Effect of sodium acetate and drying temperature on physicochemical and thermomechanical properties of gelatin films

Food Hydrocolloids 45 (2015) 140e149

Contents lists available at ScienceDirect

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

Effect of sodium acetate and drying temperature on physicochemical and thermomechanical properties of gelatin films Fei Liu, John Antoniou, Yue Li, Jianguo Ma, Fang Zhong* Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 July 2014 Accepted 10 October 2014 Available online 13 November 2014

The aim of this work was to evaluate the effect of sodium acetate (NaOAc) on the physicochemical properties of gelatin films with different leveling of triple helical structure. Films were obtained by casting method and dried at two temperatures (25 and 35
C). Drying at 25
C improved physical properties and increased the formation content of triple helix. The plasticizing effect of NaOAc was shown from typical stressestrain curves with the features of ductile materials. Dynamic mechanical thermal analysis (DMTA) also confirmed the thermomechanical properties of the films and revealed the decrease of glass transition temperature (Tg) with increasing NaOAc content. The increase of moisture content or decrease in intermolecular forces because of the existence of NaOAc might be attributed to the plasticizing effect. Films with NaOAc showed a higher capacity to absorb water compared to those without NaOAc at higher relative humidity (75% and 97%). All these gelatin films exhibited dominant elastic behavior (than viscous behavior) over the entire frequency range (0.1e100 Hz) at any loading content of NaOAc but the compactness of films was changed. X-ray diffraction (XRD) and water contact angle (WCA) revealed that the NaOAc crystals were dissolved in the gelatin matrix. These results may cause the extensive concern for the effects of salts (organic or inorganic) on films because salts exist widely. © 2014 Elsevier Ltd. All rights reserved.

Chemical compounds studied in this article: Sodium acetate (PubChem CID: 517045) Keywords: Gelatin films Sodium acetate Drying temperature Plasticizing effect Physicochemical properties Thermomechanical properties

1. Introduction Gelatin is one of the most popular biopolymers and it's obtained by a controlled degradation of the fibrous insoluble collagen which is widely present in nature as the major constituent of bones, connective tissue and skin (Karim & Bhat, 2009; Schrieber & Gareis, 2007). For many years, gelatin has been widely used in food, pharmaceutical, and photographic industries. Common industrial applications are coatings for food products, gel desserts, microencapsulation, hard and soft capsules, sealants for vascular prostheses, as well as wound dressing and adsorbent pad for surgical use (Bigi, Cojazzi, Panzavolta, Rubini, & Roveri, 2001; Bigi, Panzavolta, & Rubini, 2004). Gelatin from various sources (mammalian and fish) has gained extensive attention as a film-forming material because of its gelling properties and hence excellent film-forming ability (Arvanitoyannis, Psomiadou, Nakayama, Aiba, & Yamamoto, 1997;
mez-Estaca, Go
mez-Guille
n, Ferna
ndez-Martín, & Montero, Go

* Corresponding author. Tel.: þ86 13812536912. E-mail address: [email protected] (F. Zhong). http://dx.doi.org/10.1016/j.foodhyd.2014.10.009 0268-005X/© 2014 Elsevier Ltd. All rights reserved.

2011; Jeya Shakila, Jeevithan, Varatharajakumar, Jeyasekaran, & Sukumar, 2012). Collagen has a triple-helix structure stabilized mainly by the formation of inter-chain hydrogen bonds between carbonyl and amines groups. Although the composition of gelatin is closely similar to that of the collagen, the triple helix of gelatin is broken and a coil configuration composed of single random chains is obtained after the hydrolysis process (Rivero, García, & Pinotti, 2010; Staroszczyk, Pielichowska, Sztuka, Stangret, & Kołodziejska, 2012). However, the polymer chains can undergo a conformational coilhelix transition by reducing the temperature of the gelatin solution. Generally, gelatin films cast below their gelation temperature, gelatin solution can form partially collagen-like triple helical structures, thus the triple helix can lock in the film matrix as water evaporates. This type of gelatin films is usually referred to as “coldcast gelatin films”. On the contrary, less triple helical structures can be obtained for films cast above gelation temperature. In this case the films might remain in a primary random coil conformation and they are referred to as “hot-cast gelatin films” (Badii, MacNaughtan, Mitchell, & Farhat, 2014; Chiou et al., 2009; Chiou et al., 2008). Usually, gelatin from bovine or pig has a higher gelation

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

temperature than that from fish, especially that from cold-water species. This is mainly due to mammalian gelatin having higher concentrations of imino acids (Chiou et al., 2009). Moreover, the mechanical properties of gelatin films were highly related to the triple helix content of gelatin (Bigi, Bracci, Cojazzi, Panzavolta, & Roveri, 1998; Bigi et al., 2004). Greater tensile strength and elongation at break were observed in cold-cast than hot-cast gelatin films (Chiou et al., 2009). This means that superior mechanical properties were related to greater amount of triple helix structure. In addition, mammalian gelatin commonly has better physical properties as well as greater thermal stability than most fish gelatin, and this has been also related mainly to their higher imino
mez-Estaca, Montero, Fern

n, acid content (Go andez-Martín, Alema
mez-Guille
n, 2009). It is generally recognized that the imino & Go acids (proline and hydroxyproline) are important in the renaturation of triple helical structure during gelling (Muyonga, Cole, & Duodu, 2004). Acetic acid and sodium acetate often act as buffers to keep a relatively constant pH level. This is useful especially in biochemical applications where reactions are pH-dependent in a mildly acidic range. Sodium acetate (NaOAc) can be produced via many methods but mainly through the reaction of acetic acid and sodium carbonate or sodium hydroxide. This process is very common in applications that involve chitosan because of its inability to dissolve in water. Acetic acid solution is used frequently because it protonates the amines converting the polysaccharide to a polyelectrolyte (Jeya Shakila et al., 2012). It is necessary in many of applications of chitosan to adjust the pH to higher values (4e6). Such applications are polymer blending between chitosan and gelatin (Pereda, Ponce, Marcovich, Ruseckaite, & Martucci, 2011) and chitosan nanoparticles production through mechanisms of ionic gelation with polyanions (Hu, Wang, Li, Zeng, & Huang, 2011; Rampino, Borgogna, Blasi, Bellich, & Cesaro, 2013). In those cases sodium hydroxide is often used to adjust the pH resulting in the formation of NaOAc. In addition, our previous experimental results showing that when the pH value of pullulan-chitosan composite solution was increased from 3 to 5, the tensile strength of pullulan-chitosan blended film decreased while the elongation at break increased. Similar results were also obtained in our lab when increased the pH of gelatin-chitosan solution from 3.5 to 4.5. Generally, it is well known that the content of NaOAc increased when increased the pH of acetic acid solution. NaOAc is commonly used as a food preservative. It has the ability to suppress the growth of food-borne bacteria and protect against food deterioration. It is also used for regulating the acidity or alkalinity of food products. Sodium acetate and sodium diacetate are used frequently as flavor enhancers in bread, cakes, cheese and snack food. Furthermore, it is widely available, economical, and generally “recognized-as-safe” (Ghomi et al., 2011; Sallam, 2007). Antimicrobial ability of different percentages of NaOAc in various meat and seafood has been previously reported (Yesudhason, Lalitha, Gopal, & Ravishankar, 2014). However, direct application of antimicrobial substances, such as dipping, spraying or brushing, may result in the inactivation or evaporation of active agents and rapid migration into the bulk of the foods. Controlling surface microbial growth on food is important because it is the main source of contamination for many chilled food products. Therefore, an appropriate way is to incorporate an antimicrobial agent into biobased edible films. Manju, Jose, Srinivasa Gopal, Ravishankar, and Lalitha (2007) and Yesudhason et al. (2014) have reported that combination of packaging and NaOAc extended the shelf life and quality of fish at refrigeration temperatures. In the present study, we investigated the influence of NaOAc on the physicochemical and thermomechanical properties of gelatin films. The sol-gel and gel-sol transition temperature of gelatin was

