CN1425696A - Preparation of crust oligosaccharide and use - Google Patents
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- CN1425696A CN1425696A CN 03112619 CN03112619A CN1425696A CN 1425696 A CN1425696 A CN 1425696A CN 03112619 CN03112619 CN 03112619 CN 03112619 A CN03112619 A CN 03112619A CN 1425696 A CN1425696 A CN 1425696A
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Abstract
The present invention relates to preparation and application of crust oligosaccharide and belongs to the field of deep chitin processing technology. Non-specific enzyme is used to raise the hydrolysis degree of chitosan and the separation and purification of hydrolytic product. The key point of the present invention is that the chitosan material is produced into crust oligosaccharide powder through acid dissolving, heating, enzymolysis and hydrolysis with compoiste, enzyme comprising polysaccharide hydrolase, proteinase and lipase, deactivating enzyme, filtering, ultrafiltering, ion exchange or nanometer filtering, vacuum concentration and drying. The product is used in producing various functional health foods with the functions of immunological regulation and blood fat regulation.
Description
Technical field
The present invention relates to a kind of preparation method and application of chitin oligosaccharide, particularly relate to a kind of preparation method of the chitin oligosaccharide that adopts the combinative enzyme hydrolysis method and the application of chitin oligosaccharide, belong to chitin deep process technology field.
Background technology
Chitin oligosaccharide (chitooligosaccharides) is that a class is passed through β-1 by N-acetyl-D-amino glucose (NAG) and/or D-glucosamine (GA), the water soluble oligosaccharide that 4 glycosidic links link up can prepare by hydrolysis chitin (chitin) or chitosan (chitosan).Chitin and chitosan can obtain through acid-alkali treatment from the cell walls of the shell of shrimp crab, insect and fungi.This is a kind of renewable resource, and total resources is only second to Mierocrystalline cellulose on the earth.But its structure is compared with Mierocrystalline cellulose, and hydroxyl-removal also contains kharophen or free amine group outward, and its functional property and purposes are more important and extensive, is described as " biomaterial of 21st century ".Water insoluble because of chitin or chitosan, in human body, be not easy to be digested and assimilated, its application and some important physical activity are restricted.
Studies show that in a large number: water-soluble chitin oligosaccharide has unique physiologically active and graceful function character.It can improve the immunological competence of body, the disease resistance of enhancing body and anti-infection ability; Have strong antitumor action, the growth of tumour cell and the transfer of cancer cells are had the obvious suppression effect; It still is effectively bifidus bacillus multiplicaiton factor, can breed probiotics such as bifidus bacillus etc. in the enteron aisle.In addition, it can also promote the activation of vegetable cell, and the stimulating plant growth improves the disease resistance of plant; It also has significant germ resistance and moisture-absorbing moisture-keeping effect.Therefore, chitin oligosaccharide has broad application prospects in fields such as food, medicine, agricultural, makeup.
The common preparation method of chitin oligosaccharide has chemical method and enzyme process.Chemical method generally with chitin or chitosan with the concentrated hydrochloric acid heating hydrolysis or utilize hydrogen peroxide or HNO
2Oxidative degradation, because of reacting violent, wayward, by product is many, product separation and purification difficulty, yield is low.Enzyme process is because of the reaction conditions gentleness, and selectivity is strong, has avoided the destruction of oligose, is subjected to extensively paying attention to and research.A kind of method of enzymic degradation chitosan or chitin is to adopt specificity lytic enzyme such as chitoanase or chitin enzyme.At present, on the low side with this class enzymic activity of microorganisms producing, and exist with the enzyme system of complexity mostly, the separation and purification difficulty, the cost height is difficult to use in commercially producing of chitin oligosaccharide.Another kind of enzyme liberating mode is to adopt non-specificity lytic enzyme.This fermentoid has Degradation to chitin or chitosan, but is not to be natural substrate with it.Although non-specificity enzyme is also not fully aware of to the hydrolytic action mechanism of chitin or chitosan, but because these enzymes easily obtain from commercial, cheap, therefore replace chitin enzyme and chitoanase, a new way with commercialization value is started in the production that can be chitin oligosaccharide.
