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Analysis on the Fatty Acids and Volatile Components in Pleurotus geesteranus by HS-SPME-GC-MS
The volatile constituents and fatty acids in Pleurotus geesteranus were assayed by headspace solid-phase microextraction coupled with GC-MS. There were 5 volatile compounds in P. geesteranus that accounted for 43.43% of the total ion current peak area, and its main compounds were 2-undecanone (13.99%), 3-ethyl-2,5-dimethyl pyrazine (12.67%), and l-β-bisabolene (6.79%). Fourteen compounds were identified in the ethanol extract of P. geesteranus and 6 fatty acids were identified from the petroleum ether extract, which accounted for 93.72% and 98.48% of the total ion current peak area, the main compounds in the ethanol extract were ethyl linoleate (67.36%) and ethyl palmitate (21.83%), and the main fatty acids in the petroleum ether extract were linoleic acid (78.22%), palmitic acid (10.74%), and oleic acid (8.13%).
Pleurotus geesteranus, which belongs to the family Pleurotaceae, is native to India [1, 2]. P. geesteranus contains many types of components including proteins, fat, polysaccharides, vitamins, trace elements, and 8 essential amino acids . The volatile components of edible fungi such as Morchella esculenta , P. eryngii , Tricholoma matsutake , Boletus edulis , and Agaricus bisporus , which were detected by solid-phase microextraction (SPME), had been reported, and alcohol compounds are the main chemical components of Pleurotus mushrooms. Alcohol and ketone compounds are the key components that affect the flavor of Pleurotus mushrooms. Current studies of P. geesteranus mainly focus on cultivation techniques , preservation , and biological characteristics . A number of research studies report that P. geesteranus has antioxidant , liver protection , antitumor , hypolipidemia , and antibacterial  activities. However, the valuable medicinal components present in P. geesteranus have not been identified yet. In this study, headspace solid-phase microextraction coupled with the gas chromatography-mass spectrometry (HS-SPME-GC/MS) technique was used to assay the volatile components of P. geesteranus.
2. Materials and Methods
Dried powder of P. geesteranus was provided by Henan Longfeng Industrial Co., Ltd. (Qingfeng, Henan, China) in October 2019.
The GC/MS instrument was an Agilent 7890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a 5975 mass spectrometer (Agilent Technologies). A solid-phase microextraction (SPME) device (Supelco, USA) and the extraction head were 65 μm polydimethylsiloxane (PDMS-DVB, Supelco, USA). C6–C26 n-alkanes was purchased from Alfa Aesar, Haverhill, USA.
930 g of P. geesteranus powder was taken, and petroleum ether was added to extract 3 times at room temperature for 72 h. After filtration, the filtrate was concentrated by evaporation to obtain the petroleum ether extract with a yield of 1.39%. The residue was extracted with 70% ethanol at room temperature and then filtered. The filtrate was concentrated by evaporation to obtain the ethanol extract with a yield of 5.37%.
2.3. Methyl Esterification
Petroleum ether extract (0.5 g) was added into a 10 mL test tube with petroleum ether/ether (4/3) to 5 mL, and then 4 mL of 0.5 mol/L KOH–CH3OH solution was added under 70°C water bath for 10 min. After cooling, distilled water (10 mL) was added to sonicate and centrifuge solutions, and the supernatant was concentrated.
A manual SPME device with a fiber precoated with a 65 μm thick layer of polydimethylsiloxane/divinylbenzene (PDMS-DVB) was used for extraction. The dry powder of P. geesteranus, ethanol extract, and methylated products was placed in 5 mL vials, and then, the SPME fiber was exposed in the upper space of the sealed vial at 60°C for 30 min. After that, the fiber was withdrawn and directly inserted into the GC-MS inlet (temperature 250°C) for 1 min.
