Method for improving stability of anthocyanin by mosaic combination with silk fibroin nanofiber

Abstract

The invention relates to a method for improving the stability of anthocyanin through mosaic combination with silk fibroin nanofibers, and particularly provides a silk fibroin nanofiber-anthocyanin compound which comprises (i) silk fibroin nanofibers; and (ii) an anthocyanin; wherein the silk fibroin nanofibers and the anthocyanin are combined in an inlaid mode. Aiming at the problems of poor heat resistance and low bioavailability of the anthocyanin, silk fibroin nanofibers (such as silk fibroin nanofibers) and the anthocyanin are utilized to interact in an inlaid manner, so that the heat retention rate of the anthocyanin at 80 DEG C is maximally improved by 90%, the digestion stability is improved by 103%, and the bioavailability of the anthocyanin is improved. Therefore, the method provided by the invention can effectively improve the thermal stability and bioavailability of the anthocyanin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for improving the stability of anthocyanins by embedding with silk fibroin nanofiber. BACKGROUND

[0002] Anthocyanins, as a natural pigment derived from plants, can exhibit pink, red, orange, purple and blue colors at different pH values, and can be used for food coloring. In addition to rich colors, anthocyanins also have various biological activities such as antioxidant, anti-inflammatory, anti-tumor, etc., and as a natural functional food pigment, they have attracted the attention of consumers. At present, the new application of anthocyanins as coloring agents or functional active substances has appeared in the food, pharmaceutical and cosmetic industries, however, they are easily affected by external environment such as temperature, enzymes, etc. during extraction, operation, storage and consumption, resulting in fading and degradation, which leads to their inability to be fully utilized. At the same time, the low stability of anthocyanins also affects their bioavailability and further bioavailability, hindering their health promotion. Therefore, improving the stability of anthocyanins, especially the thermal stability and digestive stability, is of great significance to improve the food processing properties and bioavailability of anthocyanins.

[0003] Studies have shown that the stability of anthocyanins can be improved by the intermolecular interaction between proteins and anthocyanins. However, due to the influence of temperature, oxygen, acid and alkali, enzymes and other factors on the structure of proteins, the stability of protein-anthocyanin complexes is also a problem.

[0004] Therefore, there is an urgent need in the art to develop a new method for improving the binding strength of proteins and anthocyanins, improving the heat resistance and digestion resistance of the complex system, thereby enhancing the stability and bioavailability of anthocyanins. SUMMARY

[0005] The purpose of the present application is to provide a method for improving the stability of anthocyanins by embedding with silk fibroin nanofiber, realizing the improvement of the thermal stability and bioavailability of anthocyanins.

[0006] Another purpose of the present application is to provide a new method for improving the binding strength of proteins and anthocyanins, improving the heat resistance and digestion resistance of the complex system, thereby enhancing the stability and bioavailability of anthocyanins.

[0007] In a first aspect, the present application provides a silk fibroin nanofiber-anthocyanin complex, comprising

[0008] (i) silk fibroin nanofiber; and

[0009] (ii) anthocyanins;

[0010] The silk fibroin nanofiber is inlaidly combined with the anthocyanin.

[0011] In another preferred embodiment, the anthocyanin comprises black rice anthocyanin, purple cabbage anthocyanin, and radish anthocyanin.

[0012] In another preferred embodiment, the β-sheet hydrophobic cavity in the silk fibroin nanofiber and the anthocyanin form an inlaid topological structure through non-covalent bonds.

[0013] In another preferred embodiment, the non-covalent bond comprises a hydrogen bond, a hydrophobic interaction, and an electrostatic interaction.

[0014] In another preferred embodiment, in the silk fibroin nanofiber-anthocyanin complex, the mass ratio of the silk fibroin nanofiber to the anthocyanin is 1:1 to 10:1.

[0015] In another preferred embodiment, the silk fibroin nanofiber is a silk fibroin nanofiber silk.

