Variations of Human Milk Protein Preparations from Individual Milk Samples

The acid coagulability of casein from 54 individual human milk samples and the variation of coagulability of their casein preparations by rennin were examined. The casein and whey protein preparations from individual human milk samples were also compared by polyacrylamide gel electrophoresis (PAE). Casein coagulated distinctly from 22 human milk samples when the pH was adjusted to 4.6 with acid, but it did not from other 32 samples. Twenty-two samples of casein preparations coagulated distinctly by rennin in the presence of calcium ions but 19 samples just became turbid. When the classification of human casein based on PAE pattern of major six bands was applied in our preparations, type A appeared most often and type C did least. Any regular relationship was not found between variation of the PAE pattern of casein preparations from individual human milk samples and that of acid coagulability or rennin coagulability.     

Studies on human alpha(s)- and kappa-casein fractions and human caseinoglycomacropeptide

1. Fractions have been obtained from human whole casein closely resembling the α_s- and κ-fractions of cow casein. 2. The α_s-fraction (human α_s-casein) is calcium-sensitive, heterogeneous in zone analysis and inert towards rennin. 3. The κ-fraction (human κ-casein) is calcium-insensitive, heterogeneous in zone analysis, and forms a soluble glycopeptide when acted upon by rennin. 4. Human κ-casein stabilizes human α_s-casein in the presence of Ca²⁺ ions. 5. The glycopeptides released by rennin from human casein and from cow casein have been compared. There are important differences in both the peptide and non-peptide structures of the two compounds. 6. In both human and bovine glycopeptides some of the carbohydrate residues are joined to the peptide by O-glycosidic links with threonine, and possibly with serine.     

Similarity between γ-Casein and a Product of β-Casein Hydrolysis by Milk Protease

A γ-casein-like fraction (FIV) was isolated from the β-casein A hydrolyzate by milk protease and compared with γ-casein. The mobility in polyacrylamide gel electrophoresis, sedimentation coefficient, molecular weight, amino acid composition and terminal amino acids of FIV were almost coincided with those of γ-casein. It is suggested that γ-casein is possibly a product of β-casein hydrolysis by milk protease.     

Isolation of Tremerogen a-13, a Peptidal Sex Hormone of Tremella mesenterica

Bovine casein micelles were fractionated on controlled pore granule (CPG-10/3000) chromatography by size and the chemical properties of the fractionated micelles were compared. The results indicated the presence of two types of micelles distinguishable as large and small micelles. In skim milk, 72.7% of casein was calculated to be in the form of small micelles, 13.6% in the form of large micelles and 13.8% in non-micellar casein form.

The α_s1-casein content decreased, but β- and κ-casein content increased as the micelle size became smaller. κ-Casein in large micelles had a much higher sialic acid content than in small micelles. It was found that this difference in sialic acid content was due to the presence of non-glycosylated κ-casein in small micelles. In large micelles, non-glycosylated κ-casein was almost undetectable.

The addition of wheat germ lectin to micelles resulted in the formation of aggregates through intermicellar bridges between the carbohydrate chains of κ-casein located on the surface of the micelles. Both large and small micelles formed aggregates after the addition of wheat germ lectin. Large micelles were more sensitive to wheat germ lectin than small ones.     

Properties of Acid Casein and Casein Micelles from Milk Destabilized by Frozen Storage

No difference was found in calcium sensitivity, electrophoretic and optical properties between acid caseins prepared from skimmilk before and after frozen storage (up to 180 days at ?7°C).

Destabilization of casein micelles can not be explained either by the reduction of solvation of the micelles or by the liberation of κ-casein from the micelles. However, when storage period was extended (about six months), splitting of a part of κ- and β-casein from the micelles to soluble form was observed, suggesting a drastic change of structure of the destabilized casein micelles.     

Study on Protease of Dairy Bacteria

The action of intracellular proteases of lactic acid bacteria (IPLB) at pH 7 on various paracaseins was studied. Paracaseins prepared by releasing of 3~7% non casein type nitrogen (NCN) were hydrolyzed by IPLB with more difficulty than native or other paracaseins prepared by releasing of less or above 3~7% NCN. This phenomenon was not found in a case of a neutral protease of Bacillus subtilis. Hydrolyzed casein by rennin or IPLB of S. cremoris were studied by DEAE-cellulose column chromatography, starch-gel or agar-gel electrophoresis. It was estimated that not only some part of α-casein but also β-casein were hydrolyzed by IPLB of S. cremoris.     

