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The stabilisation of acidified whole milk drinks by carboxymethylcellulose
International Dairy Journal 28 (2013) 40e42
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
The stabilisation of acidified whole milk drinks by carboxymethylcellulose Juan Wu, Junliang Liu, Qiaoyu Dai, Hongbin Zhang* Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2012 Received in revised form 23 May 2012 Accepted 24 May 2012
Carboxymethylcellulose (CMC) was used as a stabiliser for acidified whole milk drinks. The stability of the acidified whole milk drinks was investigated by observation of the evolution of size and zeta potential of colloidal particles during acidification along with sedimentation measurement and was compared with the stability of acidified skim milk drinks. It was found that the presence of fat resulted in larger particle sizes, but it did not disturb the absorption of CMC onto casein micelles below pH 5.2. The absorption of CMC endows the casein micelles with electrostatic and steric repulsions, essential to the stability of the acidified milk drinks. Acidified whole milk drinks can be stabilised by CMC, which is capable of effectively preventing the unwanted creaming of fat embedded in the clusters of CMC and caseins as well as preventing the aggregation of casein micelles as it did in skim milk drinks. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Acidified milk drinks are products available worldwide in many variations, with pH values ranging from 3.6 to 4.6. Because casein micelles will aggregate and precipitate below their isoelectric point (4.6), the stabilisation of acidified milk drinks is necessary to prevent instability occurring. Acidified milk drinks are usually stabilised with pectin (Jensen, Rolin, & Ipsen, 2010; Laurent & Boulenguer, 2003; Tromp, de Kruif, van Eijk, & Rolin, 2004; Tuinier, Rolin, & de Kruif, 2002). Carboxymethylcellulose (CMC) is commonly chosen as a stabiliser instead of pectin because of its low cost in Asia, especially in China, where a long shelf life is required of many food products. Our previous work reported the stabilising effect of CMC on acidified skim milk drinks (ASMD) (Du et al., 2007, 2009). However, many commercial products actually contain whole milk, which gives a full-bodied taste because of its high fat content compared with skim milk. Whole milk products seem to be more popular in China. For acidified whole milk drinks (AWMD), creaming is a problem because of the presence of large amounts of fat. The large size and low density of fat may yield stability of AWMD different from that of ASMD. There are limited reports on the application of CMC in AWMD; therefore, there is a need to investigate and understand the CMC-induced stabilisation of AWMD. In the present work, the stabilisation of acidified whole milk by CMC was investigated by monitoring changes in particle size and zeta potential during
* Corresponding author. Tel.: þ86 21 54745005. E-mail address: [email protected] (H. Zhang). 0958-6946/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2012.05.005
acidification and sedimentation measurement and compared with acidified skim milk. 2. Materials and methods 2.1. Materials Skim and whole milk powders were obtained from Fonterra Cooperative Group Ltd. (Auckland, New Zealand). CMC sample (250 kDa, DS ¼ 0.7) was purchased from Acros Organics (Morris Plains, NJ, USA). Citric acid and other analytical grade chemicals used were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Particle size and zeta potential analysis Reconstituted whole milk (8%) was diluted at a ratio of 1e100 with simulated milk ultrafiltrate (SMUF) (Jenness & Koops, 1962). Samples for particle size and zeta potential analysis were prepared as previously reported (Du et al., 2007, 2009). The size and zeta potential of colloidal particles were determined using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) at 25 C. All measurements were performed three times. 2.3. Preparation of acidified whole milk drinks and measurement of sedimentation The samples were prepared on a pilot scale as previously reported (Du et al., 2007, 2009). Whole milk powders were dissolved
J. Wu et al. / International Dairy Journal 28 (2013) 40e42
