Structure and mechanical properties of sodium and calcium caseinate edible active films with carvacrol

Accepted Manuscript Structure and mechanical properties of sodium and calcium caseinate edible active films with carvacrol Marina P. Arrieta, Mercedes A. Peltzer, María del Carmen Garrigós, Alfonso Jiménez PII: S0260-8774(12)00428-1 DOI: http://dx.doi.org/10.1016/j.jfoodeng.2012.09.002 Reference: JFOE 7079 To appear in: Journal of Food Engineering Received Date: 4 July 2012 Revised Date: 6 September 2012 Accepted Date: 8 September 2012 Please cite this article as: Arrieta, M.P., Peltzer, M.A., Garrigós, a.d.C., Jiménez, A., Structure and mechanical properties of sodium and calcium caseinate edible active films with carvacrol, Journal of Food Engineering (2012), doi: http://dx.doi.org/10.1016/j.jfoodeng.2012.09.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Structure and mechanical properties of sodium and calcium caseinate edible 1 active films with carvacrol 2 Marina P. Arrietaa,b*, Mercedes A. Peltzera, María del Carmen Garrigósa and Alfonso Jiméneza 3 4 a Department of Analytical Chemistry, Nutrition and Food Sciences, University of Alicante, P.O. 5 Box 99, E-03080 Alicante, Spain. 6 7 b Department of Mechanical and Materials Engineering, Polytechnic University of Valencia, 8 Plaza Ferrandiz y Carbonell 1, 03801 Alcoy, Alicante Spain. 9 10 *Corresponding author. Tel: +34-965903117. Fax: +34-965903697 11 E-mail address: m[email protected], [email protected] (M.P. Arrieta) 12 Postal address: Carretera San Vicente del Raspeig s/n. P.O. Box 99, E-03080 San Vicente del 13 Raspeig, Alicante, Spain. 14 15 16 Abstract: 17 Edible active films based on sodium caseinate (SC) and calcium caseinate (CC) 18 plasticized with glycerol (G) at three different concentrations and carvacrol (CRV) as 19 active agent were prepared by solvent casting. Transparent films were obtained and 20 their surfaces were analysed by optical microscopy and scanning electron microscopy 21 (SEM). The influence of the addition of three different plasticizer concentrations was 22 studied by determining tensile properties, while Fourier transformed infrared 23 spectroscopy (FTIR) and thermogravimetic analysis (TGA) were used to evaluate the 24 structural and thermal behaviour of such films. The addition of glycerol resulted in a 25 reduction in the elastic modulus and tensile strength, while some increase in the 26 elongation at break was observed. In general terms, SC films showed flexibility higher 27 than the corresponding CC counterparts. In addition, the presence of carvacrol caused 28 further improvements in ductile properties suggesting the presence of stronger 29 interactions between the protein matrix and glycerol, as it was also observed in thermal 30 degradation studies. FTIR spectra of all films showed the characteristic bands and 31 peaks corresponding to proteins as well as to primary and secondary alcohols. In 32 summary, the best results regarding mechanical and structural properties for 33 caseinates-based films containing carvacrol were found for the formulations with high 34 glycerol concentrations. 35 36 Keywords: edible films; caseinates; glycerol; carvacrol; active packaging. 37 38 1. INTRODUCTION 39 An increasing proactive attitude of society towards a reduction on the 40 environmental impact produced by food packaging after use is currently growing. This 41 could be joined to the consumer’s demand for higher quality and longer shelf life food 42 with an increase on research in new active packaging formulations. Under this general 43 framework, the research on biopolymer-based packaging materials is being extensively 44 explored (Juvonen et al., 2011; Verbeek and Van den Berg, 2010). Another raising 45 tendency in food packaging research is the study of interactions between materials and 46 foodstuff to take advantage of the controlled migration of active additives. Some of 47 them can be extracted from essential oils obtained from aromatic plants with 48 antimicrobial activities (Chalier et al., 2007; Peltzer et al., 2009). They can be used to 49 control spoilage and pathogens proliferation in food during storage and distribution 50 (Ben Arfa et al., 2007; Moreira et al., 2011; Viuda-Martos et al., 2007). Antimicrobial 51 agents can be added by coating onto the food surface or could be incorporated into 52 food-packaging materials with controlled migration to foodstuff (Kristo et al., 2008). 