CN106928332A - A kind of preparation method of different deliquescent mussel proteins and polypeptide - Google Patents
A kind of preparation method of different deliquescent mussel proteins and polypeptide Download PDFInfo
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- CN106928332A CN106928332A CN201710015510.XA CN201710015510A CN106928332A CN 106928332 A CN106928332 A CN 106928332A CN 201710015510 A CN201710015510 A CN 201710015510A CN 106928332 A CN106928332 A CN 106928332A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
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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
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The preparation method of a kind of different dissolubility mussel proteins and polypeptide, cleans, shells, removing byssus, homogenate；Carry out myosinogen, fribrillin, the extraction of stromatin step by step successively to obtain corresponding extract solution, then carry out ultrafiltration respectively, finally be spray-dried obtaining albumen powder.Protein extract enzymolysis, the enzyme that goes out, centrifuging and taking supernatant more than in addition, ultrafiltration, again be spray-dried obtaining polypeptide powder.Myosinogen, fribrillin, stromatin are divided into according to deliquescent difference in mussel.The aqueous solution or dilute salting liquid are dissolved in using myosinogen, fribrillin is dissolved in certain density salting liquid, and matrix is insoluble in the characteristic of water, and the mixed protein in mussel has been carried out into appropriate separation.And digested with these albumen respectively, three kinds of different enzymolysis polypeptide products are obtained, improve the utilization rate of raw material.
Description
Technical field
The present invention relates to a kind of different deliquescent mussel proteins and the preparation method of polypeptide.
Background technology
Mussel is the important species in shellfish culture cause, and production quantity is also very big.Mussel dry product protein content is left 60%
The right side, economic worth is very high, also there is certain practical value.In order to more fully develop mussel, the present invention is carried using substep
The method for taking makes the protein dissolution of raw material, while also preferably according to protein dissolution sex differernce, the mixed protein in raw material
Separated, be easy to the later stage to study different albumen.
In current research, composition, content on mussel protein etc. without definite report, various groups in mussel protein
The amino acid sequence for dividing is even more and is rarely reported.This for mussel protein further investigation, with certain limitation.This limitation master
Two aspects are shown, is summarized as follows：
First, because dissolubility of the protein in different systems has certain difference, if we can be according to protein
Dissolubility classified, then in food liquid system according to architectural characteristic use different deliquescent protein, pole has
The stability of system may be improved, product quality is effectively kept.Therefore, classification is carried out to albumen according to dissolubility and extracts preparation tool
There is certain meaning.
Second, different types of protein is contained in mussel, it is that the good of biologically active peptide is obtained by biologic enzymolysis method
Protein source.Complexity yet with system is larger, has for the application that we are oriented enzyme incision technology certain
Limitation.Therefore, that mussel protein is carried out into classification system by deliquescent difference is standby, it is possible to reduce system to a certain extent
Complexity, while effective theory and technology basis, invention tool can be provided for prepared by the further separation of mussel protein
There is certain practical value.
Mussel, also known as Dan Cai ﹑ Hai Hong ﹑ seas madam, mainly there is striped Yi Bei ﹑ Mytilus galloprovincialis and Trachyostracous mussel, and mussel is in classification
On belong to Mollusca (Mollusca), lamellibranchiata (Lamellibranchia), Anisomyaria (Anisomyaria), Mytilidae
(Mytilidae) marine organisms.What China had found has nearly 50 kinds, point 18 category.Their shell is all triangular in shape, and surface has
The shinny crust of one layer of pitch-dark color.Mussel taste is extremely fresh, nutritious, and its contained Dan Bai Zhi ﹑ Dian ﹑ calcium and iron all compare many, but
Institute is fatty seldom.The maritime provinces such as Zhe Jiang ﹑ Fu Jian ﹑ Shan Dong ﹑ Liaoning are originated from, cultivation amount is very big.In seafood, low value
Mussel is simultaneously underused, therefore research and development mussel is improved its value and is just of great significance.This patent
Initial analysis is carried out to mussel from the angle of protein, to seek the utilization rate that new thinking improves mussel.
