Influence of molecular weight and degree of ...

Food Hydrocolloids 23 (2009) 1420–1426

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Influence of molecular weight and degree of substitution of carboxymethylcellulose on the stability of acidified milk drinks Baiqiao Du a, Jing Li a, Hongbin Zhang a, *, Long Huang b, Ping Chen b, Jianjun Zhou b a

Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, China b Danisco-China Co., Ltd., Kunshan 215300, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 December 2007 Accepted 13 October 2008

The influence of molecular weight (Mw, 250,000, 700,000) and degree of substitution (DS, 0.7, 0.9 and 1.2) of carboxymethylcellulose (CMC) on the diameter and z-potential of casein micelles during acidification in diluted dispersions and on the stability of acidified milk drinks was investigated. The experimental results suggested that CMC with high Mw or low DS would result in thick adsorbed layer onto casein micelles. The z-potential of CMC-coated casein micelle increased with increasing the Mw of CMC with the same DS while at a fixed Mw the z-potential for CMC with high DS (1.2) increased in comparison with those for CMC with low DS (0.7 and 0.9). Both Mw and DS of CMC influenced the stability of acidified milk drinks. CMC with high Mw increased the viscosity of acidified milk drinks significantly and therefore contributed to the stability. CMC with high DS resulted in high z-potential of CMC-coated casein micelles, increasing the electrostatic repulsion between casein particles, which prevented the phase separation in acidified milk drinks. It was also found that the amount of CMC needed for efficient coverage of casein micelles increased with increasing the Mw of CMC. Above the efficient coverage concentration, the long-term stability of acidified milk drinks with high Mw CMC was better than that with low Mw CMC. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Acidified milk drinks Carboxymethylcellulose Molecular parameter Casein micelle Stability

1. Introduction Acidified milk drinks can be described as an acidified protein liquid system with stability and viscosity similar to natural milk. Such drinks usually comprise a large range of products, from those usually prepared from fermented milk with stabilizers and sugar to those prepared by direct acidification with fruit juices and/or acids. The pH of these products ranges from 3.6 to 4.6 (Nakamura, Yoshida, Maeda, & Corredig, 2006). At neutral pH, caseins exist in the form of micelles, which are stabilized by steric repulsion due to the extended conformation of k-casein present mainly on the surface of micelles (de Kruif, 1998; Tuinier & de Kruif, 2002). During acidification, at a pH close to the isoelectric point (pH 4.6) the casein micelles aggregate mainly because of the collapse of the extended conformation of k-caseins (Holt, 1982). On account of the instability of casein in the abovementioned pH range, stabilizer needs to be added to avoid the flocculation of milk proteins and subsequent macroscopic whey separation. High methoxyl pectin (Boulenguer & Laurent, 2003; Liu, Nakamura, & Corredig, 2006; Parker, Boulenguer, & Kravtchenko, 1997) and soybean soluble polysaccharides (SSPS) (Asai et al., 1994; Nakamura, Furuta, Kato, * Corresponding author. Fax: þ86 21 54741297. E-mail address: [email protected] (H. Zhang). 0268-005X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2008.10.004

