Beef-on-dairy: Meat quality of veal and prediction of intramuscular fat using the Q-FOM™ Beef camera at the 5th-6th thoracic vertebra.

This study aims to describe the meat quality of young Holstein (HOL) beef-on-dairy heifers and bulls sired by Angus (ANG, n = 109), Charolais (CHA, n = 101) and Danish Blue (DBL, n = 127), and to investigate the performance of the handheld vision-based Q-FOM™ Beef camera in predicting the intramuscular fat concentration (IMF%) in M. longissimus thoracis from carcasses quartered at the 5th-6th thoracic vertebra. The results showed significant differences between crossbreeds and sexes on carcass characteristics and meat quality. DBL × HOL had the highest EUROP conformation scores, whereas ANG × HOL had darker meat with higher IMF% (3.52%) compared to CHA × HOL (2.99%) and DBL × HOL (2.51%). Bulls had higher EUROP conformation scores than heifers, and heifers had higher IMF% (3.70%) than bulls (2.31%). These findings indicate the potential for producing high-quality meat from beef-on-dairy heifers and ANG bulls. The IMF% prediction model for Q-FOM performed well with R2 = 0.91 and root mean squared error of cross validation, RMSECV = 1.33%. The performance of the prediction model on the beef-on-dairy veal subsample ranging from 0.9 to 7.4% IMF had lower accuracy (R2 = 0.48) and the prediction error (RMSEveal) was 1.00%. When grouping beef-on-dairy veal carcasses into three IMF% classes (2.5% IMF bins), 62.6% of the carcasses were accurately predicted. Furthermore, Q-FOM IMF% predictions and chemically determined IMF% were similar for each combination of sex and crossbreed, revealing a potential of Q-FOM IMF% predictions to be used in breeding, when aiming for higher meat quality.

Satellite cells sourced from bull calves and dairy cows differs in proliferative and myogenic capacity - Implications for cultivated meat.

Cultivated meat produced with primary muscle satellite cells (SCs) will need a continuous supply of isolated cell material from relevant animal donors. Factors such as age, sex, and breed, along with the sustainability and availability of donor animals, could determine the most appropriate donor type for an efficient production. In this study, we focus on the proliferation and differentiation of bovine SCs isolated from bull calf and dairy cow muscle samples. The proliferative performance of bull calf SCs was significantly better than SCs from dairy cows, however a dynamic differentiation assay revealed that the degree of fusion and formation of myotubes were similar between donor types. Furthermore, the proliferation of SCs from both donor types was enhanced using an in-house developed serum-free media compared to 10% FBS, which also delayed myogenic differentiation and increased final cell population density. Using gene chip transcriptomics, we identified several differentially expressed genes between the two donor types, which could help explain the observed cellular differences. This data also revealed a high biological variance between the three replicate animals within donor type, which seemed to be decreased when using our in-house serum-free media. With the use of the powerful imaging modalities of Cytation 5, we developed a novel high contrast brightfield-enabled label-free myotube quantification method along with a more efficient end-point fusion analysis using Phalloidin-staining. The results give new insights into the bovine SC biology and potential use of bull calves and dairy cows as relevant donor animals for cultivated beef cell sourcing. The newly developed differentiation assays will further enhance future research within the field of cultivated meat and SC biology.

A simple and robust serum-free media for the proliferation of muscle cells.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Recombinant production of growth factors for application in cell culture.

Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as "cellular agriculture". These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-ß1 in Escherichia coli. Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.

Bovine Satellite Cells Isolated after 2 and 5 Days of Tissue Storage Maintain the Proliferative and Myogenic Capacity Needed for Cultured Meat Production.

