Effect of Temperature and Pressure on the Stability of Protein

Nov 30, 2015 - Top Institute Food and Nutrition, P.O. Box 557 6700 AN, .... Linkam Scientific Instruments, Surrey, United Kingdom) that was ...... You...
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Effect of Temperature and Pressure on the Stability of Protein Microbubbles Tijs A.M. Rovers,†,‡ Guido Sala,†,‡,§ Erik van der Linden,†,‡ and Marcel B.J. Meinders*,†,§ †

Top Institute Food and Nutrition, P.O. Box 557 6700 AN, Wageningen, The Netherlands Laboratory of Physics and Physical Chemistry of Foods, Wageningen University and Research Centre, P.O. Box 17, 6700 AA, Wageningen, The Netherlands § Food and Biobased Research, Wageningen University and Research Centre, P.O. Box 17 6700 AA, Wageningen, The Netherlands ‡

ABSTRACT: Protein microbubbles are air bubbles with a network of interacting proteins at the air−water interface. Protein microbubbles are commonly used in medical diagnostic and therapeutic research. They have also recently gained interest in the research area of food as they can be used as structural elements to control texture, allowing for the manufacture of healthier foods with increased consumer perception. For the application of microbubbles in the food industry, it is important to gain insights into their stability under food processing conditions. In this study, we tested the stability of protein microbubbles against heating and pressurization. Microbubbles could be heated to 50 °C for 2 min or pressurized to 100 kPa overpressure for 15 s without significantly affecting their stability. At higher pressures and temperatures, the microbubbles became unstable and buckled. Buckling was observed above a critical pressure and was influenced by the shell modulus. The addition of cross-linkers like glutaraldehyde and tannic acid resulted in microbubbles that were stable against all tested temperatures and overpressures, more specifically, up to 120 °C and 470 kPa, respectively. We found a relation between the storage temperatures of microbubble dispersions (4, 10, 15, and 21 °C) and a decrease in the number of microbubbles with the highest decrease at the highest storage temperature. The average rupture time of microbubbles stored at different storage temperatures followed an Arrhenius relation with an activation energy for rupture of the shell of approximately 27 kT. This strength ensures applicability of microbubbles in food processes only at moderate temperatures and storage for a moderate period of time. After the proteins in the shell are crosslinked, the microbubbles can withstand pressures and temperatures that are representative of food processes. KEYWORDS: microbubble, stability, pressure, buckling, heating, storage temperature microbubble size and stability. Recently, Rovers et al.1 reported on the effect of preparation parameters on the yield and size of microbubbles produced using ultrasound as well as their shelf life under different storage conditions.17 For applications in food, it is essential to understand the mechanisms behind the stability of microbubbles and the material properties of the protein shell at the air−water interface under different environmental conditions. Rovers et al.1 investigated the effects of the addition of acid and small molecular weight surfactants on the stability of bovine serum albumin (BSA)-covered microbubbles. Besides the addition of food ingredients, the microbubble stability might also be influenced by processes typical of the food industry, such as heating and pressurization, but also by variations of storage conditions like storage temperature. The understanding of the effect of heat treatment on microbubble dispersions is limited. It has been reported that BSA-covered microbubbles stayed intact and formed a gel of microbubbles when subjected to a temperature of 121 °C for

1. INTRODUCTION Protein microbubbles are air bubbles covered with a shell of proteins at the air−water interface. This shell comprises of a network of protein molecules that are noncovalently linked.1 This protein network is so strong that it withstands Laplace pressure, making the microbubble stable for a long period of time and suitable for several very important applications. For example, protein microbubbles have been used as contrast agents in medical diagnostics and have been proven to be useful as carriers of drugs, the release of which can be controlled so that it can be delivered at the right time and place.2−4 Furthermore, it has been proposed that microbubbles could be used as structural ingredients in foods to create new food textures, allowing for the manufacturing of healthier foods, e.g., by replacing fat, with increased consumer preference.5−10 Protein-stabilized microbubbles can be produced using various methods, including sonication, high shear mixing, template layer-by-layer deposition, electro-hydrodynamic atomization, and pressurized gyration.11−16 Each technique has its own advantages and drawbacks. In this paper, we focus on microbubbles intended for the food industry. Important factors for applications in food are production yield as well as © 2015 American Chemical Society

Received: September 18, 2015 Accepted: November 30, 2015 Published: November 30, 2015 333

DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

Research Article

ACS Applied Materials & Interfaces 15 min.7 To our knowledge, no other results on heat treatments of microbubbles have been published. Experiments applying static pressure showed that human serum albumincovered microbubbles lost their activity as a contrast agent after 15 s at 25 kPa18 and lost 40% of their gas volume after 10 s at 137 kPa.19 When increasing the overpressure from 2 to 180 kPa for more than an hour, polyester-covered microbubbles with a diameter of 1 μm ruptured at pressures around 100 kPa.20 The onset of instability as a result of pressure variations turned out to be mainly determined by the ratio between the shell thickness and the bubble radius (d/R), and the Young’s elastic modulus (E) of the microbubble shell.20 The microbubble elastic modulus, measured with atomic force microscopy (AFM), was found to be dependent on the material used for the shell. The reported values are around 0.6 MPa for lysozyme shells of air-filled microbubbles21 and approximately 8 to 40 MPa for phospholipid shells of fluorocarbon gas-filled microbubbles.22 With regard to storage temperature, we showed in recent work that after ∼36 days all microbubbles stored at 21 °C disappeared, whereas microbubbles stored at 4 °C remained intact even after 43 days.17 When stored at 8 °C, the number of BSA microbubbles after 14 days of storage diminished in comparison with that after 2 days of storage,7 whereas microbubbles composed of BSA and dextrose were reported to be stable for more than a year when stored at 5 °C.5 In this study, we analyzed the stability of protein-covered microbubbles as a function of heating temperature (in the range 30−120 °C), overpressure (in the range 0−470 kPa), and storage temperature (in the range 4−21 °C). We did this to increase insight into the underlying mechanism of microbubble stability under changing environments to better control microbubble stability so that they can survive environmental conditions encountered in important industrial applications.

diluted with demineralized water (top layer:water = 1:4 v/v) and subsequently centrifuged for 30 min at 300g. The top layer was again collected and kept at 4 °C until cross-linking agents were added. Glutaraldehyde or tannic acid were added to the washed microbubble dispersion to a final cross-linker concentration of 1 mg/mL. After addition of the cross-linker, the microbubble dispersions were stirred for 1 h before further use. We tested the samples in such a sequence that a possible effect of the cross-linking time before measurement was eliminated. 2.4. Stability against Buckling at Different Pressures. 2.4.1. Turbidity of Microbubble Samples after Pressure Treatment. A microbubble dispersion made with BSA batch 100M1900V contained only microbubbles and no protein aggregates. As a consequence, microbubble instability caused by induced pressure could be observed by the transparency of the dispersion. Samples of 1 mL microbubble dispersion (unwashed, washed, and washed after cross-linking treatment) were transferred to 2 mL vials. These vials were placed in a pressure vessel without screw caps. In the vessel, gas overpressures of 0, 100, 200, 300, 400, and 470 kPa were applied for 15 s.It was assumed that the pressure inside the microbubble dispersion was equal to the pressure in the vessel. After the 15 s of compression, the vessel was decompressed, and photographs of the vials were taken. The transparency of the samples was quantified as the light transmission at a height of 120 mm measured with a dispersion analyzer (LUMiFuge, LUM GmbH, Berlin, Germany) at a speed of 200 rpm (∼4.7 g). 2.4.2. Quantification of the Effect of Pressure on the Disappearance of Microbubbles. Microscope (Axioskop, Carl Zeiss AG, Oberkochen, Germany) images (40× magnification) of 3.52 × 10−7 mL of the microbubble dispersions after the pressure treatment were taken with a camera (Axiocam HRc, Carl Zeiss AG, Oberkochen, Germany). From the microscopic picture, the number of microbubbles per volume was estimated by image analysis using ImageJ (1.44n, National Institutes of Health, Bethesda, MD). The stability of the microbubbles was defined as the number of microbubbles after pressurizing divided by the number of microbubbles before pressurizing. If a decay was observed, the data points were fitted to a sigmoid function: y = y0 + (y0 − y∞)/(1 + e−k(P−Pc)), in which y = N/N0, y0 = y (P0), and y∞ = y(P∞). 2.5. Stability against Buckling at Different Heating Temperatures. 2.5.1. Quantification of the Effect of Heating on the Disappearance of Microbubbles. Samples of 1 mL of microbubble dispersion (unwashed, washed, and washed after cross-linking treatment) were transferred to 8 mL glass tubes. The glass tubes were placed in an oil bath. After a preheating phase of 3 min (time required to reach the desired temperature), the samples were kept in an oil bath for 1 or 2 min at the following temperatures: 30, 50, 75, 100, and 120 °C. The microbubble dispersions were then taken out of the oil bath and cooled to ambient temperature. Subsequently, microscope (Axioskop, Carl Zeiss AG, Oberkochen, Germany) images (40× magnification) of 3.52 × 10−7 mL of the heated microbubble dispersions were taken with a camera (Axiocam HRc, Carl Zeiss AG, Oberkochen, Germany). From the microscopic picture, the number of microbubbles per volume was estimated by image analysis using ImageJ (1.44n, National Institutes of Health, Bethesda, MD). The stability of the microbubbles was defined as the number of microbubbles after heat treatment divided by the number of microbubbles before heat treatment. If a decay was observed, the data points were fitted to a sigmoid function: y = y0 + (y0 − y∞)/(1 + e−k(T−Tc)), in which y = N/N0, y0 = y(T0), and y∞ = y(T∞). 2.5.2. Heating of Samples Visualized with Light Microscope. Washed microbubble dispersions were placed on an object glass, covered with cover glass, and sealed using a 125 μL Gene Frame adhesive system (Thermo Fisher Scientific, Waltham, MA, USA). The object glasses were placed in a hot-stage (LTS120, Linkam Scientific Instruments, Surrey, United Kingdom) that was placed under a microscope (Axioskop 2 Plus, Carl Zeiss AG, Oberkochen, Germany). The temperature of the hot-stage increased from 20 to 90 °C with a heating rate of 1 °C/min. Every 5 min a picture was taken using a

