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Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: A comparative study
Accepted Manuscript Title: Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: a comparative study Authors: Mahmoud Hosseinnia, Mohammad Alizadeh Khaledabad, Hadi Almasi PII: DOI: Reference:
S0141-8130(17)30241-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.190 BIOMAC 7356
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
18-1-2017 14-3-2017 31-3-2017
Please cite this article as: Mahmoud Hosseinnia, Mohammad Alizadeh Khaledabad, Hadi Almasi, Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: a comparative study, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.190 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: a comparative study Mahmoud Hosseinnia, Mohammad Alizadeh Khaledabad*, Hadi Almasi Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran * Corresponding Author: [email protected]
Abstract The performance of whey protein isolate (WPI) and pectin as wall materials in encapsulation of Ziziphora clinopodiodes essential oil by ultrasonication method was compared. In this regard, using the response surface methodology, the influence of ultrasonication (US) power (50-150 W) and core-coating ratio (10-100%) on the properties of microcapsules was evaluated. Increasing US power and core-coating ratio, caused to increase and decrease the particle size, respectively. The polydispersity index (PDI) of WPI coated microcapsules was increased by increasing of US power. The Zeta potential values were increased by increasing of core-coating ratio. Also, the effect of core-coating ratio on encapsulation efficiency was more than US power. Morphological studies by SEM on optimized microcapsules showed regular spherical shapes. X-ray diffraction analysis revealed that the type of the wall material had no effect on the structural properties of the microparticles. FT-IR analysis confirmed the pronounced effect of electrostatic interactions in the formation of microcapsules. Keywords: Microcapsules; ; ; ; ; , Response surface methodology, Ultrasonication, Coating biopolymers, Particle size, Morphology 1. Introduction In recent years, essential oils (EOs) have been considered as natural preservatives. The most of phenolic compounds extracted from different parts of plants like leaves, stems, flowers, and roots are classified as generally recognized as safe (GRAS) [1]. For this reason, nowadays essential oils have more broad classes of applications in food industry, pharmaceutical, cosmetics etc. [2]. Ziziphora clinopodiodes (known as Kakuti) belonging to the family of Lamiaceae is the most common species from genus of Ziziphora L. that has been reported from Iran. The chemical composition of the essential oil of Z. clinopodiodes has been reported. Phenolic constituents including pulegone, 1,8-cineole, thymol, carvacrol, p-cymene and limonene have been reported as major compounds of Z. clinopodiodes EO. Also, methyl acetate, iso-neomenthol, iso-menthone and α-pinene have been reported as the most important constitutes [3,4]. Several studies have been applied on the functional properties of Z. 1
clinopodiodes EO and strong antibacterial, anthelmintic, antifungal and antiviral activities have been reported [5-7]. Using of EOs in food formulations have attracted considerable attention due to consumer awareness on the food quality and food safety. Bioactive compounds of EOs are chemically reactive substances, which can cause considerable problems when used in a complex food system. Negative effects on the physical stability or integrity of the food, poor miscibility and phase separation during storage and the thermal or chemical degradation of the bioactive compounds are the most common shortcomings of direct addition of EOs into a food [8]. A viable and efficient approach widely used for increase the physical stability of the active substances and protecting them from the interactions with the food ingredients is encapsulation of EOs. Reducing of evaporation, to promote easier handling, and release controlling of the encapsulated material during storage are the other advantages of the encapsulation of EOs [9]. The process of encapsulation consists of the formation of a multicomponent structure in the form of micro- or nano- particles consisting of two substances: the core material and the wall material. There are many techniques for encapsulation that each of them have their own advantages and disadvantages generating particles with different properties. Ultrasonication is one of the efficient encapsulation techniques. In an ultrasonic encapsulation, high intensity ultrasound waves can change the characteristics of treated matter through intensive shear forces, pressure, and temperature due to cavitation. The major benefits of this method are including lower energy consumption, smaller droplet size, lower polydispersity, and higher encapsulation efficiency [9]. The selection of a suitable wall material is critical to have a successful microencapsulation. Wide range of biopolymers and hydrocolloids has been used as wall material for microencapsulation of EOs. Whey proteins abundant by-product in the cheese-making industry, have been successfully employed as wall material. The major constituents of whey proteins are β-lactoglobulin and α-lactalbumin [10]. Both whey protein concentrate (WPC) and whey protein isolate (WPI) have been used in encapsulation studies due to their surface active properties [11-15]. Pectin as a structural heteropolysacharide is another widely used wall material. Pectin consists of a linear chain of D-galacturonic acid units joined by means of α(1-4) glycosidic linkage. The carboxylic groups of these uronic acids may be esterified by methyl groups. Galacturonic acid is replaced by α-(1-2)-linked L- rhamnose units at some distinguishing areas [16]. The potential of pectin for using as wall material in encapsulation 2
techniques, drug delivery systems and controlled release formulations is strongly dependent on its molecular weight and degree of esterification [17]. There are many reports about the use of low-methoxyl (LM) and high-methoxyl (HM) pectins in encapsulation of bioactive compounds for food and pharmaceutical uses [18-21]. To the best of our knowledge, there is no report on the encapsulation of Z. clinopodiodes EO. The aim of present study was to prepare Z. clinopodiodes EO microcapsules through ultrasonication method. The mixture of WPI and pectin has been studied recently for microand nano-encapsulation of bioactive compounds [22-24]. However, there is no report on comparison of the potential of WPI and pectin in encapsulation of EOs. The potential of WPI and pectin in encapsulation of Z. clinopodiodes EO was compared in this study. As it mentioned in literature, the core-coating ratio is an important factor that affects the properties of microcapsules [25]. On the other hand, in the production of delivery systems using ultrasonication, some variables such as power and duration of sonication can be effective upon the physical properties of microparticles [26]. Therefore, in this study, we investigated the effect of ultrasonic power and core-coating ratio on encapsulation efficiency of WPI and pectin and some physical properties of microcapsules using response surface methodology (RSM) and the optimal combination of these variables was determined to produce Z. clinopodiodes EO microcapsules by two wall materials with minimum droplet size and highest stability. Afterwards, the structural and morphological properties of microcapsules produced in optimized conditions for WPI and pectin were characterized by FT-IR, XRD and SEM analyses. 2. Materials and methods 2.1. Materials The Z. clinopodiodes leaves were harvested at flowering stage in mid-June 2016 from wild grown plants in the West-Azarbayjan province, Iran. Whey protein isolate (WPI) (85% protein) was purchased from Arla Food Ingredient (Denmark). High methoxyl pectin (degree of esterification ~60 %) and all other reagents were purchased from Sigma (Germany). 2.2 Extraction of essential oil Dried leaves of Z. clinopodiodes, were subjected to the hydro distillation for 3 h using a glass Clevenger type apparatus. The extraction yield was obtained as 2.2%. According to the literature review, the major compounds of Z. clinopodiodes EO are 1,8-cineole, thymol, carvacrol, p-cymene and limonene [3]. The obtained essential oil was stored in sterilized dark glass at 4 °C. 3
2.3. Experimental design Response surface methodology (RSM) was used to evaluate the effects of core-coating ratio (X1) and sonication power (X2) on different responses of Z. clinopodiodes EO microcapsules such as particle size (Y1), viscosity (Y2), Z-average (Y3) and PDI (Y4). Hexagonal experimental design was used to explore the linear, interaction and quadratic effects of independent variables on the studied responses. The quadratic equation for the variables was the following: 𝑌 = 𝛽0 + ∑𝑛𝑖=1 𝛽𝑖 𝑥𝑖 + ∑𝑛𝑖=1 𝛽𝑖𝑖 𝑥𝑖𝑖 + ∑𝑛𝑖=1 ∑𝑛𝑖>𝑗 𝛽𝑖𝑗 𝑥𝑖 𝑥𝑗
(1)
where Y = predicted response, 0 = a constant, i =linear coefficient, ii = squared coefficient, and ij = interaction coefficient. The software Design Expert 7.1.1 was used to analyze the results. The two factors (processing variables), levels and experimental design are given in Table 1. 2.4. Encapsulation process Hydrated solutions of pectin at different concentrations (0.5-1.5% by weight) were prepared by mixing pectin with distilled water using magnetic stirrer. In the case of WPI, their solutions were prepared by dispersing the same amounts of WPI powder into buffer solution (5 mM phosphate buffer, pH 7). The suspensions of coating materials were prepared the day before the encapsulation and kept at room temperature for 24 h to ensure complete saturation of the molecules of biopolymers. The desired amounts of Z. clinopodiodes EO was then slowly incorporated into each biopolymer suspension by mechanical stirring at 5000 rpm for 5 min in a high-speed blender (IKA T25 digital Ultra-Turrax, Selangor, Malaysia) to obtain the preemulsions with desired core to coating ratios according to experimental design (Table 1). After that, ultrasonic homogenizer (UP200Ht, Hielscher, Germany) equipped with titanium probe with diameter 14 mm was used for microencapsulation. Ultrasonic homogenization of 100 g solution was performed in 250 ml beaker using 80% power for different ultrasonic powers as mentioned in Table 1. Ultrasonication process was performed at room temperature for 2 min. Table 1 2.5. Freeze drying Immediately after encapsulation, samples were transferred to 100 ml beakers half fully and placed into freezer at about -4 C°. Frozen samples were then dried under vacuum at -50 C° and 0.007 atm for 48 h. After freeze drying process, samples were grinded by using a glass rod in order to transform them to powder form. 2.6. Particle size analysis 4
The DeBroukere mean diameter (D43) and polydispersity index (PDI) of microcapsules were determined considering the method of Zhang et al., [27]. Dynamic light scattering (DLS) technique employing a Zetasizer Nano-ZS (Malvern instruments, Worcestershire, UK) was used. Dilution with distilled water (1:100) was carried out before the size measurement. All measurements were carried out at 25 °C.
