Journal of Food Composition and Analysis 22 (2009) 102–111

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Journal of Food Composition and Analysis

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Original Article

Quantitative changes in the fatty acid profile of lipid fractions of fresh loin frompigs as affected by dietary conjugated linoleic acid and monounsaturated fattyacids during refrigerated storage

Diana Martin a, Elena Muriel a, Teresa Antequera a, Ana I. Andres b, Jorge Ruiz a,*a Tecnologia de Alimentos, Facultad de Veterinaria, Universidad de Extremadura, Avda. Universidad s/n, 10071 Caceres, Spainb Ciencia y Tecnologia de los Alimentos, Escuela de Ingenierias Agrarias, Universidad de Extremadura, Ctra. Caceres s/n, 06071 Badajoz, Spain

A R T I C L E I N F O

Article history:

Received 27 November 2007

Received in revised form 28 August 2008

Accepted 20 October 2008

Keywords:

Conjugated linoleic acid

CLA

Monounsaturated fatty acids

MUFA

Pork

Lipolysis

Refrigerated storage

Livestock feeding management

Food analysis

Food composition

A B S T R A C T

Three levels (0%, 1% and 2%) of an enriched CLA oil (28% cis-9, trans-11 and 28% trans-10, cis-12 CLA) were

combined with two levels of MUFA (low: 19%; high: 39%) for pig feeding. Changes in the amounts of fatty

acids of the neutral lipids (NL), polar lipids (PL) and free fatty acids (FFA) fractions of fresh loin as affected

by dietary CLA, MUFA and CLA �MUFA during 7 days of refrigeration were studied. Dietary CLA at 2% and

MUFA supplementation caused a decrease in SFA, MUFA, PUFA and CLA isomer contents of NL and PL

throughout the storage, whereas the increase in FFA during refrigerated storage was less marked with

dietary CLA. Release of CLA isomers of NL and PL was lower than that of non-conjugated PUFA. No

significant effect of the interaction CLA �MUFA on the change in the contents of most fatty acids

(including CLA isomers) of the lipid fractions was found.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Conjugated linoleic acid (CLA) is a collective term to describepositional and geometric isomers of linoleic acid (cis-9, cis-12octadecadienoic acid) with conjugated double bonds. The cis-9,trans-11 and trans-10, cis-12 CLA are the major CLA isomers found innature, and both have been related to positive health effects(Bhattacharya et al., 2006). Supplementation of swine feeding withCLA has also gained an increasing attention in the last decades, sinceit has been suggested as an approach for improving productive,carcass and meat quality traits and, at the same time, obtaining meatand meat products enriched in CLA (reviewed by Schmid et al., 2006).

Lipolytic enzymes, such as lipases, esterases and phospholipases,play an important role in the quality of meat during storage. Theseenzymes are responsible for the lipolysis of triacylglycerols andphospholipids. The released free fatty acids are considered moreprone to lipid oxidation and, in turn, contribute to the generation offlavour compounds (Flores et al., 1996; Alasnier et al., 2000).Nevertheless, other authors have suggested that released free fatty

* Corresponding author. Tel.: +34 927 257123; fax: +34 927 257110.

E-mail address: jruiz@unex.es (J. Ruiz).

0889-1575/$ – see front matter � 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2008.10.010

acids from phospholipids could remain in the membrane, wherethey are protected against oxidation (Muriel et al., 2007).

Several studies have found an effect of CLA on lipolyticprocesses. Park et al. (1997) reported a reduced lipoprotein lipaseactivity in skeletal muscle and fat pad of mice fed with a CLA-supplemented diet. Pariza et al. (1997) stated that CLA feedingenhanced hormone sensitive lipase and carnitine palmitoyltrans-ferase activity in mice. In 3T3-L1 preadipocytes (mouse embryonicfibroblast – adipose-like cell line), CLA increased basal lipolysis(Park et al., 1997). In a previous study (Martin et al., 2006), we alsodetected a significant effect of dietary CLA on neutral lipase andacid esterase activities of pork. Furthermore, the combination ofCLA and monounsaturated fatty acids (MUFAs) levels in pig dietsalso influenced the acid lipase, acid esterase and neutral esteraseactivities of meat.

Dietary CLA leads to increasing levels of saturated fatty acids(SFA) in animal tissue lipids, while decreasing those of MUFA(reviewed by Dugan et al., 2004; Martin et al., 2008a). Such anincrease in the ratio of SFA to unsaturated fatty acids could havenegative health implications from consumers’ standpoint (Depart-ment of Health, 1994). Thus, including high levels of MUFA in pigdiets when using dietary CLA could be a strategy for counteractingthe decrease in MUFA caused by CLA.

D. Martin et al. / Journal of Food Composition and Analysis 22 (2009) 102–111 103

The extent of the lipolytic phenomena as affected by dietaryCLA or its combination with dietary MUFA levels could be ofinterest. Therefore, the aim of our research was the study of thequantitative changes in the fatty acids of the neutral lipids (NL),polar lipids (PL) and free fatty acids (FFA) fractions of fresh lointhroughout refrigerated storage as affected by dietary CLA, MUFAand their interaction.

2. Material and methods

2.1. Animals and feeding

Three levels (0%, 1% and 2%) of commercial CLA-enriched oil(CLA-60, BASF, Dortmund, Germany) supplementation, containingapprox. 56% of CLA isomers (28% cis-9, trans-11 and 28% trans-10,cis-12) and two levels of MUFA (low: 19%, and high: 39%) werecombined for pig feedings (Table 1). One batch per experimentaldiet weighting 6000 kg was prepared. All diets were formulated toprovide similar protein and energy levels, fulfilling the advisednutritional needs for gilts at considered ages by the NationalResearch Council (NRC, 1998).

The experiment was conducted using 288 finishing gilts (Largewhite < � Landrace � Large white ,). Pigs weighing 70 kg and atabout 140 days of age were randomly allotted to the 6 differentfeeding treatments in 4 replicates of each treatment (12 pigs per

Table 1Ingredients and chemical composition of the experimental treatments.

