InsideFood Symposium, 9-12 April 2013, Leuven, Belgium 1 | P a g e
Mechanical, barrier and microstructural properties of films
based on cassava starch gelatin blends: effect of aging and lipid addition Sandra Acosta, Alberto Jimnez, Amparo Chiralt, Chelo Gonzlez-Martinez, Maite Chfer
IIAD. Instituto Universitario de Ingeniera de Alimentos para el Desarrollo. Universidad Politcnica de Valencia. C/cam de vera SN, 46022, Valencia, Espaa.
ABSTRACT Starch films exhibits low poor water barrier and poor mechanical properties due to its great hygroscopicity, fragility and brittleness. These properties can be influenced by different factors such as composition, processing conditions and storage of the films. In this work, the effect of gelatin and ester of fatty acids (E-471) addition on the mechanical and barrier properties of cassava starch films, containing glycerol as plasticizer, was analysed in both newly prepared films and those stored for 5 weeks. Microstructural analysis of the films was also studied by using SEM (Scanning electron microscopy) and AFM (Atomic force microscopy). The addition of gelatin into cassava starch film formulation improved mechanical properties and ester incorporation significantly (p<0.05) reduced the water vapour permeability of composite films. Storage time affected mechanical behavior of films, increasing stiffness and resistance and reducing their stretchability. Microstructural observations corroborated the good compatibility among both polymers (starch and gelatin) to form the film matrix and the scarce effect of ester addition in the improvement of functional properties. SEM micrographs showed compact structure of pure films. Composite films presented a more heterogeneous surface than the pure films but a good compatibility among the polymers (no separation). The E-471 integrates differently depending on the matrix: in pure starch films is included in the continuous network as amylose-lipid complexes; in composite films (50S-50G), the formation of lipid aggregates distributed throughout the film matrix can be observed. 1 Introduction The functional properties of cassava edible films are influenced by starch properties, including chain conformation, molecular bonding, crystallinity, and types and concentrations of plasticizer types. Amylose and the branching points of amylopectin form the amorphous regions while the short-branched chains in the amylopectin are the main crystalline components in granular starch. So, the higher content of amylopectin in native starch means greater crystallinity (Cheetham and Tao 1998). Tapioca starch films generally show poor mechanical properties and provide a minimal barrier to moisture. The presence of plasticizer leads to overcome the brittleness of these films, and contents between 1540 g glycerol/100 g starch are commonly used for improve these properties (Chen & Lai, 2008; Habitante, Sobral, Carvalho, Solorza-Feria, & Bergo, 2008; Mali, Sakanaka,Yamashita, & Grossmann, 2006; Talja, Hele n, Roos, & Jouppila, 2007). Properties of starch films can be further improved formulating composite films and coatings through incorporation of hydrocolloids as proteins. This incorporation to starch matrix can be a positive effect in mechanical and gas barrier properties. Gelatin is an abundant raw material, produced in the whole world at low cost and has excellent film forming properties (Gennadios et al., 1994). Hassan and Norziah (2012) evaluated the effect of fish gelatin addition on physical and mechanical properties of sago starch edible films plasticized by glycerol and sorbitol. In this work, starch:gelatin ratios of 3:1, 4:1 and 5:1 with glycerol formed good flexible films. The results obtained showed that different starch:gelatin ratios of plasticized composite films affected physical, mechanical and water vapor permeability of the films produced. Changing the ratio of two polymers can modify the strength and extensibility of the composite films. On the other hand, Vanin, Sobral, Menegalli, Carvalho, and Habitante (2005) analysed the effect of plasticizers (polyols) and their concentrations on the thermal and functional properties of gelatin-based films. In this work, authors concluded that glycerol was compatible with gelatin and showed the highest plasticizing effect on the mechanical properties of the film producing a flexible and easy handling film with no phase separation.
