Enzymatic modification of Bambara groudnut protein for the production of hydrogels

Natural polymer-based, especially plant protein hydrogels have recently been gaining attention because of their biodegradability and biocompatibility. Bambara groundnut is a potential source of protein in hydrogel production. However, the use of Bambara groundnut protein in such applications is limited because it is associated with the formation of inadequate crosslinks between polymer chains. Enzymatic treatment can potentially be used for improving the strength of Bambara groundnut protein hydrogel. In this study, the effect of laccase and transglutaminase (separately and in combination) on the microstructural, structural, rheological and mechanical properties of Bambara groundnut protein hydrogels was investigated for potential application in encapsulation and release of bioactive compounds. In the first part of this study, the effect of pH and NaCl concentration on the rheological and microstructural properties of Bambara groundnut protein gels were optimised using response surface methodology (RSM) to determine ideal starting conditions before enzymatic treatment. The effect of using crosslinking enzymes (transglutaminase and laccase) on the textural, rheological, structural and microstructural properties of Bambara groundnut protein hydrogels were then investigated. Since the effectiveness of enzymatic processes may be limited by using single enzymes, the use of a combination of enzymes was also investigated for the first time in gelation and optimised using RSM. Subsequently, encapsulation efficiency and release properties of the enzymatically crosslinked Bambara groundnut protein hydrogel were investigated using riboflavin as a model bioactive compound. The heat induced Bambara groundnut protein isolate (BPI) gels optimised for pH and NaCl prior to enzymatic treatment showed G’> G’’ over a frequency range of 0-100 rad s-1 . Although BPI gels displayed the characteristics of weak gels, slightly acidic conditions (pH 6) coupled with low NaCI concentration (0.5 M) promoted the formation of more rigid gels. These gels had the lowest water holding capacity and thiol content, suggesting the participation of disulphide linkages during network formation. Their microscopy images showed that the network was composed of porous homogeneous aggregates. Amino acid analysis showed that Bambara groundnut protein contains substantial amounts of amino acids including lysine, glutamic acid, cysteine and tyrosine with potential active sites for transglutaminase and laccase action. Laccase modification of Bambara groundnut protein caused a decrease in the gelation point temperature from approximately 85°C in the absence of laccase to 29°C at an activity of 3 U/g protein. 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. The difference between G’ and G” increased to approximately 1 log and the dependency on angular frequency reduced suggesting improvement in the strength of the formed gels. Bambara groundnut protein crosslinking by laccase, was demonstrated by the decrease in thiol and phenolic content and crosslinking of amino acids (glutathione, cysteine and lysine) in model reactions. 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 protein. Transglutaminase crosslinking at 15 U/g protein resulted in the formation of hydrogels with well-organised network structures and small pores. Gel strength improved as observed from the highest G’ (6947 Pa) and hardness (5.60 N) recorded upon use of this activity. Transglutaminase-mediated crosslinking of BPI hydrogel was demonstrated by the reduction in amine and thiol groups and the formation of a new protein band (56 kDa) in crosslinked hydrogels. The combined use of transglutaminase and laccase showed a G’ > 10G” over a frequency range of 0 – 100 rad/s suggesting the dominance of the elastic behaviour. BPI hydrogel with the highest hardness (15.96 N) and encapsulation efficiency (98.8%) was formed at 15 and 0.5 U/g protein 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 which indicated that riboflavin release was due to diffusion and swelling. Overall, modification of Bambara groundnut protein using a combination of crosslinking enzymes increased the crosslinking density and promoted the formation of strong hydrogels. The hydrogels effectively encapsulated and prevented the early release of a heat sensitive compound (riboflavin) in the stomach while making it available in the small intestines. Therefore, the optimised enzyme combination of laccase and transglutaminase is a potential strategy for application in Bambara groundnut protein gelation