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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.

Royal jelly (RJ) has been known for centuries, but in the last 5-6 decades its systematic production and consumption has increased. RJ is secreted by the hypopharyngeal and mandibular glands of worker honeybees (Apis mellifera). This thick and milky substance contains water, proteins, carbohydrates, lipids, minerals, vitamins and such bio-active compounds as acetylcholine, peptides, the hormones testosterone, progesterone, prolactin, estradiol, (hydroxydecanoic acid) (HAD), adenosine monophosphate (AMP)-N1Oxide, polyphenols, flavonoids and adenosine. Because of its bioactive compounds, RJ can be considered as a functional and nutraceutical food. The main goal of this review is to summarize and update its physicochemical properties, bio-active ingredients, storage stability and shelf life. The functional properties are antioxidative activity, insulin-like action, improvement against diabetes, liver protection, antitumoral action, neurotrophic action, antibiotic effect, anti-inflammatory action and wound healing, hypotensive effect and blood regulatory actions, anti-aging effect and skin protection, effects on the reproductive system and fertility and also fortifying, tonic action and immunomodulating and anti-alergic activity. RJ may cause allergic reactions, asthma and even fatal anaphylaxis in some humans. Therefore, RJ should be orally ingested as nutreaceutical agent or food-ingredient only after an allergy test.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The effect of chicken hydrolysates (CHs) on alcohol dehydrogenase (ADH) stability was investigated, together with further bioactivity-oriented isolation and identification of CHs. A total of 82 peptides were identified using mass spectrometry in tandem after consecutive separation by size-exclusion chromatography and high-performance liquid chromatography. The identified peptides were then subjected to in silico gastrointestinal digestion and 154 peptides were generated. The potential bioactivity, safety and applicability of the peptides were assessed using multiple predictive programs. A total of 21 among the 154 peptides were predicted to be potentially active with applicability. Four peptides (DPQYPPGPPAF, QKPVL, KPC, and APGH) obtained after in silico digestion were synthesized and validated their activity. Results showed that DPQYPPGPPAF, KPC, and APGH could stabilize ADH in a dose-dependent manner. This study further indicated that chicken hydrolysate could be a novel functional food ingredient in facilitating alcohol metabolism and protection against alcoholic liver injury., This work was supported by the National Natural Science Foundation of China (Nos. 31701539 and 31871746) and the Natural Science Foundation of Guangdong Province (No. 2017A030313127). 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. We also thank the China Scholarship Council (CSC) research program for providing funding for C.X.

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

Angiotensin I-converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC50 of 0.83 mg mL−1. To identify peptides responsible for this activity, the extract was eluted through size-exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC50 of 0.42 and 0.51 mg mL−1, respectively. These fractions were analysed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC50 of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides., This work was funded by a grant from the Ministry of Higher Education and Scientific Research of Tunisia and grant Prometeo/2012/001 from Conselleria d´Educació Cultura i Sport of Generalitat Valenciana, both are acknowledged. JAEDOC- CSIC postdoctoral contract cofounded by ESF to L.M. is also acknowledged

Angiotensin converting enzyme (ACE) inhibitory peptides have been extensively studied as an alternative to synthetic drugs for the treatment of hypertension. Recent studies have shown that dry-cured ham is an important source of naturally generated bioactive peptides, especially showing ACE inhibitory activity. However, due to their excessive degradation by digestive and brush-border enzymes, it is not clear whether these peptides resist intestinal absorption and reach the blood stream where they may exert their antihypertensive effect. Therefore, dry-cured ham extracts and specific pure peptides naturally generated during the dry-curing process, showing ACE inhibitory activity, have been studied for their stability during transepithelial transport in a Caco-2 cell monolayer. The ACE inhibitory activity of transport samples was assayed, showing the highest values in apical samples at 15 min of incubation. In concentrated basal solutions, ACE inhibition values increased during transport for purified peptides, reaching values close to 70% for AAPLAP and KPVAAP at 60 min of cellular transport. Fragments generated by cellular activity were detected by using tandem mass spectrometry techniques, showing that AAATP, AAPLAP, and KPVAAP were hydrolysed during the transport, although KPVAAP was also absorbed intact. This study highlights the potential of intact dry-cured ham peptides as well as their fragments to be absorbed across the intestinal epithelium and reach the blood stream to exert an antihypertensive action., The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement 312090 (BACCHUS). This publication reflects only the author views and the Community is not liable for any use made of the information contained therein. Grant AGL2013-47169-R from MINECO and FEDER funds and the FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. are fully acknowledged. The JAEDOC-CSIC postdoctoral contract to L.M. co-funded by the European Social Fund and BOF (Special Research Fund of Ghent University) for their financial support (Project 01B04212) are also acknowledged.

