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Influence of non-migratory metal-chelating active packaging film on food quality: Impact on physical and chemical stability of emulsions
Food Chemistry 151 (2014) 257–265
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Influence of non-migratory metal-chelating active packaging film on food quality: Impact on physical and chemical stability of emulsions Fang Tian, Eric A. Decker, D. Julian McClements, Julie M. Goddard ⇑ Department of Food Science, University of Massachusetts, Chenoweth Lab, 102 Holdsworth Way, Amherst, MA 01003, United States
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Article history: Received 22 July 2013 Received in revised form 24 September 2013 Accepted 13 November 2013 Available online 21 November 2013 Keywords: Active packaging film Metal-chelating Non-migratory Lipid oxidation Emulsion Antioxidant
a b s t r a c t Previously, we developed a novel metal-chelating packaging film (PP-g-PAA) by grafting acrylic acid (AA) monomer from polypropylene (PP) film surface, and demonstrated its potential in controlling ironpromoted lipid oxidation. Herein, we further established the industrial practicality of this active film. Specifically, the influence of film surface area-to-product volume ratio (SA/V) and product pH on the application of the film was investigated using an oil-in-water emulsion system. The films equally inhibited lipid oxidation throughout the range of SA/V ratios tested (2–8 cm2/ml). PP-g-PAA films were most effective at pH 7.0, and the activity decreased with decreasing pH. The particle size examination of emulsions indicated no adverse influence from the active film on the stability of this emulsion system. FTIR analysis suggested a non-migratory nature of PP-g-PAA films. These results provide fundamental knowledge that will facilitate the application of this effective and economical active packaging film in the food industry. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Active packaging is an innovative technology that has gained considerable interest for the purpose of controlling lipid oxidation in recent years (Byun, Bae, & Whiteside, 2012; Pereira de Abreu, Cruz, & Paseiro Losada, 2012; Tian, Decker, & Goddard, 2013). Coating active substances onto or incorporating them throughout the packaging material are the most commonly applied approaches to develop antioxidant packaging films (Lopez-de-Dicastillo et al., 2011; Unalan, Korel, & Yemenicioglu, 2011). However, covalent immobilisation of active agents onto the food contact surface of a packaging film is gaining industry attention, as the active substance is unlikely to migrate from the package into the food (Arrua, Strumia, & Nazareno, 2010; Goddard, Talbert, & Hotchkiss, 2007; Tian, Decker, & Goddard, 2012a, 2012b). A potential regulatory advantage of covalently immobilised active substances is that they may only require food contact notification approval rather than approval and labelling as a direct food additive (Anonymous, 2005; Koontz, 2012). Hence, non-migratory active packaging technologies provide food manufacturers with the possibility to obtain cleaner labels by removing synthetic additives (e.g. EDTA, etc.) from food formulations. In our previous work, a non-migratory active packaging film was successfully developed by photografting metal-chelating polymer poly(acrylic acid) (PAA) from polypropylene (PP) film surface ⇑ Corresponding author. Tel.: +1 413 545 2275; fax: +1 413 545 1262. E-mail address: [email protected] (J.M. Goddard). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.074
(Tian et al., 2012a, 2012b). The antioxidant ability of the PAA grafted PP film (PP-g-PAA) was preliminarily examined in a commercial soybean oil-in-water emulsion system, typical of many lipid containing food products (e.g. milk, beverages, dressings, sauces, desserts, etc.) (Waraho, Cardenia, Rodriguez Estrada, McClements, & Decker, 2009). PP-g-PAA films were shown to significantly extend the lag phase of lipid oxidation in the emulsion system by a factor of 4–5 times, compared to the native PP film. While our prior work focused on the synthesis and material characterisation of the film, the objective of the current work is to better establish the industrial practicality of this novel active packaging film. Specifically, the influence of film surface area to product volume ratio (SA/V), product pH, and migration of ungrafted oligomeric or polymeric chelators on the commercial application of the film were investigated using a model oil-in-water emulsion system. Herein we report on the performance of the developed PP-gPAA film in emulsion systems with SA/V ratios in the range of 2–8 cm2/ml, to better understand its commercial potential in food industry applications. Furthermore, the influence of pH on the ability of the chelating ligands to bind to metal ions was established, since this is known to have a major impact on their ability to inhibit lipid oxidation (Kontoghiorghes, Pattichis, Neocleous, & Kolnagou, 2004). Often, a chelator has one or more acidic chelating ligands (e.g. -COOH, -NOH, -SH, etc.), and the proton of these groups can be displaced by the metal ion and sequester it from solution (Kontoghiorghes et al., 2004). The activity of these acidic functional groups is therefore expected to be influenced by the
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pH of the reaction system (e.g. food product). PAA contains a large number of carboxylic acids (–COOH) as the chelating ligand, which is especially sensitive to the pH of the surrounding solution. We therefore evaluated the effectiveness of our PP-g-PAA films against oil-in-water emulsions at pH 3.0, 5.0 and 7.0, to define suitable food systems for this active packaging film. The grafting chemistry used to generate PP-g-PAA films was designed to result in nonmigratory active packaging, in which the active agent (PAA chelators) is unlikely to migrate into the packaged food. We therefore conducted a series of control experiments to ensure no residual substances transferred from the developed active film into the food systems. Finally, we examined the influence of the active film on the stability and interfacial charge of emulsion droplets at different pH. 2. Materials and methods 2.1. Materials Commercial soybean oil (Wesson, 100% natural vegetable oil) was purchased from the local grocery store; polypropylene (PP, isotactic, pellets) was purchased from Scientific Polymer Products (Ontario, NY); hydroxylamine hydrochloride, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p0 -disulfonic acid disodium salt hydrate (ferrozine, 98+%), imidazole (99%) and ethylenediaminetetraacetic acid (EDTA) were purchased from Acros Organics (Morris Plains, NJ); toluidine blue O (TBO) was purchased from MP Biomedicals (Solon, OH); 2-propanol, acetone, heptane, hexanes, ethanol, isooctane, methanol, 1-butanol, sodium acetate trihydrate, hydrochloric acid, acetic acid glacial, Brij 35 (a non-ionic surfactant), ferric chloride anhydrous, trichloroacetic acid (TCA), 4-(2-hydroxyethyl)-1piperazineethane-sulfonic acid (HEPES) and sodium hydroxide were purchased from Fisher Scientific (Fair Lawn, NJ); acrylic acid (AA, anhydrous), barium chloride dihydrate, ammonium thiocyanate, cumene hydroperoxide (80%), hexanal (98%), silicic acid (100–200 mesh, 75–150 lm, acid washed), activated charcoal (100–400 mesh) and benzophenone (BP, 99%) were purchased from Sigma–Aldrich (St. Louis, MO); all the chemicals and solvents were used without further purification. 2.2. Preparation of PP films PP pellets were cleaned by sonication in 2-propanol, acetone and deionised water (2 times per solvent, 10 min each time) sequentially. The cleaned PP pellets were dried in a desiccator overnight (25 °C, 15% RH), and then used for the preparation of PP films using a Carver Laboratory Press (Model B, Fred S. Carver Inc., NJ). PP pellets were pressed into films at 160 °C with a load force of 9000 lbs. Pressed PP films (average thickness: 225 ± 25 lm) were cut into 2 2 cm pieces and went through the same cleaning and drying process as PP pellets. 2.3. Preparation of PP-g-PAA films The chelating monomer AA was covalently grafted from PP film surface by a two-step photo-initiated graft polymerization approach. This approach adopted the procedure reported by Ma, Davis, and Bowman (2000) and Tian et al. (2012a, 2012b) with some modifications. In this work, spin coating and a Dymax light-curing system were applied (rather than dip coating and a spot UV lamp) to graft a more uniform PAA layer on the PP film surface. Briefly, 30 ll of BP solution (5 wt% in heptane) was spin-coated on each side of PP films at a speed of 2000 rpm for 10 s. The BPcoated films (2 2 cm) were then cut into 1 2 cm films, and each
