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Characterization of biodegradable film based on zein and oleic acid added with nanocarbonate/Caracterizacao de filme biodegradavel a base de zeina e acido oleico adicionado de nanocarbonato.

INTRODUCTION

The quality of a food product is directly dependent of its packing which protects it until the final destination, the customer (VILLADIEGO et al., 2005). The packages are often associated with environmental pollution problems since it has been difficult, in practice, to control their proper disposal and/or recycle according to specific programs (MAGALHAES, 2001). As legal regulations and public concern put pressure over food industries, there is a technical race in order to develop materials with dual properties to satisfy packaging purposes, while is environmentally acceptable. Biodegradability is the most desired individual characteristic for packages today, for all kinds of industries. Several materials are under intense research in order to obtain biodegradable packaging, such as starch, zein, cellulose, among others. (FELLOWS, 2006). Zein is the main corn protein and has been extensively studied because of its excellent film forming abilities. It is alcohol soluble, produces films with an efficient enough barrier against moisture and has been used commercially as degradable film for food packages (VILLADIEGO, 2005; TAVARES, 2010). However, zein based film is fragile, requiring plasticizer as glycerol and other components to improve its functional properties. Additives such as nanoparticles, can be used to improve the mechanical and barrier properties of zein films without being hazardous to both environment and human (GHANBARZADEH, 2007; MALI, GROSSMANN, YAMASHITA; 2010). On account of differentiated properties that nanoparticles exhibit, it is possible to obtain materials with superior characteristics when compared to composites made with microparticulate materials. (ASSIS et al., 2012). The objectives of this study were to produce and determine structural characteristics and functional properties of zein-oleic acid films added with different concentrations of calcium nanocarbonate (CaCO3), in order to evaluate its feasibility for uses as food packing.

MATERIAL AND METHODS

Materials

Corn-zein was obtained from Sigma-Aldrich. Analytical grade glycerol, oleic acid and ethanol (99.5 %) were supplied by Aracaprolab (Aracatuba, Brazil). Sucrose ester off fatty acids was used as emulsifier, specific for oil/water emulsion, in order to incorporate the oleic acid into the films (commercial name: SP70, Sistema, Brazil) (SOUZA, 2011). Calcium nanocarbonate were obtained as commercial name of NPCC-201.

Film composition and preparation

To prepare films with 20g of zein it was added to 100mL of ethanol at 95% (v/v). Mixtures were mechanically stirred at 65 [+ or -] 0.5[degrees]C for 5 min, then added with 70g of oleic acid, 0.875g of emulsifier, 30g of glycerol and nanocarbonate at 1, 2 or 3%, related to 100g of zein. Stirring was kept for another 10min and then, according to the casting technique, for each formulation, 6g of filmogenic solution was poured onto circular plates (43.96[cm.sup.2] of area) of polytetrafluoroethylene (Teflon[R]) to obtain a constant thickness. After drying, all films were placed in a controlled relative humidity of 75% and at ambient temperature of 20 [+ or -] 2[degrees]C and stored for prior analyses (KECHICHIAN, 2010; ARENAS, 2011; SOUZA, 2011).

Water solubility

Film water solubility was defined as the content of dried matter solubilized after 24h of immersion in water. The film was cut into 2cm diameter disk and dried in an oven at 105 [+ or -] 2[degrees]C for 24h, weighed (initial weight, wi) and immersed into 50 mL distilled water at 27 [+ or -] 2[degrees]C for 24 h under agitation in an orbital shaker MA-410 (Marconi, Brazil) at 76rpm. After 24h of immersion, disks were taken out and dried (final weight, wf) at the same conditions stated before, in order to determine the weight of dried matter that was not solubilized in water. Three replicates of each film were tested and water solubility was calculated according to Equation 1 (PENA-SERNA& LOPES-FILHO, 2013).

