Preparation method and application of composite edible film liquid containing fibroin

Abstract

The application discloses a preparation method and application of a silk fibroin-containing composite edible film liquid, relates to the field of food preservation, and raw materials of the application include silk fibroin, chitosan and a microemulsion; the microemulsion includes mixed surfactants and essential oil; the mixed surfactants include surfactants and co-surfactants. The preparation method is as follows: silk fibroin solution, chitosan solution and the microemulsion are prepared respectively; the silk fibroin solution is mixed with the chitosan solution, and then the mixture is mixed with the microemulsion. The composite edible film can be applied to food preservation. The composite edible film has good mechanical barrier property, strong adhesion and strong antibacterial activity, and can effectively delay the oxidation and aging process of food and inhibit the growth of microorganisms in food.

Description

Technical Field

[0001] This invention relates to the field of food preservation, and in particular to a method for preparing and applying a composite edible film liquid containing silk fibroin. Background Technology

[0002] Mechanical damage, physiological decay, and microbial spoilage are the main factors leading to the deterioration of the quality of fresh fruits and vegetables after harvest. Meat products are popular among consumers due to their high nutritional value and delicious taste, but they are easily contaminated by microorganisms during processing and storage, resulting in spoilage and a shortened shelf life. Therefore, the development and application of novel preservation technologies for foods such as fruits, vegetables, and meat products are imperative.

[0003] Edible films are safe and environmentally friendly. They are typically made from natural edible materials such as chitosan, pectin, soy protein, or modified starch, forming a porous network structure through intermolecular interactions. This structure can block gas exchange and microbial contamination, thus preserving freshness. However, films made from these materials often suffer from poor mechanical barrier properties and weak antibacterial activity, which can affect their effectiveness.

[0004] Silk fibroin is extracted from silkworm cocoons or silk. It is a biopolymer that has passed GRAS (Generally Recognized As Safe) certification by the US FDA. It can form a transparent, colorless, tasteless, strong, flexible, biodegradable, and water and gas barrier edible film through self-crosslinking.

[0005] Numerous studies have shown that chitosan and silk fibroin are complementary. Silk fibroin-chitosan composite films overcome the high swelling properties of pure chitosan films and exhibit better film-forming properties, mechanical properties, and stability compared to pure silk fibroin films. Furthermore, silk fibroin faces the challenge of high cost in edible film applications, while chitosan is abundant, inexpensive, and readily available. Its biocompatibility, biodegradability, and non-toxicity also make it a common ingredient in food preservation. Silk fibroin-chitosan composite films, to a certain extent, reduce the application cost of silk fibroin and are more conducive to industrial applications.

[0006] Silk fibroin membranes have good application advantages, but they also have problems such as lack of antibacterial effect and easy utilization by microorganisms (S. Ghalei, H. Handa, Materials Today Chemistry, 2022, 23:100673). Silk fibroin lacks inherent antibacterial activity and provides a good substrate for microbial growth. Studies have shown that chitosan-silk fibroin composite membranes have weak antibacterial activity and no obvious antimicrobial zone against Staphylococcus epidermidis and Escherichia coli (Guldemet Basal, Duygu Altiok, Oguz Bayraktar, Fibers And Polymers, 2010, 11(1):21-27).

[0007] Plant essential oils have strong antibacterial effects and are commonly used for preserving fruits, vegetables, and meat products. However, their application in water-soluble systems presents challenges such as low solubility and stability, which need to be addressed. Plant essential oil microemulsions can improve the water solubility of essential oils and give active ingredients a larger specific surface area and better dispersibility, making it easier for them to contact microbial cells and thus enhancing the antibacterial effect of the essential oils. Microemulsions are transparent, low-viscosity, and thermodynamically stable systems that have no significant impact on the light transmittance of edible films. Furthermore, the essential oils and surfactants in the microemulsion formulation can reduce the contact angle of the film liquid on hydrophobic surfaces, facilitating film formation on fruit and vegetable surfaces.

[0008] Therefore, those skilled in the art are dedicated to developing a composite edible film with excellent mechanical barrier properties and strong antibacterial activity. This film is safe and environmentally friendly, and its preparation and use are simple, mild, and not highly dependent on equipment. Summary of the Invention

[0009] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to develop a composite edible film with good mechanical barrier properties, strong antibacterial activity and safety and environmental protection by combining silk fibroin with chitosan and plant essential oil microemulsions. Moreover, the preparation and use methods are simple, the conditions are mild and the equipment dependence is not high.

