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Characterization of edible gum cordia film: Effects of beeswax
LWT - Food Science and Technology 68 (2016) 674e680
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Characterization of edible gum cordia film: Effects of beeswax Muhammad Abdul Haq a, *, Abid Hasnain a, Feroz Alam Jafri a, Muhammad Faheem Akbar b, Adnan Khan c a
Department of Food Science & Technology, University of Karachi, Karachi, 75270, Pakistan Department of Agriculture & Agribusiness Management, University of Karachi, Karachi, 75270, Pakistan c Department of Microbiology, University of Karachi, Karachi, 75270, Pakistan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2015 Received in revised form 4 January 2016 Accepted 5 January 2016 Available online 8 January 2016
In order to expand the applications of gum cordia films, beeswax was added into the gum and properties of resulting films were studied. Film forming emulsions were prepared by gum cordia, polyethylene glycol 400, beeswax (0.05e0.20 g g1 of gum cordia) and glycerol monostearate. The emulsions were stable during film preparation process. Lipid droplets were found to be homogenously disperse into gum cordia with the average size of about 1.3 mm. Films were prepared by emulsion casting method. Addition of beeswax reduced tensile strength, Young's modulus and elongation at break of the films. Water vapor permeability (WVP) of beeswax containing films was 0.06e0.33 1010 g m1 s1 Pa1 which is about one magnitude less than the films without wax. The activation energy of WVP was found to be higher (15 e18 kJ mol1) for the films incorporated with beeswax than of beeswax free film (6.6 kJ mol1). We also observed that addition of beeswax at the concentration of 0.05 g g1 increased oxygen permeability (OP) by a factor of about 50 (from 0.39 to 18.73 g m1 s1 Pa1). However, further increase in beeswax did not increase the OP. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Emulsified film Permeation properties Thermal properties Light scattering granulometry
1. Introduction Edible packaging provides safe, healthy, convenient and environment friendly food to the ever conscious consumer and this drives the interest of advanced research activities in this field (Janjarasskul, Rauch, McCarthy, & Krochta, 2014). A number of macromolecules (biopolymers) have been reported as edible packaging material. Proteins e.g. gelatin, casein and gluten whereas carbohydrates e.g. starch, modified cellulose and pectin have been used this purpose. Lipids are generally used as coatings rather than standalone films due to their low mechanical strength. The materials include waxes, fatty acids, and acetylated glycerides (Kester & Fennema, 1989). Biopolymers can also be combined to achieve desired property of the film as one can do in conventional plastic films (Kowalczyk & Baraniak, 2014). Combination of polysaccharide with lipid merges the high moisture barrier property of lipids with structural integrity of polysaccharides. There are two methods to achieve this combination; one is known as bi-layer and the other is emulsification. Layering method is tedious and constituted of
several cycles of casting and drying. On the other hand, the emulsified films are easy to prepare as they do not require extra casting and drying steps. A number of studies have been recently published on the incorporation of lipids such as wax into the polysaccharide film to cause the modification of several properties of hydrocolloid ska, 2015). films (Galus & Kadzin Gum cordia, an anionic polysaccharide, has been recognized to produce flexible, transparent films with excellent oxygen barrier properties (Haq, Hasnain, & Azam, 2014a). It also carries the antioxidants effectively for the protection of oxygen sensitive products (Haq, Alam, & Hasnain, 2013). Furthermore, it possesses excellent emulsifying properties (Benhura & Chidewe, 2004). However, high oxygen but low moisture barrier properties render it unsuitable for fresh commodities. As mentioned above, polysaccharide based films can be modified by blending with lipids to overcome this problem. Therefore, in order to broaden the applications of gum cordia, this work is designed to study the effects of beeswax on gum cordia film. 2. Materials and methods
* Corresponding author. E-mail address: [email protected] (M.A. Haq). http://dx.doi.org/10.1016/j.lwt.2016.01.011 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
The fruits of Cordia myxa were collected from Karachi, Pakistan and gum was extracted as described previously by our group (Haq
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et al., 2013). Other chemicals of reagent grade were purchased from Sigma (SigmaeAldrich GmbH, Sternheim, Germany).
the size distribution based on the specific surface per unit volume (Fabra, Jimenez, Atares, Talens, & Chiralt, 2009).
2.1. Film forming emulsions (FFE) and film fabrication
2.4. Microstructure of the films
Film forming emulsions (FFE) were prepared according to recipe described in Table 1. Molten beeswax and glycerol monostearate (GMS) was added to the polyethylene glycol (PEG 400) containing gum solution and the mixture was homogenized at 11,000 rpm for 10 min. During homogenization, temperature was maintained at 70 C i.e. above the melting point of the beeswax, using water bath. After degassing, FFE were poured into glass petri dishes (diameter 15.5 cm) and dried at 40 C for 48 h in an environmental chamber (Lab Tech Model LCT 1075C, Korea). The volume of FFE was adjusted to maintain a consistent mass of solids (1.6 g) in each petri dish to minimize the variation in film thickness among treatments. Thickness of the films was measured using a digital micrometer (Mitutoyo, Model MDH-25M, Japan). Five random positions around each film of six film samples were used for average thickness estimation. The samples were conditioned to 58% RH for 72 h before measurements.
Microstructure of the films was observed using a scanning electron microscope (JEOL JSM-5410, Japan). Films were dried by placing them in a desiccator containing P2O5 for 15 days. Then films were frozen in liquid N2 gently and randomly broken to investigate the cross section of the samples as describe by Fabra et al. (2009). Films were fixed on copper stubs; gold coated, and observed using an accelerating voltage of 10 kV.
2.2. Emulsion stability index Emulsion stability index (ESI) of the FFE was determined by turbidimetry as described by Kowalczyk and Baraniak (2014) with some modifications. In the freshly prepared emulsions potassium sorbate at the rate of 0.0001 g g1 of emulsion was added (to avoid microbial growth) and then emulsions were transferred into test tubes and the tubes were incubated at 40 C in water bath. 500 mL portions of the emulsions were pipetted from the bottom of the test tubes after 5 min (time zero), 24, 48 and 168 h and diluted with 0.001 g g1 sodium dodecyl sulphate (SDS) solution to obtain a dilution of 1/100. The absorbance of diluted emulsions was measured against diluted control solution (beeswax free) at 500 nm. Measurements were performed in triplicate. ESI was calculated from the following equation:
ESIð%Þ ¼
At 100 A0
where: At is the absorbance at time of 24, 48 and 168 h, and A0 is the absorbance at time zero. 2.3. Laser light scattering granulometry Laser light scattering based instrument (Malvern Mastersizer 3000, Malvern Instruments Ltd., Malvern, U.K.) was used to determined the particle size of beeswax in FFE after dilution in water or 0.001 g g1 SDS. For dried FFE i.e. films, the samples were prepared by dispersing 1 g of film in 50 mL of either deionized water or 0.001 g g1 SDS with moderate magnetic stirring. The mean particle size was recorder as the D3,2 diameter i.e., the mean diameter over
2.5. Thermal properties Glass transition temperature (Tg) of films was determined by DSC (TA instrument Q10, USA). The samples were weighed (ca. 10 mg) in aluminum pans followed by sealing with inverted lids. Reference was an empty aluminum pan sealed in a same manner. Both pans were then equilibrated at 90 C for 30 s to stabilize the baseline followed by scanning till 90 C at a heating rate of 15 C min1. Thermograms were recorded and analyzed by TA Instrument software (Universal Analysis 2000, Version 4.1D). The Tg was identified as the inflexion point of the baseline caused by the discontinuity in the specific heat of the sample. The melting point (Tm) of the films was also determined. 2.6. Tensile properties of the films Tensile properties were measured using Universal Testing Machine (Zwick Roell, Ulm, Germany) according to ASTM Standard D882 (ASTM, 1996a) using rectangular (100х30 mm) specimen with the test speed of 100 mm min1. Data was analyzed by Zwick software (Test Xpert V11.02). 2.7. Water vapor permeability Water vapor permeability (WVP) was determined by ASTM gravimetric method E-96 1996 (ASTM, 1996b). In the cup silica gel was taken to maintain 0% RH for all experiments. Test cups were then sealed with the films and placed in individual desiccators saturated with various salts (sodium bromide, sodium chloride and barium chloride) solutions, which provide the relative humidity of 58, 75 and 90% at 25 C (Greenspan, 1977). The cups were then weighed every after 2 h till 72 h. To study the effect of temperature on WVP, in the desiccators saturated sodium chloride was taken and the experiments were conducted at 5, 25 and 30 C. At these temperatures the humidity of saturated sodium chloride solution is 75.65 ± 0.27, 75.29 ± 0.12, and 75.09 ± 0.11% respectively (Greenspan, 1977). Data was analyzed for WVP as describe in our previous report (Haq et al., 2014a,Haq, Hasnain, Jamil, & Haider, 2014b).
