alexa Rosmarinus officinalis L. and Myrtus communis L. Essential Oils Treatments by Vapor Contact to Control Penicillium digitatum | OMICS International
ISSN: 2157-7110
Journal of Food Processing & Technology

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Rosmarinus officinalis L. and Myrtus communis L. Essential Oils Treatments by Vapor Contact to Control Penicillium digitatum

Ladu G1*, Cubaiu L1, d’Hallewin G1, Pintore G2, Petretto GL2 and Venditti T1

1CNR - ISPA, Trav. La Crucca, 3 - 07040 Sassari, Italy

2Department of Chemistry and Pharmacy, University of Sassari, via Muroni, 23 / A - 07100 Sassari, Italy

*Corresponding Author:
Ladu G
CNR - ISPA, Trav. La Crucca
3-07040 Sassari,Italy
E-mail: [email protected]

Received Date: June 15, 2015 Accepted Date: July 13, 2015 Published Date: July 20, 2015

Citation: Ladu G, Cubaiu L, d’Hallewin G, Pintore G, Petretto GL, et al. (2015) Rosmarinus officinalis L. and Myrtus communis L. Essential Oils Treatments by Vapor Contact to Control Penicillium digitatum. J Food Process Technol 6:492. doi: 10.4172/2157-7110.1000492

Copyright: © 2015 Ladu G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Background: The antimicrobial activity of Essential Oils (EOs) has been used for centuries and nowadays the efforts to develop natural preservatives in postharvest management have augmented interest in their possible applications.

Materials and Methods: Rosmarinus officinalis L. and Myrtus communis L. EOs, and two of their components, α- and β-pinene, have been tested in vitro against Penicillium digitatum, with the aim to assess their antifungal effects when applied as fumigation. The pathogen, inoculated on PDA dishes, was treated by EO-vapors contact and the fungal growth inhibition was recorded in order to evaluate the EOs antifungal activity.

Results: The exposure to the EOs vapors shows different ability in the control of fungal growth related to the EO concentration used and the elapsed time between the fungal inoculum and EO vapor contact. The greatest antifungal activity was observed for rosemary EO, while a less control was found for the myrtle one. Treatment performed with α-pinene showed a control of the pathogen that was similar to the myrtle, whereas control with β-pinene was very poor.

Highlight: Our finding revealed that plant EOs could be successful in controlling fungal postharvest disease in a dose and compound dependent manner, but deeper researches are needed about treatment parameters because the effectiveness seems to be affected by treatment modalities.


Antifungal; Post-harvest disease; Plant extracts; SEM


Microbial spoilage is one of the several reactions that alter the safety and the organoleptic properties of perishable products. These reactions decrease the value of food because of the destruction of functional substances, the production of off-flavors and the production of food borne illness related to unsafe food intake [1]. In the field and during postharvest several infections that occur are related to fungal diseases. Fungi are universal biological agents able to colonize fruits and vegetables, causing pathologic disorders because of their potential to synthesize a broad range of hydrolytic enzymes [2]. The high losses in quality, nutrient content and monetary value recorded during postharvest are related to the settlement of molds toxigenic strains [3,4]. Furthermore many mold species can also synthesize mycotoxin, hazardous compounds to animal and human health. Thus inhibition of fungal growth is an effective way to prevent mycotoxin accumulation [5-7]. Furthermore consumers have become more aware of potential health risks associated to synthetic fungicide residues and are increasingly interested in commodities treated with compounds from natural sources [8]. For these reasons new safe technologies, with no risk for consumers and low impact for the environment are needed.

Essential oils are secondary metabolites of plants, which have been traditionally used due to their antimicrobial/antifungal activity [9], and many of them, such as clove, oregano, thyme, basil or cinnamon are categorized as GRAS (Generally Recognized as Safe) by the USFDA (Food and Drug Administration) [10]. Indeed, essential oils and their active compounds could potentially serve as an effective alternative to conventional antimicrobial agents. The antimicrobial activity depends on the constituents and their concentration: phenolic compounds possess the highest antimicrobial properties followed by alcohols aldehydes ketones ethers and hydrocarbons [11]. The strong antimicrobial activity could be correlated to the high percentage of monoterpenes, phenols and ketones [12]. It is suggested that antimicrobial activity of EOs could be the results of disturbance in several enzymatic systems involved in energy production and structural components synthesis; also they affect and disturb genetic material functionality [13,14]. Presence of phenols with hydroxyl group (-OH) are responsible of antimicrobial properties, the interaction with the membrane enzymes and protein can cause an opposite flow of protons, affecting cellular activity [15]. Nowadays, the efforts to develop natural preservatives in postharvest management have augmented interest in their applications. The most widely used methods to determine in vitro the antimicrobial properties of EOs are agar diffusion and broth dilution, while in our study we have performed the EOs application by fumigation. Due to their bioactivity in the vapor phase [16] EOs could be used as fumigant during postharvest protection. The aim of the present investigation was to evaluate the in vitro fungi toxic activity of Rosmarinus officinalis L. and Myrtus communis L. EOs, and two of their ccomponents, α- and β-pinene, against Penicillium digitatum when applied as vapor contact.

