Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Aspergillus niger strain provided by the invention (Aspergillus niger) Li-2013-03 is through taking turns nitrosoguanidine mutagenesis by aspergillus niger (Aspergillus niger) Li-2010 of a strain cellulase-producing product of Laboratories Accession more, then mutant strain step-sizing is eliminated, finally through leavening property test screen, Aspergillus niger strain (Aspergillusniger) Li-2013-03 producing high activity cellulase is obtained to strain excellent.
The bacterial strain of the high activity cellulase of product provided by the invention is specially aspergillus niger (Aspergillus niger) Li-2013-03.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, postcode 100101) on July 15th, 2013, and preserving number is CGMCCNO.7927.
Produce the screening method of high activity cellulase bacterial strain, comprise the following steps:
1) slant culture: by original Aspergillus niger strain (Aspergillus niger) Li-2010 streak inoculation slant medium, 30 DEG C cultivate 2 ~ 3d, until mycelium ripe, produce a large amount of black spore.Described slant medium is composed as follows: 12 ° of Brix wort l000mL, pH value nature, 121 DEG C of sterilizing 20min;
2) spore suspension preparation (following steps all aseptically operate): add 15mL sterilized water to test tube slant, spore is scraped, with filter paper filtering, filtered solution poured into sterilizing and be added with in the 150mL triangular flask of 5-10 grain sterile glass beads, triangular flask is put into shaking table vibration 10-15min, spore is disperseed.
3) nitrosoguanidine (NTG) mutagenesis
A. with sterilized water spore suspension is adjusted to and is diluted to 10
6-10
7individual/mL.
B. get 10mL bacteria suspension to be transferred in 100mL triangular flask, add the NTG of 10mg, be mixed with the NTG solution that final concentration is 10mg/mL, and add 4-5 and drip acetone, be beneficial to NTG and dissolve.
C. at 30 DEG C, the centrifugal 10min of 200rpm oscillatory reaction 30min, 5000rpm collects thalline, with stroke-physiological saline solution washing several, and stopped reaction.
D. suitably spore concentration is adjusted to 10 by dilution
3individual/mL, get last dilution bacterium liquid 0.2mL, dilution spread is on Mierocrystalline cellulose-Congo red plate screening substratum.The bacterial strain 200 that after cultivating 2 ~ 3 days at 30 DEG C, picking transparent circle/colony diameter is larger.(described Mierocrystalline cellulose-Congo red plate screening substratum is composed as follows: cellulose powder 10g, Congo red 0.2g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, gelatin 2g, agar 20g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min).
E. sieve again: the 200 strain bacterium obtained are inoculated in slant medium with sterile toothpick respectively, and 30 DEG C are cultured to spore and are paved with inclined-plane.Respectively spore is equipped with 50mL and sieves again in the 250mL triangular flask of substratum ferment to be inoculated under aseptic washing, inoculum size 10%(v/v), 30 DEG C, 100r/min cultivates 96h, measures the cellulase activity of each bacterial strain respectively.(the described substratum of sieve is again composed as follows: cellulose powder 50g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min).The bacterial strain choosing cellulose enzyme vigor the highest carries out amplification test.
4) genetic stability test
Li-2013-03 bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and detects the fermentation situation after at every turn going down to posterity by the method that shaking flask is sieved again.Experiment finds, inclined-plane goes down to posterity for continuous ten times, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
5) scale-up
1. seed culture: strains A spergillus nigerLi-2013-03 the highest for cellulose enzyme vigor is accessed in 500mL triangular flask, seed culture medium loading amount 100 milliliters, 30 DEG C, 150rpm shaking table cultivation 72-96h.
3. seed tank culture: by seed liquor with 10%(v/v) inoculum size access be equipped with in the 10L fermentor tank of 7.5L fermented liquid, control ph is constant is 6.0 ± 0.2, culture temperature 30 ± 0.1 DEG C, stirring velocity 300rpm, ventilation (v/v) 1:0.8-1.2, incubation time 96h, dissolved oxygen 20-30%.Described fermention medium consists of: cellulose powder 100g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min.
After fermentation ends, get fermented supernatant fluid (crude enzyme liquid) and carry out enzyme activity detection after measured, the circumscribed beta-glucanase of strains A spergillus nigerLi-2013-03, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach 620U/mL, 1289U/mL, 456U/mL and 732U/mL respectively, improve 9.21 times, 7.43 times, 8.15 times and 10.31 times respectively than starting strain Aspergillus niger Li-2010.
