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Ozonation of cassava starch to produce biodegradable films
International Journal of Biological Macromolecules 141 (2019) 713–720
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Ozonation of cassava starch to produce biodegradable films☆ Carla I.A. La Fuente a,⁎, Andressa Tamyris de Souza b, Carmen C. Tadini b,c,d, Pedro Esteves Duarte Augusto a,d a
Department of Agri-food Industry, Food and Nutrition (LAN), Luiz de Queiroz College of Agriculture (ESALQ), University of São Paulo (USP), Piracicaba, SP, Brazil University of São Paulo, Escola Politécnica, Department of Chemical Engineering, Main Campus, 05508-010, SP, Brazil c University of São Paulo, Food Research Center (FoRC/NAPAN), SP, Brazil d Food and Nutrition Research Center (NAPAN), University of São Paulo (USP), São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 15 August 2019 Received in revised form 29 August 2019 Accepted 4 September 2019 Available online 05 September 2019 Keywords: Biodegradable films Cassava Starch Ozonation
a b s t r a c t In this study, biodegradable films were produced from cassava starch modified by ozone at different levels. The films were produced by casting technique using native and ozonated cassava starch, glycerol as the plasticizer, and water as the solvent. Films were characterized in term of their mechanical, barrier and functional properties, morphology, crystallinity, colour, and opacity. The morphology of the ozonated films was more homogeneous in comparison to the films produced with the non-modified starch and enhanced properties were achieved. Films produced with ozonated cassava starch presented higher tensile strength, Young's modulus and lower elongation. The water vapour permeation and the oxygen permeation were increased by increasing the ozonation time. Moreover, ozone processing resulted in films with a more hydrophilic surface and lower solubility after 24 h. Possible explanations and applications were discussed. In conclusion, the ozone processing showed to be a good alternative for starch based packaging production. © 2019 Elsevier B.V. All rights reserved.
1. Introduction While hundreds of millions of tons of petroleum-based plastics are produced annually all over the world, the concern about their environmental impact is increasing each day. Consequently, different renewable and biodegradable source are being studied to produce biodegradable plastics. Starch is one of the promising biopolymeric matrix for the development of biodegradable packaging [1]. Starch is one of the most important and abundant polysaccharides in nature [2,3]. They are obtained from renewable sources, presenting relatively low cost and a wide possibility of application. Consequently, starch is a very versatile material, being used in several industries such as the food, paper, textile, chemical and pharmaceutical [2]. Being the native sources limited by nature, different starch modification techniques are available in order to improve the processability, mechanical and/or barrier properties of biodegradable films. Among different possibilities of starch modification, ozone processing has gaining attention due to the demand for methods that meet safety and health standards for both consumers and the environment [4]. Ozone is a powerful oxidizing agent, which reacts with starch, increasing its carboxyl and carbonyl contents, and also reducing its molecular size and distribution [5]. Consequently, different properties are ☆ A patent related with this work was filed (BR1020190112166). ⁎ Corresponding author at: Avenida Pádua Dias, 11, Piracicaba, SP 13418-900, Brazil. E-mail address: [email protected] (C.I.A. La Fuente).
https://doi.org/10.1016/j.ijbiomac.2019.09.028 0141-8130/© 2019 Elsevier B.V. All rights reserved.
achieved due to complex relation among molecular modifications on size, charge and chemical affinity. In fact, different starch sources were already modified by ozonation, such as corn, sago, wheat, potato and tapioca/cassava [5–10]. However, different starch sources have different molecular and granule structure, being differently affected by ozonation. For instance, cassava starch had both polygonal and spherical shaped granules with size b50 μm, while potato starch contained both large and small granules, which had spherical to oval shapes [11]. Therefore, different ozonated starches present different molecular sizes and electrical charges, which affect their behaviour for reassociation. These molecular changes are competitive in relation to film production and properties. Consequently, the properties of films produced with ozone modified starches are unpredictable and, dependent on the relations among the source, reactor and processing conditions. Hence, the aim of this study was to produce films from ozonemodified cassava starch, considering different process condition and evaluating the most important mechanical, barrier and functional properties, as well as their morphology, crystallinity, colour, and opacity.
2. Material and methods This work was developed in three parts, as summarized on Fig. 1. Firstly, cassava starch was ozonated, in two conditions. Then, films based on the starches (native and the two ozonated conditions) were
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Nomenclature a⁎ A b⁎ C E E* L⁎ m OTR p P’O2 RC S t TS WVP x Y
redness/greenness CIELab parameter (−) area (m2) yellowness/blueness CIELab parameter (−) chroma (−) Young's Modulus (MPa) CIELab total color change (−) brightness/darkness CIELab parameter (−) mass (g) oxygen transmission rate (cm3·m−2·d−1) pressure (kPa) oxygen permeability (cm3/m·day·Pa) relative crystallinity solubility (%) time (h) Tensile Strength (MPa) Water Vapour Permeability (g·mm/m2·day·kPa) thickness (mm) opacity (%)
Subscripts 0 initial b black standard f final w white standard Greek Δ ε
dry basis; 1500 mL) and then ozonated for different periods under constant stirring in the same system reported by Castanha [5]. The ozone rich stream was produced from industrial oxygen by the coronal discharge method using an ozone generator unit (Ozone & Life, O&G Model RM 3.0, São Jose dos Campos, Brazil). This stream was dispersed in the sample, which was placed in a glass reactor, as small bubbles through a gas disperser. A gas flow of 1 L∙min−1 was used, with an ozone concentration in the gas current of 41 mg O3∙L−1. The amount of ozone was measured using an ozone monitor (2B Technologies Model 106-H, USA). After passing through the samples, the gas stream was led out of the reactor and converted to oxygen in a thermal ozone destroyer (Ozone & Life, São José dos Campos, Brazil). The procedures were performed at 25 ± 1 °C. Fig. 1 presents a schematic representation of the ozone processing system. Three samples were evaluated: the non-modified starch and those ozonated for 15 and 30 min. After processing, the excess of water was removed through decantation and centrifugation, and the starch samples were dried in an air circulation oven for ~ 12 h at 35 °C until moisture content of ~12 g/100 g w. b. Glycerol P.A. grade (Sigma-Aldrich, Brazil) and distilled water were also used for film production. 2.2. Film preparation
delta elongation at break (%)
formed. Finally, the obtained films were evaluated through their structure and the most important properties. 2.1. Materials The native cassava starch (Amilogill 1500) was donated by Cargill Company. This starch was suspended in distilled water (10 g/100 g in
The films were produced by casting technique, as shown in Fig. 1. A solution containing 5 g/100 g of cassava starch was homogenized in distilled water with a magnetic stirrer (GEHAKA, HU-30/2, Brazil) for 10 min. Then, the solution was heated for 45 min in a reactor with external recirculation of water at 75 °C. Glycerol was added as the plasticizer (25 g/100 g of starch) according to Souza [12] and, the suspension was heated for more 15 min under agitation. To eliminate the bubbles, the filmogenic solution was placed in an ultrasonic bath for 30 min at 70 °C (154 W, 25 kHz; UNIQUE, model USC-1850, Brazil). The solution was then poured into acrylic Petri dishes (0.15 g/cm2) for ~12 h to rest, and then dried at 35 °C and 45% HR for ~10 h in a climatic chamber (TIRACLIMA, model TCC 7034, Germany). The films were conditioned for at least 48 h in desiccators containing a saturated solution of NaCl (75% RH) before characterization at room temperature (~25 °C).
