Edible coating from citrus essential oil-loaded nanoemulsions: physicochemical characterization and preservation performance

Abstract

To improve the availability of citrus essential oil (CEOs), nanoemulsions based on chitosan nanoparticles loaded with CEOs were prepared by the emulsion-ionic gelation technique. The structural information and the coating characteristics of the nanoemulsion as well as its preservation properties on silvery pomfret were investigated. The results showed that the amount of CEOs added into a nanoemulsion reduced the stability and rheological properties of the nanoemulsion systems, which indirectly affected the physicochemical properties of the coatings. The formation of the nanoemulsion was attributed to interactions between the functional groups of chitosan and CEOs by hydrogen bonds and electrostatic interactions. Furthermore, the preservation effects on silvery pomfret by the nanoemulsion coating were better than conventional emulsion coatings, which were ascribed to more efficient preventative microorganisms and lipid oxidation from silvery pomfret by the nanoemulsion coating. These results suggested that nanoemulsion coating containing CEOs have great potential in the seafood preservation field.

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1. Introduction

Citrus fruits are very popular with consumers throughout the world due to their pleasant flavors and high nutritional value. Citrus peels, a by-product of citrus fruits, contain abundant fragrant substances, especially essential oils (EOs).¹ Those EOs consist of a mixture of terpenes, terpenoids and phenol-derived aromatic constituent that exert excellent antimicrobial and antioxidant properties with the potential to extend the shelf life of foods.^2,3 Unfortunately, the use of EOs in food industries is often limited because of their application costs and other drawbacks, such as intense aroma, low water solubility and potential toxicity.^4,5 Thus, the design of new delivery systems to optimize or even enhance the effectiveness of EOs is a great challenge in food industries.

An interesting approach to overcome those shortcomings while maintaining their effectiveness could be to incorporate these oil-based compounds into the formulation of a nanoemulsion.^6–8 EOs-in-water nanoemulsions dispersed in an aqueous solution have unique physicochemical and functional characteristics. On the one hand, nanoemulsions can be designed to have high stability to particle aggregation and gravitational separation, and therefore, they may be used to extend the shelf life of commercial products. On the other hand, it has been recently noted that nanoemulsions may enhance the transport of active ingredients through biological membranes, thus intensifying the bioavailability of bioactive compounds or the bactericidal activity of antimicrobials.^5,6,9 Although the application of nanoemulsions in drinks and beverages is growing,¹⁰ using these systems in solid foods such as fruit and fish still remains rare because of the immobilization of nano-droplets on the surface of the foods.

In this respect, a nanoemulsion in situ formed from biopolymer-based edible coatings solutions would represent an effective approach to place EOs on the surface of minimally processed foods. The incorporation of EOs to biopolymer-based edible coatings (such as chitosan coating) onto the product surface not only exhibits potential synergies antimicrobial activities with the EOs but can also act as a barrier to moisture, gas exchange and oxidative reaction during storage, further retarding food deterioration.^11,12 Among these biopolymers, chitosan (CS) is an excellent candidate for coating materials. In fact, the incorporation of EOs in CS coatings has been confirmed to be effective at extending the shelf-life of some fish and fruit, such as Japanese sea bass,¹³ Indian oil sardine,¹⁴ rainbow trout¹⁵ and strawberries.⁴ However, EOs were reported to integrate in CS-based coatings in emulsion form up to now. To the best of our knowledge, limited information has been available about the use of an EO nanoemulsion coating to preserve food product.^6,7 Furthermore, there are no studies on using such nanoemulsion coatings to extend the shelf-life of fish meat. The quantum size, mini size, surface and macro-quantum tunnel effect of nano-oil droplets could also endow unique physical and chemical features of nano-emulsion coatings.^5,6 Thus, it is hypothesized that the application of nanoemulsion coatings into a food system would be different than traditional emulsion coatings. In addition, the stability of coating-forming solutions (CFS) was influenced by the addition of the amount of EOs.³ The stability of the CFS depended on complex mechanisms, but some of the key aspects include the surface charge and the size distribution of oil droplets, which in turn both influenced the stability and the rheological properties of the CFS.^16,17 Therefore, these properties of the nanoemulsion CFS should also be considered when designing coating.

Herein, the objective of this study was to design and characterize the structural information of nano-oil droplets in nanoemulsions. The formation mechanism of nanoemulsion coating and the relevant properties of the nanoemulsion CFS (droplet size, ζ-potential and rheological properties) were investigated and related with the film properties. Additionally, the preservation effect of nanoemulsion coatings on silvery pomfret (Pampus argenteus) during refrigerated storage was also discussed in comparison with conventional emulsions coating.

