Method for simultaneously extracting fibroin and sericin based on deep eutectic solvent and application thereof

Abstract

The application discloses a method for simultaneously extracting fibroin and sericin based on a eutectic solvent and application thereof. The method comprises the following steps: S1, obtaining first process parameters for extracting fibroin and second process parameters for extracting sericin; S2, based on the second process parameters for extracting sericin, carrying out degumming treatment on cocoon to obtain degummed cocoon and a solution containing sericin; S3, using the first process parameters for extracting fibroin to carry out extraction treatment on the degummed cocoon, and obtaining fibroin after drying. The application adopts a eutectic solvent, realizes the synchronous extraction of fibroin and sericin through a double system and response surface optimization. The method can efficiently recover by-products, the obtained fibroin has the dual characteristics of small molecule peptides (easy absorption) and beta-fold dominance (stable performance), and can be used for preparing edible packaging materials, thereby expanding the application value and economic value of fibroin.

Description

Technical Field

[0001] This invention belongs to the field of protein extraction technology, specifically relating to a method and its application for simultaneously extracting silk fibroin and sericin using a eutectic solvent. Background Technology

[0002] The production and application of silk has a long history, closely related to its excellent properties, including good biocompatibility, biodegradability, and superior mechanical properties. The main components of silk include fibroin and sericin, with contents of 72%–80% and 20%–30%, respectively. As a natural high-molecular-weight fibrous protein, fibroin contains 18 amino acids, with glycine, alanine, and serine accounting for over 80% of its total composition. These amino acids can form numerous β-sheet structures, giving the material lightweight, softness, and excellent mechanical toughness and strength. These properties have led to the widespread application of fibroin in the textile, cosmetic, and biopharmaceutical industries. As of May 19, 2026, fibroin has been officially included in the public consultation phase for new food raw materials in China, indicating its potential to become a legally viable resource for use in food and dietary supplements.

[0003] Silk fibroin is insoluble in water, but soluble in strong acids (hydrochloric acid, phosphoric acid, etc.), strong bases (sodium hydroxide), enzymes (papain and trypsin, etc.), and certain neutral salt solutions. However, strong acids, strong bases, and enzyme solutions can cause silk fibroin to undergo hydrolysis, and the hydrolysis products have good solubility. Common neutral salt solutions include chloride solutions (calcium chloride solution, zinc chloride solution, lithium chloride solution, strontium chloride solution, barium chloride solution), iodide solutions, and bromide solutions. In addition, N-methylmorpholine-N-oxide solution, thiocyanate solution, ionic liquids, copper ethylenediamine solution, and hexafluoroisopropanol solution can also dissolve silk fibroin. Although chloride, iodide, bromide, and thiocyanate solutions can dissolve silk fibroin, most of these solutions often exhibit some toxicity to humans and are harmful to human health, therefore they are not suitable for dissolving silk fibroin. Non-toxic and inexpensive salts such as calcium chloride are the best choice for dissolving silk fibroin. Furthermore, the emergence of eutectic solvent technology, especially natural eutectic solvents, has provided new possibilities for the selection of silk fibroin dissolution systems.

[0004] As an organic component of silk protein, silk fibroin is coated with sericin on its surface. Therefore, it is necessary to degumme it in order to obtain silk fibroin. The current silk protein preparation technology still has the following problems: (1) The silk protein extraction system is simple and has poor safety and environmental protection, which makes it difficult to meet the inherent requirements of modern production technology for industrial production; (2) Sericin is often discarded as waste, which does not achieve the maximum resource utilization and fails to achieve the simultaneous continuous production of sericin and silk protein; (3) Silk fibroin has poor water solubility, which makes it difficult to meet the technical production requirements of multiple scenarios, and the related processing performance is still difficult to meet the technical production requirements of multiple scenarios; (4) The extraction conditions of silk fibroin are harsh, which affects its structure and function and makes it difficult to meet the subsequent processing performance requirements.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] To address the problems of difficult dissolution, poor water solubility, and environmentally unfriendly production processes associated with existing technologies for silk fibroin, this invention provides a method and application for simultaneously extracting silk fibroin and sericin using a eutectic solvent. The technical problem to be solved by this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent, comprising the following steps: S1. Obtain the first process parameters for extracting silk fibroin and the second process parameters for extracting sericin. The first process parameters include the components of the first eutectic solvent for extracting silk fibroin, the molar ratio of each component in the first eutectic solvent, the first extraction temperature, the first extraction time, and the first extraction pH value. The second process parameters include the components of the second eutectic solvent for extracting sericin, the molar ratio of each component in the second eutectic solvent, the mass concentration of the inorganic base in the second eutectic solvent, the second extraction temperature, and the second extraction time. S2. Based on the second process parameters for extracting sericin described in step S1, the silkworm cocoons are degummed to obtain degummed silkworm cocoons and a solution containing sericin; the solution containing sericin is dried to obtain sericin. S3. Using the first process parameters for extracting silk fibroin in step S1, the degummed silkworm cocoons are extracted and dried to obtain silk fibroin.

[0007] In a first aspect, the present invention provides an application of silk fibroin obtained by the above method in the preparation of edible packaging materials.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method for simultaneously extracting silk fibroin and sericin based on eutectic solvents provided by this invention achieves simultaneous and continuous production of sericin and silk fibroin by setting first and second process parameters respectively and using different eutectic solvent systems for the extraction of sericin and silk fibroin respectively. The different components of the first and second eutectic solvents, and the different selection of hydrogen bond acceptors and hydrogen bond donors, allow for optimal extraction results for sericin and silk fibroin respectively. Sericin, as a byproduct of silkworm cocoon degumming, is effectively recycled and utilized, avoiding resource waste and improving the utilization rate of silkworm cocoons.

