Process for producing fucoxanthin and/or polysaccharides from microalgae

Abstract

Provided is a process for production of fucoxanthin and/or polysaccharides from microalgae and the use of purified fucoxanthin thereof in pharmaceutical, cosmetic, nutraceutical and food compositions.

Claims

1. An improved process for producing fucoxanthin or fucoxanthin and polysaccharides from microalgae comprising the steps of: (a) cultivating the microalgae medium using inducers to enable rapid cell growth for enhanced production of fucoxanthin or fucoxanthin and polysaccharides, wherein the inducers comprise adding to the growth medium at least one brassinosteroidal hormone selected from the group consisting of auxins and cytokinins and inducing a stress condition of applying specific light wavelength of blue-green; (b) harvesting and drying the algal culture to produce a dry culture; (c) carrying out air pressure milling to crack the cell walls of the algae; (d) extracting the dry culture to produce extracts rich in fucoxanthin or fucoxanthin and polysaccharides; and (e) separating the extraction mixture into a biomass fraction and a fucoxanthin-rich oleoresin or fucoxanthin-rich oleoresin and polysaccharide-rich extract and optionally further purifying said fucoxanthin-rich oleoresin or fucoxanthin-rich oleoresin and polysaccharide-rich extract.

2. The process of claim 1, wherein the algal culture is selected from Phaeodactylum sp., Isochrysis sp., Amphora sp., Naviculla lensi, Naviculla incerta and Chaeotocerous sp.

3. The process of claim 1, wherein cultivating the microalgae is carried out using at least one additional stress condition selected from nitrogen or nitrate starvation, phosphorous starvation, adding H.sub.2O.sub.2, applying heat, using higher pH, adding chlorine ions, exposing the medium to ozone and combination of stress conditions thereof.

4. The process of claim 1, wherein harvesting the microalgae is carried out by separating the microalgae from the aqueous mixture by centrifugation and optionally re-centrifugation followed by drying.

5. The process of claim 4, wherein the algal culture is dried by at least one method selected from lyophilization, spray drying, evaporation, air or vacuum drying, exposure to hot air, refractory window belt drying, drying in an oven and combination of methods thereof.

6. The process of claim 4, wherein, the drying process is performed with addition of powdering agent or anti-caking agent selected from magnesium stearate, lactose, lecithin, talc, hydroxypropyl methylcellulose, microcrystalline cellulose, sodium alginate, sodium aluminosilicate, silicone hydroxide, chitosan and combination of powdering agents or anti-caking agents thereof.

7. The process of claim 4, wherein the harvested biomass is concentrated into a solid, washed with water in order to reduce salinity and optionally re-centrifuged to afford a product containing 10-45% weight/volume solids.

8. The process of claim 4, wherein the supernatant obtained by centrifugation and optionally re-centrifugation of the harvested biomass is used for purification of secreted polysaccharides.

9. The process of claim 1, wherein fucoxanthin is separated from the proteins by extraction using at least one organic solvent and/or supercritical fluid (SCF) liquid CO2.

10. The process of claim 9, wherein the at least one organic solvent used for extraction is selected from ethanol, isopropyl alcohol, n-butanol, diethyl ether, diisopropyl ether, n-pentane, n-hexanes, n-heptane, cyclohexane, petroleum ether, tetrahydrofuran, methyltetrahydrofuran, acetonitrile, acetone, methylethyl ketone, methylisobutyl ketone, ethyl acetate, n-propyl acetate, isopropyl acetate and mixtures of solvents thereof.

11. The process of claim 1, which affords a product containing at least 1.5% weight/weight fucoxanthin in the dry biomass and at least 10% weight/weight fucoxanthin in the extract.

12. The process of claim 9, wherein the remaining pulp after the extraction is dissolved in hot water to enable purification of polysaccharides.

13. The process of claim 9, wherein prior to or after the SCF extraction, the dried biomass is treated enzymatically using enzymes and/or extracted with hot water and/or by hot steam and/or treated by washing with buffer solution and/or treated to denature the proteins that are bound to the fucoxanthin.