2. MATERIALS AND METHODS 2.1. Materials. Bovine serum albumin (BSA, fraction V, lot no.: 100M1900V and SLBC8307V), glutaraldehyde (GA), tannic acid (TA), sodium azide, rhodamine-B, and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA). For all experiments, demineralized water was used. 2.2. Production of Microbubble Dispersions. Microbubbles were produced as previously described.17 In short, BSA was dissolved in demineralized water at a concentration of 5% (w/w), and the solution was stirred for at least 2 h. The pH of the solution was adjusted to 6.0 by the addition of 2.0 M HCl and 0.2 M HCl. The solution was divided into portions of 25 mL, which were transferred to 50 mL beakers. These specimens were heated for 10 min in a 55 °C water bath and subsequently sonicated for 3 min at power level 8 using a Branson 450 sonicator (Branson Ultrasonics Corporation, Danbury, CT) with a 20 kHz, 12.7 mm probe. The probe was placed at the air− water interface of the solution. When protein lot no. 100M1900V was used, samples were sonicated applying a duty cycle of 30%, but when protein lot no. SLBC8307V was used, a 60% duty cycle was applied. We reported earlier17 that sonicating BSA samples with a 30% duty cycle only resulted in microbubbles when protein lot no. 100M1900V was used, whereas for other batches, a higher duty cycle was required to obtain microbubbles. 2.3. Chemically Cross-Linking the Proteins in the Microbubble Shell. We chemically cross-linked the protein in the microbubble to change the microbubble shell properties. Before adding the cross-linking agents, the microbubble dispersion was washed to diminish the excess protein. After preparation, the microbubble dispersions were cooled to ambient temperature and diluted with demineralized water (top layer:water = 1:1 v/v). Subsequently, the dispersions were centrifuged for 30 min at 300g. The top layer, mainly consisting of microbubbles, was collected and 334

DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

Research Article

ACS Applied Materials & Interfaces

Figure 1. Samples containing microbubble dispersions pressurized in the pressure vessel: from left to right, increasing order of applied overpressure from 0 to 470 kPa (a). Light transmission of the microbubble dispersions as a function of the applied overpressure (b). camera (Axiocam ERc 5s, Carl Zeiss AG, Oberkochen, Germany) connected to the microscope. In all experiments, the microbubbles were in (aqueous) solution, and it was assumed that the water vapor pressure was in equilibrium with the solution. 2.6. Fluorescence Microscopy after Pressure and Heat Treatment. After preparing microbubbles as described in section 2.2, rhodamine-B was added to the microbubble dispersion to a final concentration in the solution of 0.003%. Subsequently, the microbubble dispersions were washed as described in section 2.3. To part of the washed dispersions glutaraldehyde (final concentration 0.1%) was added and to another part no cross-linking agent was added. Both type of samples were stirred for 1 h. Subsequently, they were placed in a pressure vessel to which 200 and 4.7 kPa overpressure was applied for 15 s. After decompressing the pressure vessel, the microbubble dispersions were stored at 4 °C. Micrographs of these dispersions were made with a camera (Axiocam ICc 3, Carl Zeiss AG, Oberkochen, Germany) attached to a fluorescence light microscope (Axioskop, Carl Zeiss AG, Oberkochen, Germany) illuminated with a 100 W halogen lamp. Similarly, micrographs of the dispersions, heated to 75 and 120 °C, were made using fluorescence light microscopy. 2.7. Stability against Rupture at Different Storage Temperatures. After preparation, a microbubble dispersion from BSA sample 100M1900V was cooled to ambient temperature and subsequently transferred to 20 mL vials and stored in climate chambers at 10 and 15 °C in a refrigerator (4 °C) and a thermostated lab (21 °C). At predetermined time intervals, microscope (Axioskop, Carl Zeiss AG, Oberkochen, Germany) images (40× magnification) of 3.52 × 10−7 mL of the microbubble dispersion were taken with a camera (Axiocam HRc, Carl Zeiss AG, Oberkochen, Germany). From the microscope pictures, the number of microbubbles per volume was estimated by image analysis using ImageJ (1.44n, National Institutes of Health, Bethesda, MD). The portion of intact microbubbles per volume was plotted as a function of the storage time for the different storage temperatures. 2.8. Scanning Electron Microscopy. The microbubbles from a washed microbubble dispersion without cross-linker were allowed to adhere on a surface of poly-lysine-coated glass and were subsequently submerged in glutaraldehyde to increase fixation to the glass. Thereafter, the samples were put in acetone and air-dried. The airdried samples were sputtered with platinum or iridium to form a layer of approximately 10 nm. The samples were examined with an FEI Maggalan 400 (Hillsboro, OR, USA) electron microscope at ambient temperature operating at 2 kHz.

Figure 2. N/N0 as a function of the applied overpressure (N and N0: number of microbubbles per volume after and before the pressure treatment, respectively). The dotted line data corresponds to a fitted sigmoid function with fitting parameters y0 = 0.07, y∞ = 0.04, k = 0.05 kPa−1, Pc = 140 kPa, and goodness of fit R2 = 0.90.

shows the relative number of microbubbles after pressure treatment as a function of the applied overpressure. It can be seen that the number of microbubbles decreased with increasing pressure. Dispersions to which a 200 kPa overpressure was applied contained hardly any microbubbles. We checked whether the stability could be increased by the addition of cross-linkers. The preparation of BSA gels in which the BSA molecules were cross-linked with glutaraldehyde has been reported.23 Furthermore, upon addition of the food-grade cross-linker tannic acid, protein films were shown to be strengthened, resulting in higher tensile strength and elongation at break.24 We added glutaraldehyde and tannic acid to our sample before pressure treatment. Figure 3 shows the relative number of microbubbles after the pressure treatment as a function of the applied overpressure. Samples to which a cross-linker was added did not show a decrease in the number of microbubbles upon increasing pressure. Apparently, the shell was reinforced by the crosslinkers, thereby making the microbubbles more stable during the pressure treatment as compared to microbubbles to which no cross-linking agent was added. Figure 4 shows the fluorescence micrograph of a reference sample and of dispersions containing cross-linked microbubbles that were pressurized for 15 s at 200 and 470 kPa overpressure. After disappearance of the microbubbles, remainders of the microbubbles were visible. The remainders appeared like collapsed microbubbles. These were seen as moon-shaped particles, some of which are indicated with an arrow in Figure 4. Because the shell remained intact and showed a collapsed

3. RESULTS AND DISCUSSION 3.1. Stability against Buckling at Different Pressures. With increasing applied pressure, the microbubble dispersions became more transparent, as can be seen in Figure 1. This decrease in turbidity was caused by the disappearance of the microbubbles upon pressure increase. To quantify this disappearance, the number of microbubbles was determined after pressurizing the washed microbubbles for 15 s. Figure 2 335

DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

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Figure 5. Scanning electron microscopy picture of a buckled microbubble.

