Process Biochemistry 39 (2002) 143 Á/148 www.elsevier.

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Continuous winemaking fermentation using quince-immobilized yeast at room and low temperatures
Y. Kourkoutas a, M. Douma a, A.A. Koutinas a, M. Kanellaki a, I.M. Banat b,*, R. Marchant b
a

Food Biotechnology Group, Department of Chemistry, Section of Analytical, Environmental and Applied Chemistry, University of Patras, GR-26500 Patras, Greece b Microbial Biotechnology Group, School of Biological and Environmental Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK Received 24 June 2002; received in revised form 2 October 2002; accepted 10 November 2002

Abstract Quince-supported biocatalyst, prepared by the immobilization of an alcohol resistant psychrophilic yeast Saccharomyces cerevisiae AXAZ-1 on quince pieces, was suitable for room and low-temperature continuous winemaking. Continuous fermentation was carried out for 46 days without diminution of wine and ethanol productivity (up to 750 and 71 g/l, respectively) which at 5 8C was equal to that usually obtained by traditional fermentation at 22 Á/25 8C. The total and volatile acidities were similar to those in dry wines. Methanol, ethyl acetate, propanol-1, isobutanol and amyl alcohols (2-methylbutanol-1 and 3-methylbutanol-1) were monitored. The concentrations of ethyl acetate, amyl alcohols and methanol were B/100 mg/l in all cases, indicating an improvement in the product. Preliminary sensory tests established the fruity aroma, fine taste and the overall improved quality of the produced wines. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Wine; Continuous fermentation; Quince; Immobilized cells; Yeast

1. Introduction In recent years, the use of immobilized cell systems in wine making has been an area of research interest. Various supports for immobilization of yeasts have been proposed [1 Á/5]. However, references for continuous winemaking are scarce in the literature [6 Á/8]. Continuous winemaking offers several advantages compared to the traditional fermentation by free cells such as increase in productivity and reduction in production costs. In order to utilise immobilized cells in continuous wine making the availability of a low cost support material that is abundant and preferably of food-grade quality must be assured. A host of support materials for cell immobilization for winemaking have been proposed [2 Á/8]. Many such support systems involved the use of inorganic materials

* Corresponding author. Fax: '/44-2870-324911. E-mail address: im.banat@ulster.ac.uk (I.M. Banat).

or alginate, which were later considered inappropriate for winemaking and they were eventually abandoned. The use of delignified cellulosic material as a suitable immobilization support was also reported for lowtemperature alcoholic fermentation [9]. The development of such a continuous wine production technology using immobilized cells however has been generally hampered by the lack of a suitable low-cost support material, undetermined taste and aroma quality for the produced wine and lower viability of the immobilized system. The use of fruits in developing such a biocatalyst was an obvious alternative that has been used in several food-related industries. Apple pieces were used recently as a support material for immobilization of Saccharomyces cerevisiae yeast cells for wine production using repeated batch fermentations [10] as well as in a continuous process [11] resulting in improved productivity and product aroma and taste. In the present study, we used quince pieces as support material for immobilization as it is an inexpensive,

