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Low temperature and ambient temperature wine making using freeze dried immobilized cells on gluten pellets
Process Biochemistry 37 (2002) 707– 717 www.elsevier.com/locate/procbio
Low temperature and ambient temperature wine making using freeze dried immobilized cells on gluten pellets M. Iconomopoulou, K. Psarianos, M. Kanellaki, A.A. Koutinas * Section of Analytical, En6ironmental and Applied Chemistry, Department of Chemistry, Uni6ersity of Patras, 26500, Patras, Greece Received 28 November 2000; received in revised form 28 March 2001; accepted 21 July 2001
Abstract Low temperature wine making using freeze-dried gluten supported biocatalyst (FGB) is reported to be feasible. The biocatalyst was prepared by cell immobilization of the strain AXAZ-1 on gluten pellets followed by freeze drying. Batch fermentations were performed and the effects of initial Be density, temperature and total acidity of must on kinetic parameters were examined. Improved results were shown by freeze-dried immobilized cells on gluten as compared with free freeze-dried cells (ffdc). Fermentation times obtained at low temperatures (5–15 °C) could be accepted in industry. The total acidity of grape must does not affect the fermentation time while freeze-dried immobilized cells seem to have an operational stability from batch to batch. Amyl alcohols, the main volatile, are reduced by the decrease of the temperature below 20 °C. The percentages of amyl alcohols as a function of total volatiles were reduced and those of ethyl acetate were increased by the decrease of temperature, indicating improvements in quality and nutritional value. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Freeze dried; Immobilization; Wine making; Gluten pellets; Low-temperature
1. Introduction Considerable attention has been given over the last 15 years to the cell immobilization of yeasts in wine making. However, while other prerequisites are necessary for the industrialization of immobilized cells, technical and industrial experience lead to the following conclusions. (i) The technical problems of this technique and the training in the new technology of immobilized cells mainly for small wine making enterprises are obstacles for industrialization. (ii) The new bioreactor system for easy filling and emptying by the support and (iii) the high operational stability of the bioreactor are the new prerequisites for a cost effective application of immobilized cells on an industrial scale. Cell immobilization has been studied extensively in wine making. Yeast cells fixed in gels forming materials such as sodium alginate [1], agar, k-carrageenan and pectic acid [2] have been used to produce wines. Fruit * Corresponding author. Tel.: + 3061-997104; fax: +3061-997105. E-mail address: [email protected] (A.A. Koutinas).
[3] and sparkling wines [4] were also produced by immobilized yeasts in alginate gels. Cellulosic supports covered by sodium alginate gel [5] and DEAE-cellulose [6] were also reported in wine making. Likewise, delignified cellulosic material [7] and gluten pellets [8] supported biocatalysts were proposed for low-temperature wine making. Inorganic supports proposed also in wine making [9,10], are abundant and of low cost but do not meet the prerequisite of food grade purity (due to toxic heavy metals). To accommodate the industrial application of cell immobilization in wine making freeze-dried immobilized cells are proposed. That could be produced by new enterprises and will substitute free freeze-dried wine yeasts and traditional natural fermentation. This could be successfully performed provided that freezedried immobilized cells would improve the rate of fermentation and the quality of the wine. Low-temperature fermentations improve the quality of wine and reduce toxicity [11]. Therefore, the aim of this investigation was a study of low temperature wine making by freeze dried gluten supported biocatalyst (FGB).
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2. Materials and methods
2.1. Immobilization of AXAZ-1 cells on gluten pellets AXAZ-1, an alcohol resistant and cryotolerant Saccharomyces cere6isiae strain, isolated locally in the Achaia province of Greece [12] was used in all experiments. It was grown in a complete medium and pressed wet weight cells as in the aforementioned reference were prepared and employed. Gluten pellets were used as support for cell immobilization. The preparation of gluten pellets was performed as in a previous study [8].
2.2. Preparation of freeze-dried cells 2.2.1. Preparation of freeze-dried gluten supported biocatalyst The preparation of wet gluten supported biocatalyst was made by the immobilization of cells on gluten pellets as described previously. To prepare freeze-dried immobilized cells on gluten pellets without protective media, wet immobilized cells on gluten were cooled with a cooling rate of 3 °C/min to −40 °C and then freeze-dried overnight at 15– 5 ×10 − 3 Bar and at − 50 °C in a Freeze Dry System, Freezone 4.5 (Labconco, Kansas City, MO).
cells were also freeze dried, and subsequently added into the second flask. Into the third 100 g wet gluten supported biocatalyst having also 4.2 g wet weight immobilized cells were also used. The flasks were incubated at 30 °C and the fermentations were carried out without agitation.
2.3.2. Effect of temperature The above process was repeated with must of 204 g/l initial sugar concentration. Batch fermentations were performed at 5, 10, 15, 20 and 30 °C using an initial wet weight cell concentration of 14 g/l. Likewise, a second repeated batch was fermented employing the same gluten supported biocatalyst. Before the completion of fermentation the liquid was filtered using a Buchner funnel and the biocatalyst was washed once with 200 ml grape must. 2.3.3. Effect of total acidity Most of the 204 g/l initial sugar concentration was used and batch fermentations as above were carried out at 30 °C for three different total acidities of 4, 6 and 8 g tartraric acid per l. Fermentations with free and immobilized cells were carried out by using an initial wet weight cell concentration of 11.7 g/l. 2.4. Analysis
2.2.2. Preparation of free freeze-dried cells Free freeze-dried cells were also prepared using wet biomass produced after growth in a complete medium. Free freeze-dried cells were prepared for comparative study with freeze-dried immobilized cells. 2.3. Fermentations Fermentations were performed using red grape must (Roditis) of different initial sugar concentrations. This must was sterilized in an autoclave at 130 °C for 15 min. The pH was adjusted to 3.6 and the initial total acidity was 4 g tartraric acid per l. Repeated batch fermentations of grape must were carried out separately using: (a) freeze-dried gluten supported biocatalyst (F1GB), (b) free freeze dried cells (ffdc), (c) wet gluten supported biocatalyst (WGB) and (e) wet free cells. The effects of initial sugar concentration, temperature and total acidity were examined.
