Study On Phospholipid Degradation By Intramuscular Phospholipase During The Processing Of Traditional Dry-Cured Duck

Dry-cured meat productions have been produced for many centuries that have rich aroma, resilient texture and good storability. The biochemical reactions involved in the flavor development are complex and the mechanism has not been fully understood, however, a large number of studies have confirmed that the degradation, oxidation, and further reactions of oxidation products of intramuscular lipids (especially phospholipids) are the main source of precursors of dry-cured meat flavors. Therefore, the enzymatic lipolysis of phospholipids and the flavor of dry-cured meat products were highly correlated. With dry-cured duck as the material, this thesis firstly examined the intramuscular environment by analyzing the main physical and chemical index of the ducks during the production process to learn the potential external factors that affect enzymatic lipolysis of phospholipids; Meanwhile phospholipase A2 and phospholipase C activities were tracked combined with the changes of fatty acid profiles to explicit the correlation between phospholipases and intramuscular phospholipid degradation; And further the distinct reaction products from crude enzyme extract and phospholipid model system were analyzed to indentify the enzyme which played more significant role; Finally the phospholipase was extracted and purified from duck muscle, and its enzymatic properties were investigated. The content and results are given below:1. Changes of intramuscular fat, physical-chemical index and microstructure of dry-cured duck during processingThe intramuscular fat, the proportion of neutral lipid, free fatty acid, and phospholipid, NaCl content, TB A value, water content, pH value, protein content, free amino acid content, L*, a*, b* and micro structural changes of dry-cured duck throughout the production were evaluated. Results showed that during the whole process, pH value, the proportion of neutral lipids in total lipids, and b* value did not change significantly (P>0.05), the water content, the percentage of phospholipid, L* and a* value significantly decreased (P<0.05), while significantly increase was found in NaCl content, fat content, the proportion of free fatty acid, protein content, free amino acid content, and TBA value (P<0.05). The microstructure of duck muscle displayed apparent shrinkage and increased hollows. The correlation study indicated that the percentage of phospholipids and free fatty acids was negatively correlated (r=-0.998, P<0.01), while the neutral lipid and free fatty acids was positively correlated (P<0.01), suggesting that lipolysis of phospholipids took place and gave rise to the generation of free fatty acids. The strong correlation was also found between the percentage of phospholipids and other traits such as NaCl content, protein content, fat content, TBA value, water content, the percentage of neutral lipids and free fatty acids, and free amino acids et al (P<0.01). Taking these results, it could be confirmed that intramuscular phospholipid degraded in the high salt and weak acidic environment during the processing of dry-cured duck. The destroy and shrinkage of the internal muscle structure promoted the contact between phospholipid and intercellular enzymes, and accelerated the oxidation of free fatty acids which came from the degradation of phospholipids.2. Changes in phospholipase A2, phospholipase C activities during the production of dry-cured duck and its relation with intramuscular phospholipid degradation.The activities of phospholipase A2, phospholipase C, the fatty acid profiles of neutral lipids, free fatty acids, and phospholipids were tracked in the production of dry-cured duck. It showed that the activity of phospholipase A2 gradually decreased in this process (P<0.05), but it retained 83.70%of its initial activity after 12 days of drying. The phospholipase C activity increased significantly (P<0.05) at the dry-salting stage initially, and decreased between dry salting and drying, then it markedly increased (P<0.05) at air-drying from 6 days to 12 days, and retained 86.78%of its initial activity in the end product. PUFA of FFA and neutral lipid gradually increased during the whole process and they are significantly correlated with decreased PUFA of phospholipids (r=-0.841, P<0.05, r-0.946, P<0.01). In terms of individual fatty acids, linoleic acid (C18:2) and arachidonic acid (C20:4) in phospholipid fraction declined significantly (P<0.05), whereas they remarkably increased in free fatty acids and neutral lipids (P<0.05), indicating that phospholipase A2 and phospholipase C might be involved in the lipolysis of phospholipids, leading to the generation of free fatty acids and neutral lipids. The above results preliminarily confirmed that phospholipase A2 and phospholipase C participate in the lipolysis of intramuscular phospholipids.3. Comparison of the contributions of phospholipase A2 and phospholipase C to the enzymatic lipolysis of phospholipid model systemThe model system comprised a phospholipid (C18:2/C16:0 PC) which is present in duck muscle as the substrate and the crude enzyme extract. The reaction condition was consistent with the dry-cured duck processing condition (at the temperature of below 10℃) and the properties of the end products (salt content of 8.92%, pH of 6.2). The enzymatic reaction products were analyzed with HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detector), and analysis method was established to separate these substances. The reaction products were firstly extracted with dichloromethane-methanol (2/1, V/V), then they were applied to HPLC-ELSD analysis. The column used in HPLC was XBridge C18 (250 mm×4.6 mm×5.0 μm), operating at 40℃. The mobile phase consisted of solvent A (dichloromethane), solvent B (acetonitrile) and solvent C (H2O) and the gradient program was employed. The ELSD was run at 65℃ with N2 at the pressure of 45 psi, the gain was set at 50. It performed good separation of phosphocholine (PC), lysophosphatidylcholine (LPC), fatty acid (FA), monoglyceride (MG), diglyceride (DG) and triglycerides (TG) under this condition, and the linear regression correlation coefficients was above 0.99. HPLC-ELSD analysis revealed that the initial phospholipid content in the model system was 1978.24 μg, after the reaction, it declined to 1626.35 μg. LPC and FA (49.67 μg and 42.25 μg respectively) which were the distinct reaction products by the act of phospholipase A2 were detected, but no DG was found in the reaction products which came from the lipolysis by phospholipase C. The results approved that phospholipase A2 played more important role in the lipolysis of phospholipids.4. Extraction and purification of phospholipase A2 and its enzymatic propertiesPhospholipase A2 was extracted and purified from the duck Biceps femoris muscle and its optimal temperature, optimal pH, thermal stability, kinetic equation and its enzyme activity as influenced by metal ions were studied. The crude enzyme was extracted from duck leg muscle with the buffer (50 mM Tris-HCl [pH 7.5],1 mM EDTA,3 mM MgCl2,50 mM KCl,10 mM 2-mercaptoethanol), followed by 70% ammonium sulfate precipitation, and then subjected to DEAE-Cellulose (DE-52) anion exchange chromatography eluted with 100% linear gradient, Superdex 200 gel filtration chromatography, Source 15Q anion exchange chromatography with a linear gradient of 100%equilibration buffer in 45 min (50 mM Tris-HCl [pH 7.5],1 mM EDTA,3 mM MgCl2,50 mM KCl,10 mM 2-mercaptoethanol,1 M NaCl). The electrophoretically pure phospholipase A2 was finally obtained, with molecular weight of around 50 KDa, and specific activity of 282.1 U/mg. The purity has increased by 201.5-fold, and the recovery was 1.5%. The optimal reaction temperature was at 32℃ and the optimal pH was 6.0. The enzyme had good thermal stability at 32℃, but was not tolerant to the temperature of 60℃. With L-a-l-palmitoyl-2-[14C]arachidonyl-sn-glycero-phosphatidylcholine as the substrate, the Km was 0.03 mmol/L, and the maximum reaction velocity Vraax was 1000 U/mg. Cu2+and Ca2+could stimulate the activity of the enzyme, especially Cu2+that at the concentration of 10 mM, the relative activity of phospholipase A2 increased by 51.33%. However, the Mn2+ and Zn2+had little effect on the activity of enzyme. The above results demonstrated that phospholipase A2 present in duck muscle had relatively stable activity under the dry-cured duck processing conditions (temperature, NaCl content), and the pH of duck muscle was close to its optimal pH.In conclusion, during the production of dry-cured duck, along with the changes in physical-chemical index and microstrucrure, the intramuscular phospholipid was hydrolyzed by phospholipase A2 and phospholipase C; Through the analysis of distinct reaction products from phospholipid model system and the characteristics of phospholipase A2, the results combined confirmed that phospholipase A2 played important roles in the intramuscular phospholipid lipolysis process.