Change Of Phenolic Acids In Post-Harvested Lotus Seeds During Fresh-Keeping And Research Of Proanthocyanidins From Lotus Seedpod

This research aimed to solve the fresh-keeping problem of lotus seed and the utilization of lotus seedpod, which is a by-product of lotus seed. Fresh-keeping methods for lotus seed and the structure, function and possible application of proanthocyanidins extracted from lotus seedpod (LSPCs) were studied. The microbial contamination status, nutritive preservation status, browning status and sensory characteristics were evaluated as the indicators of the quality of lotus seed treated in different conditions during storage. The change of varieties and contents of phenolic acids of lotus seed treated with the optimal methods were determined. The extraction, purification, characterization, antioxidant ability, protective effect on H2O2-induced damaged HUVECs and possible application in red wine ageing of LSPCs were respectively investigated. Main results were as follow:1. Five fresh-keeping formulas were obtained by fresh-keeping methods optimizing. They were ascorbic acid (0.25g/100 mL)+sodium bisulfate (0.25 g/100 mL), ascorbic acid (0.25 g/100 mL)+benzoic acid (0.25 g/100 mL), ascorbic acid (0.5 g/100 mL), sodium bisulfate (0.5 g/100 mL) and benzoic acid (0.5 g/100 mL) respectively. The lotus seeds were immersed in different hot fresh-keeping solutions at 80℃ for 3 min, and then air dried, germ freely packaged and stored at 25℃. The lotus seeds treated with above formulas presented better color, microbial inhibition, texture and odour.2. Nine phenolic acids were identified by HPLC-ESI-MS. They were gallic acid, chlorogenic acid, gentisic acid, caffeic acid, cinnamic acid,p-coumaric acid, ferulic acid and salicylic acid respectively. The total phenolic acid in control group decreased gradually from 62.91 mg/100 g of fresh lotus seed to 28.09 mg/100g in 10th day. The content of gallic acid in 5th day (7.32±0.09 mg/100 g) was higher than initial stage (4.23±0.03 mg/100 g) in control. For the treated groups, the total phenolic acid reached the peak values in different storage time, and the values were much higher than that of controls. Gallic acid, chlorogenic acid,p-coumaric acid, ferulic acid, salicylic acid and cinnaric acid presented most significant change in the treated groups. The content of gallic acid, chlorogenic acid,p-coumaric acid and ferulic acid in benzoic acid group were much higher than that of corresponding controls. Gallic acid reached the highest value (29.83±1.32 mg/100 g) in benzoic acid group. The contents of gallic acid (47.13±3.67 mg/100 g) and salicylic acid (43.66±2.94 mg/100 g) in sodium bisulfate group were much higher than that of fresh lotus seed. The content of gallic acid increased gradually and reached highest level in 10th day (38.95±1.68 mg/100 g). The cinnamic acid decreased markedly in different treated groups. The phenolic glycoside content decreased from 5th day to 10th day gradually.3. The purity of PCs from lotus seedpod reached 98.7% after AB-8 macroporous resin and Sephadex LH-20 purification. Twenty kinds of PCs were identified by HPLC-QTOF-MS. They were all comprised of catechin, epicatechin, gallocatechine and epigallocatechin. LSPCs mainly consisted of dimmers (74.00±8.43%) and trimmers (22.75±3.71%).4. PPO was extracted from fresh lotus seedpod by PBS at pH 7.2. The molecular weight of the lotus PPO was about 31KDa. Its secondary structure includes 59% α-helix,4.3% β-sheet,14.1% turns and 22.6% random coils by CD determination. Lotus PPO presented higher activity at 60-90℃ (the Q values are above 0.02), and has an optimal temperature of 80℃; Higher activity at alcohol concentration of 5-20%, and remained 85.1% relative enzymatic activity at 15% alcohol concentration; Higher activity at pH of 4-10, and had optimal pH of 9.0.5. LSPCs presented better FRAP, DPPH, ABTS, Fe2+/H2O2 radicals scavenging ability than positive control (ascorbic acid). PCs from mangosteen pericarp and camellia japonica flower, whose dimmer and trimmer were much lower than that of lotus seedpod, presented almost equal activity in FRAP, DPPH, ABTS, Fe2+/H2O2 radicals scavenging compared with ascorbic acid.6.150 μM H2O2 treatment for 24 h made a significant damage on HUVECs for that the morphology markedly changed compared to the control group. Pretreatment with LSPCs markedly weakened the H2O2-caused lipid peroxidation and decreased the content of MDA. LSPCs enhanced the activity of SOD and GSH-PX, increased the NO content in the cells and decreased the content of ET-1. These results revealed that LSPCs presented anti-atherosclerosis activity.7. Lotus PPO effectively catalyzed the polymerization of PCs. Compared with the content of monomers (3.12%), the content of monomers in MWS significantly increased at 0.5 h (22.47%) and 1 h (24.23%), decreased from 2 h (20.24%) to 10 h (0%). The content of dimmers gradually decreased from 0 h (74.33%) to 10 h (0%). The content of trimmers significantly decreased in 0.5 h (22.55% of 0 h to 7.13% of 0.5 h), and then gradually increased after 0.5 h, reaching highest at 10 h (100%). The conformation of PPO in MWS changed a lot. The a-helix content significantly decreased, and random coils and turns increased, revealing that the conformation was much instable. It was more easily captured by other phenolic compounds.