Increased transcriptomic studies, in conjunction

with comparative genomics, will most accurately and fully reveal the importance of plasmid-mediated functions for BSR LAB. Once more it is emphasized, that for these data to be ofutility to the brewing industry, this analysis must be performed with more frequency on BSR LAB of both same and different species. As the cost of this analysis decreases and bioinformatics tools become more sensitive (Thayer, 2014; Mardis, 2011), it will be possible to investigate the broad importance of widely conserved plasmid sequences in BSR LAB, as has been done for other niche-adapted organisms (Dziewit and Bartosik, 2014; Papadimitriou et al., 2015). Such analysis is reasonably expected to increase the number of species-independent, but beer spoilage-specific genes (and/or their transcripts) that can be screened for during quality control routines in the brewery.

Origin of BSR LAB

Phylogenetics and comparative genomics can help answer questions on the evolutionary development of BSR LAB; however, the answer to how and when these isolates emerged likely lies within the brewery itself. BSR LAB likely emerged with inclusion of hops in beer between the fifth and ninth centuries (Suzuki, 2011; Tonsmeire, 2014). Following genetic adaptation to this specific stress, BSR LAB then adapted further and have since remained tightly linked with the brewing environment (Suzuki et al., 2008a; Suzuki, 2011). Indeed, BSR LAB isolates are rarely isolated elsewhere than breweries or beer, though non-BSR LAB isolates of the same species are (Suzuki et al., 2008a; Suzuki, 2011). Breweries thus are both the selective environment and the reservoir for their own contaminants.

A recent study by Bokulich et al. (2015) investigated the distribution pattern of LAB species and putative hop tolerance genes in a brewery producing several different kinds of beer, using LAB-specific TRFLP (LAB-TRFLP) and ddPCR, respectively. The brewery involved produces ‘conventional' beer (potential BSR LAB contaminants), sour beer (potentially helpful LAB fermenters and/or BSR LAB) and coolship beer (BSR LAB and environmental microflora). The LAB-TRFLP applied in this study was found to more sensitively discriminate between species of the Lactobacillales order and most genera of the Bacillales order present in mixed culture (Bokulich and Mills, 2012a).

The LAB-TRFLP also identified organisms from other phyla not previously reported as recovered from beer, likely as a result of the fact the organisms in question are present at low abundance and are never actively selected for during detection (Bokulich et al., 2015). By applying this technique to analyse the LAB community profile throughout a brewery, Bokulich et al. (2015) were able to conclude that the brewery microbiota is probably driven by contact with raw substrates (grains, hops, yeast and beer), with this contact resulting in the profile of LAB present within a given brewery. For example, they found that wort samples contained a mixture of L. delbrueckii, L. hilgardii, L. sakei, Lacto- coccus lactis, Leuconostoc mesenteroides, Streptococcus spp., as well as a Bacillus spp., most of which were only rarely detected in other fermenting and bottled beer samples (Bokulich et al., 2015). Many of these species, while not necessarily found in finished beer, are apparently associated with grain and therefore their detection in wort is unsurprising (Bokulich and Bamforth, 2013).

Interestingly, distinct LAB profiles from specific brewery samples were detected at different sites, presumably as result of potential contact with the beer sample. For example, sour and coolship beers were dominated by Pediococcus spp. and L. lindneri, though fermenters and barrel surfaces that contacted these sour fermentations around the time of sampling exhibited similar community composition; however, L. brevis and Lactobacillus sp. were found to be more common on these surfaces then on other surfaces or in the beer. Floor and packaging area surfaces contained a more diverse composition of LAB, with the predominant organisms being L. brevis, L. delbrueckii, and L. lindneri, which were also detected in the sour wort and beer. Perhaps most interesting was the finding that only Pediococcus spp. were detected on grain samples, while L. brevis, L. lindneri and Pediococcus spp. were recovered from hop pellets. This is noted as to be potentially due to the weak amplification from grain samples as a result of either inhibition of PCR by grain polyphenols or as a function of low LAB populations (Bokulich et al., 2015). Though the data gathered are of exceptionally high detail, ultimately this work cautions against ascribing raw substrates as causing contamination of all areas or equipment that share similar microbial community compositions, as there are alternative means for microbial transfer within the environment such as fruit flies, or more likely, human activity (Bokulich et al., 2015).

Given the ubiquitous presence of LAB in and on natural sources such as plants and humans, it is likely that the introduction of specific LAB species into the brewing environment, and their prevalence and distribution throughout, is an outcome of the specific raw materials (grain, hop, water, yeast) used and is a further function of a given brewery's specific geographical location; facility history; recipe, processing, and production lines; and personnel hygiene. The individual nature of a brewery has been underscored by the analysis of LAB-contamination in Australian breweries wherein specific contamination was found to be associated more with the particular brewery, rather than with specific antimicrobial challenges present by the beer sample that they were isolated from (ethanol, pH, hops) (Menz et al., 2010). The microbiological quality and hygiene of a brewery thus is apparently dependent more on production practices and sanitation regimes than it is on the beer characteristics (e.g. highly hopped or alcoholic beers) (Menz et al., 2010).

The work presented in Bokulich et al. (2015) is a foundational study from which to model further analysis of other breweries. Though it can be restated that the presence of LAB isolates and prevalence/distribution of them in a brewery will probably be brewery specific, ultimately an understanding of where bacterial (LAB) contamination is taking place within a given brewery should allow for the identification of specific contamination sources (i.e. raw materials vs. personnel) and help to strategize how best to prevent, or treat and recover contaminated product.

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