CN1164601C - Regulating-control method for preparing different polymerization degree compound sugar by enzymatical method - Google Patents
Regulating-control method for preparing different polymerization degree compound sugar by enzymatical method Download PDFInfo
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- CN1164601C CN1164601C CNB011368411A CN01136841A CN1164601C CN 1164601 C CN1164601 C CN 1164601C CN B011368411 A CNB011368411 A CN B011368411A CN 01136841 A CN01136841 A CN 01136841A CN 1164601 C CN1164601 C CN 1164601C
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Abstract
The present invention discloses a regulation and control method for preparing oligosaccharides with different polymerization degrees by using an enzyme method. The enzymic degradation of polysaccharides is combined with membrane separation; by the selection of different membrane media and membrane pore sizes, a hollow fiber ultrafilter or a flat plate membrane ultrafilter is connected with an enzymic degradation reactor; oligosaccharides with different polymerization degrees are instantly separated; ultrafiltrate is separated by using a nanometer filter to remove small molecule compounds with low activity, such as monosaccharides, disaccharides, water, etc.; oligosaccharides with different polymerization degrees are obtained. By using the technology, various series of oligosaccharides with different polymerization degrees can be produced; the production amount of inactive monosaccharides and disaccharides is low; polysaccharides with high polymerization degrees can realize continuous operation of re-reaction and separation; since continuous operation is realized, enzymes can be recycled; the method increases the availability efficiency of degrading enzymes and is suitable for scale production.
Description
Technical field
The invention belongs to a kind of method of regulating and control the oligose polymerization degree, relate in particular to a kind of regulate and control method of method for preparing different polymerization degree compound sugar by enzymatical.
Background technology
Oligose generally is meant by 2-10 monose to be formed, and has the functional molecular of certain physiological active functions.Mainly act as: can promote bifidus bacillus propagation, suppress the growth of harmful intestinal tract, as Oligomeric manna sugar etc.; Have the inhibition tumor growth, enhancing body immunizing power reduces the ability of cholesterol in the blood, as oligomeric amino grape etc.; Also have and hinder the growth of pathogenic bacteria breeding, strengthen the defence capability of plant, as oligomeric galactose etc. disease and pest.Thereby be widely used in medicine, protective foods and the agricultural.Oligose is obtained through degraded by the polysaccharide that nature exists usually, the main at present methods such as acid degradation, oxidative degradation and enzyme liberating that adopt.Adopt acid degradation, oxidative degradation often to obtain more monose and lower oligose such as the disaccharides of the polymerization degree.The preparation of many in the world employing enzyme liberating polysaccharide has report (JP, spy open flat 5-68580) to adopt the enzyme liberating chitosan to match with ultra-fine filter after the eighties, obtain the mixture of 5-18 sugar, concentrate, after the lyophilize, handle, obtain the mixture of 7-18 sugar with methyl alcohol; Have again report (JP, clear 61-271296) with chitosan with dense HCl hydrolysis, NaOH neutralization, and with activated carbon treatment, filtration, filtrate is separated with ion-exchange membrane electrodialysis, obtains the pure product of 1-7 sugar.These methods usually run into the product polymerization degree and are difficult to control, and enzyme and substrate utilization ratio be low, how to reduce problem such as monose and disaccharides content in the product.The separation and Extraction of oligose adopts gac, diatomite, gac-sellaite or ion-exchange membrane electrodialysis technology usually.Though these methods can realize effective separation of active shell oligosaccharides, seriously polluted because trivial operations, cost is high and influence scale operation.
Summary of the invention
The regulate and control method that the purpose of this invention is to provide a kind of method for preparing different polymerization degree compound sugar by enzymatical, adopt this technology can produce the oligose of multiple different series and different polymerization degree, the monose of non-activity and disaccharides growing amount are low, and the oligose yield height of higher degrees of polymerization pollutes little, production cost is low, can realization response with separate operate continuously, be suitable for scale production, owing to realize operate continuously, enzyme is reusable, improves the service efficiency of degrading enzyme.
