Food Chemistry 145 (2014) 997–1001

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Changes of phospholipase A2 and C activities during dry-cured duck processing and their relationship with intramuscular phospholipid degradation Daoying Wang a,b,⇑, Muhan Zhang b, Huan Bian b, Weimin Xu b, Xinglian Xu a,⇑, Yongzhi Zhu b, Fang Liu b, Zhiming Geng b, Guanghong Zhou a a b

Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, PR China Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China

a r t i c l e

i n f o

Article history: Received 18 May 2013 Received in revised form 8 July 2013 Accepted 2 September 2013 Available online 11 September 2013 Keywords: Phospholipase A2 Phospholipase C Dry-cured duck Phospholipids Degradation

a b s t r a c t Phospholipid hydrolysis, as the main stage and cause of phopholipid degradation, is generally attributed to phospholipases. In this study, the changes of phospholipase A2 (PLA2) and C (PLC) activities, neutral lipid, free fatty acids and phospholipids in dry-cured duck leg muscles during processing, were examined. The composition of free fatty acids and neutral lipids increased significantly (P < 0.05) with extension of processing time while the phospholipids composition decreased. The PLA2 and PLC activity decreased in the final product, but retained 83.70% and 86.78% of their initial activities, respectively. The relative activities of both PLA2 and PLC highly correlated with the decline of phospholipids and the increase of free fatty acids. High correlations were also obtained between the relative activities of PLC and the increase of neutral lipid (P < 0.01). All these results suggest that PLA2 and PLC contribute to the degradation of intramuscular phospholipids during the processing of dry-cured duck. Ó 2013 Published by Elsevier Ltd.

1. Introduction Traditional Chinese dry-cured duck is a well known local delicacy in China and Southeast Asia, due to its tasty flavour and texture, and it has a history of over 300 years (Li, 1988). In Nanjing city alone, about five million dry-cured ducks are consumed annually (Li, 1988; Zhang et al., 2013). Similar to dry-cured Jinhua ham, dry-cured duck is produced by dry curing, marinating, piling and drying naturally, but the period of its production is shorter than that of hams (Xu, Xu, Zhou, Wang, & Li, 2008). The biochemical changes that occur during the processing of dry-cured meat are directly associated with the final flavour of the product. Flavour development in meat and meat products are reported to be associated with lipids composition, the extent of lipolysis and oxidation of lipids and free fatty acids during processing (Chizzolini, Novelli, & Zanardi, 1998). The phospholipid (PL) fraction is an important group of lipids in meat and meat products because of its high sensitivity to lipolysis and oxidation. This is due Abbreviations: PL, phospholipids; FFA, free fatty acids; PLA2, phospholipase A2; PLC, phospholipase C; PUFA, polyunsaturated fatty acids; MUFA, monounsaturated fatty acids; SFA, saturated fatty acids; RH, relative humidity. ⇑ Corresponding author. Tel./fax: 86 25 84395939. E-mail addresses: [email protected] (D. Wang), [email protected] (X. Xu). 0308-8146/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2013.09.007

to its high proportion of long chain polyunsaturated fatty acids and its close contact with catalysts of lipid lipolysis or oxidation in the aqueous phase of the muscle cell (Pérez-Palacios, Ruiz, Dewettinck, Le, & Antequera, 2010). Therefore, the research concerning the composition and changes of intramuscular phospholipids was of particular interest. Phospholipids hydrolysis, as the first stage and main cause of phopholipid degradation, is generally attributed to phospholipases. The lipolytic enzyme activities (such as neutral lipase, acid lipase and total phospholipase) have been studied in some dry-cured products, and these enzymes remained active during the entire process (Jin et al., 2010; Motilva, Toldrá, & Flores, 1992). However, the studies on the activities of the phospholipases subgroups in dry-cured meat products are rare. Phospholipases are classified into A1, A2, C and D according to the ester bonds they hydrolyse. Among them, PLA2 and PLC are more intensively studied in creatural tissues; they cleave the phospholipids into free fatty acids (FFA), lysophospholipids and diacylglycerol etc. However, the data related to muscle PLA2 and PLC and the factors affecting the activities of these enzymes are very limited. A number of methods have been applied to measure the activity of phospholipases. Titrimeric and colorimeric procedures are commonly used in studying muscle or meat samples, but they were affected by various factors, while the method using radioactive substrate could provide a more

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sensitive and accurate assay of phospholipase activities (Van den Bosch & Aarsman, 1979). The knowledge of the phospholipase activities during processing is essential for improving the quality of end products and further complicated overall lipid degradation mechanism. Therefore, the objective of this study was to evaluate the changes of phospholipase A2 and C activities during dry-cured duck processing and their relationship with intramuscular phospholipid degradation.

