Induction and chemical inactivation of Lactobacillus delbrueckii temperate bacteriophages

EBRECHT, A. C.; GUGLIELMOTTI, D. M.; REINHEIMER, J. A.; SUÁREZ, V. B.

San Miguel de Tucumán, Argentina.

Simposio; III International Symposium on Lactic Acid Bacteria. II Argentinean LAB Net Meeting International; 2009

Centro de Referencias para Lactobacilos (CERELA-CONICET)

Lysogeny in acid lactic bacteria (BAL) used as starter has a potential negative impact on fermented dairy processes. The influence of Mitomycin C (MC) concentration (0.2, 0.5 and 1.0 µg/ml) and incubation temperature (34, 37 and 43 ºC) on the prophage induction from the commercial strain L. delbrueckii subsp. lactis Cb1 was studied. Control cultures were carried out without the addition of MC to establish the spontaneous induction of the prophage. After MC addition, D.O.560nm was measured at 30 min intervals for 5 hours. Counts of phage particles released by action of MC were made using L. delbrueckii subsp. bulgaricus 342 as host strain, and compared to spontaneous liberation (without MC). For temperate phages Cb1/342 and Cb1/204, inactivation kinetics using different biocides (ethanol 10, 50, 75 and 100%, v/v, isopropanol 10, 50 and 100%, v/v, sodium hypochlorite 100, 200 y 300 ppm residual-free chlorine, and peracetic acid 0.15%, v/v) were performed. Results were expressed as concentration (PFU/ml) of viable viral particles and plotted against time. The time (min) required to achieve 99% of inactivation (T99) was calculated from the survival curves. To test the influence on phage viability, respective pH controls were carried out. For phage induction, the optimal concentration of MC was 0, 5 µg/ml.  Temperature was not determinant in MC action, since the enumerations resulted in the same log order for all of them. However, a higher number of particles were released at 43 ºC in the spontaneous induction. Peracetic acid showed a good effectiveness on the inactivation of these phages. Sodium hypochlorite inactivated completely high titers of phage Cb1/204 after 20 min (100 ppm) or after 2 min (200 ppm) of treatment. On the other hand, phage Cb1/342 filtrates were inactivated after 2 min of treatment using 300 ppm of this biocide. Ethanol 100% (v/v) was the most effective to inactivate both phages (T99 ≤ 18 min), but only Cb1/204 was completely inactivated. When isopropanol 100% (v/v) was used, T99 values were higher than 35 min. Diluted solutions did not achieve a significant loss of viability within 45 min (T99 > 45 min). The results obtained in this work give useful information regarding temperate phages, as well as to design effective treatments to minimize phage numbers in laboratories and industrial plants.