Genetic Variants Of Β-lactoglobulin Research Articles

Calcium ion binding to native and heat treated β-lactoglobulin genetic variants

Calcium ion binding to native and heat denatured β-lactoglobulin A, B and C genetic variants at pH 7.0 and 0.08 m NaCl was investigated. Native β-lactoglobulin bound calcium ions and the binding increased after denaturation. Small variations in calcium ion binding were observed for the three genetic variants. Analysis of the binding by the Scatchard model suggested about 10 binding sites in the unheated β-lactoglobulin and this decreased to about 5 in the heated samples. The association constants were low in the unheated samples and increased in the heated samples. Analysis of the binding by the Langmuir adsorption model indicated the maximum binding was about 10–13 calcium ions per β-lactoglobulin monomer in the unheated samples and this was about half in the heated samples, possibly due to aggregation making binding sites less available. The affinity constants were low for the unheated samples and increased substantially for the heated samples.

Sensitive identification of milk protein allergens using on-line combination of transient isotachophoresis/micellar electrokinetic chromatography and capillary isoelectric focusing in fused silica capillary with roughened part

A method for on-line concentration of milk proteins from large sample volumes using combination of transient isotachophoresis (tITP) and micellar electrokinetic chromatography (MEKC) in fused silica capillary with an inner roughened part has been developed. The method utilizes reversible dynamic adsorption of proteins onto a thin layer of PEG 4000 on the roughened surface of the capillary. In addition, the tITP/MEKC method was combined with capillary isoelectric focusing (CIEF) for on-line concentration, separation, identification and sensitive determination of proteins in skimmed milk. The method allows analysis of up to 50 μL of sample. This study has focused on the four important whey proteins, bovine serum albumin (BSA), α-lactalbumin (α-LA), and two genetic variants of β-lactoglobulin (β-LG A and β-LG B). The proteins were identified on the basis of their migration times and characteristic pI values. The pI values of BSA, α-LA, β-LG A, and β-LG B were determined as 4.7, 4.4, 5.1, and 5.2, respectively. Limits of detection for BSA, α-LA and both β-LG variants were found as 1.2, 1.0 and 1.0 pg mL−1, respectively. The linearity of calibration curves was characterized by the R2 = 0.9982. The method provided highly reproducible results as the relative standard deviations of the migration times and peak areas of the examined proteins did not exceed 1.6%.

Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A, B and C using 1H NMR and fluorescence quenching

The structure of β-lactoglobulin (β-LG) is well characterized, but the exact location of binding sites for retinol and (−)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic β-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and β-LG A with the most binding sites for EGCG, which include Tyr20, Phe151 and His59. Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of β-LG as nanotransporter.

Effect of fish oil and linseed supplementation on the protein composition of milk from cows with different β‐lactoglobulin phenotypes

The objective of this study was to determine the impact of fish oil and linseed (FOL) supplements on the protein fraction levels of milk from cows with different phenotypes of β-lactoglobulin. After 21 days of supplementation the study showed significantly higher concentrations of whey proteins, especially lysozyme (144% increase) and lactoferrin (45.5% increase), compared with milk from control cows (total mixed ration with no supplemented FOL). A reverse trend was demonstrated for casein, casein index and casein number (lower level). The most favourable change (higher level), in terms of lactoferrin, α-lactalbumin and bovine serum albumin contents in milk, was recorded in cows with the BB variant of β-lactoglobulin. The highest level of lysozyme was recorded in the milk of cows with the AB variant of β-lactoglobulin. The combined supplementation of fish oil and linseed had a positive impact on whey proteins in cow's milk. In addition, the phenotype of β-lactoglobulin also played a role in milk protein composition. There is therefore a clear indication that nutritional experiments should take into account not only food supplements but also the genetic variants of β-lactoglobulin.

