NL2024639B1

NL2024639B1 - Puncturing neutral protease type time-temperature indicating device

Info

Publication number: NL2024639B1
Application number: NL2024639A
Authority: NL
Inventors: Xie Jing; Huang Wenbo; Wang Jinfeng
Original assignee: Univ Shanghai Ocean
Priority date: 2019-01-20
Filing date: 2020-01-09
Publication date: 2021-02-23
Also published as: CN109706216A; NL2024639A; CN109706216B

Abstract

The disclosure provides a puncturing neutral protease type time-temperature indicating device, composed of an indicator and a color card. The indicator includes a container, which contains a color development reagent, a substrate and an enzyme, wherein the color development reagent is a Folin-Phenol reagent with a concentration of 2 mg/L, the substrate is a casein solution with a mass fraction of 2%, which is composed of a casein, 0.1 mol/L of a NaOH solution and a distilled water, and the enzyme is a neutral protease solution with a concentration of 2 mg/L. The present disclosure provides a color development system with enzyme-substrate reaction designed according to the method of determining protein concentration with Folin-Phenol, wherein the irreversible color changes can occur over time or temperature fluctuations to result in visible color changes, so that the purpose of intuitively indicating the quality change of the frozen food is realized.

Description

PUNCTURING NEUTRAL PROTEASE TYPE TIME-TEMPERATURE INDICATING DEVICE

FIELD OF TECHNOLOGY The present disclosure relates to the technical field of cold chain logistics, in particular, to a time-temperature indicating device capable of monitoring the quality status and freshness of food in the cold chain logistics process in real time.

BACKGROUND ART Fresh aquatic food requires a low temperature environment during storage and transportation. Fluctuations in storage temperature will have an important impact on the safety of such food. To ensure the safety of food, the storage temperature of food has to be effectively monitored. Thus, the principle of least-shelf-life/first-out (LSFO) can be implemented based on the actual quality of the food. Time-temperature indicating device (TTI) is a device for reflecting the temperature change history of the food experienced through the cumulative effect on time and temperature, which usually manifested as mechanical deformation or color change, and can reflect all or partial temperature history and remaining shelf life of the indicated food, so as to grasp the freshness of the food in time. At present, there are no mature commercial TTIs, and some applications focus on electronic TTIs. Research on TTIs with enzymes has considerably conducted, but they are mainly limited to theoretical research (relevant literature and patents) and have not been applied. Therefore, developing a TTI product that is easy to operate and can monitor the quality status and freshness of food in the cold chain logistics process in real time is necessary. Currently, there 1s a phenomenon of "chain scission" in cold chain logistics, so consumers lack sufficient trust in cold chain food products. Thus, developing a TTI that visually reflects the shelf life of the food can greatly increase the consumer’s confidence in the cold chain food.

SUMMARY The present disclosure provides a puncturing neutral protease type time-temperature indicating device, to overcome the deficiencies in the prior art. In the present disclosure, the method for determining protein concentration with Folin- Phenol reagent is used to design a colored system of enzyme-substrate reaction, wherein irreversible color changes may occur over time or temperature fluctuation, resulting in visible color changes. so that the purpose of indicating the quality change of the frozen food over time or temperature is realized. And also, a visual TTI packaging is provided that is easy to operate, effective immediately, and may be attached to low-temperature food packaging. The technical problem to be solved by the present disclosure is achieved by the following technical measures: A puncturing neutral protease type time-temperature indicating device is composed of an indicator and a color card, and the indicator includes a container, which is a PVC transparent cylinder with a button. the cylinder contains a color development reagent, a substrate and an enzyme, wherein an amount of the color development reagent added is between 1% and 5%. a volume amount of the substrate added is between 10% and 50%, and an amount of the enzvme added is between 2% and 8%; and a color b* value of -38 is determined to be an end point for color development of the time-temperature indicating device; (1) the color development reagent is a Folin-Phenol reagent with a concentration of 2 mg/L; (2) the substrate is a casein solution with a mass fraction of 2%, which is mixed by a casein solution, 0.1 mol/L of NaOH solution and a distilled water; (3) the enzyme is a neutral protease solution with a concentration of 2 mg/L; (4) the cylinder further comprises a layer of transparent plastic PE film, which divides the cylinder into an upper part and a lower part: (5) the cylinder has a height of 8 to 14 mm, and a bottom diameter of 56 to 66 mm; (6) the upper part of the cylinder has a height of 4 to 6 mm for containing 1.2 ml of the neutral protease solution and 7 ml of the distilled water: (7) the lower part of the cylinder has a height of 6 to 9 mm for containmg a mixture of the

0.8 ml of Folin-Phenol reagent, 9 ml of the casein solution, and the 1 ml of NaOH solution; (8) the button is composed of two white solid cylinders and a cone, wherein the flat solid cylinder has a height of 1 to 3 mm and a diameter of 56 to 66 mm, the solid cylinder between the flat solid cylinder and the cone has a height of 2 to 5 mm and a diameter of 3 to 10 mm, and the bottom diameter of the cone is greater than or equal to the diameter of the solid cylinder connected to the cone and does not exceed 10 mm; (9) the button is attached with a double-sided tape for sticking on a surface of a food packaging. Preferably, in the above solutions, the puncturing neutral protease type time-temperature indicating device has a suitable temperature of 0 to 250C. Preferably, in the above solutions, the prepared color development reagent, the substrate and the enzyme are stored at 0 oC, respectively.

