A silk fibroin-phenolic iron aggregate material, its preparation method, and its application in fruit preservation.

Abstract

This invention relates to a silk fibroin-ferric phenolate aggregate material, its preparation method, and its application in fruit preservation. The silk fibroin-ferric phenolate aggregate material comprises silk fibroin at a final concentration of 5%-15%, tris(2-carboxyethyl)phosphonic acid hydrochloride at a final concentration of 0.1%-1%, catechin at a final concentration of 1%-10%, and anhydrous ferric chloride at a final concentration of 0.1%-1%. This invention experimentally determined the optimal process parameters for the delivery system of the tris(2-carboxyethyl)phosphonic acid hydrochloride-induced silk fibroin-ferric phenolate aggregate. At a final concentration of 1% tris(2-carboxyethyl)phosphonic acid hydrochloride and an incubation temperature of 50°C, the silk fibroin aggregate exhibits the highest β-sheet aggregation degree, optimal phase separation and self-assembly efficiency, and stable aggregate structure. When sprayed onto the fruit surface and left to stand at room temperature, it spontaneously completes in-situ self-assembly to form a silk fibroin-ferric phenolate hydrogel film, achieving a long-lasting antibacterial effect and fruit preservation function.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a silk fibroin-phenolic iron aggregate material, its preparation method, and its application in fruit preservation. Background Technology

[0002] Silk fibroin is the main component of silk, accounting for 70%-80% of the total silk content. Sericin encapsulates silk fibroin, which is divided into crystalline and amorphous regions. The amorphous region gives silk its elasticity; the crystalline region mainly contains glycine-alanine-glycine-alanine-glycine-serine repeating units, which enhance its mechanical properties. During the sol-gel transition, the repeating units can form highly ordered secondary structures (such as β-sheets) to construct crystal structures. Fourier transform infrared analysis shows that the proportion of β-sheets in silk fibroin aggregates is closely related to its gel performance and protein release behavior. As a key form of intermolecular interaction of silk fibroin, the β-sheet structure indirectly affects the overall mechanical properties of the hydrogel by enhancing structural stability (such as surface stability).

[0003] Food suffers significant losses during processing, storage, and transportation. These losses and wastes lead to increased greenhouse gas emissions and wasted agricultural land. Traditional food preservation packaging materials, such as plastic cling film made of polyethylene and polyvinyl chloride, are difficult to degrade, have low recycling rates, and cause environmental harm. Furthermore, plasticizers and other chemicals in cling film can easily migrate into packaged food, posing health risks. Because of the microplastic pollution caused by these plastic cling films, which is inconsistent with the concept of sustainable development, there is a need to develop a new alternative material. Summary of the Invention

[0004] The purpose of this invention is to provide a silk fibroin-phenolic iron aggregate material, its preparation method, and its application in fruit preservation in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides a silk fibroin-ferric phenol aggregate material, which comprises silk fibroin with a final concentration of 5%-15%, tris(2-carboxyethyl)phosphine hydrochloride with a final concentration of 0.1%-1%, catechin with a final concentration of 1%-10%, and anhydrous ferric chloride with a final concentration of 0.1%-1%.

[0006] As a further optimization of the present invention, the silk fibroin-ferric condensate material comprises silk fibroin with a final concentration of 10%, tris(2-carboxyethyl)phosphine hydrochloride with a final concentration of 1%, catechin with a final concentration of 1%, and anhydrous ferric chloride with a final concentration of 0.1%.

