CN1498962A - 'Tabin' aspergillus capable of producing pectase in high yield, and application in production of solid-state fermentation - Google Patents
'Tabin' aspergillus capable of producing pectase in high yield, and application in production of solid-state fermentation Download PDFInfo
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- CN1498962A CN1498962A CNA021467749A CN02146774A CN1498962A CN 1498962 A CN1498962 A CN 1498962A CN A021467749 A CNA021467749 A CN A021467749A CN 02146774 A CN02146774 A CN 02146774A CN 1498962 A CN1498962 A CN 1498962A
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Abstract
A novel strain of Aspergillus tubingensis able to prepare the pectase with high output and its application in the solid-state fermentation of pectase are disclosed. The resultant pectase has high enzyme activity (more than 250 u/g at pH=2.5-4.2, or more than 120 u/g at pH=5.5).
Description
(1) technical field
But new bacterial strain of the Tabin aspergillus (Aspergillustubingensis Moss) that the present invention relates to a kind of high yield polygalacturonase and the application in solid-state fermentation of pectase thereof.
(2) background technology
In recent decades, because increasing sharply of population and improving constantly of standard of living, the demand of poultry and egg is also being improved year by year.Feed is the food of livestock and poultry, how to improve the utilization ratio of feed and animal growth performance, how to protect environment and ensure that the continuing of husbandry, healthy, steady progression have become the major issue that the mankind press for solution.
Complex enzyme for feed by microbial fermentation production is nontoxic, harmless, the pollution-free residual green feed additive of a class, adds an amount of zymin that is complementary with feed diet and can obviously improve efficiency of feed utilization in feed, reduces the pollution to environment.
Polygalacturonase is a kind of important zymin that is used for feed, can obviously improve the digestive utilization ratio of feed, can also reduce the mortality ratio of livestock and poultry simultaneously.Can play the effect that substitutes duomycin in early days in animal rearing.
Yet, so far for this reason, also do not have a kind of good microorganism strains that is used for the polygalacturonase preparation.The at present industrial microorganism strains that is used for the polygalacturonase preparation mainly is an aspergillus niger, before 1993, a lot of about the aspergillus niger research report of high yield polygalacturonase, limited but the fermenting enzyme vigor rises, maximum is (pH5.5 measured value) about 100u/g, basic later on not breakthrough.But so industrial new bacterial strain of microorganism that presses for a kind of high yield polygalacturonase.
(3) summary of the invention
[problem that will solve]
But the new bacterial strain of the microorganism that the purpose of this invention is to provide a kind of high yield polygalacturonase and this bacterial strain application in solid-state fermentation of pectase is produced.
[technical scheme]
The inventor is separated to a strain aspergillus tubigensis from mouldy orange skin, as starting strain, adopts mutagenic compound to handle somatic cells with this, has obtained new bacterial strain of the present invention.Concrete mutagenic and breeding process is as follows:
1. the set out screening of bacterium
Making concentration from mouldy orange skin sampling is 10
-2, 10
-3, 10
-4, 10
-5, 10
-6With 10
-7Bacteria suspension, be seeded on the solid potato agar substratum, (solid potato agar substratum proportioning is: peeling potato 100-130 gram, casein food grade 4-6 gram, wheat-flour 15-24 gram, agar 16-20 gram, distilled water 1000ml, penicillin 3-6ppm, pH 6.8-7.0,121 ℃ autoclaving 15-20 minute) 28-32 ℃ constant temperature culture 5 days, 10
-5Separate on the dilution agar plate and obtain 1 strain aspergillus.As starting strain, carry out follow-up mutagenic and breeding.
2. mutagenic and breeding
The above-mentioned bacterium that sets out is carried out mutagenesis screening and shake-flask culture, its enzymatic productivity of determination and analysis.Main analysis indexes is a polygalacturonase.
The mutagenic and breeding process: starting strain → ultraviolet mutagenesis → gamma-ray and mutagenesis → NTG is handled the single bacterium colony of spore → select.
