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Ecology of lambic beers produced in traditional and industrial lambic beer breweries

The traditional lambic beer fermentation process

Acidic lambic beers, obtained by spontaneous fermentation, are probably the oldest known beers (De Keersmaecker, 1996). They are the products of a mixed fermentation that can last up to 3 years and are traditionally fermented in wooden casks (Ver- achtert and Iserentant, 1995). Notwithstanding the increasing popularity of lambic beers, the fermentation process was dealt with only in some detail in studies performed between 1976 and 1995 by the research group of Professor-Emeritus Hubert Ver- achtert (Martens et al., 1991, 1992; Shanta Kumara and Verachtert, 1991; Spaepen et al., 1978, 1979; Van Oevelen et al., 1976, 1977; Verachtert and Iserentant, 1995). These studies were performed using biochemical methods solely and were limited in the number of isolates examined and in the taxonomi- cal information obtained (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). Since the publication of these early studies, the taxonomy of bacteria and yeasts involved in the lambic beer fermentation process underwent several changes and biochemical identification methods were shown inadequate to reliably identify these microorganisms (Cleen- werck et al., 2008; De Bruyne et al., 2008; Kampfer and Glaeser, 2012; Kurtzman and Robnett, 1998; Nhung et al., 2007). More recently, the microbial composition of these beers was updated with the current taxonomic knowledge and by the use of more advanced identification techniques (Spitaels et al., 2014c, 2015b).

Lambic beer is traditionally brewed during the cold winter months only, i.e. from October until March, because the lambic wort has to be cooled to approximately 20°C within the timeframe of one night. Lambic beer is traditionally produced using about 66% malted barley and 33% unmalted wheat. The use of at least 30% unmalted wheat in the mash is regulated by Belgian law. Traditionally, the lambic wort production starts with a turbid mash method, which is a combination of the English infusion and German decoction processes (Fig. 7.1). Hot water is added during the English infusion process to increase the temperature of the mash. During decoction, the brewer boils a part of the mash separately to rupture the starch granules and subsequently reintroduces it into the mash tun to increase the total mash temperature, ensuring the rests at the enzymes' optimal temperatures (Briggs et al., 2004). During turbid mashing, the brewer does not reintroduce the separately boiled wort (called slime) into the mash tun, so that not all of the wort passes through all temperature rests (Fig. 7.1). The use of unmalted wheat and the turbid mashing step with separate slime cooking results in a wort that is rich in malto-oligosaccharides or dextrins. These dextrins are non-fermentable by conventional Saccharomyces brewing yeasts (Shanta Kumara and Verachtert, 1991), but they can be fermented by Brettanomyces (the sporulating form of this yeast is named Dekkera) yeasts that are also present during the maturation of red-brown acidic ales of south-west Flanders (Martens et al., 1997). The wort is boiled for 3 h, which is a long period compared with other beer types, and a large amount of aged hops is added to enhance the microbiological stability of the beer without resulting in a bitter hop flavour (Verachtert and Derdelinckx, 2005; Vriesekoop et al., 2012). After wort cooking, the wort is cooled in an open vessel, called the cooling tun or coolship, which is mostly located in the attic of the brewery (Fig. 7.1). After overnight cooling in the coolship, the wort becomes exposed to the environmental microbiota that initiate fermentation. As lambic beers were originally only produced in the Senne river valley (southwest of Brussels) and in the southeast of Brussels, it was believed that the responsible microbiota were present in the air of this region (Verachtert and Iserentant, 1995).

The first reports of the microbiota and their metabolites divided the lambic beer fermentation process into four phases: the Enterobacteriaceae phase, the main fermentation phase, the acidification phase, and the maturation phase; more recent studies considered the acidification phase and maturation stage as a single period (Spitaels et al., 2014c; Van Oevelen et al., 1976, 1977; Verachtert and Iserentant, 1995). Each phase was characterized by the presence of specific microorganisms and metabolites (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). The culture media used were selected based on previous studies and the observation of increased concentrations of acetic acid and lactic acid, indicating the presence of acetic acid bacteria (AAB) and LAB (Van Oevelen et al., 1976).

Example of a brewing scheme in a traditional lambic beer brewery, making use of turbid mashing and two boiling kettles

Figure 7.1 Example of a brewing scheme in a traditional lambic beer brewery, making use of turbid mashing and two boiling kettles. The typical lambic beer fermentation process characteristics, next to the unusual mashing scheme, are underlined.

