CN103173427A - Method for producing beta-mannase by utilizing Aspergillus niger fermentation - Google Patents
Method for producing beta-mannase by utilizing Aspergillus niger fermentation Download PDFInfo
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- CN103173427A CN103173427A CN2012102402293A CN201210240229A CN103173427A CN 103173427 A CN103173427 A CN 103173427A CN 2012102402293 A CN2012102402293 A CN 2012102402293A CN 201210240229 A CN201210240229 A CN 201210240229A CN 103173427 A CN103173427 A CN 103173427A
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Abstract
The invention aims to provide a method for producing beta-mannase by utilizing Aspergillus niger fermentation. According to the method, beta-mannase can be secreted and expressed by growing the Aspergillus niger in a liquid culture medium for 96 hours; the most preferable catalytic temperature of mannase is 40 DEG C, the most preferable pH value is between 2 and 6, the catalytic activity loss of mannase cannot exceed 40% after maintaining the temperature at 70 DEG C for 20 min, and mannase has a relatively good heat resisting property; and under a shake flask condition, the fermenting activity reaches at most 1200 U/mL, and mannase has a potential prospect to be used as a feed additive.
Description
Technical field
The present invention relates to a kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase, the invention still further relates to a kind of fodder additives.
Background technology
Corn-soybean meal diet is the main daily ration that China raises monogastric animal, and in soybean, contained mannosans has very strong anti-oxidant action.At present, the annual soybean import of China is 4,000 ten thousand tons of left and right, wherein is used for fodder industry more than 80%.Mannosans is the important component of plant hemicellulose, is the wire polysaccharide that is formed by connecting by β-Isosorbide-5-Nitrae-D mannopyranose, and in dregs of beans, the content of mannosans is more than 1.2%.Mannosans has high-hydrophilic, absorb water in a large number in the digestive tube of monogastric animal, increased the viscosity of alimentary canal content, the wriggling of opposing stomach and intestine, directly affect animal to the digesting and assimilating of nutritive substance, cause plant's environmental pollution because containing more organism in movement.
The complete enzymolysis of mannosans needs the synergy of the plurality of enzymes such as 'beta '-mannase, beta-Mannosidase, beta-glucosidase enzyme.The viscosity of beta-mannase endonuclease capable efficient degradation mannosans wherein, the application in feed is more extensive.'beta '-mannase is the hemicellulose enzyme that a class can be hydrolyzed mannosans, extensively is present in animals and plants and microorganism.
The 'beta '-mannase mannosans in feed of degrading not only can be eliminated the anti-oxidant action of mannosans, and the mannooligo saccharide that generates simultaneously plays an important role in animal produces.Evidence: mannooligo saccharide can improve piglet day weight gain and feed conversion rate, and its reason may be its immunizing power that has improved piglet, has suppressed pathogenic bacteria in GI propagation.In addition, mannooligo saccharide also has certain immunogenicity, can stimulate immune response, is combined with virus, toxin, slows down the absorption of antigen, and the colony balance of regulating intestinal canal disturbs the field planting of pathogenic bacteria.Research is also found, mannooligo saccharide can significantly improve IgA, IgG and the content of IgM and the level of blood middle leukocytes Jie element-2 in aseptic piglet serum and intestinal mucosa, strengthen the activity of T lymphocyte function and small intestine lymphoblast, strengthen the interior leukocytic phagocytic activity of small intestine and promote lymphocyte to discharge IFN-gamma cells element.Shao Liang equality report, mannooligo saccharide can significantly improve the level that the piglet white corpuscle breaks up antibody CD3 by the utmost point.In recent years, more and more be subject to people's attention about the research of mannooligo saccharide absorbing mycotoxin, mannooligo saccharide can in conjunction with zearalenone, by physical adsorption or directly in conjunction with mycotoxin, and not affect other forage components yet.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase.
This aspergillus niger (Aspergillus niger) YM33182, directly fetch and come from the Cao Yunhe place, secondary source is Chen Youwei, this bacterial classification is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 31st, 2006, and deposit number is: CCTCCNo M206113.
A kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase is characterized in that, the step that described fermentation of Aspergillus niger is produced the beta-mannase enzyme method is as follows:
(1) preparation fermented bacterium: with aspergillus niger (Aspergillus niger) YM33182 of slant culture through the liquid spawn slant strains cultivate and the level liquid cultivation after, obtain fermented bacterium; Its detailed process is:
1) slant strains: use the potato substratum, to substratum, under 30-35 ℃ of condition, shake-flask seed culture medium culturing 24-72 hour is after spore covers with, under aseptic cooling water washing with the bacterial classification streak inoculation of 4 ℃ of Refrigerator stores;
2) level liquid is cultivated: with a slant tube 10ml sterilized water, the preparation spore suspension makes spore content 1 * 10
8More than CFU/ml, get the 1ml spore suspension and be inoculated in the 250ml seed culture medium, bottle was cultivated 8-48 hour under 25-35 ℃, 250rpm condition, obtained seed fermentation liquid;
(2) utilize strain fermentation obtained above to produce 'beta '-mannase:
3), to utilize the strain fermentation obtain to produce 'beta '-mannase be to adopt liquid fermentation process production, the fermentative medium formula of use comprises in parts by weight: Semen Maydis powder 35-45 part, dregs of beans 11-14 part, ammonium sulfate 1-2 part, K
2HPO
43.5-4.5 part, Rhizoma amorphophalli powder 0.5-1.0g is dissolved in 1000mL, in distilled water, regulates the pH value, autoclaving.
2, a kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase according to claim 1, its feature was measured the mannosans enzyme activity at 60-84 hour in described liquid fermentation medium, and enzyme activity is the highest; The best catalytic pH of described liquid fermentation medium enzyme has higher catalysis activity at 2-6.0;
3, method according to claim 1 is characterized in that: the enzyme that described liquid fermentation medium produces has thermotolerance preferably at the 60-70 degree;
4, method according to claim 1 is characterized in that: described potato culture medium prescription comprises in parts by weight: potato decortication chopping, 15-25 part; Sucrose, 1.5-2.5 part; Agar 1-2.0 part; 100 parts, tap water, water: raw material=1: 0.015-0.2;
5, method according to claim 1 is characterized in that: described sterilising conditions is: 115-125 ℃ autoclaving 15-25 minute;
6, a kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase according to claim 1 is characterized in that the acidic beta-mannase hydrolysis mannosans that utilizes fermentation of Aspergillus niger to obtain.
7, acidic beta-mannase hydrolysis mannosans according to claim 6, it is characterized in that: described mannosans is konjaku powder and Semen Maydis powder.
In aspergillus niger liquid medium within of the present invention, growth is 96 hours, can the secreting, expressing 'beta '-mannase.The suitableeest catalytic temperature of mannase is 40 ℃, and optimum pH in the time of 70 ℃, is incubated 20 minutes between 2-6, and the loss in catalytic activity of enzyme is no more than 40%, has thermotolerance preferably.Under the shaking flask condition, fermentative activity is up to 1200U/mL, has the potential prospect for fodder additives.
Description of drawings
Fig. 1 is the enzyme activity (U/ml) of the supernatant liquor mannase of fermentation different time points;
Fig. 2 is the relative activity (percentage ratio %) of mannase under condition of different temperatures;
Fig. 3 is the relative catalysis activity (percentage ratio %) of mannase under condition of different pH;
Embodiment
The preparation of DNS reagent: take Seignette salt 91.0g, be dissolved in 500mL water, add 3,5-dinitrosalicylic acid 3.15g, NaOH 20.0g, be no more than 50 ℃ of water-bath dissolvings; Add phenol 2.5g, sodium sulphite anhydrous 99.3 2.5g, stirring and dissolving is settled to 1000mL after cooling again; Be stored in brown bottle, place rear use of a week.
The mensuration (DNS method) that the 'beta '-mannase enzyme is lived: get solution 1mL to be measured, add 1mL 0.8% (quality percentage composition) mannan solution, react 20min in water bath with thermostatic control, then add 2.5mL DNS reagent (termination reaction).Boiling water bath boils 5min, then is cooled to room temperature, is settled to 12.5mL with distilled water, surveys OD
540
Under pH6.0 and 37 ℃ of conditions, the required enzyme amount that per minute hydrolysis from substrate polygalactomannan (Sigma, G0753) produces the seminose of 1 μ mol is defined as 1 enzyme unit that lives, and represents with U.
Embodiment 1: preparation fermentation of Aspergillus niger bacterial classification
Slant medium: potato substratum (potato decortication chopping, 20g; Sucrose, 2.0g; Agar 1.5g; Tap water 100mL, 120 ℃ of autoclavings 20 minutes); Shake-flask seed substratum: potato substratum (not containing agar).The bacterial classification of 4 ℃ of refrigerator preservations is inoculated in slant medium, cultivates 48hr for 30 ℃, after spore covers with, under aseptic cooling water washing.A slant tube 10ml sterilized water, the preparation spore suspension, the content of spore is 10
8More than CFU/ml.Inoculation 1mL spore suspension in the 250mL seed culture medium.30 ℃, 250rpm shake-flask culture 20hr obtain seed fermentation liquid.
