CN107937460B

CN107937460B - Preparation method of hydrolyzed sericin peptide

Info

Publication number: CN107937460B
Application number: CN201610898768.4A
Authority: CN
Inventors: 孙益华; 刘志军
Original assignee: Yibin Pingshan Pfizer Technology Co Ltd
Current assignee: Yibin Pingshan Pfizer Technology Co Ltd
Priority date: 2016-10-13
Filing date: 2016-10-13
Publication date: 2020-08-14
Anticipated expiration: 2036-10-13
Also published as: CN107937460A

Abstract

A preparation method of hydrolyzed sericin peptide, belonging to the technical field of protein separation and purification. The method comprises the steps of taking tussah cocoon shells or waste mulberry silk as raw materials, preparing and cleaning the tussah cocoon shells, preparing a sericin solution, preparing a hydrolyzed sericin peptide solution, assembling an activated macroporous resin decoloring system, preparing a hydrolyzed sericin peptide decoloring solution, assembling an activated MCI GEL resin system, preparing a hydrolyzed sericin peptide graded separation solution, preparing a hydrolyzed sericin peptide graded separation concentrated solution, preparing a hydrolyzed sericin peptide graded separation ethanol-removed concentrated solution and preparing a hydrolyzed sericin peptide freeze-dried powder, and finally preparing the hydrolyzed sericin peptide freeze-dried powder. The invention uses the modern chemical technologies and equipment of multi-enzyme combined hydrolysis, ultrafiltration, nanofiltration separation, macroporous resin decolorization, MCI GEL resin fractionation, low-temperature concentration, freeze drying and the like, has the characteristics of convenient operation, mild condition, high efficiency, energy conservation, low production cost and the like, and can be widely used in the fields of nutritional daily chemical products, fiber modification and the like.

Description

Preparation method of hydrolyzed sericin peptide

One, the technical field

The invention belongs to the technical field of protein separation and purification, and particularly relates to a preparation method of hydrolyzed sericin peptide.

Second, background Art

The silk is mainly composed of 20-30% of sericin and 70-80% of fibroin and is a raw material for manufacturing silk. In addition, the components sericin and fibroin are also applied to the industries of medical treatment, health care, daily chemicals, textiles, food, building materials and the like, play the roles of blood coagulation, bone setting, water retention, whitening, antibiosis, ultraviolet resistance, packaging, coating and the like, expand the application field and the dosage year by year, are natural resources with huge market potential and are worthy of development and utilization.

At present, silk sericin, fibroin and deep-processed products thereof are all prepared from waste silk or silkworm cocoon shells. In the prior art, for example, 2015 discloses a rich written major paper "research on preparation and hypoglycemic effect of tussah silk peptide", which introduces a process for preparing tussah silk peptide: the tussah silk peptide with the highest recovery rate of 75 percent is obtained by reacting for 8 hours under the conditions that the concentration of phosphoric acid is 14.67mol/L, the ratio of material to liquid is 1: 10 and the temperature is 90 ℃. The disadvantages of this method are: the use of high-concentration phosphoric acid has high price; a large amount of calcium salt and the like are needed for neutralizing phosphoric acid, a large amount of phosphate with low additional value is generated, complete separation is difficult, and the product quality is seriously influenced; the prepared tussah silk peptide is not hydrolyzed, has large molecular weight and is difficult to store stably. For another example, the invention patent with application number CN200810162579.6 and publication number CN101544685 discloses a method for preparing water-soluble sericin with different molecular weights, which respectively comprises (1) degumming in boiled distilled water; (2) placing the degumming solution in hot water at the temperature of 80-100 ℃ for treatment for 0-12 h; (3) and (4) freeze-drying the sericin solution treated by hot water. The disadvantage of this invention is that the degumming efficiency is extremely low and, in addition, no fractionation is carried out, a mixture of sericins of different molecular weights is obtained, which, due to the non-uniform molecular weight, is difficult to apply and achieve good results in specific environments.

Third, the invention

The invention aims to provide a preparation method of hydrolyzed sericin peptide aiming at the defects of the existing preparation method of hydrolyzed sericin peptide. The method makes full use of sericin and fibroin, can realize fractionation of hydrolyzed sericin peptide according to molecular weight, and prepares lyophilized powder of hydrolyzed sericin peptide with different molecular weight, so that the lyophilized powder can be better applied to different fields such as daily chemicals, and the reuse and comprehensive utilization of ethanol and decolorized solution are realized.

The principle for realizing the purpose of the invention is as follows: the silk is mainly composed of sericin and silk fibroin, the sericin is distributed around the silk fibroin, and the silk fibroin is dissolved in an alkaline solution, so that the sericin can be dissolved by dilute sodium carbonate at a certain temperature, and then the sodium carbonate solution with the sericin dissolved and the silk fibroin with the sericin removed are obtained. The protease is a biocatalyst specially used for hydrolyzing protein amido bonds, the sericin is protein formed by 18 amino acids through amido bonds, and the protease can be selectively hydrolyzed to form the amido bonds of the sericin under proper conditions to generate hydrolyzed sericin peptides with different molecular weights. The pigment substances in the silk are mainly flavonoids, and the flavonoids have a 2-phenylchromone structure and can form hydrophobic interaction with nonpolar macroporous adsorption resin with phenyl as an adsorption functional group; the sericin peptide solution hydrolyzed by the protease passes through a nonpolar macroporous adsorption resin column, so that the flavonoid pigment in the hydrolysate and the macroporous nonpolar adsorption resin form hydrophobic interaction based on a benzene ring, and the decolorization of the hydrolysate is realized. The MCI GEL resin is a separation medium which takes polystyrene or polyacrylic acid as a framework and separates solutes based on the principle of a molecular sieve, has the characteristics of realizing high-precision separation under medium pressure and in an organic medium and being reusable; and (3) passing the decolored hydrolyzed sericin peptide solution through MCI GEL resin, eluting and collecting in parts to obtain hydrolyzed sericin peptide fractional separation liquid with different molecular weight distributions. Ultrafiltration and nanofiltration are membrane separation technologies for selectively separating substances through a microporous structure on the surface of a membrane, when a solution flows through the surface of the membrane under a certain pressure, small molecular solutes permeate the membrane, and large molecular solutes are trapped, so that the concentration of large molecules in the trapped solution is gradually increased, and the separation and concentration of large and small molecules are realized; and (3) passing the hydrolyzed sericin peptide solution with different molecular weight distributions, which is subjected to MCI GEL resin fractionation, through an ultrafiltration membrane or a nanofiltration membrane, filtering solutes smaller than the diameter of the ultrafiltration membrane or the nanofiltration membrane, and intercepting solutes larger than the diameter of the nanofiltration membrane, thereby obtaining a corresponding concentrated solution. And drying the obtained concentrated solution by using a freeze dryer to obtain the freeze-dried powder of the hydrolyzed sericin peptide.

The purpose of the invention is realized as follows: a preparation method of hydrolyzed sericin peptide comprises the steps of taking tussah cocoon shells or waste mulberry silk as raw materials, preparing and cleaning the cocoon shells, preparing a sericin solution, preparing the hydrolyzed sericin peptide solution, assembling an activated macroporous resin decolorization system, preparing a hydrolyzed sericin peptide decolorization solution, assembling an activated MCI GEL resin system, preparing a hydrolyzed sericin peptide fractional separation solution, preparing a hydrolyzed sericin peptide fractional separation concentrated solution, preparing a hydrolyzed sericin peptide fractional separation ethanol removal concentrated solution and preparing a hydrolyzed sericin peptide freeze-dried powder, and finally preparing the hydrolyzed sericin peptide freeze-dried powder. The method comprises the following specific steps:

(1) preparation of cleaned silkworm cocoon shell

Firstly, impurities such as branches and leaves, silkworm chrysalis and stones on the cocoon shells are manually removed, then the manually removed cocoon shells are dispersed in a sodium carbonate aqueous solution with the mass percentage concentration of 0.2-0.5% according to the proportion (kg/L) of 1: 20-40 of the mass of the cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 0.2-0.5%, and the mixture is stirred for 30-40 min at the temperature of 60-80 ℃. Filtering, and respectively collecting the filter residue and filtrate. Pumping the collected filtrate into a wastewater tank for biochemical treatment, and discharging after reaching the standard; and preparing the collected filter residues into clean silkworm cocoon shells for the next step to prepare the degumming solution.

