CN108745216B

CN108745216B - Preparation method of silk fibroin powder for preparing SF-Cd slow-release microspheres

Info

Publication number: CN108745216B
Application number: CN201810033262.6A
Authority: CN
Inventors: 吴遵义; 曹彦美; 王文琦; 翟滢皓; 熊春华; 陈青; 姬澳淇
Original assignee: Zhejiang Gongshang University
Current assignee: Zhejiang Gongshang University
Priority date: 2018-01-14
Filing date: 2018-01-14
Publication date: 2021-05-28
Anticipated expiration: 2038-01-14
Also published as: CN108745216A

Abstract

The invention relates to a preparation method and application of silk fibroin powder for preparing SF-Cd slow-release microspheres. According to the method, mild low-temperature degassing treatment is adopted, so that the biological activity of the silk fibroin is greatly reserved. The target molecular weight silk fibroin is purified and separated by a low-temperature degumming and degradation method and an innovative overlapping dialysis method, the yield of the target product soluble silk fibroin is 24.23 percent, the target product soluble silk fibroin can be obtained by conventional component analysis, the purity of the molecular weight soluble silk fibroin prepared by a laboratory is higher, and the later-stage modification is easy.

Description

Preparation method of silk fibroin powder for preparing SF-Cd slow-release microspheres

Technical Field

The invention relates to a preparation method and application of silk fibroin powder for preparing SF-Cd slow-release microspheres.

Background

Silk Fibroin (SF) is widely applied to the pharmaceutical and food industries due to good biocompatibility and slow release performance, and meanwhile, due to good degradation efficiency, the Silk Fibroin is a sustainable biological material with high efficiency and wide application range, and cannot cause pollution to the environment. However, silk fibroin is only used as the sustained-release microsphere, which has poor hydrophilicity and is easy to explode to release the load at the initial stage of sustained release, so that the load can be quickly releasedResulting in a sudden change in the release rate affecting the sustained release effect. It has been found that microwave Maillard (Maillard Reaction by)Microwave) The reaction can effectively improve the function and the property of the silk fibroin, the method not only has the advantages of high efficiency, environmental protection, selective heating and the like, but also can effectively avoid using an organic solvent and a cross-linking agent.

At present, more methods for degumming silk are used, such as high-temperature and high-pressure boiling water degumming, urea degumming, enzyme degumming, sodium carbonate degumming and the like. Different degumming methods affect the solubility of the product, the efficiency of the later hydrolysis, the molecular weight, etc. Especially, the high-temperature high-pressure treatment method has larger influence on the stretching rate and the breaking strength of the fibroin, both the stretching rate and the breaking strength are reduced, and the whiteness of the product after the alkaline protease degumming is obviously improved.

The degummed silk fibroin is insoluble in water, and can be hydrolyzed by a salt solution or an organic solvent to obtain a silk fibroin solution. At present, most lithium bromide or ternary system degradation liquid is applied to hydrolyze silk element. Park et al^[7]The strength and stability changes of the silk fibroin fibers in the five kinds of hydrolysate are discussed. The results show that the reaction proceeds through Na₂CO₃The thermal stability of the solution degummed fibroin is significantly reduced, and the tensile strength is poorer than that of the fibroin degummed by the urea solution. The research of polyacrylamide electrophoresis experiments shows that the structure of the silk fibroin obtained by the treatment of the high-temperature urea solution and the low-temperature lithium bromide solution is similar to that in an organism. Wherein, Na₂CO₃Degumming can break peptide bonds in the silk fibroin, and differential scanning calorimeter detection shows that the higher the hydrolysis temperature, the less the peptide bonds in the silk fibroin are broken, and the higher the integrity of the protein chains is.

Disclosure of Invention

The invention aims to provide a preparation method of soluble silk fibroin powder with better emulsifying property and foaming property.

The preparation method of the silk fibroin powder for preparing the SF-Cd slow-release microspheres comprises the following steps:

1) low temperature degumming

Removing impurities in raw silkworm cocoons, shearing, weighing silk fragments with certain mass, and adding deionized water and anhydrous sodium carbonate; immersing silk completely in the solution, stirring uniformly, sealing, introducing nitrogen gas to isolate the air, degumming, placing in a constant temperature tank at 37-38 ℃, adding a rotor, and stirring at constant speed to react; taking out and filtering every 24h, cleaning the filtrate with 25% sodium carbonate solution, adding the same volume of 25% sodium carbonate solution, continuously stirring for reaction, replacing the solution every 24h, and reserving the degumming filtrate;

2) degradation of degummed fibroin

Weighing part of freeze-dried silk, and mixing silk fibroin (g) according to the volume mass ratio: solvent (mL) ═ 1: 70 adding a solvent for dissolving the silk fibroin; stirring, sealing, introducing nitrogen gas to isolate air for degradation, placing in a 37-38 deg.C constant temperature tank, adding rotor, stirring at constant speed for reaction, taking out after three days, freezing and centrifuging at low temperature of 6000r/min for 30min, and collecting supernatant;

3) separation and purification of active silk fibroin with medium molecular weight

And (3) overlapping dialysis bags with molecular weight cut-off of 20kD and 50kD respectively, wherein 50kD is in, and 20kD is out, pouring supernatant into the 50kD dialysis bag, dialyzing in deionized water for 48h, changing water every 12h, and after dialysis, carrying out freeze drying on samples in the dialysis bags to obtain the silk fibroin powder.

Preferably, the mass/g of silk in the step 1): volume of water/L: the ratio of the mass/g of the anhydrous sodium carbonate is 10: 1: 5.

preferably, the solvent for dissolving the silk fibroin in the step 2) is a solution containing 20% of ethanol and 40% of calcium chloride; solution containing 30% ethanol and 40% calcium chloride, or solution containing 40% ethanol and 40% calcium chloride

Preferably, the solvent for dissolving silk fibroin in step 2) is a solution containing 30% ethanol and 40% calcium chloride.

In addition, the invention also provides application of the silk fibroin powder in preparation of SF-Cd slow-release microspheres.

