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Release behavior of quercetin from chitosan-fish gelatin edible films influenced by electron beam irradiation
Food Control 66 (2016) 315e319
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Release behavior of quercetin from chitosan-fish gelatin edible films influenced by electron beam irradiation de ric Debeaufort* Nasreddine Benbettaïeb, Odile Chambin, Thomas Karbowiak, Fre UMR A 02-102, PAM Food Processing and Physico-Chemistry Laboratory, 1 esplanade Erasme, Universit e Bourgogne Franche-Comt e/Agrosup Dijon, F-21000 Dijon, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2015 Received in revised form 30 January 2016 Accepted 16 February 2016 Available online 18 February 2016
This work dealt with the study of the release kinetics of quercetin incorporated into chitosan-gelatin edible films after electron beam irradiation. The aim was to determine the influence of irradiation dose (at 40 and 60 kGy) on the retention of quercetin in the films, their release in a hydroalcoholic medium (30% ethanol v/v) at 25 C. Irradiation induced a reduction of the release rate for quercetin, revealing that cross-linking probably occurred during irradiation. Indeed, the content (%) of quercetin remaining in the film after the release increased from 23.4 ± 5.7% for non-irradiated sample to 33.6 ± 2.1% after a 60 kGy irradiation dose. But the effective diffusion coefficient of quercetin was not significantly modified by the irradiation process. However, it was noticed a significant increase of the lagtime (time required before the release starts) by ten times. Thus, the irradiation influenced the retention by creating new interactions or linkages between biopolymers and the quercetin, which finally led to an entrapment of a significant amount of the antioxidant. As expected, the electron beam irradiation allowed modulating the retention and then the release of the antioxidant encapsulated in the chitosangelatin matrices. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Electron beam Controlled release Quercetin Fish gelatin Chitosan Edible film Antioxidants Diffusivity and retention
1. Introduction Edible films and coatings offer the opportunity to effectively control mass transfer among different components within a food system or between the food and its surrounding environment (Debeaufort, Voilley, & Guilbert, 2002; Hernandez-Izquierdo & Krochta, 2008). Moreover, a modern trend for developing active edible films and coatings is to combine different biological polymeric materials and to incorporate various functional ingredients, such as nutritional supplements, antimicrobial or antioxidant agents (Cheng, Wang, & Weng, 2015). The most frequently used materials for edible films and coatings are polysaccharides (such as starch, cellulose derivatives, alginate, pectin and chitosan), proteins (such as gelatin, zein, gluten, milk casein, whey and soy proteins) and lipophilic materials (such as glycerides, beeswax and shellac). These materials can be used either individually or in combination to produce films and coatings (Nesterenko, Alric, Silvestre, & Durrieu, 2013). Active packaging can be achieved when functional ingredients are incorporated.
* Corresponding author. E-mail address: [email protected] (F. Debeaufort). http://dx.doi.org/10.1016/j.foodcont.2016.02.027 0956-7135/© 2016 Elsevier Ltd. All rights reserved.
Chitosan is a natural polymer obtained by the deacetylation of chitin, which is a fish industry by-product. It is among the most investigated polysaccharides for active edible films and coatings development due to its inherent antimicrobial, antifungal properties ndez-Pan, Mate , Gardrat, & and good film forming ability (Ferna Coma, 2015). Gelatin is another widely used bio-based material obtained by the controlled hydrolysis of the insoluble fibrous collagen present in the bones and skin generated by fish processing wastes. Its excellent film forming ability is well-known (Hoque, Benjakul, & Prodpran, 2010). Gelatin and chitosan based films used for coating or packaging could maintain the quality of foods during storage, due to their good barrier to oxygen, light and prevention of dehydration and lipid oxidation (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2006; Park & Zhao, 2004). In order to improve the foodprotective capacity of chitosan and/or gelatin films, various active substances including synthetic antimicrobial and antioxidant agents or natural plant extracts have been added into the film for increasing the food shelf life (Wu et al., 2015). In acidic environment (pH < pKa) the amino groups of chitosan are protonated and their positive charges can interact with polyanions such as gelatin, at a pH lower than its isoelectric point, forming a polyelectrolyte complex. Due to these characteristics, chitosan and gelatin have been widely used for
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the production of edible films (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2015a). The incorporation of antioxidants in these biodegradable edible polymers is an interesting alternative to food preservation, since oxidation is one of the major problems affecting food quality as well as biopolymer film ageing (Martins, Cerqueira, & Vicente, 2012). The use of natural, non-toxic antioxidants such as ferulic acid or atocopherol is sought in order to be consistent with the consumer health (Benbettaïeb, Karbowiak, Assifaoui, Debeaufort, & Chambin, 2015; Fabra, Hambleton, Talens, Debeaufort, & Chiralt, 2011). Very few studies have established the effects of polymer structure, in particular chitosan-gelatin films, on the retention and release properties of the antioxidant compounds (Papadokostaki, Amarantos, & Petropoulos, 1998). Besides, irradiation have been shown as a promising technique to induce cross-linking between polymers and to improve the physical and functional properties of edible coatings (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2015b; Vachon et al., 2000). However, the effects of irradiation treatment on the release mechanism of active compounds from edible films are not well-known. Lacroix et al. (2002) showed that gamma-irradiation was efficient enough for inducing cross-links in calcium caseinate edible films and could thus be envisaged for the immobilization of enzymes or active compounds. Gamma irradiation of caseinate also contributed to control the release. However, these works neither explained the mechanism involved in the release rate delay nor the impact of irradiation on the diffusivity of active molecules into the simulant media. The objectives of this study were to investigate the effects of electron beam irradiation on the release behaviour of quercetin in hydroethanolic medium. 