2&3Department of Post Harvest Process and Food Engineering, G. B. Pant University of Agriculture and Technology, Pantnagar-263145, U S Nagar, Uttarakhand, (India)
The raw grains i.e. Finger millet (Eleusinecoracana), Barley (Hordeumvulgare) oftraditional varieties, required for the study, were purchased from the local market ofHaldwani and Ramnagar District Nainital. Grains were cleaned manually and kept in moisture proof plasticcontainers for studies.Hops, the female flower clusters (commonly called seed cones or strobiles), of a hopspecies, Humuluslupulus were procured from M/s Manavbreweries Ghaziabad (UP) and used primarily as a flavoring and stability agent in beer, as they impart a bitter,tangy flavor.Yeast strains (Saccharomyces cerevisiae MTCC 170).
Preliminary experiments were conducted to identify the various affecting the fermentation process of beer production from barley and finger millet and to select the process variables and their experimental range for the study. Variables considered on the basis of review were blend ratios: Barley: Finger millet X1 (100:0, 50:50, 0:100), kilning temperature for production of malt was desirable for maintaining the quality of beer. The minimum temperature for kilning has been reported as 50°C for finger millet (Karki and Kharel 2012) and maximum temperature has been reported as 90°C. Hence, it was desired that the kilning temperature range for production of malt from finger millet and barley should be kept X2 (50, 70, 90°C), the malted grains and water ratio range was kept in the range of 1:2, 1:4, 1:6 and preliminary trails were conducted and it was observed during the experiment that the ratio 1:2 was not good as quality of wort, it was not appropriate because complete water got evaporated so wort could not be prepared. Hence, the ratios were changed to X3 (1:3, 1:5, and 1:7). The design of 17 experiments were done using Design Expert 220.127.116.11 software (Box Behenken method) (Khuri and Cornell 1987) shown in Table 1.
|Expt. No.||Coded values||Actual values|
barley: finger millet
|Kilning temperature (°C)||Slurry ratio
(malted grain: water)
Barley and finger millet were cleaned and washed thoroughly to remove immature grains, light materials, dirt and were steeped in surplus water at room temperature (28 ± 2°C) for a period of 24 hrs. The water was changed every 6 to 8 hours over a period of 24 hrs.
After soaking, for a period of 24 hrs, the water was drained off and the grains were left on stainless steel sieves for germination process for a period of 30-36 hrs. Grains were gently disturbed in order to provide aeration and to prevent from matting. During preliminary experiments, the germination was done for a period of 48 hrs but during that period mould growth was observed. The germination was observed to be best in period of 30-36 hrs.
After germination, the germinated grains (green malt) were kilned in Integrated Malting Unit at different temperature (50, 70, 90°C) for 14-20 hrs. Kilning was done till the desired moisture content was achieved (3-5% for barley and 9 ± 1% for finger millet) as reported by Karki and Kharel (2012).
The malt was crushed manuallyand breaks down barriers in the grains, giving the enzymes, full access to the carbohydrates, present in the grains, and facilitates the efficient extraction of the soluble material (extract) from the malt. This malt is called ‘malt grist’.
To prepare the wort, malted grain and water (1:3, 1:5, 1:7 slurry ratios) was boiled. Firstly, only the water as per the ratio maintained (210, 350, 490ml) was heated at the temperature 68-70°C respectively and after heating, the malted grain as per the ratio (100:0, 50:50, 0:100) kept (70gm of barley/500ml, 35gm barley+35gm finger millet/500ml and 70 gm of finger millet/500ml) were added and the total mixture was again boiled for 40 min at slow fire.
500 ml of tap water was heated at 68-70°C in another ware and sparging was done with hot water. The mash was repeatedly washed (2-3 times) so that maximum extract can be obtained. This was done with the help of strainer. This clear liquid obtained through this process is called wort.
