Identification of the main esterase involved in milk fat hydrolysis in [i]Propionibacterium Freudenreichii[/i]
Résumé
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 cheese
microorganisms. In Swiss cheese, Propionibacterium freudenreichii, a species used as ripening
culture, is the main agent of lipolysis, with 96% of FFA released during ripening resulting from its
activity. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. Since
lipolysis in Swiss cheese mainly occurs during P. freudenreichii growth, we hypothesized that it does
not result from the activity of intracellular enzymes released from lyzed cells, but from surfaceexposed
or secreted esterases. Thus, we focused the present study on previously identified
esterases, one secreted, 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. All genetically modified
strains were assessed for their lipolytic activity during their growth in a medium containing milk fat
emulsion. In parallel, the lipolytic activity of 22 wild strains was also tested under the same
conditions, and the sequences of these two targeted genes compared among strains. We showed
that mutants over‐expressing either PF#279 or PF#774 released about three times more FFA
compared to the wild‐type strain (3.5 vs 0.9 mg/g fat), demonstrating that both enzymes are
lipolytic esterases. However, only the mutant inactivated for PF#279 was affected in its lipolytic
activity. The lipolytic activity of the 22 wild strains tested varied over a large range (FFA net
production ~ 0.05 to 1.9 mg/g fat). Interestingly, the two non lipolytic strains identified exhibited
the same single deletion at the beginning of pf279 sequence, whereas pf774 sequence was highly
conserved 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.
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