Microencapsulation by Complex Coacervation Using ...

Food Bioprocess Technol DOI 10.1007/s11947-015-1521-0

ORIGINAL PAPER

Microencapsulation by Complex Coacervation Using Whey Protein Isolates and Gum Acacia: An Approach to Preserve the Functionality and Controlled Release of β-Carotene Ashay Jain 1 & Deepika Thakur 1 & Gargi Ghoshal 1 & O. P. Katare 2 & U. S. Shivhare 1

Received: 11 January 2015 / Accepted: 2 April 2015 # Springer Science+Business Media New York 2015

Abstract β-Carotene is a red–orange pigment, a known source of vitamin A and has exceptional antioxidant and free radical scavenging potential. However, uses of β-carotene in food industry are inadequate mostly because of their poor water solubility and low stability. Using the complex coacervation technique, the work is meant to fabricate the microcapsules of β-carotene, to examine the physicochemical properties of microcapsules and finally to evaluate the extent of stability improvement. The configuration of electrostatic complexes between whey protein isolate (WPI) and gum acacia (Acacia arabica, GA) was optimized as a function of pH, ionic strength, WPI/GA ratio, core material load and size of final micromolecules. The optimum process conditions were balanced by the ratio of wall materials WPI/GA 2.0/1.0 % and pH value 4.2. Morphological observations showed that microcapsules presented spherical shape, and smooth and continuous surface. The effective amount of encapsulated core was greater than 70 % for all formulations evaluated. In vitro release data indicated an initial burst release followed by sustained release behavior. The microstructure and viscoelastic properties of WPI and GA complex were studied using dynamic rheometer. The encapsulation method and the wall materials used in this work gave effective protection during storage and eventually resulted sustained release of bioactive while used in food matrix, at suitable conditions.

* Gargi Ghoshal [email protected] 1

Dr. S. S. Bhatnagar University Institute of Chemical Engineering & Technology, Panjab University, Chandigarh 160014, India

2

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

Keywords Antioxidants . β-Carotene . Complex coacervation . Gum arabic . Microcapsules . Whey protein isolate

Introduction Microencapsulation is a distinctive and versatile technique which has been extensively used in pharmaceutical, food, agricultural, pesticide, cosmetic, textile, and other related fields. It provides a controlled release and allows the protection of a wide range of active substances (Borgogna et al. 2010; Desai and Park 2007; Madene et al. 2006; Peña et al. 2012). Microencapsulation creates a microenvironment within the capsule able to control the interactions between the external part and the internal one which ensure protection of sensitive food components against nutritional loss, offer controlled-release, mask the bitter taste, and preserve fragrance. Reactive, sensitive, and volatile additives can be stabilized through microencapsulation (Hsieh et al. 2006; Jain et al. 2012; Yang et al. 2014). The same description makes microencapsulation appropriate for biomedicine and biopharmaceutics, cell therapy to drug delivery, and food industry applications, in particular for the manufacturing of high value nutraceuticals and aliments. Microencapsulation ensures the stability of certain compounds during processing and storage and prevents detrimental reactions within the food matrix, in the gastrointestinal tract and to allow a site-specific controlled release (Herrero et al. 2006; Polavarapu et al. 2011). Complex coacervation is a spontaneous phase separation phenomenon based on the simultaneous desolvation of oppositely charged polyelectrolytes in colloidal systems under defined conditions (Ma et al. 2009). The more intense polymerrich phase in colloid component is the coacervate and another very dilute polymer-deficient phase exists in equilibrium

