Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs

Abstract

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.

FIG. 1.

Yeast survival in small doughs as a function of the number of freeze-thaw cycles (−20°C and room temperature). L13 (♦) and S47 (•), wild-type industrial strains; AT2 (⋄) and AT25 (○), mutants derived from, respectively, L13 and S47. Standard errors are around 25% of the percent survival values for four independent measurements.

FIG. 2.

Loss of stress resistance during the initiation of fermentation in liquid medium. (A) Freeze stress (1 h at −30°C); (B) heat stress (15 min at 49°C). •, S47; ○, AT25. The standard error is usually between 1 and 10% and a maximum of 15% of the survival values for three independent measurements.

FIG. 3.

Effect of long-term frozen storage on RGC capacity. After 30 min of fermentation in liquid medium, samples were frozen for different time periods at −30°C. •, S47; ○, AT25; (▴) AT28. Standard errors for six independent measurements are around 3.5%.

FIG. 4.

Maintenance of gassing power in small doughs (20 g of flour and 160 mg of yeast) after freezing for different time periods at −20°C. The prefermentation time before freezing was 30 min. Gassing power after 1 day of frozen storage was taken as 100%. Absolute values for gassing power after 1 day of frozen storage (n = 3) were 120 ± 2.3 for S47, 106 ± 1.2 for AT25, and 107 ± 3.6 for AT28 (expressed in milliliters of CO₂ produced in 2 h). •, S47; ○, AT25; ▴, AT28. The standard error never exceeds 10% of the absolute values.

FIG. 5.

Effect of long-term storage on gassing power of doughs made with yeast produced on a pilot scale (20-liter fermentor). Storage was at either −3 or −30°C. •, S47; ○, AT25; ▴, AT28. Standard errors (n = 3) vary between 7.5 and 37.5 ml of CO₂.

FIG. 6.

Flow cytometric analysis of cellular DNA contents of S47 and AT25. The first peak represents cells in G₁, and the second peak represents cells in the G₂ phase of the cell cycle.

FIG. 7.

Freeze stress resistance (determined as RGC after freezing) of tetraploidized AT25. After 30 min of fermentation in liquid medium, samples were frozen for 1 day at −30°C. The strains used were S47 (tetraploid parent, a/a/a/α); AT25 (diploid a/α derived from S47); AT25^aa and AT25^α/α (diploid strains derived from AT25 through mating type switch); TAT25-1, TAT25-2, and TAT25-3 (tetraploid strains [a/a/α/α] obtained by mating of AT25^aa with AT25^αα). A representative result is shown. Compared to S47, the RGC of AT25 is 1.85 ± 0.10 (n = 2) times higher, the RGC of AT25^aa is 2.24 ± 0.02 (n = 2) times higher, the RGC of AT25^α/α is 1.39 ± 0.23 (n = 2) times higher, and the RGCs of the three tetraploid strains together are 1.31 ± 0.07 (n = 5) times higher.