In order to explore the effect of ascorbic acid on papain activity, the binding mechanism between them was investigated by molecular docking and molecular dynamics simulation. The findings demonstrated that ascorbic acid at all three concentrations tested (0.1, 0.2 and 0.3 mmol/L) could enhance papain activity with an average percentage of activation of 10%, 21%, and 28%, respectively, indicating that the higher the ascorbic acid concentration was, the more pronounced the activation effect was. The results of molecular dynamics simulation revealed that the root mean square deviation (RMSD) showed a slightly increasing trend after papain binding to ascorbic acid, and the average number of hydrogen bonds within the protease molecule decreased from 146 to 143. In addition, the total accessible surface area increased by 2.38 nm², the root mean square fluctuations of the active sites Asp158, His159 and Cys25 increased, and the gap between Cys25, which plays the primary catalytic role, and the two auxiliary amino acids decreased. Accordingly, we concluded that ascorbic acid can change the structure of papain’s active center, thereby activing the protease.

The binding mechanism of puerarin (PUE) to β-lactoglobulin (β-lg) was investigated by fluorescence spectroscopy, molecular docking, and molecular dynamics simulation. The fluorescence spectroscopy results showed that PUE statically quenched the fluorescence of β-lg. At 25, 35, and 45 ℃, the binding constants were 7.24 × 10⁴, 1.34 × 10⁵, and 2.18 × 10⁵ L/mol, and the numbers of binding sites were 1.02, 1.18, and 1.15, respectively, indicating there was only one binding site or class of binding sites. Synchronous fluorescence and three-dimensional fluorescence spectroscopy indicated that the binding of PUE to β-lg resulted in an increase in the polarity of the microenvironment of β-lg, thereby weakening the hydrophobic force. Molecular docking results showed that PUE bound to the hydrophobic cavity of β-lg, forming hydrophobic interactions with six amino acid residues of β-lg and short hydrogen bonds with five amino acid residues. The molecular dynamics results showed that the root mean square deviation (RMSD), radius of gyration (R_g), and solvent accessible surface area (SASA) of the complex were (0.17 ± 0.02) nm, (1.47 ± 0.01) nm, and (88.94 ± 2.05) nm², and those of β-lg were (0.22 ± 0.03) nm, (1.48 ± 0.01) nm, and (90.09 ± 1.73) nm², respectively. The root mean square fluctuation (RMSF) of the complex was lower than that of β-lg, suggesting that the PUE/β-lg complex has better stability at the molecular level. This study is of reference significance for increasing the bioavailability of PUE.

In order to explore the effect of combined high-pressure and thermal treatment on the lipolysis of phospholipids in pork, the phospholipid degradation kinetics of pork was studied in the 300 to 700 MPa pressure range and in the 20 to 60 ℃ temperature range. It was found that the lipolysis process followed first-order kinetics. The lipolysis rate constant (k) of polyunsaturated fatty acids (PUFA) was the highest, followed by total phospholipids (PL), and those of monounsaturated (MUFA) and saturated fatty acids (SFA) were relatively low. The average activation energies of SFA and MUFA were the highest, and the absolute values of the average activation volume were at a high level. Hence, the lipolysis rate constants of SFA and MUFA were sensitive to temperature and pressure. Kinetic models for PL lipolysis were established using the Arrhenius and Eyring equations. Both models had good fitness to the data and showed similar temperature-pressure contour curves. Accordingly, the lipolysis kinetics of different phospholipid components was different, but the lipolysis kinetics of PL was very similar to that of PUFA. The kinetic models could predict the degree of lipolysis of phospholipids under preset pressure, temperature and time conditions, which will be helpful in designing or optimizing high-pressure processing parameters to improve the quality of meat products.

To improve the storage quality and to study the changes in storage characteristics of Panlongcai, the bacteriostatic effects of rosemary extract, tea polyphenols, carvacrol, ε-poly-L-lysine (ε-PL) and nisin on spoilage bacteria isolated from Panlongcai were tested by the Oxford cup method. Panlongcai added with the preservatives with good bacteriostatic activity were stored at 4 ℃ and evaluated for microbiological, organoleptic, physicochemical and flavor qualities after 0, 5, 10, 15, 20 and 25 days. The results showed that ε-PL, nisin and carvacrol had good inhibitory effects on the dominant spoilage bacteria in Panlongcai. During the storage process, the pH decrease was smaller in the carvacrol and ε-PL addition groups, 0.11 and 0.06, respectively. Carvacrol and nisin effectively inhibited lipid oxidation as assessed by thiobarbituric acid reactive substances (TBARS) value and total volatile basic nitrogen (TVB-N) generation. The total bacterial count (TBC), pH, TBARS and TVB-N data were analyzed by the combined use of partial least squares regression (PLSR), entropy weighting method and weighted grey correlation, and it was found that the highest weighted grey correlation occurred upon the addition of nisin (0.4059) and carvacrol (0.3876) after 25 days of storage, which indicates that the addition of nisin and carvacrol could effectively improve the microbiological and physicochemical qualities of Panlongcai during the storage process. Both nisin and carvacrol groups exhibited a less porous and denser microstructure. Electronic nose analysis showed a greater difference in the flavor of Panlongcai with the addition of carvacrol compared to the control group. Sensory evaluation showed that the addition of carvacrol significantly reduced the flavor and acceptability of Panlongcai (P < 0.05). In conclusion, the addition of nisin could effectively inhibit the growth and reproduction of microorganisms and reduce the changes of physicochemical indexes during cold storage without altering the flavor of Panlongcai, and improve its storage quality.

The bacterial community composition, its dynamic changes and the dominant spoilage bacteria in Panlongcai at different storage temperatures (4 and 20 ℃ ) were studied using high-throughput sequencing (HTS). The results showed that a total 19 bacterial phyla and 166 bacterial genera were detected in all samples, with the dominant bacterial phyla being Cyanobacteria, Firmicutes, Proteobacteria and Bacteroidetes. The relative abundance of Aeromonas, unclassified Chloroplast, and Paenibacillus changed significantly. Diversity analysis showed that there were significant fluctuations in the abundance of bacteria at the genus level with extended storage, irrespective of storage temperature. The species richness of the bacterial community in Panlongcai stored at 4 ℃ showed an overall increasing trend over 40 days of storage, decreasing in the first 10 days and later increased progressively. During storage at 20 ℃ , the species richness first decreased and then increased with fluctuations, reaching its peak on day 15. Furthermore, in the samples stored at 20 ℃ , the dominant microbial community achieved a competitive advantage in a relatively short period, more pronounced changes in the microbial flora being observed. It was inferred that the spoilage organisms were Aeromonas and Acinetobacter for Panlongcai stored at 20 ℃ , and Aeromonas, Acinetobacter, and Paenibacillus at 4 ℃ . Three Pseudomonas species (P. tohonis, P. juntendi, and P. psychrotolerans) were isolated from Panlongcai under refrigerated conditions, thus presuming that Pseudomonas is potential spoilage organism.