2.7. Measurement of Zeta potential Determination of the surface charge at the interface of the particles dispersed in the aqueous solution is the purpose of Zeta potential measurement. Zeta potential of aqueous emulsion of microcapsules was measured by means of a Malvern Zeta sizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. Before analysis, emulsions were diluted to a particle concentration of 0.01% using distilled water.
2.8. Encapsulation efficiency (EE%) The amount of encapsulated EO in the microcapsules was examined by Sheikhzadeh et al., [28] method with some modifications. The EE% was measured by separation of microcapsules from 1 ml of aqueous emulsions by centrifugation at 13000 rpm for 30 min. A second the filtration of the supernatant was done using a PTFE syringe filter with 0.22 μm pore size. Afterward, the emulsions (40 μl) was diluted to 2 ml with acetone and was then used for estimations. The absorbance at 275 nm was measured with a UV/vis Spectrophotometer (Unico, S 2100 SUV, Dayton, NJ). The EO of Z. clinopodiodes had the maximum absorbance in this wavelength (λmax). Different concentrations of Z. clinopodiodes EO were used for drawing of calibration curve. Total EO content was calculated using the following linear equation based on the calibration curve: A = 0.94C + 8.525 × 10−3 (R2 = 0.9986). Where (A) is the absorbance and (C) is the concentration (mg.mL). The EE% was calculated as follow:
EE%
Total EO amount Free EO amount 100 Total EO amount
(2)
Total EO means the total loaded EO on microcapsules and free EO is released EO after centrifugation of microcapsules emulsion.
2.9. Measurement of viscosity
5
The viscosity of emulsion of microcapsules at concentration of 5% wt. were determined at 25 °C, with a Brookfield rheometer (LV DV-ΙΙΙ Ultra, Brookfield Engineering Laboratories Inc., MA, USA), equipped with a spindle LV2 at 60 rpm after 30 seconds.
2.10. Turbidity measurments The turbidity of emulsions was measured as nephelometric turbidity units (NNU) at 90◦ light scattering and 860 nm with a Nephla reader turbidometer (HKC, Germany).
2.11. Fourier transform infrared (FT-IR) spectroscopy FT-IR analysis was applied to observe the chemical structure of the powders of biopolymers and optimized microcapsules. The samples were prepared by the KBr-pellet method. The powder of samples was pressed into a small pellet about 1 mm thick. FT-IR spectra were analyzed using an FT-IR spectrometer (Nexus 670, USA) operated at a resolution of 4 cm−1 in the range from 4000 to 400 cm−1
2.12. Surface morphology of microcapsules Scanning electron microscope (SEM) was used to investigate the surface morphology and the microstructural properties of the freeze dried encapsulated powders produced in optimized conditions. Samples were coated with a thin conductive gold layer and then were observed using a scanning electron microscope (KYKY-EM3200) at an accelerating voltage from 10 to 20 KV.
2.13. X-ray diffraction XRD studies on powders of wall materials and freeze dried microcapsules of optimized samples were performed with a Siemens D5000 X-ray diffractometer (Germany), equipped with CuKα radiation source (k = 0.154 nm) operating at 40 kV and 30 mA. The samples were scanned over the range of diffraction angle 2θ=2–70° with a scanning rate of 0.5°.min-1 at room temperature. The crystallinity index, a measure of ordered orientation, was calculated by the following equation [15]: CrI (%) ( I 002 I am ) / I 002 100
(3)
where I002 is the maximum intensity of the crystal plane reflection of the biopolymers, Iam is the maximum intensity of X-ray scattering broad band due to the amorphous part of the sample (diffraction intensity at 2θ = 18°). 6
3. Results and discussion 3.1. Particle size analysis Particle size as a function of the studied factors were as follows: 𝑌1 = 145.59 + 4.08 − 2.28 ∗ 𝐵 − 22.34 ∗ 𝐴 ∗ 𝐵 + 85.07 ∗ 𝐴2
(4)
𝑌2 = 1940 − 493.33 ∗ 𝐴 + 618.19 ∗ 𝐵 − 602.77 ∗ 𝐴 ∗ 𝐵 − 151.5 ∗ 𝐴2 − 608 ∗ 𝐵2
(5)
where Y1 , Y2 ,A and B are particle size of WPI stabilized emulsions, particle size of pectin stabilized emulsions, US power and core-coating ratio, respectively.
Fig.1 (A and B) shows the DeBroukere mean diameter (D43) of microcapsules prepared by WPI and pectin as a function of US power and core-coating ratio. By comparing of particle size values in figures A and B, it can be concluded the particles obtained by WPI have a lower diameter than those obtained by pectin. This could be explained by the good emulsifying property of WPI related to the many hydrophobic and hydrophilic parts of WPI [25]. Thus, WPI has a higher ability to adsorb at the oil-water interface and can reduce the interfacial tension. However, the pectin has not an amphiphilic nature and its performance at the rapid absorption on the surface of EO droplets is less than WPI. Similar results were reported by Silva et al., [15] who compared the stabilization efficiency of WPI and modified starch in encapsulation of annatto seed oil. As shown in Fig.1B, the D43 values of pectin coated microcapsules were increased by increasing of US power. But the effect of US power on the diameter of WPI coated microcapsules was dependent on core-coating ratio (Fig.1A). At the ratios higher than 50%, increasing of US power caused to decrease the D43 of WPI coated particles, but at lower ratios, its effect was same as that observed for pectin. Perrier-Cornet et al., [29] proposed that an overly intense homogenization process can increase the size of the particles due to recoalescence, a phenomenon that is known as “over-processing”. The results of this research indicated that the intense of this phenomenon is different for various biopolymers. WPI can rapidly adsorb on the surface of the EO droplets formed during homogenization and significantly reduce the interfacial tension. It also can provide a coating that prevents the droplets from aggregating with neighboring droplets. But these effects for pectin are not as 7
effective as WPI. Therefore, reduction in the size of the encapsulated particles is not solely a function of the amount of energy supplied to the system. The lowest particle size was recorded at the middle core-coating ratio of EO and WPI (146 nm). The D43 values were increased by increasing and decreasing of EO-WPI ratios far from 50%. However, the mean diameter of pectin coated particles was decreased by increasing corecoating ratio. This means that the stabilization mechanisms of pectin and WPI are different. Steric stabilization is the dominant stabilization effect of pectin. But, the surface activity and emulsifying ability are the major functions of WPI. At the lower concentrations of WPI, the size of EO droplets was increased and at the higher EO-WPI ratios, the increasing of accumulated and aggregated WPI molecules on the surface of EO droplets causes to formation of a thick surface layer that leads to increase the diameter of microcapsules.
Fig. 1
Fig.2 shows the PDI changes of microcapsules as a function of US power and core-coating ratio. The effect of US power on PDI values was dependent on the type of biopolymer. For WPI coated microcapsules, the PDI was increased by increasing US power but increasing of ultrasonication intensity in pectin stabilized emulsions caused to decrease of PDI. The acoustic cavitation phenomenon is an effective parameter on size distribution of particles in US homogenized emulsions. This phenomenon promotes intense shear rates resulting from the formation and collapse of microbubbles associated with highly localized turbulence levels [30]. This mechanical stress is able to denature of WPI molecules and thus, the formation of protein aggregates in the emulsion may have been interpreted as larger droplets formed in sonicated emulsion in severe intensities. By increasing of US power, the greater acoustic cavitation effects were occurred and therefore, higher PDI values were expected. The increasing of temperature during sonication process can promote this phenomenon. Silva et al., [15] reported that PDI of WPI and modified starch stabilized annatto seed oil was increased by increasing ultrasonication power. They suggested that the denaturation of WPI and gelatinization of modified starch due to the acoustic cavitation phenomenon are the reasons of this increment. However, an adverse effect was observed for the emulsions stabilized with pectin and PDI was decreased significantly by increasing US power (Fig.2B). Because there is no gelatinization or denaturation behavior of pectin under the influence of ultrasonication process. As shown in Fig.2A, the PDI was decreased and uniform microparticles was formed in WPI coated samples. 8
But the effect of core-coating ratio on the PDI of pectin stabilized emulsions was not statistically significant.