As soon as the wort started boiling, 1 gm (in 500 ml of wort) of hops were added to enhance the flavor and colour of the final product and the whole mixture was kept on boiling at 100°C for further 1 hr at slow fire.
After 1 hrs boiling, hops were separated by using strainer and muslin cloth. After hops separation, the wort was cooled at a temperature of 18-20°C which was best for yeast growth in fermentation.
When the wort was cooled at the temperature of 18-20°C, the level of wort was less than 500 ml, hence the volume was made up to 500ml by adding simple tap water and then 30 ml of liquid yeast was transferred in 500ml wort in Laminar flow chamber. After transferring the yeast, the flask was closed with cotton plug and placed in dark place. After three days, cotton plug was removed and flasks were again plugged using fermentation lock for a period of 14 days.
After 14 days of fermentation, fermented liquor was centrifuged at 4000-5000 rpm for 15 minutes in order to remove all yeast cells, Supernatants was stored in refrigerator at low temperature.
pH of fermented liquor
The pH of sample was measured directly by digital pH meter (Triode India). The pH probe was calibrated using standard buffer solution (pH 4 and pH 7) prior to measurement of pH of sample at 30°C.
Ten milliliters of fermented liquor was taken in a 35 mL centrifugal tube and degassed with the help of magnetic stirrer by stirring gently. Wavelength was kept 275 nm and absorbance was set 0.000 with 2, 2, 4-trimethyl pentane as a reference blank. Twenty milliliter 2, 2, 4-trimethyl pentane and 0.5 mL HCL (6 mol/L) was taken in centrifuge tube and rotated for 15 min in centrifuge. The centrifugation was done until maximum extraction had been achieved. The absorbance of the sample was recorded for centrifuged sample and this was repeatedly done till no change in absorbance was observed. Bitterness was calculated by the formula given below:
BU = A275 × 50
Where, A is absorbance at 275 nm in a 10 mm cuvette.
Alcohol was estimated calorimetrically according to Reid and Salmon, (1955). A standard curve was prepared by using 0.0 to 8.0 mg/ml of absolute alcohol to which 2 ml standardize (0.36 N) ceric ammonium nitrate reagent was added. After 5 min of mixing, the extinction was read at 486 nm on spectrophotometer. A blank was also prepared using 5 ml of distilled water. Calibration curve was plotted taking ethanol concentration on x-axis and absorbance on y-axis.
Out of all process parameters, pH is one of the important parameter that determines the quality of beer. pH and buffering capacity of mash can influence the flavour stability. The experimental data as tabulated in Table 2 shows the pH levels of beer for different combination of the experiments conducted. Maintaining the correct pH for enzyme during the mash ensure the proper conversion of starch and degradation of haze causing protein. Data in the Table 2 shows that the least pH (5.05) of beer was observed in Experiment No. 8 which had blend ratio 0:100 (X1 = 1), kilning temperature 70°C (X2 = 0), and 1:7 slurry ratio (X3 = 1). The maximum pH (5.63) was observed for the Experiment No. 7 which had blend ratio 100:0 (X1 = -1), kilning temperature 70°C (X2 = 0), and 1:7 slurry ratio (X3 = 1).These data shows that the pH was maintained throughout the entire range of experiments i.e. ranging from 5.05 to 5.63 which shows an acceptable range as brewer’s normally look for a pH of 5.2 to 5.5.
|Exp. No.||Blend ratio||Kilning temperature||Slurry ratio||pH||Bitterness||Alcohol content|
|*Minimum and **Maximum|
pH = 5.48600-0.100X1+ 0.088750X2 - 0.033750X3- 0.027500X1X2 - 0.21250X1X3 - 0.035X2X3- 0.040500X12 -0.068X22 - 0.11800X32 .....................2
|Coeff.||P value||Coeff.||P value||Coeff.||P value|
Table 4: Total effect of individual parameters on pH.