Fig. 1. Schematic representation of the ozone processing system and the film elaboration.
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2.3. Films characterization 2.3.1. Mechanical properties The film thickness was determined using a digital micrometer with a precision of 0.001 mm (MITUTOYO, Japan) at five random positions. The mechanical properties were evaluated through a uniaxial tensile assay, according to ASTM D882-09 [13]. Tensile strength (TS) [MPa] and percent elongation at break (ε) [%] were evaluated on a texture analyzer (TAXT2i e Stable Micro Systems, UK) with a load cell of 30 kgf (294 N), using the A/TGT self-tightening roller grips fixture. The initial grip separation and the crosshead speed were set at 50 mm and 1 mm·s−1, respectively. Young's modulus (E) was calculated as the slope of the initial linear portion of the stress versus strain curve. At least ten strips (100 mm × 25 mm) were evaluated for the non-modified and modified films. Furthermore, the force and the deformation at the breaking point of the film was determined in a punctuation test [14], on a texture analyzer (TAXT2i e Stable Micro Systems, UK) with a load cell of 30 kgf (294 N). The films were fixed in a circular permeation cells (d = 0.05 m) and perforated by a 3 mm diameter probe. The puncture force (F) and the displacement of the probe (D) were obtained from forcedeformation curves, according to Fig. 2. 2.3.2. Barrier properties Water vapour permeability (WVP) of films was measured gravimetrically according to the ASTM E96-80 [15]. The films were placed in circular permeation cells containing silica gel, and then placed in a climatic chamber (TIRACLIMA, model TCC 7034, Germany) maintained at 75% RH and temperature of 25 °C. The cell weight was recorded hourly for 8 h. Water vapour permeability was calculated according to Eq. (1).
WVP ¼
Δm x 24 Δt A Δp
ð1Þ
wherein: Δm/Δt is the moisture gain per unit of time (g·h−1); x is the film thickness (mm); A is the film area exposed to permeation (1.96·10−3 m2); Δp is the vapour pressure difference (kPa) of the atmosphere over silica gel and pure water (3.168 kPa at 25 °C).
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The oxygen permeability coefficient (P’O2) of the ozonated films was measured at 23 °C and 75% RH on a 50 cm2 circular films using an oxygen permeation system (OXTRAN 2/21, Mocon, USA), according with ASTM F1927-14 [16]. The oxygen permeability reported is the mean of four films. The oxygen permeability coefficient was calculated according to Eq. (2). P’O2 ¼
OTR X p
ð2Þ
wherein: P’O2 is the oxygen permeability coefficient (cm3·m−1·d−1·Pa−1); OTR is the oxygen transmission rate (cm3·m−2·d−1); p is the partial pressure of oxygen, and X is the average thickness of the specimen (m). 2.3.3. Functional properties The film wettability was determined by the measurement of the contact angle with water, according to ASTM D7334 − 08 [17] using an OCA15- Dataphysiscs (OCA 15, Dataphysiscs, Germany). Two angle measurements were carried out (one on each drop edge) quickly to avoid changes due to water vaporization on each drop. The contact angle was defined as the average of six angles measures. The film solubility in water was evaluated with discs (diameter of 20 mm) immersed in 50 mL of water at 25 °C for 24 h, under mechanical stirring at 120 RPM, according to Gontard [18] The solubility in water was calculated as the percentage of dry matter of the solubilized film, as described in Eq. (3). S¼
m f −mo 100 mo
ð3Þ
wherein: mo and mf are the initial and the final discs' mass (g), respectively. The films moisture was determined through the gravimetric method in an oven, without recirculation, at 105 °C until a constant mass (~24 h), according to Gontard [18]. 2.3.4. Colour and opacity The film colour was determined using a colorimeter (HUNTERLAB, model ColorQuest XE, USA) with the CIELab scale (L⁎, a⁎, b⁎). The colour measurements were expressed in terms of L* (L* = 100 brightness and
Fig. 2. Diagram of the system used to determine the punctuation deformation. Adapted from Sobral [14].
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Fig. 3. Scanning electron micrographs of the surfaces and cross-sections of the films prepared with non-modified and ozonated cassava starch. Surface images at and, the cross-sections images at and, respectively. The surface and the cross-section images were obtained at 1000× and 3100×, respectively.
L* = 0 darkness); a* (+a* = redness and −a* = greenness) and b* (+b* = yellowness and −b* = blueness). The change of each of the parameters was calculated according to Eqs. (4), (5) and (6). ΔL ¼ L −L0
ð4Þ
−a0
ð5Þ
ð6Þ
Δa ¼ a
Δb ¼ b −b0
wherein: L0* = 94.04; a0* = −0.86 and b0* = 1.53 determined from a white standard pattern. The total colour change (ΔE⁎), the opacity (Y) and the Chroma (C) [19,20] were calculated using Eqs. (7), (8) and (9) respectively. h i1 2 2 ΔE ¼ ðΔL Þ þ ðΔa Þ2 þ ðΔb Þ Y¼
2
ð7Þ
2.3.6. Morphology The surface and cross-section morphology of the films were visualized by a scanning electron microscope (SEM). The films were dried in silica gel for 48 h to ensure an accurate and finer cut. They were then horizontally and vertically placed on an adhesive tape placed over circular stubs. The films were then coated with a 10-nm platinum in a high-vacuum coating system (BALTEC, model MED020, Switzerland) and the side which was not in contact with the Petri dish was observed using the microscope (FEI, model Quanta 600FEG, Netherlands) with an acceleration voltage of 15 kV and magnification ranged from 500× to 3100×. 2.4. Experimental design and statistical analyses
Yb Yw
1 2 C ¼ a2 þ b
The scanning rate was 0.02°/s with 2ϴ angles varying between 2° to 40°. The software Origin 2018b was employed for calculation (OriginLab Corporation, Massachusetts, USA).
