CN100558880C - But the aspergillus oryzae strain of high yield neutral protease and the fermentation process on solid state substrate thereof - Google Patents
But the aspergillus oryzae strain of high yield neutral protease and the fermentation process on solid state substrate thereof Download PDFInfo
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Abstract
The present invention relates to a kind of aspergillus oryzae strain and its fermentation process on solid medium.The aspergillus oryzae strain of high yield neutral protease of the present invention (Aspergillus oryza) ZW-06 is preserved in China Committee for Culture Collection of Microorganisms on July 10th, 2006, and its preserving number is CGMCC No.1754.But aspergillus oryzae strain high yield neutral protease of the present invention, the neutral protease that is produced except the enzymic activity height, the trypsin inhibitor in the meal expeller and mealsolvent raw material of also can effectively degrading.
Description
Technical field
The present invention relates to a kind of aspergillus oryzae strain and the fermentation process on solid state substrate thereof.
Background technology
Neutral protease as a kind of biological catalyst be widely used in food, brewageed, industry such as medicine.The production of neutral protease also has comparatively permanent developing history.Yet the neutral protease of domestic production at present mostly is technical grade greatly, and spray-drying process is adopted in the aftertreatment of its tunning, have in addition also prolong with the sulphur ammonium salt and technology such as analyse.A spot of food grade neutral protease, because its manufacturing process complexity, price is comparatively expensive.At present, no matter be to be applied to the prozyme of animal and fowl fodder or to be applied to the neutral protease that neutral protease contained in the prozyme of aquatic feeds adopts technical grade substantially.And form and the high reactivity neutral protease report of the feed grade of exploitation is very few at animal physiological and feed.
Trypsin inhibitor and soybean agglutinin are the antinutritional factor in the meal expeller and mealsolvent raw material.The method of eliminating this class material has physics, chemistry, biology etc. multiple.Trypsin inhibitor and soybean agglutinin also are protein in essence, existing report multiple protein enzyme can decompose the antinutritional factor in the vegetable-protein and make it the part inactivation as neutral, Sumizyme MP, but do not see and successfully utilize these proteolytic enzyme to make the aquatic feeds zymin, and can eliminate the reported in literature of vegetable-protein antinutritional factor in the aquatic feeds such as trypsin inhibitor and soybean agglutinin.Seed selection and dynamical zymotechnique with high yield neutral protein protease production strain provide the cheap aquatic feeds specific enzyme based on neutral protease, are that this product is carried out the crucial prerequisite that industrialization is produced.
Summary of the invention
The invention solves existing in prior technology the problems referred to above, a kind of aspergillus oryzae strain of high yield neutral protein proteolytic enzyme is provided.
The present invention also provides the fermentation process of a kind of above-mentioned aspergillus oryzae strain on solid state substrate.
Above-mentioned technical purpose of the present invention solves by the following technical programs:
But a kind of aspergillus oryzae strain of high yield neutral protease (Aspergillus oryza) ZW-06, CGMCCNo.1754.
Some aspergillus oryzae bacterial classifications that the contriver is introduced from some aspergillus oryzae bacterial classifications, aspergillus niger strain and from Institute of Micro-biology of the Chinese Academy of Sciences, soil institute of the Chinese Academy of Sciences, Shanghai industrial microorganism and the bacterial classification that from soil sample, separates the different Pseudomonas that obtain, through primary dcreening operation, multiple sieve, it is the highest that the aspergillus oryzae strain that obtains a strain code name and be ZW produces the neutral protein enzymic activity on selective medium.Selected this bacterial strain is as the starting strain of further mutagenic and breeding.
The seed selection of starting strain: primary dcreening operation: with the Dou face spawn culture of different Pseudomonas good after, (spore concentration is 10 to make spore suspension with sterilized water
4～10
6Individual/ml), separate application was cultivated 2-4 days for 30 ℃ on the primary dcreening operation plate culture medium, and relatively each bacterial classification is cultivated the transparent circle size that is produced at the primary dcreening operation flat board, and the bacterial classification big with transparent circle is selected in bacterial classification as primary dcreening operation.Multiple sieve: the selected Dou face bacterial classification of primary dcreening operation is made spore suspension, and (spore concentration is 10
6～10
8Individual/as ml), to insert fermentation basic medium (solid), cultivated 2-4 days for 30 ℃, to cultivate and finish the back in 35-40 ℃ of oven dry, the measuring method following by the present invention detects the neutral protein enzymic activity.Good with leavening property, produce enzyme and live the highest bacterial strain as starting strain.
