Time temperature indicator for determining whether a frozen food has been subjected to room temperature thawing and method of making and use thereof

Abstract

This invention discloses a time-temperature indicator for determining whether frozen food has undergone room temperature thawing, its preparation method, and its application. The time-temperature indicator of this invention comprises: a single-stranded DNA forming a stem-loop structure, with fluorescent and quenching groups modified at both ends respectively; an exonuclease that specifically cleaves linear single-stranded DNA; and a dispersing solvent; the single-stranded DNA forming the stem-loop structure has no dangling sequences at its 5' and 3' ends, and the fluorescent and quenching groups are close to each other, thus not producing fluorescence; in the dispersing solvent, the melting temperature T of the single-stranded DNA stem-loop is... m The temperature range is 0℃–25℃. The time-temperature indicator of this invention achieves irreversible fluorescence changes from freezing to room temperature by modifying a stem-loop structure of a single-stranded DNA sequence with fluorescent and quenching groups, as well as by the cleavage of linear single-stranded DNA by exonucleases. This allows for accurate determination of whether a frozen product has been thawed at room temperature.

Description

Technical Field

[0001] This invention relates to the field of fluorescence sensor technology. More specifically, it relates to a time-temperature indicator for determining whether frozen food has undergone room temperature thawing, its preparation method, and its application. Background Technology

[0002] Cold chain food undergoes multiple stages from production to consumption, and temperature control at each stage is crucial to product quality (Muhammad Shahar Yar, Isaiah Henry Ibeogu, et al. Trends in Food Science & Technology, 2025, 163: 105128). For foods requiring frozen transport, repeated freezing and thawing significantly reduces their nutritional value (Muhammad Shahar Yar, Isaiah Henry Ibeogu, et al. Food Chemistry, 2025, 480: 143906). However, ensuring that the temperature of frozen food does not exceed the prescribed temperature throughout the entire cold chain process presents significant challenges for temperature monitoring. Consumers often lack effective means of informed judgment to determine whether frozen food has been thawed (Shaodong Wang, Xinghai Liu, et al. Packing Technology and Science. 2015, 28: 839-867). Therefore, developing time-temperature indicators for frozen foods to determine whether they have been thawed and the cumulative thaw time is of great significance for the quality supervision of frozen foods and for helping consumers understand the storage experience of frozen foods.

[0003] Current time-temperature indicators (TTIs) are mainly based on diffusion-based (Lam Tan Hao, Minkyung Lee, et al. ACS Omega 2021, 6: 8598-8604), chemical-based (Muhammad Shahar Yar, Isaiah Henry Ibeogu, et al. Food Chemistry, 2025, 480: 143906), microbial-based (Xiaoshuan Zhang, Gege Sun, et al. Trends in Food Science & Technology, 2016, 51: 12-23), and enzymatic (Jhao Rong Jhuang, Shih Bin Lin, et al. Food Packaging and Shelf Life, 2020, 23: 100436). Among these, diffusion-based TTIs primarily rely on the diffusion process of colored substances, the rate of which is affected by temperature. The exudation of colored substances and the aging of porous materials can adversely affect the safety and accuracy of diffusion-based TTIs (Shaodong Wang, Xinghai Liu, et al. Packaging Technology and Science, 2015, 28: 839-867). Chemical-based methods, relying primarily on temperature-dependent chemical reactions, generally suffer from uncontrollable responses after activation, leading to misjudgments regarding the time elapsed at room temperature (Muhammad Shahar Yar, Isaiah Henry Ibeogu, et al. FoodChemistry, 2025, 480: 143906). Enzymatic and microbial methods, due to their typically requirement of aqueous solutions and generally operating temperatures above 0°C, are difficult to apply to quality control in frozen food transportation (Muhammad Shahar Yar, Isaiah Henry Ibeogu, et al. Trends in Food Science&Technology, 2025, 163: 105128).