ð8Þ 2
ð9Þ
wherein: Yb is the opacity on the black standard and, Yw is the opacity on the white standard. Three measurements were performed per film formulation to determine all the colour parameters. 2.3.5. Crystallinity The relative crystallinity of the films was calculated as the ratio between the crystalline area and the total area of the X-ray diffractogram (plotted from 2° to 40°), as described by Nara & Komiya [21]. An X-ray diffractometer (PANalytical, model X'Pert PRO, Netherlands), with detector X'Celerator operating at 45 mA, and voltage of 40 kV was used.
A completely randomized design was applied in three replicates. The obtained data was evaluated through analysis of variance (ANOVA), applying Tukey's test as a post-hoc test through Statgraphics Centurion XV software (StatPoint, Inc., USA). 3. Results and discussion Fig. 3 presents the morphology of the films. In general, the ozonated films present a compact structure and smooth surfaces, in comparison to the non-modified starch films. Heterogeneity and roughness were already observed on the surface of non-modified cassava films [22]. For the ozonated films, smoother surface is clearly evident in the surface of the films, while, in the cross-section, some cracks are visible, probably appeared after the film was broken. In the study performed by Biduski
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[23] although oxidized sorghum films were more homogeneous, similar fissures were also observed. According to the literature, films produced with oxidized starches allow a higher penetration of the plasticizer molecules in the starch chains, and improve the interaction between them [23–25]. Moreover, low retrogradation is expected in oxidized starches [24], promoted by the repulsion between molecules, increasing the free space between the molecules, and also increasing the homogeneity of the film. However, ozonation is more than only an oxidation process and this process also changes the molecular charges and chemical affinity, which affect the interaction among the molecules and altering the morphology of the films. Fig. 4 shows the X-ray diffractograms for the non-modified and ozonated films. Semi-crystalline characteristic (presence of amorphous and crystalline zones) was observed, with peaks located at 5.6°, 16.8°, 19.2°, 21.8°, and 25.6° (2θ). The film produced with the non-modified cassava starch showed peaks at 16.8° and 19.2°, in the same regions than those observed in the study of Fama [26] and, Jaramillo [27]. Ozonated films resulted with a B-type crystalline structure [28]. The introduction of complexing agents, like glycerol, into starch preparations disrupts double helix conformations by forming stable single chain V-conformation helices [26]. During film formation, both amylose and amylopectin macromolecules are rearranged and their linear fractions form re-associations by hydrogen bonds. Therefore, the crystallinity of starch-based films depends on several factors, such as drying and storage conditions: temperature and relative humidity and also the content of plasticizer [29]. It is important to point out that the drying and storage conditions and glycerol content were similar for all the treatments in this study. On the other hand, according to Rindlav-Westling [30] crystallinity is also dependent on the ability of the chains to form crystals as well as the mobility of the chains during the crystallization process. In fact, an increase in the Relative Crystallinity (RC) was also observed due to the ozonation process. The ozonation results in the disruption of the starch polysaccharide chains resulting in a greater number of molecules with decreased sizes and weight and, thereby, is possible to result in a facility to form crystals, thus, increasing the RC.
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As no statistical difference was observed for the film thickness, their production procedure was considered adequate and their mechanical properties could be evaluated being presented in Table 1. The results obtained for the non-modified cassava film were close to those reported in the literature for native cassava starch [12,31–33] while the films obtained from ozonated starch were stronger. The tensile strength (TS) increased according to the ozone processing time, showing that starch ozonation made stronger films. An increase of ~44% in the TS was observed between the non-modified and the 30 min ozonated films, whose Young's modulus (E) was also increased. Potato starch oxidized with sodium hypochlorite [34] and banana starch oxidized with active chlorine [35] also resulted in films with increased TS and E. According to the literature, films more rigid resulted with less elongation at break (ε) [34]. However, no statistical difference between the non-modified film and the 30 min ozonated film was observed, and only the 15 min ozonated film resulted with less elongation at break, in comparison to the non-modified film – which can reinforce the unique characteristics of ozone processing. Regarding the punctuation assay, the maximum displacement of the probe (D) (Fig. 2) was observed for the non-modified film. Moreover, an increase in the punctuation force (F) was observed due to the ozonation time, confirming that native starch films were weaker than those produced with ozonated starches. The punctuation test is not as common as the uniaxial tensile assay to determine the mechanical properties of films. As observed in this study, both analyses are related and complement each other. Therefore, this assay can be used as an alternative to evaluate the mechanical properties of this type of materials. As detailed by Maniglia [36], the ozonation results in the cleavage of the glycosidic bonds of both amylose and amylopectin molecules [5,37] and the replacement of the hydroxyl groups by carbonyl and carboxyl groups [5,24,35,38]. The cleavage of the glycosidic bonds results in a greater number of molecules with decreased sizes, which may facilitate the tendency of their re-association [37]. Therefore, a new polymeric matrix with potentially more interaction between molecules is formed, which partly explain the observed behaviour.
Fig. 4. X-ray diffraction patterns of the films prepared with non-modified and ozonated cassava starch (15 and 30 min) and their respectively Relative Crystallinity (RC).
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Table 1 Mechanical properties: Tensile strength (TS), Elongation at break (ε), Young's modulus (E), Distance (D) and, Force (F). Funtional Properties: Moisture Content (MC), WVP, and Contact angle and, parameters of colour (L*, a*, and b*), colour difference (ΔE*), chroma (C) and opacity (Y) of ozone –modified cassava starch films. Property Thickness TS ε
μm MPa %
E
MPa
D F* WVP P’O2 × 10−7 MC L* a* b* ΔE* C Y
Non modified starch
15 min ozonated
30 min ozonated
0.08 ± 0.01a 2.42 ± 0.75c 43.83 ± 6.95a
0.08 ± 0.00a 3.75 ± 1.10b 37.39 ± 6.05b
0.07 ± 0.00a 5.49 ± 1.27a 39.45 ± 6.95ab 82.04 ± 26.52a 7.55 ± 1.45b 3.68 ± 1.04b 29.95 ± 1.72a 2.22 ± 0.14a
51.75 ± 25.76b mm 12.36 ± 2.48a N 1.72 ± 0.09a g·mm/day·m2·kPa 23.77 ± 2.94b cm3/m·day·Pa 1.27 ± 0.45b g H2O/g w.b.
0.16 ± 0.016a
– – – – – –
96.85 ± 0.14b 0.06 ± 0.04ab 0.16 ± 0.08b 3.15 ± 0.14b 0.19 ± 0.06a 9.32 ± 3.99b
71.18 ± 30.59ab 7.07 ± 0.53b 3.55 ± 0.95b 26.84 ± 4.56ab 2.03 ± 0.52ab
0.192 ± 0.025a 0.182 ± 0.021a 97.21 ± 0.12a 96.85 ± 0.05b 0.10 ± 0.04b 0.03 ± 0.02a −0.12 ± 0.08a 0.14 ± 0.04b 2.80 ± 0.12a 3.16 ± 0.05b 0.16 ± 0.08a 0.15 ± 0.04b 16.12 ± 3.97a 3.69 ± 1.20c
Average ± standard deviation. Same letters in the same column are not significantly different (p N 0.05). *Statistical analyses performed at p N 0.1.
