Effect of chitosan coating containing Nepeta pogonosperma extract on shelf life of chicken fillets during chilled storage

Abstract Chicken meat is highly susceptible to microbial and chemical spoilage due to its high moisture and protein content. The use of edible coatings contains herbal extracts with antioxidant and antibacterial properties that help to extend the shelf life of meat products. In this study, the effect of chitosan coating (2%) and Nepeta pogonosperma extract (NPe) (0.2% and 0.6%) and their combination on chemical properties (pH, peroxide value (PV), thiobarbituric acid index (TBARS), total volatile basic nitrogen (TVB‐N)) and microbial (aerobic mesophilic and psychrotrophic microorganisms, lactic acid bacteria, Enterobacteriaceae and Pseudomonas sp.) of chicken fillets were studied over a 12‐day refrigerated storage period compared to the control sample. The results of NPe DPPH radical scavenging activity (DRSA) showed that IC50 and total phenolic contents values were 94.65 μg/ml and 113.53 mg GAE/g extract, respectively. Statistical results showed that the rate of increase in pH, PV, TBARS, and TVB‐N of all coated treatments were lower than control. Microbial analysis results showed a decrease in the growth of different bacteria in chitosan‐treated combined with NPe compared to the control sample during chilled storage. Chicken fillets coated with chitosan and 0.6% NPe displayed a longer shelf life compared to other samples.


| 4519
AFSHAR MEHRABI Et Al. quality of meat (Gomez & Lorenzo, 2012;Karre et al., 2013). Natural or synthetic antioxidants can reduce or inhibit the oxidation process in meat and meat products, thus enhancing the quality and shelf life of meat products. Antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tertiary butylhydroquinone (TBHQ) are synthetic antioxidants that are widely used in meat products (Shah et al., 2014). But today the use of these chemicals has been limited due to their deleterious effects on DNA and their toxicity. Recently, the tendency to use plant extracts has increased as natural additives in foods to protect them against oxidation and prevent the growth and proliferation of microorganisms (Sayari et al., 2015).
One of the Nepeta species was scientifically identified as Nepeta pogonosperma (NP) as a new species in 1984 (Jamzad & Assadi, 1984).
If the essential oil and herbal extracts are just added to food, they may alter their organoleptic properties due to their high concentration. Edible coatings can be considered as a suitable carrier for these compounds (Fernandez-Pan et al., 2014). Chitosan is the second natural polymer, cellulose being the first. One of the interesting properties of chitin and chitosan in food packaging is their antimicrobial and antifungal properties, which enhance the immunity and longevity of food products (Dhall, 2013).
However, to our knowledge, to date, the combination of chitosan and Nepeta pogonosperma extract has never been produced and tested in fresh chicken fillets. In this study, the effects of chitosan coating and Nepeta pogonosperma extract (NPe) on chemical and microbial properties of chicken fillets were studied during chilled storage.

| Materials
In this research, Nepeta pogonosperma (NP) was collected in August 2018 from Piche Bon village of Alamut region in Qazvin province (Iran) and was authenticated by a botanist. A voucher specimen of the plant has been deposited in the central herbarium (mpih.ir) with code no. 527 (MPIH). Aerial parts of NP were dried in shade at room temperature (25°C). After complete drying, the aerial parts were milled (DELMONTI-DL125) and then kept in dark glass bottles. All chemicals in analytical grades were purchased from Merck Company.
The bacterial strain cultures were obtained from the Iranian Research Organization for Science and Technology (IROST).

| Preparation of extract
20 g of the plant powder was mixed with 200 ml of methanol, and the extraction process was done by an ultrasonic bath (DT 255 H, Bandelin Co. Germany) for 2 hr (Khalighi-Sigaroodi et al., 2013). The NPe was filtered using filter paper Whatman 40 and concentrated at a low temperature (<50°C) using a vacuum rotary evaporator Flawil,Switzerland). The concentrated extract was stored in air-tight dark glass bottles and kept refrigerated (4°C) for further treatments after solvent separation (Sharifi et al., 2015).