Maeda, & Nagamatsu, 2003; Nakamura et al., 2006) are often used to achieve this, and much attention has been paid to pectin. In addition, propylene glycol alginate (PGA) and carboxymethylcellulose (CMC) are also mentioned to be able to use as stabilizers (Keiichi, 2006; Koji, Takeshi, Norihiro, & Hideaki, 2004; Mann, 2004; Masaki, Yoshitaka, Yuka, Yasuyuki, & Tatsuyuki, 2004; Murray, 2000; Nishiyama, 1978; Ogasawara, Akahoshi, Hashimoto, Yamashita, & Yamamota, 2003; Syrbe, Bauer, & Klostermeyer, 1998; Young & Bluestein, 2002). As one of the most important derivatives of cellulose, CMC is a typical anionic polysaccharide and has been widely used as a stabilizer in food. CMC chains are linear b(1 / 4)-linked glucopyranose residues. The average degree of substitution (DS) of CMC is defined as the average number of carboxymethyl groups per repeating unit and is usually in the range 0.4–1.5. CMC is generally found under sodium salt form, a water-soluble product for DS > 0.5. A maximum degree of substitution of 1.5 is permitted, but more typically DS is in the range 0.6–0.95 for food applications (Coffey, Bell, & Henderson, 2006; Murray, 2000). CMC is commonly chosen as a stabilizing agent for its low cost in acidified milk drinks instead of pectin in Asia, especially in China (Chen, Zheng, Chen, & Rao, 1996). The application and the stabilization mechanism of pectin and SSPS in acidified milk drinks have been extensively studied in recent years (Liu et al., 2006, Nakamura

B. Du et al. / Food Hydrocolloids 23 (2009) 1420–1426

et al., 2006). However, the stabilizing effects of CMC on this kind of drinks are less reported. The stability of casein micelles at low pH could be improved by CMC. In a previous work (Du et al., 2007), we found that electrosorption of CMC onto casein micelles took place below pH 5.2 and the adsorbed CMC layer on the surface of casein could prevent flocculation of casein micelles by steric repulsion. In addition, the non-adsorbed CMC increased the viscosity of serum and slowed down the sedimentation of casein particles. The adsorbed CMC layer caused a repulsive interaction between the casein micelles at low pH in the same way as k-caseins do at neutral pH. This phenomenon is related to the interaction between protein (mainly casein micelles) and CMC. The stability of acidified milk drinks depends largely on the interactions between casein and polysaccharides, which can be influenced by the concentrations of protein and polysaccharides (Tromp, de Kruif, van Eijk, & Rolin, 2004; Tuinier, Rolin, & de Kruif, 2002), pH (Nakamura et al., 2003), molecular properties of polysaccharides (Laurent & Boulenguer, 2003; Maroziene & de Kruif, 2000; Pereyra, Schmidt, & Wicker, 1997), ionic environment (Ambjerg Pedersen & Jorgensen, 1991), milk protein composition and processing (Boulenguer & Laurent, 2003; Glahn, 1982; Sedlmeyer, Brack, Rademacher, & Kulozik, 2004;), and thermal history of the sample (Horne, 1998; Lucey, Tamehana, Singh, & Munro, 1999; Zaleska, Ring, & Tomasik, 2000) etc. Although the interactions between casein micelles and CMC and the stability of the acidified milk drinks might be primarily dependent on pH and concentration of CMC as previously reported (Du et al., 2007), the molecular weight and substitution pattern of carboxymethyl groups on CMC should be emphasized because in the practical processing of acidified milk drinks the properties including stability of the drinks can be obtained by the adjustment on the molecular parameters of CMC. In the present work, we aim to investigate the influence of Mw and DS of CMC on the interaction between CMC and casein micelles and thus on the stability of acidified milk drinks. 2. Materials and methods 2.1. Materials A series of CMC with different Mw (250,000 Da and 700,000 Da) and different DS (0.7, 0.9 and 1.2) were purchased from the Acros organics (Morris Plains, New Jersey). Skim milk powders were obtained from Fonterra Co-operative Group (Wellington, New Zealand). Citric acid monohydrate was obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Preparation of samples for dynamic light scattering (DLS) and z-potential experiments The sample was made by dispersing 80 g/kg reconstituted skim milk in simulated milk ultra filtrate (SMUF) (Jenness & Koops, 1962) (1:100). SMUF contains Na, K, Ca, Mg, phosphate and citrate and was used to dilute mixtures in an environment which would simulate the salt system in milk. Then 5 g/kg CMC was added to the diluted reconstituted skim milk at about neutral pH (6.6–6.7) to obtain 800 mg/kg skim milk powders containing 400 mg/kg CMC. All solutions in this measurement were prepared with ultra-pure water with 18.2 MU/cm (Millipore, Bedford, MA. USA), and filtered through 0.22 mm membrane filters prior to use. The apparent diameter and z-potential of casein micelles were monitored during acidifying the diluted reconstituted skim milk with citric acid. 2.3. Dynamic light scattering measurement (DLS) Dynamic light scattering measurements were carried out with a Malvern Zetasizer 3000HSA (Malvern Instruments, Worcestershire,

