Lambic and Gueuze Are Belgian Beers Obtained by Spontaneous Fermentation of Wort
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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, 0·102-640,462·440-650'880·161'150-640·210'070,130'090'083'510-960·020-090-170,883,902'160·220-052-260·150'191·681-330-430'04O'll0'591'730'520'470-131'590-320-460-080·260'240'220,070-060-941·120-600-890'102-030,080-180'112·186'581-660,870'440,131'401-27I 0·140-540·390·240,100-040-080-170-100-42 30 24 18 12 6 o ClOCl<C"C18C16:1C1s:aCIa:! Fig.