Development of new active packaging film made from a soluble soybean polysaccharide incorporated Zataria multiflora Boiss and Mentha pulegium essential oils

Food Chemistry 146 (2014) 614–622

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Development of new active packaging film made from a soluble soybean polysaccharide incorporated Zataria multiflora Boiss and Mentha pulegium essential oils Davoud Salarbashi a, Sima Tajik b,1, Saeedeh Shojaee-Aliabadi c, Mehran Ghasemlou d,⇑, Hamid Moayyed e, Ramin Khaksar c, Mostafa Shahidi Noghabi f a

Department of Food Science and Technology, Sabzevar Branch, Islamic Azad University, Sabzevar, Iran Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Beheshti Avenue, Gorgan, Iran c Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran d School of Packaging, Michigan State University, East Lansing, MI 48824, USA e School of Health Sciences, Faculty of Health and Behavioural Sciences, University of Wollongong, Wollongong 2522, Australia f Department of Food Chemistry, Research Institute of Food Science and Technology, PO Box 14 91735-147, Mashhad-Quchan Highway, Mashhad, Iran b

a r t i c l e

i n f o

Article history: Received 11 May 2013 Received in revised form 6 August 2013 Accepted 3 September 2013 Available online 11 September 2013 Keywords: Soluble soybean polysaccharide Antioxidant properties Wettability Essential oil Contact angle

a b s t r a c t An active edible film from soluble soybean polysaccharide (SSPS) incorporated with different concentrations of Zataria multiflora Boiss (ZEO) and Mentha pulegium (MEO) essential oils was developed, and the film’s optical, wettability, thermal, total phenol and antioxidant characteristics were investigated, along with their antimicrobial effectiveness against Staphylococcus aureus, Bacillus cereus, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium. The film’s colour became darker and more yellowish and had a lower gloss as the levels of ZEO or MEO were increased. Antioxidant activity of the films was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric-reducing antioxidant power assays. DPPH was reduced in the range of 19.84–74.12% depending on the essential oil type and concentration. Film incorporated with 3% (v/v) ZEO showed the highest DPPH radical scavenging activity and ferric reducing antioxidant power (IC50 = 4188.60 ± 21.73 mg/l and EC50 = 8.86 ± 0.09 mg/ml, respectively), compared with the control and MEO added film. Films containing ZEO were more effective against the tested bacteria than those containing MEO. S. aureus was found to be the most sensitive bacterium to both ZEO or MEO, followed by B. cereus and E. coli. A highest inhibition zone of 387.05 mm2 was observed for S. aureus around the films incorporated with 3% (v/v) ZEO. The total inhibitory zone of 3% (v/v) MEO formulated films was 21.98 for S. typhimurium and 10.15 mm2 for P. aeruginosa. Differential scanning calorimetry (DSC) analysis revealed a single glass transition temperature (Tg) between 16 and 31 °C. The contact angle increased up to 175% and 38% as 3% (v/v) of ZEO or MEO used: it clearly shows that films with ZEO were more hydrophobic than those with MEO. The results showed that these two essential oils could be incorporated into SSPS films for food packaging. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The growing accumulation of synthetic plastic wastes, together with the difficulty of recycling most packaging, has stimulated food and packaging industries to explore new biodegradable packaging materials (Tharanathan, 2003). In recent years, there has been a growing interest in edible films and coatings, which offer several advantages over synthetic materials, such as being biodegradable

⇑ Corresponding author. Tel.: +1 517 355 9580; fax: +1 517 353 8999. 1

E-mail addresses: [email protected], [email protected] (M. Ghasemlou). This author has contributed equally as first author in this work.

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.014

and environmentally friendly (Salmieri & Lacroix, 2006). Despite increased awareness of the importance of high-level hygiene in the food supply chain, foodborne illnesses caused by microorganisms are still a large public health problem (de Moura, Mattoso, & Zucolotto, 2012). Therefore, recent decades have seen extensive investigation into antimicrobial packaging. This technology seems to be a promising alternative, since application of this method can improve food safety by inhibiting pathogenic bacteria or controlling spoilage flora using minimum amounts of active compounds (Ma, Tang, Yang, & Yin, 2013). The natural antimicrobial agents frequently employed in active packaging, include antimicrobial enzymes, bacteriocins, essential oils and phenolic compounds.

