# Heat stability of goat milks

Said Bouhallab, Ketsia Raynal-Ljutovac

► To cite this version:

Said Bouhallab, Ketsia Raynal-Ljutovac. Heat stability of goat milks. Future Of The Sheep And Goats Dairy Sector, Oct 2004, Saragosse, Spain. pp.216-225. hal-01569516

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## 4.2 Heat stability of goat milks

S. Bouhallab¹ and K. Raynal-Ljutovac²

1 UMR Science & Technologie du Lait et de l'Oeuf, INRA-Agrocampus, 85 rue de Saint-Brieuc, 35042 Rennes cedex, France. E-mail: Said.Bouhallab@rennes.inra.fr; 2 Institut Technique des Produits Laitiers Caprins (ITPLC), Avenue François Mitterrand, BP49, 17700, Surgères, France. E-mail: ketsia.raynal@itplc.asso.fr

### Abstract

Goat milk is characterized by a very low heat stability at its natural pH (i.e. pH 6.6 - 6.7). Consequently, disodium phosphate is generally used as an additive before UHT-treatment to prevent the destabilization of milk. Besides this additive, other solutions that improve the stability of heated milk can be proposed. We have shown that trisodium citrate was more efficient, mainly by ensuring stability at high pH of milk. Some steps of the UHT process must also be taken into account such as cold storage that may impair heat stability of goat milk. The heat stability of milk was also increased throughout an increase of the caseins to whey proteins (WP) ratio either through a reduction of WP content or selective concentration of casein micelles. The positive effect of high caseins to WP ratio is probably linked to the formation of a covalent complex between caseins and β-lactoglobulin. The occurrence of such a complex was evidenced in a model system by direct identification of a disulfide bound between Cys₁₆₀ of β-lactoglobulin and Cys₈₈ of κ-casein.

### 1. Introduction

Goat milk is more sensitive to UHT treatments than bovine milk and this phenomenon has been far less studied than for cow milk. The available reports indicate that pH [1-3], micelle hydration [4], genetic polymorphism of α-S1 casein [5, 6], non-protein nitrogen [7], salt balance and especially ionic calcium [2, 3] could be directly or indirectly involved in the heat sensitivity of caprine milk. Heat stability can be improved by adding substances able to bind free calcium in order to minimize calcium activity and to increase milk pH. The maximum heat stability of goat milk actually occurs at pH 6.9-7.0 [5, 8, 9]. Sodium phosphate is now used for UHT goat milk but, besides the use of different additive, a better understanding of the causes of instability such as the steps of UHT process itself is needed. Because cold storage is the most used way of preserving milk at the farm and at the arrival in the plant, the time of cold storage must be taken into account in the UHT process as a key step. On the other hand, intensity of fore warming can have an impact on solubility of minerals and whey proteins.

In the case of bovine milk, it is now generally accepted that β-lactoglobulin (β-Lg) and κ-casein (κ-Cas) interaction is an important parameter involved in the pH-dependent heat stability [10]. This protein-protein interaction could also be important in the heat sensitivity of goat milk. In the present work, the contribution of these various factors to the heat stability of goat milk is reported and discussed in the light of what is known about the composition and the physico-chemical properties of goat milks.

### 2. Material and methods

#### Goat milk samples

Mid-lactation bulk goat milk collected in a local dairy farm in Poitou Charentes Region (France) was immediately skimmed at ITPLC.

#### Evaluation of additives efficiency

Three additives were added separately on fresh skim goat milk (without cold storage) at different concentrations: Disodium hydrogenophosphate (1, 3 and 6 mM), a neutral phosphate mix with disodium hydrogenophosphate + sodium dihydrogenophosphate dihydrate (1, 3 and 6 mM) and trisodium citrate (1, 2, 3 and 4 mM). Milk with and without (blank) additives was then pasteurized (73°C/30 s) in glass tubes (15 ml) in a water bath. pH, heat stability and amount of soluble calcium and soluble proteins were determined.

#### Influence of technological parameters

The impact of different parameters was investigated : type of additive, cold storage and pasteurization intensity.

3 mM disodium hydrogenophosphate, 3 mM trisodium citrate or 6 mM phosphate mix (first experimental plan) were added to goat milk in order to obtain quite equivalent heat stability of milks. Milk was used fresh or 72 h after cold storage at 4°C. Pasteurization, 73°C/30 s or 85°C/1 min, was performed in glass tubes (15 ml) in a water bath. pH, heat stability and amount of denatured soluble proteins were determined. Milks were then sterilized in tubes (15 ml) with an equivalent UHT treatment (136°C / 6s) in an oil bath and then stored at room temperature in the dark in order to measure their stability during storage.