in distilled water to obtain 8% reconstituted whole milk, and then mixed with 0.8% CMC solution at a 1:1 (v/v) ratio. The mixture was directly acidified to pH 4.0, homogenised at 200 bar with a homogeniser (Rannie TYPE 8.30 H; APV Rannie A/S, Denmark), pasteurised at 110 C for 30 s and then stored in sealed bottles at 4 C overnight prior to stability evaluation. For stability evaluation, 10 g of AWMD were centrifuged at 1800 g for 20 min at 25 C. The sedimentable fraction was calculated by the ratio of the weight of sediment to the weight of the sample. All measurements were performed in triplicate. 3. Results and discussion 3.1. Comparison of the particle size and zeta potential in diluted whole and skim milk systems with and without CMC during acidification Fig. 1 shows the evolution of (a) particle size and (b) zeta potential during acidification of diluted whole and skim milk with different concentrations of CMC. As seen in Fig. 1a, for both kinds of milks without CMC, as the pH decreased from 5.8 to 5.2, the diameter of colloidal particles in the system decreased, which is mainly attributed to the collapse of the hairy k-casein layer on the micellar surface (Alexander & Dalgleish, 2004; Donato, Alexander, & Dalgleish, 2007). Below pH 5.2, the size greatly increased and macroscopic precipitation could be observed within a few minutes. As for the change in zeta potential for the system without CMC, a steady increase in zeta potential with increasing pH was observed. During acidification, k-caseins located outside of the micelles as flexible “hairs” collapsed (Walstra, 1990), thereby significantly weakening their steric repulsion, leading to the aggregation of caseins and, thus, the instability of the milk. Upon addition of different concentrations of CMC to the diluted whole milk, the particle size decreased as the pH was lowered from pH 5.8 to 5.2, indicating that CMC did not affect the aforementioned collapse of k-caseins. At pH below 5.2, the particle size increased slightly, unlike the dramatic increase in the system without CMC during acidification. This increase is due to the adsorption of CMC onto casein micelles, which can be also confirmed by the evolution of zeta potential, as shown in Fig. 1b. Between pH 5.2 and 4.8, the zeta potential of colloidal particles became more negative after CMC was added, which is attributed to anionic CMC molecules adsorbing onto casein micelles through electrostatic interactions (Sejersen et al., 2007). Adsorbed CMC chains presumably yielded
41
a conformation with many loops (Fujimoto & Petri, 2001), which may extend into the solution and cause repulsive interactions among casein micelles at low pH in the same way that hair-like kcaseins do at neutral pH. With a further decrease in pH, the zeta potential began to increase because the negative charges on CMC chains lessened, whereas the positive charges on casein micelles increased. Aggregates exceeding 1 mm were observed at pH 3.6 with 0.0045% CMC, but the system with 0.008% CMC was still stable until pH 3.0. Meanwhile, the zeta potential was more negative for the system with more CMC. At lower pH, the more positively charged casein micelles need to adsorb more CMC chains to maintain stability. Fig. 1 also shows that the general tendencies of particle size and zeta potential evolutions were the same for both diluted systems with or without CMC, strongly suggesting that the presence of fat does not influence the dissociation of caseins from micelles and the absorption of CMC onto the casein micelles during acidification. However, the numerical values of particle size for diluted whole milk were much larger than those for diluted skim milk, which can be reasonably related to the presence of fat globules. Milk fat globules are surrounded by a stabilising membrane, called the milk fat globule membrane (MFGM), and its composition can be changed during milk processing. McMahon, Du, McManus, and Larsen (2009) observed that proteins in casein micelles undergo dissociation, reassociation, and rearrangement during acidification, so proteins in MFGM can also go through these changes during acidification. The fat globules could attach to casein micelles through MFGM. The distribution of proteins in whole milk was previously observed at different pH with different concentrations of CMC by confocal scanning laser microscopy (Du et al., 2007). AWMD at pH 4.1 with 0.4% CMC showed a homogeneous appearance with fat uniformly distributed in protein clusters. Hence, the size analysis likely measured clusters of CMC and caseins in combination with fat globules, thus yielding larger values of particle sizes for diluted whole milk. In contrast to the particle size, the numerical difference shown in the zeta potential evolution was very small between diluted skim and whole milk. Michalski, Michel, Sainmont, and Briard (2001) measured the zeta potential of milk fat globules and found that it ranged from 13.5 mV for natural fat globules to 20 mV for strongly homogenised ones. Interestingly, fully homogenised fat globules have a zeta potential similar to that of casein micelles, indicating a high surface coverage with caseins. Therefore, similar values of zeta potential in diluted skim and whole milk during acidification could be expected.
Fig. 1. Evolution comparison of (a) particle size and (b) zeta potential for diluted whole (open symbols) and skim (closed symbols) milk in the presence of different concentrations of carboxymethylcellulose (CMC) during acidification: (,, -) no CMC; (B, C) 0.0045% CMC and (6, :) 0.008% CMC.
