Trends in Food Science & Technology 17 (2006) 164–168
Review
The role of muscle
enzymes in
dry-cured meat
products with
different drying
conditions
Fidel Toldrá*
Instituto de Agroquı́mica y Tecnologı́a de Alimentos
(CSIC), P.O. Box 73, 46100 Burjassot (Valencia), Spain
(Tel.: C34 96 3900022x2112; fax: C34 96 3636301;
e-mail: ftoldra@iata.csic.es)
Several muscle proteases (cathepsins, calpains, peptidases
and aminopeptidases) and lipases (lysosomal acid lipase,
acid phospholipase and adipose tissue lipase) are involved
in important biochemical mechanisms taking place during
the processing of dry-cured meat products which are directly
related to the final quality. These enzymes are affected by the
conditions typically found in the processing of dry-cured
meat products, being dehydration one of the most important
factors. This work is presenting the effect of different drying
conditions, typical in the processing of dry-cured meat
products, on the activity of muscle proteases and lipases as
well as its relevance for the final product quality.
Introduction
Dry-cured meats have been produced for many centuries
based on traditional practices and applying diverse
processing conditions (salting followed by drying, smok-
ing.). Today, a wide variety of dry-cured meats are
produced depending on the raw materials, processing
conditions and type of product (Toldrá, 2004a). Important
scientific advances dealing with the chemistry,
* Corresponding author.
0924-2244/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2005.08.007
biochemistry, and microbiology involved in the dry-curing
process have been reported in recent years (Toldrá, 2002,
2004b). These advances have prompted successful devel-
opments in technology which are important for quality
standardization (Toldrá, Flores, Aristoy, Navarro, & Flores,
1997; Toldrá, Gavara, & Lagarón, 2004). Proteolysis and
lipolysis constitute two of the most important biochemical
mechanisms during the processing of dry-cured ham with
relevant consequences for the final sensory quality. Several
muscle enzymes, proteases and lipases, are involved in both
groups of reactions, respectively.
Proteolysis contributes to texture by breakdown of the
muscle structure (Monin et al. 1997; Sentandreu, Coulis, &
Ouali, 2002), to taste through the generation of small
&
peptides and free amino acids and to aroma by further
degradation of some free amino acids (Toldrá & Flores,
1998). There are several consecutive stages in proteolysis
(Toldrá, 2004c): (a) breakdown of major myofibrillar
proteins by the action of calpains and cathepsins, (b)
generation of polypeptides that act as substrates for
peptidases to generate small peptides and (c) intense
generation of free amino acids by the action of
aminopeptidases.
Lipolysis constitute another important group of enzy-
matic reactions closely related to the final sensory quality,
especially aroma, of hams. There is an initial breakdown of
tri-acylglycerols and phospholipids by lipases and phos-
pholipases, respectively, followed by oxidative reactions
that produce aroma volatile compounds (Toldrá, 2002).
The muscle proteases and lipases involved in these two
groups of reactions are very important as the final sensory
quality will depend on their activity and mode of action. An
important reduction of moisture content and water activity
is typically observed in the processing of dry-cured ham.
This reduction may vary depending on drying conditions but
the decreased water activity can affect the enzyme activity.
Thus, the objective of this work is to discuss the effect of
drying processing conditions on the activity of these muscle
enzymes.
Processing conditions for dry-cured ham
There are many types of dry-cured hams and its main
characteristics depend on the pig crossbreed, age, compo-
sition of the feed and type of process. Some of the most
important hams are Spanish Iberian and Serrano hams,
Italian Parma and San Daniele prosciuttos and French
 F. Toldrá / Trends in Food Science & Technology 17 (2006) 164–168 165

