Efficient Isolation and Functional Analysis of SpontaneousStreptococcus thermophilus Bacteriophage-Insensitive Mutants

The use of bacteriophage-resistant strains that have satisfactory technological properties is essential for preventing phage infections in industrial fermentation processes. Here, we describe an improved method for the isolation of spontaneous Streptococcus thermophilus bacteriophage-insensitive mutants (BIMs). The concentration of large volumes of S. thermophilus secondary cultures, their subculture in skim milk with high titers of bacteriophages, and their inoculation with the fast milk acidifying phenotype over multiple passages were critical to increasing the probability of obtaining mutants, improving isolation efficiency, and maintaining technological performance. We obtained large quantities of BIMs after every round of screening, with isolation efficiency in excess of 85%. We analyzed nine BIMs, and these were all similar to their parent strain S. thermophilus St1 with respect to syneresis, water holding capacity, and apparent viscosity. In comparison to the parent strain, seven of the nine variants possessed equivalent acidifying activities, with the remaining two variants exhibiting excellent acidification performance. One or two new spacers from corresponding phages were found in all nine BIMs, demonstrating that the CRISPR/Cas system was responsible for the phage resistance of S. thermophilus.