Solubilization Of Colloidal Calcium Phosphate Research Articles

Impact of preacidification on the functionality and insoluble calcium levels of Low-Moisture Part-Skim Mozzarella made from high-casein milk

The use of low-concentration factor ultrafiltered (LCF-UF) milk for cheesemaking has become popular in recent years. Research on using LCF-UF milk to make low-moisture part-skim (LMPS) Mozzarella manufacture is limited due to concerns about the negative impact of the higher casein content on functional properties, such as melt and stretch. Pre-acidification (PA) (reflects acid added to milk before the addition of the starter culture) has been used in low-fat and nonfat Mozzarella cheeses to lower calcium levels and improve their texture and melting properties. We explored the use of PA to reduce the insoluble calcium content of cheese (calcium that is a crosslinking material for the casein proteins) and its impact on the functionality of LMPS Mozzarella made from high casein (4.2%) milk. Seven vats of LMPS Mozzarella, with a control (no PA), 2 pH values for PA (6.40 and 6.00) and 3 different acidification methods (use of acetic, citric, or carbonic acid from carbon dioxide) were evaluated for their impact on solubilization of colloidal calcium phosphate during LMPS manufacture. Composition, proteolysis, microstructural and functional changes in the LMPS cheeses were monitored during 90 d of refrigerated storage. All cheeses had similar composition except for the cheese made with milk PA with carbonic acid, which had a lower fat-in-dry matter probably due to foaming observed in this sample during the gas sparging. The cheese made with milk PA with citric acid to pH 6.00 had the lowest amount of total and insoluble calcium compared with other cheeses at all steps during cheese manufacture and storage, likely due to the calcium chelating ability of citric acid. The cheese that was made with milk PA with citric acid to pH 6.40 had a trend of slightly lower (not significant) total and insoluble calcium during cheese manufacture compared with other cheeses that were made with milk PA to pH 6.40. The pH value in all the cheeses remained similar throughout 90 d of storage, whereas the cheese that was made with milk PA with citric acid to pH 6.00 had the lowest pH values during storage. Treatment significantly (P < 0.05) impacted instrumental hardness, the maximum loss tangent during heating, the melting temperature and the storage modulus values measured at 70°C. Proteolysis was not impacted by treatment suggesting that typical aging should occur. Cheese that was PA with citric acid to pH 6.00 initially had lower maximum loss tangent values and higher hardness, possibly due to the lower pH value of this cheese. The performance of cheeses on pizza was assessed using Sensory Spectrum® method and quantitative descriptive analysis. The PA of cheese milk with citric acid helped to reduce the insoluble Ca level and at 30 and 90 d of storage, these cheeses had lower first chew hardness, lower chewiness, and lower strand thickness values compared with other cheeses. The use of PA could help improve the functionality of LMPS Mozzarella made from milk high in casein with citric acid being the most efficient in dissolving the insoluble calcium from casein.

Calcium balance during direct acidification of milk for Mozzarella cheese production

Calcium is an important nutrient but also contributes to the texture, taste and functionality of most dairy products. The balance between micellar and free calcium in curd and serum phase during direct acidification of bovine milk was studied by changing organic acid type, pH and acidification temperature. Concentration of free calcium increased with decreasing pH and increasing temperature. Citric acid was more efficient than lactic acid at lowering free calcium concentration, while temperature effect was larger for acidification with lactic acid. These findings are rationalized by the stronger binding of calcium to citrate compared to lactate with an almost athermal binding for citrate and an endothermal binding for lactate. Curd yield decreased by decreasing pH as solubilization of colloidal calcium phosphate causes loss of minerals into the serum phase decreasing the concentration of both calcium and phosphate left in the curd, while acidification temperature had little effect on curd yield. These results are important to improve yield and tailor the properties and functionality of dairy products, such as for example Mozzarella cheese.

Short communication: Serum composition of milk subjected to re-equilibration by dialysis at different temperatures, after pH adjustments

The objective of this work was to investigate the properties of casein micelles after pH adjustment and their re-equilibration to the original pH and serum composition. Re-equilibration was carried out by dialyzing against skim milk at 2 different temperatures (4 or 22°C). Turbidity, the average radius of the casein micelles, and the composition of the soluble phase were measured at different pH values, ranging between 5.5 and 8. Acidification led to the solubilization of colloidal calcium phosphate and decrease of the average radius of the micelles. With re-equilibration, casein dissociation occurred. In milk with pH values greater than 6.0, the average radius was recovered after re-equilibration. At pH values greater than neutral, an increase of the radius of casein micelles and increased dissociation of the casein were found. After re-equilibration, the radius of micelles and soluble protein in the serum decreased. The results were not affected by the temperature of re-equilibration. The changes to the calcium phosphate equilibrium and the dissociation of the micelles will have important consequences to the functionality of casein micelles.

