Effect of low-frequency ultrasound on the particle size, solubility and surface charge of reconstituted sodium caseinate

Accepted Manuscript Effect of Low-Frequency Ultrasound on the Particle Size, Solubility and Surface Charge of Reconstituted Sodium Caseinate Billy Lo, Elisabeth Gorczyca, Stefan Kasapis, Bogdan Zisu PII: DOI: Reference:

S1350-4177(18)31310-5 https://doi.org/10.1016/j.ultsonch.2019.03.016 ULTSON 4525

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

27 August 2018 14 March 2019 14 March 2019

Please cite this article as: B. Lo, E. Gorczyca, S. Kasapis, B. Zisu, Effect of Low-Frequency Ultrasound on the Particle Size, Solubility and Surface Charge of Reconstituted Sodium Caseinate, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.03.016

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Effect of Low-Frequency Ultrasound on the Particle Size, Solubility and Surface Charge of Reconstituted Sodium Caseinate Billy Lo, Elisabeth Gorczyca, Stefan Kasapis, Bogdan Zisu* School of Science, RMIT University, Bundoora West Campus, Plenty Road, Melbourne, Vic 3083, Australia *Corresponding author: [email protected] Abstract Low-frequency sonication (20 kHz) was applied to sodium caseinate suspensions (4%, 7% and 10% protein concentrations) at pH 4.0, 4.6, 6.7 and 9.0. Particle size, zeta potential and solubility analysis were used to evaluate the physical changes of the sodium caseinate suspensions before and after the application of ultrasound. At pH 6.7 the particle size remained between 5-7 µm for all concentrations before and after sonication (15-400 J/mL), resulting in no significant change (p>0.05). Similarly, sonication did not significantly (p>0.05) affect the solubility at pH 6.7. At this pH, the initial solubility was high at 94-98% (w/w) before sonication. At pH 9.0 for 4% and 7% concentrations, suspensions became more negatively charged and the initial particle size increased to 78-82 µm. In the presence of larger suspensions, the application of ≥15J/mL reduced the particle size to less than 2 µm. By contrast to pH 6.7, the solubility at pH 9.0 for 4% and 7% protein suspensions reached 99% before and after sonication. Viscosity was the highest (80 mPa.s at 15 sec-1) for a 10% protein concentration at pH 9.0. As the protein concentration of the sodium caseinate suspensions decreased from 10% to 4% at pH 9.0, the viscosity of the suspensions also decreased. However, application of low-frequency ultrasound had no effect on the viscosity of the sodium caseinate suspensions. Due to the absence of large insoluble aggregates in reconstituted sodium caseinate suspensions, the overall effect of low-frequency sonication were largely insignificant at native pH and only became evident at outlier pH values when the casein proteins associate.

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Highlights 

The effect of 20kHz ultrasound was examined on sodium caseinate (Na-CN) suspensions

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Particle size and solubility of Na-CN were affected by sonication

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pH and protein concentration had the greatest effect on Na-CN suspensions

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Ultrasound had little effect on non-micellear Na-CN at pH 6.7

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Ultrasound reduced the particle size of non-micellar Na-CN pH 9.0

Keywords: Sodium caseinate, Low-frequency ultrasound, Particle size, Zeta potential, Solubility, Viscosity Acknowledgement: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 1.0. Introduction Dairy ingredients play a key role in food processing due to functionalities derived from their composition and often act as foaming, stabilising and solubilising agents. Sodium caseinate (Na-CN) is one such dairy ingredient, which is commonly used in commercial food manufacturing primarily as either a fat stabiliser or an emulsifier [1]. Na-CN is derived from skim milk, where its pH is adjusted to 4.6, thereby triggering whey-casein protein separation. This enables the calcium phosphates to solubilise and subsequently be removed. Following this, minerals, lactose and whey proteins are extracted before the pH of the casein is readjusted to 7.0 through the introduction of an alkaline solution, which, in this case, is sodium hydroxide [2]. The resultant solution, which has approximately 90% protein content, is then spray dried into powder being suitable for subsequent use as a food ingredient [4]. The structural integrity of the casein micelle is supported by colloidal calcium phosphate (CCP), which consists of hydrophobic and electrostatic interactions [3]. Na-CN contains the components of the four casein fractions, namely αs1-, αs2-, β- and κ-, which have the weight proportions of 4:1:4:3 [5]. Casein: as1- and β- constitute 75% of the total casein in milk, with β-casein possessing the highest surface-active properties compared to the other casein fractions. It also contains enhanced hydrophobic properties relative to as1-casein [6].