14. The process of claim 1, wherein the polysaccharides are separated and purified by a method selected from filtration, gel-filtration on columns using natural silica beads as medium for filtration, ion-exchange chromatography, a liquid chromatography method and combination of methods thereof.

15. The process of claim 1, wherein the fucoxanthin-rich oleoresin is further refined and separated from the fatty acids by additional SCF sequences, optionally in combination with liquid chromatography to afford purified fucoxanthin.

16. The process of claim 1, wherein the at least one brassinosteroidal hormone is auxin.

17. The process of claim 4, wherein the liquid chromatography method is selected from preparative TLC and preparative HPLC.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts the fucoxanthin HPLC calibration curve using acetone/methanol/hexane as solvent.

(2) FIG. 2 depicts the fucoxanthin HPLC calibration curve using acetonitrile as solvent.

(3) FIG. 3 depicts an HPLC chromatogram of fucoxanthin solution having concentration of 3.686 mg/mL.

(4) FIG. 4 depicts the chlorophyll A calibration curve.

(5) FIG. 5 depicts the HPLC chromatogram of chlorophyll A at 450 nm.

(6) FIG. 6 depicts the chromatogram of freeze-dried extracted fucoxanthin oleoresin that includes chlorophyll A.

(7) FIG. 7 depicts the Isochrysis sp. growth curve in outdoor flat panels of 100 L under nitrate starvation, expressed as % of accumulated fucoxanthin vs. age (day).

(8) FIG. 8 depicts the Isochrysis sp. growth curve in outdoor flat panels of 100 L under nitrate starvation, expressed as dry weight (DW) vs. age (day).

(9) FIG. 9 depicts the Isochrysis sp. (induced with addition of auxin) growth curve expressed as dry weight (DW) vs. age (day).

(10) FIG. 10 depicts the Isochrysis sp. (induced with starvation of blue-green light and high pH) growth curve expressed as % of fucoxanthin vs. age (day).

(11) FIG. 11 depicts the Amphora sp. (induced with addition of auxin) growth curve expressed as dry weight (DW) vs. age (day).

(12) FIG. 12 depicts the Amphora sp. (induced with starvation of blue light and high pH) growth curve expressed as dry weight (DW) vs. age (day).

DETAILED DESCRIPTION OF THE INVENTION

(13) The present invention provides an improved process for producing fucoxanthin and/or polysaccharides in high yield and purity.

(14) According to some embodiments of the present invention, dry microalgae powder rich in fucoxanthin and/or polysaccharides and/or solid oleoresin rich in fucoxanthin and other compounds such as polyunsaturated fatty acids (PUFA) and/or solutions rich in polysaccharides and/or sulfated polysaccharides are provided by the process disclosed herein.

(15) Applicant has developed an improved process for producing fucoxanthin and/or polysaccharides from microalgae comprising the following steps: (a) Cultivating the microalgae medium using inducers to enable rapid cell growth for enhanced production of fucoxanthin and/or polysaccharides; (b) Harvesting and drying the algal culture to produce a dry culture; (c) Carrying out air pressure milling to crack the cell walls of the algae; (d) Extracting the dry culture to produce extracts rich in fucoxanthin and/or polysaccharides; and (e) Separating the extraction mixture into a biomass fraction and a fucoxanthin-rich oleoresin and/or polysaccharide-rich extract and optionally further purifying said fucoxanthin-rich oleoresin and/or polysaccharide-rich extract.

(16) According to the present invention, as used herein, the term Optical Density (O.D) relates to the absorbance measured in a spectrophotometer at wavelength of, e.g., 450 nm of e.g., fucoxanthin samples (FIGS. 1 and 2) or, e.g., chlorophyll A sample (FIG. 4) using solutions of variable concentrations.

(17) According to the present invention, as used herein, the term supernatant refers to the remaining solution or liquid obtained by centrifugation or precipitation.

(18) According to the present invention, as used herein, calibration curve is a method of determining the concentration of a substance in an unknown sample by comparing the unknown to a set of samples of known concentrations.

