- Email: [email protected]
Development and characterisation of a new biodegradable edible film made from kefiran, an exopolysaccharide obtained from kefir grains
Food Chemistry 127 (2011) 1496–1502
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Development and characterisation of a new biodegradable edible film made from kefiran, an exopolysaccharide obtained from kefir grains Mehran Ghasemlou a,⇑, Faramarz Khodaiyan a, Abdulrasoul Oromiehie b, Mohammad Saeid Yarmand a a Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, Campus of Agriculture and Natural Resources, University of Tehran, P.O. Box 4111, Karaj 31587-77871, Iran b Iran Polymer and Petrochemical Institute, Pazhoohesh Street, P.O. Box 14965/159, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 4 October 2010 Received in revised form 6 January 2011 Accepted 1 February 2011 Available online 25 February 2011 Keywords: Kefiran Edible film Plasticizer Exopolysaccharide
a b s t r a c t This study examined the feasibility of using kefiran, an exopolysaccharide obtained from kefir grains, as a new film-forming material. Kefiran-based films, with and without glycerol as plasticizer, were prepared by a casting and solvent-evaporation method. To study the impact of the incorporation of glycerol into the film matrix, physical, mechanical, and thermal properties of the films were investigated. As expected, the increase of glycerol concentration from 15% to 35% w/w increased extensibility but decreased tensile strength, implying higher mobility of polymer chains by the plasticizing effect of glycerol. Water vapour permeability of films was found to increase as the plasticizer content increased. Glass transition temperatures decreased as a result of plasticization as glycerol content increased. The properties of the films were related to their microstructure, which was observed by scanning electron microscopy. Thus, it was observed that plasticizer is a significant factor in the properties of these films and their food technology applications. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, the food and packaging industries have been joining their efforts to determine new ways to protect food from environmental conditions and mechanical stresses. The use of synthetic polymers and plastics for packaging has grown tremendously in the last century; however, this increase has created serious environmental problems due to the materials’ inability to biodegrade. Moreover, the insecurity of oil and petroleum resources – the raw materials from which such packaging is derived – is encouraging the food industry to explore the use of natural bio-based materials and polymers in packaging (Debeaufort, Quezada-Gallo, & Voilley, 1998). Edible, biodegradable films, by acting as barriers to control the transfer of moisture, oxygen, lipids, and flavours, can prevent quality deterioration and increase the shelf life of food products (Gontard, Guilbert, & Cuq, 1993). Several studies have reported the use of polysaccharides from different sources to prepare films and coatings with different properties, and have indicated that these carbohydrates are promising materials (Mali, Sakanaka, Yamashita, & Grossmann, 2005). Less attention has been paid to microbial exopolysaccharides, mainly due to their low production levels when compared to other polysaccharides, even though these materials can form gels and viscous solutions at low concentrations (Paul, Morin, & Monsan, 1986). ⇑ Corresponding author. Tel.: +98 912 598 7860; fax: +98 261 2248804. E-mail address: [email protected] (M. Ghasemlou). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.003
Kefiran, a microbial polysaccharide obtained from the flora of kefir grains, is finding increasing use in the food industry as a texturing and gelling agent. It is a water-soluble polysaccharide containing approximately equal amounts of glucose and galactose (Micheli, Uccelletti, Palleschi, & Crescenzi, 1999). Recent studies have shown that high yields of these exopolysaccharides can be easily isolated from the grains in deproteinized whey (Rimada & Abraham, 2001). Thus exopolysaccharides from kefir grains might be an affordable alternative to synthetic packaging in food applications. In addition, when compared with other polysaccharides, kefiran has several important advantages, such as antibacterial, antifungal, and antitumour properties (Maeda, Zhu, Omura, Suzuki, & Kitamura, 2004; Murofushi, Shiomi, & Aibara, 1983). The literature data and preliminary studies in our laboratory have shown that kefiran can produce films with good appearance and satisfactory mechanical properties: it appears to have excellent potential as a filmforming agent. However, to date there has been little information available on its film characteristics. In general, natural packaging films exhibit several disadvantages, such as a strong hydrophilic character and poor mechanical properties, when compared to synthetic packaging films. These drawbacks make it unsatisfactory for some applications such as packaging (Debeaufort et al., 1998). Many researchers have studied the effects of various plasticizers on film-based biopolymers as a way to overcome the films’ brittleness. Plasticizers reduce intermolecular forces and increase the
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
mobility of polymer chains, decreasing the glass transition temperature (Tg); unfortunately, they also decrease the film’s water vapour permeability (Gontard et al., 1993). Glycerol is one of the most widely used plasticizers in film-making techniques. It is a high-boiling-point plasticizer that is water-soluble, polar, and nonvolatile; these properties make glycerol a suitable plasticizer for use with a compatible water-soluble polymer (Cheng, Karim, & Seow, 2006). There is only one paper on the use of kefiran as an edible film (Piermaria, Pinotti, Garcia, & Abraham, 2009), and, to the best of our knowledge, there is no specific study on the effect of various concentrations of plasticizer on film properties. In the recent years, a major emphasis has been placed on the search for new microbial biopolymers with different compositions and properties, and several of them have been under investigation. In the current research, varying levels of glycerol were used in kefiran-based films. The aim of this work was to study some selected characteristics of glycerol plasticized kefiran films intended for use as edible or biodegradable films. These results are not available in the literature but are very important to evaluate possible applications of these films as packaging material. 2. Materials and methods 2.1. Starter culture Kefir grains, used as a starter culture in this study, were obtained from a household in Tehran, Iran. The grains were kept in skimmed milk at room temperature for short periods and the medium was exchanged daily for new culture to maintain the grains’ viability. After the culture was continued for seven subsequent days, the grains were considered active. 2.2. Isolation and purification of kefiran Exopolysaccharides in the kefir grains were extracted by the method of Piermaria et al. (2009). In brief, a weighed amount of kefir grains was treated in boiling water (1:100) for 1 h and stirred vigorously. The mixture was centrifuged (Sigma 3–16 k Frankfurt, Germany) at 10,000g for 15 min at 20 °C and an equal volume of chilled ethanol was added to precipitate the polysaccharide and kept at 20 °C overnight. The pellets were collected by centrifuging at 10,000g for 20 min at 4 °C. The precipitates were re-dissolved in hot distilled water and the precipitation method was repeated twice. The resulting solution was concentrated, yielding a crude polysaccharide. The samples were tested for the absence of other sugars and proteins by high-performance liquid chromatography and the phenol–sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), respectively. 2.3. Preparation of films Aqueous solutions of 1%, 2%, and 3% kefiran were prepared by weighing the amount of film-forming solution under constant stirring via the use of a magnetic stirrer for 15 min. Preliminary experiments had showed that filmogenic solutions containing 2% kefiran were easily removed from the plate. On the other hand, films formulated with 1% had low thickness values and were difficult to handle. Also, the films prepared without plasticizer were brittle, and cracked on the casting plates during drying. Thus, plasticizer was incorporated into the film-forming solutions to achieve more-flexible films. Glycerol (Sigma Chemical Co., St. Louis, MO, USA) was added as a plasticizer at various levels (15–35% w/w based on kefiran weight). Following the addition of plasticizers, stirring was continued for a
1497
further 15 min. The film solution was left for several minutes to naturally remove most of the air bubbles incorporated during stirring. Films were cast by pouring the mixture onto Teflon-coated plates resting on a levelled granite surface for approximately 18 h at room temperature and room relative humidity. Dried films were peeled off the casting surface and stored inside desiccators at 25 ± 1 °C until evaluation. Saturated magnesium nitrate (Merck, Darmstadt, Germany) solution was used to meet required relative humidity. 2.4. Determination of physical properties of films 2.4.1. Film thickness Thickness of the films was measured using a manual digital micrometre (Mitutoyo No. 293-766, Tokyo, Japan) to the nearest 0.001 mm. Measurements were made in at least ten random locations for each film, and an average value was calculated. The average value was used in calculations for tensile properties and WVP tests. 2.4.2. Moisture content The films’ moisture content (approximately 1 3 cm2) was determined by measuring the weight loss of films before and after drying in a laboratory oven (Blue M Electric Co., Blue Island, IL) at 103 ± 2 °C until constant weight was reached (dry sample weight). Three replications of each film treatment were used for calculating the moisture content. 2.4.3. Film solubility in water For this study, solubility in water was defined as the ratio of the water-soluble dry matter of film that is dissolved after immersion in distilled water (Gontard, Duchez, Cuq, & Guilbert, 1994). A circular film sample was cut from each film, dried at 103 ± 2 °C for 24 h in a laboratory oven, and weighed to determine the initial dry weight. The solubility in water of the different kefiran films was measured from immersion assays in 50 ml of distilled water with periodic stirring for six hours at 25 °C. After that period, the remaining pieces of films were taken out and dried at 103 ± 2 °C until constant weight (final dry weight). The percentage of the total soluble matter (% TSM) of the films was calculated using Eq.(1):
%TSM ¼ ½ðinitial dry weight final dry weightÞ=initial dry weight 100
ð1Þ
TSM tests for each type of film were carried out in three replicates. 2.4.4. Contact angle measurements The contact angle is defined as the angle between the baseline of the drop and the tangent line at the point of contact of the water droplet with the surface (Ojagh, Rezaei, Razavi, & Hosseini, 2010). Contact angle measurements were performed with water using a goniometer (Kruss G10, Germany). To perform the measurements of the contact angles, a syringe was filled with 5 ml of distilled water and a drop was placed on the film surface. For each film type, at least five measurements at different positions on the film surface were taken and the average was calculated. 2.5. Surface colour measurements Film colour was determined using a colourimeter (Minolta CR 300 Series, Minolta Camera Co., Ltd., Osaka, Japan). Film specimens were placed on a white standard plate (L⁄ = 93.49, a⁄ = 0.25 and b⁄ = 0.09) and the lightness (L) and chromaticity parameters a (red–green) and b (yellow–blue) were measured. L values range from 0 (black) to 100 (white); a values range from 80 (greenness) to 100 (redness); and b values range from 80 (blueness) to 70
1498
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
(yellowness). All measurements were performed in triplicates. Total colour difference (DE) and whiteness index (WI) were calculated using Eqs. (2) and (3) (Bolin & Huxsoll, 1991).
