Method for preparing a food freshness indicator film by tyrosinase catalyzed covalent cross-linking of myofibrillar proteins with anthocyanins

Abstract

The application discloses a method for preparing a food freshness indicating film by tyrosinase catalyzing covalent cross-linking of myofibrillar protein and anthocyanin, and has the characteristics that the method comprises the following steps: S1, preparing a myofibrillar protein solution; S2, adding tyrosinase and anthocyanin into the myofibrillar protein solution, and performing continuous stirring reaction under the condition of ice water bath and exposure to air, and adjusting the pH of the solution after the reaction is completed, so as to obtain a film-forming solution; and S3, pouring the film-forming solution into a culture dish for drying, so as to obtain the indicating film. The tyrosinase catalyzing method is used to covalently cross-link and combine the anthocyanin and the myofibrillar protein, and bacterial nanocellulose is added as a reinforcing agent, so that the indicating film with low anthocyanin release rate, high inlay stability and safe use can be prepared. In addition, the indicating film has high light stability, and the detection limit of trimethylamine can be as low as 1.5 micromoles, and the indicating film has superior trimethylamine response performance and detection stability.

Description

Technical Field

[0001] This invention belongs to the field of non-destructive food monitoring technology, specifically relating to a method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein and anthocyanins. Background Technology

[0002] Fish, rich in nutrients, is highly perishable, making accurate assessment of its freshness crucial for food safety. Traditional methods for detecting fish freshness primarily involve microbiological and chemical methods, such as volatile basic nitrogen and total bacterial count analysis, which are often time-consuming and complex. Therefore, a simple, low-cost, non-destructive, and accurate monitoring system for assessing fish product freshness is indispensable. Colorimetric smart packaging is a non-destructive monitoring technology that visually reflects the freshness of fish through changes in color hue. The principle behind this is that volatile amines released during fish spoilage raise the pH of the top air inside the packaging, causing a chemical change in the pH-sensitive dye, resulting in a color change.

[0003] Anthocyanins (ACN), as a natural, safe pigment with a wide color-changing range and significant color-changing effect, have been widely used in smart packaging. To date, most anthocyanin-based indicator films have been developed using hydrophilic polymers as carriers and are used to monitor the freshness of high-moisture foods, such as fresh fish and beef. However, hydrophilic films swell after absorbing water in high-humidity packaging environments, leading to anthocyanin leakage and limiting their indicating capabilities. Furthermore, due to the instability of natural colorants, such as anthocyanins, which are highly sensitive to light, heat, pH, oxygen, and metal ions, anthocyanins are easily degraded under light and oxygen conditions during long-term use and storage, causing the indicator film to lose its indicating effect. Additionally, since indicator films are exposed inside food packaging, they may come into direct contact with food or the human body, posing potential safety risks. While using natural and safe anthocyanins to replace chemical color developers can improve the safety of indicator films, using unsafe substrates or introducing potentially hazardous chemicals during the manufacturing process will also reduce the safety of the indicator films.

[0004] In summary, reducing the leaching of anthocyanins embedded in the indicator film, improving the stability of anthocyanins in the indicator film, and ensuring the safety of the indicator film are key issues that need to be addressed for the widespread application of anthocyanin indicator films. Existing technologies disclose relevant solutions to address these issues. For example, Chinese Patent (Application No. 202111182867.X) discloses a method for preparing anthocyanin-betaine-k-carrageenan freshness indicator film. This method enhances the pH-responsive color change sensitivity of natural pigments and improves their chemical stability by combining anthocyanins and betaine with polymers, thereby obtaining a highly efficient and stable freshness indicator film. However, this technical solution does not solve the problem of the indicator film swelling after absorbing water in a high-humidity packaging environment, leading to the leaching and leakage of anthocyanins and betaine-k.

[0005] For example, Chinese patent (application number 202211608639.9) discloses a method for preparing a low-anthocyanin leakage sodium alginate / cellulose nanocrystal / beeswax-based hydrophobic indicator membrane. This method utilizes the molecular complexation properties of cellulose nanocrystals and the water-barrier properties of beeswax to achieve low anthocyanin leakage and high hydrophobic stability, ensuring good indication performance in practical applications and extending the lifespan of the smart indicator membrane. While the above technical solution solves the problem of anthocyanin leakage caused by water absorption and swelling of the indicator membrane, the preparation method is relatively complex and uses chemical reagents such as n-hexane and polydimethylsiloxane to treat the indicator membrane. The residual issues of n-hexane and polydimethylsiloxane affect the safety of the indicator membrane in use.

[0006] For example, Chinese patent (application number 202210650331.4) discloses a method for preparing a sodium alginate-purple sweet potato peel anthocyanin smart indicator film for monitoring the freshness of chicken. It uses safe, non-toxic sodium alginate with good film-forming properties and edibility as the film-forming substrate, and utilizes purple sweet potato peel anthocyanins as an indicator to prepare an indicator film that clearly indicates the freshness of chicken breast. Although this indicator film has good safety, it does not solve the problems of easy leaching of anthocyanins and low stability.

