CN1211491C - Method of preparing chitin oligosaccharide by coupled enzymolysis and its equipment - Google Patents
Method of preparing chitin oligosaccharide by coupled enzymolysis and its equipment Download PDFInfo
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- CN1211491C CN1211491C CN 02139250 CN02139250A CN1211491C CN 1211491 C CN1211491 C CN 1211491C CN 02139250 CN02139250 CN 02139250 CN 02139250 A CN02139250 A CN 02139250A CN 1211491 C CN1211491 C CN 1211491C
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Abstract
The present invention relates to a method and a device for industrial scale production of chitin oligosaccharide needing different polymerization degrees and high activity by coupling and enzymolyzing chitosan by a multistage preprocessing and film-separating bioreactor. The method comprises the steps that the raw material of chitosan is stirred and is dissolved, and biological enzymatic degradation is carried out on the chitosan; a first-level enzymolysis product is preprocessed; secondary enzymatic degradation reaction and secondary preprocessing are carried out; a chitin oligosaccharide solution having high physiological activity is directionally separated by an ultra-filtering film technique; the ultrafiltration penetrating solution is filtered by sodium. The device comprises a dissolving and enzymatic-degradation filtering device, a secondary enzymatic-degradation filtering device and an ultrafiltration separating component which are successively connected, wherein the output end of the ultrafiltration separating component is communicated with a sodium filtering device. The present invention has the advantages of mild reaction process, easy satisfaction of reaction condition, low production cost, convenient operation, low requirement for raw materials in the production process, high device utilization rate, continuous production, high production yield, etc.
Description
Technical field
The present invention relates to multistage pre-treatment of a kind of application and film-separation biological reactor coupling enzymolysis chitosan, prepare the method and apparatus of the commercial scale production of required different polymerization degree and high reactivity chitin oligo saccharide product.
Background technology
Chitin oligo saccharide is by reducing the polymerization degree of chitosan, making it that oligo-chitosan of 4～20 glucosamines be arranged in β (1,4) glycosidic link polymerization.Because chitin oligo saccharide has fabulous physiology affinity, is widely used at present: 1, the exploitation aspect of medicine and new drug, the new drug development aspect that particularly is applied to strengthen body immunity and treats cancer; 2, the additive aspect and the daily cosmetics aspect that have the healthcare products and the heath food of obvious curative effects; 3, growth regulator and the harmless boilogical agricultural chemicals aspect of biology and farm crop; 4,, be applied to need to suppress the replacement textiles aspect of bacterium environment as the textile applications agent or be made into fiber; 5, environment protection aspect particularly is applied to the processing aspect of polluted water as flocculation agent, coagulating agent etc. and is used for degradable pollution-free food packing material aspect; Or the like.
At present, the technological approaches of available chitin oligo saccharide preparation has: acid hydrolyzation, oxidation style and enzymolysis process.Normally, its polymerization degree is reduced, adopt chromatography, ion exchange method or electroosmose process to separate then to purify and obtain chitin oligo saccharide by the degrade chitosan of relative high-polymerization degree of these three kinds of approach.Owing to these methods have the fluctuation of preparation process complexity, index big, to the large-scale industrialization production of seriously polluted, problem the limited chitin oligo saccharide product such as product yield is low, preparation cost is high, the physiologically active of product is inconsistent of environment.
Adopt conventional film-separation biological reactor technology, be used for the method and apparatus that the enzymolysis chitosan prepares chitin oligo saccharide, because requirement height to raw material, in the process of commercial scale production, to be used for secondary treatment with higher input to raw material, and in subsequent handling, because it is not separated with ash content and undegradable macromolecular substance that raw material is brought into, can seriously stop up membrane separation reactor, reduced plant factor, to such an extent as at production cost, production cycle, production unit drops into, the aspects such as recovery rate that reduce environmental pollution and product have influenced commercial scale production.
Summary of the invention
Technical problem to be solved by this invention is to propose multistage pre-treatment of a kind of application and film-separation biological reactor coupling enzymolysis chitosan, prepares method and apparatus required different polymerization degree, that have highly active large-scale industrialization production chitin oligo saccharide.Not only production process is simple for it, production cost declines to a great extent, pollution to environment is little, but also reduced the requirement of production process to material qualities such as chitosan and solution, reduced the dependence of production unit to enzyme, the recovery rate height of product, the physiologically active height has satisfied the requirement that the large-scale industrialization of chitin oligo saccharide is produced.
The technical scheme required different polymerization degree of preparation provided by the present invention, that have highly active chitin oligo saccharide method for product is:
(1) one-level enzymic hydrolysis process: the chitosan raw material is carried out stirring and dissolving and bio-enzyme degradation with acidic solution, and described biological enzyme is cellulase or hemicellulase or papoid or N,O-Diacetylmuramidase or chitoanase or chitin enzyme;
(2) pre-treatment of one-level enzymic hydrolysate: the first step to the one-level enzymic hydrolysate is filtered;
(3) reaction of secondary enzyme liberating and secondary pre-treatment: the one-level enzymolysis product through the pretreated liquid that sees through of one-level, is further carried out enzyme liberating and pre-filtering in this link;
(4) use ultrafiltration membrane technique, orientation is isolated chitin oligo saccharide solution, is meant according to the given molecular weight cutting index of product described directed the separation, selects the hyperfiltration membrane assembly with the corresponding aperture of molecular weight, carries out ultra-filtration and separation;
(5) to the nanofiltration of ultrafiltration through liquid.
