Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage

Accepted Manuscript Title: Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage Author: Behrooz Alizadeh Behbahani Fakhri Shahidi Farideh Tabatabaei Yazdi Seyed Ali Mortazavi Mohebbat Mohebbi PII: DOI: Reference:

S0141-8130(16)31791-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.10.055 BIOMAC 6635

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

27-9-2016 3-10-2016 17-10-2016

Please cite this article as: Behrooz Alizadeh Behbahani, Fakhri Shahidi, Farideh Tabatabaei Yazdi, Seyed Ali Mortazavi, Mohebbat Mohebbi, Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.10.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage

Behrooz Alizadeh Behbahani, Fakhri Shahidi*, Farideh Tabatabaei Yazdi, Seyed Ali Mortazavi, Mohebbat Mohebbi

Department of Food Science and Technology, Ferdowsi University of Mashhad, P.O. Box: 91775-1163, Mashhad, Iran Corresponding author: E-mail address: [email protected] (Fakhri Shahidi)

Highlights 

Plantago major seed mucilage (PMSM) extended the shelf life of beef.

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PMSM incorporated with Anethum graveolens essential oil extended the shelf life of beef.

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PMSM could be an effective coating material for beef.

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PMSM coatings suppressed bacteria and fungi growth in beef

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Thiobarbituric acid (TBA) and peroxide value (PV) correlated well with the microbiological data and sensory characteristics.

Abstract In this study, Plantago major seed mucilage (PMSM) was extracted from whole seeds using hot-water extraction (HWE). The dill (D) essential oil components were identified through gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) and its antioxidant properties were examined through the methods of 2,2-diphenyl-1picrylhydrazyl (DPPH), ferric reducing antioxidant potential (FRAP) and ß-carotene-linoleic acid assay (B-CL). Total phenolic content (TPC) was characterized through the FolinCiocalteu method and the antimicrobial effect was evaluated on 10 pathogenic microorganisms. PMSM edible coating incorporated were prepared in four different concentrations of essential oils, including 0, 0.5, 1 and 1.5% (w/w). The control and the coated beef samples were analyzed periodically for microbiological (total viable count,

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psychrotrophic count, Escherichia coli, Staphylococcus aureus and fungi), chemical (thiobarbituric acid, peroxide value and pH), and sensory characteristics. The IC50, FRAP, BCL and TPC of the dill essential oil were equal to 11.44 μg/mL, 9.45 mmol/g, 82.86 and 162.65 μg/ml GAE, respectively. PMSM extended the microbial shelf life of beef by 3 days, whereas the PMSM + 0.5%D, PMSM + 1 %D and PMSM + 1.5%D resulted in a significant shelf life extension of the beef by 6, 9 and 9 days, respectively, as compared to the control samples.

Keywords: Plantago major seed mucilage, Anethum graveolens, Chemical composition, Edible coating, Antimicrobial effect, Beef.

1- Introduction Plantago major, also knownas Ribwort, is a plant seed which is a species of a flowering plantin the plantaginaceae family. The plant produces a large amount of seeds. The ellipsoidal seeds are small (0.4–0.8 × 0.8–1.5 mm) and slightly bitter. They are located in capsules (8–16 per capsule) and become gummy under warm and humid conditions which can be attributed to the swelling of the polysaccharides occurring in the seed coat. These polysaccharides include xylose, arabinose, galactorunic acid, glucuronic acid, rhamnose, galactose, and glucose [1]. Dill (Anethum graveolens) is an annual plant native to the southwest and middle of Asia. Nowadays, it is raised in many regions of the world including south of Europe, Iran, Egypt, USA and China. Its natural geographical propagation in Iran has been reported in different areas such as Fars, Khuzestan, Tabriz, Khorasan and Tafresh. The pharmacological effect of this plant has also been stated among which the antiviral, antispasmodic, anti-fat effects could be mentioned. The occurrence of flavonoids and other phenolic compounds has been cited in various extracts of dill [2, 3].

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In organisms, peroxidation of the lipids present in their cell walls is one of the most important targets of free radicals. Under these conditions, not only the cell wall structure and function are influenced, but also some of oxidation products could react with biomolecules and raise cytotoxic and genotoxic effects. Therefore, the high concentration of free radicals, especially the peroxides, play a key role in developing some illnesses such as cancer, diabetes, cardiovascular diseases and many types of degenerative neurotic and pulmonary diseases [4-6]. The excessive production of free radicals (especially in chronic bacterial, viral and parasitic infections) could results in imbalance or so-called oxidative stress. Oxidative stress could cause the large biomolecules such as proteins and DNA and fats to be damaged and hence increases the development of cancers and cardiovascular diseases. It is necessary to consume sufficient amounts of antioxidants to prevent or reduce the oxidative stress [7, 8]. Infectious diseases are of the widespread and prevalent diseases throughout the world which impose high costs on societies. Not only are medicinal plants effective in the treatment of infectious diseases, but also reduce many side effects which are often accompanied by antimicrobial agents. The use of antimicrobial combinations that have synergistic or additive effects may be a strategy for inhibition or control of food-borne pathogens or food spoilage organisms [9]. Currently, the tendency towards the application of antimicrobial edible films and coatings is increasing due to environmental issues [10]. Antimicrobial edible films and coatings which contain natural herbal extracts and essential oils, have several benefits and are considered as a novelty in active biodegradable packaging [11]. Antimicrobial edible films and coatings have been developed to reduce or inhibit the microbial growth on the surface of food products. The edible coatings prepared from natural polymers such as carbohydrates and proteins not only

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are biodegradable and environmental-friendly but also do they retain the quality, increase the storage stability and minimize the loss of moisture and volatile nutraceuticals [12, 13]. Meat and meat products are prone to microbial spoilage during storage. Since meat has a high moisture content and a suitable pH as well as being rich in nitrogen compounds, fermentable carbohydrates and minerals, it could be an ideal culture medium for the growth of many spoilage and food-poisoning microorganisms [14]. The estimates made in developed countries indicate that 30% of people suffer from foodborne diseases annually [11]. Nowadays, quality and safety are two important concerns for the food industry. To the best of our knowledge, no study has gained insight in to the PMSM edible coating incorporated with Anethum graveolens essential oil. The objective of this research was to identify the components of the dill essential oil, to evaluate the antioxidant strength of the Iranian dill essential oil and to scavenge the free radicals and oxidation. This study was also aimed to examine the antibacterial and antifungal effects of the dill essential oil and PMSM containing dill essential oil on shelf life extension of beef during refrigerated storage (4 °C) were evaluated over a period of 18 days.

2- Materials and methods 2-1- Reagents, Solvents and microbial media All reagents, solvents and microbial media including Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), Sabouraud Dextrose Broth (SDB), Sabouraud Dextrose Agar (SDA) Plate Count Agar (PCA), Eosine Methylene Blue (EMB) and Manitol Salt Agar (MSA) were purchased from Merck Co. Germany and 2,2-diphenyl-1-picrylhydrazyl (DPPH), B-carotene, Linoleic acid and Butylated Hydroxy toluene (BHT) were supplied form Sigma-Aldrich, USA.

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2-2- Mucilage extraction Plantago major seeds were purchased from a local market in Mashhad, Iran. The seeds were cleaned and then wrapped in plastic bags, sealed and kept in a dry cool place. PMSM was extracted according to the method Alizadeh Behbahani et al., (2017) [1]. PMSM chemical analysis indicated that the gum contained 6.66% protein, 3.69% moisture, 6.80% ash and no fat content. The total carbohydrate content measured by the phenol-sulfuric acid method was 88.34% [1]. 2-3- Extraction of the dill essential oil and determination of chemical composition of essential oil Fresh dill was purchased form a local market (Mashhad, Khorasan Razavi, Iran) at the beginning of its vegetative period. 50 g of the powdered dill as well as 750 ml of distilled water was transferred to the glass clevenger apparatus operating based on hydrodistillation. Extraction of the essential oil was performed for 3 hrs with the distillation speed of 1 ml/min. Next, the essential oil was collected in vials which had already been weighed using a 0.0001 balance and stored at 4°C [15]. Identification of the extracted essential oil components was carried out by injecting 0.2 μl of the dill essential oil into a gas chromatograph (TRACE MS, Thermo Quest-Finnigan) equipped with the column of DB-5 (length: 30 m, internal diameter: 0.25 mm and stationary phase thickness: 0.25 μm) which was integrated with a mass spectrometer (Quadrupole). The column temperature increased from 40°C to 250°C with the rate of 2.5°C/min. Furthermore, Helium was used with the rate of 1.1 ml/min and the ionization energy of 70 EV (13). The components of the essential oil were identified through calculating their retention indexes under temperature programmed conditions for n-alkanes (C8-C20) and the essential oil on aDB-5column. After that, retention indexes of the compounds were calculated using the following equation: I=100×[n+(N-n) (log tunknown–log tn)/(log tN–log tn)], where I is Kovats retention index, N and n are the number of carbon atoms

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in the larger and the smaller n-alkanes, respectively. The individual compounds were identified through comparison of their mass spectra and retention indexes (RI) with those of the authentic samples and those given in the literature. Quantification of the individual ingredients relative amounts was conducted based on the area percentage method without regarding the calibration factor [16].