2. Materials and methods 2.1. Materials and reagents Commercial grade chitosan (CS) (France Chitine, ref 652, molecular weight of 165 kDa, low viscosity, deacetylation degree of 85%, France) and commercial grade fish gelatin (G) (Rousselot 200 FG 8, Bloom degree ¼ 180, viscosity ¼ 4 mPa s at 45 C and pH ¼ 5.4) were used as film-forming matrix. Anhydrous glycerol (GLY) (Fluka Chemical, 98% purity, Germany) was used as a plasticizer. Glacial acetic acid (Sigma, 99.85% purity) helped to improve the solubility of chitosan. Quercetin (minimum purity 99%) having a molecular weight of 302 g mol1, a melting point of 316 C, a logP value of 1.47 and a solubility in water of 0.06 g L1 (data from Chemspider) was purchased from Sigma Aldrich and used as a model of natural antioxidant molecule. 2.2. Film making A 2% (w/v) chitosan solution was prepared by dispersing the chitosan powder in 1% (v/v) aqueous acetic acid. The solution was homogenized at 1200 rpm with an Ultra Turrax (RW16 basic- IKAWERKE, Germany). Then, glycerol (10% w/w polymer dry matter) was added to the solution under stirring. A 6% (w/v) fish gelatin solution (pH ¼ 4.9 ± 0.2) was prepared in distilled water under continuous stirring and heated at 70 C for 30 min. Glycerol (10% w/ w polymer dry matter) was added to this film forming solution after dissolution of gelatin. Gelatin and chitosan film forming solutions (CS and G) were then mixed in equivalent weight proportion 50%CSe50%G (dry matter), and stirred for 30 min, while adjusting pH between 5.2 and 6. This condition aimed at obtaining a polyelectrolyte complex between chitosan and gelatin, that can only occur at a pH above the isoelectric point of gelatin (pI ¼ 4.5e5.2) to be negatively charged, and below the pKa of the amino group of chitosan (pH ¼ 6.2e6.5) to be positively charged,
and to prevent any phase separation. Quercetin was finally added to this film forming solution at a concentration of 50 mg per gram of dry matter. The aqueous dispersion was homogenized at 1200 rpm using an Ultra Turrax until complete dissolution. The film forming solution containing the antioxidant was then poured into plastic Petri dishes (13.5 cm diameter). They were then dried in a ventilated climatic chamber (KBF 240 Binder, ODIL, France) at 25 C and 45% relative humidity (RH) for 18e24 h. After drying, films were peeled off from the surface and stored up to equilibrium in a ventilated climatic chamber (KBF 240 Binder, ODIL, France) at 50% RH and 25 C before all measurements. 2.3. Radiation treatment Radiation processing was carried out at the AERIAL pilot plant (Innovation Park, Illkirch, Strasbourg, France), using a linear electron accelerator at a temperature of 20 ± 0.5 C. Dried films (65e80 mm thickness) were irradiated with electron beam carrying 2.2 MeV energy and 0.3 kGy/s dose rate. The doses used for this study were 40 and 60 kGy. One batch (with and without antioxidant) was also preserved as the non-irradiated reference. Dosimetry was performed using alanine pellet dosimeters calibrated according the international standard ISO 51607(2004E). 2.4. Release of quercetin in water/ethanol medium The release of the quercetin was carried out in triplicate using a rotating paddle dissolution apparatus (AT7 Smart type II, Sotax, Basel). 600 mg of film was immersed in 1 L of a 30% ethanol solution (v/v) under stirring (50 rpm) at 25 ± 1 C. A 3 mL sample was periodically withdrawn and assayed for quercetin release up to equilibrium. The amount of antioxidant in the release medium was determined by UVevis spectrophotometery (Biochrom Libra S22) at 376 nm. A series of standard solutions (1, 2, 4, 5, 10, 25 and 50 mg/ L) was used for calibration, according to the BeereLambert's law. The initial concentration of quercetin in the film during preparation was used to calculate the percentage of retention in the film at the end of the release kinetics by comparison with the amount released at equilibrium. The effective diffusion coefficient of quercetin in the film (D) was also determined from the release kinetics assuming a Fickian mechanism, considering the transient state of the transfer. The experimental method chosen corresponded to the case of diffusion from a stirred solution of limited volume. As the solution was constantly stirred, we assumed no boundary layer and an always uniform concentration in the solution. The initial concentration of antioxidant in the solution was equal to zero. The concentration of antioxidant in the film was assumed to be uniformly distributed within the film at time zero. We also considered a unidirectional diffusion of the antioxidant in the film, and a diffusivity which neither depended on the concentration nor on the time. Under those conditions, the following analytical solution to Fick second law was thus used (Crank, 1975):
∞ X Ct 2að1 þ aÞ Dq2n t ¼1 exp C∞ 1 þ a þ a2 q2n l2 n¼1
(1)
where Ct is the concentration of the antioxidant determined in the dissolution medium over time, C∞ is the maximum concentration of the antioxidant determined in the dissolution medium when equilibrium is achieved, a ¼ Vs =ðK Vf Þ with Vs the volume of solution (m3), Vf the volume of film (m3) and K the partition coefficient. qn are the non-zero positive roots of tanðqn Þ ¼ aqn using n values between 1 and 6. D is the effective diffusion coefficient
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Fig. 1. Release kinetics of quercetin in a 30% v/v ethanol solution (stirred medium), at 25 C, from control (0 kGy) and irradiated (40 and 60 kGy) chitosan-fish gelatin films.
