278 J. Inst. Brew., September-October, 1978, Vol. 84, pp. 278-282

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

By M. SPAEPEN, D. VAN OEVELEN AND H. VERACHTERT (Laboratory of Industrial Microbiology and Biochemistry, University of Leuven, Kardinaal Mercierlaan 92, Heverlee-Louvain, 3030, Belgium)

Received 30 January 1978 lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts._ The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. lambic and Gueuze are especially rich in caprylic (Cs) and capric (C10) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of Iambic and gueuze.

Key words: Iambic, gueuze, spontaneous fermentation, fatty EXPERIMENTAL acids, esters. Sampling Sampling of Iambic and gueuze has been described pre• viously.25.26 INTRODUCTION Determination of free fatty acids.-Free fatty acids were Free fatty acids are important in brewing for several determined after extraction and esterification accorci.ing to reasons2,5,20,21 as they exert an influence on flavour and on the MacPherson & Buckeell with some modifications. Aliquots foam properties of beer. Their impact on flavour may be of Iambic (lOO ml) were adjusted to pH 1 with concentrated direct, or indirect, through the formation of esters. Several hydrochloric acid and extracted three times with respectively reports exist on the taste and odour threshold of these com• 50 ml, 50 ml and 25 ml of a chloroform-methanol mixture pounds.5,6,8,22,27 The additive flavour effect of the C6-CI2 (3: 1). The combined extracts were then extracted with res• acids in beer often exceeds their combined flavour threshold pectively 37'5 ml, 37·5 ml and 25 ml of 10% potassium of approximately 10 ppm, causing the 'caprylic' or goaty hydroxide. The combined potassium hydroxide extracts were flavour.3,I2 NordstromI5,I6 provided a quantitative relation• acidified and extracted three times with the chloroform• ship between the amount of fatty acids and the corresponding methanol mixture (3 : 1). The final extract was dried over ethyl esters, which have a much lower threshold than the magnesium sulphate and evaporated to dryness. The residue acids. It has been reported also that lower fatty acids, when was methylated with 5 ml of boron trifluoride-methanol and added to wort in high concentration, may inhibit the syn• 10 ml anhydrous methanol. After methylation the solution thesis of ethyl acetate,I3-16 while higher fatty acids may was adjusted to 50 ml with saturated sodium chloride solu• stimulate its synthesis.I.I7.18 Unsaturated higher fatty acids tion and 0,5 ml n-decane was added. The fatty acid methyl may inhibit this process. I Also it is known that linoleic acid esters were extracted in the decane phase. Pentadecanoic acid (CI8:2) is associated with the cardboard flavour of beer.4,9 (Clf) was added to the beer as an internal standard. For gas Unsaturated higher fatty acids are drastic foam inhibitors, chromatographic analysis 0'1 fLI of the decane extract was and saturated acids are foam promotors.2 It is also generally injected on a 25 m capillary free fatty acid phase column, known that higher fatty acids may be important growth which was then operated from 100aC to 190aC at a rate of factors for yeasts, especially when membrane synthesis must SaC/min. Injection and detection blocks were at 225aC. The occur in anaerobiosis. carrier gas was hydrogen (4 mljmin). The apparatus used was With these considerations in mind it must be interesting a Carlo Erba Fractovap 2350 equipped with flame ionization to study the fatty acid composition of Iambic and gueuze. detectors and a Hewlett-Packard 3385 automatic calculator. These are beers obtained by spontaneous fermentation, in• Severe septum bleeding was always observed during tem• volving enterobacteria, Pediococcus, Kloeckera, Saccharo• perature programming, which resulted in the appearance of myces and Brettanomyces.25 The fermentation of the wort by many interfering peaks. This could be avoided by modification such a mixed flora might result in a very special spectrum of the injection port according to Tucknott & Williams.23 of higher fatty acids, which in turn might contribute to the It is surprising that such difficulties and methods to avoid special taste of Iambic and gueuze. It could also be possible them seem to be ignored by most manufacturers of GC that higher fatty acids produced by bacteria in anaerobio• apparatus. sis7,I9 might be growth promoting factors for the develop• Determination of esters of higher fatty acids.-The esters ment of yeasts and that they are partly responsible for the of higher fatty acids (ethyl C6-ethyl CID) and also phenethyl establishment of the special mixed flora of these beers. acetate were analysed by gas chromatography using the same As nothing is known of the higher fatty acid composition column as for the acids, with temperature programming from of Iambic a study of the content of fatty acids was made and 100aC to 190aC at a rate of SaC/min. They were isolated from their concentration over a two year fermentation period was Iambic by distillation of 100 ml beer until 50 ml of distillate measured. Simultaneously the concentration of the ethyl were obtained. n-Octanol was added to the beer as internal esters of the more important fatty acids was determined. standard. To the distillate 3 g of sodium bicarbonate was Finally, several micro-organisms isolated from fermenting added and the solution was extracted during 25 min with Iambic, were grown in pure culture, and their ability to syn• 0'5 ml n-heptane. For analysis 0'1 fLIof the heptane extract thesize or utilize fatty acids of wort was examined. was injected on the column. Vol. 84, 1978] SPAEPEN et al.: FATTY ACIDS AND ESTERS PRODUCED DURING FERMENTATION 279 Formation of free fatty acids by pure cu/tures.-Bacteria or old Iambic and gueuze are very rich in Cs and CIOacids. The yeasts isolated from fermenting Iambic were first inoculated acids from Cl2 to ClS' after a slow increase during the first in 10 ml of sterile Iambic wort in test tubes. After growth, months, showed a slow decrease during the later months. 3 ml of these cultures were used to inoculate 300 ml batches Nevertheless, the concentration of C12 slightly increased again of sterile Iambic wart. Enterobacteria and yeasts were grown during the three last months. The concentration of the Cl2• in erlenmeyer flasks closed with one-way valves. For Aceto• ClS acids always remained several orders of magnitude lower monas the Erlenmeyers were stoppered with cotton plugs. All flasks were shaken once a day. For Pediococcus optimal growth conditions in Iambic wort have not yet been found and they were grown and studied using the medium of Uchida & Mogi24 under a carbon dioxide atmosphere. Fatty acid production was recarded as indicated later in the text. For maintenance the different strains of yeasts, acetic acid bacteria and pediococci were kept on universal beer agarlO • supplemented with 2 % calcium carbonate and entero• bacteria on nutrient agar. Materia/s.-Fatty acids (Cg-ClS:2) and the methyl esters were obtained from Sigma (St. Louis, USA), boron tri• fluoride-methanol, octanol-1 and n-heptane from Merck (Darmstadt, W. Germany), n-decane from Koch-Light (Colnbrook, England), the ethyl esters of Cg to ClO from Aldrich Europe (Beerse, Belgium), phenethyl acetate from Fluka (Buchs, Switzerland).

