Inhibitory Mechanism Of(+)-Catechin On Squid Trimethylamine-N-oxide Demethylation And Its Effect On The Functional Properties Of Squid Protein

Squid is one of the most important marine seafood species in China and one of the important economic seafood in China.squid is a low-fat,high-protein productwithgoodeconomicvalueandmarket.Trimethylamine-N-oxidedemethylase（TMAOase）candecompose trimethylamine oxide（TMAO）into dimethylamine（DMA）and formaldehyde（FA）during cold chaintransportation or low-temperature storage,which endangers the health of consumers and affects the quality of squid quality.In this study,Illex argentinus was used as the research object,the changes of FA and TMAOase activities during storage were analyzed,and TMAOase in Illex argentinus liver was purified.At the same time,The inhibitory mechanism of（+）-catechin on squid trimethylamine-N-oxide demethylation and its effect on the functional properties of squid protein were study.The main results are as follows:1.The effect of temperature on the changes of FA and TMAOase activities during squid storage was significant.with the prolongation of storage time,FA,DMA,TMA and total number of bacteri in squid（Illex argentinus）increased.TMAO and myofibrillar protein solubility decreased.The enzyme activity increased first and then rosed gently at 4℃and 0℃.Enzyme activity increased first and then decreased slightly at-20℃.There was a significant correlation between FA content and TMAOase activity at different temperatures(R_4℃=0.791,R_0℃=0.863,R_-20℃=0.825).2.The TMAOase in Illex argentinus liver was separated and purified by DEAE-52 anion exchange column and Sephacryl S-300 gel column chromatography,and the molecular weight of TMAOase was determined by SDS-PAGE.At the same time,the amino acid composition of TMAOase was determined by an automatic amino acid analyzer.The results showed that TMAOase was purified by 205.3 fold,with a specific activity of 36.96 U/mg,a recovery of 15.3%,a molecular weight of about 25 k Da,Amino acid composition was determined,indicating that 46.5%of total amino acids were tyrosine.3.Through fluorescence spectroscopy and enzymatic kinetics,it was found that inhibitory effect of（+）-catechin on TMAOase occurred via a non-competitive mechanism.Binding of（+）-catechin to TMAOase driven by hydrogen bonding and van der Waals interactions caused changes in the conformation of TMAOase and its intrinsic fluorescence quenching.In summary,the inhibition mechanism involves the binding of（+）-catechins the inactive center of TMAOase which made the structure of TMAOase more compact,leading to a reduction in the catalytic rate.4.The squid muscle-TMAOase simulation system was constructed.The changes of physical and chemical indicators（color difference value,water holding capacity,）and protein function indicators（solubility,particle size,turbidity,gelation ability,gel strength,etc）were measured.At the same time,The effects of（+）-catechins on the functional properties of squid protein were discussed.The results showed that（+）-catechins had an inhibitory effect on TMAO catalysis in squid and significantly reduced the content of FA and DMA;Low concentration of catechins（20,40,60μmol/g）could promote the solubility and gelation of squid MP,and it could inhibit the increase of turbidity and increase of particle size.High concentrations of（+）-catechins（80,100μmol/g）could inhibit the functional properties of squid myofibrillar protein.Therefore,the optimal catechin addition for maintaining protein functional properties was60μmol/g.