The SF-Cd slow release microsphere prepared from the silk fibroin powder adopts a microwave synthesis method, and the specific method comprises the following steps:

weighing a certain mass of silk fibroin, placing the silk fibroin in a beaker, pouring a certain amount of deionized water, adjusting the pH value to 7, and preparing a solution with 10% of protein content. Adding polysaccharide, wherein the mass ratio of the silk fibroin: polysaccharide 2.5: 1, preparing a proteoglycan mixed solution. Stirring continuously by using a magnetic stirring device until the mixture is uniform in the preparation process, and then freeze-drying for later use;

grinding the powder uniformly, sieving the powder by a 150-mesh sieve, placing equivalent sieved substances in a double-frequency ultrasonic microwave combined catalytic synthesizer, adjusting the microwave power to be 500w, the ultrasonic power to be 300w and the reaction time to be 4min, taking out the powder after reaction, and finishing the reaction in ice-water bath for 1min to obtain a mixed product;

preparing proteoglycan powder with concentration ratio, treating by the same method, placing in a double-frequency ultrasonic microwave combined catalytic synthesizer for reaction, adjusting microwave power (100w, 300w, 500w, 700w and 900w) and ultrasonic power (100w, 200w and 300w), taking out after reacting for a period of time, and finishing the reaction in ice-water bath for 1 minute to obtain an SF-Cd mixed product. The invention is further illustrated below:

1) low temperature degumming

Traditional degumming methods are violent, boiling needs to be carried out for several hours under the condition of high temperature, and strong acid and strong alkali treatment is carried out, so that the space structure of the silk fibroin is damaged to a certain extent, the subsequent application performance is reduced, and even the activity of the silk fibroin is lost. The subject adopts mild low-temperature degassing treatment, and the biological activity of the silk fibroin is greatly reserved.

Removing impurities in raw silkworm cocoons, shearing, weighing silk fragments with certain mass, and adding deionized water and anhydrous sodium carbonate according to a ratio (the mass/g of silk: the volume of water/L: the mass/g of anhydrous sodium carbonate is 10: 1: 5). Immersing silk in the solution, stirring, sealing, introducing nitrogen gas to remove air, placing in 37-38 deg.C constant temperature tank, adding rotor, and stirring to react. Taking out every 24h, filtering, cleaning the filtrate with 25% sodium carbonate solution, adding equal volume of 25% sodium carbonate solution, stirring for reaction, changing the solution every 24h, collecting the degumming filtrate, and performing ultraviolet spectrum determination.

2) Degradation of degummed fibroin

Weighing part of the freeze-dried silk, and dividing into six equal parts. Silk fibroin (g) according to volume mass ratio: solvent (mL) ═ 1: 70 adding the following solvent for dissolving silk fibroin: a solution containing 20% ethanol and 40% calcium chloride; a solution containing 30% ethanol and 40% calcium chloride; or a solution containing 40% ethanol and 40% calcium chloride. Stirring, sealing, introducing nitrogen gas to isolate air for degradation, placing in 37-38 deg.C constant temperature tank, adding rotor, stirring at constant speed for reaction, taking out after three days, freezing and centrifuging at 6000r/min for 30min, and collecting supernatant.

The preliminarily separated silk fibroin has a large molecular weight range, and the folding inside molecules is complex, so that the later modification is not facilitated, and therefore, the preparation of the micromolecule silk fibroin with a specific molecular weight range is very important. The experimental innovation overlap dialysis method is used for purifying and separating the silk fibroin with the target molecular weight, and the method comprises the following steps:

the dialysis bags with the molecular weight cut-off of 20kD and 50kD are overlapped, 50kD is in, and 20kD is out, supernatant is poured into the 50kD dialysis bag, the dialysis bag is dialyzed in deionized water for 48 hours, and water changing operation is carried out every 12 hours. And after the dialysis is finished, freezing and drying the sample between the dialysis bags to obtain the silk fibroin powder.

The invention prepares silk fibroin by low-temperature degassing treatment, degradation and purification separation, takes the silk fibroin as a modified object, respectively grafts four saccharides through microwave wet Maillard reaction and screens, wherein the grafting rate of cyclodextrin (Cd) is highest, and the glycosylation graft product of the silk fibroin is prepared.

The inventor of the application explores the influence of different reaction factors on the content of free amino acid of silk fibroin and the browning degree, and simultaneously adopts various characterization means to compare the influence of microwave heating and water bath heating on the physicochemical properties, the structural functions and the like of the SF-Cd covalent compound. And finally, carrying out oral simulated slow release performance test on the fibroin protein glycosylated microspheres by taking levo carvone (Left-Carvon, L-Ca) as a slow release model substance, and determining the slow release effect. The main study content and conclusions are as follows:

1. the mild low-temperature degassing treatment is selected, so that the biological activity of the silk fibroin is greatly reserved. The target molecular weight silk fibroin is purified and separated by a low-temperature degumming and degradation method and an innovative overlapping dialysis method, the yield of the target product soluble silk fibroin is 24.23 percent, the target product soluble silk fibroin can be obtained by conventional component analysis, the purity of the molecular weight soluble silk fibroin prepared by a laboratory is higher, and the later-stage modification is easy. The emulsifying property and the foaming property of the silk fibroin are researched, the emulsifying property is increased along with the increase of the concentration of the silk fibroin at low concentration, the higher the concentration is, the better the emulsifying stability is, meanwhile, the emulsifying property is relatively higher when the pH value is 8, and different pH values have little influence on the emulsifying stability of the silk fibroin; when the concentration is 1%, the foaming ability is strongest, and when the concentration is higher than 0.5%, the foaming stability is better. The structure of the product is analyzed by means of characterization means such as FTIR, DSC, SEM and the like.

2. Taking silk fibroin as a modified object, respectively grafting beta cyclodextrin, starch, maltodextrin and agarose through a wet-process Maillard reaction, selecting a microwave Maillard method to improve the reaction rate to prepare a silk fibroin glycosylation graft product, screening out the silk fibroin graft cyclodextrin as an optimal product according to the grafting rate, researching the influence of factors such as ultrasonic power, reaction time, microwave power, substrate proportion and the like on the content of free amino acid of the silk fibroin and the browning degree, and finally obtaining the optimal reaction condition of the silk fibroin glycosylation through a response surface analysis method: and (3) preparing a substrate by using silk fibroin: β -Cd ═ 2.5: 1. the microwave power is 500w, the reaction time is 4min, and the grafting rate is 82.95% through experimental verification, so that the obtained fibroin sustained-release protein has excellent performance.

Drawings

Figure 1 effect of time on degumming effect.

FIG. 2 SDS-PAGE of the products under different hydrolysis conditions.

Figure 3 effect of concentration on emulsification performance of silk fibroin.

FIG. 4 effect of solution pH on the emulsifying properties of silk fibroin.

Figure 5 effect of concentration on emulsion stability of silk fibroin.

FIG. 6 effect of solution pH on emulsion stability of silk fibroin.

FIG. 7 influence of silk fibroin concentration on foaming ability and foaming stability.

Figure 8 infrared spectrum of silk fibroin.

Figure 9 DSC thermogram of silk fibroin.

Fig. 10 electron micrograph of silk fibroin, wherein fig. 10b is an enlarged view of the upper left corner of fig. 10 a.

FIG. 11 the effect of different ultrasound powers on the grafting yield.

FIG. 12 the effect of different ultrasound powers on the degree of browning.

