CN105348370A - Method for extracting purified glycoprotein from internal organs of squid - Google Patents
Method for extracting purified glycoprotein from internal organs of squid Download PDFInfo
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- CN105348370A CN105348370A CN201510825314.XA CN201510825314A CN105348370A CN 105348370 A CN105348370 A CN 105348370A CN 201510825314 A CN201510825314 A CN 201510825314A CN 105348370 A CN105348370 A CN 105348370A
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Abstract
The invention discloses a method for extracting purified glycoprotein from internal organs of a squid. The method mainly overcomes the problems of waste of protein resources and environmental pollution due to usage of the internal organs of the squid as a feed raw material or discarding of the internal organs of the squid in the prior art. The method comprises the following steps: with fresh or thawed frozen squid internal organs as a raw material, successively carrying out degreasing through acetone hot bath, crushing and then extraction with a sodium chloride solution so as to obtain crude glycoprotein extract; subjecting the crude glycoprotein extract to dialysis, desalination and fractional precipitation via ammonium sulfate so as to obtain a crude glycoprotein product; and subjecting the crude glycoprotein product to Sephadex G-100 gel column chromatography, then carrying out double distilled water equilibrium and elution, separating and purifying crude glycoprotein of the internal organs of the squid and subjecting eluate to freeze drying so as to obtain a purified dry squid internal organ glycoprotein product. The method provided by the invention realizes value-added utilization of the internal organs of the squid, reduces pollution of squid waste to the environment, and is reasonable in process and simple and feasible in operation; and the extracted glycoprotein has high purity and can be produced in large scale.
Description
Technical field
The present invention relates to and extract edible protein composition from other marine animals, especially a kind of method utilizing squid viscera extraction purification glycoprotein.
Background technology
The squid processing output of China is large, being processed in the processes such as dried squids, popcorn squids, Marinated Squid, can and seasonings, generally has the internal organ of 15 ~ 20%, skin, eye and eye socket the like waste to produce.These wastes contain rich in protein and other nutritive ingredient, very easily rot, and wherein containing some heavy metal, traditional treatment process it can be used as feedstuff raw material or discarded exactly, and it causes significant wastage and the environmental pollution of protein resource.
At present, narrow to the research range of squid viscera comprehensive utilization, mainly study many for its fish oil contained and albumen, such as, Liu Zhengkun etc. with fresh squid viscera for raw material, adopt light alkali hydrolysis method extract squid viscera oil (the extraction research of squid viscera oil. " Chinese oil " the 36th volume the 9th phase in 2011); Yin Qingchun etc. are that raw material extracts proteolytic enzyme with squid viscera, for multiple protein enzyme mixture (Study on extraction of squid viscera proteolytic enzyme. " modern food science and technology " the 28th volume the 3rd phase in 2012), but very few to the polysaccharide contained in squid viscera, glycoprotein isoreactivity thing Quality Research.
Summary of the invention
In order to overcome existing squid viscera as feedstuff raw material or discarded cause protein resource to waste and the deficiency of environmental pollution, the object of the present invention is to provide a kind of rational technology, operation feasible, the method utilizing squid viscera extraction purification glycoprotein that extraction glycoprotein purity is high.
The present invention solves the technical scheme that its technology adopts: a kind of method utilizing squid viscera extraction purification glycoprotein, is characterized in that: it is through following process steps:
(1) raw material is chosen and process: choose fresh squid viscera or freezing and the fresh squid viscera thawed is raw material, dirt and impurity are removed in cleaning, for subsequent use;
(2) acetone degreasing: the squid viscera quantitatively taken in step (1) after obtaining cleaning puts into distilling flask, and add acetone, wherein, the ratio of squid viscera and acetone is 1 ︰ 5 ~ 10 (m/v), connects reflux condensate device; Be placed in water-bath by distilling flask and heat, temperature controls 50 ~ 65 DEG C, until the acetone soln in flask becomes clarification again, stops heating, elimination supernatant liquor, collects filter residue, for subsequent use;
(3) broken: the filter residue obtained in step (2) is smashed to pieces to pureed, for subsequent use;
(4) lixiviate: the squid viscera obtaining smashing to pieces in step (3) is added sodium chloride solution and carries out lixiviate, obtain glycoprotein crude extract;
(5) dialyse: the glycoprotein crude extract obtained in step (4) is dialysed under 4 DEG C of conditions to without White Flocculus and occur, obtain the glycoprotein dialyzate of desalination;
(6) saltout: adopt ammonium sulfate salting-out process to carry out fractionation precipitation the glycoprotein dialyzate obtaining desalination in step (5), obtain glycoprotein crude product;
(7) purifying: SephadexG-100 gel chromatography column on the glycoprotein crude product that step (6) is obtained, with distilled water balance, wash-out, flow rate control, at 0.5 ~ 1ml/s, collects the elutriant that absorption peak is corresponding, merges, obtains elutriant;
(8) finished product: elutriant lyophilize step (7) obtained, obtains purifying squid viscera glycoprotein.