53 The coordination of both concepts (i.e. sustainability and active packaging) 54 could result in the development of edible films with antimicrobial properties to be used 55 in food packaging materials (Ponce et al., 2008; Quintavalla and Vicini, 2002). In this 56 sense, carvacrol, a volatile aromatic compound extracted from oregano and thyme 57 essential oils, is well known for its antimicrobial activity (Lu et al., 2011; Mascheroni et 58 al., 2010; Nostro et al., 2007; Viuda-Martos et al., 2010; Viuda-Martos et al., 2011; 59 Viuda-Martos et al., 2007). Oregano oil has been widely used as a dietary supplement 60 for combating infections and relieving digestive and skin-related problems (Cho et al., 61 2012). 62 The use of biopolymers as matrices in active packaging systems has been 63 relatively unexplored despite their sustainability and advantageous properties. For 64 instance, proteins are adequate for the preparation of biofilms by their high plasticity 65 and elasticity (Pereda et al., 2008; Pereda et al., 2011). In addition, they are abundant 66 in Nature and fully renewable (Ponce et al., 2008) since they can be obtained from 67 plants (corn zein, wheat gluten, soy or sunflower) and animal sources (gelatin, keratin, 68 casein or whey) (Hernandez-Izquierdo and Krochta, 2008; Verbeek and Van den Berg, 69 2010). Among them, caseinates can be considered attractive for their use in food 70 packaging, since they show numerous functional properties, such as water solubility 71 and ability to act as emulsifiers (Fabra et al., 2009; Jimenez et al., 2012; Pereda et al., 72 2010). In addition, due to the high number of polar groups in their structure, caseinates 73 also show good adhesion to different substrates making them excellent barrier to non-74 polar substances, such as oxygen, carbon dioxide and aromas (Audic et al., 2003). 75 However, due to the inherent brittleness of many biopolymers including caseinates, 76 plasticizers should be necessarily used to improve their ductile properties and to get 77 the flexibility required for films manufacturing (Martino et al., 2009). In this sense, the 78 use of glycerol has been proposed, since it contributes to the reduction in material 79 brittleness by the limitation of crosslinking and elimination of intra and intermolecular 80 hydrogen bonds (Pereda et al., 2008)). Furthermore, it should be pointed out that 81 glycerol is a by-product of biodiesel production; so, it would be positive to increase its 82 added value from a low-grade by-product to a useful plasticizer (Ye et al., 2012). 83 Some studies have been recently performed with casein and caseinates as 84 matrices for edible films. For instance, nano-biocomposites based on casein and 85 sodium montmorillonites were recently studied (Pojanavaraphan et al., 2010). Other 86 authors used modified sodium caseinate as matrix for edible films containing oleic acid-87 beeswax mixtures (Fabra et al., 2009) or tung oil (Pereda et al., 2010). Antimicrobial 88 edible films were also obtained from sodium caseinate and chitosan blends (Pereda et 89 al., 2008) or nisin (Cao-Hoang et al., 2010). In this work we compare two commercial 90 caseinates in their use as matrices form polymer edible films. Carvacrol was chosen as 91 antimicrobial agent in these formulations since this compound presents several positive 92 characteristics. It is a natural compound with antimicrobial activity against a broad 93 range of bacteria and it is categorized as GRAS (FDA Admistration US ). Carvacrol has 94 been recently used as active additive in different formulations for active packaging with 95 promising results (Gutierrez et al., 2010; Persico et al., 2009; Ramos et al., 2012). 