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To be able to attain the aforementioned functionalities, Na-CN needs to be reconstituted in water and the resultant solubility of powders must be high. The caseins in Na-CN are altered to increase its solubility, with protein concentration and pH having a significant effect on the solubility of Na-CN. The isoelectric point (IEP) of Na-CN is at pH 4.6-5.5. At this point, solubility is very low resulting in aggregation due to the decrease in the repulsive forces of the molecules, which serve as one of the primary factors that influence solubility in this case [6,7]. Na-CN has relatively high solubility at native pH (~6.5) and as the pH increases, the solubility increases in tandem [6]. This is attributable to its inherent composition and its manufacturing process, which affects the physical and chemical properties of the milk ingredients. According to literature, it is suggested that as the pH is adjusted towards a more alkaline level, the caseins change in both particle size and solubility [3]. Viscosity is also affected by pH when reconstituting the Na-CN powders in water. Minimum viscosity is observed at pH 7. As the pH of the Na-CN dispersion is adjusted and lowered to pH 5.0 or below, viscosity is observed to increase. This is due to the formation of protein aggregation, which can also establish the conditions needed for gelation resulting in a solid gel [7, 8]. Similar observations are made at extremely high pH. When the pH is adjusted to more alkaline levels (>9.0) viscosity also increases. This occurrence can be attributed to the destabilisation of the casein micelle, which increases the viscosity and can also lead to gelation [8]. Low-frequency ultrasound generates non-audible sound waves at frequencies over 20 kHz, which results in acoustic cavitation [9, 10]. Acoustic cavitation, which has been demonstrated in prior research, can generate mechanical, physical and chemical effects that are strong enough to break molecular bonds [11]. Although ultrasound has been used in various food applications, as reviewed by Chemat [14] and in numerous dairy applications [11, 12, 13], prior research in this regard has always explored the effect of low-frequency ultrasound on milk proteins, particularly on whey proteins but not caseinate powders specifically [13]. According to literature, there have been studies conducted on the effect of low-frequency ultrasound on particle size, pH, solubility and surface charge of whey protein suspensions [11, 12, 13]. Previous studies did not focus on a wide range of pH nor energy densities and the composition of ingredients other than caseinates was studied [11, 12, 13].

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For the first time, this paper will focus on the properties of sonicated non-micellar caseins in the form of sodium caseinate across a range of pH and energy densities. Without prior reported studies exploring the effects of sonication on the particle size, solubility, surface charge and viscosity, it is speculated that the technology will result in predominantly physical effects on the parameters in focus, as reported in other casein containing systems. The objectives of this study were to determine the effects low-frequency 20 kHz ultrasound (15, 30, 150, 300 and 400J/mL) on the particle size, solubility, surface charge and viscosity of Na-CN reconstituted to 4%, 7% and 10% protein at pH 4.0, 4.6, 6.7 and 9.0.

2.0.

