Lipolysis in intramuscular lipids during processing of traditional Xuanwei ham

Meat Science 71 (2005) 670–675 www.elsevier.com/locate/meatsci

Lipolysis in intramuscular lipids during processing of traditional Xuanwei ham Hongju Yang a, Changwei Ma a

a,¤

, Fadong Qiao a, Yong Song a, Min Du

b

College of Food Science & Nutrition-Engineering, China Agricultural University, Beijing 100083, China b Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA Received 15 December 2004; received in revised form 12 May 2005; accepted 12 May 2005

Abstract Lipolysis in intramuscular lipids during the processing of Chinese Xuanwei ham has been studied by analyzing the changes of glycerides, phospholipids and free fatty acids in biceps femoris muscle. 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 rapid lipolysis of phospholipids occurred during the Wrst 4 months of processing and slowed down during the rest period. A preferential hydrolysis for palmitic, linoleic and arachidonic acids in phospholipid fraction was observed. Glycerides only changed a little throughout the process, while an increase of free fatty acids during processing was observed. The results suggest that phospholipids are the main substrate of lipolysis in the intramuscular lipids of Chinese Xuanwei ham.  2005 Elsevier Ltd. All rights reserved. Keywords: Xuanwei ham; Intramuscular lipids; Lipolysis; Fatty acids

1. Introduction Xuanwei ham is a traditional dry-cured meat product in China. It is characterized by a Xavor with strong intensity that could be due to the traditional techniques used for manufacturing and also due to the pigs raised in free-range conditions. Among the components of the raw hams, it is well known that intramuscular lipids play an important role in the development of chemical and sensory characteristics of dry-cured hams (Gandemer, 2002). During processing, intramuscular lipids are progressively altered through both lipolysis and oxidation. Lipolysis causes an increase in free fatty acids content in the muscle. The autoxidation of fatty acids produces a number of diVerent hydroperoxides, in conjunction with many diVerent * Corresponding author. Tel.: +86 10 62 73 76 43; fax: +86 10 62 73 76 43. E-mail address: [email protected] (C. Ma).

0309-1740/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.05.019

decomposition pathways involved, leading to a large number of volatile compounds, such as aliphatic aldehydes, ketones, lactones, alcohols and esters (Timón, Ventanas, Carrapiso, Jurado, & García, 2001). Those volatiles produced through lipid oxidation are largely responsible for the characteristic Xavor of dry-cured ham (Bolzoni, Barbieri, & Virgili, 1996; Flores, Grimm, Toldrá, & Spanier, 1997). The characteristics of the raw ham and processing techniques (way of salting and ripening conditions) vary from one product to another. This causes the diVerences in lipid breakdown during ripening and forms a unique Xavor for each product. Several authors have studied the changes of intramuscular lipid in the typical dry-cured hams of their countries (Buscailhon, Gandemer, & Monin, 1994; Coutron-Gambotti, Gandemer, Rousset, Maestrini, & Casabianca, 1999; Mart´n, Cordoba, Ventanas, & Antequera, 1999; Vestergaard, Schivazappa, & Virgili, 2000). However, no research has been published in the dry-cured ham from China.

H. Yang et al. / Meat Science 71 (2005) 670–675

The objective of this study was to characterize the intramuscular lipids of Chinese traditional Wujin pig and lipolysis in the processing of dry-cured Xuanwei ham by analyzing the fatty acid composition of three main fractions (glycerides, phospholipids and free fatty acids) of lipids.