(m2 s1), and l is the half thickness of the film (m). This model was applied to the experimental release kinetics in order to determine the effective diffusion coefficient of antioxidant in the film. Data modeling was based on the minimisation of the sum of square of the differences between measured and predicted values, using the LevenbergeMarquardt algorithm, and taking D as adjustable parameter. This was performed using Matlab software (The Mathworks, Natick, MA). 2.5. Statistical analyses Data were analyzed using an independent sample t-test and Tukey's multiple comparison tests with the statistical software SPSS 13.0 (SPSS Inc., Chicago, IL). A standard deviation at the 95% confidence level (p-value < 0.05) was used to compare all parameters analysed between irradiated and non-irradiated films, with or without quercetin. 3. Results and discussion The release kinetics of quercetin from chitosan-gelatin based films (non-irradiated and irradiated at 40 and 60 kGy) in hydroethanolic solution (30% ethanol v/v) are given in Fig. 1. The release kinetics of quercetin exhibited the typical shape of non-time dependent and non-concentration dependent diffusion. Irradiation seemed to delay and to better retain the quercetin in the chitosan-gelatin films. Indeed, the content of quercetin remaining in the film after release increased from 6.6 ± 1.6 for non-irradiated sample to 9.5 ± 0.6 and 8.3 ± 0.7 mg/g of film, after 40 and 60 kGy irradiation doses, respectively (Table 1). This increase could be
explained by new interactions occurring between the polymer chains and the antioxidant, as generated by the radiation process. Such treatment might also probably modify the film structure and therefore affect the retention of the antioxidant by the film. Benbettaïeb et al. (2015) already reported an increase of ferulic acid or tyrosol retained in chitosan-gelatin films after irradiation. These results were also in agreement with the enhancement of mechanical, oxygen barrier and thermal properties of films containing quercetin after irradiation, as previously evidenced in another work (Benbettaïeb et al., 2015a). In particular, a 42% increase in tensile strength was noticed as well as a 65% decrease in oxygen permeability and an improvement of thermal stability after incorporation of 5 wt% of quercetin and irradiation at 60 kGy. Thus, some intermolecular linkages were very likely to establish between quercetin and polymer chains or between polymer chains once free radicals were generated during the irradiation process. Lacroix et al. (2002) also showed that gamma-irradiation at 32 kGy was efficient for inducing crosslinks in calcium caseinate edible films as evidenced by the increase of TS by more than 35% and by the increase of molecular weight distribution which was 60-fold greater than not treated films. Irradiation could similarly favor the interactions between quercetin and the biopolymers via a free radical mediated mechanism. Hence quercetin is more entrapped or linked, and consequently more protected and less mobile. Cross-linking could be favoured via bonding between the reactive site in quercetin with tyrosine and other amino acids from gelatin, through a free radical mechanism, or through esterification with hydroxyl amino acids such as serine. On another way, a too high or fast release of synthetic phenolic antioxidants (BHA (butylated hydroxyanisole) and
Table 1 Kinetics parameters of the release of quercetin from chitosan-fish gelatin films (control and irradiated) into liquid medium (30% ethanol v/v) under stirring at 25 C. Control film (0 kGy) Irradiated film (40 kGy) Theoretical content of quercetin in film prior to release (mg/g of film) Content of quercetin remaining in the film after release (mg/g of film) (and percent of the initial content, %) Time lag (min) Diffusion coefficient (1013 m2/s) Values are mean ± standard deviation. Means with the same superscript
a, b
Irradiated film (60 kGy)
47.1 ± 4.7 6.6 ± 1.6a (23.4 ± 5.7) 9.5 ± 0.6b (33.6 ± 2.1) 8.3 ± 0.7a,b (29.5 ± 2.3) 0.2 ± 0.1a 2.40 ± 1.24a R2 ¼ 0.98
1.8 ± 0.5b 2.1 ± 0.1b 1.94 ± 0.43a R2 ¼ 0.99 2.51 ± 0.42a R2 ¼ 0.99
Arabic letter in the same line are not significantly different at p < 0.05.
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Fig. 2. Modeling of the release kinetics (normalized concentration) of quercetin from 60 kGy irradiated film. Ct: concentration of the quercetin released in the aqueous dissolution medium at time t; C∞: concentration of the quercetin released at equilibrium. Symbols are experimental values (mean ± standard deviation) and solid line corresponds to data modeling using an analytical solution of the second Fick's law (Eq (1)).
PG (propyl gallate)) from PLA (polylactic acid) films into 95% ethanol don't suit with the aim of active packaging for which a lower diffusion coefficient is expected (Jamshidian, Tehrany, & Desobry, 2012). Dealing with the kinetic aspects, the time-lag, corresponding to the time during which the release was awaited, was determined from the linear regression of the concentration with respect to time, during the first 30 min. According to Table 1, the time-lag significantly increased by a factor of almost 10 times, from about 12 s up to about 120 s after 60 kGy irradiation. Such non-negligible time-lag values were thus taken into account in the determination of the effective diffusion coefficient. Modeling of experimental data was considered only once the phenomenon started after that timelag. The effective diffusion coefficient (D) of the quercetin in the films was then calculated from the corresponding kinetics by fitting experimental data using Eq (1). A typical release kinetics obtained from an irradiated film is displayed in Fig. 2, and all results are summarized in Table 1. The model fitted well to experimental data, with R2 higher than 0.98. Even though the time-lag increased, the effective diffusion coefficient of quercetin was not significantly modified by the irradiation process. Thus, the irradiation mainly influenced the retention by creating strong enough linkages or interactions between biopolymers and the quercetin, which finally led to an entrapment of a significant amount of the antioxidant (from 23 to 30%). However, the rate of release was surprisingly not affected. On the contrary, Benbettaïeb et al. (2015) displayed a significant effect of the irradiation on the release rate of ferulic acid or tyrosol from chitosan-gelatin films. Indeed, the effective diffusivity of tyrosol was in that case twice reduced for the irradiated films, and that of ferulic acid decreased by 20%. The presented results are in accordance with the effects observed on synthetic polymers subjected to similar treatments. Loo, Tan, Chow, and Lin (2010) investigated how poly(lactide-coglycolide) (PLGA) and poly(L-lactic acid) (PLLA) films produced through an electron-beam irradiated-multi-layer approach can be a viable technique to achieve controlled drug delivery. PLGA underwent pseudo surface degradation, while multi-layer PLLA degraded to a lesser extent after irradiation in a dose range of 50e200 kGy. These phenomena led for PLGA to a shorter diffusion lag-time and a
