Food Chemistry 157 (2014) 45–50
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Study on flavonoid migration from active low-density polyethylene film into aqueous food simulants Shuangling Zhang ⇑, Haiyan Zhao Food Science and Engineering College, Qingdao Agricultural University, No. 700, Changcheng Road, Qingdao 266109, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 1 September 2013 Received in revised form 15 January 2014 Accepted 4 February 2014 Available online 12 February 2014 Keywords: LDPE film Flavonoids Food simulants Migration Diffusion coefficient
a b s t r a c t The migration of flavonoids from low-density polyethylene (LDPE) film (0.4%, w/w) to aqueous food simulants over 16 weeks at 0, 15, and 30 °C was investigated. The migration amount of total flavonoids was calculated based on the rutin contents determined by high-performance liquid chromatography (HPLC). Diffusion and partition coefficients, along with the activation energy (Ea) were calculated based on Fick’s second law. The results showed that the migration of flavonoids was influenced by temperature, time and the simulants. The Ea values for flavonoid diffusion were 49.2, 55.9, and 25.8 kJ mol1 in distilled water, 4% acetic acid and 30% ethanol, respectively. This study indicated that the flavonoids in LDPE film easily migrated into food simulants; and this behaviour was related to the low Ea values of flavonoid diffusion, especially in ethanol at 0–30 °C, when the antioxidants were released from the film. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Flavonoids are the main naturally occurring compounds of pharmacological and nutritional interest, and are widely found in the flowers, leaves and seeds of many plants (Kosalec et al., 2013; Momtaz, Hussein, Ostad, Abdollahi, & Lall, 2013; Petlevski, Flajs, Kalodera, & Koncˇic´, 2013). García-Mateos, Aguilar-Santelises, Soto-Hernández, and Nieto-Angel (2013) reported that the flavonoid extracts of some Mexican flowers have antioxidant activities and their compositions were identified by HPLC-MS. Among the flavonoids, quercetin 3-O-glucoside, quercetin 3-O-rhamnoside, quercetin 3-O-rhamnosyl-(1-6)-glucoside and quercetin 3-O-rhamnosyl-(1-2)-[rhamnosyl-(1-6)]-glucoside were assigned. Li et al. (2014) reported that celery flavonoid extracts exhibited a strong total antioxidant capacity, with IC50 values of 68.0 lg/ ml1 in the DPPH assay, 0.39 lg/ml1 in the O assay and 2 48.0 lg/ml1 in the OH assay, respectively. Saroja and Annapoorani (2012) found that the flavonoid fractions of Cynodon dactylon and Terminalia catappa leaves had antioxidant activity and could potentially be used as a source of natural antioxidants. Kredy et al. (2010) researched the potential antioxidant activities of flavonols from lotus seeds. This antioxidant potential in terms of IC50 values were 5.48, 40 ± 0.14 and 0.62 ± 0.05 (dry fraction 2) lg/ml1 in the DPPH radical, hydroxyl radical and hydrogen ⇑ Corresponding author. Tel./fax: +86 0532 86080771. E-mail addresses: [email protected] (S. Zhang), xinyuyuanyin@163. com (H. Zhao). http://dx.doi.org/10.1016/j.foodchem.2014.02.018 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
peroxide assays, respectively. Flavonoids therefore have potential applications as new food additives with high antioxidant capacities. Antioxidant active packaging is a promising technology for food protection (Barbosa-Pereira et al., 2013; Bodaghi et al., 2013; Martínez-Camacho et al., 2013; Núñez-Flores et al., 2013; Salgado, López-Caballero, Gómez-Guillén, Mauri, & Montero, 2013). The major antioxidants added to films include: a-tocopherol, butylated hydroxytoluene, I-1076 and I-168. Graciano-Verdugo et al. (2010) reported that the concentration of a-tocopherol in low-density polyethylene (LDPE) films, as well as the storage temperature, directly affected the migration of a-tocopherol into corn oil and its oxidative stability. Granda-Restrepo et al. (2009) studied the migration of a-tocopherol from multilayer active packaging materials into whole milk powder. The packaging materials were made of high-density PE (HDPE), ethylene vinyl alcohol and a layer of LDPE containing the antioxidant. Nerín et al. (2006) researched the stabilization of beef using a new active packaging containing natural antioxidants and showed that the active film containing natural antioxidants efficiently enhanced the stability of both the myoglobin and fresh meat against oxidation processes. Garde, Catalá, Gavara, and Hernandez (2001) investigated the migration of antioxidants from polypropylene (PP) films into fatty food simulants and found that heptane could fully extract the antioxidants from the polymer. All the above studies provide a great deal of valuable information and promote the research and development of active packaging. However, there is little data available in scientific literature regarding the migration of flavonoids from LDPE
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films into food. In this study, the characteristics of flavonoid migration from active LDPE films into several aqueous food simulants (distilled water, 4% acetic acid, and 30% ethanol) were investigated to understand the interaction of food and plastic packaging and provide information for the development of the active packaging industry. 2. Materials and methods 2.1. Film manufacture Granular LDPE (1 kg) mixed with flavonoids (5 g) was manufactured into the experimental film using a laboratory film-blowing machine (PTF-IBS-20/28, Guangzhou, China). The running temperature of machine was 120 °C and the air pressure was 0.2 MPa. The film thickness was 4 103 cm, and the flavonoid content was 0.4% (w/w, partially loss during manufacture) as determined using the Soxhlet extraction method. Flavonoids were extracted from Chinese lotus leaves by ultrasonication. The rutin content in the flavonoid extracts was determined by high-performance liquid chromatography (HPLC, Agilent 1200, USA). And the concentration of rutin in theflavonoid extracts was 68.0%. The film was cut into pieces with dimensions of 0.5 cm 0.5 cm after being cleaned with distilled water. 2.2. Migration experiments Distilled water, 4% acetic acid and 30% ethanol were chosen as the aqueous food simulants. For each simulant, 144 LDPE film samples (16 weeks, three replicates) were accurately weighed as 0.2 g. Each sample was placed in a beaker with a cover. Fifty ml of the simulants were added to these beakers. The mixtures were stored under different temperatures (0, 15, and 30 °C). A total of 432 samples were obtained. 2.3. Quantification of flavonoids in aqueous simulants The beakers were taken out of storage and the volume was immediately made up to 50 ml. Samples (10 ml) were evaporated to dryness and the residue was redissolved in methanol (10 ml). The rutin content was determined by HPLC, and the migration of total flavonoids was calculated (68.0% of the rutin concentration in flavonoids). Three replicates were carried out for each sample; the mean values and standard deviations were calculated, denoted by X ± s. Analysis of variance and t-tests were conducted using SAS 9.13 software (SAS Institute Inc.), and pair comparisons were made using the Duncan method. 2.4. Chromatographic conditions The HPLC system (Agilent 1200, USA) was fitted with an auto sampler and diode array ultraviolet detector. Chem-station chromatographic software was used for data acquisition. Chromatographic separation was performed using a Lichrospher C18 column (25 cm 0.21 cm inner diameter). Isocratic elution was performed using acetic acid (0.4% w/w)–methanol (9/1, v/v); the column temperature was 30 °C and the flow rate was 0.3 ml/min1. 2.5. Mathematical models and determination of key parameters Migration processes in films can be fully described by the kinetics of migrant diffusion (expressed by the diffusion coefficient, D) and the chemical equilibrium (expressed by the partition coefficient, K).