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

Essential oils derived from plants appear as interesting natural compounds, and their application as natural antibacterial agents against a wide range of foodborne pathogens has gained increasing attention in the food industry (Burt, 2004). Zataria multiflora Boiss (ZEO) and Mentha pulegium (MEO), which are locally called Avishan shirazi and Pune, respectively, are aromatic and medicinal plants belonging to Labiatae family. Some medicinal and antimicrobial effects of these essential oils have been reported in the literature (Mahboubi & Haghi, 2008; Ramezani, Hosseinzadeh, & Samizadeh, 2004; Sharififar, Moshafi, Mansouri, Khodashenas, & Khoshnoodi, 2007). However, information about their possible application as components of edible films is scarcely available. The antimicrobial activity of ZEO and MEO has been attributed to their main constituents which contain terpenes: thymol and carvacrol for ZEO (Ali, Saleem, Ali, & Ahmad, 2000; Shaffiee & Javidnia, 1997) and pulegone for MEO (Mahboubi & Haghi, 2008). They received the approval from the FDA for use as food additives and could be considered as the potential alternatives to synthetic additives in antimicrobial food packaging (Shakeri, Shahidi, Beiraghi-Toosi, & Bahrami, 2011). Materials available for forming films and coating generally fall into the categories of polysaccharides, proteins and lipids. Various polysaccharides have been used for the preparation of edible films; these have included corn starch (Kuorwel, Cran, Sonneveld, Miltz, & Bigger, 2013), psyllium seed gum (Ahmadi, Kalbasi-Ashtari, Oromiehie, Yarmand, & Jahandideh, 2012), cress seed gum (Jouki, Khazaei, Ghasemlou, & HadiNezhad, 2013) and kefiran (Ghasemlou, Khodaiyan, Oromiehie, & Yarmand, 2011). However, over the past few years, there has been a renewed interest in other new resources for the production of edible and biodegradable films. Recently, a novel polysaccharide has been extracted from the cellwall material of soybean cotyledon. This polysaccharide, soluble soybean polysaccharide (SSPS), has a pectin-like structure composed of a galacturonan backbone of homogalacturonan (a-1,4galacturonan) and rhamnogalacturonan (repeating units being comprised of a-1,2-rhamunose and a-1,4-galacturonic acid) branched by b-1,4-galactan and a-1,3- or a-1,5-arabinan chains (Nakamura, Furuta, Maeda, Takao, & Nagamatsu, 2002). SSPS has been reported to provide health benefits to humans by lowering the blood cholesterol, improving laxation and reducing the risk of diabetes (Shorey, Willis, Lo, & Steinke, 1985; Tsai, Vinik, Lasichak, & Lo, 1987). Apart from its nutritional value, it has various functions such as dispersion, stabilization, emulsification and adhesion (Asai et al., 1994). In our recent paper (Tajik et al., 2013), SSPS-based film plasticized with various concentrations of glycerol were prepared and their physical, thermal, barrier and mechanical properties were studied. The results of that study suggested that SSPS can produce biodegradable films with good appearance and satisfactory mechanical properties. Although significant improvements in various properties of SSPS based films have been proposed in that study, they are still not comparable to those of the synthetic plastic films. Therefore, some functional properties of these films need to be improved. Preliminary studies in our laboratory have shown that ZEO and MEO were more compatible essential oils with the SSPS polymer matrix. It seems that their combination in SSPS films can be an appropriate way to produce antimicrobial films with improved antioxidant properties. Considering that ZEO and MEO are gaining increasing importance for their considerable antimicrobial and antioxidant activities, the goal of this study was (1) to develop an antimicrobial-antioxidant film based on SSPS and two essential oils – ZEO and MEO; (2) to study the effect of incorporating essential oils (ZEO and MEO) on the film’s antioxidant and antimicrobial properties; and (3) to evaluate some characteristics of these films, such as their wettability, thermal and optical properties, to examine potential applications as food-packaging material.

615

2. Materials and methods 2.1. Materials Soluble soybean polysaccharide (abbreviated as SSPS) was kindly donated by Fuji Oil Co. (Osaka, Japan) for this study. According to the manufacturer, the composition of the product was: 5% protein, 5.4% moisture and 8.5% ash. Glycerol and Tween 80 were purchased from Merck, Germany. ZEO and MEO were obtained from Barij Essence Pharmaceutical Co., Kashan, Iran, and stored in a dark container at 4 °C until used. Mueller–Hinton Agar (MHA) and Mueller–Hinton Broth (MHB) were bought from Merck Co. (Darmstadt, Germany). Folin–Ciocalteu reagent, sodium carbonate, standard gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), phosphate buffer, trichloroacetic acid (TCA) and potassium ferricyanide were all purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of analytical grade or the highest grade available. 2.2. Bacterial strains Staphylococcus aureus ATCC 25923; Bacillus cereus PTCC 1154, Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 27853; Salmonella typhimurium ATCC 14028 were provided by Iranian Research organisation for Science and Technology (Tehran, Iran). Stock cultures of the studied bacteria were grown in MHB at 30 °C for 24 h before the tests. 2.3. Preparation of films The films were prepared according to the so-called casting technique. A SSPS solution was prepared by dissolving 3 g of SSPS in 100 ml distilled water to obtain 3% (w/v) film-forming solutions. To achieve a complete dispersion of the SSPS, the solution was stirred constantly for 40 min using a magnetic stirrer at 300 rpm on a hot plate. The solution was then mixed with a plasticizer (50% w/ w of the polysaccharide). Preliminary studies had been performed to define the optimum level of the plasticizer to be used in the filmogenic solutions for this study. Both glycerol and sorbitol have been tested as possible plasticizers, but the former gave better results. Thus, glycerol was added to the medium as a plasticizer, and stirring was continued for a further 15 min at 82 °C. Control films (without essential oils) were cast from this solution. Composite SSPS films were achieved according the procedure previously described by Shojaee-Aliabadi et al. (2013), who had worked with carageenanbased films. The emulsions were obtained by adding ZEO and MEO to the SSPS solution to reach final concentrations of 1%, 2% and 3% v/v. Tween 80 was added as an emulsifier in quantities proportional to the essential oils (0.1%, 0.2% and 0.3% v/v). The film solutions were homogenised at 20,000 rpm for 3 min in an Ultra-Turrax T-25 homogenizer (IKA T25 Digital Ultra-Turrax, Staufen, Germany). The film solutions were left for several minutes to naturally remove most of the air bubbles incorporated during stirring. All prepared film solutions, including the control and those with essential oil, were cast by pouring the mixture onto polystyrene Petri dishes (14 cm in diameter) placed 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 solution was used to achieve the required relative humidity. 2.4. Microbiological activity of the films 2.4.1. Disc diffusion method The disc-diffusion assay was used to examine the antimicrobial characteristics of the films. SSPS films were cut into 6 mm diame-

