The use of microbial transglutaminase to improve the viscosity of common bean flour (Phaseolus vulgaris) and attach methionine to phaseolin

New and inexpensive food ingredients are needed to overcome the demand for food in the world. Common bean flour is a potential candidate to replace flour from other sources in food products, because it is a highly nutritious and abundant product. However, bean flour is deficient in sulfur-containing amino acids, such as methionine, and has only fair functional properties. Therefore, microbial transglutaminase (TG) from Streptoverticillium was utilized in an effort to both improve the viscosity and viscoelastic parameters of bean flour and covalently attach methionine to phaseolin, the major common bean protein. The first objective of this project was to identify if proteins of raw and heat-treated bean flours were crosslinked by microbial TG and if the protein cross linking improved the viscosity and viscoelastic parameters of raw navy bean flour and heat-treated bean flours. Bean flour suspensions were incubated with microbial TG at its optimum temperature for maximum activity. Viscosity, elastic moduli and loss moduli were measured with a constant stress rheometer, and the performance of protein cross linking was evaluated by SDS-PAGE. Results of experiments showed that the viscosity, elastic moduli and storage moduli of raw navy bean flour was increased more than the viscosity, elastic moduli and storage moduli of heat-treated bean flour, and the SDS-PAGE results showed that proteins in raw bean flour were more susceptible to cross linking by microbial TG than proteins in heat-treated bean flour. The second objective was to identify the cause of the low protein cross linking in heattreated bean flour. Three potential factors were studied: (1) the destruction of available lysine residues in heat-treated bean flour was evaluated by using the dinitrofluorobenzene method, (2) the potential effect of protein-starch interactions in protein cross linking was evaluated in a model system using isolated protein and starch from raw navy bean seeds, and (3) protein aggregation. Results showed that protein aggregation during the heat treatment of bean seeds prior to producing bean flour seemed to be the cause of reduced protein cross linking by microbial TG. Finally, the last objective of this study was to attach L-methionine using microbial TG to phaseolin. After initial unsuccessful test using of L-methionine, L-methionine ethyl ester hydrochloride (L-met EEHCl) was used. The results of SDS and native-PAGE analyses, size exclusion chromatography (SEC), amino acid analysis, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) showed that L-met EEHCl was not attached to phaseolin, these results differed from published studies which have reported the attachment of L-met EEHCl to soybean proteins by tissue TG. Less cross linking of phaseolin monomers occurred when L-met EEHCl was present in the solution. Therefore, it was hypothesized that L-met EEHCl was acting as an inhibitor of the microbial TG activity instead of being a substrate. To test this hypothesis, the amount of ammonia released, which is a co-product of the reactions catalyzed by microbial TG, was measured using an ion selective electrode. The results showed that the amount of ammonia released from samples incubated with both microbial TG and L-met EEHCl was lower than from samples incubated only with microbial TG. It was hypothesized that the L-met EEHCl used in this study may be a competitive inhibitor and inhibits the enzyme reaction by reacting with the thiol group of the cysteine residue in the active group. However, more work needs to be conducted to determine the specific mechanisms of inhibition of L-met EEHCl on microbial TG. The overall results of this study showed that microbial TG can be used as a potential aid to improve physical properties of common bean flours and proteins, specifically phaseolin, and more work needs to be done to identify if other kind of substrates can be used by this enzyme in order to improve the nutritional value of bean flour