Hydroxypyridinone Derivatives Containing An Oxime Ether Moiety:Synthesis,Biological Evaluation And Application In Preservation Of Shrimp

Shrimp is a widespread consumer's favorite sea food,which is a healthy and delicious food possessing a high protein and low fat content.However,the shelf life of fresh shrimp is short,due to browning and microbial spoilage during postmortem storage.Browning of shrimp is mainly caused by the melanosis initiated by the polyphenoloxidase(tyrosinase)-catalysed oxidation of tyrosine or its derivatives.Kojic acid,5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one,which can inhibit tyrosinase by chelating the copper ion in the active site of tyrosinase,has been used as an anti-browning agent in foods that rapidly change color.In this study,a series of novel hydroxypyridinone derivatives containing an oxime ether moiety were synthesized starting from kojic acid.Based on the results of their inhibitory effects on mushroom tyrosinase,compound 6f was investigated for its in vitro antioxidant ability,inhibitory effect on the growth of bacteria isolated from shrimp(Penaeus vannamei Boone),and the inhibition on polyphenoloxidase extracted from the cephalothoraxes of Penaeus vannamei Boone.At last,the potential of compound 6f as shrimp preservative was explored.The main results of this thesis are summarized as follows:1.A series of novel hydroxypyridinone derivatives containing an oxime ether moiety(6a?6f)were synthesized starting from kojic acid.All the final compounds have been fully characterized by 1H NMR,13C NMR,MS and HRMS.2.The inhibitory effect of these compounds on monophenolase of mushroom tyrosinase was determined.6f was found to possess the highest inhibitory activity,IC50 values against the monophenolase and diphenolase being 1.60 and 7.99 ?M,respectively.Investigation of the inhibitory mechanism of 6f indicated that the inhibition was reversible and of a competitive-uncompetitive mixed type.The Ki and K'i values of 6f were determined to be 18.07 and 21.34 ?M,respectively.3.The in vitro antioxidant activity assay indicated that DPPH and superoxide anion radical scavenging activities of 6f were significantly improved when compared to those of kojic acid(P<0.05).4.Six bacterial strains were isolated from Penaeus vannamei Boone,which were identified as Shewanella amazonensis,Shewanella haliotis,Microbacterium oxydans,Photobacterium damselae,Staphylococcus haemolyticus and Bacillus cereus.The results of bacterial inhibition zone,MIC and MBC indicated that 6f showed a stronger antibacterial activity than kojic acid against all of the six strains under the same concentrations(P<0.05).5.PPO was extracted from the cephalothoraxes of Penaeus vannamei Boone.Crude PPO showed the maximal activity using L-P-(3,4-dihydroxylphenyl)alanine(L-DOPA)as a substrate under the conditions of pH 6.0 and 50 ?.Based on the SDS-polyacrylamide gel electrophoresis(SDS-PAGE)assay and PPO activity staining with L-DOPA,the molecular weight of PPO was estimated to be 200?220 kDa.The Michaelis constant(Km)of PPO for the oxidation of L-DOPA was measured to be 3.02 mM.The Ki and K'i values of 6f were determined as 74.61 ?M and 146.43 ?M,respectively.6.The preservative effect of 6f on chilled Penaeus vannamei Boone was evaluated by microbiological analysis,chemical analyses(pH,TVB-N and TBARS)and sensory evaluation.The results indicated that 6f possessed a superior preservative effect on Penaeus vannamei Boone to that of kojic acid.The shelf life of shrimps treated with 0.1%6f in combination with 0.1%citric acid was extended to 12 days.