Exploring the miRNAs Profile in Dark-Cutting Beef.

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH24), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated (p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress (bta-miR-1246, bta-miR-2332, bta-miR-23b-5p, and bta-miR-2411-3p) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions (p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem, as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem. These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

High-pressure processing applied to sliced dry-cured Iberian loin: Effect of category, company, and storage temperature.

The main purpose of this study was to evaluate the effect of high-pressure processing (HPP) and storage temperature on the microbial counts, the instrumental color, and the oxidation stability of sliced dry-cured Iberian loin from two categories and two leading companies. 600 MPa for 8 min was sufficient to decrease all the microbial counts without affecting the color and the oxidation status, the effect being modulated by the loin category and company, whose effect on those variables was marked. However, the subsequent 90-day storage softened the initial effect of HPP on microorganisms and allowed a significant effect of HPP to develop on color and oxidation. In addition, the coliform counts were higher after storage at 20°C than at 4°C, suggesting that refrigeration may be needed during long-term storage to ensure loin safety.

Differences in Muscle Lipogenic Gene Expression, Carcass Traits and Fat Deposition among Three Iberian Pig Strains Finished in Two Different Feeding Systems.

The Iberian pig breed includes several well-differentiated strains. The present study evaluated carcass traits, fat deposition and muscle expression of important lipogenic genes (SCD, ME1, ACACA, FASN, EGR1, ACOX and ACLY) using 65 male pigs of 3 Iberian strains (20 Lampiño, 23 Torbiscal, and 22 Retinto) finished either in a conventional, concentrate-based system (CF) or in montanera (MF), a traditional free-range system with acorn feeding. Torbiscal had the highest ham, Longissimus thoracis and prime cuts yields, and the thinnest subcutaneous adipose tissue (SAT). Retinto had the highest monounsaturated fatty acids (MUFA) and percentage of intramuscular fat (IMF), while Lampiño had the greatest content of saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA), atherogenic (AI) and thrombogenic (TI) indexes in SAT. Conventionally finished pigs had the highest ham, L. thoracis and prime cuts yields, and SFA. Montanera-finished animals had the highest PUFA and MUFA contents, and the lowest AI, TI and n6/n3 ratio in SAT. In relation to gene expression, Retinto had the greatest SCD, FASN and ACLY levels. Most studied genes were overexpressed in CF pigs. In conclusion, MF pigs had healthier fat than CF pigs, and Retinto had the healthiest fat and the greatest lipogenic trend in muscle, supported by IMF and lipogenic gene expression.

Use of Near-Infrared Spectroscopy to Discriminate DFD Beef and Predict Meat Quality Traits in Autochthonous Breeds.

The potential of near-infrared reflectance spectroscopy (NIRS) to discriminate Normal and DFD (dark, firm, and dry) beef and predict quality traits in 129 Longissimus thoracis (LT) samples from three Spanish purebreeds, Asturiana de los Valles (AV; n = 50), Rubia Gallega (RG; n = 37), and Retinta (RE; n = 42) was assessed. The results obtained by partial least squares-discriminant analysis (PLS-DA) indicated successful discrimination between Normal and DFD samples of meat from AV and RG (with sensitivity over 93% for both and specificity of 100 and 72%, respectively), while RE and total sample sets showed poorer results. Soft independent modelling of class analogies (SIMCA) showed 100% sensitivity for DFD meat in total, AV, RG, and RE sample sets and over 90% specificity for AV, RG, and RE, while it was very low for the total sample set (19.8%). NIRS quantitative models by partial least squares regression (PLSR) allowed reliable prediction of color parameters (CIE L*, a*, b*, hue, chroma). Results from qualitative and quantitative assays are interesting in terms of early decision making in the meat production chain to avoid economic losses and food waste.

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.

Effect of Animal Age at Slaughter on the Muscle Fibres of Longissimus thoracis and Meat Quality of Fresh Loin from Iberian × Duroc Crossbred Pig under Two Production Systems.

