AU2008341009A1 - Processing of dairy ingredients by ultra-sonication - Google Patents

Description

WO 2009/079691 PCT/AU2008/001867 PROCESSING OF DAIRY INGREDIENTS BY ULTRA-SONICATION This invention relates to the processing of dairy ingredients to improve their functionality. In particular it relates to modifying protein aggregation, reducing viscosity and promoting heat stability. 5 Background to the invention The functional properties of food proteins are those physicochemical properties that affect the behaviour of proteins in food systems during preparation, processing storage or consumption. Properties of importance for dairy proteins include their 10 solubility in water (hydrophobicity), tertiary and quaternary structure (conformation) and the extent of aggregation with other proteins. In turn these properties influence the viscosity, gelation, foaming and emulsification ability of dairy systems. Of particular interest in the present case is the modulation of viscosity of dairy concentrates. Of additional interest in the present case is the relative resistance of 15 milk and dairy ingredients to thickening or coagulation after heating. This is referred to as heat stability (see Singh, International Journal of Dairy Technology Vol 57, No 2/3 May/August 2004). It is known that some high pressure or high shear processes can affect protein conformation and can lead to protein denaturation, aggregation or gelation. 20 USA patent 6511695 discloses a high pressure homogenization treatment of a protein solution preferably whey protein, to provide a protein ingredient with improved solubility and enhanced viscosity and gel firmness. The examples treat whey protein isolate (WPI) and whey protein concentrate (WPC) with high pressure homogenization and appear to be better suited to WPI. 25 USA patent 5171603 discloses the treatment of whey by ultrafiltration followed by heating under high shear conditions to provide a fat replacement protein having spherical particles mostly below 3 microns in size. A microfluidisation treatment has been used for heat denatured WPC to improve solubility (lordache, M., Jelen, P. 2003. Innovative Food Science and Emerging 30 Technologies, Vol 4, 367-376). USA patent 5932272 uses hydrostatic pressure to form a gel from a mix of proteins and polysaccharides.

WO 2009/079691 PCT/AU2008/001867 2 USA patent 6372276 to Tetra Laval discloses the treatment of milk with a combination of micro and ultrafiltration steps and a heat treatment. It is an object of this invention to improve the functional properties of dairy protein containing ingredients by modifying the protein conformation and aggregate size 5 using the unique properties of ultrasound. These modifications affect the gelation, viscosity and heat stability of the protein containing ingredients. Sonication of liquids leads to a unique combination of chemical and physical effects powered by high frequency sound waves. Ultrasonic waves generate acoustic pressure inducing motion and mixing of liquids through acoustic streaming. The 10 acoustic waves can generate microbubbles. These microbubbles grow under the influence of the ultrasonic field until they eventually collapse causing cavitation and the release of light (sonoluminescence), localized high temperatures (5000K) and pressures (up to 1000 atmospheres). As a result areas of high shear and turbulence occur at the cavitation sites. Chemical reactions can also be initiated through 15 pyrolysis and the generation of free radicals (Muthupandian Ashokkumar, Timothy J. Mason. Sonochemistry. Kirk-Othmer Encyclopedia of Chemical Technology (Online Version). Copyright @ 2007 by John Wiley & Sons, Inc.). Some other ultrasound treatments have been proposed for food ingredients in relation to different aspects of ingredient functionality 20 USA patents 4675194 and 5629037 disclose the use of sonication in the formation of cheese curd. USA patent 6294212 uses sonication in an extruder mixing fats and flour. Japanese patent 20062565512 discloses the use of sonication to form a liquid from a gel. 25 USA patent 6861080 discloses using a physical treatment such as sonication to reduce fat particle sizes in dairy emulsions. Brief description of the invention To this end the present invention provides liquid and reconstitutable dried dairy 30 ingredients that have tuneable viscosity and gelation properties and improved heat stability. These properties are obtained after subjecting the ingredients to either sonication alone or a combination of heating and sonication.

WO 2009/079691 PCT/AU2008/001867 3 The dairy ingredients contain whey proteins and are preferably selected from cheese whey, whey protein concentrates (WPCs), retentates from ultrafiltered whey, milk concentrates or milk protein concentrates that are either fresh or re-constituted from a powder form. 5 One preferred aspect of the present invention provides a method of modifying the properties of fresh and reconstituted dairy ingredients by sonication alone at an applied energy level of less than 500 J/mI and at a frequency below 213 kHz. Sonication within the specified frequency range reduces the particle size in both fresh and reconstituted dairy solutions. For solutions prepared from reconstituted io powders this leads to increased solution clarity. For both fresh and reconstituted solutions, the effect of moderate levels of sonication is to reduce viscosity. Importantly, the effect of sonication on dairy ingredients of solids content greater than 30% (w/w) solids is to immediately and significantly reduce the viscosity. When the sonicated material is subjected to further heat treatment either before or after 15 drying, gelation times and gel syneresis can be reduced and the gel strength of the product increased. Importantly, the effects on gelling are maintained after freeze drying or spray drying of the dairy ingredient and reconstitution. In a second preferred aspect the present invention provides a method of modifying the properties of dairy ingredients which includes the steps of heating the ingredient 20 to a temperature above 65*C followed by sonication at an applied energy level of less than 500 J/ml and at a frequency below 213 kHz. Sonication of a dairy ingredient preheated to above 650C disrupts heat-induced aggregates in the product and immediately reduces the viscosity of the ingredient. More importantly, sonication prevents a further increase in viscosity after a second heat treatment, i.e. 25 it improves heat stability. Gelation after a second heat treatment is also delayed or eliminated. Most importantly, these effects on gelation are maintained after freeze drying or spray drying of the dairy ingredient and reconstitution. In a third preferred aspect the present invention provides a method of modifying the properties of dairy ingredients which includes the steps of simultaneously heating the 30 ingredient to a temperature above 650C while sonicating at an applied energy level of less than 500 J/mI and at a frequency below 213 kHz. Heat-induced aggregates in the product are again disrupted and functional properties altered as in the second preferred aspect of the present invention.

