Changes in the concentrations of free fatty acid, monoacylglycerol, and diacylglycerol in the subcutaneous fat of Iberian ham during the dry-curing process

Article abstract of DOI:10.1021/jf071886u

Changes in diacylglycerols, monoacylglycerols, and free fatty acid composition of subcutaneous fat of six Iberian hams during the dry-cured process were investigated. In addition, an analytical method for simultaneous quantification of diacylglycerols, monoacylglycerols, and free fatty acid by solid-phase extraction-gas chromatography was developed. The different molecular species of free fatty acids, monoacylglycerols, and diacylglycerols and 1,2- and 1,3-isomers of diacylglycerols have been described for the first time in this type of sample. A logarithmic increase of the 1,3-diacylglycerol profile throughout the processing time has been found, reaching a balance value of 62% around 500 days. The formation of diacylglycerol isomers takes place, although the 1,3-/1,2-diacylglycerol ratio increases during the process to 1.65 due to isomerization of the 1,2-form toward the 1,3-form. The profiles of monoacyl- and diacylglycerols and free fatty acids follow the same trend. The experimental values of free fatty acid are greater than theoretical prediction, probably due to phospholipid and monoacylglycerol hydrolysis.

Full text of DOI:10.1021/jf071886u

J. Agric. Food Chem. 2007, 55, 10953–10961 10953
Changes in the Concentrations of Free Fatty Acid,
Monoacylglycerol, and Diacylglycerol in the
Subcutaneous Fat of Iberian Ham during the
Dry-Curing Process
MÓNICA NARVÁEZ-RIVAS,† ISABEL M. VICARIO,‡ E. GRACIANI CONSTANTE,† AND
MANUEL LEÓN-CAMACHO*,†
Food Characterization and Quality Department, Instituto de la Grasa (C.S.I.C.), Seville, Spain, and
Department of Food Science and Nutrition, Faculty of Pharmacy, University of Seville, Seville, Spain
Changes in diacylglycerols, monoacylglycerols, and free fatty acid composition of subcutaneous fat
of six Iberian hams during the dry-cured process were investigated. In addition, an analytical method
for simultaneous quantification of diacylglycerols, monoacylglycerols, and free fatty acid by solid-
phase extraction–gas chromatography was developed. The different molecular species of free fatty
acids, monoacylglycerols, and diacylglycerols and 1,2- and 1,3-isomers of diacylglycerols have been
described for the first time in this type of sample. A logarithmic increase of the 1,3-diacylglycerol
profile throughout the processing time has been found, reaching a balance value of 62% around 500
days. The formation of diacylglycerol isomers takes place, although the 1,3-/1,2-diacylglycerol ratio
increases during the process to 1.65 due to isomerization of the 1,2-form toward the 1,3-form. The
profiles of monoacyl- and diacylglycerols and free fatty acids follow the same trend. The experimental
values of free fatty acid are greater than theoretical prediction, probably due to phospholipid and
monoacylglycerol hydrolysis.
KEYWORDS: Iberian dry-cured ham; subcutaneous fat; free fatty acids; monoacylglycerols; diacylglyc-
erols
INTRODUCTION
the main fatty acids of the acylglycerols, but they did not differ
between mono-, di-, and triacylglycerols. Larrea et al. (7) studied
the changes in total triacylglycerols, monoacylglycerol fraction,
and the total amount of diacylglycerols. For this fraction, an
increase was reported during the ripening process. Besides this
study, a few have been conducted on the diacylglycerol fraction
of the subcutaneous fat of raw and dry-cured hams. Delgado et
al. (8) reported a decrease in the diacylglycerol fraction during
the ripening period. Coutron-Gambotti and Gandemer (9)
investigated the changes in triacylglycerols, fatty acid, diacyl-
glycerols, and monoacylglycerols of subcutaneous adipose tissue
during ripening of Corsican dry-cured ham. However, none of
these studies evaluated the different isomeric forms of diacyl-
glycerols.
Dry-cured Iberian ham is one of the few traditional meat
products. Since 1998, this product has been included in the
European Union list of the Designation of Origin (1), which
has survived due to its highly demanded characteristics, with
long taste and rich nutty flavor (2, 3). These appreciated
organoleptic properties are due to a long-term maturing process
which requires between 12 and 24 months. Among the
biochemical reactions occurring during the ripening process,
those which affect the lipid fraction are of great importance
since the degradation products contribute to the overall orga-
noleptic properties of the final product (4).
