Module 6. Heat stability of milk and condensed milk

Lesson 15

15.1 Introduction

The heat–induced coagulation of milk is one of the few major classical problems in dairy chemistry awaiting complete solution. Heat coagulation is the result of a complex series of simultaneous reactions, some of which are complementary whereas others are antagonistic. Further most research has been directed towards the heat stability of unconcentrated milk, though problems related to that of concentrated milks may be of grater commercial significance.

When milk is held at temperatures above the boiling point it eventually coagulates, the higher the temperature the sooner. Coagulation time is strongly dependent on pH. The pH at coagulation is always low (< 6.2). The heat coagulation time (HCT) at 140ºC of bulk milk at its pH of maximum stability is usually 20 to 30 min.

Casein-the major milk protein is not denaturable within the strict definition of the word because of its lack of secondary and tertiary structure. In fact, casein generally is considered to be a naturally denatured protein. However, it does undergo changes mostly hydrolytic, when subjected to severe heat treatment. The casein micelles aggregate and undergo heat coagulation unlike heat denaturation suffered by globular proteins (whey proteins). During relatively long period of heating at an elevated temperature many heat-induced changes occur. It is these changes that ultimately lead to coagulation, few of them have been investigated thoroughly and some have not been studied at all. Some heat-induced changes in milk likely to lead to protein coagulation are:

1. Decrease in pH.

2. Precipitation of calcium phosphate

3. Denaturation of whey proteins and interaction with casein

4. Maillard browning.

5. Modification of casein :

a. Dephosphorylation

b. Hydrolysis of κ-casein

c. General hydrolysis

6. Changes in micellar structure :

a. Zeta potential

b. Hydration changes

c. Association – Dissociation.

15.2 Types of Milk

15.2.1 Type A milk

Those milks which show a very pronounced maximum (~ pH 6.7) and minimum (~ pH 6.9) in stability in HCT/ pH curve. At pH>6.9, HCT increase again.

15.2.2 Type B milk

Those milks in which stability increase progressively with increasing pH and show no maximum and minimum

The precise shape of the HCT/pH curve is characteristic of individual milks and is generally similar throughout the lactation period. Presumably dietary, environmental and genetic factors are responsible for this variation. Perhaps pH is the most important single influencing factor; all bulk milks and most individual cow milks exhibit a maximum at ~ pH 6.7 and a minimum at ~ pH 6.9 in the HCT/pH curve. (Fig. 15.1).

The precise shape of the HCT/pH curve is influenced by a host of factors among which the most important are:

1. Whey proteins,

2. Other heat-denaturable proteins,

3. κ-casein,

4. Colloidal calcium phosphate,

5. Detergents,

6. Assay conditions (temperature & agitation)

7. Urea,

8. Preheat treatment,

9. aldehydes,

10. concentration

11. or dilution

Approximately 20% of individual cow milks show Type B characteristics in Canada, 1% in Australia and Ireland and 70% in Japan.

15.2.3 HCT/pH profile of buffalo milk (Temp 140ºC, pH at 5ºC)

Like Type A bovine milk, buffalo milk show maximum at pH ~ 6.6, followed by a sharp decrease in stability at pH ~ 6.8. Poor heat stability persist up to pH 7.3 to 7.7 resulting in broad minimum (Fig. 15.2). A further increase in pH leads to an increase in heat stability. Based on these data, buffalo milk is categorized as Type A milk (Table 15.1).


