2016 年 22 巻 2 号 p. 291-296
The chitosan-based active films were developed by incorporation of cinnamon essential oil (CEO, 10 g/L), pomegranate peel extract (PPE, 10 g/L) and CEO (10 g/L) + PPE (10 g/L) and their physical, antioxidant and antibacterial properties were investigated. Incorporation of CEO and CEO + PPE into the films significantly decreased the water vapor permeability. Incorporation of CEO, PPE and CEO + PPE into the films decreased the transparency, but significantly increased the antioxidant activity. Incorporation of CEO and CEO + PPE exhibited significant antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, the antibacterial activity against the Staphylococcus aureus in the film incorporated with CEO and PPE was significantly higher than that incorporated with CEO or PPE alone, suggesting that there is a synergistic action between CEO and PPE.
Active packaging technologies have greatly developed in the last decade by trying to satisfy the need for long-life processed food in addition to antioxidant/antimicrobial (AA) components in the packaging material. Given the increasing health concerns of consumers, there is an interest in films produced from natural resources due to the excellent biodegradability, biocompatibility and edibility. Plant extracts have shown great potential to be used as renewable, biodegradable and valuable sources that can be incorporated into films. Essential oils and their main constituent compounds have been extensively investigated due to their application in the food industry for improving the shelf life of perishable products (Llana-Ruiz-Cabello et al., 2015). 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 et al., 2010). Cinnamon essential oil (CEO) has been used for centuries to protect food against microorganisms, and in the last ten years CEO is also incorporated into food packaging materials as antimicrobial agent (Ojagh et al., 2010; Peng and Li, 2014; Hosseini et al., 2009; Wen et al., 2016).
Chitosan is a biodegradable, non-toxic, and biocompatible polysaccharide. Although chitosan has a great potential for applications in antimicrobial packaging, it has no significant antioxidant (Wang et al., 2013) and ambiguous antimicrobial activity (Ruiz-Navajas et al., 2013; Ojagh et al., 2010). Therefore, AA agents were incorporated into the chitosan film to expand its applications in food active packaging (Moradi et al., 2012). However, there is little work concerning the combined effects of two kinds of AA agents on the physical, antimicrobial and antioxidant properties of chitosan films. The use of combinations of natural antimicrobial (AM) agents may increase the spectrum of AM activity and also produce synergistic interactions against microorganisms (Choi et al., 2009). The objectives of the present study were to develop composite films from chitosan incorporated with CEO (10 g/L) and pomegranate peel extract (PPE, 10 g/L) alone and in combination, and to investigate the effect of these extracts on the physical, antibacterial and antioxidant properties of the films.
Materials Escherichia coliATCC8099 and Staphylococcus aureus ATCC6538 were used in the present study. Chitosan with degree of deacetylation of 90% were purchased from Shanghai Jinsui Biotechnology Company (Shanghai, China). PPE was prepared according to our previous study (Yuan et al., 2015).
Film Preparation Chitosan films were prepared by casting/solvent evaporation method. First, chitosan solution was prepared with 2% (w/w) chitosan in 1% (w/w) acetic acid at room temperature. After overnight agitation, the solution was filtered using a filter cloth to remove any insoluble particles. Glycerol (glycerol/chitosan = 0.5, w/w) and Tween 80 at 0.5% (w/w) were added into the solution and mixed with 30 min of stirring. Then, PPE and CEO were added to the chitosan solution. The different solutions were prepared: (i) control, without added agents; (ii) chitosan with 10 g/L CEO; (iii) chitosan with 10 g/L PPE; (iv) chitosan with 10 g/L CEO + 10 g/L PPE. After addition of PPE and CEO, all the solutions were homogenized at 13,000 rpm for 5 min to obtain an emulsion. Finally, the emulsions were poured into glass Petri dishes dried at incubator chamber (25 ± 2°C) for 48 h with 50 ± 2% relative humidity (RH). The films were then peeled from the plates and placed at 50 ± 2% RH at 25°C.
Film Thickness, color and opacity measurements The thickness of films was measured using a micrometer (Mitutoyo, Mitutoyo Corporation, Japan). Measurements were taken at five different locations of each film sample and the average film thickness was calculated.
The color of film samples was measured by using an automatic color difference meter (Minolta Chroma Meter CR-10, Minolta, Osaka, Japan) according to our previous study (Yuan et al., 2015). Film pieces (20 mm in diameter) were evenly distributed on a white reflector standard plate as background and L* (lightness), a* (redness) and b* (yellowness) values were measured with a halogen lamp as an illuminant. For each sample, six measurements were taken on each.