FIG. 13 effect of microwave power on grafting yield and browning level.

FIG. 14 effect of substrate concentration ratio on grafting ratio and browning level.

FIG. 15 is a graph showing the response surface analysis of the influence of the substrate ratio, microwave power and microwave time on the graft ratio.

Figure 16 effect of microwave heating on silk fibroin glycosylation intermediates.

FIG. 17 influence of solution pH on Zeta potential and particle size of the product.

Detailed Description

Example 1 preparation method of fibroin powder

1) low temperature degumming

After degumming, putting a proper amount of silkworm cocoon fragments into a small beaker, detecting degumming effect by using 1% picric acid carmine solution, immersing the sample in the picric acid carmine solution, boiling for 5min, washing with deionized water, and observing the color of silk^[72]。

2) Degradation of degummed fibroin

Weighing part of the freeze-dried silk, and dividing into six equal parts. Silk fibroin (g) according to volume mass ratio: solvent (mL) ═ 1: 70 adding the following solvent for dissolving silk fibroin: firstly, 40% of calcium chloride solution; 60% calcium chloride solution; ③ a solution containing 20 percent of ethanol and 40 percent of calcium chloride; solution containing 30% ethanol and 40% calcium chloride; a solution containing 40% ethanol and 40% calcium chloride; sixthly, 60 percent ethanol solution. Stirring, sealing, introducing nitrogen gas to isolate air for degradation, placing in 37-38 deg.C constant temperature tank, adding rotor, stirring at constant speed for reaction, taking out after three days, freezing and centrifuging at 6000r/min for 30min, and collecting supernatant.

The preliminarily separated silk fibroin has large molecular weight range and complicated folding inside molecules, and is not beneficial to later-stage modification^[14]Therefore, it is important to prepare small-molecule silk fibroin with a specific molecular weight range. The experimental innovation is that the overlapping dialysis method is used for purifying and separating the silk fibroin with the target molecular weight, and the method is as follows.

Example 2 analysis and characterization of physical and chemical indicators

(1) Determination of conventional Components

Determination of proteins: micro Kjeldahl method (GB5511-85)

Fat determination: soxhlet extraction method (GB5497-85)

And (3) measuring moisture: constant weight method at 105 ℃ (GB5512-85)

And (3) determination of ash content: dry ashing method (GB5505-85)

(2) Measurement of emulsifiability and emulsion stability

After being degraded by calcium chloride, the silk fibroin is endowed with good emulsibility, foamability and stability, and the good physicochemical properties determine the application of the silk fibroin in the fields of medical food and the like^[4]Therefore, the research on the emulsifying property, the foaming property and the stability of the silk fibroin is decisive for the improvement of the application value of the silk fibroin.

Taking a certain amount of silk fibroin respectively to prepare silk fibroin solutions with the concentrations of 0.25%, 0.5%, 1% and 2%, and adding a buffer solution to adjust the pH values to be 3, 5, 8 and 10. Respectively adding equal volume of salad oil, dispersing for 5min at 10000r/min, centrifuging for 5min with 10mL, taking out, dividing the emulsion in the centrifuge tube into three layers, namely an oil layer, an emulsifying layer and a water layer from top to bottom, and calculating the emulsifying capacity by measuring the height of the oil layer according to the following formula:

sampling and preparing according to the method, dispersing for 1min at 10000r/min, taking 10mL by using a measuring cylinder, storing at 25 ℃, taking out after 24h, and measuring the heights of an oil layer, an emulsion layer and a water layer. The emulsion stability was calculated by the following formula:

(3) measurement of foaming Property and foaming stability

Taking 10mL of the silk fibroin solution with the concentration of 0.25%, 0.5%, 1% and 2%, dispersing for 2min under the condition that the rotating speed is 10000r/min, and immediately measuring the foam height after stopping.

After the rotation had stopped for 30min, the height of the foam was measured.

(4) Ultraviolet spectrophotometry

The degummed solution obtained in step 1) of example 1 was diluted and subjected to ultraviolet spectroscopy

(5) Infrared spectrogram

Accurately weighing a proper amount of samples according to a mass ratio of 1: adding a certain amount of potassium bromide 50, grinding with mortar until the sample and potassium bromide are fully mixed into uniform powder, pressing the mixed powder into slices on a tablet machine, and using a Fourier infrared spectrophotometer as a full-wave band (4000 plus 400 cm)^-1) And (6) scanning.

(6) Differential thermal DSC scanning

Taking about 3-8mg of freeze-dried silk fibroin product, recording the accurate mass, putting the silk fibroin product into an aluminum tray, spreading a sample in the aluminum tray and compacting the sample by using a cover, setting the temperature scanning range to be 40-350 ℃, and carrying out a temperature rise program: 20 ℃/min. Carrier gas N₂. Carrier gas flow: 20 mL/min. The hollow discs were scanned as blanks prior to each experiment.

(7) Analysis by scanning electron microscope

And taking a certain amount of sample to be detected, adhering the sample to the cut conductive adhesive, spraying gold on the surface of the sample by using an ion sputtering coating machine, and putting the sample into a sample placing chamber of an electron microscope to push and observe the sample after the gold film is coated.

(8) Polypropylene gel electrophoresis

According to the literature, the following treatments are carried out:

preparing a solution with the protein concentration of 1mg/mL from the silk fibroin product, mixing a certain amount of the solution with SDS beta-mercaptoethanol in a proportion of 1: 2, boiling, and injecting sample with the sample amount of 10L. The concentrations of the separation gel and the concentration gel were 12% and 5%, respectively, and the current was 15mA, and the current was adjusted to 20mA after the sample flowed to the separation gel, and when the distance from the bottom edge was 1cm, the electrophoresis was stopped. The dyeing is carried out with Coomassie brilliant blue, and then the dyeing solution is used for decoloring, and then the mixture is soaked in distilled water overnight. The preparation method of the electrophoresis gel is shown in the following table 2-3:

TABLE 2-3 gel electrophoresis composition Table

Table 2-3 Composition of gel electrophoresis

Results and discussion

1 low temperature degumming effect

The solution retained by each filtration was diluted ten times and then its absorbance at 280nm was measured as shown in FIG. 1. It can be seen from the graph that the sericin content in the degumming filtrate of the next day is the highest. The highest sericin content appears on the next day, mainly due to the incomplete swelling of the silk by the sodium carbonate solution on the first day. Sericin in the silkworm cocoon is tightly combined with silk fibroin, and the silk fibroin are adhered layer by layer to form an irregular crossed net structure, so that the silkworm cocoon is not easy to rapidly and completely swell. After the silk was further swelled, the absorbance of the degummed solution was close to 0 on the fourth day and tended to stabilize on the fifth day as shown in the figure. Meanwhile, the color result of the silk product after degumming is yellow by detecting the picric acid carmine solution, so that the fact that the product is degummed completely can be deduced. And the degumming silk fibroin yield is 67.21 percent at the moment, which is close to the theoretical content of the silk fibroin by 70 percent. The degummed silk fibroin has fair color and better texture after being frozen and dried.