2.3.1. Effect of initial sugar concentration Three 500 ml Erlenmeyer flasks were used for each initial sugar concentration and each contained 300 ml grape must. The experiments were performed using 136, 170, 204, 238, 272 and 306 g/l initial sugar concentration. In each experiment 100 g gluten supported biocatalyst that hold 4.2 g wet weight cells were freeze dried, pretreated with grape must and added into the first Erlenmeyer flask. In a similar way 4.2 g wet weight free
Fermentation kinetics were performed by measuring the °Be density at various time intervals. Samples were collected and analyzed for ethanol concentration, residual sugar and volatile by-products. All values were the mean of three repetitions. The standard deviation for ethanol concentration was B 9 0.2, for ethanol productivity B 90.2, for residual sugar B 90.3 and conversion B 9 0.3.
2.4.1. Analysis of alcohol and residual sugar Ethanol and residual sugar concentrations were obtained by HPLC analysis. A SHIMADZU liquid chromatograph with a high pressure pump model LC-9A, a constant temperature oven C-R 6A and a refractive index detector RID-6A connected with an integrator C-R 6A was used. A column SCR-101N packed with a cationic resin was used. The temperature was set at 60 °C and an aqueous mobile phase with a flow rate of 0.8 ml/min was used. Ethanol productivity was expressed as g of ethanol per l substrate per h. 2.4.2. Analysis of 6olatiles Acetaldehyde, ethyl acetate, propanol-1, isobutyl alcohol and amyl alcohols were determined using a stainless steel column, packed with Escatro 5905 [consisting of squalene, 5%; Carbowax 300, 90% and Di-2-ethylhexyl sebacate 5% (v/v)], with N2 as carrier gas (20 ml/min). The injection port and detector temperatures
M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717
were 210 °C and the column temperature was 60 °C. The internal standard was pentanol-1 at a concentration of 0.5% v/v. Samples of 2 ml of the wine were injected directly in the column.
3. Results and discussion
3.1. Study of wine making Gluten meets the prerequisites for cost effective industrial immobilization of yeast. These prerequisites are low cost, abundance in nature, food grade purity and the support to be enough heavy material to avoid the breaking of pellets. These are more necessary in the production of freeze-dried immobilized cells in comparison with wet immobilized cells due to the further cost of lyophilization. Freeze-dried immobilized cells will lead to development of new enterprises for the exploitation of this marketable product. This will facilitate the technology of immobilized cells to be used by wine makers of low production capacity. Gluten pellets supported biocatalyst was prepared by immobilization of yeast cells on gluten pellets and subsequently lyophilized. The freeze-dried biocatalyst was employed in fermentations of grape must to examine it in wine making. So the effect of initial sugar concentration, temperature and total acidity were examined. Likewise, results by freeze-dried immobilized cells in repeated batch fermentations are also reported. The results of the aforementioned research are summarized in Tables 1–3 and Fig. 1.
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Table 1 shows the effect of initial sugar concentration on batch fermentation of grape must by freeze-dried immobilized cells in comparison with ffdc and wet gluten supported biocatalyst (WGB). The results indicate F1GB to ferment grape must be in the range of 204–272 g/l initial sugar concentration providing an alcohol concentration of 12–13.8% v/v which is the alcohol percentage of traditionally produced wines. These alcohol concentrations were obtained in a fermentation time of about 4–8 days, and are considered less than by natural fermentation of grape must. Startup of the fermentation of grape must by F1GB was about 1 day, which is equal to that of natural fermentation. However, it was less than those of ffdc. Likewise, F1GB leads to higher productivities as compared with ffdc while the alcohol concentration was about the same. WGB resulted in improved results as compared with F1GB in all parameters studied. Table 2 shows the effect of temperature on wine making by F1GB in comparison with ffdc and WGB. At all temperatures studied F1GB lead to alcohol concentrations equal to those of dry wines with low residual sugar and high conversion. Low-temperature fermentations in the range of 5–20 °C resulted in fermentation times and productivity that could be accepted by the industry. Temperatures of 10, 15, 20 °C gave fermentation times near to those of natural fermentation. Likewise, the 60 days fermentation time at 5 °C may mean that high quality wines would be produced. These results were improved when compared with those of ffdc. WGB leads to better results in comparison with F1GB. Total acidities in the range of
Fig. 1. Fermentation kinetics observed at various temperatures and 204 g/l initial sugar concentration in wine making by freeze dried gluten supported biocatalyst (FGB1,2), Free freeze dried cells (ffdc) (where 1 and 2 means first and second repeated batch). Internal figure analyses kinetics at 30 °C plots °Be density versus time.
