Selection of cassava starch carnauba wax composite edible coating used to preserve fresh-cut apples Marcela Chiumarelli a, Miriam Dupas Hubinger a
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1 Selection of cassava starch carnauba wax composite edible coating used to preserve fresh-cut apples Marcela Chiumarelli a, Miriam Dupas Hubinger a a Dept. of Food Engineering, School of Food Engineering, University of Campinas, Brazil (march@fea.unicamp.br, mhub@fea.unicamp.br) ABSTRACT The objective of this work was to select formulations for the cassava starch carnauba wax composite edible coating used to preserve fresh-cut apples. The Response Surface Methodology (RSM) was applied to determine the effect of cassava starch content ( 4% w/w), glycerol content (1 3% w/w) and carnauba wax stearic acid ratio (0:0 0.40:0.60% w/w) on barrier properties, water solubility and mechanical properties of films made with the coatings solutions. Respiration rate of apple slices with coatings and water vapor resistance were strongly influenced (p < 0.05) by carnauba wax: stearic acid ratio. All responses, mainly mechanical properties, were influenced by glycerol content and carnauba wax: stearic acid ratio. Models and response surfaces were obtained for respiration rate, coatings water vapor resistance, water solubility and elastic modulus of films. The optimized conditions were defined as cassava starch concentration of 3.0 g/ 100g of coating solution, glycerol concentration of 1.5 g/100 g of coating solution, carnauba wax concentration of 0. g/ 100 g of coating solution and stearic acid concentration of 0.8 g/ 100 g of coating solution. The models obtained in the experimental design were predictive, which was evidenced by the relative deviations of less than 10%. Thus, RSM was an effective tool for identifying optimized coating formulations. Keywords: edible coating; water vapor resistance; respiration rate; water solubility; mechanical properties. INTRODUCTION Fresh-cut fruits and vegetables are practical and convenient products, which maintain the fresh and nutritional quality of the whole product. On the other hand, the peeling and cutting operations can accelerate the metabolic activities of plant tissue, making the minimally processed product more perishable than intact fruits and vegetables. The use of edible coatings is an alternative to maintain the quality and freshness of minimally processed products and to prolong their shelf life. The coatings act as barriers to water loss and gas exchange. A range of polymers can be used in the edible coatings formulation. However, their mechanical and barrier properties are intrinsically linked to physical and chemical characteristics of their constituents, such as proteins, polysaccharides and lipids, which can be used alone or in combinations [1]. This work aimed to evaluate edible coatings/films formulated with cassava starch, glycerol, carnauba wax and stearic acid, analyzing the barrier properties of coatings applied in fresh-cut apples and the solubility and mechanical properties of films prepared with coating solutions using the Response Surface Methodology (RSM). MATERIALS & METHODS Materials The material used for coating/film formulations were: cassava starch (17% amylose content, Pilão Amidos Ltda., Guaíra, Brazil), carnauba wax type 1 (Cresal Comércio e Representações Ltda., São Paulo, Brasil), stearic acid (Synth, Diadema, Brazil) and glycerol (ECIBRA, São Paulo, Brazil). Apples (Malus domestica Borkh cv Gala ) of uniform size (10.59 ± 5.4 g), maturity stage (based on skin colour and firmness) with no physical damage were obtained from the local market (Ceasa, Campinas, Brazil). Coating/Film Preparation Cassava starch suspensions were prepared at 75 C with constant stirring and glycerol was added after the gelatinization of starch. Carnauba wax and stearic acid were melted at 85 C and homogenized in the cassava starch suspensions with a high-shear probe mixer (model MA 10, Marconi Ltda., Piracicaba, Brazil) for 3 min at 1000 rpm.
