Free amino nitrogen (FAN) levels in sorghum grain brewing are characteristically low as a direct consequence of the very low levels of FAN in unmalted sorghum grain. During fermentation, FAN provides nutritional support to the yeast, enabling optimal yeast growth and efficient fermentation. Exogenous proteolytic enzymes are required to hydrolyse sorghum grain protein into FAN. Eight commercial proteolytic enzymes were studied in terms of their efficacy to produce FAN levels during mashing considered adequate (150 mg/L) to support optimal yeast functionality and their effect on extract levels. FAN as determined by the ninhydrin assay, ranged between 25-72 mg/100 g sorghum grist (representing 0%-185% increase) when mashing was carried out at 55°C over 45 mins with the 8 different enzymes. The proteolytic enzymes varied greatly in terms of their efficacy, possibly as a result of having different optimal operating conditions. It is also suggested that the proteolytic enzymes differed in terms of their classification and exopeptidase/endopeptidase ratio. In an attempt to further increase FAN levels, the effects of the reducing agent potassium metabisulphite (KMS) and different mashing temperatures (50°C, 55°C and 60°C) were also studied. Three enzymes (Flavourzyme, Neutrase, Papain) were selected for this part of the study. The addition of KMS caused a significant increase in the FAN in mashing systems that utilized Flavourzyme (22% when mashing at 55°C) and Neutrase (14% when mashing at 55°C), but not with Papain. It is suggested that KMS reduced the stabilizing disulphide bonds present in the sorghum kafirin, thus making the kafirin more digestible. The KMS could have reduced the effectiveness of Papain because it is a sulphydryl protease which possesses stabilizing disulphide bonds which were reduced by the KMS. Flavourzyme and Papain were more active at 55°C, while Neutrase displayed maximum activity at 50°C. The effect of mashing at a low temperature (40°C) over an extended time period (7 hr) on FAN was studied using Flavourzyme. Samples treated with Flavourzyme and KMS showed a five-fold increase in FAN, while those treated with Flavourzyme only showed a four-fold increase. It is proposed that the low temperature was able to suppress the rate at which disulphide bonds were formed in kafirin, therefore improving its digestibility. The lower temperature also retarded the enzyme denaturation rate, while the long time period allowed the enzyme more contact time with the substrate. Microstructure analysis of sorghum protein bodies using transmission electron microscopy and of sorghum endosperm using confocal laser scanning microscopy revealed that the relative indigestibility of kafirin makes a significant contribution to the relatively low FAN levels. Although the action of the proteolytic enzyme and KMS on the sorghum endosperm protein matrix showed notable degradation, large fragments of undigested protein bodies were observed. This once again suggested that disulphide bond cross-linking played a major role in reducing the digestibility of sorghum protein. The addition of Flavourzyme to a complete mashing cycle was also found to result in a significant increase (9%) in extract. The ncrease in extract is attributed to the increased availability of starch to amylase hydrolysis as a result of the degradation of the protein matrix which envelopes the starch granules. The addition of KMS to the sorghum grain mashing system and the utilisation of a low temperature protein rest are effective methods of improving FAN in sorghum grain brewing.
Dissertation (MSc(Agric))--University of Pretoria, 2009.