Selection and hybridization of wine yeasts for improved winemaking properties: Fermentation rate and aroma productivity

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 77, No. 4, 428--431. 1994

Selection and Hybridization of Wine Yeasts for Improved Winemaking Properties: Fermentation Rate and Aroma Productivity TAKASHI S H I N O H A R A , * KOTARO SAITO, F U J I T O S H I YANAGIDA, AND SHOJI GOTO

Institute of Enology and Viticulture, Yamanashi University, Kitashin, Kofu, Yamanashi 400, Japan Received 26 July 1993/Accepted 19 December 1993 Three strains out of thirty-one wine yeasts (Saccharomyces cerevisiae) were selected for their good winemaking properties (fermentation rate, tolerance of sulfur dioxide, aroma productivity, wine quality) and genetic markers (KHR killer activity, gaiactose assimilation), and used for hybridization by spore to spore mating. Of the twelve hybrids produced, two, Hy17-108 (RIFY 1001 × RIFY 1067) and Hy41-308 (RIFY 1001 × RIFY 1065), were selected on the basis of a fermentation test. In experimental winemaking the two hybrids demonstrated improved aroma productivity for higher alcohols, aromatic esters and/or fatty acids, while their fermentation rate was nearly the same as that of the parental strains.

Wine yeasts have traditionally been developed by sampling and selecting wild yeasts from grapes, grape musts, wines and stock cultures. Useful yeast strains for winemaking are selected on the basis of the following characteristics: fermentation rate, fermentation at low temperature, sulfur dioxide (SO2) tolerance, high sugar tolerance, low production of volatile acid, flavor production, killer activity, and good wine quality (1, 2). The application of genetic techniques to improve the winemaking properties of wine yeasts has been reported in recent years (2-4), while useful killer wine yeasts have been bred by cell fusion and hybridization techniques for the dominance of starter yeasts in grape musts (5, 6). Enhanced flavor formation by selected and improved wine yeast strains has been investigated with the aim of producing more aromatic and tasty wines; the target properties were the productivities of glycerol (Gly) (7), organic acids (8) and flavor components (9). Higher alcohols (HA) and aromatic esters, which are the principal volatile flavor products of alcoholic fermentation by the yeast, Saccharomyces cerevisiae, contribute to wine odor; isoamyl acetate (iAA) and ethyl caproate are especially important for a pleasant fruity note (11). This paper describes the selection of wine yeasts and their improvement by hybridization for fermentation rate, aroma productivity and wine quality. Parental phenotypes, galactose assimilation and KHR killer activity were used as selection markers of the hybrids. Thirty-one strains isolated and utilized for winemaking in winegrowing regions of the world and kept at the Institute of Enology and Viticulture at Yamanashi University were used (Table 2) (10). The fermentation test was carried out in 300-ml Erlenmeyer flasks containing 200ml of Koshu grape juice (sugar content 20%) at 18°C and 12.5°C with SO2 contents of 0, 100 or 200ppm. Yeast starter was inoculated into the grape juice and the reduction of flask weight due to the release of carbon dioxide was measured during fermentation. Two fermentation rates, for the first half (vf 5-50) and for the whole period (vf 0-99), were calculated with a fermentation curve. After the fermentation, test lots were filtered and used for analysis; alcohol, pH, total

acid (T.A.), volatile acid (V.A.), SO2, Gly, and volatile aroma compounds were analyzed by the usual method (12) and gas chromatography (9). The t-test, a statistical procedure, was performed on the analytical data to evaluate the differences among the averages; the calculations were done according to the PAS Series ver. 3 system (Social Survey Research Information Co. Ltd., Tokyo). Wine quality was evaluated by sensory testing. Analytical results of the fermentation test are given in Table 1. The wine yeasts exhibited different fermentation abilities and analytical parameters. The fermentation rate differed with the yeast 'strain and was influenced by SO2 and temperature; the difference among the strains varied between two to four times. In SO2 tolerance, 31 strains were tolerant to 100 ppm and 19 to 200 ppm. The total SO2 content after fermentation differed with the strains. Aroma components, acetaldehyde (AcH), ethyl acetate (EA), HA, the A / B ratio and Gly, were affected by the fermentation conditions; their behavior was influenced by the growth and the kinetic modification of the yeast cells in response to the environment (11). The wine yeasts were classified into five groups by fermentation rate and SO2 tolerance (Table 2). Sixteen strains were selected from the original 31 on RIFY 1001(W3Y] [KHR+, GAL+]a)

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FIG. 1. Hybridization program of wine yeasts, a) marker: KHR killer activity and galactose assimilation.

