CN104099386A - Method for preparing alginate oligosaccharide through enzymatic hydrolysis - Google Patents
Method for preparing alginate oligosaccharide through enzymatic hydrolysis Download PDFInfo
- Publication number
- CN104099386A CN104099386A CN201310116610.3A CN201310116610A CN104099386A CN 104099386 A CN104099386 A CN 104099386A CN 201310116610 A CN201310116610 A CN 201310116610A CN 104099386 A CN104099386 A CN 104099386A
- Authority
- CN
- China
- Prior art keywords
- algin
- oligosaccharide
- obtains
- streptomyces
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention relates to a method for preparing alginate oligosaccharide through enzymatic hydrolysis. An employed bacterial stain is pathogenicity-free streptomyces microbe streptomyces violaceoruber. Alginate lyase secreted by the bacteria to extracellular places has relatively high degradation capability, and is capable of degrading alginate oligosaccharide produced by algin. The alginate oligosaccharide obtained by performing ethanol precipitation, centrifugation and freeze drying on the enzymatic-hydrolysis liquid has the main polymerization degree of 2-6, and has the biological activity for promoting wheat growth through experiments. The preparation method has the advantages of low cost, simple operation and technological route, less equipment and the like.
Description
Technical field
The present invention relates to a kind of enzymolysis algin oligosaccharide and preparation method thereof, belong to agricultural application field.
Background technology
Algin oligosaccharide (alginate oligosaccharides) is the low molecular weight fraction of the less and good water solubility of the polymerization degree that obtains through hydrolysis of algin.Because algin oligosaccharide has multiple physiologically active, can be widely used in the fields such as food, medicine, agricultural and makeup, therefore there is huge application and exploitation value.The preparation method of algin oligosaccharide mainly contains chemical hydrolysis and algin catenase hydrolysis method.Because algin catenase hydrolysis method has the advantages such as Substratspezifitaet is good, reaction conditions is gentle, receive much concern in recent years.Algin catenase is at occurring in nature wide material sources.At present, reported that multiple-microorganism has the activity of algin catenase, as replaced Zymomonas mobilis (Alteromonas sp.), Pseudoalteromonas (Pseudoalteromonas), vibrio marinopraesens (Vibrio sp.) etc.Yet having many in these microorganisms is pathogenic bacterium, causes septicemia, thereby limited the application of this quasi-microorganism as vibrio marinopraesens can infect Mammals.Streptomyces violaceus (Streptomyces violaceoruber) is the separated (Streptomyces violaceus (Streptomyces violaceoruber) of the streptomyces microorganism with algin catenase activity and no pathogenicity obtaining from soil, bacterial strain deposit number: CPCC200534, source: Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences, 1970 collection time).Algin catenase activity that this bacterium produces is higher and can be secreted into outside born of the same parents.The present invention be take algin polysaccharide as raw material, prepares the algin oligosaccharide of main polymerization degree 2-6, and plant experimental confirms that it has growth promoting function, can be used for preparing Plant growth-promoting effect agent.
Summary of the invention
The object of the invention is to prepare for prior art that algin oligosaccharide process is loaded down with trivial details, the indefinite defect of oligosaccharides effect, a kind of algin oligosaccharide that agricultural application is worth that has is provided, be called for short AOS(Alginate Oligosaccharides), and the preparation method who extracts above-mentioned substance is provided, be applicable to suitability for industrialized production.
For achieving the above object, the technical solution used in the present invention is:
A kind of method that the present invention relates to prepare in algin polysaccharide from enzymolysis algin oligosaccharide, comprises the following steps:
(1) produce enzyme: get bacterial classification purple strepto-(Streptomyces violaceus (Streptomyces violaceoruber), bacterial strain deposit number: CPCC200534, source: Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences, 1970 collection time) ring adds in liquid nutrient medium, 28 ℃ of-35 ℃ of shaking tables are cultivated 5-8 days, and shaking speed is 150-200 rev/min.Centrifugal removing after thalline, obtains fermentation crude enzyme liquid.
(2) enzymolysis: the algin polysaccharide soln that is 0.5%-2% by concentration, add isopyknic Streptomyces violaceus fermentation crude enzyme liquid, temperature of reaction is 45 ℃-55 ℃, continues to stir 8h-10h.