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solution. Generally, the biopolymers include a proteinaceous molecule and a polysaccharide molecule (Jun-xia et al. 2011; Ma et al. 2009). Complex coacervation is a promising and alternative to microencapsulation for sensitive and unstable compound and can achieve very high payloads with controlled release possibilities (Reineccius 1989). Microcapsules produced by coacervation method acquire admirable controlled release characteristics, heat-resistant properties, and high encapsulation efficiency therefore evident in numerous applications including processed food, cosmetics, and pharmaceuticals. There is a wide range of coating materials available for fabrication of microcapsules. Coating materials include polysaccharides, gums, proteins, and fatty acids. Gum arabic is a complex polysaccharide and a weak polyelectrolyte that carries carboxyl groups (Jayme et al. 1999). This polysaccharide presents a remarkable low viscosity and good emulsifying properties, which lead to the conglutination of macromolecules. Gum arabic is a biocompatible, biodegradable, nontoxic, and cheap polysaccharide, therefore found attractive for fundamental interest and will aid in advancing many of the emerging applications in pharmaceutical and food industry (Islam et al. 1997; Nickerson et al. 2006). Whey protein isolate is a dietary supplement and food ingredient created by separating translucent liquid part of milk that remains following the process (coagulation and curd removal) of cheese manufacturing. Whey proteins are highly bioavailable and have a high concentration of essential and branched chain amino acids (BCAAs). The bioactivities of these proteins possess many beneficial properties as well. Additionally, whey is also rich in vitamins and minerals, evident in baked goods, salad dressings, emulsifiers, infant formulas, and medical nutritional formulas (Jovanovi et al. 2005). Carotenoids are a major class of naturally occurring pigments that primarily encompass of 600 types of pigments. The most abundant are β-carotene (beta carotene), lutein, zeaxanthin, and lycopene. Carotenoids have extremely conjugated structure; therefore, extensive exposition to light heat and acids may cause isomerization of trans-carotenoids (stable form) to cis (unstable form) promoting slight loss of color, provitamin, and quenching the free radicals/singlet oxygen activity. Among the carotenoids, beta carotene (C40H56) is a strongly red–orange colored pigment and usually present in the fruits and vegetables (Franceschi et al. 2010). The main role of β-carotene in human health is as vitamin A precursor and antioxidants and therefore widely used in food, cosmetic, and pharmaceutical industry. Former studies which were made with synthetic polyelectrolytes, and systems with biopolymers currently used in food products industries have been inadequately investigated so far. The present approach Bzoom out^ the influence of varying the concentrations of the wall materials (whey protein and gum

arabic, WP/GA) and then look upon the coacervates as Bnew^ self-assembled colloidal entities as illustrated for the complex of gum arabic and whey protein containing β-carotene as a core. Study also delineates the controlled release of βcarotene and achieves a microparticulate system which provides high thermostability, long residual action of core material, and nontoxic characteristics, so as to expand the applications of β-carotene in food industry.

Materials and Methods Materials β-Carotene (BC) was procured from Sigma-Aldrich (Munich, Germany). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Fluka (Buchs, Switzerland), and gum acacia was purchased from Himedia (Mumbai, India). Whey protein isolate was purchased from NATURE’S BEST (Hauppauge, NY). Dialysis tube (MWCO 10–12 kDa), methanol, ethyl acetate, and acetonitrile were purchased from Himedia (India). All reagents were of analytical grade and were used as received. Preparation of Gum Acacia and Whey Protein Isolate Stock Dispersions One percent gum acacia dispersion was prepared by adding the weighed amounts of polysaccharide powders in distilled water under gentle stirring at 40 °C for 2 h, and the aqueous solutions of 2 % whey protein isolate were prepared by dissolving known amounts of biopolymer powders in distilled water at room temperature under gentle stirring for 2 h; then both samples were incubated at 40 °C overnight to ensure the complete hydration of the macromolecules. Microencapsulation Technique A total of 2 g whey protein isolate (WPI) was suspended in 100 ml distilled water and subjected to ultrasonication (ultrasonic bath sonicator, PCI, Mumbai, India) for 10 min at 25± 1 °C. Then, the mixture was kept over mechanical stirrer (Remi, Mumbai, India) at 40 °C for 20 min. Subsequently, 5 ml of soya oil containing 0.5 % BC was added, the mixture was homogenized at 4000 rpm for 30 min to obtain oil in water emulsions, i.e., the aqueous phase containing the WPI was the continuous phase, and the oil was the disperse phase. Then, 100 ml 1 % (w/v) GA solution was added to the emulsion, and the mixture was allowed to stir, while temperature was maintained at 40 °C. Next, to promote coacervation, the pH was lowered until the coacervates formed with 1 N HCl solution at approximately 40 °C, under constant mechanical stirring at 600 rpm (Weinbreck et al. 2003). The reaction

