CN100410370C - Process for producing bata-mannanase fermented by pseudo honey agaric and application thereof - Google Patents
Process for producing bata-mannanase fermented by pseudo honey agaric and application thereof Download PDFInfo
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- CN100410370C CN100410370C CNB2005101008745A CN200510100874A CN100410370C CN 100410370 C CN100410370 C CN 100410370C CN B2005101008745 A CNB2005101008745 A CN B2005101008745A CN 200510100874 A CN200510100874 A CN 200510100874A CN 100410370 C CN100410370 C CN 100410370C
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Abstract
The present invention provides a method for producing beta-mannase by fermenting a strain of false shoestring fungus and an application of the false shoestring fungus in the production of the beta-mannanase. The qualitative detection and the fermentation process of the false shoestring fungus for producing the beta-mannase comprises the following steps: step 1, a slant culture is inoculated in a solid culture medium containing 0.1 to 0.5% of trypan blue, a transparent hydrolysis ring can be seen after 5 to 15 days; step 2, after the false shoestring fungus cultivated by the slant culture is cultivated by first grade, second grade, third grade liquid, the false shoestring fungus is fermented for producing the beta-mannase. The production of the beta-mannase by bacterial strains produced by the present invention has simple manufacturing process, low cost and high utility.
Description
Technical field
The present invention relates to the bio-fermentation engineering field, relate in particular to a kind of armillariella tabescens and induce the method and the application in producing 'beta '-mannase thereof of producing 'beta '-mannase.
Background technology
Armillariella tabescens can produce the tart 'beta '-mannase through inducing.'beta '-mannase can decompose beta-mannase (as containing in the konjaku powder), forms manna oligosaccharide, and these oligose can be eliminated the anti-oxidant action of beta-mannase in the feed, improves the nutritive value of feed; Simultaneously, also can promote probiotics bifidobacterium growth in the human intestinal, regulate immunity system etc., therefore, the tart 'beta '-mannase has broad application prospects in feed and foodstuff additive.The tart 'beta '-mannase that armillariella tabescens produces, in application facet prospect is preferably arranged, the health food market of China has developed into the big market that annual sales amount reaches 30,000,000,000 Renminbi at present, along with approaching of aging society, China is expected to become healthcare products production and consumption state the biggest in the world, and this also provides good prospect for the market of functional oligose.
At present, because the needs of washing composition and paper-making industry, the most studies personnel concentrate on the 'beta '-mannase research of alkalescence both at home and abroad.And less to the research of acidic beta-mannase, acidic beta-mannase has broad application prospects in feed and foodstuffs industry.Animal heat is generally about 40 degree, and the small intestine pH that enzyme plays katalysis in vivo is 5.8～6.0.When selecting zymin, preferably make the suitableeest action condition of enzyme consistent, so that enzyme is brought into play its effect to greatest extent with the livestock and poultry alimentary tract condition.Between the general fungi optimal pH 4.5～5.5, and armillariella tabescens excretory 'beta '-mannase optimal pH 5.5～6.0, similar with the pH of small intestine, and this 'beta '-mannase 40 ℃ the insulation 30min still have 92.4% vigor, hold out broad prospects in application facet.It is domestic that to study at most be the bacterial classification aspergillus niger (name of patent application is " method of the aspergillus niger of a strain producing acidic beta-mannase and fermentation thereof and production mannooligo saccharide ", and number of patent application is " 01144782 ") of producing acidic beta-mannase.And the research of armillariella tabescens production 'beta '-mannase yet there are no relevant report.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art part, primary and foremost purpose of the present invention is to provide the method for a kind of armillariella tabescens (Armillariella tabescens) AS5.92 CGMCC fermentative production 'beta '-mannase.
Armillariella tabescens (Armillariella tabescens) AS5.92CGMCC of 'beta '-mannase is produced in this strain, and mutant or varient, can secrete 'beta '-mannase, the resistance toheat of 'beta '-mannase is good, operating temperature range is 30～75 ℃, 60 ℃ of optimal reactive temperatures; Reaction pH3～8, optimal pH 5.5～6.0.
Another object of the present invention is to provide the application of above-mentioned armillariella tabescens in the fermentative production 'beta '-mannase.