1421

UK) equipped with a 10 W max output He–Ne laser and at a l of 633 nm. Measurement occurred at 90 from the incident beam and gave an estimation of the particle mean diameter distribution in intensity. The temperature of the samples was controlled by a Joule– Peltier thermostat at 20 C. The stated results of each measurement were the average of 10 measurements. And all measurements were performed three times. The biggest variance of the measurement was below 10%. 2.4. z-Potential measurement The z-potential of casein micelles as a function of pH was determined using a particle electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) which measures the direction and velocity of droplet movement in applied electric field at 20 C. The z-potential provides an estimate of the net charge of a particle measured at the ‘shear plane’, which depends on the charge on the actual particle (in this case casein micelles and polysaccharides) plus the charge associated with any ions that move along with the particle in the electric field. The sample was similar to those for DLS measurements. An individual zpotential was determined from the average of three readings taken on the same sample. All measurements were performed three times. 2.5. Preparation of acidified milk drinks The 80 g/kg reconstituted skim milk was prepared by mixing skim milk powders and distilled water at 45 C for 30 min. Meanwhile, CMC and sucrose were dry mixed together and then the mixture was dissolved in distilled water at 75 C by stirring for 20 min. Stabilizer and reconstituted skim milk were mixed at a 1:1 ratio to obtain 40 g/kg skim milk powders containing 4 g/kg CMC and 80 g/kg sucrose. The pH of this mixture was directly acidified to 4.0 with 500 g/kg citric acid at 20 C. The acidified milk drinks were preheated to 65 C and then homogenized at 200 bar with a twostage value homogeniser Rannie TYPE 8.30 H (APV Rannie A/S, Denmark). All samples were stored in sealed glass bottles. The bottle of each sample (500 ml) was heated in a water bath at 90 C for 30 min. Sodium azide (0.02%) was added to prevent bacterial growth. Measurements were performed after the samples were stored at room temperature overnight. All measurements were performed three times. 2.6. Measurement of apparent viscosity for the acidified milk drinks The apparent viscosity of acidified milk drinks was measured by a Brookfield Viscometer LVDV-I, Model NDJ-79 with No. 1 rotor, at 100 rpm/min, and 25 8C. 2.7. Measurement of particle size for the acidified milk drinks The acidified milk drinks were diluted to a measurable concentration (100–200 times) with distilled water. Then the particle size was measured with a Laser Diffraction Particle Size Analyser, Malvern MasterSizer 2000 (Malvern Instruments Ltd., Worcestershire, UK). 2.8. Measurement of storage stability for the acidified milk drinks Sedimentation and serum phase occurred during storage were observed with the optical analyser TURBISCAN MA 2000 (Ramonville-St-Agne, France). The cylindrical glass tubes containing 5 ml of sample were stored at 25 C. Sedimentation and serum phase fraction as a function of time were followed after the sample was prepared for 30 days.