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mixture was then allowed to cool in ice bath under stirring. Gluteraldehyde was added to the dispersion for cross-linking of coating material and allowed to agitate for 1 h for completion of reaction. The coacervated material was stored at 7 °C overnight to promote decantation. Subsequently, the coacervats were filter and washed thrice for complete removal of unreacted cross-linking agent. To get powdered microcapsules, the coacervates was lyophilized and stored for further analysis. Determination of Coacervate Yield The effects of pH, ionic strength, and WPI/GA ratio on WPI/ GA coacervations were monitored by coacervate yield. The coacervate yield was calculated according to the following equation: Dry weight o f coacervates  100 Coacervate yieldð%Þ ¼ Total weight o f WPI and GA in the solution

PEE One hundred milligrams of dried microcapsules were moistened with adequate amount of purified water and added to 50 ml ethyl acetate and placed in a water bath at room temperature. The mixture was sonicated for 1 h with continuous stirring at 150 rpm to extract the BC completely from the microcapsules (Yang et al. 2014). Then, the insoluble substance in the mixture was removed by centrifugation at 5000 rpm, for 10 min. One milliliter of supernatants were used to determine the BC using the HPLC method. In brief, HPLC (Shimadzu, Japan) analysis was performed isocratically at room temperature (25 °C), using Merck C-18 column (150 mm×4.6 mm i.d., particle size 5 μm) and methanol/acetonitrile/ethyl acetate (80:10:10, v/v) as the mobile phase, previously degassed using bath sonicator for 15 min. The flow rate was 2 ml min−1, and the PDA detector was set at 449 nm. The injection volume was 20 μl for all the samples. Encapsulation efficiency of the microcapsules was calculated according to the formulas as follows: EEð%Þ ¼

β‐carotene encapsulated in a certain weight ofmicrocapsules  100 Total weight of β‐carotene oil used in the preparation of microcapsules

Characterization of Microencapsules

dispersed in purified water, and dispersion was analyzed. The particle size measurement and distribution of the average particles were analyzed in triplicate with suspended particles in water. Zeta Potential The zeta potential of microparticles was measured by laserbased multiple angle particle electrophoresis analyzer, Malvern Zetasizer (DTS Ver. 4.10, Malvern Instruments, England). Microparticles were dispersed in deionized water, placed in the electrophoretic cell with an electric field of 15.24 V cm−1, and the zeta potential was determined. Surface Morphology Surface morphology was determined by scanning electron microscope (SEM). The samples were prepared by lightly sprinkling the ME powder on a double adhesive tape which was stuck on an aluminum stub. The stubs were then coated with gold to a thickness of about 300 Å by using a sputter coater. All samples were examined under a scanning electron microscope (JSM-6100, JEOL, Tokyo, Japan) at an accelerated voltage of 10 kV and magnification in the range of×100– 2000. Confocal Laser Scanning Microscopy Fluorescent microcapsules (MEs-F) using Coumarin-6 marker were prepared to investigate the deposition pattern of oil within the MEs by adding 3.4×10–3 mmol of coumarrin-6 in oil instead of BC, while the other components of the formulations were kept fixed. The MEs-F were sprinkled over glass slid, covered with glass cover slip. The MEs-F was observed under a confocal laser microscope (CLSM) (Nikon C2+). Fourier Transform Infrared Spectroscopy Study FTIR spectra of GA, WPI, and microcapsules (MEs) were recorded at room temperature (28±2 °C) with a Tensor-27 spectrophotometer (Bruker, Germany) in the range of 600– 4000 cm−1 by accumulating 16 scans at 4-cm−1 resolution. For measuring the spectra of GA, WPI, and lyophilized powders of MEs, platinum ATR (attenuated total reflectance) from Bruker, Germany, was used. About 2 mg of each samples were placed on ATR for measurement. After that, the peak intensity of each sample was measured. This entire spectra acquisition procedure took 1 min per sample.