Table 1 Formulation of film forming emulsions and thickness of the resulting films.a Treatment
Recipe ratio gum: PEG 400:BW:GMS
Gum cordia (g)
PEG 400 (g)
Beeswax (g)
GMS (g)
Film thickness (mm)
Control With beeswax without GMS With beeswax and GMS
1:0.2:0.0.00:0 1:0.2:0.0.05:0 1:0.2:0.05:0.005 1:0.2:0.1:0.01 1:0.2:0.15:0.015 1:0.2:0.2:0.02
1 1 1 1 1 1
0.2 0.2 0.2 0.2 0.2 0.2
0 0.05 0.05 0.10 0.15 0.20
0 0 0.005 0.010 0.015 0.020
73.3 ND 73.7 74.8 74.6 73.8
± 1.8a ± ± ± ±
1.9a 2.1a 1.4a 1.2a
a BW ¼ beeswax, PEG 400 ¼ polyethylene glycol PEG 400, GMS ¼ glycerol monostearate, ND ¼ not determined, For film thickness data is presented as mean ± standard error and means with different superscript alphabets in the column are significantly different (P < 0.05).
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2.8. Oxygen permeability Oxygen permeability was measured using steady state technique as described by Ayranci and Tunc (2003). 2.9. Statistical analysis All tests were performed in triplicate unless otherwise specified. Data was analyzed using statistical package for social scientists (SPSS version 17). Analysis of variance (ANOVA) followed by Duncan multiple range test was used to distinguish the treatments at p < 0.05. 3. Results and discussions 3.1. Film thickness Films of uniform thickness (73.3e74.8 mm) were achieved in this study, and it was not found to vary among treatments (Table 1). In solution casting method, the amount of solids poured into the mold greatly affects the thickness of the resulting films. In previous studies of emulsified films, two different methods in terms of casted amount of film forming emulsion (FFE) have been reported. In one method constant weight or volume of FFE is casted (Bauemler, Carelli, & Martini, 2014; Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015), whereas in other method; the volume of FFE is adjusted so that the constant weight of solids is casted in all treatments (Fabra et al., 2009; Janjarasskul et al., 2014; Kowalczyk & Baraniak, 2014; Perez-Gago & Krochta, 2001). The later method was preferred in this study, because it minimizes the variation in film thickness, which may affects the permeability of the films other than treatment effect. Thickness of emulsified films may also be affected by the difference in densities of phases and their interaction. The lipid phase may impede the alignment of polymer chains and as a result, loose and unordered structure is developed which results in increase in thickness of emulsified films (Tongnuanchan et al., 2015). However, this effect was not observed in this study, probably due to low amount of lipid phase. 3.2. Emulsion stability & laser light scattering granulometry Water vapor permeability (WVP) and tensile properties of films are affected by lipid particle size (Perez-Gago & Krochta, 2001). Small and homogenous particles increase polymerelipid interactions at the lipid interfaces; result in stronger films with lower WVP. The lipid droplet size was found to be in the range of 1.31e1.34 mm with average standard deviation of about 6% (Table 2) Table 2 Mean particle Diameter D3,2 (mm) of film forming emulsion prepared from gum cordia and beeswax.a Before drying Emulsion Beeswax sample concentration Water SDS based on gum cordia weight (g g1) Without GMS With GMS
After drying Water
SDS
0.05
1.37 ± 0.08a 1.34 ± 0.08a 12.78 ± 1.03a 8.32 ± 0.98a
0.05 0.10 0.15 0.20
1.32 1.31 1.31 1.34
± ± ± ±
0.06a 0.06a 0.07a 0.08a
1.33 1.29 1.39 1.36
± ± ± ±
0.07a 0.06a 0.07a 0.07a
1.33 1.41 1.39 1.33
± ± ± ±
0.07b 0.06b 0.07b 0.09b
1.36 1.38 1.39 1.38
± ± ± ±
0.05b 0.04b 0.07b 0.07b
a Ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard deviation, n ¼ 3, Means with different superscript alphabets in the column are significantly different (P < 0.05).
Fig. 1. Representative particle size distribution curve of film forming emulsion prepared from gum cordia, PEG 400, beeswax and glycerol monostearate.
and it was not affected by the beeswax content. Furthermore, the particle size distribution was homogeneous and normal (Fig. 1). It reveals that the emulsification process was efficient and consistent (Talens & Krochta, 2005). Emulsion stability index determined by turbidimetry was found to be 100% for all the samples even after one week (data not shown). Emulsions are inherently unstable. However, destabilization is a kinetic phenomenon and FFE must be stable during drying to form homogeneous film. The primary processes leading to instability are creaming, aggregation, and coalescence. Due to difference in densities of two phases the lipid droplets move upward which is called creaming which is governs by Stokes' law. According to this law, creaming rate is directly proportional to the difference of densities of two phases and square of the diameter of lipid droplets and is inversely proportional to the viscosity of the continuous phase i.e. gum solution in our case. It means that, small particles and high viscosity favors the stable emulsion. According to Perez-Gago and Krochta (2001), lipid phase separation occurred in beeswax containing whey protein isolate (WPI) emulsion when the droplets diameter was between 1.5 and 2.0 mm. They used the 0.1 g g1 solution of WPI, which possesses the viscosity of about 2.5 mPa s. Since the viscosity of gum cordia is much higher (about 400 mPa s (Mukherjee, Dinda, & Barik, 2008)) than WPI, therefore creaming was not expected. The other mechanisms of destabilization of emulsion are aggregation and coalescence. Aggregation occurs when droplets collide, due to the prevailing attractive forces at a determined distance between them. Coalescence is the merging of droplets and hence increases in droplet size. Aggregation and coalescence can be avoided by decreasing the surface energy at two phases, which is the function of emulsifier. Dispersing the emulsion in an anionic surfactant such as SDS, allows to distinguish between coalescence and aggregation. SDS disperses the aggregates but does not affect on coalesced particles (Phan et al., 2002). Aggregation is evident when the mean particle diameter is smaller when granulometry analysis is done in the dissociating medium i.e. SDS. Since there is no difference in mean particle size in water and in SDS, therefore aggregation was not occurred before or after preparation of film (Table 2) except in case when GMS was not added during emulsification. Even this emulsion was stable before drying and the mean particle size is not different from GMS containing samples. Furthermore mean particle size in water is same before and after drying (Table 2) which reveals that coalescence was also not occurred. This shows that gum cordia has the sufficient surface
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activity to form stable emulsion with beeswax, which is consistent with the previous reports on emulsifying ability of gum cordia (Benhura & Chidewe, 2004; Mukherjee et al., 2008). Severe coalescence and aggregation was observed in the film which was prepared by emulsion without addition of GMS, which is not only evidence by particle size analysis but was also observable visually. Therefore, this film was not further characterized. This unusual behavior can be explained by the fact that the protein portion plays an important role in protein containing polysaccharide stabilized emulsions (Evans, Ratcliffe, & Williams, 2013) and this protein may undergoes conformational changes during drying step of film formation. These changes possibly alter the ability of protein to adsorb at the lipidewater interface and thus the emulsion is destabilized. Gum cordia contains about 0.12 g g1 protein (Haq et al., 2014b). Although, the role of protein portion in viscosity of gum cordia has been reported (Benhura & Chidewe, 2011), its exact function in emulsion stability is not established and therefore requires detailed study on this aspect. 