141

determined by dynamic viscoelastic measurements and differential scanning calorimetry (DSC), respectively. Two different drying temperatures (25
C and 35
C) were used to obtain gelatin films. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were used to determine the triple helix content. It is, to our knowledge, the first study on the effects of NaOAc on edible films. The existing state of NaOAc in the gelatin matrix was investigated as well as the effect of water activity on the isothermal water absorption of gelatin films with and without NaOAc. 2. Materials and methods 2.1. Materials Gelatin (type B) and sodium acetate (NaOAc) were purchased from China Medicine (Group) Shanghai Chemical Reagent Corporation (Shanghai, China). All other reagents were of analytical grade. 2.2. Film forming solution preparation Gelatin solution was prepared at concentration of 4% (w/v) by hydrating gelatin powders in distilled water for 1 h at room temperature. The solution was then heated at 65
C with continuous stirring in a magnetic stirrer until complete dissolution. Sodium acetate was obtained at concentration of 0.7% (w/v) by dissolving NaOAc powder in distilled water. 2.2.1. Dynamic viscoelastic properties Dynamic viscoelasticity of the gelatin solution was carried out on a rheometer (AR-G2, TA Instruments, USA) using a plateeplate geometry (diameter of 60 mm, gap of 1 mm). Cooling from 37.5 to 17.5
C took place at a scan rate of 0.1
C/min, a frequency of 1 Hz, and a target strain% of 0.5%. The storage modulus (G0 , Pa), loss modulus (G00 , Pa) were recorded as a function of temperature. Gelation temperature was determined from where G0 and G00 intersect with the linear temperature gradient. 2.2.2. Differential scanning calorimetry (DSC) measurements The melting behavior of gelatin gel was measured by a differential scanning calorimeter (DSC) (Netzsch DSC 204 F1, data processor Proteus® Software, Germany) according to the method of (Yoshimura et al., 2008). A 50 mL portion of gelatin solution was sealed in a 70 mL stainless steel cell and incubated at 4
C for 48 h to gel and then scanned from 20 to 40
C at a heating rate of 1
C/ min. From the DSC curve, the helix-coil transition temperature was calculated as the temperature where the endothermic peak occurs. 2.3. Film preparation Casting/solution evaporation method was used in this study for preparing films. NaOAc solution was obtained at 0.7% w/v and Table 1 Composition of film-forming solutions. Gelatin solution used for film preparation was 50 mL at concentration of 4% (w/v) in all films. NaOAc/Gelatin mass ratio

NaOAc (mL)

Water (mL)

0% 5.25% 8.75% 10.5% 12.25%

0 15 25 30 35

50 35 25 20 15

142

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

gelatin films were prepared by using various volumes of NaOAc as shown in Table 1. 50 mL of film forming solutions were casted into square Petri dishes (10 cm  10 cm) and dried at 25
C or 35
C in oven until constant weight (approximately 36 h or 16 h, respectively). After drying, films were peeled off and conditioned at 25
C and 53% of relative humidity (RH) for at least 48 h in desiccator cabinet containing Mg(NO3)2$5H2O saturated solution. 2.4. Physicochemical film characterization 2.4.1. Film thickness Film thickness was determined at 10 random points on each specimen using a micrometer (Guilin, China) and mean values were calculated. 2.4.2. Moisture content Film moisture content was determined by measuring weight loss of samples (2 cm  2 cm) after drying in an oven at 105 ± 1
C until constant weight (about 24 h). The samples were previously conditioned at 53% RH for 72 h. Films were analyzed at least in triplicate and results were expressed as a percentage of total weight. 2.4.3. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of the films were recorded in attenuated total reflection (ATR) mode using FTIR spectrometer (Nicolet IS 10, Thermo Electron, USA). Infrared spectra were recorded from 800 to 4000 cm1 and the resolution is 4 cm1. Each spectrum represented an average of 64 consecutive scans. All tests were performed at room temperature. 2.4.4. X-ray diffraction (XRD) X-ray diffraction patterns of films were recorded by an X-ray diffractometer (D8 Advance, Bruker AXS Germany) using Cu Ka radiation (1.542 Å) radiation at 40 kV and 40 mA, operated at room temperature. The relative intensity was recorded between a range (2q) of 3e60
with a step size of 0.02 and a scanning speed of 4
min1. 2.4.5. Water contact angle According to Wang et al. (2014), the contact angle of the gelatin films was determined with a contact angle meter with sessile drop method (OCA15EC, Dataphysics Co., Ltd, Germany). 10 mL distilled water was carefully dropped on films through a 2.0 mL micrometer syringe (KDL Corp, Shanghai, China) and contact angles were recorded immediately. Image analyses were carried out carefully using SCA20 software for both right and left angles of the drop. For each film, at least three replicates were tested. 2.5. Mechanical properties 2.5.1. Tensile test Tensile strength (TS), elongation at break (EB%) and stressestrain curve were determined according to ASTM standard method D882 (ASTM, 2001) with modifications using a texture analyzer (TA.XT2i, Lloyd instruments, U.K.) equipped with a tension grip system A/TG at room temperature. Film stripes of 2  8 cm were initially cut and tested using initial grip separation and crosshead speed at 50 mm and 0.5 mm/s, respectively. The curves of force (N) as a function of deformation (mm) were recorded using Texture Expert Exceed software (Version 2.64, Stable Micro Systems LTD., Godalming, UK). TS (MPa) and EB (%) were calculated using the following equations (1) and (2):

