Solvent Free-Microwave Green Extraction of Essential Oil from Orange Peel 1 ( Citrus sinensis L . ) : Effects on Shelf Life of Flavored Liquid Whole Eggs during Storage 2 under Commercial Retail Conditions

1 (Citrus sinensis L.): Effects on Shelf Life of Flavored Liquid Whole Eggs during Storage 2 under Commercial Retail Conditions 3 4 Malek Aboudaou1,2, Mohamed Amine Ferhat3, Mohamed Hazzit2, Agustín Ariño4, Djamel 5 Djenane1 6 7 1Laboratory of Food Quality and Food Safety. University Mouloud MAMMERI, P.O. Box 17 RP. Tizi-Ouzou, 8 15000. Algeria. 9 2Department of Food Science and Technology. National Institute of Agronomy, avenue Hassan Badi El-Harrach, 10 Alger, 16200. Algeria. 11 3Laboratory of Bioactive Substances and Valorization of Biomasse, Ecole Normale Supérieure, Vieux-Kouba, P.O. 12 Box 92. Alger, 16050. Algeria. 13 4Department of Animal Production and Food Science, Laboratory of Food Quality and Food Safety. Faculty of 14 Veterinary Sciences. University of Zaragoza, C/Miguel Servet, 177, 50013, Zaragoza, Spain 15 16 *Corresponding author: E-mail: djenane6@yahoo.es, Tlf.: (+213) 779 001 384; Fax.: (+213) 26 18 61 37 17 orcid.org/0000-0003-4050-4329 18 19 20 Abstract: A possible way to valorize citrus peels, which are byproducts of the juice 21 extraction industry, is to use them as natural biopreservatives. In this paper we present early 22 results from a compared Solvent Free Microwave Extraction (SFME) with Hydro-Distillation 23 (HD) and Cold Pressing (CP) of essential oils (EOs) using fresh orange peel (Citrus sinensis 24 L. var. Valencia late), a by-product in the production of orange juice in Algeria. The EOs 25 were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). All extracted 26 C. sinensis EOs were chemotype limonene (94.64 to 95.48%). SFME is performed without 27 added any solvent or water. SFME increases EO yield and eliminate wastewater treatment, 28 resulting in a great progress in terms of time and cost efficiency. In its second part, the present 29 study was conducted to evaluate “in vitro”, the antioxidant activities of Solvent Free 30 Microwave (SFM) extracted orange EO by using the DPPH• (2,2-di-phenyl-1-picrilhydrazyl) 31 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 January 2018 doi:10.20944/preprints201801.0055.v1


Introduction
In terms of international trade of orange fruits, the main citrus-producing countries, such as Brazil, the United States, China, India, Spain, Mexico, Egypt and Turkey [1] were produced in 2014, 140 Mt of these fruits, about 60% of world production.Algerian production in orange fruits estimated at 571 000 tons/year, which is the 3 rd in the Arab Maghreb Union and 19 th producers in the world [2].The climatic conditions of Algeria are favorable for the production of most citrus fruits.
Citrus by-product resulting from the citrus processing represents a source of various bioactive molecules such as EOs [3].Interest in taking profit from waste products of the juice industry, permit to minimize adverse effects of by-products in the environment.Generally, the pharmaceutical and food industries are using citrus oils as flavoring for masking other added ingredients in abundant quantity in the processed product.
Worldwide, consumers' enthusiasm for natural products and increasingly severe legislations in respect of synthetic substances, EOs can be a good alternative for the food industry.The citrus EOs and their constituents are suitable for use in foods as biological agents or for their flavor, in pharmaceutical industries for its anti-inflammatory and antimicrobial properties.In addition, great amount of these oils is also used in cosmetic industries.
During the last few years, a great expectation for green chemistry was recorded by various industries.Environmentally friendly and low cost ecological extraction techniques are becoming more attractive.Therefore, in recent years, conventional techniques, such as HD, tend to be replaced by various novel extraction techniques, such as microwave-assisted extraction (MAE).This technique is largely focused on finding technological solutions to diminish or even prevent the use of organic solvents in extraction processes to obtain more products with higher added value.Meanwhile, this technique is considered a novel with better extraction yields, decreased extraction times and prevents the degradation of thermolabile compounds and thus prevents the antioxidant activity loss of the plant extract [4].
Recently, LWE is preferred for food industry and for households for his easy and commode use.According to its biological nature, is an easily perishable animal food under conventional cooled storage, its shelf life is limited due to the oxidation and microbial development.Both considered a major concern for food technologists due to the loss of quality associated with those processes [5].The citrus EOs represents a significant source of bioactive compounds with potential prophylactic properties for the development of functional foods [6].Generally, to destroy microorganisms in liquid egg products, heat treatments were commonly employed that can improve microbial safety and increase shelf life of products, but can have negative effects on the functional properties of egg proteins, which results in technologically unattractive products [7].In this scenario, the search for new strategies and new biopreservative agents for stabilization of liquid eggs has become a central goal for food industry.EOs can be an interesting alternative to improve traditional heat treatments to reduce their intensity and therefore reduce their adverse effects on food quality.To the best of our knowledge, there is no published report regarding the biological effectiveness of SFM orange peel EO in egg products.The aims of this study were (i) to describe a fast solvent free microwave extraction of orange EO, (ii) to assess "in vitro" its antioxidant activity (DPPH method) of extracted EO, (iii) The application of this EO in a food matrix for biopreservative purposes.

