Active and sustainable materials from rice starch, fish protein and oregano essential oil for food packaging

Industrial Crops and Products 97 (2017) 268–274

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Active and sustainable materials from rice starch, fish protein and oregano essential oil for food packaging Viviane Patrícia Romani, Carlos Prentice-Hernández, Vilásia Guimarães Martins ∗ Laboratory of Food Technology, School of Chemistry and Food, Federal University of Rio Grande, Av. Itália, km 8, CEP 96203-900, Rio Grande, RS, Brazil

a r t i c l e

i n f o

Article history: Received 23 September 2016 Received in revised form 15 December 2016 Accepted 19 December 2016 Keywords: Active packaging Antioxidants Biopolymers Sustainable blends

a b s t r a c t The development of blends using sustainable raw materials promises to reach superior properties compared to biodegradable materials in general, besides the low cost and environmental appealing. This study investigates the addition of oregano essential oil (OEO) in blends from rice starch/fish protein to be used as active packaging. For this purpose, rice starch was extracted from broken grains and fish protein was recovered from Withemouth Croacker (Micropogonias furnieri). The influence of different ratios of starch/protein was confirmed by mechanical properties, solubility, water vapor permeability, opacity and color parameters. The results showed that the ratio 50/50 of starch/protein was the most suitable for packaging materials development. It had the lower solubility (8.0%), water vapor permeability (0.18 g mm kPa−1 h−1 m−2 ), and intermediate mechanical properties (Tensile strength 5.69 MPa, Elongation 85.5%). Morphology and thermal tests were related to the properties of the films and confirmed that the matrix was homogeneous and cohesive. The antioxidant tests confirmed the activity of the blends in inhibition of peroxidase suggesting its promising application as anti-browning packaging in fruits and vegetables. Based on the results, this research demonstrated that the use of rice starch and fish protein to form sustainable blends represents an interesting alternative for the production of active packaging and for the development of eco-friendly technologies. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Conventional packaging based on petroleum is used in a wide variety of application due to its durability, mechanical and barrier properties, ease processing and low cost. However, in recent years there is an increasing concern due to environmental problems because such packaging takes hundreds of years to decompose (Debiagi et al., 2014; Sun et al., 2013). Sustainable packaging from renewable resources has been widely studied for replacement of synthetic polymers. Bioplastics is a nascent market capturing plastics market at a growth rate of 30% annually (Reddy et al., 2013). A broad spectrum of materials may be used for biodegradable polymers production, such as proteins, polysaccharides and lipids (Debiagi et al., 2014; Peelman et al., 2013). Starch is used to develop films due to its high availability, low cost and its ability to form odorless and colorless polymer matrices with low oxygen permeability (Cano et al., 2014; Jiménez et al., 2012). Starch is the major chemical component of cereal grains, comprising approximately 90% of the dry weight of rice grain (Zhou

∗ Corresponding author. E-mail address: [email protected] (V.G. Martins). http://dx.doi.org/10.1016/j.indcrop.2016.12.026 0926-6690/© 2016 Elsevier B.V. All rights reserved.

et al., 2002). The rice industry produces a large quantity of byproducts, including broken grains, which are normally used as animal feed or treated as waste products incinerated for energy purposes (Li et al., 2010; Yun et al., 2005). The use of rice byproducts is feasible since the rice processing yields approximately 14% of broken grains (Dias et al., 2010). Therefore, starch extraction is an alternative to add value to broken rice grains, transforming this material into a product with increased industrial interest. One of the most interesting potential uses of starch is the production of biodegradable food packaging, contributing positively to reduce the problem of environmental pollution. Proteins are among the most promising raw materials for biodegradable polymers development due to its ability to form three-dimensional macromolecular networks, stabilized and strengthened by hydrogen bonds, hydrophobic interactions, and disulfide bonds (Thomas et al., 2013). Fish is an important source of proteins that contains considerably higher quantity of myofibrillar proteins in comparison to land animals and play significant role in three-dimensional networks formation (Lanier et al., 2005; Shiku et al., 2004). With the decline in wild fish species abundance, better utilization is called for marine by-products and underutilized fish that is currently used for animal feed (Brenner et al., 2009).

