Impact of alginate coating combined with free and nanoencapsulated Carum copticum essential oil on rainbow trout burgers

Abstract Carum copticum essential oil (CEO) is known as a valuable active food and pharmaceutical ingredient with antimicrobial and antioxidant activities. Solid lipid nanoparticles incorporated with CEO can overcome their limitations, namely low physicochemical stability and water solubility. In the current study, the antimicrobial and antioxidant activity of free and nanoencapsulated CEO were measured. The results revealed that although the nanoparticles of CEO had higher DPPH radical scavenging activity compared to free CEO, the antimicrobial activity of free CEO toward Escherichia coli and Listeria monocytogenes was higher than nanoparticles. Fish burger samples coated with free and nanoencapsulated CEO and stored for 12 days at 4°C. Alginate coating without CEO was considered as a control sample. The mean zeta potential, particle size, and polydispersity index (PDI) of nanoparticles were 19.18 ± 0.9 mV, 286.5 ± 18.2 nm, and 0.32 ± 0.01, respectively. The results revealed that lipid oxidation, microbial growth, and production of total volatile basic nitrogen in fish burger samples coated with alginate enriched with nanoencapsulated CEO were lower than free CEO. The main volatile compounds of CEO were para‐cymene, γ‐terpinene, and thymol, which were responsible for the antioxidant and antimicrobial activity of CEO. The data obtained by the current study suggest the application of alginate coating with CEO in form of nanoparticle to enhance fish burgers’ shelf life stored at 4°C.


| INTRODUC TI ON
Food preservatives are vital ingredients to improve the quality and shelf life of foods. Synthetic preservatives like nitrates, butylated hydroxytoluene (BHT), sodium benzoate, butylated hydroxyanisole (BHA), propyl gallate (PG), etc. are widely used because of abundance and low price. The adverse effects of preservatives on human health have been reported and they can increase the risk of several diseases, such as allergies, diabetes, cardiovascular diseases, and cancer (Gómez-Estaca et al., 2010;Oun et al., 2022). In recent decades, there have been growing worries regarding the use of synthetic preservatives in foods and therefore substantial studies conducted on the reconnaissance of natural preservatives (Carbone et al., 2018;Hussain et al., 2021). Essential oils (EOs) are composed of many valuable natural compounds that play vital roles in human health. EOs are natural preservatives with strong antioxidant, and antimicrobial activities employed as additives in foods, medicine, and cosmetic industries. EOs are generally recognized as safe (GRAS), and extracted from leaves, flowers, seeds, and barks of aromatic plants (Asdagh & Pirsa, 2020).
However, EOs are volatile, hydrophilic, and sensitive to light, oxygen, high temperature, and degradation throughout processing and storage (Pandit et al., 2016). Encapsulation, especially solid lipid nanoparticles (SLN), is a good strategy to improve dispersibility, physical and thermal stability, control the release rate of bioactive compounds, and retain the aroma. SLNs are colloidal carrier systems composed of high-melting-point lipids such as triglycerides, fatty acids, waxes, hard fats, and partial glycerides, as a solid core coated by surfactants (Kenari & Razavi, 2022;Pandit et al., 2016).
Biodegradable-based polymers act as a carrier of natural antioxidants and antimicrobials, and protect food from microbial, chemical, and physical corruption (Asdagh & Pirsa, 2020;Esfahani et al., 2022).
Alginate is a natural anionic polysaccharide extracted from bacteria and brown algae. Alginate has the potential to encapsulate bioactive compounds with a controlled release (Jafarzadeh et al., 2021;Razavi et al., 2020).
Fish burger is one of the secondary minced fish-based products containing a high amount of proteins, minerals, vitamins, eicosapentaenoic acid, polyunsaturated fatty acids, and docosahexaenoic acid with high bioavailability (Sharifi et al., 2017). The presence of a high concentration of unsaturated lipids is a vital factor that leads to formation of off-flavors and odors, texture changes, rancid taste, and discoloration (Ehsani et al., 2020).
The edible coatings enriched with natural antioxidants during cold storage can be efficaciously controlled or minimize lipid oxidation and microbial spoilage. Currently, no study was found considering the antimicrobial and antioxidant activity of alginate coating containing C. copticum essential oil (CEO) on fish burgers in the literature. The main aim of the research is the application of alginate and essential oil in free and encapsulated forms as a coating in fish burgers. Therefore, (1) the antimicrobial and antioxidant activity of free and nanoencapsulated CEO and (2) the effects of alginate coating incorporated with free and nanoencapsulated CEO on the microbial growth and chemical properties of coated fish burgers during refrigerated storage were evaluated.

| Materials
All reagents, chemicals, and alginic acid sodium salt from brown algae in the current study were obtained from Sigma-Aldrich (St. Louis, MO, USA). Carum copticum flower was collected in August 2020 from the surrounding areas of the South Khorasan province, Iran.

