Analysis of Lignin from Oak Casks Used for the Maturation of Scotch Whisky (*)

John M Conner, Alistair Paterson and John R Piggott Food Science Laboratories, Department of Bioscience and Biotechnology, University of Strathclyde, 131 Albion Street, Glasgow Gl ISD, UK

(Received 14 February 1992; revised version received 3 July 1999; accepted 17 July 1999)

Abstract:
Wood from four types of casks used for the maturation of Scotch whisky was analysed for free vanillin. syringaldehyde. vanillic and syringic acids and for milled wood lignin. Degradation of wood lignin by acidolysis was used to estimate the proportion of intact f-aryl ether linkages present. The yield of milled wood lignin was unaffected by repeated cask use. A comparison of acidolysis yields and the analysis of spirit matured for 3 years in each of the four types of cask indicated that the majority of -aryl ether linkages in oak lignin are resistant to spirit hydrolysis and that cask exhaustion was not accompanied by significant delignification. Significant reductions in the concentrations of free guaiacyl and syringyl compounds were recorded for used casks and proved the best indicator of cask exhaustion.
Key words:
Scotch whisky, maturation, oak, phenols, Lignin degradation, acidolysis.

INTRODUCTION

Freshly distilled spirits have pungent, unpleasant odours and sharp tastes and are traditionally matured in oak casks to improve their sensory properties. The mechanisms of maturation have not been fully elucidated, but are known to involve the loss of some distillate components, the extraction of wood components and the interaction of wood and distillate components (Nishimura et ul 1983). In the Scotch whisky industry, oak casks are reused many times (Philp 1989). With repeated use. the levels of wood components extracted into the maturing spirit decrease markedly (Reazin 1983). The improvement in the sensory properties of the spirit also decreases with repeated cask use until no satisfactory change occurs and the cask is classed as exhausted.

Lignin-related compounds are one of the most sign)ficant groups of components extracted from wood during maturation and are closely associated with the development of matured whisky flavour (Nishimura and Matsuyama 1989). They are formed from the degradation of oak lignin. either by toasting or charring prior to use. or by hydrolysis by ethanol and water during maturation. For uncharred or previously used casks' hydrolysis is thought to be the major route of formation of lignin breakdown products extracted into the spirit (Nishimura et al 1983). Studies on the delignification of cask wood samples using a flow-through reactor indicate that only a small proportion of lignin is easily soluble and consequently extracted during maturation. This lignin is thought to be solubilised by the hydrolysis of - and ,ll-aryl ether linkages, producing lower molecular weight fractions that in turn break down to monomeric cinnamyl and phenolic aldehydes (Puech and Moutounet 1990). -Aryl ether linkages are, however, stable to prolonged percolation with water at 100¡C and require the presence of strong acids for their cleavage. Under such conditions, the lignin is prone to undergo con- densation reactions leading to new C-C bonds (Nimz 1974). The -aryl ether linkage is also the most abundant interphenylpropane linkage (40 60 %) in lignin (Higuchi 1985) and cleavage of this bond would be expected to have a major effect on lignin in the wood.

Degradation of lignin by acidolysis can show whether or not there is a lower number of -aryl ether linkages in degraded lignin. The major products of acidolysis,

Hibbert's ketones. are only formed when the -aryl ether linkage is intact (Lundquist and Kirk 1971) Where this linkage is broken. as in degraded lignin. the proportion of Hibbert's ketones in the reaction products decreases (Crawford et al 1982). The work reported here investigated the extent of the degradation and loss of oak lignin in new. used and exhausted casks to identify chances in the composition of wood important in cask exhaustion.

EXPERIMENTAL

Materials

Wood samples were collected trom the staves of four types of cask used for the maturation of Scotch whisky. All casks were made from American white oak (Quercus alba) and had been charred prior to first maturation. The staves sampled came from a new. charred oak cask, a used bourbon cask (first fill cask). a used bourbon cask atter one maturation of Scotch whisky (second fill cask) and an exhausted cask. The first three samples, therefore' represented wood progressively older by one spirit maturation. and the fourth sample wood that failed to adequately improve the sensory properties of the maturing spirit. Staves were weighed and the surface area of the inner face and depth of stave measured. Samples of wood were shaved by a plane from both inner and outer faces of the stave and milled using a hammer mill (screen size: 2 mm. Culatti model DFH48. Glen Creston Ltd. Stanmore. UK).

Samples of whisky were drawn from spirit matured in casks of each type of wood for a period of 3 years. Six American standard barrels of each type were filled at two strengths (three at 634 ml ethanol litre-1 and three at 675 ml ethanol litre-1) of the same malt distillate and stored in normal conditions in a bonded warehouse. Samples were analysed as composites for each strength. The surface-to- volume ratio of the American standard barrel was taken to be 100 cm, Litre-1 (Philp 1989) and the yields are calculated from the mean stave thickness and wood density of the staves used for lignin analysis.

