Fatty Acid Methyl Ester from Neurospora Intermedia N-1 Isolated from Indonesian Red Peanut Cake (Oncom Merah)
The objective of this study was to identify the Fatty Acid Methyl Ester (FAME) from Neurospora intermedia N-1 that isolated from Indonesian red peanut cake (oncom). FAME profiles have been used as biochemical characters to study many different groups of organisms, such as bacteria and yeasts. FAME from N.intermedia N-1 was obtained by some stages of extraction the orange spores and fractination using a chromatotron. The pure compound (1) was characterized by 500 mHz NMR (1H and 13C), FTIR and LC-MS. Summarized datas of 1H and 13C NMR spectra of compound 1 contained 19 Carbon, 34 Hydrogen and 2 Oxygen (C19H34O2). The position of the double bonds at carbon number 8 and 12 were indicated in the HMBC spectrum (2D-NMR). LC-MS spectrum indicates molecular weight of the compound 1 as 294 which is visible by the presence of protonated molecular ion [M+H] at m/z 295. Methyl esters of long chain fatty acids was presented by a 3 band pattern of IR spectrum with bands near 1249, 1199 and 1172 cm-1. We suggested that the structure of the pure compound 1 is methyl octadeca-8,12-dienoate. The presence methyl octadeca-8,12-dienoate in N. intermedia is the first report.
Received: May 27, 2010;
Accepted: July 19, 2010;
Published: August 05, 2010
FAME profiles have been used as biochemical characters to study many different
groups of organisms, such as bacteria and yeasts. While fatty acids of many
filamentous fungi have been characterized, few studies have addressed taxonomic
issues. Those addressing taxonomy approach as opposed to using more rigorous
cladistic methods. The morphological data on this study suggested that during
spore development, inner walls are laid down sequentially (Bentivenga
and Morton, 1996). Mummey et al. (2002) applied
FAME biomarkers to monitor the recovery of ecosystems following surface mine
reclamation. In this study, it was found that the ratio of FAME bacterial to
fungal biomarkers reflected changes in other indicators of soil health suggesting
that this ratio is a useful indicator of reclamation progress. The use of fatty
acid methyl esters as biomarkers to determine aerobic, facultatively aerobic
and anaerobic communities in wastewater treatment systems. FAME analysis has
also been used to evaluate changes in microorganisms due to their exposure to
toxic substances. It has been proposed that specific groups of microorganisms
contain characteristic fatty acid profiles that can be used as biomarkers (Quezada
et al., 2007). Fresh soils were analyzed for their fatty acid methyl
ester (FAME) profiles by an Ester-Linked (EL) method and the method of MIDI.
With this method, microbial cells are saponified by heat and the addition of
a strong base. Once fatty acids are cleaved from lipids, they are methylated
to form FAMEs (Schutter and Dick, 2000).
In Phycomyces and Basidiomycetes, the C16 and C18 fatty acids predominate.
Approximately 70 to 80% of the total fatty acids in membrane lipids of the yeast
S. cerevisiae consist of the unsaturated fatty acids palmitoleic acid
(16 : 1) and oleic acid (18 : 1). Most other fungi contain the di- and trienoic
acids linoleic acid (18: 2) and α-linoleic acid (18: 3) (Moat
et al., 2002). Neurospora crassa synthesizes virtually all
of its fatty acid by a multifunctional cytosolic fatty acid synthase. The active
enzyme consists of two subunits, each subunit of the synthase carries out a
subset of the complete set of enzymatic reactions, leading to the biosynthesis
of the major product of the synthase, palmitate (16:0). Other fatty acids are
derived from 16:0 after its release from the fatty acid synthase, via elongation
and desaturation reactions. Trace amounts of short-chain (<16:0) fatty acids
are synthesized in mitochondria utilizing a membrane-localized acyl carrier
protein and associated proteins (Goodrich-Tanrikulu1
et al., 1999). By gas chromatography, myristic acid was identified as
the major fatty acid after hydrolysis of isolated compounds 9 and 11. This fatty
acid is efficiently synthesized in N. crassa. Minor fatty acids bound
to the C35 carotenoid alcohols are 16:0, 18:0, 18:1 and 18:3 (Sandman
et al., 2008). The isolation of an unknown lipid component (e.g.,
from a sebum sample) is indispensable for its structural elucidation. The latter
is usually performed by a combination of mass spectrometry and high resolution
NMR spectroscopy. Using these methods detailed information concerning the position
and geometry of double bonds as well as ramification points can be obtained.
