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Solid self-microemulsifying system (S-SMECS) for enhanced bioavailability and pigmentation of highly lipophilic bioactive carotenoid
Powder Technology 274 (2015) 199–204
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Solid self-microemulsifying system (S-SMECS) for enhanced bioavailability and pigmentation of highly lipophilic bioactive carotenoid Pei Yong Chow ⁎, Sue Zen Gue, Sai Kaw Leow, Lay Beng Goh Kemin Industries (Asia) Pte Limited, 12 Senoko Drive, 758200, Singapore
a r t i c l e
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Article history: Received 8 July 2014 Received in revised form 5 January 2015 Accepted 12 January 2015 Available online 15 January 2015 Keywords: Self-microemulsifying Bioavailability Bicontinuous microemulsion Pigmentation Carotenoids
a b s t r a c t The application of carotenoids as natural additives in various water-based or water-compatible formulations for the pigmentation of food and feed is seriously hampered by their insolubility in aqueous systems, resulting in low bioavailability. One way to develop the full potential of color strength and achieve a high degree of bioavailability during the gastrointestinal passage is to transform the coarse crystalline material into a nanodispersed state. Exemplified with natural carotenoid (Paprika Oleoresin), the objectives of this study were to prepare solid self-microemulsifying carotenoid system (S-SMECS) containing bicontinuous microemulsion of polyethoxylated sorbitan ester (Tween 80), water, R-(+)-limonene, ethanol, and glycerol with excellent in vivo solubilization capacity, for the delivery of bioactive carotenoid, by spraying the self-microemulsifying carotenoid system (liquid system) using colloidal silica/wheat pollard (as the inert solid carrier), and to evaluate the enhanced bioavailability and pigmentation efficacy of carotenoid from the S-SMECS. Results showed that the carotenoid solubilized better in the microemulsion and the self-microemulsifying carotenoid microemulsion is characterized by an average particle size of approximately 0.25 μm. The bioavailability study performed in layers resulted in enhanced values for the S-SMECS. The enhancement for S-SMECS was 17% above the level of carotenoid absorbed in the serum and the relative color score was 20–30% enhancement over feeding compared with the commercial product, respectively. These results demonstrated the excellent ability of S-SMECS containing bicontinuous microemulsion to enhance the bioavailability of carotenoid in vivo. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The carotenoids are a group of colored pigments which have a yellow to red hue. They are widely found in nature, and impart a characteristic color to many foodstuffs. The most important examples of this category include lutein, capsanthin, zeaxanthin and carotene. They constitute an important class of colorants that are in demand for the food and animal feed industry, as substitutes for artificial dyes. All carotenoids are water-insoluble, and slightly soluble in fats and oils. This limited solubility hinders direct use of the relatively coarse carotenoids for pigmentation, since only low color yields can be achieved. Furthermore, non-uniform particle size of the coarse carotenoid resulted in inconsistent absorption kinetics and performance [1,2]. Yolk pigmentation is an important factor in the evaluation of egg quality. The extent of the pigmentation required depends on consumers' expectations and can vary from market to market, from farm to farm. Yolk pigmentation can be achieved via synthetic ingredients, where they serve purely an aesthetic purpose. However, yolk pigmentation using natural pigments, such as those derived from marigold oleoresin and paparika oleoresin, enhances the egg as a functional food and improves ⁎ Corresponding author. Tel.: +65 64904050; fax: +65 67541266. E-mail address: [email protected] (P.Y. Chow).
http://dx.doi.org/10.1016/j.powtec.2015.01.020 0032-5910/© 2015 Elsevier B.V. All rights reserved.
its taste [3]. These natural pigments contain carotenoids which can be converted physiologically to vitamins or act as antioxidants per se [4]. Unfortunately, birds cannot synthesize carotenoids, which cannot be synthesized de novo. They need to obtain an exogenous source of carotenoids through feed ingredients such as corn, corn gluten meal, etc. [5]. To improve color yield through increased bioavailability, various methods have been devised with the objective of reducing the particle size of the active ingredient. A common approach is through the use of microemulsions. Microemulsions are thermodynamically stable, transparent, have low viscosity and isotropic dispersions consisting of oil and water, stabilized by an interfacial film of surfactant molecules, typically in conjunction with a co-surfactant. Investigations in microemulsions [6–12] generally focus on forming either water-in-oil (w/o) or oil-in-water (o/w) microemulsions, as micro-reactors where the concentrates (surfactant and oil phases) are loaded with active ingredient. However, they typically consist of ‘reverse micelles’ or ‘surfactant-in-oil phases’ that cannot be inverted into oil-in-water droplets upon simple aqueous dilution. Such a product will not be suitable as an additive, where it would be diluted and destabilized in an aqueous environment. Aqueous dilution is encountered as they enter the biological system, moving through the various stages of absorption and distribution within the animal body. Hence, such microemulsion products would have limited applications in the industry.