[0009] 2. The eutectic solvent system used in this invention comprises components such as choline chloride, betaine, citric acid, urea, and malic acid. These components are non-toxic or low-toxic and pose minimal harm to the human body. Compared to extraction systems using strong acids and alkalis, this system offers higher safety and environmental friendliness. The eutectic solvent is recyclable, reducing solvent usage and lowering production costs and environmental burden.

[0010] 3. This invention optimizes the first and second process parameters by constructing a response surface methodology. It systematically screens the optimal eutectic solvent extraction parameters using a combination of single-factor experiments and response surface analysis. The single-factor experiments first determine the optimal level range for each factor, while the response surface experiments further establish regression model equations to obtain the optimal combination of process parameters. Compared to traditional single-factor experimental methods, this approach can more comprehensively examine the interactions between factors, improving the efficiency and accuracy of process optimization. Silk fibroin and sericin can be prepared simultaneously, with a silk fibroin extraction rate as high as 15.25 ± 0.16%.

[0011] 4. The silk fibroin prepared by the extraction method provided in this invention forms a coexistence system of low molecular weight peptides and high molecular weight aggregates. The small molecular weight silk fibroin can be used in functional foods. It can be rapidly absorbed by the gastrointestinal tract without the need for proteolytic hydrolysis, and can be used for the development of functional foods. At the same time, the secondary structure of silk fibroin is mainly β-sheet (70%). This structure gives the material excellent mechanical properties and stability, ensuring its application prospects in the biopharmaceutical, food and other industries.

[0012] 4. The silk fibroin obtained by this invention can be used to prepare edible packaging materials. Silk fibroin has good film-forming properties and mechanical properties. When combined with other edible materials, it can be made into biodegradable edible packaging materials for use in the food packaging field, expanding the application scope of silk fibroin and increasing its economic value.

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is a flowchart of a method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent, provided by an embodiment of the present invention. Figure 2 This is a schematic diagram of the screening results of eutectic solvents for extracting silk fibroin provided in Example 1 of the present invention; Figure 3 This is a schematic diagram of the screening results of the eutectic solvent ratio for extracting silk fibroin provided in Example 1 of the present invention; Figure 4 This is a schematic diagram of the extraction time screening results for extracting silk fibroin provided in Example 1 of the present invention; Figure 5 This is a schematic diagram of the extraction temperature screening results for extracting silk fibroin provided in Example 1 of the present invention; Figure 6 This is a schematic diagram of the extraction pH screening results for silk fibroin provided in Example 1 of the present invention; Figure 7 This is a response surface diagram illustrating the effect of the interaction between extraction time, DES ratio, and extraction temperature on the silk fibroin extraction rate, as provided in Example 2 of this invention. Figure 8 This is a schematic diagram illustrating the analysis of the rheological properties of silk fibroin provided in Embodiment 2 of the present invention; Figure 9 This is a schematic diagram of the thermogravimetric analysis results of silk fibroin provided in Example 2 of the present invention; Figure 10 This is a schematic diagram of the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis results of silk fibroin provided in Example 2 of the present invention; Figure 11 This is a Fourier transform infrared (FTIR) characterization diagram (A) and a schematic diagram (B) of the secondary structure analysis results of silk fibroin provided in Embodiment 2 of the present invention. Detailed Implementation

[0015] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following describes in detail, with reference to the accompanying drawings and specific embodiments, a method and application for simultaneous extraction of silk fibroin and sericin based on a eutectic solvent according to the present invention.

[0016] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a more in-depth and concrete understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the technical solutions of the present invention.

[0017] It should be noted that, in this document, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified. Furthermore, the terms "comprising," "including," or any other variations are intended to cover non-exclusive inclusion, such that an article or apparatus comprising a list of elements includes not only those elements but also other elements not explicitly listed.

[0018] This invention provides a method for simultaneously extracting silk fibroin and sericin using a eutectic solvent, see [link to relevant documentation]. Figure 1 The method includes the following steps: S1. Obtain the first process parameters for extracting silk fibroin and the second process parameters for extracting sericin. The first process parameters include the components of the first eutectic solvent for extracting silk fibroin, the molar ratio of each component in the first eutectic solvent, the first extraction temperature, the first extraction time, and the first extraction pH value; the second process parameters include the components of the second eutectic solvent for extracting sericin, the molar ratio of each component in the second eutectic solvent, the mass concentration of inorganic alkali in the second eutectic solvent, the second extraction temperature, and the second extraction time.

[0019] In some examples, the components of the first eutectic solvent are choline chloride-citric acid, choline chloride-urea, or choline chloride-malic acid; The second eutectic solvent consists of betaine-citric acid, choline chloride-citric acid, or betaine-malic acid, wherein betaine and choline chloride are hydrogen bond acceptors, and citric acid, urea, and malic acid are hydrogen bond donors.

[0020] In some examples, the molar ratio of hydrogen bond acceptors to hydrogen bond donors in the first eutectic solvent is 1:(3-5); the first extraction temperature is 85-95°C; the first extraction time is 4-6 h; and the first extraction pH is 7-9.

