DESC:Field of the Invention
The present invention relates to encapsulation technologies of carotenoids, More particularly the present invention relates to specific encapsulating components for rendering Composition compatibility with respect to pH stability, non-interference of such specific carotenoid, wherein the said carotenoids can be a carotenoid or a xanthophyll, such as lutein, zeaxanthin, lycopene, beta-carotene etc, or their corresponding esters thereof.
Background of the Invention
Encapsulation refers to the process of protecting drugs and medical substances from degradation and natural damage, prevent and conserve drug potency by means of providing a protective layer, such as an organic coating or shellanted interactions by enclosing them in, to encase a drug with an outer coating. Encapsulation is performed to suit multiple requirements and/or to attain a desirable characteristic of a drug for example to enhance absorption, deliver drugs to specific target cells or tissues effects, extending and improving shelf life, reduce interactions between drugs when combined together as a single unit, or even reduction of drug side effects sometimes. Commonly used encapsulating agents are gelatin, starch, cellulose, chitosan etc. In the recent times, encapsulation has become a widely used platform for various drug delivery models. Pharmacologically active substances are enclosed in a carrier systems such as liposomes, polymeric nanoparticles, micelles, dendrimers, and solid lipid nanoparticles. Each system offers unique advantages in terms of drug stability, release kinetics, and targeting capabilities. Encapsulation helps drugs from degradation by enzymes or harsh conditions till the delivery at the target site within the internal organs, after a medication or a drug is administered. This allows for targeted delivery of the medicines thereby enabling specific objectives in medical treatment. It also makes it possible for controlled drug release over an extended period leading to sustained therapeutic effects and tailored pharmacokinetics. The inner core of an encapsulated drug system is where the active pharmaceutical ingredient (API) is located which is most of the times is embedded in a matrix of host of inactive compounds which would hold the drug stable and firm till the point of its absorption. This core is often surrounded by protective layers to shield the drug molecules from degradation and ensure their stability until reaching the target site. The outer layer of an encapsulated drug system surrounds the inner core and acts as a protective barrier. This layer can be designed to control the release of the drug, target specific tissues, or enhance stability. The interaction between the inner core and outer layer is crucial for the overall performance of the encapsulated drug system. This interaction determines the release profile, stability, and targeting capabilities of the Composition. A synergistic interaction is expected in an optimal encapsulation system, wherein both the inner core and outer core acts in harmony with each other to maximise the functionality of the drug. When encapsulating a particular drug, the choice of encapsulating agents is a critical factor that directly influences the effectiveness of drug delivery, stability, and targeted release.
Encapsulating agents need to be compatible with the drug being encapsulated to prevent chemical interactions that could alter the drug's properties or effectiveness. Specific encapsulating agents are selected based on their compatibility with the drug molecule to ensure successful encapsulation. Encapsulating agents play a crucial role in maintaining the stability of the drug during encapsulation, storage, and delivery. The encapsulating agent should provide a protective environment for the drug, shielding it from degradation, oxidation, or premature release. The solubility of the drug and the encapsulating agent can impact the encapsulation process and the release kinetics of the drug. Specific encapsulating agents are chosen based on their solubility properties to ensure efficient encapsulation and controlled release of the drug. Encapsulating agents must be biocompatible to minimize the risk of adverse reactions or toxicity in the body. Specific encapsulating agents with proven biocompatibility are preferred to ensure the safety of the encapsulated drug. Some encapsulating agents can be functionalized to enable targeted delivery of the drug to specific tissues, cells, or organs. Specific encapsulating agents with targeting ligands or surface modifications can enhance the specificity of drug delivery. The choice of encapsulating agent influences the release profile of the drug, determining whether the release is immediate, sustained, or triggered. Specific encapsulating agents with controlled release properties are selected to achieve the desired release kinetics. Encapsulating agents also contribute to the mechanical properties of the drug delivery system, such as stability, flexibility, and resistance to external stresses. Specific encapsulating agents are chosen based on the desired physical characteristics of the Composition. Different encapsulating agents offer unique properties that can be tailored to optimize the Composition for specific drug delivery requirements. The selection of specific encapsulating agents allows for customization and fine-tuning of the encapsulation process as well.
The unstable nature of carotenoids is a significant challenge that can impact their functionality, bioavailability, and shelf life. Some key factors contributing to the instability of carotenoids are light sensitivity, oxidative degradation, temperature instability, oxidative degradation, temperature instability, poor insolubility, or pH sensitivity. Carotenoids are sensitive to light, particularly blue and ultraviolet wavelengths, which can lead to degradation and loss of their antioxidant properties. This can occur during processing, storage, and even in biological systems. They are susceptible to oxidative damage when exposed to air, leading to the formation of reactive oxygen species and accelerated degradation. This can result in discoloration and a decrease in nutritional value. High temperatures during processing, cooking, or storage can accelerate the degradation of carotenoids, reducing their effectiveness. On the other hand, low temperatures can lead to crystallization, affecting their dispersibility and bioavailability. Many carotenoids have low water solubility, which can limit their absorption and bioavailability in the body, especially in aqueous Compositions like beverages and food products. The pH of the environment can impact the stability of carotenoids. Extreme pH conditions, such as highly acidic or alkaline environments, can lead to degradation and loss of bioactivity. Enzymes present in foods, as well as enzymes in the gastrointestinal tract, can contribute to the degradation of carotenoids, affecting their absorption and efficacy. Carotenoids can interact with other components in food and supplement Compositions, leading to degradation or changes in their chemical structure. Therefore, to address the instability of carotenoids, encapsulation techniques, as discussed above, have been employed to protect them from these detrimental factors and enhance their stability, solubility, and bioavailability. Encapsulation helps shield carotenoids from light, oxygen, heat, and other environmental factors, thereby preserving their beneficial properties and ensuring their efficacy in various applications.
A patent disclosure WO2004/066750 mentions novel composition comprising carotenoids for delivering carotenoids (e.g. a- and ß-carotene, lycopene) and/or other physiologically active ingredients to the colon of humans after ingestion and for producing liquid food compositions insusceptible to polyphenol-protein reactions can be obtained by encapsulating said active ingredients with pectin, in particular with low-methoxylated pectin.
Similarly, WO2019213538 discloses a carotenoid composition and uses thereof comprising carotenoids including liposomes that encapsulates carotenoids including ionizable carotenoids such as tenas-crocetin. Carotenoid-loaded liposomes are also mentioned in WO2002064110. Similarly a carotenoid Composition for increased bio-availability is provided wherein a xanthophyll carotenoid diacetate, a transition metal salt, and phospholipids, wherein the composition does not comprise micelles is not an emulsion. The phospholipids may be selected from phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphoinositides, phosphosphingolipids or combinations thereof. The xanthophyll carotenoid diacetate may comprise meso-zeaxanthin diacetate. The composition may further comprise (3R,3R’)-zeaxanthin diacetate, (3R, 3’R,6R)-lutein diacetate, or a combination thereof. The transitional metal salt may be selected from zinc oxide, cupric oxide, cuprous oxide or combinations thereof. A method of supporting eye health in subjects in need thereof using the composition are also provided in this disclosure GB2601216 for an encapsulated carotenoid with increased bioavailability.
