PRODUCTION AND CHARACTERIZATION OF ALGINATE AND PECTIN MICROPARTICLES COATED WITH ISOLATED SOY PROTEIN

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1 PRODUCTION AND CHARACTERIZATION OF ALGINATE AND PECTIN MICROPARTICLES COATED WITH ISOLATED SOY PROTEIN B.S.Costa ¹; F. L. Schmidt ², C. R. F. Grosso ³ 1- Department of Food Technology, University of Campinas, School of Food Engineering, CEP Campinas - SP- Brazil, phone: +55 (19) mail: (biancasouza09@yahoo.com.br). 2- Department of Food Technology, University of Campinas, School of Food Engineering, CEP Campinas - SP- Brazil, phone: +55 (19) mail: (schmidt@fea.unicamp.br). 3- Department of Food and Nutrition, University of Campinas, School of Food Engineering, CEP Campinas - SP- Brazil, phone: +55 (19) mail: (grosso@fea.unicamp). ABSTRACT Particles may be formed from mixtures of polysaccharides and proteins based on the interaction between these biopolymers. They can carry lipophilic bioactive compounds protecting them against gastric conditions and controlling the release of these molecules. This study aimed to produce particles of pectin and alginate coated by electrostatic interaction using isolated soy protein (ISP). These particles were evaluated for their stability by in vitro simulation of gastrointestinal conditions, ph changes and different concentrations of NaCl. The use of an ISP solution at 8% protein concentration resulted in microparticles with high protein content adsorbed (47.61%). The stability test showed that high concentrations of NaCl and low ph destabilized the protein layer with subsequent release to the environment, however, the particles remained intact. The particles coated with isolated soy protein (ISP Part ) were resistant to gastric conditions, but were disintegrated in the intestinal environment, releasing the encapsulated material. KEYWORDS: microencapsulation, ionic gelation, electrostatic interaction, isolated soy protein. 1. INTRODUCTION Alginates and pectins can associate with divalent ions through ionic interactions to form gels. These gel matrices enable the encapsulation of hydrophobic compounds, such as polyunsaturated fatty acids, however, the formed particles have porous surfaces. The combined techniques, such as ionic gelation and complexation with cationic polyelectrolytes (proteins), may reduce the porosity of the particle, improving the protection of the encapsulated oil. These particles may carry and protect lipophilic bioactive compounds against human gastrointestinal environment in order to control the release of these molecules (De Vos et al, 2010). Some researches have been conducted to obtain particles with different release forms in order to release the bioactive compound at the place of action with the required speed (Liu et al., 2007; Hoad et al., 2009). Therefore, it is necessary to study the behavior of these particles and their stability in different environments. The development of the vegetable protein application in combination with

2 polysaccharides for microencapsulation, such as the isolated soybean protein, is interesting because it is a cheap renewable raw material from a biodegradable source (Nesterenko et al., 2013). The aim of this work was to produce and characterize particles of pectin and alginate (PEC:ALG Part ) obtained by ionic gelation and coated with isolated soy protein (ISP Part ). Initially, the zeta potential of alginate-pectin solutions and ISP solutions and different ratios of mixtures were evaluated. The microparticles were characterized in terms of their morphology, size and quantity of adsorbed protein. These particles were also evaluated for their stability under in vitro simulation of gastrointestinal conditions as well as against ph changes and different concentrations of NaCl. 2. MATERIALS AND METHODS 2.1. Materials The following materials were used in this study: High molecular weight sodium alginate (ALG) with a high guluronic acid content sodium alginate (batch MANUGEL DMB, FMC Biopolymer, Campinas - SP, Brazil), citrus pectin (PEC) GENU with an amidated low-methoxyl content (CP Kelco, Limeira-SP, Brazil, content of galacturonic acid (GA) 81.3 ± 1.2%, degree of esterification (DE) 30.4 ± 1.6% and degree of amidation (DA) 10.1 ± 1.0%, FAO (2009), isolated soy protein (ISP) ( batch: , New Max Industrial, Americana-SP, Brazil, ± 1.93% protein, 5.68 ± 0.02% ash, AOAC (2006), 0.30 ± 0.01% lipids, Bligh and Dyer (1959), anhydrous calcium chloride (Dinâmica, Diadema - SP, Brazil, batch 36308), 0.2 N hydrochloric acid (Merck, Germany), sodium hydroxide (Nuclear, Diadema - SP, Brazil), sulfuric acid (Synth, Diadema e SP- Brazil), commercial sunflower oil as a model oil and all reagents used were of analytical grade Production and characterization of the microparticles The proportion of polysaccharide and protein was established based on the zeta potential from previous studies of the dilute test. These studies resulted in a proportion of 1:0.75 at ph 3 for interaction with the ISP. The encapsulated lipophilic compound was sunflower oil for all treatments. For the production of the microparticles, it was prepared one emulsion with an aqueous solution of 2% pectin and alginate (1:1 - w/w of solution), ph of 3.0 and sunflower oil (2% w/w of solution) using a Turrax at rpm/3 min (IKA, R.J., Brazil). The emulsion was atomized on a 2% (w/v) calcium chloride solution at ph 3.0 using a double fluid atomizer with diameter of 1 mm, height of 12 cm between the atomizer and calcium chloride solution, an air pressure of kgf/cm 2 and an atomization speed of 555 ml/h. The particles obtained by ionic gelation were coated with the protein by electrostatic interaction. These particles were transferred to an ISP solution at 8% protein concentration and maintained for 30 additional minutes, under agitation. The particles were washed with deionized water at ph 3.0 to remove the proteins that were not adsorbed on the particle surface. The protein and dry matter concentrations of the particles were determined according to the methods of the AOAC (2006) using nitrogen-to-protein conversion factors of The nitrogen content of the pectin particles was determined and used to correct the total nitrogen content of the

