Effect of candelilla wax on functional properties of biopolymer emulsion films – A comparative study

Effect of candelilla wax on functional properties of biopolymer emulsion films – A comparative study

Food Hydrocolloids 41 (2014) 195e209 Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd ...

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Food Hydrocolloids 41 (2014) 195e209

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Effect of candelilla wax on functional properties of biopolymer emulsion films e A comparative study Dariusz Kowalczyk*, Barbara Baraniak Department of Biochemistry and Food Chemistry, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 November 2013 Accepted 2 April 2014 Available online 19 April 2014

The overall research objective was to compare the effects of increasing concentrations of candelilla wax (CnW) on the physicochemical and morphological properties of edible films based on different biopolymers: carboxymethyl cellulose (CMC), oxidized potato starch (OPS), soy protein isolate (SPI), and gelatin (GEL). The findings are discussed in terms of the stability of emulsion formulations. CnW was incorporated into film-forming solutions at 0, 0.5, 1.0, 1.5, and 2.0%; sorbitol and Tween 40 were used as the plasticizer and surfactant, respectively. It was found that, with the exception of GEL films, wax incorporation significantly decreased the WVP of the films (from 11.1 to 41.7% compared to the controls). Regardless of the wax concentration used, OPS films had the lowest WVP compared to other films. GELbased films, in turn, were characterized by the highest mechanical resistance and elongation. The incorporation of CnW decreased both all the analyzed tensile parameters and the puncture strength of the films. Generally, as CnW concentration increased, the transparency and redness of the films decreased, while UV blocking ability and yellowness increased. The 0.5% CnW addition was the most effective in improving water barrier properties, and simultaneously had the lowest impact on the other physical properties of films. For OPS, SPI, and GEL films, sorbitol recrystallization was observed over time. Wax accelerated the sorbitol crystal growth process. CMC films, in contrast to those obtained using other polymers, were completely water soluble and did not exhibit sorbitol crystallization. The emulsion films differed from the wax-free in their surface characteristics. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Edible films CMC Oxidized starch SPI Gelatin

1. Introduction Modern food packaging has made life easier in many ways: extended product shelf life, portion control, improved food distribution (handling, opening, reclosing, use, and reuse), takeout food, ready-to-eat food, etc. Unfortunately, this convenience has contributed to a rapid increase in the quantity of packaging waste in landfills. The economic and technical problems associated with the recycling of plastic packaging waste, as well as the gradual depletion of world oil reserves, have triggered the need to develop environmentally friendly packaging. “Green” packaging can be based on a variety of environmentally sustainable materials, mainly renewable biopolymers. Depending on the formulation and production methodology, bio-based packaging can be compostable/ biodegradable or even edible, which enables new application possibilities. Edible packaging can be used wherever the application of synthetic materials is limited, e.g. layers separating various

* Corresponding author. Tel.: þ48 81 462 33 26; fax: þ48 81 462 33 24. E-mail address: [email protected] (D. Kowalczyk). http://dx.doi.org/10.1016/j.foodhyd.2014.04.004 0268-005X/Ó 2014 Elsevier Ltd. All rights reserved.

components in complex food products, edible coatings, carriers of food additives, microcapsules, etc. Edible films and coatings have the potential to improve the quality and shelf life of many different food-stuffs. In practice, however, their effectiveness is determined by mass transfer barrier properties, as well as properties of the product. Materials selection and design for structural biopolymers is becoming increasingly important for different industrial products. Edible packaging is mainly based on polysaccharides (e.g. starch, cellulose derivatives, pectin, alginate, chitosan) and proteins (e.g. collagen, gelatin, milk proteins, soy protein, albumin, gluten, zein). The principles for polymer selection for edible packaging must take into account different aspects of the raw material, such as price and accessibility, nutrition-economic aspects (utilization of some natural biopolymers, e.g. starch or soy proteins, can be problematic because of competition against food use), health safety (e.g. some proteins are known to have the capacity to cause allergies), functional properties (e.g. viscosity, solubility, and barrier, mechanical and optical properties), compatibility with additives, the possibility of interactions with food components, etc. Polysaccharides and proteins exhibit good filmogenic properties; however, due to their

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hydrophilic nature, they form poor barriers to moisture. This disadvantage, however, can be circumvented by mixing them with lipids. Currently, intensive studies are being conducted into the incorporation of fats, fatty acids and waxes into biopolymer films to improve their moisture sensitivity (Chick & Hernandez, 2002; Fabra, Jiménez, Atares, Talens, & Chiralt, 2009; Fabra, Pérez-Masiá, Talens, & Chiralt, 2011; Jiménez, Fabra, Talens, & Chiralt, 2010; Matsakidou, Biliaderis, & Kiosseoglou, 2013; Soazo, Pérez, Rubiolo, & Verdini, 2013). Among the range of different hydrophobic substances, waxes represent the best barrier to moisture (Donhowe & Fennema, 1993; Shellhammer & Krochta, 1997). From the commercial perspective, the most important waxes in the food industry are beeswax (BW), candelilla wax (CnW), and carnauba wax (CrW). These natural waxes are GRAS (generally recognized as safe) and approved by the Food and Drug Administration (FDA) for use in fruit and vegetable coatings, beverages, and confectionery. Wax emulsions, especially those containing CnW, are commonly used to prolong the shelf-life quality of some fruits (Kowalczyk & Pikula, 2010) and vegetables (Kowalczyk, 2011; Kowalczyk, Pikula, & Baraniak, 2010). CnW is derived from the leaves and stems of Candelilla shrubs (usually from the plant Euphorbia cerifera, syn. Euphorbia antisyphilitica) growing wild in northern Mexico and southern Texas. It is obtained either by boiling the plant material or by extraction with benzene. Commercially available refined CnW ranges in color between yellow and brown. It is a hard and brittle wax with a melting-point of 68.5e72.5  C. The chemical composition of CnW is about 28e29% esters, 12e14% alcohols and sterols, 50e57% hydrocarbons, 7e9% free acids, 0.5e1% moisture and 0.7% inorganic residues (FAO JECFA, 2006). The water vapor permeability (WVP) of CnW is nearly twice as low as CrW and more than three times lower than BW (Donhowe & Fennema, 1993); thus, CnW has potentially the best ability to reduce the WVP of biopolymer films. This has been proved by Chick and Hernandez (2002), who observed that incorporation of CnW decreased the WVP of casein films by 72.3%, while only a 25.8% reduction was achieved via the addition of CrW. However, Shellhammer and Krochta (1997) showed that whey protein films incorporating CnW and CrW did not differ in terms of WVP, and were more permeable than those with the addition of BW or anhydrous milk fat. As has been suggested by different authors, the lipid type and its concentration are not the only factors affecting the barrier properties of emulsion films. An important role is also played by lipid particle size in the emulsion, lipidepolymer interactions, and lipid phase distribution in the film matrix (Fabra et al., 2011; Jiménez et al., 2010; PérezGago & Krochta, 2001; Shellhammer & Krochta, 1997). The structure and stability of an emulsion is a key parameter strongly affecting barrier and mechanical properties of lipidecontaining films. If a film is cast from an unstable emulsion, the lipid may begin to separate during drying, creating a gradient of lipid concentrations across the thickness of the film. Emulsion destabilization tends to produce films with a “bilayer-like” structure, which tends to improve the functional properties of edible films. The higher the phase separation phenomena in films are, the lower the water vapor permeability (Morillon, Debeaufort, Blond, Capelle, & Voilley, 2002). Creaming is the principal process by which the disperse phase separates from an emulsion and is typically the precursor to coalescence. Its rate depends on the square of the lipid droplet diameter. According to Stoke’s law, large droplets suspended in a solution will cream more rapidly. The research by Fabra et al. (2011) showed that the bigger lipid particle size of stearic acid in filmforming dispersion resulted in a thicker lipid layer in films; thus, WVP was more efficiently reduced than when they were small. In turn, oleic acid did not form a bilayer-like structure, and thus did not offer a perpendicular resistance to moisture transfer (Fabra et al. 2011). However, the contrasting data can also be found in