[0021] In some examples, the molar ratio of hydrogen bond acceptors to hydrogen bond donors in the second eutectic solvent is 1:(0.5–2); the inorganic base in the second eutectic solvent is sodium carbonate with a mass concentration of 0.3%–0.5%; the second extraction temperature is 85–95°C; and the second extraction time is 0.5–1 h.

[0022] S2. Based on the second process parameters for extracting sericin in step S1, the silkworm cocoons are degummed to obtain degummed cocoons and a solution containing sericin. Further, the sericin-containing solution is concentrated, dialyzed with distilled water, and dried to obtain sericin solid powder.

[0023] S3. Using the first process parameters for extracting silk fibroin in step S1, extract the silk from degummed silkworm cocoons and dry it to obtain silk fibroin. Specifically, the extract obtained after extracting the degummed silkworm cocoons is concentrated, dialyzed with distilled water, and dried to obtain silk fibroin solid powder.

[0024] In one embodiment of the present invention, after step S1, a response surface model is further constructed for optimizing the first process parameter and the second process parameter. In one implementation, constructing a response surface model to optimize the first process parameter includes: Step 101: Based on the first process parameters, set up multiple corresponding single-factor experimental groups and measure the extraction rate of silk fibroin in each experiment.

[0025] Specifically, this includes: based on the first process parameters, under the same molar ratio of hydrogen bond acceptor to hydrogen bond donor, the same extraction temperature, extraction time, and extraction pH, setting up single-factor experimental groups with different eutectic solvents, detecting the extraction rate of silk fibroin, and screening to obtain the components of the optimal eutectic solvent for extracting silk fibroin.

[0026] Based on the components of the optimal eutectic solvent, under the same extraction temperature, extraction time, and extraction pH, single-factor experimental groups with different molar ratios of each component in the optimal eutectic solvent were set up to detect the extraction rate of silk fibroin and screen to obtain the molar ratio of each component in the optimal eutectic solvent for extracting silk fibroin.

[0027] Based on the components of the optimal eutectic solvent and the molar ratio of each component, single-factor experimental groups with different extraction times were set up under the same extraction temperature and extraction pH value. The extraction rate of silk fibroin was detected, and the optimal extraction time for extracting silk fibroin was screened.

[0028] Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, and the optimal extraction time, single-factor experimental groups with different extraction temperatures were set up under the same extraction pH value. The extraction rate of silk fibroin was detected, and the optimal extraction temperature for extracting silk fibroin was screened and obtained.

[0029] Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, the optimal extraction time, and the optimal extraction temperature, single-factor experimental groups with different extraction pH values ​​were set up to detect the extraction rate of silk fibroin and screen to obtain the optimal extraction pH value for extracting silk fibroin.

[0030] Step 102: Based on the extraction rate results of silk fibroin in the corresponding single-factor experimental groups, the extraction time, the molar ratio of each component in the eutectic solvent, and the extraction temperature are selected as independent variables to construct a first response surface model. Based on the regression model equation of the first response surface model, the optimized first process parameters for extracting silk fibroin are obtained; the regression model equation is shown below:

[0031] Where Y is the response variable, A is the extraction time, B is the molar ratio of each component in the eutectic solvent, and C is the extraction temperature.

[0032] The optimized first process parameters for extracting silk fibroin include: the first eutectic solvent is choline chloride-urea, with a molar ratio of choline chloride to urea of ​​1:4; the first extraction temperature is 90℃; the first extraction time is 5h; and the first extraction pH value is 8.

[0033] In one implementation, constructing a response surface model to optimize the second process parameters includes: Step 101: Based on the second process parameters, set up multiple corresponding single-factor experimental groups and measure the extraction rate of sericin in each experiment.

[0034] Specifically, this includes: based on the second process parameters, under the same molar ratio of hydrogen bond acceptor to hydrogen bond donor, the same mass concentration of inorganic base, the same extraction temperature, and the same extraction time, setting up single-factor experimental groups with different eutectic solvents, detecting the extraction rate of sericin, and screening to obtain the components of the optimal eutectic solvent for extracting sericin; Based on the components of the optimal eutectic solvent, under the same inorganic alkali concentration, the same extraction temperature, and the same extraction time, single-factor experimental groups with different molar ratios of each component in the optimal eutectic solvent were set up to detect the extraction rate of sericin and screen to obtain the molar ratio of each component in the optimal eutectic solvent for extracting the sericin. Based on the components and molar ratio of the optimal eutectic solvent, single-factor experimental groups with different extraction times were set up at the same extraction temperature and the same inorganic base concentration to detect the extraction rate of sericin and screen to obtain the optimal extraction time for extracting the sericin. Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, and the optimal extraction time, single-factor experimental groups with different extraction temperatures were set up under the same inorganic alkali concentration to detect the extraction rate of sericin and screen to obtain the optimal extraction temperature for extracting sericin. Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, the optimal extraction time, and the optimal extraction temperature, single-factor experimental groups with different inorganic alkali concentrations were set up to detect the extraction rate of sericin and screen to obtain the optimal inorganic alkali concentration for extracting sericin.

[0035] Step 102: Based on the extraction rate results of sericin in the corresponding single-factor experimental group, three parameters are selected as independent variables to construct a second response surface model. Based on the regression model equation of the second response surface model, the optimized second process parameters for extracting sericin are obtained.