Carum copticum was identified by the Department of Botany Sari
Agricultural Sciences and Natural Resources University (Sari, Iran).

| Preparation of essential oil and gas chromatography analysis
The C. copticum flowers were dried in shadow at room temperature and then ground to prepare essential oil. The powdered flowers (100 g) were hydrodistilled by a Clevenger apparatus for 3 h.
After that, the anhydrous sodium sulfate was used for removal of residual water from C. copticum EO. The EO was kept in a sealed dark bottle at 4°C (Nouri et al., 2021). The CEO was analyzed by gas chromatography-mass spectroscopy (GC6890A and MS5975, Agilent, Palo Alto, USA) equipped with a HP-5 MS capillary column (30 m 3 0.25 mm internal diameter with 0.25 m film thickness as stationary phase) using helium as carrier gas at a flow rate of 0.9 ml/ min in a split ratio of 1:20. The volume of sample injection was 0.5 μl.
The injector and detector temperatures were adjusted at 240 and 290°C, respectively. The oven temperature was programmed from 50 to 200°C at a rate of 5°C/min and then raised to 240°C at a rate of 10°C/min (Mohammadi et al., 2019).

| Formulation of the SLN
The SLN was prepared in accordance with the method previously reported by Laein et al. (2022) with slight modifications. The lipid matrix used in SLN formulation corresponded to 2% w/v of glycerol monostearate as an emulsifier and 0.5% w/v EO. Tween 80 was used as a surfactant at 1% w/v of concentration. The lipid phase melted at 70°C, and then EO at 30.0 mg/ml of concentration was added on hot plate and stirred for 2 min. Then, the water phase was added to the molten lipid matrix with gentle stirring with a magnetic stirrer.
The matrix was further dispersed with an Ultra-Turrax (T-25, Staufen, Germany) at 13,000 g for 5 min to produce the hot primary emulsion.
The hot primary emulsion at 70°C was immediately sonicated using an ultrasound bath (D-7700, Elma, Germany) at 37 kHz for 2 min to produce the nanoparticles. The final SLNs were collected in a container and allowed to recrystallize at room temperature (Laein et al., 2022).

| Nanoparticle analysis
The mean particle diameter, polydispersity index (PDI), and surface charge of nanoparticles were measured using a zeta analyzer The properties of nanoparticles were measured at room temperature.

| Preparation of coating solution
Alginate solution (3% w/v) was prepared by dissolving 3 g of alginate powder in distilled water, and then 2% glycerol as plasticizer was added and stirred for 30 min at 70°C. The CEO in both free (0.5%) and encapsulated (5%) forms were added to the solution. The mixtures were stirred for 30 min at 40°C to become clear (Sharifi et al., 2017).
Fresh rainbow trout fish was purchased from the daily market in Mashhad, Iran, and was immediately transported to the laboratory, and the fish fillet was minced after peeling, deboning, and washing.
The fish burger samples were weighed into 50 g portions and were formed into 100 × 8 mm using a burger mold. Coating solutions were sprayed uniformly on the surface of burgers using a compressed high-pressure, low-volume air gun device. Both sides of the burgers were treated with respective solutions for 30 s (about 10 ml per burger) and dried for 5 min at 25°C. Fish burgers were packed in polyethylene film of 74 mm thick and then, stored for 12 days at 4°C before analyses (Ehsani et al., 2020;Sáez et al., 2020). Table 1 shows the formulation of different treatments of fish burgers.

| pH
Briefly, 10 g of each sample was homogenized in 40 ml of distilled water for 2 min. The pH of fish burgers was monitored using a pH meter (HI 99163, Hanna, Romania).

| Thiobarbituric acid value
Briefly, 200 mg of the minced fish burger was mixed with 25 ml of butanol. Five milliliter of this solution was mixed with 5 ml of thiobarbituric acid reagent and kept at 95°C for 2 h. After cooling up to room temperature, the absorbance was recorded at 530 nm (Kiarsi et al., 2020).

| Microbial analysis
The method described by Sarvinehbaghi et al. (2021) was applied for preparation of serial dilution of fish burgers. Plate count agar using

| Statistical analysis
A two-way analysis of variance (ANOVA) with Duncan's test (post hoc) and Student's T-test were applied to analyze differences between samples. Differences between mean ± standard deviation were statistically significant at p < .05 (95% confidence level). SPSS statistical software (Inc. Chicago, IL, USA) version 20 was used for the statistical analysis.

| Composition of CEO
Essential oils are natural, volatile, and oily liquids, and their great bioactivity has been confirmed. CEO analysis revealed 97.72% of the total compounds, consisting of 19 compounds (Table 2) of C. copticum and detected 25 compounds, and the main constituents were thymol, γ-terpinene, and para-cymene, respectively (Mohammadi et al., 2019). In other studies, thymol, γ-terpinene, and para-cymene were also reported as major compounds of C.

