Rice starch active packaging films loaded with ... - Wiley Online Library

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DOI 10.1002/star.201400193

Starch/Stärke 2015, 67, 294–302

RESEARCH ARTICLE

Rice starch active packaging films loaded with antioxidants—development and characterization Bilal Ahmad Ashwar 1, Asima Shah 1, Adil Gani 1, Umar Shah 1, Asir Gani 2, Idrees Ahmed Wani 1, Sajad Mohd Wani 1 and Farooq Ahmad Masoodi 1 1 2

Department of Food Science and Technology, University of Kashmir, Srinagar, India Department of Food Technology, Guru Nanak Dev University, Amritsar, Punjab, India

Active antioxidant food packaging films were developed by the incorporation of ascorbic acid Received: October 2, 2014 (AA) and butylated hydroxytoluene (BHT) into a rice starch–glycerol matrix. BHT significantly Revised: November 11, 2014 improved the water resistance of starch films. Both AA and BHTpromoted significant increase in Accepted: November 11, 2014 the elastic modulus but a decrease in film stretchability. S/G/BHT film presented significant decrease in water vapour permeability. AA and BHT significantly affected the lightness (L) and yellow (b) color of the films with minor differences in the green (a) colour. SEM revealed smooth surface of the films. Thermal analysis showed increase in glass transition temperature and enthalpy of transition of films with the incorporation of AA and BHT. Exposure of the films to various food simulants showed that the release from the films was dependent on the type of food simulant and the antioxidant. In the aqueous food simulant, films containing ascorbic acid (S/G/ AA) produced the largest release and in the fatty food stimulant S/G/BHT film presented fast release. Keywords: Antioxidant release / Starch film / Tensile properties / Thermal analysis / WVP

1

Introduction

Due to the severe environmental pollution caused by plastic food packaging, there has been a growing amount of interest in the production of edible and biodegradable films [1]. Such films may have the ability of decreasing the amounts of non-renewable conventional synthetic polymer packaging materials, and use ingredients of agricultural derived products [2]. Edible films can be prepared from protein, polysaccharides, lipids or the combination of these components [3, 4]. Starch is the natural polysaccharide and it is described as a renewable resource, inexpensive and widely available [5]. Starch-based films exhibit appropriate physical characteristics, since these films are Correspondence: Dr. Adil Gani, Department of Food Science and Technology, University of Kashmir, Srinagar, India E-mail: [email protected] Fax: þ91-194-2425195 Abbreviations: AA, ascorbic acid; BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; SEM, scanning electron microscopy; S/G, starch/glycerol; S/G/AA, starch/glycerol/ ascorbic acid; S/G/BHT, starch/glycerol/butylated hydroxytoluene; Tg, glass transition temperature; WVP, water vapour permeability; DH, enthalpy of transition

ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

isotropic, odourless, non-toxic, biodegradable, tasteless, colourless and constitute a good barrier against oxygen transfer [1]. However starch films are brittle and addition of certain components like glycerol improves their handling properties. Glycerol imparts flexibility and is a widely used plasticizer for making starch-based films and coatings [6, 7]. These films may operate as carriers of many functional ingredients. Such ingredients may include antioxidants, antimicrobial agents, flavours, spices and colourants which improve the functionality of the packaging materials by adding novel or extra functions [8]. Application of these systems to food packaging has increased since they enable controlled release of active compounds such as antioxidants and antimicrobials from the packaging system at an appropriate rate during the storage of products, allowing protection and extension of the product’s shelf life. In this connection, several groups have reported the release of purposefully added antioxidants from packaging films. Wessling et al. [9] reported the release of butylated hydroxytoluene (BHT) and tocopherol from polyethylene films into fatty food stimulants. Granda-Restrepo et al. [10] developed polyethylene films with a-tocopherol and measured its release into milk powder. Gemili et al. [11] studied the release of ascorbic acid (AA) and L-tyrosine from a cellulose acetate-based film. Tea leaf www.starch-journal.com

