Identification of the main esterase involved in milk fat hydrolysis in [i]Propionibacterium Freudenreichii[/i]

Maria Claudia Abeijon Muksdi (1, 2) , Hélène Falentin (1) , Marie-Bernadette Maillard (1) , R.B. Medina (2) , Sandrine Parayre-Breton (1) , Anne Thierry (1)

1 STLO - Science et Technologie du Lait et de l'Oeuf
2 CONICET - Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires]

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.

Mots clés: propionibacterium freudenreichii; esterase; lait; acide gras; hydrolyse; fromage; affinage; lipolyse; protéine

Domaines: Ingénierie des aliments; Alimentation et Nutrition; Microbiologie et Parasitologie

Maria Claudia Abeijon Muksdi, Hélène Falentin, Marie-Bernadette Maillard, R.B. Medina, Sandrine Parayre-Breton, et al.. Identification of the main esterase involved in milk fat hydrolysis in [i]Propionibacterium Freudenreichii[/i]. 4. International Symposium on Lactic Acid Bacteria Food, Health and Application, Oct 2013, Tucuman, Argentina. , 2013. ⟨hal-01209701⟩