The screening of bacterium colony and product enzyme performance are measured according to following method:
Select 30-50 single bacterium colony, do and shake the pipe test, measure enzymatic productivity → selection 2-3 strain, shake flask test-→ selection 1 strain.
The power of ultraviolet lamp is 15W, and irradiation distance is 20cm, and irradiation time is 10 minutes.Prepare the pityrosporion ovale suspension with stroke-physiological saline solution, the spore count of bacteria suspension is 10
6-10
7Between individual/ml, the thickness of liquid layer is 0.3-0.5cm.Irradiation back tranquillization in enrichment liquid was cultivated 6-10 hour, and dilution is coated on the potato solid nutrient agar then, 28-32 ℃ of constant temperature culture 5 days, selected single bacterium colony, was used for next step mutagenesis.
Gamma-rays derives from Co
60, the spore count of pending bacteria suspension is 10
5-10
6Individual/ml, irradiation dose is 600-800 Curie.Dilution immediately is coated on the potato solid nutrient agar after the mutagenesis, 28-32 ℃ of constant temperature culture 5 days, selects single bacterium colony, is used for next step mutagenesis.
The concentration of NTG medicine mutagenic compound is 150-200ppm, and the spore count of pending bacteria suspension is 10
5-10
6Individual/ml, be 20-30 minute action time, and operative temperature is 30-40 ℃.After the mutagenesis in enrichment liquid tranquillization cultivated 6-10 hour, dilution is coated on the potato solid nutrient agar then, 28-32 ℃ of constant temperature culture 5 days, selects single bacterium colony, is used for next step mutagenesis.
The high yield bacterium that obtains with first round circulation is as the bacterium that sets out, carry out complex mutation, screening once more, so repeat 3 times, but the tens of strains of mutant strain of high yield polygalacturonase have finally been obtained, bacterial strain Mafic-003 preferably wherein, the pectinase activity of its solid state fermentation dry medium is 120u/g (pH is 5.5).In the pH value is the scope of 2.5-4.2, the polygalacturonase stable performance, enzyme activity remains on more than the 250u/g.
Solid slant culture based formulas: NaNO
30.2-0.4%, KCl 0.03-0.06%, KH
2PO
40.08-0.12%, FeSO
40.001-0.002%, MgSO4 0.03-0.06%, sucrose 2.0-3.5%, agar 1.8--2.0%, pH value 6.6-6.8.Culture temperature 28--30 ℃, incubation time 5-6 days.Spore is black, slant strains preservation under 4 ℃ of conditions, and switching in 6 months is once.
Enrichment culture medium prescription: NaNO
30.2-0.4%, KCl 0.03-0.06%, KH
2PO
40.08-0.12%, FeSO
40.001-0.002%, MgSO
40.03-0.06%, sucrose 2.0-3.5%, pH value 6.6-6.8.
Regulation according to patent law of china and its detailed rules for the implementation, Tabin aspergillus of the present invention (Aspergillus tubingensis Moss) the new bacterial strain of Mafic-003 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 20th, 2002, and preserving number is CGMCC 0826.
, find that all these high productive mutants all have following feature by but the bacterial strain of tens of strain high yield polygalacturonases is studied:
A. bacterium colony is grown on the Cha Shi substratum rapidly, 25 ℃ of 6 days diameter 45-60 millimeters, and smooth or approaching smooth, the edge has the mycelium of white, bacterium colony back side band yellow.
B. quality velvet shape or be with cotton-shapedly slightly, conidium is held back black.
C. conidiophore betides matrix, and the falx stem is yellow or Huang is held back look.
Therefore, but bacterial strain of the present invention comprises the microorganism strains that belongs to Tabin aspergillus (Aspergillustubinensis Moss) of high yield polygalacturonase.Especially the bacterial strain that has following feature:
A. bacterium colony is grown on the Cha Shi substratum rapidly, 25 ℃ of 6 days diameter 45-60 millimeters, and smooth or approaching smooth, the edge has the mycelium of white, bacterium colony back side band yellow.