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extract loaded rice starch coating delayed ripening effects on tomatoes during 20 days storage due to controlled release of bioactive components from starch coating [12]. The present study was focused on the development of films based on rice (Oryza sativa) starch with antioxidants AA and BHT, in order to develop an antioxidant active packaging system. To our knowledge the effects of AA and BHT antioxidants on physical, thermal, mechanical and barrier properties of starch film in one hand for its modification and in the other hand for providing an active antioxidant-based starch film have not been investigated before, so the aim of this work was to study the addition effects of antioxidants on these properties. Moreover, the antioxidant capacity of films in three different food simulants (aqueous solutions containing 0, 10 and 95% ethanol) was also evaluated by analysing 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability. The research is expected to open new ways for functional packaging development which is emerging area in the packaging science.

2

Materials and methods

2.1 Materials Rice was procured from Sher-e-Kashmir University of Agricultural Sciences and Technology, Shalimar, Srinagar, India. BHT, AA and glycerol were purchased from HiMedia. DPPH was obtained from Sigma–Aldrich. All chemicals were of analytical grade. 2.2 Starch extraction Alkaline steeping method was used for extraction of starch [13]. The rice was soaked in distilled water for 4 h and the softened rice grains were drained and soaked with 0.3% NaOH solution for 1 h. The grains were blended with alkaline solution for 5 min. The rice slurry was passed through screens. The starch suspension was allowed to stand until the crude starch precipitated. The supernatant was drained off and the sediment remaining was washed to remove the protein using 0.2% NaOH, until the yellow layer disappeared. The starch residue was then washed with distilled water, neutralized by 1 N HCl, again washed with distilled water and centrifuged at 10 000 g for 10 min. Purified rice starch was dried at room temperature, finely powdered and stored in a desiccator until used. 2.3 Proximate analysis The extracted starch was analysed for Moisture (925.10), protein (920.87), fat (920.85) and ash (923.03) according to the methods of AOAC [14]. Apparent amylose content of the starch was determined using a colorimetric method [15]. Starch sample (20 mg) was ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

taken, 10 mL of 0.5 M KOH was added and the suspension was mixed thoroughly. The dispersed sample was transferred to a 100 mL volumetric flask and the volume was made up to the mark with distilled water. An aliquot of the test starch solution (10 mL) was pipetted into a 50 mL volumetric flask and 5 mL of 0.1 M aqueous HCl was added followed by 0.5 mL of iodine reagent. The volume was diluted to 50 mL and allowed to stand for 5 min. The absorbance was measured at 625 nm (UV Spectrophotometer, U-2900, Hitachi, Tokyo, Japan). The content of amylose was determined from a standard curve developed using standard amylose and amylopectin blends from potato starch. 2.4 Film preparation Starch films, containing AA or BHT, were obtained by the casting technique. Three grams rice starch on dry weight basis (db) was mixed with 100 mL distilled water and heated at 95°C for 30 min on a heating plate while stirring with a magnetic stirrer, followed by addition of 0.6 g glycerol (20% w/w on starch dry basis) as plasticizer and further heating and mixing for 10 min. Then 0.15 g of AA or BHT (5% w/w on starch dry basis) was incorporated during the last 5 min of mixing. Film-forming solutions (100 g) were casted over the levelled glass plates (15 cm 15 cm) and dried at 45°C in a hot air oven for 8 h. Then, the dried films were carefully peeled from the plates and stored at room temperature. Each film was prepared in triplicates. 2.5 Film thickness and water content Thickness of the films was measured using a Vernier calliper. Six different positions of the samples were measured and average thickness was calculated. Water content of films was determined by a gravimetric method, whereby Film samples were dried at 105°C in a laboratory oven (NSW-143, Narang Scientific Works Pvt. Ltd., New Delhi, India) until constant weight was achieved [16]. Water content was calculated as follows: Water content ¼ M1 M=M1 100 where, M1 was the initial film mass (g) and M was the bonedry mass (g). 2.6 Film solubility The solubility of the films in water was determined by a modified method of Gontard et al. [17]. Films disks of 2 cm diameter were cut out and dried in a hot air oven at 50°C for 10 h before weighing. The dry disks were then immersed in 50 mL water at 25°C for 24 h. The samples were then dried again and weighed until constant weight. Solubility was expressed as percent weight loss of the film strips on soaking.