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Biocatalyst
Initial sugar concentration g/l
Start-up (h)
Fermentation time (h) Ethanol concentration (g/l) Ethanol productivity (g/l/h)
Residual sugar (g/l)
Conversion (%)
F1GB
136 170 204 238 272 306 136 170 204 238 272 306 136 170 204 238 272 306
18 24 24 24 24 30 24 26 48 31 48 96 0.4 0.4 0.4 0.4 0.5 0.6
57 73 100 115 170 194 84 95 110 140 240 240 36 48 54 60 69 141
5.0 8.9 2.8 12.4 51.6 121.4 3.9 5.6 7.6 22.1 65.0 105.6 0.5 3.3 3.8 8.5 13.6 42.16
96.3 94.7 98.6 94.7 81.0 60.3 97.1 96.7 96.2 90.7 76.1 65.4 99.6 98.0 98.1 96.4 95.0 86.2
ffdc
WGB
63.1 76.4 94.1 102.9 107.6 85.8 63.1 85.0 93.6 100.6 102.1 93.6 62.4 73.3 93.6 108.4 120.1 125.5
1.10 1.04 0.94 0.89 0.63 0.44 0.75 0.89 0.85 0.71 0.42 0.39 1.73 1.52 1.73 1.80 1.74 0.89
M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717
Table 1 Effect of initial sugar concentration on fermentation kinetic parameters observed at 30 °C in batch wine making by freeze dried gluten supported biocatalyst (F1GB), free freeze dried cells (ffdc) and wet gluten supported biocatalyst (WGB)
Biocatalyst
Temperature (°C)
Start-up (days)
Fermentation time (days)
Ethanol concentration (g/l)
Ethanol productivity (g/l/h)
Residual sugar (g/l)
Conversion (%)
F1GB
5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30
4 8 3 1 1 1 8 8 3 3 2 2 0.5 0.4 0.4 0.3 0.3 0.3
60 32 16 9 6 4 66 36 15 8 8 5 11.2 4.0 3.1 2.5 2.3 2.25
89.7 89.7 90.4 91.2 87.3 94.1 88.9 88.9 91.2 89.7 92.8 93.6 92.0 92.0 92.8 93.6 93.6 93.6
0.06 0.11 0.23 0.44 0.58 0.94 0.05 0.10 0.25 0.46 0.49 0.85 0.34 0.94 1.22 1.50 1.60 1.73
5.1 10.7 6.4 2.8 3.3 2.8 7.6 6.7 5.5 5.6 20.7 7.6 3.1 4.4 2.8 2.6 1.9 3.8
97.5 94.7 96.8 98.6 98.3 98.6 96.2 96.7 97.3 97.2 89.8 96.2 98.4 97.8 98.6 98.7 99.0 98.1
ffdc
WGB
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Table 2 Effect of temperature on fermentation kinetic parameters observed (204 (g/l) initial sugar concentration) in grape must fermentation by freeze dried gluten supported biocatalyst (F1GB), free freeze dried cells (ffdc) and wet gluten supported biocatalyst (WGB)
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6 g/l
8 g/l
10 g/l
Repeated Start-up (h) batch fermentations
Fermentationt Ethanol Residual ime (h) concentration sugar (g/l) (g/l)
Start-up (h)
Fermentationt Ethanol Residual ime (h) concentration sugar (g/l) (g/l)
Start-up (h)
Fermentationt Ethanol Residual ime (h) concentration sugar (g/l) (g/l)
1 2 3 4 5 6 7 Freeze dried free cells
82 78 75 80 76 80 72 102
48.0 0.6 0.5 0.5 0.4 0.4 0.4 24
81 78 78 79 82 86 74 99
48.0 0.6 0.5 0.5 0.4 0.4 0.4 24
82 79 79 82 81 90 76 102
48.0 0.6 0.5 0.5 0.4 0.4 0.4 24
90.5 91.2 91.2 89.7 92.0 92.8 89.7 92.0
5.5 6.3 6.7 6.1 3.4 2.5 2.3 2.8
93.6 91.2 94.4 86.6 66.3 89.7 88.9 92.8
3.9 8.8 4.7 9.8 – 7.6 – 3.1
93.6 92.8 88.1 85.8 93.6 88.9 89.7 92.8
4.2 6.7 6.4 11.5 3.2 2.8
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Table 3 Effect of total acidity on kinetic parameters in repeated batch fermentations by freeze-dried gluten supported biocatalyst at 30 °C and 204 g/l initial sugar concentration
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6– 10 g tartraric acid per l did not affect significantly start-up, fermentation time, ethanol concentration and residual sugar in wine making by FGB (Table 3). Repeated batch fermentations lead to a drastic drop in start-up, while the results clearly showed an operational stability in wine making from batch to batch. That improvement of the rate of fermentation from 1st to 2nd repeated batch fermentation is performed by FGB is shown in Fig. 1. This figure also shows FGB results in increased fermentation rate as compared with ffdc.
3.2. Formation of 6olatile by-products A study of the volatile by-products contained in wines and the affect on quality and nutritional value was also necessary. The effect of temperature on the formation of volatiles in repeated batch fermentations by FGB was studied, by placing the bioreactor in a low temperature incubator and adjusting the temperature successively to 30, 20, 15, 10 and 5 °C. Likewise, repeated batch fermentations were also carried out at various initial sugar concentrations. At every repeated batch fermentation, samples were collected after the end of each fermentation and analyzed for ethanol, acetaldehyde, ethyl acetate, propanol-1, isobutyl alcohol and amyl alcohols (total amount of 2-methylbutanol-1 and 3-methylbutanol-1). The results are presented in the Tables 4 and 5. Table 4 shows that the contents of acetaldehyde and isobutyl alcohol were reduced by the decrease in temperature from 15 to 5 °C. Likewise, it also decreased as the temperature was increased from 15 to 30 °C. Amyl alcohols also decreased on a decrease in temperature but the reduction started from 20 °C. Propanol-1 was not affected by the reduction in temperature. According to a previous study [7], WGB resulted in reduction of all of the aforementioned volatiles as the temperature was decreased from 27 to 5 °C. Increasing initial sugar concentrations also decreased acetaldehyde and isobutyl alcohol (Table 5). Propanol1 increased as the initial sugar concentration was increased while ethyl acetate, amyl alcohols and total volatiles increased up to 204 g/l initial sugar concentration.