[0016] In another preferred embodiment, the silk fibroin nanofiber is one or more, preferably one.

[0017] In another preferred embodiment, the diameter of the silk fibroin nanofiber is 1-20 nm, preferably 1-5 nm.

[0018] In another preferred embodiment, the method for preparing the silk fibroin nanofiber comprises:

[0019] (a) mixing a lithium bromide solution and a formic acid solution to obtain a lithium bromide-formic acid solution;

[0020] (b) providing a silk fibroin, adding the silk fibroin to the lithium bromide-formic acid solution, dialyzing, and centrifuging to obtain a supernatant;

[0021] (c) placing the supernatant obtained in step (b) in an ultrasonic microwave synergistic workstation and performing ultrasonic treatment to obtain a silk fibroin nanofiber silk solution.

[0022] In another preferred embodiment, the concentration of the lithium bromide solution is 1-15 M, preferably 2-12 M, and more preferably 6-10 M.

[0023] In another preferred embodiment, the concentration of the formic acid solution is 80-100 v / v%, preferably 90-100 v / v%, and more preferably 95-100 v / v%.

[0024] In another preferred embodiment, the step (b) further comprises a water bath heating step.

[0025] In another preferred embodiment, the water bath temperature is 60-90°C, preferably 60-80°C.

[0026] In another preferred embodiment, the water bath time is 2-6h, preferably 2-4h.

[0027] In another preferred embodiment, in step (b), the dialysis is performed in a dialysis bag with a molecular weight cut-off of 2000-4000Da (such as 3500Da).

[0028] In another preferred embodiment, in step (b), the ratio of silk fibroin to lithium bromide-formic acid mixture is 1:50-1:100.

[0029] In another preferred embodiment, in step (c), the ultrasonic power is 200-1000W, preferably 500-900W.

[0030] In another preferred embodiment, in step (c), the ultrasonic time is 5-30min, preferably 10-20min.

[0031] In another preferred embodiment, the method for preparing the silk fibroin nanofiber-anthocyanin complex comprises:

[0032] (i) providing a silk fibroin nanofiber solution and anthocyanin;

[0033] (ii) preparing the anthocyanin into an anthocyanin solution;

[0034] (iii) mixing the silk fibroin nanofiber solution with the anthocyanin solution obtained in step (iii) in equal volume, thereby obtaining the silk fibroin nanofiber-anthocyanin complex.

[0035] In another preferred embodiment, in the complex, the mass ratio of silk fibroin nanofiber to anthocyanin is 1:1-10:1.

[0036] In another preferred embodiment, the silk fibroin is intact silk fibroin or silk fibroin full-length protein, i.e. containing heavy chain, light chain, P25 protein.

[0037] The second aspect of the present application provides a method for preparing a silk fibroin nanofiber-anthocyanin complex, comprising:

[0038] (a) providing a silk fibroin nanofiber and anthocyanin;

[0039] (b) mixing the silk fibroin nanofiber with the anthocyanin, thereby obtaining the silk fibroin nanofiber-anthocyanin complex according to the first aspect of the present application.

[0040] In another preferred embodiment, in the complex, the mass ratio of silk fibroin nanofiber to anthocyanin is 1:1-10:1.

[0041] (a) mixing a lithium bromide solution with a formic acid solution, thereby obtaining a lithium bromide-formic acid solution;

[0042] (b) providing a silk fibroin, adding the silk fibroin into a lithium bromide-formic acid solution, dialysis, centrifugation, thereby obtaining a supernatant;

[0043] (c) placing the supernatant obtained in step (b) into an ultrasonic microwave synergistic workstation, ultrasonic treatment, thereby obtaining a silk fibroin nanofiber silk solution.

[0044] In another preferred embodiment, the concentration of the lithium bromide solution is 1-15 M, preferably 2-12 M, more preferably 6-10 M.

[0045] In another preferred embodiment, the concentration of the formic acid solution is 80-100 v / v%, preferably 90-100 v / v%, more preferably 95-100 v / v%.