Preferably, in the above solutions, when the present disclosure is in use. the intermediate film is punctured by pressing the button, moreover the cylinder and the button can stick together, and then one side of the button is stuck to an outer packaging of a low-temperature food to be monitored after shaking.

Preferably, in the above solutions, the film in the container is punctured by pressing the button to mix the substrate with the enzyme, and a shelf life of the product is determined by comparing the color changes under the action of the color development reagent with the color card.

The technical effects of the present disclosure are that the time-temperature indicating device is inexpensive to package, simple to operate. and easy to observe, and when the puncturing neutral protease type time-temperature indicating device is stored with the food to be monitored at the same temperature, the color of the puncturing neutral protease type time-temperature indicating device may be directly compared with a reference color using eyes to evaluate the quality of the food and the remaining shelf life.

The present disclosure is designed depending on the method for determining protein concentration with Folin-Phenol, and irreversible color changes can occur. When the temperature rises, the enzyme activity is strengthened and the enzyme reacts quickly with the substrate, which speeds up the irreversible color changes of the puncturing neutral protease type time-temperature indicating device, and then the change in food quality and the remaining shelf life can be estimated by observing the change of the color.

The system of puncturing neutral protease type time-temperature indicating device in the present disclosure has a strong stability, an inexpensive cost and is environmentally friendly. The selection of substrates and reactants for enzymatic reactions is cost-effective and convenient for mass production applications in the market.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a variation trend of b* of the puncturing neutral protease type time- temperature indicating devices with four different formula at 40C; Fig. 2 shows a variation trend of b* of the puncturing neutral protease type time- temperature indicating device with formula 1 at different temperatures; Fig. 3 shows a sensory evaluation trend of surimi at different temperatures; Fig. 4 shows a color card of the puncturing neutral protease type time-temperature indicating device; Fig. 5 shows a front view of the puncturing neutral protease type time-temperature indicating device packaging, wherein A - a bottom diameter of the cone, B - a height of the container cylinder, C - a bottom diameter of the container cylinder, D - a height of the upper part of the container cylinder for containing neutral protease solution, E - a height of the lower part of the container cylinder for containing Folin-Phenol reagent, F - a thickness of the white solid cvlinder (a height of the flat solid cylinder), G - a height of the gap between the white solid cylinder and the container cylinder (a height of the solid cylinder between the flat solid cylinder and the cone); Fig. 6 shows a top view of the puncturing neutral protease tvpe time-temperature indicating device packaging, wherein R1 - a bottom radius of the cone, R2 - a bottom radius of the container cylinder.

DETAILED DESCRIPTION OF THE EMBODIMENTS In order to fully disclose the puncturing neutral protease type time-temperature indicating device of the present disclosure, technical solutions will be clearly and completely described with the drawings.

Design a colored system of enzyme-substrate reaction depending on the method for determining protein concentration with Folin-Phenol reagent. in which irreversible color changes occur. The color changes from light yellow-green to deep green-blue. and finally to dark blue. A b value (b*) in the Lab color system is used as the criterion for color evaluation.

Through a large amount of analytical experiments with combination of refrigerating a surimi of grass carp at AoC. it is determined that an amount of the color development reagent added is between 1% and 5%, a volume amount of the substrate added is between 10% and 50%. and an amount of the enzyme added is between 2% and 8%: and the value of the b* at -38 is determined as the color development end point of the time-temperature indicating device.

The following describes the preferred embodiments of the present disclosure. The preferred embodiments described herein are only used to illustrate and explain the present disclosure, and do not limit the present disclosure.

