CN108393046B

CN108393046B - Microwave-synthesized SF-Cd slow-release microsphere and application thereof

Info

Publication number: CN108393046B
Application number: CN201810033242.9A
Authority: CN
Inventors: 熊春华; 王文琦; 曹彦美; 朱明亮; 修丽丽; 韩晓祥; 蒋胜筹
Original assignee: Zhejiang Gongshang University
Current assignee: Zhejiang Gongshang University
Priority date: 2018-01-14
Filing date: 2018-01-14
Publication date: 2020-11-20
Anticipated expiration: 2038-01-14
Also published as: CN108393046A

Abstract

The invention relates to a microwave synthesized SF-Cd slow-release microsphere and application thereof. The method takes silk fibroin as a modified object, beta-Cd is grafted through Maillard reaction to prepare the SF-Cd slow-release microsphere so as to overcome the problem of initial burst release of the silk fibroin, and meanwhile, the beta-Cd also has certain entrapment performance, so that the entrapment rate of the microsphere is increased. The space structure of silk fibroin is mostly beta-folding, and beta-Cd is a polysaccharide structure, so that the reaction sites are fewer, and the Maillard reaction time is longer. To solve this problem, the microwave Maillard method has been chosen to increase the reaction rate. The method comprises the steps of preparing silk fibroin by low-temperature degassing treatment, degradation, purification and separation, grafting four saccharides respectively by using the silk fibroin as a modified object through a microwave wet Maillard reaction, and screening, wherein the grafting rate of cyclodextrin (Cd) is highest, so that a glycosylated graft product of the silk fibroin is prepared.

Description

Microwave-synthesized SF-Cd slow-release microsphere and application thereof

Technical Field

The invention relates to a microwave synthesized SF-Cd slow-release microsphere and application thereof.

Background

The chewing gum is a leisure candy prepared by natural gum, syrup and essence perfume through a certain processing technology, and the chewing gum with good quality is usually chewed for a long time, so that the essence perfume used by the chewing gum is chewed in the mouth and is kept released for a long time. Therefore, the selection of high concentration, less soluble flavors is an important step in the chewing gum preparation process. At present, most of solid essences applied to food have no slow release performance or poor slow release capacity. Most of the existing ones are superior in productivity and costThe preparation of food essence mostly adopts a spray drying embedding method^[66]However, when the spray-dried powder is subjected to moisture in the eating process, the capsule wall is dissolved, so that the core material is easy to burst and release under the action of other components, heating and shearing force of a food system, and the slow release effect is difficult to achieve^[67]. Aiming at the problems, the silk fibroin-beta cyclodextrin microspheres are prepared by a microwave method by taking carvone as a slow release object to adsorb target carvone essence, so that the slow release essence which can be slowly released for a long time and is suitable for products such as chewing gum and the like is prepared.

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 used as the sustained-release microsphere only, the hydrophilicity of the microsphere is poor, and the sustained-release effect is influenced by the sudden change of the release rate because the sustained-release microsphere is easy to release the load in an initial period of sustained release. 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 silk fibroin is obviously reduced, and the silk fibroin is dissolved in ureaCompared with the silk fibroin subjected to liquid degumming, the silk fibroin subjected to liquid degumming has poorer tensile strength. 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 SF-Cd slow-release microspheres with good emulsifying property and foaming property.

A preparation method of 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

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;

4) screening of polysaccharide and preparation of SF-Cd slow-release microsphere

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 the power of the microwave to be 100w-500w and the power of the ultrasonic to be 100w-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.

Wherein the content of the first and second substances,

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

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

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

The polysaccharide in the step 4) is cyclodextrin, starch, maltodextrin and agarose.

In addition, the invention also provides application of the SF-Cd slow-release microspheres in carvone as slow-release substances.

The aim of the chapter is to graft beta-Cd through Maillard reaction by taking silk fibroin as a modified object to prepare SF-Cd slow-release microspheres to overcome the problem of initial burst release of the silk fibroin, and meanwhile, the beta-Cd also has certain entrapment performance, so that the entrapment rate of the microspheres can be increased. The space structure of silk fibroin is mostly beta-folding, and beta-Cd is a polysaccharide structure, so that the reaction sites are fewer, and the Maillard reaction time is longer. To solve this problem, the microwave Maillard method has been chosen to increase the reaction rate.

The method comprises the steps of preparing silk fibroin by low-temperature degassing treatment, degradation, purification and separation, grafting four saccharides respectively by using the silk fibroin as a modified object through a microwave wet Maillard reaction, and screening, wherein the grafting rate of cyclodextrin (Cd) is highest, so that a glycosylated 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.

3. The changes of the solubility, whiteness, disulfide bond, sulfydryl and other contents before and after the microwave are explored, and the influence of the microwave on the physicochemical property, the structural function and other aspects of the SF-Cd covalent compound is discussed by adopting various characterization means. The result shows that the solubility of the product after microwave processing is greatly different compared with that of the microwave front. The silk fibroin raw material and SF + Cd after water bath heating have the lowest solubility under the condition that the pH value is 4, and the SF + Cd after microwaving has the lowest solubility under the condition that the pH value is 6; the fluorescence spectrogram shows that the strongest fluorescence intensity of the emission wavelengths of silk fibroin and beta-Cd microwave glycosylation products appears at 429nm under the condition of excitation wavelength of 360nm, the fluorescence intensity of the silk fibroin subjected to microwave glycosylation is obviously higher than that of the silk fibroin heated in a water bath and untreated, and the fluorescence intensity is obviously enhanced when the reaction lasts for 3min, which indicates that the generation rate of the products is accelerated when the reaction lasts for 3-4min and the Maillard reaction is stronger; FT-IR spectrum shows that the silk fibroin and cyclodextrin react to generate glycosylation product through carbon-ammonia condensation; the DSC experiment result shows that the product has good thermal stability; SEM image observation shows that the surface roughness of the glycosylated silk fibroin is increased, which is beneficial to later adsorption application.