Low MUFA feed

0% CLA 1% CLA

Ingredient (%)

Barley 53.3 53.3

Wheat 15.0 15.0

Bran 8.0 8.0

Soybean meal 44% 16.0 16.0

Palm oil 1.6 1.1

Soy olein 0.4 0.4

Olive olein 0.0 0.0

Hydrogenated stearin palm 3.0 2.5

CLA 0.0 1.0

Carbonate 1.2 1.2

Phosphate 0.4 0.4

Salt 0.4 0.4

L-Lysine 50 0.17 0.17

L-Threonine 0.03 0.03

Coline 75 0.04 0.04

Vitamin and mineral premix 0.5 0.5

Chemical composition (%)

Dry matter 89.2 89.6

Ash 4.9 5.1

Crude fibre 4.2 4.3

Crude fat 7.7 6.9

Crude protein 16.4 16.0

Nitrogen free extractives 62.8 64.1

Fatty acid composition (%)

C14:0 0.8 0.6

C16:0 35.3 30.4

C16:1 0.1 0.1

C18:0 22.8 20.1

C18:1 n-9 18.1 18.0

C18:2 n-6 19.9 20.2

C18:3 n-3 1.8 1.7

cis-9, trans-11 CLA 0.0 3.9

trans-10, cis-12 CLA 0.0 3.7

S SFA 59.7 52.0

S MUFA 18.8 18.6

S PUFAa 21.5 21.9

CLA, conjugated linoleic acid; SFA, saturated fatty acids; MUFA, monounsaturated fattya Excluding CLA isomers.

replicate). Pigs were housed in an environmentally controlledexperimental grower/finisher shed. Pigs were group-housed (12pigs per pen) and had ad libitum access to feed (single space dryfeeders) and water (nipple drinkers) to a final average weight of107 kg. After the fattening (53 days), pigs were slaughtered at alocal slaughterhouse by electrical stunning and exsanguination.

2.2. Sampling

Representative samples of mixed diets (1 kg per batch) weretaken before the beginning of the trial to determine theirproximate chemical and fatty acid composition. The feed sampleswere ground using a laboratory mill (Ortoalresa, Madrid, Spain)prior to analysis. The analysis of the proximate composition of thefeeds was performed according to the Association of OfficialAnalytical Chemist (AOAC, 2000): crude protein (reference954.01), crude fat (reference 920.39), crude fibre (reference962.09) and ash (reference 942.05). One replicate was performedfor each analysis. Feed analysis is shown in Table 1.

Eight animals from each treatment were randomly selected forsampling. Loins from slaughtered animals were taken within10 min after bleeding and kept at 4 8C for 24 h. Two loin chops(1.5 cm thick) per animal were over-wrapped in PVC film andstored at 4 8C for 7 days. The length of the storage for 7 days waschosen according to our previous experiences (Morcuende et al.,

High MUFA feed

2% CLA 0% CLA 1% CLA 2% CLA

53.3 53.3 53.3 53.3

15.0 15.0 15.0 15.0

8.0 8.0 8.0 8.0

16.0 16.0 16.0 16.0

0.6 1.0 0.5 0.0

0.4 0.0 0.0 0.0

0.0 3.0 3.0 3.0

2.0 1.0 0.5 0.0

2.0 0.0 1.0 2.0

1.2 1.2 1.2 1.2

0.4 0.4 0.4 0.4

0.4 0.4 0.4 0.4

0.17 0.17 0.17 0.17

0.03 0.03 0.03 0.03

0.04 0.04 0.04 0.04

0.5 0.5 0.5 0.5

89.4 89.3 89.5 89.6

5.0 5.1 5.6 5.3

4.1 4.7 4.3 4.6

7.3 7.2 7.1 6.8

15.8 16.7 16.5 15.8

64.0 62.4 62.7 63.8

0.5 0.5 0.3 0.3

25.6 25.4 19.7 15.0

0.1 0.5 0.4 0.4

16.6 11.4 7.6 4.6

18.7 37.8 37.9 37.8

19.8 20.6 22.2 22.5

1.6 1.8 2.1 2.1

8.0 0.0 4.3 7.9

7.9 0.0 4.2 8.1

43.5 38.8 28.4 20.6

19.2 38.9 38.8 38.7

21.5 22.4 24.4 24.7

acids; PUFA, polyunsaturated fatty acids.

Fig. 1. Changes in the contents of total fatty acids and CLA isomers (mg/100 g

sample) from the neutral lipid fraction of loins during 7 days of refrigerated storage.

Abbreviations: CLA, conjugated linoleic acid; MUFA, monounsaturated fatty acids, t,

time of storage.

D. Martin et al. / Journal of Food Composition and Analysis 22 (2009) 102–111104

2003) on similar experiments and according to those reported inthe scientific literature (Alasnier et al., 2000), which showed thatsignificant changes on quality traits of refrigerated meat such asfatty acid content, lipid oxidation, volatile profile or instrumentalcolour, started approximately from that moment on. The sampleswere analyzed at 0, 1, 2, 4 and 7 days of storage. At each timeperiod, a small sample of each chop (5 cm2) was cut and frozen at�80 8C until analysis, while the rest of the chop was kept underrefrigeration until the next sampling time.

2.3. Fatty acid analysis

Total lipids of feeds and loins were extracted with chloroform/methanol (1:2, v/v) according to the method of Bligh and Dyer(1959). In the case of loins, each frozen 5 cm2 sample of chops fromeach time point was individually ground with a commercialgrinder and 5 g of ground sample were weighted for lipidextraction. After solvent evaporation under nitrogen, the NL, PLand FFA fractions were isolated according to the method of Ruizet al. (2004) using NH2-aminopropyl cartridges. Fatty acid methylesters (FAMEs) were obtained by acidic transesterificationfollowing the method described by Sandler and Karo (1992).FAMEs were analyzed by gas chromatography using a Hewlett-Packard HP-5890A gas chromatograph, equipped with an on-column injector and a flame ionization detector (FID). Separationwas carried out on a polyethyleneglycol capillary column(60 m � 0.32 mm i.d. � 0.25 mm film thickness) (Supelcowax-10,Supelco, Bellefonte, PA, USA). Oven temperature started at 180 8C;it was immediately raised 5 8C/min to 200 8C; held for 40 min at200 8C and, increased again at 5 8C/min to 250 8C and held for21 min at 250 8C. Injector and detector temperatures were 250 8C.Carrier gas was nitrogen at a flow rate of 0.8 mL/min. Individualcompounds were identified by comparing their retention timeswith those of standards (Sigma, St Louis, MO, USA). Tridecanoicacid was used as the internal standard. Results were expressed asmg of each fatty acid methyl ester per 100 g of sample (dry matter).