Collection of fresh peel orange fruit
Fresh oranges, variety Valencia (Citrus sinensis L.) were collected from healthy trees in full bloom during sunny days at the end of January of 2015, in province of Metidja (Algeria).
The whole fresh fruits were then extensively washed with distilled water (20 °C).Only the fresh orange peels were recovered for subsequent extraction.Citrus fruits were peeled to separate the external part of the orange (flavedo), giving a yield of  15% (w/w) of the peel with respect to the whole fruit.

List of chemicals and reagents
All reagents and solvents used in the extractions were of analytical grade.All chemicals were purchased from represented Algerian Chemical Society (Sigma-Aldrich, Chemistry-GmbH, Germany; Biochem, Chemopharma, Spain; Merck; Darmstadt, Germany).

Essential oils extraction
SFME has been performed using the "DryDist" microwave oven illustrated in Figure 1.
In a typical microwave accelerated distillation (MAD) procedure performed at atmospheric pressure, 200 g of fresh orange peels were heated using a fixed power of 200 W for 10 minutes.The extraction was continued at 100 °C until no more EO was obtained.SFME is based on a relatively simple principle; this method involves placing plant material in a microwave reactor, without any added solvent or water.The internal heating of the in situ water within the plant material distends the plant cells and leads to rupture of the glands and oleiferous receptacles.This process thus frees EO which is evaporated by the in situ water of the plant material.A cooling system outside the microwave oven condensed the distillate continuously.The excess of water was refluxed to the extraction vessel in order to restore the in situ water to the plant material.For comparison, 200 g of fresh orange peels were submitted to HD with a Clevenger-type apparatus according to the European Pharmacopoeia and extracted with 2 L of water for 3 hours (until no more EO was obtained).Another comparison has been made by CP procedure.EO is collected from 1 kg of whole orange fruit using an automated cold pressing machine.The epidermis and oil glands were lacerate by a needle, creating areas of compression in the peel, surrounded by areas of lower pressure, across which the oil flows to the exterior.The oil is carried down to a decantation vessel in a stream of water, the emulsion being collected and then separated by centrifugation.The orange peel extractions provide a liquid limpid, mobile EO of yellowish color with a very strong and persistent odor of orange.Its density at 20 ºC was  0.82 g/mL, its refraction index at 20 ºC was  1.49, while its boiling point was  52 ºC.The EOs were collected and stored in darkness in opaque sealed vials at 4 °C until further analysis and use in bioassays.

GC-FID and GC-MS analysis
The EOs were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) (Hewlett-Packard computerized system comprising a 6890 gas chromatograph coupled to a 5973A mass spectrometer) using two fused-silica-capillary columns with different stationary phases.The non-polar column was HP5MS  (30 m  0.25 mm  0.25 m film thickness) and the polar one was a Stabilwax  consisting of Carbowax  -PEG (60 m  0.2 mm  0.25 mm film thickness).GC-MS spectra were obtained using the following conditions: carrier gas He; flow rate 0.3 mL/min; split-less mode; injection volume 1 L; injection temperature 250 ºC; the oven temperature programme was 60 ºC for 8 min increased at 2 ºC/min to 250 ºC and held at 250 ºC for 5 min; the ionisation mode used was electronic impact at 70 eV.The relative percentage of the components was calculated from GC-FID peak areas.Most constituents were tentatively identified by comparison of their GC Kovats retention indices (RI), determined with reference to an homologous series of C5-C28 n-alkanes and with those of authentic standards available in the authors' laboratory.Identification was confirmed when possible by comparison of their mass spectral fragmentation patterns with those stored in the MS database (National Institute of Standards and Technology and Wiley libraries) and with mass spectra literature data [8].Then listed according to Kovat's retention index calculated in GC on apolar HP-5MS column.

