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© 2020 by the authors., In this study, orange seed proteins were hydrolyzed by Alcalase enzyme at different enzyme concentrations 1–3% (v/w) and hydrolysis times (2–5 h), to obtain bioactive peptides showing antioxidant, Angiotensin-converting enzyme (ACE) -inhibitory, and hypoglycemic activities. The highest biological activities (p < 0.05) were achieved by using a hydrolysis time of 5 h and an enzyme concentration of 2%. Orange seed protein hydrolysate (OSPH) was prepared under these conditions, and peptides were isolated and purified by using size-exclusion chromatography and high-performance liquid chromatography, respectively. The fractions that showed the highest biological activities were analyzed by mass spectrometry in tandem, and a total of 63 peptide sequences were found. Moreover, the effect of simulated gastrointestinal digestion on the bioactivity of the fractions was studied, and the novel peptide sequences generated were also identified. Overall, despite there being some differences in the profile of peptide sequences obtained, the main results showed non-significant differences in the analyzed bioactivities after simulated gastrointestinal digestion., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, and Ramón y Cajal postdoctoral contract by L.M. are acknowledged. This work was also supported by Intramural project from CSIC number 201870E006.

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

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.

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.

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

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

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

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.

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 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.

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.

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.

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.

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.

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.

[EN] The reduction of the content of sodium chloride in dry-cured ham was studied in to prevent the problems related to high sodium intake (i.e. the hypertension). One of the possibilities to reduce the sodium content is the partial replacement of sodium chloride by mixtures of potassium, magnesium and calcium chloride salts. The effect of two salting formulations (formulation II: 50% NaCl-50% KCl and formulation III: 55% NaCl, 25% KCl, 15 CaCl 2 and 5 MgCl 2) on the protease activity through the dry-curing process and on the sensory characteristics of the final product was evaluated and compared to those of control hams (formulation I, 100% NaCl). Sensory attributes were all affected in the hams containing CaCl 2 and MgCl 2 while hams containing 50% KCl and NaCl (formulation II) were better valued, except for the attribute taste probably due to the potassium contribution to bitter taste. © 2011 Elsevier Ltd., This work was supported by grant AGL2004-05064-C02-01 from Ministry of Education and Science (Madrid, Spain) and FEDER. FPI/MEC scholarship to M. Armenteros is also acknowledged.

The aim of this work was to study how nucleotide degradation during the processing of dry-cured ham is affected when using three types of salting (100% NaCl; 50% NaCl and 50% KCl; 55% NaCl, 25% KCl, 15% CaCl₂ and 5% MgCl₂). Divalent salts in the salting mixture depressed the breakdown rate from the beginning of the process (salting and post-salting) up to the ripening stage (7 months) when the inosine (Ino), hypoxanthine (Hx) and xanthine (X) concentrations matched for the three treatments. The evolution of Hx and Hx+X were analysed by HPLC and an enzyme sensor, respectively, during processing. Time and temperature conditions during the curing time did not affect Hx stability. The usefulness of the enzyme sensor was confirmed and it is a practical tool to determine Hx + X in dry-cured ham, as an index of minimum curing time. A good correlation between enzyme sensor and HPLC data was observed.

A new HPLC method based on hydrophilic interaction chromatography (HILIC) has been developed for the simultaneous analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hx) and nicotinamide adenine dinucleotide (NAD + ) in pork loin muscle. The chromatographic analysis has been developed using a zwitterionic polymeric column and it has been optimised in terms of linearity, repeatability, reproducibility and recovery. Detection limit values of 0.10, 0.15, 0.05, 0.16, 0.16, 1.72 and 0.15 μg/mL for ATP, ADP, AMP, IMP, Ino, Hx and NAD + respectively were achieved. A comparison between the pork meat concentrations of the compounds analysed by hydrophilic interaction chromatography (HILIC), using a ZIC®- p HILIC column, and ion-pair reversed-phase chromatography (IP-RP-HPLC), using a Zorbax Eclipse XDB-C18 column, has been done, concluding that the new chromatographic methodology constitutes a valid and reliable alternative to the existing methods. Finally, this method has also proved to be very effective in the isolation of guanosine, guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), creatinine (Cn) and uric acid (UA) from a standards mixture.

