Membrane interactions and lipid binding of casein oligomers and early aggregates

Caseins constitute the main protein components in mammalian milk and have critical functions in calcium transport and prevention of protein aggregation. Fibrillation and aggregation of κ-casein, a phenomenon which has only recently been detected, might be associated with malfunctions of milk secretion and amyloidosis phenomena in the mammary glands. This study employs a newly-designed chromatic biomimetic vesicle assay to investigate the occurrence and the parameters affecting membrane interactions of casein aggregates and the contribution of individual casein members to membrane binding. We show that physiological casein colloids exhibit membrane activity, as well as early globular aggregates of κ-casein, a prominent casein isoform. Furthermore, inhibition of κ-casein fibrillation through complexation with α_S-casein and β-casein, respectively, was found to go hand in hand with induction of enhanced membrane binding; these data are important in the context of casein biology since in secreted milk κ-casein is found only in assemblies containing also α_S-casein and β-casein. The chromatic experiments, complemented by transmission electron microscopy analysis and fluorescence quenching assays, also revealed significantly higher affinity early spherical aggregates of k-casein to anionic phosphatidylglycerol-lipids, as compared to zwitterionic phospholipids. Overall, this study suggests that lipid interactions play important roles in maintaining the essential physiological functions of caseins in mammalian milk.     

A Simple Device for Assuring High Reproducibility in Fluorometric Measurement of Deoxyribonucleic Acid in Yeast Cells

The heterogeneity and chemical composition were investigated in κ-casein from colostrum. The acid casein was obtained from four different Holstein cow colostra. The yield of acid casein from colostrum was higher than that from normal milk. κ-Casein from colostrum was prepared by the gel filtration method of Yaguchi et al. The gel filtration profiles differed among the four colostrum acid caseins.

Colostrum κ-casein was fractionated on a DEAE-cellulose column into one nonadsorbed and six adsorbed fractions with increasing salt concentration. Six adsorbed fractions had the same molecular weight and stabilizing ability for α_s1-casein in the presence of calcium ion. The amino acid composition and the phosphorus content of the adsorbed fractions were identical, but fractions eluted with high salt concentrations had more carbohydrates (galactose, sialic acid, glucosamine, galactosamine). Colostrum κ-casein was characterized by a higher content of carbohydrate moiety in comparison with normal κ-casein. Also glucosamine which has not been found in normal κ-casein was detected in colostrum κ-casein. The κ-casein component from colostrum contained at least one molecule of carbohydrate, though the carbo hydrate-free component was detected in normal κ-casein.     

Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus Strains improves breakdown of the pure alphas1-casein

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α(s1)- and β-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure β-casein was observed for all strains, the hydrolysis kinetics of α(s1)-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α(s1)-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.     

Influence of Molecular Weight of Chitosan on Interaction with Casein

The process of complex formation of casein from skimmed milk and purified casein with chitosan of different molecular weights was studied. It was shown that at pH 6.3 casein micelles and parts of whey proteins coagulated with positively charged chitosan molecules with molecular weights of 45.3, 25.4, 7.7 and 1.5 kDa. As a result of ionic interaction of chitosan with skimmed milk proteins the yield of target product reached 90–92%. It consisted of all forms of casein: α-casein, β-casein, κ-casein and small amount of whey proteins.     

κ-Casein terminates casein micelle build-up by its “soft” secondary structure

In our previous paper (Nagy et?al. in J Biol Chem 285:38811–38817, 2010) by using a multilayered model system, we showed that, from α-casein, aggregates (similar to natural casein micelles) can be built up step by step if Ca-phosphate nanocluster incorporation is ensured between the protein adsorption steps. It remained, however, an open question whether the growth of the aggregates can be terminated, similarly to in nature with casein micelles. Here, we show that, in the presence of Ca-phosphate nanoclusters, upon adsorbing onto earlier α-casein surfaces, the secondary structure of α-casein remains practically unaffected, but κ-casein exhibits considerable changes in its secondary structure as manifested by a shift toward having more β-structures. In the absence of Ca-phosphate, only κ-casein can still adsorb onto the underlying casein surface; this κ-casein also expresses considerable shift toward β-structures. In addition, this κ-casein cover terminates casein aggregation; no further adsorption of either α- or κ-casein can be achieved. These results, while obtained on a model system, may show that the Ca-insensitive κ-casein can, indeed, be the outer layer of the casein micelles, not only because of its “hairy” extrusion into the water phase, but because of its “softer” secondary structure, which can “occlude” the interacting motifs serving casein aggregation. We think that the revealed nature of the molecular interactions, and the growth mechanism found here, might be useful to understand the aggregation process of casein micelles also in?vivo.     