4. The carvone is used as a slow release model object, oral cavity simulation slow release performance test is carried out on the SF-Cd microsphere, and the influence of factors such as preservative, pH and the like on the slow release effect is researched. The result shows that the load rate of the microspheres loaded with carvone is 8.27 percent, the encapsulation rate is 80.65 percent, the slow release time under the conditions of oral pH and temperature can reach 30min, and the slow release rate is proper; the food additive has little influence on the slow release effect of the product and can be ignored in practical application. The slow release mechanism of the silk fibroin glycosylated microspheres is explored, and the slow release mechanism is found to accord with Fickian diffusion in the release process and have good biodegradability.

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.

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.

FIG. 18 effect of different heating patterns on disulfide bond content of the product.

FIG. 19 the effect of different heating patterns on the thiol content of the product.

FIG. 20 effect of different heating patterns on product solubility.

FIG. 21 is a fluorescence spectrum of a product under different heating conditions.

FIG. 22 is an infrared spectrum of a product of different heating modes.

FIG. 23 DSC thermograms with different heating patterns.

FIG. 24 is an electron microscope image of products under different heating modes, wherein (1) cyclodextrin (2) silk fibroin (3) is heated in water bath to obtain a product (4) and the product is heated by microwaves.

Figure 25 effect of different carvone concentrations on microsphere encapsulation and loading.

FIG. 26 in vitro sustained release performance of product microspheres at different pH conditions.

FIG. 27 in vitro sustained release performance of product microspheres at different temperature conditions.

FIG. 28 effect of microwaves on the sustained release properties of the product.

Figure 29 effect of food additives on product sustained release performance.

Detailed Description

Example 1 preparation method of fibroin powder

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

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.

After the binder removal is finished, the mixture is taken outMeasuring silkworm cocoon fragments, placing in a small beaker, detecting degumming effect with 1% picric acid carmine solution, soaking the sample in the picric acid carmine solution, boiling for 5min, washing with deionized water, and observing silk color^[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.

3) The silk fibroin primarily separated by the separation and purification of the active silk fibroin with medium molecular weight has large molecular weight range and complicated folding inside the molecule, and is not beneficial to the later 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.

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.

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 electrophoretic gel was prepared as shown in table 1:

TABLE 1 gel electrophoresis composition Table

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. The silk is formed by randomly arranging and curling soluble amino acids at the initial stage in a living body, has no obvious structural characteristics and basically no activity, and when the silk is further curled to form a higher-order structure, the fibroinThe molecules can form linear aggregates through self-assembly, and the physical shearing resistance and chemical stability of the fibroin are obviously enhanced due to the appearance of beta sheet mode and the formation of other high-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 protein content of the silk fibroin self-made in a laboratory is 92.96%, the fat content is 0.57%, the water content is 4.42%, and the ash content is 2.34%, which are shown in table 2:

TABLE 2 major Components of Silk fibroin

As can be seen from Table 2, the fibroin protein prepared by a 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 homemade in the laboratory,in addition to the presence of a relatively stable alpha-helical structure, and the presence of a beta-sheet structure, the beta-sheet of the protein enhances the stability of the product and is therefore insensitive to changes in 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 the magnification of 400 times and 2000 times is taken, and as shown in 10, the silk fibroin is spherical, the particles are clear, and no adhesion or agglomeration exists 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 Main test 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 ideally white at LWhiteness index in the ab system, in general:

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, as the reaction time is increased and the reaction progress is advanced, the browning degree is also increased, because a series of brown nitrogen-containing compounds are generated in the Maillard reaction when the Maillard reaction goes through a high-grade stage, and the substances are collectively called melanoid. 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 to select the optimal conditions, and the specific result is shown in the following figure 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]The partial amino sites of the Maillard reaction are masked. And when the power is higher than 500w, the browning degree is increased and accelerated, while the browning increase is not beneficial to the later utilization of the product, and the browning index is relatively smaller and is 0.154 when the microwave power is 500 w. Therefore, a considerable grafting rate needs to be searched under the condition of low browning degree, and therefore, 500w is selected as experimental power, so that a good effect can be achieved.

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 results are shown in the following chart.

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 4 response surface test results

TABLE 5 model regression equation analysis of variance

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 5 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 6 comparison of theory and experiment

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. Fig. 16 shows the result of the yield of the Maillard reaction intermediate product under different microwave heating times, and silk fibroin reacted under the same microwave condition is a control group, it can be seen that the rate of yield of the intermediate product increases rapidly as the heating time increases, when the heating time is just over 2min, the yield of the intermediate product tends to be stable when the heating time is over 4min, which may be due to slow folding of silk fibroin when the heating time is too long, 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 7 calculation of the whiteness values of glycosylated silk fibroin

The average whiteness is calculated from table 7 as 76.94. 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 is due toA small amount of brown stain exists in the reaction process, so the color of the powder is slightly yellow, 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.

Example 3 structural characterization of SF-Cd and the effects of microwaves on it

1.1 Experimental materials

TABLE 8 Main test materials

2 method of experiment

2.1 solubility of the product

Weighing a certain amount of product silk fibroin, adding deionized water for dissolving, preparing silk fibroin solution with different pH values, stirring at room temperature for 1h, centrifuging at 10000r/min for 10min, collecting supernatant, and determining silk fibroin content in the solution by Coomassie brilliant blue method.

2.2 fluorescence Spectroscopy

Reacting silk fibroin, beta-Cd, microwave reaction product under the same conditions for 1min, 2min, 3min, 4min, 5min, 6min, 7min respectively, wherein silk fibroin and beta-Cd are as control group. After the reaction is finished, phosphate buffer solution with pH of 7.4 is used for preparing solution with the concentration of 1mg/mL to be tested. And measuring the fluorescence emission spectrum of the copolymer in the range of 305-500 nm under the conditions that the excitation wavelength (lambda ex) is 295nm and the fluorescence emission and excitation slit width are both 5.0 nm. The reaction product generated by the Maillard reaction is characterized by an excitation wavelength of 340-370nm and an emission wavelength of 420-440 nm.