The bibliographical information of relevant chitin oligosaccharide is a lot, and domestic is survey article mostly, introduces the physiologically active and the general preparation method of chitin oligosaccharide.The external different hydrolytic actiones of once having reported papoid, cellulase, lipase to chitosan, but main research concentrates on the hydrolysis properties aspect.Using these non-specificity lytic enzymes comes the suitability for industrialized production chitin oligosaccharide also to have some difficult problems: the one, and non-specificity enzyme is limited to the hydrolysis degree of chitosan, if a kind of non-specificity enzyme of simple use, the enzyme usage quantity is big (generally more than 20% on the one hand, ratio reaches 0.2～0.3 at the bottom of the enzyme: 1), efficiency of pcr product also low (20%～40%) on the other hand, and the relative molecular mass of product is bigger than normal, usually can only reach about 10000, and the higher chitin oligosaccharide relative molecular mass of physiologically active is below 5000; The 2nd, because chitosan is water insoluble, dissolving back soltion viscosity is big in acid solution, and meltage is limited, and the concentration of substrate of general enzyme effect has only 1～3%, the separation and purification of product and the treatment capacity of concentrate drying is increased, thereby cause production cost to increase; The 3rd, though the separation purification method of hydrolysate has the application ultra-filtration and separation, can not be removed product purity and of low quality to excess acid and the excessive hydrolysate monose that brings because of the acid dissolving.Its technology is perfect inadequately, is difficult to be applied to suitability for industrialized production.
Summary of the invention
(1) technical problem that will solve
The present invention is intended on the comprehensive research both at home and abroad basis, the non-specificity enzyme of research and utilization is to the different hydrolytic actiones of chitosan emphatically, seek the cooperative compensating effect between the various enzymic hydrolysiss, improve the degree that chitosan is hydrolyzed thereby form prozyme, and reduce enzyme dosage with bigger hydrolysis effect; Simultaneously, the research form of feed pretreatment is to the raising of chitosan concentration and the favourable influence of hydrolysis degree.In addition, methods such as ion-exchange of research and utilization combining ultrafiltration or nanofiltration are come the separation and purification hydrolysate, form the production technique of chitin oligosaccharide.
(2) technical scheme
The preparation method of a kind of chitin oligosaccharide of the present invention is to be raw material with the chitosan, through mixed-acid dissolution and heat pre-treatment, with combinative enzyme hydrolysis, the enzyme that goes out, filtration, ultrafiltration, ion exchange treatment or nanofiltration processing, vacuum concentration, drying, make the product chitin oligosaccharide.Technological process such as following.
Raw materials of chitosan: with shrimp or crab shell and comprise insect shell and mycelium, behind acid-alkali treatment, obtain.Its leading indicator should meet following standard: moisture≤12%, degree of deacetylation 〉=70%, ash oontent≤3%, arsenic (in As)≤2.0mg/kg, plumbous (in Pb)≤10mg/kg.
Acid dissolving and heat pre-treatment: in retort, add 1～5% (w/w) acetum earlier, throw in chitosan while stirring, add 2～5% (W/W) hydrochloric acid soln simultaneously, and be heated to 40～90 ℃, 1～2h makes its whole dissolvings under high-speed stirring, is mixed with 5～10% (w/w) chitosan solution.
The concentration height of chitosan solution has more important economic implications to the production of chitin oligosaccharide.Because of chitosan is a macromole, after the dissolving, its viscosity increases along with the increase of concentration in acid solution, particularly concentration reaches 5% when above, and the viscosity of solution is index to be increased, and outward appearance is the viscous gel shape, be difficult to stir, this moment, chitosan was difficult to dissolve again, and concentration can not increase; In addition, if this moment, enzyme-added liquid carried out enzymolysis, will be unfavorable for the carrying out of enzyme digestion reaction because of crossing the sticking diffusion that has a strong impact on enzyme liquid.Be necessary before enzymic hydrolysis, to reduce the viscosity of system.
