Effects of the concentration of insoluble calcium phosphate associated with casein micelles on the functionality of directly acidified cheese

Directly acidified cheeses with different insoluble Ca (INS Ca) contents were made to test the hypothesis that the removal of INS Ca from casein micelles (CM) would directly contribute to the softening and flow behavior of cheese at high temperature. Skim milk was directly acidified with dilute lactic acid to pH values of 6.0, 5.8, 5.6, or 5.4 to remove INS Ca (pH trial). Lowering milk pH also reduced protein charge repulsion, which could influence melt. In a second treatment, EDTA (0, 2, 4, or 6 mM) was added to skim milk that was subsequently acidified to pH 6.0 (EDTA trial). Both types of milks were then made into directly acidified cheese. Cheese properties were determined at approximately 10 h after pressing to reduce possible confounding effects of proteolysis. The INS Ca content was determined by the acid-base titration method. Dynamic low-amplitude oscillatory rheology was used to measure the viscoelastic properties of cheese during heating from 5 to 80°C. The composition of all cheeses was as similar as possible, with cheese-making procedures being modified to obtain similar moisture contents (∼55%). Insoluble Ca contents of cheeses significantly decreased with a reduction in pH or with the addition of EDTA to skim milk. The pH values of cheeses in the pH trial varied, but all cheeses in the EDTA trial had similar pH values (∼5.73). In the pH trial, the reduction in cheese pH and consequent decrease in INS Ca content resulted in a reduction in the G′ values of cheeses at 20°C. In contrast, the G′ values at 20°C in cheeses from the EDTA trial increased with EDTA addition up to 4 mM EDTA. The G′ values at 70°C of cheeses from the pH trial decreased with a decrease in cheese pH, and a similar decrease was observed in the G′ values of cheese from the EDTA trial with an increase in EDTA concentration even though these cheeses had a similar pH value. In both trials, loss tangent (LT) values increased with temperatures >30°C and reached a maximum at approximately 70°C. In the pH trial, LT values at 70°C increased from 1.50 to 4.24 with a decrease in cheese pH from 5.78 to 5.21. The LT values increased from 1.43 to 3.23 with an increase in the concentration of added EDTA from 0 to 6 mM. In the EDTA trial, the decrease in G′ and increase in LT values at 70°C were due to the reduction in INS Ca content, because the pH values of these cheeses were the same. It can be concluded that the loss of INS Ca increases the melting in cheeses that have the same pH and gross chemical composition, and removal of INS Ca can even make cheese at high pH (∼5.73) exhibit reasonable melt characteristics.     

Measurement of ionic calcium, pH, and soluble divalent cations in milk at high temperature

Dialysis and ultrafiltration were investigated as methods for measuring pH and ionic calcium and partitioning of divalent cations of milk at high temperatures. It was found that ionic calcium, pH, and total soluble divalent cations decreased as temperature increased between 20 and 80°C in both dialysates and ultrafiltration permeates. Between 90 and 110°C, ionic calcium and pH in dialysates continued to decrease as temperature increased, and the relationship between ionic calcium and temperature was linear. The permeabilities of hydrogen and calcium ions through the dialysis tubing were not changed after the tubing was sterilized for 1 h at 120°C. There were no significant differences in pH and ionic calcium between dialysates from raw milk and those from a range of heat-treated milks. The effects of calcium chloride addition on pH and ionic calcium were measured in milk at 20°C and in dialysates collected at 110°C. Heat coagulation at 110°C occurred with addition of calcium chloride at 5.4 mM, where pH and ionic calcium of the dialysate were 6.00 and 0.43 mM, respectively. Corresponding values at 20°C were pH 6.66 and 2.10 mM.     

