CN1884478A - Aspergillus niger variant and its fermentation process in solid medium - Google Patents

Aspergillus niger variant and its fermentation process in solid medium Download PDF

Info

Publication number
CN1884478A
CN1884478A CN 200610052486 CN200610052486A CN1884478A CN 1884478 A CN1884478 A CN 1884478A CN 200610052486 CN200610052486 CN 200610052486 CN 200610052486 A CN200610052486 A CN 200610052486A CN 1884478 A CN1884478 A CN 1884478A
Authority
CN
China
Prior art keywords
enzyme
substratum
spore
micron
water
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN 200610052486
Other languages
Chinese (zh)
Inventor
许尧兴
许少春
李艳丽
姚晓红
李孝辉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Academy of Agricultural Sciences
Original Assignee
Zhejiang Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Academy of Agricultural Sciences filed Critical Zhejiang Academy of Agricultural Sciences
Priority to CN 200610052486 priority Critical patent/CN1884478A/en
Publication of CN1884478A publication Critical patent/CN1884478A/en
Pending legal-status Critical Current

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates the aspergillus Niger variant, and spore is ochre. The bacterial comprises the following biology characters: the bacterial is on the culture medium to grow for 7 days at 25Deg.C, the diameter is 45-70mm; the texture is villiform and granular; conidiospore is light brown; the reverse surface of bacterial colony is yellow. Using the aspergillus Niger variant, and adopting solid invoice method, the alpha-galactosidase is made. The enzymatic activity is 286IU/g, and the maximum is 345IU/g.