(2) Preparation of sericin solution

After the step (1), firstly dissolving anhydrous sodium carbonate into purified water according to the ratio (kg/L) of the mass of the anhydrous sodium carbonate to the volume of the purified water of 1: 20-200 to prepare a sodium carbonate aqueous solution with the mass percentage concentration of 0.5-5%; and then dispersing the cleaned silkworm cocoon shells in a sodium carbonate aqueous solution with the mass percentage concentration of 0.5-5% according to the ratio (kg/L) of the mass of the cleaned silkworm cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 0.5-5% of 1: 60-120, refluxing and degumming the sericin for 3-6 hours at 90-100 ℃ under stirring, filtering, and respectively collecting the sericin removal filter residues and the filtrate. Adding purified water which is 20-30 times of the mass of the cleaned silkworm cocoon shells into the collected degummed filtering residues, washing for 15-30 min under stirring, filtering again, respectively collecting the washed filtering residues and the washing filtrate, and combining the collected washing filtrate with the collected degummed filtrate to obtain a sericin solution which is used for preparing a hydrolyzed sericin peptide solution in the next step; and (3) preparing the degummed silk fibroin from the collected washing and filtering residues for preparing the hydrolyzed silk peptide.

(3) Preparation of hydrolyzed sericin peptide solution

And (3) after the step (2) is finished, pumping the sericin solution prepared in the step (2) into an ultrafilter with the molecular weight cutoff of 3500-10000 Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.1-0.4 MPa until the ratio (L/L) of the volume of ultrafiltration cut-off liquid to the volume of ultrafiltration filtrate is 1: 3-6. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, wherein the collected ultrafiltration filtrate contains sodium carbonate and is used for washing the silkworm cocoon shells in the next batch; pumping the collected ultrafiltration retentate into a hydrolysis kettle, heating to 50-60 ℃ at a stirring speed of 60-120 r/min, adjusting the pH to 8.0-9.5 by using dilute sodium hydroxide, adding 3.0T Novien alkaline protease or animal proteolytic enzyme according to the mass ratio (kg/kg) of the Novien alkaline protease or the animal proteolytic enzyme to the cleaned silkworm cocoon shell of 1: 100-200, stirring and hydrolyzing at a constant temperature of 50-60 ℃ at a stirring speed of 60-120 r/min for 1-2 h; then adding the Novier flavor protease or the papain according to the mass ratio (kg/kg) of the Novier flavor protease or the papain to the clean silkworm cocoon shell of 1: 80-160, and stirring and hydrolyzing for 1-2 hours at a stirring speed of 60-120 r/min and a constant temperature of 50-60 ℃. Then preparing a hydrolyzed sericin peptide solution which is used for preparing a hydrolyzed sericin peptide decoloring solution.

(4) Assembled activated macroporous resin decoloring system

Dispersing newly-purchased activated Amberlite TMXAD7HP, Amberlite TMXAD761 or HP20 resin in purified water with the volume of 3-5 times of the resin volume, pumping the resin into a medium-pressure chromatographic column by using a peristaltic pump, starting a liquid discharging valve after the resin is settled into the resin column, discharging excessive water, stopping discharging until the liquid level is 2-10 cm higher than the upper surface of the resin column, and assembling the activated Amberlite TMXAD7HP, Amberlite TMXAD761 or HP20 resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively, and assembling an activated macroporous resin decolorizing system for preparing the hydrolyzed sericin peptide decolorizing solution in the next step.

(5) Preparation of hydrolyzed sericin peptide decolorizing solution

And (4) after the step (4) is finished, pumping the hydrolyzed sericin peptide solution prepared in the step (3) into an ultrafilter with the molecular weight cutoff of 10000-30000 Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.1-0.4 MPa until the ratio (L/L) of the volume of ultrafiltration cut-off liquid to the volume of ultrafiltration filtrate is 1: 6-9. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, pumping the collected ultrafiltration trapped fluid into a wastewater tank for biochemical treatment, and discharging after reaching the standard; pumping the collected ultrafiltration filtrate into an activated Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, and carrying out decolorization treatment at the flow rate of 2-4 times the volume of the resin column per hour, wherein the ratio (L/L) of the volume of the activated Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column to the volume of the ultrafiltration filtrate is 1: 20-40. Respectively collecting column-passing effluent and pigment-adsorbing Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, and preparing hydrolyzed sericin peptide decolorization solution for preparing hydrolyzed sericin peptide fractionation solution; pumping an aqueous solution with the ethanol volume percentage concentration of 65-85% into the collected pigment adsorption Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, and carrying out elution treatment at the flow rate of 2-4 times the volume/hour of the resin column, wherein the volume ratio (L/L) of the pigment adsorption Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column to the aqueous solution with the ethanol volume percentage concentration of 65-85% is 1: 2-4. After the elution was completed, the eluted column liquid and the pigment-unloaded Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column were collected, respectively. And (3) washing the collected pigment-unloaded Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column with 0.5-1 time of the volume of the resin column, and collecting the column-passing washing liquid and the washed Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column. The collected column-passing washing liquid contains ethanol and is used for preparing ethanol eluent for washing decoloration in next batch; and (3) carrying out resin column chromatography on the collected washed Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20, namely regenerated Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, and using the regenerated Amberlite TMXAD7 or Amberlite TMXAD761 or HP20 resin column for adsorbing pigment in the next batch. Pumping the collected eluate into a rotary evaporator, and recovering ethanol until no ethanol smell exists. Respectively collecting the recovered ethanol water solution and the concentrated solution after ethanol removal, and adjusting the concentration of the collected recovered ethanol water solution for next batch of washing decoloration; pumping the collected concentrated solution without ethanol into a wastewater tank for biochemical treatment, and discharging after reaching the standard.

(6) Assembly activated MCI GEL resin system

Dispersing newly purchased MCI GEL CHP20P resin with the particle size of 37-75 microns or MCIGEL CHP20SS resin with the particle size of 63-150 microns or MCI GEL CHP2MG Y resin with the particle size of 25-35 microns in an aqueous solution with the volume percentage concentration of 30-60% of ethanol which is 3-5 times of the volume of the resin, pumping the aqueous solution into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharge valve after the resin is settled into the resin column, discharging excessive water, stopping discharging until the liquid level is 0.5-2 cm higher than the upper surface of the resin column, and assembling an activated MCI GEL CHP20P or MCI GEL CHP20SS or MCI GEL CHP2MG Y resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively to assemble an activated MCI GEL resin system for preparing the hydrolyzed sericin peptide fractional separation solution in the next step.

(7) Preparation of hydrolyzed sericin peptide fractionation liquid

And (5) after the step (6) is finished, pumping the hydrolyzed sericin peptide decolorizing solution collected in the step (5) into an activated MCI GEL CHP20P or MCI GEL CHP20SS or MCI GEL CHP2MG Y resin column, wherein the volume ratio (L/L) of the hydrolyzed sericin peptide decolorizing solution to the activated resin column is 1: 10-20, and the pumping flow rate of the hydrolyzed sericin peptide decolorizing solution is 1-3 times of the volume of the activated resin column per hour. After the completion of pumping the hydrolyzed sericin peptide decoloring solution, respectively collecting the resin column loaded with the decolored hydrolyzed sericin peptide and column-passing effluent, pumping the collected column-passing effluent containing 30-60% by volume of ethanol into a rotary evaporator, and recovering the ethanol; and pumping the collected resin column loaded with the decolored hydrolyzed sericin peptide into an aqueous solution with the ethanol volume percentage concentration of 30-60%, wherein the flow rate of the pumped aqueous solution is 1-3 times/h of the volume of the resin column. Respectively collecting the column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51min and the resin column for unloading the decolored hydrolyzed sericin peptide, and preparing a hydrolyzed sericin peptide fractional separation liquid for preparing a hydrolyzed sericin peptide fractional separation concentrated solution in the next step by collecting the collected column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51 min; the collected resin column for unloading, decoloring and hydrolyzing the sericin peptide, namely a regenerated MCI GEL CHP20P or MCI GEL CHP20SS or MCI GELCHP 2MG Y resin column, can be used for preparing a hydrolyzed sericin peptide fractionation liquid again.