2 Apple slices and cylinders were dipped in cassava starch carnauba wax stearic acid emulsions for minutes and used in analysis of respiration rate and coating water vapor resistance. For the other analysis, films were prepared by weighing an amount of emulsion that provide 130 ± µm of film thickness on a Teflon casting plate resting on a levelled surface. Films were dried until constant moisture content at 40 C and 60% RH to avoid cracking, peeled off the casting surface and conditioned at 5 C and 58% RH in desiccators with a saturated NaBr solution for 7 h prior to analysis. Experimental Design A ³ central composite rotatable design with 6 axial and 3 central points, resulting in 17 experiments, was used to obtain models for prediction of responses observed as a function of cassava starch content (CS), glycerol concentration (GLY) and carnauba wax: stearic acid ratio (CSA). The statistical design, the coded and real values of these variables are shown in Table 1. All experiments were performed randomly. Table 1. Central composite rotatable design matrix with real and coded values (in parentheses) of the variables cassava starch (CS), glycerol (GLY) and carnauba wax: stearic acid ratio (CSA) and responses to respiration rate (RR), water vapor resistance (RVA), mechanical properties (tensile strength TS, elongation at break ELO and elastic modulus EM) and solubility (sol). Run Coating/Film Ingredients CS GLY CSA RR (ml CO kg -1 h -1) RVA (s cm -1 ) Responses TS (Mpa) ELO EM (Mpa) 1.4 (-1) 1.4 (-1) 0.08:0.9 (-1) (+1) 1.4 (-1) 0.08:0.9 (-1) (-1).6 (+1) 0.08:0.9 (-1) (+1).6 (+1) 0.08:0.9 (-1) (-1) 1.4 (-1) 0.3:0.68 (+1) (+1) 1.4 (-1) 0.3:0.68 (+1) (-1).6 (+1) 0.3:0.68 (+1) (+1).6 (+1) 0.3:0.68 (+1) (-1.68).0 (0) 0.0:0.80 (0) (+1.68).0 (0) 0.0:0.80 (0) (0) 1.0 (-1.68) 0.0:0.80 (0) (0) 3.0 (+1.68) 0.0:0.80 (0) (0).0 (0) 0.00:0.00 (-1.68) (0).0 (0) 0.40:0.60 (+1.68) (0).0 (0) 0.0:0.80 (0) (0).0 (0) 0.0:0.80 (0) (0).0 (0) 0.0:0.80 (0) Sol Analyses Respiration Rate The respiration rate of coated apple slices was measured by static method, according to Bierhals et al. []. The samples (around 50 g) were placed in sealed 180 ml glass jars with silicon septum. The jars were closed and kept at 5 C during 1 h. After this time, gas sampling was carried out using an O /CO Dual Head Space Analyzer (Model PAC CHECK 35, Mocon, Minneapolis, USA), measuring the CO production. The respiration rate was determined in triplicate and expressed in ml CO kg 1 h 1. Water Vapor Resistance The determination of water vapor resistance (WVR) of coatings was carried out according to the method described by Avena-Bustillos et al. [3], using a modified Fick s equation proposed by Ben-Yehoshua et al. [4]. Apple cylinders with 5 mm diameter and 10 mm thickness were used for coatings application. Five cylinders were used for each formulation. Solubility Film solubility was determined according to the methodology described by Colla et al. [5]. Measurements were made in triplicate. Mechanical Properties Mechanical properties were determined by tensile tests (tensile strength, elongation at break and elastic modulus) using a Universal Testing Machine (model TA-TX plus, Stable Micro Systems, Surrey, England).