[0004] Therefore, the present invention aims to develop a time-temperature indicator for determining whether frozen food has been stored at room temperature and for accurately distinguishing how long it has been stored at room temperature. Summary of the Invention

[0005] One objective of this invention is to provide a time-temperature indicator for determining whether frozen food has undergone room temperature thawing, thereby solving the problem that existing time-temperature indicators still react slowly under low-temperature conditions after being turned on, which is not conducive to accurately reflecting whether frozen food has undergone room temperature thawing. A second objective of this invention is to provide a method for preparing the aforementioned time-temperature indicator.

[0006] A third objective of this invention is to provide an application of the aforementioned time-temperature indicator in the quality monitoring of frozen foods.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a time-temperature indicator for determining whether frozen food has undergone room temperature thawing, the time-temperature indicator comprising: a stem-loop structure formed by single-stranded DNA with fluorescent groups and quenching groups modified at both ends respectively, an exonuclease that specifically cleaves linear single-stranded DNA, and a dispersing solvent; The single-stranded DNA that forms the stem-loop structure does not have dangling sequences at the 5' and 3' ends, and the fluorescent group and quenching group are close to each other, so they do not produce fluorescence; To ensure better detection results, the melting temperature T of the stem-loop of single-stranded DNA in the dispersion solvent is set. m The temperature range is 0℃-25℃.

[0008] In the time-temperature indicator of this invention, the single-stranded DNA should have a suitable base sequence, forming a stem-loop structure at temperatures below 0°C. During stem-loop formation, the 5' and 3' ends of the single-stranded DNA must not have dangling sequences to avoid the action of exonucleases that specifically cleave linear single-stranded DNA. At this point, the fluorescent and quenching groups at both ends of the single-stranded DNA approach each other, thus quenching the fluorescence. When the time-temperature indicator is at room temperature, the stem-loop structure of the single-stranded DNA unwinds, the fluorescent groups move away from the quenching groups, and the fluorescence recovers. Simultaneously, the unwinded linear single-stranded DNA is cleaved by exonucleases that specifically cleave linear single-stranded DNA, completely destroying the single-stranded structure. The stem-loop structure cannot be restored under freezing conditions, and even if the time-temperature indicator is placed below 0°C again, the fluorescence will not weaken. The time-temperature indicator of this invention achieves irreversible fluorescence changes from freezing to room temperature, thereby enabling accurate differentiation of whether frozen foods have undergone room temperature storage, providing a new technology for food quality supervision.

[0009] Furthermore, exemplary, the nucleotide sequence of the single-stranded DNA forming the stem-loop structure may be: 5'-ATCATAATTATTGTTTTTTTTTTTTTTTACTATTTTTTTGAT-3', as shown in SEQ ID No: 1; 5'-TTCTATTATAATTTTTTTTTTTTTTTTTTTTATATTTTATTAGAA-3', as shown in SEQ ID No: 2; 5'-AACGTTTTATTTTTTTTTTTTTTTTTTTTTTTTTTAAATTTTTACGTT-3', as shown in SEQ ID No: 3.

[0010] Furthermore, in the dispersing solvent, the exonuclease that specifically cleaves linear single-stranded DNA refers to an exonuclease capable only of cleaving linear single-stranded DNA, and lacking cleavage activity against double-stranded DNA regions with stem-loop structures and circular single-stranded DNA regions. For example, the exonuclease that specifically cleaves linear single-stranded DNA is selected from exonuclease I (Exo I), exonuclease VII (Exo VII), exonuclease T (Exo T), and RecJ. f One of the exonucleases.

[0011] Furthermore, the dispersion solvent is composed of a buffer solution and an organic solvent. By adding a certain proportion of organic solvent to the buffer solution to lower its freezing point, it is ensured that it will not freeze under sub-zero storage conditions. That is, the dispersion solvent contains a buffer solution and an organic solvent used to lower the freezing point of the buffer solution, thereby avoiding the impact of repeated freeze-thaw cycles on DNA and exonuclease activity. Moreover, the added organic solvent itself should not damage the DNA structure and exonuclease.