616

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

ter discs using a sterile punch. Film discs were then placed on plates containing MHA which had been previously seeded with 100 ll of an overnight broth culture containing approximately 108 CFU/ml of the test bacteria. The plates were incubated at 30 °C for 24 h. A control SSPS film (without essential oil) was run in parallel. The area of the whole zone was calculated and then subtracted from the film disc area, and the difference in area was reported as the zone of inhibition. The tests were carried out in triplicate for each formulation. 2.4.2. Disc volatilization method The disc volatilization method was used to examine the film’s antimicrobial activities in vapour phase as described in our previous study (Shojaee-Aliabadi et al., 2013). Briefly, edible films with different concentration of ZEO or MEO were aseptically cut into 6 mm diameter discs and then placed on the inside surface of the upper lid of the plates, which had been previously seeded with 100 ll of an overnight broth culture containing approximately 108 CFU/ml of the test bacteria. The inoculated plates were inverted with the dish on the top of each lid containing film. Parafilm was used to tightly seal the edge of each plate to prevent leakage of essential oil vapour, and then incubated at 30 °C for 24 h. The inhibition radius free of bacterial growth on each plate was measured with a digital calliper. The values obtained were used to calculate the whole zone area in mm2. The tests were carried out in triplicate for each formulation. 2.5. Total phenolic (TP) content assay The TP content of the films was determined according to the Folin–Ciocalteu method as described by Siripatrawan and Harte (2010) with a slight modification. Twenty-five milligramme of each film sample was dissolved in 5 ml of distilled water, then the extract solution (0.1 ml), distilled water (7 ml), and Folin–Ciocalteu reagent (0.5 ml) were mixed and kept at room temperature for 8 min, after which 1.5 ml sodium carbonate (2%, w/v) and water were added to obtain a final volume of 10 ml. The mixture was stored in darkness and at room temperature for 2 h. The absorbance values were then measured at 765 nm using a spectrophotometer (Shimadzu UV–VIS 1601, Japan). A calibration curve was drawn using gallic acid in specific concentrations and the total phenolic content of the films was expressed as mg gallic acid equivalents (GAE) per gram of dried film according to the following equation:

T¼

C:V M

ð1Þ

where T is total content of phenolics compound (milligramme per gram dried film, in GAE), C is the concentration of gallic acid obtained from the calibration curve (milligramme per millilitre), V is the volume of film extract (millilitre) and M is the weight of dried film (gram).

ambient temperature for 60 min. The absorbance was read against pure methanol at 517 nm and the percentage of DPPH radical scavenging activity was calculated using the following equation:

DPPH scavenging activity ð%Þ ¼ ðAbsDPPH  Absextract =AbsDPPH Þ  100 ð2Þ where AbsDPPH is the absorbance value at 517 nm of the methanolic solution of DPPH and Absextract is the absorbance value at 517 nm for the sample extracts. Each sample was assayed at least three times. The radical scavenging activity was also expressed as the IC50 value (mg/l), the concentration required to cause 50% of DPPH inhibition. The percentage of scavenged DPPH was plotted against the extract concentration, and that required to quench 50% of initial DPPH radical was obtained from the graph by linear regression. 2.6.2. Reducing power assay The reducing power assay of different film samples was carried out as described in our previous study (Salarbashi, Fazly Bazzaz, Sahebkar, Karimkhani, & Ahmadi, 2012). Every sample of each film was dissolved in 3 ml of methanol and then 1 ml of different dilutions was added to 2.5 ml of the phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%). The mixtures were incubated at 50 °C for 30 min, after which 2.5 ml of 100 g/l trichloroacetic acid (10%) was added. An aliquot of the mixture (2.5 ml) was taken and mixed with 2.5 ml of distilled water and 0.5 ml of 1 g/l FeCl36H2O. The absorbance at 700 nm was read after allowing the solution to stand for 30 min. Analyses were performed in triplicate. The EC50 value (mg/ml) is the film concentration at which the absorbance is 0.5 for the reducing power and this was calculated from the graph of absorbance at 700 nm against film concentration. 2.7. Optical properties For measuring the colour, a Hunter Lab colourimeter (MiniScan XE Plus 45/0-L, USA) was used to determine values of L, a, and b of SSPS films. The tests were performed in accordance with ASTM D2244 (ASTM, 2011) using a D65 illuminant with an opening of 14 mm and the 10° standard observer. The colourimeter was calibrated using a standard white plate (L⁄ = 93.49, a⁄ = 0.25, b⁄ = 0.09). Then, the colour measurements were performed by placing the film specimens over the colourimeter. At least three points of each sample were selected randomly to measure the colour properties of SSPS films. The following Eqs. (3)–(5) were used to calculate the total colour difference (DE), whiteness (WI) and yellowness (YI) indexes of samples, respectively (Ghanbarzadeh, Almasi, & Entezami, 2010).

DE ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2  ðL  LÞ þ ða  aÞ2 þ ðb  bÞ

WI ¼ 100 

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ð100  LÞ2 þ a2 þ b

ð3Þ ð4Þ

2.6. Antioxidant activity 2.6.1. DPPH radical-scavenging activity The antioxidant potential of the films was measured via the in vitro determination of the free radical scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, following the methodology described by Brand-Williams, Cuvelier, and Berset (1995). Briefly, every film sample was dissolved in 5 ml of distilled water, and then film extract dilutions were made to obtain different concentrations (4000–20,000 mg/l). Then, diluted solutions were mixed with 3.9 ml of 0.1 mM methanolic solution of the DPPH (final concentration of 2.0  104 M). Then, the mixture was vortexed using a vortex mixer (Cyclo-mixer) and incubated in the dark at

YI ¼ 142:86b=L

ð5Þ

where L⁄, a⁄, and b⁄ are the colour parameter values of the standard and i, a, and b are the colour parameter values of the sample. The gloss of the films was measured at angles of 20°, 60° and 85° from the normal to the coating surface, according to the ASTM standard method D523 (ASTM, 1999) using a NOVO Gloss TRIO glossmeter (Rhopoint Instruments Ltd., East Sussex, UK). Gloss measurements were performed on the side of the film in contact with air during drying and over a black matte standard plate. For each sample at least five different points were measured and average values reported. Results are expressed as gloss units, relative to