higher release of both hydrophilic and hydrophobic active compounds due to degradation. On the contrary, PLLA chains are less degraded by irradiation and keep then a longer time lag and higher retention of active compounds. In the same trend, the results obtained by de Queiroz, Abraham and Higa (2006) showed that the use of gamma irradiation doses from 12.5 to 380 kGy on a polyethylene-co-vinyl acetate film produced a crosslinking reaction, which was maximum effect at 150 kGy. The network formed after irradiation significantly reduced the antitumor agent release from such EVA film. However, for higher doses, polymer degradation occurred. When comparing to other chemical crosslinking (non green processing), lignin-soy protein films treated by formaldehyde allowed to modify film properties so as to promote the carrying pezessentials oils and to control their release (Arancibia, Lo mez-Guille n, & Montero, 2014). Thus, further modiCaballero, Go fication is required to decelerate the release. For instance the addition of some retarding agents or the blending with other polymers can produce a more compact network that reduce the diffusivity of the active compounds and could be combined with irradiation. Irradiation, commonly used for packaging disinfection, appears therefore as an interesting strategy to modify the film structure of active packaging in order to better control the release rate of active compounds such as antioxidants when they are in contact with a food product. Acknowledgments The authors gratefully acknowledge the CNSTN Management in Tunisia and the International Atomic Energy Agency (AIEA, Austria) for the financial support of this work (AIEA TUN14011 project). The authors wish to thank the colleagues from SDRI-Direction in the CNSTN and PAM-PAPC research team for precious collaboration and help. The authors wish to sincerely thank Prof. JP Gay for English improvement. References pez-Caballero, M. E., Go mez-Guille n, M. C., & Montero, P. (2014). Arancibia, M. Y., Lo Release of volatile compounds and biodegradability of active soy protein lignin blend films with added citronella essential oil. Food Control, 44, 7e15. Benbettaïeb, N., Karbowiak, T., Assifaoui, A., Debeaufort, F., & Chambin, O. (2015). Controlled release of tyrosol and ferulic acid encapsulated in chitosan-gelatin films after electron beam irradiation. Journal of Radiation Physics and Chemistry, 118, 81e86. Benbettaïeb, N., Karbowiak, T., Brachais, C.-H., & Debeaufort, F. (2015a). Coupling tyrosol, quercetin or ferulic acid and electron beam irradiation to cross-link chitosanegelatin films: a structureefunction approach. European Polymer Journal, 67, 113e127. Benbettaïeb, N., Karbowiak, T., Brachais, C.-H., & Debeaufort, F. (2015b). Impact of electron beam irradiation on fish gelatin film properties. Food Chemistry, 195, 11e18. Cheng, S.-Y., Wang, B.-J., & Weng, Y.-M. (2015). Antioxidant and antimicrobial edible zein/chitosan composite films fabricated by incorporation of phenolic compounds and dicarboxylic acids. LWT - Food Science and Technology, 63(1), 115e121. Crank, J. (1975). Brunel University Uxbridge. The mathematics of diffusion (pp. 56e57). Ely House, London. W.I.: Oxford University Press. Debeaufort, F., Voilley, A., & Guilbert, S. (2002). The procedures of product stabilisation due to films barriers. In water in foods (pp. 549e622). Paris: Tec&Doc Lavoisier. Fabra, M. J., Hambleton, A., Talens, P., Debeaufort, F., & Chiralt, A. (2011). Effect of ferulic acid and a-tocopherol antioxidants on properties of sodium caseinate edible films. Food Hydrocolloids, 25(6), 1441e1447. , J. I., Gardrat, C., & Coma, V. (2015). Effect of chitosan moFern andez-Pan, I., Mate lecular weight on the antimicrobial activity and release rate of carvacrolenriched films. Food Hydrocolloids, 51, 60e68. Hernandez-Izquierdo, V. M., & Krochta, J. M. (2008). Thermoplastic processing of proteins for film formationda review. Journal of Food Science, 73(2), R30eR39. Hoque, M. S., Benjakul, S., & Prodpran, T. (2010). Effect of heat treatment of filmforming solution on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin. Journal of Food Engineering, 96(1), 66e73.
N. Benbettaïeb et al. / Food Control 66 (2016) 315e319 Jamshidian, M., Tehrany, E. A., & Desobry, S. (2012). Release of synthetic phenolic antioxidants from extruded poly lactic acid (PLA) film. Food Control, 28(2), 445e455. Jongjareonrak, A., Benjakul, S., Visessanguan, W., & Tanaka, M. (2006). Effects of plasticizers on the properties of edible films from skin gelatin of bigeye snapper and brownstripe red snapper. European Food Research and Technology, 222(3e4), 229e235. Lacroix, M., Le, T. C., Ouattara, B., Yu, H., Letendre, M., Sabato, S. F., et al. (2002). Use of g-irradiation to produce films from whey, casein and soya proteins: structure and functionals characteristics. Radiation Physics and Chemistry, 63(3e6), 827e832. Loo, S. C. J., Tan, Z. Y. S., Chow, Y. J., & Lin, S. L. I. (2010). Drug release from irradiated PLGA and PLLA multi-layered films. Journal of Pharmaceutical Sciences, 99(7), 3060e3071. Martins, J. T., Cerqueira, M. A., & Vicente, A. A. (2012). Influence of a-tocopherol on physicochemical properties of chitosan-based films. Food Hydrocolloids, 27(1), 220e227. Nesterenko, A., Alric, I., Silvestre, F., & Durrieu, V. (2013). Vegetable proteins in microencapsulation: a review of recent interventions and their effectiveness.
