Reagents and Chemicals
Rice bran (Giza 178) was obtained from Rice Research Center at Sakha, Agricultural Research Center, Egypt. Glycerol, Ascorbic acid, Citric acid, Sodium hydroxide (NaOH), Hydrochloric acid (HCl) ,N-hexan ,Sodium bicarbonate were purchased from El- Gomhoria Co. for Chemicals and Drugs. Teflon plates (20×30) used to cast all the films. All reagents were of analytical grade.
Rbp Extraction
RBP extraction
RB samples were treated by microwave to inhibit the enzymes action for five min. Oil was elicited from RB using n- hexane based on the method outlined by Kahlon [13]. The defatted rice bran was stored at -18oC until used. After that, defatted rice bran was suspended in distilled water (1:10 w/w). slurry’s pH was set at 9.0 using4 M NaOH solution continuously stirred for 1 h then centrifuged for 15 minutes at 12,600g. supernatant protein solution was set to pH 4.5 using4 M HCl, stirred for 30 min then left overnight at 4°C for cold precipitation. The supernatant has been siphoned off then the resulted precipitated protein was washed 3 to 4 times with distilled water. The protein slurry has been set to pH7 and lyophilized [14].
Production Deamidated Rice Bran Protein
Protein deamidation was performed as following; briefly, a weight of 0.25 g from RBP was suspended in 25 mL of 0.1 M NaHCO3 and pH was set to 8, 10, and 12 with 1 M NaOH or 1 M HCl. The solutions were then heated at 80 or 100°C for 30 or 60 min, or 120°C for 15 or 30 min. Then, samples have been neutralized, dialyzed against water at 4˚C for 48 hrs, and lyophilized.
Determination Of Protein Solubility
Fifty mg of protein sample has been suspended in 5 mL of deionized water, mixed for 30 min at room temperature, and centrifuged at 15000 g for 20 min. The supernatant has been transported to another tube, after dried at 40°C, weight of residual protein was determined based on the method outlined by Bradford [15].
Formation Of Edible Film
RBP 1% or DRBP with different concentrations (1, 2 and 3%) were dissolved in distilled water, stirred at 80˚C for 30 min. Glycerol was added and pH was adjusted. Ascorbic and citric acid were added as anti-browning agent and antimicrobial (1.5% (w/v). Film solution was left in the room temperature for 1h to remove air bubbles and cast into Teflon plates (20×30 cm) then dried at 40˚C in oven. Films have been stored at plastic bags until the measurement were performed.
Mechanical Properties (Dup: Abstract ?)
Tensile strength and Elongation
Tensile strength (TS, Mpa) and elongation percentage (%E) at break of film were determined at 22 ± 10C and RH = 31% using an Instron Universal Testing Machine (Model 1011, UK), according to Ferreira et al. [16].
Characterization Of Physical Properties
Film thickness
Thickness of film has been calculated with an accuracy of 0.01 mm using a digital micrometer (Mitutoyo digimatic indicator corporation, model: pk-1012 E, Tokyo, Japan). The mean thickness have been used to measure the water vapor permeability and tensile strength [17].
Determination Of Films Solubility And Swelling Power
Solubility and swelling degree of the formed films were measured by using the method of Riaz et al. [18] with a slight modification. The film pieces were cut into 2x 2 cm then dried at 105°C until constant weight to calculate the initial dry mass (M1). Then, those were took place in 100 mL beakers with 50 mL distilled water, covered with plastic wraps and stored at 25°C for 24 h. Later, the films have been dried using filter paper and dried at 105°C until fixed weight to determine final dry mass (M2). The following equation was used to measure film solubility:
$$\text{F}\text{i}\text{l}\text{m} \text{s}\text{o}\text{l}\text{u}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}=\left(\frac{\text{M}1-\text{M}2}{\text{M}1}\right)\text{*}100$$
films were placed in beakers (50 mL) that contain distilled water (30 ml) for 24 h at 25°C after weighing (M1). Then, wet film was dried using filter paper then weighed (M2). The swelling degree calculated with this equation:
$$\text{F}\text{i}\text{l}\text{m} \text{s}\text{w}\text{e}\text{l}\text{l}\text{i}\text{n}\text{g} \text{d}\text{e}\text{g}\text{r}\text{e}\text{e} \left(\text{\%}\right)=\left(\frac{\text{M}1-\text{M}2}{\text{M}1}\right)\text{*}100$$
Moisture Content
Moisture content was determined based on the way of Araujo-Farro et al. [19]. Moisture content was measured by Eqs. 1. film samples have been cut into squares of 2 ×2 and weighted (W1). The samples have been dried at 105°C until constant weighted (W2) obtained. Triplicates of each film samples were tested.
