Kinetics of milk coagulation: III. Mathematical modeling of the kinetics of curd formation following enzymatic hydrolysis of kappa-casein--parameter estimation

A step function model of milk micelle agglomeration is proposed to explain the observed kinetics of milk clotting following rennet addition. The model ties together the primary and secondary phases of coagulation. The basis of the model is that no micelle flocculation takes place until ca. 75% of the kappa-casein in the milk is hydrolyzed, at which time flocculation occurs rapidly and the rate limiting step for the clotting process shifts to the kappa-casein hydrolysis reaction. Using such a model, it is possible to explain the clotting kinetics for both rapidly denaturing enzymes and stable enzyme systems. The average rate of the flocculation reaction can be obtained from clotting time-versus-reciprocal-enzyme-concentration data by extrapolating the data to infinite enzyme concentration. The critical conversion required for imminent flocculation can be found by extrapolating the enzyme concentration to zero. This approach indicates that the critical conversion necessary for gelation is temperature dependent changing from a limiting value of essentially 100% hydrolysis at temperatures below 15 degrees C to only 60% conversion at temperatures above 30 degrees C.     

Kinetics of milk coagulation: I. The kinetics of kappa casein hydrolysis in the presence of enzyme deactivation

The kinetics of the primary phase of the enzymatic coagulation of milk, i.e., kappa-casein hydrolysis, was investigated in the presence and in the absence of concurrent enzyme deactivation processes. For conditions under which the enzyme is stable, the rate of hydrolysis can be described by Michaelis-Menten kinetics, as has been reported by previous investigators. A mathematical model, experimental data, and parameter estimates are provided for kappa-casein hydrolysis in the presence of concurrent deactivation of enzyme. The model accurately describes the experimental results when porcine pepsin was used as the renneting enzyme. The model and the experimental results indicate that samples of milk treated under conditions where deactivation of enzyme is significant can have fractional conversions of kappa-casein ranging from zero to unity and yet contain no active enzyme at the termination of the treatment.     

The kinetics of milk coagulation: IV. The kinetics of the gel-firming process

A mechanistic kinetic model of gel firmness development during milk gel formation is presented. The model correctly accounts for the influence of enzymatic kappa-casein hydrolysis on the rate of firmness development in renneted milk gels. The model used is based on two first-order reactions occurring in series. The first reaction is enzymatically controlled and corresponds to the formation of gel crosslink sites by kappa-casein hydrolysis. The second reaction is nonenzymatic and corresponds to the process of crosslink formation and depletion of active sites. The model successfully predicts gel firmness development in the temperature range 31-45 degrees C for a variety of initial enzyme concentrations.     

Disulphide bonds in casein micelle from milk

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.     

Localization of glycosylated kappa-casein on thin sections of casein micelles by lectin-labelled gold markers

The location of the glycosylated part of kappa-casein in bovine casein micelles was investigated using gold particles (6 nm in diameter) labelled with Ricinus communis lectin and Limulus polyphemus lectin. The pattern of marking of thin sections of micelles was similar with both lectins. Glycosylated kappa-casein was distributed uniformly throughout most micelles of all sizes. Peripheral location of glycosylated kappa-casein was observed only occasionally in some of the largest micelles. Quantitative data indicated that the concentration of the glycosylated protein was constant in micelles of increasing sizes. As larger micelles contain less total kappa-casein than smaller ones, these data indicated that a greater proportion of their kappa-casein is glycosylated. These results support models for casein micelle structure where kappa-casein is distributed throughout the micelles. They do not agree with "coat-core" structures.     

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and some effects of functional, low-phenylalanine kappa-casein expressed in the milk of transgenic rabbits

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.     

