Hybridization of Bakers' yeast by the rare-mating method to improve leavening ability in dough Yuji Oda and Kozo Ouchi

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan

Saccharomyces cerevisiae MA233 & a diploid strain that is available for breadmaking by the frozen- dough method but it has lower leavening ability than commercial Bakers' yeasts in dough with no sugar or a small amount of sugar. To improve baking quality, MA233 was hybridized with YOY34, a haploid derived from a commercial baking strain, by the rare-mating method. The hybrid YOY671 exhibited MAL-constitutive phenotype of a-glucosidase, resulting in an increased rate of C02 produc- tion from the dough without addition of sugar. YOY671 had a higher leavening ability in the dough containing 5% to 30% sucrose (based on the weight of flour) than MA233. Freeze-thaw resistance of YOY671 in dough was higher than that of YOY34, but less than that of MA233.

Keywords: Saccharomyces cerevisiae; leavening ability; constitutive maltose fermentability; rare mating

Introduction

The leavening ability of sponge dough, which is pre- pared without addition of sugar, is one of the most important properties in Bakers' yeast, and is closely related to its maltose fermentability. 1 A high CO2 pro- duction in sponge dough requires the yeast to possess both MAL-const i tu t ive (MAL ~) genes and high glyco- lytic activity. 2

We have selected 11 yeast strains available for fro- zen dough from a part of our culture collection) These strains have lower leavening ability in dough with little or no sugar than commercial Bakers' yeasts. MA233, the best strain among these yeasts, is still used in a limited number of bakeries.

In the present experiments, MA233 was hybridized with a haploid isolated from a commercial baking strain to confer the abilities of both constitutive maltose fermentation and higher dough leavening.

Materials and methods

O r g a n i s m s

Two strains of Saccharomyces cerevisiae were used for the hybridization. One was MA233, which was pre-

Address reprint requests to Dr. Oda at Tokyo Research Laborato- ries, Kyowa Hakko Kogyo Co., Ltd., Machida-shi, Tokyo 194, Japan Accepted for publication 15 January 1990

viously selected for breadmaking by the frozen-dough method. 3 From the cell size and sporulation ability of each isolate from single spores, MA233 seemed to be a homothallic diploid (MATa/a HO/HO) . Before hybrid- ization, MA233 was converted to the respiratory defi- cient mutant, MA233-RD, which had lost the ability to sporulate and could not grow on glycerol as a sole carbon source, by treatment with ethidium bromide. 4 The other strain was YOY34 (MATa M A L l c M AL3g SUC2 SUC4 gal l ) , a galactose nonfermenting haploid isolated from a commercial baking strain, s

M e d i a

The growth media used were YP (1% yeast extract, 2% peptone) supplemented with either 2% glucose (YPD), 3% glycerol (YPGIy), or 2% galactose (YPGal). The sporulation medium employed was SPO (0.1% yeast extract, 1% potassium acetate, 0.05% glucose). The media were solidified by the addition of 2% agar if necessary. For the determination of leavening ability, yeast cells were cultured in a molasses medium con- taining 3% sugar as cane molasses, 0.193% urea, and 0.046% KHzPO4. l

C o n s t r u c t i o n o f hybr id

Hybridization was carried out as shown in Figure 1. MA233-RD and YOY34 were mixed and cultured in YPD medium. A portion of the mixed culture was in- oculated into YPGly and YPGal, successively, and

© 1990 Butterworth-Heinemann Enzyme Microb. Technol., 1990, vol. 12, December 989

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MA233

I EtBr

MA233-RD

I YOY34

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YOY671

Figure 1 Procedure of hybridization between MA233 and YOY34. YPD, 1% yeast extract, 2% peptone, and 2% glucose; YPGly, 1% yeast extract, 2% peptone, and 3% glycerol; YPGal, 1% yeast extract, 2% peptone, and 2% galactose

spread on YPGIy agar. Colonies appearing on YPGly agar were picked up and tested.

Genetic analysis

Standard techniques of sporulation, dissection, and te- trad analysis were used. 4 Contour-clamped homoge- neous electric field gel electrophoresis (CHEF) was performed by the method of Chu et al. 6 with CHEF- DRII (Bio-Rad Laboratories, Richmond, CA).

Enzyme assay

The cells obtained from the molasses medium were incubated in the liquid medium containing maltose as a main carbon source, and the a-glucosidase activity of these cells was followed for up to 4 h.