The glyceridic fraction of Iberian dry-cured ham represents
a large amount of the total lipid content, and it is characterized
by a predominance of monounsaturated fatty acids, followed
by saturated fatty acids (5). Several studies have evaluated the
changes in the intramuscular and subcutaneous lipid fraction
during the ripening of Iberian dry-cured ham. Martín et al. (6)
observed that oleic (C18:1) and palmitic (C16:0) acids were
Diacylglycerols are generated by acidic and enzymatic
hydrolysis of triacylglycerols during transformation and
storage of oils and fats. During the storage of oil, the changes
in this fraction are due to an isomerization process. The 1,2-
diacylglycerols spontaneously change to 1,3-diacylglycerols
since it is a more thermodynamically stable molecular specie
(10). In olive oil, the 1,3-/1,2-diacylglycerol ratio has proved
to be useful for assessing the oil aging, conditions of storage,
and handling. Besides this, the kinetics of diacylglycerol
formation allows the estimation of the storage time of virgin
* To whom correspondence should be addressed. Telephone: ++34
954611550. Fax: ++34 954616790. E-mail: mleon@cica.es.
† Instituto de la Grasa.
‡ University of Seville.
10.1021/jf071886u CCC: $37.00
2007 American Chemical Society
Published on Web 11/27/2007

10954 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Narváez-Rivas et al.
Figure 1. Evolution of environmental temperature and relative humidity during ripening of Iberian dry-cured ham.
Figure 2. Gas chromatogram of the polar fraction from 0 to 35 min corresponding to subcutaneous fat of Iberian dry-cured ham. Labeled peaks are
identified as follows: 1, palmitic acid; 2, oleic acid, 3, 1-monoolein; 4, cholesterol; 5, 1,3-dimyristin (IS).
olive oil (11). Both the total diacylglycerol content and the
ratio between isomeric forms have also been used as
indicators of palm oil quality (12). The diacylglycerol
composition has been studied in the lipidic fraction of
different foods during its transformation or storage. A
decrease in diacylglycerol content has been observed in some
food processing, such as fermentation of pork sausage (13)
or during ice storage of sardine (Sardinella gibbosa) (14).
In other cases, a significant increase is observed, as in the
salting-fermentation process of certain fishes (15). Milk-
derived products also show a significantly higher content of
diacylglycerols than milk (16).
the quality of the fats and, consequently, of the treatment to
which they have been subjected. To our knowledge, a detailed
study of the changes occurring in this fraction during the
ripening process of dry-cured ham has not been undertaken.
The aim of this work was to investigate the changes
exclusively in free fatty acids and monoacyl- and diacyl-
glycerols of the subcutaneous fat and, hence, for a better
understanding of the Iberian ham dry-cured process. As a
previous step to achieve this objective, it was necessary to
improve the experimental fractionation procedure of the fat,
with a reliable analytical method that allows both recovery
and quantification of the free fatty acids, monoacylglycerols,
and diacylglycerols simultaneously.
Therefore, the quantitative study of the composition of
diacylglycerols appears to be of great interest to evaluate

Fatty Acid and Monoacyl/diacylglycerol in Fat of Iberian Ham
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10955
Figure 3. Gas chromatogram (expanded plot) from 20 to 30 min as taken from Figure 2. Labeled peaks are identified as follows: 1, 1,2-PP; 2, 1,2-PPo;
3, 1,2-MO; 4, 1,3-PP; 5, 1,3-PPo + 1,3-MO; 6, 1,2-PS; 7, 1,2-OP; 8, 1,2-OPo; 9, 1,2-LP; 10, 1,3-OP; 11, 1,3-OPo; 12, 1,3-LP; 13, 1,2-OS; 14, 1,2-OO;
15, 1,3-OS; 16, 1,2-OL; 17, 1,3-OO; 18, 1,2-LL; 19, 1,3-OL.
