Effects of different calcium salts on properties of milk related to heat stability

Insoluble calcium salts were added to milk to increase total calcium by 30 mM, without changing properties influencing heat stability, such as pH and ionic calcium. There were no major signs of instability associated with coagulation, sediment formation or fouling when subjected to ultra high temperature (UHT) and in‐container sterilisation. The buffering capacity was also unaltered. On the other hand, addition of soluble calcium salts reduced pH, increased ionic calcium and caused coagulation to occur. Calcium chloride showed the largest destabilising effect, followed by calcium lactate and calcium gluconate. Milk became unstable to UHT processing at lower calcium additions compared to in‐container sterilisation.     

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.     

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.     

Feasibility of using dialysis for determining calcium ion concentration and pH in calcium-fortified soymilk at high temperature

Abstract: Dialysis was performed to examine some of the properties of the soluble phase of calcium (Ca) fortified soymilk at high temperatures. Dialysates were obtained while heating soymilk at temperatures of 80 and 100 °C for 1 h and 121 °C for 15 min. It was found that the pH, total Ca, and ionic Ca of dialysates obtained at high temperature were all lower than in their corresponding nonheated Ca‐fortified soymilk. Increasing temperature from 80 to 100 °C hardly affected Ca ion concentration ([Ca²⁺]) of dialysate obtained from Ca chloride‐fortified soymilk, but it increased [Ca²⁺] in dialysates of Ca gluconate‐fortified soymilk and Ca lactate‐fortified soymilk fortified with 5 to 6 mM Ca. Dialysates obtained at 100 °C had lower pH than dialysate prepared at 80 °C. Higher Ca additions to soymilk caused a significant (P≤ 0.05) reduction in pH and an increase in [Ca²⁺] of these dialysates. When soymilk was dialyzed at 121 °C, pH, total Ca, and ionic Ca were further reduced. Freezing point depression (FPD) of dialysates increased as temperature increased but were lower than corresponding soymilk samples. This approach provides a means of estimating pH and ionic Ca in soymilks at high temperatures, in order to better understand their combined role on soymilk coagulation.     

羊奶酪蛋白热稳定性的研究

以莎能和关中羊奶为原料,通过从羊奶中提取酪蛋白,分别在不同的pH、温度以及添加不同浓度的Ca^2＋、柠檬酸钠、三聚磷酸钠、干酪素的条件下测定酪蛋白的热凝固时间（HCT）,研究其对羊奶酪蛋白热稳定性的影响。结果表明,pH在6．8时酪蛋白的热稳定性最好,高温会降低酪蛋白的热稳定性,钙离子可以降低羊奶酪蛋白的热稳定性,适量的柠檬酸钠或三聚磷酸钠可以有效提高羊奶酪蛋白的热稳定性,干酪素对酪蛋白稳定性影响不明显。     

Bovine milk composition parameters affecting the ethanol stability

The objective of the present work was to identify the compositional parameters of raw milk that affected ethanol stability at natural pH when natural milk conditions were not modified. Heat stability, measured as coagulation time (CT), was included in the analysis to verify relation to alcohol test. Statistical models were proposed for alcohol and heat (CT) stabilities. Milk samples of good hygienic quality from dairy farms were classified in two groups according to their alcohol stability. Unstable samples to ethanol (72%, v/v) presented lower values of pH, somatic cells count, casein and non-fat-solids relative to ethanol stable samples (ethanol at 78%, v/v or more); whereas freezing point, chloride, sodium and potassium concentrations were higher in the unstable group. Logistic regression and multiple regression were applied to modelling alcohol and heat stability behaviour respectively. Chloride, potassium, ionic calcium and somatic cell count were included in the alcohol regression model, whereas calcium, phosphorous, urea, pH and ionic calcium were part of CT model. Ionic calcium was the only measured variable that contributed to both models; however coagulation time was noted to be more sensitive to ionic calcium than alcohol. The relation between ionic strength and casein was found to contribute to the alcohol model but not to the CT model. However, the interaction calcium plus magnesium plus phosphorous and casein contributed only to CT model.     

