Article pubs.acs.org/JAFC
Leaching-Resistant Carrageenan-Based Colorimetric Oxygen Indicator Films for Intelligent Food Packaging Chau Hai Thai Vu and Keehoon Won* Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, 30 Pildong-ro 1-gil, Jung-gu, Seoul 100-715, Republic of Korea ABSTRACT: Visual oxygen indicators can give information on the quality and safety of packaged food in an economic and simple manner by changing color based on the amount of oxygen in the packaging, which is related to food spoilage. In particular, ultraviolet (UV)-activated oxygen indicators have the advantages of in-pack activation and irreversibility; however, these dye-based oxygen indicator films suffer from dye leaching upon contact with water. In this work, we introduce carrageenans, which are natural sulfated polysaccharides, to develop UV-activated colorimetric oxygen indicator films that are resistant to dye leakage. Carrageenan-based indicator films were fabricated using redox dyes [methylene blue (MB), azure A, and thionine], a sacrificial electron donor (glycerol), an UV-absorbing photocatalyst (TiO2), and an encapsulation polymer (carrageenan). They showed even lower dye leakage in water than conventional oxygen indicator films, owing to the electrostatic interaction of anionic carrageenan with cationic dyes. The MB/TiO2/glycerol/carrageenan oxygen indicator film was successfully bleached upon UV irradiation, and it regained color very rapidly in the presence of oxygen compared to the other waterproof oxygen indicator films. KEYWORDS: UV-activated oxygen indicators, carrageenans, water resistance, dye leakage, intelligent food packaging
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INTRODUCTION Food packaging has evolved to satisfy consumer demands for quality and safety; an intelligent packaging system senses, communicates, and monitors the conditions of packaged food to give information about food quality, safety, and history during transport and storage. This innovative packaging can provide great benefits to consumers and the food industry, and thus, it is becoming more important.1−5 Various variables related to food quality and safety are monitored in intelligent packaging: temperature, oxygen, carbon dioxide, toxins, etc.2,4,6−8 In particular, oxygen has a significant effect on the spoilage process of several products, and hence is removed in food packaging by modifying the atmosphere with gases, such as nitrogen and/or using oxygen absorbers/scavengers.4,9,10 However, the oxygen level in the package headspace can increase with time because of poor sealing, air permeation through the package materials, and package tampering or damage during storage and/or transportation. As a result, the decay of food is accelerated; therefore, the absence of oxygen should be assured by sensing the amount of oxygen in the package. Whereas conventional oxygen-detection methods require expensive instruments and trained operators, colorimetric oxygen indicators are cheap and enable consumers to sense the presence of oxygen in food packages with the naked eye.11 Among the several types of visual oxygen indicators, ultraviolet (UV)-activated oxygen indicators have been attracting great attention, because they possess many properties of ideal oxygen indicators. For example, they exhibit an irreversible response toward oxygen (i.e., preventing false indications), and they are not activated until irradiated with UV light, which allows for in-pack activation and a longer shelf life (even under aerobic conditions). They lose their color rapidly © XXXX American Chemical Society
upon exposure to UV light, remain colorless without oxygen, and regain their original color with oxygen.12,13 Typically, UVactivated oxygen indicator ink {which is composed of a redox dye [e.g., methylene blue (MB)], a sacrificial electron donor (e.g., glycerol), and an UV-absorbing photocatalyst (e.g., TiO2 nanoparticles)} is spread onto the inner side (i.e., food contact side) of food package films and then encapsulated in a polymer film (e.g., zein).11 When it contacts water contained in food, however, the oxygen indicator film suffers from the leaching of redox dyes from the film, which not only lowers the indication efficiency but also potentially leads to health problems (e.g., diarrhea, gastritis, nausea, and vomiting).14,15 Moreover, under the new regulations, intelligent packaging systems should not release their constituents into the packaged food and may be separated from the food by a barrier to prevent substances behind the barrier from migrating into the food.16 Many attempts have been made to address this problem;17−21 for example, Mills et al. employed synthetic sulfonated polystyrene and low-density PE as the coating polymer, but these polymers resulted in very slow color recovery under aerobic conditions.17,19 In this study, as the encapsulating polymer for water-resistant oxygen indicator films, we introduce carrageenans, which are natural sulfated polysaccharides extracted from edible red seaweed and used extensively in the food industry as gelling and thickening agents, edible films, and coatings.22−24 We present the carrageenan-based UV-activated oxygen indicator, which is Received: March 30, 2014 Revised: June 24, 2014 Accepted: June 30, 2014
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−Δb* = b0* − bt*
characterized by not only resistance to dye leaching but also rapid color recovery in the presence of oxygen.