[0007] The present invention also provides a method for preparing the silk fibroin-phenolic iron aggregate material as described above, comprising the following steps: Step 1: After degumming the silk fibroin raw material, degummed silk is obtained, which is then dissolved in lithium bromide solution, coarsely filtered and centrifuged, purified by dialysis, and freeze-dried to obtain freeze-dried silk fibroin powder. Step 2: Dissolve the freeze-dried silk fibroin powder in a filtered phosphate buffer solution to prepare a 10% (w / w) silk fibroin solution. Centrifuge to discard the precipitate and set aside for later use. Step 3: Dissolve tris(2-carboxyethyl)phosphine hydrochloride in filtered phosphate buffer solution to prepare a 10% (w / w) tris(2-carboxyethyl)phosphine hydrochloride mother liquor for later use; Step 4: Take a 10% silk fibroin solution and a 10% tris(2-carboxyethyl)phosphonic acid hydrochloride mother liquor and mix them in a certain proportion to obtain a mixed solution. After incubation and induction, phase-separated protein aggregates, namely silk fibroin aggregates, are obtained from the mixed solution. Step 5: Dissolve tea catechin and anhydrous ferric chloride in a filtered phosphate buffer solution, mix well, and then add silk fibroin aggregate to obtain silk fibroin-ferric chloride aggregate material.

[0008] As a further optimization of the present invention, in step one, the silk fibroin raw material is silkworm cocoon or waste silk products; The degumming process specifically involves boiling the silk fibroin raw material in a 0.01-0.05 mol / L sodium carbonate solution for 40-50 minutes at a liquor ratio of 90-110:1; after draining the solution, boiling the silk fibroin raw material again in a 0.001-0.01 mol / L sodium bicarbonate solution for 40-50 minutes at a liquor ratio of 40-60:1.

[0009] As a further optimization of the present invention, in step one, the lithium bromide solution dissolution specifically involves using a 5-15 mol / L lithium bromide solution to dissolve degummed silk at a solid-liquid volume ratio of 1 g: 5-8 mL, and the dissolution conditions are water bath heating at 60-70°C for 3-5 hours. The dialysis purification was performed using a dry dialysis bag with a pore size of DM44-3500.

[0010] As a further optimization of the present invention, in steps two, three, and five, the filtered phosphate buffer solution is a phosphate buffer solution filtered through a 0.2-0.3 μm aqueous filter membrane.

[0011] As a further optimization of the present invention, in step four, the final concentration of the tris(2-carboxyethyl)phosphine hydrochloride solution in the mixed solution is 0.1%-1%; The incubation induction specifically involves incubating at a temperature of 35℃-50℃ for 0.5-1.5 days.

[0012] The present invention also provides an application of the silk fibroin-phenolic iron aggregate material as described above in fruit preservation. The silk fibroin-phenolic iron aggregate material is sprayed onto the surface of the fruit and left to stand at room temperature. It spontaneously completes in-situ self-assembly to form a silk fibroin-phenolic iron hydrogel material film layer, which is used for antibacterial and slowing down the spoilage rate of the fruit. The amount of the silk fibroin-ferric phenol aggregate material sprayed is 0.01-0.05 g / cm³. 2 .

[0013] As a further optimization of the present invention, the fruit is a banana or a strawberry; the antibacterial bacteria are Gram-negative and Gram-positive bacteria.

[0014] The beneficial effects of this invention are as follows: 1) This invention has determined the optimal process parameters for the delivery system of tris(2-carboxyethyl)phosphine hydrochloride-induced silk fibroin-phenol iron aggregates through experiments. The final concentration of tris(2-carboxyethyl)phosphine hydrochloride is 1% and the incubation temperature is 50°C. Under these parameters, the β-sheet aggregation degree of silk fibroin is the highest, and the prepared aggregate structure is stable. When sprayed on the surface of fruit and left to stand at room temperature, the silk fibroin-phenol iron hydrogel material film is spontaneously self-assembled in situ to form a film layer, which can achieve a long-lasting antibacterial effect and fruit preservation function. 2) This invention can construct a delivery system for silk fibroin-ferric condensate by introducing tea catechin and anhydrous ferric chloride, which can further improve the mechanical properties of silk fibroin hydrogel and ensure the activity of components, thus expanding its application scenarios in food preservation. 3) The natural protein silk fibroin of the present invention undergoes spontaneous phase separation and in-situ self-assembly under the liquid surface without the need for additional cross-linking agents or external forces. The preparation process is simple and highly controllable, which greatly reduces the preparation cost. It can also load tea catechins and anhydrous ferric chloride and achieve slow release. The correlation between its structure and macroscopic properties lays the foundation for its structural reconstruction as a biomaterial, making silk fibroin a promising application in the field of biomaterials. 4) This invention uses natural silk fibroin to make a fully recyclable and degradable aggregate packaging material, which has excellent antibacterial, antioxidant and food preservation properties. The material itself is recyclable and degradable, providing a theoretical basis for the development of new preservation materials. Attached Figure Description

