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The present study aimed to evaluate actin degradation during the early

Bovine sarcoplasmic sub-proteome was studied through a straightforward gel-free pipeline supported by liquid isoelectric focusing (OFFGEL) protein fractionation coupled to liquid chromatography-mass spectrometry (LC-MS) analysis. Full-MS and data-dependent MS/MS analyses were simultaneously performed by a conventional three-dimensional ion-trap addressing targeted quantitative and untargeted qualitative research, respectively. There were unambiguously identified 47 proteins distributed along 12 OFFGEL fractions assayed. Regarding intermediate- and high-abundant peptides, bulky quantitative data processing performed by MZmine 2 freeware yielded a satisfactory linearity and coefficient of variation with r2 in the 0.95–0.99 range and about 25%, respectively. Up to 41 peptides from 20 identified proteins were relatively quantified throughout OFFGEL fractions. This reliable, flexible and affordable gel-free proteomic approach could be readily implemented by industry to improve quality assessment of protein-based food products., This work was funded by the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2014-00034-C04).

Volatile compounds from cooked and aged (0, 7, 14, 21 days) Hispano-Bretón horse meat (loin) were analyzed by solid-phase microextraction coupled to gas chromatography–mass spectrometry. A total of 77 volatile compounds were found, from which aldehydes were the predominant family. Most of the identified compounds had their origin in the degradation of lipids, with a negligible contribution of Maillard derived products. Odour impact ratios were calculated and used as indicators of the contribution of each compound to the total aroma and aldehydes were, in general, the major contributors to cooked horse meat aroma. Results revealed that ageing affected 15 of the volatile compounds detected. From them, hexadecanal and 2- and 3-methylbutanal significantly increased during ageing, presumably affecting the cooked meat odour as these have considerable odorant impact. Under the present study conditions, periods longer than 14 days would be necessary for significant changes in the volatile profile of cooked horse meat., Department of Economic Development and Infrastructures of the Basque Government is acknowledged for the doctoral fellowship of L.R.B. Lactiker Research Group (Basque Government IT944-16) funded this work as well as the contract of L.M.

The growing consumers’ interest in quality differentiated products, not only in terms of sensory and nutritional quality but also in terms of parameters such as animal welfare, product origin and production system, makes necessary to research the importance of autochthonous breeds, raised in more sustai-nable extensive systems, where welfare levels are often high. Additionally, it is well known that ageing is one of the main factors that has influence in the final quality of beef meat. Therefore, this work aimed to study the sensorial profile of organoleptic attributes of Retinta autochthonous breed from two livestock production systems (intensive and extensive) at 7 days and 14 days of ageing. Mo-reover, a consumer’s assessment was also carried out. Findings showed that the extensive production system improved the quality parameters (tenderness, juiciness and oiliness) of beef meat. The texture parameters were the first to change during the first days of ageing. Regarding the hedonic study, the consumers did not show a clear preference and/or acceptance for any sample, which could indicate that the production systems did not result in a differentiated quality at the organoleptic level by the con-sumers. It is necessary to promote higher knowledge about the organoleptic attributes which must be considered in the consumption of quality beef, so that the production system can be taken into account in the purchase decision by the consumers., Este trabajo fue financiado por el proyecto RTA 2014-00034-C04-00 (INIA-AEI) y fondos FEDER

BACKGROUND: Animal handling practices are one of the factors majorly affecting animal metabolism prior to slaughter. This phenomenon increases the occurrence of meat quality defects such as dark cutting-beef, causing high economical losses in the meat industry. Under this framework, the assessment of apoptosis onset in post mortem muscle was proposed as a novel approach to reveal biochemical characteristics in several Spanish bovine breeds (Asturiana de los Valles, Retinta and Rubia Gallega) managed under different production systems (intensive versus semi-extensive) and transport/lairage conditions (mixing versus not mixing with unfamiliar animals). To do so, the activities of initiator caspase 9 and executioner caspases 3/7 were determined in Longissimus thoracis et lumborum muscle at three early post mortem times (2, 8, and 24 h). RESULTS: Breed effect and transport/lairage conditions were the most relevant factors that influenced both caspase activities over post mortem time, showing Rubia Gallega breed a completely different behavior compared to Asturiana de los Valles and Retinta breeds. Moreover, it is postulated that apoptosis cascade is initiated via the activation of caspase 9 under hypoxic or metabolic stress followed by the activation of executioner caspases 3/7. CONCLUSIONS: Assessment of apoptosis on post mortem muscle can be a novel approach to study the influence of animal handling on muscle metabolism and post mortem cell death and its consequences on meat quality traits., Claudia Fuente-Garcia thanks the Department of Economic Development & Infrastructures of the Basque Government for the doctoral fellowship. This work was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2014-00034-C04), Agencia Estatal de Investigación (RTI2018-096162-R-C22) and FEDER funds.

