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Rennet coagulation of heated milk: A review

Michel Britten, Hélène J. Giroux

PII: S0958-6946(21)00207-7
DOI: https://doi.org/10.1016/j.idairyj.2021.105179
Reference: INDA 105179

To appear in: International Dairy Journal

Received Date: 18 June 2021

Revised Date: 30 July 2021
Accepted Date: 30 July 2021

Please cite this article as: Britten, M., Giroux, H.J., Rennet coagulation of heated milk: A review,
International Dairy Journal, https://doi.org/10.1016/j.idairyj.2021.105179.

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CRediT authorship contribution statement

Michel Britten: Writing - Review & Editing. Hélène J. Giroux: Writing - Review &

Editing.

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Rennet coagulation of heated milk: A review

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Michel Britten*, Hélène J. Giroux

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Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-Food Canada,

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3600 Casavant Boulevard West, Saint-Hyacinthe, QC, Canada, J2S 8E3

*Corresponding author. Tel.: 450-768-7981

E-mail address: michel.britten@agr.gc.ca (M. Britten)

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 1 ______________________________________________________________________________

2 ABSTRACT

4 Mild heat treatments, such as thermisation or pasteurisation, are applied to milk to prevent

5 microbiological problems occurring during or after cheese manufacture. Heating milk in more

6 severe conditions induces the denaturation of serum protein and increases cheese yields.

7 However, the rennet coagulation properties of milk are severely impaired by high heat

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8 treatments. Cheeses made from heated milk tend to have a higher moisture content and may

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9 show defective body, texture or flavour. The challenge for cheese-makers is to increase yields

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10 while minimising the undesirable changes in milk coagulation and cheese quality. This paper
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11 reviews the mechanisms of rennet-induced coagulation of milk and the detrimental effects
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12 induced by heat. Methods that have been proposed to improve renneting properties of heated

13 milk are also described.

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14 ______________________________________________________________________________
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15 Content
16
17 1. Introduction………………………………………………………………………………………………………….…….…3
18 2. Rennet coagulation of milk……………………………………………………………………………………...….…5
19 2.1. Milk proteins………………………………………………………………………………………….……….5
20 2.2. Primary phase of coagulation………………………………..……………………………....……….6
21 2.3. Secondary phase of coagulation………………………………..…………………………….………7
22 3. Effect of heat treatment on milk proteins………………………………..……………………………….…….8
23 3.1. Formation of whey protein (WP)/-casein complexes…………………………………..…8
24 3.2. Characteristics of casein micelles in heated milk………………………………..……….….11
25 3.3. Characteristics of WP/-casein complexes in the serum phase of heated milk 12
26 4. Effect of heat treatment on milk mineral equilibrium ………………………………..………………….13
27 4.1. Mineral equilibrium in milk………………………………..……………………………………………13
28 4.2. Factors influencing the mineral equilibrium in milk…………………………………….…..13

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29 4.3. Heat-induced changes to the mineral balance in milk……………………………………..14
30 5. Effect of milk heat treatment on the primary phase of coagulation………………………………..15

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31 6. Effect of milk heat treatment on the secondary phase of coagulation…………………………….16
32 6.1. Whey protein denaturation………………………………..……………………………………………17

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33 6.2. Calcium phosphate precipitation………………………………..……………………………………19
34 7. Improvement of the coagulation properties of heated milks…………………………………..……...21
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35 7.1. Modification of mineral equilibrium………………………………..……………………….….….21
36 7.2. Use of membrane separation technologies………………………………..……….….….……23
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37 7.3. High-pressure treatments………………………………..………………………………………………24

38 7.4. Other approaches…………………………………………………………………………………….………25
39 8. Cheese making using heated milk………………………………..………………………………………...……….26
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40 9. Conclusions and perspectives………………………………..……………………………………………….……….28

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43

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44 1. Introduction

45

46 The primary objective of cheese making is to convert milk into a stable product via

47 coagulation and fermentation. For the majority of cheeses, rennet is used to destabilise the

48 casein micelles and induce gel formation. The contraction of the casein network results in the

49 formation of cheese curd and the release of cheese whey. During the process, fat droplets are

50 trapped within the cheese matrix while water, lactose, some minerals and serum proteins are

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51 lost in the whey (Fox & McSweeney, 2017). For economic reasons, a great deal of research has

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52 focused on increasing cheese yields. Over the last decades, various approaches have been

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53 developed to increase the retention of serum protein in cheese (Hinrichs, 2001). Ultrafiltration
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54 of milk is commonly used to increase cheese plant efficiency, and this process also increases the
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55 concentration of serum proteins in the aqueous phase of cheese. The impact on cheese yield is

56 significant for highly concentrated milk retentates (Mistry & Maubois, 2017). Whey proteins can
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57 also be recovered from cheese whey and incorporated in cheese milk after aggregation by
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58 thermal treatment (Schenkel, Samudrala, & Hinrichs, 2011). Similarly to fat globules, whey
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59 protein aggregates are inserted inertly within the casein matrix and increase cheese yield

60 (Hinrichs, 2001; Perreault et al., 2017b). Finally, heat treatment can be applied directly to milk

61 prior to cheese making (Singh & Waungana, 2001). Heating milk is a common practice to ensure

62 microbiological safety. However, typical pasteurisation (72 °C, 15s) was shown to denature 3.2%

63 of serum protein and the extent of denaturation increases with the severity of heat treatment

64 (Guinee et al., 1998). During heat treatment, denatured serum proteins self-aggregate or form

65 complexes with -casein mainly through disulphide interactions. Both these forms of denatured

66 whey proteins are recovered in cheese (Kethireddipalli & Hill, 2015).

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67 Heating cheese milk is the simplest approach to increase cheese yield through better

68 recovery of whey proteins. However, the rennet coagulation properties of heated milk and the

69 quality of cheeses are negatively affected. The formation of whey protein/-casein complexes at

70 the surface of casein micelles or in the serum phase is the main factor responsible for the

71 inhibition of coagulation (Guyomarc'h, 2006). Heat treatment also causes the transfer of calcium

72 and phosphate from the soluble to colloidal phase of milk. This shift of the mineral balance

73 contributes to the poor coagulation properties of heated milk (Fox, Guinee, Cogan, &

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74 McSweeney, 2016). With the promise of increasing cheese yield, different strategies have been

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75 explored to restore the coagulation properties of heated milk. These strategies are based on the

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76 manipulation of mineral equilibrium, the concentration of cheese milk before or after heat
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77 treatment and the use of novel technologies, such as high-pressure treatments (Huppertz et al.,
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78 2005). Excessive moisture content is a common defect of cheeses made from heated milk, and

79 cheese-making parameters may need to be adapted to avoid this problem (Singh & Waungana,
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80 2001).
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81 This article presents an overview of the rennet-induced coagulation properties of heated

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82 milk. It complements previous review papers on the same topic (Kelly, Huppertz, & Sheehan,

83 2008; Kethireddipalli & Hill, 2015; Lucey, 1995; Singh & Waungana, 2001).

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85 2. Rennet coagulation of milk

86

87 The process of cheese making is based on the proteolytic activity of rennet on the casein

88 micelles. Various factors such as pH, temperature, milk composition and processing history

89 affect this critical reaction. Rennet coagulation of milk can be divided in two phases, which

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 90 correspond to the enzymatic cleavage of -casein and the aggregation/gelation of renneted

91 micelles.