An analytical solution of Fick’s second-law equation for diffusion in one dimension, limitless volumes of food, and lengthy contact was used for the determination of D using Eq. (1) (Crank, 1975):
" # 1 X M F;t 8 ð2n þ 1Þ2 p2 ¼1 exp Dt 2 2 MF;1 4dp n¼0 ð2n þ 1Þp2
ð1Þ
where MF,t is the amount of migrant in the food at a particular time t (s); MF,1 is the amount of migrant in the food at equilibrium; dp (cm) is the polymer thickness; D (cm2 s1) is the diffusion coefficient of migrant in the polymer and t (s) is the time. The beginning of migration process can be described by a simplified expression (Eq. (2)) (Crank, 1975):
0:5 M F;t 4 Dt ¼ MF;1 dp p
ð2Þ
To fit the data to Eq. (2), the mass of the flavonoids diffused at time t divided by the mass of the flavonoids diffused at equilibrium (Mt/M1 or Mt/Meq) was plotted versus time t (s), and then D was calculated. The MATLAB (Math Works, Natick, MA, USA) program was used to find the best fit of the data to Eq. (2), using the nlinfit (non-linear regression) function in MATLAB R2012a. The partition coefficient, K, is expressed by
K¼
C P;1 C f ;1
ð3Þ
where Cp,1 (lg g1) and Cf,1 (lg g1) are the equilibrium concentrations of the component in polymer and food, respectively (Siró et al., 2006). 2.6. Diffusion activation energy (Ea) The Ea values for the diffusion of flavonoids from films to simulants was determined using the Arrhenius equation for diffusion (Garde et al., 2001; Limm & Hollifield, 1996):
D ¼ D0 expðEa =RTÞ
ð4Þ
where Ea is the diffusion activation energy; R is the ideal gas constant (8.314 J K1 mol1); and T is the absolute temperature (K). The logarithms of D values were plotted versus the reciprocal of absolute temperature; Ea was obtained using the slope of line (=Ea/2.303R). 3. Results and discussion 3.1. Influence of temperature on migration of flavonoids The increase in the amount of flavonoids in the distilled water, 4% acetic acid, and 30% ethanol during storage at 0, 15 and 30 °C is presented in Fig. 1. It was obtained from Fig. 1 that the amount of flavonoid migration in the three simulants was lowest at 0 °C and highest at 30 °C at the same time before it reached equilibrium. For example, during the first week of storage in distilled water, the migration of flavonoids increased to 4 ± 0.01 lg, 14 ± 2.01 lg, and 29 ± 10.1 lg at 0, 15, and 30 °C, respectively. The amount of migration at 15 °C was triple that at 0 °C, and at 30 °C was twice that at 15 °C. Therefore, the temperature significantly influenced the migration of the flavonoids into the stimulants during storage. The systems reached equilibrium at almost the same percentages (70.3%, 72.5%, and 73.7%) of the amount of migration flavonoids from the total because of the similar dissolutions between the films and liquids. The equilibrium was not related to the storage temperature, as would be expected from the physicochemical properties. However, the equilibrium time had an inverse relationship with temperature and was about a week shorter when
S. Zhang, H. Zhao / Food Chemistry 157 (2014) 45–50
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storage. At 15 °C, the amount of the migrated flavonoids were 14 ± 2.01 lg in distilled water, 34 ± 14.1 lg in 4% acetic acid and 79 ± 7.07 lg in 30% ethanol, respectively, during the first week of storage. The amount of the migrated flavonoids in 30% ethanol was about twice that in 4% acetic acid, and that in 4% acetic acid was about twice that in distilled water. However, the differences were smaller after 8 weeks: 339 ± 8.03 lg, 429 ± 7.24 lg, and 499 ± 10.1 lg in distilled water, 4% acetic acid, and 30% ethanol, respectively. The equilibrium time was also influenced by the simulants, and much shorter (11 weeks) in ethanol than in distilled water (14 weeks) at 0 °C. Therefore the ethanol had a faster absorption rate than the distilled water. Some studies have reported the migration of antioxidants from polyolefin films into ethanol, but comparisons of the results show some differences. Linssen, Reitsma, and Cozijnsen (1998) observed that migration of I-168 and I-1076 from LDPE reached equilibrium after 1 day, and that the equilibrium was close to the full extraction. Lee, Franz, and Piringer (1996) studied the migration of the same antioxidants from polypropylene (PP) films, and reported that equilibrium had not been reached after 21 days, in line with results in this study. 3.3. Diffusion coefficients (D) and partition coefficients (K)
Fig. 1. Amounts of flavonoid migration in distilled water (a), 4% acetic acid (b), and 30% ethanol (c) at 0, 15, and 30 °C; the results are the means of three replicates; bars indicate standard deviations.