The present invention realizes by following technical solution: the method for armillariella tabescens fermentative production 'beta '-mannase comprises the steps:
(1) slant strains is seeded in contains in 0.1～0.5% bent sharp this blue solid medium visible transparent hydrolysis circle after 5～15 days;
(2) with the armillariella tabescens of slant culture after level liquid cultivation, secondary liquid culture, three grades of liquid culture, but the fermentative production 'beta '-mannase.
Described step (two) zymotechnique step is as follows:
1) slant culture: adding in the substratum is 1.5～1.8% agar by mass percentage, 26～27 ℃ of static cultivations 10～15 days, 4 ℃ of preservations;
2) level liquid cultivate, the secondary liquid culture: inoculum size is 5～30%, under 26～27 ℃, 140～180 rev/mins, pH5～7 conditions, cultivates in substratum 4～6 days;
3) three grades of liquid culture: inoculum size is 5～30%, under 26～27.5 ℃, 160～220 rev/mins, pH5～7 conditions, cultivates in substratum 3～6 days, and three grades of outer nutrient solutions of thalline born of the same parents are the 'beta '-mannase crude enzyme liquid.
Described bacterial classification is armillariella tabescens (Armillariella tabescens) AS5.92 CGMCC.
The culture medium prescription that described step () produces transparent hydrolysis circle is by percentage to the quality: murphy juice 10～40%, peptone 0.5～2%, konjaku powder 0.5～3%, KH
2PO
40.1 MgSO～0.5%,
40.1 vitamins B～0.5%,
10.01～0.05%, 26～27.5 ℃, static cultivation 5～15 days can be seen thalline and produce transparent hydrolysis circle on every side.
In order to realize the present invention preferably, the culture medium prescription in the described step (two) by mass percentage: murphy juice 10～40%, peptone 0.5～2%, konjaku powder 0.5～3%, KH
2PO
40.1 MgSO～0.5%,
40.1 vitamins B～0.5%,
10.01～0.05%.
In order to realize the present invention better, the culture medium prescription in the described step (two) is by mass percentage: murphy juice 25%, peptone 1%, konjaku powder 2%, KH
2PO
40.3%, MgSO
40.15%, vitamins B
10.01%.
The present invention compared with prior art has following advantage and beneficial effect:
It is raw material that the present invention adopts murphy juice, peptone, konjaku powder, induce the armillariella tabescens producing acidic beta-mannase, the acidic beta-mannase of being produced has 92.4% vigor at 40 ℃ of insulation 30min, better heat stability, optimal pH 5.5～6.0 is having broad application prospects aspect feed and the foodstuffs industry.Adopt transparent hydrolysis circle method can identify the gene that contains 'beta '-mannase in the armillariella tabescens, and this genetic expression is come out; This method fast, simply qualitative detection the 'beta '-mannase in the armillariella tabescens.
Embodiment
Below in conjunction with embodiment, the present invention is done detailed description further.
Embodiment 1
Armillariella tabescens (Armillariella tabescens) AS5.92 CGMCC.This armillariella tabescens can be secreted 'beta '-mannase, and the resistance toheat of 'beta '-mannase is good, and operating temperature range is 30～75 ℃, 60 ℃ of optimal reactive temperatures; Reaction pH3～8, optimal pH 5.5～6.0.The method of armillariella tabescens fermentative production 'beta '-mannase comprises the steps:
(1) transparent hydrolysis circle method qualitative detection armillariella tabescens can be produced 'beta '-mannase
Slant strains be seeded in contain in 0.1% bent sharp this blue solid medium, the plate culture medium prescription that produces transparent hydrolysis circle by percentage to the quality: murphy juice 25%, peptone 1%, konjaku powder 2%, KH
2PO
40.3%, MgSO
40.15%, vitamins B
10.01%.In 26.5 ℃, static cultivation 8 days can be seen thalline and produce transparent hydrolysis circle on every side.
(2) with the armillariella tabescens of slant culture after level liquid cultivation, secondary liquid culture, three grades of liquid culture, but the fermentative production 'beta '-mannase.
1) slant culture: culture medium prescription is by mass percentage: murphy juice 25%, peptone 1%, konjaku powder 2%, KH
2PO
40.3%, MgSO
40.15%, vitamins B
10.01%, agar is 1.8%, 26.5 ℃, static cultivation 12 days, 4 ℃ of preservations.
