Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions

Abstract

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.

FIG. 1.

(A and B) Evaluation of yeast gassing power (A) and dough proofing time (B) during frozen storage of large industrial doughs made with AT25 overexpressing AQY1-1 (•) or AQY2-1 (×) and with AT25 (▪) and a control strain containing an empty plasmid (○). The means and standard deviations for two independent tests are shown; the standard deviations for data points without error bars are less than 10% of the values of the points. (C) Temperature in the core of large, industrial doughs during freezing at −35°C with ventilation at Lesaffre Développement. The curve represents the average values for 64 measurements (two doughs, eight horizontal positions, and four vertical positions).

FIG. 2.

Freeze tolerance of AT25 and AT25 overexpressing AQY2-1 in small frozen doughs, either cultured under laboratory conditions and harvested from liquid medium (◊ and ○, respectively) or produced at a pilot scale and resuspended from yeast cake (⧫ and •, respectively), frozen in an ethanol bath at −30°C (A) or in a freezer at −30°C (B). Survival is expressed as the percentage of CFU isolated from two frozen doughs based on the number of CFU in two nonfrozen control doughs. The standard deviations for replicate samples are indicated; the standard deviations for data points without error bars are less than 10% of the values of the points.

FIG. 3.

Temperature courses in cell suspensions during freezing in the ethanol bath (single plastic layer [⧫] or double plastic layer [▪]) and in the freezer at −30°C (single plastic layer [▴] or double plastic layer [×]) (average of two measurements). For comparison, the first 10 min of the temperature course in the core of large, industrial dough during freezing at −35°C with ventilation at Lesaffre Développement (Fig. 1C) also is shown.