Materials and Methods

Plant material and EO extraction

Aerial parts of wild Myrtus communis L. and Rosmarinus officinalis L. were collected in April (2013) from the Sassari area, Sardinia, Italy. A sample of leaves, weighing 50 g, was subjected to hydro-distillation using a Clevenger type apparatus for 2h, according to the European Pharmacopoeia protocol (2002). The EO isolation was carried out in triplicate, the obtained EOs were collected separately, dried over anhydrous sodium sulfate (Na2SO4) and stored under nitrogen atmosphere at 4ºC, in amber glass vials, until analysis.

Gas chromatography-mass spectrometry analysis

The GC-MS analysis was carried out using a Hewlett Packard 5890 GC (Palo Alto, USA) equipped with a Hewlett Packard 5971 (Palo Alto, USA) mass selective detector (MSD, operating in the EI mode at 70 eV). The GC capillary column was a HP5 (30 m × 0.25 mm, film thickness 0.17 μm), the following temperature program was used: 60ºC hold for 3 min, then increased to 210ºC at a rate of 4ºC/min, then held at 210ºC for 15 min, then increased to 300ºC at a rate of 10ºC/min, and finally held at 300ºC for 15 min. Helium was used as carrier gas at a constant flow of 1 mL/min for both columns. Identification of the individual components was performed by comparison with the coinjected pure compounds or, when pure standard was not available, by matching the MS fragmentation patterns and retention indices with the built in libraries or literature data and commercial mass spectral libraries (NIST/EPA/NIH 1999; HP1607 purchased from Agilent Technologies).

Gas chromatography – flame ionization detector analysis

The GC analysis of the EOs was carried out using an Agilent 6890 N instrument equipped with a FID and an HP-5 capillary column (30 m × 0.25 mm, film thickness 0.17 μm). The column temperature program was the same described in GC-MS section. Injector and detector temperatures were 280°C. Helium was used as carrier gas at a flow rate of 1 mL/min. The relative proportions percentages of the HS constituents were obtained by FID peak area normalization without the use of any correction factor.

A hydrocarbon mixture of n-alkanes (C9-C22) was analyzed separately under the same chromatographic conditions used on the HP-5MS capillary columns to calculate the retention indexes with the generalized equation by Vandel Dool, Kartz [17].

As regards α- and β-pinene, these compounds were purchased from Sigma Aldrich.

Antifungal activity assay

The plant EOs as well as the α- and β-pinene activity was evaluated against a wild strain of Penicillium digitatum (Ispa-Pd1) isolated from orange fruit. PDA dishes were inoculated with 20 μL of a conidial suspension at the concentration of 1×104 conidia/mL. The dishes were then treated by vapor contact, after 0 (t 0), 24 (t 24) and 48 (t 48) hours from inoculation, with 0, 50 or 100 ppm of each compound.

Fumigations were performed at 20°C inside suitable boxes (Figure 1), equipped with a circulation fan (12 V - 0, 16 A) and a rubber septum for EOs applications. In order to obtain a gradual and controlled evaporation a heating system was constructed in our laboratory. Heat was delivered by a couple of electric resistances (10 ohms each) placed inside the chambers, connected to a stabilized power supply located outside. The power supply was provided with a voltage regulator (1-15 V of output) that allows controlling the temperature of the resistances. The established amounts of the compounds were injected through the rubber septum into a heatproof glass vessel, placed on the electric resistances. Once each dose was injected inside the vessels, the circulation fans were turned on for 45 min to circulate the compound within the chambers. The experimental design was used to examine the inhibiting impact on the pathogen of the two concentrations of each compound used. After treatments the dishes were incubated for 7 days at 25°C and the radial growths were daily recorded. Moreover, at the fourth day after treatment, some plugs from the PDA plates were taken for Scan Electron Microscopy (SEM) observations.


Figure 1: Fumigation box designed to obtain a gradual and controlled evaporation: (a) Circulation fan attached in the lid inner side; (b) Rubber septum for EOs application; (c) Heatproof glass vessel disposed over a heating system.

Data analysis

Data were reported as mean ± SD of the radial growth of nine dishes, for each concentration and compound. Analysis of Variance (ANOVA) of all data from analytical determinations was performed using the MSTAT-C software (Michigan State University, East Lansing, 1995).

