A Natural Component-Based Oxygen Indicator with In-Pack Activation

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A Natural Component-Based Oxygen Indicator with In-Pack Activation for Intelligent Food Packaging Keehoon Won, Nan Young Jang, and Junsu Jeon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04172 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Journal of Agricultural and Food Chemistry

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A Natural Component-Based Oxygen Indicator with In-Pack

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Activation for Intelligent Food Packaging

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Keehoon Won*, Nan Young Jang†, and Junsu Jeon

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Department of Chemical and Biochemical Engineering, Dongguk University-Seoul,

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30 Pildong-ro 1-gil, Jung-gu, Seoul 04620, Republic of Korea

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*Corresponding Author

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Telephone: +82 2 2260 8922. Fax: +82 2 2268 8729. E-mail: [email protected].

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†

Present address: Dermapro, 30 Bangbaejungang-ro, Seocho-gu, Seoul 06684, Republic of Korea

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ABSTRACT: Intelligent food packaging can provide consumers with reliable and correct

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information on the quality and safety of packaged foods. One of the key constituents in intelligent

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packaging is a colorimetric oxygen indicator, which is widely used to detect oxygen gas involved

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in food spoilage by means of a color change. Traditional oxygen indicators consisting of redox

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dyes and strong reducing agents have two major problems: they must be manufactured and stored

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under anaerobic conditions because air depletes the reductant, and their components are synthetic

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and toxic. In order to address both these serious problems, we have developed a natural

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component-based oxygen indicator characterized by in-pack activation in this study. The

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conventional oxygen indicator composed of synthetic and artificial components was redesigned

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using naturally occurring compounds (laccase, guaiacol, and cysteine). These natural components

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were physically separated into two compartments by a fragile barrier. Only when the barrier was

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broken for use, all the components mixed and started functioning as an oxygen indicator (i.e., in-

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pack activation). Depending on the component concentrations, the natural component-based

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oxygen indicator exhibited different response times and color differences. The rate of the color

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change was proportional to the oxygen concentration. This novel colorimetric oxygen indicator

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will contribute greatly to intelligent packaging for healthier and safer foods.

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KEYWORDS: Colorimetric oxygen indicators, in-pack activation, laccase, guaiacol, cysteine,

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intelligent food packaging

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INTRODUCTION

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Food packaging is one of the main processes for maintaining food quality during transportation

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and storage. In response to growing consumer demands over the past decades, a new food

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packaging technology has been developed: intelligent food packaging.1 According to the

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definition of the European Commission, intelligent food packaging contains components capable

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of monitoring the condition of packaged foods or the surrounding environment.2 Therefore, it can

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provide consumers with reliable and accurate information on food quality and safety, and it has

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been attracting considerable attention as demonstrated by many recent scientific publications and

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reviews.2–6 One of the key constituents in intelligent food packaging is an indicator, which

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provides qualitative or semi-quantitative information on packaged foods by means of a color

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change.4,5 Various variables related to food quality and safety (e.g., temperature, oxygen, carbon

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dioxide, and volatile amines) have been monitored using various indicators.2–10

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Oxygen is involved in microbial and biochemical spoilage of foods11 and thus is removed

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from food packaging by modifying the atmosphere with gases such as nitrogen or by using

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oxygen scavengers.3,12 However, oxygen gas can leak into the package over time because of air

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permeation through the packaging materials, poor sealing, or the package being tampered or

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damaged during transportation and storage. Therefore, the absence of oxygen should be ensured

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using visual oxygen indicators.8,13

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The most widely used oxygen indicator is a colorimetric redox dye-based indicator, which was

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commercialized (e.g., Ageless Eye® manufactured by the Mitsubishi Gas Chemical

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Company).8,13,14 This type of oxygen indicator is typically composed of a redox dye such as

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methylene blue (MB) and a reducing agent such as glucose in an alkaline (NaOH) solution. In the

