Caspase activity in post mortem muscle and its relation to cattle handling practices.

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. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterization of the Myofibrillar Proteome as a Way to Better Understand Differences in Bovine Meats Having Different Ultimate pH Values.

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.

Multiobjective optimization of liquid chromatography-triple-quadrupole mass spectrometry analysis of underivatized human urinary amino acids through chemometrics.

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.

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50-70 kDa), F2 (Mr:40-60 kDa) and F3 (Mr:10-15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.

Application of 2-D DIGE to study the effect of ageing on horse meat myofibrillar sub-proteome.

Considering the high relevance of meat tenderness for consumer acceptability, the aim of this study was to investigate post-mortem changes in myofibrillar sub-proteome in steaks from longissimus thoracis et lumborum muscle of six Hispano-Bretón horses. Indeed, the ageing process that leads to meat tenderization has been scarcely studied in this species. Steaks (n = 24) were aged (4 °C) in the dark under vacuum for 0, 7, 14 and 21 days and the myofibrillar sub-proteome was extracted. Using 2-D DIGE minimal labelling, 35 spots that were differentially abundant between 0 and 21 days aged meat were detected. Of them, 24 were analysed by LC-MS/MS, identifying a total of 29 equine proteins. These were structural and metabolic proteins, and among them, four (Actin, Troponin T and Myosin binding proteins 1 and 2) were selected for Western blot analysis, reporting changes in their abundance after 0, 7, 14 and 21 days of ageing. Results revealed that they should be further studied as potential protein biomarkers of horse meat tenderization. Additionally, several protein fragments increased after ageing, as was the case of glyceraldehyde-3-phosphate dehydrogenase. Fragments of this protein were present in four protein spots, and their study could be useful for monitoring horse meat tenderization. SIGNIFICANCE: Tenderization during ageing has been widely studied in meat from several farm animal species; however, both research and standardized ageing practices are lacking for the particular case of horse meat. In this regard, this study presents novel proteomic findings related to post-mortem evolution of horse muscle proteins. Acquired knowledge would support the development and optimization of efficient ageing practices by horse meat industry.

New insights into the search of meat quality biomarkers assisted by Orbitrap Tribrid untargeted metabolite analysis and chemometrics.

Metabolite profiles of normal and defective dry, firm and dark (DFD) meat extracts with known ultimate pH (pHu) values were determined by Orbitrap Tribrid ID-X untargeted analysis coupled to chemometrics. An intelligent MS3 AcquireXTM workflow firstly approached the unambiguous characterization of detected features that were subsequently quantified by a complementary MS1 study of biological replicates. Chemometric research revealed how threonylphenylalanine (overexpressed in normal meats) together to tetradecadienoyl- and hydroxydodecanoyl-carnitines (both overexpressed in DFD meats) appropriately grouped meat groups assayed. Robustness of such biomarkers was confirmed through a time-delayed study of a blind set of samples (unknown pHu) and evidenced limitations of pHu as an isolated parameter for accurate meat quality differentiation. Other acyl-carnitines also characterized DFD samples, suggesting interferences induced by pre-slaughter stress (PSS) on lipid catabolism that would explain accumulation of such intermediate metabolites. Results achieved can ease understanding of biochemical mechanisms underlying meat quality defects.

Early postmortem degradation of actin muscle protein in Algerian Sahraoui dromedaries.

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.

Horse meat tenderization in relation to post-mortem evolution of the myofibrillar sub-proteome.

The ageing process after animal slaughter enhances tenderness and influences the value of meat. Horse meat is becoming more popular but lacks standardized ageing practices that should be supported by a better understanding of post-mortem muscle biochemistry. Steaks from Longissimus Thoracis et Lumborum (LTL) of eight Hispano-Bretón horses were aged for 0, 7, 14 and 21 days and myofibrillar proteins were resolved by one dimensional gel electrophoresis (1-DE). Ten protein bands were found to change (p ≤ 0.05) among ageing periods. Most changes were observed between days 0 and 14, suggesting that tenderization occurred primary during the first two weeks. Liquid isoelectric focusing (OFFGEL) technology was applied to better resolve myofibrillar sub-proteome and evidenced fourteen protein bands that changed (p ≤ 0.05) between 0 and 21 days. Three of them were protein fragments coming from troponins T and I and from creatine kinase. Identified molecules could be further studied as potential markers for horse meat tenderness.

Optimization of a fluorogenic assay to determine caspase 3/7 activity in meat extracts.

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.

Effect of ageing time on the volatile compounds from cooked horse meat.

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.

A straightforward gel-free proteomics pipeline assisted by liquid isoelectric focusing (OFFGEL) and mass spectrometry analysis to study bovine meat proteome.

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.

Proteomic pipeline for biomarker hunting of defective bovine meat assisted by liquid chromatography-mass spectrometry analysis and chemometrics.

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. SIGNIFICANCE: The significance of this study was the optimization of an affordable straightforward gel-free proteomic approach addressing the differentiation of the muscle sub-proteome of normal and high pHu meat samples. This strategy allowed the study of tryptic peptides from direct meat protein extracts by combining untargeted MS1 and targeted SRM quantitative assays performed by conventional LC-MS detection. Affordability, simplicity and robustness of this methodology can facilitate its readily implementation in routine protocols for quality assessment of meat.

Protein Biomarkers of Bovine Defective Meats at a Glance: Gel-Free Hybrid Quadrupole-Orbitrap Analysis for Rapid Screening.

An understanding of biological mechanisms that could be involved in the stress response of animal cattle prior to slaughter is critical to create effective strategies aiming at the production of high-quality meat. The sarcoplasmic proteome of directly extracted samples from normal and high ultimate pH (pHu) meat groups was studied through a straightforward gel-free strategy supported by liquid chromatography hybrid quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS) analysis. A stepped proteomic pipeline combining rapid biomarker hunting supported by qualitative protein Mascot scores followed by targeted label-free peptide quantification revealed 26 descriptors that characterized meat groups assayed. The functional study of the proposed biomarkers suggested their relevant role in metabolic, chaperone/stress-related, muscle contractility/fiber organization, and transport activities. The efficiency, flexibility, rapidity, and easiness of the methodology proposed can positively contribute to the creation of innovative proteomic alternatives addressing meat quality assessment.

Muscle and Subcutaneous Fatty Acid Composition and the Evaluation of Ageing Time on Meat Quality Parameters of Hispano-Bretón Horse Breed.

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.

Assessment of Stress by Serum Biomarkers in Calves and Their Relationship to Ultimate pH as an Indicator of Meat Quality.

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.

A proteomic-based approach for detection of chicken in meat mixes.

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

Effect of ageing time on consumer preference and sensory description of foal meat.

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.

Sensory and Physicochemical Analysis of Meat from Bovine Breeds in Different Livestock Production Systems, Pre-Slaughter Handling Conditions and Ageing Time.

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.

Differences on salivary proteome at rest and in response to an acute exercise in men and women: A pilot study.

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. SIGNIFICANCE: The present study describes for the first time the changes that occur in salivary proteome detected by TMT-based proteomics in response to an acute exercise and the differences in these changes depending on sex. Of the 274 protein identified, the 87 differentially expressed proteins and their related pathways were discussed, focusing on the sex- and exercise-related differences in the salivary proteome.

El Gueddid, a traditional Algerian dried salted meat: Physicochemical, microbiological characteristics and proteolysis intensity during its manufacturing process and ripening.

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.