Opacity was determined by measuring the film absorbance at 600 nm using a UV spectrophotometer (UV-2800, Unico, USA). Each film specimen was cut into rectangular shape and directly placed on the internal side of the spectrophotometer cell. An empty test cell was used as the reference. The opacity of the films was obtained by the following equation:
 |
Where Abs600 is the value of absorbance at 600 nm and d is the film thickness (mm).
Water vapor permeability WVP was determined following ASTM method with slight modification (Kanmani and Rhim, 2014). Film samples were sealed to glass cups which had 5 cm diameter and contained water. Film-covered cups were placed in an environmental chamber set at 25°C and 50% RH. The weight of cup was measured periodically until steady state was reached. Water vapor transmission rate (WVTR) of the films was determined from the slope of the weight change of the cup vs. time curve and then used to calculate the WVP using the following equation:
 |
Where WVTR was the measured water vapor transmission rate (g/(m2 s)), L is the mean film thickness, and ΔP is the partial water vapor pressure difference (Pa) across the two sides of the film.
Mechanical Properties Tensile strength (TS) and percentage elongation at break (E %) were determined according to our previous study (Yuan et al., 2015). TS and E % were measured with a Universal Testing Machine (TA.XT plus model, Stable Micro Systems, UK) fitted with a 50 N static load cell.
Antioxidant activity The ferric reducing antioxidant power (FRAP) assay was determined according to the method of Benzie and Strain (1996). The extracts were prepared by homogenizing 25 mg film with ethanol for 5 min at 10,000 rpm and then centrifuged at 3,000 rpm for 10 min. The working FRAP reagent was prepared daily by mixing 300 mM acetate buffer (pH 3.6), 20 mM ferric chloride, and 10 mM 2,4,6-tripyridyl-S-triazine in 40 mM HCl in the ratio of 10:1:1 (v/v/v). The extracted samples (20 µL) were added to 2.8 mL of the FRAP working solution incubated at 37°C and vortexed. The absorbance was then recorded at 593 nm using a spectrophotometer (UV-2800, Unico, USA) after the mixture had been incubated at 37°C for 10 min. FRAP values were calculated from FeSO4 − 7H2O standard curves and expressed as mmol FeSO4 − 7H2O equivalents/µg of film.
Antibacterial activity The agar diffusion method was used to determine the antibacterial activity according to the method of Wang et al. (2011). The films were prepared as the above film preparation method. The nutrient agar medium in Petri dish was inoculated with 0.1 mL 105–106 cuf/mL bacteria. The prepared films were cut into 10 mm diameter disks using a hole-puncher and then placed on microbial cultures. Bacterial strains were incubated at 37°C and 50 ± 2% relative humidity for 24 h. The diameter of the zone of inhibition was measured using a caliper. The tests were performed in triplicate.
Statistical analysis Statistical analysis was performed using the SPSS package program version 11.5 (SPSS inc. Chicago, IL, USA). Data was analyzed by one-way ANOVA, followed by Turkey's HSD multiple comparison test. The values are reported as means with their standard error for all results. Differences were considered significant at P < 0.05.
Film thickness, color and opacity Table 1 shows the effects of CEO and PPE incorporation on film thickness, color and opacity. The thickness of four films varied between 0.088–0.130 mm. Adding 10 g/L CEO, 10 g/L PPE and 10 g/L CEO + 10 g/L PPE into chitosan films did not significantly affect the thickness of the films.
| Film samples | Thickness (mm) | Opacity (A mm−1) | Color | ||
|---|---|---|---|---|---|
| L* | a* | b* | |||
| Control | 0.105 ± 0.03a | 1.290 ± 0.22c | 90.43 ± 0.27a | −0.67 ± 0.23d | +11.93 ± 0.34d |
| CEO | 0.088 ± 0.02a | 2.196 ± 0.12b | 90.97 ± 0.38a | −0.08 ± 0.44c | +15. 90 ± 0.23c |
| PPE | 0.111 ± 0.02a | 2.394 ± 0.24b | 63.30 ± 0.54b | +14. 30 ± 0.22b | +54. 60 ± 0.55a |
| CEO+PPE | 0.130 ± 0.03a | 3.053 ± 0.31a | 55.60 ± 0.29c | +22. 50 ± 0.51a | +50. 50 ± 0.38b |
Mean values in each column with different lower case letters are significantly different (P < 0.05). The value of PPE was cited from our previous study (Yuan et al., 2015). PPE, pomegranate peel extract; CEO, cinnamon essential oil.
