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High cholesterolemia is a key risk factor for the development of cardiovascular diseases, which are the main cause of mortality in developed countries. Most therapies are focused on the modulation of its biosynthesis through 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) inhibitors. In this sense, food-derived bioactive peptides might act as promising health alternatives through their ability to interact with crucial enzymes involved in metabolic pathways, avoiding the adverse effects of synthetic drugs. Dry-cured ham has been widely described as an important source of naturally-generated bioactive peptides exerting ACEI-inhibitory activity, antioxidant activity, and anti-inflammatory activity between others. Based on these findings, the aim of this work was to assess, for the first time, the in vitro inhibitory activity of HMG-CoAR exerted by dipeptides generated during the manufacturing of dry-cured ham, previously described with relevant roles on other bioactivities.The in vitro inhibitory activity of the dipeptides was assessed by measuring the substrate consumption rate of the 3-hydroxy-3-methylglutaryl CoA reductase in their presence, with the following pertinent calculations.Further research was carried out to estimate the possible interactions of the most bioactive dipeptides with the enzyme by performing in silico analysis consisting of molecular docking approaches.Main findings showed DA, DD, EE, ES, and LL dipeptides as main HMG-CoAR inhibitors. Additionally, computational analysis indicated statin-like interactions of the dipeptides with HMG-CoAR.This study reveals, for the first time, the hypocholesterolemic potential of dry-cured ham-derived dipeptides and, at the same time, converges in the same vein as many reports that experimentally argue the cardiovascular benefits of dry-cured ham consumption due to its bioactive peptide content.

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.

Sweet dipeptide Ala-Ala (AA) could be one of the most abundant dipeptides naturally generated in meat products due to the high occurrence of its sequence all along endogenous proteins, which means it could play a key role in dry-cured ham flavour development and bioactivity. In this study, a mass spectrometry in tandem methodology was optimised to quantitate the dipeptide AA in dry-cured ham manufactured under different processing conditions. Dipeptide values reached 230 µg/g after 12-month of ripening, but statistically insignificant differences were observed with salt reduction. Regarding its antihypertensive activity, an IC50 value of 110.824 µM was determined from the in vitro inhibition of angiotensin-I converting (ACE-I) enzyme, and the oral administration of 1 mg of AA per kg of body weight showed a significant reduction of the systolic blood pressure (SBP) in spontaneous hypertensive rats (SHR). This study elucidates the importance of AA dipeptide and its potential role in cardiovascular health after dry-cured ham consumption., Grant AGL2017-89381-R funded by MCIN/ AEI /https://doi.org/10.13039/501100011033 by “ERDF A way of making Europe” is acknowledged. Grant PRE2018-083367 (A.H.) funded by MCIN/AEI /https://doi.org/10.13039/501100011033 and “ESF Investing in your future” and grant RYC-2016–19635 (L.M.) funded by MCIN/AEI /https://doi.org/10.13039/501100011033 and “ESF Investing in your future” are also acknowledged. Arihara funding KAKENHI (21H02348) from Japan Society for the Promotion of Science is also acknowledged.

Recently, the use of bioactive compounds in improving human health has received more attention. The aim of the present study was to hydrolyze orange seed proteins using pepsin enzyme to obtain bioactive peptides as well as to study the stability of such activity after simulated gastrointestinal digestion conditions. The method was optimized using different enzyme concentrations from 1% to 3%, hydrolysis times between 2 and 5 h, and an optimal temperature of 33 °C. Biological activities including α-glucosidase inhibition, α-amylase inhibition, Angiotensin I-Converting Enzyme (ACEI) inhibition, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated. According to the results, a significant higher value of the biological activity (p < 0.05) was observed using an enzyme ratio of 0.03 E/S and hydrolysis time of 3.5 h. After size-exclusion chromatography separation, fractions 45–49 and 50–54 showed the highest biological roles such as antioxidant, ACEI inhibitory, and hypoglycemic. Fractions with the highest biological activity were purified using RP-HPLC and analyzed using nano-liquid chromatography and mass spectrometry. The results obtained after simulated gastrointestinal digestion indicated that peptide fractions obtained after chromatographic separation significantly maintain their activity., This research was funded by grant AGL2017-89381-R, and FEDER funds from the Spanish Ministry of Economy, Industry, and Competitiveness and the Ramón y Cajal postdoctoral contract by L.M. are acknowledged.