2.4. Statistical analysis

The data structure consisted of the fatty acid amountsquantified at the five different time periods from the lipid fractionsof the intramuscular fat of pigs fed the six different treatments.Results were analyzed by a three-way mixed model repeated-measures analysis of variance by SPSS (V. 15.0) statistical software.The levels of CLA, MUFA and CLA �MUFA were the between-subject effects and time of storage was the within-subject effect.Multiple comparison Bonferroni’s test was used for comparingbetween days of storage (day 0 and day 7) within each treatment,and Tukey’s post-hoc test was used for comparing betweentreatments within each time point. Differences were consideredsignificant at p � 0.05.

3. Results

3.1. Neutral lipids

The amounts of fatty acid of the NL fraction of fresh loins (0days) and after 7 days of refrigerated storage are shown in Table 2.The values at intermediate time points of storage are not showndue to the lack of significant differences with respect to day 0. Thefatty acid profile of NL and PL of fresh loins as affected by dietaryCLA, MUFA or CLA �MUFA interaction at the beginning of the trial(day 0) has been thoroughly discussed in a previous study (Martinet al., 2008b). The basic findings of this study were a dose-dependent enrichment in cis-9, trans-11 CLA and trans-10, cis-12CLA in NL and PL of loins, the CLA accumulations of NL being higher

for high MUFA treatments. We also found that dietary CLAincreased the ratio SFA to unsaturated fatty acids of the NL and PLfractions. Moreover, we reported a CLA x MUFA interactive effecton the SFA and PUFA contents of PL of fresh loins. Our presentresearch deals with the changes in the contents of major fatty acidsof loin after 7 days of storage.

The changes in the contents of both CLA isomers detected in NLafter the storage were conditioned by the CLA level of the diet(p = 0.008 for cis-9, trans-11 CLA and p = 0.022 for trans-10, cis-12CLA) (Table 2). Thus, at 2% dietary CLA, the amounts of both CLAisomers in NL of pork chops decreased (p < 0.01) from day 0 to day7 of refrigerated storage, whereas no effects were detected for 0%or 1% CLA diets. Dietary MUFA level and the interactionCLA �MUFA had no effects on the changes in the amounts ofboth CLA isomers of NL of loins after 7 days of refrigerated storage.

Fig. 1 details the changes in the contents of cis-9, trans-11 CLA(Fig. 1B) and trans-10, cis-12 CLA (Fig. 1C) of NL of loins throughoutthe time of storage (days 0, 1, 2, 4 and 7). The level of dietary CLAaffected the changes in the amounts of CLA isomers throughout

Table 2Changes in the fatty acid content (mg/100 g sample) of the NL fraction of fresh loin after 7 days of refrigerated storage as affected by dietary CLA and MUFA levelsa,b.

CLA level MUFA level SEM p

0% 1% 2% Low High t t � CLA t �MUFA t � CLA �MUFA

Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7

C14:0 16.2 14.0 26.0 20.2 28.3 20.0** 20.2 18.7 27.1 17.4** 1.2 0.009 0.327 0.067 0.436

C14:1 n-5 3.5 3.9 3.5 4.2 4.4 3.8 3.6 4.0 4.0 3.9 0.1 0.507 0.093 0.388 0.521

C15:0 2.2 2.3 2.7 2.6 2.8 2.5 2.4 2.6 2.8 2.3 0.1 0.725 0.456 0.169 0.841

C16:0 230.6 167.2 303.7 232.0 375.2 214.7*** 263.5 213.8 342.9 195.9*** 16.0 0.000 0.239 0.060 0.221

C16:1 n-7 30.4 25.1 42.8 33.45 52.5 31.8*** 35.8 30.3 48.0 29.9** 2.3 0.002 0.058 0.142 0.420

C17:0 3.2 3.1 4.1 3.6 5.1 3.4** 3.8 3.6 4.5 3.2** 0.2 0.021 0.066 0.128 0.599

C17:1 n-7 3.2 3.2 3.7 3.4 4.0 3.2 3.5 3.5 3.8 3.1 0.1 0.162 0.345 0.205 0.921

C18:0 104.5 77.4 143.4 109.8 203.6 96.9*** 119.5 98.9 181.5 90.7*** 9.3 0.000 0.051 0.020 0.371

C18:1 n-9 351.6 262.1 394.4 312.8 497.6 274.2*** 354.9 292.6 474.2 273.9*** 22.5 0.001 0.228 0.072 0.253

C18:2 n-6 62.0 52.7 90.9 62.2* 108.9 53.0*** 75.9 56.9 98.6 55.2*** 4.7 0.000 0.036 0.120 0.080

C18:3 n-6 2.0 1.9 2.0 2.1 2.4 1.9* 2.1 2.0 2.3 1.9 0.1 0.156 0.144 0.280 0.545

C18:3 n-3 5.3 4.8 7.2 5.9 8.7 5.3** 6.1 5.5 8.0 5.2** 0.4 0.012 0.166 0.096 0.205

C20:0 3.9 3.8 3.4 4.5** 4.7 4.4 4.3 4.5 3.7 4.0 0.1 0.360 0.020 0.677 0.074

C20:1 n-9 5.3 5.3 5.2 5.9 6.9 5.1* 6.0 5.4 5.6 5.5 0.2 0.544 0.061 0.581 0.720

C20:2 n-6 3.3 3.4 3.0 3.8* 3.9 3.3 3.5 3.5 3.2 3.4 0.1 0.592 0.050 0.615 0.929

C20.3 n-6 2.6 2.1* 2.0 2.1 2.4 1.8* 2.4 2.0* 2.3 2.0 0.1 0.008 0.123 0.798 0.325

C20:4 n-6 8.3 4.4** 5.9 3.5* 5.9 3.1** 7.2 4.0*** 6.2 3.4*** 0.4 0.000 0.846 0.983 0.022