Description

A kind of black versicolor variety and the zymotechnique on solid state substrate thereof
Technical field
The present invention relates to biological technical field, particularly relate to a kind of black versicolor variety and the zymotechnique on solid state substrate thereof.
Background technology
Alpha-galactosidase is the enzyme that a kind of catalysis alpha galactosides class material decomposition becomes monose such as oligosaccharide, sucrose and fructose, extensively is present in microorganism, plant, the animal.Alpha-galactosidase has very at food, medicine industry everyway, and important use is worth.In beet sugar manufacture industry, the raffinose in the molasses decomposes through alpha-galactosidase, can not only improve productive rate, can also improve and raise the efficiency.In addition, pharmaceutically with in the modification of polysaccharide gum also having a wide range of applications.Recent study shows that this enzyme also has a wide range of applications in fodder industry as the feeding enzyme of a specific specificity external source.The existence of higher alpha galactosides class material is arranged in beans seed and grouts feed, this class material has not only reduced the nutritive value of feed, and causes easily that in the animal hindgut chyme is detained, and causes enteron aisle flatulence, digestive disorders causes diseases such as pancreas increase, diarrhea.In the feed that with soya-bean cake, the assorted dregs of rice etc. is daily ration, add alpha-galactosidase and other enzymes synergy, can effectively improve the utilization ratio of feed, eliminate the anti-oxidant action that causes by alpha galactosides class material.Seed selection and dynamical zymotechnique with highly active alpha-galactosidase production bacterium provide cheap alpha galactosides zymin, make this product carry out the crucial prerequisite that industrialization is produced.
Summary of the invention
The present invention will solve the defective of above-mentioned prior art, and a kind of black versicolor variety and the zymotechnique on solid state substrate thereof are provided.
The present invention solves the technical scheme that its technical problem adopts.Of the present invention is a kind of black versicolor variety (Aspergillusnuger v.Tiegh), and the spore tawny is preserved in China Committee for Culture Collection of Microorganisms on June 24th, 2004, and its preserving number is CGMCC No.1182.
The biological characteristics of the black versicolor variety of high reactivity alpha-galactosidase provided by the invention is, the bacterium colony appropriateness of growing on the Cha Shi substratum, 25 ℃, 7 days, diameter 45-70mm; Quality fine hair shape or be with particulate state slightly; The conidium structure is a large amount of, and is light brown, no transudate; The bacterium colony reverse side is slightly yellow.The conidial head sphere is to radiation shape, diameter 150-450 micron; Conidiophore betides matrix, falx stem 1000-3000 * 12-20 micron, and yellow or tawny, wall is level and smooth; The top capsule is spherical or subsphaeroidal, diameter 40-70 micron, and all surfaces can be educated; The conidial fructification bilayer, metulae 10-20 * 4.5-7.0 micron, bottle stalk 6-9 * 2.5-3.0 micron, conidium is spherical or subsphaeroidal, diameter 3-4.5 micron, wall is coarse.
Black versicolor variety provided by the invention adopts solid-state fermentation process, can produce highly active alpha-galactosidase.This zymotechnique has starting material and is easy to get, and technology is simple, the characteristics of enzymic activity height, unit enzyme low production cost alive.Can effectively decompose the alpha galactosides class material that is rich in the grouts feed, improve livestock and poultry production performance.
The mutagenic and breeding process of black versicolor variety of the present invention is:
1. the selection of starting strain
Our unit preserves bacterial classifications such as some aspergillus niger strains and some absidia rasmosas of introducing from Institute of Micro-biology of the Chinese Academy of Sciences, soil institute of the Chinese Academy of Sciences, Shanghai industrial microorganism, fly volume be mould and separate the bacterial classification of the different Pseudomonas that obtain from soil sample, on selective medium through primary dcreening operation, multiple sieve, it is the highest that the Aspergillus niger strain that obtains a strain code name and be R15# produces alpha-galactosidase activity, reaches the 83U/g butt.Selected this bacterial strain is as the starting strain of further mutagenic and breeding.