3.2. Zeta potential Data analysis showed that variation in zeta potential can be characterized by the following models: 𝑌3 = −18.81 − 1.34 ∗ 𝐴 + 0.84 ∗ 𝐵 + 4.1 ∗ 𝐴 ∗ 𝐵 − 3.26 ∗ 𝐴2
(6)
𝑌4 = −11.82 − 5.74 ∗ 𝐴 + 6.82 ∗ 𝐵 − 4.23 ∗ 𝐴2 − 9.75 ∗ 𝐵2
(7)
where Y3 , Y4 ,A and B are zeta potential of WPI stabilized emulsions, zeta potential of pectin stabilized emulsions, US power and core-coating ratio, respectively. The Zeta potential values of microcapsules are shown in Fig. 3 (A and B). Charged surfaces with negative potentials were recorded for microparticles stabilized by both of WPI and pectin. WPI has net negative charge at pH values higher than its isoelectric pH. Pectin also has negative charge at neutral pH due to presence of carboxyl groups on galacturonic acid units. The Zeta potential of -25.7 mV was reported by Souza et al., [22] for WPC at pH of 7 that is similar to the results of current study. Also, they reported that the Zeta potential of HM pectin is lower than LM pectin and has a decreasing trend by increasing of pH. The pH values of pure WPI and pectin solutions (measured by Metrohm pH meter, 780, Swiss) were 6.74 and 2.92, respectively. The pH was not significantly affected by core-coating ratio or US power.
Fig. 2
The results showed that increasing of US power has a decreasing effect on Zeta potential of pectin coated particles. Similar trend was also observed for WPI coated sample at higher corecoating ratios. This means that the increasing of US power causes to decrease the thickness of biopolymers on the surface of EO droplets. Therefore, the higher intensities of ultrasonication would have an adverse effect on encapsulation process. The Zeta potential values were also increased by increasing core-coating ratios and this increment was more obvious for WPI stabilized microcapsules.
3.3. Encapsulation efficiency Encapsulation efficiency was modeled as follows: 9
𝑌5 = 67.58 + 41.95 ∗ 𝐴 + 0.14 ∗ 𝐵 − 60.20 ∗ 𝐴2
(8)
𝑌6 = 48.41 + 37.4 ∗ 𝐴
(9)
where Y5 , Y6 ,A and B are encapsulation efficiency of WPI stabilized emulsions, encapsulation efficiency of pectin stabilized emulsions, US power and core-coating ratio, respectively. Fig.3 (C and D) shows the encapsulation efficiency (EE%) as a function of the US power and core-coating ratio. The EE% values obtained for WPI were more than pectin that could be attributed to the emulsifying ability of amphiphilic WPI. For example, at a same core-coating ratio (55%) and US power (100 w), the EE% of WPI was 60.9% but it was calculate as 38.4% for pectin. The effect of core-coating ratio on EE% of WPI was more than US power (Fig.3C). Also, the effect of US power on the EE% of pectin stabilized microcapsules was not significant. Fig.3C shows that the intense of ultrasonication is not an effective factor in entrapment capacity of WPI. At a constant core-coating ratio, increasing of US power had no significant effect on EE% of WPI coated microcapsules. So, it could be concluded that the effect of chemical structure and nature of wall materials on EE% is more than process parameters. Also, it shows that the US treatment has no significant disrupting effect on structure of biopolymers. Similar results were reported by Mirmajidi Hashtjin and Abbasi [26] and Yazicioglu et al., [25] for the effect of sonication on biopolymers. In the case of WPI, the EE% was increased by increasing core-coating ratio up to 70% and was decreased again at higher ratios. The highest EE% was recorded equal to 72.74% for core-coating ratio of 70%. Similar to our results, Faridi Esfanjani et al., [23] reported EE% of 84.53% for encapsulation of safranal with WPC at core-coating ratio of 75%. The EE% values were also increased by increasing of EO-pectin ratios but it was not decreased at higher ratios. This difference reveals that the encapsulation potential of pectin is more than WPI and is able to entrap a higher amount of EO especially at higher concentrations of core material.
3.4. Viscosity and turbidity The contour plots of the effects of US power and core-coating ratio on the viscosity and turbidity of Z. clinopodiodes EO emulsions stabilized by WPI and pectin are shown in Fig. S1 (Supplementary data). The viscosity of pure WPI and pectin solution at the same concentration of microcapsules (5% wt.) was 4.65 and 50.1 mPa.s respectively. Higher molecular weight and hydrophilic nature of pectin are the reasons of this difference. For both of pectin and WPI, the US power had no significant effect on viscosity of emulsions. This result shows that sonication 10
has no significant effect on structure of biopolymers and prepared microcapsules. However, as it was expected, the viscosity was decreased significantly by increasing of core-coating ratio in the emulsions stabilized by both of WPI and pectin. By increasing of core-coating ratio, the amount of biopolymers was decreased in emulsions and thus, the bonded water was decreased and caused to decrease of viscosity. Lower molecular weight of chemical compounds of EO in comparison to biopolymers, causes to decrease viscosity by increasing of EO in formulation of microcapsules. Similar trends were observed for turbidity of emulsions. US power had no significant effect on turbidity but it was increased by increasing core-coating ratio. It shows that the effect of EO on increasing of turbidity is more than biopolymers. The suspensions of pure pectin and WPI are clear with some cloudiness. Turbidity of pure WPI and pectin solutions were 2341 and 3679 NTU, respectively. Addition of EO increased the turbidity of emulsions. Z. clinopodiodes EO had a yellow color and appearance of its emulsions was milky white. This color was the reason of turbidity and was increased by increasing EO content in emulsions. Also, the EE% was decreased by increasing of core-coating ratio. The release of un-encapsulated EO from the surface of microcapsules was another reason for increasing of turbidity.