As ageing process is one of the main factors influencing the organoleptic characteristics of meat and its consumer acceptability, the objective of this research was to study the organoleptic attributes variation of meat from Asturiana de los Valles and Retinta breeds at two times of the ageing process (7 and 14 days) under different pre-slaughter transport conditions (mixing and non-mixing with unfamiliar animals). A descriptive sensory analysis was carried out with a trained panel and the acceptability and preference for ageing meat of these two breeds were analysed with 75 consumers, as well as a study on the factors that influence the purchase decision to this type of meat. The breed and mixing with unfamiliar animals before slaughter affected significantly at instrumental tenderness. The intensity of the attributes related to the texture showed significant differences among studied samples at 7 days of ageing, with an increase in tenderness, juiciness and fibrousness while at 14 days of ageing, odour, beef flavour, liver flavour and smoothness also showed significant differences. The results indicated that the organoleptic attributes changed in intensity during ageing time, therefore, we can conclude that the attributes of tenderness, juiciness, smoothness, fibrousness, odour, beef flavour and liver flavour showed significant differences among samples and could be considered sensory indicators of ageing. The type of pre-slaughter transport (with or without mixing with unknown animals) also influenced the intensity of the sensory attributes, but less markedly than the breed effect. It was also observed that consumers prefer the meat of the Asturiana de los Valles breed aged for 14 days, despite among the factors that most influence the meat purchase decision, the ageing time was not present. Regarding purchasing habits, a relationship was observed between the meat purchase format and sale point. Overall, it is necessary to promote greater knowledge among consumers about the importance of ageing in beef quality., Este trabajo fue financiado por el proyecto RTA 2014-00034-C00 (INIA-AEI) y fondos FE- DER

Seventy-eight calves from Asturiana de los Valles, Retinta, and Rubia Gallega breeds, under extensive and intensive farm systems and animal mixing and non-mixing conditions, and during the transport and lairage in slaughterhouses, were studied. This research aimed to study the effect of breed, farm system and mixing conditions on serum biomarkers (cortisol, lactate, glucose, serum amyloid A, haptoglobin, and C-reactive protein) and their relationship with pHu at slaughter time, and to evaluate the response of the serum biomarkers of calves throughout fattening period. Moreover, this study aims to evaluate the response of the biomarkers in each breed during the fattening period. At slaughter time, cortisol and lactate were affected by BreedxFarm; Retinta showed the opposite pattern to the others and revealed the highest glucose in extensive farm systems. Rubia Gallega in mixing revealed the highest Amyloid A and haptoglobin. Extensive calves in mixing conditions showed the highest glucose. There was a relationship among the variables cortisol, lactate, Amyloid A, and pHu. Slaughter time was a major stressor, and the stress response was mainly affected by breed. At slaughter, several biomarkers should be considered., This research was funded by INIA-MINECO (Spain), under RTA2014-00034-C04-00 project, and FEDER funds.

Proteome changes derived from animals that have suffered pre-slaughter stress are a fact. In this study, Proteomic analysis was carried out on 20 bovine loin samples from Asturiana de los Valles and crossbreds cattle previously classified as normal and DFD meat at 24 h post-mortem using pH measurements. Sarcoplasmic sub-proteome of Longissimus thoracis at 24 h post-mortem was fractionated by the use of liquid isoelectric focusing (OFFGEL) in the pH range 3–10, followed by SDS-PAGE analysis of each retrieved fraction. The protein fractionation profile showed high reproducibility along the different sample groups. Five protein bands showed significant differences (p, This work was funded by the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2014-00034-C04).

A full-randomized block design was used for the study of the FA composition and meat quality parameters, considering ageing time as a split-plot factor. Chemical and fatty acid composition of steaks (longissimus thoracis and lumborum muscle) from 15 month old semiextensively reared Hispano-Bretón horses were characterized (day 0), and the effect of vacuum ageing (0, 7, 14 and 21 days) on several meat quality parameters (pH, instrumental color and texture and cook loss) was determined. The average fat content of horse loin was 3.31%, and the n-3 polyunsaturated fatty acid content, although higher than in ruminant meats, suggested that the finishing on a high-grain diet limited muscle n-3 accumulation. Results revealed that ageing affected all meat quality measurements; color started to turn brownish at 14 days of ageing, with a decrease in redness but not in yellowness. Tenderness improved during the first two weeks, and the Warner-Bratzler shear force scores showed that meat aged for 7 days could be considered as ‘intermediate tender’. Under the present study conditions, an ageing period between 7 and 14 days is recommended for an optimum horse meat quality., This research was funded by the Basque Government (grant IT944-16) that also funded the postdoctoral grant of L.M. Department of Economic Development and Infrastructures of the Basque Government is acknowledged for the fellowship of L.R.B.

Usefulness of general-purpose fluorogenic assay kits to determine caspase 3/7 activity of biological extracts is highly compromised in meat-based samples due to their scarce enzyme concentration. In the present work, a straightforward protocol is presented with two main purposes: 1) to enhance sensitivity of the fluorogenic approach addressing caspase 3/7 activity in tissues showing scarce enzyme concentration such as skeletal muscle, and 2) to reduce/economize the volume of employed reagents. The enzyme extraction procedure, peptide substrate, dithiothreitol concentration and detection settings were appropriately optimized for use in microtiter-plate fluorometers. As a result, low to high enzyme activity extracts (from 10,000 to 260,000 relative fluorescence units) can be measured under developed sampling and experimental conditions. The fact that enzyme reactions took place in 96-microtiter well plates reduces the consumption of chemical compounds when analysing a high number of samples, thus contributing to environment sustainability.

A wide variety of factors prior to slaughter may affect the stress status of beef cattle, giving rise to well-known ‘dark-cutting’ defective meats characterised by a high ultimate pH (pHu). To understand the underlying mechanisms of pHu fluctuations in beef cattle there was studied the proteome changes caused by pre-slaughter stress through a gel-free proteomic approach. Comparative peptidomic analysis was carried out on 12 loin samples at 24 h post-mortem from Longissimus thoracis et lumborum bovine muscle of crossbred animals, previously sorted into two different groups according to their pHu values: normal (pHu < 6.0) and high (pHu ≥ 6.0). Tryptic peptides from direct protein extracts were approached by combining untargeted (intact mass, MS1) and targeted (Selected Reaction Monitoring, SRM) quantitative LC-MS assays followed by chemometric analysis. Seventeen peptide biomarkers belonging to 10 different proteins appropriately discriminated sample groups assayed. Results may promote the use of this simple and effective methodology towards the creation of new insights in meat quality research., Department of Economic Development and Infrastructures of the Basque Government is acknowledged for the doctoral fellowship of C.F.G. Project RTA2014-00034-C04 (Spanish Instituto Nacional de Investigación y Tecnología Agraria, INIA), RTI2018-096162-R-C22 (Spanish Agencia Estatal de Investigación) and FEDER founds partially supported this research and to uphold contract of E. Sentandreu.