Chitosan is in acid solution, and because of the katalysis of acid, its β-1,4 glycosidic link can hydrolytic cleavage take place and soltion viscosity is reduced, and its hydrolysis degree depends on acid concentration, temperature and time.Since contain free amine group in the chitosan molecule, positively charged in acid solution, and make the glycosidic link of this glycosyl be difficult to be hydrolyzed.Too low as fruit acid concentration, then the viscosity reduction is very slow, and meltage increases limited; But too high as fruit acid concentration, easily produce small molecules or the monose of excessive hydrolysate relative molecular mass below 500.Therefore, the pre-treatment purpose of present method is both to have made it that to a certain degree hydrolysis is arranged, and reduces soltion viscosity; Do not make excessive hydrolysis again simultaneously and produce small molecules monose.Through overtesting, select chitosan solution to be carried out acid dissolving and heat treated with the mixed acid solution of acetate and hydrochloride, and above-mentioned suitable acid concentration and pretreatment temperature have been determined, thereby the concentration that makes chitosan solution brings up to 10% from general 1～3%, can help the carrying out of enzymatic hydrolysis reaction like this, thereby can in industrial production, use.
Combinative enzyme hydrolysis: chitosan solution is regulated pH2.5～5.5, and prozyme joins in the chitosan solution after dissolving with less water, and the prozyme consumption is 0.3～3% (w/w) of chitosan amount, and promptly ratio is 0.003～0.03: 1 at the bottom of the enzyme.At 40～65 ℃ of stirring reaction 1～5h, thinning up to solution; Used prozyme is three kinds of prozymes that different enzymes are formed in polysaccharide hydrolase system, proteolytic enzyme, lipase three fermentoids, and it consists of (1 ± 0.2): (1 ± 0.2): (1 ± 0.2) (weight ratio).Polysaccharide hydrolase system can be a cellulase, amylase, and polygalacturonases etc., proteolytic enzyme are papoid and stomach en-.According to the requirement of hydrolysate molecular size, prozyme can select the different enzymes of three kinds in two classes wherein or three classes to form.
The relevant mechanism of non-specificity lytic enzyme degrade chitosan is also not fully aware of.There is 4 types glycosidic link in the chitosan molecule structure, i.e. NAG-NAG, NAG-GA, GA-GA, GA-NAG.When someone proposes the neutral protease degrade chitosan, interrupt the glycosidic link between NAG and the GA.We are by the viscosity change curve of research chitosan solution under different sorts enzyme difference hydrolytic action, find that its common ground is to descend fast at enzymic hydrolysis initial stage soltion viscosity, and sluggish subsequently, illustrate that these enzymes mainly act on chitosan with internal-cutting way, make β on the molecular chain-1,4 glycosidic links are interrupted, and superpolymer becomes oligopolymer.But its hydrolysis degree of different types of enzyme is different, illustrates that they may act on the glycosidic link of different sites.Wherein the compound acting in conjunction of different types of enzyme can strengthen hydrolytic action in chitosan, shows that the collaborative and complementary effect of different types of enzymic hydrolysis effect is more remarkable.
We have also compared three kinds of enzymes and have successively divided and add for 3 times and three kinds of enzyme whiles once add hydrolytic action to chitosan, see Table 1.
Collaborative and the complementary effect of three kinds of enzymic hydrolysis effects of table 1
| Sequence number | Enzyme-added number of times and enzyme amount (mg/ml) | Reducing sugar equivalent increased value (mg NAG/g chitosan) | ||||
| For the first time | For the second time | For the third time | For the first time | For the second time | For the third time | |
| ??1 | ??A(1.072) | ??B(1.179) | ??C(0.954) | ??4.12 | ??25.32 | ??71.01 |
| ??2 | ??A(1.072) ??B(1.179) ??C(0.954) | ? ??108.75 | ||||
It can be seen from the table three kinds of enzymes once add than gradation successively simultaneously and add the corresponding increase of its hydrolysis degree, and the increase degree reaches 37.7, and adding than gradation increases by 53%.
Simultaneously, also investigated the influence of enzyme dosage, condition of different pH, differing temps, differential responses time, determined appropriate condition the enzymic hydrolysis chitosan.Enzyme dosage is 3% (w/w) of chitosan, promptly is equivalent to 3mg/ml enzyme dosage, and 10% chitosan solution concentration is equivalent to about 100mg/ml amount of substrate.In the initial reaction stage 30min, hydrolysis rate is very fast, and speed of response slows down subsequently, and this is similar to single enzyme hydrolytic action characteristics; Behind reaction 5h, reaction tends to balance substantially.
Enzyme goes out: reaction solution is heated to 90 ℃, keeps being cooled to 40 ℃ again after 10 minutes.