2.3 determination of the thiol and disulfide bond content

Thiol determination by Ellman reagent method according to the literature^[53]. 39.6g of 5,5' -dithiobis (dithiol) was dissolved in 10mL of 1mol/L phosphate buffer solution having a pH value of 8, 0.02mL of the solution was added with 0.4mL of silk fibroin, 0.8mL of 1mol/L EDTA and 1% sodium lauryl sulfate, and the mixture was stored at 25 ℃ for 1 hour, followed by measurement of absorbance at 412 nm.

The molar concentration of the sulfydryl is as follows:

disulfide bond calculation formula:

wherein: a is the absorbance at 412 nm; is an extinction coefficient, which is 13600; d is the dilution multiple; c is the protein concentration.

2.4 Infrared Spectroscopy

2.5 differential thermal DSC Scan

Taking about 3-8mg of freeze-dried silk fibroin, cyclodextrin and glycosylation products of the silk fibroin before and after microwave, recording the accurate mass, putting the silk fibroin into an aluminum tray, spreading a sample in the aluminum tray and compacting the sample by 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. An empty aluminum pan was scanned as a blank control prior to each experiment.

2.6 scanning Electron microscopy analysis

3 results and discussion

3.1 thiol and disulfide bond content

It has been shown that in silk fibroin, the ratio of small molecular weight protein to large molecular weight protein is 1: 1 and the two are linked by a disulfide bond to form silk fibroin with a stable structure, and the present subject is to measure the disulfide bond and thiol content in silk fibroin and to contribute to the analysis of the state of protein internal space extension, the results of which are shown in FIG. 18.

The disulfide bond content of SF + Cd is measured under the conditions of Water Bath (WB) and Microwave (MW) respectively when the SF + Cd is heated for 1-7min, and meanwhile silk fibroin is used as a blank control experiment under the same conditions. It can be seen that the disulfide bond content decreased with the increase of the heating time. Because the heating time of the water bath is too short, the disulfide bond content of the product under the water bath condition is hardly influenced; but under the microwave condition, even if the beta-Cd is not added, the disulfide bond content is still reduced, and after the beta-Cd is added, the disulfide bond content of the product is obviously reduced.

The influence of different heating modes on the thiol content of the glycosylated silk fibroin product is shown in fig. 19, and it can be seen that the thiol content of the other three experiments increases with the increase of the heating time except the silk fibroin group heated by the water bath. The sulfhydryl content in the reaction is mainly converted from disulfide bonds, and when the reaction is over 4min, the sulfhydryl content of SF-Cd (MW) is obviously reduced, mainly because the sulfhydryl is oxidized along with the reaction, and the molecular structure of the protein is supposed not to be crosslinked, but only the beta-folding structure is opened. The water bath heating has less influence on the mercapto content, mainly because the heating time is too short.

3.2 solubility at different pH and heating regimes

From fig. 20, we can see that the solubility of the product after microwave irradiation is greatly different from that of the microwave front. SF-Cd (WB) has the lowest solubility under the condition of pH 4, while SF-Cd (MW) has the lowest solubility under the condition of pH6, which is mainly because the isoelectric point of the silk fibroin modified by glycosylation is shifted to the right, and at the isoelectric point, protein molecules exist in a zwitterion form, and because the net charges of the molecules in the protein molecules are zero and the same charges do not repel each other, the protein molecules are easy to collide with each other and aggregate to generate precipitates. Thus, protein solubility is the worst near the isoelectric point. In addition, as a whole, the solubility of the microwave glycosylated silk fibroin under pH conditions other than the isoelectric point (pH 2 to 10) was better than that of the silk fibroin under water bath heating conditions, indicating that the efficiency of the microwave heating conditions was much higher than that of the ordinary water bath heating. Meanwhile, glycosyl is introduced by microwave, hydrophobic groups existing in the silk fibroin are masked, and the solubility of the product is remarkably improved due to a large number of hydrophilic groups on the surface of the beta-Cd.

3.3 fluorescence Spectroscopy

Fluorescence spectroscopy accounts for changes in the physicochemical properties of proteins by detecting endogenous amino acid residues (mainly Trp tryptophan and Tyr tyrosine) in the protein molecules to capture structural changes in the protein. Research shows that the Maillard reaction can produce a substance with fluorescence characteristics, the excitation wavelength of the substance is 340-370nm, and the emission wavelength is 420-440nm^[84]Therefore, we can judge the progress of the Maillard reaction according to the fluorescence intensity of the product. The results are shown in the figure.

The fluorescence intensity of SF and beta-Cd in water bath for 7min and microwave time periods respectively is measured by experiment, wherein the fluorescence intensity of the monofilin protein is measured as a control group. The research finds that^[85]In the initial stage of Maillard reaction, Amadori rearrangement products formed by the rearrangement of the Schiff base by Amadori do not have fluorescence characteristics. However, these compounds can crosslink with surrounding amino acids or proteins at the middle and later stages of the Maillard reaction to produce products with fluorescent properties. The fluorescent product is produced before melanoid production^[86]. The progress of the Maillard reaction can thus be inferred by measuring the fluorescence intensity of the product, the results are shown in fig. 21. As can be seen from the figure, when the excitation wavelength is 360nm, the strongest fluorescence intensity of the emission wavelength of the SF and beta-Cd microwave glycosylation product is 429nm, and the substance is the fluorescence product generated by the Maillard reaction. The fluorescence intensity of the silk fibroin after microwave glycosylation is obviously higher than that of the silk fibroin heated in water bath and untreated, which shows that the M is heated by microwaveThe wallard reaction product increased at a significantly faster rate than the water bath heating. Meanwhile, comparing the fluorescence intensity differences of microwave heating for 1min, 3min, 4min and 7min, the fluorescence intensity is obviously enhanced when the reaction is carried out for 3min, which shows that the product generation rate is accelerated when the reaction is carried out for 3-4min, and thus the Maillard reaction is stronger. It can be concluded that the progress of the Maillard reaction can be significantly promoted by increasing the appropriate microwave power and adjusting the heating time.

