CN105399817A - Extraction and purification method of tuna myoglobins - Google Patents
Extraction and purification method of tuna myoglobins Download PDFInfo
- Publication number
- CN105399817A CN105399817A CN201510687842.3A CN201510687842A CN105399817A CN 105399817 A CN105399817 A CN 105399817A CN 201510687842 A CN201510687842 A CN 201510687842A CN 105399817 A CN105399817 A CN 105399817A
- Authority
- CN
- China
- Prior art keywords
- tuna
- myohaemoglobin
- extraction
- purification method
- myoglobins
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses an extraction and purification method of tuna myoglobins. The method is characterized in that tuna flesh is adopted as a raw material, and myoglobins are extracted from the tuna flesh; the extraction process of the myoglobins is carried out in an aqueous solution state of a carbohydrate mixture; the carbohydrate mixture contains lactose and trehalose, and a weight ratio of lactose to trehalose is 5:1-1:5. The carbohydrate mixture is used to protect, and a specific mixing ratio is selected to realize an unexpected activity keeping effect. The tuna myoglobins with high purity can be obtained by adopting the method, the yield is high, and the tuna myoglobins have high economy values. The method allows the tuna myoglobins with the advantages of good activity, high yield, high purity and high economy values to be obtained in a short time.
Description
Technical field
The present invention relates to a kind of hydrocoles body fluid protein extracting method, particularly relate to the extraction and purification method of the myohaemoglobin of tuna.
Background technology
Tuna is a kind of large-scale ocean property important goods food fish, and the yellowish pink of tuna is red, this is because contain caused by a large amount of myohaemoglobin in the muscle of tuna.Myohaemoglobin is a kind of monomer reduced hematin, is present in the muscle of some invertebratess and the heart flesh of most vertebrate and skeletal muscle.In muscle tissue sarcoplasm, the massfraction of myohaemoglobin is about 0.2% ~ 2%, and relative molecular mass is 16000 ~ 17000, is reversibly combined with oxygen, has the effect of transhipment and storage oxygen.Iron is indispensable a kind of element in human body, divides and vitamin B12, be easily absorbed by the body in the blood conjunction of tuna containing abundant iron.Often edible, can supplement iron and divide, prevention anaemia also can as the assisting therapy food of anaemia.
But existing patent rarely has report at the extraction this respect of tuna blood hemoglobin.The present invention is intended to invent a kind of myohaemoglobin that can obtain tuna in the short period of time, and yield is high.
Summary of the invention
The object of the invention is to provide a kind of for prior art and can obtain the extraction and purification method of the myohaemoglobin of the tuna of active good, yield is high, purity is high, economic worth is high tuna myohaemoglobin in the short period.
The present invention solves the problems of the technologies described above adopted technical scheme: the extraction and purification method of the myohaemoglobin of tuna, comprises and adopts tuna to be raw material, wherein: from tuna, extract myohaemoglobin; Carry out under the leaching process of myohaemoglobin is all in the aqueous solution state of carbohydrate admixture; Carbohydrate admixture contains lactose and trehalose, and lactose and trehalose weight ratio are 5:1 to 1:5.By using carbohydrate admixture as protection, specific blending ratio can obtain beyond thought activity and keep effect.Adopt present method can obtain the oxyphorase of the higher tuna of purity, and yield is high, has higher economic worth.
For optimizing technique scheme, the measure taked also comprises: have following steps:
1) accurately take certain mass tuna minced fish, carry out extracting after adding Tris-HCl damping fluid and carbohydrate admixture, after filtration, get filtrate;
2) by centrifugal in 8000-14000r/min for step 1) filtrate, removing precipitation, gets supernatant liquor;
3) by step 2) supernatant liquor 1mol/LNaOH solution adjustment pH to 7.60, add the ammonium sulfate precipitation of suitable saturation ratio, leave standstill;
4) it is centrifugal that the throw out after step 3) being saltoutd carries out 8000-14000r/min, and precipitation adds Tris-HCl Extraction buffer and dissolves, and dialyse in same damping fluid 10h to 24h, uses polyoxyethylene glycol concentrated through row;
5) work in-process after step 4) being concentrated by ion exchange column through row purifying.After above step process, the product that purity is higher can be obtained, there is beyond thought high reactivity feature.The process of the myohaemoglobin extraction and purification of tuna is all carried out in ice bath.In step 1), carbohydrate admixture accounts for the 5-30% of tuna in massfraction.Protection reagent serves the effect of well anti-oxidant, anti-sex change in whole extraction system.The condition of purification process is: chromatographic material selects DE-AE-SepharoseFF, and the NaCl solution of 0.01mol/LpH8.6Tris-HCl damping fluid and 200mmol/L does gradient elution, collects elutriant.The product of very high purity can be obtained after adopting chromatography purification, for specific end use, there is very high economic worth.It is 2000 to 100000 that dialysis treatment intercepts molecular weight.It is higher that active substance in this molecular weight area obtains rate.
