TWI715282B - Manufacturing method of time-temperature indicator by immobilized enzyme with electrospinning - Google Patents

Abstract

The enzymatic time-temperature indicator (TTI) developed by electrospinning, immobilized enzyme on electrospun fiber film, is a pioneering technique. The activation energy and coloration of enzymatic TTI can be adjusted. The enzyme activity in this enzymatic TTI prototype is temperature dependent and reversible under temperature fluctuations. These characteristics effectively expanding the applicability of enzymatic TTI and become a desirable quality indicator for accumulating temperature and time information. Electrospun fiber with high specific surface area and immobilization is to obtain stable and reusable enzymes thus can reduce the dosage of laccase. Therefore, this enzymatic TTI exhibits high accuracy, stability, temperature reversibility and low cost. The managers of food manufacturer, logistics, vendors, and consumers can respond to food quality deterioration by significant color changes from colorless to brown purple. The intelligent food packaging developed by this enzymatic TTI can reduce the food waste or the food safety risk in traditional packaged foods.

Description

Manufacturing method of time temperature sensor for preparing immobilized enzyme by electrospinning technology

The invention relates to a manufacturing method, in particular to a manufacturing method of a time and temperature sensor for preparing immobilized enzymes using electrospinning technology.

In recent years, the occurrence of food safety incidents in Taiwan has caused consumers to question whether the food can be eaten after the expiration date, or whether the food must be safe within the expiration date. Since the temperature and humidity of the storage environment will affect the quality of the food, if the storage temperature is increased, the product may fission prematurely within the expiration date. According to the “Guidelines for the Evaluation of the Effective Date of Commercial Packaged Foods” issued by the Food and Drug Administration of the Ministry of Health and Welfare, the market Packaged foods for sale shall be in accordance with Article 22, paragraph 1, paragraph 7, and Article 24, paragraph 1 of the Food Hygiene Management Act, as well as the influence of factors such as transportation, storage, and sales environment, and the preservation test shall be designed according to the aforementioned individual conditions, based on Develop a shelf life. Therefore, the expiration date is only under certain storage conditions, and the final date that the commercially available packaged food can maintain the product value. If the product deviates from the "specific storage temperature conditions" during transportation, storage and sales, whether the product quality and value can be maintained within the validity period is a risk.

The time temperature sensor (TTI) is a kind of smart packaging. Since smart food packaging includes the ability to monitor the condition of the packaged food or the environment around the food, it provides consumers with reliable and accurate information about food quality and safety. Over time, any harmful changes in temperature changes in the food supply chain (for example, above or below the reference threshold). The time temperature sensor can simply record the time and temperature history device. By monitoring the time and temperature of the entire life cycle of the food, it can evaluate whether the food is spoiled. The time temperature sensor monitors and records the time and temperature parameters of the entire food supply chain from production, storage, transportation, sales to consumers, and uses visual change indicators (color change or dye diffusion) to monitor and indicate the overall temperature History records to monitor product quality. In order to reflect the quality of food, the time and temperature sensor is generally designed as a small label, which is directly pasted on individual food packaging so that it can experience the same environment as the food product. In order to comply with the irreversibility of time advancement, the design of the time temperature sensor needs to use irreversible reactions or materials as the basis to produce irreversible color changes.

Among them, the time temperature sensor is divided into (1) Microbial TTI, (2) Polymer-based TTI, (3) Diffusion-based TTI, and (4) Microbial TTI based on different reaction mechanisms. ) Enzymatic TTI (Enzymatic TTI) and other four types. Compared with microbial and physical time temperature sensors, enzyme-based time temperature sensors have higher sensitivity and higher accuracy to environmental temperature changes. However, the current time and temperature sensors on the market are still dominated by physical products, because the high cost of enzyme-based time and temperature sensors and the instability of enzymes impose some limitations on commercial development.

