CN102586209B - Preparation method for liquid high temperature resistant acidic mannase - Google Patents
Preparation method for liquid high temperature resistant acidic mannase Download PDFInfo
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Abstract
The invention belongs to the field of enzyme preparations and particularly relates to a preparation method for liquid high temperature resistant acidic mannose. The method comprises the following steps: soaking rhizopus solid fermenting leaven; centrifuging the mixed liquid; removing residues; filtering; performing ultrafiltration concentration on the filtrate to obtain concentrated liquid; and adding preservative and a stabilizing agent in the concentrated liquid to obtain the liquid mannose. Compared with the common method for preparing the mannose with fermenting Pichia pastoris flow and methanol liquid, the method has the beneficial effects that the mannose produced by the method has high acid resistance and high heat resistance in the aspects of acid resistance and heat resistance.
Description
Technical field
The invention belongs to the zymin field, be specifically related to the making method of the high temperature resistant acid mannase of a kind of liquid.
Background technology
Mannase (endo-1,4-β-mannanase) is a kind of novel enzyme preparation, belongs to a kind of hemicellulose enzyme, and it is except the effect with general non-starch polysaccharide (NSP) enzyme---degraded NSP, reduce enteron aisle viscosity, promote outside the digestion and absorption of nutritive substance; Recently studies show that much that mannase still is a kind of multi-functional growth promoter, it can promote the secretion of quasi-insulin growthing factor I GF-I, promotes the synthetic of protein, improves lean ratio; Simultaneously, it also can eliminate the beta-mannase that is rich in the beans to the interference of glucose absorption, greatly improves the especially energy digestibility of dregs of beans of grouts.Also can find out in actual the use, add that resistibility and the reguarity of animal has raising after the mannase.Facts have proved that the effect of traditional zymin that mannase is unconventional becomes a kind of novel feed additive for promoting growth.
The mannosans that mannase can decompose in corn-dregs of beans type diet is mannooligo saccharide.Mannooligo saccharide can improve enteric microorganism and form as a kind of biological chemistry probiotics, and forming with bifidus bacillus and milk-acid bacteria is the intestinal microflora of advantage.It is reported that mannooligo saccharide can be used as nutritive substance by bifidus bacillus and lactobacillus selective fermentation utilization, and promotes its growth and breeding.But pathogenic bacterium such as intestinal bacteria, Salmonellas causes hungry death because utilizing.In addition; mannooligo saccharide can with the pathogenic bacteria cell walls on receptors bind; thereby stop the glycosyl of bacterium on animal intestinal mucosa to be combined; protect the complete of intestinal mucosa structure and function; again because mannooligo saccharide is non-digestibility material; this mixture just is excreted by gi tract smoothly, thereby plays the absorption enteric pathogenic bacteria, and animal is had function of health care.Mannooligo saccharide can not only be connected on the bacterium, and can be connected on toxin, virus, the eukaryote, can be used as the auxiliary agent of these exogenous antigens in conjunction with the back mannooligo saccharide, alleviates the absorption of antigen, the cellular immunization of enhancing body and humoral immunization.Mannooligo saccharide is not only participated in immunomodulatory directly and part substitutes microbiotic, in itself and microbiotic share, can also play the effect of reduction body tissue antibiotic remains.
After the feed course of processing and animal searched for food, zymin generally will stand the destruction of hot and humid granulation, and hydrochloric acid in gastric juice and pepsic infringement can arrive in the animal body action site and still retain active that part of enzyme work and be called effective enzyme and live.This requires corresponding enzyme to have good heat-resisting acid resistance, antipepsin, trypsinase characteristic etc.Good feed enzyme has wideer temperature action scope, and assurance can be lived at the existing higher enzyme of the temperature following table of animal heat, again can be to showing tolerance preferably in the hot and humid pelletization of feed; Can tolerate the infringement of hydrochloric acid in gastric juice, under the neutral environment of the sour environment of stomach and small intestine, also can have the performance of living of higher enzyme.
In recent years, after mannase generally adds the methanol liquid fermentation by pichia spp stream, centrifugal some the more inferior mannases of producing of Plate Filtration or video disc.Though the report mannase is also arranged by the research of aspergillus niger solid fermentation, does not also see its actual fermentative production and extract manufacturing.The present invention is by behind the head mold solid fermentation, selects for use to be fit to the link-suspended basket centrifuge separation that coarse fodder separates, and produces the resistant to elevated temperatures mannase of acid resistance.
