The Effect of Plant Essential Oil and Extracts on Fatty Acid Profile of Virgin Olive Oil Stored in Different Packaging Materials.

In this study, the combined effect of different packaging materials (transparent PET, transparent glass, glass-PET bottle and tin), some aromatic herbs (thyme, rosemary, sage and olive leaf) and also their essential oils (thyme, rosemary and sage) on fatty acid composition of virgin olive oil was investigated during storage period. The initial amounts of the main fatty acids as oleic, palmitic and linoleic acids were determined as 72.89%, 11.89% and 8.96%, respectively. The addition of aromatic plants and essential oils did not effect the fatty acid profile. Also, packaging materials had a minor influence on fatty acids. In the 6th month of storage, the oleic acid contents of olive oils showed the increase in all of samples. The highest increase was observed in olive oil stored in glass-PET (74.30-75.01%), followed by stored in glass bottle (73.41-74.82%). Generally, during the storage, the differences of fatty acid contents were in minor level. The fatty acid composition of olive oils stored under different essential oil and extract concentrations showed partial differences depending on the extract type and concentration.

essential oils of three Tunisian aromatic plants and their efficacy in preventing the oxidative changes on storage of refined oils. Antioxidants inhibit the free radical autoxidation and prevent polyunsaturated lipids from deterioration 10 . Additionally, the quality and shelf life of oil are effected from packaging material. Appropriate packaging of olive oil has importance to assure stability during distribution and marketing 11 . Generally, glass, tin or plastic bottles are used for packaging of the olive oil 12 . The objective of this study was to investigate the effect of packaging materials glass, PET, tin and glass-PET , several plants thyme, sage, rosemary and olive leaf and essential oils belonged to thyme, sage and rosemary on fatty acid composition of virgin olive oil during storage period.
from Mut and Silifke districts of Mersin in Turkey. The thyme Satureja hortensis , sage Salvia officinalis and rosemary Rosmarinus officinalis were purchased from Antalya in Turkey. Tin, PET, clear glass and glass-PET outer part PET; inner glass bottle bottle were used as packaging in this study. Extra virgin olive oil was filled in tin, PET, clear glass and glass-PET outer part PET; inner glass bottle bottles, which were separated into 4 groups and was stored for 6 months.
1. group: Control group only virgin olive oil was found in tin, PET, clear glass and glass-PET bottles . 2. group: Aromatic plants as thyme, rosemary, sage and olive leaf 1 were added to the bottled olive oil. 3. group: Essential oils of thyme, rosemary and sage 0.1 were added to the bottled olive oil. 4. group: Positive control butylated hydroxyanisole, BHA, 0.01 was added to the bottled olive oils. The fatty acid profile of olive oil samples stored in open air for 6 months were analyzed on month 1, 2, 3, 4, 5 and 6. The initial fatty acid composition of the virgin olive oil are given in Table 1.

Method 2.2.1 Extraction of essential oil and extracts
Aromatic plants were ground with grinder. Essential oils of thyme, rosemary and sage were extracted using clevenger apparatus for 6 h. The spices were dried, grounded and extracted in 90 methanol 9 water 1 acetic acid mix. The extraction duration were 24 hour. After filtration, the filtrate were evaporated under vacuum, less than 45 .

Fatty acid composition
Olive oil samples were esterificated according to ISO-5509 13 method. Fatty acid methyl esters of samples were analysed gas chromatography Shimadzu GC-2010 equipped with flame-ionization detector FID and capillary column Tecnocroma TR-CN100, 60 m 0.25 mm, film thickness: 0.20 µm . The temperature of injection block and detector was 260 . Mobile phase was nitrogen with 1.51 mL/min flow rate. Total flow rate was 80 mL/min and split rate was also 1/40. Column temperature was programmed 120 for 5 minutes and increased 240 at 4 / min and held 25 minutes at 240 . Commercial mixtures of fatty acid methyl esters were used as reference data for the relative retention times 14 .

Statistical Analyses
All analyses were carried out three times and the results were mean standard deviation MSTAT C of independent package type and extract concentrations 15 .

