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Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel
Ultrasonics Sonochemistry xxx (2015) xxx–xxx
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Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel Zhenghua Huang, Yanping Cao ⇑, Duoxia Xu, Chao Wang, Dandan Zhang Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Technology & Business University (BTBU), Beijing 100048, China Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), Beijing 100048, China
a r t i c l e
i n f o
Article history: Received 23 November 2014 Received in revised form 23 March 2015 Accepted 24 March 2015 Available online xxxx Keywords: Ultrasound Diffusion Casein Alginate–chitosan gel
a b s t r a c t The effects of ultrasound-assisted and pre-ultrasound treatment on the diffusion properties of casein imbedded by alginate–chitosan gel were investigated. The fluorescence spectrophotometry for determining the fluorescence intensity of casein was established to calculate the diffusion coefficient (De). Scanning electron microscope (SEM) was used to observe the microstructure of gel beads. The results showed that two different kinds of ultrasonic treatments had obvious distinctions on the casein diffusion. As the frequency increased, the value of De decreased from 28.56 104 m2 s1 (28 kHz) to 2.57 104 m2 s1 (135 kHz) during the ultrasound-assisted process. While, the minimum De of 8.6 104 m2 s1 was achieved at the frequency of 50 kHz for the pre-ultrasound treatment. The impact of power on the diffusion showed that De increased with the increase of ultrasound power until it reached the highest value 28.56 104 m2 s1 (0.45 W/cm2) in the ultrasound-assisted process. It would reach the maximum value (16 104 m2 s1) when the power was 0.25 W/cm2 in the pretreatment ultrasound process. SEM analysis exhibited that the gel structural changes (area ratio) were in accordance with De through different ultrasonic treatment. This was mainly due to the mechanical action and cavitation of the ultrasonic treatment. This study is important to explain the diffusion properties of large molecules and explore the mechanism of enzyme immobilization treated by ultrasound. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Enzyme immobilization is a novel technology in recent years and has attracted a wide range of interest from fundamental academic research to many different industrial applications [1]. Because of the simple preparation, mild experimental conditions and high enzyme activity, embedding immobilization becomes the most widely used in the food, pharmaceutical and biomedical application [2,3]. Many researchers have investigated the effect of embedded immobilization on enzyme properties. Di found that the gold nanoparticles and horseradish peroxidase embedded in silica sol–gel network on gold electrode surface in the presence of cysteine exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide, and the biosensor exhibited high sensitivity, rapid response, long-term stability and an excellent electrocatalytic response to the reduction of H2O2 without any mediator [4]. Wu showed that compared with free a-amylase, ⇑ Corresponding author at: No. 11, Fucheng Road, Beijing 100048, China. Tel./fax: +86 10 68985645. E-mail address: [email protected] (Y. Cao).
the immobilized a-amylase entrapped by agar retained a higher enzyme activity, good reusability and reaction stability, longer storage time and wide pH range [5]. However, the grid structure of polymer carriers may hinder the macromolecules’ diffusion and restrain the release of substance [6]. Wojcieszyn´ska reported that compared with the free state, the Km and mmax of epigallocatechin gallate dioxygenase immobilized by sodium alginate were 0.42% and 37.7%, the immobilized enzyme and substrate’s affinity increased 230 times, but the enzymatic reaction rate decreased more than 60%, which proved that the structural rigidity of sodium alginate gel blocked the substrate and the product of the reaction [7]. Therefore, to overcome the block is the essential problem of the embedding immobilization. The diffusion of macromolecules, such as casein, embedded with alginate–chitosan gel beads in liquid phase may be explained by a shrinking core model. Hsu showed that the resistance for the diffusion of solute molecules was mainly depended on the surface layer [8]. The mass transfer rate could be improved by increasing mass transfer coefficient, driving force and the contacting area [9].
http://dx.doi.org/10.1016/j.ultsonch.2015.03.015 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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At present, the research on the diffusion of protein in gels has received some progress. Gutenwik used diffusion cell to measure the effective diffusion coefficients of lysozyme and BSA [10]. Johnson studied the effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels by fluorescence recovery after photobleaching and gel chromatography. They concluded that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity are likely to result primarily from variations in equilibrium partitioning coefficients with only small contributions from the intramembrane diffusivity [11]. Ultrasound is able to produce cavitation, mechanical and heated effects, which may affect the mass transfer and diffusion of molecules in ultrasonic field [12]. Qin found that binary diffusion coefficient in the capillary was enhanced with the increase of ultrasonic power [13]. Luque–Garcia proved that ultrasonic cavitation turbulent effect caused the particles’ high speed oscillations and collision, therefore the cleaning effect of microjet and shockwave on the interfacial layer accelerated the mass transfer [14]. At present, the impact of ultrasound on macromolecular diffusion and the relationship between the microstructure of carrier and molecular diffusion were unclear. The objective of this paper was to study the effects of ultrasound-assisted and pre-ultrasound on the diffusion of casein in the alginate–chitosan gel beads by evaluating the diffusion coefficients and observing the changes of alginate–chitosan gel beads by the fluorescence spectrophotometry and scanning electron microscope techniques, to establish the relationship between the diffusion coefficients and the gel surface area ratios. 2. Materials and methods 2.1. Materials Sodium alginate (1.05–1.15 Pa s viscosity) was purchased from Tianjin City Guangfu Technology Development Co., Ltd. (Tianjin, China). Chitosan (80.0–95.0 deacetylated degree) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Anhydrous calcium chloride (>96% purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Casein (P13.5% total nitrogen) was purchased from Beijing Ao Bo Xing Bio-Tech. Co., Ltd. (Beijing, China). Three hydroxyme thyl aminomethane (Tris, P99.0% purity) was purchased from Sigma Chemical Co., Ltd. (Beijing, China). 2.2. Ultrasonic equipment An assemble ultrasonic bath system equipment with two sets of JXD-02 multi-frequencies processing system and the low temperature circulating water tank was employed (JXD-02, Beijing Jinxing Ultrasonic Equipment Technology Co., Ltd., China). The intensity of ultrasound energy could be varied at different levels by adjusting the output of frequency (28, 40, 50, 135 kHz) and power (0.05, 0.15, 0.25, 0.36, 0.45 W/cm2). The length, width and depth of the ultrasonic bath were 0.2, 0.2, and 0.15 m, respectively. This instrument is shown in Fig. 1. 2.3. Casein entrapment in the alginate–chitosan gel beads Alginate (0.57 g) and chitosan (0.03 g) were brought into good contact by dissolving them in 20 mL of 0.1 M Tris–HCl buffer solution at pH 7.0. The solutions were heated to ensure complete dispersion and dissolution. The heating temperature is 70 °C in 1 h and then cooled to 40 °C. After that, casein was added to the solution at a concentration of 10 mg/mL. Finally, the mixture was dropped into 0.6 M CaCl2 at 4 °C for 30 min to form the beads.