[0012] Furthermore, the buffer solution is one of Tris-HCl buffer solution, PBS buffer solution, and NEBuffer.

[0013] Furthermore, the organic solvent is selected from one of glycerol, mannitol, sorbitol, inositol, thiols, and polyethylene glycol.

[0014] Furthermore, according to a specific embodiment of the present invention, the dispersing solvent is composed of PBS buffer solution and glycerol, wherein the mass ratio of PBS buffer solution to glycerol is 1:1.

[0015] Furthermore, the fluorescent group is selected from one of Texas Red, FAM (Fluorescent Alcohol), and Cy5 (Cyanide 5). The quenching group is selected from one of black hole quencher 1 (BHQ1), black hole quencher 2 (BHQ2), black hole quencher 3 (BHQ3), small ditch quencher (MGB), and Dabcyl.

[0016] Secondly, the present invention provides a method for preparing a time-temperature indicator for determining whether frozen food has undergone room temperature thawing, the preparation method comprising: (1) Disperse the single-stranded DNA that can form a stem-loop structure with fluorescent groups and quenching groups at both ends in a buffer solution, anneal at 95°C, then cool to room temperature, and centrifuge to bring all the solution to the bottom of the container; (2) Add the dispersing solvent, mix well, and freeze in a refrigerator; (3) Take the exonuclease that specifically cuts linear single-stranded DNA and add it to the mixture in step (2), aliquot it, and freeze it in a refrigerator.

[0017] Furthermore, in step (2), the freezing time shall be no less than 30 minutes.

[0018] Thirdly, the present invention also protects the application of the time-temperature indicator for determining whether frozen food has undergone room temperature thawing in the quality monitoring of frozen food.

[0019] Specifically, the time-temperature indicator serves as an indicator by observing its color change under ultraviolet light. Furthermore, the longer the time is left at room temperature, the deeper the fluorescence of the time-temperature indicator becomes.

[0020] The beneficial effects of this invention are as follows: The time-temperature indicator of this invention achieves irreversible fluorescence changes from freezing to room temperature by modifying a stem-loop structure of a single-stranded DNA sequence with fluorescent and quenching groups to undergo conformational changes at freezing and room temperature, as well as by exonuclease cleavage of linear single-stranded DNA. This allows for accurate determination of whether a frozen product has undergone a room temperature thawing process.

[0021] The exonuclease selected in this invention only acts on linear single-stranded DNA in the dispersion solvent, thus thermodynamically avoiding the problem that the response process continues slowly after being returned to freezing conditions after being started at room temperature. It can roughly determine the time that frozen food has been placed at room temperature. For example, by preparing a colorimetric card, the time placed at room temperature can be roughly determined by comparing it with the colorimetric card.

[0022] This invention can adjust the conformational change temperature of the DNA sequence by changing the single-stranded DNA nucleic acid sequence and the dispersion solvent, that is, changing the melting temperature T of the stem-loop of the single-stranded DNA in the dispersion solvent. m Besides determining freezing and room temperature, it can also be applied to other scenarios. Attached Figure Description

[0023] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0024] Figure 1This diagram illustrates the mechanism of the time-temperature indicator provided by the present invention in an embodiment of the invention.

[0025] Figure 2 The fluorescence intensity of BHQ2-DNA-Texas Red in the dispersion solvent is shown as a function of temperature.

[0026] Figure 3 The fluorescence intensity of BHQ2-DNA-Texas Red in the dispersion solvent as a function of temperature is shown in three heating and cooling cycles.

[0027] Figure 4a The curves showing the change in fluorescence intensity of the time-temperature indicator over time at two -10°C locations are shown.

[0028] Figure 4b The curve showing the change in fluorescence intensity of the time-temperature indicator at 25°C over time is shown.

