SYNTHESIS OF AROMA COMPONENTS DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic is a type of Belgian beer obtained by spontaneous fermentation of wort. The fermentation in casks takes almost two years, and after that period the beer may be re-fermented in bottles and is then called gueuze. To gain insight into the many microbiological transformations occurring during this process, a qualitative and quantitative study of several components formed over a two-year period was undertaken. It was found that lambic and gueuze are characterized by high contents of ethyl acetate, ethyl lactate, lactic acid and acetic acid. Two phases of fermentation may be distinguished: a primary phase lasting for about three months during which almost all the ethanol and higher alcohols are formed and a very long secondary phase during which lactic acid is formed first, followed by ethyl lactate. Some lower fatty acids, including all the acetic acid, are made at the very beginning of the first phase. An important part of the ethyl acetate is made during the first phase but the amounts increase slowly to high levels during the second phase. Unusually low amounts of isoamyl acetate are detected. Several components are above the reported threshold levels of taste and are thus probably involved in the genesis of the typical aroma of lambic and gueuze. These results will allow a further study of the relations between the components found and the microbial populations involved in the spontaneous fermentation inherent in the brewing of lambic and gueuze.     

WORT ENTEROBACTERIA AND OTHER MICROBIAL POPULATIONS INVOLVED DURING THE FIRST MONTH OF LAMBIC FERMENTATION

Lambic is a special type of Belgian beer obtained by spontaneous fermentation. The micro-organisms occurring during the first month of lambic fermentation were counted using several selective media. During this period a mixed flora is present in which the Enterobacteriaceae predominate. They were isolated from samples of lambic of different ages originating from different breweries and identified using the minitek identification system. Much variation may occur in types of Enterobacteriaceae: Klebsiella aerogenes, Enterobacter cloacae, E. aerogenes, Escherichia coli, Citrobacter freundii, A-D group bacteria and Hafnia alvei are found during the first month of fermentation. E. cloacae is found most frequently followed by K. aerogenes. After 30 to 40 days all Enterobacteria die off because of the activities of yeasts. Lactic acid and acetic acid bacteria are found in very low numbers during the first month of fermentation; Staphylococci and Streptococci are not present.     

MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE

Lambic and Gueuze are special Belgian beers obtained by spontaneous fermentation. Micro-organisms involved in this fermentation were counted and differentiated using several selective growth media. Micro-organisms were also isolated from samples of Lambic of different age and originating from different casks and brews and identified by classical tests. The following general pattern of microbial development was observed. After 3 to 7 days the fermentation started with the development of wort Enterobacteriaceae and strains of Kloeckera apiculata. These organisms were overgrown after 3 to 4 weeks by strains of Saccharomyces cerevisiae and S. bayanus. These were responsible for the main fermentation, lasting for 3 to 4 months. This was followed by a strong bacterial activity. This period was dominated by the growth of strains of Pediococcus cerevisiae. These reached their maximal numbers during the summer months and were responsible for a fivefold increase in lactic acid concentration. In some casks they caused ropiness. After the main fermentation period Lambic is very sensitive to spoilage by acetic acid bacteria of the genus Acetomonas. The presence of air may be the determining factor for their development. After 8 months a new increase in yeast cells was noted. These belonged now mainly to the genus Brettano-myces bruxellensis and Br. lambicus. They caused a further slow decrease in residual extract and the appearance of special flavours. Oxidative yeasts of the genera Candida, Cryptococcus, Torulopsis and Pichia were also detected and may be responsible for the formation of a flim on the beer surface after the main fermentation.     

SYNTHESIS OF AROMA COMPOUNDS BY WORT ENTEROBACTERIA DURING THE FIRST STAGE OF LAMBIC FERMENTATION

Lambic is a special type of Belgian beer obtained by a spontaneous fermentation. The fermentation is initiated by a growth of enterobacteria and non-maltose fermenting yeasts. These organisms die off after one to two months. To gain a clear insight in the relations between the enterobacteria and the aroma compounds formed in wort during this first period, several bacterial isolates were studied with respect to their metabolites formed in a synthetic medium and in two different lambic worts, using aerobic or semi-anaerobic conditions. The results showed that enterobacteria, especially Enterobacter species, are responsible for the production of 2, 3-butanediol, acetic, lactic and succinic acid and lower amounts of ethyl acetate and higher alcohols which are the main aroma compounds found in 1 to 2 months old lambic. Ethanol production is mainly due to yeast activity. The results are in agreement with previous determinations of entero-bacterial species present in first phase lambic¹⁸ .     

DISTRIBUTION OF ESTERS PRODUCED DURING SUGAR FERMENTATION BETWEEN THE YEAST CELL AND THE MEDIUM

The distribution of the esters formed during sugar fermentations between the yeast cells and the medium was investigated in fermentations by 5 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum (carlsbergensis). The esters studied included the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₂ fatty acids. All of both acetates appeared in the medium. The proportion of the fatty acid ethyl esters transferred to the medium decreased with increasing chain length: all in the medium for ethyl caproate, 54–68% for ethyl caprylate, 8–17% for ethyl caprate, and all remaining in the yeast cell for ethyl laurate. A higher proportion of the esters formed appeared to remain in the cells of the S. uvarum strains than in cells of S. cerevisiae.     

