CN101078023B - Method for preparing chitin/chitosan from rind and shell of silkworm chrysalis and fly maggot - Google Patents
Method for preparing chitin/chitosan from rind and shell of silkworm chrysalis and fly maggot Download PDFInfo
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Abstract
The present invention discloses a method for producing chitin/chitosan from the shell of silkworm chrysalis, fly larvina, which comprises: to break into pieces to progress the shell of silkworm chrysalis, fly larvina by dry or wet method; React the fine grits obtained with the fat enzyme and protein enzyme from micro-organism by a enzymolysis reaction in order to to eliminate the fat and protein contained in the chrysalis / larvina raw material; bleach using two-step method; Collect the materials after bleach and to wash, dry materials to obtain chitin product; Obtain chitosan by to circulate to cast off Acetyl reaction in a Rhizopus oryzae full cell immobilization biology reaction vessel; Obtain height hydrosoluble shel oligosaccharide by liquid submerged fermentation chitosan using acid formers. The present invention improves the quality of the product and the efficiency of production; reduces greatly the energy cost, the usage of acid, alkali, oxidant and other harmful chemistry thing and the discharge of effluent, waste residue; produces byproducts of high value at the same time; utilizes the resources integratedly; and increases the benefits of production.
Description
Technical field
The invention belongs to biotechnology applications and novel material, Chemicals production technical field, relate in particular to a kind of method of from the cot of silkworm chrysalis, fly maggot and other insect, producing chitin/chitosan with biological process, specifically, be that application is total to enzymolysis with multiple microbial enzyme and the bio-transformation of immobilized whole-cell bio-reactor is produced chitin and/or chitosan from insect cot raw material.
Background technology
Chitin has another name called chitin, and formal name used at school chitin (chitin) is the natural glycosaminoglycan of a kind of straight chain, also is unique edibility animal fibre that has positive charge of finding at present.Chitin is only second to Mierocrystalline cellulose at natural reserves, and the biosynthesizing amount in annual biosphere has 10,000,000,000 tons approximately, is second largest Biological resources on the earth.As structural material, chitin mainly is distributed in fungi, protobiont and invertebrates, then mainly is distributed in the joint of joint, hoof, foot etc. and muscle and bone higher animal.For a long time, because the inertia characteristics of water insoluble, the diluted acid of chitin, diluted alkaline and most of organic solvents are subjected to that never attention should be arranged.But in recent decades; huge advance made along with life science and biotechnology; the physicochemical property of chitin; biological activity and functional characteristics are familiar with by people gradually; particularly chitin and derivative thereof as a kind of novel material and material in pharmacy; organizational project; fine chemistry industry; material and textile industry; foodstuffs industry; the using value of agricultural and all many-sides of environment protection; shown apace; get more and more people's extensive concerning; the degree of depth of its industrialization and range vigorously advance; and it is to human body and useful and harmless " green material " characteristic of environment; meet the tendency of the day especially; obtain people's favor, showed very fine market outlook.
Though chitin is a kind of good biological raw material, the biological structure composition that its conduct and protein and some inorganics are combined closely extracts and is not easy.Be used to produce chitin, the chitosan (product behind the chitin deacetylase base in the global range at present, can increase solubleness greatly) and the main flow technology of derivative be chemical method, use a large amount of soda acid and deleterious chemical reagent in its preparation process, produce a large amount of waste water waste materials, serious environment pollution is water body environment particularly; High frequency, long high-temperature process have brought huge energy consumption; The recycling difficulty of frequent washing, purifying program and diluted acid, diluted alkaline causes valuable cleaning fresh water to be wasted by mass consumption again; All this kind is for the sustainable Development and Production and the commercial application in a dark step of this " green material " have been coverd with heavy shade.Just because of this, in recent years, people begin sight has been turned to biological method, mainly be the method for biological fermentation at present, promptly by fermentation to the main raw material filamentous fungus, obtaining the mycelium of capacity, is raw material again with the mycelium, the method chitin extraction and the chitosan that adopt microbial enzyme and chemical method to combine.This method mild condition, can reduce human and material resources and energy consumption, easy to control the quality, also can alleviate environmental pollution significantly, but since in the microbe chitin content far away from reasons such as arthropods (a large amount of especially at present Crustachia and Insecta animals of using) and fungi fermentation time are very long, it is desirable not enough on output and efficient that biological fermentation process is produced chitin/chitosan, and its production cost is too high, realizes that really suitability for industrialized production also will be after some time.