Chloroform. 1.4-dioxane, 1 -methoxy- 1,1 - dimethylethane, methanol and water were HPLC grade (Rathburn Chemicals Ltd. Walkerburn. UK); hydrochloric acid and ortho-phosphoric acid were AnalaR grade

BDH Ltd, Poole, UK). Gallic acid. vanillin, vanillic acid. syringaldehyde, syringic acid and 2-naphthol were 99¡O pure (Aldrich Chemical Company Ltd, Gillingham. UK); I- hydroxy-3-(4'-hydroxy-3'-methoxyphenyl)propan-2-one, 1-(4'- hydroxy-3'methoxyphenyl)propaneI .2-dione. I -hydroxy-3-(4'- hydroxy-3'.5'-dimethoxyphenyl)propan-2-one. and 1-(4'- hydroxy-3'.5'-dimethoxphenyl)propane- 1.2-dione were supplied by Dr K Lundquist, Chalmers Tekniska Hogskola och Goteborgs Universitet, Institutionen for Orgartisk Kemi. Goteborg S-41296, Sweden.

Methods

Coarse-milled wood samples (10 g) were pre-extracted by shaking overnight with chloroform (2 x 100 ml) to remove volatile oils and free acids and aldehydes, without extracting polymeric lignin. Initial chloroform extracts were dried in vacuo and weighed. Extracts were analysed for total phenolic content (AOAC 1980) using Folin-Denis reagent with garlic acid as standard and for absorbance at 270 nm in 650 ml ethanol litre-l. Concentrations of vanillin and vanillic acids (guaiacyl) and syringaldehyde and syringic acids (syringyl) were determined by HPLC using a Kratos 400 solvent pump, Kratos 430 low pressure gradient former and Kratos 470 autosampler with 20 ~cl loop (ABI Analytical, Warrington, UK) on a Spherisorb S5 ODS2 column (Phase Separations Ltd, Clwyd, UK) using a 0.1 M orthophosphoric acid/methanol gradient (Casteele et al 1983) and a LC-UV detector (Pye Unicam Ltd, Cambridge, UK) at 300 nm with integration by a TRIO computing integrator (Trivector Systems International Ltd, Sandy, UK). Samples were redissolved in 2 ml 100 ml ethanol litre-1 and analysed in triplicate with peak areas standardised on 2-naphthol (400 ng on column). Standards of known concentration were used for calibration and gave correlation coefficients of > 0 997 and limits of detection of 0 06 and 0.03 ~g ml-1 for aldehydes and acids respectively. Variation between replicates was ~ + 5 % standardised peak area.

Pre-extracted samples were dried and then milled for 24 h using a Siebtechnik vibratory mill (Tema Machinery Ltd, Banbury, UK). Milled wood lignin (MWL) was prepared using the method of Obst and Kirk (1988). MWL samples were stored overnight over phosphorus pentoxide and weighed to give the yield of crude MWL. This crude MWL contains residual carbohydrate (Obst and Kirk 1988), so the phenolic content was assessed using Folin-Denis reagent and absorbance at 270 nm.

Samples of ethanol lignin in whisky were prepared by pre- extraction of spirit solutions (100 ml) with 1 -methoxyI, I- dimethylethane (4 x 25 ml) to remove free acids and aldehydes and the remaining solution evaporated to dryness in vacuo.

Acidolysis of wood and spirit lignin used dioxanel2 M HCI under reflux (9: I v) (Tanahashi and Higuchi 1988). Blanks of a 24 h sample extraction with dioxane/water (9: I v) were used to correct for monomer production during ball milling.

Concentrations of vanillin, syringaldehyde and vanillic and syringic acids in reaction mixtures were determined by HPLC and the concentration of Hibbert's ketones by GC-MS using a Finnegan MAT ITS40 integrated benchtop GC-MS and data analyser (Finnegan MAT. Hemel Hempstead, UK). The two major acidolysis products (1 -(4'-hydroxy- 3'methoxyphenyl)propane- 1,2dione and 1-(4'-hydroxy-3',5'- dimethoxyphenyl)

propane- 1,2-dione) were separated using a TABLE 1 Composition of the initial chloroform extracts of wood from four types of casksa 


			Yield	A270	Phenol	Guaiacyl	Syringyl
			(mg g-1)	(mg g-1)c	(umolg-1)	(umolg-1)