[0007] As can be seen from the above, the anthocyanin indicator membrane disclosed in the prior art cannot simultaneously solve the problems of anthocyanin leakage, low stability, and safety hazards of the indicator membrane; all of these issues have room for improvement. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for preparing a food freshness indicator film by covalent cross-linking myofibrillar protein and anthocyanins using tyrosinase catalysis. This invention utilizes tyrosinase catalysis to covalently cross-link anthocyanins with myofibrillar protein, while simultaneously adding bacterial nanocellulose as a reinforcing agent. This method produces an indicator film with low anthocyanin release rate, high embedding stability, and safe use. Furthermore, the indicator film of this invention exhibits high light stability and a detection limit as low as 1.5 μmol for trimethylamine, demonstrating superior trimethylamine response performance and detection stability.

[0009] The present invention is achieved through the following technical solution.

[0010] On one hand, the present invention provides a method for preparing a food freshness indicator film by covalent cross-linking of myofibrillar protein and anthocyanins catalyzed by tyrosinase, characterized by comprising the following steps:

[0011] S1. Prepare myofibrillar protein solution;

[0012] S2. Add tyrosinase and anthocyanins to the myofibrillar protein solution, and conduct a continuous stirring reaction under ice-water bath conditions while exposed to air. After the reaction is completed, adjust the pH of the solution to obtain the film-forming solution.

[0013] S3. Pour the film-forming solution into a petri dish and dry it to obtain the indicator film of the present invention.

[0014] As a preferred technical solution, step S3 specifically involves adding bacterial nanocellulose and glycerol to the film-forming solution, stirring at room temperature for 0.5 to 3 hours, then filtering to remove impurities, pouring the filtrate into a petri dish, and then drying it at 40 to 55°C to obtain the indicator membrane of the present invention.

[0015] As a preferred technical solution, the preparation method of the myofibrillar protein solution in step S1 is as follows: after thawing frozen fish paste, cut it into small pieces, then add distilled water at a ratio of 7 to 10:50 (material to liquid mass), and at the same time add 2% to 3% sodium chloride based on the mass of fish paste, and then homogenize to obtain the myofibrillar protein solution.

[0016] As a preferred technical solution, the homogenization conditions are: homogenization speed of 8000-12000 r / min and homogenization time of 100-150 s.

[0017] As a preferred technical solution, in step S2, the continuous stirring reaction time is controlled to be 3-5 hours, and the pH of the solution is adjusted to 3 after the reaction is completed.

[0018] As a preferred technical solution, in step S2, the concentration of added tyrosinase activity is controlled to be 20-35 U / mL.

[0019] As a preferred technical solution, in step S2, the amount of anthocyanin added is controlled to be 0.5% to 2% based on the mass of protein in the myofibrillar protein solution.

[0020] As a preferred technical solution, in step S3, the amount of bacterial nanocellulose added is 5% to 7% based on the mass of the protein in the myofibrillar protein solution.

[0021] As a preferred technical solution, in step S3, the amount of glycerol added is 40% based on the mass of the protein in the myofibrillar protein solution.

[0022] On the other hand, the present invention provides a food freshness indicator film, characterized in that it is prepared by any of the methods described above.

[0023] Regarding the exploration and implementation of the technical solution of this invention, since the inventors' team has long been engaged in the research and application of aquatic product processing, based on the fact that myofibrillar protein is easily obtained from aquatic product processing by-products and is edible and safe, the inventors proposed an innovative idea of ​​using myofibrillar protein extracted from fish meat as the substrate for preparing an indicator membrane. Based on the above idea, the inventors know that proteins are commonly used matrix materials for preparing anthocyanin indicator membranes, such as gelatin, soy protein, and zein. Although myofibrillar protein is also a protein material, its application in the preparation of anthocyanin indicator membrane products has not been reported. Moreover, using conventional proteins as substrates to prepare anthocyanin indicator membranes still has many problems such as easy leaching of anthocyanins, poor anthocyanin stability, and low safety. Therefore, it is unknown whether the myofibrillar protein used in this invention can successfully prepare anthocyanin indicator membrane substrate that can detect food freshness. In particular, overcoming the problems mentioned in the background technology further increases the difficulty of realizing this invention.

