CN112301080A - Preparation method of squid cartilage beta-chitosan oligosaccharide - Google Patents
Preparation method of squid cartilage beta-chitosan oligosaccharide Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention discloses a preparation method of squid cartilage beta-chitosan oligosaccharide, which solves the problems of long time consumption, more impurities, low extraction rate and low purity of the existing enzyme method for processing the beta-chitosan oligosaccharide. Which comprises the following steps: 1) extracting phosphatase; 2) extracting beta-chitosan; 3) preparing beta-chitosan oligosaccharide; 4) refining beta-chitosan; 5) spray drying; 6) and (6) decoloring. The method utilizes phosphatase and polyphosphoric acid to remove organic matters and mineral matters in squid cartilage, the residual chitin is decomposed into macromolecular beta-chitosan, the beta-chitosan is biodegraded to prepare beta-chitosan oligosaccharide, the refined beta-chitosan oligosaccharide is obtained by repeated alcohol precipitation and combined ultrafiltration and dialysis, then the beta-chitosan oligosaccharide is dried and oxidized and decolored to quantize beta-chitosan oligosaccharide micromolecules, and thus the 1200-plus-1500 Da beta-chitosan oligosaccharide product is obtained. The method has the advantages of mild and environment-friendly process, short time consumption and high efficiency; the product has high deacetylation degree and purity and good water solubility.
Description
Technical Field
The invention relates to preparation of chitin polysaccharide, in particular to a preparation method of squid cartilage beta-chitosan oligosaccharide.
Background
Chitin (Chitin) is a natural high-molecular linear polysaccharide formed by connecting N-acetyl-D-glucosamine through beta-1, 4 glycosidic bonds, exists in the form of ordered crystal microfibril, and is insoluble in water. Chitin is widely present in nature in quantities second only to natural polysaccharides of cellulose.
The squid production produces a large amount of squid cartilage which is discarded as a processing waste due to the inedibility thereof. The squid cartilage contains beta-chitin consisting of parallel sugar chains, is often combined with collagen, and has the effects of preventing three highs, inhibiting bacterial activity and discharging heavy metals. The squid cartilage is effectively utilized, waste materials are changed into valuable materials, raw material resources are fully utilized, and the application of the beta-chitin is limited because the beta-chitin is not soluble in an aqueous medium, so that an effective and simple preparation method of the beta-chitosan oligosaccharide is needed.
In the existing chitosan extraction technology, chinese patent 201510129839.X, CN110452316A discloses a preparation method of beta-chitosan oligosaccharide, which is to desalt with dilute acid solution, remove organic matter with dilute alkali solution and remove acetyl with concentrated alkali to obtain beta-chitosan oligosaccharide, but the extraction of chitin with acid-base method is not suitable for energy-saving requirement, and the process is forbidden. Chinese patent CN111171181A discloses a method for processing beta-chitosan oligosaccharide by enzyme method, which removes acetyl group by enzymolysis to obtain beta-chitosan oligosaccharide containing high protein and ash content, and the use of enzyme reduces the environmental pollution, but it takes long time, has many impurities, and reduces the production effectiveness.
