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Psychrotolerant Saccharomyces cerevisiae strains after an adaptation treatment for low temperature wine making
Process Biochemistry, Vol. 31, No. 7, pp. 639-643, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00+0.00 ELSEVIER
P! 1:S0032-9592(96)00016-7
Psychrotolerant Saccharomyces cerevisiae Strains After an Adaptation Treatment for Low Temperature Wine Making T. Argiriou, A. Kaliafas, K. Psarianos, M. Kanellaki, S. Voliotis & A. A. Koutinas* Department of Chemistry, Universityof Patras, Patras, Greece (Received 6 November 1995; revised manuscript received 25 January 1996 and accepted 10 February 1996)
Four alcohol resbtant strains isolated from a Greek vineyard plantation were examined for low temperature wine making. The strains found to be of Saccharomyces cerevisiae species. These yeasts were adapted by gradually establishing feeding and temperature conditions during yeast production. After adaptation these yeasts fermented must and prepared wines at relatively low temperatures in the range - 2 to 5°C. More specifically at -2°C the strains Visanto-1 and AXAZ-1 produced wines of 9"9 and 10% v/v alcohol at 150 and 155 days, respectively. Copyright © 1996 Elsevier Science Ltd
INTRODUCTION
tolerant have been made using genetic engineeering. 8 However, publications concerning wine making using free cells at temperatures lower than 10°C, to our knowledge, are absent in the literature. Wine making at temperatures lower than 15°C is not usual on an industrial scale since the productivity is very low. In order to decrease the temperature of fermentation below 10°C while retaining productivity, the psychrotolerant Saccharomyces cerevisiae strains Visanto-19 and AXAZ-11°'~1 were immobilised on mineral kissiris, delignified cellulosic material and gluten, respectively, so that the solid supported biocatalysts reduced the activation energy E~. The aim of this work was to isolate psychrotolerant or psychrophilic yeasts.
Manufacturers know that wines produced at low temperatures develop taste and aroma. Therefore, important products, such as the Greek semisweet vinosanto, the semi-sparkling zitsa and the famous French sparkling champagne are produced by a secondary fermentation during the winter. The improved quality of wines produced by low temperature fermentations can be attributed to the reduction of higher alcohols and to an increase of the proportioin of ethyl acetate in the total volatiles.l'2 As a result, many researchers have focused their interest on low temperature wine making. Fermentations, using free cells and performed in the temperature range 10-15°C with relatively high fermentation times, have been reported. 3-5 Likewise, recent publications reported that at temperatures higher than 25°C, higher alcohols 6 and glycerol7 are increased when compared with fermentations performed at low temperatures. For low temperature wine making attempts to modify cells to be psychro-
MATERIALS AND METHODS Isolation of strains Saccharomyces cerevisiae AXAZ-1 and AXAZ2, isolated previously 12 were examined for their alcohol resistance. AXAZ-3 was isolated by the authors in the Food Technology Laboratory,