#### Influence of caseins/whey proteins ratio

A combination of various membrane processes (microfiltration, ultrafiltration and nanofiltration) was used to modify the caseins/whey proteins ratio of goat milk [11]. By this way, caseins/whey proteins ratio was increased either by reducing whey proteins content or after selective concentration of caseins.

#### Physicochemical analyses

Soluble calcium was measured according to the IDF method (36 A, 1992) on the soluble phase obtained after addition of 0.3 ml of diluted rennet (2%v/v) into 15 ml milk, 15 min at 30°C and mild centrifugation (1000 g/ 10 min).

Soluble proteins content was calculated after preparation of total, soluble and non-protein nitrogen fractions according to Rowland [12], analysed by Kjeldahl method. Determination of denaturation rate of the individual whey proteins was performed by size exclusion chromatography on a TSK 2000XL column with phosphate buffer 100mM, pH 6.8, NaCl 150 mM with a flow 0.5 ml/min. Extent of denaturation (and/or precipitation) of soluble proteins was calculated from soluble protein values before and after pasteurization.

Heat stability of goat milk was determined according to the method of Morgan et al. [13]. Milk samples were sealed in glass-capillary tubes and heat treatment was performed in an oil bath at temperatures ranging from 120 to 150 °C for 1 min. The heat stability (HS) was defined as the maximum temperature within the range 120-150 °C at which the sample was stable during a 1-min treatment. Three replicates were carried out for measurement.

Stability of UHT goat milk during storage was evaluated until 9 weeks after UHT treatment by following pH, destabilization index (marked from 1 : no destabilization to 5 : strong destabilization) and sedimentation rate after centrifugation (1200g / 5 min) expressed as sediment weight/ milk weight (%).

### 3. Results and discussion

#### 3.1. Influence of additives and associated pH change on heat stability

##### Evaluation of additives efficiency

Stabilizing effect (Figure 1) was observed for low amount of citrate (2 mM) and disodium phosphate (3 mM). The highest heat stability was observed for trisodium citrate (149°C) compared to disodium phosphate (147°C), and neutral phosphate mix (141°C) for 3 mM concentration; coagulation temperature for milk without additives being 137°C. It corroborates findings of authors having tested sodium phosphates for goat milk [2, 3] and phosphate or citrate for cow milk [14].

This increase in heat stability can be explained by the induced increase in pH, from 0.02 to 0.15 upH according to the additive concentration, the highest increase being observed for citrate addition (Figure 1). Previous studies have shown that pH increase was beneficial for heat stability of goat milk and that the optimum was pH 6.9 (2, 3, 9).

It may influence whey proteins and caseins interactions and salt balances such as free calcium [9]. Natural pH of goat milk varies between 6.5 and 6.7. A pH decrease is associated with the induced increase in ionic calcium concentration, shielding the negative charge of the micelles and leading to aggregation of these latter. According to Montilla and Calvo [3], the ionic calcium concentration increase from 3 mM at pH 6.9 to 4.4 mM at pH 6.5 for goat milk. pH decrease of cow milk is the most important factor leading to the coagulation of milk during heating but other factors may be involved considering that pH of coagulated milk depends on initial pH of raw milk [15]. The use of additives limits the pH decrease observed for pasteurized milks (pH 6.61and 6.60 for phosphate and citrate added milks respectively vs pH 6.52 for blank (data not shown)).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Heat stability of goat milk as a function of the nature and of additives. Evolution of pH following addition to milk.</figcaption></figure>

## Nature of additive

On the other hand, phosphate mix, which did not modify milk pH, also increased milk stability, corroborating findings of Montilla and Calvo [3], but to a lesser extent than citrate and disodium phosphate. It implies that pH is the major factor but not the only one. Increase in heat stability is also due to the additive itself. Trisodium citrate was the most efficient additive for increasing heat stability. The amount of citrate is an important parameter that governs the ionic calcium level. In bovine milk, most of the ionic calcium is linked to citrate in the soluble phase. In caprine milk, citrate is 40% less concentrated than in bovine milk [16] with values of 103.7 mg.L⁻¹ and 176.8 mg.L⁻¹ respectively [17]. Thus, addition of citrate would be more efficient for goat milk than cow milk. Moreover, taking into account that phosphate is almost totally linked with calcium (in both milk), a too high addition may induce calcium phosphate precipitation. The binding mechanism and the nature of bound calcium (soluble or colloidal) seem to differ according to the additive. Soluble calcium concentration in milks was higher for citrate than for phosphate addition (Figure 2).

