CN1392265A - Process for preparing bioactive peptide-silk fibroin peptide - Google Patents
Process for preparing bioactive peptide-silk fibroin peptide Download PDFInfo
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- CN1392265A CN1392265A CN 02138016 CN02138016A CN1392265A CN 1392265 A CN1392265 A CN 1392265A CN 02138016 CN02138016 CN 02138016 CN 02138016 A CN02138016 A CN 02138016A CN 1392265 A CN1392265 A CN 1392265A
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Abstract
The present invention, the preparation process of silk fibroin peptide as one bioactive peptide, belongs to the field of food biotechnology. Silkworm cocoon leftover or waste cocoon as material is refined in Na2CO3 solution and dissolved in CaCl2 solution to obtain silk dissolving liquid; the silk dissolving liquid is enzyme hydrolyzed at pH 2.5-12.0 and 30-80 deg.c, nano filtered to elimiante high-concentration CaCl2 salt, concentrated and spray dried to obtain the product silk fibroin peptide powder. The present invention has the advantages of great treated amount, high desalting rate, mild separation condition, simple process and low power consumption. The said process may be used in industry to prepare silk fibroin peptide for functional food.
Description
Technical field
The present invention relates to a kind of preparation method of biologically active peptides-silk peptide, be specifically related to utilize the molten silk of enzymatic hydrolysis liquid, the calcium chloride that removes the enzymolysis solution middle and high concentration with nanofiltration prepares the method for silk peptide again, belongs to technical field of food biotechnology.
Background technology
Silk is a kind of scleroproein, is made up of fibroin and silk gum, and wherein fibroin accounts for 70%～80%, and silk gum accounts for 20%～30%, and silk fibroin is made up of 18 seed amino acids, and about 85% is glycine, L-Ala, Serine and tyrosine.Silk peptide (silk peptide) is the intermediate product of silk fibroin hydrolysis, and the fibroin after normally silk being come unstuck is through operations such as dissolving, dialyse, purify, degrade, concentrate, and the employing spray-drying process is made the high purity silk peptide powder.The silk peptide is identical with the amino acid composition of fibroin, but its molecular weight ratio fibroin is much smaller, and molecular conformation is based on the random coil structure, and aggregated structure is loose disordered structure.Therefore, a silk peptide has good water-solubility, good moisture-absorbing moisture-keeping function, and acid, thermostability preferably, viscosity is with advantages such as change in concentration are blunt.
Amino acid whose composition in the fibroin has very high nutritive value, along with biotechnology and other development of high-tech, both at home and abroad the edible research of fibroin is also more and more paid attention to.Animal experiment shows that glycine can reduce the concentration of cholesterol in the blood, and L-Ala has the function that promotes alcohol metabolism, and tyrosine can prevent dementia, and Serine has control skin aging, radioprotective, prevents the effect that white cell descends.Therefore, the food that is rich in silk peptide has good health protection effect, can be used as a kind of functional foodstuff, and therefore its research and development also are subjected to attention.
The production of silk peptide, generally adopt acid system hydrolysis fibroin or fibroin with after the neutral salt dissolving again through enzymic hydrolysis, control the silk peptide that certain hydrolysising condition can obtain various different molecular weights.Adopt acid system hydrolysis fibroin to produce silk peptide, though this method is simple, cheap, its shortcoming is that degree of hydrolysis is wayward, and product molecular weight distribution is in the so very wide scope of 300-5000, acid hydrolysis simultaneously all destroys tryptophane, Serine, tyrosine and Threonine partial destruction.The enzymatic hydrolysis reaction mild condition, degree of hydrolysis is easy to control, amino acid is destroyed little, and and it has specificity, and different enzymic hydrolysis silk fibroin solutions can obtain different intermediate products, and the saltiness that to also have an advantage be enzymolysis product is low.Therefore, adopt the enzymatic hydrolysis fibroin, can improve the output and the quality of silk peptide greatly.