2. Materials and methods 2.1. Processing and sampling of hams Wujin pig is a typical local breeds reared outdoors or indoors and its feeding strategy is based on the availability of natural resources (sweet potato vine, horsebean, pease, buckwheat, carrot, pumpkin, chayote and cress). When the weight reaches to 40–50 kg, pigs are fatten by adding large quantities of corn Xour and potato to the primary feed. Wujin pig grows slowly, but its body fat is high, and the quality of muscle is good. It is an ideal material for producing the high quality Xuanwei ham. Twenty-four Wujin pigs were slaughtered at 12– 14 month old, 90–100 kg. Hams were held for 12–24 h at 5–10 °C after slaughter. Dry-cured hams were made in traditional procedures. First, the hams were thoroughly rubbed with salt (containing about 89% NaCl, 10% water and 1% potassium nitrate, on the proportion of 30 g kg¡1 green legs), placed on platforms and held for 1 week at 4 °C. The hams were salted again using salt containing about 89.5% NaCl, 10% water and 0.5% potassium nitrate (20 g kg¡1 hams) and held for a further 30 days at 5–10 °C and 65–85% relative humidity. After washing to remove the excess salt on the surface of the hams, they were hung with straw strings and dried in the sunlight for 3–4 days. Then, the hams were hung on the strings to ripen for 8–12 months in a ventilated chamber (temperature ranging from 10 to 20 °C and relative humidity 60–80%). Sampling of green hams was performed 24-h post slaughter. Samples of dry-cured hams were removed at diVerent aging stages (4, 6 and 12 months). The Biceps femoris muscle was sampled, vacuum packed and kept at ¡20 °C until analysis. Six hams were used for each sampling. 2.2. Lipid analyses 2.2.1. Lipid extraction Muscles were carefully trimmed to remove adipose tissues and were minced in a blender. Lipids were extracted from samples with chloroform:methanol mixture (2:1, v/v) according to the method of Folch, Lees, and Sloane-Stanley (1957). The extracts were dried under vacuum on a rotary evaporator and Wnished with a nitrogen Xow. The total extracted lipid was weighed and expressed as g kg¡1 muscle.

671

2.2.2. Fractionation of lipid extract The total lipid extracts were fractionated into neutral lipids and phospholipids on silica cartridges (Sep-Pack®, Vac Silica 6cc, Waters Corporation, Milford, MA) following the procedure described by Juaneda and Rocquelin (1985). Neutral lipids were quantiWed by weighing and phospholipids were quantiWed by phosphorous determination (Bartlett, 1959). Free fatty acids were puriWed from the neutral lipids using an anionic-exchange resin (Amberlyst A26, Fluka Chemie GmbH, Switzerland) according to the method described by Gandemer, Morvan-mahi, Meynier, and Leperq (1991). An aliquot of 50–100 mg of lipids were dissolved in 15 ml of a mixture of acetone:methanol 2:1 (v/v). After addition of 100–200 mg of the resin, the mixture was shaken for 30 min. Non-bound lipids were removed by washing the resin with acetone/methanol 2:1 (v/v). Resin was then transferred into a dry tube for methylation. 2.2.3. Fatty acid determination The fatty acid composition of glycerides, free fatty acids and phospholipids was determined by gas–liquid chromatography. Methyl esters were prepared by treating lipids with boron Xuoride–methanol (140 g BF3 per liter methanol) according to the method of Morrison and Smith (1964). Methylation of glycerides was completed in 30 min at 100 °C, using 25% boron Xuoride–methanol, 20% benzene and 55% methanol. Phospholipids and free fatty acids were methylated at 100 °C by undiluted boron Xuoride–methanol for 10 and 2 min, respectively. The analysis of methyl esters was carried out using an Agilent 6820 system, equipped with a split and a Xame ionization detector. The methyl esters were separated using a capillary column (HP-INNOWAX, Agilent, 30 m long, 0.32 mm internal diameter, 0.25
m Wlm thickness). The temperature of the column was programmed as following: 2 min at 200 °C, increments of 2 °C min¡1 to 240 °C and held at 240 °C for 5 min. The temperature of the injector was 250 °C and the temperature of the detector was 275 °C. The Xow rate of carrier gas (N2) was set at 1.6 ml min¡1 and head pressure of the carrier gas was 14 psi. Split injection mode was used and the split Xow rate was 10:1. IdentiWcation of fatty acids was performed by comparison of their retention time with that of standards (Sigma, St. Louis, USA). The results were expressed as percent of to the total fatty acid methyl esters present. 2.2.4. Statistical analysis The eVect of time on changes of lipid characteristics was analyzed by the analysis of variance (AOV) and the student t-test was used to identify signiWcant diVerence. The statistical analyses were performed using SAS 8.1 (SAS Institute Inc., Cary, NC).

