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Certain kinds of mushrooms are considered a delicacy throughout the world. The flavor qualities are greatly dependent on the numerous volatile and semi-volatile compounds (VOC's) contained within the mushroom complex. However, others are poisonous and can be very detrimental to one's health. In addition, mushrooms have been and are currently being investigated as sources of natural products for new and better flavors, as well as those which may be used by the pharmaceutical industry in the development of life saving drugs. Analytical techniques are needed to identify and quantitate VOC's present in mushrooms, so that these techniques can be used to analyze the components which are responsible for flavor and off-flavors, as well as identify compounds which may be toxic or unique to the species of mushroom. Volatile organic compounds were collected from several kinds of edible mushrooms and analyzed by using a purge and trap technique (P&T), followed by trapping on an adsorbent resin and subsequent analysis by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS).
Samples were collected using a Scientific Instrument Services Purge & Trap System. This apparatus (Fig. 1) consists of a purge gas inlet connected to a stainless steel purging needle that is inserted through an adaptor fitting into a 10 ml test tube. A dry purge gas inlet is located at a right angle to the purge gas inlet at the top of the apparatus (Fig. 1). The purpose of the dry purge is to reduce the water vapor condensation on the adsorbent trap. This problem can be especially troublesome when isolating volatiles from high moisture samples at high temperatures. Although the adsorbent traps packed with Tenax® have a low affinity for water, it is inevitable that some water condensation will occur in the trap due to the high relative humidity of the purge gas as it exits the apparatus. When moisture condenses on the adsorbent resin, it can block the pores of the resin matrix and thereby drastically reduce the trapping of volatile organics. Opposite the dry purge inlet is the connector for the glass-lined stainless steel (GLT) desorption tube containing the adsorbent resin (Fig. 1). The Purge & Trap System also contains two ball rotameters with adjustable needle valves mounted on a stationary base and permits the visual indication and independent adjustment of the gas flow to the purge gas and dry purge inlets.
Figure 1 - Purge & Trap System
All experiments were conducted using a Scientific Instrument Services model TD-2 Short Path Thermal Desorption System accessory (1&2) connected to the injection port of an HP 5890 Series II GC interfaced to an HP 5971 Mass Selective Detector. The mass spectrometer was operated in the electron impact mode (EI) at 70eV and scanned from 35 to 400 daltons during the GC run for the total ion chromatogram.
A short 0.5 meter by 0.53 mm diameter deactivated fused silica precolumn was attached to the injection port end of a 60 meter x 0.25 mm i.d. DB-5MS capillary column containing a 0.25 µm film thickness. The GC injection port was set to 250 degrees C and direct splitless analysis was used. The head of the column was maintained at -70 degrees C using an S.I.S. Cryotrap model 951 during the desorption process in order to trap the volatiles at the front of the precolumn. The trap was then ballistically heated to 200 degrees C at the end of the desorption process and the GC oven was temperature programmed from 35 degrees C (hold for 5 minutes) to 80 degrees C at a rate of 10 degrees /min and to 280 degrees C at 4 degrees /min.
Six different kinds of edible mushrooms (Shiitake, Portabella, Agaricus bisporus, Agaricus campestris, Lactarius & puffball Calvatia) were analyzed to compare and quantify the volatile organics contained within the mushroom complex. For quantification, a deuterated cymene internal standard was spiked into the adsorbent traps after the sample had been isolated. No correction for extraction efficiency of recovery is achieved using this technique; however, it serves as a useful means of quantifying the levels of components relative from the mushroom samples. (3).
Samples sizes of 1-2 g of mushroom (pileus & stipe) were transferred into a 10 ml purge & trap tube and heated to 90 degrees C. Samples were sparged with high purity helium at 20 ml/min with an additional 25 ml/min dry purge using the S.I.S. Purge & Trap System. Volatile analytes were gas extracted and carried to a preconditioned 4.0 mm i.d. glass-lined stainless steel desorption tube packed with 100 mg of Tenax TA. Once the samples were collected, they were spiked with 200 ng of d-14 cymene internal standard by injecting 1 µl of a 200 ng/µl of a d-14 cymene stock solution in methanol by syringe injection into the Tenax matrix.
The desorption tube with sample and internal standard were then attached to the Short Path Thermal Desorption System and a syringe needle attached. The desorption tube was injected into the GC injection port and desorbed at a desorption block temperature of 250 degrees C for 5 minutes. The desorbed volatiles were trapped at the front of the GC column and subsequently eluted through the GC column for detection and identification by the mass spectrometer.
