Heat-induced interactions of beta-lactoglobulin A and kappa-casein B in a model system

The interaction of kappa-casein and beta-lactoglobulin is fundamental to all heat-induced modifications of milk product functionality, such as the heat stability of concentrated milks. Purified native kappa-casein B and beta-Ig A solutions were heated at 80 degrees C at pH 6.7 separately and in a mixture. The circular dichroism spectra in the near UV indicated irreversible changes in the disulphide bonding patterns involving both proteins. Alkaline- and SDS-PAGE of heated samples showed that, in the presence of kappa-casein, less beta-Ig was converted into beta-Ig polymers and the rate of loss of native beta-Ig was greater. When kappa-casein was added to previously heated beta-Ig and the mixture was heated, the kappa-casein reacted with the heat-induced beta-Ig polymers more readily than with the beta-Ig native monomers. The formation of beta-Ig dimers, trimers etc. was diminished. It was concluded that, when beta-Ig and kappa-casein were heated together, beta-Ig formed thiol-exposed monomers, which reacted with each other or with the native kappa-casein depending on the relative concentrations of beta-Ig and kappa-casein. The products of these reactions included some disulphide-bonded 1:1 beta-Ig:kappa-casein complexes, some monomer kappa-casein and a range of large aggregates held together by either or both disulphide bonds and hydrophobic association.     

Effects of milk protein genetic variants on milk yield and composition

Effects of genetic variants of the milk proteins, alpha S1-casein, beta-casein, kappa-casein and beta-lactoglobulin (beta-lg), on milk yield and composition, particularly the protein composition, were investigated in milk samples from 289 Jersey and 249 Friesian cows in eight commercial herds. Milk protein genotypes had no significant effect on yields over a complete lactation of milk and fat, but significant differences in fat content were detected for beta-casein (B, A1B, A2 greater than A1A2) and beta-lg (B, AB greater than A) variants. Significant differences between beta-lg variants were also found with total solids (B, AB greater than A), casein (B, AB greater than A), whey protein (A greater than AB greater than B) and beta-lg (A greater than AB, AC greater than B greater than BC) concentrations. Casein genotypes were not significantly different in total protein and casein concentrations but many differences were found in casein composition. alpha S1-Casein variants significantly affected alpha S1-casein (BC greater than B) and kappa-casein (B greater than BC) concentrations. beta-Casein variants affected concentration and proportion of beta-casein (A1B, A2B greater than A1, A1A2, A2, B), alpha S1-casein (A1, A2 greater than B) and kappa-casein (B greater than A2) and concentration of whey protein (A1 greater than most other beta-casein variants). kappa-Casein variants affected concentration and proportion of kappa-casein (B greater than AB greater than A), proportion of alpha S1-casein (A greater than AB greater than B) and concentrations of beta-lg (A greater than AB, B) and alpha-lactalbumin (A, AB greater than B). Differences in milk composition were found between breeds, herds and ages, and with stage of lactation. The potential use of milk protein genotypes as an aid in dairy cattle breeding is discussed.     

Proteolytic activity of proteinases on macropeptide isolated from kappa-casein.

Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.     

Opioid antagonist peptides derived from kappa-casein

Opioid antagonists were sought in the fragments of kappa-casein which were obtained by chemical synthesis and enzymic digestion. A synthetic bovine kappa-casein peptide (35-41), Tyr-Pro-Ser-Tyr-Gly-Leu-Asn (casoxin A) showed opioid antagonist activity at 200 microM in the guinea pig ileum assay. A synthetic peptide Tyr-Pro-Tyr-Tyr (casoxin B) which is found in bovine and human kappa-casein, also showed opioid antagonist activity at 100 microM. Another opioid antagonist peptide (casoxin C) was isolated from tryptic digests of bovine kappa-casein by reverse-phase HPLC. The structure of the peptide was Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg, which corresponded to kappa-casein (25-34). Casoxin C was active at 5 microM in the guinea pig ileum assay. Thus, bovine kappa-casein contains three potential opioid antagonist sequences.     

Heterogeneity of caprine kappa-casein macropeptide

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine kappa-casein due to the silent substitution Ile/Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8-14% and 86-92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.     

