Biopreservation of Shrimps Using Composed Edible Coatings from Chitosan Nanoparticles and Cloves Extract

Department of Fish Processing and Biotechnology, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafr el-Sheikh, Egypt Department of Biology, College of Science and Humanitarian Studies, Shaqra University, Shaqraa, Saudi Arabia Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, El-Sadat City, Egypt Department of Aquaculture, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafr el-Sheikh, Egypt


Introduction
Chitosan (Cht) is the acquired polysaccharide from deacetylated chitin, which is the major constituent of crustacean exoskeleton [1]. Both of chitin and Cht can also exist in fungi mycelia and some insects' exoskeleton [2][3][4]. Cht has numerous astonishing bioactivities including antioxidation, metal chelation, and inhibition of cancerous cells and microbial pathogens (fungi, yeast, and bacteria) [2,3,5]. Cht was stated as effectual antibacterial materials (toward both Gram positive and negative bacteria) with approved GRAS (general recognition as safe) nature [1,3].
Cht-NPs can be prepared by multiple methods; the most frequent is the ionotropic gelation between Cht and sodium tripolyphosphate (TPP). Cht/TPP-NPs were frequently used as controlled-release drug carrier and for the effectual delivery of bioactive compounds [8][9][10][11].
e most important group of Crustacea (crustaceans) is shrimps; they belong to phylum Arthropoda and order Decapoda. ey are distributed in the whole world and live in aquatic environments [12]. Due to shrimp's high water activity value, it is a highly perishable food and susceptible to fast spoilage [13]. e quality and shelf life of shrimp during storage are influenced by oxidation, microbiological activities, and enzymatic changes, which mostly correlated with the activity and growth of Gram negative aerobic bacteria [14].
Cloves (Syzygium aromaticum, Fam. Myrtaceae) are commonly used spices worldwide. e cloves oil main properties are the antifungal, antioxidant, antibacterial, and insecticidal effects; clove is also widely used for improving flavor and as antimicrobial substance in food preservation [15,16]. Cloves are supposed to be excellent sources of minerals (manganese, calcium, and magnesium), vitamin C, dietary fiber, Ω-3 fatty acids, and vitamin K [17]. S. aromaticum extract and oil contain a significant amount of carbohydrates, proteins, calcium, iron, phosphorus, sodium, potassium, and also rich in vitamins C and A [18,19]. Cloves buds extract and oil were additionally verified as potent antimicrobial agents that could hinder growth of numerous microbial pathogens including Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Shigella flexneri, Escherichia coli, many Salmonella spp., dermatophytic fungi, and Enterobacteriaceae [15,16,[19][20][21][22]. Additionally, S. aromaticum extract was suggested for natural preservation of fisheries products (fish fingers), alone or combining with other plant extracts [23].
Edible coatings (EC) were defined as "the thin layers of materials that cover food surfaces and can be eaten and considered as a part of the whole food product" [24]; the major benefits of ECs are that they can provide additional nutrients, be consumed with the food, contain quality-enhancing antimicrobials, and enhance sensory characteristics. Edible coatings also act as barrier to external elements such as oil, moisture, and vapors, to protect food, prevent dehydration, and extend the shelf-life [24][25][26].
Although various investigations evaluated the incorporation of Cht and Cht-NPs with S. aromaticum oil, for applying as ECs to preserve foodstuffs including shrimps and pork meat [26][27][28][29], it is presumed that literature could not provide sufficient researches concerning the incorporation of cloves' whole crude extract with chitosan nanoparticles and their usage as edible coatings for shrimp biopreservation. e fortification of Cht and Cht-NPs based ECs with plant extracts was recommended to augment the antimicrobial and antioxidant attributes of the composited EC [28][29][30], but cloves extract was not sufficiently investigated as a fortifier in the Cht-NPs based ECs.
Accordingly, the synthesis of Cht-NPs and their fortification with clove extract were planned for this study. e evaluation of these agents as antibacterial blends, in vitro, and as edible coatings, and their applications for biopreserving shrimps and upholding their qualities was also planned.