An enzyme sensor employing xanthine oxidase (XO), soluble or immobilised, in combination of an oxygen electrode has been developed and optimised to determinate the hypoxanthine (Hx) content in pork meat at different post-mortem times as measure of meat freshness. The amperometric signal obtained due to the oxygen depletion during the Hx oxidation was related with the consumed oxygen at 190 s in the soluble enzyme sensor or enzymatic rate at 10 s in the immobilised enzyme sensor. In both cases a linear relationship between the signal and the Hx concentration in the range 8.68–26.05 μM (R2 = 0.999) and 15.63–127 μM. (R2 = 0.995), respectively, was found. Both enzyme sensors exhibited very good working conditions and storage stability. A study of Hx oxidation was carried out in order to compare the Hx content measured by both sensors and those measured by high performance liquid chromatography (HPLC) obtaining a good agreement between both techniques. Therefore, the easy preparation and operation of both enzyme sensors suggests a reliable, rapid and an economical alternative for simple or multiple Hx measurements constituting a useful tool as quality control of meat freshness.

This work aimed to investigate the effect of pre-cure freezing Iberian hams on proteolysis phenomena throughout the ripening process. Non-protein nitrogen (NPN), peptide nitrogen (PN) and amino acid nitrogen (AN) as well as amino acid and dipeptide evolution followed the same trend in both refrigerated (R) and pre-cure frozen (F) Iberian hams during processing. At the different stages of ripening, there were no differences in the content of NPN and AN while F dry-cured hams had higher levels of PN than R hams at the final step. This seemed to be more related to the salt content (lower in F than in R hams) than to the pre-cure freezing treatment. Most amino acids and dipeptides detected showed higher concentrations in F than in R Iberian hams at the green stage, being rather similar at the intermediate phases. At the final stage, the effects of pre-cure freezing of Iberian hams were not well defined, higher levels of some amino acids and dipeptides were found in R than in F Iberian hams whereas other amino acids were lower in R than in F hams.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising new therapies for type 2 diabetes. The aim of this study was to assay DPP-IV inhibitory peptides that can be present in a water soluble extract of Spanish dry-cured ham. Such an extract was fractionated by size-exclusion chromatography and the in vitro DPP-IV inhibitory activity determined in each collected fraction. Then, several peptides previously identified in dry-cured ham extracts or known to be products of DPP IV action were synthesised and assayed for DPP-IV inhibition. Peptides KA and AAATP showed the strongest DPP-IV inhibitory activity, with IC50 values of 6.27mM and 6.47mM, respectively. Dipeptides AA, GP, PL, and carnosine, as well as peptides AAAAG, ALGGA, and LVSGM were also DPP-IV inhibitors, although at a lower degree. These findings suggest the potential of Spanish dry-cured ham as a natural precursor of DPP-IV inhibitory peptides. These biopeptides could also be used as ingredients for functional foods or pharmaceutical products against type 2 diabetes., PI Scholarship BES-2011-046096 from MINECO (Spain) to M. Gallego and grant AGL2010-16305 from MINECO and FEDER funds are fully acknowledged.

Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered. © 2013 Elsevier Ltd. All rights reserved., 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. Capital infrastructure funding from the Higher Education Funding Council of England (HEFCE) is acknowledged for purchase of the MS equipment. L.M. and P.D.F. are grateful to the EU-FP7 Marie Curie IEF scheme (FOOSAF project) for financial resources.

High-performance liquid chromatography (HPLC) was used to separate small water-soluble peptides extracted from dry-cured ham at different processing times (from 0 to 15 months). The elution profile of the deproteinised extracts after gel filtration HPLC showed five ranges of molecular mass: I (4500-2700 Da), II (2700-1200 Da), III (1200-500 Da), IV (500-375 Da) and V (375-160 Da). A clear increase of those peptides below 2700 Da was observed as dry-curing progressed confirming an intense proteolysis especially up to 3.5 months of process. Peptide profiling of deproteinized extracts by reverse phase HPLC and free solution capillary electrophoresis showed substantial changes in peptide patterns along the dry-curing. These mappings could provide valuable information regarding time of processing.