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi DE ¼ ðL LÞ2 þ ða aÞ2 þ ðb bÞ2
ð2Þ
where L⁄, a⁄, and b⁄ are the colour parameter values of the standard and L, a, and b are the colour parameter values of the sample.
WI ¼ 100
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ð100 LÞ2 þ a2 þ b
ð3Þ
2.6. Water vapour permeability The films’ water vapour permeability (WVP) was measured gravimetrically according to the standard method E96 (ASTM, 1995) and corrected for the stagnant air gap inside test cups according to the equations of Gennadios, Weller, and Gooding (1994). Special glass cups with wide rims were used to determine WVP. The cups, which contained approximately 50 g anhydrous calcium chloride desiccant (0% RH, assay cup) or nothing (control cup), were covered with films of varying plasticizer concentration. Films without pinholes or defects were cut circularly (0.00287 m2 film area) and sealed to the cup mouths using melted paraffin. Each cup was placed in a desiccator maintained at 75% RH with a sodium-chloride-saturated solution (Merck, Darmstadt, Germany). This difference in RH corresponds to a driving force of 1753.55 Pa, expressed as water vapour partial pressure. After the films were mounted, the weight gain of the whole assembly was recorded every 1 h during the first nine hours and finally after 24 h (with an accuracy of 0.0001 g). The cups were shaken horizontally after every weighing. The slope of the weight-versus-time plot (the lines’ regression coefficients were >0.998) was divided by the effective film area to obtain the water vapour transmission rate. This was multiplied by the thickness of the film and divided by the pressure difference between the inner outer surfaces to obtain the WVP (Eq. (4)).
WVP ¼
Dm X ADt Dp
ð4Þ
where Dm/Dt is the weight of moisture gain per unit of time (g/s), X is the average film thickness (mm), A is the area of the exposed film surface (m2), and Dp is the water vapour pressure difference between the two sides of the film (Pa). WVP was measured for three replicated samples for each type of film. 2.7. Determination of films’ mechanical properties The films’ mechanical properties, including tensile strength (MPa) and elongation at break (%), were determined at 25 °C using a Testometric Machine M350-10CT (Testometric Co., Ltd., Rochdale, Lancs., England) according to ASTM standard method D882 (ASTM, 2001). Films were cut in rectangular strips of 38 mm long and 5.79 mm wide. All film strips were equilibrated at 51% RH for 48 h in a desiccator using saturated magnesium nitrate solution. The films were fixed with an initial grip separation of 50 mm and stretched at a cross-head speed of 50 mm/min. A microcomputer was used to record the stress–strain curves. Tensile strength was calculated by dividing the maximum load on the film before failure by the cross-sectional area of the initial film specimen. Percentage elongation was defined as the percentage of a change in the length of the specimen when compared to the original length between the grips. At least five replicates of each film were tested.
2.8. Differential scanning calorimetry (DSC) The thermal properties of the films were carried out using DSC equipment (PL Polymer Laboratories, UK). A 10 mg film sample was cut as small pieces and placed into a sample pan of DSC equipment. An empty aluminum pan was used as reference. Samples were scanned at a heating rate of 10 °C/min between temperature ranges of 50 and 150 °C. Nitrogen was used as the purge gas at a flow rate of 20 ml/min. The glass transition temperatures (Tg) of the different films were determined from resulting thermograms as the midpoint temperature of a step-down shift in baseline, due to the discontinuity of the specific heat of the sample. The melting point (Tm) was calculated as the temperature where the peak of the endotherm occurs. The enthalpy (DHm) of the sol–gel transition was determined as the area over the endothermic peak. All these properties were determined in duplicates and the results were averaged. 2.9. Scanning electron microscopy Microstructural analysis of the surface and cross-sections of the dried films was conducted by scanning electron microscopy (Oxford Instruments INCA Penta FETX3). The films containing 25% glycerol were fractured in liquid nitrogen, mounted on aluminum stubs using a double-sided adhesive tape, and sputtered with a thin layer of gold using a BAL-TEC SCD 005 sputter coater (BALTEC AG, Balzers, Liechtenstein). All samples were examined using an accelerating voltage of 20.0 kV. Samples were photographed at an angle of 90° to the surface to allow observation of the films’ cross-section. 2.10. Statistical analysis Statistics on a completely randomized design were performed with the analysis of variance (ANOVA) procedure using SAS software (Version 9.1; Statistical Analysis System Institute Inc., Cary, NC, USA). Duncan’s multiple range tests were used to compare the difference among mean values of films’ properties at the level of 0.05. 3. Results and discussion 3.1. Physical properties of films Preliminary studies were carried out to determine the glycerol concentration range for the film formulation. The lowest effective glycerol concentration was 10% (w/w, film dry weight basis); below this concentration, the films tended to be brittle and difficult to handle, whereas films with more than 35% glycerol were flexible but sticky. This stickiness may have resulted from phase separation and diffusion of glycerol to the film surface. For a constant concentration of kefiran (2%), films prepared with different concentrations of glycerol (15%, 25%, and 35% by weight), had thickness values ranging from 0.058 to 0.067 mm (Table 1). These thickness values were higher than those reported by Piermaria et al. (2009), which may have been due to the differences in film-forming solution formulations and film-making procedures. The moisture content increased significantly from 17.95% to 37.04% as the plasticizer content increased (P < 0.05) (Table 1). Glycerol acts as a water-holding agent, with the higher number of water molecules in glycerol-plasticized films increasing plasticity (Gontard et al., 1993). The solubility of the films generally increased with increasing concentrations of glycerol. In glycerol-plasticized films, glycerol can diminish interactions between biopolymer molecules and
1499
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502 Table 1 Physical properties of kefiran films incorporated with various concentration of glycerol as plasticizer
A B
A,B
.
Glycerol conc.% (w/w) (based on kefiran content)
Thickness (mm)
Moisture content (% d.b.)
Solubility in water (%)
Water contact angle (degree)
Without plasticizer 15 25 35
0.058 ± 0.0041a 0.064 ± 0.0098a 0.062 ± 0.0070a 0.067 ± 0.0095a
17.95 ± 1.69d 23.59 ± 2.11c 32.47 ± 2.89b 37.04 ± 1.56a
21.28 ± 1.33c 25.30 ± 0.64b 27.91 ± 1.11a 28.88 ± 0.85a
106.41 ± 2.61a 103.06 ± 2.22a 97.15 ± 1.51b 95.44 ± 0.93b
Means within each column with same letters are not significantly different (P < 0.05). Data are means ± SD.