THE NATURE OF FATTY ACIDS AND CAPSANTHIN ESTERS IN PAPRIKA

SUMMARY– The triglycerides present in whole ground paprika and paprika pods were extracted and hydrolyzed. The fatty acids were methylated, separated by gas chromatography and identified by mass spectrometry. The whole paprika and pods, respectively, contained approximately 66 and 45% linoleic acid, 14 and 19% palmitic acid, 12 and 14% oleic acid and 5 and 17% linolenic acid. Small quantities of myristic and lauric acids and traces of capric, stearic and palmitoleic acids also were present. Capsanthin, which amounted to 35% of the total carotenoids, occurred as the dilaurate ester. It was isolated from paprika by thin-layer chromatography after interesterification of the triglycerides. Capsanthin dilaurate, synthesized in the laboratory, gave identical R_f value and infrared and visible spectra to those of the naturally occurring compounds.     

EFFECTS OF AERATION DURING THE CULTIVATION OF PITCHING YEAST ON ITS CHARACTERISTICS DURING THE SUBSEQUENT FERMENTATION OF WORT

The characteristics of cultivated yeast were studied after continuous aeration, aeration for the first hour and without aeration. The fatty acid content increased under aerobic conditions, while the level of storage sugars increased under anaerobic conditions. In a fermentation test, total number of cells and their viability and also the intracellular level of storage sugars and fatty acids were higher in yeast cultivated under aerobic conditions than in yeast cultivated under anaerobic conditions. Since there were no significant differences between the performance of yeast cultivated under continuous aeration and those with aeration for the first hour, the latter offer the best economic choice.     

RAPID METHODS FOR THE ANALYSIS OF THE FATTY ACIDS OF FERMENTING WORT AND BEER (C6-C10) AND YEAST (C6-C18)

Relatively rapid and simple methods for measuring the content of the free C₆-C₁₀ fatty acids in fermenting wort and beer and of the total C₆-C₁₈ fatty acids in yeast are described. The acids are extracted, after saponification in the case of yeast, using a small volume of chloroform and analysed by gas chromatography as free fatty acids. The precision of the procedures is reasonable.     

THE FORMATION OF ESTERS AND HIGHER ALCOHOLS DURING BREWERY FERMENTATION; THE EFFECT OF CARBON DIOXIDE PRESSURE

The influence of the size and geometry of brewery fermentation vessels on beer flavour and aroma formation is generally attributed to carbon dioxide pressure. In order to study this pressure effect, brewery batch fermentations were carried out on the laboratory scale with Saccharomyces cerevisiae. The formation rates and yields of esters and fusel alcohols were studied in relation to the growth of metabolically active biomass. The results indicate that the observed reduction in the formation of esters and fusel alcohols with increased carbon dioxide pressure is mainly caused by reduced yeast growth. The overall formation of fusel alcohols is less affected than the formation of esters.     

环境因子对球等鞭金藻脂肪酸含量和组成的影响

用气相色谱法分析了不同的光照强度、通气量及不同生长时间下生长的球等鞭金藻的脂肪酸组成及含量.结果表明,不同环境因素下生长的微藻,其脂肪酸组成及不饱和程度有差异.一定范围内高光照有利于总不饱和脂肪酸（TUFA）的积累,尤其有利于二十二碳六烯酸（DHA）和亚麻酸含量的增加,但是过高的光照反而不利于不饱和脂肪酸含量的增加;高的通气速率有利于TUFA及DHA含量的增加,当通气量为3 vvm时TUFA和DHA的含量均达到最大,并且DHA的含量一般在对数生长初期即达到最大,而TUFA含量则在稳定期后才达到最大.     

THE ORIGIN OF THE MEDIUM CHAIN LENGTH FATTY ACIDS PRESENT IN BEER

The medium chain length fatty acids that are excreted during fermentation are produced by synthesis and not by degradation. The fermentation of a wort supplemented with propionic acid (C₃) or valeric acid (C₅) leads to the excretion of nonanoic acid (C₉) in addition to the usual even chain acids. C₉ acid was not detected in the beer when the inoculated yeasts contained a high proportion of pentadecanoic acid (C₁₆) and heptadecanoic acid (C₁₇) or when the C₁₇ acid was added to the wort, demonstrating that a degradative route is unimportant. The content of the medium chain length fatty acids in beer varies directly with their content in yeast; thus the fatty acid composition of the beer reflects changes in the content of these acids in yeast brought about by alteration in the supply of oxygen or by the addition of C3 acid to wort.     