P a g e | 2 The poor water barrier of hydrocolloids can be enhanced formulating films with lipid components (Garcia, Martino, & Zaritzky, 2000; Jimenez et al., 2010). These investigations also showed that saturated fatty acids and a concentration above a critical ratio can increase the water vapour permeability of starch films due to the migration of oil and the decrease of the crystalline-amorphous ratio. Whatever, Bertan et al. (2005) found that the incorporation of fatty acids to gelatin films presented enhanced the water vapor barrier properties, increased the opacity and soluble matter and decreased the mechanical resistance. Finally, starch films are greatly affected by aging (defined by the storage time), inducing important physical and chemical changes in the films properties (Delville et al. 2003; Fam et al. 2007; Jimnez et al. 2012) that could affect their functionality as a consequence of the reassociation into crystalline segments and, thus, in amorphous and crystalline zones. Generally, starch-based films become stronger and stiffer but less flexible after several weeks of storage (Jimnez et al. 2012). However, Mali et al. (2006) did not report significant differences in TS and EM values of cassava, corn, and yam starch-based films containing 20 % of glycerol, though these films were less deformable as storage time increased. They concluded that the crystallinity of starch films increased the longer they were stored and glycerol-free films were more seriously affected; the plasticizer seemed to limit crystal growth and recrystallization due to the interaction with the polymeric chains. However, neither Mali et al. (2005 Y 2006) nor Jimnez et al. (2012) reported significant differences in water vapor permeability values as a consequence of the storage time. 2 Material and Methods 2.1 Materials The raw materials used were cassava starch obtained from Asia Modified Starch CO; LDT (Kalasin, Thailand), and donated by Quimidroga S.A. (Barcelona, Espaa); bovine hide type A gelatin (Bloom 220-240) donated by Sancho de Borja (Zaragoza Espaa); mono and diglicerids from fatty acids (E-471 purchased from Cargill (Barcelona-Espaa) and glycerol provided by Panreac Qumica, S.A. (Barcelona-Espaa). Cassava starch contained a 9.28% amylose and an amylose-amylopectine ratio of 1:9.78. This ratio was determinated by Amylose/Amylopectin Assay Procedure enzymatic kit which was purchased from Megazyme (Wicklow, Ireland). 2.2 Preparation of films Eight different formulations based on cassava starch (S), gelatin (G), glycerol and E-471 fatty acids (e) were prepared in order to obtain 2% (w/w) polysaccharide suspensions. Proportions (%) of starch (S):gelatin (G), used were 0:100, 50:50, 75:25 and 100:0, respectively. All formulations contained glycerol as plasticizer (ratio of 1:0.25 polymer:glycerol) and in four of formulations, a 15% of fatty acids E-471 (e) was added respect to the polymer. Starch dispersions were maintained, under stirring, at 95C for 30 min to induce starch gelatinization. Gelatin dispersions were obtained under stirring at 80C for 30 min. Composite starch-gelatin formulations, were prepared after corresponding dispersion of each polysaccharide. Afterwards, plasticizer was added and dispersions were homogenized (13,500 rpm for 1 min and at 20,500 rpm for 5 min) at 95 C, under vacuum, using a rotorestator homogenizer (Ultraturrax T25, Janke and Kunkel, Germany). In the case of emulsions containing fatty acids, these were incorporated prior to the homogenization step as this process conditions favored the lipid dispersion in the system. Newly homogenized film-forming dispersions, containing 1.5 g of total solids, were spread evenly over a Teflon casting plate (15 cm diameter). Films were formed by drying for approximately 48 h at 53% RH and 25C. These conditions were established after previous experiments to ensure that homogeneous, flawless films were obtained. Dry films could be peeled intact from the casting surface. The films were peeled off from the casting plates and conditioned at 25C and 53%RH in chambers containing Mg(NO 3 ) 2 saturated solution for 1 and 5 weeks. 2.3 Characterization of films Water vapour permeability (WVP) was analyzed following the gravimetric method (ASTM E-96-95) using Payne permeability cups (Payne, Elcometer SPRL, Hermelle/sd Argenteau, Belgium) of 3.5 cm diameter in dry films at 25C and using a RH gradient of 53-100% by using magnesium nitrate-6-hydrate and deionised 3 | P a g e water, respectively. For each type of film, WVP measurements were replicated three times and WVP was calculated according to according to Sanchez et al., (2010). Oxygen permeability (OP) was evaluated in films equilibrated at 53% RH by measuring the oxygen transference rate with an Ox-Tran 1/50 System (Mocon, Minneapolis, USA) at 25C , following the standard method (ASTM D3985-05, 2005). Temperature was 25C and 53%RH Oxygen permeability was calculated by dividing the oxygen transmission by the difference in oxygen partial pressure between the two sides of the film, and multiplying by the average film thickness. Two replicates per formulation were