Metal dependent hydrolysis of β‐casein by sIgA antibodies from human milk

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A‐Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe ≈ Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the β‐casein‐hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine‐Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease‐like β‐casein‐hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ～1.2–1.9‐fold after addition of external metal ions (Mg²⁺ > Fe²⁺ > Cu²⁺ ≥ Ca²⁺ ≥ Mn²⁺) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high β‐casein‐hydrolyzing activity in the presence of individual external metal ions (Fe²⁺ > Ca²⁺ > Co²⁺ ≥ Ni²⁺) and especially several combinations of metals: Co²⁺ + Ca²⁺ < Mg²⁺ + Ca²⁺ < Ca²⁺ + Zn²⁺ < Fe²⁺ + Zn²⁺ < Fe²⁺ + Co²⁺ < Fe²⁺ + Ca²⁺. The patterns of hydrolysis of a 22‐mer oligopeptide corresponding to one of sIgA‐dependent specific cleavage sites in β‐casein depend significantly on the metal used. Metal‐dependent sIgAs demonstrate an extreme diversity in their affinity for casein‐Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ‐ and κ‐type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

The Characterization of Rennin Action on κ-Casein Using CM-Cellulose

Rennin action on κ-Casein was studied using the CM-cellulose method which determines the amount of para-κ-casein formed during the enzymatic hydrolysis of κ-casein. The reaction rate was measured as a function of the time and enzyme concentration. A Km value of 8.9 ×10^?4m and a V value of 1.2 × 10^?4 M/min were obtained under the assay condition used in this study. The maximum initial rate of para-κ-casein formation occurred at pH 5.0 and 50°C. The present study also demonstrated that the CM-cellulose method is useful for measuring the rennin activity on κ-casein.     

人肾素转基因小鼠的建立

为研究人肾素基因在体内的功能和建立其药物干预实验的动物模型,采用显微注射法,将纯化的人肾素基因导入小鼠受精卵,再培育成转基因小鼠.通过DIG DNA印迹和PCR分析,进行转基因整合检测.在出生的13只子代鼠中,得到一只转基因阳性鼠.整合率为7.7%,有效率0.3%,转基因已稳定传代.RT-PCR显示转基因阳性鼠的肾、心和肺组织中有肾素基因表达,而在肝脏与骨骼肌中则未检测到.阳性鼠血浆肾素活性较对照鼠明显升高,而肾与心脏组织的肾素活性则无明显变化.人肾素转基因小鼠可用于研究循环或组织的RAS中肾素基因的功能及有关其药物抑制实验.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.     

Studies on κ-Casein of Bovine Milk

κ-Caseins were prepared by the calciurn-ethanol method, the Sephadex method and the urea-sulfuric acid method. Some important properties of κ-caseins were investigated using isoelectric focusing, starch gel electrophoresis, ultracentrifugation, chemical analysis, stabilization test of α_s-casein, and rennin treatment. Isoelectric focusing established that κ-casein had its isoelectric point near pH 6.0 in 6 m urea, usually accompanied by a second peak around pH 5.6. Ultracentrifugation, however, showed a single peak having a s_20,w value of 2.6 ~ 3.8 in the presence of 6 m urea and of 14.4 in the absence of such dispersing reagents. Normal contents of hexose, sialic acid, phosphorus, and nitrogen were about 1.5, 0.8, 0.2, and 14%, respectively. Relative patterns of amino acid composition were similar in all of the κ-caseins. In addition, amino acid composition in intact κ-casein and in the further purified κ-casein which formed the second peak in DEAE cellulose chromatography were almost identical, indicating that the κ-casein of the first peak is not an impurity but is one of the components which formed the original κ-casein complexes. The ability of κ-caseins to stabilize α_s-casein in the presence of calcium increased when purified by DEAE cellulose chromatography.     