Cultured meat is an emerging alternative food technology which aims to deliver a more ethical, sustainable, and healthy muscle-tissue-derived food item compared to conventional meat. As start-up companies are rapidly forming and accelerating this technology, many aspects of this multi-faceted science have still not been investigated in academia. In this study, we investigated if bovine satellite cells with the ability to proliferate and undergo myogenic differentiation could be isolated after extended tissue storage, for the purpose of increasing the practicality for cultured meat production. Proliferation of bovine satellite cells isolated on the day of arrival or after 2 and 5 days of tissue storage were analyzed by metabolic and DNA-based assays, while their myogenic characteristics were investigated using RT-qPCR and immunofluorescence. Extended tissue storage up to 5 days did not negatively affect proliferation nor the ability to undergo fusion and create myosin heavy chain-positive myotubes. The expression patterns of myogenic and muscle-specific genes were also not affected after tissue storage. In fact, the data indicated a positive trend in terms of myogenic potential after tissue storage, although it was non-significant. These results suggest that the timeframe of which viable myogenic satellite cells can be isolated and used for cultured meat production can be greatly extended by proper tissue storage.

Beef Production Systems with Steers of Dairy and Dairy × Beef Breeds Based on Forage and Semi-Natural Pastures.

This study compared animal performance and carcass characteristics in steers born to a dairy breed dam and a dairy or beef breed sire allocated to one out of two production systems, both including grazing semi-natural pastures during summer. Spring-born groups comprising 16 purebred dairy (D) steers and 16 dairy × Charolais crossbreeds (C) were allocated to moderately high indoor feed intensity with slaughter at 21 months of age (H), while two corresponding autumn-born groups of 16 D and 16 C animals were allocated to low indoor feed intensity with slaughter at 28 months of age (L). The animals were mainly fed grass-clover silage while housed. The H steers grazed semi-natural pastures for one summer, whereas the L steers grazed semi-natural pastures for two summers. From weaning to slaughter, liveweight gain was 0.94 and 0.77 kg day-1 for H and L steers, respectively (p < 0.0001), with no breed effect on weight gain. However, C carcasses had a higher weight, conformation score, and proportion of high-valued retail cuts than D carcasses (p ≤ 0.004). Moreover, conformation differed more between LC and LD than between HC and HD. From this study on forage and pasture-based beef production, we concluded that breed had no effect on the performance of living animals, but that dairy × beef crossbred steers produced heavier and higher-quality carcasses than pure-bred dairy steers.

Supplementation of sows with L-Arginine during gestating and lactation affects muscle traits of offspring related with postnatal growth and meat quality: From conception to consumption.

This study investigated the effect of dietary inclusion of 25 g/day of L-Arginine (n = 7) or iso­nitrogenous amounts of alanine (n = 6) from d 30 of gestation to d 28 of lactation of sows on performance, muscle traits and meat quality in offspring. From each litter, heaviest and smallest littermate of both sexes were reared from d 28 and slaughtered at d 140 in accordance with a 23factorial design. A response to L-Arginine were obtained on small females where L-Arginine increased birth weight, however this effect disappeared at weaning. L-Arginine increased daily gain by 7% and increased the cross-sectional area of the M. semitendinosus in small females by 14%, suggesting an increased lean ratio. Mechanistic studies showed firstly, that small female littermates had increased number of muscle fibres (myogenesis) after L-Arginine treatment (11%) and secondly increased total DNA (12%) as a consequence of satellite cell proliferation. Traits describing tenderness seem to be affected by L-Arginine but further studies are needed.

Exploration of collagen recovered from animal by-products as a precursor of bioactive peptides: Successes and challenges.

A large amount of food-grade animal by-products is annually produced during industrial processing and they are normally utilized as animal feed or other low-value purposes. These by-products are good sources of valuable proteins, including collagen or gelatin. The revalorization of collagen may lead to development of a high benefit-to-cost ratio. In this review, the major approaches for generation of collagen peptides with a wide variety of bioactivities were summarized, including antihypertensive, antioxidant and antidiabetic activities, and beneficial effects on bone, joint and skin health. The biological potentials of collagen peptides and their bioavailability were reviewed. Moreover, the unique advantages of collagen peptides over other therapeutic peptides were highlighted. In addition, the current challenges for development of collagen peptides as functional food ingredients were also discussed. This article discusses the opportunity to utilize collagen peptides as high value-added bio-functional ingredients in the food industry.

