Influence of type of dairy matrix micro- and macrostructure on in vitro lipid digestion.

Recent research indicates that the food matrix can influence digestion kinetics and uptake of nutrients, thus affecting human health. The aim of this study was to obtain knowledge on how variations in microstructure and texture of foods represented by four dairy products; (i) cheddar cheese, (ii) a homogenized cheddar cheese, (iii) a micellar casein and cream drink or (iv) a micellar casein and cream gel, all of identical nutrient ratios of protein : fat and calcium : fat, affect the in vitro digestibility kinetics of lipids. Rheology of the four dairy structures was measured at 10 °C and 37 °C before digestion, and during the gastric phase of in vitro digestion. During digestion cheddar cheese was most resistant to enzymatic and mechanical disintegration, followed by homogenized cheese, while both the drink and gel had low resistance and dissolved in the gastric juice. Particle size, fat droplet size and microstructure were assessed by light scattering and confocal microscopy during digestion. Significantly larger fat droplets were observed during digestion of the cheddar cheese sample. The release of free fatty acids during the initial intestinal digestion showed cheddar cheese to provide a significantly lower release than homogenized cheese, whereas the drink and gel both had significantly higher free fatty acid release. The results suggest that the cheese matrix resistance to degradation and its large fat droplets were responsible for a slower fat digestion.

Estimation of genetic parameters and detection of quantitative trait loci for metabolites in Danish Holstein milk.

Small components and metabolites in milk are significant for the utilization of milk, not only in dairy food production but also as disease predictors in dairy cattle. This study focused on estimation of genetic parameters and detection of quantitative trait loci for metabolites in bovine milk. For this purpose, milk samples were collected in mid lactation from 371 Danish Holstein cows in first to third parity. A total of 31 metabolites were detected and identified in bovine milk by using (1)H nuclear magnetic resonance (NMR) spectroscopy. Cows were genotyped using a bovine high-density single nucleotide polymorphism (SNP) chip. Based on the SNP data, a genomic relationship matrix was calculated and used as a random factor in a model together with 2 fixed factors (herd and lactation stage) to estimate the heritability and breeding value for individual metabolites in the milk. Heritability was in the range of 0 for lactic acid to >0.8 for orotic acid and ß-hydroxybutyrate. A single SNP association analysis revealed 7 genome-wide significant quantitative trait loci [malonate: Bos taurus autosome (BTA)2 and BTA7; galactose-1-phosphate: BTA2; cis-aconitate: BTA11; urea: BTA12; carnitine: BTA25; and glycerophosphocholine: BTA25]. These results demonstrate that selection for metabolites in bovine milk may be possible.

Nuclear magnetic resonance metabonomics reveals strong association between milk metabolites and somatic cell count in bovine milk.

Somatic cell count (SCC) is associated with changes in milk composition, including changes in proteins, lipids, and milk metabolites. Somatic cell count is normally used as an indicator of mastitis infection. The compositional changes in protein and fat affect milk coagulation properties, and also the metabolite composition is thought to contribute to differential milk properties. Milk somatic cells comprise different cell types, which may contribute to differential milk metabolite fingerprints. In this study, milk from a relatively large number of individual cows, representing significant differences in SCC, were analyzed by nuclear magnetic resonance (NMR)-based metabonomics, and the milk metabolite profiles were analyzed for differences related to SCC. Global principal component analysis performed on 876 samples from 2 Danish dairy breeds and orthogonal projection of latent structures discriminant analysis performed on a smaller subset (n=70) representing high (SCC >7.2×10(5) cells/mL) and low (SCC <1.4×10(4) cells/mL) milk SCC identified latent variables, which could be attributed to milk with elevated SCC. In addition, partial least squares regression between the NMR milk metabolite profiles and SCC revealed a strong correlation. The orthogonal projection of latent structures discriminant analysis and partial least squares regressions pinpointed specific NMR spectral regions and thereby identification of milk metabolites that differed according to SCC. Relative quantification of the identified metabolites revealed that lactate, butyrate, isoleucine, acetate, and ß-hydroxybutyrate were increased, whereas hippurate and fumarate were decreased in milk with high levels of somatic cells.