RESULTS Fatty Acid Composition of Fermenting Lambic.-Lambic was analysed for higher fatty acids during a fermentation period of from 0 days up to 20 months. The results are given in Tables I and n. A typical gas chromatographic analysis is shown in Fig. 1. Table n contains the results obtained from the analysis of casks of different age, while Table I contains results obtained during a periodic analysis of two particular casks. After 9 days of fermentation, the acids from Cs up to Clg already showed a remarkable increase in concentration, with a ratio of ClO/CS higher than one. The content of ClS and ClS:3 did not change significantly. On the 37th day the concentration of the CS-CIg acids had increased even more but the ratio of CIO/CSwas lower than one. After two months the concentration of the CIO remained fairly constant ex• cept in very old Iambic (18-20 months) where an increase was again noted. The content of Cs slowly increased. ~s a result '- TABLE 1. Content of Higher Fatty Acids (ppm) of Lambic During the First Few Days of Spontaneous Fermentation.

min Acid;~ays '-2-/-9-'-17-1-3-7Cask I ---2-/-9-/-1-7I Cask II -/-37- - ~ ---.J C, 0·102-642·440,460,133'510·210·020'070-090·160'090'080-170'880-961'150-650-643,902'161·680·150'190·222-260,880-050'240'590'220-320-460'100-080·260,080-130-180'04O'll0,070-062-030-940-891'591-331'731·120-430'520'470-606'582·180,130'440'110,871-661-271'40I 0·140-540·240,100·390-170-080-100-040-42 30 24 18 12 6 o ClOC"C18C16:1C1s:aCl

Fig. 1. The separation of the methyl esters of fatty acids (Cg• ClS:,) in gueuze. Operating Conditions: 25 m x 0,5 mm FFAP capillary column; carrier gas: hydrogen (4 mlfmin); injection tem• perature: 225°C; flame ionization detector temperature: , 225°C; column temperature: from 100°-190°C at 5°C/min. (/: not detected). Peak identification: s-solvent peak; C1s-(internal standard).

TABLE n. Content of Higher Fatty Acids (ppm) of Lambic During 20 Months of Spontaneous Fermentation.