Particle Size Rheological Studies Characterization of microparticles in terms of average size was done by laser diffraction particle size analyzer mastersizer-2000 (Malvern Instruments, UK). Samples were

The steady shear flow measurements were carried out at different shear rates (γ) of 0.1–100 s−1 using a controlled stress

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Modular compact rheometer (Anton Paar GmbH, Austria MCR 102 model), with a cone and plate fixture (75-mm diameter and 0.149-mm gap) and a Peltier system for temperature control (±0.01 °C). In the tested shear rate range, the torque values were within the specifications of rheometer (>0.1 μN m). Colloidal coacervate sample was rested between plates during 5 min at the studied temperature before conducting the measurements. Data were recorded and processed by Rheoplus software (version 3.62) provided with the instrument (Anton Paar GmbH, Germany). The fitting of shear stress vs. shear rate was carried out by means of the Hershel Buckley model, proposed for the description of flow behavior (Chenlo et al. 2011). τ ¼ τо þ K:γ

Stability Study The physical and chemical stabilities of microencapsules involved observation of different attributes, viz. possible changes in physical appearance like discoloration, change in odor, and residual content. Formulations were evaluated at three different storage conditions, i.e., in refrigerated condition (RF; 5±2 °C), room temperature (RT; 25±2 °C), and elevated temperature (HT; 40±2 °C) for a period of 3 months in terms of physical appearance and percent residual content of BC in the formulations. Antioxidant Activity by DPPH Method

n

where τ is shear stress, τo is the yield stress, γ is the shear rate, K is the consistency index, and n is the flow behavior index of the sample. For the oscillatory strain sweep investigation, the samples were exposed to increasing strain (0.1–100 %) at a constant frequency to determine the linear viscoelastic region (LVR) of the samples. Then, selected strain values in the linear region (usually 1 %) were used in the other oscillation tests. In oscillatory frequency sweep experiment, the samples were exposed to a stepwise increase of angular frequency, ω 0.1–100 rad s−1 at a constant strain (at LVR). The angular frequency (expressed in units Radian per second) and the storage (elastic) G′ and loss (viscous) G″ moduli, both in Pa, were plotted on a logarithmic scale. Graphical data were processed to determine the angular frequencies at which crossing takes place between storage modulus and loss modulus by crossover model (Angelico et al. 2013). In Vitro Release Study Dialysis tube diffusion technique was employed to assess the in vitro release of BC from the microencapsules formulations. One gram of prepared micro particles formulations located into the dialysis tube (MWCO 10–12 kDa, Hi Media, India), tied at both ends, and suspended in beakers (receptor compartment) each containing 100 ml of 0.1 N HCl and 2.0 % Tween 80. The medium was stirred (Remi, Mumbai, India) continuously at 100 rpm, and the whole system was assembled at 37± 1 °C temperature throughout the exercise. Samples were withdrawn periodically, and after each withdrawal of sample, same volume of 0.1 N HCL was added in the receptor compartment so as to maintain a constant volume throughout the study. The samples were analyzed by HPLC method reported in previous section, so as to quantify in vitro BC release (Jain et al. 2010). Same procedure has been followed to study in vitro release in different biological fluid viz. water, phosphate buffer solution, and PBS (pH 7.4).

