Control of α-Lactalbumin Aggregation by Modulation of Temperature and Concentration of Calcium and Cysteine.

The effect of free cysteine (in different concentrations) on the thermal aggregation of calcium-saturated (Ca-sat) and -depleted (Ca-dep) α-lactalbumin (α-LA) was investigated at 25, 50, and 70 °C. The temperatures chosen were below the denaturation temperature ( Td) of Ca-dep and Ca-sat α-LA (25 °C), above the Td of Ca-dep α-LA and below that of Ca-sat α-LA (50 °C), and above the Td of Ca-sat α-LA (70 °C). Size-exclusion chromatography coupled to multiangle light scattering showed that no aggregation or only minor aggregation was obtained at the investigated temperatures for both Ca-dep and Ca-sat α-LA even at extended holding times. Aggregates of Ca-sat α-LA were larger than those developed for Ca-dep α-LA. The addition of cysteine, a low-molecular-mass free thiol, resulted in increased aggregation of both Ca-sat and Ca-dep α-LA. Comparisons of SDS-PAGE run under reducing and nonreducing conditions showed that the formed cross-links were primarily disulfide bonds, but Western blots also showed small contributions from dityrosine cross-link formation. The aggregation kinetics related to monomer loss during heat treatment were determined by RP-UPLC and showed that the addition of cysteine increased the rate of aggregation. The activation energies for Ca-dep α-LA with 0.35 and 0.7 mM cysteine were found to be 59 ± 1 and 46 ± 4 kJ/mol, respectively, which showed that less energy was needed for the enhanced thermal aggregation of α-LA when the cysteine concentration was increased. This study showed that it was possible to control the aggregation size of α-LA by manipulating the incubation temperature and the cysteine concentration.

Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes.

BACKGROUND: Phytanic acid (PA) is a chlorophyll metabolite with potentials in regulating glucose metabolism, as it is a natural ligand of the peroxisome proliferator-activated receptor (PPAR) that is known to regulate hepatic glucose homeostasis. This study aimed to establish primary porcine myotubes as a model for measuring glucose uptake and glycogen synthesis, and to examine the impact of physiological amounts of PA on glucose uptake and glycogen synthesis either alone or in combination with insulin. METHODS: Porcine satellite cells were cultured into differentiated myotubes and tritiated 2-deoxyglucose (2-DOG) was used to measure glucose uptake, in relation to PA and 2-DOG exposure times and also in relation to PA and insulin concentrations. The MIXED procedure model of SAS was used for statistical analysis of data. RESULTS: PA increased glucose uptake by approximately 35%, and the presence of insulin further increased the uptake, but this further increase in uptake was non- additive and less pronounced at high insulin concentrations. There was no effect of PA alone on glycogen synthesis, while the insulin stimulation of glycogen was increased by 20% in the presence of PA. PA neither stimulated glucose uptake nor glycogen synthesis in insulin-resistant myotubes generated by excess glucose exposure. CONCLUSIONS: Primary porcine myotubes were established as a model of skeletal muscles for measuring glucose uptake and glycogen synthesis, and we showed that PA can play a role in stimulating glucose uptake at no or inadequate insulin concentrations.

Effect of feeding intensity and milking system on nutritionally relevant milk components in dairy farming systems in the North East of England.

There is increasing concern that the intensification of dairy production reduces the concentrations of nutritionally desirable compounds in milk. This study therefore compared important quality parameters (protein and fatty acid profiles; α-tocopherol and carotenoid concentrations) in milk from four dairy systems with contrasting production intensities (in terms of feeding regimens and milking systems). The concentrations of several nutritionally desirable compounds (ß-lactoglobulin, omega-3 fatty acids, omega-3/omega-6 ratio, conjugated linoleic acid c9t11, and/or carotenoids) decreased with increasing feeding intensity (organic outdoor ≥ conventional outdoor ≥ conventional indoors). Milking system intensification (use of robotic milking parlors) had a more limited effect on milk composition, but increased mastitis incidence. Multivariate analyses indicated that differences in milk quality were mainly linked to contrasting feeding regimens and that milking system and breed choice also contributed to differences in milk composition between production systems.

Fatty acid, tocopherol and carotenoid content in herbage and milk affected by sward composition and season of grazing.