[0029] Figure 5a The curves showing the change in fluorescence intensity of the time-temperature indicator at -10°C over time after 5 days of frozen storage in a refrigerator are shown.

[0030] Figure 5b The curve showing the change in fluorescence intensity of the time-temperature indicator at 25°C over time after 5 days of frozen storage in a refrigerator is shown.

[0031] Figure 6 The color differences of time-temperature indicators stored under different conditions in miniature NMR tubes are shown in the solution under 365 nm UV light excitation.

[0032] Figure 7a The color difference of the solution of the time-temperature indicator in the miniature NMR tube stored at room temperature for different times is shown under 365nm UV excitation.

[0033] Figure 7b A standard colorimetric card is shown, plotted based on the color differences of solutions containing time-temperature indicators stored at room temperature for different times under 365nm UV excitation.

[0034] Figure 8 The differences in solution fluorescence of time-temperature indicators packaged with salmon bones under different storage conditions are shown under 365 nm UV light excitation. Detailed Implementation

[0035] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0036] This invention provides a time-temperature indicator for determining whether frozen food has undergone room temperature thawing. This indicator utilizes the changes in DNA conformation caused by temperature changes and the specific cleavage of linear single-stranded DNA by exonucleases to achieve an irreversible change in fluorescence intensity from freezing conditions to room temperature conditions. Figure 1 As shown, the exonuclease that specifically cuts linear single-stranded DNA can only cut linear single-stranded DNA. When DNA forms a stem-loop structure under freezing conditions, it does not contain a linear single-stranded portion and is not affected by the exonuclease. At this time, the fluorescent group is close to the quenching group and the fluorescence is quenched. When at room temperature, the DNA stem-loop structure opens, exposing the linear single strand. Without the action of the exonuclease, the stem-loop structure can still be restored after returning to the frozen state, and the fluorescence is quenched. Thus, it is impossible to determine whether the food has undergone room temperature thawing. However, in this invention, because it contains an exonuclease that specifically cuts linear single-stranded DNA, the linear single-stranded DNA will be affected by the exonuclease at room temperature and cut. The single-stranded structure is destroyed and cannot be restored under freezing conditions. The fluorescent group is far away from the quenching group, and the fluorescence will not weaken. Therefore, it is possible to accurately determine whether frozen food has undergone room temperature thawing.

[0037] The following detailed description is based on specific embodiments.

[0038] Example 1 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 3' end, and named BHQ2-DNA-Texas Red.

[0039] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0040] 3) Anneal the 32.4 μM of the above DNA dispersed in 1×PBS at 95 °C, slowly cool to room temperature, centrifuge, and then dilute with the dispersion solvent in a cuvette to a final concentration of 200 nM. With the excitation wavelength fixed at 561 nm, measure the fluorescence spectra at different temperatures from -10 °C to 50 °C. Plot the temperature response curve of BHQ2-DNA-Texas Red with temperature on the x-axis and fluorescence intensity at 612.0 nm on the y-axis.

[0041] The temperature response curve obtained from the above steps is as follows: Figure 2 As shown in the figure. It can be seen from the figure that in the dispersion solvent, the T of DNA... m When moved to around 15°C, a stem-ring structure forms at sub-zero temperatures, quenching the fluorescence. At room temperature, the stem-ring structure opens, enhancing the fluorescence.

[0042] Example 2 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 5' end, and named BHQ2-DNA-Texas Red.

[0043] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0044] 3) Anneal the 32.4 μM of the above DNA dispersed in 1×PBS at 95 °C, slowly cool to room temperature, centrifuge, and then dilute with the dispersion solvent in a cuvette to a final concentration of 200 nM. With the excitation wavelength fixed at 561 nm, measure the fluorescence spectra at -10 °C and 25 °C three times. Plot the fluorescence intensity changes of BHQ2-DNA-Texas Red under three temperature cycles, with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0045] The fluorescence intensity changes in the three heating and cooling cycles obtained from the above steps are as follows: Figure 3 As shown, the fluorescence intensity differences between the three -10°C and three 25°C cycles were not significant, indicating that the conformational changes of the DNA sequence itself are reversible.