Described primary dcreening operation substratum: casein food grade 0.4%, Na
2PO
4.12H
2O 0.1%, KH
2PO
40.036%, BaCL
20.4%, agar 2%.
Fermention medium: wheat bran 78%, bean cake powder 18%, (NH
4)
2SO
42%, produce enzyme inducer 2%, add water and make the substratum water ratio reach 55%-60%.
The complex mutation breeding of bacterial strain: will be on potato dextrose agar (PDA) inclined-plane the fresh spore of the ZW-06# bacterial strain of growth and maturity, wash with sterilized water, and make monospore suspension (spore concentration 107-108/ml).Get 2ml monospore suspension respectively in test tube, put chamber, cobalt source and carry out radiation treatment.Treatment dosage is 0 (ck), 30,000,50,000,70,000,100,000 and 150,000 R (roentgen).After the Co60 radiation treatment, draw the suspension that each is handled, progressively dilute 101,102,103,104,105,106,107 times, coat on the PDA plate culture medium, 3 flat boards of each extent of dilution, after cultivating 48-72h under 28-30 ℃, the difform sporangium of picking falls within the PDA inclined-plane, after cultivating 72-96h under 28-30 ℃, carry out the multiple comparisons between the primary dcreening operation of the triangular flask solid fermentation of mutagenesis bacterial classification, multiple sieve and bacterial strain, obtain purpose bacterial strain ZW-06#, the neutral protease work of producing reaches the 15000U/g butt.
The concrete separation of bacterial strain of the present invention obtain into:
The bacterial strain primary dcreening operation:
Bacterial strain on the inclined-plane with 10ml aseptic water washing spore, is drawn 1ml and inserted the fermentation basic medium, and 30 ℃ of constant temperature culture 48h carry out the neutral protease enzyme activity determination.Strain number 7,8,9,23,25,26,27,50,64 bacterial strain enzyme raising amount alive all is higher than contrast more than 50%, these bacterial strains is chosen the access inclined-plane, as the bacterial strain of multiple sieve.
Bacterial strain sieves again
The product neutral protease enzyme work that primary dcreening operation is obtained is higher than the bacterium of control strain more than 50%, inserts the fermentation basic medium once more, and every strain bacterium connects three bottles, carries out enzyme activity determination, three results is averaged measurement result such as following table.Wherein 6 and No. 26 bacterial strain neutral protease enzyme activities are the highest, and comparison is according to improving more than 70%.Carry out the mitotic stability test with producing 6, No. 26 high bacterial strains of enzyme activity.
Enzyme activity determination mean value when table 1 bacterial strain sieves once more again
| Bacterial strain number | Enzyme (U/g) alive | Raising amount (%) | Bacterial strain number | Enzyme (U/g) alive | Raising amount (%) |
| Contrast 6 68 9 23 | 9012 15769 13765 13680 13390 | 74.98 52.74 51.80 48.58 | 25 26 27 50 64 | 14642 15754 13492 13653 13987 | 62.47 74.81 49.71 51.50 55.20 |
Table 2 strain passage experimental result
The gained bacterial classification is accredited as Aspergillus oryzae bacterial classification Aspergillusoryza (Ahlb.) E.Cohn through Institute of Microorganism, Academia Sinica.
The spore of gained enzyme bacterial classification is a yellow-green colour, is preserved in China Committee for Culture Collection of Microorganisms on July 10th, 2006, and its preserving number is CGMCC No.1754.
Aspergillus oryzae bacterial classification of the present invention (Aspergillus oryza) ZW-06, CGMCC No.1754 has following biological characteristics:
1, morphological feature: bacterium colony on the Cha Shi nutrient agar, 25 ℃ of 7 days diameter 43-46mm, quality velvet shape, the conidium structure is a large amount of, yellow-green colour, no transudate; Bacterium colony reverse side brown.The conidial head sphere, straight big footpath 58-110 μ m; Conidiophore is born in matrix, falx stem surface irregularity, diameter 7.6-8.7 μ m; Top capsule flask shape, diameter 23-30 μ m all surfaces can be educated; The conidial fructification individual layer, bottle stalk 8.5-11X2.6-3.9mm; The conidium sphere, tawny, smooth surface, diameter 4.6-5.4 μ m.
