food biochemistry and food processing (simpson/food biochemistry and food processing) ||...

P1: SFK/UKS P2: SFK

BLBS102-c16 BLBS102-Simpson March 21, 2012 10:54 Trim: 276mm X 219mm Printer Name: Yet to Come

16Biochemistry of Processing Meat and Poultry

Fidel Toldra

Background InformationDescription of the Muscle Enzymes

Muscle ProteasesNeutral Proteinases: CalpainsLysosomal Proteinases: CathepsinsProteasome ComplexExoproteases: PeptidasesExoproteases: Aminopeptidases and

CarboxypeptidasesLipolytic Enzymes

Muscle LipasesAdipose Tissue Lipases

Muscle Oxidative and Antioxidative EnzymesOxidative EnzymesAntioxidative Enzymes

ProteolysisProteolysis in Aged Meat and Cooked Meat ProductsProteolysis in Fermented MeatsProteolysis in Dry-Cured Ham

Nucleotide BreakdownGlycolysisLipolysis

Lipolysis in Aged Meat and CookedMeat Products

Lipolysis in Fermented MeatsLipolysis in Dry-Cured Ham

Oxidative ReactionsOxidation to Volatile CompoundsAntioxidants

References

Abstract: The biochemical changes happening during meat con-ditioning (aging) were abundantly reported during the 1970s and1980s. It has been in recent decades that more information has beenavailable for the biochemical changes in other products such ascooked, dry-fermented, and dry-cured meats. The processing andquality of these meat products have been improved based on a betterknowledge of the biochemical mechanisms involved in the genera-

tion of color, flavor, and texture. The endogenous enzyme systemsthat play important roles in these processes mainly through prote-olysis and lipolysis reactions are described in this chapter. Otherbiochemical reactions like oxidation, glycolysis, and nucleotidesbreakdown are also described.

BACKGROUND INFORMATIONThere are a wide variety of meat products that are attractiveto consumers because of their characteristic color, flavor, andtexture. This perception varies depending on local traditionsand heritage. Most of these products have been produced formany years or even centuries based on traditional practices. Forinstance, cured meat products reached America with settlers.Pork was cured in New England for consumption in the summer.Curers expanded these products by trying different recipes basedon the use of additives such salt, sugar, pepper, spices, and soforth, and smoking (Toldra 2002).

Although scientific literature on biochemical changes duringmeat conditioning (aging) and in some meat products were abun-dantly reported during the 1970s and 1980s, little informationwas available on the origin of the biochemical changes in otherproducts such as cooked, dry-fermented, and dry-cured meats.The need to improve the processing and quality of these meatproducts prompted research in the last decades on endogenousenzyme systems that play important roles in these processes,which has been later demonstrated (Toldra 2007). It is impor-tant to remember that the potential role of a certain enzyme ina specific observed or reported biochemical change can onlybe established if all the following requirements are met (Toldra1992): (1) the enzyme is present in the skeletal muscle or adi-pose tissue, (2) the enzyme is able to degrade in vitro the naturalsubstance (i.e., a protein in the case of a protease, a triacylglyc-erol in the case of a lipase, etc.), (3) the enzyme and substrate

Food Biochemistry and Food Processing, Second Edition. Edited by Benjamin K. Simpson, Leo M.L. Nollet, Fidel Toldra, Soottawat Benjakul, Gopinadhan Paliyath and Y.H. Hui.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

303

P1: SFK/UKS P2: SFK


304 Part 3: Meat, Poultry and Seafoods

are located close enough in the real meat product for an effectiveinteraction, and (4) the enzyme exhibits enough stability duringprocessing for the changes to be developed.

DESCRIPTION OF THE MUSCLEENZYMESThere are a wide variety of enzymes in the muscle. Most ofthem have an important role in the in vivo muscle functions,but they also serve an important role in biochemical changessuch as the proteolysis and lipolysis that occur in postmortemmeat, and during further processing of meat. Some enzymes arelocated in the lysosomes, while others are free in the cytosol orlinked to membranes. The muscle enzymes with most importantroles during meat processing are grouped by families and aredescribed in the succeeding sections.

Muscle Proteases

Proteases are characterized by their ability to degrade proteins,and they receive different names depending on respective modeof action (see Fig. 16.1). They are endoproteases or proteinases,when they are able to hydrolyze internal peptide bonds, but theyare exopeptidases, when they hydrolyze external peptide bonds,either at the amino termini or the carboxy termini.

Neutral Proteinases: Calpains

Calpains are cysteine endopeptidases consisting of heterodimersof 110 kDa, composed of an 80 kDa catalytic subunit and a30 kDa subunit of unknown function. They are located in thecytosol, around the Z-line area. Calpains have received differ-ent names in the scientific literature, such as calcium-activatedneutral proteinase, calcium-dependent protease, and calcium-

OOO

CC

HNC

CHN

CC

HN

O

OH

O

CC

HNC

CH3N

nOO

Endopeptidase

Dipeptidylpeptidase

CHNC

C

O

HNC

CHN

O

OH

O

CC

HNC

CH3N

n

CCHNC

O

OH

O

CC

nAminopeptidase Carboxypeptidase

Figure 16.1. Mode of action of the different types of muscleproteases.

activated factor. Calpain I is also called µ-calpain because itneeds micromolar amounts (50–70 µM) of Ca2+ for activation.Similarly, calpain II is called m-calpain because it requires mil-limolar amounts (1–5 mM) of Ca2+. Both calpains show max-imal activity around pH 7.5. Calpain activity decreases veryquickly when pH decreases to 6.0, or even reaches ineffectiveactivity at pH 5.5 (Etherington 1984). Calpains have showngood ability to degrade important myofibrillar proteins, such astitin, nebulin, troponins T and I, tropomyosin, C-protein, filamin,desmin, and vinculin, which are responsible for the fiber struc-ture. On the other hand, they are not active against myosin, actin,α-actinin and troponin C (Goll et al. 1983, Koohmaraie 1994).

The stability of calpain I in postmortem muscle is very poorbecause it is readily autolyzed, especially at high temperatures,in the presence of the released Ca2+ (Koohmaraie 1994). CalpainII appears as more stable, just 2–3 weeks before losing its activity(Koohmaraie et al. 1987). In view of this rather poor stability,the importance of calpains should be restricted to short-termprocesses. A minor contribution, just at the beginning, has beenobserved in long processes such as dry curing of hams (Roselland Toldra 1996) or in fermented meats where the acid pH valuesmakes calpain activity rather unlikely (Toldra et al. 2001).

Calpastatin is a polypeptide (between 50 and 172 kDa) actingas an endogenous reversible and competitive inhibitor of calpainin the living muscle. In postmortem muscle, calpastatin regulatesthe activity of calpains, through a calcium-dependent interaction,although only for a few days, because it is destroyed by autolysis(Koohmaraie et al. 1987). The levels of calpastatin vary withanimal species, and pork muscle has the lowest level (Valin andOuali 1992).

Lysosomal Proteinases: Cathepsins

There are several acid proteinases in the lysosomes that degradeproteins in a nonselective way. The most important are cathep-sins B, H, and L, which are cysteine proteinases, and cathepsinD, which is an aspartate proteinase. The optimal pH for activityis slightly acid (pH around 6.0) for cathepsins B and L, acid (pHaround 4.5) for cathepsin D, and neutral (pH 6.8) for cathepsinH (Toldra et al. 1992). Cathepsins require a reducing environ-ment such as that found in postmortem muscle to express theiroptimal activity (Etherington 1987). All of them are of smallsize, within the range 20–40 kDa, and are thus able to penetrateinto the myofibrillar structure. Cathepsins have shown a goodability to degrade different myofibrillar proteins. Cathepsins Dand L are very active against myosin heavy chain, titin, M and Cproteins, tropomyosin, and troponins T and I (Matsukura et al.1981, Zeece and Katoh 1989). Cathepsin L is extremely activein degrading both titin and nebulin. Cathepsin B is able to de-grade myosin heavy chain and actin (Schwartz and Bird 1977).Cathepsin H exhibits both endo- and aminopeptidase activity,and this is the reason for its classification as an aminoendopep-tidase (Okitani et al. 1981). In the muscle, there are endogenousinhibitors against cysteine peptidases. These inhibitory com-pounds, known as cystatins, are able to inhibit cathepsins B, H,and L. Cystatin C and chicken cystatin are the most well-knowncystatins.

