| China is a vast country with abundant marine fish,shrimp and shellfish resources,during these resources.Fish contains polyunsaturated fatty acids and a large number of high-quality proteins.And the high nutritional value of fish causes consumers to ignore its purine content.Existing data show that the purine content in marine fish is high,and excessive intake will lead to the increase of purine content and uric acid level in human body,which eventually induce gout.Therefore,it is important to know the purine content of marine fish and further to reduce the purine value of marine fish,in order to provide scientific guidance for the development of healthy diet and low purine food for consumers.However,at present,there is no unified national standard and method for the determination of purine content in marine fish,and the existing detection data is not perfect,which can not provide convenient guidance for consumers.And the development of low purine foods are limited to beer and legumes which contain more free purine,and the research on food with less free purine,especially fish,is less.Therefore,in this paper,single factor and orthogonal test were used to determine the appropriate purine extraction and detection methods for marine fish.At the same time,the effect of exogenous additives on purine content were detected and the removal mechanism were also explored by means of processing methods in order to provides experimental basis for improving the purine value of marine fish and establishing an appropriate,simple and highly operational method for purine removal.The main conclusions are as follows:1.Methanol-water,0.02 mol/L KH2PO4（pH=3.6）and water-methanol-glacial acetic acid-tetrabutyl ammonium hydroxide（V/V/V/V=879:100:15:6）were used as the flow phases,respectively,to optimize the flow phase ratio,pH,flow rate and column temperature.The results showed that four purine bases couldbeseparatedwhen0.02mol/LKH2PO4（pH=3.6）and water-methanol-glacialaceticacid-tetrabutylammoniumhydroxide（V/V/V/V=879:100:15:6）were used as mobile phase.The latter one showed better peak shape and resolution.Four purines could be separated at baseline within 10 minutes and saved detection time.In addition,the use of water-methanol-glacialaceticacid-tetrabutylhydrogenoxidation（V/V/V/V=879:100:15:6）avoided the tedious adjustment of pH in the process of mobile phase preparation and reduced the risk of phosphate corrosion on chromatographic column.Under the mobile phase conditions,the linear relationship of four purine bases were good within the range of 0.1-300 mg/L,and the correlation coefficient（R）was 1.0000.The detection limit of the method was 0.0465-0.1056 mg/L.The precision of the method was between 0.0220%and 0.7000%,The limits of detection are 0.0774（Adenine）,0.0178（Guanine）,0.0118（Hypoxanthine）,0.0555（Xanthine）,respectively.which met the requirements of analysis.2.Single factor and response surface analysis（RSA）were carried out on the key factors of ultrasonic purine extraction,perchlorate purine extraction and mixed acid purine extraction such as ultrasonic time,temperature,acid concentration,water bath time,temperature,etc.The optimum purine extraction method was determined by comparing the extraction percent of the three methods.The results showed that among the three purine extraction methods,ultrasonic extraction could only extract free purine from samples,and its extraction effect was inferior to acid extraction method.The total purine extraction percent of mixed acid extraction method（formic acid-trifluoroacetic acid）were better than those of perchloric acid purine extraction method.The recovery ratio of each component was more than 94.90,and the repeatability of the method ranged from 0.83%to 1.77%.The results show that the method is simple,reliable and has a low loss during the process of purine extraction.Therefore,the method was used to extract purine bases in marine fish and provided a basis for the determination of purine.3.The purine content in different parts（skin,back,abdomen,viscera and eyes）of 15 species of marine fish were determined by the optimal method.Moreover,the changes of purine content in edible parts of marine fish during storage were studied by simulating the storage conditions of marine fish.It was found that the total purine content in the tested fish ranged from 414.96 mg/100g to 1057.09 mg/100 g,in which sea bass>turbot>mackerel>sardines>yellow croaker>Brown flounder>hairtail>pedal fish>American Red Fish>cod>eel>sea catfish>rays.In addition,the purine content of edible parts of marine fish were analyzed and the content ranged from 306.40 to 812.23 mg/100g,which all belong to high purine foods.There was no significant change of adenine and guanine during storage at-18°C.The content of hypoxanthine increased rapidly in a short time and then decreased to a gentle level.Although the change of xanthine was not obvious,it also increased slowly to a gentle level.This may be related to the changes of ATP and other enzymatic reactions after the death of aquatic products.4.The effects of exogenous additives on purine removal were investigated by means of different processing methods（soaking,boiling and soaking-boiling）.The results showed that the effect of soaking-boiling was the best among the three treatments,with the total removal ratio of flesh and skin was more than70%and 35%,respectively.In addition,molecular docking results showed that allicin,the main component of garlic,could form hydrogen bonds with SER1080 and ALA 1079 of xanthine oxidase,and form hydrophobic bonds with MET 1038,PHE 914 and ALA 910,with the LibDock Score was 74.212.The results of activity determination of xanthine oxidase showed that allicin could increase its activity and promote the transformation of hypoxanthine to xanthine.Therefore,we presumed that allicin bound to xanthine oxidase through hydrogen bonding and hydrophobic interaction during immersion treatment,which enhanced the activity of xanthine oxidase and promoted the transformation of hypoxanthine to xanthine.In addition,allicin（pH<7）also provided acidic environment for the reaction and changed the thermal stability of xanthine.Therefore,the simultaneous removal of xanthine and hypoxanthine could be realized in the boiling process,and improved the final removal ratio. |