Cl

The samplesC18 were taken0-16from0,15different0'33casks. 0-37 0'42 0-23 0·33 0-28 0-26 0·14 0·10 0·12 0·17 0'13 0'13 0-08 r 0·14 Acids I 0 1'0 2-0 3-0 4'5 5'0 6'0 7-5 8'5 9-0 10'5 ll'O 12'0 13·0 18-0 19'0 20·0 280 SPAEPEN et al.: FATTY ACIDS AND ESTERS PRODUCED DURING FERMENTATION [J. Inst. Brew. than that of the Cs and CIOacids. The concentration of the analysed (results in Table Ill) it was found that it contained higher unsaturated fatty acids decreased as the Iambic be• appreciable amounts of ethyl CIO, which in general is absent came older and some disappeared completely from the wort, in lager beer or present in very low amounts in some ales. especially CIS:2and CIS:3' Formation of Free Fatty Acids by Pure Cultures of Lambic Synthesis of Ethyl Esters of Higher Fatty Acids in Ferment• Micro-organisms.-In order to find a possible relationship ing Lambic.-The concentration of the ethyl esters of the Cs• between the higher fatty acid composition of Iambic and the CIOacids during an 11 month fermentation period is given in different micro-organisms present at certain periods of Table Ill. This table also contains data on phenethyl acetate Iambic fermentation,26 several Iambic baCteria or yeasts were which was determined simultaneously. Other esters such as grown in pure culture and examined for fatty acid production. ethyl acetate, iso-amyl acetate and ethyl lactate have been Table IV shows the fatty acid composition of media after determined during previous work.25 Faint amounts of ethyl growth of Iambic bacteria. Clearly some strains of entero• lactate are recovered in the analytical procedure used here for bacteria could utilize the CS-Cl2 acids of wort and syn• the determination of the ethyl esters of the higher fatty acids. thesize CIS and the unsaturated CIS and CIS acids. Acetomonas ,Ethyl lactate thus shows a peak on a typical gas chromato• strains metabolized the Clo-Ca acids and also CIS:h gram of ethyl esters (Fig. 2). While important levels of the Cs CIS:2 and CIS:3 while forming some Cl8:I. Some strains of and CIOacid were detected after one month of fermentation, Pediococcus utilized CIS, CIS:2 and CIS:I while the con• the concentration of the corresponding ethyl esters rose signi• centration of Cs, CIS and CIS:3 was increased. Table V ficantly only after about 8 months. The concentration of shows the results for yeasts grown in Iambic wort. As shown ethyl Cs and phenethyl acetate were maximal even after one they were active in the synthesis of the CS-Cl2 acids, es• month. When gueuze (Iambic re-fermented in the bottle) was pecially Brettanomyces which accumulated high concentra• tions. Unsaturated acids showed a tendency to decrease and Cl8:2 and C18:3 were removed completely from the growth medium in some instances. Ca was lowered somewhat but C16 and CIS remained at fairly constant concentration.

DISCUSSION Table VI summarizes the results obtained with pure cultures and clearly shows that the pattern of fatty acids produced by yeasts is different from that produced by bacteria. Yeasts show the tendency to increase the concentration of the Cs• Cl2 acids, whilst bacteria mostly tend to reduce thes~ con• centrations. This may explain the rapid increase of the Cs• 4 Cl2 acids during the first month of fermentatiqn as this fer• mentation period is characterized by the rapid devdopment of Kloeckera and Saccharomyces. After a few months this results in ratios of CS/CIOof about 2/1. Similar ratios were found with pure cultures of Saccharomyces. At certain 5 other periods and especially in old Iambic (19-20 months) the ratio of CS/CIOincreased to about 3/1 and this might possibly be ascribed to Brettanomyces. This yeast appears later during fermentation and in pure culture it produced high amounts of Cs and CIO acids, in a ratio of about 3/1. As shown in Table VII, high concentrations of Cs (caprylic) and CIO (capric) acids seem to be characteristic of gueuze re-fermented in bottles. The rapid increase in Cl2 is probably due also to the development of yeasts and some higher concentrations 8 6 found in later periods may confirm the observation that Brettanomyces can synthesize this acid. With respect to Cl4 only two strains studied were found to increase its concentra• tion. These were enterobacteria. This then might explain the 7 large increase during the first month, where such bacteria are present,26 and the slow but continuous decrease during later periods (Table I and II). Bacteria as well as yeast produce CIS and its origin in Iambic may be of mixed type. Its high con• centration during the first month is certainly due to bacteria. Neither yeasts nor bacteria produce or consume much CIS' Its concentration in Iambic correcpondingly does not change significantly. Unsaturated fatty acids are taken up by pure cultures of yeasts and bacteria, except the enterobacteria. This is especially so for Cl8:2 and CIS:3• In some of the Iambic casks these two acids had already disappeared from min.. the wort after one month. In old Iambic, in general, they were 24 18 12 6 present either in very low concentration or not at all. Possibly enterobacteria may produce some of these acids during the Fig. 2. Typical gas-liquid chromatogram of esters in re-fermented first month and these are then taken up later by yeasts and gueuze. pediococci. Operating Conditions: 25 m X 0,5 mm FFAP capillary column; carrier gas: hydrogen (4 ml{min); injection tem• From the experiments with pure cultures it becomes evi• perature: 225°C; flame ionization detector temperature: dent that fatty acid metabolism may vary from strain to 225°C; column temperature: from 100°-190°C at SaC/min. strain, which may explain the variations observed in Iambic Peak identification: l-iso-amyl alcohol; 2-ethyl caproate analysis. Moreover, it must be remembered that different (C,); 3-ethyl lactate; 4-ethyl caprylate (Cs); 5-n-octanol-l casks had to be used for the sampling. Nevertheless results (internal standard); 6-ethyl caprate (ClO); 7-phenethyl acetate; 8-phenethyl alcohol. obtained do not contradict our previous studies. They con- Vol. 84, 1978] SPAEPEK et al.: FATTY ACIDS AND ESTERS PRODUCED DURING FERMENTATION 281