The L values generally increased as the concentration of glycerol increased; this increase was statistically significant (P < 0.05). The overall colour of the films was lightened with incorporation of glycerol. These results agreed with visual observation. Colour changes due to incorporation of plasticizer can be more fully described using other colour functions (Ghanbarzadeh, Almasi, & Entezami, 2010), such as DE, which indicates the degree of total colour difference from the standard colour plate, and WI, which indicates degree of whiteness. Incorporating glycerol revealed DE values that were significantly (P < 0.05) lower than that of unplasticized ones (increased clearness). In contrast, WI increased with increasing glycerol content.
increase solubility due to its hydrophilic nature, giving the polymer molecules a greater ability to attract water (Cuq, Gontard, Aymard, & Guilbert, 1997). 3.2. Water contact angle Surface-tension characterisation through contact angle measurements can be a good way to determine how hydrophilic a film is (Pearoval, Debeaufort, Desprea, & Voille, 2002). Table 1 shows the water contact angle measurements for the samples. There was significant difference among contact angles for films with different glycerol content (P < 0.05). It is accepted that water contact angle will increase with increasing surface hydrophobicity. The results in this study showed that the addition of plasticizers diminished the films’ water contact angle. Plasticization, therefore, resulted in decreasing hydrophobicity of the films. The higher hydrophilicity of the samples is attributable to the hygroscopicity (water-binding capacity) of the plasticizer. In comparison with other polysaccharide-based films, kefiran films have a higher contact angle with water, suggesting a higher surface hydrophobicity. This means that these films were less readily wetted.
3.4. Water vapour permeability (WVP) Table 3 shows the water vapour permeability (WVP) values of different kefiran films. The WVP was 4.95 1011 g s1 m1Pa s1 for the unplasticized sample (0% w/w glycerol); this increased to 5.55 and 5.88 1011 g s1 m1Pa s1 for the films containing 25% and 35% w/w glycerol, respectively. There was a significant difference between the WVP values of films made with different glycerol concentrations (P < 0.05). WVP values were in the range of those reported by other authors in films based on kefiran (Piermaria et al., 2009). An increase in glycerol concentration normally causes an increase in the WVP of hygroscopic or hydrophilic films. Glycerol is a relatively small hydrophilic molecule, which can be inserted between adjacent polymeric chains, decreasing intermolecular forces and increasing molecular mobility in the film matrix. The increased mobility results in greater free volume and segmental motions, which facilitates the migration of water vapour molecules through
3.3. Colour measurement Film colour is an important factor in a product’s acceptability to consumers. Table 2 shows the L, a, and b Hunter Lab colour values, total colour difference (DE), and whiteness index (WI) of films. The main differences in colour values among the kefiran films for different concentrations of glycerol were increased L values and decreased b values. However, no significant (P > 0.05) difference in values for a were detected.
Table 2 Hunter colour values (L, a, and b), total colour difference (DE) and whiteness index (WI) of kefiran films as a function of glycerol concentration
A B
A,B
Glycerol conc.% (w/w) (based on kefiran content)
L
a
b
DE
WI
Without plasticizer 15 25 35
77.87 ± 0.40b 79.68 ± 1.37b 82.82 ± 1.66a 84.47 ± 1.41a
0.66 ± 0.01a 0.65 ± 0.11a 0.61 ± 0.21a 0.60 ± 0.07a
2.52 ± 0.06c 2.51 ± 0.15c 2.76 ± 0.12b 3.09 ± 0.11a
15.84 ± 1.04a 14.05 ± 2.70ab 11.04 ± 2.73b 9.57 ± 1.03b
77.71 ± 0.40b 79.51 ± 1.35b 82.58 ± 1.61a 84.15 ± 1.39a
Means within each column with same letters are not significantly different (P < 0.05). Data are means ± SD.
Table 3 Effect of various concentrations of glycerol as plasticizer on the WVP and mechanical properties of kefiran films
A B
11
Glycerol conc.% (w/w) (based on kefiran content)
WVP (10
Without plasticizer 15 25 35
4.95 ± 0.13c 5.04 ± 0.18c 5.55 ± 0.21b 5.88 ± 0.10a
1 1
gm
s
pa)
A,B
.
Tensile strength (MPa)
Elongation at break (%)
11.18 ± 2.24a 8.85 ± 1.64ab 6.18 ± 2.72b 5.04 ± 2.10b
39.56 ± 11.13c 50.73 ± 10.60c 94.38 ± 3.25b 162.45 ± 6.09a
Means within each column with same letters are not significantly different (P < 0.05). Data are means ± SD.
.
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
the film (Rodriguez, Oses, Ziani, & Mate, 2006). Additionally, at a high glycerol concentration, glycerol could cluster with itself to open polymer structures, enhancing the permeability of a film to moisture (Yang & Paulson, 2000). The same behaviours were shown in research by Ghanbarzadeh et al. (2006), who found that increasing glycerol concentration, increased the WVP of zein films. Values have been reported for WVP values of a corn starch film, sodium casein film, a fish myofibrilar film, a high-density polyethylene film, a cellophane film, wheat gluten film, and a chitosan film of 8.68, 1.51, 6.4, 0.23, 8.4, 4.52, and 2.25 1011 g s1 m1Pa s1, respectively (Cuq et al., 1997; McHugh & Krochta, 1994; Ojagh et al., 2010; Smith, 1986). The WVP of the kefiran films was better than those of corn starch, cellophane, and fish myofibrilar films, but worse than those of the other films.
0% Endothermal heat flow
1500
15% 25%
35%
3.5. Mechanical properties Mechanical properties reflect films’ durability and ability to enhance the mechanical integrity of foods. Table 3 shows the effect of incorporating glycerol on the mechanical properties of kefiran films. Films without glycerol presented greater tensile strength (TS) and lower elongation at break (E). The presence of GLY in films caused significant differences in TS and E values (P < 0.05). Overall, the TS decreased as the concentration of glycerol increased. A tendency to be higher than E was observed as the concentration of glycerol increased (P < 0.05). This effect of incremental increases in plasticizer concentration on mechanical properties has been broadly discussed in the literature (Cuq et al., 1997; McHugh & Krochta, 1994). However, Lourdin, Coignard, Bizot, and Colonna (1997) have well discussed it. They stated that a small quantity of plasticizer could be easily inserted between polymer chains, producing a ‘‘cross-linker’’ effect that decreases the free volume of the polymer, causing the mechanical strength of the films to be decreased and the extensibility enhanced. The TS values of kefiran-based films were 5.04–11.18 MPa, which were lower than those of synthetic polymers such as LDPE, Low-density polyethylene, (9–17 MPa), polystyrene (35–55 MPa), and cellophane (114 MPa) (Smith, 1986). However, kefiran film plasticized with glycerol showed extremely high elongation values – higher, for example, than cellophane (20%) and polystyrene (1%) – but could not be stretched nearly as far as LDPE (500%). 3.6. Thermal properties DSC studies of the kefiran films containing different glycerol contents were performed to further understand the structure and interaction between polymer and plasticizer. Fig. 1 presents the DSC curves of these films. The glass transition temperature (Tg) is the temperature at which the material undergoes a structural transition from an amorphous solid state (glassy state) to a more viscous rubbery state (Ghanbarzadeh & Oromiehi, 2009). In all cases, only one glass transition (Tg) followed by an endothermic peak was observed. Table 4 shows the glass transition temperatures (Tg), melting temperatures (Tm), and enthalpies of fusion (DHm). All Tg values were in the range of 14 to 21 °C depending on the films’ glycerol concentrations, which are close to those of the most commonly used synthetic films (e.g. 18 °C for polypropylene, 25 °C for low-density polyethylene), but higher than the Tg of high-density polyethylene (125 °C) (Robertson, 1993) and pure glycerol liquid (93 °C) (Cherian, Gennadios, Weller, & Chinachoti, 1995). The low Tg values of kefiran films could be attributed to their inherent structural characteristics (high chain mobility) and to their relatively high hydrophilicity, which lets them absorb more water molecules than other films and thus markedly depresses the Tg due to the plasticizing effect of the absorbed water molecules
-100
-50
0
50
100
150
200
Temperature ( °C) Fig. 1. Representative examples of DSC curves of kefiran-based films without glycerol and plasticized with glycerol. The glycerol concentration (% w/w based on kefiran weight) is indicated for each curve.
