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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.

This study evaluates the effect of dietary selenium (Se) supplementation source (organic, Se-enriched yeast; SY vs. inorganic, sodium selenite; SS), dose (0.2: L vs. 0.4: H mg/kg) and the combination of Se and vitamin E (VITE + SS) for 26 days on drip loss, TBARS, colour changes, myofibrillar protein pattern and proteolysis in pork. The lowest water losses were observed in the SY-H group when compared to the others. SY-H and VITE + SS groups presented lower myofibrillar protein hydrolysis/oxidation. VITE + SS supplementation also resulted in higher PRO, TRP and PHE content at days 2 and 7, whereas the SY group showed increased GLY and CAR and tended to have higher TAU and ANS at day 2. The myofibrillar fragmentation index was not modified by the dietary treatment; however, at day 8, it tended to be higher in groups supplemented with SeY and VITE + SS. The results of the present study might indicate a possible relation between muscle proteolysis and water loss., This research was supported by projects CDTI IDI-20111017 and S2013/ABI-2913-MEDGAN-CAM.

Angiotensin I-converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC50 of 0.83 mg mL−1. To identify peptides responsible for this activity, the extract was eluted through size-exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC50 of 0.42 and 0.51 mg mL−1, respectively. These fractions were analysed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC50 of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides., This work was funded by a grant from the Ministry of Higher Education and Scientific Research of Tunisia and grant Prometeo/2012/001 from Conselleria d´Educació Cultura i Sport of Generalitat Valenciana, both are acknowledged. JAEDOC- CSIC postdoctoral contract cofounded by ESF to L.M. is also acknowledged

The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham.

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.

Recent advances in proteomics have become an indispensable tool for a fast, precise and sensitive analysis of proteins in complex biological samples at both, qualitative and quantitative level. In this study, a label-free quantitative proteomic methodology has been optimised for the relative quantitation of proteins extracted from raw pork meat. So, after the separation of proteins by one-dimensional gel electrophoresis and trypsin digestion, their identification and quantitation have been done using nanoliquid chromatography coupled to a quadrupole/time-of-flight (Q/ToF) mass spectrometer. Relative quantitation has been based on the measurement of mass spectral peak intensities, which have been described that are correlated with protein abundances. The results obtained regarding linearity, robustness, repeatability and accuracy show that this procedure could be used as a fast, simple, and reliable method to quantify changes in protein abundance in meat samples., The research leading to these results received funding from the European Union 7th 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 to L.M. is also acknowledged. FPI Scholarship BES-2011-046096 and grant AGL2010-16305 from MINECO (Spain) and FEDER funds are also acknowledged.

Coenzyme Q10 (CoQ10) or ubiquinone (2,3-dimethoxy-5-methyl-6-multiprenyl-1,4-benzoquinone) is an endogenous hydroxybenzoquinone liposoluble compound which plays important physiological roles that makes it to be considered as a bioactive compound that may be used for clinical practices and as food supplement. The purpose of this work was to analyse CoQ10 in three muscles with different oxidative patterns and determine its variability in different animal species (pork, beef, lamb and rabbit). The content of CoQ10 ranged from 4.3 to 30.9μg/g meat with the highest content in those muscles with oxidative pattern. So, more specific data on type of meat cut and proportion of muscles must be given for this nutrient when reporting its content in food composition databases., Grant PROMETEO/2012/001 from Conselleria d'Educació, Formació i Ocupació of Generalitat Valenciana (Spain) is fully acknowledged.