TS ¼

Maximum force ðNÞ Thickness ðmmÞ  Width ðmmÞ

EB% ¼

L  L0  100% L0

(1)

(2)

Where L0 is the initial length of the film and L is the length of the film when it breaks. The reported values correspond to at least five determinations. Stress-strain curves were calculated from the plot of stress (tension force/initial cross-sectional area) versus strain (extension as a fraction of the original length). 2.5.2. Dynamic mechanical thermal analysis (DMTA) The thermo-mechanical properties of films were carried out using a dynamic mechanical analyzer (DMA, Q800, TA Instruments, New Castle, USA) in tensile deformation. All the gelatin films were analyzed using the following tests:  Frequency sweep: The time-dependent behavior of the films was determined in this study. This analysis was performed at a frequency range of 0.1e100 Hz and constant temperature (25
C) by applying constant deformation amplitude of 10 mm (within the liner viscoelastic region). Storage modulus (G0 ), loss modulus (G00 ) and tan d (¼G00 /G0 ) were measured and plotted against the frequency.  Temperature ramp: The temperature-dependent behavior of the films was analyzed. This test was performed at a temperature range of 25 to 150
C with a scanning rate of 3
C/min and fixed deformation amplitude of 10 mm (within the liner viscoelastic region). All the tests were performed in a single frequency mode (1 Hz). 2.6. Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) using the SPSS 19.0 package (IBM, New York). Duncan'smultiple range test was used to determine the significant differences of the mean values (P < 0.05). 3. Results and discussion 3.1. Gelling temperature and melting temperature of gelatin The gelling and melting temperature of gelatin was measured by rheometer and DSC, respectively (Fig. 1). As the temperature decreased, G0 and G00 increased sharply at 28.0
C while tan d decreased at the described temperature (data not show), indicating the decrease in the viscous concentration to the viscoelasticity. As indicated by Lau, Tang, and Paulson (2001), the initial rise in G0 has been linked to the formation of a three-dimensional network where most of the sol fraction is converted to a gel. The temperature at 26.8
C when the G0 /G00 cross over occurred during cooling is close to the sol-gel transition point (Mhd Sarbon, Badii, & Howell, 2013). During cooling, gelation occurs by entanglement of molecules of gelatin as a result of renaturation of the triple-helix. As shown in Fig. 1b, the DSC curve of gelatin showed a single endothermic peak. Melting temperature was observed from the maximum of the endothermic peak, which is 31.9
C. During heating, a gel-sol transition occurred where gelatin remained in a primary random coil conformation. In order to evaluate the effect of NaOAc on the mechanical properties of gelatin films, two casting temperatures (25
C and 35
C) were selected. Gelatin films dried at different temperatures can produce films with different content of triple helix regardless of

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

143

Fig. 1. (a) Viscoelastic properties (G0 and G00 values) of gelatin solution on cooling from 37.5 to 17.5
C; (b) DSC curve of gelatin gels heated from 20 to 40
C.

the gelatin arrive from mammalian or fish sources (Badii et al., 2014; Chiou et al., 2009). 3.2. FTIR analysis

(a.u.)

According to Zaupa, Neffe, Pierce, Nochel, and Lendlein (2011), gelatin films dried at a temperature above the sol-gel transition temperature resulted in films with relatively lower triple helix content than that dried at lower temperatures. FTIR spectroscopy was carried out in order to explore the differences on the chemical composition and molecular conformation in resulting gelatin films (without NaOAc). Gelatin displays distinct amide bands via FTIR, which can provide information about its triple helix content (He et al., 2011; Tronci, Doyle, Russell, & Wood, 2013). As shown in Fig. 2, the main absorption peaks were related to: (i) Amide A and B bands at about 3300 and 3085 cm1, respectively, which were mainly associated with NeH stretching vibrations; OeH stretching vibration associated with hydroxyl groups. In the same region, the peaks of NeH stretching from primary amine and type II amide are overlapped. (ii) Amide I and II bands at approximately 1630 and 1540 cm1, assigned to the stretching vibrations of peptide C]O groups as well as to the NeH bending vibrations coupled to CeN stretching vibrations, respectively. (iii) Amide III band centered at about 1240 cm1, resulting from the CeN stretching and NeH bending vibrations of amide linkages, as well as wagging vibrations of CH2 groups in the glycine backbone and proline side chains.

Wavenumber (cm-1) Fig. 2. Effect of drying temperature on the FTIR spectrum of blank gelatin films (0% NaOAc/Gelatin mass ratio). ( 25
C and 35
C).

No distinct band displacements were found among the previously mentioned amide bands of the two different drying temperatures blank gelatin films, particularly the amide I band related to the native triple helix conformation (He et al., 2011; Payne & Veis, 1988). In addition to qualitative findings on unchanged band positions, FTIR absorption ratio of amide III to 1450 cm1 band (AIII/ A1450) was determined in both gelatin films in order to quantify the degree of triple helix formation (He et al., 2011; Tronci et al., 2013; Wang, An, Xin, Zhao, & Hu, 2007). The amide ratio (AIII/A1450) for films dried at 25
C was observed at 1.01 while a lower amide ratio of 0.86 was calculated when the films were dried at higher temperature (35
C). Lower values (0.59) have been reported for gelatin films by Andrews, Murali, Muralidharan, Madhulata, and Jayakumar (2003). These results suggest that gelatin films with different content of triple helical structure were obtained at different drying temperatures. 3.3. Thermomechanical properties of films 3.3.1. Tensile properties As shown in the typical stressestrain curves in Fig. 3 the NaOAc could make the films dried at 25
C and 35
C more flexible. Gelatin films containing NaOAc showed a plastic deformation which improved as the NaOAc ratio increased. Tensile strength (TS) and elongation at break (EB%) of gelatin films incorporated with various ratios of NaOAc and dried at different temperatures (25
C and 35
C) are shown in Table 2. Tensile properties (TS and EB%) of films were affected by the addition of NaOAC. At both drying temperatures, the TS of films gradually decreased while EB% increased with increasing content of NaOAC. More specifically, at drying temperature of 25
C, addition of 8.75% NaOAC decreased the TS from 88.7 MPa to 64.1 MPa while EB% increased from 5.1% to 21.2% (P < 0.05). Similarly, at the temperature of 35
C, TS of films decreased from 71.5 MPa to 57.3 MPa while EB% increased from 2.5% to 10.9% (P < 0.05). From these results, TS and EB% were inversely related, which means that films with higher elongation values often require a lower load to break (Nur Hanani, Roos, & Kerry, 2012). With increasing content of NaOAc, the TS and EB% values of the films decreased and increased further, respectively. It can be concluded that the effect of NaOAc on gelatin films was similar to that of plasticizers. Gelatin films dried at 35
C exhibited a distinctive brittle fracture mode when 5.25% of NaOAc was added. While the corresponding film dried at 25
C changed to a ductile and flexible material (Fig. 3). This could be due to the different content of triple helix within the gelatin films. The blank gelatin film dried at 25
C proved to be more stretchable compared to the film dried at 35
C which showed the typical characteristics of brittle and rigid

