Search

Proteolytic activity and physico-chemical characteristics were studied for Norwegian dry-cured ham at four different times of processing: raw hams, post-salted hams (3 months of processing), hams selected in the middle of the production (12 months of processing) and hams at the end of the processing (24 months). Cathepsin H activity decreased until negligible values after 3 months of processing, whereas cathepsins B and B + L were inactive at 12 months. AAP was the most active aminopeptidase whereas RAP and MAP were active just during the first 12 months of processing. Proteolysis index reached a value of 4.56 ± 1.03 % with non-significant differences between 12 and 24 months of ripening. Peptide identification by LC-MS/MS was done and two peptides (GVEEPPKGHKGNKK and QAISNNKDQGSY) showing a linear response with the time of processing were found. Unfreezable water content and glass transition temperature were investigated using differential scanning calorimetry (DSC) technique with non-significant differences in the temperature of glass transition for 12 and 24 months of processing., The work was supported by the Research Council of Norway (project 225262/E40 – DryMeat)

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.

Smooth hound gels (SHGs) were prepared from the viscera by heat treatment at different pH values (2.0, 5.0, 7.0 and 9.0). Their physicochemical and functional properties, microstructure and molecular forces were investigated. The pH variation was found to influence the protein molecular weight distribution resulting in different gelling properties. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles, SHG-9 prepared at pH 9.0 contained the greatest amount of high molecular weight proteins. Further, the SHG-9 exhibited the highest gel strength, fracture resistance and elasticity. On the contrary, microstructure and infrared spectra analyses showed that acidic conditions provided gel networks with unarranged secondary structure and a high hydration level, which might be attributed to the formation of hydrogen bonds between polymer molecules. In addition, the molecular forces study showed that the primary dominated intermolecular forces involved in the SHGs were hydrophobic interactions and hydrogen bonds, while the secondary forces included electrostatic interactions and disulfide bridges. These results were confirmed by the levels of free amino acids. Thus, this study identified opportunities to develop high value-added products from fish by-products that might be used to carry out the function of the desired food application.

Spanish dry-cured ham is a high quality product whose economic value is mainly given by its curing time. An intense proteolysis takes place throughout the dry-cured processing, which results in the generation of a high amount of peptides and free amino acids responsible for the final quality of dry-cured hams. In this work, a peptidomics approach has been used to study the evolution of peptides throughout the ham dry-curing process, identifying and quantifying the generated peptides in order to define potential quality biomarkers. For this purpose, dry-cured ham extracts at different processing times (0, 2, 3.5, 5, 6.5 and 9 months) were fractionated by size-exclusion chromatography and analysed by nanoliquid chromatography coupled to tandem mass spectrometry. Differences obtained in the relative quantification of peptides by using a label-free methodology were useful to establish differences between processing times, being peptides generated by the degradation of myosin light chain 1 protein mainly responsible for the observed differences during the last stages of curing. In particular, APAPAPAPPKEEKI and PAPAPAPAPAPAPAPPKE, exclusively identified at 9 months of curing, would be potential markers to control the time of curing and thus the final quality of dry-cured hams., The FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. and Grants AGL2013-47169-R from MINECO and FEDER funds are acknowledged. The research also received funding from the European Union Seventh Framework Programme FP7-PEOPLE-2013-CIG under Grant Agreement 614281 (HIGHVALFOOD). The JAEDOC-CSIC postdoctoral contract to L.M. co-funded by the European Social Fund is also acknowledged.

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.

During the last decade, the use of integrated ‘omics’ including genomics, transcriptomics, metabolomics and proteomics has provided a better knowledge of food systems. In fact, technical improvements achieved in mass spectrometry instruments together with the last developments of bioinformatics tools and hardware have allowed proteomics to be the method of choice for food control through the identification of quality biomarkers and the study of the proteome expression data. In this context, modern comparative proteomics is the basis for establishing quantitative differences in the expressed proteome of a certain sample by comparison with a reference through the relative and absolute quantification of proteins and peptides., Grant AGL2014-57367-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged. Juan de la Cierva de Incorporación postdoctoral contract to LM is also acknowledged.

