CN107937460A - A kind of preparation method for hydrolyzing sericin peptide taken - Google Patents

A kind of preparation method for hydrolyzing sericin peptide taken Download PDF

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CN107937460A
CN107937460A CN201610898768.4A CN201610898768A CN107937460A CN 107937460 A CN107937460 A CN 107937460A CN 201610898768 A CN201610898768 A CN 201610898768A CN 107937460 A CN107937460 A CN 107937460A
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CN107937460B (en
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孙益华
刘志军
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Yibin Pingshan Pfizer Oil Co Ltd
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

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Abstract

A kind of preparation method for hydrolyzing sericin peptide taken, belongs to separation and purification of protein technical field.The present invention is using tussah cocoon shell or useless mulberry silk as raw material, silkworm cocoon is cleaned by preparing, silk gum solution is prepared, prepares hydrolysis silk gum peptide solution, assembling activation macroreticular resin decoloration system, prepare hydrolysis sericin peptide taken destainer, assembling activation MCI GEL resin systems, prepare hydrolysis sericin peptide taken classification separating liquid, prepare hydrolysis sericin peptide taken classification separation concentrated solution, the step of preparing the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation and preparing hydrolysis silk gum peptide freeze-dried powder, finally prepares hydrolysis silk gum peptide freeze-dried powder.The present invention uses the modern chemical industry technology and equipments such as a variety of enzyme united hydrolysis, ultrafiltration, nanofiltration separation, macroreticular resin decoloration, the separation of MCI GEL fractionation of resins, low temperature concentration and freeze-drying, with easy to operate, mild condition, it is efficient, save the energy, the features such as production cost is low, the product prepared using the present invention can be widely used for the fields such as trophism daily chemical product and fibre modification.

Description

A kind of preparation method for hydrolyzing sericin peptide taken
First, technical field
The invention belongs to separation and purification of protein technical field, and in particular to a kind of preparation method for hydrolyzing sericin peptide taken.
2nd, background technology
Silk is mainly made of 20~30% silk gum and 70~80% fibroin, is the raw material for manufacturing silk.In addition, its Component silk gum and fibroin are also applied to the industries such as medical treatment, health care, daily use chemicals, weaving, food, building materials, play its blood coagulation, synthetism, guarantor Water, whitening, antibacterial, uvioresistant, packaging and coating etc. act on, and application field and dosage expand year by year, are that a kind of market is dived The huge natural resources of power, are worth utilization.
At present, silk silk gum, fibroin and its deep processed product are to be prepared using useless silk or silkworm cocoon as raw material.It is existing The method for preparing silk sericin peptide taken, such as the Master's thesis that meter Rui disclosed in 2015 writes《The preparation of tussah silk peptide and hypoglycemic are made With research》, the technique which prepares tussah silk peptide is：It is 14.67mol/L in phosphoric acid concentration, solid-liquid ratio 1: 10, 8h is reacted under conditions of 90 DEG C, the rate of being recycled is up to 75% tussah silk peptide.The defects of this method is：Use high concentration Phosphoric acid, price are high；Neutralising phosphoric acid then needs a large amount of calcium salts etc., generates a large amount of low value-added phosphate and is difficult to separation completely, Seriously affect product quality；The tussah silk peptide of preparation is big without hydrolysis, molecular weight, it is difficult to stablizes and preserves.In another example application number For the patent of invention of CN200810162579.6, Publication No. CN101544685, disclosure of the invention different molecular weight is water-soluble The preparation method of silk gum, is respectively that (1) carries out degumming in the distilled water boiled；(2) degumming liquid is placed in 80 DEG C~100 DEG C heat 0~12h is handled in water；(3) the silk gum liquid after hot water treatment is freeze-dried.The defects of invention is that degumming is extremely inefficient, this Outside, do not carry out classification separation, acquisition be different molecular weight silk gum mixture, due to molecular weight heterogeneity, it is difficult to specific Applied under environment and obtain good result.
3rd, the content of the invention
The purpose of the present invention is for the existing deficiency for preparing hydrolysis sericin peptide taken method, there is provided a kind of system for hydrolyzing sericin peptide taken Preparation Method.This method makes full use of silk gum and fibroin, and the classification separation of hydrolysis sericin peptide taken can be realized according to molecular weight, is prepared not With the freeze-dried powder of the hydrolysis sericin peptide taken of molecular weight, make it to be preferably applied for the different fields such as daily use chemicals, realize ethanol and take off The reuse and comprehensive utilization of color liquid.
Realizing the principle of the object of the invention is：Silk is mainly made of silk gum and fibroin albumen, and silk gum is distributed in fibroin Around, sericin is dissolved in alkaline solution, therefore, at a certain temperature can dissolve silk gum with dilute sodium carbonate, then just obtain The sodium carbonate liquor of silk gum must be dissolved with and slough the fibroin albumen of silk gum.Protease is the life of special aminosal amido link Thing catalyst, silk gum are the protein being made up of 18 kinds of amino acid amido link, and under suitable condition, protease may be selected Property hydrolysis form the amido link of silk gum, the different hydrolysis sericin peptide taken of generation molecular weight.Colors in silk are mainly Huang Ketone, flavone compound have the structure of 2- phenyl chromones, can be with the nonpolar macroporous suction rolled into a ball using phenyl as adsorption function Attached resin forms hydrophobic interaction；The silk gum peptide solution of protease hydrolytic is passed through into nonpolarity macroporous adsorptive resins chromatographic column, hydrolysis Flavonoids in liquid just form the hydrophobic interaction based on phenyl ring with macropore nonpolar adsorption resin, are then achieved that The decoloration of hydrolyzate.MCI GEL resins be one kind using polystyrene or polyacrylic acid as skeleton, based on molecular sieve principle separate it is molten The separating medium of matter, has and high-purity separation, and repeatedly used feature is realized under the conditions of middle pressure and in organic media； By the hydrolysis silk gum peptide solution of decoloration by MCI GEL resins, and eluted and fraction collection, just obtain different molecular weight point The hydrolysis sericin peptide taken classification separating liquid of cloth.Ultrafiltration and nanofiltration are that making choice property of material is separated by the microcellular structure of film surface Membrane separation technique, when solution flows through film surface under a certain pressure, small molecule solute passes through film, and macromolecular solute is then cut Stay, then macromolecular concentration gradually steps up in trapped fluid, so as to fulfill the separation and concentration of large and small molecule；MCI GEL will be passed through The hydrolysis silk gum peptide solution of the separated different molecular weight distribution of fractionation of resins is by ultrafiltration or NF membrane, less than ultrafiltration or NF membrane The solute filtration of diameter, the solute more than its diameter are trapped, and then just obtain corresponding concentrate.The concentrate of acquisition is used Freeze drier is dried, and just obtains the freeze-dried powder of hydrolysis sericin peptide taken.
The object of the present invention is achieved like this：A kind of preparation method for hydrolyzing sericin peptide taken, with tussah cocoon shell or useless silkworm Silk is raw material, cleans silkworm cocoon by preparing, prepares silk gum solution, prepares hydrolysis silk gum peptide solution, assembling activation macroreticular resin Decoloration system, prepares hydrolysis sericin peptide taken destainer, assembling activation MCI GEL resin systems, prepare hydrolysis sericin peptide taken classification separation Liquid, prepares hydrolysis sericin peptide taken classification separation concentrated solution, and preparation hydrolyzes the de- ethanol concentrate of sericin peptide taken classification separation and hydrolyzed with preparation The step of silk gum peptide freeze-dried powder, finally prepare hydrolysis silk gum peptide freeze-dried powder.It is comprised the following steps that：
(1) prepare and clean silkworm cocoon
Tree branches and leaves, silkworm chrysalis, the debris such as stone on silkworm cocoon are manually removed first, according still further to silkworm cocoon quality and quality Percentage concentration is the ratio that the ratio between 0.2~0.5% volume of aqueous sodium carbonate (kg/L) is 1: 20~40, this is manually removed Miscellaneous silkworm cocoon is scattered in the aqueous sodium carbonate that mass percentage concentration is 0.2~0.5%, the stir process 30 at 60~80 DEG C ~40min.Filtering, collected filter residue and filtered fluid respectively.To the filtered fluid of collection, it is pumped into wastewater disposal basin and carries out biochemical treatment, reach Discharged after mark；Filter residue is crossed to collection, that is, prepares clean silkworm cocoon, running sol solution is prepared for lower step.
(2) silk gum solution is prepared
It is first 1: 20~200 according to the ratio between natrium carbonicum calcinatum quality and pure water volume (kg/L) after the completion of (1) step Ratio, natrium carbonicum calcinatum is dissolved in pure water, makes the aqueous sodium carbonate that mass percentage concentration is 0.5~5%；Again It is that the ratio between 0.5~5% volume of aqueous sodium carbonate (kg/L) is 1 according to clean silkworm cocoon quality and mass percentage concentration: 60~120 ratio, clean silkworm cocoon is scattered in the aqueous sodium carbonate that mass percentage concentration is 0.5~5%, under stirring Flow back 3~6h of sericin removal in 90~100 DEG C, filtering, collects sericin removal respectively and crosses filter residue and filtered fluid.To the sericin removal mistake of collection Filter residue, adds the pure water for cleaning 20~30 times of silkworm cocoon quality, stirs 15~30min of lower washing, filters, collect respectively again Washed filter residue and washed filtrate, to the washed filtrate of collection, merge with the sericin removal filtered fluid of collection, are that silk gum is molten Liquid, hydrolysis silk gum peptide solution is prepared for lower step；Washing to collection filters slag, that is, prepares sericin removal fibroin albumen, be used for Prepare hydrolysis silk peptide.