C21:0 1.8 1.7 1.7 1.9 2.0 1.8 1.9 1.8 1.8 1.8 0.1 0.558 0.187 0.986 0.903

C20:5 n-3 1.5 1.5 2.0 1.6* 2.1 1.5** 1.9 1.5* 1.9 1.6 0.1 0.006 0.035 0.530 0.527

C22:0 2.1 2.6 2.4 2.9* 2.7 2.7 2.6 2.8 2.2 2.6* 0.1 0.021 0.272 0.293 0.697

C22:1 n-9 1.0 1.1 1.0 1.3* 1.2 1.2 1.2 1.3 1.0 1.2 0.0 0.071 0.299 0.685 0.420

C22:5 n-3 2.3 0.8*** 1.6 0.9* 2.2 0.7*** 2.2 0.9*** 1.8 0.7*** 0.1 0.000 0.149 0.510 0.028

C24:0 1.4 2.6** 2.3 3.0* 2.4 2.6 2.1 2.8** 2.2 2.7 0.2 0.002 0.377 0.084 0.470

C22:6 n-3 1.9 1.4* 1.5 1.7 1.9 1.6 1.8 1.7 1.7 1.5 0.1 0.158 0.058 0.455 0.507

C24:1 n-9 1.8 1.6 1.7 1.8 2.1 1.8 2.0 1.8 1.7 1.7 0.1 0.903 0.342 0.546 0.213

85C24:1 n-9 cis-9, trans-11 CLA 4.3 3.6 8.4 9.5 20.0 12.1*** 10.1 8.3 11.3 8.6 0.8 0.056 0.008 0.897 0.748

trans-10, cis-12 CLA 2.5 2.0 3.7 4.5 8.4 5.9** 4.4 4.2 5.1 4.1 0.3 0.169 0.022 0.563 0.941

S SFA 364.9 272.9 489.5 378.7 625.8 347.0*** 419.3 347.5 567.5 318.9*** 27.0 0.000 0.138 0.042 0.285

S MUFA 396.3 301.2 452.3 361.8 568.5 319.9** 406.9 337.7 537.8 317.9*** 25.1 0.001 0.189 0.076 0.259

S PUFA (excluding CLA) 88.8 73.0 116.1 83.7** 138.0 71.9*** 102.9 77.7* 125.7 74.7*** 5.5 0.000 0.040 0.141 0.057

a Values between 0 and 7 days of storage within the same treatment differed significantly if p � 0.05 (*), p � 0.01 (**), or p � 0.001 (***).b NL, neutral lipids; CLA, conjugated linoleic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SEM, standard error of the mean; t, time of storage.

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Fig. 2. Changes in the contents of total fatty acids and CLA isomers (mg/100 g

sample) from the polar lipid fraction of loins during 7days of refrigerated storage.

Abbreviations: CLA, conjugated linoleic acid; MUFA, monounsaturated fatty acids, t,

time of storage.

D. Martin et al. / Journal of Food Composition and Analysis 22 (2009) 102–111106

refrigerated storage (p = 0.011 for cis-9, trans-11 CLA and p = 0.028for trans-10, cis-12 CLA). Thus, a decrease in the content of CLAisomers was evident at the highest doses of CLA assayed (2%), butnot at 0% and 1% of dietary CLA. Moreover, Fig. 1B and C shows thatsuch decreases took place to a greater extent during the first daysof storage (from day 0 to day 4). MUFA level of the diet or itsinteraction with dietary CLA did not affect the changes in thecontents of CLA isomers in NL of loins throughout the refrigeratedstorage.

Regardless of the CLA and MUFA level of the diet, decreases inthe contents of most SFA, MUFA and PUFA of NL of loins from day0 to day 7 of storage were observed (Table 2). Moreover, after 7days of refrigerated storage, the change in the amount of totalPUFA (excluding CLA) was affected by CLA supplementation(p = 0.040). Thus, the decrease in contents of most PUFA from day0 to day 7 of storage was more marked at 1% and 2% of dietaryCLA. Bonferroni’s test also detected a more evident decrease inthe contents of total SFA and MUFA of NL at 2% dietary CLA.Similarly, MUFA supplementation led to a more marked decreasein contents of SFA, MUFA and PUFA of NL of loins (Table 2). Theinteraction CLA �MUFA had no effects on the changes in theamounts of total SFA, MUFA and PUFA of NL from loins after 7days of storage.

Fig. 1A shows the evolution in the contents of total fatty acids(TFAs) (including CLA isomers) of NL from loins throughout the 7days of refrigerated storage (days 0, 1, 2, 4 and 7). The amounts ofTFAs decreased during storage; this effect being more evidentduring the first days of the trial (from day 0 to day 4). Neither CLA,nor MUFA levels, nor their interaction, affected the detectedchanges in TFAs from NL of loins during the storage.

3.2. Polar lipids

The amounts of each fatty acid of the PL fraction of fresh loins (0days) and after 7 days of refrigerated storage are shown in Table 3.The values at intermediate time points of storage are not showndue to the lack of significant differences with respect to day 0.Regardless of the CLA and MUFA levels of the diet, a decrease in thecontents of both CLA isomers of PL from day 0 to day 7 of storagewas found. Such decreases were more marked at 2% dietary CLAand at high dietary MUFA level in pig diet. Nevertheless, thecombination of CLA and MUFA levels in swine feed did notinfluence the change in the amounts of CLA isomers of PL of loinsafter 7 days of refrigeration storage.

Changes in the contents of cis-9, trans-11 CLA and trans-10, cis-12 CLA of PL throughout the assay are detailed in Fig. 2B and C,respectively. The CLA level of the diet affected the change in thecontents of both CLA isomers (p < 0.001 for both CLA isomers).Thus, the highest dietary dose of CLA caused the most evidentdecrease in the amounts of CLA isomers of PL during storage,especially at the beginning of the trial (from day 0 to day 4). DietaryMUFA level or its interaction with dietary CLA did not causechanges in the contents of CLA isomers of PL of loins during therefrigerated storage.