Chitosan Preparation.
e extraction of Cht based on shells wastes of white prawn (Fenneropenaeus indicus), farmed in Aquaculture farm, Kafrelsheikh University. After shells cleaning and drying, they were immersed in 30 folds from 2.0 N NaOH solution (w/v, Sigma-Aldrich, MO) for 24 h; then after extensive washing with deionized water (DW), neutral materials were reimmersed in 30 folds from 2 N HCl solution (w/v, Sigma-Aldrich, MO) for 24 h, followed by extensive washing with DW. e resulting chitin was dried and immersed in 50% NaOH solution (with ratio of 1 g of chitin powder/13 ml of NaOH) and then heated in an oil bath at 125°C for 5 hours for obtaining chitosan. e molecular Cht weight was determined via CGP (chromatographic gel permeation), whereas its DD (deacetylation degree) measurement was based on Cht spectra using FTIR spectroscopic (Fourier transform infrared spectroscopy, FTS 45, Biorad, Germany).

Clove Extract Preparation.
Dried cloves buds (Syzygium aromaticum) were obtained from ARC (Agricultural Research Centre, Giza, Egypt). e pulverized plant materials (60 mesh size) were immersed in 8 folds (w/v) from ethanol (70%, Sigma-Aldrich, MO) and agitated for 22 h at 160 x g speed in room temperature (25 ± 2°C). e plant residues were excluded via filtration and the result cloves extract (CLE) was rotary evaporated (IKA, RV 10, Germany) at 44°C until dryness [31]. e dried CLE was dissolved in 2% Tween 80 solution (in DW) to concentration of 1 mg/ml, and this solution was applied in further experiments.

Chitosan/Clove Extract Nanoparticles Preparation.
For the synthesis of Cht-NPs and loading them with CLE, the following solutions were prepared according to Almutairi et al. [11]: Cht (1 mg/ml in 1% acetic acid solution), TPP, Sigma-Aldrich, MO (0.5 mg/ml in DW), and CLE (1 mg/ml of 2% Tween 80 solution in DW). e pH of Cht solution was adjusted to 5.2; then the TPP solution was slowly dropped onto Cht solution (while vigorously stirred at 620 x g) using a syringe needle at rate of 0.35 ml/min, until reaching equal volume from both solutions. For CLE/Cht-NPs formation, the previous steps were conducted with the addition of equal volume from CLE to Cht before TPP dropping. e formation of opalescent suspensions, after TPP dropping and persistent stirring for 110 min, indicated NPs formation. e formed NPs pellet was attained via centrifugation (SIGMA, 2-16 KL, Germany) at 11500 x g for 35 min, washing with DW and recentrifugation. e obtained NPs pellets were lyophilized and subjected to analysis.

Structural Analysis.
e morphological and organizational features of CLE/Cht-NPs (shape, size, and distribution) were appraised via spectroscopy (photon correlation, PCS, Malvern ™ Zetasizer, Malvern, UK), for determining the size, charge, and distribution of NPs, whereas the electron scanning microscopy (SEM, JEOL JEM IT-100, Tokyo, Japan) designated the CLE/Cht-NPs morphology and dispersion; the SEM imaging was conducted at 20 kV and x15000.

Bacteria Cultures.
ree standard food-borne pathogens were employed for antimicrobial screening, that is, Escherichia coli ATCC-25922, Salmonella typhimurium ATCC-14028, and Staphylococcus aureus ATCC-25923. e entire bacterial strains were maintained and subcultured onto the nutrient agar and broth (NA and NB, Difco Laboratories, Detroit, MI), aerobically at 37°C.

Qualitative Antimicrobial Assay.
e inhibitions zones (IZ), which appeared after disc diffusion test, were considered as indicators for antibacterial bioactivity of produced agents. Sterile Whatman No. 4 paper discs (6 mm diameter) were impregnated with 25 µL from 2% solutions of Cht-NPs, CLE, or CLE/Cht-NPs and placed onto freshly inoculated NA plate with each bacterial culture. e plates were then upturned incubated at 37°C for 18-24 h and the IZs diameters were measured using a precise caliper; then their triplicates means were calculated as ± standard deviation [3].