3.4 Infrared Spectroscopy

The protein and the saccharide can be obviously distinguished by a plurality of characteristic absorption peaks of the infrared spectrum, and the characteristic peaks are embodied by vibration modes of stretching, bending and the like of different characteristic groups, so that the combination condition of the protein and the saccharide can be tested by using an infrared spectrogram. The infrared result shows that the protein characteristic absorption peak of the silk fibroin is obvious and is 1650cm^-1And 1540cm^-1The peak of bending vibration of amino group appears at 1650cm^-1Characteristic absorption peak of alpha-helical structure in silk fibroin molecule, 1540cm^-1Characteristic absorption peak of beta-sheet^[87]. beta-Cd shows obvious carbohydrate absorption peak characteristic and is 3200cm in 3700-^-1A large O-H stretching vibration absorption peak of 2930cm appears^-1The C-H stretching vibration absorption peak appears at 1640cm^-1An O-H bending vibration absorption peak at 1155cm appears^-1C-O oscillation peaks appear. Wherein the beta-Cd is 855cm^-1The alpha-type glycosidic bond characteristic absorption peak shows that the beta-Cd is connected with alpha-1, 4 glycosidic bonds. After covalent grafting of silk fibroin molecules and beta-Cd, an obvious change is that the content of a hydroxyl absorption peak of 3700-. Meanwhile, the sharpness of the absorption peaks of the amino groups at 1651 and 1544 is reduced, which indicates that the protein and the saccharide have carbonyl-ammonia condensation reaction, the content of the amino groups is reduced, and Maillard reaction occurs.

3.5 differential thermal DSC Scan

The temperature dynamic function measured by Differential Scanning Calorimetry (DSC) can be representative of the course of thermal denaturation of the sample. Silk fibroin is a high molecular substance with a complex high-order structureThe heat treatment can transform the protein from an orderly folded state to a disordered unfolded state, the intramolecular force is weakened, the polypeptide chain is unfolded, the protein conformation is changed, and the recombination of covalent bonds can cause the endothermic heat absorption of the sample^[88；89]. DSC can detect the enthalpy value delta H generated by heating and unfolding the protein, and when the silk fibroin is grafted and combined with the cyclodextrin through Maillard reaction, the bond position and the structure between the protein and the sugar molecule are changed to cause the change of the enthalpy value. When the temperature reaches the unfolding (denaturation) temperature of the silk fibroin, the peak appearing on the DSC spectrum is the heat absorption peak, and the denaturation enthalpy value can be calculated from the size and the area of the peak. Along with the reaction, the structure of the silk fibroin is opened, the thermal stability is reduced, and the measured enthalpy value is reduced, so that the reaction process can be judged according to the enthalpy value.

The DSC curves of the products before and after silk fibroin, cyclodextrin and microwave are shown in figure 23.

From the change of DSC curve, the silk fibroin has a characteristic heat absorption peak at 182 ℃, the cyclodextrin has a characteristic heat absorption peak at about 136 ℃, the water bath heating products of the silk fibroin and the cyclodextrin have characteristic heat absorption peaks at 182 ℃, 173 ℃ and 135 ℃, the products after the silk fibroin and the cyclodextrin are subjected to microwave treatment have no obvious heat absorption peak at about 136 ℃, this shows that cyclodextrin in the microwave product forms Maillard reaction product with silk fibroin through grafting, which only contains one substance, the reaction rate of the water bath product is low, the reaction is not complete, so that three substances exist in the product at the same time, the figure shows that the characteristic heat absorption peak of the compound after reaction grafting appears at about 170 ℃, and is slightly shifted to the left compared with the characteristic peak of silk fibroin, this is probably because silk fibroin to which a glycosyl group is added is structurally more disordered and the folded structure is more spread out than pure silk fibroin.

3.6 scanning Electron microscope SEM analysis

Cyclodextrin, silk fibroin and glycosylation product are frozen and dried at low temperature, and then ground into powder, the external form and aggregation state are observed by a scanning electron microscope, 400-fold and 2000-fold electron microscope images of the silk fibroin, water bath glycosylation product and microwave glycosylation product are respectively taken, and the result is shown in figure 24. Observation shows that the surfaces of the silk fibroin powder particles are smooth and flat, and the particles are clear; when the product is heated in a water bath, the surface of the product begins to be rough and uneven, the particles become large, and aggregation occurs among the particles; when the silk fibroin is heated by microwave glycosylation, the surface of a heating product is rougher, and silk fibroin molecules are aggregated into larger particles by microwaves, because the thermal effect of the microwaves enables the protein micelles of the silk fibroin to be unfolded, heat energy is generated by mutual friction between polar molecules, the thermal effect is far stronger than that of water bath heating, and the aggregation is easy to occur due to the fact that the thermal effect is violently stronger than that of the water bath heating.

4 summary of this chapter

(1) The solubility of the product after microwave processing is greatly different than that of the microwave front. The solubility of the SF + Cd heated by silk fibroin and water bath is lowest under the condition that the pH is 4, and the solubility of the SF + Cd subjected to microwave treatment is lowest under the condition that the pH is about 6.

(2) The fluorescence spectrum result shows that when the excitation wavelength is 360nm, the strongest fluorescence intensity of the emission wavelength of the silk fibroin and the beta-Cd microwave glycosylation product is 429nm, the fluorescence intensity of the silk fibroin after microwave glycosylation is obviously higher than that of the silk fibroin heated in a water bath and untreated, when the reaction is carried out for 3min, the fluorescence intensity is obviously enhanced, and the product generation rate is accelerated when the reaction is carried out for 3-4min, so that the Maillard reaction is stronger.

(3) FT-IR spectrum shows that the silk fibroin and cyclodextrin react to generate glycosylation product through carbon-ammonia condensation; the DSC experiment result shows that the product has good thermal stability; SEM image observation shows that the surface roughness of the glycosylated silk fibroin is increased, which is beneficial to later adsorption application.