2. Materials and methods 2.1. Sampling and processing conditions Thirty-six lean-type Cherry Valley Pekin ducks (Anas platyrhynchos) with the average weight of approximately 2.0 kg were slaughtered humanely in a commercial meat processing plant (Jiangsu furun Food Ltd., Xuzhou, China). After chilling for 2 h, dry-cured ducks were processed as follows: duck carcasses were dry-salted for 1 day (salt content: 6.5% of carcass weight), marinated in brine for 1 day (saturated salt solution), piled for 2 days and then dried at 2–10 °C in a well-ventilated room for 6–12 days. At the end of each processing stage (including raw), six carcasses were selected for lipids and assayed for phospholipase A2 and C activities. The leg muscles were removed from the carcasses and trimmed of all visible subcutaneous fat and connective tissues, then stored at 40 °C. 2.2. Chemicals L-a-1-palmitoyl-2-[14C]arachidonyl-sn-glycero-phosphatidylcholine (2-[1-14C]AA-GPC, 58.2 mCi/mmol) was purchased from PerkinElmer (USA). Fatty acids C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, C20:2, C20:4, C20:5, C22:5 and C22:6 standards were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO); and methanol, isopropyl alcohol, chloroform, n-hexane, acetic acid, diethyl ether, 2,2-dimethoxypropane, BF3, NaCl, Triton X-100 and CaCl2 were of analytic grade. 2.3. Crude enzyme extraction and PLA2 and PLC activities assay The crude enzyme was extracted according to the method described by Jeong et al. (2011) with some modifications. Immediately after overnight storage at 40 °C, 30 g of muscle sample was homogenised with 100 ml Tris–HCl buffer (100 mM, pH 8.0) at 20000 rpm on ice using an Ultra Turrax (T25, IKA, Germany). The homogenate was then centrifuged for 20 min at 12,000g at 4 °C (Allegra 64R, Beckman, USA), the supernatant was collected and filtered through four-layer gauze. The PLA2 activity was assayed using the method of Bell, Kennerly, Standford, and Majerus (1979) and Jeong et al. (2011). The reaction was performed in 60 ll of the standard reaction buffer (150 mM Tris–HCl [pH 7.5], 0.33% Triton X-100 and 1 mM CaCl2), 40 ll of crude enzyme sample and 20 ll of the diluted substrate (2-[1-14C]AA-GPC, 0.45 nM). The mixture was incubated at 37 °C for 30 min. The assay was subsequently terminated by adding 560 ll of the terminating reagent (n-heptane/methanol/isopropyl alcoholas 4/2/1 [V/V]) and 110 ll of water. The mixture was vortexed and centrifuged at 12000g, 4 °C for 2 min (Allegra 64R, Beckman, USA). Then, 150 ll of the upper phase were transferred to a new microtube, to which 800 ll of n-heptane and silica gel (20 mg) was added. The sample was vortex-mixed and centrifuged again at 12,000g, 4 °C for 3 min. An aliquot (600 ll) of the supernatant was pooled and mixed with 3.5 ml of the b-scintillation solution (Insta Gel-XF). The radioactivity was counted with a

liquid scintillation counter (LS6500, Beckman-Coulter, Fullerton, expressed as disintegrations per minute (DPM). The PLC activity was determined by the procedures of Kurioka and Matsuda (1976) and Cao, Zheng, Wang, Rui, and Tang (2009) using p-Nitrophenylphosphorylcholine (NPPC; Aldrich) as the substrate. The reaction was carried out at 37 °C in a cuvette with a 10 mm optical path length, and the rate of NPPC hydrolysis by PLC was monitored at various time intervals by measurement of p-Nitrophenol release at 410 nm. A basal reaction mixture consisted of 1 ml of 20 mM NPPC, 1 ml of 0.25 M Tris-HCI [pH 7.2] in 60% sorbitol, and 0.5 ml of crude enzyme. The PLA2 and PLC activities were defined as the relative activity (%) as compared to that in the raw duck samples.