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93 2.1. Milk proteins

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95 Rennet coagulation of milk is the first step in cheese production. The cheese curd is

96 formed of caseins, which represent about 80% of total milk proteins, while soluble proteins are

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97 mostly lost in the whey. There are four principal types of caseins, the αS1-, αS2-, β- and -caseins.

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98 Among the caseins, -casein is the least phosphorylated and the least sensitive to precipitation

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99 by calcium (Huppertz, 2013). Furthermore, -casein is the only glycosylated casein, which
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100 confers a hydrophilic character (Horne & Lucey, 2017). In milk, the caseins are mainly present as
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101 colloidal particles: casein micelles. The casein micelles are composed of casein molecules held

102 together by calcium phosphate and hydrophobic interactions. Although it is now recognised that
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103 -casein is located at the surface of casein micelles (Dalgleish & Corredig, 2012), the αS-caseins
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104 and β-casein arrangement in the internal core of casein micelles has not been completely
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105 elucidated. Different structural models have been proposed over the years, and the models that

106 have received the most attention are: the sub-micelle model, the nanocluster model, and the

107 dual-binding model (de Kruif, Huppertz, Urban, & Petukhov, 2012; Horne, 2008). In all these

108 models, calcium phosphate plays a major role in structure maintenance (Gaucheron, 2005).

109 Soluble proteins represent about 20% of total milk proteins. Unlike caseins, soluble

110 proteins do not aggregate in the presence of rennet. They are mainly composed of β-

111 lactoglobulin (β-Lg), α-lactalbumin (α-La), along with other minor proteins: immunoglobulins,

112 serum albumin, and lactoferrin. β-Lg and α-La contain two and four intramolecular disulphide

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113 bonds respectively. Moreover, β-Lg has one free thiol group, which reacts with -casein in

114 heated milk, affecting rennet-induced coagulability (Goulding, Fox, & O’Mahony, 2020).

115

116 2.2. Primary phase of coagulation

117

118 The rennet coagulation of milk is a two-step process. The primary phase of coagulation

119 corresponds to the enzymatic hydrolysis of the -casein present at the surface of casein micelles

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120 (Corredig & Salvatore, 2016; Horne & Lucey, 2017). The C-terminal region of the -casein is

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121 mostly hydrophilic with a net negative charge at native milk pH (pH ~6.7). It protrudes from the

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122 micelle surface, forming a hairy layer (Dalgleish & Corredig, 2012). This layer provides stability to
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123 the micelles through steric hindrance and electrostatic repulsion. The chymosin from the rennet
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124 cleaves the -casein at the Phe105–Met106 position resulting in the release of

125 caseinomacropeptide (C-terminal fragment 106–169) in the serum phase of milk. It has been
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126 observed that the diameter of the casein micelles decreases by about 10 nm following the
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127 cleavage of -casein by rennet (Sandra, Alexander, & Dalgleish, 2007). The para--casein (N-
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128 terminal fragment 1–105) is positively charged at milk pH (pH ~6.7) and remains attached to the

129 casein micelles. The central region of para--casein molecule is highly hydrophobic while the

130 positively charged residues are predominantly located in the C-terminal region (Mercier,

131 Brignon, & Ribadeau-Dumas, 1973). The release of caseinomacropeptide (CMP) fragments

132 reduces the zeta potential of casein micelles from –20 mV to about –10 mV (McSweeney, 2007)

133 and as a consequence the repulsive forces decrease, allows closer approach between the

134 paracasein micelles.

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135 The rate of -casein hydrolysis is proportional to the rennet concentration (Corredig &

136 Salvatore, 2016). The optimum pH for rennet coagulation is about 5.3–5.5 (Corredig & Salvatore,

137 2016), and therefore, the proteolytic activity of rennet is increased by reducing milk pH.

138 The enzymatic hydrolysis of -casein is usually evaluated by measuring CMP release.

139 CMP is usually quantified by RP-HPLC, after acid precipitation of proteins with trichloroacetic

140 acid (Kethireddipalli, Hill, & Dalgleish, 2011).

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142 2.3. Secondary phase of coagulation

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143

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144 The secondary phase of coagulation consists in the aggregation and gelation of the
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145 destabilised micelles (Horne & Lucey, 2017). When about 80% to 90% of -casein is hydrolysed
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146 (McSweeney, 2007), the micelles begin to aggregate, via hydrophobic interactions and calcium

147 bridges, forming a gel network. The pores of the gel are relatively large and contain whey and
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148 fat globules.

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149 Aggregation occurs at a lower extent of -casein hydrolysis as the temperature and ionic
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150 calcium concentration increase, or as the pH decreases (Corredig & Salvatore, 2016;

151 McSweeney, 2007). Below 18 °C, coagulation proceeds very slowly (Bansal, Fox, & McSweeney,

152 2007). By increasing milk temperature up to 40 °C, rennet coagulation improves (McSweeney,

153 2007). However, for most cheese varieties, the renneting temperature is adjusted between 30

154 °C and 35 °C to promote starter culture growth and to maximise curd firmness (Lucey, 2011).

155 Ionic calcium is essential to the aggregation and gelation of the casein micelles. The addition of

156 ionic calcium accelerates milk coagulation and increases gel firmness (Sandra, Ho, Alexander, &

157 Corredig, 2012). The ionic calcium interacts with the negatively charged amino acids, reducing

158 the electrostatic repulsions. In addition, the formation of calcium bridges between the negative

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159 sites helps to increase the gel firming rate (Lucey, 2011). Decreasing the pH also reduces the

160 charges on the micelles, and increases the ionic calcium concentration by solubilising colloidal

161 calcium phosphate, which impairs micelle stability and promotes their aggregation. However,

162 excessive solubilisation of colloidal calcium phosphate has been shown to reduce cross-linking of

163 casein micelles and to produce weaker gels (Choi, Horne, & Lucey, 2007). Aggregation of

164 renneted micelles occurs at a lower extent of -casein hydrolysis as the casein concentration

165 increases (Lucey, 2011). Ultrafiltration of milk is a common practice to increase cheese plant

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166 efficiency and this process reduces the distance between casein micelles, and increases collision

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167 frequency (Karlsson, Ipsen, & Ardö, 2007; Kethireddipalli & Hill, 2015). However, at a given

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168 rennet concentration, complete -casein hydrolysis in concentrated milk takes longer due to
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169 lower rennet/casein ratio.
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170 Several methods have been developed over the years to evaluate rennet-induced

171 gelation of milk. The main interest is to predict the optimal time for gel cutting. According to Fox
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172 et al. (2016), cutting the gel at the optimum firmness is crucial to minimise the losses of fat and
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173 fines in whey and to maximise cheese yield. Lucey (2002) provided a chronological list of the
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174 different techniques used for continuous recording of gel formation. Most of these methods

175 monitor the changes of mechanical properties during milk coagulation, while others measure

176 the changes of optical properties, conductivity (electrical or thermal) or ultrasound propagation.