TABLE Ill. Content of Ethyl Esters of Fatty Acids (ppm) of Lambic During 11 Months of Spontaneous Fermentation.

0,13gueuze*0·230,090·250·530,440·151·58·59·00,500,450·220·420·270,54,59,00·140·320,080·350·200·070·12a-os8,50·050·180·17-0·284-5I0·080,130,071·00·260·090,490·19I 0·190-110,050·040-5 Re- EthylPhenetylEstersCIOC. fermentedacetate Ethyl C. :s:

(*: Mean of four samples; I: Not detected).

TABLE IV. Amount of Fatty Acids (ppm) in Fermentation Media (Wort or Uchida), Before and After Growth of Bacteria.

0·181·20Strain0·48Strain4-490·424·130,05I0·200·170·230·590·07medium0,690'430·026140,030·184-514,042·435·204'000,350'040·392·010,090·140-320·220·110,340'160·060,070-710,450-010·01I0-210·210'050,400,37Strain0'070'140,081·780'011-990'171·04I-5660,070·520-200·100'060'340·02Wort0-223·280'120'160'630-194'90II AABAWort-Enterobacteriaceae146PediococcusAcetomonascerevisiae Cl.:Cl.CaClOC12C16C16:Cl.:3 II2 Acids pHGrowthC. (days) Uchida

(f: Not detected).

TABLE V. Amount of Fatty Acids (ppm) in Wort, Before and After Growth of Yeasts as Pure Cultures.

.. 0·340·160·200,060·170·070,630·12cereV1SlOe0·194,90Brettanomyces0,0415·030,064,405·220,550-380·160·810·042·28Wortapiculatabruxellensis75,330,660·240·010'044·51I0'230·914·1016·50·62lambicus0,551·170·424-700'160·220,480·200,061013,05I0,53220,6814·88I0·120·060·0275-694'074,0075,690·312·966·16Brettanomyces0·260·510·130·600'054,40100·01IbayanusKloeckeraI. 74·3110 Cl.:Cl.Cl.:CaC12ClO3AcidsI2 pH Saccharomyces AttenuationGrowthC. (days)(I;;) I Sacchar?"!yces

(f: Not detected).

TABLE VI. Changes in the Fatty Compositions of Fermentation Media by the Development of Some Important Brewery Micro• Organisms.

-0,0 - T Enterobacteriaceae+++++++(+)(-)++Saccharomyces+++-++-(-)++Pediococcus(+)+++++-++(+)++-(+)+-Kloeckera-+- 0Acetomonas Cl.: 23 Ca Cl.:Cl.Cl"C16:C12CIOI I AcidsBrettanomycesI C.

(-: disappearance of fatty acids from the fermentation media; +: increase of fatty acids in the fermentation media; (+): very faint increase of fatty acids in the fermentation media; 0: unchanged). 282 SPAEPEN et al.: FATTY ACIDS AND ESTERS PRODUCED DURING FERMENTATION [J. lnst. Brew.