3.3. Microstructure of the films SEM micrographs of the surface and freeze-fractured cross section of gum cordia films with and without beeswax at different levels are shown in Fig. 2. The surface and cross section of the control film (without beeswax) was smoother and more homogeneous than those films incorporated with beeswax. In nonemulsified films, the homogeneous structure has been observed in other biopolymer e.g. casein, amaranth flour and soy protein based films (Soazo, Perez, Rubiolo, & Verdini, 2013). The homogenous structure of the control film reveals that the polymer chains were evenly and densely packed to form the film during drying which is good for mechanical properties. The microstructure of the emulsified film is affected by the arrangement of the different components in the emulsion and their interaction during drying. When FFE is dried some of the lipid particles may hang to the surface and make it rough (Tongnuanchan et al., 2015), this effect was more profoundly observed at high beeswax level (0.15 and 0.20 g g1) probably due to limited capacity of the polymer to completely coat the beeswax. However, the cross sectional structure of top (expose to air) and bottom layer (in contact with petri dish) of the film was same, showing no gross migration of beeswax during drying, which is consistence with the emulsion stability discussion. Furthermore, a layer by layer (laminar) structure was observed in the cross section of the emulsified films, which is expected to be due to the self association of the lipid fraction during
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drying. These successive lipid layers in the film matrix greatly contribute to reduce water permeability, thus improve the water barrier properties (Fabra et al., 2009). In other studies with wax containing films, this layered structure was not observed (Bauemler et al., 2014; Fabra et al., 2009; Janjarasskul et al., 2014), probably due to absence of any amphiphilic emulsifier (GMS in this work) in those studies. 3.4. Thermal properties Two different thermal transitions were observed in the films (Fig. 3). First transition (25 to 30 C) is associated with the glass transition of gum cordia while second transition around (48e53 C) is due to the combine melting of GMS and beeswax because the melting points of GMS and beeswax are 58e59 C and 62e64 C respectively. Food materials may show the first-order thermal transitions e.g. melting, crystallization and transitions between polymorphic states in fats and a second order phase transition e.g. glass transition in amorphous or partially amorphous food materials. The amorphous region of biopolymer may be present as glassy or rubbery state depending on the temperature. When glassy form of polymer is subjected to change in temperature, polymer chains may become flexible which results in large changes in physical properties. This phenomenon is referred as glass transition and corresponding temperature is called glass transition temperature (Tg). Incorporation of beeswax decreased the Tg of the gum cordia (Table 3). Low Tg indicates high mobility of polymer chains. This means that the addition of beeswax impeded the polymer chains interaction in film matrix, thereby increased their mobility. Increase in mobility of the polymer chain (plasticization effect) by lipids has been reported in other polymers and lipids systems (Talens & Krochta, 2005; Tongnuanchan et al., 2015). A combine melting peak of GMS and beeswax was also observed (second transition). The melting temperature was found to gradually decrease from 53 C in 0.2 g g1 beeswax containing film to 48 C in the film containing 0.05 g g1 beeswax (Fig. 3). This change in temperature can be explained by different crystal structure of beeswax at different level. Beeswax is a complex material containing about 300 different substances, mainly fatty acid esters and alcohols and it has been reported to crystallize upon cooling from molten state (Pinzon, Torres, Hoffmann, & Lamprecht, 2013). Lipids, in general, tend to take multiple forms in a crystal lattice (polymorphism) which exhibit different melting temperature. Apart from molecular structure, external factors like rate of cooling, pressure, impurities and shear rate influences polymorphism (Sato,
Fig. 2. SEM micrographs of surface and cross-section (1000 magnification) of films from gum cordia incorporated with beeswax at different levels. In cross-sectional images, the upper surface represents the film surface exposed to the air during film casting.
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Fig. 3. DSC thermogram of films from gum cordia incorporated with beeswax at different levels. Different lines represent the concentration of beeswax (g g1) in the film forming emulsion based on gum cordia weight. The ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax.
Table 3 Glass transition temperature (Tg) of films from gum cordia incorporated with beeswax at different levels.a Film sample
Beeswax concentration based on gum cordia weight (g g1)
Tg ( C)
Control film Emulsified film
0 0.05 0.10 0.15 0.20
18.37 25.19 26.30 27.67 29.12
± ± ± ± ±
0.48a 0.57b 0.31c 0.54d 0.49e
factors, TS and EAB may increase or decrease. The reduction in mechanical properties by addition of lipid component has been reported by several authors e.g. Bravin, Peressini, and Sensidoni (2006) observed up to 50% reduction in TS and EAB by the addition of cocoa butter emulsified with GMS. Phan et al. (2002) reported that addition of hydrogenated palm kernel Oil (HPKO) at 30% reduces TS and EAB by a factor of 0.8e3 depending on the nature of emulsifier and drying conditions. In contrast to these reports, Ma et al. (2012) have been reported that addition of olive
a Ratio of Gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard error, n ¼ 3, Means with different superscript alphabets in the column are significantly different (P < 0.05).
2001). Slow cooling results in stable polymorphic form having high melting point. Since, samples containing high level of beeswax are expected to cool slowly-heat capacity of beeswax is lower than water, therefore it is expected that beeswax formed the crystals at a slower rate in these sample and hence stable ones (high melting point). However, crystallization in emulsion is a complex phenomenon (Coupland, 2002) and high melting point of the beeswax at high level requires further investigation. 3.5. Tensile properties of the films Effects of beeswax on tensile strength (TS), Young's modulus (YM) and elongation at break (EAB) on gum cordia film are shown in Fig. 4. Addition of beeswax reduced TS, YM and EAB. This is expected to be due to the development of a global heterogeneous film structure by the incorporation of beeswax which makes the film less compact and discontinuous. Furthermore, it resulted in dilution of gum cordia polymer by the beeswax in the film matrix. Beeswax is non-polar and they have very low interaction between themselves or the polar gum cordia polymer, which resulted in poor tensile properties. In literature, both increase and decrease in mechanical properties has been reported by the addition of lipid in protein and carbohydrate films. This modification depends on several factors e.g. nature of polymer, type, physical state and level of lipid phase and film fabrication process. Depending on these
Fig. 4. Effects of beeswax on tensile strength, elongation at break and Young's modulus of films from gum cordia. The ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax ( Tensile strength, Elongation at break, Young's modulus).
oil increases TS and EAB of gelatin film. They explained this phenomenon by phase separation and possible cross linking of protein chains by the phenolic compounds present in the olive oil. As the immiscible oil phase increases the protein moves to protein rich phase and greater links between protein chains are formed thus TS
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Table 4 Activation energy (Ea) of water vapor permeability of films from gum cordia incorporated with beeswax at different levels.a Film sample
Beeswax concentration based on gum cordia weight (g g1)
Control film Emulsified film
0
Ea (kJ mol1) R2 3.75 ± 0.1
0.15 0.10 0.15 0.20
16.02 17.91 18.30 18.71
± ± ± ±
0.21 0.31 0.12 0.09
0.967 0.998 0.990 0.995 0.962
a Ratio of Gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2: x:0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard error, n ¼ 3.