From tuna, myohaemoglobin is extracted owing to present invention employs; Carry out under the leaching process of myohaemoglobin is all in the aqueous solution state of carbohydrate admixture; Carbohydrate admixture contains lactose and trehalose, and lactose and trehalose weight ratio are 5:1 to 1:5.By using carbohydrate admixture as protection, specific blending ratio can obtain beyond thought activity and keep effect.Adopt present method can obtain the oxyphorase of the higher tuna of purity, and yield is high, has higher economic worth.Thus the present invention has and can obtain the advantage of active good, yield is high, purity is high, economic worth is high tuna myohaemoglobin in the short period.
Accompanying drawing explanation
Fig. 1 is the typical curve of embodiment of the present invention oxyphorase.
Embodiment
Below in conjunction with attached embodiment, the present invention is described in further detail.
Embodiment: the extraction and purification method of the myohaemoglobin of tuna, comprises and adopts tuna to be raw material, wherein: from tuna, extract myohaemoglobin; Carry out under the leaching process of myohaemoglobin is all in the aqueous solution state of carbohydrate admixture; Carbohydrate admixture contains lactose and trehalose, and lactose and trehalose weight ratio are 5:1 to 1:5.By using carbohydrate admixture as protection, specific blending ratio can obtain beyond thought activity and keep effect.Adopt present method can obtain the oxyphorase of the higher tuna of purity, and yield is high, has higher economic worth.Comprise the steps:
1) accurately take certain mass tuna minced fish, carry out extracting after adding Tris-HCl damping fluid and carbohydrate admixture, after filtration, get filtrate;
2) by centrifugal in 8000-14000r/min for step 1) filtrate, removing precipitation, gets supernatant liquor;
3) by step 2) supernatant liquor 1mol/LNaOH solution adjustment pH to 7.60, add the ammonium sulfate precipitation of suitable saturation ratio, leave standstill;
4) it is centrifugal that the throw out after step 3) being saltoutd carries out 8000-14000r/min, and precipitation adds Tris-HCl Extraction buffer and dissolves, and dialyse in same damping fluid 10h to 24h, uses polyoxyethylene glycol concentrated through row;
5) work in-process after step 4) being concentrated by ion exchange column through row purifying.After above step process, the product that purity is higher can be obtained, there is beyond thought high reactivity feature.The process of the myohaemoglobin extraction and purification of tuna is all carried out in ice bath.In step 1), carbohydrate admixture accounts for the 5-30% of tuna in massfraction.Protection reagent serves the effect of well anti-oxidant, anti-sex change in whole extraction system.The condition of purification process is: chromatographic material selects DE-AE-SepharoseFF, and the NaCl solution of 0.01mol/LpH8.6Tris-HCl damping fluid and 200mmol/L does gradient elution, collects elutriant.The product of very high purity can be obtained after adopting chromatography purification, for specific end use, there is very high economic worth.It is 2000 to 100000 that dialysis treatment intercepts molecular weight.It is higher that active substance in this molecular weight area obtains rate.
Illustrate below by further example:
(1) suction filtration: take the Tris-HCl buffer extractions adding 40ml, 0.5mol/L, pH=8.6 in the minced fish of 5g, carry out suction filtration after extracting 1.5h, get filtrate.
(2) centrifugal: filtrate is in 14000r/min ultracentrifugation 5mim, and removing precipitation, gets supernatant liquor.
(3) saltout: supernatant liquor 1mol/LNaOH solution adjustment pH to 7.6, adds the ammonium sulfate precipitation that saturation ratio is 90%, leave standstill 1h.
(4) centrifugal: again carrying out ultracentrifugation centrifugal condition is r=14000r/min, h=3mim.