B. quality velvet shape or be with cotton-shapedly slightly, conidium is held back black.
C. conidiophore betides matrix, and the falx stem is yellow or Huang is held back look.
But microorganism strains of the present invention also comprises Tabin aspergillus mutant, varient or its various offsprings of the high yield polygalacturonase of described bacterial strain.The most preferred bacterial strain of the present invention is that the most preferred bacterial strain of the present invention is CGMCC 0826.
The present invention has adopted following method in the mensuration of carrying out pectinase activity (pH 5.5):
1. enzyme is lived and is defined
Under experiment condition, decomposing the needed enzyme amount of release 1 μ mol reducing substance (being expressed as galacturonic acid) in the per minute from substrate solution is the enzyme u of unit alive.
2. reagent
[damping fluid]
Take by weighing sodium acetate trihydrate 23.14g (being accurate to 0.01g), add glacial acetic acid 1.7ml (being accurate to 0.01ml).Be dissolved in water again, be settled to 2000ml.Measure the pH value of solution,, be adjusted to 5.5 with the acetic acid solution of 0.1mol/L or the sodium acetate solution of 0.1mol/L again if the pH value departs from 5.5.
[substrate solution]
Take by weighing 1.00 gram pectin (Fluka76280 or Sigma P9135), with the damping fluid dissolving of pH5.5, magnetic agitation 2 hours is settled to 100ml.Be stored in 4 ℃ of refrigerators validity period 2 days.
[DNS reagent]
With 400ml dissolved in distilled water 3.15 grams 3,5--dinitrosalicylic acid, progressively add 200mlNaOH solution (containing 20 gram NaOH), constantly stir simultaneously.Warm water bath (being no more than 48 ℃) is constantly stirred simultaneously, and is transparent limpid up to solution.Progressively add 91 gram Rochelle salt, 2.5g phenol and 2.5g sodium sulphite anhydrous 99.3s again, constantly stir simultaneously.Warm water bath (being no more than 48 ℃), as clear as crystal up to solution.Be settled to 1000ml with distilled water, filter with fritted glass filter.Get filtrate, be kept in the brown bottle.Can use after 5 days, validity period is 6 months.
3. typical curve
Preparation standard one water D-galacturonic acid solution.
Dissolving 1.0 grams one water galacturonic acid is settled to 100ml, and obtaining concentration is the galacturonic acid solution of 10mg/ml.Make serial dilution with damping fluid then, compound concentration is 0, the standardized solution of 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml and 0.7mg/ml.Draw 2.0ml standardized solution and 2.0ml distilled water, join in the test tube.Concussion adds 4mlDNS reagent, concussion, boiling 5 minutes.Cool off with tap water rapidly then.Be settled to 20ml with distilled water again.Concussion shakes up, with No. 6 filter paper filterings of Whatman.Get filtrate, with concentration be 0 reaction solution (the blank sample of standard) as benchmark, zeroing.Measure absorbancy at the 540nm place.Measured value is done 2 repetitions, averages.Each DNS reagent of changing, typical curve need repaint.
4. enzyme activity determination
Accurately take by weighing 1.000 gram samples with damping fluid lytic enzyme sample, be diluted to proper concn.
[enzyme sample absorbance A
X]
Draw 2.0ml enzyme diluent, join in the test tube, add 2.0ml pectin substrate solution, 40 ℃ of balances, concussion.40 ℃ of accurate water-baths 30 minutes.Add 4mlDNS reagent, mix.Cover test tube with cap stopper.In boiling water, accurately be incubated 5 minutes.Then, cool off with termination reaction with tap water rapidly.Redistilled water is settled to 20ml.With Whatman 6 filter paper filterings or under the 3000rpm condition centrifugal 10 minutes.Getting supernatant liquor, is benchmark with the blank sample of standard, zeroing, and measure absorbancy (absorbancy if surpass this scope, redefines extent of dilution between 0.15-0.7 at the 540nm place.)。Measured value is done 2 repetitions, averages.(relative error between the repetition values is no more than 8.0%, otherwise redeterminates.)
