CN1231593C - Technology for producing glucomannan using neutral beta-mannase to degradate fine konjaku flour - Google Patents
Technology for producing glucomannan using neutral beta-mannase to degradate fine konjaku flour Download PDFInfo
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- CN1231593C CN1231593C CN 01128868 CN01128868A CN1231593C CN 1231593 C CN1231593 C CN 1231593C CN 01128868 CN01128868 CN 01128868 CN 01128868 A CN01128868 A CN 01128868A CN 1231593 C CN1231593 C CN 1231593C
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- enzyme
- glucomannan
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- oligo
- mannase
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Abstract
The present invention relates to microorganism fermentation and enzyme engineering, and provides a technology using neutral beta-mannase to degrade refined konjak powder to produce oligo glucomannan. The technology comprises: Bacillus sp. M10 seed liquid is inoculated in a selected culture medium, crude enzyme liquid is obtained under the condition that the enzyme producing condition is optimized, and then, the crude enzyme liquid is used for carrying out enzymolysis to refined konjak powder, so that an oligo glucomannan syrupus product is obtained. The technology has the advantages of high enzyme activity, stable enzyme production, simple enzymolysis technology, high product physiological active substance content, high product yield, etc.
Description
The present invention relates to microbial fermentation and enzyme engineering, belong to functional oligose Production by Enzymes technology.Oligose is meant the general name of a class sugar of straight chain that 2 to 10 monosaccharide groups are formed by connecting by glycosidic link or branched chain.In recent years discover that oligo-glucomannan (or claim mannooligo saccharide) is except that having bifidobacterium factor functional, still have absorption, adhesion and remove the humans and animals enteric pathogenic bacteria, strengthen humans and animals non-specific immunity tool auxiliary agent activity, and the unique biological characteristics that promotes the hepatic secretion mannose binding protein is arranged.Be applied to healthcare products as dietetic food and foodstuff additive, be applied to fodder industry and substitute antibiotic effect as little ecological promotor and be applied to agricultural as of growth, growth, breeding, the diseases prevention and disease-resistant of a kind of novel hormonal with the regulation and control plant.Its excellent function and application DEVELOPMENT PROSPECT has caused the interest of Chinese scholars to 'beta '-mannase (β-1,4-D-mannan mannohydrolase:EC 3.2.1.78) and oligo-glucomannan production research.'beta '-mannase commodity produced in USA " with U.S. ferment " by name have entered China market.The production of functional oligose, Japan's starting is morning, and output and kind occupy prostatitis, the world.The domestic neutral 'beta '-mannase that has Institute of Microorganism, Academia Sinica to produce at the research report in this field with subtilis (Bacillus subtilis) BMG602, enzyme activity is 96IU/mL under optimum conditions, and Yunnan University has carried out separation screening and the product enzyme The Characteristic Study of screening K3-14 'beta '-mannase generation bacterium from soil.Nankai University has reported the neutral 'beta '-mannase by the bacterial strain NK-27 generation of Bacillus licheniformis (Bacillus licheniformis) mutagenic and breeding, and pure enzyme liberating 1% konjaku powder in purified back is produced functional oligo-glucomannan.Produce the research of 'beta '-mannase with Alkaliphilic bacillus, the usefulness Alkalophilic Bacillus sp.N16-5 fermentative production 'beta '-mannase of Institute of Microorganism, Academia Sinica's report, enzyme activity 160U/mL, and produce the industrialization report of mannooligo saccharide with this enzyme liberating konjaku powder.In these researchs, or exist enzyme activity not high, unstable, or complex process, cost height, as use alkaline ' beta '-mannase, and enzymolysis process carries out in the buffering system of pH9～10, and the heating glucomannan might become irreversible gel under alkaline condition, enzymolysis product need neutralize with acid, salt content height in the product increases the difficulty of desalinating process, influences the mouthfeel of product simultaneously.
The present invention's purpose is to seek the new technology with konjaku powder production oligo-glucomannan that a kind of technology is simple, cost is low.
The present invention can be achieved in the following manner:
Inoculation Bacillus sp.M10 seed liquor in culture medium selected, obtain crude enzyme liquid at suitable condition of enzyme production bottom fermentation, again this crude enzyme liquid is used for the enzymolysis konjaku powder, enzymolysis reaction heats up behind the reaction certain hour, precipitation is abandoned in filtration, filtrate is carried out secondary ions exchange desalination, decolouring, filtration sterilization behind activated carbon decolorizing, obtain the oligo-glucomannan syrupy product, will obtain the oligo-glucomannan powder-like product after the syrup spraying drying.
