Purification and Characterisation of κ-Carrageenan Oligosaccharides Prepared by κ-Carrageenase from Thalassospira sp Fjfst-332

kappa-Carrageenan oligosaccharides (KCOs) are promising agents for treating inflammatory diseases. However, the lack of purification and structural elucidation of KCOs has limited structure-function evaluation. In this study, using a system coupling medium pressure liquid chromatography (MPLC) with an evaporative light scattering detector (ELSD), four types of KCOs were separated. The total yield of the four KCO powders was similar to 5.02% after purification (KCOs/kappa-carrageenan, w/w). Their structural identities were characterised by ESI-MS, CID-MS/MS and NMR, as kappa-neocarrabiose (alpha-DA-1,3-G4Sr alpha/beta), kappa-neocarratetraose (alpha-DA-1,3-beta-G4S-1,4-alpha-DA-1,3-G4Sr alpha/beta), kappa-neocarrahexaose (alpha-DA-1,3-beta-G4S-1,4-alpha-DA-1,3-beta-G4S-1,4-alpha-DA-1,3-G4Sr alpha/beta) and heterozygous kappa/iota-neocarrahexaose (alpha-DA/DA2S-1,3-beta-G4S-1,4-alpha-DA-1,3-beta-G4S-1,4-alpha-DA-1,3-G4Sr alpha/beta). KCOs showed no cytotoxicity in RAW264.7 macrophages, and the anti-inflammatory activity was closely correlated with the degree of polymerisation and the number of sulfated groups. kappa/iota-Neocarrahexaose exhibited the highest inhibition of ROS (Reactive Oxygen Species) production in LPS-induced RAW264.7 macrophages. The MPLC-ELSD system provides a platform for large-scale fabrication of purified KCOs and affords a route to these compounds that may regulate immune defense.