Resumen de Studies of process intensification for the development of hydrophilic and hydrophobic b-carotene formulations
Nowadays the food market demands functional foods and healthy products. While the use of synthetic chemical compounds in food products is considered negatively by consumers, natural additives provide the final product with a healthy value. Carotenoids are widely used as natural additives in food products. One of the most common, abundant and used carotenoid is ß-carotene. Besides its excellent colorant properties, beta-carotene acts as precursor of retinol and retinoic acid which have an important role in human health as vitamin A precursor and as cellular regulatory signal, respectively. However, the application of ß-carotene as a natural colorant in food and nutraceutical products requires an appropriate formulation in order to protect the active compound against oxidation and degradation processes, and to overcome the low bioavailability due to the low solubility of beta -carotene in aqueous media. Because of this, the aim of this PhD Thesis is the design of different ß-carotene formulations for application as a natural colorant, developing efficient and innovative technologies for the production of these formulations. Biodegradable polymers have been used in all formulations: starches modified with the group n-octenyl succinic (OSA), poly-caprolactones and soybean lecithin.
The first processing technology developed in this thesis consists of putting into contact a hot, pressurized molecular solution of the carotenoid in a hydrophilic solvent with a cold aqueous solution of carrier material, using a mixing chamber. The process is designed in order to approximate the time scales of mixing to the time scales of particle nucleation, Thus, ß-carotene precipitation, due to antisolvent and thermal shock, occurs in a fraction of second time scale, enabling to produce a highly homogeneous product with high efficiency. The reduced process volume, due to these short processing time scales, as well as the increased throughput of the process due to the possibility of operating it continuously, leads to a considerable process intensification. In CHAPTER 1, this process is developed studying the formulation of ß-carotene by precipitation from pressurized ethyl acetate-on-water emulsions using modified OSA-starch refined from waxy maize as carrier material. Results showed that it is possible to obtain a formulation of ß-carotene with a high encapsulation efficiency of ß-carotene (over 70%) and with a particle size in the range of 300-600 nm. Different process parameters with strong influence on product properties were researched, such as concentration of surfactant (which was varied from 37 to 367 gL-1) and organic-water ratio (from 0.6 until 1.3 mL/mL). Results obtained in this chapter revealed that high concentrations of modified starch were required (over 100 gL-1) in order to obtain a high percentage of encapsulated ß-carotene and high emulsion stability. Regarding the organic-water ratio, it was shown that the best results were obtained with low ratios (between 0.65 and 0.73 mL/mL). When the organic-water ratio was increased, the particle sizes increased as well, observing a drastic increase at high organic-water ratios (over 0.85).
In the following chapters, different alternative configurations of this process are analyzed. In CHAPTER 2, the use of ethanol as water-miscible hydrophilic solvent instead of ethyl acetate, which is only partially water-miscible, is analyzed. Results obtained using ethanol as organic solvent revealed that it is possible to obtain a formulation of ß-carotene with maximum encapsulation efficiency of 30%-40% and micellar particle sizes in the range of 120 ¿ 550 nm. As in experiments with ethyl acetate, results showed an increase in the particle size when the organic-water ratio was increased. Compared with the results using ethyl acetate, lower encapsulation efficiencies were obtained in experiments with ethanol , resulting in poorer product properties.
Continuing with the analysis of the process, in CHAPTER 3, the effect of using four different OSA-starches for developing the formulation of ß-carotene by precipitation from pressurized ethyl acetate-on-water emulsions was investigated. The same process parameters as in previous studies were considered: concentration of surfactant varied from 37 to 367 gL-1 and organic-water ratio modified from 0.6 until 1.2 mL/mL. An OSA-starch derived from waxy maize blend with dried glucose syrup was not suitable for encapsulating ß-carotene due to the fact the maximum encapsulation efficiency achieved was less than 30% with particle sizes of approximately 1 µm. For the rest of OSA-starches, a minimum concentration of surfactant of 100 gL-1 was required to obtain high encapsulation efficiencies. As in results discussed in chapter 1, when the concentration of surfactant increased, the encapsulation efficiency increased as well, and at high organic-water ratios, a drastic increase in the particle size was observed, independently of the OSA-starch used. Regarding the particle size, particle sizes in the suspension of 350-760 nm were obtained. With respect to the effect of the organic-water ratio, the encapsulation efficiency was kept constant between 30-45% and particle sizes below 500 nm, when OSA-dextrin derived from waxy maize and OSA-dextrin derived from tapioca were used. Comparing these results from those shown in chapter 1, results obtained with OSA-starch refined from waxy maize presented better results, achieving maximum encapsulation efficiencies of 70-80 % with particle sizes in the nanometer range.
