ESTIMATION AND OCCURRENCE OF ACETALDEHYDE IN BEER. By G. E. Otter and L. Taylor. {Courage Limited, Southwark Bridge, London, S.E.

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1 Vol. 77, 1971] 467 ESTIMATION AND OCCURRENCE OF ACETALDEHYDE IN BEER By G. E. Otter and L. Taylor {Courage Limited, Southwark Bridge, London, S.E.I) Received 15th April, 1971 A sensitive routine quantitative method has been developed which determines acetaldehyde in beer. The method is suitable for the measurement of acetaldehyde, its bisulphite addition compound and acetal, with only limited interference from other aldehydes. The examination of a representative selection of beers shows that large differ ences of acetaldehyde can occur in beers of a similar type. Beers which have been sweetened by the addition of priming sugar vary widely in their acetaldehyde content, many being above the 25 ppm taste threshold. The acetaldehyde content has been shown to vary with both the quantity and point of addition of sulphur dioxide when this is used as a preservative or antioxidant. Introduction Acetaldehyde is produced in beer during fermentation as an intermediate stage in the formation of ethanol15 but a small amount is produced in wort1 and during the hop boil.19 Acetaldehyde has little effect on beer palate unless it is present in amounts greater than 25 ppm11 but it is a precursor of other compounds which have pronounced tastes. Ethanol will condense with acetaldehyde in acid conditions to produce acetal which can be detected by taste at 1 ppm.11 It has been suggested17 that acetoin, produced by the condensation of two molecules of acetaldehyde, is responsible for diacetyl in beer. Acetaldehyde will also react with hydrogen sulphide to produce thio-acetaldehyde7 which results in a particularly unpleasant flavour at very low concentra tions. Burger et al.6 have shown that acet aldehyde accumulates at the end of fermen tation when both yeast and air are present. Matsumoto, Oskima & Hylnka12 showed that wheat gluten treated with bisulphite pro duced an increase in -SH bonds but reaction with acetaldehyde reduced this gain, sug gesting that acetaldehyde is reacting with -SH to produce thioacetaldehyde. Similar conditions exist at the end of fermentation when malt and yeast protein are present with accumulating acetaldehyde, often in con junction with bisulphite. Acetaldehyde has been determined by a variety of methods: (a) Thin layer chromatography of the aldehyde 2-4 dinitrophenylhydrazone derivatives. (b) Gas liquid chromatography.5"11 >21 (c) Colorimetrically with various reagents, e.g., piperidine,13 p-dihydroxydiphenyl,s>2 sodium nitroprusside,9 3- methyl benzothiazolone liydrazone.16'18 Thin layer and gas liquid chromatography require specialist skills and equipment. The colorimetric reagents listed suffer from being either insensitive or subject to inter ference from other aldehydes. Arsenault & Yaphe2 have introduced a method for the determination of traces of acetaldehyde which although having some interference from n-propionaldehyde, n-butyraldehyde and n- valeraldehyde, is reasonably specific for acetaldehyde. This method has been adapted for use with beer. Experimental The direct determination of acetaldehyde in beer using a photometric method is not possible because of the high red absorbance of acid-treated beer. This difficulty can be readily overcome by distilling the sample prior to colour development. Reagents Fructose solution. Dissolve -72 g of fructose, Hopkins & Williams Bacterio logical, in 1 ml of water and dilute 1 times before use. Resorcinol solution. Dissolve -132 g of resorcinol, B.D.H. Analar, in 1 ml of water. Dilute 1 ml of this solution to 1 ml with concentrated hydrochloric acid before use. The resorcinol stock solution