Because natural fibroin is water insoluble, be difficult to by the enzyme direct hydrolysis, so use the enzymic hydrolysis fibroin, at first to prepare silk fibroin solution.Current people adopt the CaCl of high density mostly
2Solution dissolving fibroin, treat fibroin lysate cooling desalination after, promptly obtain purified silk fibroin solution colourless, tasteless, odorless, molecular weight is about 60,000.The desalination operation adopts membrane technique desalinations such as ultrafiltration, electrodialysis usually, but this method film pollution is very serious, is difficult to realize suitability for industrialized production.
Summary of the invention
The object of the invention provides a kind of preparation method of biologically active peptides-silk peptide.The production technique that traditional enzyme hydrolysis method is prepared silk peptide is improved, i.e. silk fibroin solution not desalination is earlier carried out enzymatic hydrolysis reaction but add enzyme earlier, and enzymolysis solution nanofiltration desalination then can realize commercial scale production silk peptide product.This technology yet there are no report at present at home and abroad.
The preparation method of a kind of silk peptide of the present invention, particular content relates to and utilizes the molten silk of enzymatic hydrolysis liquid, and the calcium chloride that removes the enzymolysis solution middle and high concentration with nanofiltration prepares silk peptide again.Be to be raw material, through 0.3-1.0% (w/v) Na with leftover bits and pieces silk cocoon or useless silk
2CO
3Solution is concise, concise after, with 20-60% (w/w) CaCl
2Solution is 50-140 ℃ of dissolving, and molten silk liquid is at pH2.5-12.0, carries out enzymatic hydrolysis reaction with proteolytic enzyme under 30-80 ℃, and control hydrolysis degree (DH) is 5-25%.The enzyme that goes out after reaction finishes, centrifugal, filter, filtrate is enzymolysis solution, enzymolysis solution carries out the nanofiltration desalination with nanofiltration membrane, feed liquid is carried out spraying drying after concentrating after the desalination, promptly makes the silk peptide powder.
Nanofiltration (NF) film is a kind of film between reverse osmosis (RO) film and ultrafiltration (UF) film, and its aperture, film top layer is in nano level scope (10
-9Rice).Nanofiltration membrane since molecular weight cut-off between ultrafiltration and reverse osmosis, also there is the influence of south (Donnan) ionic effect simultaneously, therefore to separating of low-molecular-weight organic matter and salt good effect being arranged, and have the biological activity that do not influence separate substance, characteristics such as energy-conservation, nuisanceless.Nanofiltration membrane can adopt polyamide membrane, poly (ether sulfone) film, cellulose acetate membrane, sulfonated polysulfone membrane or polyvinyl alcohol film etc.
In the preparation silk peptide process, make silk fibroin solution with neutral salt solution dissolving fibroin, use the molten silk of enzymic hydrolysis liquid again, remove a large amount of neutral salt in the enzymolysis solution with the nanofiltration membrane separation technology then, ratio of desalinization can reach more than 98%.
Advantage of the present invention is, adopts the molten silk of first enzymatic hydrolysis liquid, removes the CaCl of enzymolysis solution middle and high concentration again with the nanofiltration membrane separation technology
2Salt, this method have that treatment capacity is big, ratio of desalinization is high, separation condition is gentle, flow process simply reaches advantages such as energy consumption is lower, are a kind of desalting method efficiently, can realize suitability for industrialized production.
Description of drawings
Fig. 1, Fig. 2 are the silk peptide solution nanofiltration desalination experimental data curve figure of DH=12%.
Fig. 1 be concentrated solution, the specific conductivity that sees through liquid over time.
Fig. 2 be concentrated solution, the ion content that sees through liquid over time.
Fig. 3, Fig. 4 are the silk peptide solution nanofiltration desalination experimental data curve figure of DH=10%.
Fig. 3 be concentrated solution, the specific conductivity that sees through liquid over time.