Example 4 in vitro Slow Release and mechanism study

1 Experimental materials and instruments and equipment

1.1 Experimental materials

TABLE 9 Main test materials

2 method of experiment

2.1 measurement of L-Ca content

Each carvone (L-Ca) molecule contains a carbonyl group, and the carbonyl value is measured by measuring the content of the L-Ca. The carbonyl number is determined by the free hydroxylammonium method according to the following principle: the mg of KOH required to neutralize each gram of L-Ca reacted with hydroxylammonium hydrochloride. Reaction of the free hydroxylammonium liberated from the mixture of hydroxylammonium hydrochloride and KOH with carbonyl compounds leads to the formation of oximes^[90]。

Free hydroxylammonium method:

the following reagents were prepared:

bromophenol blue ethanol solution: 0.2g of bromophenol blue is dissolved in 0.1mol/l KOH ethanol solution, 10mL of absolute ethanol is added, the mixture is cooled after being boiled, and the volume is fixed to 100mL by the absolute ethanol.

Ethanol solution of hydroxylammonium hydrochloride: dissolving 50g hydroxylammonium hydrochloride in 100mL water, adding 800mL absolute ethyl alcohol and 10mL bromophenol blue ethanol solution, and using absolute ethyl alcohol to fix the volume to 1000 mL. Observing the thickness of the formed layer, if thinner, adding potassium hydroxide ethanol solution until the solution turns green; if thicker, add KOH ethanol solution until the solution turns red. And (3) detecting the effectiveness of the solution: adding a proper amount of hydrochloric acid solution to generate lemon yellow; a suitable amount of potassium hydroxide was added and a red color appeared. And standing for later use.

The measuring method comprises the following steps:

3mL of the solution containing L-Ca was added with 20mL of hydroxylamine hydrochloride ethanol solution and 15mL of 0.1mol/L KOH, and titrated with hydrochloric acid until the yellow-green color disappeared.

2.2 preparation of L-Ca Supported Slow Release microspheres

1g of silk fibroin, a water bath glycosylation product and a microwave glycosylation product are respectively put into 100mL of 10mL/L carvone ethanol solution, sealed overnight and then freeze-dried for later use.

2.3 measurement of encapsulation and Loading

3mL of adsorbed L-Ca ethanol solution is taken, filtered by a membrane, microspheres in the solution are removed, 20mL of hydroxylamine hydrochloride ethanol solution and 15mL of 0.1mol/L KOH are added, and titration is carried out by hydrochloric acid until yellow-green color disappears. The mole number of the consumed hydrochloric acid is the mole number of the L-Ca.

L-Ca encapsulation efficiency:

L-Ca loading:

2.4 in vitro Release assay

2.1 preparation of Artificial saliva

The oral cavity is used as part of human digestive system, has strong digestive function, saliva in the oral cavity is secreted by salivary gland and many small mucous glands on oral cavity wall, contains many inorganic and organic molecules and numerous oral microbial groups, and simultaneously, the continuously changing pH environment makes the oral cavity become a complex ecological environment^[91]. To obtain certain properties of saliva, many studies have formulated artificial saliva for different purposes of use to conduct experimental investigations. In the present subject, artificial saliva is prepared for the purpose of studying the sustained-release effect.

Generally, artificial saliva can be used for detecting the saliva resistance of a target product, and is an artificial synthetic reagent which is prepared by scientific proportioning according to the ingredients of human metabolic saliva strictly, and the chemical ingredients of the artificial synthetic reagent are similar to those of saliva. To test the sustained release properties of the product, the following artificial saliva was prepared: 0.126g of sodium chloride, 0.964g of potassium chloride, 189g of potassium thiocyanate, 0.655g of monopotassium phosphate, 0.2g of urea and 10mL of liquefied alpha-amylase with the activity of 10 wu/mL are respectively added into a beaker, deionized water is added for dissolution, the volume is adjusted to 1000mL, the pH value of the solution is 6.8 at the moment, and then 100g/L of hydrochloric acid and 100g/L of sodium hydroxide are used for adjusting the pH values to 4.0, 5.0, 6.8, 7.0 and 8.0. Storing in a refrigerator at 4 deg.C for use.

2.2 measurement of cumulative Release amount of L-Ca

After the reaction was started, 5mL of the solution was taken at 0,1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40min, the membrane was filtered, the carvone content of the sample after the membrane filtration was measured, and the cumulative release amount was calculated.

2.3 pH Effect on in vitro Slow Release

The pH value in the oral environment is about 6.8 generally, and the pH value fluctuates under certain conditions such as before and after eating, and in order to investigate the influence of the pH condition on the slow release performance of the glycosylation product, the following experiments are carried out: six identical Erlenmeyer flasks were charged with 100ml of artificial saliva having pH values of 4.0, 5.0, 6.8, 7.0, and 8.0, respectively, heated in a water bath at 37 ℃ for 10min, and 1g of the carvone-loaded glycosylation product was added, and the cumulative release was determined and plotted according to 5.2.2.

2.4 temperature Effect on in vitro Slow Release and Slow Release Properties

As the carvone belongs to volatile substances, the temperature control in the slow release process plays a crucial role in the slow release effect and time. In order to explore the slow release performance of the essence slow release microspheres at the temperature, particularly at the temperature of 37 ℃ of a human body, the experimental operation is as follows: taking five identical conical flasks, adding artificial saliva with pH value of 6.8 of 100ml, respectively, keeping the temperature at 4 deg.C, 10 deg.C, 37 deg.C, 50 deg.C and 80 deg.C for 10min, adding 1g of carvone-loaded glycosylation product, measuring the cumulative release amount according to 5.2.2 method, and plotting.