Disclosure of Invention
In order to overcome the defects of long time consumption, more impurities, low extraction rate and purity in the existing enzymatic method for processing the beta-chitosan oligosaccharide, the invention aims to provide the preparation method of the squid cartilage beta-chitosan oligosaccharide, and the preparation method has the advantages of mild and environment-friendly process, short time consumption, high efficiency and low cost; the product has high deacetylation degree and purity and good water solubility.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of squid cartilage beta-chitosan oligosaccharide is characterized by comprising the following process steps:
1) extracting phosphatase: taking plant root soil in phosphorus-deficient land, crushing, putting into a 200-mesh screen, leaching for 3-5 times by using leacheate with the pH value of 4-7, standing for 5-8h, taking supernatant, and freeze-drying to obtain phosphatase;
2) extracting beta-chitosan: adding the phosphatase obtained in the step 1) into a mixture of polyphosphoric acid and squid cartilage, fully and uniformly stirring, reacting at 18-45 ℃ for 5-10h, heating to 62-85 ℃, stirring for 1.5-3.2h, centrifuging at 1500-3000r/min, and taking supernatant to obtain a beta-chitosan extract solution;
3) preparing beta-chitosan oligosaccharide: preparing engineering bacteria pichia pastoris by using chitosanase secreted by aureobasidium polyclonum, collecting the chitosanase secreted by pichia pastoris, adding the chitosanase into the beta-chitosan extract solution obtained in the step 2), performing enzymolysis for 2-4h, centrifuging, and taking supernate to obtain a beta-chitosan oligosaccharide solution;
4) refining beta-chitosan oligosaccharide: taking the beta-chitosan oligosaccharide solution obtained in the step 3), adding ethanol until the mass percentage of ethanol is 65-85%, centrifuging to obtain precipitate, and repeatedly dissolving with deionized water and precipitating with ethanol for 3-5 times to obtain cleaned beta-chitosan oligosaccharide; ultrafiltering the cleaned beta-chitosan oligosaccharide, intercepting chitosan with molecular weight of 8000Da, concentrating the intercepted solution, and putting into a dialysis bag of 3000Da to remove impurities, wherein the solution in the dialysis bag is refined beta-chitosan oligosaccharide solution;
5) spray drying: spray drying the refined beta-chitosan oligosaccharide solution obtained in the step 4) to obtain beta-chitosan oligosaccharide;
6) and (3) decoloring: treating the beta-chitosan oligosaccharide obtained in the step 5) by using a supercritical carbon dioxide device, and adding an oxidant into the residual material in the extraction kettle for decoloring to obtain a finished product of the beta-chitosan oligosaccharide.
Preferably, in step 1), the eluent is composed of ammonium phosphate, phosphoric acid, acetone and water.
Preferably, in the step 2), the mass ratio of the phosphatase to the polyphosphoric acid to the squid cartilage is 1:5-15: 45-60.
Preferably, in the step 3), the water content of the chitosanase is 20%, and the weight ratio of the chitosanase to the beta-chitosan extract solution is 0.3-0.5: 100.
Preferably, in step 3), the chitosanase is induced by glycerol and methanol.
Preferably, in step 5), the spray drying conditions are that the rotating speed of the spray head is 20000-.
Preferably, in the step 6), the supercritical carbon dioxide device parameters are set to be 15-25kg/h of carbon dioxide flow rate, 25-34MPa of extraction pressure, 30-45 ℃ of extraction temperature and 60-90min of time.
Preferably, in step 2), the ultrasonic treatment is performed while stirring, and the ultrasonic treatment conditions are as follows: the ultrasonic frequency is 10-15KHz, the ultrasonic power is 0.5-2W/cm2, and the ultrasonic wave application period is 5-8 min/h.
Preferably, the leacheate comprises 4.2% of ammonium phosphate, 5-8% of phosphoric acid, 15-30% of acetone and the balance of water by mass percent.
The invention has the beneficial effects that:
(1) the invention adopts polyphosphoric acid and phosphatase to extract chitosan, utilizes the phosphatase to decompose organic matters in squid cartilage, releases phosphoric acid, starts the decomposition of polyphosphoric acid and the release of mineral matters in the squid cartilage, and then polyphosphoric acid is controllably released under the action of the phosphatase and assists in the extraction of chitosan, and the prepared chitosan has large molecular weight and good water solubility;
(2) the invention adopts alcohol precipitation combined ultrafiltration and dialysis to purify the chitosan, removes organic matters, inorganic salts and other impurities to the maximum extent, and greatly improves the extraction rate and the purity of the chitosan compared with the traditional method, wherein the extraction rate and the purity are respectively 98.98 percent and 97.62 percent;
(3) the invention adopts enzyme method combined oxidation to decompose macromolecular chitosan, prepares small molecular chitosan oligosaccharide, does not generate waste, and the obtained product has good water solubility.
