- Email: [email protected]
Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water
Food Chemistry 122 (2010) 161–166
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water Seyed Mahdi Ojagh a, Masoud Rezaei a,*, Seyed Hadi Razavi b, Seyed Mohamad Hashem Hosseini b a b
Dept. of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran Dept. of Food Science and Engineering, Faculty of Engineering and Agricultural Technology, University of Tehran, Iran
a r t i c l e
i n f o
Article history: Received 9 August 2009 Received in revised form 18 January 2010 Accepted 11 February 2010
Keywords: Chitosan film Cinnamon Antimicrobial Physical Mechanical Scanning electron microscopy
a b s t r a c t Combining antimicrobial agents such as plant essential oils directly into a food packaging is a form of active packaging. In this work chitosan-based films containing cinnamon essential oil (CEO) at level of 0.4%, .0.8%, and 1.5% and 2% (v/v) were prepared to examine their antibacterial, physical and mechanical properties. Scanning electron microscopy was carried out to explain structure–property relationships. Incorporating CEO into chitosan-based films increased antimicrobial activity. CEO decreased moisture content, solubility in water, water vapour permeability and elongation at break of chitosan films. It is postulated that the unique properties of the CEO added films could suggest the cross-linking effect of CEO components within the chitosan matrix. Electron microscopy images confirmed the results obtained in this study. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Microbial growth on food surfaces is a major cause of food spoilage. There have been remarkable developments in recent years in the polymeric packaging films incorporated with antimicrobial agents for improving the preservation of packaged foods. These films possess the potential for improving microbial stability of foods by acting on the food surface, upon contact (Cha, Choi, Chinnan, & Park, 2002). Chitosan is the deacetylated (to varying degrees) form of chitin which is the second most abundant natural biopolymer after cellulose, largely widespread in living organisms such as crustacean, insects, and fungi. It is a linear binary heteropolysaccharide composed of b-1,4-linked glucosamine and N-acetylglucosamine, and is determined as a non-toxic, a biodegradable and a biocompatible polymer (Beverlya, Janes, Prinyawiwatkula, & No, 2008; Shahidi, Arachchi, & Jeon, 1999). Interestingly some antibacterial and antifungal activities have been described for chitosan and modified chitosan such as more soluble and biodegradable polymers with the knowledge that this aminopolysaccharide was able to reduce microbial growth (Tsai, Wu, & Su, 2000). One of the reasons for
* Corresponding author. Address: Dept. of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran. Tel.: + 98 122 6253101/ 3; fax: +98 122 6253499. E-mail address: [email protected] (M. Rezaei). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.033
the antimicrobial efficacy of chitosan is its positively charged amino group which interacts with negatively charged microbial cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms (Jeon, Kamil, & Shahidi, 2002; Shahidi et al., 1999). Chitosan-based films are excellent oxygen barriers; however, due to their hydrophilic nature, they have poor moisture barrier properties (Caner, Vergano, & Wiles, 1998). Many spices and herbs and their extracts possess antimicrobial activity, which minimise questions regarding their safe use in food products. Essential oils and their constituents have wide spectra of antimicrobial action. The composition, structure as well as functional groups of the oils play an important role in determining their antimicrobial activity (Holley & Patel, 2005). Usually compounds with phenolic groups are most effective (Dorman & Deans, 2000). Among these, the oils of clove, thyme, cinnamon, rosemary, sage and vanillin have been found to be most consistently effective against microorganisms. Because of the effect of direct addition of essential oils to food on sensory characteristics of added food, incorporation of essential oils to films may have supplementary applications in food packaging (Ojagh, Rezaei, Razavi, & Hosseini 2010; Seydim & Sarikus, 2006). The overall objective of the current study was to improve antimicrobial efficacy of chitosan-based films by incorporating cinnamon essential oils (CEO). Mechanical and physical properties were characterised, and antimicrobial efficacy was assessed against five pathogenic and spoilage bacteria. The effects of CEO
162
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
on the antimicrobial, physical and mechanical properties of films were evaluated and explained in terms of their microstructures. 2. Materials and methods 2.1. Bacterial strains and maintenance The bacterial strains used in this study were Listeria monocytogenes PTCC1163, Escherichia coli PTCC1399, Lactobacillus plantarum PTCC1058, Lactobacillus sakei PTCC1272 and Pseudomonas fluorescens ATCC 17482. These bacteria were obtained from Persian Type Culture Collection (Tehran, Iran) on nutrient agar slants and kept at 4 °C. Subculturing was carried out each 14 days to maintain bacterial viability. Overnight cultures of L. plantarum and L. sakei were grown in MRS (Man-Rogosa-Sharpe) broth at 37 and 30 °C at CO2 chamber (New Brunswick Scientific Co, Edison, NJ), respectively, and L. monocytogenes, E. coli, and Ps. fluorescens were grown and agitated at 140–150 rpm in an incubator shaker for 24 h in BHI (brain heart infusion) broth at 37 °C. All the media were purchased from Merck Co (Darmstadt, Germany). The bacterial population in all the inoculated media was more than 1 109 CFU/ml after 24 h incubation. 2.2. GC–MS analysis of cinnamon essential oil Cinnamon (Cinnamomum zeylanicum) essential oil was obtained from Zardband Co. (Tehran, Iran). GC/MS analysis was carried out on a Hewlett Packard 6890 (II) coupled to an HP 5973 mass spectrometer detector with electron impact ionisation (70 eV) (Agilent, Palo Alto, CA, USA). The analysis was carried out using DB-5 fused silica capillary column (60 m, 0.25 mm I.D.; 0.25 lm film thickness, J&W Scientific Inc., Rancho Cordova, CA, USA). The temperature program was 10 min at 60 °C, then to 250 °C at 5 °C/min, held for 10 min. Other operating conditions were as follows: carrier gas, helium (99.999%), with a flow rate of 1.1 ml/min; injection volume of 1 ll and split ratio, 1:50. Essential oil components were identified by comparison of their retention indices (RI) relative to (C6–C24) n-alkanes with those of authentic compounds under the same conditions (Davies, 1990). Further identification was made by matching their mass spectra fragmentation patterns with those stored in the Wiley/NBS mass spectral library and other published mass spectra (Adams, 2001). Moreover, relative percentage of the component cinnamon oil was calculated by normalisation of the base GC–mass peak. 2.3. Determination of MIC and MBC of CEO against bacterial strains Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CEO were determined according to the method of Kim, Marshall, and Wei (1995). 2.4. Preparation of antimicrobial films Chitosan-based film was prepared by dissolving crab shell chitosan (medium molecular weight (190–310 kDa), 75–85% deacetylated, Sigma Chemical Co., St. Louis, MO., USA) in an aqueous solution (1% v/v) of glacial acetic acid to a concentration of 2% (w/v) while stirring on a magnetic stirrer/hot plate. The solution was stirred with low heat (at 40 °C) which typically required 6 h stirring. The resultant chitosan solution was filtered through a Whatman No. 3 filter paper to remove any undissolved particles. After filtration the solution was returned to the magnetic stirrer/ hot plate and glycerol (Sigma Chemical Co., St. Louis, MO, USA) was added to a level of 0.75 ml/g chitosan as a plasticiser. The plasticiser was mixed into the solution for 30 min. Then Tween 80 at
