Nitrogen limitation is one of the main causes of incomplete or protracted fermentations during brewing. In this study, the effects of sorghum type, exogenous protease enzymes and malting on the production of FAN were investigated. Further, the effects of different nitrogen sources on yeast fermentation performance, as well as the resulting changes at the phenotypic and genotypic level were studied.
With unmalted grain in the absence of exogenous proteases, white non-tannin sorghum produced substantially higher levels of FAN than white type II tannin sorghum. Similarly, in the presence of exogenous neutral proteinase enzyme, unmalted white non-tannin sorghum yielded more wort FAN than white type II tannin sorghum. Incubating unmalted W sorghum grain with neutral proteinase and amino-peptidase enzymes in combination, improved FAN production with white non-tannin sorghum. The two sorghum types produced similar FAN levels when malted, apparently as a result of the reduction in assayable tannins during malting. Malting did not influence the wort free amino acid profile. Group B amino acids which are utilized more slowly after group A amino acids have been utilized, constituted the highest percentage (42-47%).
Ammonia supplementation, in the form of diammonium phosphate, improved fermentation performance in terms of improved maltose and maltotriose uptake and more rapid reduction in specific gravity. Glycine supplementation negatively affected yeast fermentation performance when compared to the control. Lysine supplementation increased ethanol yield (up to 7.4% v/v) when compared to the control (7.1% v/v). However, it negatively affected yeast counts at the end of fermentation. Sorghum malt supplementation improved ethanol levels compared to the 100% raw sorghum control.
Serial repitching negatively influenced maltose and maltotriose uptake. Without nitrogen supplementation and with lysine supplementation, yeast cells from serially repitched sorghum worts were distorted and irregular. In contrast, yeast cells supplemented with ammonia were less affected by serial repitching.
The extent of growth supported by ammonia or lysine on brewing yeast cells was studied using phenotypic microarray. With regard to nitrogen utilization, ammonia supplementation supported more active growth under aerobic conditions than the control and lysine supplementation. Lysine did not support growth when it was used as a sole nitrogen supplement. Concerning carbon utilization, nitrogen supplementation did not affect sugar utilization except for maltotriose and xylose. Maltotriose did not support growth with the control and lysine supplementation, while xylose supported growth and this was probably an artefact. There was no genome change with serial respitching as shown by PCR fingerprinting with the (GTG)5 primer. However, relationships of the sequenced data indicated that mutation in the yeast genome occurred with lysine supplementation.
This study shows that nitrogen limitation negatively affects fermentation performance when brewing with unmalted sorghum. This is because nitrogen is required for the growth and metabolic activity of yeast cells during fermentation. Mashing white non-tannin sorghum grain and supplementing the resultant wort with ammonia in the form of diammonium phosphate are effective methods of improving assimilable nitrogen to ensure fast and complete fermentations.