The complex proteolysis in Spanish dry-cured ham processing generates large amounts of small peptides and free amino acids which are responsible for the characteristic texture and flavour of this traditional product. The aim of this work was to study the degradation of the giant protein titin throughout the dry-curing process (2, 3.5, 5, 6.5, and 9 months) through the use of proteomic tools. A total of 320 peptides have been identified by nanoliquid chromatography coupled to tandem mass spectrometry, being some of them identified only at 9 months of processing. In order to confirm the absence of these peptides at other times of processing, MALDI-TOF MS was also employed as a fast and easier technique. Only four peptides, KDEAAKPKGPIKGVAKK, KKLRPGSGGEK, KNTDKWSECAR and ISIDEGKVL, were exclusively identified at 9 months of curing by using both methodologies so that these peptides could be used as potential biomarkers of dry-cured ham processing time., FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. and grant AGL2010-16305 from MINECO and FEDER funds are fully acknowledged. JAEDOC-CSIC postdoctoral contract to L.M. is also acknowledged. The proteomic analysis was carried out in the SCSIE_University of Valencia Proteomics Unit, a member of ISCIII ProteoRed Proteomics Platform.

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.

Bioactive peptides are known to contribute relevant bioactive functions during postmortem processing of muscle foods. They can influence the metabolism and prevent diseases exerting their functions on the cardiovascular, immune, gastrointestinal, and nervous systems in human bodies. The identification and quantitation of these peptides in foods are very important for the characterization of its biological value and its benefits for consumers' health. Their importance is mainly based on the lower amount of secondary effects produced by the natural peptides, in contrast to the nondesirable effects observed when synthetic peptides are used. This article describes the main sources to obtain bioactive peptides, their most studied properties, and their occurrence in foods.

Histidine-containing dipeptides carnosine, anserine, and balenine are natural constituents in mammalians, which are known to contribute to relevant bioactive functions during postmortem processing of muscle foods. The knowledge of the content of these dipeptides in meat, poultry, and seafood is very important for the characterization of its biological value in different muscles and cuts and how its antioxidant benefits may be affected by processing or during the storage. This article is describing the importance for health, the availability, and the occurrence of histidine-containing dipeptides in meat, poultry, and seafood as well as the description of the main available methods for their analysis in foods.

The effect of the partial NaCl replacement by other salts (potassium, calcium, and magnesium chloride) on the formation of volatile compounds through the processing of dry-cured ham was studied using solid-phase microextraction (SPME). Three salt formulations were considered, namely, I (100% NaCl), II (50% NaCl and 50% KCl), and III (55% NaCl, 25% KCl, 15% CaCl(2), and 5% MgCl(2)). There was an intense formation of volatile compounds throughout the processing of dry-cured hams, particularly during the "hot-cellar" stage. The differences between treatments were found to be more remarkable at the end of the curing process. Hams from formulations I and II had significantly higher amounts of lipid-derived volatiles such as hexanal than hams from formulation III, whereas the latter had significantly higher amounts of Strecker aldehydes and alcohols. Plausible mechanisms by which salt replacement may affect the generation of volatile compounds include the influence of such replacement on lipid oxidation and proteolysis phenomena. The potential influence of the volatiles profile on the aroma of the products is also addressed in the present paper.

Several muscle compounds (creatine, creatinine, hypoxanthine, inosine, inosine 5' monophosphate, xanthine, adenosine monophosphate, guanosine, and uridine) were studied as possible biological markers of a minimum dry-cured ham processing time. A correlation between the concentration of the compounds and the time of processing was found. The ratios for some of them were calculated to study their behaviour during processing. The Hx/Ino ratio substantially increased up to 5 months of curing and then remained constant (p

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.

Sodium intake above nutritional recommendations may involve harmful consequences to health such as the increased risk of cardiovascular diseases. Dry-cured ham constitutes a product with a relatively large amount of sodium. Thus, to obtain a healthier product for consumers with reduced sodium content, two formulations containing KCl alone (formulation II) or mixed with CaCl2 and MgCl2 (formulation III) have been proposed to partially replace NaCl. Lipolysis and lipid oxidation occurring in hams processed with these formulations have been studied since they have direct influence on the final flavor. No significant differences in acid lipase activity or lipid oxidation were found at the end of the process between the alternative formulations and formulation I (control with 100% NaCl). Differences in some free fatty acids, generated along the processing, were detected among treatments and at the end of dry-curing. Data suggests a slight trend towards a major lipolysis during treatment III.