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

a highly polished surface of black glass standard with a value near to 100. 2.8. Thermal properties A differential scanning calorimetry was performed using DSC equipment (TA Instrument, New Castle, DE, USA). An empty pan was used as an inert reference and the calibration was performed using the indium standard. Samples were scanned at a heating rate of 10 °C/min between temperature ranges of 100 and 180 °C under a nitrogen atmosphere. 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. All these properties were determined in duplicate and the results were averaged. 2.9. Contact angle measurement The wettability of films was evaluated from contact angle measurements using a commercial contact angle metre (Dataphysics OCA 15plus) with an image-analysis software. A film sample (20 mm  80 mm) was put on a movable sample stage and levelled horizontally; then a drop of about 1 ll of distilled water was placed on the surface of the film using a 2 ll micro-injector. Image processing and curve fitting for contact angle measurement from a theoretical meridian drop profile was carried out, measuring the contact angle between the baseline of the drop and the tangent at the drop boundary. Video acquisition of a magnified image of the drop profile was conveyed to a computer via a CCD camera, which allowed measurement of the changes in droplet shape to be recorded as digital images over time. The contact angle was measured on both sides of the drop and averaged. Ten droplets were measured at different regions of the same piece of film. All measurements were taken in open air at a relative humidity of 30 ± 5% RH and at a room temperature of 22 ± 2 °C. 2.10. Statistical analysis The data were presented as the mean ± standard deviation of each treatment. The experiments were factorial with a completely randomised design using analysis of variance (ANOVA) in the SAS program (Version 9.1; Statistical Analysis System Institute Inc., Cary, NC, USA). Differences between the films’ properties mean values were compared using Duncan’s multiple range tests. A probability value of P < 0.05 was considered significant. 3. Results and discussion 3.1. Film formulation Preliminary studies in our laboratory showed that a SSPS concentration of less than 3% is not sufficient to obtain a strong supporting matrix, and the resulting films are soft. A 3% SSPS concentration was selected as the suitable polysaccharide concentration in the film-forming solution. The composite films formed using ZEO and MEO were visually homogeneous with no brittle areas or bubbles, and could be easily peeled from the casting plates. To determine the maximum concentration of ZEO or MEO that could be incorporated into the SSPS matrix, increasing amounts (up to 5%) were added to the film-forming dispersion. As expected, a stronger aroma was observed as the concentration of the essential oil went above 3%. Moreover, regarding the optical properties of films, it was observed that the colour intensified and

617

the transparency decreased as the concentration of both essential oils increased (data not published yet). It is important to note that no structural gradation was found, which confirms both the good dispersion of the essential oils and the lack of significant creaming during drying. 3.2. Antimicrobial activity The inhibition areas for overlay and vapour-phase diffusion for SSPS films with ZEO and MEO against S. aureus, B. cereus, E. coli O157:H7, P. aeruginosa and S. typhimurium are shown in The antimicrobial activity was tested against these microbial strains selected because of their importance in health or for being responsible for food spoilage (Table 1). SSPS film without the essential oil served as a control to determine possible antimicrobial effects of the film without additives. As expected, no inhibition area against the five bacteria tested was observed for control films in either overlay or vapour-phase assays. All composite films containing ZEO inhibited the growth of the five test bacteria in the overlay assay, with the exception of P. aeruginosa, which was not inhibited at the lowest concentration (1%). When the concentration of ZEO was increased to 2%, a significant inhibition was observed against all tested bacteria (P < 0.05). At 3%, the greatest inhibitory effect was observed on S. aureus, with a zone area of 387.05 mm2, followed by B. cereus, with a zone area of 317.93 mm2. These results are in accordance with the study of Pranoto, Salokhe, and Rakshit (2005), who reported that S. aureus and B. cereus were more sensitive to garlic oil-incorporated alginatebased film than E. coli and S. typhimurium. For all composite ZEO films, the largest inhibition zones were observed for the Gram-positive bacteria (B. cereus and S. aureus), while the smallest ones were observed for the Gram-negative bacteria (S. typhimurium, P. aeruginosa and E. coli). On the other hand, Table 1 shows that MEO at 2% concentration can form a clear inhibition zone on S. aureus and B. cereus, which are Gram-positive bacteria, while the zone was not observed for tested Gram-negative bacteria. This result, which is in agreement with our earlier studies (Aliheidari, Fazaeli, Ahmadi, Ghasemlou, & Emam-Djomeh, 2013; Shojaee-Aliabadi et al., 2013), could be attributed to the presence of an additional external membrane surrounding the cell wall in Gram-negative bacteria, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering (Burt, 2004). On the other hand, Gram-positive bacteria have a thick peptidoglycan layer that might function as a preventive barrier against essential-oil compounds (Burt, 2004). In addition to the inhibition effects through direct contact with essential-oil solutions, several authors noted that some bacteria are also susceptible to the vapours of essential oils, and could be inhibited when exposed to them. ZEO- and MEO-containing films could not form a clear zone of inhibition on all bacteria at a concentration of 1%. SSPS films containing MEO showed antimicrobial activity only against S. aureus and B. cereus (Table 1). As with the direct-contact test, the zone of inhibition due to oil vapours also rose as the oil concentration increased. The vapour-phase test results indicate that the effective levels of ZEO and MEO added to SSPS films are higher than for the overlay tests. This observation suggests that volatile components in these two essential oils diffuse more efficiently through the agar media than through the air gap. Most authors have linked the antimicrobial activity of these essential oils with their main active constituents. Recently, Saei-Dehkordi, Tajik, Moradi, and Khalighi-Sigaroodi (2010) have determined the role of thymol, carvacrol, c-terpinene, thymol methyl ether and carvacrol methyl ether as the main antimicrobial compounds of ZEO. Mahboubi and Haghi (2008) linked the antimicrobial activity of MEO to piperitone, piperitenone, terpineol and pulegone.

