selective hybridization of wine yeasts for higher yields of glycerol

Selective hybridization of wine yeasts for higher yields of glycerol

Department of Microbiology ctnd Genetics, Mussey Urziver.sitj', Pulmerston North, New Zeulctnd

Received July 15, 1986 Accepted October 24, 1986

EUSTACE, R., and R. J. THORNTON. 1987. Selective hybridization of wine yeasts for higher yields of glycerol. Can. J . Microbiol 33: 112-1 17.

Wines that are lacking in body may be improved by the presence of greater amounts of glycerol. Wine yeast strains vary in their ability to produce glycerol. A programme of hybridizing yeast strains while selecting for increased production of glycerol was undertaken. Three generations of hybridization resulted in yeast strains which produced 10- 1 1 g glycerol/L compared with 3.0-6.6 g/L produced by the wine yeast strains of the original breeding stock. Industrial-scale winemaking confirmed the ability of two hybrid strains to produce similar amounts of glycerol to those observed in laboratory-scale fermentations. The activity of .

the enzyme glycerol-3-phosphate dehydrogenase was measured in crude extracts of two breeding stock wine yeasts and in two final generation hybrid strains. The observed activities were lower in the products of the hybridization programme than in the original wine yeast strains. Two alternative explanations are suggested. ( I ) Selective hybridization may select for the alleles of the gene which codes for an alcohol dehydrogenase 1 isozyme which has a lower activity resulting in increased glycerol production. ( i i) Phospholipid synthesis is reduced and the glycerol-3-phosphate, which is the major precursor of phospholipids in yeasts, accumulates as glycerol.

EUSTACE, R., et R. J. THORNTON. 1987. Selective hybridization of wine yeasts for higher yields of glycerol. Can. J. Microbiol 33: 112-1 17.

Les vins qui manquent de corps peuvent etre ameliores par la presence de plus grandes quantites de glycerol. Les souches de levure a vin varient dans leur aptitude ri produire du glycerol. Un programme d'hybridation des souches de levure base sur la selection pour une production accrue de glycerol a etC poursuivi. Trois generations d'hybrides ont conduit ri I'obtention de souches de levure produisant 10- 1 1 g/L de glycerol par comparaison aux 3.0-6.6 g/L produits par les souches des stocks originels d'hybridation. La production de vin B I'echelle industriel a confirme I'aptitude de deux des souches hybrides B produire des quantites de glycerol semblables B celles qui furent obtenues i I'echelle des fermentations en laboratoire. L'activite de I'enzyme glycerol-3-phosphate deshydrogenase a ete mesuree dans des extraits bruts de deux levures B vin issues des stocks d'hybridation, ainsi que chez deux souches hybrides de derniere generation. Les activites observees furent inferieures dans les produits du programme d'hybridation, comparativement ri celles observees chez les souches de levure a vin d'origine. Deux explications alternatives sont suggerees: ( i ) I'hybridation selective peur proceder i la selection pour les alleles du gene qui code pour une isozyme alcool deshydrogenase I , laquelle presente une activite inferieure, ce qui se traduit par une augmentation de la production de glycerol, ( i i ) la synthese des phospholipides est reduite et le glycerol-3-phosphate, qui est le principal pricurseur des phospholipides chez les levures, s'accumule en tant que glyckrol.

[Traduit par la revue]

Introduction The possibility of improving the winemaking properties of

wine yeasts by the application of genetic techniques has been suggested by several researchers (Alikhanyan et al. 197 1 ; Thornton and Eschenbruch 1976; Cummings and Fogel 1978; Takahashi 1978; Snow 1979; Johnston and Oberman 1979; Vezinhet 198 1; S . Fogel, J . W. Welch, and M. Karin. 1982. Genetic engineering in California wine yeast. Abstracts of the 33rd Meeting of the American Society for Enology, Anaheim. CA). Several examples have been reported in recent years. Mutagenesis was utilised to isolate yeast strains suitable for the production of red wines (Avakyan and Ter-Bal yan 1976) and to isolate a leucine auxotrophic mutant which produced lower levels of higher alcohols during fermentation (Rous et al. 1983). Cell fusion and back-breeding have been used to produce wine yeast strains which have killer capability (Hara et al. 1980, 198 1). Recombinant DNA techniques were used in an attempt to improve the malic acid utilising capability of a wine yeast (Williams et al. 1984). Extensive use has been nude of selective hybridization to modify such winemaking properties as foaming (Thornton 1978u, 1978b), fermentation efficiency ( K . J . Thornton. 1980. Grape and Wine Centennial Symposium Proceedings, Davis, CA, June 1980. University of California Press, Davis, CA. pp. 97- 102). elimination of undesirable properites (Eschenbruch et al. 1982), sulphur dioxide tolerance (Thornton 1982), flocculation (Thornton 1985). and hydrogen sulphide production (Romano et a] . 1985).