firm the succession of Enterobacteria sp., Saccharomyces REFERENCEl yeasts, Pediococcus sp. and Brettanomyces yeasts. 1. Ayrapaa, T., & Lindstrom, I., Proceedings 0/ the European With respect to Iambic aroma the high concentration of Brewery Convention, Salzburg, 1973,271. Cs and ClO acids and of their ethyl esters is important. Thresh• 2. Carrington, R., Collett, R. C., Dunkin, I. R., & Halek, G., old values for these compounds may however be quite Journal 0/ the Institute 0/ Brewing, 1972, 78, 243. 3. Clapperton, J. F., Dalgliesh, C. E., & Meilgaard, M. C., different in Iambic and gueuze than in other beers, as they Journal 0/ the Institute 0/ Brewing, 1976, 82, 7. contain on the average 3 g/litre of lactic acid and 1 g/litre of 4. Drast, B. W., van Eerde, P., Hoekstra, S. F., & Strating, J., acetic acid. High concentrations of Cs and ClO anyhow Proceedings 0/ the European Brewery Convention, Estoril, provide another characteristic of gueuze re-fermented in the 1971,451. -...e. 5. Engan, S., Journal o/the Institute 0/ Brewing, 1974,80,162. bottle, which undoubtedly remains the finest product of 6. Engan, S., The Brewers Digest, 1974,49(11), 40. 1 Iambic fermentation. A comparative study on the content of 7. Goldfine, H., & Blach, K., Journal 0/ Biological Chemistry, the esters of C6-C10 in gueuze and other beers is presently 1961,236,2596. under way. 8. Harrison, G. A. F., & Collins, E., American Society 0/ Brewing Chemists, 1968,83. 9. Jamieson, A. M., & Van Gheluwe, J. E. A., American So• Acknowledgement.-The technical assistance of Mrs J. ciety 0/ Brewing Chemists, 1970, 192. Knaepen is greatly appreciated. The N.F.W.O. Foundation 10. Kozulis, J. A., & Page, H. E., American Society 0/ Brewing and the Verhelst Foundation for Brewery Research provided Chemists, 1968, 52. 11. MacPherson, J. K., & Buckee, G. K., Journal 0/ the Institute financial support. De Neve Brewery provided the oppor• 0/ Brewing, 1974, 80, 540. tunity to follow the fermentation at brewery level. 12. Meilgaard, M. C., Technical Quarterly 0/ the Master Brewers Association 0/ America, 1975,12,107. 13. Nordstrom, K., Journal 0/ the Institute 0/ Brewing, 1962, 68, 398. 14. Nordstrom, K., Journal 0/ the Institute 0/ Brewing, 1963, 69, 142. 15. Nordstrom, K., Journal 0/ the Institute 0/ Brewing, 1963, 69, 310. 16. Nordstrom, K., Journal 0/ the Institute 0/ Brewing, 1964, 70, 42. "'-. TABLE VII. Extreme Values of the Fatty Acid Concentration 17. Nordstrom, K., Journal 0/ the Institute 0/ Brewing, 1964, 70, (ppm) in Different Beers and in Gueuze. 233. 18. Nordstrom, K., Journal 0/ the Institute 0/ Brewing, 1964, 70, 328. Acids Re-fermented gueuze (3)* Beer (22)* 19. Norris, A. T., & Bloch, K., Journal 0/ Biological Chemistry, 1963,238, PC 3133. 20. Nykiinen, L., & Suomalainen, H., Teknilissen Kemian 12,40-21,85 5,30-14,59 Aikakauslehti, 1963, 20, 789. 2'30- 3,90 0,30- 1·34 21. Perkins, E. G., & Witt, P. R., American Society 0/ Brewing 0,08- 0·18 0,05- 0·24 Chemists, 1968, 145. 0'04- 0,09 0'02- 0,07 22. Salo, P., Journal 0/ Food Science, 1970,35, 95. 0,32- 0·61 0,20- 0,54 23. Tucknott, O. G., & WilIiams, A. A., Analytical Chemistry, 0,05- 0,23 0,00- 0·22 1969,418,2086. 0,13- 0·29 0,07- 0·21 24. Uchida, K., & Mogi, K., Journal 0/ General and Applied 0·22- 0'60 0,13- 0·65 0'03- 0,09 0,00- 0·20 0,00 0,00- 0·06 .... gf1hf!l1Jsfitute of1Jrf.wjng, .l~?(),Jg,)~." ....""" "_,,,__ "" 26. Van Oevelen, D., Spaepen, M., Timmermans, P., & Verach- I~-25..~i!j[cte~~f-;;:"ff.~~ll~E~£~ille,tert,H., Journal 0/ the!nstitlite F:;-&Verachte"r-t,0/ Brt!yving, 1977,83,356.H.,7our-naIl (3)*: analysis of 3 samples re-fermented gueuze; (22)*: -lrWmiams~'A.: A.., Journal o/the Institute 0/ Brewing, 1974,80, analysis of 22 samples of low and high fermentation beer). 455.

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