2.4. Determination of composition and fatty acid profiles of lipids Total lipids were extracted from muscle samples according to the method of Folch, Lees, & Sloane-Stanley (1957) with small modifications. Briefly, 10.0 g of muscle sample was homogenized with 200 ml of chloroform/methanol (2/1, V/V) solution at 1500 rpm using an Ultra Turrax. The homogenate was allowed to stand for 1 h and then passed through two layers of filters. After that, 0.2-fold of its volume of a solution containing 7.3 g/l NaCl, and 0.5 g/l CaCl2 was added to the filtrate. The mixture was centrifuged for 15 min at 3000 rpm (Allegra 64R, Beckman, USA) and the lower phase was dried under vacuum on a rotary evaporator (RE85C, Yarong, China) in a 44 °C water bath and then stored at 40 °C. Neutral lipid, FFA and phospholipids were separated from total lipids according to the procedure of Xu et al. (2008). Briefly, 100.0 mg of total lipid extract was dissolved in 2.0 ml of chloroform, and 1.0 ml of the solution was transferred into an aminopropyl-silica minicolumn (500 MG, VARIAN, USA) that was activated with 2.0 ml of chloroform before transfer. The minicolumn was washed with 5.0 ml of chloroform/2-propanol (2/1, V/V) to obtain a neutral lipid, and then FFA was eluted with 5.0 ml of 2% acetic acid in diethyl ether (V/V). Finally, phospholipids were eluted with 5.0 ml of methanol. Neutral lipid, FFA and phospholipids elute was evaporated to remove the solvent. The amounts of neutral lipid, FFA and phospholipids were gravimetrically determined and the results were expressed as a percent of total lipid weight obtained. The neutral lipid, FFA and phospholipids residue were mixed with 3.0 ml of 0.5 mol/l NaOH/methanol, then placed in a 50 °C water bath for 20 min. After that, 3.0 ml 14% BF3/methanol was added, the mixture was methylated at 60 °C for 30 min. At the same time, 0.5 ml of 2,2-dimethoxypropane was added to remove water that is produced during methylation. After cooling, 1.0 ml of water and 1.0 ml of n-hexane were added and they were shaken vigorously. The resulting mixture was allowed to stand for 1 h and then the upper organic phase was dried by rotary evaporation under N2. The residue was dissolved in 0.4 ml of hexane for GC analysis. The methylated fatty acids were analysed with a gas chromatograph (GC-2010 Plus, Shimadzu, Japan) equipped with a flame ionisation detector and a split injector. One point 5 ml of the sample was injected onto a capillary column (SP-2560, 100 m  0.25 mm  0.20 lm, Varian, USA). The oven temperature increased from 140 to 220 °C at 5 °C/min and maintained for 30 min at 220 °C. The detector temperature was maintained at 285 °C. The carrier gas used was nitrogen and its pressure was maintained at 90 KPa. The peaks were identified by comparing their retention times with those of the standards. The relative percentages of fatty acids were determined by the peak areas (Gandemer, 2002; Gandemer, Morvan-Mahi, Meynier, & Lepercq, 1991).

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2.5. Statistical analysis Relative Activity of PLA2 (%)

110

Statistical analysis of the differences between each group was evaluated by one-way analysis of variance (ANOVA) using the SPSS 18.0. The correlation coefficient was estimated with the Pearson correlation coefficient option of SPSS 18.0 (Argyrous, 2011). Differences were regarded as significant at P < 0.05. All data were expressed as mean ± standard error. 3. Results and discussion 3.1. Changes of lipid composition

a 100

a

ab bc

90

c

c

5

6

80 70 60 50 0

1

2

3

4

Processing Stages Fig. 1. Changes of the relative activities of phospholipase A2 from leg muscles through the processing of traditional Chinese dry-cured duck. 1–6: raw, dry-salted, marinated, piled, dried for 6 days, dried for 12 days.

a

150

Relative Activity of PLC (%)

