Roy James Thornton. Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand. Summary. Pure culture wine yeasts vary in ...
Journalof I'~,P~,A I~-It~l
European J Appl Microbiol Biotechnol (1982) 14:159-164
Microbiolo0y and
Biotechnology 9
S p r i n g e r - V e r l a g - 1982
Selective Hybridisation of Pure Culture Wine Yeasts II. Improvement
of Fermentation
E f f i c i e n c y and Inheritance o f S 0 2 Tolerance
Roy James Thornton Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand
Summary. Pure culture wine yeasts vary in their efficiency of conversion of grape sugar to ethanol. Selective hybridisation over three generations gave significant in fermentation efficiency, i.e. from 84% to 93%. Sulphur dioxide tolerance was found to be under the control of dominant polymeric genes. Other wine-making characteristics were monitored during the hybridisation programme and selected hybrid strains had wine-making qualities comparable with those o f the parent strains, but with increased sugar conversion efficiency.
Introduction A major factor in the production of white wines in temperate climates, e,g. New Zealand and West Germany, is the level of sugar present in the must. In poor seasons sugar levels may be too low to produce an acceptable wine and sugar addition may be permitted under controlled conditions. When sugar levels are marginal, i.e. slightly above that at which sugar addition is allowed; then it is important that the yeast is efficient in its conversion of grape sugar to ethanol during fermentation. Ethanol production b y pure culture wine yeasts is strain dependent (Rankine 1955) and should be amenable to manipulation b y genetic techniques. Sulphur dioxide is frequently added to musts to inhibit wild yeasts and bacteria, aid clarification and settling, and prevent undesirable enzyme reactions (for review see Amerine et al. 1980). Wine yeasts are tolerant of relatively high levels of S02 as the result of natural selection or acclimatisation (Schanderl 1959) and it has been suggested that S02 tolerance is genetically based (Hara et ai. 1980). This paper reports the improvement of fermentation efficiency in wine yeast strains b y selective hybridisation while retaining wine-making qualities. It supports the suggestion that
SO2 tolerance is under the control of dominant polymeric genes.
Materials and Methods Yeast Strains. Six pure culture wine yeast strains, R92, R99, R102, R103, R105, and R107 from the Ruakura collection (Thornton and Eschenbruch 1976) and one commercial strain MD26 (Montana Wines, N.Z. Ltd.) were selected as the breeding stock. All strains had previously been successfully used for the fermentation of Riesling grape juice and all are homothallic. One heterothallic haploid yeast strain X2928 3D (IC) provided by Dr. R. K. Mortimer, Yeast Genetic Stock Center, California, U.S.A. was used in the initial hybridisation. Growth Medium. All fermentations were carried out in grape juice from a single pressing of Vitis vinifera vat. Riesling Sylvaner grapes, containing 20% w/v fermentable sugar. Growth Conditions. Duplicate and triplicate fermentations were carried out in 100 ml aliquots of grape juice inoculated with 5 ml of a 24 h culture grown at 30 ~C. Small scale wine-making was carried out in 4 1 aliquots of grape juice inoculated with 20 ml of a 24 h culture grown at 30 ~ Fermentation temperature was maintained constant at 15 ~ and 1 ml samples taken at 14, 21, and 28 days; fermentation was usually complete in 21 days. SO 2 Tolerance. The SO2tolerance of yeast strains was measured by a variation of the method of Hara et ak (1980). 1 x 107 cells of a 36h culture grown at 30 ~ were inoculated into 10 ml of Riesling Sylvaner grape juice and incubated at 22 ~ Sulphur dioxide tolerance was determined as growth of cuRure and evolution of carbon dioxide in 72 hours. Hybridisation Techniques. Routine methods for hybridisation were those described previously by Thornton and Eschenbruch (1976). Assays. Glucose and fructose levels (Bergmeyer et al. 1970) and ethanol yields (Bernt and Gutmann 1970) were determined enzymatically. 0171-1741/82/0014/0159/$01.20
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R. J. Thornton: Selective Hybridisation of Pure Culture Wine Yeasts. II.
