Fish and seafood are valuable sources of important nutrients including amino acids, vitamins, unsaturated fatty acids, minerals (iron, calcium, copper, zinc and iodine) and other nutrients, which are play important roles in human health . Fishery products are extremely perishable foods compared with other fresh foodstuffs due to 65-80% water content, pH 6-7 and availability of important nutrients on the product surface . Previous studies reported that spoilage of fresh and minimally processed fish during refrigerated storage is dominated not only by microbial activities including aerobic mesophilic bacteria, psychrotrophic bacteria and coliforms but also is deeply influenced by endogenous enzymatic activity [1-3]. The microbial and chemical quality factors of untreated fresh fish including spoilage microorganism’s count as well as lipid and protein oxidation have been previously found to decline during chilled condition [1, 4], which can result in sensorial quality change of the product, and subsequently related to both economic losses and food-borne diseases . On the other hand, food-borne pathogens are causing a great number of diseases with remarkable effects on human health . Among them, Listeria monocytogenes and Staphylococcus aureus have been frequently isolated from various foodstuffs .
Shelf life extension and safety improvement of fresh fish and seafood may be performed using various preservation techniques such as active and modified atmosphere packaging (MAP), direct addition of synthetic compounds, biotechnology products and natural antimicrobials are used [3, 7-9]. Among packaging materials, chitosan (CH) has been widely studied for the development of biocompatible packaging material due to excellent properties such as intrinsic antibacterial and antifungal activities and also excellent physic-mechanical properties . CH, a linear polysaccharide, consist of β-(1–4)-2-acetamido-D-glucose and β-(1–4)-2-amino-D-glucose units . It has unique combination properties such as non-toxicity, biocompatibility, metal complexation biodegradability, good film-forming ability, stability and flexibility . Therefore, it is recognized as an eco-friendly polymer food packaging [12-14]. Some studies indicated that the application of plant essential oils and natural extracts from rosemary , cinnamon , thyme , oregano  and pomegranate peel  could improve the shelf life and safety of raw foods during storage at chilled condition. Different parts of pomegranate fruit (Punica granatum) such as arils and rinds have valuable bioactive compounds (hyrolysable tannins, anthocyanins and flavonoids) and their functional and medicinal impacts such as anti-inflammatory, anti-diabetic, anti-bacterial and anti-viral properties have been approved [18-21]. Previous studies have been evaluated the effect of pomegranate peel extract (PPE) to increasing microbial, chemical and sensory quality factors of raw, cooked and ready-to-eat food stuffs [17, 18, 22-24].
Zinc oxide nanoparticle (ZnO) is also considered as a novel compound for development of nanocomposite coatings and film materials . A recent study has been reported that carboxymethyle cellulose-sodium alginate film containing ZnO could successfully extend shelf life of minced silver carp fillets under refrigerated condition and prevent the growth of Listeria monocytogenes in a food model . There are no reported study on the application of nanocomposite film based on CH-ZnO containing PPE to control the growth of L. monocytogenes and S. aureus in fresh rainbow trout fillets. Hereto, the present study aimed to examine the impacts of CH-ZnO film enriched with methanolic PPE (0.5, 1 and 1.5%) on growth controlling of L. monocytogenes and S. aureus in stored raw rainbow trout fillets under refrigerated condition for 12 days.
MATERIALS AND METHODS
Food-grade ZnO (< 35 nm diameter and purity > 99%) was purchased from the Iranian Nanomaterials Pioneers (Razavi Khorasan, Iran). CH powder (viscosity: 200-800 cP, 75-85% deacetylated and medium molecular weight: 190-310 kDa) was purchased from Sigma-Aldrich, UK. All chemicals and culture media were of analytical grade and purchased from Merck, Germany.
Preparation of methanolic pomegranate peel extract
Pomegranates (Punica granatum L.) were purchased from a local garden in the Saveh, Tehran, Iran. The peels of the fruits cut into small pieces, cleaned, washed and dried in a dark place at ambient temperature for approximately two weeks. The extraction of methanolic PPE was performed using the previous procedures by Basiri et al.  as well as Mohebi and Shahbazi .