Background Meat has a complex physical structure and chemical composition that is very prone to oxidation. Plants are sources of biologically active compounds (antioxidants) of interest as potential raw materials for meat processing, primary as replacements for synthetic additives. Some examples are essential oils from aromatic plants that are usually unstable under common processing and storage conditions and exhibit strong smell and off flavour. Hence, stable delivery systems like encapsulation are required. Scope and approach Encapsulation, and particularly spray-drying, offers protection of active compounds, their controlled and targeted release in food products and ability to mask unacceptable odours in products. Key findings and conclusions Albeit current results are promising for microparticles and nanomaterials, more research is needed to evaluate the application of various natural ingredients in meat processing. Direction of future research should address functionality of systems, consumers’ health concerns and benefits, better sensory acceptance, reduced operating costs, scalability for industrial needs, and size of environmental footprints., This research was supported by Grant RTA 2017-00024-CO4-04 from INIA. José M. Lorenzo is member of the MARCARNE network, funded by CYTED (ref. 116RT0503)

Fat, mainly composed of triacylglycerols and phospholipids, protein, composed of amino acids, and carbohydrates, mainly lactose, constitute major components of dairy products. Other compounds of interest are vitamins, minerals and trace elements and some additional nutrients although 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.

Bioactive peptides derived from food matrices are currently a subject of intensive research, due to the multiple health benefits they can exert in the human body. However, the small size and low abundance of these peptides in complex matrices as well as the effect of food processing and gastrointestinal digestion on their structure, sequence, and functionality, are important challenges. Spanish dry-cured ham has been reported as a good and natural source of bioactive peptides mainly showing antihypertensive and antioxidant activities. In this work, the antioxidant peptide AEEEYPDL, identified from 9 months of curing dry-cured ham, showed good resistance to different heat treatments and salt contents. The bioactivity of the peptide significantly decreased after simulated gastrointestinal digestion. An optimised multiple reaction monitoring methodology was able to quantify the peptide AEEEYPDL at a concentration of 0.148 fg per g of dry-cured ham, despite the existing challenges in the quantification of bioactive peptides from complex matrices., Grant AGL2014-57367-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged. Juan de la Cierva de Incorporación postdoctoral contract to LM is also acknowledged.

Background The produced peanuts by-products are a huge challenge, but they are recognized to be a source of valuable nutrients, including natural antioxidants and antimicrobials. Antioxidants are considered as necessary ingredients in food to prevent oxidative reactions and their undesirable effects in food quality during processing and storage. However, the use of such compounds is regulated due to their harmful effects revealed by in vitro and in vivo studies. The use of natural antioxidants appears as an interesting alternative for food producers, particularly meat industries. Scope and approach Peanut skin (PS), a by-product of peanut processing in agro-industries, constitutes an under-explored source of natural antioxidants. Thus, this review was focused on both the reuse of peanuts by-products and societal health, reducing the use of synthetic antioxidants and antimicrobials. Key findings and conclusions Proanthocyanidins are the main compounds in PS that are associated with the antioxidant activity in vitro and its protective effect in meat products. Studies in recent literature strengthen the role of PS as a natural source of antioxidants wherein oxidative reactions involving mainly pigments, lipids and proteins are delayed., osé M. Lorenzo, Paulo Munekata and Marco Trindade are members of the MARCARNE network, funded by CYTED (ref. 116RT0503). A. S. Sant'Ana and R. B. Carvalho are thankful to Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) (Grant # 302763/2014-7, 305804/2017-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Grant #33003017027P1).