Dialysis: the Tris-HCl Extraction buffer that precipitation adds 40ml, 0.5mol/L, pH=8.6 dissolves, and dialyse 24h in same damping fluid.
Concentrated: after dialysis, to add the concentrated through row with polyoxyethylene glycol of 20ml.
Purifying: by ion exchange column through row purifying, the condition of purifying is that experiment chromatographic material selects DE-AE-SepharoseFF, the NaCl solution of 0.01mol/LpH8.6Tris-HCl damping fluid and 200mmol/L does gradient elution, collects elutriant (every 4min collects a pipe).
the content of oxyphorase and the mensuration of purity in two tunas
Biuret method: protein contains multiple peptide bond, can have biuret reaction.In basic solution, protein and cupric ion form red-purple complex compound, can in 540nm colorimetric estimation, and its shade is directly proportional to protein concn, and form irrelevant with the relative molecular mass of protein and amino acid.Thus can the content of oxyphorase in Fast Measurement tuna.
Concrete test experiments step:
The preparation of biuret reagent: take 1.50g copper sulfate crystal and 6.0g Seignette salt, by the water dissolution of 500ml, add down while stirring 300ml, 10% NaOH(100mg/ml) solution, be diluted with water to 1L, be stored in Plastic Bottle.
The mensuration of typical curve: get 12 test tubes and divide two groups, add 0,0.1,0.2,0.3,0.4,0.5ml standard protein solution (10mg/ml) respectively, supply 1ml with distilled water, then add 4ml biuret reagent.Shake up rear room temperature and place 30min, measure absorbancy at 540nm place.First test tube not adding protein solution does blank, and go two groups to measure mean value, take protein content as X-coordinate, absorbancy is that ordinate zou does typical curve.
The mensuration of unknown sample: get 2 test tubes is the sample after dialysis and sample after chromatographic elution respectively, with aforesaid method working sample protein concn and calculated purity.
The purity of oxyphorase in tuna
| Sample | The quality amount concentration (mg/L) of protein | The purity % of oxyphorase |
| Crude extract | 3.609 | - |
| Sample dialyzate | 4.063 | 21.78 |
| Elutriant after chromatography | 1.305 | 87.11 |
Although describe the present invention in conjunction with preferred embodiment; so itself and be not used to limit the present invention; any those skilled in the art; without departing from the spirit and scope of the present invention; can implement various change, the displacement of coordinator and amendment here to the theme listed, therefore protection scope of the present invention be as the criterion when the scope limited depending on proposed claim.
Claims (6)
1. the extraction and purification method of the myohaemoglobin of tuna, comprises and adopts tuna to be raw material, it is characterized in that: from described tuna, extract myohaemoglobin; Carry out under the leaching process of described myohaemoglobin is all in the aqueous solution state of carbohydrate admixture; Described carbohydrate admixture contains lactose and trehalose, and described lactose and trehalose weight ratio are 5:1 to 1:5.
2. the extraction and purification method of the myohaemoglobin of tuna according to claim 1, is characterized in that: comprise the steps:
1) accurately take certain mass tuna minced fish, carry out extracting after adding Tris-HCl damping fluid and described carbohydrate admixture, after filtration, get filtrate;
2) by centrifugal in 8000-14000r/min for the filtrate described in step 1), removing precipitation, gets supernatant liquor;
3) by step 2) described in supernatant liquor with 1mol/LNaOH solution adjustment pH to 7.60, add the ammonium sulfate precipitation of suitable saturation ratio, leave standstill;
4) it is centrifugal that the throw out after step 3) being saltoutd carries out 8000-14000r/min, and precipitation adds Tris-HCl Extraction buffer and dissolves, and dialyse in same damping fluid 10h to 24h, uses polyoxyethylene glycol concentrated through row;
5) work in-process after step 4) being concentrated by ion exchange column through row purifying.
3. the extraction and purification method of the myohaemoglobin of tuna according to claim 2, is characterized in that: the process of the myohaemoglobin extraction and purification of described tuna is all carried out in ice bath.
4. the extraction and purification method of the myohaemoglobin of tuna according to claim 2, is characterized in that: the carbohydrate admixture described in step 1) accounts for the 5-30% of tuna in massfraction.
5. the extraction and purification method of the myohaemoglobin of tuna according to claim 2, it is characterized in that: the condition of described purification process is: chromatographic material selects DE-AE-SepharoseFF, the NaCl solution of 0.01mol/LpH8.6Tris-HCl damping fluid and 200mmol/L does gradient elution, collects elutriant.
