Sodium caseinate enhances the reconstituability of micellar casein powder

Sodium caseinate enhances the reconstituability of micellar casein powder Erix P. Schokker 1, 2, *, Amirtha Puvanenthiran 1, & Punsandani Udabage 1 1 CSIRO Food and Nutritional Sciences, Private Bag 16, Werribee, VIC 3030, Australia. 2 FrieslandCampina Research, P.O. Box 87, 7400 AB Deventer, the Netherlands. * Corresponding author. Tel.: + +31 570 695916; E-mail address: erix.schokker@frieslandcampina.com 1

Abstract Micellar casein powders (MC) were produced where sodium caseinate (NaCas; 4% and 12% of the total protein) was added to the concentrate before and after diafiltration prior to spray drying. The reconstituability of the MC increased with increasing NaCas content, with the NaCas addition after diafiltration having the greatest impact in improving the reconstituability compared to the addition before diafiltration. A control experiment in which NaCas was dry-mixed with MC showed that the improved reconstituability of the MC could not be explained solely by the solubility of the NaCas, but was rather due to the increased amount of non-micellar casein. The non-micellar casein would delay aggregation of casein micelles by modification of the powder surface during spray-drying with the increased adsorption of the non-micellar casein, or by acting as filler between casein micelles. The strategy is novel, easily incorporated into the existing processing protocol, and more promising than the existing technological fixes, because the integrity of the native casein micelles and the high concentration of calcium are maintained with minimal changes to composition and functionality. Key words: micellar casein powder, sodium caseinate, native phosphocasein, reconstituability, solubility, air-water interface 2

1. Introduction Milk protein concentrate (MPC) and micellar casein powder (MC) are produced by membrane filtration of skim milk followed by spray-drying. For the production of MPC ultrafiltration (typically with a molecular weight cut-off of 10,000 MW) is used, and the ratio between caseins and whey proteins in the end product is the same as in the milk from which the MPC is made. Depending on the extent of diafiltration, hence removal of lactose and salts, the final protein content of the MPC ranges from 42 to 85%. At higher protein contents, e.g., 90%, the product is called milk protein isolate (MPI). For the preparation of MC (also referred to as native phosphocasein), microfiltration (0.1-0.2 µm) is used. In this process also whey proteins are removed from the milk, and a casein micelles enriched product remains (Rollema, & Muir, 2009). The reconstitution process of MPCs and MCs is slow compared to e.g. skim milk powder, especially at low temperatures and deteriorates even further on storage (Anema, Pinder, Hunter, & Hemar, 2006; Havea, 2006; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2010a). Dissolving at higher temperature or at high shear eliminates the poor reconstitution properties to some extent (Gaiani et al., 2006; Jeantet, Schuck, Six, André, & Delaplace, 2010; McKenna, 2000; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). Certain processing steps may improve the reconstituability of MPCs and MCs, e.g., lower drying temperatures (Schuck et al., 1994), the partial depletion of calcium by cation exchange chromatography, chelating agents or acidification (Bhaskar, Singh, & Blazey, 2001), the addition of NaCl before or after ultrafiltration (Carr, 2002), or the addition of whey proteins or polydextrose (Davenel, Schuck, & Marchal, 1997). Work done in our laboratories showed that the reconstitution properties of MC can be improved if the amount of non-micellar casein in the powder is increased (Schokker et al., 2010). Especially, a MC in which sodium caseinate (NaCas) was included in the preparation had improved reconstituability in cold water. Here, we explore the extent to which this novel approach could be manipulated during production of the powders and the mechanism(s) responsible for the improved reconstituability. 3

