METHOD OF FORMING ENCAPSULATED COMPOSITIONS WITH ENHANCED SOLUBILITY AND STABILITY

Abstract

A method of forming an encapsulated composition with enhanced solubility and stability. A bicontinuous or Winsor Type III microemulsion is formed using an emulsifier, a solvent and a co-emulsifier. An active composition is added to the microemulsion resulting in a micellar network of the active composition within the microemulsion. The active composition can be either water-soluble or oil-soluble or both.

Claims

1. A bicontinuous microemulsion suitable for use as a carrier for trace minerals in an animal feed composition comprising: (a) an oil phase comprising said amphiphilic or lipophilic oil-soluble material; (b) an aqueous phase comprising said amphiphilic or hydrophilic water-soluble material; and (c) a food grade emulsifier system comprising (i) an ionic or non-ionic or zwitterionic emulsifier, and (ii) a co-emulsifier; and wherein said oil phase is dispersed as particles having an average diameter of below within said aqueous phase or said aqueous phase is dispersed as particles or continuous phase having an average diameter of below within said oil phase.

2. The bicontinuous microemulsion according to claim 1, wherein the trace mineral is an organic metal.

3. The bicontinuous microemulsion according to claim 1, wherein the trace mineral is chromium propionate.

4. The bicontinuous microemulsion according to claim 1, wherein the emulsifier is selected from the group consisting of glycerol ester of fatty acids, monoglycerides, diglycerides, ethoxylated monoglycerides, polyglycerol ester of fatty acids, lecithin, glycerol ester of fatty acids, sorbitan esters of fatty acids, sucrose esters of fatty acids, and mixtures thereof.

5. The bicontinuous microemulsion according to claim 1, wherein the co-emulsifier is a water miscible alcohol or acid emulsifying agent selected from the group consisting of ethanol, propanol, propylene glycol, glycerol, acetic acid, natural vinegar and mixtures thereof.

6. The bicontinuous microemulsion according to claim 1, wherein the oil is selected from the group consisting of limonene, vegetable oils, animal oils, polyol polyesters and mixtures thereof.

7. A bicontinuous microemulsion suitable for use in animal feed or human food comprising: (a) an oil phase comprising said amphiphilic or lipophilic oil-soluble material; (b) an aqueous phase comprising said amphiphilic or hydrophilic water-soluble material; and (c) a food grade emulsifier system comprising (i) an ionic or non-ionic or zwitterionic emulsifier; (ii) a co-emulsifier; and (iii) at least one antioxidant; wherein said oil phase is dispersed as particles having an average diameter of below within said aqueous phase or said aqueous phase is dispersed as particles or continuous phase having an average diameter of below within said oil phase.

8. The bicontinuous microemulsion of claim 7, further comprising at least one antioxidant is selected from the group consisting of rosemary extract, spearmint extract, green tea extract, curcumin, ascorbic acid, annatto extract, acerola, and tocopherols.

9. The bicontinuous microemulsion according to claim 7, wherein the emulsifier is selected from the group consisting of glycerol ester of fatty acids, monoglycerides, diglycerides, ethoxylated monoglycerides, polyglycerol ester of fatty acids, lecithin, glycerol ester of fatty acids, sorbitan esters of fatty acids, sucrose esters of fatty acids, and mixtures thereof.

10. The bicontinuous microemulsion according to claim 7, wherein the co-emulsifier is a water miscible alcohol or acid emulsifying agent selected from the group consisting of ethanol, propanol, propylene glycol, glycerol, acetic acid, natural vinegar and mixtures thereof.

11. The bicontinuous microemulsion according to claim 7, wherein the oil is selected from the group consisting of limonene, vegetable oils, animal oils, polyol polyesters and mixtures thereof.

12. A method of using a bicontinuous microemulsion for extending the shelf life of oil and fats, wherein the bicontinuous microemulsion suitable for use in human food comprising: (a) an oil phase comprising said amphiphilic or lipophilic oil-soluble material; (b) an aqueous phase comprising said amphiphilic or hydrophilic water-soluble material; and (c) a food grade emulsifier system comprising (i) an ionic or non-ionic or zwitterionic emulsifier, and (ii) a co-emulsifier; and (iii) at least one plant-based extract; (d) wherein said oil phase is dispersed as particles having an average diameter of below within said aqueous phase; or (e) wherein said aqueous phase is dispersed as particles or continuous phase having an average diameter of below within said oil phase.

13. The bicontinuous microemulsion according to claim 12, wherein the emulsifier is selected from the group consisting of glycerol ester of fatty acids, monoglycerides, diglycerides, ethoxylated monoglycerides, polyglycerol ester of fatty acids, lecithin, glycerol ester of fatty acids, sorbitan esters of fatty acids, sucrose esters of fatty acids, and mixtures thereof.

14. The bicontinuous microemulsion according to claim 12, wherein the co-emulsifier is a water miscible alcohol or acid emulsifying agent selected from the group consisting of ethanol, propanol, propylene glycol, glycerol, acetic acid, natural vinegar and mixtures thereof.

15. The bicontinuous microemulsion according to claim 12, wherein the oil is selected from the group consisting of limonene, vegetable oils, animal oils, polyol polyesters and mixtures thereof.

16. The bicontinuous microemulsion according to claim 12, wherein the at least one plant-based extract is selected from the group consisting of rosemary, spearmint, green tea, curcumin and tocopherols.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a chart of the phase behavior of the transparent microemulsion region of the system composing of polysorbate 80/ethanol/limonene/glycerol/H.sub.2O; the weight ratio of limonene to ethanol and glycerol to water were fixed at 1:2 and 1:3 while that for oil to surfactant was at 1:1 along line P with increasing water content.

[0028] FIG. 2 is a chart of the changes in conductivity of microemulsions along P-line with increasing aqueous content.

[0029] FIG. 3 is a chart of the maximum solubilization of carotenoids in microemulsions consisting of polysorbate 80/ethanol/limonene/glycerol/water; parameters were plotted against the aqueous content along dilution along line P.

[0030] FIG. 4 is a chart of the UV-Vis absorption of the carotenoids-encapsulated microemulsion after storage at 25° C. for 1 month.

[0031] FIG. 5 is a chart of the UV-Vis absorption of the carotenoids-encapsulated microemulsion after storage at 25° C. and 65° C. for 1 month.

[0032] FIG. 6A is a TEM micrograph of microemulsion (without carotenoids); FIG. 6B is a TEM micrograph of carotenoid-microemulsions. FIG. 6B is a TEM micrograph of saponified carotenoids concentrate. FIG. 6C is an agglomerated TEM image of the saponified carotenoid concentrate

[0033] FIG. 7 is a chart of the particle size distribution of the carotenoid-microemulsion sample.

[0034] FIGS. 8(a) and (b) are charts of the particle size analysis of (a) Kem GLO 10 liquid precursor and (b) nanodispersed Kem GLO 10 liquid precursor.

[0035] FIG. 9 is a chart of the particle size distribution of the nanodispersed Kern GLO 10 liquid precursor at the 0th, 7th, 14th, 21st and 28th day at room temperature (25° C.).

[0036] FIG. 10 is a chart of the effect of pigment treatment on the trans-capsanthin absorption in blood plasma.

[0037] FIG. 11 is a chart of the effect of pigment treatment on the trans-capsanthin deposition in egg yolk.

[0038] FIG. 12 is a chart of the effect of pigment treatment on the YCF score of eggs.

[0039] FIG. 13 is a chart showing the stability of the SSEM of chromium propionate.

[0040] FIG. 14 is a chart showing the changes in the total polar compounds (TPC) of soybean oil with different frying cycles.

[0041] FIG. 15 is a chart showing the changes in the free fatty acid (FFA) of soybean oil with different frying cycles.

