Cider fermentation with three Williopsis saturnus yeast strains and volatile changes

Williopsis saturnus var. subsufficiens NCYC 2728, W. saturnus var. saturnus NCYC 22 and W. saturnus var. mrakii NCYC 500 were used to carry out cider fermentation to assess their impact on the volatile composition of cider. The changes of yeast cell population, °Brix and pH were similar among the three yeasts. Strain NCYC 500 grew best, with the highest cell population of 1.14 × 108 CFU ml−1, followed by strains NCYC 2728 and NCYC 22 (8 × 107 CFU ml−1 and 3.19 × 107 CFU ml−1 respectively). Esters were the most abundant volatiles produced, followed by alcohols. Among the esters, ethyl acetate, 2-phenylethyl acetate, isoamyl acetate, cis-3-hexenyl acetate and hexyl acetate were the major volatiles. The major alcohols were ethanol, isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol. The three Williopsis yeasts transformed volatile compounds during cider fermentation with significant variations in terms of volatile production and degradation. This study implied that fermentation with Williopsis yeasts could result in cider with a more complex yet fruity aroma.     

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.     

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.     

Role of cystathionine beta-lyase in catabolism of amino acids to sulfur volatiles by genetic variants of Lactobacillus helveticus CNRZ 32

Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine beta-lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine.     

Role of Cystathionine β-Lyase in Catabolism of Amino Acids to Sulfur Volatiles by Genetic Variants of Lactobacillus helveticus CNRZ 32

Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine β-lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine.     

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni

Aims:  Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine.
Methods and Results:  Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni .
Conclusion:  This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni . Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed.
Significance and impact of the study:  This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.     

Production of volatile organic sulfur compounds (VOSCs) by basidiomycetous yeasts

Thirty-seven basidiomycetous yeasts belonging to 30 species of seven genera were grown on media containing l-cysteine or l-methionine as sole nitrogen sources with the objective of evaluating volatile organic sulfur compound (VOSC) production. The headspace of yeast cultures was analyzed by the solid-phase microextraction (SPME) sampling method, and volatile compounds were quantified and identified by GC-MS techniques. Ten strains assimilating L-methionine produced the following VOSCs: 3-(methylthio)-1-propanol, methanethiol, S-methyl thioacetate, dimethyl disulfide, dimethyl trisulfide, allyl methyl sulphide and 4,5-dihydro-3(2H)-thiophenone. Production was <1 mgl(-1) except for 3-(methylthio)-1-propanol of which between 40 and 400 mgl(-1) was synthesized. Higher alcohols (isobutyl alcohol, isoamyl alcohol and active amyl alcohol) and esters (ethyl acetate, ethyl propionate, n-propyl acetate, isobutyl acetate, n-propyl propionate, n-butyl acetate, isoamyl acetate, amyl acetate, isoamyl propionate, amyl propionate and 2-phenylmethyl acetate) were also sporadically produced. This is the first report of VOSCs production by basidiomycetous yeasts. Consequently, basidiomycetous yeasts may be considered an interesting new group of microbial VOSCs producers for the flavor industry.     

Identification and characterization of a neutralizing monoclonal antibody for the epitope on HM-1 killer toxin

Killer toxin-neutralizing monoclonal antibody (nmAb-KT) against HM-1 killer toxin (HM-1) produced by yeast Williopsis saturnus var. mrakii IFO 0895 reduces both the killing and glucan synthase inhibitory activity of HM-1. nmAb-KT is classified as IgG1kappa and has been shown to be ineffective against HYI killer toxin produced by the related yeast W. saturnus var. saturnus IFO 0117. To determine the epitope for nmAb-KT, overlapping peptides were synthesized from the primary structure of HM-1. nmAb-KT reacted with peptides P5 (33NVHWMVTGGST43), P6 (39TGGSTDGKQG48) and P7 (44DGKQGCATIWEGS56), which represent the middle region of the HM-1 sequence. P6 reacted most strongly with nmAb-KT. Combined analysis by immunoblotting, surface plasmon resonance (SPR) analysis and yeast growth inhibition assay showed that nmAb-KT recognizes a specific epitope within peptide P6. The K(d) value of nmAb-KT against HM-1 and P6 were determined to be 5.48 x 10(-9) M and 1.47 x 10(-6) M by SPR analysis, respectively. These results strongly indicate that nmAb-KT binds to HM-1 at the sequence 41GSTDGK46, and not to HYI at the same position. The potential active site of HM-1 involved in the killing activity against sensitive yeast is discussed.     

