Effect of storage at 4 degrees C on the physicochemical and renneting properties of milk: a comparison of caprine, ovine and bovine milks

The effects of cold storage at 4 degrees C for 12, 24 and 48 h on the physicochemical characteristics and renneting properties of ewes', goats' and cows' milks were compared. The most important changes were observed in cows' milk. Soluble calcium concentrations were not affected in ewes' milk but were increased by 10% in cows' milk and 7% in goats' milk. More casein was dissociated on cooling cows' (+300%) than goats' (+100%) milk, and there was no change in soluble casein in ewes' milk. The coagulation characteristics of cows' milk were more impaired by cold storage than goats' or ewes' milks. Coagulation times increased by approximately 30% and whey draining capacity decreased by 40% after cows' milk was cooled, but there were no changes with ewes' milk and only a slight decrease in coagulation time with goats' milk. We propose an interpretation of these results based on the physicochemical properties of each type of milk.     

Structures and some properties of soluble protein complexes formed by the heating of reconstituted skim milk powder

The effects of heat on reconstituted skim milk powder (RSMP) at a temperature of 90 °C have been determined, with respect to the formation of soluble protein complexes. Heating at the natural pH of the RSMP forms soluble complexes in the milk serum, which can be separated by size exclusion chromatography. The diameters of the particles were found to be up to 50 nm, as shown by light scattering and by scanning electron microscopy. The formation of the soluble complexes was strongly affected by the treatment of the RSMP before heating. Treatment of the milk with small amounts of glutaraldehyde, to fix the casein micellar structure, severely inhibited the formation of the soluble complexes. The pH at which the milk was heated (in the range 6.5–7.2) also had an effect on the amounts and the sizes of the complexes, as determined by size exclusion chromatography. The amount of soluble complex present in the RSMP affected the strength of the acid gels produced from the different milks, as was shown by exchanging the sera of milks treated differently with glutaraldehyde, and by following the gelation of milks heated at different pH values. Increased amounts of soluble complexes gave stronger acid gels. However, the relationship appeared to break down if milk was heated at pH 7.2, where the soluble material appeared to change and the acid gel was much weaker.     

Changes in the surface protein of the fat globules during homogenization and heat treatment of concentrated milk

The changes in milk fat globules and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure, were examined. The average fat globule size decreased with increasing homogenization pressure. The total surface protein (mg m-2) of concentrated milk increased after homogenization, the extent of the increase being dependent on the temperature and the pressure of homogenization, as well as on the preheat treatment. The concentrates obtained from high-preheated milks had higher surface protein concentration than the concentrates obtained from low-preheated milks after homogenization. Concentrated milks heat treated at 79 degrees C either before or after homogenization had greater amounts of fat globule surface protein than concentrated milks heat treated at 50 or 65 degrees C. This was attributed to the association of whey protein with the native MFGM (milk fat globule membrane) proteins and the adsorbed skim milk proteins. Also, at the same homogenization temperature and pressure, the amount of whey protein on the fat globule surface of the concentrated milk that was heated after homogenization was greater than that of the concentrated milk that was heated before homogenization. The amounts of the major native MFGM proteins did not change during homogenization, indicating that the skim milk proteins did not displace the native MFGM proteins but adsorbed on to the newly formed surface.     

Rheological properties of milk gels formed by a combination of rennet and glucono-delta-lactone

