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 .     

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

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 (C₈) 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 lambic and gueuze.     

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 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.     

THE CONTROL OF VOLATILE ESTER SYNTHESIS DURING THE FERMENTATION OF WORT OF HIGH SPECIFIC GRAVITY

Some effects have been studied of fermenting wort of unusually high specific gravity, followed by dilution with water to give beer of normal original gravity. This procedure permits increased overall rates of beer production, but matching of flavours requires control of the level of flavour determinants. If not controlled, the concentration of volatile esters may be disproportionately increased so that, after dilution, beer flavour is markedly different. Higher alcohol production is not affected in this way. The concentration of esters can be adjusted to appropriate levels by increasing the production of yeast mass during fermentation. Unsaturated fatty acids, which increase yeast dry matter production without altering the rate of fermentation, are particularly effective in reducing the extent of ester synthesis.     

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.     

MICROBIOLOGICAL ASPECTS OF A MIXED YEAST—BACTERIAL FERMENTATION IN THE PRODUCTION OF A SPECIAL BELGIAN ACIDIC ALE

The evolution of the microbial population during the industrial fermentation of a Belgian acidic ale was followed, and the most important micro-organisms were identified. In essence, the process can be considered to be a tandem fermentation, as a period of ethanolic fermentation is followed by a period of lactic acid fermentation. The process consists of three stages. The first stage is a seven day fermentation mainly by Saccharomyces; the second is a four to five week fermentation in which Lactobacilli become important; in the third stage a twenty to twenty-four month fermentation in cask, Brettanomyces species, Lactobacilli, Pediococcus parvulus and Acetic Acid bacteria are all present. The evolution of the Brettanomyces-Pediococcus coculture during the tertiary fermentation shows a remarkable similarity with the evolution during a gueuze fermentation.     

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.     

EFFECT OF YEAST ADAPTATION TO MALTOSE UTILIZATION ON SUGAR UPTAKE DURING THE FERMENTATION OF BREWER'S WORT

Yeast adaptation to maltose utilization diminishes the repressing effect of glucose on maltose uptake. Furthermore, it affects both the rates and profiles of maltose, glucose and maltotriose uptake during high cell density brewer's wort fermentation. Yeast cells pre-grown in maltose, as sole carbon source, and harvested while the sugar is still present in the growth medium, are better adapted to utilize maltose. The adapted cells are less sensitive to glucose inhibition, and the uptake of glucose is inhibited in the early stages of fermentation. Cells grown for longer periods and harvested following maltose depletion, lose their ability to preferentially utilize maltose. In addition, they become more sensitive to glucose repression, and are able to utilize glucose faster than the cells harvested when maltose is still present in the medium. During the process of adaptation, maltose controls the induction of its own transport systems and appears to affect the biosynthesis of the glucose transport systems.     

THE EFFECT OF OSMOTIC PRESSURE ON THE INTRACELLULAR ACCUMULATION OF ETHANOL IN SACCHAROMYCES CEREVISIAE DURING FERMENTATION IN WORT

An intracellular accumulation of ethanol was observed in Saccharomyces cerevisiae during the early stages of fermentation (3 h) in wort. However, after 12 h fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure in wort by adjusting the carbohydrate concentration, from 10° to 20° plato, was observed to cause an increase in intracellular ethanol concentration and glycerol production. Although intracellular ethanol concentration increased, no adverse effect was observed on cell growth and fermentation rates. Thus, increasing the carbohydrate concentration in wort to at least 20° plato did not affect cell growth and fermentation rates, but did result in the production of more ethanol.     

THE USE OF RADIOACTIVE LABELLING TO DEMONSTRATE THE PRODUCTION OF DIMETHYL SULPHIDE FROM DIMETHYL SULPHOXIDE DURING FERMENTATION OF WORT

During fermentation, yeast reduced [¹⁴C] dimethyl sulphoxide (DMSO) to yield radioactive dimethyl sulphide (DMS) even under conditions in which no net formation of DMS could be demonstrated by GLC analysis. It was confirmed that most DMS is produced during fermentation of lager worts at 8°C.     

WORT COMPOSITION AND BEER FLAVOUR. II. THE INFLUENCE OF DIFFERENT CARBOHYDRATES ON THE FORMATION OF SOME FLAVOUR COMPONENTS DURING FERMENTATION

Wort, to which was added various amounts of solutions of glucose, fructose, sucrose or maltose, was fermented, and in the resulting beers the concentrations of the following flavour components were determined by gas chromatography: ethyl acetate, isobutyl acetate, iso-amyl acetate, 2-phenyl ethyl acetate, ethyl caproate, ethyl caprylate, n-propanol, isobutanol, amyl alcohols, 2-phenyl ethanol, caprylic acid and capric acid. The concentrations of these compounds were affected in different ways by the various amounts of sugar added, and some differences were observed etween the different carbohydrates.     