Table 5 Oxygen permeability (OP) of films from gum cordia incorporated with beeswax at different levels.a Fig. 5. Effects of beeswax on water vapor permeability of films from gum cordia. Different lines represent the relative humidity (RH) of the measurement chamber. The ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax ( 58% RH, 75% RH, 90% RH).
and EAB increases. Phan, Debeaufort, Voilley, and Luu (2009) reported, decrease in TS but increase in EAB by the addition of hydrogenated fat in agar and cassava starch film.
Film sample
Beeswax concentration based on gum cordia weight (g g1)
OP (1015 g m1 s1 Pa1)
Control Emulsified Film
0 0.05 0.10 0.15 0.20
0.39 18.73 24.37 23.41 23.10
± ± ± ± ±
0.03a 1.19b 2.30c 1.69c 1.07c
a Ratio of Gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x: 0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard error, n ¼ 3, Means with different superscript alphabets in the columns are significantly different (P < 0.05).
3.6. Water vapor permeability Water vapor permeability (WVP) of beeswax (0.05 g g1) containing film was found to be 0.08e0.24 1010 g m1 s1 Pa1 which is about one magnitude less than the films without wax (Fig. 5). However, further increase in beeswax up to 0.2 g g1 resulted in only 50% decrease in WVP and this effect was more pronounced at higher partial pressure difference (Fig. 5). For all films, WVP was found to increase by the increase in applied partial pressure difference. According to the definition; WVP is independent of applied partial pressure difference (Schwartzberg, 1986). However, the hydrophilic films show high WVP at higher partial pressure differences. This non ideal behavior is due to the assumption of linear isotherm in Fick's law which is used to derive the expression for WVP. However, edible films exhibit nonlinear isotherm. When humidity at high partial pressure side is high, water interacts with polymer matrix easily and the polymer chains become more flexible (plasticization effect of water), hence offer less resistance to mass transfer, which results in high WVP. Plasticization effect of water in edible films is well known and it was also observed in our previous study with gum cordia based edible films (Haq et al., 2014a,b). Decrease in WVP by the addition of lipid component has been previously reported for carbohydrate based films. Phan et al. (2009) reported that addition of vegetable oil reduces WVP of agar and cassava starch films by a factor of approximately 4 and 2 respectively. Yang and Paulson (2000) found that addition of beeswax reduces WVP of gellan film from 1.5 to 0.5 1010 g m1 s1 Pa1. Effects of temperature on WVP are presented in Table 4. Arrhenius equation was fitted well on experimental data (R2 > 0.90). The activation energy was also found to be higher (15e18 kJ mol1) in beeswax containing films than of control film ewithout beeswax (6.6 kJ mol1). Increase in activation energy by the addition of wax has been previously reported by Kester and Fennema (1989) for wax laminated cellulose film. However they reported about 4 times higher (59.4 kJ mol1) value than this study. The reason for this difference may be explained by the mechanism
of incorporation of wax. They used the layering technique to form the film whereas in this study the emulsification method was used.
3.7. Oxygen permeability Oxygen plays an important role in the quality of food products. Generally it is involved in deterioration such as oxidative rancidity, microbial growth and enzymatic browning. However, its presence is essential for storage of certain food commodities e.g. fruits and vegetables. These products require an optimum level of oxygen permeability (OP) of packaging for maximum shelf life (Pesis, 2005). Biopolymer based films possesses lower OP than synthetic films. In situations where high OP is required, lipophilic compounds can be combined with biopolymers. Effects of beeswax on OP of the gum cordia films are shown in Table 5. Incorporation of beeswax at the level of 0.05 g g1 increased the OP by a factor of about 50. This is expected to be due to the low oxygen barrier of non polar beeswax compared to polar polymer (Ayranci & Tunc, 2003). However, further increase in beeswax exhibited no change in OP. It can be hypothesized that beeswax created an interconnected network within the gum cordia matrix which provided the diffusion path of the lower oxygen barrier which is supported by the SEM images (Fig. 2). Furthermore, this network was fully created at lowest beeswax concentration i.e. 0.05 g g1 therefore, further increase in beeswax did not increase the OP.
4. Conclusion This study demonstrates the potential of gum cordia to combine with lipid in order to tailor the properties of the resulting film. The findings of this study will facilitate the uses of abundant but underutilized plant resource Cordia myxa. It will also provide the foundation for further development in edible packaging from gum cordia especially as coating for fresh commodities.
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References ASTM. (1996a). Test methods for tensile properties of thin plastic sheeting-method D882-91 Annual book of ASTM. Philadelphia: American Society for Testing and Materials. ASTM. (1996b). Standard test methods for water vapor transmission of materialsmethod E-96 Annual book of ASTM. Philadelphia: American Society for Testing and Materials. Ayranci, E., & Tunc, S. (2003). A method for the measurement of the oxygen permeability and the development of edible films to reduce the rate of oxidative reactions in fresh foods. Food Chemistry, 80(3), 423e431. Bauemler, E. R., Carelli, A. A., & Martini, S. (2014). Preparation and physical properties of calcium pectinate films modified with sunflower wax. European Journal of Lipid Science and Technology, 116(11), 1534e1545. Benhura, M. A. N., & Chidewe, C. K. (2004). The emulsifying properties of a polysaccharide isolated from the fruit of Cordia abyssinica. International Journal of Food Science and Technology, 39(5), 579e583. Benhura, M., & Chidewe, C. (2011). Characterization of the polysaccharide material that is isolated from the fruit of Cordia abyssinica. African Journal of Biochemistry Research, 5(3), 95e101. Bravin, B., Peressini, D., & Sensidoni, A. (2006). Development and application of polysaccharide-lipid edible coating to extend shelf-life of dry bakery products. Journal of Food Engineering, 76(3), 280e290. Coupland, J. N. (2002). Crystallization in emulsions. Current Opinion in Colloid & Interface Science, 7(5e6), 445e450. Evans, M., Ratcliffe, I., & Williams, P. A. (2013). Emulsion stabilisation using polysaccharide-protein complexes. Current Opinion in Colloid & Interface Science, 18(4), 272e282. Fabra, M. J., Jimenez, A., Atares, L., Talens, P., & Chiralt, A. (2009). Effect of fatty acids and beeswax addition on properties of sodium caseinate dispersions and films. Biomacromolecules, 10(6), 1500e1507. ska, J. (2015). Food applications of emulsion-based edible films Galus, S., & Kadzin and coatings. Trends in Food Science & Technology, 45(2), 273e283. Greenspan, L. (1977). Humidity fixed points of binary saturated aqueous solutions. Journal of Research of the National Bureau of Standards, 81(1), 89e96. Haq, M. A., Alam, M. J., & Hasnain, A. (2013). Gum cordia: a novel edible coating to increase the shelf life of Chilgoza (Pinus gerardiana). LWT e Food Science and Technology, 50(1), 306e311. Haq, M. A., Hasnain, A., & Azam, M. (2014a). Characterization of edible gum cordia film: effects of plasticizers. LWT e Food Science and Technology, 55(1), 163e169. Haq, M. A., Hasnain, A., Jamil, K., & Haider, M. S. (2014b). Extraction and characterization of gum from Cordia myxa. Asian Journal of Chemistry, 26(1), 122e126. Janjarasskul, T., Rauch, D. J., McCarthy, K. L., & Krochta, J. M. (2014). Barrier and tensile properties of whey proteinecandelilla wax film/sheet. LWT e Food Science and Technology, 56(2), 377e382.
