Combined Effect of Chitosan-Based Edible Film Containing Oregano Essential Oil on the Shelf-Life Extension of Fresh Chicken Meat
 
Fatemeh Karimnezhad; PhD¹, Vadood Razavilar; PhD¹, AmirAli Anvar; PhD¹, Shahrzad Dashtgol; PhD¹ & Atefeh Pilehvar Zavareh; DVM^*1
 
1 Department of Hygiene, Science and Research Branch, Islamic Azad University, Tehran, Iran.
 

 
Introduction

Nowadays, chicken meat is consumed widely all over the world. Chicken meat, as a raw foodstuff with no process for producing or keeping, is vulnerable to spoilage and can be an almost perfect medium for microbial growth including both spoilage and pathogenic microorganisms. Therefore, food producing factories have desire to extend shelf life of the chicken meat and its products by new packaging methods (de Azeredo, 2013). Furthermore, today's environmental contamination has increased due to the use of plastic packaging coatings and the antimicrobial resistance issue (Bohlmann, 2005). These factors have led the food production factories to use biodegradable coatings, including chitosan. Biodegradable films are thin layers of edible materials formed as coatings on foodstuffs, which can optimize the moisture and atmosphere of the food product and prevent entry of foreign materials (Dutta et al., 2009). However, biodegradable films can have antimicrobial and antioxidant properties, which are enhanced by natural compounds such as essential oil (Pranoto et al., 2005). Chitosan is a natural polysaccharide polymer, derived by deacetylation of chitin (Elsabee and Abdou, 2013). It is also considered as the generally recognized as safe (GRAS) by United States Food and Drug Administration (Kerch, 2015). Chitosan has been proved as a wide spectrum of antimicrobial activity against gram positive/gram negative bacteria and fungi. Biocompatibility, nontoxicity and biodegradability properties of chitosan make it suitable for the food preservation (Zimoch-Korzycka et al., 2016). Ample evidence shows that chitosan can be used as edible films and coatings in various meat products (Lekjing, 2016, Petrou et al., 2012).
Recently, food producing factories have a positive attitude to use the extracts and essential oils from herbs to improve the foods' sensory parameters and shelf life (Botsoglou et al., 2003). Previous studies reported that oregano essential oil had high activity against microorganisms (Chouliara et al., 2007). Considerable antimicrobial and antioxidant activity are related to phenolic monoterpens and carvacrol components (Alsaiqali et al., 2016). Recently, application of chitosan and essential oils has increased in food coating to control the microbial populations and improve the shelf life of meat products. Various studies investigated the shelf life extension by chitosan as a dipping agent incorporation of oregano essential oil on modified atmosphere packaging (MAP) for the extension of fresh poultry meat shelf life  (Petrou et al., 2012). Pomegranate juice dipping and chitosan enriched with Zataria multiflora had inhibitory effects on the microbial growth and chemical changes in chicken breast meat  (Bazargani-Gilani et al., 2015). Lekjing showed the preservative effects of chitosan-based coating with clove oil on quality and shelf life of pork sausage (Lekjing, 2016).
 Considering the above, the objective of this study was to evaluate the combined effect of chitosan-based edible film containing oregano essential oil on microbiological parameter of chicken meat.
Materials and Methods
Plant material and preparation of oregano essential oil: Origanum vulgare subsp. plants were collected from Gorgan City, Iran, from July to September 2016. Identification of voucher specimen was carried out by Tehran University Herbarium, Iran. Essential oil was obtained from 50 g of dried plant material by hydro-distillation using Clevenger's apparatus for 3 h at 70 °C. Isolated oil dried over anhydrous sodium sulphate (Merck, Germany). Oregano oil was recovered and stored in screw capped bottles at 4 °C.
Preparation of edible films: A solution of chitosan (Sigma-Aldrich, USA) was provided by dissolving chitosan of high molecular (800,000 cps; >75% deacetylation degree) weight in powder in 100 ml of acetic acid (Merck, Germany) and stirring at 45 °C for 3 h to obtain the final concentration 2% (w/v). The chitosan solution was filtered using a Whotman No. 3 filter paper and continued by vacuum filtration to remove any undissolved particles. To prepare chitosan solution with essential oil, 30% w/v of Glycerol (Sigma-Aldrich, USA) and 0.25% w/v of Tween 80 were added in film formation solutions. The pH of solution was adjusted to 5.8 by NaOH (Merck, Germany). The essential oil of oregano was added to the chitosan solution to obtain the final concentration of 1% and 2% (w/v). To prepare an emulsion, the solution was homogenized (IKA T25 basic, Staufen, Germany) at 8000 rpm for 3 min. The mixture was cast onto a flat polytetrafluoroethylene casting plate and dried at room temperature for at least 36 h. Dried films were kept under conditions 25 °C and 50% RH for 48 h (Moradi et al., 2011).
