Chemical composition and antimicrobial activity of Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) peel essential oils

Abstract The present investigation reported the chemical composition of cold pressed Gannan navel orange peel essential oil (EO) and its molecular distillation fraction (light phase EO), and examined their antimicrobial activity against spoiling and pathogenic microorganisms. Gas chromatography‐mass spectrometry analysis identified 27 and 20 different chemical constituents in cold pressed EO and light phase EO, respectively. Limonene was the major constituent, accounting for 85.32% of cold pressed EO and 60.44% of light phase EO. Both EOs and some of their constituents showed good antimicrobial activity. Compared to cold pressed EO, light phase EO exhibited the better antimicrobial activity under weak acidic and neutral conditions. The light phase EO presented a higher antimicrobial activity after thermo‐treatment at 60–100°C for 20 min than cold pressed EO. These results demonstrated that light phase EO had a potential to be used as a novel antimicrobial agent for food preservation and food processing.

component of Citrus EO, ranging from 32 to 98% of the total oil (Moufida & Marzouk, 2003). The antimicrobial activity of EO is directly correlated to the presence of its bioactive volatile constituents, although it may be varied with environmental conditions (Bakkali et al., 2008;Perczak et al., 2016). The well-known and characterized constituents of Citrus EO include limonene, linalool, and citral, which have been proved to exert potent, broad-spectrum antimicrobial capacity (Bezic, Skocibusic, & Dunkic, 2005). As Citrus EOs are generally recognized as safe (GRAS), they have been screened for antimicrobial properties against common food-borne pathogens (Bakkali et al., 2008;Burt, 2004). In addition, they have been used as natural food preservatives and accepted by consumers all over the world (Sharma et al., 2017).
In commercial practice, large-scale orange EO is mainly prepared by cold pressing method (Leão, Sampaio, Pagani, & Silva, 2014). Cold pressed orange EO was extracted by mechanical rupturing of the oil sacs in the flavedo, expressing the oil as an aqueous emulsion from which it is separated by centrifuging. It consists of terpenes, alcohols, aldehydes, ketones, esters, and acids along with some nonvolatile waxy materials, carotenoids, flavonoids, etc. However, pigment, wax, and pesticide residue in EO are disadvantageous to be used as antimicrobial agents in food. Molecular distillation is an efficient method to separate some undesired molecules without harming the natural (desirable) properties of EO (Borgarello, Mezza, Pramparo, & Gayol, 2015). Separation can be achieved based on the difference of the mean free path of different molecules. This method is characterized by a high vacuum operation that causes a decrease in the boiling point of substances (Rossi, Pramparo Mdel, Gaich, Grosso, & Nepote, 2011). Therefore, molecular distillation is very useful to separate thermal sensitive EOs and extensively used in flavor and fragrance industry.
In this study, light phase EO was separated from cold pressed Gannan navel orange peel EO by molecular distillation. Then, we investigated the chemical composition of light phase EO and cold pressed EO and examined their effectiveness in vitro on four selected spoiling and pathogenic microorganisms.

| Extraction of cold pressed EO and light phase EO
Cold pressed EO was obtained by physical extraction from the peel of Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) (Lv, Chun, Jiang, Li, & Guo, 2014). The recovery of orange oil by FOMESA extractor (Model 391, Food Machinery Espanola, S.A., Valencia, Spain) was carried out during simultaneous extraction of juice and oil. The oil cells were ruptured by pressure, and the oil was washed away with water. The oil emulsion passed through a 20-mesh shaker screen to remove most of the insolubles. Then a three-stage centrifuge system was used to separate orange oil efficiently. The first stage removed some water and insoluble solids by three-phase disk stack centrifugal separator to produce an emulsion with around 40% oil content. This emulsion was fed to the second stage centrifuge, where the oil content was concentrated to around 88%. The third stage removed the remaining water and cloudy particles to achieve complete separation of the oil phase.
Light phase EO was obtained by molecular distillation from cold pressed EO using a wiped-film molecular distillation apparatus (Pope Two Inch Laboratory Scale Wiped-Film Molecular Still & Evaporator; Pope Scientific Inc., Saukville, WI, USA). The evaporation temperature and operation pressure were 50°C and 10.0 Torr, respectively.
Orange oil was fed at room temperature, and the feeding rate was 3.0 ml/min. The rotational speed of the roller wiper was 350 rpm, and the condenser temperature was 0°C.

| GC-MS analysis
Gannan navel orange EO components were identified and quantified using an Agilent 7890B gas chromatograph coupled with an Agilent mass spectrometer detector. The GC-MS system was equipped with a DB-5 MS capillary column (30.00 m × 0.25 mm × 0.25 μm). Mass spectra were obtained by electron ionization (EI) at 70 eV with a spectra range of 50 to 500 m/z. The injector and detector temperatures were operated at 150°C and 250°C, respectively. The oven temperature was maintained at 80°C for 4 min and subsequently raised to 250°C (5°C/min) for 10 min. Helium was used as a carrier gas at a flow rate of 1.0 ml/min, at a split ratio of 100:1. Most of the components were identified by comparing their mass spectra with those of the computer mass libraries of The National Institute of Standards and Technology (2010).