The processing of dry-cured ham results in the generation of small peptides by the action of endogenous enzymes on muscle proteins. Common proteomic workflows involve previous separation techniques based on liquid chromatography which are expensive and time-consuming. In this study, a convenient proteomic approach based on MALDI-ToF is proposed for the first time for the detection of dipeptides in Spanish dry-cured ham. Dipeptides AH, AL, DD, EV, and VF were identified in hams of 18 and 24 months of dry-curing. This work provides insights on the efficiency of a new peptidomic workflow for the short peptide identification from a complex food matrix and permits to evaluate the sample in terms of the presence of taste-related and bioactive dipeptides., Grant AGL2017-89381-R funded by MCIN/ AEI /10.13039/501100011033 by “ERDF A way of making Europe”,is acknowledged. Grant PRE2018-083367 (A.H.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” are also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF.

Meat and meat products constitute an important source of nutrients and play vital roles for growth, maintenance and repair of the body. In addition to the high quality of proteins, meat is also regarded as a major resource to produce bioactive peptides. Meat processing industry also produces by-products such as bones, blood and viscera, which could be further used for the production of bioactive compounds. In the physiological analysis, meat bioactive peptides have been reported to exert antioxidant, anti-hypertensive, anti-inflammatory, anti-microbial and antitumoral activities, which endow nutritional and functional value of meat. With the objective to exert the functional effect, the bioavailability should also be considered due to the degradation by digestion enzymes and the absorption process in intestinal mucosa. In this chapter, the general source, the enzymatic hydrolysis, the physiological effects as well as the bioavailability of bioactive peptides in meat are discussed.

A large number of bioactive peptides have been reported with a variety of benefits for human health like angiotensin-converting enzyme inhibition, antioxidant, antiinflammatory, hypoglycemic, or antithrombotic activity, among others. Bioactive peptides are generated through the enzymatic breakdown of proteins either in the food itself by endogenous or microbial peptidases, or in reactors with commercial peptidases. Once consumed, gastrointestinal digestion may also contribute to further generation of bioactive peptides. Endopeptidases are those acting first on proteins followed by the action of exopeptidases on the generated polypeptides and peptides. This chapter is reporting the enzymes and mechanisms involved in the hydrolysis of food proteins for the generation of bioactive peptides.

Microenvironmental factors may influence the antioxidant efficacy of food-derived peptides. This study evaluated the in vitro antioxidant properties of peptides released during the ripening (9 to 24 months of processing) of Spanish dry-cured ham (Biceps femoris muscle) assisted by spectral-chromatographic methodologies. Results indicated that 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical-cation (ABTS●+) quenching capacity of peptide extracts significantly increased (P < 0.05) until 24 months whereas peroxyl radical (ROO●) scavenging activity increased slowly and remained with non-significant change (P > 0.05) between 15 and 24 months. However, both ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH●) scavenging ability significantly decreased (P < 0.05) at 24 months. Additionally, morphological traits of peptide extracts suggested that a prolonged ripening enabled the formation/reconstruction of intra-/inter-molecular interactions in dispersion medium. Meta-analysis of chromatographic and spectral fingerprinting proved that the non-conjugated/π-conjugated oligomers mediated by aromatic moieties probably differentiated DPPH● and ABTS●+ antioxidant performance of peptides, showing a potentially altered solvent polarization process., 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).

© 2020 by the authors., In this study, orange seed proteins were hydrolyzed by Alcalase enzyme at different enzyme concentrations 1–3% (v/w) and hydrolysis times (2–5 h), to obtain bioactive peptides showing antioxidant, Angiotensin-converting enzyme (ACE) -inhibitory, and hypoglycemic activities. The highest biological activities (p < 0.05) were achieved by using a hydrolysis time of 5 h and an enzyme concentration of 2%. Orange seed protein hydrolysate (OSPH) was prepared under these conditions, and peptides were isolated and purified by using size-exclusion chromatography and high-performance liquid chromatography, respectively. The fractions that showed the highest biological activities were analyzed by mass spectrometry in tandem, and a total of 63 peptide sequences were found. Moreover, the effect of simulated gastrointestinal digestion on the bioactivity of the fractions was studied, and the novel peptide sequences generated were also identified. Overall, despite there being some differences in the profile of peptide sequences obtained, the main results showed non-significant differences in the analyzed bioactivities after simulated gastrointestinal digestion., 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. This work was also supported by Intramural project from CSIC number 201870E006.