Effect of trisodium citrate on rheological and physical properties and microstructure of yogurt

The effect of trisodium citrate (TSC) on the rheological and physical properties and microstructure of yogurt was investigated. Reconstituted skim milk was heated at 85° C for 30 min, and various concentrations (5 to 40 mM) of TSC were added to the milk, which was then readjusted to pH 6.50. Milk was inoculated with 2% yogurt culture and incubated at 42° C until pH was 4.6. Acid-base titration was used to determine changes in the state of colloidal calcium phosphate (CCP) in milk. Total and soluble Ca contents of the milk were determined. The storage modulus (G′) and loss tangent (LT) values of yogurts were measured as a function of pH using dynamic oscillatory rheology. Large deformation rheological properties were also measured. Microstructure of yogurt was observed using confocal scanning laser microscopy, and whey separation was also determined. Addition of TSC reduced casein-bound Ca and increased the solubilization of CCP. The G′ value of gels significantly increased with addition of low levels of TSC, and highest G′ values were observed in samples with 10 to 20 mM TSC; higher ( > 20 mM) TSC concentrations resulted in a large decrease in G′ values. The LT of yogurts increased after gelation to attain a maximum at pH ∼5.1, but no maximum was observed in yogurts made with ≥ 25 mM of TSC because CCP was completely dissolved prior to gelation. Partial removal of CCP resulted in an increase in the LT value at pH 5.1. At low TSC levels, the removal of CCP crosslinks may have facilitated greater rearrangement and molecular mobility of the micelle structure, which may have helped to increase G′ and LT values of gels by increasing the formation of crosslinks between strands. At high TSC concentrations the micelles were completely disrupted and CCP crosslinks were dissolved, both of which resulted in very weak yogurt gels with large pores obvious in confocal micrographs. Gelation pH and yield stress significantly decreased with the use of high TSC levels. Lowest whey separation levels were observed in yogurt made with 20 mM TSC, and whey separation greatly increased at > 25 mM TSC. In conclusion, low concentrations of TSC improved several important yogurt characteristics, whereas the use of levels that disrupted casein micelles resulted in poor gel properties. We also conclude that the LT maximum observed in yogurts made from heated milk is due to the presence of CCP because the modification of the CCP content altered this peak and the removal of CCP eliminates this feature in the LT profiles.     

Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering

The objectives of this study were to investigate the effect of the solvent on the accuracy of casein micelle particle size determination by dynamic light scattering (DLS) at different temperatures and to establish a clear protocol for these measurements. Dynamic light scattering analyses were performed at 6, 20, and 50°C using a 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments, Holtsville, NY). Raw and pasteurized skim milk were used as sources of casein micelles. Simulated milk ultrafiltrate, ultrafiltered water, and permeate obtained by ultrafiltration of skim milk using a 10-kDa cutoff membrane were used as solvents. The pH, ionic concentration, refractive index, and viscosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a significant influence on the results of the particle size measurements. Experimental protocols were developed for accurate measurement of particle sizes in all solvents and experimental conditions. All measurements had good reproducibility, with coefficients of variation below 5%. Both the solvent and the temperature had a significant effect on the measured effective diameter of the casein micelles. When ultrafiltered permeate was used as a solvent, the particle size and polydispersity of casein micelles decreased as temperature increased. The effective diameter of casein micelles from raw skim milk diluted with ultrafiltered permeate was 176.4 ± 5.3 nm at 6°C, 177.4 ± 1.9 nm at 20°C, and 137.3 ± 2.7 nm at 50°C. This trend was justified by the increased strength of hydrophobic bonds with increasing temperature. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of casein micelles was casein-depleted ultrafiltered permeate. Dilution with water led to micelle dissociation, which significantly affected the DLS measurements, especially at 6 and 20°C. Simulated milk ultrafiltrate seemed to give accurate results only at 20°C. Results obtained in simulated milk ultrafiltrate at 6°C could not be explained based on the known effects of temperature on the casein micelle, whereas at 50°C, precipitation of amorphous calcium phosphate affected the DLS measurement.     