Changes in the physical properties, solubility, and heat stability of milk protein concentrates prepared from partially acidified milk

A limiting factor in using milk protein concentrates (MPC) as a high-quality protein source for different food applications is their poor reconstitutability. Solubilization of colloidal calcium phosphate (CCP) from casein micelles during membrane filtration (e.g., through acidification) may affect the structural organization of these protein particles and consequently the rehydration and functional properties of the resulting MPC powder. The main objective of this study was to investigate the effects of acidification of milk by glucono-δ-lactone (GDL) before ultrafiltration (UF) on the composition, physical properties, solubility, and thermal stability (after reconstitution) of MPC powders. The MPC samples were manufactured in duplicate, either by UF (65% protein, MPC65) or by UF followed by diafiltration (80% protein, MPC80), using pasteurized skim milk, at either the native milk pH (~pH 6.6) or at pH 6.0 after addition of GDL, followed by spray drying. Samples of different treatments were reconstituted at 5% (wt/wt) protein to compare their solubility and thermal stability. Powders were tested in duplicate for basic composition, calcium content, reconstitutability, particle size, particle density, and microstructure. Acidification of milk did not have any significant effect on the proximate composition, particle size, particle density, or surface morphology of the MPC powders; however, the total calcium content of MPC80 decreased significantly with acidification (from 1.84±0.03 to 1.59±0.03g/100g of powder). Calcium-depleted MPC80 powders were also more soluble than the control powders. Diafiltered dispersions were significantly less heat stable (at 120°C) than UF samples when dissolved at 5% solids. The present work contributes to a better understanding of the differences in MPC commonly observed during processing.

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 30min) 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.

Chymosin-Mediated Proteolysis, Calcium Solubilization, and Texture Development During the Ripening of Cheddar Cheese

Full fat, milled-curd Cheddar cheeses (2kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0μmol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8°C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0μmol/L of pepstatin showed complete inhibition of hydrolysis of αS1-casein (CN) at Phe23-Phe24 at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact αS1-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of αS1-CN at Phe23-Phe24 is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.

Structure and Physical Properties of Yogurt Gels: Effect of Inoculation Rate and Incubation Temperature

The effects of inoculation rates and incubation temperatures on the physical properties and microstructure of yogurt gels were investigated. A 2-factor experimental design with 3 replicates was used for data analysis. Yogurt gels were made with 0.5, 1, 2, 3, or 4% (wt/wt) inoculation rates and incubated at 40 or ∼46°C. Dynamic low amplitude oscillatory rheology was performed to monitor the formation of yogurt gels. Gel permeability and the amount of surface whey were determined. Gel structure was examined by confocal scanning laser microscopy. Higher storage modulus values were observed in yogurt gels made at higher inoculation rates and lower incubation temperatures. Gels made at higher inoculation rates and incubation temperatures exhibited higher yield stress and maximum loss tangent values, respectively. Permeability, pore size, and whey separation of yogurt gels increased with decreased inoculation rate and increased incubation temperature. An increase in the inoculation rate resulted in a decrease in the pH where the maximum in the loss tangent occurs, presumably reflecting less efficient solubilization of colloidal calcium phosphate (which is a slow process) and the need to attain a lower pH to complete the solubilization. Significant positive correlations were observed between whey separation and the value of maximum in loss tangent (r = 0.94) and permeability (r = 0.89). Whey separation was negatively correlated with storage modulus (r = −0.48). It was concluded that rearrangements of casein particles in the gel network and the rate at which the solubilization of colloidal calcium phosphate occurred were important driving forces for whey separation and weak gel.

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.

Effects of Acidification and Storage of Milk on Dissociation of Bovine Casein Micelles

The effects of acidification and storage of skim-milk on the rate and extent of solubilization of colloidal calcium phosphate and dissociation of caseins from the micelles were examined. As the pH was reduced, calcium phosphate was progressively removed from the micelles, and after each pH adjustment there was an initial rapid increase in serum concentrations of Ca and inorganic phosphate, but only a slight further increase on storage. The extent of disssociation of the caseins from the micelles on acidification was temperature-dependent. At 30 °C, concentrations of serum casein remained low, but at 20 °C, and especially at 4 °C, the levels of all caseins increased in the serum as the pH was reduced to about pH 5.5 and 5.2, respectively. After each pH adjustment, there was an initial rapid increase in the serum concentrations of the caseins and a more gradual increase on storage up to 24 h. Keywords: Calcium phosphate; casein; dissociation; pH

