Induction of Viable but Nonculturable State in Beer Spoilage Lactic Acid Bacteria

Abstract

Strong beer spoilage strains Lactobacillus lindneri DSM 20692 and Lactobacillus paracollinoides JCM 11969T 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 11969T 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 11969T in VNC states were successfully stored at −80°C with 10% dimethyl-sulfoxide 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.