THE STRUCTURAL AND STORAGE CARBOHYDRATES OF SACCHAROMYCES CEREVISIAE: CHANGES DURING FERMENTATION OF WORT AND A ROLE FOR GLYCOGEN CATABOLISM IN LIPID BIOSYNTHESIS

Detailed analysis of the structural and storage carbohydrates of Saccharomyces cerevisiae (NCYC 240) during wort fermentation showed that there were no significant changes in the amounts of trehalose or alkali-soluble glycogen. However, glucan and mannan individually increased from ca4% of the yeast dry weight at pitching to ca6% during the first 3–17 h of fermentation, butthen declined to the former level. In the first 2 h of fermentation, prior to yeast multiplication, acid-soluble glycogen was rapidly dissimilated from ca 40% to ca 6% of the yeast dry weight. During this period of oxygen uptake, wort sugars were not removed by the yeast. Glycogen, therefore, was the sole source of metabolic energy for lipid resynthesis and hexose transport appeared to require the formation of a component membrane. During the latter phase of fermentation when the yeast was not growing but expending energy for maintenance of cellular functions, glycogen reserves were slowly depleted; after a period of prolonged anaerobic storage, the content of glycogen fell well below that which was initially present in the pitching yeast.     

果蔬奶混合汁乳酸发酵过程中营养成分变化规律的探讨

为探讨果蔬奶混合汁乳酸发酵主要营养成分的变化规律，对其主要营养成了进行了测试。结果表明：ｐＨ值明显下降，含酸量增加；还原糖明显减少；Ｖｃ和β－胡罗卜素含量略有减少，总糖含量明显增加，淀粉含量明显下降。     

面团发酵过程中蛋白质和VB6变化

蛋白质和B族维生素是面粉及其制品中的重要营养成分。通过对蛋白质含量和VB6含量的测定，发现4种分离蛋白在发酵过程中均成波动变化，而不同酵母添加量对VB6含量影响不大，但在整个发酵过程中的变化趋势受微生物生长和繁殖速度的影响上下波动。     

EFFECT OF LIPIDS AND OXYGEN ON YEAST GROWTH AND THE BIOSYNTHESIS OF ACETOIN DURING FERMENTATION

Brewery yeast needs traces of oxygen for the biosynthesis of unsaturated fatty acids and ergosterol. Owing to the increase in cell mass during primary fermentation the concentrations of these essential lipids decrease and thereby affect the physiological condition of the yeast. When the sterol concentration of whole cells has decreased to 0.2 to 0.3 mg per 100 mg dry yeast, the yeast changes its metabolism. This metabolic change is revealed by a decrease in acetoin concentration. The absorption of wort nutrients and consequently the efficiency of growth is at this point also greatly reduced. The ratio between yeast growth and mole ethanol formed (i.e. the molar growth yield) decreases greatly during wort fermentation. A close correlation between molar growth yield and the change in acetoin metabolism can be observed. This metabolic change occurs when the ratio between yeast growth and ethanol formed is in the range of 8.3 to 9.1, averaging 8.7 g/mole.     

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.     

UPTAKE AND TURNOVER OF 14C GLUCOSE BY YEAST DURING WORT FERMENTATIONS

The incorporation of randomly labelled ¹⁴C glucose, or substances derived from it, into various cell components was followed during a brewery type fermentation, using a yeast polysaccharide fractionation technique in conjunction with liquid scintillation. The labelled glucose, when added to wort, was assimilated by brewer's yeast, most of it being fermented to ¹⁴C ethanol. A small but significant amount of the ¹⁴C glucose, or its metabolites, was incorporated into the polysaccharide fractions of the yeast. Employment of a simple “swop-over” technique has shown that in two of these fractions, the low molecular weight acid-soluble material and the mannan, there was a substantial turnover, reflecting respectively the transient metabolic status of the former and the special role of mannan in the structure of the outer layer of the cell wall. Glucan, as would be expected from its function as the principal structural component of the cell wall complex, was completely stable. Glycogen was also stable, and showed no evidence of turnover, even during the later stages of the fermentation. Hence it is unlikely that it could function as an energy reserve in this strain (N.C.Y.C. 240) during primary fermentation of brewer's wort.