Two production systems and several ages at slaughter were used: 12, 14 and 16 months for outdoor rearing (with the final finishing phase in the Montanera system, in which fed was based on natural resources, mainly acorns and grass) and 8, 10 and 12 months for animals reared indoors (intensive system: with feed based on commercial fodder) to evaluate their effect on the muscle fibre population and size of the Longissimus thoracis, (LT) muscle, as well as fresh loin quality traits. Animals that were older at slaughter revealed increased fibre sizes of the LT muscles in the pigs reared in the Montanera system. The LT muscles of the animals reared in intensive systems had a lower percentage of type I fibres and higher size of type IIB than those reared in the Montanera system. The approximate composition and instrumental colour of Montanera fresh loins were affected by the animal slaughter age. In the case of the intensive system, the effect of animal slaughter age had an impact on the approximate composition, instrumental colour, water loss and textural properties. Therefore, different ages at slaughter of Iberian pigs showed variations in some quality parameters in the fresh loins in both the Montanera and Intensive systems, thus proving to be a factor of variability and homogeneity of the Iberian products. The meat from Iberian pigs reared in an intensive system and slaughtered at a younger age proved to be more tender. The production system affected all the above quality traits, with the exception of water loss.

Quality Traits of Montanera Iberian Dry-Cured lomito as Affected by Pre-Cure Freezing Practice.

The seasonality to which dry-cured products from Iberian breed pigs finished in Montanera (free-range rearing system with feed based exclusively on ad libitum consumption of natural resources; acorns and grass) are subjected could be overcome by pre-cure freezing. Three sets of Montanera Iberian presas (Serratus ventralis muscle)(n = 15) were established to assess the impact of frozen storage -0, or non-frozen, 3 and 6 months-previous to the technological process of curing-on the quality traits of the dry-cured product Montanera Iberian dry-cured lomito. Similar seasoning and curing processing conditions were applied to all sets. Lower productive performance due to higher weight loss during curing, and lower colour intensity were observed in pre-frozen dry-cured lomitos. The fatty acid profile was more saturated, and the oxidative status increased as a result of pre-cure freezing. On the matter of texture, all parameters were modified, highlighting the higher values of hardness and shear force of pre-frozen dry-cured lomitos. The time that raw material was frozen exerted a slight, thus helping manufacturers to better address the gap between industry and consumer demand with minimal effect on quality traits.

Do animal slaughter age and pre-cure freezing have a significant impact on the quality of Iberian dry-cured pork loin?

The influence of various animal slaughter age and of pre-cure freezing times on the quality traits of dry-cured loins from Iberian × Duroc (IBxD) crossbreed animals was assessed. Three animal batches (n = 15) were selected (IBxD8, IBxD10 and IBxD12) to be slaughtered at the age of 8, 10 and 12 months, respectively. Following slaughter, longissimus thoracis et lumborum muscles were extracted and submitted to curing (T0) or freezing storage during three (T3) or six (T6) months prior to curing. The dry-cured loins from IBxD12 yielded lower intramuscular fat and lightness, a less saturated fatty acid profile and the highest hardness and shear force (P ≤ 0.05). The pre-cure freezing process increased weight loss, saturated fatty acids, lipid oxidation, and most of the textural parameters (P ≤ 0.05). So, both slaughter age and pre-cure freezing should be taken into account in order to obtain quality and productive performance.

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.

Effect of Breed Purity and Rearing Systems on the Stability of Sliced Iberian Dry-Cured Ham Stored in Modified Atmosphere and Vacuum Packaging.

The long-term storage stability of three quality categories of sliced Iberian dry-cured ham defined by the Spanish Iberian Quality Standard (Black, purebred Iberian reared outdoors in Montanera system; Red, Iberian × Duroc crossed (50%) pigs reared outdoors in Montanera system;, and White, Iberian × Duroc crossed (50%) pigs commercially fed) and packaged under vacuum and modified atmosphere packaging (MAP) was studied. Commercial category affected the shelf life, being Black and Red presented the highest lipid oxidation during storage, whereas the effect of packaging was not as clear as the effect of commercial category. MAP preserved more the colour and the antioxidants content than vacuum packaging, while this latter reduced lipid oxidation development and maintained better the brightness and flavour of slices than MAP.

Quality Traits of Dry-Cured Loins from Iberian Pigs Reared in Montanera System as Affected by Pre-Freezing Cure.

