Document Type : Original Research Article
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran
Edible films and coatings are becoming increasingly important in food preservation applications to maintain quality and extend shelf-life in perishable foods . Edible films keep safe from food products from mechanical damage, physical, chemical, and microbiological deteriorations . Chitosan is a natural and non-toxic polysaccharide that is of much demand for both scientific studies and the food industry owing to its numerous applications including biodegradable films, blends, coatings, composites, and nanocomposites . Compared to other edible film and coating compunds, chitosan has the advantage of being used together with functional substances such as minerals or vitamins, while it has the advantage of having antibacterial and antioxidant activity when used alone . It also possess unique properties such as non-toxicity, abundance, biocompatibility, biodegradability, excellent film-forming ability, stability, and flexibility .
Basil (Ocimum basilicum L.) belongs of the genus Ocimum and has been distributed in the tropical regions of Asia, Africa, and Central and South America . Beside its traditional application as culinary herb and flavoring agent, the outer pericarp of seeds swells into a gelatinous mass upon wetting and can be used in combination with other film-forming materials . The most important of functional properties of mucilage obtained from basil seeds including biodegradability, biocompatibility, heat-resistant, non-toxicity, low production cost, acceptability, and hydrophilic nature .
There is increasing interest about antimicrobial packaging films and coatings in the food industry. In this area, numerous studies have been focused in incorporating essential oils (EOs), bateriocins, nanofibers, and nanoparticles [9-12]. Ziziphora clinopodioides essential oil (ZEO) is defined by the U.S. Food and Drug Administration (U.S. FDA) as a generally considered as safe (GRAS) in food applications and is allowed to be used as a food additive or flavoring agent, as well as antimicrobial and antioxidant use . Recent previous studies have indicated that ZEO has a approprite antibacterial activity in perishable food products [5,11,13,14]. Nano magnesium oxide (MgO) is also considered as a GRAS material by the U.S. FDA [15-17]. It has numerous advantages including excellent reproducibility, small particle size, large surface area, simplicity, and low cost .
Gamma irradiation is also recognized as a non-thermal preservative approach and has been extensivley applied for delaying of sprouting, the growth of pathogenic and spoilage microorganisms, disinfestations, and improvement of vulnerable foodstuffs shelf-life . Previous studies indicated that gamma irradiation could improve antimicrobial activities of edible films/coatings containing antimicrobial compounds [19-21]. According to our knowledge, there were not any studies about the effects of gamma irradiation on in vitro antimicrobial property of basil seed mucilage-chitosan film. Moreover, there is a general lack of data for the in vitro antimicrobial property of basil seed mucilage-chitosan films supplemented with ZEO and MgO nanoparticles against the growth of food-borne pathogenic microorganisms. Our recent study  showed that the irradiated basil seed mucilage-chitosan films under the cobalt-60 source at 2.5 and 5 kGy doses had better tensile strength as well as lower swelling index and water vapor transmission rate compared with un-irradiated films. Moreover, rainbow trout fillets packaged with films enrichment with ZEO 2% + MgO 0.2% and ZEO 2% + MgO 0.1% had the lowest microbial property and chemical changes (P < 0.05) during prolonged refrigerated storage. Therefore, the aim of this study was to evaluate the effect of gamma irradiation at 0 and 5 KGy on in-vitro antimicrobial property of basil seed mucilage-chitosan films supplemented with ZEO and MgO nanoparticles against Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Bacillus subtilis.
MATERIALS AND METHODS
The whole part of fresh Z. clinopodioides was obtained from Gilan-e-Gharb city, Kermanshah province, west of Iran. The EO of Z. clinopodioides was acquired using hydro-distillation for 3.5 h using a Clevenger-type apparatus based on the method described in the European Pharmacopoeia . The ZEO was collected, poured in an amber glass vial, and maintained at refrigerated temperature (4 ± 1 ºC) until further use. The GC-MS analysis of the ZEO was conducted according to our previous study . Chitosan was purchased from Sigma-Aldrich, Germany (deacetylation degree = 75-85% and a medium molecular weight = 450 KDa). MgO nanoparticles (diameter < 40 nm and purity > 99%) was obtained from the Iranian Nanomaterials Pioneers company (Razavi Khorasan, Iran). Basil seeds were obtained from a local market (Kermanshah, Iran) and identified in Razi University. All chemicals and culture media were obtained from Merck, Germany.
Extraction of basil seed mucilage
Isolation of basil seed mucilage was conducted based on the published method by Luo et al., (2019)  upon wetting (1:40 w/v, basil seed: water) at 50 ± 1 °C for 3 h. The obtained mucilage was dried and kept in a dry place until required for further experiments.