New		Inner	11	5.3	0.14	0.6	1.3
		Outer	9	3.6	0.13	0.4	0.8
First		Inner	3	2.2	0.14	0.3	0.7
		Outer	5	2.3	0.12	0.5	0.9
Second		Inner	9	3.1	0.09	0.2	0.5
		Outer	11	2.8	0.10	0.3	0.6
Exhausted	Inner	21	4.8	0.18	0 1	0.1
		Outer	24	6.0	0.23	0 2	0.3
F(7.14)'		3.5	1.3	2.9	5.0	4.2

a	Values are means of three wood samples.
b	Value for extract from 1 g wood dissolved in I ml.
c	mg garlic acid equivalents g-'.
d	F ratio: significant differences at 5 % level when F > 2.8.
25 m x 0 32 mm Carbowax BP 20 column (df = 0 5,um) (sGE UK Ltd. Milton Keynes. UK). Samples were analysed in duplicate and injected on-column at an initial oven temperature of 80¡C, increasing to 120¡C at 10¡C min~' after 3 min and then to 240¡C at 4¡C min-'. The carrier pas was helium at 1 8 ml min-'. Ketones were identified by comparison of spectra and retention times with authentic compounds and quantified by calibration with known concentrations of standards. Calibration correlation coelRicients were > 0995 and the limit of detection was 0 5 ng on column. Variation was ~ _ 5 % standardised peak area.

Spirit samples were analysed by HPLC without concentration or after evaporation (50 ml) i'' tacuo and redissolution in 5 ml of 100 ml ethanol litre-l.

RESULTS

Table I gives the results of the analysis of the initial chloroform extracts. Extract yields were highest for exhausted cask samples, though analysis by HPLC showed that these extracts contained the lowest levels of guaiacyl and syringyl compounds. The highest concentrations of these compounds were found in the extracts from the new, charred casks. A sign)ficant reduction in free guaiacyl and syringyl compounds (P < 0 05) was observed between the new charred and first fill casks and represents the loss during a single maturation. A further sign)ficant reduction (P < 0 05) occurred from the second fill to the exhausted casks, though this represents an unknown number of maturations.

No significant differences in the yield of MWL~ phenolic content or absorption at 270 nm was observed between samples. Mean MWL yield was 200 mg g-' with a phenolic content of 17 mg garlic acid equivalents g-'

TABLE 2: Acidolysis yields of guaiacyl and syringyl compounds from pre-extracted ball-milled wood (umol g-1) 


			 Guaiacyl	 Syringyl

			VVA	GHK	SSA	SHK

New		Inner	21	45	24	32
		Outer	19	59	21	25
First		Inner	8	23	8	15
		Outer	8	27	14	32
Second		Inner	5	12	6	17
		Outer	4	11	6	14
Exhausted	Inner	19	63	21	54
		Outer	18	29	21	32
F (7, 14)		66	79	45	50

a	Values are means for three wood samples.
b	VVA - Yield of vanillin + vanillic acid.
c	GHK - Yield of 1-(4'-hydroxy-3'methoxyphenyl)propane-1,2-dione.
d	SSA - Yield of syringaldehyde + syringic acid.
e	SHK - Yield of 1-(4'-hydroxy-3',5'-dimethoxyphenyl)-propane-1,2-dione.
f	F ratio: significant differences at 5 % level when F > 2 8.
and absorbance of 88 units at a solution concentration 10 mg ml-l.

A decrease in the yield of all lignin aeidolysis products occurred between the new charred and second fill cask samples (Table 2). The exhausted cask samples, however, gave yields similar to those from the new, charred casks. Ratios of vanillie acid: 1-(4'-hydroxy-3'meth~xyphenyl)propane-1,2- dione and syringic acid: 1-(4'hydroxy-3',5'- dimethoxyphenyl)propane-1,2-dione did not vary significantly from means of 021 and 027, respectively.

TABLE 3: Yields of guaiacyl and syringyl compounds ~mol g ') extracted from oak barrels of different ages by a maturing spirit after a maturation period of three yeast. 


Cask type	 Guaiacyl	 Syringyl
		FAA	EL	FAA	EL

New charred	0.19	0.017	0.049	0.069
First fill	0.14	0.008	0.27	0.012
Second fill	0.08	0.003	0.12	0.009
Exhausted	0.06	0.07	0.10	0.010
F(3,4)		17.4	41.5	13.6	41.5

a	Values are means of two maturation strengths.
b	FAA - Free aldehyde and acid.
c	EL - Yield from acidolysis of ethanol lignin.
d	F ratio: significant differences at 5 % level when F > 6 6.
The yields of lignin breakdo~:n products and ethanol lignin in maturing spirit were found to decrease with each cask use (Table 3). Hibbert's ketones were present in samples of matured spirit only at trace concentrations and so could not be quantified.