Moreover, according to Zhang [38] the carbonyl groups are available to form strong hydrogen bonds with the hydroxyl groups on starch, resulting in films more rigid with reduced elongation. On the other hand, the formed carboxylic groups are known to be electronegatively charged and, thus, they cause an electrostatic repulsion between the molecules, making difficult their association [37]. Even so, the resulted network seems to be stronger, which means that the ozonation is a complex process and, the results obtained are the balance between both molecular size and charge, with different tendency of the molecules to re-association.
Herein, ozone processing improved the strength of cassava starch films, resulting in films with higher TS, E, F and, minimum D (Fig. 2) and lower ε which was evident by both assays (the tensile and punctuation). Moreover, according to Rindlav-Westling [30], films with increased TS and E and decreased ε properties also presents increased RC – thus confirming the results obtained herein. It is worth mention this result is very interesting in different applications of packages, as well as for the confection of plastic bags. Table 1 shows the results for Water Vapour Permeation (WVP) and Oxygen Permeation (P’O2). Regarding the films made with native cassava starch, the values obtained for P’O2 were similar to the reported by Llanos [32], while the WVP was in the same order of magnitude of those reported by Souza [12]. As shown in Table 1, an increase in the WVP and the P’O2 due to the ozonation time was observed. An increasing in both WVP (26%) and P’O 2 (75%) was observed for the 30 min ozonated film in comparison to the nonmodified cassava film. The functional properties of ozonated cassava films, expressed in terms of contact angle, solubility and moisture content, are presented in Fig. 5 and Table 1. Similar values are reported in the literature for non-modified cassava film, regarding the contact angle [22,31,39], the solubility [39] and the moisture content [33,39]. As shown in Fig. 5, ozonation time decreased the solubility and the water contact angle. Therefore, films less soluble, but with a more hydrophilic surface, were achieved. No statistical difference was observed for the moisture content (Table 1). The permeability and water absorption of edible films depends on several factors, such as their thickness and the glycerol content [40], besides of the starch molecules. As both thickness and the glycerol content were similar for all the treatments, the changes on barrier and functional properties can be related with the starch molecular changes due to ozonation process – and the consequent new polymeric matrix. The complex behaviour of ozonation here observed is highlighted again. The molecular charge induced by the presence of carboxyl groups results in distance among molecules, increasing the network permeability by water vapour or oxygen [23,24]. On the other hand, the smaller
Fig. 5. Functional Properties: Solubility (S) and Contact angle of ozone –modified cassava starch films. Average ± standard deviation. Same letters in the same column are not significantly different (p N 0.05).
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molecules (also with a different distribution) rearrangement makes a different network, whose connections are stronger (maybe due to the interaction between carbonyl and hydroxyl groups), as revealed in the mechanical properties. The electrostatic repulsion in general increases the solubility of starch [5,37] which is contrary to the results observed in this study for the films. A possible explanation could be that, once the molecules reassociate between them, it might reduce the ability of these molecules to linkage with water molecules of the surrounding, thus, reducing the solubility. However, the location of polar groups in the film surface can favour bonds with the first drop of water deposited onto the film, increasing its hydrophilicity. Both results are not contradictory, since this last essay (solubility) is performed for 24 h with the whole film immersed into water and not only the surface. Hence, the ozonation technology resulted in cassava films with increased strengthens, more permeable to vapour water and oxygen, less soluble, which could be an interesting or limiting result, depending on the films practical application. A possible application can be the coating of surfaces/products, in which, degradation reaction due to the water or oxygen diffusion are not important. The film colour parameters (L*, a*, b* and ΔE*), Chroma (C) and Opacity (Y) are presented in Table 1. In general, the films presented good transparency. For native cassava films, similar values of L*, a*, b* and opacity were already reported [39,41,42]. The ozonated starch films presented higher opacity (15 min of processing) or lower (30 min of processing) than the native cassava film. Once again, the different charge and size distribution of starch molecules must be highlighted, which result in the observed complex behaviour. The film opacity is an important property for packaging and coating applications. Through the results obtained herein, starch ozonation can produce films with both higher or smaller opacity. Ozonation of cassava starch for 30 min can be an interesting alternative to enhance its film transparency, which is, in general, the most desired result. 4. Final remarks The ozonation process results in the replacement of the hydroxyl groups by carbonyl and carboxyl groups, as well as the cleavage of the glycosidic bonds, which results in the depolymerisation of parts of the amylose and amylopectin molecules. Consequently, a polymeric matrix with different charge distribution was achieved. The cleavage of the glycosidic bonds results in a greater number of molecules with decreased sizes, which may facilitate the tendency of their re-association. Moreover, the carbonyl groups formed strong hydrogen bonds with the hydroxyl groups on starch. Thus, resulting in the increase of the tensile strength. On the other hand, the formed electronegative carboxyl groups promote repulsive forces among the polymer chains, resulting in an increased inter chain spacing. Thus, the permeability was increased. Therefore, a more open, but stronger polymeric matrix, was formed. The new charge distribution, might favour somehow that some polar groups migrate to the surface of the film favouring the linkage with the first drop of water, thus increasing film hydrophilicity. The results obtained in this work expand the applications of starch to produce bioplastics. 5. Conclusions This work produced and evaluated films based on ozonated cassava starch. Ozonation resulted in films enhanced mechanical properties, with increased tensile strength and Young's modulus, although decreased elongation and deformation during punctuation. This processes also increased the water vapour permeation and the oxygen permeation, also resulting in films with a more hydrophilic surface and less soluble. In general, the films presented good transparency. The
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ozonated films presented higher (15 min of processing) or lower (30 min of processing) opacity than the native cassava film, highlighting ozonation as a versatile alternative for this purpose. Finally, the morphology of the ozonated films was more homogeneous, with increased relative crystallinity. In conclusion, the ozone processing showed to be a great alternative for packaging production.
Acknowledgements The authors are grateful to the São Paulo Research Foundation (FAPESP, Brazil), for funding the project n° 2016/18052-5, the postdoctoral fellowship of CIA La Fuente (2017/05307-8) and the B.Sc. scholarship of Andressa Tamyris de Souza (2018/24291-8); and the National Council for Scientific and Technological Development (CNPq, Brazil) for funding the project of CIA La Fuente (429043/2018-0) and the productivity grants of PED Augusto (306557/2017-7) and CC Tadini (306414/2017-1).