2, cultivate to learn characteristic: the biological property of this bacterial classification: bacterium colony on the wort agar substratum, 25 ℃ of 7 days diameter 65-68mm, quality suede is cotton-shaped, the conidium structure is a large amount of, no transudate; The bacterium colony reverse side is colourless;
Aspergillus oryzae strain ZW-06 of the present invention can adopt following fermentation process to produce neutral protease:
A. with aspergillus oryzae strain (Aspergillus oryza) ZW-06, CGMCC No.1754 is seeded on the slant medium at 28-30 ℃ of following constant temperature culture 72-96h, and maturation is standby; After taking out the activation of placement room temperature during use earlier, make spore suspension with sterilized water, stand-by;
B. will produce the sub-substratum of enzyme and press the spore suspension that 1-10% (W/W) amount inserts slant strains, the solid seed culture of inoculation is based on 28-30 ℃ of following constant temperature culture 48-72h; After the bacterial strain maturation, produce zymogenic bacteria kind spore suspension, stand-by;
C. will produce the enzymic fermentation substratum and press 1-5% (W/W) inoculum size access product enzyme spore suspension, fully behind the stirring and evenly mixing, cultivate 40-56h in 30 ℃; In the fermentation culture process, in cultivating 10h, turn over Qu Yici every the 10h vibration, 3-4 the static cultivation in back continuously is until fermentation ends;
The aftertreatment of D, enzyme song: the product after the fermentation ends, put 35-40 ℃, the oven dry, sieve after the pulverizing the enzyme dry medium;
The sub-substratum of described product enzyme is: 80～120 parts in wheat bran, 5～15 parts of bean cake powders, (NH
4)
2SO
40.5～1.2 parts, add water and make the substratum water ratio reach 55%-60%; (being weight part)
Described product enzymic fermentation substratum is: 70～88 parts in wheat bran, 10～30 parts of bean cake powders, (NH
4)
2SO
41.2～3 parts, produce 1～3 part of enzyme inducer, add water and make the substratum water ratio reach 55%-60%.
As preferably, described slant medium is through the potato dextrose agar after the following processing: 200 parts of peeling potatos, add 800～1200 parts in water after being cut into small pieces, boil, after it is filtered, add 10～30 parts of glucose in the filtrate, and replenish moisture to 80～120 that lose because of evaporation part, add 10～30 parts in agar, natural pH, 0.075Mpa sterilization down.
But the high yield neutral protease aspergillus oryzae mutation that utilization of the present invention provides, adopt the solid fermentation law technology to produce neutral protease simultaneously, the enzyme of its tunning is lived on average can reach 21500U/g, reach as high as 23500U/g, than the utilization genetic engineering bacterium of domestic similar institute report, obtain liquid fermenting neutral protease 20000U/mL and want high more than 10%.And the aspergillus oryzae solid fermentation process that this technology adopted, raw material is easy to get, and zymotechnique is simple.In addition, analyze, find also to contain higher zytase and cellulase in the tunning, be very suitable for as the aquatic feeds specific enzyme by aspergillus oryzae ZW-06 having been carried out product enzyme enzyme system.Report (Yunnan Normal University's journals such as this and Xu Bo, 2005,25 (3): 51-56): " with the neutral protease functional zone of subtilis AS1.398 with comprise the leader sequence at interior full-length gene clone pichia pastoris phaff SMD1168; after methanol induction is cultivated; adopt liquid fermentation process, the neutral protease vigor in the fermented liquid reaches 20000U/M1 " are compared at zymotechnique and are produced on the enzyme performance and improve a lot.
In order to understand the characteristic of gained bacterial classification of the present invention and zymotechnique thereof, the applicant has carried out different strain produces neutral protease under zymotechnique of the present invention comparison test, has also carried out the comparison test with the product neutral protease of bacterial classification ZW-06 of the present invention on different substratum.
One, different strain adopts the comparison test of producing neutral protease under the zymotechnique of the present invention:
1) bacterial classification quoted of this test: aspergillus oryzae ZW-06# (bacterial classification of the present invention), aspergillus oryzae bacterial classification (this chamber original bacterial classification Aspergillus oryza 3.802, draw Institute of Micro-biology) from the Chinese Academy of Sciences, aspergillus niger strain (mutagenesis bacterial classification Aspergillus niger v.Tiebh mainly produces alpha-galactosidase).
2) culture of strains: the bacterial classification of being quoted is seeded in respectively on the PDA inclined-plane,, after ripe (growing plentiful spore), takes out, left with 4 ℃ of refrigerator 1-15 days in 28-30 ℃ of following constant temperature culture 72-96h.After taking out the activation of placement room temperature during use earlier, make spore suspension with sterilized water, stand-by.