[0015] Figure 1 This is a graph showing the effect of different concentrations of tri(2-carboxyethyl)phosphine hydrochloride solution on silk fibroin aggregates. Figure 2 This is a graph showing the effect of different incubation temperatures on silk fibroin aggregates. Figure 3 This is a diagram showing the self-assembly of silk fibroin aggregates loaded with rhodamine dye after 12 hours underwater. Figure 4 This is a diagram showing the self-assembly of silk fibroin-phenolic iron aggregates after 12 hours underwater. Figure 5 This is a comparative analysis of the storage modulus and loss modulus of silk fibroin hydrogel and silk fibroin-phenolic iron hydrogel as a function of angular frequency. Figure 6 The images show the antibacterial effects of phenol-iron complex and silk fibroin-phenol-iron agglomerate on Escherichia coli and Staphylococcus aureus on agar plates. Figure 7 This is a statistical chart of plate antibacterial colonies of phenol-iron complex and silk fibroin-phenol-iron aggregate against Escherichia coli and Staphylococcus aureus; Figure 8 This is a diagram showing the slow penetration of phosphate buffer (left) and silk fibroin-ferric phenolate aggregate (right) onto the surface of fruit. Figure 9 This is a morphology image of silk fibroin-phenolic iron hydrogel in a silicone mold; Figure 10 This is a morphology image of silk fibroin-phenolic iron hydrogel on banana peel; Figure 11 This is a comparison chart showing the preservation effects of using silk fibroin aggregates, phenol-iron complexes, and silk fibroin-phenol-iron aggregates on bananas on day 1 and day 8. Figure 12 This is a comparison chart showing the preservation effects of using silk fibroin aggregates, phenol-iron complexes, and silk fibroin-phenol-iron aggregates on strawberries on day 1 and day 8. Figure 13 This is a graph showing the results of cytotoxicity experiments using silk fibroin-ferric condensates at different concentration gradients. Detailed Implementation

[0016] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0017] I. Materials 1. Naturally dried silkworm cocoons: purchased from Hebei Tongxin Biotechnology Co., Ltd., 500g / pack; Tris(2-carboxyethyl)phosphonic acid hydrochloride: purchased from Aladdin Reagent (Shanghai) Co., Ltd., 20g / bottle; Epigallocatechin gallate (chagacatechin): purchased from Aladdin Reagent (Shanghai) Co., Ltd., 20mg / bottle; Anhydrous ferric chloride: iron purity (metal standard) ≥99.9%, purchased from Aladdin Reagent (Shanghai) Co., Ltd., 20g / bottle; Dry dialysis bags: purchased from Hunan Dete Biotechnology Co., Ltd., 5m / roll; Lithium bromide: purchased from Aladdin Reagent (Shanghai) Co., Ltd., 100g / bottle; Sodium carbonate: purchased from Aladdin Reagent (Shanghai) Co., Ltd., 500g / bottle; Sodium bicarbonate: purchased from Aladdin Reagent (Shanghai) Co., Ltd., 500g / bottle.

