JFS: Food Chemistry and Toxicology

Identification of Small Peptides

Generated in Spanish Dry-cured Ham
M.A. SENTANDREU, S. STOEVA, M.C. ARISTOY, K. LAIB, W. VOELTER, AND F. TOLDRÁ

ABSTRACT: A water-soluble extract of dry-cured ham was fractionated by gel filtration chromatography. Col-
lected fractions (below 1200 Da) were sensory-analyzed and subjected to amino acid analysis with and without
Food Chemistry and Toxicology

previous acid hydrolysis, in order to determine the amino acid and peptide distribution. Those fractions with the
highest peptide amount were further separated by reverse-phase and cation-exchange high-performance liquid
chromatography in order to isolate the peptides and sequence them. The results demonstrated the presence of
small peptides, mainly dipeptides. The combination of these peptides and free amino acids may contribute to the
characteristic taste in the savory fractions of dry-cured ham.
Keywords: peptides, amino acids, dry-cured ham, taste, flavor

Introduction mass spectrometry and amino acid sequencing, Stoeva and others

C URED PRODUCTS REPRESENT AN IMPORTANT PART OF PROCESSED

meat products in Spain and, in general, in the Mediterranean
area. One of the main cured products is dry-cured ham, which ex-
ists as different types in the Mediterranean countries, such as
Spanish Iberian and Serrano hams, Italian Parma and San Daniele
prosciuttos, and French Bayonne ham. Each type has its character-
istic color, texture, and flavor depending mainly on the different
raw materials and ripening conditions (Toldrá and Flores 1998).
In Spain, more than 30 million dry-cured hams are produced
every year (Toldrá 2002). Most are consumed in Spain, but during
the last decade sales of this typical product have gradually in-
creased outside the Spanish market. Actually, the dry-cured ham
industry is facing important challenges, such as demands for high-
er-quality products with adequate texture and good flavor. Many
biochemical mechanisms, taking place during the ripening period,
are responsible for the characteristic flavor. Endogenous muscle
enzymes, especially proteolytic and lipolytic ones, are mainly re-
sponsible for the postmortem changes occurring during the curing
period (Toldrá and others 1997).
In the present work, we focused on the proteolytic process. In
long-term-cured products, such as dry-cured ham, an intense pro-
teolysis is observed for sarcoplasmic and myofibrilar proteins (Bel-
lati and others 1985; Toldrá and others 1993), giving rise to an im-
portant collection of free amino acids (Córdoba and others 1994;
Toldrá and others 1997); small peptides of less than 1200 Da (Aris-
toy and Toldrá 1995; Flores and others 1997), or even less than 320
Da (Ruiz and others 1999), can directly contribute to flavor or indi-
rectly contribute as precursors of other flavor compounds. So the
presence of free lysine and tyrosine, for example, has been related