6. the extraction and purification method of the myohaemoglobin of tuna according to claim 2, is characterized in that: it is 2000 to 100000 that described dialysis treatment intercepts molecular weight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510687842.3A CN105399817A (en) | 2015-10-22 | 2015-10-22 | Extraction and purification method of tuna myoglobins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510687842.3A CN105399817A (en) | 2015-10-22 | 2015-10-22 | Extraction and purification method of tuna myoglobins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105399817A true CN105399817A (en) | 2016-03-16 |
Family
ID=55465611
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510687842.3A Pending CN105399817A (en) | 2015-10-22 | 2015-10-22 | Extraction and purification method of tuna myoglobins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105399817A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404480A (en) * | 2016-08-30 | 2017-02-15 | 宁波美康生物科技股份有限公司 | Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin |
| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800202A (en) * | 2006-01-20 | 2006-07-12 | 南京师范大学 | Method for extracting purified high ferro myoglobins from cardiac muscle |
| CN101294148A (en) * | 2008-06-06 | 2008-10-29 | 南京师范大学 | Method for extracting and purifying high ferro myohemoglobin reductase from myocardium |
-
2015
- 2015-10-22 CN CN201510687842.3A patent/CN105399817A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800202A (en) * | 2006-01-20 | 2006-07-12 | 南京师范大学 | Method for extracting purified high ferro myoglobins from cardiac muscle |
| CN101294148A (en) * | 2008-06-06 | 2008-10-29 | 南京师范大学 | Method for extracting and purifying high ferro myohemoglobin reductase from myocardium |
Non-Patent Citations (2)
| Title |
|---|
| 崔福德等: "《药剂学》", 31 January 2006, 中国医药科技出版社 * |
| 徐群英等: "金枪鱼碎鱼肉中肌红蛋白的初步提取和纯化", 《食品与生物技术学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404480A (en) * | 2016-08-30 | 2017-02-15 | 宁波美康生物科技股份有限公司 | Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin |
| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2017014287A5 (en) | Stabilization of high-pressure steam sterilized peripheral intravenous nutrition | |
| CN109761833B (en) | A kind of isolation and purification method of L-Leu | |
| CN103992321B (en) | A kind of preparation method of theophylline | |
| CN105399817A (en) | Extraction and purification method of tuna myoglobins | |
| MX2015008529A (en) | Solution for preserving vascular conduits. | |
| CN102584953B (en) | Purification method for atosiban | |
| CN102206273A (en) | Method for extracting immunoglobulin G from pig blood | |
| CN104480304A (en) | Method for reducing usage amount of ammonia gas during process of tantalum-niobium hydrometallurgical extraction | |
| MY181282A (en) | Solutions for increasing the stability and shelf life of an organ and tissue preservation solution | |
| CN103992391A (en) | Purifying method for terlipressin | |
| CN109588613A (en) | A kind of liquid nisin preparation method | |
| WO2010149572A8 (en) | Method and arrangement for preserving anatomical preparations | |
| CN103710416A (en) | Preparation method and applications of rapeseed cake-sourced metal chelating peptide and peptide metal chelate | |
| CN103695513A (en) | Method for improving yield of soybean peptide with low molecular weight | |
| CN101955512A (en) | Angiotensin I converting enzyme activity inhibiting oligopeptides as well as preparation method and application thereof | |
| CN105325702B (en) | natural antibiotic feed additive containing gentian H and preparation method and application thereof | |
| CN112592316B (en) | Method for reducing purine content in tuna anserine raw material by using membrane filtration and application thereof | |
| CN101585879A (en) | Method of quickly inducing pork liver metallothionein | |
| CN101407479A (en) | Preparation of sarcosine | |
| CN106834373A (en) | A kind of preparation method of L homophenylalanins or D homophenylalanins | |
| CN102382150B (en) | Preparation method of high-purity mixed sodium deoxyribonucleotide | |
| CN104925925A (en) | High-efficiency water quality treatment material used for aquaculture | |
| CN104187536A (en) | Method for extracting and purifying capsaicin compound from chilli extract | |
| US9896477B2 (en) | Limonin extraction method | |
| CN102864135A (en) | Pancreatic kallikrein preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160316 |
|
| RJ01 | Rejection of invention patent application after publication |