(3) hydrolysis silk gum peptide solution is prepared
After the completion of (2) step, it is 3500~10000Da that the silk gum solution that (2) step is prepared is pumped into molecular cut off Ultrafilter in, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.1~0.4MPa, until ultra-filter retentate volume and ultrafiltration are filtered Cross when the ratio between liquid product (L/L) is 1: 3~6 and stop.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, and the ultrafiltration to collection filters Liquid, containing sodium carbonate, silkworm cocoon is washed for lower batch；To the ultra-filter retentate of collection, hydrolysis kettle is pumped into, first in 60~120r/ 50~60 DEG C are warming up under the mixing speed of min, then it is 8.0~9.5 to adjust pH with dilute sodium hydroxide, believes alkali then according to Novi Property protease 3 .0T or ratio that animal proteolytic enzyme and clean silkworm cocoon mass ratio (kg/kg) are 1: 100~200, add Enter Novi letter alkali protease 3.0T or animal proteolytic enzyme, in the mixing speed of 60~120r/min and 50~60 DEG C of temperature 1~2h of the lower constant temperature stirring hydrolysis of degree；Then flavor protease or papain and clean silkworm cocoon quality are believed according still further to Novi The ratio between (kg/kg) be 1: 80~160 ratio, Novi's letter flavor protease or papain is added, in 60~120r/min Mixing speed and 50~60 DEG C at a temperature of constant temperature stirring hydrolysis 1~2h.Hydrolysis silk gum peptide solution is just prepared, is used to prepare Hydrolyze sericin peptide taken destainer.
(4) assembling activation macroreticular resin decoloration system
By activation AmberliteTMXAD7HP or AmberliteTMXAD761 or the HP20 resin dispersion newly purchased in 3~ In the pure water of 5 times of resin volumes, it is pumped into peristaltic pump in medium pressure chromatography column, tapping is opened after resin settles into resin column Valve, releases excessive moisture, until stopping tapping when liquid level is higher than 2~10 centimetres of resin column upper surface, just assembles activation AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns.Finally by the resin column respectively with peristaltic pump, UV detector and collector connection, just assemble activation macroreticular resin decoloration system, and preparing hydrolysis sericin peptide taken for lower step takes off Color liquid.
(5) hydrolysis sericin peptide taken destainer is prepared
After the completion of (4) step, by the hydrolysis silk gum peptide solution that (3) step is prepared be pumped into molecular cut off for 10000~ In the ultrafilter of 30000Da, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.1~0.4MPa, until ultra-filter retentate volume Stop when with the ratio between ultrafiltration filtered solution volume (L/L) being 1: 6~9.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to collection Ultra-filter retentate, is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard；To the ultrafiltration filtered solution of collection, activation is pumped into AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, in the stream of 2~4 times of resin column volume/hours Fast lower progress decolorization, activates AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns volume with being somebody's turn to do The ratio between ultrafiltration filtered solution volume (L/L) is 1: 20~40.Column efflux and adsorpting pigment were collected respectively AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, cross column efflux to collection, that is, prepare Sericin peptide taken destainer is hydrolyzed, is used to prepare hydrolysis sericin peptide taken classification separating liquid；To the adsorpting pigment of collection AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, be pumped into ethanol concentration expressed in percentage by volume for 65~ 85% aqueous solution, elution processing is carried out under the flow velocity of 2~4 times of resin column volume/hours, adsorpting pigment AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns and ethanol concentration expressed in percentage by volume are 65~85% The ratio between aqueous solution volume (L/L) be 1: 2~4.After the completion of elution, collect respectively and strip column liquid and unload pigment AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns.To the unloading pigment of collection AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, with the pure water of 0.5~1 times of resin column volume Washing, collected column cleaning solution and washed AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resins Column.Column cleaning solution is crossed to collection, containing ethanol, for preparing the ethanol eluate of lower batch elution pigment；To the warp of collection AmberliteTMXAD7HP or AmberliteTMXAD761 or the HP20 resin column of washing, it is namely regenerated AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, available for lower batch adsorpting pigment.To collecting Strip column liquid, be pumped into rotary evaporator, recycle ethanol, stop when no ethanol flavor.The ethanol water of recycling is collected respectively Solution and the concentrate for sloughing ethanol, can be re-used for lower batch to the ethanol water of the recycling of collection, after allotment concentration and wash Depigmentation；To the concentrate for sloughing ethanol of collection, it is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard.
(6) assembling activation MCI GEL resin systems
By the MCI GEL CHP20P resins that the particle diameter newly purchased is 37~75 μm or the MCI that particle diameter is 63~150 μm The MCI GEL CHP 2MG Y resin dispersions that GEL CHP 20SS resins or particle diameter are 25~35 μm are in 3~5 times of resin volumes Ethanol concentration expressed in percentage by volume is in 30~60% aqueous solution, is pumped into peristaltic pump in medium pressure chromatography column, treats that resin settles into tree Open bleeder valve after fat column, release excessive moisture, until stopping tapping when liquid level is higher than 0.5~2 centimetre of resin column upper surface, just Assemble activation MCI GEL CHP20P or MCI GEL CHP 20SS or MCI GEL CHP 2MG Y resin columns.Finally should Resin column is connected with peristaltic pump, UV detector and collector respectively, activation MCI GEL resin systems is just assembled, under being used for Step prepares hydrolysis sericin peptide taken classification separating liquid.
(7) hydrolysis sericin peptide taken classification separating liquid is prepared
After the completion of (6) step, by the hydrolysis sericin peptide taken destainer of (5) step collection, activation MCI GEL CHP20P are pumped into Or MCI GEL CHP 20SS or MCI GEL CHP 2MG Y resin columns, hydrolysis sericin peptide taken destainer and the activated resin cylinder The ratio between product (L/L) is 1: 10~20, hydrolysis sericin peptide taken destainer be pumped into flow velocity be 1~3 times of the activated resin column volume/it is small When.It is pumped into after the completion of hydrolysis sericin peptide taken destainer, collects the resin column of load decoloration hydrolysis sericin peptide taken respectively and cross column efflux, Column efflux is crossed to collection, containing the ethanol that concentration expressed in percentage by volume is 30~60%, rotary evaporator is pumped into, recycles ethanol； To the resin column of the load decoloration hydrolysis sericin peptide taken of collection, then the aqueous solution that ethanol concentration expressed in percentage by volume is 30~60% is pumped into, The flow velocity for being pumped into the aqueous solution is 1~3 times/hour of the resin column volume.14~21min, 26~37min are collected respectively It is right with the resin column for crossing column efflux and unloading decoloration hydrolysis sericin peptide taken for having strong absworption peak in 280nm of 42~51min 14~21min, the 26~37min and the column efflux excessively for having strong absworption peak in 280nm of 42~51min collected, Hydrolysis sericin peptide taken classification separating liquid is prepared, hydrolysis sericin peptide taken classification separation concentrated solution is prepared for lower step；Collection is unloaded Carry the resin column of decoloration hydrolysis sericin peptide taken, namely regenerated MCI GEL CHP20P or MCI GEL CHP 20SS or MCI GEL CHP 2MG Y resin columns, can be re-used for preparing hydrolysis sericin peptide taken classification separating liquid.
(8) hydrolysis sericin peptide taken classification separation concentrated solution is prepared
After the completion of (7) step, the hydrolysis sericin peptide taken classification separating liquid of (7) step collection is pumped into molecular cut off respectively is In nanofiltration/ultrafilter of 600~3500Da, nanofiltration/hyperfiltration treatment is carried out in the case where gauge pressure is 1.0~9.0MPa, until nanofiltration/super Stop when total nitrogen content reaches 3~4.5% in filter trapped fluid.Nanofiltration/ultrafiltration filtered solution and nanofiltration/ultra-filter retentate are collected respectively, To nanofiltration/ultrafiltration filtered solution of collection, containing the ethanol that concentration expressed in percentage by volume is 30~60%, it is pumped into rotary evaporator, recycles Ethanol；To nanofiltration/ultra-filter retentate of collection, respectively with sterilized 0.22 μm of filtering with microporous membrane, filtered solution is collected, i.e., Hydrolysis sericin peptide taken classification separation concentrated solution is prepared, is respectively used to prepare the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation.
(9) the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation is prepared
After the completion of (8) step, it is molten that the hydrolysis sericin peptide taken classification separation concentrated solution of (8) step preparation is pumped into low-temperature high-vacuum Agent retracting device, recycles ethanol, when no ethanol flavor under conditions of temperature is 30~45 DEG C, vacuum is 0.1~3kPa Only.Ethanol is collected respectively and sloughs the hydrolysis sericin peptide taken classification separation concentrated solution of ethanol, to the ethanol of collection, available for preparing second Alcohol concentration expressed in percentage by volume is 30~60% aqueous solution；Separation concentrated solution is classified to the hydrolysis sericin peptide taken for sloughing ethanol of collection, The de- ethanol concentrate of hydrolysis sericin peptide taken classification separation is prepared, hydrolysis silk gum peptide freeze-dried powder is prepared for lower step.