The present study aimed to investigate the effect of cooking and simulated gastrointestinal digestion on the antioxidant and alcohol dehydrogenase (ADH) stabilization activity of peptides extracted from chicken breast muscle. Results showed that cooking would not affect peptide bioactivity, whereas further digestion using gastrointestinal enzymes could lead to significant changes, producing an increase in ORAC (112.5 to 682.0 uM TE/g) and ABTS radical scavenging activities (164.0 to 848.9 uM TE/g), whereas a decrease in DPPH radical scavenging (from 36.1% to 4.4%), ferric-reducing power (OD 700 from 0.50 to 0.15) and ADH stabilization activities (from 44.1% to 20.5%) was observed. The peptidomic analysis resulted in the identification and relative quantitation of 777 peptides from 76 different parent proteins and evidenced that peptides derived from titin and collagen were mainly responsible for the differences detected in the peptide profile. The decrease of DPPH radical scavenging, ferric reducing power, and ADH stabilization activity may result from the release of inactive peptides containing oxidized residues, mainly from collagen, leading to the loss of efficacy of active sequences. The results confirmed the importance of collagen derived peptides on the antioxidant and ADH stabilization activity observed in chicken breast as well as the negative impact of oxidation on the bioactivity of generated peptides after simulated gastrointestinal digestion. Nevertheless, further work would be needed to confirm the peptide sequences responsible for the observed bioactivity., This work was supported by Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, and the National Natural Science Foundation of China (No. 31701539 and No.31871746), and the Natural Science Foundation of Guangdong Province (No. 2017A030313127). Ramón y Cajal postdoctoral contract by LM and the China Scholarship Council (CSC) research program for C. X. are also acknowledged.

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

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.jafc.9b08297, There is a wide variety of peptides released from food proteins that are able to exert a relevant benefit for human health, such as angiotensin-converting enzyme inhibition, antioxidant, anti-inflammatory, hypoglucemic, or antithrombotic activity, among others. This manuscript is reviewing the recent advances on enzymatic mechanisms for the hydrolysis of proteins from foods of animal origin, including the types of enzymes and mechanisms of action involved, the strategies followed for the isolation and identification of bioactive peptides through advanced proteomic tools, and the assessment of bioactivity and its beneficial effects. Specific applications in fermented and/or ripened foods where a significant number of bioactive peptides have been reported with relevant in vivo physiological effects on laboratory rats and humans as well as the hydrolysis of animal food proteins for the production of bioactive peptides are also reviewed., The research leading to these results received funding from Grant AGL2017-89381-R from the Spanish Ministry of Economy, Industry and Competitivity and FEDER funds. Ramón y Cajal postdoctoral contract to Leticia Mora is also acknowledged.

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.

Small peptides such as dipeptides contribute to a great extent to the characteristic taste of dry-cured hams. In this study, hydrophilic interaction liquid chromatography (HILIC) combined to tandem mass spectrometry was used to separate, identify, and quantify seven dipeptides in dry-cured hams sampled at different processing times (6, 12, 18, and 24 months). Results showed an increased concentration of dipeptides DA, DG, EE, ES, and EV with the length of processing, obtaining values up to 23 μg/g of dry-cured ham, which suggests an intense action of muscle enzymes dipeptidyl peptidases during the process. The dipeptide VG significantly decreased from 7 to 4 μg/g of dry-cured ham as the processing increased from 6 to 24 months, whereas the dipeptide PA showed low values between 380 and 550 ng/g of dry-cured ham at all the sampling times. Additionally, in silico analyses reported the sensory characteristics of the studied dipeptides, mostly giving bitter and umami taste, and predicted their allergenicity, toxicity, and physicochemical properties. These results could be useful for further studies related to the pleasant taste of dry-cured hams., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged.