3.3. Industrialization of no6el biocatalyst Most of the fermentations performed by FGB, resulted in alcohol concentrations in the range of 11.5– 13.8% v/v and fermentation times lower than that of wine making by natural fermentations. Furthermore, volatiles obtained by fermentations of grape must by FGB were in the same range as those of wet immobilized cells [11]. These results show the possibility for industrialization of freeze-dried immobilized cells on gluten. Therefore, the new marketable product facilitates the industrialization of immobilized cells.
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Most volatile by-products and especially amyl alcohols (the most abundant and toxic) were reduced as the temperature was decreased below 20 °C. The average concentration of amyl alcohols at 5 °C was only 46% of that formed at 20 °C. That means that extremely low-temperature fermentations can be performed, as by WGB, leading to a decrease in toxicity and an improvement in the aroma. That can be also seen in Fig. 2, where FGB percentages of amyl alcohols as a function of total volatiles were reduced as the temperature decreased, while the percentage of ethyl acetate on total volatiles increased. In contrast, the use of wet free cells lead to an increase in the percentage of amyl alcohol in the total volatiles and a decrease of ethyl acetate as the temperature was decreased. Ffdc percentages of amyl alcohols in total volatiles were reduced less in comparison to those of FGB, while the percentage of ethyl acetate was increased below 15 °C. FGB resulted in an increase of ethyl acetate 30–150% at the lower temperature of 20 °C compared with WGB [8]. Amyl alcohol concentrations obtained by FGB were higher than those by WGB [11]. The above discussion lead to the conclusion that FGB results in improved results in comparison with ffdc, regarding kinetic parameters as well as volatiles. Ffdc of wine yeasts are known commercial products. WGB will be also a commercial product, provided that this biocatalyst will pass successfully all technical problems of handling on an industrial scale. The total acidity of grape must did not affect wine making by FGB over a broad range of total acidities. This indicates that the biocatalyst could be treated in industry of Southern and Northern countries. The freeze-dried immobilized cells on gluten seems to have operational stability from batch to batch which is an indispensable factor for the industrialization of this technology. The FGB is also suitable for wine making at the initial °Be densities used on an industrial scale. Since freeze-dried immobilized cells on gluten formed upto 15% v/v alcohol in the fermentation of grape must, FGB will be also suitable for semi-sweet wine making. FGB will be suitable for wine making provided that the technical problems of scale up of the process can be overcome. If so, a new marketable product will be developed which substitutes freeze-dried wine yeasts.
Acknowledgements Iconomopoulou Matina in the frame of the program ‘Exploitation of Industrial Wastes’ thanks the General Secretariat of Research and Technology (G.S.R.T., Athens, Greece) for financial support.
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Temperature (°C)
Repeated batch wine making
Fermentation time (h)
Ethanol concentration (g/l)
Acetaldehyde (mg/l)
Ethyl acetate (mg/l)
Propanol-1 (mg/l)
Iso-butyl alcohol Amyl alcohols (mg/l) (mg/l)
Total volatiles were determined (mg/l)
5 10 15 20 30
1–2 1–3 1–4 1–6 1–7
1896 632 276 155 66
88.9 90.5 88.9 91.2 91.2
52.9 57.3 67.9 49.7 63.5
84.2 113.9 85.2 85.3 50.0
42.3 34.8 39.6 37.6 36.2
32.9 38.2 47.6 39.0 37.0
355.7 462.3 576.2 520.2 450.7
143.3 217.9 301.8 308.6 272.1
M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717
Table 4 Effect of temperature on formation of volatiles in repeated batch fermentations by freeze dried gluten supported biocatalyst using 204 g/l initial sugar concentration
Initial sugar Repeated batch concentration (g/l) fermentations
Fermentation time (h)
Ethanol concentration (g/l)
Acetaldehyde (mg/l)
Ethyl acetate (mg/l)
Propanol-1 (mg/l)
Iso-butyl alcohol Amyl alcohols (mg/l) (mg/l)
Total volatiles were determined (mg/l)
136 170 204 238
48 60 72 102
61.6 73.3 92.0 92.8
83.1 68.3 72.6 46.0
58.0 70.2 79.3 54.3
19.1 22.4 36.0 42.9
45.0 45.0 37.6 34.3
408.2 449.7 513.2 413.2
3 4 5 6
234.5 262.9 289.3 266.4
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Table 5 Effect of initial sugar concentration at 30 °C on formation of volatiles in repeated batch fermentations making by freeze dried gluten supported biocatalyst
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Fig. 2. Effect of temperature on percentages of amyl alcohols, iso-butyl alcohol and ethyl acetate on total volatiles were determined in wine making by freeze dried immobilized cells on gluten as compared with free freeze dried cells and wet free cells.
References [1] Shinobayashi Y, Tominaga K. Application of biotechnology in the food industry. I. Brewing of white wine by a bioreactor. Hokaidoritsu Kogyo Shikenjo Hokoku 1986;285:199 –204. [2] Nakanishi K, Yokotsuka K. Fermentation of white wine from
Koshu grape using immobilized yeast. Nippon Shokuhin Koggyo Gakkaishi 1987;34(6):362 – 9. [3] Mori S, Fruit wine or sake manufacture by bioreactor, Japanese Kokai Tokkyo Koho JP 6261,577, 18 March 1987. [4] Fumi M, Trioli G, Colagrande O. Preliminary assessment on the use of immobilized yeast cells in sodium alginate for sparkling wine processes. Biotechnol Lett 1987;9(5):339 – 42.
