CN101144097A - Method for preparing chitin and its chitosan and chitosan oligosaccharide - Google Patents
Method for preparing chitin and its chitosan and chitosan oligosaccharide Download PDFInfo
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Abstract
The present invention relates to a method for preparing chitin and chitosan oligosac charide. The method comprises the steps that usual raw materials such as the crust of shrimp and crab, the insect crust or the fungal mycelia, etc. are micronized through the dry process or wet process; the carapace material of the obtained fine powder raw material is decalcified with the chemical process, and then is defatted and deproteinized with the method of micro-organism compound enzyme coarse enzyme liquid co-enzymolysis, and the insect and fungus fine powder thereof is directly defatted and deproteinized; a whole cell immobilizing bioreactor of a chitin deacetylase high-yield producing strain is prepared, to perform the circulatory deacetylation to the chitin and then obtain chitosan with corresponding degree of deacetylatoion; obligate anaerobic acid-producing bacterium and high-yield producing chitosan bacterium are utilized, the chitosan is submerged and fermented in the liquid, to obtain chitosan oligosaccharide with high water solubility. The present invention has the advantages that the method is helpful to fully utilize the resources, and makes the waste to the worth, at the same time, the default of the manufacturing process of the chemical process can be avoided, the production efficiency is improved, the energy is saved, the consumption is reduced, the byproduct with corresponding high value added can be produced, the comprehensive economic benefits of the relative secondary industry are obviously improved, the industrial development is promoted, and the multi-win effect is attained.
Description
Technical field
The invention belongs to biological technical field, relate to the novel process that Chemicals and novel material are produced, particularly a kind of method for preparing chitin and chitosan and oligochitosan from plurality of raw materials.
Background technology
As everyone knows, (chitin is to have one of natural organic-compound the most widely on the earth chitin) to chitin, quantitatively is only second to Mierocrystalline cellulose and occupies the second, nearly 10,000,000,000 tons of annual biosynthesizing amounts; Chitin also is the nitrogenous natural organic-compound of quantity maximum outside the isolating protein on the earth; Only this 2 point just is enough to illustrate the critical role of chitin in nature and human lives.Studies show that of nearly recent decades, chitin and take off acetyl product chitosan, and both various derivatives, as novel chemical materials and organic products, in people's production and life, has using value extremely widely, relate to all respects of numerous areas such as medical and health, foodstuffs industry, light and textile industries, papermaking, detergents and cosmetic, functional materials, environment protection, energy industry, agricultural, show very fine market outlook; By chitin and chitosan hydrolyzate and oligosaccharides (crust oligosaccharides and oligochitosan), demonstrate good physiologically active, pharmacologically active especially and, become the focus of deeply developing the chitin product at present as many special benefits of new type chemical material; And, the chitin product to human body and useful and harmless " green health " and " environmental friendliness " characteristics of environment, meets the tendency of the day with it very much, thereby extremely people's favor, to continually developing and the deep discussion of related production technology of chitin and extension product thereof, just in the ascendant.
Although chitin is acknowledged as a kind of outstanding good natural organic matter raw material that is worth, the weave construction composition as various animals, microorganism and certain plants extracts and is not easy from its starting materials.Nearly recent decades, chitin extraction/chitosan and the main flow technology for preparing its derivative are chemical methods on the actual production level, find that at present there is following series of malpractice in it: 1) produce and too much use strong acid, highly basic and harmful chemical reagent in the process, produce a large amount of waste water and waste material, serious environment pollution is water body environment particularly; 2) high frequency, the huge energy consumption of high-temperature process generation for a long time; 3) produce corrosion-resistant, the high temperature resistant equipment of process need, increase production cost; 4) the non-specific katalysis of chemical catalyst can not clearly dissociate the different chemical structures composition and/or synthetic effectively, has influenced the yield and the purity of target product; 5) the violent chemical reaction condition of strong acid, highly basic and other extraction solvent has obviously restricted the stability and the quality control of target chemical ingredients; 6) the recycling difficulty of frequent washing, purifying program and diluted acid, diluted alkaline in the technological process causes a large amount of valuable cleaning fresh water to be consumed waste; 7) chemical characteristics such as the corrodibility of strong acid, highly basic and other extraction solvent and explosion hazard have constituted security threat to Working environment and HUMAN HEALTH.In view of the foregoing; can run business big and strong for guaranteeing the chitin industry; really accomplish Sustainable development; just must carry out significant improvement to existing production technology; effectively overcome above-mentioned drawback, thus develop can obviously improve target component yield, high-efficiency comprehensive utilization raw material resources, reaction conditions gentleness, save energy and equipment cost, significantly reduce chemical use reduce waste water and dregs, help environment protection, chitin/chitosan that safety in production and reducing threatens HUMAN HEALTH and the novel extraction and the preparation technology of oligochitosan.
Summary of the invention
The object of the present invention is to provide a kind of production process route based on biological process, be used for also further producing the method for chitosan and oligochitosan from current raw material chitin extraction commonly used, be intended to overcome the above-mentioned shortcoming of traditional chemical process, can obviously improve under the extraction efficiency and product quality premise of target component, significantly reduce acid, alkali, the usage quantity and the waste liquid of oxygenant and other Harmful chemicals, the discharging of waste residue, eliminate it to the pollution of environment and the threat of HUMAN HEALTH, cut down the consumption of energy and equipment cost, and can produce the high byproduct of added value simultaneously, utilize resources synthetically, improve productivity effect, thereby provide a kind of synergy joint consumption, environmental friendliness, resource-effective green the extraction and preparation technology reaches effective, promote the purpose of chitin industry development sustainably.
The invention still further relates to the chitin/chitosan that will extract and further be prepared into oligochitosan and/or crust oligosaccharides with biology and/or chemical method.