Results and Discussion
Three cultivated mushrooms (Shiitake, Portabella, Agaricus bisporus) and three edible wild mushrooms (Agaricus campestris, Lactarius sp., Calvatia sp.) were analyzed to identify, compare and quantify the volatile organics present. Table I shows the VOC's detected and the relative amounts of these compounds in each mushroom. The mushrooms were found to contain numerous middle-chain aliphatic alcohols, aldehydes and ketones, which are believed to be the degradation products of fatty acids (4). The compounds 3-methyl-butyraldehyde and 2,6 bis (1,1-dimethyl ethyl)-phenol were identified in each of the mushrooms (Fig. 2-6) with the exception of the giant puffball Calvatia. These compounds are thought to contribute to the characteristic aroma of the mushroom. Phenylacetaldehyde which appears to be an important flavor compound was present in each of the mushrooms except for A. bisporus (Figs 2,3,5-7). The flavor compound benzaldehyde was also detected in Portabella as well as A. bisporus (Figs. 3&4). Lactarius sp. contained a high concentration of the aliphatic compound 1-octen-3-ol (Fig. 6). 3-Octanone was also found in the Shiitake, Portabella and A. bisporus mushrooms (Figs. 3&4). The presence of 1-octen-3-ol and 3-octanone suggests that the activity of lipoxygenase and hydroperoxide lyase producing C8 compounds from linoleic acid was stronger in these mushrooms (4). 1-Methyl-1H-pyrrole which was detected in Shiitake was the only nitrogenous compound identified (Fig. 2). 2-formylpyrrole has also been found in the pentane extract of dried Boletus edulis (5) and in liquid cultures of Polyporous tuberasters (4). This nitrogen compound has been identified in cocoa and bread and may be produced by nonenzymatic browning reactions. In addition, the sulfur compounds 1,2,4-trithiolane, 1,2,4,6-tetrathiepane and dimethyl disulfide were identified in Shiitake (Fig. 2). This suggests that they were produced by chemical reactions in which one reactant was an amino acid containing sulfur. The meadow mushroom A. campestris and the giant puffball Calvatia were found to contain high concentrations of phenol (Fig. 5) and methoxybenzene (Fig. 7), respectively. Although similar to the button mushroom, A. bisporus, found at the grocery store, the meadow mushroom is far richer in flavor. It is superior in any recipe that calls for its relatively bland cultivated brother. Both cultivated and edible wild mushrooms possessed many common compounds although each had its own fingerprint chromatograph. However, trace amounts of the compound Ionol 2 were detected only in the cultivated mushrooms (Figs. 2-4). The presence of this compound may be due to increased pigment degradation over time in the cultivated mushrooms compared to the freshly harvested edible wild mushrooms.
Figure 2 - Shitake Mushroom - 1.169 g. Collected for 35 min At 90 Degrees C At 20 ml/min With 25 ml/min Dry Purge Thermally Desorbed At 250 Degrees C For 5 min
Figure 3 - Portabella - 1.539 g. Collected For 35 min At 90 Degrees C At 20 ml/min With 25 ml/min Dry Purge Thermally Desorbed At 250 Degrees C For 5 min
Figure 4 - A. bisporus - 1.577 g. Collected For 35 min At 90 Degrees C At 20 ml/min With 25 ml/min Dry Purge Thermally Desorbed At 250 Degrees C For 5 min
Figure 5 - A. campestris - 1.906g. Collected For 35 min At 90 Degrees C At 20 ml/min With 25 ml/min Dry Purge Thermally Desorbed At 250 Degrees C For 5 min
Figure 6 - Lactarius - 1.903 g. Collected For 35 min At 90 Degrees C At 20 ml/min With 25 ml/min Dry Purge Thermally Desorbed At 250 Degrees C For 5 min
Figure 7 - Calvatia - 1.417g. Collected For 35 min At 90 Degrees C At 20 ml/min With 25 ml/min Dry Purge Thermally Desorbed At 250 Degrees C For 5 min
Many kinds of flavors are used in the food industry, and there is a demand for new and improved ones, especially natural ones. Such a source are mushrooms which can produce a variety of flavors. In addition, mushrooms are currently being investigated as sources of natural products which can be used by the pharmaceutical industry in the development of life saving drugs. The Short Path Thermal Desorption System used in conjunction with the Purge & Trap System permits the identification and quantification of trace levels of volatile organics responsible for flavors in mushrooms, as well as those which are unique to the species of mushroom. These techniques present a tremendous improvement over the time-consuming solvent extraction techniques normally used in the laboratory, and can be easily incorporated in flavor studies, general QA/QC testing or to a lesser extent for screening of mushrooms for natural products for use in the pharmaceutical industry.
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