the literature, i.e. the smaller the lipid globule size, and the more homogeneously the globules are distributed, the lower the WVP (Debeaufort & Voilley, 1995; McHugh & Krochta, 1994b). Due to the lack of affinity of waxes to water, obtaining stable emulsions is extremely difficult. For this reason, the use of emulsifiers is often recommended. For wax-in-water emulsions, an emulsifier with an HLB value in the range of 10e16 is required (Troy & Beringer, 2006). Many polysaccharides and proteins used as filmforming agents may act as emulsifying and emulsion stabilizing agents. In the presence of polymers, wax emulsion particles can be mechanically lodged in the polymer matrix, or form surface layers at the polymer/air interface. Hydrocolloids as thickening agents can modify the rheology of the continuous phase affecting stability by changing the flocculation and creaming behavior. Lipid separation may be completely retarded if the continuous phase contains a three-dimensional network of aggregated molecules which traps the droplets and prevents them from moving. Thus the droplets (or particles of solid fats) in oil-in-water emulsions can be completely stabilized against creaming by using biopolymers which form a gel in the aqueous phase. The surface activity of some polysaccharides (e.g. arabic gum, modified starches and celluloses, some kinds of pectin and galactomannans) has a molecular origin in either (i) the non-polar character of chemical groups attached to the hydrophilic polysaccharide backbone (in hydrophobically modified starch/cellulose) or (ii) the presence of a protein component linked covalently or physically to the polysaccharide (some gums, pectins, etc.). Proteins act as emulsifier agents due to their amphiphilic nature, which allows them to reduce the interfacial tension at the oil-water interface. Soybean, milk or egg proteins are the most widely used food emulsifying agents. Gelatin, the only protein that can be properly categorized as a hydrocolloid, exhibits some emulsifying properties, but its more characteristic roles are as a colloid stabilizer and gelling agent (Dickinson, 2009). So far, there has been limited direct comparison of the stability of wax-in-water emulsions made with different biopolymers. This paper compares the wax emulsion-stabilizing properties of carboxymethyl cellulose (CMC), oxidized potato starch (OPS), soy protein isolate (SPI), and gelatin (GEL) to explain the effects of increasing concentrations of CnW on the physicochemical and morphological properties of the edible films produced using these biopolymers. The film-forming agents selected for this study are frequently used for producing edible films and coatings. However, to enable the preparation of polysaccharide and proteinaceous films under the same conditions, and mainly to obtain castable film-forming solutions, low viscosity types/derivatives of polysaccharides have been chosen. 2. Materials and methods 2.1. Materials Commercial food-grade biopolymers were used in this study: sodium carboxymethyl cellulose 30 GA (Dow-Wolff Cellulosics, , Germany); oxidized potato starch LU-1404-2 (WPPZ S.A. Lubon Poland); soy protein isolate GS5200A (Gushen Biological Technology Group Co., LTD, China); pork gelatin (McCormick-Kamis Polska S.A, Poland). Candelilla wax SP-75 was purchased from Strahl & Pitsch Inc (USA). Sorbitol (Sor) and Tween 40 were purchased from Sigma Chemical Co., St. Louis, MO (USA). 2.2. Film preparation Emulsion films were obtained from 5% (w/w) aqueous biopolymer solutions containing 3% (w/w) Sor and 0.5, 1.0, 1.5, or 2.0% (w/w) CnW. Tween 40 was added as a surfactant in a 1:0.2

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wax/surfactant weight ratio. Film-forming solutions (FFSs) based on biopolymers and a plasticizer served as the controls. Biopolymers were mixed with Sor and dispersed/hydrated in distilled water, and then heated in a water bath at 90  C for 30 min with constant stirring. In the case of OPS, the heating time was prolonged to 60 min to enable viscosity reduction. The FFSs were  cooled to 50 C and cast on leveled polycarbonate trays (12  12  1 cm). To prepare emulsified films, 5 min before the end of heating, a melted mixture of CnW and surfactant was added, and the hot solution was emulsified with a homogenizer (H-500, PolEko Aparatura, Poland) at 27,000 rpm for 2 min; then, at 20,000 rpm for 3 min. A constant amount of 1.6 g of total solids was cast onto an area of 144 cm2, in order to maintain film thickness. Films were dried at room temperature (25  1  C) for w24 h; afterward, films were peeled manually.

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2.6. Mechanical properties Test of mechanical property was carried out using the TA-XT2i texture analyzer (Stable Micro Systems, UK). The films were tested immediately after they were taken out of the chamber to minimize errors. Film samples (2  5 cm) were mounted with the aid of adhesive tape and clamped with steel grips. The initial grip separation was set to 30 mm, and films were stretched using crosshead speed of 1 mm s1. The stressestrain curves were recorded. Tensile strength (TS, MPa) and elongation at break (E, %) were calculated using the following relationships:

TS ¼ Fmax =A where Fmax is the maximum load for breaking film (N) and A is the initial specimen cross-sectional area (thickness  width, mm2)

2.3. Determination of emulsifying properties

E ¼ ðDL=LÞ$100

Emulsifying activity index (EAI) and emulsifying stability index (ESI) of the film-forming emulsions prepared as described in Section 2.2 were determined according to Pearce and Kinsella (1978) and Cameron, Weber, Idziak, Neufeld, and Cooper (1991) with some modifications. Freshly prepared emulsions were transferred into test tubes (internal diameter 10 mm, height 75 mm). 500 ml portions of the emulsions were pipetted from the bottom of the test tubes both immediately and 0.17, 0.5, 1, 3, 6, 12, 24, 48 h after homogenization. Each portion was diluted with 0.1% (w/v) SDS to obtain a dilution of 1/100. The absorbance of diluted emulsions was measured against diluted wax-free FFSs at l ¼ 500 nm. Measurements were performed in triplicate. EAI was calculated from the following equation:

where L is the initial gage length (30 mm) and DL is the difference in the length at the moment of fracture. Elastic modulus (EM, MPa) was calculated from the slope of the initial linear region of the stressestrain curve by the following formula:

 .  . EAI m2 g ¼ ð2$2:303$A0 $DFÞ ðC$l$ð1  4ÞÞ where: A0 is the absorbance at time 0 h, DF is the dilution factor (100), l is the path length of the cuvette (m), 4 is the wax volume fraction, and C is the biopolymer concentration in aqueous phase (g/m3). ESI was expressed as the percentage of the initial absorbance of diluted emulsions and was calculated using the following formula:

ESIð%Þ ¼ ðAt =A0 Þ$100 where: At is the absorbance at time of 0.17, 0.5, 1, 3, 6, 12, 24, 48 h; A0 is the absorbance at time 0 h. 2.4. Scanning electron microscopy (SEM) The morphology of FFSs and film surfaces was tested using a scanning electron microscope (Zeiss Ultra Plus, Oberkochen, Germany). Cryo-SEM was used to examine the fracture surfaces of freshly prepared FFSs. Before viewing the surfaces of freeze drying film, samples and fractured FFS specimens were dusted with gold. Imaging of samples was performed in high vacuum (5  104 Pa), using a secondary electron detector at 3 kV (film surfaces) or 7 kV (fractured FFS). 2.5. Film thickness and conditioning Film thickness was measured to the nearest 2.54 mm with a hand-held micrometer (Mitotuyo No. 7327, Tokyo, Japan). Before testing, film cut into specimens were conditioned for 48 h in a versatile environmental test chamber (MLR-350, Sanyo Electric Biomedical Co. Ltd., Japan) at 50% relative humidity (RH) and 25  C.