Although there have been a large number of researches on enzyme-type TTI and many related patents, there are few enzyme-type TTIs in commercial applications, mainly for three reasons: one is that the enzyme activity is easy to inactivate and the storage stability is poor; the other is the scope of application , It is necessary to adjust the activation energy and reaction rate of the time temperature sensor to be similar to that of the food to predict the quality of the food; the third is the cost problem. Generally, the price of high-purity enzymes is quite expensive. The current commercial TTI price needs 1~2.5 dollars. Etc., due to the high price, food manufacturers are often unwilling to apply time and temperature sensors to their products.

Therefore, how to solve the above-mentioned problems and deficiencies is the direction that the creators of the present invention and related industries engaged in this direction urgently want to study and improve.

The purpose of the present invention is to provide a method for manufacturing a time-temperature sensor for preparing immobilized enzymes using electrospinning technology, which mainly uses electrospinning technology to produce micro-nanofiber membranes with high specific surface area. The immobilized enzymes can be reused and can be used repeatedly. Reduce the amount of enzymes, while also having the characteristics of high accuracy, high stability, temperature reversibility, and low cost.

The reason is that, in order to achieve the foregoing objective, according to the present invention, a method for manufacturing a time and temperature sensor for preparing immobilized enzymes using electrospinning technology is provided, which includes:

(a) Preparation of electrospinning solution: heating and stirring a mixed polymer in a water bath to obtain an electrospinning solution;

(b) Film making step: Spinning the mixed polymer in the electrospinning solution into a micronano fiber film by electrospinning technology;

(c) Step of activating the film: immersing the micro-nano fiber film in a cross-linking agent solution to activate, to produce an activated film;

(d) Immobilization step: Immobilize the enzyme on the activated film by dipping, painting or spraying to produce an immobilized enzyme film; and

(e) Steps of preparing an enzyme-type time-temperature sensor: combine the immobilized enzyme film with the color reaction matrix solution to form an enzyme-type time-temperature sensor.

Preferably, in the step (a) preparing the electrospinning solution, the concentration of the electrospinning solution is adjusted, and the solution is heated to remove air bubbles, making it a fully dissolved and uniform electrospinning solution.

Preferably, in the step (a) preparing the electrospinning solution, the mixed polymer is selected from the group consisting of chitosan, acetic acid, polyvinyl alcohol and tetraethoxysilane.

Preferably, in the step (b), the electrospinning solution is operated with electrospinning equipment at a voltage of 15 kV-30 kV, the distance between the needle tip and the collecting plate is 15 cm -20 cm, and the feed rate is 0.3 mL /hr-1.0 mL/hr and the rotating speed of the drum are electrospinning under the conditions of 50 rpm-100 rpm to stack the fiber filaments on the transparent base film.

Preferably, in the step (c) activating the film, the cross-linking agent solution is prepared with a pH 4.5-pH 5.0 buffer solution, the micronano fiber membrane is immersed in the cross-linking agent solution, and left to stand 2 After hours, rinse with the buffer solution three times to remove the unbonded cross-linking agent solution, then place it in a drying vessel to vacuum, and finally dry at room temperature.

Preferably, in the step of (d) immobilizing enzymes, a quantitative enzyme is adsorbed on the activated film, so that the activated film adsorbs 8 μg-80 μg of enzyme, and in a constant temperature and humidity incubator at 25°C Let stand for 1 hour.

Preferably, in the step of (e) preparing an enzyme-type time-temperature sensor, a pH 4.5-pH 5.0 buffer solution is used to prepare a 5 mM-20 mM color reaction matrix solution.

More preferably, the color reaction matrix solution is 2,2-azinobis-3-ethyl benzothiazoline-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (2,2'-azinobis-3-ethyl benzothiazoline- 6-sulphonic acid, Syringaldehyde, Ferulic acid, 2,2-dimethoxy-propan (2,2-dimethoxy-propan) and guaiacol have oxidative color compound of.

Preferably, an enzyme reaction inhibitor can be added to adjust the color reaction rate and activation energy of the enzyme-type time-temperature sensor.

