Food Control 31 (2013) 1e4

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Antifungal activity of orange (Citrus sinensis var. Valencia) peel essential oil applied by direct addition or vapor contact Maria José Velázquez-Nuñez a, Raúl Avila-Sosa b, Enrique Palou a, Aurelio López-Malo a, * a b

Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Cholula, Pue. 72810, Mexico Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, 14 Sur y Av. San Claudio, Ciudad Universitaria, Puebla, Pue. 72420, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 June 2012 Received in revised form 10 September 2012 Accepted 18 September 2012

Vapor contact is an alternative when essential oils (EO’s) and microorganisms are placed separately in some sealed environment. The aim of this study was to compare the antifungal efficacy of orange peel EO at selected concentrations, applied either by vapor exposure or direct addition on the growth of Aspergillus flavus. Orange peel EO was obtained from fresh oranges (Citrus sinensis var. Valencia). EO was obtained by vapor distillation, analyzed by means of GCeMS chromatography, and applied to potatodextrose agar inoculated with A. flavus, using the techniques of direct addition to the agar or by generating EO vapors in airtight containers. Radial growth rate and lag phase were calculated using the Gompertz equation. Main compounds identified in the orange peel EO were: limonene, b-myrcene, bpinene, a-pinene, as well as citral Z and E; of which, limonene represented 96.62%. The minimum inhibitory concentration for the growth of A. flavus by direct addition was 16,000 mg l
1, while for the vapor contact was 8000 mg of EO l
1 of air. For both studied methods A. flavus growth decreased when increasing EO concentration. Although the effect of orange peel EO direct addition was faster, orange peel EO vapors were more effective, since lower concentrations were required to achieve the same antifungal effect. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Orange peel EO Vapor and direct contact assays A. flavus

1. Introduction Essential oils (EO’s) and extracts obtained from many plants have recently gained a great popularity and scientific interest. Phenolic compounds present in essential oils have been recognized as bioactive components with antimicrobial activity. Most plant phenolic compounds are classified as Generally Recognized as Safe (GRAS) substances, therefore they could be used to prevent growth of many pathogenic and spoilage microorganisms in foods (Burt, 2004; Nedorostova, Kloucek, Kokoska, Stolcova, & Pulkrabek, 2009). However, EOs antimicrobial efficacy in foods is usually achieved at higher concentrations, which many times entail a sensory impact, caused by altering the natural taste and/or odor of the food by exceeding the acceptable flavor and/or odor thresholds (Nazer, Kobilinsky, Tholozana, & Dubois-Brissonneta, 2005). Hence, for reducing EO’s sensory impact, one of the alternatives can be the use of EO’s by vapor contact instead of its direct addition. Vapor contact is an alternative when EO’s and microorganisms are placed

* Corresponding author. Tel.: þ52 222 229 2126; fax: þ52 222 229 2727. E-mail address: [email protected] (A. López-Malo). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.09.029

separately in some sealed environment and therefore microbial inhibition is achieved from a distance without direct contact of the antimicrobial agent with the food (Avila-Sosa et al., 2012). Few studies have been published regarding inhibition of microorganisms by the vapor-phase generated by EO’s (Inouye, Uchida, Maruyama, Yamaguchi, & Abe, 2006; López, Sánchez, Battle, & Nerin, 2007; Nielsen & Rios, 2000; Suppakul, Miltz, Sonneveld, & Bigger, 2003), pointing out that EOs applied in vapor phase could be effective against foodborne pathogens and spoilage microorganisms at relatively lower concentrations than when applied in liquid phase, thereby causing less effect on sensory attributes (Tyagi & Malik, 2011). Inouye (2003) reported that EO’s of cinnamon, thyme, and lavender inhibited molds more effectively by vapor contact than by direct contact in aqueous systems and explained that the diminished activity of the essential oil in aqueous media was due to the presence of detergents and lipophilic materials. Arras and Usai (2001) observed alterations in the morphology of Penicillium digitatum caused by thyme EO applied by vapor contact, and GómezSánchez, Palou, and López-Malo (2011) established the antifungal activity of Mexican oregano EO by vapor contact on Aspergillus flavus.