2.5 influence of glycosylation of silk fibroin on in vitro sustained-release performance

In order to investigate whether the sustained release performance of the glycosylated product can be improved compared with that of the product before, the sustained release performance of the protofilament protein and the glycosylated product is studied, and the experimental operation is as follows: taking three identical conical flasks, respectively adding 100ml of artificial saliva with pH value of 6.8, heating in water bath at 37 deg.C for 10min, respectively adding 1g of carvone-loaded silk fibroin, water bath glycosylation product and microwave glycosylation product, measuring cumulative release amount according to 5.2.2 method, and plotting.

2.6 Effect of food preservatives on in vitro sustained Release

Chewing gum contains a small amount of food preservative, and the principle of the food preservative is that the food preservative usually inhibits the activity of biological cells through chemical or biological action, so that the food preservative plays a role in preservation. The subject selects two preservatives commonly used in chewing gum, namely Potassium Sorbate (PS) and Sodium Benzoate (SB), and the influence of the preservatives on the slow release performance of microwave glycosylation products is measured. The following experiments were performed: taking three identical conical flasks, adding 100ml of artificial saliva with pH value of 6.8, respectively, heating in water bath at 37 deg.C for 10min, adding 0.1% of food grade preservatives, potassium sorbate and sodium benzoate, respectively, into two of the conical flasks, adding 1g of carvone-loaded microwave glycosylation product into three conical flasks, and measuring the cumulative release amount according to 5.2.2 method and plotting.

2.7 fitting of Release curves

Liu an Fang for reference^[67]The method selects a zero-order kinetic model, a first-order kinetic model, a Higuchi kinetic model and a Rigter-Peppas kinetic model to perform equation fitting on the release behavior of the L-Ca sustained-release microspheres in the artificial saliva. The specific equation is as follows:

Q_t＝K₀+C₀ (5-3)

ln(100-Q_t)＝K₁t+C₁ (5-4)

Q_t＝K_Ptⁿ (5-6)

wherein, Qt is the L-Ca release rate at t, K₀、K₁、K_H、K_PRespectively, the release rate constant, C, of the corresponding kinetic model₀、C₁、C_H、C_PRespectively, constant terms of the corresponding kinetic model.

2.5 degradability exploration

Degradation experiments three groups of experiments were set up: hydrolysis degradation liquid, hydrogen peroxide degradation liquid and double distilled water hydrolysis liquid (blank control). Wherein, in-vitro hydrolysis degradation liquid is prepared, and PBS phosphate buffer solution is composed of sodium hydrogen phosphate, potassium hydrogen phosphate and the like; the concentration of the hydrogen peroxide degradation liquid is 3 percent; the double distilled water hydrolysate is secondary distilled water. After the degradation experiments, the samples were separated through a 0.45 μm filter and the entrapped material was freeze-dried and weighed. Comparison of the reduction in Hydrogen peroxide in phosphate bufferMass loss in solutions of hydrolysate and blank control^[92]。

3 results and discussion

3.1 Effect of different L-Ca concentrations on encapsulation and Loading

The effect of L-Ca concentration on encapsulation and loading was investigated and the results are shown in FIG. 25. The graph shows that the load rate is basically in a linear increasing rule along with the increase of the L-Ca concentration, and the load rate is obviously increased before and after the glycosylation modification of the silk fibroin, which indicates that the glycosylation modification can not only improve the burst release phenomenon of the silk fibroin at the initial stage of slow release, but also increase the drug loading rate of the silk fibroin, so that the release amount per minute cannot be influenced by the extension of the slow release time in the slow release process. The trend of the encapsulation efficiency along with the increase of the concentration of the L-Ca is opposite to the load rate, and the encapsulation efficiency is lower when the concentration is higher. The subject finally selects 10mL/L as adsorption concentration, the loading rate (8.27%) and encapsulation rate (80.65%) are relatively high, and the added L-Ca is fully utilized, and relatively more loaded substances can be adsorbed, so that the subject is better applied in the later period.

3.2 in vitro release of microspheres in saliva

Saliva amylase, which is one kind of alpha-amylase, exists in human saliva and can act on alpha-1, 4-glucan such as soluble starch, cyclodextrin, glycogen and the like to hydrolyze alpha-1, 4-glycosidic bonds. In general, the normal value of salivary amylase in saliva is 607-3423u/mL, and for cost reasons, we have conducted experimental investigation using alpha-amylase instead of salivary amylase, which has the same site of action as salivary amylase and is alpha-1, 4-glycosidic bond. The enzyme activity of the alpha-amylase purchased in a laboratory is 10 ten thousand per mL, so that 10mL of the alpha-amylase is added when preparing each liter of artificial saliva.

3.2.1 pH on in vitro sustained Release Performance

The slow release performance of the microwave glycosylation product microspheres in different saliva pH environments is researched, and the result is shown in FIG. 26. As can be seen from the figure, under the condition of pH 4-5, L-Ca is exploded within 5min, and the release amounts reach 67.5% and 72.9%, respectively. When the pH value is lower, the microspheres have a large number of positive charges, the microspheres are partially denatured by the repulsion of the positive charges in molecules, and are subjected to aggregation and precipitation, so that hydrophobic groups are exposed, the clathrate compound flows out, and meanwhile, products are easily cracked in an acid solution, so that the burst release is generated at the initial slow release stage, and the cumulative release amount is larger. Under the condition that the pH value is neutral, the slow release performance of the microsphere is good, the accumulated slow release amount tends to be stable and reaches 80% in 30min, which is probably because the alpha-amylase in the artificial saliva is active at this time, so that alpha-1, 4-glycosidic bonds in the product microsphere are broken, and the L-Ca is slowly released. Meanwhile, the steric hindrance and van der waals forces in the microspheres limit the amylase action and the release of L-Ca is incomplete. When the pH value is 8, the solution is alkaline, the action effect of enzymes in saliva is poor, the slow release efficiency is poor, the release rate is too slow, the relatively fast release of the microspheres in 3min is mainly realized by the volatilization of L-Ca attached to the surfaces of the microspheres, the microspheres under the pH condition are not suitable to be used as fast-digestion leisure food additives, and the slow release effect under the normal oral cavity pH value of 6.8 is good.

