- Email: [email protected]
The role of muscle enzymes in dry-cured meat products with different drying conditions
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 Toldra´*
&
Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot (Valencia), Spain (Tel.: C34 96 3900022x2112; fax: C34 96 3636301; e-mail: [email protected]) 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, smoking.). Today, a wide variety of dry-cured meats are produced depending on the raw materials, processing conditions and type of product (Toldra´, 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 (Toldra´, 2002, 2004b). These advances have prompted successful developments in technology which are important for quality standardization (Toldra´, Flores, Aristoy, Navarro, & Flores, 1997; Toldra´, Gavara, & Lagaro´n, 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 (Toldra´ & Flores, 1998). There are several consecutive stages in proteolysis (Toldra´, 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 enzymatic 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 phospholipases, respectively, followed by oxidative reactions that produce aroma volatile compounds (Toldra´, 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, composition 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. Toldra´ / Trends in Food Science & Technology 17 (2006) 164–168
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 & Toldra´, 1993; Frentz & Zert, 1990; Parolari, 1996; Toldra´, 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 (usually below 6 8C) and the size of the ham. Ripening and drying is the following stage and a large number of variable conditions of time, temperature, relative humidity and air velocity can be found. In general, high quality hams are placed in curing chambers under mild conditions (temperatures 14–16 8C for more than 8 months) and, once they reach the expected moisture loss, are manually smeared with a layer of lard in order to prevent excessive dehydration. Poorer quality hams are subjected to more intense drying conditions for shorter periods (temperatures rising up to 25–28 8C for a few months). Final total weight loss may reach 34–36% in relation to the initial weight. The evolution of moisture content and water activity is shown in figures 1 and 2, respectively. Role of muscle proteases in dry-curing The proteolytic enzyme system in muscle is quite complex and comprises endo and exo-peptidases. Main studied endo-peptidases are calpains and cathepsins B, L, H and D while main exopeptidases are tri-peptidylpeptidases I and II and di-peptidylpeptidases I, II, III and IV as well as alanyl, arginyl, methionyl, leucyl and pyroglutamyl aminopeptidases (Toldra´, 2002). All these enzymes are involved 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 (Toldra´, 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 (Toldra´, 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; Toldra´, 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 and some unpleasant textures appear. Reasons for an excess of proteolysis are varied, including the breed types
165
Fig. 1. Evolution of the moisture content in the external muscle Semimembranosus and the internal muscle Biceps femoris along the processing of dry-cured ham (Toldra´, unpublished data).
and/or ages that have a marked influence on some enzymes (Armero, Baselga, Aristoy, & Toldra´, 1999; Armero, Barbosa, Toldra´, Baselga, & Pla, 1999). In other cases, the excess of proteolysis may be due to variations in cathepsin B activity towards high values and low salt content, which is a strong inhibitor of cathepsin activity (Parolari, Virgili, & Schivazzappa, 1994). All these variables have a deffinitive effect on the sensory quality of ham (Armero et al., 1999a,b,c). In general, muscle proteases are quite stable except calpains, that are restricted to the initial days (Rosell & Toldra´, 1996), and cathepsin D which fully inactivates by 6 months of process (Toldra´ et al., 1993). The rest of cathepsins and peptidases are very stable during the full process (Toldra´, 1992). The final products of the proteolytical chain result from the action of exopeptidases and consist in small peptides and free amino acids. A good number of peptides, mainly in the range 2700–4500 Da, have been detected during postsalting and early ripening (Rodrı´guez-Nun˜ez, Aristoy, & Toldra´, 1995) and below 2700 Da during ripening and drying
Fig. 2. Evolution of water activity in the external muscle Semimembranosus and the internal muscle Biceps femoris along the processing of dry-cured ham (Toldra´, unpublished data).
166
F. Toldra´ / 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 (Toldra´, unpublished data).
(Aristoy & Toldra´, 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 (Toldra´, 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, & Toldra´, 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; Tornquist, 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 &Toldra´, 1993). So, linoleic, arachidonic, oleic, palmitic and estearic acids from phospholipids decrease during the process (Martin, Co´rdoba, 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 (Toldra´, Flores, & Sanz, 1997; Vestergaard, Schivazzappa, & Virgili, 2000). Unsaturated fatty acids are further oxidised to aroma volatile compounds (Flores, Spanier, & Toldra´, 1998). So, this oxidation may lead to the formation of aliphatic hydrocarbons, alcohols, aldehydes and ketones while some esters
Fig. 4. Effect of different water activity levels on the activity of muscle aminopeptidases (Toldra´, unpublished data).