piece of film went through the same grafting procedure described previously. (Tian et al., 2012a, 2012b) The UV irradiation was conducted in a Dymax light-curing system (Model 5000, 320–395 nm, 200 mW/cm2, Dymax Corporation, Torrington, CT). BP-coated films were exposed to 90 s of UV irradiation, followed by rinsing in acetone (3 times, 5 min each time). PP–BP films were then submerged in 25 wt% AA in ethanol, nitrogen flushed and exposed to UV irradiation for 6 min. Finally, PP-g-PAA films were rinsed in water as described previously to remove any unreacted monomer and generated homopolymers. 2.4. Quantification of carboxylic acids The carboxylic acid density of PP-g-PAA film surface was determined by the TBO dye assay adapted from Kang, Tan, Kato, Uyama, and Ikada (1996) and Uchida, Uyama, and Ikada (1993) with some modifications. Briefly, TBO solution (0.5 mM) was prepared by dissolving TBO dye into pH 10.0 deionized water (adjusted by NaOH). PP and PP-g-PAA films were then put into the prepared TBO solution (1 piece in 5 ml, n = 6) for 2 h with shaking (25 °C) to allow TBO dye to complex with carboxylic acids. After rinsing loosely adsorbed dye, the complexed dye was desorbed by submerging films in 50 wt% acetic acid solution. After 15 min of shaking, the absorbance in the solutions was detected at 633 nm. The amount of available carboxylic acids on film surfaces was calculated using a standard curve made from TBO dye in 50 wt% acetic acid solution. 2.5. Iron chelating assay The ferric iron (Fe3+) chelating activity of the PP-g-PAA film was determined by the ferrozine colorimetric assay based on methods reported by Bou et al. (2008) and Tian et al. (2012a, 2012b) with some modifications. Iron stock solution (20 mM) was prepared by dissolving ferric chloride anhydrous in a 0.05 M HCl solution. The stock solution was then diluted to 0.08 mM by a sodium acetate/imidazole (0.05 M, pH 5.0) buffer to make the Fe3+ chelating solution. Three groups of samples were prepared for the chelation study, including Fe3+ solution without films as the control, Fe3+ solution with native PP films and Fe3+ solution with PP-g-PAA films. One piece of film (1 2 cm) was put into 10 ml of Fe3+ solution serving as a replicate, with three replicates for each determination. Samples were incubated in the dark for 24 h at room temperature (25 °C) with shaking. The Fe3+ chelating activity of films was determined by calculating the difference of Fe3+ concentration in Fe3+ chelating solution with films against the control group. The colourimetric ferrozine assay was used to measure the Fe3+ concentration. As only Fe2+ reacts with ferrozine to form a red colour complex, a reducing agent (5 wt% hydroxylamine hydrochloride, 10 wt% trichloroacetic acid) was prepared to convert Fe3+ to Fe2+. The Fe3+ chelating solution (0.5 ml) was mixed with 0.25 ml of reducing agent, followed by adding 0.25 ml of ferrozine solution (18 mM in 0.05 M HEPES buffer, pH 7.0). The reaction solution was incubated at room temperature for 1 h with shaking and the absorbance was detected at 562 nm. The Fe3+ concentration was calculated compared with a standard curve made of ferric chloride anhydrous. 2.6. Preparation of stripped soybean oil Stripped soybean oil was prepared by the method reported by Waraho et al. (2009). Chromatographic column separation was used to strip undesired compounds (e.g. a-tocopherol, free fatty acids, pigments, etc.) from commercial soybean oil with silicic acid and activated charcoal as the packing material. Silicic acid (50 g) was washed three times in deionized water to remove any small
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and damaged particles, and then filtered through a Buchner funnel. Activated charcoal (5.625 g) and washed silicic acid were dried at 110 °C for 24 h, followed by suspension in hexanes. Silicic acid (22.5 g), activated charcoal (5.625 g) and additional silicic acid (22.5 g) were packed sequentially in a chromatographic column (35 3.0 cm i.d.). Commercial soybean oil (30 g) was dissolved in hexanes (30 ml), loaded onto the column and then 270 ml of hexanes was used to eluent the triacylglycerols. Silicic acid can remove substances such as lipid oxidation products, free fatty acids and antioxidants (e.g. a-tocopherol) from the commercial oil, while activated charcoal can remove any pigments. Hexanes in the collected stripped oil was removed by a vacuum rotary evaporator (RE 111 Buchi, Flawil, Switzerland) and nitrogen flushing was used to remove the final traces of hexane residue. The obtained stripped oil, which only contains triacylglycerols, was stored at 80 °C until use. 2.7. Lipid oxidation study Previously, commercial soybean oil was used to prepare the oil-in-water emulsion system to demonstrate the effectiveness of PP-g-PAA films in controlling lipid oxidation (Tian et al., 2012a, 2012b). However, the existence of other trace components (atocopherol, phospholipids, pigments, free fatty acids, etc.) in commercial soybean oil makes the lipid oxidation process much slower and more complicated than in stripped oil, which primarily only contains triacylglycerols (Mei, Decker, & McClements, 1998; Mei, McClements, Wu, & Decker, 1998; Waraho et al., 2009). To reduce the long lag phase of lipid oxidation, and also to better understand the influence of PP-g-PAA films on the emulsion system, stripped soybean oil was used to prepare the oil-in-water emulsion system used in this work. All determinations were conducted in triplicate, and the results presented here are representative of two independent studies. Stripped soybean oil (1 wt%) and Brij 35 (0.1 wt%) were dissolved in sodium acetate/imidazole buffer (0.05 M), and the mixture was blended for 2 min by a 2-speed hand held homogenizer (Biospec Products, Inc., Bartlesville, OK) at the low speed setting to prepare the coarse emulsion. The coarse emulsion was then passed through a microfluidizer (Microfluidics, Newton, MA) four times with a pressure of 9000 psi to obtain a fine emulsion. To measure the effect of SA/V ratio, native PP and PP-g-PAA films were cut into 1 1 cm squares (with grafting on both sides, 2 cm2 of surface area for each piece of film). Films were then put into 1 ml of oil-in-water emulsion (pH 7.0) to prepare emulsion samples with different surface area (2, 4, 6 and 8 cm2). Emulsion alone (no film) and emulsions with PP or PP-g-PAA films were incubated in the dark at 25 °C for 8 days, and the formation of lipid oxidation products (lipid hydroperoxides and hexanal) were determined throughout the study as described below. To evaluate the effect of pH on the activity of PP-g-PAA films (2 cm2) in controlling lipid oxidation, emulsions with different pH (3.0, 5.0 and 7.0) were prepared by the method described above. All emulsions (1 ml) were allowed to oxidise in the dark at 25 °C for up to 20 days. Emulsions with no film as well as emulsions containing EDTA (0.01 mM) were used as negative and positive controls, respectively. The formation of lipid oxidation products (lipid hydroperoxides and hexanal), the particle size and the droplet charge (zeta potential) of emulsions were determined during the incubation. Lipid hydroperoxides, a primary lipid oxidation product, were quantified using the colourimetric ammonium thiocyanate method (Alamed, McClements, & Decker, 2006; Shantha & Decker, 1994). Briefly, 0.3 ml of emulsion was mixed with 1.5 ml of isooctane/isopropanol (3:1 v/v) by vortex (3 times, 10 s/time). The mixed solution was centrifuged at 4000 rpm for 5 min to obtain an upper phase containing hydroperoxides. The upper phase solution
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(200 ll) was then collected and added into 2.8 ml of methanol/1butanol (2:1 v/v), followed by adding 15 ll of ammonium thiocyanate (3.94 M) and 15 ll of ferrous iron solution. The clear ferrous iron solution was prepared freshly by centrifugation (1000g, 2 min) of the mixture (1:1 v/v) of BaCl2 (dissolved in 0.4 M HCl, 0.132 M) and FeSO4 (0.144 M). The absorbance was quantified at 510 nm after 20 min of incubation at room temperature. Hydroperoxide concentrations were calculated using a standard curve made from cumene hydroperoxide. The lag phase of the hydroperoxide formation in emulsions was defined as the day before the significant increase of the hydroperoxide concentration compared to time zero concentrations (p < 0.05). Formation of hexanal (a secondary lipid oxidation product) in emulsions was measured using head space gas chromatography (GC) according to the method of Panya et al. (2010). The GC (Shimadzu GC-2014, Tokyo, Japan) was equipped with an auto-injector (AOC-5000, Shimadzu, Tokyo, Japan) and a flame ionisation detector (FID). Samples were pre-incubated at 55 °C for 8 min, and a divinylbenzene/carboxen/polydimethylsiloxane (DVB/Carboxen/ PDMS) stable flex solid phase microextraction fibre (50/30 lm, Supelco, Bellefonte, PA) was then exposed to the sample headspace for 2 min to adsorb volatile compounds evaporated from the emulsion, followed by the desorption at 250 °C for 3 min in the injector at a split ratio of 1:7. A fused-silica capillary column (30 m 0.32 mm i.d. 1 lm) coated with 100% poly(dimethylsiloxane) (Equity-1, Supelco) was used to separate volatile compounds generated from the lipid oxidation with the run time of 10 min for each sample. The temperatures of the injector, oven and detector were 250, 65 and 250 °C, respectively. The hexanal concentration was calculated by comparison to a standard curve made from emulsions containing hexanal and EDTA (0.2 mM). The lag phase of the hexanal formation in emulsions was defined as the day before the significant increase of the hexanal concentration compared to zero time concentrations (p < 0.05). The particle size distribution and the electrical charge of emulsion droplets were determined by Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK). Emulsions were diluted 100-fold with the corresponding buffer before the measurement. To measure the particle size of emulsion droplets, the diluted emulsion was loaded into the instrument and equilibrated for 120 s. Each sample was measured 2 times (11 runs for each time) and the Z-average mean diameter (nm) of droplets was collected. For the electrical charge of emulsions, each sample was measured 3 times (10 runs for each time) after 60 s of equilibrating and the zeta potential (mV) of emulsion droplets was reported.