3). will produce the sub-substratum of enzyme through 121 ℃, the 30min sterilization.After the cooling, press 1-10% (W/W) amount and insert the slant strains spore suspension, last postvaccinal solid seed culture medium is in 28-30 ℃ of following constant temperature culture 48-72h.After ripe (stromal surface and inside all grow plentiful spore), inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain zymogenic bacteria kind spore suspension with double gauze, stand-by.
4). fermention medium is through 121 ℃, and the 30min sterilization after the cooling, is pressed 1-5% (W/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 40-56h in 30 ℃; In the fermentation culture process, in cultivating 10h, turn over Qu Yici every the 10h vibration, 3-4 time continuously, static cultivation behind the 48h is until fermentation ends.
5), the aftertreatment of enzyme song:
A. the product after the fermentation ends is put 35-40 ℃, under the air blast condition, dries through 15-24h.
B. the tunning after the oven dry is pulverized, and crosses 80 mesh sieves, gets the enzyme dry medium.And by described neutral protease activity test method detection.
The substratum that adopts in this process of the test:
Slant preservation substratum: be potato dextrose agar (PDA): add water 1000ml after peeling potato 200g is cut into small pieces and boil 30min, double gauze filters, add glucose 20g in the filtrate, and replenish the moisture that loses because of evaporation to 100ml, add agar 20g, nature Ph, 0.075Mpa is sterilization 20min down.
Producing the sub-substratum of enzyme is: wheat bran 90%, bean cake powder 9%, (NH
4)
2SO
41%, add water and make the substratum water ratio reach 55%-60%.(solid medium)
Producing the enzymic fermentation substratum is: wheat bran 78%, bean cake powder 18%, (NH
4)
2SO
42%, produce enzyme inducer 2%, add water and make the substratum water ratio reach 55%-60%.
This test utilization multiple comparisons method, each bacterial classification is established three repetitions, the result shows, different strain adopts the zymotechnique of present technique, in the tunning there be than big-difference the vigor of the neutral protease that produces, wherein the highest with the bacterial classification aspergillus oryzae neutral protease that ZW-06# is produced that adopts present technique, extremely remarkable with all the other two bacterial classification differences, see table 3 for details: table 3
Two, the comparison test of the product neutral protease of gained enzyme bacterial classification ZW-06 of the present invention on different substratum:
1) bacterial classification quoted of this test: aspergillus oryzae ZW-06#.
2) culture of strains: the bacterial classification of being quoted is seeded in respectively on the PDA inclined-plane,, after ripe (growing plentiful yellow-green colour spore), takes out, left with 4 ℃ of refrigerator 5-15 days in 28-30 ℃ of following constant temperature culture 72-96h.After taking out the activation of placement room temperature during use earlier, make spore suspension with sterilized water, stand-by.
3). will produce the sub-substratum of enzyme through 121 ℃, the 30min sterilization.After the cooling, press 1-10% (W/W) amount and insert the slant strains spore suspension, last postvaccinal solid seed culture medium is in 28-30 ℃ of following constant temperature culture 48-72h.After ripe (stromal surface and inside all grow plentiful yellow-green colour spore), inject the refrigerative sterilized water and fully stir, under aseptic condition, filter, obtain zymogenic bacteria kind spore suspension with double gauze, stand-by.
4). fermention medium is through 121 ℃, and the 30min sterilization after the cooling, is pressed 1-5% (W/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 40-56h in 30 ℃; In the fermentation culture process, in cultivating 10h, turn over Qu Yici every the 10h vibration, 3-4 time continuously, static cultivation behind the 48h is until fermentation ends.
5), the aftertreatment of enzyme song:
A. the product after the fermentation ends is put 35-40 ℃, under the air blast condition, dries through 15-24h.
B.. the tunning after the oven dry is pulverized, and crosses 80 mesh sieves, gets the enzyme dry medium.And by described neutral protease activity test method detection.
Substratum of the present invention:
Slant preservation substratum: be potato dextrose agar (PDA): add water 1000ml after peeling potato 200g is cut into small pieces and boil 30min, double gauze filters, add glucose 20g in the filtrate, and replenish the moisture that loses because of evaporation to 100ml, add agar 20g, nature Ph, 0.075Mpa is sterilization 20min down.
Produce the sub-substratum of enzyme: wheat bran 90%, bean cake powder 9%, (NH
4)
2SO
41%, add water and make the substratum water ratio reach 55%-60%.(all being weight percentage)
Produce the enzymic fermentation substratum:
(1) wheat bran 78%, bean cake powder 18%, (NH
4)
2SO
42%, produce enzyme inducer 2%, add water and make the substratum water ratio reach 55%-60%.This substratum is that the enzyme of sending out that present technique adopts produces the enzyme substratum.