15.2.4 Reasons for overall shape of a Type A heat stability curve

  • On the acid side of the maximum, the coagulation of casein micelles is governed solely by the calcium-induced complexation of casein. As the pH is increased, the calcium ion concentration decreases and casein micelles become more heat stable; however, after a certain degree of protein polymerization and dephosphorylation (and hydrolysis of κ-casein) have occurred, which reduce casein stability, the lower calcium ion concentration is sufficient to induce coagulation. The occurrence of the heat stability minimum (~ pH 6.9 ) is due to a combination of the formation of β-Lg / κ-casein complexes, which are sensitive to calcium phosphate precipitation, and the increase in heat – induced calcium phosphate deposition.
  • Heat produced acid may be the principal cause of coagulation at the heat stability maximum and periodic neutralization postpones coagulation very significantly.
  • The transition from the coated (by denatured whey protein) to the protein depleted (depleted of κ-casein) form, which occurs as the pH is increased above that for maximum heat stability, results in the heat stability of the micelles first increasing (the maximum), then decreasing (the minimum ) and finally increasing again over the pH range 6.4-7.0.
  • The occurrence of the minimum in the HCT/pH profile is due to the heat – induced dissociation of micellar κ-casein, which occurs when milk is heated beyond 90ºC at pH values above 6.9. The κ-casein depleted micelles are sensitive to calcium phosphate precipitation and hence the heat stability minimum occurs. The high heat stability at pH values above the heat stability minimum is probably due to a lack of available calcium ions (as a result of heat-induced precipitation of calcium phosphate) and the high net negative charge on the protein. Thus, to induce coagulation at pH values above ~7.2, a longer heating time is required to cause dissociation of sufficient κ-casein, hydrolysis of κ-casein, reduction of pH or other heat-induced changes to reduce the micellar negative charge to a value permitting coagulation.

15.3 Micro-Structural Changes Revealed by Electron Micrographs

Heating skim milk at 130 or 140ºC:

(1) Reduction in the range of micelle size and increase in the average micellar diameter.

(2) Below pH 6.7, heating cause changes in the micelle surfaces from smooth to a ragged appearance with many appendages.

(a) This is due to ß – Lg / κ-casein complexes at the micellar surfaces

(b) During heating more ß-Lg precipitated on to the surfaces.

(3) Above pH 7.0, ß-Lg / κ-casein complexes are not formed on heating milk.

(4) At the pH of maximum heat stability (pH 6.7), coagulum formed show that the casein micelles are aggregated in short chains in a relatively open network and many individual micelles persist.

(5) At the pH of minimum heat stability (pH 6.9) milk coagulum show a closed network of long chains of fused micelles.

Casein micelles joins in chains and clusters gradually build into a gel network during the last few minutes before visible coagulation, but in the coagulum just after coagulation the gel network become denser and is composed of long chains of fused micelles, indicating an abrupt change at coagulation.

15.3.1 Methods of measuring heat stability

1. Subjective heat stability test

The test is used to determine the heat stability by heating a small sample of milk (1-1.5 ml), sealed in a narrow glass tube, in a thermo statically controlled oil bath, usually at 140ºC for milk of standard concentration or at 120ºC for concentrated milks, until particles of coagulated protein are observed in the following milk.

The heat coagulation times (HCT) depend on a number of experimental factors especially

(i) Degree of tube fill

(ii) Head space gas

(iii) Rocking rate during heating

(iv) Angle of tilt and

(v) Assay temperature.

The Q 10ºC for coagulation is usually ~3.

15.3.2 Objective heat stability assay

In this test the percentage of total sedimentable nitrogen by low gravitational forces is determined after various heating intervals. The resulting "nitrogen depletion curves" show a break at the onset of visual coagulation, samples that coagulate rapidly yielding large protein flocks, show a sharp break in the curve while those that coagulate slowly show a more gradual increase in sedimentable nitrogen.

The objective method is strongly recommended if the heat stability characteristics of a sample are to be described fully.

15.4 Factors Affecting the Heat Stability of Milk

15.4.1 Species and breed of animal


(a). Buffalo milk is less stable than bovine milk and shows a maximum in HCT/pH curve similar to Type A cow's milk.

(b). Human milk is quite heat stable and shows Type B stability characteristics.

(c). Ovine and Caprine milks show a marked maximum at pH 7.0 in the HCT/pH curves, but are very unstable at all higher pH values.

(d). Sow milks are shown to have very low heat stability and show a progressive increase with increasing pH (no maximum or minimum)

(e). Mare milk is very unstable at their original pH (7.0), HCT is <10 min at 100ºC.


