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

Dec 1, 2016 - The most widely used oxygen indicator is a colorimetric redox dye-based indicator, which has been commercialized (e.g., Ageless Eye manu...
5 downloads 12 Views 715KB Size
Subscriber access provided by UNIV OF WESTERN ONTARIO

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

Journal of Agricultural and Food Chemistry

1

2

A Natural Component-Based Oxygen Indicator with In-Pack

3

Activation for Intelligent Food Packaging

4 5

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

6

7

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

8

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

9 10 11 12 13 14 15 16

*Corresponding Author

17

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

18



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

19 1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

conventional oxygen indicator composed of synthetic and artificial components was redesigned

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36 37

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

38

intelligent food packaging

39 40

2 ACS Paragon Plus Environment

Page 2 of 22

Page 3 of 22

Journal of Agricultural and Food Chemistry

41

INTRODUCTION

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

using visual oxygen indicators.8,13

59

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

60

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

61

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

62

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

63

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

64

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

Journal of Agricultural and Food Chemistry

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84 85

MATERIALS AND METHODS

86 87

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

88

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

Page 4 of 22

Page 5 of 22

Journal of Agricultural and Food Chemistry

89

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

90

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

91

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

92

(Korea) and Wowpack (Korea), respectively.

93 94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105 106

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

107

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

108

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

109

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

110

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

111

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

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 22

112

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

113

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

114

standard deviation.

115 116

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

117

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

118

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

119

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

120

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

121

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

122

difference (∆E):

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

124

(1)

125 126

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

127

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

128

averaged.

129 130

RESULTS AND DISCUSSION

131 132

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

133

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

134

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

6 ACS Paragon Plus Environment

Page 7 of 22

Journal of Agricultural and Food Chemistry

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

Journal of Agricultural and Food Chemistry

159

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

160

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

161

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

162

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

163

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

164

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

165

activation was finally produced (Figure 1).

166 167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

Page 8 of 22

Page 9 of 22

Journal of Agricultural and Food Chemistry

183

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

184

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

185

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

186

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

187

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

188

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

189

untrained eye.28

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

Journal of Agricultural and Food Chemistry

Page 10 of 22

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

shown in Figure 2.

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

for healthier and safer foods.

230 10 ACS Paragon Plus Environment

Page 11 of 22

Journal of Agricultural and Food Chemistry

231

Acknowledgements

232

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

233

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

234

11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

235 236 237 238 239 240 241

Page 12 of 22

References (1) Yam, K. L.; Takhistov, P. T.; Miltz, J. Intelligent packaging: concepts and applications. J. Food Sci. 2005, 70, R1–R10. (2) Ghaani, M.; Cozzolino, C. A.; Castelli, G.; Farris, S. An overview of the intelligent packaging technologies in the food sector. Trends Food Sci. Technol. 2016, 51, 1–11. (3) Pereira de Abreu, D. A.; Cruz, J. M.; Paseiro Losada, P. Active and intelligent packaging for the food industry. Food Rev. Int. 2012, 28, 146–187.

242

(4) Heising, J. K.; Dekker, M.; Bartels, P. V.; van Boekel, M. A. J. S. Monitoring the quality

243

of perishable foods: opportunities for intelligent packaging. Crit. Rev. Food Sci. Nutr. 2014, 54,

244

645–654.

245 246 247 248 249 250 251 252 253 254 255 256

(5) Vanderroost, M.; Ragaert, P.; Devlieghere, F.; De Meulenaer, B. Intelligent food packaging: the next generation. Trends Food Sci. Technol. 2014, 39, 47–62. (6) Realini, C. E.; Marcos, B. Active and intelligent packaging systems for a modern society. Meat Sci. 2014, 98, 404–419. (7) Kim, K.; Kim, E.; Lee, S. J. New enzymatic time-temperature integrator (TTI) that uses laccase. J. Food Eng. 2012, 113, 118–123. (8) Mills, A. Oxygen indicators and intelligent inks for packaging food. Chem. Soc. Rev. 2005, 34, 1003–1011. (9) Vu, C. H. T.; Won, K. Bioinspired molecular adhesive for water-resistant oxygen indicator films. Biotechnol. Prog. 2013, 29, 513–519. (10) Jung, J.; Lee, K.; Puligundla, P.; Ko, S. Chitosan-based carbon dioxide indicator to communicate the onset of kimchi ripening. LWT Food Sci. Technol. 2013, 54, 101–106.