(10) hydrolysis silk gum peptide freeze-dried powder is prepared
After the completion of (9) step, the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation prepared by (9) step is first put respectively 10~20h of pre-freeze in -40~-30 DEG C of low temperature refrigerator, then be transferred in freeze drier, it is -50~-40 DEG C, table in temperature Press to carry out 24~36h of freeze-drying under conditions of 20~50Pa, just prepare average molecular weight be respectively 300~450Da, 1000~1350Da and three kinds of hydrolysis silk gum peptide freeze-dried powders in 3200~3600Da sections.Three kinds of freeze-dried powders are white, average The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that molecular weight is 300~450Da is 15~15.3%, and recovery rate is silkworm cocoon quality 11~13%；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 1000~1350Da is 15~15.7%, is received Rate is the 8~10% of silkworm cocoon quality；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 3200~3600Da is 15~15.5%, recovery rate is the 6~8% of silkworm cocoon quality；Total recovery rate of three kinds of hydrolysis silk gum peptide freeze-dried powders is silkworm cocoon The 25~31% of quality.
Present invention employs mainly generate following effect after above-mentioned technological means：
1st, the method for the present invention is taken off using a variety of enzyme united hydrolysis, ultrafiltration, nanofiltration separation, macroreticular resin in process of production The modern chemical industry technology and equipments such as color, the separation of MCI GEL fractionation of resins, low temperature concentration and freeze-drying, have easy to operate, bar The features such as part is gentle, efficient, saves the energy, and production cost is low；
2nd, prepare three kinds of hydrolysis silk gum peptide freeze-dried powders prepared by the present invention.It is white, average molecular weight for 300~ The hydrolysis silk gum peptide freeze-dried powder total nitrogen content of 450Da is 15~15.3%, and recovery rate is the 11~13% of silkworm cocoon quality；It is average The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that molecular weight is 1000~1350Da is 15~15.7%, and recovery rate is silkworm cocoon quality 8~10%；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 3200~3600Da is 15~15.5%, is received Rate is the 6~8% of silkworm cocoon quality；Total recovery rate of three kinds of hydrolysis silk gum peptide freeze-dried powders is the 25~31% of silkworm cocoon quality. It may be respectively used in the fields such as trophism daily chemical product and the fibre modification that skin or hair directly absorb.
3rd, the present invention is with AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 absorption resin substitutional ions Coloring matter in exchanger resin decoloration hydrolysis sericin peptide taken, have separative efficiency it is high, suitable for acid-base solution, can Reusability etc. Advantage；Classification point is carried out to the peptide matters in different molecular weight section in hydrolysis sericin peptide taken destainer with MCI GEL resin technologies From substituting conventional reduced vacuum with Nanofiltration-membrane technique and concentrate, ethanol is recycled with low-temperature high-vacuum solvent recovering system, have point From accurate, product molecular weight distribution is homogeneous, no brown stain, colourless, saves the advantages such as energy.
4th, the waste water produced in production process of the present invention, has carried out biochemical treatment, has discharged after up to standard, produced without " three wastes " It is raw, it is typical green production process, it is easy to utilize.
4th, embodiment
With reference to embodiment, the present invention is further illustrated.
Embodiment 1
(1) prepare and clean silkworm cocoon
Tree branches and leaves, silkworm chrysalis, the debris such as stone on silkworm cocoon are manually removed first, according still further to silkworm cocoon quality and quality Percentage concentration is the ratio that the ratio between 0.2% volume of aqueous sodium carbonate (kg/L) is 1: 20, by the artificial removal of impurities silkworm cocoon It is scattered in the aqueous sodium carbonate that mass percentage concentration is 0.2%, the stir process 30min at 60 DEG C.Filtering, is collected respectively Cross filter residue and filtered fluid.To the filtered fluid of collection, it is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard；Filtering to collection Slag, that is, prepare clean silkworm cocoon, and running sol solution is prepared for lower step.
(2) silk gum solution is prepared
After the completion of (1) step, first according to the ratio between natrium carbonicum calcinatum quality and pure water volume (kg/L) for 1: 20 ratio, Natrium carbonicum calcinatum is dissolved in pure water, makes the aqueous sodium carbonate that mass percentage concentration is 5%；According still further to clean silkworm The ratio that the ratio between cocoon chitin amount and the volume of aqueous sodium carbonate that mass percentage concentration is 5% (kg/L) are 1: 60, will clean Silkworm cocoon is scattered in the aqueous sodium carbonate that mass percentage concentration is 5%, and flow back sericin removal 3h under stirring in 90 DEG C, filtering, Sericin removal is collected respectively crosses filter residue and filtered fluid.Filter residue is crossed to the sericin removal of collection, adds and cleans the pure of 20 times of silkworm cocoon quality Water purification, stirs lower washing 15min, filters again, collect washed filter residue and washed filtrate respectively, to the washing and filtering of collection Liquid, merges with the sericin removal filtered fluid of collection, is silk gum solution, and hydrolysis silk gum peptide solution is prepared for lower step；To collection Washing filtration slag, that is, prepare sericin removal fibroin albumen, be used to prepare hydrolysis silk peptide.
(3) hydrolysis silk gum peptide solution is prepared
After the completion of (2) step, the silk gum solution that (2) step is prepared is pumped into the ultrafilter that molecular cut off is 3500Da In, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.1MPa, until the ratio between ultra-filter retentate volume and ultrafiltration filtered solution volume (L/L) be 1: 3 when stop.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, and to the ultrafiltration filtered solution of collection, containing sodium carbonate, is used Silkworm cocoon is washed in lower batch；To the ultra-filter retentate of collection, hydrolysis kettle is pumped into, is first heated up under the mixing speed of 60r/min To 50 DEG C, then it is 8.0 to adjust pH with dilute sodium hydroxide, then according to Novi letter alkali protease 3.0T and clean silkworm cocoon quality The ratio between (kg/kg) be 1: 100 ratio, Novi letter alkali protease 3.0T is added, in the mixing speed of 60r/min and 50 DEG C At a temperature of constant temperature stirring hydrolysis 1h；Then it is 1 according still further to Novi's letter flavor protease and clean silkworm cocoon mass ratio (kg/kg) : 80 ratio, adds Novi's letter flavor protease, the constant temperature stirring hydrolysis at a temperature of the mixing speed of 60r/min and 50 DEG C 1h.Hydrolysis silk gum peptide solution is just prepared, is used to prepare hydrolysis sericin peptide taken destainer.
(4) assembling activation macroreticular resin decoloration system
By the activation AmberliteTMXAD7HP resin dispersions newly purchased in the pure water of 3 times of resin volumes, with wriggling It is pumped into medium pressure chromatography column, bleeder valve is opened after resin settles into resin column, release excessive moisture, until liquid level is higher than tree Stop tapping during 2 centimetres of fat column upper surface, just assemble activation AmberliteTMXAD7HP resin columns.Finally by the resin column It is connected respectively with peristaltic pump, UV detector and collector, just assembles activation macroreticular resin decoloration system, prepared for lower step Hydrolyze sericin peptide taken destainer.
(5) hydrolysis sericin peptide taken destainer is prepared
After the completion of (4) step, it is 10000Da that the hydrolysis silk gum peptide solution that (3) step is prepared is pumped into molecular cut off Ultrafilter in, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.1MPa, until ultra-filter retentate volume and ultrafiltration filtered solution The ratio between volume (L/L) is stopped when being 1: 6.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to the ultra-filter retentate of collection, are pumped into Wastewater disposal basin carries out biochemical treatment, is discharged after up to standard；To the ultrafiltration filtered solution of collection, activation AmberliteTMXAD7HP trees are pumped into Fat column, carries out decolorization under the flow velocity of 2 times of resin column volume/hours, activates AmberliteTMXAD7HP resin column volumes It is 1: 20 with the ratio between the ultrafiltration filtered solution volume (L/L).Column efflux and adsorpting pigment were collected respectively AmberliteTMXAD7HP resin columns, cross column efflux to collection, that is, prepare hydrolysis sericin peptide taken destainer, be used to prepare Hydrolyze sericin peptide taken classification separating liquid；To the AmberliteTMXAD7HP resin columns of the adsorpting pigment of collection, ethanol volume hundred is pumped into The aqueous solution for dividing concentration to be 65%, elution processing is carried out under the flow velocity of 2 times of resin column volume/hours, adsorpting pigment AmberliteTMXAD7HP resin columns and ethanol concentration expressed in percentage by volume are that the ratio between 65% aqueous solution volume (L/L) is 1: 2.Wash After the completion of de-, the AmberliteTMXAD7HP resin columns for stripping column liquid and unloading pigment are collected respectively.To the unloading color of collection The AmberliteTMXAD7HP resin columns of element, with the pure water washing of 0.5 times of resin column volume, collected column cleaning solution and warp The AmberliteTMXAD7HP resin columns of washing.Column cleaning solution is crossed to collection, containing ethanol, for preparing lower batch elution The ethanol eluate of pigment；It is namely regenerated to the washed AmberliteTMXAD7HP resin columns of collection AmberliteTMXAD7HP resin columns, available for lower batch adsorpting pigment.Column liquid is stripped to collection, is pumped into rotary evaporation In device, ethanol is recycled, is stopped when no ethanol flavor.The ethanol water of recycling is collected respectively and sloughs the concentrate of ethanol, it is right The ethanol water of the recycling of collection, lower batch elution pigment can be re-used for after allocating concentration；To the ethanol sloughed of collection Concentrate, is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard.
