- Email: [email protected]
Odour composition of the Tuber melanosporum complex
Mycol. Res. 94 (2): 201-204 (1990)
201
Printed in Great Britain
Odour composition of the Tuber melanosporum complex
GIOVANNI PACIONI Dipartimenfo di Scienze Ambientali, Universifa, 67100 L'Aquila, Ifaly
C A R L 0 BELLINA-AGOSTINONE A N D MAURO D'ANTONIO Presidio Multiwnale lgiene e Prevenzione, USSL, L'Aquila, Ifaly
Odour composition of the Tuber melanosporum complex. Mycological Research
94 ( 2 ) :201-204 (1990).
Odour composition of some taxa related to Tuber rnelanospontrn has been studied by gas chromatography coupled to a mass spectrometer (GC-MS). Although qualitative differences have been observed, the relative amounts of substances present may be more significant for their chemical taxonomy. A concept of the Tuber odour according to its ecological function is discussed. Key words: Chemotaxonomy, Tuber, Volatile metabolites, Truffle odours, Ecology. During their presumed evolution from epigeous discomycetes, active discharge into the air has been replaced by animal dispersion in hypogeous ascomycetes. Several types of animal (mammals, insects, snails) attracted by their odours feed on the sporocarps and spread the spores in the soil with their faeces (Trappe & Maser, 1977). Some hypogeous ascomycetes, commonly called truffles, are much liked by humans and are among the most expensive foods in the world. Tuber melanosporum Vitt. ( = T. nigrum Bull.: Fr. pro parte) is the most prized among the truffles and is known It forms an internationally as the 'Truffe du PCrigord'. ectomycorrhiza with forest trees, mainly oak, beech and hazel. This symbiotic growth occurs on European calcareous soils between the 40th and 47th parallel in the northern hemisphere, most frequently in France, Italy and Spain. Since the beginning of the nineteenth century truffle farming has supplemented natural production, and this experience has contributed greatly to our knowledge of the ectomycorrhizal symbiosis (Pacioni & Lalli, 1981). Around this species several taxa (species, varieties and forms) exist. Microscopically we can recognize Tuber brumale Vitt., which grows in Europe, and T. indicum Cooke, a Himalayan species ectomycorrhizal with Quercus incana. Among the other described species only Tuber moschafum Chat. and T . hiernalbum Chat. seem likely to have some taxonomic acceptance. The peculiar odour of the truffle delicacy ennobles many dishes of international cuisine. Therefore, knowledge of the chemical composition of this odour has considerable economic importance. In fact, there are several patented products of 'truffle flavouring' for both foods and liqueurs on sale. Not one of these products, however, has the same smell as the
fresh truffle to the human nose. Truffles are used fresh or preserved, although in the latter case their flavour is much less. Ethyl sulphide has often been added as a crude adulterant of canned truffles in the past (Andreotti & Casoli, 1968). The volatile substances of truffles have probably been studied in several industrial laboratories for a long time, but only a few papers exist on this topic. Ney & Freytag (1980) used steam distillation and gas-chromatography to analyse the odorous substances of fresh sporocarps of the 'Truffle of PCrigord', unusually called by them Tuber brumale var. melanosporum. They demonstrated the presence of a mixture of eight alcohols with dimethyl sulphide, isoamylamine, pand/or m-cresol. Working with another edible truffle, Tuber magnaturn Pico, and using a gas-chromatograph coupled with a mass spectrometer Fiecchi et al. (1965) found bismethyl thiomethane to be the main chemical responsible for its odour. Recently, other researchers (Bellina-Agostinone, D'Antonio & Pacioni, 1987) have shown that the odour of the summer truffle (Tuber aestivum Vitt. sensu stricto), the most widespread edible truffle, is associated with a mixture of oxygencontaining organic compounds with molecular masses between 36 and 8 8 accompanied by dimethyl sulphide (thiobismethane). In the same period Talou, Delmas & Gaset (1987) found 14 compounds, including the same substances found by us in Tuber aestivum, among the components of the Tuber melanosporum odour. They used a dynamic headspace technique with aroma volatiles trapped and then analysed, after heat desorption, by capillary gas chromatography and coupled capillary gas chromatography-mass spectrometry. Our attention has now been directed to analyse the composition of the odour of the T. rnelanosporurn complex
Odours in Tuber melanosporum
202
with the aim of using this character for taxonomic and systematic purposes.