The present study aimed to evaluate actin degradation during the early postmortem time in Longissimus Lumborum muscle according to Sahraoui dromedary's age. A sample of eight males, young (2 years old) and adult (8 years old) dromedaries, was used to investigate meat quality traits and actin proteolysis during the conversion of muscle to meat. Results demonstrated higher pH values in young compared to adult with a polyphasic pH drop profile. While, age did not affect drip loss (DL) and the values at 72 h postmortem varied from 5 to 9%. Western blot revealed that actin proteolysis occurred since 1 h postmortem and that it was affected by age and postmortem time. In particular, the 32 and 25 kDa actin fragments could be potential markers of ongoing meat tenderization.

International audience; Ahmed Ouali was born on October 4, 1948 in Tigzirt, Tizi-Ouzou, Algeria. In 1952, he moved with his parents to Montluçon, France. In 1974, he was trained and graduated with a bachelor's degree in Biochemistry at the University of Lyon. He then, in 1976, earned a joint Ph.D. in Animal Science at the University of Blaise Pascal (Clermont-Ferrand) where he studied at the National Institute of Agricultural Research (INRA, Theix). The title of his doctorate thesis was “The role of muscle proteases on meat tenderization”. Subsequently, he was employed in a private laboratory for medical analysis from 1976 to 1978 and thereafter at the Meat Research Laboratory group at INRA, Theix as a permanent researcher. In 1990, he was appointed as a research director and led the “Biochemistry and Functions of Muscle Proteins” unit for 8 years. The Meat Research Station focused their research on many topics including colour and protein oxidation; enzymology and tenderness; and muscle protein functionalities. During his entire scientific career at INRA, but before his retirement on October 2013, Ahmed was living in Clermont-Ferrand, the city of the famous volcanic chain of the Puy-de-Dôme, with his wife Anne-Marie with whom he had two lovely children: Armelle (41 years) and Gäel (38 years). In 2019, they moved to their new house in Montpellier in the South of France.

Estimates of quantitative proteomic distance between populations have not been reported to date. Here, quantitative proteomic distances between three Spanish bovine breeds (Asturiana de los Valles, AV; Retinta, RE; and Rubia Gallega, RG) were estimated from two-dimensional electrophoresis profiles of meat samples of longissimus thoracis muscle at 2 h post-mortem. Statistically significant distances were detected between AV/RG and the most genetically different RE breed, using the novel QD measure of quantitative proteomic distance. In total, 18 differentially abundant myofibrillar and sarcoplasmic proteins/isoforms contributing to proteomic distances between breeds were confidently identified by tandem mass spectrometry. The fast skeletal myosin regulatory light chain 2 followed by other five interacting proteins exhibited the most pronounced relative change between breeds. In addition, most differentially represented proteins could be associated with variations in meat tenderness. Therefore, they could be candidate biomarkers for molecular breeding programs and authentication of the three Spanish beef breeds., This work was supported by the Instituto Nacional de Investigación y Tecnología Agraria (INIA, RTA 2014-00034-C04), Spain; and by a predoctoral fellowship of the Xunta de Galicia (Spain) and the European Union (ESF) to R. Rodríguez-Vázquez.

Different bovine breeds and production systems are used worldwide, giving rise to differences in intrinsic and extrinsic characteristics of beef. In order to meet the consumer requirements, new approaches are currently being developed to guarantee tenderness, taste, and juiciness of beef. However, the final consumer perception is complex, and it is also affected by several interrelated variables. This study aimed to evaluate the physicochemical parameters and sensory profile of three Spanish cattle breeds under different livestock production systems (extensive and intensive) and pre-slaughter handling conditions (mixing and not mixing with unfamiliar individuals at pre-mortem time). Meat samples from each group were also studied at different ageing times (7 and 14 days). Regarding sensory attributes, twelve panelists assessed meat samples and an exhaustive statistical analysis was carried out. The most evident and strongest effect was the breed type, allowing a great differentiation among them using principal components and discriminant analysis. The livestock production system was the second most important parameter, significantly affecting odor, flavor, and textural profile (fibrousness). It can be concluded that there were marked differences in the traits of these beef that could be modified by other factors in order to fulfill consumer tastes., Authors are grateful to RTA 2014-00034-C04-00 (INIA-MINECO) and FEDER funds for the financial support. Special thanks for financial support from the Xunta de Galicia and the European Union (ESF) for supporting Raquel Rodríguez-Vázquez pre-doctoral scholarship. Daniel Franco and José M. Lorenzo are members of the HealthyMeat network, funded by CYTED (Ref. 119RT0568).

The aim of the present study was to evaluate the differences in salivary proteome at rest and in response to an acute exercise in men and women. For this, unstimulated whole saliva samples in rest and after a bout resistance exercise leading to failure (ELF) of both men and women (n = 5 for each sex) were subjected to isobaric Tandem Mass Tags (TMT) labelling followed by LC-MS/MS. A total of 274 proteins were identified and met the inclusion criteria. 16 proteins were modulated for the interaction sex*exercise, 6 were modulated because of the exercise, and 65 were differentially expressed between men and women at rest. In conclusion, these results indicate sex-related differences in the salivary proteome at rest and after an acute exercise, pointing out possible candidate biomarkers for sports performance and allowing further knowledge of the physiological processes occurring during ELF., LFM was granted with a predoctoral contract ‘FPU’ from the University of Murcia, Spain. AT has a post-doctoral fellowship “Ramón y Cajal” supported by the “Ministerio de Economía y Competitividad”, Spain. This work was supported by a grant from the Program for Research Groups of Excellence of the Seneca Foundation, Murcia, Spain (grant 19894/GERM/15) and European Commission ERA chair FP7 grant (VetMedZg #621394).

