IDENTIFICATION OF LAMBIC SUPERATTENUATING MICRO-ORGANISMS BY THE USE OF SELECTIVE ANTIBIOTICS

The microbial population present in Iambic beer after one year of spontaneous fermentation consists mainly of Brettanomyces yeasts, lactic acid bacteria and acetic acid bacteria. The density of the wort by that time has decreased to around 3.5° Plato. At that time a period of superattentuation is initiated, resulting in a Iambic with sometimes less than 1° Plato. Such old Iambics are used in the production of gueuze. In order to find out which organisms are really necessary for this process. Iambic attenuated to around 3.5° Plato was pasteurized and re-inoculated with a mixed microbial population obtained from fermenting Iambic. By the addition of the antibiotics actidione, pimaricin, gentamycin, oxytetracycline and nisin it was found that Brettanomyces was the main organism responsible for superattenuation, although this was less pronounced when Pediococcus was absent. Acetic acid bacteria were not involved. Bacteria alone were not really superattenuating. The process with Iambic wort which had reached the 3.5° Plato value by a natural spontaneous fermentation was slower than with a Iambic wort pre-fermented to 3.5° Plato with S. cerevisiae. It was found that Brettanomyces but not Saccharomyces survives well under the conditions normally found for a 1 year old Iambic.     

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.     

ESTERASE ACTIVITY IN THE GENUS BRETTANOMYCES

During the spontaneous fermentation of Iambic and the production of the typical Belgian beer Gueuze, yeasts of the genus Brettanomyces are always found. They are especially active in the 6 to 12 months fermentation period and during refermentation in the bottle. It was found that Brettanomyces yeasts show esterase activity towards a large number of esters but especially towards esters normally found in Gueuze: ethyl acetate, ethyl lactate, iso-amyl acetate and phenethyl acetate. The esterase activity is expressed by whole cells of all Brettanomyces strains in our collection, but not by Saccharomyces or Kloeckera. The enzyme is partially purified by ammonium sulphate precipitation. It shows optimal activity at pH 7·6 for the hydrolysis of ethyl acetete and relative good stability at around 2°C. Levels of whole cell esterase activity are dependent on growth medium, aeration rate and temperature. Most interesting is the ester-synthesising activity of this esterase. Its role and influence on the ester fraction of Iambic and Gueuze seem very likely, which may then be considered as the first confirmed function of Brettanomyces during this special fermentation process.     

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.     

PRODUCTION OF ESTERS BY DIFFERENT YEAST STRAINS IN SUGAR FERMENTATIONS

The production of the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₀ fatty acids was investigated in semiaerobic sugar fermentations by 56 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum. The S. cerevisiae yeasts generally produced more esters than the S. uvarum yeasts. Isoamyl acetate was the main component in the ester fractions examined and others in decreasing order, were ethyl caprylate, ethyl caproate, ethyl caprate and phenethyl acetate.     

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.     

Evaluation of the potential of commercial non-Saccharomyces yeast strains of Torulaspora delbrueckii and Lachancea thermotolerans in beer fermentation

This work evaluated the potential of three commercial non-Saccharomyces yeast strains Torulaspora delbrueckii (Biodiva and Prelude) and Lachancea thermotolerans (Concerto) for beer fermentation. The fermentation performance, volatile and non-volatile profiles were compared. Growth behaviours of all three yeast strains exhibited similar trends during the initial fermentation phase although a marked population decline was detected in strains Prelude and Concerto, which also showed a rapid utilisation of maltose, while strain Biodiva was unable to consume maltose and consumed lesser amounts of amino acids. Additionally, terpenoids inherently absent in the wort such as β-caryophyllene and geranyl acetone were produced in all beers, significantly higher in beers fermented with strain Prelude. For volatile profiles, Prelude and Concerto produced more ethanol and significantly higher amounts of acetate esters and long-chained ethyl esters. Strain Biodiva, on the other hand, produced higher amounts of isoamyl alcohol and ethyl butanoate.     

Evaluation of different yeast strains on the quality of beer produced from malted proso millet (Panicum miliaceum L.)

In order to investigate the influence of temperature on pH, alcohol production, carbohydrate profile and aroma compounds, a variety of top-fermenting yeast strains were used to ferment wort obtained from proso millet malt. Champagne (Saccharomyces bayanus), altbier, kölsch and ale (Saccharomyces cerevisiae), as well as Brettanomyces bruxellensis were used in two isothermal (12 and 18 °C) fermentation trials. During the fermentation, changes in pH, extract conversion and the monosaccharide profile were monitored. After 2 weeks of storage, the levels of higher alcohols and esters were also measured in the finished beers. In many cases, a strong resemblance in the behaviour of the selected strains to Saccharomyces was observed during the fermentation. All other influencing fermenting parameters like pitching rate, yeast generation, fermenting vessels geometry, amount of assimilable nitrogen, yeast nutrients, oxygen and CO₂ content and the sugar content were the same for all trials regardless of the yeast applied. However, at higher temperatures Brettanomyces showed more rapid rates of sugar conversion. Concerning the development of aroma components, no uniform picture was evident in these trials, neither with respect to temperature dependence nor to what extent the incurred values are comparable to those already known for beers brewed using barley malt wort. The values for aliphatic alcohols obtained during experimentation, ranged from 23 to 267 μg/L, and the sum of aromatic alcohols, from 2,437 to 11,844 μg/L. Values for ester content were recorded between 188 and 2,543 μg/L. Overall, wort produced using proso millet malt was found to be a substrate that lends itself to alcoholic fermentation, exhibiting a sufficient decrease in pH over the course of fermentation, as well as reasonable alcohol yield and a broad range of aroma components.     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