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

P

P

144

Fig. 3. Effect of sodium acetate on typical stressestrain curves of gelatin films dried at (a) 25
C and (b) 35
C at different NaOAc/gelatin mass ratios.

material. This result was similar to the un-plasticized gelatin type B film dried at similar temperature (37
C) reported by Rivero et al. (2010). 3.3.2. Dynamic mechanical thermal analysis (DMTA) DMTA was used to analyze film viscoelasticity and temperatureinduced transitions. It was also performed in order to verify the plasticizing effect of NaOAc on gelatin films. 3.3.2.1. Temperature ramp. The temperature dependence of storage modulus (G0 ) and tan d for gelatin based films containing NaOAc at drying temperature of 25
C and 35
C are shown in Figs. 4 and 5, respectively. With increasing temperature all samples passed through the glassy region, the glass transition region (tan d peak and storage modulus decrease) and the rubbery region. Usually, the glass transition temperature (Tg) of each film was determined as the temperature corresponding to the maximum of the tan d peak (Kristo & Biliaderis, 2006; Oliviero, Verdolotti, Maio, Aurilia, & Iannace, 2011; Thakhiew, Devahastin, & Soponronnarit, 2010) and the Tg values of gelatin films are listed in Fig. 4. It can be observed that addition of NaOAc significantly affected the Tg of the gelatin films. At both casting temperatures, increase of NaOAc content decreased the Tg of films. The Tg is characterized by an increase in the molecular mobility of the polymer chains, which changes the observed mechanical strength of the material from a hard glass to a softer rubber (Fadda et al., 2010). Therefore, the molecular mobility of gelatin films was enhanced with increasing NaOAc content. The higher Tg values in the absence of NaOAc could be related to the presence of the more gelatinegelatin interactions such as hydrogen bonding, ionic interactions or Van der Waals forces. The single Tg indicated a good compatibility between NaOAc and gelatin. This is

Table 2 Effect of sodium acetate on mechanical properties (tensile properties) of gelatin films dried at two temperatures (25
C and 35
C).a Drying temperature

NaOAc/gelatin mass ratio (%)

Thickness (mm)

25
C

0% 5.25% 8.75% 10.5% 12.25% 0% 5.25% 8.75% 10.5% 12.25%

81.0 84.7 85.8 88.6 89.4 76.9 79.3 81.0 84.6 96.6

35
C

± ± ± ± ± ± ± ± ± ±

3.3de 2.6cd 1.6bcd 2.8bc 4.2b 3.4e 2.7e 1.6de 2.0cd 3.5a

TS (MPa) 88.7 81.3 64.1 48.5 47.3 71.5 64.6 57.3 47.4 31.1

± ± ± ± ± ± ± ± ± ±

5.1a 4.8b 2.7d 5.3e 2.4e 6.4c 5.8d 3.2e 3.3e 3.1f

EB (%) 5.1 9.2 21.2 22.4 30.2 2.5 3.5 10.9 19.2 34.6

± ± ± ± ± ± ± ± ± ±

1.3%e 1.5%d 1.9%c 3.1%c 3.2%b 0.3%e 0.7%e 1.9%d 3.4%c 4.58%a

a Values are given as mean ± standard deviation. Different online letters in the same column indicate a statistically significant difference (P < 0.05). TS e tensile strength; EB e Elongation at break.

in agreement with the results of XRD and WCA analyses (Figs. 9 and 10). Furthermore, at corresponding NaOAc ratios the films dried at 25
C had higher Tg values than that dried at 35
C. The mobility of the amorphous parts in gelatin films was restricted by the more complex triple helical structure obtained at 25
C. Similar results were also reported by Badii et al. (2014). The variations in storage modulus (G0 ) as a function of temperature for gelatin films with different NaOAc content are shown in Fig. 5. Initially, the G0 decreased as the temperature increased which is so-called “softening”. The softening of the films can be explained by the molecular movements with free volume theory (Li, Li, Wang, € Ozkan, & Mao, 2010). For temperatures above 75
C the G0 increased especially for films including NaOAc. These changes might be attributed to thermal transformations as well as a packing effect resulting from water evaporation at high temperature (Lavorgna, Piscitelli, Mangiacapra, & Buonocore, 2010; Li et al., 2010; Pouplin, Redl, & Gontard, 1999). Similar observations were also mentioned by Lavorgna et al. (2010) and Li et al. (2010) for chitosan films and sweet potato roots films, respectively. The increasing range of G0 became larger as the increase of NaOAc content. These results might be attributed to the different interactions between the gelatin matrixes or to changes in the moisture content. Addition of NaOAc enlarged the free volume between the chain segments of the gelatin matrix and therefore increased its mobility. This dependence of the molecular motion on the NaOAc concentration confirms its plasticizing effect.

3.3.2.2. Frequency sweep. The viscoelastic properties of the gelatin films also changed with the loading frequency besides temperature. As shown in Fig. 6, the G0 , G00 and tan d varied as a function of oscillation frequency. Regardless of the NaOAc content and drying temperature, all samples presented higher storage modulus than loss modulus over the entire frequency range measured. This result suggests that all the gelatin films analyzed in the frequency range exhibited predominant elastic characteristic than viscous characteristic (Cespi, Bonacucina, Mencarelli, Casettari, & Palmieri, 2011; Shi, Wang, Li, & Adhikari, 2013). The frequency sweep curves of all samples showed similar profiles regardless of the drying temperature and NaOAc content. With increasing frequency, G0 slightly increased while G00 and tan d subtly decreased. This very weak frequency dependence of the G0 , G00 and tan d suggest that the films were in glassy region at the temperature tested (Shi et al., 2013). This result is in accordance with the Tg values obtained by the temperature ramp test. Moreover, smaller G0 values at low frequency, especially at 0.1 Hz, characterized slower, the more viscous, longer-range deformation of gelatin's molecular network; larger G0 values at high frequency characterized more elastic, shorter-range intra- and intermolecular interactions (Chen, Gao, & Ploehn, 2014).