Detailed Description
The invention is described in detail below with reference to specific embodiments, which are intended to facilitate the understanding and implementation of the invention and are not intended to limit the invention. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.
Example 1
A preparation method of squid cartilage beta-chitosan oligosaccharide comprises the following process steps:
1) extracting phosphatase: taking plant root soil in phosphorus-deficient land, crushing, putting into a 200-mesh screen, leaching for 4 times by using leacheate with the pH value of 5.2, standing for 7 hours, taking supernate, and freeze-drying to obtain phosphatase; wherein the leacheate consists of 4.2 percent of ammonium phosphate, 6.3 percent of phosphoric acid, 24 percent of acetone and the balance of water by mass percentage;
2) extracting beta-chitosan oligosaccharide: adding the phosphatase obtained in the step 1) into a mixture of polyphosphoric acid and squid cartilage, fully and uniformly stirring, reacting at 32 ℃ for 8 hours, heating to 80 ℃, stirring for 2.8 hours, centrifuging at 2000r/min, and taking supernatant to obtain a beta-chitosan oligosaccharide crude extract; wherein the mass ratio of phosphatase, polyphosphoric acid and squid cartilage is 1:12: 50; carrying out ultrasonic treatment while stirring, wherein the ultrasonic treatment conditions are as follows: the ultrasonic frequency is 12KHz, the ultrasonic power is 1.2W/cm2, and the ultrasonic application period is 6 min/h;
3) preparing beta-chitosan oligosaccharide: preparing engineering bacteria pichia pastoris by using chitosanase secreted by aureobasidium polyclonum, collecting the chitosanase secreted by pichia pastoris, adding the chitosanase into the beta-chitosan extract solution obtained in the step 2), performing enzymolysis for 3 hours, centrifuging, and taking supernate to obtain a beta-chitosan oligosaccharide solution; wherein, the water content of the chitosanase is 20 percent, and the weight ratio of the chitosanase to the beta-chitosan extract solution is 0.4: 100;
4) refining beta-chitosan oligosaccharide: taking the crude extract of the beta-chitosan oligosaccharide obtained in the step 2), adding ethanol until the mass percentage of the ethanol is 75%, centrifuging to obtain a precipitate, and repeatedly dissolving the precipitate with deionized water and precipitating with ethanol for 4 times to obtain the cleaned beta-chitosan oligosaccharide; carrying out ultrafiltration on the cleaned beta-chitosan oligosaccharide, intercepting chitosan with the molecular weight of 30 ten thousand Da, concentrating the intercepted solution, putting the concentrated solution into a dialysis bag with the molecular weight of 30 ten thousand Da, and removing impurities, wherein the solution in the dialysis bag is refined beta-chitosan oligosaccharide solution;
5) spray drying: spray drying the refined beta-chitosan oligosaccharide solution obtained in the step 3) to obtain beta-chitosan oligosaccharide; wherein the spray drying conditions comprise nozzle rotation speed of 21000r/min, inlet temperature of 195 deg.C, feeding speed of 150ml/min, discharge port temperature of 70 deg.C, and air speed of 2 m/s;
6) and (3) decoloring: treating the beta-chitosan oligosaccharide obtained in the step 4) by using a supercritical carbon dioxide device, setting the parameters as the flow rate of carbon dioxide of 20kg/h, the extraction pressure of 29MPa, the extraction temperature of 35 ℃ and the time of 82min, and adding an oxidant into the residual materials in the extraction kettle for decoloring to obtain the finished product of the beta-chitosan oligosaccharide.