[0046] In another preferred embodiment, the step (b) further comprises a water bath heating step.

[0047] In another preferred embodiment, the water bath temperature is 60-90℃, preferably 60-80℃.

[0048] In another preferred embodiment, the water bath time is 2-6 h, preferably 2-4 h.

[0049] In another preferred embodiment, in step (b), the dialysis is performed in a dialysis bag with a molecular weight cut-off of 2000-4000 Da (such as 3500 Da).

[0050] In another preferred embodiment, in step (b), the ratio of silk fibroin to lithium bromide-formic acid mixture is 1:50-1:100.

[0051] In another preferred embodiment, in step (c), the ultrasonic power is 200-1000 W, preferably 500-900 W.

[0052] In another preferred embodiment, in step (c), the ultrasonic time is 5-30 min, preferably 10-20 min.

[0053] The third aspect of the present application provides a use of the silk fibroin nanofiber-anthocyanin complex of the first aspect of the present application for preparing a composition or a preparation for improving the thermal stability and / or the gastrointestinal digestion stability of anthocyanins; and / or enhancing the bioavailability of anthocyanins.

[0054] In another preferred embodiment, the silk fibroin nanofiber-anthocyanin complex is further used for preparing a food additive.

[0055] The fourth aspect of the present application provides a food additive, comprising: the silk fibroin nanofiber-anthocyanin complex of the first aspect of the present application.

[0056] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The anthocyanin thermal stability results of the embodiments 1-3 and the comparative examples 1-3 of the present application are shown in the figure.

[0058] Figure 2 The anthocyanin gastrointestinal digestion stability results of the embodiments 1-3 and the comparative examples 1-3 of the present application are shown in the figure.

[0059] Figure 3 The atomic force microscope image of the silk fibroin nanofiber of Example 2.

[0060] Figure 4 The molecular simulation diagram of the silk fibroin nanofiber of Example 2 and the anthocyanin inlay type combination. DETAILED DESCRIPTION

[0061] The present inventors, through extensive and in-depth research, through a large number of experiments and screening, first accidentally discovered a silk fibroin nanofiber-anthocyanin complex. Specifically, the present application aims at the problems of poor heat resistance and low bioavailability of anthocyanin, and utilizes the inlay type interaction between silk fibroin nanofiber (such as silk fibroin nanofiber silk) and anthocyanin to make the thermal retention rate of anthocyanin at 80℃ increase by 90%, and the digestion stability increase by 103%, which shows that the method provided by the present application can effectively improve the thermal stability and bioavailability of anthocyanin. On this basis, the present application is completed.

[0062] TERMS

[0063] In order to more easily understand the present application, certain technical and scientific terms are defined in detail below. Unless otherwise specifically defined in this text, all other technical and scientific terms used in this text have the meanings generally understood by those of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present application will only be limited by the appended claims.

[0064] As used herein, the term "comprise" or its variants such as "comprises" or "comprising", etc., is understood to include the stated elements or components, without excluding other elements or components.

[0065] Silk fibroin nanofiber-anthocyanin complex

[0066] As used herein, the terms "silk fibroin nanofiber-anthocyanin complex", "the complex of the present application" are used interchangeably, and both refer to the silk fibroin nanofiber-anthocyanin complex formed by the β-sheet hydrophobic cavity in the silk fibroin nanofiber and anthocyanin through hydrogen bonding, hydrophobic interaction and other non-covalent bonds to form an inlaid topological structure according to the present application. The complex of the present application achieves the following effects: the thermal retention rate of anthocyanin at 80℃ is increased by 90%, and the digestion stability is increased by 103% by using the inlaid interaction between silk fibroin nanofiber silk and anthocyanin, which shows that the method provided by the present application can effectively improve the thermal stability and bioavailability of anthocyanin.