Table 4 DSC data of kefiran-based film as a function of various glycerol concentrations
A,B
Glycerol conc.% (w/w) (based on kefiran content)
Tg (°C)
Tm (°C)
DHm (J/g)
Without plasticizer 15 25 35
14.88 ± 1.25a 18.93 ± 0.77b 20.68 ± 0.58c 21.77 ± 0.61c
73.29 ± 1.01a 71.20 ± 1.59ab 70.84 ± 1.24ab 68.36 ± 2.67b
73.14 ± 4.29d 122.72 ± 2.08c 167.50 ± 2.12b 174.85 ± 4.83a
.
A Means within each column with same letters are not significantly different (P < 0.05). B Data are means ± SD.
(Yang & Paulson, 2000). The DSC thermograms of the studied films exhibited sharp endothermic peaks at the range of 68–73 °C. These endothermic peaks have been attributed to the melting of kefiran during retrogradation. The temperature position of the melting peak decreased significantly from 73.2 to 68.3 °C as glycerol content increased from 0% to 35% w/w kefiran. The decrease of Tg and Tm with increased plasticizer content was expected, and had been observed in other studies (Ghanbarzadeh & Oromiehi, 2009; Gontard et al., 1993). Similar results were obtained by Arvanitoyannis, Psomiadou, Nakayama, Aiba, and Yamamoto (1997) with films of gelatin/starch blends plasticized with glycerol or sorbitol. Thermograms indicated partial miscibility of glycerol and kefiran at the molecular level for any given concentration ratio. They remained homogeneous throughout the heating cycle, as no phase separation (separated glass transition temperature or melting peaks) was observed. If blends of polymer and plasticizers are immiscible, the mixture will exhibit two Tg and Tm corresponding to the two pure phases (Ghanbarzadeh et al., 2010).
3.7. Film microstructure In an attempt to study microstructural changes in the films, scanning electron microscopy was used to depict the surface
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
1501
Fig. 2. Scanning electron micrographs of the surface (left) of kefiran-based films viewed at a magnification of 500, and cross-section (right) viewed at a magnification of 1000; (A) unplasticized, (B) plasticized with 25% w/w glycerol.
and section topography of all prepared kefiran films. Microscopic views indicated smooth and uniform surface morphology without obvious cracks, breaks, or openings on the surfaces after the incorporation of glycerol as a plasticizer. Fig. 2 shows scanning electron micrographs of the outer surface (left) and crosssection (right) for both the control and glycerol-containing films. Scanning electron microscopy observations of films with different glycerol concentrations did not present any obvious differences in structure. 4. Conclusions On the basis of the results obtained in this study, kefiran could be used as a new film-forming material. The physical, mechanical, and thermal properties, which are important in food packaging applications, were examined as a function of glycerol concentration. Generally, the TS of the films decreased and % E increased with increased concentrations of glycerol. Based on DSC thermograms, the glass transition temperature (Tg) of the films decreased with the glycerol content. Kefiran film exhibited a potential application as an edible food film and coating. The properties of kefiran-based film can be further improved for food-packaging applications. The methods investigated and developed in this study are expected to be very useful for future research in this area. Acknowledgements The authors wish to express their gratitude to Iran Polymers and Petrochemical Institute and the University of Tehran for supporting the facilities for this research work, and for technical assistance.
References 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), 593–604. ASTM (1995). Standard test methods for water vapor transmission of material, E 96–95. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Material. 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. Bolin, H. R., & Huxsoll, C. C. (1991). Control of minimally processed carrot (Dascus carota) surface discoloration caused by abrasion peeling. Journal of Food Science, 56, 416–418. Cheng, L. H., Karim, A. A., & Seow, C. C. (2006). Effects of water–glycerol and water– sorbitol interactions on the physical properties of konjac glucomannan films. Journal of Food Science, 71(2), 62–67. Cherian, G., Gennadios, A., Weller, C., & Chinachoti, P. (1995). Thermomechanical behavior of wheat gluten films: Effect of sucrose, glycerin and sorbitol. Cereal Chemistry, 72(1), 1–6. Cuq, B., Gontard, N., Aymard, C., & Guilbert, S. (1997). Relative humidity and temperature effects on mechanical and water vapor barrier properties of myofibrillar protein-based films. Polymer Gels and Networks, 5(1), 1–15. Debeaufort, F., Quezada-Gallo, J. A., & Voilley, A. (1998). Edible films and coatings: Tomorrow’s packagings: A review. Critical Reviews in Food Science and Nutrition, 38(4), 299–313. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 38(3), 350–352. Gennadios, A., Weller, C. L., & Gooding, C. H. (1994). Measurement errors in water vapor permeability of highly permeable, hydrophilic edible films. Journal of Food Engineering, 21, 395–409. Ghanbarzadeh, B., & Oromiehi, A. R. (2009). Thermal and mechanical behavior of laminated protein films. Journal of Food Engineering, 90, 517–524. Ghanbarzadeh, B., Almasi, H., & Entezami, A. A. (2010). Physical properties of edible modified starch/carboxymethyl cellulose films. Innovative Food Science and Emerging Technologies. doi:10.1016/j.ifset.2010.06.001. Ghanbarzadeh, B., Musavi, M., Oromiehie, A. R., Rezayi, K., Razmi, E., & Milani, J. (2006). Investigation of water vapor permeability, hydrophobicity and morphology of zein films plasticized by polyols. Iranian Polymer Journal, 15(9), 691–700.
1502
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
Gontard, N., Duchez, C., Cuq, B., & Guilbert, S. (1994). Edible composite films of wheat gluten and lipids: Water vapour permeability and other physical properties. Food Science and Technology, 29, 39–50. Gontard, N., Guilbert, S., & Cuq, J.-L. (1993). Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58(1), 206–211. Lourdin, D., Coignard, L., Bizot, H., & Colonna, P. (1997). Influence of equilibrium relative humidity and plasticizer concentration on the water content and glass transition of starch materials. Polymer, 38, 5401–5406. Maeda, H., Zhu, X., Omura, K., Suzuki, S., & Kitamura, S. (2004). Effects of an exopolysaccharide (kefiran) on lipids, blood pressure, blood glucose, and constipation. Biofactors, 22, 197–200. Mali, S., Sakanaka, L. S., Yamashita, F., & Grossmann, M. V. E. (2005). Water sorption and mechanical properties of cassava starch films and their relation to plasticizing effect. Carbohydrate Polymers, 60, 283–289. McHugh, T. H., & Krochta, J. M. (1994). Water vapor permeability properties of edible whey protein–lipid. Journal of American Oil Chemistry Society, 71(3), 307–402. Micheli, L., Uccelletti, D., Palleschi, C., & Crescenzi, V. (1999). Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran. Applied Microbiology and Biotechnology, 53, 69–74. Murofushi, M., Shiomi, M., & Aibara, K. (1983). Effect of orally administered polysaccharide from kefir grain on delayed-type hypersensitivity and tumor growth in mice. Japanese Journal of Medical Science and Biology, 36(1), 49–53.
Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122, 161–166. Paul, F., Morin, A., & Monsan, P. (1986). Microbial polysaccharides with actual potential industrial applications. Biotechnology Advances, 4(2), 245–259. Pearoval, C., Debeaufort, F., Desprea, D., & Voille, A. (2002). Edible arabinoxylanbased films: Effects of lipid type on water vapour permeability, film structure, and other physical characteristics. Journal of Agricultural and Food Chemistry, 50, 3977–3983. Piermaria, J. A., Pinotti, A., Garcia, M. A., & Abraham, A. G. (2009). Films based on kefiran, an exopolysaccharide obtained from kefir grain: Development and characterization. Food Hydrocolloids, 23, 684–690. Rimada, P. S., & Abraham, A. G. (2001). Polysaccharide production by kefir grains during whey fermentation. Journal of Dairy Research, 68, 653–661. Robertson, G. L. (1993). Structure and related properties of plastic polymers. In Food packaging: principles and practice. New York: Marcel Dekker Inc (chap. 2). Rodriguez, M., Oses, J., Ziani, K., & Mate, J. I. (2006). Combined effect of plasticizers and surfactants on the physical properties of starch based edible film. Food Research International, 39, 840–846. Smith, S. A. (1986). Polyethylene, lowdensity. In M. Bakker (Ed.), The Wiley encyclopedia of packaging technology (pp. 514–523). New York: John Wiley and Sons. Yang, L., & Paulson, A. T. (2000). Mechanical and water vapor barrier properties of edible gellan films. Food Research International, 33, 563–570.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Development and characterisation of a new biodegradable edible film made from kefiran, an exopolysaccharide obtained from kefir grains Mehran Ghasemlou a,⇑, Faramarz Khodaiyan a, Abdulrasoul Oromiehie b, Mohammad Saeid Yarmand a a Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, Campus of Agriculture and Natural Resources, University of Tehran, P.O. Box 4111, Karaj 31587-77871, Iran b Iran Polymer and Petrochemical Institute, Pazhoohesh Street, P.O. Box 14965/159, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 4 October 2010 Received in revised form 6 January 2011 Accepted 1 February 2011 Available online 25 February 2011 Keywords: Kefiran Edible film Plasticizer Exopolysaccharide
a b s t r a c t This study examined the feasibility of using kefiran, an exopolysaccharide obtained from kefir grains, as a new film-forming material. Kefiran-based films, with and without glycerol as plasticizer, were prepared by a casting and solvent-evaporation method. To study the impact of the incorporation of glycerol into the film matrix, physical, mechanical, and thermal properties of the films were investigated. As expected, the increase of glycerol concentration from 15% to 35% w/w increased extensibility but decreased tensile strength, implying higher mobility of polymer chains by the plasticizing effect of glycerol. Water vapour permeability of films was found to increase as the plasticizer content increased. Glass transition temperatures decreased as a result of plasticization as glycerol content increased. The properties of the films were related to their microstructure, which was observed by scanning electron microscopy. Thus, it was observed that plasticizer is a significant factor in the properties of these films and their food technology applications. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, the food and packaging industries have been joining their efforts to determine new ways to protect food from environmental conditions and mechanical stresses. The use of synthetic polymers and plastics for packaging has grown tremendously in the last century; however, this increase has created serious environmental problems due to the materials’ inability to biodegrade. Moreover, the insecurity of oil and petroleum resources – the raw materials from which such packaging is derived – is encouraging the food industry to explore the use of natural bio-based materials and polymers in packaging (Debeaufort, Quezada-Gallo, & Voilley, 1998). Edible, biodegradable films, by acting as barriers to control the transfer of moisture, oxygen, lipids, and flavours, can prevent quality deterioration and increase the shelf life of food products (Gontard, Guilbert, & Cuq, 1993). Several studies have reported the use of polysaccharides from different sources to prepare films and coatings with different properties, and have indicated that these carbohydrates are promising materials (Mali, Sakanaka, Yamashita, & Grossmann, 2005). Less attention has been paid to microbial exopolysaccharides, mainly due to their low production levels when compared to other polysaccharides, even though these materials can form gels and viscous solutions at low concentrations (Paul, Morin, & Monsan, 1986). ⇑ Corresponding author. Tel.: +98 912 598 7860; fax: +98 261 2248804. E-mail address: [email protected] (M. Ghasemlou). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.003
Kefiran, a microbial polysaccharide obtained from the flora of kefir grains, is finding increasing use in the food industry as a texturing and gelling agent. It is a water-soluble polysaccharide containing approximately equal amounts of glucose and galactose (Micheli, Uccelletti, Palleschi, & Crescenzi, 1999). Recent studies have shown that high yields of these exopolysaccharides can be easily isolated from the grains in deproteinized whey (Rimada & Abraham, 2001). Thus exopolysaccharides from kefir grains might be an affordable alternative to synthetic packaging in food applications. In addition, when compared with other polysaccharides, kefiran has several important advantages, such as antibacterial, antifungal, and antitumour properties (Maeda, Zhu, Omura, Suzuki, & Kitamura, 2004; Murofushi, Shiomi, & Aibara, 1983). The literature data and preliminary studies in our laboratory have shown that kefiran can produce films with good appearance and satisfactory mechanical properties: it appears to have excellent potential as a filmforming agent. However, to date there has been little information available on its film characteristics. In general, natural packaging films exhibit several disadvantages, such as a strong hydrophilic character and poor mechanical properties, when compared to synthetic packaging films. These drawbacks make it unsatisfactory for some applications such as packaging (Debeaufort et al., 1998). Many researchers have studied the effects of various plasticizers on film-based biopolymers as a way to overcome the films’ brittleness. Plasticizers reduce intermolecular forces and increase the
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
mobility of polymer chains, decreasing the glass transition temperature (Tg); unfortunately, they also decrease the film’s water vapour permeability (Gontard et al., 1993). Glycerol is one of the most widely used plasticizers in film-making techniques. It is a high-boiling-point plasticizer that is water-soluble, polar, and nonvolatile; these properties make glycerol a suitable plasticizer for use with a compatible water-soluble polymer (Cheng, Karim, & Seow, 2006). There is only one paper on the use of kefiran as an edible film (Piermaria, Pinotti, Garcia, & Abraham, 2009), and, to the best of our knowledge, there is no specific study on the effect of various concentrations of plasticizer on film properties. In the recent years, a major emphasis has been placed on the search for new microbial biopolymers with different compositions and properties, and several of them have been under investigation. In the current research, varying levels of glycerol were used in kefiran-based films. The aim of this work was to study some selected characteristics of glycerol plasticized kefiran films intended for use as edible or biodegradable films. These results are not available in the literature but are very important to evaluate possible applications of these films as packaging material. 2. Materials and methods 2.1. Starter culture Kefir grains, used as a starter culture in this study, were obtained from a household in Tehran, Iran. The grains were kept in skimmed milk at room temperature for short periods and the medium was exchanged daily for new culture to maintain the grains’ viability. After the culture was continued for seven subsequent days, the grains were considered active. 2.2. Isolation and purification of kefiran Exopolysaccharides in the kefir grains were extracted by the method of Piermaria et al. (2009). In brief, a weighed amount of kefir grains was treated in boiling water (1:100) for 1 h and stirred vigorously. The mixture was centrifuged (Sigma 3–16 k Frankfurt, Germany) at 10,000g for 15 min at 20 °C and an equal volume of chilled ethanol was added to precipitate the polysaccharide and kept at 20 °C overnight. The pellets were collected by centrifuging at 10,000g for 20 min at 4 °C. The precipitates were re-dissolved in hot distilled water and the precipitation method was repeated twice. The resulting solution was concentrated, yielding a crude polysaccharide. The samples were tested for the absence of other sugars and proteins by high-performance liquid chromatography and the phenol–sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), respectively. 2.3. Preparation of films Aqueous solutions of 1%, 2%, and 3% kefiran were prepared by weighing the amount of film-forming solution under constant stirring via the use of a magnetic stirrer for 15 min. Preliminary experiments had showed that filmogenic solutions containing 2% kefiran were easily removed from the plate. On the other hand, films formulated with 1% had low thickness values and were difficult to handle. Also, the films prepared without plasticizer were brittle, and cracked on the casting plates during drying. Thus, plasticizer was incorporated into the film-forming solutions to achieve more-flexible films. Glycerol (Sigma Chemical Co., St. Louis, MO, USA) was added as a plasticizer at various levels (15–35% w/w based on kefiran weight). Following the addition of plasticizers, stirring was continued for a
1497
further 15 min. The film solution was left for several minutes to naturally remove most of the air bubbles incorporated during stirring. Films were cast by pouring the mixture onto Teflon-coated plates resting on a levelled granite surface for approximately 18 h at room temperature and room relative humidity. Dried films were peeled off the casting surface and stored inside desiccators at 25 ± 1 °C until evaluation. Saturated magnesium nitrate (Merck, Darmstadt, Germany) solution was used to meet required relative humidity. 2.4. Determination of physical properties of films 2.4.1. Film thickness Thickness of the films was measured using a manual digital micrometre (Mitutoyo No. 293-766, Tokyo, Japan) to the nearest 0.001 mm. Measurements were made in at least ten random locations for each film, and an average value was calculated. The average value was used in calculations for tensile properties and WVP tests. 2.4.2. Moisture content The films’ moisture content (approximately 1 3 cm2) was determined by measuring the weight loss of films before and after drying in a laboratory oven (Blue M Electric Co., Blue Island, IL) at 103 ± 2 °C until constant weight was reached (dry sample weight). Three replications of each film treatment were used for calculating the moisture content. 2.4.3. Film solubility in water For this study, solubility in water was defined as the ratio of the water-soluble dry matter of film that is dissolved after immersion in distilled water (Gontard, Duchez, Cuq, & Guilbert, 1994). A circular film sample was cut from each film, dried at 103 ± 2 °C for 24 h in a laboratory oven, and weighed to determine the initial dry weight. The solubility in water of the different kefiran films was measured from immersion assays in 50 ml of distilled water with periodic stirring for six hours at 25 °C. After that period, the remaining pieces of films were taken out and dried at 103 ± 2 °C until constant weight (final dry weight). The percentage of the total soluble matter (% TSM) of the films was calculated using Eq.(1):
%TSM ¼ ½ðinitial dry weight final dry weightÞ=initial dry weight 100
ð1Þ
TSM tests for each type of film were carried out in three replicates. 2.4.4. Contact angle measurements The contact angle is defined as the angle between the baseline of the drop and the tangent line at the point of contact of the water droplet with the surface (Ojagh, Rezaei, Razavi, & Hosseini, 2010). Contact angle measurements were performed with water using a goniometer (Kruss G10, Germany). To perform the measurements of the contact angles, a syringe was filled with 5 ml of distilled water and a drop was placed on the film surface. For each film type, at least five measurements at different positions on the film surface were taken and the average was calculated. 2.5. Surface colour measurements Film colour was determined using a colourimeter (Minolta CR 300 Series, Minolta Camera Co., Ltd., Osaka, Japan). Film specimens were placed on a white standard plate (L⁄ = 93.49, a⁄ = 0.25 and b⁄ = 0.09) and the lightness (L) and chromaticity parameters a (red–green) and b (yellow–blue) were measured. L values range from 0 (black) to 100 (white); a values range from 80 (greenness) to 100 (redness); and b values range from 80 (blueness) to 70
1498
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
(yellowness). All measurements were performed in triplicates. Total colour difference (DE) and whiteness index (WI) were calculated using Eqs. (2) and (3) (Bolin & Huxsoll, 1991).