3.2.2 temperature Effect on in vitro Slow Release

The control of the slow release temperature determines the molecular motion rate of the essence during release, thereby influencing the slow release rate of the essence and being a direct influence factor of the slow release performance of the microspheres. In order to investigate the influence of the sustained-release temperature in different environments on the in-vitro sustained-release performance of the product microsphere, the human body temperature of 37 ℃ is taken as a main research object, and a plurality of groups of control tests are simultaneously carried out to study the sustained-release performance of the essence microsphere in oral cavity under low-temperature and high-temperature conditions, and the result is shown in fig. 27. It can be seen that at the temperature below 10 ℃, the enzyme activity is low due to low temperature, the essence release is inhibited, and the release rate of the essence slow-release microspheres is almost negligible; when the temperature reaches 50 ℃, although the enzyme activity is lower than 37 ℃, the molecular movement rate is accelerated, the essence molecules are released from the special frame structure of the microspheres and need to overcome intermolecular force, the process needs to absorb heat, the temperature rise is accelerated at the moment, the release rate is accelerated, the enzyme inhibition effect and the temperature rise effect are partially offset, the release rate is still accelerated, and the temperature rise effect is more obvious at higher temperature and the release rate is faster.

3.2.3 Effect of Silk fibroin glycosylation on in vitro sustained-release property

Whether the sustained release performance of the glycosylated product can be improved compared with that of the product before is researched, and the sustained release performance of the protofilament protein and the glycosylated product is researched, and as shown in fig. 28, if silk fibroin is only used as sustained release microspheres, the load is easily burst and released 10min before sustained release, so that the sustained release effect is influenced by serious instability of the release rate, and the sustained release effect is poor when the silk fibroin is used as a carrier singly. And the physicochemical property and the sustained-release characteristic of the microsphere can be effectively improved after the beta-Cd is used for modifying the microsphere. Wherein, the microwave modification method is quick and efficient, and is better than a water bath method, and the water bath product mainly comprises a mixture of silk fibroin and cyclodextrin.

3.2.4 Effect of food additives on in vitro sustained Release Performance

Two food preservatives, Potassium Sorbate (PS) and Sodium Benzoate (SB), were explored and their effects on the sustained release properties of microwave glycosylated products were determined, and the results are shown in fig. 29. It can be seen that when the sustained-release time is 2min, the sustained-release rate of L-Ca added with potassium sorbate is increased from 58.89% to 66.35%, and the sustained-release rate of L-Ca added with sodium benzoate is increased from 58.89% to 70.90%, compared with the L-Ca without preservative. And the glycosylated silk fibroin-loaded L-Ca is not obviously changed before and after the food preservative is added, which shows that the preservative basically has no influence on the stability of the glycosylated L-Ca. Sometimes, because the glycosylated silk fibroin has a stable physical framework structure, the mechanical property of the glycosylated silk fibroin is enhanced while the physicochemical property is improved, and the glycosylated silk fibroin is not easily interfered by external chemical substances in the application process, so that the contact of L-Ca and a preservative is hindered, and the physical property can well protect the stability of a loaded substance.

3.2.5 kinetics of Release

The essence and flavor have the characteristic of easy volatilization, and the slow release and controlled release, such as trigger release, sustained release and the like, of the essence and flavor are loaded or embedded, so that the method is one of the hotspots of the existing essence research. In order to study the release mechanism of the silk fibroin glycosylated sustained release microsphere, the obtained data are fitted, and the results are as follows.

TABLE 10 Release kinetics parameters of the sustained-release microspheres

From R in the table₀ ²The values show that the experimental results are best fitted with the Rigter-Peppas model, and the model can better explain the release mechanism of the sustained-release microspheres. Since the Rigter-Peppas model is related to the magnitude of the n value, the mechanism can be further explained by the n value. When n is less than or equal to 0.5, the release mechanism of L-Ca is Fickian free diffusion, when 0.5<n<When n is more than or equal to 0.8, the slow release mechanism is the non-Fickian diffusion with the synergistic effect of free diffusion and skeleton dissolution of the slow release microspheres, and when n is more than or equal to 0.8, the slow release mechanism is the skeleton dissolution of the microspheres. In the study of the paper, the slow release index n of the silk fibroin glycosylated microsphere loaded with L-Ca is between 0.32 and 0.45, so the release kinetics conforms to Fickian diffusion.

3.3 exploratory degradability

Two-day biological rapid degradation experiments show that the quality loss in the solutions of the phosphate buffer solution, the hydrogen peroxide degradation solution and the blank control solution is compared. The mass loss after two days was 7.35%, 33.79% and 10.62%, respectively. Wherein, the product degradation rate in the hydrogen peroxide is the highest, and the products are degraded in different solutions, which shows that the product has good biodegradability.

4 summary of this chapter

(1) The influence of pH value, temperature, glycosylation and food additives on the slow release performance of the essence microspheres is explored by taking carvone as a slow release model object. The result shows that the slow release time is increased along with the increase of the pH value due to the influence of factors such as intramolecular positive charge repulsion, steric hindrance, van der Waals force, enzyme activity and the like, the slow release rate of the slow release microspheres applied to the fast-digestion food is proper under the normal pH condition of the oral cavity, and the slow release time can reach 30min to the maximum extent; the slow release rate is accelerated when the temperature is increased, and the slow release effect is good at 37 ℃; the glycosylated silk fibroin has obviously improved slow release effect compared with the pure silk fibroin; the addition of the food additives of potassium sorbate and sodium benzoate has little influence on the slow release effect of the product, and can be ignored in practical application.

(2) The slow release mechanism of the silk fibroin glycosylated microsphere is explored, and the result after data fitting shows that the release process conforms to Fickian diffusion.

(3) The degradation of different solutions in the simulated environment on the fibroin glycosylation product microspheres is shown, and the results show that the degradation is carried out in different solutions, and the biodegradability of the product is good.