Differentiation of the yeasts Williopsis, Zygowilliopsis and Komagataea by karyotypic and PCR analyses

We used polymerase chain reaction with universal and microsatellite primers, and molecular karyotyping to evaluate the extent of divergence between the genomes of the yeasts currently assigned to the heterogeneous genus Williopsis. Pulsed-field gel electrophoresis of chromosomal DNAs indicates that Zygowilliopsis californica, Komagataea pratensis, Williopsis mucosa, Williopsis salicorniae species and Williopsis sensu stricto complex have clearly different karyotypes. In contrast, the latter six species, Williopsis saturnus, W. beijerinckii, W mrakii, W. suaveolens, W. subsufficiens and W. sargentensis, show similar banding patterns and practically cannot be differentiated on the basis of their karyotypes. The data revealed that a PCR method employing the universal primer N21 is appropriate for the distinction of Williopsis, Zygowilliopsis and Komagataea yeasts. Unique fingerprints were generated with this primer for all 10 species studied while strains of the same species showed nearly identical profiles. The data of UP-PCR are in good agreement with genetic classification and provide support for the species status of the yeasts composing the Williopsis sensu stricto complex. Microsatellite primer (GTG)5 allowing molecular typing of individual strains of the same species may be useful for investigating population structure of the saturn-spored yeasts.     

Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae

Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.     

Metabolism of l-methionine linked to the biosynthesis of volatile organic sulfur-containing compounds during the submerged fermentation of Tuber melanosporum

Tuber melanosporum, known as the black diamond of cuisine, is highly appreciated for its unique and characteristic aroma, which is mainly due to its volatile organic sulfur-containing compounds (VOSCs). In this work, by adding 5 g/L?l-methionine to the fermentation medium, the activities of aminotransferase and α-ketoacid decarboxylase were significantly enhanced by 103 and 250 %, respectively, while the activities of alcohol dehydrogenase and demethiolase were decreased by 277 and 39 %. Then, the six VOSCs, i.e., methanethiol (MTL), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 3-(methylthio)propanal (methional), and 3-(methylthio)-1-propanol (methionol), were first detected in the submerged fermentation of T. melanosporum. These results indicated that the biosynthesis of VOSCs was triggered by aminotransferase and α-ketoacid decarboxylase. The production of methional and methionol increased with the increased concentrations of l-methionine (i.e., 5, 10, 15, and 20 g/L) before day 4 of the culture protocol, and methionol was the major product in the Ehrlich pathway. The production of MTL was significantly decreased after day 4 with a significantly increased DMDS, and DMDS was the major product of the demethiolation pathway. Compared with the demethiolation pathway with a total flux of sulfur of 11.33–24.32 μM, the Ehrlich pathway with a total flux of sulfur of 6,149–10,330 μM was considered the major pathway for the biosynthesis of VOSCs. This is the first report linking the metabolism of l-methionine to the biosynthesis of VOSCs by the Ehrlich and demethiolation pathways during the submerged fermentation of T. melanosporum.     

Influence of Lumbricus terrestris earthworms on the structure of the yeast community of forest litter

The taxonomic structure of yeast communities was studied in forest litter and soil, as well as in substrates transformed by the activity of Lumbricus terrestris earthworms (tree waste from the hole mouths, the gut contents, and coproliths). The activity of L. terrestris has a weak effect on the total yeast abundance but results in substantial changes in their taxonomic composition. The share of ascomycetous yeasts is significantly higher in the substrates associated with the activity of earthworms. The teleomorphic ascomycetes Williopsis saturnus were isolated from the gut contents. The effect of earthworms on the composition of the yeast community in the process of forest litter destruction is more markedly pronounced than seasonal changes.     