to flavor development in Parma ham, whereas glutamic acid has
been related to salty taste (Careri and others 1993). With respect to
peptides, some authors have identified the sequence of certain
peptides in meat, like the octapeptide Lys-Gly-Asp-Glu-Glu-Ser-
Leu-Ala (Yamasaki and Maekawa 1978), known as BMP (“beefy
meaty peptide”). This peptide has been reported to have a brothy/
umami taste, similar to that of monosodium glutamate. Nakai and
others (1995) identified in beef meat a peptide with 15 amino acids
(Ala-Pro-Pro-Pro-Pro-Ala-Glu-Val-Pro-Glu-Val-His-Glu-Glu-Val)
having significant homology with respect to troponin T and sug-
gesting its possible origin from troponin T degradation. Using
(2000) found in soluble extracts of bovine longissimus dorsi muscle
proteolytic fragments from glyceraldehyde-3-phosphate dehydro-
genase (Lys-Val-Val-Lys-Gln-Ala-Ser-Glu-Gly-Pro-Leu-Lys), tropo-
nin T (Ala-Pro-Pro-Pro-Pro-Ala-Glu-Val-Pro-Glu-Val-His-Glu-Glu-
Val-His), and creatine kinase (Asp-Pro-Ile-Ile-Gln-Asp-Arg-
His-Glu-Gly-Phe-Lys-Pro-Thr-Asp-Lys-His-Lys-Thr-Asp-Leu-Asn-
His-Glu-Asn-Leu-Lys-Gly-Gly-Asp-Asp-Leu-Asp-Pro-Asn-Tyr-Val-
Leu-Ser). Although there is evidence of the presence of peptides at
the end of the curing period (Rodríguez and others 1995), the iden-
tification of specific peptide sequences in dry-cured ham has not
been established yet. Thus, the main objective of the present work
was the identification of small peptide sequences that are present
in tasty water-soluble fractions of Spanish dry-cured ham.
Materials and methods
Materials
Serrano dry-cured ham (8 mo curing) was obtained from OMSA
Alimentación S.A. (Valencia, Spain). Sephadex G-25 and the glass
column (2.6 cm ´ 55 cm) were purchased from Pharmacia LKB
(Uppsala, Sweden). The Licrospher 60 RP-select B reverse-phase
column, 5µm (25 mm ´ 4 mm), was from Merck (Darmstadt, Ger-
many), and the Spherisorb S 10 SCX cation exchange column (10
mm ´ 250 mm) was from Waters Corp (Milford, Mass., U.S.A.). Ami-
no acid calibration standards were from Eppendorf (Hamburg, Ger-
many).
Molecular weight fractionation of the ham extract
The dry-cured ham extraction was performed in the same way as
described by Flores and others (1997), homogenizing 10 g of previ-
ously ground ham with 50 mL of 0.1 N HCl in a stomacher homog-
enizer (Seward Laboratory). After centrifugation (10,000 ´ g 20 min
at 4 °C), the supernatant of the homogenate was collected and
deproteinized by adding 3 volumes of ethanol. After 20 h at 4 °C, the
mixture was centrifuged at 10,000 ´ g for 20 min, and the superna-
tant was dried in speed-vacuum. This extract was redissolved in 10
mL of 0.01 N HCl, filtered through a 0.45-µm membrane filter (Mil-
lipore Co., Bedford, Mass., U.S.A.) and applied on a Sephadex G-25
gel filtration column. The elution was made with 0.01 N HCl at flow
rate of 30 mL/h and 4 °C. The first 103 mL was discarded, and then