(a). The values of heat stability of cow-milk from Sahiwal, Tharparkar, Red Sindhi and Crossbreds were reported to vary from 30.38 to 197 min at 120º C and 32.40 to 34.73 min at 136ºC whereas the values of heat stability of milk from Murrah buffaloes at 120ºC was from 99 to 109 min, 35.18 min at 136ºC and 32.9 min at 130ºC

(b). Milk from Jersey cows had greater maximum and natural heat stability than milk from Friesion Cows. Maximum heat stability declined with the age of the cows.

(c). It is known for long time that heat stability of buffalo milk is much lower than cow milk. But contradictory reports have appeared in the literature on heat stability of buffalo milk.

15.4.2 Genetic variants

Maximum heat stability of κ-casein is observed in genetic variants (B>AB>A) and β-lactoglobulin (B, AB>A): whereas natural heat stability is affected only by κ-casein genetic variants (B>AB>A).

a. Maximum and natural heat stability at 120ºC is correlated positively with β-casein and κ-casein concentrations and is negatively correlated with αS1- casein and β-Lg concentrations.

b. Natural and maximum heat stability is correlated positively with urea concentration.

c. The pH of skim milk samples is associated with αS1-casein, genetic variant, age of cow, stage of lactation and concentration of γ-casein.

15.4.3 Stage of lactation and dietary factors

a. Lactation

There is a general, erratic tendency for milk to become more stable with advancing lactation. Season/stage of lactation cause variability in heat stability due to differences in composition particularly urea and other factors involved in colloidal stability: Ca2+ activity, casein micelle size, phosphate content of micelles, and their voluminosity.

b. Feed

The type of feed influences heat stability characteristics. Cows produce Type A milk when grazed on good pasture but some cows produce Type B milk, or milk intermediate between A and B, when grazed on dry, withered pasture.

15.4.4 Milk constituents

a) Milk Salts

  • H+ and polyvalent cations and anions (Ca2+, Mg2+, PO43- and Citrate3-), especially the concentration of these ions in the serum phase, play an important role in the heat stability.
  • Milk salts contribute upto 65% of total buffering capacity of milk. The distribution of salts in milk results from complex temperature dependent equilibrium between dissolved and colloidal phases.
  • The soluble salts are the principal factors influencing heat stability but they apparently act only in the presence of β-Lg. However, both soluble calcium and phosphate (SP) have a major effect on the shape of the HCT/pH curve.
  • Although the HCT/pH curve can be readily altered by modifying the concentration of soluble and colloidal salts, natural variation in HCT are not correlated with the concentrations of indigenous salts.
  • Ca, P and citrate ions show negative correlation with HCT of buffalo milk and its concentrate, while Mg is found to have positive correlation with their HCT.
  • All the four polyvalent ions Ca, Mg, P and Citric acid exhibit a negative correlation with HCT of cow milk at 140 °C.

b) Caseins


(1) β-Lg / κ- casein interaction determines the shape of HCT/pH curve.

(2) The ratio of κ -casein (on the surface of casein micelles) to b -Lg determines whether a milk is of Type-A or Type-B

(3) Since buffalo casein micelles are larger than bovine micelles, it is probable that the former will have lower quantity of surface κ– casein than the latter and because buffalo milk contains 23% more b -Lg than bovine milk, a very low κ-casein / b -Lg ratio, may be partly responsible for the broad minimum in buffalo milk.

(4) Buffalo casein micelles have lower net negative charge than bovine casein micelles and this may also contribute to the broad minimum observed in buffalo milk.

(5) Increase in casein micelle diameter (from ~25 nm to above 200 nm) on heating skim bovine milk at 140°C for 10 min may be due to.

a. Precipitation of denatured whey protein on to the surface of the casein micelles via formation of b -Lg /κ– casein complexes

b. Heat – induced precipitation of calcium phosphate on to casein micelles, thus attaching small and shattered micelles to intact micelles.

c. Heat – induced aggregation of small micelles leading to the formation of larger micelles.

d. Since κ-casein is believed to be located mostly at the micellar surfaces, it is present at a higher concentration in small casein micelles than in larger casein micelles. Therefore, there are more κ-casein molecules available on the surfaces of small micelles for interaction with denatured β-Lg than on larger micelles.