DPPH radical-scavenging activity
The ability of the SFM extracted EO to scavenge 1,1-diphenyl-2 picrylhydrazyl Radical (DPPH•) was estimated.Pure EO was dissolved in 5% (vol/vol) Dimethyl Sulfoxide (DMSO), and then EO dilutions were made to obtain different concentrations (50 to 450 g/mL).Aliquots 4 ml of DPPH (100 g/L) methanol solution was taken in a test tube, and then mixed well with 4 ml of EO DMSO solution.Then, the mixture was vortexed using a vortex mixer (Cyclo-mixer) and incubated in the dark at ambient temperature (25 ± 1 ºC) for 30 min.
Decreasing of absorbance of tested mixtures was monitored every 1 min for 30 min at 517 nm using a Perkin-Elmer Lambda 25 UV/Vis spectrophotometer.The absorbance was read against pure methanol at 517 nm and the percentage of DPPH radical scavenging activity (RSA) was calculated using the following equation: RSA (%) = [(Abs (DPPH) -Abs (EO) )/Abs (DPPH)]  100 (1) where Abs(DPPH) is the absorbance value at 517 nm of the methanolic solution of DPPH and Abs(EO) is the absorbance value at 517 nm for the EO extracts.
The RSA was also expressed as the IC50 value (g/mL), the concentration required to cause 50% of DPPH inhibition.The percentage of scavenged DPPH• was plotted against the EO extract concentration, and that required to quench 50% of initial DPPH radical was obtained from the graph by linear regression.A lower IC50 value indicates greater antioxidant activity.Synthetic antioxidant reagent butylated hyroxytoluene (BHT) was used as the positive control.To standardize DPPH results, the antioxidant activity index (AAI), proposed by Scherer and Godoy [9] was calculated as follows Eq. ( 4): AAI = DPPH in reaction mixture (g/mL)/IC 50 (g/mL) (2) Studied EO was classified as showing poor antioxidant activity when AAI < 0.5, moderate antioxidant activity when 0.5 < AAI < 1.0, strong antioxidant activity when 1.0 < AAI < 2.0, and very strong when AAI > 2.0.

Biopreservative effects in LWE 2.6.1. Preparation of LWE
Eggs were surface-disinfected by immersion for 1 min in a dilute solution of ethanol (70%), washed twice by immersion in distilled and sterile water, and left in a dry place to remove excess water on the surface until using for "in vivo" assays.Just before experiments were carried out, the eggs content (separately, egg whites and egg yolks) were removed under aseptic conditions, and collected in sterile containers.When albumen was separated from yolk, care was taken to ensure that albumen was not contaminated by the yolk content.The chalaza was removed and the separated egg fractions were then homogenised for 2 min at 4000 rpm, using a commercial blender (31BL44, Waring, USA).To prepare 100 mL of LWE samples, 33.25 mL of egg yolk were mixed with 66.75 mL of egg white; this proportion is the normal average composition of whole egg and was used to attain a constant white to yolk relationship.The proportion of albumen and yolk greatly affects processing characteristics of LWE products and this proportion is mainly characterized by different moisture percentages, total solids content and fat content [10,11].
The total LWE obtained were centrifuged at 103  g for 2 min using a Heraeus Megafuge 1.0R to eliminate any residual air.EO was added directly into LWE at a final concentration between 0.1-0.5%.All samples were placed under continuous lighting (1000 lux) in display cases at 4 ± 1 °C for 8 days, simulating commercial retail/display conditions.
The positions of the samples in display cases ( 80 cm under fluorescent tube) were rotated to minimize abuse light and temperature intensities at the surface of product.
Fat contents are analyzed according to the method of Folch et al. [12].The pH of the LWE was measured using a micro pH-meter model 2001 (Crison Instruments, Barcelona, Spain) after homogenizing 3 g of the product in 27 mL of distilled water for 10 s at 1300 rpm using a Ultra-Turrax T25 macerator (Janke & Kunkel, Staufen, Germany).

Thiobarbituric Acid Assay
To measure the potential antioxidant capacity of studied EO and to evaluate the extension of the lipid oxidation on the LWE samples, the determination of the amount of the formed 2-thiobarbituric acid-reactive substances (TBARS) was undertaken, according to protocol developed by Djenane et al. [13].The results were expressed as mg of MDA/kg of LWE and calculated using a standard curve prepared with 1,1,3,3-tetramethoxypropane.The Percentage inhibition rate (IR%) respect to the control was calculated as follows: Where C is the number of TBA-RS in the untreated samples (LWE prepared without EO addition: control) and T is the number of TBA-RS in the treated samples.

Psychrotrophic bacteria analysis in LWE
A sample of 10 g was removed aseptically and transferred to a stomacher bag containing 90 mL of sterile peptone water (PW) solution (0.1%), and was homogenized at room temperature.Further serial decimal dilutions were prepared in PW solution (0.1%).The appropriate dilutions were subsequently used for enumeration of microorganisms.Total Psychrotrophic bacteria counts were determined using plate count agar (PCA) after incubation at 7 ºC for 10 days [14].The log10 CFU/mL of mean values for the counts were recorded.
Scores for "off-odor" referred to the intensity of odors associated to product spoilage: 1 = none; 2 = slight; 3 = small; 4 = moderate; and 5 = extreme.A score of 3 or higher ( 3) in both attributes denoted that LWE was unacceptable for sale or consumption.

Instrumental color evaluation
The color profile was measured using color space coordinates CIE (L*, a*, and b*) put on the surface of the LWE emulsion at six different points for each Petri dishes.The mean (n = 18) and standard error for each parameter were estimated.

