Accepted Manuscript Title: Gelatin-hydroxypropyl methylcellulose water-in-water emulsions as a new bio-based packaging material Author: Sara Esteghlal Mehrdad Niakosari Seyed Mohammad Hashem Hosseini Gholam Reza Mesbahi Gholam Hossein Yousefi PII: DOI: Reference:
S0141-8130(16)30066-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.065 BIOMAC 5755
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
27-10-2015 14-1-2016 20-1-2016
Please cite this article as: Sara Esteghlal, Mehrdad Niakosari, Seyed Mohammad Hashem Hosseini, Gholam Reza Mesbahi, Gholam Hossein Yousefi, Gelatinhydroxypropyl methylcellulose water-in-water emulsions as a new bio-based packaging material, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gelatin-hydroxypropyl methylcellulose water-in-water emulsions as a new 1 bio-based packaging material
2 3
Sara
Esteghlala,
Mehrdad
Niakosaria,
Seyed
Mohammad
Hashem 4
Hosseinia,*, Gholam Reza Mesbahia, Gholam Hossein Yousefib, c
5 6
a
Department of Food Science and Technology, School of Agriculture, Shiraz 7
University, 71441-65186 Shiraz, Iran b
8
Department of Pharmaceutics, School of Pharmacy, Shiraz University of 9
Medical Sciences, 7146864685 Shiraz, Iran c
10
Center for Nanotechnology in Drug Delivery, Shiraz University of Medical 11
Sciences, 7146864685 Shiraz, Iran
12
* Corresponding author. Tel.: +98 71 32286110; fax: +98 71 32286110
13
E-mail address: [email protected] (S. M. H. Hosseini).
14 15 16 17 18 19 20 21
1
Abstract
22
Gelatin and hydroxypropyl methylcellulose (HPMC) are two incompatible 23 and immiscible biopolymers which cannot form homogeneous composite films 24 using usual methods. In this study, to prevent phase separation, gelatin-HPMC 25 water-in-water (W/W) emulsion was utilized to from transparent composite films 26 by entrapment the HPMC dispersed droplets in gelatin continuous network. The 27 physicochemical and mechanical properties of emulsion-based films containing 28 different amounts (5-30%) of dispersed phase were determined and compared 29 with those of individual polymer-based films. Incorporating HPMC into W/W 30 emulsion-based films had no significant effect on the tensile strength. The 31 flexibility of composite films decreased at HPMC concentrations below 20%. 32 The depletion layer at the droplets interface reduced the diffusion of water 33 vapor molecules because of its hydrophobic nature, so the water vapor 34 permeability remained constant. Increasing the HPMC content in the emulsion 35 films increased the swelling and decreased the transparency. The entrapment 36 of HPMC in continuous gelatin phase decreased its solubility. Therefore, W/W 37 emulsions are capable of holding two incompatible polymers alongside each 38 other within a homogeneous film network without weakening the physical 39 properties.
40 41
Keywords: Emulsion-based film; Water-in-water emulsion; Incompatibility
42 43 44 45
2
1. Introduction
46
Increasing oil price and environmental concerns regarding petroleum- 47 based synthetic packaging materials are the driving force to find novel 48 renewable and degradable resources. Biopolymers mainly including proteins 49 and polysaccharides are suitable substitutes because of film forming properties, 50 favorable environmental advantages and potential for enhancing food quality 51 and safety [1, 2, 3, 4]. In comparison to synthetic polymers, biopolymers have 52 weaker mechanical and barrier properties resulted in limited applications. The 53 application of the mixed biopolymer systems to develop composite films is the 54 common strategy to overcome these problems [5, 6, 7]. Generally, in a 55 composite film, each biopolymer reveals its own specific characteristics so the 56 properties of the composite film are usually superior than those of individual 57 biopolymer-based films provided taking into account the compatibility between 58 the involved biopolymers. There are three possible biopolymer mixtures which 59 can have the ability to improve the properties of edible films: protein-protein, 60 polysaccharide-polysaccharide and protein-polysaccharide [8].