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2.7 Water vapour permeability

2.10 Scanning electron microscopy

Water vapour permeability (WVP) of the films was determined gravimetrically [18]. Prior to the measurement, film samples were conditioned in the same environmental chamber at 25°C and 53% relative humidity for at least 2 days. Test cups with diameter 3.7 cm and height 6.5 cm were used. The films were sealed onto test cups filled with anhydrous calcium chloride (0% RH) that was dried in a hot air oven at 120°C for 1 day initially. These cups were then placed in a desiccator containing distilled water (100% RH) and kept at 30°C. The test cups were weighed as a function of time until changes in the weight were recorded to be the nearest 0.001 g. WVP was calculated as follows:

The starch films were placed on an adhesive tape attached to a circular aluminium specimen stub. After coating with gold–palladium, the samples were photographed at an accelerator potential of 5 kV using a scanning electron microscope (Hitachi Se 300H-Tokyo, Japan).

WVP ¼ m L=A t DP where, “m” was the weight of water permeated through the film (g), “L” was the thickness of the film (m) “A” was the permeation area (m2), “t” was the time of permeation (s) and “DP” was water vapour pressure difference across the film (Pa). Five repetitions were performed for each film sample.

2.11 Thermal analysis Thermal analysis of the film samples was carried out in a Differential Scanning calorimeter (DSC Q2000, TA Instruments, New Castle, DE USA). Briefly, 50 2 mg sample conditioned at 25°C and 53% RH for 2 days was taken in aluminium pans and submitted to a temperature program, under nitrogen atmosphere. In the first scan, after cooling the sample at 10°C/min up to 60°C, it was submitted to heating at 10°C/min until 100°C. The second scan was between 60 and 250°C, at the same cooling and heating rates. The glass transition temperature (Tg) (°C) was calculated as the middle point between the onset and end temperatures caused by the discontinuity of sample specific heat. Two replicate runs were carried out for each sample.

2.8 Mechanical properties 2.12 Release of antioxidant agent from films The mechanical properties of films prepared were evaluated by conducting tensile tests (tensile strength, elongation at break and elastic modulus) according to ASTM D882–00 method [19]. Prior to the measurement, film samples were conditioned in the same environmental chamber at 25°C and 53% relative humidity for at least 2 days. Tensile strength and EAB were performed as a tension test using a Texture analyzer TA XT2 (Stable Microsystems, Surrey, UK) with a load cell of 30 kg and crosshead speed of 60 mm/min. The samples were mounted between grips with initial grip gap of 100 mm and film width of 2 cm. The tensile strength (MPa) and elongation at break (%) were calculated using the software Texture Expert (Stable Micro Systems, Surrey, UK). The elastic modulus was calculated as the slope of the initial linear portion of this curve. Each test trial per film consisted of three replicate measurements. 2.9 Film colour and transparency Colour of starch films was determined using colour flex Spectrocolorimeter (Hunter lab colorimeter D-25, Hunter Associates Laboratory, Ruston, USA) after being standardized using Hunter lab colour standards and their Hunter L (lightness), a (redness to greenness) and b (yellowness to blueness) values were measured. For each film type, four samples were measured and values averaged. The transparencies of the different films at wavelengths ranging from 400 to 800 nm were investigated as described by Salleh et al. [20]. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Release of antioxidants into different food stimulants was measured at different time intervals [21, 22]. Film samples (20 mm 20 mm) were introduced into beakers containing 100 mL of distilled water, 10% ethanol and 95% ethanol. Then the film samples were stirred magnetically at 250 rpm with a 2 cm rod coated with Teflon. One millilitre of sample at different time periods at 25°C were taken out and mixed with 4 mL 150 mmol/L DPPH in methanol. The mixture was placed at 25°C in the dark for 30 min. The absorbance of the mixture at 517 nm was measured by using a spectrophotometer (U-2900, Hitachi, Tokyo, Japan). All measurements were performed in triplicate. The release results reported in terms of the DPPH radical scavenging activity were calculated with the following equation: DPHH radical scavenging activity ð%Þ ¼ ð1 Asample Þ=Acontrol 100 where, Asample was the absorbance of test sample (4 mL DPPH plus 1 mL sample) and Acontrol was the absorbance of control solution (4 mL DPPH plus 1 mL distilled water). 2.13 Statistical analyses The data reported are averages of triplicate observations. An analysis of variance with a significance level of 5% was done and Duncan’s test applied to determine differences between means using the commercial statistical package (SPSS 16.0).


