Preparation and characterization of edible chicken skin gelatin film ...

Food Packaging and Shelf Life 15 (2018) 1–8

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Preparation and characterization of edible chicken skin gelatin film incorporated with rice flour P.Y. Soo, N.M. Sarbon

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School of Food Science and Technology, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia

A R T I C L E I N F O

A B S T R A C T

Keywords: Edible film Chicken skin gelatin Rice flour Functional properties

The novelty of this study is producing an edible composite film from chicken skin gelatin, waste of poultry processing industry with incorporation of rice flour. This alternative materials would replace many food packaging which are made of petrochemical and non-biodegradable in which generated many excessive waste which lead to environmental pollution and serious ecological problems. Thus, the aim of this study is to formulate and evaluate chicken skin gelatin films incorporated with rice flour. Characteristics such as film solubility, light transmission and transparency, water vapor permeability, thermal properties, morphology, crystallinity, tensile strength, elongation at break and Young’s Modulus were evaluated. Varying concentrations (0, 5, 10, 15, 20 and 25%, w/w) of rice flour were added to chicken skin gelatin films using casting technique. The results show that the addition of rice flour increased the water vapor permeability while decreasing film solubility (p < 0.05). Furthermore, ultraviolet and visible light transmission decreased with the transparency of films increased (p < 0.05) while thermal properties (Tm) increased with the concentration increased. The surface of films became smoother and non-porous while X-ray diffraction (XRD) analysis showed crystalline nature of films improved. The resulted films also demonstrated higher tensile strength and elongation at break (p < 0.05). Nevertheless, addition of rice flour did not significantly influence (p > 0.05) the Young’s Modulus measurement. Overall, chicken skin gelatin films with rice flour at 20%, (w/w) demonstrated better characteristics than other films.

1. Introduction

casein and mung bean protein films are certain examples of proteinbased films (Wittaya, 2012). Numerous studies have reported potential uses for whey, soy and wheat–gluten protein in protein based films, emphasizing in particular its good barrier properties (Schmid, 2013; Zink et al., 2016). However the information of using gelatin as protein based films still limited. Gelatin films have strong potential for commercial application as food packaging films due to their unique characteristics (Langmaier, Mokrejs, Kolomaznik, & Mladek, 2008; Viera, Da Silva, Santos, & Beppu, 2011; Mikkonen et al., 2012). For example, chicken skin gelatin has higher gel strength and viscous and elastic moduli. It contains higher level of alpha-helix and β-sheet type structure as well as strong hydrogen bonding (Sarbon, Badii, & Howell, 2013). Gelatin films can be applied either directly or indirectly to food surfaces to protect against microbial growth, salt rust, grease bleeding, handling abuse, water transfer, moisture loss, and oil adsorption during frying (Cutter, 2006). However, gelatin films encounter the same problem as most protein films, as they are a poor water vapor barrier, which limits applications in edible films. Therefore, the functionality of gelatin films can be

Food packaging plays an important role in the food industry. The main function of food packaging is to protect food products from the ambient environment, which may affect the quality of food (Schmid, 2013). Different types of food packaging are available in the market, such as plastic, metal, paper, and glass. Use of such food packaging may lead to increased waste generation and environmental problems (Marsh & Bugusu, 2007). Therefore, modern consumers are more strongly demanding environmentally friendly packaging to maintain the shelf life and quality of food (Rhim, Gennadios, Handa, Weller, & Hanna, 2000). Recent studies have shown interest in the development of environmental friendly packaging, such as edible films. Protein-based films are an excellent example due to their potential as a substitute for synthetic packaging in the food industry. Although protein-based films are gaining interest to researchers as alternative to traditional petroleumbased materials, their mechanical and barrier properties need to be enhanced in order to match those of the latter (Zink, Wyrobnik, Prinz, & Schmid, 2016). Collagen, gelatin, corn zein, wheat gluten, soy protein,

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Corresponding author. E-mail address: [email protected] (N.M. Sarbon).

https://doi.org/10.1016/j.fpsl.2017.12.009 Received 11 August 2017; Received in revised form 21 December 2017; Accepted 22 December 2017 2214-2894/ © 2017 Elsevier Ltd. All rights reserved.


P.Y. Soo, N.M. Sarbon

a cabinet drier at 40 °C. Completely dried skins were then ground up with a Waring blender before being defatted by using Soxhlet method (AOAC, 2006). After defatting, the dried weight of chicken skins was measured and they were kept for further use.

improved by blended with different types of polysaccharide such as chitosan, rice flour and xanthan gum (Ahmad et al., 2015; Hosseini, Rezaei, Zandi, & Ghavi, 2013; Nur Hazirah, Isa, & Sarbon, 2016). In addition, proteins are generally superior to polysaccharides in their ability to form films with high mechanical and barrier properties besides provide higher nutritional value (Zink et al., 2016). Polysaccharides are one of the polymers which can add together to form composite film. The main polysaccharides used in edible films are chitosan, starch, alginate, carragennan, modified cellulose, pectin, pullulan, gellan gum, xanthan gum, rice flour, and so on (Han & Gennadios, 2005). Rice flour is a starchy material derived from rice (Oryza sativa L.). The main chemical component of rice is starch, comprising around 90% of its dry weight, with the protein and lipid which contribute about 6.5% and 0.8%, respectively (Zhou, Robards, Helliwell, & Blanchard, 2002). Rice flour’s relative abundance, inherent biodegradability, low cost and desirable performance make it an alternative material to produce edible films (Priya, Gupta, Pathania, & Singha, 2014). Previous studies have shown that films incorporating with rice flour had lower tensile strength, elongation at break; higher water vapor permeability but lower film solubility (Ahmad et al., 2015; Dias, Muller, Larotonda, & Laurindo, 2011). Quality of films evaluated based on the functional properties, such as physical and mechanical properties. The physical properties of edible films usually evaluated in previous studies are film solubility (Tongdeesoontorn, Mauer, Wongruong, Sriburi, and Rachtanapun, 2012), light transmission and film transparency (Nur Hazirah et al., 2016), water vapor permeability (Jahit, Nazmi, Isa, & Sarbon, 2016), thermal properties (Sanyang, Sapuan, Jawaid, Ishak, & Sahari, 2015), crystallinity (Suderman, Isa, & Sarbon, 2016) and morphology of film (Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015). The mechanical properties that usually characterize edible films are tensile strength, elongation at break (Nur Hazirah et al., 2016) and Young’s Modulus (Schmid, 2013). Previous studies showed that incorporation of different types of polysaccharide into gelatin films decreased film solubility, transparency, water vapor permeability while increasing the melting temperature and crystallinity of films (Ahmad et al., 2015; Hosseini et al., 2013; Nur Hazirah et al., 2016). Additionally, combining different types of polysaccharide into gelatin films produces different surface morphologies as observed by scanning electron microscope (SEM), either with a smooth and compact surface or amorphous region (Al-Hassan & Norziah, 2012; Soliman & Furuta, 2014). In addition, according to Hammann and Schmid (2014), the qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography) and analysis of solid films (spectroscopy techniques, X-ray scattering methods). Therefore, the objective of this study is to prepare edible chicken skin gelatin films incorporated with rice flour and characterize its functional properties.