Table 1. Response Factors (RFs) Obtained for Representative
Monoacylglycerols and Fatty Acids Relative to 1,3-Dimyristin as Internal
Standard
Table 2. Repeatability Results of Diacylglycerols, Monoacylglycerol, and
Free Fatty Acid of Raw and Dry-Cured Samples Analyzed by SPE-GC
raw
dry-cured
RSD mean
RF meana
SD
RSD (%)
mean
RSD
peaka RTb
diacylglycerolsc
(n ) 6) SD (%) (n ) 6) SD (%)
1-monopalmitin
oleic acid
0.299
0.418
0.0060
0.0181
2.00
3.36
1
2
3
4
5
6
7
8
9
21.950 1,2-PPd
2.69 0.43 16.11 0.61 0.20 32.18
3.62 0.78 21.54 0.88 0.14 15.34
1.74 0.25 14.55 0.32 0.12 37.85
0.24 0.20 81.43 1.79 0.35 19.53
22.050 1,2-PPod
a Response factor (RF) calculated as the mean of four replicates.
22.101 1,2-MOd
22.240 1,3-PPd
22.347 1,3-PPO + 1,3-MOd 0.04 0.10 244.95 1.87 0.44 23.35
MATERIALS AND METHODS
23.559 1,2-PSd
23.711 1,2-OPd
23.985 1,2-OPod
24.050 1,2-PLd
4.28 0.47 11.04 0.63 0.41 64.99
28.63 1.96 6.85 10.86 0.84 7.76
4.34 0.44 10.05 4.38 0.86 19.72
4.88 0.44 9.02 0.00 0.00
Processing and Sampling of Hams. Six hams (between 8.8 and
9.1 kg) were obtained from three castrated Iberian pure f14-month-old
pig males, fattened extensively with acorns and pastured for 90 days
prior to slaughter, and were processed in an industry for 24 months.
The stages and the number of days from the beginning of the processing
were as follows: After the slaughter, hams were removed from the
carcasses after 24 h refrigerated storage at 1 °C. Then they were placed
in piles completely covered only with marine salt (they were not in
contact with each other) at low temperature (1.0 °C) and high relative
humidity (about 80%) for 9 days. After being washed to remove salt
from the surface, the hams were hung at 2.0 °C and a relative humidity
of 80% for 38 days and then at 4.0 °C and a relative humidity of 70%
to 153 days (postsalting). Then, they were taken to a dryer at
temperatures varying from 4 to 27 °C and a relative humidity ranging
from 70% to 43% for 147 days. Next, the hams were left to mature
during 430 days in a cellar at temperatures ranging from 10 to 27 °C
and 58–80% relative humidity. The environmental conditions (tem-
perature and relative humidity) were recorded continuously throughout
the whole period of maturing (Figure 1).
10 24.187 1,3-OPd
11 24.248 1,3-OPod
12 24.394 1,3-PLd
13 25.971 1,2-OSd
14 26.199 1,2-OOd
15 26.298 1,3-OSd
16 26.628 1,2-OLd
17 26.912 1,3-OOd
18 27.006 1,2-LLd
19 27.351 1,3-OLd
2.18 0.40 18.37 22.94 1.53 6.69
0.30 0.34 113.09 2.41 1.30 53.97
1.24 0.23 18.24 2.52 0.56 22.20
5.40 1.20 22.16 1.65 0.23 14.02
19.57 1.12 5.73 9.03 2.44 27.02
4.59 0.53 11.51 1.88 1.25 66.59
8.29 0.51 6.12 6.51 0.65 9.97
5.02 0.41 8.26 22.43 3.48 15.53
0.87 0.39 45.16 3.26 2.13 65.40
2.06 0.40 19.59 6.05 0.67 11.01
total diacylglycerolse 4.85 0.09 1.82 30.80 0.72 2.34
free fatty acide
monoacylglycerole
18.86 0.24 1.28 16.57 0.34 2.05
0.38 0.03 7.71 5.49 0.40 7.21
a See Figure 3. b RT ) retention time. c M ) myristic, P ) palmitic, Po )
palmitoleic, S ) stearic, O ) oleic, and L ) linoleic. d Expressed as %. e Expressed
as mg/100 g of fat.
A sample of each ham (about 1 g) was taken across of subcutaneous
adipose tissue covering the Biceps femoris muscle, without touching
it, about once a month since the animal was slaughtered (raw sample)
until the dry-cured process was finished (cured ham). Samples were
stored at -25 °C before analysis. The samples of subcutaneous fat
coming from both hams of each animal were mixed and melted in an
oven microwave (17) during 3 min at 360 W of power. Possible
isomerizations in the microwave extraction process of the samples are
not reported for subcutaneous fat of the Iberian pig. However, some
authors have made similar studies on human adipose tissue fat, using
the method of Folch as the extraction method under cold conditions
(18) and obtained mean values for 1,3-diacylglycerols around 20%,
which is greater than that found by us in raw samples (15.67%).
