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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.

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.

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.

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.