[0024] In the process of exploring the technical solution of this invention, the applicant first tried a conventional method for preparing anthocyanin indicator membranes, which involves continuously stirring and mixing myofibrillar protein and anthocyanin under anaerobic conditions to prepare a film-forming solution. However, the binding rate of anthocyanin to myofibrillar protein in the prepared film-forming solution was low, and the color stability and antioxidant stability of the anthocyanin-myofibrillar protein complex under ultraviolet irradiation were poor, as shown in the experimental data in Example 3. In order to improve the stability of the anthocyanin-myofibrillar protein complex, the inventors found through literature search that the existing strategies for stabilizing anthocyanins include co-pigmentation, acetylation, microencapsulation, and lipidation. While the above methods involve non-covalent interactions, which are typically reversible and susceptible to environmental factors, they are not suitable for preparing anthocyanin indicator films. Furthermore, existing technologies disclose that the covalent interaction between polyphenols and myofibrillar proteins can improve the antioxidant stability and gel properties of proteins. However, whether the covalent interaction between polyphenols and myofibrillar proteins will affect the stability of the anthocyanin-myofibrillar protein complex remains unpredictable or unknown. To avoid excluding the possibility of improving the stability of the anthocyanin-myofibrillar protein complex, the inventors still... Experiments were conducted to investigate the effectiveness of different covalent bonding methods in improving the color stability and antioxidant activity of the resulting anthocyanin-myofibrillar protein conjugates, as shown in the experimental data in Example 3. The inventors were pleased to discover that the anthocyanin-myofibrillar protein conjugates prepared via tyrosinase catalysis exhibited good color stability and antioxidant activity, and could be applied to the preparation of food freshness indicator films to obtain anthocyanin indicator films with good stability and high safety. Furthermore, the inventors unexpectedly discovered that because myofibrillar protein is rich in tyrosine and phenylalanine, which can absorb ultraviolet light, using myofibrillar protein as a substrate will have superior advantages. The excellent UV blocking properties can reduce anthocyanin degradation caused by UV radiation to a certain extent. In addition, to further improve the mechanical properties of the protein membrane, the inventors added bacterial nanocellulose (BNC) as a reinforcing agent to the film-forming solution. Due to the three-dimensional porous network structure of bacterial nanocellulose, with its high aspect ratio, high porosity, and high surface area, this special molecular structure makes it easy for bacterial nanocellulose to combine or link with other molecules, thereby improving the mechanical properties of the indicator membrane. Furthermore, the inventors also found that the addition of bacterial nanocellulose also improves the hydrophobicity of the indicator membrane, thus preventing anthocyanins from dissolving upon contact with water.

[0025] The beneficial effects of this invention are as follows:

[0026] 1) This invention utilizes tyrosinase catalysis to covalently crosslink anthocyanins with myofibrillar proteins, which can improve the color stability and antioxidant activity of anthocyanins. At the same time, the addition of bacterial nanocellulose as a reinforcing agent can significantly improve the hydrophobicity of the protein membrane, thereby further improving the embedding stability of anthocyanins in the protein membrane. In addition, the indicator membrane prepared by the method of this invention has high light stability and a detection limit of 1.5 μmol for trimethylamine, showing excellent trimethylamine response performance and detection stability.

[0027] 2) The indicator film of the present invention uses myofibrillar protein as the base material and adds bacterial nanocellulose as a reinforcing agent. No other chemical additives with safety risks are added during the preparation process, so that the food freshness indicator film prepared by the present invention has good safety.

[0028] 3) The indicator film prepared by the method of the present invention can significantly distinguish between fresh, semi-fresh, and spoiled grades of fresh fish. Therefore, the indicator film of the present invention can monitor and warn manufacturers and consumers of food spoilage in real time through color changes, which has good practical and promotional value. Attached Figure Description

[0029] Figure 1 This is a graph showing the effect of different covalent binding mechanisms on anthocyanin binding rate.

[0030] Figure 2 The effect of UV irradiation time on the color of anthocyanin solution, conjugates, and mixtures is shown in Figure A. Figure B shows the effect of UV irradiation time on the color of anthocyanin solution and the effect of UV irradiation time on the color of conjugates and mixtures.

[0031] Figure 3 The effect of UV irradiation time on the DPPH radical scavenging activity of the conjugates and mixtures;

[0032] Figure 4 This is a graph showing the water contact angle detection results of the indicator membrane of the present invention;

[0033] Figure 5 The X-ray diffraction pattern of the indicator film of this invention;

[0034] Figure 6 This is the Fourier transform infrared spectrum of the indicator film of the present invention;

[0035] Figure 7 This is a microstructure diagram of the thin film surface and cross-section of the indicator film of the present invention;

[0036] Figure 8 The diagram shows the color response of the indicator membrane of the present invention at different trimethylamine concentrations; where A is the visual color result diagram, B is the ΔE change trend diagram, and C is the fitting curve diagram.

[0037] Figure 9 This is a graph showing the anthocyanin release rate of the indicator membrane of the present invention;

[0038] Figure 10 This is a graph showing the results of the light stability test of the indicator film of the present invention;

[0039] Figure 11 The image shows the transmittance of the indicator films with different anthocyanin contents of the present invention in the ultraviolet light 200-400nm and visible light 400-800nm ​​regions.

[0040] Figure 12 A graph showing the changes in indicators during the storage of fresh blunt snout bream;

[0041] Figure 13 This is an image showing the changes in the membrane's color values. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. It should be noted that the following embodiments are merely illustrative examples of the present invention, but the scope of protection of the present invention is not limited thereto. All equivalent substitutions made by those skilled in the art in accordance with the spirit of the present invention fall within the scope of protection of the present invention.

[0043] Example 1

[0044] A method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein and anthocyanins, comprising the following steps:

[0045] S1. Prepare myofibrillar protein solution;

[0046] S2. Add tyrosinase and anthocyanins to the myofibrillar protein solution, and conduct a continuous stirring reaction under ice-water bath conditions while exposed to air. After the reaction is completed, adjust the pH of the solution to obtain the film-forming solution.

[0047] S3. Pour the film-forming solution into a petri dish and dry it to obtain the indicator film of the present invention.