Antioxidant activity of freeze-dried MEs was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) DPPH assay, following the method reported in the literature (Jain et al. 2013). Briefly, a stock solution (1 μg ml−1) of free BC was prepared in methanol. Freeze-dried blank MEs (without BC) and MEs loaded with BC were also dissolved in methanol to completely extract the encapsulated BC. A 100-μL portion of methanolic solution of free BC and the extracted BC from MEs were mixed with identical volume of DPPH (0.3 mM) reagent. The reaction mixture was allowed to incubate in the dark at room temperature for 30 min, and the absorbance (A) of samples was recorded at 517 nm using UV spectrophotometer (Simadzu, Japan). A control sample was prepared to calculate the radical scavenging followed by an equation:   Asample scavenging activityð%Þ ¼ 1−  100 Acontrol The antioxidant activity of free BC, plain MEs, and BC loaded MEs was determined using the above protocol at different time interval and up to 3 months to study the effect of coating over the BC.

Results and Discussion Preparation of Microencapsules Optimum pH of microencapsulation received at 4.2. Since pH may induce structural transitions of proteins and polysaccharides (Turgeon et al. 2007), the pH of the system has a paramount importance in the coacervation between WPI and GA, of which pH 4.2 resulted in the highest degree of coacervation. The procedure involves emulsification followed by phase separation and then leads to rigidization of biopolymer mix (Fig. 1). Microelectrophoretic measurements demonstrated that gum arabic is negatively charged above pH 2.2; therefore, at low pH (G′ and recommends that the system has more tendency to be transit into liquid state at lower angular

Fig. 5 a Flow curve of coacervates between apparent viscosities and shear rate under changing shear rate of 0.1–100 s−1. b Frequency sweep at 1 % strain under changing angular frequency of 0.1–100 rad s−1

Food Bioprocess Technol Fig. 6 In vitro cumulative % release of BC from microencapsules

frequencies (Fig. 5b). From the application of crossover model, 13.59 rad s−1 angular frequency was found at which crossover take place between storage and loss modulus. All the above results indicate that the microencapsules can be used in any food matrixes whether it is liquid, paste or solid viscous, viscoelastic or elastic type of food products. The rheological behavior of microencapsulated BC will also allow the food matrixes flow freely or through pipe irrespective of temperature or pH of the media. Release Profile of Microcapsules The release profile of BC microcapsules was studied in 0.1 N HCl containing 2.0 % Tween 80 (Fig. 6). This experiment somewhat emulates what would happen in GI fluid where the pH conditions might not necessarily be acidic enough for all the bioactive molecules to dissolve. The release manners of BC from the biopolymers matrix showed a biphasic pattern that is characterized by an initial burst, followed by a slower sustained release. However, the release rate of oil from microcapsules did not exactly follow the modified first-order kinetic model. It might be caused because of BC oil distributed on the surface or surface layer which was easier to release in the primary release stage or initial burst effect might be associated with the loss of wall integrity during particles production or after particle drying (Jain et al. 2010; Wang et al. 2002). Once

Fig. 7 Stability study of BC-loaded microencapsules

the oil on the surface layer released completely, thereafter, the next release stage would remain relatively slow and constant because of BC oil in microcapsule mainly released through the microcapsules wall by penetration, solubilization, and diffusion effect, diffusion of drug molecules through the biopolymeric matrix of the MEs. MEs have released BC in a sustained manner; this may be attributed to the presence of the protective coat of biopolymer mix on the droplet of oil containing BC, which interferes with the release of BC. Thus, the cumulative oil release rate was higher within the first release stage followed by slower and constant release. Moreover, finely diffused oil droplet with very high surface area would dissolve much faster in GI fluid than the micronized powder (if administered) and thus be available for absorption. This release potential may also be beneficial in the improvement of bioavailability of BC by maintaining the concentration gradient (BC concentration) between GI fluid and plasma. Stability Study The stability data indicated that formulation stored at room temperature and refrigerated conditions were stable for the time, and they had tested in terms of residual content (Fig. 7). Residual content in the formulations was determined by assuming the initial drug content to be 100 %, and it was