The concrete technical scheme that realizes the object of the invention is as follows:
A kind of method for preparing chitin and chitosan and oligochitosan, preparation process comprises the steps: raw materials commonly used such as shrimp and crab shells, insect cot or radicula byssoidea are carried out micronization processes with physical method (dry method or wet method); Gained crustaceans powder body raw material is earlier with after the chemical method decalcification, use proteolytic enzyme, lipase crude enzyme liquid composite back method degreasing, the deproteinated of enzymolysis altogether from microorganism again, insects and Mycophyta powder body are then directly used the composite back degreasing of above-mentioned crude enzyme liquid, deproteinated; In case of necessity, the material behind degreasing, the deproteinated decolours in conjunction with the method for microbial enzyme enzymolysis with oxygenant; The full cell fixation bio-reactor of preparation chitin deacetylase superior strain to the obtaining chitin deacetylation that circulates, obtains the chitosan of corresponding deacetylation; With the acid formers and the high yield chitoanase bacterium of obligate anaerobic, liquid submerged fermentation chitosan, obtain the oligochitosan of high water soluble; In case of necessity, with chemical method or enzyme process with the oligochitosan acetylize, the crust oligosaccharides.
The micronization processes of described raw material comprises dry process and wet processing: dry process is meant that mechanical mill is the powder body of 50-100 μ m to mean particle size with after the raw material thorough dryings (water content＜10%) such as shrimp and crab shells, insect cot or radicula byssoidea; Wet processing is meant and be obtains more fine-grained powder body, and above-mentioned raw materials is ground into the particle of mean particle size 150-200 μ m in advance, is made into suspension, and mechanical mill or comminution by gas stream become the powder body that mean particle size is 10-25 μ m again.
Described calcium salt, fat and the protein of removing from raw material is meant and sloughs calcareous with the dilute hydrochloric acid of 4-6% earlier for the powder body of crustaceans raw material, after precipitation is washed till neutrality, carry out common enzyme digestion reaction with the compound crude enzyme liquid that is rich in lipase and proteolytic enzyme again, remove fat and protein; For insects and Mycophyta powder body, then directly be total to enzymolysis with the microbial compound enzyme crude enzyme liquid, carry out degreasing, deproteinated.
Described microbial compound enzyme crude enzyme liquid, be meant and use isolating starting strain voluntarily, inducing and acclimating and the Candida utilis bacterial strain (preserving number CCTCC N0.M207053) of high yield lipase activity, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on April 24th, 2007, classification called after Candida utilis ODK-CC
1(Candida utilis ODK-CC
1); And high proteinase yield bacillus subtilis strain (preserving number CCTCC NO.M207028) alive, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification name subtilis ODK-BK
1(Bacillus subtilisODK-BK
1); Behind aerobic liquid submerged fermentation, the extracellular enzyme crude enzyme liquid of removing the thalline gained is mixed and forms respectively.
Described altogether enzymolysis degreasing, deproteinated reaction are meant earlier that with the powder body of raw material with 65 ℃ of warm water soaking 30min, the assorted bacterium and the irrelevant enzyme that suppress in the raw material are alive; Then, above-mentioned lipase and proteolytic enzyme crude enzyme liquid are mixed with 1: 1 volume, in reactor with the raw material powder body of handling by liquid: Gu=5: 1 proportional quantity mixes, under 40 ℃ of constant temperature, in reactor, fully mix, carry out common enzymolysis degreasing deproteinated reaction, in 18 hours reaction times, separate collecting precipitation with the centrifugal solid, liquid that carries out of 2500r/min behind the reaction terminating.
Described decolorizing process technique is meant containing the raw material of color and luster, adopts two-step approach to decolour, promptly will be total to the solid materials that enzyme digestion reaction is finished postprecipitation earlier, and add in the diluted alkaline and soak, decoct 2h, repeat 3 times behind the replacing alkali lye, filter to collect filter residue, wash 3 times after, use 5%H earlier
2O
2Boiled 1 hour, decolouring fast is again with being rich in H
2O
2Another group microorganism crude enzyme liquid of oxido-reductase system carries out enzyme digestion reaction, removes to be difficult to resolve in the raw material from pigment also successively H2O2 to be converted into nontoxic CO
2And H
2O.40 ℃ of temperature of reaction, reaction times 18h, reaction is finished centrifugal solid, liquid and is separated, and will get the white powder chitin after washing of precipitate, the drying.
The described H that is rich in
2O
2Oxido-reductase is a crude enzyme liquid, is meant to separate the high yield H of domestication voluntarily
2O
2And the Coriolus (having another name called rainbow conk) (preserving number CCTCC N0.M207024) of oxido-reductase system, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification name rainbow conk ODK-CY
1(Polystictus versicolor ODK-CY
1), behind aerobic liquid submerged fermentation, remove the extracellular enzyme crude enzyme liquid that thalline obtains.
Described deacetylation, be meant the Rhizopus oryzae bacterial strain (preserving number CCTCC NO.M207054) that will separate the high yield deacetylase of domestication earlier voluntarily, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on April 24th, 2007, classification name Rhizopus oryzae ODK-RM
1(Rhizopus oryzae ODK-RM
1), preparation becomes full cell fixation bio-reactor, again with aforementioned gained chitin with 40 ℃ of warm water suspends, behind the abundant mixing, cycling stream is added in this bio-reactor, carries out deacetylation by the hydrolytic action of deacetylase and reacts; According to the requirement of product and purposes to deacetylation, the DD value of dynamic monitoring reaction product, until reaching desired DD value standard, termination reaction, collecting reaction product, after concentrating by 1/3 volume, vacuum or lyophilize, chitosan.
The preparation of described full cell fixation bio-reactor, comprise two programs: 1) cell fixation: with quantitative 2% sodium alginate soln and Rhizopus oryzae lyophilized powder in liquid: Gu=4: 1 ratio thorough mixing, the gained suspension splashes into the CaCl of 0.2mol/L at room temperature with the speed of 10ml/min by the pin pump
2In, make the globule that forms diameter 5.0-5.5mm; After all suspensions instil and finish, the globule that forms is packed in the bioreactor device; 2) the upflowing bio-reactor is made: make cylindric stainless steel reactor main body jar, internal diameter 50.8cm, height 584cm, working volume 10L, the main body jar is pressed the design of upflowing reaction principle, be aided with that substrate flows into, product flows out, microorganism culturing becomes shunting to add and function passage such as rare gas element pressurization, is connected supporting with corresponding supply channel.
