Novel antimicrobial/antioxidant Eremurus luteus root gum coating containing rosemary essential oil nanoemulsions for extension of chicken meat shelf life

Abstract Herein, the effect of incorporation of rosemary essential oil (REO) nanoemulsions with the smallest (98.14 nm) and largest (148.04 nm) droplets' sizes at different concentrations (0%, 2%, and 4% v/v) in Eremurus luteus root gum (ELRG) coating solution on microbial, chemical, and sensory qualities of chicken fillets during cold storage was investigated. The results demonstrated a significant reduction in pH and TBA value and total viable microbial count (TVC) of chicken meat samples after using an active ELRG coating compared with the uncoated sample. Moreover, the properties of active ELRG coatings were more affected by the concentration of REO nanoemulsions than the size of their droplets. More antimicrobial and antioxidant activities were observed in coated samples containing 4% (v/v) REO nanoemulsions (L‐4 and S‐4). The highest and lowest pHs at the end of storage belonged to uncoated (6.89) and S‐4 coated (6.41) samples, respectively. Unlike the control sample (8th day), the microbial population in the active coated samples (>12th day) reached the threshold level (7 log CFU/g) later. The TBA value in the control and coated samples was 0.56 and 0.4–0.47 mg/kg after 12 days of cold storage, respectively. Increasing the REO nanoemulsion content from 2% to 4% (v/v) in the coating solution enhanced the score of sensory parameters such as odor, color, and total acceptance of the chicken meat, especially on the last day of cold storage. The obtained results suggested ELRG‐REO coatings as an effective strategy to delay the chemical and microbial deterioration of chicken meat fillets.

Therefore, extending the shelf life of raw chicken meat is a severe challenge in the food industry, and applying novel preservation methods is necessary. In recent years, the active coating containing antimicrobial or antioxidant components for meat packaging has received much attention in various studies (Ala & Shahbazi, 2019;Latou et al., 2014;Noori et al., 2018). This approach could improve meat quality and safety by reducing microbial growth and delaying lipid oxidation. Due to the increasing consumer awareness about the environmental and health hazards of using plastic packaging for food, the tendency to use biodegradable packaging made from natural and non-toxic polymers (proteins, polysaccharides, and lipids) increased (Lee et al., 2019). The coating with antimicrobial properties on the food surface through a slow release of active components during storage could limit microbial growth. Moreover, the incorporation of active components in coatings may be more beneficial than the direct use of them in food, as a reaction of antimicrobial material with food components increases or eliminates their bioactivity (Appendini & Hotchkiss, 2002). In addition, the release of active compounds from the structure of packaging on the food surface depends on different factors such as polymer type, active compounds concentration, and storage temperature (Janes & Dai, 2012). Among the compounds widely used in the preparation of active packaging, plant essential oils are the most popular.
Essential oils (EOs) are natural aromatic and volatile lipophilic plant extracts with antimicrobial and antioxidant properties and could be a suitable alternative to synthetic chemical preservatives (Prakash et al., 2015). According to previous studies, the direct incorporation of EOs in the polymeric matrix has some disadvantages, including volatilization of EOs during water evaporation because of the coarseness of its droplets and creating porous structures in coating or film (Norajit et al., 2010;Tastan et al., 2016). Accordingly, application of EOs in the form of "nanoemulsion" with tiny droplet sizes has different advantages including, high stability, enhancement of physicochemical properties, and improved biological properties through increasing the specific surface area and hence lowering the required amounts of the active component. Nanoemulsions are emulsions with particle sizes in the range of 2-200 nm that produced by high-energy or low-energy emulsification methods (McClements & Rao, 2011). Recently, some bio-based coatings containing nanoemulsion of EOs have indicated good antimicrobial activity on different food surfaces such as chitosan/ carvacrol nanoemulsions (Severino et al., 2015), alginate/citrus EO nanoemulsions (Das et al., 2020), sodium caseinate/ginger EO nanoemulsions (Noori et al., 2018), pullulan/cinnamon EO nanoemulsions (Chu et al., 2020), and chitosan/Zataria multiflora Boiss or Bunium persicum Boiss EO nanoemulsions (Keykhosravy et al., 2022).
The genus of Salvia rosmarinus is an aromatic woody perennial plant that belongs to the mint family Lamiaceae and is known as rosemary. It is found abundantly in the Mediterranean and Asia regions but can cultivate in other world regions as an ornamental and aromatic plant (Yu et al., 2013). Rosemary is commonly used as a food flavoring agent or for different pharmacological purposes. The EO of rosemary is a colorless or pale yellow liquid with a characteristic plant odor and mainly consists of monoterpenes (Shahrampour & Razavi, 2022). Recently, antioxidant and antimicrobial properties of rosemary essential oil (REO) have been reported in some studies (Chraibi et al., 2020;Hassanzad Azar et al., 2019;Nieto et al., 2018;Ramadan et al., 2020).
The Eremurus (known as Serish or Cerish in Iran) belongs to the Liliaceae family and is geographically dispersed in various regions of Asian countries such as Iran. Eremurus luteus root gum (ELRG) is a good source of low-cost and non-toxic carbohydrates. One of the main heteropolysaccharides found in ELRG is glucomannan.
According to our previous research, ELRG showed good film-forming capacity and high solubility in water .
As far as we know, the application of ELRG as a coating on food surfaces has received no attention. Moreover, the effect of particle size of active components in coating structure on food quality properties has not been investigated. Therefore, the present study aimed to evaluate the effect of ELRG coating containing different concentrations of REO nanoemulsions with various droplet sizes on microbiological, chemical, and sensory parameters of chicken breast fillet during cold storage.