Introduce this technology in detail below in conjunction with accompanying drawing:
The enzymatic production substratum is formed: konjaku powder 40.0g, yeast powder 30.0g, Na
2HPO
44.0g, KH
2PO
42.0g, (NH
4)
2SO
410.0g, Na
2CO
33.5g, CaCl
22.0g, MgCl
26H
2O0.6g, CoCl
26H
2O 0.01g, MnCl
24H
2O 1.0g adds H
2O to l liter.
Produce enzymatic process: in producing the enzyme substratum, add the 2% Bacillus sp.M10 seed liquor of cultivating 18h, 30～32 ℃, pH7.0-7.5,180r/min shaking culture 48h produces enzyme and peaks, centrifugal or the filtration of fermented liquid, supernatant liquor or filtrate are crude enzyme liquid, and the DNS method is measured enzyme activity, and the optimal reactive temperature of enzyme and pH are respectively 50 ℃ and 6.0.Fe
3+, Al
3+, EDTA, Hg
2+Enzyme there is restraining effect, and Ba
2+, Mn
2+Enzyme there is activation.Crude enzyme liquid is with 60% saturation ratio (NH
4)
2SO
4Precipitation, 4000xg frozen centrifugation 20 minutes promptly obtains thick enzyme precipitation, can be standby in room temperature preservation, thick enzyme mud uses behind the dialysis desalination.
With the above-mentioned crude enzyme liquid that obtains according to the enzyme activity thin up that records, the ratio (adding enzyme liquid is 5 to 1 with the envelope-bulk to weight ratio of konjaku powder) that adds 150u enzyme amount according to 1 gram konjaku powder adds the enzyme liquid after diluting, making concentration of substrate is 20%, at 40-50 ℃, enzymolysis is 2 hours under the pH5.0-7.0 condition, be warming up to 70-75 ℃ 15 minutes with enzymolysis reaction, sheet frame or diatomite filtration also discard precipitation, filtrate is through activated carbon decolorizing, secondary ions exchange desalination, decolouring, filtration sterilization obtains the oligo-glucomannan syrupy product, will obtain the oligo-glucomannan powder-like product after the syrup spraying drying.
The enzymolysis product of 20% concentration of substrate high-pressure liquid phase chromatograph after making with extra care shows that the relative content of forming sugar part is: monose 4.26%, disaccharides 12.17%, trisaccharide 20.39%, tetrose 17.08%, pentasaccharides 20.47%, six sugared above oligose 15.63%, glucomannan 10%, two～pentasaccharides content reach 70.11%.
The oligo-glucomannan slurry is handled 6h, 1h and 15min respectively with 85 ℃, 100 ℃, 121 ℃, the reducing sugar amount of 85 ℃ of heat treated 6h treatment solutions does not change, under 100 ℃ and 121 ℃ of conditions, the reducing sugar amount of treatment solution reduces 2%, color no change---water white transparency, oligose is degraded and light hydrogenation (aldehyde radical → alcohol radical) is only arranged, to ph stability, at pHl-12, place and measure reducing sugar amount no change in the oligo-glucomannan slurry after 7 days under the normal temperature.
The present invention has the following advantages:
1, enzyme activity height, stable, the fermentation period weak point of product enzyme are difficult for bacteria infection;
2, enzymolysis process is simple, and enzymolysis carries out in the neutral reaction system;
3, product yield is more than 75%, oligose relative content 80-90% in the product, and the physiologically active height, heat and ph stability are good;
4, technical process is short, and cost is low.
Embodiment 1
The fermention medium 100ml that in the triangular flask of two 1000ml, packs into, inoculum size is 2% to cultivate the seed liquor of 18～20h, 30～32 ℃, 180r/min shaking culture 48h, the DNS method is measured enzyme activity and is respectively 301.87u/mL, 307.16u/mL.
Embodiment 2
Take by weighing konjaku powder 20.0g, with 150u/g enzyme amount, add the crude enzyme liquid of dilution, making concentration is 20.0%, pH6.0,40-50 ℃, intermittently stirs enzymolysis 2h, intensification enzymolysis reaction.Enzymolysis product filters, filtrate is through activated carbon decolorizing, secondary ions exchange desalination, can get product 15.0g, high-pressure liquid phase chromatograph is the result show: the relative content of forming sugar part in the product is that monose 4.26%, disaccharides 12.17%, trisaccharide 20.39%, tetrose 17.08%, pentasaccharides 20.47%, the above oligose 15.63% of six sugar, glucomannan 10%, two～pentasaccharides content reach 70.11%.