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On the other hand the genus Citrus, which includes several important fruits such as oranges, mandarins, limes, and lemons, is the largest fruit crop in the world (100 million cubic tons per year) and oranges account for 60% (Oreopoulou & Tzia, 2007). Farhat et al. (2011) reports that orange peel accounts for approximately 45% of the total bulk with significant amounts of it available as a byproduct after orange processing that create environmental problems, particularly water pollution, due to the presence of biomaterials such as EO, pectin, and sugars. Citrus spp. EO’s are present in great quantities and it is known that can have an antimicrobial effect against both bacteria and fungi (Chanthaphon, Chanthachum, & Hongpattarakere, 2008; Jafari et al., 2011). Orange peel includes the epidermis covering the exocarp consisting of irregular parenchymatous cells, which are completely enclosing numerous glands or oil sacs (Lin, Sheu, Hsu, & Tsai, 2010). Nevertheless the antifungal activity of orange peel EO has been scarcely studied, Caccioni, Guizzardi, Biondi, Renda, and Ruberto (1998), reported that volatile compounds of orange and lemon peel are capable to inhibit Penicillium spp., although it is claimed (using the agar dilution technique) that are effective against the growth of some molds. The aim of this study was to compare the antifungal efficacy at selected concentrations of orange peel EO applied either by vapor exposure or direct addition on the growth of A. flavus. 2. Materials and methods 2.1. Essential oil and chemical characterization Orange peel was obtained from fresh oranges (Citrus sinensis var. Valencia) that were bought at a local market in Puebla, Mexico. Oranges were washed and peeled, then peels were cut into 1 cm square pieces, EO was obtained by vapor distillation for 4 h with a Clevenger-type apparatus. EO was analyzed with a GC Varian Star 3400cX coupled with an MS Varian Saturn 2000 (Varian Inc., Walnut Creek, CA, USA) with a splitless injector, equipped with a Zebron ZB-1 capillary column (30 m*0.32 I.D.*0.25 mm). Helium was used as the carrier gas, and the following conditions were set for the analysis: injector, 170  C, detector 225  C; the initial oven temperature was 74  C held for 4 min, followed by a ramp-up of 3  C min
1 up to 95  C, and a second ramp-up of 25  C min
1 up to 220  C, and held at the final temperature for 10 min, for a total run time of 30 min. The obtained spectra were compared with respective mass spectra of pure compounds, and also with the mass profile of the same compounds available from the US National Institute of Standard Technology (NIST) library (Adams, 1995).

Table 1 Main compounds identified in orange peel essential oil. Compound

%

Limonene b-Myrcene b-Pinene a-Pinene Citral Z Citral E

96.62 1.72 0.53 0.47 0.31 0.34

For the vapor contact assays, uncovered inoculated PDA plates were placed on a perforated plastic sheet inside hermetically closed plastic chambers (1.7-L capacity) with transparent lids, leaving sufficient headspace (each airtight chamber utilized was tested for leaks prior to experimenting with the essential oil). A glass plate containing the essential oil (500, 1000, 2000, 4000, 8000, or 16,000 mg of EO l
1 of air) was located under the perforated plastic sheet (Gómez-Sánchez et al., 2011). Dishes were incubated at 25  C. A growth control was prepared in parallel, to ensure that viable organisms were present. For both studied methods, radial growth was measured every 24 h during 20 d. The diameter of the growing mold colonies was daily measured in two directions at right angles (in the case of the vapor contact assays, through the transparent lid of the chamber in order to not disturb the internal system equilibrium). Minimal inhibitory concentration (MIC) was defined as the lower concentration tested were no fungal growth was detected. Every test was performed by triplicate. 2.3. Fungal growth modeling and statistical analysis Growth data were fitted using the modified Gompertz model as reported by Char, Guerrero, and Alzamora (2007):


 n h io Dt e ¼ Aexp
exp ymax $ ðl
tÞ þ 1 ln A Do

(1)

where: Dt (mm) is the average colony diameter at time t (d), and Do (mm) is the average colony diameter at initial time; A is the maximum mold growth achieved during the stationary phase, ymax is the maximum specific growth rate (d
1), l is the lag phase (d) and e ¼ exp (1).

2.2. Direct addition and vapor contact assay A. flavus (ATCC 18672) was obtained from Universidad de las Américas Puebla Food Microbiology Laboratory and cultivated on potato-dextrose agar plates (PDA, Merck, Mexico) for 5 days at 25  C, and then spores were harvested with 10 ml of 0.1% Tween 80 (Merck) solution sterilized through membrane (0.45-mm pore size) filtration. The number of spores present in the suspension was determined using a hemocytometer and an optical microscope (Zeiss Primo Star, Göttingen, Germany), and expressed as number of spores per milliliter. The spore suspension was adjusted with the same solution to give a final spore concentration of 106 spores ml
1 and was utilized the same day (Avila-Sosa et al., 2012). For the direct addition assays, EO was added to sterilized PDA and mixed at 45  C to achieve final concentrations of 500, 1000, 2000, 4000, 8000, or 16,000 mg l
1; plates were allowed to solidify and then 10 ml of A. flavus spore suspension was inoculated in the center of each plate.