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In recent studies [13–16], scientists have found unique mixtures of food-grade oils, which can be diluted with an aqueous phase progressively and continuously without phase separation. They can be transformed into bicontinuous structures that can be inverted into oilin-water nanodroplets upon further dilution. These unique mixtures consist of two or more food-grade nonionic hydrophilic emulsifiers that self-assemble to form mixed reverse micelles (the concentrate). Bicontinuous microemulsion [17] has been an active research topic because its unique structure lends itself well to controlled release applications. Amphiphilic molecules form bicontinuous water and oil channels, where “bicontinuous” refers to two distinct (continuous, but non-intersecting) hydrophilic regions separated by bilayers. This allows for simultaneous incorporation of water- and oil-soluble active ingredients. The phase structure also provides a tortuous diffusion pathway for controlled release of the encapsulated ingredients. Despite recent activities, there remains a gap in translation of the technique into a feasible and practical application. Difficulties include achieving a good level of product stability to provide a reasonable shelf life, manufacturing scalability, and customization using regulatory-approved material. These were hindering progress in the development of food-grade bicontinuous microemulsions into commercial products. This paper summarizes the novel development microemulsified bicontinuous carotenoid, made through the optimization of food grade U-type microemulsion production for better loading capacity, protection and reduction of carotenoids with a particle size of approximately 0.25 μm. This includes characterization of the physicochemical properties of the formulation and an evaluation on the bioavailability and effect in the pigmentation efficacy tested by monitoring the plasma level of trans-capsanthin and the yolk color (YCF) score of the eggs in an in vitro trial. 2. Materials and methods 2.1. Materials Tween 80 [polyoxyethylene (20) sorbitan monooleate], d-(+)limonene, ethanol and glycerol were of food grade [obtained from Kemin Agrifood Asia (KAA)]. All chemicals and reagents used in the analytical protocols were of analytical reagent grade. The water was double-distilled. A stabilized source of saponified red carotenoids from paprika extracts containing major natural red pigments transcapsanthin and capsorubin (available at KAA) was used as a control. 2.2. Phase diagrams and conductivity measurement of microemulsions The single-phase region of the microemulsion [10] consisting of Tween 80/ethanol/limonene/glycerol/water was determined systematically by titrating water to various compositions of Tween 80, ethanol, limonene and glycerol, in a screw-capped test tube. Each sample was vortex-mixed and allowed to equilibrate in a temperature-controlled environment at 25 °C. Stock solution of water and glycerol at a constant weight ratio of 3:1 was made. The limonene/ethanol weight ratio was held constant at 1:2. Mixtures of the surfactant/oil phase (ethanol and limonene) or mixtures of the surfactant/aqueous phase (water and glycerol) were prepared in culture tubes, sealed with screw caps at predetermined weight ratios of oil phase to surfactant, or aqueous phase to surfactant, and kept in a 25 °C (± 0.3 °C) water bath. Microemulsion areas were determined in phase diagrams by titrating either the oil phase/surfactant or aqueous phase/surfactant mixture with the aqueous phase or the oil phase, respectively. All samples were vigorously stirred. The samples were allowed to equilibrate for at least 24 h before they were examined. The microemulsion region was further classified as either oil-in-water (O/W), bicontinuous or water-in-oil (W/O) microemulsions. A rough demarcation of the bicontinuous region was further deduced from conductivity measurements [7]. Electrical conductivity measurements were
performed at 25 ± 0.3 °C on samples along the dilution line P using a conductivity meter (Extech EC500, pH/conductivity meter). Since the microemulsions were nonionic, a small quantity of an aqueous electrolyte (a solution of 0.01 M NaCl) was added. 2.3. Solubilization and preparation of self-microemulsifying carotenoid system (SMECS) To test the solubilization capacity of saponified red carotenoids in the microemulsion system, 1 mg of saponified carotenoids was added to a test tube containing 10 g of prepared microemulsion using a vortex mixer to dissolve the carotenoids for approximately 10 min. If the saponified carotenoids could dissolve in this microemulsion, the previous procedure was repeated until the mixture maintained a persistent cloudy appearance or visible grains of solid were found deposited at the bottom of the test tube after being mixed. The maximum solubility of saponified carotenoids in each microemulsion system was then determined. The carotenoid-microemulsion was prepared as follows: from the solubilization study, 0.01 wt% of the saponified carotenoids was added to a pre-mixed microemulsion sample containing 32.5 wt.% Tween 80, 32.5 wt.% limonene/ethanol and 35 wt.% water/glycerol. The mixture was vortex-mixed at 2500 rpm and uniformly dispersed with an ultrasonic probe for 2 min to obtain a persistent clear appearance mixture. The sonication also serves to remove bubbles that could be generated during vortexing. This prepared self-microemulsifying carotenoid system was subsequently used for further characterizations. 2.4. Stability measurement A stability study was conducted based on spectrophotometric determination of total carotenoids in the self-microemulsifying carotenoid system. The accelerated stability of carotenoid-microemulsions prototype over time at 75 °C for 3 days in the presence of oxygen was monitored. About 0.5 g (±0.1 mg) of the self-microemulsifying carotenoid at different time points was added to a 100 ml brown volumetric flask. The flask was filled with a mixture of hexane:ethanol:acetone:toluene (HEAT) at a ratio of 10:6:7:7 as the extracting solvent, and stirred with a magnetic stir bar for 15 min. Five milliliters of the solution was transferred by pipette to a 50 ml brown volumetric flask, diluted to the mark with HEAT, and shaken to mix the contents. A cuvette was filled with the solution and absorbance was measured at 460 nm against the extracting solvent using a spectrophotometer (UV-2401PC, Shimadzu). 2.5. Particle size analysis and transmission electron microscopy observations The particle size analysis was carried out using a particle size analyzer (HORIBA SZ-100Z). The particle size of the coarse saponified red carotenoids was also determined for comparison. Long-term stability testing involving particle size measurements was also conducted at given time intervals over 1 month storage at 25 °C. To observe morphology, the self-microemulsifying carotenoid was directly deposited onto carbon film supported by copper grids, stained with a 1% aqueous solution of osmium tetroxide (OsO4) and investigated using the transmission electron microscope (TEM) JEOL 1010. The morphology of the coarse saponified red carotenoid was also determined for comparison. 2.6. Bioavailability The bioavailability of the carotenoids was tested after the selfmicroemulsifying carotenoid (liquid system) had been transformed into beadlets of 250 μm average particle size by spray drying to produce the solid self-microemulsifying carotenoid system (S-SMECS). Both the solid self-microemulsifying carotenoid system (S-SMECS) and the non-microemulsifying (commercial product, CP) contained 5.4g/kg of
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3. Results and discussion 3.1. Phase diagram and conductivity measurement of microemulsions Fig. 1 shows the phase behavior of the transparent microemulsion region (1 phase region as denoted by blue colored outlined area) of the system composed of Tween 80/ethanol/limonene/glycerol/H2O. The shaded region represents the wide range of compositions that can be selected to form transparent microemulsions. Based on the diagram, microemulsions can be formed using aqueous content ranging from about 20 to 100 wt.%. The changes in conductivity of microemulsions along the P-line with the aqueous content are shown in Fig. 2. It shows the low conductivity of microemulsions at lower aqueous water content (b 20 wt.%), followed by a rapid increase in conductivity when the aqueous content was greater than 20 wt.%. With regards to the low conductivity for the systems containing less than 20 wt.% aqueous content, it was likely due to the formation of W/O microemulsion droplets dispersed in the oil medium. The sharp increase in conductivity for the systems containing higher than 20 wt.% aqueous content denoted the presence of numerous interconnected conducting channels, which are characteristics of bicontinuous microemulsions. An attempt has yet to be made to establish the boundary between the bicontinuous microemulsion and O/W microemulsion. The current investigation focused on microemulsions containing less than 40 wt.% aqueous content. Further studies on the boundary shall be conducted in the near future. Based on the conductivity measurements, the system containing 35 wt.% water was found to be
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Fig. 1. The phase behavior of the transparent microemulsion region of the system made up of Tween 80:ethanol/limonene:glycerol/H2O. The weight ratios of limonene to ethanol and glycerol to water were fixed at 1:2 and 1:3 respectively, while that for oil to surfactant was at 1:1 along line P with increasing water content.