2. the complex mutation breeding of bacterial strain
To eat the fresh spore of the R15# bacterial strain of growth and maturity on agar glucose (PDA) inclined-plane at Ma Ling, wash with sterilized water, and (spore concentration be 10 to make monospore suspension 7-10 8Individual/ml).Get 2ml monospore suspension respectively in test tube, put chamber, cobalt source and carry out radiation treatment.Treatment dosage is 0 (ck), 3Mraed, 5Mraed, 7Mraedt 10Mraed.Through Co 60After the radiation treatment, draw the suspension that each is handled, progressively dilute 10 1, 10 2, 10 3, 10 4, 10 5, 10 6, 10 7Doubly, coat on the PDA plate culture medium, 3 flat boards of each extent of dilution, cultivate after 72-96 hour down for 28-30 ℃, the difform sporangium of picking falls within the PDA inclined-plane, after cultivating 72-96 hour under 28-30 ℃, carries out the primary dcreening operation of the triangular flask solid fermentation of mutagenesis bacterial classification, multiple sieve, obtain purpose bacterial strain RC32#, enzyme work reaches the 125U/g butt.
Further the RC32# bacterium is carried out repeatedly uviolizing and nitrosoguanidine complex mutation, obtain a strain black versicolor variety RM45#, inulinase-producing activity reaches the 175U/g butt.
Described slant preservation substratum: for Ma Ling eats agar glucose (PDA): peeling Ma Ling eats 20 grams and adds water 100ml after being cut into small pieces and boil 30min, after four layers of filtered through gauze, adds glucose 2 grams, agar 2 grams, and supply moisture to 100ml.
Described screening with the solid fermentation substratum is: wheat bran 85%, dregs of beans 13%, inorganic salt 2%.Adding water makes water ratio reach 55%-60%.
High reactivity alpha-galactosidase black versicolor variety of the present invention can use solid fermentation process, prepares dynamical alpha galactosides zymin and is applied to fodder industry or sugar industry.
The technical solution adopted for the present invention to solve the technical problems.Utilize high reactivity alpha-galactosidase black versicolor variety provided by the invention, preparation technology's flow process that its solid fermentation is produced alpha-galactosidase is:
1. black versicolor variety RM45# of the present invention is seeded on the PDA inclined-plane,, after ripe (growing plentiful spore), makes spore suspension with sterilized water in 28-30 ℃ of following constant temperature culture 72-96 hour, stand-by.
Described PDA inclined-plane is that Ma Ling eats glucose agar medium, and it consists of: peeling Ma Ling eats 20 grams and is cut into small pieces, and adds water 100ml and boils 30min, after four layers of filtered through gauze, adds glucose 2 grams, agar 2 grams, and supply moisture to 100ml.
2. will produce the sub-substratum of enzyme through 121 ℃, the 30min sterilization.After the cooling, press the 1-5% amount and insert the slant strains spore suspension, last postvaccinal solid seed culture medium was in 28-30 ℃ of following constant temperature culture 72-96 hour.After ripe (growing plentiful spore), inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain zymogenic bacteria kind spore suspension with four layers of gauze, stand-by.
The sub-substratum of described product enzyme is: wheat bran 99%, (NH 4) 2SO 41%, add water and make the substratum water ratio reach 55%-60%.
3. fermention medium is through 121 ℃, and the 30min sterilization after the cooling, is pressed 1-5% (V/W) inoculum size and inserted product enzyme spore suspension, and fully behind the stirring and evenly mixing, the 500ml triangular flask of packing into (every bottle of 20g) is cultivated 3d in 30 ℃; In the fermentation culture process, in cultivating 10h, every the 10h vibration once, 3-4 time continuously, static cultivation behind the 50h is until fermentation ends.
1), the product after the fermentation ends described aftertreatment is:, put 40 ℃, under the air blast condition, dry through 15-24h.2). the tunning after the oven dry, pulverize, cross 80 mesh sieves, get the enzyme dry medium.And by described enzymic activity test method detection.
Described fermention medium is: wheat bran 75%, bean cake powder 15%, fish meal 3%, (NH 4) 2SO 43%, produce enzyme inducer 4%, add water and make the substratum water ratio reach 55%-60%.
The effect that the present invention is useful is: utilize high reactivity alpha-galactosidase black versicolor variety provided by the invention, adopt above-mentioned solid fermentation method to produce alpha-galactosidase, the enzyme of its tunning (dry medium) is lived on average can reach 286IU/g, reach as high as 345IU/g, ratio is like product enzyme 44-56IU/g alive in the world, exceed 5 times, the product enzyme 126IU/g alive than the similar technology of domestic report is produced exceeds 2.26 times.