2. Materials & Methods 2.1. Materials Skimmed milk was obtained from Tatura Milk Industries (Tatura, VIC 3616, Australia). Sodium caseinate 180 (NaCas) was produced by Fonterra Co Operative Ltd. (Auckland, New Zealand). 2.2. Preparation and storage of MC powders MC powders were prepared in the pilot plant at CSIRO-Food & Nutritional Sciences in June 2010. A description of the powder production is given in Figure 1. The skim milk was heat treated (15 s at 73.5 C, APV T4 plate heat exchanger), and microfiltrated (Tetra Pack Filtration Systems, Alcross Pilot M7, fitted with 1.2 µm Membralox 7P19-40 ceramic membrane module 1.7 m 2, 800 kg/h, 48 C, 30 kpa transmembrane pressure) to remove bacteria. The milk was microfiltered using a 0.1 µm membrane to remove whey proteins, lactose and salts and concentrated 3 times (Alfa-Laval MFS-1, fitted with a 0.1 µm SCT-Membralox 1P19-40 membrane module 0.24 m 2, 90 kg/h, 45 C, 30 kpa transmembrane pressure). The 3 times concentrate (3R) was subdivided in to portions of 80 L. To each of the portions 80 L equal volumes of either water, 0.5% or 1.5% sodium caseinate was added, and the mixtures were kept for 90 min at room temperature. Subsequently, the mixtures were diafiltered using another 80 L of water and concentrated to 6 times concentration (6R). Concentrate 6R1 was mixed with 6% NaCas, to match the ratio NaCas:total milk protein of 6R4 and 6R5. The concentrates were spray-dried using a Drytec Compact Laboratory Spray Dryer (Drytec, Kent, UK) fitted with a twin fluid nozzle operated at 250 kpa atomisation pressure and a peristaltic pump (inlet temperature 170 C, outlet temperature 75 C, evaporation ~3-4 kg/h, temperature feed ~45 C). A portion of powder 1 was dry-mixed with NaCas to obtain powders with similar amounts of NaCas as powders 2 and 3. In the powders the amount of protein originating from NaCas was approximately 0, 4, and 12% of the total milk protein in the powders. Each MC powder was divided into ~6 g portions and placed in a 4

desiccator containing saturated MgCl 2 solution, corresponding to an a w of 0.3, at 30 C. 2.3. Composition Protein content of the MCs was analysed by the Dumas method using a Leco FP-2000 Nitrogen/Protein Analyzer. The casein content of the powders was determined by precipitation at ph 4.6 after dissolving the MC for 24 h. Fat content was analysed using Australian Standard method AS 2300.1.3, comprising the extraction of fat of the dissolved powder with diethyl ether and petroleum spirit and measuring the extracted fat gravimetrically. Lactose content was determined by an enzymatic method according to Australian Standard method 2300.6.6. Moisture content was determined by oven drying the powder at 102 C under atmospheric pressure. Ash content was determined by incineration at 550 C. Calcium was determined by inductively coupled plasma optical emission spectrometry after acidic digestion. Water activity of the powders was determined directly after production using an Aqualab CX-2 (Decagon) water activity meter. All analyses were performed in triplicate. Composition data for NaCas was supplied by the manufacturer. 2.4. Reconstituability 5 g of MC was mixed with 95 g of milliq water at 20 ± 3 C in a 400 ml beaker. The mixture was stirred for 30 min at 600 RPM, using a Heidolph RZR 2050 electronic overhead stirrer (Heidolph Elektro Kelheim, Germany) and a flat propeller designed for dissolving powders. The solution was centrifuged 15 min at 750 g at 20 C (Beckman J2-MC Centrifuge; JA-20 rotor, 2500 rpm), and the dry matter of the dispersion and supernatant were measured by oven drying at 102 C. Reconstituability (%) = (dry matter supernatant / dry matter MC dispersion) *100 %. The reconstituability was determined in triplicate. 5