Kappa-casein micelles: structure, interaction and gelling studied by small-angle neutron scattering

Small-angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of kappa-casein micelles in a buffer at pH = 6.7 were made using the D11 diffractometer in Grenoble. Results indicate that the micelles have a dense core with a fluffy outer layer. This outer layer appears to give rise to a steeply repulsive interaction on contact. In fact, the hard-sphere model best fits the measured scattering intensities. Adding chymosin to the dispersion initiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles. This unexpected second process resembled that of spinodal demixing. The dispersion phase thus separates into a water and a protein phase on a time scale of hours. The observed phenomona contribute to the understanding of the cheese-making process.     

A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles

Casein micelles of bovine skimmed milk were fractionated by permeation chromatography on porous glass (CPG-10, 50 nm followed by CPG-10, 300 nm) at 30 degrees C. Micelles were pooled in eight eluant fractions and their size distribution was determined by electron microscopy. The composition of casein in the eight fractions was determined by quantitative hydroxyapatite chromatography. Micelle size decreased progressively with increasing elution volume, and volume-to-surface average diameter ranged from 154 nm in fraction 1 to 62 nm in fraction 8. Concurrently there was a decrease in relative proportions of alpha s- and beta-caseins and a large enrichment of kappa-casein, which changed from 4.1% total casein in fraction 1 to 12.1% total casein in fraction 8. At least half the decrease in alpha s-casein proportions was attributed to the alpha s1-casein component, but the data also suggested a decline in proportions of alpha s2-casein in the smallest micelle fractions. A plot of kappa-casein fractional content versus micelle surface-to-volume ratio gave a straight line (correlation coefficient from linear regression 0.98) from which an average kappa-casein surface coverage of 1.5 m2/mg or 47.3 nm2/molecule was obtained. If a constant surface coverage for kappa-casein is assumed, the parameters of the linear equation predict that micelle voluminosity is inversely related to micelle diameter, being approximately 30% larger in fraction 8 compared to fraction 1.     

Glycosylated kappa-caseins and the sizes of bovine casein micelles. Analysis of the different forms of kappa-casein

Fast protein liquid chromatography (FPLC) of the kappa-casein from bovine casein micelles of different sizes is used to demonstrate that the proportions of glycosylated and non-glycosylated forms of kappa-casein do not vary with micellar size. The results suggest that glycosylated kappa-casein is distributed similarly to unglycosylated kappa-casein within the micellar structure.     

Deconstructing the DGAT1 Enzyme: Membrane Interactions at Substrate Binding Sites

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.     

Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS- and beta-casein

Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate; the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.     

Purification and characterization of milk clotting enzyme from goat (Capra hircus)

Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of kappa-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat (Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 degrees C with maximum activity at 30 degrees C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 degrees C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.     

Localization of kappa-casein on thin sections of casein micelles by the gold method

The ultrastructural location of kappa-casein in bovine casein micelles was investigated by the protein A-gold method. Casein micelles, fixed in glutaraldehyde, were embedded at low temperature to enhance immunocytochemical marking of thin sections. kappa-Casein was found distributed throughout the micelles of all sizes with a higher concentration in the smaller micelles. No peripheral location of kappa-casein was observed, even in the larger micelles. These results do not agree with "coat-core" structures proposed for casein micelles. However they favor models where kappa-casein is distributed uniformly throughout the micelles.     

Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein

To enhance milk composition and milk processing efficiency by increasing the casein concentration in milk, we have introduced additional copies of the genes encoding bovine beta- and kappa-casein (CSN2 and CSN3, respectively) into female bovine fibroblasts. Nuclear transfer with four independent donor cell lines resulted in the production of 11 transgenic calves. The analysis of hormonally induced milk showed substantial expression and secretion of the transgene-derived caseins into milk. Nine cows, representing two high-expressing lines, produced milk with an 8-20% increase in beta-casein, a twofold increase in kappa-casein levels, and a markedly altered kappa-casein to total casein ratio. These results show that it is feasible to substantially alter a major component of milk in high producing dairy cows by a transgenic approach and thus to improve the functional properties of dairy milk.     

