Ida K. Straadt

Aging-induced changes in microstructure and water distribution in fresh and cooked pork in relation to water-holding capacity and cooking loss - A combined confocal laser scanning microscopy (CLSM) and low-field nuclear magnetic resonance relaxation study.

Confocal laser scanning microscopy (CLSM) and low-field nuclear magnetic resonance (LF-NMR) relaxometry were combined to characterize microstructural changes and water distribution in fresh and cooked pork during an aging period of 14 days. At day 1 (24h postmortem) a few muscle fibres, which appear swollen, were observed in both fresh and cooked meat. An identical microstructure was still apparent after 14 days, however, the number of muscle fibres showing distinguished characteristics was found to increase throughout the aging period. Hence, it was apparent that during aging the individual fibres swell and disintegrate at different rates. Development in water-holding capacity (WHC) was followed during the aging period using gravimetric methods, and an increase in the WHC in the fresh meat was observed, which resembled the amount of extramyofibrillar water measured by NMR relaxometry (T(22) population). This was consistent with the CLSM images, as a substantial increase in the number of myofibrils that appeared swollen, capable of holding more water, was observed during aging. In the cooked meat the width of the T(21c) population, reflecting the myofibrillar water in the cooked meat, was seen to decrease during the entire storage period, which corresponds to the development of a more homogeneous structure. In the CLSM data a continuous degradation during the storage period was observed, which could resemble a shift to a more homogeneous structure. Comparison of CLSM of transverse sections of fresh and cooked pork revealed a pronounced shrinkage of muscle fibres upon cooking. This resulted in large gaps between the cooked muscle fibres, which also was visible as shrinkage at the level of the individual myofibrils. This pattern was also reflected in the NMR relaxation data. The cooking-induced shrinkage of the myofibrils occurred concomitantly with a decrease in the amount of intermyofibrillar water within the individual fibre and an increase in the larger extramyofibrillar spaces between fibres, i.e. water is expelled from the myofibrillar matrix upon cooking. Accordingly, the present study demonstrated that the use of CLSM together with NMR relaxometry can provide further information on the relationship between structural characteristics of meat and resultant water distribution.

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

BackgroundCreatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach.MethodsDifferentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH.ResultsThe identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH2 oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium.ConclusionsThe explorative approach of this study combined with the determination of a decreased intracellular DCFH2 oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.

NMR‐based metabonomics reveals that plasma betaine increases upon intake of high‐fiber rye buns in hypercholesterolemic pigs

This study presents an NMR-based metabonomic approach to explore the overall endogenous biochemical effects of a rye versus wheat-based fiber-rich diet in hypercholesterolemic pigs. The pigs were fed high-fat, high-cholesterol rye- (n = 9) or wheat- (n = 8) based buns with similar levels of dietary fiber for 9-10 wk. Fasting plasma samples were collected 2 days before and after 8 and 12 days on the experimental diets, while postprandial samples taken after 58-67 days, and( 1)H NMR spectra were acquired on these. Principal component analysis on the obtained NMR spectra demonstrated clear effects of diet on the plasma metabolite profile, and partial least squares regression discriminant analysis on the spectra revealed that the intensity of the spectral region at 3.29 ppm dominated the differentiation between the two diets, as the rye diet was associated with higher spectral intensity in this region. The 3.29 ppm signal is ascribed to N(CH(3))(3) protons in betaine, which may be an important contributor to the health promoting effects of rye.

Water properties and structure of pork sausages as affected by high-pressure processing and addition of carrot fibre.

The effects of high-pressure processing (HPP) and addition of carrot fibre on pork sausages have been studied using NMR T₂ relaxometry and measurements of water-binding capacity (WBC) by centrifugation. Significant effects of temperature (raw, 40, 50, or 60 °C), holding time (1s, 3, 6, or 9 min), and addition of carrot fibre on the distribution and mobility of water were found. However, the effect of carrot fibre could not be explained by structural changes in the sausages when examined by confocal laser scanning microscopy (CLSM). Correlations between T₂ relaxation measurements and WBC determined by centrifugation revealed that T₂ relaxation times were able to explain more than 90% of the variation in WBC for both non-pressure and pressure-treated sausages. However, only 49% of the variation was explained for pressure-treated sausages with carrot fibre, indicating that combining addition of fibre and high pressure treatment causes non-coherent changes in T₂ NMR relaxation times.

Relationship between water mobility and distribution and sensory attributes in pork slaughtered at an age between 90 and 180 days

Water mobility and distribution were measured in M. longissimus dorsi from 41 pigs slaughtered at an age of 90, 140, 161 or 182 days using low-field proton NMR relaxometry, and in order to investigate the impact on sensory attributes, a sensory evaluation of the pork was performed in parallel. The sensory evaluation demonstrated a significant effect of slaughter age on juiciness of the meat, and a final juiciness score of 8.4, 7.7, 7.2 and 7.8 was obtained for meat from 90, 140, 161 and 182-day old pigs, respectively. The NMR measurements revealed that the higher juiciness in meat slaughtered at an age of 90 days could be ascribed to a longer relaxation time of the extramyofibrillar water, corresponding to more mobile water, in the fresh meat of 90-day old pigs compared with the older pigs. In the cooked meat the higher juiciness of meat from 90-day old animals could be ascribed to a more homogenous distribution of the myofibrillar water compared with meat from older pigs. In contrast, the NMR measurements showed no effects that could explain the higher juiciness in meat from pigs slaughtered at an age of 182 days compared with meat from pigs slaughtered at an age of 140 or 161 days. Possibly the increase in juiciness when the age at slaughter is increased from 161 to 182 days should be ascribed to an increase in intramuscular fat content, which was not evident in the NMR measurements.