LWE Shelf life extension
For shelf life study, chemical guidelines were used following the recommendations of Djenane et al. [5] for animal foods: TBA-RS value: limit ~ 2 mg MDA/kg.The additional microbial guidelines were also used following the recommendations of Garcia-Gonzalez et al.
[16] for whole egg: ~ 6 log10 CFU/mL for total aerobic psychrotrophic counts.are presented as the means of three independent experiments ± standard deviation with three replicates.Student's t-test was used to compare the efficacy of EO treatment and to determine any significant differences among the treatments at a 95% confidence interval (p < 0.05).

Kinetics extraction yield
The choice of the technique is the result of a compromise between efficiency and reproducibility of extraction, ease of procedure, together with considerations of cost, time, the degree of automation and safety.These shortcomings have led to the consideration of the use of new "clean" technique in EO extraction, which typically use less solvent and energy.The yields obtained by the both methods, SFME and HD, are similar but the difference is related to the extraction time.With SFME, 10 min provides yields comparable to those obtained after 3 h by HD.The values obtained are respectively 0.40% for both methods (SFME and HD), and 0.16% for CP (Table 1).The SFME is clearly quicker than its conventional counterparts.
The extraction takes 10 min., whilst 1 hour and 3 hours were required by CP and HD, respectively.For the case of CP, an additional time of centrifugation (30 min.) of the emulsion containing EO could be added to the real extraction time (1 h.).For HD or SFME, the extraction temperature is equal to water boiling temperature at atmospheric pressure (100 °C).To reach this extraction temperature (100 °C) and thus obtain the distillation of the first EO droplet, it is necessary to heat only 2 min.with SFME against 30-40 min.for HD.From point of view early formation of first droplets, the first EO droplets were observed after 3.0 min in SFME and 23.0 min in HD.Heat transfer within the samples was the most important factor for theses differences [17].The rapid increase in temperature in the case of SFME extraction is the main reason for this time reduction [18].The obtained yield of peel EO from C. sinensis was 1.89% [19].This is not in agreement with Minh Tu et al. [20] who reported a yield of 0.13%.Moreover, it has to be highlighted that one of the highest yields obtained was that in the case of Valencia Late oranges (2.3%) [21].A yield of 0.79% has been recorded for Colombian orange peel oils [22].Fresh peels of the Valencia late cultivar from Algeria yielded 0.39% of EOs [23].EOs were extracted from epicarp (waste product from India) of C. sinensis (L.) Osbeck by hydro-distillation yielded 1.8% of EO [24].Boukroufa et al. [25] found that the EO yields obtained from orange peel by Microwave Hydrodiffusion and Gravity (MHG) and steam distillation (SD) processes are comparable but a difference is only observed in the extraction time.Indeed, 15 min of extraction with this process are sufficient to extract totality from oil whereas it takes 240 min with SD, which is the one of the reference methods in EO extraction (gain of time of more than 93%).Advantages in terms of reducing time due to microwaves were also reported by other studies such as Ferhat et al. [26] for orange peel EO (12 min with microwave steam diffusion (MSD) vs. 40 min for steam diffusion (SD), Sahraoui et al. [27] for Citrus EO extraction using MSD (3 h with SD vs. 6 min with MSD), Perino-Issartier et al. [28] for lavender EO (15 min with MHG vs. 120 min with SD), and Sahraoui et al. [29] for EO extraction from lavender with MSD (6 min vs. 40 min for SD).The SFME was also reported to be the best compared to conventional HD for extracting EO from Citrus grandis peels [30].Luciardi et al. [31] found that mandarin EOs obtained by CP/SD contains more oxygenated monoterpenes and sesquiterpenes fractions than mandarin EOs obtained by CP.Probably, the SD, could remove the oils that remains in the peel and effluent after pressing and separation.
SFME is proposed an "environmentally friendly" extraction method.SFME is a very clean method, which avoids residue generation (vs.CP) and the use of large quantity of water and voluminous extraction vessels (vs.HD).The reduced cost of extraction is clearly advantageous for the proposed SFME method in terms of energy and time.Regarding environmental impact, the calculated quantity of carbon dioxide (CO2) rejected in the atmosphere is higher in the case of HD (3464 g CO2/ g of EO) than for SFME (70 g CO2/ g of EO).These calculations have been made according to literature: to obtain 1 kW h from coal or fuel, 800 g of CO2 will be rejected in the atmosphere during combustion of fossil fuel [32,33].Water is a polar solvent, which accelerates many reactions, especially reactions via carbocation as intermediates.Linalool was the main oxygenated component in the EO extracted from orange peels but the relative amounts differed for the three extraction methods.