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Ozonation of cassava starch to produce biodegradable films☆ Carla I.A. La Fuente a,⁎, Andressa Tamyris de Souza b, Carmen C. Tadini b,c,d, Pedro Esteves Duarte Augusto a,d a
Department of Agri-food Industry, Food and Nutrition (LAN), Luiz de Queiroz College of Agriculture (ESALQ), University of São Paulo (USP), Piracicaba, SP, Brazil University of São Paulo, Escola Politécnica, Department of Chemical Engineering, Main Campus, 05508-010, SP, Brazil c University of São Paulo, Food Research Center (FoRC/NAPAN), SP, Brazil d Food and Nutrition Research Center (NAPAN), University of São Paulo (USP), São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 15 August 2019 Received in revised form 29 August 2019 Accepted 4 September 2019 Available online 05 September 2019 Keywords: Biodegradable films Cassava Starch Ozonation
a b s t r a c t In this study, biodegradable films were produced from cassava starch modified by ozone at different levels. The films were produced by casting technique using native and ozonated cassava starch, glycerol as the plasticizer, and water as the solvent. Films were characterized in term of their mechanical, barrier and functional properties, morphology, crystallinity, colour, and opacity. The morphology of the ozonated films was more homogeneous in comparison to the films produced with the non-modified starch and enhanced properties were achieved. Films produced with ozonated cassava starch presented higher tensile strength, Young's modulus and lower elongation. The water vapour permeation and the oxygen permeation were increased by increasing the ozonation time. Moreover, ozone processing resulted in films with a more hydrophilic surface and lower solubility after 24 h. Possible explanations and applications were discussed. In conclusion, the ozone processing showed to be a good alternative for starch based packaging production. © 2019 Elsevier B.V. All rights reserved.
1. Introduction While hundreds of millions of tons of petroleum-based plastics are produced annually all over the world, the concern about their environmental impact is increasing each day. Consequently, different renewable and biodegradable source are being studied to produce biodegradable plastics. Starch is one of the promising biopolymeric matrix for the development of biodegradable packaging [1]. Starch is one of the most important and abundant polysaccharides in nature [2,3]. They are obtained from renewable sources, presenting relatively low cost and a wide possibility of application. Consequently, starch is a very versatile material, being used in several industries such as the food, paper, textile, chemical and pharmaceutical [2]. Being the native sources limited by nature, different starch modification techniques are available in order to improve the processability, mechanical and/or barrier properties of biodegradable films. Among different possibilities of starch modification, ozone processing has gaining attention due to the demand for methods that meet safety and health standards for both consumers and the environment [4]. Ozone is a powerful oxidizing agent, which reacts with starch, increasing its carboxyl and carbonyl contents, and also reducing its molecular size and distribution [5]. Consequently, different properties are ☆ A patent related with this work was filed (BR1020190112166). ⁎ Corresponding author at: Avenida Pádua Dias, 11, Piracicaba, SP 13418-900, Brazil. E-mail address: [email protected] (C.I.A. La Fuente).
https://doi.org/10.1016/j.ijbiomac.2019.09.028 0141-8130/© 2019 Elsevier B.V. All rights reserved.
achieved due to complex relation among molecular modifications on size, charge and chemical affinity. In fact, different starch sources were already modified by ozonation, such as corn, sago, wheat, potato and tapioca/cassava [5–10]. However, different starch sources have different molecular and granule structure, being differently affected by ozonation. For instance, cassava starch had both polygonal and spherical shaped granules with size b50 μm, while potato starch contained both large and small granules, which had spherical to oval shapes [11]. Therefore, different ozonated starches present different molecular sizes and electrical charges, which affect their behaviour for reassociation. These molecular changes are competitive in relation to film production and properties. Consequently, the properties of films produced with ozone modified starches are unpredictable and, dependent on the relations among the source, reactor and processing conditions. Hence, the aim of this study was to produce films from ozonemodified cassava starch, considering different process condition and evaluating the most important mechanical, barrier and functional properties, as well as their morphology, crystallinity, colour, and opacity.
2. Material and methods This work was developed in three parts, as summarized on Fig. 1. Firstly, cassava starch was ozonated, in two conditions. Then, films based on the starches (native and the two ozonated conditions) were
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Nomenclature a⁎ A b⁎ C E E* L⁎ m OTR p P’O2 RC S t TS WVP x Y
redness/greenness CIELab parameter (−) area (m2) yellowness/blueness CIELab parameter (−) chroma (−) Young's Modulus (MPa) CIELab total color change (−) brightness/darkness CIELab parameter (−) mass (g) oxygen transmission rate (cm3·m−2·d−1) pressure (kPa) oxygen permeability (cm3/m·day·Pa) relative crystallinity solubility (%) time (h) Tensile Strength (MPa) Water Vapour Permeability (g·mm/m2·day·kPa) thickness (mm) opacity (%)
Subscripts 0 initial b black standard f final w white standard Greek Δ ε
dry basis; 1500 mL) and then ozonated for different periods under constant stirring in the same system reported by Castanha [5]. The ozone rich stream was produced from industrial oxygen by the coronal discharge method using an ozone generator unit (Ozone & Life, O&G Model RM 3.0, São Jose dos Campos, Brazil). This stream was dispersed in the sample, which was placed in a glass reactor, as small bubbles through a gas disperser. A gas flow of 1 L∙min−1 was used, with an ozone concentration in the gas current of 41 mg O3∙L−1. The amount of ozone was measured using an ozone monitor (2B Technologies Model 106-H, USA). After passing through the samples, the gas stream was led out of the reactor and converted to oxygen in a thermal ozone destroyer (Ozone & Life, São José dos Campos, Brazil). The procedures were performed at 25 ± 1 °C. Fig. 1 presents a schematic representation of the ozone processing system. Three samples were evaluated: the non-modified starch and those ozonated for 15 and 30 min. After processing, the excess of water was removed through decantation and centrifugation, and the starch samples were dried in an air circulation oven for ~ 12 h at 35 °C until moisture content of ~12 g/100 g w. b. Glycerol P.A. grade (Sigma-Aldrich, Brazil) and distilled water were also used for film production. 2.2. Film preparation
delta elongation at break (%)
formed. Finally, the obtained films were evaluated through their structure and the most important properties. 2.1. Materials The native cassava starch (Amilogill 1500) was donated by Cargill Company. This starch was suspended in distilled water (10 g/100 g in
The films were produced by casting technique, as shown in Fig. 1. A solution containing 5 g/100 g of cassava starch was homogenized in distilled water with a magnetic stirrer (GEHAKA, HU-30/2, Brazil) for 10 min. Then, the solution was heated for 45 min in a reactor with external recirculation of water at 75 °C. Glycerol was added as the plasticizer (25 g/100 g of starch) according to Souza [12] and, the suspension was heated for more 15 min under agitation. To eliminate the bubbles, the filmogenic solution was placed in an ultrasonic bath for 30 min at 70 °C (154 W, 25 kHz; UNIQUE, model USC-1850, Brazil). The solution was then poured into acrylic Petri dishes (0.15 g/cm2) for ~12 h to rest, and then dried at 35 °C and 45% HR for ~10 h in a climatic chamber (TIRACLIMA, model TCC 7034, Germany). The films were conditioned for at least 48 h in desiccators containing a saturated solution of NaCl (75% RH) before characterization at room temperature (~25 °C).