Fig. 3
3.5. Optimization and desirability One of the main objectives of this study was to determine the optimal values of the independent variables to generate microcapsules with minimum droplet size. The Desirability Function method was used for the multi-objective optimization. The desirability function for individual responses were estimated by numerical methods and the overall desirability function was calculated. Results from statistical analysis lead to finding the optimum US power and corecoating ratio according to physical properties of microcapsules, are shown in Table 2. The optimum values of independent variables including ultrasonication power and core-coating ratio to achieve the best-predicted values of particle size (172.94 nm for WPI and 856.16 nm for pectin) were 50 W, 73.42% and 150 W, 94.99% for WPI and pectin, respectively. It should be noted that the overall desirability values of the predicted zone were equivalent to 0.842 and 0.934% for WPI and pectin, respectively. At the second step of this research, two microparticles were prepared according to the optimized conditions of WPI and pectin and the structural and morphological properties of freeze dried microcapsules were investigated. 11
Table 2
3.6. Surface morphology of optimized microcapsules Fig.4 shows the SEM micrographs of WPI (A) and pectin (B) coated microcapsules at optimized conditions. The both of freeze dried powders had regular spherical shapes without apparent pores or fractures. The morphology of the both of microparticles obtained by ultrasonication method was similar. Also, Anandharamakrishnan et al., [31], Carvalho et al., [32], Silva et al., [15] and Liu et al., [14] reported similar morphologies for WPI encapsulated EOs produced by spray drying method, indicating that the type of encapsulation method did not influence the microstructure of the particles. However, as expected, the type of biopolymer that was used as wall material yielded microparticles with different diameters. As it was shown in Fig.1 and was predicted in optimization step, microparticles obtained by pectin exhibited larger size than the WPI coated microcapsules. From the SEM micrographs, the average size of around 211 nm were calculated for WPI coated microcapsules, but it was 730 nm when pectin was used as wall material. A deviation from optimum conditions predicted by RSM method was that the PDI values predicted for WPI was larger than pectin (0.23 vs 0.19). However, as it can be seen from Fig.4, the polydispersity and size distribution in pectin coated microparticles is higher than that of WPI stabilized microcapsules and the uniformity of particle size in WPI/EO microparticles is more obvious. Fig. 4
3.7. XRD analysis X ray diffraction patterns of WPI and pectin and their powdered microparticles are presented in Fig. 5. XRD results showed a typical amorphous structure for both of WPI and pectin with a broad peak at around 2θ=25° for both of them. The crystallinity index of 26.45% and 16.11% was calculated for WPI and pectin, respectively. These are in agreement with XRD results that are reported for pectin [33] and WPI [14] in earlier researches. The results indicated that the EO incorporation had no distinct effect on crystallinity of WPI and pectin, even though the intensity of mentioned broad peaks was slightly decreased for microcapsules (crystallinity index of 17.50% and 6.42% for WPI/EO and pectin/EO microcapsules respectively). So, it could be concluded that there is not any strong interaction between EO and wall materials. This hypothesis was approved by FT-IR test (section 3.8). The observed slight decrease in crystallinity of microcapsules could be attributed to the effect of sonication on structure of 12
biopolymers [26]. Even though it was not observed any change in EE% and chemical structure (FT-IR) of WPI and pectin after sonication. Similar XRD patterns were reported for microparticles of WPI containing annatto seed oil [15], rosemary EO [34] and curcumin [14]. Fig. 5
3.8. FT-IR analysis Fig.6 presents the FT-IR spectrum of Z. clinopodiodes EO, WPI and pectin powders and their powdered microparticles loaded with EO. Fig. 6A indicates the FT-IR spectrum of the Z. clinopodiodes EO. The absorption band at 3424 cm-1 is related to OH vibrations of carvacrol, p-cymene and limonene compounds in the Z. clinopodiodes EO. The sharp peak at 2961 cm-1 is attributed to the asymmetric C-H stretching of the methyl groups that are present in most of phenolic compounds. The asymmetrical and symmetrical bending vibrations of methyl group are seen at 1460-1340 cm-1, respectively. Absorption peaks of around 1580 cm-1 are related to the vibrations of C=C groups. The pure WPI powder showed several specific absorption peaks (Fig.6B). A broad peak appeared at 667 cm-1 is related to C-N groups. The peaks ranging 10751158 cm-1 are related to N-H bonds and C-N stretching bonds (Amide type III). Amide type II structure displayed the peaks ranging 1390-1550 cm-1 related to bending groups of N-H. A sharp peak was observed at 1652 cm-1 that is related to vibratory stretching bonds of groups C=O (Amide type I). A peak at 2927 cm-1 is assigned to C-H stretching bond [35]. Fig.6D presents the FT-IR spectrum of pure pectin. C=O or C=C double bonds of pectin showed a peak at 1067 cm-1. The absorption peaks at range of 1238-1440 cm-1 and also absorption band of 1640 cm-1 are assigned to stretching bands of COO- groups. Two peaks at 1748 and 2937 cm-1 are related to carboxyl and CH2 groups of pectin, respectively [36]. The peaks ranging 3000-3600 cm-1 in spectrum of both of WPI and pectin are related to free O-H groups. By comparing of spectra obtained for WPI/EO and pectin/EO powders (Fig6.C and E) with native biopolymers, no specific new peaks were found in microparticles suggesting that no generation of new chemical bond is accrued between wall materials and EO compounds. This results confirms the formation of complexes promoted by electrostatic interaction rather than chemical reactions. Therefore, it could be concluded that the interaction between Z. clinopodiodes EO and WPI or pectin is based on electrostatic force. This type of only physical incorporation of EO in WPI or pectin matrix, preserves the intrinsic nature of the bioactive compounds of EO. However, the release rate of EO from microcapsules would be increased without accruing chemical bonding. The release profile of microcapsules bas been discussed 13
and will be published in the next manuscript. Partic et al., [37] reported similar results for microencapsulation of fish oil by gelatin.
Fig. 6
4. Conclusion The fabrication and characterization of Z. clinopodiodes EO microcapsules prepared by ultrasonication method were carried out. WPI and pectin were two wall materials and optimum values of ultrasonication power and core-coating ratio were determined by RSM method. Intensification of the ultrasonication process had negative effect in the reduction of the droplets size of microcapsules. Smaller microparticles were obtained by WPI in comparison to pectin that was attributed to amphiphilic structure and emulsifying ability of WPI. However, PDI values was lower for pectin coated microcapsules. The effect of core-coating ratio on viscosity and turbidity of emulsions was more than US power. This study showed the capability of RSM in optimization of ultrasonication process to produce Z. clinopodiodes EO microcapsules stabilized with WPI and pectin. SEM observation of optimized microcapsules, approved the predicted particle sizes. XRD test showed no structural changes of biopolymers when they were used as wall materials. FT-IR analysis revealed the importance of electrostatic interaction rather than chemical reactions in stabilization of Z. clinopodiodes EO by WPI and pectin.
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[13] A. Mohammadi, S.M. Jafari, E. Assadpour, A. Faridi Esfanjani, Int. J.Biol. Macromol. 82 (2016) 816-822. [14] W. Liu, X.D. Chen, Z. Cheng, J. Food Eng. 169 (2016) 189-195. [15] E.K. Silva, V.M. Azevedo, R.L. Cunha, M.D. Hubinger, M.A. Meireles, Food Hydrocoll. 56 (2016) 71-83. [16] P. Srivastava, R. Malviya, Indian J. Nat. Prod. Resour. 2(1) (2011) 10-18. [17] L.S. Liu, M.L. Fishman, K.B. Hicks, Cellulose, 14 (2007) 15 –24. [18] E.I. Dı́az-Rojas, R. Pacheco-Aguilar, J. Lizardi, W. Argüelles-Monal, MA. Valdez, M. Rinaudo, M.A. Goycoolea, Food Hydrocoll. 18(2) (2004) 293-304. [19] K.N.P. Humblet-Hua, N. Scheltens, E. van der Linden, L.M.C. Sagis, 2011. Food Hydrocoll. 25(4) (2011) 569-576. [20] A.M. Puga, A.C. Lima, J.F. Mano, A. Concheiro, C. Alvarez-Lorenzo, Carbohydr. Polym. 98(1) (2013) 331-340. [21] L. Aberkane, G. Roudaut, R. Saurel, Food Bioprocess Technol. 7 (2014) 1505–1517. [22] F.N. Souza, C. Gebara, M.C. Ribeiro, K.S. Chaves, M.L. Gigante, C.R.F. Grosso, Food Res, Int. 49 (2012) 560–566. [23] A. Faridi Esfanjani, S.M. Jafari, E. Assadpoor, A. Mohammadi, J. Food Eng. 165 (2015)149–155. [24] A. Mohammadi, S.M. Jafari, A. Faridi Esfanjani, A. Akhavan, Food Chem. 190 (2016) 513-519. [25] B. Yazicioglu, S. Sahin, G. Sumnu, J. Food Sci. Technol. 52(6) (2015) 3590–3597. [26] A. Mirmajidi Hashtjin, S. Abbasi, J. Food Sci. Technol. 52(5) (2015) 2679–2689. [27] H.Y. Zhang, E. Arab Tehrany, C.J.F. Kahn, M. Poncot, M. Linder F. Cleymand Carbohydr. Polym. 88 (2012) 618–627. [28] S. Sheikhzadeh, M. Alizadeh, M. Rezazad, H. Hamishehkar, Agro Food Ind. Hi Tech. 26(6) (2015) 49-52. [29] J.M. Perrier-Cornet, P. Marie, P. Gervais, J. Food Eng. 66(2) (2005) 211-217. [30] S. Kentish, T.J. Wooster, M. Ashokkumar, S. Balachandran, R. Mawson, L. Simons, Innov. Food Sci. Emerg. Technol. 9(2) (2008) 170-175. [31] C. Anandharamakrishnan, C. Rielly, A.F. Stapley, Dairy Sci. Technol. 90 (2010) 321-334. [32] A.G.S. Carvalho, V.M. Silva, M.D. Hubinger, Food Res. Int. 61 (2014) 236-245. [33] V.K. Gupta, D. Pathania, M. Asif, G. Sharma, J. Mol. Liq. 196 (2014) 107–112. [34] R.V.B. Fernandesa, S.V. Borgesa, D.A. Botrel, Carbohydr. Polym. 101 (2014) 524–532. [35] L. Ramos, I. Reinas, S.I. Silva, J.C. Fernandes, M.A. Cerqueira, R.N. Pereira, A.A. Vicente, M.F. Poças, M.E. Pintado, F.X. Malcat, Food Hydrocoll. 30 (2013) 110-122. [36] L. Shi, S. Gunasekaran, Nanoscale Res. Lett. 3 (2008) 491–495. [37] K.E. Patrick, S. Abbas, Y. Lva, I.S.B. Ntsamac, X. Zhang, Pak. J. Food Sci. 23(1) (2013) 17-25.