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where b0* is the initial b* value of samples and bt* is their b* value at a specific time t. The difference of the b* value has been successfully used as a measure of the MB color.19
MATERIALS AND METHODS
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Materials. MB, azure A (AA), thionine (Th), titanium(IV) oxide (TiO2, AEROXIDE P25, nanopowder, 21 nm particle size), glycerol, zein from maize, and kappa (κ)- and iota (ι)-carrageenans were purchased from Sigma-Aldrich (St. Louis, MO) and used as received without further purification. The film for coating the oxygen indicator ink was a commercial nylon/polyethylene (PE) vacuum packaging film of 0.15 mm thickness (Pack4U, South Korea). All experiments were conducted in triplicate. Carrageenan-Based Oxygen Indicator Film Preparation. Indicator ink consisted of 40 mg of MB, 1.2 g of TiO2, and 1.2 g of glycerol. All of these components were added to 3.2 g of aqueous ethanol solution (90%) and then mixed thoroughly by a magnetic stirrer, followed by 5 min of ultrasonic dispersion (Vibra-Cell VCX750, Sonics and Materials, Inc., Newtown, CT). The packaging film (4 × 4 cm) was spin-coated with the ink at 5000 rpm for 30 s using a spin coater (WS-400B-6NPP-LITE, Laurell Technologies Corporation, North Wales, PA) and dried. The resultant film was then dipped into ι-carrageenan solutions (0.2, 0.3, 0.4, and 0.5%, w/v) using a dip coater (KSV-DC, KSV Instruments, Bridgeport, CT) at an immersion and withdrawal speed of 85 mm/min and finally spun at 2000 rpm for 30 s. The thickness of the carrageenan-based indicator films was measured using a thickness gauge (ID-S112B, Mitutoyo, Japan) and presented with an average of three samples with eight measurements along the edge of each film. Zein-Based Oxygen Indicator Film Preparation. Zein (20%)based indicator ink was prepared by adding 40 mg of MB, 1.2 g of TiO2, 1.2 g of glycerol, and 0.8 g of zein to 3.2 g of ethanol solution (90%) and then mixing with ultrasonication. The packaging film (4 × 4 cm) was spin-coated with the ink at 5000 rpm for 30 s and then allowed to dry in the dark. Dye Leaching of Oxygen Indicator Films into Water. The oxygen indicator films were submerged in a beaker filled with distilled water for 1 day, and leached dyes were quantified at 1, 3, 6, 12, and 24 h by measuring the absorbance at λmax of each dye with a Varian Cary 50 UV−vis spectrophotometer. Dye leakage (%) was defined as a ratio of the amount of dye leaching into the water for a given time to the initial amount of dye on the film; thus, dye leakage of 100% indicates that all of the dye leached from the film into the water. The initial dye amounts were determined by measuring the absorbance after immersing the films in vigorously stirred solutions for 3 h: 70% ethanol for the zein-based films and HCl solution (2 M) for the carrageenan-based films. Activation and Recovery of Oxygen Indicator Films. For bleaching of the oxygen indicator film, UV irradiation was carried out using a CL-1000 UV cross-linker (UVP, Upland, CA) equipped with five tubes of UVC lamps (8 W each). The irradiation intensity measured with a UVX digital radiometer (UVP, Upland, CA) was 5.5 mW/cm2. For color recovery, the photobleached film was placed in ambient air, and its color change was monitored using a CM-2600d spectrophotometer (Konica Minolta, Tokyo, Japan). Color Measurement. Several systems for expressing color numerically (i.e., color space) were developed by an international organization concerned with issues of lighting and color, the International Commission on Illumination (usually abbreviated CIE for its French name, Commission Internationale de l’Éclairage). The L*a*b* color space (also referred to as CIELAB) devised in 1976 is the most widely used, owing to the uniform distribution of colors.25 In this space, L* indicates lightness, ranging from 0 (black) to 100 (white), while a* and b* are the chromaticity coordinates that indicate color directions: +a* is the red direction; −a* is the green direction; +b* is the yellow direction; and −b* is the blue direction. Because the blue MB-based indicator loses its color during photobleaching and regains the color during recovery, the main color change happens in the b* axis and can be defined as −Δb*:
RESULTS AND DISCUSSION Preparation of Carrageenan-Based Oxygen Indicator Films. To begin with, we examined whether carrageenans can interact with redox dyes. Two major classes of carrageenans were employed: κ- and ι-carrageenans. Carrageenans are linear, partially sulfated galactans that mainly consist of alternating 3linked β-D-galactopyranose (G) and 4-linked 3,6-anhydro-α-Dgalactopyranose (DA); κ-carrageenan (G4S-DA) has one sulfate group covalently coupled via ester linkage to the carbon atom C-4 of the G residue (G-4), while ι-carrageenan (G4SDA2S) possesses two sulfate groups at the G-4 and DA-2.22 Three types of thiazine dyes were used: MB, AA, and Th. When 0.2 mL of each aqueous dye solution (10 mg/mL) was added to 10 mL of ι-carrageenan solution (2.5 mg/mL), insoluble stringy aggregates were formed with all of the dyes tested; however, aggregation with κ-carrageenan was barely observed with the naked eye in all cases (data not shown). This can be explained by the difference in the sulfate content of the carrageenans; typically, commercial κ- and ι-carrageenans contain 22 and 32% (w/w) sulfate, respectively, although they vary considerably according to seaweed species and batches.23 It was demonstrated that the sulfate moiety of carrageenans is essential for the interaction with cationic dyes, and the binding activity increases when the percentage of sulfate is increased.26−28 In the present work, only ιcarrageenan was used for further study and carrageenan refers to ι-carrageenan unless otherwise mentioned. We prepared UV-activated oxygen indicator films using carrageenan as the coating polymer; the packaging film was spin-coated with ink composed of redox dyes, glycerol, and TiO2 and then dip-coated with carrageenan solutions (see the Materials and Methods). For comparison, the zein-based film was also fabricated in the same manner as the carrageenanbased film. However, little dye remained on the film because the dyes dissolved into the zein solution (90% ethanol) during the dip-coating step.20 Therefore, the indicator films using zein as the encapsulation polymer were prepared by spin-coating the packaging film with the ink comprising redox dyes, glycerol, TiO2, and zein, as described in the Materials and Methods. For reference, the carrageenan-based film could not be prepared in this manner because the addition of the dyes to the aqueous carrageenan solution led to the formation of insoluble aggregates as already mentioned. Leakage Behaviors of Redox Dyes from the Films. Because MB is readily soluble in water, without proper protective polymers, the dye in the indicator film leaches out very quickly when immersed in water.17 With the waterinsoluble zein polymer (5%), the water resistance of the MBcoated indicator film was investigated by immersing the film in water for 24 h. As shown in Figure 1 (closed circles), after MB leached out quite quickly within the first hour of immersion, the dye leakage reached 48% and then remained nearly constant. The resultant MB/TiO2/glycerol/zein (5%) film was so light-colored that it was not suitable for a colorimetric oxygen indicator. Although water-insoluble zein covered MB, it could not completely prevent the small water-soluble ionic B
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with AA and Th in the same way as with MB; for comparison, the zein (20%)-based films were also fabricated. The leakages of the three dyes after 24 h of immersion in water are shown in Figure 3: zein (open bars) and carrageenan (closed bars).
Figure 1. Dye leakage of zein (5%)-based and ι-carrageenan (0.2%)based oxygen indicator films as a function of the water immersion time.
molecules (about 16.9 Å in length, 7.4 Å in breadth, and 3.8 Å in thickness) from leaking into the water.29 The same experiment was carried out using water-soluble carrageenan (0.2%) as the encapsulating polymer; surprisingly, only 2.1% of MB on the film leached into the water (open circles in Figure 1). This high resistance to dye leakage is believed to be attributed to the electrostatic interaction of the anionic polymer and the cationic dye.26−28 Carrageenan is negatively charged because of the two sulfated groups on the repeating dimer, whereas MB, generally available as chloride salts, exists in cationic form in water. The concentration effects of each polymer were examined for the protection ability against dye leakage: 5, 10, 15, and 20% for zein and 0.2, 0.3, 0.4, and 0.5% for carrageenan. When the zein concentration was increased from 5 to 20%, the dye leakage gradually declined to around 30% but it was still high (Figure 2a). The dye leakage of the carrageenan-based film slightly decreased from 2.1 to 1.8% when the concentration was increased from 0.2 to 0.5%, indicating that carrageenan is effective enough at as low a concentration as 0.2% (Figure 2b). We examined whether carrageenan can also prevent dyes other than MB from leaching from the film into water. The carrageenan (0.2%)-based oxygen indicator films were prepared
Figure 3. Leaching behaviors of MB, AA, and Th dyes on zein (20%)based and ι-carrageenan (0.2%)-based oxygen indicator films.