[0042] FIG. 16A-C are charts showing the changes in the L* (Lightness), a* (Red/Green), b* (Yellow/Blue) color of the soybean oil with different frying cycles.

[0043] FIG. 17 is a chart showing changes in the peroxide value of the soybean oil with different frying cycles.

[0044] FIG. 18 is a chart showing changes in the p-anisidine value of the soybean oil with different frying cycles.

[0045] FIG. 19 is a chart showing changes in induction period of soybean oil during frying.

[0046] FIG. 20 is a chart showing changes in average % total polar compound (TPC) of soybean oil during frying.

[0047] FIG. 21 is a chart showing changes in average peroxide value of soybean oil during frying.

[0048] FIG. 22 is a chart showing changes in average anisidine value of soybean oil during frying.

[0049] FIG. 23 is a chart showing Changes in average % free fatty acid of soybean oil during frying.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0050] Suitable bicontinuous microemulsions can be formed when proportions of the components are respectively from about 15 to about 50% for the aqueous phase (such as glycerol/water, propylene glycol/water or water), from about 5% to about 40% for the oil phase (such as limonene, ethanol, limonene/ethanol, acetic acid, natural vinegar) and from about 10% to about 50% for the surfactants (Polysorbate 60, Polysorbate 65, Polysorbate 80, lecithin and lecithin derivatives, mono- and diglycerides, sorbitan fatty acid esters,) all percentages by weight (denoted wt % hereafter). Persons skilled in the art will understand how to combine different oil and surfactants in different ratios to achieve the desired effect on the various properties of the resulting formulation, for example, to improve the active ingredients solubilization capacity or stability of the resulting formulation.

Example 1

Materials and Methods

[0051] Materials.

[0052] Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate; TWEEN® 80), R-(+)-limonene, ethanol and glycerol were of food grade. All chemicals and reagents used in the analytical protocols were of analytical reagent grade. The water was double-distilled. The control carotenoid source used was a stabilized source of saponified yellow carotenoids from marigold extracts (OroGLO® 24 Dry, Kemin Industries, Inc.).

[0053] Phase Diagram and Electrical Conductivity.

[0054] The single-phase region of the microemulsion.sup.6 consisting of polysorbate 80/ethanol/limonene/glycerol/H.sub.2O was determined systematically by titrating water to various compositions of polysorbate 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. A stock solution of water and glycerol at a constant weight ratio of 3:1 was made. The ethanol/limonene weight ratio was held constant at 1:2. Mixtures of surfactant/oil phase (ethanol and limonene) or mixtures of 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/surfactant phase or aqueous phase/surfactant mixtures 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.

[0055] 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..sup.6 Electrical conductivity measurements were performed at 25±0.2° 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. The samples remained clear and there were no observable changes in the phase diagram.

[0056] Carotenoid-Microemulsion Preparation.

[0057] The sample was prepared as follows. Based on the formulation for the OroGLO® 24 Dry product, 38.0 g of saponified OroGLO® concentrate was added to the mixing vessel followed by 15.0 g of the pre-prepared microemulsion. The microemulsion consisted of 32.5% polysorbate 80, 32.5% limonene/ethanol (1:2), 35.0% glycerol/water (1:3). All contents were mixed until a homogeneous mixture of carotenoid-microemulsion was observed. The sample was then added with 47 g of one or more inert carriers and blended to achieve a free-flowing powder.

[0058] Centrifugal Stress Test.

[0059] The microemulsion stability of the formulation was tested by subjecting them to a centrifugal stress test. About 15 g of sample was placed in a transparent polymer tube and subjected to 24,000 g centrifugal force for 15 minutes (B. Braun Biotech Centrifuge ER 15P). The centrifuged samples were observed under fluorescent light for the degree of phase separation. The viscosity of the formulations was tested using a Brookfield viscometer model DV-I+.

[0060] Viscosity and Refractive Index Measurement.

[0061] The refractive index of the formulations was determined using an Abbe-type digital refractometer (Reichert-Jung, Abbe Mark II) by placing one drop of the formulation on the slide in triplicate at 25° C.

[0062] Solubilization Measurement.

[0063] Saponified carotenoids and limonene were first mixed. Water, glycerol, ethanol and Polysorbate 80 were then added dropwise to obtain a single-phase clear microemulsion with the desired composition. Finally, the samples were cooled and stored at 25° C. Samples that remained transparent for at least 5 days were considered to be microemulsions.

[0064] Stability Study and Spectrophotometric Determination of Total Carotenoid (SOP-10-00072).

[0065] The stability of microemulsions over time was monitored by UV/Vis absorption measurement. For unstable microemulsions, the encapsulated carotenoids would be released instantly and the UV/Vis absorption of the sample would decrease. The sample was first prepared by adding 0.5 g (+/−0.1 mg) of the carotenoid-microemulsion to a 100-ml brown volumetric flask. The flask was filled with a mixture of hexane:ethanol:acetone:toluene at a ratio of 10:6:7:7 (HEAT) as the extracting solvent, and stirred with a magnetic stir bar for 15 min. Five ml 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).

[0066] Morphology of Carotenoid-Microemulsion.

[0067] To observe the morphologies, carotenoid-microemulsions and yellow carotenoids were directly deposited onto carbon film supported by copper grids, stained with 1% aqueous solution of osmium tetroxide (OsO.sub.4) and investigated using the transmission electron microscope (TEM) JEOL 1010.

[0068] Particle Size Analysis.

[0069] The carotenoid-microemulsion sample was put through size analysis using a particle size analyzer (Horiba Particle size analyzer LA-950).

Results

[0070] Phase Diagrams and Conductivity Measurement.

[0071] FIG. 1 shows the phase behavior of the transparent microemulsion region (dotted area) of the system composing of polysorbate 80/ethanol/limonene/glycerol/H.sub.2O. 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 an aqueous content ranging from about 20 to 100 wt %.

[0072] The changes of conductivity of microemulsions along P-line with the aqueous content are shown in FIG. 2. It shows the low conductivity of microemulsion at lower aqueous water content (<20 wt %), followed by a rapid increase in conductivity when the aqueous content was greater than 20 wt %.

[0073] Based on the conductivity measurements, the system containing 35 wt % water was found to be a bicontinuous microemulsion. This was then chosen for a detailed study.

[0074] The bicontinuous carotenoid-microemulsion system was stable and able to maintain homogeneity in an emulsion-break (centrifuge) test. The viscosity was less than 100 cP (˜72.4-77.5 cP) and the refractive index of microemulsions was 1.4106.

[0075] Solubilization Capacity.

[0076] FIG. 3 shows the solubility of carotenoids in the microemulsion components at 20 wt % and 35 wt % water. The solubilization of carotenoids in microemulsions systems with 20 wt % water and 35 wt % water was ˜6 times (6630 ppm) and ˜8.4 times (10,100 ppm), respectively, higher than the solubility of carotenoids in (R)-(+)-limonene (1200 ppm).

[0077] Stability Study.

[0078] FIG. 4 shows the UV-Vis absorption of the carotenoids-encapsulated microemulsion after 1 month at room temperature (25° C.). There were no significant differences in the absorption curve of UV-Vis spectra among the microemulsions during 1-month study. No carotenoids were released from the microemulsion, and there were no signs of aggregation after 1 month.

[0079] FIG. 5 shows the changes in the concentration of carotenoids in the microemulsions over time. There was a slow degradation, resulting in 13% and 24% drop in the total carotenoids content when exposed to 25° C. and 65° C., respectively, for 1 month.

[0080] Morphology of the Carotenoid-Microemulsions.