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H. Yang et al. / Meat Science 71 (2005) 670–675

3. Results and discussion 3.1. Fatty acid composition of green ham The lipid content of the biceps femoris muscle of Wujin pig was 45 g kg¡1 fresh meat. The glycerides represented the most abundant fraction and accounted for 73.2% of total lipid content. The phospholipids represented 25.3% and the free fatty acids 2.3% of the total lipid content, respectively. These results are in agreement with values given for French White pigs by Buscailhon et al. (1994). Table 1 shows the fatty acid composition of glycerides, phospholipids and free fatty acids in green hams. In glycerides, the monounsaturated fatty acids (MUFA) were by far the most abundant (54.7% of the total fatty acids), followed by the saturated fatty acids (SFA, 34.2%) and a small proportion of polyunsaturated fatty acids (PUFA, 10.7%). Oleic, palmitic, stearic and linoleic acids were the main fatty acids in glycerides (49.4%, 22.2%, 10.4% and 9.6%, respectively). The high proportion of such fatty acids have also been reported by Petrón, Muriel, Timón, Martín, and Antequera (2004) and Tejeda, Gandemer, Antequera, Viau, and García (2002) in their studies of glyceride composition of intramuscular lipids in fresh ham from Iberian pigs. In phospholipids, PUFA were the most abundant (42.1%), followed by SFA (36.8%), and MUFA only accounted for 21.1%. These results were in agreement Table 1 Fatty acid composition of lipid fractions of Xuanwei green hams a Fatty acids

Lipid fraction Glyceridesb

C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:4 C22:5 SFA MUFA PUFA n ¡ 3/n ¡ 6e P/Sf

1.41 § 0.02 22.24 § 0.41 4.20 § 0.17 10.40 § 0.38 49.41 § 0.56 9.59 § 0.63 0.41 § 0.02 0.15 § 0.01 1.04 § 0.02 0.43 § 0.04 0.25 § 0.08 34.20 § 0.49 54.65 § 0.71 10.67 § 0.71 0.06 § 0.008 0.31 § 0.02

Phospholipidsc 0.66 § 0.09 29.02 § 0.48 2.21 § 0.20 7.03 § 0.46 18.46 § 0.68 34.04 § 1.56 0.45 § 0.03 0.17 § 0.02 0.35 § 0.04 6.58 § 0.26 1.06 § 0.03 36.81 § 1.01 21.05 § 0.78 42.14 § 1.63 0.04 § 0.002 1.15 § 0.07

Free fatty acidsd 4.94 § 0.23 23.70 § 0.67 2.49 § 0.34 11.04 § 0.27 27.41 § 0.48 20.19 § 0.12 1.20 § 0.19 0.14 § 0.02 0.80 § 0.07 6.07 § 0.07 1.35 § 0.13 39.82 § 0.55 29.70 § 1.07 28.81 § 0.29 0.10 § 0.008 0.72 § 0.01

with the results obtained by French authors (Buscailhon et al., 1994; Coutron-Gambotti et al., 1999), but diVered from other studies on Iberian ham (Cava et al., 1997; Martín et al., 1999). The latter authors found that SFA were the most abundant. This diVerence could be related to the diVerence in animal breeds and feeding regime. The composition of the free fatty acid fraction was intermediate between that of phospholipids and that of glycerides (Table 1). For example, MUFA represented 28.81% in free fatty acids, 54.7% in glycerides and 21.1% in phospholipids; PUFA represented 28.8% in free fatty acids, 10.7% in glycerides and 42.1% in phospholipids. 3.2. Changes in lipid fractions during processing The amounts of phospholipids and free fatty acids changed signiWcantly during processing, whereas glycerides did not (Table 2). There was a close relationship between the decrease in phospholipid content and the increase in free fatty acid content observed during processing, indicating that free fatty acids came basically from phospholipids. This was supported by several works conducted on changes in content and composition of free fatty acids in dry-cured hams during processing (Buscailhon et al., 1994; Martín et al., 1999). After 12 months ripening, a 77% reduction in the total quantity of phospholipids was observed by comparing phospholipid contents of raw ham and dry-cured ham (12 months). Regarding to changes in fatty acid composition of glyceride fraction during the processing, Table 3 showed the percentage of PUFA decreased and that of MUFA increased during the Wrst 4 months of processing, both remained unchanged thereafter. the SFA unchanged during whole duration of processing. Moreover, P/S ratio signiWcantly decreased during the Wrst period of processing (P < 0.001), indicating that PUFA had a higher degradation rate than SFA. The ratio n ¡ 3/n ¡ 6 displayed a signiWcant increase during the Wrst 4 months of processing (P < 0.001), caused mainly by the fact that the content of linoleic acid was signiWcantly reduced (P < 0.001), while the proportion of other fatty acids either increased or unchanged. A similar trend of change in PUFA, MUFA and SFA was observed in phospholipid fraction during processing Table 2 Changes in lipid composition of Xuanwei hams during processing a, b Time (months)