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absence of oxygen, MB is reduced to its colorless form (leuco-methylene blue) by glucose in

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NaOH solution; conversely, in the presence of oxygen, the dye is oxidized back to a highly 3 ACS Paragon Plus Environment


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colored form, indicating oxygen in the package (Scheme 1a). However, the traditional oxygen

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indicators have two major problems. One is that they must be manufactured and stored under

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anaerobic conditions because they readily react even with air and stop functioning in a few hours

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owing to the depletion of the reducing agent.3,13,15,16 This problem was addressed by two different

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approaches.6 In order to preserve oxygen indicators until use, a novel oxygen indicator is

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activated (i.e., switched on) only when UV is irradiated (Scheme 1b).15–18 As another effective

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strategy, we recently reported a simple and practical oxygen indicator the components of which

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are physically separated by an impenetrable barrier, which can be readily broken by simple

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physical methods (e.g., pressing with hands). Only when the barrier is broken for food packaging,

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all the components mix and start to function as an oxygen indicator (i.e., in-pack activation).19

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The other problem with the conventional oxygen indicators is that some components (e.g.,

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redox dyes and alkalis) are synthetic and harmful chemicals; they can contaminate packaged

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foods and thus raise safety concerns in intelligent food packaging.8,20–22 In this study, we develop

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an activation-controlled oxygen indicator all the components of which are existing in nature. The

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traditional oxygen indicator composed of synthetic and artificial components is redesigned using

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naturally occurring organic compounds [laccase, guaiacol, and cysteine (Cys)] (Scheme 1c).

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These natural components are first used in the pressure-activated compartmented oxygen

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indicator developed in our previous work. A natural component-based oxygen indicator

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characterized by in-pack activation is developed and tested in the present study.

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MATERIALS AND METHODS

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Materials. Laccase from Trametes versicolor and all chemicals were purchased from Sigma-

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Aldrich (St. Louis, MO) and used without any further purification. The amount of laccase was 4 ACS Paragon Plus Environment

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expressed in terms of catalytic activity in enzyme units. One unit (U) of laccase was defined as

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the amount of enzyme that converts 1 µmol catechol per min at pH 5 and 25°C. PET(12

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µm)/ON(15 µm)/LLDPE(30 µm) film and LDPE film (70 µm) were obtained from Sunyang

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(Korea) and Wowpack (Korea), respectively.

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Preparation of the Oxygen Indicator with In-Pack Activation. The pressure-activated

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compartmented oxygen indicator was prepared as previously described.19 The PET/ON/LLDPE

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packaging film (4.5 cm × 3 cm) was pulled by a vacuum pump to form two wells. Typically, 185

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µL of laccase solution (2 U/mL) was poured into one well (compartment I), and 185 µL of an

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aqueous mixture of guaiacol (10 mM) and Cys (10 mM) was poured into the other well

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(compartment II). All these solutions were prepared using citrate-phosphate buffer (pH 5).

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Subsequently, the rim of each well was tightly heat-sealed at 120°C for 2.4 s with the oxygen-

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permeable LDPE film (4.5 cm × 3 cm) to prevent drying and leaking. However, the barrier film

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between the two compartments was loosely heat-sealed at 70°C for 2.4 s so that it could be easily

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opened (Figure 1). After activation, the concentrations of laccase, guaiacol, and Cys became 1

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U/mL, 5 mM, and 5 mM, respectively.

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Evaluation of the Oxygen Indicator. The oxygen indicators prepared with different

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component concentrations were activated (i.e., the separator was broken with a hand) and then

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placed in a gas cell. The cell was continuously supplied with a gas mixture of O2 and N2 (1, 10,

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or 21 % oxygen) produced using an automatic gas mixing system (Sehwa Hightech Co., Korea).