The preparation technology of described oligochitosan, be meant that application separates the bifidobacterium strains of the high yield acetate of domestication (preserving number CCTCC NO.M207027) voluntarily, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification called after bifidus longum bb ODK-BS
1(Bifidobacterium longum ODK-BS
1); Lactobacterium strain (preserving number CCTCC NO.M207026) with the high yield chitoanase, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification called after plant lactobacillus ODK-LR
1(Lactobacillus plantarum ODK-LR
1), under anaerobic, ferment the altogether chitosan of aforementioned gained of mixing liquid deep layer, Monitoring and Controlling bifidus bacillus thalline biomass is 1.0 * 10
8Cfu/ml and fermented liquid acetic acid concentration are in the scope of 15-20mol/L; Fermentation later stage stream adds micro-nitrogenous source and the inorganic salt that help growth of lactobacillus and adjusts pH, reaches until Bacterium lacticum thalline biomass〉1.0 * 10
10Behind the cfu/ml, stop fermentation, fermented liquid directly concentrates after removing thalline, impurity, and vacuum or lyophilize get the oligochitosan of 80% above relative molecular weight＜8000.
The described method that obtains the crust oligosaccharides by acetylated chitooligose; be meant above-mentioned gained oligochitosan; under 0 ℃, be soaked in the mixing solutions of forming by diacetyl oxide and perchloric acid and agitation as appropriate; more than the acetylization reaction 24h; treat that the acetyl degree reaches 1.0 termination reactions when above; to neutral, use dewatering of ethanol with the frozen water washing again, vacuum-drying gets the crust oligosaccharides.
Method of the present invention is applicable to chitin extraction and preparation chitosan, oligochitosan from present representational raw material commonly used, as the crust of Crustaceans shrimp and crab in the Arthropoda, and the cot of Insecta animal silkworm chrysalis, fly maggot, blattaria; The crust of Gastropoda animal abalone and snail in the Mollusca, the crust of lamellibranchiata animal blood clam and oyster; The cell walls of fungi in the microorganism (ascomycetes, basidiomycetes, phycomycete etc.); The cell walls of certain plants (as mushroom); Or the like.Through preliminary identification, increase suitable pre-treatment step, method of the present invention also is applicable to chitin extraction and preparation chitosan, oligochitosan from the current raw material that does not generally use, as the coral polyp of the leech of the clam worm of the cuttlefish of the centipede of the locust of Insecta, Myriapoda, Cephalopoda, chaetopoda, Hirudinea, Anthozoa, diatom in little algae and mammiferous joint, hoof, foot portions, or the like.
Beneficial effect of the present invention:
1. the raw material commonly used that is used for chitin extraction at present, basically be the solid waste in some product processing, as the crust after shrimp, the crab processing, the cot that silkworm chrysalis, fly maggot stay when extracting albumen, grease, the mycelium residue of leaving over behind the fungi fermentation target product, the hoof that animal slaughtering is left, foot, joint etc.Abandoning of these wastes not only caused the waste of precious resources, and occupies expensive real estate, brings serious environmental to pollute, and influence is produced and HUMAN HEALTH.The technology of the present invention method helps to make full use of resource to the general applicability of chitin extraction raw material, turns waste into wealth, improve working condition simultaneously, administer environment, can also obviously improve the overall economic efficiency of relevant secondary industry, promote industry development, reach the effect of multi-win.
2. the objective of the invention is to the existing various drawbacks of main flow operational path-chemical process at present chitin extraction and products thereof preparation, carry out a series of technological improvement, the abundant eco-friendly biotechnology means of integrated application, relevant fermentation engineering and enzyme engineering technology means of microorganism particularly, bound fraction physics and chemical process, design is also implemented the production process route of a cover based on biological process, be used for from raw material chitin extraction commonly used at present, and further prepare chitosan and oligochitosan.The enforcement of novel process of the present invention, can overcome the multiple shortcoming that exists in traditional chemical process, mainly show: can obviously improve under the extraction efficiency and product quality premise of target component, significantly reduce the usage quantity of acid, alkali, oxygenant and other Harmful chemicals, thereby reduce waste liquid, waste sludge discharge, eliminate it the pollution of environment and the threat of HUMAN HEALTH; The technical process reaction is mild, mild condition, carry out under normal temperature, middle temperature most of working cell, reaction process does not have corrodibility and becomes dirt, intermediate product does not need a large amount of fresh water carrying out washing treatment, saved a large amount of energy consumption costs and equipment cost and precious water resource, reduced production cost; Core process be with microbial enzyme take as the leading factor biocatalytic reaction, high specificity, the selectivity height, irrelevant compositions such as the albumen in the raw material that can effectively dissociate, fat, pigment obviously improve yield, purity and the quality stability of chitin; The full cell fixation bio-reactor circulation enzyme digestion reaction that adopts takes off the acetyl novel process, not only process is simple, not pollution, and can accurately hold deacetylation, produce the chitosan of different DD values by the needs of product and purposes, be of value to the quality control of product and the diversification of kind and purposes; The high yield acetate bifidus bacillus and the high yield chitoanase Bacterium lacticum mixed fermentation chitosan novel process that adopt, the advantage of comprehensive acid hydrolysis and enzymic hydrolysis, having improved chitosan hydrolyzate greatly is the efficient and the controllability of oligochitosan, be the completely new approach that obtains to have the oligochitosan of high physiologically active with biotechnology, for the development technique of oligochitosan has increased new bright spot.In a word, the invention provides a kind of synergy joint consumption, environmental friendliness, resource-effective chitin/chitosan and green extraction of oligochitosan and preparation technology, provide new power for promoting the chitin industry development further, sustainably.