42
J. Wu et al. / International Dairy Journal 28 (2013) 40e42
Table 1 Sedimentable fractions of acidified whole milk drinks stabilized by carboxymethylcellulose (CMC) at different concentrations and pH 4.0. CMC concentration (%)
Sedimentable fraction (%)
0 0.1 0.2 0.3 0.4 0.5
10.80 9.64 4.96 4.75 2.71 2.63
0.19 0.16 0.12 0.09 0.14 0.06
3.2. The stability of acidified whole milk drinks by CMC The stability of AWMD containing 4% milk solids with 0e0.5% CMC, was characterized by sedimentation measurements as shown in Table 1. It can be seen that a higher concentration of CMC results in a lower sedimentable fraction. Low quantities of sediment generally indicate good stability of milk proteins under acidic conditions. Therefore, the stability of AWMD increased with increasing CMC concentration. With 0.1% CMC, the sedimentable fraction was almost the same as that without CMC. This is because at low CMC concentration, the amount of CMC is not sufficient to cover all particles, giving rise to the occurrence of bridging flocculation (Maroziene & de Kruif, 2000). At CMC concentrations above 0.2%, the sedimentable fraction was much smaller than those without CMC and with 0.1% CMC. The above experimental results were in good agreement with our long-term visual observation. At CMC concentrations above 0.3% CMC, almost no sedimentation or creaming could be observed, thus clearly indicating that CMC can stabilise the milk system in the presence of a large amount of fat as did in the skim milk. 4. Conclusions The behaviour of CMC-stabilised AWMD was investigated. Similar to the effect of CMC on ASMD, this cellulose ether is found to be suitable for the stabilisation of AWMD that contain a large amount of fat. The fat was suggested to exist in the clusters of CMC and caseins, the presence of which did not disturb the interaction
between CMC and caseins. Fat creaming could be restrained effectively upon addition of CMC. Acknowledgements The authors are thankful for the financial support for this work from the Shanghai Leading Academic Discipline Project (No. B202). References Alexander, M., & Dalgleish, D. G. (2004). Application of transmission diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet. Colloids and Surfaces B: Biointerfaces, 38, 83e90. Donato, L., Alexander, M., & Dalgleish, D. G. (2007). Acid gelation in heated and unheated milks: interactions between serum protein complexes and the surfaces of casein micelles. Journal of Agricultural and Food Chemistry, 55, 4160e4168. Du, B., Li, J., Zhang, H., Chen, P., Huang, L., & Zhou, J. (2007). The stabilisation mechanism of acidified milk drinks induced by carboxymethylcellulose. Lait, 87, 287e300. Du, B., Li, J., Zhang, H., Huang, L., Chen, P., & Zhou, J. (2009). Influence of molecular weight and degree of substitution of carboxymethylcellulose on the stability of acidified milk drinks. Food Hydrocolloids, 23, 1420e1426. Fujimoto, J., & Petri, D. F. S. (2001). Adsorption behavior of carboxymethylcellulose on amino-terminated surfaces. Langmuir, 17, 56e60. Jenness, R., & Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal, 16, 153e164. Jensen, S., Rolin, C., & Ipsen, R. (2010). Stabilisation of acidified skimmed milk with HM pectin. Food Hydrocolloids, 24, 291e299. Laurent, M. A., & Boulenguer, P. (2003). Stabilisation mechanism of acid dairy drinks (ADD) induced by pectin. Food Hydrocolloids, 17, 445e454. Maroziene, A., & de Kruif, C. G. (2000). Interaction of pectin and casein micelles. Food Hydrocolloids, 14, 391e394. McMahon, D. J., Du, H., McManus, W. R., & Larsen, K. M. (2009). Microstructural changes in casein supramolecules during acidification of skim milk. Journal of Dairy Science, 92, 5854e5867. Michalski, M.-C., Michel, F., Sainmont, D., & Briard, V. (2001). Apparent [zeta]potential as a tool to assess mechanical damages to the milk fat globule membrane. Colloids and Surfaces B: Biointerfaces, 23, 23e30. Sejersen, M. T., Salomonsen, T., Ipsen, R., Clark, R., Rolin, C., & Engelsen, S. B. (2007). Zeta potential of pectin-stabilised casein aggregates in acidified milk drinks. International Dairy Journal, 4, 302e307. Tromp, R. H., de Kruif, C. G., van Eijk, M., & Rolin, C. (2004). On the mechanism of stabilisation of acidified milk drinks by pectin. Food Hydrocolloids, 18, 565e572. Tuinier, R., Rolin, C., & de Kruif, C. G. (2002). Electrosorption of pectin onto casein micelles. Biomacromolecules, 3, 632e638. Walstra, P. (1990). On the stability of casein micelles. Journal of Dairy Science, 73, 1965e1979.