M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717 [5] Otsuka K, Wine making. Japanese Kokai Tokkyo Koho 80, 159,789, 12 December 1980. [6] Lommi H, Advenainen J. Method using immobilized yeast to produce ethanol and alcoholic beverages, European Patent Appl. EP 361,165, 4 April 1990. [7] Bardi EP, Koutinas AA. Immobilization of yeast on delignified cellulosic material for room temperature and low temperature wine making. J Agric Food Chem 1994;42(1):221 – 6. [8] Bardi EP, Bakoyanis V, Koutinas AA. Room temperature and low temperature wine making using yeast immobilized cells on gluten pellets. Process Biochem 1996;31(5):425 –30. [9] Ageeva NM, Merzhanian AA, Sobolev EM. Effect of yeast
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adsorption on the functional activity of the yeast cells and composition of wine. Mikrobiologiya 1985;54(5):830 – 4. [10] Bakoyianis V, Kanellaki M, Kaliafas A, Koutinas AA. Low temperature wine making by immobilized cells on mineral kissiris. J Agric Food Chem 1992;40(7):1293 – 6. [11] Bardi EP, Koutinas AA, Psarianos K, Kanellaki M. Volatile by-products formed in low temperature wine making by immobilized yeast cells. Process Biochem 1997;32(7):579 – 84. [12] Argiriou T, Kaliafas A, Psarianos K, Kana K, Kanellaki M, Koutinas AA. New alcohol resistant strains of S. Cere6isiae species for potable alcohol production using molasses. Appl Biochem Biotechnol 1992;36:153 – 61.
Low temperature and ambient temperature wine making using freeze dried immobilized cells on gluten pellets M. Iconomopoulou, K. Psarianos, M. Kanellaki, A.A. Koutinas * Section of Analytical, En6ironmental and Applied Chemistry, Department of Chemistry, Uni6ersity of Patras, 26500, Patras, Greece Received 28 November 2000; received in revised form 28 March 2001; accepted 21 July 2001
Abstract Low temperature wine making using freeze-dried gluten supported biocatalyst (FGB) is reported to be feasible. The biocatalyst was prepared by cell immobilization of the strain AXAZ-1 on gluten pellets followed by freeze drying. Batch fermentations were performed and the effects of initial Be density, temperature and total acidity of must on kinetic parameters were examined. Improved results were shown by freeze-dried immobilized cells on gluten as compared with free freeze-dried cells (ffdc). Fermentation times obtained at low temperatures (5–15 °C) could be accepted in industry. The total acidity of grape must does not affect the fermentation time while freeze-dried immobilized cells seem to have an operational stability from batch to batch. Amyl alcohols, the main volatile, are reduced by the decrease of the temperature below 20 °C. The percentages of amyl alcohols as a function of total volatiles were reduced and those of ethyl acetate were increased by the decrease of temperature, indicating improvements in quality and nutritional value. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Freeze dried; Immobilization; Wine making; Gluten pellets; Low-temperature
1. Introduction Considerable attention has been given over the last 15 years to the cell immobilization of yeasts in wine making. However, while other prerequisites are necessary for the industrialization of immobilized cells, technical and industrial experience lead to the following conclusions. (i) The technical problems of this technique and the training in the new technology of immobilized cells mainly for small wine making enterprises are obstacles for industrialization. (ii) The new bioreactor system for easy filling and emptying by the support and (iii) the high operational stability of the bioreactor are the new prerequisites for a cost effective application of immobilized cells on an industrial scale. Cell immobilization has been studied extensively in wine making. Yeast cells fixed in gels forming materials such as sodium alginate [1], agar, k-carrageenan and pectic acid [2] have been used to produce wines. Fruit * Corresponding author. Tel.: + 3061-997104; fax: +3061-997105. E-mail address: [email protected] (A.A. Koutinas).
[3] and sparkling wines [4] were also produced by immobilized yeasts in alginate gels. Cellulosic supports covered by sodium alginate gel [5] and DEAE-cellulose [6] were also reported in wine making. Likewise, delignified cellulosic material [7] and gluten pellets [8] supported biocatalysts were proposed for low-temperature wine making. Inorganic supports proposed also in wine making [9,10], are abundant and of low cost but do not meet the prerequisite of food grade purity (due to toxic heavy metals). To accommodate the industrial application of cell immobilization in wine making freeze-dried immobilized cells are proposed. That could be produced by new enterprises and will substitute free freeze-dried wine yeasts and traditional natural fermentation. This could be successfully performed provided that freezedried immobilized cells would improve the rate of fermentation and the quality of the wine. Low-temperature fermentations improve the quality of wine and reduce toxicity [11]. Therefore, the aim of this investigation was a study of low temperature wine making by freeze dried gluten supported biocatalyst (FGB).
0032-9592/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 6 6 - 7
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M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717
2. Materials and methods
2.1. Immobilization of AXAZ-1 cells on gluten pellets AXAZ-1, an alcohol resistant and cryotolerant Saccharomyces cere6isiae strain, isolated locally in the Achaia province of Greece [12] was used in all experiments. It was grown in a complete medium and pressed wet weight cells as in the aforementioned reference were prepared and employed. Gluten pellets were used as support for cell immobilization. The preparation of gluten pellets was performed as in a previous study [8].
2.2. Preparation of freeze-dried cells 2.2.1. Preparation of freeze-dried gluten supported biocatalyst The preparation of wet gluten supported biocatalyst was made by the immobilization of cells on gluten pellets as described previously. To prepare freeze-dried immobilized cells on gluten pellets without protective media, wet immobilized cells on gluten were cooled with a cooling rate of 3 °C/min to −40 °C and then freeze-dried overnight at 15– 5 ×10 − 3 Bar and at − 50 °C in a Freeze Dry System, Freezone 4.5 (Labconco, Kansas City, MO).
cells were also freeze dried, and subsequently added into the second flask. Into the third 100 g wet gluten supported biocatalyst having also 4.2 g wet weight immobilized cells were also used. The flasks were incubated at 30 °C and the fermentations were carried out without agitation.