The same results were obtained after pasteurization at 85°C/ 1 min (not shown). It corroborates results of Le Ray et al. [18] obtained for reconstituted casein micelle dispersion with sodium citrate addition (6 to 17.8 mmol/kg at pH 7.4), which increased pH (contrarily to sodium phosphate) and amount of supernatant casein and diffusible calcium and phosphorus. According to these authors, citrate induces pH increase until 8.08 by binding colloidal calcium, it increases the second apparent pK of the phosphoseryl residues and allowed groups which possess pK close to the pH of the dispersion to bind protons. It seems that this phenomenon is pronounced when buffer capacity is low. They observed less phosphorus solubilization confirming solubilization of both CCP and Ca linked to the phosphoseryl residues. Charge density increases. Citrate could induce also a solubilization of

α<sub>s1</sub> and β caseins (highly phosphorylated caseins). It would thus induce an increase in small particles number, linked to this disintegration of micelles with increase voluminosity of the latter (micelle skeleton), increasing viscosity at pH 6.7 and avoiding heat aggregation [19]. Moreover, sodium itself can have an effect since addition of NaCl (0.1-0.3 M) to casein micelle dispersion increases soluble calcium and phosphate indicating solubilization of colloidal calcium phosphate. Nevertheless, Le Graët and Brulé [20] concluded to a substitution of calcium by sodium on phosphoserines residues of caseins and no phosphore solubilization. These effects of NaCl depends on initial pH of cow milk : NaCl 20 mM lowers heat stability above pH 6.9 but increases it from pH 6.7 to 6.9 [21]. Sodium also increases solubilization of casein (mainly κ casein) during heating and it may decrease heat stability of cow skim milk [22].

Little increase in soluble calcium was observed for phosphate addition. It did not seem to displace colloidal calcium, because calcium affinity is higher for CCP and phosphoseryl residues

than for added phosphate. Phosphate binds ionic soluble calcium, which may also induce a calcium phosphate precipitation on the micelle. A destabilization of colloidal phase may then take place when a too high amount is added owing to a decrease of caprine micelle charge.

Considering native soluble proteins, their amount hardly decreased when increasing amounts of disodium phosphate (Figure 3) were added prior to the pasteurization (73°C/30s). 30% of soluble protein were precipitated for the addition of 6 mM of disodium phosphate. In contrast, about 95% of soluble protein remained in the soluble phase when citrate or phosphate mix were added and this percentage did not seem to decrease with increasing amount of citrate. Considering individual proteins, decrease of the amount of soluble protein was more accentuated for β-lactoglobulin than for α-lactalbumin owing to their different denaturation temperatures.

<figure><img src="image_3.png" /><figcaption>**Figure 3.** Effect of various concentration of additives on the precipitation rate of whey proteins after pasteurization (73°C/30s) of goat raw milk.</figcaption></figure>

<figure><img src="image_4.png" /></figure>
## 3.2 Influence of technological parameters

### Pasteurization intensity

The heat stability was found to be the same whatever the pasteurization intensity applied in this study (Table 1). Precipitation rate of soluble proteins was significantly higher for 85°C / 1min treatment than for 73°C / 30s, representative of a higher protein denaturation and a probable formation of a complex between whey proteins and casein micelles (See below) or in soluble phase following to the pH values [23]. A fore warming treatment before UHT treatment can have different effects on heat stability : negative charge of micelles increases with whey protein binding at pH 6.5 for a treatment at 80-100°C / 20 min [24] and should impair aggregation.

<table><caption>**Table 1.** Impact of technological parameters on heat stability, pH and soluble proteins of pasteurized milks.</caption><thead><tr><th rowspan="2"></th><th colspan="4">Additive</th><th colspan="3">Pasteurization intensity</th><th colspan="3">Cold storage</th></tr><tr><th>Disodium Phosphate</th><th>Phosphate Mix</th><th>Trisodium Citrate</th><th>p</th><th>73 °C 30 s</th><th>85 °C 1 min</th><th>p</th><th>without cold storage</th><th>72 h at 4°C</th><th>p</th></tr></thead><tbody><tr><td>Heat Stability (°C) sd</td><td>147.4<br>3.4</td><td>149.5<br>3.3</td><td>149.4<br>2.8</td><td>(*)</td><td>148.8<br>2.3</td><td>148.8<br>4.0</td><td>ns</td><td>151.2<br>2.2</td><td>146.3<br>2.0</td><td>***</td></tr><tr><td>pH sd</td><td>6.62<br>0.03</td><td>6.57<br>0.02</td><td>6.64<br>0.02</td><td>***</td><td>6.62 (a)<br>0.04</td><td>6.60<br>0.03</td><td>*</td><td>6.61<br>0.04</td><td>6.61<br>0.03</td><td>ns</td></tr><tr><td>Precipitation rate β-Lg (%)</td><td>44.5</td><td>45</td><td>48.5</td><td>ns</td><td>3.5</td><td>88.5</td><td>***</td><td>41</td><td>51</td><td>**</td></tr><tr><td>Precipitation rate α-La (%)</td><td>19</td><td>23</td><td>20.5</td><td>ns</td><td>3.5</td><td>38</td><td>***</td><td>16.5</td><td>25</td><td>***</td></tr></tbody></table>