*To whom correspondence should be addressed. 639
640
T. Argiriou et al.
University of Patras, Patras, Greece in October 1988. Grapes were collected from a vineyard plantation of Ano Ziria, North Peloponnisos, located at an altitude of about 500 m. Musts with high initial °Be densities (17.5 and 17.7 °Be) obtained from raisin grapes were employed. Musts of 500 ml volume were incubated at 30°C and allowed to ferment in the absence of grape skins. Just before the end of fermentation, 1 ml of fermenting mass, obtained with a sterilised pipette from the bottom, was added to 5 ml liquid medium and incubated at 30°C. The culture was purified by a pour plate technique. The strain Visanto-1 was isolated, as described above from Mandilari grapes collected on the Greek Aegean island Santorini, in September 1991. Culture media The culture medium was sterilised in an autoclave at ll0°C for 15 min and contained 2% glucose, 0.1 (NH4)aSO4, 0"1% KHaPO4, 0"5% MgSO4 and 0.4% yeast extract in distilled water. For Petri dish cultures, 2% agar was added. This synthetic culture medium was used for the preparation of the inocula employed in all fermentations carried out in this work, as well as to obtain biomass for SDS-polyacrylamide gel electrophoresis (PAGE). Identification of strains Identifications of strains were made for Visanto-1 and AXAZ-3 using an API 20 C AUX yeast identification system. To verify the identifications, protein profiles by SDSPAGE,13 and measurements of alcohol dehydrogenase (ADH) activity14 were used for all strains. Fermentations kinetics, mainly at low temperatures, were used to differentiate the strains. ADH activity was measured by a spectrophotometric method. 15 The 'ADH activity unit' term refers to the amount of enzyme that alters the absorbance by 0.001 per min, at a wavelength of 350 n m ) 4 Cell membrane and cell-free extracts were used for SDS-PAGE as described by Thomas et al. ~6
Adaptation treatment All yeast cultures were adapted to growth at low temperatures before low temperature wine making. This procedure consisted of gradually
establishing feeding and temperature conditions during yeast production, similar to those that the strain encounters during wine fermentations at low temperatures. In this case, yeasts were produced in a culture media that contained gradually increasing amounts of kerino must, as the sugar component, rather than glucose. In addition, the culture temperature was kept relatively low (not more than 20°C). Fermentations After the above adaptation procedure, fermentations were performed separately for each strain at 25, 15, 10, 5, 2, 0 and -2°C. For each strain and temperature, 250 ml must containing 20 g/litre wet weight cells were fermented in a glass cylinder and placed in a constant temperature water bath. All musts were obtained from the grape cultivar Kerino and those fermented at temperatures higher than 0°C were adjusted to an initial °Be density by the addition of a commercial concentrated must. Musts used in fermentation performed at - 2 ° C were adjusted with concentrated must without sulphur dioxide. All fermentations were carried out without stirring and the kinetics of fermentation were followed by measuring the °Be density at various time intervals. Analysis of wines After the end of fermentation, wines obtained were analysed for alcohol and residual sugar. Alcohol was determined as alcoholic degrees (milliliters of alcohol per 100 ml of wine) which were obtained after distillation of samples using a Gay-Lussac alcohol meter. Residual sugar was analysed according to the Lane-Eynon procedure. ~7
RESULTS AND DISCUSSION
To study the identity of the isolated yeast, API tests, A D H activity, protein profile by SDSPAGE and fermentation kinetics, mainly at low temperatures, were examined (Figs 1 and 2). Identification by the API test for the strains AXAZ-3 and Visanto-1 indicated by the strains are Saccharomyces cerevisiae with a probability of 97% for AXAZ-3 and 99% for Visanto-1. Protein profiles by SDS-PAGE for the strains were obtained in comparison with the baker's yeast S. cerevisiae. Chromatograms of cell free
Psychrotolerant strains for wine making
641
A D H a c t i v i t y bars
1200 1000 800
600 400 200
1
2
3
4
Strain series:
5
1. 2. 3. 4.
Baker's yeast AXAZ-I AXAZ-2 AXAZ-3
6
7
5. 6. 7. 8.
Visanto Aegio Samos Rodos
8
9
10
9. Thasos 10. Halkidiki
Fig. 1. Dehydrogenase activity of the strains AXAZ-1, AXAZ-2, AXAZ-3, Visanto-I in comparison with others isolated from other locations in Greece.
extracts and cell membranes show that the protein profiles of the isolated strains were similar to those of S. cerevisiae. The A D H activity measurements of the isolated strains were made in comparison with various strains isolated from Greek agricultural
25 °C
12:
• • • A
10 ~tk
S "5 e~
~
6
AXAZ- 1 AXAZ-2 AXAZ-3 Visanto
[\
4
"-N-.22.%~
2
5
10
15
20
25
30
35
40
45
50
T i m e (hr)
0 °C ~,
=:
I s t-
-~%.~% " ~ " ~ .