2.3.2. Effect of temperature The above process was repeated with must of 204 g/l initial sugar concentration. Batch fermentations were performed at 5, 10, 15, 20 and 30 °C using an initial wet weight cell concentration of 14 g/l. Likewise, a second repeated batch was fermented employing the same gluten supported biocatalyst. Before the completion of fermentation the liquid was filtered using a Buchner funnel and the biocatalyst was washed once with 200 ml grape must. 2.3.3. Effect of total acidity Most of the 204 g/l initial sugar concentration was used and batch fermentations as above were carried out at 30 °C for three different total acidities of 4, 6 and 8 g tartraric acid per l. Fermentations with free and immobilized cells were carried out by using an initial wet weight cell concentration of 11.7 g/l. 2.4. Analysis
2.2.2. Preparation of free freeze-dried cells Free freeze-dried cells were also prepared using wet biomass produced after growth in a complete medium. Free freeze-dried cells were prepared for comparative study with freeze-dried immobilized cells. 2.3. Fermentations Fermentations were performed using red grape must (Roditis) of different initial sugar concentrations. This must was sterilized in an autoclave at 130 °C for 15 min. The pH was adjusted to 3.6 and the initial total acidity was 4 g tartraric acid per l. Repeated batch fermentations of grape must were carried out separately using: (a) freeze-dried gluten supported biocatalyst (F1GB), (b) free freeze dried cells (ffdc), (c) wet gluten supported biocatalyst (WGB) and (e) wet free cells. The effects of initial sugar concentration, temperature and total acidity were examined.
2.3.1. Effect of initial sugar concentration Three 500 ml Erlenmeyer flasks were used for each initial sugar concentration and each contained 300 ml grape must. The experiments were performed using 136, 170, 204, 238, 272 and 306 g/l initial sugar concentration. In each experiment 100 g gluten supported biocatalyst that hold 4.2 g wet weight cells were freeze dried, pretreated with grape must and added into the first Erlenmeyer flask. In a similar way 4.2 g wet weight free
Fermentation kinetics were performed by measuring the °Be density at various time intervals. Samples were collected and analyzed for ethanol concentration, residual sugar and volatile by-products. All values were the mean of three repetitions. The standard deviation for ethanol concentration was B 9 0.2, for ethanol productivity B 90.2, for residual sugar B 90.3 and conversion B 9 0.3.
2.4.1. Analysis of alcohol and residual sugar Ethanol and residual sugar concentrations were obtained by HPLC analysis. A SHIMADZU liquid chromatograph with a high pressure pump model LC-9A, a constant temperature oven C-R 6A and a refractive index detector RID-6A connected with an integrator C-R 6A was used. A column SCR-101N packed with a cationic resin was used. The temperature was set at 60 °C and an aqueous mobile phase with a flow rate of 0.8 ml/min was used. Ethanol productivity was expressed as g of ethanol per l substrate per h. 2.4.2. Analysis of 6olatiles Acetaldehyde, ethyl acetate, propanol-1, isobutyl alcohol and amyl alcohols were determined using a stainless steel column, packed with Escatro 5905 [consisting of squalene, 5%; Carbowax 300, 90% and Di-2-ethylhexyl sebacate 5% (v/v)], with N2 as carrier gas (20 ml/min). The injection port and detector temperatures
M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717
were 210 °C and the column temperature was 60 °C. The internal standard was pentanol-1 at a concentration of 0.5% v/v. Samples of 2 ml of the wine were injected directly in the column.
3. Results and discussion
3.1. Study of wine making Gluten meets the prerequisites for cost effective industrial immobilization of yeast. These prerequisites are low cost, abundance in nature, food grade purity and the support to be enough heavy material to avoid the breaking of pellets. These are more necessary in the production of freeze-dried immobilized cells in comparison with wet immobilized cells due to the further cost of lyophilization. Freeze-dried immobilized cells will lead to development of new enterprises for the exploitation of this marketable product. This will facilitate the technology of immobilized cells to be used by wine makers of low production capacity. Gluten pellets supported biocatalyst was prepared by immobilization of yeast cells on gluten pellets and subsequently lyophilized. The freeze-dried biocatalyst was employed in fermentations of grape must to examine it in wine making. So the effect of initial sugar concentration, temperature and total acidity were examined. Likewise, results by freeze-dried immobilized cells in repeated batch fermentations are also reported. The results of the aforementioned research are summarized in Tables 1–3 and Fig. 1.
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Table 1 shows the effect of initial sugar concentration on batch fermentation of grape must by freeze-dried immobilized cells in comparison with ffdc and wet gluten supported biocatalyst (WGB). The results indicate F1GB to ferment grape must be in the range of 204–272 g/l initial sugar concentration providing an alcohol concentration of 12–13.8% v/v which is the alcohol percentage of traditionally produced wines. These alcohol concentrations were obtained in a fermentation time of about 4–8 days, and are considered less than by natural fermentation of grape must. Startup of the fermentation of grape must by F1GB was about 1 day, which is equal to that of natural fermentation. However, it was less than those of ffdc. Likewise, F1GB leads to higher productivities as compared with ffdc while the alcohol concentration was about the same. WGB resulted in improved results as compared with F1GB in all parameters studied. Table 2 shows the effect of temperature on wine making by F1GB in comparison with ffdc and WGB. At all temperatures studied F1GB lead to alcohol concentrations equal to those of dry wines with low residual sugar and high conversion. Low-temperature fermentations in the range of 5–20 °C resulted in fermentation times and productivity that could be accepted by the industry. Temperatures of 10, 15, 20 °C gave fermentation times near to those of natural fermentation. Likewise, the 60 days fermentation time at 5 °C may mean that high quality wines would be produced. These results were improved when compared with those of ffdc. WGB leads to better results in comparison with F1GB. Total acidities in the range of
Fig. 1. Fermentation kinetics observed at various temperatures and 204 g/l initial sugar concentration in wine making by freeze dried gluten supported biocatalyst (FGB1,2), Free freeze dried cells (ffdc) (where 1 and 2 means first and second repeated batch). Internal figure analyses kinetics at 30 °C plots °Be density versus time.