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
The stabilisation of acidified whole milk drinks by carboxymethylcellulose Juan Wu, Junliang Liu, Qiaoyu Dai, Hongbin Zhang* Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2012 Received in revised form 23 May 2012 Accepted 24 May 2012
Carboxymethylcellulose (CMC) was used as a stabiliser for acidified whole milk drinks. The stability of the acidified whole milk drinks was investigated by observation of the evolution of size and zeta potential of colloidal particles during acidification along with sedimentation measurement and was compared with the stability of acidified skim milk drinks. It was found that the presence of fat resulted in larger particle sizes, but it did not disturb the absorption of CMC onto casein micelles below pH 5.2. The absorption of CMC endows the casein micelles with electrostatic and steric repulsions, essential to the stability of the acidified milk drinks. Acidified whole milk drinks can be stabilised by CMC, which is capable of effectively preventing the unwanted creaming of fat embedded in the clusters of CMC and caseins as well as preventing the aggregation of casein micelles as it did in skim milk drinks. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Acidified milk drinks are products available worldwide in many variations, with pH values ranging from 3.6 to 4.6. Because casein micelles will aggregate and precipitate below their isoelectric point (4.6), the stabilisation of acidified milk drinks is necessary to prevent instability occurring. Acidified milk drinks are usually stabilised with pectin (Jensen, Rolin, & Ipsen, 2010; Laurent & Boulenguer, 2003; Tromp, de Kruif, van Eijk, & Rolin, 2004; Tuinier, Rolin, & de Kruif, 2002). Carboxymethylcellulose (CMC) is commonly chosen as a stabiliser instead of pectin because of its low cost in Asia, especially in China, where a long shelf life is required of many food products. Our previous work reported the stabilising effect of CMC on acidified skim milk drinks (ASMD) (Du et al., 2007, 2009). However, many commercial products actually contain whole milk, which gives a full-bodied taste because of its high fat content compared with skim milk. Whole milk products seem to be more popular in China. For acidified whole milk drinks (AWMD), creaming is a problem because of the presence of large amounts of fat. The large size and low density of fat may yield stability of AWMD different from that of ASMD. There are limited reports on the application of CMC in AWMD; therefore, there is a need to investigate and understand the CMC-induced stabilisation of AWMD. In the present work, the stabilisation of acidified whole milk by CMC was investigated by monitoring changes in particle size and zeta potential during
* Corresponding author. Tel.: þ86 21 54745005. E-mail address: [email protected] (H. Zhang). 0958-6946/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2012.05.005
acidification and sedimentation measurement and compared with acidified skim milk. 2. Materials and methods 2.1. Materials Skim and whole milk powders were obtained from Fonterra Cooperative Group Ltd. (Auckland, New Zealand). CMC sample (250 kDa, DS ¼ 0.7) was purchased from Acros Organics (Morris Plains, NJ, USA). Citric acid and other analytical grade chemicals used were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Particle size and zeta potential analysis Reconstituted whole milk (8%) was diluted at a ratio of 1e100 with simulated milk ultrafiltrate (SMUF) (Jenness & Koops, 1962). Samples for particle size and zeta potential analysis were prepared as previously reported (Du et al., 2007, 2009). The size and zeta potential of colloidal particles were determined using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) at 25 C. All measurements were performed three times. 2.3. Preparation of acidified whole milk drinks and measurement of sedimentation The samples were prepared on a pilot scale as previously reported (Du et al., 2007, 2009). Whole milk powders were dissolved
J. Wu et al. / International Dairy Journal 28 (2013) 40e42
in distilled water to obtain 8% reconstituted whole milk, and then mixed with 0.8% CMC solution at a 1:1 (v/v) ratio. The mixture was directly acidified to pH 4.0, homogenised at 200 bar with a homogeniser (Rannie TYPE 8.30 H; APV Rannie A/S, Denmark), pasteurised at 110 C for 30 s and then stored in sealed bottles at 4 C overnight prior to stability evaluation. For stability evaluation, 10 g of AWMD were centrifuged at 1800 g for 20 min at 25 C. The sedimentable fraction was calculated by the ratio of the weight of sediment to the weight of the sample. All measurements were performed in triplicate. 