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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.

Dry-cured ham-derived dipeptides, generated along a dry-curing process, are of high importance since they play a role in flavor development of dry-cured ham. The objective of this study was to analyze the residues of the less-studied metabotropic glutamate receptor 1 (mGluR1) implicated in the recognition of umami dry-cured ham dipeptides by molecular docking simulation using the AutoDock Suite tool. AH, DA, DG, EE, ES, EV, and VG (and glutamate) were found to attach the enzyme with inhibition constants ranging from 12.32 µM (AH) to 875.75 µM (ES) in the case if Rattus norvegicus mGluR1 and 17.44 µM (VG) to 294.68 µM (DG) in the case of Homo sapiens, in the open–open conformations. Main interactions were done with key receptor residues Tyr74, Ser186, Glu292, and Lys409; and Ser165, Ser186, and Asp318, respectively, for the two receptors in the open–open conformations. However, more residues may be involved in the complex stabilization. Specifically, AH, EE and ES relatively established a higher number of H-bonds, but AH, EV, and VG presented relatively lower Ki values in all cases. The results obtained here could provide information about structure and taste relationships and constitute a theoretical reference for the interactions of novel umami food-derived peptides., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, and Ramón y Cajal postdoctoral contract RYC-2016-19635 by L.M. 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.

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.

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.

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.

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)

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.

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

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.

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.

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

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

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

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

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

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

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

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

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.

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.

This work is focused on the determination of compounds of nutritional interest that are naturally present in pork meat and how they are affected during the processing of dry-cured ham. Such compounds are creatine, creatinine, coenzyme Q(10), glutathione, carnosine, anserine, carnitine, taurine, cystine, cysteine and the essential amino acids. Their antioxidant and antyhipertensive functions were evaluated. Of all the assayed substances, only glutathione decreased totally during processing. Carnosine, creatinine, anserine and glutathione showed antioxidant, while cysteine, glutathione and carnosine showed antyhipertensive activity. So, dry-cured ham constitutes an excellent source of essential amino acids (all essential amino acids exhibited a large increase during processing) and other nutritionally interesting compounds such as cystine, cysteine, carnosine, anserine, taurine, carnitine and coenzyme Q(10).

This study is focused on the characterization of the proteolysis occurred during the processing of Pastırma, a traditional Turkish dry-cured meat product, which is responsible for its final characteristics. Thus, the evaluation of naturally generated free amino acids and peptides present at 0, 2, 5, 10, and 21 days of processing and the bioactivity of peptide fractions have been approached. Peptides were examined by MALDI-TOF and results showed differences in the amount of generated peptides at different times of processing, and a total of 29 peptides were newly generated at Day 21 in comparison with Day 2 during processing. The water soluble fraction of Pastırma at the end of the curing period (Day 21) was also analyzed by size-exclusion chromatography and some of the collected fractions demonstrated strong ACE-inhibitory and antioxidant activities. In fact, Pastırma showed an ACE inhibitory activity higher than 86% from 220 to 270 mL corresponding to molecular masses between 900 and 1500 Da, and also a DPPH radical-scavenging activity above 60% at 250 to 300 mL corresponding to molecular masses between 700 and 2000 Da. Thus, Pastırma represents a good source of natural ACE-inhibitory and antioxidant peptides which might be due to the proteolysis occurred by endogenous enzymes and the contribution of the çemen paste used in production., Grant AGL2014-57367-R from MINECO (Spain) and FEDER funds and JAEDOC-CSIC postdoctoral contract of L.M. cofunded by European Social Funds are acknowledged. The research fellowship grant provided by Erasmus + KA1 Student Mobility Programme for Higher Education for the author Ebru Deniz is also gratefully acknowledged.

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC50 values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500–800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500–800 Da and 1200–1500 Da., This study was funded by Grant of Ministry of Science, Research and Technology of Iran and Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138). Also, a JAEDOC-CSIC postdoctoral contract was cofunded by the European Social Fund to Dr. Mora.

