Effect of glycerol, peanut oil and soybean lecithin contents on the properties of biodegradable film of improved cassava starches from Côte d’Ivoire

— Edible films have been successfully used in the food packaging industry for several decades. Today natural polysaccharides, including cassava starch,are increasingly being used in the production of such biodegradable edible films and food packaging. In Côte d'Ivoire, there are improved cassava varieties whose starches have not yet been tested in the production of biodegradable films. In thisstudy, the optical and mechanical properties and the water solubility of starch-based composite films of four improved cassava varieties withadded glycerol, peanut oil and soy lecithin were determined. Starchwas obtained by cold water extraction from native cassava from the varieties Bocou 1, Bocou 2, Yavo and TMS. Films preparation was made bycasting methodwithcassava, glycerol (25-30 %), peanutoil (5-10 %) and soybean lecithin (0-5 %). Increasing the glycerol content, increased L*color valueand elongationat break and decreased a*, b*, colourdifference (ΔE*ab) and tensilestrength of the composite films. Also, increasing the oil content from 5 to 10%, increased the opacity, b*, ΔE*ab, water solubility, elongationat break but decreased L*, a* and tensilestrength. Similarly, increasing the soy lecithin content from 0 to 5%, increased the opacity, L*, b* and ΔE*ab, but decreased a*, of the starch-based composite films. The results suggest an ideal formulation of 4% starch/25% glycerol/5% oil/5% soy lecithinfor a film with optimum mechanical properties with low solubility.

and functionality vary between cultivars within and between species (Copeland et al., 2009). Cassava (Manihot esculenta Crantz) roots are among the most important sources of starch worldwide. According to the Food and Agricultural Organisation (FA O), the global cassava production in 2015 was estimated at 281.1 million tonnes with 54 % produced in Africa (FAO, 2016). In Côte d'Ivoire cassava is the second major food crop after yam, with an estimated 5.1 million tonnes produced in 2015 (FAO, 2016).Unlike other starches, cassava starch allows obtaining transparent and flexib le films (Vicentin i and Cereda, 1999). Ho wever, for the production of edib le films and optimised application in food technology plasticizers, hydrophobic agents and emu lsifiers have to be added to the film co mposition (Bergoet al., 2010). Adding vegetable oils to hydrophilic films, for instance, reduces the water vapor barrier p roperties of films based on proteins (Fabraet al., 2009) and those based on polysaccharides (Koelschand Labuza,1992). Those emu lsion-based films with enhanced physico-chemical and mechanical properties generally arise fro m oil droplets with small diameter, a high degree of homogeneity and highly stable film-fo rming emulsions (Nilsuwan et al., 2016;Debeaufort et al., 1995). For good dispersion/homogenization of the oil in the film-forming matrix, emu lsifiers are therefore common ly added. They are nonpolar substances which bind to both water and oil, thus improving emulsificat ion and increasing the stability of the emulsion. Furthermore, soybean lecithin incorporated into protein-based emulsified films has previously shown effective in stabilizing the emu lsion film (Prodpran et al., 2007).Lip id co mpounds are used to modulate the water barrier properties of films; however, in hydrophilic suspensions they adversely affect the mechanical and optical properties of the resulting films (Yang and Paulson, 2000). The most effect ive plasticizers are generally those whose structure resembles the structure of the polymer they plasticize. For starch-based films polyols such as sorbitol and glycerol are the commonly used plasticizers (Mali et al., 2005). With increasing glycerol concentration, the breaking strain has shown to improve and the tensile strength to decrease (Alves et al., 2007).
In Côte d'Ivoire, there are improved cassava varieties whose starches have not yet been tested in the production of biodegradable films. The aim of this study is to determine the effect of plasticizer, lipid and emulsifier contents on the mechanical properties, solubility, opacity and color parameters of films based on starch from improved cassava varieties grown in Côte d'Ivoire.