NMR-based metabonomics reveals relationship between pre-slaughter exercise stress, the plasma metabolite profile at time of slaughter, and water-holding capacity in pigs.

Nuclear magnetic resonance (NMR)-based metabonomics was applied to investigate the effects of pre-slaughter exercise stress on the plasma metabolite profile at time of slaughter. The study included a total of 40 slaughter pigs, which were exposed to one of the following treatments: No pre-slaughter stress (control treatment), pre-slaughter exercise on a treadmill and subsequently 0, 1, or 3h rest prior to slaughter. NMR-based metabonomics revealed a clear difference in the plasma metabolite profile at time of slaughter between control pigs and pigs exercised without rest, which mainly could be ascribed to increased plasma lactate due to exercise. A resting period of 1 or 3h prior to slaughter reversed the stress-induced perturbations in the plasma metabolite profile. The plasma metabolite profile at time of slaughter was highly correlated with muscle temperature 1 min post-mortem, and a correlation to WHC was also demonstrated. Lactate was found to be the metabolite of importance for the association between the plasma metabolome and pH, temperature and WHC.

Rest before slaughter ameliorates pre-slaughter stress-induced increased drip loss but not stress-induced increase in the toughness of pork.

One factor affecting meat quality is pre-slaughter stress. We investigated the effects of exercise stress on drip loss and toughness in relation to resting times of 0, 1 or 3h following exercise on a treadmill. This exercise stress was regarded as combined physical and physiological stress. Exercise stress increased the muscle temperature, reduced the creatine phosphate, ATP and glycogen content of pigs slaughtered immediately after stress exposure. These conditions lead to a reduced pH early post mortem and an increased drip loss, while only 1h of rest after exercise stress normalised these effects. However, an overshooting effect was noted when pigs were rested for 1-3h before slaughter, emphasising the importance of critical control of the resting period when studying exercise stress-induced effects on meat quality. Furthermore, meat from exercise stressed pigs, irrespective of resting, had increased toughness compared to controls, indicating that the toughness was not related to drip loss in meat from exercise stressed pigs.

NMR and the water-holding issue of pork.

Consumers' awareness of food quality has never been more pronounced. Meat forms a substantial part of the food consumption, and accordingly techniques to control the quality of meat are needed. In addition, a better understanding of how basic biochemical and biophysical factors influence the final meat quality is also required for optimization of the quality. Water-holding capacity (WHC) is a major quality attribute of fresh meat. However, the exact mechanisms determining the WHC of meat are not fully understood. Especially, characteristics about proposed water populations in the meat and how they are interrelated with drip loss need to be studied further. Moreover, the distribution and mobility of water in muscle during its conversion to meat and how they are affected by intrinsic and extrinsic factors are poorly elucidated. Nuclear magnetic resonance (NMR) has during recent years gained increasing use within different areas of muscle physiology and meat science. NMR (1)H relaxation methodologies enable detection of the mobility of protons in heterogeneous materials and thereby provide possibilities for a characterization of the properties of water. The objective of this presentation is to give an overview of the use of NMR relaxation measurements to characterize the proposed water populations in meat and investigate how the distribution and mobility of the water changes postmortem. In addition, applications of NMR spectroscopy in metabolic studies will be considered.

Dietary creatine monohydrate has no effect on pork quality of Danish crossbred pigs.

Creatine content in the muscle may delay postmortem lactate formation and postpone the pH decline, hence potentially improving the water-holding capacity (WHC) as shown in a previous study including purebred Duroc pigs, although the same study did not find any effect on meat from purebred Landrace pigs. In the present study Danish D(LY) crossbreeds were supplemented with 0 or 50g creatine monohydrate (CMH)/d for five days prior to the slaughter. CMH supplementation had no effect on meat quality indicators (pH and temperature), meat quality attributes (WHC and colour) or eating quality (juiciness and tenderness) of meat from the D(LY) crossbred pigs. As a consequence the D(LY) crossbreed was classified as a non-responder to CMH supplementation.