Example 2
A preparation method of squid cartilage beta-chitosan oligosaccharide comprises the following process steps:
1) extracting phosphatase: taking plant root soil in phosphorus-deficient land, crushing, putting into a 200-mesh screen, leaching for 3 times by using leacheate with the pH value of 4, standing for 5 hours, taking supernate, and freeze-drying to obtain phosphatase; wherein the leacheate consists of 4.2 percent of ammonium phosphate, 5 percent of phosphoric acid, 15 percent of acetone and the balance of water by mass percentage;
2) extracting beta-chitosan oligosaccharide: adding the phosphatase obtained in the step 1) into a mixture of polyphosphoric acid and squid cartilage, fully and uniformly stirring, reacting at 18 ℃ for 5 hours, heating to 62 ℃, stirring for 1.5 hours, centrifuging at 1500r/min, and taking supernatant to obtain a beta-chitosan oligosaccharide crude extract; wherein the mass ratio of phosphatase to polyphosphoric acid to squid cartilage is 1:5: 45; carrying out ultrasonic treatment while stirring, wherein the ultrasonic treatment conditions are as follows: the ultrasonic frequency is 10KHz, the ultrasonic power is 0.5W/cm2, and the ultrasonic application period is 5 min/h;
3) preparing beta-chitosan oligosaccharide: preparing engineering bacteria pichia pastoris by using chitosanase secreted by aureobasidium polyclonum, collecting the chitosanase secreted by pichia pastoris, adding the chitosanase into the beta-chitosan extract solution obtained in the step 2), performing enzymolysis for 2 hours, centrifuging, and taking supernate to obtain a beta-chitosan oligosaccharide solution; wherein, the water content of the chitosanase is 20 percent, and the weight ratio of the chitosanase to the beta-chitosan extract solution is 0.5: 100;
4) refining beta-chitosan oligosaccharide: taking the crude extract of the beta-chitosan oligosaccharide obtained in the step 2), adding ethanol until the mass percentage of the ethanol is 65%, centrifuging to obtain a precipitate, and repeatedly dissolving the precipitate with deionized water and precipitating with ethanol for 3 times to obtain the cleaned beta-chitosan oligosaccharide; carrying out ultrafiltration on the cleaned beta-chitosan oligosaccharide, intercepting chitosan with the molecular weight of 30 ten thousand Da, concentrating the intercepted solution, putting the concentrated solution into a dialysis bag with the molecular weight of 30 ten thousand Da, and removing impurities, wherein the solution in the dialysis bag is refined beta-chitosan oligosaccharide solution;
5) spray drying: spray drying the refined beta-chitosan oligosaccharide solution obtained in the step 3) to obtain beta-chitosan oligosaccharide; wherein the spray drying conditions comprise nozzle rotation speed of 20000r/min, inlet temperature of 180 deg.C, feeding speed of 100ml/min, discharge port temperature of 65 deg.C, and air speed of 1 m/s;
6) and (3) decoloring: treating the beta-chitosan oligosaccharide obtained in the step 4) by using a supercritical carbon dioxide device, setting the parameters as the carbon dioxide flow rate of 15kg/h, the extraction pressure of 25MPa, the extraction temperature of 30 ℃ and the time of 60min, and adding an oxidant into the residual materials in the extraction kettle for decoloring to obtain the finished product of the beta-chitosan oligosaccharide.