Therefore, it is assumed that the microwave extraction did not produce
any appreciable isomerization. The sample of fat was then immediately
filtered through Albet filter paper ref 1300 before analysis.
Materials and Reagents. All reagents were of analytical reagent
grade, unless otherwise specified. The standards oleic and palmitic acid,
1-palmitoyl-rac-glycerol, 1,3-dimyristin, 1,2-dimyristoyl-rac-glycerol,
1,3-dipalmitin, 1,2-dipalmitoyl-rac-glycerol, 1,3-distearin, 1,2-distearoyl-
rac-glycerol, 1,2-dioleoyl-rac-glycerol, 1,3-diolein, and trilinolein were
purchased from Sigma-Aldrich (St. Louis, MO). To obtain 1,2-dilinolein
a enzymatic hydrolysis of porcine pancreas was done using lipase

10956 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Narváez-Rivas et al.
Table 3. Effect of the Dry-Curing Process on the Free Fatty Acid,
Monoacylglycerol, and Diacylglycerol Profilea
Table 4. Levels of Experimental and Calculated Free Fatty Acids
(mmol/100 g of Fat) and Experimental/Calculated Ratio of Iberian Pig
Hams during the Dry-Curing Process in Daysa
%
mg/100 g of fat
raw dry-cured
free fatty acids
diacylglycerolsb
1,2-PP
1,2-PPo
1,2-MO
1,3-PP
1,3-PPO +
1,3-MO
1,2-PS
raw
dry-cured
3.11 ( 0.61c 0.37 ( 0.11 0.07 ( 0.02 0.11 ( 0.01
3.50 ( 0.02c 0.79 ( 0.21 0.08 ( 0.04 0.24 ( 0.06
1.42 ( 0.13 0.31 ( 0.07 0.03 ( 0.01 0.09 ( 0.03
0.00 ( 0.04c 2.15 ( 0.20 0.00 ( 0.00c 0.64 ( 0.02
0.00 ( 0.00b 2.14 ( 0.29 0.00 ( 0.00c 0.64 ( 0.03
palmitic
oleic
totalexptl
totalcalcd
days
(n ) 3)
(n ) 3)
(n ) 3)
(n ) 3)
ratio
0
0.01 ( 0.00 0.03 ( 0.01 0.04 ( 0.01 0.00 ( 0.00 7.93 ( 2.04
0.01 ( 0.00 0.04 ( 0.01 0.05 ( 0.02 0.01 ( 0.01 6.28 ( 1.68
0.01 ( 0.00 0.02 ( 0.00 0.03 ( 0.01 0.00 ( 0.00 7.95 ( 2.34
0.02 ( 0.01 0.04 ( 0.01 0.06 ( 0.02 0.00 ( 0.00 11.62 ( 2.84
21
51
83
5.24 ( 2.00 0.26 ( 0.15 0.06 ( 0.00 0.08 ( 0.03
25.70 ( 3.70c 11.15 ( 0.38 0.62 ( 0.21b 3.36 ( 0.45
4.74 ( 0.51 4.92 ( 0.43 0.11 ( 0.05a 1.49 ( 0.31
4.18 ( 1.03b 0.00 ( 0.00 0.08 ( 0.02a 0.00 ( 0.00
2.00 ( 1.52b 25.17 ( 1.76 0.06 ( 0.04c 7.52 ( 0.40
1.21 ( 1.88a 1.74 ( 0.47 0.01 ( 0.02 0.53 ( 0.20
0.00 ( 0.00a 2.20 ( 0.89 0.00 ( 0.00 0.68 ( 0.31
5.38 ( 0.13b 1.69 ( 0.14 0.12 ( 0.06b 0.51 ( 0.03
24.26 ( 1.42b 8.80 ( 0.85 0.54 ( 0.25a 2.66 ( 0.55
6.85 ( 3.07 1.74 ( 0.94 0.15 ( 0.09 0.54 ( 0.34
6.73 ( 0.35 7.40 ( 0.97 0.16 ( 0.07c 2.21 ( 0.20