In vitro and in vivo studies of creatine monohydrate supplementation to Duroc and Landrace pigs.

Duroc and Landrace pigs as well as primary myotubes from these breeds were used to investigate mechanisms behind differences in their response to creatine monohydrate (CMH). Pigs were supplemented with 0, 12.5, 25 or 50g CMH/d for 5 days (n=10 per treatment and breed). Plasma levels of creatine increased dose-dependently in both breeds, while muscle-creatine phosphate content increased only in the Duroc pigs. (1)H NMR metabolic profiling showed a tendency towards clustering according to CMH supplementation only among Duroc pigs, revealing a stronger response compared to Landrace pigs. The abundance of insulin-like growth factor I and myostatin mRNA was decreased by CMH supplementation while that of type 1 IGF-receptor and creatine transporter was unaffected. Protein synthesis, increased in the myotubes from both breeds, indicating protein accretion, but no effect was observed on the mRNA abundance of IGF-I, type 1 IGF-receptor, myostatin or the creatine transporter in myotubes.

Dietary creatine monohydrate affects quality attributes of Duroc but not Landrace pork.

Increased creatine content in the muscle may delay post mortem lactate formation and postpone the pH decline, hence potentially improving the water-holding capacity (WHC). Duroc and Landrace pigs were supplemented with 0, 12.5, 25 or 50g creatine monohydrate (CMH)/d for 5 days prior to slaughter. Meat from Longissimus dorsi (LD) of Duroc pigs had a higher WHC and pH at all times, lower colour determinants; a* (redness), b* (yellowness), L* (lightness) and was more juicy compared to that of Landrace pigs. Furthermore, higher pH(2h), pH(24h) and decreased colour determinants were observed in carcass sides exposed to a faster cooling profile. Dietary supplementation with CMH increased the body weight gain of both breeds. However, only meat from Duroc pigs had higher pH(30min) and pH(45min) (at 50g CMH/d) and WHC, but reduced redness (reduced in both breeds) and juiciness when supplemented with CMH compared to non-supplemented controls.

Post mortem energy metabolism and pH development in porcine M. longissimus dorsi as affected by two different cooling regimes. A (31)P-NMR spectroscopic study.

(31)P-NMR spectroscopy was carried out on M. longissimus dorsi samples chilled by two different cooling profiles corresponding to commercial batch and tunnel chilling. The half-life of post mortem phosphocreatine (PCr) degradation was found to be significantly less in muscle samples exposed to tunnel chilling (rapid) compared with muscle samples exposed to batch chilling (soft) conditions, while no difference in the post mortem ATP degradation was found. Moreover, the post mortem pH development in the muscle samples differed considerably between the two cooling regimes. A maximum difference of approx. 0.25 pH units between the two cooling profiles was observed around 150 min post mortem. Theoretical calculations of the registered pH difference between rapid and soft chilling of muscle samples revealed that the temperature effect on the buffer capacity of muscle is the major determining factor in the detected difference in intracellular pH between the two cooling profiles, while any contribution from a temperature-induced delayed progress in the lactate formation post mortem seems negligible. Moreover, calculations on the effect of the registered pH difference between rapid and soft chilling of muscle samples resemble a 2.5 times greater denaturation of myosin in samples which were chilled softly compared with samples chilled more rapidly. Finally, the relationship to the functionality of meats from soft and rapid chilled pork carcasses is discussed.

Immunophenotyping large B-cell lymphomas. Flow cytometric pitfalls and pathologic correlation.