Example 3
A preparation method of squid cartilage beta-chitosan oligosaccharide comprises the following process steps:
1) extracting phosphatase: taking plant root soil in phosphorus-deficient land, crushing, putting into a 200-mesh screen, leaching for 5 times by using leacheate with the pH value of 7, standing for 8 hours, taking supernate, and freeze-drying to obtain phosphatase; wherein the leacheate consists of 4.2 percent of ammonium phosphate, 8 percent of phosphoric acid, 30 percent of acetone and the balance of water by mass percentage;
2) extracting beta-chitosan oligosaccharide: adding the phosphatase obtained in the step 1) into a mixture of polyphosphoric acid and squid cartilage, fully and uniformly stirring, reacting at 45 ℃ for 10 hours, heating to 85 ℃, stirring for 3.2 hours, centrifuging at 3000r/min, and taking supernatant to obtain a beta-chitosan oligosaccharide crude extract; wherein the mass ratio of phosphatase, polyphosphoric acid and squid cartilage is 1:15: 60; carrying out ultrasonic treatment while stirring, wherein the ultrasonic treatment conditions are as follows: the ultrasonic frequency is 15KHz, the ultrasonic power is 2W/cm2, and the ultrasonic application period is 8 min/h;
3) preparing beta-chitosan oligosaccharide: preparing engineering bacteria pichia pastoris by using chitosanase secreted by aureobasidium polyclonum, collecting the chitosanase secreted by pichia pastoris, adding the chitosanase into the beta-chitosan extract solution obtained in the step 2), performing enzymolysis for 4 hours, centrifuging, and taking supernate to obtain a beta-chitosan oligosaccharide solution; wherein, the water content of the chitosanase is 20 percent, and the weight ratio of the chitosanase to the beta-chitosan extract solution is 0.3: 100;
4) refining beta-chitosan oligosaccharide: taking the crude extract of the beta-chitosan oligosaccharide obtained in the step 2), adding ethanol until the mass percentage of the ethanol is 85%, centrifuging to obtain a precipitate, and repeatedly dissolving the precipitate with deionized water and precipitating with ethanol for 5 times to obtain the cleaned beta-chitosan oligosaccharide; carrying out ultrafiltration on the cleaned beta-chitosan oligosaccharide, intercepting chitosan with the molecular weight of 30 ten thousand Da, concentrating the intercepted solution, putting the concentrated solution into a dialysis bag with the molecular weight of 30 ten thousand Da, and removing impurities, wherein the solution in the dialysis bag is refined beta-chitosan oligosaccharide solution;
5) spray drying: spray drying the refined beta-chitosan oligosaccharide solution obtained in the step 3) to obtain beta-chitosan oligosaccharide; wherein the spray drying conditions comprise a nozzle rotating speed of 22000r/min, an inlet temperature of 220 ℃, a feeding speed of 180ml/min, a discharge port temperature of 80 ℃ and a wind speed of 3 m/s;
6) and (3) decoloring: treating the beta-chitosan oligosaccharide obtained in the step 4) by using a supercritical carbon dioxide device, setting the parameters to be 25kg/h of carbon dioxide flow rate, 34MPa of extraction pressure, 45 ℃ of extraction temperature and 90min of time, and adding an oxidant into the residual materials in the extraction kettle for decoloring to obtain a finished product of the beta-chitosan oligosaccharide.
Comparative example 1
In this comparative example, the process is essentially the same as in example 1, except that: polyphosphoric acid is not used in step 2).
Comparative example 2
In this comparative example, the process is essentially the same as in example 1, except that: no phosphate bacteria were inoculated in step 2).
Comparative example 3
In this comparative example, the process is essentially the same as in example 1, except that: no sonication was performed in step 2).
Comparative example 4
In this comparative example, the process is essentially the same as in example 1, except that: in step 4), alcohol precipitation is not carried out.
Comparative example 5
In this comparative example, the process is essentially the same as in example 1, except that: in step 6), the supercritical carbon dioxide impurity removal treatment is not carried out.
Comparative example 6
In this comparative example, the process is essentially the same as in example 1, except that: in step 3), no enzymatic treatment is carried out.
Comparative example 7
Example 4 of chinese patent CN110615855A was used.
The residual ash content in the extracted soluble beta-chitin is measured by a burning weighing method according to the national standard GB/T5009.4-2003 'measurement of ash content in food', and the protein content, the water binding capacity, the fat binding capacity, the relative molecular weight and the deacetylation degree of the extracted soluble beta-chitin are measured by an ultraviolet spectrophotometry according to the national standard GB/T5009.5-2010 'measurement of protein in food' according to the detection method in Chinese patent CN 110452316A. The results are shown in Table 1.