| Materials
The various media such as PCA, MRS agar, and YGC agar were purchased from Liofilchem Company. REO was prepared from Exire Gole Sorkh Company. ELRG powder was donated by Salahi et al. in June 2021 and kept in a glass container in a dark and dry place. The identified compounds of ELRG are stated in Table 1

| Preparation of REO nanoemulsions
The nanoemulsions of REO essential oil were prepared according to the method of (Fattahi et al., 2020) with some modifications. The base emulsion was first formulated by aiding dropwise of REO and Tween 80 with a ratio of 1:0.5 (v/v) to 20 mL distilled water and homogenization by an Ultra-Turrax at 10,000 g for 2 min. The nanoemulsions were obtained after sonication treatment of the emulsion at 20 kHz frequency, 200 W input power, and 30% amplitude for different times (2.5, 5 and 10 min) by an Ultrasonicator (FAPAN). The container of each nanoemulsion was kept in an ice bath to prevent increasing the temperature during sonication. All emulsions contained 10% (v/v) of REO.

| Measurement of droplet size
The average droplet size (z-average) of the REO nanoemulsions was determined by dynamic light scattering (DLS) technique (model NanoQ, Instrument VASCO). Before the analysis, nanoemulsions were diluted with deionized water to 1:100 to avoid multiple scattering effects (Noori et al., 2018). This test was done in triplicate at 25°C.

| Preparation of coating solution
1.5 g of the ELRG powder was mixed with 100 mL distilled water and continuously stirred at 85°C and 800 rpm for 30 min. Also, glycerol was added at 30% (w/w) of ELRG powder as a plasticizer agent.
Afterward, the smallest and largest droplet sizes of REO nanoemulsions at two concentrations (2% and 4%, v/v) were added with constant agitation at room temperature for 20 min. The final solutions were applied for coating chicken breast fillets in the next section.

| Coating of chicken breast fillet
The fresh chicken breast fillets were aseptically cut in the same size weighing about 20 g. The coated layer was formed on the surface of each chicken fillet's pieces after dipping into the ELRG coating solutions for 2 min and drying at room temperature for 10 min. Also, a control sample was prepared after dipping in distilled water at similar conditions. All samples were placed in polystyrene (PS) containers and kept in a refrigerator for 12 days and the following analyses were carried out on the 1st, 4th, 8th, and 12th days of storage.

| Chemical analyses
2.6.1 | pH measurement The pH was measured using AOAC (2005) method. Ten grams of each chicken fillet sample was added to 90 mL of distilled water and homogenized with a mixer for 2 min. The pH of each sample was measured by digital pH meter electrodes at room temperature.

| Thiobarbituric acid determination
Thiobarbituric acid (TBA) was estimated by using the colorimetric method as described by Majdinasab et al. (2020). Briefly, 200 mg of the homogenized sample of chicken fillet was transferred to a container containing 25 mL of 1-butanol and mixed well. TBA reagent was prepared by dissolving 200 mg of TBA in 100 mL of 1-butanol solvent and after filtration kept in a refrigerator. Then, 5 mL of previous sample mixture was added to a clean and dry test tube containing 5 mL of TBA reagent. All tubes were placed in a hot water bath at 95°C for 1 h. After cooling the tubes at ambient temperature, the absorbance at 530 nm was determined by a spectrophotometer. The control tube only contained TBA reagent. According to the following formula, the amount of TBA in terms of mg of malondialdehyde per kg of chicken fillet was calculated.
A c , absorbance of the control; A s , absorbance of the sample.

| Microbiological analyses
The counts of total mesophilic, psychrophilic, molds and yeasts, and lactic acid bacteria on chicken fillets were evaluated during cold storage. Briefly, 10 g of chicken fillets were transferred aseptically into individual stomacher bags containing 90 mL of sterile NaCl solution (8.5 g/L) and homogenized in a stomacher for 1 min.
Serial dilutions were made, and pour plate culture was prepared on PCA and MRS medium. The PCA plates for determining total viable mesophilic and psychrophilic microorganisms count were incubated at 37°C for 24 h and 10°C for 10 days, respectively. Also, the MRS plates for enumeration of lactic acid bacteria were incubated at 37°C for 2 days. The YGC plates were used to evaluate the count of molds and yeasts in samples and incubated at 25°C for 72 h. After the incubation period, the microbial load of chicken fillet samples was reported as log CFU/g.