EM ¼ ðs2  s1 Þ=ðε2  ε1 Þ where ε1 is a strain of 0.005 (0.5%), ε2 is a strain of 0.02 (2%), s1 (MPa) is the stress at ε1, and s2 (MPa) is the stress at ε2. At least eight replicates of each film type were tested for tensile properties. Puncture strength (PS) was determined by the method of Ghorpade, Li, Gennadios, and Hanna (1995), with some modifications. Films were cut into discs (50 mm diameter) and placed between a film holder consisted of two poly(methyl methacrylate) plates with a hole of 30 mm in diameter at the center of each plate and a rubber O-ring gasket to prevent slippage of the films. The plates were held tightly and a steel ball-ended (2 mm diameter) probe was moved perpendicularly at the film surface at a constant speed 1 mm s1 until it passed through the film. The PS in MPa was calculated by the following formula:

PS ¼ F=A were F is the maximum force (N) and A is the cross-sectional area of the probe (thickness  diameter of the opening of film holder, mm2). PS was measured in quadruplicate. 2.7. Moisture content (MC) and total soluble matter (TSM) Conditioned film specimens (2  2 cm) were weighed (0.0001 g) and dried in an oven at 105  C for 24 h. The MC value was determined percentage of initial film weight lost during drying and reported on wet basis. The TSM was expressed as the percentage of film dry matter solubilized after 24 h immersion in water. Dried film pieces were placed in 50 ml Falcon test tubes containing 30 ml of distilled water with sodium azide (0.02 w/v) to inhibit microbial growth. The covered tubes were shaken (150 rpm/min) at 25  1  C for 24 h. Undissolved film materials were removed from water, gently rinsed with distilled water and subsequently oven dried (105  C for 24 h) to determine solubilized dry matter. Initial dry matter values were obtained from MC measurements for the same film. Analyses were performed in quadruplicate. 2.8. Water vapor permeability The WVP (g mm m2 d1 kPa1) was calculated as:

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WVP ¼ ðWVTR$LÞ=Dp

3. Results and discussion

where WVTR (g m2 d1) is the water vapor transmission rate of films measured at 25  C and 50% RH gradient, L (mm) is the thickness of film specimens, and Dp (kPa) was the difference in partial water vapor pressure between the two sides of film specimens. WVTR was determined gravimetrically using a modification of the (PN-ISO 2528, 2000), also known as the cup method. The permeation cell (poly(methyl methacrylate) cups) had an internal diameter of 7.98 cm (exposed film area ¼ 50 cm2) and an internal depth of 2 cm. Film specimens (10 cm diameter disks) were mounted onto the open circular mouths of cups filled with distilled water (100% RH). The lid was fixed by six screws, a rubber O-ring gaskets helped to assure a good seal. The cups were placed in an environmental chamber set at 25  C and 50% RH. Weights of the cups were recorded every 2 h for a period of 10e12 h. The slopes of the steady state (linear) portion of weight grow versus time curves were used to calculate WVTR. Measurements were performed in triplicate.

3.1. Emulsifying properties

Film samples (12  12 cm) were stored in triplicate in a versatile environmental test chamber (MLR-350, Sanyo Electric Biomedical Co. Ltd., Japan) at 50% RH and 25  C for three months. The degree of crystallization was expressed as the percentage area of sorbitol crystals (white spots) appearing on the film surface. The crystallization area was estimated via analysis of pixels exposed in digital images of the films. Firstly, the color depth of photos was decreased to 2 colors (black/white), then the percentage of white pixels (tone 255) was counted using a histogram function in graphics software. Analyses were performed in duplicate.

l 8 7 5

g

e

3

e

e

a

0

2.0

c

d

CMC

All data were expressed as mean  standard deviation (n  3). Differences among data mean values were tested for statistical significance at the p < 0.05 level using analysis of variance (STATISTICA 6.0, StatSoft Inc., Tulsa, USA) and the Fisher’s test.

i

f

4 2

k

j

h

6

1

2.12. Statistical analysis

m

OPS

ab SPI

1.5

b GEL

1.0 0.5

)

2.11. Plasticizer crystallization

(%

Color values of the films were measured with a spectrophotometer (X-RiteColor 8200, X-Rite Inc., USA). Film specimens were placed on the white background (L* ¼ 95.84, a* ¼ 2.27, b* ¼ 13.28) and L*, a*, and b* color values were measured. The three color coordinates ranges were: L (0 black to 100 white), a (greenness to þ redness), and b (blueness to þ yellowness). Analyses were performed in quadruplicate.

wa x

2.10. Color

a

where A600 is the absorbance at 600 nm, T600 is the transmittance at 600 nm, and x is the film thickness (mm). Analyses were performed in quadruplicate.

lill

or  log T600 =x

de

Opacity degree ¼ A600 =x

2

The ultraviolet and visible light barrier properties of the films were measured at selected wavelengths between 200 and 800 nm, using a UV/Vis spectrophotometer (Lambda 40, PerkineElmer, Shelton, CT, USA) according to the procedure reported by Fang, Tung, Britt, Yada, and Dalgleish (2002). The opacity of the films was calculated by the following equation (Han & Floros, 1997):

EAI (m /g)

2.9. Light transmission and film opacity

Because of the high free energy of the interface between oil and water phase, most emulsions are not stable without addition another agent. The mechanism to generate the emulsion system is attributed to the adsorption of surface active agent at the interface between two phases during homogenization and the formation of a protective layer that prevent oil droplets from aggregation. Such adsorption reduces the interfacial tension between the oil and water phases thus making it easier for the droplets to be broken up into smaller entities. EAI estimates the ability of any emulsifier to aid in the formation of a newly-created emulsion by giving units of surface area of the interface that is created per unit mass of emulsifier. Fig.1 shows EAI of FFSs at various wax concentrations. For all the studied biopolymers, the EAI values of emulsions clearly increased (p < 0.05) with increased CnW concentrations. This indicated that for every gram of biopolymer the emulsions could form larger lipid surface areas in high rather than in low wax volume fractions. A similar trend has been reported for oil increasing concentrations in emulsions stabilized by bovine serum albumin and potato protein (Al-Malah, Azzam, & Omari, 2000; Guo & Mu, 2011). At 0.5% wax concentration, the highest EAI had CMC emulsion; however, at the higher wax incorporation levels (1.5 and 2.0%) the highest EAI values were found for OPS and GEL emulsions, while EAI of CMC emulsions was the lowest (p < 0.05), suggesting a poor concentration of CMC molecules on the surface of freshly formed wax droplets. Emulsion stability refers to the ability of an emulsion to resist change in its properties over time. A stable emulsion is one in which the globules retain their initial character like average size and size distribution and remain uniformly distributed throughout the continuous phase. Common mechanisms of emulsion destabilization include gravitational separation (creaming or sedimentation), flocculation, coalescence, and disproportionation. In wax-in-water emulsions, destabilization would be mainly caused by creaming. Since the dispersed wax phase is in solid state at room temperature, there would be no coalescence of the suspended globules. Coalescence, however, could occur at temperatures above wax melting point, i.e. in newly-created emulsion. Fig. 2 shows kinetic stability of the studied FFSs. For CMC, SPI and GEL emulsions the ESI decreased together with the time of storage, which implies creaming phenomenon. The highest creaming rate was observed for CMC-based FFSs. Just after 10 min of storage, their ESI was 41.6e77.6% (depending on wax

Ca n

198

Fig. 1. Profiles of emulsifying activity index (EAI) of emulsions contained the different biopolymers as a function of wax concentration.