Materials and Methods 2.1. Materials Na-CN was obtained from Murray Goulburn (MG, Australia). According to the

company’s specifications, the Na-CN powder had the following composition: 92.7% protein (dry basis), 0.2% lactose, 0.7% fat and 4.3% moisture. Analytical grade NaOH and HCl were purchased from Sigma Aldrich (St. Louis, USA). 2.2. Reconstitution of Na-CN powder The Na-CN powder was reconstituted with Milli-Q water at ambient temperature (23oC ± 1oC) to produce 4% w/w, 7% w/w, and 10% w/w protein suspensions. The powder was initially left to hydrate without stirring for 20 minutes. This was followed by magnetic stirring of the suspension at 900 rpm (9 MR Hei-Tec Stirrer + Pt1000 V4A). After 1 hour of stirring, the pH of the suspension was adjusted if necessary with NaOH (1.0 N) or HCl (1.0 N) to the required pH levels of 4.0, 4.6, 6.7 or 9.0. Magnetic stirring, as previously, was continued for a further 1 hour before overnight storage (4oC ± 1oC). Following overnight storage, the Na-CN suspension was returned to the magnetic stirrer for 10 minutes to disperse any precipitated material and to raise the temperature closer to ambient conditions before evaluation and sonication. Each treatment was prepared in triplicate. 2.3. Particle size and zeta potential of reconstituted Na-CN A Malvern Mastersizer 3000 Laser diffraction hydro MV system Malvern Instruments Ltd, (Malvern, UK) was used to determine the particle size distribution. The following 4

conditions were applied: a refractive index of 1.46 for casein proteins and 1.33 for the dispersant (deionized water), an absorbance coefficient of 0.01, and an appropriate obscuration rate. On average, three measurements were taken at ambient temperature (23oC ±1oC) for data collection, with the results expressed as average particle size D [4, 3]. The Zetasizer (Malvern Instruments Ltd, Malvern, UK) was also used to determine the particle size and zeta potential of reconstituted Na-CN. The sample was diluted 100 fold before measuring. The refractive index for protein was 1.46, and 1.33 for the dispersant (deionised water). On average, three measurements were taken at ambient temperature (23oC ±1oC) from 13 runs per measurement. 2.4. Sonication of reconstituted Na-CN The reconstituted Na-CN suspensions (100 mL) were sonicated with a 20 kHz 500W Qsonica (Sonoplus, USA) ultrasonic processor using a 12.7 mm probe at the following energy densities (ED): 15 J/mL, 30 J/mL, 150 J/mL, 300 J/mL, and 400 J/mL. To achieve these energy densities, it is required to sonicate for to the following treatment times: 26 seconds, 51 seconds, 4 minutes and 26 seconds, 8 minutes and 54 seconds, and 11 minutes and 37 seconds, respectively. The Na-CN suspension was subsequently sonicated at 5 ± 1oC, with the sample temperature being controlled with an ice bath. The ED of sonication was calculated according to the characterisation table from Contamine, Wilhelm, Berlan, and Delmas [15]. This required measuring the temperature of water (300 mL) before and after sonication at the following amplitudes: 20%, 40%, 60%, 80% and 100%. The water was sonicated for 30 seconds. The thermal difference was used to calculate the power drawn (W). The relationship between the ED (J/mL) of sonication and the power (W) applied is expressed in equation (1) [16]: ED = (W×T)/V…………………………………………………………………(1) where, T is time in seconds and V is volume in millilitres. 2.5. Solubility of reconstituted Na-CN The solubility of reconstituted Na-CN suspensions was measured according to the International Standard (ISO 8156) (IDF 129) [17] method with necessary modifications. The reconstituted samples were centrifuged at 1000xg for 10 minutes and the pellets containing insoluble material were stored in a desiccator overnight at ambient temperature (23 ± 1°C) to

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remove the remaining free moisture. On the following day, the weight of the samples was recorded.

2.6. Viscosity measurements The viscosity of reconstituted Na-CN samples was evaluated with a Thermo HAAKE Rheometer RS100 (Artisan Technology Group, USA) using a 35 mm 2o angled cylindrical geometry. The viscosity measurements of the samples were collected at shear rates of 10-800 s-1 over a period of 10 minutes. All measurements were conducted in an environment where the ambient temperature was maintained at 23 ± 1oC. 2.7. Statistical analysis The statistical analysis was conducted using IBM’s statistical software (SPSS1, version 21, IBM Corp.). To determine the significance of the difference in the results, analysis of variance (ANOVA) was performed at the 95% confidence level (p < 0.05). Significantly diverse groups were categorised using Duncan’s test.