Casein micelle dissociation in skim milk during high-pressure treatment: Effects of pressure, pH, and temperature

The effect of pH (from 5.5 to 7.5) and temperature (from 5 to 40°C) on the turbidity of reconstituted skim milk powder was investigated at ambient pressure and in situ under pressure (up to 500 MPa) by measurement of light scattering. High-pressure treatment reduced the turbidity of milk for all combinations of pH and temperature due to micelle dissociation. The turbidity profiles had a characteristic sigmoidal shape in which almost no effect on turbidity was observed at low pressures (100 MPa), followed by a stronger pressure dependency over a pressure range of 150 MPa during which turbidity decreased extremely. From the turbidity profiles, the threshold pressure for disruption of micelle integrity was determined and ranged from 150 MPa at low pH to 350-400 MPa at high pH. The threshold pressure diagram clearly showed a relationship between the barostability of casein micelles and pH, whereas almost no effect of temperature was shown. This remarkable pH effect was a consequence of pressure-induced changes in the electrostatic interactions between colloidal calcium phosphate and the caseins responsible for maintaining micellar structure and was explained by a shift in the calcium phosphate balance in the micelle-serum system. Accordingly, a mechanism for high pressure-induced disruption of micelle integrity is suggested in which the state of calcium plays a crucial role in the micelle dissociation process.     

Effect of Tetrasodium Pyrophosphate on the Physicochemical Properties of Yogurt Gels

The effect of tetrasodium pyrophosphate (TSPP) on the properties of yogurt gels was investigated. Various concentrations (0.05 to 0.2%) of TSPP were added to preheated (85°C for 30 min) reconstituted skim milk, which was readjusted to pH 6.50. Milk was inoculated with 2% starter culture and incubated at 42°C until the pH reached 4.6. Acid-base buffering profiles of milk and total and soluble calcium levels were measured. Turbidity measurements were used to indicate changes in casein dispersion. Storage modulus (G′) and loss tangent (LT) values of yogurts were monitored during fermentation using dynamic oscillatory rheology. Large deformation properties of gels were also measured. Microstructural properties of yogurt were observed using fluorescence microscopy. The addition of TSPP resulted in the disappearance of the buffering peak during acid titration at pH ∼5.1 that is due to the solubilization of colloidal calcium phosphate (CCP), and a new peak was observed at lower pH values (pH 4.0-4.5). The buffering peak at pH 6.0 during base titration virtually disappeared with addition of TSPP and a new peak appeared at pH ∼4.8. The addition of TSPP reduced the soluble Ca content of milk and increased casein-bound Ca values. The addition of up to 0.125% TSPP resulted in a reduction in turbidity because of micelle dispersion but at 0.15%, turbidity increased and these samples exhibited a time-dependent increase in turbidity because of aggregation of casein particles. Gels made with 0.20% TSPP were very weak and had a very high gelation pH (6.35), probably due to complete dispersion of the micelle structure in this sample. The LT value of gels at pH 5.1 decreased with an increase in TSPP concentration, probably due to the loss of CCP with the addition of TSPP. The G′ values at pH 4.6 of gels made with ≤0.10% TSPP were not significantly different but the addition of ≥0.125% TSPP significantly decreased G′ values. The addition of 0.05 to 0.125% TSPP to milk resulted in a reduction in the yield stress values of yogurt compared with yogurt made without TSPP. Greater TSPP levels (>0.125%) markedly reduced the yield stress values of yogurt. Lowest whey separation levels were observed in yogurts made with 0.10% TSPP. High TSPP levels (>0.10%) greatly increased the apparent pore size of gels. Addition of very low levels of TSPP to milk for yogurt manufacture may be useful in reducing the whey separation defect, but at TSPP concentrations ≥0.125% very weak gels were formed.     