Bitterness of the beer was measured over entire range of experiment 1-17. The Table 2 shows that the bitterness of beer varied from 28.50 to 32.50. As per Beer Style International Bittering Unit, ASBC, the acceptable range for bitterness of ale type of beer is reported to be 12 to 40. The values of bitterness come in acceptable range for various set of experiments. The bitterness of beer is due to addition of hops. The least bitterness value (28.5) of beer was observed for Experiment No. 9, 16 having blend ratio 50:50 (X1 = 0), kilning temperature 50°C (X2 = -1), and 1:3 slurry ratio (X3 = -1) and blend ratio 50:50 (X1 = 0), kilning temperature 70°C (X2 = 0), and 1:5 slurry ratio (X3 = 0) respectively. The maximum bitterness value (32.5) was observed for the Experiment No. 11 which had blend ratio 50:50 (X1 = 0), kilning temperature 50°C (X2 = -1), and 1:7 slurry ratio (X3 = -1). It could be seen from Table 2 that highest value of bitterness was obtained for experiment no. 11 where the pH for the same experiment is quite low. The reason behind the increase in bitterness could perhaps due to the fact that lower pH slightly decreases hop utilization and therefore, improves the quality of the bitterness, while higher pH slightly increases hop utilization and the harness of bittering compounds. The hop utilization is temperature dependent and could affect the bitterness of the beer. The coefficient of determination (R2) of the regression model for bitterness was 83.66%, which implies that the model could account for 83.66% of data. The predictive equation for estimating bitterness is given below
Bitterness = 29.1400-0.2500X1+ 0.16875X2+0.64375X3+0.062500X1X2-0.26250X1X3- 1.0X2X3- 0.20750X12 +0.6550X22 + 0.70500X32 ..........................3.
|Blending ratios (X1)||4||0.976||0.244||0.697|
|Kilning temp (X2)||4||6.056||1.514||4.325**|
|Slurry ratios (X3)||4||9.69||2.42||6.91**|
The Table 2 shows that the % alcohol varies from 4.17 to 12.08 indicating the presence of alcohol. The least alcohol content (4.17) of beer was observed in Experiment No. 07 which have blend ratio 100:0 (X1 = -1), kilning temperature 70°C (X2 = 0), and 1:7slurry ratio (X3 = 1) in the experiment. The maximum alcohol content (12.08) was observed for the Experiment No. 5 which had blend ratio 100:0 (X1 = -1), kilning temperature 70°C (X2 = 0), and 1:3 slurry ratio (X3 = -1). The maximum alcohol content 12.08% was observed for blend ratio (100:0) which shows that the barley alone could produce alcohol but the presence of alcohol as 9.70% also proves that if the finger millet is mixed with barley (50:50), the alcohol could be produced to some extent. Hence, full potential of important but underutilize source of starch, the finger millet could be explored for production of beer. The coefficient of determination (R2) of the regression model for alcohol content was 92.91%, which implies that the model could account for 92.91% of data and 7.09% variation was not explained by the model. Lack of fit was insignificant; therefore, second order model was adequate in describing alcohol content. The predictive equation is given below
Alcohol = 4.700+0.058750X1-0.46000X2-1.69625X3+0.54500X1X2+1.71250X1X3-0.050000X2X3+1.57875X12 +0.75125X22 +2.34875X32 ...................4.
Figure 1 shows the effects of slurry ratio and kilning ratio on pH at optimum points of blend ratio 0.58 (79). It was observed that the pH increases with decreases in slurry ratio while pH increases with increase in kilning temperature to point 0.20. Figure 2 shows the effects of slurry ratio and blend ratio on pH at optimum points of kilning temperature X2 = -1 (50°C). It was observed that the pH decreases with increase in blend ratio and slurry ratio.
This research was carried out in the Department of Post Harvest Process & Food Engineering under the National Fellow Project funded by the Indian Council of Agriculture Research. We sincerely thank the Research Director and Dean of the College of Technology, G.B.P.U.A. & T., Pantnagar for providing necessary support for the smooth functioning of the research work.
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