177 The coagulation profile depends on the physical properties the device is actually measuring,

178 making difficult the comparison between methods. For example, the coagulation profile was

179 determined on the same milk sample using optical densitometry and a commercial device

180 (CoaguSensTM, Chr. Hansen, Hørsholm, Denmark) and compared with the profile obtained by

181 dynamic rheology (Fig. 1; Britten, unpublished results). The optical density (Fig. 1b) increased

182 much faster than the storage modulus (Fig. 1a) and reach a plateau ~30 min after renneting. The

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183 very first signs of aggregation are efficiently captured by the change of opacity, but curd

184 strengthening (t > 30 min) is not detected. The CoaguSensTM device uses vibrations to measure

185 gel firmness. It is very sensitive to detect the first signs of aggregation (Fig. 1c) and the signal

186 continues to increase during gel strengthening phase, although at a lower rate than the storage

187 modulus measured by dynamic rheology. The infection point in the profile also occurs faster

188 (10.7 min) than observe with dynamic rheology (22.9 min). It is believed that the timescale of

189 the applied stress is shorter with the CoaguSens device, which could explain higher sensitivity in

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190 the early stage of coagulation and lower sensitivity in the later stages in comparison with

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191 dynamic rheology.

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193 3. Effect of heat treatment on milk proteins
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195 Heat treatment of milk at a temperature above 60–65 °C can denature whey proteins
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196 and expose side chain groups initially buried in their globular structure. Unfolded whey proteins
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197 are reactive and form aggregates through thiol/disulphide interchange reactions and
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198 hydrophobic interactions (Singh & Waungana, 2001). Complex formation between casein and

199 whey proteins is recognised as the main factor responsible for the poor rennet coagulation

200 properties of heated milk (Kethireddipalli & Hill, 2015).

201

202 3.1. Formation of whey protein/-casein complexes

203

204 Covalent binding through thiol/ disulphide interchange reactions is the main driver for

205 the formation of whey protein (WP)--casein complexes in heated milk (Donato & Guyomarc’h,

206 2009). Hydrophobic interactions may also be involved in the first stage of the association

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207 process (Haque & Kinsella, 1988). The exposed thiol group of -Lg forms disulphide bonds with

208 -casein with no preference between -Cys11 and -Cys88 residues (Livney & Dalgleish, 2004).

209 Kehoe et al. (2007) provided evidence that intramolecular thiol/ disulphide exchange reactions

210 in -Lg occur before linking with -casein. In heated milk, only -Cys66 and -Cys160 residues are

211 involved in the formation of complexes with -casein (Lowe et al., 2004). It was suggested that

212 -Cys160 plays a significant role in the formation of disulphide bonds with -casein in heated milk

213 (Creamer et al., 2004). The -Cys160 residue is located close to the C-terminal end of the

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214 molecule, which could explain the preferential interaction with -casein (Anema, 2019). -La

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215 also participates in the formation of WP/-casein complexes (Corredig & Dalgleish, 1999). It

216
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contains four disulphide bonds and no free thiol group. Although -La cannot initiate the
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217 reaction, once the reaction is triggered, it can be integrated in the complexes. The ratio of β-Lg
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218 to α-La in WP/-casein complexes is reported to be about 3.5 (Anema & Li, 2003b).
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219 The complexation of whey proteins depends on many variables including the time,

220 temperature, heating rate, pH and milk protein concentration (Anema, 2019). In heated milk,
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221 WP/-casein complexes are located at the surface of casein micelles and in milk serum.
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222 Adjusting the pH of milk before heating has a strong influence on the proportion of serum and

223 micelle-bound complexes. For skim milk severely heated at natural pH, about one third of

224 WP/-casein complexes are bound to the casein micelles. This proportion increases to 60% to

225 85% when milk is heated at a pH of 6.4–6.5 and decreases to 10% to 15% when milk is heated at

226 a pH of 6.9 (Anema & Li, 2003b; Kethireddipalli, Hill, & Dalgleish, 2010).

227 The origin of serum WP/-casein complexes is not clear. It is questionable whether the

228 complexes are formed at the surface of casein micelles and then dissociate, or whether -casein

229 first dissociates from the micelles and forms complexes in the serum phase. The dissociation of

230 -casein increases with increasing pH and temperature (Anema, Lee, & Klostermeyer, 2007;

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231 Kudo, 1980) and has been observed in whey protein-free milk (Anema & Li, 2000). It has also

232 been shown that -casein dissociates at lower temperatures than those inducing whey protein

233 denaturation (Anema, 2008). These observations support the view that WP/-casein complexes

234 are formed in the serum phase of milk. On the other hand, Donato, Guyomarc’h, Amiot, and

235 Dalgleish (2007) reported that -casein added to milk did not seem to interact with whey

236 protein during heating and had no effect on complex formation, supporting the initial

237 complexation of whey proteins at the surface of casein micelles. These authors suggested that

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238 the structural characteristics (surface charge, hydrophobicity, accessibility of disulphide bonds)

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239 of -casein at the surface of casein micelles were more favourable for interaction than was the

240
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case for the soluble -casein. However, in a similar study, Anema (2008) observed that adding -
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241 casein to milk significantly increased the proportion of WP/-casein complexes in the serum
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242 phase after heating. According to Anema (2019), -casein dissociates from the micelles early

243 during the heating process and denatured whey proteins subsequently interact with the -
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244 casein either in the serum or on the micelle surface. The ratio of denatured whey protein to -
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245 casein was reported to be higher in serum (~2.4) than in micelle-bound (~1.1) complexes
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246 (Anema & Li, 2003b), suggesting preferential formation of complexes in the serum phase of

247 heated milk (Anema, 2019).

248

249 3.2. Characteristics of casein micelles in heated milk

250

251 Micelle-bound complexes form a coating layer, and increase the size of casein micelles.

252 In heated milk, a linear relationship was found between the casein micelle volume and the

253 proportion of micelle-bound complexes (Anema, 2007). In conditions where the formation of

254 micelle-bound complexes was favoured (pH ~6.5), heating milk at 90 °C increased the micellar

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255 hydrodynamic diameter by 30 to 35 nm (Anema & Li, 2003b). Observation under electron

256 microscope revealed that the micelle-bound complexes protrude from the micelle as

257 filamentous appendages (Donato & Guyomarc’h, 2009; Singh & Waungana, 2001). The surface

258 hydrophobicity of casein micelles was shown to increase slightly with the intensity of heat

259 treatment (Iametti, Corredig, & Bonomi, 1993; Renan, Guyomarc'h, Chatriot, Gamerre, &

260 Famelart, 2007), while a slight decrease of zeta potential was observed (Guyomarc'h, Renan,

261 Chatriot, Gamerre, & Famelart, 2007; Li et al., 2020). Vasbinder and de Kruif (2003) suggested

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262 that the properties of casein micelles in heated milk were not only affected by the amount, but

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263 also by the homogeneity, of the denatured whey protein coating. As a consequence of the

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264 formation of WP/-casein complexes in the serum phase, the proportion of the different caseins
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265 in the micelles is altered by heat treatment. According to the data provided by Anema (2007), -
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266 casein represents 8.7% of micellar casein in non-heated milk at natural pH. After heating at 90 °C

267 for 20 min, it represents only 5.8% of micellar casein and it is further reduced to 4.4% when milk
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268 is heated at pH 7.1. Similar results have been reported for milk heated at 85 °C for 15 s (Li et al.,
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269 2020). The extensive depletion of micellar -casein in heated milk is likely to alter the rennet
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270 coagulation properties of the casein micelles.

271

272 3.3. Characteristics of WP/-casein complexes in the serum phase of heated milk

273

274 The WP/-casein complexes isolated from the serum phase of milk heated at natural pH

275 had a round shape when observed under an electron microscope (Del Angel & Dalgleish, 2006).