WO 2009/079691 PCT/AU2008/001867 4 Description of the drawings Figure 1 shows the relationship between sonication time and delivered power and solution turbidity for a reconstituted whey protein concentrate solution; 5 Figure 2 shows the particle size distribution of 5% (w/w) solids reconstituted whey protein concentrate solutions after sonication at 20 kHz and 13W delivered power to a 50 ml solution; Figure 3 shows the particle size distribution of 5% (w/w) solids reconstituted whey protein concentrate solutions after sonication at 20 kHz and 31W delivered power to 10 a 50 ml solution; Figure 4 shows the particle size distribution of 5% (w/w) solids reconstituted whey protein concentrate solutions after sonication at 20 kHz and 50W delivered power in a 50 ml solution; Figure 5 shows the effect of delivered sonication power and time at 20 kHz on the 15 particle size distribution for particles below 1 micron for a 5% (w/w) solids reconstituted whey protein concentrate solution of 50 ml; Figure 6 shows relative gel strengths against time at different delivered sonication powers at 20 kHz for a 15% (w/w) solids reconstituted whey protein concentrate solution of 50 ml; 20 Figure 7 shows the gel strength of 15% (w/w) reconstituted WPC80 at different pH values. Reconstituted WPC80 was sonicated prior to heating at 800C for 20 minutes; Figure 8 shows the relative syneresis of gels with sonication time at different delivered sonication powers at 20 kHz for a 15% (w/w) solids reconstituted whey 25 protein concentrate solution of 50 ml; Figure 9 shows TEM microscopic analysis of 15% (w/w) solids reconstituted whey protein concentrate solutions which have been sonicated at 20 kHz and then heated at 800C for 20 minutes to form gels; Figure 10 shows viscosity of whey protein retentate (54% (w/w) TS and 20% 30 protein) at 25*C. Retentate was sonicated (US) at 20 kHz using a 1kW unit at 70% power and a flow rate of 200 ml/min to achieve a residence time of 1.3 minutes. Applied energy is 210 J/ml; WO 2009/079691 PCT/AU2008/001867 5 Figure 11 shows viscosity of whey protein retentate (54% (w/w) TS and 20% protein) at 100 RPM and 25*C. Retentate was sonicated (US) at 20 kHz using a 1kW unit at various power settings and a flow rate of 200 mI/min to achieve a residence time of 1.3 minutes. Applied energy is 150, 240 and 300 J/ml; 5 Figure 12 shows viscosity of whey protein retentate (30% (w/w) TS and 11% protein) at 250C. Retentate was sonicated (US) at 20 kHz using a 1kW unit at 70% power and a flow rate of 200 ml/min to achieve a residence time of 1.3 minutes and an applied energy of 210 J/ml; Figure 13 shows viscosity of whey protein retentate (33% (w/w) TS and 27% 10 protein) at 25 0 C. Retentate was sonicated at 20 kHz using a 4kW unit at various power levels at a flow rate of 1.4 1/min; Figure 14 shows viscosity of whey protein retentate (33% (w/w) TS and 27% protein) at 250C. Retentate was sonicated at 20 kHz using a 4kW unit at various power levels and flow rates of 6 1/min, 1.4 1/min or 700 ml/min; 15 Figure 15 shows viscosity of whey protein retentate (33% (w/w) TS and 27% protein) at 25 0 C. Retentate was sonicated repeatedly at 20 kHz using a 4kW unit at 50% power at a flow rate of 1.4 1/min; Figure 16 shows gel strength of whey protein retentate (33% (w/w) TS and 27% protein) at 40C with or without sonication at 20 kHz using a 4kW unit at a flow rate of 20 1.41/min and 50% and 95% power. Retentate was gelled at 800C for 20 min; Figure 17 shows the effect on viscosity of preheating (PreH) 5% (w/w) reconstituted WPC80 solution for 1 minute, sonicating (US) at 20 kHz and 31W delivered power in a 50 ml solution, and then post heating (PostH) at 800C for 20 minutes; Figure 18 shows the effect on viscosity of preheating (PreH) 5% (w/w) reconstituted 25 WPC80 solution at 800C for 20 minutes, sonicating (US) at 20 kHz and with 31W delivered power as 50 ml solutions, and then post heating (PostH) at 800C for 20 min; Figure 19 shows the effect of preheating (PreH) 9% (w/w) reconstituted WPC80 solution for 1 minute at 800C, sonicating (US) at 20 kHz and 31W delivered power in 30 a 50 ml solution, and then post heating (PostH) at 800C for 20 minutes on the viscosity measured at a shear rate of 100s1; Figure 20 shows the effect of preheating (PreH) 12% (w/w) reconstituted WPC80 solution for 1 minute at 800C, sonicating (US) at 20 kHz and 31W delivered power in WO 2009/079691 PCT/AU2008/001867 6 a 50 ml solution, and then post heating (PostH) at 800C for 20 minutes on viscosity measured at a shear rate of 100s-1; Figure 21 shows the effect of sonicating gels; Figure 22 shows the effect on viscosity at a shear rate of 100s- and particle size of 5 preheating (PreH) a 5% (w/w) reconstituted WPC80 solution for 1 minute at 80 0 C, continuous sonication (US) in a 350 ml chamber at 20 kHz and an applied power of 300W and with a flow rate of 300 ml/min or 500 ml/min, and then post heating (PostH) at 800C for 20 minutes; Figure 23 shows the effect on viscosity at a shear rate of 100s- 1 and particle size of 10 preheating (PreH) whey protein ultrafiltration retentate diluted to 10% (w/w) solids for 1 minute at 800C, batch sonicated (US) at 20 kHz and 31W delivered power in a 50 ml solution, and then post heated (PH) at 80 0 C for 20 minutes; Figure 24 shows the effect on viscosity of whey protein ultrafiltration retentate which was diluted to 5% (w/w), preheated at 800C for 20 min and batch sonicated (US) at 15 31W delivered power and 20 kHz for 1 min then freeze dried (FD) and reconstituted to 15% (w/w); Figure 25 shows the viscosity at a shear rate of 100s-1 and particle size for a whey protein retentate which is diluted to 8% (w/w) solids and then preheated (PreH) at 800C for 1 min. This was followed by continuous sonication (US) in a 350 ml 20 chamber at an applied power of 300W and a flow rate of 300 mI/min (applied energy of 60 J/ml) before post heating at 800C for 20 min; Figure 26 shows the viscosity at a shear rate of 100s-land particle size for a whey protein retentate which is diluted to 8% (w/w) solids and then preheated (PreH) at 800C for 1 min. This was followed by continuous sonication (US) in a 350 ml 25 chamber at an applied power of 300W and at a flow rate of 300 ml/min (applied energy of 60 J/ml). The retentate is then spray dried and reconstituted to 8% (w/w) solids, before post heating (PH) at 800C for 20 min; Figure 27 shows viscosity of whey protein retentate (8% (w/w) TS and 6% protein) at 25*C. Pre heating (preH) was at 80*C for 1 min and post heating (postH) was at 30 850C for 30 min. Retentate was sonicated at 20 kHz using a 4kW unit at various power levels at a flow rate of 1.4 I/min; Figure 28 shows the viscosity of whey protein retentate (20% (w/w) TS and 7% protein) at 250C. Retentate was pre heated (PreH) at 850C for 30 seconds and WO 2009/079691 PCT/AU2008/001867 7 sonicated (US) at 20 kHz using a 1 kW unit at 70% power and a flow rate of 200 ml/min to achieve a residence time of 1.3 minutes and applied energy input of 210 J/ml; Figure 29 shows the viscosity of spray dried whey protein concentrate (WPC) 5 reconstituted to 10% (A), 15% (B) and 20% (C) (w/w) TS at 250C. Reconstituted WPC was post heated (postH) at 80 0 C for 30 min; Figure 30 shows the particle size distribution of 5% (w/w) solids reconstituted whey protein concentrate solutions after sonication (US) at 647 kHz and 13W in a 50 ml solution; 10 Figure 31 shows the change in viscosity at a shear rate of 100s 1 of 30% (w/w) reconstituted WPC80 sonicated for 1 minute at 213 kHz and a delivered power of 5, 13,31 and 51W; Figure 32 illustrates the effect of sonicating preheated (800C/1 min) 8% (w/w) reconstituted WPC80 solutions for 1 minute at 20 kHz, 213 kHz, 355 kHz and 647 15 kHz on viscosity at a shear rate of 100s 1 and particles size; Figure 33 illustrates effect of sonicating whey protein retentate for 1 minute at 20 kHz, 213 kHz, 355 kHz and 647 kHz on viscosity at a shear rate of 100s 1 and particle size; Figure 34 illustrates the effect of simultaneous heating and sonication (Sim.US) on 20 viscosity at a shear rate of 100s1 compared with preheating (PreH), cooling and sonication (US) for 10% (w/w) reconstituted WPC80. Detailed description of the invention This invention provides liquid and reconstitutable dried dairy ingredients that have 25 tuneable viscosity and gelation properties and improved heat stability. These properties are obtained after subjecting the ingredients to either sonication alone or a combination of heating and sonication. The dairy ingredients contain whey proteins and are preferably selected from cheese whey, whey protein concentrates (WPCs), retentates from ultrafiltered whey, milk 30 concentrates or milk protein concentrates that are either fresh or reconstituted from a powder form. Example 1. Sonication of reconstituted WPC80.