1422


2.9. Phase separation

D ¼ kT=3phm deff

The mixtures of 40 g/kg skim milk and CMC with different concentrations (0–5 g/kg) were acidified to pH values 4.7 0.1 and 3.8 0.1, respectively, while without acidification the pH of the mixture was ca. 6.7 0.1. 10 ml sample was placed in a graduated glass tube with lid at room temperature of 25 C. The stability of the mixture was obtained from visual observation 3 days after preparation. The sample was regarded as stable if the system was homogeneous whereas the sample was unstable if obvious phase separation took place. 3. Results and discussion 3.1. Influence of Mw and DS of CMC on the diameter of casein micelles during acidification The particle size evolution of casein micelles in the system prepared by diluting reconstituted skim milk in SMUF (1:100) was followed by DLS. The ratio of 800 mg/kg reconstituted skim milk and 400 mg/kg CMC was selected based on our previous work (Du et al., 2007), at which CMC could sufficiently form thick adsorbed layer on casein micelles and maintained their stability. Fig. 1 illustrated the effect of Mw of CMC (DS ¼ 0.9) on the particle diameter of casein micelles during acidification with citric acid. For reconstituted skim milk sample without CMC, the diameter was close to 230 10 nm, which was consistent with our previous result (Du et al., 2007) and was also in agreement with earlier DLS results regarding the size of casein micelles (de Kruif, 1992, Tuinier et al., 2002). Upon decreasing the pH from 5.8 to 5.0 the casein micelle diameter decreased due to a fraction of a- and b-casein coming out of casein micelles (Tuinier et al., 2002). Around pH 5.0, the apparent casein micelles size started to increase and exceeded 1 mm at pH 4.7 because of the formation of aggregates. The suspension became thus unstable and macroscopic flocs were observed in a few minutes. This observation was the same as those of Du et al. (2007) and Tuinier et al. (2002). When CMC was added in diluted reconstituted skim milk, a small increase in the particle diameter was found, as compared with the bare casein micelle size at neutral pH. A similar phenomenon was also observed in the size evolution during acidification of pectin/casein mixtures (Tuinier et al., 2002). The increase in size was interpreted based on the Stokes–Einstein relation as follows. 1000 skim milk

950

Mw 250000

Particle diameter/nm

900

Mw 700000

500 400 300 200 100 0 7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

pH Fig. 1. Influence of Mw of CMC (DS ¼ 0.9) on the diameter of casein micelles during acidification with citric acid. Skim milk concentration is 800 mg/kg and CMC concentration is 400 mg/kg.

(1) eff

where hm was the medium viscosity, d was the effective hydrodynamic diameter. In diluted dispersions, the self-diffusion coefficient D was related to the effective hydrodynamic diameter of the sphere, deff according to equation (1). The viscosity of SMUF was taken as the medium viscosity hm. However, upon the addition of CMC, the actual medium viscosity will increase. It was thus supposed that the increase in the particle diameter was traceable from the calculation method of DLS (Tuinier et al., 2002). Alternatively, Nakamura et al. (2006) also found that the initial diameter of skim milk increased with increasing pectin concentration at neutral