Regardless the CLA and MUFA level of the diet, a decrease in theamounts of total SFA (p = 0.009) and PUFA (p = 0.046), and a trendof decreasing contents of total MUFA (p = 0.096) from 0 to 7 days ofstorage were detected in the PL fraction (Table 3). Moreover, thechange in the contents of SFA was more marked at 2% CLA and forhigh MUFA diet. High MUFA diet also caused a more relevantdecrease in the amounts of MUFA and PUFA of PL from pork after 7days of storage. The combination of dietary CLA and MUFA did notcause changes in the amounts of any fatty acid of PL of loins after 7days of refrigerated storage.

Fig. 2A shows the evolution in the contents of TFAs of PL frompork during the trial. These changes were affected (p = 0.017) by

dietary CLA. Thus, the decrease in the contents of TFAs seemed tobe more evident when CLA was supplemented to pig diet,especially from day 0 to day 4. Nevertheless, dietary CLA didnot lead to different contents of TFAs among the treatments at anyof the tested days of storage.

3.3. Free fatty acids

The amounts of fatty acids of the FFA fraction of fresh loins (0days) and of loins after 7 days of refrigerated storage are shown inTable 4. The values at intermediate time points of storage are notshown due to the lack of significant differences with respect to day0. An increase in the contents of cis-9, trans-11 CLA and trans-10,cis-12 CLA of the FFA fraction from day 0 to day 7 of storage wasobserved, regardless the CLA and MUFA levels of the diet.Nevertheless, these increases were more marked at 1% and 2%of dietary CLA in pig diet. The MUFA level of the diet or itsinteraction with dietary CLA did not influence the change in theamounts of CLA isomers of the FFA fraction of refrigerated loins.

Table 3Changes in the fatty acid content (mg/100 g sample) of the PL fraction of fresh loin after 7 days of refrigerated storage as affected by dietary CLA and MUFA levelsa,b.

CLA level MUFA level SEM p

0% 1% 2% Low High t t � CLA t �MUFA t � CLA �MUFA

Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7

C14:0 10.2 10.2 12.7 12.9 12.2 11.1 11.3 12.6 12.1 10.2 0.5 0.666 0.951 0.104 0.087

C14:1 n-5 7.2 7.4 9.9 9.8 9.5 8.3 8.8 9.4 8.9 7.6 0.5 0.507 0.840 0.276 0.114

C15:0 2.0 2.4 2.0 2.5* 2.3 2.4 2.0 2.5** 2.2 2.3 0.1 0.026 0.404 0.171 0.776

C16:0 57.9 45.7 69.0 51.1* 69.7 51.2* 64.6 53.5 66.4 44.9** 2.2 0.001 0.905 0.260 0.954

C16:1 n-7 3.1 3.5 3.0 3.4 3.1 3.1 3.0 3.3 3.1 3.3 0.1 0.123 0.663 0.882 0.358

C17:0 2.2 2.3 2.4 2.6 2.6 2.4 2.4 2.5 2.4 2.4 0.1 0.568 0.355 0.506 0.695

C17:1 n-7 3.8 5.1 5.6 7.9 4.8 3.6 5.8 6.2 3.7 4.9 0.4 0.332 0.108 0.462 0.168

C18:0 25.2 21.5 32.5 26.5 34.1 23.4** 30.9 26.1 30.3 21.4** 1.1 0.002 0.415 0.347 0.757

C18:1 n-9 38.4 33.0 47.2 38.6 44.9 32.3* 38.9 35.1 48.1 34.1** 1.8 0.013 0.728 0.155 0.813

C18:2 n-6 91.8 85.6 121.5 107.0 127.6 94.2 117.6 111.2 109.7 79.3* 4.9 0.061 0.555 0.235 0.585

C18:3 n-6 2.3 2.5 2.4 2.4 2.8 2.4 2.4 2.5 2.6 2.4 0.1 0.941 0.352 0.516 0.832

C18:3 n-3 3.1 3.5 4.2 4.0 4.1 3.9 3.6 4.1 4.1 3.5 0.1 0.947 0.682 0.059 0.538

C20:0 2.6 3.1 2.7 3.2 3.3 3.1 2.8 3.2 3.0 3.1 0.1 0.060 0.216 0.407 0.485

C20:1 n-9 1.5 1.7 1.7 1.8 1.8 1.7 1.6 1.8 1.7 1.7 0.1 0.352 0.376 0.399 0.434

C20:2 n-6 2.2 2.1 2.7 2.4 2.9 2.5 2.6 2.6 2.6 2.1 0.1 0.199 0.882 0.211 0.641

C20.3 n-6 4.7 4.7 5.1 4.8 5.2 4.3 5.1 4.9 5.0 4.2 0.2 0.221 0.631 0.510 0.367

C20:4 n-6 32.1 27.4 36.7 31.0 38.5 28.3 37.2 32.6 34.4 25.0* 1.6 0.025 0.734 0.478 0.770

C21:0 1.4 1.6 1.5 1.7 1.8 1.5 1.5 1.7 1.6 1.6 0.1 0.253 0.081 0.349 0.659

C20:5 n-3 3.3 3.3 3.9 3.6 4.3 3.7 3.9 3.7 3.7 3.3 0.1 0.367 0.583 0.639 0.248

C22:0 2.0 2.7 2.4 2.8 2.6 2.8 2.4 2.9* 2.3 2.7 0.1 0.006 0.783 0.799 0.543

C22:1 n-9 1.0 1.2 1.2 1.2 1.2 1.2 1.1 1.2 1.1 1.2 0.0 0.132 0.441 0.759 0.587

C22:5 n-3 7.3 5.2 9.3 7.2 8.7 6.2 9.1 7.1 7.7 5.1 0.4 0.013 0.973 0.798 0.518

C24:0 3.7 2.7 2.9 3.1 3.5 2.9 3.4 3.1 3.0 2.7 0.1 – – – –

C22:6 n-3 2.6 2.5 3.6 2.9 3.7 2.8 3.4 2.9 3.2 2.6 0.1 0.086 0.342 0.816 0.451

C24:1 n-9 n.d. 1.7 1.9 2.0 1.4 1.9 1.5 2.0 2.0 1.7 0.1 – – – –

cis-9, trans-11 CLA 2.2 1.8 7.2 5.9 11.8 8.4** 7.4 6.0 6.7 4.7* 0.5 0.019 0.198 0.610 0.910

trans-10, cis-12 CLA 2.0 1.7 3.6 3.4 6.0 4.5** 3.8 3.5 3.9 2.9* 0.2 0.036 0.126 0.276 0.882