3 particles containing adsorbed proteins. The average size (d 0.5) of the moist particles was determined by laser diffraction method using the Mastersizer 2000 apparatus (Malvern, Worcestershire, WR, UK) with the Hydro 2000S sampling unit (Malvern, Worcestershire, WR, UK). The morphology of wet pectin-alginate particles with and without protein coverage was observed by optical microscopy (OM) (JENAVAL, Carl Zeiss, Toronto, Canada). The microstructure of the lyophilized particles was obtained by scanning Electron Microscopy (SEM) (JMS model e T300 Jeol, Tokyo, Japan) Stability of the microparticles under different conditions The particles were evaluated for their stability under ph changes, different concentrations of NaCl and under in vitro simulation of gastrointestinal conditions. The effect of different exposure conditions of the particles was evaluated by morphology, size and protein solubility. All tests for the analyses of protein solubility used the Kjeldahl method (total nitrogen content). These particles coated with isolated soybean protein were evaluated for their stability under different values of ph (1, 4 and 7) and different concentrations of NaCl (100 mm (0.6%), 200 mm (1.2%), 400 mm (2.4%) and 580 mm (3.5%)). In this assay, approximately two grams of particles coated with protein (ISP Part ) were suspended in two different types of solutions: 20 ml of deionized water with adjusted ph and solutions with the different NaCl aforementioned concentrations. For the simulation of gastrointestinal conditions, two grams of moist microparticles and 20 ml of simulated gastric juice (SGJ) with adjusted ph of 2 were incubated at 37 C under agitation for two hours. Subsequently, the ph was changed to 7.0 with a NaHCO 3 solution (20% (w/v)) and pancreatin was added at a concentration of 1.95 g/l and then this solution was submitted to five hours of incubation. The SGJ was a mixture of 1.12 g/l of KCl, 2 g/l of NaCl, 0.11 g/l of CaCl 2, 0.4 g/l of KH 2 PO 4, 3.5 g/l of mucin and 0.26 g/l of pepsin and HCl was added to adjust the ph (Sultana, 2000). 3. RESULTS AND DISCUSSION The particles of ionic gelation without protein coating (PEC: ALG Part ) presented an average size of ± 3.46 μm. For an 8% protein concentration in ISP solution, high levels of protein adsorption were obtained, resulting in a percentage of adsorbed protein. After the protein adsorption on the surface, using the ISP solution, the particles (ISP Part ) increased their size significantly (p <0.05), with mean size of ± 3.14 μm. The images by optical microscopy show that the particles with or without protein coating have similar morphology, spherical shape and lipid content distributed in the matrix particle (figure 1). The SEM image of the particle surface without the protein coating (PEC: ALG Part ) shows visible drops of the lipid material dispersed throughout the polysaccharide matrix, which is characterized by a discontinuous and heterogeneous surface (figure 1). The particle coated with isolated soybean protein (ISP Part ) exhibits a more continuous and homogeneous surface, with a roughness that may be associated with the protein layer adhered to the particle surface. The protein coating resulted in a dense surface that probably reduced the particle porosity, giving greater protection for the oil encapsulated within the particle. This type of morphology was

