A number of factors can influence the behavior of yeast during fermentation. Some of these factors (initial wort concentration, initial pH and percentage of corn syrup in the composition of the wort) were studied in order to determine their influence on the productivity of fermentation. Fermentations were carried out at 25ºC utilizing a 2³ factorial design of these factors. The results showed that the percentage of corn syrup had no influence on process productivity, whereas initial pH and especially initial wort concentration did. It can be concluded that using pH and initial wort concentration values higher than those utilized in this work (5.5 and 20ºP, respectively) will result in a higher productivity.
factorial design; fermentation; high-gravity process
High-gravity brewing utilizing factorial design
R. B. Almeida1* * To whom correspondence should be addressed , J. B. Almeida e Silva1, U. A. Lima2 and A. N. Assis1
1Departamento de Biotecnologia, Faculdade de Engenharia Química de Lorena,
Lorena - SP, Brazil , CP 116, CEP 12600-000,
2Instituto Mauá de Tecnologia (IMT) São Caetano do Sul - SP, Brazil
E-mail : email@example.com)
(Received: March 2, 1999 ; Accepted: March 14, 2000)
Abstract - A number of factors can influence the behavior of yeast during fermentation. Some of these factors (initial wort concentration, initial pH and percentage of corn syrup in the composition of the wort) were studied in order to determine their influence on the productivity of fermentation. Fermentations were carried out at 25ºC utilizing a 23 factorial design of these factors. The results showed that the percentage of corn syrup had no influence on process productivity, whereas initial pH and especially initial wort concentration did. It can be concluded that using pH and initial wort concentration values higher than those utilized in this work (5.5 and 20ºP, respectively) will result in a higher productivity.
Keywords: factorial design, fermentation , high-gravity process.
With the need to increase productivity and consequently competitiveness on the market, many breweries are changing their methods and processes for producing beer. In this regard, the high-gravity process has aroused a considerable interest. However, high substrate concentrations have been shown to inhibit yeast growth and fermentation performance as a result of high osmotic pressure, low water activity and the toxic effects of increased levels of ethanol and other metabolites (Jakobsen. & Piper, 1989; Stewart et al., 1988). Among the factors that influence the fermentation process are cell concentration, cell viability, fermentation temperature, wort composition and concentration, oxygenation and ethanol concentration (DAmore et al., 1990). This work utilizes a 23 factorial design to determine the influence that some factors namely wort concentration, initial pH and percentege of corn syrup, exert on process productivity.
MATERIAL AND METHODS
The yeast strain employed in this study was Saccharomyces cerevisiae obtained from a strain commercially utilized in breweries. The preinoculum was obtained from yeast cultures maintained on malt-agar slants at 4ºC and inoculated in 50mL of 15ºP wort. Cultivation was performed on a rotatory shaker utilizing 125 mL Erlenmeyer flasks at 30ºC during 24h at 120 rpm. After this period the cells were inoculated in the fermenter.
Fermentation / Maturation
Static fermentations at 25ºC were conducted in a 7L cylindrical glass fermenter containing 4.5L brewers wort prepared from barley malt utilizing conventional brewing techniques (Linko, M., 1998). The wort was aerated until saturated with mechanic agitation and then the cells were inoculated at a concentration of 5x106 cell/mL. The factors studied and the levels utilized are shown in Table 1.
The initial pH was adjusted by the addition of lactic acid or calcium carbonate to the wort. Only after the pH adjustment, was the wort aerated and were the cells inoculated.
After the main fermentation, the beer was maturated for 15 days at 4ºC. After maturation the beer was diluted to 3.5%(p/v) ethanol. To determine the end of the main fermentation, a preliminary fermentation was conducted under the same conditions prior to each experiment until the attenuation was constant. The sugar concentration obtained at the end of the preliminary fermentation was utilized to determine the end of the main fermentation.