2-4- Determination of the dill Total Phenolic Content (TPC) The method of Follin-Ciocalteu is generally used for the measurement of TPC. In order to prepare the stock solution, 0.4 g of anhydrous gallic acid was dissolved in 10 ml of ethanol 96% (Merck, Germany) in a 100-ml volumetric flask and made to the mark by distilled water. In order to plot a calibration curve, 0, 1, 2, 3, 5, 10 and 20 ml of the stock solution was transferred to 100-ml volumetric flasks and made to the volume using distilled water. The resulting solutions have the concentrations of 0, 50, 100, 250, 500 and 1000 mg gallic acid/l. In this experiment, 20 μl of the dill essential oil with the concentration of 10 g/l was mixed with 2 ml of distilled water and 100 μl of the FollinCicalteu indicator. 300 μl of the NA2CO3 solution was added to them after 3 min and the solutions were shaking for 2 h. finally, the absorbance values of the solutions were measured at 765 nm using a spectrophotometer (Sigma3-30k). After plotting the calibration curve of gallic acid, the following linear equation was obtained: y= 0.005x+0.0194; r2= 0.9977. By substituting the absorbance values for x, the gallic acid equivalent concentration of the essential oil samples was calculated. The final result was reported as mg of gallic acid / g of the dried essential oil [17].

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2-5- DPPH free radical-scavenging assay In this experiment, 3.9 ml of the prepared stock DPPH (0.004 g of DPPH in 100 ml of methanol) was transferred to a test tube and then 0.1 ml of each extract was added to it and placed in darkness for 30 min and its absorbance value was read at 517 nm. The radical scavenging percent of DPPH was calculated using the following equation: % Scavenging activity = [(Abs control − Abs sample)]/ (Abs control)] ×100 where “Abs control” is the absorbance of DPPH + ethanol; “Abs sample” represents the absorbance of DPPH radical + sample. Then, the results were stated as IC50 (amount of antioxidant required to reduce the DPPH concentration to 50% of its initial value) [18].

2-6- ß-Carotene-linoleic acid assay In this test, the antioxidant potential is assessed with the extent of oxidation prevention by linoleic acid and inhibition of the formation of volatile compounds and conjugated hydroperoxides. The B-CL was determined according to Dapkevicius et al., 1998 [19]. The average percent of inhibition was calculated from the data with the formula: % Inhibition = [(A A (120) − A C (120) / [(A C (0) − A C (120)] ×100 where AA(120) is the absorbance of the antioxidant sample after 120 min, AC(120) is the absorbance of the control after 120 min, and AC(0) is the absorbance of the control at the beginning of experiment (t=0) All determinations were run in triplicate.

2-7- Ferric reducing antioxidant potential (FRAP) assay This assay was conducted based on the method of Lim et al., 2009 [20]. According to this method, the indicator including2,4,6-tri-(2-pyridyl)-s-triozine (TPTZ) 10 mL of acetate buffer 300mM, pH 3.6, to 1.0 mL of 2,4,6-tri-(2-pyridyl)- s-triozine (TPTZ) 10mM (dissolved in HCl 40mM) and 1.0 mL of ferric chloride hexahydrate 20 Mm

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(dissolved in distilled water). In this method, the electron-donating antioxidants cause the reduction of ferric (Fe+3) to ferrous (Fe+2) at low pH. Therefore, they are able to change the colorless complex of ferric-tri-pyridyltriozine into the blue complex of ferrous-tripyridyltriozine which has the maximum absorbance value at 593 nm. In order to assess this property, 3 ml of FRAP was added to 100 μl of the obtained extract. The resulting solution was vortexed and incubated at 37°C for 10 min. the absorbance values of the solutions were read at 593 nm as compared to the blank (100 μl of distilled water along with 3 ml of FRAP). Ammonium ferrous sulfate was used as blank for comparison [20].

2-8- Preparation of the microbial strains The strains employed in this study included Pseudomonas aeruginosa ATTC 27853, Proteus vulgaris ATTC 8427, Escherichia coli ATTC 25922, Bacillus cereus ATTC 14579, Bacillus subtilis ATTC 23857, Staphylococcu aureus ATTC 25923 Streptococcus pyogenes ATTC 19615, Aspergillus fumigatus ATTC 1022 , Penicillium expansum ATTC 24692 and Candida albicans ATCC 5027which were supplied from the School of Pharmacy of Mashhad University of Medical Sciences and that of Shahid Beheshti University of Medical Sciences.

2-9- Experiments concerning the evaluation of the antimicrobial activity “in vitro” In this study, various qualitative and quantitative methods were used to evaluate the antimicrobial activity of the dill essential oil. These methods are summarized as follows:

2-9- 1- Disk Diffusion Agar (DDA)

This method is generally performed for a preliminary investigation to measure the antimicrobial activity prior to more precise tests. The kirby-bauer test of agar diffusion was used in the qualitative method during which a standard microbial suspension was cultured in the medium of MHA through the superficial method and then the blank disks (Padtanteb Co, Iran) were placed on the agar in certain distances from each other and the plate edge to

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examine the antimicrobial properties. 20 μl of the dill essential oil dilutions of 0.125, 0.25, 0.5, 1, 2, 4 and 8 mg/ml in the solution of DMSO were added on the disks. The disks of antibiotics including gentamycin, vancomycin and amphotericin B with the concentration of 10 mg/ml were utilized as positive control as well. Then, the media containing the bacteria and fungi were incubated at 37 and 27°C for 24 and 72 hrs, respectively [21]. The results were examined by measuring the diameters of the halos formed around the disks and the results achieved from antibiotics were compared with the CLSI (Clinical and Laboratory Standards Institute) tables [22]. All experiments were triplicated for each bacterial and fungal strain to get assured of each of the various concentrations of the essential oil and antibiotics.

2-9- 2- Well Diffusion Agar (WDA)

In this method, the plates containing MHA and SDA were also used which were contaminated with microorganisms. Using a sterilized pastor pipette specified for well creation, a cavity was created in the medium. Using a sampler, 20 μl of the concentrations of 0.125, 0.25, 0.5, 1, 2, 4 and 8 mg/ml of the dill essential oil in DMSO was placed on each cavity separately. Next, the media including the bacteria and fungi were incubated at 37 and 27°C for 24 and 72 hrs, respectively [23]. After that, the extent of the inhibition zone was evaluated and calculated as mm and their average value was recorded.

2-9- 3- Minimum Inhibitory Concentration (MIC)

The quantitative test for the determination of MIC was carried out in a sterilized 96-well plate with the method of broth microdilution. In the first place, 100 μl of the MHB and SDB media were added to the wells pertaining to the respective dilutions. 100 μl of the essential oil was then added to the first well and it was diluted from the second well to the seventh one. Eventually, 100 μl of the suspension (equivalent to 0.5 McFarland 1.5 × 108 CFU/ml) was

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added to each well. The media consisting of the bacteria and fungi were then incubated at 37 and 27°C for 24 and 72 hrs, respectively [24].

2-9- 4- Minimum Bactericidal and Fungicidal Concentrations (MBC& MFC)

In order to determine MBC and MFC, all opacity-free wells were separately cultured on the media of MHA and SDA. Then the media containing bacteria and fungi were incubated for 24 and 72 hrs, respectively. The essential oil concentration, in which bacteria or fungi did not grow, was reported as MBC and MFC [24]. 2-10- Preparation of PMSM coating and coating of the beef samples 5 gr of the extracted PMSM was mixed with 1.75 g of Tween 80 (35% of the PMSM dried weight) and made to 100 ml with distilled water and heated and agitated using a magnetic stirrer. The natural essential oil of dill was added to PMSM solution as a natural antimicrobial compound at 0, 0.5, 1 and 1.5% v/v. After that, 1 group out of the 5 groups of the samples was coated through being immersed in the PMSM solution for 1 min and the other groups were immersed in the PMSM solutions containing different concentrations of dill essential oil for the same period of time. A group remained uncoated as the control group [25].

2-11- Microbiological analysis 5 gr of the meat sample were mixed with 45 g of peptone water 0.1% in a Stomacher. The mixture was homogenized at a rate of 200 rpm for 1 min. Next, subsequent dilutions were prepared in the test tubes containing peptone water 0.1% and inoculated into the plates containing the culture medium. The performed microbial tests include: 1. Total viable count bacteria count in plate count agar (incubation at 37°C for 24 h). 2. Psychrotrophic count in plate count agar (at 7°C for 10 days).