Yak introgression into Tibetan Yellow cattle: evidences from mitochondrial DNA D-loop sequence analysis and β-lactoglobulin genetic variants

The objective of this study was to investigate the possible introgression of highland yak (Bos grunniens) into Tibetan Yellow cattle (Bos taurus) based on milk protein and mtDNA D-loop analysis. Tibetan Yellow cattle (43) were used to assay polymorphisms of β-lactoglobulin (β-Lg) and to extract genomic DNA for the PCR amplification of mtDNA D- loop. Native polyacrylamide gel electrophoresis (PAGE) of milk β-Lg showed that 3 milk samples exhibited the yak- specific β-Lg E variant among the 43 milk samples tested. mtDNA D-loop sequences were successfully amplified by PCR using genomic DNA extracted from milk. A total of 16 sequences of the mtDNA D-loop were obtained for analysis, which contained 133 variable sites (97 parsimony-informative sites and 36 single sites) and defined 15 different haplotypes. The 3 Tibetan Yellow cattle carrying β-Lg E variant were exactly clustered with yak when mtDNA D-loop sequences were employed for cluster analysis, while other yaks assayed (13) clustered with cattle. This research demonstrated that there existed matrilineal introgression of yak during the evolution or natural breeding of Tibetan Yellow cattle population on Qinghai-Tibet plateau.

Mapping a quantitative trait locus for the concentration of β-lactoglobulin in milk, and the effect of β-lactoglobulin genetic variants on the composition of milk from Holstein-Friesian x Jersey crossbred cows

AIM: To identify quantitative trait loci (QTL) affecting the concentration of β-lactoglobulin in milk, and to evaluate the effect of β-lactoglobulin genetic variants on the concentration of fat, protein and casein in bovine milk. METHODS: A herd of 850 F2 Holstein-Friesian x Jersey crossbred cows was produced through mating six Holstein-Friesian x Jersey F1 bulls of high genetic merit with F1 cows from the national herd. A total of 1,610 herd-test records from 556 second-parity crossbreds were analysed. The concentration of fat, protein and casein in milk was measured at peak, mid- and late lactation, during the production seasons of 2003–2004 and 2004–2005. Liveweight was measured daily. DNA from the F2 animals, their F1 dams and sires, and selected grandsires was genotyped across the genome, initially with 285 microsatellite markers, and subsequently with 6,634 single nucleotide polymorphisms (SNP). RESULTS: A highly significant QTL for the concentration of β-lactoglobulin in milk was identified, which coincided with the position of the β-lactoglobulin gene on bovine Chromosome 11. No other consistently significant QTL for the concentration of β-lactoglobulin in milk were detected. Cows with the BB β-lactoglobulin genotype produced milk with a 30% lower concentration of β-lactoglobulin than cows with the AA genotype. The β-lactoglobulin polymorphism also explained variation in the proportion of casein in total protein. In addition, the percentage of fat was higher for BB than AA animals, whereas the percentage of total protein, mean daily milk yield and liveweight did not differ between AA and BB animals. CONCLUSIONS: A significant QTL determining the concentration of β-lactoglobulin in milk was identified. Selection of animals for the β-lactoglobulin B-allele may enable the production of milk naturally enriched for casein, thus allowing a potential increase in the yield of cheese. There may be additional future value in production of bovine milk more like human milk, where decreasing the concentration of β-lactoglobulin is desirable.

Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography

In the present work, isoelectric point (p I) separation of proteins by pH-gradient ion-exchange chromatography (IEC) on packed capillary columns is demonstrated. The development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, which was an important technical challenge for adapting this technique to capillary dimensions, was solved by designing a low microliter per minute flow rate housing to a commercially available micro pH probe. Highly linear outlet pH-gradients within the pH range 8.5–4.0 were obtained when applying simple inexpensive buffers consisting solely of piperazine, N-methylpiperazine and imidazole on 10 cm×0.32 mm i.d. fused silica capillaries packed with anion-exchange poly(styrene divinylbenzene)-based macroporous materials, i.e. 10 μm Mono P from Amersham Biosciences and 10 μm PL-SAX from PolymerLabs. Furthermore, when using a pH-gradient from 6.8 to 4.3, both columns were able to baseline separate the A and B genetic variants of β-lactoglobulin, which differ with two amino acid residues only, but the PL-SAX column provided almost a two-fold decrease in peak widths compared to the Mono P column. The influence of varying the buffer concentration, injection volume and column temperature on the peak widths and resolution of the β-lactoglobulins was investigated, e.g. a 100 μl sample of dilute β-lactoglobulins was injected directly on the column with practically no increase in peak width as compared to what obtained with conventional injection volumes. Finally, a pH-gradient from 6.8 to 4.3 was used to separate proteins in skimmed bovine milk on the PL-SAX column. The milk was simply diluted 1:10 (v/v) with water and filtrated before injection.