(3) alcohol precipitation: extracting solution equal-volume adds ethanol to adjust concentration to 70-85%, after standing 12h-24h, 8000-10000 rev/min of centrifugal removal of impurities.
(4) concentrated: by liquid glucose, after rotary evaporation is concentrated, lyophilize obtains algin oligosaccharide (AOS).
The enzymolysis algin oligosaccharide of gained has following feature:
(1) physical behavior: micro-Huang or white powder, soluble in water, be insoluble to organic solvent.
(2) compositional analysis: electron spray(ES)-mass spectrum shows that its main polymerization degree is 2-6.
This enzymolysis algin oligosaccharide has the biological activity that promotes growth of wheat roots, increases wheat fresh weight.
Described algin oligosaccharide can be made into aqua, can take in use to spray or root irrigation.
Tool of the present invention has the following advantages:
Algin degradation enzyme vigor that 1 the present invention adopts is high and be extracellular enzyme, just polysaccharide can be degraded complete in the short period of time, and processing step is few, and equipment used is few, and cost is low.
The detectable polymerization degree of 2 gained algin oligosaccharide is 2-10, and the main polymerization degree is 2-6.
3 the present invention show that algin oligosaccharide has the growth of wheat roots of promotion, increases wheat fresh weight, and environmentally safe, noresidue, be the novel agricultural growth promoter of a kind of low cost, environmental protection.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Electron spray(ES)-the mass spectrum of Fig. 1 algin oligosaccharide;
The impact of Fig. 2 algin oligosaccharide on growth of wheat roots;
The impact that Fig. 3 algin oligosaccharide is long on stem and leaf of Wheat;
The impact of Fig. 4 algin oligosaccharide on wheat fresh weight.
Embodiment
The preparation method of embodiment 1 Streptomyces violaceus fermentation crude enzyme liquid, comprises the following steps:
(1) thalline activation: will be kept at-80 ℃ of Streptomyces violaceus (Streptomyces violaceus (Streptomyces violaceoruber) in the glycerine in refrigerator, bacterial strain deposit number: CPCC200534, source: Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences, 1970 collection time) draw 100 μ l, (peptone and 0.05%-0.5% yeast powder that massfraction is respectively 0.05%-0.5% are nitrogenous source evenly to coat algin plate culture medium, 0.5%-2% algin, the KH2PO4 of 0.1%-1%, the MgSO47H2O of 0.01%-0.1%, 1%-2% agar, surplus is water) in, at 30 ℃, cultivate 3 days, until bacterium colony occurs.
(2) thalline fermentation: get Streptomyces violaceus one ring in plate culture medium and add algin liquid substratum (liquid nutrient medium forms: it is nitrogenous source that massfraction is respectively 0.2% peptone and 0.3% yeast powder, 1.4% algin, 0.4%KH
2pO
4, 0.06%MgSO
47H
2o, surplus is water) in, 30 ℃ of shaking tables are cultivated 6 days, and shaking speed is 150 revs/min.4000 revs/min of centrifugal removing after thalline, obtain fermentation crude enzyme liquid.
2 one kinds of enzymolysis algin oligosaccharides of embodiment preparation method, comprise the following steps:
(1) enzymolysis: the alginate solution that is 2% by mass concentration, add isopyknic Streptomyces violaceus fermentation crude enzyme liquid, temperature of reaction is 50 ℃, continues to stir 10h.
(2) alcohol precipitation: enzymolysis solution adds ethanol to adjust the whole mass concentration of concentration ethanol to 70-85%, and after standing 12h, 8000 revs/min of centrifugal removal of impurities, obtain liquid glucose.
(3) concentrated: by liquid glucose, after rotary evaporation is concentrated, lyophilize obtains algin oligosaccharide (AOS).