P1: SFK/UKS P2: SFK


16 Biochemistry of Processing Meat and Poultry 305

Proteasome Complex

The proteasome is a multicatalytic complex with different func-tions in living muscle, even though its role in postmortem mus-cle is still not well understood. The 20S proteasome has a largemolecular mass, 700 kDa, and a cylinder structure with sev-eral subunits. Its activity is optimal at pH above 7.0, but itrapidly decreases when pH decreases, especially below 5.5. Itexhibits three major activities: (1) chymotrypsin-like activity,(2) trypsin-like activity, and (3) peptidyl-glutamyl hydrolyzingactivity (Coux et al. 1996). This multiple activity behavior is thereason why there was originally some confusion among labora-tories over its identification. The 20S proteasome concentrationis higher in oxidative muscles than in glycolytic ones (Dahlmannet al. 2001). This enzyme has shown degradation of some my-ofibrillar proteins such as troponin C and myosin light chain andcould be involved in postmortem changes in slow twitch oxida-tive muscles or in high-pH meat, where an enlargement of theZ-line with more or less density loss is observed (Sentandreuet al. 2002).

A new family of peptidases, named as caspases or apoptosis-generating peptidases, are cysteine peptidases that have beenrecently proposed to be involved in cell death and thus immediatepostmortem changes in proteins having some impact on thephases of rigor and meat aging. Three main pathways of cellulardeath development have been proposed (Herrera-Mendez et al.2006). These peptidases are active at neutral pH, and one of thelimitations to its activity in postmortem meat is the acid pH inthe postmortem muscle.

Exoproteases: Peptidases

There are several peptidases in the muscle with the ability torelease small peptides of importance for taste. Tripeptidylpep-tidases (TPPs) are enzymes capable of hydrolyzing differenttripeptides from the amino termini of peptides, while dipep-tidylpeptidases (DPPs) are able to hydrolyze different dipeptidesequences. There are two TPPs and four DPPs, and their molec-ular masses are relatively high, between 100 and 200 kDa, oreven as high as 1000 kDa for TPP II, and have different substratespecificities. TPP I is located in the lysosomes, has an optimalacid pH (4.0), and is able to hydrolyze tripeptides Gly-Pro-X,where X is an amino acid, preferentially of hydrophobic na-ture. TPP II has optimal neutral pH (6.5–7.5) and wide substratespecificity, except when Pro is present on one of both sides of thehydrolyzed bond. DPPs I and II are located in the lysosomes andhave optimal acid pH (5.5). DPP I has a special preference forhydrolyzing the dipeptides Ala-Arg and Gly-Arg, while DPPII prefers a terminal Gly-Pro sequence. DPP III is located inthe cytosol and has special preference for terminal Arg-Arg andAla-Arg sequences. DPP IV is linked to the plasma membraneand prefers a terminal Gly-Pro sequence. Both DPP III and IVhave an optimal pH around 7.5–8.0. All these peptidases havebeen purified and fully characterized in porcine skeletal muscle(Toldra 2002).

Exoproteases: Aminopeptidases and Carboxypeptidases

There are five aminopeptidases, known as leucyl, arginyl, alanyl,pyroglutamyl, and methionyl aminopeptidases, based on theirrespective preference or requirement for a specific N-terminalamino acid. They are able, however, to hydrolyze other aminoacids, although at a lower rate (Toldra 1998). Aminopeptidasesare metalloproteases with a very high molecular mass and com-plex structures. All of them are active at neutral or basic pH.Alanyl aminopeptidase, also known as the major aminopepti-dase because it exhibits very high activity, is characterized by itspreferential hydrolysis of alanine, but it is also able to act againsta wide spectrum of amino acids such as aromatic, aliphatic, andbasic aminoacyl bonds. Methionyl aminopeptidase has prefer-ence for methionine, alanine, lysine, and leucine, but also has awide spectrum of activity. This enzyme is activated by calciumions. Arginyl aminopeptidase, also known as aminopeptidase B,hydrolyzes basic amino acids such as arginine and lysine (Toldraand Flores 1998).

Carboxypeptidases are located in the lysosomes and have op-timal acid pH. They are able to release free amino acids fromthe carboxy termini of peptides and proteins. CarboxypeptidaseA has preference for hydrophobic amino acids, whereas car-boxypeptidase B has a wide spectrum of activity (McDonaldand Barrett 1986).

Lipolytic Enzymes

Lipolytic enzymes are characterized by their ability to degradelipids, and they receive different names depending on their modeof action (see Fig. 16.2). They are known as lipases when theyare able to release long-chain fatty acids from triacylglycerols,while they are know as esterases when they act on short-chainfatty acids. Lipases and esterases are located either in the skeletalmuscle or in the adipose tissue. Phospholipases, mainly foundin the skeletal muscle, hydrolyze fatty acids at positions 1 or 2in phospholipids.

OLipase

O

CR1H CO O

Phospholipase A11H2CO

OCHR2

O

CR1H CO

C

H2CO R3

H2CO

OCH

R2

CO

C

O

3

H2CO

PO

O

P

O-Phospholipase A2

OCH2CH2(CH)3

Figure 16.2. Mode of action of muscle lipases and phospholipases.

P1: SFK/UKS P2: SFK



Muscle Lipases

Lysosomal acid lipase and acid phospholipase are located inthe lysosomes. Both have an optimal acid pH (4.5–5.5) and areresponsible for the generation of long-chain free fatty acids.Lysosomal acid lipase has preference for the hydrolysis of tri-acylglycerols at positions 1 or 3 (Fowler and Brown 1984). Thisenzyme also hydrolyzes di- and monoacylglycerols but at a lowerrate (Imanaka et al. 1985, Negre et al. 1985). Acid phospholi-pase hydrolyzes phospholipids at position 1 at the water-lipidinterface.

Phospholipase A and lysophospholipase have optimal pH inthe basic region and regulate the hydrolysis of phospholipids, atpositions 1 and 2, respectively. The activities of these enzymesare higher in oxidative muscles than in glycolytic muscles, andthis fact would explain the high content of free fatty acids inoxidative muscles. The increase in activity is about 10- to 25-fold for phospholipase A and 4- to 5-fold for lysophospholipase(Alasnier and Gandemer 2000).

Acid and neutral esterases are located in the lysosomes andcytosol, respectively, and are quite stable (Motilva et al. 1992).Esterases are able to hydrolyze short-chain fatty acids from tri-,di-, and monoacylglycerols, but they exert poor action due to thelack of adequate substrate.

Adipose Tissue Lipases

Hormone-sensitive lipase is the most important enzyme presentin adipose tissue. This enzyme is responsible for the hydrolysisof stored adipocyte lipids. It has a high specificity and prefer-ence for the hydrolysis of long-chain tri- and diacylglycerols(Belfrage et al. 1984). This enzyme has positional specificitysince it hydrolyzes fatty acids at positions 1 or 3 in triacylglyc-erols four times faster than it hydrolyzes fatty acids in position2 (Belfrage et al. 1984). The hormone-sensitive lipase has amolecular mass of 84 kDa and neutral optimal pH, around 7.0.The monoacylglycerol lipase is mainly present in the adipocytes,and very little is present in stromal and vascular cells. It has amolecular mass of 32.9 kDa and hydrolyzes medium- and long-chain monoacylglycerols resulting from previous hydrolysis bythe hormone-sensitive lipase (Tornquist et al. 1978). Lipopro-tein lipase is located in the capillary endothelium and is able tohydrolyze the acylglycerol components at the luminal surfaceof the endothelium (Smith and Pownall 1984), with preferencefor fatty acids at position 1 over those at position 3 (Fieldingand Fielding 1980). Lipoprotein lipase is an acylglycerol lipaseresponsible for the degradation of lipoprotein triacylglycerol. Itsmolecular mass is around 60 kDa and it has an optimal basic pH.Unsaturated monoacylglycerols are more quickly hydrolyzedthan saturated compounds (Miller et al. 1981).

The lipolysis phenomenon in adipose tissue is not so complexas in muscle. The hormone-sensitive lipase hydrolyzes tri- anddiacylglycerols, as a rate-limiting step (Xiao et al. 2010). Theresulting monoacylglycerols from this reaction or from lipopro-tein lipase (Belfrage et al. 1984) are then further hydrolyzed bythe monoacylglycerol lipase. The end products are glycerol andfree fatty acids.