64 JOURNAL OF FOOD SCIENCE—Vol. 68, Nr. 1, 2003 © 2003 Institute of Food Technologists
Further reproduction prohibited without permission
Peptide composition of Spanish dry-cured ham . . .

Table 1—Sensory evaluation of selected gel filtration frac- matics associated with cooked meat) and ham (aromatics associat-
tions from the dry-cured ham extract ed with dry-cured ham).
Fraction Elution
nr volume (mL) Sensory perception Amino acid analysis
24 223 No taste The amino acid analysis was carried out with an Eppendorf
25 228 No taste (Hamburg, Germany), amino acid analyzer following a post-column
26 233 No taste derivatization with ninhydrin and monitoring the absorbance at
27 238 Brothy 570 nm for all the amino acids except for Pro and Hydroxy-Pro,
28 243 Sour Brothy Bitter which were monitored at 440 nm. A standard amino acid calibration
29 248 Sour Brothy Bitter mixture was run in order to correlate the response of each amino
30 253 Sour Brothy Bitter acid with its concentration. Two amino acid analyses were done per
31 258 Sour Brothy Bitter fraction, 1 to determine the free amino acid content and the other
32 263 Sour Brothy Bitter 1 to determine the total amino acid content after acid hydrolysis

Food Chemistry and Toxicology

33 268 Ham Brothy with 6 N HCl at 110 °C for 24 h. The amino acids originating from
34 273 Ham Brothy peptides were determined by calculating the difference between
35 278 Ham Brothy Salty the latter and the former.
36 283 Ham Brothy Salty
37 288 Ham Brothy Salty Separation of peptides by reverse-phase and cation-
38 293 Ham Brothy Salty exchange HPLC
39 298 Brothy Salty To isolate the peptides, gel filtration fractions were injected on
40 303 Brothy Bitter Salty both a Licrospher 60 RP-select B and a Spherisorb S 10 SCX column.
41 308 Brothy Bitter Salty For the first separation, reverse-phase high-performance liquid
42 313 Brothy Bitter chromatography (RP-HPLC), a gradient was used, starting with a
43 318 No taste 15-min isocratic step with buffer A (0.1% trifluoroacetic acid in wa-
44 323 No taste ter) and followed by a linear gradient to 53% buffer B (0.08% triflu-
oroacetic acid in 20% water:80% acetonitrile) in 60 min. The flow
rate was 1 mL/min. The second separation, cation-exchange HPLC
5-mL fractions were collected and monitored by ultra-violet (UV) (CE-HPLC), was carried out by an initial isocratic step for 10 min
absorption at 214, 225, 254, and 280 nm. Fractions eluting from 223 with 10 mM phosphate buffer, pH 3.0, followed by a linear gradient
to 323 mL (fractions 24 to 44) were dried in speed-vacuum. Then, to 500 mM NaCl in the same buffer for 30 min. The flow rate was 1
each fraction was redissolved in bidistilled water, frozen, and lyo- mL/min. Both separations were monitored at ␭= 210 nm. Promi-
philized, constituting the starting material for the amino acid and nent peaks were collected, directly frozen, lyophilized, and further
peptide analyses. redissolved in 50 mL of 0.1% trifluoroacetic acid in water for the
analyses of the amino acid composition and sequence.
Sensory analysis
Five untrained panelists took part in the sensory evaluation of Determination of the N-terminal amino acid
the different fractions from the gel filtration chromatography. The sequence of peptides
4 basic tastes were assayed: sour (taste on the tongue associated The N-terminal amino acid sequences of the collected fractions
with citric acid), salty (taste on the tongue associated with sodium were determined by automated Edman degradation, using an
and chloride ions), bitter (taste on the tongue associated with caf- Applied Biosystems (Weiterstadt, Germany) pulsed liquid se-
feine), and sweet (taste on the tongue associated with sucrose). Ad- quencer, model 473 A. Each re-dissolved fraction (5 to 25 mL) was
ditionally, the descriptors used for ham flavor were brothy (aro- applied to the cartridge filter, previously treated with polybrene.

Results and Discussion

T HE SIZE - EXCLUSION CHROMATOGRAM OF A DRY- CURED HAM

Figure 1—Gel filtration chromatography of a deproteinized
dry-cured ham. For the correspondence between fraction
number and elution volume see Table 1.
extract (Figure 1) followed a similar profile to what Aristoy and
Toldrá (1995) had reported previously. The range of fractions hav-
ing the most interesting flavor characteristics was selected, and
fractions 24 to 44 (223 to 323 mL elution volume) were sensory-test-
ed (Table 1). Fractions 28 to 29 had a high brothy flavor, although
the maximum intensity was perceived in fractions 30 to 39. The
typical ham flavor was only detected in fractions 33 to 38 (Table 1).
A sour taste above the background, due to the eluent of the gel fil-
tration chromatography, was noticed mainly in fractions 28 to 32,
just before the detection of ham flavor. Salty taste appeared in frac-
tion 35 and corresponded with the elution of NaCl from the gel fil-
tration column. The rapid decrease in brothy perception coincided
with the detection of bitter taste from fraction 40 and onwards,
which is related to the elution of phenylalanine (Table 2).
The composition in free amino acids of the nonhydrolyzed sam-
ples is shown in Table 2, whereas Table 3 shows the results ob-
tained for samples with previous acid hydrolysis. The difference in
the concentration values for each amino acid between the hydro-

Vol. 68, Nr. 1, 2003—JOURNAL OF FOOD SCIENCE 65

 Peptide composition of Spanish dry-cured ham . . .