Produce shrimp, crab shell that the most frequently used raw material of chitin is an industrial waste at present, its chemical constitution is: 40% inorganics mainly is CaCO3; 30% organism mainly is a protein; Chitin content accounts for 20-25%.Therefore, the extraction process route that adopts according to shrimp, crab shell chemical constitution characteristics is: purifying treatment-concentrated acid decalcification-diluted alkaline deproteinated-oxygenant decolouring (nonessential)-concentrated base deacetylation-chitosan.The shortcoming of this technology as described above, and also there is the restriction of the place of production and transportation cost in its raw material sources.Another kind of main raw material is the filamentous fungus mycelium, and as the mycelium waste residue that fermentation industry produces, chitin content can reach 20-22%.The advantage of its extraction process and limitation are also as described above.And biological fermentation process just obtains raw material at present, and follow-up chitin and the chitosan produced still mainly adopts chemical method, so still there is problem of environmental pollution.
In recent years, contain the fact of enriching chitin in the various insects guiding principle animal cot, cause people's very big concern.Studies confirm that, contained chitin is up to 33%-44% in the silkworm pupa skin, and the contained chitin of dried maggot skin is more up to 35%-54.8%, and contains inorganicss such as calcareous in this type of insect cot hardly, the separation and Extraction that helps chitin is ideal chitin raw materials for production rather.Yet although silkworm chrysalis and fly maggot cot have above-mentioned advantage, recent practice shows; produce chitin with chemical method from silkworm chrysalis and fly maggot and have difficulties, it is too many mainly to show as in the cot fat, and pigment is too many; the deacetylation difficulty, thereby the chitin quality product of producing is not high.In addition,, strengthened the physical/chemical strength disposal of decolouring and deacetylation, produced bigger waste liquid, waste material pollution and more energy consumption owing to increased the chemical degreasing step in the extraction process.
Therefore, how improving technology, enable to utilize the insect cot to develop high-quality chitin and related products, can effectively control energy consumption and environmental pollution again simultaneously, is the huge challenge that chitin research and development and producers face.
Summary of the invention
The technique means that the objective of the invention is to application of fermentation engineering and enzyme engineering, bound fraction physics and chemical process, design has also been implemented the chitin/chitosan preparation method of a cover based on biological process, in order to from silkworm chrysalis, obtain chitin and chitosan in fly maggot and other insect cot, be intended to overcome the above-mentioned drawback of traditional technology, under the prerequisite that obviously improves product quality and production efficiency, cut down the consumption of energy, 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, 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 reparation technology reaches effective, promote the purpose of chitin industry development sustainably.
The invention still further relates to the chitin/chitosan that to extract, further be processed into extraordinary chitosan or chitosan derivatives with biology and/or chemical process.
The technical scheme that realizes the object of the invention is as follows:
A kind of method of producing chitin/chitosan from the cot of silkworm chrysalis, fly maggot may further comprise the steps: the cot of silkworm chrysalis, fly maggot is handled by dry method or wet crushing fine-powdered; The fine powder body that obtains is carried out common enzyme digestion reaction through lipase crude enzyme liquid and proteolytic enzyme crude enzyme liquid from microorganism, fully remove fat and protein in pupa skin/maggot skin raw material; With the two-step approach decolouring, promptly earlier with H
2O
2Preliminary decolouring, again be rich in hydrogen peroxide synthetic-crude enzyme liquid of reductase enzyme system carries out enzyme process decolouring and reduction H
2O
2Collect decolouring back material, make the chitin product after washing, the drying; By the full cell fixation bio-reactor of the Rhizopus oryzae deacetylation that circulates, obtain the chitosan of different DD values.Also further applied chemistry method or biological method become extraordinary chitosan or modified chitosan with the chitin or the Preparation of Chitosan of gained.