| Microbial strains and growth conditions
The following microorganisms were purchased from China General

| Determination of minimum inhibitory concentration (MIC)
MIC values of Gannan navel orange cold pressed EO, light phase EO, and seven individual constituents against microorganisms were determined by the tube dilution method (Burt, 2004;Rota, Carraminana, Burillo, & Herrera, 2004). Serial dilutions of EOs and individual constituents were prepared with liquid nutrient media containing 5 ml of LB or YEPD medium. Each tube was inoculated with 0.5 ml of a standardized suspension of microbial test species containing 1 × 10 6 CFU/ml, then incubated at 37°C for 24 hr, except for S. cerevisiae, which was incubated at 25°C for 48 hr. The MIC was defined as the lowest concentration of EOs and individual constituents at which microorganisms failed to grow, so no visible changes were detected in the broth medium. All determinations were performed in triplicates.

| The effect of the environmental pH and temperature on antimicrobial activity of EOs
The effect of environmental acidity and alkalinity on the antimicrobial activity of EO was investigated according to the following Notes. "-" indicates not be detected. RI, retention indices determined on DB-5 column, using the homologous series of n-alkanes (C8-C20).
TA B L E 1 Chemical composition of Gannan navel orange cold pressed and light phase essential oils by GC-MS method. The agar mediums were adjusted to pH value of 5.0, 6.0, 7.0, and 8.0 with 2% NaOH solution and 50% citric acid solution, and the mixtures were autoclaved at 115°C for 30 min. Cooled autoclaved agar was distributed evenly in Petri dishes aseptically. The antimicrobial activity of both EOs was determined using the agar disk diffusion method (Xin, Liu, Zhang, & Gao, 2016). Paper disks (6 mm) were impregnated with 5 μl of cold pressed EO and light phase EO and then placed on the inoculated Petri dishes containing tested microorganisms (1 × 10 6 CFU/ml). The plates were incubated at 37°C for 24 hr, except for S. cerevisiae, which was incubated at 25°C for 48 hr. All determinations were performed in triplicates.
Antimicrobial activity was evaluated by measuring the diameter of the inhibition zones to the nearest millimeter (mm). Sterile water alone was used as the control.
The antimicrobial activity of the thermo-treated EOs was determined by the following method. Cold pressed EO and light phase EO were treated with heat at 60°C, 80°C, 100°C, and 121°C for 20 min, respectively. Then, paper disks (6 mm) were impregnated with 5 μl of heat-treated EOs and the antimicrobial activity was determined according to the method previously described. The untreated EO stored at 25°C was used as the positive control. Sterile water alone was used as the negative control.

| Statistical analysis
All data were expressed as the mean ± SD by measuring three independent replicates. Analysis of variance obtained was subjected to one-way ANOVA. Means values were separated by Duncan's multiple range tests when ANOVA was significant statistically (p < 0.05) (SPSS 18.0; Chicago, IL, USA).

| Chemical composition of essential oils
The chemical components of Gannan navel orange cold pressed EO and light phase EO were analyzed by GC-MS (

| Antimicrobial activity
Cold pressed EO and light phase EO showed a wide spectrum of antimicrobial activity in vitro ( Note. Data are presented as mean ± standard deviation, n = 3. Different superscript letters represent the significant differences at p < 0.05. Seven individual constituents were selected to test their antimicrobial activity (Table 2) These results were consistent with the previous studies that some oxygenated compounds presented a higher antimicrobial activity than nonoxygenated hydrocarbons (Burt, 2004). Fisher et al. (Fisher & Phillips, 2006) demonstrated that linalool and citral in Citrus EOs had antimicrobial effects against Campylobacter jejuni, E. coli O157, L. monocytogenes, B. cereus, and S. aureus. Moreover, some researchers have shown that carvone and limonene oxide were active against a wide spectrum of pathogenic fungi and bacteria (Aggarwal et al., 2002;Mustafa, 2015). We noticed that Gannan navel orange light phase EO had higher content of carvone and limonene oxide than cold pressed EO which might due to oxidation during distillation.
Higher activity of light phase EO might be attributable to its higher proportion of oxygenated compounds. Meanwhile, antimicrobial activity of both EOs might also be due to the synergistic, additive, and antagonistic interaction of their constituents (Burt, 2004). The investigation of the individual constituents in EO can provide useful information to fit different products or purposes with simultaneous elevation of their antimicrobial activities.

| Effect of the environmental pH and temperature on antimicrobial activity of EOs
The antimicrobial activity of cold pressed EO and light phase EO was closely related to pH value of the medium (Figure 1). The showed high antimicrobial activity under the partial acidic condition (Bakkali et al., 2008;Burt, 2004). It was probable that under acidic condition, active constituents dissolved better in the lipid phase of the bacterial membrane, and their binding abilities with membrane protein enhanced, leading to a higher antimicrobial activity (Burt, 2004;Durairaj, Srinivasan, & Lakshmanaperumalsamy, 2009  Normal University (15zb09).

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

E TH I C A L S TATEM ENT
This article does not contain any studies with human participants or animals performed by any of the authors.