Similar to MB, the leakages of AA and Th were considerable with the zein polymer, particularly Th. The reason why Th showed the highest leakage is not evident, but this could be due to the larger contact area of Th with water; Th (with no methyl group) is more hydrophilic than AA (with two methyl groups) and MB (with four methyl groups).30 In comparison to zein, carrageenan diminished the leakage of AA and Th as well as MB to a greater degree because of the electrostatic interaction of the anionic carrageenan with the cationic dyes. In addition, the dye leakage might be further lowered by additional optimization (e.g., the employment of carrageenans with higher percentages of sulfate and/or with other molecular weights).22,23 Photobleaching and Recovery Process of Carrageenan-Based Indicator Film. An UV-activated indicator film should be bleached when exposed to UV light (photobleaching step) and regain its color in the presence of oxygen (recovery step). Because the strong interaction between carrageenans and dyes might impede the photobleaching and recovery processes of the carrageenan-based indicator film, we investigated this possibility. The oxygen indicator films were prepared using MB and carrageenan (0.2, 0.3, 0.4, and 0.5%) as the redox dye and the coating polymer, respectively. When the carrageenan-coated films were irradiated with UVC light (intensity = 5.5 mW/cm2) for 4 min, all of the films were completely bleached (data not shown). The finely dispersed TiO2 nanoparticles (photocatalyst) encapsulated in the polymer generate electron−hole pairs upon UV irradiation. The photogenerated holes oxidize glycerol (a sacrificial electron donor), and the photogenerated electrons reduce MB to its colorless reduced form.12 The initial rate of photobleaching was also measured as a function of the carrageenan concentration. Figure 4a (circles) shows that the bleaching rate decreased as the polymer concentration increased. This may not be due to the increase in the thickness of the carrageenan film with the increasing concentration because the difference in the thickness was not significant, as shown in Figure 4a (bars); the reason is a question for future research. Figure 4b shows the recovery behavior of the MB/TiO2/ glycerol/carrageenan (0.2%) indicator film in air under ambient conditions in which −Δb* was plotted against recovery time. The oxygen indicator film was recovered within about 8 h, The colorless reduced form of MB on the film was reoxidized by oxygen, and the original blue color of the indicator film was
Figure 2. Effect of the polymer concentration on MB leakage after 24 h of (a) zein-based and (b) ι-carrageenan-based oxygen indicator films. C
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(20133030000300), and the Korea Carbon Capture and Sequestration (CCS) Research and Development (R&D) Center (KCRC) Program (2013M1A8A1038187) of the Ministry of Science, Information/Communications Technology (ICT), and Future Planning, Republic of Korea. Notes
The authors declare no competing financial interest.
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(1) Brody, A. L.; Bugusu, B.; Han, J. H.; Sand, C. K.; Mchugh, T. H. Innovative food packaging solutions. J. Food Sci. 2008, 73, R107− R116. (2) De Jong, A. R.; Boumans, H.; Slaghek, T.; van Veen, J.; Rijk, R.; van Zandvoort, M. Active and intelligent packaging for food: Is it the future? Food Addit. Contam. 2005, 22, 975−979. (3) Heising, J. K.; Dekker, M.; Bartels, P. V.; van Boekel, M. A. J. S. Monitoring the quality of perishable foods: Opportunities for intelligent packaging. Crit. Rev. Food Sci. Nutr. 2014, 54, 645−654. (4) 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. (5) Yam, K. L.; Takhistov, P. T.; Miltz, J. Intelligent packaging: Concepts and applications. J. Food Sci. 2005, 70, R1−R10. (6) Kim, K.; Kim, E.; Lee, S. J. New enzymatic time-temperature integrator (TTI) that uses laccase. J. Food Eng. 2012, 113, 118−123. (7) Jang, N. Y.; Won, K. New pressure-activated compartmented oxygen indicator for intelligent food packaging. Int. J. Food Sci. Technol. 2014, 49, 650−654. (8) Jung, J.; Puligundla, P.; Ko, S. Proof-of-concept study of chitosanbased carbon dioxide indicator for food packaging applications. Food Chem. 2012, 135, 2170−2174. (9) Feng, S.; Luo, Z.; Shao, S.; Wu, B.; Ying, T. Effect of relative humidity and temperature on absorption kinetics of two types of oxygen scavengers for packaged food. Int. J. Food Sci. Technol. 