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3. Mold and yeast count in sabouraud dextrose agar (incubation at 27°C for 72 h). 4. Escherichia coli count in eosin methylene blue (incubation at 37°C for 24 h). 5. Staphylococcus aureus count in manitol salt agar (incubation at 37°C for 24 h) [26].

2-12- Chemical analyses 2-12-1- Determination of 2-thiobarbituric acid (TBA)

2-Thiobarbituric acid (TBA) (mg malonaldehyde/kg beef) was measured based on the method proposed by Jouki et al., (2014a) [26]. Each TBA value denotes the average value of at least three samples taken from various treatments. 2-12-2- Estimation of peroxide value The peroxide content was determined in the total lipid extracts according to Jouki et al., (2014b) [27]. Primary lipid oxidation was evaluated by means of PV. Results were expressed in meq oxygen/kg lipid. 2-12-3- pH measurement

10 gr of the meat sample was blended with 90 ml of distilled water in 150-ml bottles and homogenized with a homogenizer at 13000 rpm for 30 s. Then, the pH value was measured using a pH meter (HI 221, Hanna Instruments, Woonsocket, RI) at room temperature [28]. 2-13- Sensory evaluation The sensory quality of the beef samples was assessed by 10 trained panelists working in the laboratory. Four beef samples representing different treatments were separately presented to each panelist in small covered porcelain dishes. The panelists were not informed of the experimental procedure and the samples were blind-coded with 3- digit random numbers. The judges scored sensory properties such as color, odor and overall acceptability using a nine-

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point hedonic scale (1, dislike extremely to 9, like extremely). The beef samples which received the overall scores of more than 4 were considered acceptable [29]. (Truelstrup Hansen et al., 1995).

2-14- Statistical analysis All experiments were done in triplicate. Microsoft Windows Excel 2016 and SPSS software (Version18.0, SPSS Inc., Chicago, USA) were used to analyze the resulting data. Data were initially evaluated by analysis of variance (ANOVA), and then a Duncan's multiple range test was employed to detect significant (p < 0.05) differences.

3- Results and discussion 3-1- The essential oil composition The yield of the essential oil was equal to 2.4 v/w and its color was yellowish. With regard to the pattern of the exhaust of the n-alkanes and comparison of the mass spectra obtained from the GC-MS apparatus and the standard ones, it was realized that each of the mass spectra was exactly associated with each component. Kovats retention index was used to confirm the identifications carried out by the mass spectra (Table 1). The results of the essential oil analysis performed by the GC-MS apparatus are shown in Table 1. A total of eighteen components were identified which accounted for 99.62% of the entire components. α-Phellandrene (34.49%) was the major component of the essential oil. In addition, other components including Limonene (31.1%), Carvone (15.09%), Dillether (11.25%), p-Cymene (3.57%) and Dillapiole (2.58%) were the main components. According to the investigations and collected information, no information exists about the essential oil components of the dill provided from Mashhad so far and the present results are the first report concerning the

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essential oil components of this valuable native medicinal plant. Hartmans et al., (1995) maintained that the constituents of the dill essential oil consisted of Carvone (40%), Limonene (32%) and α-Phellandrene (20%), respectively [30]. De Carvelha et al., (2006) claimed that Carvone was the principle compound of the dill leaf essential oil. A useful compound with the odor of caraway/dill which is extracted and purified by chemical and biotechnological methods. This substance is among the major components in black seed and mint as well [31]. The results of the present study were consistent with those of the other researches to a great extent. It should be noted that the chemical composition of each plant essential oil is different under the influence of many factors such as climate, season, herbal genotype conditions and processing conditions as well as the species to be studied [32].

Table 1. Chemical composition of the dill essential oil

3-2- Antioxidant activity and TPC In this study, the free radical scavenging activity in addition to the oxidation prevention capability of the Iranian dill essential oil was examined. The free radical scavenging activity was investigated through the DPPH assay. In this assay, as the essential oil concentration increased, its scavenging strength increased, too. The IC50 (the concentration of the essential oil which scavenges 50% of the free radicals) of the dill essential oil has been compared with BHT and ascorbic acid in Table 2. As observed, the free radical scavenging activity of the dill essential oil was equal to 11.44 μg/ml which is slightly lower than those of BHT and ascorbic acid. In this research, the TPC content of the extracts differed and was calculated in terms of gallic acid (which is a pure phenolic compound) based on its standard curve through the Follin-Cicalteu method (y= 0.005x+0.0194; r2= 0.9977). The content of the phenolic compounds of the dill essential oil was computed as 162.65 μg/ml GAE. Phenolic

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compounds have a vital role in the antioxidant potential, furthermore; these compounds are of great importance in plants because of their contribution to color, odor and flavor. These effects are due to the occurrence of large amounts of antioxidants in plants [33]. Herbal phenols and flavonoids prevent the lipid peroxidation reaction through scavenging the proxy radicals and reduction or chelation of ferric in lipoxygenase. The difference in the antioxidant potential could be concerned with the capability of scavenging proxy radicals, DPPH free radical and hydroxyl radicals [34]. In a study performed by Bahramikia et al., (2008) on the antioxidant properties of the dill extract, the IC50 of the diethyl acetate, ethyl acetate and aqueous extracts was equal to 124.1, 75.6 and 152.2 μg/ml, respectively [3]. Shyu et al., (2009) investigated the antioxidant potential of the Tai dill flower extract and claimed that the IC50 of the ethyl acetate, ethanolic and hexane extracts was 28.15, 56.83 and 399.07, respectively [35]. Bahramikia et al., (2008) stated that the high TPC content is the main reason for the high antioxidant potential. It should be noted that the phenolic compounds act as hydrogen donors effectively which is why they also act as antioxidants [3]. Golluce et al., (2007) believed that the antioxidant potential of flavonoids which are specific phenolic compounds is because of their ability of hydrogen donation [36]. The results of the FRAP test of the dill essential oil and those of BHT and ascorbic acid have been compared in Table 2. The results indicated that as the reducing power increased, the antioxidant potential increased, too. As shown in Table 2, prevention of the linoleic acid oxidation in the system of ß-Carotene-linoleic acid was equal to 82.86. This value was somewhat lower than that of the synthetic antioxidant of BHT. Shyu et al., (2009) reported the value of 72.23% for the linoleic acid oxidation prevention test of the ethanolic extract of dill with the concentration of 0.4 mg/l to which the results of the present study conformed to some extent [35].

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Table 2. Antioxidant activities of the essential oil of Anethumgraveolens and some standard antioxidants using DPPH radical-scavenging activity (IC50, μg/mL), ferric reducing antioxidant potential (FRAP, mmol/g), ß-Carotene-linoleic acid assay

3-3- Antimicrobial activity “in vitro” 3-3-1- DDA method and WDA method The effect of the dill essential oil on the studied microorganisms is depicted in Fig 1 and 2. In general, the results revealed that the dill essential oil is effective on Gram-positive and Gram-negative bacteria as well as molds and yeasts. Nevertheless, its effectiveness varied with the type of microorganism. The average microbial diameter of microbial free zone area in the DDA method was equal to 13.9 mm for the Gram-positive bacteria, 12.8 mm for vancomycin, 9.3 mm for the Gram-negative bacteria, 15.25 mm for gentamycin, 11.33mm for fungi (molds and yeast) and 12 mm for amphotericin B. based on this method, fungi were more resistant than Gram-positive bacteria and more sensitive than Gram-negative ones. The average microbial diameter of microbial free zone area for C. albicans was equal to 12.50 mm which was more than that of B. subtilis and B. cereus and less than that of S. aureus and S. pyogenes. The results of the WDA method are indicated in Fig 2. These results demonstrated that the average microbial diameter of microbial free zone area was generally the most in Gram-positive bacteria. The average microbial diameter of microbial free zone area was equal to 14.95 mm for Gram-positive bacteria, 10.43 mm for Gram-negative bacteria and 13.6 mm for fungi (molds and yeast) in the method of WDA. Fig 1. Average of inhibition zone (mm) of Anethum graveolens essential oil concentrations on some pathogenic bacteria and fungi (DDA).

Fig 2. Average of inhibition zone (mm) of Anethum graveolens essential oil concentrations on some pathogenic bacteria and fungi (WDA).