96 However, at the best of our knowledge the addition of carvacrol into sodium or calcium 97 caseinates for edible films manufacturing has not been reported. 98 The aim of the present work is the development of carvacrol edible active films 99 based on sodium and calcium caseinates plasticized with glycerol. Films were obtained 100 by solvent casting and further characterized to evaluate their viability for food 101 packaging applications, regarding their structure, mechanical and thermal properties. 102 103 2. EXPERIMENTAL 104 2.1 Materials 105 Sodium (SC) and calcium caseinates (CC) were kindly supplied in powder form 106 by Ferrer Alimentación S.A (Barcelona, Spain). Carvacrol (98%) and anhydrous 107 glycerol (99.5%) were purchased from Sigma Aldrich (Móstoles, Madrid, Spain). 108 109 2.2 Films preparation 110 Films were prepared by solvent casting. Solutions were prepared in distilled 111 water with 5 wt% of caseinate, either SC or CC. Glycerol was added to obtain 112 protein:glycerol ratios 1:0, 1:0.15, 1:0.25, 1:0.35. Solutions were then heated at 65 ºC 113 for 10 minutes under continuous stirring at 1100 rpm and further cooled at room 114 temperature. The final pH of these caseinates-glycerol solutions was 6.95. 115 Furthermore, carvacrol was added at 10 wt% resulting in a final protein:carvacrol ratio 116 1:0.10, with homogenization for 3 min at 1100 rpm. The pH of these solutions was 117 between 5.35 and 5.40. Finally, ultrasonic degasification at room temperature was 118 applied to all solutions to eliminate foams and air bubbles. 119 Films were prepared by taking 30 mL of these solutions into 15-cm diameter 120 polyethylene Petri dish containers (Distrilab S.L., Cartagena, Spain). They were 121 conditioned at 25 ± 2 ºC and 50% constant relative humidity (RH) in a Dycometal-122 CM81 climatic test chamber (Barcelona, Spain) for 48 h. 123 The average thickness of films was measured with a Digimatic Micrometer 124 Series 293 MDC-Lite (Mitutoyo, Japan) ± 0.001 mm at ten random positions over the 125 film surface. 126 127 2.3. Characterization 128 Thermogravimetric analysis (TGA) tests were carried out by using a TGA/SDTA 129 851 Mettler Toledo thermal analyzer (Schwarzenbach, Switzerland). Samples weighing 130 around 5-10 mg were heated from room temperature to 700 ºC at 10º C min-1 under 131 nitrogen atmosphere (50 mL min-1) and from 700 ºC to 900 ºC at the same heating rate 132 under oxygen atmosphere (50 mL min-1) with the aim of determining the inorganic 133 residue in each formulation. The initial degradation temperature (T0) was calculated at 134 10% mass loss, while temperatures at the maximum degradation rate (Tmax) for each 135 stage were determined from the peaks of the derivative curves (DTG). 136 Fourier transformed infrared spectroscopy (FTIR) tests were carried out by 137 using a Perkin-Elmer infrared spectrometer (Perkin Elmer Spain, S.L., Madrid Spain). 138 Samples were cut in 1cm x 1cm squares, with average thicknesses 88 ± 16 µm for SC 139 and 103 ± 11µm for CC films, and were analysed at room temperature and 50% RH. 140 Attenuated total reflectance (ATR) spectra were obtained in the 4000-600 cm-1 region, 141 using 128 scans and 4 cm-1 resolution. A blank spectrum was obtained before each 142 test to compensate the humidity effect and the presence of carbon dioxide in the air by 143 spectra subtraction. 144 Samples surfaces were observed by an optical microscopy (Olympus BH2-UMA 145 Microscope), with no further preparation, by ordinary light. 146 Scanning electronic microscopy (SEM) surface and cross section tests were 147 carried out with a JEOL JSM-840 microscope (Jeol USA Inc., Peabody, USA), 148 operated at 10 kV. 10 x 10 mm2 samples were cut and coated with gold layer (10-25 149 nm) prior to analysis in order to increase their electrical conductivity. Images were 150 registered at 1000x magnification. 