made. Mechanical properties were measured by using a Texture Analyser TA.XT plus (Stable Micro Systems, Haslemere, England) with a 50 N load cell equipped with tensile grips (A/TG model)following the standard method D882 (ASTM, 2001). Grip separation was set at 50 mm and cross-head speed was 50 mm/min. Tensile strength (TS) and percentage of elongation at break (%E), and elastic modulus (EM) were evaluated in eight samples from each type of film. SEM techniques were used to study the microstructural arrangement of film components. Previously, films were equilibrated with P2O5 for two weeks. SEM micrographs of the surface and cross-sections of the films were obtained using a scanning electron microscope (JEOL, modelo JSM-5410, Japan). To observe the sections, samples were cryofractured by immersion in liquid N 2 Films were mounted on copper stubs, gold coated, and observed using an accelerating voltage of 11 kV. The analysis of dates was performed through variance analysis (ANOVA) using the Statgraphics Plus 5.1. Program (Munugistics Corp., Rockville, MD). To discern between samples the Fisher least significant difference (LSD) at the 95% confidence level was used. 1. Results and discussion 2.4 Water vapour and oxygen permabilities Table 1 shows mean values of water vapour and oxygen permeabilities of films stored 1 and 5 weeks at 25C and 53% RH. Pure and composite films presented similar values of WVP (between 5.4-6.4). Lipid addition to films improved this property, as should be expected due to its higroscopicity and as has been described in previous works in starch films (Jimnez et al., 2012) and gelatin films (Bertan et al., 2005). This effect was especially significant (p<0.05) in starch films (100S-0G), with reductions of WVP around 60%. This can be relationed with specific interactions between the functional groups of both compounds (starch and ester) that led to major ester integration on starch matrix. Film ti= 1 weeks tf= 5 weeks ti= 1 week tf= 5 weeks ti= 1 week tf= 5 weeks ti= 1 week tf= 5 weeks ti= 1 week tf= 5 weeks 100S-0G 5,4(0,4) (BC)(x)(1) 6,8(0,5) (A)(x)(2) 0,395(0,002) (F)(x)(1) 0,253(0,0013 (G)(x)(2) 131(31) (EF)(x)(1) 770(171) (B)(x)(2) 4,1(0,7) (FG)(x)(1) 13(2) (C)(x)(2) 4,5(0,9) (E)(x)(1) 1,9(0,6) (E)(x)(2) 100S-0Ge 2,7(0,3) (D)(y)(1) 1,9(0,5) (G)(y)(2) 2,51(0,23) (A)(y)(1) 1,9(0,3) (A)(y)(1) 73(17) (F)(x)(1) 26(7) (E)(y)(2) 2,2(0,5) (G)(y)(1) 1,9(0,3) (E)(y)(1) 6(2) (DE)(x)(1) 28(5) (A)(y)(2) 75S-25G 6,2(0,7) (A)(x)(1) 6,4(0,7) (AB)(x)(1) 0,494(0,009) (F)(x)(1) 0,379(0,002) (EF)(x)(1) 354(51) (C)(x)(1) 724(57) (B)(x)(2) 11,2(1,4) (E)(x)(1) 16(3) (C)(x)(2) 15(1) (BC)(x)(1) 2,4(0,5) (E)(x)(2) 75S-25Ge 6,4(0,8) (A)(x)1) 5,2(0,3) (DE)(y)(2) 1,330(0,007) (D)(y)(1) 0,600(0,002) (CD)(y)(2) 191(22) (E)(y)(1) 544(68) (C)(y)(2) 7(1) (F)(y)(1) 10(3) (D)(y)(2) 17(3) (B)(x)(1) 2,2(0,8) (E)(x)(2) 50S-50G 5,9(0,4) (AB)(x)(1) 5,9(0,6) (BC)(x)(1) 0,52(0,09) (F)(x)(1) 0,270(0,0013) (EF)(x)(2) 627(54) (B)(x)(1) 812(119) (B)(x)(2) 24(2) (C)(x)(1) 30(3) (B)(x)(2) 14(4) (CD)(x)(1) 12(3) (C)(x)(2) 50S-50Ge 5,2(0,2) (C)(y)1) 3,8(0,2) (F)(y)(2) 1,520(0,005) (C)(y)(1) 0,860(0,003) (BC)(y)(2) 271(46) (D)(y)(1) 392(58) (D)(y)(2) 16(2) (D)(y)(1) 16(2) (C)(y)(2) 30(4) (B)(y)(1) 18(7) (B)(x)(2) 0S-100G 6,3(0,5) (A)(x)(1) 5,5(0,4) (DE)(x)(2) 0,75(0,06) (E)(x)(1) 0,49(0,23) (EF)(x)(2) 703(23) (A)(x)(1) 1119(87) (A)(x)(2) 37(2) (A)(x)(1) 46(5) (A)(x)(2) 19(2) (B)(x)(1) 8(2) (D)(x)(2) 0S-100Ge 5,3(0,2) (BC)(y)(1) 4,8 (0,5) (E)(x)(1) 1,88(0,07) (B)(y)(1) 0,98(0,23) (B)(y)(2) 336(28) (CD)(y)(1) 599 (35) (C)(y)(2) 28(5) (B)(y)(1) 31(3) (B)(y)(1) 31(5) (A)(y)(1) 17(4) (B)(y)(2) A, B, C, : Different superscripts within a column indicate significant differences between formulations (p<0,05). x, y : Different superscripts within a column indicate significant differences by ester addition for the same formulation (p<0,05). 1, 2 : Different superscripts within the same file indicate significant differences between di fferent storage ti mes for the same formul ati on. (p<0,05). Table 1: Water vapour permeability (WVP), oxygen permeability (OP), Elastic modulus (EM), tensile strength (TS) and elongation percentage (%E) of cassava starch (100S-0G), gelatin (0S- 100G) and composite films (75S-25G, 50S-50G), with E-471 ester addition (e), stored 25C and 53%RH during 1 week and 5 weeks. Mean values and (standard deviation) WVP (gmmKPa -1 h -1 m- 2 ) OP (10 -13 cm 3 m -1 seg -1 Pa -1 ) ME(MPa) TS(MPa) E(%)
Storage time showed a different behaviour between films with or without lipid addition. Lipid incorporation to matrix, reduced WVP significantly at the end of storage. This would indicate the good integration between hydrocolloids and polar groups of lipid which can be trapped in the chains of polymeric matrix. Previous works have described the protector effect of lipids due to phenomena of association between compounds (Gontard et al., 1994; Yang y Paulson 2000). Gelatin films (0S-100G) presented OP values twice greater than starch films (100S-0G), in agreement with reported values (Bertan et al., 2005; Hassan and Norziah, 2012). Composite films increased permeability as gelatin content increased.