[0081] FIGS. 6A and B show the TEM images of the bicontinuous microemulsion of 35 wt % aqueous content without and with carotenoids respectively. As shown in FIG. 6A, a micellar network formed by branched micelles was found. It was an interconnected, branched micellar network, spanning over a large space, analogous to the bicontinuous phase, where an infinite multi-connected fluid bilayer usually separates the hydrophilic region from the hydrophobic region. As seen in FIG. 6B, the particles were slightly larger than 100 nm in diameter.

[0082] Most of the particles appear spherical in shape in the well-dispersed microemulsion system. This contrasted with FIG. 6C which shows the agglomerated TEM image of the saponified carotenoid concentrate. The agglomeration was expected because there was no surfactant attached to the surface of the carotenoids to maintain them in a dispersed state.

[0083] Particle Size Analysis.

[0084] The particle size distribution of the carotenoid-microemulsion sample was as shown in the FIG. 7. The mean diameter of the carotenoid-microemulsion was relatively uniform with a particle size of ˜500 nm.

Discussion

[0085] Food-grade bicontinuous microemulsions offer unique properties of particular interest to the food and feed industry. The materials can be formed by simple combination of unique mixtures of food-grade oils and surfactants with water. In our study, carotenoids were found to be solubilized in the bicontinuous microemulsions up to 6-8 times more than their solubility in R-(+)-limonene per se. The microemulsion system has demonstrated that it can be diluted by water, an important property that will enable it to be applied across food and feed industries. In addition, this system can be diluted with an oil phase (including (R)-(+)-limonene) and, therefore, is also suitable for oil-continuous phase applications. It is essential, therefore, to construct microemulsion concentrates that are capable of dilution in both oil and water phases. The microemulsions described in this paper are unique in these properties.

[0086] As seen in FIG. 6B, the particles were slightly larger than 100 nm in diameter. This is in line with the results of particle sizing in FIG. 7, showing a uniform mean diameter of around 500 nm. 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 (as shown in the insert of FIG. 6A) 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.

[0087] The carotenoid-microemulsion had shown good stability physically and chemically, with minimal degradation of carotenoids during storage. A slightly greater loss of carotenoids occurred at 65° C. compared to 25° C. There are several possible explanations for the degradation of the carotenoids. Among them, the influence of surface area is relevant to the present study. As compared to bulk crystalline carotenoids, the surface area of carotenoids in the nanometer range is significantly larger. This may reduce the stability by providing more contact surface between the carotenoids and the aqueous environment. In one study.sup.13, it was reported that the degradation of β-carotene in multiple nanosize emulsions was rapid, leaving only 32.3% of β-carotene after 4 weeks of storage at 50° C. The significant slow degradation of carotenoids in our bicontinuous microemulsions offers an advantageous and would make it possible to develop a commercial product with an appropriate length of shelf life.

[0088] With regard 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 microemulsion.

[0089] In conclusion, we have shown that our novel system can provide enhanced solubilization of carotenoids in the microemulsions, as well as in protecting the carotenoids from fast environmental reactivity (oxidation). This novel microemulsion technology also 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.

Example 2

[0090] The objective of this example was to prepare a solid nanodispersed self-emulsifying delivery system containing bicontinuous food-grade microemulsions of polyethoxylated sorbitan ester (Tween 80), water, limonene, ethanol and glycerol with excellent solubilization capacity, as liquid phase for the delivery of bioactive carotenoids, and to evaluate the enhanced bioavailability of the carotenoids from the solid form. The bioavailability study performed in the layer trial resulted in a 2.9-fold (191%) increase in the capsanthin absorption in the bird serum and 20% increase in the capsanthin deposition in the bird eggs from the nanodispersed formulation. Furthermore, the YCF score of the eggs from the birds treated with the nanodispersed formulation compared with a current formulation showed an average score of 11.25 and 8.75, respectively. These results clearly demonstrated the excellent ability of the new solid formulation in promoting solubilization and absorption of trans-capsanthin in vivo, through the use of endogenous microemulsion and size reduction effect.

Materials and Methods

[0091] Materials.

[0092] Tween 80 (polyoxyethylene (20) sorbitan monooleate), limonene, ethanol, glycerol, wheat pollard and silica were of food-grade. 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 and Kem GLO 10 were also obtained from Kemin Animal Nutrition and Health (Asia-Pac) production. The determination of trans-capsanthin in blood serum and egg yolk were done using a standard method.

[0093] Preparation of Nanodispersed Kem GLO 10 Liquid Precursor.

[0094] The composition of bicontinuous carotenoid microemulsion was established in Example 1 which consists of tween 80:ethanol/limonene:glycerol/H2O. The weight ratio of limonene to ethanol and glycerol to water were fixed at 1:2 and 1:3, respectively. The ratio of oil/surfactant/water used were 32.5/32.5/35 (wt %) respectively, with 5.4 g/kg of trans-capsanthin. The bicontinuous carotenoid microemulsion formulation was prepared by method of Example 1. Briefly, carotenoid (37.2 wt %) was dissolved into the microemulsion mixture (15 wt %) of oil, surfactant, and co-surfactant at 25° C. in an isothermal water bath to facilitate solubilization. The resultant mixture was vortexed until a clear solution was obtained. It was then equilibrated at ambient temperature for at least 2 h and examined for signs of turbidity or phase separation prior to droplet size and optical studies.

[0095] Preparation of Nanodispersed Kem GLO 10 Dry.

[0096] A solid form of carotenoids was prepared. Briefly, silica and wheat pollard (21.8 wt %/26.0 wt %) were first added into a mixer. 52.2 wt % of nanodispersed carotenoid microemulsion containing saponified caroteniod (37.2 wt %) was then added into the mixer with constant stirring at room temperature for 15 min until homogenous mixture was obtained. The resultant powder was collected from the mixer and measured for the final trans-capsanthin content.

[0097] Characterization of Nanodispersed Kem GLO 10 Liquid Precursor.

[0098] The particle size distribution of sample was measured using an HORIBA particle size analyzer (LA-950V2). 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 one month storage at 25° C. To observe the morphology, liquid carotenoid-microemulsion was directly deposited onto carbon film supported by copper grids, stained with 1% aqueous solution of osmium tetroxide (OsO.sub.4) and investigated using the transmission electron microscope (TEM) JEOL 1010 at 100 kV. The morphology of the coarse saponified red carotenoid was also determined for comparison.

[0099] Bioavailability.

[0100] A layer trial was carried out at Genetic Improvement & Farm Technologies Sdn. Bhd., Malaysia. The trial was conducted using a control and two different treatments (nanodispersed Kem GLO 10 and current Kem GLO 10). The control diet composition listed in Table 2 was used in this trial. The two formulations were included at rate of 1 kg/ton of feed. For the two experimental treatments the concentration of trans-capsanthin in the feed was approximately 5.4 g/ton. Twenty nine weeks old Lohamann Brown hens were used. The birds were fed with the experimental diets and allowed one week for adaptation to their new environment. The birds were placed in individual wire-floored cages arranged in two tires within an open-sided house under 14 L; 10 D lighting regime. Four cages of birds were fed from a single feed trough and considered as one experimental replicate. Each experimental diet was given to eight replicates (32 birds per treatment). Feed and water were provided ad libitum throughout the experimental diet. Each week, ten eggs and blood samples from each dietary group were taken for trans-capsanthin analysis. The plasma was separated from blood and the trans-capsanthin content was quantified. A team of 8 trained observers was asked to evaluate the eggs subjectively utilizing a commercial (DSM) color fan. Data were statistically analyzed by one-way ANOVA method.

Results

[0101] Characterization of Nanodispersed Kem GLO 10 Liquid Precursor.

[0102] The composition of lipid excipients that constitutes the ternary phase of optimized nanodispersed Kem GLO 10 microemulsion is shown in Table 1. The spray dried particles of solid form had good flowability properties due to the presence of silica and wheat bran, which are regarded as suitable carriers for the solid dosage forms. The final trans-capsanthin content of the prepared solid form was 5.4 g/kg of trans-capsanthin.