61
In an aqueous dispersion, when charged amphoteric protein molecules 62 are come into contact with polysaccharides, one of four phenomena including 63 co-solubility, thermodynamic incompatibility (segregative phase separation), 64 depletion flocculation and thermodynamic compatibility (complex coacervation 65 or associative phase separation) can arise. These phenomena are mainly 66 dependent on the electrical charges on both biopolymers, and therefore on the 67 factors affecting them, such as the pH and the ionic strength. The other 68 parameters including size, molecular weight, molecular conformation, total 69 concentration of biopolymer molecules, biopolymer mixing ratio, charge 70 3
distribution and processing factors such as temperature, pressure, shearing 71 and acidification method are also involved [9, 10, 11, 12]. These phase 72 behaviors
arise
from
long-
or
short-range
interactions
between
the 73
biomacromolecules themselves, and possibly because of different affinities 74 between the biomacromolecules and the solvent too [12].
75
The associative phase separation is due to the attractive forces such as 76 electrostatic and hydrophobic interactions as well as hydrogen bonding. 77 According to Nigen et al. [13] electrostatic interactions could be mostly 78 important during the initial biopolymer complex formation, however, large-scale 79 aggregation or coacervation would be mainly driven by hydrogen bonding or 80 hydrophobic interactions, depending on the temperature. In thermodynamic 81 compatible systems, molecules spontaneously attract each other and the 82 complexation phenomenon results in the formation of a solvent-rich (biopolymer 83 depleted) phase and a phase rich in both biopolymers [14, 15]. Protein and 84 polysaccharide in the biopolymer rich phase can appear as coacervate, 85 complexes (soluble or insoluble) and gels [10]. In
thermodynamic
incompatible
systems,
86
two
non-interacting 87
macromolecules mutually segregate into two different distinct immiscible 88 aqueous phases (upon the total concentration or the molecular weight exceed a 89 certain amount), resulting in one phase mainly rich in one biopolymer and the 90 other phase mainly rich in the other biopolymer [10, 11, 16, 17, 18]. Such 91 systems have a high positive energy of mixing and usually occur when one of 92 or both biopolymers are uncharged or of similar charges. In a relatively very low 93 concentration of biopolymers or in mixed systems containing low molecular 94 weight species co-solubility is also possible.
95
4
A variety of microstructures can be formed in a segregative phase- 96 separated system by varying the preparation condition or by shearing the 97 system [19]. For example water- in- water (W/W) emulsion as a dispersed 98 system can be formed when the droplets of one immiscible aqueous phase are 99 dispersed within continuous aqueous dispersion [20]. Creating a stable 100 structure is possible using energy barriers [21]. Kinetic trapping can be 101 accomplished by gelation or phase thickening. Different types of spherical, 102 fibrous or tear-drop microstructures can be formed by applying shear forces to 103 the system during the trapping process [19]. In plastics and foods, phase- 104 separated systems are utilized to create different types of products. For 105 example, in the plastic industry mixtures of the immiscible synthetic polymers 106 are used to produce soft and hard composites [22]. In food industry, non- 107 equilibrium trapped structures arising from the mixtures of thermodynamically 108 incompatible biopolymers are applied as thickener, fat substitutes and carriers 109 of taste and nutritional components [23]. Low compatible/incompatible 110 macromolecules in food systems induce separation and partitioning of 111 components between different phases. Covering enzymes, reagents and low 112 molecular weight products of Maillard type reactions due to the fractionation, 113 influences the organoleptic perception of foods [20]. Gelatin is a protein 114 produced from the partial hydrolysis of collagen with an average molecular 115 weight of 65 - 300 kDa. Gelatin gels are transparent and easily dissolved in hot 116 water. Dry gelatin films are brittle [24]. Gelatin films exhibit strong swelling 117 behaviour in water and strong gas (O2 and CO2) barrier properties; however 118 they have poor mechanical resistance and high sensitivity to water [25, 26]. 119 Hydroxypropyl methylcellulose (HPMC) is a non-ionic cellulose derivative that 120
5
has the ability to form transparent, odorless, tasteless, oil-resistant and water 121 soluble films. They show moderate resistance to oxygen, moisture and aroma 122 transmittance, high tensile strength and low flexibility [8].
123
Many researchers have investigated the physicochemical and mechanical 124 properties of protein-polysaccharide composite films [1, 27, 28, 29, 30, 31, 32, 125 33]. However, few research projects have been performed on incompatible 126 biopolymers such as proteins and non-ionic polysaccharides [8, 33, 34]. To the 127 best of our knowledge, the physical properties of W/W emulsion-based films 128 have not been studied previously. Therefore, the objectives of the current work 129 were developing W/W emulsion from two thermodynamically incompatible 130 polymers (gelatin and HPMC) and then determining the physical properties of 131 the resulted films.