3 The measurements were acquired following the ASTM standard method D88-0 [6]. Sample films were cut into 5 mm wide strips each one at least 115 mm in length. The initial grip separation was set at 80 mm and the crosshead speed at 1.0 mm/s. At least five samples from each film were evaluated. Statistical Analysis Experimental design data were analyzed using the analysis of variance (ANOVA) and the F-test to verify the statistical significance of models at p 0.05, using the software Statistica 8.0 (StatSoft, Inc., Tulsa, USA). Models and response surfaces were generated for responses that showed F test values higher than F listed and R ² RESULTS & DISCUSSION Responses of experimental design are shown in Table 1. The model regression coefficients and F-test are shown in Table. Only the models obtained for respiration rate of coated apple slices, water vapor resistance of coatings, water solubility and elastic modulus of films were significant and predictive. These responses presented F test higher than F listed and the models showed high values of R (R 0.75). Table. Model regression coefficients and F-test for RR, WVR, Sol, TS, ELO and EM. The independent variables are coded. Source RR (ml CO kg -1 h -1 ) WVR (s cm -1 ) Sol TS (MPa) ELO EM (MPa) Mean 6.09* * 3.606* * 0.096* CS 0.478* * * CS CS * GLY -0.6*.576* * * GLY GLY 0.49* * 5.5* * 0.067* CSA 0.531* 3.783* * CSA CSA * -.654* * CS GLY * * CS CSA * * GLY CSA * 3.974* R² F test F listed *Statistically significant values (p<0.05) RR of apple slices with coatings was significantly influenced (p < 0.05) by cassava starch content, glycerol content (linear and quadratic parameters), carnauba wax: stearic acid ratio (linear and quadratic parameters) and the interaction between glycerol and wax: fatty acid ratio. The WVR of the coatings applied on apple cylinders was significantly influenced (p <0.05) by the cassava starch content (quadratic parameter), glycerol content (linear and quadratic parameters), carnauba wax: stearic acid ratio (linear and quadratic parameters) and the interaction between glycerol and wax: fatty acid ratio. The water solubility (Sol) was affected by the cassava starch content, glycerol content, besides the interactions between starch glycerol and starch wax: stearic acid ratio. The mechanical properties were mainly influenced by glycerol content, because the plasticizer affects films flexibility. The carnauba wax: stearic acid ratio also affected significantly the TS of films and the interactions cassava starch glycerol and cassava starch wax: fatty acid ratio affected the EM. The coded models used to describe the responses RR, WVR, Sol and EM are shown by Equations 1 to 4: RR = CS 0.6 GLY GLY CSA 0.4 CSA GLY CSA (1) WVR = CS +.58 GLY 4.17 GLY CSA.65 CSA GLY CSA () Sol = GLY 1.94 CSA CS GLY (3) EM = GLY GLY 0.06 CS GLY CS CSA (4) Where: RR is the respiration rate of coated apple slices (ml CO kg -1 h -1 ); WVR is the water vapor resistance of coatings (s cm -1 ); Sol is the water solubility of films ; EM is the elastic modulus of films (MPa); CS is
4 the coded cassava starch content; G is the coded glycerol content; CSA is the coded carnauba wax: stearic acid ratio. Coded independent variables ranging from to Figure 1 shows the response surfaces obtained for respiration rate of coated apple slices (RR), water vapor resistance of coatings (WVR), water solubility (Sol) and elastic modulus of films (EM). These surfaces were generated considering only the significant regression coefficients according to Table and equations 1 to 4. (a) (b) (c) (d) (e) (f) Figure 1. Response surfaces for respiration rate of coated apple slices (a), water vapor resistance of coatings (b), water solubility (c and d) and elastic modulus of films (e and f). CS = cassava starch content; GLY = glycerol content; CSA = carnauba wax: stearic acid ratio. The glycerol content was the variable that most influenced RR, WVR and EM (Figure 1a, b and e). Glycerol concentrations above the central point (.0%) resulted in reduced RR of apple slices and higher WVR of