TABLE 1 measurement results of physicochemical Properties extracted under different treatment conditions
| Group of | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 |
| Ash content% | 0.07 | 0.09 | 0.08 | 1.41 | 0.88 | 0.62 | 0.29 | 0.27 | 0.17 | 0.19 |
| Nitrogen content/%) | 7.30 | 6.85 | 6.42 | 1.37 | 6.54 | 5.29 | 6.41 | 6.38 | 6.25 | 6.31 |
| Water binding capacity/%) | 252 | 238 | 232 | 19 | 201 | 226 | 229 | 190 | 212 | 204 |
| Relative molecular weight/kDa | 1.329 | 1.496 | 1.214 | 1.433 | 1.376 | 1.341 | 1.352 | 1.427 | 431 | 1.811 |
| Degree of deacetylation/% | 98.62 | 97.11 | 98.05 | --- | 92.13 | 83.37 | 96.15 | 96.24 | 89.51 | 96.46 |
As can be seen from Table 1, polyphosphoric acid and phosphatase both have important influence on the decomposition of chitin to prepare chitosan, and comparative example 1 shows that phosphatase is a control factor of polyphosphoric acid acting on chitin, and the polyphosphoric acid and the phosphatase have synergistic effect; compared with the comparative document 2, the relative molecular weight difference of the example 1 is obvious, which shows that the phosphatase has a regulating effect on polyphosphoric acid, and is beneficial to the generation of macromolecular chitosan; comparative examples 1, 2 and 3 show that sonication, alcohol precipitation and supercritical carbon dioxide treatment all have an effect on the preparation of chitosan. The difference between the degree of deacetylation and the nitrogen content of the product is small, but the degree of deacetylation and the nitrogen content of the product are the highest in example 1, which shows that the preparation method of chitosan in example 1 can ensure high degree of deacetylation, simultaneously maintain the amino retention and increase the nitrogen content of the product molecules. Comparing example 1 with comparative example 3, it can be seen that the difference between the degree of deacetylation and the nitrogen content of the two products is significant, indicating that the degree of deacetylation and the high amino group retention of the products are significantly affected by the ultrasonic treatment. Comparison document 6 shows that chitosanase significantly affects the product molecular weight size and degree of deacetylation, and its use facilitates the production of chitosan oligosaccharide.
The extraction efficiency and purity of the beta-chitosan oligosaccharide are taken as indexes, and the extraction effect of the process is analyzed. Extraction rate (%) = extract weight 100/raw material weight; purity (%) = β -chitooligosaccharides in extract 100/total weight of extract.
TABLE 2 beta-Chitosan oligosaccharide extraction yield and purity analysis results
| Group of | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 |
| Extraction ratio/% | 98.75 | 97.94 | 98.06 | 4.12 | 75.26 | 88.91 | 97.04 | 97.55 | 97.89 | 90.42 |
| Purity/%) | 98.13 | 97.92 | 97.99 | 96.74 | 97.28 | 97.09 | 95.76 | 95.81 | 92.78 | 93.53 |
As can be seen from Table 2, the extraction rate and purity of the beta-chitosan oligosaccharide prepared by the present invention are superior to those of comparative example 7. Comparative examples 1, 2 and 3 show that phosphatase, polyphosphoric acid and ultrasonic treatment have significant influence on beta-chitosan oligosaccharide extraction, and the effect of the beta-chitosan oligosaccharide preparation process can be significantly improved by the synergy of the phosphatase, the polyphosphoric acid and the ultrasonic treatment. Chitosanase affects product purity, indicating that the use of chitosanase produces some small molecular weight impurities.