3. Results and discussion 3.1. Comparison of the particle size and zeta potential in diluted whole and skim milk systems with and without CMC during acidification Fig. 1 shows the evolution of (a) particle size and (b) zeta potential during acidification of diluted whole and skim milk with different concentrations of CMC. As seen in Fig. 1a, for both kinds of milks without CMC, as the pH decreased from 5.8 to 5.2, the diameter of colloidal particles in the system decreased, which is mainly attributed to the collapse of the hairy k-casein layer on the micellar surface (Alexander & Dalgleish, 2004; Donato, Alexander, & Dalgleish, 2007). Below pH 5.2, the size greatly increased and macroscopic precipitation could be observed within a few minutes. As for the change in zeta potential for the system without CMC, a steady increase in zeta potential with increasing pH was observed. During acidification, k-caseins located outside of the micelles as flexible “hairs” collapsed (Walstra, 1990), thereby significantly weakening their steric repulsion, leading to the aggregation of caseins and, thus, the instability of the milk. Upon addition of different concentrations of CMC to the diluted whole milk, the particle size decreased as the pH was lowered from pH 5.8 to 5.2, indicating that CMC did not affect the aforementioned collapse of k-caseins. At pH below 5.2, the particle size increased slightly, unlike the dramatic increase in the system without CMC during acidification. This increase is due to the adsorption of CMC onto casein micelles, which can be also confirmed by the evolution of zeta potential, as shown in Fig. 1b. Between pH 5.2 and 4.8, the zeta potential of colloidal particles became more negative after CMC was added, which is attributed to anionic CMC molecules adsorbing onto casein micelles through electrostatic interactions (Sejersen et al., 2007). Adsorbed CMC chains presumably yielded
41
a conformation with many loops (Fujimoto & Petri, 2001), which may extend into the solution and cause repulsive interactions among casein micelles at low pH in the same way that hair-like kcaseins do at neutral pH. With a further decrease in pH, the zeta potential began to increase because the negative charges on CMC chains lessened, whereas the positive charges on casein micelles increased. Aggregates exceeding 1 mm were observed at pH 3.6 with 0.0045% CMC, but the system with 0.008% CMC was still stable until pH 3.0. Meanwhile, the zeta potential was more negative for the system with more CMC. At lower pH, the more positively charged casein micelles need to adsorb more CMC chains to maintain stability. Fig. 1 also shows that the general tendencies of particle size and zeta potential evolutions were the same for both diluted systems with or without CMC, strongly suggesting that the presence of fat does not influence the dissociation of caseins from micelles and the absorption of CMC onto the casein micelles during acidification. However, the numerical values of particle size for diluted whole milk were much larger than those for diluted skim milk, which can be reasonably related to the presence of fat globules. Milk fat globules are surrounded by a stabilising membrane, called the milk fat globule membrane (MFGM), and its composition can be changed during milk processing. McMahon, Du, McManus, and Larsen (2009) observed that proteins in casein micelles undergo dissociation, reassociation, and rearrangement during acidification, so proteins in MFGM can also go through these changes during acidification. The fat globules could attach to casein micelles through MFGM. The distribution of proteins in whole milk was previously observed at different pH with different concentrations of CMC by confocal scanning laser microscopy (Du et al., 2007). AWMD at pH 4.1 with 0.4% CMC showed a homogeneous appearance with fat uniformly distributed in protein clusters. Hence, the size analysis likely measured clusters of CMC and caseins in combination with fat globules, thus yielding larger values of particle sizes for diluted whole milk. In contrast to the particle size, the numerical difference shown in the zeta potential evolution was very small between diluted skim and whole milk. Michalski, Michel, Sainmont, and Briard (2001) measured the zeta potential of milk fat globules and found that it ranged from 13.5 mV for natural fat globules to 20 mV for strongly homogenised ones. Interestingly, fully homogenised fat globules have a zeta potential similar to that of casein micelles, indicating a high surface coverage with caseins. Therefore, similar values of zeta potential in diluted skim and whole milk during acidification could be expected.
Fig. 1. Evolution comparison of (a) particle size and (b) zeta potential for diluted whole (open symbols) and skim (closed symbols) milk in the presence of different concentrations of carboxymethylcellulose (CMC) during acidification: (,, -) no CMC; (B, C) 0.0045% CMC and (6, :) 0.008% CMC.