[0048] Example 2

[0049] A method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein and anthocyanins, comprising the following steps:

[0050] S1. Preparation of myofibrillar protein solution: The specific method is as follows: After thawing frozen fish paste, cut it into small pieces, then add distilled water at a ratio of 7-10:50 (material to liquid mass), and simultaneously add 2%-3% sodium chloride based on the mass of the fish paste. Then homogenize at 8000-12000 r / min for 100-150 s to obtain the myofibrillar protein solution; the protein content in the myofibrillar protein solution is then determined using the Lowry method.

[0051] It should be noted that the myofibrillar protein solution used in this invention is prepared from fish paste. It can be inferred that myofibrillar proteins from other animal sources can also be used in this invention and achieve the same effect. Furthermore, the preparation form of the myofibrillar protein solution is not limited to the above. It can be extracted from fresh or frozen animal tissues, fresh or frozen animal meat paste, or obtained by reconstitution of myofibrillar protein powder.

[0052] S2. Adjust the pH of the myofibrillar protein solution prepared in step S1 to 6.5±0.5. Then, add tyrosinase and anthocyanins to the myofibrillar protein solution and expose it to air in an ice-water bath for 3-5 hours with continuous stirring. After that, adjust the pH of the solution to 3 to obtain the film-forming solution. The amount of tyrosinase added is controlled so that the enzyme activity concentration of tyrosinase is 20-35 U / mL. The amount of anthocyanins added is 0.5%-2% based on the mass of protein in the myofibrillar protein solution.

[0053] S3. Add bacterial nanocellulose and glycerol to the film-forming solution obtained in step S2, stir at room temperature for 0.5 to 3 hours, then filter to remove impurities, pour the filtrate into a polystyrene petri dish, and then dry at 40 to 55°C; wherein, the amount of bacterial nanocellulose added is 5% to 7% based on the mass of protein in the myofibrillar protein solution, and the amount of glycerol added is 40% based on the mass of protein in the myofibrillar protein solution.

[0054] Example 3

[0055] A method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein and anthocyanins, comprising the following steps:

[0056] S1. Preparation of myofibrillar protein solution: Frozen fish paste was thawed at 4℃ for 12 hours. The thawed fish paste was cut into small pieces, and 16g of fish paste was added to 84g of distilled water. At the same time, 0.4g of sodium chloride was added based on the weight of the fish paste. The mixture was then homogenized at 10000r / min for 120s to obtain the myofibrillar protein solution. The protein content in the myofibrillar protein solution was then determined using the Lowry method.

[0057] S2. Adjust the pH of the myofibrillar protein solution prepared in step S1 to 6.5±0.5. Then, add tyrosinase and anthocyanins to the myofibrillar protein solution. Under ice-water bath conditions, expose to air and continuously stir for 4 hours. Afterward, adjust the pH of the solution to 3 with 1 mol / L HCl to obtain the film-forming solution. The amount of tyrosinase added is controlled so that the enzyme activity concentration of tyrosinase is 27 U / mL. The amount of anthocyanins added is 0.5% to 2% based on the mass of protein in the myofibrillar protein solution.

[0058] S3. Add bacterial nanocellulose and glycerol to the film-forming solution obtained in step S2, stir at room temperature for 2 hours, then filter through a layer of coarse cotton cloth to remove impurities, pour the filtrate into a polystyrene petri dish, and then dry in an oven at 50°C for 36 hours. After drying, peel off the film with tweezers and store it in a desiccator at 75% relative humidity (RH) for further analysis. The amount of bacterial nanocellulose added is 6% based on the mass of protein in the myofibrillar protein solution, and the amount of glycerol added is 40% based on the mass of protein in the myofibrillar protein solution.

[0059] Example 4

[0060] Determination of the color stability and antioxidant activity of anthocyanin-myofibrillar protein complexes formed by different covalent binding methods.

[0061] I. Experimental Setup

[0062] Enzymatic conjugates: The myofibrillar protein solution was diluted to a protein concentration of 15 mg / mL using phosphate buffer containing 0.6 mol NaCl. First, the myofibrillar protein solution was adjusted to pH 6.5 ± 0.5, then anthocyanins (15 μmol / g protein) and tyrosinase (27 U / mL) were added. After mixing, the solution was continuously stirred in an ice-water bath exposed to air for 4 hours; finally, the pH of the solution was adjusted to 7.0.

[0063] Radical conjugates: A myofibrillar protein solution (15 mg / mL) was placed in a beaker, and 10 μmol FeCl3, 100 μmol ascorbic acid, and 1 mmol H2O2 were added as the oxidation system. Then, anthocyanins (15 μmol / g protein) were added, and the mixture was continuously stirred in an ice-water bath exposed to air for 4 hours. At the end of the reaction, Trolox (1 mmol / L) was added to terminate the oxidation reaction. Finally, the pH of the solution was adjusted to 7.0.

[0064] Alkaline pH shift treatment (alkaline conjugates): Anthocyanins (15 μmol / g protein) are added to a myofibrillar protein solution (15 mg / mL), and the mixture is then subjected to alkaline pH shift treatment (pH 7.0 → 9.0 → 7.0). In short, the pH of the mixture is adjusted to 9.0 with 1 mol / L NaOH, then the mixture is continuously stirred in an ice-water bath exposed to air for 4 hours, and finally the pH of the solution is adjusted to 7.0.