pH. Taking into account that the DLS measurements were carried out after the samples were extensively diluted in the corresponding ultra filtration permeate and assuming that the measurement of particle diameter was not affected by the viscosity of the pectin, they attributed this increase in size to an association of pectin to casein micelles at neutral pH, possibly because of the existence of calcium ions. In our case, the increase in diameter of casein micelles upon addition of CMC at neutral pH needed further clarification. The addition of CMC had no effect on the decrease of the casein micelles size between pH 5.8 and 5.2, which indicated that the addition of CMC did not influence the coming out of a fraction of aand b-casein from casein micelles. Upon decreasing the pH close to 5.2, it was found that the diameter of casein micelle in the mixture with CMC increased in comparison with that in the absence of CMC. This was caused by the adsorption of CMC onto casein micelles, which led to effectively larger casein micelles. The negative charge might be statistically distributed along the CMC chains, yielding a conformation with many loops like CMC chains on aminoterminated surface (Fujimoto & Petri, 2001) and pectin on casein surface (Tromp et al., 2004). For adsorbed CMC, these loops extended into the solution and caused a repulsive interaction between the casein micelles at low pH in the same way as k-casein chains did at neutral pH. The adsorption of CMC onto casein micelles took place just before casein micelles aggregation started in the absence of CMC. In the present work, the influences of Mw and DS of CMC on the diameter of casein micelles were investigated upon addition of enough CMC (400 mg/kg) and when the diameter leveled off in the low pH range 4.3–3.0. The casein micelles diameter (500 25 nm) for high Mw CMC (Mw ¼ 700,000) was larger than that (420 25 nm) for low Mw CMC (Mw ¼ 250,000). These results suggested that the thickness of adsorbed layer should be related to the Mw of CMC. CMC with high Mw would result in thick adsorbed layer onto casein micelles. When CMC was adsorbed to casein micelles, the segment of the chain between two charged points protruding from the surface of the casein micelle would be longer for high Mw CMC than that for low Mw CMC, resulting in a bigger loop conformation. Similar phenomenon was observed in other system such as the emulsion stabilized by SSPS (Nakamura, Takahashi, Yoshida, Maeda, & Corredig, 2004), in which the layer thickness of SSPS on the surface of the oil droplets increased with increasing the molecular mass of SPSS containing high protein content. Fig. 2 showed the influence of DS (0.7, 0.9 and 1.2) of CMC on the particle diameter of casein micelles for the same concentration (400 mg/kg) and Mw of CMC (250,000) during acidification with citric acid. The apparent diameter of the particles also leveled off to a maximum in low pH range (pH 4.3–3.0) (450 15 nm, 420 15 nm and 380 15 nm for DS 0.7, 0.9, and 1.2, respectively). The results indicated that the higher the charge of CMC, the smaller the particle size in low pH range. This phenomenon was probably attributed to two aspects: firstly, CMC with high DS afforded more negative charges, which could adsorb strongly onto casein micelles, while the uncharged stretches in charged blocks formed small