3.0. Results and Discussion 3.1. Particle size and solubility of reconstituted Na-CN prior to sonication Table 1 documents the particle size of the Na-CN suspensions, which were reconstituted to 4%, 7%, and 10% protein concentration before the application of ultrasound. Particle size was determined using the Mastersizer 3000. Results indicate that particle size at pH 4.0 and 4.6 could not be determined, as the pH is close to the IEP (pH 4.6) or below the IEP (pH 4.0), thereby causing coagulation and aggregation of the casein proteins at 4%, 7% and 10% protein concentration. This occurrence has been acknowledged in literature where, as the pH decreases to approximately 5.0 and below, aggregation of the casein proteins occurs due to a decrease in repulsive interaction [7,18]. This aggregation and gelling behaviour of the casein proteins is demonstrated in acidic conditions at pH 4.0 and 4.6 for 10% protein concentration (Figure ). Table 1 indicates that particle size at pH 6.7 ranges between 5 µm – 6 µm. As the suspension became more alkaline (pH 9.0), particle size increased to 78 µm and 82 µm at 4% and 7% protein, respectively. This trend of increased particle size at pH 9.0 due to swelling of 6

the casein proteins was demonstrated in prior literature [3,19]. At pH 9.0 and high protein concentration (10%), aggregation of the caseins occurred. Figure 2 shows the reconstitution of Na-CN at pH 9.0 for 4%, 7% and 10% protein concentrations, which indicates that as the pH is adjusted towards alkaline conditions the turbidity of solutions changes, particle size increases and aggregation occurs in the 10% protein sample. The caseins form aggregates, which consequently increase particle size [20]. Table 2 presents the measurements of Na-CN solubility before sonication. Although, swelling of the casein proteins occurred at pH 9.0, the highest solubility of 99.58% and 99.37%, was recorded at that pH in 4% and 7% protein solutions, respectively. Solubility was lower at neutral pH, with the lowest solubility measured at 10% protein concentration and pH 6.7 (native pH) which had a solubility of 94.37%. Significantly, higher (p < 0.05) solubility at approximately 98% was obtained when the pH was 6.7 and lower protein concentrations of 4% and 7%. The results suggest that there is a concentration effect on the solubility of reconstituted Na-CN. Moreover, these results demonstrate that as pH becomes more alkaline, solubility increases. At pH 9.0, solubility increases for all three protein concentrations. This is supported by literature where the solubility is higher as the pH is adjusted to the alkaline region [6]. As the pH becomes more acidic and close to the IEP, however, protein aggregation and coagulation occurred, thereby precluding any measurements from being taken. This is also supported in literature, where it has been stated that Na-CN starts to form aggregation at pH 4.6-5.5 and is also where solubility is at its lowest should the protein concentration be sufficiently low to remain in solution [6, 7]. 3.2. The effect of low-frequency ultrasound on the particle size and solubility of reconstituted Na-CN suspensions Low-frequency sonication was applied to the Na-CN suspensions at the specified range of ED (15, 30, 150, 300 and 400J/mL) and the particle size was measured. There was no observable particle size reduction at pH 6.7 at all three protein concentrations studied despite the increasing applied energy densities (Figures 3, 4A and 5A). Particle size remained constant at approximately 5 – 6 µm, and this agrees with previous literature suggesting that low-frequency ultrasound has no effect on the structural integrity of the casein proteins [22, 25]. Although the particle size of reconstituted Na-CN suspensions is unaffected by ultrasound, this contradicts other studies that have clearly demonstrated the particles size 7