Table 2—Free amino acid analysis, expressed as nmol/100 mg sample, of the savory fractions from the gel filtration
separation of a dry-cured ham extract
Fraction number
24 25 26 27 28-29* 30 31 32 33-34* 35-36* 37 38 39 40 41 42 43 44
Amino acid
Lysine 1.01 1.24 1.13 1.08 81.58 115.48 133.87 78.47 35.62 17.99 11.27 7.20 3.50 1.62 0.81 0.35 0.00 0.39
Histidine 0.00 0.62 0.71 0.00 0.40 2.66 22.92 30.73 13.67 8.04 6.32 5.25 4.01 3.26 2.84 1.47 0.51 0.42
b-Alanine 0.00 0.82 2.05 6.12 7.56 2.67 2.23 2.39 1.90 1.00 0.66 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Phenylalanine 0.00 0.00 0.00 0.00 0.86 0.73 0.00 0.00 0.00 0.00 0.00 2.21 10.16 22.69 68.79 88.64 69.50 114.28
Alanine 0.00 0.00 0.00 0.00 1.65 18.08 72.85 113.75 90.72 57.91 43.05 33.07 22.67 15.65 11.59 5.48 2.31 2.67
Valine 0.00 0.00 0.00 1.15 22.23 63.94 100.55 88.88 49.78 26.77 18.15 13.31 8.22 4.62 2.96 1.31 0.67 0.87
Leucine 0.00 0.00 0.00 2.38 10.82 39.53 99.86 99.73 62.33 28.33 25.28 21.52 15.10 7.85 6.27 3.04 1.26 1.51
Glutamic acid 0.00 0.00 0.00 0.00 9.42 51.18 77.81 91.39 74.63 38.70 25.79 19.71 13.75 10.20 9.12 6.73 4.73 8.04
Proline 0.00 0.00 0.00 0.00 1.10 34.35 64.42 71.41 43.80 23.64 15.12 11.07 7.01 4.00 2.36 1.00 0.33 0.65
Isoleucine 0.00 0.00 0.00 0.91 9.46 30.95 65.10 60.76 35.70 16.88 13.78 11.36 7.56 3.94 2.86 1.32 0.61 0.79
Food Chemistry and Toxicology

Glycine 0.00 0.00 0.00 0.00 1.49 0.00 0.00 5.18 51.61 55.96 42.36 37.36 31.37 29.17 30.24 20.40 10.92 13.39
Aspartic acid 0.00 0.00 0.00 0.00 3.57 14.79 19.88 27.58 55.04 29.11 19.27 15.25 11.42 9.23 8.59 6.10 5.04 9.00
Threonine 0.00 0.46 0.72 0.99 3.92 7.10 20.41 45.41 52.32 28.84 20.80 16.69 12.44 9.76 8.45 4.65 2.23 2.44
Serine 0.00 1.15 2.56 2.50 1.06 0.85 1.26 9.65 47.45 39.96 28.65 24.79 20.57 18.77 19.56 13.15 6.98 8.69
Arginine 1.40 1.98 2.06 1.62 0.00 0.00 0.00 9.63 14.10 23.79 21.56 18.21 16.31 17.55 22.98 20.49 13.23 15.81
3-Methyl-histidine 0.00 0.00 0.45 2.97 0.00 1.13 1.47 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Ornithine 0.59 1.67 3.30 5.33 4.13 12.64 16.55 9.62 3.92 2.09 1.42 1.00 0.58 0.33 0.00 0.00 0.00 0.00
Asparagine 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.39 14.93 10.99 9.86 9.08 9.67 12.13 10.26 6.58 11.46
Methionine 0.00 0.00 0.50 3.63 2.10 0.00 0.00 1.96 12.98 13.31 10.45 9.99 8.76 6.17 8.33 6.55 3.84 5.35
1-Methyl-histidine 5.78 3.52 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Glutamine 0.00 0.00 0.00 0.95 11.28 4.29 2.44 1.74 2.86 1.84 1.24 1.26 1.16 1.26 1.50 1.11 0.00 0.81
Cystine 0.00 0.00 0.39 1.45 0.82 2.11 1.26 0.83 0.33 0.29 0.00 0.00 0.00 0.00 0.00 0.00 0.24 0.35
Tyrosine 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.41
Tryptophan 4.54 3.42 0.00 0.00 0.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Hydroxyproline 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Total amino
acids 13.32 14.88 13.87 31.08 174.2 402.48 702.88 749.11 654.15 429.38 316.16 259.11 203.67 175.74 219.38 192.05 128.98 197.33
Dipeptide
Carnosine 0.84 1.43 6.01 4.04 115.07 169.85 136.95 75.66 32.17 15.69 9.99 6.60 3.59 1.81 0.99 0.42 0.00 0.00
*These fractions were pooled together.

