The effect of ultrasound on the physical and functional properties of skim milk

Innovative Food Science and Emerging Technologies 16 (2012) 251–258

Contents lists available at SciVerse ScienceDirect

Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset

The effect of ultrasound on the physical and functional properties of skim milk Akalya Shanmugam, Jayani Chandrapala, Muthupandian Ashokkumar ⁎ School of Chemistry, University of Melbourne, Victoria 3010, Australia

a r t i c l e

i n f o

Article history: Received 23 April 2012 Accepted 15 June 2012 Editor Proof Receive Date 12 July 2012 Keywords: Ultrasonication Skim milk Viscosity Particle size Turbidity Whey proteins

a b s t r a c t Pasteurized homogenized skim milk was treated with 20 kHz ultrasound at 20 and 41 W under controlled temperature conditions for different time intervals up to 60 min. There was a reduction in the turbidity, but viscosity of milk was not affected. The size of the casein micelles, fat globules and soluble particles, respectively, after 60 min of sonication were 174 nm, 157 nm and 89 nm at 20 W and 172 nm, 175 nm and 84 nm at 41 W in comparison to 178 nm, 170 nm and 136 nm for unsonicated samples. The whey proteins in the milk were denatured and formed soluble whey–whey/whey–casein aggregates, which further interacted with casein micelles to form micellar aggregates during the initial 30 min of sonication. Prolonged sonication resulted in the partial disruption of some whey proteins from these aggregates. Industrial relevance: Ultrasonic processing is a promising technique, which could be adapted in food processing sector. The minor changes to the milk, which are imparted by the shear forces of the acoustic cavitation, foresee the potential for optimizing this technique for industrial applications. The optimization process needed, depends on the functionality and the end products, such as food emulsions using bioactives. The optimization can be achieved by fine tuning the power density, temperature, processing time, etc. This study reports on the processing effect of ultrasound on a simple food matrix (milk) in view of establishing a base to extend the investigation to other complex foods. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Milk is a complex food made up of components that are essential for human health. It is composed of proteins, lipids, lactose, vitamins and minerals. The lipids in the milk are present as fat globules in emulsified form; the proteins exist as micelles in the colloidal dispersion and soluble form in the serum; the lactose and the minerals exist in solution (Keenan & Patton, 1995). Fresh milk is a highly perishable product which has a relatively short shelf life among the farm produces. The challenge for the dairy industry to provide a safe and shelf stable liquid milk with an affordable price is huge. Thermal treatment is one of the primary and traditional methods of processing, which is widely adapted to reduce the microbial spoilage and to provide a safer end product with extended shelf life. Due to the intense heat treatment on the milk components, thermal processes are always associated with many disadvantages like denaturation and modifications to the proteins (Corzo, Lopezfandino, Delgado, Ramos, & Olano, 1994; Oldfield, Singh, & Taylor, 1998; Oldfield, Singh, Taylor, & Pearce, 1998; Anema, 2009; Lan et al., 2010) and decrease in their nutritional values (AlKanhal, Al-Othman, & Hewedi, 2001; Lacroix et al., 2008). The heat treatment also affects the mineral balance (Hansen & Melo, 1977; Herzallah, Humeid, & Al-Ismail, 2005; Knudsen & Skibsted, 2010; Seiquer, Delgado-Andrade, Haro, & Navarro, 2010), vitamins ⁎ Corresponding author. Tel.: +61 3 83447090; fax: +61 3 93475180. E-mail address: [email protected] (M. Ashokkumar). 1466-8564/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2012.06.005

(Burton, 1988) and causes undesirable changes to flavour and colour of the milk (Fellows, 2000; Barbosa-Canovas & Bermudez-Aguirre, 2010). There is an enormous demand for new technologies to replace the conventional thermal processing by techniques which cause least damage to the nutritional ingredients and provide longer shelf life. A number of non-thermal techniques, such as high pressure processing, pulsed electric field, ultrasound, ohmic heating and microwave heating, have been explored for this purpose. Among these non-thermal processing techniques, ultrasound (US) processing received significant attention in recent years. The major advantages of ultrasound lies in its benign, non-toxic and environmentally friendly nature compared to other processing techniques like microwaves, gamma radiation and pulsed electric field which are considered cautiously by the general population (Kentish & Ashokkumar, 2011). US has a wide range of application in food processing, such as dehydration, drying, freezing, thawing, emulsification, tenderization of meat, crystallization of lactose, enzyme inactivation, in cleaning operations and as an analytical tool (Bhaskaracharya, Kentish, & Ashokkumar, 2009). It is also used to inactivate pathogens in food products, to enhance extraction of bioactives and to prepare emulsions (De Gennaro, Cavella, Romano, & Masi, 1999; Marchioni et al., 2009). US refers to sound waves of frequency above 20 kHz, which is undetectable by human ear and is divided into two categories, viz., power and diagnostic ultrasound. The power ultrasound, ranging from 20 kHz to ~1 MHz, has recently been explored in food processing (Ashokkumar & Mason, 2007; Patel, Carroll, & Kelly, 2008). It is