reducing effects in suspensions containing insoluble aggregates [26, 27, 28, 29]. According to literature, low-frequency sonication was able to reduce the particle size of other milk suspensions such as whey protein concentrate (WPC), whey protein isolate (WPI) and milk protein concentrate (MPC) [26, 27, 28, 29]. These studies have shown that low-frequency ultrasound was able to disrupt the large insoluble aggregates and decrease the particle size. For example, WPI had a particle size of 25 µm before sonication and after applying sonication, the particle size was reduced to 1 µm [28]. However this is not the case for sodium caseinate as shown in Figures 4, 5 and 6 at pH 6.7 where the particles size is small (5 - 6 µm) confirming the absence of large insoluble aggregates. Particle size data closely relates to the solubility of Na-CN at pH 6.7 (

Figure 3) where there is no effect on the solubility after the application of ultrasound but as the protein aggregates are absent, the solubility of the samples is high (above 96%). Previous studies have shown that sonication improves the solubility of suspensions containing aggregates and that was achieved by reducing the size of suspended insoluble aggregates [26, 27, 29). For example, according to Jambrak [26] the initial WPI suspension had 67% solubility. However, after sonication, it increased by 18% to reach 85% solubility. That was achieved by breaking down insoluble aggregates in the suspension. Comparing this to Figures 3-5, the solubility does not show a significant change as Na-CN has high solubility in the absence of insoluble aggregates. Results at pH 9.0 on the other hand differ as reported in

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Figure 3Figure 4 and

Figure 5. Data shows that particle size reduction occurs after ultrasound treatment at pH 9.0 for all protein concentrations studied. Due to swelling and association of the caseins, the particles size is significantly greater in 7% protein solutions (pH 9.0) (~80 µm) than the equivalent at pH 6.7 (~5 µm). When high pH solutions are sonicated, the physical effects of acoustic cavitation disrupt the larger size suspensions. The trend indicates that at 15 J/mL, there is a significant reduction in particle size, with further reduction after application of 30 J/mL, and at 150 J/mL, the particle size becomes constant at ~2 µm. One suggested reason for the decrease in particle size after ultrasound treatment is the influence of shear force generated by ultrasound [25]. As particle size is reduced by ultrasound, solubility of the suspension is enhanced slightly at pH 9.0 from approximately 98% to 99%. Particle size and solubility at pH 4.0 and 4.6, however, could not be determined due to aggregation and protein coagulation that occurred under these conditions. Due to the technological limitations of the Mastersizer, the particle size was also measured using the Zetasizer to observe submicron particle behaviour.

Figure 6 reports the measured sub-micron particle size of 4%, 7% and 10% reconstituted Na-CN protein solutions at pH 6.7 and 9.0 before and after sonication. In the absence of agglomerates, results showed that at pH 9.0, particle size was smaller (~150 – 200 9

nm) compared to that at pH 6.7 (300 – 400 nm) as the casein proteins were more soluble. Data also indicated that there were minimal changes in particle size in response to sonication. It is expected that there is little to no sub-micron particle size change at pH 6.7 and 9.0 in the soluble phase due to the absence of large agglomerates. Furthermore, the physical effect of 20 kHz acoustic cavitation is insufficient to disrupt the structural integrity of the caseins at applied energy densities of up to 400 J/mL [21, 25]. 3.3. The surface charge of reconstituted Na-CN prior to and after sonication Table 3 reports the zeta potential of 4%, 7% and 10% Na-CN reconstituted solutions measured before sonication. Zeta potential is the measure of the surface net charge of a solution and was measured using the Zetasizer [22]. Results indicate that Na-CN solutions have a negative charge at pH 6.7 and 9.0. The surface charge of reconstituted Na-CN solutions was approximately -17 mV at pH 6.7 and -20 mV at pH 9.0. In Figures 4 and 5, it shows that the particle size was larger as the charge became more negative prior to sonication. This demonstrates the pH dependent interactions of the soluble casein proteins affecting aggregation and particle size. These results strengthen the theory that low-frequency sonication disrupts aggregation as shown at high pH in Figures 4 and 5. Due to caseinate destabilisation and protein aggregation, the surface charge was not measured at pH 4.0 and 4.6.