3. Results The composition of the MCs and the NaCas is given in Table 1. In Table 2 the reconstituability values of the MCs directly after production and after storage for 80 d at 30 C are given. The results show that addition of NaCas to MC markedly improved the reconstituability of fresh MC, confirming previous results (Schokker et al., 2010). Even low concentrations had a positive effect. The effect of NaCas addition on the reconstituability appears proportional to its concentration (Figure 2). Mixing the NaCas to the liquid concentrate before spray-drying had a greater positive effect than if the NaCas was dry-mixed after spray-drying. Mixing the NaCas directly before spray-drying was more efficient than mixing the NaCas earlier in the process, i.e. before the diafiltration step. In the concentrated milk where NaCas is added after the diafiltration, more non-micellar casein would be retained in the concentrate than in the case when NaCas is added before diafiltration. In the case of the powder where NaCas was dry-mixed with MC, the increased reconstituability of the powder may be explained by the reconstituability of the two components. I.e., if we assume that the added NaCas dissolves almost completely within 30 min (Bastier, Dumay, & Cheftel, 1993), and the MC component reconstitutes as in the MC to which no NaCas was added, the increase in reconstituability of the MC powder would be accounted for. E.g., in the case of powder 7, containing 12% NaCas, the calculated reconstituability would be 66.0% (i.e., 0.88*61.4 + 0.12*100), as compared to 66.8% for the measured reconstituability. The increased reconstituability of the MCs to which NaCas was added to the milk concentrate (i.e. before spray-drying) is larger than just the extra dissolution of NaCas (Figure 2). This increase in reconstituability is not due to the sodium introduced with the NaCas, because this amount was less than the amount that would improve the reconstituability of MPCs (Carr, 2000). The improved reconstituability of the MC where NaCas was mixed to the milk concentrate would be due to the effect of the increased amount of non-micellar casein (Schokker et al., 2010). 6

Upon storage the reconstituability of the MCs decreased (Table 2). There was no relation between the amount of non-micellar casein in the MC and the storage stability of the MC, consistent with previous results (Schokker et al., 2010). 4. Discussion It has been reported that the loss of reconstituability in MPC and MC powder is not due to the formation of insoluble material, but is rather a decrease in the rate of release of casein micelles from the dispersed powder particles (Mimouni et al., 2010a; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2010b; Schokker et al., 2010). The loss of reconstituability is believed to be primarily due to the cross-linking between casein micelles. Cross-linking between casein micelles occurs especially between micelles at the particle surface, leading to skin formation (McKenna, 2000; Mimouni et al., 2010b). The cross-linking between casein micelles at the particle surface is possibly mediated by their adsorption and spreading at the air-water interface (Schokker et al., 2010). Casein micelles that are spread on an air-water interface are more susceptible for aggregation (Walstra, 2000). Therefore, adsorption of casein micelles at the air-water interface after atomization in the spray-drying process and the subsequent aggregation of these micelles need to be minimized to prevent extensive cross-linking between the micelles and loss of reconstituability. MPC and MC powders have 2-3 times more protein than skim milk powder; for a given volume, more proteins and less lactose are present. This would lead to an effective decrease in the distances between casein micelles. The decrease in the mean distance between two casein micelles would promote the aggregation of casein micelles leading to skin formation and loss of reconstituability. Therefore, in addition to controlling the surface composition of a powder particle, cross-linking between casein micelles, both on the powder particle surface and in the core, would be reduced if molecules that spatially separate the casein micelles in the powder are added. Processes contributing to improving the reconstituability of MPC and MC powders are: (1) addition of NaCas (this study; Schokker et al., 2010), partial depletion of 7

calcium by cation exchange chromatography, chelating agents or acidification (Bhaskar et al., 2001; Schokker et al., 2010), the addition of NaCl (Carr, 2002; Schokker et al., 2010). These processes increase the amount of non-micellar casein in the system and hence the amount of non micellar casein adsorbed on the surface. (2) The addition of whey proteins or polydextrose (Davenel et al., 1997) which act as fillers. In the case of whey proteins, the addition of whey proteins to the milk leads to a decreased amount of casein micelles adsorbed on the surface of the powder. (3) Lower drying temperatures (Schuck et al., 1994) which decreases the amount of heat denaturation and aggregation. The structural design of the powder particle and its surface is dictated by the constituents of the concentrate during drying and is responsible for the reconstituability of the fresh powders. Non-micellar casein may reduce aggregation via two mechanisms. As illustrated in Figure 3A, during spray drying, the increased amount of casein micelles in a normal milk protein concentrate (System 1) will favour a preferential adsorption of casein micelles at the air-water interface. In contrast, in a concentrate where there is increased amounts of non-micellar casein (System 2), there would be less of casein micelles being adsorbed to the interface due to the more surface active nature of non-micellar casein in comparison with micellar casein (Figure 3D). This difference in the composition of the interface will result in a powder with higher reconstituability in system 2 than in system 1. As illustrated in Figure 3B and 3E, the aggregation of the casein micelles may occur at the surface as well as in the core. In system 2 both types of aggregation occur to a lesser extent (Figure 3E) than in system 1 (Figure 3B) because the increased nonmicellar caseins in system 2 will spatially separate casein micelles, preventing crosslinking of neighbouring micelles both on the surface and in the core of the powder particle. Due to these reasons the release of the casein micelles from the dispersed powder particles would be faster in system 2 compared to system 1. The effect of non-micellar casein is apparent in the fresh MC. Non-micellar casein seems to have no preventative effect during the storage of the powders. This would suggest that the action of non-micellar casein is especially in preventing adsorption and spreading of casein micelles at the air-water interface. 8

