Enzymatic Preparation And Functional Evaluation Of Xanthine Oxidase Inhibitory Peptides From Skipjack Tuna

Hyperuricemia and gout are metabolic diseases caused by long-term high blood uric acid levels.At present,hyperuricemia is mainly treated by western medicine usually followed by severe side effects.So it is necessary to search for effective and safe uric acid-lowering bioactive compounds.Skipjack tuna,characterized by high protein and low fat,is a kind of ideal material for preparation of bioactive peptides.In the present study,XOD inhibitory peptides were prepared from the dorsal and ventral muscle of skipjack tuna through controlled enzymatic hydrolysis.Membrane separation as well as affinity ultrafiltration-mass spectrometry were used to purify and charaterize XOD inhibitory peptides.The structure-activity relationship between peptides and XOD was analyzed by molecular docking.The main results are as follows:(1)A HPLC method was established for determining the contents of carnosine and anserine.Among the six parts of skipjack tuna,the carnosine and anserine contents of the dorsal and ventral muscle were the highest,so the dorsal and ventral muscle was used as the raw material for the following research.The water content,protein,ash,and fat of the mixture of dorsal and ventral muscle were analyzed,which were 0.43%,26.83%,1.63%,and 1.36%,respectively.(2)The enzymatic hydrolysis process was optimized by investigating the effects of enzyme and its dosage,pH,temperature,time and their interactions on degree of hydrolysis,nitrogen recovery,XOD inhibition and the contents of carnosine and anserine using one-factor-at-a-time method and response surface methodology.The optimal process parameters were as follows:the suitable enzyme was neutrase,and the enzyme dosage was 489.86 U/g,the pH value was 7.08,the temperature was 49.5 ?,and the time was 5 h.Under the optimal conditions,the degree of hydrolysis was 22.38%,nitrogen recovery was 83.81%,XOD inhibition activity was 62.26%and the contents of carnosine and anserine were 0.50 mg/g and 24.50 mg/g(on a dried basis)respectively,which were all in good agreement with the predicted.The peptides obtained were mainly composed of a fraction with molecular weight less than 1000 Da.The pearson correlation analysis showed that the XOD inhibition activity of skipjack tuna muscle hydrolysate was not associated with the contents of carnosine and anserine,which maybe due to the production of other XOD inhibitory peptides during hydrolysis.(3)After membrane separation,the XOD inhibition activity of the fraction 600?1000 Da was the highest,with a IC50 value 9.18 mg/mL and the antioxidant activity of the part was also the highest,indicating that the 600?1000 Da part could also alleviate the oxidative stress damage caused by high levels of uric acid.And the angiotensin converting enzyme inhibitory activity of the fraction 300?600 Da was the highest,with a IC50 value 0.12 mg/mL.(4)A pentapeptide Ala-Cys-Glu-Cys-Asp(ACECD)was isolated and identified by affinity ultrafiltration-mass spectrometry and the synthesized ACECD demonstrated high XOD inhibition activity with a IC50 value 7.23 mg/mL.Molecular docking results reveled that ACECD could enter the active site of XOD and interacted with specific amino acid residues due to hydrogen bond,hydrophobic interaction and Van der Waals force,obstructing the interaction between xanthine and XOD.The obtained results of this research indicated that the skipjack tuna peptides had the potential for anti-hyperuricmia.