[0018] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0019] II. Methods 2.1 Preparation method of silk fibroin-phenolic iron aggregate material 2.1.1 Preparation of Lyophilized Silk Fibroin Powder The experiment in this application uses naturally dried silkworm cocoons (but is not limited to this; waste silk products can also be selected) to extract and prepare silk fibroin. The method for preparing freeze-dried silk fibroin powder from naturally dried silkworm cocoons includes the following steps: 1) Weigh 10g of naturally dried silkworm cocoons, cut them into small pieces with scissors, prepare 1L of 0.02mol / L sodium carbonate solution using ultrapure water, add the broken cocoons, stir well, and then divide into two 500mL beakers. Seal the beakers with sealing film and poke holes in them, then microwave them to a boil and maintain for 45 minutes. Prepare 500mL of 0.002mol / L sodium bicarbonate solution, pour out the sodium carbonate solution from the beakers, keep the silk, replace it with sodium bicarbonate solution, and microwave them to a boil again and maintain for 45 minutes. Take out the silk, wash it with ultrapure water, spread it on gauze and air dry at room temperature for 3 days to obtain degummed silk. 2) Set the water bath to 65°C, quickly weigh 17.02g of lithium bromide solid and dissolve it in 21mL of ultrapure water. Stir until completely dissolved and place in a 65°C water bath. Take 4g of degummed silk and add it to the lithium bromide solution in small pieces. Stir and heat at a constant temperature for 4 hours until the degummed silk is completely dissolved to obtain a silk fibroin solution. 3) After coarsely filtering the silk fibroin solution through three layers of clean gauze, collect it into a 15mL centrifuge tube, centrifuge, and take the supernatant for later use; 4) Cut a 15cm long dry dialysis bag with a pore size of DM44-3500, pre-treat it by microwaving for 45 minutes, transfer the supernatant into the dialysis bag, clamp both ends with dialysis clamps, place it in a beaker with a capacity of 2L containing 2L of ultrapure water, and dialyze for 3 days under magnetic stirring, changing the ultrapure water every 12 hours. 5) Dispense the silk fibroin solution after dialysis in step 4) into 100mL beakers, seal them with sealing film, and freeze them overnight at -80℃. Preheat the vacuum pump of the freeze dryer for 5 minutes, then pre-cool the cold trap for 5 minutes. Place the frozen sample in the freeze dryer and continue drying for 3 days after primary drying and final drying to obtain high-purity anhydrous freeze-dried silk fibroin powder.

[0020] 2.1.2 Preparation of silk fibroin aggregates 1) Use a 10mL disposable sterile syringe to draw up phosphate buffer solution, filter it through a 0.22μm aqueous filter membrane and collect it into a 15mL centrifuge tube; take 1mL of the filtered phosphate buffer solution into a 2mL EP tube, weigh 0.1g of lyophilized silk fibroin powder, divide it into small pieces and slowly dissolve it in the EP tube, centrifuge and discard the precipitate to obtain a 10% (w / w) silk fibroin solution for later use; 2) Take the filtered phosphate buffer solution into a 2 mL EP tube, weigh 0.1 g of tris(2-carboxyethyl)phosphine hydrochloride powder, dissolve it in the EP tube to obtain a 10% (w / w) tris(2-carboxyethyl)phosphine hydrochloride mother liquor for later use; 3) Take 900 μL of 10% silk fibroin solution and mix it with 100 μL of 10% tris(2-carboxyethyl)phosphine hydrochloride solution to obtain a mixed solution with a total system volume of 1 mL and a final concentration of 1% tris(2-carboxyethyl)phosphine hydrochloride. Place the mixed solution in a constant temperature mixer and incubate it at 50℃ for 1 h. The disulfide bonds between silk fibroin molecules are broken by the reduction of tris(2-carboxyethyl)phosphine hydrochloride, which induces a conformational change in silk fibroin and forms phase-separated aggregates, namely silk fibroin aggregates.

[0021] 2.1.3 Preparation of silk fibroin-phenolic iron aggregate material Add catechin and anhydrous ferric chloride (prepared by pre-concentrating anhydrous ferric chloride to obtain a 10% ferric chloride mother liquor) to the filtered phosphate buffer solution to obtain a phenol-iron complex with a final concentration of 1% catechin and a final concentration of 0.1% anhydrous ferric chloride. After mixing evenly, take 100 μL and add it to 900 μL of silk fibroin aggregate to obtain silk fibroin-phenol-iron aggregate. Let it stand upright at room temperature for 24 h to form a silk fibroin-phenol-iron aggregate that can deliver a slow-release phenol-iron complex.