Further, in described step (4), the concentration of the sodium chloride solution of squid viscera is smashed in lixiviate to pieces is 3 ~ 5%, and wherein, solid-liquid ratio is 1 ︰ 4 ~ 5, and extraction time is 0.5 ~ 1h, and extraction temperature is 70 ~ 80 DEG C.
Further, in described step (6), the concentration graded of ammonium sulfate salting-out process fractionation precipitation controls to be 20 ~ 40%, 40 ~ 60%, 60 ~ 80%.
The present invention carries out fragmentation after squid viscera adopts acetone heating bath degreasing, then adopts sodium chloride solution to carry out lixiviate, by changing solid-liquid ratio, extraction temperature, extraction time and the extracting solution salt concn in leaching process, improves glycoprotein yield; Dialysis desalting again, ammonium sulfate precipitation, obtains glycoprotein crude product; SephadexG-100 gel filtration chromatography on glycoprotein crude product, carries out separation and purification to squid viscera raw sugar albumen, obtains purifying squid viscera glycoprotein.Present invention achieves and the increment of squid viscera is utilized, improve fish production added value, reduce the pollution of sleeve-fish product production to environment.Its extracting method rational technology, easy and simple to handle feasible, the glycoprotein purity of extraction is high, is applicable to large-scale production.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
embodiment 1
Utilize a method for squid viscera extraction purification glycoprotein, it is through following process steps:
(1) raw material is chosen and process: choosing fresh squid viscera is raw material, and dirt and impurity are removed in cleaning, for subsequent use;
(2) acetone degreasing: the squid viscera quantitatively taken in step (1) after obtaining cleaning puts into distilling flask, and add acetone, wherein, the ratio of squid viscera and acetone is 1 ︰ 8 (m/v), connects reflux condensate device; Be placed in water-bath by distilling flask and heat, temperature controls 60 DEG C, until the acetone soln in flask becomes clarification again, stops heating, elimination supernatant liquor, collects filter residue, for subsequent use;
(3) broken: smash to pieces to pureed by the filter residue using-system stamp mill obtained in step (2), wherein, stamp mill arranges rotating speed 2000rpm, circulates 8 times, each grinding stops grinding 10 seconds, not occur that cotton-shaped, massive texture is as the criterion in 60 seconds;
(4) lixiviate: the squid viscera obtaining smashing to pieces in step (3) is added sodium chloride solution and carries out lixiviate, the concentration of sodium chloride solution is 4%, and solid-liquid ratio is 1 ︰ 4.5, and extraction time is 0.8h, and extraction temperature is 75 DEG C, obtains glycoprotein crude extract;
(5) dialyse: dialysed under 4 DEG C of conditions by the glycoprotein crude extract obtained in step (4), period adopts Silver Nitrate O.1mol/L to check the desalination situation of dialyzate, until occur without White Flocculus, obtains the glycoprotein dialyzate of desalination;
(6) saltout: adopt ammonium sulfate salting-out process to carry out fractionation precipitation the glycoprotein dialyzate obtaining desalination in step (5), obtain glycoprotein crude product; Wherein, ammonium sulfate concentrations stepped control is 20 ~ 40%, 40 ~ 60%, 60 ~ 80%;
(7) purifying: SephadexG-100 gel chromatography column on the glycoprotein crude product that step (6) is obtained, with distilled water balance, wash-out, flow rate control is at 0.5 ~ 1ml/s, period detects protein content with nucleic acid protein detector and with the sugared content of phenolsulfuric acid reaction detection, collect the elutriant that absorption peak is corresponding, merge, obtain elutriant;
(8) finished product: elutriant lyophilize step (7) obtained, obtains purifying squid viscera glycoprotein.