(8) Preparation of hydrolyzed sericin peptide fractionation concentrate

And (7) after the step (7) is finished, respectively pumping the hydrolyzed sericin peptide fractional separation liquid collected in the step (7) into a nanofiltration/ultrafiltration filter with the molecular weight cutoff of 600-3500 Da, and performing nanofiltration/ultrafiltration treatment under the gauge pressure of 1.0-9.0 MPa until the total nitrogen content in the nanofiltration/ultrafiltration cutoff liquid reaches 3-4.5%. Respectively collecting nanofiltration/ultrafiltration filtrate and nanofiltration/ultrafiltration retentate, pumping the collected nanofiltration/ultrafiltration filtrate containing 30-60% by volume of ethanol into a rotary evaporator, and recovering the ethanol; and filtering the collected nanofiltration/ultrafiltration trapped fluid by using sterilized microporous filter membranes with the diameter of 0.22 mu m respectively, and collecting filtered fluid, namely preparing hydrolyzed sericin peptide fractionation concentrated solution which is respectively used for preparing hydrolyzed sericin peptide fractionation ethanol-removed concentrated solution.

(9) Preparation of hydrolyzed sericin peptide fractionation ethanol-removed concentrated solution

And (5) after the step (8) is finished, pumping the hydrolyzed sericin peptide fractional separation concentrated solution prepared in the step (8) into a low-temperature high-vacuum solvent recovery device, and recovering ethanol under the conditions that the temperature is 30-45 ℃ and the vacuum degree is 0.1-3 kPa until no ethanol smell exists. Respectively collecting ethanol and the hydrolyzed sericin peptide fractionated concentrate without ethanol, wherein the collected ethanol can be used for preparing an aqueous solution with the ethanol volume percentage concentration of 30-60%; and (3) separating the collected hydrolyzed sericin peptide concentrate subjected to ethanol removal by a grading way to obtain a hydrolyzed sericin peptide concentrate subjected to graded separation and ethanol removal, which is used for preparing the hydrolyzed sericin peptide freeze-dried powder in the next step.

(10) Preparation of hydrolyzed sericin peptide lyophilized powder

After the step (9) is finished, placing the hydrolyzed sericin peptide fractional separation ethanol-removing concentrated solution prepared in the step (9) in a low-temperature refrigerator at-40 to-30 ℃ for pre-freezing for 10-20 h, transferring the pre-frozen concentrated solution into a freeze dryer, and carrying out freeze drying for 24-36 h under the conditions that the temperature is-50 to-40 ℃ and the gauge pressure is 20-50 Pa, so as to prepare three hydrolyzed sericin peptide freeze-dried powders with average molecular weights of 300-450 Da, 1000-1350 Da and 3200-3600 Da respectively. The three freeze-dried powders are white, the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 300-450 Da is 15-15.3%, and the yield is 11-13% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 1000-1350 Da is 15-15.7%, and the yield is 8-10% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 3200-3600 Da is 15-15.5%, and the yield is 6-8% of the mass of the silkworm cocoon shell; the total yield of the three hydrolyzed sericin peptide freeze-dried powders is 25-31% of the mass of the silkworm cocoon shell.

The invention mainly produces the following effects after adopting the technical means:

1. the method uses modern chemical technologies and equipment such as multi-enzyme combined hydrolysis, ultrafiltration, nanofiltration separation, macroporous resin decolorization, MCI GEL resin fractionation, low-temperature concentration, freeze drying and the like in the production process, and has the characteristics of convenient operation, mild conditions, high efficiency, energy conservation, low production cost and the like;

2. the three prepared hydrolyzed sericin peptide freeze-dried powders are prepared by the method. The hydrolyzed sericin peptide freeze-dried powder is white, the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 300-450 Da is 15-15.3%, and the yield is 11-13% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 1000-1350 Da is 15-15.7%, and the yield is 8-10% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 3200-3600 Da is 15-15.5%, and the yield is 6-8% of the mass of the silkworm cocoon shell; the total yield of the three hydrolyzed sericin peptide freeze-dried powders is 25-31% of the mass of the silkworm cocoon shell. Can be respectively used in the fields of nutritional daily chemical products directly absorbed by skin or hair, fiber modification and the like.

3. The invention uses Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 adsorption resin to replace ion exchange resin to decolor and hydrolyze pigment substances in the sericin peptide, and has the advantages of high separation efficiency, suitability for acid-base solution, reusability and the like; the MCI GEL resin technology is used for carrying out fractional separation on peptide substances in different molecular weight regions in the hydrolyzed sericin peptide decoloring solution, the nanofiltration membrane technology is used for replacing conventional reduced pressure vacuum concentration, and the low-temperature high-vacuum solvent recovery system is used for recovering ethanol.

4. The wastewater generated in the production process of the invention is subjected to biochemical treatment, is discharged after reaching the standard, does not generate three wastes, is a typical green production process, and is convenient for popularization and application.

Fourth, detailed description of the invention

The present invention will be further described with reference to the following specific embodiments.

Example 1

(1) Preparation of cleaned silkworm cocoon shell

Firstly, impurities such as branches and leaves, silkworm chrysalis, stone and the like on the cocoon shells are manually removed, then the manually removed cocoon shells are dispersed in a sodium carbonate aqueous solution with the mass percentage concentration of 0.2% according to the proportion (kg/L) of 1: 20 of the volume ratio of the mass of the cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 0.2%, and the mixture is stirred for 30min at the temperature of 60 ℃. Filtering, and respectively collecting the filter residue and filtrate. Pumping the collected filtrate into a wastewater tank for biochemical treatment, and discharging after reaching the standard; and preparing the collected filter residues into clean silkworm cocoon shells for the next step to prepare the degumming solution.

(2) Preparation of sericin solution

After the step (1), firstly dissolving anhydrous sodium carbonate into purified water according to the ratio (kg/L) of the mass of the anhydrous sodium carbonate to the volume of the purified water of 1: 20 to prepare a sodium carbonate aqueous solution with the mass percentage concentration of 5%; and then according to the proportion (kg/L) of the mass of the cleaned silkworm cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 5% as 1: 60, dispersing the cleaned silkworm cocoon shells in the sodium carbonate aqueous solution with the mass percentage concentration of 5%, refluxing and removing sericin for 3 hours at 90 ℃ under stirring, filtering, and respectively collecting the residue after filtering the sericin and the filtrate. Adding purified water with the mass 20 times of that of the clean silkworm cocoon shells into the collected sericin removal filter residues, washing for 15min under stirring, filtering again, respectively collecting the washed filter residues and the washing filtrate, and combining the collected washing filtrate with the collected sericin removal filtrate to obtain a sericin solution for preparing a hydrolyzed sericin peptide solution in the next step; and (3) preparing the degummed silk fibroin from the collected washing and filtering residues for preparing the hydrolyzed silk peptide.

(3) Preparation of hydrolyzed sericin peptide solution

And (3) after the step (2) is finished, pumping the sericin solution prepared in the step (2) into an ultrafilter with the molecular weight cutoff of 3500Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.1MPa until the ratio (L/L) of the volume of ultrafiltration cutoff liquid to the volume of ultrafiltration filtrate is 1: 3. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, wherein the collected ultrafiltration filtrate contains sodium carbonate and is used for washing the silkworm cocoon shells in the next batch; pumping the collected ultrafiltration trapped fluid into a hydrolysis kettle, heating to 50 ℃ at a stirring speed of 60r/min, adjusting the pH to 8.0 by using dilute sodium hydroxide, adding 3.0T of Novitin alkaline protease according to the mass ratio (kg/kg) of 3.0T of Novitin alkaline protease to the cleaned silkworm cocoon shell of 1: 100, and stirring at a stirring speed of 60r/min and at a constant temperature of 50 ℃ for hydrolysis for 1 h; then adding the Novier flavor protease according to the mass ratio (kg/kg) of the Novier flavor protease to the cleaned silkworm cocoon shell of 1: 80, and stirring and hydrolyzing for 1h at the constant temperature of 50 ℃ at the stirring speed of 60 r/min. Then preparing a hydrolyzed sericin peptide solution which is used for preparing a hydrolyzed sericin peptide decoloring solution.

(4) Assembled activated macroporous resin decoloring system

Dispersing newly purchased activated Amberlite TMXAD7HP resin in purified water with the volume 3 times that of the resin, pumping the resin into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharge valve after the resin is settled into the resin column, discharging excessive water, stopping discharging until the liquid level is 2 cm higher than the upper surface of the resin column, and assembling the activated Amberlite TMXAD7HP resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively, and assembling an activated macroporous resin decolorizing system for preparing the hydrolyzed sericin peptide decolorizing solution in the next step.