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Biocatalyst
Initial sugar concentration g/l
Start-up (h)
Fermentation time (h) Ethanol concentration (g/l) Ethanol productivity (g/l/h)
Residual sugar (g/l)
Conversion (%)
F1GB
136 170 204 238 272 306 136 170 204 238 272 306 136 170 204 238 272 306
18 24 24 24 24 30 24 26 48 31 48 96 0.4 0.4 0.4 0.4 0.5 0.6
57 73 100 115 170 194 84 95 110 140 240 240 36 48 54 60 69 141
5.0 8.9 2.8 12.4 51.6 121.4 3.9 5.6 7.6 22.1 65.0 105.6 0.5 3.3 3.8 8.5 13.6 42.16
96.3 94.7 98.6 94.7 81.0 60.3 97.1 96.7 96.2 90.7 76.1 65.4 99.6 98.0 98.1 96.4 95.0 86.2
ffdc
WGB
63.1 76.4 94.1 102.9 107.6 85.8 63.1 85.0 93.6 100.6 102.1 93.6 62.4 73.3 93.6 108.4 120.1 125.5
1.10 1.04 0.94 0.89 0.63 0.44 0.75 0.89 0.85 0.71 0.42 0.39 1.73 1.52 1.73 1.80 1.74 0.89
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Table 1 Effect of initial sugar concentration on fermentation kinetic parameters observed at 30 °C in batch wine making by freeze dried gluten supported biocatalyst (F1GB), free freeze dried cells (ffdc) and wet gluten supported biocatalyst (WGB)
Biocatalyst
Temperature (°C)
Start-up (days)
Fermentation time (days)
Ethanol concentration (g/l)
Ethanol productivity (g/l/h)
Residual sugar (g/l)
Conversion (%)
F1GB
5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30
4 8 3 1 1 1 8 8 3 3 2 2 0.5 0.4 0.4 0.3 0.3 0.3
60 32 16 9 6 4 66 36 15 8 8 5 11.2 4.0 3.1 2.5 2.3 2.25
89.7 89.7 90.4 91.2 87.3 94.1 88.9 88.9 91.2 89.7 92.8 93.6 92.0 92.0 92.8 93.6 93.6 93.6
0.06 0.11 0.23 0.44 0.58 0.94 0.05 0.10 0.25 0.46 0.49 0.85 0.34 0.94 1.22 1.50 1.60 1.73
5.1 10.7 6.4 2.8 3.3 2.8 7.6 6.7 5.5 5.6 20.7 7.6 3.1 4.4 2.8 2.6 1.9 3.8
97.5 94.7 96.8 98.6 98.3 98.6 96.2 96.7 97.3 97.2 89.8 96.2 98.4 97.8 98.6 98.7 99.0 98.1
ffdc
WGB
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Table 2 Effect of temperature on fermentation kinetic parameters observed (204 (g/l) initial sugar concentration) in grape must fermentation by freeze dried gluten supported biocatalyst (F1GB), free freeze dried cells (ffdc) and wet gluten supported biocatalyst (WGB)
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6 g/l
8 g/l
10 g/l
Repeated Start-up (h) batch fermentations
Fermentationt Ethanol Residual ime (h) concentration sugar (g/l) (g/l)
Start-up (h)
Fermentationt Ethanol Residual ime (h) concentration sugar (g/l) (g/l)
Start-up (h)
Fermentationt Ethanol Residual ime (h) concentration sugar (g/l) (g/l)
1 2 3 4 5 6 7 Freeze dried free cells
82 78 75 80 76 80 72 102
48.0 0.6 0.5 0.5 0.4 0.4 0.4 24
81 78 78 79 82 86 74 99
48.0 0.6 0.5 0.5 0.4 0.4 0.4 24
82 79 79 82 81 90 76 102
48.0 0.6 0.5 0.5 0.4 0.4 0.4 24
90.5 91.2 91.2 89.7 92.0 92.8 89.7 92.0
5.5 6.3 6.7 6.1 3.4 2.5 2.3 2.8
93.6 91.2 94.4 86.6 66.3 89.7 88.9 92.8
3.9 8.8 4.7 9.8 – 7.6 – 3.1
93.6 92.8 88.1 85.8 93.6 88.9 89.7 92.8
4.2 6.7 6.4 11.5 3.2 2.8
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Table 3 Effect of total acidity on kinetic parameters in repeated batch fermentations by freeze-dried gluten supported biocatalyst at 30 °C and 204 g/l initial sugar concentration
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6– 10 g tartraric acid per l did not affect significantly start-up, fermentation time, ethanol concentration and residual sugar in wine making by FGB (Table 3). Repeated batch fermentations lead to a drastic drop in start-up, while the results clearly showed an operational stability in wine making from batch to batch. That improvement of the rate of fermentation from 1st to 2nd repeated batch fermentation is performed by FGB is shown in Fig. 1. This figure also shows FGB results in increased fermentation rate as compared with ffdc.