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi DE ¼ ðL LÞ2 þ ða aÞ2 þ ðb bÞ2
ð2Þ
where L⁄, a⁄, and b⁄ are the colour parameter values of the standard and L, a, and b are the colour parameter values of the sample.
WI ¼ 100
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ð100 LÞ2 þ a2 þ b
ð3Þ
2.6. Water vapour permeability The films’ water vapour permeability (WVP) was measured gravimetrically according to the standard method E96 (ASTM, 1995) and corrected for the stagnant air gap inside test cups according to the equations of Gennadios, Weller, and Gooding (1994). Special glass cups with wide rims were used to determine WVP. The cups, which contained approximately 50 g anhydrous calcium chloride desiccant (0% RH, assay cup) or nothing (control cup), were covered with films of varying plasticizer concentration. Films without pinholes or defects were cut circularly (0.00287 m2 film area) and sealed to the cup mouths using melted paraffin. Each cup was placed in a desiccator maintained at 75% RH with a sodium-chloride-saturated solution (Merck, Darmstadt, Germany). This difference in RH corresponds to a driving force of 1753.55 Pa, expressed as water vapour partial pressure. After the films were mounted, the weight gain of the whole assembly was recorded every 1 h during the first nine hours and finally after 24 h (with an accuracy of 0.0001 g). The cups were shaken horizontally after every weighing. The slope of the weight-versus-time plot (the lines’ regression coefficients were >0.998) was divided by the effective film area to obtain the water vapour transmission rate. This was multiplied by the thickness of the film and divided by the pressure difference between the inner outer surfaces to obtain the WVP (Eq. (4)).
WVP ¼
Dm X ADt Dp
ð4Þ
where Dm/Dt is the weight of moisture gain per unit of time (g/s), X is the average film thickness (mm), A is the area of the exposed film surface (m2), and Dp is the water vapour pressure difference between the two sides of the film (Pa). WVP was measured for three replicated samples for each type of film. 2.7. Determination of films’ mechanical properties The films’ mechanical properties, including tensile strength (MPa) and elongation at break (%), were determined at 25 °C using a Testometric Machine M350-10CT (Testometric Co., Ltd., Rochdale, Lancs., England) according to ASTM standard method D882 (ASTM, 2001). Films were cut in rectangular strips of 38 mm long and 5.79 mm wide. All film strips were equilibrated at 51% RH for 48 h in a desiccator using saturated magnesium nitrate solution. The films were fixed with an initial grip separation of 50 mm and stretched at a cross-head speed of 50 mm/min. A microcomputer was used to record the stress–strain curves. Tensile strength was calculated by dividing the maximum load on the film before failure by the cross-sectional area of the initial film specimen. Percentage elongation was defined as the percentage of a change in the length of the specimen when compared to the original length between the grips. At least five replicates of each film were tested.
2.8. Differential scanning calorimetry (DSC) The thermal properties of the films were carried out using DSC equipment (PL Polymer Laboratories, UK). A 10 mg film sample was cut as small pieces and placed into a sample pan of DSC equipment. An empty aluminum pan was used as reference. Samples were scanned at a heating rate of 10 °C/min between temperature ranges of 50 and 150 °C. Nitrogen was used as the purge gas at a flow rate of 20 ml/min. The glass transition temperatures (Tg) of the different films were determined from resulting thermograms as the midpoint temperature of a step-down shift in baseline, due to the discontinuity of the specific heat of the sample. The melting point (Tm) was calculated as the temperature where the peak of the endotherm occurs. The enthalpy (DHm) of the sol–gel transition was determined as the area over the endothermic peak. All these properties were determined in duplicates and the results were averaged. 2.9. Scanning electron microscopy Microstructural analysis of the surface and cross-sections of the dried films was conducted by scanning electron microscopy (Oxford Instruments INCA Penta FETX3). The films containing 25% glycerol were fractured in liquid nitrogen, mounted on aluminum stubs using a double-sided adhesive tape, and sputtered with a thin layer of gold using a BAL-TEC SCD 005 sputter coater (BALTEC AG, Balzers, Liechtenstein). All samples were examined using an accelerating voltage of 20.0 kV. Samples were photographed at an angle of 90° to the surface to allow observation of the films’ cross-section. 2.10. Statistical analysis Statistics on a completely randomized design were performed with the analysis of variance (ANOVA) procedure using SAS software (Version 9.1; Statistical Analysis System Institute Inc., Cary, NC, USA). Duncan’s multiple range tests were used to compare the difference among mean values of films’ properties at the level of 0.05. 3. Results and discussion 3.1. Physical properties of films Preliminary studies were carried out to determine the glycerol concentration range for the film formulation. The lowest effective glycerol concentration was 10% (w/w, film dry weight basis); below this concentration, the films tended to be brittle and difficult to handle, whereas films with more than 35% glycerol were flexible but sticky. This stickiness may have resulted from phase separation and diffusion of glycerol to the film surface. For a constant concentration of kefiran (2%), films prepared with different concentrations of glycerol (15%, 25%, and 35% by weight), had thickness values ranging from 0.058 to 0.067 mm (Table 1). These thickness values were higher than those reported by Piermaria et al. (2009), which may have been due to the differences in film-forming solution formulations and film-making procedures. The moisture content increased significantly from 17.95% to 37.04% as the plasticizer content increased (P < 0.05) (Table 1). Glycerol acts as a water-holding agent, with the higher number of water molecules in glycerol-plasticized films increasing plasticity (Gontard et al., 1993). The solubility of the films generally increased with increasing concentrations of glycerol. In glycerol-plasticized films, glycerol can diminish interactions between biopolymer molecules and
1499
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502 Table 1 Physical properties of kefiran films incorporated with various concentration of glycerol as plasticizer
A B
A,B
.
Glycerol conc.% (w/w) (based on kefiran content)
Thickness (mm)
Moisture content (% d.b.)
Solubility in water (%)
Water contact angle (degree)
Without plasticizer 15 25 35
0.058 ± 0.0041a 0.064 ± 0.0098a 0.062 ± 0.0070a 0.067 ± 0.0095a
17.95 ± 1.69d 23.59 ± 2.11c 32.47 ± 2.89b 37.04 ± 1.56a
21.28 ± 1.33c 25.30 ± 0.64b 27.91 ± 1.11a 28.88 ± 0.85a
106.41 ± 2.61a 103.06 ± 2.22a 97.15 ± 1.51b 95.44 ± 0.93b
Means within each column with same letters are not significantly different (P < 0.05). Data are means ± SD.