MATERIALS A N D M E T H O D S Our analytical method followed that of Bellina-Agostinone, D'Antonio & Pacioni (1987). Fresh specimens of the black winter-truffle taxa were studied with a gas chromatograph coupled to a mass spectrometer (GC-MS), Hewlett-Packard Model 5986, computerized with HP 98293, using a 6 ft 0.1 SPlOOO on 80/100 Carbopack-C column programmed from 40 to 220 OC, rate 10°/min. Chromatogram time was 30 min. Single compounds were identified using computer methods for comparing the normalized mass spectra of specific peaks with data recorded in the computer's memory and those available in the literature. Standards for all possible compounds, except ethanol, were used. Clean and whole samples were kept singly in small air-tight bottles. Volatile substances were studied using head-space analysis, i.e. sampling the air in the bottles with a gas syringe at room temperature and then heating quickly to a temperature of SO0 with the aim of increasing the peaks of higher-boiling substances. The sampled air containing volatile substances was injected into the helium stream of the analyser system. Because of its simplicity, direct analysis on fresh specimens was chosen so as not to interfere with the truffle's metabolism. Inside the sporocarps of Tuber and other hypogeous fungi, seemingly related fermentations, by both the gleba hyphae and the guest microflora of external venae (Ozino-Marletto, 1970; Pacioni & Lalli, 1981), probably occur. Sporocarps were collected in Central Italy (Umbria, Lazio, Abruzzi) under oak trees and identified by the senior author. They were analysed as soon as possible. Seven collections of each taxon were studied during the three years since 1985, with the exception of Tuber hiemalbum, collected only once in the winter of 1988. A tentative evaluation of the quantities of constituents has been based on the assumption that these are directly proportional to the total ion signal 6f the peaks revealed by the mass spectrometer (Hamming & Foster, 1972).
RESULTS As in Tuber aestivum, the odour of the winter black truffle is a mixture of several substances. Eleven substances were identified, of which 10 are oxygen-containing organic compounds with molecular masses between 46 and 102. Thiobismethane was always present in the largest quantity in head space and must surely be the main cause of the odour. The substances with their formulae and retention times related to that of thiobismethane are shown in Table 1. Their variations of amount related to that of thiobismethane are shown in Table 2. The smell of the different species of black-winter truffles (Tuber melanosporum, T. brumale, T. brumale var. moschatum and 7. hiemalbum), is associated with a limited number of chemical compounds. Their quantitative ratios cannot be strictly quantified because they change according to the maturation conditions of sporocarps, to the taxonomic entity and often to a single sporocarp. In addition, the truffle odour is produced and sent out by living sporocarps. Therefore its amount is a function of the time and the volume of the sampling air. O n the basis of our analyses it seems that there are no qualitative differences between the four taxa examined. Only in T. brumale and var. moschatum, has the probable presence of some other compounds, generally esters, been occasionally revealed and these should be further checked. Qualitatively, a difference between T . aestivum and T. melanosporum could be that the former has the certain presence of 2-butanol and the latter has formic acid isopropyl ester. But 2-butanol has also once been found in a T. brumale specimen. In addition, the mass spectra of these two compounds are so similar that some confusion can arise.
DISCUSSION The human nose can distinguish the odours of the Tuber species treated here. Because a clear and constant difference between the chemical composition of the volatile substances of these taxa exists, we think that these differences could be significant for their chemical taxonomy. This diversity is related to different relative amounts of some specific
Table I. Principal volatile substances from fresh sporocarps of the Tuber melanosporum-T. brumale complex
Name*
Structure
Ethanol Thiobisrnethane 2-Methylpropanal 2-Butanone Formic acid isopropyl ester 2-Methyl-1-propanol 2-Methylbutanal 3-Methylbutanal Formic acid I-methyl-propyl ester 2-Methyl-I-butanol 3-Methyl-I-butanol From Chemical Abstracts. t Retention time related to thiobisrnethane (3 rnin).