Orange EOs composition
Evaluating the results of the present study with those on orange EO, the percentages of the majority of the identified compounds in the orange species EOs differ in the literature.It is therefore possible that genetic differences within species determine the expression of different metabolic pathways regardless of geographic location [34].The EOs of orange peels isolated by SFME, HD and CP are rather similar in their composition.Limonene is the main component with 94.64% for SFME, 95.48% for SD and 95.06% for CP.It is clear that microwave methods greatly accelerates the extraction process, but without causing considerable changes in the volatile oil composition.Bustamante et al. [35] found that no remarkable difference was observed in the composition of orange peel EOs extracted by HD and Microwave Assisted Hydrodistilation (MAHD).D-limonene was the most abundant chemical in both extracted EOs (96.75 and 97.38% for HD and MAHD, respectively).The yields for both samples were similar.In terms of energy consumption, the same authors found that a significant reduction was observed with comparing conventional HD to the microwave extraction (3.2 and 0.5 kWh, respectively), resulting in a great progress in terms of cost efficiency.A microwave heating causes a superheating phenomenon which facilitates the distension of the plant cells and leads to liberate the EO more quickly than in the case of conventional extraction methods [27].
However, it is worthy to note that limonene which was reported as the main component in C. sinensis EO was found in our samples.Previous studies regarding the EO of C. sinensis from different regions of the world were focused on the peel oil composition of val.cultivar.

DPPH scavenging capacity
The activity of the C. sinensis EO was found to be dose dependent and the DPPH scavenging capacity of the tested oil augmented by increasing the concentration of EO and the inhibition ranged from 21 to 81% according to the tested concentrations (Figure 3).The lowest IC50 value (highest antioxidant activity) of 89.25 g/L was obtained ( times greater than the value found for the reference compound.;BHT that was used as positive control (IC50 = 11.37 g/mL).Accordingly to the categories defined by Scherer and Godoy [9], C. sinensis EO tested in the present study presented strong antioxidant activity, when looking to its values of AAI = 1.12 g/mL.The aptitude of orange EO to scavenge free radicals could be attributed to the phenolic constituents as monoterpenes are responsible for the overall reactivity of the orange oil towards DPPH radical.Our findings are similar to those reported previously by Singh et al. [37] who examined the antioxidant activity of orange EO using the DPPH way.The relationship among the antioxidant activity and their chemical profiles was previously reported.Thus, the intensity of the antioxidant activity for the studied specie may be related mainly to the major components which are limonene (94.64-95.48%),and -myrcene (1.64-1.87%).Even the cited compounds could explain in a part the elevated antioxidant activities for this specie; it is difficult to attribute the antioxidant effect of the whole EO to one or few active compounds.Both minor and major compounds should make a significant role to the oil's activity which is the interaction result of their chemical composition.Given that limonene is the main compound of orange EO, our results confirm the study by Bacanli et al. [38] and Fancello et al. [39] which describes the antioxidant activity of this compound.In the same order of ideas, Junior et al. [40] and Singh et al. [37] found that the antioxidant nature of the EOs in terms of free radical scavenger may be due also to DL-limonene mostly present in the citrus EO.

Application in LWE
The pH values of non-treated and treated LWE were not significantly modified during chilled display (Results not shown, p > 0.05).Rossi et al. [41] and Monfort et al. [42] reported mean values of 7.5 and 7.64, respectively for raw LWE.The lipid analysis of LWE showed

Lipid oxidation (TBARS-RS)
It would seem interesting to say that EOs which are able of scavenging free radicals may play an important role in food processing industry as natural additives to replace synthetic antioxidants with natural ones.Synthetic antioxidants have successfully been used to prevent lipid oxidation in animal food products but latest statement on health declares of these synthetic chemicals have necessitated research on alternative successful compounds particularly from natural sources.EOs have been qualified as natural bioactive agents due to their ability to delay lipid oxidation in various food systems [13,45].
A large number of compounds are generated from lipid oxidation which adversely affect quality and this limits the shelf-life of food.Lipid oxidation in animal food products it is influenced by various factors such amount of polyunsaturated fatty acids (PUFA), O2, metal ions, temperature, and lighting.To prevent or delay lipid oxidation in foods, antioxidants can be applied.Today, food industry search new economical and effective natural antioxidants that can replace synthetic antioxidants.
The initial TBA-RS value of fresh whole eggs was 0.05 mg MDA/kg (Table 3).The activity of the orange EO was found to be dose dependent.The TBA values of control samples increased rapidly throughout the display.Results shows that at the 5 th day of display, samples containing higher level of orange EO (0.5%) had TBA values 69.73% lower than the TBA values of control samples.However, at the end of display, the degree of inhibition was 55.2% lower.During all period of storage, the lower treated samples (0.1%) showed a significant increment, and were more similar to the untreated controls.Our results are in conformity with those of Viuda-Martos et al. [46] who studied the effect of various plant extracts at different concentrations on lipid oxidation in egg products during storage.It was reported that the lipid oxidation values was lower at higher concentration of plant extract.
Fernandez-Lopez et al. [47] was tested the orange powder for its potential as antioxidants in beef meatballs and found them very effective against rancidity more than 12 days during storage.Lowering the concentration of EOs without compromising their antioxidant activity can also be obtained by applying them in combination with other antioxidant compounds that provide a synergistic effect [45].