level of 0.2% (v/v) of essential oil was added as an emulsifier to assist essential oil dissolution in film forming solutions. After 1 h of stirring, CEO was added to chitosan solution to reach a final concentration of 0.4%, 0.8%, 1.5% and 2% (v/v) as essential oil concentration per film in emulsifying equipment (IKA T25-Digital Ultra Turrax, Staufen, Germany) at 7000 rpm for 2 min. After cooling to room temperature, the film forming solutions were degassed under vacuum for 5 min. The film forming solutions (160 ml) were casted on the centre of 27 27 cm2 glass plates, and then dried for 30 h at ambient conditions (25 °C). Dried films were peeled and stored in a desiccator at 25 °C and 51% relative humidity until evaluation. Saturated magnesium nitrate solution was used to meet required relative humidity. 2.5. Determination of antimicrobial effects of films The agar diffusion method was used for determining the antibacterial effects of films on bacterial strains. The films were cut into 15 mm diameter discs with a circular knife. Film cuts were placed on BHI agar for L. monocytogenes, E. coli and Ps. fluorescens and on MRS agar for L. plantarum and L. sakei. Agar plates had previously been seeded with 0.1 ml of an overnight broth culture of indicator strains. The plates were incubated at appropriate temperature for 24 h in the appropriate incubation chamber. The diameter of the zone of inhibition was measured with a caliper to the nearest 0.02 mm. The whole zone area was calculated then subtracted from the film disc area and this difference in area was reported as the ‘‘zone of inhibition” (Seydim & Sarikus, 2006). 2.6. Determination of physical properties of films 2.6.1. Thickness Thickness of the films was determined using a digital coating thickness gauge (Elcometer A300 FNP 23, Elcometer Instrument Ltd., Manchester, England) to the nearest 0.001 mm. Informed values are an average of at least ten random locations of the film sheets. The means were calculated and used in the determination of mechanical and physical properties. 2.6.2. Moisture content Moisture content of films was determined measuring weight loss of films, upon drying in an oven at 110 °C until a constant weight was reached (dry sample weight). 2.6.3. Water vapour permeability coefficient (WVPC) Standard method E 96 was used to determine WVPC with a 75% relative humidity (RH) gradient at 25 °C (ASTM, 1995). Diffusion cells containing anhydrous calcium chloride desiccant (0% RH, assay cup) or nothing (control cup) were sealed by the test film (0.00287 m2 film area). To maintain a 75% RH gradient across the film, a sodium chloride-saturated solution (75% RH) was used in the desiccator. The RH inside the cell was always lower than outside. This difference in RH corresponds to a driving force of 1753.55 Pa, expressed as water vapour partial pressure. Water vapour transport was determined from the weight gain of the diffusion cell at a steady state of transfer. Changes in the weight of the cell were recorded to the nearest 0.0001 g and plotted as a function of time. The slope of the weight vs time plot was divided by the effective film area to obtain the water vapour transmission rate (WVTR). This was multiplied by the thickness of the film and divided by the pressure difference to obtain the water vapour permeability coefficient (WVPC). All WVPC values were corrected for air gap distance between calcium chloride and film surface by correcting the values of WVTR, according to the equations of Gennadios, Weller, and Gooding (1994).
163
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
2.6.4. Surface properties measurements Contact angle measurements were performed with water using a goniometer (Kruss G23, Germany). A small drop of distilled water was deposited on the film surface. The contact angle is defined as the angle between the film surface and the tangent line at the point of contact of the water droplet with the surface. For each film type, at least five measurements were made and the average was taken.
2.9. Statistical analysis The triplicate data were performed to an analysis of variance for the significance of added essential oils using MSTATC programs (version 2.10, East Lansing, MI, USA). Duncan’s multiple range tests was used to compare the difference among means at the level of 0.05. 3. Results and discussion
2.6.5. Film solubility in water Pieces of film of 1 3 cm2 were cut from each film and weighed to the nearest 0.0001 g. The solubility in water of the different chitosan films was measured from immersion assays under constant agitation in 50 ml of distilled water for 6 h at 25 °C. The remaining pieces of film after immersion were dried at 110 °C to constant weight (Final dry weight). The initial dry weight was determined by thermal processing at 110 °C to constant weight. Solubility in water (%) was calculated by using the following equation (Eq. (1)):
Solubility in water ð%Þ Initial dry weight Final dry weight ¼ 100 Initial dry weight
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðL LÞ2 ða aÞ2 ðb bÞ2
GC–MS analytical data of major compounds in CEO are shown in Table 1. E-Cinnamaldehyde was a predominant component and accounted for 60.41% of the total peak area. The other components were linalool (6.46%), ortho-methoxycinnamaldehyde (3.63%), b-caryophyllene (3.5%), 1,8-cineole (3.32%), and eugenol (3.19%). 3.2. MIC and MBC of CEO
ð1Þ
2.6.6. Surface colour measurements Film colour was determined by a colourimeter (Minolta CR 300 Series, Minolta Camera Co., Ltd., Osaka, Japan). The CIELab scale was used, lightness (L) and chromaticity parameters a (red–green) and b (yellow–blue) were measured. Measurements were performed by placing the film sample over the standard white plate (L* = 93.49, a* = 0.25 and b* = 0.09). Colour differences (DE) were calculated by the following equation (Eq. (2)):
DE ¼
3.1. Identification of volatile components from CEO
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. 2.7. Determination of mechanical properties of films The mechanical properties of films including tensile strength (MPa) and elongation at break (%) were performed at 25 °C and 51% RH. Tests were performed using a Testometric Machine M350-10CT (Testometric Co. Ltd., Rochdale, Lancs., England) according to the ASTM method D882-91 (ASTM, 1996). In preparing samples, films were cut into 1 10 cm2 strips. The films were held parallel with an initial grip separation of 5 cm and then pulled apart at a head speed of 25 mm/min. Elongation at break (deformation divided by initial grip separation and multiplying by 100) and maximum force were obtained from force versus deformation curves. Tensile strength was calculated by dividing maximum force by film cross section (thickness width).
MIC and MBC values (antibacterial properties) of CEO are presented in Table 2. The lower concentration of the essential oil could fully inhibit the growth or could almost kill L. sakei (MIC 250 lg/ mL and MBC 1000 lg/mL) and Ps. fluorescens (MIC 500 lg/mL and MBC 1000 lg/mL). However, MIC and MBC of the essential oil for the other three bacteria were 500 and more than 1500 lg/ mL, respectively. This is not supported by many other reports on the greater susceptibility of gram-positive bacteria to inhibitory effect of essential oils and their components (Oussalah, Caillet, Saucier, & Lacroix, 2007; Shan, Cai, Brooks, & Corke, 2007). Similar results have been
Table 1 Essential oil composition of Cinnamomum zeylanicum. No.
Compound
RI
% of the total peak area
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
a-Pinene
936 950 982 1001 1010 1016 1026 1086 1188 1247 1312 1334 1386 1398 1413 1431 1463 1492 1585 1736 2049
2.18 0.14 1.03 0.54 0.94 2.85 3.32 6.46 0.29 60.41 0.48 3.19 0.34 0.43 2.01 3.50 0.27 3.63 0.34 0.65 0.91
Camphene Myrcene a-Phellandrene d-3-Carene P-cymene 1,8-Cineole Linalool Z-Cinnamaldehyde E-Cinnamaldehyde Hexagerman Eugenol a-Copaene Coumarine E-Cynnamyl acetate b-Caryophyllene a-Humulene O-methoxycinnamaldehyde Caryophyllene oxide Benzyl benzoate Equilin
2.8. Scanning electron microscopy The chitosan-based films were mounted on the specimen holder with aluminium tape and then sputtered with gold in BAL-TEC SCD 005 sputter coater (BAL-TEC AG, Balzers, Liechtenstein). All the specimens were examined with a Philips XL 30 scanning electron microscope (Philips, Eindhoven, Netherlands) under high vacuum condition and at an accelerating voltage of 20.0 kV. Samples were photographed at tilt angles of 60–90° to the electron beam for the views in the cross section.
