| Nanjing cured-dry duck is one of the China traditional cured-dry products, which has long history and special flavor. Many researchers have proved that the special flavor is related with the degradation of lipids especially phospholipid, and the change of phospholipid is related with phospholipid hydrolysis enzyme. Some scholars have follow the track of the activity’s change of phospholipid hydrolysis enzyme from macro perspective during the processing of cured-dry meat products, which confirmed the function of phospholipid hydrolysis enzyme in the flavor’s form of cured-dry meat products further. Studying the activity’s change and mechanism of action is thus significant meaningful for better understanding the mechanism of the change of lipids in the processing of cured-dry meat products and revealing the relationship between the change of lipids with flavor. There were only a little researchers who have detected the total activity of phospholipases, however, the references about the extraction and mechanism of action of the phospholipid hydrolysis enzymes in cured-dry meat products from molecular level were little.This experiment of the subject purified a phospholipid hydrolysis enzyme having the phospholipase A2activity firstly from fresh duck by combining the Source15Q ion-exchange and Superdex200gel-filtration chromatography, and studied the properties of this enzyme preliminarily, which provided the theoretical basis for the studying of the mechanism of flavor’s forming in Nanjing cured-dry duck, controlling of the processing and improvement of tradition craft. The concrete studying contents and results were as follows:(1) The extraction of crude phospholipid hydrolysis enzyme from duck and analyzing of activity of various phospholipid hydrolysis enzyme. The extraction processing was optimized and the activities of various phospholipid hydrolysis enzymes (acid lipase, neutral lipase, total phospholipase, phospholipase A2, phospholipase C and phospholipase D) were detected. The results showed that the activity of phospholipase A2in biceps femoris was much higher than phospholipase A2in chest of duck, the activity of phospholipase A2was much higher than other phospholipid hydrolysis enzyme in biceps femoris, the most suitable pH for intramuscular phospholipid hydrolysis enzyme was8.0and70%saturation of ammonuium sulfate was the most suitable precipitation condition.(2) Separation and purification of phospholipase A2in duck. The experiment use the crude enzyme solution above as material and firstly tried to use Source15Q ion-exchange chromatography(1.5×5.5cm), moreover, the separation condition was optimized:mobile phase A was pH8.0,50mmol·L-1Tris-HCl and mobile phase B was phase A containing lmol·L-1NaCl, equilibrium velocity was1.0ml·min-1, elution flowing rate was1.5mL·min-1and the elution gradient was0～100%B35min and sample size was500μL. Then the activity peak was separated again by Superdex200gel-filtration chromatography and a electrophoretic homogeneity phospholipid hydrolysis enzyme with the activity of phospholipase A2was abtained finally. SDS-PAGE confirmed that the enzyme abtained was electrophoresis pure. Moreover, the activity of this enzyme was demonstrated by thin layer chromatography and high performance liquid chromatography.(3) Preliminary investigation on some enzymatic properties. SDS-PAGE measured the apparent molecular weight of the PLA2is about82.3KDa. NaF was found that it didn’t inhibit the activity of this enzyme, so it was certainly not a lipase and its optimum temperature was25℃, optimum pH was7.5and Ca2+did not affect the activity of this enzyme through studying the basic enzymatic properties. The purified enzyme solution concentrated by ultrafitration could be stored at4℃and the best storing time should be less than three days. If long-term preservation was wanted to gain, the concentrated enzyme solution needed to be freeze-dried. |
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