(6) assembling activation MCI GEL resin systems
It is 37~75 μm of MCI GEL CHP20P resin dispersions in the ethanol body of 3 times of resin volumes by the particle diameter newly purchased Product percentage concentration is in 30% aqueous solution, is pumped into medium pressure chromatography column with peristaltic pump, is opened after resin settles into resin column Bleeder valve, releases excessive moisture, until stopping tapping when liquid level is higher than 0.5 centimetre of resin column upper surface, just assembles activation MCI GEL CHP20P resin columns.Finally the resin column is connected with peristaltic pump, UV detector and collector respectively, just assembles work Change MCI GEL resin systems, hydrolysis sericin peptide taken classification separating liquid is prepared for lower step.
(7) hydrolysis sericin peptide taken classification separating liquid is prepared
After the completion of (6) step, by the hydrolysis sericin peptide taken destainer of (5) step collection, activation MCI GEL CHP20P are pumped into Resin column, hydrolysis the ratio between sericin peptide taken destainer and the activated resin column volume (L/L) are 1: 10, hydrolyze the pump of sericin peptide taken destainer Enter 1 times/hour that flow velocity is the activated resin column volume.It is pumped into after the completion of hydrolysis sericin peptide taken destainer, collects load respectively and take off Color hydrolyzes the resin column of sericin peptide taken and crosses column efflux, crosses column efflux to collection, is 30% containing concentration expressed in percentage by volume Ethanol, is pumped into rotary evaporator, recycles ethanol；To the resin column of the load decoloration hydrolysis sericin peptide taken of collection, then it is pumped into ethanol body The aqueous solution that product percentage concentration is 30%, the flow velocity for being pumped into the aqueous solution is 1 times/hour of the resin column volume.Collect respectively 14~21min, 26~37min and 42~51min 280nm have strong absworption peak cross column efflux and unloading is de- Color hydrolyzes the resin column of sericin peptide taken, to 14~21min, the 26~37min of collection and having in 280nm for 42~51min Strong absworption peak crosses column efflux, that is, prepares hydrolysis sericin peptide taken classification separating liquid, hydrolysis sericin peptide taken point is prepared for lower step Level separation concentrated solution；To collection unloading decoloration hydrolysis sericin peptide taken resin column, namely regenerated MCI GEL CHP20P resin columns, It can be re-used for preparing hydrolysis sericin peptide taken classification separating liquid.
(8) hydrolysis sericin peptide taken classification separation concentrated solution is prepared
After the completion of (7) step, the hydrolysis sericin peptide taken classification separating liquid of (7) step collection is pumped into molecular cut off respectively is In the nanofiltration device of 600Da, nanofiltration processing is carried out in the case where gauge pressure is 1.0MPa, until total nitrogen content reaches 3% in nanofiltration retentate fluid When stop.Nanofiltration filtered solution and nanofiltration retentate fluid are collected respectively, are 30% containing concentration expressed in percentage by volume to the nanofiltration filtered solution of collection Ethanol, is pumped into rotary evaporator, recycles ethanol；To the nanofiltration retentate fluid of collection, respectively with sterilized 0.22 μm of micropore Membrane filtration, collects filtered solution, that is, prepares hydrolysis sericin peptide taken classification separation concentrated solution, be respectively used to prepare hydrolysis sericin peptide taken point The de- ethanol concentrate of level separation.
(9) the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation is prepared
After the completion of (8) step, it is molten that the hydrolysis sericin peptide taken classification separation concentrated solution of (8) step preparation is pumped into low-temperature high-vacuum Agent retracting device, recycles ethanol under conditions of temperature is 30 DEG C, vacuum is 0.1kPa, stops when no ethanol flavor.Respectively Collect ethanol and slough the hydrolysis sericin peptide taken classification separation concentrated solution of ethanol, to the ethanol of collection, available for preparation ethanol volume Percentage concentration is 30% aqueous solution；Separation concentrated solution is classified to the hydrolysis sericin peptide taken for sloughing ethanol of collection, that is, prepares water outlet The de- ethanol concentrate of sericin peptide taken classification separation is solved, hydrolysis silk gum peptide freeze-dried powder is prepared for lower step.
(10) hydrolysis silk gum peptide freeze-dried powder is prepared
After the completion of (9) step, the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation prepared by (9) step is first put respectively The pre-freeze 10h in -40 DEG C of low temperature refrigerator, then be transferred in freeze drier, in the condition that temperature is -50 DEG C, gauge pressure is 20Pa Under carry out freeze-drying 24h, just prepare average molecular weight be respectively 300~450Da, 1000~1350Da and 3200~ Three kinds of hydrolysis silk gum peptide freeze-dried powders in 3600Da sections.Three kinds of freeze-dried powders are white, and average molecular weight is 300~450Da's It is 15~15.3% to hydrolyze silk gum peptide freeze-dried powder total nitrogen content, and recovery rate is the 11~13% of silkworm cocoon quality；Average molecular weight Be 15~15.7% for the hydrolysis silk gum peptide freeze-dried powder total nitrogen content of 1000~1350Da, recovery rate for silkworm cocoon quality 8~ 10%；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 3200~3600Da is 15~15.5%, and recovery rate is The 6~8% of silkworm cocoon quality；Total recovery rate of three kinds of hydrolysis silk gum peptide freeze-dried powders is the 25~31% of silkworm cocoon quality.
Embodiment 2
(1) prepare and clean silkworm cocoon
Tree branches and leaves, silkworm chrysalis, the debris such as stone on silkworm cocoon are manually removed first, according still further to silkworm cocoon quality and quality Percentage concentration is the ratio that the ratio between 0.4% volume of aqueous sodium carbonate (kg/L) is 1: 30, by the artificial removal of impurities silkworm cocoon It is scattered in the aqueous sodium carbonate that mass percentage concentration is 0.4%, the stir process 35min at 70 DEG C.Filtering, is collected respectively Cross filter residue and filtered fluid.To the filtered fluid of collection, it is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard；Filtering to collection Slag, that is, prepare clean silkworm cocoon, and running sol solution is prepared for lower step.
(2) silk gum solution is prepared
After the completion of (1) step, first according to the ratio that the ratio between natrium carbonicum calcinatum quality and pure water volume (kg/L) are 1: 100 Example, natrium carbonicum calcinatum is dissolved in pure water, makes the aqueous sodium carbonate that mass percentage concentration is 1%；According still further to washing The ratio that the ratio between net silkworm cocoon quality and the volume of aqueous sodium carbonate that mass percentage concentration is 1% (kg/L) is 1: 90, general Clean silkworm cocoon is scattered in the aqueous sodium carbonate that mass percentage concentration is 1%, and flow back sericin removal 4.5h under stirring in 95 DEG C, Filtering, collects sericin removal and crosses filter residue and filtered fluid respectively.Filter residue is crossed to the sericin removal of collection, adds and cleans 25 times of silkworm cocoon quality Pure water, stir lower washing 22min, filter again, collect washed filter residue and washed filtrate respectively, the washing to collection Filtered fluid, merges with the sericin removal filtered fluid of collection, is silk gum solution, and hydrolysis silk gum peptide solution is prepared for lower step；To receiving The washing filtration slag of collection, that is, prepare sericin removal fibroin albumen, be used to prepare hydrolysis silk peptide.
(3) hydrolysis silk gum peptide solution is prepared
After the completion of (2) step, the silk gum solution that (2) step is prepared is pumped into the ultrafilter that molecular cut off is 8000Da In, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.25MPa, until the ratio between ultra-filter retentate volume and ultrafiltration filtered solution volume (L/L) be 1: 4.5 when stop.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to the ultrafiltration filtered solution of collection, containing sodium carbonate, Silkworm cocoon is washed for lower batch；To the ultra-filter retentate of collection, hydrolysis kettle is pumped into, is first risen under the mixing speed of 90r/min Temperature is to 55 DEG C, then it is 8.8 to adjust pH with dilute sodium hydroxide, then according to Novi's letter animal proteolytic enzyme and clean silk cocoon chitin The ratio between amount (kg/kg) is 1: 150 ratio, Novi's letter animal proteolytic enzyme is added, in the mixing speed of 90r/min and 55 DEG C At a temperature of constant temperature stirring hydrolysis 1.5h；Then according still further to Novi's letter papain and clean silkworm cocoon mass ratio (kg/ Kg) the ratio for being 1: 120, adds Novi's letter papain, constant temperature stirs at a temperature of the mixing speed of 90r/min and 55 DEG C Mix hydrolysis 1.5h.Hydrolysis silk gum peptide solution is just prepared, is used to prepare hydrolysis sericin peptide taken destainer.