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Industrial Crops and Products, 42, 469e479. Papadokostaki, K. G., Amarantos, S. G., & Petropoulos, J. H. (1998). Kinetics of release of particulate solutes incorporated in cellulosic polymer matrices as a function of solute solubility and polymer swellability. I. Sparingly soluble solutes. Journal of Applied Polymer Science, 67(2), 277e287. Park, S.-i., & Zhao, Y. (2004). Incorporation of a high concentration of mineral or vitamin into chitosan-based films. Journal of Agricultural and Food Chemistry, 52(7), 1933e1939. de Queiroz, A. A., Abraham, G. A., & Higa, O. Z. (2006). Controlled release of 5-fluorouridine from radiation-crosslinked poly (ethylene-co-vinyl acetate) films. Acta Biomaterialia, 2(6), 641e650. Vachon, C., Yu, H. L., Yefsah, R., Alain, R., St-Gelais, D., & Lacroix, M. (2000). Mechanical and structural properties of milk protein edible films cross-linked by heating and g-irradiation. Journal of Agricultural and Food Chemistry, 48(8), 3202e3209. Wu, J., Liu, H., Ge, S., Wang, S., Qin, Z., Chen, L., et al. (2015). The preparation, characterization, antimicrobial stability and in vitro release evaluation of fish gelatin films incorporated with cinnamon essential oil nanoliposomes. Food Hydrocolloids, 43, 427e435.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Release behavior of quercetin from chitosan-fish gelatin edible films influenced by electron beam irradiation de ric Debeaufort* Nasreddine Benbettaïeb, Odile Chambin, Thomas Karbowiak, Fre UMR A 02-102, PAM Food Processing and Physico-Chemistry Laboratory, 1 esplanade Erasme, Universit e Bourgogne Franche-Comt e/Agrosup Dijon, F-21000 Dijon, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2015 Received in revised form 30 January 2016 Accepted 16 February 2016 Available online 18 February 2016
This work dealt with the study of the release kinetics of quercetin incorporated into chitosan-gelatin edible films after electron beam irradiation. The aim was to determine the influence of irradiation dose (at 40 and 60 kGy) on the retention of quercetin in the films, their release in a hydroalcoholic medium (30% ethanol v/v) at 25 C. Irradiation induced a reduction of the release rate for quercetin, revealing that cross-linking probably occurred during irradiation. Indeed, the content (%) of quercetin remaining in the film after the release increased from 23.4 ± 5.7% for non-irradiated sample to 33.6 ± 2.1% after a 60 kGy irradiation dose. But the effective diffusion coefficient of quercetin was not significantly modified by the irradiation process. However, it was noticed a significant increase of the lagtime (time required before the release starts) by ten times. Thus, the irradiation influenced the retention by creating new interactions or linkages between biopolymers and the quercetin, which finally led to an entrapment of a significant amount of the antioxidant. As expected, the electron beam irradiation allowed modulating the retention and then the release of the antioxidant encapsulated in the chitosangelatin matrices. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Electron beam Controlled release Quercetin Fish gelatin Chitosan Edible film Antioxidants Diffusivity and retention
1. Introduction Edible films and coatings offer the opportunity to effectively control mass transfer among different components within a food system or between the food and its surrounding environment (Debeaufort, Voilley, & Guilbert, 2002; Hernandez-Izquierdo & Krochta, 2008). Moreover, a modern trend for developing active edible films and coatings is to combine different biological polymeric materials and to incorporate various functional ingredients, such as nutritional supplements, antimicrobial or antioxidant agents (Cheng, Wang, & Weng, 2015). The most frequently used materials for edible films and coatings are polysaccharides (such as starch, cellulose derivatives, alginate, pectin and chitosan), proteins (such as gelatin, zein, gluten, milk casein, whey and soy proteins) and lipophilic materials (such as glycerides, beeswax and shellac). These materials can be used either individually or in combination to produce films and coatings (Nesterenko, Alric, Silvestre, & Durrieu, 2013). Active packaging can be achieved when functional ingredients are incorporated.
* Corresponding author. E-mail address: [email protected] (F. Debeaufort). http://dx.doi.org/10.1016/j.foodcont.2016.02.027 0956-7135/© 2016 Elsevier Ltd. All rights reserved.