Fig. 1. Plane graph of assemble ultrasonic bath system equipment (1, Thermostatic water; 2, Ultrasonic bath; 3, Sample flask; 4 and 5, Ultrasonic power supply).
2.4. Ultrasonic treatment of casein in the alginate–chitosan gel beads Casein in the alginate–chitosan gel beads (5 g) was dissolved in 300 mL of 0.1 M Tris–HCl buffer solution at pH 7.0. The solution was then treated by the ultrasonic processor at different frequencies (28, 40, 50, 135 kHz), 0.45 W/cm2, and at different powers (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2), 28 kHz, at 50 °C for different times (10, 20, 30, 40, 50, 60 min), respectively. After ultrasonic treatment, casein concentration was determined). 2.5. Ultrasonic pretreatment of casein in the alginate–chitosan gel beads Alginate–chitosan gel beads which embedded casein were pretreated by ultrasound. The treatment conditions were the same with the above 2.4. Then put these alginate–chitosan gel beads into the new Tris–HCl buffer. Casein concentration was determined every 10 min until it reached 60 min. 2.6. Casein concentration measurement Casein concentration was measured by fluorescence spectroscopy (RF 5301, Shimadzu, Co., Ltd., Tokyo, Japan). The fluorescence intensity of the sample was determined with an excitation wavelength of 280 nm and an emission wavelength of 341 nm. For a given material, when the excitation wavelength, the emission wavelength and the thickness of liquid layer are fixed, the low concentration of casein can be calculated as the following equation:
F ¼kC
ð1Þ
where F is the fluorescence intensity, k is a coefficient and C is the casein concentration. 2.7. The calculation of casein diffusion coefficient According to Estapé [15], the cumulative diffusion coefficient of casein in the alginate–chitosan gel beads can be expressed by the following equation: 2 1 X C ðtÞ 6 a ð1 þ aÞ De q2n t e R 1 ¼ 1 2 9þ9aþa C n¼1
ð2Þ
where C(t) is the concentration of release with different diffuse time. C1 means the total casein concentration. De is the effective diffusion coefficient. a is defined as the ratio of buffer volume to the volume of alginate–chitosan gel beads (a = 45.87). The number of gel beads is indicated by n = 100. R means the radius of each alginate–chitosan gel bead which is 0.25 mm.
a¼4
V
p R3 n 3
ð3Þ
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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qn is the non-zero positive solution of Eq. (4):
tan qn ¼
3 qn 3 þ a q2n
ð4Þ
After calculate Eq. (4), q 0.45
C ðtÞ In 1 1 ¼ 0:0016 De t þ 3:4 C
ð5Þ
Table 1 Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the ultrasound-assisted treatment process. Frequency (kHz)
Equations
De (104 m2 s1)
R2*
28 40 50 135
y = 0.045x + 0.294 y = 0.032x + 0.776 y = 0.014x + 0.315 y = 0.004x + 0.174
28.56 20.50 8.81 2.57
0.964 0.888 0.952 0.898
* Coefficient of determination: Used to indicate the degree of fitting the regression line and sample observation value. Values range from 0 to 1 closer to 1 better fitting degree. Better fitting degree of the independent variable on the dependent variable to explain the higher degree.
2.8. Scanning electron microscope (SEM) characterization To observe the effect of ultrasonic frequency and power on the microstructure of alginate–chitosan gels, the alginate–chitosan gels were dried after by exposure in the dryer for 24 h and then was observed by using VEGA//LSU scanning electron microscope (Tescan, Czechoslovakia), magnification was chosen as 100. The software of Image Pro Plus was applied to analysis the SEM professional image. The main parameter of the gels was Area Fraction. The function of the Image–Adjust–Threshold was used to calculated the Area Fraction. 3. Results and discussion 3.1. Effect of ultrasound-assisted treatment on the diffusion of casein in the alginate–chitosan gel beads 3.1.1. Effect of ultrasonic frequency on the diffusion coefficients of casein by the ultrasound-assisted treatment The value of De is an important parameter of the diffusion process. Casein diffused into the buffer induced by ultrasound-assisted treatment could result in the change of De. Fig. 2 shows the plots of h i C versus t during the ultrasound-assisted treatment at difIn 1 CðtÞ 1 2
ferent frequencies with a same ultrasound power 0.45 W/cm . It h i C had a linear relationship with the can be seen that the In 1 CðtÞ 1 treated time at various frequencies. This result demonstrated that the kind of diffusion process followed first-order kinetics. The parameters De was determined from the slope by plotting h i C against t were listed in Table 1. The De was obtained In 1 CðtÞ 1
treatment at 28, 40, 50, 135 kHz, respectively. It was shown that the diffusion coefficient of casein was decreased gradually with the increase of ultrasonic frequency. Therefore, ultrasonic frequency had a suppressing effect on the diffusion and the lower frequency ultrasonic field was more effective for the diffusion of casein molecules through alginate–chitosan gel. According to Gogate, the influence of ultrasonication on the molecular diffusion was extremely dependent on the cavitation effect [16]. Ultrasonic cavitation has two effects on accelerated diffusion. On one hand, the ultrasonic cavitation could further crush gel particles, and increases the reaction, then accelerates the mass transfer. On the other hand, micro jet caused by ultrasonic cavitation can make the surface of solid erosion, thereby it makes the casein which accumulating around the gel beads spread to the surrounding solution [17]. The Increase of the ultrasonic frequency might make the wave expansion phase time become shorter and the cavitation bubble collapse before it shrinked, leading to the inhibitory effect on the De [18]. 3.1.2. Effect of ultrasonic power on the diffusion coefficients of casein by the ultrasound-assisted treatment The ultrasonic power is an important factor to be considered h i C during casein diffusion. Fig. 3 shows the plots of the In 1 CðtÞ ver1 sus the time during the ultrasound-assisted process at different h i C power. The results indicated that the In 1 CðtÞ versus t plots at 1
according to the calculating formula were 28.56 10 , 20.50 104, 8.81 104, 2.57 104 m2 s1 after ultrasound-assisted
all the ultrasound-assisted powers including 0.05, 0.15, 0.25, 0.35, 0.45 W/cm2 followed linear relationships, as determined by the R2 values from 0.881 to 0.964. The De of casein were listed in Table 2 which showed that De of casein were 5.00 104, 15.19 104, 16.25 104, 17.13 104,
Fig. 2. Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the ultrasound-assisted treatment process.
Fig. 3. Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the ultrasound-assisted treatment process.