* Corresponding author. 428

NOTES

VOL. 77, 1994 TABLE 1. Analytical results of fermentation test Component

Lot

Range 0

Ferment. rateb vf 5-50 (~) :18°C 12.5°C vf0-99 (~) :18°C 12.5°C Alcohol (v/v~) :18°C 12.5°C pH :18°C 12.5°C Total acid (g//) :18°C 12.5°C Total SO2 (ppm) :18°C 12.5°C Free SO2(ppm) :18°C 12.5°C Glycerol (g//) :18°C 12.5°C AcHc :18°C 12.5°C EAc :18°C 12.5°C HAd :18°C 12.5°C A/B ratio e :18°C 12.5°C

Average* SO2 (ppm): 100 200

7.6**fl 8.0*fl 3.2 - 11.8 6.7 5.0**f3 5.1"*f3 NTs 3 . 0 - 7.3 3.4 3.5 3.3 1.7 - 4.7 2.2**f3 2.2**s NT 1 . 2 - 2.7 10.8 10.8 8 . 0 - 11.2 10.7 10.0- 11.5 11.0 *f~ 11.0 *f3 NT 3.20 3.20 3.15- 3.23 3,20 3.18 NT 3.12- 3.28 3.18 6.60 6.60 6.63 5.6 - 7.2 6 . 1 - 7.5 6,68 6.72 NT 26 80**fl 157'*".**n 5 -176 23 74 **fl NT 9 -123 5 7**fl 10**fl.**f2 4 - 17 5 6**fl NT 3 - 10 6.2 6.6*n 6.9**fl 4.8 - 8.6 4.6**n 4.7**f3 NT 3 . 6 - 5.6 26 65**" 119.*fl.**f2 11 -162 29 62**fl NT 9 -104 18 - 80 44 41 48 14 - 60 36*~ 39 NT 121 135**fl 145**ft 78 -225 113 117**f3 NT 60 -197 3.6 - 15.5 7.7 8.0 7.8 9.9*f3 10.2**f3 NT 6.1 - 15.1

a The test was conducted with 31 lots. Interrupted fermentation lots were excluded from the average calculation. Lot numbers for the calculation: 18°C-SO2 0ppm, 12.5°C-SO2 0ppm, 12.5°C-SO2 100 ppm, 31 lots; 18°C-SO2100 ppm, 29 lots; 18°C-SO2200 ppm, 19 lots. b Fermentation rate per day during from 5 to 50 percent (vf 5-50) or from zeto to 99 percent (vf 0-99) sugar consumption. ° Acetaldehyde, Ethyl acetate (mg//). d Higher alcohols: sum of n-propyl, isobutyl and isoamyl alcohols (mg/0.

c Isoamyl alcohols/isohutyl alcohol. f * and ** denote significanceat levelsP=0.05 and P=0.01, respectively. Judgement between [fl] SO2 0ppm and SO2 100ppm or 200ppm, [f2]: SO2 100ppm and SO2 200ppm, and [f3]: 18°C and 12.5°C. Not tested. the basis of their fast or moderate fermentation rate and their a r o m a productivity such as low production of AcH and EA, a n d / o r high production of H A and experimental winemaking was carded out with the selected strains using Koshu grape must (on a 7-I scale) in 1989 and 1990. Six strains, RIFY 1001 (W3), RIFY 1001 (W3Y), RIFY 1046, RIFY 1057, RIFY 1065 and RIFY 1067, were selected from the 16 strains after the analysis and tasting of the experimental white wines. The strains had such properties as good wine quality (RIFY 1001 W3, W3Y), a high fermentation rate (RIFY 1067) and aromatic ester formation ( R I F T 1046, 1057, 1065). The ratings o f the tasting are given in Table 2 (analytical data not shown). Finally, three of the six strains were selected for study o f the improvement of the fermentation rate and a r o m a productivity because they had markers such as K H R killer activity (RIFY 1001) (16) and galactose assimilation ( - ) (Biotype S. bayanus: RIFY 1065, RIFY 1067) (15), as well as good winemaking properties. The improvement program is shown in F i g . l . The spore to spore mating technique (13) was employed for hybridization, as most S. cerevisiae wine yeasts are homothallic (10) and this required an exact pairing of