Dissolving in the above-mentioned one-level enzymic hydrolysis process is meant sheet or powdery chitosan raw material, by 1%～12% weight, is dissolved in the lysate of reactor.Lysate described here can be an acetic acid, also can be citric acid, oxysuccinic acid, lactic acid, and the acid solution of other solubilized chitosans such as example hydrochloric acid; The pH value of lysate can be dissolved chitosan by selected acid solution, and does not separate out and be as the criterion.Because the degree of cleaning of the chitosan raw material in different batches, the different places of production is inconsistent, actual in the acid amount, should be adjusted between 3～6 with the pH value, the Controllable Temperature of lysate is (determining of pH value and temperature value need be as the criterion with the optimum response of selected enzyme) in 20～55 ℃ scope;
Bio-enzyme degradation in the above-mentioned one-level enzymic hydrolysis process, be meant lysed chitin oligo saccharide solution, add biological enzyme (the definite consumption that is equivalent to chitosan raw material weight 0.05～5%, activity with selected degrading enzyme requires to be as the criterion), simultaneously, before adding biological enzyme, also should further regulate the pH and the temperature value of solution in this reactor, make it to satisfy the best degraded environment of biological enzyme; Defined at present multiple biological enzyme and had Degradation chitosan, as cellulase, hemicellulase, papoid, N,O-Diacetylmuramidase, chitoanase, chitin enzyme etc., the perhaps compound biological enzyme of plurality of enzymes and variant enzyme etc., selected to these enzymes, can according to enzyme live height, steady sources, cost is low and nontoxic, environmental pollution is little etc., and principle is selected.
The pre-treatment of described one-level enzymic hydrolysate, be meant the first step of one-level enzymic hydrolysate is filtered, this process can adopt the micro-strainer of 2～10 μ m once to filter, see through liquid and enter the next stage Production Flow Chart, also can adopt secondary filtration, with 60～120 mesh filter screens thick slag is leached earlier, the liquid that sees through with the micro-strainer of 2～10 μ m enters the next stage Production Flow Chart then, leach ash content, nonsoluble and other graininess impurity of bringing into by raw materials such as chitosans to reach, reduce obstruction and damage follow-up equipment; The filtering trapped fluid of the first step turns back in the one-level enzymatic hydrolysis reaction still, so that not dissolving wherein or the chitosan that does not put in place are continued dissolving and enzymolysis; This process also can be compounded in the micro-strainer equipment and finish.
The startup of above-mentioned treating processes operation begins in the time of can solution begins to present good mobility the reactant viscosity in the one-level enzyme digestion reaction still has descended about 40～60%, still than initial value in.
Described secondary enzyme liberating reaction is meant the pretreated liquid that sees through of one-level enzymolysis product process one-level, further carries out enzyme liberating and pre-filtering in this link.The equipment of secondary enzyme liberating can be the enzyme digestion reaction still; It is any micro-strainer in 0.1～2 mu m range that pre-processing device can be selected the aperture; It sees through liquid and can enter in the first-level buffer storage tank of back or the membrane separation apparatus that directly enters next flow process separates, and trapped fluid can return secondary enzyme digestion reaction still and proceed enzyme liberating.In this stage, carry out the observing and controlling of enzyme work, pH value and temperature to the material in the reactor, make these parameters remain near the set(ting)value.
The purpose of described secondary enzyme liberating reaction and secondary preprocessing process, be to allow in the enzyme liberating reaction solution molecular weight cut to put in place or be about to the product that cutting puts in place, enter into the ultra-filtration membrane separation process of next stage as early as possible, break away from the environment that continues the cutting degraded, avoiding excessive degradation is product outside the product standard of ultra low polymerization degree (tetrose following), improves the product recovery rate; And further leach ash content, nonsoluble and other small-particle solid impurity of failing to hold back, reduce their obstruction and damages, improve plant factor the membrane separation plant in the follow-up flow process at one-level enzymolysis and pretreatment stage.