252

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

considered as an effective processing aid based on its physical effects generated in a liquid. The physical effects of US include cavitation (growth and collapse of microbubbles) which can produce high localized temperatures, pressures and turbulence (Ashokkumar & Grieser, 2007). While these are considered as the important effects of the US processing technique, they can also produce some changes to the components of food during the processing. Any new process which is developed to produce processed liquid milk is continuously researched and validated for the impact/effect of the technique on the nutritionally important components and for any alteration in their functional behaviour to make it an adaptable technique in a factory environment. Since US technology is relatively new in food processing, only a very few reports are available up-to date detailing the effects of the US technique on the denaturation of proteins and development of off-flavours, etc. in the preparation of processed liquid milk (Taylor & Richardson, 1980; Villamiel & de Jong, 2000; Riener, Noci, Cronin, Morgan, & Lyng, 2009; Chouliara, Georgogianni, Kanellopoulou, & Kontominas, 2010; Nguyen & Anema, 2010). Some of the recent studies that are reported on the liquid milk show the synergistic efficacy of pressure and thermal treatments along with US to achieve benefits like fat globule homogenization or microbial inactivation (Vercet, Oria, Marquina, Crelier, & Lopez-Buesa, 2002; Bermudez-Aguirre, Mawson, & Barbosa-Canovas, 2008; Bermudez-Aguirre, Corradini, Mawson, & Barbosa-Canovas, 2009; Bermudez-Aguirre, Mawson, Versteeg, & Barbosa-Canovas, 2009; Czank, Simmer, & Hartmann, 2010). A few researchers have studied the effects of US on the individual proteins such as lysozyme (Cavalieri, Ashokkumar, Grieser, & Caruso, 2008), bovine serum albumin (BSA) (Stathopulos et al., 2004; Gulseren, Guzey, Bruce, & Weiss, 2007), casein (Madadlou, Mousavi, Emam-Djomeh, Ehsani, & Sheehan, 2009), whey protein isolates (WPI) (Gordon & Pilosof, 2010), whey protein concentrates (WPC) (Chandrapala, Zisu, Palmer, Kentish, & Ashokkumar, 2011; Arzeni et al., 2012) and whey protein aggregates (Ashokkumar et al., 2009; Saffon, Britten, & Pouliot, 2011). A thorough search of the literature has indicated that there are no studies available on the combined effects of frequency, time and temperature control along with the different power levels of sonication on the flow behaviour and proteins of pasteurized homogenized skim milk (PHSM). Nguyen and Anema (2010) have reported a similar study using 22.5 kHz at a single power level. The outcomes of the sonication process can vary depending on the experimental conditions such as the acoustic power density, sample volume, solution temperature and other experimental conditions. Hence, the aim of the current work is to study the flow behaviour and modifications to the proteins in PHSM upon sonication at 20 kHz under two different nominal applied power levels viz., 90 and 180 W for 1 h and at controlled temperature conditions. These power levels were chosen as these were found to be optimum for preparing food emulsions (Kentish et al., 2008), which will be used in our future work. Though longer time sonication may not be practically feasible for any preservation and homogenization processes, this study will give knowledge about the process stability of milk under extreme conditions. Thus the results presented in the current study provide significant new information that is not available in the literature.

2. Materials and methods 2.1. Milk Fresh pasteurized homogenized skim milk was purchased from a local supermarket, immediately stored at 4 °C until further use. The composition of the milk was 33 g/L protein, 1.5 g/L fat, and 52 g/L lactose as determined by the manufacturer. Ultra pure (MilliQ) water was used in all experiments.

2.2. Ultrasonication Milk was sonicated in 65 ml aliquots in a glass vessel equipped with a cooling water jacket, using a 20 kHz, 450 W ultrasonic horn (12 mm diameter, Branson Sonifier, Model 102 (CE)) at 90 and 180 W of applied powers for 15, 30, 45 and 60 min. The actual powers delivered to the solution were determined to be 20 and 41 W by calorimetry, respectively. During sonication water was circulated continuously and the water temperature was maintained at 22.5± 2 °C. The solution temperatures during US treatment at 20 and 41 W were maintained between 22 to 30 °C and 22 to 37 °C, respectively. 2.3. Ultracentrifugation Milk samples were ultracentrifuged at 30,000 rpm (80,000 g) at 22 °C for 90 min using a Beckman optima L 90 K ultracentrifuge with a type Ti 50.2 rotor (Beckman Instruments Inc., Palo Alto, CA). The casein micelle-free milk serum or supernatant was carefully removed from the ultracentrifugal pellet after removing the top fat layer. Some analyses were carried out on supernatants and few on total milk as described wherever relevant. 2.4. Particle size measurement The Z-average particle sizes of the milk systems were measured with the use of Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK) after appropriate dilutions. All the measurements were performed at 25 °C using the refractive indices of the individual milk components, viz., 1.462, 1.46, 1.58 and 1.33 for the fat globules, whey proteins, casein micelle proteins and water, respectively. The size of the fat globules was measured in the total milk after fully dissociating the casein micelles by suspending the milk samples in a solution of Ethylene Diamine Tetra Acetic acid. The size of the soluble particles in the supernatants was obtained after filtering them through 0.45 μm filter syringe. The size of the casein micelles were observed as such in the total milk. 2.5. Turbidity, viscosity and pH measurements The turbidity of the milk samples was measured using a UV–VIS spectrophotometer (Carey 3E, Varian, Palo Alto, CA, USA) by transmission of light (λ=860 nm) through a 2 mm path length quartz cuvette. Viscosity of the milk samples was measured using a type 531-03/0c capillary viscometer (Schott AG) maintained at 22± 0.5 °C. The relative viscosity was determined by comparing the relative flow times and densities of the samples with those of distilled water. The pH of the milk was measured at 25 °C using a pH electrode with an integrated temperature sensor (Mettler Toledo, Australia) connected to a pH meter (Mettler Toledo, Australia). The pH probe was calibrated before each measurement at 25 °C using standard buffers at pH 4.0, 7.0 and 10.0 (Mettler Toledo, Australia). 2.6. Calcium measurement Soluble calcium was determined by measuring the total calcium contained in supernatants of ultracentrifuged milk samples. Residual protein was removed by diluting an aliquot (5 g) of supernatant sample with 20 g MilliQ water and 25 g 24% w/w trichloroacetic acid (TCA) followed by suction filtration through a Whatman No. 40 filter paper into a pre-weighed Buchner flask. The precipitated proteins were washed with 10 g of 12% w/w TCA solution and the total mass of the TCA filtrate was recorded. An aliquot of 2 g of supernatant obtained after filtration was mixed with 10 g of 10% lanthanum chloride and the diluted mass was made up to 50 g. The calcium concentration was determined by atomic absorption spectroscopy (AAS) (Varian Atomic Absorption Spectrophotometer, Mulgrave VIC 3170,

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

Australia) using an air acetylene flame and wavelength of 422.7 nm. Commercially available calcium standards for AAS were used after appropriate dilution. The concentrations of the standards were between 0 and 4 μg/g of Ca.