The polymerization degree of embodiment 3 algin oligosaccharides detects
Wherein the quality of algin oligosaccharide is measured by being furnished with electric spray ion source (ESI) liquid chromatograph-mass spectrometer.Chromatographic determination condition is: mass spectrum pattern is positive ion mode; Electric spray ion source (ESI); Spray voltage 4.5kV; 350 ℃ of transfer capillary temperature; Sheath gas (Sheath gas, N2) pressure is that 30au(1au is about 1psi), assisted gas (Aux gas, N2) pressure is 5au, ion sweep gas (Ion sweep gas, N2) pressure is 0au; Sweep limit: 150～2000.Content adopts colorimetric method for determining.Fig. 1 is shown in by its EFI thing MALDI-MS collection of illustrative plates.
After testing as shown in Figure 1, the algin oligosaccharide polymerization degree is 2-10, and its main polymerization degree is 2-6.
Embodiment 4 plant experimental are evaluated the growth-promoting activity of enzymolysis algin oligosaccharide
(1) given the test agent: algin oligosaccharide sugar, according to experiment, execute the method preparation described in example 1.
(2) experimental plant: the western agriculture 979 of wheat (Triticum aestivum L.).Culture condition is: 75% alcohol sterilizing 10min for wheat seed, after clean with distilled water flushing, seed is immersed in to the 8h that soaks seed in aseptic distilled water, in dark, germinate 3 days, choose the wheat seeding of identical growing way and in Huo Delan nutrient solution, cultivate 3 days, diurnal cycle is 12h/12h, and temperature is 25 ℃/20 ℃, relative humidity 60%, intensity of illumination is 800 μ molm
-2s
-1.
(3) test item: wheat root is long, seedling is long and fresh weight.
(4) experimental technique: set up 4 groups of processing, comprise blank group (Control, distilled water sprays), algin oligosaccharide treatment group AOS1(mass concentration 0.01%AOS, surplus is water), algin oligosaccharide treatment group AOS2(mass concentration 0.1%AOS, surplus is water) and algin oligosaccharide treatment group AOS3(mass concentration 1%AOS, surplus is water).When wheat is cultured to 3 days, with different concns algin oligosaccharide or distilled water blade spraying wheat, average every strain amount of application is 10mL, sampling and measuring after 96h.
(5) result:
The impact of algin oligosaccharide on growth of wheat roots: A represents the blank group that distilled water is processed, and B represents 1%AOS treatment group.As shown in Figure 2,3 concentration AOS all can significantly promote wheat root growth, especially best with 0.01% and 1% concentration.
The impact that algin oligosaccharide is long on stem and leaf of Wheat: as seen from Figure 3, the growing way of all AOS treatment group stem and leaf of Wheat is apparently higher than blank group.
The impact of algin oligosaccharide on wheat fresh weight: shown in Fig. 4, all AOS treatment group wheat fresh weights are all higher than blank group, and the AOS of 0.1% and 1% concentration is more obvious to the raising effect of wheat fresh weight.
The present invention finds in plant experimental, and algin oligosaccharide can promote growth of wheat roots, increase wheat fresh weight.To sum up result shows that algin oligosaccharide has the effect of Promoting plant growth.
Claims (7)
1. enzymolysis is prepared a method for algin oligosaccharide, it is characterized in that: the algin degrading enzyme degraded algin being produced by Streptomyces violaceus obtains algin oligosaccharide, and the algin oligosaccharide polymerization degree is 2-10.
2. method according to claim 1, is characterized in that: the algin oligosaccharide polymerization degree is 2-6.
3. method according to claim 1, is characterized in that:
Detailed process is: Streptomyces violaceus produces after algin catenase by fermentation, the crude enzyme liquid obtaining adds in algin polysaccharide soln degrades, and the enzymolysis solution obtaining is respectively through ethanol precipitation, centrifugal, rotary evaporation is concentrated, and vacuum freezedrying obtains algin oligosaccharide (AOS).
4. it is characterized in that in accordance with the method for claim 3:
Algin catenase used has higher enzyme activity and stability, and algin oligosaccharide degradation process is: the algin polysaccharide soln of preparation 1%-5% mass concentration; The centrifugal thalline of removing of Streptomyces violaceus fermented liquid, obtains Streptomyces violaceus fermentation crude enzyme liquid, and algin polysaccharide is mixed with equal-volume crude enzyme liquid, continues stirring at 45 ℃-55 ℃, and the 8h-10h that degrades, obtains enzymolysis solution.