Water Solubility Wi - Wf/Wi x 100 (1)

Optical Microscopy

Optical microscopy was carried out in order to determine superficial film homogeneity. The film was cut in 1[cm.sup.2] and stained with Xylidine Ponceau solution for 30min, then washed in a bath of acetic acid 2.5% for 10min and finally washed in a distilled water bath for 10min The stained film was placed on a glass microscope slide and dried in an oven for 24h at 37[degrees]C. The dried stained film was fixed with Canada balsam and covered with cover glass slide. The sample assembly was dried at room conditions for 24h and then analyzed with an optical microscope Olympus BX 60, equipped with an image capture system Olympus DP 71, using a magnification of 10x.

Scanning electron microscopy (SEM)

The surface microstructure of the films was evaluated by Scanning Electron Microscopy (SEM). The sequence for the preparation of the specimen includes the aluminum mounting brackets (stubs) and metalizing (sputtering) following GHANBARZADEH et al. (2007), where samples of the films were fixed on stubs with 12mm of diameter with double-sided conductive carbon tape. The samples were metalized with platinum for approximately 60s and then observed in a scanning electron microscope (FEI Quanta 600FEG) at 10kV

Mechanical properties

The tensile properties were obtained using a TA.TX2i Stable Micro Systems texture analyzer (Surrey-England) in accordance with ASTM D-882-91 (1996). Five sample strips (25x130mm) of each formulation were cut and clamped between tensile grips. Force (N) and deformation (mm) were recorded during extension of 0.8mm [min.sup.-1] and with an initial distance between the grips of 10mm. The parameters determined were: tensile stress (MPa) and strain at break (%).

Color

The color of the samples was measured in a spectrum wave between 400-700nm with nominal position UV filter, with viewing area of one square inch in total transmittance mode that focuses a beam of normal light to the film surface. This transmitted light was observed at the opposite side of the device sensor, which measures the intensity of wave lengths. This means that the color was obtained from measurement values for the transmitted light.

Statistical analysis

Statistical analysis was performed with software Minitab 16. The evaluated factors were nanocarbonate concentrations and the response variables water solubility, water vapor permeability, optical microcopy and scanning electron microscopy. Analysis of variance and Tukey Test were performed to detect significant differences in the properties of the films. The significance level used was 0.05.

RESULTS AND DISCUSSION

Water solubility

Standard film had the lowest water solubility, therefore being less hydrophilic because of the greater intermolecular force between proteins matrix and other components. Similar result was found by PENA-SERNA & LOPES-FILHO (2013) where higher concentrations of ethanol showed films with denser structure and less water solubility. Samples with 2% and 3% of nanocarbonate presented features visually less homogeneous (dispersed particles), which resulted in increased the solubility (Table 1). Thus, this lack of incorporation of the components in the matrix contributed to increase solubility of the sample. BATISTA et al. (2005) observed that the addition of fatty acids in pectin-based films showed a decrease in its solubility. However, no incorporation of the fatty acid in film matrix contributed to the higher exposure of the hydrocolloid matrix, promoting the solubilization of the film. All samples studied were intact, after 24 hours, immersioned in water, indicating that the protein matrix remained intact.

Scanning electrical microscopy (SEM)

The SEM micrographs of 0, 1, 2 and 3 (Figure 1) showed the surface morphology of the zein films without addition of nanocarbonate and with 1, 2 and 3% of nanocaibonate, respectively. MONTERREY-QUINTERO; SOBRAL (2000) and TAVARES (2010) suggested that the black spots observed in the micrographs were micro-bubbles embedded in the matrix or spaces occupied by glycerol previously to the drying process. ALMEIDA et al. (2010) also found black spots in electron micrographs of zein films prepared with oleic acid and glycerol. In order to identify the presence of globular deposits, the authors performed optical microscopy analysis and concluded that observations previously considered as pores in scanning electron microscopy were in fact lipid globules dispersed in the film matrix. Films without addition of nanocarbonate showed black spots (marked with yellow arrows) and those with added nanocarbonate also showed few black points (also identified with yellow arrows). It suggests that nanocarbonate was not fully solubilized in filmogenic solution. From this information and results reported in the literature, it can be concluded that these black spots are in fact micro-pores (microbubbles). The amount of micro-bubbles in the standard film may have interfered with the passage of light, making it more opaque, which was observed previously in the color analysis. For the films with nanocarbonate it was also observed light spots or agglomerates of particles showing less solubilization and/or dispersed nanoparticles in the matrix (marked with red arrows).