Novel sequences exhibiting in vitro ACE inhibitory activity as well as in vivo antihypertensive activity were identified from Spanish dry-cured ham. Water soluble peptide extracts from dry-cured ham were purified by size-exclusion chromatography and reversed-phase high performance liquid chromatography and then, further identification of sequences was carried out by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 73 peptide sequences were identified from active fractions presenting 100% homology with different Sus scrofa skeletal muscle proteins. All identified peptides showed Mr between 374 and 1610 and amino acid sequences between 5 and 14 amino acids in length. Considering the low molecular mass and structural requirements for ACE inhibition some of the identified peptides were synthesised and their IC(50) calculated. The most potent peptide was found to be AAATP (IC(50) value of 100 μM). This peptide also showed good in vivo activity because it decreased systolic blood pressure by -25.62 ± 4.5 mmHg (p

Aims The capacity of Lactobacillus sakei CRL1862 to prevent the growth of pathogens and its ability to degrade sarcoplasmic and myofibrillar proteins in pork meat systems was evaluated. In addition, basic safety aspects of Lact. sakei CRL1862 such as production of biogenic amines and antibiotic susceptibility were addressed. Methods and Results The bacteriocin-producing Lact. sakei CRL1862 showed respectively bactericide and bacteriostatic effect against Listeria monocytogenes and Staphylococcus aureus in beaker sausage assay during 9 days of storage at 22°C. The hydrolytic effect of Lact. sakei CRL1862 on protein extracts was evaluated by SDS-PAGE and reverse phase HPLC. A more pronounced proteolysis was evidenced in inoculated sarcoplasmic proteins compared with myofibrillar extracts with the generation of predominantly hydrophilic peptides and increase of total free amino acids concentration. Lactobacillus sakei CRL1862 produced neither histamine nor tyrosine and exhibited no resistance to the antibiotics assayed. Conclusions Lactobacillus sakei CRL1862 effectively controlled the growth of L. monocytogenes and Staph. aureus; moreover, it was able to hydrolyse pork meat extracts generating peptides and amino acids, which may improve hygienic and sensorial attributes of fermented meat products. Significance and Impact of the Study The use of an integrated approach to evaluate the major traits of Lact. sakei CRL1862 showed it can be applied as an autochthonous functional starter in meat fermentation. Fil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Aristoy, Maria Concepción. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España Fil: Sentandreu, Miguel Angel. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España Fil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Toldrá, Fidel. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España

The proteolytic changes taking place in muscle proteins during the processing of dry-cured ham were evaluated from the point of view of its increased nutritional quality. Sodium dodecyl sulphate polyacrylamide gel electro-phoresis (SDS-PAGE) revealed the breakdown of several muscle proteins in dry-cured ham. Peptide analysis by both reverse-phase HPLC (RP-HPLC) or free solution capillary electrophoresis (FSCE) confirmed the intense proteolysis. Dry-cured ham also showed large amounts of all the free essential amino acids: valine (221.4 mg/100g muscle), methionine (90.8 mg/100g muscle), isoleucine (155.7 mg/100g muscle), leucine (246.9 mg/100g muscle), phenylalanine (37.0 mg/100g muscle), tryptophan (25.7 mg/100g muscle) and, in particular, lysine (509.0 mg/100g muscle). Thus, dry-cured ham constitutes a concentrated source of essential amino acids of great importance when the energy intake is low or in a poor nutritional quality diet.

An attempt to decrease the NaCl content in dry-cured products through the use of three different salting treatments (II: 55% NaCl, 25% KCl, 15% CaCl(2), 5% MgCl(2); III: 45% NaCl, 25% KCl, 20% CaCl(2), 10% MgCl(2); and IV: 30% NaCl, 50% KCl, 15% CaCl(2), 5% MgCl(2)) in comparison to a control (I: 100% NaCl) was assayed to evaluate the biochemical and sensory characteristics in the final product. Most proteolytic enzyme activities from the loins submitted to the experimental salting treatments, especially treatments II and IV, remained higher than those salted traditionally (control). The higher aminopeptidase activity was also reflected in a larger release of free amino acids. Finally, a sensory paired comparison test revealed that those loins salted with the treatment II were not significantly different from the loins salted traditionally (100% NaCl), so that this treatment could be successfully used for sodium reduction.