Biodiesel constitutes an attractive source of energy because it is renewable, biodegradable, and non-polluting. Up to 20% biodiesel can be blended with fossil diesel and is being produced and used in many countries. Animal fat waste represents nearly 6% of total feedstock used to produce biodiesel through alkaline catalysis transesterification after its pretreatment. Lipase transesterification has some advantages such as the need of mild conditions, absence of pretreatment, no soap formation, simple downstream purification process and generation of high quality biodiesel. A few companies are using liquid lipase formulations and, in some cases, immobilized lipases for industrial biodiesel production, but the efficiency of the process can be further improved. Recent developments on immobilization support materials such as nanoparticles and magnetic nanomaterials have demonstrated high efficiency and potential for industrial applications. This manuscript reviews the latest advances on lipase transesterification and key operational variables for an efficient biodiesel production from animal fat waste.

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

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.

The complex proteolysis in Spanish dry-cured ham processing generates large amounts of small peptides and free amino acids which are responsible for the characteristic texture and flavour of this traditional product. The aim of this work was to study the degradation of the giant protein titin throughout the dry-curing process (2, 3.5, 5, 6.5, and 9 months) through the use of proteomic tools. A total of 320 peptides have been identified by nanoliquid chromatography coupled to tandem mass spectrometry, being some of them identified only at 9 months of processing. In order to confirm the absence of these peptides at other times of processing, MALDI-TOF MS was also employed as a fast and easier technique. Only four peptides, KDEAAKPKGPIKGVAKK, KKLRPGSGGEK, KNTDKWSECAR and ISIDEGKVL, were exclusively identified at 9 months of curing by using both methodologies so that these peptides could be used as potential biomarkers of dry-cured ham processing time., FPI Scholarship BES-2011-046096 from MINECO (Spain) to M.G. and grant AGL2010-16305 from MINECO and FEDER funds are fully acknowledged. JAEDOC-CSIC postdoctoral contract to L.M. is also acknowledged. The proteomic analysis was carried out in the SCSIE_University of Valencia Proteomics Unit, a member of ISCIII ProteoRed Proteomics Platform.

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

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

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

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

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

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

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

Background: The final characteristics of processed meats depend on many factors but one of the most important is the intense proteolysis occurred in muscle proteins due to the action of endogenous enzymes in dry-cured ham, and also microbial peptidases in the case of dry-fermented meats, that not only affects taste and flavour but also the generation of bioactive peptides. Scope and approach: In this review main difficulties in the identification of bioactive peptides in processed meats have been described. This study highlights the novel strategies used during the last years to identify naturally generated peptides, and emphasises the need of robust quantitative methodologies for the adequate characterisation of their bioavailability. In fact, the most common and well established quantitation approaches using proteomics are not adapted for peptidomics analysis, so alternative strategies need to be considered. Key findings and conclusions: The progress in the identification and characterisation of the activity of natural bioactive peptides is highly dependent on modern instruments and bioinformatics tools as well as updated protein databases. In fact, the use of in silico approaches and proteomics can be complementary tools in the identification of peptides from meat protein sources as the empirical experimental design can be simplified by using bioinformatics for computer simulation in most of the steps. Finally, Multiple Reaction Monitoring mass spectrometry methodology previously used in the quantitation of therapeutic peptides and biomarkers arises as a powerful tool for absolute quantitation or semi-quantitation of bioactive peptides., The research leading to these results received funding from the European Union 7th Framework Programme (FP7/2007–2013) under Grant Agreement 312090 (BACCHUS). This publication reflects only the author views and the Community is not liable for any use made of the information contained therein. Grant AGL2014-57367-R from MINECO and FEDER funds and the Juan de la Cierva postdoctoral contract to LM are acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, (IPT13/0001 - ISCIII-SGEFI/FEDER).