A specific disclosure of carotenoid coloring composition for cheese products is provided in WO2023/094186 for coloring foods, beverages, animal feeds, cosmetics or drugs comprising (a) dissolved carotenoid emulsified as an oil-in- water emulsion using a first hydrocolloid and (b) crystalline carotenoid encapsulated in a second hydrocolloid, making it water-dispersible and thereby miscible with the oil-in-water emulsified carotenoid fraction, mixed in a ratio of between 1 :10 to 10:1 and wherein the first hydrocolloid is native casein. Another invention WO2017/168006 is directed towards the use of granules in beverages, wherein the granules comprise (i) a milled carotenoid selected from the group consisting of lutein and zeaxanthin and any mixture thereof having the following particle size distribution: D [3,2] in the range of from 0.6 to 1.5 µm, and D [v, 0.5] in the range of from 1.1 to 3.5 µm, and (ii) a matrix comprising at least one modified food starch, a glucose syrup and sucrose, (iii) a water-soluble antioxidant, wherein the granules have the following particle size distribution: D [3,2] in the range of from 200 to 300 µm, and D [v, 0.5] in the range of from 220 to 320 µm, all D values as measured by laser diffraction according to the Fraunhofer scattering model, whereby the milled carotenoid is encapsulated by the matrix. The present invention is also directed towards the beverages and the granules as such, as well as to a process for the manufacture of the granules. Preferably the carotenoid is lutein, zeaxanthin or any mixture thereof. WO2024086307 discloses formulated food products wherein an extended release preparation including a carrier component including at least one excipient; and a payload component including a protein, a carbohydrate, a fatty acid, a lipid, a nutraceutical, a fiber, a prebiotic, a ketone, a phenolic acid, an amino acid, a peptide, a sugar, a vitamin, a mineral, an element, a salt, an electrolyte, a flavonoid, a polyphenol, an antioxidant, a metabolic intermediate, or a combination thereof, is disclosed wherein, the payload component is released in an animal over an extended period in acidic and neutral environments following ingestion of the preparation by the animal as compared to ingestion of the payload component alone.
Thus we see that a few technical interventions have been undertaken to provide stable encapsulated compositions of carotenoids, thereby multiplying the applications and benefits from such carotenoids. However, encapsulation techniques with carotenoids needs to be economically feasible to ensure affordability of carotenoid compositions as well. Numerous carotenoid end-use Compositions for oral application is available in the market today. Incorporation of encapsulated carotenoids in such oral supplements of carotenoids increases the end-use price of such carotenoid supplements. Nevertheless, if not for oral supplements a cost-effective method of encapsulation is also required for enabling easy admixture of carotenoid based food ingredients those may ensure regular consumption of carotenoid for enhanced health benefits. For example, widely used lutein compositions are available in the market today, which has limited shelf lives and specificity in their end-use Compositions. New and alternative methods of encapsulation is required for the purpose of enabling compatibility of such carotenoids or xanthophylls with other components in the Composition, wherein such other components may be pH sensitive, or selectively soluble, or may demand any specific characteristic. Hence, alternative encapsulation technology for any and all carotenoid, particularly xanthophylls are provided in this invention, which are cost effective, and easily reproducible.
Objectives of the Invention
One objective of the invention is to provide a carotenoid composition, the said composition is formed by encapsulation of the carotenoid with respective components thereof.
Another objective of the invention is to provide a novel process of encapsulation of carotenoids, of the compositions as provided in this present invention.
Further it is also an objective of the invention to increase the stability of the carotenoids of the present invention through the compositions and processes as disclosed herein.
One more objective of the present invention is to provide such encapsulated carotenoid compositions which are pH compatible with other components in end-use Compositions, which may be pH sensitive.
Further it is the objective of the invention to retain the inherent potency of such carotenoids for comparatively enhanced and prolonged durations of time.
Specifically it is also the objective of the invention that the encapsulated carotenoid compositions and process of encapsulation hereof exhibit minimal or negligible or no interference with other end-use Composition components thereby ultimately contributing to the stability of end-use Composition of the carotenoids.
Brief Description of the Figures:
Figure 1: Standard Electron Microscopy view of Composition 1 at a Electron High Tension voltage applied to the electron gun at 15.00 kV, in variable pressure secondary electron mode at gain setting of the detector at 3, enlarged at 3000 times magnification, from a working distance of 9.0 mm.
Figure 2: Standard Electron Microscopy view of Composition 2 at a Electron High Tension voltage applied to the electron gun at 15.00 kV, in variable pressure secondary electron mode at gain setting of the detector at 3, enlarged at 2000 times magnification, from a working distance of 9.0 mm.
Figure 3: Standard Electron Microscopy view of Composition 2 at a Electron High Tension voltage applied to the electron gun at 15.00 kV, in variable pressure secondary electron mode at gain setting of the detector at 3, enlarged at 3000 times magnification, from a working distance of 9.0 mm.
Figure 4: Standard electron microscopy view of Composition 5 set at high voltage of 30.0 kV enabling greater penetration, improved resolution in imaging at high surface sensitivity detector, working distance at 15.33 mm magnified at 2000 times at a field view of 104 micrometers visualized in a single frame.
Figure 5: Standard electron microscopy view of Composition 6 set at high voltage of 30.0 kV enabling greater penetration, improved resolution in imaging with high surface sensitivity detector, working distance at 15.33 mm magnified at 1500 times at a field view of 139 micrometers visualized in a single frame.

Summary of the Invention
The present invention provides stable encapsulated carotenoid composition emulsified in an oil phase and aqueous phase solution comprising:
(a) a carotenoid at a concentration range from 8.5% w/w to 45% w/w , wherein the carotenoid is selected from lutein and its esters thereof, zeaxanthin and its esters thereof, a combination of lutein and zeaxanthin and its esters thereof, any other carotenoid such as lycopene and its esters thereof, beta-carotene and its esters thereof, a xanthopylls and its esters thereof, and a combination of any of the said carotenoids and their esters thereof;
(b) an oil phase solution at a concentration range between 3% w/w to 15% w/w, wherein the said oil phase solution is selected from a marigold full spectrum extract, or a combination of a saturated fatty acid, a non-ionic emulsifier, a vegetable oil, and a non-ionic surfactant;
wherein the said saturated fatty acid of the oil phase is selected from stearic acid, palmitic acid, lauric acid, oleic acid, lauric acid, palmitic acid, and myristic acid or combinations thereof; the said non-ionic emulsifier of the oil phase being glycerol monostearate, the said vegetable oil of the oil phase is selected from coconut oil, sunflower oil, Olive oil, groundnut oil, the said non-ionic surfactant of the oil phase being selected from polysorbate 80, polysorbate 60, polysorbate 40, and polysorbate 20 or combinations thereof;
(c) an aqueous phase solution at a concentration range between 3% w/w to 21% w/w, wherein the said aqueous phase solution is selected from gum acacia or a combination of a semi-synthetic cellulose derivatives or natural gums and a sugar alcohol;
wherein the said semi-synthetic cellulose derivative selected from hydroxyethyl cellulose, hydroxy propyl methyl cellulose and hydroxymethyl cellulose, the said sugar alcohol selected from glycerol; sorbitol, mannitol, xylitol, or combinations thereof;
(d) and a carotenoid protecting antioxidant preparation at a concentration range between 2% w/w to 5% w/w, wherein the said antioxidant preparation is selected from combination of a vitamin ester and ascorbic acid or ascorbyl palmitate wherein the said vitamin ester is selected from vitamin E acetate; vitamin E palmitate, or a combination of Vitamin E, Vitamin C, coffee bean extract, or green coffee bean extract including combinations thereof;
(e) optionally including a buffer composition at a concentration between 0.1% w/w to 0.3% w/w the said buffer selected from a combination of citric acid and sodium citrate, a combination disodium hydrogen phosphate and dihydrogen sodium phosphate, or any other buffer that maintains the final pH of the composition between 4.5 to 5.5;
and
(f) a diluent filler maltodextrin at a concentration between 25% w/w to 65% w/w;
wherein the final assay content of the carotenoid in the encapsulated carotenoid composition is at least 5% to 30% purity.
