World Journal of Microbiology and Biotechnology 6, 318-322
Use of unmalted sorghum as a brewing adjunct
C.I. Owuama
Hydrolysis of three varieties of unmalted sorghum by endogenous amylases produced sufficient maltose and glucose for subsequent brewing of beer. The optimal temperature and pH of the sorghum amylaae activities varied between the three varletles./~-Amylase was optimal when used at 60~ and pH 5 for two varieties but at 60~ and pH 6 for another. The optimal :(-amylase activity was between about 72~ and 75~ for all the sorghum varieties. The aetivitles of the amylases differed considerably between the species. For French summary, see next page. This work was carried out at the Department of Microbiology, University of Nigeria, Nsukka, Nigeria. C.I. Owuama is now with the Department of Biological Sciences, Federal University of Technology, P. M. B. 2076, Yola, Gongola State, Nigeria, and N. Okafor is now with the Anambra State University of Technology, Awka Campus, P. M. B. 5025, Awka, Nigeria. C.I. Owuama is the Corresponding Author.
1990 Rapid Communications of Oxford Ltd.
318
and N. Okafor
Introduction Adjuncts in brewing processes are essentially starchy materials with very little or no protein content. They are a potential source of additional alcohol and may contribute to the colour, taste, aroma, vitamin and protein content, and head retention of the beer (Hough et al. 1971; Dhamija & Singh 1979). Unmahed materials such as bajra, sorghum, tapioca (Manihot escukntum), soy beans, and wheat and barley flours have been added to grists as adjuncts (Dhamija & Singh 1979). The presence of amylase inhibitors in wheat and sorghum grains has been reported (Novellie 1960). Earlier, adjuncts were characterized as having inadequate or zero enzyme complement, therefore their starch was believed to be digested by the enzyme present in the grist (Hough et aL 1971), but Koszyk & Lewis (1977), reported that some unmahed cereal grains (wheat) can produce as much diastatic power and protease activity as malt itself. Starch adjuncts have been reported as being acted upon only if they were gelatinized (Hudson et al. 1963). This report has, however, been contradicted by a later report (Linko et aL 1965) which stated that it was unnecessary to gelatinize the starch for the enzyme to act. Reviewing the two reports, Hough et aL (1971) suggested that the variation between them could be a reflection of differences in the fineness of grinding of the barley flour or the thickness of the mash or the quantity of enzymes present. The presence and nature of 0~- and fl-amylases in sorghum malt have been reported by Novellie (1960). Examination of both the mature and immature grains of sorghum revealed traces of amylase activity. There are soluble and insoluble fractions of the amylases. The insoluble fraction is believed to be attached to another protein and can only be solubilized when the link is broken by a protease in the grain which must be allowed to act for a long period during aqueous extraction (Novellie 1960). American varieties of sorghum have very little ct- or fl-amylase (Novellie 1962) and differ in/J-amylase content compared with the South African varieties. The use of sorghum grits as adjuncts in lager beer brewing has been reported (Hahn 1966). However, in the report, the sorghum grits were gelatinized and added to mashes made from malts. Hence the malt enzymes were responsible for sorghum starch hydrolysis.
Unmalted sorghum in brewing L'hydrolyse de trois vari6t6s de sorghum non malt6s par des amylases endog(~nes produit suffisamment de maltose et de glucose pour la fermentation ult6rleure du moQt. Les temp6ratures et pH optimum des activit6s amylolytlques du sorghum varient selon la vari~t6. Pour deux vari6t6s, I'activit6 ~-amylolytique est optimal Iors de son utilisation ~ 60~ et /z pH 5, tandis qu'elle est optimale ~z 60~ et/z pH 6 pour la troisi~me. L'activit6 ~-amylolytique est optimal ~ environ 72/! 75~ pour routes les vari6t6s de sorghum. Les activites amylolytiques varlent consld6rablement selon les esp~ces.
The aim of this report is to show the action of unmalted sorghum grain amylases during the hydrolysis of its ungelatinized starch. This knowledge, if exploited, could increase the available reducing sugar for yeast fermentation in beer brewing.
Materials and M e t h o d s Source of Sorghum The sorghum varieties used in this work were LRV, BES and L-1499 (Owuama & Okafor 1987).
Hydrolysis of Sorghum Grits This was carried out by a modification of the method of Okafor & Aniche (1980). Ten g finely ground grains were mixed with 200 ml distilled water containing 12 ml 0.1 M NH4OH at room temperature (30~ The pH of the mixture was adjusted to pH 4, pH 5, pH 6, pH 7 or pH 8. This mixture was stirred constantly for 1 h. The temperature was then raised to 40~ 50~ 60~ 70~ 72~ or 75~ and incubated for 30, 60, 90 and 120 min. The mixture was then heated to 85~ to stop further enzyme activity, and allowed to settle. The supernatant was decanted and assayed for sugars (glucose, maltose and sucrose) following the method of Somogyi (1945).
