Traditional and emerging methods for BSR LAB detection and identification
Culture methods are still the most commonly used approach for routine detection and identification of BSR LAB in the brewery, for reason of their ease of use, limited need for specialized training, relatively low monetary and space cost, and proven utility. However, culture methods have inherent disadvantages, owing to the variable nature of BSR LAB isolates, e.g. differences in their fastidious aerotoler- ance or nutritional requirements, and the different adaptive states they may exist in when isolated from beer. These factors make the primary isolation of some LAB contaminants via growth quite difficult (Deng et al., 2014; Suzuki et al., 2008). More importantly, there is no single medium that effectively screens and supports growth for all possible beer spoilage LAB (Taskila et al., 2011).
de Man Rogosa Sharpe (MRS) medium (de Man et al., 1960), which was designed for the cultivation of LAB, remains the most relied-upon medium in brewery settings (Sakamoto and Konings, 2003). There are several descriptions of supplementing MRS medium with varying concentrations of beer, expecting that the added beer enables cultivation of beer-adapted (hard-to-culture) organisms and that the nutrients provided by the MRS medium allow for more rapid growth (Haakensen et al., 2009b; Holzapfel, 1992; Suzuki et al., 2008b). Further modifications to beer-supplemented MRS include adding reducing agents to remove oxygen tension in the medium to facilitate the growth of a wide range of BSR LAB in addition to microaerophilic strains (Nishikawa and Kohgo, 1985; Taskila et al., 2010). Similarly, other developments such as the Advanced Beer Detection (ABD) medium, developed by Suzuki et al. (2008b), seek to reduce medium osmolarity with the goal of isolating hard- to-culture BSR LAB.
Often there is a need to exclude other non-LAB brewing microorganisms from growing while concurrently enriching the medium to cultivate specific or hard-to-culture LAB isolates. Enrichment culturing prior to plating is a common technique to influence the number and identity of isolates grown and is often critical for the efficiency of downstream molecular detection techniques. Thus, contaminating yeast or Gram-negative bacteria are excluded from growing in detection media by the inclusion of cycloheximide and 2-phenylethanol, respectively (Taskila et al., 2011). To select for specific or hard to cultivate BSR LAB, enrichment media are typically differentiated based on carbon sources present to exploit differences in substrate utilization between species (Endo et al., 2011). The most general substitution that can be made is removal of glucose in favour of another carbohydrate, so as to limit the growth of very fast-growing LAB, thereby ‘levelling the playing field', giving hard-to-culture isolates that are out-competed in most standard growth media a chance to grow (Endo et al., 2011). In addition, some metabolites produced by LAB, such as lactic acid or bacteriocins that have antimicrobial action, may also add to the selectivity of the enrichment cultivation (Moneke et al., 2009).
Ultimately, primary cultivation and even use of specialized culture media to detect and identify BSR LAB are not fully effective for the accurate detection of BSR LAB (Suzuki, 2011; Taskila et al., 2011). Nonetheless, culture methods remain an important area of investigation for the reason that culturing is often a preliminary step for molecular analysis and because culture-based tests traditionally have provided the most information for spoilage incidence reports, which has greatly influenced our current understanding concerning relevant BSR LAB.
Molecular methods typically have higher associated cost and the need for specialized training, and thus have different utility for research or industry interests. Further, molecular methods can often be labour-intensive despite their ascribed benefit of being ‘rapid', since pre-enrichment culture or isolation is often required before the molecular detection limit can be achieved or to remove inhibitory molecules found in beer (Back, 2005; Taskila et al., 2011). However, the allure of molecular techniques for the brewing industry is centred on their increased specificity and sensitivity in detecting and identifying BSR LAB. Recent reviews (Bokulich et al., 2012a; Bokulich and Mills 2012b) provide an extensive comparison of methodology concerning microbial community profiling in the brewing industry, and thus only a general overview of current community- or microbe-targeted molecular methods for BSR LAB analysis is presented here.