WO 2009/079691 PCT/AU2008/001867 8 Solutions (50ml) of reconstituted 5% and 15% (w/w) WPC80 were batch sonicated with a 20 kHz horn transducer for 10, 20, 40 and 60 min. The power delivered to the solution was determined calorimetrically as 13, 31 or 50 W (see T. Kimura, T. Sakamoto, J.-M. Leveque, H. Sohmiya, M. Fujita, S. Ikeda, T. Ando, Standardization 5 of ultrasonic power for sonochemical reaction, Ultrason. Sonochem. 3 (3) (1996) S157-S161 for the calorimetric method). By increasing sonication time solutions became more translucent. This effect was greater when sonicated at higher power levels (Figure 1). Through particle size and Size Exclusion Chromatography (SEC) studies, the 10 decrease in turbidity is believed to be due to the decrease in the size of the insoluble powder aggregates in solution. The size of particles in 5% (w/w) reconstituted WPC80 solutions sonicated at 13W (Figure 2), 31W (Figure 3) and 50W (Figure 4) shows a significant shift in the size of particles from the -10-100ptm region in non sonicated samples to -<0.1-1 pm when sonicated. The shift was greater with higher 15 sonication power and longer exposure. Similar results are observed for particles in the size range below 1 pm (Figure 5). The Native Polyacrylamide Gel Electrophoresis (PAGE) profile of 15% (w/w) reconstituted WPC80 solutions sonicated at 20 kHz and 13, 31 and 50W delivered power for various periods of time shows that a high molecular weight band possibly 20 corresponding to agglomerated (glycomacropeptide) GMP disappeared with an increase in sonication time. The disappearance of the GMP band was accompanied by an increase in the band corresponding to large aggregates. Sodium dodecyl sulphate (SDS) PAGE did not show significant protein changes regardless of the treatment applied to reconstituted WPC80 solutions. 25 Reverse phase High Performance Liquid Chromatography (HPLC) results for 8% (w/w) WPC80 sonicated for 0.5 min, 1 min, 2 min, 5 min, 10 min, 20 min & 60 min at 31 W, showed that the hydrophilicity of the whey proteins (both a-lactalbumin and p lactoglobulin) increased with increasing time of sonication. The time taken for reconstituted WPC80 to gel at 80 0 C was reported as the gelation point (Table 1). 30 WO 2009/079691 PCT/AU2008/001867 9 Table 1. Sonication reduces gelling time for low protein solutions. Sonication Sonication WPC Concentration pH Gel Time (min) Power Time (% w/w) No Sonication 5 6.2 No gel in 20min 31 W 60 minutes 5 6.2 12 No Sonication 15 6.2 1.55 13 W 60 minutes 15 6.2 1.5 31 W 60 minutes 15 6.2 1.4 50 W 60 minutes 15 6.2 1.4 No Sonication 15 9.5 1.25 31 W 60 minutes 15 9.5 1.25 As shown in Figure 6, gel strength (samples gelled at 80*C/20 min) of 15% (w/w) reconstituted WPC80 solutions increased with increasing sonication time. For 60 5 min sonication, there was a two fold increase in the gel strength at 13W, and at 31W and 50W the gel strength increase five and six times, respectively. The pH of 15% (w/w) reconstituted WPC80 solutions were altered prior to sonication and then gelled at 80*C/20 min. At all pH values except pH 5.0 (natural pH is 6.2), sonication increased gel strength (Figure 7). Table 2 shows that increasing pH 10 decreases gelling time and that sonication at high pH values did not affect gelling time. Table 2. The effect of pH and sonication on the gelling time of 15% (w/w) reconstituted WPC80 heated at 80 0 C. Gel Time (minutes) pH 6.2 1.55 pH 9.5 1.3 pH 9.5 with 60 min sonication at 31W 1.3 15 As shown in Figure 8 the syneresis of gels made from 15% (w/w) reconstituted WPC80 solutions increased with increasing sonication time up to 60 min when sonicated at a delivered power of 13W. At 31W, samples sonicated for 10 min showed an increase in syneresis to about 1.5 times that of unsonicated, 20 reconstituted WPC80 but a further increase in the sonication time led to a decrease WO 2009/079691 PCT/AU2008/001867 10 in the syneresis. At 50W, a decrease in the syneresis occurred after 10 min sonication. Transmission electron microscopy was used to analyse the protein structure of 15% (w/w) reconstituted WPC80 gels (Figure 9). Gels formed after sonicating for 60 min 5 at 13W and 10 min at 31W showed fewer protein aggregates than a non-sonicated sample. The space between the particles is greater and this may explain the increase in the syneresis observed in gels treated at these conditions. For 60 min at 31W, the gel network appears to be denser and made up of smaller protein aggregates that are homogeneously distributed. This compact gel network resulted io in a firmer gel with better water holding properties. Example 2. Viscosity and particle size of batch sonicated whey protein retentate containing 33% (w/w) solids and 27% protein, freeze drying and reconstitution. 15 Retentate from the ultrafiltration of cheese whey containing -33% (w/w) solids (-27% (w/w) protein) was batch sonicated using a 20 kHz horn transducer as a 50 ml solution at 31W delivered power, determined calorimetrically. The size of particles decreased with an increase in sonication time (Table 3). Particles shifted from the -1 -60pm region to <1 pm. The viscosity decreased after 20 sonication for 1, 2 and 5 min. Beyond 5 min sonication the viscosity began to increase again. The 20 min sonicated samples had a higher viscosity than 1 or 5 min sonicated samples but slightly lower viscosity than the control sample (0 min sonicated). The 60 min sonicated samples showed a further increase in viscosity. 25 Table 3. Viscosity and particle size of batch sonicated whey protein retentate containing 33% (w/w) solids and 27% protein. Sonication time Viscosity (cP) Particle size (pm) at 31W/50ml Control (0 min) 38 2.11 5 sec 2.02 1 min 33 1.75 2 min 32 5 min 31 1.22 WO 2009/079691 PCT/AU2008/001867 11 10 min 33 20 min 36 0.75 60 min 40 The batch sonicated retentates were freeze dried. The freeze dried powders were reconstituted to 5% (w/w) and 15% (w/w) TS and post heated at 80*C/20 min. Results in Table 4 show that the sonication prior to freeze drying had little effect on 5 the viscosity of the freeze dried product when reconstituted to 5% (w/w) solids for sonication times less than 20 minutes. Table 4. Changes in viscosity for 5% (w/w) reconstituted freeze dried whey retentate which was sonicated prior to freeze drying. Sonication Time Viscosity (cP) at a shear Viscosity (cP) at a shear rate of 100 sec- 1 rate of 100 sec-' after post heating at 80*C/20 min No Sonication 2.4 3.6 1 min 2.3 3.5 5 min 2.3 3.0 20 min 2.6 5.7 10 The viscosity of samples reconstituted to 15% (w/w) TS was slightly lower due to sonication. All solutions gelled when post heated at 80*C/20 min. The powders which were sonicated before freeze drying had a firmer gel than the control and gel strength increased with an increase in sonication time from 1 to 20 minutes (Table 15 5). Gel strength was at least doubled when sonicating for >5min. Table 5. Changes in viscosity and gel strength for 15% (w/w) reconstituted freeze dried whey retentate which was sonicated prior to freeze drying. Sonication Time Viscosity before postheat (cP) at a Gel strength after postheat shear rate of 1001s- (g) No Sonication 5.6 7.8 1 min 4.9 8.8 5 min 5.0 14 20 min 4.6 24 WO 2009/079691 PCT/AU2008/001867 12 Example 3. Viscosity and particle size of continuously sonicated whey protein retentate containing 33% (w/w) solids and 27% protein, spray drying and reconstitution. 5 Retentate from the ultrafiltration of cheese whey (33% (w/w) solids) was also sonicated in a continuous mode by passage through a 350 ml chamber at 300 ml/minute with sonication by a 20 kHz horn transducer and a nominal maximum power of 1kW. Results presented in Table 6 show a small reduction in particle size and solution viscosity as the applied energy input is increased. The applied energy 10 is calculated from the percentage of the nominal power amplitude applied to the unit (W), multiplied by the total flow rate through the unit (ml/min times the number of passes). Table 6. Viscosity, particle size and applied energy of continuously sonicated 15 whey protein retentate containing 33% (w/w) solids and 27% protein. Power amplitude setting Applied energy Viscosity cP at a Particle size (J/ml) shear rate of 100s" (pm) No Sonication 0 61 1.045 30% - 1 pass 60 57 1.000 60% - 1 pass 120 57 0.982 80% 160 58 0.975 60% - 2 passes 240 56 0.922 The continuous sonicated retentate was then spray dried. Samples reconstituted to 15% (w/w) total solids gelled when post heated at 80*C/20min. The powders which were sonicated before spray drying again had a firmer gel than the control (Table 7). 20 Gel strength increased with increasing number of passes through the sonication unit. Table 7. Gel strength for 15% (w/w) reconstituted spray dried retentate (gelled at 80*C/20 min). Sample ID Gel strength (g) Control (no sonication) 8.5 Sonication- 1 pass 30% 9.3 2 Pass 30% 9.7 WO 2009/079691 PCT/AU2008/001867 13 3 Pass 30% 11 Example 4. Batch sonication of reconstituted WPI. Whey protein isolate (WPI) was reconstituted at 5% and 15% (w/w) solids and batch sonicated as 50 ml solutions using a 20 kHz horn transducer. No change in solution 5 clarity was observed. Sonication at a delivered power of 31W (determined calorimetrically) only marginally reduced gelling time (Table 8). Table 8. Batch sonication of whey protein isolate at a delivered power of 31W. Concentration Sonication time Gel time (min) at 80'C 10% (w/w) No sonication 1.44 10% (w/w) 12 min 1.40 15% (w/w) No sonication 3.0 15% (w/w) 60 min 2.5 10 There was no increase in gel strength associated with sonication at 31W for 60 min. There was no difference in syneresis between reconstituted WPI 15% (w/w) with or without sonication at 31W for 60 min. These results suggest that sonication is less effective in changing the functional behaviour of whey protein isolates than in other whey protein systems. 15 Example 5. Sonication of whey protein retentate containing 54% (w/w) solids and 20% protein to reduce viscosity. Figure 10 shows a reduction in viscosity of whey protein retentate containing 54% (w/w) solids and 20% protein when sonicated at 20 kHz and 70% power using a 1kW 20 unit at a flow rate of 200 ml/min to achieve a residence time of 1.3 minutes. Figure 11 shows the viscosity of 54% retentate at a shear rate of 100 RPM after sonication at 20 kHz and 50%, 80% and 100% power amplitude to achieve a residence time of 1.3 minutes. Viscosity was reduced by >40% when sonicated at 50% power and further when sonicated at 80% power. There was no further change in viscosity 25 when sonicating at 100% power. The size of particles of the corresponding solutions also reduced in response to sonication from 8.5pm to -1 pm (Table 9).