MEAT, PROCESS EDMEATS AND POULTRY Methods to Predict Spoilage of Muscle Foods, G. Duffy, A. Dolan, and C.M. Burgess Microbial Foodborne Pathogens, M. Mataragas and E.H. Drosinos Parasites, A. Nareaho Mycotoxin Analysis in Poultry and Processed Meats, J.-D. Bailly and P. Guerre Detection of Genetically Modified Organisms in Processed Meats and Poultry, A. Germini and A. Tonelli Detection of Adulterations: Addition of Foreign Proteins, M.C. Garcia Lopez and M.L. Marina Alegre Detection of Adulterations: Identification of Animal Species, J.A. Lenstra Detection of Irradiated Ingredients, E.M. Stewart Growth Promoters, M. Reig and F. Toldra Antibiotic Residues in Muscle Tissues of Edible Animal Products, E. Verdon Determination of Persistent Organic Pollutants in Meat, A.L. Iamiceli, I. Fochi, G. Brambilla, and A. di Domenico Biogenic Amines, M.C. Vidal-Carou, M.L. Latorre-Moratalla, and S. Bover-Cid Nitrosamines, S. Rath and F.G. Reyes Reyes Polycyclic Aromatic Hydrocarbons, P. Simko FISH AND SEAFOODS Assessment of Seafood Spoilage and the Microorganisms Involved, R.E. Levin Detection of the Principal Foodborne Pathogens in Seafoods and Seafood-Related Environments, D. Rodriguez-Lazaro and M. Hernandez Parasites, J.A. Balbuena and J.A. Raga Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases, C. Pereira Dopazo and I. Bandin Marine Toxins, C. Empey Campora and Y. Hokama Detection of Adulterations: Addition of Foreign Proteins, V. Verrez-Bagnis Detection of Adulterations: Identification of Seafood Species, A. Puyet and J.M. Bautista Spectrochemical Methods for the Determination of Metals in Seafood, J. Sneddon and C.A. Thibodeaux Food Irradiation and Its Detection, Y.C. Wong, D.W.M. Sin, and W.Y. Yao Veterinary Drugs, A. Kaufmann Analysis of Dioxins in Seafood and Seafood Products, L.R. Bordajandi, B. Gomara, and M.J. Gonzalez Environmental Contaminants: Persistent Organic Pollutants, M. Perugini Biogenic Amines in Seafood Products, C. Ruiz-Capillas and F. Jimenez-Colmenero Detection of GM Ingredients in Fish Feed, K. Messens, N. Gryson, K. Audenaert, and M. Eeckhout MILK AND DAIRY PRODUCTS Microbial Flora, E. Tsakalidou Spoilage Detection, M.C. Dantas Vanetti PCR-Based Methods for Detection of Foodborne Bacterial Pathogens in Dairy Products, I. Whiting, N. Cook, M. Hernandez, D. Rodriguez-Lazaro, and M. D'Agostino Mycotoxins and Toxins, C. Soler, J.M. Soriano, and J. Manes Detection of Adulterations: Addition of Foreign Lipids and Proteins, S.M. van Ruth, M.G.E.G. Bremer, and R. Frankhuizen Detection of Adulterations: Identification of Milk Origin, G. Moatsou Analysis of Antibiotics in Milk and Its Products, J. Wang Chemical Contaminants: Phthalates, J. Zhu, S.P. Phillips, and X.-L. Cao Environmental Contaminants, S. Bogialli and A. Di Corcia Allergens, V. Tregoat and A.J. van Hengel INDEX

