Nisin-producing organisms during traditional 'Fior di latte' cheese-makingmonitored by multiplex-PCR and PFGE analyses

In this work we studied using different molecular methods the population dy namics of nisin-producing organisms and the persistence of such organisms w ithin a complex ecosystem, 'Fior di latte' cheese, a traditional high-moist ure pasta filata cheese. Using the primers targeting the eubacterial 16S-23 S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. In hibitors of Lactococcus lactis subsp. lactis DSM 20481(T) used as indicator occurred during the whole process of cheese manufacture as a significant p art of lactic microflora; however, only 12 among 109 isolates of bacterioci n producers were nisin producers. Amplification of the nisA or nisZ gene, u sing DNA extracted directly from dairy samples as templates, showed that th e nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to mo nitor nisin-producing strains during cheese manufacturing, the 12 Lactococc us lactis nis(+) strains were analysed by low frequency restriction fragmen t and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diver se. The other three isolates from curd after ripening showed the same restr iction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'. (C) 2001 Elsevier Science B.V. All rights reserved.