The effects of heat treatment of milk, and a range of rennet and glucono-delta-lactone (GDL) concentrations on the rheological properties, at small and large deformation, of milk gels were investigated. Gels were made from reconstituted skim milk at 30 degrees C, with two levels each of rennet and GDL. Together with controls this gave a total of sixteen gelation conditions, eight for unheated and eight for heated milk. Acid gels made from unheated milks had low storage moduli (G') of < 20 Pa. Heating milks at 80 degrees C for 30 min resulted in a large increase in the G' value of acid gels. Rennet-induced gels made from unheated milk had G' values in the range approximately 80-190 Pa. However, heat treatment severely impaired rennet coagulation: no gel was formed at low rennet levels and only a very weak gel was formed at high levels. In gels made with a combination of rennet and GDL unusual rheological behaviour was observed. After gelation, G' initially increased rapidly but then remained steady or even decreased, and at long ageing times G' values increased moderately or remained low. The loss tangent (tan delta) of acid gels made from heated milk increased after gelation to attain a maximum at pH approximately 5.1 but no maximum was observed in gels made from unheated milk. Gels made by a combination of rennet and GDL also exhibited a maximum in tan delta, indicating increased relaxation behaviour of the protein-protein bonds. We suggest that this maximum in tan delta was caused by a loosening of the intermolecular forces in casein particles caused by solubilization of colloidal calcium phosphate. We also suggest that in combination gels made from unheated milk a low value for the fracture stress and a high tan delta during gelation indicated an increased susceptibility of the network to excessive large scale rearrangements. In contrast. combination gels made from heated milk formed firmer gels crosslinked by denatured whey proteins and underwent fewer large scale rearrangements.     

Modifications in milk proteins induced by heat treatment and homogenization and their influence on susceptibility to proteolysis

The influence of the sequence of processing steps on protein integrity and susceptibility to proteolysis of skimmed and whole milks was examined. Laboratory-scale devices were used to homogenize and heat treat the milk. The results were confirmed in skimmed and whole milks processed in a direct ultrahigh temperature (UHT) pilot plant, with and without a homogenization step following heat treatment. The effects of homogenization varied depending on whether it preceded or followed heating. In general terms, homogenization slightly enhanced whey protein denaturation. But, although homogenization increased the concentration of micellar β-lactoglobulin (β-Lg) in whole milks, it led to high levels of soluble denatured β-Lg in skimmed milks. Homogenization after heating had a promoting effect towards proteolysis in skimmed milk, with κ-casein suffering the highest degradation. This was probably because these samples had high levels of soluble β-Lg and κ-casein that could have enhanced the susceptibility to proteolysis. Whole milk samples presented the lowest proteolysis levels, probably because of an increased complex formation between κ-casein and β-Lg.     

Effects of continuous flow microwave treatment on chemical and microbiological characteristics of milk

Raw cows' and goats' milks were heated by microwave in a continuous flow unit up to temperatures ranging from 73.1 to 96.7°C. The effects of the heat treatments were estimated by measurements of lactose isomerization, protein denaturation, inactivation of alkaline phosphatase and peroxidase and the total bacterial count. Negative phosphatase tests and low bacterial counts, together with low degrees of whey protein denaturation, were achieved under several temperature/time combinations. The results indicate that continuous microwave processing may be an efficient and mild approach for the pasteurization of milk.     

Significance of frictional heating for effects of high pressure homogenisation on milk

High pressure homogenisation (HPH) is a novel dairy processing tool, which has many effects on enzymes, microbes, fat globules and proteins in milk. The effects of HPH on milk are due to a combination of shear forces and frictional heating of the milk during processing; the relative importance of these different factors is unclear, and was the focus of this study. The effect of milk inlet temperature (in the range 10-50 degrees C) on residual plasmin, alkaline phosphatase, lactoperoxidase and lipase activities in raw whole bovine milk homogenised at 200 MPa was investigated. HPH caused significant heating of the milk; outlet temperature increased in a linear fashion (0.5887 degrees C/ degrees C, R2=0.9994) with increasing inlet temperature. As milk was held for 20 s at the final temperature before cooling, samples of the same milk were heated isothermally in glass capillary tubes for the same time/temperature combinations. Inactivation profiles of alkaline phosphatase in milk were similar for isothermal heating or HPH, indicating that loss of enzyme activity was due to heating alone. Loss of plasmin and lactoperoxidase activity in HPH milk, however, was greater than that in heated milk. Large differences in residual lipase activities in milks subjected to heating or HPH were observed due to the significant increase in lipase activity in homogenised milk. Denaturation of beta-lactoglobulin was more extensive following HPH than the equivalent heat treatment. Inactivation of plasmin was correlated with increasing fat/serum interfacial area but was not correlated with denaturation of beta-lactoglobulin. Thus, while some effects of HPH on milk are due to thermal effects alone, many are induced by the combination of forces and heating to which the milk is exposed during HPH.     