The use of dielectric spectra to detect quality defects in porcine muscle during postmortem period was evaluated. The changes in dielectric spectra during meat ageing were also studied. For these purposes, dielectric spectra were measured by using an Agilent 85070E Open-ended Coaxial Probe connected to an Agilent E8362B Vector Network Analyzer from 500 MHz to 20 GHz parallel and perpendicular to muscle fiber direction. This study reveals that dielectric properties at two punctual frequencies (0.5 GHz and 10 GHz) are a useful tool to determine meat quality classes (RFN, PSE, and DFD) soon (6 h) after slaughter. Based on these results, it is possible to develop control algorithms in order to discriminate low quality pork meats and to decide their best uses. Moreover, the evolution of dielectric spectra during meat ageing showed important variations only in DFD meats, while in PSE and RFN meats the variations were observed only at some punctual frequencies of the spectrum during the 24 h after slaughter.

The effect of partial replacement of NaCl by KCl in the dry-curing of loins has been evaluated by biochemical and sensory analysis of the final products. Endoproteolytic enzymes like cathpesins B and B + L appeared to be more active when more KCl and less NaCl were present in the curing salt while cathepsin H remained unaffected. Proteolysis was reflected by SDS–PAGE in the higher density of the sarcoplasmic proteins bands within the range 55.0–28.0 kDa. Alanyl aminopeptidase (AAP) was unaffected while arginyl and leucyl aminopeptidases were more activated by KCl. On the other hand, methionyl aminopeptidase and dipeptidylpeptidase I and III were more inhibited as KCl increased in the curing salt blends. The sensory analysis revealed no significant differences between control loins with 100% NaCl and those with up to 50% of KCl substitution. Furthermore, loins elaborated with 50% of each salt obtained the highest scores.

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.

One of the fields where the idea of developing artificial electronic mammalian sense devices might be widely applied is in food analysis. In this field, one very complex attribute yet of great importance is that of freshness. For instance, freshness is one of the most significant parameters of fish quality in most markets. The state of fish freshness can be assessed by various indicators but most of them are costly, time-consuming and lack of aptness for in situ or on site monitoring. This makes that the development of simple and undemanding systems for the evaluation of fish freshness is still an unresolved goal. Based on this demand, we focused our attention on the use of unselective sensors for the potential development of low-cost and easy-to-use devices for the estimation of freshness. After a preliminary study with a number of different electrodes, simple Au and Ag wires were used for the analysis of the evolution of sea (Sparus aurata) bream fish stored throughout time. PCA and neuronal network analyses showed that it is possible to determine the post-mortem time elapsed in minced gilthead sea bream using simple potentiometric measurements, suggesting that this method could be used for the analysis and evaluation of fish freshness. In order to somehow validate the potentiometric protocol for freshness assessment, the method has been compared with the determination of fish freshness based on the analysis of the concentration of certain bio-molecules as a function of time after the death of the fish. Among different possibilities the K1 index that simultaneously measures the variation in the ATP metabolites inosine 5′-phosphate (IMP), inosine (Ino) and hypoxanthine (Hx) was used. A remarkable correlation has been found between the K1 index determined during 7 days and the potentials of the Au and Ag electrodes.

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.

Proteolysis is a complex and dynamic process which takes place throughout the whole dry-cured processing due to the action of endogenous muscle peptidases, and results in the generation of a high number of small peptides and free amino acids responsible for the final quality of dry-cured ham. In this study, a total of sixty-eight peptides derived from the ubiquitin-60S ribosomal protein have been identified in dry-cured ham at 2, 3.5, 5, 6.5, and 9 months of processing using various chromatographic separations and a quadrupole/time-of-flight mass spectrometer in tandem. Some of the identified peptides have been detected during the whole process, whereas a total of fourteen of them were exclusively identified at 9 months of curing. The presence of any of these peptides could be a good indicative that dry-cured ham pieces have reached a minimum curing process of 9 months. The study of the generated peptides has contributed both to a better knowledge of proteolysis evolution and the endogenous enzymes participating, and to determine their potential to be used as quality markers to monitor the processing time., Grant PROMETEO/2012/001 from Conselleria d'Educació, Formació i Ocupació of Generalitat Valenciana (Spain) is fully acknowledged. FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. and JAEDOC-CSIC postdoctoral contract to L.M. are also acknowledged. Mass spectrometry analysis was performed in the SCSIE_University of Valencia Proteomics Unit, a member of ISCIII ProteoRed Proteomics Platform.