Manufacture of acid gels from skim milk using high-pressure homogenization

The effect of high-pressure homogenization (HPH) alone or in combination with a thermal treatment (TT) was investigated for the manufacture of acid gels from skim milk. Raw skim milk was subjected to HPH (0 to 350 MPa) or a TT (90°C, 5 min), or both, in the following processing combinations: 1) HPH, 2) HPH followed by TT, 3) TT followed by HPH, 4) TT, and 5) raw milk (control). After treatments, L* (lightness) values were measured, and then skim milk was acidified with 3% glucono-δ-lactone and rheological properties (G′ and gelation time), and whey holding capacity was evaluated. Treatments in which HPH and TT were combined showed greater L* values than those in which just HPH was applied. In all treatments, the L* values decreased as the pressure was increased up to 300 MPa with little change afterward. Gelation times were lower when HPH was combined with TT compared with the acid skim milk gels that were just pressure treated. The final G′ in gels obtained from skim milk subjected to the combined process (HPH and TT) was greater and pressure-dependent compared with all other gels. A maximum G′ (∼320 Pa) was observed with skim milk subjected to a combination of thermal processing before or after HPH at 350 MPa. Acid gels obtained from HPH milk at 350 MPa showed a linear decrease in whey holding capacity over time, retaining 20% more whey after centrifugation for 25 min compared with samples treated at lower pressures and all other treatments. Our results suggest that HPH in combination with TT can be used to improve the rheological properties and stability of yogurt, thus decreasing the need for additives.     

Temperature-dependent dynamics of bovine casein micelles in the range 10–40 °C

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40 °C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10–40 °C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40 min of measurement, returning to equilibrium after 16 h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10–40 °C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

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.     

Dynamics of casein micelles in skim milk during and after high pressure treatment

The effect of high hydrostatic pressure on turbidity of skim milk was measured in situ together with casein micelle size distribution. High pressure (HP) treatment reduced the turbidity of milk with a stronger pressure dependency between 50 and 300 MPa when the temperature was decreased from 20 to 5 °C, while at 30 °C (50–150 MPa) turbidity exceeded that of untreated milk. At 250 and 300 MPa turbidity decreased extremely. During pressurization of milk at 250 and 300 MPa, the turbidity initially decreased, but treatments longer than 10 min increased the turbidity progressively, indicating that re-association followed dissociation of casein micelles. Especially at 40 °C and at 250 and 300 MPa, the turbidity increased beyond untreated milk. Dynamic light scattering was used to investigate casein micelle sizes in milk immediately after long time (up to 4 h) pressurization at 250 and 300 MPa and casein micelle size distributions were bimodal with micelle sizes markedly smaller and markedly larger than those of untreated milk. Pressure modified casein micelles present after treatment of milk at 250 and 300 MPa were concluded to be highly unstable, since the larger micelles induced by pressure showed marked changes toward smaller particle sizes in milk left at ambient pressure.     

Effects of milk supplementation with skim milk powder, whey protein concentrate and sodium caseinate on acidification kinetics, rheological properties and structure of nonfat stirred yogurt

Milk supplementation with milk proteins in four different levels was used to investigate the effect on acidification and textural properties of yogurt. Commercial skim milk powder was diluted in distilled water, and the supplements were added to give different enriched-milk bases; these were heat treated at 90 °C for 5 min. These mixtures were incubated with the bacterial cultures for fermentation in a water bath, at 42 °C, until pH 4.50 was reached. Chemical changes during fermentation were followed by measuring the pH. Protein concentration measurements, microbial counts of Lactobacillus bulgaricus and Streptococcus thermophilus, and textural properties (G′, G″, yield stress and firmness) were determined after 24 h of storage at 4 °C. Yogurt made with milk supplemented with sodium caseinate resulted in significant properties changes, which were decrease in fermentation time, and increase in yield stress, storage modulus, and firmness, with increases in supplement level. Microstructure also differed from that of yogurt produced with milk supplemented with skim milk powder or sodium caseinate.     

The effect of ultrasound on casein micelle integrity

Samples of fresh skim milk, reconstituted micellar casein, and casein powder were sonicated at 20 kHz to investigate the effect of ultrasonication. For fresh skim milk, the average size of the remaining fat globules was reduced by approximately 10 nm after 60 min of sonication; however, the size of the casein micelles was determined to be unchanged. A small increase in soluble whey protein and a corresponding decrease in viscosity also occurred within the first few minutes of sonication, which could be attributed to the breakup of casein-whey protein aggregates. No measurable changes in free casein content could be detected in ultracentrifuged skim milk samples sonicated for up to 60 min. A small, temporary decrease in pH resulted from sonication; however, no measurable change in soluble calcium concentration was observed. Therefore, casein micelles in fresh skim milk were stable during the exposure to ultrasonication. Similar results were obtained for reconstituted micellar casein, whereas larger viscosity changes were observed as whey protein content was increased. Controlled application of ultrasound can be usefully applied to reverse process-induced protein aggregation without affecting the native state of casein micelles.     

