- Email: [email protected]
Continuous wine fermentation using a psychrophilic yeast immobilized on apple cuts at different temperatures
Food Microbiology, 2002, 19, 127^134 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.2001.0468
ORIGINAL ARTICLE
Continuous wine fermentation using a psychrophilic yeast immobilized on apple cuts at di¡erent temperatures Y. Kourkoutas1, A. A. Koutinas1, M. Kanellaki1, I. M. Banat2,* and R. Marchant2
Apple-supported biocatalyst prepared by immobilization of cells of an alcohol-resistant psychrophilic Saccharomyces cerevisiae AXAZ-1 on apple cuts was found to be suitable for continuous wine fermentation at temperatures between 5 and 151C.Wine productivities were much higher than those reported using repeated batch fermentation at low temperatures. Ethanol productivity at 51C was equal to that obtained by traditional fermentation at temperatures of 22^251C. The continuous fermentation bio reactor was operated for 95 days without any diminution of the ethanol productivity. Total and volatile acids were similar to those found in dry wines. The formation of methanol, ethyl acetate, propanol-1, isobutanol and amyl alcohols (2-methylbutanol-1and 3-methylbutanol-1) were also monitored. The concentrations of higher alcohols were at low levels. At temperatures 4101C the concentrations of amyl alcohols were lower than those previously reported for repeated batch fermentations. The concentration of ethyl acetate was o100 mg l 1 in all cases. Preliminary sensory tests carried out in the laboratory ascertained the fruity aroma, ¢ne taste and the overall improved quality of the wines produced compared to other commercially available wines. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Continuous processes are favoured in many ¢elds of the fermentation industry for many obvious reasons, including the economic advantages of having uninterrupted operation for longer periods (Jackson 1994). Continuous processes can generally achieve substantial improvements in e⁄ciency of the process and subsequently higher productivities and lower operating costs.
*Corresponding author. Fax: 0044 2870 324911. E-mail: [email protected] 0740 -0020/02/2^30127 +08 $35.00/0
The use of immobilized cells for continuous wine-making is an attractive process that has received great attention during the past few years (Bakoyianis et al. 1992, Iconomou et al. 1996, Maicas et al. 2001, Nedovic et al. 2000). Wine manufacturers know that low-temperature fermentation leads to higher quality products. However, only a few papers concerning continuous wine fermentation using immobilized cells at low temperatures have been published (Bakoyianis et al. 1993, 1997, Loukatos et al. 2000). The availability of a low-temperature continuous fermentation technology using immobilized yeast cells is likely to have desirable commercial applications. r 2002 Elsevier Science Ltd. All rights reserved.
Received: 28 September 2001 1 Food Biotechnology Group, Department of Chemistry, Section of Analytical, Environmental and Applied Chemistry, University of Patras, GR-26500, Patras, Greece 2 Biotechnology Group, School of Biological and Environmental Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK
128 Y. Kourkoutas et al.
The development of such a continuous wine production technology using immobilized cells has not been achieved for several reasons, including the lack of a suitable low-cost support material, unexplored taste and aroma quality for the produced wine and lower viability of the immobilized system. In the present study apple pieces were used as a support material for immobilization as apple is an inexpensive, abundant food material that has previously been successfully used for immobilization of Saccharomyces cerevisiae yeast cells for wine production using repeated batch fermentations (Kourkoutas et al. 2001). This support was used in a packed bed reactor to accomplish continuous fermentation of grape must.
Materials and Methods Yeast strain An alcohol-resistant psychrophilic S. cerevisiae AXAZ-1 yeast strain isolated from an agricultural area in Greece (Argiriou et al. 1992) was used in the present study. It was grown on medium containing (% w/v): glucose 4%, yeast extract 0?4%, (NH4 )2 SO4 0?1%, KH2 PO4 0?1% and MgSO4 0?5%. Yeast cells (15^20 g wet weight) were prepared as described by Argiriou et al. (1992) and employed directly in the fermentations.
Preparation of grape must Concentrated grape must was diluted with distilled water to a ¢nal 1Be density range 11?2^ 12?0 1Be (E19^21% w/v initial sugar concentration). The must was used without any nutrient addition or adjustments after sterilization at 1301C for 15 min.
Support and immobilization of cells The immobilization of the yeast cells on apple pieces was carried out as described previously (Kourkoutas et al. 2001). In brief, pieces of apple (425 g) were placed in a glass cylinder of 1 l and 500 ml of culture medium were added. The culture medium contained (in % w/v): 12% glucose, 0?4% yeast extract, 0?1% (NH4 )2 S04, 0?1%
KH2 P04 and 0?5% MgS04 in distilled water with no pH adjustment. A 10 g wet weight of yeast cells was added to the bioreactor and allowed to ferment until the must density was approximately 0?51Be. The fermented liquid was decanted and the support was washed twice with 400 ml of culture medium and then used for wine production.
Pilot fermentation Continuous wine, making was carried out in a 2 -l glass bioreactor (70 cm height and 3?1cm internal diameter), into which 720 ml grape must and 1200 g apple-supported biocatalyst were added. The apple-supported biocatalyst was used in six consecutive repeated batch fermentation experiments for grape must before switching into continuous mode. This was necessary to obtain a steady volume of apple as it decreased because of sugar fermentation during the ¢rst four batches of fermentation (Kourkoutas et al. 2001). Must was fed into the bioreactor in an upstream £ow by a high accuracy peristaltic pump (Cole Parmer Instruments Co., Chicago, Illinois, USA).
Experimental procedure Grape must with an initial 1Be density within the range 11?2^12?0 1Be was continuously supplied to the bioreactor. The £ow rate was reduced as the temperature was lowered, from an initial 515 to 155 ml/day. The reactor was operated for 95 days and the fermentation temperature was decreased gradually from 301C to 51C. Temperature decrease was carried out at the rate of 2^31C/day to avoid sudden changes. Samples were collected at di¡erent temperatures after 2 days of pumping to allow time to achieve steady conditions in the bioreactor and analysed for 1Be density, residual sugar, alcohol concentration, total and volatile acids, methanol and volatile by-products.
