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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.

Refers to: Chuqiao Xiao, Mouming Zhao, Feibai Zhou, Marta Gallego, Jie Gao, Fidel Toldrá, Leticia Mora. Isolation and identification of alcohol dehydrogenase stabilizing peptides from Alcalase digested chicken breast hydrolysates. Journal of Functional Foods 64:103617 (2020), Bioactive peptides have attracted extensive attention worldwide as natural alternatives to promote human health and wellness. Previous studies have shown that chicken hydrolysates could enhance alcohol dehydrogenase, and subsequently they facilitate alcohol metabolism and ameliorate alcohol-induced liver injury. The data presented in this article support the accompanying research article “Isolation and identification of alcohol dehydrogenase stabilizing peptides from Alcalase digested chicken breast hydrolysates”. Present article details all 82 peptides identified from the most active fractions of chicken hydrolysates, and 154 peptides from in silico digestion of the 82 identified peptides, together with the prediction of their potential bioactivity and applicability using several in silico assays., This work was supported by Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, the National Natural Science Foundation of China (No. 31701539 and No.31871746) and the Natural Science Foundation of Guangdong Province (No. 2017A030313127). We also thank Ramón y Cajal postdoctoral contract by L.M. and the China Scholarship Council (CSC) research program for providing funding for C. X.

A systematic insight into the structure-function properties of small bioactive peptides is of great importance. Herein, peptidomics and computational methodology were adopted to investigate the stabilization patterns and building blocks of antioxidant peptides resulting from proteolysis during the ripening of Spanish dry-cured ham (9-24 months of processing). The results showed that native peptides underwent manufacture-induced steric/redox stress, while homogeneous/heterogeneous p-π/π-π interaction significantly improved the ABTS+ inhibition activity of hydrophobic peptides. However, for more hydrophilic peptides, the intrinsic π-interaction system (i.e., cation-π and π-π packing) substantially interfered with ABTS+/DPPH scavenging events when compared to the aromatic residues. Semi-quantitative peptidomics and molecular simulation/docking revealed that VFSSQGQSELILLQK and LCPSPDGLYL are two potential antioxidant peptides at the late ripening-drying. They had distinctive self-folding destinies following solvation owing to varying charged/hydrophobic properties of termini and hydrogen atom donor, allowing different flexibility of backbone and interactive surface towards free radicals ex situ followed by electron/proton transfer., Grant AGL2017-89381-R funded by MCIN/ AEI /10.13039/501100011033 and “ERDF A way of making Europe” and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” are also acknowledged. C.L. appreciated the financial support from China Scholarship Council (CSC), China through the State Scholarship Fund for Overseas Study (No. 201806760050).

Dry-cured ham has been described as a good source of bioactive peptides and taste-active compounds. Some of them are dipeptides and tripeptides that are released in a large amount from different muscle proteins due to the action of exopeptidases during the dry-cured ham processing. The potential of dipeptides and tripeptides to exert bioactivities and impart taste characteristics to dry-cured ham has been evaluated using the BIOPEP database, since in silico approaches are a time- and cost-effective alternative to empirical approaches. Most of the studied dipeptides and tripeptides showed ACE and DPP inhibitory activities as well as imparted bitter taste. In fact, more than one bioactivity and/or taste could be assigned to a given peptide sequence, and there could be a correlation between both, like ACE inhibitory and bitter EA, EI and LG peptides. Furthermore, several dipeptides such as EK, KP, LA, PL, PP, RG, and VE, among others, were found to be multifunctional (ACE and DPP IV inhibitory) which would be determined by their structure, sequence and amino acid composition. In silico analysis evidences the relevance of dipeptides and tripeptides in the bioactivity and taste of dry-cured hams, but further empirical assays including in vitro and in vivo studies are necessary to confirm such theoretical results. Possible degradation of the small peptides during gastrointestinal digestion and intestinal absorption as well as interactions with the food matrix could reduce their bioavailability and bioaccessibility, and modify their biological activities., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged. Ramón y Cajal postdoctoral contract to LM is also acknowledged.