[0067] In the present application, the silk fibroin nanofiber-anthocyanin complex comprises:

[0068] (i) silk fibroin nanofiber; and

[0069] (ii) anthocyanin;

[0070] Wherein, the silk fibroin nanofiber and anthocyanin are inlaidly combined.

[0071] The complex of the present application is used to improve the thermal stability and gastrointestinal digestion stability of anthocyanin, and thus is conducive to improving the bioavailability of anthocyanin.

[0072] Anthocyanin

[0073] Anthocyanin is a class of flavonoids widely derived from plants. It not only provides attractive color to food, but also has high nutritional value and various physiological activities, meeting the current consumer's demand for pure natural, healthy and nutritious diet, and thus has attracted extensive attention and emphasis. However, the lack of electron characteristics of the benzopyran ring structure of anthocyanin causes it to be easily degraded in the process of food processing, transportation and storage under the influence of external environment (such as light, oxygen, heat, pH, etc.), which limits its application as a natural colorant in the food industry.

[0074] In the present application, anthocyanin includes black rice anthocyanin, purple cabbage anthocyanin and radish anthocyanin.

[0075] Silk fibroin nanofiber

[0076] The coexistence and interphase arrangement of hydrophilic and hydrophobic segments in silk fibroin make silk fibroin not only have certain solubility, but also provide a molecular level structural basis for its self-assembly in aqueous solution. Moreover, the random coil conformation of silk fibroin polypeptide chain is easily transformed into a β-pleated structure under the induction of external conditions, and further aggregated to form a fibrous self-assembly structure. Therefore, by adjusting external conditions such as electric field, pH, ions, mechanical shear, ultrasonic treatment, heating and ethanol, the silk fibroin self-assembly process can be controlled. Silk fibroin nanofibers have high specific surface area, stability and biocompatibility and other functional properties, and at the same time, more active groups are exposed during the fibration process, enhancing the binding capacity of the protein to small molecules. Therefore, fibrous silk fibroin has been used as a new type of functional material to protect active small molecules in food.

[0077] Silk fibroin

[0078] Silk fibroin is the main protein in silk, which has been certified by the U.S. Food and Drug Administration (GRN.1026), the Japanese Ministry of Health, Labor and Welfare (No. 2012-476) and the Korean Food and Drug Safety Department (No. 2012-18) and other countries, so it can be applied to food, cosmetics, medicine and other fields. The silk fibroin polypeptide chain is composed of heavy chain, light chain and P25 protein three subunits according to the molar ratio of 6:6:1, in which the heavy chain and the light chain are covalently cross-linked by disulfide bond and then connected with the P25 protein by non-covalent interaction. The molecular chain of silk fibroin presents four conformations: random coil, α-helix, β-turn and β-pleated sheet. Random coil is a metastable structure, which is easily transformed into a more mechanically stable β-pleated sheet structure under the influence of external conditions (such as temperature, pH, salt, metal ions). The silk fibroin molecular chain mainly forms an anti-parallel β-pleated sheet structure, because the hydrogen bond length formed in the anti-parallel β-pleated sheet is shorter than that in the parallel β-pleated sheet , so the structural stability is stronger. The aggregate structure of silk fibroin is related to its secondary structure composition. Silk fibroin exists in two aggregate structures: silk I and silk II. Silk I structure is mainly composed of random coil and α-helix structure, and silk II is mainly composed of anti-parallel β-pleated sheet structure. Under certain external forces, unstable Silk I structure is easily transformed into more stable silk II structure. Several adjacent anti-parallel β-pleated sheet structures are stacked through non-covalent bond interactions such as hydrogen bond, hydrophobic interaction and van der Waals force, finally forming a β-crystal structure with three-dimensional conformation. The formation of the aggregate structure of silk fibroin is closely related to external conditions. By controlling external conditions, the process of aggregate formation can be controlled, and various scale biomaterials such as nanofibers, nanoparticles, hydrogels and porous scaffolds can be prepared.