2. Material and methods

2.1 Materials

Shrimp derived chitosan (1.86 × 10⁵ Da molecular weight, 95% deacetylation) was purchased from the Qingdao Yunzhou Biochemistry Co., LTD., China. Citrus essential oil (Citrus Reticulata var. tangerine) was provided by the Ji'an Borui Perfume oil Co., LTD., China. Acetic acid, Tween 80, TPP and other analytical grade chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Fresh silvery pomfret weighing 300–320 g each with an average body length of 18–20 cm, were obtained from a fishery boat in Zhoushan Bay (Zhejiang Province, China) and were transferred to the lab in an ice box within 4 h. After that they were decapitated and filleted by hand. Two fillets were obtained from each fish and kept at 4 °C until use.

2.2 Chemical composition of citrus EOs

The chemical composition of the CEOs was analyzed by GC/MS (7890/5975, Agilent Technologies, Palo Alto, USA) with a fused silica capillary column HP-5 (30 m × 0.25 mm). The analytical conditions were as follows: injection temperature, 250 °C; injection mode split (split ratio 100 : 1); column temperature, isothermal at 70 °C for 2 min, then programmed from 70 °C to 250 °C at 3 °C min⁻¹ and held at this temperature for 10 min; ion source temperature, 230 °C; EI energy, 70 eV; and mass scan range, 40–400 amu. The identification of the individual compounds was based on the comparison of their relative retention times with those of authentic samples on the capillary column and by matching their mass spectra of peaks with available Wiley, NIST and NBS mass spectral libraries.

2.3 Preparation and characteristics of the coating-forming nanoemulsions

The nanoemulsions were prepared by the emulsion-ionic gelation technique. Briefly, CS solution (1% (w/v)) was prepared in an acetic acid (1% (v/v)) under magnetic stirring until complete dissolution. Tween 80 (0.3% v/v) was subsequently added to the CS solution with constant stirring for 1.5 h to obtain a homogeneous solution. The CEOs were then added drop wise into the CS solutions by homogenizing (20 000 rad) for 5 min to obtain an oil-in-water emulsion with different weight ratios of chitosan to CEOs of 1 : 0, 1 : 0.1, 1 : 0.5 and 1 : 1, recoded as emulsion CS, CS-CEOs-0.1, CS-CEOs-0.5 and CS-CEOs-1, respectively. Nanoemulsions were obtained by gradually dropping sodium tripolyphosphate (TPP, 2 mg ml⁻¹ (w/v)) into the above those emulsion with agitating and then the mixture were homogenized at 20 000 rpm for 2 min. Those nanoemulsion coating-forming solutions were designated as CS-Nano, CS-Nano-CEOs-0.1, CS-Nano-CEOs-0.5 and CS-Nano-CEOs-1, respectively. All solutions were degassed under vacuum and further used as coating-forming solutions to form the edible coating on the sample fish surface.

2.4 Characteristics of nanoemulsion coating

The droplet size and ζ-potential of the coating-forming solutions were measured by a Zetasizer Nano ZS90 (Malvern Instruments, UK). The morphological characteristics of the nanoemulsion were examined using a high resolution transmission electron microscope (TEM) (Tecnai F-20, Phillips Co., Netherlands).

The rheological properties of the coating-forming solutions (CFS) were studied on a controlled stress rheometer (HAKE Rheostress, Thermo Fisher Corporation, Karlsruhe, Germany) fitted with a cone-plate geometry (35 mm) with a gap of 0.104 mm. The viscosity of the nanoemulsions was measured by a steady state flow program with the shear rate ranging from 0.1 to 100 s⁻¹. Experimental flow curves were fitted to a power law model.¹⁸

The stand-alone coating was prepared by casting 40 ml of the coating-forming solutions onto an acrylic board (20 cm × 20 cm) and dried at 30 °C for 72 h. The coatings were stored in a desiccator before further use. The thickness of the coating was measured using a digital micrometer (Mitutoyo, Tokyo, Japan). The tensile strength (TS, MPa) and elongation to break (EB, %) of the stand-alone coatings were determined by a PARAM XLW (M) Auto Tensile Tester (Jinan Labthink Technology Company, China) according to ASTM standard methods.¹⁹ The WVP of the films was determined gravimetrically at 25 ± 1 °C according to ASTM E96 (ref. 20) standards.

The structure information of the stand-alone coatings was studied by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffractometer (XRD). FT-IR of the samples was carried out according to the KBr pellet method on a Thermo Fisher Nicolet in the range of 400–4000 cm⁻¹. Microstructures of the surface of the dried coating were observed by an SEM (JSM-6380 LV, Japan Electron Optics Limited, Tokyo, Japan) at 20 kV. The crystallographic structures of the samples were determined by XRD Powder XRD spectra were acquired in the angular range of 5–60° (2θ) on a Bruker AXS D8 Advance X-ray diffractometer (Bruker Inc., Germany) using Ni-filtered Cu Kα radiation.