a bicontinuous microemulsion, which was then chosen for the detailed study. The resulting bicontinuous phase offered exceptional control over the nanostructure, yielding an architecture that was well-suited for the incorporation of hydrophobic and hydrophilic active ingredients. 3.2. Solubilization and stability of self-microemulsifying carotenoid system Fig. 3 shows the solubilization pattern of the saponified red carotenoids in microemulsions. The carotenoids solubilized (~2 times) better in the W/O microemulsion than in the micellar phase consisting of only oil and surfactant without water. Moreover, enhanced solubilization (~0.01 wt.%) was observed when we increased the aqueous content to 35 wt.% to get the bicontinuous structure. A possible explanation for these dramatic increases in the solubilization of carotenoid could be related to the locus of solubilization. In systems free of water the locus of solubilization is at the micelle interface. As an aqueous phase is introduced (~ 10 wt.% aqueous), water-in-oil
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trans-capsanthin. The bioavailability of the formulation was studied by analyzing the trans-capsanthin in the blood serum of layers after oral administration of the two formulations with an average primary particle size of approximately 0.25 μm (solid self-microemulsifying carotenoid, S-SMECS) and 20 μm (commercial product, CP) respectively in an animal trial conducted by an accredited research farm in Malaysia. All procedures related to the holding and conducting an experiment with live animals are conducted under the approval of the Ethics Committee of the Genetics Improvement & Farm Technologies Sdn. Bhd., Malaysia. Twenty nine week old Lohamann Brown hens were used. The birds were fed with the experimental diets and allowed 1 week for adaptation to their new environment. The birds were placed in individual wirefloored cages arranged in two tires within an open-sided house under 14 L:10 D lighting regime. Feed and water were provided ad libitum throughout the experimental diet. The two formulations were feed to two groups of 6 replicates containing 8 carotenoid-depleted birds. The dry powders were mixed with layer feed prior to feeding. The trial was carried out in two consecutive parts. In part I, a dosage of 0.5 kg/ton (containing 2.7 g of trans-capsanthin per ton of feed) of the two formulations was given consecutively for 7 days and in Part II, the two formulations were crossed over with continuing dosing at 0.5 kg/ton daily for another 7 consecutive days. The blood levels were analyzed at 24 h, 32 h, 7th and 14th day. Similarly, the YCF scores of the eggs were analyzed at day 0, 7 and 14. The whole liquid egg color was determined by means of a commercially available yolk color fan. Where required, HPLC-based analysis of transcapsanthin equivalents using the AOAC (Association of Analytical Communities) method (method number 970.64, 1990) was carried out. Data were statistically analyzed by one-way ANOVA method using Statgraphics. Eggs were cracked without whites onto plastic Petri dishes of 6 cm in diameter and placed on a flat white background in an environment with fluorescent lighting. A team of eight trained observers was asked to evaluate the eggs utilizing a commercial yolk color fan. The transcapsanthin concentration for each formulation was determined by extracting the blood serum per treatment using a modified AOAC (Association of Analytical Communities) method (method number 970.64, 1990).
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(W/O) swollen microemulsions are formed, and the hydrophilic OH groups of the saponified carotenoids are oriented toward the aqueous phase, thus causing the molecules to insert themselves between the surfactant hydrophobic chains. This change in the locus of solubilization causes an increase in solubilization at the interface. The increase in solubilization as the aqueous phase concentration increases may be also attributed to microstructure transformations. The structural transformation from W/O to the bicontinuous microstructure (~ 35 wt.% aqueous) causes the free carotenoids to solubilize between both the hydrophobic amphiphilic chains and the interface, thus causing an increase in the solubilization. In addition, it is noted at this point that the bicontinuous morphology provides an interesting environment for loading and release properties. The domain sizes of the aqueous and oil channels can be fine-tuned by varying the microemulsion components to allow full potential for solubilization and controlled release of the active ingredients. Moreover, by customizing the specific properties of the hydrophilic and hydrophobic portions, it is possible to control their interaction with the active ingredients, offering a greater potential for tailored release properties over a broad range of applications and conditions. In general, a loss of approximately 30% of total carotenoid from the initial added amount was observed in the self-microemulsifying carotenoid sample after accelerated study conducted at 75 °C for 3 days, as shown in Fig. 4. For the saponified carotenoids solubilized in water, the losses were significantly higher (~ 42% at 3rd day). It is well known that carotenoids are sensitive to light, oxygen and heat. In a dynamic high-temperature system, the loss in the content of carotenoid in the self-microemulsifying carotenoid sample is expected but, the role of microemulsion protection on the carotenoid is more predominant resulting in a lower loss of the carotenoid during the study as compared to the unprotected carotenoids. The carotenoids pigment when solubilized and contained within the microemulsion system is better protected due to the molecular architecture of the pigment within the microemulsion matrix i.e. the size and configuration (Fig. 5). The microemulsion is hypothesized to provide a physical barrier between the pigment and the oxidation catalysts (such as oxygen) and also its light scattering property can help to reduce the intensity of light reaching the pigment entrapped within them. In addition, the smaller particle size of the carotenoid pigment achieved using microemulsion will enable it to be easily and homogeneously distributed into the interior porous passage of the carrier granules that will further help to reduce the loss caused by oxidation on the surface and enhance the stability of the product as illustrated in Fig. 6.
Fig. 4. Stability of the self-microemulsifying carotenoid system (SMECS) and commercial product (CP) under accelerated study at 75 °C for 3 days.
3.3. Particle size analysis and transmission electron microscopy observation Both the bioavailability and the color yield of the carotenoids strictly depend on the particle size distribution of the active ingredient in the final formulation. Hence, the determination of the particle size distribution is a major consideration during production for quality assurance. Figs. 7 and 8 show the corresponding result of particle size analysis and the electromicrograph for the self-microemulsifying carotenoid system and the coarse carotenoid (commercial product, CP). The particle size of the carotenoid in microemulsion is maintained to about approximately 0.25 μm at average with contrast to a particle size of 20 μm for the coarse carotenoid. Fig. 7 also shows the particle size distribution of the carotenoid-microemulsion at the 0, 7th, 14th, 21st and 28th day at room temperature (25 °C). There were no significant differences in particle size distribution for the self-microemulsifying carotenoid system during the 1 month study. The long-term stability results demonstrated that the microemulsion protected carotenoid was more stable and uniformly dispersed with no aggregation (as shown in Fig. 8). The results also suggested that the surfactant and oil phases used in this study not only influenced the formation of protective colloid responsible for establishing colloidal stability against agglomeration but also expected to help the emulsion formed in the stomach be readily restructured into a bicontinuous network even in the absence of biliary phospholipid, thereby facilitating the uptake of carotenoids during the gastrointestinal passage.