S SFA 102.7 91.0 125.3 104.1 129.0 99.4* 118.0 106.2 120.0 89.8** 3.7 0.009 0.692 0.249 0.927

S MUFA 54.7 52.9 68.8 63.4 65.3 51.2 59.1 57.8 66.7 53.7* 2.2 0.096 0.517 0.202 0.791

S PUFA (excluding CLA) 149.2 136.7 189.5 164.7 197.4 148.3 184.5 171.7 172.9 127.2* 7.3 0.046 0.611 0.280 0.594

a Values between 0 and 7 days of storage within the same treatment differed significantly if p � 0.05 (*), p � 0.01 (**), or p � 0.001 (***).b PL, polar lipids; CLA, conjugated linoleic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SEM, standard error of the mean; t, time of storage.

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Table 4Changes in the fatty acid content (mg/100 g sample) of the FFA lipid fraction of fresh loin after 7 days of refrigerated storage as affected by dietary CLA and MUFA levelsa,b.

CLA level MUFA level SEM p

0% 1% 2% Low High t t � CLA t �MUFA t � CLA �MUFA

Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7

C14:0 3.7 4.1 3.9 4.5 4.5 4.2 3.9 4.4 4.2 4.1 0.1 0.388 0.170 0.382 0.127

C14:1 n-5 1.9 2.1 2.0 2.3 2.4 2.1 2.0 2.2 2.2 2.1 0.1 0.847 0.112 0.477 0.087

C15:0 1.8 2.0 1.9 2.2 2.2 2.0 1.9 2.2 2.0 2.0 0.1 0.286 0.110 0.394 0.150

C16:0 13.2 23.3*** 14.0 23.0*** 14.7 22.7*** 13.5 22.1*** 14.4 23.9*** 0.7 0.000 0.674 0.598 0.190

C16:1 n-7 2.1 2.7* 2.4 2.7 2.5 2.4 2.3 2.6 2.3 2.7 0.1 0.138 0.178 0.567 0.124

C17:0 1.7 2.0 1.8 2.2 2.0 2.0 1.8 2.1 1.9 2.0 0.1 0.090 0.170 0.696 0.285

C17:1 n-7 1.6 1.7 1.6 1.9 2.0 1.7 1.6 1.9 1.8 1.7 0.1 0.606 0.040 0.260 0.138

C18:0 8.5 12.9** 10.0 14.5** 9.0 13.4** 9.0 14.0*** 9.3 13.3** 0.4 0.000 1.000 0.552 0.615

C18:1 n-9 10.9 18.6*** 12.2 18.6** 12.0 18.0** 11.8 17.8*** 11.6 19.0*** 0.7 0.000 0.731 0.405 0.166

C18:2 n-6 12.6 26.7*** 12.6 31.4*** 13.9 28.5*** 13.2 29.6*** 12.9 28.0*** 1.2 0.000 0.412 0.688 0.212

C18:3 n-6 1.6 2.1* 1.7 2.1* 1.9 1.9 1.7 2.0 1.8 2.0 0.1 0.036 0.151 0.900 0.473

C18:3 n-3 1.8 2.4* 2.2 2.6 2.2 2.5 2.1 2.5 2.0 2.6* 0.1 0.009 0.692 0.478 0.311

C20:0 2.7 2.9 2.8 3.3 3.3 3.0 2.8 3.2 3.0 3.0 0.1 0.498 0.171 0.376 0.153

C20:1 n-9 1.3 1.4 1.5 1.6 1.5 1.5 1.4 1.6 1.4 1.5 0.1 0.609 0.688 0.820 0.092

C20:2 n-6 1.2 1.4 1.3 1.6 1.5 1.5 1.3 1.6 1.4 1.4 0.0 0.091 0.392 0.405 0.148

C20.3 n-6 1.8 2.7*** 1.9 2.8** 1.9 2.4 1.9 2.7*** 1.8 2.5** 0.1 0.000 0.438 0.961 0.698

C20:4 n-6 4.3 9.8*** 4.3 9.6*** 4.4 8.1*** 4.4 9.4*** 4.3 9.0*** 0.4 0.000 0.310 0.752 0.391

C21:0 1.1 1.3 1.2 1.5* 1.4 1.4 1.2 1.4 1.3 1.4 0.0 0.128 0.090 0.548 0.234

C20:5 n-3 1.5 2.1* 1.7 2.3** 1.7 2.1 1.6 2.2** 1.6 2.1* 0.1 0.000 0.675 0.601 0.492

C22:0 2.3 2.6 2.4 2.9 2.8 2.7 2.4 2.8 2.6 2.6 0.1 0.288 0.133 0.348 0.151

C22:1 n-9 1.0 1.1 1.0 1.2 1.2 1.1 1.0 1.2 1.1 1.1 0.0 0.392 0.104 0.309 0.136

C22:5 n-3 1.8 3.6*** 1.5 4.1*** 1.6 3.2*** 1.5 3.8*** 1.7 3.5*** 0.2 0.000 0.194 0.265 0.470

C24:0 2.1 2.6 2.4 2.9 2.7 2.5 2.3 2.8 2.6 2.6 0.1 – – – –

C22:6 n-3 1.7 2.2* 1.8 2.7*** 1.9 2.4 1.7 2.5*** 1.9 2.3* 0.1 0.000 0.388 0.292 0.428

C24:1 n-9 1.3 1.7 1.5 1.8 1.7 1.6 1.4 1.7 1.6 1.6 0.1 0.363 0.187 0.279 0.403

cis-9, trans-11 CLA 1.3 1.5 1.7 2.8*** 2.0 3.5*** 1.6 2.6*** 1.8 2.6*** 0.1 0.000 0.009 0.475 0.291

trans-10, cis-12 CLA 1.4 1.7 1.9 2.3* 1.9 2.6** 1.7 2.2* 1.8 2.1* 0.1 0.001 0.490 0.708 0.071

S SFA 36.4 53.0*** 40.2 55.8** 42.4 53.6* 38.6 53.8*** 40.7 54.4*** 1.5 0.000 0.558 0.935 0.569

S MUFA 19.5 28.8** 22.2 29.6* 22.8 28.3 21.6 28.3* 21.4 29.5*** 0.9 0.000 0.503 0.516 0.151

S PUFA (excluding CLA) 28.4 53.0*** 28.8 59.2*** 30.9 52.6*** 29.4 56.3*** 29.4 53.4*** 2.0 0.000 0.448 0.657 0.485

a Values between 0 and 7 days of storage within the same treatment differed significantly if p � 0.05 (*), p � 0.01 (**), or p � 0.001 (***).b FFA, free fatty acids; CLA, conjugated linoleic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SEM, standard error of the mean; t, time of storage.

