermentation condition within the specific limits, does not mainly affect its growth velocity, and improving cell wall enzymes activity is further underlying condition, and the bacterial strain screened, through further biological activity determination, determines the performance of bacterial strain.
The black-koji mould that a strain selected by the present invention is new is also the bacterial strain that a class produces prozyme, except product saccharifying enzyme, also can produce amylase, cellulase etc.Under the effect of enzyme, stodgy macro-molecular protein is degraded to peptone, polypeptide and each seed amino acid, Starch Hydrolysis becomes dextrin, oligosaccharides, monose, and the anti-nutrient substance degradeds such as robust fibre can be made in auxiliary material, improve nutritive value, health-care effect and digestibility, can be widely used in brewageing, the industry such as food, fodder additives.
Black-koji mould that the present invention screens (spore content 20,000,000,000/gram, one annual survival rate >=85%), be preserved in national DSMZ, and according to great many of experiments, make and preserve and use novel form all very easily, 20000000000/gram water dispersible granules (pH value: 7.3-7.8, moisture content: 6-8%); Aspergillus niger has the function that secretion produces amylase, saccharifying enzyme, citric acid, gluconic acid, gallic acid etc.; Industrial at bio-feritlizer, aspergillus niger has cracking larger molecular organics and indissoluble inorganics, is convenient to Crop and utilizes, improve Soil structure, strengthen soil fertility, improve the effect of crop yield.From Yield Increase In Cotton effect, black-koji mould has obvious growth-promoting effect of increasing production to cotton, and after single-dose application, stimulation ratio is 13.50 ~ 20.1%.
The present invention compared with prior art, has following beneficial effect:
The present invention has possessed that high efficiency, cost are low, free from environmental pollution, microbial fertilizer notable feature to person poultry harmless.Mature production technology of the present invention, advanced technology, especially bacterial screening and formulation in there is advance, and it is cheap and easy to get to produce required raw material, and added value of product is very high, and ecological and social benefit is remarkable.
Embodiment
Below in conjunction with embodiment, the present invention is described further.
Embodiment 1
(1) actication of culture: be stained with a small amount of bacterium colony spore gently with transfering loop and be linked in wheat bran juice slant medium and cultivate, temperature remains on 30 DEG C, cultivates 72 hours;
(2) cultivation of seed liquor: the bacterial classification 1kg activated is added in 1000kg seed culture medium, 36 DEG C of constant temperature culture 48 hours, middle every 1 hour delivering oxygen once, make seed liquor;
(3) ferment: the batching 2000kg after seed liquor 200kg and sterilizing stirs and inoculates, heat-preservation fermentation, and spore is sent out in temperature adjustment, and heat-preservation fermentation temperature is 32 DEG C, and the heat-preservation fermentation time is 12h; It is 38 DEG C that spore temperature is sent out in temperature adjustment, and it is 48h that the spore time is sent out in temperature adjustment;
(4) aftertreatment: material step (3) obtained, cryodrying obtains work in-process, and 1kg work in-process dry, pulverize after adding zinc oxide 0.1g to 120 sieve meshes, and spore is collected, and rechecks, packing and get final product.
Described in step (3), preparation of batch method is as follows:
Inorganic salt solution is joined in siccative, until when the water content of batching is 53wt.%, stops adding inorganic salt solution;
Described siccative is wheat bran 1000kg, soybean cake powder 600kg and peanut straw powder 400kg;
Described inorganic salt solution is containing 2.0g KH in every premium on currency
2pO
4, 1.4g (NH
4)
2sO
4, 0.3g urea, 0.3gMgSO
47H
2o, 0.3g CaCl
2, 5.0mg FeSO
47H
2o, 1.56mg MnSO
4h
2o, 1.4mg ZnSO
47H
2o, 2.0mgCoC
l2.
Various enzyme activity analysis in table 2 in finished product.