A method for the large-scale isolation of β-casein

A method for the large-scale isolation of β-casein from renneted skim milk was developed. The curd from renneted skim milk was dispersed in hot (?70 °C) water to inactivate residual chymosin. The heated curd was subsequently recovered by centrifugation, resuspended in water and incubated at 5 °C, during which β-casein dissociated from the curd; the suspension was centrifuged and the aqueous phase lyophilised. The isolated protein consisted mainly of β-casein, containing a minor amount of γ-caseins and traces of other caseins. Unless chymosin was fully inactivated by heating, some β-casein was hydrolysed at the Leu₁₉₂–Tyr₁₉₃ bond. The yield of β-casein increased with incubation time, up to ∼20% of the β-casein present in the milk after 24 h at 5 °C. Reducing milk pH to 5.5 or 6.0, prior to renneting, caused a high level of contamination with α_s-caseins. This isolation procedure can be easily scaled-up to an industrial process and the β-casein-depleted curd may be used for the manufacture of rennet casein or processed cheese.     

Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6 s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log₁₀ BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91 ± 0.05 log₁₀ BA spores/mL of milk and the 1.4-μm membrane removed 4.50 ± 0.35 log₁₀ BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log₁₀ spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log₁₀ BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization.     

Effects of stabiliser addition and in-container sterilisation on selected properties of milk related to casein micelle stability

Different stabilising salts and calcium chloride were added to raw milk to evaluate changes in pH, ionic calcium, ethanol stability, casein micelle size and zeta potential. These milk samples were then sterilised at 121 °C for 15 min and stored for 6 months to determine how these properties changed. Addition of tri-sodium citrate (TSC) and di-sodium hydrogen phosphate (DSHP) to milk reduced ionic calcium, increased pH and increased ethanol stability in a concentration-dependent fashion. There was relatively little change in casein micelle size and a slight decrease in zeta potential. Sodium hexametaphosphate (SHMP) also reduced ionic calcium considerably, but its effect on pH was less noticeable. In contrast, sodium dihydrogen phosphate (SDHP) reduced pH but had little effect on ionic calcium. In-container sterilisation of these samples reduced pH, increased ethanol stability and increased casein micelle size, but had variable effects on ionic calcium; for DSHP and SDHP, ionic calcium decreased after sterilisation but, for SHMP, it remained little changed or increased. Milk containing 3.2 mM SHMP and more than 4.5 mM CaCl₂ coagulated upon sterilisation. All other samples were stable but there were differences in browning, which increased in intensity as milk pH increased. Heat-induced sediment was not directly related to ionic calcium concentration, so reducing ionic calcium was not the only consideration in terms of improving heat stability. After 6 months of storage, the most acceptable product, in appearance, was that containing SDHP, as this minimised browning during sterilisation and further development of browning during storage.     

Process for calcium retention during skim milk ultrafiltration

Lactose is separated from milk or other fluid dairy products for a variety of reasons. Ultrafiltration is a known process of removing lactose from these products. However, during ultrafiltration, valuable minerals, such as calcium in soluble form, are also lost into permeate. In this study, a process was developed in which first the lactose reduction in skim milk was achieved by ultrafiltration (4x volumetric concentration) using a 10-kDa membrane. Then, the calcium present in permeate was precipitated using one of three methods: 1) heat treatment, 2) pH adjustment, or 3) a combination of pH adjustment and heat treatment to permeate, then recovered by refiltering permeate. The process was first developed at laboratory scale, and then its applicability was tested at the pilot scale. Skim milk, retentates, permeates, and the treated permeates were analyzed for total solids, ash, protein, or total nitrogen, calcium, and lactose content. About 76% of the total lactose and about 16% of the calcium present in skim milk permeated through the membrane during ultrafiltration. The three treatments applied produced white precipitates and turned the clear permeates turbid. On refiltering the treated permeates approximately 42, approximately 50, and approximately 70% of the total calcium present could be recovered from 1) heat-treated, 2) pH-adjusted, and 3) pH-adjusted and heat-treated permeates, respectively. There was no marked change in the lactose content due to any of the three treatments and subsequent refiltering of the treated permeates.     