More preferably, the enzyme reaction inhibitor is sodium azide, L-cysteine, or ascorbic acid.

Accordingly, the present invention proposes a method for preparing a time-temperature sensor using electrospinning technology to immobilize enzymes, which can mainly adjust the activation energy and color reaction time of the reaction between the enzyme and the substrate. The enzyme type time-temperature sensor The activity is temperature-dependent and reversible under temperature changes, which can be used as a quality indicator for accumulating temperature and time information. The electrospun micro-nanofiber film has a high specific surface area, the immobilized enzyme can be reused, and can reduce the amount of enzyme, so it has the characteristics of high accuracy, high stability, temperature reversibility, and low cost. Food producers, logistics managers, sellers and consumers can respond to the deterioration of food quality from the obvious change from colorless to brownish-purple, and develop smart packaging to make up for most packaged foods that rely solely on expiry date judgments The problem of leftover food or food safety risks caused by the shelf sale period.

In order to make the above-mentioned features and advantages of the present invention more comprehensible, the following examples are given. And with the attached drawings, the detailed description is as follows.

The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of the preferred embodiment with reference to the drawings.

Referring to Figures 1 to 10, the embodiment of the present invention provides a method for preparing a time and temperature sensor for preparing immobilized enzymes using electrospinning technology, which mainly consists of (a) preparing the electrospinning solution, (b) preparing the membrane, and (c) The step of activating the film, (d) the step of immobilizing the enzyme, and (e) the step of preparing an enzyme-type time-temperature sensor, wherein:

The (a) step of preparing electrospinning solution: heat and stir a mixed polymer in a water bath to obtain an electrospinning solution. The concentration of the electrospinning solution is adjusted and heated to dissolve to remove bubbles and make it fully Dissolved and uniform electrospinning solution; in this embodiment, the 3% chitosan solution, 10% polyvinyl alcohol solution, 6% acetic acid and tetraethoxysilane solution are mixed at 6:4:2:3, 5:5:2:4, 5:5:2:3, 5:5:2:2, 4:6:2:3 ratio (W/W/V/V) mixed, respectively formulated to 3.9%, 4.1%, 4.3%, 4.6%, 5.0% chitosan electrospinning solution, heated and stirred at 60°C, 100 rpm for 50 minutes, followed by ultrasonic vibration and degassing for 5 minutes, but this is not a limitation, of course it can be Chitosan, zein, polyvinyl alcohol and tetraethoxysilane and other formulas.

The (b) film-making step: using electrospinning technology to spin the mixed polymer in the electrospinning solution into a micronano fiber film; in this embodiment, an electrospinning device is used with 21 gauge needles (internal Diameter 0.52 mm, outer diameter 0.82 mm) and a 5 mL adapter set for spinning, using polypropylene film with a size of 40 μm × 15.2 cm × 47.2 cm as the carrier substrate, and applying the CS/PVA/TEOS electrospinning solution to the voltage Electrospinning is performed under the conditions of 20 kV, the distance between the needle tip and the collecting plate 20cm, the feed rate 0.5 mL/hr, and the drum rotation speed 100 rpm. The spinning is stacked on the PP film to complete the single-sided electrospun coating (as shown in Figure 2 Shown), but not as a limit, as long as the electrospinning equipment is at a voltage of 15 kV-30 kV, the distance between the needle tip and the collection plate is 15 cm -20 cm, the feed rate is 0.3 mL/hr-1.0 mL/hr and the drum The speed is 50 rpm-100 rpm. Among them, the micro-nano fiber membrane has a diameter of about 300 nanometers and a specific surface area of 11.6-17.0 m ² /g, which can be effectively applied to immobilize enzymes and reduce the space barriers for the reaction of enzymes and substrates.