[0010] To achieve the above objectives, the present invention provides a method for preparing a composite edible film liquid containing silk fibroin, comprising the following steps:

[0011] Step 1: Prepare silk fibroin solution, chitosan solution and microemulsion respectively;

[0012] Step 2: Prepare the mixed membrane solution by thoroughly mixing the silk fibroin solution obtained in Step 1 with the chitosan solution to obtain the silk fibroin-chitosan mixed membrane solution; then mix the silk fibroin-chitosan mixed membrane solution with the microemulsion obtained in Step 1 to obtain the silk fibroin-chitosan-microemulsion mixed membrane solution.

[0013] Furthermore, step 1 also includes:

[0014] Step 1.1: Prepare silk fibroin solution. Degumme the silkworm silk with sodium carbonate solution, then wash it with distilled water to remove impurities and some residual sericin, and then dry it to obtain degummed silk fibroin. Dissolve the degummed silk fibroin in lithium bromide aqueous solution and heat to dissolve. After removing small molecules of lithium bromide by dialysis, ultrafiltration or electrodialysis, centrifuge to collect the supernatant to obtain silk fibroin aqueous solution, and store it in a refrigerator.

[0015] Step 1.2: Prepare chitosan solution by adding chitosan to acetic acid solution and stirring until the chitosan is completely dissolved to obtain chitosan solution;

[0016] Step 1.3: Prepare a microemulsion by mixing the surfactant and co-surfactant to obtain a mixed surfactant; then mix the mixed surfactant with the essential oil to obtain a mixed oil phase; then add deionized water dropwise to the mixed oil phase while stirring to obtain the microemulsion.

[0017] Further, in step 1.1, the sodium carbonate solution concentration is 0.01%-0.50%, the degumming temperature is 60℃-120℃, the degumming time is 20-120 min, and the lithium bromide aqueous solution concentration is 5-10 M; in step 1.2, the acetic acid solution concentration is 0.5%-1.0%, and the chitosan solution concentration is 1.0%-3.0%. In step 1.3, the surfactant is Tween 80; the co-surfactant is anhydrous ethanol; the mass ratio of surfactant to co-surfactant is 1:1-4:1; the mass ratio of mixed surfactant to essential oil is 7:3-9:1; and deionized water accounts for 75%-95% of the microemulsion composition by weight.

[0018] Further, the distilled water washing step in step 1.1 is as follows: rinse with pure water 3-7 times to remove impurities and some residual sericin; the drying step is as follows: place in a 40℃-90℃ forced-air drying oven for about 12-24 hours; the heating and dissolving step is as follows: water bath at 40℃-90℃ for 1-4 hours, shaking continuously during the dissolving process; the dialysis step is as follows: dialyze in distilled water using a dialysis bag (with a flow rate of 3-20 kDa) for 48-72 hours, changing the water every 4-8 hours; the centrifugation step is as follows: put the solution obtained after dialysis into a centrifuge tube and centrifuge at 5000-10000 rpm for 10-30 minutes.

[0019] Furthermore, step 1.2 also includes: diluting the silk fibroin solution, mixing it thoroughly with the chitosan solution, and heating and stirring to obtain a silk fibroin-chitosan mixed membrane solution.

[0020] Furthermore, the concentration of the silk fibroin solution after dilution is 0.5%-3.0%, and the volume ratio of the silk fibroin solution to the chitosan solution is 5:1-1:5; the heating and stirring are carried out by magnetic stirring in a water bath at 50℃-70℃ for 30-60 minutes.

[0021] Furthermore, in step 2, the final concentration of the microemulsion in the mixed membrane solution is 0.5%-5.0%.

[0022] Further, a composite edible film was prepared for the determination of water vapor permeability and antibacterial activity. The steps were as follows: 10 mL of film solution was slowly poured into a mold (90 mm × 15 mm), excess solution and air bubbles were removed, and the film was dried overnight on a level to obtain a film with a thickness of 0.02-0.05 mm. The film was then peeled off from the mold and placed in a sealed box for testing.

[0023] Furthermore, the drying temperature is 40℃-60℃.