0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 3 2 0 - 5

The must was used without any nutrient addition or adjustments and sterilized at 130 8C for 15 min before use. using a Shimadzu chromato- Fig. and MgSO4. Yeast cells (15 Á/20 g wet weight) were prepared and employed directly in the fermentation process. 1. Support and immobilization of cells Yeast cell immobilization was carried out on pieces of quince (400 g) in a 1 l-glass cylinder containing 500 ml of synthetic medium. The reactor was operated for 46 days and the fermentation temperature was decreased gradually from 30 to 5 8C. Kourkoutas et al. The quince pieces were cut into :/2 cm cubes.6 ( :/20% (w/v) initial sugar concentration). The decrease in temperature was carried out at the rate of 2Á/ 3 8C/day to avoid sudden changes.1% (NH4)2SO4. 1. Samples were analysed for oBe density. Preparation of grape must Concentrated grape must was diluted with distilled water to a final oBe density of 11. cerevisiae AXAZ-1 yeast strain isolated from an agricultural area in Greece [12] was used in the present study. The reactor had 2000 ml total volume to which 720 ml grape must and 1160 g quince-supported biocatalyst were added. 2. Materials and methods 2. 2.5oBe. 0.5. methanol and volatile by-products.144 Y. 0.6. residual sugar. 2.3. 0. Residual sugar was determined by high performance liquid chromatography.6oBe initial density was continuously supplied to the bioreactor. The culture medium contained in (w/v) 12% glucose. 0. . Analytical assays Alcohol concentrations were determined using a Gay Á/Lussac alcoholmeter and productivity expressed in terms of grams of ethanol per total volume produced per day was calculated by multiplying the dilution rate by ethanol concentration. 0. as shown in Fig. The fermented liquid was decanted and the biocatalyst support was washed twice with 400 ml of must before use. This support was used in a packed bed reactor to accomplish continuous fermentation of grape must at room and low temperatures. / Process Biochemistry 39 (2002) 143 Á/148 abundant food material. Wine productivity was calculated as grams of wine per litre of total volume produced per day and was calculated by multiplying the flow rate (ml/day) by the density of wine (:/1 g/l) and dividing by the total volume (2 l). 0. using a peristaltic pump (Cole Parmer Instruments Co. The quince-supported biocatalyst was used in six consecutive repeated batch fermentation experiments for grape must before switching into continuous mode. KH2PO4. This was necessary to obtain steady volume of quince as it decreased due to sugar fermentation during the first six batches of fermentation (unpublished data). Samples were collected at different temperatures after at least 2 days to allow time to achieve steady conditions. Must was fed into the bioreactor in an upstream flow Grape must with an 11.4. They were prepared aseptically after removal of seeds keeping the skin and were used immediately without further sterilization. It was grown on medium containing (in % w/v): glucose.1. total and volatile acidity. (NH4)2SO4.1% KH2PO4 and 0. Experimental procedure 2.1. 4. The flow rate was decreased as the temperature was reduced. alcohol concentration.5% MgSO4 in distilled water with no pH adjustment. from an initial 1500 to 150 ml/day. Pilot fermentation Continuous wine making was carried out in a glass bioreactor (70 cm height and 3. 2. IL). wet weight) were also added to the cylinder and allowed to ferment until the must density was approximately 0. Chicago.5%. Flow rate of must and oBe densities at different temperatures (5 Á/30 8C) during 46 days continuous fermentation wine making process. Yeast cells biomass (10 g.4.. 0. yeast extract. Yeast strain An alcohol-resistant psychrophilic S.1. 2.1 cm internal diameter).2.4% yeast extract.