Described application ultrafiltration membrane technique adopts Hollow Fiber Ultrafiltration assembly or dull and stereotyped ultra-fine filter to form, and hollow fiber membrane ultrafiltration device can be the inner pressed structure, also can be the external-compression type structure; The scale of ultra-fine filter can be determined according to production-scale size, and this ultra-filtration membrane device systems also can be made of in the mode of being connected in parallel many groups or many ultra-fine filters;
Orientation described herein is separated, be meant according to the given molecular weight cutting index of product, select the hyperfiltration membrane assembly with the corresponding aperture of molecular weight, as select for use the molecular weight separating ranges, can organize arranged in series more and carry out ultra-filtration and separation at 500～50000 multiple ultra-filtration membrane.In the process of implementing ultrafiltration, the trapped fluid of first step ultra-fine filter returns secondary enzyme liberating reaction link, continues enzymolysis; To its follow-up ultra-fine filter of arranging according to series system, carry out ultra-filtration and separation successively; Its trapped fluid returns the prime surge tank, and it sees through liquid and flows into surge tank at the corresponding levels, by the molecular weight that is cut be arranged in order from big to small, the order ultra-filtration and separation.Like this, in surge tanks at different levels, just obtained product according to different molecular weight cut offs;
Described nanofiltration process, the ultrafiltration that is meant the different molecular weight that will be obtained in the said process sees through liquid, pass through by molecular weight cut-off at the various collecting and filtering apparatus below 200 AC that is contained in the oligo-chitosan that the moisture in the feed liquid, unit molecule and two, trimerization is right, the enzyme digestion reaction liquid once more
-1And Cl
-1And Na
+, Mg
2+Leach Deng metal ion and salt, the trapped fluid that is obtained passes through desalination and spissated high reactivity chitin oligo saccharide exactly.The concentrated concentration of feed liquid can reach 20～40% or higher in this process, and the desalination rate can reach 80～95%.
Carry out drying treatment after the nanofiltration, can carry out lyophilize, low-temperature vacuum drying or spraying drying etc., to obtain to have the solid-state shape or the powdery chitin oligo saccharide product of high biological activity.
Multistage pre-treatment of application proposed by the invention and membrane separation reactor coupling enzymolysis prepare the method and apparatus of chitin oligo saccharide, the operating features of its production process, can be in batches, whole process operating process stage by stage, also can be the whole process operating process of persistence, be very suitable for commercial scale production.
The technical scheme of the required chitin oligo saccharide equipment of preparation provided by the present invention is: comprise the dissolving and enzyme liberating reaction filtration unit, secondary enzyme liberating reaction filtration unit and the ultra-filtration and separation assembly that link to each other successively, the output terminal of ultra-filtration and separation assembly is connected with nanofiltration equipment.
In the such scheme, described dissolving and enzyme liberating reaction filtration unit include dissolving-enzyme liberating reactor 1 and micro-strainer 3, by take away pump 2 they are connected, the aperture of micro-strainer is 2～10 μ m, the trapped fluid mouth 4 of micro-strainer is connected with dissolving-enzyme liberating reactor, sees through liquid mouth 5 and joins with secondary enzyme liberating reaction filtration unit; Described secondary enzyme liberating reaction filtration unit is by secondary enzyme liberating reactor 7, secondary micro-strainer 8 and see through liquid surge tank 9 and take away pump 22 is formed, the aperture of secondary micro-strainer is 0.1～2 μ m, the trapped fluid mouth of secondary micro-strainer and secondary enzyme liberating reactor are connected by take away pump, the seeing through the liquid mouth and see through the conducting of liquid surge tank of secondary micro-strainer, the liquid outlet and the ultra-filtration and separation assembly that see through the liquid surge tank are connected; Described ultra-filtration and separation assembly is made up of Hollow Fiber Ultrafiltration assembly or dull and stereotyped ultra-fine filter, according to molecular weight plural serial stage arrangement successively from big to small, the ultra-filtration and separation assembly is provided with 4-8 level ultra-filtration and separation device group usually, the assembly of every grade of ultra-filtration and separation device is equipped with storage tank and working barrel, every grade of ultra-filtration and separation device group can and connect by a plurality of ultra-filtration and separation devices and constitute, the trapped fluid of first step ultra-filtration and separation device group returns secondary enzyme liberating reaction filtration unit, the storage tank conducting of the trapped fluid mouth of every grade of ultra-filtration and separation device group and prime ultra-filtration and separation device group, the delivery port of each storage tank is connected by valve and nanofiltration equipment.
Micro-strainer of the present invention, ultra-fine filter and nanofiltration equipment can directly be bought commodity equipment, also can be designed and produced by the micro-filtration with respective aperture, ultrafiltration and nanofiltration components and parts to constitute, and the equipment that is constituted should have more perfect back flushing loop.