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

0% NaOAc/Gelatin mass ratio,

8.75% NaOAc/

P

Fig. 4. The influence of sodium acetate on tan d of gelatin films dried at (a) 25
C and (b) 35
C as a function of temperature. ( Gelatin mass ratio, 12.25% NaOAc/Gelatin mass ratio).

145

Fig. 5. The influence of sodium acetate on storage modulus of gelatin films dried at (a) 25
C and (b) 35
C as a function of temperature. ( 8.75% NaOAc/Gelatin mass ratio, 12.25% NaOAc/Gelatin mass ratio).

According to Lazaridou and Biliaderis (2002) and Paschoalick, Garcia, Sobral, and Habitante (2003), the plasticizing effect of NaOAc could be calculated from the DMTA results (G0 , G00 and tan d) at selected frequencies. The G00 and tan d both increased while the G0 decreased with increasing NaOAc content. Similar results were observed with the increase of the plasticizer content for gelatin films and for fish muscle protein films (Paschoalick et al., 2003; Thomazine, Carvalho, & Sobarl, 2005). These changes in the moduli of the films could be caused by the plasticizing effect of NaOAc and/or water which was attracted by the film matrix since strong gelatinegelatin interactions were detriment by the polymerNaOAc or polymerewater interactions. The longer-range deformation of gelatin's molecular network was elevated and the shorter-range intra- and intermolecular interactions were lowered, thus promoted the motions of the molecular network. In addition, it should be noted that there were similar cuspidal peaks of G0 , G00 and tan d for all the gelatin films. The corresponding frequency at about 79.5 Hz could be assumed to be a resonance frequency showed (Li et al., 2010).

0% NaOAc/Gelatin mass ratio,

NaOAc content (Table 2). The strong hydrophilic nature of NaOAc favors the absorption of water molecules in the film matrix and forms hydrogen bonds between gelatin matrixes, which lead to the increase of moisture content. This behavior was also reported generally in films plasticized with glycerol (Bergo, Moraes, & Sobral, 2013; Silva, Bierhalz, & Kieckbusch, 2009). Furthermore, in hydrophilic biopolymers, water can act as a plasticizer that undermines hydrogen bonds between polymer chains (Xiao, Lim, & Tong, 2012). Consequently, this resulted to the increase of EB% and the decrease of TS (Table 2 and Fig. 5). However, the moisture content was not significantly different (P > 0.05) when the content of NaOAc was lower than 5.25% for 25
C and 8.75% for 35
C while the mechanical properties were weakened. Similar results were also obtained by Godbillot, Dole, Joly, Roge, and Mathlouthi (2006) in films plasticized with glycerol. These suggest that addition of NaOAc weakened the intermolecular forces between the chains of adjacent macromolecules, thus increased the free volume and reduced the mechanical resistant. Cagri, Ustunol, and Ryser (2001) concluded that incorporation of additives other than cross linking agents could generally lower TS values.

3.4. Physicochemical properties of films 3.4.1. Moisture content Moisture content was measured to see the water absorbing capacity of the gelatin films. As shown in Fig. 7, the moisture content of gelatin films increased gradually as the NaOAc increased. When the NaOAc increased to 12.5% the moisture content increased significantly (P < 0.05) to 18.23% and 16.20% for drying temperature of 25
C and 35
C, respectively. Similarly, at both drying temperatures, the thickness of films increased slightly with the increase of

3.4.2. Isothermal water absorption When the content of NaOAc was above 10.5%, the composite films became opaque and white after three weeks of storage. NaOAc powder crystallized on the surface of gelatin films. Therefore, the stable gelatin films incorporated with 0%, 7% and 8.75% NaOAc were chosen. Fig. 8 shows the variations of moisture content of gelatin films after equilibrating at different relative humidity (11%, 33%, 53%, 75% and 97% RH). The equilibrium water content increased slowly with increasing RH up to 53%, above which there was a steep

P

P

P

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

P

146

Fig. 6. Effect of applied frequency on the mechanical parameters of storage modulus, loss modulus and tan d of gelatin films dried at (a) 25
C and (b) 35
C. ( Gelatin mass ratio, 5.25% NaOAc/Gelatin mass ratio, 8.75% NaOAc/Gelatin mass ratio, 12.25% NaOAc/Gelatin mass ratio).

NaOAc/gelatin mass ratio % Fig. 7. Effect of sodium acetate on the moisture content of gelatin films dried at ( ) 25
C and ( ) 35
C. Different lowercase letters represent a statistically significant difference (P < 0.05).

0% NaOAc/

rise in moisture content. It is notable that gelatin films with NaOAc showed a higher sensitivity to humidity compared to films without as the RH exceeded 53%. The RH increased up to 75% the water content of blank films was still very low. NaOAC, being a polar material, extended the intermolecular interactions of gelatin and loosened the compactness of the structure. Therefore, the gelatin molecules were capable to form more hydrogen bonds with water molecules increasing eventually the susceptibility to hydration of the films (Rocha, Loiko, Tondo, & Prentice, 2014). Additionally, the hydrophilic nature of NaOAc increased even further the water content of the films with NaOAc. Furthermore, amorphous materials have greater water sorption capacity than crystalline forms due to the steric restrictions, given in the ordered crystalline arrangements, which limit the location of
nez, Fabra, Talens, & Chiralt, 2013). This is in water molecules (Jime agreement with the decrease of triple helical structure with increasing NaOAc content as shown in XRD analysis (Fig. 9). However, it is interesting to note that the films dried at lower temperature (25
C) with higher triple helix had higher water content than films dried 35
C, Fig. 8. This is probably due to the formation of more hydrogen bonds with water by triple helical structures than by amorphous gelatin chains (Chiou et al., 2009).

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

Fig. 8. Effect of sodium acetate on the moisture content of gelatin films dried at (a) 25
C and (b) 35
C and equilibrated at different relative humidity. ( mass ratio, 7% NaOAc/Gelatin mass ratio, 8.75% NaOAc/Gelatin mass ratio).

147

0% NaOAc/Gelatin

Fig. 9. X-ray diffraction (XRD) patterns of gelatin films dried at (a) 25
C and (b) 35
C incorporated with various NaOAc content (0%, 8.75% and 12.25%). Diffractions are shifted vertically for clarity.