(19) According to the present invention, as used herein, supercritical fluid extraction (SFE) is a process of separating one component (the extractant) from another (the matrix) using supercritical fluids as the extracting solvent. Extraction is from a solid matrix, but can also be from liquids. SFE can be used to either strip unwanted material from a product (e.g., decaffeination) or to collect a desired product (e.g., an essential oil). Usually, liquid carbon dioxide (CO.sub.2) is one of the most used supercritical fluids.

(20) According to the present invention, as used herein, auxins are plant brassinosteroidal hormones that are added to induce higher rates of biomass acquisition.

(21) According to the present invention, as used herein, the term growth medium refers to either growth medium or culture medium that are liquid or gel mixtures that support the growth of microorganisms or cells or small plants by using specific cell types derived from plants and microbiological culture, which are used for growing, e.g., microalgae and microorganisms.

(22) According to an aspect of the present invention, the microalgae is selected from Phaeodactykum sp., Isochrysis sp., Amphora sp., Naviculla lensi, Naviculla incerta and Chaeotocerous sp.

(23) According to another aspect of the present invention, cultivating the microalgae is carried out in a medium that induces growth, thus enabling rapid cell development and mass acquisition.

(24) According to some embodiments of the present invention, cultivating the microalgae is carried out using stress conditions (that induce the synthesis and accumulation of fucoxanthin) selected form nitrogen or nitrate starvation, phosphorous starvation, light starvation, selecting specific light wavelengths, changing the light wave-length during cultivation (e.g., from blue-green to green-red), adding H.sub.2O.sub.2, applying heat, using higher pH values (up to 9.0), adding chlorine ions, adding plant hormones (such as auxins and cytokinins), exposing the medium to ozone and combination of stress conditions thereof.

(25) According to some embodiments of the present invention, cultivating microalgae is carried out in open, closed, or semi-closed systems.

(26) According to a specific embodiment of the present invention, cultivating the microalgae is carried out in an open system.

(27) According to a specific embodiment of the present invention, cultivating the microalgae is carried out in a closed system.

(28) According to a specific embodiment of the present invention, cultivating the microalgae is carried out in a semi-closed system.

(29) FIGS. 7-12 show that combinations of stress factors such as nitrate starvation, using higher temperatures (application of heat), using higher pH values (up to 9.0) and changes in the wavelengths (color) of the light (to blue-green or to green-red) cause inhibition of the growth of the algae along with induced accumulation of fucoxanthin. The accumulation of dry matter (DW) in, e.g., FIGS. 8, 11 and 12 is rapid until day 6, after which time it becomes constant.

(30) Example 3 demonstrates the impact of inducing nitrate starvation on Isochrysis sp. as well the impact of inducing changes in light wavelengths on Isochrysis sp. grown with addition of auxins, as detailed in Table 1:

(31) TABLE-US-00001 TABLE 1 Induction of nitrate Induction of changes in Parameter starvation light wavelengths Dry weight gain 0.75 g/L/day 1.4 g/L/day Fucoxanthin gain 8.38 mg/L/day 16.41 mg/L/day Fucoxanthin induction 1.57 mg/L/day 11.77 mg/L/day Harvested fucoxanthin 79.2 mg/L 176.7 mg/L

(32) Example 3 further demonstrates the impact of inducing nitrate starvation on Amphora sp. as well as the impact of inducing changes in light wavelengths and using high pH on Amphora sp. grown with auxin, as detailed in Table 2 below.

(33) TABLE-US-00002 TABLE 2 Induction of nitrate Induction of changes in Parameter starvation light wavelengths Average dry weight 0.67 g/L/day 1.16 g/L/day gain Average fucoxanthin 9.26 mg/L/day 18.52 mg/L/day gain Average fucoxanthin 2.05 mg/L/day 8.98 mg/L/day induction Average harvested 73.8 mg/L 169.3 mg/L fucoxanthin

(34) According to specific embodiment of the present invention, the addition of plant brassinosterodial hormones, such as cytokinins and auxins, during the first cultivating stage results in higher rates of biomass acquisition.

