CN1233841C - Process for preparing bioactive peptide-silk fibroin peptide - Google Patents
Process for preparing bioactive peptide-silk fibroin peptide Download PDFInfo
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- CN1233841C CN1233841C CN 02138016 CN02138016A CN1233841C CN 1233841 C CN1233841 C CN 1233841C CN 02138016 CN02138016 CN 02138016 CN 02138016 A CN02138016 A CN 02138016A CN 1233841 C CN1233841 C CN 1233841C
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- silk
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- desalination
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Abstract
The present invention relates to a process for preparing a biological active peptide, namely fibroin peptide, which belongs to the biotechnical field of food. The process comprises: silkworm cocoon screenings or waste cocoon silk is used as a raw material; after being refined with a Na2CO3 solution with the concentration of 0.3 to 1.0%(w/v), the raw material is heated and dissolved with a CaCl2 solution with the concentration of 20 to 60%(w/w) to obtain a dissolved silk solution; the enzymatic hydrolysis reaction of the dissolved silk solution with is carried out using proteinase at pH2.5 to 12.0 and 30 to 80 DEG C, CaCl2 salt with high concentration in the enzymolysis solution is eliminated by the nano filtration technology, and the concentrated solution is sprayed and dried to prepare the product of fibroin peptide powder. The process has the advantages of large treating capacity, high desalinization ratio, mild separation condition, simple process and low energy consumption; industrial production can be realized, and the process is used for preparing the fibroin peptide powder with high purity and developing 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 come into one's own.
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
Embodiment 1
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 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 ℃, 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 (7)
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, the processing condition of each step are:
1) be raw material with leftover bits and pieces silk cocoon or useless silk, with the Na of quality volume percent 0.3%-1.0%
2CO
3Solution is concise,
2) with mass concentration be the CaCla of 20%-60%
2Solution is 50-140 ℃ of dissolving,
3) molten silk liquid is at pH2.5-12.0, carries out enzymatic hydrolysis reaction under 30-80 ℃,
4) control hydrolysis degree DH is 5%-25%,
5) enzyme that goes out after reaction finishes, centrifugal, filter, filtrate be enzymolysis solution,
6) enzymolysis solution carries out the nanofiltration desalination with nanofiltration membrane, continuous thin up feed liquid in demineralising process,
7) carry out spraying drying after the feed liquid after the desalination concentrates and promptly get product silk peptide powder.
2, the preparation method of a kind of silk peptide as claimed in claim 1 is characterized in that processing condition are:
1) be raw material with leftover bits and pieces silk cocoon or useless silk, with the Na of quality volume percent 0.5%
2CO
3Solution is concise,
2) with mass concentration be 40% CaCl
2Solution is 100 ℃ of dissolvings,
3) molten silk liquid is at pH8.5, carries out enzymatic hydrolysis reaction under 60 ℃,
4) control hydrolysis degree DH is 10%-12%.
3. 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.
4. 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 nanofiltration membrane, polyether sulfone filtering film, cellulose acetate membrane, sulfonated polysulfone membrane or polyvinyl alcohol film.
5. 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%.
6. 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 ℃.
7. a nanofiltration separation The Application of Technology is characterized in that preparing in the silk peptide process, makes silk fibroin solution with neutral salt solution dissolving fibroin, uses the molten silk of enzymic hydrolysis liquid again, removes neutral salt a large amount of in the enzymolysis solution with the nanofiltration membrane separation technology again, and ratio of desalinization reaches 98%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02138016 CN1233841C (en) | 2002-07-23 | 2002-07-23 | Process for preparing bioactive peptide-silk fibroin peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02138016 CN1233841C (en) | 2002-07-23 | 2002-07-23 | Process for preparing bioactive peptide-silk fibroin peptide |
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| CN1392265A CN1392265A (en) | 2003-01-22 |
| CN1233841C true CN1233841C (en) | 2005-12-28 |
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| CN 02138016 Expired - Fee Related CN1233841C (en) | 2002-07-23 | 2002-07-23 | Process for preparing bioactive peptide-silk fibroin peptide |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Families Citing this family (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 |
| CN102219844B (en) * | 2011-04-02 | 2013-04-17 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| KR101127120B1 (en) * | 2011-06-22 | 2012-03-20 | 월드웨이(주) | Composition for enhancing immune activity, preventing and improving cancer which contains silk hydrolysate as an active ingredient |
| 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 |
| CN105385733A (en) * | 2015-11-12 | 2016-03-09 | 安康学院 | Polypeptide enzymolysis process of antioxidant activity-based natural yellow cocoon fibroin |
| 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 |
| CN109627309B (en) * | 2018-12-28 | 2020-05-26 | 浙江工业大学 | Method for preparing silk fibroin peptide by hydrolyzing fibroin with serrapeptase |
| 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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- 2002-07-23 CN CN 02138016 patent/CN1233841C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| CN1392265A (en) | 2003-01-22 |
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