Fig. 1. Effect of orange peel essential oil applied by direct addition at selected concentrations [0 (-), 500 (A), 1000 (:), 2000 (C), 4000 (,), 8000 (>) or 16,000 (D) mg l
1] on Aspergillus flavus growth. Dt is the average colony diameter at time t and Do is the average colony diameter at initial time.

M.J. Velázquez-Nuñez et al. / Food Control 31 (2013) 1e4

Fig. 2. Effect of orange peel essential oil applied by vapor contact at selected concentrations [0 (-), 500 (A), 1000 (:), 2000 (C), 4000 (,) and 8000 (>) mg of EO l
1 of air] on Aspergillus flavus growth. Dt is the average colony diameter at time t and Do is the average colony diameter at initial time.

Growth parameters were obtained by means of nonlinear regression using the software Kaleidagraph V.3.51 (Synergy Software, Reading, PA, USA). In order to compare both application methods t-Student tests were performed, and Gompertz parameters were compared with General Linear Model procedure in Minitab 16 (Minitab Inc., State College, PA, USA) with 95% of significance, different means were separated with Tukey’s test. 3. Results and discussion The main compounds in the orange peel EO (Table 1) identified by gas chromatography coupled with mass spectrometry were limonene, b-myrcene, b-pinene, a-pinene, as well as citral Z and E; of which, limonene represented 96.62%. Sawamura et al. (2004) stated that citrus essential oils are a mixture of volatile compounds that mainly consisted of monoterpene hydrocarbons compounds that can be approximated into three fractions: terpene hydrocarbons, oxygenated compounds and non-volatile compounds. The monoterpene fraction can constitute from 50 to more than 95% of the oil; however, it makes little contribution to the flavor and fragrance of the oil (Chanthaphon et al., 2008). Tyagi and Malik (2011) reported that monoterpenes concentrations in EO’s are the main components with higher antimicrobial activity in vapor phase than in direct contact and depends on their presence in gaseous form facilitating their solubilization in cell membranes.

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For both studied methods, vapor exposure or direct addition of orange peel EO, A. flavus growth (Figs. 1 and 2) decreased when increasing EO concentration. Although the effect of orange peel EO direct addition was faster, orange peel EO vapors were more effective, since lower concentrations were required to achieve the same antifungal effect. MIC value for A. flavus growth by direct addition was 16,000 mg l
1, while for the vapor contact was 8000 mg of EO l
1 of air. Sharma and Tripathi (2008) suggested that citrus EO exhibits among other properties, antifungal activity by reducing or totally inhibiting fungal growth in a doseeresponse manner, Viuda-Martos, Ruiz-Navajas, Fernández-López, and Pérez-Álvarez (2008) reported A. flavus inhibition by agar dilution method with orange EO at 7900 mg l
1, while Inouye (2003) inhibited Aspergillus fumigatus by vapor contact of yuzu (Citrus junos) EO at 4000 mg of EO l
1 of air. A. flavus growth curves were adequately adjusted by equation (1) (R2 between 0.981 and 0.999). Modified Gompertz model parameters (Table 2) demonstrated significant differences (p < 0.05) between both studied methods. Maximum mold growth (A) and maximum specific growth rate ðymax Þ show that increasing the concentration of orange peel EO decreases these parameters. In the direct addition technique, A and ymax parameters did not exhibit significant differences (p > 0.05) as compared to control parameters until 8000 mg l
1, but for vapor contact treatment, these parameters were significantly (p < 0.05) lower than control ones even at low concentrations. Lower values of A and ymax were observed for the vapor contact than for direct addition assays. Parameter A depends on the maximum growth diameter, which in our case is the Petri dish diameter. For direct addition assays, the lag phase parameter showed a fungistatic effect, since at 8000 mg l
1 its lambda value is nearly two times the value for the control. In contrast with direct addition assays, lag phase values for vapor contact assays decreased when EO concentration increased. Similar effects were reported by Bluma, Landa, and Etcheverry (2009) for A. flavus inhibition by vapor contact with boldus and poleo EO’s; moreover, they determined that EO efficacy depends on the complex interactions of tested environmental factors. Matan et al. (2006) evaluated the effects of vapor phase EO concentration (clove or cinnamon) and atmosphere composition in the number of days required for visible mold growth, they observed that higher concentrations of EO are required to delay fungal growth; and reported a synergic inhibitory effect of the surrounding gas composition of EO volatile compounds. Carson, Me, and Riley (2002) and Tyagi and Malik (2011) stated that EO causes different changes on microbial cell membrane properties and functions by increasing membrane fluidity and altering membrane permeability; while low concentrations alter its permeability, high concentrations cause

Table 2 Modified Gompertz model parametersa (mean  standard deviation) for Aspergillus flavus growth curves subjected to selected concentrations of orange peel essential oil either by direct addition or vapor contact. EO concentrationc (mg/L)

