Henry-Eric Spinnler

Production of sulfur compounds by several yeasts of technological interest for cheese ripening

Several yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, and Saccharomyces cerevisiae, were studied for their ability to generate volatile sulfur compounds. These yeasts were cultivated on a synthetic culture medium supplemented with a biosynthetic precursor. With S-methylmethionine, dimethylsulfide (DMS) was the major sulfur compound produced. l-methionine promoted the synthesis of a wider spectrum of volatile sulfur compounds, methanethiol (MTL), DMS, dimethyldisulfide (DMDS), dimethyltrisulfide (DMTS) and S-methylthioacetate (MTA). Enzymatic activities possibly involved in sulfur compound synthesis were also investigated. Important l-methionine-transaminase activities, and also l-methionine-demethiolase activities were detected in cellular extracts. Their possible role in the generation of sulfur compounds and related biosynthetic pathways are also discussed.

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

Abstract. 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.

Identification of a powerful aroma compound in munster and camembert cheeses: ethyl 3-mercaptopropionate.

With the view to investigate the presence of thiols in cheese, the use of different methods of preparation and extraction with an organomercuric compound ( p-hydroxymercuribenzoate) enabled the isolation of a new compound. The analysis of cheese extracts by gas chromatography coupled with pulse flame photometry, mass spectrometry, and olfactometry detections led to the identification of ethyl 3-mercaptopropionate in Munster and Camembert cheeses. This compound, described at low concentrations as having pleasant, fruity, grapy, rhubarb, and empyreumatic characters, has previously been reported in wine and Concord grape but was never mentioned before in cheese. A possible route for the formation of this compound in relation with the catabolism of sulfur amino acids is proposed.

Production of γ-decalactone and 4-hydroxy-decanoic acid in the genus Sporidiobolus

Within the genus Sporidiobolus, S. odorus has been widely reported as having the ability to convert castor oil or its derivatives to γ-decalactone, but other species have also shown potential for lactone production. In this work, the bioconversion of ricinoleic acid methyl ester to γ-decalactone was studied with four species of Sporidiobolus: S. salmonicolor, S. ruinenii, S. johnsonii, and S. pararoseus. With 4.1 g/l of ricinoleic acid methyl ester, only S. salmonicolor and S. ruinenii were able to produce γ-decalactone (12 and 40 mg/l respectively). S. johnsonii and S. pararoseus did not produce any lactone in spite of consuming the precursor. The four strains showed different sensitivities to lactone. Between S. salmonicolor and S. ruinenii, the latter was the best biocatalyst. During four successive batch cultivations in a 7-l bioreactor, 5.5 g/l of γ-decalactone was produced with S. ruinenii in each 10-d run. This was essentially due to its ability to produce the open form of γ-decalactone (i.e., 4-hydroxy-decanoic acid), which is far less toxic than the lactone.

Controlled production of camembert-type cheeses: part III role of the ripening microflora on free fatty acid concentrations.

Phenomena generating FFAs, important flavour precursors, are significant in cheese ripening. In Camembert-like cheeses, it was intended to establish the relationships between the dynamics of FFA concentrations changes and the succession of ripening microflora during ripening. Experimental Camembert-type cheeses were prepared in duplicate from pasteurised milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti, and Brevibacterium aurantiacum under aseptic conditions. For each cheese and each cheesy medium, concentrations of FFAs with odd-numbered carbons, except for 9:0 and 13:0, did not change over time. For long-chain FFAs, concentrations varied with the given cheese part (rind or core). K. lactis produced only short or medium-chain FFAs during its growth and had a minor influence on caproic, caprylic, capric, and lauric acids in comparison with G. candidum, the most lipolytic of the strains used here. It generated all short or medium-chain FFAs (4:0-12:0) during its exponential and slowdown growth periods and only long-chain ones (14:0-18:0) during its stationary phase. Pen. camemberti produced more long-chain FFAs (14:0-18:0) during its sporulation. Brev. aurantiacum did not generate any FFAs. The evidence of links between specific FFAs and the growth of a given microorganism is shown.