Preparation of bacterial pathogens
S. aureus ATCC 6538 and L. monocytogenes ATCC 19118 were purchased from the Iranian Research Organization for Science and Technology Institute (Tehran, Iran). For long time maintenance, the corresponding pathogens were kept in Brain Heart Infusion broth containing 20% glycerol at -18 °C. Working cultures were stored on BHI agar slants at chilled temperature (4 ± 1°C) and sub-cultured every week on the same agar. For inoculation, a single colony for each bacterium was cultured on BHI broth and incubated at 37 °C for 18 h. Then, the bacteria were diluted to 5 log CFU/ml using 0.1% peptone water before inoculation in meat samples .
Preparation of active chitosan films
CH solution (final concentration = 2%) was made using an appropriate amount of CH powder in 1% (v/v) glacial acetic acid and constantly agitated for approximately 3 h at 37 ºC on a magnetic stirrer to ensure complete dissolution. The mixture was filtered through Whatman No. 3 filter paper. Then, glycerol as a plasticizer was incorporated to a level of 0.75 ml/g CH and the solution was stirred for 30 min and Tween 80 at level of 0.2% of extract as an emulsifier was added. After 1 h of stirring, PPE (0, 0.5, 1 and 1.5%) and ZnO (0.5%) were added to CH solution. Then, 50 ml of the film forming solutions were poured on the glass petri dish and dried for 48h at ambient temperature conditions (25 ± 1ºC) .
Determination of Minimum Inhibitory Concentration (MIC) of active films
The Minimum Inhibitory Concentration (MIC) of CH-ZnO films containing different concentrations of methanolic PPE (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 0.9 and 1%) was determined according to the previously published method by other authors .
Total polyphenolic content of active films
Total polyphenolic contents of CH-ZnO films containing methanolic PPE 0.5, 1 and 1.5% were determined with Folin-Ciocalteu reagent according to the published procedures by Shahbazi using gallic acid as a standard.
Treatment of fish fillets
Fresh rainbow trout (Oncorhynchus mykiss) averaging from 900 to 980 g were purchased from a local fishery plant (Kermanshah, Iran) and immediately transported to the Food Hygiene laboratory. Upon arrival, the fishes were manually killed and filleted in aseptic condition. Then, the fillet samples dipped in beakers containing 5 log CFU/ml of S. aureus and L. monocytogenes for 30 min at room temperature. The samples were air dried for 30 min to allow attachment of bacterial pathogens onto fish fillets. After this step, fish fillets were packed in CH-ZnO films containing different concentrations of methanolic PPE (0.5, 1 and 1.5%), put in sterile bags (Intersicence, France) and kept under chilled temperature (4 ± 1 ºC) for 12 days. The experiment was conducted in triplicate.
In order to enumerate the inoculated pathogenic bacteria in raw rainbow trout fillets, Palcam Listeria selective agar (L. monocytogenes, incubated at 30 °C for 48 h) and Baird Parker agar (S. aureus, incubated at 37 °C for 48 h) were used .
Sensorial properties of un-inoculated samples including color, odor and overall acceptability was evaluated by a group of ten-members from the laboratory staff. For this purpose, ten descriptive hedonic scale (1 = extremely unacceptable and 10 = extremely acceptable) was used .
Surface morphology of films was observed using a TeScan MIRA3 SEM. Film thickness was assessed using digital micrometer (Mitutoyo, Mitutoyo Corporation, Japan) to the nearest 0.001 mm. The interaction between the components and incorporation confirming was evaluated using Fourier Transform Infrared Spectroscopy (FTIR; Bruker, model ALPHA, Germany) within the wave number range of 400–4000 cm−1. The color of film samples in terms of lightness (L), redness (a) and yellowness (b) was determined using Minolta Chroma Meter Model CR-400 (Minolta Co Ltd, Osaka, Japan). Hence, the film pieces (20 mm in diameter) were placed over the standard white plate (L* = 93.49, a* = -0.25 and b* = -0.09). Color differences (DE) were calculated by the following equation :
Where L*, a* and b* are the color parameter values of the standard and L, a and b are the color parameter values of the sample.
All experiments were conducted in triplicate. The microbiological data were expressed as log CFU/g. Analysis of variance (ANOVA) and Duncan’s multiple range test using SPSS 16 were performed for evaluation of significant differences (P < 0.05) among designated groups.
RESULTS AND DISCUSSION
The effects of incorporating PPE in the thickness of CH-ZnO films on the performance of the nanocomposite is shown in Table 1. Based on our findings, the film thickness was in the ranges of 0.082 and 0.098 mm. There were no significant differences among control and films incorporated with different concentrations of PPE (P > 0.05). Similarly, Moradi et al. reported that addition of Zataria multiflora Boiss essential oil and grape seed extract had no significant effects on the thickness property of CH film .