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Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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Table 2 Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the ultrasound-assisted treatment process. Power (W/cm2)
Equations
De (104 m2 s1)
R2
0.05 0.15 0.25 0.35 0.45
y = 0.008x + 0.461 y = 0.024x + 0.465 y = 0.026x + 0.419 y = 0.027x + 0.616 y = 0.045x + 0.294
5.00 15.19 16.25 17.13 28.56
0.957 0.959 0.881 0.902 0.964
28.56 104 m2 s1 after ultrasound-assisted treatment at 0.05, 0.15, 0.25, 0.35, 0.45 W/cm2, respectively. It can be seen that the De of casein molecules increased gradually with the increase of ultrasonic power. Therefore, the diffusion of casein was more effective in the ultrasonic field with high energy. This finding could be explained by the fact that the high intensity ultrasound could lead the polymers degradation. This is because the ultrasound accelerates the attrition between the solvent molecules and polymers molecules, so that C–C bond fraction. At the same time, high temperature and high pressure which produced by ultrasonic cavitation make the chemical bond break. Based on the above reasons, the structure of alginate–chitosan gel beads become loose, casein spread to the surround fluid environment easily, which makes De to increase [19]. Similarly, Makuta et al. reported that the bubbles produced by ultrasound were collapsed into microbubbles with diameters less than 100 lm. The number of microbubbles were increased with the increase of ultrasonic power, which improved the cavitation effect leading to the increase of the mass transfer and enhancement of the indigo carmine degradation [20].
polymer material’s C–C bond for the outer layer of the gel bead is opened, gel bead’s surface layer could be degraded gradually, which prompted the ultrasound plays another role, make the C–C bond together again [23]. As the frequency increases from 28 kHz to 50 kHz, this phenomenon is more obvious. It makes the gel beads gather. Then put the gel bead into the new buffer, diffusion of casein becomes difficult. When the pre-ultrasound treatment frequency reaches 135 kHz, the effect of ultrasound make the gel bead separation. So put the gel bead into the buffer, diffusion of casein increase again. 3.2.2. Effect of ultrasonic power on the De of casein by ultrasonic pretreatment The effect of ultrasonic power on the De of casein by ultrasonic pretreatment was shown in Table 4. In Fig. 5, it is shown that the plots of the versus the time during the ultrasonic pretreatment process at different power has a liner relationship. It showed that the De of casein were 8.9 104, 14.3 104, 16 104, 15.6 104, 15.4 104 m2 s1 after ultrasonic pretreatment at
3.2. Effect of pre-ultrasound treatment on the diffusion of casein in the alginate–chitosan gel beads 3.2.1. Effect of ultrasonic frequency on the diffusion coefficients of casein by ultrasonic pretreatment There are two different ultrasonic treatment methods including ultrasonic-assisted treatment and ultrasonic pretreatment. Because of the wide application in the food industry, it is necessary to study the impact of ultrasonic pretreatment on the molecular diffusion. In this work, the effect of ultrasonic pretreatment on the De of casein in alginate–chitosan gel was studied and the results were shown in Table 3. The plots of the versus the time during the ultrasonic pretreatment process with different frequencies is shown in Fig. 4. After ultrasonic pretreatment at 28, 40, 50, 135 kHz, the diffusion coefficients De of casein were 15.4 104, 14.1 104, 8.6 104, 13.6 104 m2 s1, respectively. It was found that the De of casein molecules decreased with the increased ultrasonic frequency. When samples were pretreated at 50 kHz, the minimum De was observed. While, it was interesting to note that the De increased with the ultrasonic pretreatment at 135 kHz. Adulkar reported that ultrasonic treatment at low frequency created weak ultrasound scattering effect [21]. According to Huang’s research, the material degradation yield increased with the increased frequency in the range of 20–500 kHz [22]. In the pre-ultrasound treatment process,
Fig. 4. Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the pre-ultrasound treatment process.
Table 3 Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the pre-ultrasound treatment process. Frequency (kHz)
Equations
De (104 m2 s1)
R2
28 40 50 135
y = 0.024x 0.075 y = 0.022x + 0.019 y = 0.013x + 0.041 y = 0.021x + 0.006
15.4 14.1 8.6 13.6
0.986 0.981 0.992 0.990
Fig. 5. Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the pre-ultrasound treatment process.
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
Z. Huang et al. / Ultrasonics Sonochemistry xxx (2015) xxx–xxx
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Table 4 Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the pre-ultrasound treatment process. Power (W/cm2)
Equations
De (104 m2 s1)
R2
0.05 0.15 0.25 0.35 0.45
y = 0.014x + 0.272 y = 0.022x + 0.169 y = 0.025x -0.066 y = 0.025x + 0.718 y = 0.024x 0.075
8.9 14.3 16 15.6 15.4
0.885 0.916 0.956 0.678 0.986
0.05, 0.15, 0.25, 0.35, 0.45 W/cm2, respectively. It should be noted that the power of the ultrasonic pretreatment significantly influenced the diffusion property of casein in the gel. With the increase of power value, the De increased initially and then decreased at higher power values. The ultrasonic pretreatment at 0.25 W/cm2 showed the fastest De. It was concluded that proper intensity of ultrasonic pretreatment was prone to diffusing the casein probably due to the increase of the local temperature and pressure in the surrounding area of collapsing bubble produced by the cavitation effect. However, the high-intensity ultrasonic pretreatment might induce the aggregation and polymerization of the alginate–chitosan gel which hindered the diffusion of casein. Similarly, Bashari et al. reported that maximum loading efficiency of calcium alginate beads was observed when ultrasonic power was 40 W/ cm2. When the ultrasonic intensity exceeded 40 W/cm2, the loading efficiency of dextranase decreased gradually with an increase of the ultrasonic power. The above mentioned behaviors were due to a formed more homogeneous system. Meanwhile, it could be explained that transient cavitation also resulted in strong
Fig. 7. Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the area ratio of alginate–chitosan gel.
acoustic streaming and reduced the mass transfer property [24]. The difference between treatment and pretreatment: the grid structure of gel particles were opened, after the pre-treatment. It is conducive to the dissolution of casein. In addition, the De of pre-ultrasound treatment has a smaller difference with ultrasound-assisted treatment in different ultrasonic power.
Fig. 6. SEM micrographs (100) of alginate–chitosan gel by different frequencies of ultrasonic pretreatment at 0.45 W/cm2, 50 °C for 60 min. The ultrasonic frequencies were (A) 28 kHz, (B) 40 kHz, (C) 50 kHz, (D) 135 kHz, respectively.
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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Fig. 8. SEM micrographs (100) of alginate–chitosan gel by different power of ultrasonic pretreatment at 28 kHz, 50 °C for 60 min. The ultrasonic power were (A) 0.05 W/ cm2, (B) 0.15 W/cm2, (C) 0.25 W/cm2, (D) 0.35 W/cm2, (E) 0.45 W/cm2, respectively.