a

Mean values § standard error. Results are expressed in percent of total fatty acid methyl esters in glycerides. c Results are expressed in percent of total fatty acid methyl esters in phospholipids. d Results are expressed in percent of total fatty acid methyl esters in free fatty acids. e n ¡ 3/n ¡ 6: (C18:3+C22:5)/(C18:2+C20:4). f P/S: PUFA/SFA. b

0 (n D 6)

4 (n D 6)

6 (n D 6)

12 (n D 6)

Glycerides 73.2 § 2.33 a 79.5 § 1.96 a 79.1 § 2.65 a 80.2 § 3.02 a Phospholipids 25.3 § 1.31 a 13.3 § 0.86 b 7.6 § 0.73 c 5.8 § 0.41 c Free fatty acids 2.3 § 0.33 c 7.8 § 0.57 b 13.3 § 1.10 a 11.2 § 1.02 a a

Results are expressed as % of total lipids. Mean values § standard error. DiVerent letters, in a line, stand for signiWcant diVerences. b

H. Yang et al. / Meat Science 71 (2005) 670–675

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Table 3 Changes in fatty acid composition of glyceride fraction in Xuanwei ham during processing (% of methyl esters) a Fatty acids

0 (n D 6) C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:4 C22:5 SFA MUFA PUFA n ¡ 3/n ¡ 6c P/Sd

Sig.b

Time (months)

1.41 § 0.02 a 22.24 § 0.41 4.20 § 0.17 b 10.40 § 0.38 49.41 § 0.56 c 9.59 § 0.63 a 0.41 § 0.02 b 0.15 § 0.01 b 1.04 § 0.02 b 0.43 § 0.04 0.25 § 0.03 34.20 § 0.49 54.65 § 0.71 b 10.67 § 0.71 a 0.06 § 0.008 c 0.31 § 0.02 a

4 (n D 6) 1.34 § 0.05 ab 22.53 § 0.67 4.73 § 0.13 b 9.74 § 0.34 54.42 § 1.07 b 4.29 § 0.24 b 0.59 § 0.02 a 0.23 § 0.01 a 1.41 § 0.11 a 0.42 § 0.10 0.31 § 0.02 33.83 § 1.04 60.56 § 1.08 a 5.61 § 0.27 b 0.19 § 0.008 b 0.17 § 0.01 b

6 (n D 6)

12 (n D 6)

1.35 § 0.07 22.84 § 0.56 5.37 § 0.34 a 8.75 § 0.10 54.84 § 0.85 b 3.92 § 0.60 b 0.76 § 0.06 a 0.19 § 0.01 ab 1.44 § 0.07 a 0.24 § 0.05 0.29 § 0.03 33.13 § 0.67 61.65 § 0.77 a 5.22 § 0.65 b 0.27 § 0.02 a 0.16 § 0.02 b

1.20 § 0.02 21.41 § 0.28 4.19 § 0.16 b 8.64 § 0.33 57.59 § 0.61 a 3.88 § 0.27 b 0.68 § 0.07 a 0.22 § 0.01 a 1.72 § 0.12 a 0.22 § 0.01 0.24 § 0.03 31.46 § 0.57 63.51 § 0.6 a 5.03 § 0.33 b 0.22 § 0.02 ab 0.16 § 0.01 b