the temperature was increased by 15 °C. The same trends were seen in all cases. 3.2. Influence of food stimulants on migration of flavonoids The properties of the food simulants had a major effect on migration of the flavonoids, especially during the first weeks of
Based on Eq. (2), the values of Mt/Meq were plotted versus t0.5 during the first 6 weeks of exposure to the simulants, and a good linear correlation was obtained (Fig. 2). The values are in good agreement with the experimental data for the three simulants and temperatures presented in Fig. 2. The values of D were calculated and are presented in Table 1. The D value describes the migration of flavonoids at a specific temperature for the film with a specific simulant (Table 1). The values of the diffusion coefficients increased with increasing storage temperature. For distilled water, D0 °C = 2.67 1013 cm2 s1 and D15 °C = 4.57 1013 cm2 s1, and for 4% acetic acid, D0 °C = 2.95 1013 cm2 s1 and D15 °C = 6.72 1013 cm2 s1. D15 °C was approximately twice D0 °C. The D values in Table 1 showed that migrations into distilled water and 4% acetic acid were slower than migration into ethanol at 0 °C; the D values of distilled water and 4% acetic acid were about 1/2 that of ethanol. This behaviour may have been caused by plasticisation of the film as a result of sorption of ethanol. There is little information available in the scientific literature regarding the D values of flavonoids from LDPE into foodstuffs. Granda-Restrepo et al. (2009) reported that the D values of a-tocopherol in migration from multilayer active packaging (HDPE + TiO2/EVOH/LDPE + 4% a-tocopherol) to whole milk powder were 2.34, 3.06 and 3.14 1011 cm2 s1 at 20, 30 and 40 °C, respectively. However, these results could not be compared with those in this study because the tested films (multi- and monolayer), fatty foods and storage conditions were different. Graciano-Verdugo et al. (2010) reported that the D values of a-tocopherol were 1.4, 7.1 and 30.3 1011 cm2 s1 at 5, 20 and 30 °C, respectively. The D values were calculated using Eq. (2) and using the same procedure as used here. However, the D values were about 100 times those presented here because the film was in contact with the corn oil. The K values were determined for the simulants using Eq. (3), assuming that equilibrium had been reached after the corresponding weeks of exposure. The K values for each simulant and temperature are presented in Table 1. As can be seen from the data in Table 1, none of the flavonoid migrations at 0, 15 and 30 °C can be regarded as full extraction because the partition equilibrium was not close to complete extraction (K > 0). Comparisons of the behaviours of the flavonoids in the three simulants show that the K values of distilled water and
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Fig. 2. Non-linear regressions of amounts of flavonoid migration versus t0.5: (a) water, (b) 4% acetic acid, and (c) 30% ethanol.
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S. Zhang, H. Zhao / Food Chemistry 157 (2014) 45–50 Table 1 Diffusion coefficients (D, 1013 cm2 s1) for transport of flavonoids from LDPE filmsa to simulants, and partition coefficients (K, flavonoid concentrations lg g1) after sufficient exposure of polymeric films (Cp,1) and simulants (Cf,1) for equilibrium. Parameter Db
Kc
T (°C) 0 15 30 0 15 30
Water 2.67 ± 0.21 h 4.57 ± 0.10 f 6.77 ± 0.13 d 0.141 ± 0.01 ab 0.133 ± 0.0025 c 0.133 ± 0.0065 c
4% acetic acid 2.95 ± 0.10 g 6.72 ± 0.28 d 8.43 ± 0.33 b 0.147 ± 0.0066 a 0.135 ± 0.0109 bc 0.147 ± 0.0128 a
30% ethanol 5.66 ± 0.23 e 8.12 ± 0.14 c 9.20 ± 0.30 a 0.121 ± 0.0109 d 0.121 ± 0.01 d 0.108 ± 0.0026 e
Table 2 Activation energiesa (Ea, kJ mol1) for transport of flavonoids from LDPE filmb to simulants. Water
4% acetic acid
30% ethanol
49.2 ± 3.79 a
55.9 ± 1.21 a
25.8 ± 1.96 b
a Values are shown as X ± s; pair comparisons were made with SAS 9.13 software, using the Duncan method. Same letters means no significant difference, P < 0.05. b Film thickness 4 103 cm.
a
Film thickness 4 103 cm. Results are shown as X ± s; pair comparisons were made with SAS 9.13 software, using the Duncan method. Different letters mean significant differences, P < 0.05. c Results are shown as X ± s; pair comparisons were made with SAS 9.13 software, using the Duncan method. Different letters mean significant differences, P < 0.05. b
4% acetic acid were higher than that of the 30% ethanol. The K value is mainly determined by the polarity of migrating species and the solubility between the film and simulant. Flavonoids were more easily dissolved in ethanol; therefore, the K values obtained were lower than those in distilled water and acetic acid. There were significant differences among the K values (P < 0.05). Some fatty food simulants can fully extract antioxidants. For example, heptane can completely extract H-SE2, I-1076, I-168 and I-168 from PP films when the exposure time is sufficient, and ethanol can totally extract I-168 from PP films (Garde et al., 2001). However, the simulants only extracted some of the antioxidant in our tests. 3.4. Diffusion activation energy (Ea) The Arrhenius plots of D in Fig. 3 are linear, and were used to calculate the Ea values. As can be seen, the theoretical curves agree with the experimental values to some extent, but the curves do not fit very well because of the complexity of the migration tests and some assumptions (one-dimension and limited volumes of packaging and food) (Crank, 1975). The slopes were used to calculate the Ea for the flavonoids in the three food simulants, and the results are presented in Table 2.