Results and Discussion
The fatty acid composition of virgin olive oil during storage are given Table 2. The main fatty acids were determined as oleic 69.41-74.38 , palmitic 10.87-13.17 and linoleic 8. 14-9.95 acids. In general, no significant change in the fatty acid composition of olive oil was observed during storage. In the first month, a decrease in the oleic acid content was observed, while there was an increase in the palmitic acid content of the samples stored in the PET and tin bottles. The results of the fatty acid composition at 2 nd month were very close to the pre-storage values of the olive oil samples. Samples stored in the pet bottle for 3 months showed a decrease in the oleic acid content at the minor level. The oleic acid amounts of especially olive oils added thyme, sage and olive leaf decreased to 69.74 , 69.41 and 70.08 , respectively. In addition, behenic acid was not detected in these oils. The significant changes were not detected in samples stored in glass, tin and glass-pet bottles. According to the results obtained at the 4 th month of storage, a minor increase in the oleic acid contents and a decrease in the amount of palmitic acid of the oils stored in the glass bottles when compared with the results of initial or 3 rd month. Stearic acid did not determine in oils added with thyme, sage and olive leaf and stored in glass-pet bottle. The content of stearic acid in olive oil added with rosemary decreased to 0.23 . No significant changes were detected in samples stored in tin and pet bottles. In the 5 th month of storage, the oils stored in PET showed a decrease in the oleic acid content and an increase in the amount of palmitic acid. The oleic acid contents of rosemary, sage and sage essential oil were determined as 70.80 , 70.86 and 70.97 , respectively. In addition, the highest palmitic acid contents were observed in samples containing thyme 13.17 and thyme volatile oil 13.04 . Samples stored in glass, tin and cam-pet bottles showed a slight decrease in oleic acid content and a minor increase in palmitic acid content compared to the results of the previous month. According to the fatty acid composition of olive oils in the 6 th month of storage, the      10.88 contents were found in olive oil added rosemary essential oil and stored in a glass-pet bottle. Moreover, significant reduction was observed in the arachidonic acid contents of the oils stored in the tin after 3 months. While fluctuations were observed in the oleic acid content of olive oils until the 5 th month of storage, a partial increase was observed in the oleic acid content of the oils stored in all packages in the 6 th month of storage. The fatty acid composition of extra virgin olive oil provides nutritional benefits because of the high amount of oleic acid and the balanced proportion of saturated and polyunsaturated fatty acids 8 . Mendez  16 also reported in broilers that dietary thymol carvacrol supplementation modified the fatty acid composition of serum and thigh muscle by reducing total SFA and increasing total PUFA and n-6 fatty acids in serum and thigh and increasing total monounsaturated fatty acids MUFA in the thigh. Vakili and Majidzadeh Heravi 17 also found that palmitic acid decreased and linolenic acid increased in egg yolk with thyme extract supplementation. The fatty acid composition of the egg yolk is closely related to the feed lipids consumed by the laying hens, and linoleic acid in yolk was increased significantly 18 .
Our results with the studies conducted have shown similarities in terms of some increase and decrease. These differences may possibly result from the composition of the material used, the applications in vitro or in vivo, and the analytical conditions. According to the fatty acid profile of one of the virgin olive oils, the oleic, linoleic and palmitic acid amounts of oils stored in plastic, glass and tin did not show an important change until 3 months of storage. However, the oleic acid contents of samples stored in plastic, glass and tin reduced from 72.4 to 70.5 ; to 71.0 ; to 71.4 , respectively after 6 months. Additionally, palmitic and linoleic acid contents of oils remained almost the same during storage period.

Conclusion
In the first month, a decrease in the oleic acid content was observed, while there was an increase in the palmitic acid content of the samples stored in the PET and tin bottles. The results of the fatty acid composition at 2 nd month were very close to the pre-storage values of the olive oil samples. While fluctuations were observed in the oleic acid content of olive oils until the 5 th month of storage, a partial increase was observed in the oleic acid content of the oils stored in all packages in the 6 th month of storage. In addition, there was a partial decrease in the linoleic acid content from the 1 st month of storage. However, this decrease remained constant from the 2 nd month of storage until the 6 th month. In general, the packaging effect was poor on the fatty acid composition of the oils. However, the effect of essential oils and extracts partially affected the fatty acid composition.