The present invention also provides a method of preparation of the encapsulated carotenoid compositions, the said method comprising the steps:
(a) preparing an oil phase solution an oil phase solution by adding the saturated fatty acid, non-ionic emulsifier, vegetable oil, and the non-ionic surfactant and heating the said solution to around 60oC to 70oC to result into a dispersion, wherein the concentration of the said oil phase solution is at a concentration range of 15% w/w and adding the carotenoid at a concentration of 45% w/w to the dispersion under stirring till a smooth, lump free paste is formed,
wherein the said saturated fatty acid of the oil phase is selected from stearic acid, palmitic acid, lauric acid, oleic acid, lauric acid, palmitic acid, and myristic acid or combinations thereof added at an individual concentration of 4% w/w; the said non-ionic emulsifier of the oil phase being glycerol monostearate added at an individual concentration of 4% w/w, the said vegetable oil of the oil phase is selected from coconut oil, sunflower oil, Olive oil, groundnut oil added at an individual concentration of 5% w/w, the said non-ionic surfactant of the oil phase being selected from polysorbate 80, polysorbate 60, polysorbate 40, and polysorbate 20 or combinations thereof added at an individual concentration of 2% w/w;
(b) simultaneously preparing a lump free aqueous phase solution by adding the semi-synthetic cellulose derivative and the sugar alcohol mixed with 80% water in to vessel, and heated to 60ºC–70ºC, and adding the aqueous phase solution at a concentration of 3 % w/w to 3.5% w/w with the paste of step (a) above, and stirred continuously to get an uniform homogenized emulsion for 30 minutes at 65 ºC,
wherein the said semi-synthetic cellulose derivative selected from hydroxyethyl cellulose, hydroxy propyl methyl cellulose and hydroxymethyl cellulose, the said sugar alcohol selected from glycerol; sorbitol, mannitol, xylitol, or combinations thereof;
(c) adding a mixture of citric acid and sodium citrate to obtain a clear solution of a buffer composition at a concentration between 0.1% w/w to 0.3%. w/w to the homogenized emulsion of step (b) above under constant stirring for at least 10 minutes such that the final pH of the composition is maintained between 4.5 to 5.5 and allowing the homogenized emulsion to cool down till the temperature of the homogenized emulsion reduces to 45o C;
(d) adding an antioxidant preparation at a concentration of 2% w/w to the homogenized emulsion of step (c) above, wherein the said antioxidant preparation is selected from combination of a vitamin ester and ascorbic acid or a combination of a vitamin ester and ascorbyl palmitate wherein the said vitamin ester is selected from vitamin E acetate; or vitamin E palmitate;
(e) adding a diluent filler to the homogenized emulsion at a concentration of 34% w/w to 35% w/w to obtain a final composition which then subjected to spray drying at the specific parameters wherein the inlet temperature is around 140oC to 180oC, outlet temperature is around 85oC to 90oC, and spray rate to be maintained accordingly to achieve the above temperature ranges to obtain the encapsulated carotenoid compositions under this present invention.
Alternatively, the present invention also provides a method of preparation of the encapsulated carotenoid compositions, the said method comprising the steps:
(a) preparing an oil phase solution, wherein the said oil phase solution is a marigold full spectrum extract and heating the said solution to around 60oC to 70oC to result into a dispersion, wherein the concentration of the said oil phase solution is at a concentration range between 3 % w/w to 5 % w/w and adding the carotenoid at a concentration between 8.5% w/w to 45% w/w to the dispersion under stirring till a smooth, lump free paste is formed;
wherein the said marigold full spectrum extract is prepared through hydroalcoholic solvent extraction at around 70oC with ethyl alcohol and water in the ratio ranging from 80 (ethyl alcohol): 20 (water) to 60 (ethyl alcohol): 40 (water) of shed dried marigold petals, followed by drying the extract in Vacuum Tray Drier at 70oC till LOD (loss on drying) is around 5% w/w to obtain a thick mass followed by subjecting the thick mass to milling using a multimill with impact forward direction to produce a desired fineness or particle size distribution of a powder called as full spectrum marigold extract according to the present invention;
(b) simultaneously preparing a lump free aqueous phase solution by adding gum acacia at a concentration range between 16% w/w to 21% w/w with the paste of step (a) above, and stirred continuously to get an uniform homogenized emulsion for 30 minutes heated to temperature between 60 ºC to 70 ºC and allowing the homogenized emulsion to cool down till the temperature of the homogenized emulsion reduces to 45o C;
(c) adding an antioxidant preparation at a concentration of 5% w/w to 6 % w/w to the homogenized emulsion of step (c) above, wherein the said antioxidant preparation is selected from combination of Vitamin E, an aqueous solution of Vitamin C, and coffee bean extract or green coffee bean extract;;
(d) adding a diluent filler to the homogenized emulsion at a concentration of 25% w/w to 44% w/w to obtain a final composition which then subjected to spray drying at the specific parameters wherein the inlet temperature is around 140oC to 180oC, outlet temperature is around 85oC to 90oC, and spray rate to be maintained accordingly to achieve the above temperature ranges to obtain the encapsulated carotenoid compositions under this present invention.
The microencapsulated carotenoid compositions of the present invention are stable for a period of at least 6 months at accelerated conditions at 40°C ? 2°C and relative humidity of 75% ? 5%.
The microencapsulated carotenoid compositions of the present invention are stable for a period of at least 36 months at real-time conditions at 25oC ± 2oC and relative humidity of 60%± 5% RH.
Detailed Description of the Invention
The invention is described in further detail in the below embodiments or working examples, which forms the essence and basis of the present invention, and those which enables a person skilled in the art to appreciate and acknowledge obvious variations of the embodiments thereof which are construed to be included within the limitations of the present invention as claimed regardless of whether specifically represented in the embodiments or not.
Example 1: Method of preparation of encapsulated carotenoid compositions
The present invention provides a method of encapsulation of carotenoids involving the above-mentioned ingredients as described below:
Preparation of Marigold Full Spectrum Extract: The marigold flowers are harvested, the petals are separated and shade-dried under controlled temperature between 30oC to 35oC. The petals are shade dried. Once dried, the petals are subjected to extraction using hydroalcoholic solvent system consisting of ethyl alcohol and water in the ratio ranging from 80 (ethyl alcohol): 20 (water) to 60 (ethyl alcohol): 40 (water). Three volumes of this extraction media with respect to the volume of the dried petals is used for the extraction at around 70oC. The extracted liquid is thereafter concentrated at 70oC to achieve a thick viscous liquid. This liquid is dried in Vacuum Tray Drier at 70oC till LOD is around 5% w/w. The obtained mass is milled using a multimill with impact forward direction wherein the impact forward direction generates a high level of energy and speed, resulting in a more efficient and effective size reduction. The multimill's design and configuration enable efficient comminution, or the breaking down of solid particles, to produce a desired fineness or particle size distribution of a powder called as full spectrum marigold extract according to the present invention. This full spectrum marigold extract is rich in palmitic, myristic esters of Marigold flower. These esters have properties similar to that of emulsifiers such as glyceryl monostearate and help in emulsifying the free Lutein and Zeaxanthin present in the microencapsulation process.