253

All the experiments were at least duplicated. The sonication of the milk was performed within two days of the stored milk samples. All measurements were performed on the same day of the sonication. 3. Results

2.7. NATIVE and SDS Gel electrophoresis and densitometry The changes to the total whey protein (including denatured whey protein) in the sonicated and unsonicated milk samples were determined using native polyacrylamide gel electrophoresis (native-PAGE). The whey proteins were separated from the milk by adjusting the pH to 4.6 and centrifuging out the precipitate using a bench-top centrifuge. The resultant supernatant (whey proteins including denatured whey proteins) obtained through acid precipitation of milk was used for analysis by native-PAGE. The changes to the soluble whey proteins in the ultracentrifugal supernatants were determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. When a milk sample is centrifuged, majority of the denatured whey proteins associated with casein micelles can be sedimented. In order to reduce the possibility of centrifuging down the soluble whey protein aggregates, the centrifugation speed chosen was at the minimum required to effectively deposit the casein micelles/associated whey proteins as a firm pellet (Anema & Li, 2003; Huppertz, Fox, & Kelly, 2004). Soluble protein particles are those that did not sediment from the milk during ultracentrifugation at 30,000 rpm (80,000g) for 90min at 22°C. Since the sample taken for our study is pasteurized homogenized skim milk (PHSM), the soluble supernatant must contain the following: whey proteins, whey protein–whey protein aggregates, smaller free caseins and whey protein-κ-casein and whey protein-αS2 casein aggregates as reported in the literature (Guyomarc'h, Law, & Dalgleish, 2003). Native-PAGE and SDS-PAGE gels were scanned using the BioRad Gel Doc XR and Imager (BioRad Laboratories, Richmond, CA). The intensities of the major protein bands were determined using the Image Lab software associated with the densitometer. Protein standards such as κ-casein, α-casein, β-casein, β-lactoglobulin (β-lac) and BSA and the unsonicated samples which were spiked with these standards were also run simultaneously. The bands were matched with the position of standards and spiked standards. The relative intensity was calculated to analyse the difference between the unsonicated and sonicated samples at both the power levels and different times of treatment. Pre-cast 12.5% criterion gels (Biorad laboratories, NSW, Australia) were used. According to manufacturer's protocol, the samples were diluted in a ratio of 1:1 with the native sample buffer (Biorad laboratories, NSW, Australia). A 1L solution of running buffer was made with Tris glycine buffer solution in the ratio of 1:10 with MilliQ water. The amount of sample injected was 25μl and the gels were run for 45min at 200V and 400mA. Then the gels were rinsed in water 3 times with 5min intervals and stained with 100ml of Coomasie G250 dye (Biorad laboratories, NSW, Australia) for 1h. The staining was made quicker by keeping it over the shaker. Later, the stain was completely removed and the gel was destained using 100ml of MilliQ water. The destaining was carried out overnight in a shaker. The same protocol was followed for the standards and the standards which were spiked with the unsonicated milk. The concentration of the protein standards was 2.5mg/ml. In the SDS PAGE, the sample was mixed with Laemmli sample buffer (Biorad laboratories, NSW, Australia) and 5% mercaptoethanol. The same protocol of Native PAGE was repeated by using the Tris glycine SDS (Biorad laboratories, NSW, Australia) as the running buffer. 2.8. Statistical analysis When necessary, one-way ANOVA with a 95% confidence level was used. The ANOVA data with pb0.05 were considered statistically significant.

