Identification of a Secreted Lipolytic Esterase in Propionibacterium freudenreichii , a Ripening Process Bacterium Involved in Emmental Cheese Lipolysis

Abstract

Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii, a species used as a ripening culture. 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 P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico. The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico, biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by P. freudenreichii.

The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

“…Consequently, the profile of released FFA by these strains differed from that of the wild-type strain, with 42.9% saturated long-chain FFA and only 6.1% short-chain FFA, versus 30.6% and 16.7%, respectively, in the wild-type strain. The results of overexpression of pf279 are in agreement with our previous results that showed that a mutant overexpressing pf279 under the control of another promoter released 5 to 8 times more FFA from milk fat than the wild-type strain (11). The results of overexpression of the pf774 gene, previously annotated as encoding a putative cell-bound esterase (11), showed that PF#774 (i) is effectively an esterase and (ii) is active on milk triglycerides.…”

“…It was grown at 37°C with agitation (200 rpm) in Luria-Bertani medium (LB) containing ampicillin (100 g ml Ϫ1 ). To determine the lipolytic activity of P. freudenreichii, cells were grown in a modified YEL medium containing 20 g/liter lactate and milk fat emulsified with a sodium caseinate solution (final fat concentration of 30% [wt/wt] in the broth medium), as previously described (11). Population levels of propionibacteria were monitored by measuring the optical density at 650 nm (OD 650 ) of a culture grown in the same medium without emulsion.…”

“…Twelve (putative) esterases were previously predicted from the genome sequence of P. freudenreichii CIRM-BIA1 T (10), all of which are expressed regardless of the presence or absence of milk fat in the culture medium (11). The majority of lipolysis takes place early during P. freudenreichii growth (5), at a stage at which the release of intracellular enzymes from lysed cells is very unlikely.…”

“…We thus targeted two putative esterases, PF#279 and PF#774, predicted to be secreted and cell wall anchored, respectively. Our previous work showed that esterase PF#279 is effectively secreted and active on milk triglycerides (11).…”

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health

“…freudenreichii also contributes in a great manner to cheese lipolysis by releasing free fatty acids from fat during cheese ripening. Two esterases, one extracellular and other surface-exposed seem to be involved in lipolysis of milk glycerides [63,64]. Furthermore, ten intracellular esterases were found in the P. freudenreichii genome that could be involved in the synthesis of the volatile esters associated with the fruity flavor of cheese [65].…”