* AxAz-2 • ^XAZ-3
/ 0
4
12
25
41
54
66
83
104
125
145
T i m e (days)
Fig. 2. Fermentation kinetics observed in the fermentation of grape must using the proposed cryotolerant S, cerevisiae strains.
areas. Figure 1 shows that the A D H activity differs between the proposed psychrotolerant strains and the other strains studied. The strains AXAZ-1 and Visanto had the higher A D H activity. The fermentation rates of the strains are relatively fast at 25°C. Fermentations performed by the strains after the adaptation treatment also gave satisfactory results at 0°C. The results show that AXAZ-1 and Visanto-1 had higher fermentation rates. Low temperature wine making with these adapted strains is shown in Tables 1 and 2. AXAZ-1, AXAZ-2, AXAZ-3 and Visanto-1 ferment at low temperatures with AXAZ-1 and Visanto-1 most suitable for fermentations at very low temperatures and more specifically at temperatures in the range lower than 5°C. The higher A D H activity observed for the strains Visanto-1 and AXAX-1 is in agreement with their higher fermentation rates and higher ethanol concentrations, obtained at low temperatures. Similarly, higher fermentation rates of AXAZ-1, as compared with these of AXAZ2 at low temperatures, agree with those reported in a previous work, 12 for room temperature fermentations. Results clearly show that, by the adaptation treatment, the strains show psychrotolerance and more activity, when no sulphur dioxide is contained in must. In addition, Visanto-1 and AXAZ-1 increased their alcohol productivities, at low temperatures, when immobilized on kissiris9 and
642
T. Argiriou et al.
Table 1. Results of fermentations performed at low temperatures
Temperature (°C) 25
15
10
5
2
0
S. cerevisiae
strain AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ- 1 AXAZ-2 AXAZ-3 Visanto
Initial density (°Be)
Final density (°Be)
12a 12 12 12 11"7 11"7 11"7 11-7 11.5 11.5 11.5 11"5 12 12 12 12 11.6 11-6 11.6 11.6 11"5 11.5 11.5 11-5
Alcohol concentration ( % v/v)
Residual sugar (g/litre)
Fermentation time (days)
11.5 12 12 11.9 12.8 11.8 12 12.7 12.2 12.2 12.5 11.8 10.8 10 11-4 11.2 10.8 9.6 10-6 10.9 9-2 4.6 3.4 5.3
2-8 3.5 3-6 3.7 Traces 4-3 2-2 Traces 6 7.4 3 9.5 8 13 4 3 9-5 23 15 12 58 119 141 95
1.8 1.8 3 3 5 5 5 5 13 12 12 14 99 99 99 99 50 112 1t2 112 147 147 147 147
0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0-8 0.9 1. I 3 7.5 8.8 5-3
a All musts contained sulphur dioxide in the range of 20-65 mg/litre.
Table 2. Results of fermentations performed at a temperature lower than 0°C
Temperature (°C) -2 - 2 - 2 - 2
S. cerevisiae
strain AXAZ-1 AXAZ-2 AXAZ-3 Visanto
Initial density (°Be)
Final density (°Be)
Alcohol concentration ( % v/v)
Residual sugar (g/litre)
Fermentation time (days)
11"5 11"5 11-5 I 1"5
0"3 0"5 1.5 0-3
10 9.1 8.5 9.9
12 16"5 32 10"5
155 155 160 150
delignified cellulosic material, 1° respectively. Low temperature fermentability of the Visanto1, isolated from the Greek island Santorini, is related to the fact that vino santo wine is produced in Santorini by completion of fermentation during winter. After adaptation, the yeasts studied (especially AXAZ-1 and Visanto-1) are suitable for low temperature wine making. These strains will be used in further studies. ACKNOWLEDGEMENTS We acknowledge the financial support of this work by the European Union and the Greek Government through the STRIDE program.
REFERENCES 1. Bakoyianis, V., Kana, K., Kaliafas, A. & Koutinas, A. A., Low-temperature wine making by kissiris-supported biocatalyst: volatile by-products. J. Agric. Food Chem., 41 (1992) 465-8. 2. Bakoyianis, V., Formation of volatile by-products in the continuous wine making by immobilized cells on mineral kissiris, ~-alumina and calcium alginates and an application in industrial scale pilot plant of a fermentation in the presence of kissiris. Doctoral Dissertation, University of Patras, Patras, Greece, 1995, pp. 140-50. 3. Nadesa, R., Production of higher alcohols during alcoholic fermentation of must. Prehrambeno-Technol. Rev., 17 (1979) 173-8. 4. Giolfi, G., Castino, M. & Distefano, R., Studies on the metabolic response of various fermentative yeast species in single must temperatures between 10°C and 40°C. Riv. Vitic. Enol., 38 (1985) 447-70.