Table 3—Amino acid analysis, expressed as nmol/100 mg sample, after acid hydrolysis of the savory fractions from the
gel filtration separation of a dry-cured ham extract
Fraction number
24 25 26 27 28-29* 30 31 32 33-34* 35-36* 37 38 39 40 41 42 43 44
Amino acid
Lysine 80.20 66.99 61.22 82.70 101.57 144.50 153.29 80.47 35.24 17.99 11.76 7.20 3.50 1.62 0.81 0.35 0.35 0.43
Histidine 17.23 25.05 32.16 49.02 103.22 128.80 146.85 88.98 40.63 16.95 16.00 9.13 6.47 3.40 2.84 1.47 0.98 0.70
ß-Alanine 13.95 20.23 25.81 63.49 121.56 130.62 128.22 56.62 23.85 10.28 9.06 5.40 3.25 1.48 0.98 0.61 0.69 0.83
Phenylalanine 0.80 0.00 0.00 0.00 0.86 0.73 0.00 0.00 0.38 0.55 0.88 2.64 10.12 25.71 68.79 88.64 89.67 114.28
Alanine 5.90 6.88 8.01 12.92 17.11 19.71 80.42 113.75 90.72 57.91 43.70 33.07 22.67 15.65 11.59 5.48 3.20 2.67
Valine 5.54 7.16 12.14 21.82 41.06 63.94 100.80 88.88 49.78 26.77 19.20 13.31 8.22 4.62 2.96 1.31 0.85 0.87
Leucine 3.38 3.94 5.50 11.20 24.58 39.53 99.86 99.73 69.03 30.56 25.28 21.52 15.10 7.85 6.27 3.04 1.77 1.51
Glutamic acid 12.63 12.39 14.96 42.82 80.17 65.33 93.77 91.39 75.99 38.96 36.04 25.23 19.30 11.71 10.40 8.39 11.46 14.08
Proline 77.24 52.37 39.64 48.61 30.64 34.35 70.81 71.41 43.80 23.64 15.04 11.07 7.01 4.00 2.36 1.00 0.78 0.69
Isoleucine 2.76 2.97 4.43 8.97 16.21 30.95 65.10 60.76 39.37 16.88 13.78 11.36 7.56 3.94 2.86 1.32 0.92 0.73
Glycine 13.22 13.72 14.82 20.90 18.23 8.78 8.73 12.72 53.30 55.96 45.99 37.36 31.37 29.17 30.24 20.40 15.08 13.39
Aspartic acid 18.34 18.90 24.27 44.85 57.61 31.41 35.00 29.70 55.04 29.16 28.60 19.83 17.12 11.74 12.43 10.48 12.81 14.38
Threonine 9.26 9.47 13.08 14.81 13.38 7.79 20.95 45.41 52.32 28.84 21.86 16.69 14.27 9.76 8.45 5.93 5.15 4.10
Serine 5.16 6.53 10.00 14.98 12.45 4.74 4.42 9.65 47.45 39.96 28.65 24.79 20.57 18.77 19.56 13.15 8.37 8.69
Arginine 2.05 1.98 2.06 1.91 1.67 1.27 3.18 10.16 14.10 23.79 21.56 18.21 16.31 17.55 22.98 20.49 15.14 15.81
3-Methyl-histidine 2.35 4.18 9.30 28.01 21.77 1.13 1.47 0.00 1.67 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Ornithine 0.85 0.71 1.07 3.34 4.88 10.27 19.20 11.23 6.07 3.69 3.14 2.25 1.71 1.11 1.23 0.96 1.19 1.21
Asparagine 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Methionine 1.11 1.43 2.89 7.35 2.62 1.03 1.53 0.58 6.11 6.12 6.89 4.95 1.36 2.72 0.74 0.77 0.00 0.53
1-Methyl-histidine 5.78 3.52 4.01 9.73 11.79 4.17 3.32 2.17 3.40 1.02 0.86 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Glutamine 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Cystine 1.30 1.38 2.84 4.14 3.88 2.11 1.26 1.29 3.83 1.31 1.18 0.31 0.31 0.00 0.00 0.00 0.00 0.00
Tyrosine 0.64 0.82 0.68 0.63 0.00 0.00 0.00 0.83 1.42 0.39 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Hydroxyproline 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Total amino
acids 279.6 260.6 288.8 492.2 685.5 731.1 1038.1 875.7 713.5 430.7 349.4 264.3 206.2 170.8 205.4 183.7 168.4 194.9
Dipeptide
Carnosine 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
*These fractions were pooled together.