Analytical assays Alcohol concentrations were determined using a Gay^Lussac Alcoholmeter and productivity (expressed in grams of ethanol per volume produced per day) was calculated by multiplying
Continuous wine fermentation 129
the dilution rate by ethanol concentration. Dilution rates were calculated by dividing the £ow rate of liquid by the total volume of the fermenter. Wine productivity was calculated as grams of wine per litre of total volume produced per day and was calculated by multiplying the £ow rate (ml/day) by the density of wine (E1 gl 1 ) and dividing by the total volume (2 l). Residual sugar was determined by high-performance liquid chromatography, using a Shimadzu chromatograph with a SCR-101N stainless steel column, a LC-9A pump, a CTO10A oven at 601C and a RID- 6A refractive index detector. Three times distilled water was used as mobile phase with a £ow rate of 0?8 ml min 1 , and butanol-1 was used as an internal standard. Samples of 0?5 ml wine and 2?5 ml of a 1% (v/v) solution of butanol-1 were diluted to 50 ml, and 40 ml was injected directly to the column.The residual sugar concentration was calculated using standard curves and expressed in terms of grams of residual sugar per litre. Total acidity was estimated by titration of samples with 0?1 M NaOH solution and volatile acidity by titration with 0?1 M NaOH of distillates obtained by steam distillation of wine samples (Zoeklein et al. 1990).
Determination of volatile by-products Acetaldehyde, ethyl acetate, propanol-1, isobutanol and amyl alcohols were determined by gas chromatography using a stainless steel column, packed with Escarto-5905 consisting of Squalene 5%, Carbowax-300 90% and diethyl-hexyl sebacate 5% (v/v) (Cabezudo et al. 1978). Nitrogen was used as a carrier gas at 20 ml min 1 . Injection port and FID detector temperatures were 2101C and 2201C, respectively. The column temperature was 701C. In all cases, the internal standard was butanol-1 at a concentration of 0?5% (v/v). Samples of 4 ml of wine were injected directly into the column and the concentrations of the above volatile compounds were determined using standard curves. Methanol was also determined by gas chromatography using Porapac S column. Nitrogen was used as a carrier gas at 40 ml min 1 . The column temperature was programmed at 120^1701C at a rate 101C/min. The temperatures of the injector and FID de-
tector were 210 and 2201C, respectively. For the methanol determination, 2 -ml samples were injected directly into the column and the concentration of methanol was determined using standard curves. Butanol-1 was used as internal standard at a concentration of 0?5% (v/v).
Preliminary taste investigation Samples of the wine produced were kept at 4^ 51C for 1 month and then tested for their aroma and taste characteristics compared to a commercial dry wine produced locally using similar must types. Ten tasters familiar with wine tastes were asked to give scores on a 0^10 scale using locally approved protocols in our laboratories for taste and aroma. The sensory evaluation was a blind test in a coloured glass under low light.
Results and Discussion Continuous fermentation The fermentation of must in a continuous bioreactor was carried out to investigate the operational stability of the immobilized yeast strain AXAZ-1 on apple pieces and the suitability for continuous process and for comparison with repeated batch fermentations with regard to wine-making. This strain had the advantage of being both alcohol-resistant and a psychrophile. These characteristics were considered advantageous for low-temperature wine-making. The use of an apple-supported biocatalyst prepared by the immobilization of a yeast strain on pieces of apple was reported to be suitable for the production of high alcohol-containing wine under batch fermentation conditions (Kourkoutas et al. 2001). Immobilization of the yeast on the support may take place as a result of hydrogen bonding, entrapment of the cells in the apple structure, Van der Waals forces or adsorption caused by charges on microbial cell walls and the apple cell mass (Bardi et al. 1996). To investigate continuous wine-making with an applesupported biocatalyst the initial 1Be density of must was kept relatively constant, and all the
130 Y. Kourkoutas et al.
Temperature (˚C)
˚Be density
must used was prepared from the same grape cultivar. The results of the continuous fermentation are summarized in Fig. 1 and Table 1. The reactor was operated over 3 months and the operational stability of the system for wine-making at low temperatures (15^51C) was observed for at least 71 days. The operational stability of the system can be attributed to the high alcohol resistance of apple-supported biocatalyst as well as to the psychrophilic nature of the yeast strain used. The apple-supported biocatalyst proved to be mechanically and chemically stable for the duration of the production. The 1Be density of the e¥uent was relatively constant during the whole experiment. The £ow rate of the must was reduced when the temperature was reduced to allow more time for complete fermentation at the lower temperatures. Wine productivities were much higher than those obtained in repeated batch fermentation, particularly at lower tempera-
12 10 8 6 4 2 0 30
Influent Effluent
Temperature
25 20 15 10 5 600
Flow rate
Flow rate (ml)
500 400 300 200 100 0
20
40
60
80
100
Time (days)
Figure 1. Flow rate of must and 1Be densities of fermentations as related to temperature changes in the range 5^301C.