Enzyme inhibition kinetics and molecular interactions of patatin peptides with angiotensin I-converting enzyme and renin.

In this work, the inhibitory effects of potato patatin-derived peptides Trp-Gly (WG) and Pro-Arg-Tyr (PRY) on angiotensin I-converting enzyme (ACE) and renin activities were investigated using kinetics, intrinsic fluorescence and molecular docking. The results indicated that PRY was a more potent ACE- and renin-inhibitory peptide than WG. Enzyme inhibition kinetics showed that WG and PRY inhibited ACE activity through mixed-type and competitive modes, respectively. The inhibitory mechanism of WG and PRY towards renin was determined to be mixed-type. PRY exhibited stronger affinity towards ACE and renin molecules, when compared to WG as determined by intrinsic fluorescence intensity. Molecular docking data confirmed that the higher inhibitory potency of PRY might be attributed to formation of more hydrogen bonds with the enzyme's active site or non-active sites that distorted the configuration necessary for catalysis.

Bioactive peptides in beef: Endogenous generation through postmortem aging.

The present research was performed to investigate endogenous release of bioactive peptides in beef during postmortem aging times (1, 10 and 20days). Gradually decreased Warner-Bratzler shear force (WBSF) values of longissimus thoracis (LT) and semitendinosus (ST) muscles were observed and the degradation of structural proteins and collagen led to release of low-molecular weight (<3kDa) peptides. These peptides exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, ACE- and renin-inhibitory activities. The peptide sequences were identified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). In silico analysis (PeptideRanker and BIOPEP) of their bioactivity potentials demonstrated peptides with the predicted bioactivity scores (>0.8) as well as collagen peptides with bioactivity scores (0.6-0.8). The present findings provide insights on development of healthy beef through postmortem aging at 4°C.

Angiotensin I-converting enzyme-inhibitory peptides from bovine collagen: insights into inhibitory mechanism and transepithelial transport.

The inhibitory mechanism and transepithelial transport of angiotensin I-converting enzyme (ACE)-inhibitory peptides (VGPV and GPRGF) derived from Alcalase®- and papain-hydrolyzed bovine collagen were investigated. The inhibitory mechanism of VGPV and GPRGF was experimentally determined to be non-competitive and the results were supported by molecular docking data. In silico and in vitro gastrointestinal digestion indicated that VGPV remained resistant to digestive enzymes, while GPRGF was degraded into smaller ACE-inhibitory peptides (GPR and GF). VGPV and GPRGF were transported across monolayers of human intestinal epithelial Caco-2 cells through paracellular pathway and retained their ACE-inhibitory activities. The present study suggests that VGPV and GPRGF may possibly be absorbed and exert antihypertensive effects in vivo.

How product trial changes quality perception of four new processed beef products.

The purpose of this paper is the quantitative analysis of the change in quality perception of four new processed beef products from pre to post trial phases. Based on the Total Food Quality Model, differences in pre and post-trial phases were measured using repeated measures technique for cue evaluation, quality evaluation and purchase motive fulfillment. For two of the tested products, trial resulted in a decline of the evaluation of cues, quality and purchase motive fulfillment compared to pre-trial expectations. For these products, positive expectations were created by giving information about ingredients and ways of processing, which were not confirmed during trial. For the other two products, evaluations on key sensory dimensions based on trial exceeded expectations, whereas the other evaluations remained unchanged. Several demographic factors influenced the pattern of results, notably age and gender, which may be due to underlying differences in previous experience. The study gives useful insights for testing of new processed meat products before market introduction.

Improving meat quality of organic pork through post mortem handling of carcasses: an innovative approach.

