the brewing industry lags behind other LAB-related fields in applying high-throughput protein analysis or sequencing techniques to solve the problem of LAB beer-spoilage as well as help characterize useful LAB (9, 92).
1.4. BSR LAB and the brewing environment
1.4.1. Niche adaptation and horizontal gene transfer
Distinction between differently adapted LAB isolates lies not only with the analysis of the LAB core genomes, but also in the investigation of chromosomal sequences that appear to have originated in another species and mobile genetic elements such as plasmids (24). The latter two genetic features are frequently acquired through horizontal gene transfer (HGT) between isolates of same or different species. By comparing recently divergent as well as ecologically distinct genomes, it is revealed that HGT is important for the transfer of sequences or clusters of sequences, and drives the existence of diversification (62, 154). In fact, HGT events are promoted by environmental stress, resulting in faster adaptation or “short-term” evolution in challenging environments (41).
For LAB, HGT events mediated by plasmids are important to a variety of industries (32). In the brewing industry, conventional genetic markers of beer-spoilage such as the exopolysaccharide gene gtf, and the hop-tolerance genes hitA, horA, and horC are all plasmid-encoded and exhibit a very high degree of sequence identity in many different species (129, 152). The existence of these markers suggests not only the occurrence and support of HGT in and by the brewery, but also the importance of investigating other plasmid-harbored genes that demarcate BSR from non-BSR LAB.
Given that the ecological diversity among LAB appears to be driven in general by genome reduction mechanisms, the acquisition of niche-specific genes through the transfer of plasmids is an important area of investigation. Indeed, recent omics-based studies support the notion that plasmids are important for conferring beer-spoilage ability. New genomic data for several L.
brevis isolates has revealed that an increased number of plasmids may correlate with the ability of isolates to withstand increasingly harsh and specific environments. For example, L. brevis
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KB290 originally isolated from a traditional Japanese fermented vegetable and also able to grow in simulated gastric and intestinal juices, has nine plasmids ranging in size from 5.8 to 42 Kb (48). Similarly, the rapid beer-spoiling isolate L. brevis BSO 464 has eight plasmids ranging from 2.3 to 85 Kb (13). These two isolates are incapable of growth in the other isolate’s niche-environment (J. Bergsveinson, unpublished), indicating that each possesses specific genetics that do not confer immediate cross-resistance to another stressful environment; as such, these isolates have niche-specific tolerance genes. In contrast, the type strain L. brevis ATCC 367T only harbors two plasmids (13 and 35 Kb) (83) and is unable to spoil beer and cannot grow in in gastric juices (J. Bergsveinson, unpublished; 48). This further suggests that increased plasmid-coding capacity likely supports the ability of L. brevis strains to infiltrate diverse environments.
This idea is supported by a recent study showing that the sequential loss of plasmids from L.
brevis BSO 464 results in loss of its original spoilage phenotype, indicating that beer-spoilage is mediated by specific plasmid-encoded functions (13). Similarly, transcriptomic analysis performed on BSR LAB P. claussenii ATCC BAA-344T revealed that several significant plasmid-based transcripts were active across its eight plasmids (ranging from 1.8 to 36 Kb) when in the beer environment, notably on the plasmid that harbored the hop-tolerance gene horA (101, 103). Collectively, these results strongly suggest that specific plasmids encode previously un-described beer-spoilage related functions and that detailed investigation of plasmid genes in relation to growth in niche environments, such as beer or the brewery, will prove useful.
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 this data to be of utility 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 (141, 87), 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 (41, 96). 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.
16 1.4.2. 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 occupied this new niche along with the inclusion of hops in beer between the 5th and 9th century. Following genetic adaptation to this specific stress, BSR LAB then adapted further and have since remained tightly linked with the brewing environment (129, 130, 132). Indeed, BSR LAB isolates are rarely isolated elsewhere than breweries or beer, though non-BSR LAB isolates of the same species are (129, 132). Breweries thus are both the selective environment and the reservoir for their own contaminants.
A recent study has investigated the distribution pattern of LAB species and putative hop-tolerance genes in a brewery producing several different kinds of beer, using LAB-specific terminal restriction fragment length polymorphism and ddPCR, respectively (20). The brewery involved produces conventional beer (potential BSR LAB contaminants), sour beer (helpful LAB fermenters or BSR LAB) and coolship beer (BSR LAB and environmental microflora). The LAB-terminal resitrction fragment length polymorphism analysis applied in this study was specifically developed for LAB isolates and was found to more sensitively discriminate between species of the Lactobacillales order and most genera of the Bacillales order present in mixed culture (20). LAB-terminal resitrction fragment length polymorphism methodology 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 (20). By applying this technique to analyze the LAB community profile throughout a brewery, it was possible to conclude that the brewery microbiota is likely 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 (20). For example, they found that wort samples contained a mixture of L. delbrueckii, L. hilgardii, L. sakei, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus spp., as well as a Bacillus spp. “A”, most of which were only rarely detected in other fermenting and bottled beer samples (20). Many of these species, while not necessarily found in finished beer, are apparently associated with grain and therefore their detection in wort is unsurprising (17).
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Interestingly, distinct LAB profiles from specific brewery samples were detected at different sites, presumably as result of potential contact with the sample. For example, sour and coolship beers were dominated by L. lindneri and Pediococcus spp., 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 LAB 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 (20). Though the data gathered is 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 (20).
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) and is a further function of a given brewery’s specific geographical location;
structural 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 starting beer sample (ethanol, hops, pH) (90). 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 (i.e., highly hopped or alcoholic beers) (90).
The work presented in (20) 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
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prevalence/distribution of them in a brewery will likely 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.