Psychrotolerant strains for wine making 5. Mauricio, J. C., Moreno, J., Medina, M. & Ortega, J., Fermentation of Pedro Ximenez musts at various temperatures and different degrees of ripeness. Belg. J. Food Chem. Biotechnol., 41 (3) (1986) 71-6. 6. Ruzic, N., Effects of temperature on yeast activity and chemical composition of wines. Zbomik Radova, 22 (1991) 35-44. 7. Gardner, N., Rodrigue, N. & Hampagne, C. P., Combined effects of sulfites, temperature and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae. Appl. Environ. Microbiol., 59 (1993) 2022-8. 8. Kishimoto, M., Fermentation characteristics of hydrid between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae. J. Ferment. B ioeng., 77 (1994) 432-5. 9. Bakoyianis, V., Kanellaki, M., Kaliafas, A. A. & Koutinas, A. A., Low temperature wine making by immobilized cells on mineral kissiris. J. Agric. Food Chem., 40 (1992) 1293-6. 10. Bardi, E. P. & Koutinas, A. A., Immobilization of yeast on delignified cellulosic material for room temperature and low-temperature wine making. J. Agric. Food Chem., 42 (1994) 221-6. 11. Bardi, E. P., Bakoyianis, V., Koutinas, A. A. & Kanellaki, M., Immobilization of yeast on gluten for room temperature and low-temperature wine making. Process Biochem., 31 (1996) 425-30.
643
12. Argiriou, T., Kaliafas, A., Psarianos, C., Kana, K., Kanellaki, M. & Koutinas, A. A., New alcohol resistant strains of Saccharomyces cerevisiae species for potable alcohol production using molasses. Appl. Biochem. Biotechnol., 36 (1992) 153-61. 13. Hantula, J., Kurki, A., Vuoriranta, P. & Bamford, D. H., Rapid classification of bacterial strains by SDSpolyacrylamide gel electrophoresis: population dynamics of the dominant dispersed phase bacteria of activated sludge. Appl. Microbiol. Biotechnol., 34 (1991) 551-5. 14. Nikolova, P. & Ward, O. P., Production of L-phenylacetyl carbinol by biotransformation product and by-product formation and activities of the key enzymes in wild-type and ADH isoenzyme mutants of Saccharomyces cerevisiae. Biotechnol. Bioeng., 38 (1991) 493-8. 15. Giolfi, G., Acetaldehyde formation in relation to yeast nitrogen nutrition during alcoholic fermentation. Riv. Vitic. Enol., 36 (1983) 431. 16. Thomas, D. S., Hossack, J. A. & Rose, A. H., Plasma membrane lipid composition and ethanol tolerance in Saccharomyces cerevisiae. Arch. Microb., 117 (1978) 239-45. 17. Egan, H., Kirk, R. S. & Sawyer, R., Pearson's Chemical Analysis of Foods, 8th ed. Churchill Livingstone, Edinburgh, 1981, pp. 150-3.
P! 1:S0032-9592(96)00016-7
Psychrotolerant Saccharomyces cerevisiae Strains After an Adaptation Treatment for Low Temperature Wine Making T. Argiriou, A. Kaliafas, K. Psarianos, M. Kanellaki, S. Voliotis & A. A. Koutinas* Department of Chemistry, Universityof Patras, Patras, Greece (Received 6 November 1995; revised manuscript received 25 January 1996 and accepted 10 February 1996)
Four alcohol resbtant strains isolated from a Greek vineyard plantation were examined for low temperature wine making. The strains found to be of Saccharomyces cerevisiae species. These yeasts were adapted by gradually establishing feeding and temperature conditions during yeast production. After adaptation these yeasts fermented must and prepared wines at relatively low temperatures in the range - 2 to 5°C. More specifically at -2°C the strains Visanto-1 and AXAZ-1 produced wines of 9"9 and 10% v/v alcohol at 150 and 155 days, respectively. Copyright © 1996 Elsevier Science Ltd
INTRODUCTION
tolerant have been made using genetic engineeering. 8 However, publications concerning wine making using free cells at temperatures lower than 10°C, to our knowledge, are absent in the literature. Wine making at temperatures lower than 15°C is not usual on an industrial scale since the productivity is very low. In order to decrease the temperature of fermentation below 10°C while retaining productivity, the psychrotolerant Saccharomyces cerevisiae strains Visanto-19 and AXAZ-11°'~1 were immobilised on mineral kissiris, delignified cellulosic material and gluten, respectively, so that the solid supported biocatalysts reduced the activation energy E~. The aim of this work was to isolate psychrotolerant or psychrophilic yeasts.