[0022] 2.2 Effects of different incubation temperatures and concentrations of tris(2-carboxyethyl)phosphine hydrochloride solutions on the β-sheet structure of silk fibroin-phenol-iron aggregates Experimental materials: 10% silk fibroin solution (prepared with phosphate buffer), 10% tris(2-carboxyethyl)phosphonic acid hydrochloride stock solution (prepared with phosphate buffer), phosphate buffer solution, Th-T thiosulfate working solution, 96-well plate, 1 ml EP tube; the specific optimization scheme is as follows: Optimization of tris(2-carboxyethyl)phosphine hydrochloride solution concentration: A 10% (w / w) silk fibroin solution and a 10% (w / w) tris(2-carboxyethyl)phosphine hydrochloride stock solution were mixed at different volume ratios to achieve final concentrations of 0.1%, 0.5%, and 1% (total solution volume 1 mL). The mixture was incubated at 37℃ for 1 h. Under light-protected conditions, 10 μL of the incubated silk fibroin and 20 μL of Th-T thiosulfate working solution were added to an EP tube, and the volume was brought up to 100 μL with phosphate buffer solution. Each concentration was repeated three times. The liquid in the EP tube was transferred to a 96-well plate, and the fluorescence emission spectrum at an excitation wavelength of 420 nm (460-600 nm) was immediately measured using a microplate reader. The experimental results are shown below. Figure 1 .

[0023] Temperature optimization: 900 μL of 10% silk fibroin was added to 100 μL of 10% tris(2-carboxyethyl)phosphonic acid hydrochloride, maintaining a final concentration of 1%. The mixture was incubated at 37℃, 45℃, and 50℃ for 1 h. Subsequent Th-T detection procedures were the same. Results are shown below. Figure 2 .

[0024] Experimental conclusions: The concentration of tris(2-carboxyethyl)phosphine hydrochloride was dependent on the concentration of silk fibroin aggregates, with the highest peak value in the 1% group. This indicates that high concentrations of tris(2-carboxyethyl)phosphine hydrochloride solution can more effectively promote the breaking of disulfide bonds in silk fibroin molecular chains, increase molecular flexibility, and accelerate β-sheet aggregation. The concentration of silk fibroin aggregates was also temperature dependent. Under the condition of 1% tris(2-carboxyethyl)phosphine hydrochloride solution, the peak value was highest in the 50℃ group, suggesting that increasing the temperature can enhance molecular thermal motion and hydrophobic interactions, which is beneficial to β-sheet conformational transition.

[0025] Figure 3 Under optimal conditions, 20 μL of silk fibroin aggregate (concentration of 10% silk fibroin and 1% tris(2-carboxyethyl)phosphine hydrochloride) loaded with 2 μL of rhodamine dye (concentration of 200 mg / L) completed self-assembly at room temperature for 12 hours below the liquid surface, as shown in the image.

[0026] Figure 4The image shows the aggregates of phenol-iron complex and silk fibroin (final concentration of 10% silk fibroin, 1% tris(2-carboxyethyl)phosphine hydrochloride, 1% catechin, and 0.1% anhydrous ferric chloride) loaded under optimal conditions after 12 hours at room temperature.

[0027] Experimental conclusion: The concentration of tris(2-carboxyethyl)phosphine hydrochloride solution and the incubation temperature have a synergistic promoting effect on the formation of β-sheets in silk fibroin aggregates. The optimal parameter combination is 1% tris(2-carboxyethyl)phosphine hydrochloride and incubation at 50℃. Under these conditions, the degree of β-sheet aggregation is the highest, and silk fibroin aggregates can complete self-assembly under the liquid surface.

[0028] 2.3 Characterization of the mechanical properties of silk fibroin hydrogel and silk fibroin-phenol iron hydrogel Silk fibroin hydrogels were prepared by incubating 10% silk fibroin and 1% tris(2-carboxyethyl)phosphonic acid hydrochloride at 50°C for 4 hours. Silk fibroin-ferric chloride hydrogels were prepared by incubating 10% silk fibroin, 1% tris(2-carboxyethyl)phosphonic acid hydrochloride, 1% catechin, and 0.1% anhydrous ferric chloride at 50°C for 4 hours. The storage modulus and loss modulus of the silk fibroin hydrogels and the silk fibroin-ferric chloride hydrogels as a function of angular frequency were measured using an advanced rheometer. The results are as follows: Figure 5 As shown.