The substratum that control experiment (2) is adopted is: wheat bran 85%, bean cake powder 13%, (NH
4)
2SO
41%, Na
2CO
31%, add water and make the substratum water ratio reach 55%-60%.
The substratum that control experiment (3) is adopted is: wheat bran 65%, bean cake powder 23%, (NH
4)
2SO
41%, KH
2PO
41%, add water and make the substratum water ratio reach 55%-60%.
This test utilization multiple comparisons method, three repetitions are established in each processing, the result shows, product neutral protease bacterial classification-aspergillus oryzae ZW-06# that present technique obtains produces on the enzymic fermentation substratum in difference, in the tunning there be than big-difference the vigor of the neutral protease that produces, wherein the highest with the neutral protease that fermention medium is produced that adopts present technique, extremely remarkable with all the other two fermention medium differences, see table 4 for details:
Table 4
The aspergillus oryzae strain of high yield neutral protease of the present invention (Aspergillus oryza) ZW-06 is preserved in China Committee for Culture Collection of Microorganisms on July 10th, 2006, and its preserving number is CGMCCNo.1754.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1:
One, spawn culture:
1, slant strains is cultivated: the aspergillus oryzae mutation ZW-06# that present technique is obtained is seeded on potato dextrose agar (PDA) inclined-plane, in 28-30 ℃ of following constant temperature culture 72-96 hour, after waiting to grow plentiful yellow-green colour spore, take out, left with 4 ℃ of refrigerator 5-15 days.Take out earlier during use place the room temperature activation after, with sterilized water make spore suspension (concentration be 107-108/ml), stand-by.
2. solid seed culture: the bean cake powder of 1.8kg wheat bran and 0.18kg fully is mixed, and then with 1: the ratio of 1.2-1.5 adds the (NH of 1% concentration
4)
2SO
4Solution behind the abundant once more mixing, is made the solid seed culture medium.This substratum is through 121 ℃, and the 30min sterilization after the cooling, press 1-5% amount (W/W) 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 is in 28-30 ℃ of following constant temperature culture 48-72h.After treating that substratum top layer and inside grow plentiful yellow-green colour 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 double gauze.Be diluted to the zymogenic bacteria kind spore suspension that spore concentration is 107-108/ml with sterilized water again.
2. fermention medium is pressed wheat bran 78%, bean cake powder 18%, (NH
4)
2SO
42%, the ratio thorough mixing of product enzyme inducer 2% 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 the ventilative semiautomatic plant of preserving moisture is with temperature, the humidity of control culturing process for aseptic ventilative square plate.
3. postvaccinal square plate is placed 30 ℃ to cultivate constant temperature cultivation 40-56h 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 35-40 ℃, 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:
Carry out three batches test as stated above altogether, every batch fermentation product is pressed sample quartering, by described neutral protein activity determination method, measures three batches enzyme and lives.The result shows, the neutral protease work in three batches of tunnings is between 21635U/g-23050U/g, and average out to 22401U/g illustrates that zymogenic bacteria kind aspergillus oryzae ZW-06# that present technique obtains under the present technique zymotechnique, produces the neutral protease quite stable.See table 5 for details: table 5
| Batch | Average enzyme (U/g) alive |
| No1 | 21635a |
| No2 | 22518a |
| No3 | 23050a |
| On average | 22401 |
Neutral protein activity determination method of the present invention-the Folin method is specific as follows in addition:
1. principle
Proteolytic enzyme is under certain temperature and pH condition, the hydrolyzed casein substrate produces the amino acid (as tyrosine, tryptophane) that contains phenolic group, under alkaline condition, Folin reagent (Folin) is reduced, generate molybdenum blue and tungsten blue, the depth of its color is directly proportional with the phenolic group aminoacids content.By measure its absorbancy at 660nm, can obtain the amino acid whose amount of phenolic group that enzymolysis produces, calculate proteinase activity, represent total enzyme activity of proteolytic enzyme with this.
2. the enzyme unit definition of living
(aspartic protease pH3.5 under (40 ± 0.2) ℃, corresponding pH condition, neutral protease pH7.0), caseinhydrolysate substrate in 1min produces the 1 microgram phenolic compound enzyme amount of (being represented by the tyrosine equivalent) that is equivalent to, be 1 enzyme unit alive, represent with u/g (u/ml).
