UBC Theses and Dissertations
Covalent attachment of limiting amino acids to wheat gluten for nutritional improvement Li-Chan, Eunice Chi Yu
The benefits of fortification of poor quality food proteins such as wheat gluten with limiting amino acids depend on the biological availability of the added amino acids and their stability with respect to processing and storage. Although simple addition of amino acids in free form is convenient, the potential improvement in nutritional quality by this method of fortification may not materialize due to possible losses during processing steps such as washing, susceptibility to degradative reactions, and different rates of absorption and utilization compared to protein-bound amino acids. In this study, covalent attachment of lysine and threonine to wheat gluten was investigated using the chemical carbodiimide reaction and the enzymatic plastein reaction. The nutritional quality of enriched products was evaluated by in vitro and microbiological tests, and susceptibility to destruction by heating in the presence of a reducing sugar was investigated. Covalent attachment of lysine ethyl ester or threonine to gluten by the plastein reaction utilizing the enzyme papain was not successful. Although lysine and threonine contents in the products were increased, these results were attributed to selective enzymatic release of other amino acids. The amino acid compositions of undialyzable "plastein" products were markedly different from the original gluten substrate, and product yields were low. The results suggest the formation of many dialyzable peptides and free amino acids, and inability for protein re-synthesis from these low molecular weight compounds. Covalent lysine and threonine contents were increased using the carbodiimide reaction. In general, the reaction was most influenced by pH, reactant concentration and type of reactant. Products enriched via primarily peptide bonds as well as products enriched via peptide and isopeptide bonds could be prepared using various starting materials. Lysine, N -acetyl lysine, N -benzylidene lysine and threonine were coupled through amide bond formation to gluten, sodium stearate-solubilized gluten, acid-solubilized gluten or pepsin-solubilized gluten. Sodium stearate solubilization did not improve extent of amino acid incorporation. Pepsin hydrolysis of gluten enhanced amino acid attachment but decreased product yields. 4.0-fold and 6.5-fold increases in lysine content resulted by reaction of pepsin-solubilized gluten with N e-benzylidene lysine and N-acetyl lysine, respectively. However, yields of these products were low (47% and 58%). 1.6-fold, 2.0-fold and 2.5-fold increases were obtained by reaction of gluten with Ne-benzylidene lysine, lysine and N£-acetyl lysine, respectively, with product yields of 90 to 95%. At least 20-fold and 5-fold increases could be achieved by reaction of 0.5N HCI and 0.05N HCI solubilized glutens respectively with lysine, with product yields of 80 to 90%. 4-fold and 2-fold increases in threonine content resulted from reaction of threonine with pepsin-solubilized gluten and gluten, respectively. Simultaneous attachment of lysine or lysine derivative and threonine was not effective. In vitro evaluation of availability and digestibility of covalently enriched products was carried out by the DNBS reaction and pepsin pancreatin digestion tests. The results indicate the formation of isopeptide bonds involving the £-amino group of lysine unless N -substituents of lysine were used. Isopeptide bonds involving the Y_carboxyl groups were indicated when gluten had been solubilized by acid treatment. Peptide bond formation predominated when N -benzylidene or N -acetyl lysine was attached to pepsin-solubilized gluten or gluten. The high hi vitro availability and digestibility values for N £-benzylidene lysine enriched products suggest lability of the Schiff's base linkage of this N £-substituent, in contrast to the stability of the amide linkage in Ne -acetyl lysine. Microbiological evaluation by a Tetrahymena bioassay confirmed the nutritional improvement of gluten by covalent attachment of lysine, Ne-acetyl lysine or Ne-benzylidene lysine. Relative nutritive value of gluten was 54, whereas covalently and freely enriched glutens had relative nutritive values similar to that of the reference casein, assigned a value of 100. Covalently and freely lysine enriched glutens were compared for color and extent of lysine destruction after baking. In general, covalently enriched products had lighter color and higher percentages of total, DNBS-available and pepsin-pancreatin-digestible lysine contents than freely enriched products. N e-Benzylidene lysine enriched gluten was particularly stable, with relative nutritive value of 88 compared to 44 for baked gluten. It is concluded that covalently attached lysine is more stable than free lysine for enrichment of food proteins susceptible to Maillard reaction.
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