Preparation of an oil phase solution along with addition of carotenoids or esters thereof with the said oil phase solution: The above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid or esters selected from lutein and its esters thereof, zeaxanthin and its esters thereof, combination of lutein and zeaxanthin and its esters thereof, or other carotenoids or extracts of carotenoid esters such as lycopene, beta-carotene or any other xanthopylls, etc. (non-exhaustive) at desired purity and concentration levels as provided in the Compositions below, preferably lutein of purity at least 70% is added to the above-mentioned marigold full spectrum extract under stirring till a smooth, lump free paste is formed.
In this present invention, the carotenoids may be added at a desired purity range according to the need and demand from the perspective of formulating end-use Composition comprising the said encapsulated carotenoid compositions of the present invention. The carotenoids are added following isolation and extraction thereof from respective sources, further at an input purity specification which ranges from 60 % to 70 %, such that the desired minimum percentage assay of the respective carotenoids added to the composition is complied during and at the end of completion of accelerated stability studies or real time stability studies with respect to the added carotenoid to the respective encapsulated carotenoid compositions.
Furthermore, the encapsulated carotenoid compositions of the present invention also includes within its ambit encapsulated carotenoids and their corresponding esters thereof prior to undergoing saponification of the said corresponding carotenoid esters.
For the purpose of enabling of the present invention, the embodiments herein below provides working embodiments with different carotenoids, specifically xanthophyll carotenoids obtained from various sources, particularly wherein lutein and/or zeaxanthin carotenoids are obtained from marigold flower Tagetes erecta, beta-carotene obtained from algae Dunaliella salina, lycopene obtained from Solanum lycopersicum or Lycopersicon esculentum. However, the carotenoids and/or esters thereof present in the encapsulated carotenoid compositions of the present invention primarily refers to the said carotenoids as such including esters thereof regardless of the source of extraction of the said carotenoids in the present invention.
For instance, the above-mentioned marigold full spectrum extract is heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid selected from marigold extract of lutein, at desired input purity specification ranges from 60% to 70% and concentration level of 45% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 3% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid selected from marigold extract of lutein, at desired input purity specification which ranges from 60% to 70 %and concentration level of 45% w/w as provided in the Compositions below, wherein preferably the desired input purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 4% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein, at desired input purity specification which ranges from 60% to 70 % and concentration level of 45% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 5% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein and zeaxanthin, at desired purity specification ranging from 60% to 70% and concentration level of 8.5% w/w as provided in the Compositions below, is added to the above-mentioned marigold full spectrum extract at a concentration range at 3% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein and zeaxanthin, at desired purity and concentration level of 8.5% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 60% to 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 4% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein and zeaxanthin, at desired purity and concentration level of 8.5% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 60% to 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 5% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein and zeaxanthin, at desired purity and concentration level of 18.5% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 60% to 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 3% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed., Carotenoids selected from marigold extract of lutein and zeaxanthin at desired input purity of 60% to 70% and concentration level of 18.5% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 4% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein and zeaxanthin, at desired input purity of 60% to 70% and concentration level of 18.5% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 5% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from beta-carotene extract obtained from algae Dunaliella salina, at desired purity and concentration level of 30% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 3% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein esters, at desired input purity of 60% to 70% and concentration level of 30% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 4% w/w under stirring till a smooth, lump free paste is formed.
For instance, the above-mentioned marigold full spectrum extract heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from lycopene obtained from Solanum lycopersicum, at desired purity and concentration level of 30% w/w as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above-mentioned marigold full spectrum extract at a concentration range at 5% w/w under stirring till a smooth, lump free paste is formed.
Alternatively, the oil phase solution is prepared wherein a vegetable oil, a saturated fatty acid, a non-ionic emulsifier, and a non-ionic surfactant is mixed and heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoids selected from marigold extract of lutein and its esters thereof, or marigold extract of zeaxanthin and its esters thereof, or marigold extract of a combination of lutein and zeaxanthin and its esters thereof, or other extracts of carotenoid esters such as lycopene, beta-carotene or any other xanthopylls, etc. (non-exhaustive) at desired purity and concentration levels as provided in the Compositions below, wherein preferably the desired purity of lutein is at least 70% is added to the above combination of the vegetable oil, saturated fatty acid, non-ionic emulsifier, and a non-ionic surfactant under stirring till a smooth, lump free paste is formed;
wherein the said saturated fatty acid of the oil phase is selected from stearic acid, palmitic acid, lauric acid, oleic acid, lauric acid, palmitic acid, and myristic acid or combinations thereof; the said non-ionic emulsifier of the oil phase being glycerol monostearate, the said vegetable oil of the oil phase is selected from coconut oil, sunflower oil, Olive oil, groundnut oil, the said non-ionic surfactant of the oil phase being selected from polysorbate 80; Polysorbate 60, Polysorbate 40, Polysorbate 20.
For instance, a vegetable oil i.e. virgin coconut oil at a concentration of 5% w/w, a saturated fatty acid stearic acid at a concentration of 4% w/w, a non-ionic emulsifier glycerol monostearate at a concentration of 4% w/w and a non-ionic surfactant polysorbate 80 at a concentration of 2% w/w is mixed and heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid lutein (at desired input purity of 70% and concentration level at 45% w/w, wherein preferably the desired purity of lutein be at least 70%) is added to the above step under stirring till a smooth, lump free paste is formed.
For instance, a vegetable oil i.e. virgin coconut oil at a concentration of 5% w/w, a saturated fatty acid stearic acid at a concentration of 4% w/w, a non-ionic emulsifier glycerol monostearate at a concentration of 4% w/w and a non-ionic surfactant polysorbate 80 at a concentration of 2% w/w is mixed and heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid lutein at desired purity and concentration level at 45% w/w, wherein preferably the desired purity of lutein be at least 70%) is added to the above step under stirring till a smooth, lump free paste is formed.
For instance, a vegetable oil i.e. virgin coconut oil at a concentration of 5% w/w, a saturated fatty acid lauric acid at a concentration of 4% w/w, a non-ionic emulsifier glycerol monostearate at a concentration of 4% w/w and a non-ionic surfactant polysorbate 80 at a concentration of 2% w/w is mixed and heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid lutein (at desired purity and concentration level at 45% w/w, wherein preferably the desired purity of lutein be at least 70%) is added to the above step under stirring till a smooth, lump free paste is formed.
For instance, a vegetable oil i.e. virgin coconut oil at a concentration of 5% w/w, a saturated fatty acid palmitic acid at a concentration of 4% w/w, a non-ionic emulsifier glycerol monostearate at a concentration of 4% w/w and a non-ionic surfactant polysorbate 80 at a concentration of 2% w/w is mixed and heated to 65ºC-70ºC under stirring till a homogenous mixture is formed. Carotenoid lutein (at desired purity and concentration level at 45% w/w, wherein preferably the desired purity of lutein be at least 70%) is added to the above step under stirring till a smooth, lump free paste is formed.