Acid and neutral esterases are also present in adipose tissue(Motilva et al. 1992). During mobilization of depot lipids, es-terases can participate by mobilizing stored cholesteryl esters.Esterases can also degrade lipoprotein cholesteryl esters takenup from the plasma (Belfrage et al. 1984).

Muscle Oxidative and Antioxidative Enzymes

Oxidative Enzymes

Lipoxygenase contains iron and catalyzes the incorporation ofmolecular oxygen into polyunsaturated fatty acids, especiallyarachidonic acid, and esters containing a Z,Z-1,4-pentadien(Marczy et al. 1995). They receive different names, 5-, 12-,or 15-lipoxygenase, depending on the position where oxygenis introduced. The final product is a conjugated hydroperoxide.They usually require millimolar concentrations of Ca+2, andtheir activity is stimulated by ATP (Yamamoto 1992). Lipoxy-genase has been found to be stable during frozen storage and isresponsible for rancidity development in chicken, especially inthe muscle Gastrocnemius (Grossman et al. 1988).

Antioxidative Enzymes

Antioxidative enzymes and their regulation in the muscle consti-tute a defense system against oxidative susceptibility (i.e., an in-creased concentration of polyunsaturated fatty acids) and physi-cal stress (Young et al. 2003). Glutathione peroxidase contains acovalently bound selenium atom that is essential for its activity.This enzyme catalyzes the dismutation of alkyl hydroperoxidesby reducing agents like phenols. Its activity has been reported tobe lower in oxidative muscles than in glycolytic muscles (Daunet al. 2001). Superoxide dismutase is a copper metalloenzyme,and catalase an iron metalloenyzme. Both enzymes catalyze thedismutation of hydrogen peroxide to less harmful hydroxides.These enzymes influence the shelf life of the meat and pro-tect against the pro-oxidative effects of chloride during furtherprocessing.

PROTEOLYSISProteolysis constitutes an important group of reactions duringthe processing of meat and meat products. In fact, proteolysis hasa high impact on texture, and thus meat tenderness, because itcontributes to the breakdown of the myofibrillar proteins respon-sible for muscle network, but proteolysis also generates peptidesand free amino acids that have a direct influence on taste andalso act as substrates for further reactions contributing to aroma(Toldra 1998, 2002). In general, proteolysis has several consec-utive stages (see Fig. 16.3) as follows: (1) action of calpains andcathepsins on major myofibrillar proteins, generating proteinfragments and intermediate size polypeptides; (2) these gener-ated fragments and polypeptides are further hydrolzyed to smallpeptides by DPPs and TPPs; and (3) dipeptidases, aminopepti-dases, and carboxypeptidases are the last proteolytic enzymesthat act on previous polypeptides and peptides to generate freeamino acids. The progress of proteolysis varies depending on

P1: SFK/UKS P2: SFK



Myofibrillar and sarcoplasmic proteinsproteins

Calpains

CathepsinsTenderness

Protein fragments and polypeptides

Tripeptidylpeptidases

Dipeptidylpeptidases

Small peptides

Aminopeptidases

Free amino acids Taste

Chl

Volatile compounds Aroma

Chemical reactions

Figure 16.3. General scheme of proteolysis during the processingof meat and meat products.

the processing conditions, the type of muscle, and the amountof endogenous proteolytic enzymes, as described later.

Proteolysis in Aged Meat and Cooked MeatProducts

During meat aging, there is proteolysis of important myofibril-lar proteins such as troponin T by calpains, with the associatedrelease of a characteristic 30 kDa fragment, which is associ-ated with meat tenderness, nebulin, desmin, titin, troponin I,myosin heavy chain, and proteins at the Z-line level (Yates et al.1983). A 95 kDa fragment is also characteristically generated(Koohmaraie 1994). Examples of cathepsin and aminopeptidaseactivity during beef aging are shown in Figures 16.4 and 16.5,respectively.

The fastest aging rate is observed in chicken, followed by pork,lamb, and beef. For instance, it has been reported that chickenmyofibrils are easily damaged by cathepsin L, whereas beefmyofibrils are much more resistant (Mikami et al. 1987). Thereasons for this difference are the differences between speciesin enzymatic activity, inhibitor content, and susceptibility toproteolysis of the myofibrillar structure. These factors are alsostrongly linked to the type of muscle metabolism, which hasa strong influence on the aging rate (Toldra 2006a). In fact,proteolysis is faster in fast-twitch white fibers (which contractrapidly) than in slow-twitch red fibers (which contract slowly),that is, aging rate increases with increasing speed of contractionbut decreases with the level of heme iron (Ouali 1991). Eventhough the effect of muscle type is estimated to be tenfold lower

14

16

18

4

6

8

10

12

14

16

Act

ivit

y (U

/g x

100

0) Cathepsin L

Cathepsin H

Cathepsin B

0

2

4

6

0 5 10 15

Time (d)

Figure 16.4. Evolution of muscle cathepsins during the aging ofbeef (Toldra, unpublished data).

than the effect of temperature, it is threefold higher than animaleffects (Dransfield 1980–1981). There are also some physicaland chemical conditions in postmortem muscle, listed in Tables16.1 and 16.2, respectively, that can affect enzyme activity. Ofthese conditions, the most significant are pH, which decreasesonce the animal is slaughtered, and osmolality, which increasesfrom 300 to around 550 mOsm within 2 days, due to the release ofions to the cytosol. The osmolality has been observed to be higherin fast-twitch muscle, which also experiences a faster agingrate (Valin and Ouali 1992). Age of the animal also decreasesthe aging rate, because collagen content as well as the cross-links that make the muscle more heat stable and mechanicallyresistant, increase with age.

Proteolysis in Fermented Meats

The progressive pH decrease by lactic acid (generated duringthe fermentation by lactic acid bacteria), the added salt (2–3%),

5

6

7

8

9

Alanyl

A B

0

1

2

3

4

0 5 10 15

Act

ivit

y (U

/g)

Alanyl

Aap B

Methionyl

0

1

0 5 10 15

Time (d)

Figure 16.5. Evolution of muscle aminopeptidases during the agingof beef (Adapted from Goransson et al. 2002).

P1: SFK/UKS P2: SFK



Table 16.1. Physical Factors Affecting Proteolytic Activity During Meat and Meat Products Processing

Factor Typical Trend Effect on Proteases

pH Near neutral pH in dry-cured ham andcooked meat products.

Favors the activity of calpain, cathepsins B and L, DPP III and IV,TPP II and aminopeptidases.

Slightly acid in aged meat Favors the activity of cathepsins B, H and LAcid pH in dry-fermented sausages Favors the activity of cathepsin D, DPP I, DPP II, and TPP I

Time Short in aged meat, cooked meat products,and fermented products

Short action for enough enzyme action except for calpains thatcontribute to tenderness

Medium in dry-fermented sausages Time allows significant biochemical changesLong in dry-cured ham Very long time for important biochemical changes

Temperature High increase in cooking andsmoking

Enzymes are strongly activated by cooking temperatures althoughstability decreases rapidly and time of cooking is short

Mild temperatures in fermentation anddry-curing

Enzymes have time enough for its activity even though the use ofmild temperatures

Water activity Slightly reduced in cooked meats Enzymes have good conditions for activitySubstantially reduced in dry-meats. Restricted enzyme activity as aw drops

Redoxpotential

Anaerobic values in postmortem meat. Most of the muscle enzymes need reducing conditions for activity

Osmolality High in fast-twitch muscle. It may increasefrom 300 to 550 mOsm within 2 d

Enhances proteases activity and these muscles are aged at fasterrate

and the heating/drying conditions during the fermentation andripening/drying affect protein solubility (Toldra and Reig 2007).The reduction may reach 50–60% in the case of myofibrillarproteins and 20–47% for sarcoplasmic proteins (Klement et al.1974). There is a variable degree of contribution to proteolysisby both endogenous and proteases and those of microbial origin.This contribution mainly depends on the raw materials, thetype of product, and the processing conditions. The contributionof microbial proteases to proteolysis also depends on the typeof strains. For instance, strains Staphylococcus carnosus andStaphylococcus simulans were reported to hydrolyze sarcoplas-mic but not myofibrillar proteins; however, no protease activitywas detected in another staphylococci but instead, some lowaminopeptidase and high esterase activity was found (Casaburiet al. 2006).