Fig. 1a shows the size of soluble particles present in the supernatant of sonicated and unsonicated milk samples at 20 and 41 W as a function of sonication time. The size of the soluble particles shows a decreasing trend with increase in sonication time at both power levels. A maximum reduction in size occurs during the first 15 min of treatment and prolonged treatment times showed a gradual decrease. The 41 W treatment shows a slightly higher impact in particle size reduction than that observed at 20 W. Fig. 1b shows the size of casein micelles of sonicated and unsonicated milk samples at 20 and 41 W as a function of sonication time. The decrease in casein micelle size is by 1 to 4 nm at 20 W and 2 to 5 nm at 41 W, when milk was sonicated for 60 min. The decrease observed in the size of casein micelles is insignificant in comparison to the size of unsonicated sample. Fig. 1c and 1d show the effect of sonication on the soluble calcium and solution pH, respectively. No significant changes to soluble calcium and solution pH can be noticed at both power levels. Fig. 1e shows that the sonication at 20 W resulted in a significant reduction of fat globule size at all treatment times. It is very obvious from the figure that the size reduction increases with increase in sonication time at 20 W. In contrast, a different pattern is observed at 41 W. A significant decrease is observed until 15 min of US treatment followed by a slight increase in size at all other treatment times. Within experimental error, the magnitude of the increase in the size of the fat globules is very small, i.e., up to 5 nm in 60 min and thus can be neglected. The turbidity of sonicated and unsonicated samples of milk at 20 and 41 W as a function of treatment time is shown in Fig. 2a. The turbidity of sonicated milk samples is significantly lower compared to the unsonicated milk at both the power levels. From the data, it is apparent that US treatment at 41 W is slightly stronger in reducing the turbidity. A photograph shown in Fig. 2b provides further support to this observation. It is observed from Fig. 3 that the change in relative viscosity of the sonicated samples follows a similar pattern at both the power treatments. Thus, the viscosity data does not show a major change on sonication when considered within the error limits of the experiments. Fig. 4 shows the changes in the relative band intensities of κ-casein, β-casein, α-casein, β-lactoglobulin (β-lac) and α-lactalbumin (α-lac) obtained from reduced SDS PAGE of supernatants from the ultracentrifuged sonicated milk samples treated at 20 W with respect to unsonicated samples. The relative band intensities of κ-casein and α-casein remain the same as the unsonicated sample. However, there is a significant change in the band intensity of β-casein upon sonication. The value has decreased in 15 min of sonication and remained at the same level on further sonication. The relative band intensities of β-lac and α-lac (major whey proteins) show a decrease until 30min of sonication and then gradually increase after 30min. Considering the error value, both at 45 and 60 min of sonication, the intensity of the whey proteins is found to be lesser than that of the unsonicated sample. It can be speculated that the amount of soluble whey proteins is significantly reduced until 30min of sonication and then increased on prolonged sonication. The changes in the relative band intensities of κ-casein, β-casein, α-casein, β-lac and α-lac obtained from reduced SDS PAGE of supernatant from the ultracentrifuged sonicated milk samples treated at 41 W with respect to unsonicated sample are shown in Fig. 5. Unlike 20 W treatment, a different trend is observed for κ-casein and α-casein soluble fractions, but the whey proteins and β-casein follow the same pattern. Both the κ-casein and α-casein show a significant

254

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

a) 185

b) Particle size (nm)

Particle size (nm)

185 160 135 110 85

180 175 170 165 160

60 0

15

30

45

60

0

15

Sonication time (min)

30

45

60

Sonication time (min)

c)

d) 14 12

40

10

30

8

pH

Soluble Ca (mg/100g)

50

6

20 4 10

2 0

0 0

15

30

45

0

60

15

30

45

60

Sonication time (min)

Sonication time (min)

e) Particle size (nm)

185 175 165 155 145 135 0

15

30

45

60

Sonication time (min) Fig. 1. a: Size of soluble particles present in the supernatants of the unsonicated and sonicated milk samples at 20 W and 41 W. b: Size of the casein micelles present in the unsonicated and sonicated milk samples at 20 W and 41 W. c: Soluble calcium in the soluble supernatant of the unsonicated and sonicated milk samples at 20 W and 41 W. d: pH of the unsonicated and sonicated milk samples at 20 W and 41 W. e: Size of fat globules present in the unsonicated and sonicated milk samples at 20 W and 41 W.

decrease in the band intensity at 15 min of sonication and within the experimental errors the trend remains constant in all the other treatment times. Among the κ-casein and α-casein, the change is prominent in κ-casein. The change in magnitude of the relative band intensities of whey proteins are higher compared to the 20 W treatment. In Fig. 6, the relative band intensity of κ-casein present in the whey protein (denatured with κ-casein) shows a similar trend i.e., it gradually increases and reaches a maximum after 30 min of treatment and then slightly decreases after 45 min of treatment at 20 W and 40 W as shown in the native PAGE. However, the trend observed for the total whey protein (including denatured whey proteins) between 20 and 41 W treatments is different until 30 min of treatment and looked similar (increasing) to each other after 30 min of treatment. Though the data until 30 min of sonication is inconclusive (in both the power treatment), it can be speculated that there is an increase in the amount of whey proteins passing inside the native PAGE. The

observation noted until 30 min of US treatment may be due to the presence of larger aggregates that did not pass through the gel. 4. Discussion The results shown in Figs. 1–6 can be summarised (4.1–4.6) as below: 4.1 The size reduction of the soluble particles present in the ultracentrifuged supernatant is significant at both power levels. 4.2 A very small decrease is observed in the size of the casein micelles at both power levels. 4.3 A small, but significant decrease in the size of fat globules is noted at both power levels. 4.4 There is a significant reduction in the values of turbidity while the viscosity of milk is not affected by sonication at both power levels.

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

a)

255

Turbidity (AU)

0.28 0.26 0.24 0.22 0.2 0.18 0.16 0

15

30

45

60

Sonication time (min)

b)

Fig. 2. a: Turbidity of sonicated and unsonicated samples of milk 20 W and different times of treatment, viz., 0 (Unsonicated), 15, 30, 45 and 60 min.

41 W. b: The supernatants obtained at 20 W. 1—Blank bottle followed by sonicated samples at

4.5 The reduced SDS PAGE of the supernatant obtained from the ultracentrifuged sonicated and unsonicated milk is compared by measuring the relative band intensities. 4.5.1. The κ-casein and α-casein show no change at 20 W, whereas a significant decrease is observed for both caseins after 15 min of sonication at 41 W. Among κ- and α-caseins, the change is prominent for κ casein. 4.5.2. The band intensity of β-casein decreases in 15 min of sonication at both 20 and 41 W and continued to remain at the same level beyond 15 min. 4.5.3. The major whey proteins show a decrease of band intensity until 30 min of sonication and then gradually increase after 30 min at both 20 and 41 W treatments. 4.6 The Native PAGE results of acid precipitated whey proteins obtained from the sonicated and unsonicated milk is compared by measuring the relative band intensities. 4.6.1 In both 20 and 41 W sonication, κ-casein present in the whey protein (denatured with κ-casein) shows a gradual

increase until 45 min of treatment followed by a slight decrease. 4.6.2 The band intensity of total whey protein (including denatured) shows an increase in the trend after 30min of treatment (prolonged sonication) at both 20 and 41 W. It also indicates the presence of larger whey protein aggregates. 4.1. Changes to the particle size and flow behaviour (summary points 4.1–4.4 and Figs. 1–3) Soluble particles in the supernatant obtained by centrifugation of the milk may comprise of native whey proteins (undenatured) in majority with some free caseins, whey protein–whey protein aggregates and/or whey protein–free casein aggregates. In our experiments, the decrease observed in the size of the soluble particles is primarily due to the shear effects generated by acoustic cavitation. At 41 W, the impact of cavitation is slightly higher compared to

Relative band intensity (AU)

200

Relative viscosity

1.05 0.95 0.85 0.75 0.65

175 150 125 100 75 50 25 0 0

0.55

0

15

30

45

60

Sonication time (min) Fig. 3. Change in relative viscosity of sonicated milk samples with respect to unsonicated milk samples 20W and 41W.

