On-line version ISSN 0104-6632
Braz. J. Chem. Eng. vol.18 no.4 São Paulo Dec. 2001
R.B.Almeida1, 3*, J.B.Almeida e Silva1, U.A.Lima2, D.P.Silva1 and A.N.Assis 1
1Departamento de Biotecnologia, Faculdade de Engenharia Química de Lorena,
FAENQUIL, Lorena - SP, Brazil, Cx.P. 116, 12600-000, Phone/Fax: (55) 12-553-3165,
2Departamento de Engenharia Química e Alimentos, Escola de Engenharia Mauá,
São Caetano do Sul - SP, Brazil
3Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, USP,
São Paulo - SP, Brazil
(Received: June 28, 2001 ; Accepted: November 7, 2001)
Abstract - A large number of advantages are obtained from the use of highly concentrated worts during the production of beer in a process referred to as "high-gravity". However, problems related to slow or stuck fermentations, which cause the lower productivity and possibility of contamination, are encountered. This study examines the influence of factors pH, percentage of corn syrup, initial wort concentration and fermentation temperature on the fermentation parameters, namely productivity, wort attenuation and the yield coefficient for sugar-to-ethanol conversion. The results show that productivity increased when the higher temperature, the higher wort concentration and the lower syrup percentage were used, while wort attenuation increased when lower wort concentration and no syrup were used. The yield coefficient for sugar-to-ethanol conversion was not influenced by any of the factors studied.
Keywords: high-gravity, beer, fermentation, factorial design, wort.
The production of beer using highly concentrated worts is the most efficient method adopted by Brazilian breweries for improving both productivity and competitiveness. This process, referred to as "high-gravity", results in a more concentrated beer, which is usually blended with water after maturation with ethanol concentration or initial wort concentration used as the parameter (Murray and Stewart, 1991). The use of the high-gravity process enhances the brewerys capacity for beer production and reduces energy consumption, working time, cleaning and effluent costs and overall costs substantially (Almeida et al., 2000a and b; Stewart and Russell, 1985; Pfisterer and Stewart, 1976). Other advantages are the production of beer with improved colloidal haze and flavor stability, higher yield coefficients for sugar-to-ethanol conversion and higher percentages of adjuncts due to the excess of enzyme caused by high wort concentrations (Stewart and Russell, 1985; Casey et al., 1984; Hackstaff, 1978). On the other hand, the high-gravity worts cause problems such as a decrease in the viability of the yeast and slow or stuck fermentations (Casey et al., 1984; Nagatani et al., 1968), mainly due to ethanol toxicity (Casey et al., 1984; Day et al., 1975), high osmotic pressure (Nagatani et al., 1968) and nutritional deficiency when corn syrup is used to increase the gravity of the wort (Casey et al., 1984). These problems decrease the possibility of reusing the yeast and stability of the foam, increase the production of contaminants during fermentation, the alter flavor and lower productivity.
In this work, a 24 factorial design was used to evaluate the infuence of factors pH, initial wort concentration, percentage of corn syrup and fermentation temperature on productivity, wort attenuation and the yield coefficient for sugar-to-ethanol conversion.
MATERIALS AND METHODS
The yeast strain employed in this study was Saccharomyces cerevisiae (lager strain), obtained from a strain used commercially in breweries. The pre-inoculum was obtained from yeast cultures maintained on malt-agar slants at 4ºC and inoculated in 50 mL of 15ºP wort (°Plato, equivalent to g of sucrose in 100 g wort). The pre-inoculum was cultivated on a rotatory shaker in 125 mL Erlenmeyer flasks at 30ºC during 24 h. After this period the cells were inoculated in the fermenter.
The brewers worts were prepared from barley malt using conventional brewing techniques. In the experiments with corn syrup, highly concentrated MOR SWEETâ 1557 syrup (Corn Products Brasil) was added to the worts as soon as they began to boil. The manufacturers carbohydrate specifications for this syrup are approximately 12% dextrose, 42% maltose, 10% maltotriose, and 23-28% other sugars (dextrins). The syrup is virtually nitrogen-free. After boiling the wort, the precipitated proteins were removed according to density and the wort was cooled to the fermentation temperature.
Fermentation and Maturation
Static fermentations were conducted in a cylindrical glass bioreactor (7 L nominal capacity) containing 4.5 L brewers wort. The wort was aerated with the addition of air to the strain and mechanic agitation until saturation and then the cells were inoculated at a concentration of 5×106 cell/mL. The factors studied and the levels used are shown in Table 1. The initial pH was adjusted by the addition of lactic acid or calcium carbonate to the wort. After the main fermentation, the beer was maturated for 15 days at 4ºC and diluted to 3.5% (p/v) ethanol. To determine when to end the main fermentation, a preliminary fermentation was conducted under the same conditions before each experiment, until attenuation became unaltered. The sugar concentration obtained at the end of each preliminary fermentation was used to determine the end of the main fermentation.
Samples were taken at specified times throughout the fermentations and centrifuged at 4000×g for 10 min at 10ºC. The supernatants were frozen for subsequent analysis. Attenuation of the fermentation process was achieved by determination of the total reducing sugars, TRS (Nelson, 1944), using maltose as the standard. Ethanol concentration was determined by a CG 3537 gas chromatograph operated at 90ºC and equipped with a 3.0 m long column packed with a Chromosorb WHP column (60/80 mesh). The ethanol was detected by a flame ionization detector operated at 150ºC. Cell concentration was determined by a Neubauer chamber.
