Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin

Abstract

The reaction mechanism of chitosan, bovine serum albumin (BSA), and gelatin with genipin (a natural crosslinking reagent) was examined with infrared, ultraviolet-visible, and 13 C NMR spectroscopies; protein-transfer reaction mass spectrometry; photon correlation spectroscopy; and dynamic oscillatory rheometry. Two reactions that proceeded at different rates led to the formation of crosslinks between primary amine groups. The fastest reaction to occur was a nucleophilic attack on genipin by a primary amine group that led to the formation of a heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA and gelatin. The second, slower, reaction was the nucleophilic substitution of the ester group possessed by genipin to form a secondary amide link with chitosan, BSA, or gelatin. A decreased crosslinking rate in the presence of deuterium oxide rather than water suggested that acid catalysis was necessary for one or both of the reactions to proceed. The behavior of the gel time with polymer concentration was consistent with second-order gelation kinetics resulting from an irreversible crosslinking process, but was complicated by the oxygen radical-induced polymerization of genipin that caused the gels to assume a blue color in the presence of air. The lower elastic modulus attained after a given time during crosslinking of the globular protein BSA as compared to the coiled protein gelatin, despite possessing more crosslinkable basic residues, demonstrated the importance of protein secondary and tertiary structures in determining the availability of sites for crosslinking with genipin in protein systems.

The physicochemical properties of legume protein isolates and their ability to stabilize oil-in-water emulsions with and without genipin

“…Although genipin has not received food-safe status in North America it is currently approved for use in food products in Japan, Korea and Taiwan (Nickerson et al 2006c). Genipin forms inter-and intra-molecular covalent bonds with primary amine groups (e.g., lysine) so as to modify protein structure (Butler et al 2003). Genipin has been extensively used to crosslink a variety of proteins including but not limited to: gelatin, bovine serum albumin (BSA), whey and gelatin-carrageenan mixtures (Butler et al 2003;Annan et al 2008;Huang et al 2009;Devi and Maji 2010).…”

“…However the choice of fixatives for use in the food industry is quite restricted. Genipin is a novel naturally derived crosslinking agent obtained from the Gardenia fruit via enzymatic hydrolysis from its parent compound, geniposide, by β-glucosidase (Butler et al 2003;Nickerson et al 2006b). Historically, Gardenia fruits have been used in traditional Chinese medicine for their anti-inflammatory, diuretic and haemostatic properties (Butler et al 2003).…”

“…Genipin is a novel naturally derived crosslinking agent obtained from the Gardenia fruit via enzymatic hydrolysis from its parent compound, geniposide, by β-glucosidase (Butler et al 2003;Nickerson et al 2006b). Historically, Gardenia fruits have been used in traditional Chinese medicine for their anti-inflammatory, diuretic and haemostatic properties (Butler et al 2003). Although genipin has not received food-safe status in North America it is currently approved for use in food products in Japan, Korea and Taiwan (Nickerson et al 2006c).…”

Characterization of Genipin Crosslinked Hydrogels Composed of Chitosan and Partially Hydrolyzed Poly(vinyl alcohol)

“…It has been reported that genipin is an effective crosslinking agent and can react spontaneously with amino groups or proteins to form dark blue pigments [10]. Genipin has also been found to be much less cytotoxic than glutaraldehyde [13].…”

“…All these compounds are chemically synthesized and are not free from the problems of physiological toxicology. Recently it has been reported that chitosan can be crosslinked with a naturally occurring cross-linking reagent, genipin [10,11].…”