Samples were taken at specified times throughout the fermentations. Attenuation of the fermentation was determined by measuring the total reducing sugars (TRS) (Nelson, 1944). Ethanol concentration was determined by using a gas chromatograph CG model 3537-D and cell concentration by using a Neubauer chamber. For the determination of the wort saturation was utilized the O2 dissolved measurer equipment Ingold model 170. The saturation was determined by the estabilization of the O2 value measured during the aeration at the fermentation temperature.
RESULTS AND DISCUSSION
Factorial design is used to analyze the factors that can really influence a given response, in our case, productivity. It is very important that interpretation of the results is able to conciliate the mathematical answer with the biotechnological meaning. An analysis of Table 2 shows that the highest productivity was achieved with an initial pH of 5.5, 30% corn syrup in the wort and initial concentration of 20ºP. The best attenuation was attained without corn syrup and with an initial concentration of 15ºP and a pH of 5.5. The highest yield coefficients of substrate to ethanol (Yp/s) were obtained with wort containing 30% corn syrup and pH 5.5 with concentrations of 15ºP and 20ºP. According to these results, it is possible to discern that for the wort fermentations at an initial pH of 5.5, the initial wort concentration had no influence on the yield coefficient of substrate to ethanol (Yp/s).
The fermentation times of the experiments as well as the total time (fermentation + maturation) can be observed in Figure 1. For all the experiments, a maturation period of fifteen days was employed. As expected, the times required for the main fermentations in the experiments utilizing worts at 15ºP were shorter than those required for the main fermentations utilizing worts at 20ºP. Despite the increase in main fermentation times, the ethanol content of the worts fermented at 20ºP caused productivity to increase. The increase in ethanol concentration resulting from the worts at 20ºP was more significant than the increase in total process time when the fermentation and maturation times used for the determination of productivity were considered together.
Table 3 shows that the initial wort concentration had a significant effect on productivity which can be confirmed by comparing the results of the calculated t and tabled t for this factor. The pH appears to be significant according to the normal probability graphic for productivity shown in Figure 2 .This is confirmed by the farthest point from the straight line in Figure 2. From the analysis of Table 3 and Figure 2 is possible to see that the factor percentage of corn syrup had no influence on the productivity of the process.
The effects were again estimated, eliminating the factor % corn syrup and analyzing only the factors initial concentration, pH and interaction between initial concentration and pH, as can be seen in Table 4. Table 5 shows the variance analysis based on the same parameters. The analysis of these tables demonstrates that the pH and mainly the initial wort concentration really exert significant influences on productivity. Equation 1 shows a mathematical model proposed for productivity including the factors that exerted significant influence and also the interaction between initial wort concentration and pH. In Figure 3 an estimated response function is shown for these factors.
In analyzing Figure 3 it is possible to observe that productivity increases when pH and initial wort concentration increase. Values of pH and initial wort concentration higher than the maximum values used in this work would increase the productivity of the process. In our experiments the initial wort concentration of 20ºP and the initial pH of 5.5 resulted in maximum productivities. It should be stressed that productivity can not be analyzed separately. Other factors wich have important economic consequences for the breweries, such as cell reutilization, percentage of attenuation of the wort, yield coefficient of substrate to ethanol and fermentation time must be considered.
Although showing the best productivities, the experiments performed with a concentration of 20ºP and a pH of 5.5 had a lower attenuation than that of the experiments performed at 15ºP. This must be taken into account when analyzing the economic viability of the utilization of more concentrated worts.
Raising the initial wort concentration above 20ºP and the initial pH values will probably result in higher productivity. Other factors are also of significance, mainly the cell reutilization, percentage of attenuation, yield coefficient of substrate to ethanol and the organoleptic characteristics of the beer produced. Future work will be conducted to define the changes in the organoleptic characteristics of the beer produced and to understand how the factors studied in this work can influence these characteristics.
We thank all of our colleagues in the Biotechnology Department of the School of Chemical Engineering at FAENQUIL in Lorena for their assistance in the preparation of this manuscript .
We thank FAPESP and CNPq for their financial support of this research
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Publication in this collection
06 July 2000
Date of issue
14 Mar 2000
02 Mar 1999