Table 1 shows the contents of the different lipid composition (neutral lipid, free fatty acids and phospholipids) expressed as percentages of the total lipids. Phospholipids accounted for 49.25% of the total lipids in raw duck muscle followed by neutral lipid and free fatty acids. The percentages of free fatty acids showed a great increase through the processing of traditional Chinese dry-cured duck which is also observed in the other dry-cured meat products (Hernandez, Navarro, & Toldra, 1999; Navarro, Nadal, Izquierdo, & Flores, 1997; Qiu, Zhao, Sun, Zhou, & Cui, 2013). The percentage of neutral lipid was found to increase significantly at the piling and drying stages (P < 0.05), in contrast, a significant decrease in the percentage of phospholipids (P < 0.05) was detected. During the whole process, the percentage of phospholipids and free fatty acids was negatively correlated (R = 0.787, P < 0.01), while the neutral lipid and free fatty acids positively correlated (P < 0.01), suggesting that the hydrolysis of phospholipids mainly occurred during this process. Both neutral lipids and phospholipids could contribute to the generation of FFA catalysed by lipases and phospholipases respectively; however in most cases, phospholipids are the main substrates for lipolysis in dry-cured meat products (Buscailhon, Gandemer, & Monin, 1994). As the muscle enzyme systems play an important role in the generation of free fatty acids (Motilva et al., 1992), the increase in the amount of free fatty acids could be the result of the action of lipolytic enzymes.

b

130 110

c

c d

90 e

70 50 0

1

2

3

4

5

6

Processing Stages Fig. 2. Changes of the relative activities of phospholipase C from leg muscles through the processing of traditional Chinese dry-cured duck. 1–6: raw, dry-salted, marinated, piled, dried for 6 days, dried for 12 days.

3.2. Changes of relative activities of phospholipase A2 and C

3.3. Changes in fatty acid profiles of different lipid fraction

Figs. 1 and 2 show the changes of phospholipase A2 and C activities during traditional Chinese dry-cured duck processing. The phospholipase A2 activities significantly decreased over the whole process (P < 0.05). 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) during air-drying from 6 to 12 days. The phospholipase activities in the final products were found to be higher than those observed by Zhou and Zhao (2007) and Jin et al. (2010) who reported the total phospholipases retained 7.56% and 9.1% of the initial activity in the end products respectively. The reduced activity of lipolitic enzymes as drying progresses might be due to the decreased water activity. Variables such as drying temperature, salt content and water activity could influence the enzyme activities in dry-cured meat products (Toldra, 2006), but up to now, compared to total phospholipases activities, very little is known about the activities of phospholipase A2 and C in skeletal muscles and the regulation of their activities during dry-cured meat processing.

Polyunsaturated fatty acids (PUFA) of FFA and neutral lipids (Tables 2 and 3) increased significantly during the entire process (P < 0.05), and it is strongly correlated with decreased PUFA of phospholipids fraction (Table 4, R = 0.841, P < 0.05, R = 0.946, P < 0.01, respectively). Concerning each single fatty acid, the linoleic (C18:2) and arachidonic acid (C20:4) of the phospholipid decreased markedly (P < 0.05), whereas they increased significantly in free fatty acids (P < 0.05), which is likely due to the lipolysis of phospholipids. It has been indicated that the enzymes have a selective activity according to carbon chain length and degree of unsaturation of fatty acids. Our result is in agreement with the study of lipids changes in dry-cured meat (Lorenzo, Bermudez, & Franco, 2013; Qiu et al., 2013; Yang, Ma, & Qiao, 2005). But some other studies indicated that lipolysis in phospholipid was not specific to the fatty acid chain length or unsaturation during the processing of dry cured hams (Buscailhon et al., 1994) and pork loins (Hernandez et al., 1999). The difference between the present study and mentioned litera-

Table 1 Changes in lipid composition during traditional Chinese dry-cured duck processing (g/100 g lipids).

Neutral lipid FFA Phospholipids a

Raw

Dry-salted

Marinated

Piled

Dried for 6 days

Dried for 12 days

45.96 ± 3.25a 4.79 ± 0.35a 49.25 ± 3.17a

46.21 ± 2.77a 5.87 ± 0.26a 47.92 ± 2.25a

46.39 ± 4.05a 6.94 ± 0.41a 46.67 ± 2.77a

46.57 ± 3.45a 8.00 ± 0.36a 45.43 ± 1.85a

46.74 ± 2.68a 9.34 ± 0.58a 43.92 ± 1.63a

47.08 ± 3.89a 10.46 ± 0.47a 42.46 ± 2.18a

Means in the same row with different letters differ significantly (P < 0.05).