Retention timet
G. Pacioni and others Table 2. Amount variations of volatile substances among different specimens of the Tuber melanosporum-T. brumale complex species compared with those of T. aestivum s.s. T. me1anosporum (T. hiemalbum)
T. brumale
7.brumak var. moschatum
T. aestivum
Ethanol Thiobismethane 2-Methylpropanal Z-Butanone 2-Butanol Formic acid isopropyl ester 2-Methyl-I-propanol 2-Methyl-I-butanal 3-Methyl-1-butanal Formic acid I-methyl-propyl ester 2-Methyl-1-butanol 3-Methyl-1-butanol Values of the total ion signal related to that of thiobismethane equal to 100 (tr, traces).
Table 3. Analyses of T. melanosporum volatile metabolites
Alcohols Ethanol I-Propano1 2-Butanol 2-Methyl-I-propanol 2-Methyl-I-butanol 3-Methyl-1-butanol 3-Octanol I-Octen-3-01 2-Nonanol 2-Phenylethanol Aldehydes Acetaldehyde 2-Methylpropanal 2-Methyl-1-butanal 3-Methyl-1-butanal
I*
2t
700 12.8
30-40 1-2 0.244 17-24 12-20 tr
-
47.4 -
393 0.4 0.3 1.8 2.2
-
3* 2.8
-
2.3 2.4 0.6
-
-
-
3-5 5-7 3-4 tr
Ketones Acetone 2-Butanone
-
5-10 2-2.5
-
Ether Anisole
-
Esters Formic acid isopropyl ester Formic acid 1-methyl propyl ester Amines 3-Methylbutylamine Sulphur compound Thiobismethane Phenol p/m-cresol
-
0.05-0.1
1
5.9 4.9
2.8
-
-
-
4.4
-
-
4.3
245
-
-
8
2-6
100
1-2
-
-
Ney & Freytag (1980): p.p.m. t Talou, Delmas & Gaset (1987):rel. %. Medium values of total ion signal at mass spectrometer related to that of thiobismethane equal to 100.
*
compounds. In fact, studying the volatile substances produced aestivum, we have observed that 2-butanone is frequently by 7. present at almost the same or up to seven times the amount as thiobismethane. However, &butanone is always much less in the black winter truffles. Within the Tuber melanosporum complex, in addition to the absence or undetectable amounts of some substances, we noted quantitative differences in others. Among the higher-oxodized compounds, in T.brumale formic acid isopropyl ester and formic acid 1-methyl-propyl ester are present at amounts much higher than 3-methyl 1butanol (amyl alcohol) and the corresponding aldehydes which characterize the head space of T. mehnosporum. The presence of other esters in var. rnoschafum requires further checking. Because only a single sporocarp of T. hiemalbum has been collected, we cannot judge its diversity of odour. Commonly immature forms of T. brumale var. moschatum have been incorrectly determined as T. hiemalbum, while the true taxon described by Chatin (1892) is characterized by a thin peridium and slightly brown gleba as well as slightly coloured spores when it is mature. The qualitative and quantitative differences between these results and those from analyses by Ney & Freytag (1980) and Talou, Delmas & Gaset (1987), are considerable. Table 3 shows our results cgmpared with these other results. Only four of the eleven substances found by Ney & Freytag (1980) and us are in common: ethanol, 2-methyl-1-propanol ( = isobutanol), 3-methyl 1-butanol ( = isopentanol) and thiobismethane ( = dimethyl sulphide). In our analyses we observed larger amounts of thiobismethane than did Ney & Freytag (1980) who detected only 8 p.p.m. compared with 700 p.p.m. of ethanol. We never found: propanol, 3-octanol, I-octen-3-01, 2-nonanol or 2-phenylethanol. These differences could depend on the different methods of analysis used as well as on a different concept of the Tuber odour. Our concept of this odour is based on the ecological significance of volatile substances emitted from sporocarps. Steam distillation can also remove high boiling substances, while the analyses by Talou, Delmas & Gaset (1987) using small pieces of sporocarps, sometimes frozen, have shown volatile substances still trapped