2.8. Migration potential of monomers and homopolymers The likelihood for migration of residual AA monomers and generated PAA homopolymers (ungrafted PAA) was tested by a control study. The two-step graft polymerization approach used here is designed to prevent the generation of PAA homopolymers that may subsequently migrate from the film surface. Nevertheless, the polymerization between AA monomers could still occur without the photoinitiator (BP) dispersed in the monomer solution with UV irradiation and an oxygen-free environment (Ma et al., 2000). As the long-term goal of this research is the development of nonmigratory active packaging films, a control experiment was designed by preparing PP-g-PAA control films using the same grafting procedure with the exception of the BP coating step. The obtained PP-g-PAA control films went through the same washing steps as standard PP-g-PAA films: stir-washing in deionized water 30 min at room temperature, 1 h at 60 °C and finally 30 min at room temperature. Films were then dried overnight in a desiccator (25 °C, 15% RH) until further use.
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Fig. 1. Lipid hydroperoxide and hexanal concentrations of stripped soybean oil-in-water emulsions (pH 7.0) with different SA/V ratios stored at 25 °C and measured over 8 days. (A), (B), (C) and (D) correspond to the lipid hydroperoxide concentrations of emulsions with SA/V ratio of 2, 4, 6 and 8 cm2/ml, respectively; (E), (F), (G) and (H) correspond to the hexanal concentrations of emulsions with SA/V ratio of 2, 4, 6 and 8 cm2/ml, respectively.
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Fig. 2. Hexanal concentrations (A), lipid hydroperoxide concentrations (B), particle size (C) and zeta potential (D) of stripped soybean oil-in-water emulsions (pH 7.0) stored at 25 °C and measured over 8 days.
The surface chemistry of native PP, PP-g-PAA and PP-g-PAA control films was analysed by a FTIR spectrometer (IRPrestige-21, Shimadzu Scientific Instruments, Inc., Kyoto, Japan) equipped with a diamond ATR crystal. Each spectrum was an average of 32 scans at a 4 cm1 resolution compared to a background spectrum, which was carried out with an ATR crystal against air. A representative spectrum of each sample was plotted with SigmaPlot 12.0 (Systat Software, Inc., Chicago, IL). The migration potentials of oligomeric or polymeric PAA chelators were assessed by comparison of the surface chemistry of the films. 2.9. Statistical analysis The data were reported as means ± standard deviation (SD). Statistical analyses were conducted using SPSS Release 17.0 (SPSS Inc., Chicago, IL). The significance of differences was determined by the one-way analysis of variance (ANOVA) with Duncan’s pairwise comparison (p < 0.05). Two-tailed Pearson correlation was performed to assess the correlation relationship between variables. 3. Results and discussion 3.1. Carboxylic acid density The TBO dye assay was used to determine the amount of available carboxylic acids on native PP and PP-g-PAA film surfaces. After the polymerized grafting of PAA, a significantly higher amount of carboxylic acids (57 ± 4 nmol/cm2, n = 6) was detected on PP-gPAA film surfaces compared to that on native PP film surfaces (0 ± 0 nmol/cm2, n = 6). As each PP-g-PAA (1 2 cm) film was
prepared independently, the small standard deviation of the determination indicated that this active film could be produced uniformly and consistently. The results also suggested that a more uniform PAA grafting could be obtained by spin coating and using a Dymax light-curing system, as opposed to films prepared with dip coating and a spot UV lamp, which resulted in significantly higher deviations (69 ± 10 nmol/cm2) (Tian et al., 2012a, 2012b). 3.2. Iron chelating activity The Fe3+ chelating activity of PP and PP-g-PAA films was measured at pH 5.0. Like most metal chelators, PAA has higher affinity to Fe3+ than Fe2+, as previously reported (Tian et al., 2012a, 2012b). In addition, with carboxylic acid groups as the chelating ligands, the PAA/Fe complex should have similar stability constants to those of citric acid/Fe complex, which are 11.85 and 3.2 for Fe3+ and Fe2+, respectively (Furia, 1972). The stability constant of a complex is a measure of the strength of the interaction between a chelator and a metal ion. Higher constants represent more stable complexes. Difficulty was encountered in attempting to release Fe3+ from the PAA/Fe3+ complex formed on the PP-g-PAA film surface using the method described in our previous work (Tian et al., 2012a, 2012b). Therefore, in this work, we determined the Fe3+ chelating activity of the films by the difference of the Fe3+ concentration in the chelating solution with and without films rather than the direct measurement of the Fe3+ released from films. PP films showed no evident activity to chelate Fe3+ (0 ± 7 nmol/cm2, n = 3), while PP-g-PAA films exhibited a significant Fe3+ chelating activity (54 ± 11 nmol/cm2, n = 3). With a carboxylic acid density of 57 ± 4 nmol/cm2, the chelating ligand (-COOH) to Fe3+ ratio of PAA grafted on PP film surface is approximately 1.
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Fig. 3. Hexanal concentrations (A), lipid hydroperoxide concentrations (B), particle size (C) and zeta potential (D) of stripped soybean oil-in-water emulsions (pH 5.0) stored at 25 °C and measured over 14 days.
3.3. Effect of SA/V ratio The influence of the ratio of film surface area to emulsion volume (SA/V ratio) was analysed by measuring the formation of oxidation products over time in a 1 ml emulsion (pH 7.0) stored in contact with varying surface areas of native PP or PP-g-PAA films (2, 4, 6 and 8 cm2) (Fig. 1). As indicated by the 1 day lag time of both lipid hydroperoxides and hexanal formation in control emulsion (i.e. emulsion stored with no film), the stripped oil oxidised rapidly due to its lack of antioxidants. Hexanal formation occurred slightly slower in emulsions stored with native PP films compared to the control emulsion, more notably at higher SA/V ratios (6 and 8 cm2/ml) (Fig. 1G and H). The apparent extension in lag phase by the native PP films may be a result of the hydrophobic PP films floating on the surface of the emulsions, and its relatively low oxygen permeability coefficient at 25 °C (Kurek et al., 2011), which may inhibit the diffusion of oxygen into emulsions. Compared to the control emulsion and emulsions with native PP films, PP-gPAA films delayed lipid oxidation in the emulsions at all tested SA/V ratios. Compared to native PP films, PP-g-PAA films extended the lag phase of lipid hydroperoxides and hexanal formation by 1 day at all examined SA/V ratios. Results indicated that increasing the SA/V ratio from 2 to 8 cm2/ml had no significant effect on the ability of PP-g-PAA films to prevent oxidative deteriorations in the tested oil-in-water emulsion system. It has been reported that the solubility of Fe3+ at pH 7.0 is approximately 4 1017 M (Zumdahl, 1989), and the concentrations of iron in emulsion systems can be on the order of parts per billion (ng/ml), which is sufficient enough to accelerate lipid oxidative reactions (Alamed et al., 2006). At an iron chelating activity of 54 ± 11 nmol/cm2, 2 cm2 of
PP-g-PAA films could chelate approximately 6 103 ng of iron, which is much higher than the amount of iron naturally existing in 1 ml of emulsion (Alamed et al., 2006). It is therefore possible that at a SA/V ratio of 2 cm2/ml, the chelation reaction between transition metals and PP-g-PAA films is already saturated, and that increasing the SA/V ratio does not further extend the lag phase of lipid oxidation. 3.4. Effect of pH A SA/V ratio of 2 cm2/ml was used to determine the effect of pH on the activity of PP-g-PAA films in inhibiting lipid oxidation in oilin-water emulsions prepared at pH values of 7.0 (Fig. 2), 5.0 (Fig. 3) and 3.0 (Fig. 4). It is worth noting that there was an observable difference between the lag phase of control emulsion (i.e. no film) recorded in the SA/V ratio study (1 day, Fig. 1E) and that observed in the pH study (3 days, Fig. 2A). This is likely a result of the variations of ambient conditions, buffer, surfactant, homogenizer, etc. during the pre-homogenisation, homogenisation and post-homogenisation processes to prepare the emulsion. The variations could induce a slight difference in the metal concentration naturally present in emulsions, which in turn influences the lipid oxidation rate. Nevertheless, the emulsion system was consistent for each study, which enabled us to make comparisons between different treatments. PP-g-PAA films exhibited the greatest activity at pH 7.0 (Fig. 2), with an extension of the lag phase of the formation of lipid hydroperoxides from 1 to 3 days (Fig. 2B) and the formation of hexanal from 3 to 6 days (Fig. 2A) compared to the control emulsion and emulsions with native PP films. At pH 5.0, PP-g-PAA films prolonged the formation of lipid hydroperoxides and hexanal by 1
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Fig. 4. Hexanal concentrations (A), lipid hydroperoxide concentrations (B), particle size (C) and zeta potential (D) of stripped soybean oil-in-water emulsions (pH 3.0) stored at 25 °C and measured over 20 days.
