Although sorghum malt is relatively rich in free amino nitrogen (FAN), the 150 mg FAN/L threshold recommended for brewing is difficult to obtain. The vitreous nature of the sorghum endosperm hinders proteolysis during brewing. Hence, exogenous proteolytic enzymes are often required to increase hydrolysis of sorghum malt protein to produce sufficient FAN in order to support rapid yeast growth during fermentation. Ten exogenous proteases were examined for their production of FAN in sorghum malt mashing. Mashing was done at 550C for 45 minutes. Levels of FAN, as determined by the ninhydrin method, showed great variation among the proteolytic enzymes, ranging from 96 in control to 182 mg/100 g malt with possibly of most effective proteolytic enzyme. The variation in FAN level was possibly due to different optimal mashing conditions of exogenous proteases used and perhaps due to low ratios of exopeptidase/endopeptidase in the enzyme preparations. Low temperature (400C) and long duration mashing for (7 hours) gave good FAN production during mashing to a total of 113 and 138 mg/100 g malt in control and the treatment with exogenous proteolytic enzyme Flavourzyme plus malt, respectively. The exogenous enzyme (Flavourzyme) plus potassium metabisulphite (PMB) increased FAN production during mashing in the ratio of 2 to 1 in a treatment where PMB was added compared to one without. Similarly, hot wort extract (HWE) increased by 8% during mashing with exogenous enzyme plus PMB compared to one without PMB, respectively. PMB was involved in destabilizing the disulphide bonds in the sorghum protein polypeptide chains allowing proteolytic enzymes better accessibility to proteins. The increase in HWE was possibly due to the starch being freed from the sorghum protein matrix. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed some oligomeric and polymeric kafirins after mashing. With transmission electron microcopy (TEM), protein bodies of varying sizes with partially degraded peripheral edges and some holes were seen after mashing. SDS-PAGE and TEM results suggest insufficient proteolysis. High protein digestibility sorghum’s potential for brewing was examined with reference to FAN production. Although during mashing FAN increased by approx. 82 and 115% for unmalted normal and high digestibility sorghums, respectively, the 150 mg FAN/L threshold, recommended for brewing was not achieved. FAN production to levels above 150 mg/L may only be realized if normal sorghum malt or high protein digestibility sorghum malt is mashed with exogenous enzymes containing sufficient exopeptidases coupled with appropriate mashing conditions. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Food Science / unrestricted

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, 2008. / Food Science / unrestricted