2013, 48, 1390−1395. (10) Lee, K.-E.; Kim, H. J.; An, D. S.; Lyu, E. S.; Lee, D. S. Effectiveness of modified atmosphere packaging in preserving a prepared ready-to-eat food. Packag. Technol. Sci. 2008, 21, 417−423. (11) Mills, A. Oxygen indicators and intelligent inks for packaging food. Chem. Soc. Rev. 2005, 34, 1003−1011. (12) Lee, S.-K.; Mills, A.; Lepre, A. An intelligence ink for oxygen. Chem. Commun. 2004, 1912−1913. (13) Mihindukulasuriya, S. D. F.; Lim, L.-T. Oxygen detection using UV-activated electrospun poly(ethylene oxide) fibers encapsulated with TiO2 nanoparticles. J. Mater. Sci. 2013, 48, 5489−5498. (14) Ghosh, D.; Bhattacharyya, K. G. Adsorption of methylene blue on kaolinite. Appl. Clay Sci. 2002, 20, 295−300. (15) Paul, P.; Kumar, G. S. Targeting ribonucleic acids by toxic small molecules: structural perturbation and energetics of interaction of phenothiazinium dyes thionine and toluidine blue O to tRNAphe. J. Hazard. Mater. 2013, 263, 735−745. (16) Restuccia, D.; Spizzirri, U. G.; Parisi, O. I.; Cirillo, G.; Curcio, M.; Iemma, F.; Puoci, F.; Vinci, G.; Picci, N. New EU regulation aspects and global market of active and intelligent packaging for food industry applications. Food Control 2010, 21, 1425−1435. (17) Mills, A.; Hazafy, D.; Lawrie, K. Novel photocatalyst-based colourimetric indicator for oxygen. Catal. Today 2011, 161, 59−63. (18) Mills, A.; Lawrie, K. Novel photocatalyst-based colourimetric indicator for oxygen: Use of a platinum catalyst for controlling response times. Sens. Actuators, B 2011, 157, 600−605. (19) Mills, A.; Lawrie, K.; Bardin, J.; Apedaile, A.; Skinner, G. A.; O’Rourke, C. An O2 smart plastic film for packaging. Analyst 2012, 137, 106−112. (20) Vu, C. H. T.; Won, K. Novel water-resistant UV-activated oxygen indicator for intelligent food packaging. Food Chem. 2013, 140, 52−56. (21) Vu, C. H. T.; Won, K. Bioinspired molecular adhesive for waterresistant oxygen indicator films. Biotechnol. Prog. 2013, 29, 513−519.
Figure 4. (a) Initial bleaching rate and coating thickness of MB/TiO2/ glycerol/ι-carrageenan oxygen indicator films as a function of the carrageenan concentration. (b) Recovery behavior of MB/TiO2/ glycerol/ι-carrageenan (0.2%) oxygen indicator film in air under ambient conditions with a sampling time of 30 min.
restored. This recovery time may not be satisfactory if compared to conventional water-susceptible indicator films (∼6 min),12 but it is appreciably short considering that the other waterproof films had recovery times of 5 days using sulfonated polystyrene17 and 2.5 days using low-density PE.19 The high recovery rate of the carrageenan-based film is probably attributed to the hydrophilic nature of carrageenans, because hydrophobic films hinder the formation of the charged species, MB+ and OH−, for color recovery.17,19 In conclusion, we have developed an UV-activated oxygen indicator film resistant to dye leaching by introducing carrageenans, which are natural sulfated polysaccharides. The dye-binding ability of carrageenans substantially lowered the leakage into water of all of the redox dyes tested (MB, AA, and Th). The MB/TiO2/glycerol/carrageenan oxygen indicator film was successfully bleached upon UV irradiation and regained its color very rapidly in the presence of oxygen. This leaching-resistant colorimetric oxygen indicator film with in-pack activation and irreversibility can be applied to verify that all oxygen is removed using oxygen absorbers/oxygen scavengers, and it can also be used as a seal and leak detector with modified atmosphere packaging; it will be essential for intelligent food packaging.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Telephone: +82-2-2260-8922. Fax: +82-2-2268-8729. E-mail:
[email protected]. Funding
This research was supported by the Agriculture Research Center Program of the Ministry of Agriculture, Food, and Rural Affairs (ARC-710003-03-4-SB120), the New and Renewable Energy of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea Government Ministry of Trade, Industry, and Energy D
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