2.5 Evaluation of the preservation property of nanoemulsion coatings on silvery pomfret

2.5.1 Treatment of fish fillet samples. Fish fillet samples were randomly assigned into six groups and soaked in distilled water (uncoated control), CS or CS-Nano or CS-CEOs-1 (emulsion) or CS-Nano-CEOs-1 (nanoemulsion) solution for 10 min. All fish samples were then drained before being individually packed in air-proof polyethylene pouches and stored at 4 ± 0.3 °C. On day 0, 1, 4, 8, 12 and 16, triplicate samples were taken out to perform the chemical, microbiological and sensory analyses.

2.5.2 Microbial analysis. Fish samples (10 g) were homogenized with 90 ml of sterile 0.9% (w/v) saline. The resulting suspensions were serially tenfold diluted in sterile saline and used for bacteriological analysis. The total microbial counts were determined according to our previous report.²¹ The data were expressed as log₁₀ CFU g⁻¹ (colony forming units per gram of fish) and performed in triplicate.

2.5.3 Physicochemical analyses. The pH values of the fish samples were measured by a Testo 205 pH meter (Testo AG, Lenzkirch, Germany) by inserting an electrode into the muscle.

The TVB-N (mg N/100 g fish meat), TBARS (mg MDA per kg fish meat) contents and K value were determined according to our previous study.^21,22 The K value was calculated as the percentage of the sum of H_xR and H_x divided by the sum of ATP determined by HPLC (Waters 2695, USA) and its degradation products as follows:

2.5.4 Sensory evaluation. The sensory evaluation of the fish samples was performed by a trained panel of seven members who were familiar with the evaluation of fish meat. The panelists were asked to evaluate the appearance, color, odor and overall acceptability of the fish samples using a nine-point hedonic scale (1 for dislike extremely to 9 for like extremely). The sensory evaluation was performed in individual booths under controlled conditions.

2.6 Statistical analysis

All of the measurements were carried out in triplicate, and the mean values were calculated and reported as the mean ± SD (n = 3). Analysis of variance (ANOVA) and Duncan's multiple range tests were performed by the SPSS software statistical analysis system (SPSS 20.0 for windows, SPSS Inc., Chicago, IL).

3. Results and discussion

3.1 Chemical composition of CEOs analysis

The chemical compositions of the CEOs are shown in Table 1. There were 34 components identified for the CEOs. Limonene was the most abundant component and accounted for 91.94% of the total peak area of the CEOs, followed by γ-terpinene (2.40%), β-myrcene (1.60%), α-pinene (0.84%) and linalool (0.90%). The concentrations of the remaining components were all below 1%. Limonene is the main volatile component of citrus fruits, including lemon, orange, bergamot and mandarin.^1,23 The antioxidant nature of the CEOs is due to the antioxidant activity of limonene, however, the antimicrobial activity of citrus EOs is not ascribable to limonene, but instead, it seems to be due to the presence of other constituent, s such as oxygenated monoterpenes.^2,24

Table 1 Chemical composition of CEOs

No.	Compound	% of the total peak area
1	α-Pinene	0.84
2	Sabinene	0.06
3	β-Pinene	0.13
4	β-Myrcene	1.60
5	D-Limonene	91.94
6	(E)-β-Ocimene	0.58
7	γ-Terpinene	2.40
8	α-Phellandrene	0.05
9	α-Terpinene	0.06
10	Nerol	0.07
11	Neryl acetate	0.02
12	β-Citronellol	0.04
13	Terpinolene	0.06
14	β-Linalool	0.05
15	Geranial	0.04
16	Neryl acetate	0.03
17	δ-Elemene	0.05
18	Linalol	0.90
19	β-Elemene	0.07
20	Germacrene	0.06
21	1-Penten-3-ol	0.01
22	(Z)-3-Hexenol	0.09
23	(E)-2-Hexenol	0.07
24	Hexanol	0.05
25	Octanol	0.10
26	Coniferyl alcohol	0.08
27	Hexanal	0.06
28	Octanal	0.11
29	Nonanal	0.05
30	Decanal	0.05
31	Undecanal	0.07
32	Hexanoic acid	0.11
33	Octanoic acid	0.06
34	p-Cymene	0.03

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3.2 Characterization of nanoemulsion

The nanoemulsions were prepared via an ionic crosslinking reaction between the protonated amino groups of the CS and the phosphoric groups of the TPP (Fig. 1). An electrostatic interaction was established in the reaction between the CS and TPP, enabling the encapsulation of an amount of CEOs within a positive matrix (CS) and using TPP as the ionic crosslinking agent with a negative charge of its phosphate groups. The acid media guarantees the protonation of the amino groups of the CS.

Fig. 1 Scheme of the preparation of nanoemulsions coating.