[0029] Experimental conclusions: In the full angular frequency range of 0.1-100 rad / s, the storage modulus (G') of both hydrogels was significantly higher than that of the loss modulus (G''), exhibiting typical solid-like viscoelastic behavior. The storage modulus of the silk fibroin-phenol iron composite hydrogel was more than an order of magnitude higher than that of the pure silk fibroin hydrogel, while the increase in loss modulus was relatively limited. This confirms the significant enhancing effect of silk fibroin-phenol iron on the elasticity and mechanical stability of the hydrogel.

[0030] 2.4 In vitro antibacterial test of silk fibroin-ferric phenol aggregates Frozen strains of *Escherichia coli* and *Staphylococcus aureus* were inoculated into 5 mL of fresh culture medium and cultured at 37°C and 220 rpm for 16 h. The bacterial concentration was adjusted to an OD600 of 0.1. 10 μL of sterile silk fibroin-ferric phenolate aggregate prepared according to the above protocol was added to 100 μL of the above bacterial culture and incubated at 37°C for 1 h. 10 μL of sterile ferric phenolate complex prepared according to the above protocol was added to 100 μL of the above bacterial culture and incubated at 37°C for 1 h. 10 μL of sterile phosphate buffer solution was added to 100 μL of the above bacterial culture as a blank control and incubated at 37°C for 1 h. The incubated bacterial culture was then diluted 10,000 times, and 100 μL of the diluted culture was plated and incubated at 37°C for 24 h. Colony morphology was observed, colony counts were performed, and photographs were taken. The results are shown in the table below. Figure 6 , Figure 7 .

[0031] Experimental conclusion: From Figure 6 , Figure 7 It is known that silk fibroin-phenol iron agglomerates have a significant inhibitory effect on both Gram-negative and Gram-positive bacteria.

[0032] 2.5. Silk fibroin-ferric phenol aggregates slowly penetrate the surface of fruits. The citrus fruit peel forms a hydrophobic interface due to the presence of a natural wax layer. This interface has low surface energy. When 20 μL of phosphate-buffered saline (PBS) is dropped onto the surface of an orange, the PBS rapidly slips and cannot achieve effective spreading and retention on the peel. However, when 20 μL of 10% silk fibroin-ferric phenolate aggregate (stained with 1 μL of 100 mg / L rhodamine solution) is dropped onto the orange surface, its high viscoelasticity, suitable surface tension, and excellent interfacial wettability effectively overcome the hydrophobic barrier of the citrus peel. After dropping, the slippage rate is significantly reduced, achieving stable spreading on the hydrophobic peel. At the same time, relying on its own molecular permeability and steric hindrance adaptability, it can gradually penetrate and fill the complex micro-nano geometric structure of the citrus peel surface, which consists of fruit spots, microcracks, and wax crystals, achieving efficient interfacial bonding with the peel microenvironment.

[0033] The experimental results are shown in Figure 8 On the left, phosphate-buffered saline (PBS) slides rapidly down an orange peel, while on the right, silk fibroin-ferric phenolate aggregates spread slowly on an orange peel.

[0034] 2.6 Morphology of silk fibroin-phenol iron hydrogel under scanning electron microscopy Silk fibroin-phenolic iron hydrogel was sprayed onto a silicone mold and the surface of a banana. After it self-assembled into a film in situ, it was gently peeled off. The silk fibroin-phenolic iron hydrogel samples were divided into sections with the cross-section facing up and those with the flat surface facing up, and were adhered to the sample stage. The samples were then scanned using a cold field emission scanning electron microscope. The morphology of the silk fibroin-phenolic iron hydrogel in the silicone mold is shown in the figure. Figure 9 The morphology of silk fibroin-phenolic iron hydrogel on banana peel is shown in the figure. Figure 10 .