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Totally, comparison between the two methods of DDA and WDA showed that the essential oil had a larger antimicrobial effect in the latter, because the essential oil is in direct contact with microorganisms in this method, while the essential oil diffuses from the disks surfaces into the medium in the DDA method. From the theoretical point of view, the diameter of the free zone area is a reaction of the concentration of the plant nutraceuticals. This phenomenon is a linear relation between the free zone area and the logarithmic concentration of the studied substance. The significant or non-significant difference between the average microbial diameters of the free zone area of various concentrations could be attributed to the content of the essential oil nutraceuticals. However, it could totally be concluded that as the concentration of the dill essential oil decreased the average microbial diameter of the free zone area decreased, too (Fig 1 and 2). The sensitivity profile of microorganisms against the dill essential oil is as follows (from the most resistant to the most sensitive): P. aeruginosa ˃ P. vulgaris ˃ E. coli ˃ B. cereus ˃ A. fumigatus ˃ P.expansum ˃ B. subtilis ˃ C. albicans ˃ S. aureus ˃S. pyogenes The dill essential oil had a proper inhibitory and microbicidal effect on the studied strains except P.aeroginosa. Dilution of the dill essential oil weakened its antimicrobial effect as the microbial free zone area was not observed in the concentrations of 0.25, 0.5 and 1 for the Gram-negative bacterium of P. aeroginosa. Neither in the case of P. vulgaris in the concentrations of 0.25 and 0.5. At the same time, the microbial free zone area was observed in the case of Gram-positive bacteria, C. albicans, A. fumigatus and P. expansum in all concentrations of the essential oil (Fig 1 and 2). C. albicans is one of the most typical human pathogens bringing about many infections and may threaten the health of people with weakened immune systems, especially those stricken by aids. A few drugs are effective on Candida infections but most of them have restrictions in terms of efficiency and side effects [37]. The antimicrobial effects of essential oils on yeasts have been discussed in detail by

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Cavanagh (2007) [38]. It is not exactly clear how essential oils inhibit yeasts growth and why some essential oils have stronger inhibitory effects on yeasts as compared to bacteria. The antimicrobial effect of an essential oil is exerted directly through the absorption of the essential oil vapor by the microorganism and indirectly through the medium which has absorbed the essential oil. Since yeasts mainly grow on the medium surface, they are more sensitive to the direct effect of the essential oil vapor, while the antimicrobial effect of an essential oil on bacteria may depend on the aggregation of its vapor inside the medium. The mechanism of the essential oils effects on yeasts has not been clearly understood yet; still, this effect is ascribed to morphologic alterations in most reports [39]. One of the principle components of the dill essential oil is Carvone (15.09%). A chemical compound similar to Carvone prevents the transformation of C. albicans form spherical to filamentous. With regard to the association of this transformation with the pathogenicity of C. albicans, this compound which is one of the major components of the dill essential oil has a good curing potential against the infection arising from this pathogenic yeast [40]. Jirovets et al., (2003) extracted the dill seeds essential oil after storing them for more than three decades and applied the essential oil against some fungi including A. niger, S. cerevisiae and C. albicans [41]. The results presented that the dill essential oil had antimicrobial activity against the above-mentioned fungi. According to chemical analyses, the antimicrobial components of essential oils primarily consist of thymol, limonene, carvone, terpenes and other compounds with phenolic nature of free hydroxyl groups all of which have been known as the most active antimicrobial compounds. These compounds occurred abundantly in the plant studied in this research. The phenolic compounds of an essential oil are produced in the phospholipid layer of the plant cell membrane. The higher the TPC contentin the essential oil, the higher the antimicrobial properties of the essential oil. Phenolic compounds have the diffusivity of the microorganism cell membrane increased [42-44].

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3-3-2- Microbial inhibition and bactericidal/fungicidal by single antimicrobials The results of MIC, MBC and MFC of Anethum graveolens essential oil are presented in Table 3. These results revealed that the minimum concentration of the dill essential oil which causes the growth inhibition and killing of microorganisms is lower for Gram-positive bacteria rather than Gram-negative ones or fungi. Many studies confirm this hypothesis of which those of Tabatabei et al (2013), Alizadeh Behbahani et al (2013) and Sureshjani et al (2014) could be pointed out [45-47].

In order to reduce the economic losses as well as the health hazards resulted from microbial pathogens, application of natural substances as antimicrobial agents is an influential approach to control the presence of pathogenic bacteria. Among these compounds, essential oils obtained from medicinal plants possess antimicrobial properties and are used as sources of antimicrobial agent against pathogens. These plants render inhibitory effects against pathogenic bacteria [48]. Arora et al., (2007) investigated the antibacterial effect of the dill aqueous extract on purified strains of S. aureus, E. coli, P. aeroginosa, S. typhimurium, S. flexneri and S. typhi. The results indicated that the extract of this plant had a significant antimicrobial effect on all these bacterial strains. These results were consistent with those of the present study [49]. Regarding the number of chemical compounds in plants essential oils, no unique mechanism could be considered for their antibacterial effects. They have numerous targets in cells. These mechanisms do not act separately and some of them are affected by the others [48]. Hydrophobicity is one of the important properties of essential oils and their components which results in the diffusion of these substances into the lipids of bacterial cell membranes and mitochondria leading to interference in their structure more diffusion. This phenomenon causes the leakage of ions and other components of the cell. Although the bacterial cell could

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sustain a limited leakage of these materials, its viability is affected and the pronounced leakage of the cell contents or the leakage of ions and vital molecules brings about the cell destruction [50-52]. In addition to the afore-mentioned reason, lipid structure and the electrical charge of the cell membrane lattice should be taken into consideration, because antimicrobial compounds may be able to pass through the cell membrane due to the similarity of their structures [53]. In general, the higher the TPC contention the essential oil the more their antibacterial properties against pathogens. The probable mechanisms of the phenolic compounds include interference in the cytoplasmic membrane, disturbance in the proton movement force and electrical current, and coalescence of the cell contents [54]. Most of the performed studies concerning the effect of essential oils on microorganisms which cause infection and poisoning, show that essential oils influence Gram-positive bacteria rather than Gram-negative ones; in the other words, Gram-positive bacteria are more susceptible to the antibacterial effect of essential oils. The lower susceptibility of Gramnegative bacteria may be due to the existence of an external membrane in their structure which restricts the diffusion of hydrophobic components of the essential oils into the lipopolysaccharide layer [45-47]. In fact, by the identification of an essential oil component, it could be realized that there is a direct correlation between its components and antimicrobial properties. However, the major compound plays the main role.

3-4- Chemical composition beef The beef used in this study contained 62.33 ± 0.50% moisture, 21.06 ± 0.45% fat, 12.64 ± 0.52% protein, and 1.5 ± 0.32% ash (the chemical composition was determined using AOAC (1990) standard methods [55]). Yin et al., (2003) reported that the contents of moisture, protein and fat in ground beef was 77.52 %, 21.31.2% and 2.60.4%, respectively [56].

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Emiroglu et al., (2010) cited that beef contained 57.90 ± 0.60% moisture, 23.91 ± 0.91 fat, 16.84 ± 0.17% protein, and 1.15 ± 0.04% ash [57]. The results revealed that the moisture, fat and protein contents of the beef used in the present study were, to some extent, different from those of the previous ones. This can be associated with the nutrition, age and size, environment, sexual variety, slaughter time and other environmental factors [58].

3-5-

Microbiological analysis

3-5-1- Total viable count (TVC)

The total viable count (TVC) of the beef stored at 4°C during storage (18 days) is depicted in figure (3a) The initial number of the TVC was equal to 3.4 log CFU/g revealing the sustainable quality of the beef sample. The results showed that on the third day of storage, there were no significant differences between the beef samples coated with PMSM, (PMSM + 0.5% D), (PMSM + 1 % D), (PMSM + 1.5% D) with the control (C), however, the slope of the bacterial growth was less steep in the coated samples in comparison with the control. The reason behind the insignificant reduction of the TVC could be attributed to the release of dill essential oil PMSM coating, as this essential oil should be able to be released from PMSM coating and influence the microorganisms present on the beef surface. A significant difference (p<0.05) was observed between the coated samples and the control on the 6th day of storage as the TVC of the control was computed as 6 log CFU/g. Nevertheless, this value was equal to 4 log CFU/g in the samples coated with (PMSM + 1.5% D). According to the International Commission of Microbiological Specifications for Foods (ICMSF), the maximal Recommended limit TVC in fresh beef is equal to 7 log CFU/g or 107 CFU/g [59]. Therefore, the TVC of the control exceeded the allowable limit after the 6th day, indicating the microbial shelf-life of the beef samples was at most 6 days. Similar to the control, the TVC of the