The must was prepared from the same grape cultivar and had the same initial oBe density in order to obtain comparable results. When the temperature was decreased. a LC-9A pump. Triple distilled water was used as mobile phase with a flow rate of 0. respectively. 2. the internal standard was butanol1 at a concentration of 0.8. or to the formation of acids during fermentation. The temperatures of the injector and FID detector were 210 and 220 8C. as carrier gas at 20 ml/min. This stability of the system can be attributed to the alcohol resistance of quince-supported biocatalyst as well as to the psychrophilic nature of the yeast strain used. Results and discussion 3. Volatile by-products The effects of temperature on the formation of the volatile compounds during continuous wine making using quince-supported biocatalyst are shown in Table 2. Preliminary taste evaluation Samples of the wine were kept at 4 Á/5 8C for 1 month and then tested for their aroma and taste characteristics compared to a commercial dry wine produced locally using similar must types. Samples of 0. Similar observations were previously reported by other researchers during fermentation of apple juices to produce cider [15]. Quince-supported biocatalyst was both mechanically and chemically stable for the duration of the fermentation. as most quince sugars were converted to ethanol and/or cells during the initial six consecutive repeated batch fermentations prior to starting the continuous fermentation leaving the nonfermentable residual cellulose.7.5 ml of a 1% (v/v) solution of butanol1 was diluted to 50 ml and 40 ml was injected directly to the column. In addition.Y. Wine and ethanol productivity at temperatures less than 10 8C were higher than those obtained under similar conditions using an apple-supported biocatalyst [11]. an CTO-10A oven at 60 8C and a RID-6A refractive index detector. isobutanol and amyl alcohols were determined by gas chromatography using a stainless steel column.5% (v/v).1 M NaOH and volatile acidity by titration of distillates obtained through steam distillation of the wine samples [13]. 3.8 ml/min and butanol-1 was used as an internal standard. Kourkoutas et al. This productivity at 5 8C is equal to that usually obtained in traditional fermentations at temperature range 22 Á/25 8C.2. 1) and results are shown in Table 1. The o Be density of the product was relatively constant during the whole experiment. Most quince acids are constituents of the wine and would contribute to the flavour of the product. Carbowax300 90% and di-ethyl-hexyl sebacate 5% (v/v) [14]. Volatile acidities in the products were similar to those found in dry wines. . Injection port and FID detector temperatures were 210 and 220 8C. These characteristics were considered advantageous for low-temperature wine making.1. Methanol was also determined by gas chromatography using Porapac S column using nitrogen as carrier gas at 40 ml/min. The sensory evaluation was a blind test in a coloured glass in the dark. propanol-1. the quince microflora has insignificant effect on the fermentation process and the quality of the final product due to the low pH. It was not necessary to carry out a pretreatment step in order to reduce the acids content of the quince before using it as support. Total acidity was estimated by titration of samples with 0. respectively. No loss of quince weight or texture was observed. high total acidity and high concentration of immobilized yeast which did not allow the growth of any other bacteria or yeasts. The column temperature was programmed at 120 Á/170 8C at a rate 10 8C/min. The total acidity of the product was slightly increased either due to the transfer of some quince acids to the produced wine at the early stages of the continuous process.5 ml wine and 2. ethyl acetate. the observed concentrations were within the normal ranges present in dry wines (4 Á/6 g of tartaric acid/l). / Process Biochemistry 39 (2002) 143 Á/148 145 graph with a SCR-101N stainless steel column. Ten tasters familiar with wine assessment tastes were asked to give scores on a 0 Á/10 scale using locally approved protocols in our laboratories for taste and aroma. Finally. Yeast immobilization within the quince pieces tissues was confirmed by electron microscopy (data not shown). The column temperature was 70 8C. This yeast had the advantage of being both alcohol resistant and a psychrophile. In all cases. 2. the flow rate was reduced in order to achieve complete fermentation of sugars. relatively high ethanol. Determination of volatile by-products Acetaldehyde. 3. Yeast cells were bound within the pieces and not only on the surface. Continuous fermentation was carried out for 46 days (Fig. The operational stability of the system for wine making at low temperatures (15 Á/5 8C) continued over 36 days. Nitrogen was used. Continuous fermentation Continuous fermentation was carried out to investigate the operational stability and suitability of the immobilized yeast strain AXAZ-1 on quince in this process. packed with Escarto-5905 consisting of Squalene 5%. Ethanol productivity of the system measured at 5 8C represented about 11% of that obtained at 30 8C.

