Abstract: The purpose of this study was to investigate the preparation of citrus low-methoxyl pectin (CLP) and caffeic acid (CaA) conjugate by a green and safe method using laccase. The conjugate was examined for its key composition, ultraviolet (UV)-visible, Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) spectroscopic characteristics and in vitro antioxidant activity, in order to clarify its formation mechanism and intermolecular bonding mode, and summarize the structural factors contributing to its antioxidant activity. The results of chemical composition and anion-exchange chromatograms showed that CLP contained 51.99% (mol%) galacturonic acid (GalA), 30.95% galactose (Gal), 7.44% rhamnose (Rha) and 9.02% glucose (Glc) as well as a trace amount of total phenolics (1.20 mg/g), with a methoxylation (DE) degree of 38.33%, suggesting that the major molecular fragments of CLP were homogalacturonan (HG) and rhamnogalacturonan type I (RG-I), whose neutral saccharide side chain was galactan. The weight-averaged molecular mass (mw) of CLP was 226.1 kDa. In contrast to CLP, CLP-CaA conjugate showed significantly increased DE and total phenolic content of 46.93% and 9.64 mg/g (P < 0.05), respectively, and an increased mw of 271.4 kDa. Moreover, CLP exhibited UV and FTIR absorption peaks characteristic of CaA at 288 nm and 1 519 cm-1, respectively. The 700 MHz 1H and 176 MHz 13C NMR spectra indicated evidently resonant peaks for α-D-GalA and β-D-Gal moieties, and partial α-L-Rha moieties of CLP, which confirmed its major structure. New resonant peaks were observed for all glucosyl carbons of CLP in CLP-CaA conjugate. The largest chemical shift occurred on the C1 and C4 atoms of the α-D-GalA moieties, the C6 (carboxyl carbon) peak was shifted to δC 175.12, and peaks characteristic of CaA appeared at δH 6–8 in the 1H spectra. To sum up, the conjugation mechanism of CLP with CaA could be laccase-mediated binding of CaA to the C6 carboxyl group of the α-D-GalA moieties in CLP at a 1:1 molar ratio, resulting in the formation of ester groups. As a result, the conjugate had excellent, multiple in vitro antioxidant activities, whose half maximal inhibitory concentration (IC50) was 0.82, 2.47, 0.92, and 0.72 mg/mL for scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation, hydroxyl radial, and superoxide anion radical, respectively. The results of this study provide a theoretical basis for applying CLP-CaA conjugate in the food industry.

Key words: low-methoxyl pectin; caffeic acid; laccase; conjugation mechanism; structural characterization; antioxidant activity