2 468 OTTER & TAYLOR: ACETALDEHYDE IN BEER [J. Inst. Brew. will keep for about 2 weeks if it is stored in a dark bottle, but it should be rejected if a pink colour develops. Procedure The following operations are carried out in subdued light. 1 ml of beer (or other suitable aliquot) is diluted to 1 ml with water and approxi mately 7 ml is distilled into a 1-ml standard flask. The delivery tube of the condenser reaches to the bottom of the collection flask which is cooled in ice. The distillate is made up to 1 ml with water and 1 ml is pipetted into a boiling tube. 1 ml of fructose solution is then added and the mixture is kept cold in ice during the addition of 1 ml of the resorcinol reagent solution. The tubes are loosely stoppered and allowed to warm to room temperature, and then heated in a water bath to 8 C for exactly 1 min followed by cooling in ice to room temperature. Simultaneously, a blank is prepared by treating 1 ml of water with 1 ml of fructose solution and 1 ml of resorcinol hydrochloric acid reagent in the same manner. The absorbance of the red colour is measured at 555 nm in a 2-cm glass cell using the reagent blank as reference. Calibration. The absorbance maximum for the reaction between acetaldehyde and resorcinol under these experimental con ditions occurs at 555 nm (Fig. 1). Standard acetaldehyde solutions are prepared by distilling 1 ml of aqueous solutions contain ing, 1, 2, 3, 4 and 5 ppm respectively. The absorbance of each is determined using the described method. The results are used to prepare a calibration chart (Fig. 2). The concentration of acetaldehyde in the beer is found by multiplying the value 2 % 4 Acetaldehyde Fig. 2. Calibration chart of the absorbance pro duced by acetaldchydc. obtained from the graph by the dilution factor of the beer. Reproducibility. To check the reproducibility of the method, six bottles of the same beers were analysed on successive days. The results are recorded in Table I. Recovery. The quantitative recovery of acetaldehyde was tested by adding known amounts of acetaldehyde to beer and analys ing the resulting solutions (Table II). Reproducibility of Acetaldehyde Analysis in Beer on Successive Days Analysis 1st day 2nd 3rd 4th 6th 6th Mean I ppm Acctaldchydc ± -3 Recovery of Acetaldehyde added to Beer II Acetalde hyde added Acetalde hyde found Acetalde hyde recovered % Recovered Wavelength(nm) Fig. 1. Absorption spectrum of the acetaldehyde colour reaction S

3 Vol. 77, 1971] OTTER & TAYLOR: ACETALDEHYDE IN BEER 469 Interference. (a) The volatile compounds which are likely to interfere in the deter mination are the lower n-aldehydes, their respective isomers and the lower ketones. Arsenault & Yaphe8 have stated that form aldehyde, aromatic aldehydes, iso-aldehydes and lower ketones do not interfere and this was confirmed. 1 mm of n-propionaldehyde, n-butyraldehyde and n-valeraldehyde were added to beer and the beer was analysed for acetaldehyde. The results shown in Table III indicate that about 2% of the higher n- aldehydes are determined as acetaldehyde. 111 Interference in Acetaldehyde Determination BY n-aldehydes n-aldehyde n-propanal n-butanal n-pcntanal Amount added to beer Apparent acetaldehyde recovered (b) Acetal. 1,1-diethoxyethane is hydrolysed under the conditions of the test to ethanol and acetaldehyde: any acetal in the beer is therefore determined as its molar equivalent of acetaldehyde. (c) Bisulphite compound. 1 ml of 1% sodium bisulphite solution was added to 25 ml of beer, known concentrations of acetaldehyde were added, and the mixture was analysed. The added acetaldehyde was recovered, showing that the acetaldehydebisulphite addition compound was quanti tatively broken down during the distillation procedure. Results A representative selection of British and other beers has been analysed and results are recorded in Table IV. These results show wide differences in acetaldehyde content, with British top fermentation beers being generally higher. Primed beers, i.e., those which have been sweetened after fermenta tion, are particularly variable with some values in excess of the 25 ppm taste threshold. The reason for this variation in primed beers was sought in a