The pH drop during the fermentation stage is very important.So, when pH drops below 5.0, the proteolytic activity of endoge-nous cathepsin D becomes very intense (Toldra et al. 2001).Several myofibrillar proteins, such as myosin and actin, aredegraded, and some fragments of 135, 38, 29 kDa, and 13 kDaare formed. The major role of cathepsin D has been confirmed inmodel systems using antibiotics and specific protease inhibitorsin order to inhibit bacterial proteinases or other endogenousmuscle proteinases (Molly et al. 1997). A minor role is played byother muscle cathepsins (B, H, and L) and bacterial proteinases.Peptides and small protein fragments are produced duringfermentation, heating (smoking), and ripening. The generationof free amino acids, as final products of proteolysis, depends onthe pH reached in the product (as aminopeptidases are affectedby low pH values), concentration of salt (these enzymes are

Table 16.2. Chemical Factors Affecting Proteolytic Activity During Meat and Meat Products Processing

Factor Typical Trend Effect on Proteases

NaCl Low in aged meat No effect on enzyme activityMedium concentration in cooked meat

productsPartial inhibition of most proteases except calpain

and aminopeptidase B that arechloride-activated at low NaCl concentration.

High concentration in dry-cured hams Strong inhibition of almost all proteasesNitrate and

nitriteConcentration around 125 ppm in cured

meat productsSlight inhibitory effect on the enzyme activity,

except cathepsin B that is activatedAscorbic acid Concentration around 500 ppm in cured

meat productsSlight inhibition of m-calpain, cathepsin H, leucyl

aminopeptidase, and aminopeptidase BGlucose Poor concentration in aged meat and dry-cured ham No effect

High concentration (up to 2 gL-1) in fermentedmeats

Slight activation of leucyl aminopeptidase andcathepsins B, H, and D

P1: SFK/UKS P2: SFK



inhibited or activated by salt), and processing conditions (time,temperature, and water activity (aw) that affect the enzymeactivity) (Toldra 1998). All these factors affect the contributionof the different aminopeptidases, as described in Tables 16.1and 16.2; thus, the pH reached at the fermentation stage (pH <

5.0) is decisive because it reduces substantially both muscle andmicrobial aminopeptidase activity (Sanz et al. 2002). So, theprocessing environmental conditions at the beginning of dryingare very important for the accumulation of free amino acids inthe final products (Roseiro et al. 2010).

Finally, it must be taken into account that some microorgan-isms, grown during fermentation, might have decarboxylase (anenzyme able to generate amines from amino acids) activity.

Proteolysis in Dry-Cured Ham

The analysis of muscle sarcoplasmic proteins and myofibrillarproteins by sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis reveals an intense proteolysis during the process.This proteolysis appears to be more intense in myofibrillar thanin sarcoplasmic proteins (Toldra and Aristoy 2010). The pat-terns for myosin heavy chain, myosin light chains 1 and 2, andtroponins C and I show a progressive disappearance during theprocessing (Toldra et al. 1993). Several fragments of 150, 95,and 16 kDa and in the ranges of 50–100 kDa and 20–45 kDa areformed (Toldra 2002). The analysis of ultrastructural changesby both scanning and transmission electron microscopy showsweakening of the Z-line as well as important damage to thefibers, especially at the end of salting (Monin et al. 1997). Anexcess of proteolysis may create unpleasant textures because ofintense structural damage. The result is a poor firmness that ispoorly rated by sensory panelists and consumers. This excess ofproteolysis is frequently due to the breed type and/or age, whichhave a marked influence on some enzymes, or just a higher levelof cathepsin B activity (Toldra 2004a). A high residual cathepsinB activity and/or low salt content, a strong inhibitor of cathepsinactivity, are correlated with the increased softness. The action ofcalpains is restricted to the initial days of processing due to theirpoor stability. Cathepsin D would contribute during the initial 6months, and cathepsins B, L, and H, which are very stable andhave an optimal pH closer to that in ham, would act during thefull process (Toldra 1992). An example of the evolution of theseenzymes is shown in Figure 16.6.

Numerous peptides are generated during processing: mainlyin the range 2700–4500 Da during postsalting and early ripen-ing, and below 2700 Da during ripening and drying (Aristoy andToldra 1995). Some of these peptides have been generated fromspecific myofibrillar and sarcoplasmic proteins degradation. Pro-teomic tools have been used for the identification of long-chainpeptides resulting from proteolysis of actin (Sentandreu et al.2007), titin, and light-chain myosin I (Mora et al. 2009a) andcreatine kinase (Mora et al. 2009b). These peptides are object offurther research for its potential bioactivity and contribution tothe nutritional properties of dry-cured ham (Jimenez-Colmeneroet al. 2010). Some of the smaller tri- and dipeptides recently havebeen sequenced. DPP I and TPP I appear to be the major enzymesinvolved in the release of di- and tripeptides, respectively, due

12

14

Cat B

4

6

8

10

12

14

Act

ivit

y (U

/g ×

100

0)

Cat B

Cat B+L

Cat H

Cat D

0

2

4

0 5 10 15

Time (m)

Figure 16.6. Evolution of cathepsins during the processing ofdry-cured ham (Toldra, unpublished data).

to their good activity, stability, and an optimal pH near to that inham. The other peptidases would play a minor role (Sentandreuand Toldra 2002). The generation of free amino acids during theprocessing of dry-cured ham is very high (Toldra 2004b). Ala-nine, leucine, valine, arginine, lysine, and glutamic and asparticacids are some of the amino acids generated in higher amounts.An example of generation is shown in Figure 16.7. The finalconcentrations depend on the length of the process and the typeof ham (Toldra et al. 2000). On the basis of the specific enzymecharacteristics and the process conditions, alanyl and methionylaminopeptidases appear to be the most important enzymes in-volved in the generation of free amino acids, while arginylaminopeptidase would mostly generate arginine and lysine(Toldra 2002).

NUCLEOTIDE BREAKDOWNThe disappearance of ATP is very fast; in fact, it only takes afew hours to reach negligible levels. Many enzymes are involvedin the degradation of nucleotides and nucleosides, as describedin Chapter 15. The main changes in the nucleotide breakdownproducts occur during a few days postmortem, as shown in Fig-ure 16.8. So, adenosine triphosphate (ATP), adenosine diphos-phate, and adenosine monophosphate, which are intermediatedegradation compounds, also disappear within 24 hours post-mortem. Inosine monophosphate reaches a maximum by 1 daypostmortem, but some substantial amount is still recovered after7 days postmortem. On the other hand, inosine and hypoxan-thine, as final products of these reactions, increase up to 7 dayspostmortem (Batlle et al. 2001).

GLYCOLYSISGlycolysis consists in the hydrolysis of carbohydrates, mainlyglucose, either that remaining in the muscle or that formed fromglycogen, to give lactic acid as the end product. As lactic acid

P1: SFK/UKS P2: SFK



2000

Glutamic acidTyrosine

80010001200140016001800

Co

nce

ntr

atio

n

(mg

/100

g d

ry w

eig

ht)

Lysine

ValineLeucine

2000

400600

0 5 10 15

Time (m)

Figure 16.7. Example of the generation of some free amino acids during the processing of dry-cured ham (Adapted from Toldra et al. 2000).

accumulates in the muscle, pH falls from neutral values to acidvalues around 5.3–5.8. The glycolytic rate, or speed of pH fall,depends on the animal species and the metabolic status. Onthe other hand, the glycolytic potential, which depends on theamount of stored carbohydrates, gives an indication of ultimatepH. The pH drop due to the lactic acid accumulation is perhapsthe main consequence of glycolysis, and it has very importanteffects on meat processing because pH affects numerous chemi-cal and biochemical (all the enzymes) reactions (Toldra 2006a).There are many enzymes involved in the glycolytic chain;some of the most important are phosphorylase, phosphofruc-tokinase, pyruvic kinase, and lactate dehydrogenase (Demeyerand Toldra 2004). Lactate dehydrogenase is involved in the laststep, which consists in the conversion of pyruvic acid into lacticacid. The contribution of glycolysis is restricted to a few hourspostmortem, although it is also important in fermented meatswhere sugar is added for microorganism growth (see Chapter18). The evolution of some glycolytic enzymes is shown inFigure 16.9.