II.
MATERIALS AND METHODS Cassava natives starches from four improved varieties Bocou 1, Bocou 2, Yavo and TMS, belonging to Centre National de Recherche Agronomique in Côte d'Ivoire were used in this study. Cassava plants were harvested at maturity, 12 months after plantation. Glycero l (bidistilled, 99.5% purity) and soybean lecithin were purchased from VW R Prolabo Chemicals (Leuven, Belgiu m). The CORA brand peanut oil used in this study was purchased at a supermarket in Belgium.
2.1. Amylose content The amy lose content of the starch samples was determined by colorimet ric reaction and subsequent measuring of the absorbance of the amylose-iodine blue complex formed (Morrison and Laignelet, 1983).

Starch paste clarity
The paste clarity of the starches was determined as previously described by Craig et al. (1989). Appro ximately 0.11 g of starch was weighed into quartz screw tubes. The mass was supplemented to 10 g with distilled water. The closed tube with the well ho mogenized content was left in aboiling water bath at 100°C for 30 min with uniform stirring. The solution obtained was cooled and the paste clarity or percent transmittance (% T) was determined using a Shimad zu UV-2401-PC (Kyoto-Japan) spectrophotometer at 650 n m against a blank samp le containing distilled water.
2.3. Film preparations and thickness measurements Film p reparation and thickness measurements were determined in accordance with previously described protocol (Adjouman et al., 2017). The emu lsified films were prepared in two steps. First, 4 g of cassava starch (w/w, starch) was mixed with glycerol (1-1.2 g ) and with two thirds of d istilled water, the final mixture being heated fro m 30°C to 75°C for 20 min. Peanut oil (0.2-0.4 g), soybean lecithin (30-60 mg ) and distilled water (a third of the total mixture) ( Table 1) was also heated together for 20 min fro m 30°C to 75°C. Both solutions were heated with constant stirring at 750 rp m/ min. Thereafter, the solution of peanut oil, soybean lecithin and distilled water was homogenised at 24,000 rp m fo r 2 min using an Ultra -Turrax T25 basic (IKA Werke, Staufen / Germany). The homogenised solution was subsequently mixed with the starch and glycerol and then heated from 75°C to 95°C at 750 rp m stirring for 25 min. A 20 g aliquot of the final solution was transferred and spread with even thickness on the surface of a Petri dish (9 cm diameter) and oven-dried in a ventilated oven model (Memmert UF-110, Schwabach, Germany) at 35°C for 24 h. The dried films were removed and stored in a desiccator at 25°C for 48 h, before testing. The thickness of all films was determined using a hand micro meter (NSK, Japan) at 10 rando m positions of the films.

Film water solubility
Film water solubility was determined as previously describe (López et al., 2008). Fro m each film p ieces of 2 x 3 cm were cut and stored in a desiccator, containing silica gel beads, for 7 days. Pieces were weighed to the nearest 0.0001 g and placed into test beakers with 80 ml deionized water. The samp les were left under constant stirring at 200 rp m for 1 h at roo m temperature. After 1 h, any remain ing pieces of film were subsequently collected by filtration, dried again in an oven at 60°C to constant weight and the proportion of total soluble matter was calculated.
2.5. Film opacity Film opacity was determined in accordance with previously described protocol (López et al., 2008). Film samples were cut into rectangles of1 x 3 cm and placed inside a spectrophotometer cell. The absorbance spectrum (400-700 n m) was recorded for each sample using a Shimad zu (UV-2401-PC Kyoto/Japon) spectrophotometer. Film opacity was determined by an integration procedure and expressed as absorbance units per nanometers (AU. nm).

Mechanical properties
Mechanical properties were studied using a TA.TX2 Stable Micro Systems Texture Analyzer. ASTM D882 -02(2002), standard test method for tensile properties of thin plastic sheeting with some modifications was used. The films were cut into 25 mm wide and 80 mm long strips, using a scalpel and mounted between the grips (A/TG) of the texture analyser. The ends of the strips were mounted between cardboard grips. Strength resistance and deformation at break were recorded in tensile mode, during extension at 10 mm min -1 , with an in itial 50-mm distance between the grips, until the specimens broke.
2.8. Statistical analysis UsingStatistica 7.1 software (Statistica), mu ltivariate analyses of variance (MANOVA) was performed, to compare the means of the various properties of the film formulat ions (cassava variety and glycerol, peanut oil and soy lecithin added). Duncan's mu ltip le range test at the 5% threshold, allowed locating the differences.