A wide variety of dry-fermented sausages are produced in European countries, where are considered valued traditional products. An intense proteolysis takes place during the processing of dry-fermented sausages due to the combined action of muscle and microbial peptidases, generating large amounts of peptides and free amino acids. These compounds participate in the development of the characteristic flavor of dry-fermented products, but some peptides can also exert certain bioactivities such as antioxidant and ACE inhibitory activities. This study has evaluated the changes in peptide profile and amino acid contents of three European dry-fermented sausages produced in Spain, Italy and Belgium, proving the intense degradation of proteins, mainly myofibrillar, and the generation of high amounts of different size peptides and free amino acids. The changes observed between the profiles of European sausages could be due to differences in product formulation, processing conditions and starter cultures used, which influence the activity of enzymes, both from muscle and bacterial origin. On the other hand, the bioactivity profile of each type of dry-fermented sausage was evaluated through the measurement of the ACE inhibitory and antioxidant activities in water-soluble peptide extracts fractionated by size-exclusion chromatography. Spanish and Belgian dry-fermented sausages showed values of ACE inhibition around 85%, whereas Belgian samples presented the highest DPPH radical-scavenging activity and ferric reducing power capacity. These results evidence the potential of Spanish, Italian and Belgian dry-fermented sausages as natural sources of bioactive peptides, giving an added-value to these traditional products., Authors acknowledge European Union FP7 under Grant Agreement 312090 (BACCHUS). This publication reflects only the authors views and the Community is not liable for any use made of the information contained therein. 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. 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).

Oxidative modifications of proteins and peptides can negatively affect nutritional, sensory and quality characteristics of dry-cured hams. The aim of this study was to use a peptidomics strategy for the identification and relative quantification of oxidised peptides in two different muscles, the external Semimembranosus (SM) and the internal Biceps femoris (BF), from dry-cured hams of 12 months of curing. The analysis by nanoliquid chromatography-tandem mass spectrometry identified peptides showing oxidation at different amino acid residues such as methionine, cysteine, histidine, tryptophan, and phenylalanine. The highest percentages of oxidised peptides identified in SM and BF muscles were from pyruvate kinase protein (16.7 and 28.6%, respectively). The relative quantitation of oxidised peptides using mass spectrometry in tandem label-free methodology evidenced significant differences between muscles, with myosin and troponin fragments as main responsible peptides for the clustering of data. The existing differences between BF and SM peptides were confirmed using MALDI imaging mass spectrometry technique. The obtained results suggest that differences in physicochemical characteristics such as water and salt contents between SM and BF muscles could affect the quantity of peptide oxidation. This peptidomics approach has allowed to analyse differences in peptide oxidation between dry-cured ham muscles as well as to improve the knowledge about the oxidative processes that occur in dry-cured hams., Grant AGL2014-57367-R from MINECO and FEDER funds are acknowledged. Ramón y Cajal 2016 postdoctoral contract to LM is 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).

Smooth-hound viscera hydrolysates (SHVHs) were prepared by treatment with Neutrase (SHVH-N) and Purafect (SHVH-P). Hydrolysates were then separated according to their molecular weight, using the ultra-filtration membrane system, into 5 fractions (≥50, 50-5, 5-3, 3-1 and ≤1kDa). Fractions showed different amino acid compositions and angiotensin I-converting enzyme (ACE) inhibitory potentials. The SHVH-P-FV (≤1kDa) and SHVH-N-FIV (3-1kDa) fractions showed the best ACE-inhibitory activities with IC50 values of 53.31 and 75.05µg/ml, respectively. According to their high ACE-inhibitory potential, FIV and FV were fractionated by RP-HPLC and then analyzed by LC-MS/MS to identify peptide sequences. A systematic peptidomic study resulted in the identification of numerous novel sequences. Furthermore, in silico data, based on the molecular docking simulation, showed that GPAGPRGPAG, AVVPPSDKM, TTMYPGIA, and VKPLPQSG could bind ACE active site with low interaction scores. Indeed, they share hydrogen bonds and Van der Waals and electrostatic interactions with ACE catalytic pockets.

In the current study, gelatin was extracted from black-barred halfbeak ( Hemiramphus far ) skin by successive alkaline and acid treatments and then hydrolyzed with Purafect ® . The black-barred halfbeak gelatin (BG) and its hydrolysate (BGH) were characterized and compared to the commercial bovine gelatin (CG). Samples were evaluated for their antioxidant, antibacterial and angiotensin I-converting enzyme (ACE) inhibitory activities. Results obtained using size exclusion chromatography showed that BG contained lower level of high molecular weight proteins, compared to CG. In addition, the amino acids composition revealed that BG contained lower level of imino acids (Pro+Hpx), compared to CG. These differences reflect the variations observed in the gel strength and gelling and melting temperatures of both skin gelatins. Furthermore, high similarities were observed between CG and BG in terms of their Fourier transform infrared (FTIR) spectra, while their amino acid compositions were quite different. BGH, with a degree of hydrolysis of 12.5%, showed high antioxidant potential that was assessed by the scavenging activity, reducing power, oxygen radical absorbance capacity, β-carotene bleaching protection and lipid per-oxidation inhibition assays. In addition, BGH sample exhibited antibacterial activity against different Gram+ and Gram- bacteria. The ACE-inhibitory activity was also investigated. BGH showed an inhibitory effect of 80.76%, while BG inhibited the ACE only by 36.51% at 1 mg/ml. Thus, black-barred halfbeak gelatin represents a promising source of antioxidant, ACE-inhibitory and antimicrobial peptides that might prevent humans from several diseases.