Table 1 Correlation relationships between the hexanal concentration and the zeta potential of emulsions during the storage at 25 °C. pH
Film treatment
Correlation coefficient (r)
Significance
7.0
Control (no film) PP PP-g-PAA Control (no film) PP PP-g-PAA Control (no film) PP PP-g-PAA
0.938 0.908 0.821 0.978 0.981 0.84 0.495 0.251 0.242
p < 0.01 p < 0.01 p < 0.05 p < 0.01 p < 0.01 p < 0.01 p > 0.05 p > 0.05 p > 0.05
5.0
3.0
and 2 days (Fig. 3B and A), respectively. Under the accelerated conditions tested (i.e. stripped oil at 25 °C), PP-g-PAA films were not able to retard lipid oxidation at pH 3.0 (Figs. 4A and B). Results indicated that the ability of the PP-g-PAA films to inhibit lipid oxidation increased with increasing pH, which is consistent with expectations. The apparent acid dissociation constant (pKa) of PAA in aqueous solution is approximately 4.7, which is similar to the pKa of the ionisable carboxylic acid group (Richter, Paschew, Klatt, Lienig, & Arndt, 2008). When the pH of the aqueous solution is higher than the pKa of PAA, most of carboxylic acid groups carried on the polymer become deprotonated, improving the ability of the PP-g-PAA to associate with positively charged metal ions. At pH below this pKa, however, most of carboxylic acid groups are protonated, hindering association with metal ions. EDTA-containing emulsions did not oxidise at all tested pH values over the storage periods.
3.5. Particle size and zeta potential of emulsions at different pH The particle size and zeta potential of emulsions at different pH were quantified throughout the storage study to demonstrate the stability of emulsified systems stored in contact with the PP-gPAA active packaging film. The particle size of the pH 7.0 emulsions was stable at particle sizes ranging from 187 to 205 nm (Fig. 2C), regardless of contact with native PP, PP-g-PAA or no film. Likewise, the particle size of the pH 5.0 emulsions remained stable throughout the study (195–240 nm, Fig. 3C). Even though emulsions at pH 3.0 exhibited high deviation in terms of particle size for all conditions (e.g. no film, native PP and PP-g-PAA), there was not a consistent increase in particle size that would cause creaming (Fig. 4C). The stability of emulsion droplets to aggregation is mainly determined by the balance of attractive (such as van der Waals) and repulsive (such as steric and electrostatic) interactions. For oil droplets coated by non-ionic surfactants, the main stabilizing mechanism is steric repulsion due to the hydrophilic head groups, but electrostatic repulsion also plays an important role if the droplets have some surface charge, e.g., due to the presence of ionic impurities (McClements & Weiss, 2005). The electrical charge of the emulsion droplets was evaluated by measuring their zeta potential. At pH 7.0, emulsions had an initial zeta potential of 4.2 ± 0.6 mV. After 8 days of incubation, the droplets in the control emulsion, emulsion with native PP and emulsion with PP-g-PAA became highly negatively charged, with zeta potentials of 29.3 ± 1.2, 25.1 ± 1.8 and 18.6 ± 1.4 mV, respectively (Fig. 2D). A sufficiently large negative charge generates a strong electrostatic repulsion between emulsion droplets, which opposes aggregation (McClements & Weiss, 2005). At pH 5.0, there was also
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aldehydes, ketones and acids (Chaiyasit, Elias, McClements, & Decker, 2007). If acid groups were formed on the fatty acids by oxidation they could be surface active and thus accumulate at the interfacial surface of emulsion droplets and increase their negative charge (Waraho et al., 2009). If the pKa of these acid groups were similar to organic acids, their pKa would be in the range of pH 4.0–5.0 (Brown, McDaniel, & Hafliger, 1955; Dippy, Hughes, & Rozanski, 1959; Richter et al., 2008). When the pH of the emulsion is higher than the pKa of the acids formed by oxidation, the emulsion droplets would become more negatively charged. At pH values below the pKa of acids formed by oxidation, the carboxylic acid groups would be protonated and thus would not change the zeta potential. This would explain the poor correlation between the zeta potential and the hexanal concentration of emulsions at pH 3.0. 3.6. Migration potential of monomers and homopolymers
Fig. 5. ATR–FTIR spectra of native PP, PP-g-PAA control and PP-g-PAA films analysed in the wavelength range of 2000–600 cm1 (A) and 4000–2000 cm1 (B). Each spectrum is representative of 6 measurements on 3 independent films.
electrostatic repulsion generated between emulsion droplets, as indicated by the zeta potential decreasing from 1.0 to 5 8 mV after 14 days of incubation (Fig. 3D). At pH 3.0, however, the zeta potentials of emulsions were around 0 mV throughout the storage period (Fig. 4D). These results suggest that the steric repulsion acting between the emulsion droplets due to their hydrophilic head groups was insufficiently strong to overcome the attractive interactions. Figs. 2C, 3C and 4C indicate that emulsion droplets remained stable after contact with PP-g-PAA films, indicating that the PAA surface grafting did not adversely affect the stability of this oil-in-water emulsion system. The results in Figs. 2D and 3D suggest that the zeta potential became more negative at the oil/water interface after lipid oxidation occurred in the emulsions. The correlation relationships between the zeta potential and the concentration of the secondary oxidation product (hexanal) of emulsions were therefore assessed by Pearson correlation analysis, in which r = + 1 indicates a strong positive correlation, r = 1 indicates an inverse correlation and r = 0 indicates no correlation (Table 1). Because emulsions prepared with EDTA did not oxidise in the course of the storage study, the correlation relationship of their parameters was not analysed. Table 1 shows a significant negative correlation between the zeta potential of the emulsion and the hexanal concentration at both of pH 7.0 and 5.0 (p < 0.05), implying that lipid oxidation has an important impact on the interfacial characteristics of emulsion droplets. The secondary oxidation products are a mixture of low molecular weight compounds produced from the decomposition of lipid hydroperoxides, including nonvolatile dimers and polymers,
A control experiment was conducted to evaluate the likelihood of migration of AA monomers and homopolymers from the PP-gPAA film surface. The PP-g-PAA control films were prepared using the same grafting and rinsing procedures as PP-g-PAA films but with no photoinitiator (BP) step. The surface chemistry of the resulting PP-g-PAA control films were analysed by ATR–FTIR and compared to that of native PP film and PP-g-PAA film (Fig. 5). After the grafting of PAA, three characteristic absorption peaks appeared in the spectrum of the PP-g-PAA film at 3500–3000, 1700–1725 and 1211–1320 cm1, corresponding to the –OH, C@O and CAO of carboxylic acid groups, respectively. No absorbance was shown at these three wavelength regions in the spectrum of the PP-g-PAA control film, indicating that the applied washing step effectively removed all the ungrafted oligomer or polymer PAA chelators generated by the homopolymerization of AA monomers during the grafting process. Throughout the whole scanned range (4000– 600 cm1), the surface chemistry of the PP-g-PAA control film showed no difference to that of the native PP film, suggesting a clean PP-g-PAA film surface without the presence of other potential migrants. 4. Conclusions The influence of SA/V ratio and pH on the performance of PPg-PAA films was investigated in a stripped soybean oil-in-water emulsion system. At the range of SA/V ratios from 2 to 8 cm2/ ml, PP-g-PAA films were equally able to control lipid oxidation in this emulsion system. PP-g-PAA films exhibited the strongest activity in preventing oxidative reactions at pH 7.0, and the activity decreased with decreasing pH. The particle size and zeta potential results indicated that the developed non-migratory active packaging film would have no adverse influence on the stability of this emulsion system. The control experiment to determine the potential for migratory ungrafted homopolymers and monomers suggested a non-migratory nature of the PAA grafting on PP-g-PAA film surface. These results provide us with more fundamental knowledge to facilitate the application of the PP-g-PAA film in the food industry. More research still needs to be done to demonstrate the effectiveness and safety of this active film in real food matrices before larger-scale commercialisation. The design and development of more effective active packaging films are also needed to control iron-promoted lipid oxidation in food products with low pH. Acknowledgements This work was supported by the United States Department of Agriculture National Institute of Food and Agriculture, and in part by UMass through the CVIP Technology Development Fund.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Influence of non-migratory metal-chelating active packaging film on food quality: Impact on physical and chemical stability of emulsions Fang Tian, Eric A. Decker, D. Julian McClements, Julie M. Goddard ⇑ Department of Food Science, University of Massachusetts, Chenoweth Lab, 102 Holdsworth Way, Amherst, MA 01003, United States