[0079] In the present application, the silk fibroin is intact silk fibroin or silk fibroin full-length protein, i.e. contains heavy chain, light chain and P25 protein.

[0080] Method for preparing silk fibroin nanofiber-anthocyanin complex

[0081] The present application provides a method for preparing a silk fibroin nanofiber-anthocyanin complex, comprising:

[0082] (a) providing a silk fibroin nanofiber and anthocyanin;

[0083] (b) mixing the silk fibroin nanofiber with the anthocyanin to obtain a silk fibroin nanofiber-anthocyanin complex.

[0084] In another preferred embodiment, the mass ratio of the silk fibroin nanofiber to the anthocyanin in the complex is 1:1 to 10:1.

[0085] In a preferred embodiment, to achieve the above-mentioned object, the present application provides the following technical solutions:

[0086] (1) mixing 8M lithium bromide solution and formic acid solution (98%) according to a mass ratio of 13.3:1 to prepare lithium bromide-formic acid solution

[0087] (2) adding silk fibroin according to a certain liquid-solid ratio into the lithium bromide-formic acid mixed solution, dissolving in a water bath under sealed conditions. After the solution is cooled to room temperature, it is loaded into a dialysis bag (with a molecular weight cut-off of 3 500 Da), and dialysis is performed at 4℃ for about 72h. After dialysis, centrifugation is performed at 12 000×g for 20min to obtain supernatant.

[0088] (3) placing the supernatant in an ultrasonic microwave synergistic workstation, and performing ultrasonic treatment to prepare a silk fibroin nanofiber solution.

[0089] (4) dissolving anthocyanin powder in deionized water, stirring in the dark, and preparing an anthocyanin solution.

[0090] (5) mixing the silk fibroin nanofiber solution obtained in step (2) and the anthocyanin solution of step (3) according to equal volume to obtain a silk fibroin nanofiber-anthocyanin complex.

[0091] Further in step (2), the liquid-solid ratio of the silk fibroin to the lithium bromide-formic acid mixed solution is 1:50 to 1:100, the water bath temperature is 60-80℃, and the water bath time is 2-4h.

[0092] Further in step (3), the ultrasonic treatment power is 500-900W, and the ultrasonic treatment time is 10-20min.

[0093] Further in the step (3), the diameter of the silk fibroin nanofiber is 1-5 nm.

[0094] Further in the step (4), the mass ratio of the silk fibroin nanofiber to the anthocyanin in the silk fibroin nanofiber-anthocyanin complex is 1:1-10:1.

[0095] The main advantages of the present application include:

[0096] (1) The present application provides a silk fibroin nanofiber-anthocyanin complex for the first time. Specifically, the present application addresses the problems of poor heat resistance and low bioavailability of anthocyanin by using silk fibroin nanofiber (such as silk fibroin nanofiber) and anthocyanin to form an inlaid interaction, which increases the heat retention rate of anthocyanin at 80℃ by 90% and improves the digestion stability by 103%, indicating that the method provided by the present application can effectively improve the heat stability and bioavailability of anthocyanin.

[0097] 2) The present application provides a method for improving the stability of anthocyanin by inlaid combination with silk fibroin nanofiber. In view of the problems of poor heat resistance and low bioavailability of anthocyanin, the present application uses silk fibroin nanofiber and anthocyanin to form an inlaid interaction, which increases the heat retention rate of anthocyanin at 80℃ by 90% and improves the digestion stability by 103%, indicating that the method provided by the present application can effectively improve the heat stability and bioavailability of anthocyanin.

[0098] (3) The present application finds that the stability of anthocyanin can be improved by the intermolecular interaction between protein and anthocyanin. However, due to the influence of temperature, oxygen, acid-base, enzyme and other factors on the structure of protein itself, the stability of the protein-anthocyanin complex is affected. Ultrasonic treatment can be used to regulate the nanoscale effect of protein, causing changes in the properties, microstructure and apparent morphology of protein materials, and forming a micro-inlaid carrier with a specific three-dimensional structure, which further improves the binding strength of protein and anthocyanin and the heat resistance and digestion resistance of the complex system, thereby enhancing the stability and bioavailability of anthocyanin.