BACKGROUND: The aim of the present work was to study to what extent grazing large amounts of white clover (WCL), red clover (RCL), lucerne (LUC) or chicory (CHI) was suitable for production of bovine milk with a high milk fat content of tocopherols, carotenoids, α-linolenic acid and conjugated linoleic acid. RESULTS: WCL, RCL, LUC and CHI swards were established to investigate the influence of herbage composition on the milk quality of grazing dairy cows at three periods. Average milk fat composition showed high concentrations of linolenic acid (12 mg g⁻¹), conjugated linoleic acid (13 mg g⁻¹), carotenoids (6 µg g⁻¹) and α-tocopherol (21 µg g⁻¹ milk fat). There were minor differences between herbage types and periods, but multivariate analysis of these data showed no clear grouping. Chemical composition of herbage varied with species as well as period, but it was not possible to relate milk and feed contents of specific fatty acids, carotenoids or tocopherols. CONCLUSION: All four herbages tested were suitable for production of milk with a high content of beneficial compounds. Thus any of these herbages could be used in production of such differentiated milk based on a large proportion of grazing in the ration.

Dityrosine, 3,4-dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on milk proteins with globular and flexible structures as a result of riboflavin-mediated photo-oxidation.

Riboflavin-mediated photo-oxidative damage to protein Tyr residues has been examined to determine whether protein structure influences competing protein oxidation pathways in single proteins and protein mixtures. EPR studies resulted in the detection of Tyr-derived o-semiquione radicals, with this species suggested to arise from oxidation of 3,4-dihydroxyphenylalanine (DOPA). The yield of this radical was lower in samples containing ß-casein than in samples containing only globular proteins. Consistent with this observation, the yield of DOPA detected on ß-casein was lower than that on two globular proteins, BSA and ß-lactoglobulin. In contrast, samples with ß-casein gave higher yields of dityrosine than samples containing BSA and ß-lactoglobulin. These results indicate that the flexible structure of ß-casein favors radical-radical termination of tyrosyl radicals to give dityrosine, whereas the less flexible structure of globular proteins decreases the propensity for tyrosyl radicals to dimerize, with this resulting in higher yields of DOPA and its secondary radical.

Effect of grazing fresh legumes or feeding silage on fatty acids and enzymes involved in the synthesis of milk fat in dairy cows.

The impact of fresh legume types or silage on the composition of milk fatty acids and transcription of enzymes involved in the synthesis of milk fat in cows was studied. Three groups of cows grazed high proportions of white clover, red clover and lucerne, respectively. A fourth group of cows was fed maize/grass silage. The cows grazing high proportions of legumes produced significantly more 18:1 trans-11, 18:2 cis9-trans11, 18:2 trans10-cis12 and 18:3 fatty acids than cows fed silage. White clover and lucerne grazing resulted in significantly lower output of 18:1 trans9 in milk than red clover grazing and maize/grass silages. Transcription of stearoyl-CoA desaturase (SCD) in mammary tissue was significantly increased by grazing high proportions of legume whereas fatty acid synthase and acetyl-CoA carboxylase were not affected by type of feeding. Furthermore, average milk fat globule diameter was correlated to daily milk fat yield but was not affected by feeding. Although the fresh forage affected the transcription of SCD in mammary tissue, the largest effects were on the trans11-based fatty acids. It is concluded that type of forage, i.e. fresh or silage, had a greater impact on rumen fermentation pattern than on transcription of enzymes involved in the synthesis of milk fat.

Modifications of amino acids during ferulic acid-mediated, laccase-catalysed cross-linking of peptides.

Mass spectral analysis demonstrated oligomerization of peptides that had been subjected to oxidation catalysed by Trametes (Coriolus) versicolor laccase. Peptide oligomerization occurred only when cysteines or tyrosines were present in the peptides. MS/MS confirmed the cross-linking in tyrosine-containing peptides to be located between tyrosine residues. Ferulic acid mediated oligomerization of cysteine-containing peptides, but prevented cross-linking of tyrosines when used in the same concentration as the peptides. This suggests an antioxidative effect of ferulic acid in relation to tyrosine oxidation, although incorporation of ferulic acid into peptide oligomers was found in some of the tyrosine-containing peptides. No other modifications to amino acid residues by laccase-catalysed oxidation were observed by mass spectroscopy. Thus, it is suggested that oxidative modifications of other amino acids observed in proteins oxidized by laccase are not major reaction products of laccase-catalysed oxidation.

Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications.