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Table 2 Changes in fatty acid profiles of neutral lipid during traditional Chinese dry-cured duck processing (%). Raw C C C C C C C C C C

14:0 14:1 16:0 16:1 18:0 18:1 18:2 20:2 20:4 20:5 R SFA R MUFA R PUFA PUFA/SFA MUFA/SFA a

Dry-salted a

1.20 ± 0.11 0.66 ± 0.05a 25.31 ± 2.18a 1.81 ± 0.15a 13.70 ± 1.11a 39.80 ± 2.18a 11.41 ± 0.89a 1.16 ± 0.06a 3.72 ± 0.15a 1.22 ± 0.09a 40.21 ± 3.40a 42.27 ± 2.38a 17.51 ± 1.19a 0.44 1.05

Marinated a

1.65 ± 0.09 0.67 ± 0.08a 25.85 ± 2.37a 1.46 ± 0.12a 13.28 ± 1.30a 39.92 ± 3.05a 11.48 ± 0.52a 1.20 ± 0.09a 3.33 ± 0.23a 1.15 ± 0.11a 40.78 ± 3.76aa 42.05 ± 3.25a 17.16 ± 0.95a 0.42 1.03

Piled a

Dried for 6 days a

1.37 ± 0.08 0.65 ± 0.06a 25.98 ± 1.96a 1.73 ± 0.07a 13.40 ± 1.25a 39.71 ± 3.12a 12.02 ± 0.93a 1.16 ± 0.05a 2.92 ± 0.21a 1.05 ± 0.05a 40.75 ± 3.29a 42.09 ± 3.25a 17.15 ± 1.24a 0.42 1.03

a

Dried for 12 days

1.66 ± 0.12 0.77 ± 0.03a 24.90 ± 2.05a 1.79 ± 0.09a 14.02 ± 0.97a 38.25 ± 2.27a 13.11 ± 0.62a 1.09 ± 0.04a 3.24 ± 0.16a 1.18 ± 0.06a 40.58 ± 3.14a 40.81 ± 2.39a 18.62 ± 0.88a 0.46 1.01

1.42 ± 0.09 0.68 ± 0.05a 23.69 ± 1.88a 1.75 ± 0.13a 14.49 ± 1.36a 38.05 ± 1.55a 14.03 ± 0.77a 0.88 ± 0.02a 3.95 ± 0.11a 1.06 ± 0.05a 39.60 ± 3.33a 40.48 ± 1.73a 19.92 ± 0.95a 0.50 1.02

1.03 ± 0.05a 0.75 ± 0.07a 24.38 ± 1.52a 1.87 ± 0.17a 14.36 ± 0.88a 37.39 ± 2.61a 14.10 ± 0.82a 0.84 ± 0.03a 4.15 ± 0.15a 1.13 ± 0.02a 39.77 ± 2.45a 40.01 ± 2.85a 20.22 ± 1.02a 0.51 1.01

Piled

Dried for 6 days

Dried for 12 days

Means in the same row with different letters differ significantly (P < 0.05).

Table 3 Changes in free fatty acid composition during traditional Chinese dry-cured duck processing (%). Raw C C C C C C C C C C C C

14:0 14:1 16:0 16:1 18:0 18:1 18:2 20:2 20:4 20:5 22:5 22:6 R SFA R MUFA R PUFA PUFA/SFA MUFA/SFA a

Dry-salted a

9.78 ± 0.05 2.93 ± 0.03a 8.33 ± 0.12a 0.25 ± 0.03a 14.55 ± 1.05a 22.87 ± 1.02a 24.74 ± 1.65a 0.59 ± 0.02a 13.62 ± 0.11a 0.53 ± 0.05a 1.08 ± 0.03a 0.72 ± 0.02a 32.66 ± 1.22a 26.05 ± 1.08a 41.28 ± 1.88a 1.23 0.74

Marinated a

7.35 ± 0.04 1.50 ± 0.02a 11.14 ± 0.11a 0.18 ± 0.01a 12.13 ± 0.64a 22.33 ± 1.55a 24.36 ± 1.76a 0.42 ± 0.02a 18.56 ± 0.13a 0.37 ± 0.03a 1.11 ± 0.05a 0.55 ± 0.02a 30.62 ± 0.79a 24.01 ± 1.58a 45.37 ± 2.01a 1.48 0.78

a

7.49 ± 0.06 1.72 ± 0.02a 12.84 ± 0.06a 0.37 ± 0.02a 12.28 ± 0.96a 22.72 ± 0.75a 25.01 ± 0.89a 0.57 ± 0.03a 14.75 ± 0.09a 0.43 ± 0.02a 1.22 ± 0.04a 0.60 ± 0.03a 32.61 ± 1.08a 24.81 ± 0.79a 42.58 ± 1.10a 1.31 0.76