III.
RESULTS AND DISCUSSION 3.1. Amylose content and paste clarity Paste clarity and amylose content of cassava varieties used in this study are presented in Table 2. The highest value of amylose was obtained with the TMS variety, followed by the variet ies Yavo, Bocou 2 and Bocou 1. In contrast, the clearest starch gel was obtained with the Bocou 1 variety, followed by Bocou 2, Yavo and TMS. Starches with low amy lose content have high paste clarity . Amylose contents are known to influence clarity of starch pastes. High amy lose content may result in more opaque starch pastes (Schmitz et al., 2006).

Film water solubility
Glycero l, o il and lecith in added showed a significant difference in the water solubility of the starch films (p < 0.05). Increasing the concentration of g lycerol fro m 25% to 30%, oil fro m 5% to 10% and lecithin fro m 0% to 5% resulted in increased water solubility of the co mposite films from all varieties ( Table 3). . The demonstrated positive correlation between the solubility of the films and the plasticizer concentration may be due to the low molecu lar weight of the glycerol which enables it to be easily inserted between the polymer chains (Cuq et al., 1997) and to the hydrophilic nature of glycero l (Lopez et al., 2008). Furthermore, the increase in the g lycerol concentration in biodegradable films was found to induce a marked decrease in the crystallinity of the starch films (Belibi, 2014). The less crystallites are formed in the films the more readily they will swell in the water and disintegrate.
Increasing the oil content from 5% to 10% increased the water solubility of the starch films of all cassava varieties which is likely to be due to hight degree of solubility of the fatty acids contained in the peanut oil. An increase in fat content increases solubility in water as long as the concentration of fatty acids is lower than the saturation level for an expected volume of water (Fakhouri et al., 2009). Previous work on gelatin films with lauric, palmit ic and stearic acids revealed indeed a positive correlation between water solubility and fatty acid content (Fakhouri et al., 2003). Another study revealed that solubility of soy protein films showed a significant increase with the addition of lauric acid (Rhim et al., 2002) supported by similar findings in wheat gluten films with added fatty acids (Gontard et al., 1994). The increase in soy lecithin content from 0 to 5% led to an increase in the water solubility of the starch films of all cassava varieties. It was hoped that the inclusion of compounds having hydrophobic characteristics (i.e. soy lecithin) could reduce the water solubility of cassava starch films; however, this behavior was not observed in this study. A study of edible composite films based on wheat gluten and lipids suggested that the increase in solubility due to addition of compounds with hydrophobic characteristics is related to the breakdown of the intermolecular bonds in the protein network and to the formation of weak interactions with hydrophobic substances (Gontard et al., 1994). The subsequent increase in the solubility of cassava-based films when the concentration of soy lecithin is being raised is likely to be related to the breakdown of the intermolecular bonds in the starch network.

Film opacity
The opacity values of the starch films of the d ifferent cassava varieties as a function of the different formu lations are presented in Table 4. At a given concentration of glycerol addition of oil allowed an increase in the opacity of the films of cassava starches. Similarly, at a given concentration of glycerol and oil, addition of soy lecithin caused an increase in the opacity of cassava starch films. Highest values were found with films fro m TMS starch, followed by Yavo, Bocou 2 and Bocou 1. Added oil and lecithin and oil-lecithin co mbination showed significant differences in the opacity of the starch films of all cassava varieties (p < 0.05).  Increase in lecithin content from 0 to 5% allo wed an increase in opacity of films fro m starches of all cassava varieties. We attribute this increase to original state of yellow soybean lecithin and a hydrophobic compound. Incorporation of the hydrophobic substances into the hydrocolloid poly mer HPM C matrix caused a decrease in the lu minance of the films wh ich resulted in an increase in their opacity in previous work (Quezada-Gallo et al., 2000). An oil-soy lecithin co mbination also allowed an increase in opacity of starch films of four cassava varieties which seems to be accumulat ion of their individual effects. Opacity was more pronounced in TMS starch films than in starch films derived fro m the three other variet ies used in this study and is related to the intrinsic characteristics of each starch variety specifically the starches paste clarity ( Table 2).

