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.

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.

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.

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.

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.

Use of liquid isoelectric focusing (OFFGEL) on the discovery of meat tenderness biomarkers.

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. SIGNIFICANCE: 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).