[0024] This invention also provides the application of a composite edible film liquid containing silk fibroin in food preservation. The raw materials of the composite film liquid include silk fibroin, chitosan and microemulsion; the microemulsion includes mixed surfactants and essential oils, and the mixed surfactants include surfactants and co-surfactants.

[0025] Further, soak the washed fruits, vegetables, or meat products in the composite edible film liquid, then remove and air dry.

[0026] Furthermore, the soaking time is 0.5-20 minutes.

[0027] In a preferred embodiment 1 of the present invention, the process of preparing chitosan film solution is described in detail and applied to strawberry preservation;

[0028] In another preferred embodiment 2 of the present invention, the process of preparing silk fibroin film solution is described in detail and applied to strawberry preservation;

[0029] In another preferred embodiment 3 of the present invention, the process of preparing the silk fibroin-microemulsion mixed membrane solution is described in detail and applied to strawberry preservation;

[0030] In another preferred embodiment 4 of the present invention, the preparation of a silk fibroin-chitosan-microemulsion mixed film solution is described in detail and applied to strawberry preservation;

[0031] In Comparative Example 1 of the present invention, the treatment process of the control group without coating preservation is described in detail;

[0032] In Examples 1-3 of the present invention, the preservation experimental process of applying chitosan membrane solution, silk fibroin membrane solution and silk fibroin-3% microemulsion mixed membrane solution separately is described in detail.

[0033] The beneficial technical effects of this invention are as follows:

[0034] This invention provides a silk fibroin-chitosan-microemulsion composite edible film liquid with excellent mechanical barrier properties, strong adhesion, and strong antibacterial activity. It can effectively delay the oxidation and aging process of food and inhibit the growth of microorganisms in food. Its application in food preservation can extend the shelf life of food and maximize the preservation of its original quality and commercial value. Furthermore, it is safe and environmentally friendly, with simple preparation and application methods, mild conditions, and low dependence on equipment. Therefore, it has high economic, social, and ecological value.

[0035] The following will further explain the concept, specific preparation process and technical effects of the present invention with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention. Attached Figure Description

[0036] Figure 1 This is a graph showing the transmittance variation of the microemulsion in Example 4 of the present invention at different temperatures;

[0037] Figure 2 The above are contact angle bar charts of the edible film liquids of Examples 1-4 of the present invention;

[0038] Figure 3 The bar chart shows the water vapor permeability of the edible films of Examples 1-4 of the present invention.

[0039] Figure 4 The bar chart shows the inhibition rate of the edible film of Example 4 of the present invention against Escherichia coli and Staphylococcus aureus.

[0040] Figure 5 These are photographs of the appearance of strawberries from Examples 1-4 and Comparative Example 1 after being stored under the same conditions for 7 days (AE represents strawberries without coating in Comparative Example 1, strawberries preserved in Examples 1-3 using chitosan film solution, silk fibroin film solution and silk fibroin-3% microemulsion mixed film solution alone, and strawberries preserved in Example 4 using silk fibroin-chitosan-3% microemulsion mixed film solution). Detailed Implementation

[0041] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0042] Example 1: Preparation of chitosan membrane solution and its application in strawberry preservation.

[0043] (1) Preparation of membrane solution: Add 5g of chitosan to 500mL of 1% acetic acid solution and stir until the chitosan is completely dissolved to prepare a 1% chitosan membrane solution. The contact angle of the membrane solution on the hydrophobic plate was then measured.

[0044] (2) Slowly pour 10 mL of 1% chitosan film solution into a mold (90 mm × 15 mm), remove excess solution and air bubbles, place it on a level and dry it overnight to obtain a film with a thickness of about 0.03 mm. Peel the film from the mold and place it in a sealed box to test the water vapor transmission rate and antibacterial activity.

[0045] (3) After washing, the strawberries were soaked in 1% chitosan film solution for 10 minutes, then removed, dried, and stored at 18±2℃. The weight loss rate, spoilage rate, and firmness of the treated strawberries were measured every 24 hours, and the preservation effect was recorded by taking pictures.

[0046] Example 2: Preparation of silk fibroin film solution and its application in strawberry preservation.

[0047] (1) Preparation of the membrane solution: The silkworm silk was degummed with 0.2% sodium carbonate solution, then washed with distilled water to remove impurities and some residual sericin, and then dried at 50°C for 12 hours; the degummed silk fibroin was dissolved in 9.3M lithium bromide aqueous solution and heated at 60°C for 1 hour; after removing lithium bromide by dialysis, the supernatant was collected by centrifugation and diluted to a silk fibroin concentration of 1% for later use. The contact angle of the membrane solution on the hydrophobic plate was measured.