OXYGEN AS A REGULATOR OF ESTER ACCUMULATION DURING THE FERMENTATION OF WORT OF HIGH SPECIFIC GRAVITY

The supply of oxygen to yeast for short periods during the fermentation of wort of high specific gravity can prevent excessive synthesis of ethyl acetate and isoamyl acetate, although it does not affect levels of higher alcohols. Oxygenation increases yeast growth and causes faster fermentation. The effect of oxygen on ester accumulation is independent of pitching rate.     

ANALYSIS OF LONG-CHAIN FATTY ACIDS IN BEER

A simple gas chromatographic method is presented for quantitative analysis of long-chain fatty acids in beer. Analytical results are quoted for ten different beers.     

不饱和脂肪酸分离技术研究概况

不饱和脂肪酸作为重要的膳食脂肪酸,具有特殊的理化性质和独特的生理功能。对目前采用的不饱和脂肪酸主要分离技术进行了综述,并分析了各自的优缺点。     

THE INFLUENCE OF LIPIDS DERIVED FROM MALT SPENT GRAINS ON YEAST METABOLISM AND FERMENTATION

The addition to wort of lipids derived from malt spent grains had a pronounced effect on yeast metabolism. The lipids allowed the fermentation of de-oxygenated wort and also stimulated yeast growth and the corresponding rate and extent of fermentation of air-saturated wort by yeast strains having a high oxygen requirement. The lipids increased the fusel alcohols content of beer and decreased the content of esters and medium chain-length fatty acids. The yeast incorporated sitosterol and unsaturated fatty acids from the spent grain lipids and the unsaturated fatty acids changed the pattern of fatty acids and sterols synthesized by the yeast. The fatty acids were present in the spent grain lipids mainly as triglycerides, free fatty acids and phospholipids. Using pure lipid compounds it was shown that the triglycerides were inactive and that the spent grain lipids exerted their effect on fermentation through the synergistic action of free unsaturated fatty acids, sitosterol and phospholipid. Phospholipid could be replaced by the detergent, Triton X-100. The effect of the lipids on the synthesis of esters, fusel alcohols and medium chain fatty acids could be explained solely by their content of unsaturated fatty acids.     

核桃果实各部位脂肪酸的组成与含量分析

为评价核桃的营养价值并探讨核桃果实各部位的脂肪酸组成结构，通过气相色谱/质谱联用技术（GC/MS）对桃核各部位（核壳、核仁、种皮及青果皮）的脂肪酸成分进行比较分析，鉴定出亚油酸、亚麻酸、棕榈酸、硬脂酸、油酸等14种脂肪酸成分及其在核桃各部分中的相对含量。分析了桃核各组成部分中不饱和脂肪酸与饱和脂肪酸所占比例及营养价值。     

洋葱汁的酶法制备及其酒精发酵特性研究

研究了洋葱汁的酶法制备工艺及其酒精发酵特性。对果胶酶处理洋葱汁最适条件进行了优化,确定的适宜条件为：酶用量0.04%;作用时间90min;作用温度35℃;最适pH3.8。并对洋葱汁酒精发酵过程中的酒度、酸度、糖度和还原糖等成分的变化规律进行了研究。     

EFFECTS OF LINOLEIC ACID SUPPLEMENTS ON THE SYNTHESIS BY YEAST OF LIPIDS AND ACETATE ESTERS

The concentrations of acetate esters in beer were reduced by up to 85% by addition of linoleic acid to the fermentation or by pitching with yeast previously enriched with this unsaturated fatty acid. Linoleic acid was rapidly incorporated into yeast lipids and was effective in reducing the rate of ethyl acetate formation within 2 h. Addition of linoleic acid altered the pattern of synthesis of fatty acids by yeast, causing a shift from medium toward long chain acids. Secondly, the amount of squalene in yeast was reduced by up to 70% whereas that of lanosterol was increased threefold. Since total yeast lipid synthesis was reduced by up to 40%, we conclude that less acetylCoA is synthesized in the presence of linoleic acid. Further, high concentrations of linoleic acid decreased the proportion of acetylCoA consumed by the synthesis of acetate esters. Therefore linoleic acid may directly decrease acetate ester synthesis in addition to its effect via reduction of acetylCoA availability.     

DETERMINATION OF FATTY ACID HYDROPEROXIDES PRODUCED DURING THE PRODUCTION OF WORT

Linoleic and linolenic acid hydroperoxides in malt, mash, or wort were determined with high sensitivity and high selectivity by the chemiluminescence-high performance liquid chromatography (CL-HPLC) method using isoluminol-microperoxidase solution as a luminescing reagent. The determination limit of this method for both hydroperoxides was 0.1 μM in mash or wort. During the mashing in a laboratory mash bath, the hydroperoxides started to increase just after mashing-in, reached a maximum at 65°C, and then decreased. Though the hydroperoxides were detected in mash just before the lautering in a pilot scale brewing, they disappeared during the lautering and could not be detected during the subsequent stages of wort production. Therefore, it was thought that the mashing process is the most important of the lipid oxidation reactions during wort production. It is also expected that the CL-HPLC method can give useful information on lipid oxidation mechanisms during wort production.