Claims (2)
1, produces the oligo-glucomannan technology with neutral beta-mannase enzyme liberating konjaku powder, it is characterized in that: consisting of konjaku powder 40.0g, yeast powder 30.0g, Na
2HPO
44.0g, KH
2PO
42.0g, (NH
4)
2SO
410.0g, Na
2CO
33.5g, CaCl
22.0g, MgCl
26H
2O 0.6g, CoCl
26H
2O 0.01g, MnCl
24H
2O 1.0g adds H
2O to 1 liter inoculation of medium Bacillus sp.M10 seed liquor, at 30～32 ℃, pH7.0-7.5, the condition of enzyme production bottom fermentation of 180r/min shaking culture 48h obtains crude enzyme liquid, again this crude enzyme liquid is used for the enzymolysis konjaku powder, enzymatic hydrolysis condition is 20% concentration of substrate, 150u/g enzyme amount, 40-50 ℃, pH5.0-7.0, react and heat up after 2 hours with enzymolysis reaction, precipitation is abandoned in filtration, filtrate is carried out secondary ions exchange desalination behind activated carbon decolorizing, decolouring, filtration sterilization, obtain the oligo-glucomannan syrupy product, will obtain the oligo-glucomannan powder-like product after the syrup spraying drying.
2, the technology of producing oligo-glucomannan according to claims 1 described neutral beta-mannase enzyme liberating konjaku powder, it is characterized in that: the character of enzyme is that optimal reactive temperature and pH are respectively 50 ℃ and 6.0.Fe
3+, Al
3+, EDTA, Hg
2+Enzyme there is restraining effect, and Ba
2+, Mn
2+Enzyme there is activation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128868 CN1231593C (en) | 2001-09-18 | 2001-09-18 | Technology for producing glucomannan using neutral beta-mannase to degradate fine konjaku flour |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128868 CN1231593C (en) | 2001-09-18 | 2001-09-18 | Technology for producing glucomannan using neutral beta-mannase to degradate fine konjaku flour |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1408879A CN1408879A (en) | 2003-04-09 |
| CN1231593C true CN1231593C (en) | 2005-12-14 |
Family
ID=4668681
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| CN 01128868 Expired - Fee Related CN1231593C (en) | 2001-09-18 | 2001-09-18 | Technology for producing glucomannan using neutral beta-mannase to degradate fine konjaku flour |
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Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008024B (en) * | 2007-01-30 | 2010-06-02 | 武汉泰运投资担保有限公司 | Process for producing high-purity kanjak mannan-oligosaccharides |
| CN101570769B (en) | 2008-04-29 | 2013-06-19 | 安琪酵母股份有限公司 | Yeast glucan and mannan and production method thereof |
| CN101861982B (en) * | 2009-04-16 | 2012-09-26 | 罗晶 | Oligosaccharide sweet potato rice and preparation method thereof |
| CN101861984B (en) * | 2009-04-16 | 2012-05-23 | 罗晶 | Oligosaccharide konjak rice and preparation method thereof |
| CN102048029B (en) * | 2010-08-10 | 2013-03-13 | 浙江省农业科学院 | Preparation method of saccharomyces-cerevisiae enriched oligo-glucomannan feed additive |
| CN102715388A (en) * | 2012-07-03 | 2012-10-10 | 江南大学 | Method for degrading konjac gum and preparing kappa-carrageenan compound colloid |
| CN103468766B (en) * | 2013-09-18 | 2015-07-01 | 恩施天天佳生物科技有限公司 | Preparation method of high-purity mannan oligosaccharide |
| CN107446969A (en) * | 2017-07-17 | 2017-12-08 | 天津科技大学 | Application of the oligosaccharides enzyme in terms of fresh konjak produces konjak oligosaccharide |
| CN107974438B (en) * | 2017-11-02 | 2020-09-29 | 中国农业大学 | Beta-mannase derived from rhizopus microsporus, and coding gene and application thereof |
| CN108851199B (en) * | 2018-05-22 | 2019-11-08 | 湖北中烟工业有限责任公司 | A kind of preparation method of enzymolysis glucomannan and the compound humectant of medicinal fungus fermentation material |
| CN111118084A (en) * | 2019-12-27 | 2020-05-08 | 苏州司克瑞特生物科技有限公司 | Preparation method of konjac mannan oligosaccharide |
| CN112587535A (en) * | 2021-01-06 | 2021-04-02 | 澳门大学 | Glucomycooligosaccharide series composition, preparation method and application thereof |
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2001
- 2001-09-18 CN CN 01128868 patent/CN1231593C/en not_active Expired - Fee Related
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