The mean droplet size and zeta potential of the nanoemulsions with different concentrations of CEOs are summarized in Table 2. The average droplet size of the conventional emulsions was in the range of 696–980 nm, and they were drastically reduced to 269–428 nm after adding TPP (p < 0.5). As can be observed, the incorporating CEOs increased the average droplet size of the nanoemulsions. Furthermore, the CEO-loaded nanoemulsion samples exhibited an increase in their droplet size from 269 to 428 nm as the CEOs content increased from 0% to 1%. This result might be attributed to the fact that the dispersed oil droplets need surfactant molecules to stabilize on the nanoparticle. Overdose effects, as an excessive oil volume fraction, could not completely be covered by the emulsifier, resulting in an increase in the size of the average droplet diameter of the nanoemulsions.¹⁶ This behavior was also confirmed by observing the droplet size distribution (PDI values, Table 2) because relatively well-distributed nanoparticles were observed in a lower CEOs concentration in the formulation. These results agreed with the findings obtained by other authors in essential oil loaded polysaccharide-based nanoemulsions.^3,25

Table 2 Mean droplet size, ζ potential and rheological parameters for all samples^a

Sample	Mean droplet, size (nm)	PDI	ζ potential, (mV)	Rheological parameters
				K	n	R^2
a Data shown are the mean ± standard deviation (n = 3). Values within each column with the different letters are significantly different (p < 0.05).
CS	—	—	57.33 ± 0.91f	0.194 ± 0.005f	0.693 ± 0.006a	0.99
CS-CEOs-0.1	688.67 ± 16.92e	0.60 ± 0.08d	52.19 ± 1.13e	0.163 ± 0.004e	0.772 ± 0.005b	0.99
CS-CEOs-0.5	860.67 ± 56.21f	0.82 ± 0.07e	47.44 ± 0.93d	0.131 ± 0.002d	0.792 ± 0.002b	0.98
CS-CEOs-1	986.34 ± 85.10g	0.96 ± 0.04f	39.63 ± 0.68b	0.076 ± 0.002b	0.873 ± 0.003c	0.97
CS-Nano	269.68 ± 14.51a	0.21 ± 0.05a	51.17 ± 0.62e	0.006 ± 0.001a	0.992 ± 0.005f	0.99
CS-Nano-CEOs-0.1	322.00 ± 19.08b	0.35 ± 0.04b	47.60 ± 1.54d	0.156 ± 0.002e	0.852 ± 0.003c	0.99
CS-Nano-CEOs-0.5	365.00 ± 12.12c	0.45 ± 0.03c	43.40 ± 0.96c	0.109 ± 0.002c	0.915 ± 0.004d	0.98
CS-Nano-CEOs-1	427.38 ± 32.50d	0.65 ± 0.06d	36.71 ± 1.03a	0.055 ± 0.007b	0.966 ± 0.003e	0.99

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The ζ-potential of the CS solution was 57.33 ± 0.91 mV (Table 2), which was due to the protonated ammonium groups of the CS in the acidic environment. The ζ-potential of all samples remained positive in the range of 36.71–57.33 mV, and the introduction of CEOs reduced the ζ-potential of the nanoemulsions. This result might be related to the electrostatic interactions between the CS nanoparticle and the CEOs components under acidic environments.

The morphologies of the nanoemulsions were observed by TEM. As depicted in Fig. 2, the unloaded CEOs of the nanoemulsions had a regular distribution and a spherical shape. Expectedly, the size of the CEO-loaded nanoparticles was larger compared to the CS nanoparticles.

Fig. 2 TEM images of (a) CS-Nano and (b) CEOs-loaded CS-Nano prepared using an initial weight ratio of CS to CEOs of 1 : 1.

The rheological properties of the CFS were investigated (Fig. 3). It was found that all solutions exhibited typical pseudoplastic behavior with marked shear-thinning areas. The rheological data were fitted to the Ostwald de Waale model and the model parameters, and the corresponding correlation coefficients (R²) of the fitting are included in Table 2. The CS solution showed a shear thinning fluid behavior that was significantly affected by the incorporation of TPP and CEOs (Table 2). The addition of TPP caused a significant decrease in the consistency index (k) and gave rise to a less marked shear thinning fluid behavior (higher n). This phenomenon may be due to the formation of nanoparticles, which reduced the affinity of the CS molecules with water, leading to the reduced viscosities.²⁶ Incorporating the CEOs in nanoemulsions also decreased the shear-thinning behavior (increasing n value). The rheological behavior of the solution was related to several factors, such as the solution concentration, particle size, distribution and shape of the particles and surface electrical charge.²⁷ The introduction of CEOs to the nanoemulsion system not only increased the average droplet size of nanoemulsions but also reduced the electrical net charge of the nanoparticles, which could lead to lower viscosities and a less obvious shear-thinning behavior of coating forming solutions.^3,16

Fig. 3 Flow curves of CFS.