429

TABLE 2. Wine yeast strains, their grouping and the rating of wine quality Strain (Origin)a Groupc WQd :Strain (Origin) Group WQ RIFYb RIFY 1001 (W3, Japan) A 5 1051(Germany) C -1001 0N3Y, Japan) A 5 1052(France) C 4 1022 (OC2, Japan) B 5 1053(France) B -1027 (U.S.A.) C _e 1054(France) E 3 1028 (U.S.A.) C -1057(South Africa) D 5 1033 (Australia) B -1058(South Africa) A -1035 (Australia) C -1059(South Africa) E -1036 (Australia) C 4 1060(South Africa) C -1038 (France) C 5 1062(Australia) A -1039 (France) C -1065 (Germany) C 4 1040 (Germany) E 5 1067(South Africa) A 5 1042 (Germany) E -1068 (Australia) B 4 1045 (Germany) E -1069(Australia) C -1046 (France) C 5 1070(Australia) B 4 1047 (Germany) A 3 1071(U.S.A.) E 3 1049 (Germany) C -a S. cerevisiae, 31 strains. b Institute of Enology and Viticulture, Yamanashi University, Japan. c Grouping by (i) fermentation rate: vf 0-99 (%/d) and (ii) SO2 tolerance (ppm) at 18°C. A: (i) >3.5, (ii) >200; B: (i) >3.5, (ii) 100200; C: (i) 3.5-2.6, (ii) >200; D: (i) 3.5-2.6, (ii) 100-200; E: (i) <2.6, (ii) 100-200. o Wine quality: 5, very good; 4, good; 3, ordinary. e Not tested.

spores. Seven and five hybrids were obtained from 600 pairings of the crosses RIFY 1001 (Gal +, Khr +) x RIFY 1067 ( G a l - , K h r - ) and of RIFY 1001 (Gal +, Khr +) x R I F T 1065 ( G a l - , K h r - ) , respectively. The hybridization was tested by tetrad analysis and by karyotype analysis (14). The chromosomal D N A patterns of the hybrids were almost identical to those of the parental strains (Fig. 2). Hybrid H y l 7 - 1 0 8 inherited a chromosomal b a n d from the strain RIFY 1001, the b a n d near to chromosome II, and doublet bands from the strain RIFY 1067, chromosomes XV and VII. Hybrid Hy41-308 had similar bands to those of RIFY 1065, except for the bands corresponding to the chromosomes VI and I of RIFY 1001. This evidence suggested the wine yeasts had successfully hybridized. Fermentation testing of the hybrids were performed using Koshu grape juice (on a 200-ml scale) as described above. The 7 hybrids from the cross RIFY 1001 x RIFY 1067 exhibited almost the same fermentation rate as the parental strains, while their S O 2 tolerance were lowered. The 5 hybrids from the cross RIFY 1001 x RIFY 1065 showed an improved rate o f fermentation over that of the parental strain RIFY 1065, while the SO2 tolerance was the same or lower than that of the parental strains (data not shown); it seemed that the fermentation rate was genetically stable and improved by the hybridization o f the wine yeasts, whereas SO2 tolerance was modified and reduced. The a r o m a productivities o f the hybrids were at almost the same levels as those o f the parental strains for AcH, EA, H A a n d 2-phenethyl alcohol (2-Ph), while for iAA, ethyl esters of fatty acids (FE) and fatty acids (FA), their productivities exceeded those o f the parental strains (data not shown). This indicates that H A formation, which is related to a m i n o acid metabolism in yeast cells, was stable in the hybrids, while lipid synthesis a n d / o r m e m b r a n e lipid, which affects F A and ester formation (11, 17), was relatively variable as a