151 Tensile tests were carried out at room temperature and 50% RH by using a 152 3344 Instron Instrument (Fareham Hants, UK) according to ASTM D882-01 Standard 153 (ASTM, 2001). Tests were performed in rectangular strips (10 x 100 mm2), initial grip 154 separation 50 mm and crosshead speed 25 mm min-1. Average percentage 155 deformation at break (εB %), elastic modulus (E) and tensile strength (TS) were 156 calculated from the resulting stress-strain curves as the average of five measurements 157 from three films of each composition. 158 159 3. RESULTS AND DISCUSSION 160 Transparent SC and CC edible films were successfully obtained by following the 161 above-described procedure. No apparent differences in transparency and colour can 162 be reported for samples containing carvacrol after comparison with the non-active 163 counterparts (Figure 1). However, samples containing carvacrol showed a slightly 164 characteristic oregano odor. Average thicknesses were 88 ± 16 µm for SC and 103 ± 165 11µm for CC films. 166 167 3.1 Thermal properties 168 Table 1 and 2 summarize the TGA results for the SC and CC edible films, 169 respectively. Pure SC and CC showed similar degradation patterns, with two main 170 thermal events. The first one was observed around 120-140 ºC corresponding to the 171 evaporation of absorbed and bound water in caseinates structure. The second stage 172 was associated to their degradation and it was observed from 189 ºC (T0) for SC and 173 198 ºC (T0) for CC, reaching Tmax at 330 ºC and 338 ºC, respectively. The residue at 174 700 ºC was around 25% in both cases. 175 Pure glycerol showed a single degradation step, with T0 146 ºC and Tmax 269 ºC. 176 No residue at 700 ºC was observed. On the other hand, carvacrol degraded in two 177 steps, with Tmax at 92 ºC and 208 ºC. The residual waste after degradation at 700 ºC 178 was negligible. 179 In general terms, TGA curves for films showed more than one degradation step. 180 Non-plasticized films (i.e. pure SC and CC) showed the same TGA pattern than the 181 original powder. Nevertheless, it should be noted that caseinate/glycerol blends 182 showed lower decomposition temperatures than non-plasticized films, indicating a 183 decrease in thermal stability caused by the presence of plasticizer. This result can be 184 explained by the influence of glycerol on the reduction in the number of inter and 185 intramolecular bonds in the protein structure, resulting in a decrease of thermal stability 186 of the whole system (Barreto et al., 2003). 187 On the other hand, plasticized films with carvacrol showed three degradation 188 steps (Figure 2). The first event was observed at temperatures around 100 ºC and it 189 was related to the loss of moisture and bound water remaining from the casting 190 process. The second stage at temperatures between 200 ºC and 250 ºC could be 191 related to the loss of glycerol and carvacrol from the material. A similar behaviour was 192 described by other authors indicating that glycerol can be easily eliminated from 193 caseinate films at temperatures between 105-239 ºC (Pereda et al., 2008). Finally, the 194 third degradation process, at temperatures above 300 ºC, was associated to the 195 protein thermal degradation. This sequential degradation of these films during heating 196 could be explained by the gradual loss of the initial ordered structure of the polymer 197 matrix, since inter and intra-molecular hydrogen bonding are broken up at raising 198 temperatures (Barreto et al., 2003). As it has been reported, the presence of additives 199 in these formulations contributes to the decrease in the number of protein-protein 200 bonds, resulting in lower thermal stability of these samples (Verbeek and Van den 201 Berg, 2010). In general CC films showed higher thermal stability than those based on 202 SC. This result could be explained by considering that divalent calcium cations in CC 203 promote cross-linking with protein chains, giving rise to a more rigid structure with 204 higher thermal stability (Fabra et al., 2010). 