Chitosan is a natural polymer obtained by the deacetylation of chitin, which is a fish industry by-product. It is among the most investigated polysaccharides for active edible films and coatings development due to its inherent antimicrobial, antifungal properties ndez-Pan, Mate , Gardrat, & and good film forming ability (Ferna Coma, 2015). Gelatin is another widely used bio-based material obtained by the controlled hydrolysis of the insoluble fibrous collagen present in the bones and skin generated by fish processing wastes. Its excellent film forming ability is well-known (Hoque, Benjakul, & Prodpran, 2010). Gelatin and chitosan based films used for coating or packaging could maintain the quality of foods during storage, due to their good barrier to oxygen, light and prevention of dehydration and lipid oxidation (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2006; Park & Zhao, 2004). In order to improve the foodprotective capacity of chitosan and/or gelatin films, various active substances including synthetic antimicrobial and antioxidant agents or natural plant extracts have been added into the film for increasing the food shelf life (Wu et al., 2015). In acidic environment (pH < pKa) the amino groups of chitosan are protonated and their positive charges can interact with polyanions such as gelatin, at a pH lower than its isoelectric point, forming a polyelectrolyte complex. Due to these characteristics, chitosan and gelatin have been widely used for
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the production of edible films (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2015a). The incorporation of antioxidants in these biodegradable edible polymers is an interesting alternative to food preservation, since oxidation is one of the major problems affecting food quality as well as biopolymer film ageing (Martins, Cerqueira, & Vicente, 2012). The use of natural, non-toxic antioxidants such as ferulic acid or atocopherol is sought in order to be consistent with the consumer health (Benbettaïeb, Karbowiak, Assifaoui, Debeaufort, & Chambin, 2015; Fabra, Hambleton, Talens, Debeaufort, & Chiralt, 2011). Very few studies have established the effects of polymer structure, in particular chitosan-gelatin films, on the retention and release properties of the antioxidant compounds (Papadokostaki, Amarantos, & Petropoulos, 1998). Besides, irradiation have been shown as a promising technique to induce cross-linking between polymers and to improve the physical and functional properties of edible coatings (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2015b; Vachon et al., 2000). However, the effects of irradiation treatment on the release mechanism of active compounds from edible films are not well-known. Lacroix et al. (2002) showed that gamma-irradiation was efficient enough for inducing cross-links in calcium caseinate edible films and could thus be envisaged for the immobilization of enzymes or active compounds. Gamma irradiation of caseinate also contributed to control the release. However, these works neither explained the mechanism involved in the release rate delay nor the impact of irradiation on the diffusivity of active molecules into the simulant media. The objectives of this study were to investigate the effects of electron beam irradiation on the release behaviour of quercetin in hydroethanolic medium. 2. Materials and methods 2.1. Materials and reagents Commercial grade chitosan (CS) (France Chitine, ref 652, molecular weight of 165 kDa, low viscosity, deacetylation degree of 85%, France) and commercial grade fish gelatin (G) (Rousselot 200 FG 8, Bloom degree ¼ 180, viscosity ¼ 4 mPa s at 45 C and pH ¼ 5.4) were used as film-forming matrix. Anhydrous glycerol (GLY) (Fluka Chemical, 98% purity, Germany) was used as a plasticizer. Glacial acetic acid (Sigma, 99.85% purity) helped to improve the solubility of chitosan. Quercetin (minimum purity 99%) having a molecular weight of 302 g mol1, a melting point of 316 C, a logP value of 1.47 and a solubility in water of 0.06 g L1 (data from Chemspider) was purchased from Sigma Aldrich and used as a model of natural antioxidant molecule. 2.2. Film making A 2% (w/v) chitosan solution was prepared by dispersing the chitosan powder in 1% (v/v) aqueous acetic acid. The solution was homogenized at 1200 rpm with an Ultra Turrax (RW16 basic- IKAWERKE, Germany). Then, glycerol (10% w/w polymer dry matter) was added to the solution under stirring. A 6% (w/v) fish gelatin solution (pH ¼ 4.9 ± 0.2) was prepared in distilled water under continuous stirring and heated at 70 C for 30 min. Glycerol (10% w/ w polymer dry matter) was added to this film forming solution after dissolution of gelatin. Gelatin and chitosan film forming solutions (CS and G) were then mixed in equivalent weight proportion 50%CSe50%G (dry matter), and stirred for 30 min, while adjusting pH between 5.2 and 6. This condition aimed at obtaining a polyelectrolyte complex between chitosan and gelatin, that can only occur at a pH above the isoelectric point of gelatin (pI ¼ 4.5e5.2) to be negatively charged, and below the pKa of the amino group of chitosan (pH ¼ 6.2e6.5) to be positively charged,
and to prevent any phase separation. Quercetin was finally added to this film forming solution at a concentration of 50 mg per gram of dry matter. The aqueous dispersion was homogenized at 1200 rpm using an Ultra Turrax until complete dissolution. The film forming solution containing the antioxidant was then poured into plastic Petri dishes (13.5 cm diameter). They were then dried in a ventilated climatic chamber (KBF 240 Binder, ODIL, France) at 25 C and 45% relative humidity (RH) for 18e24 h. After drying, films were peeled off from the surface and stored up to equilibrium in a ventilated climatic chamber (KBF 240 Binder, ODIL, France) at 50% RH and 25 C before all measurements. 2.3. Radiation treatment Radiation processing was carried out at the AERIAL pilot plant (Innovation Park, Illkirch, Strasbourg, France), using a linear electron accelerator at a temperature of 20 ± 0.5 C. Dried films (65e80 mm thickness) were irradiated with electron beam carrying 2.2 MeV energy and 0.3 kGy/s dose rate. The doses used for this study were 40 and 60 kGy. One batch (with and without antioxidant) was also preserved as the non-irradiated reference. Dosimetry was performed using alanine pellet dosimeters calibrated according the international standard ISO 51607(2004E). 2.4. Release of quercetin in water/ethanol medium The release of the quercetin was carried out in triplicate using a rotating paddle dissolution apparatus (AT7 Smart type II, Sotax, Basel). 600 mg of film was immersed in 1 L of a 30% ethanol solution (v/v) under stirring (50 rpm) at 25 ± 1 C. A 3 mL sample was periodically withdrawn and assayed for quercetin release up to equilibrium. The amount of antioxidant in the release medium was determined by UVevis spectrophotometery (Biochrom Libra S22) at 376 nm. A series of standard solutions (1, 2, 4, 5, 10, 25 and 50 mg/ L) was used for calibration, according to the BeereLambert's law. The initial concentration of quercetin in the film during preparation was used to calculate the percentage of retention in the film at the end of the release kinetics by comparison with the amount released at equilibrium. The effective diffusion coefficient of quercetin in the film (D) was also determined from the release kinetics assuming a Fickian mechanism, considering the transient state of the transfer. The experimental method chosen corresponded to the case of diffusion from a stirred solution of limited volume. As the solution was constantly stirred, we assumed no boundary layer and an always uniform concentration in the solution. The initial concentration of antioxidant in the solution was equal to zero. The concentration of antioxidant in the film was assumed to be uniformly distributed within the film at time zero. We also considered a unidirectional diffusion of the antioxidant in the film, and a diffusivity which neither depended on the concentration nor on the time. Under those conditions, the following analytical solution to Fick second law was thus used (Crank, 1975):
∞ X Ct 2að1 þ aÞ Dq2n t ¼1 exp C∞ 1 þ a þ a2 q2n l2 n¼1
(1)
where Ct is the concentration of the antioxidant determined in the dissolution medium over time, C∞ is the maximum concentration of the antioxidant determined in the dissolution medium when equilibrium is achieved, a ¼ Vs =ðK Vf Þ with Vs the volume of solution (m3), Vf the volume of film (m3) and K the partition coefficient. qn are the non-zero positive roots of tanðqn Þ ¼ aqn using n values between 1 and 6. D is the effective diffusion coefficient
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Fig. 1. Release kinetics of quercetin in a 30% v/v ethanol solution (stirred medium), at 25 C, from control (0 kGy) and irradiated (40 and 60 kGy) chitosan-fish gelatin films.