[0048] (2) Slowly pour 10 mL of 1% silk fibroin membrane solution into a mold (90 mm × 15 mm), remove excess solution and air bubbles, place it on a level and dry it overnight to obtain a film with a thickness of about 0.03 mm. Peel the film from the mold and place it in a sealed box to test the water vapor transmission rate and antibacterial activity.

[0049] (3) Soak the washed strawberries in 1% silk fibroin membrane solution for 10 minutes, and then perform the same post-treatment as in Example 1.

[0050] Example 3: A silk fibroin-microemulsion mixed membrane solution was prepared and applied to strawberry preservation.

[0051] (1) Preparation of the membrane solution: The preparation of the silk fibroin solution was the same as in Example 2. Tween 80 and anhydrous ethanol were mixed at a mass ratio of 4:1 to form a mixed surfactant. The mixed surfactant was then mixed with cinnamon essential oil at a mass ratio of 9:1 to obtain a mixed oil phase. Deionized water was then slowly added dropwise to the mixed oil phase while stirring to obtain a cinnamon essential oil microemulsion. Characterization experiments of the microemulsion were performed simultaneously. Finally, 1% silk fibroin solution was mixed with a certain amount of microemulsion to make the final concentration of the microemulsion 3%, resulting in a 1% silk fibroin-3% microemulsion mixed membrane solution for later use. The contact angle of the membrane solution on the hydrophobic plate was measured.

[0052] (2) Slowly pour 10 mL of 1% silk fibroin-3% microemulsion mixed film solution into a mold (90 mm × 15 mm), remove excess solution and air bubbles, place it on a level and dry overnight to obtain a film with a thickness of about 0.03 mm. Peel the film from the mold and place it in a sealed box to test the water vapor transmission rate and antibacterial activity.

[0053] (3) After washing, the strawberries were soaked in a 1% silk fibroin-3% microemulsion mixture for 10 minutes. The subsequent treatment was the same as in Example 1.

[0054] Example 4: A silk fibroin-chitosan-microemulsion mixed membrane solution was prepared and applied to strawberry preservation.

[0055] (1) Preparation of membrane solution: This example provides a silk fibroin-chitosan-microemulsion membrane, which is basically the same as that in Example 3. The difference is that in this example, 1% silk fibroin solution and 1% chitosan solution are first thoroughly mixed at a volume ratio of 1:1, and then magnetically stirred for 30 minutes in a 60°C water bath to obtain a silk fibroin-chitosan mixed membrane solution. Finally, this mixed membrane solution is mixed with a certain amount of microemulsion to make the final concentration of the microemulsion 3%, so as to obtain a 1% silk fibroin-1% chitosan-3% microemulsion mixed membrane solution for use. The contact angle of the membrane solution on the hydrophobic plate is measured.

[0056] (2) Slowly pour 10 mL of 1% silk fibroin-1% chitosan-3% microemulsion mixture into a mold (90 mm × 15 mm), remove excess solution and air bubbles, place it on a level and dry overnight to obtain a film with a thickness of about 0.03 mm. Peel the film from the mold and place it in a sealed box to test the water vapor transmission rate and antibacterial activity.

[0057] (3) After washing, the strawberries were soaked in a 1% silk fibroin-1% chitosan-3% microemulsion mixed film solution for 10 minutes. The subsequent treatment was the same as in Example 1.

[0058] Comparative Example 1: Strawberries were stored directly without coating.

[0059] This comparative example provides a control group for strawberry preservation experiments. The strawberries were simply washed and dried without coating, and stored directly at 18±2℃. Post-treatment was the same as in Example 1.

[0060] Specific experimental methods:

[0061] 1. Microemulsion Characterization Experiment

[0062] The cinnamon essential oil microemulsion obtained in Example 3 was subjected to microemulsion characterization experiments.

[0063] The microemulsion characterization steps are as follows:

[0064] Centrifugal stability: The appearance changes of the microemulsion were observed at rotation speeds of 2000-8000 r / min, and the transmittance of the sample after centrifugation was measured to examine the centrifugal stability of the microemulsion system.