2.2.2. Gelatin extraction Gelatin was extracted according to the method described by Sarbon et al. (2013) with some modification. Defatted dried chicken skins were dissolved into 0.15% (w/v) sodium hydroxide. The solution was shaken well and stirred at room temperature for 30 min before centrifuging at 3500 x g for 10 min. These steps were repeated when defatted chicken skins were dissolved into 0.15% (w/v) sulfuric acid and 0.7% (w/v) citric acid solutions. Each treatment was repeated three times. After that, the pellets were washed with distilled water in order to remove residual salts and centrifuged again at 3500 x g for 15 min. The final extraction was placed in distilled water and the solution was placed overnight in a water bath shaker at 45 °C. The resultant mixture was filtered in a Buchner funnel with filter paper. The volume of the solution was reduced to 1/10 at 45 °C by rotary evaporator before freezedried. The dried gelatin powder was ground, weighed, and stored for further analysis. Gelatin yield was calculated based on the dried weight of chicken skins and expressed as a percentage: Yield (%) = Weight of gelatin powder (g)/dried weight (g) × 100

2.2.3. Formation of chicken skin gelatin films Composite films were prepared by blending rice flour, chicken skin gelatin and glycerol as plasticizer together according to Al-Hassan and Norziah (2012) and Ahmad et al. (2015) with some modification. Six groups of composite films with different ratios of gelatin to rice flour were prepared: A (100/0), B (100/5), C (100/10), D (100/15), E (100/ 20) and F (100/25). Glycerol (30%, w/w total solid) was used to plasticize film formation and 100 mL distilled water was added to form a solution. Rice flour was dissolved in distilled water and heated with magnetic stirring in a water bath at 75 °C until completely gelatinized. Chicken skin gelatin powder was dissolved in distilled water at 45 °C until a clear solution was obtained. Next, gelatin solution was added into gelatinized rice flour at 45 °C and continued stirring for 30 min, followed by addition of plasticizer with constant stirring for another 30 min. The film forming solution was then cooled to room temperature. After that, the solution was cast on flat petri dish and dried in a ventilated oven at 45 °C for 3 days. To obtain uniform thickness, each petri dish was allocated with film forming solution by 25 g. The dry films obtained were peeled off and kept in a tight container for further analysis. Control films were prepared in the same way without the addition of rice flour.

2. Materials and methods

2.3. Physical properties of chicken skin gelatin films

2.1. Materials 2.3.1. Film solubility Film solubility was determined following the method of Tongdeesoontorn et al. (2012). Films with dimensions of 2 cm × 2 cm were dried in an oven at 70 °C for 24 h. Their initial dried weight was then measured. Next, the films were immersed into 50 mL screw cap tubes containing 20 mL of distilled water and 0.01 g/100 g sodium benzoate and put in a shaking water bath for 24 h at 25 ± 2 °C. Then, the film pieces and solution were passed through filter paper and dried at 70 °C in an oven for 24 h. The dried filtrate with film residues were weighed to determine their final dried weight. Tests were performed in triplicate. Solubility (%) was calculated using the following equation:

Rice flour was purchased from a local market in Kuala Nerus, while fresh chicken skins were obtained from TD Poultry Sdn. Bhd. Kuala Terengganu, Malaysia. The chicken skins were stored in ice during transportation to the laboratory. The skins were thoroughly washed and stored at −18 °C. All chemicals used for analysis were of analytical grade. 2.2. Methods 2.2.1. Chicken skin preparation The frozen chicken skins were thawed in a chiller overnight. After thoroughly rinsing with excessive water to remove impurities, the visible fat on the chicken skins was removed before overnight storage in

Film solubility(%) =

2

(Initial dried weight − Final dried weight) × 100 Initial dried weight



Modulus (YM) of the films were determined using a Texture Analyzer (Stable Microsystem, TA.XT Plus, Godalming, Surrey, United Kingdom) according to Nur Hazirah et al. (2016) and Schmid (2013) with slight modification. The films were cut into 1 cm × 7 cm from each film formulation. The film strips were placed onto grip pairs of AT/G probe which are attached to the texture analyzer with 5 kg load cell. The initial gap separation was set to 50 mm. The film strips were then stretched by moving the upper grip at head speed of 1 mm/s until the film broke. The tensile strength (TS) was calculated as follows:

2.3.2. Light transmission and film transparency Light transmission of the films in terms of ultraviolet (UV) and visible light was measured at the wavelength between 200 and 800 nm using an UV–vis spectrophotometer (Pharmaspec Shimadzu UV-1700, Kyoto, Japan) according to the method described by Nur Hazirah et al. (2016). The film samples were cut into 4 cm × 1 cm strips and placed into cuvettes. An empty cuvette was used as reference. The transparency of the film was calculated with the following equation: Transparency = −log T/x where T is the transmission (%) at 600 nm and x is the thickness of film (cm). Each sample was tested in triplicate.