Kester, J. J., & Fennema, O. (1989). An edible film of lipids and cellulose ethers: barrier properties to moisture vapor transmission and structural evaluation. Journal of Food Science, 54(6), 1383e1389. Kowalczyk, D., & Baraniak, B. (2014). Effect of candelilla wax on functional properties of biopolymer emulsion films e a comparative study. Food Hydrocolloids, 41, 195e209. Ma, W., Tang, C. H., Yin, S. W., Yang, X. Q., Wang, Q., Liu, F., et al. (2012). Characterization of gelatin-based edible films incorporated with olive oil. Food Research International, 49(1), 572e579. Mukherjee, B., Dinda, S. C., & Barik, B. B. (2008). Gum cordia: a novel matrix forming material for enteric resistant and sustained drug delivery. Aaps Pharmscitech, 9(1), 330e333. Perez-Gago, M. B., & Krochta, J. M. (2001). Lipid particle size effect on water vapor permeability and mechanical properties of whey protein/beeswax emulsion films. Journal of Agricultural and Food Chemistry, 49(2), 996e1002. Pesis, E. (2005). The role of the anaerobic metabolites, acetaldehyde and ethanol, in fruit ripening, enhancement of fruit quality and fruit deterioration. Postharvest Biology and Technology, 37(1), 1e19. roval, C., Despre , D., Courthaudon, J. L., & Voilley, A. Phan, The D., Debeaufort, F., Pe (2002). Arabinoxylanlipid-based edible films and coatings. 3. Influence of drying temperature on film structure and functional properties. Journal of Agricultural and Food Chemistry, 50(8), 2423e2428. Phan, The D., Debeaufort, F., Voilley, A., & Luu, D. (2009). Influence of hydrocolloid nature on the structure and functional properties of emulsified edible films. Food Hydrocolloids, 23(3), 691e699. Pinzon, F., Torres, A., Hoffmann, W., & Lamprecht, I. (2013). Thermoanalytical and infrared spectroscopic investigations on wax samples of native Colombian bees living in different altitudes. Engineering in Life Sciences, 13(6), 520e527. Sato, K. (2001). Crystallization behaviour of fats and lipids e a review. Chemical Engineering Science, 56(7), 2255e2265. Schwartzberg, H. G. (1986). Modeling of gas and vapor transport through hydrophilic films. In M. Mathlouthi (Ed.), Food packaging and preservation: Theory and practice (pp. 212e217). London: Elsevier Applied Science Publishing Co. Soazo, M., Perez, L. M., Rubiolo, A. C., & Verdini, R. A. (2013). Effect of freezing on physical properties of whey protein emulsion films. Food Hydrocolloids, 31(2), 256e263. Talens, P., & Krochta, J. M. (2005). Plasticizing effects of beeswax and carnauba wax on tensile and water vapor permeability properties of whey protein films. Journal of Food Science, 70(3), E239eE243. 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, 248e259. Yang, L., & Paulson, A. T. (2000). Effects of lipids on mechanical and moisture barrier properties of edible gellan film. Food Research International, 33(7), 571e578.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Characterization of edible gum cordia film: Effects of beeswax Muhammad Abdul Haq a, *, Abid Hasnain a, Feroz Alam Jafri a, Muhammad Faheem Akbar b, Adnan Khan c a
Department of Food Science & Technology, University of Karachi, Karachi, 75270, Pakistan Department of Agriculture & Agribusiness Management, University of Karachi, Karachi, 75270, Pakistan c Department of Microbiology, University of Karachi, Karachi, 75270, Pakistan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2015 Received in revised form 4 January 2016 Accepted 5 January 2016 Available online 8 January 2016
In order to expand the applications of gum cordia films, beeswax was added into the gum and properties of resulting films were studied. Film forming emulsions were prepared by gum cordia, polyethylene glycol 400, beeswax (0.05e0.20 g g1 of gum cordia) and glycerol monostearate. The emulsions were stable during film preparation process. Lipid droplets were found to be homogenously disperse into gum cordia with the average size of about 1.3 mm. Films were prepared by emulsion casting method. Addition of beeswax reduced tensile strength, Young's modulus and elongation at break of the films. Water vapor permeability (WVP) of beeswax containing films was 0.06e0.33 1010 g m1 s1 Pa1 which is about one magnitude less than the films without wax. The activation energy of WVP was found to be higher (15 e18 kJ mol1) for the films incorporated with beeswax than of beeswax free film (6.6 kJ mol1). We also observed that addition of beeswax at the concentration of 0.05 g g1 increased oxygen permeability (OP) by a factor of about 50 (from 0.39 to 18.73 g m1 s1 Pa1). However, further increase in beeswax did not increase the OP. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Emulsified film Permeation properties Thermal properties Light scattering granulometry
1. Introduction Edible packaging provides safe, healthy, convenient and environment friendly food to the ever conscious consumer and this drives the interest of advanced research activities in this field (Janjarasskul, Rauch, McCarthy, & Krochta, 2014). A number of macromolecules (biopolymers) have been reported as edible packaging material. Proteins e.g. gelatin, casein and gluten whereas carbohydrates e.g. starch, modified cellulose and pectin have been used this purpose. Lipids are generally used as coatings rather than standalone films due to their low mechanical strength. The materials include waxes, fatty acids, and acetylated glycerides (Kester & Fennema, 1989). Biopolymers can also be combined to achieve desired property of the film as one can do in conventional plastic films (Kowalczyk & Baraniak, 2014). Combination of polysaccharide with lipid merges the high moisture barrier property of lipids with structural integrity of polysaccharides. There are two methods to achieve this combination; one is known as bi-layer and the other is emulsification. Layering method is tedious and constituted of
several cycles of casting and drying. On the other hand, the emulsified films are easy to prepare as they do not require extra casting and drying steps. A number of studies have been recently published on the incorporation of lipids such as wax into the polysaccharide film to cause the modification of several properties of hydrocolloid ska, 2015). films (Galus & Kadzin Gum cordia, an anionic polysaccharide, has been recognized to produce flexible, transparent films with excellent oxygen barrier properties (Haq, Hasnain, & Azam, 2014a). It also carries the antioxidants effectively for the protection of oxygen sensitive products (Haq, Alam, & Hasnain, 2013). Furthermore, it possesses excellent emulsifying properties (Benhura & Chidewe, 2004). However, high oxygen but low moisture barrier properties render it unsuitable for fresh commodities. As mentioned above, polysaccharide based films can be modified by blending with lipids to overcome this problem. Therefore, in order to broaden the applications of gum cordia, this work is designed to study the effects of beeswax on gum cordia film. 2. Materials and methods
* Corresponding author. E-mail address: [email protected] (M.A. Haq). http://dx.doi.org/10.1016/j.lwt.2016.01.011 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
The fruits of Cordia myxa were collected from Karachi, Pakistan and gum was extracted as described previously by our group (Haq
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et al., 2013). Other chemicals of reagent grade were purchased from Sigma (SigmaeAldrich GmbH, Sternheim, Germany).
the size distribution based on the specific surface per unit volume (Fabra, Jimenez, Atares, Talens, & Chiralt, 2009).
2.1. Film forming emulsions (FFE) and film fabrication
2.4. Microstructure of the films
Film forming emulsions (FFE) were prepared according to recipe described in Table 1. Molten beeswax and glycerol monostearate (GMS) was added to the polyethylene glycol (PEG 400) containing gum solution and the mixture was homogenized at 11,000 rpm for 10 min. During homogenization, temperature was maintained at 70 C i.e. above the melting point of the beeswax, using water bath. After degassing, FFE were poured into glass petri dishes (diameter 15.5 cm) and dried at 40 C for 48 h in an environmental chamber (Lab Tech Model LCT 1075C, Korea). The volume of FFE was adjusted to maintain a consistent mass of solids (1.6 g) in each petri dish to minimize the variation in film thickness among treatments. Thickness of the films was measured using a digital micrometer (Mitutoyo, Model MDH-25M, Japan). Five random positions around each film of six film samples were used for average thickness estimation. The samples were conditioned to 58% RH for 72 h before measurements.