Preparation of basil seed mucilage-chitosan film
For preparation of basil seed mucilage-chitosan film, 2 g mucilage and 2 g chitosan individually dissolved in distilled water and acetic acid 1% for 3 and 8 h, respectively. Glycerol (0.75%, v/v of solution) was added into the solutions and mixed for 30 min. Films composed of 25 ml chitosan and 25 ml mucilage was prepared and then ZEO (1 and 2%) and MgO nanoparticles (0.1 and 0.2%) added. The film-forming solutions were casted in glass petri dishes and dried at 25 ºC for 48 h .
Gamma irradiation of basil seed mucilage-chitosan films
The gamma irradiation doses for prepared films were 0 and 5 KGy using a 60Co source with a dose rate of 4.18 kGy/h at the Atomic Energy Organization of Iran (Tehran, Iran).
Scanning electron microscopy of basil seed mucilage-chitosan films
Morphology of films and MgO nanoparticles was observed using a TeScan MIRA3 scanning electron microscope (SEM) using an accelerating voltage of 5 and 10 kV, respectively. The film samples (0.5 × 0.5 cm) were cut and prepared using dropping in liquid nitrogen. The prepared films were deposited on an aluminum holder and allowed to sputtering with gold/palladium alloy .
Bacterial strains S. aureus (ATCC 6538), L. monocytogenes (ATCC 19118), B. cereus (ATCC 11774), and B. subtilis (ATCC 6633) were acquired from the microbial archive of Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran. Bacterial strains were cultured at 37 °C for 24 h in Brain Heart Infusion broth (BHI) medium and adjusted to a final density of 9 log CFU/ml.
In vitro antimicrobial activity of basil seed mucilage-chitosan films
The antimicrobial property of basil seed mucilage-chitosan films were examined using agar disk diffusion and broth micro-dilution methods.
For agar disk diffusion assay, 15 ml molted BHI agar was poured into petri dishes with a diameter of 90 mm. Then, 100 µl of the each test bacterial suspension, containing 9 log CFU/ml, was cultured on the BHI agar by surface method. The film pieces (6 mm in diameter) were put on the surface of inoculated BHI agar. After incubation at 37 ± 1 °C for 24 h, the diameter of the inhibition zones (mm) were determined .
For broth micro-dilution assay, the 96-well sterile micro-dilution plates with U-bottom wells were prepared by adding 180 µl of film forming solutions based on basil seed mucilage-chitosan containing ZEO (1 and 2%) and MgO nanoparticles (0.1 and 0.2%), and 20 µl of bacterial suspension containing 9 log CFU/ml of the test microorganisms. The positive (BHI broth containing 0.05 µl/ml of tetracycline and microorganism) and negative (BHI broth containing tested microorganisms) controls were also considered. The plates were sealed with sterile plate sealers. The micro-dilution plates were shacked on a plate shaker for 30 s and incubated at 37 ± 1 °C for 24 h. Afterward, sampling from each well was done using ten-fold serial dilutions with BHI broth, followed by plating on BHI agar and incubation for 24 h at 37 ± 1 °C. After incubation, the number of bacterial colonies was enumerated and the findings were exhibited in terms of differences in population (DP) according to the equation :
Log DP= log = log N – log N0
Where N and N0 are the bacterial population (CFU/ml) at time t and zero, respectively.
All experiments were conducted in triplicate. The analysis was performed using SPSS 16.0 for Windows (SPSS, Chicago, IL, USA) software package. All results were subjected to one-way analysis of variance to determine the differences of samples. Significance level was considered P < 0.05 in all experimental data.
RESULTS AND DISCUSSION
Chemical composition of Ziziphora clinopodioides essential oil
As exhibited in our previous study , the main chemical composition of ZEO were determined to be carvacrol (65.22%), thymol (19.51%), ɣ-terpinene (4.63%), and p-cymene (4.86%). Ozturk and Ercisli, (2007)  indicated pulegone (31.8%), 1,8-cineole (12.2%) and limonene (10.4%) as the most corresponding compounds of ZEO. Aghajani et al., (2008)  indicated that Z. clinopodioides collected from Lorestan province contained 8.7% and 53.6% of carvacrol and thymol, respectively. In last years, Morteza-Semnani et al., (2005)  and Sonboli et al., (2010)  reported that terpineol, methyl acetate, and iso-neomenthol are the main composition of ZEO collected from various parts of Iran. In general, differences in chemical constituents of EOs obtained from medicinal herbs can be related to the herbal species, age, ecotypes, and other environmental factors .