Mean and Standard deviation (sd). ns: not significant; (*) : p<0.10; * : p<0.05; ** : p<0.01; *** : p<0.001

But if treatment intensity increases, micelles coated with denatured whey proteins expose unstable hydrophobic areas, get closer and aggregate with covalent bonds [25]. It can lead to increase in particle size especially in the case of small ruminant milks, which have low colloidal stability [26]. On the other hand, severe heating can induce the dissociation of κ-casein and/or κ-casein/ β-lactoglobulin complex that increases the proportion of small particles [10, 27]. The dissociation of κ-casein, which also occurs for high pH [8], may decrease colloidal stability. Actually, addition of κ-casein suppress the minimum of stability of cow milk.

### Cold storage prior to heat treatment

A cold storage 72 h/4°C before the pasteurization strongly depreciated heat stability of goat milk (Figure 4). This phenomenon has to be linked with modification of mineral and casein balances under cooling and/or development of microorganisms, especially psychrotrophs. Actually, the proteases of the latter are very heat stable (10% of proteolytic activity is maintained after 140°C/4s [28]) and could induce a degradation of caseins [29], decreasing colloidal stability. Cold storage induces dissociation of caseins, especially β casein [30, 31]. Nevertheless, other authors had noted higher amount of soluble caseins in small ruminant milks but a lower solubilization of the β casein under cooling than in the case of cow milk [32, 33]. Cold storage also induces solubilization of micellar calcium phosphate [34]. These modifications, which are known to impair cheese-making properties of cow milk and to a lesser extend those of goat milk [35], may lead to an increase of ionic calcium activity which favours heat coagulation of goat milk [3, 9].

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Influence of cold storage on heat stability of goat milk containing various additives: (A) 3 mM risodium citrate; (B) 3 mM disodium phosphate; (C) 6 mM phosphate mix.</figcaption></figure>

## 3.3. Influence of caseins/whey proteins ratio

We have previously reported the impact of varying caseins/whey proteins ratio on the heat stability of goat milk [11]. The results showed that increasing casein to whey proteins ratio improved the heat stability of reconstituted milks. This improvement can be achieved either by reducing the whey protein content or by selectively increasing the casein content. These two operations can be easily performed by the use of membrane processes. As illustrated in Figure 5, the heat stability of reconstituted milks was increased by about 6 °C when the caseins/whey proteins ratio was increased 1.4 times.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Dependence of the heat stability of goat milk on the caseins/whey proteins ratio.</figcaption></figure>

The effect of whey proteins on the heat stability of bovine milk has been reported in several studies. This effect is generally attributed to the interaction between β-lactoglobulin (β-Lg) and caseins, mainly κ-casein (κ-Cas), which is one of the most important heat-induced interactions occurring during bovine milk processing. However, the observed effect of β-Lg was shown to be strongly pH-dependant [36]. Higher stability at pH near 6.6 was observed in the presence of β-Lg. The mechanism of the β-Lg induced - stability at pH 6.6 - 6.7 was related either to the ability of the protein to reduce the dissociation of κ-casein from the micelles [15] or to its calcium-chelating properties [37].

In the case of goat milk, the role of whey proteins and the mechanism by which they act at the natural pH (i.e. pH 6.6) seem to be different since our results clearly show that the heat sta-
bility of goat milk is rather improved by reducing the whey protein content. Indeed, as shown in another study, the poor heat stability of caprine milk was not improved by adding whey proteins (4 g.L⁻¹) to milk before heating [9].

The observed destabilizing effect of whey proteins may be related to the formation of covalent complexes with casein micelles that we have evidenced in a subsequent study [38]. The identified complex involved a covalent bond between Cys₁₆₀ of β-Lg and Cys₈₈ of κ-Cas. Unlike the detailed studies on bovine milk, little is known regarding the formation and the nature of such complexes in caprine milk. Anema and Stanley [8] and Morgan et al. [9] hypothesized that the formation of a complex between caseins and whey proteins could explain the observed improvement of the heat stability at pH values higher than the natural pH. Hence, our results constitute the first direct evidence on the formation of a covalent complex between casein micelles and β-Lg derived from goat milk, and this at the natural pH of milk.