Comparison of heat and pressure treatments of skim milk, fortified with whey protein concentrate, for set yogurt preparation: effects on milk proteins and gel structure

Heat (85 degrees C for 20 min) and pressure (600 MPa for 15 min) treatments were applied to skim milk fortified by addition of whey protein concentrate. Both treatments caused > 90 % denaturation of beta-lactoglobulin. During heat treatment this denaturation took place in the presence of intact casein micelles; during pressure treatment it occurred while the micelles were in a highly dissociated state. As a result micelle structure and the distribution of beta-lactoglobulin were different in the two milks. Electron microscopy and immunolabelling techniques were used to examine the milks after processing and during their transition to yogurt gels. The disruption of micelles by high pressure caused a significant change in the appearance of the milk which was quantified by measurement of the colour values L*, a* and b*. Heat treatment also affected these characteristics. Casein micelles are dynamic structures, influenced by changes to their environment. This was clearly demonstrated by the transition from the clusters of small irregularly shaped micelle fragments present in cold pressure-treated milk to round, separate and compact micelles formed on warming the milk to 43 degrees C. The effect of this transition was observed as significant changes in the colour indicators. During yogurt gel formation, further changes in micelle structure, occurring in both pressure and heat-treated samples, resulted in a convergence of colour values. However, the microstructure of the gels and their rheological properties were very different. Pressure-treated milk yogurt had a much higher storage modulus but yielded more readily to large deformation than the heated milk yogurt. These changes in micelle structure during processing and yogurt preparation are discussed in terms of a recently published micelle model.     

Heat-induced and other chemical changes in commercial UHT milks

The properties of commercial directly and indirectly heated UHT milks, both after heating and during storage at room temperature for 24 weeks, were studied. Thermally induced changes were examined by changes in lactulose, furosine and acid-soluble whey proteins. The results confirmed previous reports that directly heated UHT milks suffer less heat damage than indirectly heated milk. During storage, furosine increased and bovine serum albumin in directly heat-treated milks decreased significantly. The changes in lactulose, alpha-lactalbumin and beta-lactoglobulin were not statistically significant. The data suggest that heat treatment indicators should be measured as soon as possible after processing to avoid any misinterpretations of the intensity of the heat treatment.     

Effect of increasing the colloidal calcium phosphate of milk on the texture and microstructure of yogurt

The effect of increasing the colloidal calcium phosphate (CCP) content on the physical, rheological, and microstructural properties of yogurt was investigated. The CCP content of heated (85°C for 30 min) milk was increased by increasing the pH by the addition of alkali (NaOH). Alkalized milk was dialyzed against pasteurized skim milk at approximately 4°C for 72 h to attempt to restore the original pH and soluble Ca content. By adjustment of the milk to pH values 7.45, 8.84, 10.06, and 10.73, the CCP content was increased to approximately 107, 116, 123, and 128%, respectively, relative to the concentration in heated milk. During fermentation of milk, the storage modulus (G′) and loss tangent values of yogurts were measured using dynamic oscillatory rheology. Large deformation rheological properties were also measured. The microstructure of yogurt was observed using fluorescence microscopy, and whey separation was determined. Acid-base titration was used to evaluate changes in the CCP content in milk. Total Ca and casein-bound Ca increased with an increase in the pH value of alkalization. During acidification, elevated buffering occurred in milk between pH values 6.7 to 5.2 with an increase in the pH of alkalization. When acidified milk was titrated with alkali, elevated buffering occurred in milk between pH values 5.6 to 6.4 with an increase in the pH of alkalization. The high residual pH of milk after dialysis could be responsible for the decreased contents of soluble Ca in these milks. The pH of gelation was higher in all dialyzed samples compared with the heated control milk, and the gelation pH was higher with an increase in CCP content. The sample with highest CCP content (128%) exhibited gelation at very high pH (6.3), which could be due to alkali-induced CN micellar disruption. The G′ values at pH 4.6 were similar in gels with CCP levels up to 116%; at higher CCP levels, the G′ values at pH 4.6 greatly decreased. Loss tangent values at pH 5.1 were similar in all samples except in gels with a CCP level of 128%. For dialyzed milk, the whey separation levels were similar in gels made from milk with up to 107% CCP but increased at higher CCP levels. Microstructure of yogurt gels made from milk with 100 to 107% CCP was similar but very large clusters were observed in gels made from milk with higher CCP levels. By dialyzing heated milk against pasteurized milk, we may have retained some heat-induced Ca phosphate on micelles that normally dissolves on cooling because, during dialysis, pasteurized milk provided soluble Ca ions to the heated milk system. Yogurt texture was significantly affected by increasing the casein-bound Ca (and total Ca) content of milk as well as by the alkalization procedure involved in that approach.     