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The oxygen concentration in the gas cell was checked using an oxygen sensor (CheckPoint II,

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PBI-Dansensor, Denmark). The gas cell containing the natural component-based oxygen



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indicator was incubated at 25°C for 2 h, during which the color was monitored. All the

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experiments were carried out at least in duplicate, and the data are presented as the mean and

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standard deviation.

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Analysis. The colors of the oxygen indicators were monitored using a portable

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spectrophotometer (CM-2600d; Konica Minolta, Japan), which measures the spectral distribution

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of the reflectance of samples and calculates colors. The colors were expressed as L* (lightness),

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a* (redness-greenness), and b* (yellowness-blueness); the L*a*b* color space (also referred to as

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CIELAB) is the most widely used in food color measurement owing to the uniform distribution

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of colors.23 Color changes over time were determined numerically as an index of total color

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difference (∆E):

123 ∆E = [(∆L*)2 + (∆a*)2 + (∆b*)2]1/2

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(1)

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where ∆L*, ∆a*, and ∆b* are the differences in L*a*b* between controls and samples.24 The

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controls were samples at time = 0, and the color was measured at 4 points for each sample and

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averaged.

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RESULTS AND DISCUSSION

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Development of the Natural Component-Based Oxygen Indicator with In-Pack

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Activation. In order to transform the conventional oxygen indicator into a natural component-

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based oxygen indicator, a biocatalyst (an enzyme), its natural substrate, and a biological reducing


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agent were introduced instead of traditional synthetic components. First, enzymes are biological

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catalysts with high specificity and are perceived as natural, non-toxic food components.25 The

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enzyme for oxygen indicators must convert its substrate into a product with a different color

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using molecular oxygen. Oxidases such as laccase, ascorbate oxidase, and bilirubin oxidase can

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be used as oxygen-reducing biocatalysts for oxygen indicators.26–28 Among the oxidases, laccase

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was selected because this enzyme is stable, commercialized and promising for food industry (e.g.,

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wine and beer stabilization, fruit juice processing, baking, and sugar beet pectin gelation).29 For

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example, a commercial laccase preparation named Flavourstar is marketed for use in brewing

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beer to prevent the formation of off-flavor compounds.30 Moreover, toxicological studies showed

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that there are no reasons for safety concerns when using the laccase for oral care or in food for

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human consumption.31 As a laccase substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)

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(ABTS) has been the most widely used in laccase activity assay and changes colors by the

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enzymatic reaction; however, it is artificial and synthetic. In this study, we chose guaiacol, which

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is a naturally occurring organic compound biosynthesized by a variety of organisms; it is one of

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the key flavor compounds of coffee,32 rooibos tea,33 and wine,34 and is also found in rice grains as

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one of the most abundant phenolic compounds.35 This natural compound forms a brown product

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in laccase-catalyzed reactions (the molar extinction coefficient at 470 nm = 26,600 M–1 cm–1).36

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The last component, a natural reducing agent must reduce laccase-oxidized guaiacol back to its

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original form. Several biological reductants such as glutathione, Cys, and organic acids were

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screened, and Cys was found to be the best (data not shown). Overall, the redox dye (MB) was

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replaced with a biocatalyst (laccase) and its natural substrate (guaiacol), and the reducing agents

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(glucose and NaOH) were replaced with a natural amino acid (Cys) (Scheme 1a and c).

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Since enzymatic reactions are dependent on the pH of reaction medium, the pH of the natural

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component-based oxygen indicator should be determined. The laccase from T. versicolor was 7 ACS Paragon Plus Environment


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reported to have optimal pH ranges of 3.5 to 5 for activity and 5 to 8 for stability.37,38 By taking

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both activity and stability into consideration, the pH was fixed at 5, and each component was

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dissolved in citrate-phosphate buffer (pH 5). The laccase solution was poured into one well

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(compartment I), and the aqueous mixture of guaiacol and Cys was poured into the other well

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(compartment II). By making the rim of each well tightly heat-sealed and the barrier between the

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two wells loosely heat-sealed, the natural component-based oxygen indicator with in-pack

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activation was finally produced (Figure 1).