(4) assembling activation macroreticular resin decoloration system
By the activation AmberliteTMXAD761 resin dispersions newly purchased in the pure water of 4 times of resin volumes, with wriggling It is pumped into medium pressure chromatography column, bleeder valve is opened after resin settles into resin column, release excessive moisture, until liquid level is higher than tree Stop tapping during 6 centimetres of fat column upper surface, just assemble activation AmberliteTMXAD761 resin columns.Finally by the resin column It is connected respectively with peristaltic pump, UV detector and collector, just assembles activation macroreticular resin decoloration system, prepared for lower step Hydrolyze sericin peptide taken destainer.
(5) hydrolysis sericin peptide taken destainer is prepared
After the completion of (4) step, it is 20000Da that the hydrolysis silk gum peptide solution that (3) step is prepared is pumped into molecular cut off Ultrafilter in, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.25MPa, until ultra-filter retentate volume and ultrafiltration filtered solution The ratio between volume (L/L) is stopped when being 1: 7.5.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to the ultra-filter retentate of collection, pump Enter wastewater disposal basin and carry out biochemical treatment, discharged after up to standard；To the ultrafiltration filtered solution of collection, activation AmberliteTMXAD761 is pumped into Resin column, carries out decolorization under the flow velocity of 3 times of resin column volume/hours, activates AmberliteTMXAD761 resin columns Product is 1: 30 with the ratio between the ultrafiltration filtered solution volume (L/L).Column efflux and adsorpting pigment were collected respectively AmberliteTMXAD761 resin columns, cross column efflux to collection, that is, prepare hydrolysis sericin peptide taken destainer, be used to prepare Hydrolyze sericin peptide taken classification separating liquid；To the AmberliteTMXAD761 resin columns of the adsorpting pigment of collection, ethanol volume hundred is pumped into The aqueous solution for dividing concentration to be 75%, elution processing is carried out under the flow velocity of 3 times of resin column volume/hours, adsorpting pigment AmberliteTMXAD761 resin columns and ethanol concentration expressed in percentage by volume are that the ratio between 75% aqueous solution volume (L/L) is 1: 3.Wash After the completion of de-, the AmberliteTMXAD761 resin columns for stripping column liquid and unloading pigment are collected respectively.To the unloading color of collection The AmberliteTMXAD761 resin columns of element, with the pure water washing of 0.75 times of resin column volume, collected column cleaning solution and warp The AmberliteTMXAD761 resin columns of washing.Column cleaning solution is crossed to collection, containing ethanol, for preparing lower batch elution The ethanol eluate of pigment；It is namely regenerated to the washed AmberliteTMXAD761 resin columns of collection AmberliteTMXAD761 resin columns, available for lower batch adsorpting pigment.Column liquid is stripped to collection, is pumped into rotary evaporation In device, ethanol is recycled, is stopped when no ethanol flavor.The ethanol water of recycling is collected respectively and sloughs the concentrate of ethanol, it is right The ethanol water of the recycling of collection, lower batch elution pigment can be re-used for after allocating concentration；To the ethanol sloughed of collection Concentrate, is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard.
(6) assembling activation MCI GEL resin systems
It is 63~150 μm of MCIGELCHP20SS resin dispersions in the ethanol body of 4 times of resin volumes by the particle diameter newly purchased Product percentage concentration is in 45% aqueous solution, is pumped into medium pressure chromatography column with peristaltic pump, is opened after resin settles into resin column Bleeder valve, releases excessive moisture, until stopping tapping when liquid level is higher than 1.2 centimetres of resin column upper surface, just assembles activation MCI GEL CHP 20SS resin columns.Finally the resin column is connected with peristaltic pump, UV detector and collector respectively, is just assembled MCI GEL resin systems are activated, hydrolysis sericin peptide taken classification separating liquid is prepared for lower step.
(7) hydrolysis sericin peptide taken classification separating liquid is prepared
After the completion of (6) step, by the hydrolysis sericin peptide taken destainer of (5) step collection, activation MCI GEL CHP are pumped into 20SS resin columns, hydrolysis the ratio between sericin peptide taken destainer and the activated resin column volume (L/L) are 1: 15, hydrolyze sericin peptide taken destainer Be pumped into flow velocity be the activated resin column volume 2 times/hour.It is pumped into after the completion of hydrolysis sericin peptide taken destainer, collects lotus respectively Carry the resin column of decoloration hydrolysis sericin peptide taken and cross column efflux, column efflux is crossed to collection, is containing concentration expressed in percentage by volume 45% ethanol, is pumped into rotary evaporator, recycles ethanol；To the resin column of the load decoloration hydrolysis sericin peptide taken of collection, then it is pumped into Ethanol concentration expressed in percentage by volume is 45% aqueous solution, and the flow velocity for being pumped into the aqueous solution is 2 times/hour of the resin column volume.Point Not Shou Ji 14~21min, 26~37min and 42~51min 280nm have strong absworption peak cross column efflux and The resin column of unloading decoloration hydrolysis sericin peptide taken, 14~21min, 26~37min and 42~51min to collection 280nm has the column efflux excessively of strong absworption peak, that is, prepares hydrolysis sericin peptide taken classification separating liquid, hydrolyzed-silk is prepared for lower step Glue peptide is classified separation concentrated solution；To the resin column of the unloading decoloration hydrolysis sericin peptide taken of collection, namely regenerated MCI GEL CHP 20SS resin columns, can be re-used for preparing hydrolysis sericin peptide taken classification separating liquid.
(8) hydrolysis sericin peptide taken classification separation concentrated solution is prepared
After the completion of (7) step, the hydrolysis sericin peptide taken classification separating liquid of (7) step collection is pumped into molecular cut off respectively is In the ultrafilter of 2500Da, hyperfiltration treatment is carried out in the case where gauge pressure is 5.0MPa, until total nitrogen content reaches in ultra-filter retentate Stop when 3.8%.Ultrafiltration filtered solution and ultra-filter retentate are collected respectively, to the ultrafiltration filtered solution of collection, are containing concentration expressed in percentage by volume 45% ethanol, is pumped into rotary evaporator, recycles ethanol；To the ultra-filter retentate of collection, respectively with sterilized 0.22 μm Filtering with microporous membrane, collect filtered solution, that is, prepare hydrolysis sericin peptide taken classification separation concentrated solution, be respectively used to prepare hydrolyzed-silk The de- ethanol concentrate of glue peptide classification separation.
(9) the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation is prepared
After the completion of (8) step, it is molten that the hydrolysis sericin peptide taken classification separation concentrated solution of (8) step preparation is pumped into low-temperature high-vacuum Agent retracting device, recycles ethanol under conditions of temperature is 38 DEG C, vacuum is 1.6kPa, stops when no ethanol flavor.Respectively Collect ethanol and slough the hydrolysis sericin peptide taken classification separation concentrated solution of ethanol, to the ethanol of collection, available for preparation ethanol volume Percentage concentration is 45% aqueous solution；Separation concentrated solution is classified to the hydrolysis sericin peptide taken for sloughing ethanol of collection, that is, prepares water outlet The de- ethanol concentrate of sericin peptide taken classification separation is solved, hydrolysis silk gum peptide freeze-dried powder is prepared for lower step.
(10) hydrolysis silk gum peptide freeze-dried powder is prepared
After the completion of (9) step, the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation prepared by (9) step is first put respectively The pre-freeze 15h in the low temperature refrigerator of -35C, then be transferred in freeze drier, under conditions of temperature is -45 DEG C, gauge pressure is 35Pa Freeze-drying 30h is carried out, it is respectively 300~450Da, 1000~1350Da and 3200~3600Da just to prepare average molecular weight Three kinds of hydrolysis silk gum peptide freeze-dried powders in section.Three kinds of freeze-dried powders are white, and average molecular weight is the hydrolyzed-silk of 300~450Da Glue peptide freeze-dried powder total nitrogen content is 15~15.3%, and recovery rate is the 11~13% of silkworm cocoon quality；Average molecular weight is 1000 The hydrolysis silk gum peptide freeze-dried powder total nitrogen content of~1350Da is 15~15.7%, and recovery rate is the 8~10% of silkworm cocoon quality；It is flat The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 3200~3600Da is 15~15.5%, and recovery rate is silk cocoon chitin The 6~8% of amount；Total recovery rate of three kinds of hydrolysis silk gum peptide freeze-dried powders is the 25~31% of silkworm cocoon quality.
Embodiment 3
(1) prepare and clean silkworm cocoon
Tree branches and leaves, silkworm chrysalis, the debris such as stone on silkworm cocoon are manually removed first, according still further to silkworm cocoon quality and quality Percentage concentration is the ratio that the ratio between 0.5% volume of aqueous sodium carbonate (kg/L) is 1: 40, by the artificial removal of impurities silkworm cocoon It is scattered in the aqueous sodium carbonate that mass percentage concentration is 0.5%, the stir process 40min at 80 DEG C.Filtering, is collected respectively Cross filter residue and filtered fluid.To the filtered fluid of collection, it is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard；Filtering to collection Slag, that is, prepare clean silkworm cocoon, and running sol solution is prepared for lower step.