β-Lactoglobulin genetic variants in Brown Swiss breed and its association with compositional properties and rennet clotting time of milk

β-Lactoglobulin (β-Lg) polymorphism of Holstein Friesian and Brown Swiss breeds were studied by applying native polyacrylamide gel electrophoresis. In addition, total solids, protein, fat ash, lactose and solid non-fat contents, rennet clotting time and major minerals (potassium, calcium, phosphorus and magnesium) of milk from Brown Swiss breed were examined. Phenotype and allele frequencies of β-Lg in both breeds were estimated and the association of the β-Lg genetic variants with the examined properties of Brown Swiss milk was studied. As a result of the study, β-Lg AA, AB and BB phenotypes were found in both Holstein Friesian and Brown Swiss breeds. The gene frequencies of β-Lg A and B were 0.27 and 0.73 in Holstein Friesian and 0.44 and 0.56 in Brown Swiss breeds, respectively. Therefore, β-Lg B allele gene was found more common in both breeds. In addition, significant differences were observed in β-Lg genotypes between two breeds. Also, significant relationships were found between β-Lg genetic variants and total solids (p<0.05), fat (p<0.01), calcium (p<0.05) and phosphorus (p<0.05), while no relationship was obtained for the other parameters studied in the milk of Brown Swiss breed.

Antigenic response of whey proteins and genetic variants of β-lactoglobulin — the effect of proteolysis and processing

Abstract Limited enzymatic hydrolysis trials of WPC-80 and β -lactoglobulin AA, AB and BB were performed using specific enzymes (trypsin, Neutrase, Corolase PP, Corolase PS) in a pH-stat under controlled conditions. The hydrolysates of WPC-80 and β -lactoglobulin were fractionated into high and low molecular fractions. Residual antibody binding activity of the peptides was dependent on the degree of hydrolysis (DH 2-20), but also on the enzyme used. Heat treatment affected the solubility and thereby the antigenic response. Dialysis influenced the antibody binding activity of the peptides. Tryptic hydrolysis of β -lactoglobulin AA was slower than for β -lactoglobulin BB and AB. Antigenic responses of the hydrolysates and fractions were measured by SLOT-BLOT and ELISA. SLOT-BLOT, a rapid screening method, was not able to differentiate the hydrolysates. The ELISA, being a more sensitive method, differentiated between the genetic variants, but was more time consuming. The lowest antigenicity was observed in the 1000–5000 Da fraction and β -lactoglobulin AA showed the lowest response.

Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A, B and C variants of β-lactoglobulin

Abstract The effect of fortification of reconstituted skim milk with different levels of a whey protein mixture containing a 1:2 ratio of α-lactalbumin (α-la) and different genetic variants of β-lactoglobulin (β-LG) on the rheological properties of acid milk gels, formed by acidification with glucono-δ-lactone, was investigated. Milk samples were either unheated or heated at 80°C for 30 min before acidification. Acid gels prepared from unheated skim milk had very low G′ values, long gelation times and low gelation pH. Samples prepared from heated milk had markedly higher G′ values, a reduced gelation time and an increased gelation pH. The addition of increasing levels of whey protein mixtures containing β-LG B or β-LG C to the milk prior to heating and acidification caused an almost linear increase in the G′. In contrast, whey protein mixtures containing β-LG A caused a progressive increase in the G′ with added protein levels up to about 0.7% (w/w) but little further change at higher addition levels. A mixture of the A and B variants of β-LG gave an intermediate behaviour between those of the A and B variants. In all samples, the G′ value at 5°C was approximately twice that at 30°C so that the relative differences as a result of the β-LG genetic variants were similar for the two temperatures.

The emulsifying properties of β-lactoglobulin genetic variants A , B and C

The emulsifying properties of three genetic variants of β-lactoglobulin ( β-lac) (the A, B and C variants) are investigated as a function of protein concentration. Differences in the emulsifying properties and emulsion stability are explained in view of the known differences in physico-chemical and structural/conformational properties of the β-lac variants. β-lac A forms the finest emulsion droplets, and β-lac C the largest droplets. The order of decreasing emulsifying ability (A>B>C) can be explained in terms of differences in the molecular structure, and conformational stability of the variant proteins. The creaming stability, when compared at the same particle size, is greatest for β-lac C, with β-lac A and B having a similar and lower stability. The differences in creaming stability may arise from a higher surface coverage for the β-lac C droplets at an equivalent particle size. The storage stability is lower for β-lac A than for β-lac B and C, which both show a similar behaviour. Storage stability differences are discussed in terms of differences in molecular structure, conformational stability, interfacial viscosity and surface coverage for the three variants.