Summary of the invention
Technical problem to be solved by this invention provides high temperature resistant acid mannase of a kind of liquid and preparation method thereof.
The making method of the high temperature resistant acid mannase of liquid of the present invention, its step comprises:
Soak: expect by the head mold solid fermentation is bent: the part by weight of water=1:8-16 feeds intake, and will soak in the song material input water, soaks water pH and transfers to 4.5, stirs 2-3 hour, gets mixed solution;
Centrifugal: that mixed solution is placed whizzer centrifugal 10-15min under the condition of rotating speed 800-1200rpm;
Filter: the supernatant liquor of getting after centrifugal places filter, adds the diatomite that accounts for supernatant liquor weight 7-10%, filters and obtains cleaner liquid;
Ultrafiltration and concentration: membrane molecule amount 10000 is concentrated into the 1/8-1/12 of cleaner liquid volume;
Anticorrosion stable: add sanitas and stablizer in concentrated solution, the part by weight of its adding is concentrated solution: sorbyl alcohol: glycerine: potassium sorbate=85:10:5:0.1 stirs evenly.
More than said whizzer adopt link-suspended basket centrifuge; Filter is flame filter press.
Make the high temperature resistant acid mannase of liquid also within protection scope of the present invention by aforesaid method.
Aforesaid liquid is high temperature resistant acid mannase, it contains mannase 10000U/ml.
Beneficial effect of the present invention is, compares with the common methods such as methanol liquid fermentative production mannase that added by pichia spp stream, and no matter be from acid resistance or from the thermotolerance aspect, all significantly have advantage:
On acid resistance, mannase of the present invention is than AB MP1000 acid resistance pH1.0, than with U.S. ferment acid resistance pH3.5, than 01 pichia spp acid resistance pH2.5, than BSA bacterium acid resistance pH1.5, than BSA aspergillus oryzae acid resistance pH1.0, than K pichia spp acid resistance pH2.5;
On thermotolerance, this mannase is than the high temperature resistant 3-5 of AB MP1000 ℃, than high temperature resistant about 13 ℃ with U.S. ferment, than 01 pichia spp high temperature resistant about 12 ℃, than BSA bacterium high temperature resistant 30 ℃, high temperature resistant 2 ℃ than BSA aspergillus oryzae, high temperature resistant about 13 ℃ than K pichia spp.
Embodiment
Below in conjunction with specific embodiment the present invention is done further explanation, so that those skilled in the art more understands the present invention, but therefore do not limit the present invention.
Embodiment 1
Soak: press the mixed that the bent material of head mold solid fermentation and water are pressed 1:8, soak water and transfer pH4.5 with acetic acid, stirred 3 hours;
Centrifugal: under the condition of rotating speed 1000rpm in link-suspended basket centrifuge centrifugal 10min, get supernatant liquor after centrifugal;
Plate Filtration: add 8% diatomite, filter with flame filter press and obtain cleaner liquid;
Ultrafiltration and concentration: membrane molecule amount 10000 is concentrated into 1/9.5 of cleaner liquid volume;
Anticorrosion stable: in concentrated solution: the ratio of sorbyl alcohol: glycerine: potassium sorbate=85:10:5:0.1 adds stablizer, sanitas in concentrated solution, stir evenly.
The source of material
Bent material: formed by the head mold solid fermentation
The used detection method of the present invention---enzymic activity detection method: concrete detection method is as follows:
Spectrophotometry mannosans enzyme activity determination
A.1 mannosans enzyme activity determination
A.1.1 definition: be 5.5 at pH, temperature is under 37 ℃ of conditions, per minute from concentration be mannosans (Sigma G0753) solution of 3 mg/ml degraded to discharge the needed enzyme amount of 1 μ mol reducing sugar be an enzyme activity unit u.
A.1.2 reagent and solution
A.1.2.1 sodium hydroxide solution, concentration C (NaOH) is 200 g/L:
Weighing sodium hydroxide 20.0 g are dissolved in water, and are settled to 100 ml.
A.1.2.2 acetic acid solution, concentration c (CH
3COOH) be 0.1 mol/L:
Draw glacial acetic acid 0.60 ml, be dissolved in water, be settled to 100 ml.
A.1.2.3 sodium acetate solution, concentration c (CH
3COONa) be 0.1 mol/L:
Take by weighing sodium acetate trihydrate 1.36 g, be dissolved in water, be settled to 100 ml.