Finally, the performance of this novel high-pressure emulsion processing technique was compared to that of the emulsion evaporation process commonly used in most industrial applications. This comparison is presented in CHAPTER 4, using ethyl acetate as organic solvent and OSA-starch refined from waxy maize, according to the best results obtained in the previous chapters. In addition to the conventional high-shear emulsification process, chapter 4 presents the results obtained by ultrasound emulsification, completing in both cases the processing with a last step of organic solvent removal. Different process parameters were studied by ultrasound emulsification, such as organic-water ratio (varying from 0.275 until 0.73 mL/mL), time of application of ultrasounds (from 6 until 65 minutes), amplitude (from 20 to 100 µm) and duty cicle (from 0.5 until 1). The best results were obtained with low organic-water ratios, concretely 0.275, and using 100 µm amplitude with a duty cycle of 1.0, achieving particle sizes lower than 200 nm and encapsulation efficiencies of 30%. In comparison, results obtained by high-shear emulsification showed particle sizes in the same range (less than 240 nm), but much lower encapsulation efficiencies (below 8%), probably due to the harsher conditions during emulsification. Comparing with the results obtained with the high pressure emulsion technique described in chapters 1-3, particle sizes obtained by this technique were higher (in the range of 400 nm), indicating that ultrasound emulsification induces a more efficient mixing than the mixing chamber employed in the high pressure emulsion technique. On the contrary, the much higher encapsulation efficiencies obtained by the high pressure process (70 - 80%) indicate that the very short processing times required by this process has a direct effect on a better preservation of the structure of the emulsion template during the precipitation, and therefore on the efficiency of the incorporation of beta-carotene into the carrier.
The second part of the thesis deals with the development of supercritical fluid-based technologies for the production of beta-carotene formulations. In particular the application of the PGSS (Particles from Gas Saturated Solutions) process was studied. Two different types of formulation were developed: one based on liposome-forming phospholipids, as an alternative water-soluble formulation, and the second one based on the water-insoluble, slow degrading polymer polycaprolactone, as a product aimed for a protection against degradation and a slow release of the active material.
As the first step for the development of the PGSS process, in CHAPTER 5, the solid-liquid-gas equilibrium in poly-(¿-caprolactone) + CO2 systems at high pressure (from 0.1 to 25 MPa) was determined in order to get a detailed knowledge of the phase behavior of the polymer and supercritical fluid mixtures. Experiments were carried out with three polycaprolactones with different molecular weights (4000, 10000 and 25000 gmol-1). The SLG equilibrium was developed by visual determination of the first melting point using a high pressure optical cell. The SLG equilibrium lines show a maximum temperature at low pressures (between 0.5 MPa and 1.6 MPa) and a minimum temperature at moderate pressures (between 8 MPa and 10 MPa, depending on the used polycaprolactone). The maximum reduction of melting temperature was between 12.5 and 16.0 K depending on the molecular weight of the polycaprolactone. Results obtained in this work were useful for carrying out the investigation presented in chapter 6.
In CHAPTER 6, the formulation of ß-carotene with polycaprolactones by Particles from Gas Saturated Solutions (PGSS) process was developed. The effect of different process parameters on particle size and on ß-carotene content were studied, including beta-carotene:polymer molar ratio (0.13, 0.16 and 0.25), time of homogenization in the mixing chamber (60, 120 and 240 minutes), temperature (50 and 70 ºC) and pressure (11 and 15 MPa). Two different polycaprolactones were used, obtaining particles with a particle size in the range of 270-650 µm with a ß-carotene content of up to 340 ppm when polycaprolactone with a molecular weight of 10000 g mol-1 was used while using polycaprolactone with 4000 g mol-1, particle size was reduced to 110-130 µm. The molar ratio has an important influence on particle size, obtaining an increase in particle size when molar ratio was increased as well.
In CHAPTER 7, the formulation of ß-carotene with soybean lecithin by PGSS-drying was investigated. Results showed that it was possible to obtain dry particles with a particle size in the range of 10-500 µm constituted by agglomerated spheres with encapsulation efficiencies of ß-carotene up to 60%, which can be hydrated forming ß-carotene-loaded liposomes with sizes ranging between 1-5 µm. By this rehydration process, large particles corresponding to non-encapsulated ß-carotene were also obtained. The main process parameters, which were studied their effect on the product characteristics, were the pre-expansion temperature (100-130ºC), pre-expansion pressure (8-10 MPa), gas to product ratio (21-27-32) and concentration of soybean lecithin (55-62-72 gL-1). Regarding the particle size, smaller particle sizes were obtained when pre-expansion pressure or concentration of lecithin were increased, or when pre-expansion temperature and GPR (Gas to Product Ratio) were decreased. With respect to the encapsulation efficiency, it was increased from 30% until 60% when the pre-expansion temperature was increased in the selected range of temperatures (100-130ºC).
In order to obtain basic properties for the analysis of the performance of the encapsulation process by PGSS, in CHAPTER 8, the solubility of ß-carotene in three different poly-(¿-caprolactones) with different molecular weights (4000, 10000 and 25000 gmol-1) in colloidal state was studied. The determination of the solubility of ß-carotene was carried out by two different processes: equilibration-impregnation and equilibration-de-supersaturation. The maximum ß-carotene contents achieved by equilibration impregnation process were between 87 and 191 ppm depending on the molecular weight of the polycaprolactone. These results corroborated results obtained in chapter 6, since the concentration of ß-carotene obtained by PGSS process agree well with the order of magnitude of the saturation of ß-carotene in the polymer. Regarding the equilibration de-supersaturation process, ß-carotene concentrations were considerably higher than those obtained in impregnation experiments, obtaining a maximum concentration of 8800 ppm when polycaprolactone with the highest molecular weight was used. Results showed in this chapter justify the experimental results obtained in PGSS experiments, revealing that polycaprolactones are not suitable for carrying out a formulation of ß-carotene due to the low affinity between ß-carotene and the polymer.