| Determination of DPPH radical scavenging activity
DPPH radical scavenging activity (DRSA) of NPe was evaluated by the DPPH test (2, 2-diphenyl-1-picrylhydrazyl). First, 0.5 ml of DPPH solution was mixed with 4.5 ml of methanol. Then, 0.1 ml of the extract was added at various concentrations (25-100 μg/ml) and mixed for 1 min. The mixture was incubated at room temperature for 30 min; its absorbance at 517 nm was read using a spectrometer (PerkinElmer-LAMBDA35). The DPPH scavenging activity of each sample will be calculated by Equation (1).
where A blank and A sample control and extract absorbance at 517 nm, respectively.
The EC 50 value was calculated as the concentration at which the DPPH radical scavenging activity was 50% .

| Determination of total phenolic contents
Total phenolic contents (TPC) was measured by Folin-Ciocalteu method. In this method, 20 µl of the extract was mixed with 1.16 ml distilled water and 100 µl of Folin-Ciocalteu reagent. 1 to 8 min later, 300 µl of sodium carbonate solution (20%) was added and stored at room temperature for 30 min. The absorbance was calculated at 765 nm by a spectrophotometer. The results were expressed in terms of mg Gallic Acid Equivalents per g of extract (Slinkard & Singleton, 1977).

| Disc diffusion method
Antibacterial activity of NPe was measured with agar disc diffusion assay. Microorganisms used were E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC9027), and Salmonella enterica (ATCC10708). Nutrient agar medium was prepared, autoclaved, and transferred aseptically to sterilize Petri plates. 100 µl of bacterial suspension (10 8 CFU/ml) was spread on plates, and then, circular disc (6.4 mm) was impregnated with 20 µl of NPe. The discs were placed over plates of Muller Hinton agar seeded with each bacteria, and the inoculum was adjusted to 0.5 Mc Farland turbidometry. The plates were incubated at 37°C for 24 hr.
Chloramphenicol (30 µg/disc) was applied as a positive control to determine the sensitivity of one strain in each microbial species tested.
The zones of inhibition around each of the discs were calculated by measuring the diameter in mm as a measure of the antimicrobial activity after incubation time (Prasannabalaji et al., 2012

| Preparation of coating solution containing NPe and treatment of chicken meat
Chitosan powder with medium molecular weight was dissolved in 1% acetic acid to produce a 2% solution. After filtration, 0.75% glycerol was added as a plasticizer and was stirred at room temperature on a hotplate/magnetic stirrer for one hour. Based on MIC and MBC results, the 0.2% and 0.6% of NPe mixed with 2% Tween 80 was added to the chitosan solution. The solution was stirred at room temperature for 30 min (Bazargani-Gilani et al., 2015).
Skinless and boneless chicken fillets (each slice weight 120 g) were obtained from local distributors in Qazvin, Iran. The samples were placed in a sealed cooler with a layer of ice between the samples and transported to the laboratory of Qazvin Islamic Azad University.
Fillets were divided into six groups, including control sample (chicken fillets dipped in sterile distilled water), chicken fillets dipped in 2% chitosan (Ch) solution, chicken fillets dipped in 2% chitosan solution containing 0.2% NPe, chicken fillet dipped in 2% chitosan solution containing 0.6% NPe, chicken fillet dipped in 0.2% NPe, and chicken fillet dipped in 0.6% of the NPe. Fillets were dipped in coating solutions for 1 min and then removed for 2 min and again dipped in coating solutions for 1 min. The excess solution was drained off immediately after dipping. Finally, all samples were stored in refrigeration condition (4 ± 1°C), and chemical and bacterial tests were performed on storage days 0, 3, 6, 9, and 12 (Jonaidi Jafari et al., 2018).

| Determination of pH
The pH value was recorded using a pH meter (Crison GLP 22, EEC).
10 g of the meat sample was mixed with 50 ml of distilled water and homogenized for 1 min then the pH was read (Banerjee et al., 2012).

| Determination of peroxide index (PV)
The sample (20 g) was mixed with 100 ml chloroform-methanol (2:1 V/V) in a glass tube and vortexed for 1 min. The chloroform phase was used for the solvent evaporate and fat extraction for peroxide measurement. Chloroform-acetic acid mixture was added to the fat in a ratio of 2:3. Next, 0.5 ml of a saturated solution of potassium iodide was added and kept in the dark for 5 min and after adding 75 ml of distilled water was titrated with sodium thiosulphate

| Determination of thiobarbituric acid reactive substances (TBARS)
Thiobarbituric acid reactive substances formed due to lipid peroxidation during storage were determined using the method of  where V and C volume and concentration of HCl, respectively.