Relationships of milk protein types to lifetime performance

Data from 889 cows at five research stations of Agriculture Canada were used to study the effects of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin loci on herdlife and total yield over fixed parities (one, two, and three parity) and to a fixed age (36, 48, and 61 mo). Actual yields of all cows were utilized to compute total milk regardless of lactational length. The model consisted of station, breed, year of birth, season of birth, and milk protein types with age at first calving as a covariate. Of the four milk protein types studied, only the kappa-casein locus had significant effects on fixed parity and fixed age total milk and herdlife. Cows with BB kappa-casein type outproduced those with AB or AA kappa-casein types in three parity total milk by 963 and 1657 kg, respectively. Considering total milk accumulated up to 61 mo of age in life, cows with BB kappa-casein type outperformed their counterparts with AB or AA kappa-casein types by 1050 and 1923 kg, respectively. Complete replacement of A by B allele at kappa-casein locus would result in an increase of 1657 kg in three parity total milk and an increase of 1923 kg in 61-mo total milk. The moderate gene frequency of kappa-casein B allele in the current dairy population can be increased to improve lifetime total milk to the benefit of the dairy industry.     

Structural features of a peptide corresponding to human kappa-casein residues 84-101 by 1H-nuclear magnetic resonance spectroscopy

The peptide Val-Arg-Arg-Pro-Asn-Leu-His-Pro-Ser-Phe-Ile-Ala-Ile-Pro-Pro- Lys-Lys-Ile, which corresponds to residues 84-101 of human kappa-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a beta-turn involving residues Pro87-His90 of human kappa-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human kappa-casein (which is equivalent to Arg97 in bovine kappa-casein) unavailable for interaction with Asp249 of bovine chymosin, and may partly explain why human kappa-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.     

Short communication: Application of proteomics for characterization of caseinomacropeptide isoforms before and after desialidation

Caseinomacropeptide (CMP) is an important polypeptide found in cheese whey that has various biological activities and functional properties. Because sialylations play an important role in the functional properties of CMP, the aim of the present work was to characterize CMP isoform heterogeneity in a commercial glycosylated CMP (gCMP) isolate using liquid chromatography– and gel-based proteomics before and after desialidation. Using 2-dimensional gel electrophoresis (2-DGE), we observed a shift in isoelectric point (pI) after enzymatic desialidation, indicating that sialylated gCMP were located at pI 3.0 to 3.1, but desialylated gCMP had repositioned to pI 3.7 to 3.9. We used liquid chromatography/mass spectroscopy (LC-ESI/MS) for further analysis of the glycan chains of gCMP. In total, we identified 19 CMP isoforms, of which 13 were glycosylated and 6 were nonglycosylated. We also detected 3 genetic variants (A, B, and E), with up to 3 glycosylations attached per gCMP. Further, we identified up to 4 isomers, reflecting different sites of glycosylation in the peptide backbone of these isoforms. The dominating gCMP isoform of genetic variant E appeared to contain 4 N-acetyl-neuraminic acid (NeuAc) residues, whereas the dominating gCMP isoforms of variants A and B appeared to contain 2 NeuAc. The identification revealed conversions of isoforms containing different combinations of genetic variants and degrees of glycosylation, sialylation, and phosphorylation to various desialylated counter-isoforms. The content of sialylated gCMP relative to the total CMP content was 37% (wt/wt), which decreased to 1.5% after sialidase treatment, indicating 96% effectivity of the desialidation. This approach can be valuable for future functionality studies specifically addressing the importance of NeuAc.     

Identification of kappa-casein genotype in Holstein sires: a comparison between analysis of milk samples from daughters and direct analysis of semen samples from sires by polymerase chain reaction

Two different methods were used to determine kappa-casein genotypes of Holstein sires. In the earlier procedure, genotypes of sires were deduced by analyzing frequency distribution data of kappa-casein variants obtained through typing of milk samples from daughters by electrophoresis. The second method involved direct analysis of DNA obtained from semen samples of the sires. The polymerase chain reaction was used to amplify a 99-bp region from the kappa-casein gene that contains nucleotide substitutions that are diagnostic of variants A and B. Identity of the amplified product was confirmed by sequencing. Results obtained by both methods of genotyping were similar. For the 42 sires that were analyzed by both methods, the distribution of kappa-casein genotypes were 31 AA, 11 AB, and 0 BB. The frequency of B allele for kappa-casein in the sire population studied was lower than in larger Holstein cow populations. Due to the high demand for kappa-casein B milk by the dairy industry, it might be advantageous to increase this allele in the dairy cattle population by identifying sires with homozygous kappa-casein B and using them more frequently as service sires.     