Fig. 8 Scavenging activity (%) of free BC, blank MEs, and BC-loaded MEs (BC-MEs). Each data point represents mean±SD (n=6)

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decreased with time. Microencapsules which were stored at elevated temperature have shown minimal residual content when compared to microencapsules stored at room temperature and refrigerated condition. It might be possible because rate of oxidative reactions become faster with increasing the temperature. However, microencapsules show better stability in terms of residual content. Particle size of microcapsules remained constant. However, in the succeeding months, a slight increase in particle size was ascertained. Antioxidant Activity The percent scavenging activity of free BC, freeze-dried blank MEs, and freeze-dried BC-loaded MEs is presented in Fig. 8. BC showed very strong antioxidant activity, which was retained even after its encapsulation within the protein–polysaccharide matrix. On the other hand, free freeze-dried blank MEs showed negligible scavenging activity against DPPH. In the present work, encapsulation of BC was also intended to effectively reduce the oxidative stress generated during the course of 3 months. Therefore, it was necessary to substantiate the antioxidant activity of MEs in order to confirm functional stability of BC within the MEs. DPPH colorimetric assay is a widely employed method for evaluating the functional activity of various antioxidants (Date et al. 2011). An insignificant difference in the percent scavenging activity of free BC alone or its encapsulation in MEs indicated that the functional architecture of BC was not affected after encapsulation. Notably, decreased scavenging activity of free BC after time period further confirmed that antioxidant activity of BC was solely contributed by micro-carriers which provide a safeguard to BC for the time period.

Conclusions Very recently, numerous studies have been reported for the encapsulation of various micronutrient including BC. The various encapsulation procedures by mixing different matrices including alginate-chitosan (Han et al. 2008), tapioca starchmaltodextrin (Loksuwan 2007), Furcellaran (Laos et al. 2007), kappa carrageenan-carboxymethyl cellulose, and WPI-alginate-chitosan (Muhamad et al. 2011) were employed to improve the physicochemical properties of BC. However, there are no studies reported on BC-loaded MEs which were prepared by complex coacervation technique using WPI and GA. The present study described a systematic and comprehensive study of complex coacervation phenomenon using WPI and GA and developed a novel microcapsules containing BC. However, all the perceptive of this phenomenon cannot be cognized without understanding the kind of interactions between the polyelectrolytes and the thermodynamic mechanisms concerned in the complexation. The interaction

between WPI and GA was initiated at pH 6.0, followed by the onset of phase separation at a pH value of 4.5, and subsequently, the huge number of complexes was obtained at pH 4.2. The SEM analysis showed that the microencapsules had spherical shape and smooth surface. CLSM image of fluorescent MEs revealed the internalization of oil well within the MEs. Almost 77.3 % of encapsulation efficiency was obtained when WPI/GA ratio was 2:1. In vitro studies depict the initial burst release followed by sustained release nature of formulation. Microencapsules formulation show better stability in terms of residual content when compared with free BC. MEs loaded with BC showed strong antioxidant activity, which was retained even after the course of 3 months. It was concluded that the microencapsules containing BC developed by complex coacervation approach and the encapsulation method and the wall materials used in this work provided effectual safety during storage and permissible sustained release. Acknowledgments The authors are grateful for the financial support received from ICAR, New Delhi, India.