Various enzyme activity analysis in table 2 finished product
|
Analytical procedure |
Calculating vigor |
Saccharifying enzymic activity |
GB |
98u/g |
Cellulase activity |
GB |
6872u/g |
Xylanase activity |
GB |
891u/g |
Mannosans enzyme activity |
GB |
3582u/g |
Total spore count: 35,000,000,000
Living bacteria count: 14,500,000,000
Embodiment 2
(1) actication of culture: be stained with a small amount of bacterium colony spore gently with transfering loop and be linked in wheat bran juice slant medium and cultivate, temperature remains on 28 DEG C, cultivates 70 hours;
(2) cultivation of seed liquor: the bacterial classification 1kg activated is added in 800kg seed culture medium, 33 DEG C of constant temperature culture 45 hours, middle every 1 hour delivering oxygen once, make seed liquor;
(3) ferment: the batching 2400kg after seed liquor 200kg and sterilizing stirs and inoculates, heat-preservation fermentation, and spore is sent out in temperature adjustment, and heat-preservation fermentation temperature is 36 DEG C, and the heat-preservation fermentation time is 15h; It is 32 DEG C that spore temperature is sent out in temperature adjustment, and it is 50h that the spore time is sent out in temperature adjustment;
(4) aftertreatment: material step (3) obtained, cryodrying obtains work in-process, and 1kg work in-process dry, pulverize after adding zinc oxide 0.12g to 120 sieve meshes, and spore is collected, and rechecks, packing and get final product.
Described in step (3), preparation of batch method is as follows:
Inorganic salt solution is joined in siccative, until when the water content of batching is 55wt.%, stops adding inorganic salt solution;
Described siccative is wheat bran 960kg, soybean cake powder 960kg and peanut straw powder 480kg;
Described inorganic salt solution is containing 2.0g KH in every premium on currency
2pO
4, 1.4g (NH
4)
2sO
4, 0.3g urea, 0.3gMgSO
47H
2o, 0.3g CaCl
2, 5.0mg FeSO
47H
2o, 1.56mg MnSO
4h
2o, 1.4mg ZnSO
47H
2o, 2.0mgCoCl
2.
Various enzyme activity analysis in table 3 in finished product.
Various enzyme activity analysis in table 3 finished product
|
Analytical procedure |
Calculating vigor |
Saccharifying enzymic activity |
GB |
90u/g |
Cellulase activity |
GB |
5882u/g |
Xylanase activity |
GB |
752u/g |
Mannosans enzyme activity |
GB |
3106u/g |
Total spore count: 32,000,000,000
Living bacteria count: 12,500,000,000
Embodiment 3
(1) actication of culture: be stained with a small amount of bacterium colony spore gently with transfering loop and be linked in wheat bran juice slant medium and cultivate, temperature remains on 29 DEG C, cultivates 74 hours;
(2) cultivation of seed liquor: the bacterial classification 1kg activated is added in 1200kg seed culture medium, 35 DEG C of constant temperature culture 46 hours, middle every 1 hour delivering oxygen once, make seed liquor;
(3) ferment: the batching 2200kg after seed liquor 200kg and sterilizing stirs and inoculates, heat-preservation fermentation, and spore is sent out in temperature adjustment, and heat-preservation fermentation temperature is 35 DEG C, and the heat-preservation fermentation time is 14h; It is 35 DEG C that spore temperature is sent out in temperature adjustment, and it is 49h that the spore time is sent out in temperature adjustment;
(4) aftertreatment: material step (3) obtained, cryodrying obtains work in-process, and 1kg work in-process dry, pulverize after adding zinc oxide 0.11g to 120 sieve meshes, and spore is collected, and rechecks, packing and get final product.
Described in step (3), preparation of batch method is as follows:
Inorganic salt solution is joined in siccative, until when the water content of batching is 54wt.%, stops adding inorganic salt solution;
Described siccative is wheat bran 880kg, soybean cake powder 660kg and peanut straw powder 660kg.
Described inorganic salt solution is containing 2.0g KH in every premium on currency
2pO
4, 1.4g (NH
4)
2sO
4, 0.3g urea, 0.3gMgSO
47H
2o, 0.3g CaCl
2, 5.0mg FeSO
47H
2o, 1.56mg MnSO
4h
2o, 1.4mg ZnSO
47H
2o, 2.0mgCoCl
2.
Various enzyme activity analysis in table 4 in finished product.
Various enzyme activity analysis in table 4 finished product
|
Analytical procedure |
Calculating vigor |
Saccharifying enzymic activity |
GB |
92u/g |
Cellulase activity |
GB |
6159u/g |
Xylanase activity |
GB |
786u/g |
Mannosans enzyme activity |
GB |
3386u/g |
Total spore count: 32,500,000,000
Living bacteria count: 13,000,000,000