Preparation of an Aqueous phase Solution along with addition of buffers (optional) and antioxidant preparations: The lump-free aqueous phase solution can be prepared either with the help of a semi synthetic cellulose derivative mixed with a sugar alcohol and water, or directly mixing gum acacia in water. For example, the gum acacia and water is mixed in such a concentration range such that the final Composition of the aqueous phase solution contains gum acacia within the range of 16% w/w till 21% w/w as provided in the Compositions below presented in the subsequent embodiments of the invention. This aqueous phase of gum acacia is mixed with the oil phase solution comprising the carotenoid and stirred to get an uniform gel and then subjected to form an emulsion and homogenized for 30 minutes at 65 ºC.
Alternatively, a semi-synthetic cellulose derivative and a sugar alcohol wherein the said semi-synthetic cellulose derivative selected from hydroxyethyl cellulose, hydroxy propyl methyl cellulose and hydroxymethyl cellulose, the said sugar alcohol selected from glycerol; sorbitol, mannitol, xylitol, or combinations thereof, for example Glycerin at a concentration of 3% w/w and Hydroxyethyl cellulose at a concentration of 0.2% w/w in a vessel and mixed well to form a paste without lumps, and mixed with 80% water in to vessel, and heated to 65ºC–70ºC, and then this solution is mixed with the paste of oil phase solution above, and stirred to get an uniform gel and then subjected to form an emulsion and homogenized for 30 minutes at 65 ºC. This is followed by adding a mixture of citric acid and sodium citrate to obtain a clear solution of a buffer composition at a concentration between 0.1% w/w to 0.3% w/w to the homogenized emulsion of the step above under constant stirring for at least 10 minutes such that the final pH of the composition is maintained between 4.5 to 5.5 and allowing the homogenized emulsion to cool down till the temperature of the homogenized emulsion reduces to 45o C.
Thereafter, an antioxidant preparation is added at a concentration of 2% w/w to the homogenized emulsion of step above, wherein the said antioxidant preparation is selected from combination of a vitamin ester and ascorbic acid or a combination of a vitamin ester and ascorbyl palmitate wherein the said vitamin ester is selected from vitamin E acetate; or vitamin E palmitate, Then, a diluent filler maltodextrin is added to the homogenized emulsion at a concentration of 34% w/w to 35% w/w to obtain a final composition which is then subjected to spray drying at the specific parameters wherein the inlet temperature is around 140oC to 180oC, outlet temperature is around 85oC to 90oC, and spray rate to be maintained accordingly to achieve the above temperature ranges, to obtain the encapsulated carotenoid compositions under this present invention.
Alternatively in this present invention, an antioxidant preparation is added into the encapsulated carotenoid compositions of the present invention, to ensure long term stability and to prevent the degradation of the encapsulated carotenoid is selected from combination of Vitamin E, an aqueous solution of Vitamin C, and coffee bean extract or green coffee bean extract and allowing the homogenized emulsion to cool down till the temperature of the homogenized emulsion reduces to 45o C. Then, a diluent filler maltodextrin is added to the homogenized emulsion at a concentration of 25% w/w to 44% w/w to obtain a final composition which then subjected to spray drying at specific parameters, at the specific parameters wherein the inlet temperature is around 140oC to 180oC, outlet temperature is around 85oC to 90oC, and spray rate to be maintained accordingly to achieve the above temperature ranges to obtain the encapsulated carotenoid compositions under this present invention.
Example 2: Micro encapsulated Carotenoid Compositions of the present invention
A number of compositions have been prepared according to the present invention and provided below in accordance with the process of preparation of the said microencapsulated carotenoid compositions as provided in the preceding embodiments. The various compositions are presented below Table 1 and 2.
Table 1: Different compositions of the present invention
Composition Composition 1 Concentration
% (w/w) Composition 2 Concentration % (w/w) Composition 3 Concentration % (w/w)
Carotenoid Marigold extract of lutein 45 Marigold extract of lutein 45 Marigold extract of lutein 45
Oil Phase Stearic Acid 4 Lauric Acid 4 Palmitic Acid 4
Coconut oil 5 Coconut oil 5 Coconut oil 5
Glycerol monostearate 4 Glycerol monostearate 4 Glycerol monostearate 4
Polysorbate 80 2 Polysorbate 80 2 Polysorbate 80 2
Aqueous phase Hydroxy ethyl cellulose 0.2 Hydroxy propyl methyl cellulose 0.2 Hydroxy methyl cellulose 0.2
Glycerol 3 Glycerol 3 Glycerol 3
Water q.s. Water q.s. Water q.s.
Antioxidant Vitamin E acetate 1 Vitamin E acetate 1 Vitamin E acetate 1
Ascorbic Acid 1 Ascorbic Acid 1 Ascorbic Acid 1
Buffers (pH 4.5-5.5) Citric Acid & Sodium Citrate 0.1 Citric Acid & Sodium Citrate 0.2 Na2HPO4 &NaH2PO4 0.3
Diluent Filler Maltodextrin 34.8 Maltodextrin 34.8 Maltodextrin 34.8

Table 2: Different encapsulated compositions of the present invention containing a carotenoid and marigold full-spectrum extract along with aqueous phase solutions and antioxidants including coffee bean extract
Composition Composition Concentration
% (w/w) of Composition 4 Concentration % (w/w) of Composition 5 Concentration % (w/w) of Composition 6
Carotenoid Marigold extract of lutein 45 45 45
Oil Phase Marigold full spectrum extract 3 4 5
Aqueous phase Gum Acacia 21 18.5 16
water q.s q.s. q.s.
Antioxidant Coffee Bean Extract 3.5 3.5 3.5
Vitamin C 1 1 1
Vitamin E 1 1 1
Diluent Filler Maltodextrin 25.5 27 28.5
Composition Composition Concentration
% (w/w) of Composition 7 Concentration % (w/w) of Composition 8 Concentration % (w/w) of Composition 9
Carotenoid Marigold extract of lutein & zeaxanthin 8.5 8.5 8.5 (only lutein)
Oil Phase Marigold full spectrum extract 3 4 5
Aqueous phase Gum Acacia 21 18.5 16
water q.s q.s. q.s.
Antioxidant Coffee Bean Extract 3.5 3.5 3.5
Vitamin C 1 1 1
Vitamin E 1 1 1
Diluent Filler Maltodextrin 62 63.5 65
Composition Composition Concentration
% (w/w) of Composition 10 Concentration % (w/w) of Composition 11 Concentration % (w/w) of Composition 12
Carotenoid Marigold extract of lutein & zeaxanthin 18.5 18.5 18.5
Oil Phase Marigold full spectrum extract 3 4 5
Aqueous phase Gum Acacia 21 18.5 16
water q.s q.s. q.s.
Antioxidant Coffee Bean Extract 3.5 3.5 3.5
Vitamin C 1 1 1
Vitamin E 1 1 1
Diluent Filler Maltodextrin 52 53.5 55
Composition Composition Concentration
% (w/w) of Composition 13 Concentration % (w/w) of Composition 14 Concentration % (w/w) of Composition 15
Carotenoid ß–carotene (C13);
Marigold extract of lutein esters (C14);
Lycopene (C15) 30 30 30
Oil Phase Marigold full spectrum extract 3 4 5
Aqueous phase Gum Acacia 21 18.5 16
water q.s q.s. q.s.