Embodiment 1: Four formulas of the puncturing neutral protease type time-temperature indicating device are obtained according to the parameters obtained. as shown in Table 1, and storage is conducted at 40C. Then. the change trend of b* is observed and recorded, as shown in Fig. 1. and it can be found that the change trend is regular and inversely proportional to time. The activation energies of the four formulas are tested respectively, as shown in Table 2, and it can be found that the activation energies of the four formulations for TTI are between 51 to 63 kJ/mol, which may be used as a determination of time under low-temperature transportation. Then. the color changes over time at different temperatures are observed for the puncturing neutral protease type time-temperature indicating device in Formula 1, as shown in Fig. 2, and the b* value of -38 is determined as the end point with the combination of the storage of the surimi of grass carp. As shown in Fig. 3. the resulted change trends are consistent with the change trends of sensory evaluation scores of the surimi product of grass carp under corresponding temperatures. Therefore, the prepared color 5 development system of enzyme-substrate reaction may be used as a time-temperature indicating device for the surimi of grass carp. Table 1 Four formulas of TTI No. of 9 formula | Folin casein NaOH protease distilled solution solution water of TTI Table 2 Activation energy of each formula of TTI and preexponential factors No. of formula | activation preexponential R2 of TTI energy (kJ/mol) | factor

51.465 2.89x10° 0.9969

57.014 1.77-10% 0.9979

60.175 231x101"! 0.9946

62.951 2.49x10" 0.9991 Further, to verify the applicability of the present disclosure, colors of the developed time- temperature indicating device are compared with data of shelf life for the surimi of grass carp at constant temperature and conditions of storage temperature fluctuates greatly. According to all experimental data, the deviation on the quality evaluation of the surimi of grass carp is within 5%, which proves that the present disclosure has a strong applicability in indicating the shelf life of surimi of grass carp. Embodiment 2: A system of puncturing neutral protease type time-temperature indicating device is composed of a color development reagent, a substrate and an enzyme solution; (1) the color development reagent is a 0.8 ml of Folin-Phenol reagent of with a concentration of 2 mg/L; (2) the substrate is a mixture composed of 9 ml of casein solution with a mass fraction of 2% and 1 ml of NaOH solution, wherein the casein solution is mixed by a casein, a 0.1 mol/L NaOH solution and a distilled water;

(3) the enzyme solution is 1.2 ml of neutral protease solution with a concentration of 2 mg/L and 7 ml of distilled water; (4) After adding and mixing the above according to the above concentration ratio and the order of addition, they are reacted at different temperatures, and then a correspondence between the color changes and the reaction time is observed.

The time-temperature indicating device obtained in Embodiment 2 is placed under different temperature gradients to periodically observe the color changes of the system at different temperatures (color and brightness changes and the rate of change) and to express those as RGB values. The results are shown in Table 3 and 4.

Table 3 Color codes of TTI and corresponding RGB ranges ela fo Je Jo fe Je Jo fu |

45.13 [30.13 [20.12 [| 60.95. || 55.10 [ 30.70. || 30.55. || 35.45. RGB | 0- 0- 5- 100- 0.140- || 120- 100- 80- range | 160.1 (120.1 || 110.1 [80.12 [80.11 || 50.85. || 40.80. || 40.60.

65.15 [40.14 ||30.14 || 0.135 | 5.150 | 130 120 910 0 5 0 The reaction of enzymes and proteins is related to time and temperature, and color changes occur under the action of indicator, thus changes over time and temperature may be directly reflected by the color changes and such changes are irreversible. Under different temperatures, the color change trend of the puncturing neutral protease type time-temperature indicating device over time within 10 days is shown in Table 4, wherein the RGB ranges corresponding to the color codes are shown in Table 3. Table 4 Color change trend of TTI within 10 days under different temperatures Time Color code 0°C 4°C 20°C Temperature wa [os [EE Me | B [B® LO

| ws» [ 8 | 8 ! 00 Cw [se Cw ee ew oc oc | m= [ec 1 ¢¢ 00000 ow [ce ow» J 00® J 00> 0 Ce ve Cw se Cw ee

EE Mh EFO [EE e= aa [FE Ce ew The following numbered clauses include embodiments that are contemplated and nonlimiting;

1. A puncturing neutral protease type time-temperature indicating device, wherein the device is composed of an indicator and a color card, the indicator comprises a container, which is a PVC transparent cylinder with a button, the cylinder contains a color development reagent, a substrate and an enzyme, wherein an amount of the color development reagent added is between 1% and 5%, a volume amount of the substrate added is between 10% and 50%. and an amount of the enzyme added is between 2% and 8%, a color b* value of -38 is determined to be an end point for color development of the time-temperature indicating device; (1) the color development reagent is a folin-phenol reagent with a concentration of 2 mg/L; (2) the substrate is a casein solution with a mass fraction of 2%, which is mixed by a casein solution, 0.1 mol/L of a NaOH solution and a distilled water; (3) the enzyme is a neutral protease solution with a concentration of 2 mg/L; (4) the cylinder further comprises a layer of transparent plastic PE film, which divides the cylinder into an upper part and a lower part;