Large cell lymphomas often challenge the diagnostic flow cytometrist. The purposes of this study were to improve our protocols for diagnosing large cell lymphomas and to correlate flow cytometric (FC) data with demographic and histologic features. We identified 63 cases of large B-cell lymphoma between January 1, 1995, and July 30, 1999, and reviewed the diagnostic slides and FC light scatter and staining patterns. The 51 lymphomas with adequate material for systemic review fell into 2 light scatter patterns: "clear cut," with large abnormal cells (high forward scatter relative to normal lymphocytes), 17 cases (33%); and "complex," 34 cases (67%). Clear-cut cases were more mitotically active (average of 42 vs 25 per 10 high-power fields), with higher cellularity. Apoptosis, geographic necrosis, and sclerosis were present histologically in many cases, regardless of FC findings. We conclude that morphologic features of large cell lymphomas do not predict which cases will be difficult to diagnose by FC. Gating strategies can be critical to improve the diagnostic yield.

Origin of multiexponential T(2) relaxation in muscle myowater.

To obtain a further understanding of the nature of the multiexponential T(2) relaxation seen in muscle tissue water (myowater), relaxation measurements were carried out on whole, minced, and homogenized pork of three different qualities with regard to water-holding capacity (normal, red soft exudative, and dark firm dry). Whole, minced, and homogenized pork all resulted in multiexponential T(2) relaxation (three components) independently of the quality, even though microscopic studies on homogenized meat revealed considerable disruption of the macroscopic structure. This states that the relaxation behavior in meat cannot be explained by intra-/extracellular compartmentalization of the water as suggested in earlier studies. Subsequent studies of T(2) relaxation in either whole meat, where the structure integrity was changed by the introduction of dimethyl sulfoxide (membrane disruption) or urea (protein denaturation), or minced meat with added NaCl (inter-/intraprotein interactions) lead to the suggestion that in whole meat (i) the fastest relaxation component reflects water tightly associated with macromolecules, (ii) the intermediate relaxation component reflects water located within highly organized protein structures, for example, water in tertiary and/or quaternary protein structures and spatials with high myofibrillar protein densities including actin and myosin filament structures, and (iii) the slowest relaxation component reflects the extra-myofibrillar water containing the sarcoplasmatic protein fraction. Finally, relaxation patterns in heat-set gels of superprecipitated actomyosin and bovine serum albumin similar to that identified in whole meat support the proposed nature of T(2) relaxation in muscle myowater.

Comparative study of low-field NMR relaxation measurements and two traditional methods in the determination of water holding capacity of pork.

The correlation between transverse relaxation, T(2,) and water holding capacity (WHC) determined either by Honikels bag method (Honikel, 1998) or centrifugation has been investigated in meat samples from m. longissimus dorsi (LD) 24 h post mortem from 74 pigs. Bi-exponential analysis of the transverse relaxation, T(2), showed highly significant correlations between both the two NMR time constants (T(21),T(22)) and water holding capacity determined by both Honikels bag method (r(T(21))=-0.72 and r(T(22))=0.77) and centrifugation (r(T(21))-0.50 and r(T(22))=0.75). This shows that transverse relaxation measurement is an efficient method for determination of water holding capacity in pork. Significant correlations were also found between T(21) and T(22) measured 24 h post mortem and pH measured at various time post mortem. This indicates that transverse relaxation, T(2), reflects pH-induced structural changes occurring in muscles post mortem.

Relationship between RN(-) genotype and drip loss in meat from Danish pigs.

The presence of the RN(-)-gene determined in 72 halothane negative Danish pigs, either by a direct genotyping or the glycolytic potential of the meat, in relation to drip loss, was investigated. The drip loss in the M. longissimus dorsi from RN-carriers (n=26), as determined by genotyping was 9.9% compared to 8.6% in non-carriers (n=46) (P=0.07). When a glycolytic potential of >230 µmol lactate/g meat was used to differentiate between carriers and non carriers of the RN(-)-gene, the drip loss in carriers was 10.6% (n=17) compared to 8.7% in non-carriers (n=55) (P<0.01). These results suggest that the presence of the RN(-)-gene in Danish slaughter pigs only partially explains the large variation in drip loss observed in Danish pork.