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In line with the new technologies for food packaging, the study of the incorporation of active agents in bio-based films to improve their functional and food-protective properties plays an important role (Nur Hanani et al., 2014). Natural compounds, such as plant extracts and essential oils from herbs incorporated in packaging films is a promising way to prevent or reduce deterioration of foods caused by oxidation (López-de-Dicastillo et al., 2012). Once enzymatic browning, the reaction of enzymes (polyphenol oxidase and peroxidase) and phenolic substrates that produces darker pigments on the surface (Song et al., 2007), impacts the appearance and the consumer acceptance of fruits, it is relevant to find alternatives to minimize these oxidative reactions. Oregano essential oil is well recognized for its antioxidant and antimicrobial action (Burt, 2004) and consists in an interesting compound to incorporate in bio-based materials. Some drawbacks of biodegradable plastics still limit their commercial application, such as poor mechanical and water barrier properties, which make them difficult to use in food packaging (Jeya Shakila et al., 2012). Different strategies have been used to overcome these limitations, including blending, cross-linking and composites with nanoparticles (Martucci and Ruseckaite, 2010). Blending materials has been related using many types of raw materials, such as whey protein and lotus rhizome starch (Sukhija et al., 2016), pea starch and peanut protein (Sun and Xiong, 2014), pectin and bitter vetch protein (Porta et al., 2016), among others. However, blending rice starch and fish proteins has not been related. The use of these raw materials is promising due to their sustainability and the low cost involved. Thus, this work was aimed to evaluate the influence of different ratios of rice starch/fish protein in blends incorporated with oregano essential oil and also verify its antioxidant activity against enzymatic browning. 2. Material and methods 2.1. Material Broken grains produced during rice processing were provided from Arrozeira Pelotas located in Pelotas/Brazil. Whitemouth croacker (Micropogonias furnieri) was acquired in trade from Rio Grande/Brazil. Oregano essential oil was purchased from Quinari (Ponta Grossa, Brazil) with a refractive index of 1.3521. 2.2. Protein isolation Whitemouth croacker protein was extracted similar to Nolsoe and Undeland (2009) through pH shifting process. Muscle of Whitemouth croacker was homogenized with distilled water in a ratio of 1:9 (muscle:water, w/v). Alkaline solubilization was performed at pH 11.0. After solubilization, the sample was centrifuged at 9000g for 20 min. During centrifugation, the sample was separated into three phases. The middle phase (soluble proteins) was subjected to isoelectric protein precipitation at pH 5.5 and centrifuged again at 9000g for 20 min. The precipitated protein was dehydrated in an air circulation oven (Quimis, Q342, Brazil). The Whitemouth croacker protein isolate was analyzed to determine the protein content in accordance to standard method 981.10 (AOAC, 2000), and a protein content of 96.7% was obtained.

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0.1% NaOH, followed by centrifugation. Then, the starch layer was washed with distilled water and centrifuged again. The starch was re-slurried, neutralized to pH 6.5 and centrifuged. The neutralized starch was washed twice with distilled water and dried in an air circulation oven. The amylose content was determined according to Martinez and Cuevas (1989) generating 40.8% of amylose.

2.4. Blend preparation Blends were prepared using 3% of total solids and different proportions of starch/protein (25/75, 50/50 and 75/25). Oregano essential oil was used in concentrations of 4%, 6% and 8%. The blends were prepared by casting technique as described by Rocha et al. (2013), with some modifications. Starch and protein were homogenized in distilled water and the pH of the solution was adjusted to 11.0. Glycerol was added as a plasticizer in a concentration of 25%. The solution was heated to 80 ◦ C and kept 20 min under mechanical agitation to form the film solution. After cooling to 35 ◦ C, the oregano essential oil was added, and the mixture was homogenized for 10 min. The filmogenic solutions were placed in Petri dishes (9 cm in diameter) and dried in an oven with air circulation (BIOPAR, S150BA, Brazil) at 40 ◦ C for 12 h. After drying, the blends remained 24 h in desiccators at 25 ◦ C with a relative humidity (RH) of 50%. A saturated calcium chloride solution was used to control the RH.

2.5. Blend characterization 2.5.1. Thickness Blend thickness was measured with a micrometer (Insize, IP54, precision 0.001 mm) at ten different positions for each film sample. Thickness mean values were considered in the calculations of tensile strength and water vapor permeability (WVP).