Myosinogen, fribrillin, stromatin are divided into according to deliquescent difference in mussel.Using myosinogen
The aqueous solution or dilute salting liquid are dissolved in, fribrillin is dissolved in certain density salting liquid, and matrix is insoluble in the characteristic of water, will make a gift of
Mixed protein in shellfish has carried out appropriate separation.
But the extractive technique method of the mussel protein of report has three limitation of aspect at present：
（1）Step by step arithmetic technology for three kinds of dissolubility albumen is indefinite；
（2）The recovery rate of various albumen is relatively low；
（3）The protein prepared fails by subsequent treatment, and develop into can be with direct applied product.
The medical, edible value to mussel has done substantial amounts of research work both at home and abroad, achieves many achievements, part
Applied in the production of medicine and food.But mussel is wide in China's culturing area, yield is big, no matter from medicinal angle
Degree, or it is respectively provided with wide DEVELOPMENT PROSPECT from the angle of health care dietotherapy.In China, after mussel is mainly fresh or roughing
For among the people edible, if it is possible to binding achievement, strengthen the determination of active component, separate, extract, the basis such as pharmacology and clinic
Research, develops corresponding health food and its medicine, and the not only prevention and treatment to epidemic disease are contributed, and can be produced larger
Social benefit and economic benefit.
At present, to mussel protein separation, extraction and the research of physicochemical property is rarely reported.For the research of albumen classification
It is more to concentrate on the crops protein sources such as wheat, rice, buckwheat, and the research of the classification and extraction process for mussel protein
It is rarely reported.
Digested with these albumen respectively, three kinds of different enzymolysis polypeptide products are obtained, with different application directions
And researching value, improve the utilization rate of raw material.
The content of the invention
The present invention is sufficiently utilized to raw material first, and a batch processed can obtain three albuminoids, and albumen has
Certain characteristic molecular amount.Digested with these albumen respectively, obtained three kinds of different enzymolysis polypeptide products, improve raw material
Utilization rate.
The technical proposal of the invention is realized in this way：A kind of preparation method of different dissolubility mussel proteins, cleaning,
Shell, remove byssus, be homogenized；Myosinogen, fribrillin, the extraction of stromatin is carried out step by step successively to be carried accordingly
Liquid is taken, then carries out ultrafiltration respectively, finally be spray-dried obtaining albumen powder.
Further, myosinogen extract solution is 0.05mol/L, the phosphate buffer solution of pH=7, extraction time 30-
90min, extracting times 2-3 times, 2-4 DEG C of temperature, solid-liquid ratio 1:3-1:20, it is centrifuged after extraction, final protein liquid is used
10000Da ultrafiltration membrance filters.
Further, myosinogen extract solution is 0.05mol/L, the phosphate buffer solution of pH=7, extraction time 30-
90min, extracting times 2-3 times, 2-4 DEG C of temperature, solid-liquid ratio 1:3-1:20, it is centrifuged after extraction, final protein liquid is used
10000Da film ultra-filtration filters.
Further, stromatin extract solution is the citric acid solution of 2%-5%, extraction time 60-90min, extracting times
2-4 times, 30-70 DEG C of temperature；It is centrifuged after extraction, the 10000Da film ultrafiltration of final protein liquid.
Further, using 130-170 DEG C of the inlet temperature of spray drying, 60-90 DEG C of outlet temperature is spray-dried
Before concentration can be evaporated to protein liquid, make its solid content be 15-50%.
Further, it is dried by the way of low temperature spray drying, 68-80 DEG C of EAT, leaving air temp 30-45
DEG C, solid content requirement 15-50%, the sample after drying is controlled within 5%.
In addition, the method that mussel polypeptide is prepared with above-mentioned mussel protein, in protein extract enzymolysis, the enzyme that goes out, centrifuging and taking
Clear liquid, ultrafiltration, again be spray-dried obtaining polypeptide powder.
Further, the condition of the enzymolysis is：Trypsase pH8-9,40-47 DEG C of temperature, time 2-6h；Enzyme concentration
3000-5000U/g。
Further, boiling water bath 5-15min carries out the enzyme that goes out.