Optical Microscopy

To complement SEM analysis about homogeneity of the film, it was carried out the Optical Microscopy. In this test the proteins are stained red and the fat globules were presented in white tones. The captured images for each sample are shown in figure 2 and the corresponding scale increased the photo 10 x (500pm). The optical micrographs show a lower amount of white points (fat globules) in standard film (without nanocarbonate) than the films with nanocarbonate, confirmed what was found in SEM analysis. The black spots are microbubbles air and not fat globules. As previously mentioned ALMEIDA et al. (2010) performed a comparative analysis of Optical Microscopy in zein films with xanthan gum and concluded that the decrease in size of the fat globules was due to a better homogenization of oil. Unlike observed by these authors, in the present work it was research large fat globules as nanocaibonate concentration increases. Therefore, films without added nanocaibonate formed a more homogeneous matrix and those with nanocarbonate presented a greater amount of white dots. The weak interaction between zein and the nanocarbonate resulted on glycerol exclusion. For this reason, nanocaibonate films exhibited a greasy perception at touch.

Mechanical properties

The tensile stress and strain at a break in zein films were affected by nanocarbonate content (Table 2). According to ALMEIDA et al (2010), the larger the diameter of fat globules in the matrix, the lower was the tensile strength and elongation of the film. Comparing the present research with the results found by these authors, in addition to observations of micrographs of the present study, the traction results were confirmed. Values in table 2, showed that films with nanocarbonate presented higher elongation and lower maximum tensile strength. According to MATTA JUNIOR (2009), usually, when film structure becomes less rigid, the strength/tension properties are reduced and the elongation increases. A higher elongation indicates that the film is more flexible. Therefore, according to the results obtained, films become more flexible and elastic because of nanocarbonate addition.

Color

As presented in table 3, nanocarbonate increased significantly L* parameter, indicating brightness, leaving the films lighter. Further studies on the use of higher concentrations of nanocarbonate are suggested. An analogous result can be observed with the values found for b* (to perceive blue to yellow shades, with positive values associated to yellow), where, as nanocarbonate concentration increased, the b* values decreased, indicating a reduction in yellow intensity. Table 4 shows the average values and standard deviations of the color parameter L* of the films according to nanocarbonate concentration at initial time, after 7, 14 and 30 days of storage. This shows change in color according to time. The films without nanocarbonate and those with 1% and 2% had lower values of the L* after 7 days of storage (the films became slightly darker). The luminosity of the films remained constant. The color remained constant throughout the storage period for films with 3% nanocarbonate indicating a greater color stability.

CONCLUSION

The functional and structural properties of zein-oleic acid film were affected by addition of nanocarbonate. Higher concentration of nanocarbonate allowed the improvement of water solubility, color and mechanical properties, making the film more flexible and elastic, as well as more stable related to the color. Nonetheless, there was an increase in the size of fat globules. In general, the results shown the possibilities to use the material with nanocarbonate to pack food products, when elasticity, color stability and water solubility are the concern factors.

http://dx.doi.org/10.1590/0103-8478cr20141391

ACKNOWLEDGMENTS

This study was supported by a grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES).

REFERENCES

ALMEIDA, C.B. et al. Morphological and structural characteristics of zein biofilms with added xanthan gum. Food Technology Biotechnology, Croacia, v. 48, n. 1, p. 19-27, 2010.