The preservation explanation
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center;
Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica (100080)
Preservation date: on June 24th, 2004
Deposit number (CGMCC NO): 1182
Classification name: aspergillus niger
Request preservation people: Zhejiang Academy of Agricultural Science plant protection and institute of microbiology
Embodiment
The invention will be described further below in conjunction with embodiment:
Embodiment 1:
One, spawn culture:
1, slant strains is cultivated: black versicolor variety RM45# of the present invention is seeded in Ma Ling eats on glucose agar medium (PDA) inclined-plane, in 28-30 ℃ of following constant temperature culture 72-96 hour, after waiting to grow plentiful tawny spore, (concentration is 10 to make spore suspension with sterilized water 7-10 8Individual/ml), stand-by.
2. solid seed culture: with the 1.5kg wheat bran with 1: the ratio of 1.2-1.5 adds 1% (NH 4) 2SO 4Solution fully behind the mixing, is made the solid seed culture medium.This substratum is through 121 ℃, and the 30min sterilization after the cooling, press the 1-5% amount and inserted the slant strains spore suspension, stirs, by every bottle of 500ml triangular flask substratum that 30 grams are moisture and inoculate of packing into.
3. postvaccinal solid seed culture medium was in 28-30 ℃ of following constant temperature culture 72 hours.After treating that substratum top layer and inside grow plentiful tawny spore, take out, put 4 ℃ of refrigerators, stand-by.
Two, producing enzymic fermentation cultivates:
1, gets the ripe stand-by solid seed triangular flask of cultivation, inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain the bacterial classification spore suspension with four layers of gauze.Being diluted to spore concentration with sterilized water again is 10 7-10 8The zymogenic bacteria kind spore suspension of individual/ml.
2. fermention medium is pressed wheat bran 75%, bean cake powder 15%, fish meal 3%, (NH 4) 2SO 43%, the ratio thorough mixing of product enzyme inducer 4% adds water and makes the substratum water ratio reach 55%-60%, is mixed with and produces enzymic fermentation substratum, the cloth bag of packing into and being made by two layers of cotton.This produces the enzymic fermentation substratum through 121 ℃, the 30min sterilization, after the cooling, press 1-5% (V/W) inoculum size and insert product enzyme spore suspension, fully behind the stirring and evenly mixing, packing into, (60cm * 40cm * 10cm), and be placed on the square plate with ventilative half device of preserving moisture is to control temperature, the humidity of culturing process for aseptic ventilative square plate.
3. postvaccinal square plate is placed 30 ℃ of cultivation constant temperatures to cultivate 65-72 hour down.In the fermentation culture process, in cultivating 10h, turn over Qu Yici every 10h, 3-4 time continuously, static cultivation behind the 50h is until fermentation ends.
Three, aftertreatment:
1. the product after the fermentation ends is put 40 ℃, under the air blast condition, dries through 15-24h.
2. the tunning after the oven dry is pulverized, and crosses 80 mesh sieves, gets the enzyme dry medium.And by described enzymic activity test method detection.
Four, result:
Every batch fermentation product is pressed sample quartering, and sample thief is three batches altogether, by alpha-galactosidase activity measuring method of the present invention, measures three batches enzyme and lives, and the result is as follows:
Batch enzyme live (IU/g)
No1 263
No2 251
No3 284
Average 266
Embodiment 2:
One, spawn culture:
1, slant strains is cultivated: black versicolor variety RM45# of the present invention is seeded in Ma Ling eats on glucose agar medium (PDA) inclined-plane, in 28-30 ℃ of following constant temperature culture 96 hours, after waiting to grow plentiful tawny spore, (concentration is 10 to make spore suspension with sterilized water 7-10 8Individual/ml), stand-by.
2. solid seed culture: with the 1.5kg wheat bran with 1: the ratio of 1.2-1.5 adds 1% (NH 4) 2SO 4Solution fully behind the mixing, is made the solid seed culture medium.This substratum is through 121 ℃, and the 30min sterilization after the cooling, press the 1-5% amount and inserted the slant strains spore suspension, stirs, by every bottle of 500ml triangular flask substratum that 30 grams are moisture and inoculate of packing into.