6.1 conclusion

The main findings of this paper are as follows:

(1) the subject adopts mild low-temperature degassing treatment, and the biological activity of the silk fibroin is greatly reserved. The silk fibroin with the target molecular weight is purified and separated by an 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. Study of silk fibroin emulsification and foaming properties: 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, and meanwhile, the emulsifying property is relatively higher when the pH value is 8. The foaming ability is strongest at a concentration of 1% and the foaming stability is better at a concentration higher than 0.5%. The results of various characterization means show that target soluble silk fibroin self-made in a laboratory has a relatively stable alpha-helical structure and a relatively stable beta-folded structure, has better thermal stability, and has smooth and regular particle surfaces without agglomeration.

(2) 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 83.07%. The grafting rate is 82.95%, and the relative error between the theoretical value and the actual value is small. The detection of the intermediate product shows that when the heating time is 2-4min, the MR reaction rate rises rapidly. Meanwhile, the SF-Cd microsphere is found to have flocculation phenomenon when the pH is about 5-6, and is not suitable for application in the pH range. Whiteness tests show that the glycosylated silk fibroin has yellow color compared with pure silk fibroin, but the later adsorption application is not influenced.

(3) The influence of microwave on the content of solubility, whiteness, disulfide bonds, sulfydryl and the like of the product is explored, and the change of microwave on the physicochemical property, the structural function and the like of the SF-Cd covalent compound is discussed by adopting various characterization means. The result shows that the product solubility after microwave processing is greatly different compared with the microwave front: the lowest point of the solubility of the SF-Cd (MW) after microwave treatment is shifted to the right compared with that of the pure SF and SF-Cd (WB), and the fluorescence intensity is obviously improved when the reaction is carried out for 3min, which shows that the Maillard reaction intensity reaches the highest when the reaction is carried out for 3-4 min; FT-IR spectrum shows that microwave accelerates SF and cyclodextrin to generate glycosylation product through carbon-ammonia condensation reaction; the DSC experiment result shows that the product has good thermal stability; SEM image observation shows that the surface roughness of SF-Cd (MW) is obviously higher than that of SF-Cd (WB), and the adsorption application in the later period is facilitated.

(4) The carvone is used as a slow release model, oral cavity simulation slow release performance test is carried out on the SF-Cd microsphere, 10mL/L is selected as the adsorption concentration of the carvone, and the loading rate and the encapsulation rate at the moment are 8.27% and 80.65%. The influence of pH value, temperature, glycosylation and food additives on the slow release performance of the essence microspheres is explored. The result shows that the slow release time is increased along with the increase of the pH value due to the influence of factors such as intramolecular positive charge repulsion, steric hindrance, van der Waals force, enzyme activity and the like, the slow release rate of the slow release microspheres applied to the fast-digestion food is proper under the normal pH condition of the oral cavity, and the slow release time can reach 30min to the maximum extent; the slow release rate is accelerated when the temperature is increased, and the slow release effect is good at 37 ℃; the glycosylated silk fibroin has obviously improved slow release effect compared with the pure silk fibroin; the addition of the food additives of potassium sorbate and sodium benzoate has little influence on the slow release effect of the product, and can be ignored in practical application. The slow release mechanism of the silk fibroin glycosylated microsphere is explored, and the result after data fitting shows that the release process conforms to Fickian diffusion. The degradation of different solutions in the simulated environment on the fibroin glycosylation product microspheres is shown, and the results show that the degradation is carried out in different solutions, and the biodegradability of the product is good.

6.2 prospect of

Compared with other slow release materials, the novel glycosylated silk fibroin slow release microsphere developed by the subject has the following advantages: the sustained release time is long, the sustained release tablet is nontoxic, harmless and environment-friendly, the performance is stable, the load rate of sustained release substances is high, and the like. However, because of the limited time, the research depth and breadth of the subject is expected to be improved in the later period, and the experiment still has a lot of defects and needs to be further explored in the future, if the subject is deeply researched, the following proposals are made:

(1) when the optimal process of the sustained-release microspheres is explored, various indexes of the sustained-release microsphere process can be determined through the final application sustained-release effect.

(2) The slow release microspheres developed by the subject can be widely explored to research the slow release performance of the slow release microspheres on various essences or target slow release substances.

(3) Because the process of the Maillard reaction is complex, most products are mixtures, and therefore, the Maillard reaction products can be further separated and purified so as to manufacture glycosylation products with higher utilization rate and application efficiency.

(4) The improvement of the slow release performance of the product microsphere is subjected to deep mechanism research.

Claims

1. A preparation method of SF-Cd slow-release microspheres comprises the following steps:

1) low temperature degumming

2) degradation of degummed fibroin

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, preparing a solution with 10% of protein content, and adding polysaccharide, wherein the mass ratio is that the silk fibroin: polysaccharide 2.5: 1, preparing a proteoglycan mixed solution, stirring the mixture by a magnetic stirring device continuously until the mixture is uniform in the preparation process, and then freezing and drying the mixture for later use; 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.

2. The SF-Cd slow-release microsphere of claim 1, wherein: in the step 1), the mass/g of silk: volume of water/L: the ratio of the mass/g of the anhydrous sodium carbonate is 10: 1: 5.

3. the SF-Cd slow-release microsphere of claim 1, wherein: the solvent for dissolving the silk fibroin in the step 2) is a solution containing 20 percent of ethanol and 40 percent of calcium chloride; a solution containing 30% ethanol and 40% calcium chloride, or a solution containing 40% ethanol and 40% calcium chloride.

4. The SF-Cd slow-release microsphere of claim 1, wherein: the solvent for dissolving the silk fibroin in the step 2) is a solution containing 30% of ethanol and 40% of calcium chloride.

5. The SF-Cd slow-release microsphere of claim 1, wherein: the polysaccharide in the step 4) is cyclodextrin, starch and agarose.

6. The use of the SF-Cd slow release microspheres of any one of claims 1 to 5 in carvone as a slow release formulation.