Fig. 1. Schematic representation of the ozone processing system and the film elaboration.
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2.3. Films characterization 2.3.1. Mechanical properties The film thickness was determined using a digital micrometer with a precision of 0.001 mm (MITUTOYO, Japan) at five random positions. The mechanical properties were evaluated through a uniaxial tensile assay, according to ASTM D882-09 [13]. Tensile strength (TS) [MPa] and percent elongation at break (ε) [%] were evaluated on a texture analyzer (TAXT2i e Stable Micro Systems, UK) with a load cell of 30 kgf (294 N), using the A/TGT self-tightening roller grips fixture. The initial grip separation and the crosshead speed were set at 50 mm and 1 mm·s−1, respectively. Young's modulus (E) was calculated as the slope of the initial linear portion of the stress versus strain curve. At least ten strips (100 mm × 25 mm) were evaluated for the non-modified and modified films. Furthermore, the force and the deformation at the breaking point of the film was determined in a punctuation test [14], on a texture analyzer (TAXT2i e Stable Micro Systems, UK) with a load cell of 30 kgf (294 N). The films were fixed in a circular permeation cells (d = 0.05 m) and perforated by a 3 mm diameter probe. The puncture force (F) and the displacement of the probe (D) were obtained from forcedeformation curves, according to Fig. 2. 2.3.2. Barrier properties Water vapour permeability (WVP) of films was measured gravimetrically according to the ASTM E96-80 [15]. The films were placed in circular permeation cells containing silica gel, and then placed in a climatic chamber (TIRACLIMA, model TCC 7034, Germany) maintained at 75% RH and temperature of 25 °C. The cell weight was recorded hourly for 8 h. Water vapour permeability was calculated according to Eq. (1).
WVP ¼
Δm x 24 Δt A Δp
ð1Þ
wherein: Δm/Δt is the moisture gain per unit of time (g·h−1); x is the film thickness (mm); A is the film area exposed to permeation (1.96·10−3 m2); Δp is the vapour pressure difference (kPa) of the atmosphere over silica gel and pure water (3.168 kPa at 25 °C).
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The oxygen permeability coefficient (P’O2) of the ozonated films was measured at 23 °C and 75% RH on a 50 cm2 circular films using an oxygen permeation system (OXTRAN 2/21, Mocon, USA), according with ASTM F1927-14 [16]. The oxygen permeability reported is the mean of four films. The oxygen permeability coefficient was calculated according to Eq. (2). P’O2 ¼
OTR X p
ð2Þ
wherein: P’O2 is the oxygen permeability coefficient (cm3·m−1·d−1·Pa−1); OTR is the oxygen transmission rate (cm3·m−2·d−1); p is the partial pressure of oxygen, and X is the average thickness of the specimen (m). 2.3.3. Functional properties The film wettability was determined by the measurement of the contact angle with water, according to ASTM D7334 − 08 [17] using an OCA15- Dataphysiscs (OCA 15, Dataphysiscs, Germany). Two angle measurements were carried out (one on each drop edge) quickly to avoid changes due to water vaporization on each drop. The contact angle was defined as the average of six angles measures. The film solubility in water was evaluated with discs (diameter of 20 mm) immersed in 50 mL of water at 25 °C for 24 h, under mechanical stirring at 120 RPM, according to Gontard [18] The solubility in water was calculated as the percentage of dry matter of the solubilized film, as described in Eq. (3). S¼
m f −mo 100 mo
ð3Þ
wherein: mo and mf are the initial and the final discs' mass (g), respectively. The films moisture was determined through the gravimetric method in an oven, without recirculation, at 105 °C until a constant mass (~24 h), according to Gontard [18]. 2.3.4. Colour and opacity The film colour was determined using a colorimeter (HUNTERLAB, model ColorQuest XE, USA) with the CIELab scale (L⁎, a⁎, b⁎). The colour measurements were expressed in terms of L* (L* = 100 brightness and
Fig. 2. Diagram of the system used to determine the punctuation deformation. Adapted from Sobral [14].
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Fig. 3. Scanning electron micrographs of the surfaces and cross-sections of the films prepared with non-modified and ozonated cassava starch. Surface images at and, the cross-sections images at and, respectively. The surface and the cross-section images were obtained at 1000× and 3100×, respectively.
L* = 0 darkness); a* (+a* = redness and −a* = greenness) and b* (+b* = yellowness and −b* = blueness). The change of each of the parameters was calculated according to Eqs. (4), (5) and (6). ΔL ¼ L −L0
ð4Þ
−a0
ð5Þ
ð6Þ
Δa ¼ a
Δb ¼ b −b0
wherein: L0* = 94.04; a0* = −0.86 and b0* = 1.53 determined from a white standard pattern. The total colour change (ΔE⁎), the opacity (Y) and the Chroma (C) [19,20] were calculated using Eqs. (7), (8) and (9) respectively. h i1 2 2 ΔE ¼ ðΔL Þ þ ðΔa Þ2 þ ðΔb Þ Y¼
2
ð7Þ
2.3.6. Morphology The surface and cross-section morphology of the films were visualized by a scanning electron microscope (SEM). The films were dried in silica gel for 48 h to ensure an accurate and finer cut. They were then horizontally and vertically placed on an adhesive tape placed over circular stubs. The films were then coated with a 10-nm platinum in a high-vacuum coating system (BALTEC, model MED020, Switzerland) and the side which was not in contact with the Petri dish was observed using the microscope (FEI, model Quanta 600FEG, Netherlands) with an acceleration voltage of 15 kV and magnification ranged from 500× to 3100×. 2.4. Experimental design and statistical analyses
Yb Yw
1 2 C ¼ a2 þ b
The scanning rate was 0.02°/s with 2ϴ angles varying between 2° to 40°. The software Origin 2018b was employed for calculation (OriginLab Corporation, Massachusetts, USA).