(35) According to a specific embodiment of the present invention, harvesting the microalgae is carried out by separating the microalgae from the aqueous mixture by centrifugation in, e.g., an industrial centrifuge followed by drying.

(36) According to some embodiments of the present invention, drying is carried out by at least one method selected from lyophilization, spray drying, evaporation, air or vacuum drying, exposure to hot air, refractory window belt drying, drying in an oven and combination of methods thereof.

(37) According to another aspect of the present invention, harvesting the microalgae is carried out by separating water from the microalgae by, e.g., centrifugation, scale of which depends on the microalgae species and the salinity of the cultivation medium.

(38) According to another aspect of the present invention, the harvested biomass is concentrated into a solid, then washed with water in order to reduce salinity and optionally re-centrifuged to afford a product containing at least 10% solids, preferably at least 30% solids and most preferably of at least 45% solids.

(39) According to another aspect of the present invention, the supernatant obtained by centrifugation and optionally re-centrifugation of the harvested biomass is used for purification of secreted polysaccharides.

(40) According some embodiments of the present invention, fucoxanthin is separated from the proteins and extracted using organic solvents and/or supercritical fluids (SCF) such as liquid CO.sub.2.

(41) According to some embodiments of the present invention, the solvent used for extraction is selected from ethanol, isopropyl alcohol, n-butanol, diethyl ether, diisopropyl ether, n-pentane, n-hexanes, n-heptane, cyclohexane, petroleum ether, tetrahydrofuran, methyltetrahydrofuran, acetonitrile, acetone, methylethyl ketone, methylisobutyl ketone, ethyl acetate, n-propyl acetate, isopropyl acetate and mixtures of solvents thereof.

(42) According to an aspect of the present invention, phosphoric acid is added to the solvent used for extraction.

(43) According to some embodiments of the present invention, an edible oil selected from soya oil, corn oil, sunflower oil, sesame oil and combinations thereof is used as solvent for the extraction or used as co-solvent.

(44) According to some embodiments of the present invention, the SCF solvent used for extraction of fucoxanthin-enriched oleoresin from the microalgae dry biomass is selected from liquid SCF-CO.sub.2, butane, propane, N.sub.2O and the like, preferably liquid SCF-CO.sub.2.

(45) According to another aspect of the present invention, the supercritical fluid extraction (SFE) conditions for the supercritical CO.sub.2 are above the critical temperature of 31° C. and critical pressure of 74 bars.

(46) According to another aspect of the present invention, the solvent is passed through the dried algal matrix using an extraction pressure in the range of about 500-1000 bars and a temperature in the range of about 50-100° C.

(47) According to another aspect of the present invention, the O.sub.2 to biomass ratio is between 30:1 and 500:1.

(48) According to another aspect of the present invention, the CO.sub.2 flow rate is between 100-700 Kg per hour.

(49) According some embodiments of the present invention, the drying process is optionally performed with addition of powdering agent or anti-caking agent selected from magnesium stearate, lactose, lecithin, talc, hydroxypropyl methylcellulose, micro-crystalline cellulose, sodium alginate, sodium aluminosilicate, silicone hydroxide, chitosan and the like and combination of powdering agents or anti-caking agents thereof.

(50) According to some embodiments of the present invention, the fucoxanthin-rich oleoresin is further refined by additional SCF extraction sequences (e.g., up to 5 extractions) optionally in combination with liquid chromatography.

(51) According to another aspect of the present invention, the remaining pulp after, e.g., the fifth extraction sequence is dissolved in hot water to enable purification of polysaccharides.

(52) According to preferred embodiment of the present invention, the process provided herein affords crude product containing at least about 1.5% fucoxanthin in the dry biomass and at least about 10% fucoxanthin in the extract.

(53) According to some embodiments of the present invention, prior to or after the SCF extraction, the dried biomass is treated enzymatically using enzymes such as proteases and/or extracted with hot water and/or by hot steam and/or treated by washing with buffer solution and/or treated to denature the proteins that are bound to the fucoxanthin.

(54) According to another aspect of the present invention, the remaining pulp (e.g., after the fifth extraction) is dissolved in hot water and/or in acidic aqueous solution.