42
J. Wu et al. / International Dairy Journal 28 (2013) 40e42
Table 1 Sedimentable fractions of acidified whole milk drinks stabilized by carboxymethylcellulose (CMC) at different concentrations and pH 4.0. CMC concentration (%)
Sedimentable fraction (%)
0 0.1 0.2 0.3 0.4 0.5
10.80 9.64 4.96 4.75 2.71 2.63
0.19 0.16 0.12 0.09 0.14 0.06
3.2. The stability of acidified whole milk drinks by CMC The stability of AWMD containing 4% milk solids with 0e0.5% CMC, was characterized by sedimentation measurements as shown in Table 1. It can be seen that a higher concentration of CMC results in a lower sedimentable fraction. Low quantities of sediment generally indicate good stability of milk proteins under acidic conditions. Therefore, the stability of AWMD increased with increasing CMC concentration. With 0.1% CMC, the sedimentable fraction was almost the same as that without CMC. This is because at low CMC concentration, the amount of CMC is not sufficient to cover all particles, giving rise to the occurrence of bridging flocculation (Maroziene & de Kruif, 2000). At CMC concentrations above 0.2%, the sedimentable fraction was much smaller than those without CMC and with 0.1% CMC. The above experimental results were in good agreement with our long-term visual observation. At CMC concentrations above 0.3% CMC, almost no sedimentation or creaming could be observed, thus clearly indicating that CMC can stabilise the milk system in the presence of a large amount of fat as did in the skim milk. 4. Conclusions The behaviour of CMC-stabilised AWMD was investigated. Similar to the effect of CMC on ASMD, this cellulose ether is found to be suitable for the stabilisation of AWMD that contain a large amount of fat. The fat was suggested to exist in the clusters of CMC and caseins, the presence of which did not disturb the interaction
between CMC and caseins. Fat creaming could be restrained effectively upon addition of CMC. Acknowledgements The authors are thankful for the financial support for this work from the Shanghai Leading Academic Discipline Project (No. B202). References Alexander, M., & Dalgleish, D. G. (2004). Application of transmission diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet. Colloids and Surfaces B: Biointerfaces, 38, 83e90. Donato, L., Alexander, M., & Dalgleish, D. G. (2007). Acid gelation in heated and unheated milks: interactions between serum protein complexes and the surfaces of casein micelles. Journal of Agricultural and Food Chemistry, 55, 4160e4168. Du, B., Li, J., Zhang, H., Chen, P., Huang, L., & Zhou, J. (2007). The stabilisation mechanism of acidified milk drinks induced by carboxymethylcellulose. Lait, 87, 287e300. Du, B., Li, J., Zhang, H., Huang, L., Chen, P., & Zhou, J. (2009). Influence of molecular weight and degree of substitution of carboxymethylcellulose on the stability of acidified milk drinks. Food Hydrocolloids, 23, 1420e1426. Fujimoto, J., & Petri, D. F. S. (2001). Adsorption behavior of carboxymethylcellulose on amino-terminated surfaces. Langmuir, 17, 56e60. Jenness, R., & Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal, 16, 153e164. Jensen, S., Rolin, C., & Ipsen, R. (2010). Stabilisation of acidified skimmed milk with HM pectin. Food Hydrocolloids, 24, 291e299. Laurent, M. A., & Boulenguer, P. (2003). Stabilisation mechanism of acid dairy drinks (ADD) induced by pectin. Food Hydrocolloids, 17, 445e454. Maroziene, A., & de Kruif, C. G. (2000). Interaction of pectin and casein micelles. Food Hydrocolloids, 14, 391e394. McMahon, D. J., Du, H., McManus, W. R., & Larsen, K. M. (2009). Microstructural changes in casein supramolecules during acidification of skim milk. Journal of Dairy Science, 92, 5854e5867. Michalski, M.-C., Michel, F., Sainmont, D., & Briard, V. (2001). Apparent [zeta]potential as a tool to assess mechanical damages to the milk fat globule membrane. Colloids and Surfaces B: Biointerfaces, 23, 23e30. Sejersen, M. T., Salomonsen, T., Ipsen, R., Clark, R., Rolin, C., & Engelsen, S. B. (2007). Zeta potential of pectin-stabilised casein aggregates in acidified milk drinks. International Dairy Journal, 4, 302e307. Tromp, R. H., de Kruif, C. G., van Eijk, M., & Rolin, C. (2004). On the mechanism of stabilisation of acidified milk drinks by pectin. Food Hydrocolloids, 18, 565e572. Tuinier, R., Rolin, C., & de Kruif, C. G. (2002). Electrosorption of pectin onto casein micelles. Biomacromolecules, 3, 632e638. Walstra, P. (1990). On the stability of casein micelles. Journal of Dairy Science, 73, 1965e1979.