2 degummed fibroin degradation

The silk fibroin is mainly composed of three amino acids of 18 amino acids, namely glycine, alanine and serine, and the three amino acids account for more than 80% of the total components. Silk is formed by randomly arranging and curling soluble amino acids in an initial stage in an organism, has no obvious structural characteristics and is basically inactive, silk fibroin molecules can form linear aggregates through self-assembly when being further curled to form a higher-order structure, and at the moment, the physical shearing resistance and the chemical stability of silk fibroin are obviously enhanced due to the appearance of a beta folding mode and the formation of other higher-order conformations. Research shows that the calcium chloride can effectively degrade the silk fibroin. The mechanism is that when the fibroin in a stable state is immersed in a calcium chloride solution with a certain concentration, a large amount of strong polar ions can generate strong hydration, so that a large amount of water is attached to the surface of the fibroin, the water can obviously enhance the movement of polypeptide chains in the fibroin, and van der Waals force among fibroin molecules and hydrogen bonds of amino acid residue side chains are damaged. Meanwhile, calcium chloride can directly react with polar amino acid in fibroin molecules, such as tyrosine which is a large amount of exposed amino acid residues in fibroin and is easy to react with the polar amino acid residues, and the fibroin structure and properties can be greatly damaged by the reaction due to the fact that a large amount of tyrosine exists in both a crystalline region and an amorphous region in the fibroin^[73]. In the subject, calcium chloride is used as a solvent for degrading silk fibroin, and the degradation efficiency under different proportioning concentrations of calcium chloride and ethanol is discussed.

After the reaction of six groups of degradation solutions with different proportions, observation results show that compared with a blank experiment taking distilled water as a reference, the silk fibroin of the six groups of the sixth (60% ethanol solution) is not degraded as the blank experiment; the fibroin in the first step and the second step is only slightly degraded; in the third-component system, the fibroin is degraded in a fast degradation mode, and the degradation amount of the fibroin is the largest and the speed is the fastest, so that the solution containing 30% ethanol and 40% calcium chloride is selected as the ternary degradation system of the fibroin.

As shown in FIG. 2, it is further demonstrated by polyacrylamide gel electrophoresis that hydrolysis is carried out for 1, 2 and 3 days under the conditions of (c), (d), and the results are as follows: as can be seen from the figure, the fibroin hydrolysis speed is fastest and the hydrolysis degree is thorough under the condition of the solution containing 30 percent ethanol and 40 percent calcium chloride, and the water solubility of the hydrolysate is better than that of the other two groups; the products are more in the molecular weight range of 20-50 kD. 3 preparation of soluble active silk fibroin

The molecular weight of the silk fibroin obtained by degradation is large, wherein the slow release activity of the silk peptide segment with the molecular weight of less than 10kD is poor, and the silk fibroin with the molecular weight of more than 50kD is not degraded and is not easy to modify, so that the target silk fibroin is 20-50 kD. Based on the molecular interception section, two dialysis bags of the molecular interception section are selected, and target silk fibroin is arranged between the two dialysis bags after dialysis is finished.

4-target silk fibroin yield and extraction rate

Degumming yield

The ratio of the degummed silk fibroin to the original silk fibroin is calculated as T-67.21%.

Yield of degraded silk fibroin

In the formula: g: yield of degraded silk fibroin

Mr: mass of silk fibroin after degumming

Ms: quality of silk fibroin obtained by degradation

Calculated as 96.15%

Target silk fibroin extraction rate

In the formula: k: extraction rate of target soluble silk fibroin

Mm: target soluble silk fibroin mass

Ms: quality of silk fibroin obtained by degradation

Calculated as 37.50%, the yield of the target soluble silk fibroin was 24.23% P ═ T ═ G ═ K.

5 analysis and characterization of physical and chemical indexes

Determination of conventional Components

In the research subject, the content of protein in silk fibroin self-made in a laboratory is 92.96%, the content of fat is 0.57%, the content of water is 4.42%, and the content of ash is 2.34%, which are shown in a table 2-4:

TABLE 2-4 major Components of Silk fibroin

Table 2-4 The main composition of SF

As can be seen from tables 2-4, the self-made silk fibroin in the laboratory has high protein content and high purity, and can be used for experimental research.

Measurement of emulsifiability and emulsion stability

The emulsifying properties refer to the volume of oil that can be emulsified per gram of protein^[51]. As shown in fig. 3, in the case of pH 8, the influence of different concentrations on the emulsification performance of silk fibroin was investigated, and at low concentration, the oil phase height decreased significantly with the increase of silk fibroin concentration, and the calculated emulsification capacity was in negative correlation with the oil phase height, so it can be concluded that at low concentration, the emulsification performance increased with the increase of silk fibroin concentration. However, the conclusion is that the change of the volume of the oil phase coated by the silk fibroin is not obvious at high concentration, and the reason is that when the concentration is higher than 0.5g/mL, the shearing force formed under the condition of high rotating speed causes the silk fibroin to generate gel, so that the coating capability of protein molecules is hindered, and the change of the emulsifying property is not obvious.

As shown in figure 4, the silk fibroin emulsification performance under different pH conditions is not obviously changed, and the emulsification performance is relatively high when the pH value is 8, which is mainly because the pH of the solution is close to that of the silk fibroin original solution and has good compatibility, so that the silk fibroin has good emulsification performance in a large pH range.

It can be seen from fig. 5 that at low concentration, the higher the concentration, the better the emulsion stability, and when the concentration is higher than 0.5g/mL, the emulsion stability is not improved significantly, because when the concentration reaches a certain value, the liquid emulsified by the fibroin is a highly dispersed system, and has a great surface potential energy, and at this time, the surface tension of the water-oil interface is reduced, the emulsified interface generates a membrane effect, and has a great mechanical strength, so that the stability of the system is improved, and the membrane effect is improved along with the increase of the concentration of the emulsifier. When weakly acidic oil is dissolved in a neutral silk fibroin solution, the pH value of the solution is close to the isoelectric point of silk fibroin, and a gel phenomenon is easy to occur.

As shown in FIG. 6, at a certain concentration (0.5%), different pH values did not affect the emulsion stability of fibroin much, but only slightly decreased at pH 10. The emulsification stability of the protein is related to the stability of a limiting membrane formed after high-speed shearing, and the limiting membrane formed by the silk fibroin is insensitive to the change of the pH value of the solution, so that the oil coated by the silk fibroin is not easy to overflow in the limiting membrane, and the emulsification is stable.