References Angelico, R., Carboni, M., Lampis, S., Schmidt, J., Talmon, Y., Monduzzi, M., & Murgia, S. (2013). Physicochemical and rheological properties of a novel monoolein-based vesicle gel. Soft Matter, 9(3), 921–928. Borgogna, M., Bellich, B., Zorzin, L., Lapasin, R., & Cesàro, A. (2010). Food microencapsulation of bioactive compounds: rheological and thermal characterisation of non-conventional gelling system. Food Chemistry, 122(2), 416–423. Butstraen, C., & Salaün, F. (2014). Preparation of microcapsules by complex coacervation of gum Arabic and chitosan. Carbohydrate Polymers, 99, 608–616. Chenlo, F., Moreira, R., & Silva, C. (2011). Steady-shear flow of semidilute guar gum solutions with sucrose, glucose and sodium chloride at different temperatures. Journal of Food Engineering, 107(2), 234–240. Date, A. A., Nagarsenker, M. S., Patere, S., Dhawan, V., Gude, R. P., Hassan, P. A., et al. (2011). Lecithin-based novel cationic nanocarriers (Leciplex) II: improving therapeutic efficacy of quercetin on oral administration. Molecular Pharmaceutics, 8(3), 716– 726. Desai, K. G. H., & Park, H. J. (2007). Recent developments in microencapsulation of food ingredients. Drying Technology, 23(7), 37–41. Franceschi, E., Cezaro, A. D., Ferreira, S. R. S., Kunita, M. H., Edvani, C., Rubira, A. F., & Oliveira, J. V. (2010). Co-precipitation of betacarotene and Bio-polymer using supercritical carbon dioxide as antisolvent. The Open Chemical Engineering Journal, 4, 11–20. Han, J., Guenier, A.-S., Salmieri, S., & Lacroix, M. (2008). Alginate and chitosan functionalization for micronutrient encapsulation. Journal of Agricultural and Food Chemistry, 56(7), 2528–2535. Herrero, E., Valle, E., & Galan, M. (2006). Development of a new technology for the production of microcapsules based in automization processes. Chemical Engineering Journal, 117(2), 137–342. Hsieh, W.-C., Chang, C.-P., & Gao, Y. L. (2006). Controlled release properties of chitosan encapsulated volatile citronella Oil

Food Bioprocess Technol microcapsules by thermal treatments. Colloids and Surfaces B: Biointerfaces, 53(2), 209–214. Islam, A. M., Phillips, G. O., Sljivo, A., Snowden, M. J., & Williams, P. A. (1997). A review of recent developments on the regulatory, structural and functional aspects of gum arabic. Food Hydrocolloids, 11(4), 493–505. Jain, A., Agarwal, A., Majumder, S., Lariya, N., Khaya, A., Agrawal, H., & Agrawal, G. P. (2010). Mannosylated solid lipid nanoparticles as vectors for site-specific delivery of an anti-cancer drug. Journal of Controlled Release, 148(3), 359–367. Jain, K., Kesharwani, P., Gupta, U., & Jain, N. K. (2012). A review of glycosylated carriers for drug delivery. Biomaterials, 33, 4166– 4186. Jain, S., Jain, A. K., Pohekar, M., & Thanki, K. (2013). Novel selfemulsifying formulation of quercetin for improved in vivo antioxidant potential: implications for drug-induced cardiotoxicity and nephrotoxicity. Free Radical Biology & Medicine, 65, 117–130. Jayme, M., Dunstan, D., & Gee, M. (1999). Zeta potentials of gum arabic stabilised oil in water emulsions. Food Hydrocolloids, 13(6), 459– 465. Jordan, S. L., Russo, M. R., Blessing, R. L., & Theis, A. B. (1996). Inactivation of glutaraldehyde by reaction with sodium bisulfite. Journal of Toxicology and Environmental Health, 47(3), 299–309. Jovanovi, S., Bara, M., & Ma, O. (2005). Whey proteins-properties and possibility of application. Mljekarstvo, 55(3), 215–233. Jun-xia, X., Hai-yan, Y., & Jian, Y. (2011). Microencapsulation of sweet orange oil by complex coacervation with soybean protein isolate/ gum Arabic. Food Chemistry, 125(4), 1267–1272. Laos, K., Lõugas, T., Mändmets, A., & Vokk, R. (2007). Encapsulation of β-carotene from sea buckthorn (Hippophaë rhamnoides L.) juice in furcellaran beads. Innovative Food Science & Emerging Technologies, 8(3), 395–398. Loksuwan, J. (2007). Characteristics of microencapsulated β-carotene formed by spray drying with modified tapioca starch, native tapioca starch and maltodextrin. Food Hydrocolloids, 21(5–6), 928–935. Ma, Z.-H., Yu, D.-G., Branford-White, C. J., Nie, H.-L., Fan, Z.-X., & Zhu, L.-M. (2009). Microencapsulation of tamoxifen: application to cotton fabric. Colloids and Surfaces B: Biointerfaces, 69(1), 85–90. Madene, A., Jacquot, M., Scher, J., & Desobry, S. (2006). Flavour encapsulation and controlled release - a review. International Journal of Food Science and Technology, 41(1), 1–21. Muhamad, I. I., Fen, L. S., Hui, N. H., & Mustapha, N. A. (2011). Genipin-cross-linked kappa-carrageenan/carboxymethyl cellulose beads and effects on beta-carotene release. Carbohydrate Polymers, 83(3), 1207–1212.