TABLE-US-00001 TABLE 1 Composition of optimized nanodispersed Kem GLO 10 liquid precursor and dry solid Composition (%) Vehicle Type Name Liquid Solid Oil Limonene 1.625 1.625 Surfactant Tween 80 4.875 4.875 Co-surfactant Ethanol 3.25 3.25 Aqueous phase Glycerol/water (1:3) 5.25 5.25 Carotenoid Saponified Paprika 37.2 37.2 Oleoresin Carrier Silica/Wheat pollard NA 47.8

TABLE-US-00002 TABLE 2 Composition of poultry layer mash feed Specifications 4130 Moisture (% max) 13 Ash (% max) 15 Crude Protein (% min) 17 Crude Fat (% min) 3 Crude Fiber (% max) 6 Calcium (%) 3.5-4.5 Total Phosphorus (% min) 0.5 Measured Xanthophyll in Feed 2.52 × 10.sup.−3 g/kg

[0103] FIG. 8 shows the corresponding result of particle size analysis for the nanodispersed Kern GLO 10 liquid precursor and the coarse carotenoid (Kern GLO 10 liquid precursor). The particle size of the carotenoid in microemulsion is maintained at ca. 0.5 μm on average with contrast to a particle size of ˜20 μm for the coarse carotenoid.

[0104] FIG. 9 shows the particle size distribution of the nanodispersed Kern GLO 10 liquid precursor at the 0th, 7th, 14th, 21st and 28th day at room temperature (25° C.). There were no significant differences in particle size distribution for the sample during 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(b)). It was hypothesized that the surfactant and oil phases used in this study not only influenced the formation of protective colloids responsible for establishing colloidal stability against agglomeration, but also helped the microemulsion formed in the stomach to be readily restructured into bicontinuous network. This may have occurred even in the absence of biliary phospholipid, thereby facilitating the uptake of carotenoids during the gastrointestinal passage.

[0105] Bioavailability.

[0106] The bioavailability was studied by analyzing the trans-capsanthin in blood plasma and egg yolk of layer birds, after oral administration of nanodispersed Nano Kern GLO 10 comparing with current Kern GLO 10 and control treatment. The concentration-time profiles of trans-capsanthin in blood plasma and egg yolk from the two formulations are shown in FIGS. 10 and 11. As indicated in FIG. 10, particle sizes exerted a significant influence on the relative bioavailability. The blood plasma collected from the birds treated with nanodispersed Nano Kern GLO 10 showed an average value of 0.125 ppm of capsanthin, while the samples taken from the birds treated with the control diet and current Kern GLO 10 showed average values of 0.0028 ppm and 0.043 ppm, respectively. From these results it can be seen that there is a 2.9-fold (191%) increase in the capsanthin absorption from the nanodispersed Kern GLO 10 over the Kern GLO 10. From FIG. 11, the eggs collected from the control treatment and the current Kern GLO 10 showed 0.034 ppm and 0.54 ppm of capsanthin in the egg yolk, respectively. From these results it can be seen that there was a 20% increase in the capsanthin deposition from the nanodispersed Kern GLO 10 over the current Kern GLO 10. It is also important to note that there was a ˜19-fold increase in the capsanthin deposition in the egg from the nanodispersed Kern GLO 10 over the eggs collected from the birds treated with the control diet. As for the YCF score of the eggs, as shown in FIG. 12, eggs from birds treated with the nanodispersed Kern GLO 10 showed an average score of 11.25, while samples taken from birds treated with the current Kern GLO 10 showed an average score of 8.75. There was a 28.5% improvement in the color score of the nanodispersed Kern GLO 10 over the current Kern GLO 10. It is also important to note that there was a ˜1.5 fold increase in the yolk color in the egg arising from the nanodispersed Kern GLO 10, compared to the eggs collected from birds treated with the control diet.

Discussion

[0107] From the trans-capsanthin concentration-time profiles in blood serum and egg yolk obtained for the nanodispersed Kern GLO 10 (FIGS. 10 and 11), a difference is seen compared to the results obtained for treatments using current Kern GLO 10 and control diet. This demonstrates the involvement of endogenous microemulsion and size reduction effect in promoting solubilization and absorption of trans-capsanthin in vivo. It has been reported that trans-capsanthin, like lutein, is a poorly water-soluble lipophilic compound, and follows the same route of lipid absorption.sup.14,15. 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. Furthermore, 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 bird plasma and egg yolk was possibly due to the involvement of the aforementioned absorption mechanism. Furthermore, the use of surfactants is known to help the permeability of active ingredients through perturbation of the cell membrane (transcellular permeation) and/or modifying tight junction between the cells paracellular permeation.sup.16-18.

[0108] In the nanodispersed Kem GLO 10, Tween 80 was used as an emulsifier and we hypothesized that the presented the trans-capsanthin in solubilized microemulsion form in the gastrointestinal tract, possibly enhancing uptake of the trans-capsanthin by intestinal cells. After oral administration, no further dissolution is required as such a 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. This may explain the significant improvement of the YCF score for the eggs from the nanodispersed Kem GLO 10 treatment, indicating once again that the detected difference in bioavailability is highly significant.

Conclusion

[0109] In conclusion, nanodispersed Kem GLO 10 dry containing bicontinuous microemulsion was successfully prepared for the delivery of trans-capsanthin. The droplet size analyses revealed characteristic size of liquid precursor of ˜0.5 μm compared to the coarse carotenoid of ˜20 μm. The bioavailability study performed in the layer trial resulted in a 2.9-fold (191%) increase in the trans-capsanthin absorption in the bird blood plasma and 20% increase in the trans-capsanthin deposition in the bird eggs from the nanodispersed formulation. Furthermore, the YCF score of the eggs from the birds treated with the nanodispersed formulation compared with current formulation showed an average score of 11.25 and 8.75, respectively. These results clearly demonstrated the excellent ability of the new solid formulation, with the involvement of endogenous microemulsion and size reduction effect, in promoting solubilization and absorption of trans-capsanthin in vivo.

Example 3

Materials and Methods

[0110] Materials.

[0111] Tween 80, limonene, ethanol, glycerol, wheat bran and silica were of food-grade. All chemicals and reagents used in the analytical protocols were of analytical reagent grade and double-distilled water was used. A stabilized source of saponified red carotenoids from paprika extracts and Kem GLO 10 were obtained as from Kemin Animal Health And Nutrition (Asia-Pac) production.

[0112] Preparation of Nanodispersed Kem GLO 10 Dry.

[0113] A solid form of the carotenoids was prepared. Briefly, silica and wheat pollard (21.8 wt %/26.0 wt %) were first added into a mixer. A nanodispersed carotenoid microemulsion, 52.2 wt % (as per Example 2) containing saponified caroteniod (37.2 wt %) was then added into the mixer with constant stirring at room temperature for 15 min until a homogenous mixture was obtained. The produced sample analyzed contained 12.47 g/kg of carotenoids.

[0114] Preparation of Treated Feed Meal.

[0115] The poultry layer mash feed (as per Example 2) contained 17% protein, 3% fat and not more than 6.0% crude fiber. Treated feed was prepared by a layer test facility (Genetic Improvement & Farm Technologies Sdn. Bhd., Malaysia) by adding either 0.5 kg/ton or 1.0 kg/ton nanodispersed Kem GLO 10 and Kem GLO 10 to the low carotenoids feed.

[0116] Storage of Feed Meal.

[0117] Treated feed meal was delivered to Kemin Animal Health And Nutrition (Asia-Pac) by the layer test facility and stored in open-bag at 25° C. for 3 months. The pigment content was determined according to AOAC method 970.64. Multiple analyses were performed on each sample and the resulting values were averaged.