15

30

45

60

Sonication time (min) Fig. 4. Change in the relative band intensities of κ-casein, β-casein, α-casein, β-lac and ♦ α-lac obtained from reduced SDS PAGE of supernatant from the ultracentrifuged sonicated milk samples treated at 20 W with respect to the unsonicated sample.

256

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

Relative band intensity (AU)

200 175 150 125 100 75 50 25 0 0

15

30

45

60

Sonication time (min) Fig. 5. Change in the relative band intensities of κ-casein, β-casein, α-casein, β-lac and ♦ α-lac obtained from reduced SDS PAGE of supernatant from the ultracentrifuged sonicated milk samples treated at 41 W with respect to unsonicated sample.

20 W. High-intensity ultrasound has been used as an effective technique to control the size and shape of whey protein particles. An accurate selection of the process variables allowed the control of the mean size as well as the polydispersity or even the shape of protein particles (Gordon & Pilosof, 2010). The small changes observed in the size of casein micelles may be accounted for the minor changes in the size of κ-casein and possibly due to the disruption of small fragments of κ-casein present at the outer surface of the casein micelles primarily by the cavitation forces. Colloidal calcium phosphate (CCP) is a key parameter in maintaining the intact micellar structure of casein. The pH of the milk and the soluble calcium levels (indirectly related to the solubility of CCP) are crucial parameters that need to be monitored for any disruption to the casein micelles (Anema & Klostermeyer, 1997; Chandrapala, McKinnon, Augustin, & Udabage, 2010; Orlien, Boserup, & Olsen, 2010). Hence, the pH and soluble calcium in the milk were monitored in our study to monitor the integrity of casein micelles upon sonication. From Fig. 1c and 1d, soluble calcium and solution pH in the sonicated samples did not show any significant change at both power levels. Thus, it can be concluded that the CCP in casein micelles remains stable and the micelles are held intact by the CCP and are not disrupted by sonication. Madadlou et al. (2009) have indicated that sonication at 35kHz is less sensitive in disrupting the reassembled-casein micelles (prepared using casein powder dispersed in phosphate buffer) compared to 130 kHz over a pH range of 6.35 to 11.4.

Unsonicated PHSM used in the present study is a well homogenized sample. However, a small but significant reduction in the size of fat globules was noted at 20 W and the size reduction increased with increase in sonication time as observed by Wu, Hulbert, and Mount (2000). In their work, 150 ml of milk was sonicated at 20 kHz and 450, 225 and 90 W for 1, 6 and 10 min, respectively. The change in fat globule size is mainly because of the shear effects of US (Ertugay & Sengul, 2004; Bermudez-Aguirre et al., 2008). The 41 W treatment also shows a similar behaviour until 15 min in our study. However, after 15 min the increase in size of the fat globules (175 nm at 60 min) compared to their initial size (170 nm at 0 min) is too small and hence coalescence of the globules did not occur at higher power treatment. In contrast, Kentish et al., 2008 have observed the coalescence of oil droplets during the preparation of nano emulsions of flax seed oil in water at higher power density (above 180 W nominal applied power). The turbidity data of sonicated milk samples at both power levels is consistent with the overall changes in particle size data of the milk components, viz., the soluble particles of the ultracentrifuged supernatant, fat and caseins as discussed in a review (Ashokkumar et al., 2010). Among all the components that are analysed for particle size reduction, the soluble particles showed a reduction of higher magnitude (Fig. 1a). Thus the change in turbidity upon sonication of milk may be largely due to the changes in the whey protein or their aggregates (whey protein aggregates and/or whey protein-free casein aggregates). This is further supported by a photograph, shown in Fig. 2b of the supernatants that are obtained from the sonicated milk samples at 20 W and at different treatment times. The visual clarity of the samples increases from unsonicated to sonicated treatment with an increase in treatment time from 15 to 60 min. A similar observation was also obtained for the 41 W samples. In our experiments, the viscosity of milk is not affected due to sonication at both 20 and 41 W. Usually, the process-induced changes in proteins, such as polymerisation, aggregation and hydrolysis can affect the flow behaviour and viscosity of food products (Shen, 1981). 4.2. Changes to the proteins (summary points 4.5–4.6 and Figs. 4–6) The results of the changes to the proteins, which are present in the soluble supernatant and acid precipitated whey are discussed based on the average values of the relative band intensities of reduced SDS PAGE/Native PAGE, with consideration of the error values in between the experiments. For example, RBIð1Þ ¼

Band intensity of κ−casein in the supernatant of unsonicated milk  100 Band intensity of κ−casein in the supernatant of unsonicated milk

¼ 100:

Relative band Intensity (AU)

300 RBIð2Þ ¼

250

ð1Þ

Band intensity of κ−casein in the supernatant of sonicated milk  100 : Band intensity of κ−casein in the supernatant of unsonicated milk

ð2Þ

200

RBI (1) refers to relative band intensity of κ-casein present in the supernatant of unsonicated milk (control). RBI (2) refers to relative band intensity of κ-casein present in the supernatant of sonicated milk.