tures (Kourkoutas et al. 2001). Ethanol productivity of the system measured at 51C represented about 28% of that obtained at 301C. This productivity at 51C is equal to that achieved in traditional fermentations over a temperature range of 22^251C. Free cell concentrations in the e¥uent ranged from 15 g l 1 wet weight at 301C to 5 g l 1 at 5 g l 1 wet weight. The biomass leakage was higher at high temperatures, as higher £ow rates were used.Volatile acid levels were similar to those found in dry wines. Although in most cases small amounts of residual sugar remained, it is unlikely that the volatile acidity would rise if the fermentation were completed because almost all the sugar was utilized. The total acidity of the product was slightly increased, either because of the transfer of some apple acids to the produced wine at the early stages of the continuous process, or to the formation of acids during fermentation. Similar observations were previously reported by other researchers during fermentation of apple juices to produce cider (Beech 1993). It was not necessary to pretreat the apple cuts to extract the acids before using them as support because most of the apple acids are normal constituents of the wine and may contribute positively to the £avour of the product. In addition, the observed concentrations were within the normal ranges present in dry wines (4^6 g l 1 of tartaric acid). Total acidity was in the range 3?6^6?6 g l 1 of tartaric acid at 30^151C, and decreased slightly as the temperature dropped from 10 to 51C. This reduction can be attributed to the increase of crystallization of tartrate salts with the decrease in temperature. This has been observed previously in repeated batch fermentations (Kourkoutas et al. 2001). In addition, the apple micro£ora did not a¡ect the fermentation and quality of the ¢nal product because the immobilized yeast dominated in the fermentation broth because of the low pH, the relatively high ethanol concentration and total acidity of the wine and the high immobilized yeast biomass. As apple cuts proved to be a suitable support for continuous wine-making, particularly with the advantage of being abundantly available, cheap and easy to use for immobilization, the
Continuous wine fermentation 131
Table 1. Characteristics of the wine produced by continuous fermentation of must at various temperatures using S. cerevisiae AXAZ-1 yeast cells immobilized on apple cuts Temperature Initial 1Be Wine Daily (1C) density produced wine (ml day 1 ) productivity (g l 1 )
Total Volatile Ethanol Residual Ethanol acidity acidity concen- sugar productivity tration (g l 1 ) (g l 1 day 1 ) (g l 1 tartaric (g l 1 acetic (% vol) acid) acid)
30
11?4 11?9 11?8 11?8 12?0
515 515 515 515 515
258 258 258 258 258
9?9 11?1 10?7 9?5 11?5
4?2 15?4 15?7 16?9 10?9
20 23 22 19 23
4?4 5?0 6?5 4?8 5?5
0?07 0?19 0?11 0?14 0?14
25
11?5 11?8 11?8 11?6 11?7
550 550 550 550 550
275 275 275 275 275
9?9 9?7 10?5 11?4 10?8
9?4 12?1 4?9 12?2 17?7
22 21 23 25 23
6?6 4?8 3?7 4?5 4?7
0?10 0?13 0?13 0?19 0?32
20
11?7 12?1 11?6 11?7 11?9
550 550 550 550 550
275 275 275 275 275
10?1 11?1 10?3 10?2 11?2
10?3 9?3 19?6 23?6 25?6
22 20 22 22 24
4?4 3?7 4?0 3?6 5?0
0?13 0?25 0?13 0?17 0?34
15
11?7 11?5 11?5 11?5
325 325 325 325
163 163 163 163
9?0 11?0 10?2 11?3
11?3 12?0 8?9 14?4
12 14 13 15
4?9 4?9 5?1 5?2
0?37 0?41 0?44 0?41
10
11?8 11?8 11?6 11?6 11?6
215 215 215 215 215
108 108 108 108 108
11?2 10?9 11?6 10?8 10?8
11?9 12?9 30?4 33?8 22?2
10 9 10 9 9
3?2 3?9 4?2 4?4 4?4
0?14 0?17 0?22 0?22 0?17
5
11?5 11?4 11?3 11?5 11?5
155 155 155 155 155
78 78 78 78 78
8?8 9?3 10?2 10?2 9?9
18?3 37?1 34?0 18?3 15?9
5 6 6 6 6
4?6 4?3 4?3 4?4 4?3
0?22 0?22 0?26 0?38 0?29
¢nal consideration for use of this technique was the quality of the produced product.
Volatile by-products The e¡ects of temperature on the formation of the volatile compounds during continuous wine-making, using an apple-supported biocatalyst are shown in Table 2. Table 3 also compares the volatile by-products formed during the continuous process to the volatiles of the same must be accomplished by traditional batch fermentation. The concentrations of higher alcohols (propanol-1 and isobutyl alcohol) were low. They
were in the ranges 18^40 ppm and 14^47 ppm, respectively (Jackson 1994).The concentrations of isobutyl alcohol in the wine produced by the continuous fermentation were much lower than in the wine produced by traditional batch fermentation (Table 3). The concentrations of amyl alcohols were also low at all fermentation temperatures. At temperatures 4101C the concentrations of amyl alcohols were much lower than those obtained using the repeated batch fermentation technique (Kourkoutas et al. 2001) and the traditional batch fermentation (Table 3). These results indicate that the wine product has improved quality because of lower concentrations of higher alcohols.
132 Y. Kourkoutas et al.
Table 2. E¡ect of temperature on the formation of volatile by-products during continuous wine fermentation using S. cerevisiae AXAZ-1 yeast cells immobilized on apple cuts Acetaldehyde (ppm)
Ethyl acetate (ppm)
Propanol-1 (ppm)
Isobutyl alcohol (ppm)
Amyl alcohols (ppm)
Methanol (ppm)
30
13 38 30 27 26
57 51 66 59 64
22 20 22 20 24
26 17 15 16 21
142 113 109 108 142
36 23 42 42 50
25
13 28 25 37 36
37 50 74 48 39
24 22 25 28 25
24 27 29 28 22
137 148 176 154 137
36 12 12 41 5
20
29 49 25 44 52
88 74 65 56 48
18 19 22 21 24
24 38 27 28 30
135 160 148 135 127
13 79 21 86 25
15
80 56 64 40
75 94 86 61
27 26 34 28
45 47 36 34
139 155 143 128
12 69 28 61
10
23 51 61 87 90
46 30 48 44 23
26 40 38 33 38
27 31 31 27 27
134 154 147 125 139
37 48 31 42 35
5
30 38 62 46 18
33 35 24 43 42
29 30 26 24 22
20 22 19 18 14
106 111 104 96 106
37 47 47 47 58
Temperature (1C)
Table 3. Volatile by-products formed during continuous wine fermentation using S. cerevisiae AXAZ-1 yeast cells immobilized on apple cuts in comparison with volatiles formed during traditional batch fermentation using free cells. All fermentations were carried out at 301C Wine
Continuous process Traditional batch fermentation
Acetaldehyde (ppm)
Ethyl acetate (ppm)
Propanol-1 (ppm)
Isobutyl alcohol (ppm)
Amyl alcohols (ppm)
methanol (ppm)
27 64
59 31
22 10
19 54
123 169
39 n.d.
n.d., none detected.
Ethyl acetate concentrations ranged from 23 to 94 ppm (Table 2). These concentrations are usually detected in wines and contribute to the aroma of the product.