A

0 500 1000 2000 4000 8000 16,000

2.28 2.30 2.23 2.08 1.99 1.80 _b

ymax ð1=dayÞ

Direct addition      

0.03a,A 0.01a,A 0.05a,A 0.06a,B 0.06a,B 0.05C

Vapor exposure 2.35 1.67 1.70 1.56 0.98 _b _b

    

0.08a,A 0.03b,B 0.03b,B 0.08b,C 0.02b,D

Direct addition 0.35 0.41 0.33 0.35 0.31 0.26 _b

     

0.02a,A 0.07a,A 0.06a,A 0.05a,A 0.03a,A 0.01B

l(day) Vapor exposure 0.38 0.11 0.13 0.09 0.08 _b _b

    

0.03a,A 0.01b,B 0.01b,B 0.01b,C 0.01b,C

R2

Direct addition 2.65  2.16  2.14  2.61  2.62  4.41  >20C

0.17a,A 0.51a,A 0.41a,A 0.19a,A 0.29a,A 0.21B

Vapor exposure 2.72  1.07  0.23  0.54  0.26  >20D >20D

Means followed by a different superscript letter within a row for each parameter are significantly different (p < 0.05). Means followed by a different superscript capital letter within a column for each parameter are significantly different (p < 0.05). a A: maximum mold growth in the stationary phase; ymax : maximum specific growth rate; l: lag phase. b No growth. c For direct addition assays (mg l
1); for vapor contact assays (mg of EO l
1 of air).

0.12a,A 0.19b,B 0.29b,C 0.14b,C 0.03b,C

Direct addition

Vapor exposure

0.999 0.991 0.992 0.985 0.981 0.987 0.982

0.999 0.998 0.997 0.992 0.989 0.991 0.983

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severe damage, loss of homeostasis, and death. Nychas (1995) reported that some EO’s components are able to denature the enzymes responsible for spore germination, energy production and synthesis of structural compounds or interfere with the amino acid involved in germination. Several researchers (Avila-Sosa et al., 2012; Bluma et al., 2009; Caccioni et al., 1998; Gómez-Sánchez et al., 2011; Inouye, 2003; Inouye et al., 2006; López et al., 2007; Nedorostova et al., 2009) concur that a greater antifungal activity of EO is achieved in vapor phase than in aqueous solution or agar contact. In our case although direct addition of orange peel EO had a rapid effect on A. flavus growth, exposure to orange peel EO vapors was more effective, requiring lower concentrations of EO to inhibit mold growth. Acknowledgments We acknowledge financial support from the National Council for Science and Technology of Mexico (CONACyT) for the project “Combinación de Factores Físicos y Químicos para la Inactivación de Microorganismos Relacionados con Alimentos”. Author VelázquezNúñez acknowledges financial support for her bachelor studies from CONACyT. References Adams, R. P. (1995). Identification of essential oil components by gas chromatography/ mass spectrometry. Carol Stream, IL: Allured Publishing Corporation. Arras, G., & Usai, M. (2001). Fungitoxic activity of 12 essential oils against four postharvest citrus pathogens: chemical analysis of Thymus capitatus oil and its effect in sub-atmospheric pressure conditions. Journal of Food Protection, 64, 1025e1029. Avila-Sosa, R., Palou, E., Jiménez-Munguía, M. T., Nevárez-Moorillón, G. V., NavarroCruz, A. R., & López-Malo, A. (2012). Antifungal activity by vapor contact of essential oils added to amaranth, chitosan, or starch edible films. International Journal of Food Microbiology, 153, 66e72. Bluma, R., Landa, M. F., & Etcheverry, M. (2009). Impact of volatile compounds generated by essential oils on Aspergillus section Flavi growth parameters and aflatoxin accumulation. Journal of the Science of Food and Agriculture, 89, 1473e1480. Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foods a review. International Journal of Food Microbiology, 94, 223e253. Caccioni, D. R., Guizzardi, M., Biondi, D. M., Renda, A., & Ruberto, G. (1998). Relationship between volatile components of citrus fruit essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum. International Journal of Food Microbiology, 43, 73e79. Carson, C. F., Me, B. J., & Riley, T. V. (2002). Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrobial Agents and Chemotherapy, 46, 1914e1920. Chanthaphon, S., Chanthachum, S., & Hongpattarakere, T. (2008). Antimicrobial activities of essential oils and crude extracts from tropical Citrus spp. against food-related microorganisms. Songklanakarin Journal of Science and Technology, 30, 125e131. Char, C. D., Guerrero, S., & Alzamora, S. M. (2007). Growth of Eurotium chevalieri in milk jam: influence of pH, potassium sorbate and water activity. Journal of Food Safety, 27, 1e16.

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