(m2 s1), and l is the half thickness of the film (m). This model was applied to the experimental release kinetics in order to determine the effective diffusion coefficient of antioxidant in the film. Data modeling was based on the minimisation of the sum of square of the differences between measured and predicted values, using the LevenbergeMarquardt algorithm, and taking D as adjustable parameter. This was performed using Matlab software (The Mathworks, Natick, MA). 2.5. Statistical analyses Data were analyzed using an independent sample t-test and Tukey's multiple comparison tests with the statistical software SPSS 13.0 (SPSS Inc., Chicago, IL). A standard deviation at the 95% confidence level (p-value < 0.05) was used to compare all parameters analysed between irradiated and non-irradiated films, with or without quercetin. 3. Results and discussion The release kinetics of quercetin from chitosan-gelatin based films (non-irradiated and irradiated at 40 and 60 kGy) in hydroethanolic solution (30% ethanol v/v) are given in Fig. 1. The release kinetics of quercetin exhibited the typical shape of non-time dependent and non-concentration dependent diffusion. Irradiation seemed to delay and to better retain the quercetin in the chitosan-gelatin films. Indeed, the content of quercetin remaining in the film after release increased from 6.6 ± 1.6 for non-irradiated sample to 9.5 ± 0.6 and 8.3 ± 0.7 mg/g of film, after 40 and 60 kGy irradiation doses, respectively (Table 1). This increase could be
explained by new interactions occurring between the polymer chains and the antioxidant, as generated by the radiation process. Such treatment might also probably modify the film structure and therefore affect the retention of the antioxidant by the film. Benbettaïeb et al. (2015) already reported an increase of ferulic acid or tyrosol retained in chitosan-gelatin films after irradiation. These results were also in agreement with the enhancement of mechanical, oxygen barrier and thermal properties of films containing quercetin after irradiation, as previously evidenced in another work (Benbettaïeb et al., 2015a). In particular, a 42% increase in tensile strength was noticed as well as a 65% decrease in oxygen permeability and an improvement of thermal stability after incorporation of 5 wt% of quercetin and irradiation at 60 kGy. Thus, some intermolecular linkages were very likely to establish between quercetin and polymer chains or between polymer chains once free radicals were generated during the irradiation process. Lacroix et al. (2002) also showed that gamma-irradiation at 32 kGy was efficient for inducing crosslinks in calcium caseinate edible films as evidenced by the increase of TS by more than 35% and by the increase of molecular weight distribution which was 60-fold greater than not treated films. Irradiation could similarly favor the interactions between quercetin and the biopolymers via a free radical mediated mechanism. Hence quercetin is more entrapped or linked, and consequently more protected and less mobile. Cross-linking could be favoured via bonding between the reactive site in quercetin with tyrosine and other amino acids from gelatin, through a free radical mechanism, or through esterification with hydroxyl amino acids such as serine. On another way, a too high or fast release of synthetic phenolic antioxidants (BHA (butylated hydroxyanisole) and
Table 1 Kinetics parameters of the release of quercetin from chitosan-fish gelatin films (control and irradiated) into liquid medium (30% ethanol v/v) under stirring at 25 C. Control film (0 kGy) Irradiated film (40 kGy) Theoretical content of quercetin in film prior to release (mg/g of film) Content of quercetin remaining in the film after release (mg/g of film) (and percent of the initial content, %) Time lag (min) Diffusion coefficient (1013 m2/s) Values are mean ± standard deviation. Means with the same superscript
a, b
Irradiated film (60 kGy)
47.1 ± 4.7 6.6 ± 1.6a (23.4 ± 5.7) 9.5 ± 0.6b (33.6 ± 2.1) 8.3 ± 0.7a,b (29.5 ± 2.3) 0.2 ± 0.1a 2.40 ± 1.24a R2 ¼ 0.98
1.8 ± 0.5b 2.1 ± 0.1b 1.94 ± 0.43a R2 ¼ 0.99 2.51 ± 0.42a R2 ¼ 0.99
Arabic letter in the same line are not significantly different at p < 0.05.
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Fig. 2. Modeling of the release kinetics (normalized concentration) of quercetin from 60 kGy irradiated film. Ct: concentration of the quercetin released in the aqueous dissolution medium at time t; C∞: concentration of the quercetin released at equilibrium. Symbols are experimental values (mean ± standard deviation) and solid line corresponds to data modeling using an analytical solution of the second Fick's law (Eq (1)).