Changes in Size and Composition of Protein Aggregates on Heating Reconstituted Concentrated Skim Milk at 120oC

Treatment at 120^oC caused initial transfer of protein from the serum and small micelle fraction to the medium-sized micelle fraction. More extensive heating caused transfer of protein from the medium fraction to the large-sized micelle fraction, and the serum fraction as 'soluble’casein. Protein composition of large-sized micelle fraction remained essentially constant, but for the other fractions it changed throughout the heating period. Experiments with heated milks that had been made free of colloidal calcium phosphate (CCP) showed that a proportion of the calcium phosphate in milk, whether indigenous or precipitated by heating, became less soluble. This‘insoluble’calcium phosphate was possibly involved in joining the smaller protein particles into larger-sized aggregates.     

On heating milk, the dissociation of kappa-casein from the casein micelles can precede interactions with the denatured whey proteins

When kappa-casein (kappa-CN) was added to milk, and the milk was subsequently pH adjusted (pH 6.5-6.9) and heated (90 degrees C/15 min), the serum contained considerably higher levels of denatured whey proteins than the milks without added kappa-CN. When milk at pH 6.5-6.9 was heated at 90 degrees C for different times, kappa-CN was found in the serum in the early stages of heating and before significant levels of whey proteins were denatured. kappa-CN reached its maximum level in the serum before the whey proteins were fully denatured. When milk at pH 6.5-6.9 was heated at 20-90 degrees C for 15 min, kappa-CN dissociated from the casein micelles at all temperatures, with the level in the serum increasing with the temperature and the pH at heating. kappa-CN dissociated from the micelles at temperatures below those at which significant levels of the whey proteins were denatured. When taken together, the results from these experiments strongly indicate that the dissociation of kappa-CN from the micelles can precede the interaction of the denatured whey proteins with kappa-CN, and that there is a preferential interaction of the denatured whey proteins with serum-phase kappa-CN.     

Diffusing-wave spectroscopy investigation of heated reconstituted skim milks containing calcium chloride

The effect of heat treatment on reconstituted 10 wt% skim milks containing up to 20 mM added CaCl₂ at different pH values (pH 6.0–7.2), was investigated both in situ and after cooling of the heat treated milks. Measurements of pH in situ showed that pH decreased at high temperature, that the decrease in pH increases with the increase in the initial pH and that the magnitude of the decrease in pH was greater for milks with added CaCl₂. Marked increases in the viscosity at 25 °C of heated milks indicated that milks without added CaCl₂ with initial pH ≤6.2 and milks with 10 mM added CaCl₂ with initial pH ≤6.4, heated at 90 °C were not heat stable. At a given heating temperature, it was possible to superimpose the measured viscosity of samples with or without added CaCl₂ on the same curve when these were plotted as a function of the pH at that temperature instead of the initial milk temperature. To further demonstrate this finding a DWS experimental set-up was built and in situ measurements were performed on the milk samples heated at 75 °C for 10 min. The DWS measurements showed that ηa, the product of the viscosity with particle size, can also be superimposed for all the measured samples, when plotted as a function of the pH at the heating temperatures. Both the viscosity and DWS data demonstrate the importance of the pH at the heating-temperatures in influencing heat-induced changes in milk.     