132 133
2. Materials and methods
134
2.1. Materials
135
Gelatin (type A) and dimethyl sulfoxide (DMSO, as the solvent for the 136 fluorescent dyes) were supplied by Merck Co. (Darmstadt, Germany). HPMC 137 (MW 86 kDa, methoxyl and hydroxypropyl contents of about 28.6% and 9.4%, 138 respectively),
fluoresceine
isothiocyanate
(FITC)
and
rhodamine
B 139
isothiocyanate (RITC) for biopolymers labeling were purchased from Sigma- 140 Aldrich (St. Lois, Mo, USA). Glycerol (99% purity) as a plasticizer was supplied 141 by Kimia Mavad Co. (Tehran, Iran).
142 143
6
2.2. Preparation of dispersions
144
Gelatin dispersion (10% W/W) was prepared by dispersing gelatin powder 145 into distilled water. Hydration was performed at 60 ˚C for 30 min under 146 continuous stirring [1]. HPMC was slowly added into distilled water to reach the 147 final concentration of 10% (W/W) and then allowed overnight to ensure 148 complete hydration under continuous stirring [8].
149 150
2.3. Visual observation of phase separation
151
Gelatin and HPMC dispersions have similar optical properties. Therefore, 152 FITC and RITC as fluorescent markers were used to covalently label protein 153 and polysaccharide, respectively. Labeling was performed according to the 154 method of Schmitt et al. [35] with some modifications. 5 µl of 2% (w/v) FITC or 155 RITC solution in DMSO was added to 10 ml of protein or polysaccharide 156 dispersion, respectively. To complete the process, dispersions were gently 157 stirred for 90 min at room temperature. A mixture of labeled biopolymers 158 (gelatin to HPMC mixing ratio 80:20) was prepared and then stirred for 30 min. 159 The mixture was then kept immobile to check whether phase separation occurs 160 or not.
161 162
2.4. Film formation
163
Gelatin and HPMC stock dispersions were prepared as explained 164 previously. Glycerol (30%, based on total dry weight of polymers) was added as 165 a plasticizer and stirred for 25 min. To prepare gelatin-HPMC blend 166
7
dispersions, the HPMC dispersion was added into the gelatin one in the 167 following ratios: 5:95, 10:90, 15:85, 20:80 and 30:70. In all dispersions, total 168 polymer content was kept at 10% W/W. The shear rate resulted from the 169 application of the magnetic stirrer was not enough to obtain a homogenous 170 mixture of gelatin and HPMC because of their incompatibility. During 171 preliminary experiments, casting a magnetically stirred gelatin:HPMC (70:30) 172 film forming dispersion led to a bilayer film which was delaminated after drying. 173 Therefore, W/W emulsion from two immiscible biopolymers was prepared to 174 obtain a homogenous composite film. An IKA T25 Disperser Homogenizer 175 (ULTRA-TURRAX®, Staufen, Germany) at 15000 rpm for 3 min was used to 176 prepare W/W emulsions. The shear applied by homogenizer resulted in 177 developing small droplets of HPMC phase dispersed in the continuous gelatin 178 phase. The resultant W/W emulsions were vacuum (0.02 MPa) filtered for 30 179 min to remove the air bubbles. Casting was performed onto plastic plates. 180 Plates were maintained at room temperature for 48 h. After drying, films were 181 peeled off from the plates and conditioned (temperature 24±1 °C and relative 182 humidity (RH) around 50% for 72 h) [36, 37] in a closed box containing 183 saturated salt solution prior to analysis. Control (gelatin and HPMC) films were 184 prepared and treated in the same manner.
185 186
2.5. Determining the effect of shearing on the appearance of the films
187
In order to study the effect of shear rate (magnetic stirring for 20 min at 188 500 rpm and homogenizing at 9000, 11000 and 15000 rpm for 3 min) on the 189 film appearance, the dispersed phase distribution and the shape of droplets a 190
8
FITC-labeled gelatin:HPMC (80:20) mixed dispersion was prepared. After 191 applying different shear rates, the resultant dispersions were cast onto plastic 192 plates and treated in the same manner as described previously.