Described fine-powdered is handled, be meant bright silkworm chrysalis or bright fly maggot, mechanical in advance broken skin is handled after cleaning removal of impurities, abundant draining, use such as the milling process of fluid pressure type or expeller and extrude content (described content otherwise processed), collect cot, dry to pre-freeze below moisture＜10%, 0 ℃ 6 hours, it is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order) with centrifugal collision pulverizer; As be dried pupa or dried maggot, directly pre-freeze, pulverization process become the fine powder body then to purify the back.
Described microorganism crude enzyme liquid, be meant the Candida utilis (preserving number CCTCC NO.M207053) of the high yield lipase activity that gets with different starting strain inducing and acclimatings, 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 subtilis (preserving number CCTCC NO.M207028) that high proteinase yield is lived, 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(BacillussubtilisODK-BK
1); High yield H
2O
2And the Coriolus (having another name called rainbow conk) (preserving number CCTCC NO.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), respectively behind aerobic liquid submerged fermentation, remove thalline and the extracellular enzyme crude enzyme liquid that obtains.
Described enzyme digestion reaction altogether, be meant obtaining fine powder body with solid: the proportional quantity of liquid=1: 5, earlier with 65 ℃ of warm water soaking 30min, the bacterium that suppresses to mix reaches irrelevant enzyme and lives, add lipase crude enzyme liquid and proteolytic enzyme crude enzyme liquid again with warm water volume equivalent, under 40 ℃ of constant temperature, in reactor, cot powder suspension and crude enzyme liquid are slowly mixed, fully stir, carry out common enzymolysis degreasing deproteinated reaction, reaction times 18h separates with the centrifugal solid, liquid that carries out of 2500r/min behind the reaction terminating, is dissolved in albumen and fat and degradation production method extraction collection in addition thereof in the reaction solution.
Described two-step approach decolouring is meant elder generation with the solid materials behind the enzyme digestion reaction, adds a small amount of diluted alkaline (10%NaOH) and soaks, decocts 2h, repeat after changing alkali lye, totally 3 times, thoroughly to remove small portion of residual fat and protein and remnant enzyme activity, filter to collect filter residue, wash 3 times after, use 5%H earlier
2O
2Boil 1h, decolouring fast is again with being rich in H
2O
2And H
2O
2The crude enzyme liquid of oxido-reductase system 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, centrifugal (2500r/min) finished in reaction, with washing of precipitate, drying, gets the white powder chitin.
Described deacetylation, be meant with the gained chitin with 40 ℃ of warm water suspend, fully behind the mixing, cycling stream is added on Rhizopus oryzae (preserving 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 name Rhizopus oryzae ODK-RM
1(Rhizopus oryzae ODK-RM
1); in the full cell fixation reactor; carry out deacetylation reaction, according to the requirement of product and purposes, with the DD value (deacetylation) of potentiometric titration detection of dynamic reaction product to deacetylation; until reaching desired DD value standard; termination reaction, collecting reaction product is after concentrating by 1/3 volume; vacuum or lyophilize get chitosan.
The extraordinary chitosan of described preparation, the present invention adopts biological process, promptly carries out the liquid submerged fermentation chitosan with acid formers, obtains to have the crust oligosaccharides of high water soluble.
Described preparation modified chitosan, the present invention adopts chemical method, after soon chitin will be processed as alkaline chitin colloidal solution, handle with oxyethane, Mono Chloro Acetic Acid or vinyl cyanide respectively, can generate ethoxyl chitin, carboxymethyl chitin or cyanoethyl chitin respectively.
Method of the present invention also is applicable to producing of other insect cot chitin, as cicada slough, blattaria, duster worm etc.