Contents 17.1 Introduction 287 17.2 Sample Preparation for the Analysis of Seafood Essential Amino Acids 28817.2.1 Sample Preparation for Free Essential Amino Acid Analysis 288 17.2.2 Sample Preparation for Total or Hydrolyzed Essential AminoAcid Analysis 289 17.3 Seafood Essential Amino Acid Analysis 29017.3.1 High-Performance Liquid Chromatographic Methods 291 17.3.1.1 Cation Exchange Chromatography 291 17.3.1.2 Reversed-Phase High-Performance Liquid Chromatography 29217.3.2 Gas Liquid Chromatographic Methods 298 17.3.3 Capillary Zone Electrophoretic Methods 298 17.3.4 Mass Spectrometry 29917.4 Conclusions 300 References 30017.1 Introduction Amino acids are the basic components of the muscle protein structure of seafood. However, not all proteins have the same nutritional value, because protein quality strongly depends on its amino acid composition and digestibility.1 Fish and, in general, seafood proteins are considered as highquality proteins because of their balanced content in amino acids, especially in all the essential amino acids necessary for physical and mental well-being. Amino acids may also be found in free form, which contribute to sh taste and indirectly to aroma 2,3 by generation of volatilecompounds through Maillard reactions and Strecker degradations.4 Branched-chain essential amino acids (valine, isoleucine, and leucine), sulfur-containing amino acids (methionine and cystine/cysteine), and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage.5-10us, the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood.

An intense proteolysis of muscle proteins, mainly due to the action of endogenous proteolytic enzymes, has been reported to occur during the processing of dry-cured ham. This gives rise to an important generation of free amino acids and peptides of small size that contribute directly or indirectly to flavor characteristics of the final product. The nature and properties of the free amino acids generated during postmortem proteolysis have been well established. However, little is known about the identity of peptides generated during the processing of dry-cured ham. In the present paper, we describe the isolation (by ethanol precipitation followed by size exclusion and reverse phase chromatographies) and identification (by matrix-assisted laser desorption/ionization time-of-flight MS and collision-induced dissociation MS/MS) in a Spanish dry-cured ham of the following four oligopeptides: (A) TKQEYDEAGPSIVHR, (B) ITKQEYDEAGPSIVHRK, (C) DSGDGVTHNVPIYE, and (D) DSGDGVTHNVPIYEG. This is the first time that these peptide fragments have been reported in dry-cured ham at the end of processing. Sequence homology analysis revealed that the four peptides originated from muscle actin, indicating an extensive hydrolysis of this protein during dry-curing. Some of the cleavage sites corresponding to these fragments in actin were reproduced by other authors through the incubation of this myofibrillar protein in the presence of cathepsin D (EC 3.4.23.5), thus supporting a relevant action of this enzyme during the processing of dry-cured ham.

Caracterisation de 28 parametres pour 147 echantillons de jus d'orange d'Espagne provenant de 6 usines differentes pendant deux saisons, en vue de detecter des adulterations eventuelles

A rapid, high yield, and quantitative method for determination of sugars (glucose and fructose) and acids (malic, lactic, acetic, and citric) in fresh and fermented cucumber juices is described; the procedure solves the fructose and malic acid coelution problem. Samples are prepared by passing the juice directly through a disposable C18 cartridge. Compounds are separated by liquid chromatography on an Aminex HPX-87H chromatographic column, with 0.013N H2S04 as mobile phase in the isocratic mode at 0.6 mL/min flow rate and 60°C column temperature. Compounds are then detected and quantitated by using ultraviolet and refractive index detectors connected in series, and comparing with succinic acid as internal standard. Values for both fresh and fermented cucumber juices were in good agreement with literature data.