Ageing is widely used in the meat industry to improve tenderness mainly as a result of the breakdown of muscular proteins through the action of endopeptidases during storage time. In addition, meat contains a large pool of other proteolytic enzymes, mainly exopeptidases, which cut from the outer edges of proteins and polypeptides generating a vast array of peptides and free amino acids. Some of these peptides could potentially exert bioactivities of interest for human health. In this study, ACE-inhibitory and antioxidant activities during meat ageing in chilled-storage at 4 °C for a period of time of 0, 1, 2, 3 and 4 weeks have been determined. Beef loin steak samples were analysed before and after cooking, and gastrointestinal digestion was simulated in order to study its effect on the bioactivity. Control and cooked samples showed an increase in the ACE-inhibitory activity from the third week of chilled storage whereas non-significant differences in the antioxidant activity between control and cooked samples were detected during the studied time. After gastrointestinal digestion of samples, there was a significant increment of the ACE-inhibitory and antioxidant activities in comparison with control and cooked samples at all the studied times. As a main conclusion, cooking does not significantly influence the bioactivity detected whereas the in vitro gastrointestinal digestion produces a significant increase in the ACE-inhibitory and antioxidant activities from the first week, probably due to the intense generation of small peptides as a result of the action of gastrointestinal enzymes.

Food-derived bioactive peptides are promising compounds for the prevention and treatment of cardiovascular diseases, the main cause of mortality in developed countries. The aim of this work was to determine the in vitro anti-inflammatory, antioxidant, and angiotensin I-converting enzyme (ACE-I) inhibitory activities of twenty-four peptides that were identified in Spanish dry-cured hams. For the first time, some peptides such as PSNPP, HCNKKYRSEM and FNMPLTIRITPGSKA showed anti-inflammatory activity expressed as platelet-activating factor-acetylhydrolase, autotaxin, and lipoxygenase inhibition. Peptides MDPKYR and TKYRVP were the strongest antioxidants, whereas GGVPGG, TKYRVP, and HCNKKYRSEM showed the highest ACE-I inhibitory activity. Additionally, several peptides such as KPVAAP, MDPKYR, TKYRVP, HCNKKYRSEM exerted more than one of the assayed activities, increasing their health-enhancing potential. More studies are needed to evaluate the bioavailability of such peptides and their in vivo effect. This would contribute to consider dry-cured ham as a source of peptides beneficial for cardiovascular health., Grants AGL2014-57367-R and AGL2017-89831-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness and Intramural 201870E006 grant from CSIC are acknowledged. Ramón y Cajal postdoctoral contract to LM is also acknowledged.

[EN] The interest in using food byproducts as a source of bioactive peptides has increased significantly in the recent years. The goal of this work was to determine the presence and stability of peptides showing angiotensin I-converting enzyme (ACE-I), endothelin-converting enzyme (ECE), dipeptidyl peptidase-IV (DPP-IV), and platelet-activating factor-acetylhydrolase (PAF-AH) inhibitory activity derived from dry-cured ham bones, which could exert cardiovascular health benefits. ACE-I and DPP-IV inhibitory peptides were stable against heating typically used in Mediterranean household cooking methods and also to in vitro digestion. PAF-AH inhibitory activity significantly increased following simulated gastrointestinal digestion whereas ECE inhibitory significantly decreased (P < 0.05). The mass spectrometry analysis revealed a notable degradation of hemoglobin-derived peptides after simulated digestion, and the release of a large number of dipeptides that may have contributed to the observed bioactivities. These results suggest that natural peptides from Spanish dry-cured ham bones could contribute to a positive impact on cardiovascular health., This study was funded by the Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138). A Ramon y Cajal postdoctoral contract to L.M. is acknowledged. Proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.