[0036] This invention provides a method for simultaneously extracting silk fibroin and sericin using eutectic solvents. By setting first and second process parameters and employing different eutectic solvent systems for the extraction of sericin and silk fibroin respectively, the simultaneous and continuous production of sericin and silk fibroin is achieved. The different compositions of the first and second eutectic solvents, along with the different selection of hydrogen bond acceptors and donors, optimize the extraction of sericin and silk fibroin. Sericin, a byproduct of silkworm cocoon degumming, can be effectively recycled, avoiding resource waste and enabling the high-value utilization of silk fibroin in the food industry. This also lays the foundation for the application of silk fibroin in the food industry.

[0037] The optimized first process parameters provided by this invention use a choline chloride-urea system as the first eutectic solvent, with a molar ratio of choline chloride to urea of ​​1:4, an extraction temperature of 90℃, an extraction time of 5 h, and an extraction pH of 8. This combination of process parameters, obtained through response surface methodology optimization, can effectively improve the extraction rate of silk fibroin while maintaining the structural integrity of the silk fibroin.

[0038] This invention also provides the application of the prepared silk fibroin in the preparation of edible packaging materials. Silk fibroin possesses excellent film-forming and mechanical properties, and when combined with other edible materials, it can be used to create biodegradable edible packaging materials for food packaging. This application expands the scope of silk fibroin's use and enhances its economic value.

[0039] The following specific embodiments further illustrate the method for simultaneous extraction of silk fibroin and sericin based on eutectic solvent provided by the present invention.

[0040] Example 1 Deep eutectic solvents (DES) are multi-component liquids formed by mixing hydrogen bond donors (such as urea and carboxylic acids) and hydrogen bond acceptors (such as quaternary ammonium salts) in a predetermined molar ratio. Their physicochemical properties are similar to those of ionic liquids, exhibiting low vapor pressure, high viscosity, and adjustable solubility. Therefore, DES are commonly used for extracting natural products and are often considered a greener and cheaper solvent alternative.

[0041] Single-factor experiments were conducted to screen for eutectic solvent extraction combinations of sericin and silk fibroin. This example illustrates the screening of eutectic solvents for silk fibroin.

[0042] (1) Screening of eutectic solvent systems The hydrogen bond donors used include glucose, fructose, L-proline, β-alanine, eugenol, lactic acid (LA), citric acid (CA), malic acid (MA), xylitol, ethylene glycol, glycerol (Gly), urea (Ur), and fatty acids; the hydrogen bond acceptors used include choline chloride (ChCl), betaine, thymol, menthol, and L-carnitine.

[0043] Under fixed extraction conditions of 95℃, pH 9, 5 h, and a eutectic solvent system with a hydrogen bond acceptor to hydrogen bond donor molar ratio of 1:2, single-factor experiments were conducted to determine the usable eutectic solvent extraction system for silk fibroin by varying only the combination of hydrogen bond acceptors and hydrogen bond donors. Results are shown below. Figure 2 The preferred eutectic solvent extraction systems are choline chloride / citric acid, choline chloride / urea, and choline chloride / malic acid. Among them, choline chloride / urea is the component of the optimal eutectic solvent screened in this example.

[0044] (2) The ratio of eutectic solvent system (molar ratio of hydrogen bond acceptor to hydrogen bond donor) Based on the optimized eutectic solvent system (choline chloride / urea), under fixed extraction conditions of 95℃, pH 9, and 5 h, single-factor experiments were conducted using different eutectic solvent system ratios (molar ratios of hydrogen bond acceptors to hydrogen bond donors of 1:2, 1:3, 1:4, 1:5, and 1:6) to measure the extraction rate of silk fibroin. Results are shown below. Figure 3 The optimal eutectic solvent system ratios for silk fibroin extraction are 1:3, 1:4, and 1:6, which exhibit high extraction rates. Among these, a eutectic solvent system ratio of 1:4 is the optimal ratio in this embodiment.

[0045] (3) Extraction time Based on the determined choline chloride / urea eutectic solvent system and the 1:4 molar ratio of hydrogen bond acceptors to hydrogen bond donors, under fixed extraction temperatures of 95℃ and extraction pH of 9, the effects of different extraction times (3, 4, 5, 6, and 7 h) on the extraction rate of silk fibroin were compared. (See [reference needed]). Figure 4 The optimal extraction time for silk fibroin was determined to be 4 h, 5 h, or 6 h through single-factor experiments. Among them, the extraction time of 5 h was the optimal extraction time selected in this example.

[0046] (4) Extraction temperature Based on the determined eutectic solvent system of choline chloride / urea, the molar ratio of urea to choline chloride of 1:4, and the extraction time of 5 h, the effects of different extraction temperatures (75℃, 80℃, 85℃, 90℃, and 95℃) on the extraction rate of silk fibroin were compared under the condition of a fixed extraction pH of 9. (See [reference needed]). Figure 5 The optimal extraction temperatures for silk fibroin were determined through single-factor experiments: 80℃, 90℃, or 95℃. The extraction temperature of 90℃ was the optimal extraction temperature selected in this embodiment.

[0047] (5) pH of eutectic solvent system Based on the determined eutectic solvent system of choline chloride / urea, the molar ratio of urea to choline chloride of 1:4, the extraction time of 5 h, and the extraction temperature of 90 °C, the effect of different extraction pH values ​​(pH values ​​set at 5, 6, 7, 8, and 9) on the extraction rate of silk fibroin was compared. (See [reference needed]). Figure 6 The optimal pH values ​​for silk fibroin extraction were determined to be 7, 8, and 9 through single-factor experiments.

[0048] Example 2 Based on Example 1, taking silk fibroin as an example, a response surface model was constructed based on the results of the above single-factor experimental group to further optimize the extraction parameters of silk fibroin.

