A nano-enzyme time-temperature indicator and a preparation method and application thereof

Abstract

The application belongs to the technical field of time-temperature indicator, and provides a nano-enzyme time-temperature indicator, a preparation method and application thereof, and the preparation method comprises the following steps: placing a nano-enzyme solution in an external sealing device; placing a guaiacol solution in an internal sealing device; when in use, extruding and shaking the internal sealing device to fully mix the liquids in the external sealing device and the internal sealing device. The nano-enzyme time-temperature indicator is applied to indicate the shelf life of food. The application adopts nano-enzyme with laccase activity to construct a time-temperature indicator model, has the advantages of good heat resistance, stable storage performance, can accurately indicate the change of time and temperature in the environment, and is convenient to use. The nano-enzyme time-temperature indicator prepared by the application can accurately indicate at 15-75 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of time-temperature indicator technology, and particularly relates to a nanozyme time-temperature indicator, its preparation method, and its application. Background Technology

[0002] Fruits, vegetables, meats, and seafood often lack freshness indicators. Improper storage temperatures or prolonged storage can affect their value and, in severe cases, cause foodborne illnesses, impacting consumer health. While snacks and canned goods have expiration dates, these are predicted using formulas based on existing laboratory data, lacking analysis of the specific time and temperature conditions during actual transportation and storage. Extreme conditions during transport and storage, such as prolonged transit times or malfunctioning vehicle temperature control systems, can drastically alter their internal physicochemical properties, leading to changes in shelf life. The actual shelf life will differ from the expiration date on the product packaging. Time and temperature are crucial to food freshness; therefore, it is necessary to develop smart packaging labels with time and temperature indicators to track changes in food freshness.

[0003] Traditional natural enzyme TTIs (time-temperature indicators) are prone to denaturation under extreme conditions due to their composition, limiting their applicable temperature range and affecting label accuracy. The development of time-temperature indicators requires a wider temperature tracking range to adapt to sudden environmental changes. Therefore, we propose a nanoenzyme time-temperature indicator, its preparation method, and its applications.

[0004] AMP-Cu is a typical nanozyme synthesized by coordinating adenosine monophosphate with copper and exhibits good laccase activity. Compared to laccase, this enzyme has the advantages of low cost and simple preparation, and also shows excellent stability under different storage conditions of temperature, pH, and salt concentration. Summary of the Invention

[0005] The purpose of this invention is to provide a nanozyme time-temperature indicator, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a nanozyme time-temperature indicator includes the following steps:

[0008] Step S1: Disperse the nanozyme with laccase activity in a buffer solution and place it in an externally sealed device;

[0009] Step S2: Place the guaiacol solution in the internal sealing device;

[0010] Step S3: When using, squeeze and shake the internal sealing device to fully mix the liquid in the external sealing device and the internal sealing device.

[0011] Furthermore, in step S1, the concentration of the nanoenzyme dispersion with laccase activity is 1–50 μg / mL, the concentration of the sodium tetraborate buffer solution is 50 mM, the pH is 9–10, and the volume is 4.8 ml.

[0012] Furthermore, in step S2, the concentration of the guaiacol solution is 10–100 μM and the volume is 0.8 ml.

[0013] Furthermore, the external sealing device is a transparent sealing bag, and the internal sealing device is a light-proof sealing bag.

[0014] A nanozyme time-temperature indicator prepared by the above method.

[0015] The aforementioned nanozyme time-temperature indicator is used to indicate the shelf life of food; the indication is provided by a color change in the nanozyme time-temperature indicator.

[0016] Furthermore, the color change refers to the color changing from transparent to yellow and then to brown.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. This invention uses nanoenzymes with laccase activity to construct a time-temperature indicator model, which has the advantages of good heat resistance and stable storage performance, and can accurately indicate changes in time and temperature in the environment; at the same time, it is ready to use and convenient to operate.

[0019] 2. The nanozyme time-temperature indicator prepared by this invention can provide accurate indication within the range of 15-75℃ (R 2 >0.9), which provides a wider temperature tracking range compared to existing time and temperature indicators. Attached Figure Description

[0020] Figure 1 This describes the preparation process of the nanozyme time-temperature indicator in this invention.

[0021] Figure 2 This is a flowchart illustrating the preparation and matching process of the nanozyme time-temperature indicator in this invention.

[0022] Figure 3 This is the absorbance fitting curve for the present invention at 15-75℃.

[0023] Figure 4 This is a color change diagram of the sealing device of the present invention under constant temperature.

[0024] Figure 5This is the absorbance variation curve of the sealing device of the present invention.

[0025] Figure 6 The fitted curve is for calculating the activation energy of the 10 μg / ml nanozyme AMP-Cu of this invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] An embodiment of the present invention provides a method for preparing a nanozyme-AMP-Cu time-temperature indicator, comprising the following steps:

[0029] Step S1: Dissolve AMP-Cu in sodium tetraborate buffer solution and place it in an externally sealed device;

[0030] Step S2: Place the guaiacol solution in the internal sealing device;

[0031] Step S3: When using, squeeze and shake the internal sealing device to fully mix the liquid in the external sealing device and the internal sealing device.

[0032] In a preferred embodiment of the present invention, in step S1, the sodium tetraborate buffer solution has a concentration of 50 mM, a pH of 9-10, and a volume of 4.8 ml.