The activation energy can be defined as the energy required for a migrant to move among the chains forming the polymer matrix. It is assumed that the migrant, when given sufficient energy, can ‘‘jump’’ into an adjacent space if that space is large enough to accommodate the migrant. A net diffusion flux results if another migrant molecule makes a ‘‘jump’’ into the space that was previously occupied by the first molecule (Limm & Hollifield, 1996). The energy is provided by the temperature, which has an effect on the migrant itself, on the polymer matrix, and on the medium the migrant is in contact with. In this work, the Ea of the flavonoids is lowest for LDPE in ethanol. It can be explained by that less energy was needed for the diffusion of the flavonoid molecules in an LDPE matrix, with more plasticisation compared with that needed in a matrix with less antioxidant, which may need more energy to induce a ‘‘jump’’. The Ea values of the flavonoids in LDPE film with distilled water and 4% acetic acid were similar, and were not significantly different (P < 0.05). The Ea values were in line with published data for similar systems: 105.9 kJ mol1 for a-tocopherol from LDPE films to corn oil (Graciano-Verdugo et al., 2010); 61 kJ mol1 for H-SE2, 65 kJ mol1 for I-1076 and 86 kJ mol1 for I-168 through PP films to ethanol (Garde et al., 2001). 4. Conclusions The migration data on active LDPE films containing flavonoids were obtained and effective diffusion parameters were estimated using an approximate mathematical model based on Fick’s second law. The migration of the flavonoids was influenced by temperature, time and the simulants. The flavonoids in an LDPE film easily migrated into food simulants; and this behaviour was related to
Fig. 3. Linear relationships between flavonoid diffusion coefficients (ln D) and temperature (1/T) in three food stimulants (water, 4% acetic acid, and 30% alcohol).
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the low Ea values for the diffusion of flavonoids, especially in ethanol, for the release of antioxidants from the film at 0–30 °C. Acknowledgements The project was supported by the National 12th Five-Year Research & Technology Based Plan (No. 2012BAK17B05 and 2012BAD28B03) and the 2012 Foundation Project J12LD05 of the Department of Education of Shandong Province, China. The authors gratefully acknowledge Engineering Research Center of Meat Quality Control of Shandong Province for instrument and technical assistance. References Barbosa-Pereira, L., Cruz, J. M., Sendón, R., Bernaldo de Quirós, A. R., Ares, A., CastroLópez, M., et al. (2013). Development of antioxidant active films containing tocopherols to extend the shelf life of fish. Food Control, 31(1), 236–243. Bodaghi, H., Mostofi, Y., Oromiehie, A., Zamani, Z., Ghanbarzadeh, B., Costa, C., et al. (2013). Evaluation of the photocatalytic antimicrobial effects of a TiO2 nanocomposite food packaging film by in vitro and in vivo tests. LWT – Food Science and Technology, 50(2), 702–706. Crank, J. (1975). Mathematics of diffusion (2nd ed.). London: Clarendon Press. García-Mateos, R., Aguilar-Santelises, L., Soto-Hernández, M., & Nieto-Angel, R. (2013). Flavonoids and antioxidant activity of flowers of Mexican Crataegus spp.. Natural Products Research, 27(9), 834–836. Garde, J. A., Catalá, R., Gavara, R., & Hernandez, R. J. (2001). Characterizing the migration of antioxidants from polypropylene into fatty food simulants. Food Additives and Contaminants, 18(8), 750–762. Graciano-Verdugo, A. Z., Soto-Valdez, H., Peralta, E., Cruz-Zárate, P., Islas-Rubio, A. R., Sánchez-Valdes, S., et al. (2010). Migration of a-tocopherol from LDPE films to corn oil and its effect on the oxidative stability. Food Research International, 43, 1073–1078. Granda-Restrepo, D. M., Soto-Valdez, H., Peralta, E., Troncoso-Rojas, R., VallejoCórdoba, B., Gámez-Meza, N., et al. (2009). Migration of a-tocopherol from an active multilayer film into whole milk powder. Food Research International, 42, 1396–1402. Kosalec, I., Kremer, D., Locatelli, M., Epifano, F., Genovese, S., Carlucci, G., et al. (2013). Anthraquinone profile, antioxidant and antimicrobial activity of bark
extracts of Rhamnus alaternus, R. fallax, R. intermedia, and R. Pumila. Food Chemistry, 136(2), 335–341. Kredy, H. M., Huang, D., Xie, B., He, H., Yang, E., Tian, B., et al. (2010). Flavonols of lotus (Nelumbo nucifera, Gaertn.) seed epicarp and their antioxidant potential. European Food Research and Technology, 231(3), 387–394. Lee, K.-T., Franz, R., & Piringer, O. (1996). The migration behavior of Irganox 1076, Irganox 1010, and Irgafos 168 from polyolefin plastics into ethanol and ethanol/ water mixtures as food simulants. Foods and Biotechnology, 5, 291–294. Li, P., Jia, J., Zhang, D., Xie, J., Xu, X., & Wei, D. (2014). In vitro and in vivo antioxidant activities of a flavonoid isolated from celery (Apium graveolens L. var. dulce). Food & Function, 5(1), 50–56. Limm, W., & Hollifield, C. (1996). Modelling of additive diffusion in polyolefins. Food Additives and Contaminants, 13(8), 949–967. Linssen, J. P. H., Reitsma, J. C. E., & Cozijnsen, J. L. (1998). Migration of antioxidants from polyolefins into ethanolic simulants. Packaging Technology and Science, 11, 241–245. Martínez-Camacho, A. P., Cortez-Rocha, M. O., Graciano-Verdugo, A. Z., RodríguezFélix, F., Castillo-Ortega, M. M., Burgos-Hernández, A., et al. (2013). Extruded films of blended chitosan, low density polyethylene and ethylene acrylic acid. Carbohydrate Polymers, 91(2), 666–674. Momtaz, S., Hussein, A. A., Ostad, S. N., Abdollahi, M., & Lall, N. (2013). Growth inhibition and induction of apoptosis in human cancerous HeLa cells by Maytenus procumbens. Food and Chemical Toxicology, 52, 38–45. Nerín, C., Tovar, L., Djenane, D., Camo, J., Salafranca, J., Beltran, J. A., et al. (2006). Stabilization of beef meat by a new active packaging containing natural antioxidants. Journal of Agricultural and Food