Comparison of volatile sulphur compound production by cheese-ripening yeasts from methionine and methionine-cysteine mixtures.

Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with L-methionine or L-methionine/L-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with L-methionine or L-methionine/L-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when L-cysteine was supplemented, even at a low concentration (0.2 g l(-1)). This effect was due mainly to a significant decrease in L-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by L-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.

The color of Brevibacterium linens depends on the yeast used for cheese deacidification.

The color of smear cheeses (Muenster) is traditionally thought to be due to the bacterial flora, e.g., Brevibacterium linens. This study was carried out to evaluate indirect effects of yeast on the color of B. linens. A 60% cheese medium was desacidified with Debaryomyces hansenii or Kluyveromyces marxianus until pH 5.8 was reached. After inactivation of the yeast and addition of agar-NaCl, B. linens was inoculated on the medium surface and incubated at 12 degrees C from d 2 to 28. For each bacterial biofilm, color was evaluated by L*C*h(degrees) (brightness, chroma, hue angle) spectrocolorimetry. After d 14 (D. hansenii deacidification) and d 21 (K marxianus desacidification), the color level (as a function of all 3 factors) of B. linens biofilms became maximal and remained so until d 28. Debaryomyces hansenii 304 (LGMPA) was less efficient for deacidification than K. marxianus Laf5. However, color intensity (function of chroma only) was higher when D. hansenii was used. The yeast used had an effect on the composition of the cheese medium in relation to production and consumption of metabolites during deacidification. The results concerning color are discussed with respect to this cheese medium composition.

Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus.

The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes. The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)). It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification. Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K. marxianus or by D. hansenii. A second class of volatile compounds can be represented by the ones produced by K. marxianus, which were essentially esters. Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor. A third class can be represented by the ones generated by D. hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor. The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible. Therefore, the global faded-rose odor of D. hansenii DCM can be explained by a high concentration of 2-phenylethanol.

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis.

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.

L-methionine degradation potentialities of cheese-ripening microorganisms.

Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase. The catabolism of L-methionine in these microorganisms is discussed.

Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of smear soft cheese from reconstituted milk: growth and substrate consumption dairy foods.

Experimental cheeses inoculated with Debaryomyces hansenii and Brevibacterium linens were ripened for 76 d under aseptic conditions. Triplicate cheese-making trials were similar as a result of efficient control of the atmosphere. In all trials, D. hansenii grew rapidly during the first 2 d and then slowed, but growth remained exponential until d 10 (generation time around 70 h). Total cell counts were higher than the number of viable cells, and after 10 d they remained around 3 x 10(9) yeast/g of DM. This difference resulted from the nonviability of a fraction of D. hansenii. After d 15, the pH of the rind was close to 7, and B. linens grew exponentially until d 25 (generation time around 70 h). The growth rate subsequently decreased but remained exponential (generation time around 21 d). Cell counts of D. hansenii and B. linens were correlated with the environmental technical conditions. Total D. hansenii counts were also correlated with total B. linens counts. Viable B. linens counts were related to rind lactate, and total counts depended on rind pH, internal lactate, and D. hansenii viable counts. The internal pH of the cheese depended on lactate concentrations, whereas surface pH was related to internal lactose, temperature, and relative humidity. These results suggest a determining role of the diffusion of the carbon sources in the ripening of smear soft cheese.

Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of soft smear cheese from reconstituted milk: protein degradation.

Model smear soft cheeses, prepared with Debaryomyces hansenii and Brevibacterium linens as ripening starters, were ripened under aseptic conditions. Results of the cheese-making trials, in triplicate, were similar and showed similar patterns of protein degradation. In all of the trials, the acid-soluble nitrogen and nonprotein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until d 10. The acid-soluble nitrogen and NPN of the rind then increased to 100 and 18% of total nitrogen, respectively, at d 76. The NH3 concentrations remained low until d 24 and increased until d 70, reaching about 1.8 g of NH3/kg of DM, and then remained constant. The acid-soluble nitrogen and NPN indexes and NH3 concentrations in the inner cheese mass were lower than in the rind. They showed the same evolution, reaching about 18% for acid-soluble nitrogen, 10% for NPN, and 1.5 g of NH3/kg of DM. It was shown that the inner cheese pH and populations of D. hansenii and B. linens have an effect on proteolysis. Viable cell counts of D. hansenii and B. linens were correlated with the environmental conditions and with proteolytic products. The determining role of carbon source and NH3 diffusions on the cheese ripening process were confirmed.