4 similar to the results found by Mendanha et al. (2009) that encapsulated the casein hydrolysate by complex coacervation using pectin and isolate soybean protein as wall materials, obtaining spherical shape particles. Figure 1. Morphology and microstructure of the particles of ionic gelation without protein coating (PEC: ALG Part ) and particles coated with isolated soybean protein (ISP Part ). Sample Morphology (OM) Microstructure (SEM) PEC:ALG Part ISP Part OM bar = 100 μm; SEM bar = 10 μm. In the evaluation of protein solubility of the particles at different values of ph, it was observed a significant protein solubilization in conditions of extreme acidity. These losses were significant (p <0.05) for ph 1 (15.48 ±0.97%) compared to ph 4 and ph 7, respectively 2.83 ± 0.45%, 2.53 ±0.45%. The particles coated with isolated soybean protein (ISP Part ) remained with similar particle size ( µm) when they were submitted to the ph 1 and ph 4, but showed a significant decrease size when exposed to a solution with ph 7 ( µm). In the test using different concentrations of NaCl, the amounts of solubilized protein were: 4.46 ± 0.67 %; 4.32 ± 0.5 %, 5.66 ± 0.59 %; 6.85 ± 0.45 %, for concentrations 100 mm, 200 mm, 400 mm and 540mM, respectively. These results show that particles coated with ISP have a low solubility even at high molar concentrations of salt (584mM), maintaining 93.15% of protein adhered in the particle. One possible explanation is that the globular structure and the chemical configuration of the protein provide higher input resistance of salt ions, thus hindering protein solubilization. The coated particles with ISP increased significantly when submitted to a concentration of 584 mm, resulting in a size of µm. The addition of high molar concentrations of NaCl may influence the permeability of the particle by the rupture of bonds between the polyelectrolyte of opposite charge, which may cause swelling of the particle and can result in the protein solubilization in the saline solution (SHE et al., 2010). Despite the partial protein solubilization, for different ph values and different molar concentrations of NaCl used in these tests, the particles remained intact with the withheld lipid content.

5 In the gastrointestinal assay, the pectin and alginate particles (PEC:ALG Part ) were resistant to the gastric and enteric conditions, remaining intact (figure 2). The particles coated with isolated soy protein (ISP Part ) showed high protein solubility (62.51 ± 1.68%), indicating that the particles are extremely fragile to acidic ph in the presence of pepsin. Nevertheless, the particle structure has not changed, maintaining the spherical shape with the lipid content retained inside. This particle was disintegrated in the intestinal environment, releasing their encapsulated lipid material (figure 2). Figure 2. Morphology of the particles during in vitro gastrointestinal simulation (2 hours with pepsin at ph 2 at 37 C and after 5 and 17 hours with pancreatin at ph 7). Sample ph 2 2h ph 7 5h ph 7-17h PEC:ALG Part ISP Part OM bar = 100 μm The electrostatic interaction between hydrocolloid-protein associations may have compromised some carboxylic groups-calcium ions, weakening the particles. Despite the presence of salts in the gastric conditions, they did not affect the morphological integrity of the particles. However, the ionic strength in the intestinal simulation may have contributed to the weakening of the particles. 4. CONCLUSION The results of the stability test showed that at high concentrations of NaCl and at a low ph, the protein layer was destabilized with subsequent release to the environment, but the particles remained intact with the lipid content retained inside. In the gastrointestinal assay, pectin and alginate particles (PEC:ALG Part ) uncoated were resistant to the gastric and enteric conditions, remaining intact. The particles coated with isolated soy protein (ISP Part ) were resistant to gastric conditions, but disintegrated in the intestinal environment, releasing the encapsulated material. The type of protein interaction with

6 the surface of the particle will interfere in the control of the lipid content release. This different behavior over intestinal conditions is an important factor for the type of application of such particles when it is necessary to release the lipid content quickly or slowly in the intestine, according to the required application. 5. REFERENCES AOAC. Association of official analytical chemist s official methods of analysis (2006). 16th Ed. Washington. De Vos, P., Faas, M. M., Spasojevic, M., Sikkema, J. (2010) Encapsulation for preservation of functionality and targeted delivery of bioactive food components. International Dairy Journal, 20, Hoad, C., Rayment, P., Cox, E., Wright, P., Butler, M., Spiller, R., Gowland, P. (2009). Investigation of alginate beads for gastro-intestinal functionality, Part 2: In vivo characterisation. Food Hydrocolloids, 23 (3), Liu, L., Fishman, M., Hicks, K. (2007) Pectin in controlled drug delivery a review. Cellulose, 14 (1), Mendanha, D. V., Ortiz, S. E. M., Favaro-Trindade, C. S., Mauri, A., Monterrey- Quintero, E. S., Thomazini, M. (2009). Microencapsulation of casein hydrolysate by complex coacervation with SPI/pectin. Food Research International, 42, Nesterenko, A., Alric, I., Silvestre, F., Durrieu, V. (2013). Vegetable proteins in microencapsulation: a review of recent interventions and their effectiveness. Industrial Crops and Products, 42, She, Z.; Antipina, M. N.; Li, J.; Sukhorukov, G. B. (2010). Mechanism of protein release from polyelectrolyte multilayer microcapsules. Biomacromolecules, 11, Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K. (2000). Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt. International Journal of Food Microbiology, 62,