5. Conclusions The reconstituability of MC can be enhanced by the addition of NaCas to the concentrate. We proposed that addition of NaCas increases the amount of nonmicellar casein in the concentrate, which reduces adsorption and spreading of casein micelles on the air/water interface and which acts as filler between casein micelles, thereby preventing aggregation of the micelles. The reconstituability of the MC increased with increasing NaCas content, with the NaCas addition after diafiltration having the greatest impact in improving the reconstituability compared to the addition before diafiltration. Addition of NaCas to the final concentrate has the additional advantages of postponed product differentiation, less slowing down of the microfiltration as compared to addition of NaCas to the half-concentrate, and that the viscosity of the feed for the spray-dryer remains rather low. The improvement in reconstituability with small amounts of added sodium caseinate is more appealing than the existing technological fixes since the additive is a casein, the integrity of the native casein micelles and the high concentration of calcium is maintained with minimal changes to functionality. Acknowledgements The authors wish to thank Michael Mazzonetto and Daryl Unthank for producing the micellar casein powders. 5. References 1. Anema, S.G., Pinder, D.N., Hunter, R.J., & Hemar, Y. (2006). Effects of storage temperature on the solubility of milk protein concentrate. Food Hydrocolloids, 20, 386-393. 2. Bastier, P., Dumay, E., & Cheftel, J.C. 1993. Physicochemical and functional properties of commercial caseinates. Lebensmittel-Wissenschaft und Technologie, 26, 529-537. 3. Bhaskar, G.V., Singh, H., & Blazey, N.D. (2001). Milk protein products and processes. WO01/41578. 9

4. Carr, A.J. (2002). Monovalent salt enhances solubility of milk protein concentrate. WO02/096208. 5. Davenel, A., Schuck, P., & Marchal, P. (1997). A NMR relaxometry method for determining the reconstitutability and water-holding capacity of protein-rich milk powders. Milchwissenschaft, 52, 35-39. 6. Gaiani, C., Scher, J., Schuck, P., Hardy, J., Desobry, S., & Banon, S. (2006). The dissolution behaviour of native phosphocaseinate as a function of concentration and temperature using a rheological approach. International Dairy Journal, 16, 1427-1434. 7. Havea, P. (2006). Protein interactions in milk protein concentrate powders. International Dairy Journal, 16, 415-422. 8. Jeantet, R., Schuck, P., Six, T., André, C., & Delaplace, G. (2010). The influence of stirring speed, temperature and solid concentration on the rehydration time of micellar casein powder. Dairy Science and Technology, 90, 225-236. 9. McKenna, A.B. (2000). Effect of processing and storage on the reconstitution properties of whole milk and ultrafiltered skim milk powders. PhD thesis, Massey University, Palmerston North, New Zealand. 10. Mimouni, A., Deeth, H.C., Whittaker, A.K., Gidley, M.J., & Bhandari, B.R. (2009). Rehydration process of milk protein concentrate powder monitored by static light scattering. Food Hydrocolloids, 23, 1958-1965. 11. Mimouni, A., Deeth, H.C., Whittaker, A.K., Gidley, M.J., & Bhandari, B.R. (2010a). Rehydration of high-protein-containing dairy powder: slow- and fastdissolving components and storage effects. Dairy Science and Technology, 90, 335-344. 12. Mimouni, A., Deeth, H.C., Whittaker, A.K., Gidley, M.J., & Bhandari, B.R. (2010b). Investigation of the microstructure of milk protein concentrate powders during rehydration: alterations during storage. Journal of Dairy Science, 93, 463-472. 13. Rollema, H.S., & Muir, D.D. (2009). Casein and related products. In A.Y. Tamine (Ed.), Dairy powders and concentrated products (pp. 235-254). Chichester: Wiley- Blackwell. 14. Schokker, E.P., Church, J.S., Mata, J.P., Gilbert, E.P., Puvanenthiran, A., & Udabage, P. (2010). Reconstitution properties of micellar casein powder: effects of composition and storage. International Dairy Journal, submitted. 10