Psychrotrophic bacteria analysis
Whole eggs are prone to be contaminated by Gram-positive and Gram-negative microorganisms [48], being microbial growth considerably decelerated due to the presence of natural antimicrobials.; in particular lysozyme and conalbumin, in the egg white fraction [49,50].The changes in microbiological quality in the treated LWE samples during display, at 4 ºC are presented in Figure 4.The initial Psychrotrophic bacteria counts of samples reach 2.5 log CFU/mL.Garcia-Gonzalez et al. [16] reported 4.1 log CFU/g values for the initial microbial loads of unprocessed LWE.In the first batches LWE0.1% and LWE0.2%,EO treatment was less effective in decreasing the number of Psychrotrophic bacteria counts compared to LWE0.5% treatments (p < 0.05).However, as display continued, significant changes were observed: the Psychrotrophic counts continuously increased in control and 1 st batch (0.1%) and 2 nd batch (0.2%) samples, whereas slower microbial development was observed for the 3 rd batch (0.5%).At the end of the shelf life study, the number of psychrotrophic bacteria in the LWE treated samples reached 5.63 to 7.27 log10 CFU/mL.However, at the same time values of 7.5 log10 CFU/mL were obtained for untreated samples.
High treatment (LWE0.5%) was as effective to ensure microbial stability during the entire display study.However, Psychrotrophic bacteria limit was not exceeded at the end of the storage study (< 7 log10 CFU/g) and thus acceptable for consumption even after one weeks of display for both higher treatments (0.2 and 0.5%) in accordance with the microbiological guidelines.A number of studies have demonstrated the wide spectrum of antimicrobial activity of citrus species, especially orange EO against a wide range of microorganisms [51], which has been confirmed and extended in this study.Luciardi et al. [31] showed that citrus EOs can be used to inhibit quorum sensing and virulence factors of Pseudomonas aeruginosa by producing a biofilm in food systems.Orange and lemon dry powder at 5% (w/w) were tested by Fernandez-Lopez et al. [47] for their potential as antimicrobials in beef meatballs and found them to be very effective against LAB over a 12-day period.Citrus EOs were also investigated for their ability to reduce total aerobic bacteria and psychrotrophic number in comparison to controls over a period of 90 days at refrigerated beef storage [52].Callaway et al. [53] and Muthaiyan et al. [54].have studied the antimicrobial effects against common foodborne pathogens of citrus by-products industry EOs and found that several of them have been shown to possess antimicrobial properties.Several studies have shown that Grampositive bacteria are more susceptible to Citrus EO than Gram-negative bacteria [55].
Contrary to all expectations, Lin et al. [56] showed that the orange EOs could effectively inactivate some Gram negative bacteria such as V. parahaemolyticus, S. Typhimurium, and E.
coli but not S. aureus (Gram positive), on the food contact surfaces.
Higher contents of phenolic compounds such as major monoterpene hydrocarbons present in the EO could be responsible for the higher antimicrobial activity.The antibacterial activity of studied orange EO can be assigned to several molecules, amongst other the limonene, major constituent of orange EO.It has been reported that limonene is more effective on bacterial strains [57].

Sensorial odor analysis
The production of off-flavor or strong odor limits the use of EOs as food preservatives to increase the shelf life of food products.The use of EOs as biological agents in food industry could affect the organoleptic properties of the products, so that obtaining a better biological effect in the lower concentration is essential.Thus, sensory tests by a trained panel would be necessary in some cases to elucidate this aspect.Regarding the intensity of the orange smell (Table 5), LWE 0.5% samples were given a initial score of 3 (Day 0), while LWE subjected to low treatment with 0.2 and 0.1% orange EO were given a score < 2.2 during all period of display, representative of acceptable orange smell.It appears evident that the intensity of the orange smell decreases with time of display; no more perceptible after 5 days (p < 0.05) and reached acceptable values for all samples.From a practical point of view, the dose used for processing LWE is low and acceptable in this product.The treatment with orange EO × display times had an effect on off-odor attribute sensory scores (p < 0.05).At all period of display, the untreated samples had higher (p < 0.05) off-odor scores.Generally, TBA-RS values have been correlated with consumer perception of lipid oxidation in animal products [5].Increased off-odor of untreated samples was probably due to oxidative rancidity by increased lipid oxidation as shown by the TBARS values, which could be easily perceived by the panelists'.It would also be probable that the off-odor perceived by panelists' is also due to microbial development as shown by the psychrotrophic bacteria's counts.Untreated samples were assessed by the panelists with scores rejection limit (score  4) at 5 rd day of display, whereas higher treated samples (0.2 and 0.5%) were assessed by the panelists with scores below (P < 0.05) the rejection limit during all period of display.The higher treatments with orange EO significantly (P < 0.05) extended LWE odor shelf life attributes.The antioxidant activity may be ascribed to the presence of chemical components; monoterpenes found in this EO may act as bioactive agents and to avoid loss in sensory quality.The extreme aroma of various EOs, even at low concentrations, can cause negative organoleptic effects exceeding the threshold acceptable to consumers.In the last decades, different strategies can be used to resolve this problem.One option is to use plant extracts in active packaging rather than as an ingredient in the product itself.EOs can be encapsulated in edible and biodegradable coatings polymer that provide a slow release to the headspace of food packages and consequently to the food surface [5].
The edible gelatin coating enriched with orange EO has perceptible effects on the quality and shelf life of shrimps [58].