The acetaldehyde content in wines usually ranges between 13 and 40 mg l 1 (Longo et al. 1992) and may reach 75 mg l 1 (Koutinas and Refanis 1992). In the present experiment, acet-
Continuous wine fermentation 133
Table 4. Results of aroma and taste tests of wines produced from cells of S. cerevisiae immobilized on apple cuts during continuous wine fermentation compared to a commercial wine Test
Aroma Taste
S. cerevisiae immobilized on apple cuts
Commercial wine
7?471?07 6?671?35
6?871?40 4?371?57
Values are mean (s.d.). 0, Unacceptable; 10, wonderful.
aldehyde concentrations up to 90 mg l 1 were detected, in most cases, however, they were lower (Table 2). The methanol content of the wines produced during continuous fermentation by cells immobilized on apple was o100 mg l 1.Wines in general have a methanol concentration in the range of 0?1^0?2 g l 1 . The most abundant components of the total volatiles in wine were the ethyl acetate and amyl alcohols. Their percentage, determined minus methanol, remained approximately at the same levels at all the fermentation temperatures investigated (E40^50% for amyl alcohol and 12^22% for ethyl acetate). In repeated batch fermentations an increase in the percentage of ethyl acetate and a reduction of the percentage of amyl alcohols in the total volatiles was observed in the low-temperature fermentations (Kourkoutas et al. 2001). However, although the odour of amyl alcohols may be unpleasant, it has been suggested that they contribute more to the intensity of the odour of the wine than to its quality (Etievant 1991). Preliminary sensory tests carried out in this laboratory have ascertained the fruity aroma, ¢ne taste and overall improved quality of the wines produced by continuous wine-making using immobilized cells on apple pieces compared to other commercially available wines (Table 4). The commercial wine not only produced lower scores but preference of the tasters was much more variable. The preliminary taste investigation characterized the new wines as a novel, special type of wine, with a pleasant, soft aroma and fruity taste in comparison to commercial ones. In conclusion, the use of apple-immobilized support material is suitable for continuous
yeast fermentation for the purpose of winemaking. The use in a continuous process led to higher ethanol productivities compared to immobilized batch fermentations, particularly at low temperatures, and the produced wines were of good quality with a distinctive £avour pro¢le and a pleasant taste.
Acknowledgements Thanks to the Greek Foundation of Scholarships (I.K.Y) for the grant that Y.K. has been awarded.
References Argiriou, T., Kalliafas, A., Psarianos, K., Kana, K., Kanellaki, M., Koutinas, A. A. (1992) New alcohol resistant strains of S. cerevisiae species for potable alcohol production using molasses. Appl. Biochem. Biotechnol. 36, 153^161. Bardi, E. P., Bakoyianis,V., Koutinas, A. A., Kanellaki, M. (1996). Room temperature and low temperature wine making using yeast immobilized on gluten pellets. Process Biochem. 31, 425^430. Bakoyianis, V., Kanellaki, M., Kaliafas, A., Koutinas, A. A. (1992) Low-temperature wine making by immobilized cells on mineral Kissiris. J. Agric. Food Chem. 40, 1293^1296. Bakoyianis, V., Kana, K., Kaliafas, A., Koutinas, A. A. (1993) Low-temperature wine making by Kissiris-supported biocatalyst: volatile byproducts. J. Agric. Food Chem., 41, 465^468. Bakoyianis,V., Koutinas, A. A., Agelopoulos, K., Kanellaki, M. (1997) Comparative study of Kissiris, g-Alumina, and calcium alginate as supports of cells for batch and continuous wine-making at low temperatures. J. Agric. Food Chem. 45, 4884^4888. Beech, F. W. (1993) Yeasts in cider-making. In The Yeasts (Eds A. H. Rose and J. S. Harrison) pp. 195^196. London, Academic Press. Cabezudo, M. D., Gorostiza, E. F., Herraiz, M., Fernadez-Biarange, J., Garcia-Dominguez, J. A., Molera, M. J. (1978) Mixed columns made to order in gas chromatography. IV. Isothermal selective separation of alcoholic and acetic fermentation products. J. Chromat. Sci. 16, 61^67. Etievant, X. P. (1991) Wine. In: Volatile Compounds in Foods and Beverages (Ed. H. Maarse) pp. 491. Iconomou, L., Kanellaki, M., Voliotis, S., Agelopoulos, K., Koutinas, A. A. (1996) Continuous wine making by deligni¢ed cellulosic materials supported biocatalyst. Appl. Biochem. Biotechnol. 60, 303^313.
134 Y. Kourkoutas et al.
Jackson, R. S. (1994) Principles and applications. In: Wine Science. pp. 184, 245^233. New York, Academic Press. Kourkoutas, Y., Komaitis, M., Koutinas, A. A., Kanellaki, M. (2001) Wine production using yeast immobilized on apple pieces at low and room temperatures. J. Agric. Food. Chem. 47, 1417^1425. Koutinas, A. A. and Pefanis, S. (1992) Biotechnology of Foods and Drinks, University of Patras, Greece. pp. 74 Loukatos, P., Kiaris, M., Ligas, I., Bourgos, G., Kanellaki, M., Komaitis, M., Koutinas, A. A. (2000) Continuous wine making by g-Alumina-supported biocatalyst. Appl. Biochem. Biotechnol. 89, 1^13. Longo, E., Velazquez. J. B., Siero, C., Ansado, C. J., Calo, P.,Villa,T. G. (1992) Production of higher al-
cohols, ethyl acetate, acetaldehyde and other compounds by Saccharomyces cerevisiae wine strains isolated from the same region (Salnes N. W. Spain). World J. Microbiol. Biotechnol. 8, 539^541. Maicas, S., Pardo, I., Ferrer, S. (2001) The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni. Enz. Microbial. Technol. 28, 415^419. Nedovic,V. A., Durieux, A.,Van Nedervelde, L., Rosseels, P., Vandegans, J., Plaisant, A.-M., Simon, J.-P. (2000) Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells. Enz. Microbial Technol. 26, 834^839. Zoeklein, B., Fugelsang, K., Gump, B., Nury, F. (1990) Volatile acidity. In: Production Wine Analysis pp. 105^110, New York,Van Norstrand Reinhold.