3. another outstanding beneficial effect of the present invention is fully to carry out the resource synthetic development utilization, obtains the high byproduct of a series of added values, improves overall economic efficiency.Be embodied in: 1) the micronization pre-treatment of crustaceans raw material helps follow-up with the chemical method acquisition food grade lime carbonate that output is bigger, purity is higher; 2) microbial compound enzyme enzymolysis process altogether, by fully dissociating of lipase and proteolytic enzyme, can obtain the purity height, easily extract lipid component and protein component, in order to make a series of lipids, protein and amino acids product; When 3) fermentation method prepares crude enzyme liquid, can obtain the beneficial microorganism thalline of various high-biomass, as rainbow conk, subtilis, Candida utilis, bifidus bacillus and Bacterium lacticum, be the extremely edible microorganism of safety, can also can be made into related products directly as edible or microorganism fodder microbial inoculum or probiotics.
Description of drawings
Fig. 1 represents the influence of the deacetylation cycle index of full cell fixation bio-reactor to the product deacetylation;
Fig. 2 represents the influence of the deacetylation cycle index of full cell fixation bio-reactor to the product viscosity.
Embodiment
For making purpose of the present invention, technical scheme and beneficial effect clearer, with the following Examples, the present invention is further described in detail.Should be appreciated that specific embodiment described herein only is used to explain the present invention, and be not used in qualification the present invention.
Determine the decalcification technology of crustaceans raw material:
Preparation process: shrimp, crab shell are cleaned, removal of impurities, drying, weigh after, mechanical disintegration is the powder body of 150-200 μ m to mean particle size; Crushing rear material is packed in the liquid acid-resistant container, and the HCI that immerses 4-6% soaks repeatedly, promptly proposes material every 2h, and drop acid solution 15 minutes is soaked again; The bubble (carbonic acid gas) that the question response process produces stops to emerge, and stops decalcification after continuing to soak 2h, and filtration is held back not molten material and is used for chitin extraction; To be rich in the filtrate of calcium chloride, in reactor, heat, feed CO to 45 ℃
2Constantly stir, react to filtrate substantially transparent (about 4h); Filter the lime carbonate that collecting precipitation goes out, washing, drying get pure white, fine and smooth, evengranular calcium carbonate powders, the about 30-32% of yield.
The Candida utilis of determining to ferment is produced lipase crude enzyme liquid technology:
The Candida utilis starting strain separates the home-brewed wine vinasse, by inventor's inducing and acclimating repeatedly, become high yield lipase activity bacterial strain, deposit number CCTCC NO.M207053, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on April 24th, 2007, classification called after Candida utilis ODK-CC
1(Candida utilis ODK-CC
1).
The crude enzyme liquid preparation process: Candida utilis is inoculated in the PDA slant medium and recovers to cultivate (rejuvenation) 5 days, transferred species is in the 500ml Erlenmeyer flask that the 300mlPDA liquid nutrient medium is housed, 25 ℃, 100r/min constant-temperature shaking culture 3 days, again with 5% inoculum size, with this bacteria culture fluid transferred species to the 10L stainless steel airlift fermentor of the PDA liquid nutrient medium that 6L is housed, 25 ℃, the aerobic deep-layer liquid of 100r/min was cultivated about 5 days, when recording lipase activity and peaking, stopped fermentation, remove thalline with tubular type thalline whizzer, getting its supernatant liquor is crude enzyme liquid, seals cryopreservation, is ready to use in the common enzymolysis step of this technology.
The Bacillus subtilus of determining to ferment is produced proteolytic enzyme crude enzyme liquid technology:
The Bacillus subtilus starting strain separates from the organic granule natto of Japan's product, by inventor's inducing and acclimating repeatedly, become the high proteinase yield live strain, deposit number CCTCC N0.M207028, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification name subtilis ODK-BK
1(Bacillus subtilis ODK-BK
1).
The crude enzyme liquid preparation process: the Bacillus subtilus inoculation recovers to cultivate (rejuvenation) 2 days in common solid medium, transferred species is in the 500ml Erlenmeyer flask that removes the agar liquid nutrient medium that 300ml is housed, 25 ℃, 100r/min constant-temperature shaking culture 3 days, again with 5% inoculum size, with this bacteria culture fluid transferred species to the 10L stainless steel airlift fermentor that the 6L liquid nutrient medium is housed, 25 ℃, the aerobic deep-layer liquid of 100r/min was cultivated about 3 days, when recording lipase activity and peaking, stopped fermentation, remove thalline with tubular type thalline whizzer, getting its supernatant liquor is crude enzyme liquid, seals cryopreservation, is ready to use in the common enzymolysis step of this technology.
The Coriolus of determining to ferment is produced hydrogen peroxide synthetic enzyme-reductase enzyme crude enzyme liquid technology:
Coriolus (rainbow conk) starting strain is available from Guangdong Microbes Inst DSMZ (strain number: GIM5.178), by inventor's inducing and acclimating repeatedly, become H
2O
2Oxido-reductase is a superior strain, deposit number CCTCC NO.M207024, and depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. Wuhan University, the preservation time is on March 23rd, 2007, classification name rainbow conk ODK-CY
1(Polystictus versicolor ODK-CY
1).
The crude enzyme liquid preparation process: the rainbow conk bacterial strain is inoculated in the PDA slant medium and recovers to cultivate (rejuvenation) 7 days, transferred species is in the 500ml Erlenmeyer flask that the 300mlPDA liquid nutrient medium is housed, 28 ℃, 150r/min constant-temperature shaking culture 5 days, again with 5% inoculum size, with this bacteria culture fluid transferred species to the stainless steel airlift fermentor that 6L liquid PDA substratum is housed, 30 ℃, the aerobic deep-layer liquid of 100r/min was cultivated about 7 days, when survey polyphenol peroxidase activity peaks, stopped fermentation, remove thalline with tubular type thalline whizzer, getting its supernatant liquor is crude enzyme liquid, and the sealing cryopreservation is ready to use in the decolouring step in this operational path.
