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Fat, composed mainly of triacylglycerols and phospholipids, and protein, composed of amino acids, constitute major components of meat. Carbohydrate, in the form of glycogen, is present in very low amounts. This article describes the main methodologies available for the analysis of these substances, discusses their advantages and disadvantages, and gives recommendations for the choice of methods.

Dry-cured hams are a good source of bioactive peptides, whose stability to gastrointestinal (GI) digestion determines their bioaccessibility and thus their bioavailability to exert beneficial effects. The aim of this study was to evaluate the effect of in vitro GI digestion of dry-cured hams on the peptide profiles and their antioxidant activity. Results showed that the antioxidant activity decreased in the digested samples when measured by DPPH radical scavenging activity and ferric-reducing antioxidant power methods, but increased when assayed by ABTS radical scavenging assay. Chromatographic and mass spectrometry analysis revealed changes in the peptide profiles and evidenced the degradation of proteins, mainly titin, by the digestive enzymes. The study of the peptide profiles before and after digestion of dry-cured hams would help to extend the knowledge about the influence of peptide characteristics on their antioxidant activity and resistance to hydrolysis by digestive enzymes, allowing a better understanding of the bioaccessibility of the bioactive peptides generated in dry-cured hams., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged.

Peptidyl post-translational modifications (PTMs) could influence the final quality of processed meat. In this study, the peptide oxidative phenomena in Spanish dry-cured ham (Biceps femoris muscle) was evaluated at different ripening times (9, 12, 15, 18 and 24 months of processing) evidencing interactions amongst the lipid and protein oxidation, major peptidyl PTMs and the release of free amino acids (FAAs). Results showed that 12 months of processing enabled the most abundant protein-bound carbonyls, while TBARS value was significantly favored (p < 0.001) by ripening. However, FAAs were still intensively generated during overall ripening. Peptidomics and chemometrics further revealed that proteolysis mostly hampered the oxidized peptides rather than the deamidated ones during ripening. Myosin light chain (MYL1 and MYL3) showed high oxidative susceptibility owing to peptidyl methionine and proline oxidation as well as acetaldehyde adduct formation on lysine or histidine residues., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, and Ramón y Cajal postdoctoral contract by L.M. are acknowledged. C.L. appreciated the financial support from China Scholarship Council (CSC) through the State Scholarship Fund for Overseas Study (No. 201806760050). The peptidomic analysis was performed in the proteomics facility of SCSIE University of Valencia. This proteomics laboratory is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013-2016, funded by ISCIII and ERDF

Peptides and free amino acids are naturally generated in dry-cured ham as a consequence of proteolysis phenomenon exerted by muscle peptidases. The generation of bioactive peptides in different types of dry-cured ham produced in Spain, Italy and China is reviewed in this manuscript. Major muscle proteins are extensively hydrolysed firstly by endogenous endo-peptidases followed by the successive action of exo-peptidases, mainly, tri- and di-peptidylpeptidases, aminopeptidases and carboxypeptidases. Such proteolysis is very intense and consists of the generation of large amounts of free amino acids and a good number of peptides with different sequences and lengths, some of them exerting relevant bioactivities like angiotensin converting enzyme inhibitory activity, antioxidant activity, di-peptidylpeptidase IV inhibitory activity among other and in vivo antihypertensive, hypoglycemic or anti-inflammatory activity. This manuscript reviews the recent findings showing that dry-cured ham constitutes a good source of natural bioactive peptides that have potential benefit for human health., The research leading to these results received funding from Grant AGL2017-89831-R from the Spanish Ministry of Economy, Industry and Competitivity and FEDER funds The Ramón y Cajal postdoctoral contract to LM is also acknowledged.

Angiotensin converting enzyme (ACE) inhibitory peptides have been extensively studied as an alternative to synthetic drugs for the treatment of hypertension. Recent studies have shown that dry-cured ham is an important source of naturally generated bioactive peptides, especially showing ACE inhibitory activity. However, due to their excessive degradation by digestive and brush-border enzymes, it is not clear whether these peptides resist intestinal absorption and reach the blood stream where they may exert their antihypertensive effect. Therefore, dry-cured ham extracts and specific pure peptides naturally generated during the dry-curing process, showing ACE inhibitory activity, have been studied for their stability during transepithelial transport in a Caco-2 cell monolayer. The ACE inhibitory activity of transport samples was assayed, showing the highest values in apical samples at 15 min of incubation. In concentrated basal solutions, ACE inhibition values increased during transport for purified peptides, reaching values close to 70% for AAPLAP and KPVAAP at 60 min of cellular transport. Fragments generated by cellular activity were detected by using tandem mass spectrometry techniques, showing that AAATP, AAPLAP, and KPVAAP were hydrolysed during the transport, although KPVAAP was also absorbed intact. This study highlights the potential of intact dry-cured ham peptides as well as their fragments to be absorbed across the intestinal epithelium and reach the blood stream to exert an antihypertensive action., The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement 312090 (BACCHUS). This publication reflects only the author views and the Community is not liable for any use made of the information contained therein. Grant AGL2013-47169-R from MINECO and FEDER funds and the FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. are fully acknowledged. The JAEDOC-CSIC postdoctoral contract to L.M. co-funded by the European Social Fund and BOF (Special Research Fund of Ghent University) for their financial support (Project 01B04212) are also acknowledged.

Thornback ray skin gelatin has been hydrolyzed with two different proteases in order to obtain peptides with ACE inhibitory and antioxidant activity. Hydrolysates with protease from Bacillus subtilis A26 (TRGH-A26) displayed ACE inhibitory activity with an IC 50 value of 0.94 μg/μL whereas Neutrase® hydrolysate from Bacillus amyloliquefaciens (TRGH-Neutrase) showed an IC 50 value of 2.07 μg/μL. Regarding antioxidant activity, IC 50 values of 1.98 and 21.2 μg/μL in TRGH-A26 and TRGH-Neutrase, respectively, were obtained using the DPPH radical-scavenging assay. The most active fractions identified by size-exclusion chromatography were further purified by RP-HPLC and analysed using nanoESI-LC–MS/MS to identify the sequence of the peptides. APGAP was the most active peptide inTRGH-A26 for ACE inhibitory activity with an IC 50 value of 170 μM, whereas GIPGAP showed the best ACE inhibitory activity in TRGH-Neutrase sample with an IC 50 value of 27.9 μM. The highest antioxidant activity was identified in peptide AVGAT, showing a 33% of activity at 3 mg/mL using the DPPH radical-scavenging assay. The obtained results proved the potential of thornback ray skin gelatin hydrolysates as a source of bioactive peptides. Statement of significance This study describes a peptidomic approach for the identification of ACE-inhibitory and antioxidant peptides generated from thornback ray gelatin ( Raja clavata ) hydrolysates from Bacillus subtilis A26 and Bacillus amyloliquefaciens Neutrase® enzymes and expose the potential of thornback ray gelatin hydrolysate as a source of bioactive peptides. In this sense, the decrease of systolic blood pressure is one of the main measurements considered in public health for the treatment of cardiovascular diseases, stroke and even end-stage renal disease. Traditionally, synthetic drugs such as captopril and enalapril have been used as ACE inhibitors despite their secondary effects, but the finding of new sources for the generation of natural bioactive peptides such as thornback ray muscle results is very important in the knowledge of less hostile but highly effective antihypertensive peptides as well as the development of new uses for waste and by-products generated from marine products, helping to solve the already existing environmental problem affecting this industry.

Pork meat is generally recognised as a food with relevant nutritional properties because of its content in high biological value proteins, group B vitamins, minerals especially heme iron, trace elements and other bioactive compounds. But pork meat also contributes to the intake of fat, saturated fatty acids, cholesterol, and other substances that, in inappropriate amounts, may result in negative physiologically effects. However, there are relevant factors affecting the content of many of these substances and somehow such variability should be taken into consideration. So, genetics, age and even type of muscle have a relevant influence on the amount of fat and the contents in heme iron. Also the composition in fatty acids of triacylglycerols is very sensitive to the contents of cereals in the feed; for instance, polyunsaturated fatty acids may range from 10% to 22% in pork meat. The content of other nutrients, like vitamins E and A, are also depending on the type of feed. Some bioactive substances like coenzyme Q10, taurine, glutamine, creatine, creatinine, carnosine and anserine show a large dependence on the type of muscle. This manuscript describes the main factors affecting the composition of pork meat nutrients and how these changes may affect the general food composition databases. (C) 2012 Elsevier Ltd. All rights reserved., Grant AGL2010-16305 from the Spanish Ministry of Science and Innovation (Madrid, Spain) and FEDER Funds and collaboration of Vaquero Foundation for R+D on Pork Meat (Madrid, Spain) are acknowledged. Grant PROMETEO/2012/001 from Generalitat Valenciana (Spain) is also acknowldeged. Work prepared within the Unidad Asociada IAD (UPV)-IATA (CSIC) framework.

This work is focused on the determination of compounds of nutritional interest that are naturally present in pork meat and how they are affected during the processing of dry-cured ham. Such compounds are creatine, creatinine, coenzyme Q(10), glutathione, carnosine, anserine, carnitine, taurine, cystine, cysteine and the essential amino acids. Their antioxidant and antyhipertensive functions were evaluated. Of all the assayed substances, only glutathione decreased totally during processing. Carnosine, creatinine, anserine and glutathione showed antioxidant, while cysteine, glutathione and carnosine showed antyhipertensive activity. So, dry-cured ham constitutes an excellent source of essential amino acids (all essential amino acids exhibited a large increase during processing) and other nutritionally interesting compounds such as cystine, cysteine, carnosine, anserine, taurine, carnitine and coenzyme Q(10).

This study is focused on the characterization of the proteolysis occurred during the processing of Pastırma, a traditional Turkish dry-cured meat product, which is responsible for its final characteristics. Thus, the evaluation of naturally generated free amino acids and peptides present at 0, 2, 5, 10, and 21 days of processing and the bioactivity of peptide fractions have been approached. Peptides were examined by MALDI-TOF and results showed differences in the amount of generated peptides at different times of processing, and a total of 29 peptides were newly generated at Day 21 in comparison with Day 2 during processing. The water soluble fraction of Pastırma at the end of the curing period (Day 21) was also analyzed by size-exclusion chromatography and some of the collected fractions demonstrated strong ACE-inhibitory and antioxidant activities. In fact, Pastırma showed an ACE inhibitory activity higher than 86% from 220 to 270 mL corresponding to molecular masses between 900 and 1500 Da, and also a DPPH radical-scavenging activity above 60% at 250 to 300 mL corresponding to molecular masses between 700 and 2000 Da. Thus, Pastırma represents a good source of natural ACE-inhibitory and antioxidant peptides which might be due to the proteolysis occurred by endogenous enzymes and the contribution of the çemen paste used in production., Grant AGL2014-57367-R from MINECO (Spain) and FEDER funds and JAEDOC-CSIC postdoctoral contract of L.M. cofunded by European Social Funds are acknowledged. The research fellowship grant provided by Erasmus + KA1 Student Mobility Programme for Higher Education for the author Ebru Deniz is also gratefully acknowledged.

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC50 values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500–800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500–800 Da and 1200–1500 Da., This study was funded by Grant of Ministry of Science, Research and Technology of Iran and Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138). Also, a JAEDOC-CSIC postdoctoral contract was cofunded by the European Social Fund to Dr. Mora.

Amino acids are natural constituents of proteins and peptides present in foods that are known to contribute to many relevant functions in an organism. The knowledge of the amino acid profile or the essential amino acids contents in foods and the limiting amino acids in a protein is very important for the characterization of their biological value and how their nutritional benefits may be affected by food processing or during storage. There are many available methods for the analysis of amino acids in foods that are summarized in this article.

Hydrophilic interaction chromatography has been used to investigate the retention characteristics of a group of polar compounds typically present in meat like carnosine (Car), anserine (Ans), creatine (Cr), creatinine (Cn), glutathione oxidised (GSSG), glutathione reduced (GSH) and nicotinamide adenine dinucleotide (NADH). These compounds were separated using four polar stationary phases composed by bare silica, amide and zwitterionic groups bound to porous silica and zwitterionic groups attached to a polymeric bead. Ammonium acetate salt used in the mobile phase was found to provide the best results in selectivity and reproducibility. The effect of salt concentration together with pH and acetonitrile (ACN) content of the eluent on the retention of the different polar stationary phases was also studied. A linear gradient of ACN from 80% to 30% with 10 mM ammonium acetate at neutral pH values (pH 6.0 and pH 7.8) offered retention factors k ≤ 10 while keeping a resolution higher than 1.5 for all the assayed compounds in bare silica and amide columns. The separation of NADH from GSSG was not possible under the studied conditions at any salt concentration using zwitterionic columns.

This study is focused on the effect of sodium chloride alternative salts (KCl, MgCl2 and CaCl2) on porcine muscle proteases (cathepsins, dipeptidylpeptidases and aminopeptidases). In general, KCl exerted a very similar effect to NaCl for all the studied enzymes, while the effect of divalent salts (CaCl2 and MgCl2) was more pronounced. Cathepsins, dipeptidyl peptidase III, dipeptidyl peptidase IV and alanyl aminopeptidase activities were strongly inhibited by all the chloride salts especially by divalent ones. Dipeptidyl peptidase II and leucyl aminopeptidase were little affected and methionyl aminopeptidase was only inhibited by divalent salts. Dipeptidyl peptidase I was strongly activated by low concentrations of the chloride salts except NaCl. Arginyl aminopeptidase was activated by NaCl and KCl and low amounts of MgCl2, while CaCl2 showed a strong inhibitory effect. This is very important as these enzymes play important roles in dry-cured meats and their activity is, in general, regulated by sodium chloride. Thus, reductions in the sodium concentration with subsequent increases of other alternative cations may have relevant consequences on enzyme activity that should be taken into account when processing dry-cured meats.

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.

Large amounts of different peptides are naturally generated in dry-cured meats as a consequence of the intense proteolysis mechanisms which take place during their processing. In fact, meat proteins are extensively hydrolysed by muscle endo-peptidases (mainly calpains and cathepsins) followed by exo-peptidases (mainly, tri- and di-peptidyl peptidases, dipeptidases, aminopeptidases and carboxypeptidases). The result is a large amount of released free amino acids and a pool of numerous peptides with different sequences and lengths, some of them with interesting sequences for bioactivity. This manuscript is presenting the proteomic identification of small peptides resulting from the hydrolysis of four target proteins (glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, myozenin-1 and troponin T) and discusses the enzymatic routes for their generation during the dry-curing process. The results indicate that the hydrolysis of peptides follows similar exo-peptidase mechanisms. In the case of dry-fermented sausages, most of the observed hydrolysis is the result of the combined action of muscle and microbial exo-peptidases except for the hydrolysis of di- and tri-peptides, mostly due to microbial di- and tri-peptidases, and the release of amino acids at the C-terminal that appears to be mostly due to muscle carboxypeptidases., The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant Agreement 312090 (BACCHUS). This publication reflects only the author views and the community is not liable for any use made of the information contained therein. JAEDOC-CSIC postdoctoral contract cofunded by European Social Fund to L.M. and FPI Scholarship to M. G. are also acknowledged.

All rights reserved. In this study, a peptidomic approach has been used in the identification of naturally generated peptides during a dry-curing process, showing methionine (Met) oxidation in their sequence. A total of 656 peptides derived from major myofibrillar proteins in Protected Designation of Origin (PDO) Teruel dry-cured ham have been identified by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS), including 120 peptides showing methionine oxidation. The percentage of oxidised peptides in the studied proteins ranged from 6% to 35%, being peptides derived from nebulin, titin, myosin heavy chains, and troponin I proteins, those showing the highest number of oxidised methionine. The identification of the peptide sequence incorporating the oxidised amino acid provides valuable information of neighbouring amino acids, degree of hydrolysis of the sample, and characteristics of the peptide, which might be very useful for a future better understanding of the oxidation mechanisms occurring in dry-curing processing., Grant AGL2013-47169-R from MINECO (Spain) and FEDER funds and FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. are fully acknowledged. JAEDOC-CSIC postdoctoral contract and support from European Social Funds to L.M. are also acknowledged.

Peptidomics is a necessary alternative in the analysis of naturally generated peptides in dry-fermented processing. The intense proteolysis occurred during the processing of dry-fermented sausages is due to the action of endopeptidases and exopeptidases from both, endogenous muscle origin and lactic acid bacteria (LAB) added in the starter. Sodium caseinate is frequently used as an additive in this type of products because of its emulsifying properties, and consequently influences the protein profile available during the proteolysis. In this study, a mass spectrometry approach has been used to determine the impact of added sodium caseinate in the final peptide profile as well as to analyse its possible influence in the presence of certain previously described casein-derived bioactive peptides.

A porcine kidney enzyme extract containing high levels of cathepsins B and L activities has been successfully immobilised onto porous glass beads. Cathepsins showed improved stability when immobilised, exhibiting a half-life of 97.5 h at 30 °C vs 5.6 h for the free enzyme in solution. Immobilised reactors were incubated at 30 °C and fed with a 0.2% casein solution at pH 6.0. κ-casein was rapidly hydrolysed and almost fully degraded at 24 h while the degradation of α-casein and β-casein was achieved in 24–48 h. Polypeptides of M.W. 11, 19.5 and 32 kDa were generated and further degraded, while others (18 and 21 kDa) remained undegraded. Hydrolysis was much higher than that observed with the free enzymes. A substantial number of peptides with cut-off

A water-soluble extract of dry-cured ham was fractionated by gel filtration chromatography. Collected fractions (below 1200 Da) were sensory-analyzed and subjected to amino acid analysis with and without previous acid hydrolysis, in order to determine the amino acid and peptide distribution. Those fractions with the highest peptide amount were further separated by reverse-phase and cation-exchange high-performance liquid chromatography in order to isolate the peptides and sequence them. The results demonstrated the presence of small peptides, mainly dipeptides. The combination of these peptides and free amino acids may contribute to the characteristic taste in the savory fractions of dry-cured ham.

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p -chloromercuribenzoic acid, and Hg 2+ , which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K m for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

The processing of dry-cured ham results in an intense proteolysis that leads to the generation of peptides of different sizes and composition, which have been shown to be biologically active. In this study, a total of ninety-three peptides mainly derived from actin, β-enolase, myosin heavy chain, and creatine kinase proteins have been identified from a size-exclusion chromatography (SEC) fraction that resulted as antioxidant by using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) and nESI-ion trap mass spectrometry. Several of the identified peptides have been synthesised and their antioxidant activity tested in vitro by using DPPH radical-scavenging assay and reducing power analysis. The peptide with sequence SNAAC showed the best results with an IC50 of 75.2μM in DPPH radical-scavenging assay and 205μM in ferric-reducing antioxidant power analysis, very good when comparing with the positive control 2,6-di-tert-butyl-4-methyl phenol (BHT) that showed an IC50 of 358.5μM and 90.3μM, respectively, in the different assays. These results suggest that Spanish dry-cured ham represents an important source of powerful antioxidant peptides which due to their natural characteristics may represent a highly valuable alternative in human health., HEFCE infrastructure funding is acknowledged for MS equipment to PDF. LM and PDF are grateful to the EU-FP7 Marie Curie IEF scheme (FOOSAF project) for the financial resources. FPI Scholarship from Spanish Ministry of Science and Innovation to E.E. and grant AGL2010-16305 from Spanish Ministry of Economy and Competitiveness and FEDER funds are fully acknowledged.

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.

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.

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.

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.

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.

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.

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.

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.