12 ACS Paragon Plus Environment

Page 13 of 22

257 258

Journal of Agricultural and Food Chemistry

(11) Huis in’t Veld, J. H. J. Microbial and biochemical spoilage of foods: an overview. Int. J. Food Microbiol. 1996, 33, 1–18.

259

(12) Lee, K.-E.; Kim, H. J.; An, D. S.; Lyu, E. S.; Lee, D. S. Effectiveness of modified

260

atmosphere packaging in preserving a prepared ready-to-eat food. Packag. Technol. Sci. 2008,

261

21, 417–423.

262 263 264 265 266 267 268 269 270 271 272 273 274 275

(13) Smolander, M.; Hurme, E.; Ahvenainen, R. Leak indicators for modified-atmosphere packages. Trends Food Sci. Technol. 1997, 8, 101–106. (14) Yoshikawa, Y.; Nawata, T.; Goto, M.; Fujii, Y. Oxygen indicator. US Patent 4169811, 1979. (15) Lee, S.-K.; Mills, A.; Lepre, A. An intelligence ink for oxygen. Chem. Commun. 2004, 1912–1913. (16) Lee, S.-K.; Sheridan M.; Mills, A. Novel UV-activated colorimetric oxygen indicator. Chem. Mater. 2005, 17, 2744–2751. (17) Vu, C. H. T.; Won, K. Novel water-resistant UV-activated oxygen indicator for intelligent food packaging. Food Chem. 2013, 140, 52–56. (18) Vu, C. H. T.; Won, K. Leaching-resistant carrageenan-based colorimetric oxygen indicator films for intelligent food packaging. J. Agric. Food Chem. 2014, 62, 7263–7267. (19) Jang, N. Y.; Won, K. New pressure-activated compartmented oxygen indicator for intelligent food packaging. Int. J. Food Sci. Technol. 2014, 49, 650–654.

276

(20) Gillman, P. K. CNS toxicity involving methylene blue: the exemplar for understanding

277

and predicting drug interactions that precipitate serotonin toxicity. J. Psychopharmacol. 2011, 25,

278

429–436.

13 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 22

279

(21) Paul, P.; Kumar, G. S. Targeting ribonucleic acids by toxic small molecules: structural

280

perturbation and energetics of interaction of phenothiazinium dyes thionine and toluidine blue O

281

to tRNAphe. J. Hazard Mater. 2013, 263, 735–745.

282

(22) Dainelli, D.; Gontard, N.; Spyropoulos, D.; Zondervan-van den Beuken, E.; Tobback, P.

283

Active and intelligent food packaging: legal aspects and safety concerns. Trends Food Sci.

284

Technol. 2008, 19, S103–S112.

285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302

(23) Wu, D.; Sun, D.-W. Colour measurements by computer vision for food quality control–a review. Trends Food Sci. Technol. 2013, 29, 5–20. (24) Pathare, P. B.; Opara, U. L.; Al-Said, F. A.-J. Colour measurement and analysis in fresh and processed food: a review. Food Bioprocess Technol. 2013, 6, 36–60. (25) James, J.; Simpson, B. K.; Marshall, M. R. Application of enzymes in food processing. Crit. Rev. Food Sci. Nutr. 1996, 36, 437–463. (26) Ahvenainen, R.; Pullinen, T.; Hurme, E.; Smolander, M.; Siika-Aho, M. Package for decayable foodstuffs. WO/1998/021120, 1998. (27) Sakurai, K.; Kawashima, M.; Matsuki, Y.; Ueda, S.; Takahashi, M. Oxygen indicator and packaged material. WO/2004/083360, 2004. (28) Virtanen, H.; Vehmas, K.; Erho, T.; Smolander, M. Flexographic printing of Trametes versicolor laccase for indicator applications. Packag. Technol. Sci. 2014, 27, 819–830. (29) Minussi, R. C.; Pastore, G. M.; Durán, N. Potential applications of laccase in the food industry. Trends Food Sci. Technol. 2002, 13, 205–216. (30) Osma, J. F.; Toca-Herrera, J. L.; Rodríguez-Couto, S. Uses of laccases in the food industry. Enzyme Res. 2010, 2010, 918761. (31) Brinch, D. S.; Pedersen, P. B. Toxicological studies on laccase from Myceliophthora thermophile expressed in Aspergillus oryzae. Regul. Toxicol. Pharmacol. 2002, 35, 296–307. 14 ACS Paragon Plus Environment