Embodiment 2: the aspergillus niger shake flask fermentation is produced the beta-mannase enzymatic process
Fermentative medium formula: Semen Maydis powder 40g, dregs of beans 12.5g, ammonium sulfate 1.5g, K
2HPO
44.0g Rhizoma amorphophalli powder 0.75g is dissolved in 1000mL, in distilled water, regulates pH value to 6.2,120 ℃ of autoclavings 20 minutes.500mL shaking flask liquid amount 60mL.According to the ratio of 1: 1000 inoculation seed liquor, 30 ℃, 250rpm shake continuously and cultivate 96hr, get fermented liquid 1mL every 12hr, and 12000rpm is centrifugal, gets supernatant liquor and measure the mannosans enzyme activity under pH6.0 and 37 ℃ of conditions.The results are shown in Figure 1.
Embodiment 3: the zymologic property of 'beta '-mannase
1, the mensuration of optimum temperuture: respectively 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ with 70 ℃ of conditions under measure the relative catalysis activity of enzyme.The best catalytic temperature of enzyme is 40 ℃, in 30-60 ℃ of scope, all remains on more than 60% of best catalytic activity.The results are shown in Figure 2.
2, the mensuration of suitable catalytic pH value: in 2.0,3.0,4.0,5.0,6.0,7.0 damping fluids, measure the catalytic activity of enzyme respectively.The best catalytic pH 4.0 of enzyme has higher catalysis activity in the 2.0-5.0 scope.The results are shown in Figure 3.
3, the mensuration of temperature stability: crude enzyme liquid is incubated 20 minutes at 60 ℃, 70 ℃ and 80 ℃ respectively, then measures residual enzyme under the suitableeest catalytic condition.Compare with the enzyme activity without heat treated, calculate remnant enzyme activity.This mannase also had 55% remnant enzyme activity in 20 minutes 70 ℃ of insulations, and this enzyme has thermotolerance preferably.The results are shown in Table 1.
The thermotolerance of table 1, mannase
| Treatment temp (℃) | 30 | 60 | 70 | 80 |
| Remnant enzyme activity (%) | 100 | 82 | 55 | 27 |
Embodiment 4: the crude enzyme liquid preparing manna oligosacchride with zymohydrolysis of konjaku flour
Accurately take the 10g Rhizoma amorphophalli powder, be dissolved in aqua sterilisa, be settled to 100mL, heat while stirring, make it abundant dissolving, preparation 10% solution.50mL Rhizoma amorphophalli powder solution is mixed with 10mL fermented supernatant fluid (enzyme activity 1000U/mL), is placed in 40 ℃ of water-baths insulation 5 hours, during stirred 1 time every 10 minutes.Add 10mLNa in 50mL Rhizoma amorphophalli powder solution in addition
2HPO
4-citrate buffer solution (pH6.0) is blank.After reaction is completed, the content of taking sample determination mannooligo saccharide.Result shows, approximately 70% mannosans is degraded into mannooligo saccharide.
Claims (7)
1. a method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase, is characterized in that, the step that described fermentation of Aspergillus niger is produced the beta-mannase enzyme method is as follows:
(1) preparation fermented bacterium: with aspergillus niger (Aspergillus niger) YM33182 of slant culture through the liquid spawn slant strains cultivate and the level liquid cultivation after, obtain fermented bacterium; Its detailed process is:
1) slant strains: use the potato substratum, to substratum, under 30-35 ℃ of condition, shake-flask seed culture medium culturing 24-72 hour is after spore covers with, under aseptic cooling water washing with the bacterial classification streak inoculation of 4 ℃ of Refrigerator stores;
2) level liquid is cultivated: with a slant tube 10ml sterilized water, the preparation spore suspension makes spore content 1 * 10
8More than CFU/ml, get the 1ml spore suspension and be inoculated in the 250ml seed culture medium, bottle was cultivated 8-48 hour under 25-35 ℃, 250rpm condition, obtained seed fermentation liquid;
(2) utilize strain fermentation obtained above to produce 'beta '-mannase:
3), to utilize the strain fermentation obtain to produce 'beta '-mannase be to adopt liquid fermentation process production, the fermentative medium formula of use comprises in parts by weight: Semen Maydis powder 35-45 part, dregs of beans 11-14 part, ammonium sulfate 1-2 part, K
2HPO
43.5-4.5 part, Rhizoma amorphophalli powder 0.5-1.0g is dissolved in 1000mL, in distilled water, regulates the pH value, autoclaving.