Determine high yield acetate bifidus bacillus strain reparation technology:
Bifidus longum bb separates from healthy people's fresh excreta, by inventor's inducing and acclimating repeatedly, become the acetate superior strain, deposit number CCTCC NO.M207027, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification called after bifidus longum bb ODK-BS
1(Bifidobacterium longum ODK-BS
1).
Produce the bacterial strain preparation process: pure culture master seed bacterial strain is inoculated in selectivity TPY substratum behind doubling dilution in the anaerobism glove box, and the mixed gas anaerobism is cultivated 48h, and flat board stops cultivating after being abound with colonies typical; Scrape bacterium colony in 20% skim-milk+2% sodium alginate suspension on clean bench the counting back, the bacterial classification pipe of packing into behind the mixing, and every 3ml, lyophilize is sealed preservation after vacuumizing, and bacterial strain is standby as producing.Keep sample and carry out production characteristic and API20A phenotypic evaluation.
Determine high yield chitoanase lactobacillus strains reparation technology:
Lactobacterium acidophilum separates from healthy people's fresh excreta, by inventor's inducing and acclimating repeatedly, become the chitoanase superior strain, deposit number CCTCC N0.M207026, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on March 23rd, 2007, classification called after plant lactobacillus ODK-LR
1(Lactobacillus plantarum ODK-LR
1).
Produce the bacterial strain preparation process: pure culture master seed bacterial strain is inoculated in selectivity MRS culture medium flat plate behind doubling dilution in the anaerobism glove box, and the mixed gas anaerobism is cultivated 48h, and flat board stops cultivating after being abound with colonies typical; Scrape bacterium colony in 20% skim-milk+2% sodium alginate suspension on clean bench counting back, the mixing bacterial classification pipe of packing into, and every 3ml, lyophilize is sealed preservation after vacuumizing, and bacterial strain is standby as producing.Keep sample and carry out production characteristic and API20A phenotypic evaluation.
Determine Rhizopus oryzae fermentation, immobilization reactor preparation and Deacetylation of Crab Chitin technology:
Determine Rhizopus oryzae enzymatic production processing condition: white Rhizopus oryzae starting strain is available from Guangdong Microbes Inst DSMZ (strain number: 3038), by inventor's inducing and acclimating repeatedly, become the chitin deacetylase superior strain, deposit number CCTCC NO.M207054, depositary institution is Chinese typical culture collection center, the preservation address is a China. Wuhan. and Wuhan University, the preservation time is on April 24th, 2007, classification called after Rhizopus oryzae ODK-RM
1(Rhizopus oryzae ODK-RM
1).
Rhizopus oryzae cell proliferation process: white Rhizopus oryzae is inoculated in the PDA slant medium and recovers to cultivate (rejuvenation) 7 days; transferred species is in the 500ml Erlenmeyer flask that the 300mlPDA liquid nutrient medium is housed; 25 ℃; 120r/min constant-temperature shaking culture 5 days; again with 5% inoculum size; with this bacteria culture fluid transferred species to the stainless steel airlift fermentor that 6L liquid PDA substratum is housed; 28 ℃; the aerobic deep-layer liquid of 100r/min was cultivated about 5 days; when the work of survey chitin deacetylase enzyme reaches definite value; stop fermentation, collect thalline, make the bacterium powder with freeze-drying after the protective material with tubular type thalline whizzer.
The full cell fixation bio-reactor of preparation Rhizopus oryzae:
Full cell fixation: with 2% sodium alginate soln and above-mentioned dry bacterial powder in liquid: Gu=5: 1 ratio thorough mixing, get the about 3L of suspension, under the room temperature with the speed of 10ml/min, splash into by the pin pump among the CaCL2 of 0.2mol/L, form the globule of the about 5.0-5.5mm of diameter, after all suspensions instil and finish, with the globule that the forms reactor assembly of packing into.
The upflowing bio-reactor is made: make cylindric stainless steel up-flow reactor main body, internal diameter 50.8cm, height 584cm, working volume 10L; Be aided with that substrate flows into, product flows out, bacterium culture medium stream adds and function passage such as rare gas element pressurization.
Deacetylation and effective evaluation: operational path gained chitin of the present invention; behind 40 ℃ of warm water suspensions, abundant mixing; be added on the full cell fixation reactor of Rhizopus oryzae from the substrate slow stream that enters the mouth; carry out the deacetylation reaction; after reaction solution is collected in the product outlet; be back to reactor by closed circuit channel cycle, the about 60min of each loop cycle.Bigger than normal during the conversion reaction as the reaction solution viscosity, with the rare gas element eluted product of pressurizeing.The time point of reaction terminating is complied with the requirement of product deacetylation and reactor enzyme intensity alive and is decided.The reaction cycle number of times sees the following form to the influence of deacetylation:
Immobilized cell deacetylation cycle index is to the influence of product deacetylation
| Cycle index | Deacetylation (%) | Viscosity (mPa.s) | |
| 1 2 3 4 5 6 | 42.21 56.74 68.45 76.32 85.75 92.38 | 220 380 320 230 | A small amount of molten a small amount of molten a small amount of insoluble a small amount of insoluble complete molten molten entirely |
Embodiment 1: the preparation of crab shell chitin
A. get river crab shell 10kg, clean, after the removal of impurities, drying, the HCI that immerses 4-6% soaks decalcification repeatedly, promptly proposes material every 2h, and the drop acid solution is soaked after 15 minutes again; The bubble (carbonic acid gas) that stops to emerge in the question response process stops decalcification after continuing to soak 2h again.
B. filtering separation decalcification crab shell and calcic acid solution (filtrate otherwise processed), the washing of decalcification shell material clear water is to neutral, and fully oven dry is crushed to the powder body that mean particle size is 60-100 μ m with the pin bar disintegrator.