Fig. 4 be concentrated solution, the ion content that sees through liquid over time.
Embodiment
The preparation of the silk peptide powder of degree of hydrolysis (DH)=12%
Useless silk (bourette) is through the Na of 0.5% (w/v)
2CO
3After solution is concise, place ebullient 40% (w/w) CaCl
2Dissolving (was pressed 100mlCaCl in 5 minutes 30 seconds in the solution
2Solution dissolving 14g nett silk dissolves), cooling rapidly.Molten silk liquid obtains dense molten silk liquid after gauze filters, get 8 liters of dense molten silk liquid, adds 12 premium on currency, carries out enzyme digestion reaction with the Alcalase enzyme down, employing pH-stat method control hydrolysis degree pH8.5,60 ℃.Reaction finishes the post-heating enzyme that goes out, centrifugal, filter after, filtrate use the polyamide nanofiltration membrane desalination, continuous thin up feed liquid in demineralising process is carried out spraying drying after the feed liquid after the desalination is concentrated and is promptly made the silk peptide powder.
The silk peptide solution nanofiltration desalination experiment of DH=12%
The specific conductivity that Fig. 1 shows concentrated solution, see through liquid over time, as seen from Figure 1, with the desalination time lengthening, concentrated solution, the specific conductivity that sees through liquid all descend, and desalination time when being the 4h left and right sides, and specific conductivity descends significantly, after this, with the desalination time lengthening, specific conductivity descends slowly.
As seen from Figure 2, with the desalination time lengthening, concentrated solution, the calcium ion and the chloride ion content that see through liquid all descend, and be consistent with the variation tendency of specific conductivity.
The degree of hydrolysis of enzymolysis protein matter is measured:
Under neutrality and alkaline condition, adopt the pH-stat method
Under acidic conditions, adopt the TNBS method
The silk peptide rate of recovery: feed liquid nitrogen content before feed liquid nitrogen content/desalination after the desalination
Ratio of desalinization: (feed liquid Ca after the 1--desalination
2+Feed liquid Ca before content/desalination
2+Content) * 100%
The variation of feed liquid calcium ion content before and after table 1 desalination
Feed liquid calcium ion content (g)
Desalination preceding 1335.87
After the desalination 26.04
Ratio of desalinization (%) 98.05 ratio of desalinization are higher than 98%, and the rate of recovery is higher than 80%.Concentrated solution carries out spraying drying after the desalination, drying conditions: inlet temperature: 150-220 ℃, and air outlet temperature: 60-90 ℃.
The analytical results of table 2 silk peptide product
Peptide content (%) water content (%) ash oontent (%)
85.15 5.10 9.75
Product is a pale yellow powder, and rehydration is good.
The preparation of the silk peptide powder of degree of hydrolysis (DH)=10%
Useless silk (bourette) is through the Na of 0.5% (w/v)
2CO
3After solution is concise, place ebullient 40% (w/w) CaCl
2Dissolving (was pressed 100mlCaCl in 5 minutes 30 seconds in the solution
2Solution dissolving 14g nett silk dissolves), cooling rapidly.Molten silk liquid obtains dense molten silk liquid after gauze filters, get 7 liters of dense molten silk liquid, adds 7 premium on currency, carries out enzyme digestion reaction with the Alcalase enzyme down, employing pH-stat method control hydrolysis degree pH8.5,60 ℃.Reaction finishes the post-heating enzyme that goes out, centrifugal, filter after, filtrate use the polyether sulfone filtering film desalination, continuous thin up feed liquid in demineralising process, feed liquid is carried out spraying drying after concentrated and is promptly made the silk peptide powder after the desalination.
The silk peptide solution nanofiltration desalination experiment of DH=10%
The specific conductivity that Fig. 3 shows concentrated solution, see through liquid over time, as seen from Figure 3, with the desalination time lengthening, concentrated solution, the specific conductivity that sees through liquid all descend, and desalination time when being the 100min left and right sides, and specific conductivity descends significantly, after this, with the desalination time lengthening, specific conductivity descends slowly.
