Lipolysis In Intramuscular Lipids During Processing Of Xuanwei Ham & Purification And Characterization Of Phospholipid Hydrolases From Porcine Skeletal Muscle

Xuanwei ham is a typical traditional dry-cured meat product in China. The major quality trait of this product is characterized by the unique and intense cured flavor. It's well known that lipids play an important role in the formation of flavour, since they are predecessor of flavour compounds and also act as a solvent for flavour compounds accumulated. However, intramuscular lipids of Xuanwei ham have not been studied. The aim of this research is to study the rule of lipolysis in intramuscular lipids during processing of Xuanwei ham and to purify and characterize the phospholipids hydrolases which causes the lipolysis. The work will be a base for the industrial production of Xuanwei ham.Results showed that the glycerides accounted for 73.2% of total lipid content in fresh ham, the phospholipids represented 25.3% and the free fatty acids 2.3% of the total lipid content, respectively. A reduction of 77% in the total quantity of phospholipids was observed, while Glycerides only changed a little throughout the process. The results suggest that phospholipids are the main substrate of lipolysis in the intramuscular lipids of Xuanwei ham. A selective degradation of fatty acids in the phospholipid fraction was found. The polyunsaturated fatty acids were degraded in the highest quantity (87.7%), followed by saturated (76.9%) and monounsaturated (56.3%) fatty acid. The decrease was statistically significant for linoleic (C18:2), arachidonic (C20:4) and palmitic (C16:0) acids (pO.OOl).A new method was established for detecting phospholipid hydrolase activity in skeletal muscle by assay the reduction of phospholipids. The reactive conditions were optimized. The final assay consisted of 0.9 ml muscle supernatant, added with 50 Jll 5mmol/l soybean lecithin, incubated at 40 °C for 20 min. CV of this method was 3.24% and the least LD was 2.68 nmol phospholipids. Highest recovery ratio can be obtained by adding 0.2 ml mixture of KC1/EDTA. It was found that the result in new method showed good linear correlation with that in radioactive method.The developed method could be used for detecting phospholipase A activity in eluates from chromatographic columns during purification.Two phospholipid hydrolases from porcine skeletal muscle were purified to electrophoretic homogeneity. The molecular weight of purified enzyme was about 66 kDa and 37 kDa estimated by SDS-PAGE. 66 kDa phospholipid hydrolase could hydrolyze 4-methylumbelliferyloleate, and the specific activity was 32 nmol/h/mg protein, however, 37 kDa phospholipid hydrolase did not. These two phospholipid hydrolases displayed both phospholipase A₁ and A₂ activitys analysed by gas chromatogram, and the activity of phospholipase A₁ is higher than that of phospholipase A₂. Peptide mass fingerprint showed that 66 kDa phospholipid hydrolase did not match to any protein in NCBInr database. 66 kDa phospholipid hydrolase showed optimum activity at 40℃, pH 8.5, and did not require calcium ion for activity. The enzyme of 66 kDa was inhibited by NaF whereas ATP, DTT and metal ions (Fe +, Fe⁺, Mg⁺, Cu²+, and Zn2⁺) stimulated the activity. In curing agents, NaCl strongly inhibited the activity of 66 kDa phospholipid hydrolase; NaNO₃ above 100 ppm stimulated activity and NaNO₂ between 30-150 ppm had a negligible effect.