5. it is characterized in that in accordance with the method for claim 3:
The last handling process of described enzymolysis solution is: the enzymolysis solution after degraded is added to ethanol, adjusting the whole mass concentration of ethanol is 70%-85%, standing 12h-24h, 8000-10000 rev/min of centrifugal unreacted glycan and the impurity removed, obtaining supernatant is algin oligosaccharide mixed solution, the concentrated ethanol of removing of rotary evaporation, the liquid glucose vacuum freezedrying after concentrating, obtains algin oligosaccharide powder.
6. it is characterized in that in accordance with the method for claim 3:
Streptomyces violaceus produces by fermentation and obtains crude enzyme liquid process after algin catenase and be: get bacterial classification purple strepto-one ring and add in 50mL-150mL liquid nutrient medium, 28 ℃ of-35 ℃ of shaking tables are cultivated 5-8 days, and shaking speed is 150-200 rev/min; Centrifugal removing after thalline, obtains fermentation crude enzyme liquid; Liquid nutrient medium forms: peptone and 0.05%-0.5% yeast powder that massfraction is respectively 0.05%-0.5% are nitrogenous source, 0.5%-2% algin, the KH of 0.1%-1%
2pO
4, the MgSO of 0.01%-0.1%
47H
2o, surplus is water.
7. according to the method described in claim 1 or 3, it is characterized in that:
Described Streptomyces violaceus is one of following bacterial strain:
Streptomyces violaceus (Streptomyces violaceoruber), bacterial strain deposit number: CPCC200534, source: Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences, 1970 collection time;
Bacterial strain deposit number: SCTCC200045, source: Sichuan University's school of life and health sciences, organization names: Sichuan Provincial Party committee Biological resources preservation center, is subordinate to unit: Sichuan University, collection time: 1986;
Bacterial strain deposit number: CCTCC AA92074, source: institute of microbiology of the Chinese Academy of Sciences, organization names: Chinese Typical Representative culture collection center, is subordinate to unit: Wuhan University, collection time: 1992;
Bacterial strain deposit number: ACCC40986, source: Hubei Province biological pesticide Engineering Research Center, organization names: Hubei Province biological pesticide Engineering Research Center, collection time: 2006.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310116610.3A CN104099386A (en) | 2013-04-03 | 2013-04-03 | Method for preparing alginate oligosaccharide through enzymatic hydrolysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310116610.3A CN104099386A (en) | 2013-04-03 | 2013-04-03 | Method for preparing alginate oligosaccharide through enzymatic hydrolysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104099386A true CN104099386A (en) | 2014-10-15 |
Family
ID=51667924
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310116610.3A Pending CN104099386A (en) | 2013-04-03 | 2013-04-03 | Method for preparing alginate oligosaccharide through enzymatic hydrolysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104099386A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755188A (en) * | 2016-12-08 | 2017-05-31 | 中国科学院烟台海岸带研究所 | The preparation method and brown alga oligose of a kind of brown alga oligose monomer |
| CN107135829A (en) * | 2017-05-05 | 2017-09-08 | 宁波大学 | A kind of brown alga extract for reducing fruit and vegetable residual pesticide and its application |
| CN107184966A (en) * | 2017-06-19 | 2017-09-22 | 青岛金海宝生物科技发展有限公司 | A kind of pharmaceutical composition for preventing and treating diabetes and its complication |
| CN110295209A (en) * | 2019-07-08 | 2019-10-01 | 山东德图农业科技有限公司 | A kind of enzymatic hydrolysis sargassum extracts the process of algin oligosaccharide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1445365A (en) * | 2003-04-17 | 2003-10-01 | 中国海洋大学 | Method for preparing algin lyase and its application |
| CN101423854A (en) * | 2008-12-02 | 2009-05-06 | 中国海洋大学 | Method for preparing algin oligosacchride by using algin lyase |
-
2013