Photolysis of Aryl Glycosides with Ultraviolet Irradiation

Bovine casein components (α_sl-, β-, and κ-caseins) were chemically phosphorylated and the properties of the modified components were compared with those of the native to clarify the function of the intrinsic phosphate groups of casein components in casein micelle formation. The calcium binding ability of casein components increased after chemical phosphorylation. The concentrations of calcium chloride required to precipitate modified α_sl- and β-caseins were higher than those for native components. However, phosphorylation of α_sl- and β-caseins did not affect their properties of forming micelles through interaction with κ-casein. The stabilizing ability of κ-casein for α_sl- and β caseins was impaired by its phosphorylation, but the stability was recovered by treating phosphorylated κ-casein with phosphoprotein phosphatase. The results show that the phosphate content of κ-casein must be low to form a stable casein micelle. The results also explain why the specific phosphorylation of casein components in the mammary gland is required.     

Phenotypic variations in blood and milk of the Somali camel*

132 blood samples and 54 milk samples obtained from Somali camel were analysed for red blood cell antigens with the cattle reagents and for Hb, Ca, X proteins, Tf, Alb, Am, SOD, α-La, β-Lg and casein systems respectively. Positive lytic reactions were obtained with the anti-B, -Q, -Q, -W, -F₁ and -J reagents. No biochemical polymorphism was observed except for Hb, X protein and β-Lg systems.     

Evaluation of quality changes in udder quarter milk from cows with low-to-moderate somatic cell counts

Much emphasis has been put on evaluating alterations in milk composition caused by clinical and subclinical mastitis. However, little is known about changes in milk composition during subclinical mastitis in individual udder quarters with a low-to-moderate increase in milk somatic cell count (SCC). This information is needed to decide whether milk from individual udder quarters with a moderate-to-high increase in milk SCC should be separated or not. The aim of this study was to determine how milk composition in separate udder quarters is affected when cow composite milk has low or moderately increased SCC levels. Udder quarter and cow composite milk samples were collected from 17 cows on one occasion. Milk yield was registered and samples were analyzed for SCC, fat, total protein, whey proteins, lactose, citric acid, non-protein nitrogen (NPN), lactoferrin, protein profile, free fatty acids (FFAs), lactate dehydrogenase (LDH), proteolysis, sodium and potassium. Bacteriological samples were collected twice from all four quarters of all cows. The cows were divided into three groups depending on their SCC at udder quarter level. The first group comprised healthy cows with four udder quarters with low SCC, <50 000 cells/ml; composition was equal when opposite rear and front quarters were compared. In the second and the third groups, cows had one udder quarter with 101 000 cells/ml < SCC < 600 000 cells/ml and SCC > 700 000 cells/ml, respectively. The remaining udder quarters of these cows had low SCC (<100 000 cells/ml). Despite the relatively low average cow composite SCC = 100 000 cells/ml of Group 2, milk from affected udder quarters exhibited lower casein number, content of lactose and β-casein (β-CN), while the content of whey protein, sodium, LDH and α-lactoalbumin (α-la) were higher compared to healthy opposite quarters. In addition to these changes, milk from affected udder quarters in Group 3 also exhibited lower values of potassium and αs1-casein (αs1-CN) and higher values of lactoferrin when compared to milk from opposite healthy quarters. This indicates that even when the SCC in cow composite milk is low, there might exist individual quarters for which milk composition is changed and milk quality impaired.     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

