CN102924624A - Method for preparing chondroitin sulfate from sturgeon cartilage - Google Patents
Method for preparing chondroitin sulfate from sturgeon cartilage Download PDFInfo
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- CN102924624A CN102924624A CN2012104831081A CN201210483108A CN102924624A CN 102924624 A CN102924624 A CN 102924624A CN 2012104831081 A CN2012104831081 A CN 2012104831081A CN 201210483108 A CN201210483108 A CN 201210483108A CN 102924624 A CN102924624 A CN 102924624A
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Abstract
The invention discloses a method for preparing chondroitin sulfate from sturgeon cartilage. The method comprises the following steps: (1) pretreating cartilage; (2) performing ultrasonic enzymolysis treatment: hydrolyzing the sturgeon cartilage by using trypsin, accelerating the enzymolysis process under the action of ultrasonic waves, finishing enzymolysis, deactivating enzyme, cooling and centrifuging to obtain supernate containing sturgeon chondroitin; (3) hydrolyzing and precipitating synchronously, continuously hydrolyzing the supernate in the step (2) with ethanol and NaOH solution and centrifuging to obtain white precipitate; and (4) purifying, drying and redissolving the precipitate to remove insoluble impurities, and drying to obtain a white spongy chondroitin sulfate product. The process is simple to operate and is new. Based on the steps of performing ultrasonic auxiliary enzymolysis, hydrolyzing and precipitating synchronously and purifying, the hydrolysis time can be greatly shortened, the purity and the yield of the products are increased and the production cost is reduced.
Description
Technical field
The present invention relates to a kind of method of utilizing sturgeon cartilage to prepare chondroitin sulfate, belong to the marine products processing technical field.
Background technology
Along with success and the popularization of sturgeon artificial propagation and cultural technique, the cultured area of sturgeon and cultured output cumulative year after year.The main purpose of sturgeon cultivation is to produce the sturgeon caviar of the title with black gold, and except the sturgeon seed, other position of sturgeon also has exploitation and is worth.Sturgeon is except the hone lamella of back, entire body is cartilage, cartilage occupies larger proportion in each tissue of sturgeon, this position has significantly anticancer and calcium supplementing effect, have saying of " shark fin, sturgeon bone, the establishing-Yang that makes eye bright of food is promoted longevity ", this mainly is because contain a large amount of chondroitin sulfates in the sturgeon cartilage tissue, it is reported that the chondroitin sulfate cellulose content of cartilage in sturgeon head, notochord, the fin is up to 30%.
Chondroitin sulfate (Chondroitin sulfate is called for short Chs) is that animal cartilage is organized distinctive natural acid mucopolysaccharide, has the multiple isomer such as ChsA, ChsB, ChsC, ChsD and ChsE.They all are the disaccharide polymkeric substance that alternately linked by D-Glucose aldehydic acid and N-acetyl-D-amino semi-lactosi, just sulfate group position difference.In recent years, along with to the going deep into of its physiological function and biochemical property research, find that Chs has anti-freezing, anti-inflammatory, antithrombotic, anticancer and reduce myocardial consumption of oxygen, promote coronary artery circulation, reducing blood-fat, anticoagulation and prevent the effect such as arteriosclerosis; Treatment to cardiovascular disordeies such as coronary heart disease, atherosclerosis, stenocardia, myocardial anoxia, myocardial infarctions has certain curative effect; Also neurocyte, mucomembranous cell, nephrocyte etc. had provide protection; Chs also can activate lipolytic enzyme, makes fat acid decomposition, is to prevent fat active substance therefore; Chs can be modulated into makeup for the protection of the fitness of skin with Cucumber; Chs also has water-retentivity, protects colloidality and high viscosity, and China's export all is used as foodstuff additive to the Chs of Japan, Korea S.Therefore Chs is domestic and international popular healthcare products and the important source material of makeup and pharmaceuticals.
The extracting method length how consuming time of chondroitin sulfate, extraction process complexity in traditional animal cartilage tissue, chondroitin sulfate purity is low, is unfavorable for the industrialization production of chondroitin sulfate in the sturgeon cartilage tissue.Therefore study extraction, the purifying process of Sturgon chondrine, take full advantage of the sturgeon cartilage resource, not only can improve the sturgeon added value of product, and minimizing is to the pollution of environment, also can develop the sturgeon series product, to promoting further developing of China's sturgeon cultivation industry, make the cultivation of China sturgeon and be processed to form the inexorable trend that benign cycle is development, have immeasurable meaning for the economy and the social value that improve sturgeon.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing sturgeon cartilage to prepare chondroitin sulfate, simplify the purifying process of sturgeon chondroitin sulfate, improve extraction yield and the purity of sturgeon chondroitin sulfate, promote the lifting of its economic benefit.