15
Tables Table 1: Independent variables and their levels for hexagonal design used in RSM Run 1 2 3 4 5 6 7 8 9 10
Core-coating ratio (%) (X1) 10 33 33 55 78 78 100 55 55 55
US power (W) (X2) 100 57 143 100 57 143 100 100 100 100
Table 2. Optimization of encapsulation conditions of Z. clinopodiodes EO microcapsules using WPI and pectin as wall materials. Factor name
Goal
A: core/coating ratio (%) B: US power (W) Particle size (nm)
In range In range Min
Optimized value WPI Pectin 73.42 94.99 50 150 172.94 856.16 16
Desirability (%) WPI Pectin 100 100 100 100 63.60 100
PDI Zeta potential (mV) EE (%) Viscosity (mPa.s) Turbidity (NTU)
Min Min Max None None
0.23 -22.41 74.51 3.59 3243.28
0.19 -23.19 81.65 7.41 4822.96
99.93 82.45 95.69 100 100
100 77.73 100 100 100
Figure captions
Fig. 1. Contour plots of the effects of US power and core-coating ratio on the particle size of Z. clinopodiodes EO emulsions stabilized by WPI (A) and pectin (B).
Fig. 2. Contour plots of the effects of US power and core-coating ratio on the polydispersity index of Z. clinopodiodes EO emulsions stabilized by WPI (A) and pectin (B).
Fig. 3. Contour plots of the effects of US power and core-coating ratio on properties of Z. clinopodiodes EO microcapsules. Zeta potential of microcapsules stabilized by WPI (A) and pectin (B); EE% of WPI (C) and one factor effect of core-coating ratio of pectin on EE% (D).
Fig. 4. SEM micrographs of freeze dried powders of Z. clinopodiodes EO microcapsules stabilized by WPI (A) and pectin (B) under optimized conditions. Fig. 5. XRD diffractograms of pure WPI powder (A), WPI/EO microcapsules (B), pure pectin powder (C) and pectin/EO microcapsules (D).
Fig. 6. FT-IR spectrum of Z. clinopodiodes EO (A), pure WPI powder (B), WPI/EO powdered microcapsules (C), pure pectin powder (D) and EO/pectin powdered microcapsules (E). 17
Figr-1 20
Figr-2
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Figr-3
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Figr-4
25
Figr-5
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Figr-6
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S0141-8130(17)30241-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.190 BIOMAC 7356
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
18-1-2017 14-3-2017 31-3-2017
Please cite this article as: Mahmoud Hosseinnia, Mohammad Alizadeh Khaledabad, Hadi Almasi, Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: a comparative study, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.190 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: a comparative study Mahmoud Hosseinnia, Mohammad Alizadeh Khaledabad*, Hadi Almasi Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran * Corresponding Author: [email protected]
Abstract The performance of whey protein isolate (WPI) and pectin as wall materials in encapsulation of Ziziphora clinopodiodes essential oil by ultrasonication method was compared. In this regard, using the response surface methodology, the influence of ultrasonication (US) power (50-150 W) and core-coating ratio (10-100%) on the properties of microcapsules was evaluated. Increasing US power and core-coating ratio, caused to increase and decrease the particle size, respectively. The polydispersity index (PDI) of WPI coated microcapsules was increased by increasing of US power. The Zeta potential values were increased by increasing of core-coating ratio. Also, the effect of core-coating ratio on encapsulation efficiency was more than US power. Morphological studies by SEM on optimized microcapsules showed regular spherical shapes. X-ray diffraction analysis revealed that the type of the wall material had no effect on the structural properties of the microparticles. FT-IR analysis confirmed the pronounced effect of electrostatic interactions in the formation of microcapsules. Keywords: Microcapsules; ; ; ; ; , Response surface methodology, Ultrasonication, Coating biopolymers, Particle size, Morphology 1. Introduction In recent years, essential oils (EOs) have been considered as natural preservatives. The most of phenolic compounds extracted from different parts of plants like leaves, stems, flowers, and roots are classified as generally recognized as safe (GRAS) [1]. For this reason, nowadays essential oils have more broad classes of applications in food industry, pharmaceutical, cosmetics etc. [2]. Ziziphora clinopodiodes (known as Kakuti) belonging to the family of Lamiaceae is the most common species from genus of Ziziphora L. that has been reported from Iran. The chemical composition of the essential oil of Z. clinopodiodes has been reported. Phenolic constituents including pulegone, 1,8-cineole, thymol, carvacrol, p-cymene and limonene have been reported as major compounds of Z. clinopodiodes EO. Also, methyl acetate, iso-neomenthol, iso-menthone and α-pinene have been reported as the most important constitutes [3,4]. Several studies have been applied on the functional properties of Z. 1
clinopodiodes EO and strong antibacterial, anthelmintic, antifungal and antiviral activities have been reported [5-7]. Using of EOs in food formulations have attracted considerable attention due to consumer awareness on the food quality and food safety. Bioactive compounds of EOs are chemically reactive substances, which can cause considerable problems when used in a complex food system. Negative effects on the physical stability or integrity of the food, poor miscibility and phase separation during storage and the thermal or chemical degradation of the bioactive compounds are the most common shortcomings of direct addition of EOs into a food [8]. A viable and efficient approach widely used for increase the physical stability of the active substances and protecting them from the interactions with the food ingredients is encapsulation of EOs. Reducing of evaporation, to promote easier handling, and release controlling of the encapsulated material during storage are the other advantages of the encapsulation of EOs [9]. The process of encapsulation consists of the formation of a multicomponent structure in the form of micro- or nano- particles consisting of two substances: the core material and the wall material. There are many techniques for encapsulation that each of them have their own advantages and disadvantages generating particles with different properties. Ultrasonication is one of the efficient encapsulation techniques. In an ultrasonic encapsulation, high intensity ultrasound waves can change the characteristics of treated matter through intensive shear forces, pressure, and temperature due to cavitation. The major benefits of this method are including lower energy consumption, smaller droplet size, lower polydispersity, and higher encapsulation efficiency [9]. The selection of a suitable wall material is critical to have a successful microencapsulation. Wide range of biopolymers and hydrocolloids has been used as wall material for microencapsulation of EOs. Whey proteins abundant by-product in the cheese-making industry, have been successfully employed as wall material. The major constituents of whey proteins are β-lactoglobulin and α-lactalbumin [10]. Both whey protein concentrate (WPC) and whey protein isolate (WPI) have been used in encapsulation studies due to their surface active properties [11-15]. Pectin as a structural heteropolysacharide is another widely used wall material. Pectin consists of a linear chain of D-galacturonic acid units joined by means of α(1-4) glycosidic linkage. The carboxylic groups of these uronic acids may be esterified by methyl groups. Galacturonic acid is replaced by α-(1-2)-linked L- rhamnose units at some distinguishing areas [16]. The potential of pectin for using as wall material in encapsulation 2
techniques, drug delivery systems and controlled release formulations is strongly dependent on its molecular weight and degree of esterification [17]. There are many reports about the use of low-methoxyl (LM) and high-methoxyl (HM) pectins in encapsulation of bioactive compounds for food and pharmaceutical uses [18-21]. To the best of our knowledge, there is no report on the encapsulation of Z. clinopodiodes EO. The aim of present study was to prepare Z. clinopodiodes EO microcapsules through ultrasonication method. The mixture of WPI and pectin has been studied recently for microand nano-encapsulation of bioactive compounds [22-24]. However, there is no report on comparison of the potential of WPI and pectin in encapsulation of EOs. The potential of WPI and pectin in encapsulation of Z. clinopodiodes EO was compared in this study. As it mentioned in literature, the core-coating ratio is an important factor that affects the properties of microcapsules [25]. On the other hand, in the production of delivery systems using ultrasonication, some variables such as power and duration of sonication can be effective upon the physical properties of microparticles [26]. Therefore, in this study, we investigated the effect of ultrasonic power and core-coating ratio on encapsulation efficiency of WPI and pectin and some physical properties of microcapsules using response surface methodology (RSM) and the optimal combination of these variables was determined to produce Z. clinopodiodes EO microcapsules by two wall materials with minimum droplet size and highest stability. Afterwards, the structural and morphological properties of microcapsules produced in optimized conditions for WPI and pectin were characterized by FT-IR, XRD and SEM analyses. 2. Materials and methods 2.1. Materials The Z. clinopodiodes leaves were harvested at flowering stage in mid-June 2016 from wild grown plants in the West-Azarbayjan province, Iran. Whey protein isolate (WPI) (85% protein) was purchased from Arla Food Ingredient (Denmark). High methoxyl pectin (degree of esterification ~60 %) and all other reagents were purchased from Sigma (Germany). 2.2 Extraction of essential oil Dried leaves of Z. clinopodiodes, were subjected to the hydro distillation for 3 h using a glass Clevenger type apparatus. The extraction yield was obtained as 2.2%. According to the literature review, the major compounds of Z. clinopodiodes EO are 1,8-cineole, thymol, carvacrol, p-cymene and limonene [3]. The obtained essential oil was stored in sterilized dark glass at 4 °C. 3
