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Growth, metabolism, stress and quiescence

Cropped yeast at the end of fermentation is meta- bolically in a non-growing, stationary phase. As a process, yeast storage is akin to the refrigeration of food insomuch that the lower temperature will markedly slow down metabolism and deterioration in ‘quality. Growth is not a realistic consideration at typical storage temperatures of 2-4°C, Saccharo- myces (as a mesophile) is at or below the minimum temperature for growth. Indeed, even under optimum growth conditions - which yeast storage in beer clearly fails to achieve - doubling times are 50 hours or more at 4°C (Homma et al., 2003; Murata et al., 2006) and 42-63 hours at the higher temperature of 6°C (Walsh and Martin, 1977). This would, in turn, be further extended by the presence of significant levels of ethanol, which adversely impacts on the growth rate of yeast (Sa-Correia and Van Uden, 1983).

Yeast metabolism is muted during storage, as extracellular, assimilable nutrients are few and far between in slurry held in beer or ‘barm ale'. Indeed not surprisingly, the viability of yeast declines during storage. Temperature is key with viability declining progressively more rapidly as storage temperatures increase from between 1°C to 5°C, from 5°C to 10°C, 10°C to 15°C and so on (McCaig and Bendiak, 1985). Overlaid on this, viability is linked to the concentration of extracellular ethanol, and is increasingly compromised above 7% abv (Loveridge et al., 1999). Breakdown of glycogen - the quantitatively important reserve polysaccharide - is also an inevitable consequence of storage (Quain and Tubb, 1982; McCaig and Bendiak, 1985), the rate of which is accelerated by increasing temperature. As with storage in the fermenter cone, glycogen dissimilation via fermentation results in the formation of ethanol, which adds to that present in the barm ale.

Analysis (Gibson et al., 2007) of the various stresses that brewing yeast are exposed to during fermentation and handling identified ‘cold shock' (and ethanol toxicity) as a major concern during yeast storage at 2-4°C. Of course, the size of the shift in temperature and rate of change will vary depending on fermentation temperature, cropping regime, efficacy of cone cooling and in-line cooling on transfer to storage vessel. As noted by Somani et al. (2012), ‘the impact of thermal downshift on brewing yeast has not been the subject of extensive investigation. This is surprising in light of the routine application of low-temperature storage to brewing yeast during industrial handling'. What little work has been published is directional in terms of the yeast storage process, as the insight comes from laboratory experiments with haploid yeast (Homma et al., 2003). Importantly in terms of relevance to storage, this work involved the growth - albeit slowly - of yeast aerobically at 4°C. As is the way with DNA microarray, a host of genes were either up- or down-regulated. Although reflecting growth and not a shift from (say) 30°C to 10°C, cold shock genes were typically (although not exclusively) up-regulated. Tellingly, the authors note that their work ‘suggests that long-term storage without growth would be preferable to growth at low temperature.

In a follow-up study (Murata et al., 2006), yeast was cultured at 25°C then transferred to 4°C after which gene expression was monitored over 6-48 hours. Again, the work was with a haploid laboratory strain and was aerobic. Responses to the downshift in temperature to 4°C include an upshift in the synthesis of phospholipid, trehalose, glycogen and cold shock mannoproteins located in the yeast cell wall. In response to the switch in temperature, the authors conclude that ‘trehalose and glycogen were synthesized for cold tolerance and energy preservation, and the synthesis of phospholipid and mannoproteins was for maintenance of cell membranes and permeability of cell wall.

Stored yeast is in stationary/resting phase or in cell cycle terms G0 or G zero. Such cells are also ‘quiescent', which is described as the ‘most common and, arguably, most poorly understood cell cycle state' (Allen et al., 2006). Quiescent cells are characterized as maintaining viability under growth-arrested conditions, having a decreased metabolic rate, exhibiting greater stress resistance and resuming growth when growth-promoting conditions return (Klosinska et al., 2011). Candidates for growth arresting conditions include starvation, stress and the accumulation of growth-inhibiting metabolites. Of course, such conditions sum-up those found in the stationary phase environment at the end of brewery fermentations.

However, once again, insight is at best directional as studies on quiescence in yeast are invariably in aerobic liquid cultures undergoing nutrient starvation (nitrogen, carbon and less detrimentally sulfur and phosphate) or diauxic shift from the metabolism of glucose to ethanol. Despite this there are numerous fascinating and stimulating insights into the world of quiescence. Although from the related world of an aerobic culture, it has been shown that not all yeast cells in the stationary phase of (non) growth are quiescent (Allen et al., 2006). Using density gradient centrifugation, the population was separated into (i) quiescent dense, unbudded virgin daughter cells, which are able to synchronously reproduce, and (ii) less dense, heterogeneous (budded/unbudded, mother/daughter) non-quiescent cells which lose the ability to reproduce. Further, the quiescent population contained significantly more glycogen and markedly higher viability during 28 days storage.

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