Microstructure of the films was observed using a scanning electron microscope (JEOL JSM-5410, Japan). Films were dried by placing them in a desiccator containing P2O5 for 15 days. Then films were frozen in liquid N2 gently and randomly broken to investigate the cross section of the samples as describe by Fabra et al. (2009). Films were fixed on copper stubs; gold coated, and observed using an accelerating voltage of 10 kV.
2.2. Emulsion stability index Emulsion stability index (ESI) of the FFE was determined by turbidimetry as described by Kowalczyk and Baraniak (2014) with some modifications. In the freshly prepared emulsions potassium sorbate at the rate of 0.0001 g g1 of emulsion was added (to avoid microbial growth) and then emulsions were transferred into test tubes and the tubes were incubated at 40 C in water bath. 500 mL portions of the emulsions were pipetted from the bottom of the test tubes after 5 min (time zero), 24, 48 and 168 h and diluted with 0.001 g g1 sodium dodecyl sulphate (SDS) solution to obtain a dilution of 1/100. The absorbance of diluted emulsions was measured against diluted control solution (beeswax free) at 500 nm. Measurements were performed in triplicate. ESI was calculated from the following equation:
ESIð%Þ ¼
At 100 A0
where: At is the absorbance at time of 24, 48 and 168 h, and A0 is the absorbance at time zero. 2.3. Laser light scattering granulometry Laser light scattering based instrument (Malvern Mastersizer 3000, Malvern Instruments Ltd., Malvern, U.K.) was used to determined the particle size of beeswax in FFE after dilution in water or 0.001 g g1 SDS. For dried FFE i.e. films, the samples were prepared by dispersing 1 g of film in 50 mL of either deionized water or 0.001 g g1 SDS with moderate magnetic stirring. The mean particle size was recorder as the D3,2 diameter i.e., the mean diameter over
2.5. Thermal properties Glass transition temperature (Tg) of films was determined by DSC (TA instrument Q10, USA). The samples were weighed (ca. 10 mg) in aluminum pans followed by sealing with inverted lids. Reference was an empty aluminum pan sealed in a same manner. Both pans were then equilibrated at 90 C for 30 s to stabilize the baseline followed by scanning till 90 C at a heating rate of 15 C min1. Thermograms were recorded and analyzed by TA Instrument software (Universal Analysis 2000, Version 4.1D). The Tg was identified as the inflexion point of the baseline caused by the discontinuity in the specific heat of the sample. The melting point (Tm) of the films was also determined. 2.6. Tensile properties of the films Tensile properties were measured using Universal Testing Machine (Zwick Roell, Ulm, Germany) according to ASTM Standard D882 (ASTM, 1996a) using rectangular (100х30 mm) specimen with the test speed of 100 mm min1. Data was analyzed by Zwick software (Test Xpert V11.02). 2.7. Water vapor permeability Water vapor permeability (WVP) was determined by ASTM gravimetric method E-96 1996 (ASTM, 1996b). In the cup silica gel was taken to maintain 0% RH for all experiments. Test cups were then sealed with the films and placed in individual desiccators saturated with various salts (sodium bromide, sodium chloride and barium chloride) solutions, which provide the relative humidity of 58, 75 and 90% at 25 C (Greenspan, 1977). The cups were then weighed every after 2 h till 72 h. To study the effect of temperature on WVP, in the desiccators saturated sodium chloride was taken and the experiments were conducted at 5, 25 and 30 C. At these temperatures the humidity of saturated sodium chloride solution is 75.65 ± 0.27, 75.29 ± 0.12, and 75.09 ± 0.11% respectively (Greenspan, 1977). Data was analyzed for WVP as describe in our previous report (Haq et al., 2014a,Haq, Hasnain, Jamil, & Haider, 2014b).
Table 1 Formulation of film forming emulsions and thickness of the resulting films.a Treatment
Recipe ratio gum: PEG 400:BW:GMS
Gum cordia (g)
PEG 400 (g)
Beeswax (g)
GMS (g)
Film thickness (mm)
Control With beeswax without GMS With beeswax and GMS
1:0.2:0.0.00:0 1:0.2:0.0.05:0 1:0.2:0.05:0.005 1:0.2:0.1:0.01 1:0.2:0.15:0.015 1:0.2:0.2:0.02
1 1 1 1 1 1
0.2 0.2 0.2 0.2 0.2 0.2
0 0.05 0.05 0.10 0.15 0.20
0 0 0.005 0.010 0.015 0.020
73.3 ND 73.7 74.8 74.6 73.8
± 1.8a ± ± ± ±
1.9a 2.1a 1.4a 1.2a
a BW ¼ beeswax, PEG 400 ¼ polyethylene glycol PEG 400, GMS ¼ glycerol monostearate, ND ¼ not determined, For film thickness data is presented as mean ± standard error and means with different superscript alphabets in the column are significantly different (P < 0.05).
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2.8. Oxygen permeability Oxygen permeability was measured using steady state technique as described by Ayranci and Tunc (2003). 2.9. Statistical analysis All tests were performed in triplicate unless otherwise specified. Data was analyzed using statistical package for social scientists (SPSS version 17). Analysis of variance (ANOVA) followed by Duncan multiple range test was used to distinguish the treatments at p < 0.05. 3. Results and discussions 3.1. Film thickness Films of uniform thickness (73.3e74.8 mm) were achieved in this study, and it was not found to vary among treatments (Table 1). In solution casting method, the amount of solids poured into the mold greatly affects the thickness of the resulting films. In previous studies of emulsified films, two different methods in terms of casted amount of film forming emulsion (FFE) have been reported. In one method constant weight or volume of FFE is casted (Bauemler, Carelli, & Martini, 2014; Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015), whereas in other method; the volume of FFE is adjusted so that the constant weight of solids is casted in all treatments (Fabra et al., 2009; Janjarasskul et al., 2014; Kowalczyk & Baraniak, 2014; Perez-Gago & Krochta, 2001). The later method was preferred in this study, because it minimizes the variation in film thickness, which may affects the permeability of the films other than treatment effect. Thickness of emulsified films may also be affected by the difference in densities of phases and their interaction. The lipid phase may impede the alignment of polymer chains and as a result, loose and unordered structure is developed which results in increase in thickness of emulsified films (Tongnuanchan et al., 2015). However, this effect was not observed in this study, probably due to low amount of lipid phase. 3.2. Emulsion stability & laser light scattering granulometry Water vapor permeability (WVP) and tensile properties of films are affected by lipid particle size (Perez-Gago & Krochta, 2001). Small and homogenous particles increase polymerelipid interactions at the lipid interfaces; result in stronger films with lower WVP. The lipid droplet size was found to be in the range of 1.31e1.34 mm with average standard deviation of about 6% (Table 2) Table 2 Mean particle Diameter D3,2 (mm) of film forming emulsion prepared from gum cordia and beeswax.a Before drying Emulsion Beeswax sample concentration Water SDS based on gum cordia weight (g g1) Without GMS With GMS
After drying Water
SDS
0.05
1.37 ± 0.08a 1.34 ± 0.08a 12.78 ± 1.03a 8.32 ± 0.98a
0.05 0.10 0.15 0.20
1.32 1.31 1.31 1.34
± ± ± ±
0.06a 0.06a 0.07a 0.08a
1.33 1.29 1.39 1.36
± ± ± ±
0.07a 0.06a 0.07a 0.07a
1.33 1.41 1.39 1.33
± ± ± ±
0.07b 0.06b 0.07b 0.09b
1.36 1.38 1.39 1.38
± ± ± ±
0.05b 0.04b 0.07b 0.07b
a Ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard deviation, n ¼ 3, Means with different superscript alphabets in the column are significantly different (P < 0.05).