P a g e | 4 Lipid incorporation increased significantly films OP. These results are coherent with structural irregularities observed in SEM micrographs and have also described in previous works with starch films (Jimnez et al., 2012) and gelatin films (Bertan et al., 2005). Films aging decreased OP significantly (p<0.05) due to interaction and phenomena of lipid association with polymeric matrix that appear after storage period. In films based on starch, this improvement can be due to crystal formation during retrogradation process, which acts as an oxygen barrier (Shen et al., 2010). 2.5 Mechanical properties Table 1 shows mean values of mechanical properties of films stored 1 and 5 weeks at 25C and 53% RH. Gelatin films (0S-100G) had more resistance (TS), stiffness (ME) and stretchability (%E) than cassava starch films. Gelatin addition increased these mechanical parameters (TS, ME and %E) for all films and storage times, which indicates the good integration of both polymers. In previous works, Pranoto et al. (2007) points out those macromolecular polysaccharides of large chains, as starch, can interlace with gelatin, increasing their mechanical resistance. Both polymers can form structural networks by interactions between anionic groups of polysaccharides and cationic groups of gelatin, strengthening the final structure (Linus et al. 2003). Composite films are systems with free hydroxil groups to form hydrogen bonds between gelatin, starch, glycerol and water (Hassan y Norziah, 2012). Lipid addition increased %E and decreased ME and TS. Similar results were observed by Jimnez et al. (2012) when lipis were added to starch films. This was expected since lipids introduce discontinuities in the polymer matrix which contribute to a reduction in the polymer cohesion forces and, thus, the films resistance to break. Storage time increased ME and decreased %E and TS. This behaviour can be explained by phenomena of microstructural reorganization that affect to polymeric chains and need a time to occur. In films based on starch, this result can also been attributed to starch retrogradation process that provides a higher proportion of crystalline zones (Jimnez et al., 2012). 2.6 Microstructural properties Figure 1 show cross sections of starch, gelatin and composite films obtained by SEM techniques with (b) and without ester addition (a). SEM micrographics showed compact structure of pure starch and gelatin films. Composite films presented a more heterogeneous surface than the pure films but a good compatibility among the polymers (no separation). Gelatin addition to starch matrix changes the microstructural arrangement of gelatin chains that are regrouped in helycoidal way during films drying process. Hassan and Norziah (2012) observed similar results in micrographs of starch:gelatin films. The lipid addition (E-471) integrates differently depending on the matrix: in pure starch and gelatin are included in the continuous network, forming complexes with lipids. In composite films, the formation of lipid aggregates distributed throughout the film matrix can be observed. 3 Conclusions Gelatin and lipid addition to films based on cassava starch, affected notably to physical and microstructural properties of films. Composite gelatin: starch films generated compact structures that improved mechanical properties of pure starch films. Lipid addition resulted in less permeable films to water vapor and more permeable to oxygen, for both storage times. Aging films affected significantly to mechanical properties, increasing resistance but reducing their stretchability. Global analysis of factors (starch:gelatin ratio, storage time, ester addition) and evaluated properties led to select an optimal starch:gelatin ratio of 1:1. These composite films without lipid addition, showed good mechanical and oxygen barrier properties. 5 | P a g e
Figure 1. Microstructural (SEM-cross sections) properties of cassava starch films (100S-0G), bovine gelatin films (0S- 100G) and composite of starch and gelatin (50S-50G) and their mix with E-471 (0S-100GE, 100S-0GE, 50S-50GE).
100S-0G
100S-0Ge
75S-25G
75S-25Ge
50S-50G
50S-50Ge
0S-100G
0S-100Ge
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