Antioxidant Coffee Bean Extract 3.5 3.5 3.5
Vitamin C 1 1 1
Vitamin E 1 1 1
Diluent Filler Maltodextrin 40.5 42 43.5

COMPOSITIONS FOR COMPARISON OF SYNERGISTIC ACTIVITY
Table 3.1: Carotenoid compositions devoid of either oil-phase or the aqueous phase or the antioxidants
Composition Composition Concentration
% (w/w) of Composition 16 Concentration % (w/w) of Composition 17 Concentration % (w/w) of Composition 18
Carotenoid Marigold extract of lutein 45 30 18.5
Oil Phase Marigold full spectrum extract 3 0 0
Aqueous phase Gum Acacia 0 18.5 0
water q.s q.s. q.s.
Antioxidant Coffee Bean Extract 0 0 3.5
Vitamin C 0 0 1
Vitamin E 0 0 1
Diluent Filler Maltodextrin 25.5 27 28.5

Table 3.2: Carotenoid compositions devoid of either oil-phase or the aqueous phase or the antioxidants
Composition Composition 19 Concentration
% (w/w) Composition 20 Concentration % (w/w) Composition 21 Concentration % (w/w)
Carotenoid Marigold extract of lutein 45 Marigold extract of lutein 45 Marigold extract of lutein 45
Oil Phase Stearic Acid 4 Lauric Acid 0 Palmitic Acid 0
Coconut oil 5 Coconut oil 0 Coconut oil 0
Glycerol monostearate 4 Glycerol monostearate 0 Glycerol monostearate 0
Polysorbate 80 2 Polysorbate 80 0 Polysorbate 80 0
Aqueous phase Hydroxy ethyl cellulose 0 Hydroxy propyl methyl cellulose 0.2 Hydroxy methyl cellulose 0
Glycerol 0 Glycerol 3 Glycerol 0
Water q.s Water q.s. Water q.s
Antioxidant Vitamin E acetate 0 Vitamin E acetate 0 Vitamin E acetate 1
Ascorbic Acid 0 Ascorbic Acid 0 Ascorbic Acid 1
Diluent Filler Maltodextrin 34.8 Maltodextrin 34.8 Maltodextrin 34.8

The above-mentioned compositions 16 to 21 were not encapsulated due to absence of either the oil phase, or the aqueous phase or the antioxidant. However, the content of carotenoid, measured in terms of lutein assay were prepared to analyze and establish the extent of synergism exhibited in the nature of the compositions in the present invention getting properly encapsulated that exhibits long term stability incubated at 40oC ± 2oC and 75% ± 5% RH (Accelerated stability study) for a period of 6 months and at 25oC ± 2oC and 60%± 5% RH (Real time stability study) for a period of 36 months. Contrary to the compositions as claimed in the present invention, the compositions 16 to 21 did not exhibit desirable encapsulation efficiency, showed discoloration and sensory evaluation revealed off-flavoured bad odour. These observation of lack of encapsulation were duly reflected in the stability studies of the said compositions 16 to 21, wherein the carotenoid assays of lutein in the final composition showed significant loss of purity of the carotenoids below the specified levels, as shown below in the following emobidiments.
Example 3: Stability Studies of the micro-encapsulated Compositions The samples of the encapsulated carotenoid compositions were incubated at 40oC ± 2oC and at 75% ± 5% RH (Accelerated stability study) for a period of 6 months and at 25oC ± 2oC and 60%± 5% RH (Real time stability study) for a period of 36 months. Physical, chemical and microbiological parameters were used to assess the stability. The samples were withdrawn as per protocol (0, 1, 2, 3, 6) for accelerated stability study and equal intervals of 3 months in the first year, 6 months interval after one year up to 2 years and once in 12 months thereafter (0, 3, 6, 9, 12, 18, 24months) for real time stability study and subjected to analysis and the data was compared with the initial values. The analytical data indicates that the physical, chemical parameters of the encapsulated carotenoids under this present invention remained within the specification during the stability period. The variation of the active/marker content was found to be within the specified limits. Microbiological data showed no considerable change conforms to the specification throughout the study. The product is stable physically, chemically and microbiologically throughout the accelerated stability study. Based on the same as provided in the experiments herein, it can be concluded that encapsulated carotenoid compositions under this present invention is stable for at least 36 months.
In order to establish the stability of the carotenoids after encapsulation, the purity of the respective carotenoids, preferably the purity of the lutein and zeaxanthin were accordingly assayed to ascertain the presence of minimum specifications of lutein in encapsulated state. Whereas the input purity of the carotenoids employed in this present invention for the purpose of encapsulation is within the range of 60% to 70%, the purity of the carotenoids in the final encapsulated compositions are specified to be at least 20% in case of lutein carotenoid, and in case present in combination of lutein & zeaxanthin, in that case the final assay of the carotenoids is specified to be in the ratio of 5:1 of lutein to zeaxanthin. Accordingly, the purity assay specified is at least 5% or 10% for lutein, and 1% or 2% of zeaxanthin in the final encapsulated compositions as provided in the stability data below. This enables availability of cost effective micro-encapsulated carotenoid compositions of lutein of desired purity levels ranging from 5% to 20% purity and strength of lutein, as per the requirements of end-use formulations.
Table 4: Accelerated stability study results of the encapsulated carotenoids of the present invention
Conditions: Accelerated: 40°C ? 2°C and 75% ? 5% RH
*The color, odor and appearance are not provided for compositions C16 to C21 as these are not applicable on these said compositions C16 to C21.
Sl.
No. TEST Specification Initial
Withdrawal 1st month
Withdrawal 2nd month
Withdrawal 3rd month
withdrawal 6th month
withdrawal
1. Description (color) Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder
2. Odor Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic
3. Appearance Powder Powder Powder Powder Powder Powder
4. Loss on drying < 5.0% C1 2.99 3.09 3.92 3.95 3.98
C2 3.02 3.12 3.95 3.98 4.01
C3 3.06 3.16 3.99 4.02 4.05
C4 3.84 3.01 2.91 3.87 3.88
C5 3.92 3.09 2.99 3.95 3.96
C6 4.12 3.29 3.19 4.15 4.16
C7 3.33 3.37 4.21 4.24 4.26
C8 3.29 3.39 4.22 4.25 4.28
C9 3.31 3.35 4.23 4.24 4.25
C10 3.28 3.38 4.21 4.24 4.26
C11 3.29 3.39 4.22 4.25 4.28
C14 3.39 3.49 4.32 4.35 4.38
5. Content of Carotenoid Lutein >20.0% C1 21.15 20.97 20.91 20.89 20.86
C2 21.05 20.87 20.81 20.78 20.75
C3 21.11 20.93 20.87 20.85 20.82
C4 21.11 20.93 20.87 20.85 20.82
C5 20.98 20.80 20.74 20.72 20.69
C6 21.03 20.84 20.78 20.76 20.73
Lutein > 5 % C7 5.21 5.15 5.14 5.13 5.12
Zeaxanthin > 1 % 1.07 1.04 1.03 1.03 1.02
Lutein > 5 % C8 5.20 5.16 5.14 5.14 5.13
Zeaxanthin > 1 % 1.04 1.03 1.02 1.02 1.02
Lutein > 5 % C9 5.22 5.19 5.17 5.14 5.13
Zeaxanthin > 1 % 1.08 1.05 1.04 1.04 1.03
Lutein > 10 % C10 10.49 10.45 10.43 10.36 10.37
Zeaxanthin > 2% 2.11 2.09 2.07 2.06 2.05
Lutein > 10 % C11 10.51 10.42 10.39 10.38 10.37
Zeaxanthin > 2% 2.10 2.08 2.08 2.08 2.07
Lutein Ester >20.0% C14 20.98 20.80 20.73 20.71 20.68
6. Content of Carotenoid Lutein >20.0% C16 20.13 19.45 16.34 13.09 12.23
7. Content of Carotenoid Lutein >20.0% C17 20.43 18.98 16.24 13.81 13.59
8. Content of Carotenoid Lutein >20.0% C18 20.34 19.65 19.64 19.14 18.74
9. Content of Carotenoid Lutein >20.0% C19 20.17 19.35 16.31 12.69 12.23
10. Content of Carotenoid Lutein >20.0% C20 20.15 18.74 16.21 13.45 13.29
11. Content of Carotenoid Lutein >20.0% C21 20.33 19.62 19.53 19.17 18.71
Microbiological analysis
*The microbiological analysis are not provided for compositions C16 to C21 as these are not applicable on these said compositions C16 to C21.