2.5.2. Mechanical properties Mechanical properties were determined using a texture analyzer (TA.XTplus, Stable Micro Systems, England) based on the ASTM D-882-02 method (ASTM, 2002). The blends were cut into 25 by 85 mm strips. The initial grip separation and cross-head speed were set at 50 mm and 1 mm/s, respectively. The tensile strength (MPa) was calculated dividing the maximum force by the initial cross-sectional area of the film. Elongation (%) was calculated dividing film elongation at break by the initial gauge length of the specimen.

2.5.3. Solubility in water The solubility in water (%) of the blends was measured according to method described by Gontard et al. (1994), with some modifications. The samples (2 cm in diameter) were dried in an oven (DeLeo, A15E, Brazil) at 105 ◦ C to determine the initial dry weight. The blends were immersed in 50 mL of distilled water, and the mixtures were continuously shaken (100 rpm) at 25 ◦ C for 24 h. After immersion, the blends were dried at 105 ◦ C to determine final dry weight. The film solubility (%) was defined as the ratio between the water-soluble solid content and initial solid dry content.

2.3. Starch extraction Rice starch from broken grains was extracted as previously described by Wang and Wang (2004). Rice flour was soaked in 0.1% NaOH at a ratio 1:2 (w/v) for 18 h, followed by blending, passage through a 63 ␮m screen and centrifugation at 1200g for 5 min. The soft-top layer was carefully removed, and the underlying starch layer was re-slurried. The starch layer was washed twice with

2.5.4. Water vapor permeability The water vapor permeability was gravimetrically determined according to the ASTM Standard Method E96-00 (ASTM, 2000), with some modifications. The blends were sealed on a permeation cell containing anhydrous calcium chloride (0% RH). The cells were placed in desiccators with a saturated sodium chloride solution

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(75% RH) and weighed at 1 h intervals for 8 h at 25 ◦ C. The WVP (g mm kPa−1 h−1 m−2 ) was calculated as follows: WVP =

W.L A.t.P

(1)

where W is the weight gain of the permeation cell (g); L is the film thickness (mm); A is the exposed area of blend (m2 ); t is the time of weight gain (h); and P is the vapor pressure difference across the blend (kPa). 2.5.5. Color and opacity The color was measured using a CR-400 Minolta Chroma Meter and the CIELab color space was used. The parameters analyzed in this color system are L* (lightness/brightness), a* (redness/greenness) and b* (yellowness/blueness). The color of the blends was expressed as the total difference in color (E*) obtained using Illuminant D65. The total difference in color (E*) was calculated as follows: E∗ =



(L ∗ −L)2 + (a ∗ −a)2 + (b ∗ −b)2

(2)

where L*, a* and b* are the values of the standard color parameters (L = 97.39, a* = 0.14, b* = 1.94) and L, a and b are the values of the sample color parameters. Opacity (Y, %) was determined using the same equipment used for color measurement and calculated as the relationship among the opacity of each sample in a black (Yb ) and a white (Yw ) standard. 2.5.6. Antioxidant activity The antioxidant activity of the blend incorporated with oregano essential oil was measured by the reduction in free radical 1,1diphenyl-2-picrihidrazil (DPPH) and the inhibitory effect on the peroxidase (enzyme responsible for browning in plant tissues). A mixture (blend + methanol) was maintained under horizontal stirring for 3 h at 25 ◦ C protected from light and this extract was used for the analysis. The consumption of free radical DPPH was monitored according to Herrero et al. (2005) with modifications. The measurements were performed at a wavelength of 515 nm. Aliquot of 0.7 mL of the sample extract was added to tubes containing 1.3 mL of methanol and 3.0 mL of a methanolic solution of DPPH (5.2 × 10−5 mol L−1 ). Reaction tubes were incubated at room temperature in the dark, and the color change from yellow to violet was measured after 30 min. The ability to scavenge free radicals was expressed as a percentage of the radical oxidation inhibition compared to the control. The inhibitory effect on peroxidase was evaluated similar to described by Schmidt et al. (2014). The enzyme extract was obtained from 2 g of potato (Solanum tuberosum L., Monalisa variety) with 100 mL of buffer solution pH 7 (0.1 M phosphate-citrate buffer). After 3 min of grinding in a blender, the mixture was filtered and centrifuged (10 min, 4 ◦ C, 3200g). Enzymatic browning reactions were performed using the crude enzyme extract as the enzyme source. The peroxidase enzyme activity was determined using 1 mL of enzyme extract, 1 mL of 0.08% H2 O2 , 0.5 mL of 1% guaiacol solution, 1,5 mL with buffer pH 7 and 0.5 mL of the sample as a reaction inhibitor. The absorbance was detected at 470 nm after 15 min of reaction at 25 ◦ C. The antioxidant activity was expressed as the percentage inhibition of the browning reaction compared to the control. 2.5.7. Morphology and crystallinity The microstructural characteristics of the blend surface were examined using a scanning electron microscope (Jeol, JSM-6060, Japan) at an accelerating voltage of 10 kV. Prior to visualization, the blend sample was sputtered with gold (Sputter Coater, SCDO50) making the sample conductive. The blend sample was observed at 2000× magnification.