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FIG. 2. Chromosomal DNA patterns of wine yeasts and hybrid strains. Cross Field Electrophoresis: Electroishoresis Unit AE6800 (Atto Corporation). Running conditions: 180 V; 11°C for 30 h; pulse time, 70 s. Gel: 1.5% Seakem GTG agarose (FMC BioProducts); the gel was stained with ethidium bromide. Strains: 1, YNN-295 (size marker); 2, RIFY 1067; 3, RIFY 1001; 4, Hy17-003; 5, Hy17-041; 6, Hy17-087; 7, Hy17-108; 8, Hy17-183; 9, Hy17-281; 10, Hy17-279; 11, RIFY 1065; 12, Hy41-308; 13, Hy41-488; 14, Hy41-558; 15, Hy41-596; 16, Hy41-598. TABLE 3. Analyses of experimental white wines produced by selected wine yeasts and hybrid strains AIc. (v/v %)

T.AP

V.A.

AcH

EA

HA

2Ph (mg//)

iAA

FE

FA

Koshu wines 1991 RIFY 1001 26 RIFYI067 24 Hy17-108 24 RIFYI065 30 Hy41-308 30

11.0 11.0 11.0 11.0 10.6

6.64 6.56 6.43 6.64 6.93

0.41 0.65 0.32 0.41 0.34

26 32 27 28 23

28 59 55 40 40

221 138 223 171 188

25 42 45 36 31

4.6 2.5 4.8 2.1 3.4

2.7 3.2 3.3 4.3 4.5

30.3 34.8 36.1 49.4 56.4

5 4 4 3 4

Koshu wines 1992 RIFYI001 22 RIFYI067 22 Hy17-108 28 RIFY1065 28 Hy41-308 24

11.7 11.8 12.1 12.1 12.0

6.63 7.20 6.69 6.13 7.05

0.48 0.66 0.42 0.42 0.36

4 25 22 15 3

55 62 62 49 42

221 163 240 206 243

25 49 34 37 32

8.4 2.9 8.4 4.1 6.2

3.1 2.6 2.0 3.9 4.2

40.8 34.5 23.8 52.0 53.8

5 4 4 3 4

Chardonnay wines 1992 RIFYI001 18 11.9 RIFY1067 18 11.8 Hy17-108 18 12.0 RIFYI065 18 11.7 Hy41-308 14 12.0

8.17 8.53 8.32 8.36 8.13

0.25 0.36 0.36 0.32 0.21

22 45 37 49 24

61 66 56 46 55

234 182 304 249 230

18 30 26 20 16

12.4 6.2 11.9 5.6 10.0

2.9 1.9 1.5 2.6 3.5

37.0 26.7 18.4 37.2 43.1

5 4 4 4 5

Strain

F.T a (d)

(g//)

WQc

a Fermentation time. b T.A., As tartaric acid; V.A., as acetic acid; FE, ethyl esters of fatty acids*; FA, fatty acids (* sum of caproic, caprylic and capric acids). c See the footnote of Table 2. result o f hybridization. On the basis o f the fermentation test, two hybrids, Hy17-108 and Hy41-308 were selected f r o m the original 12 for their i m p r o v e d fermentation rate and a r o m a productivity properties. Experimental white wines were made with the two hybrids and the parental strains. The fermentation times o f the hybrids were nearly the same as those o f the parental strains, t h o u g h the hybrid: Hy41-308 exhibited a faster fermentation rate than the parental strains in the 1992 winemaking (Table 3). The T . A . content in the wines was almost the same a m o n g all the strains, but the V.A. content in the wines made with the hybrid strains was the same as that o f parental strains or lower. With respect to the a r o m a components, Hy17-108 showed an ameliorated productivity for H A and i A A , but not for F E and F A ; Hy41-308 showed an ameliorated productivity for