Analysis by fast protein liquid chromatography of variants of kappa-casein and their relevance to micellar structure and renneting

Fast protein liquid chromatography was used to study the kappa-casein fraction of casein micelles from bulk milk and from milk from individual animals homozygous for kappa-caseins A and B. The extent of glycosylation of the kappa-casein appeared to have no effect on its distribution in casein micelles of different sizes, nor did it affect the rate at which kappa-casein was destroyed during renneting. The rate of breakdown of kappa-casein during renneting was also almost independent of micellar size. The results may indicate a difference between methods which analyse for intact kappa-casein or for the product macropeptide during renneting.     

The influence of acid type on self-assembly,rheological and textural properties of caseinomacropeptide

The influence of acid type (acetic, citric, lactic and hydrochloric acids) on caseinomacropeptide (CMP) self-assembly, rheological and textural properties was analysed. The acidulant selection had a large impact on self-assembly rate, gelation and the hardness of CMP gels. The highest self-assembly and gelation rates were for citric acid, followed by hydrochloric and lactic acids. CMP solutions did not gel when the pH was adjusted with acetic acid during the test period. Product development using CMP must be carefully designed from the point of view of gel properties. A product preparation with CMP gelled with lactic or acetic acids would not be recommended because these acids affect the rheological and textural properties of the gels.     

Effect of controlled kappa-casein hydrolysis on rheological properties of acid milk gels

An experimental method based on the controlled chymosin-induced kappa-casein hydrolysis of milk was proposed to modify micellar reactivity. Milk samples with a degree of kappa-casein hydrolysis of 19, 35, and 51% were obtained. The physicochemical properties of partially converted casein micelles were determined. The net negative charge of casein micelles was reduced with increasing degree of kappa-casein hydrolysis and a small but significant decrease in hydrodynamic diameter and micellar hydration were noted. Dynamic low amplitude oscillatory rheology was used to monitor the rheological properties of acid milk gels (GDL) made with partially chymosin-hydrolyzed milks in comparison with those of strictly acid and rennet gels. An increase in the gelation pH value was observed with increasing the degree of kappa-casein hydrolysis. The moduli values (G' and G') reached 2 h after the point of gel were, for all degrees of hydrolysis tested, significantly higher than those of strictly rennet and acid gels. Comparison of changes in delta G'/delta t with time indicated differences in gel formation that could be related to the increased values of G' obtained for acid gel made with chymosin-treated milk. At a given time after gelation (2 h), increasing the degree of kappa-casein hydrolysis in milk led also to an increase in the loss tangent and the serum holding capacity of acid milk gels suggesting a correlation between these two parameters.     

Caseinomacropeptide self-association is dependent on whether the peptide is free or restricted in kappa-casein

There is a general agreement that the experimentally determined molecular weight (MW) of caseinomacropeptide (CMP) is greater than the theoretical MW. Some studies suggest that this is due to a pH-dependent aggregation of monomeric CMP. How this aggregation is influenced by pH is not understood. This study was carried out to study the nature of CMP aggregates and to clarify which conditions affect aggregation of CMP. The apparent MW of CMP at different pH values was determined using size-exclusion chromatography. Caseinomacropeptide was further characterized by immunochemical analysis, sodium dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilino-naphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of κ-casein (CN), and 2 commercial CMP products. Both commercial products and CMP resulting from chymosin-hydrolysis of κ-CN (at pH 6.6) had elution volumes with a MW corresponding to 35 kDA at pH 8.0 and 3.4. Caseinomacropeptide prepared from pepsin-hydrolysis of κ-CN (at pH 2.5) eluted as multiple peaks with apparent MW of 35, 18, and 9 kDa, again independently of pH. Hydrolysis of κ-CN with chymosin or pepsin at different pH values (pH 2.5, 3.4, and 6.6) produced differently sized aggregates of CMP, largely depending on the pH of the hydrolysis. These results indicate that, whereas CMP molecules are irreversibly associated, CMP in κ-CN may associate reversibly in a pH-dependent manner. We suggest that interactions between para-κ-CN parts of the κ-CN molecules may be a requisite for the pH-dependent dissociation/association.     