Background: Diseases related to oxidative stress and food quality decay are of major concern worldwide as they can lead to economic losses in both public health and food production. The antioxidant peptides, extracted from food proteins, can be explored as natural new drug and food ingredient. Scope and approach Antioxidant peptides are extracted from non-antioxidant precursor proteins from different origin by the activity of either proteolytic microorganisms or isolated enzymes. In the present review, the main sources of bioactive peptides will be discussed. Moreover, the current strategies to obtain these compounds as well as their health benefits and in vivo biological effects will be evaluated. Considerations for further research and development of strategies to increase the knowledge about this underexplored activity of peptides will be also considered. Key findings and conclusions Bioactive peptides' content and profile differ according to the matrix studied and the method used. The utilization of fermentation processes and enzymes has been established to obtain antioxidant bioactive peptides from proteins, being isolated enzymes the most commonly used method, due to their superior control over releasing and obtaining targeted peptides. Antioxidant peptides have the ability to reduce the formation of oxidative products along with the induction of antioxidant enzymes in vivo. However, at this stage of development more in vivo studies are needed in order to evaluate the specific effects on the health of selected antioxidant peptides. In food technology, successful application in meat products strengthens the role of selected peptides as antioxidant additives, although there is a need to observe the effects of the isolated bioactive peptides in other food matrices along with studies to scale-up its production., This research was supported by Grant RTA2017-00024-CO4-04 from INIA (Spain). Acknowledgments to INIA for granting Cristina Pérez Santaescolástica with a predoctoral scholarship (grant number CPD2015-0212). José M. Lorenzo and Paulo Munekata are members of the MARCARNE network, funded by CYTED (ref. 116RT0503).

Antioxidant peptides positively regulate oxidative stress in the human body as well as delay, retard or prevent protein and lipid oxidation in food products. Spanish dry-cured ham has been reported as a good source of bioactive peptides, being SNAAC the most active antioxidant peptide identified to date. In this work, the stability of this peptide against in vitro digestion, heat treatments and different salt concentrations was evaluated using three methods for measuring antioxidant activity: β-carotene bleaching assay, ABTS radical scavenging capacity and ORAC assay. The results evidenced a marked decrease in the antioxidant activity of SNAAC after gastrointestinal digestion, and the MALDI-ToF MS analysis revealed the degradation of the peptide after the process, the generation of the fragment SNAA and the presence of a peptide dimer throughout the in vitro digestion. On the other hand, the peptide SNAAC showed good heat stability after exposure to different temperatures (50 °C, 72 °C, and 90 °C), but its antioxidant activity evaluated by ORAC assay decreased substantially when exposed to 100 °C as compared with the control at 37 °C. SNAAC remained stable in the presence of salt at concentrations ranging from 0 to 8% NaCl as well as it was able to inhibit about 40% of lipid oxidation in an emulsion system. These results reported the stability of the antioxidant peptide SNAAC to several conditions used in meat industry for the processing of dry-cured hams and ham-derived products and its effectiveness to partially prevent the lipid oxidation in these products. However, some strategies would be needed in order to increase the stability of the peptide during gastrointestinal digestion and thus improve its bioavailability to be used as functional food ingredient., Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and Juan de la Cierva postdoctoral contract to LM are acknowledged. Grant Agreement 614281 (HIGHVALFOOD) and contract to MG are also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, (IPT13/0001 - ISCIII-SGEFI/FEDER).

Thornback ray muscle hydrolysates (TRMHs) prepared by treatment with proteases from Bacillus subtilis A26 (TRMH-A26), Raja clavata crude alkaline protease extract (TRMH-Crude), Alcalase (TRMH-Alcalase) and Neutrase (TRMH-Neutrase) were elaborated and their antioxidant properties and angiotensin I-converting enzyme (ACE) inhibitory activities were tested. TRMHs showed different degrees of hydrolysis (DH from 11 to 22%) and hydrophobic/hydrophilic peptide ratio. Protein content varied from 71 to 74%. Gly, Pro, Asp and Asn were the most prominent amino acids, while hypoxanthine was the major nucleotide related compound present. The antioxidant activity was assayed using various tests. TRMH-Neutrase exhibited the highest antioxidant activity in DPPH scavenging, reducing power and inhibition of β-carotene bleaching tests. However in the total antioxidative efficacy, TRMH-Crude exhibited the highest activity. TRMH-Crude and TRMH-Neutrase were the most potent to prevent DNA oxidation by Fenton reagent. Concerning anti-ACE activity, TRMH-A26 and TRMH-Neutrase exhibited the highest activity with 87% at 5 mg/ml. The results revealed that TRMHs could be employed as a protein source in food additive processing or diets for aquatic organisms and other farmed animals. Biological significance The present study explores for the first time the elaboration of enzymatic hydrolysates from thornback ray R. clavata . The hydrolysates are well characterized and showed an interesting protein content as well as the presence of nucleotide related compounds, essential amino acids and taurine, which make them an interesting source of fish meal in aquaculture feeds. The hydrolysates were found to exhibit ACE inhibitory activity and antioxidant activity. The hydrolysates could serve also as a potential protein source for functional foods.

