Ozonation as an alternative method for the disinfection of dried herb material shows promising results concerning the microbial load reduction. However, there is not enough data about the effect of the method on the essential oil quality. The aim of this study was to investigate the effect of ozone on the essential oil content and composition of dried oregano, thyme and lemon verbena. Quantitative and qualitative essential oil measurements were performed before and after ozonation. The results showed that in cases of oregano and lemon verbena, no statistically significant difference was observed either in total essential oil content or on any of their compound concentration. However ozonation may affect the concentration of some components since in the case of thyme the concentration of 8 compounds decreased.
Aromatic plants and essential oils are widely used by food, cosmetic and
pharmaceutical industry due to their organoleptic characteristics and effective
bioactive compounds
Ozone application seems to be the most promising technique since it leaves no
residues and it is environmental friendly, in comparison to other methods developed
for the microbial load reduction of aromatic plants
Materials & Methods 2.1 Dried Plant Material Εfficient amount of dried plant material, namely oregano (Origanum vulgare ssp. hirtum), thyme (Thymus vulgaris) and lemon verbena (Aloysia triphylla syn. Lippia citriodora) was provided by local producers of Magnesia (Thessaly, Greece). The plant material was harvested during spring and summer of 2015, dried naturally under shade and was stored under ambient conditions until the experiments implementation.
The ozonation device used for the dried plant material disinfection, consisted of the oxygen tank, the ozone generator and the airtight chamber. Inside the chamber a sensor was placed in order to measure the O3 concentration. To avoid the accumulation of O3 above the treated plant material, a fan was placed in the upper side of the chamber. Three samples of 100 g from each plant species were placed inside 1 mm mesh sieves (27 cm diameter). Sieves were placed inside the chamber and ozone was produced by providing dry oxygen to the ozone generator. The ozone concentration was adjusted to 4 ppm for a time period of 30 and 60 min. These values were set in accordance to Torlak et al. (2013), who stated that ozonation with less than 3 ppm O3 (even for 90 min period) was ineffective for sufficient microbial reduction, whereas a higher concentration of 5 ppm O3 for 120 min was effective but color degradation was observed.
The essential oils of the above mentioned plant material, were extracted by watersteam distillation using Clevenger type apparatus. The distillation time and respectively the amount of plant material for each species are shown in Table 1. The volume of the essential oil yield was measured, and stored at 4˚C after the addition of the proper quantity of anhydrous Na2SO4.
The essential oils were analyzed by GC-MS on a fused silica DB-5 column, using a
Gas Chromatograph 17A Ver. 3 interfaced with a mass spectrometer Shimadzu
QP5050A supported by the GC/MS Solution Ver1.21 software, using the method
described previously
The data were statistically analyzed by analysis of variance (ANOVA) using Statgraphics Centurion XVI. Duncan’s multiple range test was used at a significance level of 0.05. 3
Essential oil content of each plant material was measured before and after O3 treatment. Ozonation for 30 min, was an adequate period to reduce the microbial load of oregano samples, whereas a period of 60 min was needed for lemon verbena and thyme samples. Distillation was performed only to the samples where the ozonation was effective in microbial load reduction. The essential oil content of the above mentioned aromatic plants was within range according to Goliaris et al. (2002), Kokkini (1997), Marzec et al. (2010) and Kizil et al. (2016). As shown in Table 2, the essential oil content of oregano and lemon verbena before and after treatment did not show a statistically significant difference. However the content of thyme essential oil was increased. This may be attributed to the reduction of the water content of the samples during ozonation, since the relative humidity inside the chamber was increased. This probably indicates that an amount of water was removed from the plant material to chambers atmosphere (Table 3). Accordingly, 40g of thyme plant material taken during sampling after the treatment possibly had lower water content and therefore higher content in essential oil. Consequently, ozonation does not seem to affect negatively the total essential oil yield of the treated plant material when applied under the above mentioned conditions. In order to verify this assumption, water content measurements of the samples must also be taken before and after O3 treatment.
Origanum vulgare ssp. hirtum Thymus vulgaris Aloysia triphylla
Essential oil content (% dry weight) 0 min 30 min 3.30±0.13 3.30±0.18 0.92±0.04a 0.61±0.03 60 min 1.35±0.02a 0.66±0.02 The results obtained were expressed as Mean ± SD, n = 3.
Mean values followed by the same letters at the same row denote statistically significant difference at a probability of P < 0.05.
The results concerning essential oil composition revealed that ozonation of dried
lemon verbena material did not affect its essential oil chemical composition (Table
4). The major constituents were β-citral ranged from 25.91 to 26.73%, α-citral ranged
from 18.55 to 19.22% , limonene ranged from 12.29 to 13.31% and 1,8 cineol
ranged from 8.32 to 8.07%, before and after the application respectively. However,
no statistically significant differences were observed between the above mentioned
values before and after treatment. Citral a and citral b contributed more than 40% of
the total essential oil content which is in agreement with Vogel et al. (1999) results.
As stated by Kizil et al. (2016), different essential oil composition, is probably due to
the different geographical and ecological factors effect. Growth stage and cultivation
methods may also affect the essential oil composition
As shown on Table 5, the main components of thyme essential oil where
pcymene ranged from 31.97 to 32.01%, thymol ranged from 30.65 to 31.5%, carvacrol
ranged from 13.11 to 14.5% and γ-terpinene 6.05%. These components contributed
more than 80% of the total essential oil content. However, the concentration of none
of these components showed significant difference after the ozonation. The
composition of thyme’s essential oil presented in this work was in accordance to that
referred by Raal et al. (2005). Consequently, this essential oil could be classified as
thymol chemotype and specifically to the subgroup p-cymene> thymol> γ-terpinene
Verbena also contained at some percentages 1,8-cineol, linalool and β-caryophyllene while oregano contained cis-sabinene hydrate, linalool, carvacrol methyl ether and βcaryophyllene. In contrast to thyme samples, no reduction was observed for these compounds. This might be attributed to the low ozonation time of oregano samples and to the differences of the plant material (leaf surface and size) among verbena and thyme samples that were treated for the same time. As a consequence, more studies need to be conducted in order to identify the appropriate O3 concentration and ozonation period so that the high quality of the essential oil can be maintained. The results obtained were expressed as Mean ± SD, n = 3.
Mean values followed by the same letters at the same row denote statistically significant difference at a probability of P < 0.05.
Essential oil analysis of oregano (Table 6), showed a typical composition of carvacrol chemotype oregano, as referred by Vokou et al. (1993). The main constituents before and after the ozone treatment were two isomeric phenols namely carvacrol ranged from 63.26 to 64.55% and thymol ranged from 5.06 to 4.58%, and their precursors namely p-cymene ranged from 13.11 to 11.9% and γ-terpinene ranged from 8.15 to 8.37%. These components contributed more than 80% of the total essential oil content. The statistical analysis did not point out any difference among the constituents before and after treatment. These results show that probably ozone treatment did not affect the chemical composition of oregano’s essential oil. The results of the present study revealed that O3 application for the disinfection of oregano and lemon verbena dried plant material did not reduce the essential oil amount and the concentration of the main constituents. In contrast, ozonation may affect the amount of some components, since in the case of thyme the concentration of 8 compounds was reduced. However, there is not enough scientific evidence that this reduction can affect the essential oil quality. More studies need to be conducted in order to identify the appropriate O3 concentration and ozonation period in order to maintain the high quality of the essential oil.