¤ NS ¤ NS ¤¤ ¤¤¤ ¤¤ ¤ ¤ NS NS NS ¤¤¤ ¤¤¤ ¤¤¤ ¤¤¤

NS, not signiWcant; ¤P < 0.05; ¤¤P < 0.01; ¤¤¤P < 0.001. a Mean values § standard error; DiVerent letters, in a line, stand for signiWcant diVerences. b Sig: SigniWcance. c n ¡ 3/n ¡ 6: (C18:3 + C22:5)/(C18:2 + C20:4). d P/S: PUFA/SFA. Table 4 Changes in fatty acid composition of phospholipid fraction in Xuanwei ham during processing (% of methyl esters)a Fatty acids

0 (n D 6) C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:4 C22:5 SFA MUFA PUFA n ¡ 3/n ¡ 6c P/Sd

Sig.b

Time (months)

0.66 § 0.09 b 29.02 § 0.48 a 2.21 § 0.20 b 7.03 § 0.46 b 18.46 § 0.68 b 34.04 § 1.56 a 0.45 § 0.03 c 0.17 § 0.02 0.35 § 0.04 c 6.58 § 0.26 a 1.06 § 0.03 b 36.81 § 1.01 21.05 § 0.78 b 42.14 § 1.63 a 0.04 § 0.002 c 1.15 § 0.07 a

4 (n D 6) 1.41 § 0.26 a 23.49 § 0.44 b 2.86 § 0.25 ab 12.07 § 0.61 a 32.97 § 2.18 a 18.26 § 1.98 b 1.09 § 0.08 b 0.20 § 0.02 0.77 § 0.06 b 4.70 § 0.42 b 2.18 § 0.26 a 37.17 § 0.25 36.59 § 2.38 a 26.24 § 2.44 b 0.14 § 0.01 b 0.71 § 0.07 b

6 (n D 6)

12 (n D 6)

1.29 § 0.14 a 22.78 § 0.51 b 3.06 § 0.16 a 11.50 § 0.44 a 36.48 § 1.76 a 15.27 § 1.38 b 1.58 § 0.07 a 0.22 § 0.02 0.96 § 0.07 b 4.56 § 0.28 b 2.31 § 0.21 a 35.79 § 0.85 40.50 § 1.81 a 23.71 § 1.44 b 0.20 § 0.02 a 0.66 § 0.04 b

1.23 § 0.05 a 22.99 § 0.62 b 2.37 § 0.12 b 12.65 § 0.28 a 36.39 § 1.16 a 15.50 § 0.68 b 1.23 § 0.16 b 0.30 § 0.06 1.40 § 0.10 a 4.15 § 0.31 b 1.79 § 0.15 a 37.17 § 0.78 40.16 § 1.22 a 22.67 § 0.89 b 0.16 § 0.01 b 0.61 § 0.02 b

¤ ¤¤¤ ¤ ¤¤¤ ¤ ¤¤¤ ¤¤¤ NS ¤¤ ¤¤¤ ¤ NS ¤¤¤ ¤¤¤ ¤¤¤ ¤¤¤

NS, not signiWcant; ¤P < 0.05; ¤¤P < 0.01; ¤¤¤P < 0.001. a Mean values § standard error. DiVerent letters, in a line, stand for signiWcant diVerences. b Sig: SigniWcance. c n ¡ 3/n ¡ 6: (C18:3 + C22:5)/(C18:2 + C20:4). d P/S: PUFA/SFA.

(Table 4). The highest decrease in fatty acids was observed during the early stage of processing. The amounts signiWcantly decreased from 29.0% to 23.5% for palmitic (C16:0), from 34.0% to 18.3% for linoleic (C18:2) and from 6.6% to 4.7% for arachidonic (C20:4) acids (P < 0.001), while the proportion of other fatty acids either increased or unchanged, suggesting that preferential hydrolysis might exist for these fatty acids. These results are consistent with the previously published data

on Iberian dry-cured ham (Martín et al., 1999). Only slight change in the fatty acid composition of phospholipids was observed in the later stage of ham processing. According to data in Tables 2 and 4, the extent of degradation of diVerent types of fatty acids was calculated as follows: the polyunsaturated fatty acids were degraded in the highest quantity (87.7%), followed by saturated (76.9%) and monounsaturated (56.3%) fatty acid. Martín et al. (1999) also found such a selective degradation when