Beneficial effect of the present invention: the present invention utilizes pupa skin, maggot skin to produce chitin, helps to administer environment, and recycling is turned waste into wealth, and drives the deep processing industry of silkworm chrysalis and fly maggot; The present invention selects for use silkworm chrysalis and fly maggot cot to produce chitin, except that its content height, is that also pupa skin chitin is mainly α-type chitin, has advantages of excellent stability, and is extremely important for the industrial applications of chitin; And fly maggot chitosan obviously is being better than shrimp, crab shell chitosan aspect low heavy metal ion content, the low ash content, and both are good chitin and produce raw material; The present invention has adopted and has removed fat, albumen and pigment technology based on enzyme process, because the highly selective of enzyme reaction, higher fatty acid, the high pigment that can overcome effectively in the insect cot extracts the difficulty that causes to chitin, obviously improves the yield and the quality of chitin; The present invention has significantly reduced acid, alkali and the application of organic solvent in reparation technology, and replace gentle microbial enzyme method as core process, not only basically eliminate the environmental pollution that causes of strong acid, highly basic, hazardous solvent waste water and waste sludge discharge, reduce relevant device and reagent cost, and stopped the residual potential health threat of bringing of chemical solvents in quality problems that strong chemical reaction causes and the product substantially; The present invention adopts full cell fixation bio-reactor circulation enzyme digestion reaction deacetylation novel process, not only process is simple, not pollution, and can accurately grasp deacetylation, need produce the chitosan of different DD values by product with purposes, be of value to the diversification of the quality control of product and kind, purposes; Another outstanding advantage of the present invention is that carry out under normal temperature, middle temperature most working cell, has significantly reduced the heat energy needs, has reduced energy consumption; In addition, also corresponding consumption and the discharge of wastewater that has reduced water of the minimizing of chemicals usage saved water; Another beneficial effect of the present invention is fully to carry out resource synthetic development, obtains the high by product of a series of added values, improves overall economic efficiency, comprising:
1) by the dissociating of lipase, can obtain the high silkworm chrysalis grease of purity,, can fully develop, as refining silkworm chrysalis oil, extract lipid acid, preparation tensio-active agent and tynex (nylon), make lubricating oil and soap etc. because of its impurity is few.
2) by the dissociating of proteolytic enzyme, can obtain high silkworm chrysalis of purity or fly maggot protein, can prepare edible or feed protein matter, produce pupa casein, preparation proteolysate and products of amino acid etc.
3) when preparing crude enzyme liquid with fermentation method, can obtain the beneficial microorganism thalline of high-biomass, wherein rainbow conk (Coriolus), subtilis, Candida utilis are 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 that puparium fine powder body is total to enzymolysis front and back lipid content and protein content compares;
Fig. 2 represents that fly shell fine powder body is total to enzymolysis front and back lipid content and protein content compares;
Fig. 3 represents the immobilized cell flow process;
Fig. 4 represents that the full cell fixation bio-reactor of upflowing Rhizopus oryzae takes off the acetyl schematic flow sheet.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with drawings and Examples.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
In order effectively to realize method of the present invention, the contriver has carried out following work:
1. 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, after inducing and acclimating is high yield lipase activity bacterial strain repeatedly by the inventor, send the typical culture collection center preservation (preserving number CCTCC NO.M207053) of Wuhan China, 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 utilisODK-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 that the 6LPDA liquid nutrient medium 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.
2. 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, after inducing and acclimating is the high proteinase yield live strain repeatedly by the inventor, deposit number CCTCC NO.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(Bacillussubtilis 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.
3. 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), tamed repeatedly to induce by the inventor and be H
2O
2After oxido-reductase is superior strain, deposit number CCTCC NO.M207024, 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-CY1 (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, seals cryopreservation, is ready to use in the decolouring step of this technology.
4. investigate the crude enzyme liquid effect of enzyme digestion reaction altogether
1) crude enzyme liquid is total to the effect that enzymolysis is removed puparium and fly shell fat and protein component
1. single factor analysis relatively is total to the lipid content (Soxhlet extraction process mensuration) of enzymolysis front and back puparium and fly shell fine powder body, sees Table 1.