3.4.3. XRD analysis of films The crystalline nature of gelatin films loaded with different concentrations of NaOAc and dried at two different temperatures (25
C and 35
C) are presented in Fig. 9. XRD patterns can verify the various types of gelatin molecular structures and especially the collagen-like helical structure content. One sharp and dominant peak at 2q ¼ 7.5
was shown at both drying temperatures (Fig. 9). This peak is related to the diameter of the triple helix and its intensity is assigned to the content of triple helix (Bigi et al., 2004; Rivero et al., 2010; Yakimets et al., 2005). No displacements of the peak occurred for different gelatin films indicating that the diameter of the triple helix structure did not change. Nevertheless, the amount of triple-helical crystalline structure (i.e. the intensity of this characteristic peak) was different. Lower drying temperature led to greater amounts of triple helix in the gelatin films compared to higher temperature. This result is consistent with the results reported by Badii et al. (2014). Higher temperature resulted in faster drying and therefore lower molecular order. Addition of NaOAc at different concentrations showed no significant changes in the XRD pattern of the films. However, the intensity of the characteristic peak decreased with increasing NaOAc. This structural change can be due to NaOAc properties. The effect of NaOAc on the XRD pattern of gelatin films was very similar to that of glycerol (Rivero et al., 2010). It can be assumed that both compounds have very similar water affinity and physical interaction in the gelatin matrix. The dispersion feature of NaOAc can also been investigated from the XRD patterns (Fig. 9). The X-ray pattern of the NaOAc powder indicated a crystalline structure (a prominent peak at 8.9
and

lower intensity peaks at, respectively, 17.8, 26.8 and 36.0
). While the XRD curves of gelatin films with or without NaOAc only showed a peak related to triple helix structure and a broad amorphous peak at 2q ¼ 18
corresponded to the distance between amino acid residues along the helix (Badii et al., 2014). The sharp diffraction peaks of NaOAc disappeared in the gelatin films containing NaOAc, which indicates that NaOAc was present as a non-crystalline “dis€llstedt, and Hedenqvist (2012) acsolved” component. Türe, Ga quired similar results with wheat gluten films incorporated with potassium sorbate. These results confirm that there was a uniform dispersion of the NaOAc into the gelatin matrix, indicating a strong interaction between the gelatin and NaOAc molecules. 3.4.4. Water contact angle (WCA) To further elucidate the dispersion state of NaOAc in gelatin films, WCA analysis was performed to determine the surface hydrophobicity and wettability, as shown in Fig. 10. Generally speaking, a high WCA means hydrophobicity, whereas a low angle indicates hydrophilicity (He et al., 2011). To our knowledge, NaOAc is a very hydrophilic and hygroscopic compound. However, the values of the WCA did not display a decreasing trend with increasing NaOAc content at both drying temperatures. More specifically, the WCA of films with 5.25%e8.75% NaOAc increased slightly compared with that of blank gelatin films (P < 0.05). Increase in the WCA has also been reported by Kokoszka, Debeaufort, Lenart, and Voilley (2010) with addition of 40% glycerol in whey protein isolate films. This could be due to the reorientation of hydrophobic moieties of gelatin molecules at the air-film interface. The existence of NaOAc in the gelatin matrix may strengthen the

148

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149

a

an amorphous state. However, addition of NaOAc exceeded 10.5% the composite film became whiter, which was caused by NaOAc particles crystallized on the film surface. Moreover, the plasticizing effect of NaOAc was closely related to the RH and it can increase the films' sensitivity to moisture. This study may raise awareness for the effects of salts on edible films since salts (organic or inorganic) exist widely in nature. Acknowledgments This work was financially supported by National 863 Program 2011BAD23B02, 2013AA102207, National Natural Science Foundation of China 31171686, 30901000, 111 project-B07029 and PCSIRT0627.

b

NaOAc/gelatin mass ratio %

References

25 °C

35 °C

0%

5.25% 8.75% 10.5% 12.25%

CN

Fig. 10. (a) Effect of sodium acetate on the water contact angles of gelatin films dried at ( ) 25
C and ( ) 35
C. (b) Shape and behavior of water droplets deposited on gelatin films as a function of NaOAc content. CN refers to the crystallization of NaOAc on the surface of films after equilibration for 3 weeks. Different lowercase letters represent a statistically significant difference (P < 0.05).

interactions between hydrophilic groups of NaOAc and gelatin because of the strong polar of NaOAc. An orientation of hydrophobic moieties was also previously described in the case of gelatin films (Białopiotrowicz & czuk, 2002) and kidney bean protein isolate films (Ma, Tang, Yang, & Yin, 2013). There was no significant difference when the content of NaOAc ranged from 8.75% to 12.25% (P > 0.05). As expected, the incorporating of NaOAc did not increase hydrophilicity. This is consistent with previous results obtained by (Kokoszka, Debeaufort, Hambleton, Lenart, and Voilley, 2010) in soy protein isolate based films plasticized with glycerol. Moreover, we have mentioned previously that NaOAc powder would crystallize on the surface of gelatin films after three weeks storage when the content of NaOAc exceeded 10.5%. The WCA values of films, dried at two temperatures, with 12.5% NaOAc after crystallization are also shown in Fig. 10. This confirmed the strong hydrophilic nature of NaOAc and significant differences were presented between films with and without precipitation of NaOAc (P < 0.05). These results are in agreement with the results of XRD, indicating a uniform dispersion of the NaOAc within the gelatin internal matrix at selected NaOAc contents. 4. Conclusions In this study, different content of triple helix structures in gelatin films were achieved by drying at two different temperatures. Regardless of triple helix content, the effect of NaOAc on the mechanical and thermal properties of gelatin films was similar to that of plasticizers, especially when the content was high. With increasing NaOAc content, the flexibility of films was improved. XRD and WCA verified the good compatibility and NaOAc presented