Foods and their industry by-products constitute very good sources of bioactive peptides, which can be naturally generated during processing but are also extensively produced through enzymatic hydrolysis, microbial fermentation, and even during gastrointestinal digestion in the human body [...]

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.

Proteins and peptides are very important nutritional and functional constituents of foods that may be subject to changes derived from either food processing or gastrointestinal digestion. Some of these changes may affect their functional properties and also their biological activities and, therefore, it is important for them to be protected. There have been numerous recent developments to ensure the delivery of bioactive peptides with good stability. Such techniques are based either on the encapsulation of bioactive peptides or on the modification of the peptide structure in order to increase their stability against any food processing treatment such as heating, cooling, pH changes, NaCl treatment and also hydrolysis during gastrointestinal digestion.

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).

Peptidomics is considered an essential tool for the identification and quantitation of naturally generated peptides present in complex matrices from animal and vegetable origin. These peptides have been generated in natural processes such as fermentation, curing, or maturation, due to the action of endogenous or microbial enzymes, and even, once ingested, from the action of gastrointestinal enzymes during the digestion. The peptides generated have a widespread range of sizes and nonspecific cleavage sites, and certain primary steps such as solid-phase extraction, ultrafiltration, or chromatography are necessary to reduce the complexity of the matrix and avoid interferences to the identification and quantification of the peptides by mass spectrometry approaches. Thus the identification and quantification of naturally generated peptides require and adaptation of the analytical and experimental protocols of previously used proteomics strategies.

© 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.

To investigate the oxidative effect of CPF on chicken breast proteins, peptidomics and proteomics analysis were used. For this, chicken breast samples were exposed to CPF and subjected to simulated gastrointestinal digestion. The identification of oxidized peptides from digested and undigested proteins were performed by LC MS/MS (nanoESI qQTOF). Prior to mass analyses undigested proteins were trypsinated. Data were analysed using MASCOT and ProteinPilot v 4.5 software. In this study, 90 and 107 oxidized peptides from digested proteins of control and exposed samples were identified, respectively. These peptides corresponding to 12 oxidized proteins. Meanwhile, 260 and 324 oxidized peptides from undigested proteins (control and exposed samples) were identified, which corresponding to 19 and 17 proteins, respectively. Collagen was protein more susceptible to oxidation promoted by chlorpyrifos in digested and undigested proteins. Identification of these oxidized proteins from simulated digestion provides an important insight about the impact of substances like certain veterinary drugs at trace concentrations on the nutritional value of chicken breast meat., 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. Also COLCIENCIAS and University of Cartagena for financial support (2019 and 2020) to the project Code 1107-711-50102 are acknowledged.

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.

Nitrite and nitrate have been traditionally used for the preservation of meat products because of the effective antimicrobial action of nitrite against Clostridium botulinum, the outgrowth of its spores as well as other bacteria. However, the use of nitrite and nitrate has been questioned in last half century due to the possible generation of N-nitrosamines through reaction of nitrite with secondary amines. Nitrite replacement strategies began in the 70s addressing these issues and instigated searches for natural alternatives to nitrate and nitrite, or for natural sources of nitrite and nitrate such as vegetable extracts. These alternatives have been considered by producers and consumers as an attractive practice even though they may also have some risks. This manuscript reviews and discusses the chemistry, safety, and regulatory considerations in the use of nitrite and nitrate from natural origin for the preservation of meat products.

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.

There is a wide range of chemicals that may be present in meat as residues either during processing or during cooking and household management. Analytical technologies are constantly being developed and improved, allowing the correct detection and determination of such compounds and contributing to a better meat safety. Some toxic compounds that may be present in meat or meat products are N-nitrosamines as a consequence of nitrite use, polycyclic aromatic hydrocarbons generated from smoking, oxidation of lipids and proteins, and, finally, other substances resulting from the raw materials used in animal production like veterinary drug residues or environmental contaminants. All these are harmful compounds and the available detection analyses are described in this chapter.

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).