The methionine sulphoxide reductase activity of the yeast dimethyl sulphoxide reductase system

Abstract Glycyl- l -methionine sulphoxide and N-acetyl- l -methionine sulphoxide were less effective inhibitors of the dimethyl sulphoxide (DMSO) reductase activity of Saccharomyces cerevisiae NCYC240 than was l -methionine (±)-sulphoxide. Methionine sulphoxide reductase and DMSO reductase activities from crude extracts co-purified over five purification steps and the two activities eluted from columns in exactly the same fractions. Both activities were sensitive to the same inhibitors. It was concluded that: (1) the DMSO reductase activity of S. cerevisiae is a property of a methionine sulphoxide reductase different from that of Black et al. [J. Biol. Chem. 235 (1960) 2910–2916], and (2) free methionine sulphoxide rather than peptidyl methionine sulphoxide is probably the enzyme's true substrate.     

Microbial production of volatile sulphur compounds in the large intestine of pigs fed two different diets

Aims: To investigate the production of volatile sulphur compounds (VSC) in the segments of the large intestine of pigs and to assess the impact of diet on this production. Methods and Results: Pigs were fed two diets based on either wheat and barley (STD) or wheat and dried distillers grains with solubles (DDGS). Net production of VSC and potential sulphate reduction rate (SRR) (sulphate saturated) along the large intestine were determined by means of in vitro incubations. The net production rate of hydrogen sulphide and potential SRR increased from caecum towards distal colon and were significantly higher in the STD group. Conversely, the net methanethiol production rate was significantly higher in the DDGS group, while no difference was observed for dimethyl sulphide. The number of sulphate‐reducing bacteria and total bacteria were determined by quantitative PCR and showed a significant increase along the large intestine, whereas no diet‐related differences were observed. Conclusion: VSC net production varies widely throughout the large intestine of pigs and the microbial processes involved in this production can be affected by diet. Significance and Impact of the Study: This first report on intestinal production of all VSC shows both spatial and dietary effects, which are relevant to both bowel disease‐ and odour mitigation research.     

YtjE from Lactococcus lactis IL1403 Is a C-S lyase with alpha, gamma-elimination activity toward methionine

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.     

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (<Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt.) and white (<Emphasis Type="Italic">Tuber magnatum</Emphasis> Pico) truffles

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated l-methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.     

Marine killer yeasts active against a yeast strain pathogenic to crab Portunus trituberculatus

Some marine yeasts have recently been recognised as pathogenic agents in crab mariculture, but may be inhibited or killed by 'killer' yeast strains. We screened multiple yeast strains from seawater, sediments, mud of salterns, guts of marine fish, and marine algae for killer activity against the yeast Metchnikowia bicuspidata WCY (pathogenic to crab Portunus trituberculatus), and found 17 strains which could secrete toxin onto the medium and kill the pathogenic yeast. Of these, 5 strains had significantly higher killing activity than the others; routine identification and molecular methods showed that these were Williopsis saturnus WC91-2, Pichia guilliermondii GZ1, Pichia anomala YF07b, Debaryomyces hansenii hcx-1 and Aureobasidium pullulans HN2.3. We found that the optimal conditions for killer toxin production and action of killer toxin produced by the marine killer yeasts were not all in agreement with those of marine environments and for crab cultivation. We found that the killer toxins produced by the killer yeast strains could kill other yeasts in addition to the pathogenic yeast, and NaCl concentration in the medium could change killing activity spectra. All the crude killer toxins produced could hydrolyze laminarin and the hydrolysis end products were monosaccharides.     

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine

Aims:  To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma.
Methods and Results:  Probiotic strains ( Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H₂S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs ( P  < 0·05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses.
Conclusions:  Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species.
Significance and Importance of the Study:  This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.     

Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from l-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher l-methionine demethiolating activity, resulting in a direct conversion of l-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.