Comparison of heat stability of goat milk subjected to ultra-high temperature and in-container sterilization

Goat milk with and without stabilizing salt was subjected to in-container and UHT sterilization. Heat stability was assessed by measuring the amount of sediment in the milk. Without stabilizing salts, goat milk usually produced less sediment when subjected to in-container sterilization compared with UHT processing. Addition of stabilizing salts up to 12.8mM resulted in a progressive increase in sediment for in-container sterilization. In contrast, adding stabilizing salts at 6.4mM initially reduced sediment formation in UHT-treated milk but addition of stabilizing salts at 12.8mM increased sediment formation. Adding stabilizing salts to goat milk increased pH, decreased ionic calcium, and increased ethanol stability. Adding up to 2mM calcium chloride increased sediment formation more after UHT treatment than after in-container sterilization. These results suggest that no single mechanism or set of reactions causes milk to produce sediment during heating and that the favored pathway is different for UHT and in-container sterilization processes. Poor heat stability could be induced both by increasing ionic calcium and by decreasing it. Ethanol stability is not a good indicator of heat stability for in-container sterilization, but it may be for UHT sterilization, if milk does not enter the region of poor heat stability found at low concentrations of ionic calcium.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

Solubility and heat stability of whey protein concentrates.

Rennet whey protein concentrates have excellent nutritional properties, but their use in fluid food systems is impaired by the poor heat stability of the protein. Heating whey protein concentrated solutions at neutral pH caused up to 70% losses in solubility. In the absence of added calcium, protein coagulation occurred. near the iso-electric zone whereas in the presence of .03 M calcium chloride, similar protein coagulation occurred in the whole pH range (pH 2 to pH 12). Tryptic hydrolysis of the protein increased the heat stability of whey protein concentrates considerably.     

Effect of soluble calcium on the renneting properties of casein micelles as measured by rheology and diffusing wave spectroscopy

Addition of calcium chloride to milk has positive effects on cheese-making because it decreases coagulation time, creates firmer gels, and increases curd yield. Although addition of calcium chloride is a widely used industrial practice, the effect of soluble calcium on the preliminary stages of gelation is not fully understood. In addition, it is not known whether the manner of addition and equilibration of the soluble calcium would affect the rennetability of the casein micelles. Therefore, the aim of this paper was to study the details of the coagulation behavior of casein micelles in the presence of additional calcium, and to elucidate whether the manner in which this cation is added (directly as calcium chloride or by gradual exchange through dialysis) affects the functionality of the micelles. Calcium was added as CaCl(2) (1 mM final added concentration) directly to skim milk or indirectly using dialysis against 50 volumes of milk. Additional soluble calcium did not affect the primary phase of the renneting reaction, as demonstrated by the analysis of the casein macropeptide (CMP) released in solution; however, it shortened the coagulation time of the micelles and increased the firmness of the gel. The turbidity parameter of samples with or without calcium showed that similar amounts of CMP were needed for particle interactions to commence. However, the amount of CMP released at the point of gelation, as indicated by rheology, was lesser for samples with added calcium, which can be attributed to a greater extent of calcium bridging on the surface or between micelles. The results also showed that the manner in which calcium was presented to the micelles did not influence the mechanism of gelation.     

Reformation of casein particles from alkaline-disrupted casein micelles

In this study, the properties of casein particles reformed from alkaline disrupted casein micelles were studied. For this purpose, micelles were disrupted completely by increasing milk pH to 10.0, and subsequently reformed by decreasing milk pH to 6.6. Reformed casein particles were smaller than native micelles and had a slightly lower zeta-potential. Levels of ionic and serum calcium, as well as rennet coagulation time did not differ between milk containing native micelles or reformed casein particles. Ethanol stability and heat stability, >pH 7.0, were lower for reformed casein particles than native micelles. Differences in heat stability, ethanol stability and zeta-potential can be explained in terms of the influence of increased concentrations of sodium and chloride ions in milk containing reformed casein particles. Hence, these results indicate that, if performed in a controlled manner, casein particles with properties closely similar to those of native micelles can be reformed from alkaline disrupted casein micelles.     