Tensile strength (MPa) = Fmax(N)/A (mm2) where Fmax is max load (N) needed to pull the sample apart and A is cross sectional area (mm2) of film sample. The elongation at break (EAB%) was calculated as follows:

2.3.3. Water vapor permeability (WVP) The water vapor permeability (WVP) of the films was determined according to method of Jahit et al. (2016). Films were cut into 3 cm × 3 cm and sealed on the plastic cups which containing 10 g of silica gel (0% RH). The cups were individually weighed and placed in desiccators with distilled water at room temperature. The cups were then weighed ( ± 0.0001 g) hourly for 8 h. The water vapor permeability (WVP) was calculated as follows:

EAB (%) = (lmax/l0) × 100 where lmax is the film elongation (mm) at the moment of rupture and l0 is the initial grip length (mm) of the sample. Young’s Modulus (YM) was calculated as follows: Young’s Modulus (MPa) = Stress (MPa)/Strain

WVP (gmm/m2 h Pa) = w × x/A t × (P2-P1)

where stress is load (N) divided by area (mm2) and strain is change in length (mm) divided by original length (mm).

where w is the weight gained by the cup (g), x is the average film thickness (mm), A is the permeation area (m2), t is the time gained (h) and P2 − P1 is the difference of partial pressure (Pa).

2.5. Statistical analysis

2.3.4. Thermal properties of film The thermal properties of films were measured by DSC Q2000 TA Instruments, Pittsburgh, PA USA. About 5 mg ( ± 0.001 mg) of each film sample was weighed and placed in an aluminum sample pan, then hermetically sealed. An empty sample pan was used as a reference. Film samples were heated at a scanning rate of 10 °C/min from 10 to 150 °C. Nitrogen gas was used to flush the DSC cell at a flow rate of 20 mL/min to create an inert environment. The melting point, Tm (°C) was determined as the temperature at which the endothermic peak occurred in the thermograms. From the melting curves obtained, the melting transition temperatures, Tm (°C) within the designated area limits were determined, which appeared as peaks (Sanyang et al., 2015).

All analysis is presented in a data of means ± standard deviations. Statistical tests were performed by using Minitab version 14 for Windows (Minitab Inc., USA). One-way ANOVA was carried out, and differences between pairs of means were assessed on the basis of confidence intervals using Fisher’s test. The level of significance was p < 0.05. 3. Results and discussion 3.1. Physical properties of chicken skin gelatin films 3.1.1. Film solubility Table 1 presents the solubility of edible chicken skin gelatin films at different formulation. The film solubility of edible chicken skin gelatin films significantly decreased (p < 0.05) from 93.66 to 82.91% with increased levels of rice flour (0 to 25%). There is a significant difference (p < 0.05) between Films C and D. However, no significant difference (p > 0.05) was noted in Films B and C, as well as Films D and E, indicating these films would soluble in water at the same extent regardless the concentration of rice flour added. Film A (control) exhibited the highest film solubility (93.66%) while Film E (20% rice flour) showed

2.3.5. Morphology of film The morphology of surface and cross-section of film samples were observed using a scanning electron microscope (SEM) (Tabletop Microscope TM1000, Tokyo, Japan). The samples were fractured under liquid nitrogen before observing the cross section of the films. Next, the samples were mounted on bronze stub and sputtered with gold to make the sample conductive. The images were captured at acceleration voltage of 10 kV with magnification ranging from 300 to 1500× (Tongnuanchan et al., 2015).

Table 1 Film solubility, water vapor permeability and melting point of edible gelatin.

2.3.6. Crystallinity of film The X-ray patterns of films were analyzed using an X-ray diffractometer (MiniFlex II, Rigaku, Japan) according to the method of Suderman et al. (2016). The X-ray diffractometer was equipped with a copper source at a voltage of 30 kV and current of 15 mA. Film samples were cut into 3 cm × 3 cm and placed on the glass slides, then secured with tapes before being placed in the diffractometer chamber for measurement. The angle diffraction ranges and scanning times were set between 2θ = 1- 60° and 2° per minute, respectively.

Film formulation

A B C D E F

2.4. Mechanical properties of chicken skin gelatin films

Film solubility (%)

93.66 88.58 88.40 84.09 82.88 82.91

± ± ± ± ± ±

1.94a 1.35b 1.62b 4.17c 2.13c 1.34c

Water vapor permeability (x 10−3) (gmm/ m2hPa) 2.14 2.46 2.63 3.33 5.00 3.65

± ± ± ± ± ±

0.14e 0.08d 0.09d 0.14c 0.14b 0.15a

Melting point (Tm) (°C) 1st peak

2nd peak

49.51 49.81 50.24 50.30 51.63 51.48

– 124.97 126.13 124.95 129.25 129.17

Film A (Control, 0% rice flour); Film B (5% rice flour); Film C (10% rice flour); Film D (15% rice flour); Film E (20% rice flour); Film F (25% rice flour); all data represent mean ± standard deviation; the different superscript letters a–c indicate significant difference (p < 0.05).

2.4.1. Tensile strength (TS), Elongation at break (EAB) and Young’s Modulus (YM) The tensile strength (TS), elongation at break (EAB) and Young’s 3



the lowest film solubility (82.88%). A lower film solubility is required for storage, while a higher solubility of film is useful when edible films are needed to coat food products for cooking (Maizura, Fazilah, Norziah, & Karim, 2007). The decreased solubility of Films B, C, D, E and F may be due to formation of hydrogen bonds between OH groups of gelatin and functional groups of rice flour, which reduced the number of available OH groups for binding water molecules. These intermolecular interactions improved the water resistance and stability of composite films (Hoque, Benjakul, & Prodpran, 2010; Limpan, Prodpran, Benjakul, & Prasarpran, 2012). Further, physical interference occurred in the gelatin polypeptide chains, which lead to a significant blockage of gelatin ability to interact with water molecules, thus reducing the solubility of films (Hosseini et al., 2013). The results in this study were consistent with a study by Ahmad et al. (2015) which reported that the film solubility of fish gelatin films decreased (94.46 to 57.96%) with an increased level of rice flour. Hosseini et al. (2013) also found a reduction of water solubility of plasticized fish gelatin film (63.81 to 29.96%) as a result of the addition of chitosan. Therefore, the addition of rice flour could decrease the solubility of chicken skin gelatin films. Film E (20% rice flour) showed the lowest film solubility.