'Author to whom reprint requests should be addressed Pr~nred In Cdndda 1 Imprlmk au Canada

Glycerol plays an enigmatic role in wine. It is the major fermentation end product after ethanol and carbon dioxide with characteristic levels between 5 and 10 g/L (Pasteur 1860). Glycerol does not contribute to wine aroma as i t is nonvolatile. It has threshold taste levles of 9 g/L in dry white wines and 13 g/L in red table wines (Berget al. 1955; Hinreineretal. 1955), although recent trials suggest a lower threshold level of 5.2 g/L in white wines (Noble and Bursick 1984). Thus, it is rarely detected as an organoleptic factor in wines produced from sound grapes. Wines which have been made from grapes which have been infected by grape moulds may contain up to 20g/L of glycerol. Since glycerol has an oily, heavy character and sweet taste, it has been suggested that glycerol contributes significantly to the body and fullness of wines (Rankine and Bridson 197 1 ), although a concentration of 25.8 g/L has been suggested as the level at which an increase in viscosity can be perceived (Noble and Bursick 1984). Rankine and Bridson's suggestion is supported if "extract" is an indication of the body and fullness of a wine. Extract is the nonvolatile component of wine and is a measure of total soluble solids which include the organic acids, tartaric, malic, lactic, and succinic, and their salts; glycerol, tannins, pigments, higher alcohols, minerals, nitrogenous com- pounds, and sugars. Amerine and Joslyn ( 1 970) suggested good quality dry table wine extract levels of 20 g/L for white wines and 25 g/L for red wines. Thus, glycerol, at characteristic levels for wines made from sound.grapes of 5- 10 g/L, lnay account for 20-50% of the wine extract.

The amount of glycerol produced during wine fermentation is influenced by many factors including temperature, pH, sulphur

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dioxide level, grape variety and ripeness (Rankine and Bridson 197 1 ; Ough et al. 1972), and level of micronutrients (Gentilini and Cappelleri 1959). Yeasts strains produce characteristic amounts of glycerol, and both high and low producers can be identified (Beckwith 1935; Hickinbotham and Ryan 1948; Keilhofer and Wurdig 196 1 ; Rankine and Bridson 197 1 ). The ability of different yeast strains to produce different amounts of glycerol under identical conditions may be related to the activity of the enzyme, glycerol-3-phosphate dehydrogenase, which catalyses the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate (Radler and Schutz 1982).

Some wines from cool-climate winemaking regions of the world have been described as lacking in body and fullness. It has been suggested that this deficiency could be alleviated by an increased level of glycerol in the wine. This paper reports the selective hybridization of wine yeasts resulting in yeast strains with enhanced levels of glycerol production under winemaking conditions and demonstrates the effectiveness of "classical" yeast genetics in the manipulation or modification of a wine yeast strain.

Methods Yeast strains

The breeding stock for the hybridization programme consisted of 1 1 strains of Saccharomyces cerevisiae which included 3 strains from the Australian Wine Research Institute (AWRI 58, AWRI 60, and AWRI 80), 2 from the Ruakura Collection (R 92, R 99), and 1 (MD 26) from Montana Wines, New Zealand. All the above strains are homothallic and have been used in commercial winemaking. The remaining five strains were deveIoped in this laboratory: H 92 and H 99 are diploid strains descended from R 92 and R 99 and are heterozygous for the homothallic character; MTA 2, MTA 4, and MTA 10 are heterothallic haploid strains obtained from MD 26 by hybridization.

Media A single crushing of Vitis vinfera var. Rhine Riesling grape juice

containing 245 g fermentable sugar/L was used throughout the laboratory hybridization programme.