a r t i c l e
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Article history: Received 22 July 2013 Received in revised form 24 September 2013 Accepted 13 November 2013 Available online 21 November 2013 Keywords: Active packaging film Metal-chelating Non-migratory Lipid oxidation Emulsion Antioxidant
a b s t r a c t Previously, we developed a novel metal-chelating packaging film (PP-g-PAA) by grafting acrylic acid (AA) monomer from polypropylene (PP) film surface, and demonstrated its potential in controlling ironpromoted lipid oxidation. Herein, we further established the industrial practicality of this active film. Specifically, the influence of film surface area-to-product volume ratio (SA/V) and product pH on the application of the film was investigated using an oil-in-water emulsion system. The films equally inhibited lipid oxidation throughout the range of SA/V ratios tested (2–8 cm2/ml). PP-g-PAA films were most effective at pH 7.0, and the activity decreased with decreasing pH. The particle size examination of emulsions indicated no adverse influence from the active film on the stability of this emulsion system. FTIR analysis suggested a non-migratory nature of PP-g-PAA films. These results provide fundamental knowledge that will facilitate the application of this effective and economical active packaging film in the food industry. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Active packaging is an innovative technology that has gained considerable interest for the purpose of controlling lipid oxidation in recent years (Byun, Bae, & Whiteside, 2012; Pereira de Abreu, Cruz, & Paseiro Losada, 2012; Tian, Decker, & Goddard, 2013). Coating active substances onto or incorporating them throughout the packaging material are the most commonly applied approaches to develop antioxidant packaging films (Lopez-de-Dicastillo et al., 2011; Unalan, Korel, & Yemenicioglu, 2011). However, covalent immobilisation of active agents onto the food contact surface of a packaging film is gaining industry attention, as the active substance is unlikely to migrate from the package into the food (Arrua, Strumia, & Nazareno, 2010; Goddard, Talbert, & Hotchkiss, 2007; Tian, Decker, & Goddard, 2012a, 2012b). A potential regulatory advantage of covalently immobilised active substances is that they may only require food contact notification approval rather than approval and labelling as a direct food additive (Anonymous, 2005; Koontz, 2012). Hence, non-migratory active packaging technologies provide food manufacturers with the possibility to obtain cleaner labels by removing synthetic additives (e.g. EDTA, etc.) from food formulations. In our previous work, a non-migratory active packaging film was successfully developed by photografting metal-chelating polymer poly(acrylic acid) (PAA) from polypropylene (PP) film surface ⇑ Corresponding author. Tel.: +1 413 545 2275; fax: +1 413 545 1262. E-mail address: [email protected] (J.M. Goddard). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.074
(Tian et al., 2012a, 2012b). The antioxidant ability of the PAA grafted PP film (PP-g-PAA) was preliminarily examined in a commercial soybean oil-in-water emulsion system, typical of many lipid containing food products (e.g. milk, beverages, dressings, sauces, desserts, etc.) (Waraho, Cardenia, Rodriguez Estrada, McClements, & Decker, 2009). PP-g-PAA films were shown to significantly extend the lag phase of lipid oxidation in the emulsion system by a factor of 4–5 times, compared to the native PP film. While our prior work focused on the synthesis and material characterisation of the film, the objective of the current work is to better establish the industrial practicality of this novel active packaging film. Specifically, the influence of film surface area to product volume ratio (SA/V), product pH, and migration of ungrafted oligomeric or polymeric chelators on the commercial application of the film were investigated using a model oil-in-water emulsion system. Herein we report on the performance of the developed PP-gPAA film in emulsion systems with SA/V ratios in the range of 2–8 cm2/ml, to better understand its commercial potential in food industry applications. Furthermore, the influence of pH on the ability of the chelating ligands to bind to metal ions was established, since this is known to have a major impact on their ability to inhibit lipid oxidation (Kontoghiorghes, Pattichis, Neocleous, & Kolnagou, 2004). Often, a chelator has one or more acidic chelating ligands (e.g. -COOH, -NOH, -SH, etc.), and the proton of these groups can be displaced by the metal ion and sequester it from solution (Kontoghiorghes et al., 2004). The activity of these acidic functional groups is therefore expected to be influenced by the
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pH of the reaction system (e.g. food product). PAA contains a large number of carboxylic acids (–COOH) as the chelating ligand, which is especially sensitive to the pH of the surrounding solution. We therefore evaluated the effectiveness of our PP-g-PAA films against oil-in-water emulsions at pH 3.0, 5.0 and 7.0, to define suitable food systems for this active packaging film. The grafting chemistry used to generate PP-g-PAA films was designed to result in nonmigratory active packaging, in which the active agent (PAA chelators) is unlikely to migrate into the packaged food. We therefore conducted a series of control experiments to ensure no residual substances transferred from the developed active film into the food systems. Finally, we examined the influence of the active film on the stability and interfacial charge of emulsion droplets at different pH. 2. Materials and methods 2.1. Materials Commercial soybean oil (Wesson, 100% natural vegetable oil) was purchased from the local grocery store; polypropylene (PP, isotactic, pellets) was purchased from Scientific Polymer Products (Ontario, NY); hydroxylamine hydrochloride, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p0 -disulfonic acid disodium salt hydrate (ferrozine, 98+%), imidazole (99%) and ethylenediaminetetraacetic acid (EDTA) were purchased from Acros Organics (Morris Plains, NJ); toluidine blue O (TBO) was purchased from MP Biomedicals (Solon, OH); 2-propanol, acetone, heptane, hexanes, ethanol, isooctane, methanol, 1-butanol, sodium acetate trihydrate, hydrochloric acid, acetic acid glacial, Brij 35 (a non-ionic surfactant), ferric chloride anhydrous, trichloroacetic acid (TCA), 4-(2-hydroxyethyl)-1piperazineethane-sulfonic acid (HEPES) and sodium hydroxide were purchased from Fisher Scientific (Fair Lawn, NJ); acrylic acid (AA, anhydrous), barium chloride dihydrate, ammonium thiocyanate, cumene hydroperoxide (80%), hexanal (98%), silicic acid (100–200 mesh, 75–150 lm, acid washed), activated charcoal (100–400 mesh) and benzophenone (BP, 99%) were purchased from Sigma–Aldrich (St. Louis, MO); all the chemicals and solvents were used without further purification. 2.2. Preparation of PP films PP pellets were cleaned by sonication in 2-propanol, acetone and deionised water (2 times per solvent, 10 min each time) sequentially. The cleaned PP pellets were dried in a desiccator overnight (25 °C, 15% RH), and then used for the preparation of PP films using a Carver Laboratory Press (Model B, Fred S. Carver Inc., NJ). PP pellets were pressed into films at 160 °C with a load force of 9000 lbs. Pressed PP films (average thickness: 225 ± 25 lm) were cut into 2 2 cm pieces and went through the same cleaning and drying process as PP pellets. 2.3. Preparation of PP-g-PAA films The chelating monomer AA was covalently grafted from PP film surface by a two-step photo-initiated graft polymerization approach. This approach adopted the procedure reported by Ma, Davis, and Bowman (2000) and Tian et al. (2012a, 2012b) with some modifications. In this work, spin coating and a Dymax light-curing system were applied (rather than dip coating and a spot UV lamp) to graft a more uniform PAA layer on the PP film surface. Briefly, 30 ll of BP solution (5 wt% in heptane) was spin-coated on each side of PP films at a speed of 2000 rpm for 10 s. The BPcoated films (2 2 cm) were then cut into 1 2 cm films, and each