i Total plate count 3000cfu/g 100cfu/g N/A N/A N/A N/A
ii Yeast and molds 100cfu/g 10cfu/g N/A N/A N/A N/A
iii E. coli Absent Absent N/A N/A N/A N/A
iv Coliform Absent Absent N/A N/A N/A N/A
v Salmonella species Absent Absent N/A N/A N/A N/A

Table 5: Encapsulated Carotenoid-Realtime stability studies at 25oC ± 2oC and 60%± 5% RH
S
No. TEST Specification Initial
Withdrawal 3rd month withdrawal 6th month
Withdrawal 9th month
Withdrawal 12th month
withdrawal 18th month
withdrawal 24th month withdrawal 36th month withdrawal
1. Description (color) Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder Orange red powder
2. Odor Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic
3. Appearance Powder Powder Powder Powder Powder Powder Powder Powder Powder
4. Loss on drying < 5.0% C1 2.99 3.94 3.97 3.98 4.1 4.16 4.20 4.25
C4 3.74 3.81 3.91 3.95 3.97 4.11 4.15 4.21
C8 3.28 3.39 4.22 4.24 4.28 4.30 4.31 4.33
C14 3.39 3.43 4.28 4.29 4.32 4.37 4.39 4.41
5. Carotenoid Lutein > 20.0% C1 20.88 20.74 20.69 20.67 20.50 20.49 20.26 20.24
C4 21.09 20.13 20.17 20.77 20.81 20.65 20.43 20.39
Lutein> 5.0 % C8 5.17 5.13 5.12 5.11 5.11 5.09 5.07 5.05
Zeaxanthin > 1.0% 1,33 1.28 1.26 1.23 1.22 1.18 1.16 1.13
Lutein Ester >20.0% C14 20.95 20.79 20.72 20.71 20.65 20.63 20.62 20.57
i. Total plate count 3000 cfu/g 100 cfu/g N/A N/A N/A 200 cfu/g N/A 300 cfu/g 300 cfu/g
ii. Yeast
and
molds 100 cfu/g 10 cfu/g N/A N/A N/A <10 cfu/g N/A <10 cfu/g <10 cfu/g
iii. E. coli Absent Absent N/A N/A N/A Absent N/A Absent Absent
iv. Coliform Absent Absent N/A N/A N/A Absent N/A Absent Absent
V Salmonella species Absent Absent N/A N/A N/A Absent N/A Absent Absent

From the stability analysis following observations were made:-
i) Description: No change in color was observed for accelerated and real time till the end of 6th month and 24th month withdrawal respectively.
ii) Loss on drying: The LOD level was found to be within the limits for accelerated as well as real time stability study.
iii) Estimation of Lutein by HPLC: According to ICH guidelines the limits of variation is ± 5% of initial label claim is allowed for active. In accelerated and real time study, Lutein content was found to be within the specified limit.
iv) HPLC Finger print: HPLC Chromatogram for Accelerated stability sample at 6th month withdrawal and 24th month withdrawal for real time stability was found to be matching with that of initially analyzed sample.
v) Microbiological Study: There was no detectable variation in the microbial profile.
Conclusion: No changes in color and appearance throughout the study. Loss on drying level was found almost constant. Lutein was found to be within the specified limit for all the withdrawals. Observed almost 4-5% variation from initial assay in 24th month (real time study). HPLC chromatogram for accelerated stability sample at 6th month withdrawal was found to be matching with that of initially analyzed sample. The HPLC chromatogram of real time stability sample was also found matching with that of initially analyzed sample. This indicates the phytochemical profile of the extract/the encapsulated composition of lutein remains the same throughout the stability study and is microbiologically stable throughout the period in the accelerated stability study. Hence, the encapsulated carotenoid compositions of the present invention is stable under accelerated stability conditions for a period of 6 months and for at least 24 months in real time conditions.
Example 4: Standard Electron Microscopy Images of Encapsulated Carotenoid Compositions of the present invention
Scanning Electron Microscopy (SEM) plays a critical role in the field of microencapsulation by providing detailed insights into the structure, morphology, and surface characteristics of microcapsules. SEM allows for high-resolution imaging of microcapsules, revealing the external and internal structures. This is essential for verifying the integrity of the microcapsule wall, detecting any cracks, pores, or deformations, and ensuring the encapsulation is effective. The surface characteristics of microcapsules, such as roughness, smoothness, or the presence of surface irregularities, can be observed using SEM. These features can influence the release profile of the encapsulated material, its stability, and its interaction with the surrounding environment. SEM enables precise measurement of the size and shape of microcapsules. This is important because the size and shape of microcapsules can affect the release rate of the encapsulated material, as well as the handling and processing of the microcapsules. By providing detailed images of the microcapsules, SEM is used in quality control to ensure consistency in production. It helps in identifying any defects or variations in the microcapsules that could affect their performance. SEM can be used to study the interaction of microcapsules with other materials, such as coating layers or embedded active agents. This can provide insights into how these interactions might affect the stability or release behavior of the microcapsules. SEM images can be used to assess the porosity of the microcapsule walls, which is crucial for determining the encapsulation efficiency and the controlled release properties of the encapsulated material. SEM is an indispensable tool in the study and development of microencapsulation technologies, offering detailed visual and quantitative information that is crucial for optimizing and controlling the encapsulation process. The SEM images of various Compositions of the present invention is provided in the Figures 1 to 5. The microencapsulated carotenoid composition samples were subjected to SEM analysis to know the surface morphology of the microencapsulated product. The SEM images show uniform, spherical particle size distribution. The images also convey uniform coating of the particles which establishes better encapsulation efficiency.