Claims (9)
1. A preparation method of squid cartilage beta-chitosan oligosaccharide is characterized by comprising the following process steps:
1) extracting phosphatase: taking plant root soil in phosphorus-deficient land, crushing, putting into a 200-mesh screen, leaching for 3-5 times by using leacheate with the pH value of 4-7, standing for 5-8h, taking supernatant, and freeze-drying to obtain phosphatase;
2) extracting beta-chitosan: adding the phosphatase obtained in the step 1) into a mixture of polyphosphoric acid and squid cartilage, fully and uniformly stirring, reacting at 18-45 ℃ for 5-10h, heating to 62-85 ℃, stirring for 1.5-3.2h, centrifuging at 1500-3000r/min, and taking supernatant to obtain a beta-chitosan extract solution;
3) preparing beta-chitosan oligosaccharide: preparing engineering bacteria pichia pastoris by using chitosanase secreted by aureobasidium polyclonum, collecting the chitosanase secreted by pichia pastoris, adding the chitosanase into the beta-chitosan extract solution obtained in the step 2), performing enzymolysis for 2-4h, centrifuging, and taking supernate to obtain a beta-chitosan oligosaccharide solution;
4) refining beta-chitosan oligosaccharide: taking the beta-chitosan oligosaccharide solution obtained in the step 3), adding ethanol until the mass percentage of ethanol is 65-85%, centrifuging to obtain precipitate, and repeatedly dissolving with deionized water and precipitating with ethanol for 3-5 times to obtain cleaned beta-chitosan oligosaccharide; ultrafiltering the cleaned beta-chitosan oligosaccharide, intercepting chitosan with molecular weight of 8000Da, concentrating the intercepted solution, and putting into a dialysis bag of 3000Da to remove impurities, wherein the solution in the dialysis bag is refined beta-chitosan oligosaccharide solution;
5) spray drying: spray drying the refined beta-chitosan oligosaccharide solution obtained in the step 4) to obtain beta-chitosan oligosaccharide;
6) and (3) decoloring: treating the beta-chitosan oligosaccharide obtained in the step 5) by using a supercritical carbon dioxide device, and adding an oxidant into the residual material in the extraction kettle for decoloring to obtain a finished product of the beta-chitosan oligosaccharide.
2. The method for preparing squid cartilage beta-chitosan oligosaccharide according to claim 1, wherein in the step 1), the leacheate is composed of ammonium phosphate, phosphoric acid, acetone and water.
3. The method for preparing squid cartilage beta-chitosan oligosaccharide according to claim 1, wherein in the step 2), the mass ratio of phosphatase, polyphosphoric acid and squid cartilage is 1:5-15: 45-60.
4. The method for preparing squid cartilage beta-chitosan oligosaccharide according to claim 1, wherein in the step 3), the water content of the chitosanase is 20%, and the weight ratio of the chitosanase to the beta-chitosan extract solution is 0.3-0.5: 100.
5. The method for preparing squid cartilage beta-chitosan oligosaccharide according to claim 1, wherein in the step 3), the chitosanase is induced by glycerol and methanol.
6. The method for preparing squid cartilage beta-chitosan oligosaccharide as claimed in claim 1, wherein in the step 5), the spray drying conditions are nozzle rotation speed 20000-.
7. The method for preparing squid cartilage beta-chitosan oligosaccharide according to claim 1, wherein in the step 6), the supercritical carbon dioxide device parameters are set to be 15-25kg/h of carbon dioxide flow rate, 25-34MPa of extraction pressure, 30-45 ℃ of extraction temperature and 60-90min of time.
8. The method for preparing squid cartilage beta-chitosan oligosaccharide according to claim 1, wherein in the step 2), the ultrasonic treatment is performed while stirring, and the ultrasonic treatment conditions are as follows: the ultrasonic frequency is 10-15KHz, the ultrasonic power is 0.5-2W/cm2, and the ultrasonic wave application period is 5-8 min/h.
9. The preparation method of squid cartilage beta-chitosan oligosaccharide according to claim 1 or 2, wherein the leacheate comprises 4.2% of ammonium phosphate, 5-8% of phosphoric acid, 15-30% of acetone and the balance of water by mass percent.
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| CN111893125A (en) * | 2020-07-01 | 2020-11-06 | 深圳润康生态环境股份有限公司 | Chitosan enzyme gene, chitosanase, preparation method and application thereof |
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