Phosphorus decomposing Bacillus megaterium is a kind in Bacillus (Bacillus).Motion, forms pod membrane, aerobic.Acid is produced from glucose, also normal from pectinose and the acid of N.F,USP MANNITOL product.Hydrolyzed starch, does not produce lecithinase, and VP is negative.The organophosphorus that can decompose in soil becomes the available rapid available phosphorus of plant.
The agent of phosphorus decomposing bacillus megaterium is provided by Cangzhou prosperous generation thing technical institute, address: liberation West Road, Hebei China Cangzhou City Canal Zone chin or cheek and Building A 1, international business affairs center district 807-812.
Rui Gu bio tech ltd, Baoding provides colloid bacillus cereus bacterium powder.
Subtilis provided by the invention (Bacillus subtilis) Li-2013-02.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute) on July 15th, 2013, and preserving number is CGMCC No.7926.
Described bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and gramstaining is positive.This bacterium can utilize Citrate trianion, and nitrate reductase, V-P test into the positive.
Described subtilis (Bacillus subtilis) Li-2013-02 is preserved in the product Thermostable α-Amylase in this laboratory subtilis Li-2013 by a strain obtains through UV-LiCl-ethyl sulfate Mutation screening, and concrete screening step is as follows:
(1) preparation of bacteria suspension
The mono-bacterium colony of Li-2013 grown after plate streaking is separated is accessed in seed culture medium, 100r/min, after 40 DEG C of cultivation 12h, after getting 1mL medium centrifugal, use brine twice, and resuspended with 9mL physiological saline.
(2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.Bacterium liquid through irradiating is coated lithium chloride flat board after gradient dilution, and contrasts to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 40 DEG C and cultivate 48h, the flat board growing bacterium colony filters out hydrolysis circle choose preserve to inclined-plane with colony diameter ratio the maximum, bacteria suspension is mixed with after purifying, fully mix with ethyl sulfate stoste after gradient dilution, and in 40 DEG C of concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board after gradient dilution.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 40 DEG C and cultivate 48h, on the flat board growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter ratio the greater, obtains three strain bacterium Li-2013-01, Li-2013-02, Li-2013-03 after purifying
(4) shake flask fermentation sieves again
By the three strain bacterium Li-2013-01 obtained, Li-2013-02, Li-2013-03 carry out shake flask fermentation in the 250mL shaking flask containing 30mL fermention medium, seed inoculum size 10% (V/V), 40 DEG C, 100r/min cultivates 72h, centrifuging and taking fermented supernatant fluid obtains crude enzyme liquid.
(5) enzyme activity determination
The definition of Mei Huo unit: 1mL crude enzyme liquid, in 105 DEG C, under pH4.2 condition, 1min liquefies 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain Li-2013-02 is stable most superior strain, and enzyme is lived and reached 30000U/mL, to live raising 1.6 times than original strain enzyme.
Described lithium chloride is dull and stereotyped: starch 1%, peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%.
Described seed culture medium: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%.
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH) 2SO40.4%, K2HPO40.8%, CaCl20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.
Fermented by bacterial strain Li-2013-02 and obtain a kind of high-temperature resistant alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature is between 105-115 DEG C, and the temperature stability of preserving below 110 DEG C is better, and more than 115 DEG C to preserve long-time temperature stability poor.
(2) this enzyme optimal reaction pH value is 4.2.High enzyme vigor is all had, the enzyme complete stability alive when pH value is 3.0 between pH value 3.0-7.0.
(3) enzymic activity: by mutant strain Li-2013-02 provided by the present invention, the Thermostable α-Amylase enzyme activity of preparation is 30000-35000U/ml.
1, the present invention uses the method for UV-LiCl-ethyl sulfate complex mutation to obtain the subtilis Li-2013-02 of a plant height product Thermostable α-Amylase, and this bacterial strain has acidproof, thermotolerance by force, produces the feature that enzyme activity is high.
2, this bacterial strain is had to produce the Thermostable α-Amylase enzyme activity of gained up to 30000-35000u/ml; Applicable temperature scope is 25-115 DEG C, optimal reactive temperature 110 DEG C, at 110 DEG C of enzymes complete stability alive; Being suitable for pH value in reaction scope is 3.0-7.0, the enzyme complete stability alive when pH value is 3.0, optimal reaction pH value is 4.2, higher than existing Thermostable α-Amylase enzyme activity, enzyme effect optimum pH wide scope, resistance to temperature is high, is particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
Embodiment 1
There is provided a kind of Green microecological compound fertilizer: parts by weight consist of: aspergillus niger culture 6 parts, colloid bud pole bacteria agent 5 parts, phosphorus decomposing Bacillus megaterium microbial inoculum 5 parts, subtilis culture 10, Aspergillus awamori culture 6, plant lactobacillus agent 3 parts.