Results

[0118] Total carotenoids losses during 3 months storage of Kem GLO 10 averaged 44.75%, compared with lower losses of 22.25% observed in the feed meal treated with nanodispersed Kem GL 10. As shown in Table 3, Kem GLO 10 lost one half of the initial carotenoids during 3-month storage period while the carotenoids stability in nanodispersed Kem GLO 10 (made with the microemulsion technology) was much improved, losing only one third of the initial carotenoids at similar dosage. Also, the relative stability of the carotenoids also decreased progressively when a greater amount of carotenoids was added to the feed (at 1.0 kg/ton). There was a further 10% and 20% drop in the carotenoids retention for Kem GLO 10 and nanodispersed Kem GLO 10, respectively compared to the lower 0.5 kg/ton addition. We also observed that the degree of carotenoids lost from feed treated with nanodispersed Kem GLO 10 is more gradual as compared to that of Kem GLO 10 suggesting it may be due to the different method of preparation and better protection efficacy (Table 4).

TABLE-US-00003 TABLE 3 Stability of the carotenoids from Kem GLO 10 and nanodispersed Kem GLO 10 added to layer feed Kem GLO 10 Nanodispersed Kem GLO 10 Initial Retention Initial Retention carotenoids after 3 carotenoids after 3 concentration months at concentration months at Dosage (g/ton) 25° C. (%) (g/ton) 25° C. (%) 0.5 kg/ton 7.02 55.25 6.24 77.75 1.0 kg/ton 14.04 43.52 12.47 60.10

TABLE-US-00004 TABLE 4 Xanthophyll Stability Test in Feed Dosage of Total Xanthophyll Total Xanthophyll Nanodispersed ORO Recovery (g/ton) Recovery (g/ton) GLO 20 in Feed (kg/ton) (Week 0) (Week 2) 0.25 4.95 4.49 0.5 9.70 9.55 0.75 14.45 14.0 1.0 20.48 15.7 Dosage of ORO GLO Total Xanthophyll Total Xanthophyll 20 in Feed (kg/ton) Recovery (Week 0) Recovery (Week 2) 0.25 7.50 5.04 0.5 10.12 10.86 0.75 13.19 8.93 1.0 19.31 11.91

Discussion

[0119] As mentioned earlier, several factors may influence the relative stability of carotenoids when added to a feed. It is known that when carotenoids are in the encapsulated form, they can be well protected from premature degradation that may be induced by light, oxygen and/or heat. The nanodispersed Kem GLO 10 had improved carotenoid retention as compared to the Kem GLO 10, perhaps because the carotenoid when solubilized and contained within the microemulsion system is better protected due to the molecular architecture of the pigment within the microemulsion matrix. 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, we also foresee that 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.

Example 4

[0120] Nanodispersions of various hydrophilic and lipophilic substances were made using the ingredients set out in Tables 5-9.

TABLE-US-00005 TABLE 5 Antimicrobial agent: Monolaurin (lipophilic) Ingredients (wt %) Ex.-1 Ex.-2 Tween 80 35.0 25.0 Limonene/Ethanol (1:2) 35.0 25.0 Glycerol/Water (1:3) 30.0 50.0 Monolaurin (ppm) 500-1000 500-1000 Ingredients (wt %) Ex.-3 Ethoxylated castor oil (EL35) 32.5 Propionic acid 32.5 Water 35.0 Monolaurin (ppm) 500-1000

TABLE-US-00006 TABLE 6 Vitamin C and antioxidant: Ascorbic acid (hydrophilic) Ingredients (wt %) Ex.-4 Ex.-5 Tween 80 45.0 35.0 Limonene/Ethanol (1:2) 45.0 35.0 Glycerol/Water (1:3) 10.0 30.0 Ascorbic acid (ppm) 200-500 500-1000

TABLE-US-00007 TABLE 7 Amino acids: L-lysine, L-arginine hydrochloride (hydrophilic) Ingredients (wt %) Ex.-6 Tween 80 32.5 Limonene/Ethanol (1:2) 32.5 Glycerol/Water (1:3) 35.0 L-Lysine Hydrochloride (wt %) 1-5 Ingredients (wt %) Ex.-7 Tween 80 32.5 Limonene/Ethanol (1:2) 32.5 Glycerol/Water (1:3) 35.0 L-Arginine Hydrochloride (wt %) 1-5

TABLE-US-00008 TABLE 8 Bile salt (amphiphilic) Ingredients (wt %) Ex.-8 Tween 80 32.5 Limonene/Ethanol (1:2) 32.5 Glycerol/Water (1:3) 35.0 Bile salt (wt %) 0.1-1

TABLE-US-00009 TABLE 9 Enzyme: Amylase (lipophilic) Ingredients (wt %) Ex.-9 Tween 80 32.5 Limonene/Ethanol (1:2) 32.5 Glycerol/Water (1:3) 35.0 Amylase liquid (wt %) 0.1-1.0

[0121] A particular application where both lipophilic and hydrophilic substances may be combined in a single microemulsion of the present invention is in the preparation of dyes for biological tissues such as is described in U.S. patent application Ser. No. 13/433,526, filed Mar. 29, 2012, and incorporated herein in its entirety by this reference. The subject application describes dyes that contain lutein or zeaxanthin, both of which are lipophilic, with traditional dyes, such as trypan blue, which often are hydrophilic.

Example 5

[0122] The microemulsions of the present invention can be used to form powders that have enhanced flowability. This is shown by the effect on the angle of repose of a pile of the material as set out in Table 10.

TABLE-US-00010 TABLE 10 Angle of Repose Comparison Nano Kem Nano Oro Kem 10 GLO 10 Oro GLO GLO GLO Dry Dry 20 Dry 20 Dry Angle of repose 19.3 Not flowable 19.69 25.27

[0123] The angle of repose is typically below 40 for a flowable product and the smaller the angle of repose the more flowable the product. The data show that the microemulsions of the present invention form powders that have enhanced flowability.

Example 6

[0124] The objective of this example was to use the novel microemulsion of the present invention to solubilize and encapsulate chromium (Cr-propionate base) using the microemulsion nanotechnology of the present invention. The bioavailability study showed no change in particle size and improved stability retained over time as shown in FIG. 13, where the particles size of Cr-propionate microemulsion ˜130 nm.

Example 7

Materials and Methods

[0125] Material.

[0126] Two compositions of FORTIUM containing the same level of rosemary extract were prepared, the microemulsified R30 and non-microemulsified R30. The R30 liquid contained 45% Rosan SF35 in sunflower oil while the microemulsified R30 was made following the same procedure except the sunflower oil was replaced with microemulsion base as shown in Table 11 below.

[0127] Treatments and Dosages.

[0128] The following treatments were prepared: (1) soybean oil (SBO) with no antioxidant (negative control), (2) soybean oil (SBO) with 250 ppm microemulsified R30 and (3) soybean oil (SBO) with 300 ppm non-microemulsified R30. The different dosages used in the study were used to prove that at a 20% reduced dosage of the microemulsified R30, an equivalent or better performance was achieved when compared to the current non-microemulsified R30. A domestic deep-fat fryer with a 2-L-volume vessel was used for the deep-fat frying. Temperature was monitored with digital thermometers. For each deep-frying cycle, after heating the oil to and maintained constantly at 180° C., chicken nuggets (100 g per batch) were added and deep fried for 5 mins for a frying cycle. After every 5 frying cycles, oil top up of 100 ml from the respective treatments were added. Samples of frying oils (50 g) after every ten frying cycles were collected (0, 10, 20 and 30) and cooled to room temperature and kept at 4° C. prior to further analyses.