WO 2009/079691 PCT/AU2008/001867 14 Table 9. Particle size by volume weighted mean (D[4,3]) of whey protein retentate (54% total solids and 20% protein) with or without sonication at 20 kHz using a I kW unit and a flow rate of 200 mlmin at various power settings to achieve a residence time of 1.3 minutes. Sample Applied energy (J/ml) Particle size D[4,3] (pm) 54% Control 0 8.5 54% US50% 200ml/min 150 1.4 54% US80% 200ml/min 240 1.0 54% US100% 200ml/min 300 1.1 5 Example 6. Sonication of whey protein retentate containing 30% (w/w) solids and 11% protein to reduce viscosity. Figure 12 shows the drop in viscosity of 30% (w/w) retentate containing 11% protein sonicated at 20 kHz using a 1 kW unit and 70% power at a flow rate of 200 ml/min to 10 achieve a residence time of 1.3 minutes. Example 7. Sonication of whey protein retentate containing 33% solids and 27% protein at <20 0 C to reduce viscosity. Whey protein retentate containing 33% (w/w) TS and 27% protein was sonicated at 15 20 KHz using a 4kW unit at a flow rate of 1.4 1/min at various power settings and with an overpressure of 0.9 to 1 Bar. Sonication reduced viscosity at all power levels (50% and 95%). A maximum drop in viscosity of approximately 7cP (11%) was achieved (Figure 13). Figure 14 shows that sonication efficiency at high power (84%-95% amplitude) is 20 influenced by the flow rate. At a fast flow rate of 6 litres/min the efficiency of sonication is lower than that at the slow flow rate of 700 ml/min where contact time is increased. This can be explained in terms of the total energy transferred to the dairy solution. At 6 litres/min only 34 J/ml of electrical energy is nominally applied to the sample. 25 The largest viscosity drop was achieved when retentate was passed repeatedly through the sonication field (Figure 15). Retentate sonicated at 50% amplitude at a flow rate of 1.4 litres/min showed a progressive drop in viscosity with each pass to achieve a maximum drop of approximately 21cP (33%) after 3 passes, with a total nominal applied energy input of 260 J/ml. 30 WO 2009/079691 PCT/AU2008/001867 15 Table 10 indicates that the size of particles in solution are gradually decreased with higher sonication power, lower flow rates and the number of passes through the sonication field supporting the concept that physical shear generated by sonication is contributing to the reduction in viscosity. 5 Table 10. Particle size by volume weighted mean (D[4,3]) of whey protein retentate (33% (w/w) TS and 27% protein) with or without sonication at 20 kHz using a 4kW unit at various flow rates and power settings. Nominal applied Particle size D[4,3] Sample energy (J/ml) (pm) 33% control 0 0.75 33% 1bar 61/min 84%amp 34 0.63 33% control 1bar 1.41/min 50%amp 86 0.46 33% control 0.5bar 1.41/min 95%amp 163 0.38 33% 0.9bar 700ml/min 95%amp 325 0.39 33% control 1 bar 1.41/min 50%amp pass 86 0.48 33% control 1bar 1.41/min 50%amp pass2 172 0.41 33% control 1 bar 1.41/min 50%amp pass3 258 0.35 10 Sonication increased the firmness of gels (Figure 16) from approximately 650g to 800-850g (-19 - 24% improvement). Sonication at 50% power (86 J/ml) was as effective at increasing gel strength as sonication at 95% power (163 J/ml). Example 8. Heat stability of reconstituted and sonicated WPC. 15 Reconstituted WPC80 solutions were preheated at 80*C for 1 min or 80 0 C for 20 min and then batch sonicated using a 20 kHz horn transducer as 50 ml solutions. Samples were then heated for a second time (post heating) at 80*C/20 min. Size exclusion chromatography showed that preheating or post heating without sonication caused an increase in soluble protein aggregate size for 5% to 12% (w/w) 20 solutions. For heated samples, the bulk of particle sizes were measured to be between -10 and 100pm depending on the severity of the treatment. The changes were less significant for 0.5% to 3% (w/w) solutions. Sonication between the preheat and post heat stages caused a decrease in the average size of these heat induced aggregates for solutions of less than 10% (w/w) 25 solids to approximately 0.1 and 10 pm even after a second heat treatment. However, for 10% and 12% (w/w) solids, a sonication time longer than 15 min led to a WO 2009/079691 PCT/AU2008/001867 16 significant increase in the size of aggregates when post heated, with the number of aggregates increasing in the 1-10pm region. As shown in Figure 17 and Table 11 preheating at 80 0 C for 1 minute increases the viscosity of the 5% (w/w) reconstituted WPC80 solution in response to the increase 5 in the size of particles. The sonicated solutions, having smaller particles, retained the same viscosity as the untreated sample even after a second heat treatment, whereas both unsonicated samples had an increase in viscosity. When preheating for 20 min (Figure 18, Table 11) similar results are obtained. For 9% (w/w) solutions (Figure 19, Table 11) the viscosity measurements behaved in 10 a similar manner however, the post heated sample without sonication gelled. Solutions sonicated for only 5 sec had a slight increase in viscosity when post heated. Similar behaviour was observed for WPC80 reconstituted to 8% solids. At 12% (w/w) reconstituted WPC80 (Figure 20, Table 11), long sonication times had an adverse effect on the viscosity when post heated. A sonication time of 40 min 15 caused gelling when post heated. Similarly a short sonication time (5 sec) caused gelling. An increase in viscosity was also recorded for samples sonicated for 15 and 20 min when post heated. This increase in the aggregate size and viscosity at high sonication times when post heated was also observed in 10% (w/w) reconstituted WPC80. 20 For 1 % and 3% (w/w) solutions (Table 11), viscosities are too close to that of water for measurable changes to be observed clearly.