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3

Results and discussion

3.1 Composition The compositional analysis of rice starch revealed moisture, protein, ash and fat were 8.01, 0.48, 0.66 and 0.28% respectively. The low protein, fat and ash contents indicate that the starch was extremely pure and the residual protein was completely removed during alkali steeping. Amylose content was found to be 24.55%. Gani et al. [23] reported amylose content of water chestnuts was in the range of 28.5–30.6%. Generally, most native starches contain 15–30% amylose [24]. This amylose is responsible for the film forming capacity of starch. Formation of starch based edible films involves two processes namely, gelatinization and retrogradation. In the gelatinization process, continuous and discontinuous phases of the viscous mass are constituted by the amylose and amylopectin contents respectively. In the retrogradation process, amylose aggregation and crystallization occurs at a rapid rate than amylopectin. The gelatinization and retrogradation processes can be interpreted as the result of forming a helical network which then aggregates into gels or retrograded materials become more rigid and difficult to disperse [25]. Amylose rich starch form amylose crystalline regions during film drying which gives stiffness and more resistance to fracture, but less stretchable films [26]. 3.2 Film thickness, water content and solubility All films were found to be flexible and easily removed from the casting plate. The basic film properties, including thickness, water content and solubility are shown in Table 1. Thickness of starch films ranged from 0.087 to 0.124 mm, and incorporation of AA and BHT did not significantly affect the resulting film thickness. Moisture contents of S/G (9.55%), S/G/AA (6.73%) and S/G/BHT (5.06%) films were significantly different. Lowest moisture content of S/G/BHT film may be due to the hydrophobic nature of BHT which can affect the ability of the film to retain water. Water solubility of biodegradable films is an important characteristic because it

can affect the water resistance of films, especially in humid environment. Solubility of biopolymer-based films assumes importance to determine their biodegradability [27]. Water insolubility of films is also important in certain potential applications wherein a prerequisite to enhance the products integrity, improve moisture barrier properties and overall shelf life is necessary [28]. High solubility was found in case of S/G (23.91%) and S/G/AA (23.88) films. Due to the hydrophilic character of glycerol, it interacts strongly with water and adds it easily to the network of the film through hydrogen bridges [29]. S/G/BHT film showed significant decrease in water solubility (20.33%) which might be a consequence of interactions between the antioxidant and the hydroxyl groups of the starch or may be due to the hydrophobic nature of BHT. Hydrophilic compounds will increase film solubility, whereas hydrophobic compounds will decrease it [30]. 3.3 Water vapour permeability Table 2 shows WVP values of starch-based films analysed at 30°C and 0–100% RH gradient. The WVP values of the S/G, S/G/AA and S/G/BHT films were 9.31 1011, 10.50 1011 and 6.86 1011 g m1 s1 pa1 respectively. Significant differences were observed between the WVP values of the films. S/G/BHT film presented lowest WVP (6.86 1011 g m1 s1 pa1). It might be due to the increased hydrophobicity of film in the presence of BHT [31]. On the other hand, S/G/AA film presented highest WVP (10.50 1011g m1 s1 pa1). As water vapour transfer process depended on the simultaneous actions of water solubility and diffusivity in a polymeric matrix [32, 33], the higher WVP for S/G/AA film can be explained by its higher water affinity. The high affinity of the acid for water (in 100% RH tested chamber) may result in its solubilization and breakage of the interaction with polymer chains, resulting in higher plasticization and subsequent increase in WVP. Zhong et al. [22] also reported high WVP of kudzu starch/AA films (9.27 1011g m1 s1 pa1). However, all the films presented high WVP which may be due to