5 coatings. Glycerol fills the empty spaces of the matrix and reduces pores and cracks in the surface of films, promoting a more effective barrier to gas exchange and moisture loss [7,8]. However, the increase of glycerol content resulted in more soluble films and low EM (Figure 1c and e). The carnauba wax: stearic acid ratio affects the matrix continuity, resulting in increase on product RR (Figure 1a). On the other hand, higher carnauba wax content improved (p 0.05) WVR of coatings (Figure 1b). Wax: fatty acid ratio between 0.1:0.9% to 0.3:0.7% associated with high concentrations of glycerol also resulted in increased solubility of the films (Figure 1d). Higher carnauba wax: stearic acid ratio lowered tensile strength and elongation, and increased elastic modulus (Figure 1f). The cassava starch content showed a positive effect on RR of the samples and on EM of films (Figure 1e and f). However, cassava starch concentration higher than 3% (w/w) in coating formulations resulted in reduction of WVR and in more soluble films (Figure 1c). The optimized conditions were defined as cassava starch concentration of 3.0 g/ 100g of coating solution, glycerol concentration of 1.5 g/100 g of coating solution, carnauba wax concentration of 0. g/ 100 g of coating solution and stearic acid concentration of 0.8 g/ 100 g of coating solution. The optimized formulation was submitted to the same analyses carried out previously and the differences observed between the experimental and predicted data were less than 10%, indicating that the models obtained in the experimental design were consistent (Table 3). Table 3. Experimental and predict values of significant responses determined to coatings and films optimized conditions. Responses Experimental Value Predict Value Relative Deviation Respiration Rate (ml CO kg -1 h -1 ) Water Vapor Resistance (s cm -1 ) Elastic Modulus (MPa) Water Solubility The optimized formulation also presented good values for tensile strenght (0.79 ± MPa) and elongation at break ( ±.78 %). CONCLUSION The RSM was an effective tool for identifying optimized formulation of coatings/films with cassava starch, glycerol, carnauba wax and stearic acid that provided better barrier and mechanical properties and lower solubility. Models and response surfaces were obtained for the respiration rate of coated apple slices, water vapor resistance of coatings, elastic modulus and solubility of films. The optimized formulation was selected and submitted to validation procedure. The models obtained in the experimental design were predictive, which was evidenced by the relative deviations of less than 10%. ACKNOWLEDGEMENTS The authors are grateful to FAPESP (process n.: 08/ and 09/5140-4) and CNPq for their financial support. REFERENCES [1] Vargas, M., Pastor, C., Chiralt, A., McClements, D. J., González-Martínez, C. (008). Recent Advances in Edible Coatings for Fresh and Minimally Processed Fruits. Critical Reviews in Food Science and Nutrition, 48(6), [] Bierhals, V. S.; Chiumarelli, M.; Hubinger, M. D. (011). Effect of Cassava Starch Coating on Quality and Shelf Life of Fresh-Cut Pineapple (Ananas comosus L. Merril cv Pérola ). Journal of Food Science, 76 (1), E6-E7. [3] Avena-Bustillos, R. J.; Krochta, J. M.; Saltveit, M. E.; Rojas-Villegas, R. J.; Sauceda-Pérez, J. (1994). Otimization of edible coating formulations on zucchini to reduce water loss. Journal of Food Engineering, 1(), [4] Ben-Yehoshua, S.; Burg, S. P.; Young, R. (1985). Resistance of citrus fruit to mass transport of water vapor and other gases. Plant Physiology, 79(4), [5] Colla, E., Sobral, P.J.D.A., Menegalli, F.C. (006). Amaranthus cruentus flour edible films: Influence of stearic acid addition, plasticizer concentration, and emulsion stirring speed on water vapor permeability and mechanical properties. Journal of Agricultural and Food Chemistry, 54 (18),
6 [6] ASTM. (005). Designation D88-0: Standard Test Method for Tensile Properties of Thin Plastic Sheeting. In: Annual Book of ASTM Standards; American Society for Testing and Materials: West Conshohocken. [7] Krochta, J. M. (00). Proteins as raw materials for films and coatings: definitions, current status, and opportunities. In: Gennadios, A. (Editor). Protein-based Films and Coatings, CRC Press: Boca Raton, p [8] Ribeiro, C.; Vicente, A. A.; Teixeira, J. A.; Miranda, C. (007). Optimization of edible coating composition to retard strawberry fruit senescence. Postharvest Biology and Technology, 44 (1),
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