[0046] Example 3 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 5' end, and named BHQ2-DNA-Texas Red.

[0047] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0048] 3) The 32.4 μM of the above DNA dispersed in 1×PBS was annealed at 95 °C, slowly cooled to room temperature, centrifuged, and then diluted with the dispersion solvent in a cuvette to a final concentration of 200 nM. It was then frozen at -10 °C for 30 min.

[0049] 4) Add 5 μL of exonuclease VII (10 U / μL) frozen at -20°C to the above cuvette, mix well, stabilize at -10°C for 30 min, fix the excitation wavelength at 561 nm, and plot the change of fluorescence intensity at -10°C with storage time as the first time with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0050] 5) Heat the system to 25°C and stabilize for 360 min. Fix the excitation wavelength at 561 nm. Plot the change in fluorescence intensity at 25°C with storage time, with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0051] 6) Cool the system to -10°C and stabilize for 30 min. Fix the excitation wavelength at 561 nm. Plot the change in fluorescence intensity at -10°C with storage time, with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0052] The fluorescence intensity versus time response curves obtained at different temperatures through the above steps are shown below. Figure 4a and Figure 4b As shown. From Figure 4a and Figure 4bIt can be seen that the fluorescence intensity stabilized at a very low value at the first -10°C, indicating that the DNA was in a stem-loop structure and that exonuclease VII did not cleave the DNA. Due to the subsequent stabilization process at room temperature (25°C), the DNA stem-loop structure opened, forming linear single strands. These strands were then cleaved by exonuclease VII, and the fluorophore and quencher groups were cleaved and released into the dispersion solvent. The fluorescence intensity gradually increased with stabilization time, eventually reaching equilibrium. Upon returning to -10°C, because the primary structure of the DNA was destroyed, a stem-loop structure could no longer form, and the quencher groups could not approach the fluorophore, resulting in a consistently high fluorescence intensity. The difference in fluorescence intensity between the two -10°C measurements demonstrates that exonuclease VII makes the change in fluorescence intensity with temperature irreversible.

[0053] Example 4 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 5' end, and named BHQ2-DNA-Texas Red.

[0054] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0055] 3) Anneal the 32.4 μM of the above DNA dispersed in 1×PBS at 95 °C, slowly cool to room temperature, dilute with dispersion solvent in centrifuge tubes to a final concentration of 200 nM, and then freeze at -20 °C for 30 min.

[0056] 4) Add 5 μL of exonuclease VII (10 U / μL) frozen at -20°C to the centrifuge tube above, mix well, and store at -20°C for 5 days.

[0057] 5) Remove the centrifuge tube and transfer the solution to a cuvette that has been stabilized at -10°C for 30 min in a fluorescence spectrometer. Then stabilize it at -10°C for 60 min, fix the excitation wavelength at 561 nm, and plot the change in fluorescence intensity at -10°C with storage time, with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0058] 6) Heat the system to 25°C and stabilize for 300 min. Fix the excitation wavelength at 561 nm. Plot the change in fluorescence intensity at 25°C with storage time, with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0059] 7) Cool the system to -10°C and stabilize for 60 min. Fix the excitation wavelength at 561 nm. Plot the change in fluorescence intensity at -10°C with storage time, with the fluorescence intensity at 612.0 nm of the emission spectrum as the ordinate.

[0060] The fluorescence intensity versus time response curves obtained at different temperatures through the above steps are shown below. Figure 5a and Figure 5b As shown, the fluorescence intensity change curve over time after 5 days of refrigerator storage is similar to the curve in Example 3, indicating that 5 days of refrigerator storage does not affect the activity of the prepared time-temperature indicator.