20

1000 skim milk

950

skim milk DS 0.7 DS 0.9

15

DS 0.7 DS 0.9 DS 1.2

900

10

DS 1.2

5

-potential/mV

Particle diameter/nm

1423

400

300

200

0 -5 -10 -15 -20

100 -25 -30

0 7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

pH

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

pH

Fig. 2. Influence of DS of CMC (Mw ¼ 250,000) on the diameter of casein micelles during acidification with citric acid. CMC concentration is 400 mg/kg. Skim milk concentration is 800 mg/kg and CMC concentration is 400 mg/kg.

Fig. 4. Influence of DS of CMC (Mw ¼ 250,000) on the z-potential of casein micelles during acidification with citric acid. CMC concentration is 400 mg/kg. Skim milk concentration is 800 mg/kg and CMC concentration is 400 mg/kg.

loops; and secondly, the already adsorbed chains might hinder the adsorption of the other arriving chains due to the electrostatic repulsion. CMC interacted with casein micelles via electrostatic interactions, and this interaction was pH-dependent. The charge of CMC molecule was fundamental in driving the adsorption of the casein micelles at low pH; however, the size of CMC chain affected the extent of the steric repulsion between particles.

CMC, those in the presence of CMC were more negative after acidification, which was due to the adsorption of CMC onto casein micelles between pH 5.4 and 4.8. Similar phenomenon was previously observed with pectin (Sejersen et al., 2007; Shaw, 1980; Surh, Decker, & McClements, 2006). Below pH 4.8, the z-potential became less negative with lowering pH. This phenomenon might be explained by the fact that the carboxylate groups on the adsorbed CMC layer were protonated in lower pH range (Thakur, Singh, & Handa, 1997). The addition of CMC to casein micelles caused some increase in the absolute magnitude of z-potential of casein micelles. And the high Mw CMC (Mw ¼ 700,000) seemed to result in a bigger increase in z-potential of CMC-coated casein micelles above pH 3.7 than low Mw CMC (Mw ¼ 250,000) (Fig. 3). Below pH 3.7, the effect of Mw of CMC on the z-potential was not perceptible. As seen in Fig. 4, the z-potential of CMC-coated casein micelles did not change so much when the DS of CMC increased from 0.7 to 0.9. Further increasing the DS to 1.2 gave rise to an increase of ca. 3 mV in z-potential. As for the effect of high and low degree of methylesterification (DE) of pectin on the z-potential of sodium caseinate emulsions, it has been found that the z-potential was independent of DE of pectin if its charge density is not too low (Surh et al., 2006). This phenomenon is attributed to the fact that once the surface charge has reached a certain value there will be a strong electrostatic repulsion between the surface and similarly charged polyelectrolyte in the aqueous solution, which limits further adsorption of the polyelectrolyte (Schonhoff, 2003). In our previous work (Du et al., 2007), the evolution of the z-potential of casein micelles during acidification without CMC and with different concentration of CMC was discussed. It was found that the evolution of the z-potential of casein micelles was independent of CMC concentration above full coverage in low pH range 4.3–3. This result also confirmed that the adsorption of CMC onto casein micelles was not only induced but also controlled by the electrostatic force. CMC with high DS resulted in a high z-potential of CMC-coated casein micelles, indicating an increase in the electrostatic repulsion between casein particles.

3.2. Influences of Mw and DS of CMC on the z-potential of casein micelles during acidification The influences of Mw and DS of CMC on the z-potential of casein micelles as a function of pH were shown in Figs. 3 and 4, respectively. The relationship between the z-potential of casein micelles and pH was complex because of the composition of casein micelles and the SMUF dilution used (Anema & Klostermeyer, 1996; Schmidt & Poll, 1986; Shaw, 1980). In this SMUF medium, the change from negative to positive z-potential was observed at pH 4.7. Fox and McSweeney (1998) reported that the casein in milk would precipitate out of solution on acidification to pH 4.6 due to a zero overall charge at this pH. In contrast to casein micelles without

20 skim milk

15

Mw 250,000

10

Mw 700,000

-potential/mV

5 0 -5 -10 -15 -20 -25 -30 7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

pH Fig. 3. Influence of Mw of CMC (DS ¼ 0.9) on the z-potential of casein micelles during acidification with citric acid. CMC concentration is 400 mg/kg. Skim milk concentration is 800 mg/kg and CMC concentration is 400 mg/kg.

3.3. Influence of Mw and DS of CMC on the stability of acidified milk drinks The stability of acidified milk drinks was expressed as the percentages of the sedimentation and serum which were measured


3.4. Influence of Mw on phase behavior of reconstituted skim milk/ CMC mixture Since Mw of CMC played an important role on the stability of acidified milk drinks, the phase behavior of reconstituted skim milk/CMC mixtures was further investigated. Fig. 5 represented the stability diagram for the mixtures, obtained from visual observation 3 days after preparation. The closed circles referred to stable states and the open circles referred to unstable. At pH 6.7, the mixtures (250,000 Mw CMC) were stable at low CMC concentration; however, higher CMC concentration (>1 g/kg) led to the loss of stability (Fig. 5a). Phase separation of mixtures with higher Mw CMC (700,000) occurred at lower CMC concentration (>0.6 g/kg, Fig. 5b). Our previous work (Du et al., 2007) showed that the interaction between casein micelles and CMC was closely related to pH. At pH 5.2–6.8, both casein and CMC are negatively charged and thus repel each other. We have previously shown that CMC started to adsorb on casein at pH 5.2; therefore, there was no CMC adsorption above pH 5.2. At these pH values, non-adsorbing CMC could lead to phase separation by depletion flocculation. The similar phenomenon was also found in other non-adsorbing polysaccharides in the system mixed with casein micelles, such as pectin above a certain pH (Maroziene & de Kruif, 2000; Tuinier