This study aims to examine the effects of non-hydrolyzed octopus (Octopus vulgaris) muscle proteins (NHOPs) and their hydrolysates (OPHs) on alloxan induced diabetes in Wistar rats (AIDR). Animals were allocated into seven groups of six rats each: control group (C), diabetic group (D) and diabetic rats treated with acarbose (D + Acar), non-hydrolyzed octopus proteins (D + NHOPs) and octopus proteins hydrolysates (D + OPHs) groups. The diabetic rats presented a significant increase in glycemic status such as α-amylase activity (in plasma, pancreas and intestine), hepatic glycogen, blood glucose and glycated hemoglobin (HbA1c) levels, as well as a significant decrease in the levels of plasma insulin and total hemoglobin compared to control group. In addition, plasma and liver contents in total cholesterol, triglycerides and LDL-cholesterol significantly increased in AIDR compared to control group. However, the daily administration of OPHs for 30 days improved the glucose tolerance test, the glycemic status of diabetic rats and corrected the lipid profiles. Further, a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase as well as in the level of plasma bilirubin on diabetic status was observed, indicating considerable hepatocellular injury. OPHs treatment was found to attenuate the increased activities of the plasma enzymes produced by diabetes and caused a subsequent recovery towards normalization compared to the control group. By contrast, the NHOPs treatment was found to increase the glucose metabolic disorders in AIDR. These beneficial effects of OPHs were confirmed by histological findings in the hepatic and pancreatic tissues of diabetic treated rats. Indeed, they avoid lipid accumulation in the hepatocytes and protect the pancreatic β-cells from degeneration. Our results thus suggest that OPHs may be helpful in the preventing from diabetic complications by reversing hepatotoxicity., This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. The authors wish to express their sincere gratitude to Pr Najiba ZEGHAL, professor in the Sfax Faculty of sciences for her comments that greatly improved the manuscript.

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.

A nitrate reductase immobilized system with an oxygen electrode has been developed and optimized to determine the nitrate content in dry-cured ham. The obtained amperometric signal was recorded at 5 s in the immobilized nitrate reductase sensor and the reaction rates (slope) were related to the nitrate content. A linear relationship between the reduction nitrate rate by action of nitrate reductase and the nitrate concentration was found within the range 10–70 μM (R2 = 0.9761). The immobilized enzyme showed a high specificity and sensibility and was stable enough to allow the reutilization of the membranes up to eight times without loss of activity. This reduces the cost of the analysis as well as the necessary equipment that is cheap and the short average measurement time for each sample. The analysis of nitrate in dry-cured ham samples with the sensor and by HPLC revealed very good agreement (R2 = 0.971). The use of this sensor may constitute an interesting and valid alternative for the quantification of nitrate in dry-cured ham., Funded by the Spanish Ministry of Economy, Industry and Competitiveness, AGL2014-57367-R and FEDER funds. Felipe Jadán enjoyed a scholarship from Secretaria de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT), Ecuador.

Aims: The present study aims to evaluate the antiobesity, hypolipidemic and cardioprotective effects of fermented sardinelle (Sardinella aurita) protein hydrolysates (FSPHs) produced with two proteolytic bacteria, Bacillus subtilis A26 (FSPH-A26) and Bacillus amyloliquefaciens An6 (FSPH-An6). Main methods: Wistar rats were fed during 10 weeks a standard laboratory diet, a high caloric diet (HCD) and a HCD coupled with the oral administration of sardinelle meat flour (SMF) or FSPHs. Key findings: HCD caused hyperlipidemia and increased body weight (BW). The daily oral administration of FSPHs or SMF reduced the total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-c) serum levels, and increased the level of high-density lipoprotein cholesterol (HDL-c). Nevertheless, FSPHs were found to be more efficient than SMF. FSPHs also lowered hepatic TC and TG content and decreased the pancreatic lipase activity. Further, the administration of FSPHs or SMF decreased the BW gain, the food intake and the relative epididymal adipose tissue weight. FSPHs exhibited a potent cardioprotective effect against heart attack, which was demonstrated by returning atherogenic indexes to their normal levels and the conservation of standard histological structure of the heart and aorta. Significance: The overall results indicate that FSPHs contained bioactive peptides which significantly attenuated hyperlipidemia, and might reduce the risk of cardiovascular disease (CVD) in rats fed HCD., This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. AGL2014-57367-R from MINECO and FEDER funds, and JAEDOC-CSIC post-doctoral contract of L.M. cofounded by the European Social Found are acknowledged. MALDI-TOF analysis was carried out by in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.