Table 1 enzymolysis front and back lipid content altogether changes relatively
2. single factor analysis relatively is total to the protein content (Kjeldahl determination mensuration) of enzymolysis front and back puparium and fly shell fine powder body, sees Table 2.
Table 2 enzymolysis front and back protein content altogether changes relatively
2) chemical method and biological process are difficult to resolve from the pigment decolorizing effect relatively the silkworm chrysalis cot
With H
2O
2The puparium depigmentation effect of oxido-reductase system's (biological process) puparium depigmentation effect and potassium permanganate-sodium bisulfite (chemical method) compares, and the results are shown in Table 3.
Table 3 potassium permanganate-sodium bisulfite and H
2O
2Oxido-reductase is that the depigmentation effect compares
5. Rhizopus oryzae fermentation, immobilization reactor preparation and Deacetylation of Crab Chitin
1) determines Rhizopus oryzae enzymatic production processing condition
White Rhizopus oryzae starting strain is available from Guangdong Microbes Inst DSMZ (strain number: 3038), tame repeatedly by the inventor and to induce to behind 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 name 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 is cultivated about 5d; 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.
2) the full cell fixation bio-reactor of preparation
1. 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 among the 0.2mol/Lde CaCL by the pin pump, 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.
2. 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.
3) 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 Table 4 to the influence of deacetylation.
Table 4 immobilized cell deacetylation cycle index is to the influence of product deacetylation
Embodiment 1: the preparation of silkworm chrysalis chitin
A. pre-treatment: collect bright silkworm pupa 10kg, clean removal of impurities, fully handle with the broken skin of mincer machinery behind the draining, extrude content (otherwise processed) with milling process (fluid pressure type or expeller), collect cot, dry to moisture＜10%, pre-freeze 6h below 0 ℃ behind the caking, is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order) with centrifugal collision pulverizer with it.
B. obtain the fine powder body earlier with 65 ℃ of hot-water soak 30min, incline to, again with solid: the proportional quantity of liquid=1: 4, the clean warm water and equivalent volumetrical lipase and the proteolytic enzyme mixing crude enzyme liquid that add 40 ℃, under 40 ℃ of constant temperature, in reactor, cot fine powder body and crude enzyme liquid are slowly mixed, carry out common enzymolysis degreasing deproteinated reaction, reaction times 18h, major part fat, protein are dissolved 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, add a small amount of diluted alkaline (10%NaOH) and soak, decoct 2h, repeat decoction process by the identical time after changing alkali lye, totally 3 times, thoroughly remove small portion of residual fat and protein, filter the filtrate otherwise processed.
D. after filter residue is washed 3 times, use 5%H earlier
2O
2Boil 1h, decolouring fast is again with being rich in H
2O
2And H
2O
2The Coriolus of oxido-reductase system goes the thalline crude enzyme liquid to carry out enzyme reaction, removes in the raw material difficult dissociated pigment and successively H2O2 is converted into nontoxic CO
2And H
2O, 40 ℃ of temperature of reaction, reaction times 18h, centrifugal (2500r/min) finished in reaction, with washing of precipitate, drying, gets the white powder chitin.
Embodiment 2: the preparation of fly maggot chitosan
A. pre-treatment: collect bright fly maggot 10kg, clean removal of impurities, fully handle with the broken skin of mincer machinery behind the draining, extrude content (otherwise processed) with milling process (fluid pressure type or expeller), collect cot, dry to moisture＜10%, pre-freeze 6h below 0 ℃ after hardening, is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order) with centrifugal collision pulverizer with it.