Table 1 Properties of the blends prepared using different ratios of starch/protein containing 8% oregano essential oil. Blend properties

Ratio of starch/protein 25/75

Thickness (mm) Tensile strength (MPa) Elongation (%) Solubility (%) WVP (g mm kPa−1 h−1 m−2 ) Color (E*) Opacity (%)

50/50

0.074 ± 0.001 6.79 ± 0.22a 108.8 ± 1.2a 22.6 ± 0.4a 0.21 ± 0.00a 3.99 ± 0.03b 9.8 ± 0.1a

a

75/25

0.074 ± 0.004 5.69 ± 0.07b 85.5 ± 2.1b 8.0 ± 1.1c 0.18 ± 0.00b 5.05 ± 0.07a 9.6 ± 0.2a

a

0.070 ± 0.001b 3.92 ± 0.06c 83.5 ± 9.6b 16.7 ± 0.5b 0.18 ± 0.00b 3.91 ± 0.02c 9.7 ± 0.3a

Values are given as mean ± standard deviation. Different superscript letters in the same line indicate a statistically significant difference (p < 0.05).

The crystallinity of the blend was analyzed using an X-ray diffractometer (Bruker, D8 Advance, USA) at a voltage of 40 kV and current of 30 mA. The sample was scanned at a scan range of 2, from 5 to 40◦ . The relative crystallinity was calculated as the area under the crystalline peaks compared with the area of the amorphous halo under the peaks. 2.5.8. Thermal degradation Thermogravimetric analysis (TGA) was performed on a thermogravimetric analyzer (Shimadzu, DTG 60, Japan) over a range of 23–550 ◦ C using aluminum pans in a static atmosphere of air at a constant flow rate of 20 mL min−1 . Differential scanning calorimetry (DSC) was performed on a calorimeter (Shimadzu TGA-60, Japan) with a nitrogen flow rate of 50 mL min−1 . The sample (3 mg ± 0.01) was hermetically sealed in aluminum pans and scanned at a heating rate of 10 ◦ C min−1 over a range of 30–200 ◦ C. The enthalpy (H) was computed from the endothermic peak observed in the thermogram. 2.6. Statistical analysis Analytical determinations were performed in triplicate and standard deviations were reported. Analysis of variance (ANOVA) was performed and Tukey’s test was used to determine the differences between the properties of blends in the 95% confidence range. 3. Results and discussion 3.1. Mechanical, barrier and color properties of the blends Results obtained with 8% oregano essential oil added in starch/protein blends were superior and therefore are shown in Table 1. Thickness of starch/protein blends was statistically lower (p < 0.05) when 75% of starch was used in the development of the blends. This behavior is probably due to the molecular reorganization during the drying process, which is different for starch and protein. The mechanical properties of biopolymer films, including tensile strength, elongation and modulus of elasticity, are extremely important because the packaging material must possess adequate mechanical strength to maintain its integrity during handling and storage (Wihodo and Moraru, 2013). Mechanical properties of the blends were strongly influenced by the different ratios of starch/protein. The blend prepared with the higher concentration of protein (25/75 starch/protein ratio) showed stronger and more flexible films. Sun and Xiong (2014) related more flexible films using higher protein content in the development of pea starch/peanut proteins blends. According to these authors, pro-