(5) the cylinder has a height of 8 to 14 mm, and the cylinder has a bottom diameter of 56 to 66 mm; (6) the upper part of the cylinder has a height of 4 to 6 mm for containing 1.2 ml of the neutral protease solution and 7 ml of the distilled water; (7) the lower part of the cylinder has a height of 6 to 9 mm for containing a mixture of 0.8 ml of the folin-phenol reagent, 9 ml of the casein solution, and 1 ml of the NaOH solution; (8) the button is composed of two white solid cylinders and a cone, wherein the flat solid cylinder has a height of 1 to 3 mm and a diameter of 56 to 66 mm, the solid cylinder between the flat solid cylinder connected and the cone has a height of 2 to 5 mm and a diameter of 3 to 10 mm, and the bottom diameter of the cone is greater than or equal to the diameter of the solid cylinder connected to the cone and does not exceed 10 mm; (9) the button is attached with a double-sided tape for sticking on a surface of a food packaging.

2. The puncturing neutral protease type time-temperature indicating device according to clause 1. wherein the transparent plastic PE film in the container is punctured by pressing the button to mix the substrate with the enzyme, and a shelf life of the product is determined by comparing color changes under the action of the color development reagent with the color card.

3. The puncturing neutral protease type time-temperature indicating device according to clause 1, wherein the time-temperature indicating device has a suitable temperature of 0 to 25°C.

4. The puncturing neutral protease type time-temperature indicating device according to clause 1, wherein the color development reagent, the substrate and the enzyme are stored at 0°C, respectively.

5. The puncturing neutral protease type time-temperature indicating device according to clause 1, wherein when in use, the transparent plastic PE film is punctured by pressing the button, the cylinder and the button stick together, and then one side of the button is stuck to an outer packaging of a low-temperature food to be monitored after shaking. The above are only preferred embodiments of the present disclosure and is not intended to limit the present disclosure. Although the present disclosure has been described in detail with reference to the foregoing embodiments. those skilled in the art can still modify the technical solutions described in all the foregoing embodiments, or equivalently replace some of the technical features. Any modifications, equivalent substitutions, improvements, and the like within the spirit and principles of the disclosure are intended to be included within the scope of the present disclosure.

Claims

Conclusions

A piercing neutral protease type time-temperature indication device, the device being composed of an indicator and a color chart; the indicator has a reservoir, which consists of a transparent PV C cylinder with a knob: the cylinder has a color development reagent, a substrate and an enzyme, an amount of the added color development reagent being between 1% and 5%, a volume amount of the substrate between 10% and 50% is added and an amount of the enzyme between 2% and 8%: a color b * value of -38 is recorded as an end point for color development of the time-temperature indicator; (1) the color development reagent is a folin phenol reagent at a concentration of 2 mg / L; (2) the substrate is a casein solution with a mass fraction of 2%, which is mixed with a casein solution. 0.1 mol / l of a NaOH solution and a distilled water; (3) the enzyme is a neutral protease solution at a concentration of 2 mg / L; (4 the cylinder further includes a layer of transparent plastic PE film, which divides the cylinder into an upper and a lower part; (5) the cylinder has a height of 8 to 14 mm and the cylinder has a bottom diameter of 56 to 66 mm (6) the upper part of the cylinder has a height of 4 to 6 mm for a content of 1.2 ml neutral protease solution and 7 ml distilled water; (7) the lower part of the cylinder has a height of 6 up to 9mm for a content of a mixture of 0.8 ml of the folin-phenol reagent. 9 ml of the casein solution and | ml of the NaOH solution; (8) the button consists of two white solid cylinders and a cone, wherein the flat solid cylinder has a height of 1 to 3 mm and a diameter of 56 to 66 mm, the solid cylinder between the connected flat solid cylinder and the cone has a height of 2 to 5 mm and a diameter of 3 to 10 mm , and the bottom diameter of the cone is greater than or equal to the diameter of the solid ci linder connected to the cone and does not exceed 10 mm; (9) the button 1s fixed with a double-sided tape to stick on a food packaging surface.

2. The piercing neutral protease tvpe time-temperature indication device according to claim 1, wherein the transparent plastic PE film in the container is pierced by pressing the button to mix the substrate with the enzyme; and the shelf life of the product is determined by comparing the color changes under the influence of the color development reagent with the color chart.

3. The piercing neutral protease type time-temperature indication device according to claim 1, wherein the time-temperature indication device has a suitable temperature of 0 to 25 ° C.

The piercing neutral protease type time-temperature indication device according to claim 1, wherein the color developing reagent, the substrate and the enzyme are stored at 0 ° C, respectively.

5. The piercing neutral protease type time-temperature indication device according to claim 1, wherein the transparent plastic PE film, when in use, is pierced by pressing the button, the cylinder and the button stick together: and then one side of the button taped to a low temperature food outer packaging, which should be checked after shaking.