The color parameter is an important property of films and may influence the consumer acceptability of a product (Ojagh et al., 2010). In general, chitosan films showed a tendency to yellow (b*). Adding CEO significantly increased b* values (yellowness/blueness) of the film in comparison with the control (P < 0.05). The present result is in agreement with those of previous studies, which found that the yellow coloration in the chitosan film was intensified after addition with CEO ranged at level from 0.4% to 2% (Ojagh et al., 2010; López-Mata et al., 2015; Peng and Li, 2014). The differences in color could be ascribed to the natural yellow of essential oils (Atarés et al., 2010). However, the coloration of films was significantly lower when CEO was incorporated at concentrations of 0.25% and 0.5% (López-Mata et al., 2015). Adding CEO + PPE into chitosan films also significantly affected L* (lightness/darkness), a* (redness/greenness) and b* values of the films when compared with the control (p < 0.05), which could be attributed to the presence of polyphenols in PPE (Yuan et al., 2015).
The high values of opacity indicate lower transparency and higher degree of opacity (Wang et al., 2013). The transparency of films incorporated with 10 g/L CEO was significantly decreased, which is consistent with the results of Ojagh et al. (2010) and Hosseini et al. (2009), who found that the transparency of chitosan films with CEO was significantly decreased. However, the present results is inconsistent with López-Mata et al. (2015), who found chitosan films with 0.5% and 1.0% CEO incorporated presented greater transparency than the control. In addition, the transparency of films supplemented with 10 g/L PPE and 10 g/L CEO +10 g/L PPE was markedly decreased in comparison with the control (Table 1), which could be caused by the presence of polyphenols in films (Yuan et al., 2015)
Water Vapor Permeability Chitosan films have been proven to present moderate oxygen barrier properties and good carbon dioxide barrier properties but high permeabilities to water vapor, due to their hydrophilic nature (Butler et al., 1996). In order to improve water barrier properties of chitosan films, hydrophobic compounds, such as lipids and essential oils are frequently incorporated (Vargas et al., 2009; Peng and Li, 2014). Incorporation of CEO alone (Ojagh et al., 2010) or the mixtures of CEO, lemon, thyme essential oils (Peng and Li, 2014) into chitosan-based films also have been proved to decrease the WVP of films. The result obtained from the present study is consistent with those findings. In the present study, the WVP of the film incorporated with 10 g/L CEO and 10 g/L CEO + 10 g/L PPE was significantly decreased when compared with the control (Fig. 1). The decrease in WVP of the chitosan films enriched with CEO could be due to the hydrophobic nature of CEO which could affect the hydrophilic/hydrophobic property of the films and the physical factors (Ojagh et al., 2010). However, other studies showed that the incorporation of 0.5 and 1.0% of CEO into chitosan films did not cause significant difference compared with the control (Hosseini et al., 2009; López-Mata et al., 2015). Different effect on WVP of chitosan films enriched CEO in the present study and those reported in literature (Hosseini et al., 2009; López-Mata et al., 2015) may be attributed to chitosan composition and suppliers, plasticizer presence and film preparation.
Water vapor permeability for chitosan films incorporated with pomegranate peel extract and cinnamon essential oil. Each data is the mean values per treatment and time point (mean ± standard error). The value of PPE was cited from our previous study (Yuan et al., 2015). PPE, pomegranate peel extract; CEO, cinnamon essential oil.
Mechanical Properties Figure 2 shows the mechanical properties of films. TS indicates the maximum tensile stress that the film can sustain, E % is the maximum change in length of a test specimen before breaking. Incorporation of 10 g/L CEO and 10 g/L CEO + 10 g/L PPE significantly increased the TS and significantly decreased of chitosan films when compared with the control. The result obtained from the present study is agreement with previous studies (Ojagh et al., 2010; Hosseini et al., 2009). The change in TS and E % could be due to the 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 (Ojagh et al., 2010; Hosseini et al., 2009).
Tensile strength and percentage elongation at break for chitosan films incorporated with pomegranate peel extract and cinnamon essential oil. Mean values in each column with different lower case letters are significantly different (P < 0.05). The value of PPE was cited from our previous study (Yuan et al., 2015). PPE, pomegranate peel extract; CEO, cinnamon essential oil.