The Inhibitory Properties of Chicken Egg White Ovomucin against Aggregation of κ-Casein by Rennin

It has been shown that ovomucin, the structural glycoprotein of chicken egg white, has the inhibitory activity against aggregation of κ-casein by rennin.

The inhibitory activity of F-ovomucin, carbohydrate rich component, was much higher than that of S-ovomucin, carbohydrate poor component. The inhibitory activity was remarkably decreased by removal of sialic acid residues from ovomucin.     

Comparing the diversity of the casein genes in the Asian mouflon and domestic sheep

We aimed to determine whether casein variants that are currently segregating in ovine populations existed before the domestication of sheep or, to the contrary, if their emergence is much more recent. To this end, we have retrieved whole-genome sequences from Iranian and domestic sheep from Africa, Europe, South and East Asia and West Asia. Population structure analysis based on 55,352,935 SNPs revealed a clear separation between Iranian mouflons and domestic sheep. Moreover, we also observed a strong genetic differentiation between Iranian mouflons sampled in geographic areas close to Tehran and Tabriz. Based on sequence data, hundreds of SNPs mapping to the casein α_S1 (CSN1S1, 248 SNPs), casein α_S2 (CSN1S2, 268 SNPs), casein ß (CSN2, 146 SNPs) and casein κ (CSN3, 112 SNPs) genes were identified. Approximately 25–63.02% of the casein variation was shared between Iranian mouflons and domestic sheep, and the four domestic sheep populations also shared 44.2–57.4% of the casein polymorphic sites. These findings suggest that an important fraction of the casein variation present in domestic sheep was already segregating in the mouflon prior to its domestication. Genomic studies performed in horses and dogs are consistent with this view, suggesting that much of the diversity that we currently detect in domestic animals comes from standing variation already segregating in their wild ancestors.     

Synthesis of α-Adenosine 5′-Monophosphate

The distributions of protein, calcium and inorganic phosphate among casein micelles of skimmilk before and after frozen storage were investigated by gel filtration through Sephadex G-200 with 6.6 m urea solution as eluant.

The results showed that the native casein micelles were fractionated into two fractions. Fast eluting fraction contained large amount of calcium and inorganic phosphorus. Slow eluting fraction contained calcium but was essentially free of inorganic phosphorus. Frozen storage caused the increase of proportion of the fast eluting fraction accompanying the increase of calcium and inorganic phosphorus contents in it. It is suggested that the salt linkage newly formed during frozen storage causes the increase of proportion of the fast eluting fraction.     

Studies on Molecular Properties of αs1-κ-Casein Complex by the Hydrodynamical Methods

Some molecular properties of α_s1-κ-casein complex, α_s1- and κ-casein polymers were examined by gel filtration, ultracentrifugation, and viscometry at pH 7.1. The Stoke’s radii of α_s1-κ-casein complex, α_s1- and κ-casein polymers were 99, 44 and 108 Å, respectively. The molecular weight of the above proteins were approximately 45 × 10⁴, 10 × 10⁴ and 80 × 10⁴, respectively. The stokes radius of α_s1-κ casein complex reduced compared with that of κ-casein polymer and the molecular weight of the complex was about half that of κ-casein polymer. These results suggest that κ-casein polymer dissociates into 4 smaller particles when α_s1-κ-casein complex is formed. The frictional coefficient and Scheraga-Mandelkern constants for each protein suggest that the molecular shape of α_s1-casein polymer is globular and that of α_s1-κ-casein complex and κ-casein polymer is rod-like.     