2.3. Experimental design Response surface methodology (RSM) was used to evaluate the effects of core-coating ratio (X1) and sonication power (X2) on different responses of Z. clinopodiodes EO microcapsules such as particle size (Y1), viscosity (Y2), Z-average (Y3) and PDI (Y4). Hexagonal experimental design was used to explore the linear, interaction and quadratic effects of independent variables on the studied responses. The quadratic equation for the variables was the following: 𝑌 = 𝛽0 + ∑𝑛𝑖=1 𝛽𝑖 𝑥𝑖 + ∑𝑛𝑖=1 𝛽𝑖𝑖 𝑥𝑖𝑖 + ∑𝑛𝑖=1 ∑𝑛𝑖>𝑗 𝛽𝑖𝑗 𝑥𝑖 𝑥𝑗
(1)
where Y = predicted response, 0 = a constant, i =linear coefficient, ii = squared coefficient, and ij = interaction coefficient. The software Design Expert 7.1.1 was used to analyze the results. The two factors (processing variables), levels and experimental design are given in Table 1. 2.4. Encapsulation process Hydrated solutions of pectin at different concentrations (0.5-1.5% by weight) were prepared by mixing pectin with distilled water using magnetic stirrer. In the case of WPI, their solutions were prepared by dispersing the same amounts of WPI powder into buffer solution (5 mM phosphate buffer, pH 7). The suspensions of coating materials were prepared the day before the encapsulation and kept at room temperature for 24 h to ensure complete saturation of the molecules of biopolymers. The desired amounts of Z. clinopodiodes EO was then slowly incorporated into each biopolymer suspension by mechanical stirring at 5000 rpm for 5 min in a high-speed blender (IKA T25 digital Ultra-Turrax, Selangor, Malaysia) to obtain the preemulsions with desired core to coating ratios according to experimental design (Table 1). After that, ultrasonic homogenizer (UP200Ht, Hielscher, Germany) equipped with titanium probe with diameter 14 mm was used for microencapsulation. Ultrasonic homogenization of 100 g solution was performed in 250 ml beaker using 80% power for different ultrasonic powers as mentioned in Table 1. Ultrasonication process was performed at room temperature for 2 min. Table 1 2.5. Freeze drying Immediately after encapsulation, samples were transferred to 100 ml beakers half fully and placed into freezer at about -4 C°. Frozen samples were then dried under vacuum at -50 C° and 0.007 atm for 48 h. After freeze drying process, samples were grinded by using a glass rod in order to transform them to powder form. 2.6. Particle size analysis 4
The DeBroukere mean diameter (D43) and polydispersity index (PDI) of microcapsules were determined considering the method of Zhang et al., [27]. Dynamic light scattering (DLS) technique employing a Zetasizer Nano-ZS (Malvern instruments, Worcestershire, UK) was used. Dilution with distilled water (1:100) was carried out before the size measurement. All measurements were carried out at 25 °C.
2.7. Measurement of Zeta potential Determination of the surface charge at the interface of the particles dispersed in the aqueous solution is the purpose of Zeta potential measurement. Zeta potential of aqueous emulsion of microcapsules was measured by means of a Malvern Zeta sizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. Before analysis, emulsions were diluted to a particle concentration of 0.01% using distilled water.
2.8. Encapsulation efficiency (EE%) The amount of encapsulated EO in the microcapsules was examined by Sheikhzadeh et al., [28] method with some modifications. The EE% was measured by separation of microcapsules from 1 ml of aqueous emulsions by centrifugation at 13000 rpm for 30 min. A second the filtration of the supernatant was done using a PTFE syringe filter with 0.22 μm pore size. Afterward, the emulsions (40 μl) was diluted to 2 ml with acetone and was then used for estimations. The absorbance at 275 nm was measured with a UV/vis Spectrophotometer (Unico, S 2100 SUV, Dayton, NJ). The EO of Z. clinopodiodes had the maximum absorbance in this wavelength (λmax). Different concentrations of Z. clinopodiodes EO were used for drawing of calibration curve. Total EO content was calculated using the following linear equation based on the calibration curve: A = 0.94C + 8.525 × 10−3 (R2 = 0.9986). Where (A) is the absorbance and (C) is the concentration (mg.mL). The EE% was calculated as follow:
EE%
Total EO amount Free EO amount 100 Total EO amount
(2)
Total EO means the total loaded EO on microcapsules and free EO is released EO after centrifugation of microcapsules emulsion.
2.9. Measurement of viscosity
5
The viscosity of emulsion of microcapsules at concentration of 5% wt. were determined at 25 °C, with a Brookfield rheometer (LV DV-ΙΙΙ Ultra, Brookfield Engineering Laboratories Inc., MA, USA), equipped with a spindle LV2 at 60 rpm after 30 seconds.
2.10. Turbidity measurments The turbidity of emulsions was measured as nephelometric turbidity units (NNU) at 90◦ light scattering and 860 nm with a Nephla reader turbidometer (HKC, Germany).
2.11. Fourier transform infrared (FT-IR) spectroscopy FT-IR analysis was applied to observe the chemical structure of the powders of biopolymers and optimized microcapsules. The samples were prepared by the KBr-pellet method. The powder of samples was pressed into a small pellet about 1 mm thick. FT-IR spectra were analyzed using an FT-IR spectrometer (Nexus 670, USA) operated at a resolution of 4 cm−1 in the range from 4000 to 400 cm−1
2.12. Surface morphology of microcapsules Scanning electron microscope (SEM) was used to investigate the surface morphology and the microstructural properties of the freeze dried encapsulated powders produced in optimized conditions. Samples were coated with a thin conductive gold layer and then were observed using a scanning electron microscope (KYKY-EM3200) at an accelerating voltage from 10 to 20 KV.
2.13. X-ray diffraction XRD studies on powders of wall materials and freeze dried microcapsules of optimized samples were performed with a Siemens D5000 X-ray diffractometer (Germany), equipped with CuKα radiation source (k = 0.154 nm) operating at 40 kV and 30 mA. The samples were scanned over the range of diffraction angle 2θ=2–70° with a scanning rate of 0.5°.min-1 at room temperature. The crystallinity index, a measure of ordered orientation, was calculated by the following equation [15]: CrI (%) ( I 002 I am ) / I 002 100
(3)
where I002 is the maximum intensity of the crystal plane reflection of the biopolymers, Iam is the maximum intensity of X-ray scattering broad band due to the amorphous part of the sample (diffraction intensity at 2θ = 18°). 6
3. Results and discussion 3.1. Particle size analysis Particle size as a function of the studied factors were as follows: 𝑌1 = 145.59 + 4.08 − 2.28 ∗ 𝐵 − 22.34 ∗ 𝐴 ∗ 𝐵 + 85.07 ∗ 𝐴2
(4)
𝑌2 = 1940 − 493.33 ∗ 𝐴 + 618.19 ∗ 𝐵 − 602.77 ∗ 𝐴 ∗ 𝐵 − 151.5 ∗ 𝐴2 − 608 ∗ 𝐵2
(5)
where Y1 , Y2 ,A and B are particle size of WPI stabilized emulsions, particle size of pectin stabilized emulsions, US power and core-coating ratio, respectively.
Fig.1 (A and B) shows the DeBroukere mean diameter (D43) of microcapsules prepared by WPI and pectin as a function of US power and core-coating ratio. By comparing of particle size values in figures A and B, it can be concluded the particles obtained by WPI have a lower diameter than those obtained by pectin. This could be explained by the good emulsifying property of WPI related to the many hydrophobic and hydrophilic parts of WPI [25]. Thus, WPI has a higher ability to adsorb at the oil-water interface and can reduce the interfacial tension. However, the pectin has not an amphiphilic nature and its performance at the rapid absorption on the surface of EO droplets is less than WPI. Similar results were reported by Silva et al., [15] who compared the stabilization efficiency of WPI and modified starch in encapsulation of annatto seed oil. As shown in Fig.1B, the D43 values of pectin coated microcapsules were increased by increasing of US power. But the effect of US power on the diameter of WPI coated microcapsules was dependent on core-coating ratio (Fig.1A). At the ratios higher than 50%, increasing of US power caused to decrease the D43 of WPI coated particles, but at lower ratios, its effect was same as that observed for pectin. Perrier-Cornet et al., [29] proposed that an overly intense homogenization process can increase the size of the particles due to recoalescence, a phenomenon that is known as “over-processing”. The results of this research indicated that the intense of this phenomenon is different for various biopolymers. WPI can rapidly adsorb on the surface of the EO droplets formed during homogenization and significantly reduce the interfacial tension. It also can provide a coating that prevents the droplets from aggregating with neighboring droplets. But these effects for pectin are not as 7
effective as WPI. Therefore, reduction in the size of the encapsulated particles is not solely a function of the amount of energy supplied to the system. The lowest particle size was recorded at the middle core-coating ratio of EO and WPI (146 nm). The D43 values were increased by increasing and decreasing of EO-WPI ratios far from 50%. However, the mean diameter of pectin coated particles was decreased by increasing corecoating ratio. This means that the stabilization mechanisms of pectin and WPI are different. Steric stabilization is the dominant stabilization effect of pectin. But, the surface activity and emulsifying ability are the major functions of WPI. At the lower concentrations of WPI, the size of EO droplets was increased and at the higher EO-WPI ratios, the increasing of accumulated and aggregated WPI molecules on the surface of EO droplets causes to formation of a thick surface layer that leads to increase the diameter of microcapsules.