Leavening ability

The leavening ability of yeast cells was determined as the volume of evolved gas from dough according to the methods described elsewhere unless otherwise stated. 1

Frozen dough was prepared by the following meth- ods. All ingredients for white bread dough, consisting of 100 g of flour, 5.0 g of sucrose, 2.0 g of salt, 0.66 g of yeast cells as dry matter, and 62 ml of water, were mixed for 2 min by a National Mixer (National Mfg. Co., Lincoln, NE) and kept 30°C for 2 h. The dough, which was punched and divided into several pieces based on 10 g of flour weight, was molded to 5-mm

thickness by hand, frozen at -20°C, wrapped with polyethylene film, and stored at -20°C. After every 7 days, the frozen dough was thawed in a proofing cabi- net at 30°C for 15 min and put through a fermentation for 2 h at 30°C to determine the leavening ability.

Trehalose content

Trehalose was extracted from the cells cultured in the molasses medium with 0.5 M trichloroacetic acid and determined by anthrone?

Results

Isolation o f rare-mated hybrids

MA233 sporulated well on SPO agar, and the asci were dissected. All the isolates from single spores had less leavening abilities in the dough after freezing and stor- age at -20°C than the original strain. The desirable properties of MA233 for frozen dough might require a set of several genes. Since classical cross-breeding or hybridization seemed to be unavailable to improve baking performance of MA233, the rare-mating method was applied to this strain as shown in Figure I. Two types of colonies, large and small, appeared on YPGly agar in 2 days at 28°C. All of the strains derived from the former type could sporulate on SPO agar and ferment galactose, and one of these was selected as a hybrid YOY671.

Genetic analysis o f YO Y671

YOY671 produced 2 to 3 spores after 3 to 5 days on SPO agar. When the asci were dissected, about 30% of ascospores were germinated. Among 129 isolates de- rived from single spores, galactose fermentation segre- gated 79+ : 50- , and mating type segregated 29 MATa phenotype, 58 MATa phenotype, 4 bisexual, and 38 non-mater. This pattern suggested that at least the chromosomes concerned were heterozygous and tri- somic.

To investigate the constitution of chromosomes without genetic markers in YOY671, chromosomal DNA was resolved by CHEF (Figure 2). The differ- ences of fluorescence among the lanes in Figure 2 were due to the different amounts loaded onto the gels. The electrophoretic mobility of most chromosomes of MA233 and YOY34 was similar but not identical. YOY671 showed the pattern similar to the mixture of chromosomal DNA, which was prepared from the sus- pension containing equal cell numbers of MA233 and YOY34, rather than either of two parents.

The above observations suggested hybrid YOY671 was a triploid derived from diploid MA233 and haploid YOY34.

Characterization o f YO Y671

Figure 3 compares o~-glucosidase activities of YOY671 and its parental strains. The enzyme activity of MA233 remained extremely low for 4 h. The initial activity of YOY671 was lower than that of YOY34, but the subse-

990 Enzyme Microb. Technol., 1990, vol. 12, December

Hybridization of yeast by rare-mating method: Y. Oda and K. Ouchi

~.~ 200 ]

(¢(~ ~ 1 0 0 ]

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Figure 3 Induction of ~-glucosidase by exogenous maltose. The cells obtained from the molasses medium were incubated in the liquid medium containing maltose as a main carbon source, and the ~-glucosidase activity of these cells was fol- lowed for up to 4 h. ([]) MA233; (e) YOY34; (=) YOY671

Figure 2 Electrophoretic karyotypes of yeasts. The mixture of chromosomal DNA was prepared from the suspension contain- ing equal cell numbers of MA233 and YOY34

quent activity was induced by exogenous maltose to reach similar level at 4 h. The constitutivity of M A L l c in YOY34 was partially dominant over the in- ducibility of M A L in MA233. Figure 4 shows the fer- mentation curves depicting the rate of CO2 production (milliliters per 5 min) from the dough without addition of sugar. The curve for YOY671 did not show two distinct peaks that were apparent in the curve for YOY34, but had a large increase based on the fermen- tation of maltose. Rapid induction of c~-glucosidase by maltose reflected the increased leavening ability in such dough.: The rate of CO2 evolution of MA233 was lower than those of other two strains, though it in- creased gradually. The leavening abilities in doughs containing from 5% to 30% sucrose (based on the weight of flour) are shown in Figure 5. The formulation of these doughs simulated that of various white bread and sweet goods. A marked difference was observed between MA233 and YOY34. YOY67! had an activity intermediate between these strains in the doughs con- taining less than 20% sucrose, while it had higher ac- tivity than the other two strains in the doughs contain- ing over 20% sucrose.