193 194
2.6. Film thickness
195
Film thickness was measured using a micrometer (Starrett Na 436-1in, 196 USA) to the nearest 0.001 inch in at least 7 random points around the testing 197 area. Average values were used to calculate mechanical (tensile strength), 198 barrier (water vapor permeability) and optical (opacity) properties.
199 200
2.7. Visual aspects
201
The appearance of the films was checked for homogeneity, color 202 uniformity and the absence of phase separating, insoluble particles and brittle 203 zones [36].
204 205
2.8. Mechanical properties
206
The tensile strength (TS) and the elongation at break (EAB) of the films 207 were measured using a Texture Analyzer (Brookfield CS3, USA) equipped with 208 a 1000 g load cell. Dumbbell shaped samples (50 mm length, 4mm width in the 209 middle and 8.5 mm width at the ends) were used in this study. The crosshead 210 speed was 50 mm/min. Test was done in at least 8 replications. TS (kPa) and 211 %EAB were measured by Eqs. (1) and (2), respectively:
212
9
TS= F/A
(1) 213
where F is the maximum force applied for tension of the sample (N), A is 214 the area of the cross section of the film (thickness × width, m2). % EAB = (∆L /L) × 100
215
(2) 216 217
where ∆L is the change in length before breakage and L is the initial 218 length.
219 220
2.9. Water vapor permeability (WVP)
221
Water vapor permeability was determined according to the standard 222 method of ASTM E95-E96 [37]. Cups were filled with 15 g anhydrous CaCl2 223 (0% RH1), covered with a specimen of conditioned film, sealed and placed in a 224 container containing saturated NaCl solution (75% RH2) at 25 °C. Samples 225 were weighed in 1 h intervals during 8 h WVP was measured using Eq. (3): WVP = (WVTR) ͯ I / P0 (RH1-RH2)
226
(3) 227
where water vapor transmission rate (WVTR) is the slope of the mass 228 change versus time curve (g/h), l is the average film thickness (mm), P0 is the 229 saturation vapor pressure at 25 °C (1753.55 Pa) and (RH1-RH2) is the 230 difference in the relative humidity of the anhydrous calcium chloride (0%) and 231 super saturated NaCl solution (0.75%).
232 233
2.10. Swelling test
234
10
Square cuts of films (2×2 cm2) were dried (at 60 °C for 24 h), weighed and 235 immersed in 0.1 M NaCl solution at room temperature. Samples were removed 236 from the solution after 30 min and placed between two dry filter papers to 237 absorb excess surface water. The swollen samples were finally weighed. 238 Swelling ratio was calculated according to Eq. (4) [38]: Swelling (%) = Mf – Mi / Mi ×100 where Mf and Mi are the weights of swollen and
239
(4) 240 dried samples, 241
respectively.
242 243
2.11. Solubility test
244
Square-shaped films (2×2 cm2) were dried in an oven at 105 °C to a 245 constant weight (Mi). Dried samples were then immersed in 50 ml distilled 246 water under slow mechanical shaking for 24 h at room temperature. After this 247 period, samples were re-dried and weighed again (Mf). Solubility ratio was 248 calculated based on Eq. (5) [39]:
249
Solubility (%) = Mi - Mf / Mi ×100
(5) 250 251
2.12. Opacity
252
A rectangular piece of film (1×5 cm2) was placed on the inner surface of a 253 spectrophotometer cell. A UV-visible spectrophotometer (UNICO UV-2100, 254 USA) was used for measuring the absorbance of the samples at 600 nm. 255 Opacity was determined using Eq. (6) [33]:
256
11
A = Absorbance600nm / X
(6) 257
where X is the average film thickness (mm).
258 259
2.13 Scanning electron microscopy (SEM)
260
The microstructure of the film network (the sample containing 20% 261 HPMC) was studied using scanning electron microscope (Vega3, TESCAN, 262 Brno, Czech). The sample was mounted on the specimen holder with aluminum 263 tape and then coated with gold sputter coater (Dsr1, Nanostructured Co, Iran). 264 Morphological observation of the film surface was performed at an accelerating 265 voltage of 20.0 kV [33].