Laccase-catalyzed oxidation was able to induce intermolecular cross-links in beta-lactoglobulin, and ferulic acid-mediated laccase-catalyzed oxidation was able to induce intermolecular cross-links in alpha-casein, whereas transglutaminase cross-linked only alpha-casein. In addition, different patterns of laccase-induced oxidative modifications were detected, including dityrosine formation, formation of fluorescent tryptophan oxidation products, and carbonyls derived from histidine, tryptophan, and methionine. Laccase-catalyzed oxidation as well as transglutaminase induced only minor changes in surface tension of the proteins, and the changes could not be correlated to protein cross-linking. The presence of ferulic acid was found to influence the effect of laccase, allowing laccase to form irreducible intermolecular cross-links in beta-lactoglobulin and resulting in proteins exercising higher surface tensions due to cross-linking as well as other oxidative modifications. The outcome of using ferulic acid-mediated laccase-catalyzed oxidation to modify the functional properties of proteinaceous food components or other biosystems is expected to be highly dependent on the protein composition, resulting in different changes of the functional properties.

Short-term effects of selenium supplementation of cows' feed on the content and distribution of selenium, copper and zinc in bovine milk, whey and blood plasma.

The effect of selenium supplementation of feed on the Se content in bovine milk, whey and plasma, and on the distribution of Se, Zn and Cu in whey and plasma was investigated. In a cross-over study two groups of cows were given a basal feed with 0.16 ppm selenite (approx. 3 mg Se/d) with or without 25 mg yeast Se/d for 2 weeks. In the supplemented group the Se content increased 10-fold in milk, 10-fold in whey and 2-fold in plasma, and after the cessation of the supplementation, selenium in milk decreased with a calculated half-life of 3.5 d. In another experiment, two groups of cows were given either 100 mg yeast Se/d for 1 week or only the basal feed. The increase in Se content in both whole and defatted milk was 40-50-fold, and in whey it was approx. 20-fold. Size-exclusion chromatography of whey using inductively coupled plasma mass spectrometry for detection showed that supplementation increased the proportion of Se in the beta-lactoglobulin-alpha-lactalbumin fraction. Distribution of Cu and Zn was essentially unaffected. In plasma, supplementation increased the Se content in all major Se fractions like selenoprotein P, albumin and low-molecular-weight compounds, but the distribution profiles of Zn and Cu underwent no major changes. The study showed for the first time the rapid kinetics of the Se increase and decrease in milk after the initiation and cessation of supplementation, respectively, and the preferential appearance of Se in the beta-lactoglobulin-alpha-lactalbumin fraction of whey. Milk highly enriched in selenium will be a useful tool for different research purposes.

Changes in structures of milk proteins upon photo-oxidation.

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, and methionine, as well as formation of dityrosine, was observed for all proteins studied, although at very different levels. Generally, the increment was highest for alpha- and beta-casein and was lowest for lactoferrin. Loss of tryptophan because of photo-oxidation was well-correlated with the formation of the tryptophan oxidation products, N-formylkynurenine and kynurenine. Changes at the tertiary protein structure level were observed after photo-oxidation of the globular proteins, where tryptophan fluorescence emission indicated unfolding of alpha-lactalbumin and beta-lactoglobulin, whereas lactoferrin achieved a more compact tertiary structure. Changes in secondary structure were observed for alpha-lactalbumin and beta-lactoglobulin, whereas the secondary structure of lactoferrin did not change. Polymerization of alpha- and beta-casein and of lactoferrin was observed, whereas kappa-casein, alpha-lactalbumin, and beta-lactoglobulin showed little tendency to polymerize after photo-oxidation. Lability toward photo-oxidation is discussed according to the structural stabilities of the globular proteins.

Characterization of reaction products formed in a model reaction between pentanal and lysine-containing oligopeptides.

Aldehydes formed as a result of lipid oxidation form fluorophores after binding to proteins. The structure of the fluorophores formed by reaction between saturated aldehydes and lysine has not yet been identified. The reaction products formed in the reaction between pentanal and oligopeptides were studied by fluorescence spectroscopy and mass spectrometry. The emission spectra showed an increase in fluorescence intensity with incubation time, and the rates were linear with the concentration of pentanal. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the reaction products suggested a molar relation for peptide:pentanal of 1:4. Further tandem mass spectrometry analysis of one of the modified peptides (Pro-Thr-His-Ile-Lys-Trp-Gly-Asp) strongly suggested binding of one pentanal molecule to the amino terminal proline and three pentanal molecules bound to the lysine residue. The latter species is suggested to be the actual fluorophore, through the formation of conjugated double bonds, and a possible reaction pathway through a combination of aldol condensation of pentanal and Schiff base formation with the lysine is suggested.