Each chemical and physico-chemical information is highly valuable in order to
confine the number of conceivable structures prior to a mass- or NMR spectrometric
analysis (Bodoprost and Rosemeyer, 2007).
The fermented food, oncom found exclusively in West Java, Indonesia is important
because it is a common and very low cost protein food derived from fermentation
of legume waste residues by Neurospora sp., a fungus well-known in biochemical
genetics. In oncom production, the producers deliberately inoculate each batch
of cakes with conidia from the previous culture. Both mating types and a variety
of genotypes affecting visible traits have been recovered from the same oncom
cake (Perkins and Turner, 1988). On this study, FAME
from Neurospora intermedia N-1 which isolated from Indonesian red peanut
cake (oncom) was obtained by some stages of extraction the orange spores and
fractination using chromatotron. The pure compound was characterized by NMR
(1H and 13C), FTIR and LC-MS.
MATERIALS AND METHODS
This study was conducted in laboratory of Natural Products and Pharmaceutical, Research Center for Chemistry, Indonesian Institute of Sciences and School of Pharmacy, Bandung Institute of Technology on 2009.
Materials: A wild type strain of Neurospora intermedia N-1 was isolated from red oncom samples and is collected by Research Center for Chemistry-Indonesian Institute of Sciences, was used in the present study. It was maintained in Potato Dextrose Agar (PDA) slants. Dried solid waste of tofu processing was used for N. intermedia N-1 fermentation.
Solid substrates fermentation of N. intermedia N-1: Solid waste of tofu processing was dried at 50°C overnight and grinded by a grind blender. 100 g of this substrate was mixed with 200 mL aquadest (2:1), then autoclaved at 121°C for 15 min. One tube agar slant of Neurospora intermedia N-1 strain was suspended by 50 mL of sterile water and mixed with the autoclaved substrate. Fermentation was carried out in two aluminium holed trays (25x35 cm) and incubated at 30°C for 3-5 days. The spores were cultivated and the substrate was discarded.
Extraction and isolation: Extraction of spores was carried out by shaking
10 g spores with 100 mL 95% of ethanol at 150 rpm for an hour. Suspension was
filtered with suction through a Buchner funnel. The residue was grinded and
rinsed with cold ethanol. Extraction and filtration were repeated 3-4 times.
The pigment extract was evaporated in a vacuum rotary evaporator at 35°C.
The dry residue (±500 mg) was then dissolved in few drops of acetone.
100 mL of n-hexane was placed in a 500 mL separatory funnel with a teflon stop-cock
and this acetone solution was added. Fifty milliliter the distillated water
was added slowly by flowing along the walls of the funnel. The lower phase,
aqueous-acetone phase was discarded. 10% methanolic KOH was added to the upper
remaining n-hexane phase and was left in the dark at room temperature overnight.
The methanolic phase was discarded and the upper n-hexane phase was washed with
water to remove the alkali. n-hexane phase was dried with anhydrous sodium sulfate
and evaporated to dryness. The residue was dried additionally by nitrogen purge
(Amaya, 2001). Three hundred milligram of dry residue
was dissolved in few drops of chloroform and filtered before being introduced
to the rotating plate of chromatotron. This plate was coated with the Silica
Gel-Gypsum (contains 254 nm fluorescent indicator) 1 mm the thickness of coating
material (depends on the quantity of sample). Before the sample solution was
introduced, the sorbent layer was completely wetted with n-hexane and at least
5 min to allow the equilibration. Sample solution was filtered by suction into
the chromatotron plate. Elution of the sample was carried out by 50 mL of 0,
20, 40, 60, 80 and 100% chloroform in hexane and continued with 10, 20, 40,
60, 80 and 100% methanol in chloroform. Flow rates of the rotating plate were
adjusted to 2-4 mL min-1. UV lamp was used to detect the UV absorbing
fractions ring. The fractions were collected and their purity examined by TLC.
The pure compound (1) was characterized by one and two dimension of 500 mHz
NMR 1H and 13C (JEOL ECA), FTIR (FT/IR-4200typeA) and
LC-MS (Mariner Biospectrometry Hitachi L6200, system ESI).
Neurospora intermedia N-1 was fermented on waste solid tofu production
for at 30°C for 3-5 days. The orange spores was harvested and extracted
for spectrometric identification of fatty acid methyl ester in this fungus.