[0099] (4) The present application finds that the silk fibroin nanofiber and anthocyanin form an inlaid topological combination, forming a cavity structure with anthocyanin as the core, which avoids the hydration and nucleophilic attack of water molecules on anthocyanin, thereby improving the stability of anthocyanin. The present application uses the inlaid interaction between anthocyanin and silk fibroin nanofiber to form a more stable complex system, which further improves the heat stability and digestion stability of anthocyanin in the complex system, thereby helping to improve the processing applicability and bioactivity of anthocyanin.

[0100] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0101] The materials and reagents used in the examples are commercially available unless otherwise specified.

[0102] Example 1:

[0103] (1) The lithium bromide-formic acid solution was prepared by mixing 8M lithium bromide solution and formic acid solution (98%) according to a mass ratio of 13.3:1.

[0104] (2) The silk fibroin (purchased from Suzhou Simi Biotech Co., Ltd.) was added into the lithium bromide-formic acid solution according to a material-liquid ratio of 1:50, and dissolved by heating in a water bath at 60°C under sealed conditions for 4h. After the solution was cooled to room temperature, it was loaded into a dialysis bag (molecular weight cut-off 3 500Da), and dialyzed at 4°C for about 72h. After dialysis, centrifugation was performed at 12 000xg for 20min, and the supernatant was obtained, which was freeze-dried to obtain a freeze-dried powder for use.

[0105] (3) The freeze-dried powder in step (2) was dissolved in deionized water, and after complete dissolution by low-speed stirring, it was placed in an ultrasonic microwave synergistic workstation, and ultrasonic treatment was performed at a power of 500W for 15min to prepare a silk fibroin nanofiber silk solution at 6mg / mL. The diameter of the obtained silk fibroin nanofiber was 5nm, which was consistent with the diameter of the single silk fibroin nanofiber in natural cocoon (3-5nm).

[0106] (4) The anthocyanin powder (purchased from Shanghai Zishi Biotechnology Co., Ltd.) was dissolved in deionized water under stirring in the dark to prepare an anthocyanin solution at 2mg / mL.

[0107] (5) The silk fibroin nanofiber silk solution obtained in step (2) was mixed with the anthocyanin solution in step (3) according to equal volume to obtain a silk fibroin nanofiber silk-anthocyanin complex, and the mass ratio of silk fibroin nanofiber silk to anthocyanin was 3:1.

[0108] Example 2:

[0109] (1) The lithium bromide-formic acid solution was prepared by mixing 8M lithium bromide solution and formic acid solution (98%) according to a mass ratio of 13.3:1.

[0110] (2) The silk fibroin was added to the lithium bromide-formic acid mixed solution solvent according to the material liquid ratio of 1:50, and was dissolved in a sealed condition at 60°C water bath for 4h. After the dissolved solution was cooled to room temperature, it was loaded into a dialysis bag (molecular weight cut-off 3 500Da), and was dialyzed at 4°C for about 72h. After dialysis, it was centrifuged at 12 000xg for 20min to obtain the supernatant, which was freeze-dried to obtain a freeze-dried powder for standby.

[0111] (3) The freeze-dried powder in step (2) was dissolved in deionized water, and was stirred at low speed until completely dissolved. Then, it was placed in an ultrasonic microwave cooperative workstation, and was ultrasonically treated at a power of 700W for 15min to prepare a silk fibroin nanofiber silk solution of 8mg / mL. The atomic force microscopic structure of the nanofiber is shown in Figure 3 , and the fiber diameter is 3.6nm, which is consistent with the diameter of single silk fibroin nanofiber in natural cocoon (3-5nm).

[0112] (4) The anthocyanin powder was dissolved in deionized water, and was stirred in the dark to prepare an anthocyanin solution of 2mg / mL.