A.1.2.4 acetic acid---sodium acetate buffer solution, c(CH
3COOH-CH
3COONa) be 0.1 mol/L, the pH value is 5.5:
Take by weighing sodium acetate trihydrate 23.14 g, add glacial acetic acid 1.70 ml.Be dissolved in water again, be settled to 2000 ml.Measure the pH value of solution.If the pH value departs from 5.5, use acetic acid solution (A.1.2.2) or sodium acetate solution (A.1.2.3) to be adjusted to 5.5 again.
A.1.2.5 mannose solution, C(C
6H
12O
6) be 10.0mg/ml:
Take by weighing anhydrous D-seminose 1.000g, be dissolved in water, be settled to 100ml.
A.1.2.6 mannan solution 0.6%(w/v):
Take by weighing mannosans (Sigma G0753) 0.60g, add the 80ml acetate-sodium acetate buffer.Magnetic agitation, slowly heating is dissolved (notes: can add an amount of damping fluid in the process in stirring heating, but the cumulative volume of solution can not surpass 100ml fully until mannosans simultaneously.）。Stop heating then, continue to stir 30min, be settled to 100ml with acetate-sodium acetate buffer.Mannan solution can use immediately, suitably shakes up before the use.4 ℃ keep in Dark Place, and validity period is 3 days.
A.1.2.7DNS reagent
Take by weighing 3,5-dinitrosalicylic acid 3.15g(chemical pure), add water 500 ml, stir 5 s, water-bath to 45 ℃.Progressively add 100 ml sodium hydroxide solutions (A.1.2.1) then, constantly stir simultaneously, as clear as crystal (note: adding in the sodium hydroxide process, solution temperature is not above 48 ℃ up to solution.）。Progressively add Rochelle salt 91.0 g, phenol 2.50 g and sodium sulphite anhydrous 99.3 2.50 g again.Continue 45 ℃ of heating in water bath, add water 300 ml simultaneously, constantly stir, dissolve fully up to the material that adds.Stop the heating, be cooled to room temperature after, water is settled to 1000 ml.Filter with fritted glass filter.Get filtrate, be stored in the brown bottle, keep in Dark Place.Deposit under the room temperature after 7 days and can use, validity period is 6 months.
A.1.3 instrument, equipment
A.1.3.1 the laboratory is used the sample pulverizer or is ground alms bowl.
A.1.3.2 sub-sieve: the aperture is 0.25 mm(60 order).
A.1.3.3 analytical balance: sensibility reciprocal 0.001 g.
A.1.3.4 pH meter: be accurate to 0.01.
A.1.3.5 magnetic stirring apparatus: additional heat function.
A.1.3.6 hertz oscilltor.
A.1.3.7 fritted glass filter: the aperture is 0.45 μ m.
A.1.3.8 whizzer: 4000 r/min.
A.1.3.9 thermostat water bath: temperature controlling range is between 30-60 ℃, and precision is 0.1 ℃.
A.1.3.10 stopwatch: per hour error is no more than 5s.
A.1.3.11 spectrophotometer: the absorbancy scope that can detect 350-800nm.
A.1.4 analytical procedure
A.1.4.1 the making of typical curve
Draw damping fluid (A.1.2.4) 4.0ml, add DNS reagent 5.0ml, boiling water bath heating 5min.Be cooled to room temperature with tap water, water is settled to 25.0ml, the blank sample of the standard of making.
Draw mannose solution (A.1.2.5) 1.00,2.00,3.00,4.00,5.00,6.00 and 7.00ml respectively, use damping fluid (A.1.2.4) to be settled to 100ml respectively, being mixed with concentration is 0.10-0.70mg/ml D-seminose standardized solution.
Each 2.00ml(of seminose standardized solution that draws above-mentioned concentration series respectively do two parallel), join respectively in the scale test tube, add 2ml water and 5mlDNS reagent more respectively.Electromagnetic oscillation 3s, boiling water bath heating 5min.Use the tap water cool to room temperature then, water is settled to 25ml again.Serve as the contrast zeroing with the blank sample of standard, in the mensuration absorbancy OD of 540nm place value.
Be that Y-axis, absorbancy OD value are X-axis with the mannose concentration, the drawing standard curve.Each new preparation DNS reagent all needs to repaint typical curve.