- 2013-04-03 CN CN201310116610.3A patent/CN104099386A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1445365A (en) * | 2003-04-17 | 2003-10-01 | 中国海洋大学 | Method for preparing algin lyase and its application |
| CN101423854A (en) * | 2008-12-02 | 2009-05-06 | 中国海洋大学 | Method for preparing algin oligosacchride by using algin lyase |
Non-Patent Citations (2)
| Title |
|---|
| 刘翼翔等: "褐藻胶裂解酶在制备海洋寡糖中的应用", 《食品工业科技》 * |
| 刘航等: "紫色链霉菌产褐藻胶裂解酶培养条件的初步研究", 《安徽农业科学》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755188A (en) * | 2016-12-08 | 2017-05-31 | 中国科学院烟台海岸带研究所 | The preparation method and brown alga oligose of a kind of brown alga oligose monomer |
| CN106755188B (en) * | 2016-12-08 | 2020-04-14 | 中国科学院烟台海岸带研究所 | Preparation method of brown algae oligosaccharide monomer and brown algae oligosaccharide |
| CN107135829A (en) * | 2017-05-05 | 2017-09-08 | 宁波大学 | A kind of brown alga extract for reducing fruit and vegetable residual pesticide and its application |
| CN107184966A (en) * | 2017-06-19 | 2017-09-22 | 青岛金海宝生物科技发展有限公司 | A kind of pharmaceutical composition for preventing and treating diabetes and its complication |
| CN110295209A (en) * | 2019-07-08 | 2019-10-01 | 山东德图农业科技有限公司 | A kind of enzymatic hydrolysis sargassum extracts the process of algin oligosaccharide |
| CN110295209B (en) * | 2019-07-08 | 2023-06-16 | 山东德图农业科技有限公司 | Technological method for extracting alginate oligosaccharides by enzymatic hydrolysis of gulfweed |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111534439B (en) | Penicillium oxalicum SDF-25 strain and application thereof | |
| Hariprasad et al. | Diversity of cultivable rhizobacteria across tomato growing regions of Karnataka | |
| CN113061555A (en) | Screening and application of bacillus strain for producing cellulase | |
| CN105647832A (en) | High-temperature-resistant garden waste decomposition bacterium FHM1 and application thereof | |
| CN113502250B (en) | Ralstonia strain and application, acquisition and control effect evaluation method thereof | |
| CN104099386A (en) | Method for preparing alginate oligosaccharide through enzymatic hydrolysis | |
| CN108753642B (en) | Flavobacterium johnsonii strain for producing alginate lyase | |
| CN109112069B (en) | Biocontrol endophytic fungus and application thereof | |
| CN110438036B (en) | Nitrogen-fixing bacterium N24 with nitrogen-fixing effect and application thereof | |
| CN110564625B (en) | Saline-alkali resistant aspergillus flavus and separation method and application thereof | |
| CN107964516B (en) | Acinetobacter and application thereof in degrading quorum sensing signal molecule DSF | |
| CN110564624A (en) | high-salt-and-alkali-resistance penicillium chrysogenum and separation method and application thereof | |
| CN107467075B (en) | Application of bacillus pumilus as rice growth promoter | |
| CN113684139A (en) | Ralstonia bainieri and screening method thereof and application of Ralstonia bainieri in degradation of forest waste | |
| CN101875905B (en) | Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof | |
| CN114990020B (en) | Biocontrol strain and application thereof | |
| CN102086439B (en) | Method for preparing ginseng fungus and ginseng fungus extract and application of ginseng fungus extract | |
| CN113234601B (en) | Novel marine fungus strain and application thereof in drought stress resistance of plants | |
| Aini et al. | Identification of potential pathogen bacteria causing tuber rot in porang (Amorphophallus muelleri Blume) | |
| CN110066755B (en) | Burdock endophytic streptomycete, microbial agent containing endophytic streptomycete and application | |
| CN105567608A (en) | High-temperature-resistant garden waste decomposing bacteria ST1 and application thereof | |
| CN110878271A (en) | Bacillus licheniformis MES816 and product and application thereof | |
| CN113812422B (en) | Application of paenibacillus polymyxa and/or fermentation liquor thereof | |
| CN115340961B (en) | Streptomyces griseus for antagonizing rice bacterial strip spot bacteria and application thereof | |
| CN117431191B (en) | Bacillus pseudomycoides HDCP1 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20141015 |