Interactions in Heated Skim Milk between Genetic Variants of β-Lactoglobulin and κ-Casein

Bovine skim milk samples with different phenotypes of κ-casein (AB and BB; κ-cn) and β-lactoglobulin (AA, AB, and BB; β-lg) were heat-treated at 90 °C for different times (1-10 min). The residual native whey proteins in the ultracentrifugal supernatants were determined by fast performance liquid chromatography using a MonoQ HR5/5 column at pH 6.2, and the loss of native β-lg was recorded. The rate of heat-induced loss of native β-lg, expressed as the inverted half-life (1/t 1/2 ) of the reaction, was calculated. The reaction did not follow true first- or second-order kinetics, varying between the different genetic combinations. The highest 1/t 1/2 values were found in milk from cows homozygous for κ-cn B or β-lg B. The same was true after adjustment for differences in casein number. Both the β-lg and κ-cn genetic variants were found to significantly influence the heat-induced aggregation reaction; with β-lg having the greatest effect. Statistical analysis showed that the two loci for β-lg and κ-cn accounted for more than half of the phenotypic variance in the experimental groups.

Conformation of β-Lactoglobulin Studied by FTIR: Effect of pH, Temperature, and Adsorption to the Oil–Water Interface

The structure of mixed A and B genetic variants of β-lactoglobulin (β-lg) in its native and denatured states has been studied by FTIR. The denaturation was achieved through either heating at various temperatures at pH 6 and 7 or by adsorption to the surfaces of oil droplets in oil-in-water emulsions. The time dependence of the structural changes of the adsorbed protein was also studied. It was found that β-lg was more resistant to heat denaturation at pH 6 than at pH 7. Conversely, the adsorbed protein appeared to be more deformable at pH 6 than at pH 7. Although heating at lower temperatures (70 and 80°C) for a longer time causes extensive denaturation, as does heating at higher temperature (90°C) for a short time, the structure of the denatured protein was different in the two cases. When denatured at lower temperatures, more β-structure remained as indicated by the presence of bands around 1632 cm−1in the IR spectrum, whereas evidence for extensive intermolecular β-structure was found on the sample heated at higher temperature, indicated by the strong adsorption band at 1684 cm−1. This explains that although gelation of the protein can be achieved in both cases, the gel structure may be different. Denaturation of β-lg during heat treatment and upon adsorption appears to occur via similar intermediate structures, which begin with the loss of β-sheet structure, but whereas heat denaturation generates more intermolecular β-sheet and a small amount of unordered structure, adsorption of the protein to the oil–water interface induces a larger amount of unordered structure and a small amount of intermolecular β-sheet. The denaturation of β-lg on the interface is a much slower process compared to heat denaturation; even though some changes are detectable shortly after the adsorption of the protein, more extensive denaturation occurs during storage of the emulsions for 72 h.

Interactions between Different Genetic Variants of β-Lactoglobulin and κ-Casein A during Heating of Skim Milk

Milk samples (5 mL) from cows, homozygous for κ-casein A and either homozygous A (n = 14) or B (n = 12) or heterozygous AB (n = 14) for β-lactoglobulin (β-lg), were heated at 90 °C for 0-10 min, followed by ultracentrifugation at 100000g. The native whey proteins in the supernatant were thereafter analyzed by fast performance liquid chromatography (FPLC), and the decrease of whey proteins was recorded. The reaction orders for the heat-induced loss of β-lg were found to be 0.53, 0.65, and 0.91 for β-lg A, β-lg A+B, and β-lg B, respectively, in milk containing the A variant of κ-casein. The rate of heat-induced loss, expressed as the half-life (t 1/2 ) of the reaction, was found to be 340 and 270 s for β-lg A and B, respectively, in milk from homozygous cows. In milk from heterozygous cows (β-lg A+B), this value was 330 s. After correction for the casein number, the t 1/2 value for β-lg B was still notably lower than for β-lg A. The results, therefore, showed that the concentration of β-lg B decreased more rapidly than β-lg A in skim milk containing κ-casein A, probably due to differences in hydrophobicity between the genetic variants of β-lg.

