This work investigated the stabilization mechanisms of annatto seed oil emulsions, rich in δ-tocotrienol, by employing biopolymers such as gum arabic (GA), whey protein isolate (WPI), modified starch (Hi-Cap and SF), polyethylene glycol (PEG) and inulin as emulsifiers/stabilizers. The effects of emulsification by high-intensity ultrasound were evaluated and compared to emulsification by mechanical stirring. The research in this study was focused on the ζ-potential and pH of the biopolymer suspensions, the dynamic tension on the interface between the annatto seed oil and water, the viscosity and rheological behavior, the size distribution and average diameter of the droplets, the optical microscopy and the creaming index of the emulsions. The steric stabilization mechanism predominated over electrostatic in the emulsions obtained with GA, WPI, Hi-Cap and SF. The stabilization of the emulsions with PEG or inulin was associated with a reduction in the oil droplet size and an increase in the viscosity of the continuous phase. In general, the emulsification efficiency achieved using ultrasound was greater than that of the mechanical stirring process for all biopolymers because it resulted in the formation of more stable emulsions over time as well as monomodal and narrow size distributions with smaller droplets, thus indicating uniform processing.47 113(2000) Approved methods of the American Association of Cereal Chemists, , American Association of Cereal Chemists, Saint PaulAbbas, S., Bashari, M., Akhtar, W., Li, W.W., Zhang, X., Process optimization of ultrasound-assisted curcumin nanoemulsions stabilized by OSA-modified starch (2014) Ultrasonics Sonochemistry, 21 (4), pp. 1265-1274Albuquerque, C.L.C., Meireles, M.A.A., Defatting of annatto seeds using supercritical carbon dioxide as a pretreatment for the production of bixin: experimental, modeling and economic evaluation of the process (2012) Journal of Supercritical Fluids, 66, pp. 86-94Álvarez Cerimedo, M.S., Iriart, C.H., Candal, R.J., Herrera, M.L., Stability of emulsions formulated with high concentrations of sodium caseinate and trehalose (2010) Food Research International, 43 (5), pp. 1482-1493Amine, C., Dreher, J., Helgason, T., Tadros, T., Investigation of emulsifying properties and emulsion stability of plant and milk proteins using interfacial tension and interfacial elasticity (2014) Food Hydrocolloids, 39, pp. 180-186(2000) Official methods of analysis of the Association of the Official Analytical Chemists, p. 2200. , Association of Official Analytical Chemists, Arlington(2004) Standard test method for determination of water in petroleum products, lubricating oils, and additives by coulometric Karl Fischer titration, , ASTM D 6304-04, American Society Standard Testing and Materials, PhiladelphiaBohmer, M.R., Evers, O.A., Scheutjens, J.M.H.M., Weak polyelectrolytes between two surfaces. Adsorption and stabilization (1990) Macromolecules, 23 (8), pp. 2288-2301Bos, M.A., Van Vliet, T., Interfacial rheological properties of adsorbed protein layers and surfactants: a review (2001) Advances in Colloid and Interface Science, 91 (3), pp. 437-471Bot, A., Erle, U., Vreeker, R., Agterof, W.G.M., Influence of crystallisation conditions on the large deformation rheology of inulin gels (2004) Food Hydrocolloids, 18 (4), pp. 547-556Bouyer, E., Mekhloufi, G., Huang, N., Rosilio, V., Agnely, F., β-Lactoglobulin, gum arabic, and xanthan gum for emulsifying sweetalmond oil: formulation and stabilization mechanisms of pharmaceutical emulsions (2013) Colloids and Surfaces A: Physicochemical and Engineering Aspects, 433, pp. 77-87Bouyer, E., Mekhloufi, G., Potier, I.L., Kerdaniel, T., Grossiord, J.-L., Rosilio, V., Stabilization mechanism of oil-in-water emulsions by β-lactoglobulin and gum arabic (2011) Journal of Colloid and Interface Science, 354 (2), pp. 467-477Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J.-L., Agnely, F., Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: alternatives to synthetic surfactants in the pharmaceutical field? (2012) International Journal of Pharmaceutics, 436 (1-2), pp. 359-378Brigelius-Flohé, R., Traber, M.G., Vitamin E: function and metabolism (1999) FASEB Journal, 13 (10), pp. 1145-1155Camino, N.A., Pilosof, A.M.R., Hydroxypropylmethylcellulose at the oil-water interface. Part II. Submicron-emulsions as affected by pH (2011) Food Hydrocolloids, 25 (5), pp. 1051-1062Carneiro, H.C.F., Tonon, R.V., Grosso, C.R.F., Hubinger, M.D., Encapsulation efficiency and oxidative stability of flaxseed oil microencapsulated by spray drying using different combinations of wall materials (2013) Journal of Food