6 Wine produced (ml/day) 1500 1500 1500 1500 1500 520 520 520 520 520 320 320 320 150 150 150 Daily wine productiv.7 2.6 11.9 1.7 5.6 11.9 Volatile acidity (g of acetic acid/l) 0.6 11.20 0.14 0.9 11.0 4.9 3.32 Y.1 4.8 0.146 Table 1 Characteristics of the wine produced by continuous fermentation of must at room and low temperatures Temperature (8C) 30 Initial oBe density 11.2 3.1 3.4 9. / Process Biochemistry 39 (2002) 143 Á/148 15 10 5 .4 11.18 0.6 11.13 0.6 11.17 0.1 11.6 2.2 1.6 11.32 0.6 11.Ethanol concentration ity (g/l) (% vol) 750 750 750 750 750 260 260 260 260 260 160 160 160 75 75 75 10.0 5.0 11.4 0.7 4.3 Residual sugar (g/l) 0.7 4.6 11.8 5.1 5. Kourkoutas et al.6 11.22 0.6 11.14 0.6 11.6 11.7 1.9 Ethanol productivity (g/l/day) 64 65 66 71 66 24 23 23 22 23 12 12 15 7 7 7 Total acidity (g of tartaric acid/l) 4.1 1.20 0.0 11.6 11.0 10.26 0.5 9.9 5.17 0.4 4.29 0.3 4.6 11.5 4.8 4.6 11.6 11.22 0.0 4.8 11.9 4.16 0.6 11.1 4.6 11.28 0.9 11.4 2.1 4.0 2.8 11.1 12.

Higher alcohols The concentrations of higher alcohols (propanol-1 and isobutyl alcohol) were low in the ranges (11 Á/41 ppm) and (15 Á/32 ppm) (Table 2). In the present experiment. acetaldehyde concentrations up to 106 mg/l were detected. These results show that the product has an improved quality due to the low concentrations of higher and amyl alcohols. However. their percentage on total volatiles in our wine produced on immobilized cells on quince remained between 16 and 29% at 10 Á/30 8C (Fig. / Process Biochemistry 39 (2002) 143 Á/148 Table 2 Effect of temperature on formation of volatiles in the continuous fermentation using quince-supported biocatalyst 147 Temperature ( 8C) Acetaldahyde (ppm) Ethyl acetate (ppm) Propanol-1 (ppm) Isobutyl alcohol (ppm) Amyl alcohols (ppm) Methanol (ppm) 30 30 32 54 47 46 72 83 74 74 73 83 106 84 26 26 25 80 74 70 54 66 86 52 90 76 71 48 14 72 70 82 80 26 25 26 22 25 29 18 41 16 17 11 16 18 26 22 21 22 18 18 15 15 20 29 32 27 27 18 20 25 19 17 17 79 73 74 72 73 81 95 99 90 87 56 51 80 26 25 34 28 33 34 34 31 34 30 44 63 47 38 48 56 67 54 29 15 10 5 3. 3. while amyl alcohols Fig. special type wine. Kourkoutas et al.2.3. The preliminary taste investigation characterised the new wine as a novel.2. 2). The concentrations of amyl alcohols were also low (B/100 ppm) at all fermentation temperatures. .1. The methanol content in the our product was B/100 mg/l which is lower than the usual range in wines (100 Á/200 mg/l). With regard to the most abundant by-products in wines which are ethyl acetate and amyl alcohols. Other volatiles Acetaldehyde concentrations in wines are usually in the range 13Á/40 mg/l [16] and occasionally may reach 75 mg/l [17].Y. 3. The commercial wine not only produced lower scores but the preference of the tasters was much more variable. These concentrations are usually observed in wines and contribute to the aroma of the product. 2. soft aroma and fruity taste. were significantly reduced ( :/13%). Preliminary sensory tests carried out in the laboratory have established the fruity aroma and the fine taste of the wines produced by continuous fermentation using yeast immobilized on quince pieces at low temperatures (Table 3). The reduction of amyl alcohols percentage at low temperatures (5 Á/ 10 8C) leads to a high quality product.2. At 5 8C the percentage of ethyl acetate was higher ( :/35%). Percentage concentration of ethyl acetate and amyl alcohols on total volatiles determined in the wine product at different temperatures. in most cases they were lower (Table 2). with a pleasant.2. The low concentration of methanol is considered advantageous as this compound is undesirable. Ethyl acetate Ethyl acetate concentrations ranged from 14 up to 90 ppm (Table 2).

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