66 JOURNAL OF FOOD SCIENCE—Vol. 68, Nr. 1, 2003

Peptide composition of Spanish dry-cured ham . . .

Table 4—Principal sequencing results obtained from the fractions 33 to 38, in which ham flavor was detected (Table 1). It can
peptide fractions obtained by gel filtration chromatogra- be assumed that these amino acids contribute in some way to the
phy of a Spanish dry-cured ham extract
typical flavor generation in dry-cured ham. Much bigger amounts
Sequence/ of Lys, His, b-Ala, Glu, and Asp were found in the hydrolyzed sam-
Peaks composition ples. According to Figure 2, the differences for Lys, Asp, and Glu were
Fraction also mainly observed for fractions 24 to 31. The contents of b-Ala
28 to 29: Reverse phase Peak 1: Free amino acids and His differed a great deal between hydrolyzed and nonhydro-
Peak 2: Val-Glu
lyzed samples. Small amounts of free His and b-Ala were detected
Peak 3: Free amino acids
Peak 4: Ile-Val ; Leu-Glu (Table 2), although large quantities of these amino acids originate
Peak 5: Ile-Asp ; Ala-Met ; from peptides (Table 3), probably from carnosine (␤-Ala-His), bale-
Gly-Glu nine (␤-Ala-3-methl-His), and anserine (b-Ala-1-methyl-His),
Peak 6: Glu, Val, Asp integrating dipeptides that are naturally present in dry-cured ham in high
a tripeptide
amounts. In fact, the distribution of carnosine along the gel-filtra-

Food Chemistry and Toxicology

Peak 7: Asp as free amino acid
Peak 10: Asp-Ala-Gly-Asp
Peak 11: Glu, Val, Leu integrating
a tripeptide
Peak 12: Glu-Arg ; Pro-Leu
Peak 13: Gly-Ser
Fraction 31: Reverse phase
¯ balenine content in dry-cured ham is much lower compared to that
Cation exchange Peak 9: Asp-Val ; Ser-Lys
lyzed and nonhydrolyzed samples was calculated for each of the
analyzed fractions and gave an idea of the amino acid content dis-
tribution originating from peptides (Figure 2).
Remarkable aspects can be observed in the distribution of indi-
vidual amino acids along the gel filtration fractions from dry-cured
ham extract. Phe, Ala, and Val, some of the amino acids appearing
in higher concentrations, gave the same distribution profile with
similar amounts, both in the hydrolyzed and nonhydrolyzed sam-
ples (Table 2 and 3), indicating that these amino acids are mainly
present as such in dry-cured ham. A similar distribution pattern was
observed for Leu and Ile when nonhydrolyzed and hydrolyzed sam-
ples were compared. It should be noted that incomplete acid hy-
drolysis of the peptide bonds containing Val and Ile (Blackburn
1978) could cause the underestimation of these amino acids in the
hydrolyzed samples. Pro, Thr, Gly, and Ser contents were slightly
higher in the hydrolyzed preparations of the fractions around 28 to
29, which is in accordance with the predominance of the total amino
acid content originating from peptides (Figure 2). In addition, max-
imal concentrations for Gly, Ser, and Met (Table 2) were found in
tion fractions in the nonhydrolyzed samples (fractions 28 to 31)
coincided with its disappearance and the apparition of large
amounts of b-Ala and free His in the same samples after hydroly-
ses. 1-methyl-His and 3-methyl-His also followed this pattern, al-
though they were found in lower amounts. In fact, the anserine and
of carnosine (Table 2).
The distribution of Met and Arg was quite irregular, possibly due
to interactions with the Sephadex matrix. In the case of Met, lower
amounts were found in some hydrolyzed samples, indicating par-
tial or total destruction during acid hydrolysis. Asn and Gln gave
also an irregular distribution, being only present in the nonhydro-
lyzed samples, as they are transformed to Asp and Glu, respective-
ly, during acid hydrolysis. Tyr eluted after fraction 44, coinciding
with the highest absorbance peak at 280 nm (Figure 1). In the hy-
drolyzed samples, Tyr appeared in very low amounts (Table 3).
The distribution of the total amino acid content along fractions
24 to 44 is shown in Figure 2. The fractions with the highest concen-
trations in free amino acids were 30 to 37 (253 to 288 mL), being
maximal in fraction 32 (263 mL). The distribution of the total ami-
no acid content ran in parallel with the distribution of free amino
acids from fraction 35 onwards, indicating an almost negligible
presence of peptides. Only small amounts of amino acids originat-
ing from peptides were observed in 2 fractions, mainly due to the