Mukai-Corrêa, R., Prata, A. S., & GROSSO, C. R. F. (2003). Microcapsules obtained by ionic polymerization. In Release of casein encapsulated in alginate and pectin. World Aquaculture, 2. Nickerson, M. T., Patel, J., Heyd, D. V., Rousseau, D., & Paulson, A. T. (2006). Kinetic and mechanistic considerations in the gelation of genipin-crosslinked gelatin. International Journal of Biological Macromolecules, 39(4–5), 298–302. Peña, B., Panisello, C., Aresté, G., Garcia-Valls, R., & Gumí, T. (2012). Preparation and characterization of polysulfone microcapsules for perfume release. Chemical Engineering Journal, 179, 394–403. Polavarapu, S., Oliver, C. M., Ajlouni, S., & Augustin, M. A. (2011). Physicochemical characterisation and oxidative stability of fish oil and fish oil–extra virgin olive oil microencapsulated by sugar beet pectin. Food Chemistry, 127(4), 1694–1705. Reineccius, G. A. (1989). Flavor encapsulation. Food Reviews International, 5(2), 147–176. Schmitt, C. (2000). Effect of protein aggregates on the complex coacervation between β-lactoglobulin and acacia gum at pH 4.2. Food Hydrocolloids, 14(3), 403–413. Schmitt, C., Sanchez, C., Thomas, F., & Hardy, J. (1999). Complex coacervation between β-lactoglobulin and acacia gum in aqueous medium. Food Hydrocolloids, 13(6), 483–496. Turgeon, S. L., Schmitt, C., & Sanchez, C. (2007). Proteinpolysaccharide complexes and coacervates. Current Opinion in Colloid and Interface Science, 12(4–5), 166–178. Wang, J., Wang, B. M., & Schwendeman, S. P. (2002). Characterization of the initial burst release of a model peptide from poly(D, L-lactideco-glycolide) microspheres. Journal of Controlled Release, 82(2–3), 289–307. Wang, X., Lee, J., Wang, Y.-W., & Huang, Q. (2007). Composition and rheological properties of beta-Lactoglobulin/pectin coacervates: effects of salt concentration and initial protein/polysaccharide ratio. Biomacromolecules, 8(3), 992–997. Weinbreck, F., de Vries, R., Schrooyen, P., & de Kruif, C. G. (2003). Complex coacervation of whey proteins and gum arabic. Biomacromolecules, 4(2), 293–303. Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & De Kruif, C. G. (2004). Complexation of whey proteins with carrageenan. Journal of Agricultural and Food Chemistry, 52(11), 3550–3555. Yang, Z., Peng, Z., Li, J., Li, S., Kong, L., Li, P., & Wang, Q. (2014). Development and evaluation of novel flavour microcapsules containing vanilla oil using complex coacervation approach. Food Chemistry, 145, 272–277.