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teins have the ability to form denser matrix. Mehyar et al. (2012) attributed this behavior to the strong hydrostatic intermolecular interactions and disulfide bonds in matrices composed of proteins. Munoz et al. (2012) reported that the protein network has the ability to accommodate polysaccharide chains resulting in a continuous phase and dense matrix responsible for the improved barrier and mechanical properties of films. Higher elongation for the ratio 25/75 starch/protein (108.8%) could also reflect the decrease of amylose content (responsible for the crystallinity of starch) in the films prepared with higher protein concentration. Smaller starch concentration leads to lower crystallinity, consequently increasing flexibility and elasticity. According to Bourtoom and Chinnan (2008) and Gontard et al. (1992), a high tensile strength is generally required, but the deformation values must be adjusted according to the intended application of the polymer. For food packaging, the material used must provide integrity and reinforce structure to protect the product. Solubility in water was significantly different (p < 0.05) for the ratios of rice starch/fish protein. However, all solubility results suggest that the films have good stability in water. Sukhija et al. (2016) suggested that low solubility might results from interaction among free hydroxyl groups of starch and free sulfidryl groups of protein as well as formation of hydrogen and ester bonds between hydroxyl and carboxyl groups of starch. The lowest solubility value (8.0%) might suggest that the ratio 50/50 of starch/protein is the most adequate for materials development when the intended application requires insolubility. Its behavior is probably due to the higher density of bonds between starch and protein, suggesting its better interaction in this ratio. Solubility is an important property of films for applications in food protection in high water activity products, for example to avoid exudation of frozen or fresh foods during processing and to maintain the product integrity (Gontard et al., 1992). Potential applications of biodegradable films may demand low solubility or insolubility to enhance moisture barrier properties and shelf-life stability (Zavareze et al., 2012). Higher protein contents are, in general, responsible for lower water vapor permeability. In the present study, opposite behavior was observed in the ratio 25/75 of starch/protein, which was significantly higher (p < 0.05). According to Davanc¸o et al. (2007), increased WVP is associated with the polarity of chains, suggesting the presence of polar chains in the protein. The oregano essential oil also plays an important role in WVP of films. Thus, this behavior can also be attributed to the better distribution of oregano essential oil in the ratios 50/50 and 75/25 of starch/protein, decreasing the free volume in network and making difficult the water diffusion. Sukhija et al. (2016) related that good WVP of blends could be attributed to the formation of cross-links between starch and protein, as well the accommodation of starch as filler in the polymeric matrix. The fish protein presence imparted yellow color and consequently influenced E*, which was significantly superior (p < 0.05) for the higher protein contents, consistent with visual observations. Chinma et al. (2012) and Sun et al. (2013) found results in line with the present study when preparing composite films. These authors suggested that increase in E* may be the result of yellow coloration associated with protein-aldehyde interaction due to intermediate or final products of Maillard reaction occurring between rice starch and the amino groups of fish protein during heating. Color parameters of biodegradable packaging films are important in terms of appearance and consumer acceptability. The opacity was not influenced by different ratios of starch and proteins used in the present study. This behavior is in agreement to Al-Hassan and Norziah (2012) in the preparation of blends using different proportions of sago starch and fish gelatin. Fakhoury et al. (2012) reported a higher starch content in films with superior opac-

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ity due to the crystallinity of this compound, which functions as a barrier to light, whereas the chemical structure of protein does not permit crystallization. The opacity is important while processing fatty foods to extend their shelf-life, as these foods are susceptible to oxidative degradation catalyzed by light. The materials used in the current study showed promising properties in comparison to other blends studies. Similar range of tensile strength (3.06–5.44 MPa) and solubility (9.78–22.31%) was observed by Sun et al. (2013) in pea starch/peanut protein blends. Sukhija et al. (2016) observed the same trend in composites of whey protein and lotus rhizome starch (tensile strength 6.06 MPa and solubility 15.90%). Wang et al. (2010) reported higher tensile strength (6.06–10.77 MPa) for whey protein + gelatin + sodium alginate films. However, all authors cited above developed materials with lower elongation (20.56–98.12%, 37.19% and 12.55–29.42%, respectively) in comparison to the present study. The higher elongation values of these blends might be associated to the essential oil addition. The addition of lipid components in protein or starchbased films may hinder polymer chain-to-chain interactions and provide flexible domains within the film (Limpisophon et al., 2010). Beside the low cost of these raw materials, rice starch/fish protein blends are interesting biopolymers to develop sustainable alternative materials for food packaging. The blend prepared with the ratio 50/50 of rice starch/fish protein showed better properties related to its lower WVP and water solubility and promising mechanical properties. Thus, this formulation was chosen to a more detailed study in its antioxidant capacity, morphological and thermal properties.