Pork, one of the most consumed meats worldwide, has been facing major challenges regarding its low sensory quality and unhealthy image of fat. This chapter addresses current feeding strategies to ameliorate pork sensory attributes and nutritional quality by increasing intramuscular fat deposition and improving fatty acid composition, respectively. Dietary protein reduction, alone or combined with some components, contributes to satisfy consumer requirements and enhances the competitiveness of the meat industry with higher pork quality and lower production costs. In addition, feeding sources of n-3 polyunsaturated fatty acids to pigs, mainly from marine origin (rich in eicosapentaenoic and docosahexaenoic acids), increases their content in pork, thus improving the health value of its fatty acid profile. In the near future, the inclusion of microalgae and seaweeds in feed represents a promising approach for the maintenance and development of the livestock sector, as an environmental friendly alternative to balance food and feed industries.

Peptides with a similar antioxidant and ACE-inhibitory activity of royal jelly (RJ) generated from Alcalase hydrolysated pollen (AHP) were predicted by Response Surface Methodology (RSM). The model equations were proposed according to the effects of time and enzyme concentration on the antioxidant and ACE-inhibitory activity. The optimum values for Alcalase concentration and hydrolysis time were 1.5% and 4 h, respectively. Later, AHP was prepared and deproteinised to be further analysed using size-exclusion chromatography (SEC). After SEC separation, fractions with the highest activity of ACE-inhibitory, DPPH radical scavenging and ferric-reducing power were purified by RP-HPLC. The highest ACE-inhibitory and DPPH scavenging activity of fractions was found 100% and 66.61%, respectively. The most active fractions were analysed by nano-liquid chromatography and mass spectrometry in tandem (nLC-MS/MS) and a total of 195 peptide sequences were identified. The origins of all peptides were herbal proteins and certain coincidences with previously described bioactive sequences were discussed., This work was funded by the Ministry of Science, Research and Technology of Iran and the Grant AGL2014-57367-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness. Ramón y Cajal postdoctoral contract to LM is acknowledged. LC-MS/MS analysis was carried out by in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII Proteo Red Proteomics Platform.

In the present study, response surface method was used to optimize hydrolysis condition to generate potential bioactive peptides from pollen protein using pepsin (pepsin hydrolysated pollen—PHP) and trypsin (trypsin hydrolysated pollen—THP). Then PHP and THP prepared under optimized conditions were analyzed by size-exclusion chromatography. The fractions possessing the maximum ACE-inhibitory, DPPH radical scavenging, and ferric-reducing power were further purified by RP-HPLC. A heterogeneous composition of hydrophobic and hydrophilic peptides in both fractions was obtained. Finally, peptide sequences in active fractions of PHP and THP were identified by mass spectrometry in tandem. All the identified peptides had herbal protein origins. These were 6–21 amino acids in length, and Glycine and Alanine were two main hydrophobic amino acids present in their sequences. The results proved that using controlled enzymatic hydrolysis of pollen protein is possible to generate bioactive peptides with high ACE-inhibitory and antioxidant activity in final product. Practical applications: Pollen is well-known as an interesting protein source. Compared to other types of hydrolysis, enzymatic hydrolysis of vegetable proteins has few or no undesirable side reactions or products. In this study, controlled enzymatic hydrolysis of pollen protein was applied as a suitable method to produce bioactive peptide. The results proved that using controlled enzymatic hydrolysis of pollen protein is possible to generate bioactive peptides with high ACE-inhibitory and antioxidant activity in final product. This product can be used as functional and health promoting ingredient in different food formulations., This work was financially supported by the Ministry of Science, Research and Technology of Iran, Grant AGL2014‐57367‐R and FEDER funds from the Spanish Ministry of Economy, Industry, and Competitiveness. Ramón y Cajal postdoctoral contract of LM is also acknowledged. MS/MS analysis was conducted in SCSIE University of Valencia Proteomics Unit (Spain) which is registered in ISCIII ProteoRed Proteomics Platform.

Enzymes are proteins with the ability to catalyze specific reactions using proteins, lipids or carbohydrates as substrate and generating a particular product. Enzymes are widely used in food processing and preservation. This chapter describes sources of enzymes, its safety in use and the wide range of applications of enzymes in foods.

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.