Preparation of chicken meat samples: Fresh chicken fillet was provided by a slaughter house in Tehran province, Iran (samples weighing 400 g, 15 cm × 7 cm each). All meat samples were packaged by chitosan based films for further treatment. The samples were assigned and treated as follows:    
1) Chitosan group: chicken fillet coated with chitosan free essential oil.
2) & 3) Treated groups with oregano essential oil: chicken fillet coated with chitosan containing 1% and 2% concentrations of oregano, respectively.
4) Control group: Chicken fillet with no package.
All meat samples were kept at 4 °C for a period of 12 days. Sampling was carried out on 0, 3, 6, 9, and 12 days of storage.
Microbiological examinations: Aerobic plate counts (APC), coliform counts (CC), and total psychrophilic counts (TPC) were determined as follows: a 10% suspension of each sample was prepared by homogenizing in peptone water (Merck, Germany) using stomacher (IUL Instrument, Spain). Serial tenfold dilution was made from 10^-2 to 10^-6 in sterile peptone water diluent. Later, 0.1 ml of each dilution was inoculated into selective agar plates. Aerobic plate counts were determined by the spread -plate method using plate count agar (Merck, Germany) and incubated (Binder, Germany) at 37 °C for 24 h and 48 h  (Association, 1992). Coliform counting was assessed on the basis of pour plate method; the sample was spread into Violet Red Bill agar (Merck, Germany) and incubated at 35 ± 2 °C for 48 h. Suspected colonies were cultured into the Brilliant Green Broth (Merck, Germany) following the incubation at 35 ± 2 °C for 24-48 h (Association, 1992). Furthermore, TPC was done similar to APC but the plates were incubated at 7 °C for 7-10 days  (Association, 1992). The counts were showed as log CFU/g.
Data analysis: All tests were done three times. Data were presented as mean ± Standard Deviation (SD) and analyzed using SPSS (SPSS Inc, Chicago, IL. v. 22.0). Significance levels (P < 0.05) were assessed by ANOVA and supplementary Tukey HSD tests.
Results
Changes in APC: Table 1 shows the number of chicken fillet APC in the control (untreated) and treated groups during the storage period for 12 days. The initial APC counts ranged from 3.91 to 8.32 log CFU/g and the counts increased in the control and treated groups throughout the storage time. The increase rate of aerobic bacteria counts in groups of chicken fillet treated with chitosan, with 1% oregano essential oil, as well as with chitosan and 2% oregano were significantly lower comparable to the control group. Statistically significant differences were observed for the APC of chicken fillet on 3, 6, 9, and 12 days of storage period between the control and treated groups (two different concentrations of oregano essential oil).
Changes in TPC: Table 2 shows the TPC rates of chicken fillet in the control and treated groups during the storage. The total psychrophilic counts ranged from 4.2 ± 0.13 to 8.65 ± 0.28 log CFU/g. We observed that the counts gradually increased the control and chitosan groups during the storage period. However, the increase levels were lower in the treated samples than the control sample. Statistically significant differences were observed for the TPC of chicken fillet between the
control and treated groups across the entire storage periods.
Changes in CC: Table 3 represents the coliform count of chitosan fillet in the control and treated groups during the storage period. The numbers of total coliform bacteria were in the range of 1.89 ± 0.1 to 5.62 ± 0.16 log CFU/g and the count consistently increased in the control and treated groups throughout the storage duration. However, the rate of increase was slower for chicken fillet treated by chitosan enriched with 1% and 2% concentrations of oregano essential oil compared to the control group. In group of fillet chitosan treated with 2% oregano, CC decreased in days 3 and 9, while it increased in days 6 and 12 throughout the storage period. Since the CC levels increased gradually in the group treated with 1% essential oil. Statistically significant differences were observed in chicken fillet CC in days 3, 6, 9, and 12 of storage between the control and treated groups.