Andrews, M. E., Murali, J., Muralidharan, C., Madhulata, W., & Jayakumar, R. (2003). Interaction of collagen with corilagin. Colloid & Polymer Science, 281(8), 766e770. Arvanitoyannis, I., Psomiadou, E., Nakayama, A., Aiba, S., & Yamamoto, N. (1997). Edible films made from gelatin, soluble starch and polyols, Part 3. Food Chemistry, 60(4), 593e604. ASTM. (2001). Standard test method for tensile properties of thin plastic sheeting. Standard D882. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Materials. Badii, F., MacNaughtan, W., Mitchell, J. R., & Farhat, I. A. (2014). The effect of drying temperature on physical properties of thin gelatin films. Drying Technology, 32(1), 30e38. Bergo, P., Moraes, I. C. F., & Sobral, P. J. A. (2013). Effects of plasticizer concentration and type on moisture content in gelatin films. Food Hydrocolloids, 32(2), 412e415. Białopiotrowicz, T., & czuk, B. J. (2002). Surface properties of gelatin films. Langmuir, 18, 9462e9468. Bigi, A., Bracci, B., Cojazzi, G., Panzavolta, S., & Roveri, N. (1998). Drawn gelatin films with improved mechanical properties. Biomaterials, 19, 2335e2340. Bigi, A., Cojazzi, G., Panzavolta, S., Rubini, K., & Roveri, N. (2001). Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials, 22, 763e768. Bigi, A., Panzavolta, S., & Rubini, K. (2004). Relationship between triple-helix content and mechanical properties of gelatin films. Biomaterials, 25(25), 5675e5680. Cagri, A., Ustunol, Z., & Ryser, E. (2001). Antimicrobial, mechanical, and moisture barrier properties of low pH whey Protein-based edible films containing paminobenzoic or sorbic acids. Journal of Food Science, 66(6), 865e870. Cespi, M., Bonacucina, G., Mencarelli, G., Casettari, L., & Palmieri, G. F. (2011). Dynamic mechanical thermal analysis of hypromellose 2910 free films. European Journal of Pharmaceutics and Biopharmaceutics, 79(2), 458e463. Chen, X., Gao, H., & Ploehn, H. J. (2014). Montmorillonite-levan nanocomposites with improved thermal and mechanical properties. Carbohydrate Polymers, 101, 565e573. Chiou, B.-S., Avena-Bustillos, R. J., Bechtel, P. J., Imam, S. H., Glenn, G. M., & Orts, W. J. (2009). Effects of drying temperature on barrier and mechanical properties of cold-water fish gelatin films. Journal of Food Engineering, 95(2), 327e331. Chiou, B.-S., Avena-Bustillos, R. J., Bechtel, P. J., Jafri, H., Narayan, R., Imam, S. H., et al. (2008). Cold water fish gelatin films: effects of cross-linking on thermal, mechanical, barrier, and biodegradation properties. European Polymer Journal, 44(11), 3748e3753. Fadda, H. M., Khanna, M., Santos, J. C., Osman, D., Gaisford, S., & Basit, A. W. (2010). The use of dynamic mechanical analysis (DMA) to evaluate plasticization of acrylic polymer films under simulated gastrointestinal conditions. European Journal of Pharmaceutics and Biopharmaceutics, 76(3), 493e497.
mez-Estaca, J., Go
mez-Guille
n, M. C., Fern
Go andez-Martín, F., & Montero, P. (2011). Effects of gelatin origin, bovine-hide and tuna-skin, on the properties of compound gelatinechitosan films. Food Hydrocolloids, 25(6), 1461e1469.
mez-Estaca, J., Montero, P., Fern

mezGo andez-Martín, F., Alem
an, A., & Go
n, M. C. (2009). Physical and chemical properties of tuna-skin and bovineGuille hide gelatin films with added aqueous oregano and rosemary extracts. Food Hydrocolloids, 23(5), 1334e1341. Ghomi, M. R., Nikoo, M., Heshmatipour, Z., Jannati, A. A., Ovissipour, M., Benjakul, S., et al. (2011). Effect of sodium acetate and nisin on microbiological and chemical changes of cultured grass carp (Ctenopharyngodon Idella) during refrigerated storage. Journal of Food Safety, 31(2), 169e175. Godbillot, L., Dole, P., Joly, C., Roge, B., & Mathlouthi, M. (2006). Analysis of water binding in starch plasticized films. Food Chemistry, 96(3), 380e386. He, L., Mu, C., Shi, J., Zhang, Q., Shi, B., & Lin, W. (2011). Modification of collagen with a natural cross-linker, procyanidin. International Journal of Biological Macromolecules, 48(2), 354e359.

F. Liu et al. / Food Hydrocolloids 45 (2015) 140e149 Hu, B., Wang, S. S., Li, J., Zeng, X. X., & Huang, Q. R. (2011). Assembly of bioactive peptide-chitosan nanocomplexes. Journal of Physical Chemistry B, 115(23), 7515e7523. Jeya Shakila, R., Jeevithan, E., Varatharajakumar, A., Jeyasekaran, G., & Sukumar, D. (2012). Comparison of the properties of multi-composite fish gelatin films with that of mammalian gelatin films. Food Chemistry, 135(4), 2260e2267.
nez, A., Fabra, M. J., Talens, P., & Chiralt, A. (2013). Phase transitions in starch Jime based films containing fatty acids. Effect on water sorption and mechanical behaviour. Food Hydrocolloids, 30(1), 408e418. Karim, A. A., & Bhat, R. (2009). Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocolloids, 23(3), 563e576. Kokoszka, S., Debeaufort, F., Hambleton, A., Lenart, A., & Voilley, A. (2010). Protein and glycerol contents affect physico-chemical properties of soy protein isolatebased edible films. Innovative Food Science & Emerging Technologies, 11(3), 503e510. Kokoszka, S., Debeaufort, F., Lenart, A., & Voilley, A. (2010). Water vapour permeability, thermal and wetting properties of whey protein isolate based edible films. International Dairy Journal, 20(1), 53e60. Kristo, E., & Biliaderis, C. G. (2006). Water sorption and thermo-mechanical properties of water/sorbitol-plasticized composite biopolymer films: caseinateepullulan bilayers and blends. Food Hydrocolloids, 20(7), 1057e1071. Lau, M. H., Tang, J., & Paulson, A. T. (2001). Effect of polymer ratio and calcium concentration on gelation properties of gellan/gelatin mixed gels. Food Research International, 34, 879e886. Lavorgna, M., Piscitelli, F., Mangiacapra, P., & Buonocore, G. G. (2010). Study of the combined effect of both clay and glycerol plasticizer on the properties of chitosan films. Carbohydrate Polymers, 82(2), 291e298. Lazaridou, A., & Biliaderis, C. G. (2002). Thermophysical properties of chitosan, chitosan-starch and chitosan-pullulan films near the glass transition. Carbohydrate Polymers, 48, 179e190. € Li, Q., Li, D., Wang, L.-J., Ozkan, N., & Mao, Z.-H. (2010). Dynamic viscoelastic properties of sweet potato studied by dynamic mechanical analyzer. Carbohydrate Polymers, 79(3), 520e525. Ma, W., Tang, C.-H., Yang, X.-Q., & Yin, S.-W. (2013). Fabrication and characterization of kidney bean (Phaseolus vulgaris L.) protein isolateechitosan composite films at acidic pH. Food Hydrocolloids, 31(2), 237e247. Manju, S., Jose, L., Srinivasa Gopal, T., Ravishankar, C., & Lalitha, K. (2007). Effects of sodium acetate dip treatment and vacuum-packaging on chemical, microbiological, textural and sensory changes of pearlspot (Etroplus suratensis) during chill storage. Food Chemistry, 102(1), 27e35. Mhd Sarbon, N., Badii, F., & Howell, N. K. (2013). Preparation and characterisation of chicken skin gelatin as an alternative to mammalian gelatin. Food Hydrocolloids, 30(1), 143e151. Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004). Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloids, 18(4), 581e592. Nur Hanani, Z. A., Roos, Y. H., & Kerry, J. P. (2012). Use of beef, pork and fish gelatin sources in the manufacture of films and assessment of their composition and mechanical properties. Food Hydrocolloids, 29(1), 144e151. Oliviero, M., Verdolotti, L., Maio, E. D., Aurilia, M., & Iannace, S. (2011). Effect of supramolecular structures on thermoplastic zein-lignin bionanocomposites. Journal of Agricultural and Food Chemistry, 59, 10062e10070. Paschoalick, T. M., Garcia, F. T., Sobral, P. J. A., & Habitante, A. M. Q. B. (2003). Characterization of some functional properties of edible films based on muscle proteins of Nile Tilapia. Food Hydrocolloids, 17(4), 419e427. Payne, K. J., & Veis, A. (1988). Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies. Biopolymers, 27, 1749e1760. Pereda, M., Ponce, A. G., Marcovich, N. E., Ruseckaite, R. A., & Martucci, J. F. (2011). Chitosan-gelatin composites and bi-layer films with potential antimicrobial activity. Food Hydrocolloids, 25(5), 1372e1381.