[0049] Based on the results of the single-factor experiments, as shown in Table 1, three factors that significantly affect the extraction rate of silk fibroin were selected (that is, three parameters with a high impact on the extraction rate): extraction time (A), eutectic solvent (DES) ratio (B), and extraction temperature (C). Each factor was tested at three levels, and the response surface methodology was designed as shown in Table 1 below.

[0050] Table 1. Factors and levels in response surface methodology

[0051] Based on the independent variables (extraction time, DES ratio, and extraction temperature) in Table 1, the response surface methodology and results for silk fibroin extraction are shown in Table 2, and the analysis of variance for the response surface methodology is shown in Table 3.

[0052] Table 2. Response Surface Experiment Design and Results

[0053] Table 3. Analysis of variance of the response surface

[0054] Table 3 shows that the significance test P < 0.05, indicating that the response surface methodology (RSM) model is statistically significant. The F-value analyzes the significance of each variable's influence on the response value. The order of influence of each variable on the silk fibroin extraction rate is: C (extraction temperature) > A (extraction time) > B (DES ratio). The coefficient of determination R0 of the RSM model is... 2 =0.9490, Corrected coefficient of determination R 2 The adj=0.8573 value and the lack-of-fit term are not significant, indicating that the response surface model fits the original data well. Therefore, the response surface model can be used to analyze the optimal process conditions for silk fibroin extraction. Using Design-Expert 13.0 software to analyze the data in Table 3, the regression equations for extraction rate (Y) on extraction time (A), DES ratio (B), and extraction temperature (C) are as follows:

[0055] Based on the above regression equation, the optimal extraction process for silk fibroin was determined to be: choline chloride / urea as the eutectic solvent, a DES ratio (molar ratio of urea to choline chloride) of 1:4, an extraction time of 5 h, a temperature of 90℃, and a pH of 8. Under these conditions, three replicate experiments were conducted, and the extraction rate was 15.25 ± 0.16%.

[0056] like Figure 7 As shown, in order to further analyze the effects of extraction time, DES ratio and extraction temperature on the silk fibroin extraction process, response surfaces of the interaction between extraction time and DES ratio on the silk fibroin extraction rate were plotted (a), (b), and (c) respectively.

[0057] Figure 7 It can be seen that the interaction between extraction time, DES ratio and extraction temperature has a significant impact on the extraction rate of silk fibroin, and the overall trend is "first increase and then decrease", indicating that there are optimal process conditions to achieve the peak extraction rate. Figure 7 (a) shows the effect of the interaction between extraction time and DES ratio on the extraction rate of silk fibroin. The slope of the curve is relatively gentle, indicating that the interaction between the two factors is weak. When the extraction time or DES ratio is too low or too high, the extraction rate decreases significantly, indicating that the key to extraction is that each factor is within a reasonable range. Figure 7 (b) shows the effect of the interaction between extraction time and extraction temperature on the extraction rate of silk fibroin. The steep slope of the surface indicates that these two factors are more sensitive to the extraction rate. When the temperature increases or the time is prolonged, the extraction rate rises rapidly and reaches a peak. However, if the temperature continues to increase, the extraction rate drops sharply, and the contour lines are obviously elliptical, indicating that there is a significant interaction between the two factors. This suggests that excessively high temperature or excessively long time will lead to the degradation of silk fibroin, thereby reducing the extraction rate. Figure 7 Figure (c) shows the effect of the interaction between DES ratio and extraction temperature on the silk fibroin extraction rate. The steep slope of the curve indicates that the interaction between DES ratio and temperature is also significant. Too low a DES ratio cannot effectively dissolve silk fibroin, while too high a DES ratio or temperature may exacerbate silk fibroin denaturation or degradation. Therefore, synergistic optimization of both is necessary to avoid the negative impact of excessively increasing any single factor. In summary, the interaction of the three factors is significant. Process optimization should focus on balancing the interactions between temperature and time, and temperature and DES ratio, to obtain the highest silk fibroin extraction rate.

[0058] Silk fibroin performance characterization: According to the extraction parameters selected in this embodiment, choline chloride / urea is used as a eutectic solvent, the DES ratio (molar ratio of choline chloride to urea) is 1:4, the extraction time is 5 h, the temperature is 90℃, and the pH value is 8. Silk fibroin is prepared, and a silk fibroin sample is obtained. The performance of the silk fibroin sample is then tested.

[0059] 1. Solubility and processing characteristics of silk fibroin As shown in Table 4, the soluble protein content in the silk fibroin sample was 73.54%. The solubility of the silk fibroin sample in distilled water was 0.953 g / mL, and in choline chloride / urea solution was 0.826 g / mL, indicating that the DES system has high compatibility for extracting silk fibroin samples. It also exhibits a solubility of 0.016 g / mL in 0.5% sodium carbonate solution and is insoluble in ethanol, making it suitable for the separation and purification of silk fibroin samples. Furthermore, the silk fibroin sample showed a foaming capacity (Fc) of 0.05%, foam stability (Fs) of 40%, oil retention capacity (OHC) of 190.85%, emulsifying ability (EAI) of 50%, and emulsifying stability (ES) of 37.5%, indicating moderate emulsifying performance. Silk fibroin can be used in functional foods to supplement nutrition, control weight, protect bones, and regulate blood pressure and lipids. Based on the aforementioned solubility and processing characteristics, silk fibroin has broad application prospects in the food, cosmetic, and biopharmaceutical fields.