Odours in Tuber melanosporum inside the sporocarp. This fact could reflect in qualitative and quantitative differences, particularly in those concerning the relative amount of thiobismethane and aldehydes. According to our research which is still in progress, the oxygencontaining compounds seem to play an important role in the plant and microbial control of mycorrhizosphere and in the attraction of hydnophagous animals, including dogs. Because of their function and toxicity, their concentration should of course be different between the outside and the micro-aerobic environment of the 'venae externae' inside Tuber sporocarps. The use of an atmosphere of helium prevents, in part, a natural oxidation of alcohols. Secretion of thiobismethane may have a function in detoxification of the internal sporocarp and thus its concentration is lower inside the sporocarp. Ney & Freytag (1980) and Talou, Delmas & Gaset (1987) analysing sporocarps, not the air around the sporocarps, found lower quantities of this metabolite. In the case of isoamylamine (3-methylbutylamine), a compound absent in our analyses but found by Ney & Freytag (1980), it could have been produced by a decomposition process occurring in some of their sporocarps, collected in France and then analysed in Germany. For a long time, in fact, it has been known that such a compound is produced by enzymic decarboxylation of the aminoacid I-leucine (Fruton & Simrnonds, 19-58), but it can be produced by the normal metabolism of some odoriferous fungi (Wrigh & Vinning, 1976). Steam distillation might also cause damage to natural materials, including protein degradation (Fenaroli, 1975). As regards the presence of aromatic substances with one aromatic ring, found by Ney & Freytag (1980), p-/m-cresol, and Talou, Delmas & Gaset (1987), anisole, they are probably not compounds of the odour, but of other organoleptic qualities of T . melanosporum sporocarps. We thank Prof. A. Fiecchi for his valuable suggestions on sulphide metabolism in Tuber species. (Received for publication 26 January 1989)
204
REFERENCES ANDREOTTI, R. & CASOLI, U. (1968). Composizione chimica e tecnologia di conservazione del tartufo. In Atti Convegno Internazionale sul Tartufo, 55-66. Spoleto, Italy: Ente Rocca di Spoleto. BELLINA-AGOSTINONE, C., D'ANTONIO, M. & PACIONI, G. (1987). Odour composition of the summer truffle, Tuber aestivum. Transactions of the British Mycological Society 88, 568-569. CHATIN, A. (1892). La Trufle. Impr. CrCte, Corbeil. FENAROLI, G. (1975). Handbook of Flavor Ingredients. Cleveland: CRC. FIECCHI, A., GALLI-KIENLE, M., SCALA, A. & CABELLA, P. (1967). Bis-methylthiomethane, an odorous substance from white truffle, Tuber magnaturn Pico. Tetrahedron Letters 18, 1681-1682. FRUTON, J. S. & SIMMONDS, S. (1958). General Biochemistry. New York: Wiley. HAMMING, M. C. & FOSTER, N. G. (1972). Interpretation of Mass Spectra of Organic Compounds. London, U.K.:Academic Press. NEY, K. H. & FREYTAG, W. G. (1980). Truffle-Aroma. Gordian 80, 214. OZINO-MARLETTO, 0. (1970). Osservazioni su due schizomiceti isolati da sporocarpi di Tuber magnafum Pico e di Tuber macrosporum Vitt. Allionia 16, 87-90. PACIONI, G. & LALLI, G. (1981). Biologia ed ecologia del tartufo nero (Tuber melanosporum Vitt.). In Atti Symposium lnfemazionale di Micologia, pp. 69-86. Borgo Val di Taro. TALOU, T., DELMAS, M. & GASET, A. (1987).Principal constituents of black truffle (Tuber melanosporum) aroma. Iournal of Agn'cultural and Food Chemistry 35, 774-777. TRAPPE, J. M. & MASER, C. (1977). Ectomycorrhizal fungi: interactions of mushrooms and truffles with beasts and trees. In Mushrooms and Man, an Interdisciplinary Approach to Mycology (ed. T. Walters), pp. 165-179. Corvallis, U.S.A.: U.S.D.A. Forest Service. WRIGH, J. L. C. & VINING, L. C. (1976). Secondary metabolites derived from non-aromatic amino acids. In The Filamentous Fungi, 11. Biosynthesis and Metabolism (ed. J . E. Smith & D. R. Berry), pp. 475-502. London: Arnold.