[0061] Example 5 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 5' end, and named BHQ2-DNA-Texas Red.

[0062] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0063] 3) Anneal the 32.4 μM of the above DNA dispersed in 1×PBS at 95 °C, slowly cool to room temperature, dilute with dispersion solvent in centrifuge tubes to a final concentration of 200 nM, and then freeze at -20 °C for 30 min.

[0064] 4) Add 5 μL of exonuclease VII (10 U / μL) frozen at -20°C to the centrifuge tube above, mix well, and store at -20°C for 30 min.

[0065] 5) Cut the NMR tube with a glass cutter into two smaller NMR tubes, and seal both ends with NMR tube caps. Transfer 200 μL of the solution from the centrifuge tube to each of the smaller NMR tubes. Store one NMR tube frozen at -20°C and the other at room temperature. After 5 hours, remove the smaller NMR tubes and irradiate them with a 365 nm UV lamp to observe the color difference.

[0066] The color differences in the small NMR tube obtained by the above steps are as follows: Figure 6 As shown, under 365nm UV excitation, the solution in the miniature NMR tube at room temperature turns pink, while the solution in the miniature NMR tube frozen at -20°C does not turn pink. This indicates that a small amount of the time-temperature indicator can be placed in the miniature NMR tube to reduce the amount used and to be easily applied to the surface of frozen food. The color change observed under 365nm UV light can then differentiate between room temperature and frozen storage conditions.

[0067] Example 6 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 5' end, and named BHQ2-DNA-Texas Red.

[0068] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0069] 3) Anneal the 32.4 μM of the above DNA dispersed in 1×PBS at 95 °C, slowly cool to room temperature, dilute with dispersion solvent in centrifuge tubes to a final concentration of 200 nM, and then freeze at -20 °C for 30 min.

[0070] 4) Add 5 μL of exonuclease VII (10 U / μL) frozen at -20°C to the centrifuge tube above, mix well, and store at -20°C for 30 min.

[0071] 5) Cut the NMR tubes into five smaller NMR tubes using a glass cutter, and cap both ends with NMR tube caps. Every hour, transfer 200 μL of the solution from the centrifuge tube to one of the smaller NMR tubes and store at room temperature. After each transfer, return the centrifuge tube to -20°C. After filling the fifth NMR tube, place all five NMR tubes together at -20°C for half an hour. Then, remove them together and irradiate them with a 365 nm UV lamp to observe the color differences at different room temperatures after storage time.

[0072] 6) Draw a standard color chart based on the obtained color differences to reflect the room temperature storage time corresponding to different colors.

[0073] The color differences in the five small NMR tubes obtained by the above steps are as follows: Figure 7a As shown, the longer the storage time at room temperature, the deeper the pink color of the solution, indicating that the time-temperature indicator can distinguish between different storage times at room temperature. The resulting standard colorimetric card is shown below. Figure 7b As shown.

[0074] Example 7 1) Design and prepare the following DNA sequences: 5'-ATCATAATTATTGTTTTTTTTTTTTTACTATTTTTTTGAT-3' (SEQ ID No: 1); According to predictions, under 1×PBS, the T of DNA m The temperature was 22.2℃. The DNA strand was modified with the fluorescent group Texas Red at the 3' end and the quenching group BHQ2 at the 5' end, and named BHQ2-DNA-Texas Red.

[0075] 2) Prepare 1×PBS buffer (1.06mM potassium dihydrogen phosphate, 2.97mM disodium hydrogen phosphate heptahydrate, 155.17mM sodium chloride, pH=7.20) precisely, and then mix the 1×PBS with glycerol in equal mass to obtain the dispersion solvent.

[0076] 3) Anneal the 32.4 μM of the above DNA dispersed in 1×PBS at 95 °C, slowly cool to room temperature, dilute with dispersion solvent in centrifuge tubes to a final concentration of 200 nM, and then freeze at -20 °C for 30 min.