WO 2009/079691 PCT/AU2008/001867 E 0 ~-0 C E- mc m L 00 n n n 0 0 a E 00 LAc 0 0) A D ' ~ A t 6 .- ~ "4 W .-4 o) ID00 Un( M~ r' Z 0 C.) CL 0 0 ~ ~ 0 k N 0LA 0 ~ c; .4 I r4 (Ni ei C (N U) LA 00 m (N A WO 2009/079691 PCT/AU2008/001867 18 WPC80 reconstituted to 15% (w/w) gelled when preheated at 72*C/3 min due to the high protein content (Table 12). Preheating increased the gelling time, however, when the preheated samples were sonicated, the gelling time decreased. 5 Table 12. Effect of preheating and sonication on gel time. Gel Time at 80 0 C (min) Preheat 72 0 C/3min 3 Preheat + US 10 sec 31W 2.3 Preheat + US 3 min 31W 1.5 Preheat + US 2 hr 31W 1.3 The preheated solution was then further heated until a paste was formed. This paste was then sonicated, causing it to return to a liquid state. As shown in Figure 21 the fluidity was demonstrated by tilting the glass plate. The preheated (a) and 10 sonicated paste (c) sample was then gelled at 800C. Gel times were 3.0 min for (a) and 1.2 min for (c). Without preheating, sonication of the 5% (w/w) reconstituted WPC80 solutions causes gelation to occur during post heating for extended periods at 800C (Table 13) where as the unsonicated solutions remained in liquid form. The preheated 5% 15 (w/w) reconstituted WPC80 solutions show some aggregation but without gelation. However, the preheated sample that has been treated with ultrasound showed no presence of large aggregates and remained in solution even after 30 min of heating at 800C (Table13). Although it appears that preheating and sonication can inhibit aggregation and 20 gelation, severe heat treatment e.g., 100*C/40 min, causes gelation. 25 WO 2009/079691 PCT/AU2008/001867 19 Table 13. Gel behaviour of reconstituted and sonicated WPC80. 5% (w/w) reconstituted WPC80 Gel behaviour at 80*C Gel behaviour at 1OOC No treatment No gel in 30 min No gel in 40 min Sonication 31W, 60 min 14 min Gels Preheat (80C/1 min) Some aggregation but no Gels gel in 30 min Preheat + sonication 31W, 60 No gel in 30 min Gels min Example 9. Heat stability of reconstituted and continuously sonicated WPC80. WPC80 reconstituted to 5% (w/w) was also continuously sonicated at 300W applied 5 power in a 350 ml chamber using a 20 kHz horn transducer and a flow rate of 300 ml/min or 500 ml/min. Solutions were preheated at 80*C/1min and post heated at 80*C/20min. As shown in Figure 22 heating increased the size of particles in solution and viscosity. Continuous sonication reduced both the size of particles and viscosity 10 and these effects were maintained after post heating. Solutions processed at 300 ml/min had a lower viscosity than those processed at 500 ml/min, reflecting the longer sonication residence time. This is a clear indication that the combined effects of heating and sonication improves heat stability. Note that the size of particles in the control solution were a measure of insoluble powder aggregates whereas in 15 heat treated samples were a measure of heat induced protein aggregates. Example 10. Heat stability of batch sonicated retentate before and after freeze drying. Whey protein retentate was diluted to 5% (w/w) and 10% (w/w) solutions, pre 20 heated at 80*C/1 min, batch sonicated at 20 kHz, with a delivered power of 31W (determined calorimetrically) for 1 min in a 50 ml cell and then post heated at 80*C/20 min. The viscosity was measured and is shown in Figure 23 for the 10 % (w/w) solids case. Preheating and post heating caused an increase in the particle size of protein 25 aggregates thereby increasing viscosity. Heat induced aggregates ranged from WO 2009/079691 PCT/AU2008/001867 20 -10-120pm, when sonicated the particle size range reduced to -<0.1-100pm. This effect was enhanced at 10% (w/w). Sonication of preheated retentate also reduced the viscosity to similar levels as control (no treatment). This reduction in viscosity and size of particles due to 5 sonication remained effective even after post heating at 80*C/20 min indicating improved heat stability. Some retentates, which had been diluted to 5% (w/w), pre-heated at 80*C/20 min and batch sonicated at 31W and 20 kHz for 1 min, were then freeze dried and reconstituted to 5% and 15% (w/w) before post heating at 80*C/20 min. Viscosity 10 results on these reconstituted freeze dried powders indicates that improved heat stability as a result of heating and sonication is maintained after freeze drying (Figure 24). The size of particles in solutions reconstituted to 5% (w/w) ranged from -<0.1-30pm, -10->1OOpm for heated samples and -1-30pm for sonicated samples after heating. 15 The particle size range for powders reconstituted to 15% (w/w) was -<0.1-30pm, -1 >100pm for heated samples and 1-100pm for sonicated and heated samples. 15% (w/w) solutions gelled when post heated at 80*C/20 min due to the high protein content, however, gel strength was significantly lower than the unsonicated example. 20 Example 11. Heat stability of continuously sonicated retentate before and after spray drying. Whey protein retentate was diluted to 5% and 8% (w/w), pre-heated at 80*C/1 min and continuously sonicated at 20 kHz and 300W at a flow rate of 300 ml/min in a 350 ml chamber. As was observed with batch sonication, the increase in particle size 25 and viscosity associated with a heat treatment of 8% (w/w) retentate were reversed by preheating and continuous sonication (Figure 25). These trends were also observed at 5% (w/w). A further set of retentate samples diluted to 8% (w/w), preheated at 80*C/1 min and continuously sonicated at 20 kHz and 300W at a flow rate of 300 ml/min. They were 30 then spray dried and reconstituted to 5%, 8%, 10% and 15% (w/w) before post heating at 80*C/20 min. As shown in Figure 26, aggregate size and viscosity of solutions reconstituted at 8% (w/w) with preheating and sonication are lower than those for post heated alone.