7

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Figure 4 Time course change of C02 production rate from sponge dough. The rate of CO2 gas production from the sponge dough based on 20 g of four was recorded automatically with the Fermograph (Atto Co. Ltd., Tokyo, Japan) at 30°C. Symbols are the same as in Figure 3

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Figure 6 Leavening ability in dough after freezing and storage at -20°C. Symbols are the same as in Figure 3

The leavening abilities of three strains in the frozen dough were more or less reduced during the storage period (Figure 6). YOY34 was highly susceptible to freezing injury and its activity decreased rapidly after 14 days. The freeze-thaw resistance of YOY671 was lower than that of MA233, but higher than that of YOY34. The trehalose content (mg g-1 dry basis) was 150 in MA233, 32 in YOY34, and I12 in YOY671.

Discussion Several methods have been developed to introduce constitutive maltose fermentability into the yeast.

MAL-constitutive mutants were isolated by the re- version of maltose nonfermenting mutant or the direct mutation of MAL-inducible strains.7 MAL-constitutive strains were readily distinguished from MAL-inducible ones by the utilization of e-methyl glucoside or melezi- t o s e . 7,8

Recombinant DNA techniques have been applied in this field. 9 Osinga et al.10 constructed integrating plas- mids containing genes coding maltose permease and maltase under strong promoters. In the transformants by these plasmids, both enzyme activities increased severalfold, resulting in enhanced CO2 production from dough without addition of sugar. Although the presence of bacterial DNA is generally considered un- desirable when yeasts are used in the manufacture of food and beverage, 11,~2 some methods for stable gene transfer in most industrial strains of S. cerevisiae with- out incorporation of bacterial plasmid sequences have been provided. 11,12

To improve baking quality, MA233 should acquire not only constitutive maltose fermentability but also high glycolytic activity in dough, z Glycolysis, which causes dough leavening and ethanol production, con- sists of a sequence of 12 reactions.13 The enzymes with the lowest activity were considered to limit the rate of overall reaction. ~3 However, overprodution of eight

different enzymes in glycolysis had no effect on the rate of fermentation. TM An increase in the glycolytic flux might need simultaneous overproduction of multi- ple glycolytic enzymes, but this would hardly be possi- ble by the current recombinant DNA techniques.

To overcome these problems, we adopted hybrid- ization with a haploid derived from a commercial bak- ing strain by the rare-mating method, which was easier than protoplast fusion. Rare mating is usually em- ployed when normal hybridization procedures are in- effective.~5 Since mutation of YOY34 might impair the essential properties required for Bakers' yeasts, the gall phenotype which was contained intrinsically in this strain was used as a selectable maker.

As shown in Figure 3, the resulting hybrid YOY671 seemed to have the MAL-constitutive phenotype by the included M A L l ' derived from YOY34, but the rea- son why the expression of a-glucosidase activity was delayed was not explained by the present work.

MA233 was one of the strains that were selected for bread-making by the frozen-dough method? Since these strains possessed a common ability to accumu- late a higher trehalose content than did the commercial baking strain in the molasses medium, we assumed this property was related to the higher viability of the selected strains in frozen dough? In YOY671, freeze- thaw resistance in dough was retained but not fully developed. This corresponded to the intracellular tre- halose content of YOY671, which accumulated more than YOY34 and less than MA233 in the molasses me- dium.

Spencer et al.16 hybridized S. cerevisiae and Zygo- sacchromyces rouxii by protoplast fusion and obtained the segregants from several asci formed by these hy- brids. Some of these segregants had a much higher leavening ability than both the original hybrid and a commercial Bakers' yeast. However, this was not the case in the present experiments, because none of 129 isolates derived from single spores had better proper- ties than YOY671 (data not shown). The expression of leavening ability and freeze-thaw resistance in dough might be highly complex in YOY671. A contribution of mitochondrial genome derived from YOY34 to the baking performance of YOY671 ~v would not be ex- cluded.

Acknowledgement We thank K. Ishii for technical assistance.

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