This study was conducted to examine the best combination of post mortem chilling, suspension and ageing in order to optimize tenderness of organic pork at slaughter, which may be tougher than conventionally produced pork, because of lower daily gain. Combinations of stepwise chilling with a holding period of 6h at 10°C or traditional blast tunnel chilling, suspension in the pelvic bone or Achilles Tendon and ageing 2 or 4 days post mortem were tested. Stepwise chilling and ageing improved tenderness of the loin, and the effects were additive, whereas pelvic suspension was less effective in texture improvements, and non-additive to stepwise chilling. Stepwise chilling improved tenderness to a similar degree as can be obtained within 2-4 days of extended ageing, however, the minimum temperature during the holding period seems to be crucial in order to obtain a positive effect of stepwise chilling, and it should be above 7.5°C.

Sensory profiling of textural properties of meat from dairy cows exposed to a compensatory finishing strategy.

A compensatory finishing strategy was evaluated to improve the quality of meat from dairy cows. The experiment included ten pairs of Holstein Friesian dairy cows. Each pair was the progeny of the same sire, in the same parity, and approximately at the same number of days in lactation before entering the experiment. Within each pair, one cow was allocated to a compensatory finishing strategy, dried off for 4 days, and further restricted in energy intake for another 17 days followed by 6 weeks of ad libitum feeding. The strategy improved the sensory texture and flavour of both M. longissimus dorsi (LD) and M. semimembranosus (SM). This was supported by lower shear force in both muscles (P<0.02). The effect can be ascribed to various factors; in LD an increased amount of IMF plays a significant role, whereas in SM an increased protein turnover is suggested to be the dominating factor.

Compensatory growth response as a strategy to enhance tenderness in entire male and female pork M. longissimus thoracis.

The effects of restrictive feeding strategies aimed at promoting a compensatory growth response were investigated with respect to tenderness improvement in pork M. longissimus thoracis. Compensatory growth response is defined by increased weight in pigs fed ad libitum after a period with restricted feeding compared to pigs fed ad libitum the entire fattening period. Specifically, the aim was to study the sensory textural characteristics after female and entire male pigs have been exposed to restrictions in either energy, protein or both in specific growth periods. It was found that textural differences were caused by feeding strategies and not related to variation in intramuscular fat, which did not vary significantly between genders. The female pigs demonstrated compensatory growth response and the texture was significantly (P<0.001) improved by low dietary level of energy from day 50 to 90 and normal dietary level of protein during the entire feeding period. For the entire male pigs, low level of protein in the late feeding period significantly (P<0.05) improved the texture. Also, low level of protein and normal level of energy in the early feeding period resulted in improved tenderness. However, these texture improvements were deemed not to be an effect of compensatory growth since the entire male pigs did not compensate for the feeding restriction in the early feeding period. The tenderness enhancement for the entire male pigs compromised the production results in terms of 7-15% lower carcass weight.

In vitro study to evaluate the degradation of bovine muscle proteins post-mortem by proteasome and µ-calpain.

The degradation of bovine muscle proteins by proteasome and ubiquitous calpains was explored via 2D gel proteome analysis by inhibition of the physiological level of the proteases by specific inhibitors. The inhibition of the proteasome chymotrypsin- and trypsin-like activity results in the lack of degradation of several fragments of structural proteins such as actin, troponin T, myosin light chain and nebulin. In addition the degradation of several sarcoplasmatic proteins was eliminated when proteasome was inhibited. The inhibition of the ubiquitous calpain only resulted in minor changes in the degradation pattern, which might indicate that p94, which is not inhibited by calpastatin, is involved in the degradation post-mortem. The results of the present study indicate a sequential degradation of the structural proteins post-mortem, where calpain initiates the disruption and destabilisation of the myofibrillar structure, and thereby allows the proteasome to act.

Feeding strategy for improving tenderness has opposite effects in two different muscles.