doi:10.1006/fmic.2001.0468
ORIGINAL ARTICLE
Continuous wine fermentation using a psychrophilic yeast immobilized on apple cuts at di¡erent temperatures Y. Kourkoutas1, A. A. Koutinas1, M. Kanellaki1, I. M. Banat2,* and R. Marchant2
Apple-supported biocatalyst prepared by immobilization of cells of an alcohol-resistant psychrophilic Saccharomyces cerevisiae AXAZ-1 on apple cuts was found to be suitable for continuous wine fermentation at temperatures between 5 and 151C.Wine productivities were much higher than those reported using repeated batch fermentation at low temperatures. Ethanol productivity at 51C was equal to that obtained by traditional fermentation at temperatures of 22^251C. The continuous fermentation bio reactor was operated for 95 days without any diminution of the ethanol productivity. Total and volatile acids were similar to those found in dry wines. The formation of methanol, ethyl acetate, propanol-1, isobutanol and amyl alcohols (2-methylbutanol-1and 3-methylbutanol-1) were also monitored. The concentrations of higher alcohols were at low levels. At temperatures 4101C the concentrations of amyl alcohols were lower than those previously reported for repeated batch fermentations. The concentration of ethyl acetate was o100 mg l 1 in all cases. Preliminary sensory tests carried out in the laboratory ascertained the fruity aroma, ¢ne taste and the overall improved quality of the wines produced compared to other commercially available wines. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Continuous processes are favoured in many ¢elds of the fermentation industry for many obvious reasons, including the economic advantages of having uninterrupted operation for longer periods (Jackson 1994). Continuous processes can generally achieve substantial improvements in e⁄ciency of the process and subsequently higher productivities and lower operating costs.
*Corresponding author. Fax: 0044 2870 324911. E-mail: [email protected] 0740 -0020/02/2^30127 +08 $35.00/0
The use of immobilized cells for continuous wine-making is an attractive process that has received great attention during the past few years (Bakoyianis et al. 1992, Iconomou et al. 1996, Maicas et al. 2001, Nedovic et al. 2000). Wine manufacturers know that low-temperature fermentation leads to higher quality products. However, only a few papers concerning continuous wine fermentation using immobilized cells at low temperatures have been published (Bakoyianis et al. 1993, 1997, Loukatos et al. 2000). The availability of a low-temperature continuous fermentation technology using immobilized yeast cells is likely to have desirable commercial applications. r 2002 Elsevier Science Ltd. All rights reserved.
Received: 28 September 2001 1 Food Biotechnology Group, Department of Chemistry, Section of Analytical, Environmental and Applied Chemistry, University of Patras, GR-26500, Patras, Greece 2 Biotechnology Group, School of Biological and Environmental Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK
128 Y. Kourkoutas et al.
The development of such a continuous wine production technology using immobilized cells has not been achieved for several reasons, including the lack of a suitable low-cost support material, unexplored taste and aroma quality for the produced wine and lower viability of the immobilized system. In the present study apple pieces were used as a support material for immobilization as apple is an inexpensive, abundant food material that has previously been successfully used for immobilization of Saccharomyces cerevisiae yeast cells for wine production using repeated batch fermentations (Kourkoutas et al. 2001). This support was used in a packed bed reactor to accomplish continuous fermentation of grape must.
Materials and Methods Yeast strain An alcohol-resistant psychrophilic S. cerevisiae AXAZ-1 yeast strain isolated from an agricultural area in Greece (Argiriou et al. 1992) was used in the present study. It was grown on medium containing (% w/v): glucose 4%, yeast extract 0?4%, (NH4 )2 SO4 0?1%, KH2 PO4 0?1% and MgSO4 0?5%. Yeast cells (15^20 g wet weight) were prepared as described by Argiriou et al. (1992) and employed directly in the fermentations.
Preparation of grape must Concentrated grape must was diluted with distilled water to a ¢nal 1Be density range 11?2^ 12?0 1Be (E19^21% w/v initial sugar concentration). The must was used without any nutrient addition or adjustments after sterilization at 1301C for 15 min.
Support and immobilization of cells The immobilization of the yeast cells on apple pieces was carried out as described previously (Kourkoutas et al. 2001). In brief, pieces of apple (425 g) were placed in a glass cylinder of 1 l and 500 ml of culture medium were added. The culture medium contained (in % w/v): 12% glucose, 0?4% yeast extract, 0?1% (NH4 )2 S04, 0?1%
KH2 P04 and 0?5% MgS04 in distilled water with no pH adjustment. A 10 g wet weight of yeast cells was added to the bioreactor and allowed to ferment until the must density was approximately 0?51Be. The fermented liquid was decanted and the support was washed twice with 400 ml of culture medium and then used for wine production.
Pilot fermentation Continuous wine, making was carried out in a 2 -l glass bioreactor (70 cm height and 3?1cm internal diameter), into which 720 ml grape must and 1200 g apple-supported biocatalyst were added. The apple-supported biocatalyst was used in six consecutive repeated batch fermentation experiments for grape must before switching into continuous mode. This was necessary to obtain a steady volume of apple as it decreased because of sugar fermentation during the ¢rst four batches of fermentation (Kourkoutas et al. 2001). Must was fed into the bioreactor in an upstream £ow by a high accuracy peristaltic pump (Cole Parmer Instruments Co., Chicago, Illinois, USA).
Experimental procedure Grape must with an initial 1Be density within the range 11?2^12?0 1Be was continuously supplied to the bioreactor. The £ow rate was reduced as the temperature was lowered, from an initial 515 to 155 ml/day. The reactor was operated for 95 days and the fermentation temperature was decreased gradually from 301C to 51C. Temperature decrease was carried out at the rate of 2^31C/day to avoid sudden changes. Samples were collected at di¡erent temperatures after 2 days of pumping to allow time to achieve steady conditions in the bioreactor and analysed for 1Be density, residual sugar, alcohol concentration, total and volatile acids, methanol and volatile by-products.