Table 1. Thickness, water content and solubility of starch/ glycerol (S/G), starch/glycerol/ascorbic acid (S/G/AA) and starch/glycerol/BHT (S/G/BHT) films

Table 2. Water vapour permeability (WVP), tensile strength (TS), elongation at break (EAB) and elastic modulus (EM) of starch/glycerol (S/G), starch/glycerol/ascorbic acid (S/G/AA) and starch/glycerol/BHT (S/G/BHT) films

Films

Films

S/G S/G/AA S/G/BHT

Thickness (mm)

Moisture content (%)

Solubility (%)

0.087 0.04a 0.124 0.00a 0.091 0.02a

9.55 0.01c 6.73 0.16b 5.06 0.04a

23.91 0.01b 23.88 0.02b 20.33 0.14a

Means are three replicates plus or minus the standard deviation. Significant difference (p 0.05) is represented by different alphabetic superscript letters within a column.


WVP (1011g m1 s1 pa1)

S/G 9.31 0.10b S/G/AA 10.50 0.04c S/G/BHT 6.86 0.23a

TS (MPa)

EAB (%)

EM (MPa)

1.94 0.29a 14.59 3.48c 0.19 0.01a 5.47 0.97b 1.98 0.50a 3.19 0.06b 6.44 0.20c 2.48 0.41b 3.25 0.19b

Means are three replicates plus or minus the standard deviation. Significant difference (p 0.05) is represented by different alphabetic superscript letters within a column.


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hydrophilic nature of both starch and glycerol. Hydrophilic plasticizers like glycerol is known to enhance the water vapour permeability of starch-based films. Cerqueira et al. [34] reported plasticizer increases the free volume and chain movements, reducing the rigidity and increasing the molecular mobility of the films, allowing higher water vapour transport through their structure. Arvanitoyannis et al. [35] reported that increasing the total plasticizer content (water and polyols) in the polymer matrix results in a proportional increase of water vapour transfer rate. 3.4 Mechanical properties Tensile properties of the starch-based films are shown in Table 2. S/G films showed lowest tensile strength (1.94 MPa). Glycerol decreased the tensile strength of starch films due to its hygroscopic character that tends to provide additional water into the film matrix [36, 37]. Significant increase in TS was found in S/G/AA (5.47 MPa) and S/G/BHT films (6.44 MPa). Jongjareonrak et al. [31] reported that the hydrogen bond formation between BHT and gelatin could be responsible for the increased TS of fish skin gelatin films added with BHT. The increased TS of S/G/AA and S/G/BHT films might also be due to the hydrogen bonding between the antioxidants (AA and BHT) and starch molecules. S/G films showed significantly high % EAB (14.59). Thus S/G films were stretchable and flexible due to the plasticization effect of glycerol that increases the mobility of polymer chains. Plasticizers have been used to overcome the brittleness of films resulting from high intermolecular forces by increasing the mobility of polymer chains which makes films stretchable and flexible [38]. With increasing amounts of glycerol, cassava starch and gelatin films presented decreases in tensile strength and increases in the elongation at break [39]. Lower values of EAB of S/G/AA (1.98) and S/G/BHT (2.48) films may be due to high intermolecular forces. The addition of AA and BHT to the films may have altered structure of starch films and decreased the movement of the macromolecules in the film matrix, leading to the decrease of EAB. Elastic modulus, a measure of intrinsic film stiffness, is the slope of the linear range of the stress– strain plot [40]. Young’s modulus of S/G films (0.19) was significantly lower than S/G/AA (3.19) and S/G/BHT (3.25). Higher TS and lower % EAB of S/G/AA and S/G/BHT films resulted in their higher Young’s modulus. 3.5 Film colour and transparency Colour and transparency of the packaging film are important indexes in terms of general appearance and consumer acceptance. The colour and % transmittance of the starchbased films were recorded in Table 3. The starch–glycerol blend film without any antioxidant (S/G) was used as a reference film. The addition of the two antioxidants ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Starch/Stärke 2015, 67, 294–302 Table 3. Colour, % transmittance and thermal properties of starch/glycerol (S/G), starch/glycerol/ascorbic acid (S/G/AA) and starch/glycerol/BHT (S/G/BHT) films