Bioavailability of lead from various milk diets studied in a suckling rat model

The bioavailability of lead from various milk diets was studied in 14 day old suckling rats. Human milk, infant formula, cow's milk, rat milk and deionized water labeled with ²⁰³Pb were given to rat pups by gastric intubation. Animals were killed after 2 or 6 h and the radioactivity in the tissues was measured. At 2 h after administration the lead bioavailability, defined as lead uptake in the body, excluding the gastrointestinal tract, was 47% from water, 42% from human milk, 40% from infant formula, 31% from cow's milk and 11% from rat milk. After 6 h the bioavailability of lead was about 50% from water and human milk, 45% from infant formula and cow's milk, and 36% from rat milk. The blood lead levels in the pups reflected the total body uptake and were also correlated to the brain lead levels. Thus, rat pups given lead in human milk had approximately twice as high lead levels in blood and brain than pups given lead in rat milk. The intestinal absorption of lead was dependent on the milk diet given to the sucklings. In duodenum, the highest uptake of lead was found in rats given water or human milk, whereas in rats given rat or cow's milk the highest uptake of lead was found in ileum. The distribution of lead in cream, whey and casein fractions of the milk diets after in vitro labeling with ²⁰³Pb was also studied. The casein fraction in cow's and rat milk contained 90–96% of the total amount of lead in the diet. In infant formula and human milk, 77 and 56% lead was found in the casein fraction, respectively. The higher lead bioavailability observed in the suckling rat fed human milk than in those fed rat and cow's milk may partly be explained by a lower proportion of lead bound to casein in human milk.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Intracellular protease (IPLB) of Streptococcus cremoris was extracted from the cells, which were cultivated in liquid media, by momentarily disrupting between two disks by high pressure.

The hydrolyzing modes of α_s-, crude k-, β-, and whole casein by IPLB of Str. cremoris or rennet were observed through the released amounts of tyrosine, sialic acid, NPN, and calcium insensitive substance. Relative specific turbidity of casein solution and dissymmetry coefficient of casein were measured. Particle weight and UV absorption spectrum of each high molecular hydrolyzate of whole casein were also determined.

Among four kinds of casein fractions, α_s- or crude k-casein was most easily hydrolyzed by IPLB of Str. cremoris or rennet. Relative specific turbidity of crude k-casein solution was remarkably, but those of α_s-, β-, and whole casein slightly increased by the action of IPLB of Str. cremoris or of rennet. Changes of dissymmetry coefficients were negligibly induced by these two enzymes. Absorption spectrum of IPLB-Str. cremoris-casein showed some conformational change.

It was recognized that intracellular protease (IPLB) of L. bulgaricus, L. helveticus or Str. lactis, all together, more easily hydrolyzed α_s-casein than crude k-, β-, and whole casein. By the actions of three IPLBs, relative specific turbidity of crude k-casein solution remarkably but those of α_s-, β-, and whole casein slightly increased, and dissymmetry coefficients of these casein fractions changed negligibly.

Particle weight of whole casein hydrolyzed by each IPLB for five days was larger than that of control casein. UV absorption of each whole casein hydrolyzed by a IPLB increased at the wave length range of 280~250 mμ.     

Studies of camel casein micelles: Treatment with soluble and immobilized neuraminidase

Camel casein micelles were obtained from raw uncooled skim milk by ultracentrifugation, washing and then resuspending in UF-skim milk permeate. They were treated with neuraminidase, in both soluble and immobilized forms, to study the location and distribution of the glycosylated portion of camel casein micelle. Kinetic release of sialic acid, soluble in 12% TCA, was studied. Camel casein micelle contains 7·35 mg sialic acid per g casein; 99·3% of it was released with soluble neuraminidase, whereas only 90% was released with immobilized neuraminidase. This implies that about 90% of the glycosylated portion of camel casein micelle (glyco-k-casein-like component) is on the surface of the micelle.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Enrichment of n-Alkane Assimilation-Deficient Mutants of Candida Yeast by Synergistic Effect of Nystatin and Pyrrolnitrin

In order to clarify further the relationship between the heat stability of casein micelles and the formation of soluble casein upon heating concentrated milk, the effect of formaldehyde was examined. The addition of formaldehyde up to 20 mM markedly increased the heat stability of both concentrated skim milk and concentrated whey protein-free (WPF) milk. The stabilizing effect of formaldehyde was greater for concentrated skim milk than for concentrated WPF milk. The addition of formaldehyde depressed the formation of soluble casein upon heating concentrated milk. No soluble casein was formed on the addition of 20 mM formaldehyde. It was confirmed by Sephadex G-200 gel filtration in the presence of 6.6 M urea that cross-links among the casein components were formed in heated concentrated WPF milk containing formaldehyde. These facts suggest that formaldehyde may introduce cross-links among the casein components and prevent the formation of soluble casein accompanying the release of K-casein from micelles, thus stabilizing the casein micelles.