Sonodisruption of re-assembled casein micelles at different pH values

Ultrasonics Sonochemistry 16 (2009) 644–648

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Sonodisruption of re-assembled casein micelles at different pH values Ashkan Madadlou a, Mohammad Ebrahimzadeh Mousavi a,*, Zahra Emam-djomeh a, Mohammadreza Ehsani a, David Sheehan b a b

Department of Food Science and Engineering, Faculty of Biosystem Engineering, Campus of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Department of Biochemistry, Faculty of Science, University College Cork, Cork, Ireland

a r t i c l e

i n f o

Article history: Received 25 November 2008 Received in revised form 16 December 2008 Accepted 24 December 2008 Available online 6 January 2009 Keywords: Casein Ultrasound Dissociation Cavitation

a b s t r a c t Casein solutions with different pH values were sonicated at a frequency of 35 kHz and increasing acoustic powers. As the sonication power increased, turbidity of solutions and particle diameter decreased at any given pH value, suggesting particles disruption due to the ultrasonic treatment. The magnitude of decrease in re-assembled micelles diameter was greater at a higher pH, indicating an interaction between pH and sonication power in sonodissociation. This interaction is attributed to a looser structure of micelles at higher pH values which increases the efficiency of ultrasonic disruption and not directly to the increased cavitation efficiency. We argue that increased cavitation efficiency with increasing sonication power, which enhances shear forces is the most likely reason for sonodisruption of re-assembled casein micelles. Ó 2009 Elsevier B.V. All rights reserved.

1. Introduction Ultrasound is defined as any sound of a frequency greater than the upper limit of human hearing [1] i.e., above 16 kHz [2] up to 10 MHz. They have velocities in a liquid between 1000 and 1600 m/s [3] and wavelengths of 10–0.01 cm. These are not molecular dimensions and the chemical effects of ultrasound do not come from direct interaction with molecular species. Instead, sonochemical and power ultrasounds’ effects derive principally from acoustic cavitations [4]. This is the violent collapse of microbubbles generated in a fluid exposed to high intensity ultrasound [5]. Sudden collapse of the bubble results in an inrush of liquid to fill the void producing strong shear forces in the surrounding bulk liquid [6]. Bubble collapse can occur so rapidly (even less than a microsecond) and nearly adiabatically, to create a localized hot spot [5]. These hot spots have temperatures of approximately 5000 °C, pressures of about 500 atm, and heating and cooling rates greater than 109 K/s. In aqueous solutions, water vapor molecules and volatiles inside cavities are thermolysed to generate highly reactive hydroxyl, hydrogen and organic radical species [2,7]. The major sonolytic products are hydroxyl radicals and hydrogen peroxide formed from recombination of hydroxyl radicals with each other [7]. Other mechanisms for ultrasound effects such as microstreaming, microcurrents and liquid jets originate from cavitation and cavities circulation. * Corresponding author. Fax: +98 2612248804. E-mail addresses: [email protected] (A. Madadlou), [email protected] (M.E. Mousavi). 1350-4177/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2008.12.018

Several applications have been reported for ultrasound in milk and dairy processing: enhancing whey ultrafiltration and cleaning of whey-fouled membranes [8]; reduction of yogurt’s total fermentation time and increase in its water-holding capacity and viscosity [9]; and manufacturing yogurts with superior rheological properties [10]. There are also some reports on the influence of high intensity ultrasound on chemical components of milk and dairy suspensions. Villamiel and de Jong [11] studied the effect of an ultrasonic probe (20 kHz frequency, 120 lm amplitude, 150 W nominal power) in a continuous flow mode of milk with different residence times on fat, protein and native enzymes of milk. Hardly any effect on enzymes was observed when ultrasound was applied without heat generation. Interestingly, sonication was able to denature both a-lactalbumin and b-lactoglobulin but, according to peak areas in electrophoregrams, no changes on casein were observed after sonoprocessing. Those researchers hypothesized that sonication modifies the quaternary and/or tertiary structure of casein but does not fully disrupt casein micelle. Low (500 kHz bath) and high intensity (20 kHz probe and 40 kHz bath) ultrasounds were used by Jambrak et al. [12] to improve the functional properties of whey protein suspensions. They found that ultrasound effected the solubility and foaming ability of whey proteins and attributed this to the changes in conformation and structure of proteins. Casein micelles, self associated colloids in milk [13] although are stable [14] but not fixed structures. Changes in temperature, pH, ionic strength, water activity and imposition of high pressures lead to changes in casein micelles size distribution [15,16] and probably the proportion of sub-structures. This is attributed to their lack of a rigid 3-dimensional tertiary conformation [17].