The present invention compared with prior art, the beneficial effect that has is:
(1) method that adopts multistage pre-treatment and membrane separation reactor coupling enzymolysis to prepare chitin oligo saccharide makes production process decrease to the requirement of raw material, no matter is the chitin oligo saccharide raw material of pulvis or tablet, and the present invention can both adapt to;
(2) use multistage preprocessing process in the method that multistage pre-treatment and membrane separation reactor coupling enzymolysis prepare chitin oligo saccharide, not only reduced the blockage problem of ultrafiltration apparatus effectively, improved the utilization ratio of follow-up equipment, the prime that feeds back to enzyme after also will separating effectively continues to reuse, and has improved the utilization ratio of enzyme significantly;
(3) because multistage pre-treatment of application and membrane separation reactor coupling enzymolysis prepare the method for chitin oligo saccharide, can realize the serialization production of product; And multistage echelon degraded, multi-stage separation process are arranged in this process, thereby the product that can effectively degraded be put in place in time separates, make its disengaging might meet with the continuation degraded environment of biological enzyme to qualified product, its result has improved the recovery rate of product effectively;
(4), can have with the ultra-filtration membrane in the corresponding different apertures of molecular weight by selection reaction solution is separated, with the variation that realizes product, the requirement of seriation for the product of required different molecular weight;
(5) the reaction solution flow process of whole process of production all is to carry out in the good pipeline of stopping property, reactor and storage tank, has isolated the interference of outside atmosphere, helps realizing meeting the production requirement condition of high hygienic standard and helping environmental protection requirement;
In sum, owing to use the method and apparatus that multistage pre-treatment and membrane separation reactor coupling enzymolysis prepare chitin oligo saccharide, not only has the reaction process gentleness, reaction conditions satisfies easily, production cost is low, easy to operate, the advantage that facility investment is not high, and also have in process of production to raw material require low, plant factor is high, can serialization production, product recovery rate height, the directional trend that satisfies product, seriation, variation require, the production process sanitary condition is good, help advantage such as environmental protection.Using the method and apparatus that multistage pre-treatment and membrane separation reactor coupling enzymolysis prepare chitin oligo saccharide, is effective ways and device that the big industrial scale of a kind of easy acquisition is produced.
Description of drawings
Fig. 1 is the structural representation of an embodiment of present device.
Fig. 2 is the structural representation of enzyme liberating reaction filtration unit among the embodiment of present device.
Fig. 3 is reaction solution viscosity change curve diagram among the preparation method of the present invention.
Embodiment
Further specify embodiments of the invention below in conjunction with accompanying drawing.
Embodiment 1: select for use the hemicellulose prozyme as the chitosan degrading enzyme, the optimum environment of enzyme liberating is: pH=5.3, and temperature t=47 ℃, enzyme concn is 0.1%, concentration of substrate 10%; By above-mentioned technical parameter, in dissolving-enzyme liberating reactor 1, add 1000L (liter) pure water, add a certain amount of acetic acid again, the pH value of solution in the still is adjusted to 5.3, still internal reaction liquid temp is in the time of 47 ℃ ± 1 ℃, add the 100kg chitosan again, stirring and dissolving after 1 hour, is checked the pH value of feed liquid in the still, if change, need to add corresponding acid or alkali and adjust, qualified after, disperse to add ready biological enzyme 1000mL in the still, under the condition of keeping still internal reaction liquid temp, stir degraded, after 2 hours, the reaction solution viscosity degradation 40%, after 3～4 hours, still internal reaction fluid viscosity has descended about 60%; Time-the viscograph that records solubilizing reaction still internal reaction liquid with viscometer as shown in Figure 3.Still internal reaction fluid viscosity has descended after 40～60% than initial value, just can open take away pump 2, feed liquid is successfully through micro-strainer 3, (also can pass through strainer 6 of wire cloth earlier herein, or through primary filter screen 23, flow into micro-strainer 3 again, as Fig. 2, shown in Figure 1), feed liquid is divided into two-way flows to: the one tunnel is trapped fluid, returns former dissolving-enzyme liberating reactor 1 and continues degraded, another road enters next stage flow process secondary enzyme liberating reactor 7 processing links for seeing through liquid;
About about 60 minutes time, handle through above-mentioned pre-filtering, there is 70% reaction solution to be transferred to secondary enzyme liberating reactor 7 nearly, the enzyme that the records this moment content of living is higher than the content in the enzyme liberating reactor one time; Under the condition that continues to keep pH value in the reactor 7 and temperature value, continue to stir degraded about 10 minutes, can open take away pump 22 and secondary pre-treatment and filter equipment 8, still internal reaction liquid is filtered once more; See through liquid and can flow into surge tank 9; Also can be directly join, carry out ultra-filtration and separation with follow-up membrane sepn ultra-fine filter inlet; Trapped fluid then can return dissolving one enzyme liberating reactor 1 and continue degraded, also can return secondary enzyme liberating still 7 and continue degraded; After testing, reaction solution after this link is handled, contained ash content only be in the raw material contained ash content about 5%, the reaction solution molecular weight is basically all below 50000, compare with the reaction solution of handling without described secondary enzyme liberating pre-filtering, improved the blockage problem of back level ultra-filtration membrane device to a great extent;