Figure 3. N/N0 as a function of the applied overpressure for microbubble dispersions with added glutaraldehyde (circles) and tannic acid (squares). N and N0: number of microbubbles per volume after and before the pressure treatment, respectively. The horizontal lines (dotted: with added tannic acid; dashed: with added glutaraldehyde) serve as a guide to the eye and correspond to the average value.

means that the Young’s modulus of the shell was between 23 and 31 MPa. With the addition of cross-linkers (both tannic acid and glutaraldehyde), the microbubbles were stable up to the highest overpressure applied of 470 kPa. This implies that the elastic modulus of the shell was at least 52 MPa, which represents at least a 2-fold increase compared to the shell modulus without cross-linkers. High internal phase emulsions (HIPE) in which the emulsion droplets were covered with BSA nanoparticles were reported to show solid-like behavior.27 A common feature of HIPEs is that the elastic modulus of the HIPE is determined by the elastic modulus of the emulsion droplets, which in turn is determined by that of the interface shell. Upon addition of glutaraldehyde (12.5% as weight percentage of the protein weight), the storage (G′) and loss modulus (G″) of HIPE showed a 5-fold increase.27 In our study, the glutaraldehyde amount was 20% of the weight of the protein content. A 5-fold increase of the modulus (assumed to be at least 23 MPa) of our microbubbles would lead to a modulus that could withstand an overpressure of approximately 1.3 MPa. We investigated whether the microbubbles with added glutaraldehyde were also stable when pressurized for a longer period of time (1, 5, and 30 min). It was found that after 30 min at 200 kPa overpressure all of the microbubbles remained intact and that more than 50% of the microbubbles could withstand 30 min at 470 kPa. When we applied pressure on a microbubble dispersion by screwing a cap on a vial with an excess volume of microbubble dispersion, the dispersion turned from turbid to almost transparent (Figure 6). No difference in turbidity was observed when the dispersion was depressurized by unscrewing the cap.

morphology, we propose that as a result of pressure the microbubbles buckled. After buckling, the air of the microbubble dissolved into the surrounding solution. Figure 5 shows typical examples of buckled microbubbles observed in a former experiment using scanning electron microscopy. Pressure increase results in an increase in air solubility. Therefore, the air from the microbubbles will have a tendency to dissolve and leave the bubble. This will result in an external pressure on the bubble shell. This pressure can be estimated by the sum of the Laplace pressure and the applied overpressure. It has been reported that when this pressure exceeds a critical pressure, Pcrit, the shell will buckle25,26

Pcrit =

2E Bd 2 R2 3(1 − ν 2)

where d is the shell thickness (∼50 nm), ν is the Poisson ratio (assumed to be 0.5), R is the bubble radius (∼500 nm), and EB is the bulk Young’s modulus of the protein shell. On the basis of the parameters typical of our microbubbles, we obtained Pcrit/EB ≈ 0.013. The Laplace pressure was estimated to be 200 kPa assuming a surface tension of 50 mN m−1. In Figure 2, it can be seen that the number of microbubbles disappeared between 100 and 200 kPa overpressure, implying that the critical pressure has a value between 300 and 400 kPa. This

Figure 4. Fluorescence micrographs of a reference washed microbubble dispersion (a) and of dispersions that were pressurized for 15 s at 200 kPa overpressure (b) and for 15 s at 470 kPa overpressure (c). The light circles in the top panel represent the microbubbles; the dark irregular shapes in the middle and bottom panel represent the remainders of the shell after releasing air. The arrows indicate moon-shaped particles. 336

DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

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ACS Applied Materials & Interfaces