region (350–800 nm). There was a significant difference (p < 0.05) between Films A and F. The interaction between gelatin and hydrocolloids such as rice flour in the film matrix and the presence of amylose and amylopectin in rice flour absorbed the energy of incoming light, thus reducing the visible light transmission through the films (Ahmad et al., 2015; Prodpran, Benjakul, Vittayanont, & Nalinanon, 2013). Ahmad et al. (2015) also demonstrated that the addition of rice flour provided a barrier to visible light, since visible light transmission decreased in the fish gelatin films. In contrast, Hosseini et al. (2013) found that visible light transmission increased as the concentration of chitosan increased in the fish gelatin films. The transparency values in edible chicken skin gelatin films increased significantly (p < 0.05) from 1.94 to 3.06 with the increased levels of rice flour. These findings strongly suggest that the higher concentrations of rice flour decreased the transparency of gelatin films. There is a significant difference (p < 0.05) between Films A and F. However, no significant difference (p > 0.05) was noted in Films B, C, D and E, indicating that these films had similar transparency. The increased transparency values in this study may be due to the crosslinking reaction between gelatin and rice flour, contributed to the compactness of film matrix, thus decreased the transparency of gelatin films. These findings were consistent with a study by Nur Hazirah et al. (2016) which reported that the transparency values of gelatin-CMC films increased after the addition of xanthan gum. Therefore, the addition of rice flour influenced the UV and visible light transmission and transparency of gelatin films. Thus, Film F (25% rice flour) showed the highest light transmission barrier in UV and visible range, but was less transparent than Film A (control).

3.1.2. Light transmission and film transparency Table 2 shows the transmission of UV (200–280 nm) and visible light (350–800 nm) the transparency values (600 nm) of edible chicken skin gelatin films at different concentrations of rice flour. The UV light transmission of edible chicken skin gelatin films decreased from 0.16 to 0.06% at 200 nm and from 5.89 to 0.14% at 280 nm with an increase in rice flour concentration. There was a significant difference (p < 0.05) noted in Films A and F. However, no significant difference (p > 0.05) was noted in Films A and B; Film D and C; Film E and F, showed that these films had the similar potential in barrier properties against UV light. The decreased in UV light transmission was probably due to the presence of aromatic amino acids (Tyr, Phe and Trp) in gelatin molecules and rice flour. This finding has been supported by studies by Guerrero, Nur Hanani, Kerry, and de le Caba (2011), in which sensitive chromophores were shown to absorb light at wavelengths below 300 nm. These findings parallel a study of Ahmad et al. (2015) which reported the UV light transmission at 280 nm decreased (50.08 to 10.31%) as rice flour concentration increased. Nur Hazirah et al. (2016) also showed that addition of xanthan gum significantly decreased (p < 0.05) light transmission at 200 (0.09 to 0.02%) and 280 nm (2.39 to 0.09%), indicating that xanthan gum improved the barrier properties of blended films against UV light. For visible light transmission (350–800 nm), values were similar to those for UV light transmission, as an increasing content of rice flour in the film formulation significantly lowered (p < 0.05) the transmission of visible light. Film A (control) exhibited the highest visible light transmission (41.86 to 51.97%), while Film F (25% rice flour) showed the lowest visible light transmission (15.29 to 40.95%) in the visible

3.1.3. Water vapor permeability (WVP) Table 1 shows the values of WVP of edible gelatin films at different formulation of rice flour. The WVP values of edible chicken skin gelatin films were significantly increased (p < 0.05) from 2.14 to 5.00 × 10−3 gmm/m2hPa with the increased levels of rice flour. There is a significant difference (p < 0.05) between Films A and E. However, no significant difference (p > 0.05) was noted in Films B and C, indicating that these films possessed similar degree of hydrophilicity. Film A (control) exhibited the lowest WVP value (2.14 × 10−3 gmm/ m2hPa), while Film E (20% rice flour) showed the highest WVP value (5.00 × 10−3 gmm/m2hPa). WVP is strongly dependent on the relative polarity of the polymer used. Water can interact with polymer matrix if the films are cationic and strongly hydrophilic, thus increasing the values of WVP (Rawdkuen, Sai-Ut, & Benjakul, 2010). The lowest WVP value shown in Film A (control) may be due to the presence of hydrophobic amino acids in the structure of gelatin causes it to attract less water (Al-Hassan & Norziah, 2012). The increase in WVP values from Film A to F is most probably because of the greater water affinity and hydrophilic nature of rice flour, which promotes the migration of water vapor molecules through the gelatin films (Ahmad et al., 2015). Additionally, films containing linear polymeric chains such as gelatin molecules can be

Table 2 Light transmission and film transparency of edible gelatin films at different formulation of rice flour. Film formulation

Light transmission at different wavelength (%) 200 nm

A B C D E F

0.16 0.15 0.11 0.11 0.08 0.06

± ± ± ± ± ±

280 nm 0.03a 0.02a 0.06ab 0.05ab 0.02b 0.03b

5.89 3.89 1.25 0.38 0.28 0.14

± ± ± ± ± ±

350 nm 0.21a 0.27b 0.18c 0.10d 0.07d 0.05d

41.86 36.88 27.57 24.09 20.16 15.29

± ± ± ± ± ±

Transparency values at 600 nm 400 nm

3.08a 1.43b 4.04c 2.01cd 1.93d 1.55e

45.14 42.93 34.35 33.62 30.33 24.62

± ± ± ± ± ±

500 nm 2.15a 0.97a 3.03b 1.39b 1.98b 2.22c

45.78 45.77 39.71 38.15 37.37 31.77

± ± ± ± ± ±

600 nm 2.07a 0.72a 1.15b 3.66b 1.21b 3.07c

48.36 47.53 43.39 41.55 38.17 35.96

± ± ± ± ± ±

700 nm 0.65a 1.98a 1.25b 0.90b 1.13c 3.52c

49.76 48.77 45.65 43.89 39.31 38.38

± ± ± ± ± ±

800 nm 0.58a 2.04ab 1.04bc 0.81c 1.26d 3.74d

51.97 50.96 48.01 46.62 42.56 40.95

± ± ± ± ± ±

0.51a 2.10a 2.84ab 0.78b 2.84c 3.97c

1.94 2.45 2.55 2.63 2.80 3.06

± ± ± ± ± ±

0.20b 0.36ab 0.33ab 0.21ab 0.30ab 0.52a

Film A (Control, 0% rice flour); Film B (5% rice flour); Film C (10% rice flour); Film D (15% rice flour); Film E (20% rice flour); Film F (25% rice flour); all data represent mean ± standard deviation; the different superscript letter a–e in the same column indicate significant difference (p < 0.05).