(55) According to some embodiments of the present invention, fucoxanthin is separated from the polysaccharides and purified by a method selected from filtration, gel-filtration on columns, using for example natural silica beads as medium for filtration, ion-exchange chromatography, liquid chromatography methods including preparative TLC or preparative HPLC and combination of methods thereof to afford highly pure fucoxanthin.

(56) According to some embodiments of the present invention, highly pure fucoxanthin, obtained by, e.g., liquid chromatography, is used alone or in combination with other active ingredients as dietary supplement or as active pharmaceutical ingredient in formulations for preventing, ameliorating or treating a condition or disease selected from cancer, metabolic syndrome including overweight, obesity, high blood cholesterol LDL, high-blood triglycerides, diabetes type II, insulin resistant diabetes, high-blood sucrose, atherosclerosis, dementia, Alzheimer's disease, loss of memory, multiple-sclerosis, depression including environmental-stress, heat stress and general neuroprotection

(57) The present invention provides pharmaceutical compositions that contain highly pure fucoxanthin, obtained as described herein, in admixture with pharmaceutically acceptable excipients and, optionally, other therapeutic agents.

(58) According to some embodiments, the pharmaceutical compositions of the present invention that are, e.g., formulated dosage forms, are administered, for example, as tablets, pills, powders, granules, dragees, liquids, suspensions, emulsions, granules, capsules, suppositories, injection preparations (solutions and suspensions), patches and the like.

(59) According to some embodiments of the present invention, the highly pure fucoxanthin is used alone or in combination with other active ingredients in cosmetic preparations such as ointments, gels, creams, solutions, emulsions, lotions and the like for topical or other forms of administration to be used as anti-aging, skin-whitening, skin protection and other cosmetic uses.

(60) In some embodiments, pharmaceutical compositions comprising the fucoxanthin of the present invention are prepared by mixing said fucoxanthin with at least one additional active ingredient selected from absorption accelerators, binders, bulking agents, carriers, coating agents, diluents, disintegrants, extenders, fillers, flavoring agents, lubricants, surface-active agents, wetting agents and the like.

(61) According to some embodiments of the present invention, nutraceuticals, dietary supplements or food preparations comprising the fucoxanthin and/or the polysaccharides of the present invention are prepared by mixing said fucoxanthin and/or polysaccharides with food ingredients such as sugars and starches, dietary fibers, lipids, amino acids, proteins such as protein isolates or protein hydtrolyzates, lactic acid, vitamins, minerals and other ingredients that are commonly used in such preparations.

(62) According to some embodiments of the present invention, the nutraceuticals, dietary supplements or food preparations of the present invention may include, for example, a beverage, a soup, a snack, a dairy product and the like.

(63) Reference is now made to the following examples, which, together with the above description, serve to illustrate the invention without limiting its scope. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art.

EXAMPLES

Example 1

(64) This example details the cultivation of the microalgae Phaeodactylum, wild type strain 646.

(65) The microalgae Phaeodactylum, wild type strain 646, was cultivated under artificial light in agar plates and tubes and transferred into Erlenmeyer flasks and 5 liter round glass flasks. Cultures were then transferred into 3 liter columns and cultivated. The correlation between illumination conditions (shade vs. full sunlight), dry weight accumulation and fucoxanthin accumulation have been studied. Table 3 below details the growth medium compounds that were added to the liquid stock that had initial concentration of 30 g/L and final concentration of 30 mg/L.

(66) A blend of 34 g of salts was added to 980 mL de-ionized water along with potassium nitrate, potassium dihydrogen phosphate and a blend of microelements. The volume was adjusted to 1 L and the mixture was autoclaved. After cooling, 1 mL each of ferric citrate and 20 mM Tris buffer (pH 7.6) were added. Table 3 below details the initial concentrations of the compounds that were added.