c) a s2 -Casein

Addition of a s2-casein to milk has no significant effect on heat stability at pH values acid to the maximum but causes slight destabilization in the minimum pH range and extends the minimum over a wider pH range.

d) a s1 – and b -caseins

b -casein plays an important role in micellar structure and heat stability although natural variation in the a s1: b - ratios are relatively small.

e) Whey proteins

Serum proteins exercise a major effect on the shape of the HCT/pH curve.

f) b -lg

b -lg is one of the major whey proteins found to exist in dimeric form in milk. Under the action of heat, dimer is cleaved into monomeric form, and there is formation of reactive free SH-groups on account of marginal group containing cysteine/cystine. The cleavage of b -lg favours polymerization and also the reaction between b -lg and κ-casein. The other observations are:

(a) The interaction between b -kg & κ-casein is confined to a narrow pH range of 6.7 to 7.0.

(b) At lower pH, direct denaturation of whey proteins occurs.

(c) In addition to disulphide bonds, hydrophobic bonds also take part in the interaction between κ-casein & b -lg in the colloidal phase thus, enhancing the heat stability of milk in the pH range of 6.7 to 7.0.

(d) An increase in pH of milk (below pH 6.8) could be induced by the addition of b -lg during heating. Thus it is possible to counteract the reduction of pH caused by heating, and increase in heat stability.

(e) The ratio of b -lg to κ-casein may be more important in heat stability than the concentration of these proteins, suggesting that surface characteristics of the micelle are critical in heat stability.

(f) The maximum in the HCT/pH curve is a consequence of low stability at pH ~6.9 which is due to heat-induced precipitation of calcium phosphate on casein micelles sensitized by complexation with heat-denatured whey protein.

g) a -la

Although a -la contains no sulphydryl groups, it does possess 4 disulphide bonds per mole. a -la and b -lg have essentially the same effect on heat stability and are about equally effective on a weight bases. a -la & b -lg can form intermolecular complexes, presumably via disulphide interchange.

h) Ovalbumin

It causes destabilization of milk in the pH range 6.5 -7.4 and 6.4 – 7.6 respectively.

i) Lysozyme

It markedly reduces the stability of milk at unadjusted pH values.

j) Colloidal calcium phosphate

Colloidal calcium phosphate (CCP) is an integrating factor in the casein micelle. Native micellar structure is necessary for the occurrence of a typical maximum/ minimum in the HCT/pH curve. Calcium ion and CCP play at least partially similar and interchangeable roles in heat stability.

  • Removal of CCP increases stability at all values throughout the pH range 6.3-7.0 and the minimum in the HCT/pH curve disappear when greater than 30% of the CCP is removed. The effect is reversible provided not more than 30% of the CCP is solubilized.
  • The destabilizing influence of CCP below pH 7.1 may be due to masking of organic phosphate groups.

k) Lactose

  • Enrichment of milk with lactose decrease stability. Lactose acts as a destabilizing factor by virtue of acting as a source of heat-induced acids, in the presence of oxygen. Lactose reduces the stability and modifies the HCT / pH curve. It is involved in Maillard browning and apparently urea increases stability only in the presence of lactose.
  • Protein-carbohydrate interactions, on the contrary, increase the heat stability of milk according to the reactivity of the carbonyl group attached to the carbohydrate moiety
  • When 50-70% hydrolysis of the lactose in milk using the enzyme b -galactosidase is carried out, heat stability increase is maximized.

l) Urea

  • The seasonal variation in heat stability is significantly correlated to changes in the naturally occurring level of urea in milk. Between 72 and 90% of the variation of heat coagulation time, measured at the original pH of the milk, is accounted for by changes in milk urea alone.
  • The lesser amount of urea in buffalo milk (17.5 mg / 100 ml) as compared to cow milk (40 mg/100 ml) is one of the factors responsible for lower heat stability of buffalo milk.

15.5 Additives

a) Urea and other amides

● Addition of urea prior to heating has a marked stabilizing effect. At low concentrations (< 7mM) urea does not alter stability in the pH region of minimum stability (Type A) but does stabilize throughout the pH range 6.4- 7.4 at higher concentration (>15mM). Type B milks are stabilized throughout the pH range even by low concentrations of urea.