[0113] (5) The silk fibroin nanofiber silk solution obtained in step (2) was mixed with the anthocyanin solution of step (3) according to equal volume to obtain a silk fibroin nanofiber silk-anthocyanin complex, and the mass ratio of silk fibroin nanofiber silk to anthocyanin is 4:1. The mosaic structure of the complex is shown in Figure 4 A and B.

[0114] Example 3:

[0115] (1) The 8M lithium bromide solution was mixed with the formic acid solution (98%) according to the mass ratio of 13.3:1 to prepare a lithium bromide-formic acid solution

[0116] (2) The silk fibroin was added to the lithium bromide-formic acid mixed solution solvent according to the material liquid ratio of 1:50, and was dissolved in a sealed condition at 60°C water bath for 4h. After the dissolved solution was cooled to room temperature, it was loaded into a dialysis bag (molecular weight cut-off 3 500Da), and was dialyzed at 4°C for about 72h. After dialysis, it was centrifuged at 12 000xg for 20min to obtain the supernatant, which was freeze-dried to obtain a freeze-dried powder for standby.

[0117] (3) The freeze-dried powder in step (2) was dissolved in deionized water, and was stirred at low speed until completely dissolved. Then, it was placed in an ultrasonic microwave cooperative workstation, and was ultrasonically treated at a power of 900W for 15min to prepare a silk fibroin nanofiber silk solution of 10mg / mL. Due to the high ultrasonic power, the fiber aggregation diameter increased, and the fiber diameter was 10nm.

[0118] (4) The anthocyanin powder was dissolved in deionized water, and was stirred in the dark to prepare an anthocyanin solution of 2mg / mL.

[0119] (5) The silk fibroin nanofiber solution obtained in step (2) is mixed with the anthocyanin solution of step (3) in equal volumes to obtain a silk fibroin nanofiber- anthocyanin complex, with a mass ratio of silk fibroin nanofiber to anthocyanin of 5:1.

[0120] Comparative Example 1:

[0121] (1) Anthocyanin powder is dissolved in deionized water, stirred in the dark, and prepared into an anthocyanin solution of 2 mg / mL.

[0122] (2) Water is mixed with the above-prepared 2 mg / mL anthocyanin solution in equal volumes to obtain an anthocyanin solution.

[0123] Comparative Example 2:

[0124] (1) The specific scheme is as in the example, except that there is no ultrasonic treatment. The rest is consistent with the example.

[0125] Comparative Example 3:

[0126] (1) Silk fibroin is prepared into a solution of 6 mg / mL

[0127] (2) Anthocyanin powder is dissolved in deionized water, stirred in the dark, and prepared into an anthocyanin solution of 2 mg / mL.

[0128] (3) The above silk fibroin solution and anthocyanin solution are mixed in equal volumes to obtain a silk fibroin-anthocyanin complex, with a mass ratio of silk fibroin to anthocyanin of 3:1.

[0129] After the above samples of Examples 1-3 and Comparative Examples 1-3 are taken in a constant-temperature water bath at 80°C for 1 h, they are cooled in an ice bath, and the total anthocyanin content in each group of samples is determined by pH differential method. The retention rate of anthocyanin is calculated according to the following formula.

[0130]

[0131] In the formula, C0and C t (mg / L) are the contents of black rice anthocyanin after 0 and 1 h of water bath heating, respectively.

[0132] The above samples of Examples 1-3 and Comparative Examples 1-3 are taken, an equal volume of simulated gastric juice is added, and after being placed in a 37°C water bath for 1 h of oscillation reaction, the pH is adjusted to 7.5 with 1M sodium bicarbonate, an equal volume of simulated intestinal juice is added, and after being taken out after 1 h of oscillation reaction in a 37°C water bath, it is stored at 4°C for testing. The monomeric anthocyanin content in each sample to be tested is detected by HPLC detection method.