4.99 ( 2.10b 21.39 ( 1.76 0.17 ( 0.11c 6.39 ( 0.27
0.06 ( 0.39 2.04 ( 1.02 0.01 ( 0.01 0.64 ( 0.37
0.62 ( 1.19b 5.73 ( 0.40 0.05 ( 0.03b 1.73 ( 0.31
84.33 ( 2.92c 37.74 ( 1.74 1.89 ( 0.73b 11.37 ( 1.81
15.67 ( 2.92c 62.26 ( 1.74 0.44 ( 0.26c 18.67 ( 1.73
2.33 ( 0.99c 30.04 ( 3.49
114 0.02 ( 0.00 0.05 ( 0.01 0.07 ( 0.01 0.01 ( 0.00 6.23 ( 0.56
153 0.04 ( 0.03 0.12 ( 0.09 0.17 ( 0.11 0.10 ( 0.07 1.74 ( 0.30
178 0.04 ( 0.03 0.11 ( 0.05 0.15 ( 0.08 0.10 ( 0.07 1.57 ( 0.52
212 0.03 ( 0.02 0.10 ( 0.06 0.13 ( 0.08 0.12 ( 0.08 1.17 ( 0.05
244 0.02 ( 0.00 0.09 ( 0.01 0.11 ( 0.01 0.09 ( 0.04 1.42 ( 0.53
275 0.02 ( 0.01 0.07 ( 0.02 0.09 ( 0.02 0.08 ( 0.03 1.26 ( 0.62
308 0.04 ( 0.02 0.09 ( 0.06 0.12 ( 0.08 0.11 ( 0.02 1.08 ( 0.49
338 0.04 ( 0.01 0.15 ( 0.02 0.19 ( 0.03 0.12 ( 0.02 1.58 ( 0.29
386 0.02 ( 0.02 0.12 ( 0.07 0.14 ( 0.09 0.16 ( 0.08 0.83 ( 0.15
427 0.06 ( 0.02 0.18 ( 0.03 0.24 ( 0.04 0.13 ( 0.02 1.78 ( 0.31
458 0.02 ( 0.00 0.07 ( 0.01 0.10 ( 0.00 0.03 ( 0.01 3.64 ( 1.25
499 0.03 ( 0.01 0.08 ( 0.01 0.10 ( 0.02 0.03 ( 0.01 2.92 ( 0.37
524 0.03 ( 0.01 0.08 ( 0.02 0.11 ( 0.03 0.03 ( 0.01 3.34 ( 0.27
567 0.03 ( 0.01 0.09 ( 0.01 0.12 ( 0.02 0.03 ( 0.01 3.69 ( 0.74
597 0.05 ( 0.00 0.12 ( 0.01 0.16 ( 0.01 0.04 ( 0.01 3.87 ( 0.48
626 0.04 ( 0.02 0.10 ( 0.01 0.14 ( 0.03 0.05 ( 0.01 3.42 ( 1.59
660 0.02 ( 0.01 0.10 ( 0.03 0.12 ( 0.04 0.09 ( 0.05 1.58 ( 1.04
687 0.01 ( 0.00 0.04 ( 0.01 0.04 ( 0.01 0.08 ( 0.01 0.55 ( 0.03
1,2-OP
1,2-OPo
1,2-PL
1,3-OP
1,3-OPo
1,3-PL
1,2-OS
1,2-OO
1,3-OS
1,2-OL
1,3-OO
1,2-LL
1,3-OL
total 1, 2-DGs
total 1, 3-DGs
total diacylglycerols
monoacylglycerols
palmitic acid
oleic acid
total free fatty acid
0.80 ( 0.47c 5.06 ( 2.00
4.15 ( 0.68b 2.66 ( 0.33
a Data are the means ( standard deviation (n ) 3).
14.54 ( 3.00 14.09 ( 2.49
18.69 ( 3.54 16.75 ( 2.76
solution (200 µL) (1.02 mg mL-1) and 500 µL of fat solution in
n-hexane (0.2 mg mL-1) were applied to the column, and the solvent
was pulled through, leaving the standard and the sample on the column.
A volume of 6 mL of hexane-methylene chloride-ethyl ether
(89:10:1 v/v) was applied to the column, and a first fraction was
collected. Subsequently, 4 mL of chloroform-methanol (2:1 v/v) was
applied to the column and a second fraction collected. This fraction
was evaporated to dryness in a rotary evaporator under reduced pressure.