Measurement of foaming Property and foaming stability

As shown in fig. 7, the influence of silk fibroin concentration on the foaming capacity is obviously enhanced with the increase of the concentration, and the foaming capacity of silk fibroin is reduced when the concentration is higher than 1%, which is mainly due to the beta-sheet structure contained in the high-concentration silk fibroin solution. Compared with other proteins, the foaming capacity of silk fibroin under the same conditions is slightly lower than that of casein, because fewer hydrophobic groups are contained in silk fibroin molecules than that of casein, and the foaming capacity of the protein can be effectively influenced by the number of the hydrophobic groups.

The effect of silk fibroin concentration on foaming stability is shown in the figure, the foaming stability is poor at low concentration, and the foaming stability is good and tends to be stable at concentration higher than 0.5%, because the protein at high concentration only partially unfolds a folded structure, and the desorption rate at the formed interface is slow, so that the silk fibroin-based foaming agent can form a more stable foam structure compared with the protein at low concentration. Proteins with a folded structure will form a cyclic non-covalent structure with strong interactions and will extend into the aqueous phase, promoting the formation of a stable network of proteins.

Infrared spectrogram

The infrared result shows that the thickness of the film is 1650cm^-1And 1540cm^-1The infrared absorption peak of amino group appears, wherein 1650cm^-1Characteristic absorption peak of alpha-helical structure in silk fibroin molecule, 1540cm^-1Characteristic absorption peaks for the beta-sheet. This shows that in the target soluble silk fibroin home-made in the laboratory, in addition to the existence of a relatively stable alpha-helix structure, a beta-sheet structure also exists, and the beta-sheet of the protein enhances the stability of the product, so that the protein is not sensitive to the change of external conditions.

Differential thermal DSC scanning

The DSC thermogram of silk fibroin is shown in FIG. 9. As can be seen, the thermal reaction of the silk fibroin is concentrated in the small range of 180-185 ℃, and the thermal stability can be better embodied in specific applications. From the primary structure analysis of silk fibroin, it is composed of 18 amino acids, among the 18 amino acids, Thr, Ser, Arg, Pro are the least stable amino acids, His and Asn are the less stable amino acids, and the more unstable amino acids, the more unstable silk fibroin is^[76]. When the amino acid composing the silk fibroin is analyzed, the unstable amino acid only accounts for 8.8 percent of the total amino acid component of the silk fibroin^[77]This explains to some extent the pyrolysis of silk fibroin at higher temperatures. In addition, silk fibroin contains more crystal regions, and in the heating process, because peptide chains are directionally bound by the crystal regions, the free activity degree of the chains is low, the thermal movement of molecules and the pyrolysis difficulty of amino acid are high, which indicates that the thermal stability of the silk fibroin is good. In addition, the heat absorption peak of the silk fibroin is fine and sharp, and the heat absorption temperature range is narrow, which indicates that the process is concentrated and discontinuous.

Analysis by scanning electron microscope

An electron microscope image of silk fibroin with 400 and 2000 times of the weight is taken, as shown in figure 10, silk fibroin is spherical and has clear particles, and no adhesion and agglomeration exist among the particles.

And (4) conclusion:

(1) according to the method, mild low-temperature degassing treatment is adopted, so that the biological activity of the silk fibroin is greatly reserved. The silk fibroin with the target molecular weight is purified and separated by an innovative overlapping dialysis method after low-temperature degumming and silk fibroin degradation after degumming, the yield of the target soluble silk fibroin is 24.23 percent at the moment, the target soluble silk fibroin can be obtained by conventional component analysis, the purity of the molecular weight soluble silk fibroin prepared by a laboratory is higher, and the later-stage modification is easy.

(2) The silk fibroin emulsifying and foaming properties were studied. At low concentration, the emulsifying property is increased along with the increase of the concentration of the silk fibroin, the higher the concentration is, the better the emulsifying stability is, meanwhile, the emulsifying property is relatively higher when the pH value is 8, and different pH values have little influence on the emulsifying stability of the silk fibroin; the foaming ability is strongest at a concentration of 1%, and the foaming stability is better at a concentration higher than 0.5%.

(3) The results of various characterization means show that, in target soluble silk fibroin self-made in a laboratory: relatively stable alpha-helix structures and beta-sheet structures coexist; the thermal stability is better; the surface of the particles is smooth and regular without agglomeration.

4) Microwave synthesis

Experimental Material

TABLE 3-1 Main test materials

Table 3-1 Main experiment materials

2 method of experiment

2.1 screening of polysaccharide and preparation of SF-Cd Slow-Release microspheres

Weighing a certain mass of silk fibroin, placing the silk fibroin in a beaker, pouring a certain amount of deionized water, adjusting the pH value to 7, and preparing a solution with 10% of protein content. Adding different polysaccharides (cyclodextrin, starch, maltodextrin and agarose) into the mixed solution, wherein the mass ratio of the fibroin protein: polysaccharide 2.5: 1, preparing a proteoglycan mixed solution. Stirring with magnetic stirring device continuously until uniform, and freeze drying.

Grinding the four different powders uniformly, sieving the powders with a 150-mesh sieve, placing the equivalent amount of sieved substances in a double-frequency ultrasonic microwave combined catalytic synthesizer, adjusting the microwave power to be 500w, the ultrasonic power to be 300w and the reaction time to be 4min, taking out the powders after reaction, and finishing the reaction in ice-water bath for 1min to obtain a mixed product. And (3) determining the amount of free amino acid in the four products according to a method 2.2, calculating to obtain the grafting ratio, and selecting the proteoglycan combination with the highest grafting ratio to continue the experiment.

Preparing proteoglycan powder with different substrate concentration ratios, treating the proteoglycan powder by the same method, placing the proteoglycan powder into a double-frequency ultrasonic and microwave combined catalytic synthesizer for reaction, adjusting microwave power (100w, 300w, 500w, 700w and 900w) and ultrasonic power (100w, 200w and 300w), taking out the proteoglycan powder after reacting for a period of time, and finishing the reaction in ice-water bath for 1 minute to obtain an SF-Cd mixed product.

2.2 calculation of the graft ratio for determination of free amino groups

The protein and the saccharide are mainly reacted through the free amino group in the protein and the carboxyl group of the saccharide, and the lower the number of the free amino group, the higher the grafting ratio and the higher the reaction degree, therefore, the grafting ratio can be calculated through the measured number of the free amino acid.

The OPA method is used herein^[55]The number of free amino groups is determined by the following specific procedures:

two reagents were prepared, CA reagent and CB reagent, respectively.

CA reagent: 0.04g of o-phthalaldehyde (OPA) is dissolved in 1mL of methanol and 3mL of deionized water and stored in a brown reagent bottle for later use.