The process of the directed cutting and separating chitosan of enforcement ultra-filtration membrane as shown in drawings.Present embodiment adopts the directed cutting and separating chitosan of level Four ultra-filtration membrane, and the water flux of ultra-fine filter device is 5T/h; Among the figure, 9 see through liquid buffering storage tank for the prime reaction solution; 10 is first step ultra-filtration and separation device group, and molecular weight cut-off selected in the present embodiment is 10000; 11 is second stage ultra-filtration and separation device group, and molecular weight cut-off selected in the present embodiment is 6000; 12 is third stage ultra-filtration and separation device group, and molecular weight cut-off selected in the present embodiment is 3000; 13 is fourth stage ultra-filtration and separation device group, and molecular weight cut-off selected in the present embodiment is 1000; 14 is first step storage tank, stores to be the chitin oligo saccharide product solution of molecular weight in 10000～6000 scopes; 15 is second stage storage tank, stores to be the chitin oligo saccharide product solution of molecular weight in 6000～3000 scopes; 16 is third stage storage tank, stores to be the chitin oligo saccharide product solution of molecular weight in 3000～1000 scopes; 17 is fourth stage storage tank, store for molecular weight less than 1000 chitin oligo saccharide product solution; The molecular weight cutting and separating scope of selecting for use in the present embodiment is carried out ultra-filtration and separation at 10000,6000,3000,1,000 four kinds of hyperfiltration membrane assembly equipment according to the many successively from big to small group arranged in series of molecular weight.In the process of implementing ultrafiltration, the trapped fluid of first step ultra-fine filter returns secondary enzyme liberating reaction link, continues enzymolysis; To its follow-up ultra-fine filter of arranging according to series system, carry out ultra-filtration and separation successively; Its trapped fluid returns the prime surge tank, and it sees through liquid and flows into surge tank at the corresponding levels.After testing, obtain 1# product 214L (liter), 2# product 178L, 3# product 233L, 4# product 211L adds up to 836L.The feed liquid total recovery is 83.6%; , handle by the chitin oligo saccharide solution of membrane sepn, can obtain the chitin oligo saccharide concentrated solution of high density and high cleanliness by nanofiltration.Carry out lyophilize, low-temperature vacuum drying or spraying drying again, to obtain to have the solid-state shape or the powdery chitin oligo saccharide product of high biological activity.
Detect at the product to the embodiment 1 after and the lyophilize concentrated through nanofiltration equipment, the acquisition result is:
| Project | Lyophilize product (kg) | Final product/raw material recovery rate (%) |
| 1# product (molecular weight ∈ (10000,6000)) | 22.3 | 22.3 |
| 2# product (molecular weight ∈ (6000,3000)) | 18.1 | 18.1 |
| 3# product (molecular weight ∈ (3000,1000)) | 24.3 | 24.3 |
| 4# product (molecular weight ∈ (1000,300)) | 20.9 | 20.9 |
| Total yield of products (%) | 85.6kg | 85.6％ |
Require to have carried out multiple batches of enforcement through the processing parameter to embodiment 1, the undulating quantity of the index of product is all less than 2%, and consistence is good, is suitable for commercial scale production.
Embodiment 2: the appointed condition that adopts embodiment 1, select for use by a certain percentage papoid, polygalacturonase, cellulase and N,O-Diacetylmuramidase to be mixed with a certain amount of prozyme, through test, the hydrolysis optimum environment parameter that obtains this prozyme is: pH=4.5, temperature t=42 ℃; Enzyme concn is 2% in the reaction solution, and concentration of substrate is 3～4.5%;
By above-mentioned desired technical parameter, in dissolving one enzyme liberating reactor 1, add the 1000L pure water, add a certain amount of acetic acid again, the pH value of solution in the still is adjusted to 4.5, still internal reaction liquid temp is in the time of 42 ℃ ± 1 ℃, add the 30kg chitosan again, stirring and dissolving is mixed with concentration of substrate and is 3%, the reaction solution of PH=4.5.After 1～2 hour, check the pH value of feed liquid in the still,, need corresponding acid of adding or alkali to adjust if change, after qualified, ready compound biological enzyme 10L is disperseed to add in the still, under the condition of keeping still internal reaction liquid temp, stir degraded, after 4 hours, the reaction solution viscosity degradation after 15%, 18～24 hours, still internal reaction fluid viscosity has descended about 40%; Degraded 15 hours at still internal reaction liquid, viscosity of sludge has descended after 30% than initial value, just can open take away pump 2, and feed liquid is successfully through micro-strainer 3, (also can pass through strainer 6 of wire cloth earlier herein, or, flow into micro-strainer 3 again through primary filter screen 23, as Fig. 2, shown in Figure 1), feed liquid is divided into two-way to be flowed to: the one tunnel is trapped fluid, return dissolving-enzyme liberating reactor 1 and continue degraded, another road enters next stage flow process secondary enzyme liberating reactor 7 processing links for seeing through liquid;
Other disposition is identical with embodiment 1.