3.3 Characterization of the coatings

The physical and mechanical properties of the stand-alone coatings, comprising thickness, WVP, TS, and E%, are shown in Table 3. The additional amount of TPP exhibited negligible differences in the thickness of the coatings (p > 0.05), while the incorporation of CEOs significantly increased the thickness of the coatings (p < 0.05). The addition of CEOs led to a significant reduction (p < 0.05) in the WVP, and this changing trend was in line with the increase in the CEOs additives. This behavior is expected as an increase in the hydrophobic compound fraction (essential oils as lipid compounds) usually leads to an improvement in the water barrier properties of polymer-based films.²⁸ On the other hand, CEOs caused hydrogen and covalent bonds to form between the functional groups of the CS chains, leading to a decrease in the availability of –OH and –NH₂, limiting CS chain–water interactions by hydrogen bonding and resulting in a decrease of the moisture content value of the edible coating.^3,29 Similar findings were reported by other researchers when they incorporated a hydrophobic agent such as α-tocopherol or olive oil into CS-based films.^30,31 In addition, the WVP of the nanoemulsion coating was better than that of the emulsions coating, which was attributed to more crosslinking between polymers in the matrix after adding TPP. The formed nanoparticles reduced the intermolecular spacing within the coating, hindered the diffusion of water molecules through the coating and created a diffusive tortuosity effect on the transport of molecules through the coating, thus reducing the WVP through the coating.^32,33

Table 3 Mechanical and physical properties of coating in the presence of different CEOs^a

Sample	Thickness (mm)	WVP (g Pa^−1 s^−1 m^−1) 10^−11	TS (MPa)	E (%)
a Different superscripts indicate significant differences according to ANOVA test (p < 0.05).
CS	0.079 ± 0.001^a	26.410 ± 0.738^f	25.740 ± 0.491^d	15.086 ± 0.949^a
CS-CEOs-0.1	0.085 ± 0.002^b	23.047 ± 0.948^d	22.910 ± 0.282^c	16.260 ± 0.892^b
CS-CEOs-0.5	0.094 ± 0.003^c	20.603 ± 0.454^c	17.790 ± 0.578^b	18.857 ± 0.352^c
CS-CEOs-1	0.099 ± 0.002^d	18.493 ± 0.438^b	12.547 ± 1.080^a	22.540 ± 0.600^d
CS-Nano	0.078 ± 0.003^a	24.310 ± 0.393^e	28.210 ± 0.472^e	14.397 ± 1.028^a
CS-Nano-CEOs-0.1	0.086 ± 0.001^b	19.747 ± 0.342^c	25.970 ± 0.327^d	14.940 ± 0.598^a
CS-Nano-CEOs-0.5	0.095 ± 0.001^c	18.047 ± 0.077^b	22.047 ± 0.818^c	15.477 ± 0.563^ab
CS-Nano-CEOs-1	0.100 ± 0.003^d	16.747 ± 0.334^a	17.727 ± 0.904^b	19.490 ± 0.510^c

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The incorporation of CEOs into the nanoemulsion system caused a significant reduction in the TS of the nanoemulsion coating (p < 0.05) compared to the CS-Nano coating. On the other hand, the E% of the CS-based nanoemulsion coating did not significantly change when the CEOs concentration was increased from 0% to 0.5% (p > 0.05) but significantly increased when the concentration reached 1% (p < 0.05). The addition of EOs to polysaccharide-based film/coatings may hinder the polymer chain-to-chain interactions and provide flexible domains within the film/coating. As a consequence, the TS decreased as the concomitant increased in the E% of the corresponding film/coatings. In addition, incorporating high concentrations of CEOs to the nanoemulsion system resulted in a heterogeneous and irregular structure in the film matrix, which decreased the TS of the nanoemulsion coating. These results were in line with the outcome of the works by.^28,29,34

To examine the possible molecular interactions between components of the nanoemulsion systems, the structural properties of the dry stand-alone coating were characterized by FT-IR (Fig. 4a), XRD (Fig. 4b) and SEM (Fig. 4c) analyses.

Fig. 4 FT-IR spectra (a), XRD spectra (b) and SEM images (c) of stand-alone coating.

Compared to the FT-IR spectrum of the CS, the peak of the amide I (–NH₂ bending) of CS-Nano shifted from 1627 to 1615 cm⁻¹, and the intensity of amide I at 1639 cm⁻¹ decreased, which could be attributed to the electrostatic interaction between the P₃O₁₀⁵⁻ of TPP and the NH³⁺ of CS-Nano. A new peak appeared at 1210 cm⁻¹ because of P=O stretching in CS-Nano, which was absent in bulk chitosan, implying the formation of nanoparticles. In the case of CEO-loaded nanoemulsion coatings, the main spectra of the nanoemulsions were similar to the bands of CS-Nano, except for a new band (at 1735 cm⁻¹) and changes (broadening) in the bands of CS-Nano spectrum (in the region 2850–2950 cm⁻¹, representing aliphatic –CH₂ asymmetric and symmetric stretching vibration). These changes increased with the amount of CEOs incorporated in the nanoemulsion coating system. This variation may be due to hydrogen bonding and the electrostatic interaction between the functional groups in the CEO ingredients and the –NH³⁺ and –OH groups in CS-Nano. Furthermore, the addition of CEOs in nanoemulsion coating could increase the hydrophobicity of the coating, as evidenced by the existing peak at wavenumbers of 2854 cm⁻¹, 2924 cm⁻¹ and 1735 cm⁻¹. This result was supported by the decrease of the WVP of the coatings incorporated with CEOs (Table 3).