The effects of β-lactoglobulin genetic variants A and B on the functional properties of whey under different conditions

Partially delactosated and demineralized whey protein powders were purified from pooled milks of individual cows homozygous for the β-lactoglobulin ( β-Lg) genetic variants A and B ( whey A and whey B). The protein composition of the two samples was similar. The two whey protein powders were dissolved in water at different pH (2, 4.5, 6.8) and NaCl concentrations (150 and 400 mmol/dm 3), and emulsions were made utilizing grape-seed oil. Emulsion stability (ES), emulsion activity index (EAI) and emulsion capacity (EC) were examined. The results showed the influence of the two genetic variants of β-Lg on the functional behaviour of whey. Emulsion obtained from B powder solutions showed the higher stability at the different test conditions. The best results occurred at pH 6.8 with no significant differences between the two tested NaCl concentrations. At pH2 and 400 mmol/dm 3 NaCl, both samples did not demonstrate any stability. Data on emulsion capacity indicated that whey B is less affected by the different physicochemical conditions tested (pH, NaCl concentration, heat treatment at 90°Cl5 min), while whey A gave the highest and the lowest EC values depending on the experimental conditions.

Chromatographic separation of whey proteins: Outinen, M., Harju, M., Tossavainen, O. and Antila, P. (Valio Oy, FIN-00100 Helsinki, Finland) PCT International Patent Application WO 95/ 19714

A perfusion reversed-phase (RP) HPLC method was developed for the rapid separation of the main bovine whey proteins: α-lactalbumin (α-LA), serum albumin (BSA) and the genetic variants of β-lactoglobulin (A and B) (β-LG A and β-LG B). For the method development, the influence of factors favouring structural changes of proteins (temperature and organic acid concentration in the mobile phase), gradient and other chromatographic conditions and the mass of protein injected was examined. The optimized method allowed the separation of proteins in about 1.5 min (cycle time 3.5 min) with resolution around 1.0 for the β-lactoglobulins. The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determinations was ⩽3.75 mg per 100 ml (S.D.). With respect to the accuracy, errors ⩽7.0% in the determination on α-LA, β-LG A and β-LG B were obtained, compared with an RP-HPLC reference method. However, higher errors in the quantification of BSA were found owing to the lack of purity of the peak assigned. In addition, the proposed method has proved to be very useful in the detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.

Aggregation of β-lactoglobulin studied by in situ light scattering

SummaryIn situ light scattering, where light scattered from a sample is measured directly while the sample is heated in the instrument, is presented as a simple and effective technique for studying the heat-induced aggregation of β-lactoglobulin. This technique was shown to be applicable not only to monitoring the initial aggregation steps, but to following the overall aggregation process with time. The experiments gave results similar to measurements carried out after a heat-quench treatment, but were more informative. From experiments on a standard NIZO β-lactoglobulin sample, a strongly desalted standard NIZO sample, different genetic variants of β-lactoglobulin and a mixture of these, we concluded that the standard NIZO sample was suitable for studying heat-induced aggregation. This sample has been investigated more extensively. Results with β-lactoglobulin (10–100 g/1) at 65 °C fitted a kinetic model for the denaturation and aggregation of β-lactoglobulin. This model, which held for β-lactoglobulin dissolved in water at near neutral pH and at 60–75 °C, recognizes an initiation, propagation and termination reaction, by analogy with polymer radical chemistry. It gave a quantitatively correct description of the dependence of the scattering intensity on the initial β-lactoglobulin concentration. Salt composition, pH and temperature strongly influenced the aggregation of β-lactoglobulin. Particle size increased with salt concentration in the range studied (up to 20 mM-NaCl and 1·0 mM-CaCl2). When the pH increased from 6°9 to 8·0 particle size was strongly reduced, whereas it strongly increased when pH was lowered to 6·2. Between 61·5 and 70 °C temperature did not affect particle size, whereas aggregation rate strongly increased. These effects could be incorporated in the kinetic model via the reaction constants of the reaction kinetic pathway.