piece of film went through the same grafting procedure described previously. (Tian et al., 2012a, 2012b) The UV irradiation was conducted in a Dymax light-curing system (Model 5000, 320–395 nm, 200 mW/cm2, Dymax Corporation, Torrington, CT). BP-coated films were exposed to 90 s of UV irradiation, followed by rinsing in acetone (3 times, 5 min each time). PP–BP films were then submerged in 25 wt% AA in ethanol, nitrogen flushed and exposed to UV irradiation for 6 min. Finally, PP-g-PAA films were rinsed in water as described previously to remove any unreacted monomer and generated homopolymers. 2.4. Quantification of carboxylic acids The carboxylic acid density of PP-g-PAA film surface was determined by the TBO dye assay adapted from Kang, Tan, Kato, Uyama, and Ikada (1996) and Uchida, Uyama, and Ikada (1993) with some modifications. Briefly, TBO solution (0.5 mM) was prepared by dissolving TBO dye into pH 10.0 deionized water (adjusted by NaOH). PP and PP-g-PAA films were then put into the prepared TBO solution (1 piece in 5 ml, n = 6) for 2 h with shaking (25 °C) to allow TBO dye to complex with carboxylic acids. After rinsing loosely adsorbed dye, the complexed dye was desorbed by submerging films in 50 wt% acetic acid solution. After 15 min of shaking, the absorbance in the solutions was detected at 633 nm. The amount of available carboxylic acids on film surfaces was calculated using a standard curve made from TBO dye in 50 wt% acetic acid solution. 2.5. Iron chelating assay The ferric iron (Fe3+) chelating activity of the PP-g-PAA film was determined by the ferrozine colorimetric assay based on methods reported by Bou et al. (2008) and Tian et al. (2012a, 2012b) with some modifications. Iron stock solution (20 mM) was prepared by dissolving ferric chloride anhydrous in a 0.05 M HCl solution. The stock solution was then diluted to 0.08 mM by a sodium acetate/imidazole (0.05 M, pH 5.0) buffer to make the Fe3+ chelating solution. Three groups of samples were prepared for the chelation study, including Fe3+ solution without films as the control, Fe3+ solution with native PP films and Fe3+ solution with PP-g-PAA films. One piece of film (1 2 cm) was put into 10 ml of Fe3+ solution serving as a replicate, with three replicates for each determination. Samples were incubated in the dark for 24 h at room temperature (25 °C) with shaking. The Fe3+ chelating activity of films was determined by calculating the difference of Fe3+ concentration in Fe3+ chelating solution with films against the control group. The colourimetric ferrozine assay was used to measure the Fe3+ concentration. As only Fe2+ reacts with ferrozine to form a red colour complex, a reducing agent (5 wt% hydroxylamine hydrochloride, 10 wt% trichloroacetic acid) was prepared to convert Fe3+ to Fe2+. The Fe3+ chelating solution (0.5 ml) was mixed with 0.25 ml of reducing agent, followed by adding 0.25 ml of ferrozine solution (18 mM in 0.05 M HEPES buffer, pH 7.0). The reaction solution was incubated at room temperature for 1 h with shaking and the absorbance was detected at 562 nm. The Fe3+ concentration was calculated compared with a standard curve made of ferric chloride anhydrous. 2.6. Preparation of stripped soybean oil Stripped soybean oil was prepared by the method reported by Waraho et al. (2009). Chromatographic column separation was used to strip undesired compounds (e.g. a-tocopherol, free fatty acids, pigments, etc.) from commercial soybean oil with silicic acid and activated charcoal as the packing material. Silicic acid (50 g) was washed three times in deionized water to remove any small
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and damaged particles, and then filtered through a Buchner funnel. Activated charcoal (5.625 g) and washed silicic acid were dried at 110 °C for 24 h, followed by suspension in hexanes. Silicic acid (22.5 g), activated charcoal (5.625 g) and additional silicic acid (22.5 g) were packed sequentially in a chromatographic column (35 3.0 cm i.d.). Commercial soybean oil (30 g) was dissolved in hexanes (30 ml), loaded onto the column and then 270 ml of hexanes was used to eluent the triacylglycerols. Silicic acid can remove substances such as lipid oxidation products, free fatty acids and antioxidants (e.g. a-tocopherol) from the commercial oil, while activated charcoal can remove any pigments. Hexanes in the collected stripped oil was removed by a vacuum rotary evaporator (RE 111 Buchi, Flawil, Switzerland) and nitrogen flushing was used to remove the final traces of hexane residue. The obtained stripped oil, which only contains triacylglycerols, was stored at 80 °C until use. 2.7. Lipid oxidation study Previously, commercial soybean oil was used to prepare the oil-in-water emulsion system to demonstrate the effectiveness of PP-g-PAA films in controlling lipid oxidation (Tian et al., 2012a, 2012b). However, the existence of other trace components (atocopherol, phospholipids, pigments, free fatty acids, etc.) in commercial soybean oil makes the lipid oxidation process much slower and more complicated than in stripped oil, which primarily only contains triacylglycerols (Mei, Decker, & McClements, 1998; Mei, McClements, Wu, & Decker, 1998; Waraho et al., 2009). To reduce the long lag phase of lipid oxidation, and also to better understand the influence of PP-g-PAA films on the emulsion system, stripped soybean oil was used to prepare the oil-in-water emulsion system used in this work. All determinations were conducted in triplicate, and the results presented here are representative of two independent studies. Stripped soybean oil (1 wt%) and Brij 35 (0.1 wt%) were dissolved in sodium acetate/imidazole buffer (0.05 M), and the mixture was blended for 2 min by a 2-speed hand held homogenizer (Biospec Products, Inc., Bartlesville, OK) at the low speed setting to prepare the coarse emulsion. The coarse emulsion was then passed through a microfluidizer (Microfluidics, Newton, MA) four times with a pressure of 9000 psi to obtain a fine emulsion. To measure the effect of SA/V ratio, native PP and PP-g-PAA films were cut into 1 1 cm squares (with grafting on both sides, 2 cm2 of surface area for each piece of film). Films were then put into 1 ml of oil-in-water emulsion (pH 7.0) to prepare emulsion samples with different surface area (2, 4, 6 and 8 cm2). Emulsion alone (no film) and emulsions with PP or PP-g-PAA films were incubated in the dark at 25 °C for 8 days, and the formation of lipid oxidation products (lipid hydroperoxides and hexanal) were determined throughout the study as described below. To evaluate the effect of pH on the activity of PP-g-PAA films (2 cm2) in controlling lipid oxidation, emulsions with different pH (3.0, 5.0 and 7.0) were prepared by the method described above. All emulsions (1 ml) were allowed to oxidise in the dark at 25 °C for up to 20 days. Emulsions with no film as well as emulsions containing EDTA (0.01 mM) were used as negative and positive controls, respectively. The formation of lipid oxidation products (lipid hydroperoxides and hexanal), the particle size and the droplet charge (zeta potential) of emulsions were determined during the incubation. Lipid hydroperoxides, a primary lipid oxidation product, were quantified using the colourimetric ammonium thiocyanate method (Alamed, McClements, & Decker, 2006; Shantha & Decker, 1994). Briefly, 0.3 ml of emulsion was mixed with 1.5 ml of isooctane/isopropanol (3:1 v/v) by vortex (3 times, 10 s/time). The mixed solution was centrifuged at 4000 rpm for 5 min to obtain an upper phase containing hydroperoxides. The upper phase solution
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(200 ll) was then collected and added into 2.8 ml of methanol/1butanol (2:1 v/v), followed by adding 15 ll of ammonium thiocyanate (3.94 M) and 15 ll of ferrous iron solution. The clear ferrous iron solution was prepared freshly by centrifugation (1000g, 2 min) of the mixture (1:1 v/v) of BaCl2 (dissolved in 0.4 M HCl, 0.132 M) and FeSO4 (0.144 M). The absorbance was quantified at 510 nm after 20 min of incubation at room temperature. Hydroperoxide concentrations were calculated using a standard curve made from cumene hydroperoxide. The lag phase of the hydroperoxide formation in emulsions was defined as the day before the significant increase of the hydroperoxide concentration compared to time zero concentrations (p < 0.05). Formation of hexanal (a secondary lipid oxidation product) in emulsions was measured using head space gas chromatography (GC) according to the method of Panya et al. (2010). The GC (Shimadzu GC-2014, Tokyo, Japan) was equipped with an auto-injector (AOC-5000, Shimadzu, Tokyo, Japan) and a flame ionisation detector (FID). Samples were pre-incubated at 55 °C for 8 min, and a divinylbenzene/carboxen/polydimethylsiloxane (DVB/Carboxen/ PDMS) stable flex solid phase microextraction fibre (50/30 lm, Supelco, Bellefonte, PA) was then exposed to the sample headspace for 2 min to adsorb volatile compounds evaporated from the emulsion, followed by the desorption at 250 °C for 3 min in the injector at a split ratio of 1:7. A fused-silica capillary column (30 m 0.32 mm i.d. 1 lm) coated with 100% poly(dimethylsiloxane) (Equity-1, Supelco) was used to separate volatile compounds generated from the lipid oxidation with the run time of 10 min for each sample. The temperatures of the injector, oven and detector were 250, 65 and 250 °C, respectively. The hexanal concentration was calculated by comparison to a standard curve made from emulsions containing hexanal and EDTA (0.2 mM). The lag phase of the hexanal formation in emulsions was defined as the day before the significant increase of the hexanal concentration compared to zero time concentrations (p < 0.05). The particle size distribution and the electrical charge of emulsion droplets were determined by Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK). Emulsions were diluted 100-fold with the corresponding buffer before the measurement. To measure the particle size of emulsion droplets, the diluted emulsion was loaded into the instrument and equilibrated for 120 s. Each sample was measured 2 times (11 runs for each time) and the Z-average mean diameter (nm) of droplets was collected. For the electrical charge of emulsions, each sample was measured 3 times (10 runs for each time) after 60 s of equilibrating and the zeta potential (mV) of emulsion droplets was reported.