Parameters

S/G

Colour L 90.68 a 0.37 b 4.89 % Transmittance 400 nm 34.56 500 nm 36.36 600 nm 44.30 700 nm 54.23 800 nm 54.83 Thermal properties Tg(°C) 73.17 DH(J g1) 47.87

S/G/AA

0.20c 0.05a 0.70a

0.33c 0.28c 0.29b 0.20b 0.20b

0.01a 0.01a

S/G/BHT

85.38 0.19a 0.58 0.08a 15.25 0.34c 18.13 20.86 33.13 34.23 34.98

0.26a 0.65a 0.26a 0.33a 0.10a

76.60 0.04b 167.28 0.06b

87.30 0.76b 0.50 0.25a 7.35 1.39b 21.96 22.91 33.17 34.13 35.09

0.44b 0.21b 0.33a 0.26a 0.33a

76.87 0.00c 174.44 0.35c

Means are three replicates plus or minus the standard deviation. Significant difference (p 0.05) is represented by different alphabetic superscript letters within a row.

significantly affected the lightness (L) and yellow (b) colour of the starch– glycerol blend films with no significant difference in the green (a) colour. The yellow colour observed in the S/G/BHT film could be attributed to the presence of quinones, produced due to the oxidization of BHT during processing. Under thermo-oxidative conditions, BHT forms coloured by-products such as quinone methide and stilbene bis(quinone) (tetra-bis(tert-benzoquinone)), which could be responsible for small colour change of the PLA-BHT films [41]. The yellow colour in the S/G/AA film could be due to the oxidative degradation of AA and browning development. The first reaction of AA degradation leads to hydrolyzes of the ascorbic acid-lactone ring to render 2-ketoL-gulonic acid (KGA). Once KGA appears, this reactive molecule (a, b-unsaturated carbonyl, b-hydroxy carboxyl) suffers successive dehydrations producing other compounds (b-hydroxy carbonyl-) which are also easily dehydrated, or decarboxylated (in case of b-keto carboxylic acids), rendering 3-deoxy-L-pentosone (3-DP), 3,4-dideoxy-pentosulos-3-ene (3,4-DDP) and furfural [42]. The % transmittance of the S/G film was high. Improved transmittance of S/G film at all wavelengths might be because of reduced number of ghost granules. Low transmittance in S/G/AA and S/G/BHT films may be because of the antioxidant compounds in the film matrix. Colour analysis also showed decreased lightness and development of yellow colour in case of S/G/AA and S/G/BHT films, which may be responsible for their low transmittance. 3.6 Scanning electron microscopy The Scanning electron microscopy (SEM) pictures of S/G, S/ G/AA and S/G/BHT films are shown in Fig. 1. It was clearly www.starch-journal.com

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Figure 1. SEM micrographs of (A) starch/glycerol (S/G), (B) starch/glycerol/ascorbic acid (S/G/AA) and (C) starch/glycerol/BHT (S/G/BHT) films.