[0065] Ultrasonic conjugates: Anthocyanins (15 μmol / g protein) were added to a myofibrillar protein solution (15 mg / mL), and the mixture was ultrasonicated at 400 W for 20 min in an ice-water bath. The mixture was then continuously stirred in an ice-water bath exposed to air for 4 hours, and finally the pH of the solution was adjusted to 7.0.

[0066] In comparison, the conventional treatment method for the film-forming solution in the preparation of the indicator membrane was adopted, namely, mixing myofibrillar protein solution with anthocyanins, stirring continuously under anaerobic conditions for 4 hours, and finally adjusting the pH of the solution to 7.0 to obtain myofibrillar protein-anthocyanin complexes.

[0067] In the above method, the most representative anthocyanin in food, cyanidin-3-O-glucoside (C3G), was purchased from Chengdu Ruifenside Biotechnology Co., Ltd.; tyrosinase (1350u / mg) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0068] II. Detection Methods

[0069] Anthocyanin binding rate was determined using the pH differential method. The protein concentration of the sample was diluted to 2 mg / mL with phosphate buffer, and then 1 mL of protein denaturing agent (20% trichloroacetic acid and 0.4% phosphotungstic acid) was added. The sample was incubated at 4°C for 30 minutes. After incubation, the sample was centrifuged at 12000 rpm at 4°C for 15 minutes. The supernatant was removed, the precipitate was washed, and then reconstituted with 0.1 mol / L NaOH. The anthocyanin content in the protein was determined using the pH differential method. The formula for calculating the anthocyanin binding rate is shown below:

[0070] Anthocyanin binding rate (%) = (Anthocyanin binding amount / Total anthocyanin content) × 100%

[0071] The method for determining color stability is as follows: the prepared sample is exposed to ultraviolet light for 48 hours using an ultraviolet analyzer, with an irradiance of 400 μW / cm². 2The wavelength was 365 nm, and samples were collected at 24 h and 48 h for colorimetric change analysis. The colorimetric values ​​of the samples treated with ultraviolet radiation were measured using a Hunter Lab UltraScan VIS colorimeter.

[0072] The antioxidant properties were determined by diluting the protein concentration of the sample to 0.5 mg / mL with phosphate buffer containing 0.6 mol NaCl. 2 mL of the sample solution was mixed with 2 mL of DPPH ethanol solution and reacted at room temperature in the dark for 1 h. The absorbance was then measured at 517 nm using a spectrophotometer. The DPPH free radical scavenging rate was calculated using the following formula: DPPH free radical scavenging rate (%) = [A0 – (A – B)] / A0 × 100%, where A is the absorbance of the sample, B is the absorbance of the control (ethanol was added to the sample instead of DPPH), and A0 is the absorbance of the blank control (distilled water was added to DPPH instead of the sample).

[0073] II. Results Analysis

[0074] The anthocyanin binding rate, color stability, and antioxidant properties of film-forming solutions obtained by different covalent binding methods and conventional treatment methods were determined. The results are shown in the table below. Figure 1 , Figure 2 And Table 1, Figure 3 .

[0075] Table 1. Effects of UV irradiation time on colorimetric parameters of anthocyanin solution, myofibrillar protein-anthocyanin conjugate, and myofibrillar protein-anthocyanin mixture.

[0076]

[0077] Note: Different lowercase letters indicate significant differences (P<0.05).

[0078] like Figure 1 As shown, the myofibrillar protein-anthocyanin conjugate exhibits a higher binding rate than the myofibrillar protein-anthocyanin mixture, indicating that covalent interactions are stronger than non-covalent interactions. The results demonstrate that the tyrosinase-catalyzed method is more effective than other methods in binding myofibrillar proteins to anthocyanins.

[0079] like Figure 2 As shown in Figure A, after 24 hours and 48 hours of UV irradiation, the color of the myofibrillar protein-anthocyanin mixture solution changed from purplish-red to light pink, and then to yellow. Meanwhile, as shown in Table 1, the L* and b* values ​​of the myofibrillar protein-anthocyanin mixture solution increased significantly, while the a* value decreased significantly, indicating that the anthocyanins in the myofibrillar protein-anthocyanin mixture solution underwent photolysis and fading under UV irradiation. Figure 2Tables B and 1 show the effect of different UV irradiation times on the color stability of myofibrillar protein-anthocyanin conjugates and mixtures. After UV irradiation, the ΔE value of anthocyanins bound to proteins was significantly lower (P<0.05) compared to the untreated myofibrillar protein-anthocyanin mixture solution, indicating that myofibrillar protein protects against anthocyanin degradation after UV irradiation. Furthermore, after 24 h of UV irradiation, the fading of the myofibrillar protein-anthocyanin conjugate was not as pronounced as that of the mixture, indicating that the covalent binding of anthocyanins to proteins contributes to the color stability of anthocyanins. After 48 h of UV irradiation, only the tyrosinase-catalyzed conjugate had the lowest ΔE value, indicating that UV irradiation had the least effect on it.