Figure 7. Zeta Potential before and after sonication (0, 15, 30, 150, 300 and 400J/mL) of 10% (A), 7% (B) and 4% (C) Na-CNreports the zeta potential measurements before and after sonication. As anticipated, there were no changes in the zeta potential after the application of low-frequency ultrasound, hence the resultant acoustic cavitation of the procedure has no effect on the chemical properties of the protein solutions [12]. 3.4. Viscosity of reconstituted Na-CN solutions The shear rate viscosity profiles of reconstituted Na-CN solutions are reported in Error! Reference source not found. (A and B). Viscosity was dependent on the protein concentration and pH, an observation supported by literature [23]. The greater the protein content, the higher the viscosity. At pH 9.0 and 10% protein, the viscosity was significantly greater (75 mPa.s at 100 sec-1) than at 7% protein (10 mPa.s at 100 sec-1) and 4% protein (3 mPa.s at 100 sec-1) at the same pH. 10

Solutions at pH 9.0 had a greater viscosity compared to solutions of the same concentrations at pH 6.7. The 10% protein solution at pH 9.0 was the most viscous prior to sonication. These findings are supported by literature where the viscosity was demonstrated to be high when the pH exceeds 9.0 [7, 8]. This is caused by casein protein aggregation, which then leads to an increase in viscosity. As the protein concentration at pH 9.0 is reduced, viscosity also decreases [8, 20]. At pH 6.7, prior to sonication, the viscosity of the suspension was at its lowest. That was also reported by other researchers [8]. Sonication at 20 kHz and applied ED of 150 J/mL shows no measurable viscosity reduction and this is vastly different to the viscosity reduction reported in whey protein solutions [11,13, 24]. In whey protein studies, the viscosity of solutions was reduced by approximately 10% after an applied ED as low as 31 J/mL. The viscosity lowering effect in whey protein solutions was attributed to the disruption of large aggregates, which are largely absent in Na-CN solutions. As low-frequency ultrasound disrupts the intramolecular bonds of the system which results in a physical effect, parameters such as viscosity can be modified. However, since Na-CN does not contain many large insoluble aggregates, our results demonstrate that ultrasound does not affect the viscosity of reconstituted Na-CN 4.0. Conclusions Low-frequency ultrasound and the acoustic cavitation effects associated with 20 kHz sonication had no physical influence on reconstituted Na-CN solutions at native pH 6.7. Due to the high solubility of Na-CN and absence of large agglomerates, sonication had little effect on the particle size, solubility and viscosity at pH 6.7. At pH 9.0 or below the IEP (pH 4.6), there was a noticeable increase in the particle size of Na-CN solutions. In acidic conditions and at high protein concentrations, the casein proteins aggregated. In alkali conditions at pH 9.0, the larger particles were reduced in size and solubility increased after ultrasonic treatment at applied energy densities of 15 J/mL to 150 J/mL. Particle size and solubility were stabilised and remained constant at energy densities above 150 J/mL. Viscosity of the Na-CN suspension was dependent on pH and protein concentration, and it was the greatest at pH 9.0 at higher protein concentrations. Solutions containing 10% protein had the highest viscosity whereas it was the lowest for 4% protein at pH 6.7. However, in the absence of large agglomerates, sonication did not influence the viscosity of Na-CN solutions at an applied ED of 150 J/mL.