Analytical assays Alcohol concentrations were determined using a Gay^Lussac Alcoholmeter and productivity (expressed in grams of ethanol per volume produced per day) was calculated by multiplying
Continuous wine fermentation 129
the dilution rate by ethanol concentration. Dilution rates were calculated by dividing the £ow rate of liquid by the total volume of the fermenter. Wine productivity was calculated as grams of wine per litre of total volume produced per day and was calculated by multiplying the £ow rate (ml/day) by the density of wine (E1 gl 1 ) and dividing by the total volume (2 l). Residual sugar was determined by high-performance liquid chromatography, using a Shimadzu chromatograph with a SCR-101N stainless steel column, a LC-9A pump, a CTO10A oven at 601C and a RID- 6A refractive index detector. Three times distilled water was used as mobile phase with a £ow rate of 0?8 ml min 1 , and butanol-1 was used as an internal standard. Samples of 0?5 ml wine and 2?5 ml of a 1% (v/v) solution of butanol-1 were diluted to 50 ml, and 40 ml was injected directly to the column.The residual sugar concentration was calculated using standard curves and expressed in terms of grams of residual sugar per litre. Total acidity was estimated by titration of samples with 0?1 M NaOH solution and volatile acidity by titration with 0?1 M NaOH of distillates obtained by steam distillation of wine samples (Zoeklein et al. 1990).
Determination of volatile by-products Acetaldehyde, ethyl acetate, propanol-1, isobutanol and amyl alcohols were determined by gas chromatography using a stainless steel column, packed with Escarto-5905 consisting of Squalene 5%, Carbowax-300 90% and diethyl-hexyl sebacate 5% (v/v) (Cabezudo et al. 1978). Nitrogen was used as a carrier gas at 20 ml min 1 . Injection port and FID detector temperatures were 2101C and 2201C, respectively. The column temperature was 701C. In all cases, the internal standard was butanol-1 at a concentration of 0?5% (v/v). Samples of 4 ml of wine were injected directly into the column and the concentrations of the above volatile compounds were determined using standard curves. Methanol was also determined by gas chromatography using Porapac S column. Nitrogen was used as a carrier gas at 40 ml min 1 . The column temperature was programmed at 120^1701C at a rate 101C/min. The temperatures of the injector and FID de-
tector were 210 and 2201C, respectively. For the methanol determination, 2 -ml samples were injected directly into the column and the concentration of methanol was determined using standard curves. Butanol-1 was used as internal standard at a concentration of 0?5% (v/v).
Preliminary taste investigation Samples of the wine produced were kept at 4^ 51C for 1 month and then tested for their aroma and taste characteristics compared to a commercial dry wine produced locally using similar must types. Ten tasters familiar with wine tastes were asked to give scores on a 0^10 scale using locally approved protocols in our laboratories for taste and aroma. The sensory evaluation was a blind test in a coloured glass under low light.
Results and Discussion Continuous fermentation The fermentation of must in a continuous bioreactor was carried out to investigate the operational stability of the immobilized yeast strain AXAZ-1 on apple pieces and the suitability for continuous process and for comparison with repeated batch fermentations with regard to wine-making. This strain had the advantage of being both alcohol-resistant and a psychrophile. These characteristics were considered advantageous for low-temperature wine-making. The use of an apple-supported biocatalyst prepared by the immobilization of a yeast strain on pieces of apple was reported to be suitable for the production of high alcohol-containing wine under batch fermentation conditions (Kourkoutas et al. 2001). Immobilization of the yeast on the support may take place as a result of hydrogen bonding, entrapment of the cells in the apple structure, Van der Waals forces or adsorption caused by charges on microbial cell walls and the apple cell mass (Bardi et al. 1996). To investigate continuous wine-making with an applesupported biocatalyst the initial 1Be density of must was kept relatively constant, and all the
130 Y. Kourkoutas et al.
Temperature (˚C)
˚Be density
must used was prepared from the same grape cultivar. The results of the continuous fermentation are summarized in Fig. 1 and Table 1. The reactor was operated over 3 months and the operational stability of the system for wine-making at low temperatures (15^51C) was observed for at least 71 days. The operational stability of the system can be attributed to the high alcohol resistance of apple-supported biocatalyst as well as to the psychrophilic nature of the yeast strain used. The apple-supported biocatalyst proved to be mechanically and chemically stable for the duration of the production. The 1Be density of the e¥uent was relatively constant during the whole experiment. The £ow rate of the must was reduced when the temperature was reduced to allow more time for complete fermentation at the lower temperatures. Wine productivities were much higher than those obtained in repeated batch fermentation, particularly at lower tempera-
12 10 8 6 4 2 0 30
Influent Effluent
Temperature
25 20 15 10 5 600
Flow rate
Flow rate (ml)
500 400 300 200 100 0
20
40
60
80
100
Time (days)
Figure 1. Flow rate of must and 1Be densities of fermentations as related to temperature changes in the range 5^301C.
