Impacts of Kafirin Allelic Diversity, Starch Content, and Protein Digestibility on Ethanol Conversion Efficiency in Grain Sorghum

Abstract

Cereal Chem. 91(3):218-227Seed protein and starch composition determine the efficiency of the fermentation process in the production of grain-based ethanol. Sorghum, a highly water-and nutrient-efficient plant, provides an alternative to fuel crops with greater irrigation and fertilizer requirements, such as maize. However, sorghum grain is generally less digestible because of extensive disulfide cross-linking among sulfur-rich storage proteins in the proteinstarch matrix. Thus, the fine structure and composition of the seed endosperm directly impact grain end use, including fermentation performance. To test the hypothesis that kafirin (prolamin) seed storage proteins specifically influence the efficiency of ethanol production from sorghum, 10 diverse genetic lines with allelic variation in the β-, γ-, and δ-kafirins, including three β-kafirin null mutants, were tested for ethanol yield and fermentation efficiency. Our selected lines showed wide variation in grain biochemical features, including total protein (9.96-16.47%), starch (65.52-74.29%), and free amino nitrogen (FAN) (32.84-73.51 mg/L). Total ethanol yield (ranging from 384 to 426 L/metric ton), was positively correlated to starch content (R 2 = 0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (R 2 = 0.52). Increases in FAN content enhanced fermentation efficiency (R 2 = 0.65). The highest ethanol producer was elite staygreen breeding line B923296, and the line with the highest fermentation efficiency at the 72 h time point was inbred BT×623. A large-seeded genotype, KS115, carrying a novel γ-kafirin allele, was rich in FAN and exhibited excellent short-term fermentation efficiency at 85.68% at the 20 h time point. However, the overall ethanol yield from this line was comparatively low at 384 L/metric ton, because of insufficient starch, low digestibility, and high crude protein.Multivariate analysis indicated an association between the β-kafirin allele and variation in grain digestibility (P = 0.042) and FAN (P = 0.036), with subsequent effects on ethanol yield. Reversed-phase HPLC profiling of the alcohol-soluble kafirin protein fraction revealed diversity in protein content and composition across the lines, with similarities in peak distribution profiles among β-kafirin null mutants compared with normal lines.

Genetic Basis of Protein Digestibility in Grain Sorghum

“…The 45-kDa oligomer is present in both the sorghum and maize prolamin profiles, but unlike in sorghum, the maize oligomer is susceptible to depolymerization by reducing agents both in cooked and uncooked samples (Nunes et al, 2005). Cremer et al (2014b) measured raw and cooked in vitro protein digestibility of three lines containing the b-kafirin null gene and reported that protein digestibility of the null lines was not different from other lines tested. In monomeric forms, kafirins are relatively readily digestible, with a-kafirin being the most easily digestible (Nunes et al, 2004), but because of its interior location within the protein body, a-kafirin is the last to be digested and the most abundant fraction in an undigested residual protein as demonstrated by in vitro digestion experiments (Oria et al, 1995b;Nunes et al, 2004) and transmission electron microscopy observations (Taylor et al, 1985a;Oria et al, 1995b).…”

“…Grain sorghum protein digestibility is influenced by several factors, some of which are related to grain physicochemical properties, but is controlled, to a large degree, by kafirin composition and endosperm protein body structure (Hamaker et al, 1995;Oria et al, 2000;Duodu et al, 2002Duodu et al, , 2003Bean et al, 2011;Cremer et al, 2014b). In other words, protein structure, composition, and protein-protein interactions have far greater influence on sorghum protein digestibility than nonprotein factors and protein-nonprotein interactions.…”

“…These oligomers were subsequently detected in other experiments as pepsin-resistant kafirin fractions (Italianskaya et al, 2009;Elkonin et al, 2013). Natural sorghum mutant lines containing a null b-kafirin gene have been reported (Laidlaw et al, 2010;Cremer et al, 2014b). Belton et al (2006) suggests that these oligomers are probably stabilized by non-disulfide bonds that form during wet cooking of sorghum flour and could contribute to the indigestibility of sorghum protein and may hinder digestion by forming physical barrier around monomeric proteins.…”

Grain Proteomics of Sorghum: Improving Digestibility, Nutritional Quality and Starch Conversion to Ethanol

“…Chip profiles indicate that several cultivars in addition to the β-kafirin null mutants contain relatively low levels of β-kafirin, including 296B, a highly digestible line (189). LOC analysis also revealed the presence of a small peak visible at 46kDa in alcohol-soluble samples, which could represent a high Mw kafirin or kafirin dimer.…”

“…S2) protein fractions across 28 sorghum lines using RP-HPLC revealed a range of diversity, particularly in the alcoholsoluble kafirin-containing fraction. Variation observed across wild-type and mutant lines could contribute to phenotypic differences, such as increased flour viscosity and fermentation efficiency (188,189). Among the β-kafirin null lines QL12, IS17214 and RTx2737, a high degree of similarity was observed in the alcohol-soluble peak distributions compared to lines with functional β-kafirin…”