2) level liquid cultivation, secondary liquid culture: inoculum size is 30%, and culture medium prescription is by mass percentage: murphy juice 25%, peptone 1%, konjaku powder 2%, KH
2PO
40.3%, MgSO
40.15%, vitamins B
10.01%, 26.5 ℃ of shake-flask culture, 160 rev/mins, pH6.0,5 days.
3) three grades of liquid culture: inoculum size is 30%, and culture medium prescription is by mass percentage: murphy juice 25%, peptone 1%, konjaku powder 2%, KH
2PO
40.3%, MgSO
40.15%, vitamins B
10.01%, 27 ℃ of shake-flask culture, 200 rev/mins, pH6.0,4 days.Three grades of outer nutrient solutions of thalline born of the same parents are the 'beta '-mannase crude enzyme liquid.
The vitality test of 'beta '-mannase
PH5.50.02M citric acid-0.04M phosphoric acid buffer is towards the konjaku powder substrate of liquid preparation 0.5%, get 0.92ml in test tube, add enzyme liquid 80ul, replace enzyme liquid alive to compare with inactivator liquid, behind 60 ℃ of accurate response 15min, add 1.5mlDNS (3, the 5-dinitrosalicylic acid), behind the boiling water bath 10min, be settled to 10ml, measure absorbance value in the 540nm place.Under this experiment condition, the vigor definition: it is 1 enzyme unit alive that every milliliter of enzyme liquid per minute produces 1 μ m seminose.
Embodiment 2
The method of armillariella tabescens fermentative production 'beta '-mannase comprises the steps:
(1) transparent hydrolysis circle method qualitative detection armillariella tabescens can be produced 'beta '-mannase
Slant strains be seeded in contain in 0.5% bent sharp this blue solid medium, the plate culture medium prescription that produces transparent hydrolysis circle by percentage to the quality: murphy juice 10%, peptone 0.5%, konjaku powder 3%, KH
2PO
40.5%, MgSO
40.1%, vitamins B
10.02%.In 26.5 ℃, static cultivation 5 days can be seen thalline and produce transparent hydrolysis circle on every side.
(2) with the armillariella tabescens of slant culture after level liquid cultivation, secondary liquid culture, three grades of liquid culture, but the fermentative production 'beta '-mannase.
1) slant culture: culture medium prescription is by mass percentage: murphy juice 10%, peptone 0.5%, konjaku powder 3%, KH
2PO
40.5%, MgSO
40.1%, vitamins B
10.02%, agar is 1.5%, 26 ℃, static cultivation 10 days, 4 ℃ of preservations.
2) level liquid cultivation, secondary liquid culture: inoculum size is 20%, and culture medium prescription is by mass percentage: murphy juice 10%, peptone 0.5%, konjaku powder 3%, KH
2PO
40.5%, MgSO
40.1%, vitamins B
10.02%, 26 ℃ of shake-flask culture, 180 rev/mins, pH5.0,4 days.
3) three grades of liquid culture: inoculum size is 20%, and culture medium prescription is by mass percentage: murphy juice 10%, peptone 0.5%, konjaku powder 3%, KH
2PO
40.5%, MgSO
40.1%, vitamins B
10.02%, 27.5 ℃ of shake-flask culture, 220 rev/mins, pH5.0,3 days.Three grades of outer nutrient solutions of thalline born of the same parents are the 'beta '-mannase crude enzyme liquid.
Embodiment 3
The method of armillariella tabescens fermentative production 'beta '-mannase comprises the steps:
(1) transparent hydrolysis circle method qualitative detection armillariella tabescens can be produced 'beta '-mannase
Slant strains be seeded in contain in 0.3% bent sharp this blue solid medium, the plate culture medium prescription that produces transparent hydrolysis circle by percentage to the quality: murphy juice 40%, peptone 2%, konjaku powder 0.5%, KH
2PO
40.1%, MgSO
40.5%, vitamins B
10.05%.In 26.5 ℃, static cultivation 15 days can be seen thalline and produce transparent hydrolysis circle on every side.
(2) with the armillariella tabescens of slant culture after level liquid cultivation, secondary liquid culture, three grades of liquid culture, but the fermentative production 'beta '-mannase.
1) slant culture: culture medium prescription is by mass percentage: murphy juice 40%, peptone 2%, konjaku powder 0.5%, KH
2PO
40.1%, MgSO
40.5%, vitamins B
10.05%, agar is 1.6%, 27 ℃, static cultivation 15 days, 4 ℃ of preservations.