Considering from the viewpoint of energy saving, ultrasonic pretreatment has more broad prospects in industrial production. 3.3. Microstructure of alginate–chitosan gel by ultrasonic pretreatment 3.3.1. Effect of the ultrasonic frequency To obtain the microstructure of alginate–chitosan gel by ultrasonic pretreatment, the dried samples were detected by SEM with
100 magnification. SEM micrographs of alginate–chitosan gel by ultrasonic pretreatment at different frequency (28, 40, 50, 13 5 kHz) and 0.45 W/cm2, 50 °C for 60 min were presented in Fig. 6. After treatment at 28 kHz, the alginate–chitosan gel showed a rather uneven surface. With the increase of the frequency, the extent and amount of the uneven surfaces were gradually reduced. When treated frequency was 135 kHz, the amount of the uneven surface of the gel became much more and denser. Meanwhile, the 135 kHz treated sample showed quite numerous
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
Z. Huang et al. / Ultrasonics Sonochemistry xxx (2015) xxx–xxx
small wrinkles. Therefore, 135 kHz treatment showed an obviously different microstructure compared with other treatment. Ultrasonic cavitation prompted the interaction between the liquid molecules and gel beads, which made the surface of the gel bead produced many fold. With the increase of frequency, cavitation effect gradually weakened, which made the attrition between buffer and the gel beads surface also decreased, resulting in the amount of surface folds down. When the frequency reached 135 kHz, ultrasonic became extremely strong and caused damage to the surface of the gel structure [25]. In order to further analysis the microstructure, the software of Image Pro Plus was applied to analysis the SEM professional image. The main parameters of the gels were total area and area fraction. The function of the Image–Adjust–Threshold was used to calculate the area fraction. The area ratio of alginate–chitosan gel as a function of the treated ultrasonic frequency was shown in Fig. 7. After treatment at 28 kHz, the alginate–chitosan gel showed an area ratio of 15.42%. With the increase of ultrasonic frequency, the area ratio of alginate–chitosan gel decreased initially and then increased at 135 kHz. This conclusion was in good agreement with the previous diffusion results. The higher the area ratio was, the bigger the De would be. It indicated that the diffusion was impacted by the alginate–chitosan gel surface.
3.3.2. Effect of ultrasonic power The impact of ultrasonic power on the microstructure of alginate–chitosan gel was also investigated. Fig. 8 showed the SEM micrographs of alginate–chitosan gel at varying ultrasonic power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2) and 28 kHz, 50 °C for 60 min. It was found that the extent and amount of the uneven surfaces were gradually increased but then reduced with the increase of the ultrasonic power. When ultrasonic power was 0.25 W/cm2, a maximum amount of the uneven surface was found. Also, as shown, excess energy brought less changes of the gel surface. With the increase of ultrasound power, the friction between gel bead and liquid became more and more acute, which made the amount of fold on the gel bead increased. But when the power of ultrasound excessed a critical value, the layer of gel bead was degraded, until the gel bead’s structure became loose, which makes the number of fold of gel bead decreased [26]. The area ratio of alginate–chitosan gel as a function of the treated ultrasonic power was shown in Fig. 9. With the increase of
Fig. 9. Effect of different ultrasonic powers with 28 kHz frequency on the area ratio of alginate–chitosan gel.
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ultrasonic power, the area ratio of alginate–chitosan gel increased initially and decreased at high power values. The area ratio showed a maximum of 37.22% at 0.25 W/cm2. It was interesting to find that a close relationship existed between microstructure and diffusion property for the effect of ultrasonic power. Alginate–chitosan gel undergoing much more microstructure changes at the surface tended to exhibit a higher De due to the proper energy input by ultrasound. 4. Conclusion Ultrasound treatment of alginate–chitosan was confirmed to impact the diffusion of casein. The effects of ultrasound conditions on the casein diffusion were different for the two different treatment method, ultrasound-assisted and pre-ultrasound. The treatment condition of the optimum De of casein was 28 kHz, 0.45 W/ cm2 for the ultrasound-assisted treatment. While, the optimum pre-ultrasound treatment condition was 28 kHz, 0.25 W/cm2. The uneven surfaces of alginate–chitosan gel were observed after ultrasound treatment. It was interesting to find that there was a close relationship between micro structure and diffusion property for the effect of ultrasonic frequency and power. The mechanism of molecular diffusion impact by ultrasound should be examined further and this study is under way and will be reported in the near future. Acknowledgements This research was funded by the National Key Technology R&D Program of the National Natural Science Foundation of China (31371722). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ultsonch.2015.03. 015. References [1] Q. Li, Study of application and development of immobilization technique in food industry, Sichuan Food Ferment 132 (2006) 16–19. [2] Z.N. Yin, Immobilized enzyme and its research progress in food and biological applications, Life Sci. Instrum. 7 (2009) 11–15. [3] H.Z. Liu, Y.Y. Zhang, G.Z. Zhang, B.H. Niu, Research progress on preparation of immobilized enzyme, Chem. Ind. Eng. 30 (2009) 21–23. [4] J.W. Di, C.P. Shen, S.H. Peng, Y.F. Tu, S.J. Li, A one-step method to construct a third-generation biosensor based on horseradish peroxidase and gold nanoparticles embedded in silica sol–gel network on gold modified electrode, Anal. Chim. Acta 553 (2005) 196–200. [5] H.L. Wu, L.N. Wei, Z.Q. Zhou, G.X. Yuan, Z. Yan, H.P. Pan, K.Y. Lin, X.B. Jiang, Y.Q. Chen, W.F. Hu, Optimization of immobilization conditions for protease and its application in the fermentation of soy sauce, Mod. Food Sci. Technol. 29 (2003) 1080–1084. [6] P.X. Jia, X.Y. Liu, J.L. Liu, L.M. Jiao, K.Y. Tang, Immobilized enzyme and its application in fields including chemical engineering, Leather Sci. Eng. 14 (2004) 31–37. [7] D. Wojcieszyn´ska, K. Hupert-Kocurek, A. Jankowska, U. Guzik, Properties of catechol 2,3-dioxygenase from crude extract of Stenotrophomonas maltophilia strain KB2 immobilized in calcium alginate hydrogels, Biochem. Eng. J. 66 (2012) 1–7. [8] W.L. Hsu, M.J. Lin, J.P. Hsu, Dissolution of solid particles in liquids: a shrinking core model, World Acad. Sci. Eng. Technol. 3 (2009) 801–805. [9] Z.P. Zhao, C.H. Chen, Mass transfer processes by using ultrasonic, Chem. Des. 6 (1997) 30–35. [10] J. Gutenwik, B. Nilsson, A. Axelsson, Determination of protein diffusion coefficients in agarose gel with a diffusion cell, Biochem. Eng. J. 19 (2004) 1–7. [11] E.M. Johnson, D.A. Berk, R.K. Jain, W.M. Deen, Diffusion and partitioning of proteins in charged agarose gels, Biophys. J. 68 (1995) 1561–1568. [12] T.J. Mason, J.P. Lorimer, Applied Sonochemistry: The Uses of Power Ultrasound in Chemistry and Processing, Wiley VCH Weinheim, Germany, 2002. [13] W. Qin, Y. Zhang, Y.Y. Dai, Ultrasonic field effect on electrolyte diffusion coefficient in the capillary, J. Tsinghua Univ. (Nat. Sci. Ed.) 41 (2001) 41–43.
