Comparative Study of Fatty Acids Profile of Wild Mushroom Species from Turkey

A comparative study was enhanced on the fatty acids profile of wild mushroom species; namely, Armillaria tabescens, Leucopaxillus gentianeus, Pleurotus eryngii and Suillus granulatus growing in Turkey. A total of fifteen fatty acids were identified and quantified by GC-FID and GC-MS. The main fatty acid was linoleic acid in L. gentianeus, P. eryngii, and S. granulatus varied from 40.92 to 68.24% while oleic acid was major fatty acid in A. tabescens (35.92%). The other major fatty acids were palmitic and stearic acids in studied mushrooms. The concentration of total saturated, monounsaturated and polyunsaturated fatty acids ranged from 16.71 to 31.90%, 12.04 to 40.41%, 27.64 to 68.24%, respectively. The present study showed that unsaturated fatty acids concentrations were higher than saturated ones. Since unsaturated fatty acids are valuable healthy compounds for human nutrition, mushrooms can be considered as a rich dietary natural source.


INTRODUCTION
Since ancient times, mushrooms have been considered as valuable healthy foods because of their taste, flavour, high nutritional values, and various biological activities such as antioxidant, antifungal, anti-immunomodulatory, antitumor, antibacterial, antidiabetic, anticholesterol, anti-coagulant, antiviral, anti-inflammatory and cytotoxic [1][2][3][4].Mushrooms are nutritionally rich in vitamins, proteins, minerals, fibers and moisture as well as comprise considerable levels of essential fatty acids and phenolic compounds.In addition, they are low in calories and fats [5].Fatty acids are important constituents of mushrooms.It is known that fatty acids, especially polyunsaturated ones the omega-3, and -6 series are necessary human health for preventing and treatment of hypertension, coronary artery disease, diabetes, osteoporosis, arthritis, cancer, other inflammatory and autoimmune disorders [6].The research of the fatty acid compositions of mushrooms has interested by the researchers due to having various useful effects on health.Especially, the linoleic acid (18:2) and linolenic acid (18:3) are polyunsaturated acids that are fundamental in human diets [7].Additionally, the linoleic acid is the precursor of arachidonic acid and it has a role in prostaglandins biosynthesis [8].So far, some studies are reported on the fatty acids of mushrooms [5,[9][10][11][12][13][14].Because of important properties of polyunsaturated fatty acids, scientific studies about mushrooms including polyunsaturated fatty acids have increased with each passing day.The aim of this work was to investigate the fatty acid compositions of four mushrooms namely; Armillaria tabescens (Scop.)Emel, Leucopaxillus gentianeus (Quél.)Kotl, Pleurotus eryngii var.eryngii (DC.)Quél and Suillus granulatus (L.) Roussel growing in Turkey using GC-FID and GC-MS.

Mushroom materials
Mushroom species were collected from Uşak, province of Turkey and were identified by Dr. Aziz Türkoğlu, Muğla Sıtkı Koçman University.They were deposited in the Fungarium of Department of Biology, Muğla Sıtkı Koçman University and were stored at 18 ºC until they were used.The species names, collection localities and dates, family, edibility and fungarium numbers of four mushroom species are given in Table 1.

Extraction
Each mushroom was extracted individually with 500 mL n-hexane for three times (24 h x 3) at room temperature (298 K), then filtered and evaporated to dryness under vacuum.They stored at refrigerator until they were methylated with 14 % BF3:MeOH solution.

Derivatization of fatty acids with GC and GC-MS
The n-hexane extract (100 mg) was dissolved in 0.5 M NaOH (2 mL) in a 25 mL flask.After the flask was heated in a water bath (50 °C), then 2 mL BF3:MeOH was added.The mixture was boiled for 2 minutes, and then the mixture was left until it cooled down, and then the volume was completed to 25 mL with saturated NaCl solution.Esters were extracted with n-hexane; thus, the organic layer was separated.The hexane layer was washed with a potassium bicarbonate solution (4 mL, 2 %) and dried with anhydrous Na2SO4 and filtered.The organic solvent was removed under reduced pressure by a rotary evaporator to give methyl esters.