Engineering, 115 (4), pp. 443-451Chandrapala, J., Oliver, C., Kentish, S., Ashokkumar, M., Ultrasonics in food processing (2012) Ultrasonics Sonochemistry, 19 (5), pp. 975-983Chemat, F., Zill e, H., Khan, M.K., Applications of ultrasound in food technology: processing, preservation and extraction (2011) Ultrasonics Sonochemistry, 18 (4), pp. 813-835Damodaran, S., Protein stabilization of emulsions and foams (2005) Journal of Food Science, 70 (3), pp. R54-R66Desrumaux, A., Marcand, J., Formation of sunflower oil emulsions stabilized by whey proteins with high-pressure homogenization (up to 350MPa): effect of pressure on emulsion characteristics (2002) International Journal of Food Science and Technology, 37 (3), pp. 263-269Dickinson, E., Hydrocolloids at interfaces and the influence on the properties of dispersed systems (2003) Food Hydrocolloids, 17 (1), pp. 25-39Dickinson, E., Radford, S.J., Golding, M., Stability and rheology of emulsions containing sodium caseinate: combined effects of ionic calcium and non-ionic surfactant (2003) Food Hydrocolloids, 17 (2), pp. 211-220Dokić, L., Krstonošić, V., Nikolić, I., Physicochemical characteristics and stability of oil-in-water emulsions stabilized by OSA starch (2012) Food Hydrocolloids, 29 (1), pp. 185-192Dubey, R.R., Parikh, R.H., Studies of PLGA microspheres (2004) Pharmaceutical Technology Europe, 16 (5), pp. 23-34Flanagan, J., Singh, H., Microemulsions: a potential delivery system for bioactives in food (2006) Critical Reviews in Food Science and Nutrition, 46 (3), pp. 221-237Floury, J., Desrumaux, A., Axelos, M.A.V., Legrand, J., Effect of high pressure homogenisation on methylcellulose as food emulsifier (2003) Journal of Food Engineering, 58 (3), pp. 227-238Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R., Applications of spray-drying in microencapsulation of food ingredients: an overview (2007) Food Research International, 40 (9), pp. 1107-1121Glibowski, P., Effect of thermal and mechanical factors on rheological properties of high performance inulin gels and spreads (2010) Journal of Food Engineering, 99 (1), pp. 106-113Glibowski, P., Pikus, S., Jurek, J., Kotowoda, M., Factors affecting inulin crystallization after its complete dissolution (2014) Carbohydrate Polymers, 110, pp. 107-112Gonnet, M., Lethuaut, L., Boury, F., New trends in encapsulation of liposoluble vitamins (2010) Journal of Controlled Release, 146 (3), pp. 276-290Gottlieb, N., Schwartzbach, C., Development of an internal mixing two-fluid nozzle by systematic variation of internal parts (2004) Proceedings of the Americas, , Institute for Liquid Atomization and Spray Systems Conference, NottinghamGouin, S., Microencapsulation: industrial appraisal of existing technologies and trends (2004) Trends in Food Science & Technology, 15 (7-8), pp. 330-347Hunter, R.J., (1981) Zeta potential in colloid science, , Academic Press, LondonIdris, O.H.M., Williams, P.A., Phillips, G.O., Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography (1998) Food Hydrocolloids, 12 (4), pp. 379-388Jafari, S.M., Assadpoor, E., He, Y., Bhandari, B., Encapsulation efficiency of food flavours and oils during spray drying (2008) Drying Technology, 26 (7), pp. 816-835Jafari, S.M., Assadpoor, E., He, Y., Bhandari, B., Re-coalescence of emulsion droplets during high-energy emulsification (2008) Food Hydrocolloids, 22 (7), pp. 1191-1202Jafari, S.M., Beheshti, P., Assadpoor, E., Rheological behavior and stability of d-limonene emulsions made by a novel hydrocolloid (Angum gum) compared with Arabic gum (2012) Journal of Food Engineering, 109 (1), pp. 1-8Jafari, S.M., He, Y., Bhandari, B., Nano-emulsion production by sonication and microfluidization - a comparison (2006) International Journal of Food Properties, 9 (3), pp. 475-485Jafari, S.M., He, Y., Bhandari, B., Production of sub-micron emulsions by ultrasound and microfluidization techniques (2007) Journal of Food Engineering, 82 (4), pp. 478-488Jayme, M.L., Dunstan, D.E., Gee, M.L., Zeta potentials of gum arabic stabilised oil in water emulsions (1999) Food Hydrocolloids, 13 (6), pp. 459-465Kaltsa, O., Michon, C., Yanniotis, S., Mandala, I., Ultrasonic energy input influence on the production of sub-micron o/w emulsions containing whey protein and common stabilizers (2013) Ultrasonics Sonochemistry, 20 (3), pp. 881-891Kentish, S., Wooster, T.J., Ashokkumar, M., Balachandran, S., Mawson, R., Simons, L., The use of ultrasonics for nanoemulsion preparation (2008) Innovative Food Science & Emerging Technologies, 9 (2), pp. 170-175Kim, Y., Faqih, M.N., Wang, S.S., Factors affecting gel formation of inulin (2001) Carbohydrate Polymers, 46 (2), pp. 135-145Koocheki, A., Kadkhodaee, R., Mortazavi, S.A., Shahidi, F., Taherian, A.R., Influence of Alyssum homolocarpum seed gum on the stability and flow properties of O/W emulsion prepared by high intensity ultrasound (2009) Food Hydrocolloids, 23 (8), pp. 2416-2424Kuhn, K.R., Silva, F., Netto, F.M., Cunha, R., Assessing the potential of flaxseed protein as an emulsifier combined with whey protein isolate (2014) Food Research International, 58, pp. 89-97Li, M.K., Fogler, H.S., Acoustic emulsification - 1. The instability of the oil-water interface to form the initial droplets (1978) Journal of Fluid Mechanics, 88, pp. 499-511Li, M.K., Fogler, H.S., Acoustic emulsification - 2. Breakup of the large primary oil droplets in a water medium (1978) Journal of Fluid Mechanics, 88, pp. 513-528Linse, P., Adsorption of weakly charged polyelectrolytes at oppositely charged surfaces (1996) Macromolecules, 29 (1), pp. 326-336McClements, D.J., (1999) Food emulsions. Principles, practice and techniques, , CRC Press, New YorkMcClements, D.J., Protein-stabilized emulsions (2004) Current Opinion in Colloid & Interface Science, 9 (5), pp. 305-313McClements, D.J., Decker, E.A., Weiss, J., Emulsion-based delivery systems for lipophilic bioactive components (2007) Journal of Food Science, 72 (8), pp. R109-R124Mizuta, S., Parish, M., Bowen, H.K., Dispersion of BaTiO3 powders (part II) (1984) Ceramics International, 10 (3), pp. 83-86O'Brien, W.D., Ultrasound-biophysics mechanisms (2007) Progress in Biophysics and Molecular Biology, 93 (1-3), pp. 212-255Paraskevopoulou, A., Boskou, D., Kiosseoglou, V., Stabilization of olive oil-lemon juice emulsion with polysaccharides (2005) Food Chemistry, 90 (4), pp. 627-634Pierpaoli, E., Viola, V., Barucca, A., Orlando, F., Galli, F., Provinciali, M., Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice (2013) Carcinogenesis, 34 (6), pp. 1352-1360Rabrenović, B.B., Dimić, E.B., Novaković, M.M., Tešević, V.V., Basić, Z.N., The most important bioactive components of cold pressed oil from different pumpkin (Cucurbita pepo L.) seeds (2014) LWT - Food Science and Technology, 55 (2), pp. 521-527Shanmugama, A., Ashokkumar, M., Ultrasonic preparation of stable flax seed oil emulsions in dairy systems - physicochemical characterization (2014) Food Hydrocolloids, 39, pp. 151-162Silva, G.F., Gamarra, F.M.C., Oliveira, A.L., Cabral, F.A., Extraction of bixin from annatto seeds using supercritical carbon dioxide (2008) Brazilian Journal of Chemical Engineering, 25 (2), pp. 419-426Soottitantawat, A., Bigeard, F., Yoshii, H., Furuta, T., Ohkawara, M., Linko, P., Influence of emulsion and powder size on the stability of encapsulated d-limonene by spray drying (2005) Innovative Food Science & Emerging Technologies, 6 (1), pp. 107-114Soottitantawat, A., Yoshii, H., Furuta, T., Ohkawara, M., Linko, P., Microencapsulation by spray drying: influence of emulsion size on the retention of volatile compounds (2003) Journal of Food Science, 68 (7), pp. 2256-2262Steffe, J.F., (1996) Rheological methods in food process engineering, , Freeman Press, East LansingStratulat, I., Britten, M., Salmieri, S., Fustier, P., St-Gelais, D., Champagne, C.P., Enrichment of cheese with bioactive lipophilic compounds (2014) Journal of Functional Foods, 6, pp. 48-59Sun, C., Gunasekaran, S., Richards, M.P., Effect of xanthan gum on physicochemical properties of whey protein isolate stabilized oil-in-water emulsions (2007) Food Hydrocolloids, 21 (4), pp. 555-564Sylvester, P.W., Shah, S.J., Mechanisms mediating the antiproliferative and apoptotic effects of vitamin E in mammary cancer cells (2005) Frontiers in Bioscience: A Journal and Virtual Library, 10, pp. 699-709Tadros, T.F., Emulsion science and technology: a general introduction (2009) Emulsion science and technology, pp. 1-56. , Wiley-VCH Verlag GmbH and Co. KGaA, WeinheimTaylor, T.M., Davidson, P.M., Bruce, B.D., Weiss, J., Liposomal nanocapsules in food science and agriculture (2005) Critical Reviews in Food Science and Nutrition, 45 (7-8), pp. 587-605Tesch, S., Gerhards, C., Schubert, H., Stabilization of emulsions by OSA starches (2002) Journal of Food Engineering, 54 (2), pp. 167-174Van Duynhoven, J.P.M., Kulik, A.S., Jonker, H.R.A., Haverkamp, J., Solid-like components in carbohydrate gels probed by NMR spectroscopy (1999) Carbohydrate Polymers, 40 (3), pp. 211-219Xu, K., Yao, P., Stable oil-in-water emulsions prepared from soy protein-dextran conjugates (2009) Langmuir, 25 (17), pp. 9714-9720Ye, A., Singh, H., Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: influence of polysaccharides (2006) Food Hydrocolloids, 20 (2-3), pp. 269-276