Aims: To develop a detection medium for hard‐to‐culture beer‐spoilage lactic acid bacteria (LAB).
Methods and Results: Four hard‐to‐culture beer‐spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild‐type strains in beer. To develop a countermeasure against these hard‐to‐culture beer‐spoilage LAB, a beer‐based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard‐to‐culture beer‐spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer‐based medium. Further comparative study was performed with five other media widely used for the detection of beer‐spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer‐spoiler detection (ABD) medium, possessed superior sensitivity for hard‐to‐culture beer‐spoilage LAB and comparable sensitivity with easy‐to‐culture beer‐spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro‐organisms, and thereby allow the selective growth of beer‐spoilage LAB.
Conclusions: Advanced beer‐spoiler detection medium is considered as an effective tool for comprehensive detection of beer‐spoilage LAB in breweries.
Significance and Impact of the Study: The detection by ABD medium can be used as an indicator for differentiating the beer‐spoilage ability of LAB without further confirmatory tests in breweries.
Strong beer spoilage strains Lactobacillus lindneri DSM 20692 and Lactobacillus paracollinoides JCM 11969 T were repeatedly subcultured in degassed beer and their culturability on MRS agar was examined. As a result, the two strains were found to show decreased culturability, suggesting that the prolonged contact with beer reduces the culturability of beer spoilage lactic acid bacteria (LAB). After 30 subcultures in degassed beer, both strains were subjected to sublethal heat treatment. As a consequence, L. lindneri DSM 20692 and L. paracollinoides JCM 11969 T were no longer detectable on MRS agar despite the presence of 460 viable cells, indicating that the viable but nonculturable (VNC) states were induced for both strains. Problematically, the heat treated VNC strains were shown to exhibit beer spoilage ability, suggesting that spoilage incidents can occur without detection by culture media. It was also shown that, once acquired, the VNC states are stably maintained in beer without further heat treatment. These results suggest the possibility that beer spoilage LAB strains remain hidden in pitching yeast and work-in-process products without detection. Furthermore L. lindneri DSM 20692 and L. paracollinoides JCM 11969 T in VNC states were successfully stored at -80°C with 10% dimethylsulfoxide as a cryoprotectant and reconstituted in degassed beer without losing VNC characteristics. Taken together, these findings show that valuable bioresources can be acquired from culturable beer spoilage LAB strains and maintained for long-term storage as frozen culture stocks.
Four beer-spoilage strains, LA20, LA21, LA22 and LA23, were isolated from brewery environments. Based on the 16S rRNA gene sequence, LA20 was identified as Pediococcus inopinatus and the remaining three were identified as Lactobacillus backi. The homologs of horA and horC, the hop resistance genes originally identified in L. brevis ABBC45, were detected simultaneously in LA22 and LA23, while only a horA homolog and a horC homolog were found in LA20 and LA21 respectively. The 5.6 kb DNA regions containing the horA homolog in LA20 and LA22 were almost 99% identical with the corresponding region of ABBC45. Similarly the 8.2 kb regions containing the horC homolog in LA21 and LA22 were more than 99% identical with that of ABBC45. Interestingly the horA-containing 5.6 kb regions in LA20 and LA22 were found to be completely identical despite the distinct genus status. Coupled with the fact that LA20 and LA22 were isolated from the same sampling site, these results, taken collectively, reinforce our hypothesis that horA and horC genes were acquired by beer-spoilage species through horizontal gene transfer and confirm the usefulness of horA and horC as genetic markers for the species-independent determination of beer-spoilage ability in lactic acid bacteria.
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