Enterobacteriaceae were isolated from the cooled wort in the cooling tun and the cask (Martens et al., 1991; Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). The Enterobacteriaceae phase was reported to start after 3-7 days of fermentation, when bacterial counts reached up to 108 CFU/ml, to proceed for 30 to 40 days, and to be characterized by Enterobacter cloacae and Klebsiella pneumoniae (Klebsiella aerogenes) (Brisse et al., 2006) as the predominantly isolated Enterobacteriaceae species (Martens et al., 1991). Enterobacter aerogenes, Cit- robacter freundii, Escherichia coli and Hafnia alvei were additionally isolated (Martens et al., 1991). The number of Enterobacteriaceae cells present in brewery air is however low, and it has been hypothesized that wort inoculation during cooling is not homogeneous and bacteria are probably adsorbed to particles present in the air (Martens et al., 1991).

Oxidative yeasts, such as the cycloheximide- resistant Hanseniaspora uvarum [its asexual form is named Kloeckera apiculata (Meyer et al., 1978)] and Naumovia dairensis (previously known as Sac- charomyces dairensis) (Kurtzman, 2003) as well as Saccharomyces uvarum [previously known as S. globosus (Nguyen and Gaillardin, 2005)] are the main yeast species present during the enterobacterial phase of the lambic beer fermentation process (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). H. uvarum has a low fermentative capacity and is commonly found during the spontaneous fermentation of wines and ciders, where its contribution to flavour complexity is increasingly appreciated (Bezerra-Bussoli et al., 2013). Similar to B. bruxellensis, H. uvarum is capable of producing ethyl esters, and was long considered as a wine spoilage yeast (Moreira et al., 2011; Romano et al., 2003). During this phase, the pH dropped one value, and considerable levels of butanediol and dimethyl sulfide were formed, along with formic acid, acetic acid, lactic acid, and ethanol (Verachtert and Iserentant, 1995). The disappearance of the Enterobacteriaceae after about one month of fermentation is explained by the depletion of glucose, the increase in ethanol concentration and the decreased pH of the wort (Martens et al., 1991).

A similar start of the lambic beer fermentation was found during a more recent study of a traditional lambic beer brewing process (Spitaels et al., 2014c). A fast dereplication technique based on MALDI-TOF MS enabled the processing of more isolates from several phases during lambic beer production. Overnight cooled wort already contained high counts of Enterobacteriaceae in the cooling tun (106-107 CFU/ml). No yeasts were found in the lambic beer samples from the cooling tun (Spitaels et al., 2014c). The counts of the Enterobacteriaceae were the highest (108 CFU/ml) after 1-2 weeks. E. coli was again isolated, whereas Hafnia paralvei [an opportunistic human and animal pathogen (Huys et al., 2010)] was isolated instead of H. alvei. It is likely that isolates from previous studies were in fact also H. paralvei, as this species was separated from H. alvei only very recently (Huys et al., 2010). Other isolates in the Enterobacteriaceae phase were identified as Enterobacter hormaechei, Enterobacter kobei, Klebsiella oxytoca, Citrobacter gillenii and Raoultella terrigena (Spitaels et al., 2014c). All these species are coliform bacteria and thus indicator microorganisms for faecal contamination of surface waters and foods. Although these species are considered to be opportunistic pathogens, they are commonly found in various spontaneously fermented foods and beverages, and some were previously isolated from lambic beer as well (Chao et al., 2013; Martens et al., 1991). Remarkably, most of these microorganisms have also been reported as spoilage microorganisms in sweet unfermented wort and pitching yeast (Bokulich and Bamforth, 2013; Van Vuuren and Priest, 2003; Vriesekoop et al., 2012) (see Chapter 10).

Debaryomyces hansenii and S. cerevisiae were two yeast species isolated immediately after the transfer of the wort into the cask. S. pastorianus and Naumovia castelii were subsequently isolated from a 1-week-old wort sample. Debaryomyces hansenii is a known beer spoilage yeast (Bokulich and Bamforth, 2013), whereas N. castellii was previously known as Saccharomyces castellii and part of the Saccharo- myces sensu stricto group (Kurtzman, 2003). DNA originating from H. uvarum was detected in the community profiles of wort samples of traditionally produced lambic beer, but this species was not isolated (Spitaels et al., 2014c). Hanseniaspora uvarum originates from the fruit surfaces when thriving in wine and cider fermentation processes (Beltran et al., 2002), but it is known as an environmental contaminant in ales and lagers (Bokulich and Bamforth, 2013; Manzano et al., 2011).