[0080] like Figure 3 As shown, the antioxidant capacity of the myofibrillar protein-anthocyanin conjugate was significantly higher than that of the mixture, which is consistent with the results of anthocyanin binding rate. The sample prepared by the tyrosinase-catalyzed method exhibited the highest DPPH free radical scavenging activity, likely due to the higher binding efficiency of the enzymatic method. These results indicate that the myofibrillar protein-anthocyanin conjugate prepared by the tyrosinase-catalyzed method possesses better antioxidant activity, thereby delaying the degradation of anthocyanins under UV irradiation.

[0081] In summary, the anthocyanin-myofibrillar protein conjugate prepared by tyrosinase catalysis exhibits good color stability and antioxidant activity, and can be applied to the preparation of food freshness indicator films to obtain anthocyanin indicator films with good stability and high safety in use.

[0082] Example 5

[0083] Performance testing of the indicator membrane prepared in this invention.

[0084] 1. Sample preparation

[0085] MP, the indicator membrane was prepared using the method of Example 3, but without the addition of bacterial nanocellulose (BNC) and anthocyanins (ACN);

[0086] MP / BNC, the indicator membrane was prepared using the method of Example 3, but without the addition of anthocyanins (ACN);

[0087] MP / BNC / ACN0.5, the indicator membrane was prepared using the method of Example 3, wherein the amount of anthocyanin added in step S2 was controlled to be 0.5%;

[0088] MP / BNC / ACN1, the indicator membrane was prepared using the method of Example 3, wherein the amount of anthocyanin added in step S2 was controlled to be 1%;

[0089] The MP / BNC / ACN2 indicator membrane was prepared using the method described in Example 3, wherein the amount of anthocyanin added in step S2 was controlled to be 2%.

[0090] 2. Detection methods and results

[0091] 2.1 Mechanical properties of the indicator membrane

[0092] The mechanical properties of the prepared samples were tested. The mechanical properties of the indicator film were determined according to the method of the national standard "Determination of Tensile Properties of Plastics" (GB / T1040.1-2018). The results are shown in Table 2.

[0093] Table 2 Results of Mechanical Property Tests

[0094] MP <![CDATA[5.28±0.36 d ]]> <![CDATA[60.63±2.86 a ]]> MP / BNC <![CDATA[7.62±0.13 c ]]> <![CDATA[41.18±1.32 b ]]> MP / BNC / ACN0.5 <![CDATA[8.06±0.21 b ]]> <![CDATA[30.12±2.31 c ]]> MP / BNC / ACN1 <![CDATA[8.53±0.11 a ]]> <![CDATA[24.85±1.38 d ]]> MP / BNC / ACN2 <![CDATA[8.22±0.15 b ]]> <![CDATA[23.57±1.88 d ]]>

[0095] As shown in the table above, the addition of bacterial nanocellulose (BNC) to the protein membrane significantly increased the tensile strength of the membrane while decreasing the elongation at break. After covalent cross-linking with anthocyanins, the tensile strength of the membrane further increased. When the anthocyanin concentration increased to 2%, the tensile strength of the membrane showed a decreasing trend.

[0096] 2.2 Water contact angle of the indicator membrane

[0097] The water contact angle of the indicator membrane was measured using an OCA20 contact angle meter, and the results are shown in [Figure number missing]. Figure 4 .Depend on Figure 4 It can be seen that the addition of bacterial nanocellulose significantly improved the hydrophobicity of the membrane. With the increase of anthocyanin concentration, the contact angle of the membrane gradually increased to a maximum of 60.6° at an anthocyanin concentration of 1%, and then decreased at an anthocyanin concentration of 2%.

[0098] 2.3 X-ray diffraction of the indicator film

[0099] The X-ray diffraction pattern of the film was determined using an X-ray diffractometer, and the results are shown in [Figure number missing]. Figure 5 .Depend on Figure 5 It can be seen that after adding bacterial nanocellulose to the protein polymer matrix, a new peak appeared in the X-ray diffraction pattern of the membrane, confirming the presence of bacterial nanocellulose in the polymer substrate.

[0100] 2.4 Fourier transform infrared image of the indicator membrane

[0101] The Fourier transform infrared spectrum of the membrane was measured using a Fourier transform infrared spectrometer, and the results are shown in [Figure number missing]. Figure 6 .Depend on Figure 6 It is known that anthocyanins can bind to myofibrillar proteins through hydrogen bonds, hydrophobic interactions, electrostatic interactions, and covalent bonds. This is consistent with the experimental data in Example 5, proving that the tyrosinase catalytic method can more effectively bind myofibrillar proteins to anthocyanins.

[0102] 2.5 Scanning electron microscopy observation of the indicator membrane

[0103] The microstructure of the thin film surface and cross-section was observed using scanning electron microscopy, and the results are shown in [Figure number missing]. Figure 7 .Depend on Figure 7 It is evident that the surface of the myofibrillar protein membrane is uneven, and the cross-section also exhibits protrusions. After the addition of bacterial nanocellulose, the surface roughness of the MP / BNC membrane significantly decreased, but the number of cross-sectional cracks increased. With the addition of anthocyanins, the membrane's microstructure became denser, and the cross-section showed no obvious pores. With further increases in anthocyanin content, aggregates and cracks formed on the surface and cross-section of the MP / BNC / ACN2 membrane.