4



incorporated with 25% rice flour. The purpose of choosing Film B and F is to compare the contrast in adding the least and the most concentration of rice flour into the films. For the surface section, Film A (control) had rough and bumpy surface. After addition of rice flour, Film B (5% rice flour) showed compact, flat and homogenous surface. No distinction separation was observed in the matrix of composite film. However, a relatively rough, slightly irregular and protruded surface was found in Film F (25% rice flour). The compact, flat and homogenous surface showed in Film B may be due to the intermolecular interaction in the film matrix were favored by gelatin and 5% rice flour while the entanglement of gelatin and 25% rice flour chains via covalent and non-covalent bonding promoted the roughness of the surface of Film F (Arfat, Benjakul, Prodpran, & Osako, 2014; Shakila, Jeevithan, Varatharajakumar, Jeyasekaran, & Sukumar, 2012). Dias, Muller, Larotonda, and Laurindo (2010) also reported that the surface of films prepared with rice flour were more irregular due to the presence of more than one macromolecule in the polymeric matrix, such as starch, protein and lipid. For cross section, Film A (control) exhibited a rough laminated surface. A porous and rough network was found in Film B (5% rice flour). However, a non-porous and smooth cross section was noticeable in Film F (25% rice flour), indicating addition of rice flour could promote the smoother cross section in gelatin films. This may be due to the homogenous network and good compatibility between gelatin and rice flour (Ahmad et al., 2015). The observations were in the agreement with Pranoto et al., (2007) who found that addition of gellan could eliminate the cracks in the fish gelatin films, thus providing a more compact cross-sectional appearance. Ahmad et al. (2015) also supported that addition of rice flour into fish gelatin films could produce a smooth and non-porous cross section appearance. However, Al-Hassan and Norziah (2012) observed that discontinuous zones and cracks randomly distributed along the network in the fish gelatin films after addition of sago starch. Thus, the addition of rice flour could influence the surface and cross section of chicken skin gelatin films, as Film F (25% rice flour) had a smooth and non-porous cross section with some protruded surface in this study.

firmly packed, whereas polymeric molecules with voluminous chains such as polysaccharide molecules are more loosely packed, showing greater permeability (Lee & Yoo, 2011). The findings in this study are consistent with a previous study by Nur Hazirah et al. (2016), which reported that values of WVP increased (24.40 to 36.38 gmm/m2dkPa) after incorporation of xanthan gum. Additionally, Ahmad et al. (2015) observed that the values of WVP increased (3.05 to 4.42 × 10−10 gs−1m−1Pa−1) after addition of rice flour. Nevertheless, Hosseini, et al. (2013) obtained contradict readings which the values of WVP decreased (0.826 to 0.410 gmm/kPahm2) in gelatin films when the chitosan content increased. Thus, the incorporation of rice flour influenced the WVP of gelatin films and film E (20% rice flour) presented the highest WVP value compared to the others. The WVP values that obtained in this study showed higher values as compared to the WVP values of protein based films from whey protein isolate (WPI) and wheat of gluten (WG) as reviewed by Zink et al. (2016). Referring to the barrier properties as described by Schmid et al. (2012), the studied film’s properties had a similar barrier properties as whey based-layer. In which, the dense crosslinked protein network provided high barrier properties and thus confirmed the potential of protein based films to replace EVOH with average ethylene contents. 3.1.4. Thermal properties of film The melting temperature (Tm) of edible chicken skin gelatin films at different rice flour concentrations are shown in Table 1. The melting temperature increased from 49.51 to 51.63 °C at 1st peak and from 124.97 to 129.25 °C at 2nd peak as the rice flour concentration increased in chicken skin gelatin films. Film A (control) exhibited a single endothermic peak (Tm) at 49.51 °C, while Films B to F displayed two separated endothermic peaks (Tm). The appearance of endothermic peak was mostly due to the breakage of hydrogen bond, followed by the overlapping of different process such as water evaporation, melting and recrystallization, as well as the imperfections in the gelatin crystallites and association of glass transition of α-amino acid blocks in the polypeptide chain (Dai, Chen, & Liu, 2006; Langmaier et al., 2008). Increased in the melting temperature (Tm) of chicken skin gelatin films in this study may be due to the addition of rice flour promoted the crosslinking reaction in the film matrix and reduced the mobility of biopolymer chains, thus producing highly heat stable films (Zolfi, Khodaiyan, Mousavi, & Hashemi, 2014). A single endothermic peak shown in Film A was related to the melting crystalline domains of gelatin and the absence of rice flour (Hoque et al., 2010). Furthermore, the two separated endothermic peaks displayed in Films B to F were probably due to two different ordered structures in the composite film matrix, one dominated by chicken skin gelatin (at low Tm) and other governed by rice flour (at higher Tm), caused by the partial immiscibility of their molecules (Ahmad et al., 2015). The findings in this study were parallel with Pranoto, Lee and Park, (2007) who showed that addition of gellan and carrageenan increased the melting temperature (Tm) of gelatin films from 24.55 to 39.20 °C and from 24.55 to 28.55 °C, respectively. Besides, Ahmad et al. (2015) observed that there were two separated endothermic peaks at Tm of 64.88 °C and 279.03 °C displayed in the rice flour-fish gelatin composite film, which the low Tm was dominated by fish gelatin and high Tm was contributed by rice flour. Therefore, the addition of rice flour improved the thermal properties of chicken skin gelatin films, especially Film E (20% rice flour), which showed the highest melting temperature (Tm) amongst all films.

3.1.6. Crystallinity of film X-Ray diffraction (XRD) diffractograms of edible chicken skin gelatin films at varying formulations of rice flour are presented in Fig. 1. All films, including Film A (control), possessed an amorphous state. The intensity of peaks increased when the amount of rice flour increased in gelatin films, indicating the crystalline nature of films were improved. Film A presented amorphous characteristics, with a broad peak located at 2θ = 20.32°, while Film F had a semi-crystalline region, as observed in Fig. 1. The amorphous state showed in all films in this study were likely the result of structural contributions from the principle components such as gelatin and rice flour which are in agreement with Nur Hazirah et al. (2016). Besides, lack of re-crystallization and slower water evaporation process during the production of films promotes an amorphous state in films (Bergo & Sobral, 2007; Flores, Fama, Rojas, Goyanes & Gerschenson 2007). Moreover, the intensity of peaks increased when the concentration of rice flour increased, Thus, Film F had a more semicrystalline region compared to control (Jahit et al., 2016). The strong networks formed between anionic domains of polysaccharides and cationic domains of gelatin can produce a semi-crystalline region (Soliman & Furuta, 2014). The results in this study are in agreement with the studies of Rivero, Garcia, and Pinotti (2010) and Shehap, Mahmoud, Abd El-Kader, and ElBasheer (2015) which reported that the gelatin film possessed a broad peak located at 2θ = 20°. Jahit et al. (2016) also showed that the films’ crystalline nature improved with increased intensity after addition of chitosan into gelatin films. Therefore, the addition of rice flour could enhance the crystallinity of chicken skin gelatin films. Film F (25% rice flour) showed the highest crystalline structure amongst all films.