Chemistry, 54, 7840–7846. Núñez-Flores, R., Giménez, B., Fernández-Martín, F., López-Caballero, M. E., Montero, M. P., & Gómez-Guillén, M. C. (2013). Physical and functional characterization of active fish gelatin films incorporated with lignin. Food Hydrocolloids, 20(1), 163–172. Petlevski, R., Flajs, D., Kalodera, Z., & Koncˇic´, M. Z. (2013). Composition and antioxidant activity of aqueous and ethanolic Pelargonium radula extracts. South African Journal of Botany, 85, 17–22. Salgado, P. R., López-Caballero, M. E., Gómez-Guillén, M. C., Mauri, A. N., & Montero, M. P. (2013). Sunflower protein films incorporated with clove essential oil have potential application for the preservation of fish patties. Food Hydrocolloids, 33(1), 74–84. Saroja, M., & Annapoorani, S. (2012). In vitro antioxidant activity of flavonoid fractions of cynodon dactylon and terminalia catappa leaves. International Research Journal of Pharmacy, 3(8), 209–211. Siró, I., Fenyvesi, B., Szente, L., Meulenaer, B., Devlieghere, F., Orgovanyi, J., et al. (2006). Release of a-tocopherol from antioxidative low-density polyethylene film into fatty food simulant: Influence of complexation in b-cyclodextrin. Food Additives and Contaminants, 23(8), 845–853.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Study on flavonoid migration from active low-density polyethylene film into aqueous food simulants Shuangling Zhang ⇑, Haiyan Zhao Food Science and Engineering College, Qingdao Agricultural University, No. 700, Changcheng Road, Qingdao 266109, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 1 September 2013 Received in revised form 15 January 2014 Accepted 4 February 2014 Available online 12 February 2014 Keywords: LDPE film Flavonoids Food simulants Migration Diffusion coefficient
a b s t r a c t The migration of flavonoids from low-density polyethylene (LDPE) film (0.4%, w/w) to aqueous food simulants over 16 weeks at 0, 15, and 30 °C was investigated. The migration amount of total flavonoids was calculated based on the rutin contents determined by high-performance liquid chromatography (HPLC). Diffusion and partition coefficients, along with the activation energy (Ea) were calculated based on Fick’s second law. The results showed that the migration of flavonoids was influenced by temperature, time and the simulants. The Ea values for flavonoid diffusion were 49.2, 55.9, and 25.8 kJ mol1 in distilled water, 4% acetic acid and 30% ethanol, respectively. This study indicated that the flavonoids in LDPE film easily migrated into food simulants; and this behaviour was related to the low Ea values of flavonoid diffusion, especially in ethanol at 0–30 °C, when the antioxidants were released from the film. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Flavonoids are the main naturally occurring compounds of pharmacological and nutritional interest, and are widely found in the flowers, leaves and seeds of many plants (Kosalec et al., 2013; Momtaz, Hussein, Ostad, Abdollahi, & Lall, 2013; Petlevski, Flajs, Kalodera, & Koncˇic´, 2013). García-Mateos, Aguilar-Santelises, Soto-Hernández, and Nieto-Angel (2013) reported that the flavonoid extracts of some Mexican flowers have antioxidant activities and their compositions were identified by HPLC-MS. Among the flavonoids, quercetin 3-O-glucoside, quercetin 3-O-rhamnoside, quercetin 3-O-rhamnosyl-(1-6)-glucoside and quercetin 3-O-rhamnosyl-(1-2)-[rhamnosyl-(1-6)]-glucoside were assigned. Li et al. (2014) reported that celery flavonoid extracts exhibited a strong total antioxidant capacity, with IC50 values of 68.0 lg/ ml1 in the DPPH assay, 0.39 lg/ml1 in the O assay and 2 48.0 lg/ml1 in the OH assay, respectively. Saroja and Annapoorani (2012) found that the flavonoid fractions of Cynodon dactylon and Terminalia catappa leaves had antioxidant activity and could potentially be used as a source of natural antioxidants. Kredy et al. (2010) researched the potential antioxidant activities of flavonols from lotus seeds. This antioxidant potential in terms of IC50 values were 5.48, 40 ± 0.14 and 0.62 ± 0.05 (dry fraction 2) lg/ml1 in the DPPH radical, hydroxyl radical and hydrogen ⇑ Corresponding author. Tel./fax: +86 0532 86080771. E-mail addresses: [email protected] (S. Zhang), xinyuyuanyin@163. com (H. Zhao). http://dx.doi.org/10.1016/j.foodchem.2014.02.018 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
peroxide assays, respectively. Flavonoids therefore have potential applications as new food additives with high antioxidant capacities. Antioxidant active packaging is a promising technology for food protection (Barbosa-Pereira et al., 2013; Bodaghi et al., 2013; Martínez-Camacho et al., 2013; Núñez-Flores et al., 2013; Salgado, López-Caballero, Gómez-Guillén, Mauri, & Montero, 2013). The major antioxidants added to films include: a-tocopherol, butylated hydroxytoluene, I-1076 and I-168. Graciano-Verdugo et al. (2010) reported that the concentration of a-tocopherol in low-density polyethylene (LDPE) films, as well as the storage temperature, directly affected the migration of a-tocopherol into corn oil and its oxidative stability. Granda-Restrepo et al. (2009) studied the migration of a-tocopherol from multilayer active packaging materials into whole milk powder. The packaging materials were made of high-density PE (HDPE), ethylene vinyl alcohol and a layer of LDPE containing the antioxidant. Nerín et al. (2006) researched the stabilization of beef using a new active packaging containing natural antioxidants and showed that the active film containing natural antioxidants efficiently enhanced the stability of both the myoglobin and fresh meat against oxidation processes. Garde, Catalá, Gavara, and Hernandez (2001) investigated the migration of antioxidants from polypropylene (PP) films into fatty food simulants and found that heptane could fully extract the antioxidants from the polymer. All the above studies provide a great deal of valuable information and promote the research and development of active packaging. However, there is little data available in scientific literature regarding the migration of flavonoids from LDPE