ARENAS, A.M.Z. Filme biodegradavel a base de fecula de mandioca como potencial indicador de mudanca de pH. 2012. 131 f. Dissertacao (Mestrado em Engenharia Quimica)--Escola Politecnica, Universidade de Sao Paulo, Sao Paulo, SP

ASSIS, L.M et al. Caracteristicas de nanoparticulas e potenciais aplicacoes em alimentos. Jornal Brasileiro de Tecnologia de Alimentos, Campinas, v. 15, n. 2, p. 99-109, 2012.

BATISTA, J.A. et al. Efeito da adicao de acidos graxos em filmes a base de pectina. Ciencia e Tecnologia de Alimentos, v. 25, n. 4, p. 781-788, 2005.

FELLOWS, P.J. Tecnologia do processamento de alimentos: principios e praticas. 2. ed. Trad. F.C. Oliveira et al. Porto Alegre: Artmed, 2006. p. 602.

GHANBARZADEH, B. et al. Effect of plasticizing sugars on water vapor permeability, surface energy and microstructure properties of zein films. Food Science and Technology, v. 40, p.1191-1197, 2007.

KECHICHIAN, V., et al., Natural antimicrobial ingredients incorporated in biodegradable films based on cassava starch, LWT --Food Science and Technology, v. 43, p 1088-1094, 2010.

MAGALHAES, M. A. Tempo de degradacao de materiais descartados no meio ambiente. Jornal do Centro Mineiro para Conservacao da Natureza (CMCN). Vicosa-MG, ano 08, n.37, jan/fev/mar 2001.

MALI, S. et al. Filmes de amido: producao, propriedades e potencial de utilizacao. Revisoes Ciencias Agrarias, Londrina, v. 31, n. 1, p. 137-156, 2010.

MATTA JUNIOR, M. D. Caracterizacao de biofilmes obtidos a partir de amido de ervilha (Pisum sativum) associado a goma xantana e glicerol. 2009. 110 f. Dissertacao (Mestrado em Ciencia e Tecnologia de Alimentos) - Programa de Pos-graduacao em Ciencias e Tecnologia de Alimentos, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de Sao Paulo, Piracicaba, SP.

MONTERREY-QUINTERO, E.S.; SOBRAL, PJ.A. Preparo e caracterizacao de proteinas miofibrilares de tilapia-do-nilo para elaboracao de biofilmes. Pesquisa Agropecuaria Brasileira, Brasilia, v.35, n. 1, p.179-189, 2000.

PENA-SERNA, C.; LOPES-FILHO, J.F. Influence of ethanol and glycerol concentration over functional and structural properties of zein-oleic acid films. Materials Chemistry and Physics, n. 142, p. 580-585, 2013.

SOUZA, A. C. Desenvolvimento de embalagem biodegradavel ativa a base de fecula de mandioca e agentes antimicrobianos naturais. 2011. 137 f. Tese (Doutorado em Engenharia Quimica)--Escola Politecnica, Universidade de Sao Paulo, Sao Paulo, SP.

TAVARES, L.L. Efeito da adicao de argilas modificadas nas propriedades de biofilmes produzidos a base de zeina. 2010. 74 p. Dissertacao (Mestrado)--Universidade Estadual Paulista, Instituto de Biociencias, Letras e Ciencias Exatas, Sao Jose do Rio Preto, 2010.

VILLADIEGO, A. M. D. et al. Filmes e revestimentos comestiveis na conservacao de produtos alimenticios. Revista CERES, v. 52, n, 300, p. 221-243, 2005.

Wanessa Ximenes Ribeiro (I) * Jose Francisco Lopes Filho (I) Monica Souza Cortes (I)

(I) Departamento de Engenharia e Tecnologia de Alimentos, Universidade Estadual Paulista "Julio de Mesquita Filho" (UNESP), 15054-000, Sao Jose do Rio Preto, SP, Brasil. E-mail: wa_ribeiro@yahoo.com.br. Corresponding author.