276 The size of these complexes increased with the intensity of the heat treatment and values

277 ranging from 25 to 170 nm were reported (Del Angel & Dalgleish, 2006; Donato & Guyomarc’h,

278 2009; Li et al., 2020). The zeta potential of serum complexes was found to be similar to that of

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279 casein micelles, and the apparent isoelectric point was around 4.5 (Guyomarc'h et al., 2007;

280 Jean, Renan, Famelart, & Guyomarc’h, 2006). WP/-casein complexes formed in the serum

281 phase of heated milk are highly hydrophobic particles (Jean et al., 2006). According to

282 Guyomarc'h et al. (2007), the surface hydrophobicity of these particles is much higher than that

283 of casein micelles isolated from heated milk. The -casein in serum complexes is sensitive to

284 chymosin (Mollé, Jean, & Guyomarc'h, 2006), which is likely to further increase the

285 hydrophobicity of these particles when heated milk is renneted.

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286

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287 4. Effect of heat treatment on milk mineral equilibrium

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289 Although minerals account for a relatively small portion of milk solids, they play a critical
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290 role in milk coagulation. Heat treatments affect the salt balance in milk, and can alter rennet-

291 induced gel formation and characteristics.

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292
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293 4.1. Mineral equilibrium in milk

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295 Salts are distributed between the colloidal and the soluble phases of milk. Sodium,

296 potassium and chloride are almost totally dissolved in the aqueous phase of milk, while portions

297 of calcium, phosphorus, magnesium and citrate are bound to the casein micelles (Gaucheron,

298 2005). Insoluble minerals, present in the form of nanoclusters, act as cross-linking agents in the

299 casein micelle structure (Holt, 2004). Casein molecules contain phosphoserine residues, which

300 are the binding sites for calcium phosphate nanoclusters. Calcium and magnesium can also bind

301 directly to phosphoserine and glutamic and aspartic residues (Bauland et al., 2020). In the

302 soluble phase of milk, calcium forms complexes with phosphates and citrates, according to the

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303 association constants and the solubility of the various salts (Gaucheron, 2005). At native milk pH

304 (~ 6.7), 80% of soluble calcium is in the complexed form, and the concentration of ionic calcium

305 is ~2.0 mM (Chandrapala, McKinnon, Augustin, & Udabage, 2010).

306

307 4.2. Factors influencing the mineral equilibrium in milk

308

309 A change in the pH of milk or the addition of various salts influences the mineral balance

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310 in milk. As a result of decreasing pH, the acido-basic groups in milk become more protonated,

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311 causing the solubilisation of colloidal calcium phosphate to restore the equilibrium. In milk, the

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312 colloidal calcium phosphate is completely solubilised at a pH of about 5.2. A lower pH (~3.5) is
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313 required to release calcium that is directly bound to phosphoserine residues of casein (Le Graet
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314 & Brûlé, 1993). The pH-induced changes in salt equilibria are irreversible, and the original

315 structure of calcium phosphate nanoclusters cannot be restored after the acidified milk is
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316 neutralised (Gaucheron, 2011). The addition of salts can alter the mineral equilibrium in milk.
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317 Calcium chloride is commonly added to cheese milk at a low concentration to enhance
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318 coagulation properties. Part of the added calcium reacts with soluble phosphates in the aqueous

319 phase, forming insoluble calcium phosphates and releasing protons, which results in a decrease

320 of pH (Gaucheron, 2011). Adding sodium chloride to milk increases the concentration of soluble

321 calcium and also reduces the pH of milk. Added sodium participates in ion exchange reactions

322 with calcium and protons bound to negatively charged residues of caseins. However, according

323 to Cooke and McSweeney (2017), calcium phosphate nanoclusters are not affected by the

324 addition of sodium chloride to milk.

325

326 4.3. Heat-induced changes to the mineral balance in milk

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327

328 During heating, there is a transfer of soluble calcium and phosphate to the colloidal

329 phase of milk through precipitation as calcium phosphate. This transfer takes place in the first

330 minutes of heating, and intensifies with increasing temperature and pH (Boiani, Fenelon,

331 FitzGerald, & Kelly, 2018; Chandrapala et al., 2010). An increase in temperature promotes the

332 dissociation of phosphoric acids in the aqueous phase of milk (H2PO4- → HPO42- + H+). The

333 dissociated forms of inorganic phosphates have a high affinity for ionic calcium, promoting the

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334 precipitation of calcium phosphate salts (Ca2+ + HPO42- → CaHPO4) on casein micelles

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335 (Gaucheron, 2011). The release of protons during the formation of calcium phosphate is

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336 responsible for the slight decrease of pH after milk is heated (Kethireddipalli & Hill, 2015). The
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337 alteration of the mineral equilibrium of milk heated to temperatures below ~90 °C is almost
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338 totally reversed upon cooling, provided there is enough time for equilibration (de la Fuente,

339 1998). However, more severe heat treatment causes irreversible modifications in the salt
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340 partition (Gaucheron, 2005; Holt, 1995). Although the salt balance is restored after cooling
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341 moderately heated milk, the organisation of micellar calcium phosphate is altered. Heat-
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342 precipitated calcium phosphate is different from native micellar calcium phosphate and is less

343 able to maintain the micellar structure (Raynal & Remeuf, 1998). It is also less soluble than the

344 native micellar calcium phosphate (van Hooydonk, de Koester, & Boerringter, 1987) and, during

345 cooling, the solubilisation of native micellar calcium phosphate is favoured. As a result, native

346 micellar calcium phosphate is partially replaced by precipitated calcium phosphate after heated

347 milk is cooled, with potential impacts on milk coagulation (Choi et al., 2007).

348

349 5. Effect of milk heat treatment on the primary phase of coagulation

350

16
351

352 Heat treatment at temperatures allowing for whey protein denaturation is known to

353 impair the rennet coagulation properties of milk. Heated milk has a longer coagulation time and

354 forms a softer gel than unheated milk (Dinkov & Dushkova, 2007; Kethireddipalli et al., 2010;

355 Singh & Waungana, 2001). It has been suggested that the hydrolysis of -casein in heated milk is

356 significantly inhibited or incomplete. The accessibility of the rennet sensitive bond of -casein

357 (Phe105–Met106) could be reduced by the formation of complexes with whey proteins or by

of
358 precipitation of calcium phosphate resulting from heat treatment. However, it has been

ro
359 observed that heating milk at temperatures below 100 °C alters the kinetics of CMP release after

-p
360 renneting only slightly (Leaver, Law, Horne, & Banks, 1995). Vasbinder, Rollema, and de Kruif
re
361 (2003) reported that heating milk at 90 °C for 10 min reduced the amount of CMP released by
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362 8% compared with unheated milk, while a 10% to 15% reduction was reported by Sandra and

363 Dalgleish (2007) for milk heated at 85 °C for 10 min. It has been observed that heating whey
na

364 protein-free milk (90 °C for 10 min) had no effect on the release of CMP. This excludes the
ur

365 precipitation of calcium phosphate as a factor influencing the enzymatic hydrolysis of -casein
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366 (Vasbinder et al., 2003). Consequently, the effect of heat treatment on the kinetics of -casein

367 hydrolysis has been attributed to the denaturation of whey protein. Ferron-Baumy, Maubois,

368 Garric, & Quiblier (1991) and Calvo, Law, and Leaver (1995) also reported a partial inhibition of

369 CMP release in heated milk. On the other hand, Anema et al. (2007) reported identical kinetics

370 of CMP release for non-heated milk and milk heated at different pH levels. Kethireddipalli et al.

