The effect of active and edible starch-chitosan composite film incorporated with thymus kotschyanus essential oil and punica granatum peel extracts on shelf life of meat during storage

Meat is sensitive to microbial spoilage and chemical oxidation, so it is favorable to use a natural preservative with antioxidant and antimicrobial effects. The objective of this study was to prepare composite films from chitosan and starch containing pomegranate peel extract (PPE) and Thymus kotschyanus EO alone and in combination to evaluate their effects on shelf-life characteristics, including physico-chemical (pH, aw and lipid oxidation), sensorial (color, odor and general appearance) and microbial (lactic acid bacteria, aerobic mesophiles, pseudomonas) on meat at 4°C for 21 days.
Fresh pomegranate fruits (Punica granatum) were purchased from a local hypermarket. After separation, the peels were washed and dried. Thus 700 ml alcohol and 300 ml distilled water were added to 250g of powdered peel. The mixture was left in a shaker for 24 hr. After cooling and filtration using a paper filter, the solvent was removed in a rotary evaporator and the extract was stored at 4 °C until use. The Thymus kotschyanus was purchased a local market in Urmia. Hydrodistilation of dried parts of plant was performed in a Clevenger-type apparatus for 3 hr. Sodium sulfate was used for dehydration of the oil, then oil was filtered using 0.22 μm filters and stored in colored glass tubes in the dark at refrigerated temperature. Chitosan film solution was prepared by dissolving 1.5% (w/v) of chitosan in 1% (v/v) of glacial acetic acid solution with constant agitation using a magnetic stir plate during 24 h at room temperature. As a plasticizer, glycerol was added in the proportion of 30% (w/w) to chitosan powder, and the system was agitated for 5 min to complete homogenization. Starch solutions with concentrations of 3.5 % (w/v) were prepared by dispersing 27% amylose corn starch (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in distilled water and heating the mixtures on hotplates 95 ˚C during 30 min with stirring until it gelatinized, and then cooling to 40 ˚C. Composite films were prepared by mixing 100 mL of 2% chitosan solution with 100 mL of 3.5% starch solutions. For the films incorporated with the essential oils, tween 80 at a level of 0.2% w/v of essential oil was used as emulsifier. EO or hydro-alcoholic extracts were incorporated to the system at the level of 1% (v/v) of film form solution and homogenized in magnetic stir plate during more 5 min at room temperature. For microbial tests ten grams of each sample was diluted in 90 ml sterile 0.1 % ml peptone water (Merck, Darmstadt, Germany) and homogenized using shaker for 3 min at room temperature. Then, 0.1 mL of serial dilutions (0.1 % peptone water) of beef meat homogenates were transferred to agar plates. Pseudomonas were counted on Pseudomonas agar supplemented with CFC at 25 °C for 48 h (CFC, Merck, Darmstadt, Germany). Lactobacillus were enumerated on MRS agar (Merck, Darmstadt, Germany, Oxoid) incubated at 30 °C for 48 h. Finally, total mesophilic counts were determined using Plate Count Agar (PCA, Merck, Darmstadt, Germany), after kept for 2 days at 30°C. Level of lipid oxidation was investigated using thiobarbituric acid reactive substances (TBARS) method as described by Pikul et al (1989). Ten grams of sample was homogenized with 1 mL BHT (1 mg/mL) and 35 mL trichloroacetic acid (5%) in a blender. The mixture was filtered using Whatman no 1. Five mL TBA solution (0.02 M) of filtrate solution was added to 5 mL of filtrate solution and kept in a water bath at 100°C for 60 min to expand the malondialdehyde–TBA complex. After cooling the tube, the absorbance of the samples were determined at 532 nm. The pH values were determined using a digital pH meter. A 5 g of beef meat sample containing 25 ml of distilled water was homogenized for up to 1 min. The sensory analysis of meat samples was carried out with 10 trained PhD students. After preparation of treatments, the samples were offered to each panelist separately. Fresh beef meat was used as a control. Panelists were asked to evaluate texture, color, flavor and odor and overall acceptance on a nine-point Hedonic scale, with 9 being so good and 1 being so poor.
The incorporation of PPE and EO into composite film showed that aerobic mesophiles and lactic acid bacteria were the most sensitive and resistant groups to films by 7.11 and 6.92 log cycles reduction after 21 days’ storage, respectively. In addition, meat wrapped by 1% PPE films had the lowest degrees of lipid oxidation (1.11 mg MDA/kg samples) which was 61% lower than the control samples (2.90 mg MDA/kg samples). This study showed that starch-chitosan composite films containing of pomegranate peel extract and Thymus kotschyanus EO was significantly antibacterial properties, while shows the higher antioxidant effect. PPE also improves some physical properties of the film. Our results indicated that incorporation of pomegranate peel extract (PPE) and Thymus kotschyanus EO as a natural antibacterial agents have a potential to prolong the shelf-life of meat.