Effect of milk solids concentration on the gels formed by the acidification of heated pH-adjusted skim milk

Reconstituted skim milk of 10–25% total solids was adjusted to pH values between about 6.2 and 7.1 and heated at 80 °C for 30 min. Gels were formed from the heated milks by slow acidification to pH 4.2 and the gelation process and final gels were analyzed for their rheological properties. At each milk concentration, the final acid gel firmness (final G′) and breaking stress could be changed markedly by manipulation of the pH during heating. The final gel firmness and breaking stress could also be modified by changing the concentration of the milk solids prior to heating and acidification. The results indicated that similar gel firmness and breaking stress could be achieved over a range of milk concentrations by control of the pH of the milk during heating. When expressed as a percentage change in final G′ or breaking stress relative to that obtained at the natural pH, plots of the change in final G′ or breaking stress versus pH fell close to a single curve, indicating that the same mechanism may influence the gelation properties at all milk concentrations. The final G′ and breaking stress were related to the denaturation and interaction of the whey proteins with the casein micelles, and the formation of non-sedimentable casein when the milk was heated.     

Efficiency of serum protein removal from skim milk with ceramic and polymeric membranes at 50°C

Raw milk (2,710 kg) was separated at 4°C, the skim milk was pasteurized (72°C, 16 s), split into 3 batches, and microfiltered using pilot-scale ceramic uniform transmembrane pressure (UTP; Membralox model EP1940GL0.1μA, 0.1 μm alumina, Pall Corp., East Hills, NY), ceramic graded permeability (GP; Membralox model EP1940GL0.1μAGP1020, 0.1 μm alumina, Pall Corp.), and polymeric spiral-wound (SW; model FG7838-OS0x-S, 0.3 μm polyvinylidene fluoride, Parker-Hannifin, Process Advanced Filtration Division, Tell City, IN) membranes. There were differences in flux among ceramic UTP, ceramic GP, and polymeric SW microfiltration membranes (54.08, 71.79, and 16.21 kg/m² per hour, respectively) when processing skim milk at 50°C in a continuous bleed-and-feed 3× process. These differences in flux among the membranes would influence the amount of membrane surface area required to process a given volume of milk in a given time. Further work is needed to determine if these differences in flux are maintained over longer processing times. The true protein contents of the microfiltration permeates from UTP and GP membranes were higher than from SW membranes (0.57, 0.56, and 0.38%, respectively). Sodium-dodecyl-sulfate-PAGE gels for permeates revealed a higher casein proportion in GP and SW permeate than in UTP permeate, with the highest passage of casein through the GP membrane under the operational conditions used in this study. The slight cloudiness of the permeates produced using the GP and SW systems may have been due to the presence of a small amount of casein, which may present an obstacle in their use in applications when clarity is an important functional characteristic. More β-lactoglobulin passed through the ceramic membranes than through the polymeric membrane. The efficiency of removal of serum proteins in a continuous bleed-and-feed 3× process at 50°C was 64.40% for UTP, 61.04% for GP, and 38.62% for SW microfiltration membranes. The SW polymeric membranes had a much higher rejection of serum proteins than did the ceramic membranes, consistent with the sodium-dodecyl-sulfate PAGE data. Multiple stages and diafiltration would be required to produce a 60 to 65% serum protein reduced micellar casein concentrate with SW membranes, whereas only one stage would be needed for the ceramic membranes used in this study.     

Cryo-transmission electron tomography of native casein micelles from bovine milk

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (∼20 to 30 nm in diameter), channels (diameter greater than ∼5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed.