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Thirty-six carcasses from 6-month-old pigs were classified in different exudative groups based on measurements of pH2h, pH24h, the colour parameter L∗ and drip loss. A fraction containing polypeptides between 66 and 21 kDa was analysed by reverse phase chromatography at 2-h post-mortem and the evolution of 8 polypeptide fractions followed during ageing and related to meat quality. Three polypeptide (fractions P2, P3 and P4) at 2-h post-mortem showed significant lowest area values in the dark firm and dry class. During ageing, the higher content of P4 in exudative meats at 8-h post-mortem could be due to activation of the cathepsin system. On the other hand, P3 and P4 increased in DFD meats during the first 96-h post-mortem probably due to higher calpain activity. Few differences in polypeptides were related to meat qualities although they are important as precursors of small peptides and free amino acids.

The content of nucleotide metabolites in the muscle L.dorsi from 9 exudative and 9 acceptable quality pork carcasses was determined during 7 d of aging in order to establish a period postmortem for detecting exudative meats. Hypoxanthine and inosine levels in exudative meats were different (p < 0.05) from those of normal meats up to 3 days of aging, while 5′-inosine monophosphate (IMP) and 5′-adenosine triphosphate (ATP) contents only differed during the 1st 4 and 6 h postmortem, respectively. The IMP/ATP ratio was only different (p < 0.05) at 2 h. Thus, 2 h postmortem is proposed as the optimal sampling time for the detection of exudative pork meats.

Muscle proteins, peptides and free amino acids were analysed at 2 h post-mortem as potential markers of different pork meat qualities. The meat quality classification was based on the measurements: pH2h, pH24h, the colour parameter L, and drip loss. The measurement of proteolytic fragments by SDS-PAGE revealed that the nebulin band was different among classes being more defined in the dark, firm and dry meat class. The detection and characterisation of small peptide fragments resulting from protein breakdown was done by isolation through cationic and reverse-phase chromatography. The analysis of peptide fractions isolated by reverse-phase chromatography revealed that could be used as potential markers of meat quality. So, peptide fraction 1 could be used to distinguish exudative from non-exudative meats and peptide fraction 4 distinguished dark, firm and dry meat from the rest. The concentration of free amino acids present in muscle was not different among qualities, probably due to the early post-mortem time (2 h) used in the study.

The content of nucleotide metabolites in the muscle L. dorsi was analyzed for the detection of pale, soft and exudative (PSE) pork meats at just 2 h postmortem. PSE meat was characterized by significantly lower (p < 0.05) amounts of adenosine 5′-triphosphate (ATP) and higher adenosine 5′-monophosphate (AMP), inosine 5′-monophosphate (IMP), inosine (ino) and hypoxanthine (hyp) than normal meat. IMP and ino classified all the samples from both groups with statistical significance (p < 0.05). The K0 value, R′ value and IMP/ATP ratios were also useful indicators for full distinction of PSE meats. Thus, the assay of any of these nucleotide metabolites may allow a good detection of PSE meats at very short, only 2 h, postmortem time.

The activity of muscle aminopeptidases (alanyl, arginyl, leucyl and pyroglutamyl aminopeptidases) have been assayed along the processing of dry-cured ham. The generation of free amino acids resulting from aminopeptidase action on N-terminal of proteins and peptides has been also analyzed. The assayed aminopeptidases, except pyroglutamyl aminopeptidase, showed good stability. Alanyl and arginyl aminopeptidases have optimal neutral pH near the pH in ham and, in addition, their spectrum of activity against terminal amino acids is in coincidence with the observed release of free amino acids in ham. So, both aminopeptidases appear to be the main contributors to the generation of free amino acids during the processing of dry-cured ham.

Strains of Lactobacillus plantarum originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening, L. plantarum CRL 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE. The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine. In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE.

Lactobacillus casei CRL 705 was screened, among other meat isolates, for its proteinase and aminopeptidase activities toward synthetic substrates and, according to that, selected for specific assays on muscle proteins. The hydrolytic effects of whole cells, cell free extracts (CFE), and the combination of both on muscle sarcoplasmic and myofibrillar protein extracts was evaluated by SDS-PAGE and reverse phase HPLC analyses. The proteinase activity of whole cells caused the degradation of a great number of sarcoplasmic protein bands. A partial hydrolysis was also associated with CFE that when combined with whole cells showed an important additional degradation. Peptide profiles from sarcoplasmic protein extracts were greatly modified regardless of the addition of whole cells or CFE, although their combination intensified these changes. The generation of free amino acids was remarkable when whole cells and CFE were incorporated together to sarcoplasmic protein extracts.

Five sire types, namely Danish Duroc (DU), Dutch Large White (LWD), English Large White (LWE), Belgian Landrace × Landrace (BL × LR) and Belgian Landrace (BL), were mated with LR × LW crossbred sows. Dipeptidylpeptidase and aminopeptidase activities and free amino acid and natural dipeptide contents were compared between the offspring of each sire type. Regarding the sire type effect, significant differences were detected in dipeptidylpeptidase I and IV and alanyl and pyroglutamyl aminopeptidase activities, with activity being lowest in the offspring of BL sires. Significant differences were also observed between sire types in the content of aspartic acid, glutamic acid, serine, glycine, glutamine, histidine and arginine. Taking into account the role of exopeptidases and free amino acids in taste development, the meat from BL-sired pigs may be considered as inferior in generating flavour precursors and thus not appropriate to undergo a dry-curing process. © 1999 Society of Chemical Industry

Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were selected on the basis of their proteolytic activities against synthetic substrates. Further, the effects of whole cells, cell extracts, and a combination of both enzymatic sources on muscle sarcoplasmic proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography analyses. Strains of both species displayed proteinase activities on five sarcoplasmic proteins. The inoculation of whole cells caused a degradation of peptides, whereas the addition of cell extracts resulted in the generation of both hydrophilic and hydrophobic peptides. This phenomenon was remarkably more pronounced when L. curvatus was involved. Whole cells also consumed a great amount of free amino acids, while the addition of intracellular enzymes contributed to their generation. L. sake accounted for a greater release of free amino acids. In general, cell viability and also proteolytic events were promoted when cell suspensions were provided with cell extracts as an extra source of enzymes.

[EN] Sodium intake exceeds the nutritional recommendations in most industrialized countries becoming one concern for public health. This elimination or reduction is not simple due to its role in final food sensory, quality and safety. The aim of this work is to review the possibilities of magnesium ion, due to its healthy properties, to become a partial substitute of sodium in the production of fish and meat products, and a particular case for Spanish dry-cured ham and loin. Magnesium diffusion into different muscle based foods such as ham or loin, and its effect in the most important characteristics of the final product (microbiology, physico-chemical and sensory properties) has been analyzed. Results show that magnesium has more difficulty to penetrate inside the muscle and slightly modifies the water-holding capacity of proteins, their solubility and the enzymatic activity. Salty taste, bitterness and off-flavor are the most affected characteristics. However, these effects could be compensated by using longer post-salting periods and by employing masking agents. It is possible to reduce the sodium content in fish and meat products using magnesium as one of the ingredients, allowing to obtain new products with similar physicochemical characteristics and safety conditions., Grant AGL2007-65379-C02-01 & 02 and AGL2010-16305 from Ministry of Science and Innovation (Spain) and FEDER funds are acknowledged.

Foods and especially food hydrolyzates, may contain a large amount of peptides and some interfering substances that require their primary separation prior to MS identification. In other cases, some target peptides might be present in very low concentration, requiring a pre-concentration of the sample before further analysis by MS. These are essential steps for optimal results. The peptide purification process should be as simple as possible and may contain a combination of two different types of chromatography. This chapter is focused on the chromatographic methods used in the primary separation of peptides.

[EN] While muscle foods are the more commonly consumed portion of an animal, meat by-products such as the entrails and internal organs are also widely consumed. Considered high-priced delicacies or waste material to be tossed away, the use and value of offal-edible and inedible meat by-products depend entirely on the culture and country in question. The skin, blood, bones, meat trimmings, fatty tissues, horns, hoofs, feet, skull, and internal organs of harvested animals comprise a wide variety of products including human or pet food or processed materials in animal feed, fertilizer, or fuel. Industry is using science and innovation to add value to animal by-products far beyond its usual profitability. Regardless of the final product's destination, it is still necessary to employ the most up-to-date and effective tools to analyze these products for nutritional properties, to search for key active molecules in nutrition like bioactive peptides, food safety (antimicrobial peptides), medicine, cosmetics or other fields, to develop new technological applications and to continue innovation towards advanced value-addition of meat by-products. © 2012 Elsevier Ltd., Grant from Vaquero Foundation for R + D on Pork Meat (Madrid, Spain), grant AGL2010-16305 from the Spanish Ministry of Science and Innovation and FEDER funds are acknowledged. Work was prepared within the Unidad Asociada IAD (UPV)-IATA (CSIC) framework.

[EN] Simultaneous brine thawing/salting process was applied as an alternative to traditional pile salting process using 51 frozen Iberian hams. The effect of this type of salting process on endogenous enzyme activity and sensory quality of Iberian dry-cured hams was analysed. The frozen hams were simultaneously thawed and salted with saturated brine, with and without vacuum pulses, and were compared to hams thawed under refrigeration and traditionally salted. The peptidase and lipase activities were measured at the end of salting and post-salting stages. The activity of cathepsin B+L was reduced in the two brine salted batches while few differences among batches were observed for the other peptidases. Several lipase activities were significantly reduced in the two brine salted batches. The brine thawed processing affected the free fatty acid content at the different stages although the differences were more appreciated at the beginning of the process and no differences were observed at the end. The long ripening time makes these differences negligible and the consumer did not appreciate any differences between the sensory quality of Iberian brine/thawed hams and traditional Iberian thawed pile salted hams. (C) 2011 Elsevier Ltd. All rights reserved., Financial support from PETRI by the Ministry of Science and Innovation (PTR-1995-0754-OP-03) is fully acknowledged. Work was carried out under the Unidad Asociada IIAD (UPV) - IATA (CSIC) framework. The authors are grateful to M. Lozano and MI. Nadal for technical assistance.

HPLC methods based on hydrophilic interaction chromatography (HILIC) have been recently applied to the analysis of biochemical compounds, related to the quality, in meat and poultry as well as meat products like cooked ham and dry-cured ham. This type of chromatography has also been successfully used for the analysis of antibiotic residues in meat and poultry. HILIC chromatography has an increasing relevance for the analysis of polar compounds in foods due to its good retention without need for derivatisation. In general, HILIC-based methods have shown good performance for the analysis of polar biochemical compounds like dipeptides, creatine and creatinine, nucleotides and nucleosides, sphingolipids and residues, mainly antibiotics, in rather complex matrices such as muscles. Thus, this manuscript presents a review of recent applications of HILIC methods for the analysis of quality-related biochemical compounds and residues in meat, poultry and processed meats. © 2010 Springer Science+Business Media, LLC., Grant AGL2007-65379-C02-01 from Ministry of Science and Innovation (Spain) and FEDER funds are fully acknowledged. FPU Scholarship from Ministerio de Ciencia y Educacion (Spain) to L. M. and scholarship from Consejo Nacional de Ciencia y Tecnologia (CONACyT) and Colegio de Postgraduados (Mexico) to A. S. H-C. are fully acknowledged. This work was performed under the Associated Unit IAD (UPV)-IATA (CSIC).

This work describes the correlation found along 10 days between potentiometric measurements obtained by using an electronic tongue and the variation in certain physicochemical, microbial and biochemical parameters measured on a whole piece of pork loin stored under refrigeration. The electronic tongue consists of a set of six electrodes made of Au, Ag, Cu, Pb, Zn and C, and a reference electrode. Through the use of various multivariate analysis techniques, such as: PCA and two types of artificial neural networks (i.e. multilayer perceptron (MLP) and fuzzy ARTMAP) it was found that it is possible to determine the time elapsed in relation to the degradation of the loin by using simple potentiometric measurements. Additionally, in the same pork sample used to measure redox potentials with the electronic tongue, the following parameters were also determined; pH, microbial count, concentrations of inosine 5'-monophosphate (IMP), inosine (Ino) and hypoxanthine (Hx). Through the use of PLS analysis, it was found a rather good correlation between pH and the potentiometric data. Also a remarkable correlation was observed between the measures carried out with the electronic tongue and the so-called K-index that simultaneously measures the variation in the adenosine triphosphate (ATP) degradation products. These results suggest that this simple, or a similar electronic tongue, could be useful for the undemanding qualitative or semi-quantitative evaluation of freshness in meat samples in a wide range of situations. © 2010 Elsevier Ltd. All rights reserved., We would like to thanks the Spanish Government for support (Project CTQ2006-15456-C04-01/BQU and AGL2007-65379-C02-01 and 02/ALI). This work has been partially carried out under Associated Unit framework between IIAD (UPV) and IATA (CSIC).

Freshness in one of the main quality attributes for fish commercialization and consumption. The traditional method for fish freshness evaluation is sensory analysis. However, instrumental methods such as electrical, texture and colour measurements, image analysis, VIS spectroscopy and electronic noses have been widely studied as objective alternatives. Each of these methods has advantages and disadvantages, but none of them can be universally proposed for defining and measuring fish freshness. This work evaluated the correlation of potentiometric measurements, obtained with gold and silver electrodes, with physicochemical, microbiological and biochemical analyses of sea bream stored under refrigeration. Results showed a strong correlation of the potentiometric measurements with the determined changes in fish, and an important correlation with the K1 index, dependent on the nucleoside degradation, which is used as a good indicator of post-mortem time and freshness.

In a previous study, the brine thawing/salting operation using frozen hams as raw material was studied as a valid alternative for the accelerated processing of dry-cured hams. But no information was available on how this treatment could affect some important biochemical mechanisms and the sensory quality of hams. The aim of this work was to study the influence of the simultaneous brine thawing/salting operation on proteolysis and sensory acceptability of the produced dry-cured hams. The results confirm that dry-cured hams can be produced by using brine thawing/salting with a substantial reduction in the thawing and salting time needed. This accelerated process resulted in similar or even better sensory preferences than hams produced through the traditional method. However, the preference of consumers based on the appearance was lower for most of the hams than when using the traditional method, probably due to a wider slice section of the brined hams that can be corrected by adequate pressure during the salting. Thus, this treatment can be used without affecting the quality of dry-cured hams.

The effects of the addition of a combined cell-free extract from Lactobacillus sakei and Debaryomyces hansenii (D + L) or just a D. hansenii cell-free extract (D) to the initial formulation of a dry-fermented sausage were evaluated. The differences found among batches in the main microbial populations, pH, moisture content and global proteolytic and lipolytic indexes (total free amino acids, non protein nitrogen, acidity and tiobarbituric acid index) were not significant. Only, the acidity value of batch D was significantly higher (p < 0.05) than that of batch D + L. Thus, cell-free extract from D. hansenii accelerated the lipolysis. Moreover, there were some significant differences (p < 0.05) in the amino acid profile and, especially, in the aroma profile. The combination D + L and D promoted the generation of volatile compounds derived from lipid oxidation and carbohydrate fermentation. In batch D, the production of volatile compounds derived from amino acid catabolism and microbial fermentation was also enhanced. The overall quality was improved by both treatments (D + L, D) and also the aroma by addition of the combination of extracts (D + L). It is concluded that the addition of cell-free extracts from D. hansenii and, particularly, D. hansenii plus L. sakei could be useful to improve the final quality of fermented sausages.

The protease B (PrB; EC. 3.4.21.48) of Debaryomyces hansenii CECT 12487 was purified by selective fractionation with protamine sulfate followed by three chromatographic separations. The whole procedure resulted in 324-fold purification with a recovery yield of 1.0%. PrB was active at neutral-basic pH ranging from 6.0 to 12.0 with an optimum at pH 8.0. The molecular mass of the denatured enzyme was 30 kDa. Polyclonal-antibodies raised against PrB from Saccharomyces cerevisiae cross-reacted with the corresponding 30-kDa protein from D. hansenii. The serine protease inhibitor 3,4-DCI and sulphydryl group reagents markedly reduced the enzyme activity. The Km against N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was 1.79 mM. The presence of endogenous inhibitor for PrB was detected in cell-free extracts of D. hansenii although their inhibitory effect was lost after incubation at 25 °C for 20 h. PrB was able to hydrolyze muscle sarcoplasmic proteins by in vitro assays. This is the first endopeptidase purified and characterized from the yeast D. hansenii, whose possible contributions to meat fermentation processes are discussed.

The flavor of a food is one of the principle factors involved in a consumer’s purchase decision; meat is no exception. This makes it necessary for food technologists to have a thorough understanding of flavors and changes in flavors if they are to prepare products that consumers will purchase repeatedly. Such knowledge is particularly important in meat and meat products, since the development and deterioration of meat flavor is a continual process (Spanier et al., 1988, Spanier et al.,1990, Spanier et al.,1992a, Spanier et al.,1993; St. Angelo et al., 1988) that involves both the generation and loss of desirable flavor components (St. Angelo et al., 1988; Drumm and Spanier 1991; Spanier and Drumm-Boylston 1994) and the formation of off-flavor compounds (Bailey 1988; Spanier et al., 1992b; St. Angelo et al., 1992; Timms and Watts 1958). The development of many of these flavor producing components is associated with the process of postmortem aging (Spanier et al., 1997), with the end-point cooking temperature (Spanier et al., 1996), and with the process of lipid oxidation (St. Angelo 1992). Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned

A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides. The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.

Strains of Debaryomyces hansenii originally isolated from sausages were screened for proteinase and aminopeptidase activity towards synthetic substrates. On the basis of these results, D. hansenii CT12487 was selected for further assays. The activities of the whole cells (WC), cell-free extracts (CFE) and a combination of both from the selected strain on pork muscle sarcoplasmic protein extracts were determined by protein, peptide and free amino acid analyses. There was a pronounced hydrolysis of protein bands of 110 kDa and 27-64 kDa regardless the incorporation of WC, CFE or a combination of both. The proteolytic activity also resulted in the generation of polar and non-polar peptides showing noticeable differences depending on the addition of WC or CFE. Whole cells generated greater amounts of free amino acids than the cell-free extracts.

Twenty pork carcasses were classified in different pork meat qualities: red, firm and non-exudative (RFN), pale, soft and exudative (PSE), red, soft and exudative (RSE) and dark, firm and dry (DFD) meat. The content of peptides and free amino acids during the ageing process was analysed and compared within quality classes. Four peptide fractions were isolated through cation-exchange and reverse-phase chromatography. The main significant differences among qualities were obtained for peptide fractions 3 and 4. Peptide fraction 3 at 4 days and peptide fraction 4 at 2 h postmortem were higher in the ideal pork quality (RFN) than in the other quality classes. The ageing of pork meats produced a general increase in all free amino acid concentrations for the studied quality classes except for Gln, β-Ala, Taurine and Orn and the dipeptides carnosine and anserine. The DFD class showed higher increases in Lys, Ala and Met probably due to the activation of neutral aminopeptidases.

Muscle longissimus dorsi excised at 2hr post mortem was utilised to study the effect of post-mortem meat quality, and their content of nucleotide metabolites, on the sensory characteristics of cooked loin. Four different post mortem meat qualities were defined: pale soft exudative (PSE), red soft exudative (RSE), red firm non-exudative (RFN) and dark firm dry (DFD). The DFD meat quality class showed low sour and salty tastes and a high sweet taste, low astringency and high juiciness and tenderness, giving the best overall quality. On the other hand, the RSE quality class produced a meat with a low juiciness and high hardness, and a low overall quality although these differences were not significant (p0.05) against RFN and PSE meats. The effect of nucleotide metabolite contents on the sensory meat properties were most significant on taste although, the different meat qualities were only significantly different (p0.05) in the content of hypoxanthine, guanosine, GDP and IDP.

Free amino acids and natural dipeptides, have been analyzed in pork muscles having different metabolic type (masseter, trapezius, semimembranosus and longissimus dorsi). Carnosine and anserine showed significantly higher (p < 0.05) concentrations with the glycolytic activity of the muscle while taurine and glutamine were significantly higher in the oxidative muscles. Non-essential free amino acids also showed significant (p < 0.05) increased content in the oxidative muscles.

The activity of muscle aminopeptidase B, alanyl aminopeptidase, acid lipase and acid esterase were assayed in pork muscles of different metabolic type. The muscles were Masseter, Trapezius, Semimembranosus, Biceps Femoris and Longissimus dorsi and some significant differences (P > 0.05) were found in enzyme activities. Alanyl aminopeptidase showed lower activity in Masseter and Biceps femoris while aminopeptidase B had lower activity in Biceps femoris. Acid lipase and acid esterase showed lower activity in Trapezius and higher in Masseter. However, no clear relationship could be established between the assayed enzyme activities and the metabolism of the muscles.

Proteolytic events taking place during prok meat ageing and curing processes are described. Muscle cathepsins and calpains provide suitable substrate for muscle peptidases and aminopeptidases which ultimately generate small peptides and free amino acids. The levels of small peptides and free amino acids in all meats are higher after processing than before as a result of intense action of those proteases. Increases of the free essential amino acids such as valine, methionine, isoleucine, leucine, phenylalanine, tryptophan and lysine are observed along the conditioning of meat and are especially large in the processing of dry-cured ham. Thus, aged meat but very especially dry-cured ham have an important nutritional significance when the energy intake is low or in poor nutritional quality diet.

Many flavor peptides in raw pork meat and dry-cured ham are generated as a consequence of muscle protein degradation. Low molecular mass peptides (Mr

Alanyl and arginyl aminopeptidases from porcine skeletal muscle were purified, and the effect of free amino acids on their activities was studied. Both aminopeptidases were inhibited by free amino acids via the competitive mode and were more strongly inhibited in the pure form than when they were in the meat extract. Arginyl aminopeptidase (RAP)was less susceptible to inhibition by free amino acids than alanyl aminopeptidase (AAP), as shown especially by the strong inhibitory effect of hydrophobic amino acids on AAP activity in the meat extract. The free amino acid concentration present in meat products such as dry-cured loin and dry-cured ham produced a strong inhibitory effect on AAP and RAP activity. The concentration of free amino acids in cooked ham mainly affected the activity of AAP. These facts can explain the differences obtained in free amino acid concentration depending on the kind of process or product. Keywords: Aminopeptidase; muscle exopeptidases; peptidase inhibitor; amino acid

The effect of curing agents (salt, nitrite, nitrate, ascorbic acid, glucose and polyphosphates) on the main pure aminopeptidases involved in the release of amino acids during the processing of cured meat products has been studied. Salt strongly inhibited alanyl aminopeptidase and pyroglutamyl aminopeptidase: about 40–50% of their original activities were recovered in the presence of 4–5% salt, the typical concentration in dry-cured meat products, while leucyl aminopeptidase was not affected. However, arginyl aminopeptidase was activated to 2–3 times its control level. Nitrate and nitrite did not affect aminopeptidase activity. Ascorbic acid (500 mg/l) slightly inhibited arginyl, alanyl and pyroglutamyl aminopeptidases whereas glucose (20 g/l) activated (to almost 5 times its control level) leucyl aminopeptidase activity. Polyphosphates inhibited (25%) alanyl and arginyl aminopeptidases.

Alanyl aminopeptidase from porcine skeletal muscle was purified and its activity against alanine oligopeptides studied using capillary electrophoresis. The purpose was to determine the effect of the chain length of alanine oligopeptides on aminopeptidase activity. The enzyme showed high specificity against penta- and tetraalanine and absence of activity against dialanine. In this way, dipeptides and free amino acids would be generated in meat processing. Thus, the presence of a specific amino acid on the N-terminal end of a peptide is determinant for the enzyme activity and the length of that peptide also affects the hydrolysis rate.

An enzyme sensor employing diamine oxidase from porcine kidney in combination with an oxygen electrode has been developed and optimised to estimate the content of total amines in dry-fermented sausages. The enzyme DAO was immobilised on a preactivated Immunodyne membrane using glutaraldehyde as cross-linking agent. The enzymatic determination was based on the measurement of the consumed O2 in a platinum electrode poised at −600 mV versus Ag/AgCl. The reaction was started by the direct injection of either the standard or meat extract on the enzymatic membrane. The immobilised enzyme could be used up to 30 analyses along the day without significant loss of sensitivity and could be stored at 4 °C showing good stability for at least 6 weeks. A good correlation was observed when comparing data obtained with the enzyme sensor to those obtained with a standard HPLC method. Thus, this sensor may be a reliable screening method to detect the presence of biogenic amines in dry-fermented sausages that can constitute a useful tool for quality control in the meat industry.

Samples of muscle Semimembranosus were removed from light and heavy pork hams 3 days after slaughtering and analysed for chemical composition and proteolytic and lipolytic enzyme activity. Light hams were higher in moisture content and cathepsin (B and B+L) activity, and lower in peptidase (pyroglutamyl and dipetidyl IV) activity; the absence of further differences in proteolytic and in lipolytic enzymes could be due to large variation coefficients found within both ham classes. The use of principal components analysis displayed distinct patterns for light and heavy samples, with the latter being mainly characterised by greater peptidase-to-proteinase ratio and, to a lesser extent, by higher lipase activity. It was concluded that different chemical and enzyme patterns in fresh meat might be the cause of the wide variations in protein and lipid breakdown mechanisms that are often encountered in dry-cured pork derivatives.

The effect of biogenic polyamines (agmatine, cadaverine, putrescine, spermidine, and spermine) on the activity of pork muscle aminopeptidase and alanyl aminopeptidase was investigated. Agmatine (5 mM) showed a powerful inhibitory effect for both enzymes (more than 50% inhibition). Cadaverine and less intensely putrescine showed similar inhibitory effects for both enzymes. All assayed polyamines inhibited activity of both aminopeptidases except spermine and spermidine which did not affect aminopeptidase B. Thus, one of these inhibitors could be used to avoid the interference of alanyl aminopeptidase when assaying aminopeptidase B in muscle extracts.

Following long dry-curing periods, Spanish “Serrano” ham develops a specific “dry-cured ham” flavor. To determine the effects of length of curing on flavor generation, replicate hams were processed for 7 or 12 mo prior to amino acid, peptide, and flavor evaluation by reverse phase HPLC, capillary zone electrophoresis, and sensory analysis, respectively. Generation of dry-cured and pork flavors correlated with the accumulation of amino acids. The relation of these components with sensory descriptors was examined by factor analysis. Results indicated that combinations and proportions of taste-active components produced the specific dry-cured flavor characteristic rather than an accumulation of any single flavor component.

The effect of endogenous dipeptides (carnosine and anserine) and peptidic fractions from meat extracts on aminopeptidase activity, alanyl and arginyl aminopeptidases purified from porcine skeletal muscle, was studied. Carnosine inhibited the activity of porcine arginyl aminopeptidase (RAP); the inhibition being stronger with the purified form of the enzyme than in the muscle extract. The RAP inhibition was competitive. One of the peptidic fractions isolated from meat extracts inhibited RAP activity But the degree of inhibition depended on the extent of purification. The major aminopeptidase present in muscle, alanyl aminopeptidase (AAP), was neither inhibited by natural dipeptides nor arty of the peptidic fractions. The study of meat inhibitory peptides provides a better understanding of the action of proteolytic enzymes during meat processing.