CB reagent: 2.5mL of 20% Sodium Dodecyl Sulfate (SDS), 25mL of 0.1mol/L borax and 0.1mL of beta-mercaptoethanol are mixed, added into a 50mL volumetric flask and then fixed to the volume for standby.

And (3) putting 0.3mL of the prepared solution CA reagent and 7mL of the prepared solution CB reagent into a test tube, uniformly mixing, adding 0.2mL of a sample solution containing 0.01g/mL of silk fibroin, uniformly mixing, reacting for 2min at 35 ℃, measuring the light absorption value at 340nm, simultaneously preparing a blank solution (adding an equivalent amount of water instead of the sample), and measuring the difference of the two values to obtain the net light absorption value of the free amino. The degree of reaction was determined by calculating the free amino acid content C from the standard curve (lysine) and the net absorbance and calculating the grafting. The calculation formula is as follows:

in the formula: DG: graft ratio

C0: free amino content before reaction

Ct: free amino content in the reaction t

2.3 measurement of the degree of browning of the reaction product

As the glycosylation reaction proceeds, melanoidins are produced and deepen as the reaction time increases. And grinding the silk fibroin-polysaccharide sample obtained after reaction and cooling until the silk fibroin-polysaccharide sample is crushed, putting the sample into a refrigerated centrifuge for 5min at 4000r/min, taking out the sample, diluting the sample by 20 times by using a Sodium Dodecyl Sulfate (SDS) solution with the mass concentration of 0.1%, and measuring the light absorption value of the sample at the position of 420nm by using the SDS solution as a blank control. And expressing the browning degree by using a light absorption value, wherein the larger the light absorption value is, the higher the browning degree is, and the more thorough the reaction is.

2.4 determination of intermediates

According to the high power^[78]The method of (3) determining the intermediate product of the Maillard reaction. Taking out a certain amount of reaction products, dissolving the reaction products in phosphate buffer solution with the pH value of 7.0, putting the phosphate buffer solution into a refrigerated centrifuge 4000r/min for centrifugation for 5min, taking out samples, diluting the samples by 100 times by using Sodium Dodecyl Sulfate (SDS) solution with the mass concentration of 0.1%, and measuring the light absorption value of the samples at 294nm by using the SDS solution as a blank control. To detect the intermediates of the Maillard reaction, the absorbance at 294nm is generally determined. The greater the absorbance, the more intermediate.

2.5 measurement of whiteness

The fried sweet potato slices are measured by a portable color difference meter HP-2132 produced by Shanghai Hanpu photoelectric technology limited company, and three indexes of L, a and b are obtained, wherein L represents lightness [0,100], a represents red/green difference [127, -128], and b represents yellow/blue difference [127, -128 ]. The color and luster of the sample are judged by taking the L value and the b value as judgment standards. Each sample was measured 10 times, averaged, and the whiteness value calculated by Hunter (Hunter) whiteness formula. The formula defines the whiteness of the fully reflective diffuser as 100 and compares the whiteness of the sample with the whiteness of the fully reflective diffuser to evaluate the whiteness of the sample by calculating the color difference.

In the formula, K₁Is a constant, typically a value of 1. a is_p，b_PIs the whiteness index of an ideal white in a Lab system, and generally:

measurement of samples without fluorescence a_p＝0.00，b_P＝0.00；

Measurement of samples with fluorescence a_p＝50，b_P＝-15.87：

2.6 particle size and distribution analysis

Adding the silk fibroin glycosylation product under different preparation conditions into a cuvette, detecting the size and distribution of the particle size by a particle size analyzer at 25 ℃, and simultaneously measuring the Zeta potential.

3 results and discussion

1 Effect of parameters in the microwave field on the products

1.1 ultrasonic Power and time

Researches show that the Maillard reaction rate is accelerated along with the increase of the reaction ultrasonic power, when the ultrasonic power is increased by 50w, the reaction rate can be increased by 1-2 times, because the increase of the ultrasonic power causes the vibration of protein and polysaccharide to be intensified, so that the structure is expanded, more reactive groups are exposed, the active sites (carbonyl and amino) of the Maillard reaction are increased, the reaction is accelerated, and the grafting rate is increased^[79]. Meanwhile, with the increase of the reaction time and the advance of the reaction process, the browning degree also increases, because a series of brown nitrogen-containing compounds are generated in the Maillard reaction when the Maillard reaction goes through a high-grade stageThe substances are collectively referred to as melanoids. The melanoid mainly comprises amino acid and saccharide, the melanoid is accumulated along with the increase of the reaction time, the color of a reaction product is deepened, the Maillard reaction speed can be judged by detecting the browning degree, the browning degree value can be obtained by the absorbance of the Maillard reaction product at 420nm, and the generation amount of the melanoid is determined by the reaction condition^[54]. In this study, the microwave conditions (mainly time and power) after the reaction, the relationship between the grafting ratio and the browning degree should be reconsidered because the microwave conditions are added.

The ultrasonic power and the reaction time have great influence on the protein glycosylation reaction, and mainly influence the intermediate products and the final products of the reaction process. In the experiment, the reaction microwave power is controlled to be 400w, the set power is constant, the relative humidity is 79%, and the concentration ratio of reaction substrates is silk fibroin: cyclodextrin ═ 2.5: 1; and meanwhile, controlling ultrasonic power and time variables, ultrasonic power (100w, 200w and 300w) and time (1min, 2min, 3min, 4min, 5min, 6min and 7min), and researching the change of glycosylation grafting rate and browning degree under different ultrasonic power and reaction time conditions to select optimal conditions, wherein the specific result is shown in the figure.

From FIG. 11 we can see the effect of different ultrasound powers and reaction times on the grafting yield. Along with the increase of the microwave time, the grafting rate increases rapidly along with the increase of the microwave time in 2-16h, and the grafting rate at 300w is increased from 51% at 1min to 86.18% at 4 min; and as the microwave time continues to increase, the grafting rate slowly decreases and finally approaches to equilibrium, and the grafting rate is 81.95% in 7 min. This is probably because the active sites of silk fibroin are gradually opened with time to undergo grafting reaction when the microwave time is less than 4min, and when the microwave time reaches 4min, the reaction is substantially completed, the active sites are substantially completely reacted, and the reaction is prolonged to allow the reactants and reaction products to undergo hydrolysis to some extent^[54]. Therefore, as the microwave time continues to increase, the grafting rate slowly decreases, so 4min was selected as the microwave time.

From the ultrasonic power, when the reaction time is 4min, the reaction is carried out under three powers of 100w, 200w and 300w, and the grafting rates are 69.45%, 830% and 86.18% respectively. It can be seen that the grafting rate is highest at 300w, which is probably due to the crimping of silk fibroin with polysaccharide structures at 100w-200w, the reaction structure is not easily exposed, the effect of the ultrasonic power increase on the reaction is large, which helps to open the active sites, and when the power is increased to a certain level, the effect is reduced but still has an effect.