Crystallographic structure of CS, CS-Nano, emulsion, and nanoemulsion coating were illustrated in Fig. 4c. As expect, chitosan exhibited two typical peaks around 2θ = 15.10° and 20.77°, indicating the high degree of crystallinity. After ionic cross-linking with TPP, a shift of peak positions, reduction of peak intensity, and broadness of peaks were observed, reflecting the destruction of the native CS structure. These phenomena might be attributed to a modification in the arrangement of molecules in the crystal lattice induced by ionic interaction.^35,36 As compared with CS-Nano, the peak intensity at 2θ of 15.43° disappeared, while the peak at 2θ of 22.57° was markedly enhanced and broadened in diffraction spectrum of nanoemulsion coating. This implied that the incorporation of CEOs resulted in a change in CS-Nano structure, which was also confirmed the presentence of CEOs in CS-Nano.

The surfaces of neat CS, CS-Nano, emulsion, and nanoemulsion coating were observed by SEM (Fig. 4c). Pure CS film displayed smooth and homogeneous surfaces without pores and with excellent structural integrity (Fig. 4c-1). Different surface morphology was observed after TPP was added into the CS coating. The surface morphology of the CS-Nano coating was smooth with a small spherical shape nanoparticle (Fig. 4c-2). Compared with the CS-Nano coating, the presence of CEOs in the nanoemulsion coatings caused a roughness and opaqueness surface structure in which oil droplets were entrapped in the continuous CS-Nano network (Fig. 4c-4). The size of the nanoparticle in the surface nanoemulsion coatings was larger than the CS-Nano coating. This could decrease the TS of the coating.

3.4 Preservation properties of nanoemulsion coatings on silvery pomfret

After being coated by CS-based coatings, the silvery pomfret samples were stored at 4 ± 0.2 °C, and the preservation performance indices are shown in Fig. 5.

Fig. 5 Preservation performance indices of silvery pomfret (Pampus argenteus) during refrigerated storage. (a) TVC (b) pH; (c) TVB-N; (d) TBARS; (e) K value; (f) the sensory score.

As shown in Fig. 5a, the total viable counts (TVC) values of all samples increased with the storage time, especially for the control group. The initial TVC of the control was 2.99 log CFU g⁻¹ and then improved to 7.58 log CFU g⁻¹ after 8 days. This value exceeded the upper acceptability limit (7 log CFU g⁻¹) for raw marine species.³⁷ The control sample only had a shelf life of 8 days. The groups treated with CS-based coating had a significantly lower value (p < 0.05) than the control group after 3d. The TVC of those treated with CS, CS Nano and emulsions coating reached 8.13, 7.64 and 7.16 log CFU g⁻¹, respectively, at the end of storage, while the samples treated with nanoemulsion coatings decreased to 6.46 log CFU g⁻¹ on day 16 (p < 0.05), indicating that the shelf-life of the fish samples treated with nanoemulsion coatings could be extended to more than 16 days of storage. The incorporation of CEOs into CS coating enhanced its antimicrobial activity because the oxygenated monoterpenes ingredients of CEOs could disrupt and penetrate the lipid structure of the bacteria cell membrane and damage the enzyme systems of bacterial to inhibit the growth of the microorganism.^2,38 The nanoemulsions exhibited a better effect on the preservation of the fish samples compared to the emulsions treatments, which could be because of the nanoparticle's larger surface area and higher affinity with bacteria cells, which yields a quantum-size effect.^7,39

As shown in Fig. 5b, the pH values of all fish samples decreased and then increased gradually with the storage time. The initial decrease in the pH might be related to the accumulation of lactic acid produced by glycolysis, while the increase was attributed to the growth of bacteria in the fish with the formation of alkaline autolysis compounds (nitrogenous compounds), ammonia and other volatile bases compounds during the postmortem period. Similar increases in the pH have also been obtained for other marine fish species stored at cold temperatures.^13,40 Compared with the control sample, the samples showed lower pH values when treated with CS-based coatings, especially for nanoemulsion coatings (which had the highest quality). However, no significant difference was found between the pH of the CS-Nano, emulsion and nanoemulsion coatings samples during refrigerated storage.

The TVB-N content of all samples increased steadily with the storage period, as shown in Fig. 5c. The increase of the TVB-N values was attributable to proteolysis, indicating the activities of spoilage bacteria and endogenous enzymes. Apart from the initial amount of TVB-N, the control sample had a significantly higher value than that of the CS-based coated samples (p < 0.05), while the nanoemulsion coating group had lower TVB-N values among the CS-based coating. According to other researchers,⁴¹ 30 mg N/100 g were taken as the limit of acceptability for seafood products. In this study, the TVB-N content in the control group exceeds this limit of acceptability at 8 days. Moreover, treated groups with CS, CS-Nano and emulsions reached the maximum borderline of the acceptable level after 12, 12 and 16 d, respectively. The TVB-N value in the nanoemulsion coating group did not surpass the borderline of the acceptable level at day 16, indicating that the nanoemulsion coating has the ability to extend the shelf life of fish during refrigerated storage.