Detect at the product to the embodiment 2 after and the lyophilize concentrated through nanofiltration equipment, the acquisition result is:
| Project | Lyophilize product (kg) | Final product/raw material recovery rate (%) |
| 1# product (molecular weight ∈ (10000,6000)) | 5.036 | 16.8 |
| 2# product (molecular weight ∈ (6000,3000)) | 5.28 | 17.6 |
| 3# product (molecular weight ∈ (3000,1000)) | 7.08 | 23.6 |
| 4# product (molecular weight ∈ (1000,300)) | 5.49 | 18.3 |
| Total yield of products (%) | 22.886kg | 76.3％ |
Embodiment 3: adopt the appointed condition of embodiment 1, this example selects for use the hemicellulose prozyme as the chitosan degrading enzyme, and the optimum environment of enzyme liberating is: pH=5.3, temperature t=45 ℃; The enzyme concn of reaction solution preparation is 0.2%, and the concentration of substrate of preparing according to inventory is 5%;
By above-mentioned desired technical parameter, in dissolving-enzyme liberating reactor 1, add the 1000L pure water, add a certain amount of acetic acid again, the pH value of solution in the still is adjusted to 5.3, still internal reaction liquid temp is in the time of 45 ℃ ± 1 ℃, add the 50kg chitosan again, stirring and dissolving is mixed with concentration of substrate and is 5%, the reaction solution of PH=5.3.0.5 after hour, check the pH value of feed liquid in the still,, need corresponding acid of adding or alkali to adjust if change, after qualified, ready hemicellulose biological enzyme 2000mL is disperseed to add in the still, under the condition of keeping still internal reaction liquid temp, stir degraded, 1.5 after hour, the reaction solution viscosity degradation after 25%, 2～3 hours, still internal reaction fluid viscosity has descended about 40%; Degraded 2 hours at still internal reaction liquid, viscosity of sludge has descended after 40% than initial value, just can open take away pump 2, feed liquid is successfully through micro-strainer 3, feed liquid is divided into two-way to be flowed to: the one tunnel is trapped fluid, return dissolving-enzyme liberating reactor 1 and continue degraded, another road enters next stage flow process secondary enzyme liberating reactor 7 processing links for seeing through liquid;
Other disposition is identical with embodiment 1.
Detect at the product to the embodiment 3 after and the lyophilize concentrated through nanofiltration equipment, the acquisition result is:
| Project | Lyophilize product (kg) | Final product/raw material recovery rate (%) |
| 1# product (molecular weight ∈ (10000,6000)) | 9.35 | 18.7 |
| 2# product (molecular weight ∈ (6000,3000)) | 9.19 | 18.4 |
| 3# product (molecular weight ∈ (3000,1000)) | 14.15 | 28.3 |
| 4# product (molecular weight ∈ (1000,300)) | 10.65 | 21.3 |
| Total yield of products (%) | 43.34kg | 86.7％ |
The present invention prepares embodiment such as Fig. 1 of required chitin oligo saccharide equipment, shown in 2, include dissolving and enzyme liberating reaction filtration unit, it includes dissolving-enzyme liberating reactor 1 and micro-strainer 3, the aperture of micro-strainer is 2～10 μ m, dissolving-enzyme liberating reactor is connected with micro-strainer by take away pump 2, the trapped fluid mouth 4 of micro-strainer is connected with dissolving-enzyme liberating reactor, it sees through liquid mouth 5 and joins with secondary enzyme liberating reaction filtration unit, be anti-clogging plug, can primary filter screen 23 be set at the admission port place of micro-strainer, perhaps between dissolving-enzyme liberating reactor and micro-strainer, be connected in series strainer 6 of wire cloth, filter screen and strainer adopt the 60-100 order, the liquid mouth that sees through of a strainer joins through take away pump 2 and micro-strainer, and the trapped fluid mouth of a strainer links to each other with dissolving-enzyme liberating reactor;
Secondary enzyme liberating reaction filtration unit is by secondary enzyme liberating reactor 7, secondary micro-strainer 8 and see through liquid surge tank 9 and take away pump 22 is formed, the aperture of secondary micro-strainer is 0.1～2 μ m, the trapped fluid mouth of secondary micro-strainer and secondary enzyme liberating reactor are connected, and also can be conducted with dissolving-enzyme liberating reactor 1 simultaneously; Secondary enzyme liberating reactor and secondary micro-strainer are connected by take away pump 22, the seeing through the liquid mouth and see through the conducting of liquid surge tank of secondary micro-strainer, and the liquid outlet and the ultra-filtration and separation assembly that see through the liquid surge tank are connected;
The ultra-filtration and separation assembly is made up of Hollow Fiber Ultrafiltration assembly or dull and stereotyped ultra-fine filter, Hollow Fiber Ultrafiltration can be inner pressed structure or external-compression type structure, between molecular weight separating ranges 1000-10000 from big to small, select for use level Four ultra-filtration and separation device group to be connected in series mutually, the assembly of every grade of ultra-filtration and separation device is equipped with product storage tank and working barrel, every grade of ultra-filtration and separation device group can and connect by a plurality of ultra-filtration and separation devices and constitute, and wherein first step ultra-filtration and separation device group 10 is made of 6 ultra-filtration and separation devices, and molecular weight cut-off is 10000; Trapped fluid mouth and secondary enzyme liberating reactor 7 join, see through the liquid mouth and connect first step storage tank 14, the delivery port of first step storage tank joins through working barrel and second stage ultra-filtration and separation device group, second stage ultra-filtration and separation device group 11 is made of 6 ultra-filtration and separation devices, molecular weight cut-off is 6000, trapped fluid mouth and first step storage tank 14 join, see through the liquid mouth and connect second stage storage tank 15, the delivery port of second stage storage tank joins through working barrel and third stage ultra-filtration and separation device group, third stage ultra-filtration and separation device group 12 is made of 6 ultra-filtration and separation devices, molecular weight cut-off is 3000, trapped fluid mouth and second stage storage tank 15 join, see through the liquid mouth and connect third stage storage tank 16, the delivery port of third stage storage tank joins through working barrel and fourth stage ultra-filtration and separation device group, fourth stage ultra-filtration and separation device group 13 is made of 6 ultra-filtration and separation devices, molecular weight cut-off is 1000, trapped fluid mouth and third stage storage tank 16 join, see through the liquid mouth and connect fourth stage storage tank 17, the storage tank conducting of the trapped fluid mouth of every grade of ultra-filtration and separation device group and prime ultra-filtration and separation device group, the delivery port of each storage tank is connected by valve and nanofiltration equipment.Nanofiltration equipment 18 is provided with input aperture 20, concentrated solution mouth 19 and filtrate port 21, nanofiltration equipment 18 with the mode of connection of the chitin oligo saccharide solution storage tank of each different molecular weight on, can directly be connected with the chitin oligo saccharide storage tank of different molecular weight a plurality of nanofiltration equipment are man-to-man, constitute the parallel running mode of many nanofiltration device equipment; Also can adopt respectively chitosan solution to be connected, constitute the tandem working mode of separate unit nanofiltration equipment with a nanofiltration equipment with different molecular weight.