Filter: enzyme digestion reaction liquid is removed by filter insolubles.
Chitosan is in sour dissolution process and enzymolysis process, a certain amount of acid, alkali and zymin have been added, this part additive can influence the quality index of chitin oligosaccharide, therefore be necessary chitin oligosaccharide solution is carried out separation and purification, remove materials such as excessive acid, salt and a small amount of unhydrolysed macromole, to guarantee the purity of chitin oligosaccharide.
Ultrafiltration: filtered liquid separates with ultrafiltration.Membrane molecule amount cutoff value is 5000～10000, to remove deproteinize and macromole.Ultrafiltration apparatus is simple to operate, and is convenient and practical, and floor space is little, do not have the variation of thing phase in the ultra-filtration process, can not bring any side effect to product, can carry out the separation of material in molecular level.Adopt membrane molecule cutoff value 5000 and 10000 pairs of chitosan hydrolyzate liquid to remove protein, measure the protein content that sees through in liquid and the trapped fluid respectively, calculate the protein decreasing ratio and reach 92.2% and 88.5% respectively, show that ultrafiltration can remove protein effectively, can the purifying chitin oligosaccharide.
Ion exchange treatment: ultrafiltrated at room temperature carries out ion-exchange with strongly basic anion exchange resin as 717 types with ion exchange column, makes the pH value of effluent liquid reach 5.5～6.5, wash with water break away from sub-exchange resin after, carry out resin regeneration with acid ﹠ alkali liquid again.Use the hydrolyzed solution that enzyme process makes, owing to added a certain amount of acid in the preprocessing process, these acid can combine salify with the free amine group in the chitin oligosaccharide molecule, and the acid of rest part is free acid.Adopting ion-exchange-resin process that ultrafiltrated is further handled, mainly is in order to remove free acid and anionic part.Analysis revealed, product chitin oligosaccharide retention rate after treatment reaches more than 89%, and the negatively charged ion decreasing ratio reaches 88%, and free acid removes substantially.Ion-exchange-resin process is industrial a kind of separation method the most commonly used, and simple to operate, cost is low.Therefore, select for use this method that chitosan hydrolyzate liquid is removed negatively charged ion and desalting and purifying processing, also can be removed a small amount of protein residual in the ultrafiltrated simultaneously by resin absorption.
Vacuum concentration: the ion-exchange effluent liquid sucked in the thin-film evaporator with vacuum concentrate, reach 20%～40% until the soluble solid concentration of concentrated solution.
Dry: concentrated solution is carried out thermal dehydration in drying machine with centrifugal spray, the control flow velocity obtains product chitin oligosaccharide powder.160～180 ℃ of inlet temperature, 70～80 ℃ of air outlet temperatures.
If without ion exchange resin treatment, also can adopt nanofiltration that ultrafiltrated is handled in the above-mentioned purge process.Nanofiltration is emerging Physical Separation Technology in the membrane sepn field, and its separation performance is to develop one of membrane sepn kind faster in recent years in the world between reverse osmosis and ultrafiltration.The pore diameter range of nanofiltration membrane is about several nanometers, its trapped molecular weight is generally 200～1000 dalton, under pressure-driven, allow some inorganic salt and small organic molecule to see through film, larger molecular organics is separated with small organic molecule, organism is separated with inorganics, and the double organism that makes concentrates.Handling equally with nanofiltration can desalination, remove free acid and anionic part, and can also remove the small molecules of relative molecular mass below 500 such as monose.Get the nanofiltration trapped fluid and carry out vacuum concentration, drying, can obtain the very high chitin oligosaccharide of purity.The processing condition of chitin oligosaccharide nanofiltration purifying are: working pressure: 1.5～2.0MPa, temperature: 25～50 ℃.
Chitin oligosaccharide with the inventive method preparation, it mainly to be applied as with the chitosan be main raw material, through enzymolysis, filter, concentrate, dry gained be that the pulvis of main component is filled into capsule shell or compressing tablet and makes and have functional foodstuff or the protective foods of regulating immunologic function and regulating blood fat effect with the chitin oligosaccharide.In addition, chitin oligosaccharide purity height, quality height through this method preparation also can be applicable in the industries such as medicine, makeup.