(2) silk gum solution is prepared
After the completion of (1) step, first according to the ratio that the ratio between natrium carbonicum calcinatum quality and pure water volume (kg/L) are 1: 200 Example, natrium carbonicum calcinatum is dissolved in pure water, makes the aqueous sodium carbonate that mass percentage concentration is 0.5%；According still further to Clean silkworm cocoon quality and the ratio that mass percentage concentration is that the ratio between 0.5% volume of aqueous sodium carbonate (kg/L) is 1: 120 Example, clean silkworm cocoon is scattered in the aqueous sodium carbonate that mass percentage concentration is 0.5%, de- in 100 DEG C of reflux under stirring Silk gum 6h, filtering, collects sericin removal and crosses filter residue and filtered fluid respectively.Filter residue is crossed to the sericin removal of collection, adds and cleans silkworm cocoon The pure water that 30 times of quality, stirs lower washing 30min, filters again, collect washed filter residue and washed filtrate respectively, to receiving The washed filtrate of collection, merges with the sericin removal filtered fluid of collection, is silk gum solution, and it is molten to prepare hydrolysis sericin peptide taken for lower step Liquid；Washing to collection filters slag, that is, prepares sericin removal fibroin albumen, be used to prepare hydrolysis silk peptide.
(3) hydrolysis silk gum peptide solution is prepared
After the completion of (2) step, the silk gum solution that (2) step is prepared is pumped into the ultrafiltration that molecular cut off is 10000Da In device, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.4MPa, until ultra-filter retentate volume and ultrafiltration filtered solution volume it Than (L/L) be 1: 6 when stop.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to the ultrafiltration filtered solution of collection, containing sodium carbonate, Silkworm cocoon is washed for lower batch；To the ultra-filter retentate of collection, hydrolysis kettle is pumped into, is first risen under the mixing speed of 120r/min Temperature is to 60 DEG C, then it is 9.5 to adjust pH with dilute sodium hydroxide, then according to Novi letter alkali protease 3.0T and clean silk cocoon chitin The ratio between amount (kg/kg) is 1: 200 ratio, Novi letter alkali protease 3.0T is added, in the mixing speed of 120r/min and 60 Constant temperature stirring hydrolysis 2h at a temperature of DEG C；Then according still further to Novi's letter flavor protease and clean silkworm cocoon mass ratio (kg/ Kg) the ratio for being 1: 160, adds Novi's letter flavor protease, the constant temperature at a temperature of the mixing speed of 120r/min and 60 DEG C Stirring hydrolysis 2h.Hydrolysis silk gum peptide solution is just prepared, is used to prepare hydrolysis sericin peptide taken destainer.
(4) assembling activation macroreticular resin decoloration system
By the activation HP20 resin dispersions newly purchased in the pure water of 5 times of resin volumes, middle laminate layer is pumped into peristaltic pump Analyse in column, bleeder valve is opened after resin settles into resin column, release excessive moisture, until liquid level is higher than resin column upper surface 10 Centimetre when stop tapping, just assemble activation HP20 resin columns.Finally by the resin column respectively with peristaltic pump, UV detector and Collector connects, and just assembles activation macroreticular resin decoloration system, hydrolysis sericin peptide taken destainer is prepared for lower step.
(5) hydrolysis sericin peptide taken destainer is prepared
After the completion of (4) step, it is 30000Da that the hydrolysis silk gum peptide solution that (3) step is prepared is pumped into molecular cut off Ultrafilter in, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.4MPa, until ultra-filter retentate volume and ultrafiltration filtered solution The ratio between volume (L/L) is stopped when being 1: 9.Ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to the ultra-filter retentate of collection, are pumped into Wastewater disposal basin carries out biochemical treatment, is discharged after up to standard；To the ultrafiltration filtered solution of collection, activation HP20 resin columns are pumped into, in 4 times of resins Decolorization, activation the ratio between HP20 resin columns volume and the ultrafiltration filtered solution volume (L/L) are carried out under the flow velocity of column volume/hour For 1: 40.The HP20 resin columns of column efflux and adsorpting pigment were collected respectively, and column efflux is crossed to collection, that is, prepares water outlet Sericin peptide taken destainer is solved, is used to prepare hydrolysis sericin peptide taken classification separating liquid；To the HP20 resin columns of the adsorpting pigment of collection, it is pumped into Ethanol concentration expressed in percentage by volume is 85% aqueous solution, and elution processing is carried out under the flow velocity of 4 times of resin column volume/hours, is adsorbed The HP20 resin columns of pigment and ethanol concentration expressed in percentage by volume are that the ratio between 85% aqueous solution volume (L/L) is 1: 4.Elution is completed Afterwards, the HP20 resin columns for stripping column liquid and unloading pigment are collected respectively.To the HP20 resin columns of the unloading pigment of collection, with 1 The pure water washing of times resin column volume, collected column cleaning solution and washed HP20 resin columns.Column washing is crossed to collection Liquid, containing ethanol, for preparing the ethanol eluate of lower batch elution pigment；To the washed HP20 resin columns of collection, i.e., To regenerate HP20 resin columns, available for lower batch adsorpting pigment.Column liquid is stripped to collection, is pumped into rotary evaporator, is returned Ethanol is received, is stopped when no ethanol flavor.The ethanol water of recycling is collected respectively and sloughs the concentrate of ethanol, and collection is returned The ethanol water of receipts, lower batch elution pigment can be re-used for after allocating concentration；To the concentrate for sloughing ethanol of collection, pump Enter wastewater disposal basin and carry out biochemical treatment, discharged after up to standard.
(6) assembling activation MCI GEL resin systems
It is 25~35 μm of MCI GEL CHP 2MG Y resin dispersions in the second of 5 times of resin volumes by the particle diameter newly purchased Alcohol concentration expressed in percentage by volume is in 60% aqueous solution, is pumped into peristaltic pump in medium pressure chromatography column, after resin settles into resin column Bleeder valve is opened, releases excessive moisture, until stopping tapping when liquid level is higher than 2 centimetres of resin column upper surface, just assembles activation MCI GEL CHP2MG Y resin columns.Finally the resin column is connected with peristaltic pump, UV detector and collector respectively, is just filled Activation MCI GEL resin systems are allotted, hydrolysis sericin peptide taken classification separating liquid is prepared for lower step.
(7) hydrolysis sericin peptide taken classification separating liquid is prepared
After the completion of (6) step, by the hydrolysis sericin peptide taken destainer of (5) step collection, activation MCI GEL CHP 2MG are pumped into Y resin columns, hydrolysis the ratio between sericin peptide taken destainer and the activated resin column volume (L/L) are 1: 20, hydrolysis sericin peptide taken destainer It is pumped into 3 times/hour that flow velocity is the activated resin column volume.It is pumped into after the completion of hydrolysis sericin peptide taken destainer, collects load respectively Decoloration hydrolyzes the resin column of sericin peptide taken and crosses column efflux, crosses column efflux to collection, is 60% containing concentration expressed in percentage by volume Ethanol, be pumped into rotary evaporator, recycle ethanol；To the resin column of the load decoloration hydrolysis sericin peptide taken of collection, then it is pumped into ethanol Concentration expressed in percentage by volume is 60% aqueous solution, and the flow velocity for being pumped into the aqueous solution is 3 times/hour of the resin column volume.Receive respectively Collect column efflux and the unloading excessively that have strong absworption peak in 280nm of 14~21min, 26~37min and 42~51min The resin column of decoloration hydrolysis sericin peptide taken, 14~21min, 26~37min and 42~51min to collection in 280nm Have strong absworption peak crosses column efflux, that is, prepares hydrolysis sericin peptide taken classification separating liquid, hydrolysis sericin peptide taken is prepared for lower step It is classified separation concentrated solution；To the resin column of the unloading decoloration hydrolysis sericin peptide taken of collection, namely regenerated MCI GEL CHP 2MG Y trees Fat column, can be re-used for preparing hydrolysis sericin peptide taken classification separating liquid.
(8) hydrolysis sericin peptide taken classification separation concentrated solution is prepared
After the completion of (7) step, the hydrolysis sericin peptide taken classification separating liquid of (7) step collection is pumped into molecular cut off respectively is In the ultrafilter of 3500Da, hyperfiltration treatment is carried out in the case where gauge pressure is 9.0MPa, until total nitrogen content reaches in ultra-filter retentate Stop when 4.5%.Ultrafiltration filtered solution and ultra-filter retentate are collected respectively, to the ultrafiltration filtered solution of collection, are containing concentration expressed in percentage by volume 60% ethanol, is pumped into rotary evaporator, recycles ethanol；To the ultra-filter retentate of collection, respectively with sterilized 0.22 μm Filtering with microporous membrane, collect filtered solution, that is, prepare hydrolysis sericin peptide taken classification separation concentrated solution, be respectively used to prepare hydrolyzed-silk The de- ethanol concentrate of glue peptide classification separation.