For achieving the above object, the invention provides a kind of method of utilizing sturgeon cartilage to prepare chondroitin sulfate, with sturgeon cartilage micronizing or crushed after being dried, under hyperacoustic assisting, carry out Enzymatic Extraction, described ultrasonic subsidiary conditions are frequency 35kHz, and power is 280 ~ 360W, and the adjusting temperature is 40～50 ℃ and carries out enzymolysis 20～50min, then in 95 ℃ of enzyme 5min that go out, get supernatant liquor in 0～4 ℃ of lower frozen centrifugation 10min; By after hydrolysis and the precipitation synchronous processing, namely get platinum sponge shape chondroitin sulfate product through redissolution removal of impurities, drying treatment again.
Described sturgeon cartilage pretreatment process is: get the sturgeon cartilage tissue, in 80 ℃ of-90 ℃ of water bath processing 10～20min, remove the impurity such as surperficial muscle and fat, soybean dietary fiber or drying and crushing particle diameter are 80～120 orders.
Described chondroitin sulfate leaching process is: cartilage sample and phosphate buffer soln 0.1M, and the ratio of pH6.5～7.0 is that 1:6～10(m/v), the used enzyme of enzymolysis is trypsinase, its addition is that every gram sturgeon cartilage sample adds 1000 ~ 2000U.
The concrete steps of hydrolysis and precipitation slowly join after the mixing precooling in the supernatant liquor of enzymolysis gained for NaOH is joined in the ethanol solution, and hydrolysis and precipitation temperature are normal temperature, and the time is 30～40min.Described hydrolysis and precipitation process are: the dehydrated alcohol volume is 0.8～1.4 times (V/V) of supernatant liquor, and NaOH concentration is 0.4～0.8M.
Described purification process process is: the water that will precipitate with 2～3 times redissolves, and the acetic acid with 40% is neutralized to neutrality, and 0～4 ℃ centrifugal, and vacuum lyophilization namely gets platinum sponge shape chondroitin sulfate product.
Can reach following effect after adopting the present invention: the present invention carries out enzymolysis under the ultrasonic wave subsidiary conditions, promotes that protein decomposes in the cartilaginous tissue, and effectively the chondroitin sulfate of combination discharges with it, shortens extraction time; Alkaline hydrolysis and one step of settling step are processed, shortened the purifying time, improve the extraction yield of sturgeon chondroitin sulfate; Improve product purity by purification step.Product colour is the platinum sponge shape, and the product yield is 26～31%, and purity surpasses 75%, reaches as high as 81.3%.
Embodiment
Embodiment 1
The sturgeon chondroitin sulfate extracting and purifying method that present embodiment provides, may further comprise the steps: 1) sample pre-treatments: get the sturgeon cartilage tissue, in 80 ℃ of water bath processing 20min, remove the impurity such as surperficial muscle and fat, it is the wet samples of 100 purposes that soybean dietary fiber is made particle diameter.
2) the sturgeon chondroitin sulfate extracts: the wet sample of cartilage sample is used and phosphate buffer soln 0.1M, and pH6.5 mass volume ratio example is that 10(g/ml), the used enzyme of enzymolysis is trypsinase, and its addition is that every gram sturgeon cartilage sample adds 1000U.Described ultrasonic assistance enzymolysis condition is frequency 35kHz, and power is 280W, and the adjusting temperature is 50 ℃ and carries out enzymolysis 50min, then in 95 ℃ of enzyme 5min that go out, gets supernatant liquor in 4 ℃ of lower frozen centrifugation 10min.
3) hydrolysis of sturgeon chondroitin sulfate and purifying: the dehydrated alcohol volume is 0.8 times (V/V) of supernatant liquor, and NaOH concentration is 0.8M.NaOH is joined in the ethanol solution, slowly join after the mixing precooling in the supernatant liquor of enzymolysis gained, hydrolysis and precipitation temperature are normal temperature, and the time is 30min.Precipitation with the redissolution of 2 times water, is neutralized to neutrality with the acetic acid of mass percentage concentration 40%, and 4 ℃ centrifugal, and vacuum lyophilization namely gets platinum sponge shape chondroitin sulfate product.The product yield is 28%, and purity surpasses 79.2%.
Embodiment 2
The sturgeon chondroitin sulfate extracting and purifying method that present embodiment provides, may further comprise the steps: 1) sample pre-treatments: get the sturgeon cartilage tissue, in 90 ℃ of water bath processing 10min, remove the impurity such as surperficial muscle and fat, drying and crushing, drying temperature is 50 ℃, and pulverizing and making particle diameter is 80 purpose dry samples.