[0035] Experimental conclusion: From Figure 9 , Figure 10 It can be seen that the silk fibroin-phenol iron aggregate has good surface and interface self-adaptability, and can penetrate into complex geometric environments and self-assemble to form a silk fibroin-phenol iron hydrogel film.

[0036] 2.7 Fruit Preservation Experiment Using Silk Fiber Protein-Phenolic Iron Aggregates Twenty strawberries of similar ripeness were selected and divided into four groups of five strawberries each. Twelve bananas of similar ripeness were also divided into four groups of three bananas each. One group served as a blank control group, receiving no treatment. Another group was sprayed with a silk fibroin aggregate (10% silk fibroin, 1% tris(2-carboxyethyl)phosphonic acid hydrochloride, spraying amount 0.02 g / cm³). 2 Within 30 minutes, silk fibroin aggregates self-assembled in situ on the fruit surface to form a silk fibroin hydrogel film, which was the silk fibroin aggregate experimental group; another group was coated with a phenol-iron complex (3% catechin, 0.1% anhydrous ferric chloride) using a small spray bottle, with a coating amount of 0.02 g / cm³. 2 One group consisted of phenol-iron complexes; the other group was coated with a silk fibroin-phenol-iron aggregate (10% silk fibroin, 1% tris(2-carboxyethyl)phosphine hydrochloride, 3% catechin, and 0.1% anhydrous ferric chloride) using a small spray bottle, with a coating amount of 0.02 g / cm³. 2 After waiting for 1 hour, the silk fibroin-ferric phenol aggregates were allowed to self-assemble in situ on the fruit surface to form a film, creating a silk fibroin-ferric phenol hydrogel film layer, which was designated as the silk fibroin-ferric phenol aggregate experimental group. Fruits from both the control and experimental groups were placed in a clean room at 25°C and left to stand for 8 days. The condition of the fruit on day 1 and day 8 was photographed and recorded. The results showed that the preservation effect of the bananas was [see attached image]. Figure 11 See the preservation effect of strawberries Figure 12 .

[0037] Experimental conclusion: From Figure 11 , Figure 12 It is known that silk fibroin-ferric phenolate aggregate has a long-lasting preservation effect on fruits.

[0038] 2.8 Thiazol blue cytotoxicity test of silk fibroin-ferric condensate HEK293T cells were stored at a density of 3-5 × 10⁶ cells per well. 3Cells were seeded at a density of 1000 cells / well in 96-well cell culture plates and then incubated overnight at 37°C with 5% CO2. After cell attachment, silk fibroin-ferric ether aggregates were added to the plates at a predetermined concentration gradient: 0%, 0.1%, 0.5%, 1%, and 10%, with five replicates for each concentration. The treated cells were then cultured for another 24 hours at 37°C with 5% CO2. The culture medium without cell seeding served as a blank control, and phosphate-buffered saline (PBFS) was added as a negative control when adding the test sample. After 24 hours of culture, 10 μL of thiazolyl blue solution was added to each well of the 96-well plate, and the plates were incubated for another 1 hour at 37°C with 5% CO2. All culture medium was then gently aspirated and discarded. 150 μL of dimethyl sulfoxide was added to dissolve formazan, and the solution was then transferred to a SpectraMaxPlus 384 microplate. The absorbance at 570 nm was read using an ELISA reader; each experiment was repeated three times, cell viability was calculated using Origin 8.5 software, and the significance of the difference between the experimental group and the control group was determined by one-way ANOVA.

[0039] Experimental conclusion: From Figure 13 It is known that the silk fibroin-phenol iron aggregate has no cytotoxicity and good biocompatibility.

[0040] In summary, the silk fibroin-phenolic iron aggregate material has good in-situ self-assembly film-forming properties, excellent antibacterial and food preservation properties, and the material itself is recyclable and degradable, providing a theoretical basis for the development of new preservation materials.

[0041] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A silk fibroin-phenolic iron aggregate material, characterized in that, The silk fibroin-ferric condensate material comprises silk fibroin with a final concentration of 5%-15%, tris(2-carboxyethyl)phosphonic acid hydrochloride with a final concentration of 0.1%-1%, catechin with a final concentration of 1%-10%, and anhydrous ferric chloride with a final concentration of 0.1%-1%.