150 100 50 0

0

15

30

45

60

Sonication time (min) Fig. 6. Native PAGE of resultant supernatants obtained through acid precipitation of milk showing changes in the relative band intensity of κ-casein at 20 W and 41 W with respect to unsonicated sample and change in relative band intensity of total whey proteins at 20 W and ■ 41 W with respect to unsonicated sample.

From the summary point 4.5.1, the amounts of free κ- and α‐caseins and caseins in casein–whey aggregates present in soluble supernatant were decreased with higher power US treatment. A possible explanation for this observation may be due to the reaggregation of the free caseins with casein micelles caused by stronger shear forces of the acoustic cavitation. The turbulent conditions generated by the ultrasound treatment have been shown to increase particle mobility and promote the formation of aggregates (Walstra, 1983). The same reason can

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

be extended to the decrease which was observed in the band intensity of β-casein at both power levels (summary point 4.5.2). Since β-casein is the most hydrophobic among all the caseins, and the vigorous conditions generated by acoustic cavitation may have increased the interaction between β-casein molecules and re-association of β-casein with the micelles. Among the soluble proteins, the whey proteins are affected by US to a larger extent, a prominent change in band intensity with the initial decrease until 30 min of sonication (summary point 4.5.3). It is suggested that the decrease in the amount of whey protein aggregates or whey proteins (already present in PHSM) may be due to their denaturation and aggregation with the casein micelles. In the current study, the possibility of denaturation of the globular whey proteins under the influence of high shear forces is significantly high (Walstra, 2001; Wierenga, Egmond, Voragen, & de Jongh, 2006). Also, the new surfaces which are created constantly during the transient cavitation can easily damage the proteins at the bubble solution interface causing denaturation (Thomas & Geer, 2011). The bubble-solution interface possesses a hydrophobic character, where the partially denatured monomers interact with each other resulting in the formation of aggregates (Sluzky, Klibanov, & Langer, 1992). The denaturation of soluble whey proteins followed by the self-aggregation (summary point 4.6.2) and aggregation with other free caseins (summary points 4.5.1 and 4.5.2) are also observed. From summary point 4.6.1, it is also clear that these newly formed denatured/aggregated soluble whey proteins interact with κ-caseins present on the surface of the casein micelles and form micellar aggregates. These micellar aggregates might have been formed by the thiol-disulphide exchange reactions between the denatured whey proteins and the κ-caseins of the micelles (Fox & McSweeney, 1998; Anema & Li, 2003). Hence, the amount of whey proteins present in the soluble supernatant are reduced upon 30 min of sonication compared to unsonicated sample (Figs. 4 and 5). From summary point 4.5.3, the increase in the relative band intensity of whey proteins (β-lac and α-lac) noted during the prolonged sonication can be due to the disruption of whey proteins from the newly formed micellar aggregates. The sonication for a longer period of time remains as a possible reason for this disruption. It is apparent from Figs. 4 and 5 that the dissociation is incomplete within the error limits of the experiment. This may possibly be due to the compensating effect of shear over aggregation, since the surface available for aggregation is decreasing towards 30 min of sonication and the continuous shear produced by the US dissociated some of the whey particles from the surface of the micellar aggregates. The partial disruption can also be supported by Fig. 6 and summary point 4.6.2 showing the increase in the band intensity of total whey proteins. This provides indirect evidence that the disruption of some of the whey proteins from the aggregates causes them to enter the gel in native PAGE. 5. Conclusions PHSM was treated with 20 kHz US at 20 W and 41 W for up to 60 min. Whey proteins and whey–whey aggregates present in the milk were denatured and formed soluble whey–whey/whey–casein aggregates which further interacted with casein micelles to form micellar aggregates during the initial 30 min of sonication. Prolonged sonication resulted in the partial disruption of some whey proteins from these aggregates. The physical forces of acoustic cavitation did not affect the intact structure of the casein micelles. A small decrease in casein micelle size can be accounted for the disruption of κ-casein fragments present in the outer surface of the micelle. The minor changes to the proteins, caused by sonication, did not alter the viscosity of the milk though there was an overall particle size reduction of fat globules and soluble particles in the supernatant. It can also be concluded from our data that the changes to the flow behaviour and the proteins of PHSM were perceived to be minimum. The shear