Instrumental color
At positive values, the CIE a* index indicates reddish colors, however, at negative values, colors are green.CIE b* index take positive values for yellow and negative for blue.
Finally, the CIE L* parameter is an approximate measure of luminosity.Each of the color parameters mentioned above is directly associated with certain quality criteria of the product in question, for example, the presence of fundamental compounds in the product, pH, water retention capacity and texture.The color of the LWE is a subject of practical importance for the egg-processing industry, which requires egg products with an appropriate and a homogeneous distribution of the color to satisfy the demand of the food industry.Customers may not accept discoloration caused by long term storage, considering the LWE as being of low-quality.For this reason, synthetic antioxidants are usually added to the egg products to reach the desired color and make the products more attractive for consumers and more appropriate for the egg-processing industry.Color perception highly depends on the chemical and microbial properties of the LWE components.It has been described that denaturation of certain proteins.;lipids and pigments, which occurs during storage, explains the main color changes that occurred in stored animal food products [5,59].
The most obvious changes during the storage of liquid egg products are typically related to color.Results concerning orange EO treatments are recorded on Table 6.After the treatments, red (CIE a*) and yellow (CIE b*) coordinates increased moderately for LWE0.5% during display if compared with other treatments, resulting in more orange products.Also, the CIE L* value decreased with the exposure time, and results for untreated samples are comparatively darker than the others.In similar products, de Souza and Fernandez [59] found a comparable tendency for the CIE Lab color coordinates.The results obtained instrumentally would justify the physical-chemical and microbial tests results for displayed fresh LWE, being the oxidation reaction and microbial development likely responsible for the observed differences.

Shelf-life determination
The shelf life of LWE is determined based on sensory analysis (attribute scoring: A score < 3 in "off odor" parameter denoted that product was acceptable, chemical (TBA-RS value: limit ~ 1.5 mg MDA/kg), and microbiological (psychrotrophic aerobic count: limit ~ 7 log10 CFU/g) properties.The shelf life is defined as "the period between raw LWE scale laboratory preparation and the storage sampling, during which the product is in a state of satisfactory quality in terms of chemical, physical, microbiological and sensory attributes".The display of LWE demonstrated that orange EO was needed for obtaining a significant increase of retail shelf life.It was evident that the LWE quality attributes during the display period depended on the concentration of orange EO added in the product.The long term chemical, physical, microbial and sensory stabilities of the raw LWE during display were positively influenced by EO treatments and shelf-life was up to 1 week.The quality stability of LWE during display was higher at higher orange EO treatment.
It is evident that the constituents separately isolated from EOs may play an important role in the biological activity of the latter.However, the main obstacle for using separated constituents as food preservatives is that they are most frequently not potent as separate use, and they cause negative organoleptic effects when added in sufficient amounts to provide a biological effect.
The food industry primarily uses EOs as flavorings.However, application of EOs as food preservatives requires detailed knowledge about their properties, i.e., the range of target organisms, the mode of action, and the effect of food matrix components on their antimicrobial and antioxidant properties.A range of EO components have been accepted by the European Commission for their intended use as flavorings'' in food products such as limonene which is considered to present no risk to the health of the consumer.
The United States Food and Drug Administration (FDA) also classify these substances as generally recognized as safe (GRAS).The high value of LD50 of the C. sinensis oil through oral administration on mice indicates their non-mammalian toxicity [37] and recommended that EO of C. sinensis and DL-limonene can be used as potent shelf life enhancement of stored food.In addition, the various constituents may interact, causing synergistic, antagonistic, indifferent and additive effects.A comparative study of EO constituents may help to understand the key points of the biological activity of EOs, acting alone or in combination with other food preservation techniques.