Surface charge and conformational properties of phaseolin,the major globulin in red kidney bean (Phaseolus vulgaris L): effect of pH

The aim of this investigation was to explore the pH‐induced conformational changes of phaseolin using differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD) spectra. The surface charges at the varying pH and ionic strength were also determined to probe the possible relationship between the surface charges of phaseolin and its conformation. The ζ‐potential of phaseolin decreased from 27.9 mV at pH 3.0 to –44 mV at pH 9.4, and the isoelectric point (iep) occurred at pH 4.21, which were comparable with the theoretical estimations derived from amino acid composition and pK values. The iep of phaseolin was independent of ionic strength, while the magnitude of ζ‐potential decreased as the ionic strength increased. The near‐UV CD, DSC and fluorescence spectra analyses indicated highly acidic and alkaline pH conditions induced the loss of tertiary conformation of phaseolin. The far‐UV CD spectra analysis confirmed pH‐shift led to the decreases in ordered secondary structure compositions. The present results suggested the loss of secondary and/or tertiary conformation of phaseolin as a result of the varying pattern in electrostatic interactions as pH shifted from 7.0 to acidic or alkalic pH.     

Action of rennin on kappa-casein.

The action of rennin on kappa-casein was studied as a function of time using several methods to measure activity. The first indication of rennin cleavage of kappa-casein is precipitability in .1 M acetate buffer at pH 5.2 and 5 C. A longer exposure to rennin is required to alter kappa-casein so that it forms a precipitate with calcium ions and loses its ability to stabilize alpha s-casein. The least sensitive indication of rennin activity is measurement of nitrogen soluble in 2% trichloroacetic acid. Electrophoresis experiments showed that these methods detect various stages in the conversion of kappa-casein para-kappa-casein.     

Effect of pepsin-treated bovine and goat caseinomacropeptide on Escherichia coli and Lactobacillus rhamnosus in acidic conditions

Caseinomacropeptide (CMP) is a 7-kDa phosphoglycopolypeptide released from κ-casein during milk digestion and in the cheesemaking process. The objective of the study was to analyze the effect of pepsin-treated CMP from cow and goat milk on the resistance of Escherichia coli and Lactobacillus rhamnosus during acid stress. Bacterial cells in the exponential growth phase were suspended in acidified phosphate buffered saline with or without pepsin-treated CMP. Viability was determined during a 90-min incubation period. Pepsin-treated CMP exhibited bactericidal activity at pH 3.5 when added in a dose-dependent manner to E. coli, decreasing survival by more than 90% within 15 min at 0.25 mg/mL. At pH >4.5, the bactericidal activity disappeared, indicating that pepsin-treated CMP was efficient at low pH only. The effectiveness of pepsin-treated CMP at pH 3.5 was not affected by the presence of glycoconjugates linked to CMP or by the bovine or caprine origin of milk. In contrast, L. rhamnosus, a probiotic, was more resistant to acid stress when pepsin-treated bovine or caprine CMP was added to the media. Viability reached 50% after 60 min of incubation at pH 3 compared with 5% survival in the media without added pepsin-treated CMP. Neither glycosylation extent nor sequence variations between CMP from bovine milk and caprine milk affected the protective activity of hydrolyzed CMP toward L. rhamnosus. This suggests that encrypted bioactive peptides released by the pepsin treatment of CMP had an antibacterial effect on E. coli in acidic media, but improved the resistance of L. rhamnosus to acid stress. The peptide fragment accountable for bactericidal activity is the N-terminal region κ-casein f(106-124).     

Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates

This work evaluated the angiotensin-converting enzyme (ACE)-inhibitory activities of bovine, ovine, and caprine kappa-casein macropeptides (CMPs) and their tryptic hydrolysates. The results obtained indicate that bovine, ovine, and caprine CMPs exhibited moderate in vitro ACE-inhibitory activities that increased considerably after digestion under simulated gastrointestinal conditions. Active peptides could also be produced from CMPs via proteolysis with trypsin, with tryptic hydrolysates exhibiting a more extensive ACE-inhibitory activity than intact CMPs during simulated gastrointestinal digestion. Two active fractions were chromatographically separated from the tryptic hydrolysate of the bovine CMP, but their complexity hampered the assignment of the ACE-inhibitory activity to specific peptide sequences. Evidence for the release of the strong ACE-inhibitory tripeptide IPP was found upon simulation of the gastrointestinal digestion of peptides released by trypsin from the CMP sequence. These findings might help to promote further exploitation of cheese whey in the preparation of nutraceuticals for inclusion in the composition of functional food products with high added values.     