ð8Þ 2
ð9Þ
wherein: Yb is the opacity on the black standard and, Yw is the opacity on the white standard. Three measurements were performed per film formulation to determine all the colour parameters. 2.3.5. Crystallinity The relative crystallinity of the films was calculated as the ratio between the crystalline area and the total area of the X-ray diffractogram (plotted from 2° to 40°), as described by Nara & Komiya [21]. An X-ray diffractometer (PANalytical, model X'Pert PRO, Netherlands), with detector X'Celerator operating at 45 mA, and voltage of 40 kV was used.
A completely randomized design was applied in three replicates. The obtained data was evaluated through analysis of variance (ANOVA), applying Tukey's test as a post-hoc test through Statgraphics Centurion XV software (StatPoint, Inc., USA). 3. Results and discussion Fig. 3 presents the morphology of the films. In general, the ozonated films present a compact structure and smooth surfaces, in comparison to the non-modified starch films. Heterogeneity and roughness were already observed on the surface of non-modified cassava films [22]. For the ozonated films, smoother surface is clearly evident in the surface of the films, while, in the cross-section, some cracks are visible, probably appeared after the film was broken. In the study performed by Biduski
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[23] although oxidized sorghum films were more homogeneous, similar fissures were also observed. According to the literature, films produced with oxidized starches allow a higher penetration of the plasticizer molecules in the starch chains, and improve the interaction between them [23–25]. Moreover, low retrogradation is expected in oxidized starches [24], promoted by the repulsion between molecules, increasing the free space between the molecules, and also increasing the homogeneity of the film. However, ozonation is more than only an oxidation process and this process also changes the molecular charges and chemical affinity, which affect the interaction among the molecules and altering the morphology of the films. Fig. 4 shows the X-ray diffractograms for the non-modified and ozonated films. Semi-crystalline characteristic (presence of amorphous and crystalline zones) was observed, with peaks located at 5.6°, 16.8°, 19.2°, 21.8°, and 25.6° (2θ). The film produced with the non-modified cassava starch showed peaks at 16.8° and 19.2°, in the same regions than those observed in the study of Fama [26] and, Jaramillo [27]. Ozonated films resulted with a B-type crystalline structure [28]. The introduction of complexing agents, like glycerol, into starch preparations disrupts double helix conformations by forming stable single chain V-conformation helices [26]. During film formation, both amylose and amylopectin macromolecules are rearranged and their linear fractions form re-associations by hydrogen bonds. Therefore, the crystallinity of starch-based films depends on several factors, such as drying and storage conditions: temperature and relative humidity and also the content of plasticizer [29]. It is important to point out that the drying and storage conditions and glycerol content were similar for all the treatments in this study. On the other hand, according to Rindlav-Westling [30] crystallinity is also dependent on the ability of the chains to form crystals as well as the mobility of the chains during the crystallization process. In fact, an increase in the Relative Crystallinity (RC) was also observed due to the ozonation process. The ozonation results in the disruption of the starch polysaccharide chains resulting in a greater number of molecules with decreased sizes and weight and, thereby, is possible to result in a facility to form crystals, thus, increasing the RC.
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As no statistical difference was observed for the film thickness, their production procedure was considered adequate and their mechanical properties could be evaluated being presented in Table 1. The results obtained for the non-modified cassava film were close to those reported in the literature for native cassava starch [12,31–33] while the films obtained from ozonated starch were stronger. The tensile strength (TS) increased according to the ozone processing time, showing that starch ozonation made stronger films. An increase of ~44% in the TS was observed between the non-modified and the 30 min ozonated films, whose Young's modulus (E) was also increased. Potato starch oxidized with sodium hypochlorite [34] and banana starch oxidized with active chlorine [35] also resulted in films with increased TS and E. According to the literature, films more rigid resulted with less elongation at break (ε) [34]. However, no statistical difference between the non-modified film and the 30 min ozonated film was observed, and only the 15 min ozonated film resulted with less elongation at break, in comparison to the non-modified film – which can reinforce the unique characteristics of ozone processing. Regarding the punctuation assay, the maximum displacement of the probe (D) (Fig. 2) was observed for the non-modified film. Moreover, an increase in the punctuation force (F) was observed due to the ozonation time, confirming that native starch films were weaker than those produced with ozonated starches. The punctuation test is not as common as the uniaxial tensile assay to determine the mechanical properties of films. As observed in this study, both analyses are related and complement each other. Therefore, this assay can be used as an alternative to evaluate the mechanical properties of this type of materials. As detailed by Maniglia [36], the ozonation results in the cleavage of the glycosidic bonds of both amylose and amylopectin molecules [5,37] and the replacement of the hydroxyl groups by carbonyl and carboxyl groups [5,24,35,38]. The cleavage of the glycosidic bonds results in a greater number of molecules with decreased sizes, which may facilitate the tendency of their re-association [37]. Therefore, a new polymeric matrix with potentially more interaction between molecules is formed, which partly explain the observed behaviour.
Fig. 4. X-ray diffraction patterns of the films prepared with non-modified and ozonated cassava starch (15 and 30 min) and their respectively Relative Crystallinity (RC).
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Table 1 Mechanical properties: Tensile strength (TS), Elongation at break (ε), Young's modulus (E), Distance (D) and, Force (F). Funtional Properties: Moisture Content (MC), WVP, and Contact angle and, parameters of colour (L*, a*, and b*), colour difference (ΔE*), chroma (C) and opacity (Y) of ozone –modified cassava starch films. Property Thickness TS ε
μm MPa %
E
MPa
D F* WVP P’O2 × 10−7 MC L* a* b* ΔE* C Y
Non modified starch
15 min ozonated
30 min ozonated
0.08 ± 0.01a 2.42 ± 0.75c 43.83 ± 6.95a
0.08 ± 0.00a 3.75 ± 1.10b 37.39 ± 6.05b
0.07 ± 0.00a 5.49 ± 1.27a 39.45 ± 6.95ab 82.04 ± 26.52a 7.55 ± 1.45b 3.68 ± 1.04b 29.95 ± 1.72a 2.22 ± 0.14a
51.75 ± 25.76b mm 12.36 ± 2.48a N 1.72 ± 0.09a g·mm/day·m2·kPa 23.77 ± 2.94b cm3/m·day·Pa 1.27 ± 0.45b g H2O/g w.b.
0.16 ± 0.016a
– – – – – –
96.85 ± 0.14b 0.06 ± 0.04ab 0.16 ± 0.08b 3.15 ± 0.14b 0.19 ± 0.06a 9.32 ± 3.99b
71.18 ± 30.59ab 7.07 ± 0.53b 3.55 ± 0.95b 26.84 ± 4.56ab 2.03 ± 0.52ab
0.192 ± 0.025a 0.182 ± 0.021a 97.21 ± 0.12a 96.85 ± 0.05b 0.10 ± 0.04b 0.03 ± 0.02a −0.12 ± 0.08a 0.14 ± 0.04b 2.80 ± 0.12a 3.16 ± 0.05b 0.16 ± 0.08a 0.15 ± 0.04b 16.12 ± 3.97a 3.69 ± 1.20c
Average ± standard deviation. Same letters in the same column are not significantly different (p N 0.05). *Statistical analyses performed at p N 0.1.