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.

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.

The simultaneous brine thawing/salting operation has been proposed as an effective alternative for the accelerated processing of frozen cured hams. The aim of the present study was to study the effect of this new technology on the lipolytic and proteolytic enzymes responsible for the generation of flavour precursors during the salting and post-salting stages of the manufacturing of Spanish dry-cured ham. The effect of the frozen and thawed process produced a higher proteolytic and lipolytic activity than in the fresh traditional salted hams (FPS) that was detected by a higher concentration in free amino acids and free fatty acids. On the other hand, the brine thawed/salted treatments, at atmospheric pressure (BTS) and with vacuum impregnation (BTS-TP), produced an acceleration of the myofibrillar degradation that occurred in the BTS and BTS-TP hams in comparison to FPS. However, the lipolysis was affected by the frozen treatment but not by the brine thawed treatments, as few differences in free fatty acids and lipase activity were detected among the frozen and brine thawed hams at the end of post-salting stage.

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

The present work is focused on the use of the relative amounts of carnosine and anserine for detecting the presence of proteins of mammalian origin in feeds for ruminants. The methodology is cheap and simple and may serve for the rapid screening of feeds. Animal protein additions to feeds as low as 0.5% can be easily detected based on the cation exchange HPLC determination of the carnosine and/or anserine dipeptides. Furthermore, the molar ratio of these dipeptides was found to be characteristic of the animal species and could be used to discriminate between mammalian and non-mammalian species eventually added to feeds. Feeds having carnosine/anserine molar ratios higher than 0.3 were strongly suspected of containing banned mammalian proteins. Those suspicious feeds might be later confirmed by more specific techniques. The exact source of feeds containing mixtures of animal proteins from different species could not be identified by this method even though the method was able to detect its mammalian origin.

A simple methodology, consisting in cation exchange HPLC separation combined with OPA postcolumn detection, has been succesfully applied to the analysis and detection of histidine dipeptides in feeds. The use of the proposed methodology, which is very simple and relatively fast, allows the detection of 2.63 ppm and 0.58 ppm of carnosine and anserine, respectively, in cattle feed. As balenine (3-methylhistidine) was not commercialy available, it was isolated from pork muscle and used as standard for its quantitation in feeds. These dipeptides have shown good stability against intense heat treatments, even at temperatures as high as 120 °C for 20 min. So, these histidine dipeptides can be feasible used as markers of animal protein content in feeds.

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.

Aims: The effect of the common curing conditions used during the manufacture of dry fermented sausage on the proteolytic activity of Lactobacillus casei CRL705 against meat proteins was investigated. Methods and Results: Hydrolysis of pork muscle sarcoplasmic and myofibrillar proteins was evaluated by SDS-PAGE and reverse phase-HPLC analysis. Ascorbic acid exerted a stimulatory effect on both sarcoplasmic and myofibrillar protein breakdown by Lact. casei CRL705 with the release of hydrophilic peptides and free amino acids, while NaCl and NaNO2 mainly stimulated myofibrillar degradation. Conclusions: Even when processing temperature (25°C) did not positively affect bacterial protein hydrolysis, the presence of curing salts accounted for a remarkable increase in the non-volatile components that constitute taste-active compounds that strongly influence the final flavour of the product. Significance and Impact of the Study: To predict the suitability of Lact. casei CRL705 and its proteolytic enzymes as a starter culture for the dry processing of dry fermented sausages. Fil: Fadda, Silvina G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Aristoy, M. C.. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España Fil: Oliver, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Toldrá, F.. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España

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.