Optimization of instrumental settings of a triple-quadrupole mass analyzer was performed by Box-Behnken design, support vector machines, and a Pareto-optimality approach. This time-saving, stepped chemometric strategy was used to model the signal response of underivatized human urinary amino acids. Drying gas flow, nebulizer pressure, sheath gas flow, and capillary voltage settings were exhaustively studied beyond the parameters conventionally optimized in triple-quadrupole devices (multiple reaction monitoring transitions, fragmentor and collision energy voltages). The results indicate that the best signal response for high-abundance and low-abundance underivatized amino acids was achieved with drying gas flow of 9 L/min, nebulizer pressure of 60 psi, sheath gas flow of 13 L/min, and capillary voltage of 3000 V. Compared with the widely standardized settings tested, chemometric analysis led to signal intensities 74% and 68% higher for high-abundance and low-abundance amino acids, respectively. The flexibility, speed, and efficiency of this method allows its affordable implementation in all mass spectrometry-based research to obtain superior results compared with those obtained with conventionally optimized mass spectrometry instrumental parameters.

In this study, the production, fractionation, purification and identification of ACE (angiotensin-I-converting enzyme) inhibitory peptides from wild almond (Amygdalus scoparia) proteins were investigated. Wild almond proteins were hydrolyzed using 5 different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme) and assayed for their ACE inhibitory activities. The degree of ACE inhibiting activity obtained after hydrolysis was found to be in the following order: alcalase > chymotrypsin > trypsin/pepsin > flavourzyme. The hydrolysates obtained from alcalase (IC50 = 0.8 mg/mL) were fractionated by sequential ultrafiltration at 10 and 3 kDa cutoff values and the most active fraction (

This study was designed within the scope of industrial exploitation of antioxidant extracts from Hibiscus sabdariffa. Efficiency in the extraction of antioxidants (total phenolics, flavonoids and anthocyanins) from calyces was assessed through different processes involving solvents (water, 80% acetone and 80% methanol), microwaves and ultrasounds. Results indicated that microwave extraction with 80% acetone gave the highest yield for total phenolics, expressed as gallic acid equivalents (GAE) per g dry weight (DW) (3.73 GAE/100 g DW), while the highest results for total flavonoids and anthocyanins were found in the microwaved aqueous extracts. Antioxidant activity of the extracts reported the highest reducing power for aqueous extracts with microwave extraction, while 80% acetone and methanol samples exhibited the highest antioxidant activity by high-performance thin-layer chromatography (HPTLC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. HPTLC performed for screening of phenolic compounds revealed the occurrence of anthocyanins in dimer-like bands. Laboratory tests showed high protection against lipid oxidation of the whey spread emulsion (22.32 h) incorporated with the sonicated aqueous extract. Results of peptide sequencing data by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, revealed protein–polyphenols interactions by the formation of dimers between polyphenols and protein/peptides. These interactions were found without effect on the oxidative stability of the emulsions. These study suggest that H. sabdariffa’s calyces have the potential to be used like food antioxidants.

A consumer test (n = 120) was performed in Vitoria-Gasteiz (northern Spain) in order to study the effect of ageing time (0, 7, 14 and 21 days) on the sensory quality of Hispano-Bretón foal meat. Steaks (Longissimus thoracis et lumborum) were wet aged and evaluated in-mouth and visually. In both cases, acceptability was scored using a hedonic scale, and sensory drivers related to ageing were characterised by applying check-all-that-apply method in meat. For both, in-mouth and visual acceptability, meat aged for 7 days obtained higher scores than non-aged meat, whereas longer ageing periods did not improve consumer acceptability. Check-all-that-apply method showed to be able to discriminate among samples, both in-mouth and visually. Results revealed that texture related attributes were the most discriminant ones in the in-mouth evaluation, being non-aged meat related to ‘dry‘, ‘high residue‘, ‘tough‘ and ‘chewy‘ terms, whereas aged meat was associated to ‘juicy‘, ‘tender‘ and ‘easily dissolving‘ terms. Visually, consumers perceived that, after 14 days of ageing, meat colour changed to ‘brownish‘. Under present study conditions, the establishment of a period of 7 days of ageing would be recommended., Department of Economic Development and Infrastructures of the Basque Government is acknowledged for the doctoral fellowship of L.R. Beldarrain. Lactiker Research Group (Basque Government IT944-16) funded this work as well as the contract of L. Morán.