3. reagent and solution
Unless otherwise prescribed, agents useful for same is analytical pure in the test, and institute's water is distilled water or deionized water.
3.11mol/L and 0.1mol/L hydrochloric acid soln
Get concentrated hydrochloric acid 85ml, thin up also is settled to 1000ml, is the 1mol/L hydrochloric acid soln; Get the 100ml1mol/L hydrochloric acid soln, be settled to 1000ml, be the 0.1mol/L hydrochloric acid soln.
3.21mg/ml L-tyrosine standard stock solution
Accurately take by weighing in advance, after 1 dissolving of 20ml left and right sides 1mol/L hydrochloric acid, be settled to 100ml with distilled water again, be 1mg/ml tyrosine standardized solution prior to 105 ℃ of L-tyrosine 0.1000g that are dried to constant weight.
3.3L-the tyrosine standard is used solution
Draw 1mg/ml tyrosine standardized solution 10.0ml and be settled to 100ml, promptly obtain 100 μ g/ml L-tyrosine reference liquids with 0.1mol/L hydrochloric acid.
Draw 100 μ g/ml L-tyrosine reference liquid 0ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, 6.0ml respectively in the 10ml volumetric flask, be settled to scale with distilled water, shake up, make the standard use solution that every ml contains L-tyrosine 0 μ g, 10 μ g, 20 μ g, 30 μ g, 40 μ g, 50 μ g, 60 μ g respectively.
3.40.4mol/L yellow soda ash (Na
2CO
3) solution
Take by weighing anhydrous sodium carbonate (Na
2CO
3) 42.4g, with dissolved in distilled water and be settled to 1000ml.
3.5 forint (Folin) reagent
In 2000ml ground reflux, add sodium wolframate (Na
2WO
4.2H
2O) 100g, Sodium orthomolybdate (Na
2MoO
4.2H
2O) 25g, distilled water 700ml, 85% phosphoric acid 50ml, concentrated hydrochloric acid 100ml.Little fiery boiling reflux 10h takes off reflux cooler, adds Lithium Sulphate (Li in ventilating kitchen
2SO
4) 50g, adding distil water 50ml and several dense (99%) bromine waters, little again 15min that boils, to remove unnecessary bromine (still have green need add bromine water again after cold, boil and remove excessive bromine), cooling back adding distil water is settled to 1000ml.Mixing, filtration.It is golden yellow that reagent should be, and is stored in the brown bottle.During use,, make rare Folin reagent with 1 part of former Folin reagent and 2 parts of distilled water mixings.
3.6 phosphoric acid buffer (pH=7.0) is applicable to neutral protease
First liquid: take by weighing Sodium phosphate dibasic (Na
2HPO
4.12H
2O) 71.64g uses dissolved in distilled water, and is settled to 1000ml.
Second liquid: take by weighing SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
4.2H2O) 31.21g uses dissolved in distilled water, and is settled to 1000ml..
Use solution: get first liquid 610ml, add second liquid 390ml and mix, proofread and correct to pH with pH meter and be (7.0 ± 0.05), standby.
3.71.0% casein solution
Take by weighing casein 1.0000g, after elder generation used a small amount of (3ml-4ml) dense lactic acid moistening, (if neutral protease is a small amount of 0.5mol/L sodium hydroxide solution) added the about 80ml of corresponding pH damping fluid again, heats while stirring in boiling water bath until dissolving fully.Change over to after the cooling in the 100ml volumetric flask, be settled to scale with corresponding damping fluid.This solution is at the 4C freezer storage, and validity period is 3 days.
3.80.4mol/L trichoroacetic acid(TCA) (CCl
3-COOH) solution (TCL precipitation reagent)
Take by weighing trichoroacetic acid(TCA) 65.4g, with dissolved in distilled water and be settled to 1000ml.
4. instrument and equipment
Except that the common lab instrument, also should have:
4.1 thermostat water bath (40 ± 0.2) ℃.
4.2 spectrophotometer
4.3 acidometer precision ± 0.01pH.
4.4 analytical balance sensibility reciprocal 0.1mg.
4.5 stopwatch or timing watch.
5 determination steps
5.1 typical curve is drawn
Press Table A .1, draw standard tyrosine standard respectively and use solution, 0.4mol/L sodium carbonate solution and rare Folin reagent in each pipe (every pipe number 3 samples of parallel work), mixing.