a

8

7

phase separation 6

pH

by turbimetry after the sample was prepared for 30 days. The results in Table 1 showed that there was an obvious phase separation in reconstituted skim milk without addition of CMC. Proteins were completely flocculated and settled down at the bottom (sedimentation fraction was 16.8%). The large fraction (83.2%) was the clear serum phase. The addition of CMC increased the stability of reconstituted skim milk at low pH with both a less serum fraction and a sedimentation fraction (Table 1). When compared the effect of Mw of CMC on stability of acidified milk drinks, it was found that CMC with higher Mw resulted in a more stable acidified milk drink system than CMC with lower Mw. Previous work indicated that the stability of acidified milk drinks increased with increasing the concentration of CMC (Du et al., 2007). The better stability of the sample with high Mw (700,000) could be explained by that the stability of acidified milk drinks was related not only to the stability of casein micelles themselves, but also to the serum viscosity. When CMC concentration was sufficient to cover the casein micelles efficiently, the redundantly nonadsorbed CMC increased the viscosity of acidified milk drinks and thus contributed to the stability of acidified milk drinks. The particle size and apparent viscosity of casein in acidified milk drinks stabilized by CMC with different Mw and DS were also summarized in Table 1. The apparent viscosity (35.9 0.1 cp) of acidified milk drinks stabilized by CMC with high Mw (700,000) was much higher than that with low Mw (250,000) (24.8 0.2 cp), thus consequently contributed to the stability of acidified milk drinks. There was no population of aggregates in all samples because the CMC was sufficient to completely cover the casein micelles. The value of the particle size of casein in acidified milk drinks containing CMC with Mw of 700,000 and DS of 0.9, 0.68 0.12 mm, was slightly larger than that (0.60 0.22 mm) of the sample containing CMC with Mw of 250,000 and the same DS; however, the sample containing high Mw CMC was more stable (less serum fraction and sedimentation fraction). This suggested that the higher viscosity endued by the margin of the non-adsorbed high Mw CMC should contribute to the higher stability of the system. The influence of DS of CMC on the stability of acidified milk drinks could also be seen in Table 1. The addition of CMC with DS 0.7 and DS 0.9 had no obvious difference on the stability of acidified milk drinks. The serum fraction (ca. 40%) of acidified milk drinks with DS 0.9 was a little higher than that (ca. 36%) with DS 0.7, and the sedimentations (ca. 14%) were similar for both samples. There was no large aggregation of particles in the system, in which the particle size was below 1 mm with different DS of CMC. The apparent viscosity of acidified milk drinks containing CMC (Mw ¼ 250,000) with various DS was similar. The reason was that the rheological behavior of an aqueous CMC solution was mainly influenced by Mw of CMC and its concentration (Kulicke, Kull, Kull, & Thielking, 1996). The stability of acidified milk drinks induced by CMC with DS 1.2 was better than that with DS 0.7 and 0.9. This improved stability might be related to the increase electrorepulsion which generated by the high z-potential of casein micelles with high DS of CMC, as indicated in Fig. 4.

5

bridging 4

3

0

1

2

3

4

5

4

5

CMC concentration g/kg

b

8

7 phase separation 6

pH

1424

5 bridging

Table 1 Influence of Mw and DS of CMC on the apparent viscosity, particle size and stability of acidified milk drinks (40 g/kg MSNF, 4 g/kg CMC, and 80 g/kg sucrose, pH 4.0). CMC sample added Without CMC Mw 250,000; DS 0.7 Mw 250,000; DS 0.9 Mw 250,000; DS 1.2 Mw 700,000; DS 0.9

Apparent viscosity/cp – 25.7 0.2 24.8 0.2 25.5 0.1 35.9 0.1

Particle size D[4,3]/mm >10 0.87 0.30 0.60 0.22 0.38 0.20 0.68 0.12

Serum fraction/% 83.2 0.1 36.4 2.2 40.8 2.9 11.9 2.5 6.8 1.0

Sedimentation fraction/% 16.8 1.0 14.2 2.8 14.6 2.1 10.3 2.4 5.7 1.5

4

3 0

1

2

3

CMC concentration g/kg Fig. 5. Influence of CMC Mw (DS ¼ 0.9), CMC concentration, and pH on the phase separation of mixtures of reconstituted skim milk (40 g/kg MSNF) and CMC. (a) Mw ¼ 250,000; (b) Mw ¼ 700,000. Open circle indicated unstable system, and closed circle indicated stable system.