A.1.4.2 the processing of sample solution
Solid sample should be pulverized or fully pulverize, and crosses 60 mesh sieves (aperture is 0.25 mm) then, takes by weighing two parts on sample according to the sample weighting amount of advising among the appendix B, is accurate to 0.001 g.Add 50 ml acetate-sodium acetate buffer (A.1.2.4).Magnetic agitation 30 min use buffered soln (A.1.2.4) to be settled to 100 ml again, and 24h keeps in Dark Place under 4 ℃ of conditions.Centrifugal 3 min of last whizzer.Get the 0.5ml supernatant liquor, use buffered soln (A.1.2.4) to do suitable dilution again.(the mannosans enzyme activity preferably can be controlled between 0.04-0.08 u/ml in the enzyme liquid to be measured after the dilution).
Liquid sample can be directly with buffered soln (A.1.2.4) dilute, constant volume.The mannosans enzyme activity preferably can be controlled between 0.04-0.08 u/ml in the enzyme liquid to be measured after the dilution).
A.1.4.3 determination step
Draw 10.0ml mannan solution (A.1.2.6), 37 ℃ of balance 10min.
Draw the enzyme liquid of 10.0ml through suitably diluting, 37 ℃ of balance 10min.
Draw the enzyme liquid (through 37 ℃ balances) of 2.00ml through suitably diluting, join in the scale test tube, add 5mlDNS reagent again, electromagnetic oscillation 3s.Add 2.0ml mannan solution (A.1.2.6) then, 37 ℃ of insulation 30min, boiling water bath heating 5min.Be cooled to room temperature with tap water, add water and be settled to 25ml, electromagnetic oscillation 3s.Be blank with the blank sample of standard, at the mensuration absorbance A B of 540nm place.
Draw the enzyme liquid (through 37 ℃ balances) of 2.0ml through suitably diluting, join in the scale test tube, add 2.0ml mannan solution (A.1.2.6) (through 37 ℃ of balances) again, electromagnetic oscillation 3s, 37 ℃ of accurate insulation 30min.Add 5.0mlDNS reagent, electromagnetic oscillation 3s, enzyme digestion reaction.Boiling water bath heating 5min is cooled to room temperature with tap water, adds water and is settled to 25ml, electromagnetic oscillation 3s.Be blank with the blank sample of standard, at the mensuration absorbance A E of 540nm place.
A.1.5 calculate
XD = [（AE - AB）×K + CO] × 1000 /M/t------------------------（1）
X = XD·Df -------------------------（2）
In the formula:
The mannosans enzyme activity of XD-sample diluent, U/ml;
The absorbancy of AE-enzyme reaction solution;
The absorbancy of the blank sample of AB-enzyme;
The slope of K-typical curve;
The intercept of CO-typical curve;
The molecular weight of M-seminose (180.2);
T-enzyme digestion reaction time, min;
1000-transforming factor, 1mmol=1000 μ mol.
The XD value should be between 0.04-0.08 U/ml.If not in this scope, should reselect the extent of dilution of enzyme liquid, carry out assay determination again.
The vigor of mannase in X-sample, U/g or U/ml;
Total extension rate of Df-sample.
A.1.6 permissible variation as a result
The relative error of two replicate(determination) values of same sample is no more than 8.0%, and the mean value of the two is final enzyme activity determination value (keeping three position effective digitals).
Equipment used in the present invention is industry common equipment, comprises steeping tank, link-suspended basket centrifuge, mixes flow container, sheet frame, clear liquid jar, ultra-fine filter.
PH character
Preparation A solution: the citric acid solution of 0.05mol/l, the disodium phosphate soln of B solution: 0.1mol/l mixes two kinds of solution of A.B by a certain percentage, and adjusts pH to 2.5,3.0,3.5,4.0 with A or B ... 8.5 damping fluid.
Prepare substrate solution with distilled water.