Determination of A mylase Activity Ten g ground grains were mixed with 100 ml distilled water containing 6 ml of 0.1 m NH4OH. The mixture was stirred constantly for 1 h at room temperature (30~ The solution was centrifuged. The supernatant was taken as the enzyme extract (Zamora & Fields 1973). A 1% (w/v) soluble starch solution was made by boiling the starch in each of two buffers, pH 5, pH 6. The buffers were prepared by mixing required amounts of 0.1 M citric acid and 0.2M NaeHPO 4 (Zamora & Fields 1978), 0.5 ml aqueous extract from each variety of sorghum was added to 0.5 ml 1% (w/v) buffered soluble starch solution at pH values 4 to 8. These samples were incubated at 40~ 50~ 60~ 70"C, 72~ and 75~ for 120 min. The pH and temperature optima for amylase activities were determined by monitoring the amount of reducing sugar produced (Miller 1959; Novellie 1962). The activity of 0.5 ml purified enzyme extract on 0.5 ml 1% buffered soluble starch was regarded as 100%.
Determination of o~- and fl-Amylase Activities The enzyme extract was purified and the ~- and fl-amylase fractions separated following the method of Srivastava et al. (1984). The optimal temperature and pH activities of the enzyme were determined (Srivastava et al. 1984). Results
and Discussion
The results of these experiments are the average of three observations. The sugars observed in the sorghum varieties during processing were sucrose, maltose and glucose. This agrees with the report of Owuama & Okafor (1987). The sucrose concentration remained virtually the same at 0.3 mg/ml but the maltose and glucose values altered with temperature, pH and time of hydrolysis. Amylase (~ and fi) activity varied between the three sorghum varieties, L-1499, LRV and BES showing the highest activity at 50~ and 72~ at pH 5 or 6 after 2 h (Fig. 1). No further changes were observed after 2 h. The highest glucose plus maltose concentrations were observed at pH 5 or 6. The amylase activities at other pH values examined, namely 4, 7 and 8, were lower than those at pH 5 and 6. The optimal pH for amylase activity apparently agrees with that of sorghum malt (Owuama & Okafor 1987). The variation in the quantity of reducing sugars (maltose plus glucose) produced
319
C.I. Owuama and N . Okafor
1.4
i.4
1.3 15
c
1.2
1.2
1.1
1.1
1.0
1.0
\
0.9
0.9
0.8 ~
Use of unmalted sorghum as a brewing adjunct
C.I. Owuama
Hydrolysis of three varieties of unmalted sorghum by endogenous amylases produced sufficient maltose and glucose for subsequent brewing of beer. The optimal temperature and pH of the sorghum amylaae activities varied between the three varletles./~-Amylase was optimal when used at 60~ and pH 5 for two varieties but at 60~ and pH 6 for another. The optimal :(-amylase activity was between about 72~ and 75~ for all the sorghum varieties. The aetivitles of the amylases differed considerably between the species. For French summary, see next page. This work was carried out at the Department of Microbiology, University of Nigeria, Nsukka, Nigeria. C.I. Owuama is now with the Department of Biological Sciences, Federal University of Technology, P. M. B. 2076, Yola, Gongola State, Nigeria, and N. Okafor is now with the Anambra State University of Technology, Awka Campus, P. M. B. 5025, Awka, Nigeria. C.I. Owuama is the Corresponding Author.
1990 Rapid Communications of Oxford Ltd.
318
and N. Okafor
Introduction Adjuncts in brewing processes are essentially starchy materials with very little or no protein content. They are a potential source of additional alcohol and may contribute to the colour, taste, aroma, vitamin and protein content, and head retention of the beer (Hough et al. 1971; Dhamija & Singh 1979). Unmahed materials such as bajra, sorghum, tapioca (Manihot escukntum), soy beans, and wheat and barley flours have been added to grists as adjuncts (Dhamija & Singh 1979). The presence of amylase inhibitors in wheat and sorghum grains has been reported (Novellie 1960). Earlier, adjuncts were characterized as having inadequate or zero enzyme complement, therefore their starch was believed to be digested by the enzyme present in the grist (Hough et aL 1971), but Koszyk & Lewis (1977), reported that some unmahed cereal grains (wheat) can produce as much diastatic power and protease activity as malt itself. Starch adjuncts have been reported as being acted upon only if they were gelatinized (Hudson et al. 1963). This report has, however, been contradicted by a later report (Linko et aL 1965) which stated that it was unnecessary to gelatinize the starch for the enzyme to act. Reviewing the two reports, Hough et aL (1971) suggested that the variation between them could be a reflection of differences in the fineness of grinding of the barley flour or the thickness of the mash or the quantity of enzymes present. The presence and nature of 0~- and fl-amylases in sorghum malt have been reported by Novellie (1960). Examination of both the mature and immature grains of sorghum revealed traces of amylase activity. There are soluble and insoluble fractions of the amylases. The insoluble fraction is believed to be attached to another protein and can only be solubilized when the link is broken by a protease in the grain which must be allowed to act for a long period during aqueous extraction (Novellie 1960). American varieties of sorghum have very little ct- or fl-amylase (Novellie 1962) and differ in/J-amylase content compared with the South African varieties. The use of sorghum grits as adjuncts in lager beer brewing has been reported (Hahn 1966). However, in the report, the sorghum grits were gelatinized and added to mashes made from malts. Hence the malt enzymes were responsible for sorghum starch hydrolysis.