Table 2 MIC and MBC values of CEO against five tested bacterial strains. Bacteria
L. monocytogenes
E. coli
Ps. fluorescens
L. plantarum
L. sakei
MIC (ppm) MBC (ppm)
500 >1500
500 >1500
500 1000
500 >1500
250 1000
164
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
reported by Kim et al. (1995) who found that L. monocytogenes was more resistant to the inhibitory effects of eleven essential oil constitutes than the gram-negative bacteria tested under the same condition, including E. coli, E. coli O157:H7, Salmonella typhimurium and Vibrio vulnificus. It seems that the variability of the inhibitory effect of essential oil may be due to differences between strains of the same bacterial species. This hypothesis was confirmed by Sivropoulou et al. (1996) with two strains of Staphylococcus aureus in the presence of carvacrol and thymol.
3.3. Antimicrobial properties of films Effects of CEO addition on antimicrobial properties of chitosanbased films are shown in Table 3. When antimicrobial agents are incorporated into films, these materials diffuse through agar gel and result in clear zone around the film cuts. Incorporation of CEO into chitosan-based films at higher than 0.4% (v/v) exhibited a clear inhibitory zone by the absence of bacterial growth around the film cuts. At CEO concentration of 0.4% (v/ v) the clear zone of inhibition was not observed with L. plantarum. As the concentration increased, the zone of inhibition also increased significantly (p < 0.05). However, increasing level of CEO at its high concentration did not reveal significant an increased inhibitory. It was generally caused by the compactness of chitosan film network containing CEO because of the occurrence of functional groups interaction phenomenon. The active component of CEO is cinnamaldehyde (Matan et al., 2006). As shown in Table 1, cinnamaldehyde constitutes more than 60% of CEO. Sublethal concentrations of cinnamaldehyde have been found to inhibit production of amylase and proteases by Bacillus cereus (Thoroski, Blank, & Biliaderis, 1989). Chitosan control films did not show inhibitory zone in bacterial strains tested. Despite antimicrobial activity of chitosan because of its innate characteristic, this effect of chitosan occurred without
migration of active agents. Chitosan does not diffuse through the adjacent agar media in agar diffusion test method; so that only organisms in direct contact with the active sites of chitosan are inhibited (Coma et al., 2002). 3.4. Physical properties of films The effects of incorporating CEO on the physical properties of chitosan films are shown in Table 4. Thin films were easily removed from the cast plate. Thickness of films varied between 0.095 and 0.107 mm, as shown in Table 4. In general, the moisture content value decreased as CEO was incorporated into chitosan-based film, which is attributed to compactness of film network. CEO caused to formation covalent bonds between the functional groups of chitosan chains, leading to a decrease in the availability of hydroxyl and amino groups and limiting polysaccharide–water interactions by hydrogen bonding and resulting in a decrease of moisture content value of edible films (Park & Zhao, 2004). As CEO concentration increased, the moisture content of films decreased significantly (p < 0.05). Chitosan control films were compact, and the film surface had a smooth surface without pores or cracks (Fig. 1A and B). Control film had water vapour permeability coefficient (WVPC) value 2.250 1010 g s1 m1 Pa s1. Same results were obtained by Wong, Gastineau, Gregorski, Tillin, and Pavlath (1992), Caner et al. (1998) and Seydim and Sarikus (2006). Incorporation of CEO into chitosan-based films decreased WVPC of films. Beside the hydrophobic nature of CEO which could affect the hydrophilic/hydrophobic property of the films, the physical factors had an influence on the WVPC of the films enriched with CEO. When CEO was incorporated into the chitosan film formulation, the sur-
Table 4 Physical properties of chitosan films incorporated with CEO.
Table 3 Antibacterial activity (inhibitory zone) of chitosan films incorporated with CEO against gram-positive and gram-negative bacteria.
*
Physical properties*
CEO conc. (v/v) in film solution (%)
Thickness (mm)
Control 0.4 0.8 1.5 2
0.095 ± 0.0025b 0.098 ± 0.0024b 0.104 ± 0.0029a 0.105 ± 0.0032a 0.107 ± 0.0039a
Bacteria
CEO conc. (v/v) in film solution (%)
Inhibitory zone* (mm2)
L. monocytogenes (Gram +)
Control 0.4 0.8 1.5 2
0c 38.17 ± 1.00b 39.80 ± 0.94b 53.05 ± 1.84a 52.31 ± 2.65a
Moisture content (%)
Control 0.4 0.8 1.5 2
20.82 ± 1.82a 18.72 ± 2.11a 14.04 ± 0.93b 10.82 ± 1.47c 8.47 ± 1.53c
L. plantarum (Gram +)
Control 0.4 0.8 1.5 2
0c 0c 43.6 ± 0.94b 49.80 ± 2.77a 53.64 ± 3.07a
Water vapor permeability coefficient (g s1 m1 Pa s1 1010)
Control 0.4 0.8 1.5 2
2.250 ± 0.074a 1.352 ± 0.152b 1.234 ± 0.040b 1.014 ± 0.040c 1.003 ± 0.067c
L. sakei (Gram +)
Control 0.4 0.8 1.5 2
0c 39.60 ± 2.36b 41.08 ± 1.44b 56.69 ± 2.82a 57.23 ± 1.50a
Water contact angle (°)
Control 0.4 0.8 1.5 2
37.3 ± 2.25d 39.6 ± 1.89d 45.3 ± 1.38c 59.2 ± 2.77b 70.3 ± 1.42a
Ps. fluorescens (Gram )
Control 0.4 0.8 1.5 2
0c 34.68 ± 1.71b 36.91 ± 1.32b 41.60 ± 2.09a 43.60 ± 1.21a
Solubility in water (%)
Control 0.4 0.8 1.5 2
23.2 ± 1.09a 21.6 ± 0.65a 16.8 ± 0.85b 13.6 ± 1.55c 10.4 ± 0.94d
E. coli (Gram )
Control 0.4 0.8 1.5 2
0d 36.31 ± 0.77c 38.90 ± 0.94b 51.72 ± 1.15a 51.04 ± 2.12a
Total colour difference (DE)
Control 0.4 0.8 1.5 2
17.35 ± 0.98d 17.83 ± 0.47d 21.29 ± 0.55c 23.35 ± 0.87b 25.62 ± 0.58a
Means in each column with different superscript letters are significantly different (p < 0.05). Control is a film disc containing no essential oil.
*
Means in each column with different superscript letters are significantly different (p < 0.05).
165
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
Fig. 1. Scanning electronic microscopic images of chitosan control film (A) and (B), and film containing CEO at level of 1.5% (C) and (D) surfaces and cross sections, respectively.
face view suggested a sheet like and dense structure, while the cross section revealed the sheets stacked in compact layers (Fig. 1C and D), which showed CEO incorporated uniformly in the matrix. The value of the contact angle with water indicates how hydrophobic the surface is. It is well-known that the water contact angle will increase with increasing surface hydrophobicity. The higher hydrophilicity of the control film is attributed to the water binding capacity of plasticiser (glycerol) and functional groups of chitosan. According to Table 4, addition of CEO increased water contact angle of films and resulted in decreasing hydrophilicity of the chitosan films which might be due to the loss of free functional groups (amino and hydroxyl groups). Chitosan control film had low solubility in water below 24% at 25 °C after 6 h dipping. Same result was reported by García, Pinotti, Martino, and Zaritzky (2004). Control film in general was very hydrophilic thus absorbed water quickly and resulted in swelling. Incorporation of CEO into the chitosan film formulation at level of 1.5% and 2% (v/v) led to 41% and 55% reduction in solubility in water respectively. This phenomenon is due to the cross-linking effects of CEO components leading to esters and/or amide groups. Cross-linking in the chitosan film matrix resulted in a decrease in solubility in water and produced films with low affinity toward water which is beneficial when product integrity and water resistance are intended. Cross-linking of chitosan-hydroxy propyl methyl cellulose based films using citric acid (polycarboxylic acid) as the cross-linking agent, was reported by Möller, Grelier, Pardon, and Coma (2004). The colour of the films may influence the consumer acceptability of a product (Sivarooban, Hettiarachchy, & Johnson, 2008). Total colour differences (DE) of chitosan films are shown in Table 4. Visually, chitosan films had a slightly yellow appearance. Control film had DE value 17.35. Its transparency was reduced as the CEO was incorporated. Incorporating CEO revealed DE values, which were significantly higher than that of the control. The values of chromaticity parameter b of chitosan films incorporated with CEO were higher than that of control film (data not shown). These
results agreed with visual observations. Nevertheless, the sensory studies should be conducted for evaluating consumer acceptability of this colour change.