Fig. 1
Fig.2 shows the PDI changes of microcapsules as a function of US power and core-coating ratio. The effect of US power on PDI values was dependent on the type of biopolymer. For WPI coated microcapsules, the PDI was increased by increasing US power but increasing of ultrasonication intensity in pectin stabilized emulsions caused to decrease of PDI. The acoustic cavitation phenomenon is an effective parameter on size distribution of particles in US homogenized emulsions. This phenomenon promotes intense shear rates resulting from the formation and collapse of microbubbles associated with highly localized turbulence levels [30]. This mechanical stress is able to denature of WPI molecules and thus, the formation of protein aggregates in the emulsion may have been interpreted as larger droplets formed in sonicated emulsion in severe intensities. By increasing of US power, the greater acoustic cavitation effects were occurred and therefore, higher PDI values were expected. The increasing of temperature during sonication process can promote this phenomenon. Silva et al., [15] reported that PDI of WPI and modified starch stabilized annatto seed oil was increased by increasing ultrasonication power. They suggested that the denaturation of WPI and gelatinization of modified starch due to the acoustic cavitation phenomenon are the reasons of this increment. However, an adverse effect was observed for the emulsions stabilized with pectin and PDI was decreased significantly by increasing US power (Fig.2B). Because there is no gelatinization or denaturation behavior of pectin under the influence of ultrasonication process. As shown in Fig.2A, the PDI was decreased and uniform microparticles was formed in WPI coated samples. 8
But the effect of core-coating ratio on the PDI of pectin stabilized emulsions was not statistically significant.
3.2. Zeta potential Data analysis showed that variation in zeta potential can be characterized by the following models: 𝑌3 = −18.81 − 1.34 ∗ 𝐴 + 0.84 ∗ 𝐵 + 4.1 ∗ 𝐴 ∗ 𝐵 − 3.26 ∗ 𝐴2
(6)
𝑌4 = −11.82 − 5.74 ∗ 𝐴 + 6.82 ∗ 𝐵 − 4.23 ∗ 𝐴2 − 9.75 ∗ 𝐵2
(7)
where Y3 , Y4 ,A and B are zeta potential of WPI stabilized emulsions, zeta potential of pectin stabilized emulsions, US power and core-coating ratio, respectively. The Zeta potential values of microcapsules are shown in Fig. 3 (A and B). Charged surfaces with negative potentials were recorded for microparticles stabilized by both of WPI and pectin. WPI has net negative charge at pH values higher than its isoelectric pH. Pectin also has negative charge at neutral pH due to presence of carboxyl groups on galacturonic acid units. The Zeta potential of -25.7 mV was reported by Souza et al., [22] for WPC at pH of 7 that is similar to the results of current study. Also, they reported that the Zeta potential of HM pectin is lower than LM pectin and has a decreasing trend by increasing of pH. The pH values of pure WPI and pectin solutions (measured by Metrohm pH meter, 780, Swiss) were 6.74 and 2.92, respectively. The pH was not significantly affected by core-coating ratio or US power.
Fig. 2
The results showed that increasing of US power has a decreasing effect on Zeta potential of pectin coated particles. Similar trend was also observed for WPI coated sample at higher corecoating ratios. This means that the increasing of US power causes to decrease the thickness of biopolymers on the surface of EO droplets. Therefore, the higher intensities of ultrasonication would have an adverse effect on encapsulation process. The Zeta potential values were also increased by increasing core-coating ratios and this increment was more obvious for WPI stabilized microcapsules.
3.3. Encapsulation efficiency Encapsulation efficiency was modeled as follows: 9
𝑌5 = 67.58 + 41.95 ∗ 𝐴 + 0.14 ∗ 𝐵 − 60.20 ∗ 𝐴2
(8)
𝑌6 = 48.41 + 37.4 ∗ 𝐴
(9)
where Y5 , Y6 ,A and B are encapsulation efficiency of WPI stabilized emulsions, encapsulation efficiency of pectin stabilized emulsions, US power and core-coating ratio, respectively. Fig.3 (C and D) shows the encapsulation efficiency (EE%) as a function of the US power and core-coating ratio. The EE% values obtained for WPI were more than pectin that could be attributed to the emulsifying ability of amphiphilic WPI. For example, at a same core-coating ratio (55%) and US power (100 w), the EE% of WPI was 60.9% but it was calculate as 38.4% for pectin. The effect of core-coating ratio on EE% of WPI was more than US power (Fig.3C). Also, the effect of US power on the EE% of pectin stabilized microcapsules was not significant. Fig.3C shows that the intense of ultrasonication is not an effective factor in entrapment capacity of WPI. At a constant core-coating ratio, increasing of US power had no significant effect on EE% of WPI coated microcapsules. So, it could be concluded that the effect of chemical structure and nature of wall materials on EE% is more than process parameters. Also, it shows that the US treatment has no significant disrupting effect on structure of biopolymers. Similar results were reported by Mirmajidi Hashtjin and Abbasi [26] and Yazicioglu et al., [25] for the effect of sonication on biopolymers. In the case of WPI, the EE% was increased by increasing core-coating ratio up to 70% and was decreased again at higher ratios. The highest EE% was recorded equal to 72.74% for core-coating ratio of 70%. Similar to our results, Faridi Esfanjani et al., [23] reported EE% of 84.53% for encapsulation of safranal with WPC at core-coating ratio of 75%. The EE% values were also increased by increasing of EO-pectin ratios but it was not decreased at higher ratios. This difference reveals that the encapsulation potential of pectin is more than WPI and is able to entrap a higher amount of EO especially at higher concentrations of core material.
3.4. Viscosity and turbidity The contour plots of the effects of US power and core-coating ratio on the viscosity and turbidity of Z. clinopodiodes EO emulsions stabilized by WPI and pectin are shown in Fig. S1 (Supplementary data). The viscosity of pure WPI and pectin solution at the same concentration of microcapsules (5% wt.) was 4.65 and 50.1 mPa.s respectively. Higher molecular weight and hydrophilic nature of pectin are the reasons of this difference. For both of pectin and WPI, the US power had no significant effect on viscosity of emulsions. This result shows that sonication 10
has no significant effect on structure of biopolymers and prepared microcapsules. However, as it was expected, the viscosity was decreased significantly by increasing of core-coating ratio in the emulsions stabilized by both of WPI and pectin. By increasing of core-coating ratio, the amount of biopolymers was decreased in emulsions and thus, the bonded water was decreased and caused to decrease of viscosity. Lower molecular weight of chemical compounds of EO in comparison to biopolymers, causes to decrease viscosity by increasing of EO in formulation of microcapsules. Similar trends were observed for turbidity of emulsions. US power had no significant effect on turbidity but it was increased by increasing core-coating ratio. It shows that the effect of EO on increasing of turbidity is more than biopolymers. The suspensions of pure pectin and WPI are clear with some cloudiness. Turbidity of pure WPI and pectin solutions were 2341 and 3679 NTU, respectively. Addition of EO increased the turbidity of emulsions. Z. clinopodiodes EO had a yellow color and appearance of its emulsions was milky white. This color was the reason of turbidity and was increased by increasing EO content in emulsions. Also, the EE% was decreased by increasing of core-coating ratio. The release of un-encapsulated EO from the surface of microcapsules was another reason for increasing of turbidity.