Based on the results of the color property (Table 2), yellowness (b* value) reduced significantly with incorporation of the PPE, whereas control CH film had the most positive value (29.11 ± 0.2). The a* and L* values were higher and lower in films incorporated with PPE compared to control group, which is could be related to internal interaction between film compounds and phenolic constituents of PPE during film drying . Previous study reported that the droplet size of film compounds had the important effect on the color properties of films made from them. Similar trend was found by incorporation of plant extracts in soy protein  and pea starch .
Through comparison of Figs. 1a-c which show the surfaces of CH films without or with ZnO and PPE, homogenous structure (without any insoluble particles) was observed in the CH film. From Fig. 2 SEM analysis, it was observed that the ZnO size in the nanocomposite films was lower than 35 nm. As it can be seen, high degree of ZnO agglomeration was observed in the nanocomposite film, which is in consistent with the findings of Rezaei and Shahbazi  and Rahman et al. .
The FTIR spectra of ZnO, CH, CH + ZnO and CH + ZnO + PPE 0.5% films are presented in Fig. 3. The most important peaks of pure CH film were as follow: 3428.59 cm−1 (stretching vibrations of O-H and N-H), 2927.26 cm−1 (aliphatic C-H stretching vibration), 3068.88 cm−1 (CH2 stretching vibration), 3027.81 cm−1 (CH3 stretching vibration), 1702.25 cm−1 (C=O stretching of amide I), 1664.02 cm−1 (NH of amide II stretching vibration), 1110.29 cm−1 (C3-OH stretching vibration) and 1039.48 cm−1 (C6-OH stretching vibration) . For the FTIR of ZnO, two peaks including 548 cm−1 (Zn–O stretching) and 3254 cm-1 (OH group) were observed . Based on our findings, there were no new bands for designated nanocomposite film without PPE, suggesting ZnO physically included to the film matrix. The result of the present study is in consistent with those reported for polyvinyl chloride-ZnO  and fish protein isolate-fish skin gelatin-ZnO  based films. For film containing PPE, peaks around 1000-1800 cm-1 might be attributed to the stretching of C=O, -C=C-C=O, -C=C- [(in-ring) aromatic] and -C-C- [(in-ring) aromatic] found in the phenolic components of PPE .
Total phenolic content of active films
In the current work (Fig. 4), total phenolic contents of CH-ZnO films containing PPE (0.5, 1 and 1.5%) was recorded to be 72-139 mg gallic acid/g film. Based on our findings, pure CH had a total phenolic contents of 8 mg gallic acid/g film. It might be related to the production of chromogens during the expriment, because of the reaction of Folin-Ciocalteu reagent with other reducing constituents which determined spectrophotometrically . According to the previous studies [18, 20, 21, 39, 40], phenolic compounds are the main constituents in PPE. Berizi et al.  found that the total phenolic content of pomegranate extract obtained from Shiraz, Iran (Rabab variety) was 70.83 mg tannic acid/g PPE. Turgut et al. reported total phenolic content of 165.4 mg gallic acid /g PPE for pomegranate obtained from Isparta, Turkey. Özdemir et al. indicated that phenolic content of aqueous pomegranate extract was 140 mg catechin/g and 158.5 mg gallic acid/g, respectively. In another studies [39, 40], the total phenolic contents of PPE were also reported 258.2 mg/g and 508.8 mg/g PPE, respectively. It has been generally accepted that the variability of total phenolic contents of fruit extracts in different studies are greatly depends on growth stage and strain of fruits and also climate change, seasonal variation and geographical condition .
Antibacterial effect of active films
Based on the findings of the current study, the MICs of active films containing PPE for both S. aureus and L. monocytogenes were found to be 0.5%.
As it can be seen in Fig. 5, for untreated samples, the initially recorded population of 5 log CFU/g of L. monocytogenes reached 5.36 log CFU/g at the end of designated study period. There were significant differences between samples packed with CH-ZnO films enriched with PPE at concentrations of 0.5, 1 and 1.5% and untreated one (P < 0.05). In both samples packed with 1 and 1.5% PPE, the final population of L. monocytogenes were reached below 1 log CFU/g on days 12 of storage, while the final count of treated samples with 0.5% PPE was found as 3.06 log CFU/g at the end of refrigerated storage.