Table 1. Ethanol yields of wine yeasts in 100 ml fermentations Wine yeast strain number
Ethanol, % w/v
R92 R99 R102 R103 R105 RIO'I MD26
8.0 7.9 8.0 7.9 8.0 8.1 8.4
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Sulphur Dioxide Levels. Sulphur dioxide content was determined by the aspiration technique (Rankine and Pocock 1970)
Results
A. Fermentation Efficiency Seven pure culture wine yeast gave ethanol yields ranging from 7 . 9 - 8 . 4 % w/v ethanol (Table 1). Strains R 99, R102, and MD26, all homothallic diploids, were sporulated and single spores mated with single heterothallic cells of strain X2928 3D (IC). The resulting hybrid diploid strains HT99, HT102, and HM26 yielded 8.0, 7.7, and 8.2% w/v ethanol respectively. Seventy-three heterothallic haploid segregants, 32 from HT99, 15 from HT102, and 26 from HM26 were isolated and tested for ethanol production, yields ranged from 2.2-8.3% w/v ethanol (Fig. 1). Ten high yielding segregants, five of HT99, two of HT102, and three of HM26, were cross bred to produce fifteen diploid hybrid strains designated R M 3 - 1 7 . The RM hybrids were examined for ethanol yield, spore via-
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Fig. 1. Ethanol yields of 73 haploid segregant strains from hybrids HT99, HT102, and HM26
bility and wine-making qualities (Table 2). The average ethanol yield of the RM hybrids was 8.2% which compared favourably with the 8.0% of the original seven wine yeast strains. Ascus formation was observed in all the RM hybrids but only eight hybrids produced viable
Table 2. RM hybrid yeast strain characters RM strain number
Parents
Ethanol % w/v
Compact yeast pad
Spore viability
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
HM26 HT99 HM26 HT99 HM26 HT99 HM26 HT99 HM26 HT99 HM26 HT99 HM26 HT99 HM26 HT102 HM26 HT102 HM26 HT102 HM26 HT102 HT99 HT102 HT99 HT102 HT99 HT102 HT99 HT102
8.3 8.5 8.3 8.1 8.2 8.3 8.3 8.1 8.3 8.0 8.3 8.0 8.4 7.7 8.2
+ + + + + + + +
+ + + + + + + +
Taste panel ranking in order of preference acetaldehyde 1st 6fh = ethylacetate 3rd = 2nd 5th 6th = 10th = 10th = 3rd = 10th = 10th = 6th = 9th
R. J. Thornton: Selective Hybridisation of Pure Culture Wine Yeasts. II.
ascospores. A very compact yeast pad was observed in eight hybrids. The yeast pad in the remaining 7 hybrids was acceptable and a clear wine was produced. The performance of the RM hybrids could not be correlated with the parents of the hybrid. The ethanol yield, spore viability, yeast sedimentation and sensory qualities were used to select RM4, RM10, and RM13 for the isolation of haploid segregants. Hybrid RM17 was included as the best cross of HT99 and HT102 parents. The ethanol yields of 123 haploid segregants from the 4 RM hybrid strains ranged from 0-8.9% w/v ethanol (Fig. 2.). The spread of yields gave further support to the suggestion of a multigenic system for fermentation since all the RM hybrids had high ethanol yielding parents. More supportive evidence came from the observation in 6 tetrads of hybrid RM17 of 2: 2 segregation for high: low ethanol production. Twenty high ethanol producing segregants, 9 from RM4, 6 from RM10, 4 from RM13, and 1 from RM17, were used to breed 27 diploid hybrids designated RR. A marked increase was observed in the average ethanol
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9 Fig. 2. Ethanot yields of 123 haploid segregant strains from hybrids RM4, RM10, RM13, and RM17
Table 3. RR hybrid yeast strain characters RR strain number
1 2 3 4 5 6 8 11 12 13 16 17 20 21 22 25 26 27 28 29 30 31 32 34 35 38 42 ND = not done
Parents RM strains
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 10 10 l0 10 10 10 10 4 4 4 13
Ethanol
% w/v
10 4 4 4 4 4 17 10 10 10 13 4 10 10 10 13 10 4 4 4 4 4 17 10 10 10 10
8.4 8,6 8,5 8.6 8.7 8.8 8.6 8.7 8.6 8.6 8.8 8.4 8.8 8.7 8.5 8.7 8.7 8.8 8.4 8.9 8.9 8.9 8.8 8.9 8.6 8.9 8.4
Compact yeast pad
+ + + + + + + + +
% Spore viability
38.8 87.5 97.2 83.3 77.7 86.1 86.1 83.3 72.2 92.5 94.4 91.7 82.5 86.1 97.2 97.2 100 62.5 62.5 67.5 97.5 91.7 77.5 90.0 83.3 86.1 82.5
Taste panel ranking in order of preference ND ND 1st -ND ND ND H2S ND 5th ND 4th ND slight foaming slight foaming ND H2S 6th 3rd H2S 1st = ethylaeetate acetaldehyde ND ND ND ethylacetate H2S
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R. J. Thornton: Selective Hybridisation of Pure Culture Wine Yeasts. II. Table 4. Ethanol yields and evaluation of wines made by commercial and hybrid wine yeasts in small-scalewine fermentations l
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Strain number
Ethanol % w/v
MD26 HM26 RM4 RM13 RM17 RR16 RR20 RR26 RR29 RR35 RR38
8.6 8.6 9.0 9.2 9.0 9.5 9.1 8.7 8.9 9.0 9.0
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