Manufacturers know that wines produced at low temperatures develop taste and aroma. Therefore, important products, such as the Greek semisweet vinosanto, the semi-sparkling zitsa and the famous French sparkling champagne are produced by a secondary fermentation during the winter. The improved quality of wines produced by low temperature fermentations can be attributed to the reduction of higher alcohols and to an increase of the proportioin of ethyl acetate in the total volatiles.l'2 As a result, many researchers have focused their interest on low temperature wine making. Fermentations, using free cells and performed in the temperature range 10-15°C with relatively high fermentation times, have been reported. 3-5 Likewise, recent publications reported that at temperatures higher than 25°C, higher alcohols 6 and glycerol7 are increased when compared with fermentations performed at low temperatures. For low temperature wine making attempts to modify cells to be psychro-
MATERIALS AND METHODS Isolation of strains Saccharomyces cerevisiae AXAZ-1 and AXAZ2, isolated previously 12 were examined for their alcohol resistance. AXAZ-3 was isolated by the authors in the Food Technology Laboratory,
*To whom correspondence should be addressed. 639
640
T. Argiriou et al.
University of Patras, Patras, Greece in October 1988. Grapes were collected from a vineyard plantation of Ano Ziria, North Peloponnisos, located at an altitude of about 500 m. Musts with high initial °Be densities (17.5 and 17.7 °Be) obtained from raisin grapes were employed. Musts of 500 ml volume were incubated at 30°C and allowed to ferment in the absence of grape skins. Just before the end of fermentation, 1 ml of fermenting mass, obtained with a sterilised pipette from the bottom, was added to 5 ml liquid medium and incubated at 30°C. The culture was purified by a pour plate technique. The strain Visanto-1 was isolated, as described above from Mandilari grapes collected on the Greek Aegean island Santorini, in September 1991. Culture media The culture medium was sterilised in an autoclave at ll0°C for 15 min and contained 2% glucose, 0.1 (NH4)aSO4, 0"1% KHaPO4, 0"5% MgSO4 and 0.4% yeast extract in distilled water. For Petri dish cultures, 2% agar was added. This synthetic culture medium was used for the preparation of the inocula employed in all fermentations carried out in this work, as well as to obtain biomass for SDS-polyacrylamide gel electrophoresis (PAGE). Identification of strains Identifications of strains were made for Visanto-1 and AXAZ-3 using an API 20 C AUX yeast identification system. To verify the identifications, protein profiles by SDSPAGE,13 and measurements of alcohol dehydrogenase (ADH) activity14 were used for all strains. Fermentations kinetics, mainly at low temperatures, were used to differentiate the strains. ADH activity was measured by a spectrophotometric method. 15 The 'ADH activity unit' term refers to the amount of enzyme that alters the absorbance by 0.001 per min, at a wavelength of 350 n m ) 4 Cell membrane and cell-free extracts were used for SDS-PAGE as described by Thomas et al. ~6
Adaptation treatment All yeast cultures were adapted to growth at low temperatures before low temperature wine making. This procedure consisted of gradually
establishing feeding and temperature conditions during yeast production, similar to those that the strain encounters during wine fermentations at low temperatures. In this case, yeasts were produced in a culture media that contained gradually increasing amounts of kerino must, as the sugar component, rather than glucose. In addition, the culture temperature was kept relatively low (not more than 20°C). Fermentations After the above adaptation procedure, fermentations were performed separately for each strain at 25, 15, 10, 5, 2, 0 and -2°C. For each strain and temperature, 250 ml must containing 20 g/litre wet weight cells were fermented in a glass cylinder and placed in a constant temperature water bath. All musts were obtained from the grape cultivar Kerino and those fermented at temperatures higher than 0°C were adjusted to an initial °Be density by the addition of a commercial concentrated must. Musts used in fermentation performed at - 2 ° C were adjusted with concentrated must without sulphur dioxide. All fermentations were carried out without stirring and the kinetics of fermentation were followed by measuring the °Be density at various time intervals. Analysis of wines After the end of fermentation, wines obtained were analysed for alcohol and residual sugar. Alcohol was determined as alcoholic degrees (milliliters of alcohol per 100 ml of wine) which were obtained after distillation of samples using a Gay-Lussac alcohol meter. Residual sugar was analysed according to the Lane-Eynon procedure. ~7
RESULTS AND DISCUSSION
To study the identity of the isolated yeast, API tests, A D H activity, protein profile by SDSPAGE and fermentation kinetics, mainly at low temperatures, were examined (Figs 1 and 2). Identification by the API test for the strains AXAZ-3 and Visanto-1 indicated by the strains are Saccharomyces cerevisiae with a probability of 97% for AXAZ-3 and 99% for Visanto-1. Protein profiles by SDS-PAGE for the strains were obtained in comparison with the baker's yeast S. cerevisiae. Chromatograms of cell free
Psychrotolerant strains for wine making
641
A D H a c t i v i t y bars
1200 1000 800
600 400 200
1
2
3
4
Strain series:
5
1. 2. 3. 4.
