Background Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage.Methodology/Principal FindingsThe circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters.Conclusions/SignificanceWith the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.

This study followed the progression of lipolysis in Emmental cheese by quantifying the concentrations of individual free fatty acids (FFA) released during ripening in each of the different rooms: 12 days at 12 degrees C, 28 days at 21 degrees C, and 8 days at 4 degrees C. Lipolysis, which corresponded to 1.56% of fat, mainly occurred in the 21 and 4 degrees C rooms, with 68 and 16.5% of total FFA, respectively. The nonselectivity of lipolytic enzymes was evidenced: all fatty acids were released with level of > or =1%. Differential scanning calorimetry experiments showed that the thermal properties of cheese were affected by (i) lipolysis of fat, that is, the monoacylglycerols, diacylglycerols, and FFA that may be localized at the fat/whey interface, and/or by (ii) hydrolysis of high-melting-point triacylglycerols constituted mainly by long-chain saturated fatty acids (e.g., palmitic acid). Analysis of the cheese microstructure was performed using confocal laser scanning microscopy. Fat globules were mainly disrupted after pressing of curd grains, leading to the release of the milk fat globule membrane (MFGM); fat inclusions were surrounded by pockets of whey, delimited by casein strands. Moreover, colonies of bacteria were preferentially localized in situ at the fat/protein interface. This study showed that both the localization of bacteria and the supramolecular organization of fat which was not protected by the MFGM can help the accessibility of milk fat to lipolytic enzymes and then contribute to the quality of cheese.

-Cheese flavour is the result of a complex mixture of volatile compounds, originating mainly from the enzymatic degradation of curd components by cheese microflora during cheese ripening. Directing cheese flavour development requires knowledge on inter-and intra-species contributions to flavour development, i.e. identification of the volatile (flavour) compounds produced by each strain. The aim of this study was to identify the volatile compounds produced in Swiss cheese by Propionibacterium freudenreichii, one of the species essential for the development of the characteristic flavour of this type of cheese. The volatile profile of compounds obtained from smallscale (1/100) Swiss cheeses, with or without P. freudenreichii, were compared (three strains tested, in association with three thermophilic lactic starters, i.e. twelve cheeses, manufactured in duplicate). Neutral volatile compounds, extracted by dynamic headspace, and free fatty acids were identified using gas chromatography-mass spectrometry. The concentrations of all carboxylic acids and 14 of 58 neutral compounds were significantly higher in the presence of propionibacteria (PAB). The three PAB strains tested produced the same volatile compounds, but observed quantitative differences were strain-dependent. Propionic acid and four propionate esters were detected only in the presence of PAB. Moreover, cheeses with PAB contained two-to three-fold higher levels of free fatty acids derived from lipolysis and five-to fifty-fold higher levels of branched-chain compounds derived from isoleucine catabolism (2-methylbutanal, 2-methylbutanol and 2-methylbutanoic acid) and from leucine catabolism (3-methylbutanoic acid). Lactic starters induced significant variations in the concentrations of some of the compounds produced by PAB, such as methylbutanoic acids and free fatty acids, which varied by 2.0 and 1.4, respectively, as a function of the lactobacilli strains. PAB strains affect the concentration of varied volatile compounds and could therefore have distinct contributions to the formation of Swiss cheese flavour.