[0033] In a preferred embodiment of the present invention, in step S2, the concentration of the guaiacol solution is 10 μM and the volume is 0.8 ml.

[0034] In this embodiment of the invention, the user can choose an appropriate dosage according to their own needs.

[0035] In a preferred embodiment of the present invention, the external sealing device is a transparent sealing bag, and the internal sealing device is a light-proof sealing bag.

[0036] A nanozyme time-temperature indicator prepared by the above method.

[0037] The aforementioned nanozyme time-temperature indicator is used to indicate the shelf life of food; the indication is provided by a color change in the nanozyme time-temperature indicator.

[0038] In a preferred embodiment of the present invention, the color change refers to the color changing from transparent to yellow and then to brown.

[0039] Example 1, see Figure 2 The method for matching nanozyme time-temperature indicators with food indicators is as follows: Nanozyme and guaiacol solutions are prepared according to a specific concentration ratio, mixed thoroughly, and their color changes are observed and characterized. Based on the time and color change indicators, a fitting curve is constructed, a kinetic model is built, and the corresponding activation energy is obtained according to the Arrhenius equation. According to the matching principle, if the difference between the two is less than 25 kJ / mol, it is determined whether a match is possible; otherwise, the reaction is adjusted (by changing the enzyme concentration, adding an inhibitor, or an accelerator).

[0040] AMP-Cu was dissolved in sodium tetraborate buffer and placed in an outer sealed device, while guaiacol was placed in an inner sealed device.

[0041] like Figure 3 As shown, the reaction system exhibits significant color changes when reacted at temperatures ranging from 15 to 75°C. These changes follow a zero-order reaction, and the higher the temperature, the faster the color change. The slope of the fitted curve is also greater.

[0042] like Figure 4 As shown, when the reaction system is reacted at 15-37℃, obvious color changes are observed, namely colorless, yellow, and brown. After reaching brown, the color no longer changes significantly, thus achieving the purpose of visualizing the shelf life of food.

[0043] A. Color change measurement: The reaction system was placed at 15 / 27 / 37℃ respectively, and its absorbance was measured with an ELISA reader at regular intervals.

[0044] B. Determination of kinetic parameters: The absorbance curves as a function of time under constant temperature conditions of 15 / 27 / 37℃ were plotted respectively, and the zero-order reaction equation (1) was used for fitting to calculate the corresponding k. 15℃ k 27℃ k 37℃ Calculate the corresponding activation energy according to the Arrhenius equation (2).

[0045] y = kt + y0 (1)

[0046]

[0047] In formula (1), t is time (h), and y is the absorbance at time t. The calculation results are shown in Table 1. In formula (2), R is a constant (8.314 × 10⁻⁶). -3 kJ mol -1 K -1 T is the temperature (K), and the calculation results are shown in Table 2; the reaction system is at 15-75℃, and k and R are fitted according to the zero-order reaction equation. 2 As shown in Table 3.

[0048] Table 1 shows the absorbance-time fitting results of the time-temperature indicator.

[0049]

[0050] The absorbance curve of the reaction system is as follows Figure 5 As shown, in the initial stage of the reaction, the color change follows the zero-order reaction equation, and the reaction stabilizes after reaching the endpoint. Moreover, the higher the temperature, the faster the color change.

[0051] Table 2 shows the activation energy fitting results of the time-temperature indicator.

[0052]

[0053] According to formula (2), with the reciprocal of temperature as the abscissa and the logarithm of the slope of absorbance change as the ordinate, the fitted curve is as follows: Figure 6 As shown, the activation energy is 43.48915 kJ / mol.

[0054] Table 3. Absorbance variation trend of AMP-Cu at 15-75℃

[0055]

[0056] It can be seen that R 2 The value >0.9 indicates that the AMP-Cu time-temperature indicator prepared by this invention has excellent thermal stability and can provide accurate indication at 15-75℃.

[0057] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a nanozyme time-temperature indicator, characterized in that, Includes the following steps: Step S1: Disperse the nanozyme with laccase activity in a buffer solution and place it in an externally sealed device; Step S2: Place the guaiacol solution in the internal sealing device; Step S3: When using, squeeze and shake the internal sealing device to fully mix the liquid in the external sealing device and the internal sealing device.

2. The method for preparing the nanozyme time-temperature indicator according to claim 1, characterized in that, In step S1, the concentration of the nanoenzyme dispersion with laccase activity is 1–50 μg / mL, the concentration of the sodium tetraborate buffer solution is 50 mM, the pH is 9–10, and the volume is 4.8 ml.

3. The method for preparing the nanozyme time-temperature indicator according to claim 1, characterized in that, In step S2, the concentration of the guaiacol solution is 10-100 μM and the volume is 0.8 ml.

4. The method for preparing the nanozyme time-temperature indicator according to claim 1, characterized in that, The external sealing device is a transparent sealing bag, and the internal sealing device is a light-proof sealing bag.

5. A nanozyme time-temperature indicator prepared by the method according to any one of claims 1-4.

6. A nanozyme time-temperature indicator according to claim 5 is used to indicate the shelf life of food; the indication is made by a color change of the nanozyme time-temperature indicator.

7. The application according to claim 6, characterized in that, The color change refers to the color changing from transparent to yellow and then to brown.

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