embodiment 2
Utilize a method for squid viscera extraction purification glycoprotein, it is through following process steps:
(1) raw material is chosen and process: choose freezing and the fresh squid viscera thawed is raw material, and dirt and impurity are removed in cleaning, for subsequent use;
(2) acetone degreasing: the squid viscera quantitatively taken in step (1) after obtaining cleaning puts into distilling flask, and add acetone, wherein, the ratio of squid viscera and acetone is 1 ︰ 10 (m/v), connects reflux condensate device; Be placed in water-bath by distilling flask and heat, temperature controls 50 DEG C, until the acetone soln in flask becomes clarification again, stops heating, elimination supernatant liquor, collects filter residue, for subsequent use;
(3) broken: the filter residue using-system stamp mill obtained in step (2) to be smashed to pieces to pureed, not occur that cotton-shaped, massive texture is as the criterion;
(4) lixiviate: the squid viscera obtaining smashing to pieces in step (3) is added sodium chloride solution and carries out lixiviate, the concentration of sodium chloride solution is 5%, and solid-liquid ratio is 1 ︰ 4, and extraction time is 0.5h, and extraction temperature is 80 DEG C, obtains glycoprotein crude extract;
(5) dialyse: dialysed under 4 DEG C of conditions by the glycoprotein crude extract obtained in step (4), period adopts Silver Nitrate O.1mol/L to check the desalination situation of dialyzate, until occur without White Flocculus, obtains the glycoprotein dialyzate of desalination;
(6) saltout: adopt ammonium sulfate salting-out process to carry out fractionation precipitation the glycoprotein dialyzate obtaining desalination in step (5), obtain glycoprotein crude product; Wherein, ammonium sulfate concentrations stepped control is 20 ~ 40%, 40 ~ 60%, 60 ~ 80%;
(7) purifying: SephadexG-100 gel chromatography column on the glycoprotein crude product that step (6) is obtained, with distilled water balance, wash-out, flow rate control is at 1ml/s, period detects protein content with nucleic acid protein detector and with the sugared content of phenolsulfuric acid reaction detection, collect the elutriant that absorption peak is corresponding, merge, obtain elutriant;
(8) finished product: elutriant lyophilize step (7) obtained, obtains purifying squid viscera glycoprotein.
embodiment 3
Utilize a method for squid viscera extraction purification glycoprotein, it is through following process steps:
(1) raw material is chosen and process: choosing fresh squid viscera is raw material, and dirt and impurity are removed in cleaning, for subsequent use;
(2) acetone degreasing: the squid viscera quantitatively taken in step (1) after obtaining cleaning puts into distilling flask, and add acetone, wherein, the ratio of squid viscera and acetone is 1 ︰ 5 (m/v), connects reflux condensate device; Be placed in water-bath by distilling flask and heat, temperature controls 65 DEG C, until the acetone soln in flask becomes clarification again, stops heating, elimination supernatant liquor, collects filter residue, for subsequent use;
(3) broken: the filter residue using-system stamp mill obtained in step (2) is smashed to pieces to pureed, for subsequent use;
(4) lixiviate: the squid viscera obtaining smashing to pieces in step (3) is added sodium chloride solution and carries out lixiviate, the concentration of sodium chloride solution is 3%, and solid-liquid ratio is 1 ︰ 5, and extraction time is 1h, and extraction temperature is 70 DEG C, obtains glycoprotein crude extract;
(5) dialyse: dialysed under 4 DEG C of conditions by the glycoprotein crude extract obtained in step (4), period adopts Silver Nitrate O.1mol/L to check the desalination situation of dialyzate, until occur without White Flocculus, obtains the glycoprotein dialyzate of desalination;
(6) saltout: adopt ammonium sulfate salting-out process to carry out fractionation precipitation the glycoprotein dialyzate obtaining desalination in step (5), obtain glycoprotein crude product; Wherein, ammonium sulfate concentrations stepped control is 20 ~ 40%, 40 ~ 60%, 60 ~ 80%;
(7) purifying: SephadexG-100 gel chromatography column on the glycoprotein crude product that step (6) is obtained, with distilled water balance, wash-out, flow rate control is at 0.5ml/s, period detects protein content with nucleic acid protein detector and with the sugared content of phenolsulfuric acid reaction detection, collect the elutriant that absorption peak is corresponding, merge, obtain elutriant;
(8) finished product: elutriant lyophilize step (7) obtained, obtains purifying squid viscera glycoprotein.