From fig. 12 we can see the effect of different ultrasound powers and reaction times on the degree of browning. Browning substances generated by Maillard reaction interfere the purity and color of reaction products and influence later-stage application. When the reaction time was 4min, the effect of different ultrasonic powers on the browning degree was compared. It can be seen that the browning index at 200w is relatively low, 0.144. With reference to fig. 11, an appropriate condition is found to ensure that the glycosylation product reaches a considerable grafting degree under the condition of a small browning degree, so that the microwave time is determined to be 4min, the ultrasonic power is 200w, and the reaction product effect can reach relatively optimal at the moment.

1.2 microwave power

The microwave power of the reaction has great influence on the glycosylation reaction of the protein, and mainly influences the intermediate products and the final products of the reaction process. In the experiment, the ultrasonic power is controlled to be 200w, the reaction time is 4min, the editing power is constant, the relative humidity is 79%, and the concentration ratio of reaction substrates is silk fibroin: cyclodextrin ═ 2.5: 1; meanwhile, the microwave power variables (100w, 300w, 500w, 700w and 900w) are controlled, the change of the glycosylation grafting rate and the browning degree under different microwave power conditions is researched, so that the optimal conditions are selected, and the specific result is shown in fig. 13.

From FIG. 13 we can see the effect of microwave power on the degree of grafting and the degree of browning. As can be seen, the grafting rate increases significantly with increasing power from 100w to 500w, and increases and decreases with increasing power above 500 w. The reason is that when the power is lower, the power is increased to be beneficial to the temperature rise of a reaction system, reaction sites are opened, the reaction rate is increased, and when the power exceeds 500w, the physical agglomeration occurs among silk fibroin molecules in a microwave field^[75]Masking the Maillard reaction

1.3 substrate ratio

The substrate concentration ratio has great influence on the rate and the progress of the glycosylation reaction, and the grafting rate and the browning degree are both reflected. In the experiment, the ultrasonic power is controlled to be 200w, the reaction time is 4min, the microwave power is 500w, the editing temperature is constant, the relative humidity is 79%, and the substrate mixture ratio concentration is preliminarily selected to be silk fibroin: cyclodextrin (1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1), and the change of glycosylation grafting rate and browning degree under different substrate concentration proportioning conditions is studied to select the optimal conditions, and the specific result is shown in fig. 14.

From fig. 14, it can be seen that the degree of browning increases continuously with the increase of the substrate ratio concentration (silk fibroin: cyclodextrin) and the accumulation rate of melanoid increases, while in terms of the degree of grafting, with the increase of the substrate ratio concentration, the degree of browning increases at a rate of 2.5: peak at 1, then slowly decline, we select 2.5: the substrate mixture concentration of 1 was used as the experimental condition.

2 optimization of the grafting reaction conditions

2.1 model regression equation analysis

In order to verify the correctness of the conclusion by combining the experimental conditions screened by the single-factor experiment, the response surface software Design-Expert 9 is used for carrying out the multiple regression analysis of the data. According to the result of the single-factor experiment, the microwave reaction temperature, time and power are selected as influencing factors, and the grafting rate is used as a response value. Using the Box-Benhnken model to design experiments, we obtained independent variables: a (microwave power), B (substrate ratio), C (microwave time), response surface experimental results and model regression equation variance analysis are as follows:

TABLE 3-3 response surface test results

Table 3-3 The results of the Response Sursace Design

TABLE 3-4 analysis of variance of model regression equation

Table 3-4 ANOVA for Response Surface Quadratic Model Analysis of variance table

And (3) carrying out regression analysis on the grafting rate Y (%) response value by using a response surface program, and finally obtaining a regression equation as follows:

Y＝81.86-0.066A-33B-0.24C+0.077AB+0.88AC+1.34BC-11.46A²-2.53B²-5.85C²(3-4)

as can be seen from the analysis of variance table 3-4 of the model regression equation, in the influence of each single factor on the response value, the substrate ratio and the microwave time are not significant, the nonlinear relation is realized, the microwave power P value is less than 0.05, and the influence on the response value is large; the influence of the pairwise interaction of the three single factors on the response value is not obvious, which shows that the interaction of each single factor has small influence on the change of the response value; the P value of the model regression equation is less than 0.05, which shows that the fitting degree of the equation is good; meanwhile, the P value of each quadratic term is smaller than 0.05, which shows that the quadratic terms of each single factor have larger influence on the experimental result; meanwhile, the correlation coefficient R of the regression model²The value is 0.9502, indicating that the model data is reliable.

2.2 visual analysis of response surface

Interaction effects among variables were explored by response surface visual analysis software Design-Expert 9 to determine optimal reaction conditions. The results are shown in FIG. 15. The response surface map can intuitively reflect the interactive influence of various factors on the response value Y (grafting rate). When the gradient of the response curved surface is slow and the gradient is small, the variable has little influence on the response value; and when the gradient of the response surface is steeper, the variable has large influence on the response value, and the response value is greatly influenced when the variable is adjusted.

The regression equation and the response surface analysis are combined, and the optimized result shows that: (a) the influence of the substrate proportion and the microwave power on the grafting ratio is increased along with the increase of the microwave power, and the substrate proportion is 2.5: the highest graft ratio was obtained in case 1. (b) The influence of the substrate ratio and the microwave time on the grafting rate is controlled in the following steps of 4min, 2.5: the grafting yield reached a maximum at 1. (c) The influence of the microwave power and the microwave time on the grafting rate is increased along with the increase of the microwave power, and the grafting rate is highest when the microwave time is 4 min.

2.3 reaction optimization conditions

The theoretical optimal conditions obtained by model fitting are that silk fibroin is matched by a substrate: β -Cd ═ 2.5: 1. the microwave power was 500w and the reaction time was 4min, under which the graft ratio was estimated to be 807%. The theoretical value is verified through experiments, the obtained grafting rate is 82.95%, and the relative error between the theoretical value and the actual value is small, so that the equation of the optimized process parameter can guide the actual synthesis.

TABLE 3-5 comparison of theory and experiment

Table 3-5 The contrast of the experimental value and the theoretical value

3 intermediate product

To detect intermediates of the Maillard reaction, the absorbance at 294nm is generally determined^[80]. The greater the absorbance, the more intermediate. The absorption value measured at the wavelength is generated by a plurality of aldehyde ketone small molecular substances, and the larger the absorption value is, the more intermediate products are, and the faster the reaction is. The results of the production of Maillard reaction intermediate products under different microwave heating times are shown in FIG. 16, and silk fibroin reacted under the same microwave conditions is a control group, and it can be seen that the glycosylation intermediate products of silk fibroin increase with the increase of heating time, when the heating time just exceeds 2min, the production rate of the intermediate products rapidly increases, and when the heating time exceeds 4min, the production of the intermediate products tends to trend toThis is probably because when the heating time is too long, the silk fibroin is slowly folded, which is not favorable for further reaction.