SEM Imaging of Treated Bacteria.
e morphological variations in S. typhimurium cell surfaces, after exposure to CLE/Cht-NPs solution (2.0%, w/v), were screened via SEM imaging, after 0, 5, and 10 h of exposure and incubation at 37°C, as formerly described [32]. e SEM micrographs were captured at 20 kV and x10000, depending on the appeared distortions in bacterial cells layouts.

Edible Coating (EC) Preparation.
For the EC solutions preparation, 1.5% Cht-NPs (w/v) was slowly dissolved in DW (at 100°C for 45 min, with stirring at 450 x g); the solution was cooled to 45°C during stirring; then 1% (v/v) acetic acid and 0.25 mL from the plasticizer glycerol per gram of Cht-NPs were added [33]. After stirring for additional 30 minute (during which the temperature of the coating solution decreased to approximately 37°C), clove extract dispersed in 2% Tween 80 solution was added to the EC solution by gradual concentrations (w/v).

Application of Coatings on Peeled Shrimp.
Freshly harvested shrimps (F. indicus), after manual deheading, deshelling, and cleaning with DW, were divided into five groups (each of them consisted of 25 shrimps with weight of∼10 ± 1 g/shrimp). e first group was the uncoated control (C), and the other 4 groups of shrimp were immersed into Cht-NPs based EC solutions incorporated with clove extract (CLE) with the following order: group (Ch) contained Cht-NPs only and groups Ch-Cl 0.5, Ch-Cl 1.0, and Ch-Cl 1.5 were fortified with 0.5, 1.0, and 1.5% (w/v) from CLE, respectively. e shrimps coating was performed through dipping of samples in EC solutions (for 30 min at 4°C), at 1 : 2 ratio (w/v) from shrimp/EC solution; then coated shrimps were drained for 5 min at 25 ± 2°C. Samples were then packaged in polystyrene trays and each tray was wrapped with plastic films and held at 4 ± 1°C for 10 days and at RH (relative humidity) of 65 ± 3% and sampled at 2 days intervals for further analyses [34,35].

Sensory Analysis.
A well-trained panelist team (13 members; 8 females and 5 males), with experience in seafood judgments, was involved in evaluating the sensory attributes of EC-treated shrimps. e panelists were inquired to assess the samples appearance, odor, color, and texture, using a ranged hedonic scale from 1 (extremely poor) to 9 (extremely good) [31].

Statistical Analysis.
e entire experiments were triplicated, with presented data as means ± SD. e SPSS package (SPSS V-11.5, Chicago, IL, USA) was applied for statistically analyzed data using t-test and one-way ANOVA at p < 0.05.

Results and Discussion
Chitosan was efficaciously extracted from shrimp wastes; the produced Cht powder had a creamy-white color, a 38.3 kDa molecular weight (MW), and a DD of 89.4%. e DD of Journal of Food Quality ≥70% and the MW of produced Cht confirmed the transformation of shrimp chitin to low MW chitosan [40].

Physiognomies of Synthesized Clove Extract/Chitosan Nanoparticles
is an important characteristic parameter for nanosuspensions [41]. Particles size distribution of synthesized clove extract/chitosan nanoparticles is shown in Figure 1 Morphology and microstructure of CLE/Cht-NPs were evaluated by SEM (Figure 1(b)). Electron micrograph of the sample demonstrates presence of spherical structures, absence of cracks, and creation of a continuous layer onto NPs surfaces. e SEM image for clove extract chitosan nanoparticles illustrates spherical shape and regular distribution, which seems separated and well stable during the preparation process [42,43].

Fourier Transform Infrared Spectroscopy.
e FTIR spectra of Cht-NPs and composited CLE/Cht-NPs are given in  [7,44]. Also, in Cht-NPs spectrum, the band at 3450 cm −1 , which has lower wideness than bulk Cht indicates reduced hydrogen bonding. e reduced intensity of hydrogen bonding in the cross-linked NPs complexes is owing to more open structure caused from crosslinking with TPP [44].
A new sharp peak at 1625 cm −1 appeared, the amine bending intensity at 1625 cm −1 goes down, which assumingly attributed to linkage between TPP and the ammonium group of Cht-NPs [9,10]. FTIR analysis of combined CLE with Cht-NPs showed a band at 3450 cm −1 , which could indicate the reduced hydrogen bonding in Cht-NPs and/or the O−H groups of alcoholic CLE. Another band at 1687.5 cm −1 represented ester group frequency patterns C−O or the aromatic ketone group C�O that combines with more than ring. At the wavenumber of 1562.5 cm −1 , the detected band designates matched patterns to the aromatic carbonyl group belonging to quinine [45], which is close to the aromatic group (C�C). e bands at 780 cm −1 and 1330 cm −1 could represent the CH 2 group frequency pattern [46].