was large due to the inaccuracy of sampling and analysis inherent to the method used. However, the applied method was still much more accurate than others that are commonly used to measure the number of microbubbles in a microbubble suspension, such as turbidity measurements that cannot discriminate microbubbles from aggregates.17 However, despite the large error, we could still conclude that for temperatures up to 55 °C the number of microbubbles was comparable to the number of microbubbles at 20 °C. At temperatures above 55 °C, the number of microbubbles decreased by more than 50% (compared to the number at 20 °C). The temperatures at which the number of microbubbles decreased coincided with the temperature at which the microbubble volume increased. In Figure 8, we can also see that a large part of the remainder was bigger in size than the (air-filled) microbubbles. This suggests that the air disappeared after the microbubble expanded due to heating. We regard the microbubble as an air bubble for which the Laplace pressure is opposed by the stress in the microbubble shell. Three factors that influence the microbubble volume upon heating were considered: (1) thermal expansion following the ideal gas law, (2) an increase in the number of air molecules in the microbubble caused by a decrease of air solubility in water at elevated temperatures (air solubility decreases from 0.020 mL air/mL water at 20 °C to 0.015 mL air/mL at 75 °C), and (3) elastic stresses in the microbubble shell limiting microbubble expansion. Assuming that (i) the microbubble does not disproportionate, (ii) the shell modulus decreases by 40% upon a temperature increase from 20 to 75 °C,28 (iii) the difference in the total amount of dissolved air between an equilibrated dispersion at 20 °C and at elevated temperature of 75 °C will go to the microbubble, the contributions of the three factors described above on the increase in microbubble volume could be estimated using the ideal gas law and rheological properties of a sphere under a homogeneous (under)pressure.26 We found that the volume increase was ∼15% upon a temperature increase from 20 to 75 °C. From the pictures in Figure 8, it can be seen that, after the heat treatment and subsequent cooling, air had disappeared from the microbubble, leaving moon-shaped particles behind. Upon cooling, the volume of gas in the microbubble decreased again, mainly as a result of the increase in air solubility. The temperature of the microbubble decreased from 75 to 20 °C within approximately 2 s. It seems that the elastic modulus of the shell was too low to withstand the stresses due to the fast volume decrease of air, resulting in buckling of the shell quickly after cooling. The samples to which glutaraldehyde or tannic acid were added were also placed in an oil bath for 4 or 5 min at temperatures ranging from 30 and 120 °C. Figure 9 shows the relative number of microbubbles after heat treatment as a function of the treatment temperature for these samples. In contrast to the samples without cross-linkers, the samples with glutaraldehyde could be heated to 120 °C without a significant decrease in the number of microbubbles compared to the sample at 30 °C. As mentioned above, upon addition of glutaraldehyde, the modulus of the shell is expected to increase approximately 5×. A shell with increased elastic modulus is able to withstand higher pressures before it starts to buckle. Furthermore, an increased modulus restricts the microbubble extension and expansion. Not only the increased modulus but also the nature of the bonds between protein molecules make the microbubble less sensitive to temperature change. The interactions in the shell before cross-linking are of a

Figure 6. Bottle with an excess volume of microbubble dispersion (a) and the same bottle after it had been closed with a cap (b).

This implies that the microbubbles disappeared during compression and not during decompression. 3.2. Stability against Buckling upon Increasing Temperature. Washed microbubble samples to which no cross-linkers were added were heated for 4 and 5 min at temperatures ranging from 30 to 120 °C. Figure 7 shows the relative number of microbubbles as a function of the temperature during the heat treatment.

Figure 7. N/N0 as a function of the heating temperature. N and N0: number of microbubbles per volume after and before the heat treatment, respectively. Closed symbols: 4 min heating; open symbols: 5 min heating. The dotted (4 min) and dashed (5 min) lines correspond to a fitted sigmoid function with fitting parameters y0 = 0.9, y∞ = 0.01, k = 0.9 °C−1, and Tc = 76 °C and y0 = 0.8, y∞ = 0.01, k = 0.8 °C−1, and Tc = 75 °C for the dotted and dashed lines, respectively. The goodness of fit are R2 = 0.92 and 0.78, respectively.

It can be seen that the number of microbubbles did not change significantly when the microbubble dispersion was heated at 30 and 50 °C. However, when heated at 100 and 120 °C, all microbubbles disappeared. At 70 °C, the sample heated for 5 min lost more than half of the microbubbles, whereas the same sample heated for 4 min retained more than half of the microbubbles. After the air dissolved from the microbubbles, the protein shells remained in the suspension. This can clearly be seen in Figure 8, where we show fluorescence micrographs of washed microbubble dispersions heated for 4 min at 75 and 120 °C. To better understand how the microbubbles behave at elevated temperatures, a microbubble dispersion was heated under the microscope from 20 to 90 °C using a hot-stage. Micrographs were made after increasing temperature steps of 5 °C, and the number of microbubbles was estimated using image analysis (results not shown). The error in the measurements 337

DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

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ACS Applied Materials & Interfaces

Figure 8. Fluorescence micrographs of washed microbubble dispersions heated for 4 min at 75 (a) or 120 °C (b). The light circles in panel (a) represent the microbubbles, and the dark irregular shapes in both panels represent the remainder of the shell after releasing air. The arrows indicate moon-shaped particles.