| Color measurement
The color of chicken fillets was measured by using a Hunter laboratory Instrument (TES)(135A-Taiwan). L* (lightness) represents the brightness on a scale of (dark) to 100 (white), a* (redness) scale ranges from negative values for green to positive values for red and b* (yellowness) scale ranges from negative values for blue to positive values for yellow .

| Microbiological analysis
Chicken fillet (10 g) was mixed with 90 ml of buffered water in a sterile plastic bag and homogenized in the stomacher (Seward Ltd) for 60 s. Appropriate dilutions were prepared in tubes containing 0.1% buffered water and cultured by the pour plate method. Culture media of Aerobic mesophilic bacteria, Enterobacteriaceae, psychrotrophic bacteria, Lactic acid bacteria, Pseudomonas sp. were plate count agar, VRBG, plate count agar, MRS, Pseudomonas agar base, respectively (Fernández-Pan et al., 2014).

| Statistical analysis
All experiments were conducted in triplicate, and analysis of variance was performed using SPSS software version 21. The least significant difference at p < .05 was calculated using the Duncan multiple range test.

| DPPH radical scavenging activity (DRSA) and total phenolic contents (TPC) of NPe
The results of DRSA evaluation of NPe different concentrations re- In recent years, there has been a global trend toward the use of the natural substances present in medicinal plants with high antioxidant activity and much research has been done by various researchers to evaluate the antioxidant properties of medicinal plants (Lee et al., 2005). The results of Shahsavari et al. (2008) indicated the IC 50 of Zataria multiflora Boiss extract was 2.22 ± 0.04 mg/ml (Shahsavari et al., 2008). The IC 50 value of the Nepeta cataria methanol extract was 171.98 μg/ml (Adiguzel et al., 2009). According to the results and compared with previous studies, NPe has high antioxidant activity.
The amount of phenolic compounds of NPe was 113.53 mg GAE/g extract. Phenolic compounds are secondary metabolites of plants. These compounds have high antioxidant potential and are effective in removing and preventing free radicals (Wong et al., 2006).

| Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of NPe
NPe showed good antibacterial activity against tested microorganisms. The MIC values of the NPe against the E. coli, S. entrica, and P. aeruginosa were found in the range of 0.78-6.25 mg/ml (Table 1).
The results obtained from the disc diffusion method indicated that the highest (12.0 ± 9.5 mm) and the lowest (8.0 ± 7.89 mm) zone of inhibition diameters belonged to E. coli and S. entrica, respectively ( Figure 1). There is a significant difference in the zone of inhibition values for the three bacterial strains (p < .05).

| pH
On day zero of storage, pH values of the treatments varied from 5.57 to 5.54, and no significant differences were observed between the samples at a level of 0.05. Over time, pH values of the samples increased slightly during all days of storage (p < .05) ( Table 2). On the 12th day of storage, the control and chitosan treatment containing 0.6% of NPe had the highest (6.17 ± 0.005) and the lowest bactria of alkaline compounds such as trimethylamine and ammonia by bacteria (Hassanzadeh et al., 2018;Kostaki et al., 2009). The low pH in chicken meat coated with chitosan and NPe during storage can also be attributed to their microorganisms growth inhibitory potential and protease enzymes (Fan et al., 2009;Pabast et al., 2018) and acidic pH of chitosan (Hassanzadeh et al., 2017).