Iberian dry-cured loins from pigs fattened in Montanera are usually subjected to seasonal production, which could be overcome through freezing the loin before the curing process. Hence, three homogeneous batches (n = 15 per batch) were established to evaluate the effect of different pre-freezing cure (-20 ± 2 °C) times (three and six months) of raw material on main quality characteristics of dry-cured loins in comparison to those elaborated from unfrozen meat. All samples were subjected to similar seasoning and same curing length to obtain dry-cured loins. Results showed a decrease in lightness, redness, chrome and hue values, polyunsaturated fatty acids (PUFA), higher oxidative phenomena, and changes in rheological properties in fresh loins on account on freezing (p ≤ 0.05). Some quality parameters of dry-cured loins were affected by freezing, weight loss, and hardness being higher as well as salt content being lower. PUFA and Warner-Braztler Shear Force (p ≤ 0.05) also showed higher values in dry-cured loins from frozen meat than those elaborated from unfrozen counterparts.

Effect of Two Organic Production Strategies and Ageing Time on Textural Characteristics of Beef from the Retinta Breed.

The primary aim of this paper is to determine the influence of two organic production systems, organic grass-fed (OG) and organic concentrate-fed (OC), vs. a conventional concentrate-fed (CC) system; the second aim is to determine the influence of the ageing period on the physical parameters and texture properties of beef from the Retinta breed. Muscle samples from Longissimus thoracis were stored at 2-4 ± 1 °C for 0, 7, 14, and 21 days for the purposes of ageing. Analyses of pH, water losses (drip loss and cooking loss), Warner-Bratzler shear force, texture profile analysis (TPA), and histological analysis of muscle fibre were carried out. The results revealed that organic meat experienced lower drip loss and higher cooking loss than conventional meat. Although the meat of organic grass-fed animals was tougher initially, it showed a higher tenderisation speed in the first ageing days than OC and CC meats. The sarcomere length increased during the ageing period, which showed a negative correlation to shear force. According to its texture characteristics, the Retinta meat produced in organic systems could be recommended by its quality.

Quality traits of fresh and dry-cured loin from Iberian x Duroc crossbred pig in the Montanera system according to slaughtering age.

Iberian x Duroc cross grower pigs of similar weight, but 12, 10 and 8 months of age, were finished using the Montanera system to study effects on fresh and dry cured loin quality. Pigs were slaughtered simultaneously at 16, 14 and 12 months of age (IBxD16, IBxD14 and IBxD12) and longissimus thoracis et lumborum were removed and processed into dry-cured loin. Fresh and dry cured loins from the oldest animals (IBxD16) had higher tocopherols, C18:2 n-6 and PUFA as well as higher insoluble collagen, which led to greater hardness. Fresh loin from the youngest animals (IBxD12) had the lowest myoglobin content, redness (a*), chroma and hue angle. No differences in proximate composition or instrumental colour were found in dry-cured loins.

Stability of fatty acid composition of intramuscular fat from pasture- and grain-fed young bulls during the first 7 d postmortem.

In order to study the effect of different amounts of concentrate feed and the effectiveness of natural antioxidants on the fatty acid stability of intramuscular fat during the first days postmortem, 75 young bulls of the Retinta breed were divided in three groups: 30 were grazed, 30 were fed on medium concentrate diets, and 15 were fed on high-concentrate diets. Young bulls were slaughtered at commercial weight, around a 500 kg final body weight. Samples from Longissimus lumborum muscle were assigned to two ageing periods (0 and 7 d) and were vacuum packaged in vacuum bags ( O 2 permeability: 9.3  mL   O 2 / m 2 per 24  h at 0  ∘ C ) using an EGARVAC ® sealer. Beef from grass-fed bulls showed a higher polyunsaturated fatty acid content than concentrate-fed bulls. During the first 7 d postmortem, no changes in the fatty acids profile were observed, because α -tocopherol content was optimal to prevent lipid oxidation. The higher level of natural antioxidants in grass than in grain resulted in the stability of the fatty acid profile. This study shows that the anti-oxidative potential of natural antioxidants in meat plays an important role during the first 7 d postmortem.

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.

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.