371 (2011) reported only a marginal effect for milk heated at pH 6.3 and 7.1. While there is still

372 some disagreement regarding the effect of heating on the rate of CMP release, it is generally

373 accepted that the slight inhibition of enzymatic activity cannot explain the poor clotting

374 properties of heated milk (Kethireddipalli & Hill, 2015; Vasbinder et al., 2003). The formation of

17
375 WP/-casein complexes at the surface of casein micelles or in the serum of heated milk is likely

376 to interfere with micelle fusion and casein network contraction, which would have more

377 intensive effect on the secondary phase of coagulation (Donato & Guyomarc’h, 2009).

378

379 6. Effect of milk heat treatment on the secondary phase of coagulation

380

381 Milk heated at temperatures above ~75 °C takes a longer time to clot and forms weak

of
382 gels. Depending on the intensity, heat treatments can partially or totally inhibit the aggregation

ro
383 of renneted micelles and the formation of a self-supporting network (Raynal & Remeuf, 1998).

-p
384 The poor coagulability of heated milk relative to that of unheated milk is due to the
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385 denaturation of whey proteins and the heat-induced alteration of mineral equilibrium.
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386

387 6.1. Whey protein denaturation

na

388
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389 A correlation was observed between the level of whey protein denaturation and the
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390 deterioration of renneting properties of heated milk (Guinee et al., 1997; Waungana, Singh, &

391 Bennett, 1996). The gelation time gradually increased with the level of whey protein

392 denaturation, and the gel firmness decreased. In comparison with unheated milk, the gelation

393 time of heated milk with 30% whey protein denaturation increased by 9% while the curd firming

394 rate and storage modulus at 60 min (G’60) decreased by 40% and 30% respectively (Giroux,

395 Dupont, Villeneuve, & Britten, 2020). Total inhibition of rennet gel formation was reported

396 when the level of whey protein denaturation reached ~70% (Waungana et al., 1996).

397 The formation of WP/-casein complexes at the surface of casein micelles appears to be

398 the main factor responsible for the adverse effect on the renneting properties of heated milk.

18
399 Even after complete release of CMP, the presence of complexes attached to the surface of the

400 micelles is believed to cause steric hindrance, which prevents aggregation (Singh & Waungana,

401 2001; Vasbinder & de Kruif, 2003). Considering that heating milk at a higher pH reduces the

402 proportion of micelle-bound complexes (Anema & Li, 2003a), it was expected that alkalinisation

403 before heating would improve the coagulation properties of heated milk (Ménard, Camier, &

404 Guyomarc'h, 2005). Working on dilute suspensions of skim milk heated at various pH values,

405 Anema, Lee, and Klostermeyer (2011) confirmed that micelles with low levels of bound

of
406 complexes aggregated faster than micelles with higher levels of bound complexes. However, in

ro
407 non-diluted milk, the deterioration of coagulation properties seems to be independent of milk

-p
408 heating pH (Anema et al., 2007; Giroux et al., 2020; Kethireddipalli et al., 2010). It was suggested
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409 that in milk heated at higher pH, micelles depleted in WP/-casein complexes rapidly aggregate
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410 after renneting, but that the gradual incorporation of complexes from the serum phase slows

411 down the aggregation process and prevents gelation (Anema et al., 2011). This mechanism is
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412 supported by the results of Kethireddipalli et al. (2011) which confirmed the interaction
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413 between renneted micelles and WP/-casein complexes from the serum phase. According to
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414 Lin, Kelly, O'Mahony, and Guinee (2018), the extensive depletion of micellar -casein in milk

415 heated at high pH also impairs the coagulation process. Renneted micelles with a lower content

416 of para--casein are likely to have reduced hydrophobic interactions and to exhibit a lower

417 capacity to build up the casein network.

418 It is widely accepted that WP/-casein complexes are responsible for the poor

419 coagulation properties of heated milk. In an attempt to differentiate the contributions of WP/-

420 casein complexes bound to casein micelles from those in the serum phase, Kethireddipalli et al.

421 (2010) conducted a series of serum exchange experiments (Fig. 2). In a first set of trials, micelles

422 collected from milk heated at different pH values were suspended in serum from non-heated

19
423 milk. As expected, the coagulation properties of micelles from milk heated at pH 6.3 (~80%

424 micelle-bound WP) did not recover when suspended in native serum. Interestingly, the

425 coagulation properties of micelles from milk heated at pH 7.1 (~2% micelle-bound WP), were

426 only partially recovered when suspended in native serum (free of WP/-casein complexes).

427 Compared with the gel from non-heated milk, maximum firmness was still 85% lower. As

428 previously proposed, the depletion of micellar -casein seems to be largely responsible for the

429 poor coagulation properties of milk heated at high pH (Lin et al., 2018).

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430 In a second set of trials, native micelles collected from non-heated milks were

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431 suspended in the serum from milks heated at different pH levels. The coagulation of native

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432 casein micelles was totally inhibited when suspended in the serum from milk heated at pH 7.1
re
433 (rich in WP/-casein complexes), a result that supports the idea that WP/-casein complexes in
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434 the serum phase interact with renneted micelles and interfere with the coagulation process

435 (Kethireddipalli et al., 2011). However, it was also observed that the dialysis of heated serum
na

436 against non-heated milk significantly reduces its negative impact on the coagulation of native
ur

437 micelle. Apparently, the minerals are involved in the clotting impairment of casein micelles by
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438 WP/-casein complexes, but the mechanism is still poorly understood (Kethireddipalli & Hill,

439 2015).

440

441 6.2. Calcium phosphate precipitation

442

443 The effect of heat-induced changes of mineral distribution on the rennet coagulation

444 properties of milk is certainly less pronounced than the effect of whey protein denaturation

445 (Guyomarc'h, 2006). Nevertheless, it is generally accepted that these changes can modulate

446 rennet gel formation. Mild treatment, such as preheating milk at ~65 °C, was reported to

20
447 improve rennet coagulation properties due to the slight decrease of pH resulting from calcium

448 phosphate precipitation (Fox et al., 2016). However, the change in the mineral balance

449 associated with more severe treatments contributes to the degradation of milk coagulability. It

450 was suggested that the decrease of soluble calcium concentration in heated milk retarded

451 rennet gel formation, due to the increase in the net negative charge of casein micelles

452 (Guyomarc'h, 2006). On the other hand, it has been reported that heating whey protein-free

453 milk at 90 °C for 10 min had no effect on the onset of renneted micelles aggregation (Vasbinder

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454 et al., 2003). According to these authors, the loss of up to 15% of soluble calcium in heated milk

ro
455 does not significantly increase clotting time, but is likely to reduce the gel strength.

-p
456 Heat treatment induces the precipitation of calcium phosphate, which alters the micelle
re
457 structure and its gel forming properties (Kethireddipalli & Hill, 2015). Heat-precipitated calcium
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458 phosphate differs from native colloidal calcium phosphate: it is less soluble and has no ability to

459 cross-link caseins (de la Fuente, 1998). When heated milk is cooled back to room temperature,
na

460 the native colloidal calcium phosphate is preferentially solubilised (Raynal & Remeuf, 1998),
ur

461 resulting in a lower casein cross-linking capacity. Choi et al. (2007) clearly showed that reducing
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462 the concentration of native colloidal calcium phosphate in milk significantly reduces the rennet

463 gel firming rate and the final gel firmness. It was observed that cold storage of heated milk for

464 several hours further deteriorates gelation properties (Lucey, 1995). This phenomenon, known

465 as rennet hysteresis, is mainly attributed to further solubilisation of native micellar calcium

466 phosphate at low temperature (Remeuf & Raynal, 2001). The slight increase of pH

467 accompanying calcium phosphate solubilisation (Fox et al., 2016) and possibly some continuing

468 structural changes in the WP/-casein complexes (Lucey, 2011) may also contribute to the

469 rennet hysteresis of heated milk.