3.5. Mechanical properties of films The effect of incorporating CEO on mechanical properties of chitosan films is presented in Table 5. Chitosan control film had tensile strength value 10.97 MPa. Incorporation of CEO into chitosan films increased tensile strength values significantly (p < 0.05). As previously mentioned, a strong interaction between the polymer and the CEO produced a cross-linker effect, which decreases the free volume and the molecular mobility of the polymer. This phenomenon led to a sheet like structure (Fig. 1C). Arrangement of stacking layers of CEO added chitosan sheets (Fig. 1D) means that in these films a compact structure was present thus increased continuities within the polysaccharide network leading to decrease in elongation at break. There are also possibilities such as decrease in moisture content of films incorporated with CEO during the film production, thus leading to the decrease in strain and the increase
Table 5 Mechanical properties of chitosan films incorporated with CEO.
*
Mechanical properties*
CEO conc. (v/v) in film solution (%)
Tensile strength (MPa)
Control 0.4 0.8 1.5 2
10.97 ± 0.54e 13.35 ± 1.23d 17.43 ± 1.08c 24.10 ± 1.47b 29.23 ± 2.25a
Elongation at break (%)
Control 0.4 0.8 1.5 2
24.73 ± 1.86a 16.57 ± 0.77b 11.26 ± 1.39c 6.42 ± 0.63d 3.58 ± 0.35e
Means in each column with different superscript letters are significantly different (p < 0.05).
166
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
in tensile strength. The results of moisture content measurements are shown in the Table 4. According to Gontard, Guilbert, and Cuq (1993) and Pouplin, Redl, and Gontard (1999) water is the most ubiquitous and uncontrollable plasticiser for most hydrocolloidbased films because of its ability to modify the structure of natural polymers. Thus, the plasticisation effect of water could not be negligible for these films, and their plasticisation efficiency does not only come from intrinsic plasticiser action of glycerol. Differences between data of chitosan films obtained in the current study and those of reported in literature (Caner et al., 1998; García et al., 2004) may be attributed to chitosan composition and suppliers, plasticiser presence and film preparation. 4. Conclusions Chitosan is a promising biopolymer for active food packaging. Its sensitivity to moisture can be offset by blending it with CEO. The results of this study showed that a unique compatibility can be achieved between chitosan and CEO. The incorporation of CEO improved the antibacterial properties of chitosan. Films containing CEO are useful for coating of highly perishable foods such as fish and poultry. The other edible films may be ruptured upon contact with wet surfaces. Functional groups interaction phenomenon in edible films has critical effect on their antibacterial, physical and mechanical properties which are important in food packaging applications. Moreover, the antimicrobial effect of CEO enriched films should be determined on an entire model food. References Adams, R. P. (2001). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Allured, Carol Stream, IL. 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 (1996). Standard test methods for tensile properties of thin plastic sheeting, D882-91. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Material. Beverlya, R. L., Janes, M. E., Prinyawiwatkula, W., & No, H. K. (2008). Edible chitosan films on ready-to-eat roast beef for the control of Listeria monocytogenes. Food Microbiology, 25, 534–537. Caner, C., Vergano, P. J., & Wiles, J. L. (1998). Chitosan film mechanical and permeation properties as affected by acid, plasticizer and storage. Journal of Food Science, 63, 1049–1053. Cha, D. S., Choi, J. H., Chinnan, M. S., & Park, H. J. (2002). Antimicrobial films based on Na-alginate and j-carrageenan. Lebensmittel-Wissenschaft und -Technologie, 35, 715–719. Coma, V., Martial-Gros, A., Garreau, S., Copinet, A., Salin, F., & Deschamps, A. (2002). Edible antimicrobial films based on chitosan matrix. Journal of Food Science, 67, 1162–1169. Davies, N. W. (1990). Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone on Carbowax 20M phases. Journal of Chromatography, 503, 1–24. Dorman, H. J., & Deans, S. G. (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308–316.
García, M. A., Pinotti, A., Martino, M. N., & Zaritzky, N. E. (2004). Characterization of composite hydrocolloid films. Carbohydrate Polymers, 56, 339–345. 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. Gontard, N., Guilbert, S., & Cuq, J. (1993). Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58, 206–211. Holley, R. A., & Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials: A review. Food Microbiology, 22, 273–292. Jeon, Y.-I., Kamil, J. Y. V. A., & Shahidi, F. (2002). Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod. Journal of Agricultural and Food Chemistry, 20, 5167–5178. Kim, J., Marshall, M. R., & Wei, C. (1995). Antimicrobial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry, 43, 2839–2845. Matan, N., Rimkeeree, H., Mawson, A. J., Chompreeda, P., Haruthaithanasan, V., & Parker, M. (2006). Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. International Journal of Food Microbiology, 107, 180–185. Möller, H., Grelier, S., Pardon, P., & Coma, V. (2004). Antimicrobial and physicochemical properties of chitosan-HPMC-based films. Journal of Agricultural and Food Chemistry, 52, 6585–6591. Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. Food Chemistry, 120, 193–198. Oussalah, M., Caillet, S., Saucier, L., & Lacroix, M. (2007). Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control, 18, 414–420. Park, S., & Zhao, Y. (2004). Incorporation of a high concentration of mineral or vitamin into chitosan-based films. Journal of Agricultural and Food Chemistry, 52, 1933–1939. 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, 538–543. Seydim, A. C., & Sarikus, G. (2006). Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Research International, 39, 639–644. Shahidi, F., Arachchi, J. K. V., & Jeon, Y. J. (1999). Food application of chitin and chitosans. Trends in Food Science and Technology, 10, 37–51. Shan, B., Cai, Y., Brooks, J. D., & Corke, H. (2007). Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum burmannii): Activity against foodborne pathogenic bacteria. Journal of Agricultural and Food Chemistry, 55, 5484–5490. Sivarooban, T., Hettiarachchy, N. S., & Johnson, M. G. (2008). Physical and antimicrobial properties of grape seed extract, nisin, and EDTA incorporated soy protein edible films. Food Research International, 41, 781–785. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T., & Arsenakis, M. (1996). Antimicrobial and cytotoxic activity of origanum essential oils. Journal of Agricultural and Food Chemistry, 44, 1202–1205. Thoroski, J., Blank, G., & Biliaderis, C. (1989). Eugenol induced inhibition of extracellular enzyme production by Bacillus cereus. Journal of Food Protection, 52, 399–403. Tsai, G. I., Wu, Z. Y., & Su, W. H. (2000). Antimicrobial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. Journal of Food Protection, 63, 747–752. Wong, D. W. S., Gastineau, F. A., Gregorski, K. S., Tillin, S. J., & Pavlath, A. E. (1992). Chitosan lipid films: Microstructure and surface energy. Journal of Agricultural and Food Chemistry, 40, 540–544.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water Seyed Mahdi Ojagh a, Masoud Rezaei a,*, Seyed Hadi Razavi b, Seyed Mohamad Hashem Hosseini b a b
Dept. of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran Dept. of Food Science and Engineering, Faculty of Engineering and Agricultural Technology, University of Tehran, Iran
a r t i c l e
i n f o
Article history: Received 9 August 2009 Received in revised form 18 January 2010 Accepted 11 February 2010
Keywords: Chitosan film Cinnamon Antimicrobial Physical Mechanical Scanning electron microscopy