WO 2009/079691 PCT/AU2008/001867 21 These trends are replicated at 10% (w/w) but are difficult to observe at 5% (w/w) due to the low solids level. Example 12. Heat stability of whey protein retentate containing 8% (w/w) solids 5 and 6% protein. Whey protein retentate containing 8% (w/w) solids and 6% protein, was pre heated (preH) at 80 0 C for 1 minute, cooled to <20*C, sonicated at 20 kHz using a 4kW unit with 0.5 to 1 bar overpressure at various power levels at a flow rate of 1.4 litres/min and post heated (postH) at 85 0 C for 30 minutes. The viscosity of heated retentate 10 (preH or postH) increased due to heat induced aggregation of whey proteins (Figure 27). The viscosity of heated retentate was reduced from >5OcP to below 5cP when sonicated. When post heated, the viscosity of samples sonicated at 50%, 80% and 95% power remained low (<5cP) indicating that heat stability was greatly improved. 15 Example 13. Heat stability of spray dried and reconstituted whey protein retentate. Whey protein retentate containing 20% (w/w) solids and 7% protein was pre heated at 85 0 C for 30 seconds to denature whey proteins. The increase in viscosity 20 indicates that whey proteins were denatured to form heat induced aggregates (Figure 28). Denatured whey protein aggregates were sonicated at 20 kHz using a 1 kW unit at 70% power and a flow rate of 200 ml/min to achieve a residence time of 1.3 minutes and applied energy input of 210 J/ml. The sonication treatment disrupted heat induced whey protein aggregates resulting in a drop in viscosity 25 comparable to that of the control. Control and pre heated and sonicated retentates were immediately spray dried. Spray dried powders were reconstituted to 10%, 15%, and 20% (w/w) TS and post heated at 80 0 C for 30 minutes to test for heat stability (Figure 29). Reconstituted control powders were heat unstable at all concentrations as indicated by the increase in viscosity after heating. Pre heated 30 and sonicated powders reconstituted to 10%, 15% and 20% TS (w/w) were heat stable as indicated by the viscosity remaining low after heating.