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samples coated with (PMSM + 0.5% D), (PMSM + 1 % D) and (PMSM + 1.5% D) increased as the storage time increased; nonetheless, this increase had a less steep slope. The growth of microorganisms was inhibited most in the case of the beef samples coated with (PMSM + 1% D) and (PMSM + 1.5% D). The TVC was lower than the allowable limit on the 15th day of storage; however, it was slightly higher (7.1 log CFU/g) than the allowable limit (7 log CFU/g) in the samples coated with (PMSM + 1.5% D) on the 18th day. This difference was not statistically significant. The results suggested that PMSM adequately inhibited the total bacterial growth in the beef samples as the shelf-life of this treatment was 3 days longer than that of the control. Alizadeh Behbahani et al., (2017) stated that the total phenol content and the total flavonoid content of PMSM were equal to 76.79 and 97.80 mg/g dry weight, respectively. Furthermore, the antioxidant activity of PMSM was measured as 915.54 µg/ml; as a result, the antimicrobial activity of PMSM can be ascribed to its phenolic and flavonoid constituents [1]. Zinoviadou et al., (2009) examined the effect of the addition of oregano essential oil to whey protein film on beef shelf-life during 12 days of storage at 5°C. They came to conclusion that the addition of oregano essential oil had no effect on the film water vapor permeability. Coating the meat with the film reduced the total viable count and pseudomonas count of the samples and completely prevented lactic acid bacteria form growing. Their results exhibited that the application of the why protein film containing 1.5% oregano essential oil doubled the shelf-life of the beef samples as compared with the control [60]. Pohlman et al., (2009) investigated the effect of a gelatin coating containing potassium lactate on the pathogens growth in beef steak. Their results demonstrated that the gelatinous coating, alone or combined with potassium lactate, could prevent the growth of pathogens and enhance the safety and shelf-life of the beef steak compared to the control [61].

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3-5-2- Psychrotrophic count (PTC)

The Psychrotrophic count of the beef samples stored at 4°C during storage (18 days) is illustrated in figure (3b) The initial number of the bacteria was 2.1 log CFU/g. The increase in the PTC of the samples coated with PMSM had a less steep slope than the control, as the PTC of the sample coated with PMSM had reached 7.7 log CFU/g on the 18 th day of storage, whereas this value was equal to 9 log CFU/g for the control on the same day. This could be ascribed to the inhibition of oxygen diffusion into the coated samples compared with the control. The results indicated that the increase in the PTC of the samples coated with (PMSM + 1.5% D) was less pronounced than that of the other samples and this increase differed significantly (p<0.05) from that of the control. Pseudomonas spp. are extremely aerobic and cannot survive in the absence of oxygen. PMSM coating, as an oxygen barrier, prevented pseudomonas from growing. Alghooneh et al (2015) verified the effect of Satureja bachtiarica on the population of Pseudomonas aeruginosa occurring in Frankfurter. Their results revealed that Satureja bachtiarica extract at different concentrations (0, 2000, 4000, 6000 and 8000 ppm) inhibited the growth of Pseudomonas aeruginosa properly [24].

3-5-3- Escherichia coli and Staphylococcus aureus count

The E. coli and S. aureus count of the beef stored at 4°C during storage (18 days) is displayed in figure (3c & d). The results suggested the increase in the number of these bacteria was less pronounced in the samples coated with (PMSM + 1% D) and (PMSM + 1.5% D) in comparison with the other ones. This increase was significantly (p<0.05) different from that of the control and the sample coated only with PMSM on the 18th day of storage. Oussalah et al., (2004) examined the effect of a whey protein edible film containing oregano and pimento essential oils on the shelf-life of beef slices stored at 4°C for 7 days. They realized that these films influenced the growth of Escherichia coli and Pseudomonas aeruginosa [25].

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3-5-4- Total yeast and mold (fungi) count

The total yeast and mold (fungi) count of the beef stored at 4°C during storage (18 days) is exhibited in figure (3e). The results showed that the total fungi count of the beef samples was 0.5 log CFU/g on the 0th day. Since fungi are aerobic and grow on meat surface and PMSM, as a barrier, reduced the oxygen concentration on the samples surfaces, the fungi growth was well prevented in the coated samples. The obtained results demonstrated that the increase in total fungi count was equal to 2.5 log CFU/g in the samples coated with (PMSM + 1.5% D) on the 18th day, while this value was equal to 3.9 and 4.9 log CFU/g for the sample coated with PMSM and the control, respectively. The statistical analysis of the fungi growth on different samples indicated that there was no significant (p>0.05) difference between the samples coated with (PMSM + 0.5% D), (PMSM + 1% D) and (PMSM + 1.5% D) and the ones merely coated with PMSM. At the same time, the increase in total fungi count had a less steep slope in the case of the samples containing various concentrations of dill essential oil as compared with the ones free of dill essential oil.

3-6-

Chemical analysis

3-6-1- Thiobarbituric acid (TBA) The results of TBA concentration in the beef samples, representing the malonaldehyde concentration, are summarized in figure (4a). 2-Thiobarbituric acid (TBA) indicates lipid oxidation. It is often employed as an indicator for measuring the extent of secondary lipid oxidation [62]. The initial TBA concentration was 0.09 mg MDA/kg at the 0th day of storage. It remained stable in the control between the 3rd and 6th days, followed by a dramatic increase to 0.79 mg MDA/kg at the 18th day. The TBA values of the samples coated with PMSM and (PMSM + 0.5% D), (PMSM + 1% D) and (PMSM + 1.5% D) increased as the storage time increased, nonetheless, the ones coated with (PMSM + 1.5 %D) reached a significantly lower

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TBA value (0.31 mg MDA/kg) of tissue compared with the control or the PMSM-coated samples, which attained a higher level of TBA (0.79 and 0.64 mg MDA/kg) of tissue, respectively. TBA value is widely considered as an indicator of lipid oxidation in meat products during storage. Tarladgis et al., (1960) realized that 0.5-1 was the TBA value range at which the rancid odor was first sensed [63]. This threshold guides researchers for interpreting TBA test results. Over the 18 days of storage in our study, both of the samples coated with (PMSM + 1 %D) and (PMSM + 1.5 %D) had TBA values less than 0.5 mg MDA/kg. Alizadeh Behbahani et al., (2017) reported that PMSM contains phenolic and flavonoid compounds and its antioxidant activity is equal to 915.54 µg/ml [1]. on the other hand, based on the results of the present study, it was found out that dill essential oil contained phenolic compounds (162.65 µg/ml GAE) and its antioxidant activity was 11.44 µg/ml. Therefore, the delay in the oxidation of the beef samples during storage (18 days) could be attributed to the phenolic compounds and antioxidant activity of PMSM and dill essential oil which could have been able to inhibit the meat spoilage competently. Juki et al., (2014a) suggested the antioxidant activity of PMSM films as the reason behind the delay in the spoilage of the trout samples stored at 4°C [26]. Ojagh et al., (2010) also ascribed the delay in the oxidation of the fish samples coated with the chitosan film containing cinnamon extract to the film antioxidant activity and lack of oxygen permeability [64]. The findings of the present study are consistent with those realized by these researchers.

3-6-2- Peroxide value (PV)

The effect of PMSM on the changes in the PV of the beef lipids is depicted in figure (4b). Primary lipid oxidation was assessed by means of PV. The results revealed that the PV of the

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control increased from 0.75 meq peroxide/kg beef sample to 8.8 meq peroxide/kg beef sample on the 0th day. Likewise, the results indicated that the PV of all of the treatments increased during storage (18 days). The increase in the PV of the samples coated with PMSM had a steeper slope than that of the samples coated with (PMSM + 0.5% D), (PMSM + 1 % D) and (PMSM + 1.5% D). Furthermore, the results demonstrated that the PV of the PMSMcoated sample was 7.1 meq peroxide/kg beef sample on the 18th day which was significantly (p<0.05) different from that of the control. The comparison between the PVs of the samples coated with (PMSM + 0.5% D), (PMSM + 1 % D) and (PMSM + 1.5% D) exhibited that the one coated with (PMSM + 1.5% D) had the lowest PV (4.7 meq peroxide/kg beef sample) which significantly (p<0.05) differed from that of the others except for the sample coated with (PMSM + 1 % D). The results also revealed that the PV of the control decreased slightly from the 9th day to the 12th day. This could be attributed to the breakdown of hydroperoxides or their reaction with the proteins present on the meat surface, conforming to the findings of Jiun et al., (2002) [62].

