Identification of Streptococcus infantarius subsp. infantarius as the species primarily responsible for acid production in Izmir Brined Tulum Cheese from the Aegean Region of Türkiye

Abstract

Izmir Brined Tulum (IBT) Cheese is a traditional semi hard cheese produced in the Aegean region of Türkiye. Lactic acid bacteria (LAB) isolates from IBT cheese samples taken during manufacture and from mature IBT cheeses were investigated for their acid producing capability with the aim of detecting LAB strains responsible for acid production in IBT cheese. Forty two out of 216 isolates decreased the pH of milk to 5.0 or below in 18 h at 37 °C or 42 °C. 16S rRNA Sanger sequencing revealed the presence of LAB species that had not been detected in IBT cheese previously and, indeed, were identified for the first time as the primary acid producers. The majority of these acid producing isolates were identified as putative Streptococcus lutetiensis/Streptococcus infantarius subsp. infantarius (Sii). Further analysis by sequencing the groES/groEL genes of these isolates established that they were Sii. The remaining isolates from cheese samples taken during manufacture were identified as Streptococcus macedonicus, S. thermophilus, Lactococcus lactis subsp. lactis, Lactobacillus delbrueckii subsp. sunkii and L. delbrueckii subsp. indicus and, from mature cheeses, as Enterococcus faecalis and L. delbrueckii subsp. sunkii. Pulsed-field gel electrophoresis (PFGE) results revealed a large genetic diversity amongst the Sii isolates recovered from the IBT cheeses. It was also established that the Sii strains exhibited efficient and consistent acidification ability equivalent to S. thermophilus. Whole-genome sequencing (WGS) and comparative genome analysis of the representative Sii AYB210 strain provided further insights. More specifically, the genome of AYB210 differed from the previously sequenced African dairy isolate Sii CJ18 and the human isolate ATCC®BAA-102™. Modifications in the lactose operon, which may be an indicator of dairy adaptation, were identified and a high number of CRISPR spacers and putative bacteriocin, virulence factor and antibiotic resistance genes were also detected.

Keywords: Fast-slow differential agar; Lactic acid bacteria; Starter culture; Streptococcus lutetiensis; Streptococcus macedonicus; Whole-genome sequencing (WGS).