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

Pork meat is generally recognised as a food with relevant nutritional properties because of its content in high biological value proteins, group B vitamins, minerals especially heme iron, trace elements and other bioactive compounds. But pork meat also contributes to the intake of fat, saturated fatty acids, cholesterol, and other substances that, in inappropriate amounts, may result in negative physiologically effects. However, there are relevant factors affecting the content of many of these substances and somehow such variability should be taken into consideration. So, genetics, age and even type of muscle have a relevant influence on the amount of fat and the contents in heme iron. Also the composition in fatty acids of triacylglycerols is very sensitive to the contents of cereals in the feed; for instance, polyunsaturated fatty acids may range from 10% to 22% in pork meat. The content of other nutrients, like vitamins E and A, are also depending on the type of feed. Some bioactive substances like coenzyme Q10, taurine, glutamine, creatine, creatinine, carnosine and anserine show a large dependence on the type of muscle. This manuscript describes the main factors affecting the composition of pork meat nutrients and how these changes may affect the general food composition databases. (C) 2012 Elsevier Ltd. All rights reserved., Grant AGL2010-16305 from the Spanish Ministry of Science and Innovation (Madrid, Spain) and FEDER Funds and collaboration of Vaquero Foundation for R+D on Pork Meat (Madrid, Spain) are acknowledged. Grant PROMETEO/2012/001 from Generalitat Valenciana (Spain) is also acknowldeged. Work prepared within the Unidad Asociada IAD (UPV)-IATA (CSIC) framework.

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.

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

Antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities of sardinelle (Sardinella aurita) protein hydrolysates (SPHs) obtained by fermentation with Bacillus subtilis A26 (SPH-A26) and Bacillus amyloliquefaciens An6 (SPH-An6) were investigated. Both hydrolysates showed dose-dependent antioxidant activities evaluated using various in vitro antioxidant assays. Further, they were found to exhibit ACE-inhibitory activity. Peptides from SPH-A26 and SPH-An6 were analyzed by nESI-LC–MS/MS and approximately 800 peptides were identified. Identified peptides derived mainly from myosin (43% and 31% in SPH-An6 and SPH-A26, respectively). Several peptides identified in both hydrolysates were found to share sequences with previously identified antioxidant and ACE-inhibitory peptides based on Biopep database. Some of these peptides were selected for synthesis and their biological activities were evaluated. Among the synthesized peptides, NVPVYEGY and ITALAPSTM were found to be the most effective ACE-inhibitors with IC50 values of 0.21 and 0.23 mM, respectively. On the other hand, NVPVYEGY, which exhibited the highest ACE-inhibitory activity, showed the highest reducing power and peroxyl radical scavenging activities, followed by SLEAQAEKY and GTEDELDKY. The results of this study suggest that fermented sardinelle protein hydrolysates are a good source of natural antioxidant peptides and could have the potential to act as hypotensive nutraceutical ingredients., This work was funded by grant AGL2014-57367-R from MINECO and FEDER funds as well as the Ministry of Higher Education and Scientific Research-Tunisia and the Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138). JAEDOC-CSIC postdoctoral contract of L.M. cofunded by the European Social Found is also acknowledged. LC-MS/MS analysis was carried out in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.

The present study investigates the antibacterial activity of zebra blenny (Salaria basilisca) protein hydrolysates obtained by fermentation with a proteolytic bacterium, Bacillus mojavensis A21. The fermentative zebra blenny protein hydrolysate (FZPH), with a degree of hydrolysis (DH) of 17.35%, was fractionated by size exclusion chromatography on a Sephadex G-25 into six major fractions (F1-F6). Fraction F2, which exhibited antibacterial activity against several Gram-positive and Gram-negative bacteria, was further fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Fractions A and B from RP-HPLC exhibiting the highest antibacterial activity, were analysed using nano ESI-LC–MS/MS to identify the sequences of the peptides. A total of 28 and 41 peptides, containing from 8 to 31 amino acid residues, were identified in sub-fractions A and B, respectively. Further, identified bioactive peptides sharing sequences with previously identified peptides were reported. The results of this study suggest that FZPH is a good source of natural antimicrobial peptides and therefore, they could serve as a beneficial ingredient for nutraceuticals., This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. Grant AGL2014-57367-R from MINECO (Spain) and FEDER funds and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.

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.

Sodium intake above nutritional recommendations may involve harmful consequences to health such as the increased risk of cardiovascular diseases. Dry-cured ham constitutes a product with a relatively large amount of sodium. Thus, to obtain a healthier product for consumers with reduced sodium content, two formulations containing KCl alone (formulation II) or mixed with CaCl2 and MgCl2 (formulation III) have been proposed to partially replace NaCl. Lipolysis and lipid oxidation occurring in hams processed with these formulations have been studied since they have direct influence on the final flavor. No significant differences in acid lipase activity or lipid oxidation were found at the end of the process between the alternative formulations and formulation I (control with 100% NaCl). Differences in some free fatty acids, generated along the processing, were detected among treatments and at the end of dry-curing. Data suggests a slight trend towards a major lipolysis during treatment III.