[the blank sample absorbance A of enzyme
O]
Draw the 2.0ml pectin solution, accurately be incubated 30 minutes at 40 ℃.Add 4mlDNS reagent, concussion.The diluent that adds the 2.0ml enzyme mixes.Accurate boiling is 5 minutes in boiling water.Cool off with termination reaction with tap water rapidly then.Be settled to 20ml with distilled water,, get supernatant liquor with Whatman 6 filter paper filterings or under the 3000rpm condition centrifugal 10 minutes.With the blank sample of standard is benchmark, and absorbancy is measured in zeroing at the 540nm place.Measured value is done 2 repetitions, averages.
5. enzyme work is calculated
Enzyme A=[A alive
X-A
O] * K * 1000 * D
f/ W * t (u/g)
A
X: the absorbancy of enzyme sample;
A
O: the absorbancy of the blank sample of enzyme;
K: the slope of typical curve;
1000: conversion factor 1mmol=1000 μ mol;
D
f: extension rate;
W: the molecular weight of a water galacturonic acid (212.16g/mol);
T: the reaction times (minute).
Microorganism strains of the present invention can be used for the solid state fermentation of polygalacturonase and produce.The solid state fermentation of polygalacturonase is used for strain fermentation substratum of the present invention and is not particularly limited in producing, and all is applicable to bacterial strain of the present invention as long as contain the substratum of assimilable carbon source, nitrogenous source and essential growth factor.Can be used to cultivate microorganism of the present invention nutrition source comprise carbon source, nitrogenous source, inorganic salt and trace nutrient, wherein carbon source comprises D-glucose, D-fructose, D-sorbyl alcohol, D-seminose, amylum hydrolysate of the sugar, starch etc.; Nitrogenous source comprises organonitrogen and inorganic nitrogen, and organonitrogen such as corn steep liquor, yeast extract paste, dry yeast, fish meal, meat extract, peptone etc., inorganic nitrogen comprise ammoniacal liquor, ammonia, sulfuric acid amine, ammonium nitrate, ammonium chloride, amine carbonate, phosphamide etc.; In addition, also contain each metal ion species, VITAMIN, amino acid and necessary other materials of peculiar microorganism growth in the substratum.These compositions can disposable in advance adding substratum in, perhaps intermittently or add in the substratum continuously.
For microorganism strains of the present invention, preferably can adopt following solid-state fermentation culture medium prescription: wheat bran 90%, ammonium phosphate 4.0%, beet pulp 4.0%, orange skin powder 2.0%.Ammonium phosphate is dissolving earlier, mixes with beet pulp, orange skin powder and wheat bran then, regulates the pH value between the 6.5-7.0.
Solid ferment process comprises sterilization, inoculation, and triangular flask constant temperature culture, culture temperature are 30-35 ℃.After fermentation proceeded to 30 hours, the speed of growth of thalline descended gradually, and the fermentation heat production also reduces, and thalline begins to produce spore, and enzyme is lived, and beginning is rapid to be increased.Ferment after 48 hours, the rate of growth that enzyme is lived descends gradually, and enzyme is lived after 60 hours increases seldom.
[beneficial effect]
With the stable performance in the pH value is the scope of 2.5-4.2 of the polygalacturonase of bacterial strain production of the present invention, enzyme activity (40 ℃) can be stablized and remains on more than the 250u/g.Be that enzyme activity (40 ℃) reaches more than the 120u/g under 5.5 the condition in the pH value.Fermentation time is about 60 hours, and production cost is no more than 8 yuan/kg (dry medium).