Fig. 5. Schematic arrangement of the self-assembled surfactant, oil and alcohol in the bicontinuous phase facilitates the entrapment of carotenoids pigment within the core or at its interface.
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Fig. 6. Schematic arrangement of S-SMECS within the internal passage of the carrier particle.
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Fig. 8. (a) TEM micrograph of commercial product (CP) (SMECS) and (b) TEM micrograph of self-microemulsifying carotenoid system (SMECS).
of feeding with S-SMECS, the trans-capsanthin in blood level increased from virtually zero to approximately 0.038 ppm, which is approximately 17% above the level achieved by the commercial product. After feed cross-over at day 7, the blood plasma collected from the birds originally treated with S-SMECS showed a drop of the trans-capsanthin from approximately 0.038 ppm to 0.017 ppm and was significantly lower than the blood plasma collected from the birds originally treated with the commercial product. A similar trend was observed for the YCF score of the egg yolk after the feed cross-over as shown in Fig. 10, the score of Total Trans-capsanthin in blood plasma (ppm)
The bioavailability of the formulation was studied by analyzing the trans-capsanthin in blood serum of layer birds after oral administration of the solid self-microemulsifying carotenoid system (S-SMECS) and the non-microemulsifying commercial product (CP) with an average primary particle size of approximately 0.25 μm and 20 μm respectively. The plasma concentration–time profile of trans-capsanthin from these products is shown in Fig. 9. As indicated in Fig. 9, carotenoid particle sizes exerted a significant influence on the relative bioavailability. It has been reported that trans-capsanthin, like lutein, is a poorly watersoluble lipophilic compound, that follows the same route of absorption as lipids. The plasma concentrations of trans-capsanthin were measurable in layers with 0.038 ppm and 0.017 ppm for S-SMECS and CP, respectively. This implies the involvement of endogenous emulsifiers in promoting solubilization and absorption of carotenoids in vivo. Although the exact mechanism of the absorption is not yet fully understood, trans-capsanthin has been thought to be absorbed through enterocytes by simple diffusion or receptor-mediated transport. Specifically, trans-capsanthin is emulsified into small lipid droplets in the stomach and further incorporated into mixed micelles by the action of bile salts and biliary phospholipids, after which mixed micelles are taken up by enterocytes. Thus, the appearance of relatively low concentrations of trans-capsanthin in layer plasma was possibly due to the involvement of the aforementioned absorption mechanism. After 7 days
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Fig. 9. Influence of particle size on the bioavailability of trans-capsanthin. Oral administration to carotenoid-depleted layers with solid self-microemulsifying carotenoid system (S-SMECS) and commercial product (CP).
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administration, significantly exceeding that of the commercial product containing the coarse carotenoids. Furthermore from the trial result, it supported our hypothesis that a desired yolk color score and carotenoid bioavailability can now be achievable at a significantly lower inclusion rate when carotenoid molecules are self-micromulsified. This novel microemulsion technology offers greatly enhanced flexibility for product development efforts, the capability to tailor different active ingredients loading of bicontinuous phases, and the controlled tolerance of bicontinuous phases for other ingredients. Overall, the information obtained from this study shall facilitate the rational design and fabrication of self-microemulsifying delivery systems for many ingredients. References
S-SMECS
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Fig. 10. Influence of particle size of solid self-microemulsifying carotenoid system (S-SMECS) and commercial product (CP) on the yolk-color score for the layer's egg yolk.
the eggs taken from the birds that treated with the S-SMECS improved from 7.5 to 9.75, which is a 30% enhancement over feeding the commercial product. Apart from an increase in pigment absorption, color intensity of the egg yolk is possibly dependent also on the pigment particle size. Perhaps at the nanometer range, the number of carotenoid molecules that can be packed at the surface has increased, leading to an increase in light absorption and scattering coefficient. After oral administration, no further dissolution is required as such the trans-capsanthin would be maintained in a fully solubilized state, after the bicontinuous microemulsion pre-concentrate self-emulsifies on contact with gastric fluid in the stomach. The already small and uniform bicontinuous arrays containing the trans-capsanthin may be further emulsified by the bile/ lecithin micelles in the intestinal fluids, digested by enzymes and converted into even smaller lipid particles. This process of digestion would greatly increase the surface area of trans-capsanthin for transfer to the intestinal epithelium. A similar observation was reported by Fu et al. [18]. This may explain why a richer redness was observed for the egg yolks obtained from layers treated with the self-microemulsifying carotenoids system. 4. Conclusions The described microemulsion process, which is based on using water soluble and toxicologically safe food-grade ingredients, offers new opportunity to produce a large variety of self-microemulsifying formulations. With proper choice of the surfactant, oils and water, bicontinuous microemulsions can be formed to enhance solubilization and protection of active ingredients. With a characteristic size of the active ingredient of the S-SMECS of about 0.25 μm, the preparation exhibits high bioavailability and better yolk pigmentation after oral
[1] A. Amar, A. Aserin, N. Garti, Solubilization of lutein and lutein esters in food grade nonionic microemulsions, J. Agric. Food Chem. 51 (2003) 4775–4781. [2] A. Alves-Rodrigues, A. Shao, The science behind lutein, Toxicol. Lett. 150 (2004) 57–83. [3] Y. Nys, Dietary carotenoids and egg yolk coloration—a review, Arch. Geflugelk. 64 (2000) 45–54. [4] D. Dutta, U.R. Chaudhuri, R. Chakraborty, Structure, health benefits, antioxidant property and processing and storage of carotenoids, Afr. J. Biotechnol. 4 (13) (2005) 1510–1520. [5] J. Irwandi, N. Dedi, F.H. Reno, O. Fitri, Carotenoids: sources, medicinal properties and their application in food and nutraceutical industry, J. Med. Plants Res. 5 (33) (2011) 7119–7131. [6] C. Solans, R. Pons, H. Kunieda, in: C. Solans, H. Kunieda (Eds.), Overview of basic aspects of microemulsions, Industrial Applications of Microemulsions, 66, Dekker, New York, 1997, pp. 1–17. [7] S.R. Dungan, Microemulsions in foods: properties and applications, 2001. 