The L values generally increased as the concentration of glycerol increased; this increase was statistically significant (P < 0.05). The overall colour of the films was lightened with incorporation of glycerol. These results agreed with visual observation. Colour changes due to incorporation of plasticizer can be more fully described using other colour functions (Ghanbarzadeh, Almasi, & Entezami, 2010), such as DE, which indicates the degree of total colour difference from the standard colour plate, and WI, which indicates degree of whiteness. Incorporating glycerol revealed DE values that were significantly (P < 0.05) lower than that of unplasticized ones (increased clearness). In contrast, WI increased with increasing glycerol content.
increase solubility due to its hydrophilic nature, giving the polymer molecules a greater ability to attract water (Cuq, Gontard, Aymard, & Guilbert, 1997). 3.2. Water contact angle Surface-tension characterisation through contact angle measurements can be a good way to determine how hydrophilic a film is (Pearoval, Debeaufort, Desprea, & Voille, 2002). Table 1 shows the water contact angle measurements for the samples. There was significant difference among contact angles for films with different glycerol content (P < 0.05). It is accepted that water contact angle will increase with increasing surface hydrophobicity. The results in this study showed that the addition of plasticizers diminished the films’ water contact angle. Plasticization, therefore, resulted in decreasing hydrophobicity of the films. The higher hydrophilicity of the samples is attributable to the hygroscopicity (water-binding capacity) of the plasticizer. In comparison with other polysaccharide-based films, kefiran films have a higher contact angle with water, suggesting a higher surface hydrophobicity. This means that these films were less readily wetted.
3.4. Water vapour permeability (WVP) Table 3 shows the water vapour permeability (WVP) values of different kefiran films. The WVP was 4.95 1011 g s1 m1Pa s1 for the unplasticized sample (0% w/w glycerol); this increased to 5.55 and 5.88 1011 g s1 m1Pa s1 for the films containing 25% and 35% w/w glycerol, respectively. There was a significant difference between the WVP values of films made with different glycerol concentrations (P < 0.05). WVP values were in the range of those reported by other authors in films based on kefiran (Piermaria et al., 2009). An increase in glycerol concentration normally causes an increase in the WVP of hygroscopic or hydrophilic films. Glycerol is a relatively small hydrophilic molecule, which can be inserted between adjacent polymeric chains, decreasing intermolecular forces and increasing molecular mobility in the film matrix. The increased mobility results in greater free volume and segmental motions, which facilitates the migration of water vapour molecules through
3.3. Colour measurement Film colour is an important factor in a product’s acceptability to consumers. Table 2 shows the L, a, and b Hunter Lab colour values, total colour difference (DE), and whiteness index (WI) of films. The main differences in colour values among the kefiran films for different concentrations of glycerol were increased L values and decreased b values. However, no significant (P > 0.05) difference in values for a were detected.
Table 2 Hunter colour values (L, a, and b), total colour difference (DE) and whiteness index (WI) of kefiran films as a function of glycerol concentration
A B
A,B
Glycerol conc.% (w/w) (based on kefiran content)
L
a
b
DE
WI
Without plasticizer 15 25 35
77.87 ± 0.40b 79.68 ± 1.37b 82.82 ± 1.66a 84.47 ± 1.41a
0.66 ± 0.01a 0.65 ± 0.11a 0.61 ± 0.21a 0.60 ± 0.07a
2.52 ± 0.06c 2.51 ± 0.15c 2.76 ± 0.12b 3.09 ± 0.11a
15.84 ± 1.04a 14.05 ± 2.70ab 11.04 ± 2.73b 9.57 ± 1.03b
77.71 ± 0.40b 79.51 ± 1.35b 82.58 ± 1.61a 84.15 ± 1.39a
Means within each column with same letters are not significantly different (P < 0.05). Data are means ± SD.
Table 3 Effect of various concentrations of glycerol as plasticizer on the WVP and mechanical properties of kefiran films
A B
11
Glycerol conc.% (w/w) (based on kefiran content)
WVP (10
Without plasticizer 15 25 35
4.95 ± 0.13c 5.04 ± 0.18c 5.55 ± 0.21b 5.88 ± 0.10a
1 1
gm
s
pa)
A,B
.
Tensile strength (MPa)
Elongation at break (%)
11.18 ± 2.24a 8.85 ± 1.64ab 6.18 ± 2.72b 5.04 ± 2.10b
39.56 ± 11.13c 50.73 ± 10.60c 94.38 ± 3.25b 162.45 ± 6.09a
Means within each column with same letters are not significantly different (P < 0.05). Data are means ± SD.
.
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
the film (Rodriguez, Oses, Ziani, & Mate, 2006). Additionally, at a high glycerol concentration, glycerol could cluster with itself to open polymer structures, enhancing the permeability of a film to moisture (Yang & Paulson, 2000). The same behaviours were shown in research by Ghanbarzadeh et al. (2006), who found that increasing glycerol concentration, increased the WVP of zein films. Values have been reported for WVP values of a corn starch film, sodium casein film, a fish myofibrilar film, a high-density polyethylene film, a cellophane film, wheat gluten film, and a chitosan film of 8.68, 1.51, 6.4, 0.23, 8.4, 4.52, and 2.25 1011 g s1 m1Pa s1, respectively (Cuq et al., 1997; McHugh & Krochta, 1994; Ojagh et al., 2010; Smith, 1986). The WVP of the kefiran films was better than those of corn starch, cellophane, and fish myofibrilar films, but worse than those of the other films.
0% Endothermal heat flow
1500
15% 25%
35%
3.5. Mechanical properties Mechanical properties reflect films’ durability and ability to enhance the mechanical integrity of foods. Table 3 shows the effect of incorporating glycerol on the mechanical properties of kefiran films. Films without glycerol presented greater tensile strength (TS) and lower elongation at break (E). The presence of GLY in films caused significant differences in TS and E values (P < 0.05). Overall, the TS decreased as the concentration of glycerol increased. A tendency to be higher than E was observed as the concentration of glycerol increased (P < 0.05). This effect of incremental increases in plasticizer concentration on mechanical properties has been broadly discussed in the literature (Cuq et al., 1997; McHugh & Krochta, 1994). However, Lourdin, Coignard, Bizot, and Colonna (1997) have well discussed it. They stated that a small quantity of plasticizer could be easily inserted between polymer chains, producing a ‘‘cross-linker’’ effect that decreases the free volume of the polymer, causing the mechanical strength of the films to be decreased and the extensibility enhanced. The TS values of kefiran-based films were 5.04–11.18 MPa, which were lower than those of synthetic polymers such as LDPE, Low-density polyethylene, (9–17 MPa), polystyrene (35–55 MPa), and cellophane (114 MPa) (Smith, 1986). However, kefiran film plasticized with glycerol showed extremely high elongation values – higher, for example, than cellophane (20%) and polystyrene (1%) – but could not be stretched nearly as far as LDPE (500%). 3.6. Thermal properties DSC studies of the kefiran films containing different glycerol contents were performed to further understand the structure and interaction between polymer and plasticizer. Fig. 1 presents the DSC curves of these films. The glass transition temperature (Tg) is the temperature at which the material undergoes a structural transition from an amorphous solid state (glassy state) to a more viscous rubbery state (Ghanbarzadeh & Oromiehi, 2009). In all cases, only one glass transition (Tg) followed by an endothermic peak was observed. Table 4 shows the glass transition temperatures (Tg), melting temperatures (Tm), and enthalpies of fusion (DHm). All Tg values were in the range of 14 to 21 °C depending on the films’ glycerol concentrations, which are close to those of the most commonly used synthetic films (e.g. 18 °C for polypropylene, 25 °C for low-density polyethylene), but higher than the Tg of high-density polyethylene (125 °C) (Robertson, 1993) and pure glycerol liquid (93 °C) (Cherian, Gennadios, Weller, & Chinachoti, 1995). The low Tg values of kefiran films could be attributed to their inherent structural characteristics (high chain mobility) and to their relatively high hydrophilicity, which lets them absorb more water molecules than other films and thus markedly depresses the Tg due to the plasticizing effect of the absorbed water molecules
-100
-50
0
50
100
150
200
Temperature ( °C) Fig. 1. Representative examples of DSC curves of kefiran-based films without glycerol and plasticized with glycerol. The glycerol concentration (% w/w based on kefiran weight) is indicated for each curve.