2.8. Migration potential of monomers and homopolymers The likelihood for migration of residual AA monomers and generated PAA homopolymers (ungrafted PAA) was tested by a control study. The two-step graft polymerization approach used here is designed to prevent the generation of PAA homopolymers that may subsequently migrate from the film surface. Nevertheless, the polymerization between AA monomers could still occur without the photoinitiator (BP) dispersed in the monomer solution with UV irradiation and an oxygen-free environment (Ma et al., 2000). As the long-term goal of this research is the development of nonmigratory active packaging films, a control experiment was designed by preparing PP-g-PAA control films using the same grafting procedure with the exception of the BP coating step. The obtained PP-g-PAA control films went through the same washing steps as standard PP-g-PAA films: stir-washing in deionized water 30 min at room temperature, 1 h at 60 °C and finally 30 min at room temperature. Films were then dried overnight in a desiccator (25 °C, 15% RH) until further use.
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Fig. 1. Lipid hydroperoxide and hexanal concentrations of stripped soybean oil-in-water emulsions (pH 7.0) with different SA/V ratios stored at 25 °C and measured over 8 days. (A), (B), (C) and (D) correspond to the lipid hydroperoxide concentrations of emulsions with SA/V ratio of 2, 4, 6 and 8 cm2/ml, respectively; (E), (F), (G) and (H) correspond to the hexanal concentrations of emulsions with SA/V ratio of 2, 4, 6 and 8 cm2/ml, respectively.
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Fig. 2. Hexanal concentrations (A), lipid hydroperoxide concentrations (B), particle size (C) and zeta potential (D) of stripped soybean oil-in-water emulsions (pH 7.0) stored at 25 °C and measured over 8 days.
The surface chemistry of native PP, PP-g-PAA and PP-g-PAA control films was analysed by a FTIR spectrometer (IRPrestige-21, Shimadzu Scientific Instruments, Inc., Kyoto, Japan) equipped with a diamond ATR crystal. Each spectrum was an average of 32 scans at a 4 cm1 resolution compared to a background spectrum, which was carried out with an ATR crystal against air. A representative spectrum of each sample was plotted with SigmaPlot 12.0 (Systat Software, Inc., Chicago, IL). The migration potentials of oligomeric or polymeric PAA chelators were assessed by comparison of the surface chemistry of the films. 2.9. Statistical analysis The data were reported as means ± standard deviation (SD). Statistical analyses were conducted using SPSS Release 17.0 (SPSS Inc., Chicago, IL). The significance of differences was determined by the one-way analysis of variance (ANOVA) with Duncan’s pairwise comparison (p < 0.05). Two-tailed Pearson correlation was performed to assess the correlation relationship between variables. 3. Results and discussion 3.1. Carboxylic acid density The TBO dye assay was used to determine the amount of available carboxylic acids on native PP and PP-g-PAA film surfaces. After the polymerized grafting of PAA, a significantly higher amount of carboxylic acids (57 ± 4 nmol/cm2, n = 6) was detected on PP-gPAA film surfaces compared to that on native PP film surfaces (0 ± 0 nmol/cm2, n = 6). As each PP-g-PAA (1 2 cm) film was
prepared independently, the small standard deviation of the determination indicated that this active film could be produced uniformly and consistently. The results also suggested that a more uniform PAA grafting could be obtained by spin coating and using a Dymax light-curing system, as opposed to films prepared with dip coating and a spot UV lamp, which resulted in significantly higher deviations (69 ± 10 nmol/cm2) (Tian et al., 2012a, 2012b). 3.2. Iron chelating activity The Fe3+ chelating activity of PP and PP-g-PAA films was measured at pH 5.0. Like most metal chelators, PAA has higher affinity to Fe3+ than Fe2+, as previously reported (Tian et al., 2012a, 2012b). In addition, with carboxylic acid groups as the chelating ligands, the PAA/Fe complex should have similar stability constants to those of citric acid/Fe complex, which are 11.85 and 3.2 for Fe3+ and Fe2+, respectively (Furia, 1972). The stability constant of a complex is a measure of the strength of the interaction between a chelator and a metal ion. Higher constants represent more stable complexes. Difficulty was encountered in attempting to release Fe3+ from the PAA/Fe3+ complex formed on the PP-g-PAA film surface using the method described in our previous work (Tian et al., 2012a, 2012b). Therefore, in this work, we determined the Fe3+ chelating activity of the films by the difference of the Fe3+ concentration in the chelating solution with and without films rather than the direct measurement of the Fe3+ released from films. PP films showed no evident activity to chelate Fe3+ (0 ± 7 nmol/cm2, n = 3), while PP-g-PAA films exhibited a significant Fe3+ chelating activity (54 ± 11 nmol/cm2, n = 3). With a carboxylic acid density of 57 ± 4 nmol/cm2, the chelating ligand (-COOH) to Fe3+ ratio of PAA grafted on PP film surface is approximately 1.
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Fig. 3. Hexanal concentrations (A), lipid hydroperoxide concentrations (B), particle size (C) and zeta potential (D) of stripped soybean oil-in-water emulsions (pH 5.0) stored at 25 °C and measured over 14 days.
3.3. Effect of SA/V ratio The influence of the ratio of film surface area to emulsion volume (SA/V ratio) was analysed by measuring the formation of oxidation products over time in a 1 ml emulsion (pH 7.0) stored in contact with varying surface areas of native PP or PP-g-PAA films (2, 4, 6 and 8 cm2) (Fig. 1). As indicated by the 1 day lag time of both lipid hydroperoxides and hexanal formation in control emulsion (i.e. emulsion stored with no film), the stripped oil oxidised rapidly due to its lack of antioxidants. Hexanal formation occurred slightly slower in emulsions stored with native PP films compared to the control emulsion, more notably at higher SA/V ratios (6 and 8 cm2/ml) (Fig. 1G and H). The apparent extension in lag phase by the native PP films may be a result of the hydrophobic PP films floating on the surface of the emulsions, and its relatively low oxygen permeability coefficient at 25 °C (Kurek et al., 2011), which may inhibit the diffusion of oxygen into emulsions. Compared to the control emulsion and emulsions with native PP films, PP-gPAA films delayed lipid oxidation in the emulsions at all tested SA/V ratios. Compared to native PP films, PP-g-PAA films extended the lag phase of lipid hydroperoxides and hexanal formation by 1 day at all examined SA/V ratios. Results indicated that increasing the SA/V ratio from 2 to 8 cm2/ml had no significant effect on the ability of PP-g-PAA films to prevent oxidative deteriorations in the tested oil-in-water emulsion system. It has been reported that the solubility of Fe3+ at pH 7.0 is approximately 4 1017 M (Zumdahl, 1989), and the concentrations of iron in emulsion systems can be on the order of parts per billion (ng/ml), which is sufficient enough to accelerate lipid oxidative reactions (Alamed et al., 2006). At an iron chelating activity of 54 ± 11 nmol/cm2, 2 cm2 of
PP-g-PAA films could chelate approximately 6 103 ng of iron, which is much higher than the amount of iron naturally existing in 1 ml of emulsion (Alamed et al., 2006). It is therefore possible that at a SA/V ratio of 2 cm2/ml, the chelation reaction between transition metals and PP-g-PAA films is already saturated, and that increasing the SA/V ratio does not further extend the lag phase of lipid oxidation. 3.4. Effect of pH A SA/V ratio of 2 cm2/ml was used to determine the effect of pH on the activity of PP-g-PAA films in inhibiting lipid oxidation in oilin-water emulsions prepared at pH values of 7.0 (Fig. 2), 5.0 (Fig. 3) and 3.0 (Fig. 4). It is worth noting that there was an observable difference between the lag phase of control emulsion (i.e. no film) recorded in the SA/V ratio study (1 day, Fig. 1E) and that observed in the pH study (3 days, Fig. 2A). This is likely a result of the variations of ambient conditions, buffer, surfactant, homogenizer, etc. during the pre-homogenisation, homogenisation and post-homogenisation processes to prepare the emulsion. The variations could induce a slight difference in the metal concentration naturally present in emulsions, which in turn influences the lipid oxidation rate. Nevertheless, the emulsion system was consistent for each study, which enabled us to make comparisons between different treatments. PP-g-PAA films exhibited the greatest activity at pH 7.0 (Fig. 2), with an extension of the lag phase of the formation of lipid hydroperoxides from 1 to 3 days (Fig. 2B) and the formation of hexanal from 3 to 6 days (Fig. 2A) compared to the control emulsion and emulsions with native PP films. At pH 5.0, PP-g-PAA films prolonged the formation of lipid hydroperoxides and hexanal by 1
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Fig. 4. Hexanal concentrations (A), lipid hydroperoxide concentrations (B), particle size (C) and zeta potential (D) of stripped soybean oil-in-water emulsions (pH 3.0) stored at 25 °C and measured over 20 days.