Figure 2—Distribution of the amino acid contents along Figure 3—Reverse-phase chromatography of fraction 28
fractions 24 to 44 (223 to 323 mL) resulting from the gel- to 29 resulting from the gel filtration separation of a dry-
filtration separation of a dry-cured ham extract cured ham extract

Vol. 68, Nr. 1, 2003—JOURNAL OF FOOD SCIENCE 67

 Peptide composition of Spanish dry-cured ham . . .
content in Glu, Asp, Gly, and Val (fraction 37) and Asp, Ser, Glu, Gly,
Phe, and Arg (fraction 43). On the contrary, no free amino acids
were found before fractions 28 to 29. The content of amino acids
originating from peptides concentrated on fractions 27 to 31 (238 to
258 mL), reaching maximum value in fractions 28 to 29 (243 to 248
mL). This maximum coincided with the first absorbance peak at
254 and 280 nm, as shown in Figure 1.
In addition to the amino acid analysis, the same fractions were
separated on reverse-phase HPLC. The results confirmed, as sug-
gested, that, indeed, fractions 28 to 29, containing approximately
9.1% of soluble non-protein nitrogen, were the most promising
ones for the isolation of peptides (Figure 3). In the chromatograms
of subsequent fractions, the intensity of the peaks shown in Figure
Food Chemistry and Toxicology
3 decreased drastically (data not shown). Only a new and intense
peak corresponding to Phe appeared (Tables 2 and 3). The main
sequencing results from the collected peak fractions are summa-
rized in Table 4. From RP-HPLC peaks 6, 8, 9, and 11 (fractions 28 to
29), it was not possible to get reliable sequencing data by Edman
degradation. However, the amino acid analysis after hydrolysis of
peak 6 revealed the presence of Asp, Glu, and Val, but not in the
nonhydrolyzed one. A similar result was obtained from peak 11; after
hydrolysis Glu, Val, and Leu might be determined. So, the presence
of a N-terminally modified tripeptide is suggested in both cases.
The injection (not retained on the column) peak of fraction 31, con-
taining 13.8% of soluble non-protein nitrogen, was collected after
RP-HPLC and rechromatographed on a cation exchange-column.
Several fractions were obtained, most of them containing free ami-
no acids, as expected. Only in 1 of the fractions the peptides Asp-
Val and Ser-Lys were found (Table 4).
Conclusions
T HE MAIN OBJECTIVE OF THIS WORK WAS TO DEMONSTRATE THE
presence of peptides in dry-cured ham at the end of the ripen-
ing period. The results demonstrate the existence of specific pep-
tides, mainly dipeptides, in dry-cured ham. The occurrence of
dipeptides could be attributed to the enzymatic action of dipepti-
dylpeptidases (DPP), a group of 4 muscle enzymes (DPP I, II, III,
and IV ) directly involved in the release of dipeptides from the
amino termini of longer peptides and protein fragments (Sentan-
dreu and Toldrá 1998). Peptidyl dipeptidases and cathepsin B can
also release dipeptides, but from the carboxy- termini of peptides
(Barrett and others 1998). DPPs have been purified recently and
characterized from porcine skeletal muscle, and their participation
during the processing of dry-cured ham being feasible, especially
DPP I and DPP IV (Sentandreu and Toldrá 2001). Apart from dipep-
tides, considerable amounts of free amino acids were determined
(Figure 2), as previously reported (Aristoy and Toldrá 1991; Toldrá
and others 1992; Toldrá and others 1997). The observed presence
of free amino acids and small peptides in dry-cured ham, produced
as a result of an intense proteolysis during ripening, is a process
closely related to the biological significance of the proteolytic chain
in the living cell. Coffey and De Duve (1968) observed that the con-
certed action of peptidases from rat liver was able, at acidic pH, to
degrade an acid-denatured globin substrate to small fragments,
42% of them being free amino acids and 30% dipeptides. This accu-
mulation of dipeptides was later attributed to the action of dipep-