Moreover, according to Zhang [38] the carbonyl groups are available to form strong hydrogen bonds with the hydroxyl groups on starch, resulting in films more rigid with reduced elongation. On the other hand, the formed carboxylic groups are known to be electronegatively charged and, thus, they cause an electrostatic repulsion between the molecules, making difficult their association [37]. Even so, the resulted network seems to be stronger, which means that the ozonation is a complex process and, the results obtained are the balance between both molecular size and charge, with different tendency of the molecules to re-association.
Herein, ozone processing improved the strength of cassava starch films, resulting in films with higher TS, E, F and, minimum D (Fig. 2) and lower ε which was evident by both assays (the tensile and punctuation). Moreover, according to Rindlav-Westling [30], films with increased TS and E and decreased ε properties also presents increased RC – thus confirming the results obtained herein. It is worth mention this result is very interesting in different applications of packages, as well as for the confection of plastic bags. Table 1 shows the results for Water Vapour Permeation (WVP) and Oxygen Permeation (P’O2). Regarding the films made with native cassava starch, the values obtained for P’O2 were similar to the reported by Llanos [32], while the WVP was in the same order of magnitude of those reported by Souza [12]. As shown in Table 1, an increase in the WVP and the P’O2 due to the ozonation time was observed. An increasing in both WVP (26%) and P’O 2 (75%) was observed for the 30 min ozonated film in comparison to the nonmodified cassava film. The functional properties of ozonated cassava films, expressed in terms of contact angle, solubility and moisture content, are presented in Fig. 5 and Table 1. Similar values are reported in the literature for non-modified cassava film, regarding the contact angle [22,31,39], the solubility [39] and the moisture content [33,39]. As shown in Fig. 5, ozonation time decreased the solubility and the water contact angle. Therefore, films less soluble, but with a more hydrophilic surface, were achieved. No statistical difference was observed for the moisture content (Table 1). The permeability and water absorption of edible films depends on several factors, such as their thickness and the glycerol content [40], besides of the starch molecules. As both thickness and the glycerol content were similar for all the treatments, the changes on barrier and functional properties can be related with the starch molecular changes due to ozonation process – and the consequent new polymeric matrix. The complex behaviour of ozonation here observed is highlighted again. The molecular charge induced by the presence of carboxyl groups results in distance among molecules, increasing the network permeability by water vapour or oxygen [23,24]. On the other hand, the smaller
Fig. 5. Functional Properties: Solubility (S) and Contact angle of ozone –modified cassava starch films. Average ± standard deviation. Same letters in the same column are not significantly different (p N 0.05).
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molecules (also with a different distribution) rearrangement makes a different network, whose connections are stronger (maybe due to the interaction between carbonyl and hydroxyl groups), as revealed in the mechanical properties. The electrostatic repulsion in general increases the solubility of starch [5,37] which is contrary to the results observed in this study for the films. A possible explanation could be that, once the molecules reassociate between them, it might reduce the ability of these molecules to linkage with water molecules of the surrounding, thus, reducing the solubility. However, the location of polar groups in the film surface can favour bonds with the first drop of water deposited onto the film, increasing its hydrophilicity. Both results are not contradictory, since this last essay (solubility) is performed for 24 h with the whole film immersed into water and not only the surface. Hence, the ozonation technology resulted in cassava films with increased strengthens, more permeable to vapour water and oxygen, less soluble, which could be an interesting or limiting result, depending on the films practical application. A possible application can be the coating of surfaces/products, in which, degradation reaction due to the water or oxygen diffusion are not important. The film colour parameters (L*, a*, b* and ΔE*), Chroma (C) and Opacity (Y) are presented in Table 1. In general, the films presented good transparency. For native cassava films, similar values of L*, a*, b* and opacity were already reported [39,41,42]. The ozonated starch films presented higher opacity (15 min of processing) or lower (30 min of processing) than the native cassava film. Once again, the different charge and size distribution of starch molecules must be highlighted, which result in the observed complex behaviour. The film opacity is an important property for packaging and coating applications. Through the results obtained herein, starch ozonation can produce films with both higher or smaller opacity. Ozonation of cassava starch for 30 min can be an interesting alternative to enhance its film transparency, which is, in general, the most desired result. 4. Final remarks The ozonation process results in the replacement of the hydroxyl groups by carbonyl and carboxyl groups, as well as the cleavage of the glycosidic bonds, which results in the depolymerisation of parts of the amylose and amylopectin molecules. Consequently, a polymeric matrix with different charge distribution was achieved. The cleavage of the glycosidic bonds results in a greater number of molecules with decreased sizes, which may facilitate the tendency of their re-association. Moreover, the carbonyl groups formed strong hydrogen bonds with the hydroxyl groups on starch. Thus, resulting in the increase of the tensile strength. On the other hand, the formed electronegative carboxyl groups promote repulsive forces among the polymer chains, resulting in an increased inter chain spacing. Thus, the permeability was increased. Therefore, a more open, but stronger polymeric matrix, was formed. The new charge distribution, might favour somehow that some polar groups migrate to the surface of the film favouring the linkage with the first drop of water, thus increasing film hydrophilicity. The results obtained in this work expand the applications of starch to produce bioplastics. 5. Conclusions This work produced and evaluated films based on ozonated cassava starch. Ozonation resulted in films enhanced mechanical properties, with increased tensile strength and Young's modulus, although decreased elongation and deformation during punctuation. This processes also increased the water vapour permeation and the oxygen permeation, also resulting in films with a more hydrophilic surface and less soluble. In general, the films presented good transparency. The
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ozonated films presented higher (15 min of processing) or lower (30 min of processing) opacity than the native cassava film, highlighting ozonation as a versatile alternative for this purpose. Finally, the morphology of the ozonated films was more homogeneous, with increased relative crystallinity. In conclusion, the ozone processing showed to be a great alternative for packaging production.
Acknowledgements The authors are grateful to the São Paulo Research Foundation (FAPESP, Brazil), for funding the project n° 2016/18052-5, the postdoctoral fellowship of CIA La Fuente (2017/05307-8) and the B.Sc. scholarship of Andressa Tamyris de Souza (2018/24291-8); and the National Council for Scientific and Technological Development (CNPq, Brazil) for funding the project of CIA La Fuente (429043/2018-0) and the productivity grants of PED Augusto (306557/2017-7) and CC Tadini (306414/2017-1).
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