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D. Martin et al. / Journal of Food Composition and Analysis 22 (2009) 102–111 109

Fig. 3B and C shows the changes in the contents of both CLAisomers of the FFA fraction from pork during the trial. The increasein the amounts of CLA isomers was specially marked during the lastdays of the assay (from day 4 to day 7). Moreover, increasesthroughout the trial seemed to be higher with the highest level ofdietary CLA, this effect being significant in the case of the cis-9,trans-11 isomer (p < 0.001).

The contents of total SFA, MUFA and PUFA of the FFA fraction ofthe loins increased from day 0 to day 7 of storage, regardless theCLA or MUFA level of the diet (Table 4). Nevertheless, the increasein SFA, MUFA and PUFA contents with the storage seemed to be lessmarked as the level of dietary CLA increased.

The change in the contents of TFAs of the FFA fractionthroughout the storage is detailed in Fig. 3A. The highest increasein total TFAs was obtained from day 4 to day 7 of refrigeratedstorage. Dietary CLA seemed to affect the change in the contents oftotal TFAs throughout storage (p = 0.059). Thus, it seems that alower increase in TFAs took place at the highest level of dietaryCLA.

Fig. 3. Changes in the contents of total fatty acids and CLA isomers (mg/100 g

sample) from the free fatty acid lipid fraction of loins during 7 days of refrigerated

storage. Abbreviations: CLA, conjugated linoleic acid; MUFA, monounsaturated fatty

acids, t, time of storage.

4. Discussion

4.1. Quantitative changes in the fatty acid contents (excluding CLA

isomers) of the NL, PL and FFA fractions

In general, the refrigerated storage of fresh loins during 7 daysled to a decrease in the contents of fatty acids from NL (mainlycomposed by triacylglycerols) and PL (mainly composed byphospholipids) as well as a parallel increase in the FFA fraction.Such an increase in FFA evidenced the lipolytic phenomena thattook place during meat storage, whereas the lost of fatty acids fromtriacylglycerols and phospholipids could be due either to lipidoxidation or to lipolytic processes, but most likely to the latterphenomena (Bermudez et al., 1993).

Supplementation of swine diets with CLA seemed to favourdecreases in the contents of most fatty acids from bothtriacylglycerols and phospholipids of pork during refrigeratedstorage. Nevertheless, most of the significant decreases in thecontents of fatty acids were detected at the highest levels of dietaryCLA (2%) and after 7 days of storage (Tables 2 and 3).

The more marked decrease in fatty acids from triacylglycerolsand phospholipids at high doses of dietary CLA might suggest astimulation of lipolysis of these lipid fractions by dietary CLA.Therefore, a subsequent increase in FFA with dietary CLA should bealso detected. However, we detected a less marked increase in FFAwith increasing levels of dietary CLA (Table 4). Thus, from day 0 today 7 of storage, the contents of SFA in the FFA fraction increased16.9 mg/100 g sample at 0% CLA and 11.1 mg/100 g sample at 2%CLA; the contents of MUFA increased 9.3 mg/100 g sample at 0%CLA and 5.5 mg/100 g sample at 2% CLA, and the contents of PUFAincreased 24.6 mg/100 mg sample at 0% CLA and 21.7 mg/100 mgsample at 2% CLA.

In a previous work (Martin et al., 2006), the lipolytic activity ofpork as affected by dietary CLA and MUFA was studied. In thatexperiment, dietary CLA and CLA �MUFA interaction did not showrelevant effects on the lipolytic activity, and both increasing anddecreasing activities occurred depending on the CLA and MUFAlevels of the diet. However, the lipolytic activity was only studiedfor fresh meat and changes in the activity of lipolytic enzymesduring storage might take place due to the modification of theenvironmental conditions, such as the release of FFA to themedium (Pafumi et al., 2002). In the study of Martin et al. (2006),the in vitro activity of lipases after CLA addition (as a free fatty acid)to the enzymatic medium was also assayed. Decreasing lipaseactivities with increasing concentrations of added free CLA werefound. Therefore, in the present work, it would be possible that thefree CLA isomers hydrolyzed from the lipid fractions acted asinhibitors of the lipolytic enzymes of pork, protecting lipidfractions against lipolysis. This would explain the lower releaseof FFA with dietary CLA.

The CLA isomers have been suggested as antioxidants or pro-oxidants in different studies, but no effect of CLA on oxidativereactions has been also found (reviewed by Hur et al., 2007). In thepresent work, the more marked decrease in the contents of fattyacids from triacylglycerols and phospholipids at high doses ofdietary CLA might also be related to a stimulated oxidation of theselipid fractions by dietary CLA. However, in a previous work (Martinet al., 2008b), a lack of effect of dietary CLA on susceptibility tooxidation of the same samples studied in the present experimentwas concluded. Moreover, the low susceptibility to lipid oxidationof triacylglycerols is well documented (Nawar, 1996) due to theirlower proportion of PUFA and their location in adipocytes, whichare not close to pro-oxidant systems. Furthermore, if oxidation oftriacylglycerols had been relevant, the decrease in PUFA shouldhave been more marked than the decrease in MUFA and SFA, due tohigher susceptibility to oxidation of PUFA. However, decreases in

D. Martin et al. / Journal of Food Composition and Analysis 22 (2009) 102–111110

the contents of SFA, MUFA and PUFA of NL from day 0 to day 7 ofstorage were quite similar (at 2% CLA, such decreases were 46% forSFA, 45% for MUFA and 48% for PUFA) (Table 2). Regardingphospholipids, these lipids are more susceptible to oxidationcompared to triacylglycerols (Igene et al., 1980), due to theirlocation in membranes close to heme pigments and oxidantsystems, and due to their higher PUFA contents (Gray and Pearson,1987). In our research, decrease in the contents of PUFA ofphospholipids was similar to that of MUFA and SFA (at 2% CLA, suchdecreases were 23% for SFA, 22% for MUFA and 25% for PUFA),similar to the case of triacylglycerols. Therefore, an enhancedoxidation of triacylglycerols and phospholipids by dietary CLAcould not be evidenced.