3.1.5. Morphology of film SEM micrographs of surface and cross section of edible chicken skin gelatin films at different formulation of rice flour are presented in Table 3. Films A, B and F were chosen to demonstrate the difference in surface and cross section structure morphology between gelatin film, gelatin film incorporated with 5% rice flour and gelatin film 5



Table 3 SEM micrographs of surface and cross-section of edible gelatin films at different formulation of rice flour. Film Formulation

A

B

F

Surface

Cross section

Film A (Control, 0% rice flour); Film B (5% rice flour); Film F (25% rice flour).

3.2. Mechanical properties of chicken skin gelatin films

Table 4 Tensile strength, elongation at break and Young’s Modulus of edible gelatin films at different formulation of rice flour.

3.2.1. Tensile strength (TS) and elongation at break (EAB) Values of tensile strength (TS) and elongation at break (EAB) of edible chicken skin gelatin films at different formulation of rice flour are summarized in Table 4. The TS and EAB values were significantly increased (p < 0.05) from 1.54 to 2.91 MPa and 48.33 to 79.31%, respectively, for Films A and E. The TS and EAB values decreased from 2.91 to 2.30 MPa and 79.31 to 58.45%, respectively for Film F. There is significant difference (p < 0.05) found in Film A and E. However, no significant difference (p > 0.05) was noted in Films B, C and D as well as Films D and E, indicating that these films performed the similar tensile strength and extensibility, regardless of the percentage of rice flour added. Film E (20% rice flour) possessed the highest TS (2.91 MPa) and EAB (79.31%), while Film A (control) exhibited the lowest TS (1.54 MPa) and EAB (48.33%). Generally, increased TS values followed by a decreased in EAB values (Tong, Xiao, & Lim, 2008). A packaging film typically requires adequate mechanical strength and extensibility to resist external stress

Film formulation

Tensile strength (MPa)

A B C D E F

1.54 2.08 2.17 2.40 2.91 2.30

± ± ± ± ± ±

0.37b 0.16ab 0.06ab 0.25ab 0.43a 0.82ab

Elongation at break (%) 48.33 61.36 73.71 75.76 79.31 58.45

± ± ± ± ± ±

3.55c 3.32bc 1.77ab 6.35a 5.46a 9.14c

Young’s Modulus (MPa) 3.20 3.39 2.94 3.19 3.74 3.15

± ± ± ± ± ±

0.76a 0.07a 1.45a 0.44a 0.71a 0.86a

Film A (Control, 0% rice flour); Film B (5% rice flour); Film C (10% rice flour); Film D (15% rice flour); Film E (20% rice flour); Film F (25% rice flour); all data represent mean ± standard deviation; the different superscript letter a−e in the same column indicate significant difference (p < 0.05).

and maintain its integrity and barrier properties (Rao, Kanatt, Chawla, & Sharma, 2010). The increased TS and EAB values in this study may be due to the desire amount of rice flour added into gelatin polymer chains Fig. 1. XRD diffractograms of edible gelatin films at different formulation of rice flour Film A (Control, 0% rice flour); Film B (5% rice flour); Film C (10% rice flour); Film D (15% rice flour); Film E (20% rice flour); Film F (25% rice flour).

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and reacted with the available hydroxyl groups in rice flour, thus formation of strong hydrogen bond, lowered the molecular mobility and resulted in higher chain entanglement as well as higher molecular slippage upon tensile deformation (Arfat et al., 2014; Ahmad et al., 2015). Besides, excess levels of rice flour added into films interfered with the interaction with gelatin molecules in the film matrix. For example, starch intra-molecular hydrogen bonds rather than inter-molecular hydrogen bonds are formed, resulting in a phase separation between the two main components and non-uniform network, thus decreasing TS and EAB in Film F (Prodpran et al., 2013; Xu, Kim, Hanna, & Nag, 2005). The findings in this study are supported by a study by Pranoto et al., (2007), which found that incorporation of gellan and k-carrageenan into fish gelatin films increased TS from 101.23 to 109.76 MPa and 101.23 to 104.48 MPa and EAB values from 5.08 to 6.24% and 5.08 to 6.81%, respectively. Ahmad et al. (2015) also reported that incorporation of rice flour into fish gelatin films caused a significant decreased in TS from 25.33 to 13.26 MPa and EAB values from 14.72 to 6.46%. However, Hosseini et al. (2013) obtained contradictory readings in which the TS values increased from 2.17 to 16.60 MPa and EAB values decreased from 82.6 to 25.3%, respectively after addition of chitosan into fish gelatin films. As compared to the mechanical properties of heat treated SPI and WG protein based films as reviewed by Zink et al. (2016), the chicken skin gelatin −rice flour composite films showed low mechanical properties.Therefore, the addition of rice flour influenced the TS and EAB of chicken skin gelatin film. Thus, Film E (20% rice flour) had the greatest tensile strength and extensibility properties as compared to other films.