PG (propyl gallate)) from PLA (polylactic acid) films into 95% ethanol don't suit with the aim of active packaging for which a lower diffusion coefficient is expected (Jamshidian, Tehrany, & Desobry, 2012). Dealing with the kinetic aspects, the time-lag, corresponding to the time during which the release was awaited, was determined from the linear regression of the concentration with respect to time, during the first 30 min. According to Table 1, the time-lag significantly increased by a factor of almost 10 times, from about 12 s up to about 120 s after 60 kGy irradiation. Such non-negligible time-lag values were thus taken into account in the determination of the effective diffusion coefficient. Modeling of experimental data was considered only once the phenomenon started after that timelag. The effective diffusion coefficient (D) of the quercetin in the films was then calculated from the corresponding kinetics by fitting experimental data using Eq (1). A typical release kinetics obtained from an irradiated film is displayed in Fig. 2, and all results are summarized in Table 1. The model fitted well to experimental data, with R2 higher than 0.98. Even though the time-lag increased, the effective diffusion coefficient of quercetin was not significantly modified by the irradiation process. Thus, the irradiation mainly influenced the retention by creating strong enough linkages or interactions between biopolymers and the quercetin, which finally led to an entrapment of a significant amount of the antioxidant (from 23 to 30%). However, the rate of release was surprisingly not affected. On the contrary, Benbettaïeb et al. (2015) displayed a significant effect of the irradiation on the release rate of ferulic acid or tyrosol from chitosan-gelatin films. Indeed, the effective diffusivity of tyrosol was in that case twice reduced for the irradiated films, and that of ferulic acid decreased by 20%. The presented results are in accordance with the effects observed on synthetic polymers subjected to similar treatments. Loo, Tan, Chow, and Lin (2010) investigated how poly(lactide-coglycolide) (PLGA) and poly(L-lactic acid) (PLLA) films produced through an electron-beam irradiated-multi-layer approach can be a viable technique to achieve controlled drug delivery. PLGA underwent pseudo surface degradation, while multi-layer PLLA degraded to a lesser extent after irradiation in a dose range of 50e200 kGy. These phenomena led for PLGA to a shorter diffusion lag-time and a
higher release of both hydrophilic and hydrophobic active compounds due to degradation. On the contrary, PLLA chains are less degraded by irradiation and keep then a longer time lag and higher retention of active compounds. In the same trend, the results obtained by de Queiroz, Abraham and Higa (2006) showed that the use of gamma irradiation doses from 12.5 to 380 kGy on a polyethylene-co-vinyl acetate film produced a crosslinking reaction, which was maximum effect at 150 kGy. The network formed after irradiation significantly reduced the antitumor agent release from such EVA film. However, for higher doses, polymer degradation occurred. When comparing to other chemical crosslinking (non green processing), lignin-soy protein films treated by formaldehyde allowed to modify film properties so as to promote the carrying pezessentials oils and to control their release (Arancibia, Lo mez-Guille n, & Montero, 2014). Thus, further modiCaballero, Go fication is required to decelerate the release. For instance the addition of some retarding agents or the blending with other polymers can produce a more compact network that reduce the diffusivity of the active compounds and could be combined with irradiation. Irradiation, commonly used for packaging disinfection, appears therefore as an interesting strategy to modify the film structure of active packaging in order to better control the release rate of active compounds such as antioxidants when they are in contact with a food product. Acknowledgments The authors gratefully acknowledge the CNSTN Management in Tunisia and the International Atomic Energy Agency (AIEA, Austria) for the financial support of this work (AIEA TUN14011 project). The authors wish to thank the colleagues from SDRI-Direction in the CNSTN and PAM-PAPC research team for precious collaboration and help. The authors wish to sincerely thank Prof. JP Gay for English improvement. References pez-Caballero, M. E., Go mez-Guille n, M. C., & Montero, P. (2014). Arancibia, M. Y., Lo Release of volatile compounds and biodegradability of active soy protein lignin blend films with added citronella essential oil. Food Control, 44, 7e15. Benbettaïeb, N., Karbowiak, T., Assifaoui, A., Debeaufort, F., & Chambin, O. (2015). Controlled release of tyrosol and ferulic acid encapsulated in chitosan-gelatin films after electron beam irradiation. Journal of Radiation Physics and Chemistry, 118, 81e86. Benbettaïeb, N., Karbowiak, T., Brachais, C.-H., & Debeaufort, F. (2015a). Coupling tyrosol, quercetin or ferulic acid and electron beam irradiation to cross-link chitosanegelatin films: a structureefunction approach. European Polymer Journal, 67, 113e127. Benbettaïeb, N., Karbowiak, T., Brachais, C.-H., & Debeaufort, F. (2015b). Impact of electron beam irradiation on fish gelatin film properties. Food Chemistry, 195, 11e18. Cheng, S.-Y., Wang, B.-J., & Weng, Y.-M. (2015). Antioxidant and antimicrobial edible zein/chitosan composite films fabricated by incorporation of phenolic compounds and dicarboxylic acids. LWT - Food Science and Technology, 63(1), 115e121. Crank, J. (1975). Brunel University Uxbridge. The mathematics of diffusion (pp. 56e57). Ely House, London. W.I.: Oxford University Press. Debeaufort, F., Voilley, A., & Guilbert, S. (2002). The procedures of product stabilisation due to films barriers. In water in foods (pp. 549e622). Paris: Tec&Doc Lavoisier. Fabra, M. J., Hambleton, A., Talens, P., Debeaufort, F., & Chiralt, A. (2011). Effect of ferulic acid and a-tocopherol antioxidants on properties of sodium caseinate edible films. Food Hydrocolloids, 25(6), 1441e1447. , J. I., Gardrat, C., & Coma, V. (2015). Effect of chitosan moFern andez-Pan, I., Mate lecular weight on the antimicrobial activity and release rate of carvacrolenriched films. Food Hydrocolloids, 51, 60e68. Hernandez-Izquierdo, V. M., & Krochta, J. M. (2008). Thermoplastic processing of proteins for film formationda review. Journal of Food Science, 73(2), R30eR39. Hoque, M. S., Benjakul, S., & Prodpran, T. (2010). Effect of heat treatment of filmforming solution on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin. Journal of Food Engineering, 96(1), 66e73.
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