Establishment of bacteriophages in an immobilized cells system used for continuous inoculation of lactococci

Abstract

Lactococcus lactis ssp. lactis CRA-1 cells were immobilized in K-carrageenan-locust bean gum gel beads, and the beads used for continuous inoculation of milk in a bioreactor. The effect of dilution rate (10, 20 and 30 h −1 ) and medium composition on the development of bacteriophages in the system was examined. Milk coagulation, due to local acidification, was observed in the reactor vessel, mostly in the exit filters, and two types of bioreactors had to be designed to tentatively solve this problem. Following an initial contamination of 10 2 pfu ml −1 , phage populations ranging from 10 6 and 10 8 pfu ml −1 were reached in the milk effluent after only 6 h of incubation, as a function of dilution rate (10–30 h −1 ), and stabilized at about 5×10 7 pfu ml −1 after 48 h of incubation for all dilution rates tested. After 48 h of fermentation in a synthetic medium without acid-coagulating proteins, the phage population in the effluent medium (6.8×10 8 pfu ml −1 ) was higher than in the milk-based fermentations. The phage count in the beads was approximately 2 log units higher than the free phage count in the medium.The phage-resistant (PR) lactococci in the beads represented between 0.9 and 1.9% of the total bacterial population, 48 h after the phage contamination, but data for free-cell release suggested that the surface population contained 10–23% of PR cells. Even though total bacterial population in the milk effluent was not influenced by phage contamination, subsequent acidification of the phage-contaminated culture was markedly delayed. The bioreactor-inoculated milk showed an acidifying activity related to its PR bacterial population.