Thom Huppertz

Effects of high pressure on constituents and properties of milk

High pressure (HP) treatment has significant and, in many cases, unique effects on many constituents of milk. The structure of casein micelles is disrupted and the whey proteins, alpha-lactalbumin and beta-lactoglobulin, are denatured, with the former being more resistant to pressure than the latter. Pressure-induced shifts in the mineral balance in milk also occur and moderately high pressures (100-400 MPa) induce the crystallisation of milk fat. However, milk enzymes seem to be quite resistant to pressure. As a result of pressure-induced effects on individual milk constituents, many properties of milk are affected. HP treatment increases the pH of milk, reduces its turbidity, changes its appearance, and can reduce the rennet coagulation time of milk and increase cheese yield, thereby indicating potential applications in cheese technology. However, to fully understand the effects of HP treatment on milk and to evaluate the full potential of this process in dairy technology, further research is required in several areas, including the reversibility of pressure-induced changes in milk and the physical stability of HP-treated milk

High pressure treatment of bovine milk: effects on casein micelles and whey proteins

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.

High pressure-induced denaturation of α-lactalbumin and β-lactoglobulin in bovine milk and whey: a possible mechanism

In this study, high pressure (HP)-induced denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in dairy systems was examined. In both milk and whey, beta-lg was less baroresistant than alpha-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of alpha-la and beta-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KlO3, to milk or whey increased HP-induced denaturation of beta-lg, but reduced the denaturation of alpha-la. Denaturation of both alpha-la and beta-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of alpha-la and beta-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of alpha-la and beta-lg significantly. The higher level of HP-induced denaturation of alpha-la and beta-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of alpha-la and beta-lg in milk.

Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk

Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of β-lactoglobulin, whereas β-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of κ-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine κ-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.

Casein Micelles: Size Distribution in Milks from Individual Cows

The size distribution and protein composition of casein micelles in the milk of Holstein-Friesian cows was determined as a function of stage and number of lactations. Protein composition did not vary significantly between the milks of different cows or as a function of lactation stage. Differences in the size and polydispersity of the casein micelles were observed between the milks of different cows, but not as a function of stage of milking or stage of lactation and not even over successive lactations periods. Modal radii varied from 55 to 70 nm, whereas hydrodynamic radii at a scattering angle of 73° (Q² = 350 μm⁻²) varied from 77 to 115 nm and polydispersity varied from 0.27 to 0.41, in a log-normal distribution. Casein micelle size in the milks of individual cows was not correlated with age, milk production, or lactation stage of the cows or fat or protein content of the milk.

Heat and ethanol stabilities of high-pressure-treated bovine milk

Despite having well-documented effects on several constituents of milk, high pressure (HP) had little overall effect on the heat stability of raw, pre-heated or serum protein-free skim milk at pH values in the range 6.1-7.0. HP treatment at 600 MPa considerably increased the heat stability of concentrated skim milk at pH values > 6.7. The ethanol stability of raw skim milk was reduced by HP treatment, shifting the somewhat sigmoidal ethanol stability versus pH profile to more alkaline values; however, these changes were partially reversible during subsequent storage at 5degreesC for 24 h. Ethanol-mediated temperature-induced dissociation of casein micelles occurred in both untreated and HP-treated milk and was not dependent on storage of milk subsequent to HP treatment. These findings provide further insight into how the HP-induced changes in the constituents of milk influence milk properties

High pressure-induced changes in the creaming properties of bovine milk

Abstract Creaming of raw whole bovine milk at refrigeration temperatures is generally regarded as an undesirable phenomenon; traditionally, creaming is prevented by homogenising the milk. The effects of high pressure (HP) processing on the creaming of raw whole bovine milk were examined in this study. HP treatment at pressures ≤250 MPa increased the rate and level of creaming, whereas treatment at ≥400 MPa reduced both of these parameters. On treatment at 200 or 600 MPa, creaming increased or decreased, respectively, with increasing treatment time. At 400 MPa, HP-induced changes in the rate and level of creaming were strongly treatment time-dependent. Treatment at 100–600 MPa for 0–60 min had little effect on milk fat globule size, whereas the viscosity of skimmed milk increased with increasing pressure and treatment time. The amount of milk protein associated with the milk fat globules was increased by HP treatment, the extent of the increase being maximal at 200 MPa. Although increased viscosity and the level of protein associated with the fat globules may partially explain the reduced rate and level of creaming, HP-induced aggregation and denaturation of agglutinins and lipoproteins are likely to have significant effects on HP-induced changes in the creaming characteristics of milk.

Disruption and reassociation of casein micelles during high pressure treatment: influence of whey proteins.

In the study presented in this article, the influence of added alpha-lactalbumin and beta-lactoglobulin on the changes that occur in casein micelles at 250 and 300 MPa were investigated by in-situ measurement of light transmission. Light transmission of a serum protein-free casein micelle suspension initially increased with increasing treatment time, indicating disruption of micelles, but prolonged holding of micelles at high pressure partially reversed HP-induced increases in light transmission, suggesting reformation of micellar particles of colloidal dimensions. The presence of alpha-la and/or beta-lg did not influence the rate and extent of micellar disruption and the rate and extent of reformation of casein particles. These data indicate that reformation of casein particles during prolonged HP treatment occurs as a result of a solvent-mediated association of the micellar fragments. During the final stages of reformation, kappa-casein, with or without denatured whey proteins attached, associates on the surface of the reformed particle to provide steric stabilisation.

Effects of high pressure on some constituents and properties of buffalo milk

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100-600MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by approximately 35%. Levels of non-micellar alpha(S1)and beta-caseins were increased by treatment > or = 250MPa, and were highest after treatment at 400-800MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from approximately 82 for untreated milk to approximately 65 for milk treated at 800 MPa. Milk pH was increased by approximately 0.07 units after treatment at 100-800 MPa, with no significant difference between treatment pressures. Denaturation of alpha-lactalbumin occurred at pressures > or = 400 MPa, and reached >90% after treatment at 800 MPa, whereas beta-lactoglobulin (beta-Ig) was denatured > 100 MPa, reaching approximately 100% after treatment at 400MPa; after treatment > or = 400MPa, all beta-Ig was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250-800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.

Coacervates of Lactotransferrin and β- or κ-Casein: Structure Determined Using SAXS

Lactotransferrin (LF) is a large globular protein in milk with immune-regulatory and bactericidal properties. At pH 6.5, LF (M = 78 kDa) carries a net (calculated) charge of +21. β-Casein (BCN) and κ-casein (KCN) are part of the casein micelle complex in milk. Both BCN and KCN are amphiphillic proteins with a molar mass of 24 and 19 kDa and carry net charges of -14 and -4, respectively. Both BCN and KCN form soap-like micelles, with 40 and 65 monomers, respectively. The net negative charges are located in the corona of the micelles. On mixing LF with the caseins, coacervates are formed. We analyzed the structure of these coarcervates using SAXS. It was found that LF binds to the corona of the micellar structures, at the charge neutrality point. BCN/LF and KCN/LF ratios at the charge neutrality point were found to be ~1.2 and ~5, respectively. We think that the findings are relevant for the protection mechanism of globular proteins in bodily fluids where unstructured proteins are abundant (saliva). The complexes will prevent docking of enzymes on specific charged groups on the globular protein.