(9) the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation is prepared
After the completion of (8) step, it is molten that the hydrolysis sericin peptide taken classification separation concentrated solution of (8) step preparation is pumped into low-temperature high-vacuum Agent retracting device, recycles ethanol under conditions of temperature is 45 DEG C, vacuum is 3kPa, stops when no ethanol flavor.Receive respectively Collection ethanol and the hydrolysis sericin peptide taken classification separation concentrated solution for sloughing ethanol, to the ethanol of collection, available for preparation ethanol volume hundred Divide the aqueous solution that concentration is 60%；Separation concentrated solution is classified to the hydrolysis sericin peptide taken for sloughing ethanol of collection, that is, prepares hydrolysis The de- ethanol concentrate of sericin peptide taken classification separation, hydrolysis silk gum peptide freeze-dried powder is prepared for lower step.
(10) hydrolysis silk gum peptide freeze-dried powder is prepared
After the completion of (9) step, the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation prepared by (9) step is first put respectively The pre-freeze 20h in -30 DEG C of low temperature refrigerator, then be transferred in freeze drier, in the condition that temperature is -40 DEG C, gauge pressure is 50Pa Under carry out freeze-drying 36h, just prepare average molecular weight be respectively 300~450Da, 1000~1350Da and 3200~ Three kinds of hydrolysis silk gum peptide freeze-dried powders in 3600Da sections.Three kinds of freeze-dried powders are white, and average molecular weight is 300~450Da's It is 15~15.3% to hydrolyze silk gum peptide freeze-dried powder total nitrogen content, and recovery rate is the 11~13% of silkworm cocoon quality；Average molecular weight Be 15~15.7% for the hydrolysis silk gum peptide freeze-dried powder total nitrogen content of 1000~1350Da, recovery rate for silkworm cocoon quality 8~ 10%；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 3200~3600Da is 15~15.5%, and recovery rate is The 6~8% of silkworm cocoon quality；Total recovery rate of three kinds of hydrolysis silk gum peptide freeze-dried powders is the 25~31% of silkworm cocoon quality.

Claims (1)

1. a kind of preparation method for hydrolyzing sericin peptide taken, it is characterised in that specific processing step is as follows：
(1) prepare and clean silkworm cocoon
Tree branches and leaves, silkworm chrysalis, the debris such as stone on silkworm cocoon are manually removed first, according still further to silkworm cocoon quality and quality percentage Concentration is the ratio that the ratio between 0.2~0.5% volume of aqueous sodium carbonate (kg/L) is 1: 20~40, by the artificial removal of impurities silkworm Cocoon shell be scattered in mass percentage concentration be 0.2~0.5% aqueous sodium carbonate in, at 60~80 DEG C stir process 30~ 40min, filtering, collected filter residue and filtered fluid respectively, to the filtered fluid of collection, is pumped into wastewater disposal basin and carries out biochemical treatment, up to standard After discharge；Filter residue is crossed to collection, that is, prepares clean silkworm cocoon, running sol solution is prepared for lower step；
(2) silk gum solution is prepared
After the completion of (1) step, first according to the ratio that the ratio between natrium carbonicum calcinatum quality and pure water volume (kg/L) are 1: 20~200 Example, natrium carbonicum calcinatum is dissolved in pure water, makes the aqueous sodium carbonate that mass percentage concentration is 0.5~5%；Press again It is that the ratio between 0.5~5% volume of aqueous sodium carbonate (kg/L) is 1: 60 according to clean silkworm cocoon quality and mass percentage concentration ~120 ratio, by clean silkworm cocoon be scattered in mass percentage concentration be 0.5~5% aqueous sodium carbonate in, under stirring in 90~100 DEG C of reflux 3~6h of sericin removal, filtering, collects sericin removal and crosses filter residue and filtered fluid, the sericin removal of collection is filtered respectively Slag, adds the pure water for cleaning 20~30 times of silkworm cocoon quality, stirs 15~30min of lower washing, filters again, collects wash respectively Filter residue and washed filtrate were washed, to the washed filtrate of collection, was merged with the sericin removal filtered fluid of collection, is that silk gum is molten Liquid, hydrolysis silk gum peptide solution is prepared for lower step；Washing to collection filters slag, that is, prepares sericin removal fibroin albumen, be used for Prepare hydrolysis silk peptide；
(3) hydrolysis silk gum peptide solution is prepared
After the completion of (2) step, it is the super of 3500~10000Da that the silk gum solution that (2) step is prepared is pumped into molecular cut off In filter, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.1~0.4MPa, until ultra-filter retentate volume and ultrafiltration filtered solution The ratio between volume (L/L) is stopped when being 1: 3~6, collects ultra-filter retentate and ultrafiltration filtered solution respectively, to the ultrafiltration filtered solution of collection, Containing sodium carbonate, silkworm cocoon is washed for lower batch；To the ultra-filter retentate of collection, hydrolysis kettle is pumped into, first in 60~120r/min Mixing speed under be warming up to 50~60 DEG C, then it is 8.0~9.5 to adjust pH with dilute sodium hydroxide, believes alkalescence then according to Novi Protease 3 .0T or animal proteolytic enzyme and the ratio that clean silkworm cocoon mass ratio (kg/kg) is 1: 100~200, add Novi believes alkali protease 3.0T or animal proteolytic enzyme, in the mixing speed of 60~120r/min and 50~60 DEG C of temperature 1~2h of lower constant temperature stirring hydrolysis；Then according still further to Novi believe flavor protease or papain and clean silkworm cocoon quality it Than the ratio that (kg/kg) is 1: 80~160, Novi's letter flavor protease or papain are added, 60~120r/min's Constant temperature stirring 1~2h of hydrolysis at a temperature of mixing speed and 50~60 DEG C, just prepares hydrolysis silk gum peptide solution, is used to prepare water Solve sericin peptide taken destainer；
(4) assembling activation macroreticular resin decoloration system
By activation AmberliteTMXAD7HP or AmberliteTMXAD761 or the HP20 resin dispersion newly purchased in 3~5 times In the pure water of resin volume, it is pumped into peristaltic pump in medium pressure chromatography column, opens bleeder valve after resin settles into resin column, put Go out excessive moisture, until stopping tapping when liquid level is higher than 2~10 centimetres of resin column upper surface, just assemble activation AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, finally by the resin column respectively with peristaltic pump, UV detector and collector connection, just assemble activation macroreticular resin decoloration system, and preparing hydrolysis sericin peptide taken for lower step takes off Color liquid；
(5) hydrolysis sericin peptide taken destainer is prepared
After the completion of (4) step, by the hydrolysis silk gum peptide solution that (3) step is prepared be pumped into molecular cut off for 10000~ In the ultrafilter of 30000Da, hyperfiltration treatment is carried out under conditions of gauge pressure is 0.1~0.4MPa, until ultra-filter retentate volume Stop when with the ratio between ultrafiltration filtered solution volume (L/L) being 1: 6~9, ultra-filter retentate and ultrafiltration filtered solution are collected respectively, to collection Ultra-filter retentate, is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard；To the ultrafiltration filtered solution of collection, activation is pumped into AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, in the stream of 2~4 times of resin column volume/hours Fast lower progress decolorization, activates AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns volume with being somebody's turn to do The ratio between ultrafiltration filtered solution volume (L/L) is 1: 20~40, collects column efflux and adsorpting pigment respectively AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, cross column efflux to collection, that is, prepare Sericin peptide taken destainer is hydrolyzed, is used to prepare hydrolysis sericin peptide taken classification separating liquid；To the adsorpting pigment of collection AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, be pumped into ethanol concentration expressed in percentage by volume for 65~ 85% aqueous solution, elution processing is carried out under the flow velocity of 2~4 times of resin column volume/hours, adsorpting pigment AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns and ethanol concentration expressed in percentage by volume are 65~85% The ratio between aqueous solution volume (L/L) be 1: 2~4, after the completion of elution, collect respectively and strip column liquid and unload pigment AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, to the unloading pigment of collection AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, with the pure water of 0.5~1 times of resin column volume Washing, collected column cleaning solution and washed AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resins Column, column cleaning solution is crossed to collection, containing ethanol, for preparing the ethanol eluate of lower batch elution pigment；To the warp of collection AmberliteTMXAD7HP or AmberliteTMXAD761 or the HP20 resin column of washing, it is namely regenerated AmberliteTMXAD7HP or AmberliteTMXAD761 or HP20 resin columns, available for lower batch adsorpting pigment, to collecting Strip column liquid, be pumped into rotary evaporator, recycle ethanol, stop when no ethanol flavor, collect the ethanol water of recycling respectively Solution and the concentrate for sloughing ethanol, can be re-used for lower batch to the ethanol water of the recycling of collection, after allotment concentration and wash Depigmentation；To the concentrate for sloughing ethanol of collection, it is pumped into wastewater disposal basin and carries out biochemical treatment, discharged after up to standard；