Further, after the sample after the enzyme that goes out is cooled to room temperature, it is centrifuged, rotating speed 8000-10000g/min, time
5-15min。
Further, after centrifugation by supernatant by ultrafiltration, collect the enzymolysis liquid of below molecular weight 3K, remove macromolecular complex
Matter.
Beneficial effects of the present invention are：It is contemplated that with mussel as raw material, the technology extracted respectively using three steps is made a gift of
Most albumen in shellfish.The low problem of protein extracting ratio is not only solved, mixed protein is suitably separated.Simultaneously
The mode such as digest again, prepare new mussel polypeptide.It is open in can be widely applied to the products such as health food, functional food
Extensive market prospects.Using hyperfiltration technique and spray drying technology, the pure of albumen and polypeptide products is ensure that to greatest extent
Degree problem, so as to ensure that its final activation plays to maximum（Size distribution, moisture content, bioactivity, dissolubility, color, taste
Deng）.
Specific embodiment
Explanation is further explained to the present invention with reference to specific embodiment.
The preparation method of the mussel protein of embodiment 1：
1）Pre-treatment：Clean, shell, removing byssus, homogenate 5000-8000rpm/min, time 15-30min.
2）Myosinogen extract solution is 0.05mol/L, the phosphate buffer solution of pH=7, extraction time 30-90min, extraction
Number of times 2-3 times, 2-4 DEG C of temperature, solid-liquid ratio 1:3-1:20. centrifugal conditions：8000-13000g, 10-20min, 4 DEG C.Final albumen
Liquid 10000Da films ultrafiltration 2-6 times.
3）Fribrillin extract solution is 0.1mol/L, phosphate buffer solutions of the pH=7 containing 0.6mol/L sodium chloride；Leaching
Carry time 30-60min；Extracting times 2-3 times；2-4 DEG C of temperature；Solid-liquid ratio 1:3-1:20；Centrifugal condition：8000-13000g,
10-20min, 4 DEG C.Final protein liquid 10000Da films ultrafiltration 2-6 times.
4）Stromatin extract solution is the citric acid solution of 2%-5%, extraction time 60-90min, extracting times 2-4 times, temperature
30-70 DEG C of degree；Centrifugal condition：8000-10000rpm, 20-40min, 4 DEG C.Final protein liquid 10000Da films ultrafiltration 2-6 times.
It is to remove the salinity in protein solution using hyperfiltration technique purpose.Sample is centrifuged twice before ultrafiltration, to seek quickness
Fast ultrafiltration, prevents from blocking, and extends the service life of film.
5）Mussel protein is dried using spray drying technology, to the inlet temperature 130-170 using spray drying
DEG C, 60-90 DEG C of outlet temperature can be evaporated concentration before being spray-dried to protein liquid, make its solid content be
15-50%。
Related assays method
The measure of protein content：
（1）Using the method for kjeldahl determination.
Well-mixed solid sample 0.2-2g is weighed, 0.001g is accurate to and is moved into dry 250ml or 500ml nitrogen fixing bottles
In, add 0.2g cupric sulfate pentahydrates, 6g potassium sulfates, the 20ml concentrated sulfuric acids to be put into after shaking up in digesting and digested, selected as follows
Program：220 DEG C for the treatment of 60min, 440 DEG C for the treatment of 90min.After the completion of be put into Protein Analyzer and processed, boric acid is inhaled
Free ammonia is received, is titrated with the standard hydrochloric acid that concentration is 0.0995-0.1005mol/L.Consumption according to acid is multiplied by and changes
Calculate coefficient 6.25, the as content of protein.
（2）Using the method for Coomassie brilliant blue
Configuration solution：Standard protein liquid：0.1g bovine serum albumins are with ultrapure water dissolves and are diluted to 1L；Coomassie brilliant G-250
（0.01%）Dye liquor：Weigh 0.1g Coomassie brilliant G-250s to be dissolved in 50mL95% ethanol, add 100mL SPAs（85%）Plus
Distilled water is settled to 1000mL.