[0065] Thermal stability: The transmittance of the microemulsion was measured after heating at different temperatures (40℃-90℃) for 30 min to examine the stability of the microemulsion at different temperatures.

[0066] The formula for calculating light transmittance is:

[0067] T(%) = A0 / A1 × 100%

[0068] Where T is the transmittance; A0 is the absorbance of the microemulsion before centrifugation / heating; and A1 is the absorbance of the microemulsion after centrifugation / heating.

[0069] The results of the centrifugation stability test are shown in Table 1. It can be seen that the microemulsion did not show any turbidity or stratification after centrifugation at different speeds and the transmittance was above 100%.

[0070] Table 1. Microemulsion stability at different centrifugation rates

[0071]

[0072] Thermal stability test results are as follows Figure 1 As shown, when the temperature is below 80℃, the microemulsion remains clear and transparent, with a stable transmittance above 100%, indicating good thermal stability. When the heating temperature reaches 80℃, the microemulsion becomes turbid and separates into layers, with the transmittance rapidly dropping to around 20%. However, when the turbid sample is allowed to cool naturally at room temperature, the solution quickly returns to a clear and transparent state. This is because, for polyoxyethylene nonionic surfactants, at higher temperatures, the hydrogen bonds between water and the polyoxyethylene chain break, causing the surfactant's solubility in water to decrease rapidly and precipitate out. The system changes from clear to turbid or separates into layers; this phenomenon is called condensation. The above results indicate that this microemulsion possesses good thermal and centrifugal stability.

[0073] 2. Membrane contact angle experiment

[0074] The contact angle experiments were conducted on the 1% chitosan film solution, 1% silk fibroin film solution, 1% silk fibroin-3% microemulsion mixed film solution and 1% silk fibroin-1% chitosan-3% microemulsion mixed film solution obtained in Examples 1-4, respectively.

[0075] The contact angle test procedure is as follows:

[0076] The contact angle of the membrane solution was measured using the seated drop method to evaluate its surface hydrophobicity. 5 μL of membrane solution was carefully dropped onto a hydrophobic plate with a polypropylene surface material, which was then placed on a substrate surface. Digital images were then captured using a high-resolution digital camera, and the contact angles of the images were analyzed using ImageJ software. Triple replicates were performed for each membrane solution.

[0077] Contact angle test results are as follows Figure 2 As shown, the 1% chitosan film solution obtained in Example 1 and the 1% silk fibroin film solution obtained in Example 2 have relatively high contact angles, close to the contact angle of pure water (approximately 90°). This indicates that these film solutions easily drip like water on hydrophobic surfaces, making it difficult to form a film on the fruit surface. In contrast, the contact angles of the 1% silk fibroin-3% microemulsion mixed film solution obtained in Example 3 and the 1% silk fibroin-1% chitosan-3% microemulsion mixed film solution obtained in Example 4 are significantly reduced, indicating that the film solutions exhibit strong hydrophobic properties. This may be because essential oils are primarily non-polar substances, thus increasing the hydrophobicity of the film solution and enhancing its adhesion to the fruit surface.

[0078] 3. Thin film water vapor transmission rate and antibacterial activity test

[0079] The water vapor transmission rate and antibacterial activity of the 1% chitosan film, 1% silk fibroin film, 1% silk fibroin-3% microemulsion mixed film, and 1% silk fibroin-1% chitosan-3% microemulsion mixed film obtained in Examples 1-4 were tested, and the steps are as follows:

[0080] Water vapor transmission rate: The permeability of the membrane to water vapor is measured according to ASTM E96-95a.

[0081] Antibacterial activity: *Escherichia coli* ATCC 25922 and *Staphylococcus aureus* ATCC 29213 were used as test strains. 0.20 g of each membrane was weighed and placed in 10 mL of the corresponding liquid culture medium, and incubated at room temperature for 12 h. 100 μL of 1×10⁻⁶ medium was aseptically pipetted. 5CFU / mL test bacterial suspension was introduced into each group of liquid media and incubated at 37℃ for 24 h. Each sample was serially diluted with sterile water, and 100 μL of each serial dilution was spread onto the corresponding solid culture medium and incubated at 37℃ for 12 h. Finally, after determining the number of viable bacteria according to GB / T 4789.2-2016, the inhibition rate against the test bacterial species was calculated.