Claims (3)
1. utilize a method for squid viscera extraction purification glycoprotein, it is characterized in that: it is through following process steps:
(1) raw material is chosen and process: choose fresh squid viscera or freezing and the fresh squid viscera thawed is raw material, dirt and impurity are removed in cleaning, for subsequent use;
(2) acetone degreasing: the squid viscera quantitatively taken in step (1) after obtaining cleaning puts into distilling flask, and add acetone, wherein, the ratio of squid viscera and acetone is 1 ︰ 5 ~ 10 (m/v), connects reflux condensate device; Be placed in water-bath by distilling flask and heat, temperature controls 50 ~ 65 DEG C, until the acetone soln in flask becomes clarification again, stops heating, elimination supernatant liquor, collects filter residue, for subsequent use;
(3) broken: the filter residue obtained in step (2) is smashed to pieces to pureed, for subsequent use;
(4) lixiviate: the squid viscera obtaining smashing to pieces in step (3) is added sodium chloride solution and carries out lixiviate, obtain glycoprotein crude extract;
(5) dialyse: the glycoprotein crude extract obtained in step (4) is dialysed under 4 DEG C of conditions to without White Flocculus and occur, obtain the glycoprotein dialyzate of desalination;
(6) saltout: adopt ammonium sulfate salting-out process to carry out fractionation precipitation the glycoprotein dialyzate obtaining desalination in step (5), obtain glycoprotein crude product;
(7) purifying: SephadexG-100 gel chromatography column on the glycoprotein crude product that step (6) is obtained, with distilled water balance, wash-out, flow rate control, at 0.5 ~ 1ml/s, collects the elutriant that absorption peak is corresponding, merges, obtains elutriant;
(8) finished product: elutriant lyophilize step (7) obtained, obtains purifying squid viscera glycoprotein.
2. a kind of method utilizing squid viscera extraction purification glycoprotein according to claim 1, it is characterized in that: in described step (4), the concentration of the sodium chloride solution of squid viscera is smashed in lixiviate to pieces is 3 ~ 5%, wherein, solid-liquid ratio is 1 ︰ 4 ~ 5, extraction time is 0.5 ~ 1h, and extraction temperature is 70 ~ 80 DEG C.
3. a kind of method utilizing squid viscera extraction purification glycoprotein according to claim 1, is characterized in that: in described step (6), the concentration graded of ammonium sulfate salting-out process fractionation precipitation controls to be 20 ~ 40%, 40 ~ 60%, 60 ~ 80%.
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| CN201510825314.XA CN105348370A (en) | 2015-11-24 | 2015-11-24 | Method for extracting purified glycoprotein from internal organs of squid |
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| CN201510825314.XA CN105348370A (en) | 2015-11-24 | 2015-11-24 | Method for extracting purified glycoprotein from internal organs of squid |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105852092A (en) * | 2016-04-12 | 2016-08-17 | 上海海洋大学 | Nutritional formula food for tumor patients and for special medical purposes, and production method thereof |
| CN108148109A (en) * | 2017-12-12 | 2018-06-12 | 浙江海洋大学 | A kind of preparation method of the preparation method of squid sexual gland saccharide complex and its ferrous complex of hydrolysate |
| CN108676610A (en) * | 2018-05-16 | 2018-10-19 | 浙江海润达科技有限公司 | A method of obtaining active material from Squid By-products |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005018337A1 (en) * | 2003-08-22 | 2005-03-03 | Sergey Marovich Dudkin | Mehtod for producing enzymatic fish protein hydrolysate |
| CN103113462A (en) * | 2011-11-16 | 2013-05-22 | 浙江海洋学院 | Shark glycoprotein, and preparation method and application thereof |
-
2015
- 2015-11-24 CN CN201510825314.XA patent/CN105348370A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005018337A1 (en) * | 2003-08-22 | 2005-03-03 | Sergey Marovich Dudkin | Mehtod for producing enzymatic fish protein hydrolysate |
| CN103113462A (en) * | 2011-11-16 | 2013-05-22 | 浙江海洋学院 | Shark glycoprotein, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| YUTING SU，ET AL.: "Study on the extraction and purifi cation of glycoprotein from the yellow seahorse,Hippocampus kuda Bleeker", 《FOOD SCIENCE & NUTRITION》 * |
| 章建设: "鱿鱼内脏糖蛋白提取分离及其免疫调节作用的研究", 《万方学术期刊数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105852092A (en) * | 2016-04-12 | 2016-08-17 | 上海海洋大学 | Nutritional formula food for tumor patients and for special medical purposes, and production method thereof |
| CN108148109A (en) * | 2017-12-12 | 2018-06-12 | 浙江海洋大学 | A kind of preparation method of the preparation method of squid sexual gland saccharide complex and its ferrous complex of hydrolysate |
| CN108676610A (en) * | 2018-05-16 | 2018-10-19 | 浙江海润达科技有限公司 | A method of obtaining active material from Squid By-products |
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