tidylpeptidases (McDonald and Schwabe 1977). So, the whole se-
ries of peptidases (endo- and exopeptidases) represents an
efficient enzymatic system being able to degrade proteins, main-
ly down to free amino acids and dipeptides.
Considering the studies carried out by other authors in relation
to individual taste imparted by amino acids and some peptides, it
should be possible to make some predictions for the peptides col-
68 JOURNAL OF FOOD SCIENCE—Vol. 68, Nr. 1, 2003
lected in Table 4. So, it has been observed that almost all peptides
containing hydrophobic amino acids, L-Phe, L-Tyr, L-Trp, L-Leu, L-
Val, and L-Ile, impart bitter taste, as do their respective free forms.
According to Ney (1971), it is possible to predict whether a certain
peptide structure can provide a bitter taste or not, depending on
its hydrophobic value. This value is obtained by adding the hydro-
phobic values of each individual amino acid constituting the pep-
tide and dividing the sum by the number of amino acid residues.
This rule can be applied to almost all peptides. In our case, the
peptides that should provide bitter taste would be Ile-Val, Leu-Glu,
Ile-Asp, and Pro-Leu (Table 4). This prediction agrees with the bit-
ter taste found for fractions 28 to 29 and near-by ones (Table 1).
The presence of glutamic and aspartic acids, in a free form and dis-
sociated state, provides a sour taste (Nishimura and Kato 1988).
Kirimura and others (1969) observed that dipeptides containing
Glu and/or Asp also provide a sour taste, and among the described
specific sequences were Val-Glu and Gly-Glu, which are present in
dry-cured ham (Table 4). The same authors did not make comments
on the peptides isolated in this work, mentioned in Table 4 (Asp-
Ala-Gly-Asp and Asp-Val), but they detected a sour taste for similar
sequences like Asp-Ala, Gly-Asp, or Val-Asp. So, it can be suggest-
ed that our peptides might also provide sour taste. This would be
in agreement with the sour taste detected for fractions 28 to 32 by
the sensory analysis (Table 1). On the other hand, Glu and Asp, as
sodium salts, provide brothy/umami taste (Nishimura and Kato
1988). In our study, fractions providing the highest brothy/umami
taste (fractions 30 to 39 in Table 1) were coincident with those hav-
ing higher concentrations of free Glu and Asp (Table 2). Arai and
others (1973) observed that some dipeptides with L-Glu in N-termi-
nal position, although they give a sour taste, can also provide
brothy taste in aqueous solution containing NaCl at pH 6.0. Accord-
ing to this, the isolated Glu-Arg (Table 4) could provide certain
umami taste to the ham extract. In summary, this is the first time
that several peptides have been separated and identified in Span-
ish dry-cured ham. The presence of some of these peptides with
specific flavors in dry-cured ham soluble fraction indicates that they
may make an important contribution to taste or even interact with
volatile compounds to affect the whole flavor. However, it should be
realized that other compounds, mainly free amino acids, are more
important and may be major contributors to the final dry-cured
ham flavor.
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MS 20020061 Submitted 1/28/02, Revised 3/12/02, Accepted 4/18/02, Received
4/23/02
This work was supported by grant AGL 2001-1141 from Comisión Interministerial de Ciencia
y Tecnología (CICyT, Spain). A FPI/MEC scholarship to M. A. Sentandreu is also acknowl-
edged.
Authors Sentandreu, Aristoy, and Toldrá are with the Instituto de Agroquímica
Food Chemistry and Toxicology
y Tecnología de Alimentos (CSIC). Apartado 73, 46100 Burjassot, Valencia,
Spain. Authors Stoeva, Laib, and Voelter are with the Abteilung für
Physikalische Biochemie, Physiologisch-Chemisches Institut der Univ.
Hoppe-Seyler-Strabe 4. D-72076 Tübingen, Germany. Direct inquiries to
author Toldrá (E-mail: ftoldra@iata.csic.es).
Vol. 68, Nr. 1, 2003—JOURNAL OF FOOD SCIENCE 69