C. M. DELAHUNTY, J. M. CONNER, J. R. PIGGOTT and A. PATERSON
University of Strathclyde, Centre for Food Quality, Department of Bioscience and Biotechnology, 131 Albion Street, Glasgow Cl ISD
Received 23 January 1993
Standard solutions of N-heterocyclic compounds in ethanol are extracted with pentane at a range of pHs. Pentane extractions are also performed on N- heterocyclic standards in ethanol with addition of Limousin Oak extract at a range of concentrations. The effects observed on extraction are collated with sensory assessments. The results obtained would suggest that pyridines are absent from the aroma of a mature whisky due to its acidity, and that interaction with wood constituents in the cask does not occur.
Key Words: Aciditv, oak wood constituents, flavour analysis
In general, the odours of pyrazines are associated with pleasant roasted foods2. The terms burnt, roasted and nutty12, and nutty, roasted, earthy, fruity and woody10, have been used as descriptors for these most often. Nutty, green and roasted have been used to describe thiazoles. Thiazole itself is considered to smell like pyridine12. For pyrazines, thresholds and aroma notes conferred vary through differences in side chains12.
The odours of the pyridines are less pleasant. Descriptive terminologies such as green, bitter, astringent, roasted or burnt were used as pyridine evaluators14,15,16,17. In a study comparing the odour qualities of nine chemicals3, pyridine was described as pyridine-like, pungent, solvent, and fishy, none of which could be considered desirable. The flavour properties of pyridines identified in whiskies have been described as astringent, green, hazelnut, earthy, buttery, caramel, rubbery, roasted, fatty and bitter6 and it has been found that in sensory tests, both the odour and taste of whiskies improved as pyridine content decreased13. On addition of several pyridines, known to be present in low concentrations, to English beer and subsequent evaluation of the associated sensory properties, aroma and taste sensations such as estery, cardboard, oxidised and harsh, most of which would be considered objectionable, have been recorded4.
In work carried out to try and qualify the sensory importance of the heterocyclic nitrogen compounds to whisky flavour 10,13 findings to date have been tentative. This is due mainly to the difficulties in their extraction and isolation, whisky being a highly complex material. This work seeks to gain a better understanding of the nature of the N-heterocyclic compounds in a mature whisky, and in doing so provide a firmer base on which the sensory importance of these compounds can be assessed.
Analyses were performed on a Finnegan-MAT ITS-40 GCMS with septum programmable injector (temp. 230 deg C) (Finnegan- MAT, Hemel Hempstead, UK). The column used was a 30 m x 0.25 mm Carbowax BP20 (df = 0.25 um) (SGE (UK) Ltd., Milton Keynes, UK) with Helium at 1.8 ml min-1 as carrier gas. The GC-MS was calibrated with the pentane standard at known concentrations in the range from 1 ug ml-1 to 100 ug ml-1. Samples were analysed in duplicate with an initial co~umn temperature of 60 deg C increasing after 5 min to 180 deg C at 8 deg C min -1. Octan-1-ol (Aldrich Chemical Co. Ltd., Gillingham. UK) (20 ng on column) was used as an internal standard.
Extraction of the N-heterocyclic compounds in ethanol was performed by a simple liquid/liquid method in which 10 ml standard was gently shaken overnight with 10 ml pentane in 20 ml screw cap culture tubes which contained teflon coated seals. It was found that this simple method gave the most consistently reproducible results. The solvent extract was separated in a separatory funnel and for each analysis 1 ul was injected. The amount of each compound extracted was calculated from the calibration curve. The pH of the control standard was 6.65 (S.D. = 0.04).
The extraction was then investigated at pHs from 6.0 to 3.5. The pH was controlled with addition of 0.1 M phosphate buffer to standard solutions and was monitored continuously with a Whatman PHA 300 pH meter. A second extraction was subsequently performed on the same sample set having firstly raised their pH to 6.5. thus reflecting that of the control. The total of each compound extracted was also examined with additions of Limousin Oak extract (International Flavour and Fragrances (GB) Ltd., Haverhill, UK) at a range of concentrations from 2 mg ml-1 to 12 mg ml-1; the effects of such additions on pH were monitored in parallel.