tures (Kourkoutas et al. 2001). Ethanol productivity of the system measured at 51C represented about 28% of that obtained at 301C. This productivity at 51C is equal to that achieved in traditional fermentations over a temperature range of 22^251C. Free cell concentrations in the e¥uent ranged from 15 g l 1 wet weight at 301C to 5 g l 1 at 5 g l 1 wet weight. The biomass leakage was higher at high temperatures, as higher £ow rates were used.Volatile acid levels were similar to those found in dry wines. Although in most cases small amounts of residual sugar remained, it is unlikely that the volatile acidity would rise if the fermentation were completed because almost all the sugar was utilized. The total acidity of the product was slightly increased, either because of the transfer of some apple acids to the produced wine at the early stages of the continuous process, or to the formation of acids during fermentation. Similar observations were previously reported by other researchers during fermentation of apple juices to produce cider (Beech 1993). It was not necessary to pretreat the apple cuts to extract the acids before using them as support because most of the apple acids are normal constituents of the wine and may contribute positively to the £avour of the product. In addition, the observed concentrations were within the normal ranges present in dry wines (4^6 g l 1 of tartaric acid). Total acidity was in the range 3?6^6?6 g l 1 of tartaric acid at 30^151C, and decreased slightly as the temperature dropped from 10 to 51C. This reduction can be attributed to the increase of crystallization of tartrate salts with the decrease in temperature. This has been observed previously in repeated batch fermentations (Kourkoutas et al. 2001). In addition, the apple micro£ora did not a¡ect the fermentation and quality of the ¢nal product because the immobilized yeast dominated in the fermentation broth because of the low pH, the relatively high ethanol concentration and total acidity of the wine and the high immobilized yeast biomass. As apple cuts proved to be a suitable support for continuous wine-making, particularly with the advantage of being abundantly available, cheap and easy to use for immobilization, the
Continuous wine fermentation 131
Table 1. Characteristics of the wine produced by continuous fermentation of must at various temperatures using S. cerevisiae AXAZ-1 yeast cells immobilized on apple cuts Temperature Initial 1Be Wine Daily (1C) density produced wine (ml day 1 ) productivity (g l 1 )
Total Volatile Ethanol Residual Ethanol acidity acidity concen- sugar productivity tration (g l 1 ) (g l 1 day 1 ) (g l 1 tartaric (g l 1 acetic (% vol) acid) acid)
30
11?4 11?9 11?8 11?8 12?0
515 515 515 515 515
258 258 258 258 258
9?9 11?1 10?7 9?5 11?5
4?2 15?4 15?7 16?9 10?9
20 23 22 19 23
4?4 5?0 6?5 4?8 5?5
0?07 0?19 0?11 0?14 0?14
25
11?5 11?8 11?8 11?6 11?7
550 550 550 550 550
275 275 275 275 275
9?9 9?7 10?5 11?4 10?8
9?4 12?1 4?9 12?2 17?7
22 21 23 25 23
6?6 4?8 3?7 4?5 4?7
0?10 0?13 0?13 0?19 0?32
20
11?7 12?1 11?6 11?7 11?9
550 550 550 550 550
275 275 275 275 275
10?1 11?1 10?3 10?2 11?2
10?3 9?3 19?6 23?6 25?6
22 20 22 22 24
4?4 3?7 4?0 3?6 5?0
0?13 0?25 0?13 0?17 0?34
15
11?7 11?5 11?5 11?5
325 325 325 325
163 163 163 163
9?0 11?0 10?2 11?3
11?3 12?0 8?9 14?4
12 14 13 15
4?9 4?9 5?1 5?2
0?37 0?41 0?44 0?41
10
11?8 11?8 11?6 11?6 11?6
215 215 215 215 215
108 108 108 108 108
11?2 10?9 11?6 10?8 10?8
11?9 12?9 30?4 33?8 22?2
10 9 10 9 9
3?2 3?9 4?2 4?4 4?4
0?14 0?17 0?22 0?22 0?17
5
11?5 11?4 11?3 11?5 11?5
155 155 155 155 155
78 78 78 78 78
8?8 9?3 10?2 10?2 9?9
18?3 37?1 34?0 18?3 15?9
5 6 6 6 6
4?6 4?3 4?3 4?4 4?3
0?22 0?22 0?26 0?38 0?29
¢nal consideration for use of this technique was the quality of the produced product.
Volatile by-products The e¡ects of temperature on the formation of the volatile compounds during continuous wine-making, using an apple-supported biocatalyst are shown in Table 2. Table 3 also compares the volatile by-products formed during the continuous process to the volatiles of the same must be accomplished by traditional batch fermentation. The concentrations of higher alcohols (propanol-1 and isobutyl alcohol) were low. They
were in the ranges 18^40 ppm and 14^47 ppm, respectively (Jackson 1994).The concentrations of isobutyl alcohol in the wine produced by the continuous fermentation were much lower than in the wine produced by traditional batch fermentation (Table 3). The concentrations of amyl alcohols were also low at all fermentation temperatures. At temperatures 4101C the concentrations of amyl alcohols were much lower than those obtained using the repeated batch fermentation technique (Kourkoutas et al. 2001) and the traditional batch fermentation (Table 3). These results indicate that the wine product has improved quality because of lower concentrations of higher alcohols.
132 Y. Kourkoutas et al.
Table 2. E¡ect of temperature on the formation of volatile by-products during continuous wine fermentation using S. cerevisiae AXAZ-1 yeast cells immobilized on apple cuts Acetaldehyde (ppm)
Ethyl acetate (ppm)
Propanol-1 (ppm)
Isobutyl alcohol (ppm)
Amyl alcohols (ppm)
Methanol (ppm)
30
13 38 30 27 26
57 51 66 59 64
22 20 22 20 24
26 17 15 16 21
142 113 109 108 142
36 23 42 42 50
25
13 28 25 37 36
37 50 74 48 39
24 22 25 28 25
24 27 29 28 22
137 148 176 154 137
36 12 12 41 5
20
29 49 25 44 52
88 74 65 56 48
18 19 22 21 24
24 38 27 28 30
135 160 148 135 127
13 79 21 86 25
15
80 56 64 40
75 94 86 61
27 26 34 28
45 47 36 34
139 155 143 128
12 69 28 61
10
23 51 61 87 90
46 30 48 44 23
26 40 38 33 38
27 31 31 27 27
134 154 147 125 139
37 48 31 42 35
5
30 38 62 46 18
33 35 24 43 42
29 30 26 24 22
20 22 19 18 14
106 111 104 96 106
37 47 47 47 58
Temperature (1C)
Table 3. Volatile by-products formed during continuous wine fermentation using S. cerevisiae AXAZ-1 yeast cells immobilized on apple cuts in comparison with volatiles formed during traditional batch fermentation using free cells. All fermentations were carried out at 301C Wine
Continuous process Traditional batch fermentation
Acetaldehyde (ppm)
Ethyl acetate (ppm)
Propanol-1 (ppm)
Isobutyl alcohol (ppm)
Amyl alcohols (ppm)
methanol (ppm)
27 64
59 31
22 10
19 54
123 169
39 n.d.
n.d., none detected.
Ethyl acetate concentrations ranged from 23 to 94 ppm (Table 2). These concentrations are usually detected in wines and contribute to the aroma of the product.