Measurement of ionic calcium in milk

There are many reports in the literature regarding the effects of ionic calcium on reactions related to casein micelle stability, such as heat stability, ethanol stability and susceptibility to gelation, sediment formation and fouling. However, experimental evidence supporting these assertions is much less readily available.
This paper evaluates three selective ion electrode systems for measuring ionic calcium directly in milk as well as looking at the effects on pH reduction and addition of calcium chloride.
The best electrode system was the Ciba Corning 634 system, which was designed for blood but has been modified for milk. This was found to be reproducible and stable when calibrated daily and allowed direct measurements to be taken on milk in 70 s. This has been found to perform well now for 3 years. The other systems were not so useful, as they took longer to stabilize, but may be useful for higher ionic calcium concentrations, which are found in acidified milk products.
Reducing the pH increased ionic calcium and reduced ethanol stability. Calcium chloride addition reduced pH, increased ionic calcium and reduced the ethanol stability. Readjusting the pH to its value before calcium addition reduced the ionic calcium, but not back to its original value. Milks from individual cows showed wide variations in their ionic calcium concentrations.
This establishes the methodology for a more detailed investigation on measurement of ionic calcium in milks from individual cows and from bulk milks, to allow a better understanding of its role in casein micelle stability.     

Effect of NaCl on some physico-chemical properties of concentrated bovine milk

The influence of added NaCl (75–850 mmol L⁻¹) on some physicochemical properties of 2× or 3× concentrated milk (18 or 27%, w/v, total solids, respectively) was investigated. Adding NaCl did not influence average casein micelle size, but reduced the net-negative charge on the casein micelles and milk pH. The level of soluble and ionic calcium was increased by addition of NaCl, but the level of soluble inorganic phosphorus was not influenced. Addition of NaCl shifted the maximum in the pH–heat coagulation time (HCT) profile of 2× or 3× concentrated milk to a higher pH value and certain concentrations increased the maximum HCT, probably due to the fact that NaCl reduced the extent of heat-induced dissociation of κ-casein. Added NaCl reduced the ethanol stability, with the extent of this effect increasing with the concentration of NaCl. The key-mechanism though which added NaCl induces changes in the physico-chemical stability of casein micelles appears to be through changes in the charge on the casein micelles.     

Effects of calcium-chelating agents and pasteurisation on certain properties of calcium-fortified soy milk

Addition of 25 mM calcium chloride to soy milk reduced pH, increased ionic calcium and caused it to coagulate. The effects of different chelating agents were investigated on selected physicochemical properties of soy milk and on preventing coagulation. The soy milks were then pasteurised to examine how heat treatment changed some of these properties as well as to evaluate their effects on heat stability.     

Influence of added calcium chloride on the heat stability of unconcentrated and concentrated bovine milk

The influence of added calcium chloride (1–10 mmol/L) on the heat-induced coagulation of skim bovine milk was examined. Unconcentrated milk displayed a pH-heat coagulation time (HCT) profile with a maximum at pH 6.6 and minimum at pH 7.0. Adding calcium chloride to unconcentrated milk progressively reduced the HCT at the maximum, increased the pH at which the maximum occurred and reduced the HCT at pH > 7.0. For concentrated milk, the shape of the pH-HCT profile, that is, a maximum at pH 6.6, was not altered by added calcium chloride, but HCT was reduced progressively with increasing concentration of calcium chloride. Preheating (90°C for 10 min) shifted the maximum in the pH-HCT profile of unconcentrated milk to a more acidic pH, and addition of 5 mmol/L calcium chloride to preheated milk induced changes in heat stability similar to those noted for unheated milk. Addition of calcium chloride to milk prior to preheating strongly reduced the stability of milk against heat-induced coagulation. These data suggest that calcium has a strong destabilizing effect on the stability of milk systems against heat-induced coagulation.     

Heat‐induced coagulation of whole milk by high levels of calcium chloride

This study investigated the interaction of calcium ions and milk proteins during heat‐induced coagulation of milk. Addition of 20–200 mM calcium chloride to milk caused coagulation on heating to 70 °C. Preheating milk at 90 °C for 10 min or ultra‐high temperature treatment at 140 °C for 6 s increased the sensitivity of milk proteins to coagulation. The former treatment was more effective than the latter in coagulating proteins. A maximum of 98% of the protein in milk preheated at 90 °C for 10 min was coagulated by 50 mM added calcium chloride at 70 °C with holding for 5 min.     