[0060] Table 4. Silk fibroin content, solubility, and processing characteristics

[0061] 2. Results of Rheological Properties Analysis of Silk Fibroin For silk fibroin samples with different concentrations (50 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL), the effects of shear rate on viscosity and shear stress, as well as the effects of temperature on viscosity and shear stress, were studied using a rheometer. Figure 8 a indicates that as the shear rate increases, the viscosity decreases and tends to stabilize, and the higher the concentration of silk fibroin, the greater the initial viscosity. Figure 8 b indicates that the higher the concentration, the greater the shear stress at the same shear rate, reflecting the increase in flow resistance with increasing concentration. To further clarify the fluid type of the silk fibroin solution, a power-law equation was used to fit the curve of shear rate versus shear force. Figure 8 (b and Table 5) The flow behavior index n increases with silk fibroin concentration by 0.9736, 0.9631, 0.9797, 0.9892, 0.9860, and 0.9783, respectively, all less than 1. This indicates that silk fibroin solutions of different concentrations exhibit typical pseudoplastic fluid characteristics with shear-thinning properties. Overall, the n value increases slightly with increasing concentration, indicating that the shear-thinning degree of the solution weakens slightly with increasing silk fibroin concentration, becoming closer to a Newtonian fluid, but still maintaining obvious pseudoplastic characteristics. The consistency coefficient K increases with silk fibroin concentration by 1.3426, 1.9078, 2.1305, 2.7312, 3.2381, and 3.5958, showing a significant increasing trend, indicating that the apparent viscosity of the solution increases with increasing concentration. (Concentration curves R...) 2 The value >0.998 indicates that the power-law equation can effectively describe the shear stress-shear rate relationship of silk fibroin solution, providing a reliable basis for the quantitative analysis of its rheological behavior. Figure 8 c indicates that the viscosity decreases significantly with increasing temperature. The higher the temperature, the more intense the molecular thermal motion, which leads to a decrease in solution viscosity. Figure 8 Figure d shows that the shear stress continuously decreases with increasing temperature, and the stress difference between high and low concentrations gradually narrows with increasing temperature, indicating that high temperature can weaken the effect of concentration on flow resistance. These results provide a basis for temperature control in the industrial molding process of silk fibroin solutions, enabling optimization of process parameters, solving processing challenges in high-concentration systems, adapting to different production scenarios, and reducing energy costs.

[0062] Table 5. Fitting results of power law for silk fibroin (SF) solution

[0063] 3. Thermal stability of silk fibroin like Figure 9As shown, the thermal decomposition process of the silk fibroin sample mainly consists of three stages: 50–250℃, 250–400℃, and 400–700℃. In the 50–250℃ stage, the mass of the silk fibroin sample remained relatively stable with no significant weight loss, indicating that free and bound water in the sample had been largely removed before testing. As the temperature gradually increased to 400℃, the silk fibroin sample underwent drastic thermal weight loss, with the TG curve (thermogravimetric curve) dropping sharply. The DTG curve (derivative thermogravimetric curve) showed a significant weight loss rate peak around 300℃, corresponding to the breakage of peptide bonds, thermal degradation of the molecular backbone, and destruction of secondary structures. The weight loss rate in this stage was approximately 50%. Above 400℃, the sample entered a slow carbonization stage, with a significant decrease in the thermal weight loss rate. Overall, the silk fibroin exhibits good thermal stability and can be stored for extended periods at room temperature or under refrigeration.

[0064] 4. Analysis of the processing characteristics of silk fibroin (1) SDS-PAGE analysis results See Figure 10 Under reducing conditions, the disulfide bonds of silk fibroin were completely broken by the reducing agent (β-mercaptoethanol), and the macromolecular chains depolymerized. Electrophoretic bands were diffusely distributed in the 10–50 kDa low molecular weight region, with no obvious high molecular weight bands. This indicates that after the disulfide bonds were broken under reducing conditions, the originally cross-linked silk fibroin macromolecules were dissociated into low molecular weight peptides. Under non-reducing conditions, the disulfide bonds were not broken, and the molecules were cross-linked or aggregated through disulfide bonds. The band remained at the top of the separating gel, presenting as a wide and diffuse high molecular weight band, indicating the presence of a large number of macromolecular aggregates formed by disulfide bond cross-linking in the solution, which were difficult to separate. The results show that silk fibroin exhibits disulfide bond cross-linking, which degrades during extraction, forming a system where low molecular weight peptides and high molecular weight aggregates coexist. Small molecular weight silk fibroin can be used in functional foods, as it can be rapidly absorbed by the gastrointestinal tract without enzymatic hydrolysis, and can be used in the development of functional foods.

[0065] (2) Fourier transform infrared spectroscopy characterization like Figure 11 As shown, Fourier transform infrared spectroscopy (FTIR) is a commonly used structural characterization technique for silk fibroin. Amide I, II, and III regions are characteristic signal regions of the protein, with amide I (1600–1700 cm⁻¹) being the most prominent. - ¹) It can be used for secondary structure analysis of silk fibroin ( Figure 11 (a). This embodiment uses deconvolution, second derivative, and peak fitting to deconvolve the amide I region, and then assigns and quantifies the characteristic peaks of each secondary structure. Its secondary structures mainly include four types: β-sheets, random coils, α-helices, and β-turns. Silk fibroin is predominantly β-sheet ( Figure 11(b) This structure endows the material with excellent mechanical properties and stability, and also enables it to be used to make wound dressings, which can accelerate wound healing and prevent secondary damage.