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.

Consumed for centuries in many countries, dry-cured meats are characterized by a typical flavor, color, and texture that make them a high-quality product. This chapter presents the proteolysis phenomena involving muscle and microbial proteases and their resulting products, and the latest proteomic tools available for the study of proteolysis in dry-cured meats. There are three important groups of endopeptidases, or proteinases: cathepsins, calpains, and proteasomes. Traditionally, proteomics has been used to identify proteins from a mix or an extract, obtaining the characterization of the protein profile of the sample. Peptidomics is an area of science that focuses on the comprehensive characterization of peptides present in a biological sample by studying their structure, composition, interactions, and properties. Several bioactive peptides exerting antihypertensive, antioxidant, and antimicrobial activities have been identified in Spanish dry-cured ham. The use of a peptidomic approach permitted the study of the differences between different genotypes in the final peptide profile of dry-cured ham.

11.1 A Global Perspective on Functional Foods 276 11.2 Options to Design Functional Meat and Meat Products 277 11.3 Animal Production Strategies for Improving the Nutritional Profiles of Raw Meat 279 11.3.1 Genetics for the Reduction of Animal Fats 279 11.3.2 Dietary Feed Formulation for the Modification of Animal Fats 280 11.3.2.1 Manipulation of Fatty Acids in Pigs 280 11.3.2.2 Manipulation of Fatty Acids in Ruminants 283 11.3.3 Dietary Enrichment in Compounds of Specific Nutritional Interest 285 11.3.3.1 Vitamins and Antioxidants 285 11.3.3.2 Minerals 285 11.4 Processing Strategies for Developing Functional Meat Products 286 11.4.1 Reduction of Specific Unhealthy Compounds 287 11.4.1.1 Fat and Cholesterol 287 11.4.1.2 Sodium 288 11.4.1.3 Allergens 291 11.4.2 Promoting the Presence of Specific Healthy Compounds 291 11.4.2.1 Fat and Fatty Acid Profile 291 11.4.2.2 Proteins, Peptides, and Amino Acids 293 11.4.2.3 Prebiotics and Probiotics 294 DK3813.book Page 275 Friday, February 10, 2006 10:33 AM

In this study, tomato seeds were obtained as by-products and submitted to fermentation with the proteolytic strain Bacillus subtilis A14h. The resulting peptide mixture was fractionated and purified through different chromatographic steps. Fractions were assayed for antioxidant and angiotensin converting enzyme (ACE)-inhibitory activities and peptides were identified by using nano-liquid chromatography coupled to mass spectrometry in tandem (nLC-MS/MS). Most of the identified peptides were smaller than 1000 Da and had different aromatic and hydrophobic amino acid residues. Their sequences were novel but some of them showed active domains previously reported in other bioactive peptides. The hexapeptide DGVVYY showed an IC50 value of 2 µM in angiotensin-I converting enzyme (ACE-I) inhibitory activity, whereas the pentapeptide GQVPP displayed a 97% of DPPH activity at 0.4 mM. The results revealed that B. subtilis fermentation of tomato by-products could be a good strategy for obtaining added-value peptides that might be used as an ingredient in functional foods and nutraceuticals., This work was funded by Grant of Ministry of Science, Research and Technology of Iran, Research Council of University of Tehran (Tehran, Iran) and Emerging Research Group Grant from Generalitat Valenciana (Valencia, Spain) (GV/2015/138). Also a Juan de la Cierva de Incorporación postdoctoral contract to Dr. Mora is acknowledged. Q-ToF nLC-MS/MS was carried out in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.