(67) TABLE-US-00003 TABLE 3 Compound Initial concentration Salts blend 34 g/L KNO.sub.3 2 g/L KH.sub.2PO.sub.4 70 mg/L Ferric citrate 1 ml/L Citric acid 42.8 μM Na.sub.2SiO.sub.3 9H.sub.2O 1 ml/L Microelements blend 1 ml/l Vitamins (soluble) 0.5 ml/L

(68) Table 4 below details the quantities and initial concentrations of the microelements that were added to the 1 L solution:

(69) TABLE-US-00004 TABLE 4 Compound Quantity (g) Initial concentration, μM ZnSO.sub.4 7H.sub.2O 0.22 0.77 CuSO.sub.4 5 H.sub.2O 0.08 0.31 Na.sub.2MoO.sub.4 2 H.sub.2O 0.39 1.61 H.sub.3BO.sub.3 2.86 46.3 MnCl.sub.2 4 H.sub.2O 1.81 9.15 CO(NO.sub.3).sub.2 6 H.sub.2O 0.05 0.17

(70) Table 5 below details the quantities and initial concentrations of the vitamins that were added:

(71) TABLE-US-00005 TABLE 5 Compound Quantity Initial concentration Vitamin B12 1 ml 1 g/L Biotin 1 ml 1 g/L Thiamine HCl 200 mg 0.2 g/L

Example 2

(72) This example details the production of fucoxanthin in columns.

(73) Four columns were loaded each with initial concentration of 0.4 g/L stock solution. Column No. 1 was 50-60% shaded every day until 17:00 hours. Column No. 2 had the light scattered to the “green” wavelength group by filtering through a green-red filter. Column No. 3 had the light scattered to the “blue” wavelength group by filtering through a blue filter. Column No. 4 was not shaded and instead it was subjected to full sunlight. Cultivation was carried out at temperatures of 24-25° C. with a flux of 1% CO.sub.2. The following Table 6 below includes data of results measured after two weeks of cultivation.

(74) TABLE-US-00006 TABLE 6 Light Dry weight Fucoxanthin Fucoxanthin Column No. scattering (g/L) (mg/L) (%) 1 Shade 5.3 46.1 0.87 2 Green 5.7 68.4 1.2 3 Blue 7.1 99.4 1.4 4 Full sunlight 6.5 40.9 0.63

Example 3

(75) This example describes the cultivation of the microalgae Isochrysis sp, wild type.

(76) The microalgae Isochrysis sp. wild type, was cultivated under artificial light in agar plates and tubes and transferred into Erlenmeyer flasks and 5 L round glass flasks. Then, the cultures were transferred into 7 L plastic sleeves to form solutions with maximal concentration of 6 g/L and cultivated under full sunlight. The cultures were collected and diluted to a concentration of 2 g/L and seeded in plastic bags. The correlation between the illumination conditions (wavelength), dry weight accumulation and fucoxanthin production have been studied.

(77) Table 7 below details the content of the growth medium, wherein the initial stock concentration was 30 g/L and the final stock medium concentration was 30 mg/L.

(78) TABLE-US-00007 TABLE 7 Compound Initial concentration Blend of salts 34 g/L KNO.sub.3 0.1 g/L KH.sub.2PO.sub.4 35 mg/L Ferric citrate 1 mL/L Na.sub.2SiO.sub.3 9H.sub.2O 1 mL/L Microelements blend 1 mL/L Vitamins, soluble 0.5 mL/L

(79) 34 g of salts blend was added to 980 mL of de-ionized water along with potassium nitrate (KNO.sub.3) and potassium dihydrogen phosphate (KH.sub.2PO.sub.4) and microelements blend. The volume was adjusted to 1 L and the mixture was autoclaved. After cooling, 1 mL each of ferric citrate and 20 mM Tris buffer (pH 7.6) were added.

Example 4

(80) This example demonstrates the cultivation of Isochrysis sp. and of Amphora sp.

(81) Colonies of Isochrysis sp. and of Amphora sp. were grown on agar, in petri dishes. The material was transferred into tubes containing artificial sea water medium. Cultures were diluted from the test tubes into the flasks and further to polyethylene sleeves and grown under artificial light and injection of filtered air enriched with 1% CO.sub.2.

