Abstract
Bioconversion of chitosan into soluble anti-inflammatory chitosan oligosaccharides (CHOS) using a Bacillus chitosanase, BsCsn46A, was investigated, including food-grade approaches. After 48 h of enzymatic reaction, most of the final products were dimers and trimers. None of the CHOS products showed toxicity to human fibroblasts. Analysis of CHOS bioactivity against LPS-induced inflammation of human macrophages indicated that CHOS generated from different bioconversion processes have anti-inflammatory activity, the magnitude of which depends on the type of substrate and production process. Both lactic acid and HCl can be used to dissolve chitosan; however, the product generated from lactic acid solution was highly hygroscopic after lyophilization, hence not suitable for long-term storage. Downstream processes, i.e., centrifugation and filtration, affect its anti-inflammatory activity. Analysis of standard CHOS with known structure showed that an acetyl group at the reducing end and the degree of polymerization (DP) are critical for biological activity. Importantly, when applied at levels above the optimal concentrations, certain standard CHOS and CHOS mixtures could induce inflammation. These results support the potential of CHOS as anti-inflammatory agents but reveal batch-to-batch variation and possible side effects, indicating that careful quality assurance of CHOS preparations is essential.