148–170 (ibid). [8] K. Holmberg, Quarter century progress and new horizons in microemulsions, in: O. Shah (Ed.), Micelles, Microemulsions and Monolayers, Dekker, New York, 1998, pp. 161–191. [9] N. Garti, Microemulsions, emulsions, double emulsions and emulsions in food, Formulation Science (Proceeding from Formulation Forum '97-Association of Formulation Chemists), 1998, pp. 185–246. [10] S. Ezrahi, A. Aserin, N. Garti, in: P. Kumar, K.L. Mittal (Eds.), Microemulsions— fundamental and applied aspects, Aggregation Behavior in One-Phase (Winsor IV) SystemsMarcel Dekker, Inc., New York, 1999, pp. 185–246. [11] N. Garti, V. Clement, M. Leser, A. Aserin, M. Fanun, Sucrose esters microemulsions, J. Mol. Liq. (1999) 253–296. [12] N. Anton, J.P. Benoit, P. Saulnier, Design and production of nanoparticles formulated from nano-emulsion templates a review, J. Control. Release (2008) 185–199. [13] A. Spernath, A. Yaghmur, A. Aserin, R.E. Hoffman, N. Garti, Food-grade microemulsions based on nonionic emulsifiers: media to enhance lycopene solubilization, J. Agric. Food Chem. (2002) 6917–6922. [14] A. Spernath, A. Yaghmur, A. Aserin, R.E. Hoffman, N. Garti, Self-diffusion nuclear magnetic resonance, microstructure transitions, and solubilization capacity of phytosterols and cholesterol in Winsor IV food-grade microemulsions, J. Agric. Food Chem. (2003) 2359–2364. [15] A. Yaghmur, A. Aserin, I. Tiunova, N. Garti, Sub-zero temperature behaviour of nonionic microemulsions in the presence of propylene glycol by DSC, J. Therm. Anal. Calorim. (2002) 163–177. [16] A. Yaghmur, A. Aserin, B. Antalek, N. Garti, Microstructure considerations of new five-component Winsor IV food-grade microemulsions studied by pulsed gradient spin-echo NMR, conductivity, and viscosity, Langmuir 19 (2003) 1063–1068. [17] P.Y. Chow, L.M. Gan, Microemulsion polymerizations and reactions, Adv. Polym. Sci. (2005) 257–298. [18] J.M. Fu, Y. Li, J.L. Guo, Optical behavior of organic pigments in aqueous dispersions and its application, J. Colloid Interface Sci. (1998) 450–455.
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Solid self-microemulsifying system (S-SMECS) for enhanced bioavailability and pigmentation of highly lipophilic bioactive carotenoid Pei Yong Chow ⁎, Sue Zen Gue, Sai Kaw Leow, Lay Beng Goh Kemin Industries (Asia) Pte Limited, 12 Senoko Drive, 758200, Singapore
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 5 January 2015 Accepted 12 January 2015 Available online 15 January 2015 Keywords: Self-microemulsifying Bioavailability Bicontinuous microemulsion Pigmentation Carotenoids
a b s t r a c t The application of carotenoids as natural additives in various water-based or water-compatible formulations for the pigmentation of food and feed is seriously hampered by their insolubility in aqueous systems, resulting in low bioavailability. One way to develop the full potential of color strength and achieve a high degree of bioavailability during the gastrointestinal passage is to transform the coarse crystalline material into a nanodispersed state. Exemplified with natural carotenoid (Paprika Oleoresin), the objectives of this study were to prepare solid self-microemulsifying carotenoid system (S-SMECS) containing bicontinuous microemulsion of polyethoxylated sorbitan ester (Tween 80), water, R-(+)-limonene, ethanol, and glycerol with excellent in vivo solubilization capacity, for the delivery of bioactive carotenoid, by spraying the self-microemulsifying carotenoid system (liquid system) using colloidal silica/wheat pollard (as the inert solid carrier), and to evaluate the enhanced bioavailability and pigmentation efficacy of carotenoid from the S-SMECS. Results showed that the carotenoid solubilized better in the microemulsion and the self-microemulsifying carotenoid microemulsion is characterized by an average particle size of approximately 0.25 μm. The bioavailability study performed in layers resulted in enhanced values for the S-SMECS. The enhancement for S-SMECS was 17% above the level of carotenoid absorbed in the serum and the relative color score was 20–30% enhancement over feeding compared with the commercial product, respectively. These results demonstrated the excellent ability of S-SMECS containing bicontinuous microemulsion to enhance the bioavailability of carotenoid in vivo. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The carotenoids are a group of colored pigments which have a yellow to red hue. They are widely found in nature, and impart a characteristic color to many foodstuffs. The most important examples of this category include lutein, capsanthin, zeaxanthin and carotene. They constitute an important class of colorants that are in demand for the food and animal feed industry, as substitutes for artificial dyes. All carotenoids are water-insoluble, and slightly soluble in fats and oils. This limited solubility hinders direct use of the relatively coarse carotenoids for pigmentation, since only low color yields can be achieved. Furthermore, non-uniform particle size of the coarse carotenoid resulted in inconsistent absorption kinetics and performance [1,2]. Yolk pigmentation is an important factor in the evaluation of egg quality. The extent of the pigmentation required depends on consumers' expectations and can vary from market to market, from farm to farm. Yolk pigmentation can be achieved via synthetic ingredients, where they serve purely an aesthetic purpose. However, yolk pigmentation using natural pigments, such as those derived from marigold oleoresin and paparika oleoresin, enhances the egg as a functional food and improves ⁎ Corresponding author. Tel.: +65 64904050; fax: +65 67541266. E-mail address: [email protected] (P.Y. Chow).
http://dx.doi.org/10.1016/j.powtec.2015.01.020 0032-5910/© 2015 Elsevier B.V. All rights reserved.
its taste [3]. These natural pigments contain carotenoids which can be converted physiologically to vitamins or act as antioxidants per se [4]. Unfortunately, birds cannot synthesize carotenoids, which cannot be synthesized de novo. They need to obtain an exogenous source of carotenoids through feed ingredients such as corn, corn gluten meal, etc. [5]. To improve color yield through increased bioavailability, various methods have been devised with the objective of reducing the particle size of the active ingredient. A common approach is through the use of microemulsions. Microemulsions are thermodynamically stable, transparent, have low viscosity and isotropic dispersions consisting of oil and water, stabilized by an interfacial film of surfactant molecules, typically in conjunction with a co-surfactant. Investigations in microemulsions [6–12] generally focus on forming either water-in-oil (w/o) or oil-in-water (o/w) microemulsions, as micro-reactors where the concentrates (surfactant and oil phases) are loaded with active ingredient. However, they typically consist of ‘reverse micelles’ or ‘surfactant-in-oil phases’ that cannot be inverted into oil-in-water droplets upon simple aqueous dilution. Such a product will not be suitable as an additive, where it would be diluted and destabilized in an aqueous environment. Aqueous dilution is encountered as they enter the biological system, moving through the various stages of absorption and distribution within the animal body. Hence, such microemulsion products would have limited applications in the industry.
