Abstract: In this research, the final products resulting from the hydrolysis of agar by recombinant agarase AgaP438 were isolated and purified by gel filtration chromatography to investigate the moisturizing activity and potential mechanism of neoagaro-oligosaccharide with single degree of polymerization. The purified products were subjected to analysis, with purity evaluated via ion exchange chromatography (IEC) and the relative molecular mass determined using electrospray time-of-flight mass spectrometry (ESI-TOF-MS). The impact of neoagarotetraose on the viability of keratinocytes was assessed using the CCK-8 assay, while ELISA and immunofluorescence techniques were employed to examine its effects on the expression of filaggrin (FLG), aquaporin-3 (AQP3), and zonula occludens-1 (ZO-1) in keratinocytes. The study findings revealed that the isolation of neoagarotetraose with a relative molecular mass of 630 and a purity of 96.53% was accomplished via Bio-Gel P2 fine gel column, with a yield of 78.4%. Cell experiments demonstrated that neoagarotetraose had no significant impact on the viability of keratinocytes within the mass concentration range of 0.0625－1 mg/mL, and exhibited good biocompatibility. Neoagarotetraose significantly increased the expression of moisturizing-related proteins FLG, AQP3, and ZO-1 in keratinocytes at the mass concentration of 0.125 mg/mL and 0.5 mg/mL (P<0.05). These findings highlighted the development and utilization potential of neoagarotetraose in moisturizing cosmetics, which provided a new direction for the development of cosmetic raw materials.

Key words: neoagarotetraose, moisturizing, filaggrin, aquaporin-3, zonula occludens-1