3-6-3- pH

The pH variations of the beef samples stored at 4°C is indicated in figure (4c). The initial pH value of the samples was equal to 5.8. ANOVA demonstrated that the pH values of the samples coated with different concentrations of dill essential oil were lower than that of the control during the 18 days of storage. As time goes by, beef texture is degraded due to the enzymatic activity of the microorganism existing in it, which is accompanied by the dissociation of protein constituents and the production of nitrogen compounds, increasing the pH value of the meat. The PMSM coating (containing or free of dill essential oil) on beef reduced the microorganisms growth; consequently, the meat texture degraded more slowly. Meat respiration and microbial activity produce carbon dioxide. Bifani et al., (2009) reported that the type and concentration of the extract and essential oil used in coatings affect their

25

carbon dioxide permeability [65]. The presence of some components such as herbal essential oils and extracts in edible films could decrease or increase the solubility of carbon dioxide, thus influencing the permeability of this gas. It seems that the presence of dill essential oil in the PMSM coating reduced its carbon dioxide permeability, hence increasing the concentration of this gas and consequently decreasing the pH value which can in turn be effective on the reduction of the meat microflora. Fig. 4. Changes in TBA values (A), Peroxide values (B) and pH of beef samples during refrigerated storage.

3-7-

Sensory evaluation

The results of the sensory evaluation (color, odor and overall acceptability) of the beef samples are displayed in figure (5 and 6). The strong odor of dill essential oil is the main obstacle to its usage as a food preservative. Application of dill essential oil in the film or coating network could be offered as a solution to this problem. In the present study, the PMSM containing dill essential oil did not have any negative effects on the sensory properties of the samples from the panelists` points of view. The beef samples with a score higher than 4 can be accepted [66, 26a]. The control and the PMSM-coated samples acquired a sensory (odor) score of less than 4 after 6 and 12 days of storage, respectively. Dinty et al., (1983) stated that pseudomonas caused off-odor in the unpackaged meat samples [67]. After 6 days of storage, the odor of the blank samples became unacceptable, whereas it was acceptable for the coated ones. According to the sensory tests (overall acceptability), the shelf-lives of the blank and the samples coated with PMSM, (PMSM + 0.5% D), (PMSM + 1 % D) and (PMSM + 1.5% D) were obtained as 6, 15 and 18 days, respectively. The comparison between the results of sensory evaluation and total viable count signified that there was a suitable correlation between them in the case of the control as in terms of total viable count, the shelf-life of the control was similar to that (6 days) determined by the

26

judges. The results also implied that the shelf-life of the sample coated with (PMSM + 1.5% D) was equal to 15 and 18 days in terms of total viable count and sensory evaluation, respectively. Indeed, there was a 3-day interval between the total viable count and the sensory evaluation. It can be maintained that the sensory results were not properly correlated with those of the total viable count in the case of the samples coated with (PMSM + 1.5% D). Similar results were obtained by Kykkidou et al., (2009) for the Mediterranean swordfish fillets. They observed that the sensory results were not adequately correlated with the microbial ones for the samples coated with thyme essential oil and there was 4-day interval between them [68]. Fig. 5. Sensory evaluation (Color (A), Odor (B), and Overall acceptability (C)) of beef during storage at 4 °C for 18 days. Bars represent the standard deviation (n = 3).

Fig. 6. Effect of PMSM +1.5%D coating on changes of color of beef after 18 days of storage at 4◦C.

3-8-

Correlation among microbiological, chemical and sensory attributes of beef

Correlation coefficient expresses the linear correlation between two variables. It can be either perfect or imperfect. If the linear curve passes through all of the points, the correlation between the two variables is perfect and if the curve does not pass through all of the points, the correlation is imperfect. Correlation coefficient always varies from -1 to 1. The values of 1 and -1 show the perfect correlation and the values between them indicate the imperfect correlation. According to table 4, it was found out that the correlation coefficients between all variables were higher than 0.8 which are acceptable. The linear correlation between color, odor and overall acceptability with the other variables was negative, suggesting the reverse correlation between these variables and the others. The results showed that the lowest

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correlation coefficient (0.802) was associated with pH and PTC and the highest one (0.992) was related to TVC and PTC. Table 4. Correlation matrix (Pearson) among microbiological, chemical and sensory attributes of beef

Conclusion Our results revealed that the dill essential oil had a considerable antimicrobial and antioxidant potential. It should be pointed out that the content of antioxidants, including phenolic compounds, varies with respect to various genetic factors, post-harvest conditions and different environmental factors. Regarding the chemical compounds identified in the dill essential oil, these components could be employed as an important economical source in pharmaceutical and chemical industries. This study showed that the PMSM containing dill essential oil enhanced the shelf life of beef by preventing lipid oxidation and microbial spoilage. The results indicated that the effect of PMSM+ 1.5%D wrapping on beef was to retain their good quality characteristics and extend the shelf life during storage, which was supported by the results of microbiological, chemical, and sensory evaluation. Thus, PMSM containing dill essential oil could be used as an active packaging to maintain quality and extend the shelf life of the beef at 4 °C.

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References [1] B. Alizadeh Behbahani, F.Tabatabaei Yazdi, F. Shahidi, M.A. Hesarinejad, S.A. Mortazavi, M. Mohebbi, Plantago major seed mucilage: Optimization of extraction and some physicochemical and rheological aspects, Carbohydrate Polymers 155 (2017) 68-77. [2] G. Singh, S. Maurya, M. Lampasona, C. Catalan, Chemical constituents, antimicrobial investigations, and antioxidative potentials of Anethum graveolens L. essential oil and acetone extract: Part 52, Journal of food science 70(4) (2005) M208-M215. [3] S. Bahramikia, R. Yazdanparast, Antioxidant and free radical scavenging activities of different fractions of Anethum graveolens leaves using in vitro models, Pharmacology online 2 (2008) 233-219. [4] R. Borneo, A. León, A. Aguirre, P. Ribotta, J. Cantero, Antioxidant capacity of medicinal plants from the Province of Córdoba (Argentina) and their in vitro testing in a model food system, Food Chemistry 112(3) (2009) 664-670. [5] I. Stoilova, A. Krastanov, A. Stoyanova, P. Denev, S. Gargova, Antioxidant activity of a ginger extract (Zingiber officinale), Food chemistry 102(3) (2007) 764-770. [6] S.R. Kanatt, R. Chander, A. Sharma, Antioxidant potential of mint (Mentha spicata L.) in radiation-processed lamb meat, Food Chemistry 100(2) (2007) 451-458. [7] R.H. Liu, J.H. Hotchkiss, Potential genotoxicity of chronically elevated nitric oxide: a review, Mutation Research/Reviews in Genetic Toxicology 339(2) (1995) 73-89. [8] R.H. Liu, Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals, The American journal of clinical nutrition 78(3) (2003) 517S-520S. [9] S.S. Saei‐ Dehkordi, A.A. Fallah, S.S. Saei‐ Dehkordi, S. Kousha, Chemical Composition and Antioxidative Activity of Echinophora platyloba DC. Essential Oil, and Its Interaction with Natural Antimicrobials against Food‐ Borne Pathogens and Spoilage Organisms, Journal of food science 77(11) (2012) M631-M637. [10] S. Guilbert, N. Gontard, B. Cuq, Technology and applications of edible protective films, Packaging Technology and Science 8(6) (1995) 339-346. [11] L. Cao, J.Y. Si, Y. Liu, H. Sun, W. Jin, Z. Li, X.H. Zhao, R. Le Pan, Essential oil composition, antimicrobial and antioxidant properties of Mosla chinensis Maxim, Food Chemistry 115(3) (2009) 801-805. [12] T. Bourtoom, Edible films and coatings: characteristics and properties, International Food Research Journal 15(3) (2008) 237-248. [13] X.L. Shen, J.M. Wu, Y. Chen, G. Zhao, Antimicrobial and physical properties of sweet potato starch films incorporated with potassium sorbate or chitosan, Food Hydrocolloids 24(4) (2010) 285-290.