seen from the micrographs that the surface of starch films was smooth and homogeneous without pores or cracks. Addition of AA and BHTdid not affect the morphology of films. Absence of distinct separation of the different components in the micrographs indicates adequate mixing of different constituents during preparation. 3.7 Thermal analysis Thermal properties are recorded in Table 3. The S/G film had a glass transition temperature (Tg) value of 73.17°C (at 9.55% moisture) and enthalpy of transition (DH) value of 47.87 J g1. Al-Hassan et al. [43] reported greater values of Tg (84.04°C) and DH (219.63) of unplasticized sago starch films. Incorporation of glycerol reduced intermolecular forces and increased the mobility of polymer chains. In this way, plasticizers like glycerol decrease the glass transition temperature and DH value of these materials and improve their flexibility [44]. The structure of the film is glassy, hard and brittle below glass transition temperature (Tg) and rubbery, soft and flexible above it. Tg and DH values of starch films were significantly increased with the incorporation of AA and BHT. The increase in Tg and DH values of S/G/AA and S/G/BHT films probably may be due to the formation of hydrogen bonding between the antioxidants (AA and BHT) and the starch which strengthened the film network and limited the molecular movement of the films. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.8 Release of antioxidant agent from films DPPH radical is one of the stable organic nitrogen radicals and its scavenging activity assay is considered as one of the most standardized methods for the determination of antioxidant capacity [45]. To display release of antioxidant substances from films, DPPH radical scavenging abilities were studied in certain period of time into three food simulants (water was used as an aqueous food simulant, 10% ethanol as an alcoholic food simulant, and 95% ethanol as fatty food simulant) (Fig. 2). The DPPH radical scavenging activity of the control film was lowest and its scavenging activity was hardly detected. The presence of AA and BHT in starch films significantly increased DPPH radical scavenging ability. S/G/BHT film showed the highest DPPH radical scavenging activity (95.7%). As for the release in different food simulants, the extent and rate of release depended on the type of food simulant and the antioxidant agent. AA was released to a large extent into water and 10% ethanol but its release into 95% ethanol was restricted. This may be due to its great solubility in water and 10% ethanol. The equilibration time (i.e. the time reaching the highest scavenging activity) of AA in distilled water was 150 min, while that in 10% ethanol it was 330 min. Carol et al. [46] also reported largest release of AA from ethylene vinyl alcohol copolymer matrix into water and 10% ethanol as compared to 95% ethanol. BHT was released to a large extent into 95% ethanol due to its great solubility in ethanol.


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BHT from extruded poly lactic acid (PLA) film into 95% ethanol and its small release into 10% ethanol. Buonocore et al. [48] reported that the release of an active compound from a polymeric network took place in several steps. At first, solvent molecules penetrated from the outer solution into the polymer matrix and led to network swelling. These changes in the film structure allowed the diffusion of the active compound through the polymer matrix into the outer solution until thermodynamic equilibrium was achieved. Thus, the release of antioxidants from starch films into different food simulants could be related to some factors such as nature of antioxidant compound, solvent, polymer solubility and swelling properties and intermolecular interactions between antioxidant and starch.

4

Conclusions

Active packaging films based on starch and antioxidants (AA and BHT) were successfully developed by the casting technique. Incorporation of these antioxidants into the starch film greatly modified their properties. BHT significantly improved the water resistance of starch films. Both AA and BHT promoted an increase in the elastic modulus but a decrease in film stretchability. The addition of BHT to the film may have altered starch film structure and decreased the movement of the macromolecules in the film matrix, leading to a decrease of EAB and increase in the elastic modulus. S/G/BHT film presented significant decrease in WVP which may be due to the hydrophobic nature of BHT. SEM revealed smooth surface of the films. DSC analysis indicated that there was a strong interaction in the film matrix with the incorporation of AA and BHT. Exposure of the films to various food simulants showed that the release from the films was dependent on the type of food simulant and the antioxidant incorporated. In the aqueous food simulant, films containing AA produced the largest release and in the fatty food simulant, S/G/BHT film presented the fast release. Authors are thankful to Department of Biotechnology, Govt. of India, for their financial support. The authors have declared no conflict of interest.

Figure 2. The release of antioxidant agents from starch/glycerol (S/ G), starch/glycerol/ascorbic acid (S/G/AA) and starch/glycerol/ BHT (S/G/BHT) films in (A) distilled water, (B) 10% ethanol and (C) 95% ethanol.

In contrast, its release into 10% ethanol and water was restricted by its low solubility. The equilibration time of BHT in 95% ethanol and 10% ethanol was 180 and 390 min respectively. Majid et al. [47] reported total and fast release of ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

5

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