The TBARS value reflects the degree of lipid oxidation of fish meat, which is another index for the assessment of the quality of seafood products during refrigerated storage.⁴² Based on the results of TBARS determined in the present study (Fig. 5d), the TBARS value in CS-based coating samples were significantly (p < 0.05) lower than those of the control samples during storage (p < 0.05). Additionally, the emulsions and nanoemulsions coatings had significantly lower TBARS values than other CS-based coating after 4 d (p < 0.05), while there was no significant difference between the emulsions and nanoemulsions groups (p > 0.05). This result was due to limonene, which, as a major ingredient of CEOs, had the ability to scavenge free radicals and prevent lipid peroxidation,⁴³ thus enhancing the antioxidant activity of the emulsions and nanoemulsions coatings.

As for the K value, the trend was similar for all fish samples, and the values increased continuously with the storage time over a period of 16 days (Fig. 5e). On sampling day 0, no significant difference was obtained. However, on that day, CS-based coatings significantly (p < 0.05) affected the K values of the fish samples compared to that of the control. In addition, there was no significant difference in the K value after sampling day 12 between the groups of the CS-Nano and emulsions coating. However, the fish samples treated with nanoemulsion coating showed lower K values (p < 0.05) than those of the other coatings on days 12 and 16. This result indicated that the nanoemulsion coating could effectively inhibit microbial activity to degrade ATP and extended the shelf life of fish samples.

The sensory qualities of the fish samples were evaluated in terms of their appearance, color, odor and overall acceptability using a nine-point hedonic scale (1, dislike extremely, to 9, like extremely). As exhibited in Fig. 5f, there was no significant difference in the overall acceptability among all treatment groups during the initial 4 d. However, compared with the CS-based coating fish samples, the overall acceptability of the control samples decreased sharply from days 8 to 16 with significantly lower scores (p < 0.05). Moreover, the fish treated by nanoemulsion coating had the highest sensory scores after 12 d of storage, which were well in agreement with the microbial and chemical value (TVB-N, TBARS and K value) analyses.

4. Conclusions

CEOs-loaded CS-based nanoemulsions were successfully developed. The incorporation amount of the CEOs influenced the nano-oil droplet size, stability and rheological properties of the nanoemulsion systems, which directly affected the physicochemical properties of the final nanoemulsion coating. The formation of nanoemulsion was attributed to interactions between the functional groups of the CS, TPP and CEOs by hydrogen bonds and electrostatic interaction. Compared to conventional emulsions coating, the nanoemulsion coating was effective in inhibiting the growth of microorganisms and changes in the biochemistry of the silvery pomfret during refrigerated storage, thus extending the shelf life of the fish from 12 days to 16 days. These results suggest that nanoemulsion coating might be an effective preservation material for marine fish species and has great potential in the seafood industry.

Acknowledgements

The authors would like to thank the support of Key Project of National Science and Technology Ministry of China (2012BAD38B09).

