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

Dec 1, 2016 - detect oxygen gas involved in food spoilage by means of a color change. ... KEYWORDS: colorimetric oxygen indicators, 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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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):

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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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(42) Jiang, C.; Ling, S.; Wang, P.; Liang, A.; Chen, B.; Wen, G.; Jiang, Z. A new and

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sensitive catalytic resonance scattering spectral assay for the detection of laccase using guaiacol

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as substrate. Luminescence 2011, 26, 500–505.

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(43) Trujillo, M.; Alvarez, B.; Radi, R. One- and two-electron oxidation of thiols: mechanisms, kinetics and biological fates. Free Radic. Res. 2016, 50, 150–171.

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(44) Valent, I.; Topolská, D.; Katarína Valachová, K.; Bujdák, J.; Šoltés, L. Kinetics of ABTS

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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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(a)

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(b)

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(c)

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

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

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indicator developed in this study.

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

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indicator with in-pack activation.

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(a) 50

∆E

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20 1 mM Cysteine 2 mM Cysteine 5 mM Cysteine

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0 60

(b) 50

∆E

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1 mM Guaiacol 2 mM Guaiacol 5 mM Guaiacol

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0 60

(c) 50

∆E

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20

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1 U/mL Laccase 2 U/mL Laccase

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0.5

1.0

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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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∆E after 2 h

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0 0

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Oxygen Concentration (%)

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

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

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

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value of 2.

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Table of Contents Graphics

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