properties such as melting temperature (Tm) and crystallinity, while decreasing the film solubility and light transmission of gelatin films. The characteristics of a packaging material vary with its intended use and thus have to be customized specifically according to potential application. Rice flour can be used as an alternative material to produce edible film for potential application in the food industry. References AOAC (2006). Official methods of analysis of AOAC international (18th). Virginia, USA: Association of Official and Analytical Chemists International. Ahmad, M., Hani, N., Nirmal, N., Fazial, F., Mohtar, N., & Romli, S. (2015). Optical and thermo-mechanical properties of composite films based on fish gelatin/rice flour fabricated by casting technique. Progress in Organic Coatings, 84, 115–127. Al-Hassan, A., & Norziah, M. (2012). Starch–gelatin edible films: Water vapor permeability and mechanical properties as affected by plasticizers. Food Hydrocolloids, 26(1), 108–117. Alves, V. D., Mali, S., Beleia, A., & Grossman, M. V. E. (2007). Effect of glycerol and amylose enrichment on cassava starch film properties. Journal of Food Engineering, 78, 941–946. Arfat, Y. A., Benjakul, S., Prodpran, T., & Osako, K. (2014). Development and characteristics of blend films based on fish protein isolate and fish skin gelatin. Food Hydrocolloids, 39, 58–67. Bergo, P., & Sobral, P. (2007). Effects of plasticizer on physical properties of pigskin gelatin films. Food Hydrocolloids, 21(8), 1285–1289. Campo, C., Costa, T., Rios, A., & Flôres, S. (2016). Effect of incorporation of nutraceutical capsule waste of safflower oil in the mechanical characteristics of corn starch films. Food Science and Technology, 36, 33–36 Campinas. Cutter, C. (2006). Opportunities for bio-based packaging technologies to improve the quality and safety of fresh and further processed muscle foods. Meat Science, 74(1), 131–142. Dai, C. A., Chen, Y. F., & Liu, M. W. (2006). Thermal properties measurements of renatured gelatin using conventional and temperature modulated differential scanning calorimetry. Journal of Applied Polymer Science, 99, 1795–1801. Dias, A., Müller, C., Larotonda, F., & Laurindo, J. (2010). Biodegradable films based on rice starch and rice flour. Journal of Cereal Science, 51(2), 213–219. Dias, A., Müller, C., Larotonda, F., & Laurindo, J. (2011). Mechanical and barrier properties of composite films based on rice flour and cellulose fibers. LWT − Food Science and Technology, 44(2), 535–542. Endres, H., & Siebert-Raths, A. (2011). Engineering biopolymers. Cincinnati: Hanser Publishers. Flores, S., Fama, L., Rojas, A. M., Goyanes, S., & Gerschenson, L. (2007). Physical properties of tapioca-starch edible films: Influence of film making and potassium sorbate. Food Research International, 40, 257–265. Guerrero, P., Nur Hanani, Z. A., Kerry, J. P., & de la Caba, K. (2011). Characterization of soy protein-based films prepared with acids and oils by compression. Journal of Food Engineering, 107, 41–79. Hammann, F., & Schmid, M. (2014). Determination and quantification of molecular interactions in protein films: A review. Materials, 7(12), 7975–7996. Han, J. H., & Gennadios, A. (2005). Edible films and coatings: A review. In J. Han (Ed.). Innovations in food packaging (pp. 239–259). Elsevier Science & Technology Books. Hoque, M. S., Benjakul, S., & Prodpran, T. (2010). Effect of heat treatment of film forming solution on the properties of film from cuttlefish (Septa pharaonts) skin gelatin. Journal of Food Engineering, 96, 66–73. Hosseini, S., Rezaei, M., Zandi, M., & Ghavi, F. (2013). Preparation and functional properties of fish gelatin–chitosan blend edible films. Food Chemistry, 136(3–4), 1490–1495. Jahit, I. S., Nazmi, N. N. M., Isa, M. I. N., & Sarbon, N. M. (2016). Preparation and physical properties of gelatin/CMC/chitosan composite films as affected by drying temperature. International Food Research Journal, 23(3), 1068–1074. Langmaier, F., Mokrejs, P., Kolomaznik, K., & Mladek, M. (2008). Biodegradable packaging materials from hydrolysates of collagen waste proteins. Waste Management, 28(3), 549–556. Lee, H. L., & Yoo, B. (2011). Effect of hydroxypropylation on physical and rheological properties of sweet potato starch. L.W.T. Food Science and Technology, 44, 765–770. Limpan, N., Prodpran, T., Benjakul, S., & Prasarpran, S. (2012). Influences of degree of hydrolysis and molecular weight of poly (vinyl alcohol) (PVA) on properties of fish myofibrillar protein/PVA blend films. Food Hydrocolloids, 29, 226–233. Maizura, M., Fazilah, A., Norziah, M. H., & Karim, A. A. (2007). Antibacterial activity and mechanical properties of partially hydrolyzed sago starch-alginate edible film containing lemongrass oil. Journal of Food Science, 72, 324–330. Majzoobi, M., Pesaran, Y., Mesbahi, G., Golmakani, M., & Farahnaky, A. (2015). Physical properties of biodegradable films from heat-moisture-treated rice flour and rice starch. Starch − Stärke, 67(11–12), 1053–1060. Marsh, K., & Bugusu, B. (2007). Food packaging −roles, materials and environmental issues. Journal of Food Science, 72(3), 39–55. Mikkonen, K. S., Pitkanen, L., Liljestrom, V., Bergstrom, E. M., Serimaa, R., Salmen, L., et al. (2012). Arabinoxylan structure affects the reinforcement of films by microfibrillated cellulose. Cellulose, 19(2), 467–480. Nur Hazirah, M. A. S. P., Isa, M. I. N., & Sarbon, N. M. (2016). Effect of xanthan gum on the physical and mechanical properties of gelatin-carboxymethyl cellulose film blends. Food Packaging and Shelf Life, 9, 55–63. Pranoto, Y., Lee, C. M., & Park, H. J. (2007). Characterizations of fish gelatin films added