Measurements of pH were made on spirit samples supplied by Chivas Brothers Ltd (Keith AB5 3B5, Scotland) after 0, 6, 12, 18, 24, 30, 36, 42 and 48 months maturation. The samples were composite drawn from casks filled with the same batch of Longmorn new distillate. The cask types used were new charred or uncharred (new charred; new plain), casks previously used in the USA for the maturation of Bourbon whisky (Type 1), Bourbon casks used at least once for maturation of Scotch malt whisky (Type 2) and casks used several times for the maturation of Scotch whisky (Type 3).
Sensory analysis, for correlative purposes, was performed on a 12-year old mature Scotch malt whisky purchased locally. Two sample sets were prepared (23% v/v), with the pH of the whisky adjusted to a range of values from 3.5 to
FIG. 6. Loadings of decriptive terms on principal component one from PCA of
sensory data. Pu = pungent; So = solvent; Spi = spicy; Grai = grainy; Ma = malty;
Mo = mouldy; FE = fruity (eatery); FO = fruity (other); Flo = floral; Smo = smooth; Va
= vanilla; Soa = Soapy; So = sour; Nu = nutty; Bu = buttery; Gras = grassy; Phe =
phenolic; 0i = oily; Wo = woody; Mea = meaty; Su = sulphury; Ca = catty; Fi = fishy;
Swe = sweet.
range 0-2. As the pH was lowered to and beyond the pKas of 4-ethylpyridine and 5-
ethyl-2-methylpyridine the percentage existing as free bases decreased and at pH
3.5 they exist almost exclusively as protonated cations. These forms are insoluble in
the pentane extractant. Conversely, at this pH, both of the pyrazines, 4-
methylpyrazine and 2,5-dimethylpyrazine, and the thiazole, 4-methyl-5-vinylthiazole,
exist almost exclusively in the non-ionised form which are soluble in pentane.
Peppard and Halsey8, in attempting to detect pyridines in beer, found solvent
extracts of the basic fraction of the beer dominated by pyrazines, which made
identification and detection of the pyridines very difficult. These authors exploited
variation in pKa values for separation by means of a cation-exchange resin, but in
doing so extraction efficiency of the N-heterocyclic compounds decreased. Usage of
the variable extraction rates as pH is altered and thus separation using solvent may
be a more successful method.
The results obtained from the standard extractions, knowing the pH of a mature
whisky to be in the range 4.0 to 4.5, suggests that the pyridines, being ionised at
the pH of mature whisky would be absent from the headspace and thus the aroma
of the whisky. The results obtained from sensory analysis support this hypothesis. A
recent study carried out in these laboratories attempted to correlate nitrogen
containing heterocyclic compounds isolated with whisky flavour10. Whiskies, of 22
different types; were chosen, representative of 5 major groupings (Islay, Speyside,
Irish, Scotch grain and Bourbon). A trained sensory panel examined the whiskies
using descriptive sensory analysis and solvent extracts were also analysed by GC-
MS and by High Resolution (HR) GCsniffport to obtain aromagrams. PCA of the
composition data showed that groups of whiskies could be distinguished on the
basis of N-heterocyclic compound content and broadly similar groups were found in
PCA of sensory data. Pyrazines and 4-methyl-5-vinylthiazole could be linked with
burnt, roasted and nutty flavour notes. In the case of pyridines, no obvious links with
flavour were observed. The present findings could explain for this.