Fig. 1. Representative particle size distribution curve of film forming emulsion prepared from gum cordia, PEG 400, beeswax and glycerol monostearate.
and it was not affected by the beeswax content. Furthermore, the particle size distribution was homogeneous and normal (Fig. 1). It reveals that the emulsification process was efficient and consistent (Talens & Krochta, 2005). Emulsion stability index determined by turbidimetry was found to be 100% for all the samples even after one week (data not shown). Emulsions are inherently unstable. However, destabilization is a kinetic phenomenon and FFE must be stable during drying to form homogeneous film. The primary processes leading to instability are creaming, aggregation, and coalescence. Due to difference in densities of two phases the lipid droplets move upward which is called creaming which is governs by Stokes' law. According to this law, creaming rate is directly proportional to the difference of densities of two phases and square of the diameter of lipid droplets and is inversely proportional to the viscosity of the continuous phase i.e. gum solution in our case. It means that, small particles and high viscosity favors the stable emulsion. According to Perez-Gago and Krochta (2001), lipid phase separation occurred in beeswax containing whey protein isolate (WPI) emulsion when the droplets diameter was between 1.5 and 2.0 mm. They used the 0.1 g g1 solution of WPI, which possesses the viscosity of about 2.5 mPa s. Since the viscosity of gum cordia is much higher (about 400 mPa s (Mukherjee, Dinda, & Barik, 2008)) than WPI, therefore creaming was not expected. The other mechanisms of destabilization of emulsion are aggregation and coalescence. Aggregation occurs when droplets collide, due to the prevailing attractive forces at a determined distance between them. Coalescence is the merging of droplets and hence increases in droplet size. Aggregation and coalescence can be avoided by decreasing the surface energy at two phases, which is the function of emulsifier. Dispersing the emulsion in an anionic surfactant such as SDS, allows to distinguish between coalescence and aggregation. SDS disperses the aggregates but does not affect on coalesced particles (Phan et al., 2002). Aggregation is evident when the mean particle diameter is smaller when granulometry analysis is done in the dissociating medium i.e. SDS. Since there is no difference in mean particle size in water and in SDS, therefore aggregation was not occurred before or after preparation of film (Table 2) except in case when GMS was not added during emulsification. Even this emulsion was stable before drying and the mean particle size is not different from GMS containing samples. Furthermore mean particle size in water is same before and after drying (Table 2) which reveals that coalescence was also not occurred. This shows that gum cordia has the sufficient surface
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activity to form stable emulsion with beeswax, which is consistent with the previous reports on emulsifying ability of gum cordia (Benhura & Chidewe, 2004; Mukherjee et al., 2008). Severe coalescence and aggregation was observed in the film which was prepared by emulsion without addition of GMS, which is not only evidence by particle size analysis but was also observable visually. Therefore, this film was not further characterized. This unusual behavior can be explained by the fact that the protein portion plays an important role in protein containing polysaccharide stabilized emulsions (Evans, Ratcliffe, & Williams, 2013) and this protein may undergoes conformational changes during drying step of film formation. These changes possibly alter the ability of protein to adsorb at the lipidewater interface and thus the emulsion is destabilized. Gum cordia contains about 0.12 g g1 protein (Haq et al., 2014b). Although, the role of protein portion in viscosity of gum cordia has been reported (Benhura & Chidewe, 2011), its exact function in emulsion stability is not established and therefore requires detailed study on this aspect. 3.3. Microstructure of the films SEM micrographs of the surface and freeze-fractured cross section of gum cordia films with and without beeswax at different levels are shown in Fig. 2. The surface and cross section of the control film (without beeswax) was smoother and more homogeneous than those films incorporated with beeswax. In nonemulsified films, the homogeneous structure has been observed in other biopolymer e.g. casein, amaranth flour and soy protein based films (Soazo, Perez, Rubiolo, & Verdini, 2013). The homogenous structure of the control film reveals that the polymer chains were evenly and densely packed to form the film during drying which is good for mechanical properties. The microstructure of the emulsified film is affected by the arrangement of the different components in the emulsion and their interaction during drying. When FFE is dried some of the lipid particles may hang to the surface and make it rough (Tongnuanchan et al., 2015), this effect was more profoundly observed at high beeswax level (0.15 and 0.20 g g1) probably due to limited capacity of the polymer to completely coat the beeswax. However, the cross sectional structure of top (expose to air) and bottom layer (in contact with petri dish) of the film was same, showing no gross migration of beeswax during drying, which is consistence with the emulsion stability discussion. Furthermore, a layer by layer (laminar) structure was observed in the cross section of the emulsified films, which is expected to be due to the self association of the lipid fraction during
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drying. These successive lipid layers in the film matrix greatly contribute to reduce water permeability, thus improve the water barrier properties (Fabra et al., 2009). In other studies with wax containing films, this layered structure was not observed (Bauemler et al., 2014; Fabra et al., 2009; Janjarasskul et al., 2014), probably due to absence of any amphiphilic emulsifier (GMS in this work) in those studies. 3.4. Thermal properties Two different thermal transitions were observed in the films (Fig. 3). First transition (25 to 30 C) is associated with the glass transition of gum cordia while second transition around (48e53 C) is due to the combine melting of GMS and beeswax because the melting points of GMS and beeswax are 58e59 C and 62e64 C respectively. Food materials may show the first-order thermal transitions e.g. melting, crystallization and transitions between polymorphic states in fats and a second order phase transition e.g. glass transition in amorphous or partially amorphous food materials. The amorphous region of biopolymer may be present as glassy or rubbery state depending on the temperature. When glassy form of polymer is subjected to change in temperature, polymer chains may become flexible which results in large changes in physical properties. This phenomenon is referred as glass transition and corresponding temperature is called glass transition temperature (Tg). Incorporation of beeswax decreased the Tg of the gum cordia (Table 3). Low Tg indicates high mobility of polymer chains. This means that the addition of beeswax impeded the polymer chains interaction in film matrix, thereby increased their mobility. Increase in mobility of the polymer chain (plasticization effect) by lipids has been reported in other polymers and lipids systems (Talens & Krochta, 2005; Tongnuanchan et al., 2015). A combine melting peak of GMS and beeswax was also observed (second transition). The melting temperature was found to gradually decrease from 53 C in 0.2 g g1 beeswax containing film to 48 C in the film containing 0.05 g g1 beeswax (Fig. 3). This change in temperature can be explained by different crystal structure of beeswax at different level. Beeswax is a complex material containing about 300 different substances, mainly fatty acid esters and alcohols and it has been reported to crystallize upon cooling from molten state (Pinzon, Torres, Hoffmann, & Lamprecht, 2013). Lipids, in general, tend to take multiple forms in a crystal lattice (polymorphism) which exhibit different melting temperature. Apart from molecular structure, external factors like rate of cooling, pressure, impurities and shear rate influences polymorphism (Sato,
Fig. 2. SEM micrographs of surface and cross-section (1000 magnification) of films from gum cordia incorporated with beeswax at different levels. In cross-sectional images, the upper surface represents the film surface exposed to the air during film casting.
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Fig. 3. DSC thermogram of films from gum cordia incorporated with beeswax at different levels. Different lines represent the concentration of beeswax (g g1) in the film forming emulsion based on gum cordia weight. The ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax.