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199

Fig. 2. Profiles of emulsifying stability index (ESI) of emulsions contained the different biopolymers as a function of wax concentration.

concentration). Generally, as the CnW concentration increased, the ESI decreased. The high instability of CMC emulsions could be explained by a low viscosity-increasing effect. The stabilizers’ mechanism for improving the stability of emulsions is increasing the viscosity of the medium. Cellulosic gums are available in a wide range of viscosity grades and the CMC 30 GA used in this study represent one of the lowest obtainable viscosities. GEL produced fairly stable emulsions; the ESI0.5h reached 82.1e89.1%. Solidification of GEL-based FFSs made it impossible to carry out the ESI tests over 0.5 h, but it could be assumed that the change in the physical state prevented further emulsion destabilization process. The emulsifying properties of GEL result from the fact that it forms a high viscosity solution/gel that decelerates lipid particle movements (Surh, Decker, & McClements, 2006). It is also known that altering the sequence of relatively polar and non-polar tripeptides promotes the adsorption of gelatin molecules at hydrophobic/hydrophilic interfaces (Thomas, Kellaway, & Jones, 1985). Proteins, due to their amphiphilic character, show surface activity; thus, they are widely used as emulsifying agents in foods to produce oil-inwater emulsions with improved stability. As can be seen, the SPI emulsions also had high stability (ESI48 h ¼ 75.4e86.2%); however, only at higher CnW concentrations (1.0e2.0%). For both proteinaceous film forming agents, emulsions at the lowest lipid addition level were less stable than the emulsions at higher one. Similar tendency has been shown by other authors (Sun & Gunasekaran, 2009). It seems that at higher oil concentration, the packing fraction of oil droplets will increase which in turn will enhance the emulsion viscosity and delay the creaming rate. Clearly, further

study is required to identify the influence of CnW concentration on rheological properties of the biopolymer-stabilized emulsions. The wax concentration played an important role in the stability of OPS emulsion. At concentrations of 0.5 and 1.0% sedimentation was observed (downward movement of wax droplets/particles resulted in increased ESI values), whereas at 1.5 and 2.0% of CnW globules tended to creaming (ESI48 h ¼ 90.5 and w81.4% respectively) (Fig. 2). The reason for the different droplet loss mechanisms of OPS emulsions is not clear, but it might be related to the extent of OPSCnW interactions. Starch constituents, especially amylose, accumulate at the waxewater interface (Fanta, Felker, Shogren, & Knutson, 2001). Formation of complexes with lipids modifies the properties of starch, for example reducing starch solubility in water (Tang & Copeland, 2007). It is possible that settling process in OPS emulsions was attributed to formation of starch-wax aggregates that displayed a reduced capacity for hydration and, therefore, lower water solubility, and simply collapsed under their own weight. Heavy interfacial starch coatings increase wax droplets density and thus inhibit their rapid rise to the surface or (if they are enough heavy) cause them to precipitate (Fanta et al., 2001). Formation of complexes between starch and lipid depends on ratio of lipid to starch. Increasing concentration of water-insoluble lipids above a critical concentration led to a decrease in complex formation, due to the lipids self-associating in preference to binding with amylose (Tang & Copeland, 2007). Thus, it could be speculated that at high concentration of CnW self-association of the lipid into micellar structures contributed to opposite instability process (i.e. creaming).

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Fig. 3. SEM micrographs of film-forming solutions without (a) and with addition of 2.0% candelilla wax (b).

3.2. Microstructure The microscope technique is popular for estimation of droplet size distribution of emulsions. Its drawback, however, is that only small part of the sample is analyzed, therefore, information deduced from cryo-SEM images is not exactly reliable. Nevertheless, when carried out correctly, the microscopic methods provide extremely valuable information about the arrangement and interactions of lipid droplets witch each other and the other structural entities found in food emulsions. Fig. 3 presents SEM images of the fracture surfaces of FFSs both without and containing 2.0% CnW. The porous structures created by freeze-dried FFSs enabled observation of solidified wax globules lodged in the polymer

matrix. Different magnifications were needed to visualize the lipid phase in different emulsion systems. The largest wax particles were observed in CMC emulsion (w5e20 mm). The maximum diameter of wax globules in other FFSs was much smaller and ranged from <1 mm (protein-based emulsions) to w1e2 mm (OPS-based emulsion). In emulsions, larger particles cream out at a faster rate than the smaller ones. It can therefore be assumed that the large wax globule size in CMC-based FFS (at 2.0% of CnW) was responsible for their high instability (Fig. 2). SEM images revealed differences in the surface morphology of the studied films (Fig. 4). Generally, the surface structure of control wax-free films was fairly smooth and homogenous. Nevertheless, apparent morphological differences could be seen. The surface of

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201

Fig. 4. SEM micrographs (2000 magnification) of surfaces of the wax-free control films (a) and films with 2.0% candelilla wax (b).

the CMC film was covered with numerous dimples, OPS film had a surface with slight inequalities, the surface of SPI film was covered with dimples and pinholes, whereas the GEL film were smooth and uniform. As expected, the incorporation of CnW affected the film surface characteristics. Due to the presence of wax droplets, the surface of emulsion films was rough and uneven. Similar observations have been reported for casein films with incorporated CnW and CrW (Chick & Hernandez, 2002), and emulsion films with incorporated oil bodies (Matsakidou et al., 2013). The largest globules were observed for the film based on SPI (max. diameter w20 mm) and CMC (max. diameter w8 mm), the smallest CnW particles were found on the surface of OPS and GEL films

(diameter  1 mm). It could be speculated that the differences in the sizes of wax globules on film surfaces were due to differences in FFS consistency. Those emulsions containing SPI were characterized by the lowest viscosity among all the obtained emulsions (data not shown). In this way, the possibility of wax coalescence in SPI emulsions was the highest. In freshly prepared hot emulsion, the melted wax droplets easily bump into each other and combine to form a larger droplet, so the average droplet size increased over time. In turn, for emulsion containing thickening agents such as CMC, OPS and GEL, the higher viscosity of continuous phase probably limited coalescence; thus, the average size of the wax globules was smaller.