Fermentation Trial fermentations were carried out in duplicate or triplicate in the

following manner. Grape juice (4 mL) was inoculated with 1 mL of a 24-h culture which had been incubated in liquid MYGP medium (0.3% malt extract, 0.3% yeast extract, 0.570 peptone, 1% glucose) at 25°C. After 24 h incubation, 95 mL grape juice was inoculated with the 5-mL culture and incubated semianaerobically (fermentation traps without shaking) at 15°C. Fermentations were usually complete (i .e. , no fermentable sugar remaining) in 20-21 days. Samples were taken and analysed for glycerol, ethanol, and fermentable sugar.

Commercial-scale fermentations of Vitis vinfera var. Rhine Ries- ling grape juice (22" Brix; volume, 10 000 L) were carried out at temperatures between 12 and 18°C by a major New Zealand wine company.

Analysis Glycerol, ethanol, and fermentable sugar were determined enzymat-

ically (Bergmeyer et al. 1970).

Enzyme activity The activity of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) in

crude extract was determined by the method of Nader et al. ( 1 979) and expressed as micromoles NAD produced per minute per milligram protein. Protein was determined by the method of Ehresmann et al. (1973).

Hybridization Conventional techniques were used for hybridization (Mortimer and

Hawthorne 1969). Spore-cell matings and zygote and haploid spore isolations were carried out by micromanipulation.

TABLE 1. Glycerol and ethanol yields of breeding stock wine yeast

Glycerol, Ethanol, Wine yeast g/L g/L

Homothallic diploid MD 26

AWRI 58 AWRI 60 AWRI 80

R 92 R 99 H 92 H 99

Heterothallic haploid MTA 2 MTA 4 MTA 10

TABLE 2. Glycerol production by six diploid yeast strains from the initial hybridization

Diploid Glycerol, hybrid Parent strains g/L

XGL 3 MTA 10XAWRI 60 5.5 X G L 4 MTA 4XAWRI60 5.2 XGL 6 MTA 2X AWRI 80 3.8 XGL 7 MTA 2XAWRI 60 4.6 X G L 8 MTAlOXAWRI80 7.6 XGL 9 MTA 4XAWRI 80 5 .O

Results Screening trials a n d selection of breeding stock yeast strains

Eleven yeast strains were screened for their ability to ferment grape juice to dryness, i.e., no fermentable sugar, and for their yields of glycerol and ethanol (Table 1). When fermentation ended, glucose and fructose were not detected and the yield of glycerol ranged from 3 to 7 g/L and that of ethanol from 72 to 86g/L. Strains AWRI 58, AWRI 60, and AWRI 80, all homothallic diploids, were sporulated and single spores were mated with single cells of the haploid heterothallic strains MTA 2, MTA 4, and MTA 10. Strain AWRI 58 sporulated by the spores were nonviable; this strain was eliminated from the programme.

First-generation diploid hybrid s t ra ins Six diploid strains, XGL 3, XGL 4, XGL 6 , XGL 7, XGL 8,

and XGL 9 , were isolated from the above matings and examined for glycerol yields (Table 2). Seventy-one heterothallic haploid segregants , 28 from XGL 3, 8 from XGL 4, 8 from XGL 6 , 1 5 from XGL 7, 3 from XGL 8, and 9 from XGL 9 , were isolated and tested for glycerol and ethanol production; yields ranged from 2.7 to 10.3 g glycerol/L (Fig. 1) and 66 to 92 g/L for ethanol (Table 3).

Second-generation diploid hybrid s t ra ins Fifteen high glycerol yielding segregants (Table 4) were

crossbred to produce 53 diploid hybrid strains. Some of the segregant strains were included in this crossbreeding in an endeavour to maintain the gene pool as large as possible instead of selecting only on the basis of fermentation performance, e.g., XGL 615 and XGL 914. Glycerol yields of the 53 diploid hybrid strain ranged from 3.6 to 9.3 g/L in fermentations (Fig. 2). The

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NUMBER OF SEGREGANTS

FIG. 1. The production of glycerol by 71 haploid offspring of six diploid strains of the first generation of hybridization.

