Comparing the Effect of HPP on the Structure and Stability of Soluble and Membrane-Bound Polyphenol Oxidase from 'Lijiang Snow' Peach: Multispectroscopic and Molecular Dynamics Simulation.

Polyphenol oxidase (PPO) easily causes fruits and vegetables to lose their color and nutritional value. As a non-thermal process, high-pressure processing (HPP) showed different inactivation effects on endogenous enzymes. In this work, soluble PPO (sPPO) and membrane-bound PPO (mPPO) from 'Lijiang snow' peaches were purified, and then the effect of high pressure on the conformation of sPPO and mPPO was investigated and compared at the molecular level. The maximum activation of sPPO and mPPO by 11.2% and 4.8% was observed after HPP at 200 MPa, while their activities both gradually decreased at 400 MPa and 600 MPa; in particular, the residual activities of sPPO and mPPO at 600 MPa for 50 min were 41.42% and 72.95%, respectively. The spectroscopic results indicated that the secondary structure of PPOs was little affected by HPP, but HPP led to obvious changes in their tertiary structure. The simulations showed that the decreasing distance between the copper ion and His residue in the copper-binding region of two PPOs at 200 MPa was favorable to catalytic activity, while the increasing distance between copper ions and His residues and the disordered movement of the loop region above 400 MPa were unfavorable. In addition, the structure of sPPO was relatively looser than that of mPPO, and high pressure showed a more significant effect on the conformation of sPPO than that of mPPO. This study clarified the effect of HPP on PPO's structure and the relationship between its structure and activity and provided a basis for the prevention of enzymatic browning.

Comparison on inhibitory effect and mechanism of inhibitors on sPPO and mPPO purified from 'Lijiang snow' peach by combining multispectroscopic analysis, molecular docking and molecular dynamics simulation.

The inhibition mechanisms of soluble PPO (sPPO) by l-cysteine, reduced glutathione and thiourea, and membrane-bound (mPPO) by l-cysteine, reduced glutathione, thiourea, anisaldehyde and cinnamaldehyde were investigated by combining multispectroscopic analysis and computational simulations. Reduced glutathione showed the strongest inhibitory effect, with IC50 of 0.46 and 0.94 mM, respectively. The multispectral results showed that all inhibitors inhibited activity by destroying the secondary and tertiary structure, and the structure of sPPO were more easily affected. Docking showed that hydrogen bond and metal contact were the main driving force for inhibitors binding to sPPO and mPPO, respectively. Simulation showed that sPPO-inhibitor system had more fluctuation than mPPO-inhibitor system, indicating easier inhibition of sPPO activity. This work revealed that the structural differences between sPPO and mPPO led to different inhibition mechanisms of PPOs by inhibitors at the molecular level, which could provide the guidance for the selection of inhibitors in fruit and vegetable processing.

Comparison of the Effects of High Pressure Processing, Pasteurization and High Temperature Short Time on the Physicochemical Attributes, Nutritional Quality, Aroma Profile and Sensory Characteristics of Passion Fruit Purée.

The study investigated the effects of high-pressure processing (HPP) (600 MPa/5 min), pasteurization (PT) (85 °C/30 s), and high-temperature short time (HTST) (110 °C/8.6 s) on physicochemical parameters (sugar, acid, pH, TSS), sensory-related attributes (color, aroma compounds), antioxidants (phenolics, vitamin C, carotenoids, antioxidant capacity), and sensory attributes of yellow passion fruit purée (PFP). Compared to the PT and HTST, HPP obtained the PFP with better color, sugar, and organic acid profiles. Although PT was equally effective preservation of antioxidants and antioxidant capacity of PFP compared to HPP, high temperature inevitable resulted in the greater degradation of the aroma profile. The amounts of esters, alcohols, and hydrocarbon in PFP were significantly increased by 11.3%, 21.3%, and 30.0% after HPP, respectively. All samples were evaluated by a panel comprising 30 panelists according to standard QDA (quantitative descriptive analysis) procedure, and the result showed that HPP-treated PFP was rated the highest overall intensity score with 7.06 for its sensory attributes, followed by control (6.96), HTST (6.17), and PT (6.16). Thus, HPP is a suitable alternative technology for achieving the good sensory quality of PFP without compromising their nutritional properties.

Structural studies and molecular dynamic simulations of polyphenol oxidase treated by high pressure processing.

High pressure processing (HPP) exhibited different effect on polyphenol oxidase (PPO), but the conformational changes was not clear yet. In this study, molecular dynamics simulation combined with spectroscopic experiments were used to explore PPO conformational changes under high pressure at the molecular level. The simulation results showed that high pressure decreased volume and hydrogen bonds, induced changes in active center and movement of loop. Especially, the conformational changes under 200 and above 400 MPa were different. Under 200 MPa, the distance between His 61 and Cu decreased by 0.4 Å, active pocket was exposed, substrate channel became larger. However, the distance increased by 6.1 Å under 600 MPa, active pocket moved inward, substrate channel became narrower. Docking results of 200 and 600 MPa had the highest and lowest binding affinity, whose T-score was 4.657 and 4.130, respectively. These results were consistent with spectroscopic experiments of PPO after HHP.