205 However, in CC films the presence of carvacrol had no influence on protein-206 protein bonds since the maximum degradation temperature remained nearly constant 207 for all films. As expected, it was observed that the T0 decreased with the addition of 208 higher amounts of glycerol in SC and CC films indicating a good incorporation of the 209 plasticizer to the polymer matrix. 210 211 3.2 FTIR analysis 212 3.2.1 Raw materials 213 Molecular interactions in blends were studied by obtaining their FTIR spectra. 214 The analysis of films obtained from raw materials was performed and results for 215 glycerol and carvacrol as well as for SC and CC in powder form are shown in Figure 3. 216 The spectrum for glycerol (Figure 3a) shows the typical bands for alcohols, with the 217 stretching absorption associated with the hydroxyl groups (-OH) in the 3600-3000 cm-1 218 range, while the carbon-oxygen (C-O) absorption peaks characteristic of primary and 219 secondary alcohols were observed at 1030 and 1100 cm-1, respectively. In addition, 220 peaks for CH-OH bending were observed at 1125-1100 cm-1, while CH2-OH bending 221 bands appeared at 1075-1000 cm-1. Bands in the 1400-1200 cm-1 range can be 222 assigned to the in-plane bending of the hydroxyl group. Finally, asymmetric and 223 formulations, where stable emulsions are formed. Regarding mechanical properties, 391 SC edible films showed higher flexibility than their CC counterparts. Ductile properties 392 of films improved with the addition of glycerol, but some caution should be necessary to 393 avoid phase separation and the consequent migration of plasticizer to foodstuff. 394 Therefore, it can be concluded that glycerol and carvacrol showed good 395 compatibility with caseinates to form homogeneous films. Among all the tested 396 formulations, the best results were found for materials with carvacrol incorporated at 10 397 wt% and glycerol at 35 wt %. This formulation ensures conditions for film processing as 398 well as the significant presence of an antimicrobial additive to get an active packaging 399 system. Consequently, these edible films show potential for their future use in fresh 400 food preservation. Furthermore, more studies on functional properties related to food 401 contact materials (i.e. permeability to gases, water vapour permeability, migration in 402 different environments and antimicrobial properties) as well as the biodegradable 403 characteristics of these formulations should be necessary and are currently ongoing. 404 405 Acknowledgments 406 Marina Patricia Arrieta thanks Fundación MAPFRE for “Ignacio Hernando de 407 Larramendi 2009Medio Ambiente” fellowship (MAPFRE-IHL-01). The Spanish 408 Ministry of Economy and Competitiveness is acknowledged by financial support 409 (project Ref. MAT2011-28468-C02-01). 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Comparison between FTIR amide (1630 nm) and alcohol (1030 nm) bands of SC and CC samples SC:G:CRV 1:0.15:0 1:0.25:0 1:0.35:0 1:0.15:0.10 1:0.25:0.10 1:0.35:0.10 Amide I : alcohol 1º 1:0.23 1:0.36 1:0.49 1:0.54 1:0.61 1:0.83 CC:G:CRV 1:0.15:0 1:0.25:0 1:0.35:0 1:0.15:0.10 1:0.25:0.10 1:0.35:0.10 Amide I : alcohol 1º 1:0.36 1:0.71 1:0.84 1:2.02 1:2.13 1:2.17 Table 4. Tensile properties of SC edible films (n=5) SC:G:CRV Tensile properties εB (%) E (MPa) TS (MPa) 1:0.15:0 17 ± 6 178 ± 80 1.50 ± 0.20 1:0.25:0 32 ± 1 31 ± 10 0.62 ± 0.19 1:0.35:0 79 ± 11 7 ± 1 0.14 ± 0.06 1:0.15:0.10 27 ± 5 82 ± 15 0.73 ± 0.08 1:0.25:0.10 81 ± 3 29 ± 8 0.42 ± 0.15 1:0.35:0.10 62 ± 11 8 ± 2 0.09 ± 0.02 Table 5. Tensile properties of CC edible films (n=5) CC:G:CRV Tensile properties εB (%) E (MPa) TS (MPa) 1:0.15:0 6 ± 2 424 ± 15 1.78 ± 0.09 1:0.25:0 9 ± 2 272 ± 6 0.54 ± 0.01 1:0.35:0 36 ± 2 39 ± 3 0.50 ± 0.02 1:0.15:0.10 5 ± 2 137 ± 2 3.02 ± 0.04 1:0.25:0.10 20 ± 3 61 ± 7 1.20 ± 0.05 1:0.35:0.10 33 ± 8 36 ± 6 0.26 ± 0.03  Transparent and homogeneous edible active films based on caseinates were obtained.  Sodium Caseinate films showed higher flexibility than the Calcium Caseinate ones.  Glycerol and carvacrol showed good compatibility with caseinates to form films. Highlights