The food additive nitrite (E249, E250) is commonly used in meat curing as a food preservation method. Because of potential negative health effects of nitrite, its use is strictly regulated. In an earlier study we have shown that the calculated intake of nitrite in children can exceed the acceptable daily intake (ADI) when conversion from dietary nitrate to nitrite is included. This study examined time-dependent changes in nitrite levels in four Swedish meat products frequently eaten by children: pork/beef sausage, liver paté and two types of chicken sausage, and how the production process, storage and also boiling (e.g., simmering in salted water) and frying affect the initial added nitrite level. The results showed a steep decrease in nitrite level between the point of addition to the product and the first sampling of the product 24 h later. After this time, residual nitrite levels continued to decrease, but much more slowly, until the recommended use-by date. Interestingly, this continuing decrease in nitrite was much smaller in the chicken products than in the pork/beef products. In a pilot study on pork/beef sausage, we found no effects of boiling on residual nitrite levels, but frying decreased nitrite levels by 50%. In scenarios of time-dependent depletion of nitrite using the data obtained for sausages to represent all cured meat products and including conversion from dietary nitrate, calculated nitrite intake in 4-year-old children generally exceeded the ADI. Moreover, the actual intake of nitrite from cured meat is dependent on the type of meat source, with a higher residual nitrite levels in chicken products compared with pork/beef products. This may result in increased nitrite exposure among consumers shifting their consumption pattern of processed meats from red to white meat products.

An enzymatic sensor employing lysine oxidase (LOx) with the immobilised enzyme system by crosslinking with glutaraldehyde using an immunodyne ABC nylon membrane, in combination with an oxygen electrode, has been optimised to determine the lysine content in dry-cured ham and dry-fermented sausage at different cured times. The amperometric signal obtained due to the oxygen depletion (consumed oxygen) during the lysine oxidation was recorded at 5 s in the immobilised enzyme sensor, and the reaction rates (slope) were related to the lysine content. A linear relationship between the consumed oxygen as a function of time (mg O2/l/s) and the lysine concentration in the range 10–250 μM (R2 = 0.9946) for the immobilised enzyme system was found. The immobilised enzyme sensor showed a high specificity and sensibility. Nevertheless, the stability of the immobilised enzyme at the assay temperature was very poor, and thus, a new membrane was required for each analysis. The analysis of lysine with the immobilised enzyme system in cured meat samples revealed very good agreement with the determination performed through standard HPLC methodology, which validated the use of this sensor as an alternative technique to evaluate cured meat quality., This study was funded by Grant Prometeo 2012/001 from Conselleria d’Educació of Generalitat Valenciana (Valencia, Spain) and the scholarship to F. A. Jadán Piedra from Secretaria de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT) of Ecuador.

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

The aim of this study was to investigate the antioxidant, anti-ACE and antibacterial activities of smooth hound viscera hydrolysates (SHVHs). Viscera, as by-product, were hydrolyzed with crude alkaline protease extract from the same species, three commercial proteases and a combination endogenous and exogenous preparations. Hydrolysates showed different degrees of hydrolysis and the digestion with endogenous enzymes in combination with commercial proteases was found to enhance protein hydrolysis. The resulting SHVHs had high protein content (70.89%–89.06%) and they were mainly constituted by Gly, Glu and Gln, while Tau was the major free amino acid. In addition, they contained high amounts of UMP, Uridine, GMP and Guanosine, while undigested proteins were mainly rich in IMP and Xanthine. Furthermore, SHVHs showed different molecular mass distribution and RP-HPLC profiles proving their molecular mass and hydrophilic/hydrophobic peptide heterogeneity. All the SHVHs exhibited antioxidant activity, in terms of radical-scavenging activity, reducing power, metal chelating activity, β-carotene protection, lipid peroxidation inhibition and DNA breakage assay, and it was found that a medium degree of hydrolysis was appropriate to obtain hydrolysates with good antioxidant capacity. In addition, the SHVHs antioxidant activity was improved after simulated gastrointestinal digestion. Besides their antibacterial effect against several Gram-positive and Gram-negative bacteria, all the hydrolysates showed varying degrees of ACE inhibitory activities and the highest one was achieved by the Purafect hydrolysate (IC50 = 75 μg/mL). The overall data suggested that the SHVHs could be used as potential source of natural antioxidant, antimicrobial and anti-ACE peptides to formulate functional foods., 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.

Amino acids are natural constituents of proteins and peptides present in foods that are known to contribute to many relevant functions in an organism. The knowledge of the amino acid profile or the essential amino acids contents in foods and the limiting amino acids in a protein is very important for the characterization of their biological value and how their nutritional benefits may be affected by food processing or during storage. There are many available methods for the analysis of amino acids in foods that are summarized in this article.