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3.3. Mechanical properties Table 1 shows the mechanical parameters of the wax-free and emulsion films with different wax levels. As can be seen, GEL films, regardless of CnW concentration, had the highest values of TS, PS and E among tested films. This fact is most likely attributed to the continuous fibrillar three-dimensional network of GEL films. GEL is obtained by thermal or chemical dissociation of collagen, the principal fibrous protein constituent in fascias, cartilages, ligaments, tendons, bones and skin. Collagen is known for its great tensile strength which is attributed to its triple-stranded helical structure. Despite the degraded state, GEL still has a capacity to form a large variety of supermolecular structures, from the simplest globular structures, typical of amorphous polymers, to a wellorganized fibrillar structures (Kozlov, 1983). Renaturation of the GEL is observed in solutions at low temperatures. Studies on GEL films have revealed that, in films cast at room temperatures and lower, the GEL macromolecules exhibit a developed tertiary structure, while in films prepared from aqueous solutions by evaporating the solvent off at temperatures above 35  C GEL chains assume the conformation of a statistical coil with no indications of ordering (Bradbury & Martin, 1952; Robinson, 1953). The great mechanical resistance of GEL-based films has also been demonstrated in other studies, i.e. based on a comparison with methyl hydroxyethyl cellulose, starch, acacia gum, and SPI films (Cao, Fu, & He, 2007; Denavi et al., 2009; Healey, Rubinstein, & Walters, 1974). The results of this study showed that the TS of wax-free GEL films was about 1.5-, 3-, and 6-times higher than counterpart films based on CMC, OPS and SPI, respectively (Table 1). It should be noted that relatively high mechanical strength values have also been determined for CMC films. Similar to the collagen in animals, cellulose is the structural material of plant cell walls. The ability of cellulose chains to form intrachain and interchain H-bonds gives them their unique properties of mechanical resistance and chemical stability. CMC is a cellulose derivative with carboxymethyl groups bound to some of the hydroxyl groups of the glucose monomers. As shown in Table 1, the Sor plasticized CMC gels formed relatively tough and stiff films, likely due to the numerous H-bonds holding the CMC chains firmly together. Despite the structural similarity between

Table 1 Effect of candelilla wax concentration on the mechanical properties of biopolimeric films: tensile strength (TS), elongation at break (E), elastic modulus (EM), and puncture strength (PS). Films

CnW (%)

TS (MPa)

CMC

0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0

19.0 14.7 11.9 10.5 9.4 9.8 4.2 3.8 3.7 3.6 5.4 4.5 4.3 3.9 3.7 31.2 23.1 20.0 21.3 18.2

OPS

SPI

GEL

                   

1.5fg 1.0e 1.7cd 1.1cd 1.2c 0.5c 0.2ab 0.3a 0.3a 0.4a 0.2b 0.2ab 0.1ab 0.2a 0.3a 2.5j 1.7i 1.7gh 2.1h 1.9f

E (%) 23.0 16.2 12.5 12.2 10.6 76.0 71.2 41.7 42.2 41.3 55.5 39.0 30.9 30.7 35.0 83.5 82.7 73.9 73.6 109.2

EM (MPa)                    

3.7bc 1.9ab 2.3a 2.6a 2.0a 5.0ghi 8.7g 10.7e 5.2e 7.9e 5.8f 12.2de 5.6cd 3.9cd 4.2de 11.5i 9.0hi 11.9gh 10.2gh 9.0j

172.5 106.5 88.0 75.8 75.9 81.8 22.5 26.5 15.6 21.5 51.2 32.3 33.2 35.6 43.5 181.0 119.8 109.3 92.2 67.8

                   

11.7l 5.1j 7.7hi 3.6fg 8.5g 4.5gh 6.7ab 7.0bc 3.6a 5.3ab 4.9e 6.4cd 4.4cd 7.0d 5.6e 10.3m 5.3k 8.3j 4.2i 7.8f

PS (MPa) 8.2  0.3h 3.9  0.1f 3.6  0.1f 2.6  0.1e 2.3  0.3de 3.8  0.1f 1.8  0.0bc 1.8  0.0bc 1.6  0.0ab 1.4  0.1a 2.2  0.1d 1.9  0.1bc 1.8  0.2bc 1.9  0.1ab 1.6  0.1abc 14.3  0.4l 12.6  0.2k 11.5  0.2j 10.2  0.8i 7.8  0.5g

Values are given as mean  standard derivation. Values with the same superscript letters within a column are not significantly different (p < 0.05).

cellulose and starch, the films based on starch exhibited significantly lower tensile strength and higher elongation at the break than those of cellulose gum. This is consistent with the results of other authors (Goswami, Anandjiwala, & Hall, 2004; Sun et al., 2010; Tongdeesoontorn, Mauer, Wongruong, Sriburi, & Rachtanapun, 2011). As has been previously suggested, the predominantly intramolecular bonded helical structures in starch, with water mainly located inside the helix, may be more likely to rupture than linear cellulose (Sun et al., 2010). The more linear three-dimensional structure of cellulose (and its derivatives) offers the possibility of a more extensive and indeed cooperative behavior of the hydrogen bonds along the chains than in starch, which is a mixture of linear amylose (10e20%) and branched amylopectin (80e90%). Branching makes the polymers less dense and results in low tensile strength. For example, it has been found that that amylose molecules formed stronger films than those of amylopectin (Myllärinen, Partanen, Seppala, & Forssell, 2002). From the results above, it seems that highly ordered polymer structures exhibit higher tensile yield strength than heterogeneous polymers. SPI is a complex mixture of proteins, mainly globulins. Thus, a less organized network was the most likely reason for the weakest mechanical strength of SPI films (Table 1). Whatever the type of biopolymer used, the presence of wax significantly decreased the mechanical strength of the films. The highest reduction in TS and EM was observed after the incorporation of CnW into OPS-based films (the addition of 0.5% CnW resulted in a reduction of TS by 57.1% and EM by 72.5% compared to films without wax). The TS of emulsion films based on OPS and SPI was not affected by CnW concentration (p > 0.05), while for films formed from CMC and GEL the TS generally decreased as the CnW content increased (p < 0.05). The PS of the films exhibited a similar trend as seen for TS, with the exception that the highest decreases in puncture resistance (w52.5% at addition of 0.5% CnW) were observed both for OPS and CMC films. Decreases in film toughness (lower values of TS and EM) after wax introduction have also been reported by other authors (Perez-Gago & Krochta, 2000; Shellhammer & Krochta, 1997; Sohail, Wang, Biswas, & Oh, 2006; Soazo et al., 2013; Tanaka, Ishizaki, Suzuki, & Takai, 2001). Generally, lipids including waxes exhibit a plasticizing effect on biopolymer-based films. This is related to the development of heterogeneous structure, where lipid particles lead to discontinuities in the polymer network. The results of this study indicated that the plasticizing/weakening effect of lipids may strongly vary with the matrix of the films (Table 1). Based on puncture testing, which is a precise and reliable method to measure film mechanical strength  (Kowalczyk, Gustaw, Swieca, & Baraniak, 2013), the addition of CnW into films caused a greater reduction in the mechanical resistance of polysaccharide films than in protein films. For example, the PS of polysaccharide films casted from FFSs contained 2.0%, which was about 3.5- (CMC) and 2.6-times (OPS) lower compared to control films, while the PS of SPI and GEL films was respectively 1.4- and 1.8-times lower than films without wax. A similar dependence was observed for TS values, however only at higher CnW concentrations (1.5 and 2.0%). The lesser impact of CnW on the mechanical resistance of protein films may be attributed to the protein-wax interactions resulting in more coherent film matrix. The addition of CnW caused a loss in E; however, in the case of CMC, OPS and GEL films, significant differences were observed at wax levels exceeding 0.5% (p < 0.05). In general, the CnW concentration had no effect on E; the exceptions were the OPS film containing 0.5% CnW (which showed the highest extensibility among OPS-based emulsion films) and GEL film containing 2% CnW. For the latter improved extensibility compared to other GEL films (p < 0.05) was observed, which was most likely attributed to