2) level liquid cultivation, secondary liquid culture: inoculum size is 5%, and culture medium prescription is by mass percentage: murphy juice 40%, peptone 2%, konjaku powder 0.5%, KH
2PO
40.1%, MgSO
40.5%, vitamins B
10.05%, 27 ℃ of shake-flask culture, 140 rev/mins, pH7.0,6 days.
3) three grades of liquid culture: inoculum size is 5%, and culture medium prescription is by mass percentage: murphy juice 40%, peptone 2%, konjaku powder 0.5%, KH
2PO
40.1%, MgSO
40.5%, vitamins B
10.05%, 26 ℃ of shake-flask culture, 160 rev/mins, pH7.0,6 days.Three grades of outer nutrient solutions of thalline born of the same parents are the 'beta '-mannase crude enzyme liquid.
As mentioned above, can realize the present invention preferably.
Claims (4)
1. the method for an armillariella tabescens fermentative production 'beta '-mannase comprises following processing step:
(1) slant strains is seeded in contains in 0.1～0.5% bent sharp this blue solid medium visible transparent hydrolysis circle after 5～15 days;
(2) with the armillariella tabescens of slant culture after level liquid cultivation, secondary liquid culture, three grades of liquid culture, but the fermentative production 'beta '-mannase;
1) slant culture: adding in the substratum is 1.5～1.8% agar by mass percentage, 26～27 ℃ of static cultivations 10～15 days, 4 ℃ of preservations;
2) level liquid cultivate, the secondary liquid culture: inoculum size is 5～30%, under 26～27 ℃, 140～180 rev/mins, pH5～7 conditions, cultivates in substratum 4～6 days;
3) three grades of liquid culture: inoculum size is 5～30%, under 26～27.5 ℃, 160～220 rev/mins, pH5～7 conditions, cultivates in substratum 3～6 days, and three grades of outer nutrient solutions of thalline born of the same parents are the 'beta '-mannase crude enzyme liquid;
Described bacterial classification is armillariella tabescens (Armillariella tabescens) AS5.92 CGMCC.
2. the method for armillariella tabescens fermentative production 'beta '-mannase according to claim 1 is characterized in that: the culture medium prescription that described step () produces transparent hydrolysis circle by mass percentage: murphy juice 10～40%, peptone 0.5～2%, konjaku powder 0.5～3%, KH
2PO
40.1 MgSO～0.5%,
40.1 vitamins B～0.5%,
10.01～0.05%, 26～27.5 ℃ of static cultivations 5～15 days produce transparent hydrolysis circle around can seeing thalline.
3. the method for armillariella tabescens fermentative production 'beta '-mannase according to claim 1 is characterized in that: the culture medium prescription in the described step (two) by mass percentage: murphy juice 10～40%, peptone 0.5～2%, konjaku powder 0.5～3%, KH
2PO
40.1 MgSO～0.5%,
40.1 vitamins B～0.5%,
10.01～0.05%.
4. the method for armillariella tabescens fermentative production 'beta '-mannase according to claim 1 is characterized in that: the culture medium prescription in the described step (two) by mass percentage: murphy juice 25%, peptone 1%, konjaku powder 2%, KH
2PO
40.3%, MgSO
40.15%, vitamins B
10.01%.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (6)
| Title |
|---|
| Structural features of two antitumor polysaccharides from thefruiting bodies of Armillariella tabescens. Kiho T.Chemical and Pharmaceutical Bulletin.,Vol.40 No.8. 1992 |
| Structural features of two antitumor polysaccharides from thefruiting bodies of Armillariella tabescens. Kiho T.Chemical and Pharmaceutical Bulletin.,Vol.40 No.8. 1992 * |
| 假蜜环菌液体深层发酵条件的研究. 刘大岭等.生物技术，第14卷第3期. 2004 |
| 假蜜环菌液体深层发酵条件的研究. 刘大岭等.生物技术，第14卷第3期. 2004 * |
| 发酵法生产β－甘露聚糖酶的研究现状. 罗强，孙启玲.四川食品与发酵，第1期. 2000 |
| 发酵法生产β－甘露聚糖酶的研究现状. 罗强，孙启玲.四川食品与发酵，第1期. 2000 * |
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