[0077] 4) Add 5 μL of exonuclease VII (10 U / μL) frozen at -20°C to the centrifuge tube above, mix well, and store at -20°C for 30 min.

[0078] 5) Cut the NMR tubes into two smaller NMR tubes using a glass cutter, and seal both ends with NMR tube caps. Transfer 200 μL of the solution from each centrifuge tube to the smaller NMR tubes, and seal each tube with salmon bones in a vacuum-sealed bag. Store one set at -20°C and the other at room temperature for 5 hours, then return to -20°C for 30 minutes. Afterward, remove both sets and observe the color difference under a 365 nm UV lamp.

[0079] The color differences obtained from the above steps are as follows: Figure 8 As shown, the salmon bone group that had been thawed at room temperature showed a pink color in the time-temperature indicator solution, while the salmon bone group that had been kept frozen did not show a noticeable pink color in the time-temperature indicator solution. This indicates that the time-temperature indicator can help distinguish whether frozen food has been thawed at room temperature.

[0080] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A time-temperature indicator for determining whether frozen food has undergone room temperature thawing, characterized in that, The time-temperature indicator comprises: a stem-loop structure-forming single-stranded DNA with fluorescent and quenching groups modified at both ends, an exonuclease that specifically cleaves linear single-stranded DNA, and a dispersing solvent; The single-stranded DNA that forms the stem-loop structure does not have dangling sequences at the 5' and 3' ends, and the fluorescent group and quenching group are close to each other, so no fluorescence is produced; In the dispersion solvent, the melting temperature T of the stem-loop of single-stranded DNA is... m The temperature range is 0℃-25℃.

2. The time-temperature indicator according to claim 1, characterized in that, The nucleotide sequence of the single-stranded DNA forming the stem-loop structure is shown in SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No:

3.

3. The time-temperature indicator according to claim 1, characterized in that, The exonuclease that specifically cleaves linear single-stranded DNA is selected from exonuclease I, exonuclease VII, exonuclease T, and RecJ. f One of the exonucleases.

4. The time-temperature indicator according to claim 1, characterized in that, The dispersion solvent consists of a buffer solution and an organic solvent; Preferably, the buffer solvent is selected from one of Tris-HCl buffer solution, PBS buffer solution, and NEBuffer; Preferably, the organic solvent is selected from one of glycerol, mannitol, sorbitol, inositol, thiols, and polyethylene glycol.

5. The time-temperature indicator according to claim 4, characterized in that, The dispersion solvent consists of PBS buffer solution and glycerol; Preferably, the mass ratio of the PBS buffer solvent to glycerol is 1:

1.

6. The time-temperature indicator according to claim 1, characterized in that, The fluorescent group is selected from one of Texas Red, Carboxyfluorescein, and Anthocyanin Dye 5; The quenching group is selected from one of black hole quencher 1, black hole quencher 2, black hole quencher 3, small ditch quencher, and Dabcyl.

7. The method for preparing the time-temperature indicator for determining whether frozen food has undergone room temperature thawing as described in any one of claims 1-6, characterized in that, The preparation method includes: (1) Disperse single-stranded DNA with fluorescent and quenching groups at both ends that can form stem-loop structures in buffer solution, anneal at 95°C, then cool to room temperature and centrifuge; (2) Add the dispersing solvent, mix well, and freeze in a refrigerator; (3) Take the exonuclease that specifically cuts linear single-stranded DNA and add it to the mixture in step (2), aliquot it, and freeze it in a refrigerator.

8. The preparation method according to claim 6, characterized in that, In step (2), the freezing time shall be no less than 30 minutes.

9. The application of the time-temperature indicator for determining whether frozen food has undergone room temperature thawing as described in any one of claims 1-6 in the quality monitoring of frozen food.

10. The application according to claim 9, characterized in that, The time-temperature indicator serves as an indicator by observing its color change under ultraviolet conditions.

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