● Added urea has been reported to have no effect or produce a small decrease in heat stability in concentrated bovine milk systems.

Thus, urea may perform at least two functions:

(i) To react chemically to prevent cross-link formation and aggregation

(ii) To diminish pH drift, thereby modifying the rate of formation of chemical cross-links.

b) Other amides

Urea, biuret, triuret, methyl- and ethyl-urea have similar effects on the HCT/pH curve and are about equally effective on an equimolar bases. They all are

(i) Capable of forming homocitrulline from lysine

(ii) Capable of involvement in Maillard browning with lactose and

(iii) They reduce the rate of pH decline during heating

However, pH buffering is considered to be the most likely mechanism for heat stabilization.

c) Aldehydes and sugars

Formaldehyde, glyoxal, glycol aldehyde, glyceraldehyde, erythrose and 2–deoxyribose stabilize milk and concentrated milk but several hexoses have no effect. In contrast to urea, which has no effect on the stability of concentrated milk, aldehydes are effective on both standard and concentrated milk. The reaction of aldehydes with ε-NH2 groups or the cross-linking of polypeptide chains may be involved.

  • Most of the sugars increased stability over the pH range 6.5 – 7.1, but at pH 7.2 – 7.4 had no effect.
  • Glyoxal, as well as simple aldehydes, is a very effective stabilizer of concentrated milk.
  • Urea and glyceraldehydes have synergistic effect of on the HCT – pH curve of buffalo milk.

d) Gums & Alginates

κ-Carrageenan at concentrations upto 0.05% (w/v) stabilizes milk in the region of the minimum without a significant effect on stability at the maximum pH. Higher concentrations (especially >0.4%) cause marked destabilization throughout the pH range 6.4 – 7.4. Other hydrocolloids, e.g. carageenan K-100, CMC and Manucol esters, have little effect on heat stability at pH values below ~ 7 but cause destabilization at higher pH values.

f) Detergents

  • Sodium dodecyl suphate: Increasing additions of SDS upto 0.5% progressively shift the HCT / pH curve of milk to more acidic values without significantly altering its shape, and markedly increase maximum stability but have very little effect on stability in the minimum pH range. Casein micelles dissociate at higher concentrations of SDS.
  • Cationic detergents: Shift the HCT / pH curves to more alkaline values without significantly altering the shape but causing slight stabilization. Cetyltrimethyl ammonium bromide is particularly effective. The hydrophobic bonding may be involved.
  • The non – ionic detergents : Triton X and Tween 60 added to milk at levels upto 3% (w/v) have no significant effect on heat stability throughout the pH range 6.4 – 7.4.

g) Acid casein

Casein addition does not have stabilizing effect on the fluid milk due to its effect on the salt balance.

h) Chemical stabilizers (salts)

Salt balance is regarded as one of the most important factor in heat stability of milk. The effects of different salts on heat stability of cow & buffalo milk are:

  • Primary effect of stabilizers is a consequence of their influence on the pH of milk.
  • The heat stability of buffalo milk could be increased either by the addition of phosphate or citrate which reduce the ionic calcium content or by the replacement of 15% of calcium by sodium through electro metathesis.
  • Calcium has a drastic effect in destabilizing milk.
  • Addition of calcium chloride, sodium chloride and potassium phosphate significantly decrease the heat stability of buffalo milk.
  • Na2HPO4 cause a significant decrease in the heat stability of buffalo milk, which is attributed to a significant increase in milk pH. On the other hand, addition of Na2HPO4 to acidic milk restores its pH and heat stability. Sodium phosphate causes a shift in the pH of maximum stability towards the acidic side of fresh milk.
  • Ortho phosphates are generally the most effective stabilizers and CaCl2, which reduces intrinsic heat stability, should be used in moderation.
  • The point in the process at which stabilizers are added is not important and no single predictive test for the minimum level of stabilizer or combination of stabilizing salts required to optimize heat stability is apparent.

Last modified: Monday, 22 October 2012, 5:44 AM