The effect of a beef meat coating prepared using cuttlefish skin gelatin with henna aqueous extract (HAE) as natural preservative was studied during chilled storage of meat at 1, 3, 6 and 8 days. The combined coating increased the shelf-life of meat by decreasing the total and psychrophilic vial bacteria counts at the end of storage. In addition, the lipid oxidation of meat decreased during the period of storage in gelatin-HAE coated samples measured using the thiobarbituric acid reactive substances test. Furthermore, coating significantly preserved color properties in comparison with uncoated samples. According to free amino acid contents, coating decreased the rate of proteolysis process, whereas it was faster in uncoated samples. Thus, the gelatin coatings offer an excellent protection to avoid meat deterioration, which was significantly improved by the addition of HAE to the gelatin gel., The Ministry of Higher Education and Scientific Research of Tunisia is acknowledged. The research leading to these results has also received funding from the Spanish Ministry of Economy and Competitiveness, AGL2014-57367-R, and FEDER funds. Juan de la Cierva de Incorporación postdoctoral contract (L.M.) funded by the Spanish Ministry of Economy and Competitiveness is fully acknowledged.

Sardinelle protein hydrolysate (SPH), prepared by treatment with Bacillus subtilis A26 proteases, was found to exhibit antibacterial, antioxidant and ACE-inhibitory activities. SPH, with a degree of hydrolysis of 4%, was fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (F1–F5). F2, which exhibited the highest antibacterial and ACE-inhibitory activities, and F4, which exhibited the highest antibacterial and antioxidant activities, were further fractionated by reverse phase-high performance liquid chromatography (RP-HPLC) and then analysed using nano-ESI-LC-MS/MS to identify the sequences of peptides. Eight peptides were identified in the sub-fraction F2-A, nine peptides in the sub-fraction F4-B, and 45 peptides in F4-C. Identified peptides were found to share sequences with previously described bioactive peptides based on Biopep database. The results of this study suggest that SPH is a good source of natural bioactive peptides. Hence, it can be used as a potential ingredient in nutraceutical field., This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. Grant AGL2014-57367-R from MINECO (Spain) and FEDER funds and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged. LC-MS/MS analysis was carried out in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.

In the present study, it was hypothesized that the incorporation of fatty acids is distinct among ruminant tissues and that it could be modulated by diet composition. To test this hypothesis, fatty acid composition, including conjugated linoleic acid isomers, of the most relevant beef by-products (brain, heart, kidney, liver, pancreas and tongue) from young bulls those fed distinct silage levels was assessed. Data indicated a large variation in fatty acid profile and conjugated linoleic acid composition among edible by-products. The most abundant fatty acids were C16:0 (kidney), C18:0 (heart and liver) and C18:1c9 (brain, pancreas and tongue) followed by C20:4n-6, except in brain (C22:6n-3 predominates). Brain, as shown by principal component analysis, presents a distinct fatty acid composition compared to the other beef by-products analysed. In addition, high silage diet relative to low silage diet promoted an increase of n-3 polyunsaturated fatty acid, t11,t13 and t11,c13 conjugated linoleic acid in heart, kidney, liver and pancreas. Overall, the data suggested that beef by-products had, in general, high contents of cholesterol, saturated fatty acid and trans fatty acid, as well as high levels of conjugated linoleic acid. Therefore, from a nutritional point of view they are recommended only in small amounts as part of a balanced diet., Individual fellowships to Susana Alves (SFRH/BPD/76836/2011) and Marta Madeira (SFRH/BPD/97432/2013) and financial support from Fundação para a Ciência e a Tecnologia (Lisboa, Portugal) grants (PTDC/CVT/2006/66114 and UID/CVT/00276/2013) are acknowledged.