Claims (9)
1, a kind of coupling enzymolysis prepares the method for chitin oligo saccharide, it is characterized in that
(1) one-level enzymic hydrolysis process: the chitosan raw material is carried out stirring and dissolving and bio-enzyme degradation with acidic solution, and described biological enzyme is cellulase or hemicellulase or papoid or N,O-Diacetylmuramidase or chitoanase or chitin enzyme;
(2) pre-treatment of one-level enzymic hydrolysate: the first step to the one-level enzymic hydrolysate is filtered;
(3) reaction of secondary enzyme liberating and secondary pre-treatment: the one-level enzymolysis product through the pretreated liquid that sees through of one-level, is further carried out enzyme liberating and pre-filtering in this link;
(4) use ultrafiltration membrane technique, orientation is isolated chitin oligo saccharide solution, is meant according to the given molecular weight cutting index of product described directed the separation, selects the hyperfiltration membrane assembly with the corresponding aperture of molecular weight, carries out ultra-filtration and separation;
(5) to the nanofiltration of ultrafiltration through liquid.
2, by the described method for preparing chitin oligo saccharide of claim 1, it is characterized in that the dissolving in the described one-level enzymic hydrolysis process, be meant sheet or powdery chitosan raw material, by 1%～12% weight, be dissolved in the acidic solution of reactor, described acidic solution, be acetic acid or citric acid or oxysuccinic acid or lactic acid or hydrochloric acid, the pH value of lysate, can dissolve chitosan by selected acid solution, and not separate out and be as the criterion, actual in the acid amount, be adjusted between 3～6 with the pH value, the temperature of lysate is controlled in 20～55 ℃ the scope.
3, by claim 1 or the 2 described methods that prepare chitin oligo saccharide, it is characterized in that the bio-enzyme degradation in the described one-level enzymic hydrolysis process, be meant lysed chitin oligo saccharide solution, add the biological enzyme that is equivalent to chitosan raw material weight 0.05～5%, simultaneously, before adding biological enzyme, also should further regulate the pH and the temperature value of solution in this reactor, make it to satisfy the degraded environment of biological enzyme.
4, by claim 1 or the 2 described methods that prepare chitin oligo saccharide, it is characterized in that the pre-treatment of described one-level enzymic hydrolysate, be meant the first step of one-level enzymic hydrolysate is filtered, this process adopts the micro-strainer of 2～10 μ m once to filter, see through liquid and enter the next stage Production Flow Chart, or the employing secondary filtration, thick slag leaching earlier with 60～120 mesh filter screens, the liquid that sees through with the micro-strainer of 2～10 μ m enters the next stage Production Flow Chart then.
5, by claim 1 or the 2 described methods that prepare chitin oligo saccharide, it is characterized in that described secondary enzyme liberating reaction and secondary pre-treatment, be meant the one-level enzymolysis product through the pretreated liquid that sees through of one-level, further carry out enzyme liberating and pre-filtering in this link, it is any micro-strainer in 0.1～2 mu m range that the aperture is selected in the secondary pre-treatment; It sees through liquid and enters in the first-level buffer storage tank of back or the membrane separation apparatus that directly enters next flow process separates, and trapped fluid returns secondary enzyme digestion reaction still and proceeds enzyme liberating.