2.5. Determination of Fatty Acids and Volatile Components in Pleurotus geesteranus
The fatty acids and volatile constituents were analyzed using the GC/MS instrument. The GC was fitted with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies). High-purity helium (99.999%) was used as the carrier gas at a flow rate of 1.0 mL/min. The inlet temperature was 250°C. The temperature program was as follows: the initial column temperature was 50°C for 2.0 min, then programmed to 120°C at a rate of 8°C/min, and held for 2 min, and programmed finally to 220°C at a rate of 4°C/min and held at 220°C for 5 min. Split injection with a split ratio of 10 : 1 was used. The MS was operated in the SCAN mode (m/z 30–400) with electron impact ionization at an ionization energy of 80 eV, the ion source temperature was 230°C, the quadrupole temperature was 150°C, transmission line temperature was 280°C, and electron multiplier voltage was 1635 V. According to the previous reports in the literature [17, 18], the Kovats retention index (KI) was calculated by using the retention times of C6–C26n-alkanes that were injected under the same chromatographic conditions. The Kovats retention index calculation formula was as follows:where n and n + 1 are the number of normal alkane carbon atoms before and after the outflow; tRn and tRn+1 are the retention times of the corresponding normal alkane, respectively; and tR is the retention time of the unknown substance in the gas chromatography (tRn < tR < tRn+1).
3. Results and Discussion
According to the above conditions, the components of P. geesteranus powder, ethanol extract, and petroleum ether extract were analyzed by GC-MS, and their total ion flow chromatograms were obtained, respectively. The fatty acids and volatile constituents were identified by their mass spectra with the Rtlpest3.L, Nist08.L spectral library, combined with retention index published in the literature  and related websites (http://www.vcf-online.nl). Relative percentage amounts of the separated compounds were calculated automatically from peak areas of the total ion chromatograms.
Five compounds accounting for 43.43% were identified from the powder of P. geesteranus, 14 compounds accounting for 93.72% were identified from ethanol extracts, and 6 fatty acids accounting for 98.48% were identified from petroleum ether extracts after methylation. The specific results are presented in Table 1 and Table 2.
As shown in Table 1 and Figure 1, the main compounds in P. geesteranus powder were 2-undecanone (13.99%), 3-ethyl-2, 5-dimethylpyrazine (12.67%), l-β-bisabolene (6.79%), and 2-Phenylcrotonaldehyde (6.08%). Among them, the content of 2-undecanone (13.99%) was the highest. It has been reported that it is not only an important aroma component of P. geesteranus, but also has an insect repellent effect . In addition, 3-ethyl-2,5-dimethylpyrazine is a common aroma active substance, and l-β-bisabolene is mainly used as an edible flavor.
In Table 2 and Figure 2, we could see that the main components in the ethanol extract were ethyl linoleate (67.36%) and ethyl palmitate (21.83%). Compounds in the petroleum ether extract after methylation were linoleate (78.22%), palmitate (10.74%), and elaidate (8.13%) as shown in Table 2 and Figure 3. There were four common compounds: estragole, cis-anethol, elaidate, and linoleate, and the content in the petroleum ether extract was higher than that in the ethanol extract. Among the ethanol extract components, ethyl linoleate had the highest content, and it has a variety of pharmacological effects, such as anti-inflammatory , antioxidant , and lowering human cholesterol . The relative content of ethyl palmitate in ethanol extracts was also high, not only as a vasodilator factor for lowering blood pressure , but also for preventing nonalcoholic steatohepatitis , further having anti-inflammatory  and antifibrosis effects . Linoleate was the component with the highest content in petroleum ether extracts and has anti-inflammatory , antithrombotic , anticancer, and antiatherosclerotic effects .
Previous studies have found that the main fatty acid components of edible fungi such as Lentinus edodes, Dictyophora indusiata, and Auricularia auricula are linoleic acid, palmitic acid, and linolenic acid [31, 32]. In this study, we also found that linoleic acid is the main fatty acid in P. geesteranus. Liu  analyzed the volatile components of P. geesteranus by the HP-SPME-GC-MS method, 19 compounds were identified, and the major ones are 3-octanol (55.12%), 1-octen-3-ol (20.03%), and 3-octanone (19.22%), which is different from our research.
The volatile constituents and fatty acids in P. geesteranus were assayed by the HS-SPME-GC-MS method. There were 5 volatile constituents in P. geesteranus, 14 compounds were identified from the ethanol extract, and 6 fatty acids were identified from the petroleum ether extract. It was found that the main volatile component is 2-undecanone and the main fatty acid is linoleic acid.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
All authors declare that there are no conflicts of interest regarding this study.
This work was funded by the National Key R&D Program of China (2018YFD0400200) and Key Project in Science and Technology Agency of Henan Province (202102110283).
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