The residue was treated with 200 µL of the silylating reagent and left
at room temperature for 15 min.
a Data are the means ( standard deviation (n ) 3). Different letters indicate
significant differences between raw and dry-cured data (a for p < 0.01, b for p <
0.001, and c for p < 0.0001). b M ) myristic, P ) palmitic, Po ) palmitoleic, S
) stearic, O ) oleic, and L ) linoleic.
One microliter of the silylated fraction was injected into the gas
chromatographic system. The chromatographic analysis was performed
using a Varian 3800 (Varian Co., Palo Alto, CA) equipped with a split/
splitless injector and a flame ionization detector; a fused silica capillary
DB-17HT column (30 m × 0.32 mm i.d., 0.15 µm film thickness) and
Varian 8100 automatic injector were used. Hydrogen was used as carrier
gas at a column constant flow of 4.0 mL min-1
.
Injector conditions were as follows: split/splitless mode, split ratio
1:40, and temperature 300 °C. The liner used for split mode injection
in the 1079 type injector was a Gooseneck-type liner (i.d. ) 3.4 mm)
which was deactivated and packed with a glass frit.
Figure 4. Evolution of total diacylglycerols (1,2 and 1,3) and monoacylg-
lycerol amount (mmol/100 g of fat) during the dry-curing process.
The oven conditions were as follows: The initial oven temperature
was kept at 100 °C and was then raised to 180 °C at a rate of 15.0 °C
min-1. Next it was raised to 296 °C at a rate of 7.0 °C min-1 and held
isothermally for 13.10 min. While the detector temperature was 360
according to a procedure previously described in the literature (19).
After a short reaction time, 1,2-dilinolein was isolated by thin-layer
chromatography in silica gel impregnated with boric acid to prevent
the isomerization (20). 1,3-Dilinolein was purchased from Nuchek-
Prep (Elysian, MN). The solid-phase extraction cartridge (3 mL), packed
with a diol-bonded phase, was purchased from Supelco (Bellefonte,
PA). Silylating reagent was prepared by adding 3 mL of hexamethyl-
disilazane and 1 mL of trimethylchlorosilane to 9 mL of anhydrous
pyridine.
Free Fatty Acid and Mono- and Diacylglycerol Analysis. Free
fatty acids and mono- and diacylglycerols form a part of the fat polar
fraction, and consequently, their analysis can be carried out following
a methodology for suitable separation and quantification (21).
Free fatty acid and mono- and diacylglycerol fractions were isolated
by solid-phase extraction (22, 23). The solid-phase extraction column
was placed in a vacuum elution apparatus and washed under vacuum
with 4 mL of hexane. The vacuum was released immediately after the
wash to prevent the column from becoming dry. 1,3-Dimyristin standard
°C, air and hydrogen with flow rates of 300 and 30 mL min-1
,
respectively, were used for the detector, which had an auxiliary flow
of 30 mL min-1 nitrogen.
A representative chromatogram report of subcutaneous fat and the
corresponding peak identification is shown in Figure 2. Figure 3 shows
more detail of the diacylglycerol profile shown in Figure 2.
For quantification of the diacylglycerols, 1,3-dimyristin was used
as internal standard. The internal standard was selected because no
components eluted were found in this region. For quantification of the
free fatty acids and monoacylglycerols, 1,3-dimyristin was used, too;
then the relative response factors were determined using solutions of
1.140 and 1.012 mg mL-1 pure standard of oleic acid and 1-mono-
palmitin, respectively, following the derivatization procedure described
above. Relative response factors determined for free fatty acids and
monoacylglycerols are listed in Table 1. An accurate determination of

Fatty Acid and Monoacyl/diacylglycerol in Fat of Iberian Ham
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10957
Figure 5. Plot of the relationship of total diacylglycerol (1,2 and 1,3) amount vs monoacylglycerols (mmol/100 g of fat) during the dry-curing process.
the response factors was necessary for obtaining a good quantification
of the different components.
In order to verify the absence of contamination by high molecular
weight compounds or decomposition products, we have injected
silylating reagent between injections of samples, observing that these
compounds do not elute.