The antioxidant activity Figure 3 shows the antioxidant activity of the films. Incorporation of 10 g/L PPE, 10 g/L CEO and 10 g/L CEO + 10 g/L PPE significantly increased the antioxidant activity of the films when compared with the control. Previous studies have reported that CEO has considerable antioxidant activity and addition with CEO in the chitosan film could exert antioxidant activity (Nanasombat and Wimuttigosol, 2011; Mathew and Abraham, 2006). This activity could be ascribed to the presence of the main CEO antioxidant, which is eugenol; however, it could also be due to the presence of cinnamaldehyde, which has been reported to have a lower activity than eugenol, or to a synergetic effect among chitosan, eugenol, and cinnamaldehyde (Mathew and Abraham, 2006).
The antioxidant activity for chitosan films incorporated with pomegranate peel extract and cinnamon essential oil. Each data is the mean values per treatment and time point (mean ± standard error). The value of PPE was cited from our previous study (Yuan et. al, 2015). PPE, pomegranate peel extract; CEO, cinnamon essential oil.
In addition, the antioxidant activity of the film with combined addition of CEO and PPE was significantly higher than that incorporated with CEO or PPE alone, suggesting that there is an additive action between CEO and PPE. The higher antioxidant activity of the film with combined addition of CEO and PPE was plausibly due to the higher amount of added antioxidants. According to the studies by Gomez-Estaca et al. (2009) and Moradi et al. (2012), the degree of antioxidant power of edible film, generally is proportional to the amount of added antioxidant additives. The result obtained from the present study is consistent with those findings.
The antibacterial activity Table 2 shows the antibacterial activity of the films. Incorporation of 10 g/L CEO and 10 g/L CEO + 10 g/L PPE have significantly inhibitory activity against Gram-negative E. coli, and those incorporation of 10 g/L PPE, 10 g/L CEO and 10 g/L CEO + 10 g/L PPE also have significantly inhibitory activity against Gram-positive S. aureus.
| Film samples | Inhibition zone (mm) | |
|---|---|---|
| S. aureus | E. coli | |
| Control | ND | ND |
| CEO | 11.00 ± 0.58b | 15.50 ± 0.29a |
| PPE | 2.50 ± 0.29c | ND |
| CEO+PPE | 15.00 ± 1.00a | 15.67 ± 0.88a |
Mean values in each column with different lower case letters are significantly different (P < 0.05). The value of PPE was cited from our previous study (Yuan et al., 2015). PPE, pomegranate peel extract; CEO, cinnamon essential oil; ND: not detected.
The antibacterial properties of essential oils have been known for many centuries. In recent years, a large number of essential oils and their constituents have been investigated for their antimicrobial properties against some bacteria and fungi (Vergis et al., 2015). The antibacterial activity of CEO-incorporated chitosan film against E. coli and S. aureus was previously reported. Incorporation of CEO into chitosan films showed significant antibacterial activity against S. aureus and E. coli in the present study, which matches the previous studies (Ojagh et al., 2010; Peng and Li, 2014; Hosseini et al., 2009). The antibacterial mechanism of CEO could be able to disrupt and penetrate the lipid structure of the bacteria cell membrane, leading to its destruction (Turina et al., 2006).
Essential oils containing carvacrol, cinnamaldehyde, cinnamic acid, eugenol and thymol can have a synergistic effect in combination with other antibacterial agents (Langeveld et al., 2014; Bassolé and Juliani, 2012). The present study showed that the antibacterial activity against the S. aureus in the film incorporated with CEO + PPE was significantly higher than that incorporated with CEO or PPE alone, suggesting there is a synergistic action between CEO and PPE. Similar with our previous study, the degree of antibacterial activity of edible film could be proportional to the amount of antibacterial agents (Yuan et al., 2015). As a general conclusion, the incorporation of CEO and PPE to chitosan films conferred some antimicrobial activity against bacterial strains potentially present in food to the films.
This study indicated that active films from chitosan-based films could be prepared by incorporation with CEO, PPE and CEO + PPE. Incorporation of CEO, PPE and CEO + PPE decreased the transparency, but significantly increased the antioxidant activity of the films. Incorporation of CEO and CEO + PPE significantly decreased the WVP of the films. Incorporation of CEO and CEO + PPE exhibited significant antibacterial activity against E. coli and S. aureus. In addition, a synergistic action in the antibacterial activity against the S. aureus was found in the film with combined incorporation of CEO and PPE. The chitosan films incorporated with PPE and CEO shows potential to be used as active films. Nevertheless, further studies are warranted before using these films as active packaging for food products.
Acknowledgments This work was supported by Key Laboratory of Health Risk Factors for Seafood of Zhejiang Province (No. 2014E10002).