149

Pouplin, M., Redl, A., & Gontard, N. (1999). Glass transition of wheat gluten plasticized with water, glycerol or sorbitol. Journal of Agricultural and Food Chemistry, 47, 538e543. Rampino, A., Borgogna, M., Blasi, P., Bellich, B., & Cesaro, A. (2013). Chitosan nanoparticles: preparation, size evolution and stability. International Journal of Pharmaceutics, 455(1e2), 219e228. Rivero, S., García, M. A., & Pinotti, A. (2010). Correlations between structural, barrier, thermal and mechanical properties of plasticized gelatin films. Innovative Food Science & Emerging Technologies, 11(2), 369e375. Rocha, M. D., Loiko, M. R., Tondo, E. C., & Prentice, C. (2014). Physical, mechanical and antimicrobial properties of argentine anchovy (Engraulis anchoita) protein films incorporated with organic acids. Food Hydrocolloids, 37, 213e220. Sallam, K. I. (2007). Antimicrobial and antioxidant effects of sodium acetate, sodium lactate, and sodium citrate in refrigerated sliced salmon. Food Control, 18(5), 566e575. Schrieber, R., & Gareis, H. (2007). Gelatine handbook. Weinhem: Wiley-VCH GmbH & Co. Shi, A. M., Wang, L. J., Li, D., & Adhikari, B. (2013). Characterization of starch films containing starch nanoparticles. Part 2: viscoelasticity and creep properties. Carbohydrate Polymers, 96(2), 602e610. Silva, M. A. d, Bierhalz, A. C. K., & Kieckbusch, T. G. (2009). Alginate and pectin composite films crosslinked with Ca2þ ions: effect of the plasticizer concentration. Carbohydrate Polymers, 77(4), 736e742. Staroszczyk, H., Pielichowska, J., Sztuka, K., Stangret, J., & Kołodziejska, I. (2012). Molecular and structural characteristics of cod gelatin films modified with EDC and TGase. Food Chemistry, 130(2), 335e343. €llstedt, M., & Hedenqvist, M. S. (2012). Antimicrobial compressionTüre, H., Ga moulded wheat gluten films containing potassium sorbate. Food Research International, 45(1), 109e115. Thakhiew, W., Devahastin, S., & Soponronnarit, S. (2010). Effects of drying methods and plasticizer concentration on some physical and mechanical properties of edible chitosan films. Journal of Food Engineering, 99(2), 216e224. Thomazine, M., Carvalho, R., & Sobarl, A. A. (2005). Physical properties of gelatin films plasticized by blends of glycerol and sorbitol. Journal of Food Science, 70, 172e176. Tronci, G., Doyle, A., Russell, S. J., & Wood, D. J. (2013). Triple-helical collagen hydrogels via covalent aromatic functionalisation with 1,3-phenylenediacetic acid. Journal of Materials Chemistry B, 1(40), 5478. Wang, L., An, X., Xin, Z., Zhao, L., & Hu, Q. (2007). Isolation and characterization of collagen from the skin of deep-sea redfish (Sebastes mentella). Journal of Food Science, 72(8), E450eE455. Wang, Z., Zhou, J., Wang, X.-X., Zhang, N., Sun, X.-X., & Ma, Z.-S. (2014). The effects of ultrasonic/microwave assisted treatment on the water vapor barrier properties of soybean protein isolate-based oleic acid/stearic acid blend edible films. Food Hydrocolloids, 35, 51e58. Xiao, Q., Lim, L.-T., & Tong, Q. (2012). Properties of pullulan-based blend films as affected by alginate content and relative humidity. Carbohydrate Polymers, 87(1), 227e234. Yakimets, I., Wellner, N., Smith, A. C., Wilson, R. H., Farhat, I., & Mitchell, J. (2005). Mechanical properties with respect to water content of gelatin films in glassy state. Polymer, 46(26), 12577e12585. Yesudhason, P., Lalitha, K., Gopal, T., & Ravishankar, C. (2014). Retention of shelf life and microbial quality of seer fish stored in modified atmosphere packaging and sodium acetate pretreatment. Food Packaging and Shelf Life, 1(2), 123e130. Yoshimura, K., Terashima, M., Hozan, D., Ebato, T., Nomura, Y., Ishii, Y., et al. (2008). Physical properties of shark gelatin compared with pig gelatin. Journal of Agricultural and Food Chemistry, 48(6), 2023e2027. Zaupa, A., Neffe, A. T., Pierce, B. F., Nochel, U., & Lendlein, A. (2011). Influence of tyrosine-derived moieties and drying conditions on the formation of helices in gelatin. Biomacromolecules, 12, 75e81.