618

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

Table 1 Antimicrobial activities of different concentrations of ZEO and MEO incorporated into SSPS films in over lay and vapour phase testa. Film

Essential oil conc. (% v/v)

S. aureus

B. cereus

E. coli

S. typhimurium

P. aeruginosa

Overlay Control ZEO ZEO ZEO MEO MEO MEO

0 1 2 3 1 2 3

0.00f 50.24 ± 4.14d 148.37 ± 10.67b 387.05 ± 24.78a 0.00f 21.98 ± 1.32e 107.40 ± 8.17c

0.00e 10.96 ± 0.97d 125.60 ± 6.89b 317.93 ± 21.24a 0.00e 10.24 ± 1.36d 84.78 ± 9.83c

0.00e 10.21 ± 1.7d 104.41 ± 11.23b 255.13 ± 21.19a 0.00e 0.00e 38.37 ± 4.11c

0.00e 35.32 ± 3.42c 84.78 ± 11.29b 198.61 ± 18.52a 0.00e 0.00e 21.98 ± 2.47d

0.00d 0.00d 35.32 ± 1.01b 94.98 ± 14.35a 0.00d 0.00d 10.15 ± 1.07c

Vapour phase Control ZEO ZEO ZEO MEO MEO MEO

0 1 2 3 1 2 3

0.00d 0.00d 94.85 ± 5.23b 226.75 ± 12.37a 0.00d 37.72 ± 3.52c 88.65 ± 7.52b

0.00d 0.00d 50.14 ± 1.67b 176.86 ± 9.54a 0.00d 10.25 ± 1.19c 59.51 ± 3.74b

0.00c 0.00c 35.47 ± 6.49b 148.32 ± 11.42a 0.00c 0.00c 0.00c

0.00c 0.00c 21.63 ± 1.41b 106.16 ± 6.38a 0.00c 0.00c 0.00c

0.00b 0.00b 0.00b 94.98 ± 8.12a 0.00b 0.00b 0.00b

Data reported are mean values and standard deviations. Values within each column with different letters are significantly different (P < 0.05).

90

d

8 e 6 f f

4

50 d 40

f 20 g Control

MEO2

0 ZEO3

0 ZEO2

10 ZEO1

2 Control

e

30

MEO3

c

10

MEO2

12

c 60

MEO1

14

b

70

ZEO2

b

(b)

a

80 DPPH scavenging activity (%)

16

MEO1

mg gallic acid/g SSPS film

18

ZEO3

(a)

a

ZEO1

20

MEO3

a

Inhibition zone (mm2)

Fig. 1. Total phenol content (a) and DPPH scavenging activities (b) of SSPS films with ZEO and MEO.

3.3. Total phenolic content and antioxidant activity Fig. 1 shows the total phenolic (TP) content of SSPS films incorporating ZEO and MEO. The TP content of pure SSPS used in this study was 11.3 mg GAE/100 g SSPS. TP content in the composite film significantly increased (P < 0.05) with increasing ZEO or MEO concentration (Fig. 1). The TP content was higher in the films with ZEO than those with MEO. Since the antioxidant capacity of food is determined by a mixture of different antioxidants with different action mechanisms, it is necessary to combine more than one method in order to determine in vitro, the antioxidant capacity of foodstuffs (Pérez-Jiménez et al., 2008). Therefore, the antioxidant activities of the composite SSPS films were investigated using the DPPH radical scavenging and ferric reducing power assay. A DPPH scavenging assay was used to indicate the film’s antioxidant activity. This method has become a general test method for measurement of free radical scavenging ability of films added with plant extracts. Being rapid, simple and independent of sample polarity, the DPPH assay is very convenient for the quick screening of many samples for determination of radical scavenging activity. SSPS films showed a slight scavenging activity (5.56%), with a IC50 value of

19395.15 ± 49.56 mg/l, which was considerably improved by adding either ZEO or MEO. At concentration of 1%, SSPS films incorporating ZEO could better reduce the stable radical DPPH (IC50 = 4731.43 ± 11.45 mg/l) than MEO-containing films (IC50 = 5877.20 ± 41.45 mg/l). At higher concentrations, antioxidant activity values varied from 68.71% (IC50 = 4621.55 ± 10.86 mg/l) to 79.12% (IC50 = 4188.60 ± 21.73 mg/l) for films with ZEO, and from 28.38% (IC50 = 5641.41 ± 23.01 mg/l) to 41.77% (IC50 = 5225.24 ± 15.77 mg/l) for those with MEO. The lowest IC50 value (highest antioxidant activity) was obtained for those films containing 3% (v/v) ZEO. The ferric reducing power assay is also commonly used to study the antioxidant capacity of plant materials. The antioxidant capacity of films in this test is determined by the ability of the antioxidants in these extracts to reduce ferric to ferrous iron in its reagent. Fig. 2 shows the ferric reducing capacity of SSPS films incorporating ZEO and MEO. A concentration-dependent ferric reducing capacity was found for both ZEO and MEO. A lower EC50 value indicates a higher antioxidant activity. SSPS film showed a slight ability to reduce ferric to ferrous iron (EC50 = 30.42 ± 0.25 mg/ml). As for DPPH, Fig. 2 suggests that the ferric reducing power ability of those films containing ZEO were higher than those of

619

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

Fig. 3 shows the gloss values of the films measured at different incidence angles. At lower angles, samples with high gloss can be better differentiated, whereas high incidence angles are better for distinguishing among low-gloss surfaces, due to the enhancement of specular reflection with an increased incidence angle (Hutchings, 1999). Similar behaviour at 20° and 60° incidence angles was observed, though this only changed slightly at 85°, where there was much less capacity to differentiate samples because of the scarce reproducibility of the measurements. The addition of the essential oil to the film matrix led to lower gloss, especially for MEO, regardless of the oil concentration. The decrease in gloss value could be explained by an increase of the surface roughness of the composite films. This roughness appears to be the consequence of the migration of oil droplets to the top of the film during drying, decreasing the specular reflectance in the air-film interface.