a b s t r a c t Combining antimicrobial agents such as plant essential oils directly into a food packaging is a form of active packaging. In this work chitosan-based films containing cinnamon essential oil (CEO) at level of 0.4%, .0.8%, and 1.5% and 2% (v/v) were prepared to examine their antibacterial, physical and mechanical properties. Scanning electron microscopy was carried out to explain structure–property relationships. Incorporating CEO into chitosan-based films increased antimicrobial activity. CEO decreased moisture content, solubility in water, water vapour permeability and elongation at break of chitosan films. It is postulated that the unique properties of the CEO added films could suggest the cross-linking effect of CEO components within the chitosan matrix. Electron microscopy images confirmed the results obtained in this study. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Microbial growth on food surfaces is a major cause of food spoilage. There have been remarkable developments in recent years in the polymeric packaging films incorporated with antimicrobial agents for improving the preservation of packaged foods. These films possess the potential for improving microbial stability of foods by acting on the food surface, upon contact (Cha, Choi, Chinnan, & Park, 2002). Chitosan is the deacetylated (to varying degrees) form of chitin which is the second most abundant natural biopolymer after cellulose, largely widespread in living organisms such as crustacean, insects, and fungi. It is a linear binary heteropolysaccharide composed of b-1,4-linked glucosamine and N-acetylglucosamine, and is determined as a non-toxic, a biodegradable and a biocompatible polymer (Beverlya, Janes, Prinyawiwatkula, & No, 2008; Shahidi, Arachchi, & Jeon, 1999). Interestingly some antibacterial and antifungal activities have been described for chitosan and modified chitosan such as more soluble and biodegradable polymers with the knowledge that this aminopolysaccharide was able to reduce microbial growth (Tsai, Wu, & Su, 2000). One of the reasons for
* Corresponding author. Address: Dept. of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran. Tel.: + 98 122 6253101/ 3; fax: +98 122 6253499. E-mail address: [email protected] (M. Rezaei). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.033
the antimicrobial efficacy of chitosan is its positively charged amino group which interacts with negatively charged microbial cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms (Jeon, Kamil, & Shahidi, 2002; Shahidi et al., 1999). Chitosan-based films are excellent oxygen barriers; however, due to their hydrophilic nature, they have poor moisture barrier properties (Caner, Vergano, & Wiles, 1998). Many spices and herbs and their extracts possess antimicrobial activity, which minimise questions regarding their safe use in food products. Essential oils and their constituents have wide spectra of antimicrobial action. The composition, structure as well as functional groups of the oils play an important role in determining their antimicrobial activity (Holley & Patel, 2005). Usually compounds with phenolic groups are most effective (Dorman & Deans, 2000). Among these, the oils of clove, thyme, cinnamon, rosemary, sage and vanillin have been found to be most consistently effective against microorganisms. Because of the effect of direct addition of essential oils to food on sensory characteristics of added food, incorporation of essential oils to films may have supplementary applications in food packaging (Ojagh, Rezaei, Razavi, & Hosseini 2010; Seydim & Sarikus, 2006). The overall objective of the current study was to improve antimicrobial efficacy of chitosan-based films by incorporating cinnamon essential oils (CEO). Mechanical and physical properties were characterised, and antimicrobial efficacy was assessed against five pathogenic and spoilage bacteria. The effects of CEO
162
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
on the antimicrobial, physical and mechanical properties of films were evaluated and explained in terms of their microstructures. 2. Materials and methods 2.1. Bacterial strains and maintenance The bacterial strains used in this study were Listeria monocytogenes PTCC1163, Escherichia coli PTCC1399, Lactobacillus plantarum PTCC1058, Lactobacillus sakei PTCC1272 and Pseudomonas fluorescens ATCC 17482. These bacteria were obtained from Persian Type Culture Collection (Tehran, Iran) on nutrient agar slants and kept at 4 °C. Subculturing was carried out each 14 days to maintain bacterial viability. Overnight cultures of L. plantarum and L. sakei were grown in MRS (Man-Rogosa-Sharpe) broth at 37 and 30 °C at CO2 chamber (New Brunswick Scientific Co, Edison, NJ), respectively, and L. monocytogenes, E. coli, and Ps. fluorescens were grown and agitated at 140–150 rpm in an incubator shaker for 24 h in BHI (brain heart infusion) broth at 37 °C. All the media were purchased from Merck Co (Darmstadt, Germany). The bacterial population in all the inoculated media was more than 1 109 CFU/ml after 24 h incubation. 2.2. GC–MS analysis of cinnamon essential oil Cinnamon (Cinnamomum zeylanicum) essential oil was obtained from Zardband Co. (Tehran, Iran). GC/MS analysis was carried out on a Hewlett Packard 6890 (II) coupled to an HP 5973 mass spectrometer detector with electron impact ionisation (70 eV) (Agilent, Palo Alto, CA, USA). The analysis was carried out using DB-5 fused silica capillary column (60 m, 0.25 mm I.D.; 0.25 lm film thickness, J&W Scientific Inc., Rancho Cordova, CA, USA). The temperature program was 10 min at 60 °C, then to 250 °C at 5 °C/min, held for 10 min. Other operating conditions were as follows: carrier gas, helium (99.999%), with a flow rate of 1.1 ml/min; injection volume of 1 ll and split ratio, 1:50. Essential oil components were identified by comparison of their retention indices (RI) relative to (C6–C24) n-alkanes with those of authentic compounds under the same conditions (Davies, 1990). Further identification was made by matching their mass spectra fragmentation patterns with those stored in the Wiley/NBS mass spectral library and other published mass spectra (Adams, 2001). Moreover, relative percentage of the component cinnamon oil was calculated by normalisation of the base GC–mass peak. 2.3. Determination of MIC and MBC of CEO against bacterial strains Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CEO were determined according to the method of Kim, Marshall, and Wei (1995). 2.4. Preparation of antimicrobial films Chitosan-based film was prepared by dissolving crab shell chitosan (medium molecular weight (190–310 kDa), 75–85% deacetylated, Sigma Chemical Co., St. Louis, MO., USA) in an aqueous solution (1% v/v) of glacial acetic acid to a concentration of 2% (w/v) while stirring on a magnetic stirrer/hot plate. The solution was stirred with low heat (at 40 °C) which typically required 6 h stirring. The resultant chitosan solution was filtered through a Whatman No. 3 filter paper to remove any undissolved particles. After filtration the solution was returned to the magnetic stirrer/ hot plate and glycerol (Sigma Chemical Co., St. Louis, MO, USA) was added to a level of 0.75 ml/g chitosan as a plasticiser. The plasticiser was mixed into the solution for 30 min. Then Tween 80 at