Accurately take by weighing enzyme sample 1.0g in the 50ml volumetric flask, add 40ml distilled water, magnetic agitation 30min leaves standstill for some time, uses the distilled water constant volume.Do suitably dilution with the damping fluid of different pH again, measure enzyme according to measuring method and live.
| pH | AB MP1000 | With U.S. ferment | 01 pichia spp | This mannase | The BSA bacterium | The BSA aspergillus oryzae | The K pichia spp |
| 2.5 | 29556 | 0 | 0 | 2449 | 0 | 9774 | 0 |
| 3.0 | 37072 | 0 | 0 | 2733 | 0 | 12344 | 0 |
| 3.5 | 44634 | 3.5 | 785 | 2810 | 0 | 14328 | 0 |
| 4.0 | 50413 | 16 | 5084 | 2855 | 289 | 15369 | 29 |
| 4.5 | 57693 | 30 | 10805 | 2953 | 1545 | 16372 | 1633 |
| 5.0 | 57784 | 43 | 23068 | 2778 | 2611 | 16497 | 4604 |
| 5.5 | 59422 | 72 | 27116 | 2578 | 3235 | 18347 | 9083 |
| 6.0 | 58876 | 134 | 28301 | 2366 | 3278 | 18064 | 9649 |
| 6.5 | 50695 | 269 | 36842 | 1889 | 3157 | 15188 | 14014 |
| 7.0 | 32378 | 591 | 37821 | 1541 | 2583 | 10609 | 15452 |
| 7.5 | 20128 | 978 | 23634 | 1298 | 1437 | 6009 | 13661 |
| 8.0 | 19609 | 1066 | 19685 | 747 | 732 | 3842 | 10090 |
| 8.5 | 10137 | 926 | 12643 | 476 | 393 | 2795 | 6060 |
Be 100% to be converted to per-cent with vertex:
| pH | AB MP1000 | With U.S. ferment | 01 pichia spp | This mannase | The BSA bacterium | The BSA aspergillus oryzae | The K pichia spp |
| 2.5 | 50 | 0 | 0 | 83 | 0 | 53 | 0 |
| 3.0 | 62 | 0 | 0 | 93 | 0 | 67 | 0 |
| 3.5 | 75 | 0.3 | 2 | 95 | 0 | 78 | 0 |
| 4.0 | 85 | 1.5 | 13 | 97 | 9 | 84 | 0.2 |
| 4.5 | 97 | 2.8 | 29 | 100 | 47 | 89 | 11 |
| 5.0 | 97 | 4 | 61 | 94 | 80 | 90 | 30 |
| 5.5 | 100 | 6.7 | 72 | 87 | 99 | 100 | 59 |
| 6.0 | 99 | 13 | 75 | 80 | 100 | 98 | 62 |
| 6.5 | 85 | 25 | 97 | 64 | 96 | 83 | 91 |
| 7.0 | 54 | 55 | 100 | 52 | 79 | 58 | 100 |
| 7.5 | 34 | 92 | 62 | 44 | 44 | 33 | 88 |
| 8.0 | 33 | 100 | 52 | 25 | 22 | 21 | 65 |
| 8.5 | 17 | 87 | 33 | 16 | 12 | 15 | 39 |
Can find out that from last table this mannase is than AB MP1000 acid resistance pH1.0, ratio and U.S. ferment acid resistance pH3.5 are than 01 pichia spp acid resistance pH2.5, than BSA bacterium acid resistance pH1.5, than BSA aspergillus oryzae acid resistance pH1.0, than K pichia spp acid resistance pH2.5.
Thermotolerance
1g(ml) enzyme/100ml pH5.5 acetic acid-sodium acetate buffer stirred 30 minutes, and it is stand-by to get supernatant liquor.
The test tube that fills the 9ml damping fluid put preheating 10min in 35,45,55,65,75,80 ℃ the water-bath respectively, add respectively that 1.00ml enzyme supernatant liquor shakes up rapidly and accurately timing 10 minutes, take out in ice-water bath rapidly and cool off.Live in cooling back further dilution survey enzyme.