(5) Preparation of hydrolyzed sericin peptide decolorizing solution

And (4) after the step (4) is finished, pumping the hydrolyzed sericin peptide solution prepared in the step (3) into an ultrafilter with the molecular weight cutoff of 10000Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.1MPa until the ratio (L/L) of the volume of ultrafiltration cutoff liquid to the volume of ultrafiltration filtrate is 1: 6. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, pumping the collected ultrafiltration trapped fluid into a wastewater tank for biochemical treatment, and discharging after reaching the standard; the collected ultrafiltrate was pumped into an activated Amberlite TMXAD7HP resin column and decolorized at a flow rate of 2 times the volume of the resin column/hour, and the ratio (L/L) of the volume of the activated Amberlite TMXAD7HP resin column to the volume of the ultrafiltrate was 1: 20. Respectively collecting column-passing effluent and an Amberlite TMXAD7HP resin column for adsorbing pigment, and preparing hydrolyzed sericin peptide decolorization solution for preparing hydrolyzed sericin peptide fractionation solution for the collected column-passing effluent; pumping 65% ethanol solution at 2 times of resin column volume/hr into collected pigment adsorbing Amberlite TMXAD7HP resin column, and eluting with pigment adsorbing Amberlite TMXAD7HP resin column and 65% ethanol solution at a volume ratio (L/L) of 1: 2. After the elution was complete, the eluted column liquid and the pigment-unloaded Amberlite TMXAD7HP resin column were collected separately. The collected pigment-off-loaded Amberlite TMXAD7HP resin column was washed with 0.5 column volume of purified water, and the column wash and the washed Amberlite TMXAD7HP resin column were collected. The collected column-passing washing liquid contains ethanol and is used for preparing ethanol eluent for washing decoloration in next batch; and (3) carrying out resin column washing on the collected Amberlite TMXAD7HP, namely regenerating Amberlite TMXAD7HP resin column, and allowing the regenerated Amberlite TMXAD7 to be used for adsorbing pigment in the next batch. Pumping the collected eluate into a rotary evaporator, and recovering ethanol until no ethanol smell exists. Respectively collecting the recovered ethanol water solution and the concentrated solution after ethanol removal, and adjusting the concentration of the collected recovered ethanol water solution for next batch of washing decoloration; pumping the collected concentrated solution without ethanol into a wastewater tank for biochemical treatment, and discharging after reaching the standard.

(6) Assembly activated MCI GEL resin system

Dispersing MCI GEL CHP20P resin with the particle size of 37-75 mu m, which is newly purchased, in an aqueous solution which is 3 times of the volume of the resin and has the ethanol volume percentage concentration of 30%, pumping the aqueous solution into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharging valve after the resin is settled into the resin column, discharging excessive water, stopping discharging the liquid until the liquid level is 0.5 cm higher than the upper surface of the resin column, and assembling the activated MCIGEL CHP20P resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively to assemble an activated MCI GEL resin system for preparing the hydrolyzed sericin peptide fractional separation solution in the next step.

(7) Preparation of hydrolyzed sericin peptide fractionation liquid

And (4) after the step (6) is finished, pumping the hydrolyzed sericin peptide decolorizing solution collected in the step (5) into an activated MCI GEL CHP20P resin column, wherein the volume ratio (L/L) of the hydrolyzed sericin peptide decolorizing solution to the activated resin column is 1: 10, and the pumping flow rate of the hydrolyzed sericin peptide decolorizing solution is 1 time/h of the volume of the activated resin column. After the completion of the pumping of the hydrolyzed sericin peptide decolorization solution, respectively collecting the resin column loaded with the decolorized and hydrolyzed sericin peptide and the column-passing effluent, pumping the collected column-passing effluent containing 30% by volume of ethanol into a rotary evaporator, and recovering the ethanol; and pumping the collected resin column loaded with the decolored hydrolyzed sericin peptide into an aqueous solution with the ethanol volume percentage concentration of 30%, wherein the flow rate of the aqueous solution is 1 time/hour of the volume of the resin column. Respectively collecting the column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51min and the resin column for unloading the decolored hydrolyzed sericin peptide, and preparing a hydrolyzed sericin peptide fractional separation liquid for preparing a hydrolyzed sericin peptide fractional separation concentrated solution in the next step by collecting the collected column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51 min; the collected resin column for unloading, decoloring and hydrolyzing the sericin peptide is a regenerated MCI GEL CHP20P resin column, and can be used for preparing a hydrolyzed sericin peptide fractionation liquid again.

(8) Preparation of hydrolyzed sericin peptide fractionation concentrate

And (7) after the step (7) is finished, respectively pumping the hydrolyzed sericin peptide fractional separation liquid collected in the step (7) into nano filters with the molecular weight cutoff of 600Da, and performing nanofiltration treatment under the gauge pressure of 1.0MPa until the total nitrogen content in the nanofiltration cutoff liquid reaches 3%. Collecting nanofiltration filtrate and nanofiltration trapped fluid respectively, pumping the collected nanofiltration filtrate containing 30% ethanol by volume percentage into a rotary evaporator, and recovering ethanol; and filtering the collected nanofiltration trapped fluid by using sterilized microporous filter membranes with the diameter of 0.22 mu m respectively, and collecting filtered fluid, namely preparing hydrolyzed sericin peptide fractionation concentrated solution which is respectively used for preparing hydrolyzed sericin peptide fractionation ethanol-removed concentrated solution.

And (4) after the step (8) is finished, pumping the hydrolyzed sericin peptide fractional separation concentrated solution prepared in the step (8) into a low-temperature high-vacuum solvent recovery device, and recovering ethanol under the conditions that the temperature is 30 ℃ and the vacuum degree is 0.1kPa until no ethanol smell exists. Respectively collecting ethanol and the hydrolyzed sericin peptide fractionated concentrate without ethanol, wherein the collected ethanol can be used for preparing an aqueous solution with the ethanol volume percentage concentration of 30%; and (3) separating the collected hydrolyzed sericin peptide concentrate subjected to ethanol removal by a grading way to obtain a hydrolyzed sericin peptide concentrate subjected to graded separation and ethanol removal, which is used for preparing the hydrolyzed sericin peptide freeze-dried powder in the next step.

(10) Preparation of hydrolyzed sericin peptide lyophilized powder

After the step (9) is finished, placing the hydrolyzed sericin peptide fractional separation ethanol-removing concentrated solution prepared in the step (9) in a low-temperature refrigerator at-40 ℃ for pre-freezing for 10 hours, transferring the pre-frozen concentrated solution into a freeze dryer, and carrying out freeze drying for 24 hours under the conditions that the temperature is-50 ℃ and the gauge pressure is 20Pa, so as to prepare three kinds of hydrolyzed sericin peptide freeze-dried powders with average molecular weights of 300-450 Da, 1000-1350 Da and 3200-3600 Da respectively. The three freeze-dried powders are white, the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 300-450 Da is 15-15.3%, and the yield is 11-13% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 1000-1350 Da is 15-15.7%, and the yield is 8-10% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 3200-3600 Da is 15-15.5%, and the yield is 6-8% of the mass of the silkworm cocoon shell; the total yield of the three hydrolyzed sericin peptide freeze-dried powders is 25-31% of the mass of the silkworm cocoon shell.

Example 2

(1) Preparation of cleaned silkworm cocoon shell

Firstly, impurities such as branches and leaves, silkworm chrysalis, stone and the like on the cocoon shells are manually removed, then the manually removed cocoon shells are dispersed in a sodium carbonate aqueous solution with the mass percentage concentration of 0.4% according to the proportion that the volume ratio (kg/L) of the mass of the cocoon shells to the sodium carbonate aqueous solution with the mass percentage concentration of 0.4% is 1: 30, and the mixture is stirred for 35min at the temperature of 70 ℃. Filtering, and respectively collecting the filter residue and filtrate. Pumping the collected filtrate into a wastewater tank for biochemical treatment, and discharging after reaching the standard; and preparing the collected filter residues into clean silkworm cocoon shells for the next step to prepare the degumming solution.