level of 0.2% (v/v) of essential oil was added as an emulsifier to assist essential oil dissolution in film forming solutions. After 1 h of stirring, CEO was added to chitosan solution to reach a final concentration of 0.4%, 0.8%, 1.5% and 2% (v/v) as essential oil concentration per film in emulsifying equipment (IKA T25-Digital Ultra Turrax, Staufen, Germany) at 7000 rpm for 2 min. After cooling to room temperature, the film forming solutions were degassed under vacuum for 5 min. The film forming solutions (160 ml) were casted on the centre of 27 27 cm2 glass plates, and then dried for 30 h at ambient conditions (25 °C). Dried films were peeled and stored in a desiccator at 25 °C and 51% relative humidity until evaluation. Saturated magnesium nitrate solution was used to meet required relative humidity. 2.5. Determination of antimicrobial effects of films The agar diffusion method was used for determining the antibacterial effects of films on bacterial strains. The films were cut into 15 mm diameter discs with a circular knife. Film cuts were placed on BHI agar for L. monocytogenes, E. coli and Ps. fluorescens and on MRS agar for L. plantarum and L. sakei. Agar plates had previously been seeded with 0.1 ml of an overnight broth culture of indicator strains. The plates were incubated at appropriate temperature for 24 h in the appropriate incubation chamber. The diameter of the zone of inhibition was measured with a caliper to the nearest 0.02 mm. The whole zone area was calculated then subtracted from the film disc area and this difference in area was reported as the ‘‘zone of inhibition” (Seydim & Sarikus, 2006). 2.6. Determination of physical properties of films 2.6.1. Thickness Thickness of the films was determined using a digital coating thickness gauge (Elcometer A300 FNP 23, Elcometer Instrument Ltd., Manchester, England) to the nearest 0.001 mm. Informed values are an average of at least ten random locations of the film sheets. The means were calculated and used in the determination of mechanical and physical properties. 2.6.2. Moisture content Moisture content of films was determined measuring weight loss of films, upon drying in an oven at 110 °C until a constant weight was reached (dry sample weight). 2.6.3. Water vapour permeability coefficient (WVPC) Standard method E 96 was used to determine WVPC with a 75% relative humidity (RH) gradient at 25 °C (ASTM, 1995). Diffusion cells containing anhydrous calcium chloride desiccant (0% RH, assay cup) or nothing (control cup) were sealed by the test film (0.00287 m2 film area). To maintain a 75% RH gradient across the film, a sodium chloride-saturated solution (75% RH) was used in the desiccator. The RH inside the cell was always lower than outside. This difference in RH corresponds to a driving force of 1753.55 Pa, expressed as water vapour partial pressure. Water vapour transport was determined from the weight gain of the diffusion cell at a steady state of transfer. Changes in the weight of the cell were recorded to the nearest 0.0001 g and plotted as a function of time. The slope of the weight vs time plot was divided by the effective film area to obtain the water vapour transmission rate (WVTR). This was multiplied by the thickness of the film and divided by the pressure difference to obtain the water vapour permeability coefficient (WVPC). All WVPC values were corrected for air gap distance between calcium chloride and film surface by correcting the values of WVTR, according to the equations of Gennadios, Weller, and Gooding (1994).
163
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
2.6.4. Surface properties measurements Contact angle measurements were performed with water using a goniometer (Kruss G23, Germany). A small drop of distilled water was deposited on the film surface. The contact angle is defined as the angle between the film surface and the tangent line at the point of contact of the water droplet with the surface. For each film type, at least five measurements were made and the average was taken.
2.9. Statistical analysis The triplicate data were performed to an analysis of variance for the significance of added essential oils using MSTATC programs (version 2.10, East Lansing, MI, USA). Duncan’s multiple range tests was used to compare the difference among means at the level of 0.05. 3. Results and discussion
2.6.5. Film solubility in water Pieces of film of 1 3 cm2 were cut from each film and weighed to the nearest 0.0001 g. The solubility in water of the different chitosan films was measured from immersion assays under constant agitation in 50 ml of distilled water for 6 h at 25 °C. The remaining pieces of film after immersion were dried at 110 °C to constant weight (Final dry weight). The initial dry weight was determined by thermal processing at 110 °C to constant weight. Solubility in water (%) was calculated by using the following equation (Eq. (1)):
Solubility in water ð%Þ Initial dry weight Final dry weight ¼ 100 Initial dry weight
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðL LÞ2 ða aÞ2 ðb bÞ2
GC–MS analytical data of major compounds in CEO are shown in Table 1. E-Cinnamaldehyde was a predominant component and accounted for 60.41% of the total peak area. The other components were linalool (6.46%), ortho-methoxycinnamaldehyde (3.63%), b-caryophyllene (3.5%), 1,8-cineole (3.32%), and eugenol (3.19%). 3.2. MIC and MBC of CEO
ð1Þ
2.6.6. Surface colour measurements Film colour was determined by a colourimeter (Minolta CR 300 Series, Minolta Camera Co., Ltd., Osaka, Japan). The CIELab scale was used, lightness (L) and chromaticity parameters a (red–green) and b (yellow–blue) were measured. Measurements were performed by placing the film sample over the standard white plate (L* = 93.49, a* = 0.25 and b* = 0.09). Colour differences (DE) were calculated by the following equation (Eq. (2)):
DE ¼
3.1. Identification of volatile components from CEO
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. 2.7. Determination of mechanical properties of films The mechanical properties of films including tensile strength (MPa) and elongation at break (%) were performed at 25 °C and 51% RH. Tests were performed using a Testometric Machine M350-10CT (Testometric Co. Ltd., Rochdale, Lancs., England) according to the ASTM method D882-91 (ASTM, 1996). In preparing samples, films were cut into 1 10 cm2 strips. The films were held parallel with an initial grip separation of 5 cm and then pulled apart at a head speed of 25 mm/min. Elongation at break (deformation divided by initial grip separation and multiplying by 100) and maximum force were obtained from force versus deformation curves. Tensile strength was calculated by dividing maximum force by film cross section (thickness width).
MIC and MBC values (antibacterial properties) of CEO are presented in Table 2. The lower concentration of the essential oil could fully inhibit the growth or could almost kill L. sakei (MIC 250 lg/ mL and MBC 1000 lg/mL) and Ps. fluorescens (MIC 500 lg/mL and MBC 1000 lg/mL). However, MIC and MBC of the essential oil for the other three bacteria were 500 and more than 1500 lg/ mL, respectively. This is not supported by many other reports on the greater susceptibility of gram-positive bacteria to inhibitory effect of essential oils and their components (Oussalah, Caillet, Saucier, & Lacroix, 2007; Shan, Cai, Brooks, & Corke, 2007). Similar results have been
Table 1 Essential oil composition of Cinnamomum zeylanicum. No.
Compound
RI
% of the total peak area
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
a-Pinene
936 950 982 1001 1010 1016 1026 1086 1188 1247 1312 1334 1386 1398 1413 1431 1463 1492 1585 1736 2049
2.18 0.14 1.03 0.54 0.94 2.85 3.32 6.46 0.29 60.41 0.48 3.19 0.34 0.43 2.01 3.50 0.27 3.63 0.34 0.65 0.91
Camphene Myrcene a-Phellandrene d-3-Carene P-cymene 1,8-Cineole Linalool Z-Cinnamaldehyde E-Cinnamaldehyde Hexagerman Eugenol a-Copaene Coumarine E-Cynnamyl acetate b-Caryophyllene a-Humulene O-methoxycinnamaldehyde Caryophyllene oxide Benzyl benzoate Equilin
2.8. Scanning electron microscopy The chitosan-based films were mounted on the specimen holder with aluminium tape and then sputtered with gold in BAL-TEC SCD 005 sputter coater (BAL-TEC AG, Balzers, Liechtenstein). All the specimens were examined with a Philips XL 30 scanning electron microscope (Philips, Eindhoven, Netherlands) under high vacuum condition and at an accelerating voltage of 20.0 kV. Samples were photographed at tilt angles of 60–90° to the electron beam for the views in the cross section.