The acetaldehyde content in wines usually ranges between 13 and 40 mg l 1 (Longo et al. 1992) and may reach 75 mg l 1 (Koutinas and Refanis 1992). In the present experiment, acet-
Continuous wine fermentation 133
Table 4. Results of aroma and taste tests of wines produced from cells of S. cerevisiae immobilized on apple cuts during continuous wine fermentation compared to a commercial wine Test
Aroma Taste
S. cerevisiae immobilized on apple cuts
Commercial wine
7?471?07 6?671?35
6?871?40 4?371?57
Values are mean (s.d.). 0, Unacceptable; 10, wonderful.
aldehyde concentrations up to 90 mg l 1 were detected, in most cases, however, they were lower (Table 2). The methanol content of the wines produced during continuous fermentation by cells immobilized on apple was o100 mg l 1.Wines in general have a methanol concentration in the range of 0?1^0?2 g l 1 . The most abundant components of the total volatiles in wine were the ethyl acetate and amyl alcohols. Their percentage, determined minus methanol, remained approximately at the same levels at all the fermentation temperatures investigated (E40^50% for amyl alcohol and 12^22% for ethyl acetate). In repeated batch fermentations an increase in the percentage of ethyl acetate and a reduction of the percentage of amyl alcohols in the total volatiles was observed in the low-temperature fermentations (Kourkoutas et al. 2001). However, although the odour of amyl alcohols may be unpleasant, it has been suggested that they contribute more to the intensity of the odour of the wine than to its quality (Etievant 1991). Preliminary sensory tests carried out in this laboratory have ascertained the fruity aroma, ¢ne taste and overall improved quality of the wines produced by continuous wine-making using immobilized cells on apple pieces compared to other commercially available wines (Table 4). The commercial wine not only produced lower scores but preference of the tasters was much more variable. The preliminary taste investigation characterized the new wines as a novel, special type of wine, with a pleasant, soft aroma and fruity taste in comparison to commercial ones. In conclusion, the use of apple-immobilized support material is suitable for continuous
yeast fermentation for the purpose of winemaking. The use in a continuous process led to higher ethanol productivities compared to immobilized batch fermentations, particularly at low temperatures, and the produced wines were of good quality with a distinctive £avour pro¢le and a pleasant taste.
Acknowledgements Thanks to the Greek Foundation of Scholarships (I.K.Y) for the grant that Y.K. has been awarded.
References Argiriou, T., Kalliafas, A., Psarianos, K., Kana, K., Kanellaki, M., Koutinas, A. A. (1992) New alcohol resistant strains of S. cerevisiae species for potable alcohol production using molasses. Appl. Biochem. Biotechnol. 36, 153^161. Bardi, E. P., Bakoyianis,V., Koutinas, A. A., Kanellaki, M. (1996). Room temperature and low temperature wine making using yeast immobilized on gluten pellets. Process Biochem. 31, 425^430. Bakoyianis, V., Kanellaki, M., Kaliafas, A., Koutinas, A. A. (1992) Low-temperature wine making by immobilized cells on mineral Kissiris. J. Agric. Food Chem. 40, 1293^1296. Bakoyianis, V., Kana, K., Kaliafas, A., Koutinas, A. A. (1993) Low-temperature wine making by Kissiris-supported biocatalyst: volatile byproducts. J. Agric. Food Chem., 41, 465^468. Bakoyianis,V., Koutinas, A. A., Agelopoulos, K., Kanellaki, M. (1997) Comparative study of Kissiris, g-Alumina, and calcium alginate as supports of cells for batch and continuous wine-making at low temperatures. J. Agric. Food Chem. 45, 4884^4888. Beech, F. W. (1993) Yeasts in cider-making. In The Yeasts (Eds A. H. Rose and J. S. Harrison) pp. 195^196. London, Academic Press. Cabezudo, M. D., Gorostiza, E. F., Herraiz, M., Fernadez-Biarange, J., Garcia-Dominguez, J. A., Molera, M. J. (1978) Mixed columns made to order in gas chromatography. IV. Isothermal selective separation of alcoholic and acetic fermentation products. J. Chromat. Sci. 16, 61^67. Etievant, X. P. (1991) Wine. In: Volatile Compounds in Foods and Beverages (Ed. H. Maarse) pp. 491. Iconomou, L., Kanellaki, M., Voliotis, S., Agelopoulos, K., Koutinas, A. A. (1996) Continuous wine making by deligni¢ed cellulosic materials supported biocatalyst. Appl. Biochem. Biotechnol. 60, 303^313.
134 Y. Kourkoutas et al.
Jackson, R. S. (1994) Principles and applications. In: Wine Science. pp. 184, 245^233. New York, Academic Press. Kourkoutas, Y., Komaitis, M., Koutinas, A. A., Kanellaki, M. (2001) Wine production using yeast immobilized on apple pieces at low and room temperatures. J. Agric. Food. Chem. 47, 1417^1425. Koutinas, A. A. and Pefanis, S. (1992) Biotechnology of Foods and Drinks, University of Patras, Greece. pp. 74 Loukatos, P., Kiaris, M., Ligas, I., Bourgos, G., Kanellaki, M., Komaitis, M., Koutinas, A. A. (2000) Continuous wine making by g-Alumina-supported biocatalyst. Appl. Biochem. Biotechnol. 89, 1^13. Longo, E., Velazquez. J. B., Siero, C., Ansado, C. J., Calo, P.,Villa,T. G. (1992) Production of higher al-
cohols, ethyl acetate, acetaldehyde and other compounds by Saccharomyces cerevisiae wine strains isolated from the same region (Salnes N. W. Spain). World J. Microbiol. Biotechnol. 8, 539^541. Maicas, S., Pardo, I., Ferrer, S. (2001) The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni. Enz. Microbial. Technol. 28, 415^419. Nedovic,V. A., Durieux, A.,Van Nedervelde, L., Rosseels, P., Vandegans, J., Plaisant, A.-M., Simon, J.-P. (2000) Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells. Enz. Microbial Technol. 26, 834^839. Zoeklein, B., Fugelsang, K., Gump, B., Nury, F. (1990) Volatile acidity. In: Production Wine Analysis pp. 105^110, New York,Van Norstrand Reinhold.