(82) Cultures grown in sleeves were transferred into outdoor 100 liter flat panels, or sleeves containing 10-15 liters. The day of transferring the cultures into 100 liter flat panels was designated as culture age day 0.

(83) The cultures were sampled daily and their dry weight and fucoxanthin concentration were determined. The growth was generally divided into two stages. First the growth conditions were set to support optimal and fast growth and acquisition of biomass. After 5-7 days of fast growth, the algae were transferred into the second production stage, which was the fucoxanthin induction stage.

Example 5

(84) This example details the production of fucoxanthin in sleeves.

(85) Four sleeves were seeded at initial concentration of 2 g/L. The first sleeve was 50-70% shaded every day until 17:00 hours. The second sleeve had light scattered to the “green” wavelength group by filtering through a green-red filter. The third sleeve had light scattered to the “blue” wavelength group by filtering through a blue filter. The fourth was not shaded and instead it was subjected to full sunlight. Table 8 below details the cultivation data after 10 days of growth:

(86) TABLE-US-00008 TABLE 8 Light Dry Weight Fucoxanthin Fucoxanthin Sleeve No. scattering (g/L) (mg/L) (%) 1 Shade 4.4 57.2 1.3 2 Green 5.5 121 2.2 3 Blue 5.9 141.6 2.4 4 Full sunlight 4.8 52.8 1.1

Example 6

(87) This example details the harvest, post processing and extraction.

(88) The algae cultures were collected and spin down using a centrifuge. The pelleted algae paste, containing about 30% solids, was washed with fresh water and re-suspended in water while mixing, frozen and freeze-dried. After freeze drying, 1% of silicon dioxide was added to the biomass powder which was further refined and homogenized by air pressure milling. The dry biomass powder (4.2 Kg), containing 2% fucoxanthin (84 g), was extracted by super-critical-fluid CO.sub.2 extraction.

(89) Table 9 below summarizes the SCF-CO.sub.2 extraction results:

(90) TABLE-US-00009 TABLE 9 Initial quantities Separator 1 Separator 2 Total Weight Fuco Weight g % Weight g % g Recovery % 4.2 Kg 84 g 606 g 74.8 12.3 95.7 4.0 4.2 78.8 93.8 Fuco = Fucoxanthin

(91) The SCF extraction of 4.2 Kg of biomass powder containing 84 g (2%) fucoxanthin resulted in 606 g fucoxanthin oleoresin (separator 1)+95.7 g fucoxanthin oleoresin (separator 2)=701.7 grams of fucoxanthin oleoresin containing 11.2% fucoxanthin. Thus, the recovery of fucoxanthin was: 74.8 g (separator 1)+4.0 g (separator 2)=78.8 g fucoxanthin (93.8%).

Example 7

(92) This example details the extraction of fucoxanthin using an organic solvent.

(93) The biomass powder was incubated twice in absolute ethanol for 4 hours at ambient temperature and extracted. The extract was partitioned by liquid/liquid extraction with hexane. The solvents were removed by vacuum distillation.

Example 8

(94) This example details the HPLC method for the analysis of fucoxanthin.

(95) Samples were dissolved in a 1:1:1 solvent mixture of acetone/methanol/hexane or in acetonitrile and diluted in same solvent mixture or solvent respectively. Measurements of O.D values were carried out at peak absorbance level of fucoxanthin (450 nm).

(96) Data Relating to Fucoxanthin Separation by HPLC: Column: C18 250×4.6 Wave lengths: 450 nm Flow rate: 0.8 mL/min. Sampler temperature: 15° C.; Column temperature: 28° C. Mobile phase: (A)—methanol 85%+0.5M ammonium acetate; (B)—acetonitrile: water (90:10); (C)—ethyl acetate. Run time: 35 min.

(97) Table 10 below details the HPLC gradient used:

(98) TABLE-US-00010 TABLE 10 Time (min.) A (%) B (%) C (%) 0 60 40 0 2 0 100 0 7 0 80 20 17 0 50 50 21 0 30 70 29.5 0 100 0 30 60 40 0

(99) All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

(100) The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(101) Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.