As seen from Figure 4, with the desalination time lengthening, concentrated solution, the calcium ion and the chloride ion content that see through liquid all descend, and be consistent with the variation tendency of specific conductivity.
The variation of feed liquid calcium ion content before and after table 3 desalination
Feed liquid calcium ion content (g)
Desalination preceding 742.33
After the desalination 11.92
Ratio of desalinization (%) 98.39
Ratio of desalinization is higher than 98%, and the rate of recovery is higher than 80%.
Concentrated solution carries out spraying drying after the desalination, drying conditions: inlet temperature: 150-220 ℃, and air outlet temperature: 60-90 ℃.
Product composition and table 2 are basic identical.
Claims (6)
1. the preparation method of a silk peptide is characterized in that utilizing the molten silk of enzymatic hydrolysis liquid, removes the CaCl of enzymolysis solution middle and high concentration again with nanofiltration
2Salt, concrete steps are: be raw material with leftover bits and pieces silk cocoon or useless silk 1), with the Na of 0.3-1.0% (w/v)
2CO
3Solution is concise, 2) with the CaCl of 20-60% (w/w)
2Solution is 50-140 ℃ of dissolving, 3) molten silk liquid is at pH2.5-12.0, carry out enzymatic hydrolysis reaction under 30-80 ℃, 4) enzyme that goes out after control hydrolysis degree DH is 5-25%, 5) reaction finishes, centrifugal, filter, getting filtrate is enzymolysis solution, 6) enzymolysis solution carries out the nanofiltration desalination with nanofiltration membrane, continuous thin up feed liquid in demineralising process, 7) feed liquid after the desalination carries out spraying drying after concentrating and promptly gets product silk peptide powder.
2. the preparation method of a kind of silk peptide as claimed in claim 1 is characterized in that enzymatic hydrolysis reaction, and used enzyme is a proteolytic enzyme.
3. the preparation method of a kind of silk peptide as claimed in claim 1 is characterized in that the nanofiltration desalination of enzymolysis solution, and used nanofiltration membrane is polyamide membrane, poly (ether sulfone) film, cellulose acetate membrane, sulfonated polysulfone membrane or polyvinyl alcohol film.
4. the preparation method of a kind of silk peptide as claimed in claim 1 is characterized in that enzymolysis solution carries out the nanofiltration desalination with nanofiltration membrane, compares the feed liquid of desalination front and back, and its silk peptide rate of recovery reaches 80%, and ratio of desalinization reaches 98%.
5. the preparation method of a kind of silk peptide as claimed in claim 1, it is characterized in that desalination after concentrated solution carry out spraying drying, drying conditions is: inlet temperature 150-220 ℃, air outlet temperature 60-90 ℃.
6. nanofiltration membrane separation The Application of Technology, it is characterized in that preparing in the silk peptide process, make silk fibroin solution, use the molten silk of enzymic hydrolysis liquid again with neutral salt solution dissolving fibroin, remove neutral salt a large amount of in the enzymolysis solution with the nanofiltration membrane separation technology again, ratio of desalinization can reach 98%.