The effect of ultrasound (US) pre-treatment on the evolution of Maillard reaction (MR), induced between low molecular weight (LMW) peptides and sucrose, was studied. LMW peptides (, This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.

A large number of peptides are generated during the processing of dry-cured ham that can be affected by post-translational modifications (PTM). One of the most studied PTMs is the oxidation of susceptible residues but other modifications such as deamidation have been scarcely reported in the literature. This work has been focused on the impact of deamidation in those peptides generated in 12-months dry-cured hams. The results obtained in Biceps femoris and Semimembranosus muscles showed that 52% and 48% of the identified peptides, respectively, were affected by PTMs, with a total of 277 and 282 sequences showing deamidation in both muscles, respectively. It appears that deamidation is not influenced by the type of muscle but is probably favored by characteristic processing conditions like temperature, pH, and salting of hams. The knowledge of the affected sequences provides novel information for a better characterisation of proteolysis phenomena during the processing of dry-cured ham., The research leading to these results has received funding from the Spanish Ministry of Economy, Industry and Competitiveness, AGL2014-57367-R and FEDER funds. FPI Scholarship to MC and Juan de la Cierva de Incorporación postdoctoral contract to LM are acknowledged.

Smooth-hound viscera proteins were enzymatically hydrolyzed, using Esperase®, and the resulting hydrolysate was fractionated by ultrafiltration through four membranes with decreasing molecular weight (MW) cut-offs. Five fractions were obtained, FI (>50 kDa), FII (5–50 kDa), FIII (3–5 kDa), FIV (1–3 kDa) and FV (, This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and Juan de la Cierva de Incorporación postdoctoral contract of L.M. are acknowledged. The proteomic analysis was carried out by the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform. Authors want to acknowledge also the Laboratory of Animal Ecophysiology (University of Sfax, Faculty of Sciences of Sfax) for rats care and treatment.

In this chapter, several factors that should be considered for selecting and developing suitable analytical methods for determining nitrite/nitrate are presented. Nitrite and nitrate occurrence and suitability are a controversial issue. Nitrite is an approved additive considered a foremost curing ingredient for the preservation of meat products. Nitrate is a natural constituent of the human diet that, however, raises fears for its suggested potential harmfulness related to carcinogenesis and environmental contamination. Chemical, regulatory, and analytical aspects are discussed in the light of the need to obtain reliable data of nitrite and nitrate for law enforcement purposes, exposure estimates, and investigation of their physiological role in the human body. In addition, current metrological aspects to ensure the "fitness for purpose" of the selected method are suggested and discussed.

Dry-cured ham by-products have been traditionally used in Mediterranean household cooking of broths and stews. The aim of this work was to evaluate the effect of cooking treatments and in vitro gastrointestinal digestion on the antioxidant activity of natural peptides found in bones from Spanish dry-cured hams. The antioxidant activity was tested using five different assays and results demonstrated that cooking using conventional household methods increased the antioxidant activity of ham by-products when assessed using different antioxidant assays with the exception of the ABTS radical scavenging measurement assay. Simulated gastrointestinal digestion showed no significant effect on the antioxidant activity of ham by-products and antioxidant activity decreased when assessed using the ORAC and β-carotene bleaching assays. Analysis by MALDI-TOF MS revealed a considerable breakdown of peptides due to the action of gastrointestinal enzymes, mainly in samples cooked at 100 °C for 1 h. In addition, 459 peptides derived from 57 proteins were identified and quantified using mass spectrometry in tandem, evidencing that peptides derived from collagen protein were responsible for the differences in antioxidant activities observed between the uncooked and cooked samples after digestion. The results show the potential of dry-cured ham bones as a source of antioxidant peptides that retain their bioactivity after household cooking preparations and gastrointestinal digestion., Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and Juan de la Cierva postdoctoral contract to LM are acknowledged. Grant Agreement 614281 (HIGHVALFOOD) and contract to MG are also acknowledged.