201
Printed in Great Britain
Odour composition of the Tuber melanosporum complex
GIOVANNI PACIONI Dipartimenfo di Scienze Ambientali, Universifa, 67100 L'Aquila, Ifaly
C A R L 0 BELLINA-AGOSTINONE A N D MAURO D'ANTONIO Presidio Multiwnale lgiene e Prevenzione, USSL, L'Aquila, Ifaly
Odour composition of the Tuber melanosporum complex. Mycological Research
94 ( 2 ) :201-204 (1990).
Odour composition of some taxa related to Tuber rnelanospontrn has been studied by gas chromatography coupled to a mass spectrometer (GC-MS). Although qualitative differences have been observed, the relative amounts of substances present may be more significant for their chemical taxonomy. A concept of the Tuber odour according to its ecological function is discussed. Key words: Chemotaxonomy, Tuber, Volatile metabolites, Truffle odours, Ecology. During their presumed evolution from epigeous discomycetes, active discharge into the air has been replaced by animal dispersion in hypogeous ascomycetes. Several types of animal (mammals, insects, snails) attracted by their odours feed on the sporocarps and spread the spores in the soil with their faeces (Trappe & Maser, 1977). Some hypogeous ascomycetes, commonly called truffles, are much liked by humans and are among the most expensive foods in the world. Tuber melanosporum Vitt. ( = T. nigrum Bull.: Fr. pro parte) is the most prized among the truffles and is known It forms an internationally as the 'Truffe du PCrigord'. ectomycorrhiza with forest trees, mainly oak, beech and hazel. This symbiotic growth occurs on European calcareous soils between the 40th and 47th parallel in the northern hemisphere, most frequently in France, Italy and Spain. Since the beginning of the nineteenth century truffle farming has supplemented natural production, and this experience has contributed greatly to our knowledge of the ectomycorrhizal symbiosis (Pacioni & Lalli, 1981). Around this species several taxa (species, varieties and forms) exist. Microscopically we can recognize Tuber brumale Vitt., which grows in Europe, and T. indicum Cooke, a Himalayan species ectomycorrhizal with Quercus incana. Among the other described species only Tuber moschafum Chat. and T . hiernalbum Chat. seem likely to have some taxonomic acceptance. The peculiar odour of the truffle delicacy ennobles many dishes of international cuisine. Therefore, knowledge of the chemical composition of this odour has considerable economic importance. In fact, there are several patented products of 'truffle flavouring' for both foods and liqueurs on sale. Not one of these products, however, has the same smell as the
fresh truffle to the human nose. Truffles are used fresh or preserved, although in the latter case their flavour is much less. Ethyl sulphide has often been added as a crude adulterant of canned truffles in the past (Andreotti & Casoli, 1968). The volatile substances of truffles have probably been studied in several industrial laboratories for a long time, but only a few papers exist on this topic. Ney & Freytag (1980) used steam distillation and gas-chromatography to analyse the odorous substances of fresh sporocarps of the 'Truffle of PCrigord', unusually called by them Tuber brumale var. melanosporum. They demonstrated the presence of a mixture of eight alcohols with dimethyl sulphide, isoamylamine, pand/or m-cresol. Working with another edible truffle, Tuber magnaturn Pico, and using a gas-chromatograph coupled with a mass spectrometer Fiecchi et al. (1965) found bismethyl thiomethane to be the main chemical responsible for its odour. Recently, other researchers (Bellina-Agostinone, D'Antonio & Pacioni, 1987) have shown that the odour of the summer truffle (Tuber aestivum Vitt. sensu stricto), the most widespread edible truffle, is associated with a mixture of oxygencontaining organic compounds with molecular masses between 36 and 8 8 accompanied by dimethyl sulphide (thiobismethane). In the same period Talou, Delmas & Gaset (1987) found 14 compounds, including the same substances found by us in Tuber aestivum, among the components of the Tuber melanosporum odour. They used a dynamic headspace technique with aroma volatiles trapped and then analysed, after heat desorption, by capillary gas chromatography and coupled capillary gas chromatography-mass spectrometry. Our attention has now been directed to analyse the composition of the odour of the T. rnelanosporurn complex
Odours in Tuber melanosporum
202
with the aim of using this character for taxonomic and systematic purposes.