LIPOLYSISLipolysis makes an important contribution to the quality of meatproducts by the generation of free fatty acids, some of whichhave a direct influence on flavor, and others that, with polyun-saturations, may be oxidized to volatile aromatic compounds,acting as flavor precursors. The general scheme for lipolysis inmuscle and adipose tissue is shown in Figure 16.10. In addition,the breakdown of triacylglycerols affects the texture of the adi-pose tissue, and an excess of lipolysis/oxidation may contributeto the development of rancid aromas or yellowish colors in fat(Toldra 1998).

Lipolysis in Aged Meat and CookedMeat Products

The relative amounts of released free fatty acids depends not onlyon the enzyme preference, but also on many other factors suchas raw materials (especially affected by feed composition), type

8

3

4

5

6

7

Co

nce

ntr

atio

n (

um

ol/g

)

ATP

ADP

AMP

IMP

Inosine

0

1

2

0 5 10 15

Time (d)

IMP

Inosine

Hypoxanthine

Figure 16.8. Evolution of nucleotides and nucleosides during the aging of pork meat. (Adapted from Batlle et al. 2001.)

P1: SFK/UKS P2: SFK



0 2

0.25

0.3

0.35

0

0.05

0.1

0.15

0.2

0.25

0 20 40 60 8

Act

ivit

y (U

/g ×

100

0)

Glucuronidase

N acetyl glucosamidase

080

Time (d)

N

Figure 16.9. Evolution of β-glucuronidase andN-acetyl-β-glucosaminidase during the processing of adry-fermented sausage (Toldra, unpublished data).

of process, and extent of cooking (Toldra 2002). The generationrate observed during in vitro incubations of muscle lipases withpure phospholipids is the following (in order of importance):linoleic > oleic > linolenic > palmitic > stearic > arachidonicacid. Lipolysis may also vary depending on the type of muscle, asoxidative and glucolytic muscles exhibit different lipase activity(Flores et al. 1996). The generation of free short-chain fatty acidsis very low due to the lack of adequate substrate (Motilva et al.1993b). Studies comparing pale, soft, exudative (PSE) versusnormal pork reported that phospholipase A2 activity was higherand well correlated with the occurrence of the PSE syndrome(Chen et al. 2010). Reverse correlation was also reported for

the antioxidant activity of glutathione peroxidase (Chen et al.2010). The physical and chemical conditions in the muscle andadipose tissue, especially during cooking, may affect the enzymeactivity (see Tables 16.3 and 16.4). The evolution of musclelipases during aging of pork meat is shown in Figure 16.11.

Lipolysis in Fermented Meats

Depending on the raw materials, type of product, and processingconditions, the degree of contribution of endogenous and micro-bial origin lipases will vary (Toldra 2007). The relative impor-tance of both enzyme systems has been checked with mixturesof antibiotics and antimycotics used in sterile meat model sys-tems ( Molly et al. 1997). The results showed a minimal effect ofantibiotics, and it was concluded that lipolysis is mainly broughtabout by muscle and adipose tissue lipases (60–80% of totalfree fatty acids generated), even though some variability mightbe found, depending on the batch and the presence of specificstrains (Molly et al. 1997). Other authors have also observedthat fatty acids are released in higher amounts when starters areadded, but that there is significant lipolysis in the absence of mi-crobial starters (Montel et al. 1993, Hierro et al. 1997). When pHdrops during fermentation, the action of muscle lysosomal acidlipase and acid phospholipase becomes very important. Somestrains are selected as starters based on their contribution tolipolysis. So, Micrococcaceae present a highly variable amountof extra- and intracellular lipolytic enzymes, dependent on thestrain and type of substrate (Casaburi et al. 2008). The action ofthe extracellular enzymes on the hydrolysis of triacylglycerolsbecomes more important after 15–20 days of ripening (Ordonezet al. 1999). Other microorganisms used as starters are Staphy-lococcus warneri, which gives the highest lipolytic activity, and

Triacylglycerols

In muscle and adipose tissue: In muscle tissue:

Diacylglycerols

PhospholipidsLipase

Phospholipases

Monoacylglycerols

LysophospholipidsLipase

Lysophospholipase

Freefatty acids

Freefatty acids

Monoacylglycerol lipase

Oxidation

Volatile compounds

Volatile compoundsOxidation

Figure 16.10. General scheme of lipolysis during the processing of meat and meat products.

P1: SFK/UKS P2: SFK



Table 16.3. Physical Factors Affecting Lipolytic Activity During Meat and Meat Products Processing

Factor Typical Trend Effect on Lipolytic Enzymes

pH Near neutral pH in dry-cured hamand cooked meat products.

Favors the activity of neutral lipase, neutral esterase andhormone-sensitive lipase.

Slightly acid in aged meat Favors the activity of lysosomal acid lipase, acid esterase and acidphospholipase

Acid pH in dry-fermented sausages Favors the activity of lysosomal acid lipase, acid esterase and acidphospholipase

Time Short in aged meat, cooked meatproducts and fermented products

Short action for enough enzyme action but significant oxidation ofthe released fatty acids

Medium in dry-fermented sausagesand long in dry-cured ham

Time allows significant biochemical changes in dry sausages andvery important in dry-cured ham

Temperature High increase in cooking andsmoking

Enzymes are strongly activated by cooking temperatures althoughfor short time

Mild temperatures in fermentationand dry-curing

Enzymes have time enough for its activity even though the use ofmild temperatures. Lipases are active even during freezingstorage of the raw meats

Water activity Slightly reduced in cooked meats Enzymes have very good conditions for activitySubstantially reduced in dry-meats. Muscle neutral lipase and esterases are affected. Rest of lipolytic

enzymes remain unaffected by aw dropRedox

potentialAnaerobic values in postmortem

meat.Slight effect on lysosomal acid lipase and phospholipase. More

intense for rest of lipases

Staphylococcus saprophyticus; S. carnosus and Staphylococcusxylosus present poor and variable lipolytic activity (Montel et al.1993). Lactic acid bacteria have poor lipolytic activity, mostlyintracellular. Many molds and yeasts such as Candida, Debary-omyces, Cryptococcus, and Trichosporum have been isolatedfrom fermented sausages, and all of them exhibit lipolytic ac-tivity (Ordonez et al. 1999, Ludemann et al. 2004, Sunesen andStahnke 2004).

The increases in the levels of free fatty acids show a greatvariability depending on the raw materials, type of sausage, andprocessing conditions (Toldra et al. 2002). The increase mayreach 2.5–5% of the total fatty acids, and the rate of releasedecreases in the following order: oleic > palmitic > stearic >

linoleic (Demeyer et al. 2000). The release of polyunsaturatedfatty acids from phospholipids is more pronounced during ripen-

ing (Navarro et al. 1997). Some short-chain fatty acids such asacetic acid may increase, especially during early stages of ripen-ing. The generation of volatile compounds with impact on thearoma of fermented sausages depends on the type of processingand the starter culture added (Talon et al. 2002, Tjener et al.2004). Some of the most important are aliphatic saturated andunsaturated aldehydes, ketones, methyl-branched aldehydes andacids, free short-chain fatty acids, sulfur compounds, some alco-hols, terpenes (from spices), and some nitrogen-derived volatilecompounds (Stahnke 2002).