Baker's yeast AXAZ-I AXAZ-2 AXAZ-3
6
7
5. 6. 7. 8.
Visanto Aegio Samos Rodos
8
9
10
9. Thasos 10. Halkidiki
Fig. 1. Dehydrogenase activity of the strains AXAZ-1, AXAZ-2, AXAZ-3, Visanto-I in comparison with others isolated from other locations in Greece.
extracts and cell membranes show that the protein profiles of the isolated strains were similar to those of S. cerevisiae. The A D H activity measurements of the isolated strains were made in comparison with various strains isolated from Greek agricultural
25 °C
12:
• • • A
10 ~tk
S "5 e~
~
6
AXAZ- 1 AXAZ-2 AXAZ-3 Visanto
[\
4
"-N-.22.%~
2
5
10
15
20
25
30
35
40
45
50
T i m e (hr)
0 °C ~,
=:
I s t-
-~%.~% " ~ " ~ .
* AxAz-2 • ^XAZ-3
/ 0
4
12
25
41
54
66
83
104
125
145
T i m e (days)
Fig. 2. Fermentation kinetics observed in the fermentation of grape must using the proposed cryotolerant S, cerevisiae strains.
areas. Figure 1 shows that the A D H activity differs between the proposed psychrotolerant strains and the other strains studied. The strains AXAZ-1 and Visanto had the higher A D H activity. The fermentation rates of the strains are relatively fast at 25°C. Fermentations performed by the strains after the adaptation treatment also gave satisfactory results at 0°C. The results show that AXAZ-1 and Visanto-1 had higher fermentation rates. Low temperature wine making with these adapted strains is shown in Tables 1 and 2. AXAZ-1, AXAZ-2, AXAZ-3 and Visanto-1 ferment at low temperatures with AXAZ-1 and Visanto-1 most suitable for fermentations at very low temperatures and more specifically at temperatures in the range lower than 5°C. The higher A D H activity observed for the strains Visanto-1 and AXAX-1 is in agreement with their higher fermentation rates and higher ethanol concentrations, obtained at low temperatures. Similarly, higher fermentation rates of AXAZ-1, as compared with these of AXAZ2 at low temperatures, agree with those reported in a previous work, 12 for room temperature fermentations. Results clearly show that, by the adaptation treatment, the strains show psychrotolerance and more activity, when no sulphur dioxide is contained in must. In addition, Visanto-1 and AXAZ-1 increased their alcohol productivities, at low temperatures, when immobilized on kissiris9 and