(6) assembling activation MCI GEL resin systems
By the MCI GEL CHP20P resins that the particle diameter newly purchased is 37~75 μm or the MCI GEL that particle diameter is 63~150 μm CHP20SS resins or particle diameter are 25~35 μm of MCI GEL CHP2MGY resin dispersions in the ethanol body of 3~5 times of resin volumes Product percentage concentration is in 30~60% aqueous solution, is pumped into peristaltic pump in medium pressure chromatography column, after resin settles into resin column Bleeder valve is opened, releases excessive moisture, until stopping tapping when liquid level is higher than 0.5~2 centimetre of resin column upper surface, is just assembled MCI GEL CHP20P or MCI GEL CHP 20SS or MCI GEL CHP 2MG Y resin columns are activated, finally by the resin column It is connected respectively with peristaltic pump, UV detector and collector, just assembles activation MCI GEL resin systems, prepared for lower step Hydrolyze sericin peptide taken classification separating liquid；
(7) hydrolysis sericin peptide taken classification separating liquid is prepared
After the completion of (6) step, by the hydrolysis sericin peptide taken destainer of (5) step collection, activation MCI GEL CHP20P or MCI are pumped into GEL CHP 20SS or MCI GEL CHP 2MG Y resin columns, hydrolyze the ratio between sericin peptide taken destainer and the activated resin column volume (L/L) it is 1: 10~20, hydrolysis sericin peptide taken destainer is pumped into 1~3 times/hour that flow velocity is the activated resin column volume, pump After the completion of entering to hydrolyze sericin peptide taken destainer, the resin column of load decoloration hydrolysis sericin peptide taken is collected respectively and crosses column efflux, to receiving Collection crosses column efflux, containing the ethanol that concentration expressed in percentage by volume is 30~60%, is pumped into rotary evaporator, recycles ethanol；To receiving The resin column of the load decoloration hydrolysis sericin peptide taken of collection, then the aqueous solution that ethanol concentration expressed in percentage by volume is 30~60% is pumped into, it is pumped into The flow velocity of the aqueous solution is 1~3 times/hour of the resin column volume, collects 14~21min, 26~37min and the respectively The resin column for crossing column efflux and unloading decoloration hydrolysis sericin peptide taken for having strong absworption peak in 280nm of 42~51min, to collecting 14~21min, 26~37min and 42~51min 280nm have strong absworption peak cross column efflux, that is, make It is standby to go out to hydrolyze sericin peptide taken classification separating liquid, prepare hydrolysis sericin peptide taken classification separation concentrated solution for lower step；Unloading to collection takes off Color hydrolyzes the resin column of sericin peptide taken, namely regenerated MCI GEL CHP20P or MCI GEL CHP 20SS or MCI GEL CHP 2MG Y resin columns, can be re-used for preparing hydrolysis sericin peptide taken classification separating liquid；
(8) hydrolysis sericin peptide taken classification separation concentrated solution is prepared
After the completion of (7) step, the hydrolysis sericin peptide taken classification separating liquid that (7) step is collected is pumped into molecular cut off as 600 respectively In nanofiltration/ultrafilter of~3500Da, nanofiltration/hyperfiltration treatment is carried out in the case where gauge pressure is 1.0~9.0MPa, until nanofiltration/ultrafiltration Stop when total nitrogen content reaches 3~4.5% in trapped fluid, collect nanofiltration/ultrafiltration filtered solution and nanofiltration/ultra-filter retentate respectively, it is right The nanofiltration of collection/ultrafiltration filtered solution, containing the ethanol that concentration expressed in percentage by volume is 30~60%, is pumped into rotary evaporator, recycles second Alcohol；To nanofiltration/ultra-filter retentate of collection, respectively with sterilized 0.22 μm of filtering with microporous membrane, filtered solution is collected, that is, is made It is standby to go out to hydrolyze sericin peptide taken classification separation concentrated solution, it is respectively used to prepare the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation；
(9) the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation is prepared
After the completion of (8) step, hydrolysis sericin peptide taken classification separation concentrated solution prepared by (8) step is pumped into low-temperature high-vacuum solvent time Receiving apparatus, recycles ethanol under conditions of temperature is 30~45 DEG C, vacuum is 0.1~3kPa, stops when no ethanol flavor, point Ethanol and the hydrolysis sericin peptide taken classification separation concentrated solution of ethanol Shou Ji not be sloughed, to the ethanol of collection, available for preparing ethanol body The aqueous solution that product percentage concentration is 30~60%；Separation concentrated solution is classified to the hydrolysis sericin peptide taken for sloughing ethanol of collection, that is, is made It is standby to go out to hydrolyze the de- ethanol concentrate of sericin peptide taken classification separation, prepare hydrolysis silk gum peptide freeze-dried powder for lower step；
(10) hydrolysis silk gum peptide freeze-dried powder is prepared
After the completion of (9) step, the de- ethanol concentrate of hydrolysis sericin peptide taken classification separation prepared by (9) step is first placed in -40 respectively 10~20h of pre-freeze in~-30 DEG C of low temperature refrigerator, then be transferred in freeze drier, it is -50~-40 DEG C, gauge pressure 20 in temperature 24~36h of freeze-drying is carried out under conditions of~50Pa, just prepare average molecular weight be respectively 300~450Da, 1000~ 1350Da and three kinds of hydrolysis silk gum peptide freeze-dried powders in 3200~3600Da sections, three kinds of freeze-dried powders are white, average molecular weight Be 15~15.3% for the hydrolysis silk gum peptide freeze-dried powder total nitrogen content of 300~450Da, recovery rate for silkworm cocoon quality 11~ 13%；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 1000~1350Da is 15~15.7%, and recovery rate is The 8~10% of silkworm cocoon quality；The hydrolysis silk gum peptide freeze-dried powder total nitrogen content that average molecular weight is 3200~3600Da is 15~ 15.5%, recovery rate is the 6~8% of silkworm cocoon quality；Total recovery rate of three kinds of hydrolysis silk gum peptide freeze-dried powders is silkworm cocoon quality 25~31%.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108743446A (en) * 2018-06-28 2018-11-06 中国科学院新疆理化技术研究所 A kind of ultrafiltration membrane preparation method of cocoon sericin protein and its application
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
CN110179127A (en) * 2019-06-25 2019-08-30 南通大学 A kind of nutrition fortifier and preparation method promoting iron zinc calcium uptake
CN111528480A (en) * 2020-05-21 2020-08-14 南通大学 Calcium nutritional supplement and preparation method thereof
CN113318049A (en) * 2021-06-22 2021-08-31 广东省农业科学院蚕业与农产品加工研究所 Anthocyanin sustained-release microcapsule, preparation method thereof and skin care product
CN113372426A (en) * 2020-03-10 2021-09-10 森田生医股份有限公司 Sericin extract, preparation method and use thereof, and antioxidant composition comprising the same
CN114404568A (en) * 2022-01-16 2022-04-29 重庆理工大学 Sericin polypeptide injection and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080124473A1 (en) * 2002-06-19 2008-05-29 National Institute Of Agrobiological Sciences Biodegradable biopolymers, method for their preparation and functional materials constituted by these biopolymers
CN103829029A (en) * 2014-03-11 2014-06-04 安徽省农业科学院蚕桑研究所 Production method of non-denatured pure sericin stock solution
CN104876843A (en) * 2015-05-22 2015-09-02 广州六顺生物科技有限公司 Method for preparing high-purity sulforaphene from carmine radish seeds
CN103393671B (en) * 2013-08-21 2015-12-02 上海信谊百路达药业有限公司 The extracting and purifying method of bilobalide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080124473A1 (en) * 2002-06-19 2008-05-29 National Institute Of Agrobiological Sciences Biodegradable biopolymers, method for their preparation and functional materials constituted by these biopolymers
CN103393671B (en) * 2013-08-21 2015-12-02 上海信谊百路达药业有限公司 The extracting and purifying method of bilobalide
CN103829029A (en) * 2014-03-11 2014-06-04 安徽省农业科学院蚕桑研究所 Production method of non-denatured pure sericin stock solution
CN104876843A (en) * 2015-05-22 2015-09-02 广州六顺生物科技有限公司 Method for preparing high-purity sulforaphene from carmine radish seeds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈文兴等: "从茧衣、茧壳、废丝中提取丝胶多肽", 《技术与市场》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108743446A (en) * 2018-06-28 2018-11-06 中国科学院新疆理化技术研究所 A kind of ultrafiltration membrane preparation method of cocoon sericin protein and its application
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
CN110179127A (en) * 2019-06-25 2019-08-30 南通大学 A kind of nutrition fortifier and preparation method promoting iron zinc calcium uptake
CN110179127B (en) * 2019-06-25 2022-04-22 南通大学 Nutrient supplement for promoting absorption of iron, zinc and calcium and preparation method thereof
CN113372426A (en) * 2020-03-10 2021-09-10 森田生医股份有限公司 Sericin extract, preparation method and use thereof, and antioxidant composition comprising the same
CN111528480A (en) * 2020-05-21 2020-08-14 南通大学 Calcium nutritional supplement and preparation method thereof
CN111528480B (en) * 2020-05-21 2023-07-04 南通大学 Calcium nutrition supplement and preparation method thereof
CN113318049A (en) * 2021-06-22 2021-08-31 广东省农业科学院蚕业与农产品加工研究所 Anthocyanin sustained-release microcapsule, preparation method thereof and skin care product
CN114404568A (en) * 2022-01-16 2022-04-29 重庆理工大学 Sericin polypeptide injection and application thereof
CN114404568B (en) * 2022-01-16 2023-12-26 重庆理工大学 Sericin polypeptide injection preparation and application thereof

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