3. postvaccinal solid seed culture medium was in 28-30 ℃ of following constant temperature culture 72 hours.After treating that substratum top layer and inside grow plentiful tawny spore, take out, put 4 ℃ of refrigerators, stand-by.
Two, producing enzymic fermentation cultivates:
1, gets the ripe stand-by solid seed triangular flask of cultivation, inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain the bacterial classification spore suspension with four layers of gauze.Being diluted to spore concentration with sterilized water again is 10 7-10 8The zymogenic bacteria kind spore suspension of individual/ml.
2. fermention medium is pressed wheat bran 75%, bean cake powder 15%, fish meal 3%, (NH 4) 2SO 43%, the ratio thorough mixing of product enzyme inducer 4% adds water and makes the substratum water ratio reach 55%-60%, is mixed with and produces enzymic fermentation substratum, the cloth bag of packing into and being made by two layers of cotton.This produces the enzymic fermentation substratum through 121 ℃, 30mi n sterilization, after the cooling, press 1-5% (V/W) inoculum size and insert product enzyme spore suspension, fully behind the stirring and evenly mixing, packing into, (60cm * 40cm * 10cm), and be placed on the square plate with the air permeation device of preserving moisture is with temperature, the humidity of control culturing process for aseptic ventilative square plate.
3. postvaccinal square plate is placed 30 ℃ of cultivation constant temperatures to cultivate 65-72 hour down.In the fermentation culture process, in cultivating 10h, turn over Qu Yici every 10h, 3-4 time continuously, static cultivation behind the 50h is until fermentation ends.
Three, aftertreatment:
1. the product after the fermentation ends is put 40 ℃, under the air blast condition, dries through 15-24h.
2. the tunning after the oven dry is pulverized, and crosses 80 mesh sieves, gets the enzyme dry medium.And by described enzymic activity test method detection.
Four, result:
Every batch fermentation product is pressed sample quartering, and sample thief is three batches altogether, by alpha-galactosidase activity measuring method of the present invention, measures three batches enzyme and lives, and the result is as follows:
Batch enzyme live (IU/g)
No1 285
No2 345
No3 312
Average 314
Embodiment 3:
One, spawn culture:
1, slant strains is cultivated: black versicolor variety RM45# of the present invention is seeded in Ma Ling eats on glucose agar medium (PDA) inclined-plane, in 28-30 ℃ of following constant temperature culture 72-96 hour, after waiting to grow plentiful tawny spore, (concentration is 10 to make spore suspension with sterilized water 7-10 8Individual/ml), stand-by.
2. solid seed culture: with the 1.5kg wheat bran with 1: the ratio of 1.2-1.5 adds 1% (NH 4) 2SO 4Solution fully behind the mixing, is made the solid seed culture medium.This substratum is through 121 ℃, and the 30min sterilization after the cooling, press the 1-5% amount and inserted the slant strains spore suspension, stirs, by every bottle of 500ml triangular flask substratum that 30 grams are moisture and inoculate of packing into.
3. postvaccinal solid seed culture medium was in 28-30 ℃ of following constant temperature culture 72 hours.After treating that substratum top layer and inside grow plentiful tawny spore, take out, put 4 ℃ of refrigerators, stand-by.
Two, producing enzymic fermentation cultivates:
1, gets the ripe stand-by solid seed triangular flask of cultivation, inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain the bacterial classification spore suspension with four layers of gauze.Being diluted to spore concentration with sterilized water again is 10 7-10 8The zymogenic bacteria kind spore suspension of individual/ml.
2. fermention medium is pressed wheat bran 75%, bean cake powder 15%, fish meal 3%, (NH 4) 2SO 43%, the ratio thorough mixing of product enzyme inducer 4% adds water and makes the substratum water ratio reach 55%-60%, is mixed with and produces enzymic fermentation substratum, the cloth bag of packing into and being made by two layers of cotton.This produces the enzymic fermentation substratum through 121 ℃, the 30min sterilization, after the cooling, press 1-5% (V/W) inoculum size and insert product enzyme spore suspension, fully behind the stirring and evenly mixing, packing into, (60cm * 40cm * 10cm), and be placed on the square plate with ventilative half device of preserving moisture is to control temperature, the humidity of culturing process for aseptic ventilative square plate.
3. postvaccinal square plate is placed 30 ℃ of cultivation constant temperatures to cultivate 65-72 hour down.In the fermentation culture process, in cultivating 10h, turn over Qu Yici every 10h, 3-4 time continuously, static cultivation behind the 50h is until fermentation ends.