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[12] J.Chandrapala, B. Zisu, M. Palmer, S. Kentish, M. Askhokkumar. Effect of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate. Ultrasonics Sonochemistry, 18 (2011) 951-957. [13] B. Zisu, J. Lee, J. Chandrapala, R. Bhaskarachary, M. Palmer, S. Kentish, M. Ashokkumar, Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. Journal of Dairy Research 78 (2011) 226–232. [14] F. Chemat, , Zill-e-Huma, M. K. Khan,. Applications of ultrasound in food technology: Processing, preservation and extraction. Ultrasonics Sonochemistry, 18 (2011) 813-835. [15] F. R. Contamine, A. M. Wilhelm, J. Berlan, H. Delmas, Power measurement in Sonochemistry, Ultrasonics Sonochemistry, (1994) 43-47. [16] B. Bhandari, B. Zisu, Effect of Ultrasound Treatment on the Evolution of Solubility of Milk Protein Concentrate Powder. Handbook of Ultrasonics and Sonochemistry, (2016) 1383-1401 [17] International Dairy Federation (ISO 8156) (IDF 129) (2005). Dried milk and dried milk products - Determination of insolubility index . [18] A. HadjSadok, A. Pitkowski, T. Nicolai, L. Benyahia, N. Moulai-Mostefa, Characterisation of sodium caseinate as a function of ionic strength, pH and temperature using static and dynamic light scattering. Food Hydrocolloids, 22(2007) 1460–1466. [19] T. Huppertz, B. Vaia, M. A. Smiddy, Reformation of casein particles from alkalinedisrupted casein micelles. Journal of Dairy Research, 75 (2008), 44-47 [20] B. Vaia, M. A. Smiddy, A. L. Kelly, T. Huppertz, Solvent-Mediated Disruption of Bovine Casein Micelles at Alkaline pH. Journal of Agricultural and Food Chemistry, 54 (2006), 8288-8293. [21] J. Chandrapala, G. J. Martin, B. Zisu, S. Kentish, M. Askhokkumar, The effect of ultrasound on casein micelle integrity. Journal of Dairy Science, 95 (2012) 6882–6890. [22] R. J. Hunter, R. H. Ottewill, R. L. Rowell, Chapter 2 - Charge and Potential Distribution at Interfaces. Zeta Potential in Colloid Science., 1981, pp. 11-58

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[23] H. S. Rollema, D. D. Muir, (2009). Casein and Related Products. A. Y. Tamime (1st Ed.), Dairy Powders and Concentrated Products, United Kingdom: John Wiley & Sons. 2009, pp. 235-255. [24] B. Zisu, R. Bhaskaracharya, S. Kentish, & M. Ashokkumar, Ultrasonic processing of dairy systems in large scale reactors. Ultrasonics Sonochemistry, 17 (2010) 1075–1081. [25] Madadlou, A., Mousavi, M. E., Emam-djomeh, Z., Ehsani, M., & Sheehan, D. (2009). Sonodisruption of re-assembled casein micelles at different pH values. Ultrasonics Sonochemistry, 16 (2009), 644–648. [26] Jambrak, A. R., Mason, T. J., Lelas, V., Herceg, Z., & Herceg, I. L., (2007). Effect of ultrasound treatment on solubility and foaming properties of whey protein suspensions. Journal of Food Engineering, 86(2), 281–287. [27] Jambrak, A. R., Mason, T. J., Lelas, V., Paniwnyk, L., & Herceg, Z., (2013). Effect of ultrasound treatment on particle size and molecular weight of whey proteins. Journal of Food Engineering, 121 (2014), 15-23. [28] Zisu, B., Lee, J., Chandrapala, J., Bhaskaracharya, R., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. Journal of Dairy Research, 78 (2011), 226–232. [29] Chandrapala, J., Martin, G. J. O., Kentish, S. E., & Ashokkumar, M. (2014). Dissolution and reconstitution of casein micelle containing dairy powders by high shear using ultrasonic and physical methods. Ultrasonics Sonochemistry, 21(2014), 1658–1665.

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A

B

Figure 1. Na-CN reconstituted to 10% protein concentration: A: pH 4.0; B: pH 4.6.

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A

B

C

Figure 2. Na-CN reconstituted to pH 9.0: A: 4% protein concentration, B: 7% protein concentration, C: 10% protein concentration.