Conclusion
Microwave energy is a key enabling technology in achieving the objective of sustainable "clean" production for research, teaching and commercial applications.It has been shown that solvent-free conditions are especially suited to microwave-assisted organic synthesis, as reactions can be run safely under atmospheric pressure in the presence of significant amounts of products.SFME has been conceived following the concepts of SFM synthesis.When coupled to microwave radiation, solvent free techniques have proved to be of special efficiency as clean and economic procedures.Major improvements and simplifications over conventional methods originate from their rapidity, their enhancement in yields and product purities.Orange EO is also shown to be a particularly interesting field for applications within the food industries.The long term oxidative, microbial and sensory stability of the LWE was positively influenced by orange EO treatments and shelf-life was up to 1 week of refrigerated display.Therefore, the results obtained confirm EO treatment as a promising technology to lengthen the commercial shelf-life of liquid egg products.1.00 ± 0.00 aA 1.00 ± 0.00 aA 2.17 ± 0.41 aB 3.00 ± 0.00 aC 1.00 ± 0.00 aA 1.00 ± 0.00 aA 2.17 ± 0.41 aB 2.83 ± 0.41 aC 1.00 ± 0.00 aA 1.00 ± 0.00 aA 1.17 ± 0.41 bA 1.33 ± 0.52 bAB 1.00 ± 0.00 aA 1.00 ± 0.00 aA 1.00 ± 0.00 bA 1.17 ± 0.
Portable type, USA) in accordance with the recommendations of the International Commission on Illumination[15].L (lightness), a (redness), b (yellowness) values were recorded on the LWE emulsion of all samples.The instrument was calibrated using black and white tile, provided with the instrument.The LWE samples were kept inside the glass Petri dishes (diameter and height is 100 × 15 mm) in triplicate, and then instrument was directly Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 8 January 2018 doi:10.20944/preprints201801.0055.v1 All data were analyzed by the general linear model (GLM) procedure of the Statistical Package for the Social Sciences software (SPSS version 21, IBM Corporation, USA).Results 59. de Souza, P.M.; Fernandez, A. Effects of UV-C on physicochemical quality attributes and Salmonella enteritidis inactivation in liquid egg products.Food Control 2011, 22, 1385-1392.

Figure captions Fig. 1 Fig. 2 3 4
Figure captionsFig.1Solvent-FreeMicrowave Extraction System Fig.2Characteristics of the microwave heating Fig.3Free radical scavenging activity (%) of the orange EO Fig.4Total psychrotrophic bacteria counts (log 10 CFU/mL ± SD) of LWE treated with orange EO during cold storage.Letters show significant differences among the groups at p < 0.05

Table 1
lists the grouped compounds in EO: oxygenated and non-oxygenated fractions and composition of chemical families of orange EOs obtained by different extraction methods.The oxygenated fraction in EO samples from SFME (1.6%) was 40% higher than HD (0.82%) and 30% higher than CP (0.95%).The monoterpene hydrocarbons (i.e., limonene) are present in larger amounts in the hydro-distilled EO (95.48%) than the SFME EO (94.64%), but the extract obtained by SFME is more concentrated in oxygenated compounds (1.6 vs. 0.85-0.95%).The greater proportion of oxygenated compounds in the SFME EO is probably due to the diminution of thermal and hydrolytic effects, compared with CP which uses a large quantity of water and with HD which is time and energy consuming.

Table 1
Extraction time, yields and chemical compositions of EOs obtained by SFME, HD, 812 and CP extraction from Valencia late (Citrus sinensis L.) peels.

Table 2
DPPH scavenging method of SFME EO of Citrus sinensis L. (Mean values  standard deviation) Preprints (www.

Table 3
TBA values (mg MDA/kg of meat) and IR%* (values between parenthesis) of LWE containing orange EO during cold display.The Percentage inhibition rate (IR%) respect to the control was calculated as follows:IR% = [(TBA-RS C -TBA-RS T )/TBA-RS C ]  100.Where C is the number of TBA-RS in the untreated samples (control) and T is the number of TBA-RS in the treated samples.a-d Within each row, different superscript lowercase letters show differences between treatment groups (p < 0.05).-Z Within each column, different superscript uppercase letters show differences between the storage times within same treatment group (p < 0.05).LWE0.1%:Liquid Whole Eggs treated with 0.1%of orange EO.LWE0.3%:Liquid Whole Eggs treated with 0.3%of orange EO.LWE0.5%:Liquid Whole Eggs treated with 0.5% of orange EO. *W

Table 4
CIELAB L*, a* and b* color coordinates in LWE treated with orange EO.Each sample was measured in 10 different positions; results are the mean of three independent replications.
L* (lightness); a* (redness); b* (yellowness) a-c Within each column for each color coordinates, different superscript uppercase letters show differences between the storage times within treatment groups (p < 0.05).Preprints (www.

Table 5
Sensory scores* (Mean ± SD) for EO Citrus sinensis L. odor and off-odor of LWE during display.A score < 3 in any of the parameters denoted that LWE was acceptable.When the score  3 in any of the parameters denoted that LWE can be rejected (End of the shelf life).
*a-d Means of the same row (between days of display) with different letters differ significantly (p < 0.05).A-C Means for Citrus sinensis L. odor of the same column (between treatments) with different letters differ significantly (p < 0.05).w-yMeansfor off-odor of the same column (between treatments) with different letters differ significantly (p < 0.05).**Off-odor:referred to the intensity of odors associated to LWE oxidation and microbial development: 1 = none; 2 = slight; 3 = small; 4 = moderate; and 5 = extreme.