Although Limousin Oak extract caused a decrease in the extraction of the pyridines,
and of the pyrazines, the highest level of the added wood extract is in excess of
those reported to occur in the cask during maturation. The total solid content, or
residue, of a mature whisky is between 0.1 mg ml-1 and 2.5 mg ml-1, with whiskies
matured in new casks having the most residue1,11
Pyridines would thus be predicted to have little direct impact on the aroma of a
mature whisky. This can only be seen as desirable given their general reputation as
found in the literature. However, it should be noted. whisky being a highly complex
material, that there are many other factors which could have an important bearing
on the contribution of the pyridine compounds to flavour. It should also be noted
that flavour is a combination of odour, taste and preception in the mouth. Thus
absence from the headspace does not necessarily imply that they have no role to
play in the overall character of the whisky. There is thus a need for work on the
release of flavour in the mouth, as a whisky is being consumed, in order to elucidate
further flavour perception.
Acknowledgements. The authors wish to thank the Agricultural and Food Research
Council and Chivas Brothers Ltd. For financial support and technical assistance. Mr.
Delahunty would like to thank the National Universitv of Ireland for financial support
and Mrs. Francis Jack for assistance with the sensory data.
2. Buttery, R. G.. Ling, L. C., Teranishi, R. & Mon, T. R. Journal of Agricultural and
Food Chemistry, 1977, 25, 1227.
3. Dravnieks, A., McDaniel, H. C. & Powers, J. J. Journal of Agricultural and Food
Chemistry, 1979, 27, 336.
4. Harding, R. J.. Nursten, H. E. & Wren, J. J. Journal of the Science of Food and
Agriculture, 1977, 28, 225.
5. ter Heide, R. Food Flavours Part B. The Flavour of Alcoholic Beverages, ed.
Morton, I. D. & MacLeod, A. J., Elsevier, Amsterdam, 1986. 239.
6. Maga, J. A. Journal of Agricultural and Food Chemistry, 1981, 29, 895.
7. Nykanen, K. & Suomalainen, H. Aroma of Beer, Wine and Distilled Beverages.
Reidel, Dordrecht, 1983.
8. Peppard, T. L. & Halsey, S. A. Journal of Chromatography, 1980, 202, 271.
9. Piggott, J. R. Sensory Science Theory and Applications in Foods, ed. Lawless H.
. & Klein, B. P., Marcel Dekker, New York, 1991, 339.
10. Piggott, J. R., Paterson, A., Conner, J. M. & Haack, G. Recent Developments in
Food Science and Nutrition; Proceedings of the 7th International Flavour
Conference, ed. Charalambous, G., Elsevier, Amsterdam, 1993, in press.
11. Sharp, R. Personal communication.
12. Vernin, G. & Vernin, G. The Chemistry of Heterocyclic Flavouring and Aroma
Compounds, ed. Vernin, G., Ellis Horwood, Chichester, 1982, 72.
13. Viro, M. Flavour Research of Alcoholic Beverages, ed. Nykanen, L. & Lehtonen,
P., Foundation for Biotechnical and Industrial Fermentation Research, Helsinki,
1984, Vol. 3, 227.
14. Winter, M., Gautschi, F., Flament, I., Stoll, M. & Goldman, I. M. US Patent
3702253, Nov. 7, 1972.
15. Winter, M., Gautschi, F., Flament, I., Stoll, M. & Goldman, I. M. US Patent
3900582, Aug. 19, 1975.
16. Winter, M., Gautschi, F., Flament, I. & Stoll, M. US Patent 3931245, Jan. 6,
1976.
17. Winter, M., Gautschi, F., Flament, I. & Stoll, M. US Patent 3931246, Jan. 6,
1976.
CONCLUSIONS
Pyridines are widely believed to produce unpleasant odours. During whisky
maturation spirit pH decreases to that where the pyridine concentration in the
headspace is considerably reduced. Headspace concentrations of the pyrazines
and the thiazoles are virtually unaffected. Extractions performed following addition
of wood extract showed the effect to be pH dependent and unaffected by wood
constituents at levels reached during typical maturations.
REFERENCES
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