F. Toldra´ / Trends in Food Science & Technology 17 (2006) 164–168
167
Fig. 5. Effect of different water activity levels on the activity of muscle acid and neutral lipases and acid phospholipase (Toldra´, unpublished data).
may be derived from the interaction of those free fatty acids with alcohols formed by oxidation (Toldra´ & 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 (Toldra´, 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 drycured 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
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, Toldra´, Nieto, & Flores, 1993; Toldra´, 1998; Toldra´ et al., 2000) and even some residual activity may be found by to 2 years (Toldra´, 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 (Toldra´, 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 (Toldra´, 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 unsaturated 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 2004-05064C02-01 and AGL2001-1141 from McyT (Spain) are acknowledged. References Aristoy, M. C., & Toldra´, F. (1995). Isolation of flavor peptides from raw pork meat and dry-cured ham. In G. Charalambous (Ed.),
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F. Toldra´ / Trends in Food Science & Technology 17 (2006) 164–168
Food flavors: Generation, analysis and process influence (pp. 1323–1344). Amsterdam, The Netherlands: Elsevier Science Publication. Armero, E., Barbosa, J. A., Toldra´, F., Baselga, M., & Pla, M. (1999a). Effects of the terminal sire type and sex on pork muscle cathepsins (B, BCL and H), cysteine proteinase inhibitors and lipolytic enzyme activities. Meat Science, 51, 185–189. Armero, E., Baselga, M., Aristoy, M. C., & Toldra´, F. (1999b). Effects of sire types and sex on pork muscle exopeptidase activity and the content of natural dipeptides and free amino acids. Journal of the Science of Food and Agriculture, 79, 1280–1284. Armero, E., Flores, M., Toldra´, F., Barbosa, J. A., Olivet, J., Pla, M., et al. (1999c). Effects of pig sire types and sex on carcass traits, meat quality and sensory quality of dry-cured ham. Journal of the Science of Food and Agriculture, 79, 1147–1154. Buscailhon, S., Gandemer, G., & Monin, G. (1994a). Time-related changes in intramuscular lipids of French dry-cured ham. Meat Science, 37, 245–255. Buscailhon, S., Monin, G., Cornet, M., & Bousset, J. (1994b). Timerelated changes in nitrogen fractions and free amino acids of lean tissue of French dry-cured ham. Meat Science, 37, 449– 456. Flores, J., & Toldra´, F. (1993). Curing: Processes and applications. In R. MacCrae, R. Robinson, M. Sadle, & G. Fullerlove (Eds.), Encyclopedia of food science, food technology and nutrition (pp. 1277–1282). London (UK): Academic Press. Flores, M., Spanier, A. M., & Toldra´, F. (1998). Flavour analysis of dry-cured ham. In F. Shahidi (Ed.), Flavor of meat, meat products and seafoods (pp. 320–341). London, UK: Blackie Academic & Professional. Fowler, S. D., & Brown, W. J. (1984). Lysosomal acid lipase. In B. Borgstro¨m, & H. L. Brockman (Eds.), Lipases (pp. 329–364). London (UK): Elsevier Science Publication. Frentz, J. C., & Zert, P. (1990). L’ encyclope´die de la charcuterie Dictionnaire encyclope´dique de la charcuterie (3rd ed.). Orly, Paris: Soussana p. 435. Imanaka, T., Yamaguchi, M., Ahkuma, S., & Takano, T. (1985). Positional specificity of lysosomal acid lipase purified from rabbit liver. Journal of Biochemistry, 98, 927–931. Martin, L., Co´rdoba, J. J., Ventanas, J., & Antequera, T. (1999). Changes in intramuscular lipids during ripening of Iberian drycured ham. Meat Science, 51, 129–134. Monin, G., Marinova, P., Talmant, A., Martin, J. F., Cornet, M., Lanore, D., et al. (1997). Chemical and structural changes in drycured hams (Bayonne hams) during processing and effects of the dehairing technique. Meat Science, 47, 29–47. Motilva, M. J., & Toldra´, F. (1993). Effect of curing agents and water activity on pork muscle and adipose subcutaneous tissue lipolytic activity. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung, 196, 228–231. Motilva, M. J., Toldra´, F., & Flores, J. (1992). Assay of lipase and esterase activities in fresh pork meat and dry-cured ham. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung, 195, 446–450. Motilva, M. J., Toldra´, F., Nieto, P., & Flores, J. (1993). Muscle lipolysis phenomena in the processing of dry-cured ham. Food Chemistry, 48, 121–125. Parolari, G. (1996). Review: Achievements, needs and perspectives in dry-cured ham technology: The example of Parma ham. Food Science and Technology International, 2, 69–78. Parolari, G., Virgili, R., & Schivazzappa, C. (1994). Relationship between cathepsin B activity and compositional parameters in dry-cured hams of normal and defective texture. Meat Science, 38, 117–122.