| Sensory evaluation
The sensory attributes of chicken fillet samples were analyzed by seven members trained panel. The sensory properties of different treatments such as color, odor, and general acceptance were scored using a 5-point descriptive hedonic scale (1 = dislike extremely and 5 = like extremely).

| Statistical analysis
All results were presented as the mean ± standard deviation. The statistical analysis of data was performed by analysis of variance (ANOVA), and the comparison of means was done by Duncan test at a significance level of 5% using SPSS software (version 21).

| Droplet size of REO nanoemulsions
The droplet size of REO emulsions measured by DLS is depicted in in chicken fillets (Shahrampour & Razavi, 2022). In addition, the polysaccharide-based coating with its inhibitory properties against oxygen gas can play a role in delaying the oxidation of lipids. In another study, an increase in MDA in chicken fillet samples coated with sodium caseinate was reported and the lowest level of TBA on the 12th day of cold storage was 0.12 mg/kg related to a coating containing 2.5% cinnamon EO (Ranjbaryan et al., 2018). In the study of In general, the antioxidant activity of essential oils is related to various mechanisms such as preventing the formation of free radicals, limiting the transfer of metal ion catalysts, decomposing peroxides, and interacting with free radicals. Cold storage was also reported as a factor in delaying the fat oxidation process in all samples. Fernandez et al. (1997) stated that the TBA index could probably report at incorrectly rate due to several interactions of MDA with amino acids, proteins and glucose, and other compounds in meat products.

| Microbial quality of chicken fillet samples
The effect of active ELRG coating on the microbial quality of chicken fillets during the storage time is presented in Figure 4. The count of 7 log CFU/g was reported as a critical microbial load for the start of microbial food spoilage (Senter et al., 2000).  Also, a significant increase was observed in the count of lactic acid bacteria (LAB) in all stored chicken fillets after 4 days (Figure 4c).

F I G U R E 4
Effect of ELRG coating containing REO nanoemulsions on microbial count (CFU/g) in chicken fillets during cold storage: (a) Total viable mesophilic count (TVC), (b) psychrophilic, (c) lactic acid bacteria, and (d) molds and yeasts.
(Control: sample without coating, ELRG: sample coated with Eremurus luteus root gum, L-2: sample coated with ELRG containing the largest droplet size of nanoemulsion with 2% v/v concentration, L-4: sample coated with ELRG containing the largest droplet size of nanoemulsion with 4% v/v concentration, S-2: sample coated with ELRG containing the smallest droplet size of nanoemulsion with 2% v/v concentration, S-4: sample coated with ELRG containing the smallest droplet size of nanoemulsion with 4% v/v concentration). Anyway, the count of these bacteria was less than 7 log CFU/g on 12th day in control and coating samples. By increasing REO nanoemulsions content in ELRG coating to 4%, the LAB count of chicken meat reduced by 13.32%-13.63% compared with control sample. Contrary to these results, Fernández-Pan et al. (2014) reported no effect of whey protein isolate coating containing 2% oregano or clove essential oil on reducing a load of LAB of chicken fillets to less than 7 CFU/g at the end of the 13th day of storage at 4°C.
In addition, the population of molds and yeasts demonstrated similar trends during cold storage. Unlike the control and ELRG sample, the count of molds and yeasts was less than 5 logs CFU/ml in the

| Sensory attributes of chicken fillet samples
The sensory properties of foods are an important factor in consumer acceptance. Microbial spoilage and lipid oxidation cause adverse changes in the smell, color, and taste of meats, which reduce their shelf life (Brannan, 2009). The sensory properties of stored chicken meat samples such as color, odor, and overall acceptance scored by trained panelists. This analysis showed a reduction in sensory scores with increasing cold storage time ( Figure 5). On the first day of evaluation, there was no significant difference in the color properties of various active coated samples (Figure 5a). The color score of con-    (Majdinasab et al., 2020). According to Zhou et al. (2021) research, the sensory attributes of chicken fillet improved after the use of 3.5% of camellia EO in konjac glucomannan/carrageenan coating and its shelf life was prolonged up to at least 10 days.

| Correlation between chemical and microbial parameters
As indicated in Table 2, a strong correlation was found between mi-  TA B L E 2 Pearson's correlation coefficients (r) among chemical and microbiological parameters of chicken fillet samples coated by ELRG/REO nanoemulsions.

ACK N OWLED G M ENTS
Postdoc research project. Also, part of the research was funded by the Iran National Science Foundation (Grant No. 96015540). The financial supports are gratefully acknowledged.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest regarding the publication of this paper.

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.

E TH I C A L A PPROVA L
Ethics approval was not required for this research.