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Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel Zhenghua Huang, Yanping Cao ⇑, Duoxia Xu, Chao Wang, Dandan Zhang Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Technology & Business University (BTBU), Beijing 100048, China Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), Beijing 100048, China
a r t i c l e
i n f o
Article history: Received 23 November 2014 Received in revised form 23 March 2015 Accepted 24 March 2015 Available online xxxx Keywords: Ultrasound Diffusion Casein Alginate–chitosan gel
a b s t r a c t The effects of ultrasound-assisted and pre-ultrasound treatment on the diffusion properties of casein imbedded by alginate–chitosan gel were investigated. The fluorescence spectrophotometry for determining the fluorescence intensity of casein was established to calculate the diffusion coefficient (De). Scanning electron microscope (SEM) was used to observe the microstructure of gel beads. The results showed that two different kinds of ultrasonic treatments had obvious distinctions on the casein diffusion. As the frequency increased, the value of De decreased from 28.56 104 m2 s1 (28 kHz) to 2.57 104 m2 s1 (135 kHz) during the ultrasound-assisted process. While, the minimum De of 8.6 104 m2 s1 was achieved at the frequency of 50 kHz for the pre-ultrasound treatment. The impact of power on the diffusion showed that De increased with the increase of ultrasound power until it reached the highest value 28.56 104 m2 s1 (0.45 W/cm2) in the ultrasound-assisted process. It would reach the maximum value (16 104 m2 s1) when the power was 0.25 W/cm2 in the pretreatment ultrasound process. SEM analysis exhibited that the gel structural changes (area ratio) were in accordance with De through different ultrasonic treatment. This was mainly due to the mechanical action and cavitation of the ultrasonic treatment. This study is important to explain the diffusion properties of large molecules and explore the mechanism of enzyme immobilization treated by ultrasound. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Enzyme immobilization is a novel technology in recent years and has attracted a wide range of interest from fundamental academic research to many different industrial applications [1]. Because of the simple preparation, mild experimental conditions and high enzyme activity, embedding immobilization becomes the most widely used in the food, pharmaceutical and biomedical application [2,3]. Many researchers have investigated the effect of embedded immobilization on enzyme properties. Di found that the gold nanoparticles and horseradish peroxidase embedded in silica sol–gel network on gold electrode surface in the presence of cysteine exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide, and the biosensor exhibited high sensitivity, rapid response, long-term stability and an excellent electrocatalytic response to the reduction of H2O2 without any mediator [4]. Wu showed that compared with free a-amylase, ⇑ Corresponding author at: No. 11, Fucheng Road, Beijing 100048, China. Tel./fax: +86 10 68985645. E-mail address: [email protected] (Y. Cao).
the immobilized a-amylase entrapped by agar retained a higher enzyme activity, good reusability and reaction stability, longer storage time and wide pH range [5]. However, the grid structure of polymer carriers may hinder the macromolecules’ diffusion and restrain the release of substance [6]. Wojcieszyn´ska reported that compared with the free state, the Km and mmax of epigallocatechin gallate dioxygenase immobilized by sodium alginate were 0.42% and 37.7%, the immobilized enzyme and substrate’s affinity increased 230 times, but the enzymatic reaction rate decreased more than 60%, which proved that the structural rigidity of sodium alginate gel blocked the substrate and the product of the reaction [7]. Therefore, to overcome the block is the essential problem of the embedding immobilization. The diffusion of macromolecules, such as casein, embedded with alginate–chitosan gel beads in liquid phase may be explained by a shrinking core model. Hsu showed that the resistance for the diffusion of solute molecules was mainly depended on the surface layer [8]. The mass transfer rate could be improved by increasing mass transfer coefficient, driving force and the contacting area [9].
http://dx.doi.org/10.1016/j.ultsonch.2015.03.015 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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At present, the research on the diffusion of protein in gels has received some progress. Gutenwik used diffusion cell to measure the effective diffusion coefficients of lysozyme and BSA [10]. Johnson studied the effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels by fluorescence recovery after photobleaching and gel chromatography. They concluded that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity are likely to result primarily from variations in equilibrium partitioning coefficients with only small contributions from the intramembrane diffusivity [11]. Ultrasound is able to produce cavitation, mechanical and heated effects, which may affect the mass transfer and diffusion of molecules in ultrasonic field [12]. Qin found that binary diffusion coefficient in the capillary was enhanced with the increase of ultrasonic power [13]. Luque–Garcia proved that ultrasonic cavitation turbulent effect caused the particles’ high speed oscillations and collision, therefore the cleaning effect of microjet and shockwave on the interfacial layer accelerated the mass transfer [14]. At present, the impact of ultrasound on macromolecular diffusion and the relationship between the microstructure of carrier and molecular diffusion were unclear. The objective of this paper was to study the effects of ultrasound-assisted and pre-ultrasound on the diffusion of casein in the alginate–chitosan gel beads by evaluating the diffusion coefficients and observing the changes of alginate–chitosan gel beads by the fluorescence spectrophotometry and scanning electron microscope techniques, to establish the relationship between the diffusion coefficients and the gel surface area ratios. 2. Materials and methods 2.1. Materials Sodium alginate (1.05–1.15 Pa s viscosity) was purchased from Tianjin City Guangfu Technology Development Co., Ltd. (Tianjin, China). Chitosan (80.0–95.0 deacetylated degree) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Anhydrous calcium chloride (>96% purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Casein (P13.5% total nitrogen) was purchased from Beijing Ao Bo Xing Bio-Tech. Co., Ltd. (Beijing, China). Three hydroxyme thyl aminomethane (Tris, P99.0% purity) was purchased from Sigma Chemical Co., Ltd. (Beijing, China). 2.2. Ultrasonic equipment An assemble ultrasonic bath system equipment with two sets of JXD-02 multi-frequencies processing system and the low temperature circulating water tank was employed (JXD-02, Beijing Jinxing Ultrasonic Equipment Technology Co., Ltd., China). The intensity of ultrasound energy could be varied at different levels by adjusting the output of frequency (28, 40, 50, 135 kHz) and power (0.05, 0.15, 0.25, 0.36, 0.45 W/cm2). The length, width and depth of the ultrasonic bath were 0.2, 0.2, and 0.15 m, respectively. This instrument is shown in Fig. 1. 2.3. Casein entrapment in the alginate–chitosan gel beads Alginate (0.57 g) and chitosan (0.03 g) were brought into good contact by dissolving them in 20 mL of 0.1 M Tris–HCl buffer solution at pH 7.0. The solutions were heated to ensure complete dispersion and dissolution. The heating temperature is 70 °C in 1 h and then cooled to 40 °C. After that, casein was added to the solution at a concentration of 10 mg/mL. Finally, the mixture was dropped into 0.6 M CaCl2 at 4 °C for 30 min to form the beads.
Fig. 1. Plane graph of assemble ultrasonic bath system equipment (1, Thermostatic water; 2, Ultrasonic bath; 3, Sample flask; 4 and 5, Ultrasonic power supply).