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S. Zhang, H. Zhao / Food Chemistry 157 (2014) 45–50
films into food. In this study, the characteristics of flavonoid migration from active LDPE films into several aqueous food simulants (distilled water, 4% acetic acid, and 30% ethanol) were investigated to understand the interaction of food and plastic packaging and provide information for the development of the active packaging industry. 2. Materials and methods 2.1. Film manufacture Granular LDPE (1 kg) mixed with flavonoids (5 g) was manufactured into the experimental film using a laboratory film-blowing machine (PTF-IBS-20/28, Guangzhou, China). The running temperature of machine was 120 °C and the air pressure was 0.2 MPa. The film thickness was 4 103 cm, and the flavonoid content was 0.4% (w/w, partially loss during manufacture) as determined using the Soxhlet extraction method. Flavonoids were extracted from Chinese lotus leaves by ultrasonication. The rutin content in the flavonoid extracts was determined by high-performance liquid chromatography (HPLC, Agilent 1200, USA). And the concentration of rutin in theflavonoid extracts was 68.0%. The film was cut into pieces with dimensions of 0.5 cm 0.5 cm after being cleaned with distilled water. 2.2. Migration experiments Distilled water, 4% acetic acid and 30% ethanol were chosen as the aqueous food simulants. For each simulant, 144 LDPE film samples (16 weeks, three replicates) were accurately weighed as 0.2 g. Each sample was placed in a beaker with a cover. Fifty ml of the simulants were added to these beakers. The mixtures were stored under different temperatures (0, 15, and 30 °C). A total of 432 samples were obtained. 2.3. Quantification of flavonoids in aqueous simulants The beakers were taken out of storage and the volume was immediately made up to 50 ml. Samples (10 ml) were evaporated to dryness and the residue was redissolved in methanol (10 ml). The rutin content was determined by HPLC, and the migration of total flavonoids was calculated (68.0% of the rutin concentration in flavonoids). Three replicates were carried out for each sample; the mean values and standard deviations were calculated, denoted by X ± s. Analysis of variance and t-tests were conducted using SAS 9.13 software (SAS Institute Inc.), and pair comparisons were made using the Duncan method. 2.4. Chromatographic conditions The HPLC system (Agilent 1200, USA) was fitted with an auto sampler and diode array ultraviolet detector. Chem-station chromatographic software was used for data acquisition. Chromatographic separation was performed using a Lichrospher C18 column (25 cm 0.21 cm inner diameter). Isocratic elution was performed using acetic acid (0.4% w/w)–methanol (9/1, v/v); the column temperature was 30 °C and the flow rate was 0.3 ml/min1. 2.5. Mathematical models and determination of key parameters Migration processes in films can be fully described by the kinetics of migrant diffusion (expressed by the diffusion coefficient, D) and the chemical equilibrium (expressed by the partition coefficient, K).
An analytical solution of Fick’s second-law equation for diffusion in one dimension, limitless volumes of food, and lengthy contact was used for the determination of D using Eq. (1) (Crank, 1975):
" # 1 X M F;t 8 ð2n þ 1Þ2 p2 ¼1 exp Dt 2 2 MF;1 4dp n¼0 ð2n þ 1Þp2
ð1Þ
where MF,t is the amount of migrant in the food at a particular time t (s); MF,1 is the amount of migrant in the food at equilibrium; dp (cm) is the polymer thickness; D (cm2 s1) is the diffusion coefficient of migrant in the polymer and t (s) is the time. The beginning of migration process can be described by a simplified expression (Eq. (2)) (Crank, 1975):
0:5 M F;t 4 Dt ¼ MF;1 dp p
ð2Þ
To fit the data to Eq. (2), the mass of the flavonoids diffused at time t divided by the mass of the flavonoids diffused at equilibrium (Mt/M1 or Mt/Meq) was plotted versus time t (s), and then D was calculated. The MATLAB (Math Works, Natick, MA, USA) program was used to find the best fit of the data to Eq. (2), using the nlinfit (non-linear regression) function in MATLAB R2012a. The partition coefficient, K, is expressed by
K¼
C P;1 C f ;1
ð3Þ
where Cp,1 (lg g1) and Cf,1 (lg g1) are the equilibrium concentrations of the component in polymer and food, respectively (Siró et al., 2006). 2.6. Diffusion activation energy (Ea) The Ea values for the diffusion of flavonoids from films to simulants was determined using the Arrhenius equation for diffusion (Garde et al., 2001; Limm & Hollifield, 1996):
D ¼ D0 expðEa =RTÞ
ð4Þ
where Ea is the diffusion activation energy; R is the ideal gas constant (8.314 J K1 mol1); and T is the absolute temperature (K). The logarithms of D values were plotted versus the reciprocal of absolute temperature; Ea was obtained using the slope of line (=Ea/2.303R). 3. Results and discussion 3.1. Influence of temperature on migration of flavonoids The increase in the amount of flavonoids in the distilled water, 4% acetic acid, and 30% ethanol during storage at 0, 15 and 30 °C is presented in Fig. 1. It was obtained from Fig. 1 that the amount of flavonoid migration in the three simulants was lowest at 0 °C and highest at 30 °C at the same time before it reached equilibrium. For example, during the first week of storage in distilled water, the migration of flavonoids increased to 4 ± 0.01 lg, 14 ± 2.01 lg, and 29 ± 10.1 lg at 0, 15, and 30 °C, respectively. The amount of migration at 15 °C was triple that at 0 °C, and at 30 °C was twice that at 15 °C. Therefore, the temperature significantly influenced the migration of the flavonoids into the stimulants during storage. The systems reached equilibrium at almost the same percentages (70.3%, 72.5%, and 73.7%) of the amount of migration flavonoids from the total because of the similar dissolutions between the films and liquids. The equilibrium was not related to the storage temperature, as would be expected from the physicochemical properties. However, the equilibrium time had an inverse relationship with temperature and was about a week shorter when