El Gueddid is a traditional salted and dried meat with high popularity in Algeria. It is used as an ingredient in various dishes. In this study, different samples of El Gueddid were analyzed at different processing times to follow up their microbiological and physicochemical properties. Changes in the protein profile were also demonstrated by electrophoretic study of myofibrillar proteins. Microbiological determinations included the total viable count, coliforms, Staphylococci, lactic acid bacteria, yeasts, and molds, whereas physicochemical properties were characterized by pH, moisture, salt content and water activity. The results showed that microbial profiles were elevated for all the studied micro-organisms. Staphylococci and lactic acid bacteria were the most abundant micro-organisms in the product. Total coliforms were found in low numbers in fresh meat, being eliminated at the post salting stage of process. The physicochemical characteristics showed that the moisture content decreased in the product during the drying period. The pH also decreased during the drying period, then remained almost unchanged during the rest of the ripening period. Moreover, El Gueddid showed low water activity and high salt content. One of the most important changes in the profile of myofibrillar proteins was a reduction in the myosin heavy chain content., Project AGL2012-32146 from the Spanish Ministry of Economy and Competitiveness is fully acknowledged

This is the peer reviewed version of the following article: Fuente-Garcia, C.; Sentandreu, E.; Aldai, N.; Oliván, M.; Sentandreu, M.Á. Characterization of the Myofibrillar Proteome as a Way to Better Understand Differences in Bovine Meats Having Different Ultimate pH Values. Proteomics DOI:10.1002/pmic.202000012 (2020), which has been published in final form at https://doi.org/10.1002/pmic.202000012. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions, Influence of ultimate pH (pHu) on the occurrence of defective meats known as dark, firm, and dry (DFD) meats has been studied through a proteomic approach at early post‐mortem times. The myofibrillar sub‐proteome of longissimus thoracis et lumborum muscle from twelve loin samples from Asturiana de los Valles x Friesian yearling bulls, previously classified into two groups of six samples according to their pH values (normal, pHu < 6.0 and high, pHu ≥ 6.0), is analyzed at 24 h post‐mortem. Fractionation/enrichment of muscle samples is carried out by combining OFFGEL fractionation in the pH range 4–7 followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) of the retrieved liquid fractions. Four protein bands satisfactorily discriminate between meat samples with normal and high pHu. These bands are quantified by image analysis, and further identified by liquid chromatography‐mass spectrometry as desmin, pyruvate kinase, myosin light chain, and myosin heavy chain‐1 and ‐2. Coupling OFFGEL and SDS‐PAGE separation with MS provides detailed and reproducible myofibrillar protein profiles enabling comparison among the sample groups assayed. This makes feasible to identify biomarkers capable to better understand pre‐slaughter stress condition susceptible to give DFD meats with high pHu values., This work was funded by the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2014‐00034‐C04) and FEDER funds.

Fermented sausage technology is currently compromised in decreasing the addition of NaCl. Use of starter cultures with peptidogenic potential could be a valuable strategy that can mask or hide off flavors produced by the use of NaCl substituents. In the present work, the peptidogenic potential of four lactic acid bacteria species was evaluated in a low-sodium beaker sausage (BS) model. Using a peptidomic approach, a total of 86 low molecular weight (LMW) peptides were accurately identified, mostly derived from myofibrillar proteins, especially actin, which generated 53 peptides. The BS inoculated with L. curvatus CRL705 generated 56 LMW peptides, followed by Enterococcus (E.) mundtii CRL35 with 43 peptides. In addition, BS inoculated with Lactobacillus (L.) plantarum and with L. sakei produced higher amino acid amounts over time as compared to the rest of BS models, highlighting the importance of both, time and sample effect on the overall free amino acid generation. The presence of each LAB strain in BS models generated a unique profile of small peptides and amino acids that could serve as a distinctive biochemical trait to differentiate specific fermented products. According to these results, E. mundtii and L. sakei, which are compatible between them, are proposed as the most efficiently adapted to low-sodium conditions. The use of selected strains during the processing of low-sodium fermented sausages could have a positive effect on the production of small peptides and free amino acids. Fil: de Almeida, Marcio Aurélio. Universidade de Sao Paulo; Brasil Fil: Saldaña, Erick. Universidade de Sao Paulo; Brasil Fil: da Silva Pinto, Jair Sebastião. Universidade de Sao Paulo; Brasil Fil: Palacios, Jorge Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina Fil: Contreras-Castillo, Carmen J.. Universidade de Sao Paulo; Brasil Fil: Sentandreu, Miguel Angel. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España Fil: Fadda, Silvina Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Although Peptidomics is a discipline complementary to proteomics, since nowadays both mainly rely on analytical strategies based on mass spectrometry, there are fundamental differences. In this chapter, we discuss these differences along with the application of these technologies for the study of the different stages of meat production, from storage to processing to unravel mechanisms that will allow reaching high quality and safer meat products. The use of Peptidomics and the related high throughput technologies, now relying on mass spectrometry but once also on N-terminal sequencing, are discussed. Clear examples are provided dealing with relevant studies on meat proteolysis and peptide generation occurring during ageing, as well as those produced during ripening of meat products by endogenous and microbial enzymes. Also the involvement of this phenomenon in the development of taste active compounds is addressed. Finally, the application of novel Omics technologies on bacterial identification in food for diagnosis and safety purposes is presented, putting emphasis on their potential advantages and future perspectives. Fil: Fadda, Silvina G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sentandreu, Enrique. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España Fil: Sentandreu Vicente, Miguel Angel. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España