Zinc binding in bovine milk

About 90% of the Zn in bovine skim milk was sedimented by ultracentrifugation at 100,000 g for 1 h. About half of the non-sedimentable Zn was non-dialysable, indicating that it was associated with protein, probably non-sedimented casein micelles. Casein micelles incorporated considerable amounts of Zn added to skim milk as ZnCl2, and at Zn concentrations greater than or equal to 16 mM coagulation of casein micelles occurred. Ca was displaced from casein micelles by increasing ZnCl2 concentration and approximately 40% of micellar Ca was displaced by 16 mM-ZnCl2. Micellar Zn, Ca and Pi were gradually rendered soluble as the pH of milk was lowered and at pH 4.6 greater than 95% of the Zn, Ca and Pi were non-sedimentable. These changes were largely reversible by readjustment of the pH to 6.7. About 40% of the total Zn in skim milk was non-sedimentable at 0.2 mM-EDTA and most of the remainder was gradually rendered soluble by EDTA over the concentration range 1-50 mM. This indicates that there are two distinct micellar Zn fractions. No micellar Ca or Pi was solubilized at EDTA concentrations up to 1.0 mM, indicating that both colloidal calcium phosphate (CCP) and casein micelles remained intact under conditions where the more loosely bound micellar Zn fraction dissolved. Depletion of casein micelles of colloidal Ca and Pi by acidification and equilibrium dialysis resulted in removal of Zn, and in colloidal Pi-free milk non-dialysable Zn was reduced to 1.2 mg/l (approximately 32% of the original Zn). Thus, approximately 32% of the Zn in skim milk is directly bound to caseins, while approximately 63% is associated with CCP. Over 80% of the Zn in colloidal Pi-free milk was rendered soluble by 0.2 mM-EDTA, indicating that the casein-bound Zn is the loosely bound Zn fraction in casein micelles. A considerable fraction of the Zn in acid whey (pH 4.6) co-precipitated with Ca and Pi on raising the pH to 6.7 and heating for 2 h at 40 degrees C, indicating that insoluble Zn phosphate complexes form readily under these conditions. Studies on dialysis of milk against water, or dilution of milk or casein micelles with water, showed that CCP and its associated Zn is very stable and dissolves only very slowly at pH 6.6. The nature of Zn binding in casein micelles may help to explain the lower nutritional bioavailability of Zn in bovine milk and infant formulae compared with human milk.     

The composition of interfacial material from skim milk foams

The foaming properties of skim milk vary with temperature of foaming in the range from 5 to 85°C, with foams of maximum stability being formed at approximately 45°C. This paper reports the significance of different milk fractions in the foam and concludes that the micellar casein fraction plays an important role in stabilization of milk foam formed at higher temperatures. This finding was supported by the fact that added calcium chloride increased and calcium-chelating agents decreased foam stability. These effects were attributed to the increase and decrease, respectively, in the amount of micellar casein in the milk. Furthermore, bubble ghost material sedimented by low-speed centrifugation of foam was found to contain predominantly caseins, and electron micrographs of foams formed at 45°C clearly showed casein micelles spread over the interface. However, other structures observed in the electron micrographs suggest that soluble milk proteins and possibly polar lipids are also present in the foams and play a role in formation of milk foams.     

Effects of calcium chloride and sodium hexametaphosphate on certain chemical and physical properties of soymilk

ABSTRACT:  Soymilks with sodium hexametaphosphate (SHMP) (0% to 1.2%) and calcium chloride (12.50, 18.75, and 25.00 mM Ca) were analyzed for total Ca, Ca ion concentration, pH, kinematic viscosity, particle diameter, and sediment after pasteurization. Higher added Ca led to significant ( P ≤ 0.05) increases in Ca ion concentration and significant ( P ≤ 0.05) decreases in pH. At certain levels of SHMP, higher concentrations of added Ca significantly increased ( P ≤ 0.05) kinematic viscosity, particle diameter, and sediment. Increasing SHMP concentration reduced Ca ion concentration, particle diameter, and dry sediment content, but reduced kinematic viscosity of samples ( P ≤ 0.05). Adding SHMP up to 0.7% influenced pH of soymilk in different ways, depending on the level of Ca addition. When the pH of Ca-fortified soymilk was adjusted to a higher level, ionic Ca decreased as pH increased. There was a negative linear relationship between the logarithm of ionic Ca concentration and the adjusted pH of the soymilk. Ionic Ca appeared to be a good indicator of thermally induced sediment formation, with little sediment being produced if ionic Ca was maintained below 0.4 mM.