. 2000. Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process ef®ciency. Here, it is shown that¯uorescent stains and¯ow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by¯ow cytometry, populations of rehydrated yeasts. A strong relationship (r 2 0Á99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r 2 r 0Á83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with¯ow cytometry con®rmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of¯ow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.

Four strains of bakers' yeast were analysed for their hyperosmotic responses when in media that mimic conditions occurring in bread doughs. Two of the strains produced strong fermentative activity in medium with low osmotic stress, but produced considerably less ethanol in high sucrose concentration medium. Two other strains produced more similar fermentation activities across the range of media tested. The strains that were inhibited by high sucrose concentration were unable to produce significant amounts of glycerol under hyperosmotic conditions. By contrast, the yeasts that were not inhibited significantly by high sucrose produced a considerable amount of glycerol. The strains that produced significant glycerol exhibited efficient expression of the glycerol‐3‐phosphate dehydrogenase gene GPD1. These novel data on the molecular responses of industrially relevant strains of bakers' yeasts are prerequisite to designing strategies for improving the performance of industrial yeasts in high sugar concentration media.

Strains of Sacchammyces cerevisiae that exhibit varied capacities for accumulation of trehalose were tested for intrinsic thermotolerance. Yeast that accumulated trehalose rapidly in early respiratory phase showed equally rapid attainment of thermotolerance, whereas a strain unable to accumulate trehalose a t this stage of culture showed markedly delayed appearance of thermotolerance. These results were obtained using closely related but nonisogenic diploids and so it is possible that variable factors other than trehalose were responsible for the observed thermotolerance effects. Therefore, a pair of isogenic diploid 5. cerevisiae strains was generated to facilitate further testing of whether trehalose functions in intrinsic stress tolerance. Both isogenic strains inherited a partially reverted c i f l phenotype, designated CPR, from the trehalose-deficient progenitor that had been used in construction of the non-isogenic strains. The CPR phenotype permitted growth on glucose but not accumulation of trehalose, indicating that not all cifl-related deficiencies were suppressed in the CPR strains. However, one of the isogenic CPR pair was c i f l l c i f l and failed to accumulate trehalose, whilst the other was cifllCIF1 and was able to accumulate this sugar. The trehalose-prof icient strain showed intrinsic stress tolerance whereas the trehalose-def icient strain was sensitive to heat stress during early respiratory growth. These results suggest that one or more functions of CIFl, not operating in the cifl/cifl(CPR) strains, are important for intrinsic thermotolerance of yeast in early respiratory phase. When considering these results with those of others whose work has indicated a role for trehalose in protection of proteins and membranes, it is reasonable to hypothesize that the trehalose deficiency associated with cifl/cifl(CPR) strains could be a key factor in their intrinsic thermosensitivity. However, if this is the case the importance of trehalose, relative to other stress tolerance factors, appears to vary with growth phase and culture status.

The transcriptional responses of the osmotically induced genes ALD2, CTT1, ENA1, GPD1, HSP12 and HSP104, were studied in Saccharomyces cerevisiae strains differing in CIF1 gene function following application of osmotic stress. The CIF1 gene (allelic to GGS1 and TPS1) encodes a subunit of the trehalose synthase complex that affects trehalose synthesis. Recent work has implicated this gene in various signalling events in the cell, including transcriptional response to heat-shock treatment. Because many genetic factors can influence S. cerevisiae osmoresponse, we have compared the expression of osmotically induced genes and glycerol production in isogenic strains differing only in functionality of CIF1, growing logarithmically on galactose medium. When cultures were exposed to 0.8 M NaCl or 1.5 M sorbitol the cif1 strain showed greatly reduced transcription of osmotically induced genes compared to the wild type. These treatments did not affect viability of the yeast strains. Treatment with 0.3 M NaCl produced no significant differences in transcription of these genes in CIF1 or cif1 strains. Treatment with 0.6 M sorbitol induced small but reproducible differences, with gene expression higher in the CIF1 strain compared to the cif1 mutant. When cultures were treated with 0.3 M NaCl or 0.6 M sorbitol for 1 h, glycerol production was similar for both strains, but after 3 h of the same treatment, total glycerol production was higher in the CIF1 strain. When cultures were treated with 0.8 M NaCl for 3 h, the wild type strain produced more glycerol than the mutant strain. Both strains produced similar amounts of glycerol following exposure to 1.5 M sorbitol for 3 h, although the wild type strain showed enhanced ability to retain glycerol inside the cell. The results are discussed in the context of the possible role that the CIF1 gene product has in response to osmotic stress.