An NMR-based metabolomics study of pork from different crossbreeds and relation to sensory perception

Meat extracts from five different pig crossbreeds including Duroc/Landrace/Yorkshire (DLY), Iberian/Duroc (ID), Iberian/Duroc/Landrace (ILY), Mangalitza/Duroc (MD), and Mangalitza/Landrace/Yorkshire (MLY) were analysed by nuclear magnetic resonance (NMR)-based metabolomics. The results were compared with technological traits and sensory analyses in order to elucidate the potential of NMR-based metabolomics to highlight meat metabolites of importance for technological and sensory attributes of meat. Amino acids including alanine, carnosine, isoleucine, methionine, phenylalanine, and valine, as well as lactate, inosine monophosphate (IMP), inosine, glycerol and choline-containing compounds were found to be significantly affected by crossbreed. The breed-specific differences in the metabolome were ascribed to differences in ante mortem metabolism, differences in the membrane properties and glycolytic potential of muscle fibres and differences in lipolysis and proteolysis. A high content of carnosine in the meat was associated with a low value of many sensory attributes related to meat flavor/taste, while IMP and inosine were in general not correlated with sensory attributes related to meat flavor/taste.

Assessment of meat quality by NMR--an investigation of pork products originating from different breeds.

In the present study, meat obtained from uncommon and novel pig crossings between the rare Iberian and Mangalitza pigs and the more frequent Duroc and Landrace/Yorkshire pigs was characterized by time‐domain proton NMR relaxometry and high‐resolution proton NMR spectroscopy to elucidate the potential of NMR to assess the meat quality of new‐introduced pig breeds. Multivariate data analysis of proton NMR T2 relaxation curves obtained on fresh meat samples revealed differences in the T2 relaxation pattern of the different breeds included in the study. Comparison of NMR T2 relaxation data with gravimetric determination of water‐holding capacity (WHC) indicated that this should be ascribed to differences in the WHC of the different meats, and that NMR T2 relaxation in accordance with previous studies provides unique information about WHC, which may be ascribed to the fact that NMR T2 relaxation reflects information about intrinsic meat structure. High‐resolution proton NMR spectroscopy of freeze exudate and meat extracts also revealed differences in the metabolite profile of the meat between the different breeds studied. The effects of breed on the amount of lactate in the freeze exudate were observed, which could be linked to WHC of the meat. In conclusion, the different NMR techniques applied could provide complementary information about biophysical and biochemical factors of importance for meat quality assessment. Copyright

Sensory and consumer evaluation of pork loins from crossbreeds between Danish Landrace, Yorkshire, Duroc, Iberian and Mangalitza

Iberian (I) and Mangalitza (M) boars were crossed with Duroc (D) and Landrace/Yorkshire (LY) sows and compared with the traditional crossbreed DLY. The sensory attributes were analysed, and consumers evaluated the fried loins. Gas chromatography (GC) aroma analysis was performed on the fried meat, and fatty acid composition was determined on the loin and the subcutaneous fat, respectively. No marked differences were found in odour, appearance or flavour/taste between the alternative crossbreeds and DLY; however, the alternative crossbreeds had improved textural properties. Consumer evaluation revealed that the alternative crossbreeds obtained positive associations compared with DLY, and MD was ranked best or second best by almost 60% of the consumers. No pronounced differences were found in the aroma compounds between the different crossbreeds. Correlations were found between the fatty acid composition and sensory attributes. In conclusion, by crossing the alternative breeds Iberian and Mangalitza with traditional Danish pig breeds it is possible to produce pork with attractive sensory attributes.

Any link between integrin degradation and water-holding capacity in pork?

In order to investigate a possible relationship between integrin degradation and water-holding capacity (WHC), integrin was quantified using western blot, and water mobility and distribution was measured by proton NMR T(2) relaxometry at 24h postmortem in pork (n=30) with a large variation in WHC (drip loss varying from 2.8% to 11.3%). Regression analyses revealed correlation coefficients of r=-0.32 (P=0.08) and r=0.40 (P=0.03) for the correlations between the content of integrin determined by western blot analysis and WHC determined as either drip loss or by NMR, respectively. Water mobility and distribution was also measured in 18 meat samples upon 7 days of aging, which revealed a correlation (r=0.54) between integrin content determined by western blot analysis 24h postmortem and the mobility of the myofibrillar water (T(21) relaxation time) at day 7. In contrast, no correlation could be established between integrin content 24h postmortem and WHC at day 7 determined by NMR as the amount of extramyofibrillar water (T(22) population) (r=-0.01). In conclusion, both visualisation by CLSM and quantification of integrin by western blot analyses of suggested that a strong link between integrin degradation and WHC in pork is questionable, whereas integrin degradation seems to have impact on the succeeding development in the mobility of the myofibrillar water.

NMR-Based Metabonomic Investigation of Heat Stress in Myotubes Reveals a Time-Dependent Change in the Metabolites

NMR-based metabonomics was applied to elucidate the time-dependent stress responses in mouse myotubes after heat exposure of either 42 or 45 degrees C for 1 h. Principal component analysis (PCA) revealed that the gradual time-dependent changes in metabolites contributing to the clustering and separation of the control samples from the different time points after heat stress primarily are in the metabolites glucose, leucine, lysine, phenylalanine, creatine, glutamine, and acetate. In addition, PC scores revealed a maximum change in metabolite composition 4 h after the stress exposure; thereafter, samples returned toward control samples, however, without reaching the control samples even 10 h after stress. The results also indicate that the myotubes efficiently regulate the pH level by release of lactate to the culture medium at a heat stress level of 42 degrees C, which is a temperature level reached in muscles of pigs during exposure to slaughter stress.