RESULTS AND DISCUSSION
Table 2 shows the results of the experiments, including three repetition experiments used to estimate the experimental error of the process. The best attenuation value (87.38%) was obtained with a wort concentration of 15ºP at 25ºC and pH 5.5 without corn syrup. The highest yield coefficient for sugar-to-ethanol conversion (Yp/s) was 0.418 g ethanol /g TRS (consumed), obtained in the experiment employing wort of 15ºP at 15ºC and pH 5.5 without corn syrup in the wort composition. The highest productivity was 0.143 g/L.h, attained with a wort concentration of 20ºP at 25ºC and pH 5.5 without corn syrup, considering for this the significant influence of the factors according to estimate of the effects (Tables 3, 4 and 5). As can be observed, the best results were those obtained when there was no corn syrup in the wort composition.
Tables 3, 4 and 5 show the statistical analysis of the results obtained with the parameters studied. Table 3 shows that the factors wort concentration and percentage of corn syrup in the wort composition had a significant effect on wort attenuation at a probability level below 5%. The effect of interaction between the factors wort concentration and fermentation temperature was significant at the same probability level. A decrease in wort attenuation within the range studied was evident not only when the wort concentration increased, but also when malt was partially replaced by corn syrup, which contains no nutritional substances other than sugars (Jones and Pierce, 1964). This nutritional deficiency is mainly due to the lack of nitrogen components (Kirsop, 1982), which are used as growing factors, causing the fermentations to become slow or stuck.
Interaction between the factors wort concentration and fermentation temperature (Figure 1) reveals that the increase in wort concentration did not affect wort attenuation when fermentation temperature was kept at 15ºC. On the other hand, at 25ºC wort attenuation decreased, probably due to the toxic effects of ethanol at higher temperatures, as described in the literature (Stewart et al., 1988).
Table 4 shows the results of statistical analysis of the effects of the factors studied on the yield coefficient for TRS-to-ethanol conversion. As can be seen, none of the factors was significant at a probability level below 5%.
Table 5 shows the effects of the factors studied on volumetric productivity. Wort concentration, percentage of corn syrup and fermentation temperature were significant at a probability level below 5%. Interaction between percentage of corn syrup and fermentation temperature was also significant at the same probability level. The increase in wort concentration within the range considered resulted in an increase in the volumetric productivity of the process. Although fermentation time was increased, the increase in ethanol concentration was higher, thereby enhancing productivity. The increase in the percentage of corn syrup in the wort composition interfered with productivity, confirming that nutritional supplementation is required by high-gravity worts, as described in the literature (Casey et al., 1983). Raising the temperature decreased fermentation time and improved productivity.
By the interaction between temperature and percentage of corn syrup (Figure 2), it can be seen that when temperature was fixed at the maximum level (25ºC), the addition of syrup did not change productivity. On the other hand, when temperature was fixed at the minimum level (15ºC), the addition of syrup reduced productivity by 15%.
Based on the fermentation parameters studied and on the analysis performed in this work, and considering for this the significant influence of the factors according to estimate of the effects (Tables 3, 4 and 5) and not the punctual values obtained (Table 2), use of 100% malt worts was shown to result in the best attenuations and the highest productivities, having no significant influence on the TRS/ethanol yield coefficient. On the other hand, it is known that when 100% malt worts are used, it is more difficult to obtain very highly concentrated worts with traditional equipment, as is the case with the lauter tun, which utilizes malt husk to filter the wort. Overloading the lauter tun causes the malt to become more compact and this, in turn, interferes with filter efficiency. In that case, the use of worts with high concentration obtained with 100% malt, can cause excessive load in the mashing process and wort filtration, decreasing the productivity and quality. To avoid this problem, some large breweries are using press filters. Another point to be considered is that the syrup is easier to manipulate than rice and corn gritz, which makes it more attractive than other cereals. The highest attenuation values were achieved with the lower wort concentration (15ºP), although productivity decreased. The increase in attenuation without affecting productivity was only possible with the use of nutritional supplementation. Casey et al. (1983) attained a 64% increase in productivity when the high-gravity worts were supplemented with yeast extract, ergosterol and oleic acid.
In our work, the pH value had no significant influence on the parameters studied at a probability level below 5%. The pH range from 4.5 to 5.5 did not influence productivity, attenuation or Yp/s. Breweries traditionally use initial pH from 5 to 5.5 to produce "lager" beer in Brazil.
The higher temperature (25ºC) resulted in the highest attenuation and productivity values and did not influence the Yp/s. In Almeida et al. (2000a), applying only 25°C, the percentage of corn syrup also had no influence on process productivity, whereas initial pH and especially initial wort concentration had influence on productivity. However, according to Casey et al. (1984), in fermentations at 25 and 30ºC, the viability and the reuse of the yeast decreased drastically. Moreover, the increase in production of esther and fusel alcohols at temperatures above 14ºC, as described by Engan and Aubert (1977), is also relevant.
The best results of the fermentation parameters evaluated were obtained with 100% malt worts fermented at 25°C. The use of 15ºP and 20ºP worts provided the best attenuation and the highest productivity, respectively. However, other factors, such as brewery facilities (mainly the lauter tun), economic viability of adjunct manipulation and flavor alterations must also be taken into account in further studies.
The authors are grateful to FAPESP and CNPq for their financial support and to Maria Eunice Machado Coelho for her assistance in writing this paper.
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