(2) Preparation of sericin solution

After the step (1), firstly dissolving anhydrous sodium carbonate into purified water according to the ratio (kg/L) of the mass of the anhydrous sodium carbonate to the volume of the purified water of 1: 100 to prepare a sodium carbonate aqueous solution with the mass percentage concentration of 1%; and then according to the proportion (kg/L) of the mass of the clean silkworm cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 1% as 1: 90, dispersing the clean silkworm cocoon shells in the sodium carbonate aqueous solution with the mass percentage concentration of 1%, refluxing and removing sericin for 4.5 hours at 95 ℃ under stirring, filtering, and respectively collecting the removed sericin filter residues and the filtrate. Adding purified water with the mass 25 times of that of the clean silkworm cocoon shells into the collected sericin removal filter residues, washing for 22min under stirring, filtering again, respectively collecting the washed filter residues and the washing filtrate, and combining the collected washing filtrate with the collected sericin removal filtrate to obtain a sericin solution for preparing a hydrolyzed sericin peptide solution in the next step; and (3) preparing the degummed silk fibroin from the collected washing and filtering residues for preparing the hydrolyzed silk peptide.

(3) Preparation of hydrolyzed sericin peptide solution

And (3) after the step (2) is finished, pumping the sericin solution prepared in the step (2) into an ultrafilter with the molecular weight cutoff of 8000Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.25MPa until the ratio (L/L) of the volume of ultrafiltration cutoff liquid to the volume of ultrafiltration filtrate is 1: 4.5. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, wherein the collected ultrafiltration filtrate contains sodium carbonate and is used for washing the silkworm cocoon shells in the next batch; pumping the collected ultrafiltration trapped fluid into a hydrolysis kettle, heating to 55 ℃ at a stirring speed of 90r/min, adjusting the pH to 8.8 by using dilute sodium hydroxide, adding the novacin animal protein hydrolase according to the mass ratio (kg/kg) of the novacin animal protein hydrolase to the cleaned silkworm cocoon shell of 1: 150, and stirring and hydrolyzing at a constant temperature of 55 ℃ at a stirring speed of 90r/min for 1.5 hours; then adding the Novitin papain according to the mass ratio (kg/kg) of the Novitin papain to the cleaned silkworm cocoon shell of 1: 120, stirring at the stirring speed of 90r/min and at the constant temperature of 55 ℃ and hydrolyzing for 1.5 h. Then preparing a hydrolyzed sericin peptide solution which is used for preparing a hydrolyzed sericin peptide decoloring solution.

(4) Assembled activated macroporous resin decoloring system

Dispersing newly purchased activated Amberlite TMXAD761 resin in purified water with the volume 4 times that of the resin, pumping the resin into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharge valve after the resin is settled into the resin column, discharging excessive water, stopping discharging the liquid until the liquid level is 6 cm higher than the upper surface of the resin column, and assembling the activated Amberlite TMXAD761 resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively, and assembling an activated macroporous resin decolorizing system for preparing the hydrolyzed sericin peptide decolorizing solution in the next step.

(5) Preparation of hydrolyzed sericin peptide decolorizing solution

And (4) after the step (4) is finished, pumping the hydrolyzed sericin peptide solution prepared in the step (3) into an ultrafilter with the molecular weight cutoff of 20000Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.25MPa until the ratio (L/L) of the volume of ultrafiltration cutoff liquid to the volume of ultrafiltration filtrate is 1: 7.5. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, pumping the collected ultrafiltration trapped fluid into a wastewater tank for biochemical treatment, and discharging after reaching the standard; the collected ultrafiltrate was pumped into an activated Amberlite TMXAD761 resin column and decolorized at a flow rate of 3 times the volume of the resin column/hour, and the ratio (L/L) of the volume of the activated Amberlite TMXAD761 resin column to the volume of the ultrafiltrate was 1: 30. Respectively collecting column-passing effluent and an Amberlite TMXAD761 resin column for adsorbing pigment, and preparing hydrolyzed sericin peptide decolorization solution for preparing hydrolyzed sericin peptide fractionation solution for the collected column-passing effluent; pumping 75% ethanol solution at 3 times of resin column volume/hr, and eluting with pigment adsorbing Amberlite TMXAD761 resin column at a volume ratio (L/L) of 1: 3. After the elution was completed, the eluted column liquid and the pigment-unloaded Amberlite TMXAD761 resin column were collected separately. The collected pigment-off-loaded amberlite (tm) xad761 resin column was washed with 0.75 column volume of purified water, and the column wash and the washed amberlite (tm) xad761 resin column were collected. The collected column-passing washing liquid contains ethanol and is used for preparing ethanol eluent for washing decoloration in next batch; and (3) carrying out column chromatography on the collected washed Amberlite TMXAD761 resin, namely the regenerated Amberlite TMXAD761 resin column, and allowing the regenerated Amberlite TMXAD761 resin column to be used for adsorbing pigments in the next batch. Pumping the collected eluate into a rotary evaporator, and recovering ethanol until no ethanol smell exists. Respectively collecting the recovered ethanol water solution and the concentrated solution after ethanol removal, and adjusting the concentration of the collected recovered ethanol water solution for next batch of washing decoloration; pumping the collected concentrated solution without ethanol into a wastewater tank for biochemical treatment, and discharging after reaching the standard.

(6) Assembly activated MCI GEL resin system

Dispersing MCIGELCHP20SS resin with the particle size of 63-150 mu m, which is newly purchased, in an aqueous solution with the ethanol volume percentage concentration of 45% and the volume of 4 times of the resin volume, pumping the aqueous solution into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharging valve after the resin is settled into the resin column, discharging excessive water, stopping discharging the liquid until the liquid level is 1.2 cm higher than the upper surface of the resin column, and assembling the activated MCIGEL CHP20SS resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively to assemble an activated MCI GEL resin system for preparing the hydrolyzed sericin peptide fractional separation solution in the next step.

(7) Preparation of hydrolyzed sericin peptide fractionation liquid

And (4) after the step (6) is finished, pumping the hydrolyzed sericin peptide decolorizing solution collected in the step (5) into an activated MCI GEL CHP20SS resin column, wherein the volume ratio (L/L) of the hydrolyzed sericin peptide decolorizing solution to the activated resin column is 1: 15, and the pumping flow rate of the hydrolyzed sericin peptide decolorizing solution is 2 times of the volume of the activated resin column per hour. After the completion of the pumping of the hydrolyzed sericin peptide decolorization solution, respectively collecting the resin column loaded with the decolorized and hydrolyzed sericin peptide and the column-passing effluent, pumping the collected column-passing effluent containing 45 volume percent of ethanol into a rotary evaporator, and recovering the ethanol; and pumping an aqueous solution with the ethanol volume percentage concentration of 45% into the collected resin column loaded with the decolored hydrolyzed sericin peptide, wherein the flow rate of the aqueous solution is 2 times of the volume of the resin column per hour. Respectively collecting the column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51min and the resin column for unloading the decolored hydrolyzed sericin peptide, and preparing a hydrolyzed sericin peptide fractional separation liquid for preparing a hydrolyzed sericin peptide fractional separation concentrated solution in the next step by collecting the collected column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51 min; the collected resin column for unloading, decoloring and hydrolyzing the sericin peptide is a regenerated MCI GEL CHP20SS resin column, and can be used for preparing a hydrolyzed sericin peptide fractionation liquid again.

(8) Preparation of hydrolyzed sericin peptide fractionation concentrate

And (7) after the step (7) is finished, respectively pumping the hydrolyzed sericin peptide fractionation liquid collected in the step (7) into ultrafilters with the molecular weight cutoff of 2500Da, and carrying out ultrafiltration treatment under the gauge pressure of 5.0MPa until the total nitrogen content in the ultrafiltration cutoff liquid reaches 3.8%. Respectively collecting ultrafiltration filtrate and ultrafiltration retentate, pumping the collected ultrafiltration filtrate containing 45% ethanol by volume into a rotary evaporator, and recovering ethanol; and filtering the collected ultrafiltration trapped fluid by using sterilized 0.22 mu m microporous filter membranes respectively, and collecting the filtered fluid, namely preparing hydrolyzed sericin peptide fractionation concentrated solution which is respectively used for preparing hydrolyzed sericin peptide fractionation ethanol-removed concentrated solution.

And (4) after the step (8) is finished, pumping the hydrolyzed sericin peptide fractional separation concentrated solution prepared in the step (8) into a low-temperature high-vacuum solvent recovery device, and recovering ethanol under the conditions that the temperature is 38 ℃ and the vacuum degree is 1.6kPa until no ethanol smell exists. Respectively collecting ethanol and the hydrolyzed sericin peptide fractionated concentrate without ethanol, wherein the collected ethanol can be used for preparing an aqueous solution with the ethanol volume percentage concentration of 45%; and (3) separating the collected hydrolyzed sericin peptide concentrate subjected to ethanol removal by a grading way to obtain a hydrolyzed sericin peptide concentrate subjected to graded separation and ethanol removal, which is used for preparing the hydrolyzed sericin peptide freeze-dried powder in the next step.