642
T. Argiriou et al.
Table 1. Results of fermentations performed at low temperatures
Temperature (°C) 25
15
10
5
2
0
S. cerevisiae
strain AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ-1 AXAZ-2 AXAZ-3 Visanto AXAZ- 1 AXAZ-2 AXAZ-3 Visanto
Initial density (°Be)
Final density (°Be)
12a 12 12 12 11"7 11"7 11"7 11-7 11.5 11.5 11.5 11"5 12 12 12 12 11.6 11-6 11.6 11.6 11"5 11.5 11.5 11-5
Alcohol concentration ( % v/v)
Residual sugar (g/litre)
Fermentation time (days)
11.5 12 12 11.9 12.8 11.8 12 12.7 12.2 12.2 12.5 11.8 10.8 10 11-4 11.2 10.8 9.6 10-6 10.9 9-2 4.6 3.4 5.3
2-8 3.5 3-6 3.7 Traces 4-3 2-2 Traces 6 7.4 3 9.5 8 13 4 3 9-5 23 15 12 58 119 141 95
1.8 1.8 3 3 5 5 5 5 13 12 12 14 99 99 99 99 50 112 1t2 112 147 147 147 147
0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0-8 0.9 1. I 3 7.5 8.8 5-3
a All musts contained sulphur dioxide in the range of 20-65 mg/litre.
Table 2. Results of fermentations performed at a temperature lower than 0°C
Temperature (°C) -2 - 2 - 2 - 2
S. cerevisiae
strain AXAZ-1 AXAZ-2 AXAZ-3 Visanto
Initial density (°Be)
Final density (°Be)
Alcohol concentration ( % v/v)
Residual sugar (g/litre)
Fermentation time (days)
11"5 11"5 11-5 I 1"5
0"3 0"5 1.5 0-3
10 9.1 8.5 9.9
12 16"5 32 10"5
155 155 160 150
delignified cellulosic material, 1° respectively. Low temperature fermentability of the Visanto1, isolated from the Greek island Santorini, is related to the fact that vino santo wine is produced in Santorini by completion of fermentation during winter. After adaptation, the yeasts studied (especially AXAZ-1 and Visanto-1) are suitable for low temperature wine making. These strains will be used in further studies. ACKNOWLEDGEMENTS We acknowledge the financial support of this work by the European Union and the Greek Government through the STRIDE program.
REFERENCES 1. Bakoyianis, V., Kana, K., Kaliafas, A. & Koutinas, A. A., Low-temperature wine making by kissiris-supported biocatalyst: volatile by-products. J. Agric. Food Chem., 41 (1992) 465-8. 2. Bakoyianis, V., Formation of volatile by-products in the continuous wine making by immobilized cells on mineral kissiris, ~-alumina and calcium alginates and an application in industrial scale pilot plant of a fermentation in the presence of kissiris. Doctoral Dissertation, University of Patras, Patras, Greece, 1995, pp. 140-50. 3. Nadesa, R., Production of higher alcohols during alcoholic fermentation of must. Prehrambeno-Technol. Rev., 17 (1979) 173-8. 4. Giolfi, G., Castino, M. & Distefano, R., Studies on the metabolic response of various fermentative yeast species in single must temperatures between 10°C and 40°C. Riv. Vitic. Enol., 38 (1985) 447-70.
Psychrotolerant strains for wine making 5. Mauricio, J. C., Moreno, J., Medina, M. & Ortega, J., Fermentation of Pedro Ximenez musts at various temperatures and different degrees of ripeness. Belg. J. Food Chem. Biotechnol., 41 (3) (1986) 71-6. 6. Ruzic, N., Effects of temperature on yeast activity and chemical composition of wines. Zbomik Radova, 22 (1991) 35-44. 7. Gardner, N., Rodrigue, N. & Hampagne, C. P., Combined effects of sulfites, temperature and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae. Appl. Environ. Microbiol., 59 (1993) 2022-8. 8. Kishimoto, M., Fermentation characteristics of hydrid between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae. J. Ferment. B ioeng., 77 (1994) 432-5. 9. Bakoyianis, V., Kanellaki, M., Kaliafas, A. A. & Koutinas, A. A., Low temperature wine making by immobilized cells on mineral kissiris. J. Agric. Food Chem., 40 (1992) 1293-6. 10. Bardi, E. P. & Koutinas, A. A., Immobilization of yeast on delignified cellulosic material for room temperature and low-temperature wine making. J. Agric. Food Chem., 42 (1994) 221-6. 11. Bardi, E. P., Bakoyianis, V., Koutinas, A. A. & Kanellaki, M., Immobilization of yeast on gluten for room temperature and low-temperature wine making. Process Biochem., 31 (1996) 425-30.
643
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