2.4. Ultrasonic treatment of casein in the alginate–chitosan gel beads Casein in the alginate–chitosan gel beads (5 g) was dissolved in 300 mL of 0.1 M Tris–HCl buffer solution at pH 7.0. The solution was then treated by the ultrasonic processor at different frequencies (28, 40, 50, 135 kHz), 0.45 W/cm2, and at different powers (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2), 28 kHz, at 50 °C for different times (10, 20, 30, 40, 50, 60 min), respectively. After ultrasonic treatment, casein concentration was determined). 2.5. Ultrasonic pretreatment of casein in the alginate–chitosan gel beads Alginate–chitosan gel beads which embedded casein were pretreated by ultrasound. The treatment conditions were the same with the above 2.4. Then put these alginate–chitosan gel beads into the new Tris–HCl buffer. Casein concentration was determined every 10 min until it reached 60 min. 2.6. Casein concentration measurement Casein concentration was measured by fluorescence spectroscopy (RF 5301, Shimadzu, Co., Ltd., Tokyo, Japan). The fluorescence intensity of the sample was determined with an excitation wavelength of 280 nm and an emission wavelength of 341 nm. For a given material, when the excitation wavelength, the emission wavelength and the thickness of liquid layer are fixed, the low concentration of casein can be calculated as the following equation:
F ¼kC
ð1Þ
where F is the fluorescence intensity, k is a coefficient and C is the casein concentration. 2.7. The calculation of casein diffusion coefficient According to Estapé [15], the cumulative diffusion coefficient of casein in the alginate–chitosan gel beads can be expressed by the following equation: 2 1 X C ðtÞ 6 a ð1 þ aÞ De q2n t e R 1 ¼ 1 2 9þ9aþa C n¼1
ð2Þ
where C(t) is the concentration of release with different diffuse time. C1 means the total casein concentration. De is the effective diffusion coefficient. a is defined as the ratio of buffer volume to the volume of alginate–chitosan gel beads (a = 45.87). The number of gel beads is indicated by n = 100. R means the radius of each alginate–chitosan gel bead which is 0.25 mm.
a¼4
V
p R3 n 3
ð3Þ
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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qn is the non-zero positive solution of Eq. (4):
tan qn ¼
3 qn 3 þ a q2n
ð4Þ
After calculate Eq. (4), q 0.45
C ðtÞ In 1 1 ¼ 0:0016 De t þ 3:4 C
ð5Þ
Table 1 Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the ultrasound-assisted treatment process. Frequency (kHz)
Equations
De (104 m2 s1)
R2*
28 40 50 135
y = 0.045x + 0.294 y = 0.032x + 0.776 y = 0.014x + 0.315 y = 0.004x + 0.174
28.56 20.50 8.81 2.57
0.964 0.888 0.952 0.898
* Coefficient of determination: Used to indicate the degree of fitting the regression line and sample observation value. Values range from 0 to 1 closer to 1 better fitting degree. Better fitting degree of the independent variable on the dependent variable to explain the higher degree.
2.8. Scanning electron microscope (SEM) characterization To observe the effect of ultrasonic frequency and power on the microstructure of alginate–chitosan gels, the alginate–chitosan gels were dried after by exposure in the dryer for 24 h and then was observed by using VEGA//LSU scanning electron microscope (Tescan, Czechoslovakia), magnification was chosen as 100. The software of Image Pro Plus was applied to analysis the SEM professional image. The main parameter of the gels was Area Fraction. The function of the Image–Adjust–Threshold was used to calculated the Area Fraction. 3. Results and discussion 3.1. Effect of ultrasound-assisted treatment on the diffusion of casein in the alginate–chitosan gel beads 3.1.1. Effect of ultrasonic frequency on the diffusion coefficients of casein by the ultrasound-assisted treatment The value of De is an important parameter of the diffusion process. Casein diffused into the buffer induced by ultrasound-assisted treatment could result in the change of De. Fig. 2 shows the plots of h i C versus t during the ultrasound-assisted treatment at difIn 1 CðtÞ 1 2
ferent frequencies with a same ultrasound power 0.45 W/cm . It h i C had a linear relationship with the can be seen that the In 1 CðtÞ 1 treated time at various frequencies. This result demonstrated that the kind of diffusion process followed first-order kinetics. The parameters De was determined from the slope by plotting h i C against t were listed in Table 1. The De was obtained In 1 CðtÞ 1
treatment at 28, 40, 50, 135 kHz, respectively. It was shown that the diffusion coefficient of casein was decreased gradually with the increase of ultrasonic frequency. Therefore, ultrasonic frequency had a suppressing effect on the diffusion and the lower frequency ultrasonic field was more effective for the diffusion of casein molecules through alginate–chitosan gel. According to Gogate, the influence of ultrasonication on the molecular diffusion was extremely dependent on the cavitation effect [16]. Ultrasonic cavitation has two effects on accelerated diffusion. On one hand, the ultrasonic cavitation could further crush gel particles, and increases the reaction, then accelerates the mass transfer. On the other hand, micro jet caused by ultrasonic cavitation can make the surface of solid erosion, thereby it makes the casein which accumulating around the gel beads spread to the surrounding solution [17]. The Increase of the ultrasonic frequency might make the wave expansion phase time become shorter and the cavitation bubble collapse before it shrinked, leading to the inhibitory effect on the De [18]. 3.1.2. Effect of ultrasonic power on the diffusion coefficients of casein by the ultrasound-assisted treatment The ultrasonic power is an important factor to be considered h i C during casein diffusion. Fig. 3 shows the plots of the In 1 CðtÞ ver1 sus the time during the ultrasound-assisted process at different h i C power. The results indicated that the In 1 CðtÞ versus t plots at 1
according to the calculating formula were 28.56 10 , 20.50 104, 8.81 104, 2.57 104 m2 s1 after ultrasound-assisted
all the ultrasound-assisted powers including 0.05, 0.15, 0.25, 0.35, 0.45 W/cm2 followed linear relationships, as determined by the R2 values from 0.881 to 0.964. The De of casein were listed in Table 2 which showed that De of casein were 5.00 104, 15.19 104, 16.25 104, 17.13 104,
Fig. 2. Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the ultrasound-assisted treatment process.
Fig. 3. Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the ultrasound-assisted treatment process.