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Performance of the Oxygen Indicator. We examined the performance of the natural

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component-based oxygen indicators with different concentrations of each component. Firstly, the

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effects of reductant concentration were investigated; the final concentration (i.e., the

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concentration after the two separated solutions were mixed by activation) of Cys was varied at 1,

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2, and 5 mM, while the final concentrations of laccase and guaiacol were fixed at 1 U/mL and 5

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mM, respectively. The natural component-based oxygen indicators were activated by rupturing

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the impervious barrier with a hand (Figure 1). Immediately after the activation, the oxygen

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indicators were incubated in a gas cell with 21% oxygen, and the ∆E values were monitored

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using the portable spectrophotometer. For reference, the oxygen indicators activated were

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initially colorless because of the reductant even though it had been stored and activated in air.

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Figure 2a shows the time-course profiles of the ∆E values. At a low concentration (1 mM) of Cys,

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the ∆E value increased rapidly and reached a constant value within 0.5 h. The color change over

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time may be attributed to the colored oxidation products of guaiacol. Enzymatic one-electron

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oxidation of guaiacol yields a phenoxyl radical, and the reactive radical subsequently forms a

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colored mixture of dimers, trimers, and tetramers.39–42 As the Cys concentration was increased to

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5 mM, the ∆E value increased slowly and took longer to approach the final value. This is because 8 ACS Paragon Plus Environment

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Cys reduced the phenoxyl radical of guaiacol generated by laccase; biologically derived thiols

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such as Cys can scavenge various radicals including phenoxyl radicals and thus play a key role in

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protecting cells from oxidative damage.43,44 The reduction of the phenoxyl radical by Cys

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accelerates with increasing the Cys concentration. On the other hand, the final ∆E value of about

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50 indicates a considerable color change. Generally, ∆E values below 1 are extremely small for

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detecting color changes with the naked eye, whereas values above 2 are noticeable even to an

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untrained eye.28

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In Figure 2b, the effects of guaiacol concentration are shown. The final concentrations of

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laccase and Cys were fixed at 1 U/mL and 5 mM, respectively, while the final concentration of

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guaiacol was varied at 1, 2, and 5 mM. As the guaiacol concentration was decreased from 5 mM

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to 1 mM, the rate of color formation declined, and the final ∆E value also dropped. Since the Km

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value (the substrate concentration that gives a velocity equal to one-half the maximal velocity) of

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T. versicolor laccase with guaiacol is 0.3 mM,37 the formation rate of the colored oxidation

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products can decrease as the guaiacol concentration decreases from 5 mM to 1 mM. In addition,

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the final ∆E value that indicates the final concentration of the colored product was proportional to

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the substrate concentration. Regarding the effects of enzyme concentration, when the laccase

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concentration was doubled with the final concentrations of 5 mM Cys and 5 mM guaiacol, the ∆E

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value rose sharply and took shorter to reach the final value (from 80 min to 45 min) as shown in

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Figure 2c. This is because of the increased rate of enzymatic guaiacol oxidation. This feature

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makes it possible to control the response time toward oxygen by adjusting the concentration of

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oxygen reduction biocatalyst, laccase. In this respect, the oxygen indicator in this study is in

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contrast to other oxygen indicators with in-pack activation (e.g., UV-activated oxygen indicators),

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which do not have a catalyst for oxygen reduction. The final ∆E value remained unchanged

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because a catalyst affects a reaction rate, but has no effect on chemical equilibrium. 9 ACS Paragon Plus Environment


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The dependence of the natural component-based oxygen indicator on oxygen concentration

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was also investigated. After the activation, the natural component-based oxygen indicator was

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placed in a gas cell with varying oxygen concentration (1, 10, and 21%), and its color was

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monitored over time. The ∆E values after 2 h are shown in Figure 3. The rate of the color change

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was proportional to the oxygen concentration as observed with other oxygen indicators,16,45

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because oxygen is also a substrate of the chromogenic reaction catalyzed by laccase (Scheme 1c).