a

6.32 ± 0.11 1.26 ± 0.05a 11.55 ± 0.09a 0.23 ± 0.02a 12.29 ± 0.15a 21.58 ± 1.23a 28.26 ± 1.17a 0.25 ± 0.02a 16.10 ± 0.07a 0.29 ± 0.03a 1.26 ± 0.05a 0.63 ± 0.06a 30.15 ± 0.35a 23.07 ± 1.30a 46.79 ± 1.40a 1.55 0.77

a

5.23 ± 0.10 1.05 ± 0.03a 11.20 ± 0.05a 0.12 ± 0.04a 13.64 ± 0.09a 21.20 ± 0.86a 28.03 ± 1.85a 0.24 ± 0.03a 17.51 ± 0.09a 0.20 ± 0.02a 0.87 ± 0.02a 0.72 ± 0.05a 30.07 ± 0.24a 22.37 ± 0.93a 47.57 ± 2.06a 1.61 0.77

5.80 ± 0.08a 0.99 ± 0.06a 10.37 ± 0.08a 0.10 ± 0.02a 13.83 ± 0.07a 21.37 ± 1.15a 26.61 ± 1.26a 0.20 ± 0.02a 17.94 ± 0.15a 0.46 ± 0.02a 1.42 ± 0.05a 0.91 ± 0.03a 30.00 ± 0.23a 22.46 ± 1.23a 47.54 ± 1.53a 1.58 0.75

Means in the same row with different letters differ significantly (P < 0.05).

Table 4 Changes in fatty acid profiles of phospholipids during traditional Chinese dry-cured duck processing (%).

C C C C C C C C C C C C

14:0 14:1 16:0 16:1 18:0 18:1 18:2 20:2 20:4 20:5 22:5 22:6 R SFA R MUFA R PUFA PUFA/SFA MUFA/SFA a

Raw

Dry-salted

Marinated

Piled

Dried for 6 days

Dried for 12 days

5.28 ± 0.57a 1.23 ± 0.21a 16.15 ± 1.51a 0.15 ± 0.08a 22.04 ± 2.18a 16.85 ± 1.55a 8.71 ± 0.56a 0.55 ± 0.11a 22.78 ± 2.25a 0.32 ± 0.07a 2.17 ± 0.22a 3.77 ± 0.33a 43.47 ± 4.26a 18.23 ± 1.84a 38.30 ± 3.54a 0.88 0.42

5.29 ± 0.66a 1.20 ± 0.15a 16.66 ± 0.85a 0.14 ± 0.06a 23.05 ± 1.33a 17.14 ± 1.68a 7.99 ± 1.15a 0.54 ± 0.20a 22.04 ± 1.18a 0.30 ± 0.12a 2.07 ± 0.15a 3.59 ± 0.18a 44.99 ± 2.84a 18.48 ± 1.89a 36.53 ± 2.98a 0.81 0.41

5.17 ± 0.24a 1.32 ± 0.35a 16.72 ± 1.36a 0.25 ± 0.09a 23.19 ± 2.78a 18.62 ± 1.26a 7.16 ± 0.64a 0.49 ± 0.18a 21.34 ± 0.94a 0.29 ± 0.11a 1.98 ± 0.16a 3.47 ± 0.25a 45.08 ± 4.38a 20.19 ± 1.70a 34.73 ± 2.28a 0.77 0.45

4.87 ± 0.37a 1.30 ± 0.26a 18.42 ± 1.89a 0.22 ± 0.05a 23.86 ± 1.56a 18.96 ± 1.77a 6.94 ± 0.39a 0.35 ± 0.15a 19.95 ± 1.86a 0.28 ± 0.05a 1.75 ± 0.09a 3.09 ± 0.27a 47.15 ± 3.82a 20.48 ± 2.08a 32.37 ± 2.81a 0.69 0.43

4.88 ± 0.41a 1.24 ± 0.33a 20.62 ± 2.05a 0.14 ± 0.06a 24.89 ± 1.89a 20.47 ± 1.95a 7.40 ± 0.45a 0.29 ± 0.05a 16.07 ± 1.02a 0.14 ± 0.06a 1.24 ± 0.10a 2.61 ± 0.16a 50.39 ± 4.35a 21.85 ± 2.34a 27.75 ± 1.84a 0.55 0.43