Reconstitution of Human Casein Micelle and Its Properties

Human casein micelles were reconstituted from isolated κ- and β-caseins and calcium ions. Micelle formation was recognized in the presence of calcium chloride even at the low concentration of 5mM. At pH levels ranging from 5.5 to 8.0, the re-formed micelles were quite stable so that precipitation of β-casein was not observed. The large micelles were constituted by a higher ratio of β-casein to κ-casein (16:1 by weight) than the small micelles (3: 1). The κ-casein in the small micelles contained carbohydrates to about 43% (w/w) in the molecule, whereas that in the large micelles was only about 25%. When the casein micelles were re-formed from κ-easein and fractionated β-casein components, the extent of phosphorylation of the β-casein component was found to influence the micelle formation; i.e., the β-casein component with no phosphate (the 0-P form) was disadvantageous to form micelles, but the component with 5 phosphates (the 5-P form) formed micelles most easily.     

K-Casein Suppresses Melanogenesis in Cultured Pigment Cells

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk—including β-lactoglobulin, α-lactalbumin, α-, β-, and k-casein—only K-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of K-casein, glycomacropeptide, did not show this activity. Also, K-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that K-casein acts as a melanogenesis-suppressing modulator.     

A lactational study of the composition and integrity of casein micelles from the milk of the tammar wallaby (Macropus eugenii)

The amount of casein found in the milk of the tammar wallaby increases as lactation progresses. The increase is due to increasing amounts of β-casein; the α-casein remains largely constant. The α-casein is the more highly phosphorylated; the most abundant form is the 10-P, throughout lactation. The level of phosphorylation of β-casein shifts to lower average values in late lactation, possibly indicating the enzymatic reaction is overloaded by the increasing amounts of β-casein. Unlike bovine casein micelles, the wallaby micelles are not completely disrupted at pH 7.0 by sequestration of their calcium content with ethylene diamine tetraacetic acid (EDTA). Complete disruption only follows the addition of sodium dodecyl sulphate, indicating considerably greater importance for hydrophobic bonds in maintaining their integrity. This micellar behaviour indicates that, despite the evolutionary divergence of marsupials millennia ago, the caseins of wallaby milk assemble into micelles in much the same fashion as in bovine milk.     

Conversion of amino acids into aroma compounds by cell-free extracts of Lactobacillus helveticus

AIMS: Lactobacillus helveticus is an essential starter in Swiss-type cheeses such as Emmental. This study was to determine whether cell-free extracts of Lact. helveticus were able to convert free amino acids into neutral volatile aroma compounds at the pH and temperature occurring in cheese. METHODS AND RESULTS: A mix of branched-chain (Leu, Ile, Val), aromatic (Tyr, Phe) and sulphur (Met) amino acids was incubated for 7 days, at pH 5.7 and 24 degrees C, with cell-free extracts of six strains. The amino acids were all transaminated into the corresponding keto acids when an amino group acceptor (alpha-ketoglutaric acid) was provided. Phe and Tyr were transaminated the most efficiently, followed by Leu, Met, Ile and Val. Three major volatile compounds were detected by GC-MS: benzaldehyde, dimethyl disulphide and 2-methyl propanol. Whatever the strain, benzaldehyde was produced in the highest quantity (0.25-1 micromol l(-1) mg(-1) protein). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus intracellular enzymes could significantly contribute to the production of aroma compounds from amino acid catabolism.     

草地藏系绵羊乳游离氨基酸质量分数及蛋白遗传多态性研究

用反相高效液相色谱法检测草地藏系绵羊乳游离氨基酸质量分数,用聚丙烯酰胺凝胶电泳法研究乳蛋白组分及其遗传多态性。结果表明:草地藏系绵羊乳中检测到17种游离氨基酸,其中质量分数最高的为Arg;与金堂黑山羊乳比较17种游离氨基酸中,甲硫氨酸的质量分数极显著高于金堂黑山羊(p<0.01),而天冬氨酸、甘氨酸、赖氨酸、谷氨酸、苏氨酸、丙氨酸和缬氨酸的质量分数均显著低于金堂黑山羊(p<0.05),其余9种游离氨基酸质量分数未发现明显差异,但两种羊乳中的必需氨基酸总量基本相同,差异不明显(p>0.05)。草地藏系绵羊乳蛋白组分主要包括α-La、β-Lg、CN、IgG等,CN的相对质量分数约50%~52%;研究还发现4种分子量类型的乳上皮粘蛋白MUC1,分子量分别为214kD、209kD、207kD、205kD;CN、β-Lg均未检测到多态性,说明草地藏系绵羊乳蛋白遗传多态性较为贫乏。     