Adopt bacterial strain of the present invention to carry out solid state fermentation and prepare polygalacturonase and have less investment, characteristics such as easy to operate.Calculate with unit volume, the vigor of solid state fermentation polygalacturonase than the high 5-6 of liquid state fermentation doubly.Raw materials for production mainly are agricultural byproducts such as wheat bran, orange skin powder and beet pulp.
The present invention is the breach with seed selection complex enzyme for feed high-yield strains, adopts the solid state fermentation that tallies with the national condition to carry out large-scale production; Not only help improving the fodder industry economic benefit, also help preserving the ecological environment simultaneously, promote lasting, healthy, the development stably of China's livestock industry.
(4) embodiment
Further specify the present invention with non-limiting example below.
Embodiment 1
Get one of Tabin aspergillus (Aspergillus tubingensis Moss) CGMCC 0826 slant strains, add the 5ml sterilized water, washing tower guest aspergillar spore.Draw spore liquid then, be inoculated in the 50g seed culture medium of forming by wheat bran (96%), ammonium sulfate (2.0%) and potassium primary phosphate (2.0%) (pH6.0, water content are 50%), cultivate after 48 hours for 30 ℃, in the 2kg solid medium that access is made up of wheat bran (90%), ammonium sulfate (2.0%), potassium primary phosphate (2.0%) and orange skin powder (6.3%) (pH6.8, water content is 60%).Cultivated 58 hours down at 28 ℃, pectinase activity reaches 122.4u/g in the substratum.
Embodiment 2
Get one of Tabin aspergillus (Aspergillus tubingensis Moss) CGMCC 0826 slant strains, add the 10ml sterilized water, washing tower guest aspergillar spore.Draw spore liquid 5ml then, be inoculated in the 50g seed culture medium of forming by wheat bran (96%), ammonium sulfate (2.0%) and potassium primary phosphate (2.0%) (pH6.0, water content are 50%), cultivate after 48 hours for 30 ℃, in the 2kg solid medium that access is made up of wheat bran (90%), ammonium sulfate (2.0%), potassium primary phosphate (2.0%), orange skin powder (2.0%) and beet pulp (4.0%) (pH6.7, water content is 60%).Cultivated 60 hours down at 28 ℃, pectinase activity reaches 132.2u/g in the substratum.
Embodiment 3
Get one of Tabin aspergillus (Aspergillus tubingensis Moss) CGMCC 0826 slant strains, add the 5ml sterilized water, washing tower guest aspergillar spore.Draw spore liquid 3ml then, be inoculated into the 20g solid medium (pH6.8 that forms by wheat bran (90%), ammonium sulfate (2.0%), potassium primary phosphate (2.0%), orange skin powder (2.0%) and beet pulp (4.0%), water content is 50%) in, substratum is contained in the 250ml triangular flask 30 ℃ and cultivates after 62 hours, and pectinase activity reaches 120.5u/g in the substratum.
Claims (5)
1. but the microorganism strains that belongs to Tabin aspergillus (Aspergillus tubingensisMoss) of a high yield polygalacturonase.
2. according to the bacterial strain of claim 1, it has following feature:
A. bacterium colony is grown on the Cha Shi substratum rapidly, 25 ℃ of following 6 days diameter 45-60 millimeters, and smooth or approaching smooth, the edge has the mycelium of white, bacterium colony back side band yellow;
B. quality velvet shape or be with cotton-shapedly slightly, conidium is held back black;
C. conidiophore betides matrix, and the falx stem is yellow or Huang is held back look.
3. press the bacterial strain of claim 2, but it is Tabin aspergillus mutant, varient or its various offsprings, the derivative of high yield polygalacturonase.
4. by the bacterial strain of claim 2, it is Tabin aspergillus CGMCC 0826.