| Temperature | AB MP1000 | With U.S. ferment | 01 pichia spp | This mannase | The BSA bacterium | The BSA aspergillus oryzae | The K pichia spp |
| 35℃ | 58678 | 76 | 33940 | 3036 | 4396 | 18889 | 12463 |
| 45℃ | 66643 | 94 | 37302 | 3122 | 3878 | 20412 | 12958 |
| 55℃ | 63594 | 81 | 36938 | 3042 | 2295 | 20593 | 13018 |
| 65℃ | 63485 | 0 | 4116 | 3040 | 222 | 19481 | 115 |
| 75℃ | 25254 | 0 | 404 | 1886 | 0 | 7845 | 73 |
| 80℃ | 1064 | 0 | 516 | 613 | 0 | 430 | 0 |
Be 100% to be converted to per-cent with vertex:
| Temperature | AB MP1000 | With U.S. ferment | 01 pichia spp | This mannase | The BSA bacterium | The BSA aspergillus oryzae | The K pichia spp |
| 35℃ | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 45℃ | 100 | 100 | 100 | 100 | 88 | 100 | 100 |
| 55℃ | 100 | 100 | 100 | 100 | 52 | 100 | 100 |
| 65℃ | 100 | 0 | 11 | 100 | 5 | 100 | 0.9 |
| 75℃ | 40 | 0 | 1 | 62 | 0 | 40 | 0.6 |
| 80℃ | 1.7 | 0 | 1 | 20 | 0 | 2.2 | 0 |
Can find out that from last table this mannase is than the high temperature resistant 3-5 of AB MP1000 ℃, high temperature resistant about 13 ℃ of ratio and U.S. ferment, high temperature resistant about 12 ℃ than 01 pichia spp, high temperature resistant 30 ℃ than BSA bacterium, than BSA aspergillus oryzae high temperature resistant 2 ℃, high temperature resistant about 13 ℃ than K pichia spp.
Embodiment 2
Soak: press the mixed that the bent material of head mold solid fermentation and water are pressed 1:13, soak water and transfer pH4.5 with acetic acid, stirred 3 hours;
Centrifugal: under the condition of rotating speed 1000rpm in link-suspended basket centrifuge centrifugal 15min, get supernatant liquor after centrifugal;
Plate Filtration: add 7% diatomite, filter with flame filter press and obtain cleaner liquid;
Ultrafiltration and concentration: membrane molecule amount 10000 is concentrated into 1/12 of cleaner liquid volume;
Anticorrosion stable: in concentrated solution: the ratio of sorbyl alcohol: glycerine: potassium sorbate=85:10:5:0.1 adds stablizer, sanitas in concentrated solution, stir evenly.
Embodiment 3
Soak: press the mixed that the bent material of head mold solid fermentation and water are pressed 1:16, soak water and transfer pH4.5 with acetic acid, stirred 2 hours;
Centrifugal: under the condition of rotating speed 1200rpm in link-suspended basket centrifuge centrifugal 12min, get supernatant liquor after centrifugal;
Plate Filtration: add 10% diatomite, filter with flame filter press and obtain cleaner liquid;
Ultrafiltration and concentration: membrane molecule amount 10000 is concentrated into 1/8 of cleaner liquid volume;
Anticorrosion stable: in concentrated solution: the ratio of sorbyl alcohol: glycerine: potassium sorbate=85:10:5:0.1 adds stablizer, sanitas in concentrated solution, stir evenly.
Claims (3)
1. the making method of the high temperature resistant acid mannase of liquid comprises that the bent material of head mold solid fermentation soaks, centrifugal, filtration, ultrafiltration, anticorrosion stable, and the actual conditions of each step is as follows:
Soak: expect by the head mold solid fermentation is bent: the part by weight of water=1:8-16 feeds intake, and will soak in the song material input water, soaks water pH and transfers to 4.5, stirs 2-3 hour, gets mixed solution;
Centrifugal: that mixed solution is placed whizzer centrifugal 10-15min under the condition of rotating speed 800-1200rpm;
Filter: the supernatant liquor of getting after centrifugal places filter, adds the diatomite that accounts for supernatant liquor weight 7-10%, filters and obtains cleaner liquid;
Ultrafiltration and concentration: membrane molecule amount 10000 is concentrated into the 1/12-1/8 of cleaner liquid volume;
Anticorrosion stable: add sanitas and stablizer in concentrated solution, the part by weight of its adding is concentrated solution: sorbyl alcohol: glycerine: potassium sorbate=85:10:5:0.1 stirs evenly.
2. according to the making method of the high temperature resistant acid mannase of the described liquid of claim 1, wherein said whizzer is link-suspended basket centrifuge.
3. according to the making method of the high temperature resistant acid mannase of the described liquid of claim 1, wherein said filter is flame filter press.
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| CN101067130A (en) * | 2007-05-17 | 2007-11-07 | 江南大学 | Process of producing beta-mannase with Aspergillus niger |
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| CN1428415A (en) * | 2001-12-25 | 2003-07-09 | 北京锐思嘉业饲料应用技术研究中心 | Aspergillus niger capable of producing acidic beta-mannase and its fermentation and method for producing manno-oligose |
| CN101067130A (en) * | 2007-05-17 | 2007-11-07 | 江南大学 | Process of producing beta-mannase with Aspergillus niger |
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