Distribution of Iron Between Caseins and Whey Proteins in Acidified Milk

The association of iron with casein and whey proteins was studied by fortifying pasteurized skim milk with 100 mg of iron (Fe3+)/L and adjusting the pH to 6.7, 6.2, 5.8, 5.3, 4.5, and 4.0. Samples were fractionated by ultracentrifugation, and the pellets and serum collected and analyzed for iron, calcium, and phosphorus contents, and for protein profile. At pH 6.7, 6.2, 5.8, and 5.3, some casein, particularly β-casein was not sedimented. More iron was sedimented at pH≤4. However, expressed on a protein basis, the pellets had lower iron contents, probably because denatured whey proteins have lower iron-binding affinities. As pH decreased, the calcium and phosphorus dissociated from casein micelles, with less solubilization of colloidal calcium phosphate occurring in the iron-fortified samples at pH 6.2, 5.8 and 5.3. In yogurt fortified with casein-chelated iron, iron remained bound to casein and was distributed within the micelles. Yogurt fortified with whey protein-chelated iron showed iron being distributed throughout the nonmicellar portion of the yogurt. Fortification with FeCl3resulted in iron being preferentially located within the casein micelles.

Effect of acidification and neutralization of milk on some physico-chemical properties of casein micelles

Abstract Acidification of milk, at low temperatures, to pH 5.0 or 4.6, followed by neutralization to pH 6.6 (reformed milk), resulted in a reduction in the buffering maximum of milk at pH ~5.1; this buffering peak is caused by the solubilization of colloidal calcium phosphate (CCP). The reduced buffering in reformed milk suggests that little reformation of CCP occurs on neutralization; reformed milks also had an elevated Ca 2+ activity. Acidification of milk to pH > 5.5, followed by neutralization to pH 6.6, hardly reduced buffering (at pH ~5.1), suggesting that either little CCP dissolved on acidification or that reformation of CCP occurred on neutralization. Acidification of milk to low pH values and neutralization resulted in improved renneting properties and a reduction in rennet coagulation time (RCT). Dialysis of reformed milk resulted in a reduction in its renneting properties which became inferior to those of control milk, possibly due to its reduced CCP content or to structural changes in the micelles caused by removal of CCP. Addition of low concentrations of CaCl 2 to milk, at a constant pH (6.6), improved its renneting properties. Electron micrographs of milk acidified to pH values ⩽5.5 prior to neutralization showed increased clustering of casein particles, presumably caused by the reduction in electrostatic repulsion between casein particles during acidification to low pH values. The original micellar appearance was not restored on neutralization or dialysis of reformed milk. It is concluded that the micellar system is not readily reversible; once disintegrated by acidification, micelles do not reform on neutralization.

Effects of heat treatment and acidification on the dissociation of bovine casein micelles

SummaryThe effects of heat treatment and subsequent acidification of milk on the distribution of proteins, Ca and Pi, between the serum and micellar phases were examined using ultracentrifugation. After heating milk at 85 °C for 10 min, and storing for 22 h at 4, 20 or 30 °C, there was a marked increase in the concentration of κ-casein in the serum. At 4 and 20 °C there was also slightly more β-casein in the serum from heat-treated milk than in that from the corresponding raw milk. The whey proteins were extensively denatured, and were almost equally distributed between the supernatants and micellar pellets. After storage for 22 h the distribution of Ca and Pibetween soluble and colloidal phases in heat-treated milk was similar to that in raw milk. After acidifying heat-treated milk by the addition of glucono-δ-lactone and storing for 22 h at 4, 20 or 30 °C there was progressive solubilization of colloidal calcium phosphate with decreasing pH, and at pH 5·0 almost all of the Ca and Piwas present in the serum. At 20 °C, and even more so at 4 °C, serum concentrations of the individual caseins increased considerably with decreasing pH, reaching maximum levels of about 25 and 40% of the total casein at pH 5·7 and 5·5 respectively, and then decreasing rapidly at lower pH. Compared with raw milk, maximum dissociation in heat-treated milks stored at 4 and 20 °C occurred at higher pH, and the overall levels of dissociation of individual caseins from the micelles were lower. At 30 °C, the concentrations of individual caseins in the serum of heat-treated milk decreased steadily as the pH was reduced, and did not show the slight increase found previously for raw milk. The role of the denatured whey proteins in interacting with κ-casein and in promoting aggregation of the micelles on acidification is discussed.