[0066] This embodiment uses response surface methodology to analyze the parameter selection for silk fibroin extraction using a eutectic solvent (choline chloride / urea, choline chloride to urea molar ratio of 1:4, extraction temperature of 90℃, extraction time of 5h, pH of 8, corresponding to a silk fibroin extraction rate of 15.25%±0.16%). The extracted silk fibroin exhibits good water solubility, reaching 0.95±0.13 g / mL, while maintaining the high molecular weight characteristics of silk fibroin. This avoids the industry problem of silk fibroin molecule decomposition caused by other related extraction technologies. It demonstrates good performance in processing-related properties such as water solubility, foaming and foam stability, oil holding capacity, emulsification and emulsion stability, rheology, and thermal stability, and is expected to be applied as a new food resource in the food industry and other fields. The silk fibroin extraction scheme provided by this invention can promote technological innovation in the sericulture industry, ensure the healthy and sustainable development of the industry, and lay a solid theoretical and technical foundation for the preparation and derivative development of silk fibroin, thereby driving the development of the sericulture industry through new consumption growth points.

[0067] It should be noted that Examples 1 and 2 use silk fibroin as an example to screen extraction conditions. The same screening process can be used to optimize the extraction parameters of eutectic solvent for sericin.

[0068] Example 3 This embodiment uses the silk fibroin extraction conditions selected in Example 2 to prepare edible packaging materials. Specifically, it includes: 1. Raw material preparation: Silk fibroin, silkworm cocoons were degummed (betaine / citric acid eutectic solvent, pH adjusted to 8 with Na2CO3, extracted at 90℃ for 1 h), after separating the sericin, silk fibroin was extracted using choline chloride / urea as the eutectic solvent at a ratio of 1:4, at a time of 5 h, a temperature of 90℃, and a pH of 8, respectively. The extracted silk fibroin was obtained by dialysis and freeze-drying (stored at 4℃ for later use); glycerol (food grade).

[0069] 2. Preparation steps: Silk fibroin dissolution: Add silk fibroin to distilled water at a solid-liquid ratio (mass-volume ratio) of 1:10, stir overnight to ensure dissolution effect, and obtain silk fibroin solution.

[0070] Preparation of film-forming solution: Take 20 mL of silk fibroin solution, add glycerol to the silk fibroin solution, the mass ratio of glycerol to silk fibroin (dry weight) is 1:4; stir for 30 minutes, degas by sonication to obtain film-forming solution.

[0071] Casting: Pour the film-forming liquid onto a food-grade polytetrafluoroethylene plate and dry it in a 50°C constant temperature and humidity oven for 24 hours.

[0072] Post-processing: The dried membrane was immersed in a 50% ethanol aqueous solution for 30 minutes to induce the transformation of silk fibroin to a β-sheet structure (enhancing mechanical strength). After removal, it was dried again at room temperature to obtain a silk fibroin membrane.

[0073] Silk fibroin membrane pretreatment: Store at room temperature (15-30℃) and 50% relative humidity for no less than 3 days.

[0074] 3. Performance Test Results (1) Basic properties: Thickness is 0.035±0.001 mm; optical properties are transparent film with the ability to block ultraviolet rays; it absorbs water and swells when placed in water at room temperature, but completely dissolves in water within 24 hours.

[0075] (2) Mechanical properties: tensile strength was 25.27±2.15 MPa; elongation at break was 58.55±3.38%; water vapor transmission rate was 1.16±0.14×10 –1 2 g·m / (Pa·s·m 2 ).

[0076] (3) Food safety: Tested according to GB 4806.8-2022 standard, the total migration (4% acetic acid, 60℃, 2h) was 2.5 mg / dm³. 2 Well below 10 mg / dm 2 It meets national standards and requirements for direct contact with food.

[0077] (4) Applying coating technology extends the shelf life of strawberries by about 7 days, and reduces weight loss and nutrient loss; applying coating technology extends the shelf life of fresh-cut potatoes by 3 days, prevents browning, and reduces weight loss and nutrient loss.

[0078] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously extracting silk fibroin and sericin using a eutectic solvent, characterized in that, Includes the following steps: S1. Obtain the first process parameters for extracting silk fibroin and the second process parameters for extracting sericin. The first process parameters include the components of the first eutectic solvent for extracting silk fibroin, the molar ratio of each component in the first eutectic solvent, the first extraction temperature, the first extraction time, and the first extraction pH value. The second process parameters include the components of the second eutectic solvent for extracting sericin, the molar ratio of each component in the second eutectic solvent, the mass concentration of the inorganic base in the second eutectic solvent, the second extraction temperature, and the second extraction time. S2. Based on the second process parameters for extracting sericin described in step S1, the silkworm cocoons are degummed to obtain degummed silkworm cocoons and a solution containing sericin; the solution containing sericin is dried to obtain sericin. S3. Using the first process parameters for extracting silk fibroin in step S1, the degummed silkworm cocoons are extracted and dried to obtain silk fibroin.

2. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 1, characterized in that, The components of the first eutectic solvent are choline chloride-citric acid, choline chloride-urea, or choline chloride-malic acid; The second eutectic solvent is composed of betaine-citric acid, choline chloride-citric acid, or betaine-malic acid, wherein betaine and choline chloride are hydrogen bond acceptors, and citric acid, urea, and malic acid are hydrogen bond donors.

3. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 2, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor in the first eutectic solvent is 1:(3-5); the first extraction temperature is 85-95℃; the first extraction time is 4-6h; and the first extraction pH value is 7-9.

4. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 2, characterized in that, The molar ratio of hydrogen bond acceptors to hydrogen bond donors in the second eutectic solvent is 1:(0.5-2); the inorganic base in the second eutectic solvent is sodium carbonate, and the mass concentration of sodium carbonate is 0.3%-0.5%; the second extraction temperature is 85-95℃; and the second extraction time is 0.5-2h.

5. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to any one of claims 1-4, characterized in that, After step S1, the method further includes constructing a response surface model for optimizing the first process parameters and the second process parameters. The construction of the response surface model includes: Step 101: Based on the first process parameters, set up multiple corresponding single-factor experimental groups and measure the extraction rate of silk fibroin in each experiment. Based on the second process parameters, multiple corresponding single-factor experimental groups were set up, and the extraction rate of sericin in each experiment was measured. Step 102: Based on the extraction rate results of silk fibroin in the corresponding single-factor experimental group, three parameters are selected as independent variables to construct the first response surface model. Based on the regression model equation of the first response surface model, the optimized first process parameters for extracting silk fibroin are obtained. Based on the extraction rate results of sericin in the corresponding single-factor experimental group, three parameters were selected as independent variables to construct a second response surface model. The optimized second process parameters for extracting sericin were obtained based on the regression model equation of the second response surface model.

6. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 5, characterized in that, In step S101, based on the first process parameters, multiple corresponding single-factor experimental groups are set up, including: Based on the first process parameters, under the same hydrogen bond acceptor to hydrogen bond donor molar ratio, the same extraction temperature, extraction time, and extraction pH, single-factor experimental groups with different eutectic solvents were set up to detect the extraction rate of silk fibroin and screen the components of the optimal eutectic solvent for extracting the silk fibroin. Based on the components of the optimal eutectic solvent, under the same extraction temperature, extraction time, and extraction pH, single-factor experimental groups with different molar ratios of each component in the optimal eutectic solvent were set up to detect the extraction rate of silk fibroin and screen to obtain the molar ratio of each component in the optimal eutectic solvent for extracting the silk fibroin. Based on the components and molar ratio of the optimal eutectic solvent, single-factor experimental groups with different extraction times were set up under the same extraction temperature and the same extraction pH value. The extraction rate of silk fibroin was detected, and the optimal extraction time for extracting the silk fibroin was screened. Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, and the optimal extraction time, single-factor experimental groups with different extraction temperatures were set up at the same extraction pH value. The extraction rate of silk fibroin was detected, and the optimal extraction temperature for extracting the silk fibroin was screened. Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, the optimal extraction time, and the optimal extraction temperature, single-factor experimental groups with different extraction pH values ​​were set up to detect the extraction rate of silk fibroin and screen to obtain the optimal extraction pH value for extracting the silk fibroin.

7. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 6, characterized in that, Step 102 includes, based on the extraction rate results of silk fibroin in the corresponding single-factor experimental group, screening extraction time, the molar ratio of each component in the eutectic solvent, and extraction temperature as independent variables to construct a first response surface model, and obtaining the optimized first process parameters for extracting silk fibroin based on the regression model equation of the first response surface model; the regression model equation is shown below: Where Y is the response variable, A is the extraction time, B is the molar ratio of each component in the eutectic solvent, and C is the extraction temperature.

8. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 7, characterized in that, The optimized first process parameters for extracting silk fibroin include: the first eutectic solvent is choline chloride-urea, with a molar ratio of choline chloride to urea of ​​1:4; the first extraction temperature is 90℃; the first extraction time is 5h; and the first extraction pH value is 8.

9. The method for simultaneously extracting silk fibroin and sericin based on a eutectic solvent according to claim 5, characterized in that, In step S101, based on the second process parameters, multiple corresponding single-factor experimental groups are set up, including: Based on the second process parameters, under the same molar ratio of hydrogen bond acceptor to hydrogen bond donor, the same mass concentration of inorganic base, the same extraction temperature, and the same extraction time, single-factor experimental groups with different eutectic solvents were set up to detect the extraction rate of sericin and screen the components of the optimal eutectic solvent for extracting the sericin. Based on the components of the optimal eutectic solvent, under the same inorganic alkali concentration, the same extraction temperature, and the same extraction time, single-factor experimental groups with different molar ratios of each component in the optimal eutectic solvent were set up to detect the extraction rate of sericin and screen to obtain the molar ratio of each component in the optimal eutectic solvent for extracting the sericin. Based on the components and molar ratio of the optimal eutectic solvent, single-factor experimental groups with different extraction times were set up at the same extraction temperature and the same inorganic base concentration to detect the extraction rate of sericin and screen to obtain the optimal extraction time for extracting the sericin. Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, and the optimal extraction time, single-factor experimental groups with different extraction temperatures were set up under the same inorganic alkali concentration to detect the extraction rate of sericin and screen to obtain the optimal extraction temperature for extracting sericin. Based on the components of the optimal eutectic solvent, the molar ratio of each component in the optimal eutectic solvent, the optimal extraction time, and the optimal extraction temperature, single-factor experimental groups with different inorganic alkali concentrations were set up to detect the extraction rate of sericin and screen to obtain the optimal inorganic alkali concentration for extracting sericin.

10. The use of the silk fibroin extracted by the method of any one of claims 1-9 in the preparation of edible packaging materials.

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