Table 2 MIC and MBC values of CEO against five tested bacterial strains. Bacteria
L. monocytogenes
E. coli
Ps. fluorescens
L. plantarum
L. sakei
MIC (ppm) MBC (ppm)
500 >1500
500 >1500
500 1000
500 >1500
250 1000
164
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
reported by Kim et al. (1995) who found that L. monocytogenes was more resistant to the inhibitory effects of eleven essential oil constitutes than the gram-negative bacteria tested under the same condition, including E. coli, E. coli O157:H7, Salmonella typhimurium and Vibrio vulnificus. It seems that the variability of the inhibitory effect of essential oil may be due to differences between strains of the same bacterial species. This hypothesis was confirmed by Sivropoulou et al. (1996) with two strains of Staphylococcus aureus in the presence of carvacrol and thymol.
3.3. Antimicrobial properties of films Effects of CEO addition on antimicrobial properties of chitosanbased films are shown in Table 3. When antimicrobial agents are incorporated into films, these materials diffuse through agar gel and result in clear zone around the film cuts. Incorporation of CEO into chitosan-based films at higher than 0.4% (v/v) exhibited a clear inhibitory zone by the absence of bacterial growth around the film cuts. At CEO concentration of 0.4% (v/ v) the clear zone of inhibition was not observed with L. plantarum. As the concentration increased, the zone of inhibition also increased significantly (p < 0.05). However, increasing level of CEO at its high concentration did not reveal significant an increased inhibitory. It was generally caused by the compactness of chitosan film network containing CEO because of the occurrence of functional groups interaction phenomenon. The active component of CEO is cinnamaldehyde (Matan et al., 2006). As shown in Table 1, cinnamaldehyde constitutes more than 60% of CEO. Sublethal concentrations of cinnamaldehyde have been found to inhibit production of amylase and proteases by Bacillus cereus (Thoroski, Blank, & Biliaderis, 1989). Chitosan control films did not show inhibitory zone in bacterial strains tested. Despite antimicrobial activity of chitosan because of its innate characteristic, this effect of chitosan occurred without
migration of active agents. Chitosan does not diffuse through the adjacent agar media in agar diffusion test method; so that only organisms in direct contact with the active sites of chitosan are inhibited (Coma et al., 2002). 3.4. Physical properties of films The effects of incorporating CEO on the physical properties of chitosan films are shown in Table 4. Thin films were easily removed from the cast plate. Thickness of films varied between 0.095 and 0.107 mm, as shown in Table 4. In general, the moisture content value decreased as CEO was incorporated into chitosan-based film, which is attributed to compactness of film network. CEO caused to formation covalent bonds between the functional groups of chitosan chains, leading to a decrease in the availability of hydroxyl and amino groups and limiting polysaccharide–water interactions by hydrogen bonding and resulting in a decrease of moisture content value of edible films (Park & Zhao, 2004). As CEO concentration increased, the moisture content of films decreased significantly (p < 0.05). Chitosan control films were compact, and the film surface had a smooth surface without pores or cracks (Fig. 1A and B). Control film had water vapour permeability coefficient (WVPC) value 2.250 1010 g s1 m1 Pa s1. Same results were obtained by Wong, Gastineau, Gregorski, Tillin, and Pavlath (1992), Caner et al. (1998) and Seydim and Sarikus (2006). Incorporation of CEO into chitosan-based films decreased WVPC of films. Beside the hydrophobic nature of CEO which could affect the hydrophilic/hydrophobic property of the films, the physical factors had an influence on the WVPC of the films enriched with CEO. When CEO was incorporated into the chitosan film formulation, the sur-
Table 4 Physical properties of chitosan films incorporated with CEO.
Table 3 Antibacterial activity (inhibitory zone) of chitosan films incorporated with CEO against gram-positive and gram-negative bacteria.
*
Physical properties*
CEO conc. (v/v) in film solution (%)
Thickness (mm)
Control 0.4 0.8 1.5 2
0.095 ± 0.0025b 0.098 ± 0.0024b 0.104 ± 0.0029a 0.105 ± 0.0032a 0.107 ± 0.0039a
Bacteria
CEO conc. (v/v) in film solution (%)
Inhibitory zone* (mm2)
L. monocytogenes (Gram +)
Control 0.4 0.8 1.5 2
0c 38.17 ± 1.00b 39.80 ± 0.94b 53.05 ± 1.84a 52.31 ± 2.65a
Moisture content (%)
Control 0.4 0.8 1.5 2
20.82 ± 1.82a 18.72 ± 2.11a 14.04 ± 0.93b 10.82 ± 1.47c 8.47 ± 1.53c
L. plantarum (Gram +)
Control 0.4 0.8 1.5 2
0c 0c 43.6 ± 0.94b 49.80 ± 2.77a 53.64 ± 3.07a
Water vapor permeability coefficient (g s1 m1 Pa s1 1010)
Control 0.4 0.8 1.5 2
2.250 ± 0.074a 1.352 ± 0.152b 1.234 ± 0.040b 1.014 ± 0.040c 1.003 ± 0.067c
L. sakei (Gram +)
Control 0.4 0.8 1.5 2
0c 39.60 ± 2.36b 41.08 ± 1.44b 56.69 ± 2.82a 57.23 ± 1.50a
Water contact angle (°)
Control 0.4 0.8 1.5 2
37.3 ± 2.25d 39.6 ± 1.89d 45.3 ± 1.38c 59.2 ± 2.77b 70.3 ± 1.42a
Ps. fluorescens (Gram )
Control 0.4 0.8 1.5 2
0c 34.68 ± 1.71b 36.91 ± 1.32b 41.60 ± 2.09a 43.60 ± 1.21a
Solubility in water (%)
Control 0.4 0.8 1.5 2
23.2 ± 1.09a 21.6 ± 0.65a 16.8 ± 0.85b 13.6 ± 1.55c 10.4 ± 0.94d
E. coli (Gram )
Control 0.4 0.8 1.5 2
0d 36.31 ± 0.77c 38.90 ± 0.94b 51.72 ± 1.15a 51.04 ± 2.12a
Total colour difference (DE)
Control 0.4 0.8 1.5 2
17.35 ± 0.98d 17.83 ± 0.47d 21.29 ± 0.55c 23.35 ± 0.87b 25.62 ± 0.58a
Means in each column with different superscript letters are significantly different (p < 0.05). Control is a film disc containing no essential oil.
*
Means in each column with different superscript letters are significantly different (p < 0.05).
165
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
Fig. 1. Scanning electronic microscopic images of chitosan control film (A) and (B), and film containing CEO at level of 1.5% (C) and (D) surfaces and cross sections, respectively.
face view suggested a sheet like and dense structure, while the cross section revealed the sheets stacked in compact layers (Fig. 1C and D), which showed CEO incorporated uniformly in the matrix. The value of the contact angle with water indicates how hydrophobic the surface is. It is well-known that the water contact angle will increase with increasing surface hydrophobicity. The higher hydrophilicity of the control film is attributed to the water binding capacity of plasticiser (glycerol) and functional groups of chitosan. According to Table 4, addition of CEO increased water contact angle of films and resulted in decreasing hydrophilicity of the chitosan films which might be due to the loss of free functional groups (amino and hydroxyl groups). Chitosan control film had low solubility in water below 24% at 25 °C after 6 h dipping. Same result was reported by García, Pinotti, Martino, and Zaritzky (2004). Control film in general was very hydrophilic thus absorbed water quickly and resulted in swelling. Incorporation of CEO into the chitosan film formulation at level of 1.5% and 2% (v/v) led to 41% and 55% reduction in solubility in water respectively. This phenomenon is due to the cross-linking effects of CEO components leading to esters and/or amide groups. Cross-linking in the chitosan film matrix resulted in a decrease in solubility in water and produced films with low affinity toward water which is beneficial when product integrity and water resistance are intended. Cross-linking of chitosan-hydroxy propyl methyl cellulose based films using citric acid (polycarboxylic acid) as the cross-linking agent, was reported by Möller, Grelier, Pardon, and Coma (2004). The colour of the films may influence the consumer acceptability of a product (Sivarooban, Hettiarachchy, & Johnson, 2008). Total colour differences (DE) of chitosan films are shown in Table 4. Visually, chitosan films had a slightly yellow appearance. Control film had DE value 17.35. Its transparency was reduced as the CEO was incorporated. Incorporating CEO revealed DE values, which were significantly higher than that of the control. The values of chromaticity parameter b of chitosan films incorporated with CEO were higher than that of control film (data not shown). These
results agreed with visual observations. Nevertheless, the sensory studies should be conducted for evaluating consumer acceptability of this colour change.