### 3.4. Influence of technological parameters on UHT milk stability during storage
During storage at room temperature, destabilization occurred for all UHT milks and was characterized by the appearance of a translucent phase at the top of the tube and a sediment phase with a thickness varying between few mm to 2 cm (to compare with a total liquid phase of 10 cm high). Sedimentation of skim goat milk was affected by each of the studied factors (Table 2).

<table><caption>**Table 2.** Impact of technological parameters on UHT milks stability during storage.</caption><thead><tr><th rowspan="2"></th><th colspan="4">Additive</th><th colspan="3">Pasteurization intensity</th><th colspan="3">Cold storage</th></tr><tr><th>Disodium Phosphate</th><th>Phosphate Mix</th><th>Trisodium Citrate</th><th>p</th><th>73 °C 30 s</th><th>85 °C 1 min</th><th>p</th><th>without</th><th>72 h at 4°C</th><th>p</th></tr></thead><tbody><tr><td>Sedimentation rate (%)</td><td>1.20</td><td>1.58</td><td>1.65</td><td>ns</td><td>1.11</td><td>1.89</td><td>***</td><td>0.94</td><td>2.08</td><td>***</td></tr><tr><td>sd</td><td>0.91</td><td>1.62</td><td>1.96</td><td></td><td>0.81</td><td>2.04</td><td></td><td>0.49</td><td>2.06</td><td></td></tr><tr><td>Destabilization Index (mark/5)</td><td>3.18</td><td>2.94</td><td>2.64</td><td>*</td><td>2.92</td><td>2.95</td><td>ns</td><td>2.77</td><td>3.11</td><td>(*)</td></tr><tr><td>sd</td><td>1.33</td><td>1.32</td><td>1.09</td><td></td><td>1.25</td><td>1.29</td><td></td><td>1.46</td><td>0.99</td><td></td></tr></tbody></table>

Mean and Standard deviation (sd). ns: non significant; (*) : p<0.10; * : p<0.05; ** : p<0.01; *** : p<0.001

A significant effect of additive was observed after 6 and 9 week storage. Trisodium citrate induced less sedimentation than phosphates during the considered period. It could be then an interesting alternative to the use of phosphates. Due to sterilization, pH slightly decreased from 6,62 to 6.51 and from 6,64 to 6,56 for disodium phosphate and trisodium citrate added milk respectively, then pH remained unchanged during storage whatever the additive was. Thus, the higher pH of citrate added milks before and after UHT treatment can contribute to an improved stability with less sediment during storage [2].

Destabilization was limited when intensity of pasteurization was low and when stabilizer was added to the milk after pasteurization.

Cold storage of goat milk at 4°C during 72 h before heat treatment led to an early and strong sedimentation compared to non stored milks (Figure 6). The effects of cold storage on the stability of UHT milks could be linked with heat resistant proteolytic enzymes of psychrotrophic bacteria. These enzymes can then slowly degrade casein during storage of UHT milks.

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Influence of cold storage of goat raw milk on UHT milk stability during subsequent storage at room temperature.</figcaption></figure>

## 4. Conclusion

The main differences between goat and cow milks are the size, mineralization and hydration of their casein micelles and more generally the mineral balances. If some work has shown quite good intramicellar stability under cold storage, with less solubilization of colloidal calcium and casein than for cow milk [35], the high propensity to coagulate under the action of rennet on raw or pasteurized milk (no delay for coagulation time after pasteurization [26]) or under more severe heating treatments such as UHT (heat coagulation) reveals a low colloidal stability of goat milk.

Some means were given in this study to better control or to limit heat aggregation of goat skim milk. We have worked on skim milk and did not include possible impact of fat globule but destabilization of goat milk during UHT treatment and storage is known to occur for both skim and whole milk.

Besides the use of other additives such as phenolic compounds [39], the use of membrane technologies alone or in combination with the use of additives could constitutes an interesting way to improve the heat stability of goat milks. Moreover, if these solutions have a great beneficial impact on heat stability, sedimentation phenomenon during storage may still occur and need a deeper investigation.

## Acknowledgement:

Part of this work was supported by a grant from the French Ministry of Agriculture and Fisheries (program "Aliment-Qualité-Sécurité" TQ 42, Décision N° 99P0391).

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