Measurement of 4 whiteness

The quality of the whiteness of the sample takes the L value, the a value and the b value as judgment standards. The measurement was performed 10 times for each sample, and after obtaining L, a, and b values, the whiteness value W was calculated by Hunter whiteness formula^[40]And taking an average value.

TABLE 3-6 calculation of the whiteness values of glycosylated silk fibroin

Table 3-6 The whiteness value calculation of SF-Cd

An average value of the whiteness of 76.94 was calculated from tables 3-6. The whiteness value W is one of important indexes for judging the properties of the glycosylated silk fibroin, and the closer the W value is to 100, the whiter the W value is^[81]The product has little browning in the reaction process, so the powder has yellow color, but the later application is not influenced.

Influence of 5pH on Zeta potential and particle size of product

The experiment determines the Zeta potential and the particle size change of the microwave glycosylation silk fibroin surface under different environmental pH values, and is helpful for guiding the application condition of the product in different environments, and the result is shown in figure 17. The result shows that the Zeta potential of the microwave glycosylated silk fibroin surface changes from 22mV when the pH value is 3 to-35.7 mV when the pH value is 9, and shows a monotonous decreasing trend, and the potential 0 point appears between the

pH value

5 and 6. When the pH value is 7-9, the potential is-19.3 mV, -29.5mV, -35.7mV respectively. The pH is far away from the isoelectric point band, the particles are strongly negatively charged, and the electrostatic repulsive force among the glycosylated silk fibroin particles ensures that the mutual extrusion particle size among the particles is small, and no aggregation is generated at the moment. The potentials at pH5 and 6 were 4.2mV and-13 mV, respectively, with particle sizes of 1.81 μm and 1.69 μm, respectively, significantly higher than the particle sizes at the other pH values, with significant agglomeration. This is because the solution approaches the isoelectric point and the interparticle forces are mainly van der waals forces. When the pH value is 3 and 4, the Zeta potential on the surface of the microwave glycosylated silk fibroin is 22.8mV and 15.3mV respectively, almost no flocculation is generated, the absolute potential value of the particles at the time is close to that of the pH value of 8, but the particle diameter is far larger than that of the particles at the pH value of 8, because the pH value of the solution is adjusted by taking the pH value of 7 as a starting point, and irreversible flocculation is performed when the pH value is adjusted to 3-4 to be about 5, so that the particle diameter at the pH value of 3-4 is larger than the theoretical value.

4 summary of this chapter

(1) The silk fibroin is used as a modified object, beta-Cd is grafted through Maillard reaction, and a microwave Maillard method is selected to improve the reaction rate, so that a glycosylation graft product of the silk fibroin is prepared. The influence of factors such as ultrasonic power, reaction time, microwave power, substrate proportion and the like on the content of free amino acid of silk fibroin and the browning degree is explored, and meanwhile, the optimal reaction condition of glycosylation of the silk fibroin obtained by a response surface analysis method is as follows: and (3) preparing a substrate by using silk fibroin: β -Cd ═ 2.5: 1. the microwave power is 500w, the reaction time is 4min, and the grafting rate obtained by optimization is 807%. The grafting rate is 82.95%, and the relative error between the theoretical value and the actual value is small.

(2) The silk fibroin glycosylation intermediate product increases with the increase of heating time, when the heating time is 2min, the production rate of the intermediate product rapidly rises, and when the heating time exceeds 4min, the production amount of the intermediate product tends to be stable.

(3) The results of investigating the influence of pH on the particle size of the product show that flocculation occurs at a pH around 5 to 6, and the application is not suitable in this pH range.

(4) Compared with pure silk fibroin, the glycosylated silk fibroin has yellow color, but does not affect the later adsorption application.

Claims

1. The application of the silk fibroin powder for preparing the silk fibroin powder-cyclodextrin sustained-release microspheres adopts a microwave synthesis method, and comprises the following steps:

1) low temperature degumming

Removing impurities in raw silkworm cocoons, shearing, weighing silk fragments with certain mass, adding deionized water and anhydrous sodium carbonate, wherein the mass/g of silk: volume of water/L: the ratio of the mass/g of the anhydrous sodium carbonate is 10: 1: 5; immersing silk completely in the solution, stirring uniformly, sealing, introducing nitrogen gas to isolate the air, degumming, placing in a constant temperature tank at 37-38 ℃, adding a rotor, and stirring at constant speed to react; taking out and filtering every 24h, cleaning the filtrate with 25% sodium carbonate solution, adding the same volume of 25% sodium carbonate solution, continuously stirring for reaction, replacing the solution every 24h, and reserving the degumming filtrate;

2) degradation of degummed fibroin

Weighing part of freeze-dried silk, and mixing silk fibroin (g) according to the volume mass ratio: solvent (mL) = 1: 70 adding a solvent for dissolving silk fibroin, wherein the solvent for dissolving silk fibroin is a solution containing 30% of ethanol and 40% of calcium chloride; stirring, sealing, introducing nitrogen gas to isolate air for degradation, placing in a 37-38 deg.C constant temperature tank, adding rotor, stirring at constant speed for reaction, taking out after three days, freezing and centrifuging at low temperature of 6000r/min for 30min, and collecting supernatant;

Superposing dialysis bags with cut-off molecular weights of 20kD and 50kD respectively, pouring supernatant into the 50kD dialysis bag, dialyzing in deionized water for 48h, changing water every 12h, and freeze-drying a sample in the dialysis bags after dialysis to obtain fibroin protein powder; the yield of the soluble silk fibroin is 24.23 percent;

4) preparation of silk fibroin powder-cyclodextrin sustained-release microspheres

Weighing a certain mass of silk fibroin, placing the silk fibroin in a beaker, pouring a certain amount of deionized water, adjusting the pH value to 7, and preparing a solution with 10% of protein content; adding cyclodextrin, wherein the mass ratio of the silk fibroin: cyclodextrin = 2.5: 1, preparing a protein cyclodextrin mixed solution; stirring continuously by using a magnetic stirring device until the mixture is uniform in the preparation process, and then freeze-drying;

grinding uniformly, sieving with a 150-mesh sieve, placing the equivalent amount of sieved substances in a double-frequency ultrasonic microwave combined catalytic synthesizer, adjusting the microwave power to 500W, the ultrasonic power to 300W and the reaction time to 4min, taking out after reaction, and finishing the reaction in ice-water bath for 1min to obtain a mixed product.