Received 09.17.14 Approved 01.15.15 Returned by the author 05.15.15

CR-2014-1391.R1

Table 1--Water solubility of zein-nanocarbonate films.

Nanocarbonate (%)      Water solubility (%)

0                   0.4243 [+ or -] 0.1717 (b)
1                   1.5186 [+ or -] 1.1625 (b)
2                   3.8851 [+ or -] 0.1339 (a)
3                   4.0889 [+ or -] 0.3484 (a)

* Mean [+ or -] standard deviation of each analysis.
(a,b) Different letter indicate a statistically significant
difference (P< 0.05).

Table 2--Values of mechanical properties of zein-nanocarbonate
films.

Nanocarbonate      Tensile stress (MPa)        Strain at break (%)
(%)

0               1.7583 [+ or -] 0.0479 (a)   1088.8 [+ or -] 36.2 (b)
1               1.6731 [+ or -] 0.1647 (b)   1445.0 [+ or -] 43.2 (a)
2               1.6656 [+ or -] 0.0677 (b)   1421.9 [+ or -] 56.6 (a)
3               1.6545 [+ or -] 0.0565 (b)   1403.6 [+ or -] 60.9 (a)

* Mean [+ or -] standard deviation of each analysis.
(a,b) Different letter indicate a statistically
significant difference (P < 0.05).

Table 3--Parameters of color [L.sup.*], [a.sup.*] e [b.sup.*]
of zein-nanocarbonate films.

Nanocarbonate (%)     [L.sup.*]         [a.sup.*]

0                   87.638 [+ or -]   -0.882 [+ or -]
                    1.406 (d)         0.279 (a)
1                   90.509 [+ or -]   -1.356 [+ or -]
                    1.749 (b)         0.314 (c)
2                   89.675 [+ or -]   -1.078 [+ or -]
                    1.483 (c)         0.257 (b)
3                   92.057 [+ or -]   -1.370 [+ or -]
                    0.881 (a)         0.264 (c)

Nanocarbonate (%)     [b.sup.*]

0                   40.682 [+ or -]
                    3.400 (a)
1                   35.249 [+ or -]
                    4.952 (b)
2                   35.138 [+ or -]
                    4.464 (b)
3                   28.142 [+ or -]
                    3.840 (c)

Mean [+ or -] standard deviation of each analysis.
(a,b,c,d) Different letter indicate a statistically
significant difference (P < 0.05).

Table 4--Parameters of color L* of zein-nanocarbonate
films according to the time.

Time                         Nanocarbonate (%)
(days)

                        0                              1

0        88.474 [+ or -] 1.255 (a)         91.682 [+ or -] 1.182 (a)
7        86.803 [+ or -] 1.007 (b)         89.336 [+ or -] 1.410 (b)
14       86.026 [+ or -] 0.781 (b), (c)    89.840 [+ or -] 1.758 (b)
30       85.685 [+ or -] 0.945 (c)         89.647 [+ or -] 0.918 (b)

Time                        Nanocarbonate (%)
(days)

                     2                           3

0        90.570 [+ or -] 1.187 (a)   92.148 [+ or -] 1.009 (a)
7        89.008 [+ or -] 1.214 (b)   91.817 [+ or -] 0.964 (a)
14       88.408 [+ or -] 1.706 (b)   91.945 [+ or -] 0.906 (a)
30       88.195 [+ or -] 1.053 (b)   91.574 [+ or -] 1.149 (a)

Mean [+ or -] standard deviation of each analysis. (a,b,c) Different
letter indicate a statistically significant difference (P < 0.05).
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Title Annotation:tecnologia de los alimentos; texto en ingles
Author:Ribeiro, Wanessa Ximenes; Filho, Jose Francisco Lopes; Cortes, Monica Souza
Publication:Ciencia Rural
Date:Oct 1, 2015
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