266 267
2.14. Statistical analysis
268
All experiments were performed at least in triplicates. Analysis of variance 269 (ANOVA) was performed to determine significant differences between the 270 means. Duncan multiple range test (P
S0141-8130(16)30066-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.065 BIOMAC 5755
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
27-10-2015 14-1-2016 20-1-2016
Please cite this article as: Sara Esteghlal, Mehrdad Niakosari, Seyed Mohammad Hashem Hosseini, Gholam Reza Mesbahi, Gholam Hossein Yousefi, Gelatinhydroxypropyl methylcellulose water-in-water emulsions as a new bio-based packaging material, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gelatin-hydroxypropyl methylcellulose water-in-water emulsions as a new 1 bio-based packaging material
2 3
Sara
Esteghlala,
Mehrdad
Niakosaria,
Seyed
Mohammad
Hashem 4
Hosseinia,*, Gholam Reza Mesbahia, Gholam Hossein Yousefib, c
5 6
a
Department of Food Science and Technology, School of Agriculture, Shiraz 7
University, 71441-65186 Shiraz, Iran b
8
Department of Pharmaceutics, School of Pharmacy, Shiraz University of 9
Medical Sciences, 7146864685 Shiraz, Iran c
10
Center for Nanotechnology in Drug Delivery, Shiraz University of Medical 11
Sciences, 7146864685 Shiraz, Iran
12
* Corresponding author. Tel.: +98 71 32286110; fax: +98 71 32286110
13
E-mail address: [email protected] (S. M. H. Hosseini).
14 15 16 17 18 19 20 21
1
Abstract
22
Gelatin and hydroxypropyl methylcellulose (HPMC) are two incompatible 23 and immiscible biopolymers which cannot form homogeneous composite films 24 using usual methods. In this study, to prevent phase separation, gelatin-HPMC 25 water-in-water (W/W) emulsion was utilized to from transparent composite films 26 by entrapment the HPMC dispersed droplets in gelatin continuous network. The 27 physicochemical and mechanical properties of emulsion-based films containing 28 different amounts (5-30%) of dispersed phase were determined and compared 29 with those of individual polymer-based films. Incorporating HPMC into W/W 30 emulsion-based films had no significant effect on the tensile strength. The 31 flexibility of composite films decreased at HPMC concentrations below 20%. 32 The depletion layer at the droplets interface reduced the diffusion of water 33 vapor molecules because of its hydrophobic nature, so the water vapor 34 permeability remained constant. Increasing the HPMC content in the emulsion 35 films increased the swelling and decreased the transparency. The entrapment 36 of HPMC in continuous gelatin phase decreased its solubility. Therefore, W/W 37 emulsions are capable of holding two incompatible polymers alongside each 38 other within a homogeneous film network without weakening the physical 39 properties.
40 41
Keywords: Emulsion-based film; Water-in-water emulsion; Incompatibility
42 43 44 45
2
1. Introduction
46
Increasing oil price and environmental concerns regarding petroleum- 47 based synthetic packaging materials are the driving force to find novel 48 renewable and degradable resources. Biopolymers mainly including proteins 49 and polysaccharides are suitable substitutes because of film forming properties, 50 favorable environmental advantages and potential for enhancing food quality 51 and safety [1, 2, 3, 4]. In comparison to synthetic polymers, biopolymers have 52 weaker mechanical and barrier properties resulted in limited applications. The 53 application of the mixed biopolymer systems to develop composite films is the 54 common strategy to overcome these problems [5, 6, 7]. Generally, in a 55 composite film, each biopolymer reveals its own specific characteristics so the 56 properties of the composite film are usually superior than those of individual 57 biopolymer-based films provided taking into account the compatibility between 58 the involved biopolymers. There are three possible biopolymer mixtures which 59 can have the ability to improve the properties of edible films: protein-protein, 60 polysaccharide-polysaccharide and protein-polysaccharide [8].