Angiotensin I-converting enzyme (ACE) inhibitory activity of peptides derived from the hydrolysis of sarcoplasmic and myofibrillar porcine proteins by the action of Lactobacillus sakei CRL1862 and Lactobacillus curvatus CRL705 (whole cells + cell free extracts) was investigated at 30 °C for 36 h. The protein hydrolysates were subjected to RP-HPLC in order to fractionate the extracts for further evaluate the ACE inhibitory activity of the collected peptide fractions. Bioactive fractions were only found from the hydrolysis of sarcoplasmic proteins by both assayed lactic acid bacteria. Identification of peptides contained in these bioactive fractions was carried out by tandem mass spectrometry using a nanoLC-ESI-QTOF instrument and the mascot seach engine. A total of eighteen peptides were characterized from the two most active fractions obtained from the hydrolysis made by L. sakei CRL1862. Regarding L. curvatus CRL705, its proteolytic action was able to generate thirty and twenty peptides, which were identified in the two most active fractions, respectively. It is worth noting that the sequence FISNHAY was generated by the proteolytic action of the two species. Sequence similarity analyses between the peptides identified in this study and those previously identified as ACE inhibitory peptides and detailed in the BIOPEP database were outlined. Results suggest that lactic acid bacteria selected in this study were able to generate peptides with ACE inhibitory activity, thus having potential to be used in the development of functional fermented products. Fil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina; Fil: Aristoy, María Concepción. Consejo Superior de Investigaciones Cientificas. Instituto de Ciencia y Teconologia de Alimentos y Nutricion; España; Fil: Sentandreu, Miguel Ángel. Consejo Superior de Investigaciones Cientificas. Instituto de Ciencia y Teconologia de Alimentos y Nutricion; España; Fil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina; Fil: Toldrá, Fidel. Consejo Superior de Investigaciones Cientificas. Instituto de Ciencia y Teconologia de Alimentos y Nutricion; España

The angiotensin I-converting enzyme (ACE) inhibitory activities and antioxidant properties of thornback ray gelatin hydrolysates (TRGHs) prepared by treatment with proteolytic proteases from Bacillus subtilis A26, Raja clavata crude alkaline protease extract, Alcalase and Neutrase were investigated. All gelatin hydrolysates showed different degrees of hydrolysis and hydrophobic/hydrophilic peptides ratio. Moreover, they possess high protein content (70.04 ± 0.55–74.14 ± 0.28%). The antioxidant activity was assayed using various in vitro tests. The highest antioxidant activity was observed with hydrolysate obtained by treatment with A26 proteases (TRGH-A26) which exhibited a 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity with a concentration that produces 50% of inhibition (IC50) of 1.98 ± 0.02 mg/ml of sample, reduced the ferric ions with an absorbance at 700 nm of 0.962 ± 0.07, prevents bleaching of β-carotene with 73.02 ± 1.90% inhibition and gave an antioxidative efficacy of 180 ± 0.08 µmol/ml α-tocopherol equivalents at 5 mg/ml in the phosphomolybdenum assay. However, gelatin hydrolysate treated with Alcalase (TRGH-Alcalase) was the most potent to prevent DNA oxidation. For the ACE inhibitory activity, all hydrolysates displayed ACE-inhibitory activity. TRGH-A26 and TRGH-Alcalase exhibited the highest activity with 85 ± 0.65 and 82 ± 0.49%, respectively, at 5 mg/ml. The results revealed that TRGHs could be used as ingredients to formulate functional foods.

A soluble alanyl aminopeptidase (EC 3.4.11.14) from porcine skeletal muscle was purified by ammonium sulfate fractionation and anion-exchange HPLC. The enzyme eluted at 0.31 M NaCl, had a relative molecular mass of 106 000 (by SDS−polyacrylamide gel electrophoresis), and was markedly stimulated by sulfhydryl compounds, Co2+ and Ca2+. The enzyme exhibited maximum activity at pH 6.5 and 50 °C and showed a broad substrate specificity hydrolyzing aromatic, aliphatic, and basic aminoacyl bonds, but did not show endopeptidase activity. The affinity of the enzyme toward dipeptides was increased in the presence of an aromatic amino acid and the C-terminal side. Inhibition of enzyme activity was obtained in the presence of metal-chelating agents, sulfhydryl reagents, bestatin, amastatin, and puromycin. The enzyme was stable at temperatures below 15 °C and had a higher stability, 60% of initial activity after 3.5 months of incubation at −25 °C, in the presence of 50% ethylene glycol. Keywords: Alanyl aminopeptidase...