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Figure 3. Particle size and solubility of reconstituted and sonicated (0, 15, 30, 150, 300 and 400J/mL) Na-CN at 10% protein concentration and pH 6.7. (n=3, Average ±SD)

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A

B

Figure 4. Particle size and solubility of reconstituted and sonicated (0, 15, 30, 150, 300 and 400J/mL) Na-CN at 7% protein concentration. A: pH 6.7, B: pH 9.0. (n=3, Average ±SD)

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B

A

Figure 5. Particle size and solubility of reconstituted and sonicated (0, 15, 30, 150, 300 and 400J/mL) Na-CN at 4% protein concentration. A: pH 6.7, B: pH 9.0. (n=3, Average ±SD)

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B

A

C

Figure 6. Sub-micron particle size of 10% (A), 7% (B) and 4% (C) Na-CN protein solutions sonicated (0, 15, 30, 150, 300 and 400J/mL) at pH 6.7 and 9.0. (n=3, Average ±SD)

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B

A

C

Figure 7. Zeta Potential before and after sonication (0, 15, 30, 150, 300 and 400J/mL) of 10% (A), 7% (B) and 4% (C) Na-CN reconstituted to pH 6.7 and 9.0. (n=3, Average ±SD)

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B

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Figure 8. Shear rate viscosity profile of Na-CN reconstituted to 4%, 7% and 10% protein at pH 6.7 and 9.0 prior to and after /mL sonication at 150J/mL: A: Full profile. B: y-axis set to 20 mPa.S.

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Table 1. Average* particle size (µm) of un-sonicated Na-CN suspensions at 4, 7 and 10% protein concentration and pH 4.0, 4.6, 6.7 and 9.0. pH

Protein (w/w) 4%

7%

10%

9.0

81.75 ± 11.68

78.20 ± 35.35

ND**

6.7

5.02 ± 0.11b***

4.97 ± 0.23b

6.81 ± 0.16a

4.6

ND

ND

ND

4.0

ND

ND

ND

*(n=3, Average ±SD) **Not determined (ND) due to sample aggregation and gelling. ***Mean values with different uppercase letter superscripts (a-c) in the same row are significantly different (p < 0.05).

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Table 2. Average* solubility (%) of un-sonicated Na-CN suspensions at 4, 7 and 10% protein concentration and pH 4.0, 4.6, 6.7 and 9.0. pH

Protein (w/w) 4%

7%

10%

9.0

99.58 ± 0.26

99.37 ± 0.04

ND**

6.7

98.38 ± 0.03b***

97.34 ± 0.18b

94.37 ± 1.09a

4.6

ND

ND

ND

4.0

ND

ND

ND

*(n=3, Average ±SD) **(ND) Not determined due to sample aggregation and gelling. ***Mean values with different letter superscripts (a-c) in the same row are significantly different (p < 0.05).

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Table 3. Average* zeta potential (mV) of un-sonicated Na-CN suspensions at 4, 7 and 10% protein concentration and pH 4.0, 4.6, 6.7 and 9.0. pH

Protein (w/w) 4%

9.0

-20.50 ± 1.09a***

10%

-20.09 ± 0.93a

21.87 ± 2.60a

-17.40 ± 0.45a

-17.80 ± 1.05a

ND

ND

ND

ND

-

6.7

17.40± 0.68a

7%

ND** 4.6 ND 4.0 *(n=3, Average ±SD) **(ND) Not determined due to sample aggregation and gelling. ***Mean values with different letter superscripts (a-c) at the same row are significantly different (p < 0.05).

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Highlights 

The effect of 20kHz ultrasound was examined on sodium caseinate (Na-CN) suspensions



Particle size, zeta potential, viscosity and solubility of Na-CN were affected



pH and protein concentration had the greatest effect on Na-CN suspensions



Ultrasound had little effect on non-micellear Na-CN at pH 6.7



Ultrasound reduced the particle size of non-micellar Na-CN pH 9.0

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