Three, aftertreatment:
1. the product after the fermentation ends is put 40 ℃, under the air blast condition, dries through 15-24h.
2. the tunning after the oven dry is pulverized, and crosses 80 mesh sieves, gets the enzyme dry medium.And by described enzymic activity test method detection.
Four, result:
Every batch fermentation product is pressed sample quartering, and sample thief is three batches altogether, by alpha-galactosidase activity measuring method of the present invention, measures three batches enzyme and lives, and the result is as follows:
Batch enzyme live (IU/g)
No1 279
No2 265
No3 296
Average 280
Adopt the method for the alpha-galactosidase activity in the p-NP method mensuration gained alpha galactosides zymin as follows:
One, enzyme unit definition alive:
Under pH4.0,40 ℃ of conditions, the enzyme amount that per minute degraded p-nitrophenol α-D-galactopyranoside discharges 1 μ mol p-nitrophenol is 1 alpha-galactosidase unit that lives, and abbreviates IU as.
Two, plant and instrument:
UV1100 ultraviolet-visible spectrophotometer (Beijing Rayleigh analytical instrument factory), thermostat water bath, acidometer (precision ± 0.01pH), analytical balance (sensibility reciprocal 0.1mg), stopwatch or timing watch.
Three, reagent:
1, p-nitrophenol (p-NPH) standardized solution:
Accurately take by weighing p-nitrophenol (be called for short p-NPH, Sigma company produces, lot number: 117H5057) 100mg, use dissolved in distilled water, and be settled to 100ml.Be mixed with concentration then and be respectively 0,10 μ g.mL -1, 20 μ g.mL -1, 30 μ g.mL -1, 40 μ g.mL -1, 50 μ g.mL -1The p-nitrophenol standardized solution to make typical curve.
2, p-nitrophenol-α-D-galactopyranose (p-NPG) solution (Fluka company produces, lot number WA13251):
Accurately take by weighing p-nitrophenol-α-D-galactopyranose 150.5mg, use dissolved in distilled water, be settled to 100ml.Freezing.
3,0.5M Na 2CO 3Solution: accurately take by weighing anhydrous sodium carbonate 53 grams, use dissolved in distilled water, be settled to 1000ml.
4, pH 4.0 Sodium phosphate dibasics-citrate buffer solution:
First liquid: take by weighing Sodium phosphate dibasic (Na 2HPO 4.2H 2O) 35.61g uses dissolved in distilled water, and is settled to 1000ml.
Second liquid: take by weighing citric acid (C 6H 8O 7.H 2O) 21.01g uses dissolved in distilled water, and is settled to 1000ml..
Use solution: get first liquid 385.5ml, add second liquid 614.5ml and mix, proofread and correct to pH with pH meter and be (4.0 ± 0.05), standby.
5, enzyme liquid preparation
Take by weighing enzyme dry medium 2.000 gram, adding distil water 40ml soaks 30min in 40 ℃, during stir 3-4 time.Be settled to 100ml with distilled water then, No. 1 filter paper filtering of usefulness Xinhua is got filtrate being used to and is measured enzyme work.
Four, measuring method:
1, the p-nitrophenol typical curve is drawn:
Draw the p-nitrophenol standardized solution 0.5ml (3 parallel pipes of every number work) of different concns respectively, add pH4.0 damping fluid 1.5ml, 0.5M Na 2CO 3Solution 2.0ml.Fully send out shake up after, replacing the test tube of p-nitrophenol standardized solution with 0.5Ma distilled water is blank, is the 400nm place in spectrophotometer in absorbancy, usefulness 5mm cuvette colorimetric.With absorbancy (OD value) is abscissa, is ordinate zou with p-nitrophenol concentration, drawing standard curve, or calculating K value.
2, determination step
The enzyme liquid 0.5ml to be measured that draws through suitably dilution adds pH4.0 damping fluid 1.0ml, and in 40 ℃ of water-bath preheating 5min, substrate is preheating simultaneously also.The p-NPG substrate 0.5ml that adds 5m mol/L in each test tube, timing immediately.Accurately reaction 10min adds 0.5M Na immediately 2CO 3Solution 2.0ml stopped reaction.Take out, in spectrophotometer 400nm place colorimetric. (using the 5mm cuvette).Blank pipe promptly adds 2ml 0.5M Na after adding enzyme liquid and damping fluid 2CO 3, reaction adds the 0.5ml substrate after finishing again.
1.3.2.3 calculate: survey ABS (OD) value that obtains by colorimetric, with the regression Calculation method, (or with K value) is calculated as follows alpha galactosides enzyme activity A2 (IU.g -1)
A = [ ( A x - A 0 ) × K + C 0 ] × N t × W × 139
In the formula: A x----sample absorbancy OD value, A 0----blank pipe absorbancy OD value,
K----p-nitrophenol slope of standard curve, C 0---p-nitrophenol typical curve intercept,
The total extension rate of N---sample, the t----reaction times,
W: take by weighing the weight (g) of zymin, 139----p-nitrophenol molecular weight.