Antibacterial Activity of CLE/Cht-NPs against Food-
Borne Pathogens 3.2.1. Qualitative Assay. e antibacterial activities of produced natural products, i.e., clove extract (CLE), nanochitosan (Cht-NPs), and clove extract with nanochitosan (CLE/Cht-NPs), expressed as IZ diameters, are shown in (Table 1). e antibacterial activities, toward the entire food-borne pathogens, were significantly more forceful from CLE/Cht-NPs and then from Cht-NPs and CLE, respectively. S. typhimurium was the most susceptible strain to screened compounds compared to E. coli and S. aureus, as evidenced from the wider IZs. Larger IZ typically indicates higher antibacterial activity of tested solutions against challenged microbial species [20].
e Cht-NPs antimicrobial potentialities were recurrently confirmed from many investigations [4,6,9,44]; they indicated that Cht-NPs had more forceful action than native Cht for inhibiting numerous pathogenic microorganisms.
e Cht-NPs antibacterial action was principally attributed to their increased positive charges, which enable them to interact with microbial cells' surface and interior vital components (DNA, RNA, enzymes, etc.) and thus prohibit microbial growth and survival. e alcoholic CLE contains plenty of bioactive compounds belonging to revealed existence of flavonoids, tannins, alkaloids, glycoside, steroids, terpenoids, ketones, aldehydes, and phenolics; more than 46 phenolic constituents were detected and quantified in CLE [22,23], several compounds from these phytochemicals possess antimicrobial potentiality. Additionally, the high CLE content from tannins (10-19%) was also verified to augment its antimicrobial activity [21]. e major detected phytoconstituents in CLE and oil are eugenol, a-and ß-caryophyllene, eugenyl acetate, limonene, a-copaene, and a-terpinolene [47]. Eugenol and caryophyllene (the highest occurring constituents in CLE and oil) were verified to possess strong antimicrobial (antibacterial, antimycotic, and antifungal) properties toward numerous pathogenic species [17]; the crude ethanolic CLE exhibited also comparable antimicrobial powers against several food-borne strains as the essential oil [16]. e synergistic action of the composited Cht-NPs and CLE was stronger than each of the individual agents; this is because the microbial pathogen cannot resist a composite from different antimicrobial agents with diverse action modes [9,31].