[0082] The experimental results of water vapor transmission rate are as follows: Figure 3 As shown, the water vapor transmission rates of the 1% silk fibroin film obtained in Example 2 and the 1% silk fibroin-3% microemulsion mixed film obtained in Example 3 are not significantly different, and the former is slightly better than the 1% silk fibroin-1% chitosan-3% microemulsion mixed film obtained in Example 4. The water vapor transmission rates of the films obtained in Examples 2, 3, and 4 are all significantly better than the 1% chitosan film obtained in Example 1. This indicates that the introduction of microemulsion has little effect on the mechanical barrier properties of the silk fibroin film. The mechanical barrier properties of the silk fibroin-chitosan-microemulsion composite film are slightly lower than those of the silk fibroin film and the silk fibroin-microemulsion film, but are still significantly better than those of the chitosan film.

[0083] The experimental results of antibacterial activity are as follows: Figure 4 As shown, the initial colony count in the system was approximately 3log. 10 The 1% silk fibroin solution obtained in Example 2, at CFU / mL, not only showed no antibacterial activity but also exhibited a certain degree of proliferation of both types of bacteria. In contrast, the membranes in Examples 1, 3, and 4 showed varying degrees of inhibitory effects on the test bacterial species. Specifically, the residual colony counts of *Escherichia coli* and *Staphylococcus aureus* in the pure chitosan membrane were 2.74 ± 0.06 and 2.88 ± 0.06 log, respectively. 10 The CFU / mL count indicates that pure chitosan has a certain inhibitory effect on bacteria; while the residual colony count in the composite membrane containing microemulsions ranged from 0.76 ± 0.04 log₂O₅. 10 CFU / mL ~0.99±0.12log 10 The CFU / mL concentration indicates that the composite membrane not only has antibacterial properties but also a good bactericidal effect. This may be related to the large specific surface area and good dispersibility of the essential oil components in the microemulsion system, which facilitates contact with microbial cells. This result is consistent with the spoilage rate of strawberries after coating preservation in Examples 1, 3, and 4 in Table 2.

[0084] The above results indicate that the edible film liquid prepared by the present invention utilizes the complementarity of chitosan and silk fibroin and the antibacterial properties of microemulsions, exhibiting good adhesion to food surfaces and possessing good mechanical barrier and antibacterial activity after film formation.

[0085] 4. Strawberry preservation experiment

[0086] Strawberries treated with a 1% silk fibroin-1% chitosan-3% microemulsion mixed film solution in step (3) of Example 4, strawberries without coating treatment in Comparative Example 1, and strawberries coated with chitosan film solution, silk fibroin film solution and silk fibroin-3% microemulsion mixed film solution in Examples 1-3 were stored under the same conditions for 7 days. Their appearance, weight loss rate, spoilage rate and hardness were observed.

[0087] The steps are as follows:

[0088] Weight loss rate: The difference between the initial mass of the strawberries and the mass of the same group at each measurement is defined as the mass loss of the strawberries. The weight loss rate is calculated as a percentage of the initial mass loss. The formula for calculating the weight loss rate is as follows:

[0089] Weight loss rate (%) = (m0 - m) n ) / m n ×100%

[0090] Where m0 is the initial mass of the strawberry, and m n Let be the mass of the strawberries on day n. The experiment was repeated 3 times.

[0091] Rot rate: Strawberries were divided into four grades based on the size of the rotten area, and a rot index was calculated. Grade 0: No rotten strawberries; Grade 1: Rotten area less than 25%; Grade 2: Rotten area 25%-50%; Grade 3: Rotten area more than 50%. Each example set up three parallel groups, with 10 strawberries in each group. The rot rate calculation formula is as follows:

[0092] Corruption rate (%) = (ΣY1×a) / (Y2×b)×100%

[0093] Where Y1 is the rot level, Y2 is the highest rot level, a is the number of rotten fruits, and b is the total number of fruits.

[0094] Hardness: Three strawberries were randomly selected from each group, and the hardness of the strawberries was tested using an Adeberg GY-4 fruit hardness tester.

[0095] The specific measurement results are shown in Table 2.

[0096] Table 2. Preservation results of strawberries after 7 days of storage in different examples.