Claims (4)

1, a kind of black versicolor variety is characterized in that, is a kind of black versicolor variety (Aspergillus nuger v.Tiegh) that the spore tawny is preserved in China Committee for Culture Collection of Microorganisms on June 24th, 2004, and its preserving number is CGMCCNo.1182.
2, high reactivity alpha-galactosidase black versicolor variety according to claim 1 is characterized in that, the biological characteristics of this high reactivity alpha-galactosidase black versicolor variety is: bacterium colony is grown appropriate on the Cha Shi substratum, and 25 ℃, 7 days, diameter 45-70mm; Quality fine hair shape or be with particulate state slightly; The conidium structure is a large amount of, and is light brown, no transudate; The bacterium colony reverse side is slightly yellow.The conidial head sphere is to radiation shape, diameter 150-450 micron; Conidiophore betides matrix, falx stem 1000-3000 * 12-20 micron, and yellow or tawny, wall is level and smooth; The top capsule is spherical or subsphaeroidal, diameter 40-70 micron, and all surfaces can be educated; The conidial fructification bilayer, metulae 10-20 * 4.5-7.0 micron, bottle stalk 6-9 * 2.5-3.0 micron, conidium is spherical or subsphaeroidal, diameter 3-4.5 micron, wall is coarse.
3, a kind of according to the zymotechnique of the described black versicolor variety of claim 1 on solid state substrate, it is characterized in that:
1), above-mentioned black versicolor variety is seeded on the PDA inclined-plane,, grow after plentiful spore is maturation, make spore suspension with sterilized water in 28-30 ℃ of following constant temperature culture 72-96 hour, stand-by; Wherein the PDA inclined-plane is that Ma Ling eats glucose agar medium, and it consists of: peeling Ma Ling eats 20 grams and is cut into small pieces, and adds water 100ml and boils 30min, after four layers of filtered through gauze, adds glucose 2 grams, agar 2 grams, and supply moisture to 100ml;
2), will produce the sub-substratum of enzyme through 121 ℃, the 30min sterilization; After the cooling, press the 1-5% amount and insert the slant strains spore suspension, last postvaccinal solid seed culture medium was in 28-30 ℃ of following constant temperature culture 72-96 hour; Grow after plentiful spore is maturation, inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain zymogenic bacteria kind spore suspension with four layers of gauze, stand-by; The sub-substratum of described product enzyme is: wheat bran 99%, (NH 4) 2SO 41%, add water and make the substratum water ratio reach 55%-60%;
3), fermention medium is through 121 ℃, the 30min sterilization after the cooling, press 1-5% (V/W) inoculum size and is inserted and produce the enzyme spore suspension, fully behind the stirring and evenly mixing, the 500ml triangular flask of packing into is in 30 ℃ of cultivation 3d; In the fermentation culture process, in cultivating 10h, every the 10h vibration once, 3-4 time continuously, static cultivation behind the 50h is until fermentation ends; Fermention medium wherein is: wheat bran 75%, bean cake powder 15%, fish meal 3%, (NH 4) 2SO 43%, produce enzyme inducer 4%, add water and make the substratum water ratio reach 55%-60%.
1), the product after the fermentation ends 4, according to the zymotechnique of the described black versicolor variety of claim 3 on solid state substrate, it is characterized in that: carry out postprocessing working procedures, be specially:, put 40 ℃, under the air blast condition, dry through 15-24h; 2), the tunning after the oven dry, pulverize, cross 80 mesh sieves, the enzyme dry medium; And by described enzymic activity test method detection.
CN 200610052486 2006-07-10 2006-07-10 Aspergillus niger variant and its fermentation process in solid medium Pending CN1884478A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200610052486 CN1884478A (en) 2006-07-10 2006-07-10 Aspergillus niger variant and its fermentation process in solid medium