Standard curve making：Test tube numbering 0123456
100ug/ml standard proteins（mL） 0.0 0.1 0.2 0.3 0.4 0.5 0.6
Ultra-pure water（mL） 1 0.9 0.8 0.7 0.6 0.5 0.4
Coomassie brilliant blue reagent（mL）5555555 shake up, and are stored at room temperature 3min
It is blank with No. 1 pipe, the colorimetric at 595nm, at least three groups of the drafting of standard curve is parallel, makes R*R>0.9980
Sample 1mL adds 5ml coomassie reagents, mixes, and is stored at room temperature 3min with No. 1 pipe in standard curve as blank,
The colorimetric at 595nm.Bring into standard curve, calculate sample protein content.
The basic index of the mussel protein that the method is obtained is：
1. mussel protein finally presents powdered
2. albumen moisture≤5%
3. three kinds of protein contents of mussel protein（In terms of N）≥80%
4. the distribution of myosinogen band is relatively broad, scope in 90-40KDa, wherein based on 45KDa albumen, muscle fibril egg
White molecular weight ranges 70-120KDa, wherein based on 102KDa albumen, stromatin molecular weight is substantially distributed as 103,87,72,
17KDa.Three kinds of albumen respectively have obvious characteristic bands.Total protein extraction rate scope is 75-85%.
5. the protein content of albumen powder is obtained after three kinds of protein solution ultrafiltration in the range of 65-90%, hence it is evident that improve albumen
Purity.Be conducive to the research work of latter step.
The mussel protein obtained with the method for embodiment 1 is further digested, the enzyme that goes out, centrifuging and taking supernatant, ultrafiltration, again
Be spray-dried and obtain polypeptide powder, specific processing step and condition are：
1）Enzymatic hydrolysis condition：Trypsase pH8-9,40-47 DEG C of temperature, time 2-6h；Enzyme concentration 3000-5000U/g
2）Go out enzyme：Boiling water bath 5-15min
3）Centrifugation：After the sample after the enzyme that goes out is cooled to room temperature, it is centrifuged, rotating speed 8000-10000g/min, time 5-
15min。
4）Ultrafiltration：By supernatant by ultrafiltration, the enzymolysis liquid of below molecular weight 3K is collected, remove macromolecular substances.
5）Spray drying：Mussel protein is dried using spray drying technology, 130-170 DEG C of inlet temperature, exported
60-90 DEG C of temperature, can be concentrated to protein liquid before being spray-dried makes its solid content be 15-50%（According to
The sample rheological properties of needs of production and unit type determine the suitable solid content of tool.Also cold nebulization can be used
Dry mode is dried, 68-80 DEG C of EAT, 30-45 DEG C of leaving air temp, solid content requirement 15-50%.Dry
Sample afterwards is controlled within 5%.
The molecular weight distribution that preparation method of the invention obtains polypeptide is：700-2700Da, wherein<The ratio for accounting for of 1000Da
Example be：21.74%；<The ratio of 2000Da is：85.51%.Polypeptide yield is：60%-80%, three kinds of polypeptide moisture≤5%.Three
Plant polypeptide protein content（In terms of N）≥95%.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (11)
1. a kind of preparation method of different dissolubility mussel proteins, it is characterised in that clean, shell, removing byssus, is homogenized；Successively
Substep carries out myosinogen, fribrillin, the extraction of stromatin and obtains corresponding extract solution, then carry out ultrafiltration respectively,
Finally be spray-dried obtaining albumen powder.
2. the preparation method of different dissolubility mussel proteins according to claim 1, it is characterised in that myosinogen is extracted
Liquid is 0.05mol/L, the phosphate buffer solution of pH=7, extraction time 30-90min, extracting times 2-3 times, 2-4 DEG C of temperature, material
Liquor ratio 1:3-1:20, it is centrifuged after extraction, final protein liquid 10000Da ultrafiltration membrance filters.