The (c) activating film step: immersing the micronanofiber film in a crosslinking agent solution to activate, to produce an activated film; in this embodiment, acetic acid and sodium acetate (Sodium acetate, Sigma-Aldrich) Prepare 1 mM acetic acid-sodium acetate buffer solution of pH 4.5-pH 5.0, and prepare 3% glutaraldehyde (Glutaraldehyde, Nihon Shiyaku) with 1 mM acetic acid-sodium acetate buffer solution of pH 4.5-pH 5.0 to complete electrospinning. Micro-nano fiber membranes (CS/PVA/TEOS/PP) are immersed in 3% glutaraldehyde solution and allowed to stand for 2 hours to make the aldehyde group of glutaraldehyde and the amine group on the electrospun fiber covalently bond After the film is left standing, take it out and repeatedly rinse with buffer solution three times to remove the unbonded glutaraldehyde, place it in a drying vessel and vacuum, and dry at room temperature to complete the film activation.

The (d) step of immobilizing enzyme: Immobilize the enzyme on the activation film by dipping, painting or spraying to produce an immobilized enzyme film. This step can stabilize the activity and function of the enzyme, and in (d) fixation In the enzyme step, a quantitative enzyme is adsorbed in the activated film, so that the activated film adsorbs 8 μg -80 μg enzyme; in this embodiment, laccase (Laccase, Lacc) powder (Sigma-Aldrich, production source) To Yunzhi (Trametes versicolor, ≧0.5 U/mg, lot result 1.07 U/mg), add 1 mM acetic acid-sodium acetate buffer solution of pH 4.5 to prepare laccase solutions of different concentrations, and store them in a refrigerator at -20℃. Take 50 μL of laccase solution of different concentrations and drop it on the activated 1 cm ² electrospun small film, so that the laccase solution is evenly distributed on the activated film, and there are 8, 10, 15, 20 and 25 on each square centimeter of the film. μg laccase, put it in a constant temperature and humidity incubator at 25°C for 1 hour to complete immobilization and drying, which is the immobilized enzyme film (as shown in Figure 3). The structure at this time is due to immobilization Enzyme, fiber diameter increased to about 500 nanometers.

The step (e) of preparing an enzyme-type time-temperature sensor: combine the immobilized enzyme film with the color reaction substrate solution to form an enzyme-type time-temperature sensor; in this embodiment, the pH is 4.5-5.0 The buffer solution is prepared into a 5-20 mM color reaction matrix solution, wherein the color reaction matrix solution is 2,2-diazide-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt ( 2,2'-azinobis-3-ethyl benzothiazoline-6-sulphonic acid), Syringaldehyde (Syringaldehyde), Ferulic acid (Ferulic acid), 2,2-Dimethoxy-propan (2,2-dimethoxy-propan) And guaiacol (guaiacol) has oxidized and colored compounds.

其中，V ₀為未固定化酵素反應速率、V ₁為固定化酵素反應速率。此固定化酵素於4℃存10天後，相對活性仍維持在95%左右(如圖4所示)。 The color reaction substrate solution of the enzyme at a pH value of 4.5-5.0 is reacted with the immobilized enzyme film at 30°C, and the color absorption value is measured every 10 minutes with a full spectrum analyzer, and the color absorption value is measured continuously for 90 minutes, and then calculated The reaction rate is measured and calculated with a fixed amount of immobilized enzyme as a control group to calculate the relative activity of the immobilized enzyme:

Among them, V ₀ is the reaction rate of unimmobilized enzymes, and V ₁ is the reaction rate of immobilized enzymes. After the immobilized enzyme was stored at 4°C for 10 days, the relative activity remained at about 95% (as shown in Figure 4).

Please refer to Figures 5 to 8, respectively, at different temperatures (4°C-37°C) for constant temperature reaction for different time, the color development rate increases with the increase of the fixed amount of enzyme and the higher the temperature. The activation energy can also be adjusted by adjusting the fixed amount of enzyme, the pH of the reaction environment, and adding an enzyme reaction inhibitor. Taking the addition of an enzyme reaction inhibitor as an example, the activation energy range is shown in Figure 9, where the enzyme reaction inhibitor is azide Sodium sulfide, L-cysteine, or ascorbic acid, but not limited to this. The color reaction is carried out at dynamic temperature (intermittently fluctuating temperature of 4°C and 25°C) to confirm that the enzyme will not be irreversibly deactivated (as shown in Figure 10).