(10) Preparation of hydrolyzed sericin peptide lyophilized powder

After the step (9) is finished, placing the hydrolyzed sericin peptide fractional separation ethanol-removing concentrated solution prepared in the step (9) in a low-temperature refrigerator of-35C for pre-freezing for 15h, transferring the pre-frozen hydrolyzed sericin peptide fractional separation ethanol-removing concentrated solution into a freeze dryer, and carrying out freeze drying for 30h under the conditions that the temperature is-45 ℃ and the gauge pressure is 35Pa, so as to prepare three kinds of hydrolyzed sericin peptide freeze-dried powders with average molecular weights of 300-450 Da, 1000-1350 Da and 3200-3600 Da respectively. The three freeze-dried powders are white, the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 300-450 Da is 15-15.3%, and the yield is 11-13% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 1000-1350 Da is 15-15.7%, and the yield is 8-10% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 3200-3600 Da is 15-15.5%, and the yield is 6-8% of the mass of the silkworm cocoon shell; the total yield of the three hydrolyzed sericin peptide freeze-dried powders is 25-31% of the mass of the silkworm cocoon shell.

Example 3

(1) Preparation of cleaned silkworm cocoon shell

Firstly, impurities such as branches and leaves, silkworm chrysalis, stones and the like on the cocoon shells are manually removed, then the manually removed cocoon shells are dispersed in a sodium carbonate aqueous solution with the mass percentage concentration of 0.5% according to the proportion that the volume ratio (kg/L) of the mass of the cocoon shells to the sodium carbonate aqueous solution with the mass percentage concentration of 0.5% is 1: 40, and the mixture is stirred for 40min at the temperature of 80 ℃. Filtering, and respectively collecting the filter residue and filtrate. Pumping the collected filtrate into a wastewater tank for biochemical treatment, and discharging after reaching the standard; and preparing the collected filter residues into clean silkworm cocoon shells for the next step to prepare the degumming solution.

(2) Preparation of sericin solution

After the step (1), firstly dissolving anhydrous sodium carbonate into purified water according to the ratio (kg/L) of the mass of the anhydrous sodium carbonate to the volume of the purified water of 1: 200 to prepare a sodium carbonate aqueous solution with the mass percentage concentration of 0.5%; and then according to the proportion that the volume ratio (kg/L) of the mass of the clean silkworm cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 0.5% is 1: 120, dispersing the clean silkworm cocoon shells in the sodium carbonate aqueous solution with the mass percentage concentration of 0.5%, refluxing and removing sericin for 6 hours at 100 ℃ under stirring, filtering, and respectively collecting the filtered residue of the removed sericin and the filtrate. Adding purified water with the mass 30 times of that of the clean silkworm cocoon shells into the collected sericin removal filter residues, washing for 30min under stirring, filtering again, respectively collecting the washed filter residues and washing filtrate, and combining the collected washing filtrate with the collected sericin removal filtrate to obtain a sericin solution for preparing a hydrolyzed sericin peptide solution in the next step; and (3) preparing the degummed silk fibroin from the collected washing and filtering residues for preparing the hydrolyzed silk peptide.

(3) Preparation of hydrolyzed sericin peptide solution

And (3) after the step (2) is finished, pumping the sericin solution prepared in the step (2) into an ultrafilter with the molecular weight cutoff of 10000Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.4MPa until the ratio (L/L) of the volume of ultrafiltration cutoff liquid to the volume of ultrafiltration filtrate is 1: 6. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, wherein the collected ultrafiltration filtrate contains sodium carbonate and is used for washing the silkworm cocoon shells in the next batch; pumping the collected ultrafiltration trapped fluid into a hydrolysis kettle, heating to 60 ℃ at a stirring speed of 120r/min, adjusting the pH to 9.5 by using dilute sodium hydroxide, adding 3.0T of Novitin alkaline protease according to the mass ratio (kg/kg) of 3.0T of Novitin alkaline protease to the cleaned silkworm cocoon shell of 1: 200, and stirring and hydrolyzing at a constant temperature of 60 ℃ at a stirring speed of 120r/min for 2 hours; then adding the Novier flavor protease according to the mass ratio (kg/kg) of the Novier flavor protease to the cleaned silkworm cocoon shell of 1: 160, stirring and hydrolyzing for 2h at the constant temperature of 60 ℃ at the stirring speed of 120 r/min. Then preparing a hydrolyzed sericin peptide solution which is used for preparing a hydrolyzed sericin peptide decoloring solution.

(4) Assembled activated macroporous resin decoloring system

Dispersing newly purchased activated HP20 resin in purified water with the volume 5 times that of the resin, pumping the resin into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharge valve after the resin is settled into the resin column, discharging redundant water, stopping discharging the liquid until the liquid level is 10 cm higher than the upper surface of the resin column, and assembling the activated HP20 resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively, and assembling an activated macroporous resin decolorizing system for preparing the hydrolyzed sericin peptide decolorizing solution in the next step.

(5) Preparation of hydrolyzed sericin peptide decolorizing solution

And (4) after the step (4) is finished, pumping the hydrolyzed sericin peptide solution prepared in the step (3) into an ultrafilter with the molecular weight cutoff of 30000Da, and carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.4MPa until the ratio (L/L) of the volume of ultrafiltration cutoff liquid to the volume of ultrafiltration filtrate is 1: 9. Respectively collecting ultrafiltration trapped fluid and ultrafiltration filtrate, pumping the collected ultrafiltration trapped fluid into a wastewater tank for biochemical treatment, and discharging after reaching the standard; the collected ultrafiltrate was pumped into an activated HP20 resin column and decolorized at a flow rate of 4 times the volume of the resin column per hour, the ratio (L/L) of the volume of the activated HP20 resin column to the volume of the ultrafiltrate was 1: 40. Respectively collecting column-passing effluent and an HP20 resin column for adsorbing pigment, and preparing hydrolyzed sericin peptide decolorization solution for preparing hydrolyzed sericin peptide fractionation solution; pumping the collected pigment adsorbing HP20 resin column into 85% ethanol water solution, eluting at flow rate 4 times the volume of resin column/hr, wherein the ratio (L/L) of pigment adsorbing HP20 resin column to 85% ethanol water solution is 1: 4. After the elution was completed, the eluted column liquid and the HP20 resin column from which the pigment was unloaded were collected separately. The collected pigment-off-loaded HP20 resin column was washed with 1 resin column volume of purified water, and the column wash and washed HP20 resin column were collected. The collected column-passing washing liquid contains ethanol and is used for preparing ethanol eluent for washing decoloration in next batch; and (3) carrying out resin column chromatography on the collected washed HP20, namely a regenerated HP20 resin column, and allowing the regenerated HP20 resin column to be used for adsorbing pigments in the next batch. Pumping the collected eluate into a rotary evaporator, and recovering ethanol until no ethanol smell exists. Respectively collecting the recovered ethanol water solution and the concentrated solution after ethanol removal, and adjusting the concentration of the collected recovered ethanol water solution for next batch of washing decoloration; pumping the collected concentrated solution without ethanol into a wastewater tank for biochemical treatment, and discharging after reaching the standard.

(6) Assembly activated MCI GEL resin system

Dispersing newly purchased MCI GEL CHP2MG Y resin with the particle size of 25-35 mu m into an aqueous solution with the volume percentage concentration of 60 percent of ethanol and the volume of 5 times of the resin volume, pumping the aqueous solution into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharging valve after the resin is settled into the resin column, discharging redundant water, stopping discharging the liquid until the liquid level is 2 cm higher than the upper surface of the resin column, and assembling the activated MCI GEL CHP2MG Y resin column. And finally, connecting the resin column with a peristaltic pump, an ultraviolet detector and a collector respectively to assemble an activated MCI GEL resin system for preparing the hydrolyzed sericin peptide fractional separation solution in the next step.