3. the preparation method of different dissolubility mussel proteins according to claim 1, it is characterised in that myosinogen is extracted
Liquid is 0.05mol/L, the phosphate buffer solution of pH=7, extraction time 30-90min, extracting times 2-3 times, 2-4 DEG C of temperature, material
Liquor ratio 1:3-1:20, it is centrifuged after extraction, final protein liquid 10000Da film ultra-filtration filters.
4. the preparation method of different dissolubility mussel proteins according to claim 1, it is characterised in that stromatin is extracted
Liquid is the citric acid solution of 2%-5%, extraction time 60-90min, extracting times 2-4 times, 30-70 DEG C of temperature；Carried out after extraction from
The heart, the 10000Da film ultrafiltration of final protein liquid.
5. the preparation method of different dissolubility mussel proteins according to claim 1, it is characterised in that using spray drying
130-170 DEG C of inlet temperature, 60-90 DEG C of outlet temperature can be evaporated concentration before being spray-dried to protein liquid,
Its solid content is set to be 15-50%.
6. the preparation method of different dissolubility mussel proteins according to claim 5, it is characterised in that use cold nebulization
Dry mode is dried, 68-80 DEG C of EAT, 30-45 DEG C of leaving air temp, solid content requirement 15-50%, dries
Sample afterwards is controlled within 5%.
7. the method that the mussel protein as described in claim any one of 1-6 prepares mussel polypeptide, it is characterised in that carried to albumen
Take liquid enzymolysis, the enzyme that goes out, centrifuging and taking supernatant, ultrafiltration, again be spray-dried obtaining polypeptide powder.
8. the method for preparing mussel polypeptide according to claim 7, it is characterised in that the condition of the enzymolysis is：Pancreas egg
White enzyme pH8-9,40-47 DEG C of temperature, time 2-6h；Enzyme concentration 3000-5000U/g.
9. it is according to claim 7 prepare mussel polypeptide method, it is characterised in that boiling water bath 5-15min carries out the enzyme that goes out.
10. it is according to claim 1 prepare mussel polypeptide method, it is characterised in that be cooled to wait the sample after the enzyme that goes out
After room temperature, it is centrifuged, rotating speed 8000-10000g/min, time 5-15min.
The preparation method of 11. different dissolubility mussel proteins according to claim 1, it is characterised in that will be upper after centrifugation
Clear liquid collects the enzymolysis liquid of below molecular weight 3K by ultrafiltration, removes macromolecular substances.
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Cited By (4)
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| CN110604313A (en) * | 2019-09-30 | 2019-12-24 | 浙江海洋大学 | Method for preparing high-protein high-calcium supplement by using mussel waste materials |
| CN113040264A (en) * | 2021-04-12 | 2021-06-29 | 大连工业大学 | Method for modifying mussel protein through high-pressure homogenization treatment |
| CN113841784A (en) * | 2021-09-17 | 2021-12-28 | 大连工业大学 | Method for modifying mussel myofibrillar protein |
| CN114574535A (en) * | 2022-03-08 | 2022-06-03 | 西南民族大学 | Preparation method of sarcoplasmic protein peptide glycosylated graft and application of sarcoplasmic protein peptide glycosylated graft in preparation of fermented meat floss sauce |
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| CN113040264A (en) * | 2021-04-12 | 2021-06-29 | 大连工业大学 | Method for modifying mussel protein through high-pressure homogenization treatment |
| CN113841784A (en) * | 2021-09-17 | 2021-12-28 | 大连工业大学 | Method for modifying mussel myofibrillar protein |
| CN114574535A (en) * | 2022-03-08 | 2022-06-03 | 西南民族大学 | Preparation method of sarcoplasmic protein peptide glycosylated graft and application of sarcoplasmic protein peptide glycosylated graft in preparation of fermented meat floss sauce |
| CN114574535B (en) * | 2022-03-08 | 2023-06-30 | 西南民族大学 | Preparation method of sarcoplasmic protein peptide glycosylation graft and application of sarcoplasmic protein peptide glycosylation graft in preparation of fermented dried meat floss sauce |
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