Figure 9. N/N0 as a function of the heating temperature for samples with glutaraldehyde (a) and tannic acid (b) added. N and N0: number of microbubbles per volume after and before the heat treatment, respectively. Closed symbols: 4 min heating; open symbols: 5 min heating; asterisks: precipitation. In panel (a), no clear trend is observed, and the horizontal lines (dotted: 4 min; dashed: 5 min) serve as a guide to the eye and correspond to the average value. In panel (b), the dotted (4 min) and dashed (5 min) lines correspond to a fitted sigmoid function with fitting parameters y0 = 0.9, y∞ = 0.002, k = 0.3 °C−1, and Tc = 74 °C and y0 = 0.9, y∞ = 5 × 10−7, k = 1 °C−1, and Tc = 74 °C for the dotted and dashed lines, respectively. The goodness of fits are R2 = 0.74 and 0.70, respectively.

hydrophobic nature.1 Addition of glutaraldehyde results in covalent bonds, which are less sensitive to temperature. For an elastic modulus increased by a factor of 5 and similar assumptions as mentioned above, the volume of the microbubbles was expected to increase by approximately 3% instead of 15%. The results for the sample with tannic acid were comparable to those of the sample without cross-linkers. The samples with tannic acid heated to 100 and 120 °C formed large aggregates. It has been reported that, at high temperatures, when the degree of attachment of tannic acid to protein is high, complexes are formed and precipitate.29 3.3. Stability against Rupture at Different Storage Temperatures. Microbubble dispersions were stored at different temperatures. In time, the number of microbubbles was measured. The results are shown in Figure 10. As reported previously,17 at higher storage temperatures the number of microbubbles decreased faster. In Figure 10, we see that the stability of samples stored at 10 and 15 °C was higher than that of samples stored at 21 °C and lower than that of samples stored at 4 °C. The difference between the samples stored at 10 and 15 °C was small. We plotted the natural logarithm of the average rupture time (defined as tb = Σn1(ΔNn· tn)/Σn1ΔNn, in which ΔNn = Nn − Nn−1, with Nn and Nn−1 as the number of microbubbles per volume at time tn and tn−1) as a function of the inverse storage temperature (Figure 11).

Figure 10. N/N0 as a function of the storage time (N and N0: number of microbubbles per volume after and before the storage, respectively) for different storage temperatures of 4 °C (diamonds), 10 °C (triangles), 15 °C (squares), and 21 °C (circles). The dotted lines serve as guides to the eye and correspond to fitted Weibull functions (Rovers et al.1). The values of the goodness of fits were R2 > 0.97 for all storage temperatures.

Assuming tb ∼ eEact/kT, with Eact the activation energy to break a molecular bond in the shell, we found from the slope of Figure 11 that Eact = 27 kT. Transferring this value into an energy per characteristic molecular volume (4 × 10−27 m3) leads to a value of 25 MPa. This compares reasonably well with 338

DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

Research Article

ACS Applied Materials & Interfaces

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Figure 11. Natural logarithm of the average breaking time, tb, as a function of the inverse temperature (1/T). The dotted line corresponds to a fitted linear function (goodness of fit R2 = 0.98).

the Young’s modulus of 23−30 MPa estimated on the basis of the expression of the critical pressure for buckling.

4. CONCLUSIONS The stability of protein microbubbles upon pressure increase depends on the elastic modulus of the microbubble shell. A temperature decrease after heating also results in an increase of pressure on the microbubble due to the fast volume decrease of the enclosed air. When the pressure exceeds a certain critical value, the microbubble shell buckles. The critical value is related to the elastic modulus of the shell at buckling. Cross-linking of the proteins in the shell causes an increase in the elastic modulus of the microbubble shell and significantly increases the microbubbles stability. Similarly, the increased microbubble stability as a result of a decrease in storage temperature can also be explained by an increase in the shell elastic modulus. The elastic modulus of the shell at buckling could be estimated from the activation energy of rupture, which could be obtained from the average rupture time of the microbubbles as a function of the storage temperatures.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +31 317 480165, e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340

Research Article

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DOI: 10.1021/acsami.5b08527 ACS Appl. Mater. Interfaces 2016, 8, 333−340