DISCUSSION

The analysis of wood from new. used and exhausted casks found no sign)ficant differences in the yield of MWL or in the ratios of the major acidolysis products. This suggests that. during maturation, only a very small portion of the polymeric lignin present is degraded and extracted by the maturing spirit. Considering the larger differences between the yields from acidolysis and spirit extraction, it may be concluded that the majority of -aryl ether linkages in lignin are resistant to spirit hydrolysis and that the decrease in acidolysis yields observed from the new charred to second fill casks are not the result of degradation and loss of lignin into the maturing spirit. The decrease in the yield of Hibbert's ketones may be the result of an increase in the proportion of bonds in the lignin resistant to acidolysis. Acidolysis would then produce a higher proportion of dimers, trimers and oligomers which were not measured in these exper~ments.

In contrast to polymeric lignin, the wood concentrations of free guaiacyl and syringyl acids and aldehydes decreased sign)ficantly with repeated cask use. For the first two spirit maturations. decreases in the inner face concentrations corresponded closely to the amounts detected in the matured spirit, indicating that direct extraction of these components was their major route of entry to the maturing spirit. For the exhausted cask samples, however, extraction during maturation accounted for 100% of the inner face wood content of syringaldehyde and syringic acid. This suggests that diffusion of these compounds from deeper layers of the wood or spirit hydrolysis of a small portion of labile lignin during maturation occurred to maintain the extraction gradient. Low wood concentrations of free guaiacyl and syringyl acids and aldehydes were the best indicator of an exhausted cask.

CONCLUSIONS

The analysis of oak lignin from new and used whisky casks indicated that cask exhaustion was not accompanied by significant delignification of cask wood. Degradation of cask lignin by acidolysis showed that the majority of -aryl ether linkages were resistant to spirit hydrolysis. Decreases in the wood concentrations of free guaiacyl and syringyl acids and aldehydes were consistent with repeated cask use and so provided the best indicator of cask exhaustion.

ACKNOWLEDGEMENTS

This research is supported by the Agricultural and Food Research Council. London. UK and Chivas Brothers Ltd. Keith. UK. Dr Knut Lundquist kindly provided the standards t'or the GC-MS analysis of Hibbert's ketones.

REFERENCES

AOAC 1980 Official Methods of Analysis (9.098-9.100). Association of Official Analytical Chemists, Washington DC. USA. p 158.

Casteele K V. Geiger H.van Sumere C F 1983 Separation of phenolics and coumarins by reversed-phase high- performance liquid chromatography. J Chromatogr 258 I I I- 174.

Crawford D L. Barder M J. Pometto A L, Crawford R L 1982 Chemistry of softwood lignin degradation by Streptomyces viridosporus. Arch Micobiol 131 140-145.

Higuchi T 1985 Biosynthesis of lignin. In: Biosynthesis and Biodegradation of Wood components' ed Higuchi T. Academic Press. London. UK. pp 141-160.

Lundquist K. Kirk T K 1971 Acid degradation of lignin. IV. Analysis of lignin acidolysis products by gas chromatography, using trimethylsilyl derivatives. Acta Cllem Scand 25 889-894.

Nimz H 1974 Beech lignin - Proposal of a constitutional scheme. .4ngeir Chem Int Edi' 13 313-321.

Nishimura K. Matsuyama R 1989 Maturation and maturation chemistry. In: The Science and Technologl, of Whiskies. ed Piggott J R. Sharp R. Duncan R E B. Longman, Harlow. Essex. UK. pp 235-263.

Nishimura K. Ohnishi M. Masuda M, Koga K, Matsuyama R 1983 Reactions of wood components during maturation. In: Flut our of Distilled Beverages. Origin and Development. ed Piggott J R. Ellis Horwood. Chichester, UK, pp 241-255.

Obst J R. Kirk T K 1988 Isolation of lignin. In: Methods in Enzymology vol 161 Biomass Part B Lignin, Pectin and Chitin. ed Wood W A. Kellogg S T. Academic Press. London. UK. pp 3-12.

Philp J 1989 Cask quality and warehouse conditions. In: The Science and Technology of Whiskies, ed Piggott J R, Sharp R. Duncan R E B. Longman. Harlow, Essex. UK. pp 264 294.

Puech J-L, Moutounet M 1990 Oakwood chemistry and extractable substances. In: Proceedings of the Third Aviemore Conference on Malting Brewing and Distilling ed Campbell 1. Institute of Brewing. London. UK. pp209-"5.

Reazin G H 1983 Chemical analysis of whisky maturation. In:

Flavour of Distilled Bevereges. Origin and Derelopment. ed Piggott J R. Ellis Horwood. Chichester. UK. pp 225-240.

Tanahashi M, Higuchi T 1988 Chemical degradation methods for characterisation of lignins. In: Methods in Enzymlogy vol 161 Biomass Part B Lignin. Pectin and Chitin. ed Wood W A. Kellogg S T. Academic Press. London. UK. pp 101-109.

* Based on material presented at the Third Aviemore Con- ference on Malting. Brewing and Distilling, Aviemore, UK. 1990.