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 200610052486 CN1884478A (en) 2006-07-10 2006-07-10 Aspergillus niger variant and its fermentation process in solid medium

Publications (1)

Publication Number Publication Date
CN1884478A true CN1884478A (en) 2006-12-27

Family

ID=37582785

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200610052486 Pending CN1884478A (en) 2006-07-10 2006-07-10 Aspergillus niger variant and its fermentation process in solid medium

Country Status (1)

Country Link
CN (1) CN1884478A (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899398B (en) * 2009-05-25 2012-05-30 中国石油化工股份有限公司 Aspergillusniger strain and application thereof
CN103330060A (en) * 2013-05-21 2013-10-02 张永根 Method for producing novel milk cow forage by mixed bacterium fermentation of potato slag
CN110066780A (en) * 2019-03-29 2019-07-30 上海国龙生物技术集团有限公司 A kind of feeding alpha-galactosidase solid fermenting production technology
CN110331115A (en) * 2019-08-08 2019-10-15 河南省科学院生物研究所有限责任公司 A kind of culture medium quickly screened for alpha-galactosidase producing strains and its application
CN110699263A (en) * 2019-10-29 2020-01-17 浙江工业大学 Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium
CN111036647A (en) * 2019-12-12 2020-04-21 安徽省农业科学院水产研究所 Freshwater fish viscera recycling method based on mixed strain solid state fermentation

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899398B (en) * 2009-05-25 2012-05-30 中国石油化工股份有限公司 Aspergillusniger strain and application thereof
CN103330060A (en) * 2013-05-21 2013-10-02 张永根 Method for producing novel milk cow forage by mixed bacterium fermentation of potato slag
CN110066780A (en) * 2019-03-29 2019-07-30 上海国龙生物技术集团有限公司 A kind of feeding alpha-galactosidase solid fermenting production technology
CN110331115A (en) * 2019-08-08 2019-10-15 河南省科学院生物研究所有限责任公司 A kind of culture medium quickly screened for alpha-galactosidase producing strains and its application
CN110699263A (en) * 2019-10-29 2020-01-17 浙江工业大学 Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium
CN111036647A (en) * 2019-12-12 2020-04-21 安徽省农业科学院水产研究所 Freshwater fish viscera recycling method based on mixed strain solid state fermentation
CN111036647B (en) * 2019-12-12 2021-07-27 安徽省农业科学院水产研究所 Freshwater fish viscera recycling method based on mixed strain solid state fermentation

Similar Documents

Publication Publication Date Title
CN101028006A (en) Production of trichodermin agricultural chemicals
CN102870600B (en) Cordyceps militaris fruit body cultivation technology for stabilizing cordycepin content
CN108865953A (en) One plant of wide spectrum inhibits bacillus and its composite bacteria preparation of aquatic products vibrio pathogen
CN1966670A (en) Bacillus laterosporus and soil inoculation agent prepared from the strain
CN1884478A (en) Aspergillus niger variant and its fermentation process in solid medium
CN1740327A (en) Animal plant and microbial cell wall lytic enzyme reactive liquid and its application
CN107602215A (en) A kind of gourd, fruit and vegetable plantation Nutrition Soil and preparation method thereof
CN1185336C (en) Li's Trichoderma strains and use thereof
CN110734346A (en) Fermentation process and preparation method of agricultural enzymes
CN1955276A (en) Organic microbial composite and use
CN103387428A (en) Preparation method for organic material decomposition agent
CN1163446C (en) Microbial streptomycete fertilizer and production process
CN101695255B (en) Method for cultivating cordyceps sinensis stroma by using hirsutella sinensis
CN1693445A (en) Black aspergillus mutation of high active alpha-galactosldase and its ferment tech. on solid substrate
CN101638645A (en) Method for producing xylanase by solid mechanical fermentation
CN102040403A (en) Production of trichoderma spp. microbial fertilizer by waste water of kitchen garbage
CN1498865A (en) Micro ecological agent for supporting water in fishing use and its prepn. method
CN1742607A (en) Micro-ecological feed additive and solid fermentation preparing technology
CN100999713A (en) Black aspergillus strain and preparation process of its NSP enzyme
CN1225172C (en) Batch production process of biological herbicide
Bankole et al. Use of agro-wastes for tissue culture process and spawn production of oyster mushroom (Pleurotus florida)
CN104620817B (en) A kind of Semen avenae nudae Biological control disease-resistant growth-promoting method
CN107227261A (en) A kind of edible fungi residue promotees rotten composite bacteria agent and preparation method thereof
CN1563352A (en) Zymogen agent for treating agricultural wastes and preparation method
TWI580779B (en) Solid matrix medium for antrodia cinnamomea and method of culturing antrodia cinnamomea

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C12 Rejection of a patent application after its publication
RJ01 Rejection of invention patent application after publication