(7) Preparation of hydrolyzed sericin peptide fractionation liquid

And (5) after the step (6) is finished, pumping the hydrolyzed sericin peptide decoloration solution collected in the step (5) into an activated MCI GEL CHP 2MGY resin column, wherein the volume ratio (L/L) of the hydrolyzed sericin peptide decoloration solution to the activated resin column is 1: 20, and the pumping flow rate of the hydrolyzed sericin peptide decoloration solution is 3 times of the volume of the activated resin column per hour. After the completion of the pumping of the hydrolyzed sericin peptide decolorization solution, respectively collecting the resin column loaded with the decolorized and hydrolyzed sericin peptide and the column-passing effluent, pumping the collected column-passing effluent containing 60% ethanol by volume percentage into a rotary evaporator, and recovering the ethanol; and pumping the collected resin column loaded with the decolored hydrolyzed sericin peptide into an aqueous solution with the ethanol volume percentage concentration of 60%, wherein the flow rate of the aqueous solution is 3 times of the volume of the resin column per hour. Respectively collecting the column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51min and the resin column for unloading the decolored hydrolyzed sericin peptide, and preparing a hydrolyzed sericin peptide fractional separation liquid for preparing a hydrolyzed sericin peptide fractional separation concentrated solution in the next step by collecting the collected column-passing effluent liquid with strong absorption peaks at 280nm from 14 to 21min, 26 to 37min and 42 to 51 min; the collected resin column for unloading, decoloring and hydrolyzing the sericin peptide is a regenerated MCI GEL CHP2MG Y resin column, and can be used for preparing a hydrolyzed sericin peptide fractionation liquid again.

(8) Preparation of hydrolyzed sericin peptide fractionation concentrate

And (7) after the step (7) is finished, respectively pumping the hydrolyzed sericin peptide fractionation liquid collected in the step (7) into ultrafilters with molecular weight cutoff of 3500Da, and carrying out ultrafiltration treatment under the gauge pressure of 9.0MPa until the total nitrogen content in the ultrafiltration cutoff liquid reaches 4.5%. Respectively collecting ultrafiltration filtrate and ultrafiltration retentate, pumping the collected ultrafiltration filtrate containing 60% ethanol by volume percentage into a rotary evaporator, and recovering ethanol; and filtering the collected ultrafiltration trapped fluid by using sterilized 0.22 mu m microporous filter membranes respectively, and collecting the filtered fluid, namely preparing hydrolyzed sericin peptide fractionation concentrated solution which is respectively used for preparing hydrolyzed sericin peptide fractionation ethanol-removed concentrated solution.

And (4) after the step (8) is finished, pumping the hydrolyzed sericin peptide fractional separation concentrated solution prepared in the step (8) into a low-temperature high-vacuum solvent recovery device, and recovering ethanol under the conditions that the temperature is 45 ℃ and the vacuum degree is 3kPa until no ethanol smell exists. Respectively collecting ethanol and the hydrolyzed sericin peptide fractionated concentrate without ethanol, wherein the collected ethanol can be used for preparing an aqueous solution with ethanol volume percentage concentration of 60%; and (3) separating the collected hydrolyzed sericin peptide concentrate subjected to ethanol removal by a grading way to obtain a hydrolyzed sericin peptide concentrate subjected to graded separation and ethanol removal, which is used for preparing the hydrolyzed sericin peptide freeze-dried powder in the next step.

(10) Preparation of hydrolyzed sericin peptide lyophilized powder

After the step (9) is finished, placing the hydrolyzed sericin peptide fractional separation ethanol-removing concentrated solution prepared in the step (9) in a low-temperature refrigerator at-30 ℃ for pre-freezing for 20 hours, transferring the frozen sericin peptide fractional separation ethanol-removing concentrated solution into a freeze dryer, and carrying out freeze drying for 36 hours under the conditions that the temperature is-40 ℃ and the gauge pressure is 50Pa, so as to prepare three kinds of hydrolyzed sericin peptide freeze-dried powders with average molecular weights of 300-450 Da, 1000-1350 Da and 3200-3600 Da respectively. The three freeze-dried powders are white, the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 300-450 Da is 15-15.3%, and the yield is 11-13% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 1000-1350 Da is 15-15.7%, and the yield is 8-10% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 3200-3600 Da is 15-15.5%, and the yield is 6-8% of the mass of the silkworm cocoon shell; the total yield of the three hydrolyzed sericin peptide freeze-dried powders is 25-31% of the mass of the silkworm cocoon shell.

Claims

1. A preparation method of hydrolyzed sericin peptide is characterized by comprising the following specific process steps:

(1) preparation of cleaned silkworm cocoon shell

Firstly, manually removing impurities on cocoon shells, then dispersing the manually-removed cocoon shells in a sodium carbonate aqueous solution with the mass percentage concentration of 0.2-0.5% according to the proportion that the volume ratio of the mass of the cocoon shells to the volume of the sodium carbonate aqueous solution with the mass percentage concentration of 0.2-0.5% is 1: 20-40 kg/L, stirring for 30-40 min at the temperature of 60-80 ℃, filtering, respectively collecting filter residues and filtrate, pumping the collected filtrate into a waste water tank for biochemical treatment, and discharging after reaching the standard; preparing clean silkworm cocoon shells from the collected filter residues for preparing a degumming solution in the next step;

(2) preparation of sericin solution

After the step (1), firstly dissolving anhydrous sodium carbonate into purified water according to the ratio of the mass of the anhydrous sodium carbonate to the volume of the purified water of 1: 20-200 kg/L to prepare a sodium carbonate aqueous solution with the mass percentage concentration of 0.5-5%; dispersing the cleaned silkworm cocoon shells into a 0.5-5% by mass sodium carbonate aqueous solution according to the volume ratio of 1: 60-120 kg/L of the mass of the cleaned silkworm cocoon shells to the 0.5-5% by mass sodium carbonate aqueous solution, refluxing and degumming for 3-6 h at 90-100 ℃ under stirring, filtering, respectively collecting the sericin-removed filter residues and filtrate, adding purified water 20-30 times the mass of the cleaned silkworm cocoon shells into the collected sericin-removed filter residues, washing for 15-30 min under stirring, filtering again, respectively collecting the washed filter residues and the washed filtrate, combining the collected washed filtrate with the collected sericin-removed filtrate to obtain a sericin solution, and using the sericin solution to prepare a hydrolyzed sericin peptide solution in the next step; the collected washing and filtering residues are used for preparing the degummed silk fibroin which is used for preparing the hydrolyzed silk peptide;

(3) preparation of hydrolyzed sericin peptide solution

After the step (2) is finished, pumping the sericin solution prepared in the step (2) into an ultrafilter with the molecular weight cutoff of 3500-10000 Da, carrying out ultrafiltration treatment under the condition that the gauge pressure is 0.1-0.4 MPa until the ratio of the volume of ultrafiltration retentate to the volume of ultrafiltration filtrate is 1: 3-6L/L, respectively collecting the ultrafiltration retentate and the ultrafiltration filtrate, and washing the silkworm cocoon shells in the next batch by using the collected ultrafiltration filtrate containing sodium carbonate; pumping the collected ultrafiltration retentate into a hydrolysis kettle, heating to 50-60 ℃ at a stirring speed of 60-120 r/min, adjusting the pH to 8.0-9.5 by using dilute sodium hydroxide, adding 3.0T Novien alkaline protease or animal proteolytic enzyme according to the mass ratio of 1: 100-200 kg/kg of Novien alkaline protease or animal proteolytic enzyme to the cleaned silkworm cocoon shell, and stirring and hydrolyzing at a constant temperature of 50-60 ℃ at a stirring speed of 60-120 r/min for 1-2 h; then adding the Novier flavor protease or the papain according to the mass ratio of the Novier flavor protease or the papain to the cleaned silkworm cocoon shell of 1: 80-160 kg/kg, stirring and hydrolyzing at the stirring speed of 60-120 r/min and the constant temperature of 50-60 ℃ for 1-2 h to prepare a hydrolyzed sericin peptide solution for preparing a hydrolyzed sericin peptide decoloring solution;

(4) assembled activated macroporous resin decoloring system

Dispersing newly-purchased activated Amberlite TMXAD7HP, Amberlite TMXAD761 or HP20 resin in purified water with the volume of 3-5 times of that of the resin, pumping the resin into a medium-pressure chromatographic column by using a peristaltic pump, starting a liquid discharge valve after the resin is settled into the resin column, discharging redundant water, stopping discharging the liquid until the liquid level is 2-10 cm higher than the upper surface of the resin column, assembling an activated Amberlite TMXAD7HP, Amberlite TMXAD761 or HP20 resin column, and finally connecting the resin column with the peristaltic pump, an ultraviolet detector and a collector respectively, and assembling an activated macroporous resin decolorizing system for preparing hydrolyzed sericin peptide liquid in the next step;