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In recent studies [13–16], scientists have found unique mixtures of food-grade oils, which can be diluted with an aqueous phase progressively and continuously without phase separation. They can be transformed into bicontinuous structures that can be inverted into oilin-water nanodroplets upon further dilution. These unique mixtures consist of two or more food-grade nonionic hydrophilic emulsifiers that self-assemble to form mixed reverse micelles (the concentrate). Bicontinuous microemulsion [17] has been an active research topic because its unique structure lends itself well to controlled release applications. Amphiphilic molecules form bicontinuous water and oil channels, where “bicontinuous” refers to two distinct (continuous, but non-intersecting) hydrophilic regions separated by bilayers. This allows for simultaneous incorporation of water- and oil-soluble active ingredients. The phase structure also provides a tortuous diffusion pathway for controlled release of the encapsulated ingredients. Despite recent activities, there remains a gap in translation of the technique into a feasible and practical application. Difficulties include achieving a good level of product stability to provide a reasonable shelf life, manufacturing scalability, and customization using regulatory-approved material. These were hindering progress in the development of food-grade bicontinuous microemulsions into commercial products. This paper summarizes the novel development microemulsified bicontinuous carotenoid, made through the optimization of food grade U-type microemulsion production for better loading capacity, protection and reduction of carotenoids with a particle size of approximately 0.25 μm. This includes characterization of the physicochemical properties of the formulation and an evaluation on the bioavailability and effect in the pigmentation efficacy tested by monitoring the plasma level of trans-capsanthin and the yolk color (YCF) score of the eggs in an in vitro trial. 2. Materials and methods 2.1. Materials Tween 80 [polyoxyethylene (20) sorbitan monooleate], d-(+)limonene, ethanol and glycerol were of food grade [obtained from Kemin Agrifood Asia (KAA)]. All chemicals and reagents used in the analytical protocols were of analytical reagent grade. The water was double-distilled. A stabilized source of saponified red carotenoids from paprika extracts containing major natural red pigments transcapsanthin and capsorubin (available at KAA) was used as a control. 2.2. Phase diagrams and conductivity measurement of microemulsions The single-phase region of the microemulsion [10] consisting of Tween 80/ethanol/limonene/glycerol/water was determined systematically by titrating water to various compositions of Tween 80, ethanol, limonene and glycerol, in a screw-capped test tube. Each sample was vortex-mixed and allowed to equilibrate in a temperature-controlled environment at 25 °C. Stock solution of water and glycerol at a constant weight ratio of 3:1 was made. The limonene/ethanol weight ratio was held constant at 1:2. Mixtures of the surfactant/oil phase (ethanol and limonene) or mixtures of the surfactant/aqueous phase (water and glycerol) were prepared in culture tubes, sealed with screw caps at predetermined weight ratios of oil phase to surfactant, or aqueous phase to surfactant, and kept in a 25 °C (± 0.3 °C) water bath. Microemulsion areas were determined in phase diagrams by titrating either the oil phase/surfactant or aqueous phase/surfactant mixture with the aqueous phase or the oil phase, respectively. All samples were vigorously stirred. The samples were allowed to equilibrate for at least 24 h before they were examined. The microemulsion region was further classified as either oil-in-water (O/W), bicontinuous or water-in-oil (W/O) microemulsions. A rough demarcation of the bicontinuous region was further deduced from conductivity measurements [7]. Electrical conductivity measurements were
performed at 25 ± 0.3 °C on samples along the dilution line P using a conductivity meter (Extech EC500, pH/conductivity meter). Since the microemulsions were nonionic, a small quantity of an aqueous electrolyte (a solution of 0.01 M NaCl) was added. 2.3. Solubilization and preparation of self-microemulsifying carotenoid system (SMECS) To test the solubilization capacity of saponified red carotenoids in the microemulsion system, 1 mg of saponified carotenoids was added to a test tube containing 10 g of prepared microemulsion using a vortex mixer to dissolve the carotenoids for approximately 10 min. If the saponified carotenoids could dissolve in this microemulsion, the previous procedure was repeated until the mixture maintained a persistent cloudy appearance or visible grains of solid were found deposited at the bottom of the test tube after being mixed. The maximum solubility of saponified carotenoids in each microemulsion system was then determined. The carotenoid-microemulsion was prepared as follows: from the solubilization study, 0.01 wt% of the saponified carotenoids was added to a pre-mixed microemulsion sample containing 32.5 wt.% Tween 80, 32.5 wt.% limonene/ethanol and 35 wt.% water/glycerol. The mixture was vortex-mixed at 2500 rpm and uniformly dispersed with an ultrasonic probe for 2 min to obtain a persistent clear appearance mixture. The sonication also serves to remove bubbles that could be generated during vortexing. This prepared self-microemulsifying carotenoid system was subsequently used for further characterizations. 2.4. Stability measurement A stability study was conducted based on spectrophotometric determination of total carotenoids in the self-microemulsifying carotenoid system. The accelerated stability of carotenoid-microemulsions prototype over time at 75 °C for 3 days in the presence of oxygen was monitored. About 0.5 g (±0.1 mg) of the self-microemulsifying carotenoid at different time points was added to a 100 ml brown volumetric flask. The flask was filled with a mixture of hexane:ethanol:acetone:toluene (HEAT) at a ratio of 10:6:7:7 as the extracting solvent, and stirred with a magnetic stir bar for 15 min. Five milliliters of the solution was transferred by pipette to a 50 ml brown volumetric flask, diluted to the mark with HEAT, and shaken to mix the contents. A cuvette was filled with the solution and absorbance was measured at 460 nm against the extracting solvent using a spectrophotometer (UV-2401PC, Shimadzu). 2.5. Particle size analysis and transmission electron microscopy observations The particle size analysis was carried out using a particle size analyzer (HORIBA SZ-100Z). The particle size of the coarse saponified red carotenoids was also determined for comparison. Long-term stability testing involving particle size measurements was also conducted at given time intervals over 1 month storage at 25 °C. To observe morphology, the self-microemulsifying carotenoid was directly deposited onto carbon film supported by copper grids, stained with a 1% aqueous solution of osmium tetroxide (OsO4) and investigated using the transmission electron microscope (TEM) JEOL 1010. The morphology of the coarse saponified red carotenoid was also determined for comparison. 2.6. Bioavailability The bioavailability of the carotenoids was tested after the selfmicroemulsifying carotenoid (liquid system) had been transformed into beadlets of 250 μm average particle size by spray drying to produce the solid self-microemulsifying carotenoid system (S-SMECS). Both the solid self-microemulsifying carotenoid system (S-SMECS) and the non-microemulsifying (commercial product, CP) contained 5.4g/kg of