Datas 1H and 13C NMR spectra of compound 1 contained
19 Carbon, 34 Hydrogen and 2 Oxygen, which were tabulated in Table
1. This datas was confirmed by DEPT 13C NMR spectra (Fig.
1a, b), observed one of the methyl group; 12 of CH2 atoms and
4 of CH atoms.
For a detailed assignment of both proton and carbon spectra we used 1H/13C 2D HMQC and HMBC spectroscopy. HMQC and HMBC of this compound were analyzed to elaborate the direct and long-range correlations between the 1H and 13C NMR, respectively. Figure 2 shows the HMBC and HMQC spectra.
LC-MS spectrum indicates molecular weight of the compound 1 as 294 which is
visible by the presence of protonated molecular ion [M+H] at m/z 295 and the
presence of sodium adducts at m/z 317, respectively (Fig. 3).
IR spectra shown that the present of =CH group indicated by absorption at wave
number 3413 cm-1, -CH stretching vibration at 2927 cm-1,
C = O absorption band at 1739 cm-1. Methyl esters of long chain fatty
acids was presented by a 3 band pattern with bands near 1249, 1199 and 1172
cm-1, the band 1172 cm-1 is the strongest. IR spectra
indicates present of a trans C = C which gives a strong C-H bending band near
970 cm-1 (Fig. 3a, b).
|| (a, b) 500 MHz 1H and DEPT of compound 1 in CDCl3
|| HMBC and HMQC of compound 1 in CDCl3
|| NMR data of compound 1 in CDCl3
|| (a, b) Mass spectra and IR of compound 1
|| The elucidated structure of compound 1
Based on NMR spectra, shielding at δH 3.66 (s, H3) indicates
presence of one methoxy group. Mello et al. (2008)
observed three different calibration curves based on 1H-NMR spectroscopy
(300 MHz) were used for quantifying the reaction yield during biodiesel synthesis
by esterification of fatty acids mixtures and methanol. The integrated intensities
of the hydrogens of the ester methoxy group (3.67 ppm) were correlated with
the areas related to the various protons of the alkyl chain.
Carbonyl group was shown in 13C NMR at 174.50. The location of the
carbonyl carbon (C1) was confirmed from the HMBC correlation from C1,
C2 and C3. Shielding at δH 5.36 (m, H 3,296) indicates presence
of two un-conjugated double bonds. The position of the double bonds at carbon
number 8 and 12 were indicated in the HMBC spectrum. 13C-NMR was
very characteristic in the olefinic signal region, namely δ 128 to δ
131 (Shiao and Shiao, 1989). In food products from ruminants,
c-9, t-11 is the most prevalent one comprising 80 to 90% of total Conjugated
Linoleic Acid (CLA). Isomerization the double bond at carbon-12 position is
transferred to carbon-11 position forming c-9, t-11 CLA. It is followed by the
rapid hydrogenation of cis-9 bond leaving TVA. Both these steps are carried
out by a group a bacteria. The enzyme responsible for the conjugation of cis-9,
cis-9 double bonds was identified as linoleic acid isomerase (EC 220.127.116.11). it
is a particulate enzyme bound to the bacterial cell membrane (Khanal
and Dhiman, 2004). Migration of the double bond leads to shift of signals,
especially when the double bond approaches one of end of the chain. This effect
was discovered in early NMR studies of a full series of cis-octadecenoic
and some acetylenic fatty acids. Prior work demonstrated this effect for double
bonds near the terminal methyl group. The shifts of the olefinic protons did
not differ significantly (Knothe, 2005). The sequence
of compound 1 was deduced from a COSY experiment. Thus, the methyl signal at
δH 0.88 (C18) was coupled to the methylene signals at δH
1.30. Another COSY experiment indicates presence of the sequence for protons
with signals at δH 1.30 (C17), 5.36 (C13) and 1.62 (C3). COSY
spectrum which showed the connectivity between proton signals and its HMBC correlation
are shown in Fig. 4.
The molecular ion peak of a methyl ester of a straight-chain aliphatic acid
is usually can be distinguished by a mass spectra. The ion R-C = O+
gives an excellent diagnostic peak for esters. In methyl esters it occurs at
M-31 (Silverstain et al., 1981). On this study,
molecular ion peak of methyl ester is visible by the presence peak at m/z 263.5.