Page 15 of 22

Journal of Agricultural and Food Chemistry

303

(32) Dorfner, R.; Ferge, T.; Kettrup, A.; Zimmermann, R.; Yeretzian, C. Real-time monitoring

304

of 4-vinylguaiacol, guaiacol, and phenol during coffee roasting by resonant laser ionization time-

305

of-flight mass spectrometry, J. Agric. Food Chem. 2003, 51, 5768–5773.

306 307

(33) Lasekan, O.; Lasekan, A. Flavour chemistry of mate and some common herbal teas. Trends Food Sci. Technol. 2012, 27, 37–46.

308

(34) Escudero, A.; Campo, E.; Fariña, L.; Cacho, J.; Ferreira, V. Analytical characterization of

309

the aroma of five premium red wines. Insights into the role of odor families and the concept of

310

fruitiness of wines. J. Agric. Food Chem. 2007, 55, 4501–4510.

311 312 313 314 315 316 317 318 319 320 321 322

(35) Setyaningsih, W.; Saputro, I. E.; Palma, M.; Barroso, C. G. Pressurized liquid extraction of phenolic compounds from rice (Oryza sativa) grains. Food Chem. 2016, 192, 452–459. (36) Roy, J. J.; Abraham, T. E. Preparation and characterization of cross-linked enzyme crystals of laccase. J. Mol. Catal. B: Enzym. 2006, 38, 31–36. (37) Eichlerová, I.; Šnajdr, J.; Baldrian, P. Laccase activity in soils: considerations for the measurement of enzyme activity. Chemosphere 2012, 88, 1154–1160. (38) Kurniawati, S.; Nicell, J. A. Characterization of Trametes versicolor laccase for the transformation of aqueous phenol. Bioresour. Technol. 2008, 99, 7825–7834. (39) Doerge, D. R.; Divi, R. L.; Churchwell, M. I. Identification of the colored guaiacol oxidation product produced by peroxidases. Anal. Biochem. 1997, 250, 10–17. (40) Hwang, S.; Lee, C.-H.; Ahn, I.-S. Product identification of guaiacol oxidation catalyzed by manganese peroxidase. J. Ind. Eng. Chem. 2008, 14, 487–492.

323

(41) Moshtaghioun, S. M.; Haghbeen, K.; Sahebghadam, A. L.; Legge, R. L.;

324

Khoshneviszadeh, R.; Farhadi, S. Direct spectrophotometric assay of laccase using diazo

325

derivatives of guaiacol. Anal. Chem. 2011, 83, 4200–4205.

15 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 22

326

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

327

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

328

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

329 330

(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.

331

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

332

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

333

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

334

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

335

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

336

Sens. Actuators B 2015, 213, 322–328.

337 338

16 ACS Paragon Plus Environment

Page 17 of 22

Journal of Agricultural and Food Chemistry

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.

347

17 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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.

356 357

18 ACS Paragon Plus Environment

Page 19 of 22

Journal of Agricultural and Food Chemistry

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

358

0.5

1.0

1.5

Time (h) 19 ACS Paragon Plus Environment

2.0

2.5

Journal of Agricultural and Food Chemistry

Page 20 of 22

359 360

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

361

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

362 363

20 ACS Paragon Plus Environment

Page 21 of 22

Journal of Agricultural and Food Chemistry

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

21 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

373 374

Table of Contents Graphics

375

376

22 ACS Paragon Plus Environment

Page 22 of 22