35 a 30

20

b

c

d

MEO3

g

MEO2

f

MEO1

e

ZEO3

10

ZEO2

15

ZEO1

EC50 (mg/ml)

25

5

Control

0

3.5. Thermal properties

Fig. 2. The ferric reducing power capacity of SSPS films with ZEO and MEO.

MEO. The SSPS films including ZEO showed a higher (P < 0.05) ability to reduce ferric to ferrous ion activity, at all concentrations assayed, than MEO. These differences may be attributed to the main phenolic and terpenoid compounds present in ZEO (thymol, carvacrol and c-terpinene) and MEO (pulegone, menthone and piperitone) and the intensity of their possible interactions with SSPS chains, which seems to be greater in the case of ZEO (Kamkar, Javan, Asadi, & Kamalinejad, 2010; Saei-Dehkordi et al., 2010).

3.4. Optical properties Table 2 shows the L, a and b values, total colour difference (DE), whiteness index (WI) and yellowness index (YI) for pure and composite (essential oil-containing) SSPS-based films. Generally, films with ZEO showed higher L and WI values but lower b and YI values than those with MEO; MEO gave the films a darker appearance with a light yellowish tint. Table 2 shows that L values slightly decreased (P < 0.05) when 1% ZEO was added, whilst b values did not significantly change (P > 0.05). There were no obvious differences (P < 0.05) in L and b values when the ZEO concentrations increased from 1% to 3%. Regardless of type and concentration of essential oils, no significant (P > 0.05) difference in values for a were detected. The DE value also tended to vary similarly to WI. This is consistent with our previous findings (Shojaee-Aliabadi et al., 2013) that emulsified carrageenan films turned more opaque with the incorporation of an essential oil, probably due to the increase in diffuse reflectance provoked by light scattering in the lipid droplets; the lower the light-scattering intensity, the lower the film’s whiteness index.

DSC analysis was carried out to determine the thermal transitions of the films. Initial analyses of the samples (not shown) were affected by the presence of solvent residues, expressed as an endotherm that partially hid the endset of Tg and the beginning of the melting process. Subsequently, samples were rigorously dried under vacuum before analysis. The typical DSC thermograms (data shown in Supplementary contents) and Table 2 summarizes the main information obtained from the DSC analysis. The pure SSPS film exhibited a Tg around 31 °C and an endothermic peak at about 99 °C. The reduced Tm of SSPS films incorporating ZEO was slightly lower than those containing MEO. The addition of 1% MEO caused a decrease in Tg to 23 °C. This effect was even more apparent for the films with high levels of the essential oil. This may be due to the molecular structure of the essential oil, which has an effect on the overall chain mobility in the SSPS film. The increase of essential-oil concentration led to an increase in DHm values (data not shown). As a result, the observed single glass transition followed by an endothermic peak for all composite films was attributed to the whole polymer matrix. The films remained homogeneous throughout the heating cycle, as no phase separation was observed. 3.6. Wettability properties The wettability of SSPS films was evaluated via the water contact angle on the film surface using the sessile drop method. The dynamic contact angle was determined by recording the process from the drop’s initial contact with the film surface to its transitory equilibrium on the film. Table 3 shows the contact angle data. The films with ZEO had an increased water contact angle, indicating an increase in hydrophobicity. The contact angle of the films with MEO was lower than for films with ZEO, indicating less water resis-

Table 2 Colour and thermal properties of various SSPS film samples.a

a

Film type

Essential oil conc. (% v/v)

L

a

b

DE

WI

YI

Tg (°C)

Tm (°C)

Control ZEO ZEO ZEO MEO MEO MEO

0 1 2 3 1 2 3

92.24 ± 2.66a 86.69 ± 0.75c 85.20 ± 1.75 cd 84.02 ± 1.54c 89.09 ± 0.50b 83.35 ± 1.56d 80.40 ± 0.74e

1.42 ± 0.15a 1.43 ± 0.06 a 1.44 ± 0.04a 1.48 ± 0.07a 1.45 ± 0.03a 1.44 ± 0.01a 1.44 ± 0.03a

17.86 ± 1.64c 15.56 ± 1.05c 14.53 ± 2.71b 20.87 ± 1.06b 15.47 ± 0.65c 21.47 ± 2.39b 24.07 ± 1.21a

17.56 ± 2.04d 17.11 ± 1.24 d 20.89 ± 0.55 c 23.05 ± 1.30b 16.21 ± 0.51d 23.90 ± 2.11b 27.52 ± 0.70a

80.91 ± 3.01 a 79.46 ± 1.25 a 75.83 ± 0.76 b 73.65 ± 1.43c 80.95 ± 0.30a 72.73 ± 1.87c 68.90 ± 0.47d

26.41 ± 1.07d 25.66 ± 1.94d 31.83 ± 1.13c 35.50 ± 2.12bc 24.83 ± 0.93d 36.79 ± 3.93b 42.75 ± 1.77a

31.17 ± 0.36a 30.40 ± 0.63a 29.76 ± 1.79a 28.84 ± 1.05a 23.92 ± 2.60b 17.20 ± 0.92c 16.38 ± 2.02c

99.63 ± 0.83a 94.63 ± 0.52c 94.29 ± 1.57c 91.48 ± 0.94d 97.41 ± 1.28b 97.96 ± 1.25ab 94.62 ± 1.05c

Data reported are mean values and standard deviations. Values within each column with different letters are significantly different (P < 0.05).