Standard pipe is placed 40 ℃ of water-baths simultaneously, and reaction 20min takes out, and is cooled to room temperature rapidly, use the 10mm cuvette, with blank pipe (No. 0 pipe) demodulating apparatus zero point, in spectrophotometer wavelength 660nm place survey absorbancy.With the tyrosine amount is abscissa, is ordinate with the absorbancy, and the drawing standard curve obtains equation of linear regression.Tyrosine amount when absorbancy is 1 (μ g) is colorimetric constant K value.
Linear regression coeffficient (r) should can use (otherwise must reform) when above 0.9990.Folin reagent to each new preparation is made new typical curve.
Table 1 tyrosine typical curve
5.2 the mensuration of sample
5.2.1 the preparation of enzyme liquid to be measured
The solid enzyme: enzyme is lived big or small per sample, take by weighing 2g-10g solid enzyme sample, be accurate to 0.1mg, add 40-200ml distilled water, the rearmounted 40 ℃ of water-bath lixiviate 30min of dissolving use filter paper filtering, again enzymic activity size per sample, secondary dilution is to suitable concentration (dropping between the 0.2-0.4 difference of the absorbancy that supplies test liquid and blank solution, down together).Liquid enzymes: accurately measure the some ml of liquid enzymes, enzyme activity size per sample, directly with distilled water diluting to suitable concentration, to be measured.
5.2.2 mensuration program
---get three 15 * 150mm test tubes (a blank pipe, two sample hoses).
---in three test tubes, accurately add the good enzyme liquid 1.0ml to be measured of dilution respectively.
---three test tubes are put into (40 ± 0.2) ℃ water-bath preheating 5min, will measure substrate (1% casein solution) simultaneously and put preheating 5min in the same temperature water-bath.
---in two sample hoses, add 1.0ml 1% casein solution respectively, accurately timing, reaction 10min takes out.
---rapidly, accurately in three test tubes, add 2ml TCL (precipitation reagent), in blank pipe, add the 1.0ml1% tyrosine solution, shake up.Three test tubes are continued to put in (40 ± 0.2) ℃ water-bath place 10min, take out, be cooled to room temperature rapidly.
---reaction solution filters with filter paper (No. 1 filter paper of Whatman1 filter paper or Xinhua) in three test tubes.
---other gets the test tube (blank, two sample hoses) of three 18 * 180mm and draws above-mentioned corresponding filtrate 1.0ml, 0.4mol/L sodium carbonate solution 5.0ml respectively, rare Foiln reagent 1.0ml shakes up, and puts in (40 ± 0.2) ℃ water-bath and places 20min, take out, be cooled to room temperature rapidly.
---with blank pipe (contrast) demodulating apparatus zero point, under spectrophotometer wavelength 660nm, use the 10mm cuvette, survey the absorbancy of sample liquid in two sample hoses respectively, average.By looking into typical curve or obtaining the tyrosine contents of generation with equation of linear regression.
6. enzyme activity calculates
According to following formula (A.1) calculation sample proteinase activity:
In the formula:
The X1---proteinase activity, u/g;
A---sample and barren absorbancy are poor
K---colorimetric constant;
The amount (ml) that adds entry during the V---sample extraction;
4---enzyme reaction system cumulative volume (ml);
Extension rate during N---sample secondary dilution;
1---participates in the enzyme amount (ml) of reaction;
W---example weight (g/ml);
The 10---reaction times (min).
7. precision
The absolute difference of twice test result of same sample must not surpass 10% of arithmetical av.
The neutral protease that aspergillus oryzae strain of the present invention produced, except the inulinase-producing activity height, the trypsin inhibitor in the meal expeller and mealsolvent raw material of also can effectively degrading.Further investigated the passivation of aspergillus oryzae neutral protease that ZW-06 produces to trypsin inhibitor in the dregs of beans.The result shows: in dregs of beans, add the proteolytic enzyme of 1: 500 mass ratio just can the passivation dregs of beans in 40% trypsin inhibitor, temperature is very little to the influence of proteolytic enzyme passivation trypsin inhibitor, the general arrestin enzyme of the adding of metal ion is to the degraded of trypsin inhibitor, and enzymolysis time just can reach maximum suppression effect at 30min.
Strain fermentation gained neutral protease of the present invention active high reaches 23000U/g, and has the trypsin inhibitor in the meal expeller and mealsolvent raw material is had stronger passivation.Therefore be very suitable for as the aquatic feeds specific enzyme.