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.

berian dry-cured ham is a meat product known as a source of different bioactive peptides due to its high protein content and intense hydrolysis during its processing. In this study, the potential of α-glucosidase inhibitory peptides generated in Iberian dry-cured ham was evaluated for the first time. After size-exclusion chromatography and high-performance liquid chromatography using different stationary phases, the characterization and identification of the peptide sequences contained in the most active fractions was done using MALDI-ToF mass spectrometry and Q-ToF mass spectrometry in tandem. A total of 16 and 47 sequences of peptides were identified in the two most active fractions. On the other hand, the α-glucosidase inhibitory activity of previously identified bioactive peptides in Spanish dry-cured ham such as AEEEYPDL and LGVGG was established, confirming their multifunctionality., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, and Ramón y Cajal postdoctoral contract by LM are acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.

Meat and meat products have been described as a very good source of angiotensin I converting enzyme (ACEI)-inhibitory peptides. The generation of bioactive peptides can occur through the action of endogenous muscular enzymes during processing, gastrointestinal digestion, or by using commercial enzymes in laboratory or industry under controlled conditions. Studies of bioavailability are necessary in order to prove the positive health effect of bioactive peptides in the body as they should resist gastrointestinal digestion, cross the intestinal barrier, and reach blood stream and target organs. However, in order to better understand their effect, interactions, and bioavailability, it is necessary to consider food matrix interactions and continue the development of quantitative methodologies in order to obtain more data that will enable advances in the field of bioactive peptides and the determination of their influence on health., This research was funded by grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Science, Innovation, and Universities. Ramón y Cajal postdoctoral contract to L.M. is also acknowledged.

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: 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.

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.

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.

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.

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.

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).

This study investigated the potential effects of fermented sardinelle protein hydrolysates (FSPHs) obtained by two proteolytic bacteria, Bacillus subtilis A26 (FSPH-A26) and Bacillus amyloliquefaciens An6 (FSPH-An6), on hypercaloric diet (HCD) induced hyperglycemia and oxidative stress in rats. Effects of FSPHs on blood glucose level, glucose tolerance, α-amylase activity and hepatic glycogen content were investigated, as well as their effect on the oxidative stress state. Biochemical findings revealed that, while undigested sardinelle proteins did not exhibit hypoglycemic activity, oral administration of FSPHs to HCD-fed rats reduced significantly α-amylase activity as well as glycemia and hepatic glycogen levels. Further, the treatment with FSPHs improved the redox status by decreasing the levels of lipid peroxidation products and increasing the activities of the antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) and the level of glutathione in the liver and kidneys, as compared to those of HCD-fed rats. FSPHs were also found to exert significant protective effects on liver and kidney functions, evidenced by a marked decrease in alkaline phosphatase activity and a modulation of creatinine and uric acid contents. These results indicated the beneficial effect of FSPHs on the prevention from hyperglycemia and oxidative stress., 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.

Dry-cured meat products experience an intense proteolysis phenomenon during their processing. Products like dry-cured ham, that experience processing times longer than 10 months, show an extensive breakdown of major proteins and the generation of a high number of small peptides and finally, large amounts of free amino acids. Proteomic techniques have been successfully applied to the identification of the generated peptides and their sequencing. These data are essential for a better understanding of proteolytic enzymes and their reaction during processing. This approach should reveal key biomarker peptides controlling the process and establish strategies to drive and optimise enzyme reactions for the production of optimal quality products.

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.

The objective of this study was to purify and identify antioxidant peptides present in a water soluble extract of Spanish dry-cured ham. The initial extract was loaded into a Sephadex G25 column and fractions showing antioxidant activity were collected, pooled together and subjected to reversed-phase chromatography for further purification. Using a nano-LC-MS/MS analysis, 27 peptides were identified in these fractions. Several key peptides were selected for synthesis and the determination of their antioxidant properties using the DPPH radical-scavenging assay and reducing power analysis. The strongest radical-scavenging activity was observed with peptide SAGNPN which showed 50% antioxidant activity at a concentration of 1.5mg/ml. On the other hand, the peptide GLAGA showed the higher reducing power with 0.5 units of absorbance at 700 nm at a concentration of 1mg/ml. Other synthesised sequences showed lower antioxidant activity. The results indicate the potential of Spanish dry-cured ham as a source of antioxidant peptides naturally generated during the dry-curing process.