61
In an aqueous dispersion, when charged amphoteric protein molecules 62 are come into contact with polysaccharides, one of four phenomena including 63 co-solubility, thermodynamic incompatibility (segregative phase separation), 64 depletion flocculation and thermodynamic compatibility (complex coacervation 65 or associative phase separation) can arise. These phenomena are mainly 66 dependent on the electrical charges on both biopolymers, and therefore on the 67 factors affecting them, such as the pH and the ionic strength. The other 68 parameters including size, molecular weight, molecular conformation, total 69 concentration of biopolymer molecules, biopolymer mixing ratio, charge 70 3
distribution and processing factors such as temperature, pressure, shearing 71 and acidification method are also involved [9, 10, 11, 12]. These phase 72 behaviors
arise
from
long-
or
short-range
interactions
between
the 73
biomacromolecules themselves, and possibly because of different affinities 74 between the biomacromolecules and the solvent too [12].
75
The associative phase separation is due to the attractive forces such as 76 electrostatic and hydrophobic interactions as well as hydrogen bonding. 77 According to Nigen et al. [13] electrostatic interactions could be mostly 78 important during the initial biopolymer complex formation, however, large-scale 79 aggregation or coacervation would be mainly driven by hydrogen bonding or 80 hydrophobic interactions, depending on the temperature. In thermodynamic 81 compatible systems, molecules spontaneously attract each other and the 82 complexation phenomenon results in the formation of a solvent-rich (biopolymer 83 depleted) phase and a phase rich in both biopolymers [14, 15]. Protein and 84 polysaccharide in the biopolymer rich phase can appear as coacervate, 85 complexes (soluble or insoluble) and gels [10]. In
thermodynamic
incompatible
systems,
86
two
non-interacting 87
macromolecules mutually segregate into two different distinct immiscible 88 aqueous phases (upon the total concentration or the molecular weight exceed a 89 certain amount), resulting in one phase mainly rich in one biopolymer and the 90 other phase mainly rich in the other biopolymer [10, 11, 16, 17, 18]. Such 91 systems have a high positive energy of mixing and usually occur when one of 92 or both biopolymers are uncharged or of similar charges. In a relatively very low 93 concentration of biopolymers or in mixed systems containing low molecular 94 weight species co-solubility is also possible.
95
4
A variety of microstructures can be formed in a segregative phase- 96 separated system by varying the preparation condition or by shearing the 97 system [19]. For example water- in- water (W/W) emulsion as a dispersed 98 system can be formed when the droplets of one immiscible aqueous phase are 99 dispersed within continuous aqueous dispersion [20]. Creating a stable 100 structure is possible using energy barriers [21]. Kinetic trapping can be 101 accomplished by gelation or phase thickening. Different types of spherical, 102 fibrous or tear-drop microstructures can be formed by applying shear forces to 103 the system during the trapping process [19]. In plastics and foods, phase- 104 separated systems are utilized to create different types of products. For 105 example, in the plastic industry mixtures of the immiscible synthetic polymers 106 are used to produce soft and hard composites [22]. In food industry, non- 107 equilibrium trapped structures arising from the mixtures of thermodynamically 108 incompatible biopolymers are applied as thickener, fat substitutes and carriers 109 of taste and nutritional components [23]. Low compatible/incompatible 110 macromolecules in food systems induce separation and partitioning of 111 components between different phases. Covering enzymes, reagents and low 112 molecular weight products of Maillard type reactions due to the fractionation, 113 influences the organoleptic perception of foods [20]. Gelatin is a protein 114 produced from the partial hydrolysis of collagen with an average molecular 115 weight of 65 - 300 kDa. Gelatin gels are transparent and easily dissolved in hot 116 water. Dry gelatin films are brittle [24]. Gelatin films exhibit strong swelling 117 behaviour in water and strong gas (O2 and CO2) barrier properties; however 118 they have poor mechanical resistance and high sensitivity to water [25, 26]. 119 Hydroxypropyl methylcellulose (HPMC) is a non-ionic cellulose derivative that 120
5
has the ability to form transparent, odorless, tasteless, oil-resistant and water 121 soluble films. They show moderate resistance to oxygen, moisture and aroma 122 transmittance, high tensile strength and low flexibility [8].
123
Many researchers have investigated the physicochemical and mechanical 124 properties of protein-polysaccharide composite films [1, 27, 28, 29, 30, 31, 32, 125 33]. However, few research projects have been performed on incompatible 126 biopolymers such as proteins and non-ionic polysaccharides [8, 33, 34]. To the 127 best of our knowledge, the physical properties of W/W emulsion-based films 128 have not been studied previously. Therefore, the objectives of the current work 129 were developing W/W emulsion from two thermodynamically incompatible 130 polymers (gelatin and HPMC) and then determining the physical properties of 131 the resulted films.