[0129] Oxidative Stability Measurement.

[0130] A preliminary assessment of the antioxidant activity of microemulsified and non-microemuslified R30 was measured using the Oxidative Stability Instrument (OSI).

[0131] Analysis of Total Polar Compounds (TPC).

[0132] The temperature of sample oils was maintained at 175-180° C. and TPC of samples were measured using a Testo 270 cooking oil tester according to the manufacturer operation guide.

[0133] Measurement of Free Fatty Acids (FFA).

[0134] Free fatty acids, as oleic acid percentages in oil samples were measured according to well-known methods.

[0135] Color Measurement.

[0136] The color of the oil was measured using the Hunter Lab Colorimeter10. L*—degree of lightness or darkness of sample extended from 0 (black) to 100 (white), a*—degree of redness (+) to greenness (−) and b*—degree of yellowness (+) to blueness (−)

[0137] Analysis of Peroxide Value.

[0138] The peroxide value (PV) of all samples was measured according to industry practice and well-known methods.

[0139] Measurement of p-Anisidine Value.

[0140] p-Anisidine value was determined for each of the samples.

[0141] Characterization of Microemulsified and Non-Microemulsified R30 Liquid.

[0142] The composition of lipid excipients that constitutes the ternary phase of optimized microemulsified R30 and the specifications comparison between the two formulations are shown in Table 11.

TABLE-US-00011 TABLE 11 Comparison of microemulsified and non-microemulsified R30 Non-microemulsified Microemulsified R30 R30 Sample Rosan SF 35 45.0 45.0 Sunflower oil 55.0 — Microemulsion base — 55.0 Specifications Color Dark brown Dark brown Odour Herbal Citrus Specific gravity 0.930-0.960 0.970-0.990 Protection Factor 1.00-1.30 1.00-1.30 Microemulsion Composition (wt %) Tween 80 32.5 Limonene/Ethanol (1:3) 32.5 Glycerol/Water (1:3) 35.0

[0143] The antioxidant activity comparison, determined using the OSI instrument, is shown in Table 12. The results indicate that the samples of sunflower oil containing 250 ppm of microemulsified R30 showed the least oxidation and from the induction period it showed that improved resistance to oxidative rancidity compared to non-microemulsified R30 and control oil.

TABLE-US-00012 TABLE 12 Antioxidant activity using the oxidative stability index (OSI) method at 100° C. Induction Protection Sample period.sup.a (h) factor.sup.b Soybean oil (SBO) with no antioxidant 12.35 ± 0.00 — SBO + 300 ppm non-microemulsified R30 13.18 ± 0.11 1.07 SBO + 250 ppm microemulsified R30 13.28 ± 0.17 1.08 .sup.aValues are mean ± standard deviation, .sup.bProtection factor = (induction period for stabilized oil)/(induction period for unstabilized oil)

[0144] Effect of Total Polar Compounds (TPCs) During Deep Frying.

[0145] Determination of polar compounds in used oils and fats is a well-accepted method due to its accuracy and reproducibility. It provides the most reliable measure of the extent of deterioration in frying oils and fats in most situations. TPCs were found to increase with the frying time for all the oils. The rate of increase was gradual for sample containing microemulsified R30 as compared to non-microemulsified R30 added samples at the end of the frying period. These results show that the addition of microemulsified R30 effectively reduced the formation of polar compounds as compared to non-microemulsified R30 control oil sample. The microemulsified R30 at 250 ppm had least value of TPC (13.5%) after 30th batches of frying as compared to non-microemulsified R30 (14.0%). The variation of TPC with frying cycle is presented in the (FIG. 14). Although the TPC value for the control oil at the end of the 30th frying cycle is also at 13.5% but it already showed the extensive degradation within the 10th frying cycle.

[0146] Changes in the Free Fatty Acid (FFA) Content.

[0147] The amount of FFA in fats and oils can be used to indicate the extent of its deterioration due to hydrolysis of triacylglycerol (TAG) and/or cleavage and oxidation of fatty acid double bonds. Free fatty acid (FFA) is an important fat quality indicator during each stage of fats and oils processing and is generally accepted as a regular quality parameter in frying oil industry. The changes of FFA with frying cycle is presented in the (FIG. 15). As shown in FIG. 15, there was a linear increase in the values of FFA with different frying cycles. Based on the information obtained from these frying experiments, at the end of the frying cycle, the total change in FFA values from the initial to end of the frying cycle, the lowest were found to be in oil with microemulsified R30, followed by control and highest value was found in non-microemulsified R30 treated oil sample. This data does indicate that the microemulsified R30 could be used in place of non-microemulsified R30 for better controlling the FFA of oil.

[0148] Color Changes in Frying Oil.

[0149] Color is widely used in the industry as an important parameter to understand an index of oil quality during deep fat frying. The oil rapidly changes from a light yellow to brown color during frying. This is the combined result of oxidation, polymerization and other chemical changes which also result in an increase in viscosity of the frying oil. The comparative analysis of color of frying oil at different frying batch is presented in the FIGS. 16A-C with L* as the measure of the oil lightness/darkness, a* as the measure of redness/greenness and the b* as the measure of the yellowness/blueness. As seen from the (FIGS. 16A-C), with increasing frying batches, the color of the oil degrades to brown as compared to initial batch oil. However, in case of microemulsified R30, there is least degradation of color. The microemuslified R30 treated frying oil tends to have lower value (a*, b*) except for a higher, improved L* compared to the non-microemulsified R30 treated and control frying oil.

[0150] Change in Peroxide Value (PV) and p-Anisidine (AnV) in Frying Oil.

[0151] Thermo-oxidation of frying oils involves both primary and secondary oxidation. But, secondary oxidation continues because of the least stability of peroxides at frying temperature. Oxidation further proceeds to the formation of minor compounds, including aldehydes, ketones, and dienes. The PV and AnV values of oils treated with different antioxidants have shown in (FIGS. 17 and 18) respectively. PV and AnV values showed extensive degradation of the control sample throughout the whole frying cycles. But oils added with both the microemulsified and non-microemulsified R30 showed resistance towards primary and secondary oxidation with the microemulsified R30 showing a potent antioxidant capacity towards the primary and secondary oxidation even at 20% lower dosage.

[0152] Results.

[0153] This study was performed to test the feasibility and effect of including rosemary-based natural plant extract antioxidants of different forms: microemulsified against non-microemuslified in controlling the soybean oil deterioration during the frying of chicken nuggets. In terms of the TPCs, FFAs, PV and AnV, the study showed that the oil treated with microemulsified R30 had lower values compared to the control oil and the oil with the addition of non-microemulsified R30. The microemulsified R30 also showed slightly better antioxidative effects at a much lower concentration of active ingredient compared to the non-microemulsified R30. This study showed that the efficacy of rosemary extract was enhanced when incorporated into the microemulsion system as an active ingredient.

Example 8

[0154] A frying trial was set up using soybean oil for frying chicken nuggets using microemulsified R30 (R30ME) against non-microemulsified liquid R30 (R30) treated at 500 ppm, 1000 ppm and 1500 ppm to compare the frying performance in terms of number of extra frying cycles and % improvement. This trial demonstrated that the microemulsion nanotechnology enhanced the efficacy of the rosemary extract.

Materials and Methods

[0155] Materials.

[0156] Two samples were prepared, R30ME and R30, containing the same level of rosemary extract. The R30 liquid contained 45% Rosan SF35 in sunflower oil while microemulsified R30 was made following the same procedure except the sunflower oil was replaced with microemulsion liquid.

[0157] Treatments and Dosages.