3.2. Antioxidant activity of active blends on DPPH and peroxidase inhibition Despite the essential oil of Origanum vulgare L. has been widely studied in relation to its antimicrobial and antioxidant activity, its effectiveness in peroxidase inhibition is not reported in the literature. The DPPH method is the most commonly used to evaluate antioxidant properties of active compounds. Thus, it was used in comparison to the enzymatic method to evaluate the blends incorporated with 8% of oregano essential oil. The blend prepared with 50/50 of starch/protein and 8% of oregano essential oil showed 4.2% inhibition using the DPPH method. Higher antioxidant activity was observed in enzymatic method (63.1%), suggesting that the blend effectively inhibited the action of peroxidase, one of the main enzymes responsible for enzymatic browning in foods. These active properties attributed to oregano essential oil are mostly due to the presence of two phenols, carvacrol, and thymol (major constituents of oregano essential oil) and the monoterpene hydrocarbons pcymene and ␥-terpinene that are present at low concentrations, as mentioned by Burt (2004) and Hosseini et al. (2013). It is worth noting that the ability of oregano essential oil to inhibit enzymatic browning was evaluated when incorporated in the blends. Thus, the biopolymers used to prepare the blend, rice starch and fish protein, were suitable for incorporation of OEO. According to Toivonen and Brummell (2008) and Zhang et al. (2015), the enzymatic browning is a problem in fruits and vegetables, such as potato, apple and pear. It is responsible for the adverse effects on appearance, aroma, flavor and nutritional value, which reduces the shelf life and consumer acceptance of these products. Thus, the blend prepared in the present study showed potential for application in the development of materials for active packaging, increasing the shelf-life of fruits and vegetables in which enzymatic browning occurs.

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Fig. 1. Scanning electron micrograph (magnification 2000x) of blend surface microstructure (50/50 starch/protein and 8% oregano essential oil).

Fig. 2. X-ray diffractogram of blends of 50/50 starch/protein with 8% of oregano essential oil.

3.3. Structural analysis and thermal degradation The surface microstructure of blend incorporated with oregano essential oil suggested the fine compatibility of oregano essential oil, rice starch and fish protein (Fig. 1). The SEM image revealed a smooth and homogeneous surface without cracks. According to Atarés et al. (2011) essential oils could cause discontinuities due to the presence of droplets, giving rise to a more open structure and thicker films. Oil droplets could also be noticed by SEM on the surface of the films, which was not observed in the present study. The structure of the films greatly influences the physical properties of these. Morphology studies of the blends are in agreement with mechanical and water vapor permeability properties. More structural integrity consequently leads to good mechanical properties like tensile strength and elongation at break. This behavior might be attributed to strong intermolecular interaction, entanglement and formation of continuous phase of polymeric matrix and improved interfacial interactions between the blending components and the essential oil. The X-ray diffractogram (Fig. 2) shows the crystalline regions as defined peaks in the 2 region between 10 and 35◦ (specifically centered at 12.11, 19.76 and 30.23◦ ). Similar range was observed by Fakhouri et al. (2013), who detected peaks between 15 and 25◦ . Corradini et al. (2007) suggested that these peaks indicate a type of crystalline structure, called V type, related to the crystallization of amylose. These V complexes are responsible for the formation of