[0104] 2.6 Color response of the indicator membrane to trimethylamine

[0105] Place the membrane (10mm × 10mm) in a sealed test chamber with a relative humidity of 75%. Inject the analyte solution into the test chamber using a micro-syringe. Calculate the concentration of trimethylamine in the test chamber using the following formula:

[0106]

[0107] Where C (mol / L) is the concentration of trimethylamine in the test chamber; ρ s Vs is the density of the trimethylamine solution (g / mL); Vs is the volume of the trimethylamine solution (mL); W is the mass fraction of trimethylamine in the trimethylamine solution; M is the molar mass of trimethylamine (g / mol); V is the volume of the test chamber (L).

[0108] After the membrane was kept in the test chamber for 3 hours to reach reaction equilibrium, the image and colorimetric values ​​of the membrane were recorded using a scanner and a colorimeter, respectively. The color difference value ΔE was calculated according to the following formula:

[0109]

[0110] Among them, L * a * and b * These represent the color values ​​of the film after trimethylamine treatment. and The colorimetric value represents the film before trimethylamine treatment; the results are shown in [reference needed]. Figure 8 .

[0111] Depend on Figure 8 As shown in A, the color of the MP / BNC / ACN film exposed to a trimethylamine atmosphere changes from red to bluish-gray, and the color gradually deepens with increasing anthocyanin content, indicating that the anthocyanin content in the indicator film has a certain influence on the sensitivity of the trimethylamine color response. Figure 8As shown in B, the color difference values ​​of the indicator film exhibit a good linear relationship when the trimethylamine concentration is between 2.5 and 10 μmol. Through fitting calculations, it can be concluded that... Figure 8 As shown in C, the limits of detection (LOD) for trimethylamine by MP / BNC / ACN0.5, MP / BNC / ACN1, and MP / BNC / ACN2 are 1.5, 2.0, and 2.2 μmol, respectively, all of which are much lower than the previously reported limit of detection for trimethylamine of 5.6 μmol (Valdez, Gupta, Lozano, & Mao. ForceSpun polydiacetylene nanofibers ascolorimetric sensor for food spoilage detection. Sensors and Actuators B-Chemical. 2019), thus indicating that the present invention has high trimethylamine reaction sensitivity.

[0112] 2.7 Anthocyanin release characteristics in indicator membranes

[0113] Immerse the prepared film (20mm × 20mm) in a test tube containing 50mL of deionized water. Then, transfer the test tube to a shaking incubator and set the shaking speed to 100r / min and the temperature to 25℃ in the dark. The percentage of anthocyanin released within the corresponding time period can be calculated based on the standard curve, using the following formula:

[0114]

[0115] Where M t M0 and M0 represent the amount of anthocyanins released at time t and the total amount of anthocyanins in the membrane, respectively. Measurements were taken every 2 hours. After each measurement, the test sample was immediately poured back into the original solution to maintain a constant total volume. The results are shown in [Figure number missing]. Figure 9 .

[0116] Depend on Figure 9 It can be seen that due to the covalent cross-linking of anthocyanins with myofibrillar proteins, the indicator membrane has a low anthocyanin release rate. The cumulative release rate of the three indicator membrane samples, MP / BNC / ACN0.5, MP / BNC / ACN1, and MP / BNC / ACN2, was 12.32% to 14.33% after 12 hours, and the release rate tended to stabilize with the extension of time, which was much lower than the 33.7% reported in the invention patent with patent number 202211608639.9.

[0117] 2.8 Determination of the light stability of the indicator film

[0118] The film was exposed to ultraviolet light using a UV analyzer at an irradiance of 400 μW / cm². 2The wavelength was 365 nm, and images and colorimetric values ​​of the thin film were recorded every 16 hours using an optical scanner and colorimeter. The thin film was also illuminated with a fluorescent lamp at a distance of 20 cm from the light source, and images and colorimetric values ​​were recorded every 5 days using an optical scanner and colorimeter. The measurement results are shown below. Figure 10 .

[0119] Depend on Figure 10 It can be seen that after 30 days of visible light irradiation and 96 hours of ultraviolet irradiation at 25℃, the MP / BNC / ACN1 and MP / BNC / ACN2 indicator film samples showed small ΔE and slight color changes, indicating that the MP / BNC / ACN1 and MP / BNC / ACN2 indicator films have high light stability.