Scanning electron microscopy of basil seed mucilage-chitosan films
SEM images of non-irradiated (0 KGy) and irradiated films with 5 KGy are shown in Fig. 1a-d and 2a-d, respectively. A smooth, compact, and homogeneous surface without grainy and porous structure was observed in the pure films (Fig. 1a and 2a). Moreover, MgO nanoparticles completely incorporated in the film matrices (Fig. 1b and 2b). Sanuja et al., (2014)  also found that MgO nanoparticles completely dispersed in the chitosan film. Pores and cavities were found in the films containing ZEO (Fig. 1c-d and 2c-d), which may result from the volatility of the ZEO constituents [34,35]. These results are in general agreement with those reported for EO obtained from bergamot, lemongrass , rosemary, cinnamon , thyme , and clove . Moreover, based on SEM images, gamma irradiation with 5 KGy had not observed remarkable effects on the morphology of prepared films. Moreover, the SEM image of MgO nanoparticles are shown in Fig. 3.
In vitro antimicrobial activity of basil seed mucilage-chitosan films
The results of in vitro antimicrobial activity of basil seed mucilage-chitosan films are presented in Tables 1 and 2. Based on our findings presented in Table 1, for pure basil seed mucilage-chitosan film as a control group (without ZEO and MgO nanoparticles) and films containing MgO nanoparticles (0.1 and 0.2%) no inhibition zone was observed. After removing the films from agar plate, the pathogenic microorganisms did not growth in the agar plates. These results are in good agreement with those reported for chitosan film/coating with inherent antimicrobial property [2,38]. Based on the results of broth-micro dilution assay (Table 2), the log DP of pure basil seed mucilage-chitosan film, film + MgO 0.1%, and film + MgO 0.2% were found to be -0.18 - -0.41, -0.35 - -0.89, and -0.54 - -0.97, respectively. Recent studies indicated that the antimicrobial property of chitosan is probably due to the interaction of its positively charged amino group with negatively charged bacterial cell membranes, which is cause the releasing of critical constituents of bacterial cell and kill them [12,39]. It has been suggested that reactive oxygen species released from MgO nanoparticles can attack to the bacteria such as E. coli O157:H7, S. aureus, and Salmonella Stanley and lead to cell death via disrupting the cell wall [15,40,41].
The inhibition zone and log DP of films containing ZEO 1% and 2% against bacteria were recorded to be 2.3-7 mm and -0.79 - - 1.86, respectively (Tables 1 and 2). The in vitro antimicrobial activity of ZEO is also attributed to the main phenolic compounds which they release its proton increases the delocalization of double bonds and ATP in bacteria, also disrupt their cell wall and finally cell death . The in vitro antibacterial activity of ZEO has been also conducted [24,28,43]. Behravan et al., (2007)  showed that the MICs of ZEO against E. coli, S. aureus, Pseudomonas aeruginosa, S. Typhimurium, and Klebsiella pneumoniae were 0.003, 0.033, 0.033, 0.067, and 0.067% (v/v), respectively. Ozturk & Ercisli, (2007)  indicated that ZEO had broad-spectrum antibacterial activity against Bacillus spp., Moraxella catarrhali, and Enterobacter sakazakii in broth micro-dilution assay. In this study, the highest antimicrobial activity was found for film containing ZEO 2% + MgO nanoparticles 0.2% with inhibition zone and log DP of 5-7.9 mm and -1.76 - -3.34, respectively. There are numerous pathway for combined effects of antimicrobial compounds such as suppressing the crucial enzymes, combinations of cell wall active agents, and application of cell wall active agents to increasing the receiving of other antimicrobials . Previous studies also indicated that the antimicrobial properties of films varies depending on the properties of incorporated nanomaterials, molecular weight of polymers, preparation method, type of target microorganisms and solvents, and the diameter size of prepared films. It has been reported that the antibacterial activity of films with increasing size of diameters resulting in increased antibacterial property [45,46].
Our findings also showed that gamma irradiation of prepared films with 5 KGy has not effect on antimicrobial property of designated films. Zantar et al., (2015)  indicated that low dose of gamma irradiation (10 KGy) did not any effect on the antimicrobial activity of Thymus vulgaris and Mentha pulegium EOs, which is in agreement with our findings.
The main chemical composition of ZEO were found to be carvacrol (65.22%), thymol (19.51%), ɣ-terpinene (4.63%), and p-cymene (4.86%). According to our results, it may be recommended that basil seed mucilage-chitosan films containing ZEO and MgO nanoparticles can be used for increasing of shelf-life of stored food commodities.
CONFLICT OF INTEREST
There is no conflict of interest among the authors.