MATERIALS A N D M E T H O D S Our analytical method followed that of Bellina-Agostinone, D'Antonio & Pacioni (1987). Fresh specimens of the black winter-truffle taxa were studied with a gas chromatograph coupled to a mass spectrometer (GC-MS), Hewlett-Packard Model 5986, computerized with HP 98293, using a 6 ft 0.1 SPlOOO on 80/100 Carbopack-C column programmed from 40 to 220 OC, rate 10°/min. Chromatogram time was 30 min. Single compounds were identified using computer methods for comparing the normalized mass spectra of specific peaks with data recorded in the computer's memory and those available in the literature. Standards for all possible compounds, except ethanol, were used. Clean and whole samples were kept singly in small air-tight bottles. Volatile substances were studied using head-space analysis, i.e. sampling the air in the bottles with a gas syringe at room temperature and then heating quickly to a temperature of SO0 with the aim of increasing the peaks of higher-boiling substances. The sampled air containing volatile substances was injected into the helium stream of the analyser system. Because of its simplicity, direct analysis on fresh specimens was chosen so as not to interfere with the truffle's metabolism. Inside the sporocarps of Tuber and other hypogeous fungi, seemingly related fermentations, by both the gleba hyphae and the guest microflora of external venae (Ozino-Marletto, 1970; Pacioni & Lalli, 1981), probably occur. Sporocarps were collected in Central Italy (Umbria, Lazio, Abruzzi) under oak trees and identified by the senior author. They were analysed as soon as possible. Seven collections of each taxon were studied during the three years since 1985, with the exception of Tuber hiemalbum, collected only once in the winter of 1988. A tentative evaluation of the quantities of constituents has been based on the assumption that these are directly proportional to the total ion signal 6f the peaks revealed by the mass spectrometer (Hamming & Foster, 1972).
RESULTS As in Tuber aestivum, the odour of the winter black truffle is a mixture of several substances. Eleven substances were identified, of which 10 are oxygen-containing organic compounds with molecular masses between 46 and 102. Thiobismethane was always present in the largest quantity in head space and must surely be the main cause of the odour. The substances with their formulae and retention times related to that of thiobismethane are shown in Table 1. Their variations of amount related to that of thiobismethane are shown in Table 2. The smell of the different species of black-winter truffles (Tuber melanosporum, T. brumale, T. brumale var. moschatum and 7. hiemalbum), is associated with a limited number of chemical compounds. Their quantitative ratios cannot be strictly quantified because they change according to the maturation conditions of sporocarps, to the taxonomic entity and often to a single sporocarp. In addition, the truffle odour is produced and sent out by living sporocarps. Therefore its amount is a function of the time and the volume of the sampling air. O n the basis of our analyses it seems that there are no qualitative differences between the four taxa examined. Only in T. brumale and var. moschatum, has the probable presence of some other compounds, generally esters, been occasionally revealed and these should be further checked. Qualitatively, a difference between T . aestivum and T. melanosporum could be that the former has the certain presence of 2-butanol and the latter has formic acid isopropyl ester. But 2-butanol has also once been found in a T. brumale specimen. In addition, the mass spectra of these two compounds are so similar that some confusion can arise.
DISCUSSION The human nose can distinguish the odours of the Tuber species treated here. Because a clear and constant difference between the chemical composition of the volatile substances of these taxa exists, we think that these differences could be significant for their chemical taxonomy. This diversity is related to different relative amounts of some specific
Table I. Principal volatile substances from fresh sporocarps of the Tuber melanosporum-T. brumale complex
Name*
Structure
Ethanol Thiobisrnethane 2-Methylpropanal 2-Butanone Formic acid isopropyl ester 2-Methyl-1-propanol 2-Methylbutanal 3-Methylbutanal Formic acid I-methyl-propyl ester 2-Methyl-I-butanol 3-Methyl-I-butanol From Chemical Abstracts. t Retention time related to thiobisrnethane (3 rnin).