(3) beneficial effect
The raw material of producing chitin oligosaccharide is chitosan or chitin, extracts to obtain from shrimp crab processing fent.There is very long shoreline in China, and marine fishery and the development of fresh water culture fishery are very fast, and shrimp crab crust resource is very abundant.Since the nineties, many chitins source mill has all been built up in China's Coastal Areas and all parts of the country, and annual production is estimated nearly 100,000 tons.At present chitin perhaps only is processed into the glucosamine salt mainly as chemical industry or medical material for export, and added value is low, and the overall technology level is not high.And research is the raw material production chitin oligosaccharide with the chitosan, and this added value of product is big, and is with high content of technology.So the present invention improves China's chitin industrial level to effectively utilizing the chitin resource, promote China's chitin industrial expansion to have great importance; Simultaneously, also make contributions for the development of Chinese national economy and community service.
From commercial zymin, find several non-specificity enzymes that chitosan is had hydrolytic action, and find that a few class prozymes of being made up of these enzymes have significant cooperative compensating hydrolytic action, have determined the optimum condition of these combinative enzyme hydrolysis chitosans.Under suitable hydrolysising condition, can improve the hydrolysis degree of chitosan with these several prozymes greatly, the relative molecular mass that makes product is below 5000, and the consumption of enzyme is reduced to below 3%, provides feasibility for commercially producing chitin oligosaccharide.
Chitosan is dissolved in the acetate and hydrochloride mixed acid solution, adopt gentle pretreatment mode, the viscosity of chitosan solution is reduced, meltage increases, thereby the concentration that makes chitosan solution brings up to 10% from general 1～3%, and the enzyme digestion reaction concentration of substrate has been increased more than 3 times.Under such treatment condition, contents of monosaccharides in the enzymic hydrolysate＜5%.Since the raising of reactant concn, thus the energy can be saved, reduce production costs.
Adopt the separation purification method of combining ultrafiltration ion exchange resin or nanofiltration, can remove protein in the chitin oligosaccharide, salt and monose, make the purity of chitin oligosaccharide reach (in glucosamine content) more than 96%.
With the chitosan is raw material, and the prozyme that adopts the commercial enzyme preparation to form is hydrolyzed, and has formed the enzymatic production process of chitin oligosaccharide, has passed through the pilot scale of 500L, and chitin oligosaccharide product yield reaches 83%.Product manager's fractional analysis is measured, and is water-soluble 99.9%, contents of monosaccharides 3.5%, and moisture 4.5%, ash content 0.46%, degree of deacetylation 81%, the polymerization degree is 2～15, molecular weight distribution 500～3000.
Description of drawings
Preparation technology's FB(flow block) of accompanying drawing chitin oligosaccharide.
Embodiment
Embodiment 1
Be dissolved in chitosan raw material 30kg among the hydrochloric acid soln 500L of 3% acetic acid and 2%, be heated to 60～90 ℃, constantly stir and dissolve up to chitosan.Chitosan solution is transferred pH3.5, and the prozyme composition joins in the chitosan solution 40 ℃ of stirring reaction 5h after dissolving with less water with polygalacturonase, stomach en-, each 0.3kg of lipase, enzyme digestion reaction finishes, and the enzyme that goes out filters, filtrate is separated with ultrafiltration, and the membrane molecule cutoff value is 5000.Ultrafiltrated at room temperature carries out ion-exchange with ion exchange column with strongly basic anion exchange resin 717 types, makes the pH value of effluent liquid reach 5.5～6.5.The ion-exchange effluent liquid carries out vacuum concentration, drying, makes product chitin oligosaccharide powder.
Embodiment 2
Chitosan raw material 50kg when acid dissolving and heat pre-treatment, dissolves chitosan with 3% acetum earlier, adds 4% hydrochloric acid soln simultaneously, and heat temperature raising to 90 ℃, reduces chitosan solution viscosity, and all the other conditions are with embodiment 1.
Embodiment 3
Prozyme is formed with cellulase, papoid, lipase, and all the other are with embodiment 1.
Embodiment 4
Prozyme is formed with amylase, proteolytic enzyme, lipase, and all the other are with embodiment 1.
Embodiment 5
Prozyme is formed with polygalacturonase, stomach en-, papoid, and all the other are with embodiment 1.
