Isolation and characterization of spontaneous phage-resistant mutants of Lactobacillus paracasei

Abstract

Spontaneous phage-resistant mutants were isolated from Lactobacillus paracasei LPC by agar plate (AP) and secondary-culture (SC) methods. They were characterized by cell and colony morphologies, carbohydrate fermentation patterns, phage resistance stability, efficiency of plaquing (EOP), acidifying and milk acidification kinetics. Only 98 out of 175 isolates (56%) proved to be true phage-resistant mutants and the SC method was more efficient than the AP method. Although the phage resistance stability varied among the mutants isolated, the EOP values were mostly very high (<10−11). Three phage-resistant strains were selected based on their extreme resistance capacity to the phage ΦT25 (EOP<10−10) and their nonlysogenic property. Acidifying activity did not differ between phage-sensitive strains and their three respective phage-resistant derivatives. Most of the mutants were completely or partially unable to adsorb phage particles. Restriction-modification type systems were not detected in all phage-resistant derivatives. All three selected mutants were identical to the corresponding parent strain of L. paracasei LPC in the cell and colony morphologies. The comparison of random amplified polymorphic DNA profiles obtained with four arbitrary primers also revealed the highest similarity coefficient (87%) among the parent strain and the three mutants, indicating that each mutant has been derived from this parent strain. A good performance during milk fermentation and the subsequent refrigerated storage were obtained when these mutants were employed. These phage-resistant derivatives could be used as improved strains or for strain rotation programs when commercial strains become sensitive to the phages present in industrial environments.