Retention timet
G. Pacioni and others Table 2. Amount variations of volatile substances among different specimens of the Tuber melanosporum-T. brumale complex species compared with those of T. aestivum s.s. T. me1anosporum (T. hiemalbum)
T. brumale
7.brumak var. moschatum
T. aestivum
Ethanol Thiobismethane 2-Methylpropanal Z-Butanone 2-Butanol Formic acid isopropyl ester 2-Methyl-I-propanol 2-Methyl-I-butanal 3-Methyl-1-butanal Formic acid I-methyl-propyl ester 2-Methyl-1-butanol 3-Methyl-1-butanol Values of the total ion signal related to that of thiobismethane equal to 100 (tr, traces).
Table 3. Analyses of T. melanosporum volatile metabolites
Alcohols Ethanol I-Propano1 2-Butanol 2-Methyl-I-propanol 2-Methyl-I-butanol 3-Methyl-1-butanol 3-Octanol I-Octen-3-01 2-Nonanol 2-Phenylethanol Aldehydes Acetaldehyde 2-Methylpropanal 2-Methyl-1-butanal 3-Methyl-1-butanal
I*
2t
700 12.8
30-40 1-2 0.244 17-24 12-20 tr
-
47.4 -
393 0.4 0.3 1.8 2.2
-
3* 2.8
-
2.3 2.4 0.6
-
-
-
3-5 5-7 3-4 tr
Ketones Acetone 2-Butanone
-
5-10 2-2.5
-
Ether Anisole
-
Esters Formic acid isopropyl ester Formic acid 1-methyl propyl ester Amines 3-Methylbutylamine Sulphur compound Thiobismethane Phenol p/m-cresol
-
0.05-0.1
1
5.9 4.9
2.8
-
-
-
4.4
-
-
4.3
245
-
-
8
2-6
100
1-2
-
-
Ney & Freytag (1980): p.p.m. t Talou, Delmas & Gaset (1987):rel. %. Medium values of total ion signal at mass spectrometer related to that of thiobismethane equal to 100.
*
compounds. In fact, studying the volatile substances produced aestivum, we have observed that 2-butanone is frequently by 7. present at almost the same or up to seven times the amount as thiobismethane. However, &butanone is always much less in the black winter truffles. Within the Tuber melanosporum complex, in addition to the absence or undetectable amounts of some substances, we noted quantitative differences in others. Among the higher-oxodized compounds, in T.brumale formic acid isopropyl ester and formic acid 1-methyl-propyl ester are present at amounts much higher than 3-methyl 1butanol (amyl alcohol) and the corresponding aldehydes which characterize the head space of T. mehnosporum. The presence of other esters in var. rnoschafum requires further checking. Because only a single sporocarp of T. hiemalbum has been collected, we cannot judge its diversity of odour. Commonly immature forms of T. brumale var. moschatum have been incorrectly determined as T. hiemalbum, while the true taxon described by Chatin (1892) is characterized by a thin peridium and slightly brown gleba as well as slightly coloured spores when it is mature. The qualitative and quantitative differences between these results and those from analyses by Ney & Freytag (1980) and Talou, Delmas & Gaset (1987), are considerable. Table 3 shows our results cgmpared with these other results. Only four of the eleven substances found by Ney & Freytag (1980) and us are in common: ethanol, 2-methyl-1-propanol ( = isobutanol), 3-methyl 1-butanol ( = isopentanol) and thiobismethane ( = dimethyl sulphide). In our analyses we observed larger amounts of thiobismethane than did Ney & Freytag (1980) who detected only 8 p.p.m. compared with 700 p.p.m. of ethanol. We never found: propanol, 3-octanol, I-octen-3-01, 2-nonanol or 2-phenylethanol. These differences could depend on the different methods of analysis used as well as on a different concept of the Tuber odour. Our concept of this odour is based on the ecological significance of volatile substances emitted from sporocarps. Steam distillation can also remove high boiling substances, while the analyses by Talou, Delmas & Gaset (1987) using small pieces of sporocarps, sometimes frozen, have shown volatile substances still trapped