4
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Table 2 Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the ultrasound-assisted treatment process. Power (W/cm2)
Equations
De (104 m2 s1)
R2
0.05 0.15 0.25 0.35 0.45
y = 0.008x + 0.461 y = 0.024x + 0.465 y = 0.026x + 0.419 y = 0.027x + 0.616 y = 0.045x + 0.294
5.00 15.19 16.25 17.13 28.56
0.957 0.959 0.881 0.902 0.964
28.56 104 m2 s1 after ultrasound-assisted treatment at 0.05, 0.15, 0.25, 0.35, 0.45 W/cm2, respectively. It can be seen that the De of casein molecules increased gradually with the increase of ultrasonic power. Therefore, the diffusion of casein was more effective in the ultrasonic field with high energy. This finding could be explained by the fact that the high intensity ultrasound could lead the polymers degradation. This is because the ultrasound accelerates the attrition between the solvent molecules and polymers molecules, so that C–C bond fraction. At the same time, high temperature and high pressure which produced by ultrasonic cavitation make the chemical bond break. Based on the above reasons, the structure of alginate–chitosan gel beads become loose, casein spread to the surround fluid environment easily, which makes De to increase [19]. Similarly, Makuta et al. reported that the bubbles produced by ultrasound were collapsed into microbubbles with diameters less than 100 lm. The number of microbubbles were increased with the increase of ultrasonic power, which improved the cavitation effect leading to the increase of the mass transfer and enhancement of the indigo carmine degradation [20].
polymer material’s C–C bond for the outer layer of the gel bead is opened, gel bead’s surface layer could be degraded gradually, which prompted the ultrasound plays another role, make the C–C bond together again [23]. As the frequency increases from 28 kHz to 50 kHz, this phenomenon is more obvious. It makes the gel beads gather. Then put the gel bead into the new buffer, diffusion of casein becomes difficult. When the pre-ultrasound treatment frequency reaches 135 kHz, the effect of ultrasound make the gel bead separation. So put the gel bead into the buffer, diffusion of casein increase again. 3.2.2. Effect of ultrasonic power on the De of casein by ultrasonic pretreatment The effect of ultrasonic power on the De of casein by ultrasonic pretreatment was shown in Table 4. In Fig. 5, it is shown that the plots of the versus the time during the ultrasonic pretreatment process at different power has a liner relationship. It showed that the De of casein were 8.9 104, 14.3 104, 16 104, 15.6 104, 15.4 104 m2 s1 after ultrasonic pretreatment at
3.2. Effect of pre-ultrasound treatment on the diffusion of casein in the alginate–chitosan gel beads 3.2.1. Effect of ultrasonic frequency on the diffusion coefficients of casein by ultrasonic pretreatment There are two different ultrasonic treatment methods including ultrasonic-assisted treatment and ultrasonic pretreatment. Because of the wide application in the food industry, it is necessary to study the impact of ultrasonic pretreatment on the molecular diffusion. In this work, the effect of ultrasonic pretreatment on the De of casein in alginate–chitosan gel was studied and the results were shown in Table 3. The plots of the versus the time during the ultrasonic pretreatment process with different frequencies is shown in Fig. 4. After ultrasonic pretreatment at 28, 40, 50, 135 kHz, the diffusion coefficients De of casein were 15.4 104, 14.1 104, 8.6 104, 13.6 104 m2 s1, respectively. It was found that the De of casein molecules decreased with the increased ultrasonic frequency. When samples were pretreated at 50 kHz, the minimum De was observed. While, it was interesting to note that the De increased with the ultrasonic pretreatment at 135 kHz. Adulkar reported that ultrasonic treatment at low frequency created weak ultrasound scattering effect [21]. According to Huang’s research, the material degradation yield increased with the increased frequency in the range of 20–500 kHz [22]. In the pre-ultrasound treatment process,
Fig. 4. Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the pre-ultrasound treatment process.
Table 3 Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the molecular De during the pre-ultrasound treatment process. Frequency (kHz)
Equations
De (104 m2 s1)
R2
28 40 50 135
y = 0.024x 0.075 y = 0.022x + 0.019 y = 0.013x + 0.041 y = 0.021x + 0.006
15.4 14.1 8.6 13.6
0.986 0.981 0.992 0.990
Fig. 5. Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the pre-ultrasound treatment process.
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
Z. Huang et al. / Ultrasonics Sonochemistry xxx (2015) xxx–xxx
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Table 4 Effect of different ultrasonic powers with 28 kHz frequency on the molecular De during the pre-ultrasound treatment process. Power (W/cm2)
Equations
De (104 m2 s1)
R2
0.05 0.15 0.25 0.35 0.45
y = 0.014x + 0.272 y = 0.022x + 0.169 y = 0.025x -0.066 y = 0.025x + 0.718 y = 0.024x 0.075
8.9 14.3 16 15.6 15.4
0.885 0.916 0.956 0.678 0.986
0.05, 0.15, 0.25, 0.35, 0.45 W/cm2, respectively. It should be noted that the power of the ultrasonic pretreatment significantly influenced the diffusion property of casein in the gel. With the increase of power value, the De increased initially and then decreased at higher power values. The ultrasonic pretreatment at 0.25 W/cm2 showed the fastest De. It was concluded that proper intensity of ultrasonic pretreatment was prone to diffusing the casein probably due to the increase of the local temperature and pressure in the surrounding area of collapsing bubble produced by the cavitation effect. However, the high-intensity ultrasonic pretreatment might induce the aggregation and polymerization of the alginate–chitosan gel which hindered the diffusion of casein. Similarly, Bashari et al. reported that maximum loading efficiency of calcium alginate beads was observed when ultrasonic power was 40 W/ cm2. When the ultrasonic intensity exceeded 40 W/cm2, the loading efficiency of dextranase decreased gradually with an increase of the ultrasonic power. The above mentioned behaviors were due to a formed more homogeneous system. Meanwhile, it could be explained that transient cavitation also resulted in strong
Fig. 7. Effect of different ultrasonic frequencies with 0.45 W/cm2 power on the area ratio of alginate–chitosan gel.
acoustic streaming and reduced the mass transfer property [24]. The difference between treatment and pretreatment: the grid structure of gel particles were opened, after the pre-treatment. It is conducive to the dissolution of casein. In addition, the De of pre-ultrasound treatment has a smaller difference with ultrasound-assisted treatment in different ultrasonic power.
Fig. 6. SEM micrographs (100) of alginate–chitosan gel by different frequencies of ultrasonic pretreatment at 0.45 W/cm2, 50 °C for 60 min. The ultrasonic frequencies were (A) 28 kHz, (B) 40 kHz, (C) 50 kHz, (D) 135 kHz, respectively.
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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Fig. 8. SEM micrographs (100) of alginate–chitosan gel by different power of ultrasonic pretreatment at 28 kHz, 50 °C for 60 min. The ultrasonic power were (A) 0.05 W/ cm2, (B) 0.15 W/cm2, (C) 0.25 W/cm2, (D) 0.35 W/cm2, (E) 0.45 W/cm2, respectively.