Claims (2)
1, the fermentation process of a kind of aspergillus oryzae strain on solid state substrate may further comprise the steps:
A. with aspergillus oryzae strain (Aspergillus oryza) ZW-06, CGMCC No.1754 is seeded on the slant medium at 28-30 ℃ of following constant temperature culture 72-96h, and maturation is standby; After taking out the activation of placement room temperature during use earlier, make spore suspension with sterilized water, stand-by;
B. will produce enzyme solid seed culture medium and press the spore suspension that 1-10% (W/W) amount inserts slant strains, the product enzyme solid seed culture of inoculation is based on 28-30 ℃ of following constant temperature culture 48-72h; After the bacterial strain maturation, produce zymogenic bacteria kind spore suspension, stand-by;
C. will produce the enzymic fermentation substratum and press 1-5% (W/W) inoculum size access zymogenic bacteria kind spore suspension, fully behind the stirring and evenly mixing, cultivate 40-56h in 30 ℃; In the fermentation culture process, in cultivating 10h, turn over Qu Yici every the 10h vibration, 3-4 the static cultivation in back continuously is until fermentation ends;
The aftertreatment of D, enzyme song: the product after the fermentation ends, put 35-40 ℃, the oven dry, sieve after the pulverizing the enzyme dry medium;
Described product enzyme solid seed culture medium is: 80～120 parts in wheat bran, 5～15 parts of bean cake powders, (NH
4)
2SO
40.5～1.2 parts, add water and make the substratum water ratio reach 55%-60%;
Described product enzymic fermentation substratum is: 70～88 parts in wheat bran, 10～30 parts of bean cake powders, (NH
4)
2SO
41.2～3 parts, produce 1～3 part of enzyme inducer, add water and make the substratum water ratio reach 55%-60%.
2, the fermentation process of aspergillus oryzae strain according to claim 1 on solid state substrate, it is characterized in that the potato dextrose agar after described slant medium is for the following processing of process: 200 parts of peeling potatos, add 800～1200 parts in water after being cut into small pieces, boil, after it is filtered, add 10～30 parts of glucose in the filtrate, and replenish moisture to 80～120 that lose because of evaporation part, add 10～30 parts in agar, natural pH, 0.075Mpa sterilization down.
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| CN103013844B (en) * | 2012-12-20 | 2014-08-27 | 山东隆科特酶制剂有限公司 | Aspergillus oryzae bacterial strain giving high yield of neutral protease and liquid fermentation method thereof |
| CN103642695B (en) * | 2013-08-28 | 2015-10-28 | 浙江工业大学 | One Aspergillus oryzae and the application prepared at fermentable in fodder additives thereof |
| CN103555594B (en) * | 2013-11-08 | 2015-05-13 | 佛山市海天调味食品股份有限公司 | Aspergillus oryzae and application thereof |
| CN104711242B (en) * | 2013-12-12 | 2017-11-10 | 青岛蔚蓝生物集团有限公司 | A kind of neutral proteinase and its application |
| CN105950481B (en) * | 2016-05-30 | 2019-05-03 | 湖北工业大学 | One Aspergillus oryzae bacterial strain and its protease of generation are produced applied to yeast extract |
| CN106922952B (en) * | 2017-03-24 | 2020-11-13 | 江苏天种牧业股份有限公司 | Method for preparing polypeptide-rich protein feed additive by solid state fermentation of aspergillus oryzae |
| KR102213300B1 (en) * | 2019-02-22 | 2021-02-09 | 코스맥스 주식회사 | Strain of Aspergillus oryzae and composition for improving skin condition comprising a fermenttion production thereof |
| CN110885762B (en) * | 2019-12-06 | 2020-09-08 | 鹤山市东古调味食品有限公司 | Preparation method of aspergillus oryzae fermentation liquor |
| CN110904083A (en) * | 2019-12-31 | 2020-03-24 | 沂源康源生物科技有限公司 | Production process for producing acid protease by aspergillus niger solid state fermentation |
| CN111909857B (en) * | 2020-08-21 | 2023-08-08 | 四川润格生物科技有限公司 | Technical method for producing aspergillus oryzae by using white spirit vinasse |
| CN115404173B (en) * | 2022-09-15 | 2023-10-20 | 佛山市海天(高明)调味食品有限公司 | Aspergillus oryzae ZA216 and application thereof |
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Application publication date: 20080319 Assignee: Anhui Rongxin Bioengineering Co., Ltd. Assignor: Zhejiang Academy of Agricultural Sciences Contract record no.: 2014330000061 Denomination of invention: Aspergillus zryzae strain capable of highly producing neutral proteinase and fermentation method thereof on solid state substrate Granted publication date: 20091111 License type: Exclusive License Record date: 20140331 |
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