Table 4 DSC data of kefiran-based film as a function of various glycerol concentrations
A,B
Glycerol conc.% (w/w) (based on kefiran content)
Tg (°C)
Tm (°C)
DHm (J/g)
Without plasticizer 15 25 35
14.88 ± 1.25a 18.93 ± 0.77b 20.68 ± 0.58c 21.77 ± 0.61c
73.29 ± 1.01a 71.20 ± 1.59ab 70.84 ± 1.24ab 68.36 ± 2.67b
73.14 ± 4.29d 122.72 ± 2.08c 167.50 ± 2.12b 174.85 ± 4.83a
.
A Means within each column with same letters are not significantly different (P < 0.05). B Data are means ± SD.
(Yang & Paulson, 2000). The DSC thermograms of the studied films exhibited sharp endothermic peaks at the range of 68–73 °C. These endothermic peaks have been attributed to the melting of kefiran during retrogradation. The temperature position of the melting peak decreased significantly from 73.2 to 68.3 °C as glycerol content increased from 0% to 35% w/w kefiran. The decrease of Tg and Tm with increased plasticizer content was expected, and had been observed in other studies (Ghanbarzadeh & Oromiehi, 2009; Gontard et al., 1993). Similar results were obtained by Arvanitoyannis, Psomiadou, Nakayama, Aiba, and Yamamoto (1997) with films of gelatin/starch blends plasticized with glycerol or sorbitol. Thermograms indicated partial miscibility of glycerol and kefiran at the molecular level for any given concentration ratio. They remained homogeneous throughout the heating cycle, as no phase separation (separated glass transition temperature or melting peaks) was observed. If blends of polymer and plasticizers are immiscible, the mixture will exhibit two Tg and Tm corresponding to the two pure phases (Ghanbarzadeh et al., 2010).
3.7. Film microstructure In an attempt to study microstructural changes in the films, scanning electron microscopy was used to depict the surface
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
1501
Fig. 2. Scanning electron micrographs of the surface (left) of kefiran-based films viewed at a magnification of 500, and cross-section (right) viewed at a magnification of 1000; (A) unplasticized, (B) plasticized with 25% w/w glycerol.
and section topography of all prepared kefiran films. Microscopic views indicated smooth and uniform surface morphology without obvious cracks, breaks, or openings on the surfaces after the incorporation of glycerol as a plasticizer. Fig. 2 shows scanning electron micrographs of the outer surface (left) and crosssection (right) for both the control and glycerol-containing films. Scanning electron microscopy observations of films with different glycerol concentrations did not present any obvious differences in structure. 4. Conclusions On the basis of the results obtained in this study, kefiran could be used as a new film-forming material. The physical, mechanical, and thermal properties, which are important in food packaging applications, were examined as a function of glycerol concentration. Generally, the TS of the films decreased and % E increased with increased concentrations of glycerol. Based on DSC thermograms, the glass transition temperature (Tg) of the films decreased with the glycerol content. Kefiran film exhibited a potential application as an edible food film and coating. The properties of kefiran-based film can be further improved for food-packaging applications. The methods investigated and developed in this study are expected to be very useful for future research in this area. Acknowledgements The authors wish to express their gratitude to Iran Polymers and Petrochemical Institute and the University of Tehran for supporting the facilities for this research work, and for technical assistance.
References 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), 593–604. ASTM (1995). Standard test methods for water vapor transmission of material, E 96–95. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Material. 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. Bolin, H. R., & Huxsoll, C. C. (1991). Control of minimally processed carrot (Dascus carota) surface discoloration caused by abrasion peeling. Journal of Food Science, 56, 416–418. Cheng, L. H., Karim, A. A., & Seow, C. C. (2006). Effects of water–glycerol and water– sorbitol interactions on the physical properties of konjac glucomannan films. Journal of Food Science, 71(2), 62–67. Cherian, G., Gennadios, A., Weller, C., & Chinachoti, P. (1995). Thermomechanical behavior of wheat gluten films: Effect of sucrose, glycerin and sorbitol. Cereal Chemistry, 72(1), 1–6. Cuq, B., Gontard, N., Aymard, C., & Guilbert, S. (1997). Relative humidity and temperature effects on mechanical and water vapor barrier properties of myofibrillar protein-based films. Polymer Gels and Networks, 5(1), 1–15. Debeaufort, F., Quezada-Gallo, J. A., & Voilley, A. (1998). Edible films and coatings: Tomorrow’s packagings: A review. Critical Reviews in Food Science and Nutrition, 38(4), 299–313. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 38(3), 350–352. Gennadios, A., Weller, C. L., & Gooding, C. H. (1994). Measurement errors in water vapor permeability of highly permeable, hydrophilic edible films. Journal of Food Engineering, 21, 395–409. Ghanbarzadeh, B., & Oromiehi, A. R. (2009). Thermal and mechanical behavior of laminated protein films. Journal of Food Engineering, 90, 517–524. Ghanbarzadeh, B., Almasi, H., & Entezami, A. A. (2010). Physical properties of edible modified starch/carboxymethyl cellulose films. Innovative Food Science and Emerging Technologies. doi:10.1016/j.ifset.2010.06.001. Ghanbarzadeh, B., Musavi, M., Oromiehie, A. R., Rezayi, K., Razmi, E., & Milani, J. (2006). Investigation of water vapor permeability, hydrophobicity and morphology of zein films plasticized by polyols. Iranian Polymer Journal, 15(9), 691–700.
1502
M. Ghasemlou et al. / Food Chemistry 127 (2011) 1496–1502
Gontard, N., Duchez, C., Cuq, B., & Guilbert, S. (1994). Edible composite films of wheat gluten and lipids: Water vapour permeability and other physical properties. Food Science and Technology, 29, 39–50. Gontard, N., Guilbert, S., & Cuq, J.-L. (1993). Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58(1), 206–211. Lourdin, D., Coignard, L., Bizot, H., & Colonna, P. (1997). Influence of equilibrium relative humidity and plasticizer concentration on the water content and glass transition of starch materials. Polymer, 38, 5401–5406. Maeda, H., Zhu, X., Omura, K., Suzuki, S., & Kitamura, S. (2004). Effects of an exopolysaccharide (kefiran) on lipids, blood pressure, blood glucose, and constipation. Biofactors, 22, 197–200. Mali, S., Sakanaka, L. S., Yamashita, F., & Grossmann, M. V. E. (2005). Water sorption and mechanical properties of cassava starch films and their relation to plasticizing effect. Carbohydrate Polymers, 60, 283–289. McHugh, T. H., & Krochta, J. M. (1994). Water vapor permeability properties of edible whey protein–lipid. Journal of American Oil Chemistry Society, 71(3), 307–402. Micheli, L., Uccelletti, D., Palleschi, C., & Crescenzi, V. (1999). Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran. Applied Microbiology and Biotechnology, 53, 69–74. Murofushi, M., Shiomi, M., & Aibara, K. (1983). Effect of orally administered polysaccharide from kefir grain on delayed-type hypersensitivity and tumor growth in mice. Japanese Journal of Medical Science and Biology, 36(1), 49–53.
Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122, 161–166. Paul, F., Morin, A., & Monsan, P. (1986). Microbial polysaccharides with actual potential industrial applications. Biotechnology Advances, 4(2), 245–259. Pearoval, C., Debeaufort, F., Desprea, D., & Voille, A. (2002). Edible arabinoxylanbased films: Effects of lipid type on water vapour permeability, film structure, and other physical characteristics. Journal of Agricultural and Food Chemistry, 50, 3977–3983. Piermaria, J. A., Pinotti, A., Garcia, M. A., & Abraham, A. G. (2009). Films based on kefiran, an exopolysaccharide obtained from kefir grain: Development and characterization. Food Hydrocolloids, 23, 684–690. Rimada, P. S., & Abraham, A. G. (2001). Polysaccharide production by kefir grains during whey fermentation. Journal of Dairy Research, 68, 653–661. Robertson, G. L. (1993). Structure and related properties of plastic polymers. In Food packaging: principles and practice. New York: Marcel Dekker Inc (chap. 2). Rodriguez, M., Oses, J., Ziani, K., & Mate, J. I. (2006). Combined effect of plasticizers and surfactants on the physical properties of starch based edible film. Food Research International, 39, 840–846. Smith, S. A. (1986). Polyethylene, lowdensity. In M. Bakker (Ed.), The Wiley encyclopedia of packaging technology (pp. 514–523). New York: John Wiley and Sons. Yang, L., & Paulson, A. T. (2000). Mechanical and water vapor barrier properties of edible gellan films. Food Research International, 33, 563–570.