21
470 Heating milk at temperatures above 100 °C severely impairs rennet-induced gelation

471 properties, but in these conditions, the contribution of changes in the mineral balance is difficult

472 to determine due to the overriding effect of whey protein denaturation. In a series of

473 experiments, Bulca, Leder, and Kulozik (2004) measured the coagulation properties of milks with

474 reduced whey protein contents. They reported that for a similar level of whey protein

475 denaturation, the deterioration of milk coagulation properties increased with an increase in

476 heating temperature from 100 °C to 140 °C. A similar study was performed on milk after total

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477 removal of whey proteins (Schreiber, 2001) and a strong correlation was observed between the

ro
478 precipitation of calcium and the reduction of gel strength. In this study, gel strength was

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479 reduced by half after heating whey protein-free milk at 100 °C for 400 s. For comparison
re
480 purposes, a similar decrease of gel strength was observed after heating regular milk (containing
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481 whey proteins) at the same temperature for only 4 s (Singh & Waungana, 2001). These results

482 confirm the predominant role of whey protein denaturation as an explanation for the heat-
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483 induced deterioration of milk renneting properties and suggest that the changes in the mineral
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484 balance make a modest contribution.

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485

486 7. Improvement of the coagulation properties of heated milks

487

488 The negative effect of heat treatment on the rennet coagulation of milk can be partially

489 reversed. Various approaches have been proposed to reduce milk clotting time and increase the

490 gel firmness. These approaches are based on the modification of the mineral equilibrium, the

491 use of membrane separation technologies and high-pressure treatments.

492

493 7.1. Modification of mineral equilibrium

22
494

495 The mineral equilibrium affects all aspects of cheese production, including the

496 coagulation of milk after renneting. Adding calcium chloride is a common practice to speed up

497 milk clotting and increase gel firmness. It can also improve the coagulation properties of heated

498 milk, provided that the intensity of the heat treatment is not too severe (Lucey, 1995). Ionic

499 calcium screens the charges on casein micelles and lowers milk pH slightly; both of these factors

500 reduce inter-particle repulsions and promote aggregation (Guyomarc'h, 2006; Singh &

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501 Waungana, 2001). Calcium has been shown to bind to renneted micelles and it was suggested

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502 that calcium binding strengthened short range interactions between and increased gel firmness

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503 (Corredig & Salvatore, 2016). The exact mechanism is not yet established, but it has been
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504 hypothesised that calcium changes the conformation of para--casein which increases the
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505 exhibition of hydrophobic binding sites (Bringe & Kinsella, 1986). The concentration of calcium

506 chloride required to restore the coagulation properties of heated milk was shown to increase
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507 with the intensity of the heat treatment (Lucey, 1995). There is, however, a limit to the amount
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508 of calcium chloride that can be added to heated milk. In excess of ~1 g L-1 CaCl2, binding of
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509 calcium to casein micelles can increase the positive charge and interparticle repulsions,

510 inhibiting rennet gel formation (Sandra et al., 2012). For milk heated at 80 °C for 1 min, Remeuf

511 and Raynal (2001) observed a total recovery of the coagulation properties after the addition of

512 CaCl2. However, when holding time at 80 °C was increased to 10 min, only limited recovery was

513 observed and rennet gel firmness was 80% lower in comparison with gel from non-heated milk.

514 Acidification is another approach that can be used to improve the coagulation

515 properties of heated milk. Faster clotting and firmer gels were observed after the pH of heated

516 milk was reduced (Remeuf & Raynal, 2001; Singh & Waungana, 2001). Maximum gel strength

517 was observed at a pH of around 6.2 (Lucey, 1995). The improvement of gelation properties is

23
518 attributed to the reduced electrostatic repulsions between casein micelles at lower pH and the

519 increased ionic calcium concentration resulting from the solubilisation of colloidal and heat-

520 precipitated calcium phosphate (Kethireddipalli & Hill, 2015). Increased rennet activity at lower

521 pH may also help to reduce the clotting time of heated milk (Fox et al., 2016). The main

522 drawbacks of the milk acidification step in cheese making are the increased retention of rennet

523 in cheese curd and the decreased production of lactic acid by starter bacteria. These factors can

524 negatively affect flavour development during ripening (Guyomarc'h, 2006). As an alternative, it

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525 has been proposed that acidified milk be neutralised just before renneting (Singh & Waungana,

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526 2001). This so-called pH-cycling treatment requires that the pH of the heated milk be reduced to

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527 about 5.5 and then neutralised to renneting pH (~6.6). Upon neutralisation, only a portion of the
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528 calcium phosphate solubilised during the acidification step returns to the colloidal phase. The
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529 concentration of ionic calcium increases after this treatment (Singh, 1988). It was also suggested

530 that the composition and properties of reformed colloidal calcium phosphate are closer to those
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531 of native colloidal calcium phosphate (van Hooydonk et al., 1987). The improvement in
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532 coagulation properties is attributed to both factors: the reformed colloidal calcium phosphate
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533 and the increased concentration of ionic calcium. Increasing the holding time at low pH, to 24 h

534 at 4 °C, was shown to further increase the efficiency of the pH-cycling treatment (Lucey, 1995).

535 Total recovery of milk clotting time and partial recovery of gel firmness were reported after pH-

536 cycling of milk heated for 10 min at temperatures of up to 100 °C; however, more severely

537 heated milk (120 °C for 10 min) showed no signs of coagulation after the pH-cycling treatment

538 (Singh & Waungana, 2001).

539

540 7.2. Use of membrane separation technologies

541

24
542 Membrane separation technologies are widely used in the cheese industry to

543 standardise the composition of cheese milk (Mistry & Maubois, 2017). Ultrafiltration is used to

544 concentrate total milk proteins, while microfiltration selectively concentrates micellar casein.

545 These processes can be applied before or after heat treatment to modulate the coagulation

546 properties of milk.

547 The main purpose of heating milk at temperatures above pasteurisation is to increase

548 whey protein retention in cheese. However, severe heat treatment can also be applied to

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549 prevent problems with sporulating bacteria (Schreiber, 2001) or to produce milk powders with

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550 improved cheese-making properties (Garem, Schuck, & Maubois, 2000). In these applications,

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551 the removal of whey protein before heating may well be justified. Microfiltration can be used to
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552 concentrate caseins and remove a portion of whey proteins from milk (Soodam & Guinee, 2018;
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553 Xia et al., 2020). When used in diafiltration mode, microfiltration can almost completely remove

554 whey proteins (Bulca et al., 2004; Schreiber, 2001). Whey protein denaturation is the main
na

555 factor responsible for the poor coagulability of heated milk and, as expected, heating whey
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556 protein-free milk at 90 °C for 15 s (Xia et al., 2021) or between 110 °C and 140 °C for 10 s (Bulca
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557 et al., 2004) showed good coagulation properties. However, longer holding times (up to 10 min)

558 at temperatures above 100 °C, significantly reduced rennet gel strength (Schreiber, 2001). This

559 effect was attributed to the lower concentration of soluble calcium after heating. The addition

560 of calcium chloride or pH-cycling may increase rennet gel strength; however, to our knowledge,

561 these corrective treatments have not yet been tested on severely heated whey protein-free

562 milk.