2. The silk fibroin-ferric condensate material according to claim 1, characterized in that, The silk fibroin-ferric phenol aggregate material comprises silk fibroin with a final concentration of 10%, tris(2-carboxyethyl)phosphonic acid hydrochloride with a final concentration of 1%, catechin with a final concentration of 1%, and anhydrous ferric chloride with a final concentration of 0.1%.

3. A method for preparing the silk fibroin-phenolic iron aggregate material as described in any one of claims 1-2, characterized in that, Includes the following steps: Step 1: After degumming the silk fibroin raw material, degummed silk is obtained, which is then dissolved in lithium bromide solution, coarsely filtered and centrifuged, purified by dialysis, and freeze-dried to obtain freeze-dried silk fibroin powder. Step 2: Dissolve the freeze-dried silk fibroin powder in a filtered phosphate buffer solution to prepare a 10% (w / w) silk fibroin solution. Centrifuge to discard the precipitate and set aside for later use. Step 3: Dissolve tris(2-carboxyethyl)phosphine hydrochloride in filtered phosphate buffer solution to prepare a 10% (w / w) tris(2-carboxyethyl)phosphine hydrochloride mother liquor for later use; Step 4: Take a 10% silk fibroin solution and a 10% tris(2-carboxyethyl)phosphonic acid hydrochloride mother liquor and mix them in a certain proportion to obtain a mixed solution. After incubation and induction, phase-separated protein aggregates, namely silk fibroin aggregates, are obtained from the mixed solution. Step 5: Dissolve tea catechin and anhydrous ferric chloride in a filtered phosphate buffer solution, mix well, and then add silk fibroin aggregate to obtain silk fibroin-ferric chloride aggregate material.

4. The method for preparing a silk fibroin-phenolic iron aggregate material according to claim 3, characterized in that, In step one, the silk fibroin raw material is silkworm cocoon or waste silk products; The degumming process specifically involves boiling the silk fibroin raw material in a 0.01-0.05 mol / L sodium carbonate solution for 40-50 minutes at a liquor ratio of 90-110:1; after draining the solution, boiling the silk fibroin raw material again in a 0.001-0.01 mol / L sodium bicarbonate solution for 40-50 minutes at a liquor ratio of 40-60:

1.

5. The method for preparing a silk fibroin-phenolic iron aggregate material according to claim 3, characterized in that, In step one, the lithium bromide solution dissolution specifically involves using a 5-15 mol / L lithium bromide solution to dissolve degummed silk at a solid-liquid volume ratio of 1 g: 5-8 mL, under the following conditions: heating in a water bath at 60-70°C for 3-5 hours. The dialysis purification was performed using a dry dialysis bag with a pore size of MD44-3500.

6. The method for preparing a silk fibroin-phenolic iron aggregate material according to claim 3, characterized in that, In steps two, three, and five, the filtered phosphate buffer solution is a phosphate buffer solution filtered through a 0.2-0.3 μm aqueous filter membrane.

7. The method for preparing a silk fibroin-phenolic iron aggregate material according to claim 3, characterized in that, In step four, the final concentration of the tris(2-carboxyethyl)phosphine hydrochloride solution in the mixed solution is 0.1%-1%; The incubation induction process specifically involves incubating at a temperature of 35℃-50℃ for 0.5-1.5 hours.

8. The application of a silk fibroin-ferric phenol aggregate material as described in any one of claims 1-2 in fruit preservation, characterized in that, Spraying the silk fibroin-phenolic iron aggregate material onto the surface of fruit and letting it stand at room temperature allows it to spontaneously complete in-situ self-assembly to form a silk fibroin-phenolic iron hydrogel material film, which is used for antibacterial purposes and to slow down the rate of fruit spoilage. The amount of the silk fibroin-ferric phenol aggregate material sprayed is 0.01-0.05 g / cm³. 2 .

9. The application according to claim 8, characterized in that, The fruits are bananas and strawberries; the antibacterial bacteria are Gram-negative and Gram-positive bacteria.

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