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.

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.

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.

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.

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.

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.

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.

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.

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.

Search for proteomic biomarkers related to bovine pre-slaughter stress using liquid isoelectric focusing (OFFGEL) and mass spectrometry.

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 < 0.05) between the two groups, allowing discrimination between them. Proteins present in these bands, which were identified by LC-MS, were actin, phosphoglucomutase-1, alpha-crystallin B, heat shock protein beta-6 and heat shock protein beta-1. SIGNIFICANCE: The significance of this study relies on the optimization of OFFGEL fractionation as a promising technology to search for reliable biomarkers of pre-slaughter stress. This method separates proteins along different liquid fractions according to their isoelectric point; the obtained fractions can be further characterized by SDS-PAGE or directly identified by LC-MS. This achievement stands out as an alternative to the use of 2-DE electrophoresis in protein separation and analysis.

A peptidomic approach of meat protein degradation in a low-sodium fermented sausage model using autochthonous starter cultures.

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.

Identification of Potent ACE Inhibitory Peptides from Wild Almond Proteins.

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 (<3 kDa) was further separated using reversed phase high-performance liquid chromatography (RP-HPLC). Peptide sequence identifications were carried out on highly potential fractions obtained from RP-HPLC by means of liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Sequencing of ACE inhibitory peptides present in the fraction 26 of RP-HPLC resulted in the identification of 3 peptide sequences (VVNE, VVTR, and VVGVD) not reported previously in the literature. Sequence identification of fractions 40 and 42 from RP-HPLC, which showed the highest ACE inhibitory activities (84.1% and 86.9%, respectively), resulted in the identification of more than 40 potential ACE inhibitory sequences. The results indicate that wild almond protein is a rich source of potential antihypertensive peptides and can be suggested for applications in functional foods and drinks with respect to hindrance and mitigation of hypertension after in vivo assessment. PRACTICAL APPLICATION: This 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.

Caspases and Thrombin Activity Regulation by Specific Serpin Inhibitors in Bovine Skeletal Muscle.

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.

Peptides with angiotensin I converting enzyme (ACE) inhibitory activity generated from porcine skeletal muscle proteins by the action of meat-borne Lactobacillus.

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 evaluation of ACE inhibitory activity. Bioactive fractions were only found from the hydrolysis of sarcoplasmic proteins by both assayed lactobacilli strains. Identification of peptides contained in the bioactive fractions was carried out by tandem mass spectrometry using a nanoLC-ESI-QTOF instrument and the mascot search engine. From the four most active fractions obtained, a total of eighteen and fifty peptides were characterized from L. sakei CRL1862 and L. curvatus CRL705 protein hydrolysates, respectively. The sequence FISNHAY was generated by the proteolytic activity of the two lactobacilli 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 meat-borne Lactobacillus were able to generate peptides with ACE inhibitory activity, highlighting their potential to be used in the development of functional fermented products. BIOLOGICAL SIGNIFICANCE: The results of this study would enable the obtention of porcine functional foods by applying lactic acid bacteria generating bioactive peptides. ACE inhibitory peptides obtained by the hydrolytic action of L. curvatus CRL705 and L. sakei CRL1862 on sarcoplasmic proteins were analyzed. Among them, the peptide FISNHAY exhibited the highest activity and its sequence has not yet been reported.

Muscle and meat: new horizons and applications for proteomics on a farm to fork perspective.

Meat consumption is an important part of human diet with strong implications in health, economy and culture worldwide. Meat is a proteinaceous product and therefore proteomics holds a considerable value to the study of the protein events underlying meat production and processing. In this article we will review this subject in an integrated "farm to fork" perspective, i.e. focusing on all the major levels of the meat producing chain: farm, abattoir and transformation industry. We will focus on the use, importance and applications of proteomics, providing clear examples of the most relevant studies in the field. A special attention will be given to meat production, as well as quality control. In the latter, a particular emphasis will be given to microbial safety and the detection of frauds.

Antihypertensive activity of peptides identified in the in vitro gastrointestinal digest of pork meat.

This study investigated the in vivo antihypertensive activity of three novel peptides identified in the in vitro digest of pork meat. These peptides were RPR, KAPVA and PTPVP and all of them showed significant antihypertensive activity after oral administration to spontaneously hypertensive rats, RPR being the peptide with the greatest in vivo activity. To our knowledge, this is the first report showing the in vivo antihypertensive action of the three peptides from nebulin (RPR) and titin (KAPVA and PTPVP), thus confirming their reported in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. These findings suggest that pork meat could constitute a source of bioactive constituents that could be utilized in functional foods or nutraceuticals.