A. Madadlou et al. / Ultrasonics Sonochemistry 16 (2009) 644–648

Casein micelles can also be re-assembled in vitro from caseinate and casein [18–20] even in the absence of calcium [21]. This is because of self-assembly tendency of casein monomers [22]. The overall morphology and size of the re-assembled micelles are never likely nor expected to be identical to native casein micelles [23,20]. They are however, similar to those of naturally occurring micelles [18] and the results obtained for these systems can be generalized for native casein-based foods. Because of the shear forces generated by bubble implosion and/ or microstreaming, it is reasonable to expect that multimolecular arrangements in milk (mainly casein micelles) can be broken to smaller structures [10]. The aim of the present work was to explore the influence of sonication (35 kHz frequency) at different pH values on turbidity, electrical conductivity, and particle size of casein solutions. As the structure of re-assembled micelles is loosen at alkaline conditions [24] pH of casein solutions was up warded to enhance the assumed disruption of micelles by ultrasound and assay the possibility of casein chains sonolysis.

2. Experimental 2.1. Materials and ultrasound equipment Casein, methanol and phosphate buffer pH 7.0 (di-sodium hydrogen phosphate/potassium dihydrogen phosphate) were purchased from Merck (Darmstadt, Germany). Sodium tetraborate, sodium dodecyl sulfate (SDS), o-phthaldialdehyde (OPA) and b-mercaptoethanol were from Sigma–Aldrich (Dorset, UK). A double frequency ultrasonic bath model TI-H 10 (Elmasonics, Singen, Germany) of internal dimensions 30  24  15 cm provided continuous low frequency ultrasound (35 kHz) using four disc transducers with a total nominal output power of 200 W. The power output can be set from 10% up to 100% by adjustment of the power level. 2.2. Sample preparation and sonoprocessing Casein dispersion (3%) was prepared by adding 12 g casein powder to 0.5 M phosphate buffer (prepared by deionized water), stirring it at 4600 rpm for 60 min at room temperature and making it up to the final volume of 400 mL; sodium azide (100 mg L1) was added to prevent microbial growth. The dispersion was stored at 4 °C for 10 h to allow complete hydration [19]. Dispersion (100 mL) was transferred to a 250 mL Erlenmeyer at its original pH value (6.35 ± 0.05). The pH value of the remainder was then adjusted to 8.0 ± 0.05, 9.7 ± 0.05 and 11.4 ± 0.05 by slow addition of sodium hydroxide solution to well-stirred dispersion. Each time, immediately after the desired pH was reached, 100 mL solution was transferred to a 250 mL Erlenmeyer. Very strong sodium hydroxide (20 M) was used for increasing pH value in order to prevent solution dilution. The Erlenmeyers were placed in the centre of the bath and rested for 1 h to equilibrate with the surrounding water (30 °C). They were then sonicated for 6 h, during which samples temperature was maintained at 30.5 ± 1.5 °C by tap water circulation around the containers in the bath (except for power measurements). The rise in temperature of casein solution during 120 s was used to calculate the actual power dissipated to the solution from the following equation [12,25]:

P ¼ mC p ðdT=dtÞ

ð1Þ

where P is power (W), Cp is the specific heat capacity of the water (4.18 J °C1 g1), m is the mass of water (grams), dT/dt is the temperature difference over the 120 s sonication time. The actual values for power dissipated to the solutions were 0.0, 2.0, 4.1 and 6.6 W for nominal power levels of off, 40%, 70% and 100%, respectively.

645

2.3. Turbidity and electric conductivity measurements Turbidity measurements were carried out with a turbidimeter (WTW, 350 IR., West Chester, PA, USA) by three readings for each sample at 30 °C immediately after sonoprocessing. Banon and Hardy [26] used a similar method for turbidity measurement of milk samples. Electrical conductivity of casein solutions was determined after 6 h processing by a digital conductivity meter (Hanna, model HI 8633, Hanna Instruments Inc., Bedfordshire, UK) to monitor the changes in the concentration of ions at the solution. 2.4. Particle analysis Mean particle diameter and size distribution of casein dispersions were measured with a laser-diffraction based particle size analyzer (Malvern Master Sizer Hydro 2000 S, Malvern Instruments Ltd., Malvern, UK) operated with program Mastersizer 2000 version 5.22. Experiments were performed on 5-fold diluted dispersions over the range 20 nm–15 lm. Just before measurements, samples were diluted with deionized water having the same pH of sample (adjusted by sodium hydroxide addition) to prevent their foaming during stirring and pumping in the instrument. Water was used as the dispersant with the refractive index of 1.330. Particle characteristics of dispersions are reported by volume-weighted mean (D4,3) and span. The latter is an index of particles polydispersity [27] and expressed by