further investigation. Acelaldehyde in primed beer. A sweet stout was tested for acetaldehyde, from the start of fermentation through the various stages of production to the final product. The results (Table V) show that the acet aldehyde reached 8-8 ppm at the end of fermentation and then increased rapidly after the addition of the priming solution. This figure then remained constant during cold storage, filtering and bottling. After 6 weeks in storage, no significant change in the acetaldehyde content had taken place. The priming sugar was a commercial sucrose solution with -1% of sulphur dioxide added as a preservative. The con clusion from these findings is that either the sucrose or the sulphur dioxide was respons ible for the increased acetaldehyde. The effect of different sugars on acetaldehyde production. 2% samples of pure fructose, glucose, sucrose and maltose were added respectively to four portions of a "rough" primarily-fermented beer and to a further three samples of the beer were added re spectively, 2% of caramel, priming sugar and malt wort. Secondary fermentation was allowed to proceed and the acetaldehyde content of each was determined after 24 h. Table VI IV ACETALDEKYDE CONTENT OF BEER Beer No. of samples examined Acetaldehyde content Range of values Mean value No. of samples over 26 ppm taste threshold British lager Lager from other countries.. Light and pale ales Primed beers

4 47 OTTER & TAYLOR: ACETALDEHYDE IN BEER [J. Inst. Brew. V Increase of Acetaldehyde Content in a Primed Beer During Production Sampling point Yeast presence Time in hours AcetaUloliydc Wort before addition of yeast. 1 day fermentation 2»,,,... 3,,,, End of fermentation After addition of priming sugar and to bottling store Sample ex receiving tank Transfer to chilling tank.. End of chilling Bright filtered beer Bottled After pasteurization After weeks storage in bottle dc livery absent present abb cnt ior. 4:i-r> :t shows that although there was a general rise in acetaldehyde the priming sugar induced a far greater increase. These results show that sucrose is not responsible for the increase and suggest that the sulphur dioxide added to the priming sugar is blocking the normal fermentation reaction of sugar -> acetaldehyde -> alcohol. The sulphur dioxide is reacting with the acetaldehyde as it is formed to give the acetaldehyde bisulphite addition compound. Acetaldehyde Produced by Different Sugars Sugar (2%) addition to beer containing yeast Beer without added sugar Pure fructose glucose,, sucrose,, maltose Priming sugar Malt wort.. Caramel (sucriase).. VI Acctaldchydc after 24 h IOC The effect of sulphur dioxide on acet aldehyde production in beer. A sample of beer was taken prior to racking, and filtered, and -2 lb/barrel of yeast and 2% of pure sucrose was added. This beer was divided into four equal portions and treated with, 5, 25 and 5 ppm of sulphur dioxide respectively. Acetaldehyde content was determined after 18 and 42 h. Table VII shows that, over the range studied, the acetaldehyde increase is approximately proportional to the sulphur dioxide addition, confirming that the high acetaldehyde in the beer was due to the presence of sulphur dioxide. Three beers which had been treated differently with sulphur dioxide during production were examined to find out if the method of addition of sulphur dioxide affected the acetaldehyde level (Table VIII). VII AcETALDEHYDE PRODUCED BY THE FERMENTATION of Sucrose in the Presence of SO, Beer containing -2 lb/barrel yeast and 2% pure sucrose Initial beer 5 ppm SO, 25 ppm SOS SO ppm SOS 18 h S 42 h M! 4 In both cases where sulphur dioxide was present with active yeast, the acetaldehyde rose to a comparable level. In the third case, where the sulphur dioxide was added with sugar and yeast under cold conditions, the acetaldehyde content was low. These results confirm that the yeast must be actively fermenting in the presence of sulphur dioxide for high levels of acetaldehyde to appear in the beer.