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.

The in vivo wound-healing potential of gels and films based on cuttlefish skin gelatin (CSG) incorporated with aqueous henna extract (AHE) (at 50 and 500 μg/ml) was studied. As wound healing is related to reactive oxygen species (ROS), this study was first conducted to evaluate the antioxidant activity of AHE by DPPH radical-scavenging, β-carotene-linoleate bleaching, and reducing power assays. The major volatile compounds of AHE were lawsone, dimethyl sulfoxide, dimethyl sulfide, eugenol and maltol. The finding of the study showed that the incorporation of AHE into gelatin gels and films enhanced the antioxidant activity in a dose-dependent manner. In the wound-healing study, topical application of gelatin hydrogels or films enriched by AHE, on the wound site in a rat model, enhanced significantly wound healing activity and helped to prevent from inflammation damage, when compared to the control and CICAFLORA®-treated groups. Further, all preparations were found to improve the antioxidant status of treated rats as was evidenced by increased antioxidant enzymatic activities and reduced malondialdehyde (MDA) content. The increase in the activities of catalase (CAT), superoxide dismutase (SOD) and gluthatione peroxydase (GPx) in wound tissues revealed that some bioactive compounds in AHE are able to enhance the healing of wounds by reducing the damage caused by ROS. Wound-healing activity was confirmed by histopathology studies. The obtained results showed that the addition of AHE to the gelatin matrix could improve the wound healing activity., 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.

Organophosphate pesticides are frequently used to eliminate or prevent insects in poultry. However, their residues may continue in meat after slaughtering. In this study, proteomics and peptidomics approaches were used to evaluate their oxidative impact on myosin and chicken breast proteins under in vitro conditions. Myosin protein was exposed to diazinon and chlorpyrifos showing an increase in its oxidation by increasing times, especially with chlorpyrifos. Then, chicken breast was contaminated with chlorpyrifos to evaluate carbonylation and the effect of simulated gastrointestinal digestion. Proteins were isolated using size-exclusion-chromatography and identified by mass spectrometry in tandem. Myosin, β-enolase, CK-M-type and actin were identified as main proteins susceptible to oxidation. Also, oxidised peptides obtained before and after simulated gastrointestinal digestion were identified. Collagen peptides the most susceptible to oxidation. These results suggest that the presence of chlorpyrifos residues on meat could have a negative effect on its final quality and nutritional value., 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. Also COLCIENCIAS and University of Cartagena for financial support (2019 and 2020) to the project Code 1107-711-50102 are acknowledged. J.M.L. thanks to Colciencias for the scholarship No 647-2014.

The worldwide consumption of high-protein food has notoriously increased in recent years. Meat industry generates substantial quantities of protein-rich raw material, which are often discarded as low-value by-products. However, several bioactive compounds can be isolated from these products giving an added value to them. In addition to conventional extraction methods, emerging technologies, including high-pressure processing (HPP), ultrasound (US), pulsed electric fields (PEF) can be used for peptide isolation from meat by-products allowing to maintain the functional properties of these compounds. Antioxidant and antimicrobial activities are between the properties associated with peptides, what would enable their introduction in foods as ingredient and preservative. This review is focused to gather accurate information about the entire extraction process, from the source used until the final peptides obtained. In this sequence are included the pretreatment of the by-product, the extraction procedure, the fractionation and purification, as well as the assay used for the determination of their antioxidant and antimicrobial activities., This research was supported by Grant RTA 2017-00024-CO4-04 from INIA (Spain) and Grant AGL2017-89381-R from Spanish Ministry of Economy, Industry and Competitiveness and Fondo Europeo de Desarrollo Regional (FEDER). Acknowledgments to INIA for granting Paula Borrajo with a predoctoral scholarship (grant number CPD2016-0030). LM was supported by Ramón y Cajal postdoctoral contract. José M. Lorenzo is member of the HealthyMeat network, funded by CYTED (ref. 119RT0568). Thanks to GAIN (Axencia Galega de Innovación) for supporting this research (grant number IN607A2019/01).