Variation in milk protein concentrations associated with genetic polymorphism and environmental factors

Concentrations of alpha s-casein, beta-casein, kappa-casein, beta-lactoglobulin, alpha-lactalbumin, serum albumin, and immunoglobulin in milk from 1888 Holstein cows were determined monthly over the lactation period. Cows were phenotyped for genetic variants of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Least squares analyses showed variations in individual proteins due to parity number, month of test, stage of lactation, somatic cell count, fat content, milk yield, and phenotypes of cows for milk proteins. beta-Casein declined and serum proteins increased with advancing age of cows. Concentration of individual proteins decreased during the first 2 to 3 mo in lactation and then increased as lactation progressed. alpha s1-Casein variants significantly affected concentrations of alpha s-casein (BC greater than BB greater than AB) and beta-lactoglobulin (AB greater than BB greater than BC). Variant B for beta-casein is associated with lower alpha s-casein, beta-lactoglobulin, immunoglobulins, and higher beta-casein and alpha-lactalbumin concentrations than variant A1, A2, or A3. Milk from BB kappa-casein, and BB beta-lactoglobulin cows contained more alpha s-casein, kappa-casein, and less beta-lactoglobulin than milk from AA cows for the two proteins. Concentrations of all proteins were negatively correlated with milk production. Increased somatic cell counts were associated with lower beta-casein and higher concentrations of other proteins. Fat content of milk was positively correlated with the three casein fractions and beta-lactoglobulin.     

Purification of disulphide-linked alpha s2- and kappa-casein from bovine milk

Naturally occurring disulphide-linked alpha s2- and kappa-casein in bovine milk were purified by gel chromatography on a column of Sepharose CL-6B. Four fractions (A-D) were obtained by elution with ammonium acetate-urea buffer. Fractions A and B, identified by SDS gel electrophoresis and amino acid sequence analysis, corresponded to disulphide-linked kappa-casein and alpha s2-casein respectively. Fraction C consisted of a mixture of alpha s1-, alpha s2-, and beta-casein. Separation of fraction C into its components was achieved by reversed-phase HPLC. The stability of the disulphide bridges in alpha s2- and kappa-casein was shown to differ with respect to reducing agents (dithioerythritol and 2-mercaptoethanol).     

Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids.

A study was done to determine if various organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted Escherichia coli O157:H7 cells. E. coli O157:H7 strain EO139, isolated from venison jerky, was grown in tryptic soy broth (TSB) and in TSB supplemented with 1% glucose (TSBG) for 18 h at 37 degrees C, then plated on tryptic soy agar (TSA) acidified with malic, citric, lactic, or acetic acid at pH 5.4, 5.1, 4.8, 4.5, 4.2, and 3.9. Regardless of whether cells were grown in TSB or TSBG, visible colonies were not formed when plated on TSA acidified with acetic, lactic, malic, or citric acids at pH values of < or =5.4, < or =4.5, < or =4.2, or < or =4.2, respectively. Cells not adapted to reduced pH did not form colonies on TSA acidified with lactic acid (pH 3.9) or acetic acid (pH 3.9 and 4.2); however, a portion of acid-adapted cells remained viable on TSA containing lactic acid (pH 3.9) or acetic acid (pH 4.2) and could be recovered in TSB. Inactivation of acid-adapted cells was less than that of unadapted cells in TSB acidified at pH 3.9 with citric, lactic, or acetic acid and at pH 3.4 with malic acid. Significantly (P< or =0.05) higher numbers of acid-adapted cells, compared with unadapted cells, were detected 12 h after inoculation of TSB acidified with acetic acid at pH 3.9; in TSB containing lactic acid (pH 3.9), the number of acid-adapted cells was higher than the number of unadapted cells after 5 h. In TSB acidified at pH 3.9 with citric acid or pH 3.4 with malic acid, significantly higher numbers of acid-adapted cells survived. This study shows that organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted E. coli O157:H7 cells, and acid-adapted cells are more tolerant than unadapted cells when subsequently exposed to reduced pH caused by these acids.