2 1 1 2 3 4 5 6

NUMBER OF HYBRID STRAINS

FIG. 2. The production of glycerol by 53 diploid yeast strains of the second generation of hybridization.

major New Zealand wine company using strains XGL 50 and XGL 5 1. Strain XGL 50 produced 8.4 g glycerol/L and strain XGL 5 1 produced 8.5 g/L. Both strains produced 12.3% (w/v)

TABLE 3. Range and average glycerol and ethanol production of 1 ethanol and 2 g residual sugar/L was detected. The yields of haploid segregants from six diploid hybrid strains glycerol are similar to the results of the laboratory-scale

fermentations. Glycerol, g/L Ethanol, g/L

Parent diploid No. of strains segregants Average Range Average Range

XGL 3 2 8 6.0 4.8-9.0 85 79-92 XGL 4 8 6.3 5.9-7.0 84 81-87 XGL 6 8 4.6 3.2-6.5 80 66-92 XGL 7 15 5.3 4.2-6.8 84 79-90 XGL 8 3 4.6 4.0-5.2 85 75-91 XGL 9 9 6.1 2.7-10.3 84 74-92

ethanol production ranged from 68 to 91 g/L. The average ethanol yield of 83 g/L was slightly lower than that of the previous generation of haploid strains, i.e., 84g/L. The five highest glycerol yielding strains produced above average amounts of ethanol (Table 5).

Ninety-five haploid segregants, 20 each from diploid strains XGL 50, XGL 51, and XGL 56, 19 from XGL 17, and 16 from XGL 1 1, gave glycerol yields ranging from 5.0 to 12.2 g/L after fermentation (Fig. 3). Ethanol yields also increased (Table 6). Significant increases in the average yields of glycerol, 8.1 g/L, and ethanol, 88g/L, were observed. Some segregants of diploid strains XGL 50 and XGL 5 1 produced as much as 1 1 and 12 g glycerol/L.

Third-generation diploid hybrid strains Nine haploid segregants of strains XGL 50 and XGL 5 1 , six a

mating type and three a mating type, were mated and 16 diploid hybrid strains isolated. The range of glycerol yields by those 16 hybrid strains was quite small, from 9.9 to 1 1 .O g/L, but the average yield showed a marked increase from the 8.1 g/L of the previous generation of strains to 10.6 g/L (Table 7). A decrease from 89 to 84 g/L was observed in the average ethanol yield.

Commercial-scale winemaking trials A commercial-scale fermentation trial was carried out by a

Specific activity of glycerol-3-phosphate dehydrogenase in four yeast strains

The specific activity of the enzyme, glycerol-3-phosphate dehydrogenase, was determined in four yeast strains in order to examine the possible mehanism of increased glycerol production (Table 8). The two yeast strains, AWRI 60 and AWRI 80, from the beginning of the hybridization programme had similar specific activities which were significantly higher than those of strains XGL 78 and XGL 8 1 from the final generation of hybrid strains.

Discussion The results show that the level of glycerol production by wine

yeasts can be increased by selective hybridization. The average glycerol yield was raised from 5.1 g/L in the original five breeding stock strains to 10.6g/L in the final generation of diploid hybrids, an improvement of 108%. Individual diploid hybrids gave yields of up to 11.4 g/L compared with 6.6 g/L of ,the best glycerol producing original wine yeast strain (AWRI 58), an increase of 72%. No obvious segregation pattern with respect to glycerol production could be detected when complete tetrads were available for analysis, as in the case of strains XGL 17, XGL 50, XGL 51, and XGL 56. The variations in performance of haploid segregants of diploid hybrid strains would support the suggestion that glycerol production is under the control of more than one gene, i.e., polygenic.

The average ethanol yield showed a slight increase during the hybridization programme from an original value of 83 g/L to a final value of 84 g/L. Specific selection for ethanol yield was not a major objective; however, segregants with low ethanol yields were eliminated from the hybridization programme. Similar variations in the production of ethanol were observed both in this study and in previous experiments (Thornton 1982), supporting the suggestion that ethanol production is also under polygenic control (Kusewicz and Johnston 1980).