$$\text{M}\text{o}\text{i}\text{s}\text{t}\text{u}\text{r}\text{e} \text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t} \left(\text{\%}\right)=\left(\frac{\text{W}1-\text{W}2}{\text{W}1}\right)\text{*}100$$
Water Vapor Permeability (Wvp)
Modification of the ASTM E96-92 gravimetric method to measure the relative humidity (RH) at the film underside was used for measuring WVP [20]. Distinct glass cups have been used with 4 cm diameter then filled with anhydrous CaCl2 then film samples (5×5 cm) have been sealed above each glass cup. The cups have been placed in a desiccators at 75% RH which was maintained by a saturated sodium chloride solution. The glass cups have been then weighted change against time was determined by linear regression.
The WVP was measured by the following equation
$$\text{W}\text{V}\text{P}=\left(\frac{\text{S}\text{l}\text{o}\text{p}\text{e}\times \text{L} }{\text{A}\times \varDelta \text{P}}\right)$$
Where L is the average film thickness (m), A is the transfer area (m2) and ∆P the partial water vapor pressure difference.
Exposure To Light Properties
Haze and Transparency
Haze and transparency of samples were determined by Hazemeter (BYK- Gardner Gmbh model haze-grad plus, Germany, according to ASTM., (2001).
Film Gloss
Gloss is a way to measure of the ability of the film to reflect incident light at angle 450. The sample’s gloss has been measured by glossmeter (BYK- Gardner Gmbh model micro- gloss 45, Germany, according to ASTM., (1997). Standard measure for tensile properties of thin plastic sheeting. D882- 97, Annual book of American Standard Testing Methods, ASTM, Philadelphia, PA. Gloss values measurement based on 6 random positions per sample from double faces and three samples per film.
Films Opacity
Spectrophotometer (Model PU 8800 UV/VIS, Pye Unicam Ltd., Cambridge, UK) has been used to determine film opacity as described by Sun et al. [21]. Three strips (1 x 4 cm) were cut from the soaked films and placed on the inner side of a transparent plastic cuvette. The adsorption spectrum was determined with a wavelength domain of 400–800 nm, and opacity was taken as the area under the curve, as determined through an integration procedure, and termed as Absorbance Units in nanometers (A nm).
Characterization Of Rbp Films
Analysis of SEM
Scanning electron microscopy (SEM, SU8010, Hitachi, Japan) at 10 K V has been used to study film surface morphology. The films were cut in pieces ( 10 x 10 mm) then, dried and mounted on aluminum stubs using a double – sided carbon tape and sputter- coated with gold [21].
Analysis Of Ft- Ir
Fourier Transform – Infrared (FT- IR) spectrometry ( Nicolet IR200, Thermo, USA) was used to study prepared films structure through KBr module. FT- IR spectra were recorded in the frequency range of 4000 to 400 cm− 1.
Differential Scanning Calorimetry ( Dsc)
DSC analysis was performed using a Q1000 DSC system ( TA instruments, USA) following the way of Akhtar et al. [22]. film pieces (10 mg) were sealed in a standard aluminum pan and heated at a constant rate of 10°C / min from 0 to 450°C at a nitrogen atmosphere.
X-ray Diffraction
The crystal structures of films was explored following the way of Ali et al. [17] using x-ray diffractometer (D8 Advance, Bruker. USA) at a voltage of 40kV and 100 Ma.The scattered radiation was detected in the angular range 2Ɵ= 10–40º with scanning speed of 5 º/ min.
Statistical analysis
Data were analyzed statistically by SPSS 20.0. Analysis of the data (ANOVA) assessed the difference between factors and levels. To identify the significance of differences among mean values. Tukey's multiple range tests were executed. Differences were considered significant when p < 0.05.