CONTROL OF FERULIC ACID AND 4-VINYL GUAIACOL IN BREWING

Phenolic acids in beer are important because they can be decarboxylated to phenols, which usually impart off-flavours. An improved high performance liquid chromatographic system was used to monitor phenolic acids and phenols during the brewing process. Ferulic acid was the most significant phenolic acid found in beers prepared from malted barley. Extraction of ferulic acid from malt involved an enzymatic release mechanism with an optimum temperature about 45°C. Mashing-in at 65°C significantly decreased the release of free ferulic acid into the wort. Wort boiling produced 4-vinyl guaiacol by thermal decarboxylation, in amounts (0.3 mg/L) close to its taste threshold, from worts that contained high contents of free ferulic acid (> 6 mg/L). The capacity of yeasts to decarboxylate phenolic acids (Pof⁺ phenotype) was strong in wild strains of Saccharomyces and absent in all lager brewing yeast and most ale brewing yeasts. Some top-fermenting strains, especially those used in wheat beer production, possessed a weak decarboxylating activity (i.e. Pof^δ). During storage of beers there were appreciable temperature-dependent losses of 4-vinyl guaiacol. These results indicated that the production of 4-vinyl guaiacol is amenable to close technological control.     

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.     

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.     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

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.     

CAPRYLIC FLAVOUR AS A FEATURE OF BEER FLAVOUR

Caprylic flavour is part of the distinctive overall flavour of a large proportion of beers in trade. The flavour occurs in the majority of lager beers and in about 20% of ales. It is correlated with the levels of octanoic acid and decanoic acid present in beer. Lager yeasts (S. uvarum) tend to liberate larger amounts of these two fatty acids during fermentation than do ale yeasts (S. cerevisiae). The flavour significance of these acids has been determined by correlating the results of sensory and instrumental analyses and also by adding the purified acids to beer. Implications of these two methods for assessing the flavour significance of chemical constituents of a complex product are discussed.     

Molecular and biochemical aspects of Brettanomyces in brewing

Brettanomyces is a semi‐domesticated yeast that is a crucial component of lambic beers and is increasingly attracting the attention of the brewing industry. Brettanomyces display Saccharomyces‐like features, such as a positive Crabtree effect, ethanol synthesis and tolerance to harsh environments. Additionally, Brettanomyces exhibit β‐glucosidase and esterase activities, the production of phenolic compounds and tetrahydropyridines, together with the ability to ferment dextrins and breakdown cellobiose from wooden casks. Although the importance of Brettanomyces species is documented in the production of different beer styles, the molecular and biochemical features of these species required for brewing are poorly understood. Therefore, this work reviews the current knowledge of the molecular biology and biochemistry underlying the performance of Brettanomyces in the brewing industry. © 2019 The Institute of Brewing & Distilling     

Degradation of 5-hydroxymethylfurfural during yeast fermentation

5-Hydroxymethyl furfural (HMF) may occur in malt in high quantities depending on roasting conditions. However, the HMF content of different types of beers is relatively low, indicating its potential for degradation during fermentation. This study investigates the degradation kinetics of HMF in wort during fermentation by Saccharomyces cerevisiae. The results indicated that HMF decreased exponentially as fermentation progressed. The first-order degradation rate of HMF was 0.693?×?10^?2 and 1.397?×?10^–2?min^?1 for wort and sweet wort, respectively, indicating that sugar enhances the activity of yeasts. In wort, HMF was converted into hydroxymethyl furfuryl alcohol by yeasts with a high yield (79–84% conversion). Glucose and fructose were utilised more rapidly by the yeasts in dark roasted malt than in pale malt (p?<?0.05). The conversion of HMF into hydroxymethyl furfuryl alcohol seems to be a primary activity of yeast cells, and presence of sugars in the fermentation medium increases this activity.     

Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking

Since the large occurrence of Brettanomyces yeasts in strict anaerobiosis environments (sparkling wines) has been found without an increase in acetic acid content, we evaluated the influence of the oxygen concentration on acetic acid production. Results showed that the oxygen concentration exerted a strong influence on both growth and acetic acid production by Brettanomyces yeasts in winemaking. Full aerobiosis lead to a large production of acetic acid causing a block of metabolic activity. Semi-aerobiosis resulted in the best condition for alcoholic fermentation (Custers effect) combined with acetic acid production. In anaerobic condition Brettanomyces yeasts did not result in high acetic acid production and a pure, even if slow, alcoholic fermentation occurred. The absence of an increase in acetic acid in wines, does not exclude the active presence of Brettanomyces yeast since the characteristic ‘high acetic producer’ in Brettanomyces yeast is linked to the presence of oxygen. ©1997 SCI