Rodrı´guez-Nun˜ez, E., Aristoy, M. C., & Toldra´, F. (1995). Peptide generation in the processing of dry-cured ham. Food Chemistry, 53, 187–190. Rosell, C. M., & Toldra´, F. (1996). Effect of curing agents on m-calpain activity throughout the curing process. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung, 203, 320–325. Sentandreu, M. A., Coulis, G., & Ouali, A. (2002). Role of muscle endopeptidases and their inhibitors in meat tenderness. Trends in Food Science and Technology, 13, 398–419. Sentandreu, M. A., Stoeva, S., Aristoy, M. C., Laib, K., Voelter, W., & Toldra´, F. (2003). Identification of taste related peptides in Spanish Serrano dry-cured hams. Journal of Food Science, 68, 64–69. Toldra´, F. (1992). The enzymology of dry-curing of meat products. In F. J. M. Smulders, F. Toldra´, J. Flores, & M. Prieto (Eds.), New technologies for meat and meat products (pp. 209–231). Nijmegen, The Netherlands: Audet. Toldra´, F. (1998). Proteolysis and lipolysis in flavour development of dry-cured meat products. Meat Science, 49, s101–s110. Toldra´, F. (2002). Dry-cured meat products pp. 1–238. Trumbull, CT: Food & Nutrition Press. Toldra´, F. (2004a). Ethnic meat products: Mediterranean. In W. Jensen, C. Devine, & M. Dikemann (Eds.), Encyclopedia of meat sciences (pp. 451–453). London, UK: Elsevier Science Ltd. Toldra´, F. (2004b). Dry-cured ham. In Y. H. Hui, L. M. Goddik, J. Josephsen, P. S. Stanfield, A. S. Hansen, W. K. Nip, et al. (Eds.), Handbook of food and beverage fermentation technology (pp. 369–384). New York: Marcel Dekker Inc.. Toldra´, F. (2004c). Curing: Dry. In W. Jensen, C. Devine, & M. Dikemann (Eds.), Encyclopedia of meat sciences (pp. 360–365). London, UK: Elsevier Science Ltd. Toldra´, F., Aristoy, M. C., & Flores, M. (2000). Contribution of muscle aminopeptidases to flavor development in dry-cured ham. Food Research International, 33, 181–185. Toldra´, F., & Flores, M. (1998). The role of muscle proteases and lipases in flavor development during the processing of dry-cured ham. CRC Critical Reviews in Food Science and Nutrition, 38, 331–352. Toldra´, F., Flores, M., Aristoy, M.-C., Navarro, J.-L., & Flores, J. (1997). New developments in dry-cured ham. In A. M. Spanier, M. Tamura, H. Okai, & O. Mills (Eds.), Chemistry of novel foods (pp. 259–272). Carol Stream, IL: Allured Pub. Co. Inc.. Toldra´, F., Flores, M., & Sanz, Y. (1997). Dry-cured ham flavour: Enzymatic generation and process influence. Food Chemistry, 59, 523–530. Toldra´, F., Gavara, G., & Lagaro´n, J. M. (2004). Packaging and quality control. In Y. H. Hui, L. M. Goddik, J. Josephsen, P. S. Stanfield, A. S. Hansen, W. K. Nip, et al. (Eds.), Handbook of food and beverage fermentation technology (pp. 445–458). New York: Marcel Dekker Inc.. Toldra´, F., Miralles, M. C., & Flores, J. (1992). Protein extractability in dry-cured ham. Food Chemistry, 44, 391–394. Toldra´, F., Rico, E., & Flores, J. (1992). Activities of pork muscle proteases in cured meats. Biochimie, 74, 291–296. Toldra´, F., Rico, E., & Flores, J. (1993). Cathepsin B, D, H and L activity in the processing of dry-cured-ham. Journal of the Science of Food and Agriculture, 62, 157–161. Tornquist, H., Nilsson-Ehle, P., & Belfrage, P. (1978). Enzymes catalyzing the hydrolysis of long-chain monoacylglycerols in rat adipose tissue. Biochimica et Biophysica Acta, 530, 474–486. Vestergaard, C. S., Schivazzappa, C., & Virgili, R. (2000). Lipolysis in dry-cured ham maturation. Meat Science, 55, 1–5. Yuan, W., Quinn, D. M., Sigler, P. B., & Gelb, M. H. (1990). Kinetic and inhibition studies of phospholipase A2 with short chain substrates and inhibitors. Biochemistry, 29, 6082–6094.