Results and Discussion

The EOs of rosemary and myrtle used in this study were obtained by steam distillation of wild plants collected in Sardinia, Italy. The qualitative analysis was performed by Gas Chromatography-Mass Spectrometry (GC-MS), whereas their percentage composition (semiquantitative analysis) was achieved by GC-FID technique. The chemical composition of rosemary and myrtle EOs are reported in Tables 1 and 2, respectively and data are consistent with those reported in previous papers [18-20]. The GC-MS was also used to perform a dynamic study of the system, aimed to determine the atmosphere composition inside the boxes during the experiment (data not shown).

Compound Composition (%) SD RI ID
tricyclene 0,34 0,03 927 MS, RI
α-thujene 0,23 0,01 928 MS, RI
α-pinene 30,89 0,15 938 MS, RI, STD
camphene 6,76 0,51 951 MS, RI
verbenene 0,97 0,11 963 MS, RI, STD
α-pinene 2,61 0,21 974 MS, RI, STD
myrcene 1,79 0,07 993 MS, RI, STD
α-phellandrene 0,42 0,00 999 MS, RI, STD
α-terpinene 0,47 0,02 1014 MS, RI, STD
p-cymene 1,78 0,05 1026 MS, RI, STD
3-carene 0,45 0,03 1031 MS, RI, STD
limonene 4,19 0,08 1035 MS, RI, STD
cineole 1,8 13,26 0,03 1040 MS, RI, STD
ocimene beta (z) 0,17 0,00 1050 MS, RI
α–terpinene 0,78 0,02 1087 MS, RI, STD
terpinolene 1,01 0,00 1108 MS, RI, STD
linalool 0,88 0,04 1138 MS, RI, STD
chrysantenone 0,28 0,03 1122 MS, RI
capholenal 0,36 0,01 1128 MS, RI
camphor 7,19 0,34 1142 MS, RI, STD
pinocamphone trans 0,16 0,00 1163 MS, RI
pinocarvone 0,18 0,01 1168 MS, RI
borneol 3,80 0,02 1171 MS, RI, STD
terpinen 4-ol 0,81 0,02 1192 MS, RI, STD
α-terpineol 0,61 0,75 1197 MS, RI, STD
myrtenol 0,69 0,63 1197 MS, RI, STD
verbenone 2,78 3,65 1201 MS, RI, STD
trans carveol 2,86 3,92 1215 MS, RI
bornyl acetate 8,94 0,38 1280 MS, RI, STD
pinocarvyl acetate 0,08 0,01 1300 MS, RI
α-caryophyllene 1,52 0,05 1420 MS, RI, STD
α–humulene 0,15 0,01 1453 MS, RI, STD

Table 1: Percentage composition of Rosmarinus officinalis EO.

Compound Composition (%) SD RI ID
propyl butanoate 2,97 0,67 918 MS, RI
α-thujene 0,22 0,07 928 MS, RI
α-pinene 35,01 2,81 938 MS, RI, STD
β-pinene 0,41 0,12 974 MS, RI, STD
myrcene 0,38 0,10 993 MS, RI, STD
propanoic acid 2 methyl 2 methylpropyl 0,62 0,03 1005 MS, RI
butanoic acid 2 methyl 2 methylpropyl 0,36 0,03 1019 MS, RI
p-cymene 0,13 0,09 1026 MS, RI, STD
cineole 1,8 28,37 2,68 1040 MS, RI, STD
ocimene beta (z) 0,23 0,23 1050 MS, RI
ocimene beta (E) 0,33 0,07 1058 MS, RI
γ-terpinene 0,18 0,11 1087 MS, RI, STD
terpinolene 0,23 0,01 1108 MS, RI, STD
linalool 15,81 2,78 1138 MS, RI, STD
pinocarveol cis 0,08 0,03 1177 MS, RI
terpinen 4-ol 0,20 0,04 1192 MS, RI, STD
α-terpineol 2,57 0,51 1197 MS, RI, STD
linalyl acetate 2,38 0,58 1258 MS, RI, STD
pinocarvyl acetate 0,06 0,04 1300 MS, RI
myrtenil acetate 0,08 0,08 1343 MS, RI
terpinil acetate 0,86 0,15 1350 MS, RI, STD
neryl acetate 1,08 1,46 1367 MS, RI, STD
geranyl acetate 2,59 1,87 1387 MS, RI, STD
methyl eugenol 1,16 0,46 1408 MS, RI
α-humulene 0,25 0,09 1453 MS, RI, STD
flavesone 0,41 0,19 1545 MS, RI
caryophyllene oxide 0,24 0,11 1584 MS, RI, STD
humulene epoxide 0,57 0,22 1610 MS, RI
Leptospremone (not ident isom) 0,11 0,07 1621 MS, RI
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