Gas chromatography (GC)
GC analyses of the methyl derivatives of fatty acids were performed by Shimadzu GC-17 AAF, V3, 230 V series gas chromatography (Japan) coupled with a Flame Ionisation Detector (FID) and a DB-1 fused silica capillary non-polar column (30 m x 0.25 id., film thickness 0.25 µm).Injector and detector temperatures were 250 and 270 °C, respectively, carrier gas was He at a flow rate of 1.4 mL/min; sample size, 1.0 µL; split ratio, 50:1.The initial oven temperature was held at 100 °C for 5 min, then increased up to 238 ºC with 3 ºC/min increments and held at this temperature for 9 min.The relative percentages of separated compounds were calculated by using GC Solution computer program.

Gas chromatography-Mass spectrometry (GC-MS)
GC-MS analyses of the methyl derivatives of fatty acids using Varian Saturn 2100T (USA) coupled with an ion trap mass spectrometer (IT-MSD) and a DB-1 MS fused silica nonpolar capillary column (30 m x 0.25 mm ID, film thickness 0.25 µm).For GC-MSD detection, an electron ionization system with ionization energy of 70 eV was used.Carrier gas was helium (15 psi) at a flow rate of 1.3 mL/min.Injector and MS transfer line temperatures were set at 250 and 200 °C, respectively.The oven temperature was held at 100 °C for 5 min, then increased up to 238 °C with 3 °C/min increments and held at this temperature for 9 min.Diluted samples (1/25, w/v, in hexane) of 0.2 µL were injected manually in the split mode.Split ratio was 50:1.EI-MS were taken at 70 eV ionization energy.Mass range was from m/z 28 to 650 amu.Scan time 0.5 sec with 0.1 inters scan delays.The library search was carried out using NIST and Wiley 2005 (Gas Chromatography-Mass Spectrometry) GC-MS libraries.FAME (Fatty acid Methyl Ester) mixture (Supelco™ 37, Catalog no: 47885-U) were identified by comparing their retention times with those of the pure FAMEs standards.

Statistical Analysis
All data on the fatty acid composition were the averages of three parallel sample measurements.The data were recorded as the mean ± S.E.M. Significant differences between the means were determined by student's-t test, and p values <0.05 were regarded as significant.
As shown in Table 2, linoleic acid, which is the most abundant fatty acid, was determined between 27.64 -68.24% in the studied mushrooms.This fatty acid is very important for human diet.Likewise, it is known that linoleic acid is the precursor of 1-octen-3-ol, known as the alcohol of mushroom, which is the principal aromatic compound in most mushrooms and might contribute to mushrooms flavour [21].Oleic acid, which is the main monounsaturated fatty acid, was also major fatty acid ranged from 10.05 to 39.78% in studied mushrooms.Both linoleic and oleic acids decrease the risk of cardiovascular disease; thus, mushrooms are recommended people in the human diet for the high blood cholesterol [22].
The total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA) and unsaturated fatty acid (UFA) compositions of the studied mushroom species were ranged from 16.71 to 31.90%, 12.04 to 40.41%, 27.64 to 68.24% and 68.05 to 83.26%, respectively.As result of high level of palmitic acid in A. tabescens, SFA content (31.90%) was higher than other studied species.PUFA content was present in highest level in P. eryngii due to linoleic acid (68.24%).Unsaturated fatty acids increase nutritional values of mushrooms.As given in Table 2, the ratio of linoleic acid to oleic acid (L/O) was found between 0.77-6.79.The linoleic: oleic acid ratio provides an important criterion from a chemotaxonomic viewpoint for the upcoming studies and is useful for the taxonomical differentiation between species of the same genus.

CONCLUSIONS
Mushrooms can be considered as healthy foods because of their low-fat composition, low calories and high essential fatty acid levels especially linoleic acid.Low-fat diets and low calorie are recommended by people with high blood cholesterol.Therefore, mushrooms are perfect food in human diet.The present study indicates that unsaturated fatty acids are higher than saturated ones.Since mushrooms contain high percentage of unsaturated and essential fatty acids, the findings of results support their potential uses as food supplements and nutraceuticals.

Table 1 .
Collection localities and dates, family, edibility and fungarium numbers of studied mushroom species

Table 2 .
The fatty acid compositions (%) of the mushroom species a