C. after powder body boiling water being boiled 15min, drain, again with solid: the proportional quantity of liquid=1: 4, add 40 ℃ clean warm water, behind the suspendible in reactor, slowly mix with lipase and proteolytic enzyme mixing crude enzyme liquid by 1: 1 proportional quantity, carry out common enzymolysis degreasing deproteinated reaction, reaction times 18h is dissolved in major part fat, protein in the reaction solution, centrifugal behind the reaction terminating (2500r/min) carries out solid, liquid and separates, and is dissolved in albumen and the method extraction in addition of fatty product in the reaction solution.
D. collect the solid materials behind the enzyme digestion reaction, wash 3 times after, use 5%H earlier
2O
2Boil 1h, decolouring fast is again with being rich in H
2O
2The Coriolus of oxido-reductase system goes the thalline crude enzyme liquid to carry out enzyme reaction, removes difficult dissociated pigment in the raw material, and successively with H
2O
2Be converted into nontoxic CO
2And H
2O, 38 ℃ of temperature of reaction, reaction times 18h, centrifugal (2500r/min) finished in reaction, with washing of precipitate, drying, gets white powder crab shell chitin.
Embodiment 2: the preparation of maggot shell chitosan
A. pre-treatment: collect fresh fly maggot 30kg, the broken skin of machinery is handled after cleaning removal of impurities, abundant draining, extrude content (otherwise processed) with squeezing machine, collect surplus cot, dry to moisture＜10%, pre-freeze 6h below 0 ℃ after hardening, is crushed to the powder body that mean particle size is 150-250 μ m (a 60-100 order) with centrifugal collision pulverizer with it.
B. after obtaining powder body boiling water and boiling 15min, drain, again with solid: the proportional quantity of liquid=1: 4, add 40 ℃ clean warm water, behind the suspendible in reactor, slowly mix with lipase and proteolytic enzyme mixing crude enzyme liquid by 1: 1 proportional quantity, carry out common enzymolysis degreasing deproteinated reaction, reaction times 18h is dissolved in major part fat, protein in the reaction solution, centrifugal behind the reaction terminating (2500r/min) carries out solid, liquid and separates, and is dissolved in albumen and the method extraction in addition of fatty product in the reaction solution.
C. collect the solid materials behind the enzyme digestion reaction, 40 ℃ of Warm Wash 3 times drain, and use 5%H earlier
2O
2Boil 1h, decolouring fast is again with being rich in H
2O
2The Coriolus of oxido-reductase system goes the thalline crude enzyme liquid to carry out the enzyme process decolouring, 38 ℃ of temperature of reaction, and reaction times 18h, reaction Bi Jinhang centrifugal (2500r/min) with washing of precipitate, drying, gets the white powder chitin.
D. the throw out after will washing is by solid: the proportional quantity of liquid=1: 4 or the exsiccant chitin by solid: the proportional quantity of liquid=1: 8; be mixed into suspension with 40 ℃ of warm water; fully behind the mixing; measuring cycling stream in accordance with regulations is added in the full cell fixation reactor of upflowing Rhizopus oryzae; carry out the deacetylation reaction; with the DD value (deacetylation) of potentiometric titration detection of dynamic reaction product, until the DD of reaction solution sample value〉85%, viscosity is lower than 100 * 10
-3During Pas, termination reaction.
F. wash-out, collecting reaction product concentrate final vacuum or lyophilize by 1/3 volume decompression, get high deacetylized, low-viscosity chitosan.
Embodiment 3: the preparation of black-koji mould filament oligochitosan
A. pre-treatment: citric acid waste residue (mainly the containing the black-koji mould filament) 50kg that foodstuff additive factory collects, the clear water drop is wet to reach 70% to humidity, and grinding to the material mean particle size with colloidal mill is the mycelia powder body of 20-60 μ m.
B. the mycelia powder body is earlier with 65 ℃ of hot-water soak 30min, incline to, again with solid: the proportional quantity of liquid=1: 4, the clean warm water that adds 40 ℃, in reactor with (lipase: proteolytic enzyme=1: 6) mix, intermittently stirring and evenly mixing carries out common enzymolysis degreasing deproteinated reaction with mycelia suspension equivalent volumetrical lipase and proteolytic enzyme crude enzyme liquid, reaction times 18h, centrifugal behind the reaction terminating (2500r/min) carry out solid, liquid to be separated.
C. collect solid substance, 40 ℃ of Warm Wash 3 times drain, and use 5%H earlier
2O
2Boil 1h, decolouring fast is again with being rich in H
2O
2The Coriolus that oxido-reductase is goes the thalline crude enzyme liquid to carry out the enzyme process decolouring, 40 ℃ of temperature of reaction, and reaction times 18h, centrifugal (2500r/min) finished in reaction, and throw out washing, drying get the white powder chitin.
D. with obtaining chitin by solid: the proportional quantity of liquid=1: 8; again suspend with 40 ℃ of warm water; quantitative cycling stream is added in the full cell fixation reactor of upflowing Rhizopus oryzae behind the mixing; carry out the deacetylation reaction; the DD value (deacetylation) of potentiometric titration detection of dynamic reaction product; until the DD of reaction solution sample value〉70%, termination reaction.
F. collect reaction solution, constant volume is decided after the concentration as carbon source and fermentation substrate, adding 1% multivalence peptone is nitrogenous source and small amounts of inorganic salt, inoculate inventor's inducing and acclimating, can be the bifidus bacillus and the Bacterium lacticum of nutritive substance by 2% inoculum size with the chitosan, the mixed fermentation of anaerobism deep-layer liquid reaches at least until the thalline biomass〉1.0 * 10
9Behind the cfu/ml, stop fermentation, remove thalline with special thalline separating machine, fermented liquid directly concentrates, and vacuum or lyophilize get Powdered, the oligochitosan of 80% relative molecular weight＜8000 wherein of solid translucent.