Compensatory growth has been shown to affect rate of muscle protein turnover and may potentially improve tenderness of beef. Thus, a study of tenderness in relation to feeding regime and slaughtering at a time with maximal muscle protein degradation was performed. Friesian bull calves (5-month-old) were fed either ad libitum (n=6) (AA) or restrictively for 3 months followed by re-alimentation for 6 weeks (n=6) (RA) before slaughter at 10 months. At slaughter the fractional breakdown rate of muscle protein was 2.4% in RA compared with 1.6% in AA (P<0.06). Sensory profiling revealed superior texture of M. semimembranosus from RA compared with AA, whereas M. longissimus was superior in texture from AA compared with RA, with no difference in proteolysis and shear force. In conclusion it was clear that different muscles in terms of tenderness responded very differently to the nutritional manipulation.

Proteome analysis of early post-mortem changes in two bovine muscle types: M. longissimus dorsi and M. semitendinosis.

To study early post-mortem changes in muscle tissues from bull calves, cytosole proteins from two muscles: M. longissimus dorsi (LD) and M. semitendinosis (ST) at 0 and 24 h after slaughter were analysed by 2-DE. Principal component analysis (PCA) and rotation testing were used to analyse the protein patterns in the two muscles in order to select protein spots that were significantly different at the two time-points. Selected proteins were identified by MALDI-TOF/TOF. Five proteins, namely cofilin, lactoylglutathione lyase, substrate protein of mitochondrial ATP-dependent proteinase SP-22, HSP 27 and HSP20, were changed in both LD and ST muscles during post-mortem storage. Fifteen additional protein changes were observed in either LD or ST muscles, and some of these changes have not previously been observed to change during post-mortem storage of bovine muscles. Further studies will reveal the relevance of these biomarkers for meat quality.

Effects of a compensatory growth strategy on sensory and physical properties of meat from young bulls.

The objective of the present study was to investigate the sensory properties, with special emphasis on tenderness, of meat from strategically fed young bulls (13 months of age) slaughtered when a plateau in protein turnover was observed. Twelve Holstein Friesian young bulls were divided into two feeding strategies. One group of young bulls (n=6) were fed ad libitum throughout the rearing period (AD) whereas the second group (n=6) was subjected to a compensatory growth feeding strategy (CO). Sensory profiling of beef longissimus dorsi (LD), semimembranosus (SM) and supraspinatus (SS) was performed in addition to physical measurements (shear force) and content of intramuscular fat of LD. The data was analysed using a regression-based multivariate data analytical strategy. In relation to predictivity of the various shear force measurement constituents for sensory texture, it was determined that a number of the responses collected (e.g. max(mm)), in addition to the commonly used max((N)) may be utilised to predict subtlety in the sensory texture differences of the samples (e.g. Crumbliness) with respect to compensatory feeding. Through profiling LD and SS were found to have enhanced texture and flavour properties when the young bulls were fed ad libitum during rearing. However, the SM samples were found to improve in characteristics regarding texture and appearance when the young bulls had been fed compensatorily. Thus, compensatory feeding as a texture improvement strategy proved to be highly dependant on muscle type. Of note, LD and SS were found to develop an off-flavour designated as 'steer taint' when derived from compensatory feeding. This was postulated as potentially not a problem for the consumer, in LD as the level was not significant, and in SS due to sensory masking when commonly prepared as a stew or casserole. Considering the different preparation methods used for meat from LD, SM and SS, compensatory feeding may be considered to have improved the texture and elevated the eating quality where it was most relevant, namely in SM roasts.

Feeding and meat quality - a future approach.

The continuous demand for high standards of quality assurance in the meat production of today and tomorrow calls for development of new tools capable of meeting such demands. The present paper aims to re-think the traditional way of using feeding as a quality control tool in the production of meat and to introduce the potential of a nutrigenomic approach as a first step in the development of pro-active quality control systems which fulfil future demands from industry and consumers. A few chosen examples present how specific feeding strategies can manipulate (i) muscle protein turnover and thereby meat tenderness as well as the cost and sustainability of the production and (ii) muscle energy levels at slaughter and thereby the pH decline, water-holding capacity and the sensory characteristics of meats. The examples are discussed in relation to exploiting essential and basic understanding of physiological and physical processes, which can subsequently be included in a systems biology line of thought of importance for development of unique decision support systems in future meat production.