3.2.2. Young’ modulus measurement Table 4 shows the Young’s Modulus (YM) values for edible chicken skin gelatin films with different formulations of rice flour. Addition of rice flour into gelatin films had no effect on Young’s Modulus measurement. No significant difference (p > 0.05) was noted in all film formulations. Film E (20% rice flour) had the highest value of YM (3.74 MPa) which was parallel with the results of tensile strength (TS) and elongation at break (EAB) in this study. Films with higher values of YM indicated that they are less flexible and more rigid compared to those with lower values of YM (Campo, Costa, Rios, & Flores, 2016; Endres & Siebert-Raths, 2011). The values obtained in this study may be due to the existence of irregularities at microstructure level and the presence of proteins and lipids in rice flour were not able to form a cohesive and continuous matrix; thus, more flexible films were produced (Majzoobi, Pesaran, Meshabi, Golmakani, & Farahnaky, 2015). Besides, the amylose in rice flour contributes more significantly to Young’s Modulus of films than amylopectin (Alves, Mali, Beleia, & Grossman, 2007). This is because the linear chains of amylose are able to interact via hydrogen bonds to a higher extent and be more easily entangled than the branched amylopectin chains, which act as self-reinforcement (Wittaya, 2012). The findings in this study are in agreement with the study of AlHassan and Norziah (2012) which reported that addition of sago starch into fish gelatin films plasticized with glycerol was not significantly influenced (p > 0.05) the Young’s Modulus. Besides, Schmid (2013) showed that hydrolyzed whey protein isolate content did not significantly influence the Young’s Modulus of whey protein isolate films plasticized with glycerol. Therefore, the addition of rice flour had no effect on the Young’s Modulus measurement on chicken skin gelatin films in this study. 4. Conclusion In conclusion, the data and analysis from this study showed that varying concentrations of rice flour influence the physical and mechanical properties of gelatin films. Increasing the rice flour content improved transparency values, water vapor permeability, and thermal 7



of mammalian gelatin films. Food Chemistry, 135(4), 2260–2267. Shehap, A. M., Mahmoud, Kh. H., Abd El-Kader, M. F. H., & El-Basheer, T. M. (2015). Preparation and thermal properties of gelatin/TGS composite films. Middle East Journal of Applied Sciences, 5(1), 157–170. Soliman, E., & Furuta, M. (2014). Influence of phase behavior and miscibility on mechanical, thermal and micro-Structure of soluble starch-gelatin thermoplastic biodegradable blend films. Fns, 05(11), 1040–1055. Suderman, N., Isa, M. I. N., & Sarbon, N. M. (2016). Effect of drying temperature on the functional properties of biodegradable CMC-based film for potential food packaging. International Food Research Journal, 23(3), 1075–1084. Tong, Q., Xiao, Q., & Lim, L. (2008). Preparation and properties of pullulan-alginatecarboxymethyl cellulose blend films. Food Research International, 4, 1007–1014. Tongdeesoontorn, W., Mauer, L., Wongruong, S., Sriburi, P., & Rachtanapun, P. (2012). Mechanical and physical properties of cassava starch-Gelatin composite films. International Journal of Polymeric Materials, 61(10), 778–792. Tongnuanchan, P., Benjakul, S., Prodpran, T., & Nilsuwan, K. (2015). Emulsion film based on fish skin gelatin and palm oil: Physical, structural and thermal properties. Food Hydrocolloids, 48, 248–259. Viera, M. G. A., Da Silva, M. A., Santos, L. O., & Beppu, M. M. (2011). Natural-based plasticizers and biopolymer films: A review. European Polymer Journal, 47(3), 254–263. Wittaya, T. (2012). Protein-Based edible films: Characteristics and improvement of properties. Structure and Function of Food Engineering, 43–71. Xu, X. Y., Kim, K. M., Hanna, M. A., & Nag, D. (2005). Chitosan-starch composite film: Preparation and characterization, Industrial crops and products. International Journal, 21, 85–192. Zhou, Z., Robards, K., Helliwell, S., & Blanchard, C. (2002). Composition and functional properties of rice. International Journal of Food Science & Technology, 37, 849–868. Zink, J., Wyrobnik, T., Prinz, T., & Schmid, M. (2016). Physical, chemical and biochemical modifications of protein-Based films and coatings: An extensive review. International Journal of Molecular Sciences, 17(9), 1376. Zolfi, M., Khodaiyan, F., Mousavi, M., & Hashemi, M. (2014). Characterization of the new biodegradable WPI/clay nanocomposite films based on kefiran exopolysaccharide. Journal of Food Science & Technology, 52(6), 3485–3493.

with gellan and k-carrageenan. Food Science and Technology, 40(5), 766–774. Priya, B., Gupta, V. K., Pathania, D., & Singha, A. S. (2014). Synthesis: Characterization and antibacterial activity of biodegradable starch/PVA composite films reinforced with cellulosic fiber. Carbohydrate Polymer, 109, 171–179. Prodpran, T., Benjakul, S., Vittayanont, M., & Nalinanon, S. (2013). Physico-chemical properties of gelatin films incorporated with different hydrocolloids. International Conference on Nutrition and Food Sciences, 53(16), 82–86. Rao, M. S., Kanatt, S. R., Chawla, S. P., & Sharma, A. (2010). Chitosan and guar gum composite films: Preparation, physical, mechanical and antimicrobial properties. Carbohydrate Polymers, 82, 1243–1247. Rawdkuen, S., Sai-Ut, S., & Benjakul, S. (2010). Properties of gelatin films from giant catfish skin and bovine bone: A comparative study. European Food Research Technology, 231(6), 907–916. Rhim, J. W., Gennadios, A., Handa, A., Weller, C. L., & Hanna, M. A. (2000). Solubility, tensile and color properties of modified soy protein isolate films. Journal of Agricultural and Food Chemistry, 48, 4937–4941. Rivero, S., García, M., & Pinotti, A. (2010). Correlations between structural, barrier, thermal and mechanical properties of plasticized gelatin films. Innovative Food Science & Emerging Technologies, 11(2), 369–375. Sanyang, M. L., Sapuan, S. M., Jawaid, M., Ishak, M. R., & Sahari, J. (2015). Effect of plasticizer type and concentration on tensile: Thermal and barrier properties of biodegradable films based on sugar palm (Arenga pinnata) starch. Journal Polymers, 7, 1106–1124. Sarbon, N. M., Badii, F., & Howell, N. K. (2013). Preparation and characterization of chicken skin gelatin as an alternative to mammalian gelatin. Food Hydrocolloids, 30, 143–151. Schmid, M., Dallmann, K., Bugnicourt, E., Cordoni, D., Wild, F., Lazzeri, A., et al. (2012). Properties of whey protein coated films and laminates as novel recyclable food packaging materials with excellent barrier properties. International Journal of Polymer Science, 2012, 7. Schmid, M. (2013). Properties of cast films made from different ratios of whey protein isolate: Hydrolysed whey protein isolate and glycerol. Materials, 6, 3254–3269. Shakila, R. J., Jeevithan, E., Varatharajakumar, A., Jeyasekaran, G., & Sukumar, D. (2012). Comparison of the properties of multi-composite fish gelatin films with that

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