Most of the nonmeat portion of an animal carcass is biologically and nutritionally able to be consumed and can be made microbiologically safe for human consumption. Liver constitutes a good example of a very nutritional tissue in an animal carcass. Depending on the traditions, culture, and country of origin, certain by-products may be considered as a delicacy, even with high prices. So, the popularity of consumption of these by-products may vary by countries even though in general, there is a decreasing trend in spite of the increase in the world meat consumption. This chapter is dealing with the most usual consumed edible animal by-products and briefly discusses the specific products, their yield and further processing, and main uses given by the consumer.

Background: The final characteristics of processed meats depend on many factors but one of the most important is the intense proteolysis occurred in muscle proteins due to the action of endogenous enzymes in dry-cured ham, and also microbial peptidases in the case of dry-fermented meats, that not only affects taste and flavour but also the generation of bioactive peptides. Scope and approach: In this review main difficulties in the identification of bioactive peptides in processed meats have been described. This study highlights the novel strategies used during the last years to identify naturally generated peptides, and emphasises the need of robust quantitative methodologies for the adequate characterisation of their bioavailability. In fact, the most common and well established quantitation approaches using proteomics are not adapted for peptidomics analysis, so alternative strategies need to be considered. Key findings and conclusions: The progress in the identification and characterisation of the activity of natural bioactive peptides is highly dependent on modern instruments and bioinformatics tools as well as updated protein databases. In fact, the use of in silico approaches and proteomics can be complementary tools in the identification of peptides from meat protein sources as the empirical experimental design can be simplified by using bioinformatics for computer simulation in most of the steps. Finally, Multiple Reaction Monitoring mass spectrometry methodology previously used in the quantitation of therapeutic peptides and biomarkers arises as a powerful tool for absolute quantitation or semi-quantitation of bioactive peptides., 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. Grant AGL2014-57367-R from MINECO and FEDER funds and the Juan de la Cierva postdoctoral contract to LM are acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, (IPT13/0001 - ISCIII-SGEFI/FEDER).

Establishing health effects of bioactive compounds from dry-cured meat is an active area of clinical research. The present study aims to investigate whether consuming dry-cured ham with biopeptides, among other bioactive compounds, modifies blood pressure (BP) and improves other risk factors for cardiovascular disease in humans. This two-arm, cross-over, randomised controlled trial involved 38 healthy subjects with pre-hypertension. Participants received 80 g/day dry-cured pork ham or 100 g/day cooked ham (control product). A daily intake of 80 g dry-cured ham did not impair BP or 24 h sodium excretion. Total cholesterol, LDL and basal glucose levels dropped after dry-cured ham consumption (p = 0.00019, p = 0.021 and p = 0.014, respectively). Cooked ham did not affect any of the clinical and biochemical markers. Dry-cured ham components could exert a plethora of activities over the cardiovascular system including lipid and glucose metabolism. Additional studies are needed to confirm the effects of dry-cured meat biopeptides on diverse risk factors in pathological conditions. Ciencias de la Alimentación Farmacia Medicina

Sensory and nutritional properties of dry-cured ham can be negatively affected due to oxidative modifications of muscle proteins during its processing. In this study, a peptidomic approach has been used in order to study the evolution of oxidised peptides generated throughout the dry-curing process (0, 2, 3.5, 5, 6.5 and 9 months), focusing on those derived from major myofibrillar proteins. A total of 67 peptides showing methionine, proline, and tryptophan oxidations were identified in common in all samples by nano liquid chromatography coupled to tandem mass spectrometry for their relative quantification using a label-free methodology, showing the hydrolysis of some of the peptides during the process. So, the peptidomics strategy used in this work has resulted to be very useful as a complementary tool to the methods currently used to study protein oxidation, allowing a better understanding of the oxidation at peptide level and the influence of ham processing conditions., Grant AGL2014-57367-R from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER funds are acknowledged. Juan de la Cierva de Incorporación postdoctoral contract to LM is also acknowledged. The peptidomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII (PT13/0001-ISCIII-SGEFI/FEDER).