Lipid oxidation in algae oil-in-water emulsions stabilized by bovine and caprine caseins.

Caseins (alpha(s1)-, alpha(s2)-, and beta-casein) are phosphoproteins that are capable of binding transition metals and scavenging free radicals; this property makes them good candidates to be used as natural antioxidants in oil-in-water emulsions. Caprine casein exhibits variability in alpha(s1)-casein content generated by genetic polymorphism. This variability in composition could lead to altered antioxidant properties. Thus, the ability of two caprine caseins differing in alpha(s1)-casein content to inhibit lipid oxidation in algae oil-in-water emulsions at 5% oil was investigated and compared to bovine caseinate. All caseins inhibited the formation of lipid oxidation at pH 7.0 as determined by lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). However, caprine caseins were in general more effective inhibitors of lipid oxidation than the bovine caseins, which may be attributed to their altered casein amino acid content and/or metal binding capabilities. The combination of the carotenoids with bovine and caprine caseins was highly effective at repressing oxidation leading to the speculation that the caseins may inhibit the loss of the carotenoids and/or react with and enhance the carotenoid activity; again some differences between bovine and caprine caseins were observed with caprine caseins being slightly more effective in the presence of carotenoids.

Influence of bovine and caprine casein phosphopeptides differing in alphas1-casein content in determining the absorption of calcium from bovine and caprine calcium-fortified milks in rats.

Bovine and caprine milks have a similar overall gross composition, but vary considerably in the ratios of their casein components. These differences cause significant changes in the ability of caseins to bind and stabilize calcium (Ca). It might be expected that these in vitro variations, which are thought to be due to differences in casein phosphopeptides (CPP) content, could lead to in vivo differences in the digestion and absorption of Ca. To test this hypothesis three milks with different casein ratios [bovine (B), caprine high in alphas1-casein (CH) and caprine low in alphas1-casein (CL)] were compared with regard to Ca absorption and deposition in growing male rats. For comparison, each milk was Ca-fortified (BCa-milk, CHCa-milk, and CLCa-milk) and CPP, prepared by enzymatic hydrolysis from the respective caseins (extrinsic CPP), were added to both native and Ca-milks. The effects of added CPP (extrinsic) could then be compared with intrinsic CPP released from the gastrointestinal digestion of caseins. Total gastric Ca was sampled at 15, 30 and 60 min after ingestion. No differences were found among the native milks with or without CPP, but the Ca from all Ca-milks (regardless of casein type) appeared to clear the stomach more rapidly and this was enhanced by the extrinsic CPP. The total intestinal Ca was not different among the native milks+/-CPP, however, it rose more rapidly with Ca fortification, and was higher at 30 min for all CPP-Ca-milks. At 60 min the total intestinal Ca level fell for the CPP-Ca-milks while all others continued to rise. These observations suggest that the CPP in Ca-milks enhance gastric clearance and uptake from the intestine. Ca availability from BCa-milk, CHCa-milk, and CLCa-milk with and without CPP was estimated by both plasma and femur uptake of 45Ca. Ca availability was enhanced at 5 h in the plasma in each case by added CPP. In all cases CPP stimulated Ca availability in the femur, but the CL-CPP was higher (P<0.05) than that of either CH-CPP or B-CPP (extrinsic CPP). Based on the results of this study we can conclude that the addition of CPP will have beneficial effect on the absorption of Ca in growing rats from CaCO3 added to bovine and caprine milks.

Reconstituted micelle formation using reduced, carboxymethylated bovine kappa-casein and human beta-casein.

In milk, kappa-casein, a mixture of disulfide-bonded polymers, stabilizes and regulates the size of the unique colloidal complex of protein, Ca2+ and inorganic phosphate (Pi) termed the casein (CN) micelle. However, reduced, carboxymethylated bovine kappa-CN (RCM-kappa) forms fibrils at 37 degrees C and its micelle-forming ability is in question. Here, the doubly- and quadruply-phosphorylated human beta-CN forms and 1:1 (wt:wt) mixtures were combined with RCM-kappa at different beta/kappa weight ratios. Turbidity (OD(400 nm)) and a lack of precipitation up to 37 degrees C were used as an index of micelle formation. Studies were with 0, 5 and 10 mM Ca2+ and 4 and 8 mM Pi. The RCM-kappa does form concentration-dependent micelles. Also, beta-CN phosphorylation level influences micelle formation. Complexes were low-temperature reversible and RCM-kappa fibrils were seen. There appears to be equilibrium between fibrillar and soluble forms since the solution still stabilized after fibril removal. The RCM-kappa stabilized better than native bovine kappa-CN.

Implication of C-terminal deletion on the structure and stability of bovine beta-casein.

Bovine beta-casein (beta-CN) with its C-terminal truncated by chymosin digestion, beta-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in beta-CN-(f1-192) relative to its parent protein,beta-CN, in the temperature range (5 degrees to 70 degrees C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in beta-CN-(f1-192) compared to whole beta-casein at 2 degrees -37 degrees C. Furthermore, binding experiments with the common hydrophobic probe - 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that beta-CN-(f1-192) is nearly incapable of binding to ANS relative to whole beta-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide beta-CN-(f193-209) is important in maintaining the hydrophobic core of beta-CN but the residual association observed argues for a minor role for other sites as well.

Thermal and alkaline denaturation of bovine beta-casein.

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.

Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures.

The caseins of milk form a unique calcium-phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of kappa-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the kappa-casein (90 degrees C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high-molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem. 17: 73-84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-K). Surprisingly, at only 37 degrees C, the RCM-kappa-casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37 degrees C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM-kappa-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and beta turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turn-sheet motif in kappa-casein (residues 15-65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM-kappa-casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.