Odours in Tuber melanosporum inside the sporocarp. This fact could reflect in qualitative and quantitative differences, particularly in those concerning the relative amount of thiobismethane and aldehydes. According to our research which is still in progress, the oxygencontaining compounds seem to play an important role in the plant and microbial control of mycorrhizosphere and in the attraction of hydnophagous animals, including dogs. Because of their function and toxicity, their concentration should of course be different between the outside and the micro-aerobic environment of the 'venae externae' inside Tuber sporocarps. The use of an atmosphere of helium prevents, in part, a natural oxidation of alcohols. Secretion of thiobismethane may have a function in detoxification of the internal sporocarp and thus its concentration is lower inside the sporocarp. Ney & Freytag (1980) and Talou, Delmas & Gaset (1987) analysing sporocarps, not the air around the sporocarps, found lower quantities of this metabolite. In the case of isoamylamine (3-methylbutylamine), a compound absent in our analyses but found by Ney & Freytag (1980), it could have been produced by a decomposition process occurring in some of their sporocarps, collected in France and then analysed in Germany. For a long time, in fact, it has been known that such a compound is produced by enzymic decarboxylation of the aminoacid I-leucine (Fruton & Simrnonds, 19-58), but it can be produced by the normal metabolism of some odoriferous fungi (Wrigh & Vinning, 1976). Steam distillation might also cause damage to natural materials, including protein degradation (Fenaroli, 1975). As regards the presence of aromatic substances with one aromatic ring, found by Ney & Freytag (1980), p-/m-cresol, and Talou, Delmas & Gaset (1987), anisole, they are probably not compounds of the odour, but of other organoleptic qualities of T . melanosporum sporocarps. We thank Prof. A. Fiecchi for his valuable suggestions on sulphide metabolism in Tuber species. (Received for publication 26 January 1989)
204
REFERENCES ANDREOTTI, R. & CASOLI, U. (1968). Composizione chimica e tecnologia di conservazione del tartufo. In Atti Convegno Internazionale sul Tartufo, 55-66. Spoleto, Italy: Ente Rocca di Spoleto. BELLINA-AGOSTINONE, C., D'ANTONIO, M. & PACIONI, G. (1987). Odour composition of the summer truffle, Tuber aestivum. Transactions of the British Mycological Society 88, 568-569. CHATIN, A. (1892). La Trufle. Impr. CrCte, Corbeil. FENAROLI, G. (1975). Handbook of Flavor Ingredients. Cleveland: CRC. FIECCHI, A., GALLI-KIENLE, M., SCALA, A. & CABELLA, P. (1967). Bis-methylthiomethane, an odorous substance from white truffle, Tuber magnaturn Pico. Tetrahedron Letters 18, 1681-1682. FRUTON, J. S. & SIMMONDS, S. (1958). General Biochemistry. New York: Wiley. HAMMING, M. C. & FOSTER, N. G. (1972). Interpretation of Mass Spectra of Organic Compounds. London, U.K.:Academic Press. NEY, K. H. & FREYTAG, W. G. (1980). Truffle-Aroma. Gordian 80, 214. OZINO-MARLETTO, 0. (1970). Osservazioni su due schizomiceti isolati da sporocarpi di Tuber magnafum Pico e di Tuber macrosporum Vitt. Allionia 16, 87-90. PACIONI, G. & LALLI, G. (1981). Biologia ed ecologia del tartufo nero (Tuber melanosporum Vitt.). In Atti Symposium lnfemazionale di Micologia, pp. 69-86. Borgo Val di Taro. TALOU, T., DELMAS, M. & GASET, A. (1987).Principal constituents of black truffle (Tuber melanosporum) aroma. Iournal of Agn'cultural and Food Chemistry 35, 774-777. TRAPPE, J. M. & MASER, C. (1977). Ectomycorrhizal fungi: interactions of mushrooms and truffles with beasts and trees. In Mushrooms and Man, an Interdisciplinary Approach to Mycology (ed. T. Walters), pp. 165-179. Corvallis, U.S.A.: U.S.D.A. Forest Service. WRIGH, J. L. C. & VINING, L. C. (1976). Secondary metabolites derived from non-aromatic amino acids. In The Filamentous Fungi, 11. Biosynthesis and Metabolism (ed. J . E. Smith & D. R. Berry), pp. 475-502. London: Arnold.