620

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

50

7

60º Gloss

20 Gloss

ab

ab

ab bc

35

ab

4 3

a

40

a

a

5

a

45

a

6

a

ab

c

30 25 20 15

2

10 1

ab

5 MEO3

MEO2

MEO1

ZEO3

ZEO1

Control

MEO3

MEO2

MEO1

ZEO3

ZEO2

ZEO1

Control

ZEO2

0

0

60 55

a

a

50

85 Gloss

a

a

a a a

45 40 35 30 25

MEO3

MEO2

MEO1

ZEO3

ZEO2

ZEO1

Control

20

Fig. 3. Gloss values at different incidence angles as a function of ZEO and MEO concentrations in SSPS films.

Table 3 Results of contact angle measurement of SSPS films containing ZEO or MEO.a

a b

Film type

Essential oil conc. (% v/v)

Left angle (deg)

Right angle (deg)

Average contact angle (deg)

Wetting energyb (mJ/m2)

Control ZEO ZEO MEO MEO

0 1 3 1 3

28.7 ± 1.5d 51.8 ± 0.6b 79.9 ± 0.5a 29.5 ± 1.6d 41.2 ± 1.13c

29.2 ± 1.4d 48.05 ± 1.06b 79.4 ± 2.26a 29.65 ± 1.48d 40.3 ± 1.97c

28.95 ± 1.48d 49.9 ± 0.84b 79.6 ± 1.41a 29.55 ± 1.62d 40.7 ± 1.55c

63.66 ± 0.87a 46.88 ± 0.82c 13.10 ± 1.75d 63.26 ± 1.02a 55.14 ± 1.28b

Data reported are mean values and standard deviations. Values within each column with different letters are significantly different (P < 0.05). Wetting energy = surface tension of a probe liquid (72.8 mJ/m2 for water)  cos h, where h is an average contact angle measured.

tance. The more-hydrophobic surface of ZEO films results in a lower wetting energy, as shown in Table 3. Fig. 4 presents the kinetics observed for water droplets, with images acquired at 0, 15, 30, 45 and 60 s. When a water droplet is deposited onto a solid surface, two mechanisms are involved in the decrease of contact angle over time: evaporation, due to the difference of water vapour pressure between the droplet and the surrounding atmosphere, and absorption inside the material. Generally, over a short time, evaporation from the film is slower than water absorption (Han & Krochta, 1999). For the films containing ZEO or MEO, a slight decrease of contact angle was observed, which can be attributed to a slight absorption by the films. This effect was more pronounced on MEO films. This phenomenon could be attributed to the reorientation of SSPS molecules when the essential oil is introduced in the system. The oil component could also increase the hydrophobic

interaction with the SSPS inside the system, with polar groups rejected toward the surface of the film.

4. Conclusion This study was the first to explore the antimicrobial, antioxidant, thermal and optical properties of SSPS-based films containing ZEO or MEO. Both oils exhibited a large inhibitory effect on all microorganisms tested, although for MEO the results were less striking. The study demonstrated that ZEO or MEO can be successfully incorporated onto SSPS films, giving them excellent antioxidant activities. A reduction of the Tg, as determined by DSC analysis, was caused by the addition of oil into the film matrix. Overall, this study suggests that SSPS films with ZEO or MEO show

621

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

t=0

t=15

t=30

t=45

t=60

ZEO1

ZEO3

MEO1

MEO3

Fig. 4. Volume kinetics of water droplets deposited on surface of SSPS films with different levels of ZEO or MEO.

good potential to be used as active films. Nevertheless, further studies are required before using such films as a form of packaging for food products. Acknowledgements The authors are thankful to Fuji Oil Company for supplying raw material for this research. This work was financially supported by a research grant (Grant no.: 41591054) from Neyshabur Technology Incubator (a branch of Khorasan Science and Technology Park (KSTP)). The authors also acknowledge technical support from the National Nutrition and Food Technology Research Institute (NNFTRI) of Iran. The first author is deeply grateful to Dr. Mahdi Khadang Nikfarjam and Dr. Mohamad Mahdi Karimkhani for their technical assistance with this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 09.014. References Ahmadi, R., Kalbasi-Ashtari, A., Oromiehie, A., Yarmand, M. S., & Jahandideh, F. (2012). Development and characterization of a novel biodegradable edible film obtained from psyllium seed (Plantago ovata Forsk). Journal of Food Engineering, 109, 745–751.

Ali, M. S., Saleem, M., Ali, Z., & Ahmad, V. U. (2000). Chemistry of Zataria multiflora (Lamiaceae). Phytochemistry, 55, 933–936. Aliheidari, N., Fazaeli, M., Ahmadi, R., Ghasemlou, M., & Emam-Djomeh, Z. (2013). Comparative evaluation on fatty acid and Matricaria recutita essential oil incorporated into casein-based film. International Journal of Biological Macromolecules, 56, 69–75. Asai, I., Watari, Y., Iida, H., Masutake, K., Ochi, T., Ohashi, S., et al. (1994). Effect of soluble soybean polysaccharide on dispersion stability of acidified milk protein. In K. Nishinari & E. Doi (Eds.), Food hydrocolloids: Structure, properties and functions (pp. 151–156). New York: Plenum Press. ASTM (1999). Standard test method for specular gloss. Standard designation: D523. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Materials. ASTM (2011). Standard practice for calculation of colour tolerances and colour differences from instrumentally measured colour coordinates: D2244. Annual book of ASTM. Philadelphia, PA: American Society for Testing and Materials. Brand-Williams, W., Cuvelier, M., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28(1), 25–30. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods: A review. International Journal of Food Microbiology, 94, 223–253. de Moura, M. R., Mattoso, L. H. C., & Zucolotto, V. (2012). Development of cellulosebased bactericidal nanocomposites containing silver nanoparticles and their use as active food packaging. Journal of Food Engineering, 109, 520–524. Ghanbarzadeh, B., Almasi, H., & Entezami, A. A. (2010). Physical properties of edible modified starch/carboxymethyl cellulose films. Innovative Food Science and Emerging Technologies, 11, 697–702. Ghasemlou, M., Khodaiyan, F., Oromiehie, A., & Yarmand, M. S. (2011). Development and characterization of a new biodegradable edible film made from kefiran, an exopolysaccharide obtained from kefir grains. Food Chemistry, 127, 1496–1502. Han, J. H., & Krochta, J. M. (n and Krochta1 1999). Wetting properties and water vapor permeability of whey–protein-coated paper. Transactions of the ASAE, 42, 1375–1382. Hutchings, J. B. (1999). Food colour and appearance (2nd ed.). Gaithersburg, Maryland, USA: Aspen Publishers, Inc..