SEM Detection of Morphological Alterations in CLE/ Cht-NPs-Treated Bacteria.
e SEM image shows how bacterial pathogen (S. typhimurium) was influenced by CLE/ Cht-NPs treatment (Figure 3). At zero time of treatment, there is no apparent change in bacterial cells wall/membrane; cells appeared with regular shapes, smooth and uniformed surfaces (Figure 3(c)). After 5 h of CLE/Cht-NPs exposure, S. typhimurium cell appeared with swollen and partially lyses manifestation (Figure 3, 5 h), many NPs were appeared to attach/interact with exposed bacterial cells. With prolongation of CLE/Cht-NPs exposure to10 h, most of bacterial cells were severely damaged; the lyses/explosion signs in their walls became obvious, the interior cellular components were released and composited with NPs (Figure 3, 10 h). e CLE/Cht-NPs composite had powerful activity to deteriorate S. typhimurium cells; this is assumed to result from the combined bioactivities of CLE and Cht-NPs. e bioactivity of eugenol (the major component of CLE) was stated to enable it for deteriorating bacterial membrane through serial mechanisms that alter cell morphology, prompt leakage of cellular constituents, and increase permeability [48].
In addition, the electrostatic interaction between the positively charged Cht and negatively charged S. typhimurium walls/membranes could increase Cht-NPs attachment to bacteria and their penetration into their interior organelles; thus they can severely alter the negative-charged bacterial cell walls/membranes (generated from their constituents of lipopolysaccharide and teichoic acid) [49], and interfere with cells' vital functions and components to stop them, hinder cells progress, and disrupt their outer membranes [1,3,6,50]. e bactericidal actions of Cht, resulting from its adsorption onto microbial surfaces and induction of cell walls' lyses, were described and stated [3,9,31,51]; the intensity of absorbed particles depended on Cht concentration, its MW, contact time, challenged microorganisms, and the charges on their cells. Many of preceding investigations stated the disruption/explosion of microbial cells as a consequence after their exposure to Cht [1][2][3]. e wide antimicrobial spectrum (antibacterial, antimycotic, and antifungal) from S. aromaticum oil, crude extract, and their constituents, was indicated [15,52]; they suggested that S. aromaticum antimicrobial actions could include the interaction with microbial cells, proteins' denaturation and reactions with phospholipids in cell membranes, which suggested to affect microbial membranes' permeability.
e treated cells of pathogenic Gram-negative bacteria (S. enteritidis and E. coli) with ethanolic CLE appeared with uniform, coarse surfaces, and withered and irregular morphology, with formed adhesions and aggregations, which suggest that CLE led to severe damages in bacterial cell membranes [53]. with CLE at percentages of 0.5, 1.0, and 1.5% (w/v), on the microbial load during refrigerated storage at 4 ± 1°C, are illustrated ( Figure 4). While all examined microbial groups (Total aerobic microorganisms, E. coli, Enterobacteriaceae and coagulase-positive staphylococci) tended to sharply increase, in control (uncoated) groups, with storage time prolongation, the inhibitory effects of ECs on these microbial groups were very remarkable. e ECs formulation could significantly reduce the microbial loads in treated shrimps, compared with uncoated control samples. Generally, the EC antimicrobial efficacy increased with CLE percentage increment; the most effectual EC mixture contained 1.5% w/v from Cht-NPs with 1.0 or 1.5% from CLE. ese combinations could reduce the counts of E. coli and coagulase-positive staphylococci to zero after 4 and 6 days of cold storage, respectively. e Cht-NPs based EC was remarkably effective in hindering microbial growth, and its efficacy notably increased with CLE fortification. e strong antimicrobial potentialities (of both Cht and Cht-NPs) were proved and confirmed against numerous species from bacteria (Gram positives and negatives), fungi, and yeasts [1-3, 6, 9, 49, 51], either using in vitro evaluation or in food models [5,24,32,33,54]. ese antimicrobial potentialities of Cht were attributed to its superficial bioactive positive charges, high chelation, and antioxidant capabilities, and its strong interactions with microbial cells components.

Biopreservation of Shrimp through CLE/Cht-NPs Edible
e Cht-NPs were reported to have extra bioactivities and powers than bulk Cht due to their interacting surface area and their minute size that lead to more effectual interactions with microbial cells [4,6,8,43]. e microbial inhibitory action of Cht-NPs coating, especially toward aerobic microorganisms, is assumingly attributed to oxygen parrying capability of Cht coating films that prevents the essential O 2 penetration for microbial breathing [23,24,33]. e antimicrobial consequences generated from S. aromaticum extract and oil were recurrently stated toward wide pathogenic microorganisms varieties, especially foodborne species [15,16,19,22,47]; they mainly attributed these microbicidal impacts to CLE and oil contents from bioactive constituents, particularly eugenol, caryophyllene, and other phenolics. e high amounts from TPC (total phenolic compounds) in CLE were suggested as the main reason for its influential antimicrobial activity [47,53]. e combined synergistic antimicrobial effect of CLE and Cht-NPs persisted for the duration of storage time, and that was illustrated for many formulated EC from Cht and other plant extracts [24,28,34,35,55].