For achieving the above object, the regulate and control method of a kind of method for preparing different polymerization degree compound sugar by enzymatical provided by the invention, the enzymic degradation polysaccharide is combined with membrane sepn, by selecting the different film mediums and the pore size of film, tubular fibre or flat membrane ultrafiltration device are connected with the enzyme liberating reactor, oligose to different polymerization degree separates immediately, molecule greater than molecular weight cut-off returns reactor continuation degraded, ultrafiltrated separates through the nanofiltration device again, remove micromolecular compounds such as SA monose, disaccharides and water, obtain the oligose of different polymerization degree.
Concrete scheme is that the polysaccharide material dissolution is become solution, adding degrading enzyme by 0.5-3.0% (weight percent) degrades, the polysaccharide raw material degraded that the present invention refers to comprises pectin, carrageenin, agar-agar, algin, xanthan gum, sesbania gum, dextran, xylan, mannosans, heparin, the biological degradation of raw materials such as sulfated polysaccharide, degrading enzyme can be a chitinase, cellulase, bromeline, N,O-Diacetylmuramidase, lipase, chitoanase, choose any one kind of them in the polygalacturonase etc. or several, temperature of reaction 15-80 ℃, pH=3.0-10.0, viscosity degradation begin ultrafiltration to 40-60%; Tubular fibre, flat membrane ultrafiltration device are connected with the enzyme liberating reactor, earlier solution is pumped into tubular fibre, flat membrane ultrafiltration device, oligose to different polymerization degree separates, the aperture of film is a criterion with the oligose that sees through corresponding molecular weight, but must hold back the enzyme molecule, and adopting the molecular weight that dams usually is 2,000-100,000 film sees through film less than the part of molecular weight cut-off, returns reactor greater than the molecule of molecular weight cut-off and continues degraded; Ultrafiltrated pumps into rolling flat sheet membrane nanofiltration device to be separated, and the nanofiltration membrane molecular weight that dams is 100-2, and 000, its selection principle is as the criterion to see through monose, disaccharides and water, and selecting for use usually the MgSO4 rejection is the film of 80-95%; Through above step, remove micromolecular compounds such as SA monose, disaccharides and water, obtain the oligose of different polymerization degree.
Reaction Separation coupling operation mode of the present invention can be operate continuously or periodical operation, and the raw material arbitrary way can be a fed-batch, also can be continuous feeding.Fed-batch be membrane separation apparatus discharge a certain amount of after, in reactor, add isopyknic stock liquid, continuous feeding drips stock liquid in reactor when membrane separation apparatus leaches, but add speed must with leach speed and equate.
Chromatographic separation relatively has following advantage after the present invention and the existing batch of degraded:
1, the present invention adopts enzymic degradation chitosan and membrane sepn to be coupled the oligose of generation in time to be separated, stop its further degraded effectively.
2, degrading enzyme can be reused continuously, has improved the service efficiency of enzyme.
3, the control manipulation condition of the film by selecting different apertures can obtain the oligose of different polymerization degree.
4, process of the present invention is simple and direct, has reduced the pollution that sepn process causes, has improved separation efficiency, and process can be carried out continuously, helps realizing suitability for industrialized production.
Description of drawings
Fig. 1 is a main technique schema of the present invention.
Embodiment
Provide embodiments of the invention below, and in conjunction with the accompanying drawings the present invention is described in detail, except that specified otherwise was arranged, described concentration all was weight percentage.