Table 1 Correlation relationships between the hexanal concentration and the zeta potential of emulsions during the storage at 25 °C. pH
Film treatment
Correlation coefficient (r)
Significance
7.0
Control (no film) PP PP-g-PAA Control (no film) PP PP-g-PAA Control (no film) PP PP-g-PAA
0.938 0.908 0.821 0.978 0.981 0.84 0.495 0.251 0.242
p < 0.01 p < 0.01 p < 0.05 p < 0.01 p < 0.01 p < 0.01 p > 0.05 p > 0.05 p > 0.05
5.0
3.0
and 2 days (Fig. 3B and A), respectively. Under the accelerated conditions tested (i.e. stripped oil at 25 °C), PP-g-PAA films were not able to retard lipid oxidation at pH 3.0 (Figs. 4A and B). Results indicated that the ability of the PP-g-PAA films to inhibit lipid oxidation increased with increasing pH, which is consistent with expectations. The apparent acid dissociation constant (pKa) of PAA in aqueous solution is approximately 4.7, which is similar to the pKa of the ionisable carboxylic acid group (Richter, Paschew, Klatt, Lienig, & Arndt, 2008). When the pH of the aqueous solution is higher than the pKa of PAA, most of carboxylic acid groups carried on the polymer become deprotonated, improving the ability of the PP-g-PAA to associate with positively charged metal ions. At pH below this pKa, however, most of carboxylic acid groups are protonated, hindering association with metal ions. EDTA-containing emulsions did not oxidise at all tested pH values over the storage periods.
3.5. Particle size and zeta potential of emulsions at different pH The particle size and zeta potential of emulsions at different pH were quantified throughout the storage study to demonstrate the stability of emulsified systems stored in contact with the PP-gPAA active packaging film. The particle size of the pH 7.0 emulsions was stable at particle sizes ranging from 187 to 205 nm (Fig. 2C), regardless of contact with native PP, PP-g-PAA or no film. Likewise, the particle size of the pH 5.0 emulsions remained stable throughout the study (195–240 nm, Fig. 3C). Even though emulsions at pH 3.0 exhibited high deviation in terms of particle size for all conditions (e.g. no film, native PP and PP-g-PAA), there was not a consistent increase in particle size that would cause creaming (Fig. 4C). The stability of emulsion droplets to aggregation is mainly determined by the balance of attractive (such as van der Waals) and repulsive (such as steric and electrostatic) interactions. For oil droplets coated by non-ionic surfactants, the main stabilizing mechanism is steric repulsion due to the hydrophilic head groups, but electrostatic repulsion also plays an important role if the droplets have some surface charge, e.g., due to the presence of ionic impurities (McClements & Weiss, 2005). The electrical charge of the emulsion droplets was evaluated by measuring their zeta potential. At pH 7.0, emulsions had an initial zeta potential of 4.2 ± 0.6 mV. After 8 days of incubation, the droplets in the control emulsion, emulsion with native PP and emulsion with PP-g-PAA became highly negatively charged, with zeta potentials of 29.3 ± 1.2, 25.1 ± 1.8 and 18.6 ± 1.4 mV, respectively (Fig. 2D). A sufficiently large negative charge generates a strong electrostatic repulsion between emulsion droplets, which opposes aggregation (McClements & Weiss, 2005). At pH 5.0, there was also
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aldehydes, ketones and acids (Chaiyasit, Elias, McClements, & Decker, 2007). If acid groups were formed on the fatty acids by oxidation they could be surface active and thus accumulate at the interfacial surface of emulsion droplets and increase their negative charge (Waraho et al., 2009). If the pKa of these acid groups were similar to organic acids, their pKa would be in the range of pH 4.0–5.0 (Brown, McDaniel, & Hafliger, 1955; Dippy, Hughes, & Rozanski, 1959; Richter et al., 2008). When the pH of the emulsion is higher than the pKa of the acids formed by oxidation, the emulsion droplets would become more negatively charged. At pH values below the pKa of acids formed by oxidation, the carboxylic acid groups would be protonated and thus would not change the zeta potential. This would explain the poor correlation between the zeta potential and the hexanal concentration of emulsions at pH 3.0. 3.6. Migration potential of monomers and homopolymers
Fig. 5. ATR–FTIR spectra of native PP, PP-g-PAA control and PP-g-PAA films analysed in the wavelength range of 2000–600 cm1 (A) and 4000–2000 cm1 (B). Each spectrum is representative of 6 measurements on 3 independent films.
electrostatic repulsion generated between emulsion droplets, as indicated by the zeta potential decreasing from 1.0 to 5 8 mV after 14 days of incubation (Fig. 3D). At pH 3.0, however, the zeta potentials of emulsions were around 0 mV throughout the storage period (Fig. 4D). These results suggest that the steric repulsion acting between the emulsion droplets due to their hydrophilic head groups was insufficiently strong to overcome the attractive interactions. Figs. 2C, 3C and 4C indicate that emulsion droplets remained stable after contact with PP-g-PAA films, indicating that the PAA surface grafting did not adversely affect the stability of this oil-in-water emulsion system. The results in Figs. 2D and 3D suggest that the zeta potential became more negative at the oil/water interface after lipid oxidation occurred in the emulsions. The correlation relationships between the zeta potential and the concentration of the secondary oxidation product (hexanal) of emulsions were therefore assessed by Pearson correlation analysis, in which r = + 1 indicates a strong positive correlation, r = 1 indicates an inverse correlation and r = 0 indicates no correlation (Table 1). Because emulsions prepared with EDTA did not oxidise in the course of the storage study, the correlation relationship of their parameters was not analysed. Table 1 shows a significant negative correlation between the zeta potential of the emulsion and the hexanal concentration at both of pH 7.0 and 5.0 (p < 0.05), implying that lipid oxidation has an important impact on the interfacial characteristics of emulsion droplets. The secondary oxidation products are a mixture of low molecular weight compounds produced from the decomposition of lipid hydroperoxides, including nonvolatile dimers and polymers,
A control experiment was conducted to evaluate the likelihood of migration of AA monomers and homopolymers from the PP-gPAA film surface. The PP-g-PAA control films were prepared using the same grafting and rinsing procedures as PP-g-PAA films but with no photoinitiator (BP) step. The surface chemistry of the resulting PP-g-PAA control films were analysed by ATR–FTIR and compared to that of native PP film and PP-g-PAA film (Fig. 5). After the grafting of PAA, three characteristic absorption peaks appeared in the spectrum of the PP-g-PAA film at 3500–3000, 1700–1725 and 1211–1320 cm1, corresponding to the –OH, C@O and CAO of carboxylic acid groups, respectively. No absorbance was shown at these three wavelength regions in the spectrum of the PP-g-PAA control film, indicating that the applied washing step effectively removed all the ungrafted oligomer or polymer PAA chelators generated by the homopolymerization of AA monomers during the grafting process. Throughout the whole scanned range (4000– 600 cm1), the surface chemistry of the PP-g-PAA control film showed no difference to that of the native PP film, suggesting a clean PP-g-PAA film surface without the presence of other potential migrants. 4. Conclusions The influence of SA/V ratio and pH on the performance of PPg-PAA films was investigated in a stripped soybean oil-in-water emulsion system. At the range of SA/V ratios from 2 to 8 cm2/ ml, PP-g-PAA films were equally able to control lipid oxidation in this emulsion system. PP-g-PAA films exhibited the strongest activity in preventing oxidative reactions at pH 7.0, and the activity decreased with decreasing pH. The particle size and zeta potential results indicated that the developed non-migratory active packaging film would have no adverse influence on the stability of this emulsion system. The control experiment to determine the potential for migratory ungrafted homopolymers and monomers suggested a non-migratory nature of the PAA grafting on PP-g-PAA film surface. These results provide us with more fundamental knowledge to facilitate the application of the PP-g-PAA film in the food industry. More research still needs to be done to demonstrate the effectiveness and safety of this active film in real food matrices before larger-scale commercialisation. The design and development of more effective active packaging films are also needed to control iron-promoted lipid oxidation in food products with low pH. Acknowledgements This work was supported by the United States Department of Agriculture National Institute of Food and Agriculture, and in part by UMass through the CVIP Technology Development Fund.
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