Application of crosslinking enzymes for the improvement of Bambara groundnut Vigna subterranea protein hydrogels

Abstract: Bambara groundnut is a pulse of African origin and a good protein source, which can be applied in hydrogel production. However, proteins derived from such pulses usually produce weak gels due to inadequate crosslinks between polymer chains. Therefore, in this study, two crosslinking enzymes, laccase (0-3 U/g protein) and transglutaminase (0-15 U/g protein) were employed (separately and in combination) in Bambara groundnut protein isolate (BPI) modification for gel enhancement. Structural modification, rheological and release properties were investigated. Model reactions were used to predict possible reaction mechanisms within the protein matrix. Laccase modification of BPI decreased the gelation point temperature from approximately 85°C (0 U/g) to 29°C at 3 U/g. Laccase treated samples showed a sharp increase in the G’ and G” values during the heating ramp as well as a wider gap between the moduli, suggesting the formation of a more established network structure. Microscopy images of the gel showed an increase in homogeneity and compactness of the lath sheet-like structure with increase in laccase activity up to 2 U/g. Transglutaminase crosslinking at 15 U/g resulted in strong gels (G’ 6947 Pa and hardness 5.60 N) with well-organised network structures and small pores. Crosslinking was demonstrated by reduction in amine and thiol groups, formation of a new protein band (56 kDa) and crosslinking of reactive amino acids in model reactions. BPI hydrogel with the highest hardness (15.96 N) and encapsulation efficiency (98.8%) was formed at 15 and 0.5 U/g of transglutaminase and laccase activities, respectively. The lowest swelling capacity recorded in this hydrogel contributed to the lowest release kinetic constants in both simulated gastric fluid (0.51) and simulated intestinal fluid (0.73) in the presence of digestive enzymes indicating that riboflavin release was due to diffusion and swelling. The optimised enzyme combination is a potential strategy for application in BPI gelation.