The Enterobacteriaceae phase was followed by the main fermentation phase or alcoholic fermentation phase, which started after 3 to 4 weeks. Saccharomyces spp. dominated the fermentation process from month 1 until month 4 (Spitaels et al., 2014c; Van Oevelen et al., 1977; Verachtert and Iserentant,

1995). S. cerevisiae, S. bayanus/pastorianus and S. uvarum were identified as the main actors during this stage (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). High counts of S. cerevisiae and S. pastorianus were present at the start of the main fermentation phase, but after 3 months of fermentation most isolates were identified as S. pastorianus (Spitaels et al., 2014c). Previous studies only reported the presence of S. cerevisiae, S. bayanus/S. pastori- anus and S. uvarum in the main fermentation phase and did not provide detailed information for different sampling moments. It is however not clear why S. pastorianus can outlive S. cerevisiae in the lambic beer fermentation process of a traditional brewery. The genomic background of the hybrid species S. pastorianus was recently elucidated (Libkind et al., 2011). In Saccharomyces, hybridization events between cryotolerant and non-cryotolerant Sac- charomyces species offered a benefit for the resulting hybrids, because of the capacity of these hybrids to ferment at lower temperature (Peris et al., 2012). Consequently, all commonly used lager-type yeasts are domesticated strains of the initial pastorianus and bayanus hybrids (Libkind et al., 2011). The ambient temperature of the rooms where lambic beers are fermenting is rarely 20°C or higher during the first fermentation months, which may explain the predominance of S. pastorianus in a traditional lambic beer brewery process. Vidgren et al. (2010) reported that ale (generally S. cerevisiae) and lager (generally S. pastorianus or S. bayanus) strains exhibit a similar maltose transport activity at 20°C, but at 0°C the activity of lager strains is higher by the expression of cryotolerant maltose and maltotriose transporters. The different temperature sensitivity of the maltose and maltotriose transporters could have an influence on the survival of different Saccharomyces hybrids, since the transport of these molecules is assumed to be the rate-limiting step in the utilization of these saccharides (Cousseau et al., 2013).

After the main fermentation phase, oxidative yeasts, i.e. Cryptococcus spp., Candida spp., Pichia spp. and Torulopsis spp. form a pellicle at the top of the liquid and serve as an oxygen barrier (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995).

Pediococcus damnosus (Pediococcus cerevisiae) was commonly isolated during and after this main fermentation phase, in addition to low counts of AAB [Acetobacter spp. and Gluconobacter (Acetomonas) spp.], which were isolated irregularly (Spitaels et al., 2014c; Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). Acetobacter lambici (Spitaels et al., 2014a) and Gluconobacter cerevisiae (Spitaels et al., 2014b), two newly described AAB species, were occasionally isolated during the lambic beer fermentation process. From 2 months onwards, P. damnosus was consistently present. During this phase, the majority of the ethanol present in the lambic beers was produced, and the level of dimethyl sulfide (produced during the Entero- bacteriaceae phase) decreased, driven off by CO2 bubbles produced by the yeasts still present from the fermenting lambic beer (Verachtert and Iserentant, 1995). Simultaneously, higher alcohols, fatty acids and esters, including hexanoate, octanoate, decanoate and their ethyl esters, respectively, were formed as well (Spaepen et al., 1978; Van Oevelen et al., 1976; Verachtert and Iserentant, 1995).

After 2-3 months of main fermentation and the depletion of the carbon sources that can be fermented by Saccharomyces spp. (simple saccharides up to maltotriose), an acidification phase has been reported that was characterized by the increasing isolation of Pediococcus and occasionally Lactobacillus strains (only in breweries with large casks), while Brettanomyces strains became prevalent after 4 to 8 months of fermentation. Simultaneously, the number of Saccharomyces yeasts decreased (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). The acidification was characterized by a strong increase in lactic acid and ethyl lactate concentrations, which are typical metabolites of lambic beers (Van Oevelen et al., 1976; Verachtert and Iserentant, 1995). During the warm summer months, LAB can also cause slime in the fermenting lambic beer, which is undesirable (Van Oevelen et al., 1977; Van Oevelen and Verachtert, 1979).

Brettanomyces and LAB species have a synergistic effect on beer attenuation (Andrews and Gilliland, 1952; Shanta Kumara and Verachtert, 1991). Brettanomyces spp. in combination with LAB degrade the residual dextrins that are not fermented by Saccharomyces spp. (Shanta Kumara and Verachtert, 1991). Lambic beers reach a high attenuation during the maturation phase, resulting in a residual gravity that may be below 1°P (Shanta Kumara and Verachtert, 1991; Verachtert and Iserentant, 1995). Super-attenuation or over-attenuation was already described by Andrews and Gilliland (1952), who demonstrated that a primary attenuation limit, typical for an axenic S. cerevisiae culture, and a secondary attenuation limit, typical for an axenic B. bruxellensis culture, can still be overcome by the use of a mixed culture of yeasts and bacteria (Andrews and Gilliland, 1952). Hence, there is probably a synergistic effect of the yeast and bacterial cultures during the degradation of dextrins and starch (Andrews and Gilliland, 1952). A similar finding was reported by Shanta Kumara and Verachtert (1991), who demonstrated that Brettanomyces is the main contributor to the super-attenuation of lambic beers, but its effect is more pronounced in a mixed culture with Pediococcus (Shanta Kumara and Verachtert, 1991). Brettanomyces produces an a-glucosidase, an enzyme capable of dextrin degradation (Shanta Kumara and Verachtert, 1991). This a-glucosidase shows intracellular as well as extracellular activities and acts by removing a single glucose molecule from the dextrin polymer (De Cort et al., 1994; Shanta Kumara et al., 1993). The enzyme is fast-acting, as under optimal conditions malto- oligosaccharides shorter than maltotetraose are not found in the presence of the enzyme (Shanta Kumara et al., 1993). The low pH of lambic beers, however, may explain the slow process of overattenuation in situ in lambic beers (Shanta Kumara et al., 1993).