insoluble materials when blends are prepared with starch and other polymers. During the cooling step, amylose forms helices involving other constituents of the polymeric matrix, such as proteins, lipids or plasticizers. The relative crystallinity of the starch and protein blend was 35.1% compared with the amorphous phase. Rindlav-Westling et al. (2002) and Fakhouri et al. (2013) have reported similar relative crystallinity and they also attributed it to the crystallization of amylose. These results are consistent with the values of the mechanical properties of tensile strength and elongation. Liu et al. (2013) explained that the strong interactions between the polymer chains of starch favor crystallization, thereby increasing the stiffness of the chain, which is responsible for increased tensile strength. The broad amorphous peaks observed in the X-ray diffractogram demonstrates molecular miscibility and the interaction among essential oil, starch and protein. This observation corroborated with the microstructure of the blend. Similar observations of broad amorphous peaks have been earlier reported for whey protein and lotus rhizome starch blends (Sukhija et al., 2016), cassava starch, chitosan and gelatin blend films (Zhong and Xia, 2008) and pullulan-chitosan blend films (Wu et al., 2013). The thermal degradation of the blend produced with renewable sources and oregano essential oil was observed mainly in three stages (Fig. 3). Initially, there was a slight weight loss around 100 ◦ C, probably due to loss of water molecules linked through hydrogen bonds to hydrophilic components, such as starch, and some protein chains (Mariani et al., 2009). Higher thermal degradation (46.8% weight loss) was observed between 218 and 312 ◦ C. This second stage probably reflects the loss of glycerol from the blend and protein degradation (Kadam et al., 2013). The depolymerization of amylose, characterizing the beginning of the thermal degradation of the starch, probably also occurs at this stage (Bhat et al., 2013). The third degradation stage probably corresponds to the oxidation of starch, that was partially decomposed in an earlier step, and some fractions of proteins more tightly bound to the matrix (Arfat et al., 2014). The thermogram showed that the rice starch/fish protein blend is stable up to nearly 190 ◦ C, and this result might be associated with the presence of oregano essential oil. Tongnuanchan et al. (2013) explained that the addition of essential oils is likely correlated with the discontinuity or interrupted starch-protein interactions. The maximum rate of degradation was approximately 245 ◦ C, and the total weight loss was 84.1%. The residual mass is probably due to the presence of inorganic compounds after thermal degradation (Fakhouri et al., 2013; Mariani et al., 2009). One main peak and a broad peak can be observed of mass loss in derivative of the mass curve, with maximum at 245 ◦ C and 274 ◦ C. These two stages of mass loss could be associated with the degradation of starch/protein and the evaporation/oxidation of oregano essential oil separately. Similar trend was observed by Morelli et al. (2015) in the development of active incorporation copaiba oil in paper and poly(lactic acid). The DSC thermogram of rice starch/fish protein film is presented in Fig. 4. A single endothermic peak between 123 and 177 ◦ C indicates the homogeneity of the blend as well as miscibility of components. This endothermic peak has been associated with the melting of crystalline starch domains reorganized during retrogradation (Almasi et al., 2010). The DSC analysis presented melting point at 138 ◦ C and enthalpy (H) of 73.25 J g−1 . Al-Hassan and Norziah (2012) obtained a melting point between 133 and 157 ◦ C and enthalpies of 161–177 J g−1 when preparing sago starch and fish protein blends. Similar results have been also reported in cassava starch/gelatin blends (Tongdeesoontom et al., 2012) and whey protein/lotus rhizome starch (Sukhija et al., 2016). The glass transition temperature (Tg) was 133 ◦ C, and the mobility of the polymer chains, passing from a glassy state to a less

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Fig. 3. Thermogravimetric analysis of 50/50 starch/protein blend incorporated with 8% of oregano essential oil.

thermal properties presented. The active polymer showed 63.1% of inhibition of enzyme peroxidase, thus it is appropriate to develop materials for foods that enzymatic browning is undesirable, such as fruits and vegetables. Besides the important characteristics of the blends prepared in the present study, further research on the evaluation of the physical stability and application in food systems are also required.

Acknowledgments

Fig. 4. DSC thermogram of 50/50 stach/protein blend incorporated with 8% of oregano essential oil.

ordered state, also initiated at this temperature. This result is consistent with the X-ray diffraction, confirming the presence of crystalline regions in the matrix. According to Su et al. (2010) the presence of only one Tg indicates that the blend components are miscible and compatible, as observed in the film of the present study. 4. Conclusion Rice broken grains and the Whitemouth croaker fish were good sources to obtain starch and muscle proteins, respectively. These raw materials were appropriate to develop blends for eco-friendly and low cost food packaging due to their ability to form cohesive films. Different ratios of rice starch/fish protein strongly influenced mechanical properties, solubility and color parameters of the blends. Lower influence was observed in water vapor permeability and opacity. Fish proteins were responsible to form films with higher mechanical properties, solubility and WVP. The ratio 50/50 of starch/protein presented the most suitable results, lower WVP and water solubility and promising mechanical properties, then this blend was studied as its antioxidant activity, morphology and thermal properties. The incorporation of oregano essential oil into 50/50 rice starch/fish protein blend was successfully performed to obtain active blends due to the smooth and homogeneous structure and

This work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil. The authors are grateful to CEME-SUL/FURG (Centro de Microscopia Eletrônica do Sul/Universidade Federal do Rio Grande do Sul/Brazil) for SEM analysis.

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