The above is the configuration description of the main components of the embodiment of the present invention. As for the efficacy of the present invention, the description is as follows:

The enzyme-type time temperature sensor immobilizes a very small amount of enzyme (0.009-0.086 activity unit), which can show a reasonable color change, and can be designed and changed according to the quality deterioration rate and activation energy of various foods during storage The preparation conditions of the enzyme-type time-temperature sensor (fixed amount of enzyme, reaction pH and addition of enzyme inhibitors, etc.) are used to adjust the color rendering rate and activation energy. Fixing the enzyme-type time-temperature sensor on the outer packaging of the product becomes an intelligent packaging with a time-temperature sensor, whereby the observer can visually evaluate the quality change of the product under the temperature and time accumulation.

In addition, when the previous technology uses laccase to prepare an enzyme-type time temperature sensor, since the enzyme is not immobilized, it is necessary to use 0.104 to 0.650 active units of laccase to observe a reasonable color change, and the activation energy is between 43.9 to 46.9 kJ /mol, the enzyme usage is much higher than the immobilized enzyme of the present invention. The electrospun micro nanofiber film of the present invention has a high specific surface area, the immobilized enzyme can be reused, and the amount of enzyme can be reduced. At the same time, the present invention also has the characteristics of high accuracy, high stability, temperature reversibility, and low cost, and can effectively expand the activation energy range and color rendering rate of the time temperature sensor by adjusting the preparation conditions, and only needs to monitor the food If needed, choosing different amounts of immobilized enzymes can be suitable for monitoring the quality of the food.

In summary, the above-mentioned embodiments and drawings are only preferred embodiments of the present invention. When they cannot be used to limit the scope of implementation of the present invention, all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention shall belong to This invention patent covers the scope.

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Fig. 1 is a flow chart of preparing an immobilized enzyme film using electrospinning technology to produce an enzyme-type time-temperature sensor in an embodiment of the present invention. Fig. 2 is a SEM microstructure diagram of the electrospun micro-nano fiber film according to an embodiment of the present invention. Fig. 3 is an SEM microstructure diagram of an embodiment of the present invention after the electrospun micronanofiber film is immobilized with enzyme. Fig. 4 is a graph showing the enzyme stability of the electrospun film immobilized and unimmobilized enzymes (50μg/cm2) stored at 4°C for 10 days. Fig. 5 is a color change diagram of the enzyme-type time-temperature sensor (enzyme 8-25 μg/cm ² immobilized on the electrospun film) stored at 4°C in the embodiment of the present invention. Fig. 6 is a color change diagram of the enzyme-type time-temperature sensor (enzyme 8-25 μg/cm ² immobilized on the electrospun film) stored at 15°C in the embodiment of the present invention. Fig. 7 is a color change diagram of the enzyme-type time-temperature sensor (enzyme 8-25 μg/cm ² immobilized on the electrospun film) of the embodiment of the present invention stored at 25°C. Fig. 8 is a color change diagram of the enzyme-type time-temperature sensor (enzyme 8-25 μg/cm ² immobilized on the electrospun film) of the embodiment of the present invention stored at 37°C. Fig. 9 shows the enzyme-type time-temperature sensor with a fixed amount of 0.021 active unit of enzyme according to the embodiment of the present invention, adding enzyme reaction inhibitors at concentrations of 0.01, 0.05, and 0.1 mM. The pH=4.5 reaction rate constant and Arrhenius activation energy ( Ea) data graph. Figure 10 shows the variation of the color absorbance value of the enzyme-type time-temperature sensor (fixed enzyme 8-25 μg/cm ² ) stored at dynamic temperature (4°C and 25°C each for 8 hours, a total of 3 cycles) of the present invention Graph.