Long-lived radicals of amino acids induced by X-ray-radiation are the source of hydrogen peroxide in aqueous medium

The formation of long-lived radicals in the solutions of casein and its hydrolysate with an equimolar mixture of amino acids was compared by measuring the X-ray-induced chemiluminescence. It was shown that free amino acids constituting the protein produce long-lived radicals. It was demonstrated that some amino acids (Leu, Ile, Val, Ser, Trp, Met, Pro, Arg, Gly, Phe) emit light of visible spectrum over a long period of time after the irradiation, which indicates the generation of long-lived radicals of these amino acids. The half-life times of these radicals are several hours. Dissolving irradiated dry amino acids capable of luminescing over a long time gives rise to the formation of hydrogen peroxide in aqueous medium.     

One-Step Lipophilization of Proteins Using Fatty Acyl Anhydride

Nucleotide sequences of mRNAs were compared between major calcium-sensitive caseins of cow (α_s1-casein) and rat (α-casein). A best fit alignment of the two sequences showed homology of 81% and 69% for the 5′- and 3′-untranslated regions, respectively. Homology in the comparable coding region of the mature asl-casein (76% of total codons) was remarkably lower at amino acid level (46%) than at nucleotide level (69%). The low conservation at amino acid level is explained by the unusual nucleotide substitution pattern (random at all three positions of codons) in contrast to synonymous substitutions at the third position revealed on comparison of other related proteins. The evolutionary distances among the number of the casein family were estimated by comparing known nucleotide sequences of the signal peptides which were the most conserved coding regions in the family. The divergence time for most distantly related caseins (both rat α-casein/rat β-casein and rat α-casein/mouse ε-casein) was estimated to be about 170 million years.     

Suitability of animals' purified milk caseins and their subunit kappa-caseins as substrates for subtilisin and trypsin.

Acid casein and kappa-casein were purified from different species of animal's milk, such as cow, sheep, goat, and water buffalo. These caseins were used as substrates for commercially available subtilisin and trypsin. It was established that, when acid caseins were used as a substrate for subtilisin, cow acid casein was found to be a better substrate for the enzymes, compared to other animals' milk casein. It was suggested that this acid casein has significantly more aromatic amino acids, as compared to arginine and lysine. K(M) and Vmax values, which were obtained for cow kappa-casein, showed that cow kappa-casein was a better susbstrate for trypsin than the others, suggesting that cow kappa-casein has a rich content of lysine, arginine, and aromatic amino acids by comparison with the others. The calculated C/N ratio also supports this suggestion.     

Nutritional,antimicrobial and medicinal properties of Camel’s milk: A review

Camel’s milk is an important part of staple diet in several parts of the world, particularly in the arid and semi-arid zones. Camel’s milk is rich in health-beneficial substances, such as bioactive peptides, lactoferrin, zinc, and mono and polyunsaturated fatty acids. These substances could help in the treatment of some important human diseases like tuberculosis, asthma, gastrointestinal diseases, and jaundice. Camel’s milk composition is more variable compared to cow’s milk. The effects of feed, breed, age, and lactation stage on milk composition are more significant in camel. Region and season significantly change the ratio of compounds in camel’s milk. Camel’s whey protein is not only composed of numerous soluble proteins, but also has indigenous proteases such as chymotrypsin A and cathepsin D. In addition to their high nutritional value, these whey proteins have unique characteristics, including physical, chemical, physiological, functional, and technological features that are useful in the food application. The hydrolysis of camel’s milk proteins leads to the formation of bioactive peptides, which affect major organ systems of the body and impart physiological functions to these systems. The camel’s milk has antioxidant, antimicrobial, angiotensin-I-converting enzyme (ACE)-inhibitory peptides, antidiabetic as well as anticholesterol activities.