Dual influence of the carbon source and l-methionine on the synthesis of sulphur compounds in the cheese-ripening yeast Geotrichum candidum

The effect of the carbon source and l-methionine on the ability of Geotrichum candidum to produce volatile sulphur compounds (VSC) was studied. This yeast was cultivated in a synthetic medium supplemented with various carbon sources and l-methionine at different concentrations. Both glycerol and glucose significantly increased VSC production by G. candidum. Unlike the effect on the l-methionine- and 4-methylthio-2-oxobutyric acid-demethiolating activities, the supply of a carbon source had a dramatic effect on the activity of aminotransferase, a key enzyme in l-methionine catabolism. An increase in the initial concentration of l-methionine resulted in a rise in the production of sulphur compounds (VSC, 4-methylthio-2-oxobutyric acid) but had limited effect on l-methionine-catabolising enzyme activities. Evidence for the existence of a dual effect of the carbon source and l-methionine on VSC biosynthesis was obtained in this study.

Growth and colour development of some surface ripening bacteria with Debaryomyces hansenii on aseptic cheese curd.

The growth of five bacteria isolated from red-smear cheeses, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variabile, Microbacterium gubbeenense and Staphylococcus saprophyticus in mixed cultures with Debaryomyces hansenii on aseptic model cheese curd at 10 and 14 degrees C was investigated. At both temperatures, C. casei and Micro. gubbeenense had a longer lag phase than C. variabile, Brevi. aurantiacum and Staph. saprophyticus. In all cultures, lactose was utilised first and was consumed more rapidly at 14 degrees C than at 10 degrees C, i.e., 6 d at 14 degrees C and 10 d at 10 degrees C. This utilisation coincided with the exponential growth of Deb. hansenii on the cheese surface. Lactate was also used as a carbon source and was totally consumed after 21 d at 14 degrees C and approximately 90% was consumed after 21 d at 10 degrees C regardless of the ripening culture. Small differences (<0.5 pH unit) in the surface-pH during ripening were noticeable between ripening cultures. Differences in the colour development of the mixed cultures with the yeast control were only noticeable after 15 d for Brevi. aurantiacum and after 21 d for the other bacteria. Regardless of the organisms tested, colour development and colour intensity were also greater at 14 degrees C than at 10 degrees C. This study has provided useful information on the growth and contribution to colour development of these bacteria on cheese.

Metabolism of phenylalanine and biosynthesis of styrene in Penicillium camemberti

The occurrence of styrene in food may be an important aroma defect (celluloid odour), even at very low concentrations (Miltz et al. 1980) causing consumer rejection and is therefore a problem for the food industry. We examined the biosynthetic pathway leading to styrene formation by Penicillium camemberti using labelled compounds. As styrene is strongly hydrophobic and volatile, we first had to develop a continuous extraction process. Using resins XAD2 it was reasonable to suspect phenylalanine (Phe) as the precursor. The addition of Phe marked with 13C on the ring provokes the accumulation of labelled styrene. The enzyme activities involved were also tentatively measured. Styrene appears to be synthesized from phenylalanine by phenylalanine ammonia-lyase activity followed by a decarboxylation catalysed by a cinnamic acid decarboxylase.

Suprathreshold intensity and odour quality of sulphides and thioesters

Abstract Odour intensity ratings over a range of concentrations were determined for 13 sulphur compounds, in this case, sulphides and thioesters. Odour intensities were assessed by an olfactory intra-modal intensity matching procedure in which a concentration series of isoamyl acetate was used as a reference intensity scale. The relationship between perceived intensity and concentration for each sulphur compound was approximated by power functions on an individual and panel level. Relationships between equally intense smelling concentrations of sulphur compounds and isoamyl acetate led to power function exponents that ranged from 0.83 (dimethyltrisulphide) to 1.42 ( S -methyl thioisobutanoate). Derived exponents of the suprathreshold intensity functions, obtained through calibration with the parameters of the isoamyl acetate intensity function, ranged from 0.22 to 0.38. Exponents differed significantly between compounds as well as between subjects. Besides, the subjects described, with their own vocabulary, the odour quality of each compound. The odorants differed in the nature and the number of qualities used for their description.