S. Zhang, H. Zhao / Food Chemistry 157 (2014) 45–50
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storage. At 15 °C, the amount of the migrated flavonoids were 14 ± 2.01 lg in distilled water, 34 ± 14.1 lg in 4% acetic acid and 79 ± 7.07 lg in 30% ethanol, respectively, during the first week of storage. The amount of the migrated flavonoids in 30% ethanol was about twice that in 4% acetic acid, and that in 4% acetic acid was about twice that in distilled water. However, the differences were smaller after 8 weeks: 339 ± 8.03 lg, 429 ± 7.24 lg, and 499 ± 10.1 lg in distilled water, 4% acetic acid, and 30% ethanol, respectively. The equilibrium time was also influenced by the simulants, and much shorter (11 weeks) in ethanol than in distilled water (14 weeks) at 0 °C. Therefore the ethanol had a faster absorption rate than the distilled water. Some studies have reported the migration of antioxidants from polyolefin films into ethanol, but comparisons of the results show some differences. Linssen, Reitsma, and Cozijnsen (1998) observed that migration of I-168 and I-1076 from LDPE reached equilibrium after 1 day, and that the equilibrium was close to the full extraction. Lee, Franz, and Piringer (1996) studied the migration of the same antioxidants from polypropylene (PP) films, and reported that equilibrium had not been reached after 21 days, in line with results in this study. 3.3. Diffusion coefficients (D) and partition coefficients (K)
Fig. 1. Amounts of flavonoid migration in distilled water (a), 4% acetic acid (b), and 30% ethanol (c) at 0, 15, and 30 °C; the results are the means of three replicates; bars indicate standard deviations.
the temperature was increased by 15 °C. The same trends were seen in all cases. 3.2. Influence of food stimulants on migration of flavonoids The properties of the food simulants had a major effect on migration of the flavonoids, especially during the first weeks of
Based on Eq. (2), the values of Mt/Meq were plotted versus t0.5 during the first 6 weeks of exposure to the simulants, and a good linear correlation was obtained (Fig. 2). The values are in good agreement with the experimental data for the three simulants and temperatures presented in Fig. 2. The values of D were calculated and are presented in Table 1. The D value describes the migration of flavonoids at a specific temperature for the film with a specific simulant (Table 1). The values of the diffusion coefficients increased with increasing storage temperature. For distilled water, D0 °C = 2.67 1013 cm2 s1 and D15 °C = 4.57 1013 cm2 s1, and for 4% acetic acid, D0 °C = 2.95 1013 cm2 s1 and D15 °C = 6.72 1013 cm2 s1. D15 °C was approximately twice D0 °C. The D values in Table 1 showed that migrations into distilled water and 4% acetic acid were slower than migration into ethanol at 0 °C; the D values of distilled water and 4% acetic acid were about 1/2 that of ethanol. This behaviour may have been caused by plasticisation of the film as a result of sorption of ethanol. There is little information available in the scientific literature regarding the D values of flavonoids from LDPE into foodstuffs. Granda-Restrepo et al. (2009) reported that the D values of a-tocopherol in migration from multilayer active packaging (HDPE + TiO2/EVOH/LDPE + 4% a-tocopherol) to whole milk powder were 2.34, 3.06 and 3.14 1011 cm2 s1 at 20, 30 and 40 °C, respectively. However, these results could not be compared with those in this study because the tested films (multi- and monolayer), fatty foods and storage conditions were different. Graciano-Verdugo et al. (2010) reported that the D values of a-tocopherol were 1.4, 7.1 and 30.3 1011 cm2 s1 at 5, 20 and 30 °C, respectively. The D values were calculated using Eq. (2) and using the same procedure as used here. However, the D values were about 100 times those presented here because the film was in contact with the corn oil. The K values were determined for the simulants using Eq. (3), assuming that equilibrium had been reached after the corresponding weeks of exposure. The K values for each simulant and temperature are presented in Table 1. As can be seen from the data in Table 1, none of the flavonoid migrations at 0, 15 and 30 °C can be regarded as full extraction because the partition equilibrium was not close to complete extraction (K > 0). Comparisons of the behaviours of the flavonoids in the three simulants show that the K values of distilled water and
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Fig. 2. Non-linear regressions of amounts of flavonoid migration versus t0.5: (a) water, (b) 4% acetic acid, and (c) 30% ethanol.
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S. Zhang, H. Zhao / Food Chemistry 157 (2014) 45–50 Table 1 Diffusion coefficients (D, 1013 cm2 s1) for transport of flavonoids from LDPE filmsa to simulants, and partition coefficients (K, flavonoid concentrations lg g1) after sufficient exposure of polymeric films (Cp,1) and simulants (Cf,1) for equilibrium. Parameter Db
Kc
T (°C) 0 15 30 0 15 30
Water 2.67 ± 0.21 h 4.57 ± 0.10 f 6.77 ± 0.13 d 0.141 ± 0.01 ab 0.133 ± 0.0025 c 0.133 ± 0.0065 c
4% acetic acid 2.95 ± 0.10 g 6.72 ± 0.28 d 8.43 ± 0.33 b 0.147 ± 0.0066 a 0.135 ± 0.0109 bc 0.147 ± 0.0128 a
30% ethanol 5.66 ± 0.23 e 8.12 ± 0.14 c 9.20 ± 0.30 a 0.121 ± 0.0109 d 0.121 ± 0.01 d 0.108 ± 0.0026 e
Table 2 Activation energiesa (Ea, kJ mol1) for transport of flavonoids from LDPE filmb to simulants. Water
4% acetic acid
30% ethanol
49.2 ± 3.79 a
55.9 ± 1.21 a
25.8 ± 1.96 b
a Values are shown as X ± s; pair comparisons were made with SAS 9.13 software, using the Duncan method. Same letters means no significant difference, P < 0.05. b Film thickness 4 103 cm.