Table 3 Glass transition temperature (Tg) of films from gum cordia incorporated with beeswax at different levels.a Film sample
Beeswax concentration based on gum cordia weight (g g1)
Tg ( C)
Control film Emulsified film
0 0.05 0.10 0.15 0.20
18.37 25.19 26.30 27.67 29.12
± ± ± ± ±
0.48a 0.57b 0.31c 0.54d 0.49e
factors, TS and EAB may increase or decrease. The reduction in mechanical properties by addition of lipid component has been reported by several authors e.g. Bravin, Peressini, and Sensidoni (2006) observed up to 50% reduction in TS and EAB by the addition of cocoa butter emulsified with GMS. Phan et al. (2002) reported that addition of hydrogenated palm kernel Oil (HPKO) at 30% reduces TS and EAB by a factor of 0.8e3 depending on the nature of emulsifier and drying conditions. In contrast to these reports, Ma et al. (2012) have been reported that addition of olive
a Ratio of Gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard error, n ¼ 3, Means with different superscript alphabets in the column are significantly different (P < 0.05).
2001). Slow cooling results in stable polymorphic form having high melting point. Since, samples containing high level of beeswax are expected to cool slowly-heat capacity of beeswax is lower than water, therefore it is expected that beeswax formed the crystals at a slower rate in these sample and hence stable ones (high melting point). However, crystallization in emulsion is a complex phenomenon (Coupland, 2002) and high melting point of the beeswax at high level requires further investigation. 3.5. Tensile properties of the films Effects of beeswax on tensile strength (TS), Young's modulus (YM) and elongation at break (EAB) on gum cordia film are shown in Fig. 4. Addition of beeswax reduced TS, YM and EAB. This is expected to be due to the development of a global heterogeneous film structure by the incorporation of beeswax which makes the film less compact and discontinuous. Furthermore, it resulted in dilution of gum cordia polymer by the beeswax in the film matrix. Beeswax is non-polar and they have very low interaction between themselves or the polar gum cordia polymer, which resulted in poor tensile properties. In literature, both increase and decrease in mechanical properties has been reported by the addition of lipid in protein and carbohydrate films. This modification depends on several factors e.g. nature of polymer, type, physical state and level of lipid phase and film fabrication process. Depending on these
Fig. 4. Effects of beeswax on tensile strength, elongation at break and Young's modulus of films from gum cordia. The ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax ( Tensile strength, Elongation at break, Young's modulus).
oil increases TS and EAB of gelatin film. They explained this phenomenon by phase separation and possible cross linking of protein chains by the phenolic compounds present in the olive oil. As the immiscible oil phase increases the protein moves to protein rich phase and greater links between protein chains are formed thus TS
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Table 4 Activation energy (Ea) of water vapor permeability of films from gum cordia incorporated with beeswax at different levels.a Film sample
Beeswax concentration based on gum cordia weight (g g1)
Control film Emulsified film
0
Ea (kJ mol1) R2 3.75 ± 0.1
0.15 0.10 0.15 0.20
16.02 17.91 18.30 18.71
± ± ± ±
0.21 0.31 0.12 0.09
0.967 0.998 0.990 0.995 0.962
a Ratio of Gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2: x:0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard error, n ¼ 3.
Table 5 Oxygen permeability (OP) of films from gum cordia incorporated with beeswax at different levels.a Fig. 5. Effects of beeswax on water vapor permeability of films from gum cordia. Different lines represent the relative humidity (RH) of the measurement chamber. The ratio of gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x:0.1x, where x represent the concentration of beeswax ( 58% RH, 75% RH, 90% RH).
and EAB increases. Phan, Debeaufort, Voilley, and Luu (2009) reported, decrease in TS but increase in EAB by the addition of hydrogenated fat in agar and cassava starch film.
Film sample
Beeswax concentration based on gum cordia weight (g g1)
OP (1015 g m1 s1 Pa1)
Control Emulsified Film
0 0.05 0.10 0.15 0.20
0.39 18.73 24.37 23.41 23.10
± ± ± ± ±
0.03a 1.19b 2.30c 1.69c 1.07c
a Ratio of Gum cordia: PEG 400: beeswax: glycerol monostearate is 1:0.2:x: 0.1x, where x represent the concentration of beeswax, Data is presented as mean ± standard error, n ¼ 3, Means with different superscript alphabets in the columns are significantly different (P < 0.05).
3.6. Water vapor permeability Water vapor permeability (WVP) of beeswax (0.05 g g1) containing film was found to be 0.08e0.24 1010 g m1 s1 Pa1 which is about one magnitude less than the films without wax (Fig. 5). However, further increase in beeswax up to 0.2 g g1 resulted in only 50% decrease in WVP and this effect was more pronounced at higher partial pressure difference (Fig. 5). For all films, WVP was found to increase by the increase in applied partial pressure difference. According to the definition; WVP is independent of applied partial pressure difference (Schwartzberg, 1986). However, the hydrophilic films show high WVP at higher partial pressure differences. This non ideal behavior is due to the assumption of linear isotherm in Fick's law which is used to derive the expression for WVP. However, edible films exhibit nonlinear isotherm. When humidity at high partial pressure side is high, water interacts with polymer matrix easily and the polymer chains become more flexible (plasticization effect of water), hence offer less resistance to mass transfer, which results in high WVP. Plasticization effect of water in edible films is well known and it was also observed in our previous study with gum cordia based edible films (Haq et al., 2014a,b). Decrease in WVP by the addition of lipid component has been previously reported for carbohydrate based films. Phan et al. (2009) reported that addition of vegetable oil reduces WVP of agar and cassava starch films by a factor of approximately 4 and 2 respectively. Yang and Paulson (2000) found that addition of beeswax reduces WVP of gellan film from 1.5 to 0.5 1010 g m1 s1 Pa1. Effects of temperature on WVP are presented in Table 4. Arrhenius equation was fitted well on experimental data (R2 > 0.90). The activation energy was also found to be higher (15e18 kJ mol1) in beeswax containing films than of control film ewithout beeswax (6.6 kJ mol1). Increase in activation energy by the addition of wax has been previously reported by Kester and Fennema (1989) for wax laminated cellulose film. However they reported about 4 times higher (59.4 kJ mol1) value than this study. The reason for this difference may be explained by the mechanism
of incorporation of wax. They used the layering technique to form the film whereas in this study the emulsification method was used.
3.7. Oxygen permeability Oxygen plays an important role in the quality of food products. Generally it is involved in deterioration such as oxidative rancidity, microbial growth and enzymatic browning. However, its presence is essential for storage of certain food commodities e.g. fruits and vegetables. These products require an optimum level of oxygen permeability (OP) of packaging for maximum shelf life (Pesis, 2005). Biopolymer based films possesses lower OP than synthetic films. In situations where high OP is required, lipophilic compounds can be combined with biopolymers. Effects of beeswax on OP of the gum cordia films are shown in Table 5. Incorporation of beeswax at the level of 0.05 g g1 increased the OP by a factor of about 50. This is expected to be due to the low oxygen barrier of non polar beeswax compared to polar polymer (Ayranci & Tunc, 2003). However, further increase in beeswax exhibited no change in OP. It can be hypothesized that beeswax created an interconnected network within the gum cordia matrix which provided the diffusion path of the lower oxygen barrier which is supported by the SEM images (Fig. 2). Furthermore, this network was fully created at lowest beeswax concentration i.e. 0.05 g g1 therefore, further increase in beeswax did not increase the OP.
4. Conclusion This study demonstrates the potential of gum cordia to combine with lipid in order to tailor the properties of the resulting film. The findings of this study will facilitate the uses of abundant but underutilized plant resource Cordia myxa. It will also provide the foundation for further development in edible packaging from gum cordia especially as coating for fresh commodities.
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