Western Blot Analysis Uncovers Clues to Prolamin Digestibility in Raw and Cooked Meal from Sorghum and Corn

摘要

Digestibility of cereal grain protein is conventionally expressed as percentage of protein digested by pepsin in a defined period. Sorghum has the lowest protein digestibility among cereals especially after wet cooking. If additional details were known as to which proteins are resistant to digestion, it should be possible to find means to improve this property. In this paper, two approaches are described to extract and identify proteins undigested by pepsin in uncooked and cooked meal from numerous sorghum and corn varieties. The first, one-solvent approach involves extracting residues undigested after 2 h with Borate-SDS-ME and separating by PAGE. Improved separation of undigested sorghum proteins was achieved using NuPAGE Bis-Tris gels. Western blots with antibodies against particular zeins and kafirins, used to monitor fates of different kafirins, revealed differential digestion rates. A second approach involved extracting undigested residues sequentially with 60% r-butanol, 60% t-butanol-ME andBorate-SDS-ME. The second, three-solvent approach, coupled with western analysis, revealed the following, (i) Oligomeric forms of certain kafirins exist that differ in the degree of their susceptibility to pepsin digestion, (ii) Effect of cooking on theformation and digestion of the oligomers, (iii) Cross-linked forms of most alpha-kafirins became more resistant to digestion after cooking, (iv) Most alpha-kafirins are preferentially extracted in 60% gmma-butanol-ME while most gamma-kafirins are extracted in Borate-SDS-ME buffer, (v) Monomeric gamma-kafirins are resistant to pepsin digestion, (vi) gamma-Kafirins form a series of oligomers that exhibit differential resistance to digestion. Our results suggest that the presently described systematic approach to analyzing the digestion by pepsin of sorghum prolamins should lead to greater insights into the digestion of specific types of sorghum grain proteins.