References

1. C. Liu, Y. Cheng, H. Zhang, X. Deng, F. Chen and J. Xu, J. Agric. Food Chem., 2012, 60, 2617–2628.
2. W. Randazzo, A. Jiménez-Belenguer, L. Settanni, A. Perdones, M. Moschetti, E. Palazzolo, V. Guarrasi, M. Vargas, M. A. Germanà and G. Moschetti, Food Control, 2016, 59, 750–758.
3. Y. Peng and Y. Li, Food Hydrocolloids, 2014, 36, 287–293.
4. A. Perdones, L. Sánchez-González, A. Chiralt and M. Vargas, Postharvest Biol. Technol., 2012, 70, 32–41.
5. H. Majeed, Y.-Y. Bian, B. Ali, A. Jamil, U. Majeed, Q. F. Khan, K. J. Iqbal, C. F. Shoemaker and Z. Fang, RSC Adv., 2015, 5, 58449–58463.
6. D. J. McClements, Soft Matter, 2011, 7, 2297–2316.
7. L. Salvia-Trujillo, M. A. Rojas-Graü, R. Soliva-Fortuny and O. Martín-Belloso, Postharvest Biol. Technol., 2015, 105, 8–16.
8. A. M. Díez-Pascual and A. L. Díez-Vicente, J. Mater. Chem. B, 2015, 3, 4458–4471.
9. F. Shakeel, G. A. Shazly, M. Raish, A. Ahmad, M. A. Kalam, N. Ali, M. A. Ansari and G. M. Elosaily, RSC Adv., 2015, 5, 105206–105217.
10. H. D. Silva, M. Â. Cerqueira and A. A. Vicente, Food Bioprocess Technol., 2011, 5, 854–867.
11. M. Jouki, F. T. Yazdi, S. A. Mortazavi and A. Koocheki, Food Hydrocolloids, 2014, 36, 9–19.
12. M. Pereda, G. Amica and N. E. Marcovich, Carbohydr. Polym., 2012, 87, 1318–1325.
13. X. Qiu, S. Chen, G. Liu and Q. Yang, Food Chem., 2014, 162, 156–160.
14. C. O. Mohan, C. N. Ravishankar, K. V. Lalitha and T. K. Srinivasa Gopal, Food Hydrocolloids, 2012, 26, 167–174.
15. S. M. Ojagh, M. Rezaei, S. H. Razavi and S. M. H. Hosseini, Food Chem., 2010, 120, 193–198.
16. R. Liang, S. Xu, C. F. Shoemaker, Y. Li, F. Zhong and Q. Huang, J. Agric. Food Chem., 2012, 60, 7548–7555.
17. Y. Li, W. Yokoyama, J. Wu, J. Ma and F. Zhong, RSC Adv., 2015, 5, 105844–105850.
18. H. S. Melito and C. R. Daubert, Annu. Rev. Food Sci. Technol., 2011, 2, 153–179.
19. A. S. D882-09, ASTM International, 2009.
20. A. S. E96/E96M-05, ASTM International, 2005.
21. Y. Cao, W. Gu, J. Zhang, Y. Chu, X. Ye, Y. Hu and J. Chen, Food Chem., 2013, 141, 1655–1660.
22. Y. Hu, J. Zhang, K. Ebitani and K. Kunihiko, Nippon Suisan Gakkaishi, 2013, 79, 219–225.
23. M. G. Volpe, F. Siano, M. Paolucci, A. Sacco, A. Sorrentino, M. Malinconico and E. Varricchio, LWT–Food Sci. Technol., 2015, 60, 615–622.
24. M. Chutia, P. Deka Bhuyan, M. G. Pathak, T. C. Sarma and P. Boruah, LWT–Food Sci. Technol., 2009, 42, 777–780.
25. J. Rao and D. J. McClements, Food Hydrocolloids, 2011, 25, 1413–1423.
26. J. Li and Q. Huang, Carbohydr. Polym., 2012, 87, 1670–1677.
27. M. A. Rao, J. Texture Stud., 1977, 8, 135–168 %@ 1745–4603.
28. L. Sánchez-González, C. González-Martínez, A. Chiralt and M. Cháfer, J. Food Eng., 2010, 98, 443–452.
29. Q. Ma, Y. Zhang, F. Critzer, P. M. Davidson, S. Zivanovic and Q. Zhong, Food Hydrocolloids, 2016, 52, 533–542 %@ 0268-0005X.
30. M. Pereda, G. Amica and N. E. Marcovich, Carbohydr. Polym., 2012, 87, 1318–1325 %@ 0144–8617.
31. J. T. Martins, M. A. Cerqueira and A. A. Vicente, Food Hydrocolloids, 2012, 27, 220–227 %@ 0268-0005X.
32. S. F. Hosseini, M. Rezaei, M. Zandi and F. Farahmandghavi, Food Hydrocolloids, 2015, 44, 172–182.
33. M. R. de Moura, L. H. C. Mattoso and V. Zucolotto, J. Food Eng., 2012, 109, 520–524.
34. Á. Perdones, M. Vargas, L. Atarés and A. Chiralt, Food Hydrocolloids, 2014, 36, 256–264.
35. A. Esmaeili and A. Asgari, Int. J. Biol. Macromol., 2015, 81, 283–290.
36. R. Yoksan, J. Jirawutthiwongchai and K. Arpo, Colloids Surf., B, 2010, 76, 292–297.
37. ICMSF, 1986.
38. N. Metoui, S. Gargouri, I. Amri, T. Fezzani, B. Jamoussi and L. Hamrouni, Nat. Prod. Res., 2015, 1–4,  DOI:10.1080/14786419.2015.1007136.
39. J. Hu, X. Wang, Z. Xiao and W. Bi, LWT–Food Sci. Technol., 2015, 63, 519–526.
40. J. Duan, G. Cherian and Y. Zhao, Food Chem., 2010, 119, 524–532.
41. V. Ocaño-Higuera, A. Maeda-Martínez, E. Marquez-Ríos, D. Canizales-Rodríguez, F. Castillo-Yáñez, E. Ruíz-Bustos, A. Graciano-Verdugo and M. Plascencia-Jatomea, Food Chem., 2011, 125, 49–54.
42. B. W. S. Souza, M. A. Cerqueira, H. c. A. Ruiz, J. T. Martins, A. Casariego, J. A. Teixeira and A. n. A. Vicente, J. Agric. Food Chem., 2010, 58, 11456–11462.
43. P. Singh, R. Shukla, B. Prakash, A. Kumar, S. Singh, P. K. Mishra and N. K. Dubey, Food Chem. Toxicol., 2010, 48, 1734–1740.

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