563 The concentration of total milk protein by ultrafiltration increases the rennet gel firming

564 rate and final gel strength. This effect is directly related to the increase of casein concentration

565 (Mistry & Maubois, 2017). Ultrafiltration can therefore be used to compensate for the negative

25
566 effect of heat treatment on the coagulation properties of milk (Singh & Waungana, 2001).

567 Ultrafiltration can be applied before or after heat treatment of milk; however it has been shown

568 that stronger rennet gels are obtained when ultrafiltration is performed before heat treatment

569 (Waungana, Singh, & Bennett, 1998). The ratio of soluble minerals to casein is reduced by milk

570 ultrafiltration, which is likely to reduce the amount of calcium phosphate precipitated onto

571 casein micelles after thermal treatment. One limitation of using ultrafiltered milk for cheese

572 making is the narrow window for cutting the curd at the optimal firmness (Panthi et al., 2019).

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573 This is critical for large and automated cheese vats, where the time required for cutting the curd

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574 is 5 to 10 min. Optimal combinations of ultrafiltration and heating conditions have been shown

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575 to produce heated milk retentates with curd firming rate similar to those of pasteurised milk
re
576 (Bulca, Tolkach, Kupfer, & Kulozik, 2010). This approach can be used to maximise the benefits of
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577 processing high protein milk with increased whey protein recovery.

578
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579 7.3. High-pressure treatments

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580
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581 High hydrostatic pressure (HHP) treatment influences the coagulation and cheese-

582 making properties of milk (López-Fandiño, Ramos, & Olano, 1997; Needs, Stenning, Gill,

583 Ferragut, & Rich, 2000). HHP treatment alters milk proteins by two simultaneous mechanisms:

584 the disruption of casein micelles and the denaturation of whey proteins (Huppertz et al., 2005).

585 In the low-pressure range (100–300 MPa), the fragmentation of casein micelles dominates and is

586 responsible for the reduced clotting time and increased gel firmness (Zobrist, Huppertz, Uniacke,

587 Fox, & Kelly, 2005). This effect is attributed to the larger surface area available for inter-micellar

588 interactions, which promotes aggregation (Corredig & Salvatore, 2016). HHP treatment of

589 heated milk significantly improves the rennet coagulation properties. The coagulation properties

26
590 of milk heated at 90 °C for 10 min were fully recovered after HPP treatment at pressure > 250

591 MPa (Huppertz et al., 2005). In comparison with non-heated milk, the rennet clotting time was

592 even shorter and the gel firmer. HPP is an efficient treatment for improving the coagulation

593 properties of heated milk. However, the limited size and high cost of the available processing

594 units are major drawbacks for applications in large cheese plants (Masotti, Cattaneo, Stuknytė,

595 & De Noni, 2017).

596

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597 7.4. Other approaches

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598

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599 Alternative approaches have been investigated for maintaining the coagulation
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600 properties of heated milk while taking advantage of the retention of denatured whey proteins in
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601 cheese. Zoon (1994) proposed that severely heated milk be mixed with non-heated milk, so that

602 unaffected casein micelles can drive the coagulation process. This approach was recently
na

603 revisited and compared with heating milk at 80 °C for different lengths of time (Giroux et al.,
ur

604 2020). However, for similar levels of whey protein denaturation, mixing heated milk with non-
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605 heated milk showed a more detrimental effect on rennet-induced coagulation kinetics and

606 higher moisture retention in cheese.

607 The poor coagulation properties of heated milk is correlated with the formation of

608 complexes between denatured serum proteins and -casein (Singh & Waungana, 2001). To

609 avoid the formation of these complexes, it has been proposed that heat treatment be applied

610 only to the whey protein fraction of milk and that this fraction be added to non-heated cheese

611 milk (Banks & Muir, 1985). The whey protein fraction can be recovered by the ultrafiltration of

612 cheese whey (Hinrichs, 2001) or milk microfiltration permeate (Maubois, Fauquant, Famelart, &

613 Caussin, 2001). The whey protein concentrate is then treated using a combination of heating

27
614 and shearing (microparticulation process) to produce aggregates of optimal size for inclusion

615 and retention in the cheese matrix (Hinrichs, 2001). Denatured whey protein aggregates are

616 often considered as non-interacting filler particles; however, they have been shown to impair

617 rennet coagulation properties when added to milk (Perreault, Turcotte, Morin, Pouliot, &

618 Britten, 2016). Subsequent investigations have demonstrated that a portion of heat-denatured

619 whey proteins exists as soluble complexes after treatment (Perreault, Morin, Pouliot, & Britten,

620 2017a). It is believed that these complexes can interact with renneted micelles and interfere

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621 with gel formation (Giroux, Lanouette, & Britten, 2015). Compared with heat treatment of milk,

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622 adding denatured whey proteins to non-heated milk entails additional steps for the separation,

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623 concentration and thermomechanical treatment of whey proteins. However, the detrimental
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624 effect on rennet coagulation properties and the increase of cheese moisture are reduced
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625 significantly. For similar concentrations of denatured whey protein retained in cheese, the

626 increase in moisture was found to be four times lower with the addition of denatured whey
na

627 proteins compared with the use of heated milk (Fig. 3; Britten, 2018, unpublished).
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628
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629 8. Cheese making using heated milk

630

631 Heat treatments impair the rennet coagulation properties of milk, and by extension,

632 they alter the characteristics of cheese. Depending on the severity of the treatment, cheese

633 composition, texture and flavour development can be affected.

634 Heat treatment of milk increases protein recovery in cheese due to the denaturation of

635 serum proteins. The formation of WP--casein complexes at the surface of casein micelles or in

636 the serum phase interferes with curd contraction, slows down curd syneresis and increases

637 moisture retention (Giroux et al., 2020). The high water binding capacity of denatured whey

28
638 protein also contributes to moisture retention (Perreault et al., 2017b). The effect of

639 denaturation rate of whey protein in cheese milk on both the coagulation properties and some

640 cheese characteristics is summarised in Fig. 4. With higher moisture content, the amount of

641 residual lactose in cheese increases, which promotes lactic acid production by starter cultures.

642 Higher moisture content also lowers the concentration of casein and colloidal minerals, resulting

643 in a lower buffering capacity. These two factors lead to excessive acid production and lower

644 cheese pH compared with cheese made from non-heated milk (Kethireddipalli & Hill, 2015).

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645 The microstructure and mechanical properties of rennet gel network from heated milk

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646 may differ significantly from those of unheated milk. The rennet gel from pasteurised milk is

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647 characterised by large void spaces and thick protein cross links, while a dense and compact
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648 network, with smaller void spaces is observed in gels from milk heated at 90 °C for 5 min
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649 (Miloradovic et al., 2020). Curds from heat-treated milks tend to be soggy and ragged in

650 appearance and show poor matting ability (Singh & Waungana, 2001). Impaired fusion of curds
na

651 leads to cheese with a coarser and less homogeneous matrix, containing numerous small holes
ur

652 and cracks. Hard cheeses made from heated milk are generally soft and crumbly. The functional
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653 properties of cheeses may also be adversely affected by milk heat treatment. Poor melting and

654 stretching properties were observed in cheeses such as Cheddar and mozzarella (Banks, 1990).