Span ¼ ðd0:9  d0:1 Þ=ðd0:5 Þ

ð2Þ

where d0.9, d0.1 and d0.5 are the diameters at 90%, 10% and 50% cumulative volume of particles, respectively. 2.5. Primary amine content The content of primary amines was measured to follow probable lysis of peptide bonds due to sonication. OPA was prepared as described by Church et al. [28]. The reagent was made by combining the following materials and diluting to a final volume of 50 mL with deionized water daily before analysis: 25 mL of 0.1 M sodium tetraborate; 2.5 mL of 20% (w/w) SDS; 40 mg of OPA dissolved in 1 mL methanol; and 100 lL of b-mercaptoethanol. To assay probable proteolysis 60 lL of 10-fold diluted sample in 0.5 phosphate buffer was added to 2 mL of OPA reagent. This solution was briefly stirred and the absorbance at 340 nm read after 2-min incubation at room temperature. A standard curve was obtained using L-leucine as a reference compound. Reference samples were prepared in the concentration range 0.3–3 lM mL1 in 0.5 M phosphate buffer. The obtained absorbance value for each sample could be converted in amino groups concentration using equation of L-leucine standard curve. 2.6. Statistical analysis The experiment was replicated 3 times in a complete randomized design. The effect of pH value and ultrasonic treatment on tested parameters was determined by analysis of variance (ANOVA) using GraphPad Prism 5.0 for windows version 5.00 (GraphPad Software Inc.). Two-way ANOVA at 5% significant level (a = 0.05) was carried out to assess whether different treatments resulted in statistically significant differences in variables evaluated.

3. Results and discussion Ultrasonic experiments were conducted for 6 h at three different power levels of off, 40%, 70% and 100%. The actual power dissi-

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pated to the medium was measured calorimetrically (as described in experimental section) and does not exceed 0.0, 2.0, 4.1 and 6.6 W for the respective power levels used. In some processes, only 1–20% of supplied energy in the liquid is converted to cavitational activity [29]. The results of experiments on solution turbidity and particle diameter are illustrated in Fig. 1. Casein solution turbidity decreased rapidly as pH increased from 6.35 to 8.0 with a marked but more modest linear-like decrease with increasing pH above 8.0. The higher the pH, the lower was the turbidity value for each treatment. This decrease in turbidity was concomitant with increasing particles diameter (Fig. 1 right axis). The mechanism underlying the expansion of re-assembled casein micelles is discussed elsewhere and based on the electrostatic repulsion among casein molecules in micelles and the increased solvent quality of serum phase [24]. It is clear that increasing the sonication power resulted in a decrease in solution turbidity and particle diameter at any given pH value. It has been reported that higher breakage is favored at higher sonication power for aluminum oxide particles with nominal size of 150 lm [30]. The magnitude of decrease in particle diameter was greater at a higher pH. Two-way ANOVA revealed this interaction between pH and sonication power in sonodisruption of particles also. It should be noted that pH does not have any direct effect on the cavitational intensity in terms of the number of cavitation events or the pressure/temperature generated due to cavity collapse [31]. However, higher pH values loosen the micelles structure leading to a more efficient influence of cavitating forces on micelles. The decreased particle diameter and, consequently, increased surface area available for light scattering account for the role of ultrasound in decreasing dispersion turbidity. Conductivity was decreased as sonication power increased at any given pH value (Fig. 2). Jambrak et al. [12] observed that the conductivity of whey protein suspensions increased for samples treated with an ultrasound probe but decreased for those treated in ultrasound baths. They hypothesized that increased conductivity is due to the formation of hydroxyl radicals during sonication and decreased conductivity is due to the presence of ion aggregates combined with an increase in viscosity. It is however, important to remember that radicals are highly reactive species and most of them react rapidly inside the collapsing bubble or at the bubble-liquid interface either with each other or with solutes diffused into the cavity. Although a considerable number may also enter the liquid phase where they can attack a given solute [6,7,32], they do not survive in the medium to participate in increased conductivity of the suspension after processing. The decrease in conductivity of

6

3 6

→ 350

350 300

7

8

9

10

11

12

pH Fig. 2. Electric conductivities of casein solutions as a result of pH and acoustic power. Dispersions sonicated at acoustic power, 2.0 W (–s–), 4.1 W (–j–) and 6.6 W (–D–). Error bars indicate standard deviations.