Chelator,metal ion and buffer studies for protein C separation

Protein C (PC) is the pivotal anticoagulant and antithrombotic in the human coagulation cascade. PC deficiency can result in major medical problems such as deep vein thrombosis (DVT), leading to tissue oxygen deprivation. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed. One source of PC is transgenic animal milk. The major components in the milk, such as alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin, are proteins that must be separated from PC. Immobilized metal affinity chromatography (IMAC) is an inexpensive separation technology with relatively high specificity, and it has great potential for difficult protein separations. After systematic studies of different chelator, metal ion and buffers, the combination of iminodiacetic acid (IDA) and Fe was found to be effective to separate PC from major milk components. alpha-Lactalbumin and beta-lactoglobulin fell through the column in the starting buffer. PC was eluted. alpha-Casein, beta-casein, kappa-casein remained bound in the column after PC elution. This technology might be applied for PC separation from transgenic animal milk. It is very important for PC production in large quantities and at low cost to treat PC-deficient patients.     

The identification of the kappa-casein genotype in Holstein dairy cattle using the polymerase chain reaction

Summary The polymerase chain reaction (PCR) was used to amplify a 99-bp region from the kappa-casein gene of Holstein dairy cattle which contains nucleotide substitutions that are diagnostic of the two major protein variants of kappa-casein. Identity of the amplified product was confirmed by direct sequencing. Digestion of the PCR product with MboII (A-variant specific) or TaqI (B-variant specific) allowed direct determination of the genotype of the animal (homozygous or heterozygous). A total of 58 lactating cows with known kappa-casein phenotype were tested using PCR. In all cases, the measured genotype confirmed the phenotype. We have also tested the genotype of 42 sires that were top ranked for milk yield by the CIAQ (Centre d'insemination artificielle du Quebec). The B-allele of kappa-casein which occurred at a frequency of 0.13 among the proven bulls is associated with superior milk for industrial applications. Identification of the kappa-casein genotype by PCR in bulls and calves would provide a means for rapidly changing the frequency of the B-allele in the breeding population by selection.     

Restriction fragment length polymorphism analysis of the kappa-casein locus in cattle

The two common genetic variants (A and B) of bovine kappa-casein originate from two point mutations in the codons for the aminoacids in position 136 and 148. These mutations give rise to polymorphic sites for the restriction endonucleases Hin dIII, AluI, HinfI, Mbo II and TaqI. We have examined DNAs of several Italian Friesian cows and bulls of known and unknown genotype by Southern analyses using kappa-casein cDNA probes. Restriction fragment length polymorphisms (RFLPs) specific for the A and B alleles were identified for each of the above enzymes, except for AluI, which has a non-polymorphic site 12bp away from the polymorphic one. We have also found two new polymorphic sites for MboII and TaqI in the non-coding regions. These sites differentiate the A allele into two new variants, named A1 and A2. The RFLP analysis permits the characterization of kappa-casein alleles even in the absence of their expression. This should facilitate selective breeding programmes aimed at increasing the frequency of the kappa-casein B allele whose product improves the cheesemaking properties of milk.     

Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology

The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.     

Kinetics of the micelle-to-vesicle transition: aqueous lecithin-bile salt mixtures

Important routes to lipid vesicles (liposomes) are detergent removal techniques, such as dialysis or dilution. Although they are widely applied, there has been only limited understanding about the structural evolution during the formation of vesicles and the parameters that determine their properties. We use time-resolved static and dynamic light scattering to study vesicle formation in aqueous lecithin-bile salt mixtures. The kinetic rates and vesicle sizes are found to strongly depend on total amphiphile concentration and, even more pronounced, on ionic strength. The observed trends contradict equilibrium calculations, but are in agreement with a kinetic model that we present. This model identifies the key kinetic steps during vesicle formation: rapid formation of disk-like intermediate micelles, growth of these metastable micelles, and their closure to form vesicles once line tension dominates bending energy. A comparison of the rates of growth and closure provides a kinetic criterion for the critical size at which disks close and thus for the vesicle size. The model suggests that liposomes are nonequilibrium, kinetically trapped structures of very long lifetime. Their properties are hence controlled by kinetics rather than thermodynamics.