,CLAIMS:We claim:
1. A micro-encapsulated carotenoid composition emulsified in an oil phase and aqueous phase solution comprising:
(a) a carotenoid at a concentration range from 8.5% w/w to 45% w/w , wherein the carotenoid is selected from lutein and its esters thereof, zeaxanthin and its esters thereof, a combination of lutein and zeaxanthin and its esters thereof, any other carotenoid such as lycopene and its esters thereof, beta-carotene and its esters thereof, a xanthopylls and its esters thereof, and a combination of any of the said carotenoids and their esters thereof;
(b) an oil phase solution at a concentration range between 3% w/w to 15% w/w, wherein the said oil phase solution is selected from a marigold full spectrum extract, or a combination of a saturated fatty acid, a non-ionic emulsifier, a vegetable oil, and a non-ionic surfactant;
wherein the said saturated fatty acid of the oil phase is selected from stearic acid, palmitic acid, lauric acid, oleic acid, lauric acid, palmitic acid, and myristic acid or combinations thereof; the said non-ionic emulsifier of the oil phase being glycerol monostearate, the said vegetable oil of the oil phase is selected from coconut oil, sunflower oil, Olive oil, groundnut oil, the said non-ionic surfactant of the oil phase being selected from polysorbate 80, polysorbate 60, polysorbate 40, and polysorbate 20 or combinations thereof;
(c) an aqueous phase solution at a concentration range between 3% w/w to 21% w/w, wherein the said aqueous phase solution is selected from gum acacia or a combination of a semi-synthetic cellulose derivatives or natural gums and a sugar alcohol;
wherein the said semi-synthetic cellulose derivative selected from hydroxyethyl cellulose, hydroxy propyl methyl cellulose and hydroxymethyl cellulose, the said sugar alcohol selected from glycerol; sorbitol, mannitol, xylitol, or combinations thereof;
(d) and a carotenoid protecting antioxidant preparation at a concentration range between 2% w/w to 5% w/w, wherein the said antioxidant preparation is selected from combination of a vitamin ester and ascorbic acid or ascorbyl palmitate wherein the said vitamin ester is selected from vitamin E acetate; vitamin E palmitate, or a combination of Vitamin E, Vitamin C, coffee bean extract, or green coffee bean extract including combinations thereof;
(e) optionally including a buffer composition at a concentration between 0.1% w/w to 0.3% w/w the said buffer selected from a combination of citric acid and sodium citrate, a combination disodium hydrogen phosphate and dihydrogen sodium phosphate, or any other buffer that maintains the final pH of the composition between 4.5 to 5.5;
and
(f) a diluent filler maltodextrin at a concentration between 25% w/w to 65% w/w;
wherein the final assay content of the carotenoid in the encapsulated carotenoid composition is at least 5% to 30% purity.
2. The micro-encapsulated carotenoid composition of claim 1, wherein the said composition is prepared a method of preparation of the encapsulated carotenoid compositions, the said method comprising the steps:
(a) preparing an oil phase solution an oil phase solution by adding the saturated fatty acid, non-ionic emulsifier, vegetable oil, and the non-ionic surfactant and heating the said solution to around 60oC to 70oC to result into a dispersion, wherein the concentration of the said oil phase solution is at a concentration range of 15% w/w and adding the carotenoid at a concentration of 45% w/w to the dispersion under stirring till a smooth, lump free paste is formed,
wherein the said saturated fatty acid of the oil phase is selected from stearic acid, palmitic acid, lauric acid, oleic acid, lauric acid, palmitic acid, and myristic acid or combinations thereof added at an individual concentration of 4% w/w; the said non-ionic emulsifier of the oil phase being glycerol monostearate added at an individual concentration of 4% w/w, the said vegetable oil of the oil phase is selected from coconut oil, sunflower oil, Olive oil, groundnut oil added at an individual concentration of 5% w/w, the said non-ionic surfactant of the oil phase being selected from polysorbate 80, polysorbate 60, polysorbate 40, and polysorbate 20 or combinations thereof added at an individual concentration of 2% w/w;
(b) simultaneously preparing a lump free aqueous phase solution by adding the semi-synthetic cellulose derivative and the sugar alcohol mixed with 80% water in to vessel, and heated to 60ºC–70ºC, and adding the aqueous phase solution at a concentration of 3 % w/w to 3.5% w/w with the paste of step (a) above, and stirred continuously to get an uniform homogenized emulsion for 30 minutes at 65 ºC,
wherein the said semi-synthetic cellulose derivative selected from hydroxyethyl cellulose, hydroxy propyl methyl cellulose and hydroxymethyl cellulose, the said sugar alcohol selected from glycerol; sorbitol, mannitol, xylitol, or combinations thereof;
(c) adding a mixture of citric acid and sodium citrate to obtain a clear solution of a buffer composition at a concentration between 0.1% w/w to 0.3%. w/w to the homogenized emulsion of step (b) above under constant stirring for at least 10 minutes such that the final pH of the composition is maintained between 4.5 to 5.5 and allowing the homogenized emulsion to cool down till the temperature of the homogenized emulsion reduces to 45o C;
(d) adding an antioxidant preparation at a concentration of 2% w/w to the homogenized emulsion of step (c) above, wherein the said antioxidant preparation is selected from combination of a vitamin ester and ascorbic acid or a combination of a vitamin ester and ascorbyl palmitate wherein the said vitamin ester is selected from vitamin E acetate; or vitamin E palmitate;
(e) adding a diluent filler to the homogenized emulsion at a concentration of 34% w/w to 35% w/w to obtain a final composition which then subjected to spray drying at the specific parameters wherein the inlet temperature is around 140oC to 180oC, outlet temperature is around 85oC to 90oC, and spray rate to be maintained accordingly to achieve the above temperature ranges to obtain the encapsulated carotenoid compositions under this present invention.
3. The micro-encapsulated carotenoid compositions of claim 1, wherein the said micro-encapsulated carotenoid compositions is prepared by a method of preparation of the encapsulated carotenoid compositions, the said method comprising the steps:
(a) preparing an oil phase solution, wherein the said oil phase solution is a marigold full spectrum extract and heating the said solution to around 60oC to 70oC to result into a dispersion, wherein the concentration of the said oil phase solution is at a concentration range between 3 % w/w to 5 % w/w and adding the carotenoid at a concentration between 8.5% w/w to 45% w/w to the dispersion under stirring till a smooth, lump free paste is formed;
wherein the said marigold full spectrum extract is prepared through hydroalcoholic solvent extraction at around 70oC with ethyl alcohol and water in the ratio ranging from 80 (ethyl alcohol): 20 (water) to 60 (ethyl alcohol): 40 (water) of shed dried marigold petals, followed by drying the extract in Vacuum Tray Drier at 70oC till LOD (loss on drying) is around 5% w/w to obtain a thick mass followed by subjecting the thick mass to milling using a multimill with impact forward direction to produce a desired fineness or particle size distribution of a powder called as full spectrum marigold extract according to the present invention;
(b) simultaneously preparing a lump free aqueous phase solution by adding gum acacia at a concentration range between 16% w/w to 21% w/w with the paste of step (a) above, and stirred continuously to get an uniform homogenized emulsion for 30 minutes heated to temperature between 60 ºC to 70 ºC and allowing the homogenized emulsion to cool down till the temperature of the homogenized emulsion reduces to 45o C;
(c) adding an antioxidant preparation at a concentration of 5% w/w to 6 % w/w to the homogenized emulsion of step (c) above, wherein the said antioxidant preparation is selected from combination of Vitamin E, an aqueous solution of Vitamin C, and coffee bean extract or green coffee bean extract;;
(d) adding a diluent filler to the homogenized emulsion at a concentration of 25% w/w to 44% w/w to obtain a final composition which then subjected to spray drying at the specific parameters wherein the inlet temperature is around 140oC to 180oC, outlet temperature is around 85oC to 90oC, and spray rate to be maintained accordingly to achieve the above temperature ranges to obtain the encapsulated carotenoid compositions under this present invention.
4. The microencapsulated carotenoid compositions of claim 1. are stable for a period of at least 6 months at accelerated conditions at 40°C ? 2°C and relative humidity of 75% ? 5%.
5. The microencapsulated carotenoid compositions of claim 1, are stable for a period of at least 36 months at real-time conditions at 25oC ± 2oC and relative humidity of 60%± 5%.