655 Cheese melting is driven by the decrease of the number and strength of casein-casein

656 interactions. Hydrophobic attractive forces increase in strength with temperature which causes

657 the contraction of individual casein molecules. This contraction provides fluidity by reducing the

658 contact area between caseins (Lucey, Johnson, & Horne, 2003). It is accompanied by the release

659 of water, which acts as a lubricant and promotes cheese fluidity (Guinee, 2016). Electrostatic

660 repulsions also increase with temperature (Bryant & McClements, 1998) and contribute to

661 cheese melting. In cheese produced from heated milk, cross links between -casein and β-

29
662 lactoglobulin, through disulphide bonds, reduce the contraction capacity of casein molecules

663 and restrain cheese meltability (Lucey et al., 2003). Furthermore, the lower pH of cheeses made

664 from heated milk (Rynne, Beresford, Kelly, & Guinee, 2007) is likely to reduce electrostatic

665 repulsions between caseins and negatively affect melting and stretching properties.

666 Heat treatment of cheese milk can also affect the maturation and the flavour profiles of

667 cheeses. Indigenous milk microflora and enzymes contribute to flavour development; they are

668 partially or totally inactivated by heat treatments. Furthermore, changes such as increased

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669 moisture content, reduced pH, and altered mineral balance, all influence the cheese ripening

ro
670 process (Kethireddipalli & Hill, 2015). Cheese maturation involves an extremely complex set of

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671 biochemical reactions, and changes induced by milk heat treatment can cause an atypical
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672 flavour profile. The breakdown of caseins during ripening not only affects the cheese flavour but
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673 also the texture and functional properties of cheese (McMahon & Oberg, 2017).

674 The characteristics of cheese produced from thermally treated milk vary according to
na

675 the heating conditions. Cheese-making parameters can be optimised to improve cheese quality.
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676 Corrections, such as reducing renneting pH or increasing the coagulation and cooking
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677 temperatures, were used to avoid excessive moisture content (Singh & Waungana, 2001). Other

678 corrective treatments have been proposed to better control the composition, functionality, and

679 organoleptic qualities of cheeses made from heated milk. An extensive review and listing of

680 these restorative treatments can be found elsewhere (Kethireddipalli & Hill, 2015).

681

682 9. Conclusions and perspectives

683

684 Heating milk offers the opportunity to increase whey protein retention in cheese but

685 producing good quality cheese from thermally processed milk is not simple. Complex

30
686 physicochemical changes occur during heat treatment, altering milk coagulation properties and

687 cheese characteristics. It is necessary to adapt the cheese-making parameters and correct the

688 mineral balance to partially reverse the negative effects of heat treatment and better control

689 cheese moisture. Combining ultrafiltration and heat treatment is a suitable approach for

690 maximising the benefits of better protein recovery and increased plant efficiency. High

691 hydrostatic pressure processing can fully restore the coagulation properties of heated milk, but

692 large-scale applications are limited at the present time. Considering that HPP treatment of raw

of
693 milk ensures microbiological safety, induces whey protein denaturation and increases cheese

ro
694 yield, there may be no additional benefit from applying it to heated milk. The reincorporation of

-p
695 heat-treated whey proteins into cheese milk entails additional processing steps but appears to
re
696 be less deleterious to cheese quality when compared with directly heating cheese milk.
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697 Significant progress has been made in gaining a better understanding of the effect of

698 heat treatments on the coagulation properties of milk, and corrective measures have been
na

699 developed to improve the quality of cheeses made from heated milk. At the same time, it must
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700 be emphasised that even after correction, the characteristics of heated milk differ from those of
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701 non-heated milk. Production of existing types of cheese from heated milk is a challenge and

702 depending on the variety concerned, is limited to a certain degree of whey protein

703 denaturation. However, the use of heated milk is more appropriate for the development of new

704 cheese products with different textural and functional characteristics.

705

References

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Anema, S. G. (2007). Role of -casein in the association of denatured whey proteins with casein

micelles in heated reconstituted skim milk. Journal of Agricultural and Food Chemistry,

55, 3635–3642.

Anema, S. G. (2008). On heating milk, the dissociation of -casein from the casein micelles can

precede interactions with the denatured whey proteins. Journal of Dairy Research, 75,

415–421.

Anema, S. G. (2019). The whey proteins in milk: Thermal denaturation, physical interactions, and

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effects on the functional properties of milk. In M. Boland & H. Singh (Eds.), Milk

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Figure legends

Fig. 1. Coagulation profiles of pasteurised milk (3.33% protein, 3.96% fat) at 32 °C and pH 6.5 after

rennet addition (0.01%, v/w). Coagulation was monitored using dynamic rheometry (a) optical

densitometry ( = 650 nm) (b) and CoaguSensTM (c). Dashed line is the first derivative or the

coagulation curve and, the hatched zone corresponds to the optimal time for gel cutting (cutting

window).

f
oo
Fig. 2. Rennet gel firmness of mixtures of heated and non-heated milk fractions. Milk was heated at

90 °C for 10 min at pH 6.3 () and 7.1 (). Gel firmness (G’ measured 3.5 h after renneting) is

r
-p
reported relative to the firmness of gel from non-heated milk (control). Adapted from Kethireddipalli
re
et al. (2010).
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Fig. 3. Effect of denatured whey protein on cheese moisture content. Denatured whey protein
na

(DWP) was incorporated in cheese by heating milk at 80 °C for various times prior to cheese making
ur

() or by adding various amounts of denatured whey protein concentrate to cheese milk ().
Jo

Fig. 4. Effect of whey protein denaturation rate in milk on rennet coagulation parameters (a) and

cheese properties (b). Results are reported relative to those obtained with non-heated milk

(control). Rennet clotting time (), Gel firming rate (  ), Gel firmness ( ), Cheese moisture (),

Total milk protein recovery (), Cheese yield (). Adapted from Giroux et al. (2020).
 120 0.06
a
100 0.05

80 0.04
G' (Pa)

dG'/dt
60 0.03

40 0.02

20 0.01

0 0

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0 10 20 30 40 50 60
Time (min)

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1 0.0020
0.9 b 0.0018

-p
0.8 0.0016
0.7 0.0014
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0.6 0.0012

dOD650/dt
OD650

0.5 0.0010
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0.4 0.0008
0.3 0.0006
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0.2 0.0004
0.1 0.0002
0 0.0000
ur

0 10 20 30 40 50 60
Time (min)
Jo

300 0.7
c 0.6
250
0.5
Firmness F (Pa)

200
0.4
dF/dt

150
0.3
100
0.2
50 0.1

0 0
0 10 20 30 40 50 60
Time (min)

Figure 1
 1.0
Gel firmness relative to control

0.8

0.6

0.4

of
0.2

ro
0.0
Heated milk

-p
Heated micelles/ Native micelles/ Native micelles/
non-heated serum serum from heated dialyzed serum from
re
milk heated milk
lP
na
ur
Jo

Figure 2
 57

Cheese moisture (%) 55

53

51

of
ro
49

-p
re
47
lP

0 1 2 3 4 5 6
DWP in cheese (%)
na
ur
Jo

Figure 3
 1.20
a
1.00
Coagulation parameter
(relative to control)

0.80

0.60

0.40

0.20
0 5 10 15 20 25 30 35 40 45

of
Whey protein denaturation (%)

ro
1.20 b
(relative to control)

1.15

-p
Cheese property

1.10
re
1.05
lP

1.00
na

0.95
0 5 10 15 20 25 30 35 40 45
Whey protein denaturation (%)
ur
Jo

Figure 4
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

Both authors declare that they have no conflict of interest.

of
ro
-p
re
lP
na
ur
Jo