[EN] The use of antibiotics in the European Union was banned since the 1st of January 2006. At present, and even though such ban, residues of drugs used in animal therapy or, illegally, as growth promoters might be found in food. Such residues may cause adverse health effects on consumers like allergic reactions in sensitive individuals develop transmissible antimicrobial resistance in food microflora, and have technological implications in the manufacture of fermented products. This is the reason why to check food for the presence of antibiotics residues is still necessary. ELISA test constitutes an attractive screening technique due to its simplicity, large number of samples per kit and possibility of automation. The aim of this manuscript is to provide a review of the numerous patents and applications for the analysis of antibiotic residues in foods., Projects A-05/08 and A-01/09 of Platform on Food Safety from Conselleria de Sanitat (Generalitat Valenciana, Spain) are fully acknowledged. Work prepared within the Unidad Asociada IAD (UPV)-IATA (CSIC) framework.

The main objective of this work was to identify and characterize the peptides generated by simulated gastrointestinal digestion of pork meat (longissimus dorsi) by the sequential action of pepsin and pancreatin. The obtained hydrolysate was analyzed by liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer equipped with a nanoelectrospray ionization source (nano LC-ESI-MS/MS). Using this technique 51 different peptides were identified in the hydrolysate, corresponding to fragments of the main structural muscle proteins and some well-known sarcoplasmic proteins. To the best of our knowledge, this constitutes the highest number of peptides identified in pork meat digests. Peptide fragment size ranged from six to sixteen amino acids, being rich in proline residues and thus making them more resistant to further degradation by digestive enzymes. The present study constitutes a clear evidence of the extensive degradation that pork muscle proteins would undergo after gastrointestinal digestion, giving rise to a wide variety of short peptides. So, the use of in vitro digestion contributes to a better knowledge about the generation of peptides from diets with high protein quality.

A proteomic approach has been carried out to investigate the extensive proteolysis occurring in the processing of Serrano ham. In this study, a total of 14 peptide fragments derived from myosin light chain I and titin have been identified for the first time. Nine of these peptides originated from myosin light chain I protein, with the loss of dipeptides at the N-terminal position observed in some of them. This suggests that dipeptidyl peptidases are involved in the generation of dipeptides, which contribute to the generation of the characteristic taste associated with Serrano ham. The other five peptides came from the PEVK region of the titin protein. This region is believed to confer elasticity to the sarcomere as well as the ability to bind calpains. The hypothetical action of mu-calpain and calpain 3 enzymes over this region would make these enzymes potentially responsible for protein breakdown during the early dry-curing stage.

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.

In this study, two strains of Staphylococcus xylosus isolated from traditional fermented sausages of Vallo di Diano (Southern Italy) were used in combination with an acidifying strain of Lactobacillus curvatus as starter culture for the production of fermented sausages. Two starter formulation were developed combining the proteolytic but not lipolytic (prt + , lip − ) S. xylosus CVS11 with the L. curvatus AVL3 (starter S1) and the S. xylosus FVS21 (prt − , lip + ) with the same strain of L. curvatus (starter S2). Proteolysis and lipolysis were observed during ripening by the increase in total free amino acids (FAA) and free fatty acids (FFA), respectively. Such activities were observed in both started and non started sausages (control). Moreover, the proteolytic and lipolytic activities were detected in products started by both formulations irrespective of the presence of such activities in the strains used. Therefore, it was not possible to conclude whether the effect of proteolysis and lipolysis during ripening of the started fermented sausages was due to the activity of the starter cultures or to the action of meat endogenous enzymes.

A simultaneous actomyosin and actin isolation procedure from post-rigor porcine muscle was developed, based on differential solubility, gel filtration chromatography and extraction steps. The isolation process was evaluated by SDS-PAGE analysis and silver staining. Actomyosin and actin were isolated in a simultaneous process yielding 0.14 mg and 2.5 mg/g of meat, respectively, using a shorter purification process than others reported in the literature but with similar recoveries. Furthermore, actin preserves its polymerisation ability and both proteins, actomyosin and actin, could be used in further studies.