Embodiment 1: in enzyme liberating reactor 1 0.5% chitosan is dissolved in pH5.6, concentration is in the sodium-acetate buffer of 0.05M, filter cleaner, adding 1% chitoanase is degrading enzyme, 35 ℃ of temperature of reaction, 4 hours reaction times, degrade chitosan treats that soltion viscosity drops to 40-60% and can carry out ultrafiltration.Hollow-fibre membrane ultra-fine filter rolling flat sheet membrane nanofiltration device is assembled into a membrane module 2 and is connected with enzyme liberating reactor 1 by infusion pump 3, and the oligosaccharide of different polymerization degree is separated, and the hollow fiber ultrafiltration membrane surface-area is 2m
2, molecular weight cut off is 2,000-10, and 000, see through film less than the part of molecular weight cut-off, obtain ultrafiltrated, return enzyme liberating reactor 1 greater than the molecule of molecular weight cut-off through control valve 4 and continue degraded.This example adopts the continuous feeding mode.The ultrafiltrated that obtains through hollow fiber ultrafiltration membrane pumps into 2 inches rolling flat sheet membrane nanofiltration devices separation, its membrane area 0.5m again
2, the molecular weight that dams is 100-2,000, remove SA monose, disaccharides and big water gaging, and obtain the oligochitosan that the polymerization degree is 3-20, and collect by product collector 5.
Embodiment 2: 2% pectin is dissolved in the aqueous solution of 20L, adds 3.0% polygalacturonase, 50 ℃ of temperature of reaction, 1 hour reaction times, pH3.0, the ultrafiltration of flat membrane ultrafiltration device, adopt fed-batch, all the other are operated with embodiment 1, obtain the pectin oligosaccharide that the polymerization degree is 3-20.
Add 0.5% lipase in the carrageenin aqueous solution of embodiment 3:3%, 80 ℃ of temperature of reaction, pH10, all the other are operated with embodiment 1, and obtaining the polymerization degree is the carrageenin oligosaccharide of 3-18.
Embodiment 4: add chitinase in the agar-agar aqueous solution, and 15 ℃ of temperature of reaction, all the other are operated with embodiment 1, and obtaining the polymerization degree is the agar-agar oligosaccharide of 3-18.
Embodiment 5: add N,O-Diacetylmuramidase in the glue solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the brown alga oligosaccharide of 3-18
Embodiment 6: add bromeline in the xanthan gum solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the xanthan gum oligosaccharide of 3-18.
Embodiment 7: add cellulase in the sesbania gum aqueous solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the sesbania gum oligosaccharide of 3-18.
Embodiment 8: add the plain enzyme of polygalacturonase and pineapple in the glucan aqueous solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the poly-oligosaccharide in Portugal of 3-20
Embodiment 9: add cellulase and N,O-Diacetylmuramidase in the sugar aqueous solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the poly-oligosaccharide of wood of 3-20.
Embodiment 10: add polygalacturonase in the mannosans aqueous solution, all the other are operated with embodiment 1, obtain the manna oligosaccharide that the polymerization degree is 3-20.
Embodiment 11: add polygalacturonase in the heparin solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the heparin oligosaccharide of 3-18.
Embodiment 12: add polygalacturonase in the sulfated polysaccharide aqueous solution, all the other are operated with embodiment 1, and obtaining the polymerization degree is the sulfuric acid oligosaccharide of 3-18.
Embodiment 13: the enzyme liberating reaction of chitosan
1% chitosan is dissolved in pH=5.6, in the acetate buffer solution of 0.05M, presses enzyme: substrate=add enzyme at 1: 20 45 ℃ of reactions down, characterizes the degrading activity of various enzymes with the percentage of reaction solution viscosity degradation.12 hours afterreaction liquid of initial and degraded is by t fall time of measuring section with the Ping Shi viscometer determining
0, t
1, then the percentage of chitosan viscosity degradation is t
0-t
1/ t
0
Table 1: various enzyme liberating chitosan activity:
Used enzyme | Viscosity degradation percentage (%) |
Lipase cellulase chitoanase bromeline N,O-Diacetylmuramidase | 70.4 75.3 94.6 90.4 80.2 |
Embodiment 14: fed-batch and continuous feeding experiment
Reaction cumulative volume 20L, test conditions is identical with embodiment 1, began ultrafiltration after feeding intake 1 hour, simultaneously add raw material and acetic acid continuously with constant speed, ultrafiltration filtrate 60L gets concentrated solution 1.8L through nanofiltration, in fed-batch and continuous feeding, different polymerization degree chitosan proportion is as shown in table 2, and continuous feeding reduces 3-4 sugar relative quantity, and 6-8 sugar relative quantity increases.