Bayonne ham. Other hams as the Country-style ham in USA

and Westphalia ham in Germany are smoked and cooked
before consumption.
The process can be summarised as follows (Flores &
Toldrá, 1993; Frentz & Zert, 1990; Parolari, 1996; Toldrá,
2002, 2004c): Initially, hams are bledded on a steel belt with
pressing rollers and receive the adequate amount of cure salt
(saltCpotassium nitrate) that is rubbed on the outer surface.
During salting, that is kept under refrigeration (below 4 8C),
hams are placed in plastic or stainless steel shelves and are
given a certain amount of salt. They are left to stand for
10–14 days to allow salt and nitrate penetration into the ham.
The main objective of the next stage, named post-salting, is
for salt and nitrate diffusion into the full piece. This stage may
last between 40 and 60 days, depending on the temperature Fig. 1. Evolution of the moisture content in the external muscle
(usually below 6 8C) and the size of the ham. Ripening and Semimembranosus and the internal muscle Biceps femoris along the
drying is the following stage and a large number of variable processing of dry-cured ham (Toldrá, unpublished data).
conditions of time, temperature, relative humidity and air
and/or ages that have a marked influence on some enzymes
velocity can be found. In general, high quality hams are
(Armero, Baselga, Aristoy, & Toldrá, 1999; Armero,
placed in curing chambers under mild conditions (tempera-
Barbosa, Toldrá, Baselga, & Pla, 1999). In other cases,
tures 14–16 8C for more than 8 months) and, once they reach
the excess of proteolysis may be due to variations in
the expected moisture loss, are manually smeared with a
cathepsin B activity towards high values and low salt
layer of lard in order to prevent excessive dehydration.
content, which is a strong inhibitor of cathepsin activity
Poorer quality hams are subjected to more intense drying
(Parolari, Virgili, & Schivazzappa, 1994). All these
conditions for shorter periods (temperatures rising up to
variables have a deffinitive effect on the sensory quality of
25–28 8C for a few months). Final total weight loss may reach
34–36% in relation to the initial weight. The evolution of ham (Armero et al., 1999a,b,c).
moisture content and water activity is shown in figures 1 and In general, muscle proteases are quite stable except
2, respectively. calpains, that are restricted to the initial days (Rosell &
Toldrá, 1996), and cathepsin D which fully inactivates by 6
months of process (Toldrá et al., 1993). The rest of
Role of muscle proteases in dry-curing cathepsins and peptidases are very stable during the full
The proteolytic enzyme system in muscle is quite process (Toldrá, 1992).
complex and comprises endo and exo-peptidases. Main The final products of the proteolytical chain result from
studied endo-peptidases are calpains and cathepsins B, L, H the action of exopeptidases and consist in small peptides and
and D while main exopeptidases are tri-peptidylpeptidases I free amino acids. A good number of peptides, mainly in the
and II and di-peptidylpeptidases I, II, III and IV as well as range 2700–4500 Da, have been detected during postsalting
alanyl, arginyl, methionyl, leucyl and pyroglutamyl amino- and early ripening (Rodrı́guez-Nuñez, Aristoy, & Toldrá,
peptidases (Toldrá, 2002). All these enzymes are involved 1995) and below 2700 Da during ripening and drying
in the successive stages of the proteolytical chain.
Intense changes in the profiles of muscle sarcoplasmic
proteins and myofibrillar proteins, have been detected by
SDS-polyacrylamide electrophoresis along the processing
of dry-cured ham (Toldrá, Miralles, & Flores, 1992). This
proteolysis is specially evident in some myofibrillar
proteins, that progressively disappear along the processing,
like myosin heavy chain and myosin light chains 1 and 2,
troponins C and I (Toldrá, Rico, & Flores, 1993) while some
fragments of 150, 95 and 16 kDa and in the ranges 50–
100 kDa and 20–45 kDa are formed (Buscailhon, Monin,
Cornet, & Bousset, 1994a; Toldrá, 2002). Most of the
damage to the ultrastructure is detected by the end of salting
and is mainly focused on the Z-line regions as well as
through the fibers (Monin et al., 1997). When the extent of
proteolysis is exceeded, the structure is severely damaged Fig. 2. Evolution of water activity in the external muscle
and some unpleasant textures appear. Reasons for an Semimembranosus and the internal muscle Biceps femoris along
excess of proteolysis are varied, including the breed types the processing of dry-cured ham (Toldrá, unpublished data).
166 F. Toldrá / Trends in Food Science & Technology 17 (2006) 164–168

Fig. 3. Effect of different water activity levels on the activity of muscle cathepsins and calpain (Toldrá, unpublished data).