3.2. Formation of 6olatile by-products A study of the volatile by-products contained in wines and the affect on quality and nutritional value was also necessary. The effect of temperature on the formation of volatiles in repeated batch fermentations by FGB was studied, by placing the bioreactor in a low temperature incubator and adjusting the temperature successively to 30, 20, 15, 10 and 5 °C. Likewise, repeated batch fermentations were also carried out at various initial sugar concentrations. At every repeated batch fermentation, samples were collected after the end of each fermentation and analyzed for ethanol, acetaldehyde, ethyl acetate, propanol-1, isobutyl alcohol and amyl alcohols (total amount of 2-methylbutanol-1 and 3-methylbutanol-1). The results are presented in the Tables 4 and 5. Table 4 shows that the contents of acetaldehyde and isobutyl alcohol were reduced by the decrease in temperature from 15 to 5 °C. Likewise, it also decreased as the temperature was increased from 15 to 30 °C. Amyl alcohols also decreased on a decrease in temperature but the reduction started from 20 °C. Propanol-1 was not affected by the reduction in temperature. According to a previous study [7], WGB resulted in reduction of all of the aforementioned volatiles as the temperature was decreased from 27 to 5 °C. Increasing initial sugar concentrations also decreased acetaldehyde and isobutyl alcohol (Table 5). Propanol1 increased as the initial sugar concentration was increased while ethyl acetate, amyl alcohols and total volatiles increased up to 204 g/l initial sugar concentration.
3.3. Industrialization of no6el biocatalyst Most of the fermentations performed by FGB, resulted in alcohol concentrations in the range of 11.5– 13.8% v/v and fermentation times lower than that of wine making by natural fermentations. Furthermore, volatiles obtained by fermentations of grape must by FGB were in the same range as those of wet immobilized cells [11]. These results show the possibility for industrialization of freeze-dried immobilized cells on gluten. Therefore, the new marketable product facilitates the industrialization of immobilized cells.
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Most volatile by-products and especially amyl alcohols (the most abundant and toxic) were reduced as the temperature was decreased below 20 °C. The average concentration of amyl alcohols at 5 °C was only 46% of that formed at 20 °C. That means that extremely low-temperature fermentations can be performed, as by WGB, leading to a decrease in toxicity and an improvement in the aroma. That can be also seen in Fig. 2, where FGB percentages of amyl alcohols as a function of total volatiles were reduced as the temperature decreased, while the percentage of ethyl acetate on total volatiles increased. In contrast, the use of wet free cells lead to an increase in the percentage of amyl alcohol in the total volatiles and a decrease of ethyl acetate as the temperature was decreased. Ffdc percentages of amyl alcohols in total volatiles were reduced less in comparison to those of FGB, while the percentage of ethyl acetate was increased below 15 °C. FGB resulted in an increase of ethyl acetate 30–150% at the lower temperature of 20 °C compared with WGB [8]. Amyl alcohol concentrations obtained by FGB were higher than those by WGB [11]. The above discussion lead to the conclusion that FGB results in improved results in comparison with ffdc, regarding kinetic parameters as well as volatiles. Ffdc of wine yeasts are known commercial products. WGB will be also a commercial product, provided that this biocatalyst will pass successfully all technical problems of handling on an industrial scale. The total acidity of grape must did not affect wine making by FGB over a broad range of total acidities. This indicates that the biocatalyst could be treated in industry of Southern and Northern countries. The freeze-dried immobilized cells on gluten seems to have operational stability from batch to batch which is an indispensable factor for the industrialization of this technology. The FGB is also suitable for wine making at the initial °Be densities used on an industrial scale. Since freeze-dried immobilized cells on gluten formed upto 15% v/v alcohol in the fermentation of grape must, FGB will be also suitable for semi-sweet wine making. FGB will be suitable for wine making provided that the technical problems of scale up of the process can be overcome. If so, a new marketable product will be developed which substitutes freeze-dried wine yeasts.
Acknowledgements Iconomopoulou Matina in the frame of the program ‘Exploitation of Industrial Wastes’ thanks the General Secretariat of Research and Technology (G.S.R.T., Athens, Greece) for financial support.
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Temperature (°C)
Repeated batch wine making
Fermentation time (h)
Ethanol concentration (g/l)
Acetaldehyde (mg/l)
Ethyl acetate (mg/l)
Propanol-1 (mg/l)
Iso-butyl alcohol Amyl alcohols (mg/l) (mg/l)
Total volatiles were determined (mg/l)
5 10 15 20 30
1–2 1–3 1–4 1–6 1–7
1896 632 276 155 66
88.9 90.5 88.9 91.2 91.2
52.9 57.3 67.9 49.7 63.5
84.2 113.9 85.2 85.3 50.0
42.3 34.8 39.6 37.6 36.2
32.9 38.2 47.6 39.0 37.0
355.7 462.3 576.2 520.2 450.7
143.3 217.9 301.8 308.6 272.1
M. Iconomopoulou et al. / Process Biochemistry 37 (2002) 707–717
Table 4 Effect of temperature on formation of volatiles in repeated batch fermentations by freeze dried gluten supported biocatalyst using 204 g/l initial sugar concentration
Initial sugar Repeated batch concentration (g/l) fermentations
Fermentation time (h)
Ethanol concentration (g/l)
Acetaldehyde (mg/l)
Ethyl acetate (mg/l)
Propanol-1 (mg/l)
Iso-butyl alcohol Amyl alcohols (mg/l) (mg/l)
Total volatiles were determined (mg/l)
136 170 204 238
48 60 72 102
61.6 73.3 92.0 92.8
83.1 68.3 72.6 46.0
58.0 70.2 79.3 54.3
19.1 22.4 36.0 42.9
45.0 45.0 37.6 34.3
408.2 449.7 513.2 413.2
3 4 5 6
234.5 262.9 289.3 266.4
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Table 5 Effect of initial sugar concentration at 30 °C on formation of volatiles in repeated batch fermentations making by freeze dried gluten supported biocatalyst
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Fig. 2. Effect of temperature on percentages of amyl alcohols, iso-butyl alcohol and ethyl acetate on total volatiles were determined in wine making by freeze dried immobilized cells on gluten as compared with free freeze dried cells and wet free cells.
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