Considering from the viewpoint of energy saving, ultrasonic pretreatment has more broad prospects in industrial production. 3.3. Microstructure of alginate–chitosan gel by ultrasonic pretreatment 3.3.1. Effect of the ultrasonic frequency To obtain the microstructure of alginate–chitosan gel by ultrasonic pretreatment, the dried samples were detected by SEM with
100 magnification. SEM micrographs of alginate–chitosan gel by ultrasonic pretreatment at different frequency (28, 40, 50, 13 5 kHz) and 0.45 W/cm2, 50 °C for 60 min were presented in Fig. 6. After treatment at 28 kHz, the alginate–chitosan gel showed a rather uneven surface. With the increase of the frequency, the extent and amount of the uneven surfaces were gradually reduced. When treated frequency was 135 kHz, the amount of the uneven surface of the gel became much more and denser. Meanwhile, the 135 kHz treated sample showed quite numerous
Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015
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small wrinkles. Therefore, 135 kHz treatment showed an obviously different microstructure compared with other treatment. Ultrasonic cavitation prompted the interaction between the liquid molecules and gel beads, which made the surface of the gel bead produced many fold. With the increase of frequency, cavitation effect gradually weakened, which made the attrition between buffer and the gel beads surface also decreased, resulting in the amount of surface folds down. When the frequency reached 135 kHz, ultrasonic became extremely strong and caused damage to the surface of the gel structure [25]. In order to further analysis the microstructure, the software of Image Pro Plus was applied to analysis the SEM professional image. The main parameters of the gels were total area and area fraction. The function of the Image–Adjust–Threshold was used to calculate the area fraction. The area ratio of alginate–chitosan gel as a function of the treated ultrasonic frequency was shown in Fig. 7. After treatment at 28 kHz, the alginate–chitosan gel showed an area ratio of 15.42%. With the increase of ultrasonic frequency, the area ratio of alginate–chitosan gel decreased initially and then increased at 135 kHz. This conclusion was in good agreement with the previous diffusion results. The higher the area ratio was, the bigger the De would be. It indicated that the diffusion was impacted by the alginate–chitosan gel surface.
3.3.2. Effect of ultrasonic power The impact of ultrasonic power on the microstructure of alginate–chitosan gel was also investigated. Fig. 8 showed the SEM micrographs of alginate–chitosan gel at varying ultrasonic power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2) and 28 kHz, 50 °C for 60 min. It was found that the extent and amount of the uneven surfaces were gradually increased but then reduced with the increase of the ultrasonic power. When ultrasonic power was 0.25 W/cm2, a maximum amount of the uneven surface was found. Also, as shown, excess energy brought less changes of the gel surface. With the increase of ultrasound power, the friction between gel bead and liquid became more and more acute, which made the amount of fold on the gel bead increased. But when the power of ultrasound excessed a critical value, the layer of gel bead was degraded, until the gel bead’s structure became loose, which makes the number of fold of gel bead decreased [26]. The area ratio of alginate–chitosan gel as a function of the treated ultrasonic power was shown in Fig. 9. With the increase of
Fig. 9. Effect of different ultrasonic powers with 28 kHz frequency on the area ratio of alginate–chitosan gel.
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ultrasonic power, the area ratio of alginate–chitosan gel increased initially and decreased at high power values. The area ratio showed a maximum of 37.22% at 0.25 W/cm2. It was interesting to find that a close relationship existed between microstructure and diffusion property for the effect of ultrasonic power. Alginate–chitosan gel undergoing much more microstructure changes at the surface tended to exhibit a higher De due to the proper energy input by ultrasound. 4. Conclusion Ultrasound treatment of alginate–chitosan was confirmed to impact the diffusion of casein. The effects of ultrasound conditions on the casein diffusion were different for the two different treatment method, ultrasound-assisted and pre-ultrasound. The treatment condition of the optimum De of casein was 28 kHz, 0.45 W/ cm2 for the ultrasound-assisted treatment. While, the optimum pre-ultrasound treatment condition was 28 kHz, 0.25 W/cm2. The uneven surfaces of alginate–chitosan gel were observed after ultrasound treatment. It was interesting to find that there was a close relationship between micro structure and diffusion property for the effect of ultrasonic frequency and power. The mechanism of molecular diffusion impact by ultrasound should be examined further and this study is under way and will be reported in the near future. Acknowledgements This research was funded by the National Key Technology R&D Program of the National Natural Science Foundation of China (31371722). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ultsonch.2015.03. 015. References [1] Q. Li, Study of application and development of immobilization technique in food industry, Sichuan Food Ferment 132 (2006) 16–19. [2] Z.N. Yin, Immobilized enzyme and its research progress in food and biological applications, Life Sci. Instrum. 7 (2009) 11–15. [3] H.Z. Liu, Y.Y. Zhang, G.Z. Zhang, B.H. Niu, Research progress on preparation of immobilized enzyme, Chem. Ind. Eng. 30 (2009) 21–23. [4] J.W. Di, C.P. Shen, S.H. Peng, Y.F. Tu, S.J. Li, A one-step method to construct a third-generation biosensor based on horseradish peroxidase and gold nanoparticles embedded in silica sol–gel network on gold modified electrode, Anal. Chim. Acta 553 (2005) 196–200. [5] H.L. Wu, L.N. Wei, Z.Q. Zhou, G.X. Yuan, Z. Yan, H.P. Pan, K.Y. Lin, X.B. Jiang, Y.Q. Chen, W.F. Hu, Optimization of immobilization conditions for protease and its application in the fermentation of soy sauce, Mod. Food Sci. Technol. 29 (2003) 1080–1084. [6] P.X. Jia, X.Y. Liu, J.L. Liu, L.M. Jiao, K.Y. Tang, Immobilized enzyme and its application in fields including chemical engineering, Leather Sci. Eng. 14 (2004) 31–37. [7] D. Wojcieszyn´ska, K. Hupert-Kocurek, A. Jankowska, U. Guzik, Properties of catechol 2,3-dioxygenase from crude extract of Stenotrophomonas maltophilia strain KB2 immobilized in calcium alginate hydrogels, Biochem. Eng. J. 66 (2012) 1–7. [8] W.L. Hsu, M.J. Lin, J.P. Hsu, Dissolution of solid particles in liquids: a shrinking core model, World Acad. Sci. Eng. Technol. 3 (2009) 801–805. [9] Z.P. Zhao, C.H. Chen, Mass transfer processes by using ultrasonic, Chem. Des. 6 (1997) 30–35. [10] J. Gutenwik, B. Nilsson, A. Axelsson, Determination of protein diffusion coefficients in agarose gel with a diffusion cell, Biochem. Eng. J. 19 (2004) 1–7. [11] E.M. Johnson, D.A. Berk, R.K. Jain, W.M. Deen, Diffusion and partitioning of proteins in charged agarose gels, Biophys. J. 68 (1995) 1561–1568. [12] T.J. Mason, J.P. Lorimer, Applied Sonochemistry: The Uses of Power Ultrasound in Chemistry and Processing, Wiley VCH Weinheim, Germany, 2002. [13] W. Qin, Y. Zhang, Y.Y. Dai, Ultrasonic field effect on electrolyte diffusion coefficient in the capillary, J. Tsinghua Univ. (Nat. Sci. Ed.) 41 (2001) 41–43.
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Please cite this article in press as: Z. Huang et al., Effect of ultrasound on the diffusion properties of casein entrapped in alginate–chitosan gel, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.03.015