From the NMR, LC-MS and IR datas, the elucidated structure of compound
1 was methyl octadeca-8,12-dienoat. The presence of methyl octadeca-8,12-dienoat
or methyl linoleate in N. intermedia is the first report. In the fungus
Penicillium javanicum, linoleic acid was preferentially synthesized in
the younger cells. Linoleic acid was the major fatty acid in the spores of P.
atrovenetum. The increase in the fatty acids during the period of spore
germination and early lag phase (Van Etten and Gottlieb,
1965). According to Bentivenga and Morton (1996) FAME
can be used as biochemical character of organisms. We suggested that methyl
octadeca-8,12-dienoate can be used as a specific biochemical character or marker
of Neurospora intermedia.
This research was supported by Natural Products, Food and Pharmaceuticals Division- Research Center for Chemistry LIPI, Bandung Institute of Technology and State Ministry of Research and Technology, Indonesia. Thank you to Dr. Sokolowski, Dr. Wuryani and Dr. Iqbal for their valuable suggestions.
1: Amaya, D.B.R., 2001. A Guide to Carotenoids Analysis in Foods. International Life Sciences Institute, Brasil.
2: Bentivenga, P.S. and J.B. Morton, 1996. Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae. Proc. Natl. Acad. Sci. USA., 93: 5659-5662.
Direct Link |
3: Knothe, G., 2005. 1H-NMR spectroscopy of fatty acids and their derivatives-non-conjugated double bonds. The Lipid Library, http://lipidlibrary.aocs.org/nmr/1NMRdbs/index.htm.
4: Goodrich-Tanrikulu1, M., D.J. Jacobson, A.E. Stafford, J.T. Lin and T.A. McKeon, 1999. Characterization of Neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase. Curr. Genet., 36: 147-152.
5: Van Etten, J.L. and D. Gottlieb, 1965. Biochemical changes during the growth of fungi 11. ergosterol and fatty acids in Penicillium Atrovenetum. J. Bacteriol., 89: 409-414.
6: Bodoprost, J. and H. Rosemeyer, 2007. Analysis of phenacylester derivatives of fatty acids from human skin surface sebum by reversed-phase HPLC: Chromatographic mobility as a function of physico-chemical properties. Int. J. Mol. Sci., 8: 1111-1124.
Direct Link |
7: Khanal, R.C. and T.R. Dhiman, 2004. Biosynthesis of conjugated linoleic acid (CLA): A review. Pak. J. Nutr., 3: 72-81.
CrossRef | Direct Link |
8: Quezada, M., G. Buitrón, I. Moreno-Andrade, G. Moreno and L.M. López-Marín, 2007. The use of fattyacidmethyl esters as biomarkers to determine aerobic, facultativelyaerobic and anaerobic communities in wastewater treatment systems. FEMS Microbiol. Lett., 266: 75-82.
9: Schutter, M.E. and R.P. Dick, 2000. Comparison of fatty acid methyl ester (FAME) methods for characterizing microbial communities. Soil Sci. Soc. Am. J., 64: 1659-1668.
10: Moat, A.G., J.W. Foster and M.P. Spector, 2002. Lipid and Sterol in Microbial Physiology. 4th Edn., Wiley and Sons Inc., New York, pp: 450-464.
11: Mummey, D.L., P.D. Stahl and J.S. Buyer, 2002. Microbial biomarkers as an indicator of ecosystem recovery following surface mine reclamation. Appl. Soil Ecol., 21: 251-259.
12: Perkins, D.D. and B.C. Turner, 1988. Neurospora from natural population: toward the population biology of a haploid eukaryote. Exp. Mycoll., 12: 91-131.
Direct Link |
13: Sandman, G., S. Takaichi and P.D. Fraser, 2008. C35-apocarotenoids in the yellow mutant Neurospora crassa YLO. Phytochemistry, 69: 2886-2890.
14: Silverstain, R.M., G.C. Bassler and T.C. Morril, 1981. Spectrometric Identification of Organic Compounds. 4th Edn., John Wiley and Son, New York, pp: 95-105.
15: Shiao, T.Y. and M.S. Shiao, 1989. Determination of fatty acid compositions of triacylglycerols by high resolution NMR spectroscopy. Bot. Bull. Academia Sinica, 30: 191-199.
16: Mello, V.M., F.C. Oliveira, W.G. Fraga, C.J. do Nascimento and P.A. Suarez, 2008. Determination of the content of fatty acid methyl esters (FAME) in biodiesel samples obtained by esterification using 1H-NMR spectroscopy. Magnetic Resonance Chem., 46: 1051-1054.