The assignment of the chromatographic peaks was done by means
of standards of 1,2- and 1,3-isomers of dimyristin, diplamitin, diolein,
distearin, and dilinolein, which allowed to deduce the carbon number
of the components associated with each peak group, as well as the
difference between the retention times of the 1,2- and 1,3-isomers
(Table 2).
OP and 1,2-OO while in the cured sample are the isomeric forms
1,3-OP and 1,3-OO. Considering the total isomeric forms, the
1,2-diacylglycerols predominate in the raw sample while the
1,3-diacylglycerols are the most abundant in the dry-cured one.
No data are available in relation to the diacylglycerol profile of
raw or dry-cured hams. However, the same changes have been
detected in olive oil during the storage process. It has been
suggested that 1,2-diacylglycerols isomerize toward 1,3-dia-
cylglycerols, which are more thermodynamically stable molec-
ular species (10, 11).
Evolution of Free Fatty Acid, Monoacylglycerol, and
Diacylglycerol Fractions during the Dry-Cured Process. As
mentioned above, the lipids of the subcutaneous adipose tissue
of the Iberian ham were subjected to an intense chemical and
enzymatic hydrolytic process. Some authors suggest the pos-
sibility that the hydrolysis processes of subcutaneous fat lipids
are due to the activity of endogenous lipase, which has not yet
been studied (23). Concerning the present results, Figure 4
shows the evolution of the total diacylglycerols and monoa-
cylglycerols (expressed as mmol/100 g of fat). It can be observed
that during the postsalting stage (up to 150 days) the levels of
the polar acylglycerols remained at a low level, indicating that
they both have a slow formation rate (Figure 4). Although
previous research (9, 24) indicates that the highest lypolytic
enzyme activity occurs during this stage, it has also been
emphasized that there is a temperature dependence for the
enzyme activity. The low temperatures during the postsalting
period could be a limitation factor for the enzyme activity.
During the drying period there is an intense increase in the levels
of both acylglycerols, which could be related to an increase in
the enzyme activity due to a rise in temperature up to 15–25
°C, which also favors the chemical hydrolysis. This increase
remains during drying and the first 150 days of the cellar stage.
These results are in agreement with previous research (9). The
highest values of diacylglycerols, monoacylglycerols, and free
fatty acids were obtained during this period of time (Figure 4
and Table 4). From this point (450 days), it can be observed
that both fractions suffer a sharp decrease, but with an increasing
tendency toward the end of the process. This fact is in agreement
RESULTS AND DISCUSSION
Recovery and Repeatability of the Method. The recovery
has been previously described, because the SPE procedure for
isolating the polar glyceridic fraction was used by other authors
(22). This is complete (104%), and therefore, the separation
obtained with the SPE diol column is adequate.
The repeatability of the method was studied using a raw and
a dry-cured sample and a new SPE column for each replicate.
These results are shown in Table 2; they indicate a good
repeatability for the assay.
Free Fatty Acid, Monoacylglycerol, and Diacylglycerol
Profile in Raw and Cured Ham. Table 3 shows the mean
values of the diacylglycerols analyzed in the subcutaneous fat
as a relative percentage of total diacylglycerols and as mg/100
g of fat they correspond to in the initial and final stages of the
dry-curing process. Several interesting observations can be
deduced from these data. First, the diacylglycerol content in
the raw sample is significantly (p < 0.0001) lower than in the
dry-cured ham. This result is in agreement with previosly
published data (6, 8). They could be explained by the intense
hydrolysis of the triacylglycerol fraction during the dry-curing
process (9). At the same time, diacylglycerols are also affected
by hydrolysis to monoglyceride, as supported by the increase
in the monoacylglycerol content observed in the dry-cured ham
vs the raw ham. The only specie of monoglyceride found was
the monoolein. Second, the major diacylglycerols (considered
as a relative percentage of the total) in the raw sample are 1,2-

10958 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Narváez-Rivas et al.
Figure 6. Evolution of total diacylglycerols (1,2 and 1,3) as a relative percentage of total diacylglycerols vs time of dry-curing (in days).
with the disappearance of lipoprotein lipase activity described
in previous papers (9, 24) and a remaining chemical hydrolysis.
Figure 5 shows the concentration of the diacylglycerols versus
monoacylglycerols during the whole process. From this figure,
the relative formation rate between these species can be deduced.