B. obtain the fine powder body earlier with 65 ℃ of hot-water soak 30min, incline to, again with solid: the proportional quantity of liquid=1: 4, the clean warm water and equivalent volumetrical lipase and the proteolytic enzyme mixing crude enzyme liquid that add 40 ℃, under 40 ℃ of constant temperature, in reactor, cot fine powder body and crude enzyme liquid are slowly mixed, carry out common enzymolysis degreasing deproteinated reaction, reaction times 18h, major part fat, protein are dissolved 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 solid materials, 40 ℃ of Warm Wash 3 times drain, and boil 1h with 5%H2O2 earlier, and decolouring fast is again with being rich in H
2O
2And H
2O
2The Coriolus of oxido-reductase system goes the thalline crude enzyme liquid to carry out the enzyme process decolouring, 40 ℃ 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,
Quantitatively cycling stream 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%, when viscosity is lower than 100 * 10-3Pas, termination reaction.
F. collecting reaction product, after concentrating by 1/3 volume decompression, vacuum or lyophilize, high deacetylized, low-viscosity chitosan.
Embodiment 3: lactobacillus ferment prepares chitin oligosaccharide
A. pre-treatment: after silkworm pupa 10kg cleans removal of impurities, broken skin processing, milling process is extruded content, collects cot, dries to moisture＜10%, after the pre-freeze, it is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order) below 0 ℃ with centrifugal collision pulverizer.
B. the fine 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 and equivalent volumetrical lipase and the proteolytic enzyme mixing crude enzyme liquid that add 40 ℃ under 40 ℃ of constant temperature, slowly mix in reactor, intermittently stir, carry out common enzymolysis degreasing deproteinated reaction, reaction times 18h, centrifugal behind the reaction terminating (2500r/min) carry out solid, liquid to be separated.
C. collect solid materials, 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
2And 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 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 reaction substrate, add 1% multivalence peptone and be nitrogenous source (can directly from byproduct pupa albumen and hydrolysate thereof) and small amounts of inorganic salt, inoculate inventor's inducing and acclimating, can be the Bacterium lacticum of nutritive substance with the chitosan by 2% inoculum size, the anaerobism deep fermentation, until the thalline biomass reach at least＞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 solid translucent crust oligosaccharides Powdered, wherein 80% relative molecular weight＜3000.
Embodiment 4: the preparation of carboxymethyl chitin
A. pre-treatment: after bright fly maggot 10kg cleans removal of impurities, broken skin processing, milling process is extruded content, collects cot, dries to moisture＜10%, after the pre-freeze, it is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order) below 0 ℃ with centrifugal collision pulverizer.
B. the fine 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 and equivalent volumetrical lipase and the proteolytic enzyme mixing crude enzyme liquid that add 40 ℃, in reactor, carry out common enzymolysis degreasing deproteinated reaction under 40 ℃ of constant temperature, time 18h, centrifugal behind the reaction terminating (2500r/min) carry out solid, liquid to be separated.
C. collect solid materials, drain behind 40 ℃ of Warm Wash 3 times, use 5%H earlier
2O
2Boil 1h, decolouring fast is again with being rich in H
2O
2And H
2O
2The crude enzyme liquid that oxido-reductase is carries out the enzyme process decolouring, 40 ℃ of temperature of reaction, and reaction times 18h, centrifugal (3500r/min) finished in reaction, and throw out washing, drying get the white powder chitin.
D. the chitin that obtains add earlier in the Virahol of 10 times of amounts and stir swelling, add 45% about 5 times sodium hydroxide solution of chitin quality after draining again, continue to soak 3h below 15 ℃, intermittently stir, reaction finishes back elimination residual reaction liquid, must be equivalent to the alkaline chitin colloidal solution of 3 times of chitin quality approximately; Take by weighing the solid Mono Chloro Acetic Acid with 1.2 times of mass ratios to chitin, gradation adds in this colloidal solution, each space-number minute, finish, promote temperature to 70 ℃, reaction 3h, get water-soluble cm-chitosan, add 5 times of dissolved in distilled water, with 70% washing with alcohol, drying, get the white powder cm-chitosan behind Glacial acetic acid accent pH to 7.0, the suction filtration.