| Antibacterial activity
In order to investigate antibacterial properties of C. copticum in both free and nanoencapsulated forms, MBC and MIC values were assessed toward L. monocytogenes and E. coli (

| Characteristics of nanoparticle (NANO)
The particle size, zeta potential, and PDI are the most important parameters associated with the quality, stability, and other macroscopic properties of nanoparticles. The results of DLS showed that the mean particle size, zeta potential, and PDI of nanoparticles were 286.5 ± 18.2 nm, −19.18 ± 0.9 mV, and 0.32 ± 0.01, respectively. The Therefore, the nanoemulsion must dry quickly to prevent Ostwald ripening and aggregation. The negative zeta potential of nanoparticles could be due to the presence of acidic polyphenol groups. The negative zeta potential of plant essential oil in different coatings was also reported in previous literature (Sayyari et al., 2021). PDI value showed the homogeny of droplet size distribution in emulsion systems, and it ranged from 0 to 1.  Nasseri et al. (2016) stated that the particle size of Zataria multiflora EO solid lipid nanoparticle was about 255.5 nm. They also reported the PDI and zeta potential as 0.369 and −37.8 mV, respectively, which is in line with the data obtained in our study (Nasseri et al., 2016).
The CEO surface morphology loaded in SLN was assessed by transmission electron microscopy at two magnifications (Figure 1a

| pH changes
The pH values of different fish burgers stored at 4°C were observed in Figure 2a. An increasing trend was observed for all samples related to the volatile basic compounds produced by microbial enzymes and endogenous. The sample NANO showed an acceptable pH value (pH) of <6.5 up to day 12, which was due to the gradual release of phenols during storage. Alginate coating individually did not have an effect on pH values of fish burger samples, while the coatings enriched with EO showed a lower pH than control. These results were in line with other studies which described that the pH value of fish products increased over time while samples coated with a coating containing essential oil showed a lower pH (Karamkhani et al., 2018;Kazemi & Rezaei, 2015;Sayyari et al., 2021).

| Thiobarbituric acid (TBA) changes
Lipid oxidation one is of the major chemical reactions that limit the seafood shelf life containing high amounts of polyunsaturated fatty acids. Peroxides as initial lipid oxidation products are unstable and decompose into secondary products. Therefore, estimating TBA value to investigate the extension of lipid oxidation TA B L E 4 DPPH radical scavenging activity of free and encapsulated Carum copticum essential oil (%).

| Microbial growth
The microbial growth results of different fish burger samples are depicted in Figure 3a, b. The primary count of TVC and PTC ranged

(b)
CONT FREE NANO from 3.05 to 3.65 and 2.60 to 3.55 log CFU/g, respectively, which indicates the acceptable microbial quality of fish burgers. The increasing microbial growth with a statistically significant difference (p < .05) was observed during chilled storage. These results are in accordance with Sáez et al. (2020), who stated an increasing trend for PTC of rainbow trout fillet coated with alginate enriched with tannin. Also, they stated that fish fillets without preservatives had higher microbial growth (Sáez et al., 2020).
The fish burger sample without CEO showed a higher TVC and PTC. The efficiency of coating incorporated with CEO in SLN form was higher than coating with free CEO. Nanoencapsulation of essential oil caused the gradual release of phenols from the coating and  (Kazemi & Rezaei, 2015).
Listeria monocytogenes is one of the challenging foodborne pathogens that generally contaminates seafood products (Sharifi et al., 2017). The results of L. monocytogenes growth stored for 12 days at 4°C are shown in Figure 5a. On the first day, the count of L. monocytogenes was between 2.12 and 2.39 log CFU/g. It significantly increased in the control sample, while in the samples coated with free and encapsulated EO, the reducing trend was observed up to days 9 and 6, respectively, and then the bacterial counts increased. This result is inconsistent with Sharifi et al. (2017), who reported a significant increase in L. monocytogenes count (Sharifi et al., 2017). Gas chromatography-mass spectrograph analysis of CEO showed that the para-cymene, thymol, and γ-terpinene are the major components of the essential oil. These compounds released from the coating matrix and caused to eliminate bacterial growth.
The hydroxyl group increased hydrophilic characteristics of phenolic compounds and accelerated the dissolving of the phenolic compounds in microbial membranes and therefore, eliminating the bacteria. indirectly reduce the TVN. The previous study, also reported that applying the plant EOs and extracts due to the preservative effects of phenolic compounds can reduce the TVN value of fish products (Sayyari et al., 2021;Zarei et al., 2015).

| CON CLUS ION
In this study, the antimicrobial and antioxidant activity of alginate coating containing free and nanoencapsulated Carum copticum

ACK N OWLED G M ENTS
The authors would like to express their sincere appreciation to Ahvaz Jundishapur University of Medical Sciences and NIMAD.

FU N D I N G I N FO R M ATI O N
This study was supported financially by Jundishapur University of Medical Sciences (grant number: U-98108) and NIMAD.

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

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available upon reasonable request.

E TH I C S S TATEM ENT
The current study has been approved by the ethics committee of