Myofibrillar proteins are extensively degraded by muscle endo- and exopeptidases during the ageing of meat and the processing of meat products. One of the most studied products is dry-cured ham. This degradation implies changes in the product in terms of texture (mainly due to calpains and cathepsins endopeptidases) and flavour (due to the action of exopeptidases) and defines its final quality. During the last decade, naturally generated peptides from the myofibrillar proteins titin, myosin light chain, troponin T, LIM domain-binding protein 3, and actin have been identified using peptidomic approaches, also showing the potential to act as bioactives in the human body when ingested. In this study, tandem mass spectrometry has been used for the identification of the peptides generated during the proteolysis of myosin heavy chain protein after 9 months of Spanish dry-cured ham processing. The size, sequence, and properties of some of the peptides showed their potential to act as bioactives., Grant AGL2014-57367-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged. Ramón y Cajal postdoctoral contract to LM is also acknowledged.

The present study aims to characterize black-barred (Hemiramphus far) skin gelatin (BG) gel enriched or not with its hydrolysate (BGH) in terms of their rheological and micro-structural properties, as well as to evaluate their effect on smooth hound fillet quality during chilled storage. The results of the rheological properties showed that peptides addition influenced elastic and loss modulus as observed from temperature sweep. Based on the microstructure, BG gel had a fine network with small voids that were filled by the presence of BGH peptides. On the other hand, when used as coating agents for smooth hound fillets, BG and BG-BGH showed that, in comparison to control group, fish spoilage was significantly delayed in coated samples. In fact, after 8 days of storage, samples coated with BG and BG+BGH showed the lowest weight loss levels, and preserved their initial water activity (a) values. In addition, fillet coating was able to significantly reduce TVB-N content and oxidation process, as reflected by the low malondialdehyde and carbonyl contents. Furthermore, compared to the control group, all the treatments were found to reduce the degree of microbial deterioration of the fillets, leading to low the free amino acids and nucleotides contents in wrapped fish samples. Therefore, gelatin coating helps to prevent fish fillets from deterioration and oxidation processes., This work was funded and financially supported by the Ministry of Higher Education and Scientific Research, Tunisia (Grant no. 18PJEC06-03).

In this study, the antihypertensive activity of Purafect®-smooth hound viscera protein hydrolysate (VPH) and its peptide fraction with molecular weight (MW) below 1 kDa (VPH-I) was investigated. In addition, the lipase inhibitory activity, as well the anticoagulant potential, in vitro, were assessed. The antihypertensive effects of VPH and VPH-I were studied during 24 h (short-term effect) and 30 days (long-term effect) using high-salt (18% NaCl) and -fructose (10%) diet (HSFD)-induced hypertension. Data showed that, 4 h post-administration of VPH and VPH-I (200 mg/kg BW), the systolic blood pressure of rats was reduced by about 6 and 9 mmHg, respectively. These effects were similar to that obtained with Captopril (~9 mmHg at t = 4 h). On the other hand, exposing the rats to daily to HSFD, coupled to the administration of viscera peptides, was found to attenuate hypertension. In addition, the proteins’ treatments were able to correct lipid and glycemic disorders, by reducing the total cholesterol and triglyceride contents and resorting to the plasma glucose level, compared to the HSFD group. Overall, the present findings demonstrated the preventive effect of VPH-peptides from hypertension complications, as a result of their biological properties., This research was funded by the Ministry of Higher Education and Scientific Research, Tunisia, We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

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.