132 133
2. Materials and methods
134
2.1. Materials
135
Gelatin (type A) and dimethyl sulfoxide (DMSO, as the solvent for the 136 fluorescent dyes) were supplied by Merck Co. (Darmstadt, Germany). HPMC 137 (MW 86 kDa, methoxyl and hydroxypropyl contents of about 28.6% and 9.4%, 138 respectively),
fluoresceine
isothiocyanate
(FITC)
and
rhodamine
B 139
isothiocyanate (RITC) for biopolymers labeling were purchased from Sigma- 140 Aldrich (St. Lois, Mo, USA). Glycerol (99% purity) as a plasticizer was supplied 141 by Kimia Mavad Co. (Tehran, Iran).
142 143
6
2.2. Preparation of dispersions
144
Gelatin dispersion (10% W/W) was prepared by dispersing gelatin powder 145 into distilled water. Hydration was performed at 60 ˚C for 30 min under 146 continuous stirring [1]. HPMC was slowly added into distilled water to reach the 147 final concentration of 10% (W/W) and then allowed overnight to ensure 148 complete hydration under continuous stirring [8].
149 150
2.3. Visual observation of phase separation
151
Gelatin and HPMC dispersions have similar optical properties. Therefore, 152 FITC and RITC as fluorescent markers were used to covalently label protein 153 and polysaccharide, respectively. Labeling was performed according to the 154 method of Schmitt et al. [35] with some modifications. 5 µl of 2% (w/v) FITC or 155 RITC solution in DMSO was added to 10 ml of protein or polysaccharide 156 dispersion, respectively. To complete the process, dispersions were gently 157 stirred for 90 min at room temperature. A mixture of labeled biopolymers 158 (gelatin to HPMC mixing ratio 80:20) was prepared and then stirred for 30 min. 159 The mixture was then kept immobile to check whether phase separation occurs 160 or not.
161 162
2.4. Film formation
163
Gelatin and HPMC stock dispersions were prepared as explained 164 previously. Glycerol (30%, based on total dry weight of polymers) was added as 165 a plasticizer and stirred for 25 min. To prepare gelatin-HPMC blend 166
7
dispersions, the HPMC dispersion was added into the gelatin one in the 167 following ratios: 5:95, 10:90, 15:85, 20:80 and 30:70. In all dispersions, total 168 polymer content was kept at 10% W/W. The shear rate resulted from the 169 application of the magnetic stirrer was not enough to obtain a homogenous 170 mixture of gelatin and HPMC because of their incompatibility. During 171 preliminary experiments, casting a magnetically stirred gelatin:HPMC (70:30) 172 film forming dispersion led to a bilayer film which was delaminated after drying. 173 Therefore, W/W emulsion from two immiscible biopolymers was prepared to 174 obtain a homogenous composite film. An IKA T25 Disperser Homogenizer 175 (ULTRA-TURRAX®, Staufen, Germany) at 15000 rpm for 3 min was used to 176 prepare W/W emulsions. The shear applied by homogenizer resulted in 177 developing small droplets of HPMC phase dispersed in the continuous gelatin 178 phase. The resultant W/W emulsions were vacuum (0.02 MPa) filtered for 30 179 min to remove the air bubbles. Casting was performed onto plastic plates. 180 Plates were maintained at room temperature for 48 h. After drying, films were 181 peeled off from the plates and conditioned (temperature 24±1 °C and relative 182 humidity (RH) around 50% for 72 h) [36, 37] in a closed box containing 183 saturated salt solution prior to analysis. Control (gelatin and HPMC) films were 184 prepared and treated in the same manner.
185 186
2.5. Determining the effect of shearing on the appearance of the films
187
In order to study the effect of shear rate (magnetic stirring for 20 min at 188 500 rpm and homogenizing at 9000, 11000 and 15000 rpm for 3 min) on the 189 film appearance, the dispersed phase distribution and the shape of droplets a 190
8
FITC-labeled gelatin:HPMC (80:20) mixed dispersion was prepared. After 191 applying different shear rates, the resultant dispersions were cast onto plastic 192 plates and treated in the same manner as described previously.