Embodiment 5: the greasy extraction of silkworm chrysalis
A. pre-treatment: after silkworm pupa 30kg cleans removal of impurities, broken skin processing, it is stand-by that milling process is extruded content, cot is dried to pre-freeze below moisture＜10%, 0 ℃, with centrifugal collision pulverizer it is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order).
B. the fine 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 and equivalent volumetrical lipase and the proteolytic enzyme mixing crude enzyme liquid that add 40 ℃, in reactor, carry out common enzymolysis degreasing deproteinated reaction under 40 ℃ of constant temperature, 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 volume, with the contents mixed that had before squeezed out, continues to be concentrated into paste.
D. carrying out grease with lixiviation process extracts: above-mentioned enriched material is moved into batch extractor, tinning amount 85%, adopt adverse current immersion type extract technology, with the soybean extracting solvent no.6 is leach solvent, pot group type (totally 3 jars) leaching mode is carried out the grease extracting, after mixing oil is put jar, press down, steam on the 145kPa steam with 98kPa steam, residual solvent in the pupa dregs of rice is taken off in steaming, after detecting residual fused lattice, discharge the degreasing pupa dregs of rice, continue on for proteins extraction by distiller grains outlet.
E. to the mixing oil that leaches gained evaporate, stripping, remove the leaching solvent in the mixing oil and easily bring into play component, the silkworm chrysalis crude oil, solvent residual amount＜0.05% in the control crude oil, or 220 ℃ of flash-points.Crude oil can continue refining and be refining pupal fat.
Embodiment 6: the extraction of fly maggot protein
A. pre-treatment: after fly maggot 20kg cleans removal of impurities, broken skin processing, it is stand-by that milling process is extruded content, cot is dried to pre-freeze below moisture＜10%, 0 ℃, with centrifugal collision pulverizer it is crushed to the fine powder body that mean particle size is 150-250 μ m (a 60-100 order).
B. the fine 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 ℃, and equivalent volumetrical lipase and proteolytic enzyme mixing crude enzyme liquid, carry out common enzymolysis degreasing deproteinated reaction under 40 ℃ of constant temperature in reactor, 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 volume, with the contents mixed that had before squeezed out, continues to be concentrated into paste.
D. with of the sherwood oil submergence of maggot cream with 3 times of amounts, 60 ℃ of leachings 4 times, each 1h soaks and finishes that centrifugal (3500r/min 20min) carries out solid-liquid separation.
E. isolating liquid mixing oil is used for maggot oil and extracts.With the grey degreasing maggot dregs of rice of solid precipitation, 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, precipitation 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 maggot protein powder.
Claims (7)
1. a method of producing chitin/chitosan from the cot of silkworm chrysalis, fly maggot is characterized in that may further comprise the steps: the cot of silkworm chrysalis, fly maggot is handled by dry method or wet crushing fine-powdered; The fine powder body that obtains is carried out common enzyme digestion reaction through lipase crude enzyme liquid and proteolytic enzyme crude enzyme liquid from microorganism, fully remove fat and protein in pupa skin/maggot skin raw material; With the two-step approach decolouring, promptly earlier with H
2O
2Preliminary decolouring is carried out enzyme process decolouring and reduction H with the crude enzyme liquid that is rich in hydrogen peroxide oxidation-reductase enzyme system again
2O
2Collect decolouring back material, make the chitin product after washing, the drying; By the full cell fixation bio-reactor of the Rhizopus oryzae deacetylation that circulates, obtain the chitosan that difference is taken off acetylation value; Described microorganism crude enzyme liquid is meant Candida utilis (Candida utilis) ODK-CC of the high yield lipase activity that gets with different starting strain inducing and acclimatings
1, preserving number CCTCC NO.M207053, subtilis (Bacillussubtilis) ODK-BK that high proteinase yield is lived
1, preserving number CCTCC NO.M207028, high yield H
2O
2Rainbow conk (Polystictus versicolor) ODK-CY of oxido-reductase system
1, preserving number CCTCC NO.M207024 respectively behind aerobic liquid submerged fermentation, removes thalline and the extracellular enzyme crude enzyme liquid that obtains; Described Rhizopus oryzae is Rhizopus oryzae (Rhizopus oryzae) ODK-RM
1, preserving number CCTCC NO.M207054.