Claims (10)

A manufacturing method of a time and temperature sensor for preparing immobilized chemical enzymes by electrospinning technology, comprising: (a) preparing an electrospinning solution: heating and stirring a mixed polymer in a water bath to obtain an electrospinning solution; (b) Membrane production step: spin the mixed polymer in the electrospinning solution into a micro nanofiber film by electrospinning technology; (c) activation film step: soak the micro nanofiber film in a crosslinking agent solution Activation to produce an activated film; (d) step of immobilizing enzyme: immobilize the enzyme on the activated film by dipping, smearing or spraying to produce an immobilized enzyme film; and (e) preparing enzyme type time temperature sensor Device step: combine the immobilized enzyme film with the color reaction substrate solution to form an enzyme-type time and temperature sensor. As described in claim 1, the method for preparing a time-temperature sensor for preparing immobilized enzymes by electrospinning technology, wherein in the step (a) preparing the electrospinning solution, the electrospinning solution is adjusted in concentration and heated to dissolve Air bubbles are removed to make the electrospinning solution fully dissolved and uniform. The method for preparing a time temperature sensor using electrospinning technology to immobilize enzymes as described in claim 1, wherein in the step (a) preparing electrospinning solution, the mixed polymer is selected from the group consisting of: chitin Glycan, acetic acid, polyvinyl alcohol and tetraethoxysilane. The method for preparing a time temperature sensor for preparing immobilized enzymes using electrospinning technology as described in claim 1, wherein in the (b) film forming step, the electrospinning solution is used with electrospinning equipment at a voltage of 15kV-30kV and a needle tip The distance to the collection plate is 15cm-20cm, the feed rate is 0.3mL/hr-1.0mL/hr and the drum speed Electrospinning is performed under the condition of 50 rpm-100 rpm to stack the fiber filaments on the transparent base film. The method for preparing a time-temperature sensor using electrospinning technology to immobilize enzymes as described in claim 1, wherein in the step (c) activating the film, the crosslinking agent solution is prepared with a pH 4.5-pH 5.0 buffer solution, Soak the micro-nano fiber membrane in the cross-linking agent solution, let it stand for 2 hours, and then repeatedly rinse with the buffer solution three times to remove the unbonded cross-linking agent solution, and then place it in a desiccator to vacuum , And finally dry at room temperature. The method for preparing a time-temperature sensor for preparing immobilized enzymes using electrospinning technology as described in claim 1, wherein in the step (d) immobilizing enzymes, a quantitative enzyme is adsorbed on the activated film to make the activated film Adsorbed with 8μg-80μg enzyme, and placed in a constant temperature and humidity incubator at 25°C for 1 hour. The method for preparing a time-temperature sensor using electrospinning technology as described in claim 1, wherein in the step (e) preparing the enzyme-type time-temperature sensor, it is prepared with a pH 4.5-pH 5.0 buffer solution 5mM-20mM of the color reaction matrix solution. The method for preparing a time-temperature sensor using electrospinning technology to immobilize enzymes as described in claim 7, wherein the color reaction matrix solution is 2,2-diazo-bis(3-ethyl-benzothiazole-6 -Sulfonic acid) diammonium salt (2,2'-azinobis-3-ethyl benzothiazoline-6-sulphonic acid), syringaldehyde (Syringaldehyde), ferulic acid (Ferulic acid), 2,2-dimethoxypropane ( 2,2-dimethoxy-propan) and guaiacol (guaiacol) have oxidized and colored compounds. The method for preparing a time-temperature sensor using electrospinning technology as described in claim 1, wherein an enzyme reaction inhibitor can be added to adjust the color reaction rate and activation energy of the enzyme-type time-temperature sensor. Use electrospinning technology to immobilize enzyme preparation time temperature sensor as described in claim 9 The preparation method of, wherein the enzyme reaction inhibitor is sodium azide, L-cysteine, or ascorbic acid.

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