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Considering that ∆E values above 2 (represented by the dashed line in Figure 3) indicate color

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differences that are noticeable to an untrained eye, even the presence of 1% oxygen in the

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package can be detected easily within 2 h using the novel oxygen indicator. The response rate

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toward oxygen can be tuned by changing the concentrations of guaiacol, Cys, and/or laccase as

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shown in Figure 2.

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In conclusion, the natural component-based oxygen indicator with in-pack activation has been

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developed for the first time. The conventional oxygen indicator composed of a redox dye, a

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reducing agent, and an alkaline compound (e.g., MB, glucose, and NaOH) was completely

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redesigned using natural compounds (laccase, guaiacol, and Cys). These natural components

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were physically separated into two compartments. When the barrier to separate the two

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compartments was ruptured, the oxygen indicator was activated and functioned as expected; it

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changed colors in the presence of oxygen. Depending on the component and oxygen

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concentrations, the natural component-based oxygen indicator exhibited different response times

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and color differences. This novel, simple, and practical oxygen indicator addresses the problems

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of traditional oxygen indicators. Hence the natural component-based colorimetric oxygen

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indicator characterized by in-pack activation will contribute substantially to intelligent packaging

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for healthier and safer foods.

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Acknowledgements

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This research was supported by the Agriculture Research Center Program of the Ministry of

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Agriculture, Food and Rural Affairs, Republic of Korea (ARC, 710003-03).

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derived radical cation scavenging by bucillamine, cysteine, and glutathione. Catalytic effect of

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Cu2+ ions. Biophys. Chem. 2016, 212, 9–16.

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(45) Son, E. J.; Lee, J. S.; Lee, M.; Vu, C. H. T.; Lee, H.; Won, K.; Park, C. B. Self-adhesive

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graphene oxide-wrapped TiO2 nanoparticles for UV-activated colorimetric oxygen detection.

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Sens. Actuators B 2015, 213, 322–328.

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339

(a)

340

(b)

341

(c)

342 343 344

Scheme 1. Schematic illustrations of the working principles of (a) the traditional oxygen

345

indicator, (b) the UV-activated oxygen indicator, and (c) the natural component-based oxygen

346

indicator developed in this study.

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Page 18 of 22

348 349 350 351

352 353 354

Figure 1. Schematic diagram and working process of the natural component-based oxygen

355

indicator with in-pack activation.

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60

(a) 50

∆E

40

30

20 1 mM Cysteine 2 mM Cysteine 5 mM Cysteine

10

0 60

(b) 50

∆E

40

1 mM Guaiacol 2 mM Guaiacol 5 mM Guaiacol

30

20

10

0 60

(c) 50

∆E

40

30

20

10

1 U/mL Laccase 2 U/mL Laccase

0 0.0

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0.5

1.0

1.5

Time (h) 19 ACS Paragon Plus Environment

2.0

2.5


Page 20 of 22

359 360

Figure 2. Effects of the concentrations of (a) cysteine, (b) guaiacol, and (c) laccase on the total

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color difference (∆E) changes of the oxygen indicator over time in 21% oxygen.

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364 365

60

∆E after 2 h

50

40

30

20

10

0 0

5

10

15

20

25

Oxygen Concentration (%)

366 367 368

Figure 3. Total color difference (∆E) values of the oxygen indicator 2 h after activation with

369

different concentrations of oxygen (1, 10, and 21 %). The final concentrations of laccase,

370

guaiacol, and Cys were 1 U/mL, 5 mM, and 5 mM, respectively. The dashed line represents a ∆E

371

value of 2.

372



373 374

Table of Contents Graphics

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A Natural Component-Based Oxygen Indicator with In-Pack Activation

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