Review
The role of muscle enzymes in dry-cured meat products with different drying conditions Fidel Toldra´*
&
Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot (Valencia), Spain (Tel.: C34 96 3900022x2112; fax: C34 96 3636301; e-mail: [email protected]) 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, smoking.). Today, a wide variety of dry-cured meats are produced depending on the raw materials, processing conditions and type of product (Toldra´, 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 (Toldra´, 2002, 2004b). These advances have prompted successful developments in technology which are important for quality standardization (Toldra´, Flores, Aristoy, Navarro, & Flores, 1997; Toldra´, Gavara, & Lagaro´n, 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 (Toldra´ & Flores, 1998). There are several consecutive stages in proteolysis (Toldra´, 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 enzymatic 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 phospholipases, respectively, followed by oxidative reactions that produce aroma volatile compounds (Toldra´, 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, composition 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. Toldra´ / Trends in Food Science & Technology 17 (2006) 164–168
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 & Toldra´, 1993; Frentz & Zert, 1990; Parolari, 1996; Toldra´, 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 (usually below 6 8C) and the size of the ham. Ripening and drying is the following stage and a large number of variable conditions of time, temperature, relative humidity and air velocity can be found. In general, high quality hams are placed in curing chambers under mild conditions (temperatures 14–16 8C for more than 8 months) and, once they reach the expected moisture loss, are manually smeared with a layer of lard in order to prevent excessive dehydration. Poorer quality hams are subjected to more intense drying conditions for shorter periods (temperatures rising up to 25–28 8C for a few months). Final total weight loss may reach 34–36% in relation to the initial weight. The evolution of moisture content and water activity is shown in figures 1 and 2, respectively. Role of muscle proteases in dry-curing The proteolytic enzyme system in muscle is quite complex and comprises endo and exo-peptidases. Main studied endo-peptidases are calpains and cathepsins B, L, H and D while main exopeptidases are tri-peptidylpeptidases I and II and di-peptidylpeptidases I, II, III and IV as well as alanyl, arginyl, methionyl, leucyl and pyroglutamyl aminopeptidases (Toldra´, 2002). All these enzymes are involved 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 (Toldra´, 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 (Toldra´, 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; Toldra´, 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 and some unpleasant textures appear. Reasons for an excess of proteolysis are varied, including the breed types
165
Fig. 1. Evolution of the moisture content in the external muscle Semimembranosus and the internal muscle Biceps femoris along the processing of dry-cured ham (Toldra´, unpublished data).
and/or ages that have a marked influence on some enzymes (Armero, Baselga, Aristoy, & Toldra´, 1999; Armero, Barbosa, Toldra´, Baselga, & Pla, 1999). In other cases, the excess of proteolysis may be due to variations in cathepsin B activity towards high values and low salt content, which is a strong inhibitor of cathepsin activity (Parolari, Virgili, & Schivazzappa, 1994). All these variables have a deffinitive effect on the sensory quality of ham (Armero et al., 1999a,b,c). In general, muscle proteases are quite stable except calpains, that are restricted to the initial days (Rosell & Toldra´, 1996), and cathepsin D which fully inactivates by 6 months of process (Toldra´ et al., 1993). The rest of cathepsins and peptidases are very stable during the full process (Toldra´, 1992). The final products of the proteolytical chain result from the action of exopeptidases and consist in small peptides and free amino acids. A good number of peptides, mainly in the range 2700–4500 Da, have been detected during postsalting and early ripening (Rodrı´guez-Nun˜ez, Aristoy, & Toldra´, 1995) and below 2700 Da during ripening and drying
Fig. 2. Evolution of water activity in the external muscle Semimembranosus and the internal muscle Biceps femoris along the processing of dry-cured ham (Toldra´, unpublished data).
166
F. Toldra´ / 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 (Toldra´, unpublished data).
(Aristoy & Toldra´, 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 (Toldra´, 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, & Toldra´, 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; Tornquist, 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 &Toldra´, 1993). So, linoleic, arachidonic, oleic, palmitic and estearic acids from phospholipids decrease during the process (Martin, Co´rdoba, 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 (Toldra´, Flores, & Sanz, 1997; Vestergaard, Schivazzappa, & Virgili, 2000). Unsaturated fatty acids are further oxidised to aroma volatile compounds (Flores, Spanier, & Toldra´, 1998). So, this oxidation may lead to the formation of aliphatic hydrocarbons, alcohols, aldehydes and ketones while some esters
Fig. 4. Effect of different water activity levels on the activity of muscle aminopeptidases (Toldra´, unpublished data).
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Fig. 5. Effect of different water activity levels on the activity of muscle acid and neutral lipases and acid phospholipase (Toldra´, unpublished data).
may be derived from the interaction of those free fatty acids with alcohols formed by oxidation (Toldra´ & 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 (Toldra´, 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 drycured 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
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, Toldra´, Nieto, & Flores, 1993; Toldra´, 1998; Toldra´ et al., 2000) and even some residual activity may be found by to 2 years (Toldra´, 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 (Toldra´, 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 (Toldra´, 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 unsaturated 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 2004-05064C02-01 and AGL2001-1141 from McyT (Spain) are acknowledged. References Aristoy, M. C., & Toldra´, F. (1995). Isolation of flavor peptides from raw pork meat and dry-cured ham. In G. Charalambous (Ed.),
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