2. a kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase according to claim 1, its feature was measured the mannosans enzyme activity at 60-84 hour in described liquid fermentation medium, and enzyme activity is the highest; The best catalytic pH of described liquid fermentation medium enzyme has higher catalysis activity at 2-6.0;
3. method according to claim 1 is characterized in that: the enzyme that described liquid fermentation medium produces has thermotolerance preferably at the 60-70 degree;
4. method according to claim 1, it is characterized in that: described potato culture medium prescription comprises in parts by weight: potato decortication chopping, 15-25 part; Sucrose, 1.5-2.5 part; Agar 1-2.0 part; 100 parts, tap water, water: raw material=1: 0.015-0.2;
5. method according to claim 1, it is characterized in that: described sterilising conditions is: 115-125 ℃ autoclaving 15-25 minute;
6. a kind of method of utilizing fermentation of Aspergillus niger to produce 'beta '-mannase according to claim 1, is characterized in that the acidic beta-mannase hydrolysis mannosans that utilizes fermentation of Aspergillus niger to obtain.
7. acidic beta-mannase according to claim 6 is hydrolyzed mannosans, and it is characterized in that: described mannosans is konjaku powder and Semen Maydis powder.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820421A (en) * | 2014-02-25 | 2014-05-28 | 济南天天香有限公司 | Doublet mannanase and preparation method |
| CN104651338A (en) * | 2015-02-27 | 2015-05-27 | 江苏奕农生物工程有限公司 | Acidic temperature-resistance B-seminase as well as gene and application thereof |
| CN105087521A (en) * | 2014-05-21 | 2015-11-25 | 东莞泛亚太生物科技有限公司 | Mannase capable of improving enzyme yield and activity |
| CN105838697A (en) * | 2016-01-18 | 2016-08-10 | 浙江大学舟山海洋研究中心 | Fermentation method for producing salt-tolerant cellulase from marine aspergillus niger |
| CN112715763A (en) * | 2020-11-11 | 2021-04-30 | 贵州省畜牧兽医研究所 | Guizhou local pig breeding method with high intramuscular fat content |
| CN114350638A (en) * | 2022-01-14 | 2022-04-15 | 山东隆科特酶制剂有限公司 | Method for producing high-temperature-resistant acidic beta-mannase |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820421A (en) * | 2014-02-25 | 2014-05-28 | 济南天天香有限公司 | Doublet mannanase and preparation method |
| CN103820421B (en) * | 2014-02-25 | 2016-07-13 | 济南天天香有限公司 | A kind of bimodal mannase and preparation method thereof |
| CN105087521A (en) * | 2014-05-21 | 2015-11-25 | 东莞泛亚太生物科技有限公司 | Mannase capable of improving enzyme yield and activity |
| CN105087521B (en) * | 2014-05-21 | 2018-02-09 | 东莞泛亚太生物科技有限公司 | The mannase of tool lifting production of enzyme and activity |
| CN104651338A (en) * | 2015-02-27 | 2015-05-27 | 江苏奕农生物工程有限公司 | Acidic temperature-resistance B-seminase as well as gene and application thereof |
| CN105838697A (en) * | 2016-01-18 | 2016-08-10 | 浙江大学舟山海洋研究中心 | Fermentation method for producing salt-tolerant cellulase from marine aspergillus niger |
| CN112715763A (en) * | 2020-11-11 | 2021-04-30 | 贵州省畜牧兽医研究所 | Guizhou local pig breeding method with high intramuscular fat content |
| CN112715763B (en) * | 2020-11-11 | 2022-12-27 | 贵州省畜牧兽医研究所 | Guizhou local pig breeding method with high intramuscular fat content |
| CN114350638A (en) * | 2022-01-14 | 2022-04-15 | 山东隆科特酶制剂有限公司 | Method for producing high-temperature-resistant acidic beta-mannase |
| CN114350638B (en) * | 2022-01-14 | 2023-06-27 | 山东隆科特酶制剂有限公司 | Method for producing high-temperature-resistant acidic beta-mannase |
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Application publication date: 20130626 |