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the plasticizing effect of wax. Some researchers have also reported that the presence of lipids in a film matrix can enhance the E (Bertan, Tanada-Palmu, Siani, & Grosso, 2005; Colla, Do Amaral Sobral, & Menegalli, 2006). However, many studies have reported the opposite effect (Chick & Hernandez, 2002; Han, Seo, Park, Kim, & Lee, 2006; Kim, Hwang, Weller, & Hanna, 2002; Tanaka et al., 2001; Zahedi, Ghanbarzadeh, & Sedaghat, 2010). 3.4. Moisture content (MC) Table 2 shows MC of the biopolymer films and changes in this parameter as a function of CnW incorporation. Among control films, the CMC film exhibited the highest MC (10.8%) while OPS film the lowest (7.4%) (p < 0.05). The MC of protein-based films did not differ statistically and was 8.9 and 9.5% for SPI and GEL films, respectively. The observed order of the MC of the films is in agreement with the water absorbability of film components determined by Tongdeesoontorn, Mauer, Wongruong, and Rachtanapun (2009). The higher MC of CMC film compared to that of starch has also been presented by Goswami et al. (2004). The observed differences in the film humidity values reflect the primarily different moisture-binding abilities of hydrocolloids. The high MC of CMC films was easy to predict because CMC is known for its excellent water binding and moisture sorption properties, which are attributed to the numerous hydrophilic groups (hydroxyl and carboxylic) in its structure. The MC of the films was decreased by introduction of CnW; however, 1.0 or 1.5% levels of incorporation were needed to lead to a statistically significant reduction (Table 2). A decrease in the MC of edible films after a certain level of lipid incorporation has also been observed in other studies (Shih, Daigle, & Champagne, 2011; Soazo, Rubiolo, & Verdini, 2011; Zahedi et al., 2010). The addition of lipids reduces the equilibrium water content due to the fact that lipids correspond to fractions of solids with small water uptake capacity. The decrease in MC was polymer-dependent. At 1.5% CnW addition, the MC of CMC, OPS, SPI, and GEL films was reduced by 29.9, 13.5, 16.6, and 14.7%, respectively. The increasing CnW level in FFS from 1.5 to 2.0% did not result in any further reduction in the MC of the films (p > 0.05). Regardless of the wax incorporation level, CMCTable 2 Effect of candelilla wax concentration on the properties of biopolimeric films: moisture content (MC), total soluble matter (TSM), water vapor permeability (WVP), and opacity (Op). Films

CnW (%)

MC (%)

CMC

0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0

10.8 10.8 10.3 9.0 9.4 7.4 6.8 6.5 6.3 6.1 8.9 8.1 8.0 6.3 6.3 9.5 9.8 9.8 8.2 7.6

OPS

SPI

GEL

                   

WVP (g mm m2 d1 kPa1)

TSM (%) 0.2j 0.3j 0.8ij 0.3g 0.5gh 0.3cd 0.1bc 0.3bc 0.9ab 0.4a 0.4g 0.9fg 0.4def 0.3ab 0.1ab 0.3gh 0.3hi 0.5hi 0.3f 0.4def

100.0 100.0 100.0 100.0 100.0 45.5 41.4 42.9 38.7 37.1 46.1 43.0 40.0 38.7 39.5 54.5 48.3 44.3 43.4 39.6

                   

0.0j 0.0j 0.0j 0.0j 0.0j 0.6fg 0.4cd 0.6de 1.1ab 0.5a 1.6g 1.0de 2.0bc 1.7ab 1.0b 1.1i 2.2h 1.1ef 2.1e 1.7b

39.3 26.2 32.1 35.4 35.6 37.8 22.0 26.9 29.5 25.7 41.6 37.0 36.1 34.8 34.8 36.0 37.1 36.9 37.1 41.2

                   

0.6h 0.2b 2.0d 1.5ef 1.6ef 0.3gh 0.2a 0.1b 1.4c 0.8b 1.1i 0.8fg 1.8efg 1.0ef 1.8ef 0.2efg 1.8fg 1.1fg 0.5fg 0.7i

Op (A600/mm) 0.8 6.4 9.0 11.5 12.4 1.7 2.0 2.5 3.6 5.8 1.0 3.9 1.0 2.8 6.9 0.9 1.3 1.8 3.5 4.1

                   

0.1a 0.2hi 0.6j 0.6k 1.0k 0.1bcd 0.1cd 0.3de 0.3fg 0.4h 0.1abc 0.4fg 0.1abc 0.1def 0.6i 0.1ab 0.2abc 0.3bcd 0.1efg 0.2g

Values are given as mean  standard derivation. Values with the same superscript letters within a column are not significantly different (p < 0.05).

203

based films had the highest MC while OPS and SPI films exhibited the lowest MC. 3.5. Total soluble matter (TSM) The usefulness of biopolymer films in many technological applications is critically dependent on the solubility parameter. Use of edible films as protective layers on high water activity food products requires these materials to be water resistant. On the other hand, water soluble biopolymer films (in the form of microcapsules, bags, sachets, pouches) could provide a convenient, safe and economical delivery system for a wide range of products including laundry detergents and cleaners, concentrate additives, pigments, biocides, and others. As can be seen in Table 2, the CMC-based films were 100% soluble in water, whereas the other polymer films were only partially soluble (the TSM of OPS, SPI and GEL control films were 45.5, 46.1, and 54.5%, respectively). The differences in film dissolution behavior could be due to differences in the water resistance of the polymers, and in consequence differences in diffusion, permeation, and segmental integrity of the resulted films. Because of the presence of numerous polar groups, CMC is strongly hydratable, which explains the rapid dissolution of CMC film pieces after coming into contact with water. The partial solubility of films based on OPS and GEL may be due to fact that these polymers only swell in water at 25  C and heat is required for their complete dissolution. In this case, the dry matter solubilized in water was probably composed of small molecules, such as plasticizers and small fragments of polymer chains, e.g. small peptides (GEL films) or pyrodextrins (OPS films). The limited TSM of SPI films was most likely due to heat-induced cross-linking of proteins (the film samples prior to immersion in water were dried in an aircirculating oven). Improved integrity of dehydrothermo-treated protein films throughout the film-soaking procedure has been reported (Kim, Weller, Hanna, & Gennadios, 2002). With the exception of CMC films, incorporation of CnW reduced (p < 0.05) the TSM of the films. At 0.5% of CnW incorporation level, the reduction in TSM ranged from 6.8 to 11.4% as compared to films without wax. A further increase in wax concentration up to 2.0% decreased film solubility by 14.4e27.2%, depending on the polymer type. Regardless of the wax concentration, CnW had the best efficiency in the reduction of the TSM of GEL films (Table 2). The reduction/slowdown in film solubility due to the incorporation of waxes (BW or CnW) into edible films were reported by Kim and Ustunol (2001b), and Soazo et al. (2013). These authors considered that, because the total solid levels remained constant in the formulation, the incorporation of wax reduced the soluble matter present in the films, and consequently the solubility. However, as was shown in this study (Table 2) as well as by Kim and Ustunol (2001b), lipid addition is an ineffective way to decrease the solubility of films with initial TSM of 100%. 3.6. Water vapor permeability (WVP) Comparison of control films showed that, despite the use of different polymers, the permeability of the films was relatively similar (Table 2). However, the SPI-based film exhibited significantly higher WVP (41.6 g mm m2 d1 kPa1) compared to the other films (36.0e39.3 g mm m2 d1 kPa1) (p < 0.05). The low barrier properties of SPI films might be explained in terms of their microstructure. As is already known, the gas and water vapor barrier properties of edible films and coatings vary greatly not only in composition, but also in terms of the presence of bubbles, pinholes or cracks of the films (Pascat, 1986). SEM images revealed that SPI films, unlike others, contained numerous pinholes (Fig. 4) that likely provide routes for water transfer.