Preparative membrane adsorber chromatography for the isolation of cow milk components

Preparative membrane adsorber (MA) chromatography was used to process milk fractions such as the whey and the permeate commonly obtained during lactose production in modern dairies. In MA systems the fluid-dynamic and mass-transfer properties are superior to conventional HPLC or fast protein liquid chromatography (FPLC) columns. Since the flow resistance caused by the MA stacks is quite low, high throughputs can be realized without loss in resolution. Feed sizes were varied from the laboratory scale (several ml) up to batches of 10 l during the investigations. MAs based on modified cellulose filtration membranes (average single layer thickness 200 μm, average pore size 5.0 μm) were used for the small-scale experiments. The MAs are functionalized by covalent linkage of strong and weak ion exchanger groups to their surface. Three commercially available types were used [strong ion exchanger: MA Q15 (3 layers of 5 cm 2) and MA Q100 (5 layers of 20 cm 2); weak ion exchanger: MA D15 (3 layers of 5 cm 2); all Sartorius, Germany]. For the large-scale work a dead-end filtration unit containing up to 1300 cm 2 of MA-area was used. Here MAs based on a synthetic co-polymer, that were prepared from cut-out sheets, were inserted. Chromatographic conditions were transferable from the cellulose- to the polymer-based MA carrying the same functional groups. The influence of the flow-rate and the pH of the mobile phase on the separation was investigated. The flow-rate could be raised to the limit of the respective chromatographic systems and/or MA modules without loss in resolution. The use of the strong anion exchangere MA together with a mobile phase pH of 6.0 and a fine-tuned gradient allowed the separation of BSA, α-lactalbumin and the genetic variants of β-lactoglobulin, even though no baseline separation was possible in the latter case. The use of coupled modules rather than a single one is shown to improve the separation considerably.

Perfusion liquid chromatography of whey proteins

A perfusion reversed-phase (RP) HPLC method was developed for the rapid separation of the main bovine whey proteins: α-lactalbumin (α-LA), serum albumin (BSA) and the genetic variants of β-lactoglobulin (A and B) (β-LG A and β-LG B). For the method development, the influence of factors favouring structural changes of proteins (temperature and organic acid concentration in the mobile phase), gradient and other chromatographic conditions and the mass of protein injected was examined. The optimized method allowed the separation of proteins in about 1.5 min (cycle time 3.5 min) with resolution around 1.0 for the β-lactoglobulins. The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determinations was ⩽3.75 mg per 100 ml (S.D.). With respect to the accuracy, errors ⩽7.0% in the determination on α-LA, β-LG A and β-LG B were obtained, compared with an RP-HPLC reference method. However, higher errors in the quantification of BSA were found owing to the lack of purity of the peak assigned. In addition, the proposed method has proved to be very useful in the detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.

Influence of κ-casein and β-lactoglobulin genetic variants on the heat stability of milk

SUMMARYHeat coagulation time-pH curves at 140°C were obtained for 43 blended skim milk samples from Holstein cows to determine the effects of genetic variants of κ-casein and β-lactoglobulin on milk heat stability. The blended milk samples were similar in terms of protein content and milk salts, but were genotypically different for κ-casein (AA, AB) and β-lactoglobulin (AA, AB, BB). Type A curves were obtained for all milks. Maximum heat stability was affected by the κ-casein genotype (AB &gt; AA, P &lt; 0·01) but the influence of the β-lactoglobulin genotype was only significant when the κ-casein AA genotype was present (β-lactoglobulin AA &gt; BB, P &lt; 0·0001). Minimum heat stability was significantly higher (P &lt; 0·0001) for milk genotyped κ-casein AB:β-lactoglobulin BB. The effects of milk genotyped κ-casein BB on maximum and minimum heat stability were determined by analysing individual milks: κ-casein BB:β-lactoglobulin AB (n=8) and reconstituted milks: κ-casein BB:β-lactoglobulin AA, AB and BB (n = 17). Type B curves were obtained on three occasions for individual κ-casein BB:β-lactoglobulin AB milk and on five occasions in the case of reconstituted milks with κ-casein BB:β-lactoglobulin AA, AB and BB. This suggests a relationship between the type B curve and the κ-casein B genetic variant. Comparison of the mean values of heat stability at the pH of maximum heat stability of each individual and reconstituted milk genotype suggested that the best genotype for κ-casein in terms of heat stability was BB.