After 10 months, the bacterial counts decrease and a new phase in the lambic beer fermentation process is initiated by the increase of Brettanomyces spp. During this phase, cell-bound esterases of Brettanomyces yeasts can form and degrade several esters in the fermenting lambic beer (Spaepen and Verachtert, 1982) and several metabolites and flavour compounds are produced by the synergistic action of LAB and Brettanomyces yeasts (Shanta Kumara and Verachtert, 1991; Van Oevelen et al., 1976, 1977; Verachtert and Iserentant, 1995). These include the esters ethyl acetate and ethyl lactate as well as the long-chain fatty acids and their esters such as ethyl caprylate and ethyl caprate (Spaepen et al., 1978). Only minimal concentrations of ethyl caprate are present in most other beers and this can thus be considered as a typical aroma compound of lambic beers (Spaepen et al., 1978). However, a beer produced by the mixed fermentation of a LAB-harbouring pitching yeast with a secondary cask fermentation contains comparable concentrations of these long-chain fatty acids and their esters (Spaepen et al., 1979). At the end ofthis phase, after about 2 years, the number of LAB and Brettanomyces yeasts was reported to decrease (Van Oevelen et al., 1977; Verachtert and Iserentant, 1995). AAB were also isolated during these phases.

However, as mentioned above, a detailed analysis of the microbiota at 3 and 6 months of fermentation in a more recent study could not discriminate between the acidification and maturation phases (Spitaels et al., 2014c). A gradual decrease of Saccharomyces yeasts and a consecutive increase of Brettanomyces yeasts was not found, which is characteristic for the acidification phase. The number of LAB was elevated in the 6-month-old sample compared to the 3-month old sample, and reached counts that were comparable to those of Brettanomyces yeasts. Consequently, the acidification probably occurred very rapidly between the sampling at 3 and 6 months and it was therefore considered as a part of the long maturation phase. Indeed, yeast isolates from the 3-month-old sample were identified as Saccharomyces spp., while those of the 6-month-old sample were identified as Bret- tanomyces spp.

The ambient temperature of the cask storage room can influence the pace of fermentation and the ability of B. bruxellensis to dominate the fermentation, as shown by a batch-dependent species distribution during the fermentation phase (Spi- taels et al., 2014c). This phase can be dominated by B. bruxellensis or can have a more complex microbiota and include several other yeast species, such as Candida patagonica, Brettanomyces anomalus, Pichia membranifaciens, Priceomyces carsonii, and Wickerhamomyces anomalus (Spitaels et al., 2014c).

LAB were isolated from all samples after the Enterobacteriaceae phase. In contrast to previous studies, P. damnosus was the only LAB species found (Spitaels et al., 2014c). It is unclear why P damnosus was the only LAB species isolated, while other studies reported the general presence of Lactobacillus spp., including Lactobacillus brevis, a well-known beer spoilage bacterium (De Cort et al., 1994; Shanta Kumara and Verachtert, 1991; Vriesekoop et al., 2012).

The microbial communities present in 3-year-old lambic beer were highly similar to those present in the 2-year-old lambic beer and consisted of P dam- nosus, A. lambici, P. membranifaciens, B. bruxellensis, B. anomalus, C. patagonica and W anomalus (Spitaels et al., 2015a). This contrasted with the results of Verachtert and Iserentant (1995), who reported a decrease in the counts of LAB and yeasts towards the end of the fermentation process and suggested that this microbiota is highly adapted to growth and survival in lambic beer.

Besides lambic beers, lambic beer brewers produce gueuze and fruit lambic beers, while gueuze blenders buy lambic beers from lambic beer brewers to produce their own beers. Gueuze beers are produced by the re-fermentation of a mixture of young lambic beer that contains a lot ofdextrins and old lambic beer that contains dextrin-hydrolysing microorganisms (Verachtert and Iserentant, 1995). The pellicle yeasts survive in the initial stages of the re-fermentation process, although they do not multiply (Verachtert and Iserentant, 1995). Their presence can be explained by breaking of the pellicle during emptying of the casks.

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