Table 2: in fed-batch and the continuous feeding, the relative quantity of 3-10 sugar
The polymerization degree | Relative content (%) fed-batch continuous feeding |
3 sugar | 41.3 32.0 |
4 sugar | 23.3 20.8 |
5 sugar | 15.0 14.9 |
6 sugar | 12.1 16.5 |
7 sugar | 4.0 6.0 |
8 sugar | 2.4 4.4 |
9 sugar | 1.1 3.1 |
10 sugar | 0.8 2.3 |
Claims (3)
1, a kind of regulate and control method of method for preparing different polymerization degree compound sugar by enzymatical comprises the steps:
The polysaccharide material dissolution is become solution, add 0.5-3.0% (weight percent) degrading enzyme, temperature of reaction 15-80 ℃, pH=3.0-10.0, degradation solution viscosity degradation begin ultrafiltration to 40-60%; Tubular fibre, flat membrane ultrafiltration device are connected with the enzyme liberating reactor, with operate continuously or andnon-continuous operation manner, solution is pumped into tubular fibre, flat membrane ultrafiltration device, the oligose of different polymerization degree is separated, the enzyme molecule is held back with the oligose that sees through corresponding molecular weight in the aperture of film; See through film less than the part of molecular weight cut-off, return reactor greater than the molecule of molecular weight cut-off and continue degraded; Ultrafiltrated pumps into rolling flat sheet membrane nanofiltration device to be separated, nanofiltration membrane is dammed molecular weight selection principle to be as the criterion through monose, disaccharides and water, its ultra-fine filter molecular weight that dams is 2,000-100,000, return reactor greater than the molecule of molecular weight cut-off and continue degraded, ultrafiltrated separates through rolling flat sheet membrane nanofiltration device again, the nanofiltration membrane molecular weight that dams is 100-2,000, obtain the oligose of polymerization degree 3-20.
2, the regulate and control method of method for preparing different polymerization degree compound sugar by enzymatical according to claim 1, it is characterized in that wherein the polysaccharide degraded refers to comprise the biological degradation of pectin, carrageenin, agar-agar, algin, xanthan gum, sesbania gum, dextran, xylan, mannosans, heparin, sulfated polysaccharide raw material.
3, the regulate and control method of method for preparing different polymerization degree compound sugar by enzymatical according to claim 1, it is characterized in that wherein degrading enzyme can be to choose any one kind of them in chitinase, cellulase, bromeline, N,O-Diacetylmuramidase, lipase, chitoanase, the polygalacturonase or several.
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CN103333264B (en) * | 2008-05-30 | 2016-06-08 | 动量制药公司 | The method of sugar structure and manufacture and this class formation of use |
CN103589811B (en) * | 2012-08-15 | 2017-06-23 | 天津天士力现代中药资源有限公司 | Monose, the isolation and purification method of disaccharides in a kind of compound Danshen Root medicinal extract |
WO2014192958A1 (en) * | 2013-05-31 | 2014-12-04 | アクテイブ株式会社 | Glucose production method, and glucose produced using said method |
CN104004799B (en) * | 2014-06-12 | 2016-04-13 | 南京工业大学 | Method for continuously preparing galacto-oligosaccharide |
CN104177450A (en) * | 2014-09-17 | 2014-12-03 | 江苏天晟药业有限公司 | Oligosaccharide for preventing and treating fungous plant diseases and insect pests of liquorice and preparation method and application of oligosaccharide |
CN105695528A (en) * | 2016-03-29 | 2016-06-22 | 江南大学 | Method for selectively removing monosaccharides and producing oligosaccharides by using yeast culture |
CN109134676B (en) * | 2018-07-25 | 2020-08-18 | 广东省农业科学院蚕业与农产品加工研究所 | Oligosaccharide and preparation method and application thereof |
CN111197065A (en) * | 2020-02-24 | 2020-05-26 | 江南大学 | Method for producing algin hydrolysate |
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