Embodiment 4: the preparation of crab shell food grade lime carbonate
A. river crab shell 10kg cleans, after the removal of impurities, drying, and mechanical disintegration is the powder body of 150-200 μ m to mean particle size.
B. above-mentioned powder body is packed in the saturating liquid container of acid proof, the HCI that immerses 4-6% soaks repeatedly, promptly mentions the pickling material every 2h, and drop acid solution 15 minutes is soaked again; After the bubble (carbonic acid gas) that the question response process produces stops to emerge, stop decalcification after continuing to soak 2h, filtration is held back undissolved solid materials and is used for chitin extraction again.
C. collect the filtrate of being rich in calcium chloride, in reactor, heat, feed CO2 and constantly stir, react to filtrate substantially transparent (about 4h), stop ventilation and stirring to 45 ℃.
D. filter the lime carbonate that collecting precipitation goes out, washing, drying get pure white, fine and smooth, evengranular calcium carbonate powders, and yield is about the 30-32% of crab shell dry weight.
Embodiment 5: the extraction of pupa albumen
A. pre-treatment: fresh silkworm chrysalis 20kg cleans removal of impurities, and it is stand-by that milling process is extruded content, and cot is dried to pre-freeze below moisture＜10%, 0 ℃, with centrifugal collision pulverizer it is crushed to the powder body that mean particle size is 150-250 μ m (a 60-100 order).
B. powder body is earlier with 65 ℃ of hot-water soak 30min, incline to, again with solid: the proportional quantity of liquid=1: 4, the clean warm water that adds 40 ℃, in reactor, carry out common enzymolysis degreasing deproteinated reaction under 40 ℃ of constant temperature with equivalent volumetrical lipase and proteolytic enzyme mixing crude enzyme liquid, time 18h, centrifugal behind the reaction terminating (2500r/min) carry out solid, liquid to be separated.
C. solid is used for the chitin extraction.The separating liquid of lipid protein matter is rich in collection, is evaporated to 1/4 former volume, with the contents mixed that had before squeezed out, continues to be concentrated into paste.
D. with the sherwood oil submergence of pupa cream with 3 times of amounts, 60 ℃ leach 4 times, and each 1h soaks and finishes centrifugal (3500r/min, 20min) solid, liquid separation.
E. the liquid mixing oil after separating is used for pupal fat and extracts.With the sedimentary gray solid degreasing pupa dregs of rice, adding distil water stirs into homogenate, uses the 0.25%NaOH solubilising protein, filter and remove solid substance, filtrate is transferred pH to 4.5 with HCL, leaves standstill 3h, add saturated ammonium sulphate solution, leave standstill, centrifugal, throw out is protein, with this pulpous state albumen precipitation dialysis tubing of packing into, use activated carbon decolorizing behind the dialysis desalination, 95% ethanol extracting, vacuum-drying must be made with extra care the pupa albumen powder.
Embodiment 6: the extraction of mushroom handle Mierocrystalline cellulose, xylogen
A. collect the processing mushroom surplus mushroom handle 30kg in back, clean, removal of impurities, oven dry, section, mechanical disintegration is to mean particle size 100-150 μ m, and comminution by gas stream is the powder body of 10-25 μ m to mean particle size again.
B. above-mentioned powder body with 40 ℃ of water purification by solid: behind liquid=1: 4 suspendible, in reactor, be that crude enzyme liquid carries out common enzyme digestion reaction with isometric(al) ready-formed lignin-degrading enzymes system and cellulose degrading enzyme, 40 ℃ of temperature of reaction, reaction times 24h.
C. reaction finishes, lyophilize immediately, and dry back powder body adds capacity 2.0%NaOH and removes albumen, heat 95 ℃, and 2h is chilled to the centrifugal 15min of 4000r/min after the room temperature, and inclining contains the albumen supernatant, precipitates to be washed to neutrality.
D. precipitation is by solid: the proportional quantity of liquid=1: 8; again suspend with 40 ℃ of warm water; cycling stream is added in the full cell fixation reactor of upflowing Rhizopus oryzae; carry out the deacetylation reaction, the DD value (deacetylation) of potentiometric titration detection of dynamic reaction product〉70%, termination reaction; collecting the reaction solution solid, liquid separates; the centrifugal 15min of 4000r/min, supernatant is a chitosan solution, is precipitated as xylogen and Mierocrystalline cellulose.
E. will precipitate with 80% ethanol in solid: after the abundant lixiviate of ratio of liquid=1: 10, the 2500r/min low-speed centrifugal is collected supernatant, the alcohol reflux lixiviate, and totally 3 times, each 30min merges vat liquor, and ethanol is reclaimed in suction filtration, distillation; PH transfers to 4.0 with concentrated solution, and fully the post precipitation finish-drying gets lignin product, and wherein content of lignin is not less than 90%.
F. collect the precipitation of 2500r/min after centrifugal, the alcohol reflux lixiviate, totally 3 times, each 30min collects insoluble precipitate, press solid with 18% sodium hydroxide solution: liquid=1: 20 mixed, be heated to 120 ℃, stir 45min, wash 3 times after thorough drying, get cellulose prods, wherein content of cellulose is not less than 90%.
Claims (8)
1. a method for preparing chitin and chitosan and oligochitosan is characterized in that, comprises the steps:
Shrimp and crab shells, insect cot or hypha,hyphae body and function dry method or wet method are carried out micronization processes make the powder body raw material, wherein said dry process is meant that with shrimp and crab shells, insect cot or radicula byssoidea thorough drying to water content＜10%, mechanical mill is the powder body of 50-100 μ m to mean particle size; Described wet processing is meant the particle that described raw material is ground in advance 150-200 μ m, is made into suspension, and mechanical mill or comminution by gas stream become the powder body that mean particle size is 10-25 μ m again;
For the crustaceans powder body, slough earlier with the dilute hydrochloric acid of 4-6% calcareous, precipitation is washed till neutrality after, the usefulness crude enzyme liquid that is rich in lipase and proteolytic enzyme carries out common enzyme digestion reaction again, removes fat and protein, makes chitin; For insects and Mycophyta powder body, then directly be total to enzymolysis with the microbial compound enzyme crude enzyme liquid, carry out degreasing, deproteinated, make chitin.