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In the literature, different comparative overviews of the water vapor barrier properties of biopolymer films have been made (Krochta, 2002; McHugh & Krochta, 1994a). In most comparisons, data values come from different research, thus materials differ in their composition (e.g. different film forming agent concentration, different plasticizer types and their concentrations), formation method, modification treatments, and methodologies used for determining WVP. As a consequence, WVP values of different edible films vary significantly, e.g. from lower than 0.1 to higher than 100 g mm m2 d1 kPa1 (McHugh & Krochta, 1994a). This study indicated that if the ingredient proportions (1:0.6 biopolymer/ plasticizer weight ratio) and experimental test conditions employed for the testing of permeability (25 C, 50/100% RH) are the same, the WVPs of different biopolymer films are comparable. A similar result was observed in the study by Chen and Nussinovitch (2001) where the WVP values of different hydrocolloid coatings (xanthan, locus bean and guar gum) did not differ statistically. It was found that, with the exception of GEL films, CnW incorporation significantly decreased the WVP of films. The different impact of CnW on the possibility of reducing WVP may be explained in terms of the different structural arrangement of the lipid phase in the film matrix. GEL with excellent wax phase trapping capacity (Fig. 2) formed emulsion films with unchanged water barrier properties with respect to the control film (p > 0.05). In turn, polymers with more time-dependent emulsion stabilizing properties produced films with improved water vapor barrier properties. It has previously been demonstrated that lipid creaming toward the evaporation surface contributed to a decrease in permeability because the structure tended to resemble a bilayer film (Morillon et al., 2002). Waxes, because of their low specific gravity relative to water, show a preferential location on the film side exposed to air during drying (Chick & Hernandez, 2002; Soazo et al., 2013). However, as previously reported, in presence of starch the heavy specific gravity of the starch-coated wax droplets can also cause them to precipitate (Fanta et al. 2001, Fig. 2). It could be speculated that in the emulsion films prepared from CMC, OPS and SPI, wax molecules formed bilayer-like structures, which acted as effective barriers to the transfer of water vapor. In turn, in fast solidified emulsion films based on GEL, the homogeneous distribution of CnW did not offer a successful barrier to the mass transfer. In this case, moisture most likely still easily penetrated between lipid globules trapped inside the continuous hydrocolloid network. Depending on the polymer type and CnW addition level, the reduction in WVP ranged from 9.3 to 41.6% compared to wax-free samples (Table 2). Films based on OPS, regardless of the wax concentration used, had statistically better barrier efficiency compared to other films. This result may be partially explained in terms of the low moisture absorption capacity of OPS films (Table 2). The water vapor transport includes basic phenomena such as adsorptione desorption, diffusion, condensation. If the physical adsorption on the surface is low, the water vapor flow will be low too. The addition of 0.5% CnW was the most effective in improving water barrier properties. At this CnW concentration, the WVP of OPS, CMC and SPI films was decreased by 41.6, 34.0, and 11.4%, respectively. The high reduction in WVP observed for films at the lowest wax concentration used could by connected with the amount of emulsion taken for the film preparation. In this study, a constant amount of total solids was cast onto the film-forming area. In this way, the mass of FFS that were cast gradually decreased as CnW content increased. Consequently, the FFS containing 0.5% wax required a longer time for solvent evaporation and, as was shown by Debeaufort and Voilley (1995), longer drying times of film-forming wax emulsions produce films with better water vapor barrier properties. The study showed that an increase in CnW concentration above 0.5% resulted in increased WVP of OPS and CMC films

(p < 0.05), but did not affect the WVP of SPI films (p > 0.05) (Table 2). A decrease in water vapor barrier properties for amounts of lipid above the critical value was also observed by Sapru and Labuza (1994) in methylcellulose-stearic acid films. It has been suggested that the incorporation of solid-state apolar components up to a certain quantity might cause more disruption in the film matrix, creating an increased number of void spaces at the polymerewax interface, which facilitate the transfer of water molecules inside the film (Chick & Hernandez, 2002; Martin-Polo, Mauguin, & Voilley, 1992). In the literature can also be found data that suggest that incorporation of lipid material above certain levels may have no effect on the reduction of WVP. For example, Bravin, Peressini, and Sensidoni (2004) observed that the proportion of oil in excess of 10% (relative to polymer) did not contribute to any further improvement in edible film barrier properties, because, as suggested, the effect of polar groups of polymers and plasticizers can only be partially reduced by the presence of apolar compounds. 3.7. Light transmission and transparency In food packaging, transparency of material is an advantage because it allows consumers to see the product before buying, and products with an attractive appearance could be better presented by sellers. On the other hand, packaging materials should protect products from the negative effects of UV light. This study demonstrated that all the tested control films were transparent; however, only SPI films exhibited excellent barrier properties to UV light. The transmittance (T%) of these films noted at the middle ultraviolet spectrum (200e280 nm) did not exceed 0.1% (Fig. 5). For comparison, the T% values of CMC, OPS and GEL films in the same light region reached 63.6, 46.9 and 22.1%, respectively. The significant barrier properties of proteins against UV radiation are associated with the presence of UV-absorbing chromophores, especially aromatic amino acids e tyrosine and tryptophan and to a lesser extent, phenylalanine and disulfide bonds (Aitken & Learmonth, 2000). GEL films were not as good UV barriers as those films based on SPI, apparently due to their unusual amino acid composition (the predominant content of glycine, proline and hydroxyproline). In the visible range, GEL and CMC films exhibited the highest light transmission (T% z 80.0e88.0%), while the T% of SPI and OPS films were lower (64.3e84.7 and 58.8e64.2%, respectively). Introduction of CnW in all the studied films resulted in decreased light transmission. Generally, as the wax concentration increased, the T% successively decreased (Fig. 5). This could be easily explained in terms of increasing light reflection by increasing numbers of wax particles; thereby, at higher CnW incorporation levels, the films were more opaque and less permeable for UV light. However, one exception was observed. At the 0.5% CnW addition level, the light transmission of SPI film was lower than that obtained at a concentration of 1.0 or 1.5%. In this one case, wax bloom was visible to the naked eye. The explanation for this phenomenon might lie in the emulsifying properties of SPI. As was shown in Fig. 2, the stability of SPI emulsions containing 0.5% of CnW was lower than that of those with higher wax concentrations. As a consequence, the CnW was not homogenously distributed in the film matrix; thus, wax haze limited light transmission. Table 2 presents the opacity degree of tested films. All films without CnW were perceived to be clear and translucent; however, OPS-based films exhibited significantly higher (p < 0.05) opacity (1.9 A600/mm) than other films (0.8e1.0 A600/mm). The presence of CnW, regardless of its concentration, increased the opacity of CMC films, and their transparency reduced significantly (p < 0.05) as CnW levels increased. In the case of other tested films, CnW had less effect on film opacity, and only for concentrations of 1.5 and

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205

Fig. 5. Light transmission of biopolimeric films with various candelilla wax contents.

