This study aims to investigate the molecular characteristics, spectral characteristics, and in vitro antioxidant and immunomodulatory activities of laccase-mediated passion fruit high-methoxyl pectin (PFP) derivatives with ferulic acid (FA) or p-coumaric acid (CA) in comparison with those of its derivative with ascorbic acid (PFP-L-VC) (without linkage with quinones formed from phenolic oxidation). The contents of total phenolic compounds (TPC) of the derivatives PFP-L-FA and PFP-L-CA were significantly higher than those of PFP and PFP-L-VC (P < 0.05). The weight-average molecular mass (m_w) of PFP-L-CA increased to 256.5 kDa, while the m_w of PFP-L-FA did not change obviously, and the m_w of PFP-L-VC decreased compared with that of PFP (190.5 kDa). Both phenolic acid derivatives showed significantly smaller Mark-Houwink-Sakurada exponent (α), radius of gyration (R_g), and anisotropic conformation parameter (ρ), implying a random-coil, flexible, dense, spherical conformation. Fourier transform infrared spectra displayed that the peak area at 1625 cm^-1 (free carboxyl group) decreased and the peak area at 1739 cm^-1 (esterified carboxyl group) increased, suggesting the formation of ester linkages between PFP’s carboxyl groups and phenolic acids. Compared with PFP, both PFP-phenolic acid derivatives had significantly improved in vitro antioxidant activities (in scavenging hydroxyl and superoxide anion radical, chelating Fe²⁺ and reducing power) and immunological activities (macrophage proliferation rate, NO release and neutral red phagocytosis), which could stimulate NO release from mouse macrophages and neutral red phagocytosis by mouse macrophages. In terms of scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, PFP-L-CA was superior to PFP-L-FA and PFP-L-VC, which may be related to phenolic acid structure. This study has demonstrated that phenolic acids can endow PFP derivatives with improved in vitro antioxidant and immunomodulatory activities, which will provide a theoretical basis for the application of phenolic acid-conjugated pectin as food gums and dietary fibers.

This study was executed to investigate the non-enzymatic browning kinetics and key phenolic composition of tamarind seed coat proanthocyanidins extract (TSPA) with different food formulation factors in order to elucidate the effects of food formulation factors and thermal storage on the key proanthocyanidin compounds, and to infer the possible mechanism of non-enzymatic browning. TSPA, prepared with 55% (V/V) ethanol, had total phenolic content of (397.1 ± 3.1) mg/g, mainly consisting of total proanthocyanidins (tannins) at (218.3 ± 3.9) mg/g. The ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) results indicated that the main proanthocyanidin components of TSPA were procyanidins B2 (PCB2), epicatechin (EC), protocatechuic acid (PCA), and caffeic acid hexoside (CAH). The results of zero-order browning kinetics showed that under different conditions of TSPA concentration, ethanol concentration, pH, and food additives (metal ions except Cu²⁺, thickening gums, and protectants), the browning rate constant (k₀) of TSPA was 0.0207–0.1581 d^-1 (R² = 0.969–0.999) during storage at 55 ℃ for 6 days. Addition of 1.0 mg/mL ginseng flower extract significantly (P < 0.05) reduced the browning degree of TSPA (at 1 mg/mL in 5% (V/V) ethanol). At pH 3.0–3.8 browning was reduced. Addition of 5 mmol/L metal ions significantly reduced the content of phenolic compounds in TSPA; Ca²⁺ and Cu²⁺ reduced the contents of PCB2 and EC by 96%–98%, decreased PCA content by 33% and 38%, respectively, and Na⁺ and Zn²⁺ reduced phenolic content by 10%–25%. The results of principal component analysis indicated that (-)-epicatechin-3,5-digallate (ECdG), PCA, PCB2 and EC were key for browning. Possible responses related to changes in the phenolic composition of TSPA were roughly deduced. This study may provide a theoretical basis for understanding the non-enzymatic browning mechanism of TSPA and controlling it.

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 (m_w) 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 m_w of 271.4 kDa. Moreover, CLP exhibited UV and FTIR absorption peaks characteristic of CaA at 288 nm and 1519 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 δ_C 175.12, and peaks characteristic of CaA appeared at δ_H 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.