622

D. Salarbashi et al. / Food Chemistry 146 (2014) 614–622

Jouki, M., Khazaei, N., Ghasemlou, M., & HadiNezhad, M. (2013). Effect of glycerol concentration on edible film production from cress seed carbohydrate gum. Carbohydrate Polymers, 96, 39–46. Kamkar, A., Javan, A. J., Asadi, F., & Kamalinejad, M. (2010). The antioxidative effect of Iranian Mentha pulegium extracts and essential oil in sunflower oil. Food and Chemical Toxicology, 48(7), 1796–1800. Kuorwel, K. K., Cran, M. J., Sonneveld, K., Miltz, J., & Bigger, S. W. (2013). Migration of antimicrobial agents from starch-based films into a food stimulant. LWT-Food Science and Technology, 50, 432–438. Ma, W., Tang, C.-H., Yang, X.-Q., & Yin, S.-W. (2013). Fabrication and characterization of kidney bean (Phaseolus vulgaris L.) protein isolate–chitosan composite films at acidic pH. Food Hydrocolloids, 31(2), 237–247. Mahboubi, M., & Haghi, G. (2008). Antimicrobial activity and chemical composition of Mentha pulegium L. essential oil. Journal of Ethnopharmacology, 119, 325–327. Nakamura, A., Furuta, H., Maeda, H., Takao, T., & Nagamatsu, N. (2002). Structural studies by stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan. Bioscience, Biotechnology, and Biochemistry, 66, 1301–1313. Pérez-Jiménez, J., Arranz, S., Tabernero, M., Díaz-Rubio, M. E., Serrano, J., Goñi, I., et al. (2008). Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results. Food Research International, 41(3), 274–285. Pranoto, Y., Salokhe, V. M., & Rakshit, S. K. (2005). Physical and antibacterial properties of alginate-based edible film incorporated with garlic oil. Food Research International, 38, 267–272. Ramezani, M., Hosseinzadeh, H., & Samizadeh, S. (2004). Antinociceptive effects of Zataria multiflora Boiss fractions in mice. Journal of Ethnopharmacology, 91, 167–170. Saei-Dehkordi, S. S., Tajik, H., Moradi, M., & Khalighi-Sigaroodi, F. (2010). Chemical composition of essential oils in Zataria multiflora Boiss. from different parts of Iran and their radical scavenging and antimicrobial activity. Food and Chemical Toxicology, 48(6), 1562–1567. Salarbashi, D., Fazly Bazzaz, B. S., Sahebkar, A., Karimkhani, M. M., & Ahmadi, A. (2012). Investigation of optimal extraction, antioxidant, and antimicrobial

activities of Achillea biebersteinii and A. wilhelmsii. Pharmaceutical Biology, 50(9), 1168–1176. Salmieri, S., & Lacroix, M. (2006). Physicochemical properties of alginate/ polycaprolactone-based films containing essential oils. Journal of Agricultural and Food Chemistry, 54, 10205–10214. Shaffiee, A., & Javidnia, K. (1997). Composition of essential oil of Zataria multiflora. Planta Medica, 63, 371–372. Shakeri, M. S., Shahidi, F., Beiraghi-Toosi, S., & Bahrami, A. (2011). Antimicrobial activity of Zataria multiflora Boiss. essential oil incorporated with whey protein based films on pathogenic and probiotic bacteria. International Journal of Food Science and Technology, 46(3), 549–554. Sharififar, F., Moshafi, M. H., Mansouri, S. H., Khodashenas, M., & Khoshnoodi, M. (2007). In vitro evaluation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss. Food Control, 18, 800–805. Shojaee-Aliabadi, S., Hosseini, H., Mohammadifar, M. A., Mohammadi, A., Ghasemlou, M., Ojagh, S. M., et al. (2013). Characterization of antioxidantantimicrobial j-carrageenan films containing Satureja hortensis essential oil. International Journal of Biological Macromolecules, 52(1), 116–124. Shorey, R. L., Willis, R. A., Lo, G. S., & Steinke, F. H. (1985). Effect of soybean polysaccharides on plasma lipids. Journal of the American Dietetic Association, 85, 1460–1465. Siripatrawan, U., & Harte, B. R. (2010). Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract. Food Hydrocolloids, 24(8), 770–775. Tajik, S., Maghsoudlou, Y., Khodaiyan, F., Jafari, S. M., Ghasemlou, M., & Aalami, M. (2013). Soluble soybean polysaccharide: A new carbohydrate to make a biodegradable film for sustainable green packaging. Carbohydrate Polymers, 97(2), 817–824. Tharanathan, R. N. (2003). Biodegradable films and composite coatings: past, present and future. Trends in Food Science and Technology, 14, 71–78. Tsai, A. C., Vinik, A. I., Lasichak, A., & Lo, G. S. (1987). Effects of soy polysaccharides on postprandial plasma glucose, insulin, glucagon, pancreatic polypeptide, somatostatin, and triglyceride in obese diabetic patients. The American Journal of Clinical Nutrition, 45, 596–601.