WO 2009/079691 PCT/AU2008/001867 22 Example 14. Further results on the effects of preheating prior to sonication. Whey protein retentate was diluted to contain 8% (w/w) TS, preheated (80C/1 min), sonicated at 20 kHz and then either spray dried or freeze dried. Table 14 shows the viscosity of freeze dried (FD) or spray dried (SD) powders reconstituted to 5%, 10%, 5 20%, 30%, 35% and 40% (w/w). Sonication reduced the viscosity of samples, particularly at high solids concentrations and such changes were not affected during further processing into powders by freeze drying or spray drying, i.e. functional properties were maintained after drying and reconstitution. 10 Table 14. Viscosity measured for reconstituted freeze dried (FD) or spray dried (SD) powders made from preheated and sonicated retentate. Viscosity (cP) Viscosity (cP) Solids (% Control Pre-H Pre-H + Control Pre-H Pre-H + consti ution FD FD US + SD SD US + FD SD 5 2 2 2 2 2 2 10 3 4 3 2 4 3 20 10 34 17 8 33 20 30 51 382 145 28 481 182 35 190 1823 535 66 1509 574 40 477 5308 1099 155 4263 1720 Example 15. Effects of sonication at frequencies greater than 20 kHz. Sonication of whey systems at frequencies greater than 213 kHz was ineffective at 15 reducing the size of particles in solution and the corresponding viscosities. Frequencies of 213 kHz, 355 kHz and 647 kHz did not produce sufficient turbulence to physically disrupt insoluble aggregates in reconstituted WPC80 solutions. As an example, reconstituted 5% (w/w) WPC80 was batch sonicated as 50 ml solutions at 647 kHz and with delivered power of 50W for up to 2 hours. No change 20 is solution clarity was observed. As shown in Figure 30 there was little change to the size of aggregates in solution. A small shift in the size of particles from the -10- WO 2009/079691 PCT/AU2008/001867 23 100l m region to the -0.1-1pm region occurred when sonicated at 50W for 20, 40 and 60 min. Sonication for 2 h did not shorten gelling time as observed at 20 kHz. Figure 31 shows that 30% (w/w) reconstituted WPC80 sonicated for 1 minute at a 5 high frequency of 213 kHz and delivered powers of 3W, 13W, 31W or 51W did not show significant change in viscosity. The viscosity remained unaltered. There is clear evidence of free radical formation at this frequency (chemiluminsence) indicating that cavitation is definitely occurring. However, sonication at high frequency did not produce enough mechanical shear as seen at 20 kHz to cause a 10 decrease in viscosity of WPC80 solution. Figure 32 shows the viscosity and size of particles of WPC80 reconstituted to 8% (w/w) and preheated at 80*C for 1 minute. Samples were sonicated for 1 minute at delivered powers of 3W, 13W, 31W or 51W and at 20 kHz, 213 kHz, 355 kHz and 647 kHz. A slight decrease in viscosity observed at 213 kHz and 51W suggests that 15 effects can be seen at this frequency by increasing overall power levels, but 20 kHz is far more efficient. The viscosity and particle size of whey protein retentate (-33% (w/w) TS) sonicated for 1 minute at 20 kHz, 213 kHz, 355 kHz and 647 kHz at various power levels is shown in Figure 33. At frequencies greater than 20 kHz sonication of retentate is 20 ineffective at reducing the size of particles in solution and the corresponding viscosity. Example 16. Effect of simultaneous preheating and sonication. Figure 34 shows the viscosity of WPC80 reconstituted to 10% (w/w), heated to 50 0 C, 25 65 0 C, 75 0 C or 85 0 C for 1 min and sonicated at 20 kHz and 51W for 1 min while hot. Results from this study indicate that sonicating hot samples has the same effect of reducing viscosity as for preheated samples. Therefore there is no need to cool samples between heating and sonication. It also shows that for aggregates to form, the preheat temperature must be > 65 0 C. 30 From the above it can be seen that this invention provides a method of treating dairy ingredients containing dairy proteins that produce ingredients with improved functionality.