6, by claim 1 or the 2 described methods that prepare chitin oligo saccharide, it is characterized in that be to select for use the molecular weight separating ranges at 500～50000 multiple ultra-filtration membrane described directed the separation, many group arranged in series are carried out ultra-filtration and separation, the trapped fluid of first step ultra-fine filter, return secondary enzyme liberating reaction link, continue enzymolysis; To its follow-up ultra-fine filter of arranging according to series system, carry out ultra-filtration and separation successively; Its trapped fluid returns the prime surge tank, and it sees through liquid and flows into surge tank at the corresponding levels, by the molecular weight that is cut be arranged in order from big to small, the order ultra-filtration and separation, like this, in surge tanks at different levels, just obtained product according to different molecular weight cut offs.
7, by claim 1 or the 2 described methods that prepare chitin oligo saccharide, it is characterized in that described nanofiltration process, the ultrafiltration that is meant the different molecular weight that will be obtained in the said process sees through liquid, pass through once more by molecular weight cut-off at the various collecting and filtering apparatus below 200, the metal ion and the salt that are contained in the oligo-chitosan that moisture in the feed liquid, unit molecule and two, trimerization is right, the enzyme digestion reaction liquid leach, and the trapped fluid that is obtained passes through desalination and spissated high reactivity chitin oligo saccharide exactly.
8, a kind of coupling enzymolysis prepares the equipment of chitin oligo saccharide, it is characterized in that comprising the dissolving and enzyme liberating reaction filtration unit, secondary enzyme liberating reaction filtration unit and the ultra-filtration and separation assembly that link to each other successively, and the output terminal of ultra-filtration and separation assembly is connected with nanofiltration equipment.
9, press the equipment of the described preparation chitin oligo saccharide of claim 8, it is characterized in that described dissolving and enzyme liberating reaction filtration unit includes dissolving-enzyme liberating reactor (1) and micro-strainer (3), by take away pump (2) they are connected, the aperture of micro-strainer is 2～10 μ m, the trapped fluid mouth (4) of micro-strainer is connected with dissolving-enzyme liberating reactor, sees through liquid mouth (5) and joins with secondary enzyme liberating reaction filtration unit; Described secondary enzyme liberating reaction filtration unit is by secondary enzyme liberating reactor (7), secondary micro-strainer (8) and see through liquid surge tank (9) and take away pump (22) is formed, the aperture of secondary micro-strainer is 0.1～2 μ m, the trapped fluid mouth of secondary micro-strainer and secondary enzyme liberating reactor are connected by take away pump, the seeing through the liquid mouth and see through the conducting of liquid surge tank of secondary micro-strainer, the liquid outlet and the ultra-filtration and separation assembly that see through the liquid surge tank are connected; Described ultra-filtration and separation assembly is made up of Hollow Fiber Ultrafiltration assembly or dull and stereotyped ultra-fine filter, according to molecular weight plural serial stage arrangement successively from big to small, the ultra-filtration and separation assembly is provided with 4-8 level ultra-filtration and separation device group usually, the assembly of every grade of ultra-filtration and separation device is equipped with storage tank and working barrel, every grade of ultra-filtration and separation device group is by a plurality of ultra-filtration and separation devices and connect and constitute, the trapped fluid of first step ultra-filtration and separation device group returns secondary enzyme liberating reaction filtration unit, the storage tank conducting of the trapped fluid mouth of every grade of ultra-filtration and separation device group and prime ultra-filtration and separation device group, the delivery port of each storage tank is connected by valve and nanofiltration equipment.
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| CN 02139250 CN1211491C (en) | 2002-11-07 | 2002-11-07 | Method of preparing chitin oligosaccharide by coupled enzymolysis and its equipment |
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| CN 02139250 CN1211491C (en) | 2002-11-07 | 2002-11-07 | Method of preparing chitin oligosaccharide by coupled enzymolysis and its equipment |
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| CN100402658C (en) * | 2005-03-21 | 2008-07-16 | 中国科学院过程工程研究所 | Method and apparatus for preparing reducing sugar by enzymolysis of steam-exploded straw with membrane reactor |
| CN103958678B (en) * | 2011-11-21 | 2016-01-13 | 东丽株式会社 | The manufacture method of cellulase and device thereof |
| CN102978263B (en) * | 2012-12-12 | 2014-07-16 | 石狮市华宝海洋生物化工有限公司 | Method for producing high-purity N-acetylglucosamine |
| CN103194510A (en) * | 2013-04-17 | 2013-07-10 | 肇庆长龙生物科技有限公司 | Preparation method of chitosan oligosaccharide for paddy rice seed soaking and foliar spraying |
| CN106432815A (en) * | 2016-09-07 | 2017-02-22 | 华东师范大学 | Biological nano-composite and preparation method and application thereof |
| CN107235534B (en) * | 2017-06-06 | 2023-04-25 | 南京蔚华膜科技有限公司 | Nanofiltration separation method of monovalent salt mixed solution |
| CN108552534A (en) * | 2018-04-08 | 2018-09-21 | 大连军门保健食品有限公司 | With the complex polysaccharide electuary and preparation method thereof for adjusting intestinal flora function |
| CN113088443A (en) * | 2021-03-11 | 2021-07-09 | 三峡大学 | Separation coupling system and method for multi-enzyme cascade reaction |
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