[0158] The different treatments used for the frying experiment using soybean oil were prepared as shown in Table 13. A domestic deep-fat fryer with a 2-L-volume vessel was used for the deep-fat frying. Temperature was monitored with digital thermometers. For each deep-frying cycle, after heating the oil to and maintained constantly at 180° C., chicken nuggets (100 g per batch) was added and deep fried for 5 mins for a frying cycle. After every 5 frying cycles, oil top up of 100 ml from the respective treatments were added. Samples of frying oils (50 g) after every ten frying cycles were collected (0, 10, 20, 30, 40 and 50) and cooled to room temperature before storing at 4° C. prior to further analyses. Frying trials were conducted in duplicates (n=2).

TABLE-US-00013 TABLE 13 Treatments used in frying and their inclusion rate Treatment Inclusion rate (ppm) NC Soybean oil (without antioxidant) T1 Soybean oil with 500 ppm R30 T2 Soybean oil with 1000 ppm R30 T3 Soybean oil with 1500 ppm R30 T4 Soybean oil with 500 ppm R30ME T5 Soybean oil with 1000 ppm R30ME T6 Soybean oil with 1500 ppm R30ME

[0159] Physico-Chemical Analysis of Oil.

[0160] Peroxide value (PV), p-anisidine value (AV), free fatty acid (FFA), and induction period were calculated. The total polar compounds (TPC) were determined using TESTO 270 cooking oil tester.

[0161] Statistical Analysis.

[0162] Analysis of variance (ANOVA) and multiple range tests were conducted using Statgraphics Plus version 5.0 software package.

[0163] Comparison of Frying Performance.

[0164] Frying performance of soybean oil was measured in terms of number of extra frying cycles and % improvement offered by microemulsified R30 liquid against untreated control and regular R30 liquid for all quality parameters. For each quality parameter, number of extra frying cycles provided by microemulsified R30 liquid with respect to untreated control and regular R30 liquid was calculated by subtracting frying cycle of untreated control and R30 liquid from microemulsified R30 liquid. Similarly, the % improvement in frying performance offered by microemulsified R30 liquid with respect to untreated control and regular R30 liquid was measured by finding the percentage of number of extra frying cycles with respect to the frying cycle of untreated control and regular R30 liquid.

Results

[0165] Induction Period.

[0166] Induction period (OSI) is a direct evidence for changes in oxidative resistance. Induction period of frying oils were measured at 100° C. There was a decrease in induction period observed in all the treatments due to deterioration of oil with increase in number of frying cycles (FIG. 19). Table 2 shows that for the regular R30 treated oil, at 0th and 10th frying cycle, there was a significant (p<0.05) difference observed between the treatments but for the microemulsified R30 treated oil, there was significant difference observed between the treatments at 0th-40th. Microemulsified R30 treated oil showed longer induction periods compared to untreated control and regular R30 treated oil providing extra oxidative stability. From FIG. 19, it can be estimated that frying performance of untreated oil in terms of OSI at 10th frying cycle matched the same of regular R30 and microemulsified R30 treated oil: 500 ppm at 10th and 20th frying cycle, 1000 ppm at 15th and 40th frying cycle and 1500 ppm at 30th and 50th frying cycle respectively.

TABLE-US-00014 TABLE 14 Changes in induction period (hours at 100° C.) of soybean oil during frying Frying Frying systems Characteristic cycles NC T1 T2 T3 Induction 0 13.00 ± 0.14.sup.a  16.30 ± 0.07.sup.b  18.70 ± 0.14.sup.c  21.45 ± 0.21.sup.c  period (h)at 10 8.65 ± 0.14.sup.a .sup. 9.23 ± 0.11.sup.ab 9.50 ± 0.28.sup.b 10.45 ± 0.57.sup.b  100° C. 20 8.60 ± 0.28.sup.a 8.75 ± 0.42.sup.a  9.03 ± 0.39.sup.ab .sup. 9.23 ± 0.11.sup.ab 30 8.23 ± 0.18.sup.a .sup. 8.50 ± 0.07.sup.ab  8.88 ± 0.04.sup.bc .sup. 9.13 ± 0.04.sup.cd 40 8.00 ± 0.07.sup.a 7.88 ± 0.18.sup.a 8.60 ± 0.07.sup.b 9.00 ± 0.07.sup.c 50 7.75 ± 0.07.sup.a 8.23 ± 0.11.sup.b 8.38 ± 0.04.sup.b 8.88 ± 0.11.sup.c Frying Frying systems Characteristic cycles NC T4 T5 T6 Induction 0 13.00 ± 0.14.sup.a  16.68 ± 0.11.sup.e  19.60 ± 0.14.sup.f   22.30 ± 0.21.sup.g  period (h)at 10 8.65 ± 0.14.sup.a 9.38 ± 0.04.sup.c 10.23 ± 0.04.sup.c  10.68 ± 0.46.sup.c  100° C. 20 8.60 ± 0.28.sup.a  9.23 ± 0.18.sup.ab .sup. 9.53 ± 0.32.sup.bc 9.93 ± 0.11.sup.c 30 8.23 ± 0.18.sup.a  8.78 ± 0.11.sup.bc 9.33 ± 0.39.sup.d 9.75 ± 0.07.sup.e 40 8.00 ± 0.07.sup.a 8.35 ± 0.07.sup.b 9.20 ± 0.14.sup.c 9.58 ± 0.11.sup.d 50 7.75 ± 0.07.sup.a 8.28 ± 0.04.sup.b 8.98 ± 0.32.sup.c 9.15 ± 0.00.sup.c .sup.a-gmeans within a row (between treatments) with different letters are significantly different (p < 0.05)

[0167] Total Polar Compound.

[0168] Total polar content is one of the key quality parameter to judge the quality of cooking oil or frying oil. The polar compounds are results of oxidation of fat or oil during deep fat frying. As oxidation progresses, polarity of byproducts of oxidation increases and it results in fat deterioration. The maximum level of polar content should not exceed 25 g/100 g oil (i.e. 25%). Table 15 showed that TPC increases with frying time in all the treatments but none of the treatments reached the 25% limit. It is estimated from FIG. 20 that frying performance of untreated oil in terms of TPC at 20th frying cycle matched the regular R30 and microemulsified R30 treated oil: 500 ppm at 19th and 23th frying cycle, 1000 ppm at 20th and 25th frying cycle and 1500 ppm at 26th and 30th frying cycle respectively.

TABLE-US-00015 TABLE 15 Changes in total polar compound of soybean oil during frying Frying Frying systems Characteristic cycles NC T1 T2 T3 Total polar 0 10.50 ± 0.00.sup.b 10.50 ± 0.00.sup.b 10.00 ± 0.00.sup.b 10.50 ± 0.35.sup.a compound 10 11.75 ± 0.4.sup.ab  11.50 ± 0.00.sup.a 12.75 ± 0.00.sup.c 11.50 ± 0.00.sup.c 20 13.50 ± 0.00.sup.c 13.75 ± 0.40.sup.c .sup. 13.50 ± 0.00.sup.bc .sup. 12.75 ± 0.35.sup.bc 30 14.50 ± 0.00.sup.c 14.50 ± 0.00.sup.c 15.25 ± 0.40.sup.c 14.00 ± 0.00.sup.d 40 15.00 ± 0.00.sup.c 15.50 ± 0.00.sup.d 15.50 ± 0.00.sup.b 15.00 ± 0.00.sup.d 50 17.50 ± 0.00.sup.d 16.25 ± 0.40.sup.c 16.50 ± 0.40.sup.a 16.25 ± 0.35.sup.c Frying Frying systems Characteristic cycles NC T4 T5 T6 Total polar 0 10.50 ± 0.00.sup.b 10.50 ± 0.00.sup.b 10.50 ± 0.00.sup.b  9.75 ± 0.35.sup.a compound 10 11.75 ± 0.4.sup.ab   12.50 ± 0.70.sup.bc 12.25 ± 0.40.sup.a 11.25 ± 0.35.sup.a 20 13.50 ± 0.00.sup.c 13.00 ± 0.40.sup.b .sup. 13.00 ± 0.40.sup.ab 12.50 ± 0.00.sup.a 30 14.50 ± 0.00.sup.c 14.75 ± 0.00.sup.b 14.00 ± 0.40.sup.b 13.50 ± 0.00.sup.a 40 15.00 ± 0.00.sup.c 14.50 ± 0.70.sup.d 15.50 ± 0.70.sup.c 14.00 ± 0.00.sup.a 50 17.50 ± 0.00.sup.d  15.25 ± 0.70.sup.bc 16.00 ± 0.70.sup.c .sup. 15.50 ± 0.00.sup.ab .sup.a-dmeans within a row (between treatments) with different letters are significantly different (p < 0.05)

[0169] Peroxide Value (PV), P-Anisidine Value (AV) and TOTOX Value.