[0133] After detection, the thermal stability of anthocyanin is as shown in Table 1, and the digestion stability is as shown in Table 2. Figure 1 Figure 2 ​as shown.

[0134] In Example 1, after the silk fibroin nanofiber-sinensin complex was heated at 80℃ for 1h, the retention rate of sinensin in the complex was 58%, and after 2h of gastrointestinal continuous digestion, the retention rate of sinensin was 51%.

[0135] In Example 2, after the silk fibroin nanofiber-sinensin complex was heated at 80℃ for 1h, the retention rate of sinensin in the complex was 65%, and after 2h of gastrointestinal continuous digestion, the retention rate of sinensin was 63%.

[0136] In Example 3, after the silk fibroin nanofiber-sinensin complex was heated at 80℃ for 1h, the retention rate of sinensin in the complex was 55%, and after 2h of gastrointestinal continuous digestion, the retention rate of sinensin was 46%.

[0137] In Comparative Example 1, after the sinensin solution was heated at 80℃ for 1h, the retention rate of sinensin was 34%, and after 2h of gastrointestinal continuous digestion, the retention rate of sinensin was 25%.

[0138] In Comparative Example 2, after the sinensin solution was heated at 80℃ for 1h, the retention rate of sinensin was 53%, and after 2h of gastrointestinal continuous digestion, the retention rate of sinensin was 39%.

[0139] In Comparative Example 3, after the sinensin solution was heated at 80℃ for 1h, the retention rate of sinensin was 41%, and after 2h of gastrointestinal continuous digestion, the retention rate of sinensin was 31%.

[0140] It can be seen that the method for improving the stability of sinensin by embedding the sinensin with the silk fibroin nanofiber is provided.The silk fibroin nanofiber and the sinensin are embedded to interact with each other, so that the heat retention rate of the sinensin at 80℃ is increased by 90%, and the digestion stability is increased by 103%, which shows that the method can effectively improve the heat stability and the bioavailability of the sinensin.

[0141] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. In addition, it should be understood that various changes and modifications can be made to the present application by those skilled in the art upon reading the above description of the present application, and such equivalent forms are also within the scope of the appended claims.

Claims

1. A silk fibroin nanofiber-anthocyanin complex, characterized in that, Comprise (i) silk fibroin nanofiber; and (ii) anthocyanin; Wherein, the silk fibroin nanofiber and anthocyanin are inlayed combination.

2. The composite of claim 1, wherein, The anthocyanin includes black rice anthocyanin, purple cabbage anthocyanin, and radish anthocyanin.

3. The composite of claim 1, wherein, The β-sheet hydrophobic cavity in the silk fibroin nanofiber and anthocyanin form inlayed topological structure through non-covalent bond.

4. The complex according to claim 3, wherein The non-covalent bond includes hydrogen bond, hydrophobic interaction, and electrostatic interaction.

5. The composite of claim 1, wherein, In the silk fibroin nanofiber-anthocyanin complex, the mass ratio of silk fibroin nanofiber to anthocyanin is 1:1-10:

1.

6. The composite of claim 1, wherein, The silk fibroin nanofiber is silk fibroin nanofiber silk.

7. The composite of claim 1, wherein, The diameter of the silk fibroin nanofiber is 1-20 nm, preferably 1-5 nm.

8. A method of preparing a silk fibroin nanofiber- anthocyanin complex, characterized by, Comprise (a) providing a silk fibroin nanofiber and anthocyanin; (b) mixing the silk fibroin nanofiber and anthocyanin, thereby obtaining the silk fibroin nanofiber-anthocyanin complex of claim 1.

9. Use of the silk fibroin nanofiber- anthocyanin complex of claim 1, characterized in that, For preparing a composition or preparation for improving the thermal stability, and / or gastrointestinal digestion stability of anthocyanin; and / or improving the bioavailability of anthocyanin.

10. A food additive, characterized by, Comprise The silk fibroin nanofiber-anthocyanin complex of claim 1.

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