Lipolysis in Dry-Cured Ham

The generation of free fatty acids in the muscle is correlatedwith the period of maximal phospholipid degradation (Motilva

Table 16.4. Chemical Factors Affecting Lipolytic Activity During Meat and Meat Products Processing

Factor Typical Trend Effect on Lipolytic Enzymes

NaCl Low in aged meat No effect on enzyme activityMedium concentration in cooked meat products Partial inhibition of muscle neutral lipase and

esterases. Adipose tissue lipases not affected.Activation of lysosomal acid lipase and acidphospholipase

High concentration in dry-cured hams Slight activation of lysosomal acid lipase andacid phospholipase

Nitrate and nitrite Concentration around 125 ppm in cured meat products No significant effectAscorbic acid Concentration around 500 ppm in cured meat products Slight inhibition of all lipolytic enzymesGlucose Poor concentration in aged meat and dry-cured ham No effect

High concentration (up to 2 gL-1) in fermented meats No effect

P1: SFK/UKS P2: SFK



1

1.2

Lysosomal acid lipaseAcid esterase

0

0.2

0.4

0.6

0.8

Act

ivit

y (U

/g)

PhospholipaseLysosomal acid lipaseAcid esteraseNeutral esterase

0

0.2

0 5 10 15

Time (m)

Figure 16.11. Evolution of muscle lipases along the processing ofdry-cured ham (Toldra, unpublished data).

et al. 1993b, Buscailhon et al. 1994). Furthermore, a decreasein linoleic, arachidonic, oleic, palmitic, and stearic acids fromphospholipids is observed at early stages of processing (Martinet al. 1999). This fact corroborates muscle phospholipases asthe most important enzymes involved in muscle lipolysis. Theamount of generated free fatty acids increases with aging time,up to 6 months of processing (see Fig. 16.12) and is higher inthe external muscle (Semimembranosus), which contains moresalt and is more dehydrated, than in the internal muscle (Bicepsfemoris). As lipase activity is mainly influenced by pH, saltconcentration, and aw (Motilva and Toldra 1993), it appears thatthe observed lipid hydrolysis is favored by the same variables(salt increase and aw reduction), as shown in Tables 16.3 and16.4, that enhance enzyme activity in vitro (Vestergaard et al.2000, Toldra et al. 2004).

In the case of the adipose tissue, triacylglycerols form the ma-jor part of this tissue (around 90%) and are mostly hydrolyzedby neutral lipases to di- and monoacylglycerols and free fatty

6

Oleic acidLinoleic acid

2

3

4

5

Fre

e fa

tty

acid

co

nc

(g/1

00g

)

StearicOleic acidLinoleic acidLinolenic acid

0

1

0 2 4 6 8 10 12

Time (m)

Stearic acid

Figure 16.12. Example of the generation of some free fatty acids inthe adipose tissue during the processing of dry-cured ham (Adaptedfrom Motilva et al. 1993b).

acids (see Fig. 16.12), especially up to 6 months of processing(Motilva et al. 1993a). Hormone-sensitive lipase was reportedto be more stable than the adipose tissue neutral triglyceridelipase (Xiao et al. 2010). The amount of triacylglycerols de-creases from about 90% to 76% (Coutron-Gambotti and Gande-mer 1999). There is a preferential hydrolysis of polyunsaturatedfatty acids, although some of them may not accumulate due tofurther oxidation during processing. Triacylglycerols that arerich in oleic and linoleic acids and are liquid at 14–18◦C morehydrolyzed than triacylglycerols that are rich in saturated fattyacids such as palmitic acid and are solid at those temperatures(Coutron-Gambotti and Gandemer 1999). This means that thephysical state of the triacylglycerols would increase the lipolysisrate by favoring the action of lipases at the water–oil interface.The rate of release of individual fatty acids is as follows: linoleic> oleic > palmitic > stearic > arachidonic (Toldra 1992). Thegeneration rate remains high up to 10 months of processing,when the accumulation of free fatty acids remains asymptotic oreven decreases as a consequence of further oxidative reactions.Oleic, linoleic, stearic, and palmitic acids are those accumulatedin higher amounts because they are present in great amounts inthe triacylglycerols and have a better stability against oxidation.Similar results for generation of oleic, linoleic and palmitic acidswere reported for Chinese Xuanwei ham (Xiao et al. 2010).

OXIDATIVE REACTIONSThe lipolysis and generation of free polyunsaturated fatty acids,susceptible to oxidation, constitute a key stage in flavor gener-ation. The susceptibility of fatty acids to oxidation and the rateof oxidation depend on their unsaturation (Shahidi 1998a). So,linolenic acid (C 18:3) is more susceptible than linoleic acid (C18:2), which is more susceptible than oleic acid (C 18:1). Theanimal species have different susceptibility to autoxidation inthe following order: poultry > pork > beef > lamb (Tichiva-gana and Morrisey 1985). Oxidation has three consecutive stages(Shahidi 1998b). The first stage, initiation, consists in the for-mation of a free radical. This reaction can be enzymaticallycatalyzed by muscle lipoxygenase or chemically catalyzed bylight, moisture, heat, and/or metallic cations. The second stage,propagation, consists in the formation of peroxide radicals by re-action of the free radicals with oxygen. When peroxide radicalsreact with double bonds, they form primary oxidation products,or hydroxyperoxides, that are very unstable. Their breakdownproduces many types of secondary oxidation products by a freeradical mechanism. Some of them are potent flavor-active com-pounds that can impart off-flavor to meat products during cook-ing or storage. The oxidative reactions finish by inactivation offree radicals when they react with each other (last stage). Thus,the result of these oxidative reactions consists in the generationof volatile compounds responsible of final product aroma. Itis important to have a good control of these reactions becausesometimes, oxidation may give undesirable volatile compoundswith unpleasant off-flavors.

Muscle proteins may also experience some oxidative re-actions during the processing of dry-cured ham. Dry-curedham presented an intermediate oxidation when compared to

P1: SFK/UKS P2: SFK



other dry-cured and cooked meat products when based on to-tal protein carbonyls and fluorescence corresponding to proteincarbonyls. But a higher oxidation was reported in dry-curedham when using α-aminoadipic semialdehyde and γ -glutamicsemialdehyde as indicators of protein oxidation (Armenteroset al. 2009a).

Oxidation to Volatile Compounds

As mentioned previously, some oxidation is needed to gener-ate volatile compounds with desirable flavor properties. For in-stance, a characteristic aroma of dry-cured meat products is cor-related with the initiation of lipid oxidation (Buscailhon et al.1994, Flores et al. 1998). However, an excess of oxidation maylead to off-flavors, rancidity, and yellow colors in fat.

The primary oxidation products, or hydroperoxides, are fla-vorless, but the secondary oxidation products have a clear contri-bution to flavor. There are a wide variety of volatile compoundsformed by oxidation of the unsaturated fatty acids. The mostimportant are (1) aliphatic hydrocarbons that result from autoxi-dation of the lipids; (2) alcohols, mainly originated by oxidativedecomposition of certain lipids; (3) aldehydes, which can reactwith other components to produce flavor compounds; and (4)ketones produced through either β-keto acid decarboxylationor fatty acid β-oxidation. Other compounds, like esters, maycontribute to characteristic aromas (Shahidi et al. 1987).

Oxidation rates may vary depending on the type of productor the processing conditions. For instance, TBA (thiobarbituricacid), a chemical index used as an indication of oxidation, in-creases more markedly in products such as Spanish chorizo thanin French saucisson or Italian salami (Chasco et al. 1993). Onthe other hand, processing conditions such as curing or smokingalso give a characteristic flavor to the product (Toldra 2006b).

Antioxidants

The use of spices such as paprika and garlic, which are richin natural antioxidants, protects the product from certain oxida-tions. The same applies to antioxidants such as vitamin E that areadded in the feed to prevent undesirable oxidative reactions inpolyunsaturated fatty acids. Nitrite constitutes a typical curingagent that generally retards the formation of off-flavor volatilesthat can mask the flavor of the product, and allows extendedstorage of the product (Shahidi 1998). Nitrite acts against lipidoxidation through different mechanisms: (1) binding of hemeand prevention of the release of the catalytic iron, (2) binding ofheme and nonheme iron and inhibition of catalysis, and (3) stabi-lization of lipids against oxidation. Smoking also contains someantioxidant compounds such as phenols that protect the externalpart of the product against undesirable oxidations. The muscleantioxidative enzymes also exert some contribution to the lipidstability against oxidation. In the case of fermented meats, themicrobial enzyme catalase degrades the peroxides formed dur-ing the processing of fermented sausages (Toldra et al. 2001).Thus, this enzyme contributes to stabilizing the color and fla-vor of the final sausage. Catalase increases its activity with cellgrowth to a maximum at the onset or during the stationary phase,

but it is mainly formed during the ripening stage. Large amountsof salt exert an inhibitory effect on catalase activity, especiallyat low pH values. The catalase activity is different depending onthe strain. For instance, S. carnosus has a high catalase activ-ity in anaerobic conditions, while S. warneri has a low catalaseactivity (Talon et al. 1999).

REFERENCES

Alasnier C, Gandemer G. 2000. Activities of phospholipase A andlysophospholipases in glycolytic and oxidative skeletal musclesin the rabbit. J Sci Food Agric 80: 698–704.