2. the method for preparing chitin and chitosan and oligochitosan according to claim 1, it is characterized in that: described microbial compound enzyme crude enzyme liquid is meant uses isolating starting strain voluntarily, inducing and acclimating and the Candida utilis bacterial strain of high yield lipase activity, its preserving number CCTCC NO.M207053, with high proteinase yield bacillus subtilis bacterial strain alive, its preserving number CCTCC NO.M207028, behind aerobic liquid submerged fermentation, the extracellular enzyme crude enzyme liquid of removing the thalline gained is mixed and forms respectively; Described altogether enzymolysis degreasing, deproteinated reaction be earlier with the raw material powder body with 65 ℃ of warm water soaking 30min, the assorted bacterium and the irrelevant enzyme that suppress in the raw material are alive; After above-mentioned lipase and proteolytic enzyme crude enzyme liquid be mixed with 1: 1 volume, in reactor with the raw material powder body by liquid: Gu=5: 1 proportional quantity mixes, under 40 ℃ of constant temperature, in reactor, fully mix, carry out common enzymolysis degreasing deproteinated reaction, 18 hours reaction times, separate collecting precipitation with the centrifugal solid, liquid that carries out of 2500r/min behind the reaction terminating.
3. the method for preparing chitin and chitosan and oligochitosan according to claim 1 and 2, it is characterized in that: the chitin that makes behind described degreasing, the deproteinated further decolours in conjunction with the method for microbial enzyme enzymolysis with oxygenant, described decolouring is handled and is adopted the two-step approach decolouring, promptly elder generation is with the solid materials of enzyme digestion reaction postprecipitation, add in the diluted alkaline and soak, decoct 2h, repeat 3 times behind the replacing alkali lye, filter and collect filter residue, after washing 3 times, use 5%H
2O
2Boiled 1 hour, decolouring fast is again with being rich in H
2O
2Oxido-reductase is that crude enzyme liquid carries out enzyme digestion reaction, removes to be difficult to resolve in the raw material from pigment and successively with H
2O
2Be converted into nontoxic CO
2And H
2O, 40 ℃ of temperature of reaction, reaction times 18h, reaction is finished centrifugal solid, liquid and is separated, and will get the white powder chitin after washing of precipitate, the drying.
4. the method for preparing chitin and chitosan and oligochitosan according to claim 3 is characterized in that: described H
2O
2Oxido-reductase is that crude enzyme liquid is to separate the high yield H of domestication voluntarily
2O
2And the Coriolus of oxido-reductase system, its preserving number CCTCC NO.M207024 behind aerobic liquid submerged fermentation, removes the extracellular enzyme crude enzyme liquid that thalline obtains.
5. the method for preparing chitin and chitosan and oligochitosan according to claim 1 and 2, it is characterized in that: the full cell fixation bio-reactor of preparation chitin deacetylase superior strain, to the chitin deacetylation that circulates, described deacetylation is the Rhizopus oryzae bacterial strain that will separate the high yield deacetylase of domestication earlier voluntarily, its preserving number CCTCC NO.M207054, preparation becomes full cell fixation bio-reactor, again aforementioned gained chitin is suspended with 40 ℃ of warm water, fully behind the mixing, cycling stream is added in this reactor, carries out the deacetylation reaction by the hydrolytic action of deacetylase; According to the requirement of product and purposes to deacetylation, the DD value of dynamic monitoring reaction product, until reaching desired DD value standard, termination reaction, collecting reaction product, after concentrating by 1/3 volume, vacuum or lyophilize, chitosan.
6. the method for preparing chitin and chitosan and oligochitosan according to claim 5, it is characterized in that: the preparation of described full cell fixation bio-reactor comprises: 1) cell fixation: with quantitative 2% sodium alginate soln and Rhizopus oryzae lyophilized powder in liquid: Gu=4: 1 ratio thorough mixing, the gained suspension splashes into the CaCl of 0.2mol/L at room temperature with the speed of 10ml/min by the pin pump
2In, make the globule that forms diameter 5.0-5.5mm, after all suspensions instil and finish, the globule that forms is packed in the bioreactor device; 2) make cylindric stainless steel reactor main body jar, internal diameter 50.8cm, height 584cm, working volume 10L, main body jar press the design of upflowing reaction principle, are aided with that substrate flows into, product flows out, microorganism culturing becomes to shunt and add and rare gas element pressurization function passage.
7. the method for preparing chitin and chitosan and oligochitosan according to claim 5, it is characterized in that: use the bifidus bacillus strain that separates the high yield acetate of domestication voluntarily, its preserving number CCTCCNO.M207027, lactobacillus strains with the high yield chitoanase, its preserving number CCTCC NO.M207026, the chitosan of mixing liquid submerged fermentation under anaerobic, control bifidus bacillus thalline biomass is 1.0 * 10
8Cfu/ml and fermented liquid acetic acid concentration are in the scope of 15-20mol/L; The fermentation later stage stream add micro-nitrogenous source and the inorganic salt that help growth of lactobacillus and adjust pH, until Bacterium lacticum thalline biomass reach＞1.0 * 10
10Behind the cfu/ml, stop fermentation, fermented liquid directly concentrates after removing thalline, and vacuum or lyophilize get the oligochitosan of 80% above relative molecular weight＜8000.
8. the method for preparing chitin and chitosan and oligochitosan according to claim 7; it is characterized in that: under 0 ℃, be soaked in the mixing solutions of forming by diacetyl oxide and perchloric acid above-mentioned gained oligochitosan and agitation as appropriate; more than the acetylization reaction 24h; treat that the acetyl degree reaches 1.0 termination reactions when above; extremely neutral with the frozen water washing; use dewatering of ethanol again, vacuum-drying gets the crust oligosaccharides.
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