Comparison of Assimilation of Fermentable Sugars in Wort by the Immobilized Yeast in Alginate Gel and the Free Yeast in High Gravity Brewing
Total Page：16
File Type：pdf, Size：1020Kb
TAÏP CHÍ PHAÙT TRIEÅN KH&CN, TAÄP 16, SOÁ K3- 2013 Comparison of assimilation of fermentable sugars in wort by the immobilized yeast in alginate gel and the free yeast in high gravity brewing Tran Quoc Hien Le Van Viet Man University of Technology, VNU-HCM Hoang Kim Anh The Institute of Tropical Biology (Manuscript Received on January 21st, 2013, Manuscript Revised October 18th, 2013) ABSTRACT: In this work, the assimilation of different cells was also observed during the first hours fermentable sugars by the immobilized yeast of fermentation while the free cells only in alginate gel and the free yeast under high started assimilating maltotriose after the first gravity brewing conditions was investigated 48 h. High osmotic pressure at the start of and compared. Both immobilized and free the fermentation changed cellular yeast utilized glucose, fructose, sucrose and morphology and that could decelerate the maltose in wort from the beginning of the maltotriose uptake of the free yeast. fermentation. Maltotriose uptake of the fixed Key words: fermentation kinetics, high gravity brewing, immobilization, morphology, ccharomyces cerevisiae 1. INTRODUCTION Wort contains different sugars, including were taken up more and less simultaneously. fructose, glucose, sucrose, maltose and Completion of assimilation of glucose was maltotriose as well as other carbohydrates, in followed by utilization of maltose, the major wort varying concentrations [2,7]. During the sugar. Maltotriose was utilized last after all fermentation, the utilization of wort utilization of maltose [2]. Meanwhile, according carbohydrates, especially the utilization of to D’Amore et al., when glucose and fructose are maltose, glucose and fructose by the yeast is a fermented separately, both sugars are utilized at major determinant of fermentation efficiency and similar rates. However, when fermentations are final beer quality. According to Boulton et al., conducted in media containing an equal sucrose was utilized first and the resultant concentration of glucose and fructose, glucose is hydrolysis causes a transient increase in the utilized at approximately twice the rate of concentration of fructose. Fructose and glucose fructose. The preferential utilization of glucose Trang 93 SCIENCE & TECHNOLOGY DEVELOPMENT, Vol 16, No.K3- 2013 also occurred when sucrose, which was first Sodium alginate was supplied by rapidly hydrolyzed into glucose and fructose by Biotechnology Center, Nha Trang University. the enzyme invertase, was employed as a The ratio of mannuronic acid to guluronic acid substrate [3]. was 1.2. The viscosity of 2% alginate solution at o Phaweni et al. stated that the sequential uptake 25 C was 423.6cp. of sugars in all-malt wort by S. cerevisiae 2036 Barley malt was supplied by Duong Malt was as follow: sucrose, glucose, fructose, maltose Company; the extraction yield was 80%. High and maltotriose. This result was also valid in the maltose syrup supplied by Bibica Company was fermentation if maltose was used as the total wort used as adjunct; the dextrose equivalent was adjunct. Meanwhile, when glucose was used as approximately 42. the total adjunct, glucose utilization was retarded 2.2. Experimental methods and consequently fructose utilization was Preparation of high gravity wort blocked [7]. The wort was prepared from barley malt using During the last decades, high gravity brewing conventional brewing techniques [2]. In this has been widely applied to brewing industry. study, the 24oPt wort was used. The extract of High gravity brewing involves preparation and 24oPt wort was originated from 70% barley malt fermentation of wort with a specific gravity and 30% maltose syrup. o higher than 12 Plato. By increasing the wort Inoculum preparation specific gravity, the higher levels of the ethanol Inoculum was prepared from 8% (w/w) malt per given plant capacity can be achieved and wort medium according to the procedure substantial savings can be attained by the brewer; previously described elsewhere [11]. the plant efficiency and capacity are increased; Yeast immobilization in alginate gel labour, energy and capital costs are reduced [6]. On the other hand, application of immobilized A volume of yeast suspension yeast to brewing has been considered as a very (1.0×108cell/mL) was added to an equal volume promising technology. The use of immobilized of sodium alginate solution (50g/L) and yeast in the fermentation process and their homogenized. This mixture was then dropped potential advantages over the free yeast systems into a 3% (w/v) CaCl2 solution for formation of have been widely investigated and reviewed [4]. calcium alginate gel beads. The cell concentration in the gel bead was approximately The assimilation of fermentable sugars by 5.0×107cell/cm3. The residence time of the gel immobilized and free yeast in high gravity beads in CaCl2 solution was 4h at 4oC for brewing has not been clearly considered. The increasing the