Unmalted sorghum in brewing L'hydrolyse de trois vari6t6s de sorghum non malt6s par des amylases endog(~nes produit suffisamment de maltose et de glucose pour la fermentation ult6rleure du moQt. Les temp6ratures et pH optimum des activit6s amylolytlques du sorghum varient selon la vari~t6. Pour deux vari6t6s, I'activit6 ~-amylolytique est optimal Iors de son utilisation ~ 60~ et /z pH 5, tandis qu'elle est optimale ~z 60~ et/z pH 6 pour la troisi~me. L'activit6 ~-amylolytique est optimal ~ environ 72/! 75~ pour routes les vari6t6s de sorghum. Les activites amylolytiques varlent consld6rablement selon les esp~ces.
The aim of this report is to show the action of unmalted sorghum grain amylases during the hydrolysis of its ungelatinized starch. This knowledge, if exploited, could increase the available reducing sugar for yeast fermentation in beer brewing.
Materials and M e t h o d s Source of Sorghum The sorghum varieties used in this work were LRV, BES and L-1499 (Owuama & Okafor 1987).
Hydrolysis of Sorghum Grits This was carried out by a modification of the method of Okafor & Aniche (1980). Ten g finely ground grains were mixed with 200 ml distilled water containing 12 ml 0.1 M NH4OH at room temperature (30~ The pH of the mixture was adjusted to pH 4, pH 5, pH 6, pH 7 or pH 8. This mixture was stirred constantly for 1 h. The temperature was then raised to 40~ 50~ 60~ 70~ 72~ or 75~ and incubated for 30, 60, 90 and 120 min. The mixture was then heated to 85~ to stop further enzyme activity, and allowed to settle. The supernatant was decanted and assayed for sugars (glucose, maltose and sucrose) following the method of Somogyi (1945).
Determination of A mylase Activity Ten g ground grains were mixed with 100 ml distilled water containing 6 ml of 0.1 m NH4OH. The mixture was stirred constantly for 1 h at room temperature (30~ The solution was centrifuged. The supernatant was taken as the enzyme extract (Zamora & Fields 1973). A 1% (w/v) soluble starch solution was made by boiling the starch in each of two buffers, pH 5, pH 6. The buffers were prepared by mixing required amounts of 0.1 M citric acid and 0.2M NaeHPO 4 (Zamora & Fields 1978), 0.5 ml aqueous extract from each variety of sorghum was added to 0.5 ml 1% (w/v) buffered soluble starch solution at pH values 4 to 8. These samples were incubated at 40~ 50~ 60~ 70"C, 72~ and 75~ for 120 min. The pH and temperature optima for amylase activities were determined by monitoring the amount of reducing sugar produced (Miller 1959; Novellie 1962). The activity of 0.5 ml purified enzyme extract on 0.5 ml 1% buffered soluble starch was regarded as 100%.
Determination of o~- and fl-Amylase Activities The enzyme extract was purified and the ~- and fl-amylase fractions separated following the method of Srivastava et al. (1984). The optimal temperature and pH activities of the enzyme were determined (Srivastava et al. 1984). Results
and Discussion
The results of these experiments are the average of three observations. The sugars observed in the sorghum varieties during processing were sucrose, maltose and glucose. This agrees with the report of Owuama & Okafor (1987). The sucrose concentration remained virtually the same at 0.3 mg/ml but the maltose and glucose values altered with temperature, pH and time of hydrolysis. Amylase (~ and fi) activity varied between the three sorghum varieties, L-1499, LRV and BES showing the highest activity at 50~ and 72~ at pH 5 or 6 after 2 h (Fig. 1). No further changes were observed after 2 h. The highest glucose plus maltose concentrations were observed at pH 5 or 6. The amylase activities at other pH values examined, namely 4, 7 and 8, were lower than those at pH 5 and 6. The optimal pH for amylase activity apparently agrees with that of sorghum malt (Owuama & Okafor 1987). The variation in the quantity of reducing sugars (maltose plus glucose) produced
319
C.I. Owuama and N . Okafor
1.4
i.4
1.3 15
c
1.2
1.2
1.1
1.1
1.0
1.0
\
0.9
0.9
0.8 ~