Pressurisation of raw skim milk and of a dispersion of phosphocaseinate at 9 degrees C or 20 degrees C: effects on the distribution of minerals and proteins between colloidal and soluble phases

The effects of high pressure treatments (100-300 MPa; 15 min; 9 degrees C or 20 degrees C) on the distribution of minerals and proteins of raw skim milk (RSM) and of a dispersion of industrial phosphocaseinate (PC) were studied after separation of the micellar and soluble phases by ultracentrifugation (UCF). Whatever the temperature of high pressure treatments, the pressure-induced dissociation of the casein micelles was accompanied by calcium (Ca), phosphorus (P) and casein release from the micelles. The released Ca and P were or became bound to soluble proteins since progressive increases in Ca and P concentrations were observed in the UCF supernatants of RSM and of the PC dispersion but not in the ultrafiltrates from these UCF supernatants (free of soluble proteins). Simultaneously, alpha(S1-), alpha(S2-), beta- and kappa-caseins were progressively released from the micelles, as seen by electrophoretic analysis. The pressure-induced solubilisation of alpha(S1-) and alpha(S2-)caseins, essentially located in the core of the micelles, suggests that high pressure de-stabilized micelles including their internal structure.     

Plasmin activity in pressurized milk

The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activity of milk were investigated by means of plasmin activity, plasmin-derived activity after plasminogen activation and their distribution in different milk fractions, micelle microstructure, beta-LG denaturation, and casein susceptibility to proteolytic attack. The pressure conditions assayed did not lead to plasmin inactivation and only decreased around 20 to 30% total plasmin activity after plasminogen activation. However, pressure caused severe disruption of the micellar structure, releasing high levels of caseins, plasmin, and plasminogen to the soluble fraction of milk. High levels of soluble denatured beta-LG were also found in the ultracentrifugation supernatants of pressurized milks, particularly in those treated at 400 MPa. Probably as a result of micellar disintegration, caseins became more susceptible to proteolysis by exogenous plasmin. However, no enhanced proteolytic degradation was observed when we compared the evolution of pressurized and unpressurized milks during refrigerated storage. Serum-liberated plasmin may become more vulnerable to the action of proteinase inhibitors leading to a reduced proteolysis on refrigerated storage, despite the increased susceptibility of caseins to proteinase action.     

Influence of denaturation and aggregation of β-lactoglobulin on rennet coagulation properties of skim milk and ultrafiltered milk

Skim milk and ultrafiltered milk were heated at temperatures in the range 80–140 °C for 4s in a UHT plate heat exchanger. The extent of denaturation of β-lactoglobulin and its association with the casein micelles increased with increase in heating temperature; the extent of association was markedly less than the amount that denatured. There was no noticeable difference in the extent of denaturation and association of β-lactoglobulin between skim milk and the corresponding ultrafiltered milk. The coagulation properties of renneted heated milks were measured by dynamic oscillation rheometry. Gelation times and storage modulus (G′) could be related to the extent of denaturation of β-lactoglobulin and its association with the casein micelles. Denaturation of β-lactoglobulin up to 60% had no effect on the gelation time, but additional denaturation markedly increased the gelation times. In contrast, G' decreased gradually with increase in the extent of denaturation of β-lactoglobulin and its association with casein micelles. The effects of β-lactoglobulin denaturation and association on coagulation properties were less pronounced in ultrafiltered milk. G' values for ultrafiltered milk were markedly higher than the corresponding skim milks under all heating conditions.     