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|---|---|---|---|
| CN 02138016 CN1233841C (en) | 2002-07-23 | 2002-07-23 | Process for preparing bioactive peptide-silk fibroin peptide |
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|---|---|---|---|
| CN 02138016 CN1233841C (en) | 2002-07-23 | 2002-07-23 | Process for preparing bioactive peptide-silk fibroin peptide |
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| CN1392265A true CN1392265A (en) | 2003-01-22 |
| CN1233841C CN1233841C (en) | 2005-12-28 |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102172257A (en) * | 2011-01-20 | 2011-09-07 | 肖仲君 | Method for making superfine silk peptide powder |
| CN102219844A (en) * | 2011-04-02 | 2011-10-19 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN102836415A (en) * | 2011-06-22 | 2012-12-26 | 吾儿得伟有限公司 | Composition for enhancing immune activity, medical composition for preventing and improving cancer |
| CN102965423A (en) * | 2012-11-07 | 2013-03-13 | 华南理工大学 | Preparation method of silk fibroin peptide with bacteriostatic activity |
| CN103243145A (en) * | 2013-05-30 | 2013-08-14 | 苏州大学 | Preparation method of high-purity silk fibroin peptide |
| CN105385733A (en) * | 2015-11-12 | 2016-03-09 | 安康学院 | Polypeptide enzymolysis process of antioxidant activity-based natural yellow cocoon fibroin |
| CN106048766A (en) * | 2016-05-27 | 2016-10-26 | 东莞市联洲知识产权运营管理有限公司 | Anti-corrosive granulation-promoting fabric containing silk protein and preparation method thereof |
| CN107326057A (en) * | 2017-07-28 | 2017-11-07 | 浙江理工大学 | The method that a kind of utilization ionic liquid and protease extract Bombyx silk albumen |
| CN109627309A (en) * | 2018-12-28 | 2019-04-16 | 浙江工业大学 | A method of silk peptide is prepared using serrapeptase hydrolysis fibroin |
| CN110184321A (en) * | 2019-06-26 | 2019-08-30 | 湖州珍露生物制品有限公司 | A kind of preparation method of silk peptide powder |
Families Citing this family (1)
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| CN100376170C (en) * | 2006-04-06 | 2008-03-26 | 浙江省农业科学院 | Edible watural silk protein powder and preparation method of natural silk extract |
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2002
- 2002-07-23 CN CN 02138016 patent/CN1233841C/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102172257A (en) * | 2011-01-20 | 2011-09-07 | 肖仲君 | Method for making superfine silk peptide powder |
| CN102219844A (en) * | 2011-04-02 | 2011-10-19 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN102219844B (en) * | 2011-04-02 | 2013-04-17 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN102836415A (en) * | 2011-06-22 | 2012-12-26 | 吾儿得伟有限公司 | Composition for enhancing immune activity, medical composition for preventing and improving cancer |
| CN102965423A (en) * | 2012-11-07 | 2013-03-13 | 华南理工大学 | Preparation method of silk fibroin peptide with bacteriostatic activity |
| CN103243145B (en) * | 2013-05-30 | 2015-03-25 | 苏州大学 | Preparation method of high-purity silk fibroin peptide |
| CN103243145A (en) * | 2013-05-30 | 2013-08-14 | 苏州大学 | Preparation method of high-purity silk fibroin peptide |
| CN105385733A (en) * | 2015-11-12 | 2016-03-09 | 安康学院 | Polypeptide enzymolysis process of antioxidant activity-based natural yellow cocoon fibroin |
| CN106048766A (en) * | 2016-05-27 | 2016-10-26 | 东莞市联洲知识产权运营管理有限公司 | Anti-corrosive granulation-promoting fabric containing silk protein and preparation method thereof |
| CN106048766B (en) * | 2016-05-27 | 2018-06-15 | 东莞市澳莉莎家用纺织品有限公司 | A kind of erosion prevention and muscle growing fabric containing fibroin and preparation method thereof |
| CN107326057A (en) * | 2017-07-28 | 2017-11-07 | 浙江理工大学 | The method that a kind of utilization ionic liquid and protease extract Bombyx silk albumen |
| CN109627309A (en) * | 2018-12-28 | 2019-04-16 | 浙江工业大学 | A method of silk peptide is prepared using serrapeptase hydrolysis fibroin |
| CN110184321A (en) * | 2019-06-26 | 2019-08-30 | 湖州珍露生物制品有限公司 | A kind of preparation method of silk peptide powder |
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|---|---|
| CN1233841C (en) | 2005-12-28 |
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