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TABLE 4. Glycerol and alcohol production by 15 segregants selected for further hybridization

a mating type a mating type

Segregant Glycerol, Ethanol, Segregant Glycerol, Ethanol, no. g/L g/L no. g/L g/L

XGL 311 7.5 8 8 XGL 317 XGL 313 7.0 8 2 XGL 3/ 14 XGL 3/10 7.2 84 XGL 412 XGL 615 5.9 77 XGL 413 XGL 917 10.3 8 7 XGL 617 XGL 9/ 1 1 7.1 92 XGL 713

XGL 719 XGL 914

TABLE 5. Five highest glycerol producing second-generation diploid strains and their parents

Glycerol, Ethanol, Parents g/L gIL

XGL 11 XGL 313, XGL 3/14 9.3 85 XGL 17 XGL 3/ 1, XGL 719 7.5 86 XGL 50 XGL 917, XGL 317 8.1 7 7 XGL 51 XGL9/7, XGL3/14 8.8 84 XGL 56 XGL 917, XGL 617 8.3 90

NUMBER OF SEGREGANTS

FIG. 3. The variation in glycerol production by 95 haploid offspring of five diploid strains of the second generation of hybridization.

Radler and Schutz (1982) suggested that glycerol formation is the result of competition between two enzymes, glycerol- 3-phosphate dehydrogenase and alcohol dehydrogenase, for the reduced coenzyme NADH2. An increase in the specfic activity of the enzyme, glycerol-3-phosphate dehydrogenase, would increase the yield of glycerol. However, the specific activity of glycerol-3-phosphate dehydrogenase was higher in the original strains AWRI 60 and AWRI 80 than in the products of the hybridization programme, e .g . , XGL 78 and XGL 8 1. Thus, it must be argued that some other event(s) must be taking place.

TABLE 6. Glycerol and ethanol production by 95 segregants of five second-generation hybrid diploid strains

Glycerol, g/L Ethanol, g/L Parent diploid No. of

strains segregants Average Range Average Range

XGL 11 16 7.8 6.6-8.6 85 82-91 XGL 17 19 7.2 5.8-9.3 85 79-91 XGL 50 20 8.7 5.2-12.2 87 80-99 XGL 5 1 20 9.3 6.6-1 1.6 88 77-97 XGL 56 20 7.5 5.0-10.0 94 85-104

TABLE 7. Glycerol and ethanol production by 16 third-generation hybrid strains

Strain no. Glycerol, Ethanol, XGL g/L g/L

Several forms of alcohol dehydrogenase I (ADH I) are found in Saccharomyces cerevisiae. ADH I is a constitutive enzyme and is not repressed by glucose. Saccharomyces cerevisiae strains which have a low specific activity of ADH I, or are ADH I deficient, produce more glycerol during fermentation (Ciriacy 1975; Johansson and Sjostrom 1984). It is suggested that the selective hybridization programme has selected for a gene which codes for an ADH I isozyme which has low activity. The increase in the ratio of glycerol to ethanol yield during the hybridization programme is consistent with this suggestion (Table 9). However, it is interesting to note that the yield of

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TABLE 8. Specific activity of glycerol-3-phosphate dehydrogenase of four yeast strains

Glycerol produced, Specific activity, Yeast strain g/L kmol NAD produced. min-' . mg protein ' AWRI 60 4.7 AWRI 80 5.6 XGL 78 11.0 XGL 81 11.0

TABLE 9. Changes in the glycerol/ethanol ratio during selective hybridization

Glycerol/ethanol ratio Breeding Yeast No. of

generation description strains Average Range

0 Homothallic diploids Heterothallic haploids

1 Diploid hybrids Haploid segregants

2 Diploid hybrids Selected hybrids Haploid segregants

3 Diploid hybrids

ethanol did not decrease. The net effect is an increase in the efficiency of conversion of fermentable sugar to glycerol.

Reduction of phospholipid biosynthesis may be an alternative explanation of these results. Glycerol-3-phosphate is the major phospholipid precursor in yeast (Henry 1983). If phospholipid synthesis were reduced, e .g . , by selection of enzymes with a lower activity, then glycerol-3-phosphate might accumulate as glycerol. Investigations are currently being conducted to examine both these possible explanations.

The ability of the hybrid strains to produce higher levels of glycerol under large-scale winemaking conditions was confirmed by the trial fermentations carried out with strains XGL 50 and XGL 5 1. The wines made in these fermentations were not

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vers

ity o

f P.

E.I

. on

11/1

9/14

For

pers

onal

use

onl

y.

selective hybridization of wine yeasts for higher yields of glycerol

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