| Peroxide value (PV)
Lipid oxidation in meat leads to off-flavor and degraded quality.
Peroxide is formed in the early stages of oxidation as a result of oxygen binding to double bonds of unsaturated fatty acids. Therefore, initial lipid oxidation can be assessed by measuring PV . According to Table 3, there was no significant difference between the treatments on day zero of storage. With increasing storage time, however, PVs increased in all samples, particularly in the control sample (p < .05), with the highest PV (7.11 ± 0.04 meq/ kg) in the control sample on the 12th day of storage. On the 12th day, peroxide levels were significantly lower in all coated treatments than the control treatment, with the chitosan coating having 0.6% of NPe containing the lowest level (4.47 ± 0.09 meq/kg).
During the storage period, there was no significant difference between the treatment with chitosan coating and that containing 0.2% NPe. Results show the advantage of pure NPe coatings and it can be concluded that pure NP coating could reduce the production of hydroperoxides and decelerate the oxidation process, the same as 2% chitosan solution whose antioxidant activity was proven in various studies (Darmadji & Izumimoto, 1994;Inanli et al., 2020;Ojagh et al., 2010;Zhang et al., 2020). Plant extracts, including NPe, cease oxidative chain reactions by donating hydrogen to free radicals, thereby exerting their antioxidant effects (Abdou et al., 2018).
In line with the results of this study, Ojagh et al. (2010) reported that PVs increased in all treatments of coated rainbow trout meat, but this increase was lower in chitosan and chitosan plus cinnamon essential oil treatments during the storage period (Ojagh et al., 2010).

| Thiobarbituric acid reactive substances (TBARS)
Changes in the TBARS index during the storage period generally  changes in TBARS were observed in chitosan-coated samples con-

| Total volatile basic nitrogen (TVB-N)
TVB-N as one of the indicators of fresh meat detection includes a wide range of volatile compounds such as ammonia, methylamine, dimethylamine, trimethylamine, and other similar compounds produced during the storage of meat in cold conditions due to microbial activity (Anderson, 2008;Rodríguez et al., 2008). According to On the 12th day, the highest (67.23 ± 0.08 mg/100 g) and the lowest (32.12 ± 0.7 mg/100 g) amounts of TVB-N were recorded, respectively, in the control sample and the chitosan treatment contained 0.6% of NPe. A maximum permissible amount of TVB-N as 28 mg/100 g was announced by the Veterinary Organization of Iran. Accordingly, the control sample with a TVB-N amount of 39.33 ± 1.4 mg/100 g could not be consumed on the 6th day of storage whereas all the coated treatments were within the allowable limit in terms of TVB-N levels until the end of the 6th day. On the 9th day, however, TVB-N levels in all treatments were outside the standard permissible range, and the chitosan-coated treatment containing 0.6% of NPe with a TVB-N amount of 25.5 ± 0.8 mg/100 g was only consumable on this day.
The reductions of TVB-N changes in samples with chitosan coating, NPe, and their combination can be attributed to the antimicrobial properties of chitosan and NPe (Aziz & Karboune, 2018;Goy et al., 2016;Kong et al., 2010), as well as the combined performance and synergistic effect of chitosan coating and the extract . Consistent with our findings, Mojaddar Langroodi

| Brightness factor (L* value)
The results of color changes in chicken fillets during the storage period are shown in Figure 4.