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3. Results and discussion 3.1. Phase diagram and conductivity measurement of microemulsions Fig. 1 shows the phase behavior of the transparent microemulsion region (1 phase region as denoted by blue colored outlined area) of the system composed of Tween 80/ethanol/limonene/glycerol/H2O. The shaded region represents the wide range of compositions that can be selected to form transparent microemulsions. Based on the diagram, microemulsions can be formed using aqueous content ranging from about 20 to 100 wt.%. The changes in conductivity of microemulsions along the P-line with the aqueous content are shown in Fig. 2. It shows the low conductivity of microemulsions at lower aqueous water content (b 20 wt.%), followed by a rapid increase in conductivity when the aqueous content was greater than 20 wt.%. With regards to the low conductivity for the systems containing less than 20 wt.% aqueous content, it was likely due to the formation of W/O microemulsion droplets dispersed in the oil medium. The sharp increase in conductivity for the systems containing higher than 20 wt.% aqueous content denoted the presence of numerous interconnected conducting channels, which are characteristics of bicontinuous microemulsions. An attempt has yet to be made to establish the boundary between the bicontinuous microemulsion and O/W microemulsion. The current investigation focused on microemulsions containing less than 40 wt.% aqueous content. Further studies on the boundary shall be conducted in the near future. Based on the conductivity measurements, the system containing 35 wt.% water was found to be
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a bicontinuous microemulsion, which was then chosen for the detailed study. The resulting bicontinuous phase offered exceptional control over the nanostructure, yielding an architecture that was well-suited for the incorporation of hydrophobic and hydrophilic active ingredients. 3.2. Solubilization and stability of self-microemulsifying carotenoid system Fig. 3 shows the solubilization pattern of the saponified red carotenoids in microemulsions. The carotenoids solubilized (~2 times) better in the W/O microemulsion than in the micellar phase consisting of only oil and surfactant without water. Moreover, enhanced solubilization (~0.01 wt.%) was observed when we increased the aqueous content to 35 wt.% to get the bicontinuous structure. A possible explanation for these dramatic increases in the solubilization of carotenoid could be related to the locus of solubilization. In systems free of water the locus of solubilization is at the micelle interface. As an aqueous phase is introduced (~ 10 wt.% aqueous), water-in-oil
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trans-capsanthin. The bioavailability of the formulation was studied by analyzing the trans-capsanthin in the blood serum of layers after oral administration of the two formulations with an average primary particle size of approximately 0.25 μm (solid self-microemulsifying carotenoid, S-SMECS) and 20 μm (commercial product, CP) respectively in an animal trial conducted by an accredited research farm in Malaysia. All procedures related to the holding and conducting an experiment with live animals are conducted under the approval of the Ethics Committee of the Genetics Improvement & Farm Technologies Sdn. Bhd., Malaysia. Twenty nine week old Lohamann Brown hens were used. The birds were fed with the experimental diets and allowed 1 week for adaptation to their new environment. The birds were placed in individual wirefloored cages arranged in two tires within an open-sided house under 14 L:10 D lighting regime. Feed and water were provided ad libitum throughout the experimental diet. The two formulations were feed to two groups of 6 replicates containing 8 carotenoid-depleted birds. The dry powders were mixed with layer feed prior to feeding. The trial was carried out in two consecutive parts. In part I, a dosage of 0.5 kg/ton (containing 2.7 g of trans-capsanthin per ton of feed) of the two formulations was given consecutively for 7 days and in Part II, the two formulations were crossed over with continuing dosing at 0.5 kg/ton daily for another 7 consecutive days. The blood levels were analyzed at 24 h, 32 h, 7th and 14th day. Similarly, the YCF scores of the eggs were analyzed at day 0, 7 and 14. The whole liquid egg color was determined by means of a commercially available yolk color fan. Where required, HPLC-based analysis of transcapsanthin equivalents using the AOAC (Association of Analytical Communities) method (method number 970.64, 1990) was carried out. Data were statistically analyzed by one-way ANOVA method using Statgraphics. Eggs were cracked without whites onto plastic Petri dishes of 6 cm in diameter and placed on a flat white background in an environment with fluorescent lighting. A team of eight trained observers was asked to evaluate the eggs utilizing a commercial yolk color fan. The transcapsanthin concentration for each formulation was determined by extracting the blood serum per treatment using a modified AOAC (Association of Analytical Communities) method (method number 970.64, 1990).
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(W/O) swollen microemulsions are formed, and the hydrophilic OH groups of the saponified carotenoids are oriented toward the aqueous phase, thus causing the molecules to insert themselves between the surfactant hydrophobic chains. This change in the locus of solubilization causes an increase in solubilization at the interface. The increase in solubilization as the aqueous phase concentration increases may be also attributed to microstructure transformations. The structural transformation from W/O to the bicontinuous microstructure (~ 35 wt.% aqueous) causes the free carotenoids to solubilize between both the hydrophobic amphiphilic chains and the interface, thus causing an increase in the solubilization. In addition, it is noted at this point that the bicontinuous morphology provides an interesting environment for loading and release properties. The domain sizes of the aqueous and oil channels can be fine-tuned by varying the microemulsion components to allow full potential for solubilization and controlled release of the active ingredients. Moreover, by customizing the specific properties of the hydrophilic and hydrophobic portions, it is possible to control their interaction with the active ingredients, offering a greater potential for tailored release properties over a broad range of applications and conditions. In general, a loss of approximately 30% of total carotenoid from the initial added amount was observed in the self-microemulsifying carotenoid sample after accelerated study conducted at 75 °C for 3 days, as shown in Fig. 4. For the saponified carotenoids solubilized in water, the losses were significantly higher (~ 42% at 3rd day). It is well known that carotenoids are sensitive to light, oxygen and heat. In a dynamic high-temperature system, the loss in the content of carotenoid in the self-microemulsifying carotenoid sample is expected but, the role of microemulsion protection on the carotenoid is more predominant resulting in a lower loss of the carotenoid during the study as compared to the unprotected carotenoids. The carotenoids pigment when solubilized and contained within the microemulsion system is better protected due to the molecular architecture of the pigment within the microemulsion matrix i.e. the size and configuration (Fig. 5). The microemulsion is hypothesized to provide a physical barrier between the pigment and the oxidation catalysts (such as oxygen) and also its light scattering property can help to reduce the intensity of light reaching the pigment entrapped within them. In addition, the smaller particle size of the carotenoid pigment achieved using microemulsion will enable it to be easily and homogeneously distributed into the interior porous passage of the carrier granules that will further help to reduce the loss caused by oxidation on the surface and enhance the stability of the product as illustrated in Fig. 6.