Tenderness is an important organoleptic trait that determines bovine meat quality and consumer satisfaction. Therefore, it is essential to understand the biochemi-cal processes taking place in the conversion of muscle into meat. In fact, this ageing process is determinant for the tenderness of the final product.This article will summarize the decisive role of proteins in the ageing process, and the opportunities that proteomic techniques provide in order to analyze and predict meat tenderness, which is profitable for beef industry. In this sense, a recently developed approach will be introduced, which is based on liquid isoelectric focusing (O FFGEL) and electrophoresis. Seven bands capable to discriminate between tender and tough samples were identified and measured in a study performed on beef from Maine Anjou bovine breed. Therefore, this methodology constitutes a promising alter-native for the study of muscle proteome.; Samurtasuna behi-haragiaren kalitatean eragiten duen ezaugarri garrantzitsua da, kontsumitzaileak produktua aukeratzeko orduan gehien balioesten duenen artean dagoena. Hori dela eta, muskulua haragi bihurtu bitarteko prozesu biokimikoaren azterketak berebiziko garrantzia dauka. Izan ere, ontze-prozesu honetan garatzen da neurri handi batean amaierako produktuaren samurtasuna.Prozesu honetan proteinek daukaten garrantzizko papera laburbilduko da artikulu honetan, bai eta teknika proteomikoek haragiaren kalitatea aztertzeko eta optimizatzeko eskaintzen dituzten aukerak ere, haragi-industriari begira probetxuzkoak izan daitezkeenak. Zentzu honetan, gure taldeak garatutako estrategia proteomiko berria aurkeztuko da, isoelektrofokatze likidoa (OFFGEL) eta elektroforesia oinarri dituena. Maine Anjou behi arrazako animalien haragian burututako esperimentuan samurtasunaren adierazgarri diren 7 banda identifikatu eta neurtu dira, muskuluaren proteoma ikertzeko metodologia berritzaile honen potentziala agerian utziz.

Epub 2018 May 9; International audience; Protein biomarkers of meat tenderness are known to be of primary importance for the prediction of meat quality, and hence, industry profitability. Proteome analysis was performed on meat from 8 Main Anjou beef cattle, previously classified as tender or tough meats by Warner Bratzler shear force measurements. Myofibrillar fraction of Longissimus thoracis muscle was separated by a novel fractionation approach based on liquid isoelectric focusing (OFFGEL) and further analyzed by SDS-PAGE and liquid chromatography coupled to tandem mass spectrometry. Obtained OFFGEL fraction profiles were reproducible allowing the comparison of both meat qualities and revealing 7 protein bands capable to discriminate between tender and tough samples. The proteins present in these bands were troponin T, Heat Shock protein beta-1, creatine kinase, actin, troponin C, myosins 1 and 2 and myozenin-1. The latter protein has not been previously reported as a marker of meat tenderness. This study introduces an innovative proteomic approach for the study of muscle proteome. The fact of obtaining fractions in liquid state after OFFGEL fractionation allows for a faster analysis of proteins by mass spectrometry, being an interesting alternative to more classical proteomic approaches based on two dimensional gel electrophoresis (2-DE).

Summary A protein extract from wild almond was hydrolysed using five different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme). The hydrolysates were then assayed for their antioxidant activities. The highest extent of proteolysis was obtained with alcalase (0.35; determined as the change in the absorbance at 340 nm, ΔA340) and the lowest was with pepsin (ΔA340 = 0.12). Radical scavenging activities obtained by 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) and ferric-reducing abilities of the hydrolysates demonstrated that the hydrolysate from alcalase had significantly (P

This study focuses on meat protein degradation by different starter cultures. Sausage modelsinoculated with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 alone and asa mixture were incubated 10 days at 22◦C. Low molecular weight peptides (

In this study, the production, fractionation, purification and identification of ACE (angiotensin-I-converting enzyme) inhibitory peptides from wild almond (Amygdalus scoparia) proteins were investigated. Wild almond proteins were hydrolyzed using 5 different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme) and assayed for their ACE inhibitory activities. The degree of ACE inhibiting activity obtained after hydrolysis was found to be in the following order: alcalasechymotrypsintrypsin/pepsinflavourzyme. The hydrolysates obtained from alcalase (ICThis study has shown the potential of wild almond proteins as good sources for producing ACE-inhibitory active peptides. According to this finding, peptides with higher ACE inhibitory activities could be released during the gastrointestinal digestion and contribute to the health- promoting activities of this natural protein source.

More than ever, people today demand clear and reliable information about the food they consume. This has a great impact on the economy since the consumer's choice is greatly influenced by the food composition detailed in labelling. In the case of processed meat products this is going to be especially important because a simple visual inspection would not allow us to discriminate between the different components so easily as in the case of fresh meat. In order to assure fair trade, food safety and freedom of choice, honest and accurate food label is a requisite that must be assured by legal authorities. To do that, robust and reliable methodologies of analysis must be implemented in control laboratories. In relation to this, the present review intends to give an overview of the different analytical strategies that have been traditionally used by control laboratories to assess meat authenticity or that could be interesting alternatives for fraud detection in near future.

The effect of the muscle type [semimembranosus (SM) and biceps femoris (BF)] on the evolution of free amino acids and sarcoplasmic and myofibrillar proteins throughout the manufacture of Celta dry-cured ham was studied. A total of thirty Celta hams were analyzed, 5 in the green stage, 5 after the end of the salting stage, 5 after 120 days of post-salting, 5 after the end of the drying–ripening stage, 5 after 165 days and 5 after 330 days of “bodega” step. Possibly due to its higher moisture values, the BF muscle underwent higher protein degradation than the SM muscle. The average of total FAA content increased significantly (P

International audience; Biomarkers of the meat quality are of prime importance for meat industry, which has to satisfy consumers' expectations and, for them, meat tenderness is and will remain the primary and most important quality attribute. The tenderization of meat starts immediately after animal death with the onset of apoptosis followed by a cooperative action of endogenous proteolytic systems. Before consideration of the biomarkers identified so far, we present here some new features on the apoptotic process. Among them, the most important is the recent discovery of a complex family of serpins capable to inhibit, in a pseudo-irreversible manner, caspases, the major enzymes responsible of cell dismantling during apoptosis. The biomarkers so far identified have been then sorted and grouped according to their common biological functions. All of them refer to a series of biological pathways including glycolytic and oxidative energy production, cell detoxification, protease inhibition and production of Heat Shock Proteins. Some unusual biomarkers are also presented: annexins, galectins and peroxiredoxins. On this basis, a detailed analysis of these metabolic pathways allowed us to identify some domains of interest for future investigations. It was thus emphasized that mitochondria, an important organelle in the production of energy from carbohydrates, lipids and proteins are a central element in the initiation and development of apoptosis. It was therefore stressed forward that, in fact, very little is known about the postmortem fate of these organelles and their multiple associated activities. Other topics discussed here would provide avenues for the future in the context of identifying reliable predictors of the ultimate meat tenderness. (C) 2013 Elsevier Ltd. All rights reserved.