A linear trend is observed which indicates that the formation
rate of diacylglycerols is almost double (1.87) that of mono-
acylglycerols.

Fatty Acid and Monoacyl/diacylglycerol in Fat of Iberian Ham
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10959
Figure 7. Effect of dry-curing time on the total 1,3-diacylglycerol contents, as a relative percentage of total diacylglycerols, as found in Iberian pig
hams.
Figure 8. Evolution of 1,3-diacylglycerol/1,2-diacylglycerol ratio as a function of dry-curing time (in days).
Table 4 shows the values in mmol/100 g of fat of palmitic
and oleic acids for each of the samplings carried out during the
whole dry-curing process. It can be observed that the free fatty
acids show a similar tendency as that described for diacylglyc-
erols and monoacylglycerols. The higher levels are observed
during the dryer stage, according to the intense hydrolytic
activity, as previously suggested.
However, the final acidity value is not significantly
different from the value obtained for the raw sample. The
decrease in free fatty acid content in the dry-cured ham
compared to the raw sample can be explained in part by the
preferred loss of free fatty acids with respect to mono-, di-,
and triacylglycerols by the sweated process observed during
curing that really happens. Also, Table 4 depicts the total
amount of free fatty acids formed during the ripening process
and the total calculated amount according to the equation
FFA (mmol/100 g) ) 2(monoacylglycerol) (mmol/100 g) +
diacylglycerol (mmol/100 g); this equation is only applied
to the enzymatic hydrolytic process. It can be observed that
the stoichiometric ratio of free fatty acids (experimental/
calculated) is greater than 1 in almost all of the measuring
points. This can be explained considering that, besides the
enzymatic hydrolysis of tri- and diacylglycerols, there is a
chemical hydrolysis of monoacylglycerols and other chemical
compounds, such as phospholipids, cholesterol esters, and
tocopherol esters. However, the phospholipid level in sub-
cutaneous fat is very low, about 0.46 g/100 g of fat (5, 25),
and their hydrolysis is more relevant in the intramuscular

10960 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Narváez-Rivas et al.
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fat (7). The high experimental values found here could also
be due to the hydrolysis of monoacylglycerols, which could
be important in the last stage (450 days) of curing, where
both diacylglycerols and monoglyceride decrease.
Isomerization of Diacylglycerols. As mentioned before, the
diacylglycerols formed by the hydrolysis of triacylglycerols are
affected by an isomerization process. Figure 6 shows the
evolution (in relative percentage) of the diacylglycerols during
the whole process (from 0 to 687 days). The two isomeric forms,
the 1,2- and 1,3-diacylglycerols, are represented in the same
figure. In general, it can be observed that the percentages of
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Figure 7 shows the evolution of the relative percentage of
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rithmic increase can be observed up to a value of approximately
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isomerization process is rather quick, reaching an equilibrium
in a short time.
Figure 8 represents the molar relationships between 1,3-
diacylglycerols and 1,2-diacylglycerols during the process.
It can be observed that there is an increase from 0.20 (initial
value) to 1.65 at the end of the process. This increase in the
relative formation rate cannot be considered linear along the
whole process as found in olive fruit (10). The higher increase
is observed during postsalting and dryer stages (about 300
days), and then a moderate increase occurs during the cellar
period. At about 450 days, this ratio reaches a possible
equilibrium. This fact can be explained, in agreement with
the previous reports found in the literature, which reports
besides the 1,3-diacylglycerol formation from triacylglycerols
hydrolysis there is a isomerization from 1,2-diacylglycerols
to 1,3-diacylglycerols. This process is an acid-catalyzed
reaction by the free fatty acids liberated from triacylglycerol
hydrolysis (10, 11).
In conclusion, from the results above mentioned, it is deduced
that the analytical method applied is a good alternative to
quantify this fraction. Also, the dry-curing of the Iberian ham
process is characterized by a least three simultaneous reactions:
(a) triacylglycerol hydrolysis to form diacylglycerols; (b)
diacylglycerol isomerization to give an important increase of
1,3-diacylglycerols, and (c) diacylglycerol hydrolysis. However,
other competitive reactions such as oxidation of acylglycerols
and fatty acids and hydrolysis of monoacylglycerols cannot be
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Received for review June 26, 2007. Revised manuscript received
October 8, 2007. Accepted October 10, 2007.
JF071886U