Aristoy MC, Toldra F. 1995. Isolation of flavor peptides from rawpork meat and dry-cured ham. In: G Charalambous (ed.) FoodFlavors: Generation, Analysis and Process Influence. ElsevierScience Pub., Amsterdam, pp. 1323–1344

Armenteros M et al. 2009a. Analysis of protein carbonyls in meatproducts by using the DNPH method, fluorescence spectroscopyand liquid chromatography-electrospray lonisation-mass spec-trometry (LC-ESI-MS). Meat Sci 83: 104–112.

Batlle N et al. 2001. ATP metabolites during aging of exudative andnonexudative pork meats. J Food Sci 66: 68–71.

Belfrage P et al. 1984. Adipose tissue lipases. In: B Borgstrom,HL Brockman (eds.) Lipases. Elsevier Science Pub., London,pp. 365–416.

Buscailhon S et al. 1994. Time-related changes in intramuscularlipids of French dry-cured ham. Meat Sci 37: 245–255.

Casaburi A et al. 2006. Protease and esterase activity of staphylo-cocci. Int J Food Microbiol 112: 223–229.

Casaburi A et al. 2008. Proteolytic and lipolytic starter culturesand their effect on traditional fermented sausages ripening andsensory traits. Food Microbiol 25: 335–347.

Chasco J et al. 1993. A study of changes in the fat content of somevarieties of dry sausage during the curing process. Meat Sci 34:191–204.

Chen T, et al. 2010. Phospholipase A2 and antioxidant enzymeactivities in normal and PSE pork. Meat Sci 84: 143–146.

Coutron-Gambotti C, Gandemer G. 1999. Lipolysis and oxidationin subcutaneous adipose tissue during dry-cured ham processing.Food Chem 64: 95–101.

Coux O et al. 1996. Structure and function of the 20S and 26Sproteasomes. Ann Rev Biochem 65: 801–847.

Dahlmann et al. 2001. Subtypes of 20S proteasomes from skeletalmuscle. Biochimie 83: 295–299.

Daun C et al. 2001. Glutathione peroxidase activity, tissue andsoluble selenium content in beef and pork in relation to meataging and pig RN phenotype. Food Chem 73: 313–319.

Demeyer DI, Toldra F. 2004. Fermentation. In: W Jensen, C Devine,M Dikemann (eds.) Encyclopedia of Meat Sciences. Elsevier Sci-ence, Ltd., London, pp. 467–474

Demeyer DI et al. 2000. Control of bioflavor and safety in fermentedsausages: First results of a European project. Food Research Int33: 171–180.

Dransfield E et al. 1980–1981. Quantifying changes in tendernessduring storage of beef meat. Meat Sci 5: 131–137.

Etherington DJ. 1984. The contribution of proteolytic enzymes topostmortem changes in muscle. J Anim Sci 59: 1644–1650.

Etherington DJ. 1987. Conditioning of meat factors influencingprotease activity. In: A Romita, C Valin, AA Taylor (eds.) Ac-

P1: SFK/UKS P2: SFK



celerated Processing of Meat. Elsevier Applied Sci., London,pp. 21–28.

Fielding CJ, Fielding PE. 1980. Characteristics of triacylglyceroland partial acylglycerol hydrolysis by human plasma lipoproteinlipase. Biochim Biophys Acta 620: 440–446.

Flores M et al. 1996. Activity of aminopeptidase and lipolytic en-zymes in five skeletal muscles with various oxidative patterns. JSci Food Agric 70: 127–130.

Flores M et al. 1998. Flavour analysis of dry-cured ham. In: FShahidi (ed.) Flavor of Meat, Meat Products and Seafoods.Blackie Academic and Professional, London, pp. 320–341.

Fowler SD, Brown WJ. 1984. Lysosomal acid lipase. In: BBorgstrom, HL Brockman (eds.) Lipases. Elsevier Science Pub.,London, pp. 329–364.

Goll DE et al. 1983. Role of muscle proteinases in maintenance ofmuscle integrity and mass. J Food Biochem 7: 137–177.

Goransson A et al. 2002. Effect of electrical stimulation on theactivity of muscle exoproteases during beef aging. Food Sci TechInt 8: 285–289.

Grossman S et al. 1988. Lipoxygenase in chicken muscle. J AgricFood Chem 36: 1268–1270.

Herrera-Mendez CH et al. 2006. Meat aging: Reconsideration ofthe current concept. Trends Food Sci Technol 17: 394–405.

Hierro E et al. 1997. Contribution of microbial and meat endogenousenzymes to the lipolysis of dry fermented sausages. J Agric FoodChem 45: 2989–2995.

Imanaka T et al. 1985. Positional specificity of lysosomal acid lipasepurified from rabbit liver. J Biochem 98: 927–931

Jimenez-Colmenero J et al. 2010. Nutritional composition ofdry-cured ham and its role in a healthy diet. Meat Sci 84:585–593.

Klement JT et al. 1974. The effect of bacterial fermentation onprotein solubility in a sausage model system. J Food Sci 39:833–835.

Koohmaraie M. 1994. Muscle proteinases and meat aging. Meat Sci36: 93–104.

Koohmaraie M et al. 1987. Effect of postmortem storage on Ca2+-dependent proteases, their inhibitor and myofibril fragmentation.Meat Sci 19: 187–196.

Ludemann V et al. 2004. Determination of growth characteristicsand lipolytic and proteolytic activities of Penicillium strains iso-lated from Argentinian salami. Int J Food Microbiol 96: 13–18.

Marczy JS et al. 1995. Comparative study on the lipoxygenaseactivities of some soybean cultivars. J Agric Food Chem 43:313–315.

Martin L et al. 1999. Changes in intramuscular lipids during ripeningof Iberian dry-cured ham. Meat Sci 51: 129–134.

Matsukura U et al. 1981. Mode of degradation of myofibrillar pro-teins by an endogenous protease, cathepsin L. Biochim BiophysActa 662: 41–47.

McDonald JK, Barrett AJ (eds.). 1986. Mammalian proteases:A glossary and bibliography, vol 2, Exopeptidases. AcademicPress, London.

Mikami M et al. 1987. Degradation of myofibrils from rabbit,chicken and beef by cathepsin L and lysosomal lysates. MeatSci 21: 81–87.

Miller CH et al. 1981. Specificity of lipoprotein lipase and hepatic li-pase towards monoacylglycerols varying in the acyl composition.Biochim Biophys Acta 665: 385–392.

Molly K et al. 1997. The importance of meat enzymes in ripeningand flavor generation in dry fermented sausages. First results ofa European project. Food Chem 54: 539–545.

Monin G et al. 1997. Chemical and structural changes in dry-curedhams (Bayonne hams) during processing and effects of the de-hairing technique. Meat Sci 47: 29–47.

Montel MC et al. 1993. Effects of starter cultures on the biochemicalcharacteristics of French dry sausages. Meat Sci 35: 229–240.

Mora L et al. 2009a. Naturally generated small peptides derivedfrom myofibrillar proteins in serrano dry-Cured Ham. J AgricFood Chem 57: 3228–3234

Mora L et al. 2009b. The degradation of creatin kinase in dry-CuredHam. J Agric Food Chem 57: 8982–8988.

Motilva MJ, Toldra F. 1993. Effect of curing agents and wateractivity on pork muscle and adipose subcutaneous tissue lipolyticactivity. Z Lebensm Unters Forsch 196: 228–231.

Motilva MJ et al. 1992. Assay of lipase and esterase activities infresh pork meat and dry-cured ham. Z Lebensm Unters Forsch195: 446–450.

Motilva MJ et al. 1993a. Subcutaneous adipose tissue lipoly-sis in the processing of dry-cured ham. J Food Biochem 16:323–335.

Motilva MJ et al. 1993b. Muscle lipolysis phenomena in the pro-cessing of dry-cured ham. Food Chem 48: 121–125.

Navarro JL et al. 1997. Lipolysis in dry-cured sausages as affectedby processing conditions. Meat Sci 45: 161–168.

Negre AE et al. 1985. New fluorimetric assay of lysosomalacid lipase and its application to the diagnosis of Wolmanand cholesteryl ester storage diseases. Clin Chim Acta 149:81–88.

