J. Agric. Food Chem. 1996, 44, 463−467
463
Immobilization of Yeast on Delignified Cellulosic Material for Low Temperature Brewing E. P. Bardi, A. A. Koutinas, M. J. Soupioni, and M. E. Kanellaki* Department of Chemistry, Section of Analytical, Environmental and Applied Chemistry, University of Patras, GR-26500 Patras, Greece
A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on delignified cellulosic (D.C.) material and studied in the fermentation of wort for batch and continuous brewing. The immobilized yeast gave an important operational stability without decrease of its activity, even at low temperatures (0-5 °C), compared with free cells. Batch fermentations at various temperatures were faster than those of free cells and those usual in commercial brewing. A traditional bottom fermentation takes 8-10 days. Specifically, at 0 °C, the fermentation rate was 4-5 times higher than that of free cells. Diacetyl and polyphenol contents as well as bitterness and pH were lower than those when free cells were used for fermentations; at 0 °C, polyphenol content was ∼30% and bitterness 50% of the values noted when free cells were used for fermentations. The alcohol concentration at 0 °C was ∼20% higher than that of free cells. The continuous system was operated continuously for 3 months with relatively high productivity. However, polyphenols and bitterness were higher than those obtained in batch fermentations with immobilized cells. Preliminary taste tests indicated that the beer produced by the immobilized cell process had an acceptable clarity, aroma, and taste. Keywords: Cell immobilization; delignified cellulosic material; Saccharomyces cerevisiae; brewing; beer INTRODUCTION
Beer was known to early civilizations, but the brewing was an art and mystery. Although explanation for fermentation was not available until the nineteenth century, the steady improvement in manufacturing techniques was not impeded. The use of adsorbed yeast cells for the continuous production of beer was first attempted in 1899 by Barbet. Recently, a number of studies have been published on the production of beer by batch fermentation with immobilized cells on polyethylene film (Kolpakchi et al., 1976), ceramic or polyethylene rings (Kolpakchi et al., 1980), alginate gel (Pardonova et al., 1982), and hollow PVA gel beads (Shindo et al., 1990). Also, a number of studies have been published on brewing by continuous fermentation with immobilized yeast cells on PVC and porous bricks (Corrieu et al., 1976; Navarro et al., 1976), diatoms, PVC and plastic (Moll, 1977), pieces of brick (Moll and Duteurtre, 1979), Ca alginates (Godtfredsen et al., 1981; Linko and Linko, 1981; Onaka et al., 1985), and ceramics (Nakanishi et al., 1989). The use of an immobilized yeast cell system for alcoholic fermentation is an attractive and rapidly expanding research area because of its additional technical and economical advantages compared with the free cell system (Stewart and Russell, 1986; Margaritis and Merchant, 1984). However, for industrial application, further research is needed to obtain cell immobilization on a support that is more hygienic for food, cheap and abundant in nature, and suitable for low temperature fermentation in brewing. Manufacturers of beer know that the low temperature fermentation results in a product with improved aroma and taste. Finally, it has been reported that immobilized cells on * Author to whom correspondence should be addressed (fax 0030 61 997105). 0021-8561/96/1444-0463$12.00/0
delignified cellulose (D.C. material are suitable for lowtemperature wine making (Bardi and Koutinas, 1994) and increased productivity. The aim of this investigation was to evaluate the use of D.C. material-supported biocatalyst in the brewing fermentation of brewing beer. MATERIALS AND METHODS The biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae strain AXAZ-1 on D.C. material as described previously (Bardi and Koutinas, 1994). Briefly, D.C. material was prepared after lignin removal from sawdust with sodium hydroxide solution. AXAZ-1, an alcohol-resistant and psychrophile S. cerevisiae strain isolated (Argiriou et al., 1992) from the Greek agricultural area, was grown on the complete medium used in the previous study (Bardi and Koutinas, 1994). Pressed wet cells were prepared as in the aforementioned reference and employed directly in the fermentations. Wort was obtained from Athenian Brewery S.A., hopped, filtered, and sterilized. The pH of the wort was 5.1-5.2 and the °Be density was 6.9. The values of the percent original extract of the wort are shown in Table 3. Alcoholic degrees were obtained, after distillation of samples, with a Gay-Lussac alcohol meter. The determination of ethanol enabled us to calculate the ethanol productivity, which is defined as the grams of ethanol per liter liquid volume produced per day. Beer productivity was calculated as grams of beer per liter total volume produced per day. Total carbohydrates were determined in all samples by the LaneEynon method (Egan et al., 1981). Apparent extract percent, polyphenols and diacetyl content, as well as bitterness, color, and refractive index (20 °C) were determined by well-known methods (Hough et al., 1982). Original and real extract percent were determined from a nomograph furnished by the Athenian Brewery S.A. Wet free cell concentrations were determined by the absorbance experimental procedure (Klein and Kressdorf, 1983; Bajpai and Margaritis, 1986) and are given in grams of wet weight per liter, as determined with standard curves. Repeated Batch Fermentations at Room and Low Temperatures. For repeated batch fermentations, an amount
© 1996 American Chemical Society
464 J. Agric. Food Chem., Vol. 44, No. 2, 1996
Bardi et al.
Table 1. Kinetic Parameters Obtained in the Repeated Batch Fermentations at Various Temperatures (0-30 °C) with D.C. Material-Supported Biocatalyst temp (°C)
repeated fermentn batche
fermentn time (h)
total carbohydrates (g/L)
EtOH concn (% v/v)
EtOH productivity (g/L/d)
beer productivity × 103 (g/L/d)
30 30 30 15 15 15 15 10 10 10 10 10 7 7 7 5 5 0 0 0 0
1 4 6 8 9 10 11 12 14 15 16 17 19 20 21 22 23 24 25 26 27
11 12 14 27 24 22 27 25 72 72 72 72 120 144 144 216 216 264 312 288 288
12.8 14.4 11.2 16.0 16.0 24.0 19.2 12.8 11.2 4.8 8.0 8.0 12.8 6.4 4.8 11.2 9.6 1.6 1.6 0.0 1.6
4.9 5.0 5.2 5.6 5.6 5.0 5.3 5.0 5.6 5.6 5.6 5.4 5.2 6.1 6.2 5.9 6.2 6.6 6.2 6.6 6.3
59.0 55.1 49.2 28.2 30.9 30.0 26.0 26.5 10.3 10.3 10.3 9.9 5.7 5.6 5.7 3.6 3.8 3.3 2.6 3.0 2.9
1.505 1.379 1.182 0.613 0.690 0.752 0.613 0.662 0.230 0.230 0.230 0.230 0.138 0.115 0.115 0.077 0.077 0.063 0.053 0.057 0.057
of D.C. material-supported biocatalyst (∼170 g) was introduced into 400 mL of wort in a 1-L glass cylinder. The glass cylinder for each fermentation batch was incubated at the temperatures indicated in Table 1. The fermentations were carried out without agitation. Before the fermentation was completed, the liquid was filtered through a Bu¨chner funnel, and the support was washed three times, each time with 400 mL of wort. The biocatalyst was pressed on the funnel to remove the liquid. After that, the biocatalyst was used for the next fermentation batch. To compare the fermentation times and the other parameters obtained in the presence of the D.C. material-supported biocatalyst with those of free cells, similar runs were carried out simultaneously with the same cell concentration. Yeast cells immobilized on D.C. material were determined in a recent study (Bardi and Koutinas, 1994). All values were the mean of three repeats. The standard deviation for ethanol concentration was