Regarding the effects of MUFA supplementation, MUFA-enriched diets seemed to favour the mobilization of fatty acidsfrom triacylglycerols and phospholipids during the storage.Enhanced fat catabolism and reduced unsaturated fatty acidsynthesis have been reported to occur in rats fed unsaturated fattyacid-enriched diets compared with rats fed diets enriched with SFA(Shimomura et al., 1990). In the previous study about the lipolyticactivity of the same samples that we used in the present research(Martin et al., 2006), dietary MUFA did not exert any effect on theactivity of any of the lipolytic enzymes assayed in muscle tissue.Other studies as well have found no difference between the effectsof saturated or unsaturated fats on lipolysis in swine adipose tissue(Mersman et al., 1992).

Despite the enhanced mobilization of fatty acids due to MUFAsupplementation in our study, the combination of dietary CLA andMUFA levels in pig diet had no relevant effects on the changes inthe contents of most fatty acids throughout refrigerated storage.

4.2. Quantitative changes in the contents of CLA isomers of the NL, PL

and FFA fractions

The extent of the loss of CLA isomers from triacylglycerols andphospholipids with the storage seemed to be lower than that of therest of PUFA, especially when compared to the non-conjugatedisomer of C18:2 n-6. The content of C18:2 n-6 linearly decreasedduring 7 days of refrigerated storage with increasing levels ofdietary CLA, both in NL (decreases of 15% at 0% CLA, 32% at 1% CLAand 51% at 2% CLA) and in PL (decreases of 7% at 0% CLA, 12% at 1%CLA and 26% at 2% CLA). However, this linear behaviour was notobserved for the change in the contents of CLA isomers from bothlipid fractions.

The conjugated structure of the CLA isomers is considered themain reason that determines most of the particular characteristicsof this group of fatty acids. Thus, the less susceptibility of CLA tofree radical attacks or to oxidative changes found by severalauthors has been attributed to the presence of conjugated doublebonds (Shantha et al., 1994; Du et al., 2000). The conjugatedstructure of CLA might also explain the lower mobilization of thisfatty acid from triacylglycerols and phospholipids by lipolyticenzymes compared to other PUFAs. As Raclot (2003) suggested, themobilization of fatty acids from triacylglycerols seems to depend,among other factors, on the positional isomerism of the fatty acids.

The location of the CLA isomers within the glycerol backbonealso might be related to the lower mobilization of CLA fromtriacylglycerols and phospholipids. One of the most importantenzymes implied in the mobilization of fatty acids fromtriacylglycerols (hormone sensitive lipase) preferentially cleavesthe sn-1 and sn-3 positions of triacylglycerols (Belfrage et al.,1984). Thus, if CLA isomers were mobilized from triacylglycerols ina lower extent compared with the non-conjugated C18:2 n-6, thismight point to a preferential location of CLA isomers in the sn-2position of triacylglycerols. The postulated location of CLA isomersin the sn-2 position of triacylglycerols of loins would be in

agreement with the results found by King et al. (2004). Theseauthors detected a greater enrichment of cis-9, trans-11 CLA andtrans-10, cis-12 CLA isomers in the sn-2 than in the sn-1 or sn-3positions of triacylglycerols of subcutaneous adipose tissue fromCLA-fed pigs.

Curiously, despite the lower mobilization of CLA isomers fromtriacylglycerols and phospholipids with dietary CLA compared toother PUFA, it should be noted that the amounts of CLA in the FFAfraction increase linearly with dietary CLA (from day 0 to day 7,total CLA increased 0.5 mg/100 g sample at 0% CLA, 1.5 mg/100 gsample at 1% CLA and 2.2 mg/100 g sample at 2% CLA in the FFAfraction). Ha et al. (1989) have suggested that linoleic acid radicalsderived from oxidative reactions might influence CLA concentra-tions by conversion of those radicals to CLA by hydrogen donors.

The MUFA level of the pig diet as well as the CLA �MUFAinteraction effects were not relevant in the change in the amountsof CLA isomers of the loin lipids with the storage.

5. Conclusions

The effects of dietary CLA on the change in the amounts of fattyacids of pork lipids during refrigerated storage is not conclusive.CLA supplementation seems to lead to a higher release of fattyacids from the lipid fractions, especially at high doses of CLA in thefeed. However, evidences of a protective effect of dietary CLAagainst lipolysis of lipid fractions were also found.

A high MUFA diet, regardless of the included CLA level in thefeed, seems also to cause higher losses of fatty acids from the lipidfractions of stored meat. Nevertheless, the combination of CLA andhigh MUFA levels in pig diet does not seem to influence the changein the contents of fatty acids (including CLA isomers) of pork lipidsduring refrigerated storage.

The release of CLA isomers from the lipid fractions with thestorage seems to be less marked than that of other PUFA, especiallythe non-conjugated linoleic acid. The combination of CLA withMUFA enrichment of pig diet would not damage the achieved CLAenrichment of meat, even during refrigerated storage.

Acknowledgments

This research was supported by the Ministerio de Educacion yCiencia, Spain (AGL 2003-03538). CLA was generously provided byBASF. The valuable cooperation of Dr. Clemente Lopez-Bote andDra. Elena Gonzalez as well as the collaboration of I+DAgropecuaria in designing the experimental diets, sampling andpig management are also acknowledged. Diana Martin thanks theMinisterio de Educacion y Ciencia for funding her research.

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