2.0%, as well as at 0.5% in the case of SPI films (as commented on above), was a significant decrease in film transparency observed (p < 0.05). An increase in film opacity after solid fat incorporation, including wax, has been reported by different authors (Fabra et al., 2009; Fabra et al., 2011; Wang et al., 2014; Zahedi et al., 2010). It seems that, due to their physical state, particles of solid lipids cause morphological inhomogeneity of emulsion films, and thus visible light scatter through the film, causing its opaqueness (Kim & Ustunol, 2001a). Differences in the transparency of different biopolymer films are related to their different internal structure (Villalobos, Chanona, Hernandez, Gutierrez, & Chiralt, 2005). According to Fabra, Talens, and Chiralt (2009), in lipid-containing films this structure is greatly affected by the initial structure of the emulsions (volume fraction of the dispersed lipids and size of lipid aggregates) and their development throughout the drying process due to destabilization processes. In the present study, it was found that CMC-based emulsion films significantly differ from the other tested films in respect of transparency properties. The reason could be the large size of wax globules in emulsions contained CMC (Fig. 3). It is known that bigger particles give rise to greater light dispersion, and thus decrease film transparency (Fabra et al. 2011; Jiménez et al., 2010). However, the final size of CnW particles on the CMC film surface was not the largest (Fig. 4). This suggests that the effect of the size of lipid aggregates on optical properties for the determined volume fraction of the dispersed phase is difficult to predict due to the complexity of the interactions between particle size, lipid layer size produced during film drying and light scattering (Fabra, Talens, et al., 2009).

3.8. Color The color of edible films is highly relevant to their functionality due to their direct impact on the appearance of the coated or packaged food. In general, edible/biodegradable films should be as close to colorless as possible to simulate the appearance of common polymer materials (Rhim, Gennadios, Weller, & Hanna, 2002). The hunter color parameters (L*, a*, and b*) of the tested films are summarized in Fig. 6. The CMC-, OPS- and GEL-based films were colorless, while SPI ones were yellowish (bþ), darker (higher L*) and more green (lower a*) compared to other films. The appreciable yellowness of SPI films has also been revealed in previous studies via comparison with fish gelatin and casein films (Denavi et al., 2009; Monedero, Fabra, Talens, & Chiralt, 2010). The yellow color of soybean proteins is most likely attributed to the presence of pigments, such as anthocyanins, that were found in SPI from hexane defatted meal (Eldrige, 1972). The incorporation of CnW into FFSs, regardless of the polymer type, resulted in lower a* and higher b* color values, and as CnW concentrations increased the film becomes more greenish-yellow. This effect was likely due to the inherent yellow color of CnW. 3.9. Plasticizer crystallization The use of Sor as a plasticizer results in films with higher mechanical resistance and lower permeability compared to other plasticizers (Al-Hassan & Norziah, 2012; Kowalczyk & Baraniak, 2011; Kowalczyk et al. 2013). For this reason, Sor was chosen in

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Fig. 6. Color parameters of biopolimeric films with various candelilla wax contents. Means with different superscripts are significantly different (p < 0.05).

this study. Since it has been previously proved that Sor has tendency to recrystallize over time (Kowalczyk et al. 2013), we evaluated the possibility of its crystallization in the different biopolymer matrices. From the data in Fig. 7, it is apparent that the Sor crystallization greatly depended on the type of the film forming agent. Regardless of the time storage and CnW concentration, crystallization did not occur in CMC films. In turn, initially transparent films based on OPS, SPI and GEL, turned into unattractive films, covered in white spots. The longer the storage time, the higher the degree of crystallization was observed. The fastest progress of Sor crystallization was noticed in films based on starch. This phenomenon could be partially attributed to their low water content (Table 2). It is also possible that starch crystal formed during the retrogradation process acted as a growth promoter for Sor crystals. The most interesting finding was that despite the fact that both starch and cellulose are composed of glucose, Sor (glucose derivative) showed a completely different behavior (i.e. it did not crystalize) in CMC matrix. The causes behind this may lie in the fact that CMC acts as a crystallization controller. It reduces or retards sugar crystal growth in food products, as well as ice-crystal growth during the storage of ice-cream (Glicksman, 1963). Wax-free films had a lower degree of crystallization than emulsion films (Fig. 7). For example, OPS-based emulsion films crystalized (0.3e0.5%) within one week of their preparation, while in control films the first signs of crystallization (0.2%) were found after two weeks of storage. It is well known that any additives, foreign substances or impurities can accelerate or decelerate the

crystal growth process. In honey, for example, impurities such as pollen, propolis or beeswax are platforms on which sugar crystals can start to form (Assil, Sterling, & Sporns, 1991). From the results of this study, it can be concluded that wax particles served as nuclei for Sor crystallization. A similar effect was observed in the study by Mantzari, Raphaelides, and Exarhopoulos (2010), where the presence of solid fatty acids in starch dispersions containing sorbitol (30%) caused the formation of free Sor crystals on the sample’s surface, while in lipid-free systems Sor crystallization did not appear. The presence of wax accelerated the rate of the Sor crystal growth process; however, no clear trend was observed between its concentration and crystallization (Fig. 7). After the end of the storage experiment, the surface of emulsion OPS films was almost 100% covered by Sor crystals, while the degree of Sor crystallization for SPI- and GEL-based emulsion films, depending on the wax concentration, amounted to 58.3e64.7% and 37.7e65.4%, respectively. Additional extended tests showed further progress in Sor recrystallization. After about one year of storage (at ambient conditions), all the tested films based on OPS, SPI, and GEL became white (100% Sor crystallization), while there were still no signs of crystallization in CMC films. 4. Conclusion Gelatin and CMC, derivatives of structural polymers, produced films with higher mechanical strength and stiffness compared with plant storage polymers (starch and soy proteins). The moisture

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207

Fig. 7. Degree of sorbitol crystallization found in biopolimeric films during storage at 25  C and 50% RH.

transfer properties of different wax-free (Sor-plasticized) biopolymer films were comparable. The addition of CnW into film formulations allowed the improvement of the moisture barrier properties of films but only those based on CMC, OPS and SPI emulsions, where was possibility of migration and aggregation of wax particles over a longer period of time. Solidification of GEL avoided creaming of casted emulsions and created the network that had unaffected vapor resistance. This proves that phase separation phenomena in emulsion films are a key parameter strongly affecting WVP. The combination of CnW with proteins, most likely due to their amphiphilic character, provoked a lower decrease in mechanical properties than the addition of wax into polysaccharide films. The 0.5% CnW incorporation level resulted in the most effective reduction in WVP, and at the same time had the lowest impact on mechanical properties, as well as film transparency (with the exception of SPI films). Thus, it could be summarized that the 0.5% wax concentration used should be applied to produce films with optimal functional properties. OPS films, regardless of the CnW content, had the lowest WVP compared to other films. In turn, SPI films exhibited the best barrier properties to UV light, which suggests that these films could help to prevent the degradation of UV-sensitive food ingredients. Sor is not a suitable plasticizer for films based on OPS, SPI, and GEL because of its fast recrystallization over time. The presence of wax particles in the film matrix accelerated the Sor crystal growth process. Considering the possibility of applying biopolymer films to the food process, Sor-plasticized CMC emulsion films can be recommended for use as packaging material. These films exhibit relatively good mechanical resistance, improved water vapor barrier properties, and do not show sorbitol

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