[0097] Comparative Example 1 7.01±0.44 53.33±3.33 0.69±0.07 Example 1 6.59±0.80 41.11±1.92 1.95±0.02 Example 2 2.51±0.46 54.44±5.09 1.69±0.07 Example 3 2.20±0.56 4.44±5.09 4.52±0.04 Example 4 2.11±0.48 2.22±1.92 4.49±0.05

[0098] The appearance of strawberries in different embodiments is as follows Figure 5As shown, A, E represent strawberries stored under the same conditions for 7 days after preservation: strawberries without coating treatment in Comparative Example 1, strawberries preserved using chitosan film solution, silk fibroin film solution, and a mixture of silk fibroin-3% microemulsion film solution alone in Examples 1-3, and strawberries preserved using a mixture of 1% silk fibroin-1% chitosan-3% microemulsion film solution in Example 4. Strawberries are perishable fruits. In Examples 1-2, the chitosan film solution and silk fibroin film solution alone had no significant preservation effect on strawberries, and the spoilage rate after 7 days was not significantly different from the control group. In Example 3, the mixture of silk fibroin-3% microemulsion film solution alone and in Example 4, the mixture of 1% silk fibroin-1% chitosan-3% microemulsion film solution showed excellent preservation effects, with spoilage rates below 5% after 7 days of storage. Furthermore, since chitosan is abundant, inexpensive, and readily available, it can reduce the cost of composite edible films to a certain extent; therefore, the method in Example 4 also has higher economic value.

[0099] Based on the above experimental results, the silk fibroin-chitosan-microemulsion composite edible film liquid involved in this invention has the characteristics of good mechanical barrier properties, strong adhesion, and strong antibacterial activity, which can more effectively delay the oxidation, aging and microbial spoilage process of food and further extend the shelf life of the product.

[0100] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a composite edible film liquid containing silk fibroin, characterized in that, The method includes the following steps: Step 1: Prepare silk fibroin solution, chitosan solution and microemulsion respectively; Step 2: Prepare the mixed membrane solution by thoroughly mixing the silk fibroin solution obtained in Step 1 with the chitosan solution to obtain a silk fibroin-chitosan mixed membrane solution; then mix the silk fibroin-chitosan mixed membrane solution with the microemulsion obtained in Step 1 to obtain a silk fibroin-chitosan-microemulsion mixed membrane solution, wherein the final concentration of the microemulsion in the mixed membrane solution is 0.5%-5.0%. Step 1 further includes: Step 1.1: Prepare the silk fibroin solution. Degumme the silkworm silk with sodium carbonate solution, then wash it with distilled water to remove impurities and some residual sericin, and then dry it to obtain degummed silk fibroin. Dissolve the degummed silk fibroin in lithium bromide aqueous solution and heat to dissolve. Remove the lithium bromide by dialysis, ultrafiltration or electrodialysis, centrifuge to collect the supernatant, and store the silk fibroin aqueous solution in a refrigerator. The concentration of the sodium carbonate solution is 0.01%-0.50%, the degumming temperature is 60℃-120℃, the degumming time is 20-120 min, and the concentration of the lithium bromide aqueous solution is 5-10 M. Step 1.2: Prepare the chitosan solution by adding the chitosan to an acetic acid solution and stirring until the chitosan is completely dissolved to obtain the chitosan solution; the concentration of the acetic acid solution is 0.5%-1.0%, and the concentration of the chitosan solution is 1.0%-3.0%. Step 1.3: Prepare the microemulsion by mixing the surfactant and co-surfactant to obtain a mixed surfactant; then mix the mixed surfactant with the essential oil to obtain a mixed oil phase; then add deionized water dropwise to the mixed oil phase while stirring to obtain the microemulsion; wherein the surfactant is Tween 80, the co-surfactant is anhydrous ethanol; the mass ratio of the surfactant to the co-surfactant is 1:1-4:1; the mass ratio of the mixed surfactant to the essential oil is 7:3-9:1; and the deionized water accounts for 75%-95% of the composition of the microemulsion by weight percentage.

2. The preparation method according to claim 1, characterized in that, Step 2 further includes: diluting the silk fibroin solution, mixing it thoroughly with the chitosan solution, and heating and stirring to obtain the silk fibroin-chitosan mixed membrane solution.

3. The preparation method according to claim 2, characterized in that, The concentration of the silk fibroin solution after dilution is 0.5%-3.0%, and the volume ratio of the silk fibroin solution to the chitosan solution is 5:1-1:5; the heating and stirring are performed by magnetic stirring in a water bath at 50℃-70℃ for 30-60 min.

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