FE, F A and i A A , but not for H A . The white wines o f hybrid strain Hy17-108 had as g o o d a flavor as the wines o f the parental strains and the wines o f Hy41-308 had an ameliorated aroma. Naturally, more experimental winemaking with different grape varieties is required to evaluate m o r e fully the winemaking properties of the hybrid strains. The study demonstrated the process o f breeding wine yeasts for winemaking properties such as fermentation rate, SO2 tolerance and a r o m a productivity, and the results showed that the hybrid strains have higher productivity o f H A , aromatic esters a n d / o r FA . We would like to thank Mr. H. Sasaki for his assistance in part of this study.

VOL. 77, 1994

NOTES

REFERENCES 1. Goto, S.: Yeasts for winemaking, p. 73-87. In Akiyama, H. (ed.), Utilization and development of yeast. Gakukai-Shuppan Center, Tokyo (1979). (in Japanese) 2. Goto, S.: Characterization and improvement of wine yeasts. Nippon Nogeikagaku Kaishi, 63, 1885-1887 (1989). (in Japanese) 3. Vezinhet, F.: Quelques applications de la g6n6tique des levures en oenologie. Bull. L'O. I. V., 54, 830-842 (1981~; 4. Snow, R.: Genetic improvement of wine yeast, p. 439--459. In Spencer, J. F. T., Spencer, D.M., and Smith, A. R. W. (ed.), Yeast genetics. Spring-Verlag, New York (1983). 5. Hara, S., limura, Y., and Otsukn, K.: Breeding of useful killer wine yeasts. Am. J. Enol. Viticult., 31, 28-33 (1980). 6. Shimizu, K.: Breeding of wine yeast. J. Brew. Soc. Jpn., 83, 667-673 (1988). (in Japanese) 7. Eutaee, R. and Thornton, R.J.: Selective hybridization of wine yeasts for higher yields of glycerol. Can. J. Microbiol., 33, 112-117 (1987). 8. lino, S., Watanabe, S., and Goto, S.: Winemaking property of excessive acid-producing yeasts. J. Brew. Soc. Jpn., 84, 651 (1989). (in Japanese) 9. Shinohara, T.: L'importanee des substances volatiles du vin. Formation et effets sur la qualit6. Bull. L'O. I. V., 57, 606-618 (1984).

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I0. Shinohara, T., Yanagida, F., and Goto, S.: On thallism, ploidy and killer property of wine yeasts. J. Inst. Enol. Vitic., Yamanashi Univ., 27, 7-11 (1992). (in Japanese) 11. Slflnohara, T.: An aspect of aroma formation by the wine yeasts. Nippon Nogeikagaku Kaishi, 63, 1900-1903 (1989). (in Japanese) 12. Murakami, H. (ed.): Standard methods for analysis established by the National Tax Administration Agency. Brewing Society of Japan, Tokyo (1981). (in Japanese) 13. Yanagida, T. (ed.): Laboratory manual for microbiology, p. 324-328. Kodansha-scientific, Tokyo (1990). (in Japanese) 14. Yanagida, F., Oshida, A., Shinohara, T., and Gore, S.: Chromosomal DNA patterns of wine yeasts and wild yeasts by pulsed field gel electrophoresis. J. Inst. Enol. Vitic., Yamanashi Univ., 27, 13-19 (1992). (in Japanese) 15. van der Walt, J.P. and Yarrow, D.: Method for the isolation, maintenance, classification and identification of yeasts, p. 45104. In Kreger-van Rij, N. J. W. (ed.), The yeasts, a taxonomic study, 3rd ed. Elsevier Science Publishers, Amsterdam (1984). 16. Kitano, K., Totsuka, A., and Hara, S.: Killer phenomenon independent of RNA plasmid, p. 1-8. In Hirano, T. (ed.), Biotechnology in yeasts. Gakukal-Shuppan Center, Tokyo (1988). (in Japanese) 17. Peddle, H.: Ester formation in brewery fermentations. J. Inst. Brew., 96, 327-331 (1990).