Rheological properties of concentrated skim milk: importance of soluble minerals in the changes in viscosity during storage

Properties of condensed milks prior to spray drying dictate to a large extent the functionality of the resulting milk powder. Rheological properties of concentrated skim milk, with total solids content of 45% but different mineral content, were studied as a function of shear rate and storage time at 50 degrees C. These milks are proposed as a model to study the effects of minerals on rheology and age gellation of condensed milk prior to drying. During storage of the concentrated milk, the apparent viscosity, particularly after 4 h, increased markedly at all shear rates studied. The yield stress also increased steeply after 4 h of storage at 50 degrees C. The changes in apparent viscosity of concentrated milk stored for up to 4 h were largely reversible under high shear, but irreversible in samples stored for longer time. The appearance of yield stress suggested the presence of reversible flocculation arising from weak attraction between casein micelles, with a transition from reversible to irreversible aggregation during storage. Particle size analysis confirmed irreversible aggregation and fusion of casein micelles during storage. Gradual reduction of mineral content of concentrated milks resulted in a marked decrease in the apparent viscosity and casein micelle aggregation during storage, while addition of minerals to milk had the opposite effect. The results demonstrated that the soluble mineral content is very important in controlling the storage-induced changes in the rheology of concentrated milks.     

Heat resistance of Bacillus cereus spores: effects of milk constituents and stabilizing additives.

Heat resistance of Bacillus cereus spores (ATCC 7004, 4342, and 9818) heated in different types of milk (skim, whole, and concentrated skim milk), skim milk containing stabilizing additives (sodium citrate, monopotassium phosphate, or disodium phosphate, 0.1%), and cream was investigated. Thermal resistance experiments were performed at temperatures within the range of 92 to 115 degrees C under continuous monitoring of pH. For strain 4342 no significant differences (P < 0.05) in D values were detected in any case. For strains 7004 and 9818 higher D values of about 20% were obtained in whole and concentrated skim milk than those calculated in skim milk. From all stabilizing additives tested, only sodium citrate and sodium phosphate increased the heat resistance for strain 9818. However, when the menstruum pH was measured at the treatment temperature, different pH values were found between the heating media. The differences in heat resistance observed could be due to a pH effect rather than to the difference in the substrates in which spores were heated. In contrast, when cream (fat content 20%) was used, lower D values were obtained, especially for strains 7004 and 9818. z values were not significantly modified by the milk composition, with an average z value of 7.95+/-0.20 degrees C for strain 7004, 7.88+/-0.10 degrees C for strain 4342, and 9.13+/-0.16 degrees C for strain 9818.     

The use of the chromogenic Limulus amoebocyte lysate assay to distinguish between ultra high temperature and in-container sterilized milks

The chromogenic Limulus amoebocyte lysate (LAL) assay may be used to distinguish between ultra high temperature (UHT) and in-container sterilized (ICS) milks, when applied to samples which have received an additional heat treatment of 100°C for 10 minutes. This heat treatment resulted in a significant increase (p < 0.001) in the availability of lipopolysaccharides in ICS milks which did not occur in UHT milk samples. This method for distinguishing between these milk types is, however, limited to samples whose sera require extensive dilution prior to testing. This possibly reflects the calcium levels present.     

Effects of pH,calcium-complexing agents and milk solids concentration on formation of soluble protein aggregates in heated reconstituted skim milk

The proportion of protein present in the supernatant after centrifugation and the formation of soluble protein aggregates in heated (90 °C for 10 min) reconstituted skim milk were investigated as a function of (a) pH, (b) milk concentration 9–21%, w/w (milk solids non-fat, MSNF) and (c) addition of calcium-complexing agents (orthophosphate or EDTA). Compositional changes in milk, resulting from pH adjustment or salt addition, altered the distribution of caseins between the serum and micellar phases of milk prior to heating, and influenced the amount and composition of soluble aggregates formed during heat treatment. Although milk pH was a good predictor of the aggregation behaviour of the proteins during heating of milk of different solids concentrations, neither the proportion of protein in the supernatant prior to heating nor the pH alone could predict the aggregation behaviour when the composition was adjusted with mineral salts.