193 194
2.6. Film thickness
195
Film thickness was measured using a micrometer (Starrett Na 436-1in, 196 USA) to the nearest 0.001 inch in at least 7 random points around the testing 197 area. Average values were used to calculate mechanical (tensile strength), 198 barrier (water vapor permeability) and optical (opacity) properties.
199 200
2.7. Visual aspects
201
The appearance of the films was checked for homogeneity, color 202 uniformity and the absence of phase separating, insoluble particles and brittle 203 zones [36].
204 205
2.8. Mechanical properties
206
The tensile strength (TS) and the elongation at break (EAB) of the films 207 were measured using a Texture Analyzer (Brookfield CS3, USA) equipped with 208 a 1000 g load cell. Dumbbell shaped samples (50 mm length, 4mm width in the 209 middle and 8.5 mm width at the ends) were used in this study. The crosshead 210 speed was 50 mm/min. Test was done in at least 8 replications. TS (kPa) and 211 %EAB were measured by Eqs. (1) and (2), respectively:
212
9
TS= F/A
(1) 213
where F is the maximum force applied for tension of the sample (N), A is 214 the area of the cross section of the film (thickness × width, m2). % EAB = (∆L /L) × 100
215
(2) 216 217
where ∆L is the change in length before breakage and L is the initial 218 length.
219 220
2.9. Water vapor permeability (WVP)
221
Water vapor permeability was determined according to the standard 222 method of ASTM E95-E96 [37]. Cups were filled with 15 g anhydrous CaCl2 223 (0% RH1), covered with a specimen of conditioned film, sealed and placed in a 224 container containing saturated NaCl solution (75% RH2) at 25 °C. Samples 225 were weighed in 1 h intervals during 8 h WVP was measured using Eq. (3): WVP = (WVTR) ͯ I / P0 (RH1-RH2)
226
(3) 227
where water vapor transmission rate (WVTR) is the slope of the mass 228 change versus time curve (g/h), l is the average film thickness (mm), P0 is the 229 saturation vapor pressure at 25 °C (1753.55 Pa) and (RH1-RH2) is the 230 difference in the relative humidity of the anhydrous calcium chloride (0%) and 231 super saturated NaCl solution (0.75%).
232 233
2.10. Swelling test
234
10
Square cuts of films (2×2 cm2) were dried (at 60 °C for 24 h), weighed and 235 immersed in 0.1 M NaCl solution at room temperature. Samples were removed 236 from the solution after 30 min and placed between two dry filter papers to 237 absorb excess surface water. The swollen samples were finally weighed. 238 Swelling ratio was calculated according to Eq. (4) [38]: Swelling (%) = Mf – Mi / Mi ×100 where Mf and Mi are the weights of swollen and
239
(4) 240 dried samples, 241
respectively.
242 243
2.11. Solubility test
244
Square-shaped films (2×2 cm2) were dried in an oven at 105 °C to a 245 constant weight (Mi). Dried samples were then immersed in 50 ml distilled 246 water under slow mechanical shaking for 24 h at room temperature. After this 247 period, samples were re-dried and weighed again (Mf). Solubility ratio was 248 calculated based on Eq. (5) [39]:
249
Solubility (%) = Mi - Mf / Mi ×100
(5) 250 251
2.12. Opacity
252
A rectangular piece of film (1×5 cm2) was placed on the inner surface of a 253 spectrophotometer cell. A UV-visible spectrophotometer (UNICO UV-2100, 254 USA) was used for measuring the absorbance of the samples at 600 nm. 255 Opacity was determined using Eq. (6) [33]:
256
11
A = Absorbance600nm / X
(6) 257
where X is the average film thickness (mm).
258 259
2.13 Scanning electron microscopy (SEM)
260
The microstructure of the film network (the sample containing 20% 261 HPMC) was studied using scanning electron microscope (Vega3, TESCAN, 262 Brno, Czech). The sample was mounted on the specimen holder with aluminum 263 tape and then coated with gold sputter coater (Dsr1, Nanostructured Co, Iran). 264 Morphological observation of the film surface was performed at an accelerating 265 voltage of 20.0 kV [33].
266 267
2.14. Statistical analysis
268
All experiments were performed at least in triplicates. Analysis of variance 269 (ANOVA) was performed to determine significant differences between the 270 means. Duncan multiple range test (P