| Microbial analysis
Microbial analysis of samples during storage at 4°C indicated an increasing trend in the populations of aerobic mesophilic, psychrotrophic, Pseudomonas, lactic acid, and Enterobacteriaceae bacteria ( Figures 5-9). The control treatment and the chitosan treatment containing 0.6% of NPe, respectively, contained the highest and lowest microbial populations during the storage period, except on day zero.
A comparison of 0.6% NPe with pure chitosan coating treatments also showed no significant difference in the former treatment on some days of the storage period. When the total number of bacteria in the meat exceeds 7 logarithmic cycles, the meat begins to spoil and changes occur in its organoleptic properties (Eldaly et al., 2018).
According to Figure 5, the number of mesophiles in the control treatment reached 7.50 Log CFU/g on the 6th day of storage, while the microbial load of coated treatments was still less than seven logarithmic cycles on the same day when the highest effect of declined F I G U R E 4 Effect of chitosan (Ch) and Nepeta pogonosperma extract (NPe) coating on the brightness factor (L* value) of chicken fillets during storage at 4°C (Different letters of each bar indicate significant difference between the storage time within same analysis group (lowercase) and differences between treatment groups within same analysis day (uppercase) at p < .05 )   aB  bB  cB  dB  eB   aA  bA  cA  dA  eA  aC  bB  bB  cB  dB   aCD  bC  cC  dC  eC  aD  bD  cD  dD  eD  aE  bE  cE  dE  microbial growth was also observed in the chitosan treatment containing 0.6% NPe. In this treatment, the number of mesophiles (6.01 ± 0.16 Log CFU/g) shows that the chitosan coating containing 0.6% of NPe was able to reduce the microbial load of fillets by approx. 1.5 logarithmic cycles. On the 9th day of storage, the number of mesophiles in all samples was above the allowable limit (seven logarithmic cycles), except in chitosan treatment containing 0.6% of the extract, meaning that this treatment was able to maintain its microbial quality until the end of the 9th day of storage. In a study concerning the effect of chitosan coating at different concentrations (1, 1.5, and 2%) on the microbial characteristics of chicken fillets during 15 days of storage at refrigerator temperature, the total number of aerobic bacteria reached 6.87 Log CFU/g in the control treatment on the 3rd day of storage, while that of coated samples was 5.99-6.97 Log CFU/g at the end of the 12th day of storage (Eldaly et al., 2018).
The growth inhibition of aerobic microorganisms by chitosan was at- Pseudomonas is a gram-negative, aerobic rod bacterium that grows rapidly in refrigerated conditions (Lu et al., 2016). Due to the strong proteolytic properties of these bacteria, the signs of spoilage appear in fresh meat when the number of these bacteria reaches about 7-8 logarithmic cycles (Mehdizadeh & Mojaddar Langroodi, 2019). In this study, the number of Pseudomonas ranged from 2.30 to 2.43 CFU/g in all treatments on day zero, which increased during the refrigerated storage. The highest (8.29 ± 0.04 CFU/g) population of Pseudomonas was observed in the control treatment on the last day of storage, and the sample coated with chitosan containing 0.6% NPe was the only treatment in which Pseudomonas population was 6.89 ± 0.03 CFU/g by the end of the 12th day and did not exceed seven logarithmic cycles. In addition, the results demonstrated that Pseudomonas population decreased with increasing the extract concentration from 0.2 to 0.6 in all test days except day zero, suggesting the elevated antibacterial property of the extract with increasing the concentration ( Figure 7). In agreement with this study, Lu et al. (2016) examined the antimicrobial properties of eucalyptus essential oil in vitro and reported a significant decrease in Pseudomonas population compared with control treatment after adding 4% eucalyptus concentration to pork (Lu et al., 2016).
The number of lactic acid bacteria also showed a rising trend during storage at refrigerated temperatures, so that the highest (8.58 ± 0.03 CFU/g) and the lowest (7.18 ± 0.05 CFU/g) populations were present in the control sample and the chitosan treatment containing 0.6% of NPe on the 12th day ( Figure 8). These findings are in line with those reported for chicken fillets treated with niacincontaining sodium alginate and cinnamon and rosemary essential oils (Raeisi et al., 2016).
The population of Enterobacteriaceae, as facultative anaerobic bacteria, also showed a significant increase with time ( Figure 9).
This upward trend was observed with much greater intensity in all treatments on the last days of storage, which corresponds to Cai et al. (2018) who studied the effect of combined chitosan coating and herbal (lemon and thyme) essential oils on the fillet shelf life of grass carp (Ctenopharyngodon idella) (Cai et al., 2018). Overall, the results of this study demonstrated the antimicrobial properties of chitosan and NPe. Herbal extracts and essential oils have been reported to disrupt the cytoplasmic membrane activity, proton motive force electron flow, and active transport, leading to coagulation of bacterial cell content and consequently death (Nikmaram et al., 2018). The strongest treatment against microbial changes was chitosan treatment containing 0.6% of NPe, which indicates a more effective synergistic effect of chitosan coating and NPe.

| CON CLUS ION
The results showed that coating chicken fillets led to decreased pH, peroxide value, thiobarbituric acid index, total volatile basic nitrogen, and microbial counts (aerobic mesophilic, psychrotrophic, lactic acid, Pseudomonas, and Enterobacteriaceae bacteria) during the storage period. The chicken fillet sample coated with chitosan containing 0.6% of Nepeta pogonosperma extract could better retain its chemical and microbial qualities during storage than the other treatments.
Considering the potential antioxidant activity and high levels of phenolic compounds in Nepeta pogonosperma extract, it can be used in the pharmaceutical and food industries instead of synthetic antioxidants and other chemical preservatives to delay lipid oxidation and inhibit the growth of microorganisms.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
This article does not contain any studies with human participants or animals performed by any of the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, upon reasonable request.