29

[14] M. Garriga, N. Grebol, M. Aymerich, J. Monfort, M. Hugas, Microbial inactivation after high-pressure processing at 600 MPa in commercial meat products over its shelf life, Innovative Food Science & Emerging Technologies 5(4) (2004) 451-457. [15] N. Zandi-Sohani, M. Hojjati, Á.A. Carbonell-Barrachina, Insecticidal and repellent activities of the essential oil of Callistemon citrinus (Myrtaceae) against Callosobruchus maculatus (F.)(Coleoptera: Bruchidae), Neotropical entomology 42(1) (2013) 89-94. [16] F.Z. Kucukbay, E. Kuyumcu, S. Çelen, A.D. Azaz, T. Arabac, Chemical composition of the essential oils of three Thymus taxa from Turkey with antimicrobial and antioxidant activities, Rec Nat Prod 8(2) (2014) 110-120. [17] M.P. Kähkönen, A.I. Hopia, H.J. Vuorela, J.-P. Rauha, K. Pihlaja, T.S. Kujala, M. Heinonen, Antioxidant activity of plant extracts containing phenolic compounds, Journal of agricultural and food chemistry 47(10) (1999) 3954-3962. [18] W. Brand-Williams, M.-E. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, LWT-Food science and Technology 28(1) (1995) 25-30. [19] A. Dapkevicius, R. Venskutonis, T.A. van Beek, J.P. Linssen, Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania, Journal of the Science of Food and Agriculture 77(1) (1998) 140-146. [20] T.Y. Lim, Y.Y. Lim, C.M. Yule, Evaluation of antioxidant, antibacterial and antityrosinase activities of four Macaranga species, Food Chemistry 114(2) (2009) 594-599. [21] M. Jacobs, Y. Mithal, R. Robins-Browne, M. Gaspar, H. Koornhof, Antimicrobial susceptibility testing of pneumococci: determination of Kirby-Bauer breakpoints for penicillin G, erythromycin, clindamycin, tetracycline, chloramphenicol, and rifampin, Antimicrobial agents and chemotherapy 16(2) (1979) 190-197. [22] M. Pfaller, D. Andes, D. Diekema, A. Espinel-Ingroff, D. Sheehan, C.S.f.A.S. Testing, Wild-type MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods, Drug Resistance Updates 13(6) (2010) 180-195. [23] M. Afsharzadeh, M. Naderinasab, Z. Tayarani Najaran, M. Barzin, S.A. Emami, In-vitro antimicrobial activities of some iranian conifers, Iranian Journal of Pharmaceutical Research 12(1) (2012) 63-74. [24] A. Alghooneh, B. Alizadeh Behbahani, H. Noorbakhsh, F. Tabatabaei Yazdi, Application of intelligent modeling to predict the population dynamics of Pseudomonas aeruginosa in Frankfurter sausage containing Satureja bachtiarica extracts, Microbial pathogenesis 85 (2015) 58-65. [25] M. Oussalah, S. Caillet, S. Salmiéri, L. Saucier, M. Lacroix, Antimicrobial and antioxidant effects of milk protein-based film containing essential oils for the preservation of whole beef muscle, Journal of Agricultural and Food Chemistry 52(18) (2004) 5598-5605.

30

[26] M. Jouki, F. Tabatabaei Yazdi, S.A. Mortazavi, A. Koocheki, N. Khazaei, Effect of quince seed mucilage edible films incorporated with oregano or thyme essential oil on shelf life extension of refrigerated rainbow trout fillets, International journal of food microbiology 174 (2014a) 88-97. [27] M. Jouki, S.A. Mortazavi, F. Tabatabaei Yazdi, A. Koocheki, N. Khazaei, Use of quince seed mucilage edible films containing natural preservatives to enhance physico-chemical quality of rainbow trout fillets during cold storage, Food Science and Human Wellness 3(2) (2014b) 65-72.

[28] K.I. Sallam, K. Samejima, Microbiological and chemical quality of ground beef treated with sodium lactate and sodium chloride during refrigerated storage, LWT-Food Science and Technology 37(8) (2004) 865-871. [29] L.T. Hansen, T. Gill, H.H. Hussa, Effects of salt and storage temperature on chemical, microbiological and sensory changes in cold-smoked salmon, Food Research International 28(2) (1995) 123-130. [30] K.J. Hartmans, P. Diepenhorst, W. Bakker, L.G. Gorris, The use of carvone in agriculture: sprout suppression of potatoes and antifungal activity against potato tuber and other plant diseases, Industrial Crops and Products 4(1) (1995) 3-13. [31] C.C. de Carvalho, M.M.R. da Fonseca, Carvone: Why and how should one bother to produce this terpene, Food Chemistry 95(3) (2006) 413-422. [32] D.J. Daferera, B.N. Ziogas, M.G. Polissiou, GC-MS analysis of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum, Journal of Agricultural and Food Chemistry 48(6) (2000) 2576-2581. [33] N. Deepa, C. Kaur, B. George, B. Singh, H. Kapoor, Antioxidant constituents in some sweet pepper (Capsicum annuum L.) genotypes during maturity, LWT-Food Science and Technology 40(1) (2007) 121-129. [34] G. Singh, S. Maurya, M. Lampasona, C. Catalan, Chemical constituents, antimicrobial investigations, and antioxidative potentials of Anethum graveolens L. essential oil and acetone extract: Part 52, Journal of food science 70(4) (2005) M208-M215. [35] Y.-S. Shyu, J.-T. Lin, Y.-T. Chang, C.-J. Chiang, D.-J. Yang, Evaluation of antioxidant ability of ethanolic extract from dill (Anethum graveolens L.) flower, Food Chemistry 115(2) (2009) 515-521. [36] M. Gulluce, F. Sahin, M. Sokmen, H. Ozer, D. Daferera, A. Sokmen, M. Polissiou, A. Adiguzel, H. Ozkan, Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. ssp. longifolia, Food chemistry 103(4) (2007) 1449-1456. [37] A. Radford, Quantitation analysis of polysaccharids and glycoprotein fractions in Echinacea purpurea and Echinacea anngustifolia by HPLC-ELSD for quality control of raw material, J Pharmacol Biomed 45(4) (2007) 115-20.

31

[38] H. Cavanagh, Antifungal activity of the volatile phase of essential oils: a brief review, Natural Product Communications 2(12) (2007) 1297-1302. [39] S. Inouye, T. Takizawa, H. Yamaguchi, Antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact, Journal of antimicrobial chemotherapy 47(5) (2001) 565-573. [40] P. McGeady, D.L. Wansley, D.A. Logan, Carvone and Perillaldehyde Interfere with the Serum-Induced Formation of Filamentous Structures in Candida a lbicans at Substantially Lower Concentrations than Those Causing Significant Inhibition of Growth, Journal of natural products 65(7) (2002) 953-955. [41] L. Jirovetz, G. Buchbauer, A.S. Stoyanova, E.V. Georgiev, S.T. Damianova, Composition, quality control, and antimicrobial activity of the essential oil of long-time stored dill (Anethum graveolens L.) seeds from Bulgaria, Journal of agricultural and food chemistry 51(13) (2003) 3854-3857. [42] M. Moreira, A. Ponce, C. Del Valle, S. Roura, Inhibitory parameters of essential oils to reduce a foodborne pathogen, LWT-Food Science and Technology 38(5) (2005) 565-570. [43] N. Singh, R. Singh, A. Bhunia, R. Stroshine, Efficacy of chlorine dioxide, ozone, and thyme essential oil or a sequential washing in killing Escherichia coli O157: H7 on lettuce and baby carrots, LWT-Food Science and Technology 35(8) (2002) 720-729. [44] C. Bagamboula, M. Uyttendaele, J. Debevere, Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri, Food microbiology 21(1) (2004) 33-42. [45] F. Tabatabaei Yazdi, B. AlizadehBehbahani, A. Vasiee, S.A. Mortazavi, F.T. Yazdi, An investigation on the effect of alcoholic and aqueous extracts of Dorema aucheri (Bilhar) on some pathogenic bacteria in vitro, Journal of Paramedical Sciences 6(1) (2015) 58-64. [46] B. Alizadeh Behbahani, F. Tabatabaei Yazdi, A. Mortazavi, F. Zendeboodi, M.M. Gholian, Effect of aqueous and ethanolic extract of Eucalyptus camaldulensis L, Journal of Paramedical Sciences 4(3) (2013) 89-99. [47] M.H. Sureshjani, F. Tabatabaei Yazdi, S.A. Mortazavi, B. Alizadeh Behbahani, F. Shahidi, Antimicrobial effects of Kelussia odoratissima extracts against food borne and food spoilage bacteria" in vitro", Journal of Paramedical Sciences 5(2) (2014) 115-120. [48] S. Burt, Essential oils: their antibacterial properties and potential applications in foods— a review, International journal of food microbiology 94(3) (2004) 223-253. [49] D.S. Arora, G.J. Kaur, Antibacterial activity of some Indian medicinal plants, Journal of natural medicines 61(3) (2007) 313-317. [50] J. Morris, A. Khettry, E. Seitz, Antimicrobial activity of aroma chemicals and essential oils, Journal of the American Oil Chemists’ Society 56(5) (1979) 595-603.