2) the sturgeon chondroitin sulfate extracts: cartilage sample dry sample and phosphate buffer soln 0.1M, and pH7.0 mass volume ratio example is that 10(g/ml), the used enzyme of enzymolysis is trypsinase, its addition is that every gram sturgeon cartilage sample adds 2000U.Described ultrasonic assistance enzymolysis condition is frequency 35kHz, and power is 360W, and the adjusting temperature is 40 ℃ and carries out enzymolysis 20min, then in 95 ℃ of enzyme 5min that go out, gets supernatant liquor in 3 ℃ of lower frozen centrifugation 10min.
3) sturgeon chondroitin sulfate purifying: described hydrolysis and precipitate 1.2 times (V/V) that used dehydrated alcohol volume is supernatant liquor, NaOH concentration is 0.6M.The concrete steps of hydrolysis and precipitation slowly join after the mixing precooling in the supernatant liquor of enzymolysis gained for NaOH solution is joined in the quantitative ethanol solution, and hydrolysis and precipitation temperature are normal temperature, and the time is 35min.The water of precipitation with 3 times is redissolved, and the acetic acid with 40% is neutralized to neutrality, and 2 ℃ centrifugal, and vacuum lyophilization namely gets platinum sponge shape chondroitin sulfate product.The product yield is 29.61%, purity 76.3%.
Embodiment 3
The sturgeon chondroitin sulfate extracting and purifying method that present embodiment provides may further comprise the steps:
1) sample pre-treatments: get the sturgeon cartilage tissue, in 85 ℃ of water bath processing 20min, remove the impurity such as surperficial muscle and fat, drying temperature is 48 ℃, and powder particle diameter is 120 orders.
2) the sturgeon chondroitin sulfate extracts: cartilage sample and phosphate buffer soln 0.1M, and the mass volume ratio example of pH7.0 is that 10(g/ml), the used enzyme of enzymolysis is trypsinase, its addition is that every gram sturgeon cartilage sample adds 2000U.Described ultrasonic assistance enzymolysis condition is frequency 35kHz, and power is 360W, and the adjusting temperature is 45 ℃ and carries out enzymolysis 30min, then in 95 ℃ of enzyme 5min that go out, gets supernatant liquor in 0 ℃ of lower frozen centrifugation 10min.
3) sturgeon chondroitin sulfate purifying: described hydrolysis and precipitate 1.0 times (V/V) that used dehydrated alcohol volume is supernatant liquor, NaOH concentration is 0.8M.The concrete steps of hydrolysis and precipitation slowly join after the mixing precooling in the supernatant liquor of enzymolysis gained for NaOH is joined in the ethanol solution, and hydrolysis and precipitation temperature are normal temperature, and the time is 40min.The water of precipitation with 3 times is redissolved, and the acetic acid with 40% is neutralized to neutrality, and 4 ℃ centrifugal, and vacuum lyophilization namely gets platinum sponge shape chondroitin sulfate product.The product yield is 31.0%, purity 81.3%.
Embodiments of the present invention are not limited to this; according to foregoing of the present invention; ordinary skill knowledge and customary means according to this area; do not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite; can also make modification, replacement or the change of other various ways, all drop within the rights protection scope of the present invention.
Claims (9)
1. method of utilizing sturgeon cartilage to prepare chondroitin sulfate is characterized in that: may further comprise the steps:
(1) sturgeon cartilage is processed: with sturgeon cartilage soybean dietary fiber or drying and crushing;
(2) the ultrasonic wave enzymolysis is processed: sturgeon cartilage is dissolved in the buffered soln, adds trypsinase, mixing, carry out ultrasonic assistance enzymolysis, described ultrasonic enzymatic hydrolysis condition is frequency 35kHz, and power is 280 ~ 360W, and the adjusting temperature is 40～50 ℃ and carries out enzymolysis 20～50min; In 95 ℃ of enzyme 5min that go out, get supernatant liquor in 0～4 ℃ of lower frozen centrifugation 10min after enzymolysis is complete;
(3) hydrolysis and precipitation synchronous processing: step 2 gained supernatant liquor is continued hydrolysis with dehydrated alcohol and NaOH mixed solution, the centrifugal white precipitate that gets;
(4) purifying, drying: insoluble impurities is removed in the redissolution of step 3 gained throw out water, processed namely getting platinum sponge shape chondroitin sulfate product through lyophilize.
2. prepare the method for chondroitin sulfate described in according to claim 1, it is characterized in that in the step (1) sturgeon cartilage being carried out pre-treatment: sturgeon cartilage in 80 ℃～90 ℃ water bath processing 10～20min, is removed muscle and the fat on surface.
3. prepare the method for chondroitin sulfate described in according to claim 1, it is characterized in that the micronizing particle diameter is 80～100 orders described in the step (1).
4. prepare the method for chondroitin sulfate described in according to claim 1, it is characterized in that the drying and crushing temperature is 40～50 ℃ described in the step (1), powder particle diameter is 80～120 orders.