4.64 ± 0.39a 1.26 ± 0.19a 20.94 ± 2.31a 0.12 ± 0.09a 25.62 ± 2.27a 20.53 ± 1.85a 6.96 ± 0.28a 0.27 ± 0.09a 16.08 ± 1.33a 0.21 ± 0.08a 1.17 ± 0.05a 2.21 ± 0.11a 51.20 ± 4.97a 21.91 ± 2.13a 26.90 ± 1.94a 0.53 0.43

Means in the same row with different letters differ significantly (P < 0.05).

ture is probably due to the different origins of experimental materials and also to the different processing conditions. Linoleic acid (C18:2) is among the moderately mobilized fatty acids and arachidonic acid (C20:4) is classified as a highly mobilized fatty acid, which is proposed by Raclot when he studied lipolysis in Adipose tissue (Raclot, 2003).

3.4. The relationship among fatty acid profiles, PLA2 and PLC activities and intramuscular phospholipid degradation The relative activities of PLA2 highly correlated with the decline of phospholipids (R = 0.996, P < 0.01), and the increase of free fatty acids (R = 0.967, P < 0.01). Significant correlations were also

D. Wang et al. / Food Chemistry 145 (2014) 997–1001

obtained between the relative activities of PLC and the decrease of phospholipids (R = 0.958, P < 0.05), the increase of neutral lipid (R = 0.990, P < 0.01). The slowing-down decrease in phospholipids and increase in FFA corresponded to the decreased activity of PLA2. The increasing rate of neutral lipids content became slower in the early processing stages and faster between 6 and 12 days of drying, which might result from the changes in PLC activity. Correlation analysis indicated that PLA2 and PLC may contribute to the degradation of intramuscular phospholipids during the processing of traditional Chinese dry-cured duck. PLA2 comprise a set of extracellular or intracellular enzymes and catalyze the selective hydrolysis of the sn-2 acyl ester bond in 1,2-diacyl-sn-glycero-3 phospholipids, resulting in the formation of free fatty acids such as arachidonic acid (C20:4) and lysophospholipids (Burke & Dennis, 2009). There is in fact a cytosolic PLA2 that preferentially hydrolyses phospholipids with arachidonic acid in the sn-2 position (Balsinde, Winstead, & Dennis, 2002). In our study, a distinct decline (P < 0.05) of the percentages of arachidonic acid in phospholipids was observed, concomitant with a marked increase (P < 0.05) in free fatty acids. This further confirmed that phospholipids were hydrolysed by PLA2 during the traditional Chinese dry-cured duck processing. PLC cleaves the phosphodiester bond of membrane phospholipids, resulting in the formation of diacylglycerol (Bunney & Katan, 2011; Rebecchi & Pentyala, 2000). Since PUFA is high in phospholipids, the diacylglycerol which is derived from the lipolysis of phospholipids would also have a high content of PUFA. We found an obvious increase (P < 0.05) of the percentages of PUFA in neutral lipid, which was similar to the study by Qiu et al. (2013). In combination with the results of Gandemer, (2002), who reported that the percentages of diacylglycerol increased during the processing of Serrano dry cured ham, the increased diacylglycerol content in dry-cured duck could attribute to PLC on the lipolysis of intramuscular phospholipids. In summary, lipolysis of phospholipids was observed during the processing of dry-cured duck. PLA2 and PLC activities declined with the processing time, but they retained 83.70% and 86.78% of their initial activities respectively at the end of the drying process. PUFA of FFA and neutral lipid gradually increased during the whole process whereas PUFA of phospholipids decreased, suggesting the production of FFA and diacylglycerol catalyzed by PLA2 and PLC. The significant correlation between phospholipase activities and phospholipids degradation and increase of FFA further confirmed that PLA2 and PLC play important roles in intramuscular phospholipids lipolysis. Acknowledgement This study was supported by National Natural Science Foundation of China (31271891), Natural Science Foundation Program of Jiangsu Province (BK2012785), Innovation of Agricultural Science and Technology of Jiangsu Province (CX(11)4028) and the Postgraduate Education Innovation Project of Jiangsu province in China (CXLX11-0673). References Argyrous, G. (2011). Statistics for research: with a guide to SPSS (3rd ed.). London: SAGE Publications Ltd. Sage.

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Changes of phospholipase A₂ and C activities during dry-cured duck processing and their relationship with intramuscular phospholipid degradation.

Phospholipid hydrolysis, as the main stage and cause of phopholipid degradation, is generally attributed to phospholipases. In this study, the changes...
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