Okitani A et al. 1981. Characterization of hydrolase H, a new muscleprotease possessing aminoendopeptidase activity. Eur J Biochem115: 269–274.

Ordonez JA et al. 1999. Changes in the components of dry-fermented sausages during ripening. Crit Rev Food Sci Nutr 39:329–367.

Ouali A. 1991. Sensory quality of meat as affected by muscle bio-chemistry and modern technologies. In: LO Fiems, BG Cottyn,(eds.) Animal Biotechnology and the Quality of Meat Production.Elsevier Science Pub., B.V., Amsterdam, pp. 85–105.

Roseiro LC et al. 2010 Effect of processing on proteolysis and bio-genic amines formation in a Portuguese traditional dry-fermentedripened sausage Chourico Grosso Estremoz e Borba PGI. MeatSci 84: 172–179.

Rosell CM, Toldra F. 1996. Effect of curing agents on m-calpainactivity throughout the curing process. Z Lebensm Unters Forchs203: 320–325.

Sanz Y et al. 2002. Role of muscle and bacterial exopeptidases inmeat fermentation. In: F Toldra (ed.) Research Advances in theQuality of Meat and Meat Products. Research Signpost, Trivan-drum, pp. 143–155

Schwartz WN, Bird JWC. 1977. Degradation of myofibrillar pro-teins by cathepsins B and D. Biochem J 167: 811–820.

Sentandreu MA, Toldra F. 2002. Dipeptidylpeptidase activitiesalong the processing of Serrano dry-cured ham. Eur Food ResTechnol 213: 83–87.

Sentandreu MA et al. 2002. Role of muscle endopeptidases andtheir inhibitors in meat tenderness. Trends Food Sci Technol 13:398–419.

P1: SFK/UKS P2: SFK



Sentandreu MA et al. 2007. Proteomic identification of actin-derived oligopeptides in dry-cured ham. J Agric Food Chem 55:3613–3619.

Shahidi F. 1987. Assessment of lipid oxidation and off-flcour devel-opment in meat, meat products and seafoods. In: F Shahidi (ed)Flavour of meat, meat products and seafoods, 2nd edn. London:Blackie Academic & Professional, pp. 373–419.

Shahidi F. 1998a. Assessment of lipid oxidation and off-flavour de-velopment in meat, meat products and seafoods. In: F Shahidi(ed.) Flavor of Meat, Meat Products and Seafoods. Blackie Aca-demic and Professional, London, pp. 373–394.

Shahidi F. 1998b. Flavour of muscle foods—An overview. In:F Shahidi (ed.) Flavor of Meat, Meat Products and Seafoods.Blackie Academic and Professional, London, pp. 1–4.

Smith LC, Pownall HJ. 1984. Lipoprotein lipase. In: B Borgstrom,HL Brockman (eds.) Lipases. Elsevier Science Pub., London,pp. 263–305.

Stahnke LH. 2002. Flavour formation in fermented sausage. In: FToldra (ed.) Research Advances in the Quality of Meat and MeatProducts. Research Signpost, Trivandrum, pp. 193–223.

Sunesen LO, Stahnke LH. 2004. Mould starter cultures fordry sausages: Selection, application and effects. Meat Sci 65:935–948.

Talon R et al. 1999. Effect of nitrate and incubation conditions onthe production of catalase and nitrate reductase by staphylococci.Int J Food Microbiol 52: 47–56.

Talon R et al. 2002. Bacterial starters involved in the quality offermented meat products. In: F Toldra (ed.) Research Advancesin the Quality of Meat and Meat Products. Research Signpost,Trivandrum, pp. 175–191.

Tichivagana JZ, Morrisey PA. 1985. Metmyoglobin and inorganicmetals as prooxidants in raw and cooked muscle systems. MeatSci 15: 107–116.

Tjener K et al. 2004. Growth and production of volatiles by Staphy-lococcus carnosus in dry sausages: Influence of inoculation leveland ripening time. Meat Sci 67: 447–452.

Toldra F. 1992. The enzymology of dry-curing of meat products.In: FJM Smulders et al. (eds.) New Technologies for Meat andMeat Products. Audet, Nijmegen, pp. 209–231.

Toldra F. 1998. Proteolysis and lipolysis in flavour development ofdry-cured meat products. Meat Sci 49: s101–s110.

Toldra F. 2002. Dry-Cured Meat Products. Food and Nutrition Press,Trumbull, CT, pp. 1–238.

Toldra F. 2004a. Dry-cured ham. In: YH Hui et al. (eds.) Handbookof Food and Beverage Fermentation Technology. Marcel Dekker,New York, pp. 369–384.

Toldra F. 2004b. Curing: Dry. In: W Jensen, (eds.) Ency-clopedia of Meat Sciences. Elsevier Science, Ltd., London,pp. 360–365.

Toldra F. 2006a. Meat: Chemistry and biochemistry. In: YH Huiet al. (eds.) Handbook of Food Science, Technology and Engi-neering. CRC Press, Boca Raton, FL, vol 4, pp. 28–1 a 28–18.

Toldra F. 2006b. Dry-cured ham. In: YH Hui et al. (eds.) Handbookof Food Science, Technology, and Engineering. CRC Press, BocaRaton, FL, vol. 4, pp. 164–1 a 164–11.

Toldra F. 2007. Biochemistry of muscle and fat. In: F Toldra et al.(eds.) Handbook of fermented meat and poultry. Blackwell Pub-lishing, Ames, IA, pp. 51–58.

Toldra F, Aristoy MC. 2010. Dry-cured ham. In: F Toldra (ed.)Handbook of Meat Processing. Wiley-Blackwell, Ames, IA,pp. 351–362.

Toldra F, Flores M. 1998. The role of muscle proteases and lipasesin flavor development during the processing of dry-cured ham.CRC Crit Rev Food Sci Nutr 38: 331–352.

Toldra F, Reig M. 2007. Sausages. In: YH Hui et al. (eds.) Handbookof Food Product Manufacturing, vol 2. John Wiley Interscience,New York, pp. 249–262.

Toldra F et al. 1992. Activities of pork muscle proteases in curedmeats. Biochimie 74: 291–296.

Toldra F et al. 1993. Cathepsin B, D, H and L activity in the pro-cessing of dry-cured-ham. J Sci Food Agric 62: 157–161.

Toldra F, et al. 2000. Contribution of muscle aminopeptidases toflavor development in dry-cured ham. Food Res Internat 33:181–185.

Toldra F et al. 2001. Meat fermentation technology. In: YH Hui,WK Nip, RW Rogers, OA Young (eds.) Meat Science and Appli-cations. Marcel Dekker, New York, pp. 537–561.

Toldra F et al. 2004. Packaging and quality control. In: YH Hui et al.(eds.) Handbook of Food and Beverage Fermentation Technology.Marcel Dekker, New York, pp. 445–458.

Tornquist H et al. 1978. Enzymes catalyzing the hydrolysis of long-chain monoacylglycerols in rat adipose tissue. Biochim BiophysActa 530: 474–486.

Valin C, Ouali A. 1992. Proteolytic muscle enzymes and post-mortem meat tenderisation. In: FJM Smulders et al. (eds.) NewTechnologies for Meat and Meat Products. Audet, Nijmegen,pp. 163–179.

Vestergaard CS et al. 2000. Lipolysis in dry-cured ham maturation.Meat Sci 55: 1–5.

Xiao S et al. 2010. Changes of hormone-sensitive lipase (HSL),adipose tissue triglyceride lipase (ATGL) and free fatty acids insubcutaneous adipose tissue throughout the ripening process ofdry-cured ham. Food Chem 121: 191–195.

Yamamoto S. 1992. Mammalian lipoxygenases: Molecular struc-tures and functions. Biochim Biophys Acta 1128: 117–131.

Yates LD et al. 1983. Effect of temperature and pH on thepost-mortem degradation of myofibrillar proteins. Meat Sci 9:157–179.

Young JF et al. 2003. Significance of preslaughter stress anddifferent tissue PUFA levels on the oxidative status and sta-bility of porcine muscle and meat. J Agric Food Chem 51:6877–6881.

Zeece MG, Katoh K. 1989. Cathepsin D and its effects on myofib-rillar proteins: A review. J Food Biochem 13: 157–161.

food biochemistry and food processing (simpson/food biochemistry and food processing) ||...

Documents