Identification of the main esterase involved in milk fat hydrolysis in Propionibacterium Freudenreichii

Free fatty acids (FFA) are important flavor compounds in cheese, where they contribute to pungent,rancid, cheesy, and fruity notes. They mainly result from the lipolytic activity of cheesemicroorganisms. In Swiss cheese, Propionibacterium freudenreichii, a species used as ripeningculture, is the main agent of lipolysis, with 96% of FFA released during ripening resulting from itsactivity. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. Sincelipolysis in Swiss cheese mainly occurs during P. freudenreichii growth, we hypothesized that it doesnot result from the activity of intracellular enzymes released from lyzed cells, but from surfaceexposedor secreted esterases. Thus, we focused the present study on previously identifiedesterases, one secreted, PF#279, and one putative esterase predicted to be anchored in the plasmamembrane, 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. All genetically modifiedstrains were assessed for their lipolytic activity during their growth in a medium containing milk fatemulsion. In parallel, the lipolytic activity of 22 wild strains was also tested under the sameconditions, and the sequences of these two targeted genes compared among strains. We showedthat mutants over‐expressing either PF#279 or PF#774 released about three times more FFAcompared to the wild‐type strain (3.5 vs 0.9 mg/g fat), demonstrating that both enzymes arelipolytic esterases. However, only the mutant inactivated for PF#279 was affected in its lipolyticactivity. The lipolytic activity of the 22 wild strains tested varied over a large range (FFA netproduction ~ 0.05 to 1.9 mg/g fat). Interestingly, the two non lipolytic strains identified exhibitedthe same single deletion at the beginning of pf279 sequence, whereas pf774 sequence was highlyconserved among strains, confirming the prominent role of PF#279 in lipolysis. Taken together,these results show that PF#279 is the main lipolytic esterase involved in milk fat hydrolysis in P.freudenreichii and is likely a key component in Swiss cheese lipolysis.