Impact on Sensorial Quality of Stored Shrimps.
e influences of shrimp coating with 1.5% solution from Cht-NPs and their fortified blends with CLE (at 0.5, 1.0 and 1.5% concentrations), on the sensorial attributes after refrigerated storage (4 ± 1°C) for 7 days, are presented in Figure 5.
Whereas, in the beginning of the EC experiment, no significant differences were observed for the examined sensory attributes of all samples (data not included), the panelists scores for the examined attributes (appearance, odor, color, and texture) revealed that the control (uncoated) sampled became inconsumable after this time, regarding the acceptance level of 5/9 as the limiting value. e entire sensorial scores for the control group were significantly much lower than EC treated samples. e best composition of EC blends, to preserve appearance, odor, and color of stored shrimps, contained 1.5% from Cht/NPs and 1.0% from CLE, whereas the best for texture attribute contained 1.5% from both agents. Table 1: Antibacterial activity of produced natural products; clove extract (CLE), nanochitosan (Cht-NPs), and their composites (CLE/Cht-NPs) against examined food-borne pathogens.

Antibacterial agent
Inhibition zone (mm) * Salmonella typhimurium Staphylococcus aureus E. coli CLE 8.9 ± 0.4 a 8.4 ± 0.6 a 8.7 ± 0.7 a Cht-NPs 9.3 ± 0.3 a 8.8 ± 0.5 a 9.0 ± 0.6 a CLE/Cht-NPs 12.7 ± 0.9 b 12.1 ± 0.8 b 11.4 ± 1.1 b * Results represent means of triplicates ± SD. * * Different superscript letters in the same column indicate significant difference at p < 0.05.  Journal of Food Quality e quality upholding of appearance, color, and texture attributes, in EC treated shrimps, was notably evidenced, compared with non-EC (control) shrimps ( Figure 6). Many health benefits from seafood are principally attributed to their elevated contents from beneficial lipids, especially omega 3 and long-chain PUFA (poly unsaturated fatty acids) [56]. But, these valued constituents in seafood are extremely susceptible to oxidation (resulting from autoxidation, photosensitized oxidation, peroxidation, lipoxygenase, or microsomal enzymes), which lead to off-flavors emergence, at any storage conditions. e application of Cht-NPs and CLE-Cht-NPs based ECs for shrimps biopreservation led to sensory quality maintenance through the protection from oxidation. Cht was reported as powerful antioxidant material and chelating agent; this is assumed to make it able to bind with oxidation enzymes and inhibit their effects [32]. e oxygen parrying effect of Cht-NPs-based EC also protected shrimps from oxidative reactions that regularly happened [23,24,32,35,55]. Additionally, the efficacy of Cht to control lipids oxidation in cooked cod (Gadus morhua) was attributed to the influential capability of it for metal chelation [57]. e sensorial quality maintenance, in CLE/Cht-NPs coated shrimps, is noticeably correlated with their lowered microbial load after EC treatments, which is assumed to
Many investigations recommended the application of Cht coatings and their blends with plant extracts/oils for shrimp biopreservation [24,28,34,35,55]. e achieved results here, for the microbiological and sensorial qualities, auspiciously exceeded those reported in these studies, which may be because of the usage of Cht-NPs (with higher antimicrobial and antioxidant activity than bulk Cht) in EC blends and the effectiveness of CLE for strengthen the NPs bioactivities. e CLE and oils convincingly stated to possess numerous phytoconstituents with high antimicrobial and antioxidant potentialities [16,17,19,22,47,53]; these bioactivities are proved here to protect stored shrimps from spoilage signs and to augment the activity of Cht-NPs based coatings to uphold the shrimps' qualities during cold storage.

Conclusion
Toward the elimination of pathogenic food-borne bacterial pathogens and preserving the microbiological and sensorial qualities of stored shrimps, the bioactive formulations from nanochitosan (Cht-NPs) and cloves buds extract (CLE) were innovatively composited. e structural physiognomies of CLE/Cht-NPs were proved and their remarkable antimicrobial potentialities against bacterial pathogens were confirmed, along with their potential action modes. e coating treatment of unpeeled shrimps with CLE/Cht-NPs solutions resulted in sharp decrease in the microbial loads and the upholding of stored shrimps sensorial qualities during cold storage for 7 days. e CLE/Cht-NPs could be impressively recommended as effectual natural composites for the biopreservation of shrimp during cold storage.
Future investigations are suggested for further appraisal of CLE/Cht-NPs biopreservation actions in different foodstuffs and their biomedical consequences.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.