samples observed in the present study is most probably due to a lower number of hydroxyl ions in the dispersion after sonoprocessing because of reaction with hydroxyl and hydrogen radicals generated during sonication. Random measurements revealed that pH decreased for all samples upon ultrasonic treatment by a value of 0.1–0.2. The proposed chemical equations for the abovementioned reactions are given below:

OH þ H ! H2 O

ð3Þ

OH þ OH ! H2 O2

ð4Þ

It is also noteworthy that oxidation of nitrogen molecules dissolved in water and subsequent generation of nitric oxide likely played a role in decrease of pH value too [33]. As sonication power increased, polydispersity of casein particles decreased at any given pH value (Fig. 3). This indicates that increased homogeneity of particles occurred due to sonication. The ability of ultrasound to produce a food product with narrower molecular weight distribution has been previously reported for dextran [34]. These observations could be attributed to the increased cavitation efficiency, which enhances shear forces, combined with rapidly changing pressure. A possible role for microstreaming should also be noted. This can specifically occur as a result of oscillations of acoustically-driven bubbles in a sound field and can disrupt DNA or disaggregate bacteria [1]. Bubble collapse near an extended surface can also produce localized high-speed jets of liquid that

1.4

1.2

Span

← 400

5

4

400

particle size (nm)

Turbidity (NTU)

450

7

EC (mVS)

646

1.0

0.8

0.6

300 6

7

8

9

10

11

12

pH 6 Fig. 1. Turbidity of casein solutions (left Y axis) and diameter of re-assembled casein micelles (right Y axis) as a result of acoustic power. Solutions sonicated at acoustic power, 2.0 W (–s–), 4.1 W (–j–) and 6.6 W (–D–). Arrows compare the reduction in particle size at different pH values. Error bars indicate standard deviations.

7

8

9

10

11

12

pH Fig. 3. Polydispersity index (span) of casein solutions as a result of pH and acoustic power. Dispersions sonicated at acoustic power, 2.0 W (–s–), 4.1 W (–j–) and 6.6 W (–D–).

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dard curve (Fig. 5) slope [39]. It resulted in 0.6633 lM mg1 casein which is close to the amount previously reported by Dinnella et al. [39]. Measurements in the present study revealed that the total amount of primary amines was not affected by ultrasound power. This means that ultrasound did not split the peptide bond in casein chains. More powerful sonicators and different frequencies are probably needed to sonolyse protein chains.

2

0.6

3

0.5

4

0.4

Abs 340 nm

647

5

0.3

6

0.2

4. Conclusion 8

0.1

9

0.0

0 0.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Casein concentration (mg mL-1) Fig. 4. Relationship between absorbance value at 340 nm and casein concentration. The relationship is defined by the following equation: 0.016336714 + 0.18616633x; R = 0.99409319.

impinging on the surface [35]. Casein nanoparticles, however, are too small to perturb the ultrasonic filed. At 35 kHz the collapsing bubble will have a diameter larger than 100 lm; solid particles smaller than this size cannot cause microjet formation [34]. Upon the implosion of a cavity, a high strain rate directly proportional to bubble wall velocity and the radius of bubble is generated [36]. For a given ultrasound frequency, the number of cavitating bubbles [37] and their maximum size increase with acoustic power. This increase in maximum size could result in a higher strain rate [36] and an increase in the maximum collapse temperature. The former would enhance the shearing effect of imploding bubbles; while, the latter one may increase the amount of primary radicals [33]. A direct relationship between liberation of hydroxyl radicals and the magnitude of power dissipation has been observed [31]. Barteri et al. [38] pointed out that hydroxyl and hydrogen radicals generated by sonolysis of water molecules can extensively degrade proteins and other biopolymers. In the presence of b-mercaptoethanol, OPA reacts with primary amines forming 1-alkylthio-2-alkylisoindole. The product heavily absorbs at 340 nm and this has been used in spectrophotometric determination of amounts of peptides and amino acids. The primary amount of OPA reactive amino groups was calculated as the ratio between the casein curve (Fig. 4) slope and L-leucine stan-

0

0.9

5

Abs 340 nm

0.7

0

0.6

5

0.4

0

0.3

5

0.1

0 0.0

0.0

0.5

1.1

1.6

2.2

2.7

3.2

-1

L-leucine (µM mL ) Fig. 5. Relationship between absorbance value at 340 nm and using L-leucine concentration. The relationship is defined by the following equation: 0.0190389440 + 0.2806628x; R = 0.99622734.

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