Meat fraud implies many illegal procedures affecting the composition of meat and meat products, something that is commonly done with the aim to increase profit. These practices need to be controlled by legal authorities by means of robust, accurate and sensitive methodologies capable to assure that fraudulent or accidental mislabelling does not arise. Common strategies traditionally used to assess meat authenticity have been based on methods such as chemometric analysis of a large set of data analysis, immunoassays or DNA analysis. The identification of peptide biomarkers specific of a particular meat species, tissue or ingredient by proteomic technologies constitutes an interesting and promising alternative to existing methodologies due to its high discriminating power, robustness and sensitivity. The possibility to develop standardized protein extraction protocols, together with the considerably higher resistance of peptide sequences to food processing as compared to DNA sequences, would overcome some of the limitations currently existing for quantitative determinations of highly processed food samples. The use of routine mass spectrometry equipment would make the technology suitable for control laboratories.

Troponin T (TnT) is a myofibrillar protein present in striated muscle that develops important structural and regulatory functions. This protein is easily degraded during post-mortem ageing but details of the TnT hydrolysis occurring during much longer processes, e.g. the dry-cured processing, as well as the specific sequences of the generated fragments, still remain undetermined. In the present study, the isolation and identification of 27 peptides, generated from TnT during dry-cured processing, are reported for the first time. This study has been developed using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) and it evidences the extensive degradation of TnT occurring during the processing of dry-cured ham. Some of the cleavage sites, corresponding to these sequenced fragments, confirm the sites reported by other authors both in vitro and during post-mortem ageing of meat.

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.

International audience; Thrombin/antithrombin III (AT-III) proteolytic system is well known for its function in blood coagulation. Thrombin is expressed in skeletal muscle but nothing is known about the presence of AT-III in the tissue. In postmortem muscle this system has been and is still totally ignored. We therefore successfully attempted to purify AT-III from bovine skeletal muscle and characterized the purified protein (identified as AT-III by N-ter sequencing and mass spectrometry finger print) for its physicochemical and inhibitory properties. As the human blood serpin, muscle AT-III is thermolabile and stable only at alkaline pH (pH 910). The muscle serpin inhibits strongly thrombin in a heparin dependent manner and trypsin. Phosphatidylserine (PS) externalization demonstrated in the present work suggested that prothrombin can be activated to thrombin through binding of the activator complex on the external PS groups. PS externalization is concomitant with shrinkage of muscle fibers indicating that muscle cells are engaged in the cell death program known as apoptosis few minutes after death. We then discussed the potential role of this proteolytic system in postmortem apoptosis development as well as in the control of low voltage electrical stimulation efficiency.

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.

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.

Dipeptidyl peptidases (DPP) constitute a group of proteolytic enzymes able to release a good number of dipeptides from the N-terminus of both synthetic substrates and natural polypeptides. Specific sequences generated by the action of DPP purified from porcine skeletal muscle have been assayed to evaluate their capacity to inhibit the activity of angiotensin-I converting enzyme (ACE; EC 3.4.15.1). A fluorimetric assay based on the hydrolysis of the internally quenched fluorescent substrate o -aminobenzoylglycyl- p -nitrophenylalanylproline by the action of ACE was used for this purpose. The generated fluorescence of the product (the o -aminobenzoylglycine group) was continuously monitored in a microtiter-plate multiscan fluorometer. Among the assayed dipeptides, Arg-Pro showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 25 μM. Dipeptides Lys-Ala, Gly-Pro and Ala-Ala also demonstrated to be effective ACE inhibitors, although at a lower degree. Dipeptides Ala-Arg and Gly-Arg caused a more moderate inhibition of ACE activity, and even lower was the inhibition exerted by Arg-Arg. From the data obtained, it is suggested that the proteolytic action of DPP along the ripening period of dry-cured meat products could contribute to the generation of antihypertensive peptides.