(Aristoy & Toldrá, 1995). Some tri- and di-peptides have
been analysed and sequenced (Sentandreu et al., 2003). The
generation of free amino acids, especially alanine, leucine,
valine, arginine, lysine, glutamic and aspartic acids, is very
high, mainly depending on the aminopeptidase levels,
length of the process and type of ham (Toldrá, Aristoy, &
Flores, 2000).
Role of muscle lipases in dry-curing
Phospholipids and triacylglycerols are hydrolysed by
muscle phospholipases and lipases, respectively. Acid
lipase and acid phospholipase have optimal acid pH and
are located in the lysosomes (Motilva, Flores, & Toldrá,
1992). Lysosomal acid lipase and neutral lipase preferently
hydrolyse fatty acids at positions 1 or 3 of tryacylglycerols
(Fowler & Brown, 1984) although the acid lipase can also
hydrolyse di- and mono-acylglycerols at a lower rate
(Imanaka, Yamaguchi, Ahkuma, & Takano, 1985; Torn-
quist, Nilsson-Ehle, & Belfrage, 1978). Acid phospholipase
hydrolyses phospholipids at position 1 at the water-lipid
interface (Yuan, Quinn, Sigler, & Gelb, 1990).
There is an important generation of free fatty acids
during the processing of dry-cured ham which is correlated
with phospholipid degradation (Buscailhon, Gandemer, &
Monin, 1994b; Motilva &Toldrá, 1993). So, linoleic,
arachidonic, oleic, palmitic and estearic acids from
phospholipids decrease during the process (Martin, Cór-
doba, Ventanas, & Antequera, 1999), confirming the
importance of muscle phospholipases in lipolysis. The
main increase of free fatty acids, especially oleic, estearic,
linoleic and palmitic is observed up to 6 months of
processing. This increase is higher in the external muscle
Semimembranosus, which is more dehydrated than the
internal muscle Biceps femoris (Toldrá, Flores, & Sanz,
1997; Vestergaard, Schivazzappa, & Virgili, 2000). Unsa-
turated fatty acids are further oxidised to aroma volatile
compounds (Flores, Spanier, & Toldrá, 1998). So, this
oxidation may lead to the formation of aliphatic hydro-
carbons, alcohols, aldehydes and ketones while some esters

Fig. 4. Effect of different water activity levels on the activity of muscle aminopeptidases (Toldrá, unpublished data).
 F. Toldrá / Trends in Food Science & Technology 17 (2006) 164–168
Fig. 5. Effect of different water activity levels on the activity of muscle acid and neutral lipases and acid phospholipase (Toldrá, unpublished data).
may be derived from the interaction of those free fatty acids
with alcohols formed by oxidation (Toldrá & Flores, 1998).
Effect of different drying conditions on the activity
of muscle proteases and lipases
The dry-curing process can be considered as a mild
process since temperatures are below 4 8C during the salting
stage, below 6 8C during the post-salting stage and do not
usually exceed 30 8C during drying. Furthermore, the pH
keeps relatively constant along the processing of dry-cured
ham starting at 5.6–5.8 and finishing at 6.3–6.4 by the end of
the process (Toldrá, 2002). However, hams experience an
intense dehydration process during the ripening/drying stage
(see Fig. 1), reaching moisture contents around 60% in the
inner part of the ham (region of the muscle Biceps femoris)
and around 50% in the outer part (region of the muscle
Semimembranosus). Accordingly, water activity decreases
during drying and its values can be reduced to near 0.90 in the
inner part and around 0.85 in the outer part of the ham (see
Fig. 2). Water activity inside the dry-cured meats is important
in controlling the enzyme actvity, especially when the
ripening/drying progresses and aw approaches 0.90 or even
lower values. The effect of water activity on the muscle
proteolytic and lipolytic enzymes is shown in Figs. 3–5. As
can be observed, most of the assayed muscle enzymes
(cathepsins, aminopeptidases and neutral lipase) are strongly
affected by the decrease in aw along the processing of dry-
cured meats with few exceptions. So, calpain appears to be
slightly affected by aw reduction as can be observed in Fig. 3.
In a similar way, acid lipase and acid phospholipase appear to
be quite insensitive to aw reduction (Fig. 5).
The length of the process is also important for the
activity of the enzymes. As longer is the process, a more
pronounced action of the enzymes can be observed,
especially if the drying conditions are mild. Thus, a more
intense proteolysis and lipolysis can be expected when
167
longer is the ripening/drying time and milder are the
conditions. Most of the muscle enzymes are very stable and
exhibit activity during the full process (Motilva, Toldrá,
Nieto, & Flores, 1993; Toldrá, 1998; Toldrá et al., 2000)
and even some residual activity may be found by to 2 years
(Toldrá, 2002). This allows a prolonged enzyme action
during the full process even though its activity is reduced
as drying progresses due to lower water activity values.
Calpain and pyroglutamylaminopeptidase constitute an
exception since they exhibit reduced stability and their
activities are detectable only in the initial weeks of process
(Toldrá, 2004c). Finally, other factors like salt and other
curing agents must be taken into account because they
exert inhibitory or activation effects on the muscle
enzymes (Toldrá, Rico, & Flores, 1992).
Conclusions
In general, long processes with mild ripening/drying
conditions allow a relatively higher enzyme activity and
thus a higher generation of free amino acids and free fatty
acids. Free amino acids contribute directly to taste, and
indirectly to certain aroma compounds, while free unsatu-
rated fatty acids are further oxidised to aroma volatile
compounds.
This increased activity is in agreement with the better
flavour development usually observed in long-processed
hams with milder ripening/drying conditions.
Acknowledgements
Grants AIR CT93-1757 from EU, AGL 2004-05064-
C02-01 and AGL2001-1141 from McyT (Spain) are
acknowledged.
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