(5) preparation of hydrolyzed sericin peptide decolorizing solution

After the step (4) is finished, pumping the hydrolyzed sericin peptide solution prepared in the step (3) into an ultrafilter with the molecular weight cutoff of 10000-30000 Da, performing ultrafiltration treatment under the condition that the gauge pressure is 0.1-0.4 MPa until the ratio of the volume of ultrafiltration retentate to the volume of ultrafiltration filtrate is 1: 6-9L/L, respectively collecting the ultrafiltration retentate and the ultrafiltration filtrate, pumping the collected ultrafiltration retentate into a wastewater tank for biochemical treatment, and discharging after reaching the standard; pumping the collected ultrafiltration filtrate into an activated Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, carrying out decolorization treatment at the flow rate of 2-4 times the volume of the resin column per hour, wherein the ratio of the volume of the activated Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column to the volume of the ultrafiltration filtrate is 1: 20-40L/L, respectively collecting column effluent and pigment-adsorbing Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, and preparing hydrolyzed sericin decolorization solution for the collected column effluent; pumping an aqueous solution with the ethanol volume percentage concentration of 65-85% into a collected pigment adsorbing Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, carrying out elution treatment at the flow rate of 2-4 times the volume/hour of the resin column, wherein the volume ratio of the pigment adsorbing Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column to the aqueous solution with the ethanol volume percentage concentration of 65-85% is 1: 2-4L/L, after the elution is finished, respectively collecting an eluted column liquid and pigment unloading Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, washing the collected pigment unloading Amberlite TMXAD7 or Amberlite XAD761 or HP20 resin column with the volume of 0.5-1 time of the resin column, collecting a column washing liquid and a washed Amberlite XAD7 or TMberlite XAD HP or HP20 resin column containing ethanol, and preparing an ethanol-containing eluent in batches, wherein the eluted column is 0.5-1 time; collecting washed Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, namely regenerated Amberlite TMXAD7HP or Amberlite TMXAD761 or HP20 resin column, which can be used for adsorbing pigment in the next batch, pumping collected eluted column liquid into a rotary evaporator, recovering ethanol until no ethanol smell exists, respectively collecting recovered ethanol water solution and ethanol-removed concentrated solution, and adjusting the concentration of the collected recovered ethanol water solution and then using the recovered ethanol water solution for washing decolorant in the next batch; pumping the collected concentrated solution without ethanol into a wastewater tank for biochemical treatment, and discharging after reaching the standard;

(6) assembly activated MCI GEL resin system

Dispersing newly purchased MCI GEL CHP20P resin with the particle size of 37-75 microns or MCI GELCHP20SS resin with the particle size of 63-150 microns or MCI GEL CHP2MG Y resin with the particle size of 25-35 microns in an aqueous solution with the volume percentage concentration of 30-60% of ethanol which is 3-5 times of the volume of the resin, pumping the aqueous solution into a medium-pressure chromatographic column by using a peristaltic pump, opening a liquid discharge valve after the resin is settled into a resin column, discharging excessive water, stopping discharging until the liquid level is 0.5-2 cm higher than the upper surface of the resin column, assembling an activated MCI GEL CHP20P or MCI GEL CHP20SS or MCI GEL CHP2MG Y resin column, and finally connecting the resin column with the peristaltic pump, an ultraviolet detector and a collector respectively to assemble an activated MCI GEL resin system for preparing a hydrolyzed sericin peptide fractional separation liquid in the next step;

(7) preparation of hydrolyzed sericin peptide fractionation liquid

After the step (6) is finished, pumping the hydrolyzed sericin peptide decolorizing solution collected in the step (5) into an activated MCI GEL CHP20P or MCIGEL CHP20SS or MCI GEL CHP2MG Y resin column, wherein the volume ratio of the hydrolyzed sericin peptide decolorizing solution to the activated resin column is 1: 10-20L/L, the pumping flow rate of the hydrolyzed sericin peptide decolorizing solution is 1-3 times of the volume of the activated resin column per hour, after the hydrolyzed sericin peptide decolorizing solution is pumped, respectively collecting resin column loaded with decolorized hydrolyzed sericin peptide and column-passing effluent, pumping the collected column-passing effluent containing 30-60% by volume of ethanol into a rotary evaporator, and recovering the ethanol; pumping the collected resin column loaded with the decolored hydrolyzed sericin peptide into an aqueous solution with the ethanol volume percentage concentration of 30-60%, wherein the flow rate of the pumped aqueous solution is 1-3 times of the volume of the resin column per hour, respectively collecting column-passing effluent liquid with strong absorption peaks at 280nm and resin column unloading decolored hydrolyzed sericin peptide at 14-21 min, 26-37 min and 42-51 min, and collecting column-passing effluent liquid with strong absorption peaks at 280nm at 14-21 min, 26-37 min and 42-51 min, namely preparing hydrolyzed sericin peptide fractional separation liquid for preparing a hydrolyzed sericin peptide fractional separation concentrated solution in the next step; the collected resin column for unloading, decoloring and hydrolyzing the sericin peptide is a regenerated MCI GEL CHP20P or MCI GEL CHP20SS or MCI GEL CHP 2MGY resin column, and can be used for preparing a hydrolyzed sericin peptide fractionation liquid again;

(8) preparation of hydrolyzed sericin peptide fractionation concentrate

After the step (7) is finished, respectively pumping the hydrolyzed sericin peptide fractional separation liquid collected in the step (7) into a nanofiltration/ultrafiltration filter with the molecular weight cutoff of 600-3500 Da, performing nanofiltration/ultrafiltration treatment under the gauge pressure of 1.0-9.0 MPa until the total nitrogen content in the nanofiltration/ultrafiltration trapped fluid reaches 3-4.5%, respectively collecting nanofiltration/ultrafiltration filtrate and nanofiltration/ultrafiltration trapped fluid, pumping the collected nanofiltration/ultrafiltration filtrate containing 30-60% ethanol by volume percentage concentration into a rotary evaporator, and recovering the ethanol; filtering the collected nanofiltration/ultrafiltration trapped fluid by sterilized microporous filter membranes with the diameter of 0.22 mu m respectively, and collecting the filtered fluid, namely preparing hydrolyzed sericin peptide fractionation concentrated solution which is respectively used for preparing hydrolyzed sericin peptide fractionation ethanol-removed concentrated solution;

After the step (8) is finished, pumping the hydrolyzed sericin peptide fractional separation concentrated solution prepared in the step (8) into a low-temperature high-vacuum solvent recovery device, recovering ethanol under the conditions that the temperature is 30-45 ℃ and the vacuum degree is 0.1-3 kPa until no ethanol smell exists, respectively collecting ethanol and the hydrolyzed sericin peptide fractional separation concentrated solution without ethanol, wherein the collected ethanol can be used for preparing an aqueous solution with the ethanol volume percentage concentration of 30-60%; separating the collected hydrolyzed sericin peptide concentrate subjected to ethanol removal by a grading method to obtain a hydrolyzed sericin peptide concentrate subjected to graded separation and ethanol removal for preparing hydrolyzed sericin peptide freeze-dried powder;

(10) preparation of hydrolyzed sericin peptide lyophilized powder

After the step (9) is finished, respectively pre-freezing the hydrolyzed sericin peptide fractional separation ethanol-removing concentrated solution prepared in the step (9) in a low-temperature refrigerator at-40 to-30 ℃ for 10-20 h, then transferring the obtained product into a freeze dryer, and carrying out freeze drying for 24-36 h under the conditions that the temperature is-50 to-40 ℃ and the gauge pressure is 20-50 Pa, so as to prepare three kinds of hydrolyzed sericin peptide freeze-dried powders with average molecular weights of 300-450 Da, 1000-1350 Da and 3200-3600 Da respectively, wherein the three kinds of freeze-dried powders are white, the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powders with the average molecular weights of 300-450 Da is 15-15.3%, and the yield is 11-13% of the mass of the cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 1000-1350 Da is 15-15.7%, and the yield is 8-10% of the mass of the silkworm cocoon shell; the total nitrogen content of the hydrolyzed sericin peptide freeze-dried powder with the average molecular weight of 3200-3600 Da is 15-15.5%, and the yield is 6-8% of the mass of the silkworm cocoon shell; the total yield of the three hydrolyzed sericin peptide freeze-dried powders is 25-31% of the mass of the silkworm cocoon shell.