3.5. Mechanical properties of films The effect of incorporating CEO on mechanical properties of chitosan films is presented in Table 5. Chitosan control film had tensile strength value 10.97 MPa. Incorporation of CEO into chitosan films increased tensile strength values significantly (p < 0.05). As previously mentioned, a strong interaction between the polymer and the CEO produced a cross-linker effect, which decreases the free volume and the molecular mobility of the polymer. This phenomenon led to a sheet like structure (Fig. 1C). Arrangement of stacking layers of CEO added chitosan sheets (Fig. 1D) means that in these films a compact structure was present thus increased continuities within the polysaccharide network leading to decrease in elongation at break. There are also possibilities such as decrease in moisture content of films incorporated with CEO during the film production, thus leading to the decrease in strain and the increase
Table 5 Mechanical properties of chitosan films incorporated with CEO.
*
Mechanical properties*
CEO conc. (v/v) in film solution (%)
Tensile strength (MPa)
Control 0.4 0.8 1.5 2
10.97 ± 0.54e 13.35 ± 1.23d 17.43 ± 1.08c 24.10 ± 1.47b 29.23 ± 2.25a
Elongation at break (%)
Control 0.4 0.8 1.5 2
24.73 ± 1.86a 16.57 ± 0.77b 11.26 ± 1.39c 6.42 ± 0.63d 3.58 ± 0.35e
Means in each column with different superscript letters are significantly different (p < 0.05).
166
S.M. Ojagh et al. / Food Chemistry 122 (2010) 161–166
in tensile strength. The results of moisture content measurements are shown in the Table 4. According to Gontard, Guilbert, and Cuq (1993) and Pouplin, Redl, and Gontard (1999) water is the most ubiquitous and uncontrollable plasticiser for most hydrocolloidbased films because of its ability to modify the structure of natural polymers. Thus, the plasticisation effect of water could not be negligible for these films, and their plasticisation efficiency does not only come from intrinsic plasticiser action of glycerol. Differences between data of chitosan films obtained in the current study and those of reported in literature (Caner et al., 1998; García et al., 2004) may be attributed to chitosan composition and suppliers, plasticiser presence and film preparation. 4. Conclusions Chitosan is a promising biopolymer for active food packaging. Its sensitivity to moisture can be offset by blending it with CEO. The results of this study showed that a unique compatibility can be achieved between chitosan and CEO. The incorporation of CEO improved the antibacterial properties of chitosan. Films containing CEO are useful for coating of highly perishable foods such as fish and poultry. The other edible films may be ruptured upon contact with wet surfaces. Functional groups interaction phenomenon in edible films has critical effect on their antibacterial, physical and mechanical properties which are important in food packaging applications. Moreover, the antimicrobial effect of CEO enriched films should be determined on an entire model food. References Adams, R. P. (2001). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Allured, Carol Stream, IL. 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 (1996). Standard test methods for tensile properties of thin plastic sheeting, D882-91. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Material. Beverlya, R. L., Janes, M. E., Prinyawiwatkula, W., & No, H. K. (2008). Edible chitosan films on ready-to-eat roast beef for the control of Listeria monocytogenes. Food Microbiology, 25, 534–537. Caner, C., Vergano, P. J., & Wiles, J. L. (1998). Chitosan film mechanical and permeation properties as affected by acid, plasticizer and storage. Journal of Food Science, 63, 1049–1053. Cha, D. S., Choi, J. H., Chinnan, M. S., & Park, H. J. (2002). Antimicrobial films based on Na-alginate and j-carrageenan. Lebensmittel-Wissenschaft und -Technologie, 35, 715–719. Coma, V., Martial-Gros, A., Garreau, S., Copinet, A., Salin, F., & Deschamps, A. (2002). Edible antimicrobial films based on chitosan matrix. Journal of Food Science, 67, 1162–1169. Davies, N. W. (1990). Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone on Carbowax 20M phases. Journal of Chromatography, 503, 1–24. Dorman, H. J., & Deans, S. G. (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308–316.
García, M. A., Pinotti, A., Martino, M. N., & Zaritzky, N. E. (2004). Characterization of composite hydrocolloid films. Carbohydrate Polymers, 56, 339–345. 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. Gontard, N., Guilbert, S., & Cuq, J. (1993). Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58, 206–211. Holley, R. A., & Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials: A review. Food Microbiology, 22, 273–292. Jeon, Y.-I., Kamil, J. Y. V. A., & Shahidi, F. (2002). Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod. Journal of Agricultural and Food Chemistry, 20, 5167–5178. Kim, J., Marshall, M. R., & Wei, C. (1995). Antimicrobial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry, 43, 2839–2845. Matan, N., Rimkeeree, H., Mawson, A. J., Chompreeda, P., Haruthaithanasan, V., & Parker, M. (2006). Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. International Journal of Food Microbiology, 107, 180–185. Möller, H., Grelier, S., Pardon, P., & Coma, V. (2004). Antimicrobial and physicochemical properties of chitosan-HPMC-based films. Journal of Agricultural and Food Chemistry, 52, 6585–6591. Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. Food Chemistry, 120, 193–198. Oussalah, M., Caillet, S., Saucier, L., & Lacroix, M. (2007). Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control, 18, 414–420. Park, S., & Zhao, Y. (2004). Incorporation of a high concentration of mineral or vitamin into chitosan-based films. Journal of Agricultural and Food Chemistry, 52, 1933–1939. 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, 538–543. Seydim, A. C., & Sarikus, G. (2006). Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Research International, 39, 639–644. Shahidi, F., Arachchi, J. K. V., & Jeon, Y. J. (1999). Food application of chitin and chitosans. Trends in Food Science and Technology, 10, 37–51. Shan, B., Cai, Y., Brooks, J. D., & Corke, H. (2007). Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum burmannii): Activity against foodborne pathogenic bacteria. Journal of Agricultural and Food Chemistry, 55, 5484–5490. Sivarooban, T., Hettiarachchy, N. S., & Johnson, M. G. (2008). Physical and antimicrobial properties of grape seed extract, nisin, and EDTA incorporated soy protein edible films. Food Research International, 41, 781–785. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T., & Arsenakis, M. (1996). Antimicrobial and cytotoxic activity of origanum essential oils. Journal of Agricultural and Food Chemistry, 44, 1202–1205. Thoroski, J., Blank, G., & Biliaderis, C. (1989). Eugenol induced inhibition of extracellular enzyme production by Bacillus cereus. Journal of Food Protection, 52, 399–403. Tsai, G. I., Wu, Z. Y., & Su, W. H. (2000). Antimicrobial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. Journal of Food Protection, 63, 747–752. Wong, D. W. S., Gastineau, F. A., Gregorski, K. S., Tillin, S. J., & Pavlath, A. E. (1992). Chitosan lipid films: Microstructure and surface energy. Journal of Agricultural and Food Chemistry, 40, 540–544.