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Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.

Tracing experiments were carried out to identify volatile and nonvolatile l -methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens , both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the l -methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and α-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of l -methionine catabolism. However, the l -methionine degradation pathways differ in the first steps of l -methionine degradation. l -Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens , whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum . Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens .

International audience; Three cheese-ripening yeasts Kluyveromyces lactis, Geotrichum candidum and Debaryomyces hansenii were grown in a cheese slurry either individually or in association with the cheese-ripening bacterium Brevibacterium linens. Alcohols, aldehydes and esters were produced by the yeasts, and appeared in the early stages of ripening, whereas volatile sulphur compounds (VSCs) were produced late during the ripening process. The production of VSCs was greatly improved when the yeasts D. hansenii and K. lactis were associated with B. linens. In contrast to G. candidum, these yeasts efficiently neutralised the cheese slurry, which promoted the growth of B. linens. Our results gave evidence of metabolic synergies between the yeasts and B. linens. For instance, generation of S-methyl thioacetate was improved when a yeast that was able to produce esters and thus to accumulate acyl CoA was associated with a bacterium that was able to form large amounts of methanethiol.

International audience; The aromatic potential of various cocultures of yeasts, Brevibacterium linens and lactic acid bacteria (LAB) was studied in cheese-based medium. Three yeasts (Debaryomyces hansenii, Geotrichum candidum and Kluyveromyces lactis) were cultivated in association with B. linens, in the presence or in the absence of LAB - added as the commercial lactic acid starter Flora Danica®. Various parameters were analysed such as aroma compound production, the growth of each microorganism and lactose/lactate degradation. All tested yeasts could grow in all the associations regardless of the presence or the absence of LAB. LAB enhanced the growth of B. linens in D. hansenii associations, but they reduced B. linens' growth when associated with K. lactis. When cultivated alone, LAB produced very few aroma compounds and in lesser amount than the yeast-B. linens associations. In pure cultures of LAB, ethanol was the major volatile compound, and only scanty amounts of other volatile compounds were produced. The K. lactis-B. linens association exhibited the most diversified aroma compound profile with high quantities of S-methyl thioacetate and ethyl acetate. LAB promoted the synthesis of volatile sulphur compounds in this association.; Contribution de plusieurs associations microbiennes à l'affinage d'un milieu fromager. Le potentiel aromatisant de plusieurs co-cultures constituées de levures, de Brevibacterium linens et de bactéries lactiques (BL), a été étudié dans un milieu fromager. Trois levures (Debaryomyces hansenii, Geotrichum candidum et Kluyveromyces lactis) ont été cultivées en association avec B. linens, avec ou sans BL - ajoutées sous forme du levain lactique Flora Danica®. Plusieurs paramètres ont été analysés parmi lesquels la production de composés d'arôme, la croissance de chaque micro-organisme et la dégradation du lactose/lactate. Toutes les levures étudiées se sont développées que ce soit en présence ou en l'absence de BL. La présence de BL a favorisé la croissance de B. linens dans les associations avec D. hansenii, alors que la croissance de B. linens s'est trouvée réduite dans les associations avec K. lactis. Lorsqu'elles ont été cultivées seules, les BL ont produit qualitativement et quantitativement moins de composés d'arôme que les associations levures- B. linens. Les cultures pures de BL ont produit majoritairement de l'éthanol et seulement de très faibles quantités d'autres composés volatils. L'association K. lactis-B. linens a permis d'obtenir la plus grande diversité de composés d'arôme, avec des quantités importantes de S-methylthioacétate et d'acétate d'éthyle. Les BL ont permis d'augmenter les quantités de composés soufrés volatils produites dans cette association.

l-Methionine degradation products and catabolic enzymatic activities involved in methanethiol generation were investigated in Geotrichum candidum GcG. l-Methionine was easily degraded by G. candidum and the transamination product, 4-methylthio-2-oxobutyric acid (KMBA), was found to transiently accumulate. In parallel, considerable l-methionine aminotransferase activity was found in this microorganism. l-Methionine and KMBA demethiolating activities were also detected. The degradation of KMBA corresponded to an overall increase in the production of volatile sulfur compounds. These results show that the transamination pathway is of major importance in the initial breakdown of l-methionine by this cheese-ripening microorganism.

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters – ethylacetate (EA) being the major one – and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA – a common precursor of MTA and EA – but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.