Evaluation of cooling strategies for pumping of milk - impact of fatty acid composition on free fatty acid levels.

Cooling strategies for pumping of raw milk were evaluated. Milk was pumped for 450 s at 31 degrees C, or pumped after cooling to 4 degrees C and subsequently subjected to various incubation times. Two types of milk were used; i.e. milk from cows fed a diet high in saturated fat supplements resulting in significantly larger milk fat globules than the other type of milk which comes from cows fed a low-fat diet that stimulates high de novo fat synthesis. The content of liquid fat was determined by low-field 1H NMR, which showed that milk from cows given the saturated fat diet also contained less liquid fat at both 4 degrees and 31 degrees C than the other type of milk. This can be ascribed to the differences in the fatty acid composition of the milk as a result of the fatty acid composition of the diets. After pumping of the milk at 31 degrees C, measurement of fat globule size distribution revealed a significant coalescence of milk fat globules in the milk obtained from the saturated fat diet due to pumping. Pumping at 4 degrees C or pumping the other type of milk did not result in coalescence of milk fat globules. Formation of free fatty acids increased significantly in both types of milk by pumping at 31 degrees C. Cooling the milk to 4 degrees C immediately before pumping inhibited an increased content of free fatty acids. However, when the milk was incubated at 4 degrees C for 60 min after cooling and then subjected to pumping, a significant increase in the formation of free fatty acids was observed in both types of milk. It is suggested that this increase in free fatty acids is caused by transition of polymorphic crystal forms or higher level of attached lipoprotein lipases to the milk fat globule before pumping.

Laccase-catalyzed polymerization of tyrosine-containing peptides.

Laccase-catalyzed polymerization of tyrosine and tyrosine-containing peptides was studied in the presence and absence of ferulic acid (FA). Advanced spectroscopic methods such as MALDI-TOF MS, EPR, FTIR microscopy and HPLC-fluorescence, as well as more conventional analytical tools: oxygen consumption measurements and SDS/PAGE were used in the reaction mechanism studies. Laccase was found to oxidize tyrosine and tyrosine-containing peptides, with consequent polymerization of the compounds. The covalent linkage connecting the compounds was found to be an ether bond. Only small amounts of dityrosine bonds were detected in the polymers. When FA was added to the reaction mixtures, it was found to be incorporated into the polymer structure. Thus, in addition to homopolymers, different heteropolymers containing two or four FA residues were formed in the reactions.

Significance of vitamin E supplementation, dietary content of polyunsaturated fatty acids, and preslaughter stress on oxidative status in pig as reflected in cell integrity and antioxidative enzyme activities in porcine muscle.

The present study investigates the combined effects of feed-induced increase in polyunsaturated fatty acids (PUFA) content and/or alpha-tocopherol content in pig muscles and preslaughter stress on cell integrity. Cell integrity was determined by plasma lactate dehydrogenase (LDH) activity, and antioxidative status of muscle was measured by activities of the antioxidative enzymes catalase, superoxide dismutase, and glutathione peroxidase. Preslaughter stress increased LDH activity, reflecting loss in cell membrane integrity independent of increased content of PUFA and/or alpha-tocopherol. However, feed-induced increase of PUFA decreased the LDH activity in plasma immediately after slaughter. Catalase activity in the muscle tissue increased as a consequence of the high-PUFA diet, which may indicate an increased demand caused by introduction of oxidative labile PUFA.

The influence of oxidation on proteolysis in raw milk.

The link between oxidation and increased proteolysis in raw milk was studied. To accelerate oxidation, H2O2 (1 mM) was added to raw milk, resulting in enhanced proteolysis by up to 11.2% after 24 h incubation at 5 degrees C. Addition of Cu2+ (10 microM) to milk or exposure of milk to light (60 min) likewise increased proteolysis. To explain the mechanism responsible for increased proteolysis as a result of oxidation, the effect of lipid oxidation products on plasmin-induced proteolysis was tested. Addition of malondialdehyde to skim milk increased the formation of gamma-caseins, a proteolysis product from plasmin hydrolysis of beta-casein. The same observation was made in a model system containing 4.5 g beta-casein/l sodium tetraborate buffer at pH 8 and plasmin. Addition of a plasmin inhibitor blocked the formation of gamma-casein. The results indicate that aldehydes accumulated from lipid oxidation can modify beta-casein and thereby increase susceptibility of the proteins to proteolysis. Furthermore, the data suggest that proteolysis in raw milk may be connected to oxidative processes.