5. the application of arbitrary bacterial strain on solid-state fermentation of pectase of claim 1 to 4 is characterized in that described bacterial strain is used for the solid state fermentation production of polygalacturonase.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102998269A (en) * | 2012-11-29 | 2013-03-27 | 武汉新华扬生物股份有限公司 | DNS (dinitrosalicylic acid) detection method for fodder pectinase |
| CN103114046A (en) * | 2013-03-01 | 2013-05-22 | 淮海工学院 | Aspergillus tubingensis SZX-6 and fermentation liquid, fermentation liquid extract and application thereof |
| CN104745559A (en) * | 2015-04-09 | 2015-07-01 | 广西靖西梁鹏食品有限公司 | Pectinase for hawthorn fruit wine and preparation method thereof |
| CN105907649A (en) * | 2016-05-23 | 2016-08-31 | 广西中烟工业有限责任公司 | Fungus for degrading pectin and cellulose in tobacco stems and application of fungus |
| CN106222096A (en) * | 2016-08-18 | 2016-12-14 | 滨州学院 | One strain Tabin aspergillus bacterium CT1 and the application in terms of the phosphorus decomposing of salt-soda soil thereof |
| CN106434600A (en) * | 2016-09-28 | 2017-02-22 | 集美大学 | Method for preparing pectinesterase through fermentation of aspergillus tubingensis |
| CN107048472A (en) * | 2017-05-26 | 2017-08-18 | 河南中烟工业有限责任公司 | Porous ceramics immobilization pectase and its application in reconstituted tobacoo process |
| CN107151631A (en) * | 2017-03-16 | 2017-09-12 | 西北农林科技大学 | A kind of high yield pectase bacterial strain and its application |
-
2002
- 2002-11-08 CN CN 02146774 patent/CN1229490C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102998269A (en) * | 2012-11-29 | 2013-03-27 | 武汉新华扬生物股份有限公司 | DNS (dinitrosalicylic acid) detection method for fodder pectinase |
| CN103114046A (en) * | 2013-03-01 | 2013-05-22 | 淮海工学院 | Aspergillus tubingensis SZX-6 and fermentation liquid, fermentation liquid extract and application thereof |
| CN104745559A (en) * | 2015-04-09 | 2015-07-01 | 广西靖西梁鹏食品有限公司 | Pectinase for hawthorn fruit wine and preparation method thereof |
| CN105907649A (en) * | 2016-05-23 | 2016-08-31 | 广西中烟工业有限责任公司 | Fungus for degrading pectin and cellulose in tobacco stems and application of fungus |
| CN106222096A (en) * | 2016-08-18 | 2016-12-14 | 滨州学院 | One strain Tabin aspergillus bacterium CT1 and the application in terms of the phosphorus decomposing of salt-soda soil thereof |
| CN106222096B (en) * | 2016-08-18 | 2018-02-23 | 滨州学院 | One plant of Tabin aspergillus bacterium CT1 and its application in terms of the phosphorus decomposing of salt-soda soil |
| CN106434600A (en) * | 2016-09-28 | 2017-02-22 | 集美大学 | Method for preparing pectinesterase through fermentation of aspergillus tubingensis |
| CN106434600B (en) * | 2016-09-28 | 2019-08-09 | 集美大学 | A kind of method of Tabin aspergillus fermenting and producing pectinesterase |
| CN107151631A (en) * | 2017-03-16 | 2017-09-12 | 西北农林科技大学 | A kind of high yield pectase bacterial strain and its application |
| CN107048472A (en) * | 2017-05-26 | 2017-08-18 | 河南中烟工业有限责任公司 | Porous ceramics immobilization pectase and its application in reconstituted tobacoo process |
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| Publication number | Publication date |
|---|---|
| CN1229490C (en) | 2005-11-30 |
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Assignee: Beijing Longke Fangzhou Biological Engineering Technology Center Assignor: China Agricultural University Contract fulfillment period: 2007.11.1 to 2015.12.31 contract change Contract record no.: 2008110000140 Denomination of invention: 'Tabin' aspergillus capable of producing pectase in high yield, and application in production of solid-state fermentation Granted publication date: 20051130 License type: Exclusive license Record date: 20081128 |
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