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A Scottish Brewing Revolution
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Good condition, outside of spine shows some minor wear, but inside of book looks like it hasn't even been read. Several DNA bands of the bacterial community fingerprints were assigned to yeast taxa Table S1A and S1B , confirming that the V3 primers were not specific for bacteria  , . The yeast band classes 5 and 6 were assigned to the genus Saccharomyces Table S1C and S1D and were present throughout the fermentation. A total of bacterial and yeast isolates were obtained from the 2 batches. The freshly boiled wort did not allow microbial growth. All cooling tun isolates 48 from batch 1 [ Fig.
The identification of isolates belonging to the Enterobacteriaceae are reported to the species level, when reliable identification by housekeeping gene sequences could be obtained. The number of isolates is given between brackets. A total of isolates from batch 1 samples taken during the first month were identified. In batch 2, from which a total of isolates were identified, a similar evolution was found: the major occurrence of H.
The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer
However, batch 2 Enterobacteriaceae were more diverse and included also Citrobacter gillenii and Raoultella terrigena data not shown. From months 2 until 24, Pediococcus damnosus was consistently the only micro-organism isolated from MRS agar batch 1 [ Fig. AAM counts of the samples up to 3 months of fermentation were influenced by the presence of yeasts, which was due to the apparent loss of activity of amphotericin B under acidic conditions .
Amphotericin B was also reported to be unstable in other media with a composition similar to AAM . A combination of amphotericin B and cycloheximide was subsequently found to be more effective in inhibiting yeast growth under all isolation conditions used. All but one of the isolates were identified as a novel Acetobacter species, for which the name Acetobacter lambici has been proposed .
One batch 2 isolate represented a novel Gluconobacter species, for which the name Gluconobacter cerevisiae has been proposed . An overview of the identified yeast species of batch 1 is graphically represented in Fig. Isolation and accurate enumeration of yeasts during the first two weeks of fermentation of batch 1 was not possible, due to an insufficient suppression of bacterial growth. In batch 2 samples data not shown yeasts could not be detected in the wort after one night in the cooling tun, but increased in numbers directly after the wort was transferred into the casks not more than 15 min after the cooling tun was sampled.
The relative number of S. After 3 months, S. The same trend occurred during the first three months of fermentation of batch 1 Fig. The few DYPAIX isolates that were obtained from samples after 2 months batch 2 or 3 months batches 1 and 2 failed to grow on the same growth agar medium upon subculture, indicating that there were no cycloheximide-resistant yeast species present in these samples Fig.
Saccharomyces spp. The cultivated yeast diversity in batch 2 was low compared to batch 1 see below and the three yeast media yielded the same species diversity from 6 months onwards. The yeast species distribution in batch 1 samples after 6 months of fermentation Fig. The most frequently cultivated species were D. In contrast to batch 2 where the three yeast agar isolation media yielded the same species diversity from 6 months onwards, the species diversity recovered from different yeast agar isolation media in batch 1 was not comparable.
For example, D.
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S3 , of which the latter has never been associated with a beer fermentation process. None of the directly plated samples yielded growth. A total of isolates from the brewery environment were picked up from the bacterial and yeast agar isolation media after enrichment and were identified through MALDI-TOF MS and sequence analysis of 16S rRNA genes or other molecular markers as described above Table 2. Several species or taxa that were previously isolated during the fermentation process as described above were also found in environmental samples. Raoultella terrigena , Pichia membranifaciens , Debaryomyces marama and Db.
The latter species was also isolated from the ceiling, the attic and cellar air, along with S. The latter species was also found on the outside of a cask. A considerable number of additional micro-organisms that were not detected during the fermentation process were also isolated from environmental samples. These included species previously related to beverage fermentation or spoilage, such as Brettanomyces custersianus  , Pediococcus pentosaceus  , Lactobacillus malefermentans  and Acetobacter cerevisiae .
25 Essential Belgian Beers
Serious limitations of the few available microbiological studies of the lambic beer fermentation process are the rather low numbers of isolates identified using biochemical methods only  , . Recent polyphasic taxonomic studies revealed that phenotypic identification approaches alone have an inadequate taxonomical resolution for the accurate species level identification of these micro-organisms  ,  —  , .
Therefore, the present study revisited the microbiology of the lambic beer fermentation process of the most traditional lambic brewery Cantillon in Belgium and identified and monitored the microbiota using MALDI-TOF MS as a high-throughput dereplication technique. This allowed to compare numerous fingerprints and to reduce these isolates to a non-redundant set of different species that were further identified using an array of DNA sequence-based methods  , .
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This approach allowed a more in depth analysis of the culturable microbiota of this ecosystem and resulted in the isolation and description of two novel AAB species, i. The former species was even the most frequently isolated AAB species during the lambic fermentation process of Cantillon. The present study also used DGGE profiles of variable prokaryotic and eukaryotic rRNA gene regions to identify and monitor the microbial communities in two batches of lambic beer during a two-year fermentation period at Cantillon.
In both lambic batches, members of the Enterobacteriaceae were isolated during the first month, which corresponded to previous studies on Belgian lambic and American coolship ales  ,  , . The bacteria identified included E.