Oxidative stability of buttermilk as influenced by the fatty acid composition of cows' milk manipulated by diet.

Milk from cows fed a low-fat diet high in cereals designed to stimulate fat synthesis de novo was lower in unsaturated fatty acids (21.3%) than milk from cows fed a diet high in fat, mainly from roasted soy beans (41.3% unsaturated fatty acids). Buttermilk from the more unsaturated milk was less oxidatively stable during storage (at 4 degrees C, followed for 11 d) than buttermilk from the more saturated milk, as monitored both by primary lipid oxidation products (lipid hydroperoxides) and by the secondary lipid oxidation product, hexanal. Fat-soluble antioxidants, beta-carotene and alpha-tocopherol, analysed by HPLC, were not consumed during storage for either of the two types of buttermilk. In contrast, the antioxidative capacity of the serum phase decreased during storage as evaluated in a radical scavenging assay based on the semi-stable water-soluble radical nitrosodisulphonate (Fremy's salt). The time course for the decrease in water-soluble antioxidants was very similar for the two types of buttermilk suggesting that oxidation is initiated in the serum phase independently of fatty acid composition.

Significance of preslaughter stress and different tissue PUFA levels on the oxidative status and stability of porcine muscle and meat.

Polyunsaturated fatty acids (PUFAs) and exercise-induced stress are known to increase the oxidative susceptibility of lipids in muscle tissue. In contrast, antioxidative enzymes, e.g., catalase, superoxide dismutase, and glutathione peroxidase, are known to help sustain the delicate oxidative balance in biological tissue upon the application of stressors. The present study investigates the combined effect of different diet-induced muscle PUFA contents and preslaughter stress on the activity of antioxidative muscle enzymes and the oxidative stability of cooked meat. An increased content of unsaturated fatty acids in the tissue led to a decreased activity of lactate dehydrogenase in the plasma, indicating increased cell integrity. Catalase activity in the muscle tissue increased with increasing PUFA levels. However, this upregulation in antioxidative status of the muscle could not counteract the subsequent development of accelerated lipid oxidation in cooked meat as measured in terms of thiobarbituric acid reactive substances. Moreover, preslaughter stress induced increasing oxidative changes with elevated PUFA levels in the muscle tissue.

Aldehyde-induced xanthine oxidase activity in raw milk.

In the present study, the aldehyde-induced pro-oxidative activity of xanthine oxidase was followed in an accelerated raw milk system using spin-trap electron spin resonance (ESR) spectroscopy. The aldehydes acetaldehyde, propanal, hexanal, trans-2-hexenal, trans-2-heptenal, trans-2-nonenal, and 3-methyl-2-butenal were all found to initiate radical reactions when added to milk. Formation of superoxide through aldehyde-induced xanthine oxidase activity is suggested as the initial reaction, as all tested aldehydes were shown to trigger superoxide formation in an ultrahigh temperature (UHT) milk model system with added xanthine oxidase. It was found that addition of aldehydes to milk initially increased the ascorbyl radical concentration with a subsequent decay due to ascorbate depletion, which renders the formation of superoxide in milk with added aldehyde. The present study shows for the first time potential acceleration of oxidative events in milk through aldehyde-induced xanthine oxidase activity.

Chicken model for studying dietary antioxidants reveals that apple (Cox's Orange)/broccoli (Brassica oleracea L. var. italica) stabilizes erythrocytes and reduces oxidation of insoluble muscle proteins and lipids in cooked liver.

A chicken model for studying the effects of antioxidants in the diet on oxidative status was set up. Chickens fed a semi-synthetic diet low in antioxidants showed a remarkable decrease in erythrocyte stability toward H(2)O(2) or 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), but increases in catalase activity in liver, carbonyls in insoluble muscle proteins, and enhanced lipid oxidation in heat-treated liver samples compared to that of conventionally fed chickens. Thus, this chicken model proved to be more susceptible to oxidative changes than conventionally fed chickens, reflecting a low antioxidative defense. Supplementing this low antioxidant diet with 10% apple/broccoli mixture counteracted these changes, except for activity of catalase in the liver and AAPH-induced lysis of erythrocytes. Supplementation with 10% sweet corn only reduced the carbonyl content in insoluble proteins. However, neither low antioxidant diet nor vegetable supplements affected selected antioxidative enzymes or oxidative stability of lipids in heat-treated muscle tissue.