5 Vol. 77, 1971] OTTER & TAYLOR: ACETALDEHYDE IN BEER '471 acbtaldehyde in beers from different breweries where sulphur dioxide had been Added in Different Manners VIII Brewery Method of SO: addition SO, Acetaldehyde Beer transported to bottling store with yeast and SOS present SO, and sugar added to chilled beer SO, and sugar added with yeast present: held several hours at ambient temperature Other factors which have been reported to influence the levels of acetaldehyde are yeast strain and oxidation. The effect of yeast strain on acetaldehyde production. Drews el a/.1 found that both acetaldehyde and diacetyl varied with differ ent yeast strains. Using five different strains of Saccharomyces cerevisiae and two of Saccharomyces carlsbergensis to ferment samples of the same wort in similar condi tions, we were unable to find significant differences in the acetaldehyde content. The effect of aeration on acetaldehyde content. Ahrenst-Larsen & Levin-Hansen1 showed that beer which had been artificially aged by storing at 45 C for 5 days had increased acetaldehyde. Burger et al.6 and Cowland & Maule9 showed that high air content during processing also produced high acetaldehyde. Although we have not investigated this aspect in detail, it was noted that the acetaldehyde remained con stant in those beers which had been bottled with negligible air contents even after having been stored for several months. Beers which were contained in tank or bottle with high air had variable and inconsistent acetaldehyde contents. Taste threshold of acetaldchyde-bisulphite addition compound. Taste testing of beers which contained increasing quantities of acetaldehyde, both in the presence of and without sulphur dioxide, showed that the taste threshold was 25 ppm in both cases. This is in agreement with the work of Harri son11 but there was no clear description of the flavour produced at this level. Discussion This method for the determination of acetaldehyde in beer is more specific than most other colorimetric methods, the inter ference from other aldehydes being reduced. The sensitivity of the method is such that -1 ppm of acetaldehyde can be determined in beer, wines or spirits. Acetaldehyde is present in varying amounts in beer and the cause of the high content in primed beer containing SO2 is evident from these investigations. Neuberg et al.1* have shown that in the presence of sodium sulphite the normal fermentation pathway is blocked. When acetaldehyde is bound by sulphite it can no longer act as the hydrogen acceptor for nicotinamide adenine dinucleotide in its reduced form (NADH) and is therefore not reduced to ethanol. Dihydroxy acetone phosphate replaces acetaldehyde as sub strate for NADH and glycerol is the main fermentation product. Sulphur dioxide added to beer containing actively fermenting yeast results in a partial blockage of the metabolic conversion of acetaldehyde to ethanol and leads to a high content of acetaldehyde-bisulphite addition compound. The only other cause of high and variable acetaldehyde levels found was aeration. It is a common practice to add sulphur dioxide as a preservative and antioxidant to beer which contains active yeast. If sulphur dioxide is to be used for this purpose and high acetaldehyde levels avoided, the sulphur dioxide should be added after fermentation is completed and the yeast has been removed or inactivated. Acknowledgement. The authors wish to thank the Directors of Courage Limited for permission to publish this work and Mr. D. J. Silvester for technical assistance.

6 472 OTTER & TAYLOR: ACETALDEHYDE IN BEER [J. Inst. Brew. References 1. Ahrcnst-Larsen. B., & Levin-Hanson, H., Wallerstein Laboratories Communications, 1964, 27, No. 92, Arsenault, G. P.. & Yaphe, W., Analytical Chemistry, , Barker, S. B., & Summcrson, W. H., Journal of Biological Chemistry, 1941, 138, Bavisotto, V. S., & Roche, L. A., Proceedings of the American Society of Brewing Chemists, 196, Bavisotto, V. S., Roche, L. A., & Hcinisch, B., Proceedings ofthe A merican Society of Brewing Chemists, 1961, Burger, M., Glenistcr, P. R., & Becker, K., Brewers Digest, 19G5, 3, Brenner, M. W.. Owades, J. L., & Fazio, T., Proceedings ofthe American Society ofbrewing Chemists, 1956, Clancy, D. J., & Kramni, D. E., Analytical Chemistry, 1963, 35, Cowland, T. W., & Maule, D. R., Journal of the Institute of Brewing, 1966, 72, Drews, B., Barwald, G., & Niefind, H. J., Technical Quarterly Master Brewers Associa tion of America, 199, 6, Harrison, G. A. F., Proceedings of the European Brewery Convention Congress, 13, Matsumoto, H., Oskiroa, I., & Hylnka, I., Cereal Chemistry, 196, 3, Misnelhom, K., Branntweinwintschaft, 1963, 13, Ncuberg, C. et at.. The Chemistry and Biology of Yeasts, Ed. A. H. Cook. Academic Press, 1958, G. Nordstrom, K., Ada Chemica Scandinavica, 1966, 2, Owades, j. L., & Dons, J. M., Journal of the Association of Official Agricultural Chemists, 1968, 51, Sandegren, E., & Enebo, L., Wallerstein Laboratories Communications, 1961, 24, Sawicki, E., Hauscr, T. R., Stanley, T. W., & Elbert, W., Analytical Chemistry, 191, 33, Siefker, J., & Pollock, G., Proceedings of the American Society of Brewing Chemists, 196, Stotz, E., Journal ofbiological Chemistry, , Van der Kloot, A. P., & Wilcox, F. A., Pro ceedings of the American Society of Brewing Chemists, 1969, 7; 191, 24.