The bacterial classification adopted is as follows:
Subtilis (Bacillus subtilis subsp) CGMCC7926
Plant lactobacillus (Lactobacillus plantarum) CICC20764
Aspergillus awamori (Aspergillus awamori) CGMCC3.6484
Aspergillus niger (Aspergillus niger) Li-2013-03 preserving number is CGMCC NO.7927.
The preparation method of Aspergillus awamori microbial inoculum:
Technical scheme is as follows:
Slant strains activation culture: be transferred on slant medium by Aspergillus awamori slant strains, cultivates 3 days for 27 DEG C.
Solid first order seed is cultivated: the access of picking Aspergillus awamori slant strains is equipped with in 500 milliliters of triangular flasks of 100 grams of substratum and is carried out seed culture, cultivates 3 days for 30 DEG C.
Solid secondary seed is cultivated: stirred by above-mentioned cultured solid first order seed and carry out seed culture, culture condition for adding in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed after fragment: cultivate 3 days for 30 DEG C.
Solid fermentation is cultivated: pulverized by second-level shake flask seed, add in the fermentation vat or pallet that sterilising medium is housed and mix rear cultivation, and bent material culture temperature controls at 26-35 DEG C, humidity 80-90%, every 10 hours stirrings once, and incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat culture material cover with mycelia can terminate cultivate, substratum in advance through high temperature steaming sterilising treatment, sterilising conditions control temperature 121 DEG C, 1 hour time.
Drying and crushing: fermentation ends culture material carries out drying on fluidized-bed or other drying plants, drying temperature controls, at 60 DEG C, to be dried to moisture content below 10%, then to be pulverized by solid culture medium, and crushing material aperture is more than 60 orders.
Substratum forms: solid material: wheat bran 80%, soybean cake powder 10%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
The preparation method of subtilis culture:
1. the acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid;
(1) first order seed is cultivated: subtilis slant strains accessed in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05Mpa, incubation time 24 hours;
(5) fermentation culture: it is 1.5 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 10% inoculum size, fermention medium loading amount 1 ton, culture condition culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05Mpa, incubation time 24 hours.
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO31%, pH6.8.
The preparation method of plant lactobacillus agent:
(1) first order seed is cultivated: accessed by plant lactobacillus bacterial classification in 500 ml shake flasks, substratum loading amount 100 milliliters, culture temperature 30 DEG C, incubation time 24 hours;
(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 5% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, tank pressure 0.05Mpa, incubation time 18 hours;
(5) fermentor cultivation: it is 3 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 5% inoculum size, fermention medium loading amount 2 tons, culture condition culture temperature 30 DEG C, tank pressure 0.05Mpa, incubation time 22 hours.The complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% of original volume, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin.Fluidised bed drying, drying temperature 50 DEG C.
Substratum consists of: casein peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 0.5%, sodium acetate 0.5%, citric acid diamines 0.2%, Tween800.1%, K2HPO40.2%, MgSO4.7H2O0.02%, MnSO4.H2O0.005%, CaCO32%, agar 1.5%, pH6.8.
Embodiment 2
Basic with embodiment 1
There is provided a kind of Green microecological compound fertilizer: parts by weight consist of: aspergillus niger culture 5 parts, colloid bud pole bacteria agent 7 parts, phosphorus decomposing Bacillus megaterium microbial inoculum 6 parts, subtilis culture 12, Aspergillus awamori culture 6, plant lactobacillus agent 2 parts.
Product effect experimental
Selection experimental field and test design: test and carry out in Ba Bao village, dapple pond town, Yanchi county Ningxia 20 days-September 30 March in 2009.
Experimental plot reaches field maize planting 10 mu, using product every mu of the present invention 0.7 kilogram respectively, emerging about 1 month and using invention product 0.6 kilogram by loose ground mode when planting, and control group uses common fertilizer.
Invention product uses corn field corn yield to reach 650 kilograms, and control group reaches 510 kilograms; This plot was at the 2nd year plant spring wheat, and spring wheat production reaches 400 kilograms, improves 20% than control group per unit area yield.And experimental plot Soil structure is good, without bulk with harden.