Embodiment 6
Enzyme digestion reaction and processing before are with embodiment 2.The separation and purification treatment condition adopts ultrafiltration and nanofiltration to handle, the pressure 1.5～2.0MPa of nanofiltration operation, flux 5～10L/min, 35～50 ℃ of temperature.In order to improve purification effect, can add the pure water dilution several times.Along with increasing progressively of purifying multiple, small molecules monose, salt constantly reduce in the liquid that dams, and chitin oligosaccharide purity improves constantly, and the finished product contents of monosaccharides is less than 1%, and ash content is less than 0.3%.
Claims (5)
1, a kind of preparation method of chitin oligosaccharide is characterized in that with the chitosan being raw material, through mixed-acid dissolution and heat pre-treatment, uses combinative enzyme hydrolysis, and the enzyme that goes out filters, and ultrafiltration, ion-exchange or nanofiltration are handled, vacuum concentration, and drying makes the product chitin oligosaccharide;
(1) raw materials of chitosan: with shrimp or crab shell and comprise insect shell and mycelium, behind acid-alkali treatment, obtain;
(2) acid dissolving and heat pre-treatment: in retort, add dilute acetic acid solution earlier, throw in chitosan while stirring, add dilute hydrochloric acid solution simultaneously, and be heated to 40～90 ℃, under high-speed stirring, chitosan is all dissolved, be mixed with 5～10% (w/w) chitosan solution;
(3) combinative enzyme hydrolysis: chitosan solution is regulated pH2.5～5.5, and prozyme joins in the chitosan solution after dissolving with less water, and the prozyme consumption is 0.3～3% (w/w) of chitosan amount, and is at 40～65 ℃ of stirring reaction 1～5h, thinning up to solution; Used prozyme is the prozyme that the different enzymes of three kinds in polysaccharide hydrolase system, proteolytic enzyme, lipase three fermentoids are formed, and it consists of (1 ± 0.2): (1 ± 0.2): (1 ± 0.2) (weight ratio);
(4) enzyme that goes out: reaction solution is heated to 90 ℃, keeps being cooled to 40 ℃ again after 10 minutes;
(5) filter: enzyme digestion reaction liquid is filtered, remove insolubles;
(6) ultrafiltration: filtered liquid separates with ultrafiltration, and membrane molecule amount cutoff value is 5000～10000, to remove deproteinize and macromole;
(7) ion-exchange: ultrafiltrated at room temperature carries out ion-exchange with ion exchange column with strongly basic anion exchange resin 717 types, make the pH value of effluent liquid reach 5.5～6.5, to remove free acid, salt and anionic part, after washing the disengaging sub-exchange resin with water, carry out resin regeneration with acid ﹠ alkali liquid again;
(8) vacuum concentration: the ion-exchange effluent liquid sucked in the thin-film evaporator with vacuum concentrate, reach 20%～40% until the soluble solid concentration of concentrated solution;
(9) drying: concentrated solution is carried out thermal dehydration in drying machine with centrifugal spray, obtain product chitin oligosaccharide powder.
2, the preparation method of a kind of chitin oligosaccharide according to claim 1 adopts 1～5% (W/W) acetic acid and 2～5% (W/W) hydrochloric acid soln when it is characterized in that acid dissolving and heat pre-treatment.
3, the preparation method of a kind of chitin oligosaccharide according to claim 1, it is characterized in that used prozyme can be cellulase, amylase, the polygalacturonase of polysaccharide hydrolase system, the papoid of proteolytic enzyme, stomach en-and lipase, according to the requirement of hydrolysate molecular size, prozyme can select the different enzymes of three kinds in two classes wherein or three classes to form.
4, the preparation method of a kind of chitin oligosaccharide according to claim 1, it is characterized in that filtrate that the product separation purifying can be later with ultrafiltration directly carries out nanofiltration and handle, carry out separation and purification with nanofiltration membrane, the processing condition of nanofiltration purifying are: working pressure: 1.5～2.0MPa, temperature: 25～50 ℃.
5, a kind of application of chitin oligosaccharide, it is characterized in that with the chitosan being main raw material, through enzymolysis, filter, concentrate, dry gained be that the pulvis of main component is filled into capsule shell or compressing tablet and makes and have functional foodstuff or the protective foods of regulating immunologic function and regulating blood fat effect with the chitin oligosaccharide.
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