The hydrolysis of myofibrillar proteins during fermentation of sausage models by an autochthonous starter culture was investigated. In order to provide a whole map of the generated products, proteomic and peptidomic were used andcomplemented with the amino acid profile. Beaker sausages (BS) were used as models which were inoculated or not with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 as starter cultures. The hydrolysis of actin, myosin light chain 1/3 (MLC 1/3), myosin regulatory light chain-2 (MRLC-2) and myosin heavy chain (MHC) were evidenced by two-dimensional gel electrophoresis (2-DE). In addition, a total of 33 peptides arisen from troponin T, MRLC-2 and particularly from actin were identified by LC-MS/MS. These results showed that the starter culture significantly enhanced the proteolysis of the proteins named above, even when the endogenous enzymes induced a clear breakdown. L. curvatus CRL705 highly enriched both peptide pattern and amino acid concentrations. When the autochthonous starter culture was inoculated, although proteolysis was remarkably reinforced, a reduction in peptide and amino acid composition was observed. Regarding actin primary structure, three regions of this proteinwere highly susceptible to degradation by the starter culture. Additionally, the essential role of exopeptidases- from meat and bacteria- in diversity of actin peptides during fermentation was shown. This study improved the knowledge of the proteolysis of myofibrillar proteins and the involved enzymes, as well as, completed the previously reported degradation of sarcoplasmic proteins by the same autochthonous starter culture. The singular peptides and amino acids pattern generated might contribute to the uniqueness of produced fermented sausages while they may be used as quality markers. Fil: López, Constanza María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sentandreu Vicente, 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: Fadda, Silvina G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Postmortem (PM) meat tenderization is a complex biochemical process that involves multiple endogenous proteolytic enzymes and that is not completely understood. Cell death by apoptosis is recently proposed as a novel mechanism in this process. The signification of the caspases implication in muscle proteins degradation is that the cells are dying through apoptosis, a major cell death programme. Our purpose was therefore to verify this statement using different well known hallmarks of the apoptotic process including cell shrinkage, phosphatidylserine (PS) externalization. Cells start to shrink few minutes after animal death and reach a maximum shrinkage about 24 h postmortem while they showed the typical rounding appearance of apoptotic cells. Externalization of PS is detectable within 1 h postmortem and increased gradually with time. The present work is therefore the first to provide direct evidence supporting the onset of apoptosis in post-mortem muscle. The present findings should lead us to reconsider as a whole, the mechanisms contributing of the development of meat qualities (tenderness, flavor, juiciness …) by a detailed analysis of the apoptosis associated biochemical and physiological modifications taking place within the cells.

International audience; In living cells, after activation, protein inhibitors constitute the last step of proteases activity regulation. This review intends to provide original information about a group of bovine muscle serine proteases inhibitors belonging to the Serpin superfamily and characterized at the gene and protein level. This report is the only one and the first to provide much information on this group of proteases inhibitors of the serpin type and their potential biological functions. Amongst the eight genes identified in bovine, three serpins were purified from the muscle tissue and characterized. These are two members of the bovSERPINA3 family, i.e., bovSERPINA3-1 and A3-3, and the last one is antithrombin III (AT-III or BovSERPINC1). BovSERPINA3 family comprises at least eight protein members encoded by different genes mapped on chromosome 7q23–q26 cluster. BovSERPINA3-1 and A3-3 were shown to locate within muscle cells and are cross-class inhibitors strongly active against trypsin as well as against human initiator and effector caspases 8 and 3. They constitute a key apoptosis control in mammals. They were thus expressed in proliferating and confluent myoblasts phases where cells must be alive but not in myotubes. Antithrombin III inhibits trypsin and, in a heparin dependent manner, thrombin. AT-III and its mRNA were expressed in muscle cells and in differentiating primary myoblasts in culture.

The 20S proteasome is a large complex (700 kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9 ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331 μg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213 μg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209 μg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203 μg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the μg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation coefficient of 0.99.

The conversion of muscle into meat is a complex process in which all mechanisms responsible for the development of meat qualities are very likely interdependent. Colour and flavour are thus both dependent on oxidative mechanisms. Oxidation and proteolysis are probably two processes involved in the development of meat tenderness. This paper reviewed the consequences of programmed cell death or apoptosis on muscle cells structure and biochemistry and on meat qualities as well. We therefore look at different new hypothesis susceptible to highlight the meat science field and provide new supports for a more dynamic meat research. One of them which would have appeared evident for our purpose since a decade, deals with the fact that, after animal bleeding, muscle cells have no other alternative to only enter the programmed cell death procedure or apoptosis. If we introduce an early phase corresponding to apoptosis, taking place before the rigor onset and overlapping it, we will see that the known consequences of that process bring forward possible answers to still unexplained observations. After an overview of the actual state-of-the-art in meat science, we will introduce the programmed cell death and its underlying mechanisms. We then described the strong analogies between the known consequences of apoptosis and the postmortem changes affecting a set of different muscle characteristics.

Dipeptidyl peptidases (DPP) are a group of enzymes capable to liberate dipeptides from the N-terminal side of longer peptides. Four different DPP activities have been described in skeletal muscle, each one of them having different biochemical characteristics. The four enzymes have proved to remain active during meat ageing and during processing of cured meat products. So, the fact that dipeptides are present at the end of the curing process could be related to the action of DPP during this period. In the present work we investigated DPP action on the hydrolysis of oligopeptides and its ability to generate dipeptides with antihypertensive properties by studying its inhibitory effect on angiotensin-I converting enzyme. Among the assayed dipeptides, Val-Tyr showed the strongest ACE inhibitory activity with an IC50 value of 4.6 μM under our assay conditions. In a lower degree, Arg-Ser also proved to be a strong ACE inhibitor. The effect of Arg-Phe, Gly-Phe, Met-Ala and Val-Gly was lower though still remarkable. The present results stress forward the importance of DPP in the generation of antihypertensive peptides during the processing of meat products.

Non-specific binding (NSB) of compounds other than the capture antigen or antibody to the solid support is one of the most important error sources occurring in ELISA. Undesirable NSB may be minimized by saturating the remaining binding sites of the plastic surface with different protein compounds (blocking agents) that must not interfere with the immunoassay. This work had as main objective the evaluation of different blocking agents for their ability to avoid unwanted NSB in ELISA using bovine muscle crude extracts and a polyclonal antibody raised against purified cathepsin L. Results obtained showed that skimmed milk, casein, BSA, goat and rabbit serum proved to be efficient in suppressing NSB. In the case of skimmed milk and casein, an effective blocking effect was observed even in absence of the surcoating step. However, these two compounds and BSA interfered considerably the immunochemical reaction, making their use inadequate. Of the two assayed serums, rabbit serum would be a more suitable blocking agent than goat serum when working with bovine crude extracts because it did not interfere in great manner with the antigen–antibody reaction and, at the same time, did not increase background.