5. prepare the method for chondroitin sulfate described in according to claim 1, it is characterized in that the buffered soln described in the step (2) is phosphate buffer soln 0.1M, pH6.5～7.0, the mass/volume ratio of cartilage and buffered soln is 1:6～10 m/v.
6. prepare the method for chondroitin sulfate described in according to claim 1, it is characterized in that the tryptic addition described in the step (2) is to add 1000 ~ 2000U in every gram sturgeon cartilage sample.
7. the method for preparing chondroitin sulfate described in according to claim 1, it is characterized in that the adition process of the dehydrated alcohol described in the step (3) and alkali is for joining NaOH in the dehydrated alcohol first, slowly join after the mixing precooling in the supernatant liquor of step (2) gained, hydrolysis and precipitation temperature are room temperature, and the time is 30 ~ 40min.
8. according to claim 1 or prepare the method for chondroitin sulfate described in 7, it is characterized in that: the hydrolysis described in the step (3) and precipitate 0.8～1.4 times (V/V) that used dehydrated alcohol volume is supernatant liquor, NaOH concentration is 0.4～0.8M.
9. the method for preparing chondroitin sulfate described in according to claim 1, it is characterized in that: the purification process process described in the step (4) is: the water of throw out with 2～3 times is redissolved, acetic acid with 40% is neutralized to neutrality, 0～4 ℃ centrifugal, and vacuum lyophilization namely gets platinum sponge shape chondroitin sulfate product.
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Cited By (8)
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| CN103739742A (en) * | 2013-12-26 | 2014-04-23 | 安徽宝迪肉类食品有限公司 | Preparation method of extracting chondroitin sulfate by buffer liquor |
| CN104140472A (en) * | 2013-05-08 | 2014-11-12 | 清华大学 | Excellent chondroitin sulfates A and C and method for preparing excellent chondroitin sulfates A and C |
| CN106397629A (en) * | 2016-08-30 | 2017-02-15 | 集美大学 | Method for extracting chondroitin sulfate from sturgeon bones, chondroitin sulfate extracted through method, and application of chondroitin sulfate |
| CN107114793A (en) * | 2017-04-13 | 2017-09-01 | 大连工业大学 | It is a kind of to comprehensively utilize the method that sturgeon bone prepares calcium and chondroitin sulfate |
| CN107158360A (en) * | 2017-04-21 | 2017-09-15 | 浙江海洋大学 | A kind of joint repair composition based on sturgeon cartilage active component and preparation method thereof |
| CN107216407A (en) * | 2017-06-12 | 2017-09-29 | 四川菲德力制药有限公司 | The preparation method of sodium chondroitin sulfate |
| CN111471120A (en) * | 2020-06-16 | 2020-07-31 | 北京赫尔默技术有限公司 | Preparation method and application of chondroitin sulfate |
| CN114010654A (en) * | 2021-11-12 | 2022-02-08 | 江苏大学 | Composition for improving obesity comprising sturgeon skull extract |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104140472A (en) * | 2013-05-08 | 2014-11-12 | 清华大学 | Excellent chondroitin sulfates A and C and method for preparing excellent chondroitin sulfates A and C |
| CN103739742A (en) * | 2013-12-26 | 2014-04-23 | 安徽宝迪肉类食品有限公司 | Preparation method of extracting chondroitin sulfate by buffer liquor |
| CN106397629A (en) * | 2016-08-30 | 2017-02-15 | 集美大学 | Method for extracting chondroitin sulfate from sturgeon bones, chondroitin sulfate extracted through method, and application of chondroitin sulfate |
| CN106397629B (en) * | 2016-08-30 | 2019-08-20 | 集美大学 | Method, the chondroitin sulfate by this method extraction and the application of chondroitin sulfate are extracted from sturgeon fish-bone |
| CN107114793A (en) * | 2017-04-13 | 2017-09-01 | 大连工业大学 | It is a kind of to comprehensively utilize the method that sturgeon bone prepares calcium and chondroitin sulfate |
| CN107158360A (en) * | 2017-04-21 | 2017-09-15 | 浙江海洋大学 | A kind of joint repair composition based on sturgeon cartilage active component and preparation method thereof |
| CN107216407A (en) * | 2017-06-12 | 2017-09-29 | 四川菲德力制药有限公司 | The preparation method of sodium chondroitin sulfate |
| CN111471120A (en) * | 2020-06-16 | 2020-07-31 | 北京赫尔默技术有限公司 | Preparation method and application of chondroitin sulfate |
| CN114010654A (en) * | 2021-11-12 | 2022-02-08 | 江苏大学 | Composition for improving obesity comprising sturgeon skull extract |
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Application publication date: 20130213 |