WO 2009/079691 PCT/AU2008/001867 24 Those skilled in the art will realise that this invention may be implemented in embodiments other than those described without departing from the core teachings of this invention. 5

Claims (8)

1. A method of modifying the properties of dairy ingredients which includes sonication at an applied energy of less than 500J/ml and at a frequency below 5 213 kHz.

2. A method of modifying the properties of dairy ingredients which includes the steps of heating the ingredient to a temperature above 65*C followed by sonication at an applied energy of less than 500J/ml and at a frequency below 10 213 kHz.

3. A method of modifying the properties of dairy ingredients which includes the steps of simultaneously heating the ingredient to a temperature above 65 0 C and sonicating at an applied energy of less than 500J/ml and at a frequency 15 below 213 kHz.

4. A method as claimed in any preceding claim in which the ingredient is any ingredient containing dairy proteins, but preferably selected from cheese whey, whey protein concentrates (WPCs), retentates from ultrafiltered whey, 20 milk concentrates or milk protein concentrates that are either fresh or re constituted from a powder form.

5. A method as claimed in any preceding claim in which the sonication is at a power of at least 10 Watts. 25

6. A method as claimed in any preceding claim in which the treated ingredient is then spray dried or freeze dried.

7. A method as claimed in any preceding claim in which the sonicated ingredient 30 is subjected to a further heat treatment prior to drying.

8. A method as claimed in any preceding claim in which the sonicated ingredient is subjected to a further heat treatment after drying and reconstitution to a liquid form. 35