Film color
The color parameters (Fig. 1) o f the films were significantly influenced by the addition of glycero l, oil and lecithin (p < 0.05). Increasing the glycerol content, led to increased L* (Fig. 1a) and decreased a* (Fig. 1b), b* (Fig.  1c) and ΔE*ab (Fig. 1d), of starch films fro m four cassava varieties. Conversely, increasing the oil content, resulted in a decrease in L* and a*, and an increase in b* and ΔE* ab, of the starch films formulated using cassava varieties. Lecithin addition, resulted in an increased L*, b* and ΔE*ab, but decreased a*, of starch films of cassava varieties. The increase in b*, resulted in an increase in ΔE*ab of the resulting films. //dx.doi.org/10.22161/ijeab/3.4.39  ISSN: 2456-1878 www.ijeab.com Page | 1437 The study on HPMC films plasticized with 15 and 30% glycerol was showed a slight increase in L* lu minance but not a decrease in a*, b* and ΔE*ab indexes (Al Mahdi, 2006). However, our results concerning effect of oil on color parameters are consistent with studies on emu lsified films based on gelatine of fish shell and palm oil (Tongnuanchan et al., 2015) and on the stability of the emu lsion and the properties of gelatin films affected by palm o il and emu lsifiers (Nilsuwan et al. 2016). Previous studies in films with soy lecithin as emu lsifier reported highest b* and ΔE*ab values compared with other emu lsifiers (Nilsuwan et al., 2016). Therefore, the colors of peanut oil and soy lecith in directly determined the co lor of the cassava starch films in this study. Table 5 shows values of tensile strength and elongation at break of co mposite films based on cassava starch. Increasing glycerol content from 25% to 30% resulted in a decrease in tensile strength at break and an increase in the elongation at break of films fro m cassava starches varieties. It has already been pointed out by several authors that increase in level of p lasticizer such as glycerol in starch films leads to a decrease in tensile strength and an increase in elongation at break (Alves et al., 2007;Bertuzzi et al., 2012;Sanyang et al., 2015). It has been shown that glycerol reduces the rigidity of the starch network, lead ing to a less orderly film structure and increasing the capacity of the poly mer chain movement (Sothornvit and Krochta, 2005). These properties render glycerol a suitable plasticizer able to reduce intramolecular fo rces between starch chains and to promote hydrogen bond formation between p lasticizer and starch molecu les. An increase in elongation of films may be related to the fact that plasticizers decrease intermolecular bonds between amylose, amylopectin and amy lopectin amylose phases of the starch and substituting them by hydrogen bonds formed between starch mo lecules and plasticizer (Sanyang et al., 2015). Such perturbation and reconstruction of starch molecular chains reduces stiffness and promotes film flexibility by allo wing greater mobility of the chain. Zavareze et al. At 25% glycero l, addition of soybean lecithin resulted in an increase in tensile strength and an increase in elongation at break for all variet ies; however, at 30% the addition resulted in decrease in tensile strength and increases an elongation at break of the starch-based films. This behavior observed at 30% glycero l when the soy lecithin is added could be due an apparent synergistic effect between glycerol and emu lsifiers (Rodriguez et al., 2006). This interaction means that the films with glycerol in the presence of an emulsifier mechanically behave in the form of films with a larger amount of plasticizer. At 25% glycero l, an increase in tensile strength was observed that is likely to be due to the good dispersion and distribution of the added oil in the film caused by the addition of 5% lecithin. It is well known that mechanical properties of emu lsified films are improved when the hydrophobic compound is s mall and distributed more homogeneously (Debeaufort and Vo illey, 1995). Resistance and elongation at break of starch films fro m four cassava vary between varieties. This difference in mechanical properties of films may be exp lained by the difference in amy lose content of starches constituting these films. Indeed, as the amylose content of the starch increases at constant glycerol concentration, the tensile strength at break of the corresponding films increases while the elongation at break decreases (Alves et al., 2007).

IV.
CONCLUSION Starches of imp roved cassava varieties in Côte d'Ivoire can be used in the production of biodegradable films with plasticizer, lip id and emu lsifier. Effect of plasticizer, lipid and emu lsifier concentration on films fro m four improved cassava varieties was evaluated. The increased levels of glycerol, oil and soybean lecithin influenced the properties of cassava-based starch films. The resultant films fro m imp roved cassava starch with glycerol, peanut