[0120] 2.9 Monitoring the freshness of blunt snout bream using indicator membranes

[0121] The transmittance of the membrane was determined using a UV-Vis spectrophotometer in the range of 200–800 nm. The membrane was cut into rectangular strips of 10 mm × 30 mm, and the strips were placed in cuvettes for measurement. An empty cuvette served as a control. Six samples were measured for each membrane. The results are shown below. Figure 11 ;

[0122] The transmittance of indicator films reflects their light-blocking properties; low transmittance indicates high barrier properties, such as... Figure 11 As shown, the myofibrillar protein membrane (MP) exhibits low transmittance in the 200–320 nm range, indicating that the MP membrane itself has good UV blocking properties. This may be related to the fact that myofibrillar protein is rich in tyrosine and phenylalanine, which can absorb UV radiation. Therefore, using myofibrillar protein as the substrate for preparing the indicator membrane can help protect anthocyanins from UV degradation. Furthermore, when anthocyanins are added to the myofibrillar protein membrane, i.e., after covalent cross-linking of myofibrillar protein and anthocyanins via tyrosinase catalysis, the transmittance of the indicator membrane is much lower than that of the myofibrillar protein membrane (MP). Moreover, the transmittance of the indicator membrane decreases with increasing anthocyanin content, and it exhibits extremely low transmittance throughout the entire UV region. This is because the abundant aromatic rings in the anthocyanin structure can absorb UV-Vis radiation.

[0123] 2.10 Monitoring the freshness of blunt snout bream using indicator membranes

[0124] Fresh blunt snout bream were sealed in sterile petri dishes. A prepared smart indicator film (20mm × 20mm) was affixed to the inner surface of the lid, ensuring it did not contact the fish. The dishes were then stored at 4℃ for 10 days. Every two days, the image and colorimetric values ​​of the smart indicator film were recorded using a camera and a colorimeter. The results are shown below. Figure 9During this period, the changes in pH, volatile basic nitrogen content (TVB-N), total bacterial count (TVC), and thiobarbituric acid value (TBARS) of fish meat were measured according to the operating requirements of GB5009.237-2016 "Determination of pH value in food", GB5009.228-2016 "Determination of volatile basic nitrogen in food", GB4789.2-2016 "Microbiological examination of food - determination of total bacterial count", and GB5009.181-2016 "Determination of malondialdehyde in food". The results are shown in [the table below]. Figure 8 .

[0125] Depend on Figure 12 It was observed that the pH of the fish meat initially decreased and then increased during storage at 4℃. The total bacterial count, thiobarbituric acid value, and volatile basic nitrogen of the fish meat gradually increased, reaching 8.171g CFU / g, 1.08mg MDA / kg, and 25.02mg / 100g, respectively, on day eight. It is generally believed that an unpleasant rancid taste will occur when the thiobarbituric acid value exceeds 1.0mg MDA / kg. These values ​​significantly exceeded the International Committee for Microbiological Standards (ICMSF) food microbial limits of 10. 7 The concentration of 1g CFU / g and the upper limit of 20mg / 100g according to the Chinese national standard (GB2733-2015) indicate that the fish should not be consumed after 8 days. Figure 13 It can be seen that during the storage and spoilage process of fish, the colorimetric membrane generally changes from red to grayish-black, which can intuitively reflect the color change of fish from fresh to rotten. Among them, the MP / BNC / ACN1 indicator membrane is the most sensitive and accurate to changes in the freshness of fish. When the indicator membrane is red, it indicates that the fish is relatively fresh; when the membrane is light red, it indicates that the fish is in a slightly less fresh state; when the membrane is grayish-black, it indicates that the fish has obviously spoiled.

Claims

1. A method for preparing a food freshness indicator film by covalent cross-linking of myofibrillar protein and anthocyanins catalyzed by tyrosinase, characterized in that, Includes the following steps: S1. Prepare myofibrillar protein solution; S2. Add tyrosinase and anthocyanins to the myofibrillar protein solution, and conduct a continuous stirring reaction under ice-water bath conditions while exposed to air. After the reaction is completed, adjust the pH of the solution to obtain the film-forming solution. S3. Add bacterial nanocellulose and glycerol to the film-forming solution, stir at room temperature for 0.5-3 h, then filter to remove impurities, pour the filtrate into a petri dish, and then dry at 40-55ºC to obtain the food freshness indicator film.

2. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 1, characterized in that, The method for preparing the myofibrillar protein solution in step S1 is as follows: after thawing frozen fish paste, cut it into small pieces, then add distilled water at a ratio of 7-10:50 (material to liquid mass), and at the same time add 2%-3% sodium chloride based on the mass of fish paste. After homogenization, the myofibrillar protein solution is obtained.

3. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 2, characterized in that, The homogenization conditions are: homogenization speed of 8000-12000 r / min and homogenization time of 100-150 s.

4. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 1, characterized in that, In step S2, the continuous stirring reaction time is controlled to be 3-5 h, and the pH of the solution is adjusted to 3 after the reaction is completed.

5. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 1, characterized in that, In step S2, the concentration of added tyrosinase activity is controlled to be 20–35 U / mL.

6. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 1, characterized in that, In step S2, the amount of anthocyanin added is controlled to be 0.5% to 2% based on the mass of protein in the myofibrillar protein solution.

7. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 1, characterized in that, In step S3, the bacterial nanocellulose is added at a rate of 5% to 7% based on the mass of the protein in the myofibrillar protein solution.

8. The method for preparing a food freshness indicator film by tyrosinase-catalyzed covalent cross-linking of myofibrillar protein-anthocyanin as described in claim 1, characterized in that, In step S3, the amount of glycerol added is 40% based on the mass of the protein in the myofibrillar protein solution.

9. A food freshness indicator film, characterized in that, It is prepared by any one of the methods described in claims 1 to 8.

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