The final characteristics of texture and flavour in dry-cured ham are strongly influenced by the intense proteolysis occurred during its processing. However, the proteolysis phenomena depend on both the enzymatic activity of endogenous enzymes which varies with process conditions such as temperature, humidity, or time of curing, and the raw material characteristics such as genetics. In this study, the influence of three different genotypes in the final peptide profile of dry-cured ham has been evaluated through the identification of the peptide fragments generated at the end of processing. The peptide sequences have been identified by peptidomics using LC–MS/MS and a total of 2266 common peptides from 75 proteins have been relatively quantified with a label-free methodology showing three main statistically different groups. The most influential peptides were identified from the proteins myosin light chain 1, creatine kinase, myoglobin, troponin T, and myosin heavy chain 7. The differential peptide sequences have also been investigated, proving the differences in the action of endogenous enzymes between genetics as different peptide sequences were obtained at the end of the dry-curing period., AGL2014-57367-R from MINECO and FEDER funds and funding from the European Union 7th Framework Programme FP7-PEOPLE-2013-CIG under Grant Agreement 614281 (HIGHVALFOOD) are acknowledged. Incarlopsa enterprise (Tarancón, Spain) that processed and supplied the dry-cured hams and JAEDOC-CSIC postdoctoral contract of L.M. cofunded by the European Social Found are acknowledged.

Nitrate and nitrite are commonly added to dry-cured ham to provide protection against pathogen microorganisms, especially Clostridium botulinum. Both nitrate and nitrite were monitored with ion chromatography in dry-cured hams salted with different NaCl formulations (NaCl partially replaced by KCl and/or CaCl2, and MgCl2). Nitrate, that is more stable than nitrite, diffuses into the ham and acts as a reservoir for nitrite generation. A correct nitrate and nitrite penetration was detected from the surface to the inner zones of the hams throughout its processing, independently of the salt formulation. Nitrate and nitrite achieved similar concentrations, around 37 and 2.2 ppm, respectively in the inner zones of the ham for the three assayed salt formulations at the end of the process, which are in compliance with European regulations.

This study examined the antihypertensive and antioxidant activities of water soluble fractions of a Spanish dry-cured ham extract. Antihypertensive activity of a fractionated peptide extract, by size-exclusion chromatography was determined by measuring changes in systolic blood pressure of spontaneously hypertensive rats after oral administration. Every sample exhibited antihypertensive activity, pooled fractions corresponding to 1700 Da or lower were the most antihypertensive with a decrease of 38.38 mm Hg in systolic blood pressure. In vitro experiments revealed marked in vitro angiotensin I-converting enzyme inhibitory activity in fractions corresponding to these elution volumes. Some of the fractions exhibited great 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, ranging from 39% to 92% as well as superoxide ion extinguishing ability with values ranging from 41.67% to 50.27% of the antioxidant activity, suggesting the presence of peptides with antioxidant activity. These findings suggest that Spanish dry-cured ham contains peptides with antioxidant and antihypertensive activities.

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

Hydrophilic interaction chromatography has been used to investigate the retention characteristics of a group of polar compounds typically present in meat like carnosine (Car), anserine (Ans), creatine (Cr), creatinine (Cn), glutathione oxidised (GSSG), glutathione reduced (GSH) and nicotinamide adenine dinucleotide (NADH). These compounds were separated using four polar stationary phases composed by bare silica, amide and zwitterionic groups bound to porous silica and zwitterionic groups attached to a polymeric bead. Ammonium acetate salt used in the mobile phase was found to provide the best results in selectivity and reproducibility. The effect of salt concentration together with pH and acetonitrile (ACN) content of the eluent on the retention of the different polar stationary phases was also studied. A linear gradient of ACN from 80% to 30% with 10 mM ammonium acetate at neutral pH values (pH 6.0 and pH 7.8) offered retention factors k ≤ 10 while keeping a resolution higher than 1.5 for all the assayed compounds in bare silica and amide columns. The separation of NADH from GSSG was not possible under the studied conditions at any salt concentration using zwitterionic columns.