Fig. 3
3.5. Optimization and desirability One of the main objectives of this study was to determine the optimal values of the independent variables to generate microcapsules with minimum droplet size. The Desirability Function method was used for the multi-objective optimization. The desirability function for individual responses were estimated by numerical methods and the overall desirability function was calculated. Results from statistical analysis lead to finding the optimum US power and corecoating ratio according to physical properties of microcapsules, are shown in Table 2. The optimum values of independent variables including ultrasonication power and core-coating ratio to achieve the best-predicted values of particle size (172.94 nm for WPI and 856.16 nm for pectin) were 50 W, 73.42% and 150 W, 94.99% for WPI and pectin, respectively. It should be noted that the overall desirability values of the predicted zone were equivalent to 0.842 and 0.934% for WPI and pectin, respectively. At the second step of this research, two microparticles were prepared according to the optimized conditions of WPI and pectin and the structural and morphological properties of freeze dried microcapsules were investigated. 11
Table 2
3.6. Surface morphology of optimized microcapsules Fig.4 shows the SEM micrographs of WPI (A) and pectin (B) coated microcapsules at optimized conditions. The both of freeze dried powders had regular spherical shapes without apparent pores or fractures. The morphology of the both of microparticles obtained by ultrasonication method was similar. Also, Anandharamakrishnan et al., [31], Carvalho et al., [32], Silva et al., [15] and Liu et al., [14] reported similar morphologies for WPI encapsulated EOs produced by spray drying method, indicating that the type of encapsulation method did not influence the microstructure of the particles. However, as expected, the type of biopolymer that was used as wall material yielded microparticles with different diameters. As it was shown in Fig.1 and was predicted in optimization step, microparticles obtained by pectin exhibited larger size than the WPI coated microcapsules. From the SEM micrographs, the average size of around 211 nm were calculated for WPI coated microcapsules, but it was 730 nm when pectin was used as wall material. A deviation from optimum conditions predicted by RSM method was that the PDI values predicted for WPI was larger than pectin (0.23 vs 0.19). However, as it can be seen from Fig.4, the polydispersity and size distribution in pectin coated microparticles is higher than that of WPI stabilized microcapsules and the uniformity of particle size in WPI/EO microparticles is more obvious. Fig. 4
3.7. XRD analysis X ray diffraction patterns of WPI and pectin and their powdered microparticles are presented in Fig. 5. XRD results showed a typical amorphous structure for both of WPI and pectin with a broad peak at around 2θ=25° for both of them. The crystallinity index of 26.45% and 16.11% was calculated for WPI and pectin, respectively. These are in agreement with XRD results that are reported for pectin [33] and WPI [14] in earlier researches. The results indicated that the EO incorporation had no distinct effect on crystallinity of WPI and pectin, even though the intensity of mentioned broad peaks was slightly decreased for microcapsules (crystallinity index of 17.50% and 6.42% for WPI/EO and pectin/EO microcapsules respectively). So, it could be concluded that there is not any strong interaction between EO and wall materials. This hypothesis was approved by FT-IR test (section 3.8). The observed slight decrease in crystallinity of microcapsules could be attributed to the effect of sonication on structure of 12
biopolymers [26]. Even though it was not observed any change in EE% and chemical structure (FT-IR) of WPI and pectin after sonication. Similar XRD patterns were reported for microparticles of WPI containing annatto seed oil [15], rosemary EO [34] and curcumin [14]. Fig. 5
3.8. FT-IR analysis Fig.6 presents the FT-IR spectrum of Z. clinopodiodes EO, WPI and pectin powders and their powdered microparticles loaded with EO. Fig. 6A indicates the FT-IR spectrum of the Z. clinopodiodes EO. The absorption band at 3424 cm-1 is related to OH vibrations of carvacrol, p-cymene and limonene compounds in the Z. clinopodiodes EO. The sharp peak at 2961 cm-1 is attributed to the asymmetric C-H stretching of the methyl groups that are present in most of phenolic compounds. The asymmetrical and symmetrical bending vibrations of methyl group are seen at 1460-1340 cm-1, respectively. Absorption peaks of around 1580 cm-1 are related to the vibrations of C=C groups. The pure WPI powder showed several specific absorption peaks (Fig.6B). A broad peak appeared at 667 cm-1 is related to C-N groups. The peaks ranging 10751158 cm-1 are related to N-H bonds and C-N stretching bonds (Amide type III). Amide type II structure displayed the peaks ranging 1390-1550 cm-1 related to bending groups of N-H. A sharp peak was observed at 1652 cm-1 that is related to vibratory stretching bonds of groups C=O (Amide type I). A peak at 2927 cm-1 is assigned to C-H stretching bond [35]. Fig.6D presents the FT-IR spectrum of pure pectin. C=O or C=C double bonds of pectin showed a peak at 1067 cm-1. The absorption peaks at range of 1238-1440 cm-1 and also absorption band of 1640 cm-1 are assigned to stretching bands of COO- groups. Two peaks at 1748 and 2937 cm-1 are related to carboxyl and CH2 groups of pectin, respectively [36]. The peaks ranging 3000-3600 cm-1 in spectrum of both of WPI and pectin are related to free O-H groups. By comparing of spectra obtained for WPI/EO and pectin/EO powders (Fig6.C and E) with native biopolymers, no specific new peaks were found in microparticles suggesting that no generation of new chemical bond is accrued between wall materials and EO compounds. This results confirms the formation of complexes promoted by electrostatic interaction rather than chemical reactions. Therefore, it could be concluded that the interaction between Z. clinopodiodes EO and WPI or pectin is based on electrostatic force. This type of only physical incorporation of EO in WPI or pectin matrix, preserves the intrinsic nature of the bioactive compounds of EO. However, the release rate of EO from microcapsules would be increased without accruing chemical bonding. The release profile of microcapsules bas been discussed 13
and will be published in the next manuscript. Partic et al., [37] reported similar results for microencapsulation of fish oil by gelatin.
Fig. 6
4. Conclusion The fabrication and characterization of Z. clinopodiodes EO microcapsules prepared by ultrasonication method were carried out. WPI and pectin were two wall materials and optimum values of ultrasonication power and core-coating ratio were determined by RSM method. Intensification of the ultrasonication process had negative effect in the reduction of the droplets size of microcapsules. Smaller microparticles were obtained by WPI in comparison to pectin that was attributed to amphiphilic structure and emulsifying ability of WPI. However, PDI values was lower for pectin coated microcapsules. The effect of core-coating ratio on viscosity and turbidity of emulsions was more than US power. This study showed the capability of RSM in optimization of ultrasonication process to produce Z. clinopodiodes EO microcapsules stabilized with WPI and pectin. SEM observation of optimized microcapsules, approved the predicted particle sizes. XRD test showed no structural changes of biopolymers when they were used as wall materials. FT-IR analysis revealed the importance of electrostatic interaction rather than chemical reactions in stabilization of Z. clinopodiodes EO by WPI and pectin.
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15
Tables Table 1: Independent variables and their levels for hexagonal design used in RSM Run 1 2 3 4 5 6 7 8 9 10
Core-coating ratio (%) (X1) 10 33 33 55 78 78 100 55 55 55
US power (W) (X2) 100 57 143 100 57 143 100 100 100 100
Table 2. Optimization of encapsulation conditions of Z. clinopodiodes EO microcapsules using WPI and pectin as wall materials. Factor name
Goal
A: core/coating ratio (%) B: US power (W) Particle size (nm)
In range In range Min
Optimized value WPI Pectin 73.42 94.99 50 150 172.94 856.16 16
Desirability (%) WPI Pectin 100 100 100 100 63.60 100
PDI Zeta potential (mV) EE (%) Viscosity (mPa.s) Turbidity (NTU)
Min Min Max None None
0.23 -22.41 74.51 3.59 3243.28
0.19 -23.19 81.65 7.41 4822.96
99.93 82.45 95.69 100 100
100 77.73 100 100 100
Figure captions
Fig. 1. Contour plots of the effects of US power and core-coating ratio on the particle size of Z. clinopodiodes EO emulsions stabilized by WPI (A) and pectin (B).
Fig. 2. Contour plots of the effects of US power and core-coating ratio on the polydispersity index of Z. clinopodiodes EO emulsions stabilized by WPI (A) and pectin (B).
Fig. 3. Contour plots of the effects of US power and core-coating ratio on properties of Z. clinopodiodes EO microcapsules. Zeta potential of microcapsules stabilized by WPI (A) and pectin (B); EE% of WPI (C) and one factor effect of core-coating ratio of pectin on EE% (D).
Fig. 4. SEM micrographs of freeze dried powders of Z. clinopodiodes EO microcapsules stabilized by WPI (A) and pectin (B) under optimized conditions. Fig. 5. XRD diffractograms of pure WPI powder (A), WPI/EO microcapsules (B), pure pectin powder (C) and pectin/EO microcapsules (D).
Fig. 6. FT-IR spectrum of Z. clinopodiodes EO (A), pure WPI powder (B), WPI/EO powdered microcapsules (C), pure pectin powder (D) and EO/pectin powdered microcapsules (E). 17
Figr-1 20
Figr-2
22
Figr-3
24
Figr-4
25
Figr-5
26
Figr-6
27
28