The rheological and structural properties of fish collagen cross-linked by N-hydroxysuccinimide activated adipic acid

Fish skin collagen, with a concentration (6 mg/ml) higher than its critical aggregation concentration (∼0.5 mg/ml), was cross-linked using N-hydroxysuccinimide activated adipic acid (NHS-AA). Dynamic rheological measurement indicated that the storage modulus, loss modulus, complex viscosity and dynamic denaturation temperature increased while the loss tangent decreased with increasing the degree of cross-linking (DC). In addition, collagens with different DC exhibited a significant difference in their activation energies. It was found that collagens with DC in three intervals (DC < ∼25%, ∼25% < DC < ∼50% and DC > ∼50%, respectively) possessed remarkably different rheological behaviors each other. That is, collagens with DC < ∼25% exhibited a liquid-like behavior, while collagens with DC > ∼25% tended to exhibit a solid-like behavior, and especially a sudden loss of flow was observed for collagen with DC > ∼50%. Similar trends were observed from the features in differential scanning calorimetry (DSC) thermograms of collagen. That is, denaturation temperature (Td) was slightly increased and the endothermic peak became broader as DC < ∼25%, Td was increased gradually as DC was further increased, while a sudden increase in Td was observed and the endothermic peak became sharp for collagen with DC > ∼50%. The three-step changes in the rheological behaviors and DSC thermograms of collagens might be due to the unique aggregation behavior of collagen in solution.

(a) In collagen solution of 6 mg/ml, many of the collagen molecules were in an aggregation state. (b) As a small amount of NHS-AA was added into collagen solution, cross-linking reaction would mainly occur between adjacent collagen molecules in the same one aggregate. (c) As more NHS-AA was used, cross-linking reaction would occur between collagen molecules in different aggregates. (d) As NHS-AA was further added, most of the aggregates might be linked each other to form a whole network structure.Figure optionsDownload as PowerPoint slideHighlights
► The critical aggregation concentration of fish skin collagen was determined.
► Change in structural properties of cross-linked collagens was in a three-step manner.
► Collagen solution became gel in a sudden as cross-linking degree reached ∼50%.
► This three-step change might be due to the unique aggregation behavior of collagen.