257

forces generated by acoustic cavitation are responsible for the observed changes, particularly to the protein particles. Acknowledgements Akalya Shanmugam would like to thank the University of Melbourne for providing the Melbourne International Fee Remission and Melbourne International Research scholarships. References AlKanhal, H. A., Al-Othman, A. A., & Hewedi, F. M. (2001). Changes in protein nutritional quality in fresh and recombined ultra high temperature treated milk during storage. International Journal of Food Sciences and Nutrition, 52, 509–514. Anema, S. G. (2009). The whey proteins in milk: Thermal denaturation, physical interactions and effects on the functional properties of milk. In A. Thompson, M. Boland, & H. Singh (Eds.), Milk proteins — From expression to food (pp. 239–281). Elsevier. Anema, S. G., & Klostermeyer, H. (1997). Heat-induced, pH-dependent dissociation of casein micelles on heating reconstituted skim milk at temperatures below 100 degrees C. Journal of Agricultural and Food Chemistry, 45, 1108–1115. Anema, S. G., & Li, Y. M. (2003). Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size. The Journal of Dairy Research, 70, 73–83. Arzeni, C., Martinez, K., Zema, P., Arias, A., Perez, O. E., & Pilosof, A. M. R. (2012). Comparative study of high intensity ultrasound effects on food proteins functionality. Journal of Food Engineering, 108, 463–472. Ashokkumar, M., Bhaskaracharya, R., Kentish, S., Lee, J., Palmer, M., & Zisu, B. (2010). The ultrasonic processing of dairy products — An overview. Dairy Science & Technology, 90, 147–168. Ashokkumar, M., & Grieser, F. (2007). The effect of surface active solutes on bubbles in an acoustic field. Physical Chemistry Chemical Physics (PCCP), 9, 5631–5643. Ashokkumar, M., Lee, J., Zisu, B., Bhaskarcharya, R., Palmer, M., & Kentish, S. (2009). Hot topic: Sonication increases the heat stability of whey proteins. Journal of Dairy Science, 92, 5353–5356. Ashokkumar, M., & Mason, T. J. (2007). Sonochemistry Kirk-Othmer Encyclopedia of Chemical Technology: John Wiley & Sons, Inc. Barbosa-Canovas, G., & Bermudez-Aguirre, D. (2010). Other novel milk preservation technologies: ultrasound, irradiation, microwave, radio frequency, ohmic heating, ultraviolet light and bacteriocins. In M. W. Griffiths (Ed.), Improving the safety and quality of milk, Volume 1: Milk production and processing (pp. 420–450). Bermudez-Aguirre, D., Corradini, M. G., Mawson, R., & Barbosa-Canovas, G. V. (2009a). Modeling the inactivation of Listeria innocua in raw whole milk treated under thermo-sonication. Innovative Food Science & Emerging Technologies, 10, 172–178. Bermudez-Aguirre, D., Mawson, R., & Barbosa-Canovas, G. V. (2008). Microstructure of fat globules in whole milk after thermosonication treatment. Journal of Food Science, 73, E325–E332. Bermudez-Aguirre, D., Mawson, R., Versteeg, K., & Barbosa-Canovas, G. V. (2009b). Composition properties, physicochemical characteristics and shelf life of whole milk after thermal and thermo-sonication treatments. Journal of Food Quality, 32, 283–302. Bhaskaracharya, R. K., Kentish, S., & Ashokkumar, M. (2009). Selected applications of ultrasonics in food processing. Food Engineering Reviews, 1, 31–49. Burton, H. (1988). Ultra-high-temperature processing of milk and milk products: London. New York: Elsevier Applied Science. Cavalieri, F., Ashokkumar, M., Grieser, F., & Caruso, F. (2008). Ultrasonic synthesis of stable, functional lysozyme microbubbles. Langmuir, 24, 10078–10083. Chandrapala, J., McKinnon, I., Augustin, M. A., & Udabage, P. (2010). The influence of milk composition on pH and calcium activity measured in situ during heat treatment of reconstituted skim milk. The Journal Of Dairy Research, 77, 257–264. Chandrapala, J., Zisu, B., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate. Ultrasonics Sonochemistry, 18, 951–957. Chouliara, E., Georgogianni, K. G., Kanellopoulou, N., & Kontominas, M. G. (2010). Effect of ultrasonication on microbiological, chemical and sensory properties of raw, thermized and pasteurized milk. International Dairy Journal, 20, 307–313. Corzo, N., Lopezfandino, R., Delgado, T., Ramos, M., & Olano, A. (1994). Changes in furosine and proteins of UHT-treated milks stored at high ambient-temperatures. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung, 198, 302–306. Czank, C., Simmer, K., & Hartmann, P. E. (2010). Simultaneous pasteurization and homogenization of human milk by combining heat and ultrasound: Effect on milk quality. The Journal of Dairy Research, 77, 183–189. De Gennaro, L., Cavella, S., Romano, R., & Masi, P. (1999). The use of ultrasound in food technology I: Inactivation of peroxidase by thermosonication. Journal of Food Engineering, 39, 401–407. Ertugay, M. F., & Sengul, M. (2004). Effect of ultrasound treatment on milk homogenisation and particle size distribution of fat. Turkish Journal of Veterinary and Animal Sciences, 28, 303–308. Fellows, P. J. (2000). Food processing technology — principles and practice (2 ed.): Woodhead Publishing. Fox, P. F., & McSweeney, P. L. H. (1998). Dairy chemistry and biochemistry: Springer — Verlag. Gordon, L., & Pilosof, A. M. R. (2010). Application of high-intensity ultrasounds to control the size of whey proteins particles. Food Biophysics, 5, 203–210.