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H. Yang et al. / Meat Science 71 (2005) 670–675

Table 5 Changes in fatty acid composition of free fatty acid fraction in Xuanwei ham during processing (% of methyl esters)a Fatty acids

0 (n D 6) C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:4 C22:5 SFA MUFA PUFA n ¡ 3/n ¡ 6c P/Sd

Sig.b

Time (months)

4.94 § 0.23 a 23.70 § 0.67 a 2.49 § 0.34 11.04 § 0.27 b 27.41 § 0.49 ab 20.19 § 0.12 1.20 § 0.20 c 0.14 § 0.02 0.80 § 0.07 6.07 § 0.07 1.35 § 0.13 c 39.82 § 0.55 a 29.70 § 1.07 a 28.81 § 0.29 0.10 § 0.008 b 0.72 § 0.01 b

4 (n D 6) 1.54 § 0.14 b 22.17 § 0.48 ab 2.41 § 0.21 13.86 § 0.66 a 25.02 § 1.65 b 23.32 § 2.62 1.84 § 0.14 b 0.18 § 0.02 0.92 § 0.10 6.61 § 0.85 2.14 § 0.09 b 37.75 § 1.03 a 26.51 § 1.07 b 33.91 § 2.02 0.14 § 0.01 b 0.91 § 0.07 a

6 (n D 6) 1.60 § 0.08 b 19.89 § 0.62 b 2.73 § 0.24 13.47 § 0.39 a 28.31 § 1.40 ab 20.72 § 1.66 2.52 § 0.12 a 0.12 § 0.01 0.89 § 0.06 6.99 § 0.32 2.75 § 0.17 a 35.08 § 0.66 b 31.93 § 1.64 a 32.99 § 1.54 0.19 § 0.02 a 0.94 § 0.05 a

12 (n D 6) 1.40 § 0.10 b 21.26 § 0.51 b 2.48 § 0.10 11.40 § 0.40 b 30.21 § 0.79 a 21.39 § 0.51 1.77 § 0.18 b 0.13 § 0.02 1.05 § 0.08 6.97 § 0.34 1.93 § 0.17 b 34.29 § 0.69 b 33.74 § 0.85 a 32.01 § 0.58 0.13 § 0.02 b 0.94 § 0.02 a

¤¤¤ ¤¤ NS ¤ ¤ NS ¤¤ NS NS NS ¤¤¤ ¤ ¤¤ NS ¤¤ ¤

NS, not signiWcant; ¤P < 0.05; ¤¤P < 0.01; ¤¤¤P < 0.001. a Mean values § standard error; DiVerent letters, in a line, stand for signiWcant diVerences. b Sig: SigniWcance. c n ¡ 3/n ¡ 6: (C18:3+C22:5)/(C18:2+C20:4). d P/S: PUFA/SFA.

they studied lipolysis in Iberian hams. However, Buscailhon et al. (1994) found that lipolysis in phospholipid was not speciWc to fatty acid chain length or unsaturation. Due to a marked decrease of PUFA in glycerides and especially in phospholipids during the processing, an increase of PUFA portion in free fatty acids was expected. However, it remained unchanged (Table 5). Buscailhon et al. (1994) and Martín et al. (1999) even found that PUFA in free fatty acid fraction decreased during the curing or the whole duration of processing. This fact could be explained by their high susceptibility to oxidation, which converted them to other compounds. In fact, in our study on volatile Xavor compounds from Xuanwei ham, some compounds such as unsaturated aldehydes with long carbon chain, unsaturated hydrocarbons, alcohols and esters, increased signiWcantly (data unpublished).

4. Conclusion A signiWcant change in fatty acids during the processing of Xuanwei ham was observed. Results in the present work support the hypothesis that free fatty acids come mostly from the hydrolysis of phospholipids. Moreover, a selective degradation of fatty acids in the phospholipid fraction was found.

Acknowledgments This work was carried out with the Wnancial support of the Yunnan province project ‘analysis and improve-

ment of the quality of traditional Xuanwei ham’ (2001 FBBMA00D016).

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