et al., 2002) and guar gum at neutral condition (Tuinier, ten Grotenhuis, & de Kruif, 2000). In Fig. 5, CMC concentration at the phase boundary decreased with increasing CMC Mw. This was consistent with the Vrij’s theory that could be used to calculate the phase boundary from the size of the casein micelles and the size and molar mass of the non-adsorbed polysaccharides (Mao, Cates, & Lekkerkerker, 1995; Tuinier, ten Grotenhuis, & de Kruif, 2000; Vrij, 1976), i.e., increasing the chain length of the polysaccharides causing the phase boundary to move to lower polysaccharide concentrations. Below pH 5.2, at pH 4.7 and 3.8, CMC could adsorb onto casein micelles. Here, pH 4.7 and 3.8 were selected because pH 4.7 was near the pI of casein micelle and pH 3.8 was in the range of acidified milk drinks. In these cases, the state diagrams were quite different from that at the pH at which the adsorption of CMC could not take place. In contrast to the mixtures at pH 6.7, the system at low pH was phase separated. When CMC adsorbs onto casein micelles it could cause either bridging flocculation or steric stabilization depending on its concentration. Bridging flocculation occurred at a CMC concentration that was not enough for full coverage of the casein micelles. A single polymer chain may adsorb onto two or more particles with low polymer concentration, thereby connecting the particles. The system may thus separate into one phase concentrated in complexes and the other phase in solvent (Maroziene & de Kruif, 2000; Syrbe et al., 1998; Tuinier et al., 2002). More CMC was required for stabilization at pH 3.8, which was attributed to the increased positive charges on casein micelles. The more positively charged casein micelles would adsorb more CMC to form thick layer to maintain the stability of casein micelles. Therefore CMC concentration at which the system was stable shifted to higher CMC concentration at lower pH. In the case of pH 3.8, for Mw ¼ 700,000 the boundary concentration of CMC, 3.4 g/kg, required to stable the acidified milk drinks, was higher than that for Mw ¼ 250,000, i.e., 2.6 g/kg. It was supposed that when the amount of CMC with high and low Mw was the same, CMC with high Mw would have a smaller chain number than that with low Mw. Thus, in order to obtain the stability for the same amount of acidified milk drinks, relatively more molecular chains of CMC with high Mw (i.e., higher concentration) were required than that with low Mw, to cover the protein particles efficiently. However, it should be noted that beyond this boundary concentration, the long-term stability of the acidified milk drinks with high Mw CMC was better than that with low Mw as described in Table 1. 4. Conclusions Both Mw and DS of CMC influenced the interaction between CMC and casein micelles and thus the stability of acidified milk drinks. At pH 6.7, there was no interaction between caseins and CMC due to charge repulsion and mixtures of casein and CMC were stable at low CMC concentrations. Above a certain CMC concentration, depletion flocculation occurred leading to phase separation. Below pH 5.2 CMC adsorbed onto casein micelles. In the case of low CMC concentrations, CMC/casein micelles mixture was phase separated via bridging flocculation. With increasing CMC concentrations, the casein micelles were effectively coated and consequently sterically stabilized. The amount of CMC needed for effective coverage onto casein micelles increased with increasing the Mw of CMC. It was found that the high Mw CMC or low DS CMC led to a thick adsorbed layer onto casein micelles. However, this thick layer was not directly related to the long-term stability of acidified milk drinks because the stability of acidified milk drinks was also related to the z-potential of casein micelles, the particle size of casein micelles, and the viscosity of the system. Acidified milk drinks with high Mw CMC showed good stability due to both

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