258

A. Shanmugam et al. / Innovative Food Science and Emerging Technologies 16 (2012) 251–258

Gulseren, I., Guzey, D., Bruce, B. D., & Weiss, J. (2007). Structural and functional changes in ultrasonicated bovine serum albumin solutions. Ultrasonics Sonochemistry, 14, 173–183. Guyomarc'h, F., Law, A. J. R., & Dalgleish, D. G. (2003). Formation of soluble and micelle-bound protein aggregates in heated milk. [Article]. Journal of Agricultural and Food Chemistry, 51, 4652–4660. Hansen, A. P., & Melo, T. S. (1977). Effect of ultra-high-temperature steam injection upon constituents of skim milk. Journal of Dairy Science, 60, 1368–1373. Herzallah, S. M., Humeid, M. A., & Al-Ismail, K. M. (2005). Effect of heating and processing methods of milk and dairy products on conjugated linoleic acid and trans fatty acid isomer content. Journal of Dairy Science, 88, 1301–1310. Huppertz, T., Fox, P. F., & Kelly, A. L. (2004). High pressure treatment of bovine milk: Effects on casein micelles and whey proteins. The Journal of Dairy Research, 71, 97–106. Keenan, T. W., & Patton, S. (1995). The structure of milk: Implications for sampling and storage. In R. G. Jensen (Ed.), The milk lipid globule membrane. Handbook of Milk Composition. (pp. 420–450) New York: Academic Press. Kentish, S., & Ashokkumar, M. (2011). The physical and chemical effects of ultrasound. In H. Feng, G. Barbosa-Canovas, & J. Weiss (Eds.), Ultrasound technologies for food and bioprocessing (pp. 1–12). New York: Springer. Kentish, S., Wooster, T. J., Ashokkumar, A., Balachandran, S., Mawson, R., & Simons, L. (2008). The use of ultrasonics for nanoemulsion preparation. Innovative Food Science & Emerging Technologies, 9, 170–175. Knudsen, J. C., & Skibsted, L. H. (2010). High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy. Food Chemistry, 119, 202–208. Lacroix, M., Bon, C., Bos, C., Leonil, J., Benamouzig, R., Luengo, C., et al. (2008). Ultra high temperature treatment, but not pasteurization, affects the postprandial kinetics of milk proteins in humans. Journal of Nutrition, 138, 2342–2347. Lan, X. Y., Wang, J. Q., Bu, D. P., Shen, J. S., Zheng, N., & Sun, P. (2010). Effects of heating temperatures and addition of reconstituted milk on the heat indicators in milk. Journal of Food Science, 75, C653–C658. Madadlou, A., Mousavi, M. E., Emam-Djomeh, Z., Ehsani, M., & Sheehan, D. (2009). Comparison of pH-dependent sonodisruption of re-assembled casein micelles by 35 and 130 kHz ultrasounds. Journal of Food Engineering, 95, 505–509. Marchioni, C., Riccardi, E., Spinelli, S., Dell'Unto, F., Grimaldi, P., Bedini, A., et al. (2009). Structural changes induced in proteins by therapeutic ultrasounds. Ultrasonics, 49, 569–576. Nguyen, N. H. A., & Anema, S. G. (2010). Effect of ultrasonication on the properties of skim milk used in the formation of acid gels. Innovative Food Science & Emerging Technologies, 11, 616–622. Oldfield, D. J., Singh, H., & Taylor, M. W. (1998a). Association of beta-lactoglobulin and alpha-lactalbumin with the casein micelles in skim milk heated in an ultra-high temperature plant 8, 770. Oldfield, D. J., Singh, H., Taylor, M. W., & Pearce, K. N. (1998b). Kinetics of denaturation and aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot plant. International Dairy Journal, 8, 311–318.

Orlien, V., Boserup, L., & Olsen, K. (2010). Casein micelle dissociation in skim milk during high-pressure treatment: Effects of pressure, pH, and temperature. Journal of Dairy Science, 93, 12–18. Patel, H. A., Carroll, T., & Kelly, A. L. (2008). Nonthermal preservation technologies for dairy applications. In R. C. Chandan, A. Kilara, & N. P. Shah (Eds.), Dairy processing & quality assurance (pp. 467). Ames, Iowa: Wiley-Blackwell. Riener, J., Noci, F., Cronin, D. A., Morgan, D. J., & Lyng, J. G. (2009). Characterisation of volatile compounds generated in milk by high intensity ultrasound. International Dairy Journal, 19, 269–272. Saffon, M., Britten, M., & Pouliot, Y. (2011). Thermal aggregation of whey proteins in the presence of buttermilk concentrate. Journal of Food Engineering, 103, 244–250. Seiquer, I., Delgado-Andrade, C., Haro, A., & Navarro, M. P. (2010). Assessing the effects of severe heat treatment of milk on calcium bioavailability: In vitro and in vivo studies. Journal of Dairy Science, 93, 5635–5643. Shen, J. L. S. (1981). Protein functionality in foods. In J. P. Cherry (Ed.), Protein functionality in foods (pp. 89–109). Washington, DC: American Chemical Society. Sluzky, V., Klibanov, A. M., & Langer, R. (1992). Mechanism of insulin aggregation and stabilization in agitated aqueous solutions. Biotechnology And Bioengineering, 40, 895–903. Stathopulos, P. B., Scholz, G. A., Hwang, Y. M., Rumfeldt, J. A. O., Lepock, J. R., & Meiering, E. M. (2004). Sonication of proteins causes formation of aggregates that resemble amyloid. Protein Science, 13, 3017–3027. Taylor, M. J., & Richardson, T. (1980). Antioxidant activity of skim milk — Effect of heat and resultant sulfhydryl-groups. Journal of Dairy Science, 63, 1783–1795. Thomas, C. R., & Geer, D. (2011). Effects of shear on proteins in solution. [Review]. Biotechnology Letters, 33, 443–456. Vercet, A., Oria, R., Marquina, P., Crelier, S., & Lopez-Buesa, P. (2002). Rheological properties of yoghurt made with milk submitted to manothermosonication. Journal of Agricultural and Food Chemistry, 50, 6165–6171. Villamiel, M., & de Jong, P. (2000). Influence of high-intensity ultrasound and heat treatment in continuous flow on fat, proteins, and native enzymes of milk. Journal of Agricultural and Food Chemistry, 48, 472–478. Walstra, P. (1983). Formation of emulsion. Encyclopedia of emulsion technology: basic theory, vol. 1. (pp. 57–127)New York: Marcel Dekker Inc. Walstra, P. (2001). Effect of agitation on proteins. In E. Dickinson, & R. Miller (Eds.), Food colloids: Fundamentals of formulation (pp. 245–254). Cambridge: Royal Society of Chemistry. Wierenga, P. A., Egmond, M. R., Voragen, A. G. J., & de Jongh, H. H. J. (2006). The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state. Journal Of Colloid And Interface Science, 299, 850–857. Wu, H., Hulbert, G. J., & Mount, J. R. (2000). Effects of ultrasound on milk homogenization and fermentation with yogurt starter. Innovative Food Science & Emerging Technologies, 1, 211–218.