2. the method for producing chitin/chitosan from the cot of silkworm chrysalis, fly maggot according to claim 1 is characterized in that: further carry out the liquid submerged fermentation chitosan with acid formers, obtain to have the crust oligosaccharides of high water soluble.
3. the method for from the cot of silkworm chrysalis, fly maggot, producing chitin/chitosan according to claim 1, it is characterized in that: after further chitin being processed as alkaline chitin colloidal solution, handle with oxyethane, Mono Chloro Acetic Acid or vinyl cyanide respectively, generate ethoxyl chitin, carboxymethyl chitin or cyanoethyl chitin respectively.
4. according to each described method of from the cot of silkworm chrysalis, fly maggot, producing chitin/chitosan of claim 1-3, it is characterized in that: described fine-powdered is handled, be meant if bright silkworm chrysalis or bright fly maggot, mechanical in advance broken skin is handled after then cleaning removal of impurities, abundant draining, milling process with fluid pressure type or expeller is extruded content, collect cot, dry to moisture＜10%, pre-freeze below 0 ℃ 6 hours is crushed to the fine powder body that mean particle size is 150-250 μ m with centrifugal collision pulverizer with it; If dried pupa or dried maggot, directly pre-freeze, pulverization process become the fine powder body then to purify the back.
5. each is described from silkworm chrysalis according to claim 1-3, produce the method for chitin/chitosan in the cot of fly maggot, it is characterized in that: described enzyme digestion reaction altogether, be meant obtaining fine powder body with solid: the proportional quantity of liquid=1: 5, earlier with 65 ℃ of warm water soaking 30min, the bacterium that suppresses to mix reaches irrelevant enzyme and lives, add again and the lipase crude enzyme liquid of warm water volume equivalent and the mixed solution that the blend of proteolytic enzyme crude enzyme liquid forms, under 40 ℃ of constant temperature, in reactor, cot powder suspension and crude enzyme liquid are slowly mixed, fully stir, carry out common enzymolysis degreasing, the deproteinated reaction, in 18 hours reaction times, consolidate so that 2500r/min is centrifugal behind the reaction terminating, liquid separates.
6. according to each described method of from the cot of silkworm chrysalis, fly maggot, producing chitin/chitosan of claim 1-3, it is characterized in that: described two-step approach decolouring, be meant earlier the solid materials behind the enzyme digestion reaction, add in a small amount of diluted alkaline and soak, decoct 2h, repeat 3 times after changing 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
2The crude enzyme liquid of oxido-reductase system 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, centrifugation is finished in reaction, with washing of precipitate, the dry white powder chitin that gets.
7. each is described from silkworm chrysalis according to claim 1-3; produce the method for chitin/chitosan in the cot of fly maggot; it is characterized in that: described deacetylation; be meant the gained chitin is suspended with 40 ℃ of warm water; fully behind the mixing; cycling stream is added in the full cell fixation reactor of Rhizopus oryzae; carry out the deacetylation reaction; according to the requirement of product and purposes to deacetylation; deacetylation with potentiometric titration detection of dynamic reaction product; until reaching desired deacetylation standard; termination reaction, collecting reaction product is after concentrating by 1/3 volume; vacuum or lyophilize get chitosan.
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| CN102993334B (en) * | 2010-03-30 | 2015-01-14 | 甘肃农业大学 | Preparation technology of fly maggot chitin and chitosan |
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| KR102393124B1 (en) * | 2021-01-13 | 2022-05-03 | 농업회사법인 (주)한국유용곤충연구소 | Method for preparing fermented insect feed additive comprising Ptecticus tenebrifer and insect chitin, and fermented insect feed additive prepared by the same |
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| CN114230629B (en) * | 2021-12-09 | 2023-03-31 | 山东凤凰生物科技股份有限公司 | Preparation method of 18-alpha glycyrrhetinic acid |
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