32

[51] K. Knobloch, A. Pauli, B. Iberl, H. Weigand, N. Weis, Antibacterial and antifungal properties of essential oil components, Journal of Essential Oil Research 1(3) (1989) 119128. [52] A. Smith-Palmer, J. Stewart, L. Fyfe, The potential application of plant essential oils as natural food preservatives in soft cheese, Food Microbiology 18(4) (2001) 463-470. [53] D. Trombetta, F. Castelli, M.G. Sarpietro, V. Venuti, M. Cristani, C. Daniele, A. Saija, G. Mazzanti, G. Bisignano, Mechanisms of antibacterial action of three monoterpenes, Antimicrobial agents and chemotherapy 49(6) (2005) 2474-2478. [54] M. Tajkarimi, S.A. Ibrahim, D. Cliver, Antimicrobial herb and spice compounds in food, Food control 21(9) (2010) 1199-1218. [55] AOAC. (1990). Official methods of analysis (13th ed.). Washington,DC: Association of Official Analytical Chemists. [56] M.-c. Yin, W.-s. Cheng, Antioxidant and antimicrobial effects of four garlic-derived organosulfur compounds in ground beef, Meat Science 63(1) (2003) 23-28. [57] Z.K. Emiroğlu, G.P. Yemiş, B.K. Coşkun, K. Candoğan, Antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties, Meat science 86(2) (2010) 283-288. [58] E. González-Fandos, A. Villarino-Rodrıguez, M. Garcıa-Linares, M. Garcıa-Arias, M. Garcıa-Fernandez, Microbiological safety and sensory characteristics of salmon slices processed by the sous vide method, Food Control 16(1) (2005) 77-85.

[59] ICMSF ‘‘International Commission on Microbiological Specification for Foods’’. Microorganisms in foods 2. Sampling for microbiological analysis: Principles and specific applications. 2nd ed. Toronto, Canada: University of Toronto Press; 1986. [60] K.G. Zinoviadou, K.P. Koutsoumanis, C.G. Biliaderis, Physico-chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef, Meat Science 82(3) (2009) 338-345. [61] F. Pohlman, A. Brown Jr, P. Dias-Morse, L. McKenzie, T. Rojas, L. Mehall, Evaluation of Potassium Lactate Incorporated Gelatin Coating as an Antimicrobial Intervention on Microbial Properties of Beef Steaks, Arkansas Animal Science Department Report (2007) 117-119. [62] Y.-J. Jeon, J.Y. Kamil, F. Shahidi, Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod, Journal of Agricultural and Food Chemistry 50(18) (2002) 5167-5178. [63] B.G. Tarladgis, B.M. Watts, M.T. Younathan, L. Dugan Jr, A distillation method for the quantitative determination of malonaldehyde in rancid foods, Journal of the American Oil Chemists Society 37(1) (1960) 44-48.

33

[64] S.M. Ojagh, M. Rezaei, S.H. Razavi, S.M.H. Hosseini, Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water, Food Chemistry 122(1) (2010) 161-166. [65] V. Bifani, C. Ramírez, M. Ihl, M. Rubilar, A. García, N. Zaritzky, Effects of murta (Ugni molinae Turcz) extract on gas and water vapor permeability of carboxymethylcellulose-based edible films, LWT-Food Science and Technology 40(8) (2007) 1473-1481. [66] S. Mexis, E. Chouliara, M. Kontominas, Combined effect of an oxygen absorber and oregano essential oil on shelf life extension of rainbow trout fillets stored at 4 C, Food microbiology 26(6) (2009) 598-605. [67] R. Dainty, R. Edwards, C. Hibbard, J. Marnewick, Volatile compounds associated with microbial growth on normal and high pH beef stored at chill temperatures, Journal of applied bacteriology 66(4) (1989) 281-289.7 [68] S. Kykkidou, V. Giatrakou, A. Papavergou, M. Kontominas, I. Savvaidis, Effect of thyme essential oil and packaging treatments on fresh Mediterranean swordfish fillets during storage at 4 C, Food Chemistry 115(1) (2009) 169-175.

34

Average diameter (mm) of microbial free zone area

p.aueruginase 20

P.Vulgaris

18

E.coli

16

B.cereus

14

B.subtilis

12

S.aureus

10

S.pyogenes

8

A.fumigatus

6

P.expansum

4

C.albicans

2 0 -2

8

4

2

1

0.5

0.25

Anethum graveolens essential oil concentrations

Fig. 1. Average of inhibition zone (mm) of Anethum graveolens essential oil concentrations on some pathogenic bacteria and fungi (DDA).

35

Average diameter (mm) of microbial free zone area

p.aueruginase P.Vulgaris

20

E.coli B.cereus

15

B.subtilis S.aureus 10

S.pyogenes A.fumigatus P.expansum

5

C.albicans 0 8

4

2

1

0.5

0.25

Anethum graveolens essential oil concentrations -5

Fig. 2. Average of inhibition zone (mm) of Anethum graveolens essential oil concentrations on some pathogenic bacteria and fungi (WDA).

36

37

38

39

Fig. 3. Total viable count (A), Psychrophilic bacterial count (B), Escherichia coli (C), Staphylococcus aureus (D) and Fungi (E) of beef without and with PMSM during storage at 4°C for 18 days. Control: unwrapped samples, PMSM: samples wrapped with PMSM, PMSM + D: samples wrapped with PMSM incorporated with dill essential oil. Bars represent the standard deviation (n = 3).

40

41

Fig. 4. Changes in TBA values (A), Peroxide values (B) and pH of beef samples during refrigerated storage.

42

43

Fig. 5. Sensory evaluation (Color (A), Odor (B), and Overall acceptability (C)) of beef during storage at 4 °C for 18 days. Bars represent the standard deviation (n = 3).

44

Fig. 6. Effect of PMSM +1.5%D coating on changes of color of beef after 18 days of storage at 4◦C.

45

Table 1. Chemical composition of the dill essential oil NO 1

Compound name α-thujene

%

0.04

KIa 925

2

α-Pinene

0.59

933

3

Sabinene

0.02

972

4

β-Pinene

0.02

976

5

β-Myrcene

0.04

989

6

α-Phellandrene

34.49

1010

7

α-Terpinene

0.04

1017

8

p-Cymene

3.57

1025

9

Limonene

31.1

1031

10

α-Terpinolene

0.03

1088

11

Dillether

11.25

1192

12

cis-Dihydrocarvone

0.12

1202

13

trans-Dihydrocarvone

0.29

1209

14

Pulegone

0.06

1243

15

Carvone

15.09

1251

16

Anethole

0.03

1288

17

Germacrene D

0.03

1484

18

Myristicin

0.03

1525

19

Dillapiole

2.58

1630

KI: The Kovats retention indices relative to C8-C20 n-alkanes were determinedon DB5column capillary column

46

Table 2. Antioxidant activities of the essential oil of Anethum graveolens and some standard antioxidants using DPPH radical-scavenging activity (IC50, μg/mL), ferric reducing antioxidant potential (FRAP, mmol/g), ß-Carotene-linoleic acid assay Sample

DPPH

FRAP

ß-CL

Anethumgraveolens essential oil

11.44

9.45

82.86

Butylatedhydroxytoluene (BHT)

9.20

0.48

94.65

Ascorbic acid

4.42

1.62

23.34

47

Table 3. MIC and MBC/MFC of Anethum graveolens essential oil on some pathogenic bacteria and fungi

MIC

MBC/MFC

2 2 1

4 4 2

0/5 0/25 0/25 0/125

1 0/5 0/5 0/25

0/5 0/25 0/125

1 0/5 0/25

Microorganism Gram negative P. aeruginosa P. vulgaris E. coli Gram positive B. cereus B. subtilis S. aureus S. pyogenes Fungi A. fumigatus P. expansum C. albicans

48

Table 4. Correlation matrix (Pearson) among microbiological, chemical and sensory attributes of beef

Variables

TVC

PTC

E. coli

S. aureus

Fungi

TBA

PV

PH

Color

Odor

Overall acceptability

TVC

1

0.992

0.981

0.932

0.979

0.921

0.974

0.819

-0.878

-0.921

-0.933

PTC

0.992

1

0.986

0.944

0.976

0.917

0.976

0.802

-0.849

-0.908

-0.911

E.coli

0.981

0.986

1

0.957

0.974

0.920

0.983

0.846

-0.871

-0.913

-0.926

S.aureus

0.932

0.944

0.957

1

0.903

0.889

0.964

0.886

-0.857

-0.899

-0.910

Fungi

0.979

0.976

0.974

0.903

1

0.919

0.951

0.802

-0.858

-0.897

-0.911

TBA

0.921

0.917

0.920

0.889

0.919

1

0.908

0.860

-0.817

-0.865

-0.873

PV

0.974

0.976

0.983

0.964

0.951

0.908

1

0.861

-0.883

-0.904

-0.926

PH

0.819

0.802

0.846

0.886

0.802

0.860

0.861

1

-0.893

-0.843

-0.903

Color

-0.878

-0.849

-0.871

-0.857

-0.858

-0.817

-0.883

-0.893

1

0.858

0.966

Odor

-0.921

-0.908

-0.913

-0.899

-0.897

-0.865

-0.904

-0.843

0.858

1

0.961

-0.911 -0.926 -0.910 Overall acceptability -0.933 Values in bold are different from 0 with a significance level alpha=0.05

-0.911

-0.873

-0.926

-0.903

0.966

0.961

1

49