Aim: The polysaccharide composition of the Saccharomyces cerevisiae cell wall was measured under various growth conditions and was compared with the cell wall structure. Methods and Results: Chemical and enzymatic methods were used to determine levels of b-1,3-glucan and 1,6-glucan, mannan and chitin of the yeast cell wall, whereas the structure/resistance of the wall was qualitatively assessed by the sensibility to the lytic action by zymolyase. It was found that the dry mass and polysaccharides content of the cell wall could vary by more than 50% with the nature of the carbon source, nitrogen limitation, pH, temperature and aeration, and with the mode of cell cultivation (shake flasks vs controlled fermentors). While no obvious correlation could be found between b-glucan or mannan levels and the susceptibility of whole yeast cells to zymolyase, increase of b-1,6-glucan levels, albeit modest with respect to the growth conditions investigated, and to a lesser extent that of chitin, was associated with decreased sensitivity of yeast cells to the lytic action by zymolyase. Significance and Impact of the Study: Our results indicate that the cell wall structure is merely determined by cross-linking between cell wall polymers, pointed out the role of b-1,6-glucan in this process. Hence, this study reinforces the idea that enzymes involved in these cross-linking reactions are potential targets for antifungal drugs.

Cell-wall damage caused by mutations of cell-wall-related genes triggers a compensatory mechanism which eventually results in hyperaccumulation of chitin reaching 20% of the cell-wall dry mass. We show that activation of chitin synthesis is accompanied by a rise, from 1.3-fold to 3.5-fold according to the gene mutation, in the expression of most of the genes encoding enzymes of the chitin metabolic pathways. Evidence that GFA1, which encodes glutamine-fructose-6-Phosphate amidotransferase (Gfa1p), the first committed enzyme of this pathway, plays a major role in this process was as follows. Activation of chitin synthesis in the cell-wall mutants correlated with activation of GFA1 and with a proportional increase in Gfa1p activity. Overexpression of GFA1 caused an approximately threefold increase in chitin in the transformed cells, whereas chitin content was barely affected by the joint overexpression of CHS3 and CHS7. Introduction of a gfa1-97 allele mutation in the cellwall-defective gas1D mutant or cultivation of this mutant in a hyperosmotic medium resulted in reduction in chitin synthesis that was proportional to the decrease in Gfa1p activity. Finally, the stimulation of chitin production was also accompanied by an increase in pools of fructose 6-Phosphate, a substrate of Gfa1p. In quantitative terms, we estimated the flux-coefficient control of Gfa1p to be in the range of 0.90, and found that regulation of the chitin metabolic pathway was mainly hierarchical, i.e. dominated by regulation of the amount of newly synthesized GFA1 protein. In the search for the mechanism by which GFA1 is activated in response to cell-wall perturbations, we could only show that neither MCM1 nor RLM1, which encode two transcriptional factors of the MADS box family that are required for expression of cell-cycle and cell-wall-related genes, was involved in this process.

This study investigated the cancer chemopreventive, the antiradical, and the antiproliferative properties of polysaccharides extracts from cell wall of Saccharomyces boulardii and Kluyveromyces marxianus. β-glucan, mannan, and chitin were also quantified to identify the most important extract responsible for these biological properties. Soluble and insoluble glucans as well as mannoprotein were extracted from cell wall using single hot-alkaline method. Superoxide anion scavenging (antiradical capacity), NAD(P)H: quinone reductase (QR) (EC 1.6.99.2) induction, and antiproliferative assays were done for the evaluation of biological properties of those extracts. The insoluble glucan from S. boulardii revealed the most relevant biological properties by increasing QR activity and exhibiting the highest growth inhibition against colorectal cancer cells. Moreover, high amount of glucan, high glucan/total sugars ratios, and low chitin/glucan ratios were shown to have an impact on enhancing cancer chemopreventive and antiproliferative properties. To our knowledge, this is the first study that demonstrates QR activity by yeast cell wall components in a dose-dependent manner.

Ochratoxin A (OTA) produced by mycotoxigenic fungi (Aspergillus and Penicillium spp.) is an extremely toxic and carcinogenic metabolite. The use of cold plasma to inhibit toxin-producing microorganisms in coffee could be an important alternative to avoid proliferation of mycotoxigenic fungi. Roasted coffee samples were artificially inoculated with A. westerdijikiae, A. steynii, A. versicolor, and A. niger, and incubated at 27 °C over 21 days for OTA production. Samples were cold plasma treated at 30 W input power and 850 V output voltage with helium at 1.5 L/min flow. OTA production in coffee was analyzed by high performance liquid chromatography coupled to a mass spectrometer (HPLC-MS). After 6 min of treatment with cold plasma, fungi were completely inhibited (4 log reduction). Cold plasma reduces 50% of OTA content after 30 min of treatment. Toxicity was estimated for extracts of artificially contaminated roasted coffee samples using the brine shrimp (Artemia salina) lethality assay. Toxicity for untreated roasted coffee was shown to be “toxic”, while toxicity for cold plasma treated coffee was reduced to “slightly toxic”. These results suggested that cold plasma may be considered as an alternative method for the degradation and reduction of toxin production by mycotoxigenic fungi in the processing of foods and feedstuffs.