15. Schuck, P., Piat, M., Méjean, S., Le Graet, Y., Fauquant, J., Brulé, G., & Maubais, J.L. (1994). Déshydratation par atomisation de phosphocaséinate natif obtenu par microfiltration sur membrane. Lait, 74, 375-388. 16. Walstra, P. (1990). On the stability of casein micelles. Journal of Dairy Science, 73, 1965-1979. 11

Table 1: Composition of micellar casein powders and NaCas. Powder Fat Protein Casein Lactose Ash Calcium Sodium Moisture A w g/100g powder 1 1.4 85.3 72.4 2.1 8.2 2.6 0.05 3.4 0.20 2 1.3 86.6 73.5 1.8 7.7 2.3 0.16 3.0 0.21 3 1.1 85.7 71.4 1.9 7.9 2.4 0.09 3.3 0.22 4 1.3 85.8 71.2 1.6 7.9 2.5 0.05 3.4 0.19 5 1.3 86.0 71.9 1.6 7.4 2.3 0.07 3.7 0.20 NaCas 0.7 92.3 NA 0.0 3.4 NA 1.4 4.8 NA NA: Data not available 12

Table 2: Reconstituability of micellar casein powders. Powder Directly after production After 80 d at 30 C 1 61.4±0.9 20.5±1.1 2 67.3±1.4 33.3±0.5 3 75.4±1.8 27.4±.07 4 64.6±0.7 21.9±1.5 5 68.9±2.2 31.8±2.0 6 63.2±0.2 24.0 * 7 66.8±0.5 30.2 * * single measurement 13

Skimmed milk Pasteurisation and Microfiltration 1.2µm Microfiltration 0.1 µm Concentrate (3R) Mix 1:1 with water Diafiltration 0.1 µm Mix 1:1 with 0.5% NaCas Diafiltration 0.1 µm Mix 1:1 with 1.5% NaCas Diafiltration 0.1 µm Concentrate (6R1) Concentrate (6R4) Concentrate (6R5) Spray-dry Powder 1 Mix 2.5:1 with 6% NaCas (6R2) Mix 7.5:1 with 6% NaCas (6R3) Dry mix with NaCas Spray-dry Spray-dry Spray-dry Spray-dry Powders 6 & 7 Powder 2 Powder 3 Powder 4 Powder 5 Figure 1: Production of micellar casein powders 14

80 Reconstituability (%) 70 60 0 5 10 15 Proportion of NaCas (%) Figure 2: Reconstituability of micellar casein powders containing sodium caseinate (NaCas). : NaCas mixed directly before spray-drying (powders 2 and 3); : NaCas mixed before diafiltration (powders 4 and 5); : NaCas dry-mixed with MC (powders 6 and 7). : Micellar casein powder without extra NaCas (powder 1). 15

System 1 A B C Storage Solubilization System 2 D E F Storage Solubilization Casein Micelles Non-micellar casein Figure 3: Proposed mechanism for the improvement of reconstituability of micellar casein powder with an increased concentration of non-micellar casein, e.g. by addition of sodium caseinate. 16