0-5.0 (Table 1). Interestingly, significant AP26113 in vitro concentrations of tyramine (50 μM, 2.5 nmol mL-1 min-1) and putrescine (13 μM, 0.65 nmol mL-1 min-1) were observed in the samples exposed to pH 1.8 in the presence

of the two BA precursors, even though only 1.7 × 101 CFU mL-1 were detected at the end of the assay. This suggests that the inoculum was able to synthesise a substantial quantity of tyrosine decarboxylase during the test before cell death and lysis occurred, and that probably the tyrosine decarboxylase remained substantially active in the dead cells and cell lysate. The tyrosine decarboxylase of IOEB 9809 is active in a range of pH 2.0-8.0 in cell-free extract [24]. Figure 2 Detection of live-dead bacteria by confocal microscopy. Observation by confocal microscopy of L. brevis IOEB 9809 after gastric stress to pH 5.0 in absence of BA precursors (A) or in presence of: agmatine (B), tyrosine (C) or agmatine plus tyrosine (D). Green cells represent live bacteria, while red cells are bacteria with damaged membrane. When we simulated the gastric environment, in addition to the action of lysozyme, the bacteria were subjected to multiple stress stimuli: decreasing pH, proteolytic activity of pepsin and heat shock at 37°C. Griswold et al. [25] (2006), propose that the agdi operon could be part of a

general stress response pathway in BMN-673 Streptococcus mutans. The agmatine deimination, by forming ammonia and providing ATP, would result

in mild deacidification of the medium, metabolic C646 energy release and degradation of toxic compounds [25]. Here, the Rutecarpine maximum levels of putrescine (around 40 μM) production by L. brevis were observed between pH 5.0-4.1 for cultures supplemented with agmatine (Table 1), which accords with that reported for Lactobacillus hilgardii at pH 4.5 [26] and for Streptococcus mutans at pH 4.0 [27]. There is evidence suggesting that BA production enables producing organisms to survive at low pH [28]. Our results show that at pH 5.0 the presence of agmatine, tyrosine or both precursors enhanced the cell survival two-, three- and four-fold respectively compared to controls (Figure 1). At pH 4.1, the beneficial effect on viability was even more pronounced (4- and 6-fold increase in the presence of tyrosine, and tyrosine plus agmatine); however, it has no beneficial effect at more acidic pHs (Figure 1). Thus, it seems that the beneficial effect of the putrescine and tyramine biosynthetic pathways is restricted only to mild acidic conditions. Transcriptional analysis of tyrDC and aguA1 genes The above results indicated that an increase of BA production occurred under saliva and mild gastric stresses, presumably due either to a physiological effect, or to increased gene expression.