Lipolysis by Propionibacterium freudenreichii: Identification of two lipolytic esterases.

ABEIJON MUKDSI, M.C., FALENTIN, H., MAILLARD, M-B., MEDINA, R.B., PARAYRE, S., LORTAL, S., THIERRY, A.

Madison, Wisconsin

Simposio; IDF Cheese Ripening & Technology Symposium; 2012

Free fatty acids are important flavor compounds in cheese, where they contribute to pungent, rancid, cheese, and fruity notes. They mainly result from the lipolytic activity of cheese micro-organisms. Propionibacterium freudenreichii, a species used as a ripening culture in Swiss cheese, is the main agent of Swiss cheese lipolysis, with 96% of the free fatty acids released during the ripening resulting from P. freudenreichii activity. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii. Since cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that is does not result from the activity of intracellular enzymes released from lyzed cells, and thus we focused our study on surface-exposed or secreted esterases. Out of the twelve putative esterases previously predicted from the genome sequence of P. freudenreichii CIRM-BIA1T, we identified one secreted esterase, PF#279, and one putative esterase predicted to be anchored in the plasma membrane, PF#774. To evaluate the respective role of these two proteins in lipolysis, P. freudenreichii CIRM-BIA1T was knocked out for the genes encoding these two proteins, separately. Each of these genes was also over expressed in the same strain. These genetically modified strains were assessed for their lipolytic activity during their growth in a medium containing an emulsion of milk fat. Results showed that the mutants over-expressing either PF#279 or PF#774 released three times more free fatty acids compared to the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, only the mutants inactivated for PF#279 were affected in their lipolytic activity, suggesting that PF#279 is the main lipolytic esterase involved in milk fat hydrolysis in P. freudenreichii CIRM-BIA1 and is a key component in Swiss cheese lipolysis.