-The catabolism of amino acids by cheese micro-organisms results in the production of various volatile flavour compounds. It was recently shown to be a rate-limiting factor in the formation of cheese flavour, leading to an increased interest in elucidating the pathways and the flora involved. This paper reviews the ability of propionibacteria (PAB) to produce flavour compounds deriving from branched-chain, aromatic and sulphur-containing amino acids. In culture media, PAB produced volatile compounds derived from Leu, Ile, Met and Phe. In cheese, the presence of PAB is positively correlated to the amount of acids, alcohols and/or aldehydes derived from Leu or Ile. The metabolic pathways of amino acid conversion to flavour compounds by PAB have been only partly elucidated. Aminotransferase(s) catalyse the first step of conversion of branched-chain, aromatic amino acids and methionine, with a higher activity for branched-chain amino acids. The α-keto acids resulting from transamination are further degraded to various compounds by resting cells of PAB. So α-ketoisocaproic acid, derived from Leu, is essentially converted to isovaleric acid by a ketoacid dehydrogenase complex; phenylpyruvic acid, derived from Phe, is converted to phenyllactic acid, phenylacetic acid, benzoic acid and benzaldehyde. Methionine can also be directly degraded by α, γ -elimination, leading to methanethiol. The amino acid catabolism pathways in PAB share similarities with those of lactic acid bacteria but PAB seem to produce higher amounts of branched-chain acids, which are important flavour compounds in cheese.propionibacteria / flavour compound / amino acid / catabolism / cheese ripening Résumé -Production de composés d'arôme du fromage issus du catabolisme des acides aminés par Propionibacterium freudenreichii. Le catabolisme des acides aminés par les micro-organismes du fromage entraîne la formation de composés volatils variés. C'est une étape limitante de la formation de la flaveur du fromage, et les recherches visant à déterminer les voies métaboliques et les flores impliquées se sont récemment multipliées. Cette revue fait le point sur le catabolisme des acides aminés en composés d'arôme chez les bactéries propioniques (PAB). Les PAB sont impliquées dans la formation des composés volatils dérivant de Leu, Ile, Phe et Met, par des voies métaboliques qui ne sont que partiellement connues. La première étape de conversion des acides aminés ramifiés, aromatiques et de la méthionine est catalysée par une(des) aminotransférase(s). Les cétoacides résul-tant de la transamination sont ensuite dégradés en différents composés. Ainsi, l'acide α-cétoisoca-proïque, issu de Leu, est pour l'essentiel converti en acide isovalérique par un complexe cétoacide déshydrogénase ; l'acide phénylpyruvique, issu de Phe, est converti en acide phényllactique, acide phénylacétique, acide benzoïque et benzaldéhyde. La méthionine peut également être directement dégradée par α, γ -élimination, formant du méthanethiol. Ces voies cataboliques présentent des si...

Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of L-leucine, ␣-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced ␣-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal nor␣-hydroxyisocaproic acid was detected. L-Isoleucine and L-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening.The development of cheese flavor during ripening results from the enzymatic breakdown of curd components into sapid and aroma compounds (11). Catabolism of branched-chain (BC), aromatic, and S-containing amino acids has recently received attention because these amino acids are precursors of various volatile compounds, such as acids, aldehydes, alcohols, esters, and thiols, which can contribute to the development of flavor or off-flavor in cheese depending on their levels and the type of cheese (7, 23). In Swiss cheese, the following BC compounds have been identified as flavor impact compounds: 3-methylbutanal, 2-methylbutanal, 3-methylbutanoic acid (isovaleric acid), and the ethyl ester of 3-methylbutanoic acid. These compounds are probably produced from enzymatic conversion of BC amino acids (BCAAs), particularly leucine, but the flora and the pathways involved in their formation in Swiss cheese remain unclear. Therefore, a better understanding of the origin of these compounds is needed in order to control and accelerate the formation of cheese flavor.Dairy propionic acid bacteria (PAB) constitute one of the major floras that grow during the ripening of Swiss type cheeses and are commonly used as secondary starters (26). They are involved in formation of the characteristic flavor and openings of Swiss cheese via fermentation of lactate into acetic acid, propionic, acid and CO 2 (18). While the presence of PAB has been associated with the presence of isovaleric acid in controlled-flora Swiss cheese (28), the ability of these organisms to generate fla...

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Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, including modulation of bifidobacteria, colon epithelial cell proliferation and apoptosis, and intestinal inflammation, rely on active metabolism in situ. Survival and activity are thus key factors determining its efficacy, creating stress adaptation and tolerance bottlenecks for probiotic applications. Growth media and growth conditions determine tolerance acquisition. We investigated the possibility of using sweet whey, a dairy by-product, to sustain P. freudenreichii growth. It was used at different concentrations (dry matter) as a culture medium. Using hyperconcentrated sweet whey led to enhanced multistress tolerance acquisition, overexpression of key stress proteins, and accumulation of intracellular storage molecules and compatible solutes, as well as enhanced survival upon spray drying. A simplified process from growth to spray drying of propionibacteria was developed using sweet whey as a 2-in-1 medium to both culture P. freudenreichii and protect it from heat and osmotic injury without harvesting and washing steps. As spray drying is far cheaper and more energy efficient than freeze-drying, this work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness. IMPORTANCEIn this study, we demonstrate that sweet whey, a dairy industry by-product, not only allows the growth of probiotic dairy propionibacteria, but also triggers a multitolerance response through osmoadaptation and general stress response. We also show that propionibacteria accumulate compatible solutes under these culture conditions, which might account for the limited loss of viability after spray drying. This work opens new perspectives for more energy-efficient production of dairy starters and probiotics.

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