Fig. 4. Stability of the self-microemulsifying carotenoid system (SMECS) and commercial product (CP) under accelerated study at 75 °C for 3 days.
3.3. Particle size analysis and transmission electron microscopy observation Both the bioavailability and the color yield of the carotenoids strictly depend on the particle size distribution of the active ingredient in the final formulation. Hence, the determination of the particle size distribution is a major consideration during production for quality assurance. Figs. 7 and 8 show the corresponding result of particle size analysis and the electromicrograph for the self-microemulsifying carotenoid system and the coarse carotenoid (commercial product, CP). The particle size of the carotenoid in microemulsion is maintained to about approximately 0.25 μm at average with contrast to a particle size of 20 μm for the coarse carotenoid. Fig. 7 also shows the particle size distribution of the carotenoid-microemulsion at the 0, 7th, 14th, 21st and 28th day at room temperature (25 °C). There were no significant differences in particle size distribution for the self-microemulsifying carotenoid system during the 1 month study. The long-term stability results demonstrated that the microemulsion protected carotenoid was more stable and uniformly dispersed with no aggregation (as shown in Fig. 8). The results also suggested that the surfactant and oil phases used in this study not only influenced the formation of protective colloid responsible for establishing colloidal stability against agglomeration but also expected to help the emulsion formed in the stomach be readily restructured into a bicontinuous network even in the absence of biliary phospholipid, thereby facilitating the uptake of carotenoids during the gastrointestinal passage.
Fig. 5. Schematic arrangement of the self-assembled surfactant, oil and alcohol in the bicontinuous phase facilitates the entrapment of carotenoids pigment within the core or at its interface.
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Fig. 6. Schematic arrangement of S-SMECS within the internal passage of the carrier particle.
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of feeding with S-SMECS, the trans-capsanthin in blood level increased from virtually zero to approximately 0.038 ppm, which is approximately 17% above the level achieved by the commercial product. After feed cross-over at day 7, the blood plasma collected from the birds originally treated with S-SMECS showed a drop of the trans-capsanthin from approximately 0.038 ppm to 0.017 ppm and was significantly lower than the blood plasma collected from the birds originally treated with the commercial product. A similar trend was observed for the YCF score of the egg yolk after the feed cross-over as shown in Fig. 10, the score of Total Trans-capsanthin in blood plasma (ppm)
The bioavailability of the formulation was studied by analyzing the trans-capsanthin in blood serum of layer birds after oral administration of the solid self-microemulsifying carotenoid system (S-SMECS) and the non-microemulsifying commercial product (CP) with an average primary particle size of approximately 0.25 μm and 20 μm respectively. The plasma concentration–time profile of trans-capsanthin from these products is shown in Fig. 9. As indicated in Fig. 9, carotenoid particle sizes exerted a significant influence on the relative bioavailability. It has been reported that trans-capsanthin, like lutein, is a poorly watersoluble lipophilic compound, that follows the same route of absorption as lipids. The plasma concentrations of trans-capsanthin were measurable in layers with 0.038 ppm and 0.017 ppm for S-SMECS and CP, respectively. This implies the involvement of endogenous emulsifiers in promoting solubilization and absorption of carotenoids in vivo. Although the exact mechanism of the absorption is not yet fully understood, trans-capsanthin has been thought to be absorbed through enterocytes by simple diffusion or receptor-mediated transport. Specifically, trans-capsanthin is emulsified into small lipid droplets in the stomach and further incorporated into mixed micelles by the action of bile salts and biliary phospholipids, after which mixed micelles are taken up by enterocytes. Thus, the appearance of relatively low concentrations of trans-capsanthin in layer plasma was possibly due to the involvement of the aforementioned absorption mechanism. After 7 days
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Fig. 9. Influence of particle size on the bioavailability of trans-capsanthin. Oral administration to carotenoid-depleted layers with solid self-microemulsifying carotenoid system (S-SMECS) and commercial product (CP).
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administration, significantly exceeding that of the commercial product containing the coarse carotenoids. Furthermore from the trial result, it supported our hypothesis that a desired yolk color score and carotenoid bioavailability can now be achievable at a significantly lower inclusion rate when carotenoid molecules are self-micromulsified. This novel microemulsion technology offers greatly enhanced flexibility for product development efforts, the capability to tailor different active ingredients loading of bicontinuous phases, and the controlled tolerance of bicontinuous phases for other ingredients. Overall, the information obtained from this study shall facilitate the rational design and fabrication of self-microemulsifying delivery systems for many ingredients. References
S-SMECS
CP
Fig. 10. Influence of particle size of solid self-microemulsifying carotenoid system (S-SMECS) and commercial product (CP) on the yolk-color score for the layer's egg yolk.
the eggs taken from the birds that treated with the S-SMECS improved from 7.5 to 9.75, which is a 30% enhancement over feeding the commercial product. Apart from an increase in pigment absorption, color intensity of the egg yolk is possibly dependent also on the pigment particle size. Perhaps at the nanometer range, the number of carotenoid molecules that can be packed at the surface has increased, leading to an increase in light absorption and scattering coefficient. After oral administration, no further dissolution is required as such the trans-capsanthin would be maintained in a fully solubilized state, after the bicontinuous microemulsion pre-concentrate self-emulsifies on contact with gastric fluid in the stomach. The already small and uniform bicontinuous arrays containing the trans-capsanthin may be further emulsified by the bile/ lecithin micelles in the intestinal fluids, digested by enzymes and converted into even smaller lipid particles. This process of digestion would greatly increase the surface area of trans-capsanthin for transfer to the intestinal epithelium. A similar observation was reported by Fu et al. [18]. This may explain why a richer redness was observed for the egg yolks obtained from layers treated with the self-microemulsifying carotenoids system. 4. Conclusions The described microemulsion process, which is based on using water soluble and toxicologically safe food-grade ingredients, offers new opportunity to produce a large variety of self-microemulsifying formulations. With proper choice of the surfactant, oils and water, bicontinuous microemulsions can be formed to enhance solubilization and protection of active ingredients. With a characteristic size of the active ingredient of the S-SMECS of about 0.25 μm, the preparation exhibits high bioavailability and better yolk pigmentation after oral
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