gel strength. The gel beads were objective of this work was to investigate and then washed with sterile water [11]. compare the uptake of the fermentable sugars in Beer fermentation by immobilized yeast in wort by the immobilized yeast in alginate gel and alginate gel the free yeast in high gravity brewing. Batch fermentation was carried out in 2.5L 2. MATERIALS AND METHODS stainless steel fermenters containing 2.0L of 2.1. Materials 24oPt wort without stirring. The fermentation Saccharomyces cerevisiae (lager strain) used temperature was 17oC. The inoculation rate was in this study was supplied by Tien Giang Foster 1.0×106 cells/mL of wort. The fermentation was Company. considered as completed when the degree of Trang 94 TAÏP CHÍ PHAÙT TRIEÅN KH&CN, TAÄP 16, SOÁ K3- 2013 attenuation reached 75%. The degree of Sucrose uptake attenuation was calculated by the reducing sugar The wort used in this study had low sucrose content in the initial wort and in the green beer level (3.5 g/L). After the first 12 h of [6]. fermentation, the sucrose concentration in the Control sample was simultaneously performed immobilized and free yeast cultures remained with the free yeast under the same conditions. 7.8% and 22.9% of that in the initial wort, Change in morphology of yeast cells under high respectively. Thus, the immobilized yeast osmotic stress assimilated sucrose faster than the free yeast The immobilized and free yeast cells were during the first 12 h of fermentation. Patel et al. soaked in 24% (w/v) sorbitol solution at ambient suggested that sucrose hydrolyzed into glucose temperature for 30 min. The cells were then and fructose by the enzyme invertase on the removed for morphological observation under cytoplasmic membrane of yeast and the yeast scanned electronic microscope (SEM). easily assimilated the substrates [5]. Figure 1 and 2.3. Analytical methods 2 shows that after 24 h of fermentation, the sucrose was disappeared from the wort in both Fermentable sugars were determined by high- cases. performance liquid chromatography (Shimadzu corporation) using a detector RID-10A. A RNM- Glucose uptake carbohydrate Na+ column (7.8×300) at 65oC was The glucose concentration in the free yeast used with H2O as eluant. The flow rate was culture was nearly unchanged during the first 24 1mL/min, and the injection volume was 20µL h of fermentation. During this period, the sucrose [7]. was completely hydrolyzed but the glucose level SEM was used for morphological observation in the fermented wort did not increase further. of yeast cells. The immobilized and free cells We suggested that the hydrolytic rate of sucrose were fixed by 1% glutaraldehyde in cocadylic was similar to the uptake rate of glucose by the buffer, dehydrated and prepared for observation free yeast during the first 24 h of fermentation. under SEM (JEOL Co., JSM-7401F) according to D'Amore et al. also observed the same rule in o a standard procedure described by Van Neerven 16 Pt wort fermentation with the free yeast [3]. et al. [12]. In our study after 24 h fermentation, the glucose concentration in the free yeast culture started 2.4. Statistical analysis decreasing; and the glucose was exhausted after All experiments were performed in triplicate. 72 h. For the immobilized yeast, the glucose Mean values were considered significantly content in wort gradually reduced during the first different when P < 0.05. Analysis of variance hours of fermentation although the hydrolysis of was performed using the software Statgraphics sucrose occurred during this period. After 48 h, plus (version 3.2, StatPoint technologies, Inc., the glucose in the immobilized yeast culture was USA). disappeared. Thus, the alginate gel increased the 3. RESULTS AND DISCUSSION glucose uptake rate of the immobilized yeast. The The kinetics of sugar assimilation of the assimilation time of glucose by the fixed yeast in immobilized yeast in alginate gel and the free alginate gel was 24 h shorter than that by the free yeast are presented in Figure 1 and 2, yeast. respectively. Trang 95 SCIENCE & TECHNOLOGY DEVELOPMENT, Vol 16, No.K3- 2013 Fructose uptake was reduced. Consequently, the immobilized The fructose level in the two cultures was cells in alginate gel utilized fructose faster than unchanged during the first 12 h of fermentation. the free cells. Disappearance of fructose from the Due to sucrose hydrolysis during this period, we wort fermented by the fixed and the free yeast concluded that both immobilized and free yeast was observed after 84 h and 108 h, respectively. utilized fructose from the beginning of the It should be noted that fructose uptake was fermentation. From the 12th to the 24th hour, significantly slower than glucose uptake by both slight increase in fructose content was observed immobilized and free yeast in high gravity in the free yeast culture whereas the fructose brewing. concentration in the immobilized yeast culture Figure 1. Assimilation of fermentable sugars of the free yeast during the primary fermentation in high gravity brewing (The 24oPt wort extract was originated from 70% barley malt and 30% maltose syrup) Figure 2.