[0170] PV is a measure of the amount of peroxides formed in the fats and oils throughout the oxidation process. However, peroxides in oxidized oils are unstable intermediates, which decompose into various carbonyls and other secondary oxidation products, principally 2-alkenals and 2, 4-dienals. Typically, when used on oils during frying, PV can be very misleading as peroxides are destroyed under frying conditions and the AN is a more meaningful test than PV for oils during frying because it measures aldehydes which are less easily destroyed under these conditions. The PV and AN results obtained in this trial are shown in FIGS. 21 and 22, respectively. It is evident that peroxide formation is erratic under these conditions and has a large experimental error and the concentration of secondary products of oxidation was significantly (p<0.05) increasing with frying time. As such, it would be more appropriate to compare the quality of the oxidized oil using the TOTOX value which is defined as 2×PV+AV. Microemulsified R30 and regular R30 liquid at 500, 1000 and 1500 ppm showed lower TOTOX values as compared to untreated oil throughout the frying process (Table 16) which indicates the improvement in the frying performance of antioxidant treated oil. From FIG. 22, it is estimated that on comparison with untreated oil at 30th frying cycle which has p-anisidine value of ˜67 whereas regular R30 and microemulsified R30 treated oil: 500 ppm withstand until 36th and 39th frying cycle and both 1000 ppm and 1500 ppm withstand until 49th and 50th frying cycle respectively for the same level of p-anisidine values respectively.

TABLE-US-00016 TABLE 16 Changes in TOTOX value of soybean oil during frying Frying Frying systems Characteristic cycles NC T1 T2 T3 TOTOX 0 7.02 7.71 8.06 9.35 value 10 44.27 43.29 48.15 46.00 20 68.33 62.48 60.33 57.78 30 78.17 74.00 70.65 65.69 40 88.18 83.67 77.00 71.98 50 93.75 82.74 80.09 80.08 Frying Frying systems Characteristic cycles NC T4 T5 T6 TOTOX 0 7.02 9.07 9.78 9.78 value 10 44.27 44.43 41.85 41.04 20 68.33 53.79 55.14 56.92 30 78.17 70.42 68.73 65.19 40 88.18 78.76 77.43 72.96 50 93.75 80.72 77.82 76.72

[0171] Free Fatty Acid.

[0172] Most of the lipids undergo hydrolysis liberating free fatty acids resulting in hydrolytic rancidity. Table 17 and FIG. 23 show that the FFA content for all treatments significantly (p<0.05) increased throughout the frying cycles. Also, there was no significant (p<0.05) difference between regular R30 in comparison to the microemulsified R30 liquid. Generally, the increase in FFA content could be caused by an increase in rate of hydrolysis when moisture in the substrate is introduced into frying system during frying. It showed that either R30 or microemulsified R30 liquid has no significant role in controlling the hydrolytic rancidity to prevent formation of free fatty acids. Table 18 shows comparison of frying performance in terms of number of extra frying cycles and % improvement by microemulsified R30 against untreated control and regular R30 in soybean oil.

TABLE-US-00017 TABLE 17 Changes in % free fatty acid of soybean oil during frying Frying Frying systems Characteristic cycles NC T1 T2 T3 Free fatty 0 0.04 ± 0.001.sup.ab .sup. 0.05 ± 0.0012.sup.abc  0.05 ± 0.0019.sup.bc  0.04 ± 0.0005.sup.ab acid (%) 10 0.26 ± 0.006.sup.e  0.22 ± 0.0081.sup.c 0.24 ± 0.0007.sup.d 0.17 ± 0.0011.sup.b 20 0.35 ± 0.001.sup.cd 0.35 ± 0.0003.sup.d  0.35 ± 0.0001.sup.cd 0.26 ± 0.0217.sup.b 30 0.42 ± 0.002.sup.c  .sup. 0.42 ± 0.0014.sup.cd 0.42 ± 0.0055.sup.c 0.32 ± 0.0006.sup.b 40 0.58 ± 0.0003.sup.f 0.53 ± 0.0001.sup.e 0.51 ± 0.001.sup.c  0.38 ± 0.0013.sup.b 50 .sup. 0.62 ± 0.0002.sup.c 0.58 ± 0.001.sup.b  0.58 ± 0.0020.sup.b 0.45 ± 0.0064.sup.a Frying Frying systems Characteristic cycles NC T4 T5 T6 Free fatty 0 0.04 ± 0.001.sup.ab .sup. 0.04 ± 0.0006.sup.abc 0.05 ± 0.0027.sup.c 0.04 ± 0.0022.sup.a acid (%) 10 0.26 ± 0.006.sup.e  0.21 ± 0.0008.sup.c 0.22 ± 0.0001.sup.c 0.16 ± 0.0000.sup.a 20 0.35 ± 0.001.sup.cd  0.33 ± 0.0.000.sup.c 0.33 ± 0.0010.sup.c 0.24 ± 0.0001.sup.a 30 0.42 ± 0.002.sup.c  0.41 ± 0.0013.sup.c 0.41 ± 0.0009.sup.c 0.31 ± 0.0000.sup.a 40 0.58 ± 0.0003.sup.f 0.53 ± 0.0001.sup.e 0.51 ± 0.0004.sup.d 0.38 ± 0.0016.sup.a 50 .sup. 0.62 ± 0.0002.sup.c 0.58 ± 0.0001.sup.b 0.58 ± 0.0001.sup.b 0.45 ± 0.0002.sup.a .sup.a-fmeans within a row (between treatments) with different letters are significantly different (p < 0.05)

TABLE-US-00018 TABLE 18 Frying performance of microemulsified R30 liquid vs. untreated control and regular R30 liquid in soybean oil Treatment 500 ppm Vs. Untreated Control Vs. R30 liquid (%) No. of % No. of % extra frying Improve- extra frying Improve- Quality Parameters cycles ment cycles ment Induction period 10 50 10 50 Total polar 3 15 4 20 compound (%) TOTOX value 9 45 3 15 Treatment 1000 ppm Vs. Untreated Control Vs. R30 liquid (%) No. of % No. of % extra frying Improve- extra frying Improve- Quality Parameters cycles ment cycles ment Induction period 30 150 25 125 Total polar 5 25 5 25 compound (%) TOTOX value 20 100 1 5 Treatment 1500 ppm Vs. Untreated Control Vs. R30 liquid (%) No. of % No. of % extra frying Improve- extra frying Improve- Quality Parameters cycles ment cycles ment Induction period 40 200 20 100 Total polar 10 50 4 20 compound (%) TOTOX value 20 100 1 5

[0173] Discussion.

[0174] The results of this study shows that soybean oil treated with microemulsified R30 liquid have better frying performance and oxidative stability over the soybean oil treated with similar dosage of regular R30 liquid throughout the frying process which can be attributed to the microemulsion technology.

[0175] The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

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