The changes of S. aureus in untreated and treated raw trout fillets with designated films enriched with PPE at concentrations of 0.5, 1 and 1.5% are shown in Fig. 6. In control group, the initial bacterial count of 5 log CFU/g decreased up to 3.03 log CFU/g after 12 days of refrigerated storage. All samples packed with CH-ZnO films incorporated with PPE significantly prevented S. aureus growth during refrigerated period as compared to control group (P < 0.05). The group treated with CH-ZnO + PPE 1.5% showed the maximum antibacterial impact against the microorganism (P < 0.05). Like L. monocytogenes, CH-ZnO + PPE 0.5% could not inhibit the growth of bacterial strain; as the final bacterial population in treated sample with PPE 0.5% was found to be 2 log CFU/g at the end of storage day.
Control CH-ZnO film exhibited antibacterial effect against both food-borne pathogens, however, prevention of their growth was significantly lower that designated films enriched with PPE. In general, the positively charged amino group of CH is interacted with negatively charged bacterial cell membranes and it is considered as the most important reason of antibacterial effect of CH film. This phenomena cause the leakage of intracellular essential constituents of the bacterial pathogens and ultimately killed them [27, 41]. These results are similar with those published by Ojagh et al.  and Mohebi and Shahbazi  who indicated that CH film was success in delaying growth of bacteria in food models compared to untreated ones. Moreover, previous studies reported that Zn+2 ions from the powder could react with the microorganism surfaces and interior constituents, irreversibly damage the cell membrane and cause leakage of essential process in the bacterial cell [25, 34]. Previous studies have been reported that antimicrobial activities and many other benefits of essential oils and natural extracts are related to their phenolic compounds . It has been demonstrated that PPE has excellent antibacterial effects against some pathogenic bacteria including Escherichia coli, Pseudomonas aeruginosa and S. aureus [43-46]. A previous study , reported that MIC of methanolic PPE against L. monocytogenes was 0.5%. They also reported that PPE was effective (MIC 2%) against two strains of S. aureus including S. aureus ATCC 6538 and methicillin-resistant S. aureus. Regarding the results of disk diffusion method, PPE was a good inhibitor for L. monocytogenes, S. aureus, E. coli O157:H7 and Yersinia enterocolitica by inhibition zones of 3.68, 14.42, 4.12 and 5.32 mm, respectively . Variation in findings among studies were found on PPE with MIC determinations against L. monocytogenes and S. aureus [48-50]. The antibacterial effect differences of PPE might be described with differences of fruit species, bacterial inoculation level, culture media and the used methods for evaluation of antibacterial efficacy . Our previous study  showed that CH and gelatin films containing Ziziphora clinopodioides essential oil (0 and 1%) alone and in combination with PPE (0 and 1%) were very success to increase shelf life of peeled shrimp during chilled storage. Moreover, we found that all shrimp groups treated with the aforementioned designated films contained 2-3 log CFU/g reduction of L. monocytogenes as compared to control group. In another study , treating the peeled shrimp with PPE could significantly retard the microbial spoilage counts. Moreover, previous studies indicated that the incorporation of plant essential oils and natural extracts caused a decrease of 2-4 log CFU/g in the numbers of L. monocytogenes and S. aureus in different food models [9, 16, 51, 52]. Khan and Hanee  have also found the role of bioactive compounds of PPE such as anthocyanins, hydrolysable tannins, flavan-3-ols and flavonoids as the most active compounds against bacteria. According to their results, these compounds were also responsible for its antifungal and antioxidant properties.
The impact of CH-ZnO + PPE films on the general aspect of raw rainbow trout fillets after 12 days of storage during chilled condition is shown in Fig. 7. Our findings indicated that PPE 1 and 1.5% had no negative impacts on the sensorial characteristics of treated samples in comparison control samples. The reason of lower acceptability scores of untreated samples was higher microbial spoilage growth and chemical changes.
The findings of the current work indicate a potential use of CH-ZnO films enriched with PPE as an excellent types of biodegradable compounds to inhibit the growth of L. monocytogenes and S. aureus in raw rainbow trout fillets. Both samples containing 1 and 1.5% of PPE had the highest scores of sensory evaluation until day 12 of storage.
CONFLICT OF INTERESTS
The author claims that there is no conflict of interest.
The author acknowledges Razi University for providing their instrumentations.
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