a
Film thickness 4 103 cm. Results are shown as X ± s; pair comparisons were made with SAS 9.13 software, using the Duncan method. Different letters mean significant differences, P < 0.05. c Results are shown as X ± s; pair comparisons were made with SAS 9.13 software, using the Duncan method. Different letters mean significant differences, P < 0.05. b
4% acetic acid were higher than that of the 30% ethanol. The K value is mainly determined by the polarity of migrating species and the solubility between the film and simulant. Flavonoids were more easily dissolved in ethanol; therefore, the K values obtained were lower than those in distilled water and acetic acid. There were significant differences among the K values (P < 0.05). Some fatty food simulants can fully extract antioxidants. For example, heptane can completely extract H-SE2, I-1076, I-168 and I-168 from PP films when the exposure time is sufficient, and ethanol can totally extract I-168 from PP films (Garde et al., 2001). However, the simulants only extracted some of the antioxidant in our tests. 3.4. Diffusion activation energy (Ea) The Arrhenius plots of D in Fig. 3 are linear, and were used to calculate the Ea values. As can be seen, the theoretical curves agree with the experimental values to some extent, but the curves do not fit very well because of the complexity of the migration tests and some assumptions (one-dimension and limited volumes of packaging and food) (Crank, 1975). The slopes were used to calculate the Ea for the flavonoids in the three food simulants, and the results are presented in Table 2.
The activation energy can be defined as the energy required for a migrant to move among the chains forming the polymer matrix. It is assumed that the migrant, when given sufficient energy, can ‘‘jump’’ into an adjacent space if that space is large enough to accommodate the migrant. A net diffusion flux results if another migrant molecule makes a ‘‘jump’’ into the space that was previously occupied by the first molecule (Limm & Hollifield, 1996). The energy is provided by the temperature, which has an effect on the migrant itself, on the polymer matrix, and on the medium the migrant is in contact with. In this work, the Ea of the flavonoids is lowest for LDPE in ethanol. It can be explained by that less energy was needed for the diffusion of the flavonoid molecules in an LDPE matrix, with more plasticisation compared with that needed in a matrix with less antioxidant, which may need more energy to induce a ‘‘jump’’. The Ea values of the flavonoids in LDPE film with distilled water and 4% acetic acid were similar, and were not significantly different (P < 0.05). The Ea values were in line with published data for similar systems: 105.9 kJ mol1 for a-tocopherol from LDPE films to corn oil (Graciano-Verdugo et al., 2010); 61 kJ mol1 for H-SE2, 65 kJ mol1 for I-1076 and 86 kJ mol1 for I-168 through PP films to ethanol (Garde et al., 2001). 4. Conclusions The migration data on active LDPE films containing flavonoids were obtained and effective diffusion parameters were estimated using an approximate mathematical model based on Fick’s second law. The migration of the flavonoids was influenced by temperature, time and the simulants. The flavonoids in an LDPE film easily migrated into food simulants; and this behaviour was related to
Fig. 3. Linear relationships between flavonoid diffusion coefficients (ln D) and temperature (1/T) in three food stimulants (water, 4% acetic acid, and 30% alcohol).
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the low Ea values for the diffusion of flavonoids, especially in ethanol, for the release of antioxidants from the film at 0–30 °C. Acknowledgements The project was supported by the National 12th Five-Year Research & Technology Based Plan (No. 2012BAK17B05 and 2012BAD28B03) and the 2012 Foundation Project J12LD05 of the Department of Education of Shandong Province, China. The authors gratefully acknowledge Engineering Research Center of Meat Quality Control of Shandong Province for instrument and technical assistance. References Barbosa-Pereira, L., Cruz, J. M., Sendón, R., Bernaldo de Quirós, A. R., Ares, A., CastroLópez, M., et al. (2013). Development of antioxidant active films containing tocopherols to extend the shelf life of fish. Food Control, 31(1), 236–243. Bodaghi, H., Mostofi, Y., Oromiehie, A., Zamani, Z., Ghanbarzadeh, B., Costa, C., et al. (2013). Evaluation of the photocatalytic antimicrobial effects of a TiO2 nanocomposite food packaging film by in vitro and in vivo tests. LWT – Food Science and Technology, 50(2), 702–706. Crank, J. (1975). Mathematics of diffusion (2nd ed.). London: Clarendon Press. García-Mateos, R., Aguilar-Santelises, L., Soto-Hernández, M., & Nieto-Angel, R. (2013). Flavonoids and antioxidant activity of flowers of Mexican Crataegus spp.. Natural Products Research, 27(9), 834–836. Garde, J. A., Catalá, R., Gavara, R., & Hernandez, R. J. (2001). Characterizing the migration of antioxidants from polypropylene into fatty food simulants. Food Additives and Contaminants, 18(8), 750–762. Graciano-Verdugo, A. Z., Soto-Valdez, H., Peralta, E., Cruz-Zárate, P., Islas-Rubio, A. R., Sánchez-Valdes, S., et al. (2010). Migration of a-tocopherol from LDPE films to corn oil and its effect on the oxidative stability. Food Research International, 43, 1073–1078. Granda-Restrepo, D. M., Soto-Valdez, H., Peralta, E., Troncoso-Rojas, R., VallejoCórdoba, B., Gámez-Meza, N., et al. (2009). Migration of a-tocopherol from an active multilayer film into whole milk powder. Food Research International, 42, 1396–1402. Kosalec, I., Kremer, D., Locatelli, M., Epifano, F., Genovese, S., Carlucci, G., et al. (2013). Anthraquinone profile, antioxidant and antimicrobial activity of bark
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